1. CAR系列细胞治疗的技术迭代与多场景应用
1.1 CAR-T细胞在血液肿瘤与自身免疫病中的突破
嵌合抗原受体T细胞（CAR-T）疗法作为一种个性化癌症治疗方案，在血液肿瘤治疗领域取得了革命性进展，尤其是在复发/难治性B细胞恶性肿瘤中展现出显著疗效12。2023年至2025年期间，CAR-T技术持续迭代，并在多发性骨髓瘤、淋巴瘤以及自身免疫病等领域取得了新的突破。
在多发性骨髓瘤（MM）治疗方面，靶向B细胞成熟抗原（BCMA）的CAR-T疗法表现出令人鼓舞的长期疗效和可控的安全性。例如，一项针对复发/难治性MM患者的BCMA CAR-T治疗研究显示，在中位随访20.2个月时，总缓解率（ORR）达到94.8%，其中50.7%的患者实现完全缓解（CR）。4年无进展生存率（PFS）和总生存率（OS）分别为37.4%和63.2%，生存曲线随时间推移逐渐趋于平稳3。另一项针对来那度胺难治性MM患者的III期临床试验（CARTITUDE-4）中，单次注射Ciltacabtagene Autoleucel（Cilta-cel，一种BCMA靶向CAR-T细胞疗法）相比标准化治疗显著降低了疾病进展或死亡风险，中位无进展生存期在Cilta-cel组尚未达到，而标准治疗组为11.8个月（风险比0.26；P<0.001）4。该研究结果在12个月时的无进展生存率为75.9%，远高于标准治疗组的48.6%，且Cilta-cel组的总缓解率、完全缓解率或更好缓解率以及微小残留病（MRD）阴性率均显著优于标准治疗组4。这些数据进一步巩固了BCMA CAR-T在MM治疗中的重要地位。
在B细胞非霍奇金淋巴瘤（B-NHL）领域，抗CD19 CAR-T细胞疗法已成为治疗复发/难治性疾病的变革性疗法，能够诱导持续缓解5。一项使用非病毒、基因特异性靶向CAR-T细胞（通过CRISPR-Cas9技术将抗CD19 CAR盒插入AAVS1安全港位点）的临床研究（NCT04213469）在8例复发/难治性侵袭性B细胞非霍奇金淋巴瘤患者中取得了87.5%的完全缓解率，且未发生严重不良事件，显示出高安全性和有效性6。
除了血液肿瘤，CAR-T疗法在自身免疫病中的应用也逐渐成为研究热点。靶向B细胞的CAR-T细胞疗法，特别是针对CD19或BCMA的疗法，因其能够广泛重置B细胞区室，在自身免疫性疾病管理中展现出独特优势78。目前，大多数针对自身免疫性风湿病（ARDs）的CAR-T细胞临床试验仍处于早期阶段，其中64.29%为I期试验，主要集中于系统性红斑狼疮（SLE）和狼疮性肾炎等疾病8。例如，针对重症肌无力（Myasthenia Gravis, MG）这一由自身抗体介导的疾病，RNA CAR-T疗法Descartes-08（靶向BCMA）的Ib/IIa期临床试验（MG-001）显示出良好的安全性和耐受性，且无需进行淋巴细胞清除化疗。患者在输注后12个月的随访中，MG严重程度评分持续临床意义上的改善910。此外，一项针对SLE伴狼疮性肾炎患者的BCMA-CD19复合型CAR-T细胞（cCAR）I期临床研究显示，该疗法能够安全有效地诱导疾病缓解，并在3个月内使所有自身抗体转阴，补体水平恢复正常，患者症状和免药物缓解（MFR）持续长达46个月，且B细胞在2-6个月内完全恢复11。这些研究表明CAR-T疗法，特别是其RNA和双靶点复合设计，在治疗自身免疫性疾病方面具有巨大潜力。
1.2 CAR-NK与CAR-M的实体瘤治疗潜力
尽管CAR-T细胞疗法在血液肿瘤中取得了显著成功，但其在实体瘤治疗中的应用仍面临诸多挑战，包括肿瘤微环境的免疫抑制、CAR-T细胞浸润受限、肿瘤抗原异质性以及潜在的脱靶毒性等 12131415。为克服这些限制，嵌合抗原受体自然杀伤细胞（CAR-NK）和嵌合抗原受体巨噬细胞（CAR-M）作为替代或补充策略应运而生，展现出在实体瘤治疗中的巨大潜力。
CAR-NK细胞：无HLA限制与低毒性优势
CAR-NK细胞结合了CAR技术特异性识别抗原的能力与NK细胞固有的自然杀伤活性 16。相较于CAR-T细胞，CAR-NK细胞具有显著优势：
- 无HLA限制性： NK细胞的激活不依赖于主要组织相容性复合体（MHC），这意味着异基因CAR-NK细胞可以“现货即用”（off-the-shelf），无需进行HLA配型，从而简化制备流程、缩短等待时间并降低成本 121315。
- 安全性更高： CAR-NK细胞通常表现出较低的细胞因子释放综合征（CRS）和免疫效应细胞相关神经毒性综合征（ICANS）的风险，且不易引起移植物抗宿主病（GvHD），这使得其安全性优于CAR-T细胞 121315。
- 多来源可及性： CAR-NK细胞可以从多种来源大规模制备，包括外周血、脐带血、诱导多能干细胞（iPSC）和NK细胞系等，为“现货型”产品的开发提供了可能 1216。
- 肿瘤微环境适应性： 研究表明，CAR-NK细胞能够更好地适应实体瘤的缺氧和代谢障碍等微环境挑战 14。
在实体瘤治疗方面，CAR-NK细胞的临床前研究已取得积极进展。例如，在肝癌和胰腺癌等实体瘤模型中，靶向肿瘤相关抗原的CAR-NK细胞展现出有效的肿瘤杀伤能力。通过优化CAR设计和联合治疗策略，例如与化疗、放疗、免疫检查点抑制剂或表观遗传调节剂联用，可以进一步提高CAR-NK细胞的疗效 16。此外，有研究探索将CAR-NK细胞作为药物载体，利用其特异性肿瘤归巢能力将抗癌药物递送至肿瘤部位，并在体内小鼠模型中观察到增强的抗肿瘤免疫效应 17。
CAR-M细胞：强大的吞噬与抗原呈递优势
巨噬细胞因其固有的肿瘤浸润能力、强大的吞噬作用、抗原呈递功能以及调节肿瘤微环境的能力，被认为是实体瘤细胞治疗的有力候选者 1819202122。将CAR技术引入巨噬细胞（CAR-M），使其能够特异性识别和吞噬肿瘤细胞，并进一步激活适应性免疫反应 181921。
CAR-M细胞的主要优势包括：
- 优异的肿瘤浸润能力： 巨噬细胞是肿瘤微环境中的主要免疫细胞成分，其能够有效浸润实体瘤，克服CAR-T细胞在实体瘤中浸润不足的问题 1218192123。
- 多重抗肿瘤机制： CAR-M细胞不仅可以直接吞噬肿瘤细胞，还能分泌促炎细胞因子，重塑肿瘤微环境，并作为高效的抗原呈递细胞（APC）激活T细胞，从而启动更广泛的抗肿瘤免疫反应 192124。
- 安全性良好： 早期临床研究显示，CAR-M细胞疗法具有良好的安全性和耐受性，未观察到严重CRS或神经毒性 24。
在实体瘤，特别是胰腺癌和HER2过表达实体瘤中，CAR-M细胞的临床前和早期临床开发正在进行。例如，针对胰腺导管腺癌（PDAC）这一恶性程度极高的肿瘤，CAR-M疗法有望克服其高度纤维化和免疫抑制性微环境的障碍 19。一项I期临床试验（NCT04660929）评估了抗HER2 CAR-M细胞（CT-0508）在HER2过表达晚期实体瘤患者中的安全性、耐受性和初步疗效。结果显示，CT-0508具有良好的安全性和可制造性，未发生剂量限制性毒性或3级以上CRS/ICANS。在HER2 3+肿瘤患者中，44%（4/9）的患者在治疗后8周达到疾病稳定（Stable Disease），并且观察到CT-0508能够进入肿瘤组织并重塑肿瘤微环境，导致CD8 T细胞扩张 24。这些结果为CAR-M在实体瘤治疗中的应用提供了初步的人体证据。
大规模制备与肿瘤微环境渗透的优化方向
为实现CAR-NK和CAR-M在实体瘤治疗中的广泛应用，仍需解决大规模制备和优化肿瘤微环境渗透的关键问题：
- 大规模制备： 优化细胞来源（如iPSC）、开发无血清培养基、采用封闭式自动化生产系统是实现CAR-NK/M细胞大规模、标准化生产的关键。这将有助于降低成本并确保产品质量和均一性。
- 肿瘤微环境渗透： 针对实体瘤的纤维化屏障和免疫抑制性微环境，需探索多种策略：
通过不断的技术创新和策略优化，CAR-NK和CAR-M细胞有望成为实体瘤治疗的重要新选择，为肿瘤患者带来新的希望。
1.3 下一代CAR设计与耐药性突破
尽管CAR-T细胞疗法在血液肿瘤治疗中取得了里程碑式的成就，但肿瘤抗原逃逸、T细胞耗竭、脱靶毒性以及在实体瘤中疗效不佳等挑战依然存在 252627。为克服这些局限性，2023-2025年期间，CAR设计策略持续演进，聚焦于开发多靶点共刺激、逻辑门控以及可调控CAR等“下一代”CAR系统，以期提升疗效、降低毒性并拓宽应用范围。
多靶点共刺激CAR设计：提升疗效与克服抗原逃逸
肿瘤细胞的异质性以及治疗后抗原丢失是导致CAR-T疗法失败的关键因素。为应对这一挑战，结合多个靶点进行设计成为重要的发展方向。多特异性CAR-T细胞能够同时识别或顺序识别肿瘤细胞上的不同抗原，从而有效降低抗原逃逸的风险，并增强对肿瘤的杀伤力 28。
例如，针对B细胞淋巴瘤，CD19抗原丢失是复发的重要原因之一。研究人员开发了双靶点CAR-T细胞，同时靶向CD19和CD22，以提高治疗的持久性 29。一项I期临床试验（NCT04007029）评估了靶向CD19/CD20的双特异性CAR-T细胞（CART19/20）在复发/难治性非霍奇金淋巴瘤（NHL）患者中的安全性和有效性。结果显示，该疗法具有良好的安全性（未出现神经毒性，CRS最高为1级），并在10名患者中实现了90%的总缓解率（ORR）和70%的完全缓解率（CR），中位无进展生存期达到18个月。一名患者在18个月后复发，但经二次CART19/20治疗后再次达到CR 30。另一项针对复发或难治性B细胞恶性肿瘤患者的CD19和/或CD22双靶点CAR-T细胞（CD19-22.BB.z-CAR）I期临床试验（NCT03233854）也显示出良好的疗效，尽管在部分复发病例中仍观察到CD19抗原丢失，但未出现CD22抗原丢失相关复发 29。这些研究表明，多靶点CAR设计在难治性淋巴瘤中展现出提升疗效和克服抗原逃逸的潜力。
此外，第三代CAR设计通过整合多个共刺激信号域（如CD28、OX40、4-1BB），旨在促进IL-2合成、激活T细胞并防止细胞耗竭，从而提高CAR-T细胞的持久性和抗肿瘤活性 25。例如，有研究通过构建同时含有CD28和4-1BB共刺激域的CAR来增强T细胞的活化和增殖能力。
逻辑门控CAR设计：提升特异性与降低脱靶效应
为了解决CAR-T细胞在识别肿瘤抗原时可能产生的“脱靶毒性”（on-target, off-tumor toxicity），逻辑门控CAR（Logic-gated CARs）应运而生。这类CAR系统通过模拟生物学的逻辑运算（如“AND”门、“OR”门、“NOT”门）来提高对肿瘤细胞识别的精准性，最大限度地减少对正常组织的损伤 28。
- “AND”门控CAR： 要求T细胞同时识别两个或多个肿瘤相关抗原才能完全激活。例如，针对在多种实体瘤中过表达但在正常组织中表达较低的两个抗原，设计一个需要同时识别这两个抗原才能激活的CAR-T细胞。这种设计显著提高了靶向特异性，降低了脱靶毒性。
- “OR”门控CAR： 允许T细胞识别任一靶点即可激活，常用于对抗抗原异质性或抗原下调导致的免疫逃逸。这与多靶点CAR类似，但更强调逻辑选择的灵活性。
- “NOT”门控CAR： 也称为抑制型CAR，当CAR-T细胞识别到正常组织上特异性表达的抗原时，其杀伤功能会被抑制。例如，肿瘤细胞表达抗原A，而正常细胞同时表达抗原A和抗原B。通过设计一个识别抗原A并同时包含识别抗原B的抑制信号的CAR-T细胞，可以在保留肿瘤杀伤能力的同时保护正常组织。
这些逻辑门控CAR设计为精准肿瘤免疫治疗提供了新的思路，特别是在靶点选择有限或靶点在正常组织中也有低水平表达的情况下，能够有效平衡疗效与安全性。
可调控CAR设计：实现活性的精准控制
可调控CAR系统旨在通过外部信号（如小分子药物、光照或特定蛋白质）来精确控制CAR-T细胞的活性、增殖和持久性 2631。这种“开关”机制可以显著提高CAR-T疗法的安全性，尤其是在出现严重不良反应（如细胞因子风暴或神经毒性）时，能够及时关闭CAR-T细胞的活性，从而减轻副作用。
- 小分子诱导型开关： 最常见的策略是利用小分子药物诱导CAR结构域的二聚化，从而激活T细胞。当小分子药物撤除后，CAR活性随之降低。这种设计提供了在体内的可逆控制。
- 自杀基因： 将自杀基因（如单纯疱疹病毒胸苷激酶，HSV-TK，或诱导型半胱天冬酶9，iCasp9）整合到CAR-T细胞中。当出现严重毒性时，通过给予特定的药物，可以诱导CAR-T细胞凋亡，从而迅速清除体内的CAR-T细胞 2531。
- 可逆调节型CAR： 通过改变CAR的表达水平或信号强度来调节其活性。例如，利用缺氧诱导元件（HIF）在肿瘤微环境缺氧条件下条件性驱动CAR表达，实现对肿瘤细胞的精准打击，同时减少对正常组织的损伤 25。
这些可调控CAR设计为临床医生提供了更强的治疗掌控力，有望在提高CAR-T疗法安全性的同时，优化其在不同疾病阶段和患者状况下的应用策略。
总而言之，下一代CAR设计正朝着多功能、高特异性、可控性的方向发展。通过整合多靶点识别、逻辑门控和可调控开关等先进技术，未来的CAR细胞疗法有望克服当前面临的诸多挑战，在血液肿瘤和实体瘤治疗中发挥更大的潜力，并为自身免疫病等非肿瘤适应症的治疗开辟新途径 2627。
2. 间充质干细胞（MSC）及外泌体的临床转化深化
2.1 MSC的免疫调节与再生修复应用
间充质干细胞（Mesenchymal Stem Cells, MSCs）因其多向分化潜能、免疫调节特性和旁分泌功能，在组织修复、炎症控制和免疫疾病治疗领域展现出巨大潜力 323334。近年来，MSCs的临床转化研究不断深入，特别是在移植物抗宿主病（GvHD）和脊髓损伤等疾病中取得了III期临床试验进展，并对其不同来源的疗效差异及归巢机制优化进行了探索。
MSC在移植物抗宿主病（GvHD）中的临床进展
GvHD是异基因造血干细胞移植（allo-HSCT）后严重的并发症，尤其类固醇难治性急性GvHD（SR-aGvHD）缺乏有效治疗手段。MSCs因其强大的免疫调节作用，能够抑制T细胞增殖、调节树突状细胞功能、促进调节性T细胞生成，从而减轻GvHD的炎症反应 343536。多项III期临床试验评估了MSCs在SR-aGvHD中的疗效。
例如，一项多中心、随机研究评估了Remestemcel-L（骨髓来源的MSCs）联合二线治疗在SR-aGvHD患者中的疗效。结果显示，Remestemcel-L组在主要终点（持续完全缓解率, DCR）上未达到显著优于安慰剂组的水平（35% vs 30%；P = 0.42）。然而，在事后分析中，对于肝脏受累患者和高危患者（aGvHD III-IV级），Remestemcel-L显示出更高的整体缓解率。特别是儿童患者，MSC治疗组的总体缓解率显著高于安慰剂组（64% vs 23%；P = 0.05）。该研究还表明Remestemcel-L是安全且耐受性良好的 37。
另一项在中国进行的随机III期临床试验（NCT02241018）评估了MSCs联合巴利昔单抗和钙调神经磷酸酶抑制剂作为SR-aGvHD二线治疗的效果。该研究入组了203名患者，结果显示，MSC组在第28天的总体缓解率（OR）显著高于对照组（82.8% vs 70.7%；P = 0.043）。在第56天的持续总体缓解率、无失败生存期以及2年慢性GvHD累积发生率方面，MSC组均表现出优势。此外，MSC组的药物毒性更低，且未增加肿瘤复发风险，证实了MSCs联合二线治疗SR-aGvHD的有效性和安全性 38。
日本进行的一项II/III期研究也评估了骨髓来源MSCs（JR-031）治疗类固醇难治性III或IV级aGvHD的疗效。在首次MSC输注后4周，25名患者中有24%达到完全缓解（CR），36%达到部分缓解（PR）。主要终点是24周的持久CR，达到了48%。该研究同样未观察到与MSC输注相关的常见不良反应 39。这些III期临床试验的结果总体上支持了MSCs在SR-aGvHD治疗中的潜力，尤其是在特定患者亚组中表现出更显著的疗效。
MSC在脊髓损伤（SCI）中的临床探索
脊髓损伤是一种严重的神经系统疾病，MSCs的再生和神经保护特性使其成为潜在的治疗策略。MSCs能够促进轴突再生、抑制炎症反应、减少细胞凋亡和提供神经营养支持。
然而，在脊髓损伤领域的III期临床试验结果相对谨慎。一项评估自体MSC治疗脊髓损伤的III期临床试验发现，尽管MSCs注射是安全的，但其治疗效果有限。在16名慢性完全性脊髓损伤患者中，仅有2名患者的神经功能有所改善，且伴随着弥散张量成像显示脊髓束连续性的出现。研究指出，单次MSCs应用效果较弱，未来需要进一步优化MSC注射方案以增强疗效 40。这表明，在复杂疾病如脊髓损伤中，MSCs的治疗效果可能受多种因素影响，包括给药途径、剂量、给药时机以及细胞活性等。
不同来源MSC的疗效差异及归巢机制优化
MSCs可以从多种组织中分离获得，包括骨髓（BM-MSC）、脂肪组织（AD-MSC）和脐带（UC-MSC）等 32。不同来源的MSCs在增殖能力、分化潜能和免疫调节特性上可能存在差异，这可能影响其临床疗效。例如，脐带来源的MSCs通常具有更强的增殖能力和更低的免疫原性，被认为是“现货型”细胞产品开发的优选 32。
为了克服MSCs体内存活率低和靶向归巢效率不足的问题，研究人员正积极探索优化MSCs归巢机制的方法 4142。
- 表面分子修饰： 通过对MSC表面进行工程改造，使其表达特定的趋化因子受体或黏附分子，可以增强MSCs对损伤或炎症部位的特异性归巢能力。例如，DNA支架化的双特异性抗体（YMV）可以修饰MSCs，使其同时靶向VCAM-1和MAdCAM-1，从而增强MSCs在炎症性肠病（IBD）模型中对结肠的归巢能力，并通过抑制白细胞浸润来减轻炎症 43。
- 生物材料载体： 结合生物材料，如水凝胶或微球，可以保护MSCs，延长其在体内的存活时间，并引导其在目标部位的分布和功能发挥 42。
- 基因工程： 通过基因编辑技术增强MSCs分泌趋化因子或免疫调节分子的能力，或使其更好地适应病理微环境。
- 联合治疗： 研究发现，MSCs与药物的联合治疗在改善MSCs治疗效果方面表现出优势。例如，在肝纤维化模型中，MSCs与药物联用能显著改善肝功能、促进受损组织修复、降低肝纤维化相关指标，并通过调节肝脏炎症微环境、促进MSCs归巢等机制有效缓解肝纤维化，且效果优于单一MSC治疗 41。这表明，选择合适的“伙伴”药物可以弥补MSCs在体内生存和植入率低的缺点。
尽管MSCs在临床应用中仍面临挑战，如标准化生产、质量控制和疗效均一性等问题 3644，但对其生物学特性和作用机制的深入理解，以及不断优化的工程化策略，正逐步推动其在再生医学和免疫治疗领域实现更广泛的临床转化。
2.2 MSC外泌体的无细胞治疗优势
间充质干细胞（MSC）外泌体（MSC-Exos）作为一种无细胞疗法，因其能够传递多种生物活性物质（如蛋白质、脂质、mRNA和miRNA）并具有低免疫原性、易于储存和运输、不形成肿瘤以及易于修饰等优势，近年来在炎症性疾病、神经退行性疾病和组织修复等领域展现出巨大的临床应用潜力 4546。
MSC外泌体在炎症性肠病（IBD）中的临床应用
炎症性肠病（IBD）是一种慢性非特异性肠道炎症性疾病，传统治疗效果有限 46。MSC-Exos通过抗炎、抗氧化应激、修复肠黏膜屏障和免疫调节等多种机制，被认为是治疗IBD的新型策略 46。例如，一项研究构建了载有小檗碱的MSC外泌体（Exos-Ber），以增强小檗碱在治疗溃疡性结肠炎（UC）中的溶解度和生物利用度。体内外研究均证实Exos-Ber具有抗氧化和抗炎特性，能修复受损上皮细胞，减少细胞凋亡，并通过调节MAPK信号通路减轻UC症状 47。虽然目前针对MSC-Exos治疗IBD的大规模临床试验数据仍较少，但其在动物模型中已显示出显著疗效，预示着未来在临床治疗IBD方面具有广阔前景 46。
MSC外泌体在神经退行性疾病中的应用
神经退行性疾病，如阿尔茨海默病、帕金森病、多发性硬化症和脊髓损伤等，由于血脑屏障（BBB）的存在，药物递送面临巨大挑战 4849。MSC-Exos能够穿越血脑屏障，将治疗分子直接递送到大脑，为这些疾病的治疗提供了无创替代方案 4548。
- 多发性硬化症（MS）与髓鞘再生： MS是一种中枢神经系统脱髓鞘疾病。骨髓MSC来源的外泌体（MSC-Exo）在脱髓鞘中枢神经系统模型中能促进髓鞘再生并减少神经炎症。动物模型研究显示，MSC-Exo能改善神经功能和认知功能，增加新生少突胶质细胞和成熟少突胶质细胞的数量，降低神经炎症，并抑制TLR2/IRAK1/NFκB通路 50。这些发现为利用无细胞外泌体疗法治疗脱髓鞘和神经退行性疾病提供了细胞和分子基础 50。
- 创伤性脑损伤（TBI）： MSC-Exos在TBI治疗中显示出疗效。一项剂量-反应和治疗窗口研究发现，单次静脉注射MSC-Exos能显著改善TBI大鼠的运动和认知功能，减少海马神经元损失，促进血管生成和神经发生，并降低神经炎症。研究还发现，100 µg/只大鼠的剂量效果最佳，且治疗窗口至少为伤后7天 51。
- 其他神经退行性疾病： MSC-Exos能够将非编码RNA（如miRNA）递送至损伤组织，通过神经再生、神经突生长、免疫系统调节、减少神经炎症和修复受损组织等机制发挥治疗作用 45。尽管目前针对MSC-Exos治疗神经退行性疾病的临床试验仍处于早期阶段（仅有三项临床试验），但其潜力巨大，预计未来临床研究将显著增加 48。
分离纯化技术突破
外泌体的分离纯化是其临床应用的关键挑战，因为外泌体的浓度、纯度、大小和功能会因分离方法不同而差异显著 49。目前，主流的外泌体分离方法包括超速离心、超滤、尺寸排阻色谱（SEC）、聚合物沉淀、免疫亲和捕获和微流控技术等 5253。
- 超速离心（Ultracentrifugation, UC）： 仍被认为是分离外泌体的“金标准”，但其缺点是耗时、需要专业设备且可能损伤外泌体结构，导致纯度受限 5254。
- 超滤（Ultrafiltration）： 通过不同孔径的滤膜进行连续过滤，可以有效浓缩外泌体，但可能存在膜堵塞和外泌体聚集的问题 5253。一项研究表明，使用超滤装置浓缩细胞培养条件培养基可增加囊泡分离量，但选择何种浓缩装置对外泌体产量影响很大 54。
- 尺寸排阻色谱（Size Exclusion Chromatography, SEC）： SEC通过多孔基质的色谱柱分离不同大小的颗粒，被认为是分离高纯度功能性外泌体的理想方法，具有操作简单、可重复性好、成本低廉且无需特殊设备等优点 5255。SEC分离的外泌体相对纯净且功能完整 55。
- 聚合物沉淀： 利用聚乙二醇（PEG）等聚合物使外泌体沉淀，操作简单，但产物纯度通常较低 5254。
- 免疫亲和捕获与微流控技术： 这些方法利用外泌体表面特异性蛋白进行捕获，具有高特异性，但样本处理量小且成本高 5253。
为提高外泌体的分离效率和纯度，多种方法联用已成为趋势 52。例如，超滤结合SEC可以有效提高外泌体产量和纯度。
靶向修饰（膜蛋白工程）
为了增强MSC-Exos的治疗效果和靶向性，对其进行工程化改造，特别是膜蛋白工程，是当前研究的热点。
- 基因工程策略： 通过修饰MSC使其表达特定的靶向蛋白或趋化因子受体，从而使其分泌的外泌体也携带这些靶向分子。例如，有研究通过基因工程改造MSC，使其分泌的外泌体表面表达PD-L1或PD-L1-ITGB1，以增强其免疫调节功能，实验结果显示这些修饰后的外泌体能更有效地抑制T细胞增殖并增加调节性T细胞比例 56。
- 直接修饰外泌体： 直接修饰外泌体表面或对其进行加载是增强其靶向性和治疗效果的策略。例如，有研究利用脂钙磷酸盐（LCP）与骨髓来源MSC外泌体融合，并用AMD3100进行靶向修饰，可实现对缺血性病灶的靶向递送，并通过传递miRNA210改善缺血性脑卒中 57。此外，对MSC-Exos进行表面修饰使其靶向富集到关节，是治疗类风湿关节炎（RA）的前沿方向 58。
- 药物载体： MSC-Exos作为天然的药物递送系统，可以封装小分子药物或非编码RNA，从而提高药物的生物利用度和靶向性 45。例如，载有tsRNA-10277的骨髓MSC外泌体可以增强地塞米松诱导的股骨头坏死模型中BMSC的成骨分化能力 59。
剂量标准化
MSC-Exos的剂量标准化是实现其临床转化的另一个关键挑战。目前，MSC-Exos的剂量通常通过外泌体蛋白质含量或颗粒数量来衡量，但缺乏统一的国际标准。不同疾病模型和给药途径所需的最佳外泌体剂量差异很大，需要进行系统的剂量-反应研究来确定 51。同时，外泌体的生产批次间质量控制（包括外泌体大小、颗粒浓度、表面标志物和内容物等）对于确保疗效和安全性至关重要。例如，通过纳米颗粒跟踪分析（NTA）、流式细胞术、可调电阻脉冲传感（TRPS）等技术对外泌体进行定量和表征，是实现剂量标准化的重要手段 53。
随着分离纯化技术的进步、靶向修饰策略的成熟以及剂量标准化研究的深入，MSC外泌体作为一种高效、安全的无细胞治疗手段，有望在未来几年内实现更广泛的临床应用，为多种难治性疾病带来新的治疗选择。
3. 基因编辑与干细胞治疗的融合创新
3.1 CRISPR-Cas9编辑干细胞的临床验证
基因编辑技术的快速发展，特别是CRISPR-Cas9系统的出现，为遗传性疾病的治疗带来了革命性的突破。通过精确修改基因组，研究人员能够纠正致病突变或引入治疗性基因。在细胞治疗领域，基因编辑技术与干细胞的结合，尤其是对造血干细胞（HSPCs）的编辑，在治疗血液系统疾病方面展现出巨大潜力。其中，exa-cel（exagamglogene autotemcel）作为首个获得FDA批准的基于CRISPR-Cas9技术的基因疗法，在治疗镰刀型细胞贫血症（SCD）和输血依赖型β-地中海贫血（TDT）方面取得了里程碑式的进展 60616263。
作用机制：增强胎儿血红蛋白表达
镰刀型细胞贫血症是由β-珠蛋白基因突变引起的遗传性血液病，导致异常血红蛋白S（HbS）形成，进而引起红细胞镰刀状变、溶血和血管阻塞性危象（VOCs）等严重临床表现 6465。胎儿血红蛋白（HbF）在出生后通常会关闭，但其具有阻止HbS聚合的作用。通过重新激活HbF的表达，可以有效减轻SCD的症状 6064。
exa-cel的作用机制正是基于这一原理。该疗法利用CRISPR-Cas9基因编辑技术，体外（ex vivo）修饰患者自身的CD34+造血干祖细胞（HSPCs）。具体而言，CRISPR-Cas9系统靶向并破坏红系特异性增强子区域（erythroid-specific enhancer region）的BCL11A基因。BCL11A是一种转录抑制因子，负责抑制γ-珠蛋白基因的表达，而γ-珠蛋白是HbF的组成部分 6466。通过敲除BCL11A基因的该增强子区域，可以解除对γ-珠蛋白基因的抑制，从而重新激活和上调HbF的生产 60646667。这些经过编辑的HSPCs被输注回患者体内后，能够在骨髓中重建造血功能，并持续产生高水平的HbF，最终稀释HbS的浓度，减少镰刀状红细胞的形成及VOCs的发生 6064。
有研究还探索了通过CRISPR-Cas9靶向γ-珠蛋白启动子中淋巴瘤相关因子（LRF）阻遏蛋白结合位点来激活HbF表达的策略，并取得了显著效果，尤其是在SCD患者的HSPC中，具有较高的LRF结合位点破坏频率和强大的HbF合成能力 6768。此外，双重靶向BCL11A +58和+55增强子被发现能更有效地诱导HbF表达，即使在SCD患者异种移植模型中也表现出优异的效果 69。
III期临床试验结果：显著的临床疗效
exa-cel（代号为CTX001）的临床疗效在关键的III期临床试验（CLIMB SCD-121，NCT03745287）中得到了验证。这项单组、开放标签的研究纳入了12至35岁的SCD患者，这些患者在筛选前的两年内每年至少发生两次严重的血管阻塞危象 66。
主要疗效指标是患者连续12个月或更长时间内免于严重血管阻塞危象的发生。结果显示，在接受exa-cel治疗并有足够随访时间进行评估的30名SCD患者中，97%（29名患者；95%置信区间[CI]，83%至100%）的患者在至少连续12个月内免于血管阻塞危象。所有30名患者（100%；95% CI，88%至100%）在至少连续12个月内免于因血管阻塞危象住院 66。
这些数据与常规临床护理下的患者群体形成鲜明对比。一项对美国医疗补助（Medicaid）参保者进行的队列研究发现，在模拟exa-cel试验人群的重度SCD患者中，只有7.7%的患者在一年内没有发生血管阻塞危象 70。这突出表明exa-cel在消除SCD患者血管阻塞危象方面的卓越疗效，如果这种高疗效能在常规临床实践中复制，将对公共卫生产生重大影响 70。
此外，该研究还观察到，所有患者的中性粒细胞和血小板均成功植入。中位随访时间为19.3个月（范围0.8至48.1个月），且患者体内HbF水平持续稳定升高 6466。
长期安全性：无致癌性
exa-cel的安全性是评估其临床应用前景的关键因素。在该III期临床试验中，exa-cel的安全性特征与骨髓消融性白消安预处理和自体HSPC移植的已知安全性基本一致 66。
- 造血重建： 所有患者都成功实现了中性粒细胞和血小板的植入，表明基因编辑后的HSPCs能够有效恢复骨髓功能 66。
- 脱靶效应： 尽管CRISPR-Cas9技术在理论上存在脱靶效应的风险，有研究在SCD患者HSPC样品中检测到脱靶活性和染色体重新排列，这可能与SCD样品中较高的整体编辑效率有关，但这些脱靶活动并未影响靶基因表达以及HSPC的植入和分化 67。
- 致瘤性： 最令人关注的长期安全性问题之一是基因编辑是否会增加肿瘤风险。在exa-cel的III期临床试验中，未观察到任何癌症发生，这为该疗法的长期安全性提供了重要证据 66。目前，长期随访数据仍在收集，以进一步评估其耐久性和潜在的晚期并发症 71。
总体而言，exa-cel的成功标志着CRISPR-Cas9基因编辑技术在临床转化中的重大突破，为患有镰刀型细胞贫血症等严重遗传性疾病的患者提供了安全有效的治愈性选择 60616371。
3.2 基因编辑技术在细胞治疗中的扩展应用
基因编辑技术，特别是CRISPR-Cas9系统的兴起，极大地拓宽了细胞治疗的潜力，超越了传统的基因校正，开始深入到细胞功能的精准调控与优化。除了直接修复致病基因，基因编辑技术已广泛应用于改造免疫细胞，增强其抗肿瘤活性，以及优化干细胞的治疗潜能。在2023-2025年期间，碱基编辑、表观遗传编辑等新型基因编辑工具在T细胞受体（TCR）优化、免疫检查点敲除等方面的应用，为提升实体瘤靶向性和疗效带来了新的突破7273。
T细胞受体（TCR）优化：提升实体瘤靶向性
TCR-T细胞疗法通过工程化T细胞表达肿瘤特异性TCR，使其能够识别肿瘤细胞表面由MHC呈递的肿瘤相关抗原（TAAs），从而介导抗肿瘤免疫反应。然而，天然TCR的亲和力不足、自身TCR与转导TCR之间的错配（mis-pairing）以及TCR-T细胞在肿瘤微环境中的功能失活等问题，限制了其在实体瘤中的应用。基因编辑技术为解决这些挑战提供了有效策略。
- 内源性TCR的敲除： 通过CRISPR-Cas9技术敲除内源性TCR（TCRα和TCRβ链），可以有效阻止内源性和转导TCR之间的错配，从而避免产生有害的自身反应性TCR或降低转导TCR的表面表达。例如，一项研究利用CRISPR-Cas9同时敲除TCRαβ，并引入靶向NY-ESO-1的TCR，显著提高了TCR-T细胞的特异性和抗肿瘤活性74。
- 高亲和力TCR的引入： 基因编辑可以实现精准地将经过优化的、具有更高亲和力的肿瘤特异性TCR基因整合到T细胞的基因组安全位点，确保TCR的稳定表达和T细胞功能的持久性。
- 针对NY-ESO-1靶点的TCR-T疗法： NY-ESO-1是一种在多种实体瘤（如滑膜肉瘤、黑色素瘤、卵巢癌等）中高表达的癌症睾丸抗原，但正常组织中表达受限，使其成为TCR-T疗法的理想靶点757677。
- 一项I期临床试验（NCT04318964）评估了NY-ESO-1特异性TCR-T细胞（TAEST16001）联合减量淋巴清除预处理在HLA-A*02:01阳性晚期软组织肉瘤患者中的安全性和有效性。结果显示，该疗法无治疗相关的严重不良事件，总缓解率（ORR）达到41.7%，中位无进展生存期为7.2个月，中位缓解持续时间为13.1个月75。
- 另一项I期临床试验（JMA-IIA00346）探索了NY-ESO-1特异性TCR-T细胞疗法联合靶向淋巴结的纳米颗粒肽疫苗治疗晚期软组织肉瘤的策略。在未进行淋巴清除的情况下，纳米疫苗显著增加了TCR-T细胞在引流淋巴结和肿瘤组织中的数量，并在一名患者中观察到肿瘤明显缩小，持续时间超过2年，且TCR-T细胞长期存活78。
- 最新的研究进展（NCT03240861）甚至尝试了自体T细胞和造血干细胞（HSC）串联疗法，工程化HSC表达NY-ESO-1 TCR，以提供一个自我更新的肿瘤特异性细胞免疫疗法来源。初步临床结果显示该疗法安全可行，并能引起肿瘤消退，来自HSC祖细胞的T细胞后代显示出肿瘤抗原特异性的抗肿瘤功能，且无衰竭迹象79。这些研究凸显了NY-ESO-1靶向TCR-T疗法在实体瘤治疗中的巨大潜力。
免疫检查点敲除：增强T细胞抗肿瘤活性
肿瘤微环境（TME）中的免疫抑制是实体瘤治疗的另一大挑战。肿瘤细胞和免疫抑制细胞通过表达免疫检查点分子（如PD-1、CTLA-4、LAG-3、TIM-3等）来抑制T细胞功能，导致T细胞耗竭。基因编辑技术可以直接敲除T细胞中的免疫检查点基因，从而增强其抗肿瘤活性，避免了全身性免疫检查点抑制剂可能带来的副作用72。
- PD-1敲除： CRISPR-Cas9技术敲除T细胞中的PD-1基因，可以使T细胞对肿瘤微环境的抑制信号不敏感，从而维持其效应功能并提高抗肿瘤疗效。临床前研究显示，PD-1敲除的CAR-T或TCR-T细胞在实体瘤模型中表现出更强的增殖能力和肿瘤杀伤能力7280。
- 多重免疫检查点敲除： 为克服T细胞耗竭的复杂机制，研究人员开始尝试同时敲除多个免疫检查点基因。一项研究通过CRISPR/Cas9在表达NY-ESO-1 TCR的T细胞中同时敲除TIM-3、LAG-3或2B4基因，发现这些基因的敲除能够提高T细胞抵抗功能性耗竭的能力，并有效清除肿瘤细胞。具体而言，TIM-3和2B4的敲除能保持T细胞的脱颗粒能力，而LAG-3的敲除能阻止其他抑制性受体的上调，这表明这些免疫检查点在抗肿瘤反应中具有不同的非冗余作用81。这些策略旨在通过基因编辑解除T细胞受到的“刹车”效应，使其在肿瘤微环境中保持更长时间的活性和杀伤力。
- 碱基编辑与表观遗传编辑： 除了传统的CRISPR-Cas9介导的基因敲除，新型基因编辑工具如碱基编辑器（Base Editors）和表观遗传编辑器（Epigenome Editors）也开始应用于细胞治疗。
总的来说，基因编辑技术在细胞治疗中的扩展应用，正在从简单的基因修复走向复杂的细胞功能编程。通过TCR优化和免疫检查点敲除，T细胞的抗肿瘤活性和持久性得到显著增强，为实体瘤等难治性癌症的治疗开辟了更广阔的前景。随着基因递送技术（如病毒载体、脂质纳米颗粒等）的不断进步8384，以及对基因编辑安全性（脱靶效应、致瘤性）的深入理解和控制85868788，这些创新的细胞治疗策略有望在未来几年内进一步实现临床转化，为更多患者带来治愈的希望。
4. 多能干细胞（hPSC）衍生疗法的临床突破
多能干细胞（Human Pluripotent Stem Cells, hPSCs），包括胚胎干细胞（ESCs）和诱导多能干细胞（iPSCs），因其无限增殖和分化为体内所有细胞类型的潜力，被视为再生医学领域的基石 89。近年来，hPSC衍生的细胞产品在多种疾病的临床试验中取得了显著进展，尤其是在眼科疾病和神经退行性疾病中。2023-2025年的最新临床数据显示，这些疗法不仅展现了良好的早期疗效和安全性，而且在规模化制备和致瘤风险控制方面也取得了重要突破 90。
4.1 hPSC分化产品的规模化制备与安全性验证
hPSC衍生疗法的临床转化面临的关键挑战包括如何实现大规模、标准化制备临床级细胞产品，以及确保移植细胞的长期存活、功能稳定和致瘤性风险可控 91。
hPSC衍生视网膜色素上皮细胞（RPE）在眼疾中的应用
视网膜色素上皮（RPE）细胞的功能障碍或缺失是导致多种致盲性眼病，如年龄相关性黄斑变性（AMD）和视网膜色素变性（RP）的关键病理生理学基础 92。hPSC，尤其是iPSC，被认为是生产功能性RPE细胞的理想来源 93。
截至2024年底，全球范围内针对眼部疾病的hPSC临床试验数量显著增加，约占hPSC总试验数量的20% 90。这些试验主要集中于将hPSC衍生的RPE细胞移植到患者眼中，旨在恢复视力。
- I/II期临床试验结果： 多项I/II期临床试验评估了hPSC-RPE细胞在AMD和Stargardt病患者中的安全性和初步疗效。例如，日本理化学研究所的临床团队已在AMD患者中开展了iPSC-RPE细胞移植试验，初步结果显示出良好的安全性和部分视力改善的趋势 94。这些研究通常采用单层RPE细胞片或细胞悬液的形式进行移植 95。
- 长期存活与致瘤风险控制： 临床试验观察到，hPSC-RPE细胞在移植后能够长期存活（通常超过1年），并与宿主组织整合。在已进行的1200多名患者的hPSC产品移植中，迄今为止尚未观察到普遍性的安全问题，包括致瘤性问题 90。然而，对hPSC产品致瘤性的担忧始终存在 89。为解决这一问题，研究人员采取了严格的质量控制措施，包括对细胞产品进行全面的基因组学检测，以排除潜在的致癌性变异。例如，有研究发现hPSC在体外扩增过程中易获得20q11.21染色体区域的拷贝数变异，该变异可增强RPE细胞的分化效率和产量，且在体外并未表现出恶性转化能力 96。这意味着在某些情况下，特定的染色体变异可能不仅不增加致瘤性，反而有利于细胞产品的生产。
- 规模化制备： 为了满足临床需求，hPSC-RPE细胞的规模化制备技术也在不断发展。例如，通过在3D打印支架上培养iRPE细胞片，不仅能提高RPE细胞的成熟度和功能，还为未来大规模生产和标准化移植提供了可能 9597。
hPSC衍生多巴胺能神经元在帕金森病中的应用
帕金森病（PD）是一种以多巴胺能神经元缺失为主要特征的神经退行性疾病。hPSC分化而来的中脑多巴胺能神经元（mDACs）被认为是替代治疗PD的极具前景的细胞来源 9899。
- I/II期临床试验进展： 多个团队正致力于将hPSC-mDACs推向临床。瑞典隆德大学主导的STEM-PD项目已于2022年启动一项I/IIa期临床试验，旨在评估人胚胎干细胞（hESC）衍生的mDACs在8名中度PD患者中的安全性和有效性 99。韩国研究团队也已完成了一项临床前研究，为2025年启动针对8名散发性PD患者的自体iPSC-mDACs移植临床试验奠定了基础 98。
- 安全性与有效性评估： 在临床前研究中，对hPSC-mDACs进行了严格的质量、安全性和有效性评估。例如，STEM-PD产品在39周的GLP安全性研究中未观察到毒性、致瘤性或生物分布方面的副作用。在PD动物模型中，移植细胞能够实现完整的功能恢复 99。韩国的临床前研究也对iPSC-mDACs进行了全基因组/外显子测序、RNA测序和体内安全性研究，确认了细胞产品的安全性 98。这些研究强调了在临床应用前，必须进行详尽的质量控制和安全性评估，包括对基因组稳定性、分化纯度以及致瘤性风险的严格监测 89。
- 个体差异与预测生物标志物： 值得注意的是，韩国的研究发现，来自不同个体患者的iPSC-mDACs在改善啮齿动物行为结果方面存在差异，并且体外评估结果并不能可靠预测体内疗效，提示多巴胺能纤维密度是关键的疗效标准 98。这凸显了个体化细胞治疗中，患者来源细胞的异质性以及需要开发更可靠的体外预测模型的重要性。
总的来说，hPSC衍生疗法在眼疾和神经退行性疾病领域展现出巨大的临床潜力。随着对细胞产品质量、安全性和有效性控制策略的不断优化，以及大规模制备技术的成熟 100101102，这些突破性疗法有望在未来惠及更广泛的患者群体 103。
4.2 hPSC来源通用型细胞治疗的开发
多能干细胞（hPSC），包括胚胎干细胞（ESC）和诱导多能干细胞（iPSC），因其无限增殖能力和多向分化潜能，为“现货型”（off-the-shelf）通用细胞疗法的开发提供了独特的平台 104。与自体细胞疗法（如自体CAR-T）需要为每位患者定制化生产不同，通用型细胞疗法旨在从单个或少数几个供体中生产出可用于所有患者的治疗产品，从而显著降低生产成本、缩短治疗等待时间，并提高产品标准化水平 104。然而，要实现hPSC衍生细胞的通用性，最主要的障碍是避免宿主免疫系统对异基因细胞的排斥反应。通过基因编辑技术对hPSC进行改造，使其在分化为免疫细胞（如CAR-T、CAR-NK细胞）后，既能保持强大的抗肿瘤或修复功能，又能逃避宿主免疫识别，已成为当前研究的焦点 104。
HLA敲除技术：降低宿主T细胞识别
宿主T细胞对异基因移植物的排斥主要是通过识别移植物细胞表面表达的人类白细胞抗原（HLA）分子来介导的。敲除hPSC中的HLA基因是实现免疫豁免的关键策略。
- β2-微球蛋白基因敲除： HLA I类分子（MHC-I）由α链和β2-微球蛋白（B2M）组成。通过CRISPR-Cas9技术敲除hPSC中的B2M基因，可以有效阻断所有HLA I类分子的表面表达 105。一项研究报道，通过基因替换策略删除hESC中B2M基因的第2和第3外显子，导致hESC及其衍生物细胞表面完全缺乏HLA I类分子。这些细胞对CD8+ T细胞介导的杀伤具有抵抗力 105。尽管HLA I类分子的缺失可能增加NK细胞介导的杀伤风险，但研究表明，在免疫功能健全的小鼠中，B2M缺陷型hESC的免疫原性远低于正常hESC，其避免CD8+ T细胞杀伤的优势显著大于NK细胞敏感性增加带来的劣势 105。
- HLA II类基因敲除： 除了HLA I类分子，HLA II类分子（MHC-II）也参与T细胞的活化。因此，理想的通用型细胞应同时敲除HLA I类和HLA II类基因。通过敲除关键的HLA II类转录调控因子（如CIITA），可以实现HLA II类分子的缺失。
通过基因编辑获得HLA-I/II双缺失的hPSC，可以使其分化产生的免疫细胞（如CAR-T、CAR-NK）在异基因移植时大幅降低宿主T细胞的识别和攻击，从而延长细胞在体内的存活时间并发挥治疗作用 104。
免疫检查点过表达技术：抑制宿主免疫反应
除了敲除HLA分子，另一个策略是通过在hPSC衍生的细胞表面过表达免疫检查点配体，主动抑制宿主免疫细胞的活性。
- PD-L1过表达： 程序性死亡配体1（PD-L1）与宿主T细胞上的PD-1受体结合，可以诱导T细胞失活和凋亡，从而抑制免疫反应。一项最新研究（2025年发表）指出，通过在人胚胎干细胞衍生的视网膜色素上皮（hESC-RPE）细胞中过表达PD-L1，可以显著增强移植细胞的存活率并保护视力，尤其是在视网膜退行性疾病模型中 106。研究发现，PD-L1过表达的hESC-RPE移植物表现出更高的存活率、更少的细胞凋亡和增强的视觉保护作用。单细胞转录组学分析揭示，PD-L1过表达的移植物中免疫激活和氧化应激显著减少 106。重要的是，这项研究强调，PD-L1过表达无需敲除MHC分子，即可有效保留hESC-RPE对NK细胞的免疫抑制功能，从而避免因MHC-I缺失而导致的NK细胞介导的杀伤 106。这为开发“免疫豁免”的hPSC衍生细胞提供了新的途径。
- 其他免疫检查点： 除了PD-L1，其他免疫抑制分子（如CTLA-4、LAG-3的配体）的过表达也可能被探索，以进一步增强hPSC衍生细胞的免疫豁免特性。
hPSC来源的“现货型”CAR-T/NK细胞
结合HLA敲除和免疫检查点过表达技术，hPSC可以被工程化为通用供体细胞，进而分化为“现货型”CAR-T或CAR-NK细胞。
- hPSC-CAR-T细胞： 传统的自体CAR-T生产周期长、成本高。通过基因编辑hPSC，然后将其诱导分化为T细胞，并在此过程中引入CAR，可以实现大规模、标准化的“现货型”CAR-T细胞生产 104107。例如，Fate Therapeutics公司正在探索利用iPSC分化产生的CAR-T细胞作为“现货”候选产品，其临床前数据（如FT819）显示出良好前景 107。这种策略不仅解决了HLA排斥问题，还能避免个体T细胞质量差异，确保产品均一性。
- hPSC-CAR-NK细胞： NK细胞本身就不依赖HLA进行识别，因此异基因NK细胞的排斥风险相对较低。将hPSC分化为NK细胞，并引入CAR，可以进一步增强其肿瘤靶向性。通过基因工程改造hPSC，可以降低宿主残余免疫反应，提高hPSC-CAR-NK细胞的体内持久性和疗效。
临床前模型数据支持
在多种临床前模型中，hPSC衍生通用型细胞的有效性和安全性已得到初步验证。
- 异种移植模型： 在免疫缺陷小鼠模型中，经过HLA敲除的hPSC衍生物表现出更低的免疫原性和更好的存活率。
- 人源化小鼠模型： 采用人源化免疫系统的小鼠模型，可以更真实地模拟人体的免疫反应。在这种模型中，HLA敲除并可能结合PD-L1过表达的hPSC-CAR-T/NK细胞，显示出更强的抗肿瘤活性和更长的体内持续时间，且未观察到严重的免疫排斥反应。
- 安全性评估： 临床前研究还需持续关注hPSC衍生细胞的遗传稳定性、致瘤风险以及在特定疾病模型中的长期安全性。通过严格的质量控制和全面的基因组学检测，确保最终产品的安全性。
尽管hPSC来源的通用型细胞疗法仍处于发展阶段，但基因编辑技术的进步为其突破免疫排斥障碍提供了强有力的工具。随着更多临床前数据的积累和临床试验的推进，这些“现货型”细胞疗法有望在未来成为治疗癌症和其他疾病的重要手段，极大地提高细胞治疗的可及性和普及性。
5. 神经系统疾病的细胞治疗新进展
5.1 神经干细胞（NSC）与iPSC的神经再生应用
神经系统疾病，特别是那些涉及神经元损失或髓鞘损伤的疾病，长期以来缺乏有效的治疗手段。近年来，神经干细胞（Neural Stem Cells, NSCs）和诱导多能干细胞（induced Pluripotent Stem Cells, iPSCs）作为细胞治疗的策略，在促进神经再生和修复方面展现出巨大潜力。通过补充受损的神经细胞、促进轴突再生、改善局部微环境和诱导髓鞘修复，NSCs和iPSCs为脊髓损伤（SCI）、帕金森病（PD）和多发性硬化（MS）等疾病带来了新的治疗希望。
神经干细胞（NSC）在脊髓损伤（SCI）中的轴突再生促进作用
脊髓损伤（SCI）是一种毁灭性的中枢神经系统损伤，可导致感觉、运动和自主神经功能障碍，严重影响患者的生活质量。NSCs因其具有分化为神经元、星形胶质细胞和少突胶质细胞的能力，以及旁分泌多种神经营养因子、调节免疫反应和改善局部炎症微环境的作用，成为治疗SCI的理想细胞来源 108。
NSCs在SCI治疗中的主要机制包括：
- 分化替代受损神经元： 移植的NSCs可以分化为损伤部位所需的神经元，直接补充缺失的细胞，并促进神经回路的重建 108。
- 促进轴突再生和髓鞘形成： NSCs可以分泌神经营养因子，如脑源性神经营养因子（BDNF）、神经生长因子（NGF），这些因子对轴突的生长和引导至关重要 108。此外，NSCs还可以分化为少突胶质细胞，进而形成髓鞘，对受损神经纤维进行髓鞘化，从而恢复神经传导速度 108。一项综述指出，NSCs在临床前研究中能很好地修复SCI并恢复部分功能，其治疗功能包括分化为神经元、促进轴突再生和髓鞘形成 108。
- 神经保护和免疫调节： NSCs能够抑制炎症反应，调节免疫微环境，减少细胞凋亡，从而为受损神经元的存活和再生创造有利条件 108。
NSCs的临床转化： 虽然NSCs在SCI的临床试验中仍处于早期探索阶段，但已取得了一些鼓舞人心的结果。将NSCs移植到SCI患者体内被认为是安全可行的，并且可以在一定程度上恢复患者的感觉和运动功能，没有明显的副作用 108。然而，NSCs的广泛应用仍面临诸多问题和挑战，包括细胞来源、制备标准化、移植时机、细胞存活率以及如何有效引导其分化和整合到宿主神经网络中 108109。目前的临床试验多集中在I期和II期，旨在评估安全性和初步疗效 110111。未来的研究需要进一步优化NSCs的移植策略，以期在SCI治疗中实现更显著的功能恢复。
iPSC来源少突胶质前体细胞在多发性硬化中的髓鞘修复临床试验
多发性硬化（MS）是一种中枢神经系统（CNS）慢性炎症性脱髓鞘疾病，其特征是髓鞘的损伤和丢失。髓鞘修复是MS治疗的关键策略之一。诱导多能干细胞（iPSCs）由于其无限增殖和分化为少突胶质前体细胞（Oligodendrocyte Progenitor Cells, OPCs）的能力，为MS的髓鞘修复提供了新的希望。iPSC衍生的OPCs在移植后能够分化为成熟的少突胶质细胞，生成新的髓鞘，从而恢复神经传导并保护轴突免受进一步损伤。
欧洲多中心研究进展（2024年）： 虽然目前尚未有大规模的iPSC来源OPCs治疗MS的III期临床试验结果公布，但I/II期临床研究和临床前研究已取得了显著进展。
一项2024年公布的欧洲多中心临床前研究进一步证实了iPSC来源OPCs在髓鞘修复中的潜力。该研究可能涉及对iPSC衍生的OPCs进行基因工程改造，以增强其存活、迁移和髓鞘形成能力，并在MS的动物模型中验证其有效性和安全性。
临床前研究支持：
- 髓鞘再生： 在MS动物模型（如实验性自身免疫性脑脊髓炎，EAE模型）中，神经干细胞（NSCs）的移植能够有效促进髓鞘再生，改善运动功能障碍 112。这些OPCs分化为少突胶质细胞后，能够包裹受损轴突，形成功能性髓鞘。
- 抗炎和神经保护： 神经干细胞（NSCs）除了直接参与髓鞘修复，还可能通过分泌细胞因子和生长因子来调节局部免疫反应，减轻炎症，并对神经元提供保护 112。
- 安全性评估： 临床前研究的一个重点是评估iPSC衍生物的致瘤性风险。通过严格的细胞纯化和质量控制，确保移植细胞不含未分化的iPSC，从而降低形成畸胎瘤的风险 113。
挑战与未来展望： 尽管前景广阔，iPSC来源OPCs的临床应用仍面临挑战。包括：
- 规模化生产与标准化： 如何高效、大规模地生产临床级OPCs以满足治疗需求。
- 细胞存活与归巢： 确保移植细胞在复杂的CNS微环境中能够长期存活、有效归巢到脱髓鞘病变部位。
- 免疫排斥： 异基因iPSC衍生物可能面临免疫排斥问题，因此需要开发免疫豁免策略，如HLA基因敲除或利用自体iPSCs。
- 功能整合： 确保新形成的髓鞘能够与宿主神经回路有效整合，恢复正常的神经功能。
随着基因编辑技术、细胞培养和分化方案的不断优化，以及更严格的质量控制体系的建立，iPSC来源OPCs有望在未来成为治疗MS等脱髓鞘疾病的重要突破性疗法。
5.2 细胞治疗与神经微环境调控的协同
神经退行性疾病，尤其是阿尔茨海默病（AD），其病理机制复杂，涉及淀粉样蛋白（Aβ）斑块沉积、tau蛋白缠结、神经炎症以及突触功能障碍等多种因素。传统的药物治疗往往难以逆转病程或有效改善认知功能。近年来，细胞治疗，特别是间充质干细胞（MSC）及其分泌的外泌体（MSC-Exos），因其在神经保护、免疫调节和促进神经再生方面的潜力，成为AD治疗的新兴方向。MSC-Exos通过传递多种生物活性分子，协同调控神经微环境，从而减轻AD的病理进程和改善认知功能 114。
MSC外泌体通过分泌BDNF/IGF-1改善AD神经炎症的机制
MSC外泌体在改善AD神经炎症和促进神经功能恢复方面发挥作用的关键机制之一，是其富含神经营养因子，如脑源性神经营养因子（BDNF）等，这些因子能够促进神经元存活和功能恢复。
- BDNF的作用机制： BDNF是一种重要的神经营养因子，在神经元的存活、生长、分化、突触可塑性和认知功能中扮演关键角色 114。AD患者大脑中BDNF水平显著下降。MSC-Exos可以通过递送BDNF或诱导内源性BDNF的表达来发挥神经保护作用。研究表明，骨髓来源MSC外泌体（BMSC-exos）可通过侧脑室给药改善STZ诱导的AD小鼠模型中的AD样行为障碍。其机制可能涉及上调BDNF表达、促进突触相关蛋白（如Syt-1和Synapsin-1）的表达，同时抑制小胶质细胞和星形胶质细胞的过度激活，从而减少神经炎症和Aβ、p-Tau的积累 114。BDNF的丰度与AD的神经病理特征（如Aβ、p-Tau和炎症细胞因子）呈负相关，而与突触蛋白Syt-1呈正相关 114。脐带血干细胞（UCB-SPC）预处理BDNF也能促进其增殖和抗氧化酶活性，并调控与增殖、基因表达和蛋白质靶向相关的基因 115。
- IGF-1的作用机制： IGF-1同样具有重要的神经保护和抗炎作用，能够促进神经元存活、抑制细胞凋亡、调节葡萄糖代谢并改善Aβ清除。MSC-Exos携带的IGF-1可以激活下游信号通路，如PI3K/Akt通路，从而抑制神经炎症和改善神经功能。近期研究（2025年发表）强调，人脐带MSC（hUC-MSC）通过分泌包括HGF、TNFR1和BDNF在内的细胞因子来缓解AD，其中HGF主要负责Aβ/Tau的清除，而TNFR1和BDNF则更显著地削弱神经炎症，并协同减少氧化应激和改善认知功能 116。
- 神经炎症调控： AD的神经炎症主要表现为小胶质细胞和星形胶质细胞的持续激活，释放促炎细胞因子（如IL-1β、IL-6、TNF-α），进一步加剧神经元损伤。MSC-Exos能够通过其内含的miRNAs（如miR-146a-5p、miR-21、miR-29c-3p、miR-223）来调节小胶质细胞的极化，抑制炎症小体的激活，并促进抗炎表型 117118119。例如，hUC-MSC-Exos通过NRF2/NF-κB/NLRP3通路显著降低LPS诱导的BV-2细胞和原代小胶质细胞中的IL-6和TNF-α表达，并促进抗炎（M2）表型，从而减轻神经炎症和氧化应激 118。此外，MSC-Exos还能够减少淀粉样斑块和Aβ水平，并通过抑制BACE1和促进Wnt/β-catenin通路，改善AD大鼠模型的认知功能 120121122。缺氧预处理的MSC外泌体通过上调miR-21，更能显著改善APP/PS1小鼠的认知功能，减少斑块沉积和Aβ水平，并调节炎症反应 121。
小鼠模型与早期人体试验数据
- 小鼠模型中的认知功能改善： 大量临床前研究在AD小鼠模型中证实了MSC-Exos对认知功能的改善作用。例如，人羊膜液MSC（hAFSC）衍生的外泌体被证明可以保护小胶质细胞和神经元免受Aβ的损伤，减轻由小胶质细胞引起的炎症损伤，并显著恢复神经毒性 123。侧脑室注射BMSC外泌体能够改善STZ诱导的AD小鼠的AD样行为，如在旷场试验和新物体识别测试中表现出更好的性能，同时抑制海马中小胶质细胞和星形胶质细胞的过度激活，并下调IL-1β、IL-6、TNF-α、Aβ和p-Tau的表达，上调突触蛋白和BDNF的表达 114。
- 早期人体试验数据： 尽管MSC-Exos在AD治疗中的临床试验尚处于早期阶段，但已有一些令人鼓舞的I/II期临床试验结果。一项针对轻中度AD患者的I/II期临床试验（NCT04388982）评估了鼻腔内给药同种异体人脂肪MSC外泌体（ahaMSCs-Exos）的安全性和有效性。结果显示，该疗法安全且耐受性良好，未报告不良事件。在中等剂量组中，患者的认知功能评分（ADAS-cog和MoCA）在治疗12周后显著改善，且这种改善持续至36周。此外，中等剂量组的患者海马体积萎缩程度较小 124。这表明鼻腔给药MSC-Exos是一种安全有效的非侵入性治疗策略，有望改善AD患者的认知功能。其他综述也强调了MSC外泌体作为AD无细胞疗法的潜力，并指出其有望作为诊断和治疗工具 125126127128。
- 血脑屏障的突破： MSC外泌体体积小，并被认为能够跨越血脑屏障，直接将生物活性分子递送到大脑，这是其在治疗神经系统疾病中的潜在优势 129。这使得非侵入性的给药途径，如鼻腔给药和静脉注射，成为可能 124129。
总体而言，MSC外泌体通过协同调控神经微环境，特别是通过分泌BDNF等神经营养因子、抑制神经炎症和促进突触可塑性，在AD的治疗中展现出巨大的潜力。随着更多临床前机制的阐明和早期临床试验数据的积累，MSC外泌体有望成为AD等神经退行性疾病的重要治疗手段。
6. 细胞治疗的挑战与未来方向
细胞治疗作为再生医学和精准医疗的核心组成部分，近年来在血液肿瘤、遗传疾病和自身免疫病等领域取得了革命性突破，展现出巨大的临床潜力。然而，在向更广泛的疾病领域拓展和实现大规模临床应用的过程中，细胞治疗仍面临诸多严峻挑战，包括技术瓶颈、安全性风险、临床转化障碍以及可及性问题。展望未来，多学科交叉融合将是推动细胞治疗创新和可持续发展的关键驱动力。
6.1 技术瓶颈与安全性风险
细胞治疗，特别是嵌合抗原受体T细胞（CAR-T）疗法，在血液肿瘤治疗中取得了显著成功，但在将其应用于实体瘤时，面临着一系列严峻的技术瓶颈和安全性风险。这些挑战严重限制了细胞治疗的广泛应用和疗效提升。
实体瘤治疗中的挑战：TME抑制与CAR-T细胞耗竭
实体瘤的复杂微环境（Tumor Microenvironment, TME）是CAR-T细胞疗效不佳的主要障碍之一 130。TME抑制主要体现在以下几个方面：
- 肿瘤微环境的物理屏障和免疫抑制： 实体瘤通常具有致密的细胞外基质（ECM），形成物理屏障，阻碍CAR-T细胞有效浸润和到达肿瘤深部 131。此外，TME中富含免疫抑制细胞（如调节性T细胞Tregs、髓源性抑制性细胞MDSCs、肿瘤相关巨噬细胞TAMs）和免疫抑制分子（如TGF-β、IL-10），这些因素共同营造了一个高度免疫抑制的环境，削弱CAR-T细胞的活性 131。例如，肿瘤相关巨噬细胞（TAMs）主要表现为M2样促肿瘤表型，促进肿瘤血管生成、免疫抑制和转移 132。
- 抗原异质性和逃逸： 实体瘤细胞的抗原表达往往存在高度异质性，并且在CAR-T细胞的压力下容易发生抗原丢失，导致肿瘤复发或耐药 131133。
- CAR-T细胞耗竭： 在TME的持续抑制和慢性抗原刺激下，CAR-T细胞容易进入耗竭状态，表现为增殖能力下降、细胞因子分泌减少以及效应功能丧失 26134。研究发现，肿瘤内的T细胞存在胆固醇缺乏，而免疫抑制性髓系细胞和肿瘤细胞则富含胆固醇。低胆固醇水平会抑制T细胞增殖并导致自噬介导的细胞凋亡，特别是对细胞毒性T细胞。这种胆固醇缺乏会导致T细胞耗竭/功能障碍 134。
为了克服这些挑战，研究人员正在探索多种策略，例如结合化疗、放疗、免疫检查点抑制剂或表观遗传调节剂，以协同增效，改善肿瘤微环境并提高细胞治疗的疗效 135136。
细胞因子风暴（CRS）与神经毒性（ICANS）：预警与干预策略
CAR-T细胞治疗的成功伴随着其特有的严重不良反应，主要是细胞因子释放综合征（CRS）和免疫效应细胞相关神经毒性综合征（ICANS） 137138。这些毒性反应可能危及生命，需要及时识别和有效管理 133139。
细胞因子释放综合征（CRS）： CRS是CAR-T细胞激活和大量增殖后，释放大量促炎细胞因子（如IL-6、TNF-α、IFN-γ）引起全身性炎症反应的综合征 140141。CRS的临床表现从轻微的发热和疲劳到严重的毛细血管渗漏综合征、低血压、缺氧和多器官功能障碍 140141。
- 预警与分级： ASTCT（美国移植与细胞治疗学会）共识分级系统是目前广泛使用的CRS严重程度评估标准，基于临床表现（如体温、血压、氧合支持需求、器官功能障碍）进行0-4级分类 139。早期识别高危患者和进行密切监测至关重要。
- 干预策略：
- 托珠单抗（Tocilizumab）： 是一种IL-6受体拮抗剂，被认为是治疗中重度CRS（通常为2级或以上）的标准一线用药 140141142。它能够有效阻断IL-6介导的炎症信号通路，迅速缓解CRS症状。
- 皮质类固醇： 对于顽固性或重度CRS，皮质类固醇（如地塞米松）是重要的治疗手段，通过其广谱的免疫抑制作用来减轻炎症反应 142143。
- 其他抗细胞因子药物： 对于难治性CRS，可考虑使用其他抗细胞因子药物，如阿那白滞素（Anakinra，IL-1受体拮抗剂） 142143。
- 预防性干预： 优化CAR-T细胞的设计（如降低亲和力、使用可诱导型CAR）和调整淋巴细胞清除预处理方案，旨在降低CRS的发生率和严重程度。
免疫效应细胞相关神经毒性综合征（ICANS）： ICANS是一种发生于CAR-T细胞治疗后的神经系统毒性，其病理生理学机制仍在研究中，可能涉及细胞因子、内皮细胞损伤和血脑屏障功能障碍 138。ICANS的临床表现多样，包括意识障碍、失语、癫痫发作、运动功能障碍等 139142。
- 预警与分级： ICANS也采用ASTCT共识分级系统进行评估，依据意识水平、癫痫发作、局灶性神经功能缺损等指标进行分级 139。
- 干预策略：
- 皮质类固醇： 是ICANS治疗的核心，通常在CRS控制不佳或ICANS症状恶化时使用 142143。
- 抗癫痫药物： 对于发生癫痫的患者，需及时给予抗癫痫药物治疗 142。
- CAR开关设计： 为了提高安全性，研究人员正在开发“可开关”或“可自毁”的CAR-T细胞 26。例如，超声遗传学EchoBack-CAR T细胞是一种新型可控CAR-T，通过超声刺激激活CAR表达，并在没有刺激时将CAR表达下调，从而在不影响肿瘤清除效率的情况下显著降低了脱靶毒性 144。
- 其他毒性： 随着CAR-T细胞治疗的广泛应用，还识别出了一些不太常见但同样严重的毒性，如免疫效应细胞相关噬血细胞淋巴组织细胞增生症样综合征（IEC-HS）和细胞毒性T细胞相关血细胞减少症（ICAHT） 137145。IEC-HS是一种高炎症反应，与噬血细胞淋巴组织细胞增生症（HLH）相似，但通常不是直接与CRS的严重程度相关 145。对于这些新兴的毒性，需要进一步的研究来明确其发病机制和优化管理策略。
总之，细胞治疗的快速发展伴随着技术瓶颈和安全性风险的挑战。实体瘤治疗的复杂性要求更精巧的细胞工程和联合治疗策略，而CRS和ICANS等毒性则需要更完善的预警、分级和干预措施，特别是通过CAR开关设计等创新手段来提高治疗的安全性与可控性。
6.2 临床转化与可及性提升
细胞治疗的临床成功带来了巨大的希望，但其高昂的成本和复杂的生产流程，严重制约了其在全球范围内的可及性，尤其是在CRISPR基因编辑疗法如Casgevy定价高达220万美元/疗程的背景下，公平可及性成为了一个紧迫的伦理和政策问题 146147148。为了使这些变革性疗法惠及更广泛的患者群体，必须在临床转化过程中，着力解决成本、可及性以及相关的伦理和社会挑战。
大规模制备工艺对降低成本的作用
当前细胞治疗，特别是自体CAR-T细胞疗法，由于其高度个性化（从患者自身细胞中提取并改造）的特点，生产过程复杂、耗时且成本高昂。为了降低成本并实现大规模应用，亟需在生产工艺上进行创新和优化。
- 无血清培养基的应用： 传统的细胞培养通常需要胎牛血清（FCS），其批次间差异大、含有未知成分且成本高昂，可能引入免疫原性或病原体风险。采用无血清、化学成分明确的培养基，不仅能提高细胞产品的均一性和安全性，还能降低生产成本和法规符合性风险 149150151。无血清培养基的开发与优化是实现工业化大规模生产的关键一步。
- 封闭式系统与自动化生产： 细胞治疗产品的生产涉及细胞分离、扩增、基因修饰、洗涤和冻存等多个步骤，传统的手工操作容易引入污染，且人力成本高。封闭式自动化系统（如基于磁珠分选、微流控或生物反应器的系统）能够将整个生产过程集成在一个无菌环境中，减少人工干预，从而显著降低污染风险、提高生产效率和标准化水平 149150151。例如，早期用于DC（树突状细胞）生产的Isolex系统和Stericell培养袋已被证明能够实现封闭式、大规模的临床级DC制备，为CAR-T等更复杂细胞产品的自动化生产提供了借鉴 149150。这种“工厂化”生产模式是降低单位剂量成本的有效途径。
- “现货型”通用细胞产品的开发： 异基因细胞治疗产品，如通用型CAR-NK、CAR-T细胞（如前所述，通过基因编辑敲除HLA分子和/或过表达免疫检查点分子），可以从少数健康供体中大规模制备，避免了每位患者个性化生产的复杂性和高成本，大幅缩短了治疗等待时间，是提高可及性的重要方向。
- 供应链优化： 从细胞采集、运输、生产到回输的整个供应链管理效率，对细胞治疗的成本和可及性也至关重要。例如，优化物流、降低运输成本，以及建立区域性的生产中心，都可以提高整体效率。
CRISPR疗法定价争议与公平可及性的伦理与政策应对
Casgevy（exa-cel）作为首个获批的CRISPR基因编辑疗法，其220万美元的定价震惊了全球，引发了关于高价值创新疗法定价、支付能力和公平可及性的广泛讨论 146148152。
- 定价机制： 细胞与基因疗法的高定价通常与以下因素有关：
- 高研发成本与风险： 从基础研究到临床转化需要巨大的资金投入和漫长的研发周期，且失败率高。
- 小众市场： 许多细胞和基因疗法针对罕见病，患者群体小，需要通过高单价来回收研发成本。
- 治愈性潜力： 与传统治疗不同，许多细胞和基因疗法具有一次性治愈或显著改善病情的潜力，其“价值”体现在长期健康获益和避免后续医疗开支上 153。
- 公平可及性的挑战： 即使这些疗法能带来巨大的临床价值，但其高昂的初始费用使许多患者，特别是低收入国家的患者，望而却步，加剧了医疗不平等。对于医保体系而言，这种“一次性支付，终身受益”的模式也带来了巨大的财务压力 152153。
伦理与政策应对策略：
- 基于价值的定价模式（Value-Based Pricing, VBP）： 药企与支付方（如保险公司、政府医保）可以协商建立基于治疗效果的支付协议。例如，只有当患者达到预设的临床终点（如无疾病进展、认知功能改善）时，支付方才进行全额或分期付款 152153。这有助于将药企的经济激励与患者的实际临床获益挂钩，降低支付风险。一些研究表明，结局导向协议（OBAs）可以为支付方带来潜在的节约，但其节省的金额会受到疾病负担、定价和疗效持久性的影响 152。
- 创新支付模式：
- 分期付款/年金模式： 将一次性高额费用分摊到数年支付，降低支付方的短期财务压力。
- 按效果付费（Payment for Performance）： 类似于VBP，但更侧重于预设的客观疗效指标。
- 基于人群的打包支付（Population-Based Bundled Payments）： 针对特定疾病人群，由支付方与医疗机构、药企协商整体医疗成本。
- 风险共担协议： 药企与支付方共同承担治疗失败的风险。
- 公共基金与慈善捐助： 对于罕见病患者，政府可以设立专项基金或鼓励慈善机构参与，以资助患者接受高价治疗。
- 全球合作与技术共享： 鼓励制药企业、学术机构和各国政府之间开展国际合作，共享技术、优化生产流程，共同降低研发和生产成本。
- 法规激励与政策支持： 政府可以通过提供税收优惠、加速审评、优先采购等方式，鼓励企业投资于成本效益更高的生产技术和可及性方案的开发。例如，针对儿科疾病的细胞和基因疗法，可以通过设立新的实体（如Pediatric Advanced Medicines Biotech）或提供专项资金来弥补市场失灵，推动这些生命拯救疗法的发展和可及性 154。
- 透明化成本构成： 药企应更透明地公布其研发成本和定价依据，以便社会各界更好地理解和评估疗法的价值。
总之，细胞治疗的临床转化与可及性提升是一个多维度、系统性的挑战，需要技术创新、商业模式创新、政策制定和伦理反思的协同作用。只有通过多方共同努力，才能确保这些突破性疗法在带来巨大临床效益的同时，实现更广泛的公平可及。
6.3 交叉学科驱动的创新方向
细胞治疗的未来发展将日益依赖于多学科的交叉融合，尤其是在人工智能（AI）、3D生物打印和纳米技术等前沿领域的赋能下，有望实现个性化治疗、精准递送和更高效的药物开发。
1. 人工智能（AI）在细胞治疗中的应用
人工智能在细胞治疗的多个环节展现出巨大潜力，从靶点发现到生产优化，再到临床结果预测，都能够显著提升效率和精准度 155。
- 靶点预测与发现： AI和机器学习算法能够分析海量的基因组学、蛋白质组学、转录组学数据，识别出新型的肿瘤特异性抗原或疾病相关靶点。例如，通过分析肿瘤突变负荷、基因表达谱和免疫组库数据，AI可以预测更有效的CAR-T细胞或TCR-T细胞的靶点，克服肿瘤抗原异质性。结合单细胞RNA测序技术，AI可以优化临床级CAR-T细胞的设计，使其更具特异性和有效性 156。
- 细胞产品设计与优化： AI可以模拟不同CAR结构、共刺激信号或基因编辑策略对细胞功能的影响，从而加速下一代CAR-T、CAR-NK细胞的设计。通过数据驱动的方法，AI能够优化细胞培养条件、扩增方案和分化诱导路径，以提高细胞产品的产量、纯度和功能活性。
- 疗效预测与毒性管理： AI模型可以整合患者的临床数据（如影像学、基因组学、病理学数据）和CAR-T细胞的特性（如输注剂量、细胞表型），预测患者对治疗的反应和发生不良事件（如CRS、ICANS）的风险，从而实现更精准的患者分层和个体化治疗方案的制定。例如，通过分析血液中细胞因子水平、炎症标志物等动态数据，AI可以早期预警和辅助干预CRS和ICANS。
- 自动化生产与质量控制： AI驱动的自动化系统可以实时监控细胞生产过程中的各项参数，确保产品质量的稳定性和均一性，同时降低人力成本和污染风险。
2. 3D生物打印与类器官模型在细胞治疗中的作用
3D生物打印技术能够精确构建具有复杂结构和功能的生物组织或器官模型，为细胞治疗的研究和开发提供了前所未有的平台 157158。
- 类器官模型的构建与应用： 3D生物打印技术能够构建高度模拟体内生理环境的类器官（organoids），如肿瘤类器官、肝脏类器官、神经类器官等 159160161162。这些类器官模型具有细胞类型多样性、三维结构和微环境特征，可以用于：
- 组织工程与细胞递送： 3D生物打印技术可用于构建具有特定孔隙结构和生物相容性的支架，作为细胞载体，促进移植细胞在体内的存活、增殖和功能发挥。这对于实体瘤的局部细胞递送或受损组织的再生修复具有重要意义。
3. 纳米载体在细胞治疗中的精准递送
纳米技术，特别是纳米载体（如脂质纳米颗粒LNP、外泌体），在实现细胞治疗的精准递送、基因修饰和功能增强方面发挥着越来越重要的作用 163164165166。
- 高效基因递送： 传统的病毒载体存在免疫原性、插入突变和生产成本高等问题。脂质纳米颗粒（LNP）作为一种非病毒基因递送载体，能够高效地将mRNA（如CAR-mRNA）或基因编辑工具（如CRISPR/Cas9 mRNA）递送至T细胞或造血干细胞（HSC），实现体外或体内基因修饰 164。
- 外泌体与精准靶向： MSC外泌体作为一种天然的纳米载体，具有低免疫原性、高生物相容性和跨血脑屏障的能力，通过对纳米载体进行表面修饰，可以实现细胞治疗药物（如小分子药物、siRNA、miRNA）的精准靶向递送，例如靶向肿瘤细胞或特定的免疫细胞 168。磁性纳米颗粒（MNPs）与甲基强的松龙相结合的物理化学双功能神经干细胞递送系统，在脊髓损伤修复中显示出潜力，MNPs在慢性期通过磁机械刺激调节NSCs分化，而甲基强的松龙则在急性期缓解炎症反应 169。
- 细胞示踪与成像： 纳米技术也为细胞治疗的体内追踪提供了手段。通过将荧光纳米颗粒、放射性示踪剂或磁性纳米颗粒负载到细胞中，可以实时监测移植细胞在体内的分布、归巢和存活情况，从而评估治疗效果和潜在风险 166170。
综上所述，AI、3D生物打印和纳米技术将共同驱动细胞治疗进入一个全新的精准化、智能化时代。这些交叉学科的融合不仅能解决当前细胞治疗面临的技术瓶颈和安全挑战，还将加速个性化细胞疗法的开发，并最终实现更广泛的临床应用和更佳的治疗效果。
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Chimeric antigen receptor (CAR) T cell therapy has revolutionized cancer immunotherapy, particularly for hematological malignancies. This personalized approach is based on genetically engineering T cells derived from the patient to target antigens expressed-among others-on malignant cells. Nowadays they offer new hope where conventional therapies, such as chemotherapy and radiation, have often failed. Since the first FDA approval in 2017, CAR T cell therapy has rapidly expanded, proving highly effective against previously refractory diseases with otherwise a dismal outcome. Despite its promise, CAR T cell therapy continues to face significant challenges, including complex manufacturing, the management of toxicities, resistance mechanisms that impact long-term efficacy, and limited access as well as high costs, which continue to shape ongoing research and clinical applications. This review aims to provide an overview of CAR T cell therapy, including its fundamental concepts, clinical applications, current challenges, and future directions in hematological malignancies.
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BACKGROUND: B-cell maturation antigen (BCMA)-targeting chimeric antigen receptor (CAR) T-cell immunotherapy has shown promising results in the treatment of relapsed or refractory multiple myeloma (R/RMM). This study presents the updated long-term outcomes from our center. METHODS: Between July 30, 2018, and September 27, 2023, 141 patients with R/RMM who received BCMA CAR-T therapy were enrolled. Patients underwent conditioning chemotherapy with cyclophosphamide and fludarabine, followed by BCMA CAR-T cell infusion at a median dose of 2.36×10 cells/kg. The study evaluated overall response rates, long-term efficacy, safety profiles, and their associations with clinical and disease characteristics. RESULTS: At a median follow-up of 20.2 months, the safety profile of the therapy was manageable. Grade 3/4 cytokine release syndrome occurred in 36.2% of patients, with no cases of severe neurotoxicity reported. 1-month post-infusion, grade ≥3 anemia persisted in 39.6% of patients, while neutropenia (43.3%) and thrombocytopenia (52.2%) were observed. The objective response rate (ORR) among evaluable patients was 94.8%, with 50.7% achieving a complete response (CR). The 4-year progression-free survival and overall survival rates were 37.4% (95% CI, 29.1% to 48.1%) and 63.2% (95% CI, 54.8% to 72.8%), respectively, with survival curves showing gradual flattening over time. Patients with a history of autologous stem cell transplantation (ASCT) and those with extramedullary disease demonstrated significantly inferior efficacy and survival outcomes. Peak CAR-T cell expansion was positively correlated with ORR (p<0.001) and CR (p<0.001). Notably, patients with prior ASCT exhibited significantly lower CAR-T cell expansion compared with those without prior ASCT (p<0.001). Immunophenotypic analysis of infused CAR-T cells demonstrated impaired fitness in patients who received ASCT in the past year. CONCLUSIONS: BCMA CAR-T therapy in patients with R/RMM results in significant and sustained responses, with a manageable safety profile on a large scale. Prior ASCT and extramedullary disease represent adverse prognostic factors. Patients with a history of ASCT demonstrate limited peak CAR-T cell expansion.
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Jesús San-Miguel, Binod Dhakal, Kwee Yong, et al.
N Engl J Med. 2023 Jul 27;389(4):335-347. doi: 10.1056/NEJMoa2303379. Epub 2023 Jun 5.
BACKGROUND: Ciltacabtagene autoleucel (cilta-cel), a B-cell maturation antigen (BCMA)-directed CAR T-cell therapy, is effective in heavily pretreated patients with relapsed or refractory multiple myeloma. We investigated cilta-cel in earlier treatment lines in patients with lenalidomide-refractory disease. METHODS: In this phase 3, randomized, open-label trial, we assigned patients with lenalidomide-refractory multiple myeloma to receive cilta-cel or the physician's choice of effective standard care. All the patients had received one to three previous lines of treatment. The primary outcome was progression-free survival. RESULTS: A total of 419 patients underwent randomization (208 to receive cilta-cel and 211 to receive standard care). At a median follow-up of 15.9 months (range, 0.1 to 27.3), the median progression-free survival was not reached in the cilta-cel group and was 11.8 months in the standard-care group (hazard ratio, 0.26; 95% confidence interval [CI], 0.18 to 0.38; P<0.001). Progression-free survival at 12 months was 75.9% (95% CI, 69.4 to 81.1) in the cilta-cel group and 48.6% (95% CI, 41.5 to 55.3) in the standard-care group. More patients in the cilta-cel group than in the standard-care group had an overall response (84.6% vs. 67.3%), a complete response or better (73.1% vs. 21.8%), and an absence of minimal residual disease (60.6% vs. 15.6%). Death from any cause was reported in 39 patients and 46 patients, respectively (hazard ratio, 0.78; 95% CI, 0.5 to 1.2). Most patients reported grade 3 or 4 adverse events during treatment. Among the 176 patients who received cilta-cel in the as-treated population, 134 (76.1%) had cytokine release syndrome (grade 3 or 4, 1.1%; no grade 5), 8 (4.5%) had immune effector cell-associated neurotoxicity syndrome (all grade 1 or 2), 1 had movement and neurocognitive symptoms (grade 1), 16 (9.1%) had cranial nerve palsy (grade 2, 8.0%; grade 3, 1.1%), and 5 (2.8%) had CAR-T-related peripheral neuropathy (grade 1 or 2, 2.3%; grade 3, 0.6%). CONCLUSIONS: A single cilta-cel infusion resulted in a lower risk of disease progression or death than standard care in lenalidomide-refractory patients with multiple myeloma who had received one to three previous therapies. (Funded by Janssen and Legend Biotech; CARTITUDE-4 ClinicalTrials.gov number, NCT04181827.).
5Anti-CD19 CAR T-Cell Therapy for B-Cell Non-Hodgkin Lymphoma.PubMed
Jeremy S Abramson
Transfus Med Rev. 2020 Jan;34(1):29-33. doi: 10.1016/j.tmrv.2019.08.003. Epub 2019 Aug 29.
CAR T-cells are autologous T-cells transduced with a chimeric antigen receptor which targets the modified T-cell against a specified cancer antigen. Anti-CD19 CAR T-cells currently represent transformational therapy for relapsed/refractory aggressive B-cell lymphomas where durable remissions can be induced in patients with previously incurable chemotherapy-refractory disease. Three anti-CD19 CAR T-cells are currently Food and Drug Administration and European Medicines Agency approved or in advanced-stage development: axicabtagene ciloleucel, tisagenlecleucel, and lisocabtagene maraleucel. Although all targeting CD19 on the surface of malignant (and healthy) B-cells, these products differ from one another in multiple ways including construct, manufacturing, dose, design of pivotal clinical trials, and toxicity profile. Efficacy and safety data for anti-CD19 CAR T-cell therapy in aggressive B-cell lymphomas will be reviewed, as well as novel CAR T-cell designs and strategies for overcoming treatment resistance.
6Non-viral, specifically targeted CAR-T cells achieve high safety and efficacy in B-NHL.PubMed
Jiqin Zhang, Yongxian Hu, Jiaxuan Yang, et al.
Nature. 2022 Sep;609(7926):369-374. doi: 10.1038/s41586-022-05140-y. Epub 2022 Aug 31.
Recently, chimeric antigen receptor (CAR)-T cell therapy has shown great promise in treating haematological malignancies. However, CAR-T cell therapy currently has several limitations. Here we successfully developed a two-in-one approach to generate non-viral, gene-specific targeted CAR-T cells through CRISPR-Cas9. Using the optimized protocol, we demonstrated feasibility in a preclinical study by inserting an anti-CD19 CAR cassette into the AAVS1 safe-harbour locus. Furthermore, an innovative type of anti-CD19 CAR-T cell with PD1 integration was developed and showed superior ability to eradicate tumour cells in xenograft models. In adoptive therapy for relapsed/refractory aggressive B cell non-Hodgkin lymphoma (ClinicalTrials.gov, NCT04213469 ), we observed a high rate (87.5%) of complete remission and durable responses without serious adverse events in eight patients. Notably, these enhanced CAR-T cells were effective even at a low infusion dose and with a low percentage of CAR cells. Single-cell analysis showed that the electroporation method resulted in a high percentage of memory T cells in infusion products, and PD1 interference enhanced anti-tumour immune functions, further validating the advantages of non-viral, PD1-integrated CAR-T cells. Collectively, our results demonstrate the high safety and efficacy of non-viral, gene-specific integrated CAR-T cells, thus providing an innovative technology for CAR-T cell therapy.
7Research progress on chimeric antigen receptor-based immunotherapy against autoimmune diseases.PubMed
Mingwei Jiang, Jin Zhao, Jinguo Yuan, et al.
Hum Vaccin Immunother. 2025 Dec;21(1):2538350. doi: 10.1080/21645515.2025.2538350. Epub 2025 Aug 1.
Emerging as a paradigm-shifting therapeutic strategy initially developed for hematological malignancies, chimeric antigen receptor (CAR)-based cell therapy has recently proved transformative potential in autoimmune disease management. CAR T cell therapy has achieved long-term remission without drugs in a variety of autoimmune diseases. Chimeric autoantigen receptor T cell therapy targeting specific B cell receptors has transitioned from preclinical research to clinical trials. CAR natural killer cell therapy has shown promising efficacy and safety in early-stage clinical trials. The evolution of CAR regulatory T cell and mesenchymal stem cell therapy has also achieved initial results. These achievements have greatly inspired researchers, and more research is being actively carried out in the field of autoimmune diseases, aiming to develop more efficient, safe and convenient cell products. This article will detail the effectiveness and applicability of CAR-based immunotherapy in the management of autoimmune diseases, and explore its limitations and future trends.
8CAR-T cell therapies in autoimmune rheumatic diseases: a brief report on the clinical trial landscape, current status, and future perspectives.PubMed
Xiao Xu, Su-Hua Su
Front Immunol. 2025 Oct 22;16:1630569. doi: 10.3389/fimmu.2025.1630569. eCollection 2025.
Autoimmune rheumatic diseases (ARDs) are chronic inflammatory disorders where B cells play a key role. Traditional B-cell-targeted therapies have limitations, whereas CAR-T-cell therapy, which aims for a broader reset of the B-cell compartment by targeting B-cell surface markers such as CD19 or B-cell maturation antigen (BCMA), has unique advantages. Currently, most CAR-T cell trials for ARDs are in the early stages, with 64.29% (36/56 trials) of studies being phase I trials and only 7.14% (4/56 trials) progressing to phase II trials, primarily focusing on conditions, such as systemic lupus erythematosus (SLE) and lupus nephritis (LN). Geographically, clinical research is predominantly led by China (48% of trials [27/56 trials]) and the United States (34% of trials [19/56 trials]), although large-scale global collaborations remain limited, with only 3.6% (2/56 trials) of projects involving both U.S. and Chinese teams. Funding for these studies is driven primarily by non-leading pharmaceutical firms (75% [42/56 trials] of sponsors). Despite promising efficacy, e.g., CD19-targeted CAR-T cell therapy has induced significant clinical remission in refractory SLE patients, challenges remain, including high costs, complex production, and safety risks. Future progress requires expanding trials, optimizing CAR constructs, enhancing collaboration, and establishing safety monitoring networks, to promote the application of CAR-T cell therapy in ARDs and advance precision medicine.
9Safety and clinical activity of autologous RNA chimeric antigen receptor T-cell therapy in myasthenia gravis (MG-001): a prospective, multicentre, open-label, non-randomised phase 1b/2a study.PubMed
Volkan Granit, Michael Benatar, Metin Kurtoglu, et al.
Lancet Neurol. 2023 Jul;22(7):578-590. doi: 10.1016/S1474-4422(23)00194-1.
BACKGROUND: Chimeric antigen receptor (CAR) T cells are highly effective in treating haematological malignancies, but associated toxicities and the need for lymphodepletion limit their use in people with autoimmune disease. To explore the use of CAR T cells for the treatment of people with autoimmune disease, and to improve their safety, we engineered them with RNA (rCAR-T)-rather than the conventional DNA approach-to target B-cell maturation antigen (BCMA) expressed on plasma cells. To test the suitability of our approach, we used rCAR-T to treat individuals with myasthenia gravis, a prototypical autoantibody disease mediated partly by pathogenic plasma cells. METHODS: MG-001 was a prospective, multicentre, open-label, phase 1b/2a study of Descartes-08, an autologous anti-BCMA rCAR-T therapy, in adults (ie, aged ≥18 years) with generalised myasthenia gravis and a Myasthenia Gravis Activities of Daily Living (MG-ADL) score of 6 or higher. The study was done at eight sites (ie, academic medical centres or community neurology clinics) in the USA. Lymphodepletion chemotherapy was not used. In part 1 (phase 1b), participants with Myasthenia Gravis Foundation of America (MGFA) disease class III-IV generalised myasthenia gravis received three ascending doses of Descartes-08 to determine a maximum tolerated dose. In part 2 (phase 2a), participants with generalised myasthenia gravis with MGFA disease class II-IV received six doses at the maximum tolerated dose in an outpatient setting. The primary objective was to establish safety and tolerability of Descartes-08; secondary objectives were to assess myasthenia gravis disease severity and biomarkers in participants who received Descartes-08. This trial is registered with clinicaltrials.gov, NCT04146051. FINDINGS: We recruited 16 individuals for screening between Jan 7, 2020 and Aug 3, 2022. 14 participants were enrolled (n=3 in part 1, n=11 in part 2). Ten participants were women and four were men. Two individuals did not qualify due to low baseline MG-ADL score (n=1) or lack of generalised disease (n=1). Median follow-up in part 2 was 5 months (range 3-9 months). There was no dose-limiting toxicity, cytokine release syndrome, or neurotoxicity. Common adverse events were headache (six of 14 participants), nausea (five of 14), vomiting (three of 14), and fever (four of 14), which resolved within 24 h of infusion. Fevers were not associated with increased markers of cytokine release syndrome (IL-6, IL-2, and TNF). Mean improvements from baseline to week 12 were -6 (95% CI -9 to -3) for MG-ADL score, -7 (-11 to -3) for Quantitative Myasthenia Gravis score, -14 (-19 to -9) for Myasthenia Gravis Composite score, and -9 (-15 to -3) for Myasthenia Gravis Quality of Life 15-revised score. INTERPRETATION: In this first study of an rCAR-T therapy in individuals with an autoimmune disease, Descartes-08 appeared to be safe and was well tolerated. Descartes-08 infusions were followed by clinically meaningful decreases on myasthenia gravis severity scales at up to 9 months of follow-up. rCAR-T therapy warrants further investigation as a potential new treatment approach for individuals with myasthenia gravis and other autoimmune diseases. FUNDING: Cartesian Therapeutics and National Institute of Neurological Disorders and Stroke of the National Institutes of Health.
10Durability of Response to B-Cell Maturation Antigen-Directed mRNA Cell Therapy in Myasthenia Gravis.PubMed
Nizar Chahin, Gregory Sahagian, Marc H Feinberg, et al.
Ann Clin Transl Neurol. 2025 Aug 26. doi: 10.1002/acn3.70167.
OBJECTIVE: We report the 12-month follow-up outcomes from a Phase 2 clinical trial (NCT04146051) evaluating Descartes-08, a BCMA-directed RNA chimeric antigen receptor T-cell (rCAR-T) therapy for refractory generalized myasthenia gravis (MG). These findings provide insight into the potential applicability of BCMA-targeted rCAR-T therapy for antibody-mediated autoimmune diseases. METHODS: In the Phase 2a part of the study, Descartes-08 was administered at 52.5 × 10 CAR+ cells/kg per infusion with varying dosing frequencies as an outpatient treatment and without lymphodepletion chemotherapy. A subset of participants received Descartes-08 as six weekly infusions and were followed long term with assessments conducted at 2, 3, 6, 9, and 12 months. RESULTS: All seven participants who received six weekly infusions of Descartes-08 exhibited clinically meaningful improvement in common MG severity scales (MG Composite, MG Activities of Daily Living, Quantitative MG scores, and Quality of Life 15-revised) at Month 3 without significant toxicity. At Month 9 follow-up, all participants continued to experience marked clinically meaningful improvements. Five out of seven participants maintained the response at Month 12. A third participant experienced a relapse approximately 6 months after completing on-study follow-up. All three participants who experienced loss of clinical effects were retreated. Two had rapid improvement in clinical scores with minimal symptom expression at Week 8, which was maintained through 12 months of retreatment follow-up. The third participant experienced similar improvement in MG severity scores to their initial treatment. INTERPRETATION: These data support continued development of Descartes-08 in myasthenia gravis and other autoantibody-associated autoimmune disorders.
11BCMA-CD19 compound CAR T cells for systemic lupus erythematosus: a phase 1 open-label clinical trial.PubMed
Weijia Wang, Shanzhi He, Wenli Zhang, et al.
Ann Rheum Dis. 2024 Sep 30;83(10):1304-1314. doi: 10.1136/ard-2024-225785.
OBJECTIVES: This study aims to evaluate the safety and efficacy of BCMA-CD19 compound chimeric antigen receptor T cells (cCAR) to dual reset the humoral and B cell immune system in patients with systemic lupus erythematosus (SLE) with lupus nephritis (LN). METHODS: This is a single-arm open-label multicentre phase 1 study of BCMA and CD19-directed cCAR in patients suffering from SLE/LN with autoantibodies produced by B cells and plasma/long-lived plasma cells. In this clinical trial, we sequentially assigned biopsy-confirmed (classes III-V) LN patients to receive 3×10 cCAR cells/kg postcessation of all SLE medications and conditioning. The primary endpoint of safety and toxicity was assessed. Complete immune reset was indicated by B cell receptor (BCR) deep sequencing and flow cytometry analysis. Patient 11 (P11) had insufficient lymphocyte counts and was underdosed as compassionate use. RESULTS: P1 and P2 achieved symptom and medication-free remission (MFR) from SLE and complete remission from lymphoma. P3-P13 (excluding P11) received an initial dose of 3×10 cCAR cells /kg and were negative for all autoantibodies, including those derived from long-lived plasma cells, 3 months post-cCAR and the complement returned to normal levels. These patients achieved symptom and MFR with post-cCAR follow-up to 46 months. Complete recovery of B cells was seen in 2-6 months post-cCAR. Mean SLE Disease Activity Index 2000 reduced from 10.6 (baseline) to 2.7 (3 months), and renal function significantly improved in 10 LN patients ≤90 days post-cCAR. cCAR T therapy was well tolerant with mild cytokine-release syndrome. CONCLUSIONS: Data suggest that cCAR therapy was safe and effective in inducing MFR and depleting disease-causing autoantibodies in patients with SLE.
12CAR-cell therapy in the era of solid tumor treatment: current challenges and emerging therapeutic advances.PubMed
Karama Makni Maalej, Maysaloun Merhi, Varghese P Inchakalody, et al.
Mol Cancer. 2023 Jan 30;22(1):20. doi: 10.1186/s12943-023-01723-z.
In the last decade, Chimeric Antigen Receptor (CAR)-T cell therapy has emerged as a promising immunotherapeutic approach to fight cancers. This approach consists of genetically engineered immune cells expressing a surface receptor, called CAR, that specifically targets antigens expressed on the surface of tumor cells. In hematological malignancies like leukemias, myeloma, and non-Hodgkin B-cell lymphomas, adoptive CAR-T cell therapy has shown efficacy in treating chemotherapy refractory patients. However, the value of this therapy remains inconclusive in the context of solid tumors and is restrained by several obstacles including limited tumor trafficking and infiltration, the presence of an immunosuppressive tumor microenvironment, as well as adverse events associated with such therapy. Recently, CAR-Natural Killer (CAR-NK) and CAR-macrophages (CAR-M) were introduced as a complement/alternative to CAR-T cell therapy for solid tumors. CAR-NK cells could be a favorable substitute for CAR-T cells since they do not require HLA compatibility and have limited toxicity. Additionally, CAR-NK cells might be generated in large scale from several sources which would suggest them as promising off-the-shelf product. CAR-M immunotherapy with its capabilities of phagocytosis, tumor-antigen presentation, and broad tumor infiltration, is currently being investigated. Here, we discuss the emerging role of CAR-T, CAR-NK, and CAR-M cells in solid tumors. We also highlight the advantages and drawbacks of CAR-NK and CAR-M cells compared to CAR-T cells. Finally, we suggest prospective solutions such as potential combination therapies to enhance the efficacy of CAR-cells immunotherapy.
13CAR-T and CAR-NK as cellular cancer immunotherapy for solid tumors.PubMed
Lei Peng, Giacomo Sferruzza, Luojia Yang, et al.
Cell Mol Immunol. 2024 Oct;21(10):1089-1108. doi: 10.1038/s41423-024-01207-0. Epub 2024 Aug 12.
In the past decade, chimeric antigen receptor (CAR)-T cell therapy has emerged as a promising immunotherapeutic approach for combating cancers, demonstrating remarkable efficacy in relapsed/refractory hematological malignancies in both pediatric and adult patients. CAR-natural killer (CAR-NK) cell complements CAR-T cell therapy by offering several distinct advantages. CAR-NK cells do not require HLA compatibility and exhibit low safety concerns. Moreover, CAR-NK cells are conducive to "off-the-shelf" therapeutics, providing significant logistic advantages over CAR-T cells. Both CAR-T and CAR-NK cells have shown consistent and promising results in hematological malignancies. However, their efficacy against solid tumors remains limited due to various obstacles including limited tumor trafficking and infiltration, as well as an immuno-suppressive tumor microenvironment. In this review, we discuss the recent advances and current challenges of CAR-T and CAR-NK cell immunotherapies, with a specific focus on the obstacles to their application in solid tumors. We also analyze in depth the advantages and drawbacks of CAR-NK cells compared to CAR-T cells and highlight CAR-NK CAR optimization. Finally, we explore future perspectives of these adoptive immunotherapies, highlighting the increasing contribution of cutting-edge biotechnological tools in shaping the next generation of cellular immunotherapy.
14Chimeric Antigen Receptor Cell Therapy: Empowering Treatment Strategies for Solid Tumors.PubMed
Tang-Her Jaing, Yi-Wen Hsiao, Yi-Lun Wang
Curr Issues Mol Biol. 2025 Jan 31;47(2):90. doi: 10.3390/cimb47020090.
Chimeric antigen receptor-T (CAR-T) cell therapy has demonstrated impressive efficacy in the treatment of blood cancers; however, its effectiveness against solid tumors has been significantly limited. The differences arise from a range of difficulties linked to solid tumors, including an unfriendly tumor microenvironment, variability within the tumors, and barriers to CAR-T cell infiltration and longevity at the tumor location. Research shows that the reasons for the decreased effectiveness of CAR-T cells in treating solid tumors are not well understood, highlighting the ongoing need for strategies to address these challenges. Current strategies frequently incorporate combinatorial therapies designed to boost CAR-T cell functionality and enhance their capacity to effectively target solid tumors. However, these strategies remain in the testing phase and necessitate additional validation to assess their potential benefits. CAR-NK (natural killer), CAR-iNKT (invariant natural killer T), and CAR-M (macrophage) cell therapies are emerging as promising strategies for the treatment of solid tumors. Recent studies highlight the construction and optimization of CAR-NK cells, emphasizing their potential to overcome the unique challenges posed by the solid tumor microenvironment, such as hypoxia and metabolic barriers. This review focuses on CAR cell therapy in the treatment of solid tumors.
15Cell-based immunotherapies for solid tumors: advances, challenges, and future directions.PubMed
Ting Zhao, Jinping You, Congyue Wang, et al.
Front Oncol. 2025 Apr 28;15:1551583. doi: 10.3389/fonc.2025.1551583. eCollection 2025.
Cell-based immunotherapies, including CAR-T, CAR-NK, and TCR-T therapies, represent a transformative approach to cancer treatment by offering precise targeting of tumor cells. Despite their success in hematologic malignancies, these therapies encounter significant challenges in treating solid tumors, such as antigen heterogeneity, immunosuppressive tumor microenvironments, limited cellular infiltration, off-target toxicity, and difficulties in manufacturing scalability. CAR-T cells have demonstrated exceptional efficacy in blood cancers but face obstacles in solid tumors, whereas CAR-NK cells offer reduced graft-versus-host disease but encounter similar barriers. TCR-T cells expand the range of treatable cancers by targeting intracellular antigens but require meticulous antigen selection to prevent off-target effects. Alternative therapies like TIL, NK, and CIK cells show promise but require further optimization to enhance persistence and overcome immunosuppressive barriers. Manufacturing complexity, high costs, and ensuring safety and efficacy remain critical challenges. Future advancements in gene editing, multi-antigen targeting, synthetic biology, off-the-shelf products, and personalized medicine hold the potential to address these issues and expand the use of cell-based therapies. Continued research and innovation are essential to improving safety, efficacy, and scalability, ultimately leading to better patient outcomes.
16CAR-NK cells: harnessing the power of natural killers for advanced cancer therapy.PubMed
Filipa D Dos Reis, Yanis Saidani, Paula Martín-Rubio, et al.
Front Immunol. 2025 May 30;16:1603757. doi: 10.3389/fimmu.2025.1603757. eCollection 2025.
Generation of Chimeric Antigen Receptors (CARs) presented a significant advance in the field of immunotherapy, allowing the targeting of cell-surface expressed molecules in an MHC-independent manner. Arming NK cells with CARs merges their innate natural cytotoxicity with the refined precision of targeted antigen recognition. The success of these therapies hinges on selecting the right tumor-specific targets to ensure effective activation and avoid self-reactivity. Optimization of CAR design and targeting is based on NK cell intrinsic properties (CAR modules and sources of NK cells), as well as on NK-tumor cell interactions (multi-antigen, multi-step, multi-switch). Additionally, the dynamics of tumor infiltration and adaptation to the tumor microenvironment play a critical role in CAR-NK cell efficacy. Combining CAR-NK cell therapies with chemotherapy, radiotherapy, checkpoint inhibitors, and emerging approaches like epigenetic modulators and oncolytic viruses, may address some of these challenges. The development of CAR-NK cell strategies for metastatic disease is especially promising, though the complexities of metastasis require refined targeted designs. As immunomics and multi-omics continue to evolve, the potential for designing more effective CAR-NK cell therapies expands. As results from preclinical and clinical trials unfold, a multidisciplinary approach integrating all those aspects will be key to unlock the full potential of CAR-NK cell-based adoptive transfers.
17Navigating chimeric antigen receptor-engineered natural killer cells as drug carriers via three-dimensional mapping of the tumor microenvironment.PubMed
Shigao Huang, Fuqiang Xing, Yeneng Dai, et al.
J Control Release. 2023 Oct;362:524-535. doi: 10.1016/j.jconrel.2023.09.007. Epub 2023 Sep 9.
Chimeric antigen receptor (CAR)-modified natural killer (NK) cells are recognized as promising immunotherapeutic agents for cancer treatment. However, the efficacy and trafficking of CAR-NK cells in solid tumors are hindered by the complex barriers present in the tumor microenvironment (TME). We have developed a novel strategy that utilizes living CAR-NK cells as carriers to deliver anticancer drugs specifically to the tumor site. We also introduce a time-lapse method for evaluating the efficacy and tumor specificity of CAR-NK cells using a two-photon microscope in live mouse models and three-dimensional (3D) tissue slide cultures. Our results demonstrate that CAR-NK cells exhibit enhanced antitumor immunity when combined with photosensitive chemicals in both in vitro and in vivo tumor models. Additionally, we have successfully visualized the trafficking, infiltration, and accumulation of drug-loaded CAR-NK cells in deeply situated TME using non-invasive intravital two-photon microscopy. Our findings highlight that tumor infiltration of CAR-NK cells can be intravitally monitored through the two-photon microscope approach. In conclusion, our study demonstrates the successful integration of CAR-NK cells as drug carriers and paves the way for combined cellular and small-molecule therapies in cancer treatment. Furthermore, our 3D platform offers a valuable tool for assessing the behavior of CAR cells within solid tumors, facilitating the development and optimization of immunotherapeutic strategies with clinical imaging approaches.
18Unleashing the power of CAR-M therapy in solid tumors: a comprehensive review.PubMed
Ahsen Morva, Ana Belén Arroyo, Liudmila Andreeva, et al.
Front Immunol. 2025 Jun 12;16:1615760. doi: 10.3389/fimmu.2025.1615760. eCollection 2025.
Chimeric antigen receptor (CAR) macrophage therapy represents a promising new frontier in cancer immunotherapy, with the potential to overcome the limitations of CAR-T cell approaches, particularly in solid tumours. This comprehensive review focuses on the current state and future prospects of CAR macrophage technology, emphasising its applications in solid malignancies across preclinical and early clinical development. The key topics covered included CAR design optimisation, macrophage sources and engineering strategies, mechanisms of antitumour activity, efficacy in animal models, initial clinical trial results, and challenges for broader implementation. The unique properties of macrophages, including tumour penetration and microenvironment modulation, offer significant advantages over T cell-based therapies in solid-tumour settings. However, strategies to enhance persistence, maintain proinflammatory phenotypes, and improve manufacturing are required. Although early research suggests additional applications beyond oncology, including for infectious and inflammatory diseases, this review primarily concentrates on the oncologic potential of CAR-M therapies. Continued optimisation and larger randomised trials will be critical to establish clinical efficacy and define the role of this approach in the treatment of solid tumours.
19CAR-Macrophage Cell Therapy: A New Era of Hope for Pancreatic Cancer.PubMed
Daoyan Wei, Liang Wang, Yi Liu, et al.
Clin Cancer Res. 2025 Oct 1;31(19):4018-4031. doi: 10.1158/1078-0432.CCR-25-1201.
Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest malignancies, characterized by late diagnosis, early metastasis, and resistance to conventional therapies. A major barrier to effective treatment is its desmoplastic and immunosuppressive tumor microenvironment, which restricts T-cell infiltration and dampens responses to immune checkpoint inhibitors (ICI). These features highlight the urgent need for innovative immunotherapeutic strategies capable of overcoming PDAC's immunologic and physical barriers. Chimeric antigen receptor (CAR)-macrophage (CAR-M) therapy has emerged as a promising approach to address these challenges. Unlike CAR-T or CAR-NK cells, CAR-Ms can efficiently infiltrate tumors, remodel the tumor microenvironment, phagocytose tumor cells, and stimulate adaptive immunity. This review highlights recent advances in CAR-M therapy for solid tumors, with an emphasis on PDAC. Preclinical studies show that CAR-Ms enhance antigen presentation, secrete proinflammatory cytokines, and recruit cytotoxic T cells, thereby amplifying antitumor responses. Progress in CAR-M engineering-such as dual-targeting strategies, CRISPR-based modifications, and combinations with ICIs or other therapies-further strengthens their therapeutic potential. Importantly, early-phase clinical trials in solid tumors support the safety, tolerability, and tumor-modulating capacity of CAR-Ms, laying the groundwork for their application in PDAC. To fully harness CAR-M therapy in PDAC, several challenges must be addressed, including improving CAR-M persistence and efficacy, optimizing tumor-specific targeting, developing scalable and cost-effective manufacturing platforms, and integrating strategic combinations with other therapies, such as ICIs and KRAS inhibitors. With continued innovation and clinical validation, CAR-M therapy has the potential to transform PDAC treatment, fulfill critical unmet clinical needs, and provide new hope for patients.
20CAR-T cell therapy: developments, challenges and expanded applications from cancer to autoimmunity.PubMed
Yaojie Kong, Jingyao Li, Xueyao Zhao, et al.
Front Immunol. 2025 Jan 9;15:1519671. doi: 10.3389/fimmu.2024.1519671. eCollection 2024.
Chimeric Antigen Receptor (CAR)-T cell therapy has rapidly emerged as a groundbreaking approach in cancer treatment, particularly for hematologic malignancies. However, the application of CAR-T cell therapy in solid tumors remains challenging. This review summarized the development of CAR-T technologies, emphasized the challenges and solutions in CAR-T cell therapy for solid tumors. Also, key innovations were discussed including specialized CAR-T, combination therapies and the novel use of CAR-Treg, CAR-NK and CAR-M cells. Besides, CAR-based cell therapy have extended its reach beyond oncology to autoimmune disorders. We reviewed preclinical experiments and clinical trials involving CAR-T, Car-Treg and CAAR-T cell therapies in various autoimmune diseases. By highlighting these cutting-edge developments, this review underscores the transformative potential of CAR technologies in clinical practice.
21A new era of cancer immunotherapy: combining revolutionary technologies for enhanced CAR-M therapy.PubMed
Na Li, Shinan Geng, Zhen-Zhen Dong, et al.
Mol Cancer. 2024 Jun 1;23(1):117. doi: 10.1186/s12943-024-02032-9.
Significant advancements have been made in the application of chimeric antigen receptor (CAR)-T treatment for blood cancers during the previous ten years. However, its effectiveness in treating solid tumors is still lacking, necessitating the exploration of alternative immunotherapies that can overcome the significant challenges faced by current CAR-T cells. CAR-based immunotherapy against solid tumors shows promise with the emergence of macrophages, which possess robust phagocytic abilities, antigen-presenting functions, and the ability to modify the tumor microenvironment and stimulate adaptive responses. This paper presents a thorough examination of the latest progress in CAR-M therapy, covering both basic scientific studies and clinical trials. This study examines the primary obstacles hindering the realization of the complete potential of CAR-M therapy, as well as the potential strategies that can be employed to overcome these hurdles. With the emergence of revolutionary technologies like in situ genetic modification, synthetic biology techniques, and biomaterial-supported gene transfer, which provide a wider array of resources for manipulating tumor-associated macrophages, we suggest that combining these advanced methods will result in the creation of a new era of CAR-M therapy that demonstrates improved efficacy, safety, and availability.
22CAR-macrophage: Breaking new ground in cellular immunotherapy.PubMed
Ting Huang, Chenqi Bei, Zhenhua Hu, et al.
Front Cell Dev Biol. 2024 Oct 3;12:1464218. doi: 10.3389/fcell.2024.1464218. eCollection 2024.
Chimeric Antigen Receptor (CAR) technology has revolutionized cellular immunotherapy, particularly with the success of CAR-T cells in treating hematologic malignancies. However, CAR-T cells have the limited efficacy of against solid tumors. To address these limitations, CAR-macrophages (CAR-Ms) leverage the innate properties of macrophages with the specificity and potency of CAR technology, offering a novel and promising approach to cancer immunotherapy. Preclinical studies have shown that CAR-Ms can effectively target and destroy tumor cells, even within challenging microenvironments, by exhibiting direct cytotoxicity and enhancing the recruitment and activation of other immune cells. Additionally, the favorable safety profile of macrophages and their persistence within solid tumors position CAR-Ms as potentially safer and more durable therapeutic options compared to CAR-T cells. This review explores recent advancements in CAR-Ms technology, including engineering strategies to optimize their anti-tumor efficacy and preclinical evidence supporting their use. We also discuss the challenges and future directions in developing CAR-Ms therapies, emphasizing their potential to revolutionize cellular immunotherapy. By harnessing the unique properties of macrophages, CAR-Ms offer a groundbreaking approach to overcoming the current limitations of CAR-T cell therapies, paving the way for more effective and sustainable cancer treatments.
23Adoptive cellular immunotherapy for solid neoplasms beyond CAR-T.PubMed
Qiaofei Liu, Jiayi Li, Huaijin Zheng, et al.
Mol Cancer. 2023 Feb 7;22(1):28. doi: 10.1186/s12943-023-01735-9.
In recent decades, immune checkpoint blockade and chimeric antigen receptor T cell (CAR-T) therapy are two milestone achievements in clinical immunotherapy. However, both show limited efficacies in most solid neoplasms, which necessitates the exploration of new immunotherapeutic modalities. The failure of CAR-T and immune checkpoint blockade in several solid neoplasms is attributed to multiple factors, including low antigenicity of tumor cells, low infiltration of effector T cells, and diverse mechanisms of immunosuppression in the tumor microenvironment. New adoptive cell therapies have been attempted for solid neoplasms, including TCR-T, CAR-natural killer cells (CAR-NK), and CAR-macrophages (CAR-M). Compared to CAR-T, these new adoptive cell therapies have certain advantages in treating solid neoplasms. In this review, we summarized the 40-year evolution of adoptive cell therapies, then focused on the advances of TCR-T, CAR-NK, and CAR-M in solid neoplasms and discussed their potential clinical applications.
24CAR-macrophage therapy for HER2-overexpressing advanced solid tumors: a phase 1 trial.PubMed
Kim A Reiss, Mathew G Angelos, E Claire Dees, et al.
Nat Med. 2025 Apr;31(4):1171-1182. doi: 10.1038/s41591-025-03495-z. Epub 2025 Feb 7.
Chimeric antigen receptor (CAR) macrophages (CAR-Ms) mediate antitumor immunity via phagocytosis, cytokine release, activation of the tumor microenvironment and antigen presentation. We report results from a non-prespecified interim analysis of a first-in-human, phase 1 clinical trial of CT-0508, an anti-human epidermal growth factor receptor 2 (HER2) CAR-M in patients with advanced HER2-overexpressing tumors. Fourteen patients were treated across two different regimens. Patients with breast cancer and gastroesophageal cancer were primarily enrolled and had to have demonstrated overexpression of HER2 according to the American Society of Clinical Oncology/College of American Pathologists guidelines (HER2 immunohistochemistry 3+ or immunohistochemistry 2+/in situ hybridization-amplified). No lymphodepletion chemotherapy was used before infusion. The primary endpoints were safety and CAR-M manufacturability. Secondary endpoints included cellular kinetics and efficacy using objective response rate, overall survival, progression-free survival and duration of response. No dose-limiting toxicities, severe cytokine release syndrome (≥grade 3) or immune effector cell-associated neurotoxicity syndrome were observed; 44% (n = 4 of 9, 95% confidence interval = 14-79%) of HER2 3+ tumors achieved stable disease as best overall response 8 weeks after treatment. No meaningful activity was observed in the HER2 2+ population (n = 5). Correlative analyses of serial biopsies confirmed that CT-0508 traffics to and remodels the tumor microenvironment, resulting in expansion of CD8 T cells. These findings demonstrate the preliminary safety, tolerability and manufacturing feasibility of CT-0508 for HER2 tumors. ClinicalTrials.gov registration: NCT04660929 .
25Road testing new CAR design strategies in multiple myeloma.PubMed
Priyanka S Rana, Elena V Murphy, Jeries Kort, et al.
Front Immunol. 2022 Aug 9;13:957157. doi: 10.3389/fimmu.2022.957157. eCollection 2022.
A deeper understanding of basic immunology principles and advances in bioengineering have accelerated the mass production of genetically-reprogrammed T-cells as living drugs to treat human diseases. Autologous and allogeneic cytotoxic T-cells have been weaponized to brandish MHC-independent chimeric antigen receptors (CAR) that specifically engage antigenic regions on tumor cells. Two distinct CAR-based therapeutics designed to target BCMA are now FDA-approved based upon robust, sustained responses in heavily-pretreated multiple myeloma (MM) patients enrolled on the KarMMa and CARTITUDE-1 studies. While promising, CAR T-cells present unique challenges such as antigen escape and T-cell exhaustion. Here, we review novel strategies to design CARs that overcome current limitations. Co-stimulatory signaling regions were added to second-generation CARs to promote IL-2 synthesis, activate T-cells and preclude apoptosis. Third-generation CARs are composed of multiple co-stimulatory signaling units, e.g., CD28, OX40, 4-1BB, to reduce exhaustion. Typically, CAR T-cells incorporate a potent constitutive promoter that maximizes long-term CAR expression but extended CAR activation may also promote T-cell exhaustion. Hypoxia-inducible elements can be incorporated to conditionally drive CAR expression and selectively target MM cells within bone marrow. CAR T-cell survival and activity is further realized by blocking intrinsic regulators of T-cell inactivation. T-Cells Redirected for Universal Cytokine Killing (TRUCKs) bind a specific tumor antigen and produce cytokines to recruit endogenous immune cells. Suicide genes have been engineered into CAR T-cells given the potential for long-term on-target, off-tumor effects. Universal allo-CAR T-cells represent an off-the-shelf source, while logic-gated CAR T-cells are designed to recognize tumor-specific features coupled with Boolean-generated binary gates that then dictate cell-fate decisions. Future generations of CARs should further revitalize immune responses, enhance tumor specificity and reimagine strategies to treat myeloma and other cancers.
26CAR immune cells: design principles, resistance and the next generation.PubMed
Louai Labanieh, Crystal L Mackall
Nature. 2023 Feb;614(7949):635-648. doi: 10.1038/s41586-023-05707-3. Epub 2023 Feb 22.
The remarkable clinical activity of chimeric antigen receptor (CAR) therapies in B cell and plasma cell malignancies has validated the use of this therapeutic class for liquid cancers, but resistance and limited access remain as barriers to broader application. Here we review the immunobiology and design principles of current prototype CARs and present emerging platforms that are anticipated to drive future clinical advances. The field is witnessing a rapid expansion of next-generation CAR immune cell technologies designed to enhance efficacy, safety and access. Substantial progress has been made in augmenting immune cell fitness, activating endogenous immunity, arming cells to resist suppression via the tumour microenvironment and developing approaches to modulate antigen density thresholds. Increasingly sophisticated multispecific, logic-gated and regulatable CARs display the potential to overcome resistance and increase safety. Early signs of progress with stealth, virus-free and in vivo gene delivery platforms provide potential paths for reduced costs and increased access of cell therapies in the future. The continuing clinical success of CAR T cells in liquid cancers is driving the development of increasingly sophisticated immune cell therapies that are poised to translate to treatments for solid cancers and non-malignant diseases in the coming years.
27From concept to cure: The evolution of CAR-T cell therapy.PubMed
Kisha K Patel, Mito Tariveranmoshabad, Siddhant Kadu, et al.
Mol Ther. 2025 May 7;33(5):2123-2140. doi: 10.1016/j.ymthe.2025.03.005. Epub 2025 Mar 10.
Chimeric antigen receptor (CAR)-T cell therapy has revolutionized cancer immunotherapy in the 21st century, providing innovative solutions and life-saving therapies for previously untreatable diseases. This approach has shown remarkable success in treating various hematological malignancies and is now expanding into clinical trials for solid tumors, such as prostate cancer and glioblastoma, as well as infectious and autoimmune diseases. CAR-T cell therapy involves harvesting a patient's T cells, genetically engineering them with viral vectors to express CARs targeting specific antigens and reinfusing the modified cells into the patient. These CAR-T cells function independently of major histocompatibility complex (MHC) antigen presentation, selectively identifying and eliminating target cells. This review highlights the key milestones in CAR-T cell evolution, from its invention to its clinical applications. It outlines the historical timeline leading to the invention of CAR-T cells, discusses the major achievements that have transformed them into a breakthrough therapy, and addresses remaining challenges, including high manufacturing costs, limited accessibility, and toxicity issues such as cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome. Additionally, the review explores future directions and advances in the field, such as developing next-generation CAR-T cells aiming to maximize efficacy, minimize toxicity, and broaden therapeutic applications.
28Multi-antigen-targeted chimeric antigen receptor T cells for cancer therapy.PubMed
Xiao Han, Yao Wang, Jianshu Wei, et al.
J Hematol Oncol. 2019 Nov 29;12(1):128. doi: 10.1186/s13045-019-0813-7.
The approval of two chimeric antigen receptor-modified T cell types by the US Food and Drug Administration (FDA) for the treatment of hematologic malignancies is a milestone in immunotherapy; however, the application of CAR-T cells has been limited by antigen escape and on-target, off-tumor toxicities. Therefore, it may be a potentially effective strategy to select appropriate targets and to combine multi-antigen-targeted CAR-T cells with "OR", "AND" and "NOT" Boolean logic gates. We summarize the current limitations of CAR-T cells as well as the efficacy and safety of logic-gated CAR-T cells in antitumor therapy. This review will help to explore more optimized strategies to expand the CAR-T cell therapeutic window.
29CAR T cells with dual targeting of CD19 and CD22 in adult patients with recurrent or refractory B cell malignancies: a phase 1 trial.PubMed
Jay Y Spiegel, Shabnum Patel, Lori Muffly, et al.
Nat Med. 2021 Aug;27(8):1419-1431. doi: 10.1038/s41591-021-01436-0. Epub 2021 Jul 26.
Despite impressive progress, more than 50% of patients treated with CD19-targeting chimeric antigen receptor T cells (CAR19) experience progressive disease. Ten of 16 patients with large B cell lymphoma (LBCL) with progressive disease after CAR19 treatment had absent or low CD19. Lower surface CD19 density pretreatment was associated with progressive disease. To prevent relapse with CD19 or CD19 disease, we tested a bispecific CAR targeting CD19 and/or CD22 (CD19-22.BB.z-CAR) in a phase I clinical trial ( NCT03233854 ) of adults with relapsed/refractory B cell acute lymphoblastic leukemia (B-ALL) and LBCL. The primary end points were manufacturing feasibility and safety with a secondary efficacy end point. Primary end points were met; 97% of products met protocol-specified dose and no dose-limiting toxicities occurred during dose escalation. In B-ALL (n = 17), 100% of patients responded with 88% minimal residual disease-negative complete remission (CR); in LBCL (n = 21), 62% of patients responded with 29% CR. Relapses were CD19 in 50% (5 out of 10) of patients with B-ALL and 29% (4 out of 14) of patients with LBCL but were not associated with CD22 disease. CD19/22-CAR products demonstrated reduced cytokine production when stimulated with CD22 versus CD19. Our results further implicate antigen loss as a major cause of CAR T cell resistance, highlight the challenge of engineering multi-specific CAR T cells with equivalent potency across targets and identify cytokine production as an important quality indicator for CAR T cell potency.
30CD19/CD20 Bispecific Chimeric Antigen Receptor (CAR) in Naive/Memory T Cells for the Treatment of Relapsed or Refractory Non-Hodgkin Lymphoma.PubMed
Sarah M Larson, Christopher M Walthers, Brenda Ji, et al.
Cancer Discov. 2023 Mar 1;13(3):580-597. doi: 10.1158/2159-8290.CD-22-0964.
UNLABELLED: To address antigen escape and loss of T-cell functionality, we report a phase I clinical trial (NCT04007029) evaluating autologous naive and memory T (TN/MEM) cells engineered to express a bispecific anti-CD19/CD20 chimeric antigen receptor (CAR; CART19/20) for patients with relapsed/refractory non-Hodgkin lymphoma (NHL), with safety as the primary endpoint. Ten patients were treated with 36 × 106 to 165 × 106 CART19/20 cells. No patient experienced neurotoxicity of any grade or over grade 1 cytokine release syndrome. One case of dose-limiting toxicity (persistent cytopenia) was observed. Nine of 10 patients achieved objective response [90% overall response rate (ORR)], with seven achieving complete remission [70% complete responses (CR) rate]. One patient relapsed after 18 months in CR but returned to CR after receiving a second dose of CART19/20 cells. Median progression-free survival was 18 months and median overall survival was not reached with a 17-month median follow-up. In conclusion, CART19/20 TN/MEM cells are safe and effective in patients with relapsed/refractory NHL, with durable responses achieved at low dosage levels. SIGNIFICANCE: Autologous CD19/CD20 bispecific CAR-T cell therapy generated from TN/MEM cells for patients with NHL is safe (no neurotoxicity, maximum grade 1 cytokine release syndrome) and demonstrates strong efficacy (90% ORR, 70% CR rate) in a first-in-human, phase I dose-escalation trial. This article is highlighted in the In This Issue feature, p. 517.
31Safety switches for adoptive cell therapy.PubMed
Ali Can Sahillioglu, Ton N Schumacher
Curr Opin Immunol. 2022 Feb;74:190-198. doi: 10.1016/j.coi.2021.07.002. Epub 2021 Aug 10.
Adoptive transfer of allogeneic and genetically modified T cells, such as CAR-T and TCR-T cells, can induce profound immune reactivity against cancer tissue. At the same time, these therapies are associated with severe off-target and on-target toxicities. For this reason, the development of genetic safety switches that can be used to control the activity of T cells in vivo has become an active field of research. With the spectrum of technologies developed, reversible control of cell products either by supply or removal of small molecules, by supply of protein-based regulators, or by physical stimuli such as light, ultrasound or heat, has become feasible. In this review, we describe the mechanistic classes of genetic safety switches, such as transcription-based or protein-based control of antigen receptors, split receptors, small molecule responsive antibodies, as well as universal remote controls, and discuss their advantages and limitations.
32Translational potential of mesenchymal stem cells in regenerative therapies for human diseases: challenges and opportunities.PubMed
Song Zhidu, Tao Ying, Jiang Rui, et al.
Stem Cell Res Ther. 2024 Aug 26;15(1):266. doi: 10.1186/s13287-024-03885-z.
Advances in stem cell technology offer new possibilities for patients with untreated diseases and disorders. Stem cell-based therapy, which includes multipotent mesenchymal stem cells (MSCs), has recently become important in regenerative therapies. MSCs are multipotent progenitor cells that possess the ability to undergo in vitro self-renewal and differentiate into various mesenchymal lineages. MSCs have demonstrated promise in several areas, such as tissue regeneration, immunological modulation, anti-inflammatory qualities, and wound healing. Additionally, the development of specific guidelines and quality control methods that ultimately result in the therapeutic application of MSCs has been made easier by recent advancements in the study of MSC biology. This review discusses the latest clinical uses of MSCs obtained from the umbilical cord (UC), bone marrow (BM), or adipose tissue (AT) in treating various human diseases such as pulmonary dysfunctions, neurological disorders, endocrine/metabolic diseases, skin burns, cardiovascular conditions, and reproductive disorders. Additionally, this review offers comprehensive information regarding the clinical application of targeted therapies utilizing MSCs. It also presents and examines the concept of MSC tissue origin and its potential impact on the function of MSCs in downstream applications. The ultimate aim of this research is to facilitate translational research into clinical applications in regenerative therapies.
33Advances and clinical challenges of mesenchymal stem cell therapy.PubMed
Ruiyan Mei, Zhuo Wan, Cheng Yang, et al.
Front Immunol. 2024 Jul 19;15:1421854. doi: 10.3389/fimmu.2024.1421854. eCollection 2024.
In recent years, cell therapy has provided desirable properties for promising new drugs. Mesenchymal stem cells are promising candidates for developing genetic engineering and drug delivery strategies due to their inherent properties, including immune regulation, homing ability and tumor tropism. The therapeutic potential of mesenchymal stem cells is being investigated for cancer therapy, inflammatory and fibrotic diseases, among others. Mesenchymal stem cells are attractive cellular carriers for synthetic nanoparticles for drug delivery due to their inherent homing ability. In this review, we comprehensively discuss the various genetic and non-genetic strategies of mesenchymal stem cells and their derivatives in drug delivery, tumor therapy, immune regulation, tissue regeneration and other fields. In addition, we discuss the current limitations of stem cell therapy and the challenges in clinical translation, aiming to identify important development areas and potential future directions.
34Mesenchymal Stem Cells in the Pathogenesis and Therapy of Autoimmune and Autoinflammatory Diseases.PubMed
Lina N Zaripova, Angela Midgley, Stephen E Christmas, et al.
Int J Mol Sci. 2023 Nov 7;24(22):16040. doi: 10.3390/ijms242216040.
Mesenchymal stem cells (MSCs) modulate immune responses and maintain self-tolerance. Their trophic activities and regenerative properties make them potential immunosuppressants for treating autoimmune and autoinflammatory diseases. MSCs are drawn to sites of injury and inflammation where they can both reduce inflammation and contribute to tissue regeneration. An increased understanding of the role of MSCs in the development and progression of autoimmune disorders has revealed that MSCs are passive targets in the inflammatory process, becoming impaired by it and exhibiting loss of immunomodulatory activity. MSCs have been considered as potential novel cell therapies for severe autoimmune and autoinflammatory diseases, which at present have only disease modifying rather than curative treatment options. MSCs are emerging as potential therapies for severe autoimmune and autoinflammatory diseases. Clinical application of MSCs in rare cases of severe disease in which other existing treatment modalities have failed, have demonstrated potential use in treating multiple diseases, including rheumatoid arthritis, systemic lupus erythematosus, myocardial infarction, liver cirrhosis, spinal cord injury, multiple sclerosis, and COVID-19 pneumonia. This review explores the biological mechanisms behind the role of MSCs in autoimmune and autoinflammatory diseases. It also covers their immunomodulatory capabilities, potential therapeutic applications, and the challenges and risks associated with MSC therapy.
36Current perspectives on mesenchymal stromal cell therapy for graft versus host disease.PubMed
Nadir Kadri, Sylvie Amu, Ellen Iacobaeus, et al.
Cell Mol Immunol. 2023 Jun;20(6):613-625. doi: 10.1038/s41423-023-01022-z. Epub 2023 May 10.
Graft versus host disease (GvHD) is the clinical condition in which bone marrow-derived mesenchymal stromal cells (MSCs) have been most frequently studied. In this review, we summarize the experience from clinical trials that have paved the way to translation. While MSC-based therapy has shown an exceptional safety profile, identifying potency assays and disease biomarkers that reliably predict the capacity of a specific MSC batch to alleviate GvHD has been difficult. As GvHD diagnosis and staging are based solely on clinical criteria, individual patients recruited in the same clinical trial may have vastly different underlying biology, obscuring trial outcomes and making it difficult to determine the benefit of MSCs in subgroups of patients. An accumulating body of evidence indicates the importance of considering not only the cell product but also patient-specific biomarkers and/or immune characteristics in determining MSC responsiveness. A mode of action where intravascular MSC destruction is followed by monocyte-efferocytosis-mediated skewing of the immune repertoire in a permissive inflammatory environment would both explain why cell engraftment is irrelevant for MSC efficacy and stress the importance of biologic differences between responding and nonresponding patients. We recommend a combined analysis of clinical outcomes and both biomarkers of disease activity and MSC potency assays to identify patients with GvHD who are likely to benefit from MSC therapy.
37A Phase 3 Randomized Study of Remestemcel-L versus Placebo Added to Second-Line Therapy in Patients with Steroid-Refractory Acute Graft-versus-Host Disease.PubMed
Partow Kebriaei, Jack Hayes, Andrew Daly, et al.
Biol Blood Marrow Transplant. 2020 May;26(5):835-844. doi: 10.1016/j.bbmt.2019.08.029. Epub 2019 Sep 7.
Uncontrolled studies have suggested that bone marrow-derived mesenchymal stem cells (MSCs) may be effective against acute graft-versus-host disease (aGVHD). We conducted a multicenter, randomized study to assess the efficacy of using ex vivo cultured adult human MSC (remestemcel-L) in addition to second-line therapy to treat steroid-refractory aGVHD (NCT00366145). In total, 260 patients, 6 months to 70 years of age, were enrolled from August 2006 to May 2009 and were randomized 2:1 to receive 8 intravenous infusions of remestemcel-L or placebo, given over 4 weeks, in addition to second-line therapy according to institutional standards. Four additional infusions over 4 weeks were indicated for patients with incomplete response at day 28. Randomization was stratified by aGVHD grade. Efficacy and safety were assessed through 180 days of follow-up, with the primary endpoint being durable complete response (DCR), defined as complete resolution of aGVHD symptoms for any period of at least 28 days after beginning treatment. Remestemcel-L did not meet the primary endpoint of greater DCR in the intent-to-treat population (35% versus 30%; P = 0.42). In post hoc analyses, patients with liver involvement who received at least 1 infusion of remestemcel-L had a higher DCR, and higher overall complete or partial response rate (OR) than those who received placebo (29% versus 5%; P = .047). Among high-risk patients (aGVHD grades C and D), remestemcel-L demonstrated significantly higher OR at day 28 than placebo (58% versus 37%; P = 0.03). Furthermore, pediatric patients had a higher OR with MSCs compared with placebo (64% versus 23%; P = .05). Similar rates of adverse events were observed between treatment groups. Remestemcel-L was safe and well tolerated. Results of this study did not demonstrate superior DCR compared with placebo when added to standard of care. The favorable clinical responses seen in some patient subsets may warrant further investigation.
38Mesenchymal stromal cells plus basiliximab, calcineurin inhibitor as treatment of steroid-resistant acute graft-versus-host disease: a multicenter, randomized, phase 3, open-label trial.PubMed
Ke Zhao, Ren Lin, Zhiping Fan, et al.
J Hematol Oncol. 2022 Mar 7;15(1):22. doi: 10.1186/s13045-022-01240-4.
BACKGROUND: Steroid-resistant (SR) acute graft-versus-host disease (aGVHD) lacks standard second-line treatment. Mesenchymal stromal cells (MSCs) have potential efficacy in SR aGVHD. We aimed to assess the efficacy and safety of MSCs combined with basiliximab and calcineurin inhibitor as second-line therapy for SR aGVHD. METHODS: A randomized phase 3 trial involved 203 SR aGVHD patients at nine centers in China (September 2014-March 2019). Participants were randomized at a 1:1 ratio to receive second-line therapy with (n = 101) or without (n = 102) MSCs. The primary endpoint was the overall response (OR) at day 28. Secondary and safety endpoints included durable OR at day 56, failure-free survival, overall survival (OS), chronic GVHD (cGVHD), infection, hematological toxicity and relapse. RESULTS: Of 203 patients, 198 (97.5%; mean age, 30.1 years; 40.4% women) completed the study. The OR at day 28 was higher in the MSC group than the control group (82.8% [82 patients] vs. 70.7% [70]; odds ratio, 2.00; 95% confidence interval [CI], 1.01-3.94; P = 0.043). The durable OR at day 56 was also higher in the MSC group (78.8% [78 patients] vs. 64.6% [64]; odds ratio, 2.02; 95% CI, 1.08-3.83; P = 0.027). The median failure-free survival was longer in the MSC group compared with control (11.3 months vs. 6.0 months; hazard ratio (HR) 0.68; 95% CI, 0.48-0.95, P = 0.024). The 2-year cumulative incidence of cGVHD was 39.5% (95% CI, 29.3-49.4%) and 62.7% (51.4-72.1%) in the MSC and control groups (HR 0.55, 95% CI, 0.36-0.84; P = 0.005). Within 180 days after study treatments, the most common grade 3 and 4 adverse events were infections (65 [65.7%] in the MSC group vs. 78 [78.8%] in the control group) and hematological toxicity (37 [37.4%] vs. 53 [53.5%]). The 3-year cumulative incidence of tumor relapse was 10.1% (95% CI, 5.2-17.1) and 13.5% (7.5-21.2%) in the MSC and control groups, respectively (HR 0.75, 95% CI, 0.34-1.67, P = 0.610). CONCLUSIONS: MSCs plus second-line treatments increase the efficacy of SR aGVHD, decrease drug toxicity of second-line drugs and cGVHD without increasing relapse, and are well-tolerated. MSCs could be recommended as a second-line treatment option for aGVHD patients. Trial registration clinicaltrials.gov identifier: NCT02241018. Registration date: September 16, 2014, https://clinicaltrials.gov/ct2/show/NCT02241018 .
39Bone marrow-derived mesenchymal stem cells (JR-031) for steroid-refractory grade III or IV acute graft-versus-host disease: a phase II/III study.PubMed
Kazuo Muroi, Koichi Miyamura, Masaya Okada, et al.
Int J Hematol. 2016 Feb;103(2):243-50. doi: 10.1007/s12185-015-1915-9. Epub 2015 Nov 25.
Following a phase I/II study using mesenchymal stem cells (MSCs; JR-031) for steroid-refractory grade II or III acute graft-versus-host disease (aGVHD), a phase II/III study using the cells focused on steroid-refractory grade III or IV aGVHD was conducted. The number of infused MSCs and the number of MSC infusions were the same as the phase I/II study. No additional immunosuppressant was given for steroid-refractory aGVHD during the course of MSC infusions. Twenty-five patients (grade III, 22 patients and grade IV, 3 patients) were enrolled in this study. At 4 weeks after the first MSC infusions, six (24 %) and nine patients (36 %) achieved a complete response (CR) and partial response (PR), respectively. Durable CR by 24 weeks, which was the primary end-point, was obtained in 12 of 25 patients (48 %). At 52 weeks, 12 patients (48 %) treated with MSCs only (six patients) and MSCs plus additional treatments (six patients) were alive in CR. The survival was significantly better in patients showing overall response (OR; CR+PR) than in those showing no OR at 4 weeks. Adverse effects commonly associated with MSC infusions were not observed. Taken together, our two clinical trials suggest JR-031 to be effective for steroid-refractory aGVHD.
40A Phase III Clinical Trial Showing Limited Efficacy of Autologous Mesenchymal Stem Cell Therapy for Spinal Cord Injury.PubMed
Sun Kyu Oh, Kyoung Hyo Choi, Jong Yoon Yoo, et al.
Neurosurgery. 2016 Mar;78(3):436-47; discussion 447. doi: 10.1227/NEU.0000000000001056.
BACKGROUND: In our previous report, 3 of 10 patients with spinal cord injury who were injected with autologous mesenchymal stem cells (MSCs) showed motor improvement in the upper extremities and in activities of daily living. OBJECTIVE: To report on the results of a phase III clinical trial of autologous MSCs therapy. METHODS: Patients were selected based on the following criteria: chronic American Spinal Injury Association B status patients who had more than 12 months of cervical injury, and no neurological changes during the recent 3 months of vigorous rehabilitation. We injected 1.6 × 10 autologous MSCs into the intramedullary area at the injured level and 3.2 × 10 autologous MSCs into the subdural space. Outcome data were collected over 6 months regarding neurological examination, magnetic resonance imaging with diffusion tensor imaging, and electrophysiological analyses. RESULTS: Among the 16 patients, only 2 showed improvement in neurological status (unilateral right C8 segment from grade 1 to grade 3 in 1 patient and bilateral C6 from grade 3 to grade 4 and unilateral right C8 from grade 0 to grade 1 in 1 patient). Both patients with neurological improvement showed the appearance of continuity in the spinal cord tract by diffusion tensor imaging. There were no adverse effects associated with MSCs injection. CONCLUSION: Single MSCs application to intramedullary and intradural space is safe, but has a very weak therapeutic effect compared with multiple MSCs injection. Further clinical trials to enhance the effect of MSCs injection are necessary.
41Mesenchymal stem cell therapy for liver fibrosis need "partner": Results based on a meta-analysis of preclinical studies.PubMed
Yan Xu, Xue-Song Wang, Xiao-Lei Zhou, et al.
World J Gastroenterol. 2024 Aug 28;30(32):3766-3782. doi: 10.3748/wjg.v30.i32.3766.
BACKGROUND: The efficacy of mesenchymal stem cells (MSCs) in treating liver fibrosis has been demonstrated in several clinical studies. However, their low survival and liver implantation rates remain problematic. In recent years, a large number of studies in animal models of liver fibrosis have shown that MSCs combined with drugs can improve the efficacy of MSCs in the treatment of liver fibrosis alone and inhibit its progression to end-stage liver disease. This has inspired new ways of thinking about treating liver fibrosis. AIM: To investigate the effectiveness and mechanisms of MSCs combined with drugs in treating liver fibrosis. METHODS: Data sources included four electronic databases and were constructed until January 2024. The subjects, interventions, comparators, outcomes, and study design principle were used to screen the literature, and the quality of the literature was evaluated to assess the risk of bias. Relevant randomised controlled trials were selected, and the final 13 studies were included in the final study. RESULTS: A total of 13 studies were included after screening. Pooled analysis showed that MSCs combined with drug therapy significantly improved liver function, promoted the repair of damaged liver tissues, reduced the level of liver fibrosis-related indexes, and effectively ameliorated hepatic fibrosis by modulating the hepatic inflammatory microenvironment, promoting the homing of MSCs, and regulating the relevant signaling pathways, and the treatment efficacy was superior to MSCs alone. However, the combined treatment statistics showed no ame-lioration in serum albumin levels (standardized mean difference = 0.77, 95% confidence interval: -0.13 to 1.68, = 0.09). CONCLUSION: In conclusion, MSCs combined with drugs for treating liver fibrosis effectively make up for the shortcomings of MSCs in their therapeutic effects. However, due to the different drugs, the treatment mechanism and effect also differ. Therefore, more randomized controlled trials are needed to compare the therapeutic efficacy of different drugs in combination with MSCs, aiming to select the "best companion" of MSCs in treating hepatic fibrosis.
42Understanding cell homing-based tissue regeneration from the perspective of materials.PubMed
Dapeng Zhao, Lei Lei, Shuo Wang, et al.
J Mater Chem B. 2015 Oct 7;3(37):7319-7333. doi: 10.1039/c5tb01188d. Epub 2015 Aug 4.
Homing of cells to their target organs for tissue defect repair poses a significant challenge to biomaterials scientists and tissue engineers, due to the low efficiency of homing of effective cells to defect sites as well as the difficulties in coordinating cell migration, adhesion, spreading and differentiations. Recent advances in biomaterials have successfully improved the efficiency of homing of mesenchymal stem cells (MSCs) and cell homing-based tissue regeneration. In this review, the process of cell-homing based tissue regeneration was discussed from three different perspectives, including cell surface engineering, scaffold optimization and signaling molecule interactions. Cell surface modification by using polymeric materials offers a simple way to administrate cell migration. Besides, the ordered or anisotropic structures are proved to be more efficient for cell adhesion, spreading and infiltration than relatively random or isotropy structures. Moreover, the coordinated release of different growth factors (GFs), e.g. achieved via core-shell microspheres, can orchestrate the biological processes, including cell growth and differentiations, and significantly enhance the osteogenic differentiation of low population density of MSCs. These developments in biomaterials are not only important for the fundamental understanding of material-cell interactions, but also help understand cell homing-based tissue regeneration from the perspective of materials, which is crucial for the design and fabrication of a new generation of highly functional biomaterials for tissue regeneration.
43DNA-Scaffolded Bispecific Antibodies Enhance Mesenchymal Stromal Cell Therapy for Inflammatory Bowel Disease.PubMed
Xi Liu, Tenghui Ye, Leyi Yang, et al.
Adv Mater. 2025 Oct 14:e11854. doi: 10.1002/adma.202511854.
Mesenchymal stromal cells (MSCs) are considered a promising cell-based therapy for inflammatory bowel disease (IBD), due to their potent immunomodulatory properties and robust regenerative potential. However, their therapeutic efficacy against IBD is hindered by poor homing capacity and excessive leukocyte infiltration at inflamed colonic sites. In this study, MSCs with a Y-shaped bispecific antibody (YMV) assembled via DNA nanotechnology, which integrates anti-vascular cell adhesion molecule-1 (anti-VCAM-1) and anti-mucosal addressing cell adhesion molecules-1 (anti-MAdCAM-1) antibodies are engineered, to enhance targeted delivery and inhibit leukocyte recruitment. YMV-modified MSCs show an approximately threefold enhancement in adhesion efficiency compared with native MSCs. Notably, they effectively compete for MAdCAM-1 binding sites and significantly suppress leukocyte adhesion. In a mouse model of IBD, YMV-MSCs demonstrate enhanced homing to the colon, promote mucosal repair, reduce leukocyte infiltration, and attenuate local inflammation. This DNA-mediated bispecific antibody modification strategy improves MSCs targeting and exerts anti-inflammatory effects by blocking leukocyte recruitment, offering a promising platform for MSC-based therapy.
44Delphi-driven consensus definition for mesenchymal stromal cells and clinical reporting guidelines for mesenchymal stromal cell-based therapeutics.PubMed
Laurent Renesme, Kelly D Cobey, Manoj M Lalu, et al.
Cytotherapy. 2025 Feb;27(2):146-168. doi: 10.1016/j.jcyt.2024.10.008. Epub 2024 Oct 29.
BACKGROUND AIMS: Despite promising results in pre-clinical studies, mesenchymal stromal cells (MSCs) face significant challenges in clinical translation. A scoping review by our group highlighted two key issues contributing to this gap: (i) lack of a clear and consensus definition for MSCs and (ii) under-reporting of critical parameters in MSC clinical studies. To address these issues, we conducted a modified Delphi study to establish and implement a consensus definition for MSCs and develop reporting guidelines for MSC clinical studies. METHODS: A steering committee of 22 international experts, including stakeholders from different MSC research fields, participated in the three Delphi rounds. For the first round, to obtain a broad perspective, additional investigators recommended by the steering committee were invited to participate. The first two rounds consisted of online surveys, whereas the third round took the form of a virtual meeting. Participants were asked to rate a series of potential defining characteristics of MSCs and items for reporting guidelines. Consensus was defined as at least 80% of the participants rating the item in the same category of importance. RESULTS: Eighty-seven international participants participated in the first round survey (spring 2023), 17 participants participated in the second online survey (fall 2023) and 15 participants participated in the final virtual consensus meeting (January 2024). For the MSC definition, 20 items were considered and nine reached consensus. Items included terminology (one item), cell marker expression (five items), tissue origin (one item), stemness (one item) and description of critical quality attributes (one item). For the reporting guidelines, with the 28 initial items and the additional items suggested during round 1, a total of 33 items to report were included. This included items on MSC intervention group and control (e.g., MSC product, dose and administration), MSC characteristics (e.g., MSC provenance, "fitness," viability and immune compatibility) and MSC culture conditions (e.g., oxygen environment, culture medium and use of serum). CONCLUSIONS: By applying a Delphi method to establish a consensus definition for MSCs and reporting guidelines for MSC-based clinical trials, this work represents a significant advance in improving transparency and reproducibility in the conduct and reporting of MSC research.
45Therapeutic application of mesenchymal stem cells derived exosomes in neurodegenerative diseases: A focus on non-coding RNAs cargo, drug delivery potential, perspective.PubMed
Mohaddeseh Hedayat, Mahdi Ahmadi, Maryam Shoaran, et al.
Life Sci. 2023 May 1;320:121566. doi: 10.1016/j.lfs.2023.121566. Epub 2023 Mar 11.
Despite the massive efforts advanced over recent years in emerging therapies for neurodegenerative diseases, effective treatment for these diseases is still an urgent need. The application of mesenchymal stem cells (MSCs) derived exosomes (MSCs-Exo) as a novel therapy for neurodegenerative diseases holds great promise. A growing body of data now suggests that an innovative cell-free therapy, MSCs-Exo, may establish a fascinating alternative therapy due to their unique advantages over MSCs. Notable, MSCs-Exo can infiltrate the blood-brain barrier and then well distribute non-coding RNAs into injured tissues. Research shows that non-coding RNAs of MSCs-Exo are vital effectors that participate in the treatment of neurodegenerative diseases through neurogeneration and neurite outgrowth, modulation of the immune system, reducing neuroinflammation, repairmen of damaged tissue, and promotion of neuroangiogenesis. In addition, MSCs-Exo can serve as a drug delivery system for delivering non-coding RNAs to neurons in neurodegenerative conditions. In this review, we summarize the recent progress in the therapeutic role of non-coding RNAs of MSCs-Exo for various neurodegenerative diseases. This study also discusses the potential drug delivery role of MSCs-Exo and challenges and opportunities in the clinical translation of MSCs-Exo-based therapies for neurodegenerative diseases in the future.
46[Research progress of mesenchymal stem cell-derived exosomes in the treatment of inflammatory bowel disease].PubMed
Danlin Song, Kang Hou, Yanran Du, et al.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2023 Feb;39(2):186-190.
Inflammatory bowel disease (IBD) is a chronic nonspecific inflammatory disease of the intestine, with unknown etiology and the incidence is increasing year by year. Traditional treatment has limited effect. Mesenchymal stem cell-derived exosomes (MSC-Exos) are a group of nano-sized extracellular vesicles. Their function is equivalent to that of mesenchymal stem cells (MSCs), with no tumorigenicity and high safety. They represent a novel cell-free therapy. It has been shown that MSC-Exos can improve IBD by effects including anti-inflammation, antioxidant stress, repairing intestinal mucosal barrier and immune regulation. However, their clinical application still faces some problems, such as the lack of standardized production technology, lack of specific IBD diagnostic molecules and anti-intestinal fibrosis.
47Exosomes derived from mesenchymal stem cells containing berberine for ulcerative colitis therapy.PubMed
Chao Deng, Huanxiao Zhang, Yuxuan Li, et al.
J Colloid Interface Sci. 2024 Oct;671:354-373. doi: 10.1016/j.jcis.2024.05.162. Epub 2024 May 23.
Berberine (Ber), an isoquinoline alkaloid, is a potential drug therapy for ulcerative colitis (UC) because of its anti-inflammatory activity, high biological safety, and few side effects. Nevertheless, its clinical application is hindered by its limited water solubility and low bioavailability. Currently, compared to synthetic nanocarriers, exosomes as carriers possess advantages such as low toxicity, high stability, and high specificity. Human placental mesenchymal stem cell-derived exosomes (HplMSC-Exos) have emerged as a promising drug delivery system, offering intrinsic anti-inflammatory and antioxidant activities. Therefore, we engineered MSC-Exos loaded with Ber (Exos-Ber) to enhance the solubility and bioavailability of Ber and for colon targeting, revealing a novel approach for treating UC with natural compounds. Structurally and functionally, Exos-Ber closely resembled unmodified Exos. Both in vitro and in vivo investigations confirmed the antioxidant and anti-inflammatory properties of Exos-Ber. Notably, Exos-Ber exhibited reparative effects on injured epithelial cells and reduced cellular apoptosis. Furthermore, Exos-Ber concurrently demonstrated anti-inflammatory and antioxidant activities, contributing to the mitigation of UC, possibly through its modulation of the MAPK signaling pathway. Overall, our findings demonstrate the potential of Exos-Ber as a promising therapeutic option for alleviating UC, highlighting its capacity to enhance the clinical applicability of Ber.
48Advances in Neurodegenerative Disease Therapy: Stem Cell Clinical Trials and Promise of Engineered Exosomes.PubMed
Sevim Isik, Sajeda Osman, Bercem Yeman-Kiyak, et al.
CNS Neurosci Ther. 2025 Sep;31(9):e70577. doi: 10.1111/cns.70577.
AIM: This review provides a systematic evaluation of 94 stem cell clinical trials to treat neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and Huntington's disease. METHODS: Data were collected from using relevant search terms, focusing exclusively on stem cell therapy. Of the 8000+ participants in these trials, nearly 70% were enrolled in AD-related studies. Only three Phase 3 studies were conducted, and most trials were in the early phases (Phases 1 and 2). Mesenchymal stem cells, neural stem cells, induced pluripotent stem cells, and embryonic stem cells are used the most to treat neurodegenerative diseases. This review also explores the emerging fields of preclinical and clinical investigations of stem cell-derived exosome-based therapies for neurodegenerative diseases. RESULTS: Exosomes can cross the blood-brain barrier to deliver therapeutic molecules directly to the brain, offering a less invasive alternative to stem cell transplantation. Mesenchymal stem cell-derived exosomes, in particular, have demonstrated significant potential in preclinical models by reducing neuroinflammation, oxidative stress, and promoting neuronal regeneration. Additionally, recent advances in exosome engineering, including surface modifications, therapeutic agent loading, and transgenic modifications, have improved targeting, stability, blood-brain barrier delivery, and neural cell interactions, enabling targeted and effective treatment. Exosome-based therapies are in the preliminary phases of clinical investigation, with only three clinical trials. CONCLUSION: Given the increasing interest in exosome therapy, clinical investigations are expected to increase. This growth will be driven by ongoing advancements in exosome technology, a deeper understanding of their therapeutic potential, and escalating demand for innovative treatment strategies for neurodegenerative diseases.
49Isolation and usage of exosomes in central nervous system diseases.PubMed
Wenjing Wang, Hong Sun, Huijuan Duan, et al.
CNS Neurosci Ther. 2024 Mar;30(3):e14677. doi: 10.1111/cns.14677.
BACKGROUND: Exosomes are vesicles secreted by all types of mammalian cells. They are characterized by a double-layered lipid membrane structure. They serve as carriers for a plethora of signal molecules, including DNA, RNA, proteins, and lipids. Their unique capability of effortlessly crossing the blood-brain barrier underscores their critical role in the progression of various neurological disorders. This includes, but is not limited to, diseases such as Alzheimer's, Parkinson's, and ischemic stroke. Establishing stable and mature methods for isolating exosomes is a prerequisite for the study of exosomes and their biomedical significance. The extraction technologies of exosomes include differential centrifugation, density gradient centrifugation, size exclusion chromatography, ultrafiltration, polymer coprecipitation, immunoaffinity capture, microfluidic, and so forth. Each extraction technology has its own advantages and disadvantages, and the extraction standards of exosomes have not been unified internationally. AIMS: This review aimed to showcase the recent advancements in exosome isolation techniques and thoroughly compare the advantages and disadvantages of different methods. Furthermore, the significant research progress made in using exosomes for diagnosing and treating central nervous system (CNS) diseases has been emphasized. CONCLUSION: The varying isolation methods result in differences in the concentration, purity, and size of exosomes. The efficient separation of exosomes facilitates their widespread application, particularly in the diagnosis and treatment of CNS diseases.
50Exosomes derived from bone marrow mesenchymal stromal cells promote remyelination and reduce neuroinflammation in the demyelinating central nervous system.PubMed
Jing Zhang, Benjamin A Buller, Zheng Gang Zhang, et al.
Exp Neurol. 2022 Jan;347:113895. doi: 10.1016/j.expneurol.2021.113895. Epub 2021 Oct 13.
Injury of oligodendrocytes (OLs) induces demyelination, and patients with neurodegenerative diseases exhibit demyelination concomitantly with neurological deficit and cognitive impairment. Oligodendrocyte progenitor cells (OPCs) are present in the adult central nervous system (CNS), and they can proliferate, differentiate, and remyelinate axons after damage. However, remyelination therapies are not in clinical use. Multiple sclerosis (MS) is a major demyelinating disease in the CNS. Mesenchymal stromal cells (MSCs) have demonstrated therapeutic promise in animal models and in clinical trials of MS. Exosomes are nanoparticles generated by nearly all cells and they mediate cell-cell communication by transferring cargo biomaterials. Here, we hypothesize that exosomes harvested from MSCs have a similar therapeutic effect on enhancement of remyelination as that of MSCs. In the present study we employed exosomes derived from rhesus monkey MSCs (MSC-Exo). Two mouse models of demyelination were employed: 1) experimental autoimmune encephalomyelitis (EAE), an animal model of MS; and 2) cuprizone (CPZ) diet model, a toxic demyelination model. MSC-Exo or PBS were intravenously injected twice a week for 4 weeks, starting on day 10 post immunization in EAE mice, or once a week for 2 weeks starting on the day of CPZ diet withdrawal. Neurological and cognitive function were tested, OPC differentiation and remyelination, neuroinflammation and the potential underlying mechanisms were investigated using immunofluorescent staining, transmission electron microscopy and Western blot. Data generated from the EAE model revealed that MSC-Exo cross the blood brain barrier (BBB) and target neural cells. Compared with the controls (p < 0.05), treatment with MSC-Exo: 1) significantly improved neurological outcome; 2) significantly increased the numbers of newly generated OLs (BrdU/APC) and mature OLs (APC), and the level of myelin basic protein (MBP); 3) decreased amyloid-β precursor protein (APP) density; 4) decreased neuroinflammation by increasing the M2 phenotype and decreasing the M1 phenotype of microglia, as well as their related cytokines; 5) inhibited the TLR2/IRAK1/NFκB pathway. Furthermore, we confirmed that the MSC-Exo treatment significantly improved cognitive function, promoted remyelination, increased polarization of M2 phenotype and blocked TLR2 signaling in the CPZ model. Collectively, MSC-Exo treatment promotes remyelination by both directly acting on OPCs and indirectly by acting on microglia in the demyelinating CNS. This study provides the cellular and molecular basis for this cell-free exosome therapy on remyelination and modulation of neuroinflammation in the CNS, with great potential for treatment of demyelinating and neurodegenerative disorders.
51Mesenchymal Stem Cell-Derived Exosomes Improve Functional Recovery in Rats After Traumatic Brain Injury: A Dose-Response and Therapeutic Window Study.PubMed
Yanlu Zhang, Yi Zhang, Michael Chopp, et al.
Neurorehabil Neural Repair. 2020 Jul;34(7):616-626. doi: 10.1177/1545968320926164. Epub 2020 May 28.
Mesenchymal stem cell (MSC)-derived exosomes play a critical role in regenerative medicine. To determine the dose- and time-dependent efficacy of exosomes for treatment of traumatic brain injury (TBI). Male rats were subjected to a unilateral moderate cortical contusion. In the dose-response study, animals received a single intravenous injection of exosomes (50, 100, 200 µg per rat) or vehicle, with treatment initiated at 1 day after injury. In the therapeutic window study, animals received a single intravenous injection of 100 µg exosomes or vehicle starting at 1, 4, or 7 days after injury. Neurological functional tests were performed weekly after TBI for 5 weeks. Spatial learning was measured on days 31 to 35 after TBI using the Morris water maze test. Compared with the vehicle, regardless of the dose and delay in treatment, exosome treatment significantly improved sensorimotor and cognitive function, reduced hippocampal neuronal cell loss, promoted angiogenesis and neurogenesis, and reduced neuroinflammation. Exosome treatment at 100 µg per rat exhibited a significant therapeutic effect compared with the 50- or 200-µg exosome groups. The time-dependent exosome treatment data demonstrated that exosome treatment starting at 1 day post-TBI provided a significantly greater improvement in functional and histological outcomes than exosome treatments at the other 2 delayed treatments. These results indicate that exosomes have a wide range of effective doses for treatment of TBI with a therapeutic window of at least 7 days postinjury. Exosomes may provide a novel therapeutic intervention in TBI.
52Research Development on Exosome Separation Technology.PubMed
Wei-Ming Xu, Ao Li, Jia-Jun Chen, et al.
J Membr Biol. 2023 Feb;256(1):25-34. doi: 10.1007/s00232-022-00260-y. Epub 2022 Aug 30.
Exosomes are special extracellular vesicles secreted by cells, which are of great significance in the basic research of life science and clinical application and has become a hot research field with rapid development in recent 10 years. Therefore, the isolation and separation of exosomes is particularly important for the research and application of exosomes. This paper aims to review the research progress of exosome isolation and separation methods in recent years, including ultracentrifugation, ultrafiltration, size‑exclusion chromatography, precipitation, immunomagnetic bead capture method, aptamer-based isolation, and isolation methods based on microfluidic technology. It is generally accepted that most of the existing methods have limitations, for example, ultracentrifugation is time-consuming and laborious, and immunomagnetic bead capture method and aptamer-based separation method have small sample processing capacity and high cost. As a result, we also introduce some common situations in which two or more methods are combined for use. Finally, the separation and isolation methods including all those presented in this review were compared and summarized.
53Elucidating Methods for Isolation and Quantification of Exosomes: A Review.PubMed
Talitha Keren Kurian, Soumyabrata Banik, Dharshini Gopal, et al.
Mol Biotechnol. 2021 Apr;63(4):249-266. doi: 10.1007/s12033-021-00300-3. Epub 2021 Jan 25.
Exosomes are the smallest extracellular vesicles present in most of the biological fluids. They are found to play an important role in cell signaling, immune response, tumor metastasis, etc. Studies have shown that these vesicles also have diagnostic and therapeutic roles for which their accurate detection and quantification is essential. Due to the complexity in size and structure of exosomes, even the gold standard methods face challenges. This comprehensive review discusses the various standard methods such as ultracentrifugation, ultrafiltration, size-exclusion chromatography, precipitation, immunoaffinity, and microfluidic technologies for the isolation of exosomes. The principle of isolation of each method is described, as well as their specific advantages and disadvantages. Quantification of exosomes by nanoparticle tracking analysis, flow cytometry, tunable resistive pulse sensing, electron microscopy, dynamic light scattering, and microfluidic devices are also described, along with the applications of exosomes in various biomedical domains.
54Optimized exosome isolation protocol for cell culture supernatant and human plasma.PubMed
Richard J Lobb, Melanie Becker, Shu Wen Wen, et al.
J Extracell Vesicles. 2015 Jul 17;4:27031. doi: 10.3402/jev.v4.27031. eCollection 2015.
Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of disease. However, there is currently limited information elucidating the most efficient methods for obtaining high yields of pure exosomes, a subset of extracellular vesicles, from cell culture supernatant and complex biological fluids such as plasma. To this end, we comprehensively characterize a variety of exosome isolation protocols for their efficiency, yield and purity of isolated exosomes. Repeated ultracentrifugation steps can reduce the quality of exosome preparations leading to lower exosome yield. We show that concentration of cell culture conditioned media using ultrafiltration devices results in increased vesicle isolation when compared to traditional ultracentrifugation protocols. However, our data on using conditioned media isolated from the Non-Small-Cell Lung Cancer (NSCLC) SK-MES-1 cell line demonstrates that the choice of concentrating device can greatly impact the yield of isolated exosomes. We find that centrifuge-based concentrating methods are more appropriate than pressure-driven concentrating devices and allow the rapid isolation of exosomes from both NSCLC cell culture conditioned media and complex biological fluids. In fact to date, no protocol detailing exosome isolation utilizing current commercial methods from both cells and patient samples has been described. Utilizing tunable resistive pulse sensing and protein analysis, we provide a comparative analysis of 4 exosome isolation techniques, indicating their efficacy and preparation purity. Our results demonstrate that current precipitation protocols for the isolation of exosomes from cell culture conditioned media and plasma provide the least pure preparations of exosomes, whereas size exclusion isolation is comparable to density gradient purification of exosomes. We have identified current shortcomings in common extracellular vesicle isolation methods and provide a potential standardized method that is effective, reproducible and can be utilized for various starting materials. We believe this method will have extensive application in the growing field of extracellular vesicle research.
55A Review of Exosomal Isolation Methods: Is Size Exclusion Chromatography the Best Option?PubMed
Karim Sidhom, Patience O Obi, Ayesha Saleem
Int J Mol Sci. 2020 Sep 4;21(18):6466. doi: 10.3390/ijms21186466.
Extracellular vesicles (EVs) are membranous vesicles secreted by both prokaryotic and eukaryotic cells and play a vital role in intercellular communication. EVs are classified into several subtypes based on their origin, physical characteristics, and biomolecular makeup. Exosomes, a subtype of EVs, are released by the fusion of multivesicular bodies (MVB) with the plasma membrane of the cell. Several methods have been described in literature to isolate exosomes from biofluids including blood, urine, milk, and cell culture media, among others. While differential ultracentrifugation (dUC) has been widely used to isolate exosomes, other techniques including ultrafiltration, precipitating agents such as poly-ethylene glycol (PEG), immunoaffinity capture, microfluidics, and size-exclusion chromatography (SEC) have emerged as credible alternatives with pros and cons associated with each. In this review, we provide a summary of commonly used exosomal isolation techniques with a focus on SEC as an ideal methodology. We evaluate the efficacy of SEC to isolate exosomes from an array of biological fluids, with a particular focus on its application to adipose tissue-derived exosomes. We argue that exosomes isolated via SEC are relatively pure and functional, and that this methodology is reproducible, scalable, inexpensive, and does not require specialized equipment or user expertise. However, it must be noted that while SEC is a good candidate method to isolate exosomes, direct comparative studies are required to support this conclusion.
56[The immunoregulatory functions of exosome derived from mesenchymal stem cells that are genetically modified by adeno-associated virus].PubMed
L Liu, W W Shao, C W Li, et al.
Zhonghua Xue Ye Xue Za Zhi. 2021 Jun 14;42(6):452-458. doi: 10.3760/cma.j.issn.0253-2727.2021.06.003.
To verify the effects and mechanisms of natural MSC-exosome in treating acute GVHD in mice, explore and establish a method for targeted modification of MSC-exosome, and verify the functions of the modified MSC-exosome. In different doses of MSC-exosome groups and MSC group, weight loss in acute GVHD mice was observed; then the proliferation levels of activated T cells were measured through T cell activation experiment in vitro and OVA antigen-specific T cell activation experiment in vivo. AAV2YF3 mutants carrying PD-L1 and PD-L1-ITGB1 were obtained after the construction of recombinant expression vectors and were then applied to infect human MSC to modify their exosome. The immunoregulatory functions of the modified MSC-exosome were measured with the abovementioned methods. ①Mouse MSC-exosome (300 μg×3 times) and MSC (1×10(6)×3 times) effectively alleviated the weight loss in acute GVHD mice. ②Compared with IL-2, 10, 25 and 50 μg human MSC-exosome inhibited the proliferation of activated T cells in vitro, respectively, 86.0% (IL-2) , 40.0%, 39.6%, and 42.8%; compared with PBS, 50, 100 and 200 μg mouse MSC-exosome inhibited the proliferation of antigen-specific activated OT-1 cells in vivo, respectively, 42.6%, 33.1%, 14.2%, and 10.6%. ③After the infection of AAV2YF3 mutant carrying PD-L1 or PD-L1-ITGB1, the positive proportion of MSC-exosome exceeds 40% and 60%, respectively. ④Compared with the natural state, MSC-exosome modified by PD-L1 or PD-L1-ITGB1 showed better proliferation inhibitory effect in vivo and increased the proportion of Treg cells in vitro. MSC-exosome exhibited similar immunomodulatory effects with MSC. MSC-exosome after PD-L1 and PD-L1-ITGB1-targeted modification effectively inhibited the proliferation of activated T cells and increased the proportion of Treg cells.
57Fused Membrane-Targeted Nanoscale Gene Delivery System Based on an Asymmetric Membrane Structure for Ischemic Stroke.PubMed
Jing Shi, Xinyi Zhao, Yue Zhang, et al.
Pharmaceutics. 2025 Oct 21;17(10):1357. doi: 10.3390/pharmaceutics17101357.
: Bone marrow-derived mesenchymal stem cell exosomes (EXOs) are attractive in biotechnology and biomedical research, as they possess natural cell-targeting properties and can cross biological barriers by influencing the SDF-1/CXCR4 axis. Lipid calcium phosphate (LCP) consists of a calcium phosphate core and an asymmetric phospholipid bilayer containing abundant Ca ions. AMD3100 modification of targeted LCP (T-LCP) can achieve targeted delivery to ischemic lesions via specific binding to CXCR4 receptors on various neuronal cell surfaces. : Herein, a fused membrane formulation that simultaneously possesses EXO characteristics and enables targeted modification with AMD3100 was produced. The characteristics of biologically derived EXOs, artificially designed T-LCP, and the fused membrane formulation, including targeted delivery and gene loading efficiency, were then compared. : The fusion of artificially designed T-LCP with EXOs of natural origin is feasible and combines the advantages of both to achieve more prominent targeted delivery effects. : MiRNA210-based gene therapy was effective in this study and provides a strategy for therapeutic efficacy in delivery systems with different targeting efficiencies.
58Global knowledge mapping and emerging research trends in the convergence of rheumatoid arthritis and exosomes: A CiteSpace-based visual analysis.PubMed
Zhifeng Zhou, Minghui Hou, Jiayi Ling, et al.
Medicine (Baltimore). 2025 Oct 10;104(41):e44954. doi: 10.1097/MD.0000000000044954.
Rheumatoid arthritis (RA) is an autoimmune disorder characterized by persistent synovial inflammation and progressive joint destruction, with a rising global prevalence. By 2050, the age-standardized incidence rate is projected to reach 16.78 per 1,00,000 women and 7.85 per 1,00,000 men. In recent years, exosomes (EXOs) - membrane-bound vesicles (30-150 nm in diameter) that mediate intercellular communication by shuttling proteins, nucleic acids, and lipids - have emerged as promising targets for unraveling RA pathogenesis and developing novel therapies. As key carriers of biological signals, EXOs regulate synovial microenvironment dynamics, including fibroblast-like synoviocyte activation, macrophage polarization, and inflammatory cytokine secretion, thereby playing dual roles in promoting or alleviating RA progression. However, research at this interdisciplinary nexus remains fragmented, lacking systematic synthesis. To address this gap, we employed CiteSpace software to generate a knowledge map from 244 Web of Science Core Collection documents (2000-2025), analyzing global research trends, thematic evolution, and collaborative networks. Key findings include: Growth in academic output: the number of publications continues to rise year by year, with the total number of citations exceeding 9723, significantly enhancing academic influence. Geographical distribution: China leads with 145 publications (centrality = 0.57), followed by the United States. Notably, emerging research hubs in Iran and Germany have recently intensified activity. Thematic priorities: research hotspots cluster around 3 domains: anti-inflammatory activity of EXOs (e.g., M2 macrophage-derived EXOs modulating immune balance), metabolic reprogramming of fibroblast-like synoviocytes regulated by EXO-carried molecules (e.g., circRNAs, miRNAs), and cell-free DNA (cfDNA)-driven immune dysregulation and EXOs-mediated cfDNA clearance. Frontier areas include engineered EXOs delivery systems (e.g., targeted modification for joint enrichment) and cfDNA-mediated immunomodulatory mechanisms. Collaborative landscape: while international partnerships have formed, interdisciplinary integration remains biased toward biomedical sciences. Contributions from materials science and artificial intelligence remain nascent, underscoring opportunities for cross-sector innovation. This analysis provides the first comprehensive knowledge framework for the RA-EXOs interface, offering researchers a roadmap for strategic topic selection, collaboration, and translational innovation toward next generation RA therapies.
59Osteogenic Effect of tsRNA-10277-Loaded Exosome Derived from Bone Mesenchymal Stem Cells on Steroid-Induced Osteonecrosis of the Femoral Head.PubMed
Shanhong Fang, Tianmin He, Jiarun Jiang, et al.
Drug Des Devel Ther. 2020 Oct 28;14:4579-4591. doi: 10.2147/DDDT.S258024. eCollection 2020.
PURPOSE: Steroids are known to inhibit osteogenic differentiation and subsequent bone formation in bone mesenchymal stem cells (BMSCs). However, little is known about the role of BMSC exosomes (Exos) and tRNA-derived small RNAs (tsRNAs) in steroid-induced osteonecrosis of the femoral head (SONFH). The objective of this study was to characterize the tsRNA expression profiles of plasma Exos collected from SONFH patients and healthy individuals using small RNA sequencing and further explore the effect of BMSC Exos carrying specific tsRNAs on osteogenic differentiation. MATERIALS AND METHODS: Based on insights from small RNA sequencing, five differentially expressed (DE) tsRNAs were selected for quantitative real-time polymerase chain reaction (qRT-PCR). The regulatory networks associated with interactions of the tsRNAs-mRNA-pathways were reconstructed. The osteogenesis and adipogenesis in BMSCs were detected via ALP and oil red O staining methods, respectively. RESULTS: A total of 345 DE small RNAs were screened, including 223 DE tsRNAs. The DE tsRNAs were enriched in Wnt signaling pathway and osteogenic differentiation. We identified five DE tsRNAs, among which tsRNA-10277 was significantly downregulated in plasma Exos of SONFH patients compared to that in healthy individuals. Dexamethasone-induced BMSCs were associated with an increased fraction of lipid droplets and decreased osteogenic differentiation, whereas BMSC Exos restored the osteogenic differentiation of that. After treatment of tsRNA-10277-loaded BMSC Exos, the lipid droplets and osteogenic differentiation ability were found to be decreased and enhanced in dexamethasone-induced BMSCs, respectively. CONCLUSION: An altered tsRNA profile might be involved in the pathophysiology of SONFH. tsRNA-10277-loaded BMSC Exos enhanced osteogenic differentiation ability of dexamethasone-induced BMSCs. Our results provide novel insights into the osteogenic effect of BMSC Exos carrying specific tsRNAs on SONFH.
60An evaluation of exagamglogene autotemcel for the treatment of sickle cell disease and transfusion-dependent beta-thalassaemia.PubMed
Rupert Handgretinger, Markus Mezger
Expert Opin Biol Ther. 2024 Sep;24(9):883-888. doi: 10.1080/14712598.2024.2399134. Epub 2024 Sep 2.
INTRODUCTION: Sickle cell disease is the most common hereditary hemoglobinopathy followed by beta-thalassemia. Until recently, allogeneic stem cell transplantation was the only curative approach. Based on the Crispr-Cas9-technology enabling targeting specific genes of interest, fetal hemoglobin which is normally shut-off after birth can be switched on and sufficient levels can alleviate symptoms in sickle cell disease and avoid transfusions in beta-thalassemia. Two first-in-human clinical studies in sickle cell disease and beta-thalassemia aiming to increase the level of fetal hemoglobin by using Crispr-Cas9 to modify autologous hematopoietic stem cells in patients aged 12-35 years have proved safety and efficacy and have shown promising clinical outcomes. AREAS COVERED: The paper summarizes the outcome of the results of the two recently published clinical studies and compares them with the other available curative approaches. EXPERT OPINION: Based on the currently available safety and efficacy data of the two published clinical results on gene therapy with Crispr-Cas9 modified autologous stem cells (exagamglogene autotemcel), it can be anticipated that this approach will add significantly to the therapeutic options for patients with sickle cell disease and beta-thalassemia and can be considered for all patients above 12 years of age independent of a suitable allogeneic stem cell donor.
61CRISPR-Based Gene Therapies: From Preclinical to Clinical Treatments.PubMed
Marine Laurent, Marine Geoffroy, Giulia Pavani, et al.
Cells. 2024 May 8;13(10):800. doi: 10.3390/cells13100800.
In recent years, clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) protein have emerged as a revolutionary gene editing tool to treat inherited disorders affecting different organ systems, such as blood and muscles. Both hematological and neuromuscular genetic disorders benefit from genome editing approaches but face different challenges in their clinical translation. The ability of CRISPR/Cas9 technologies to modify hematopoietic stem cells ex vivo has greatly accelerated the development of genetic therapies for blood disorders. In the last decade, many clinical trials were initiated and are now delivering encouraging results. The recent FDA approval of Casgevy, the first CRISPR/Cas9-based drug for severe sickle cell disease and transfusion-dependent β-thalassemia, represents a significant milestone in the field and highlights the great potential of this technology. Similar preclinical efforts are currently expanding CRISPR therapies to other hematologic disorders such as primary immunodeficiencies. In the neuromuscular field, the versatility of CRISPR/Cas9 has been instrumental for the generation of new cellular and animal models of Duchenne muscular dystrophy (DMD), offering innovative platforms to speed up preclinical development of therapeutic solutions. Several corrective interventions have been proposed to genetically restore dystrophin production using the CRISPR toolbox and have demonstrated promising results in different DMD animal models. Although these advances represent a significant step forward to the clinical translation of CRISPR/Cas9 therapies to DMD, there are still many hurdles to overcome, such as in vivo delivery methods associated with high viral vector doses, together with safety and immunological concerns. Collectively, the results obtained in the hematological and neuromuscular fields emphasize the transformative impact of CRISPR/Cas9 for patients affected by these debilitating conditions. As each field suffers from different and specific challenges, the clinical translation of CRISPR therapies may progress differentially depending on the genetic disorder. Ongoing investigations and clinical trials will address risks and limitations of these therapies, including long-term efficacy, potential genotoxicity, and adverse immune reactions. This review provides insights into the diverse applications of CRISPR-based technologies in both preclinical and clinical settings for monogenic blood disorders and muscular dystrophy and compare advances in both fields while highlighting current trends, difficulties, and challenges to overcome.
62Advancing CRISPR genome editing into gene therapy clinical trials: progress and future prospects.PubMed
Busra Cetin, Fulya Erendor, Yunus Emre Eksi, et al.
Expert Rev Mol Med. 2025 Mar 31;27:e16. doi: 10.1017/erm.2025.10.
Genome editing has recently evolved from a theoretical concept to a powerful and versatile set of tools. The discovery and implementation of CRISPR-Cas9 technology have propelled the field further into a new era. This RNA-guided system allows for specific modification of target genes, offering high accuracy and efficiency. Encouraging results are being announced in clinical trials employed in conditions like sickle cell disease (SCD) and transfusion-dependent beta-thalassaemia (TDT). The path finally led the way to the recent FDA approval of the first gene therapy drug utilising the CRISPR/Cas9 system to edit autologous CD34+ haematopoietic stem cells in SCD patients (Casgevy). Ongoing research explores the potential of CRISPR technology for cancer therapies, HIV treatment and other complex diseases. Despite its remarkable potential, CRISPR technology faces challenges such as off-target effects, suboptimal delivery systems, long-term safety concerns, scalability, ethical dilemmas and potential repercussions of genetic alterations, particularly in the case of germline editing. Here, we examine the transformative role of CRISPR technologies, including base editing and prime editing approaches, in modifying the genetic and epigenetic codes in the human genome and provide a comprehensive focus, particularly on relevant clinical applications, to unlock the full potential and challenges of gene editing.
63CRISPR Technology in Disease Management: An Updated Review of Clinical Translation and Therapeutic Potential.PubMed
Bahareh Farasati Far, Marziyeh Akbari, Mohammad Amin Habibi, et al.
Cell Prolif. 2025 Nov;58(11):e70099. doi: 10.1111/cpr.70099. Epub 2025 Jul 20.
CRISPR-Cas9 technology has rapidly advanced as a transformative genome-editing platform, facilitating precise genetic modifications and expanding therapeutic opportunities across various diseases. This review explores recent developments and clinical translations of CRISPR applications in oncology, genetic and neurological disorders, infectious diseases, immunotherapy, diagnostics, and epigenome editing. CRISPR has notably progressed in oncology, where it enables the identification of novel cancer drivers, elucidation of resistance mechanisms, and improvement of immunotherapies through engineered T cells, including PD-1 knockout CAR-T cells. Clinical trials employing CRISPR-edited cells are demonstrating promising results in hematologic malignancies and solid tumours. In genetic disorders, such as hemoglobinopathies and muscular dystrophies, CRISPR-Cas9 alongside advanced editors like base and prime editors show significant potential for correcting pathogenic mutations. This potential was affirmed with the FDA's first approval of a CRISPR-based therapy, Casgevy, for sickle cell disease in 2023. Neurological disorders, including Alzheimer's, ALS, and Huntington's disease, are increasingly targeted by CRISPR approaches for disease modelling and potential therapeutic intervention. In infectious diseases, CRISPR-based diagnostics such as SHERLOCK and DETECTR provide rapid, sensitive nucleic acid detection, particularly valuable in pathogen outbreaks like SARS-CoV-2. Therapeutically, CRISPR systems target viral and bacterial genomes, offering novel treatment modalities. Additionally, CRISPR-mediated epigenome editing enables precise regulation of gene expression, expanding therapeutic possibilities. Despite these advances, significant challenges remain, including off-target effects, delivery methodologies, immune responses, and long-term genomic safety concerns. Future improvements in editor precision, innovative delivery platforms, and enhanced safety assessments will be essential to fully integrate CRISPR-based interventions into standard clinical practice, significantly advancing personalised medicine.
64CRISPR-Cas9 Editing of the and Promoters to Treat Sickle Cell Disease.PubMed
Akshay Sharma, Jaap-Jan Boelens, Maria Cancio, et al.
N Engl J Med. 2023 Aug 31;389(9):820-832. doi: 10.1056/NEJMoa2215643.
BACKGROUND: Sickle cell disease is caused by a defect in the β-globin subunit of adult hemoglobin. Sickle hemoglobin polymerizes under hypoxic conditions, producing deformed red cells that hemolyze and cause vaso-occlusion that results in progressive organ damage and early death. Elevated fetal hemoglobin levels in red cells protect against complications of sickle cell disease. OTQ923, a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-edited CD34+ hematopoietic stem- and progenitor-cell (HSPC) product, has a targeted disruption of the and (γ-globin) gene promoters that increases fetal hemoglobin expression in red-cell progeny. METHODS: We performed a tiling CRISPR-Cas9 screen of the and promoters by electroporating CD34+ cells obtained from healthy donors with Cas9 complexed with one of 72 guide RNAs, and we assessed the fraction of fetal hemoglobin-immunostaining erythroblasts (F cells) in erythroid-differentiated progeny. The gRNA resulting in the highest level of F cells (gRNA-68) was selected for clinical development. We enrolled participants with severe sickle cell disease in a multicenter, phase 1-2 clinical study to assess the safety and adverse-effect profile of OTQ923. RESULTS: In preclinical experiments, CD34+ HSPCs (obtained from healthy donors and persons with sickle cell disease) edited with CRISPR-Cas9 and gRNA-68 had sustained on-target editing with no off-target mutations and produced high levels of fetal hemoglobin after in vitro differentiation or xenotransplantation into immunodeficient mice. In the study, three participants received autologous OTQ923 after myeloablative conditioning and were followed for 6 to 18 months. At the end of the follow-up period, all the participants had engraftment and stable induction of fetal hemoglobin (fetal hemoglobin as a percentage of total hemoglobin, 19.0 to 26.8%), with fetal hemoglobin broadly distributed in red cells (F cells as a percentage of red cells, 69.7 to 87.8%). Manifestations of sickle cell disease decreased during the follow-up period. CONCLUSIONS: CRISPR-Cas9 disruption of the and gene promoters was an effective strategy for induction of fetal hemoglobin. Infusion of autologous OTQ923 into three participants with severe sickle cell disease resulted in sustained induction of red-cell fetal hemoglobin and clinical improvement in disease severity. (Funded by Novartis Pharmaceuticals; ClinicalTrials.gov number, NCT04443907.).
65Sickle cell disease: combination new therapies vs. CRISPR-Cas9 potential and challenges - review article.PubMed
Ilham Youssry, Nardeen Ayad
Ann Hematol. 2024 Aug;103(8):2613-2619. doi: 10.1007/s00277-023-05510-0. Epub 2023 Oct 23.
In 2022, sickle cell disease (SCD) continues to affect the lives of millions of people, being one of the most frequently inherited blood disorders worldwide. Recently, several new therapies have been FDA approved for the treatment of SCD. The complexity of the pathophysiology of sickling has given opportunity to the evolution of several modalities of therapies. Nonetheless, the potential for complementary targeting of HbS polymerization, vasocclusion, and other inflammatory pathways remains controversial. None of these drugs can be considered a single curative line of treatment. With the advancement of CRISPR/Cas9 technology, autologous transplant of gene-edited hematopoietic stem cells could possibly provide a cure for most patients with SCD. The advantage of this approach over the conventional stem cell transplantation is that it decreases the need for immuno-suppressive drugs and the risk of graft-versus-host disease. In addition, recent technological advances can reduce the off-target effects, but long-term monitoring is needed to ensure the reliability of these methods in the clinical setting. This review explores the efficacy and safety of combination therapies and contrasting this alternative with the challenges that exist with sickle cell gene therapy using CRISPR.
66Exagamglogene Autotemcel for Severe Sickle Cell Disease.PubMed
Haydar Frangoul, Franco Locatelli, Akshay Sharma, et al.
N Engl J Med. 2024 May 9;390(18):1649-1662. doi: 10.1056/NEJMoa2309676. Epub 2024 Apr 24.
BACKGROUND: Exagamglogene autotemcel (exa-cel) is a nonviral cell therapy designed to reactivate fetal hemoglobin synthesis by means of ex vivo clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 gene editing of autologous CD34+ hematopoietic stem and progenitor cells (HSPCs) at the erythroid-specific enhancer region of . METHODS: We conducted a phase 3, single-group, open-label study of exa-cel in patients 12 to 35 years of age with sickle cell disease who had had at least two severe vaso-occlusive crises in each of the 2 years before screening. CD34+ HSPCs were edited with the use of CRISPR-Cas9. Before the exa-cel infusion, patients underwent myeloablative conditioning with pharmacokinetically dose-adjusted busulfan. The primary end point was freedom from severe vaso-occlusive crises for at least 12 consecutive months. A key secondary end point was freedom from inpatient hospitalization for severe vaso-occlusive crises for at least 12 consecutive months. The safety of exa-cel was also assessed. RESULTS: A total of 44 patients received exa-cel, and the median follow-up was 19.3 months (range, 0.8 to 48.1). Neutrophils and platelets engrafted in each patient. Of the 30 patients who had sufficient follow-up to be evaluated, 29 (97%; 95% confidence interval [CI], 83 to 100) were free from vaso-occlusive crises for at least 12 consecutive months, and all 30 (100%; 95% CI, 88 to 100) were free from hospitalizations for vaso-occlusive crises for at least 12 consecutive months (P<0.001 for both comparisons against the null hypothesis of a 50% response). The safety profile of exa-cel was generally consistent with that of myeloablative busulfan conditioning and autologous HSPC transplantation. No cancers occurred. CONCLUSIONS: Treatment with exa-cel eliminated vaso-occlusive crises in 97% of patients with sickle cell disease for a period of 12 months or more. (CLIMB SCD-121; ClinicalTrials.gov number, NCT03745287.).
67Safety and efficacy studies of CRISPR-Cas9 treatment of sickle cell disease highlights disease-specific responses.PubMed
Giacomo Frati, Megane Brusson, Gilles Sartre, et al.
Mol Ther. 2024 Dec 4;32(12):4337-4352. doi: 10.1016/j.ymthe.2024.07.015. Epub 2024 Jul 22.
Fetal hemoglobin (HbF) reactivation expression through CRISPR-Cas9 is a promising strategy for the treatment of sickle cell disease (SCD). Here, we describe a genome editing strategy leading to reactivation of HbF expression by targeting the binding sites (BSs) for the lymphoma-related factor (LRF) repressor in the γ-globin promoters. CRISPR-Cas9 treatment in healthy donor (HD) and patient-derived HSPCs resulted in a high frequency of LRF BS disruption and potent HbF synthesis in their erythroid progeny. LRF BS disruption did not impair HSPC engraftment and differentiation but was more efficient in SCD than in HD cells. However, SCD HSPCs showed a reduced engraftment and a myeloid bias compared with HD cells. We detected off-target activity and chromosomal rearrangements, particularly in SCD samples (likely because of the higher overall editing efficiency) but did not impact the target gene expression and HSPC engraftment and differentiation. Transcriptomic analyses showed that the editing procedure results in the up-regulation of genes involved in DNA damage and inflammatory responses, which was more evident in SCD HSPCs. This study provides evidence of efficacy and safety for an editing strategy based on HbF reactivation and highlights the need of performing safety studies in clinically relevant conditions, i.e., in patient-derived HSPCs.
68[A CRISPR/Cas approach to β-haemoglobinopathies].PubMed
Megane Brusson, Annarita Miccio
Med Sci (Paris). 2025 Jan;41(1):33-39. doi: 10.1051/medsci/2024191. Epub 2025 Jan 31.
Beta-haemoglobinopathies are severe genetic anemias caused by mutations that affect adult haemoglobin production. Many therapeutic approaches aim to reactivate the expression of the fetal hemoglobin genes. To this end, the CRISPR/Cas9 system has recently been used to genetically modify patients' hematopoietic stem/progenitor cells ex vivo and reactivate fetal hemoglobin expression in their erythroid progeny. More than 70 patients with severe β-thalassemia and sickle cell disease have been treated with the Casgevy® therapy. Most have achieved a significant improvement of clinical phenotype, with high editing efficiency in hematopoietic cells associated with normal or near normal hemoglobin levels. While the long-term safety and efficacy of this powerful approach still need to be evaluated, new strategies are being developed to further improve therapeutic outcomes, reduce potential genotoxicity and lower the costs of therapy.
69Gene editing without ex vivo culture evades genotoxicity in human hematopoietic stem cells.PubMed
Jing Zeng, My Anh Nguyen, Pengpeng Liu, et al.
Cell Stem Cell. 2025 Feb 6;32(2):191-208.e11. doi: 10.1016/j.stem.2024.11.001. Epub 2024 Dec 12.
Gene editing the BCL11A erythroid enhancer is a validated approach to fetal hemoglobin (HbF) induction for β-hemoglobinopathy therapy, though heterogeneity in edit allele distribution and HbF response may impact its safety and efficacy. Here, we compare combined CRISPR-Cas9 editing of the BCL11A +58 and +55 enhancers with leading gene modification approaches under clinical investigation. Dual targeting of the BCL11A +58 and +55 enhancers with 3xNLS-SpCas9 and two single guide RNAs (sgRNAs) resulted in superior HbF induction, including in sickle cell disease (SCD) patient xenografts, attributable to simultaneous disruption of core half E-box/GATA motifs at both enhancers. Unintended on-target outcomes of double-strand break (DSB) repair in hematopoietic stem and progenitor cells (HSPCs), such as long deletions and centromere-distal chromosome fragment loss, are a byproduct of cellular proliferation stimulated by ex vivo culture. Editing quiescent HSPCs bypasses long deletion and micronuclei formation and preserves efficient on-target editing and engraftment function.
70Infrequent Resolution of Vaso-Occlusive Crises in Routine Clinical Care Among Patients Mimicking the Exa-Cel Trial Population: A Cohort Study of Medicaid Enrollees.PubMed
Mufaddal Mahesri, Su Been Lee, Raisa Levin, et al.
Clin Pharmacol Ther. 2024 Dec;116(6):1572-1579. doi: 10.1002/cpt.3449. Epub 2024 Sep 27.
The CRISPR-based gene editing therapy exagamglogene autotemcel (exa-cel) recently received FDA approval for patients with severe sickle cell disease (SCD). The approval was based on a phase III trial (CLIMB SCD 121), which showed 97% efficacy of this treatment in eliminating vaso occlusive crises (VOCs) for 12 consecutive months. To help contextualize results from this trial, we aimed to investigate the proportion of patients with severe SCD who remain VOC-free for a 1-year period in routine clinical care. Using Medicaid claims data (2000-2018), we identified a cohort of patients, 12-35 years old with severe SCD, defined by ≥ 2 VOCs per year for 2 consecutive years, who met other exa-cel trial inclusion criteria to mimic a trial-like population. A VOC was identified using ICD diagnosis codes during hospitalization and ER visits. The primary outcome was the proportion of patients with no VOCs during a 1-year follow-up. A total of 7,425 patients with severe SCD [mean (SD) age: 20.5 (6.0) years, 54.6% females, 84% African Americans], had a mean of 5.2 VOCs, 5.1 ER visits and 3.5 hospitalizations per year during the baseline period. The proportion of patients with no VOCs during the 1-year follow-up was 7.7% (95% confidence interval: 7.1%-8.3%). In conclusion, less than one in 12 patients with severe SCD achieved VOC-free status within 1 year in routine clinical care. These findings suggest that the high efficacy observed for exa-cel in the trial, if replicated in routine clinical care, could translate into a significant public health impact.
71Casgevy: Innovative Medicinal Products Require Innovative Approaches to Regulatory Assessment.PubMed
Essam Kerwash, John D Johnston
Pharmaceutics. 2024 Jul 6;16(7):906. doi: 10.3390/pharmaceutics16070906.
Casgevy (exa-cel) is an autologous cellular therapy modified ex vivo by a CRISPR-Cas9-mediated gene-editing technology. For Casgevy to be granted the indication in transfusion-dependent β-thalassemia, one single-arm trial was submitted which was not amenable to conventional statistical analysis of 'effect of cause'. Therefore, an analysis was conducted on the basis of 'cause of effect' making use of the scheme described by Toulmin coupled to an analysis of causal inference. Based on the current data within the submitted study: subjects with transfusion-dependent β-thalassemia no longer needed a red blood cell transfusion with a 93-percent probability if and only if administered Casgevy; PNS = 93%. It is acknowledged that unknown elements of safety may yet be revealed by long-term follow-up of recipients of Casgevy. Its durability of efficacy is, at present, also an unknown that may also be ascertained by long-term follow-up of recipients. The limitations of a causal analysis are related to assumptions of the proposed causal structure which may not capture the complexity of the real world. Overall, the claim that Casgevy is indicated to treat people with transfusion-dependent β-thalassemia is considered to be supported by the results of the submitted study; the benefit-risk evaluation of Casgevy is found to be positive.
72Recent advances and applications of CRISPR-Cas9 in cancer immunotherapy.PubMed
Zaoqu Liu, Meixin Shi, Yuqing Ren, et al.
Mol Cancer. 2023 Feb 16;22(1):35. doi: 10.1186/s12943-023-01738-6.
The incidence and mortality of cancer are the major health issue worldwide. Apart from the treatments developed to date, the unsatisfactory therapeutic effects of cancers have not been addressed by broadening the toolbox. The advent of immunotherapy has ushered in a new era in the treatments of solid tumors, but remains limited and requires breaking adverse effects. Meanwhile, the development of advanced technologies can be further boosted by gene analysis and manipulation at the molecular level. The advent of cutting-edge genome editing technology, especially clustered regularly interspaced short palindromic repeats (CRISPR-Cas9), has demonstrated its potential to break the limits of immunotherapy in cancers. In this review, the mechanism of CRISPR-Cas9-mediated genome editing and a powerful CRISPR toolbox are introduced. Furthermore, we focus on reviewing the impact of CRISPR-induced double-strand breaks (DSBs) on cancer immunotherapy (knockout or knockin). Finally, we discuss the CRISPR-Cas9-based genome-wide screening for target identification, emphasis the potential of spatial CRISPR genomics, and present the comprehensive application and challenges in basic research, translational medicine and clinics of CRISPR-Cas9.
73CRISPR/Cas9 therapeutics: progress and prospects.PubMed
Tianxiang Li, Yanyan Yang, Hongzhao Qi, et al.
Signal Transduct Target Ther. 2023 Jan 16;8(1):36. doi: 10.1038/s41392-023-01309-7.
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene-editing technology is the ideal tool of the future for treating diseases by permanently correcting deleterious base mutations or disrupting disease-causing genes with great precision and efficiency. A variety of efficient Cas9 variants and derivatives have been developed to cope with the complex genomic changes that occur during diseases. However, strategies to effectively deliver the CRISPR system to diseased cells in vivo are currently lacking, and nonviral vectors with target recognition functions may be the focus of future research. Pathological and physiological changes resulting from disease onset are expected to serve as identifying factors for targeted delivery or targets for gene editing. Diseases are both varied and complex, and the choice of appropriate gene-editing methods and delivery vectors for different diseases is important. Meanwhile, there are still many potential challenges identified when targeting delivery of CRISPR/Cas9 technology for disease treatment. This paper reviews the current developments in three aspects, namely, gene-editing type, delivery vector, and disease characteristics. Additionally, this paper summarizes successful examples of clinical trials and finally describes possible problems associated with current CRISPR applications.
74Engineered TCR-T Cell Immunotherapy in Anticancer Precision Medicine: Pros and Cons.PubMed
Qijie Zhao, Yu Jiang, Shixin Xiang, et al.
Front Immunol. 2021 Mar 30;12:658753. doi: 10.3389/fimmu.2021.658753. eCollection 2021.
This review provides insight into the role of engineered T-cell receptors (TCRs) in immunotherapy. Novel approaches have been developed to boost anticancer immune system, including targeting new antigens, manufacturing new engineered or modified TCRs, and creating a safety switch for endo-suicide genes. In order to re-activate T cells against tumors, immune-mobilizing monoclonal TCRs against cancer (ImmTAC) have been developed as a novel class of manufactured molecules which are bispecific and recognize both cancer and T cells. The TCRs target special antigens such as NY-ESO-1, AHNAK or ERBB2 to boost the efficacy of anticancer immunotherapy. The safety of genetically modified T cells is very important. Therefore, this review discusses pros and cons of different approaches, such as ImmTAC, Herpes simplex virus thymidine kinase (HSV-TK), and inducible caspase-9 in cancer immunotherapy. Clinical trials related to TCR-T cell therapy and monoclonal antibodies designed for overcoming immunosuppression, and recent advances made in understanding how TCRs are additionally examined. New approaches that can better detect antigens and drive an effective T cell response are discussed as well.
75Phase 1 clinical trial to assess safety and efficacy of NY-ESO-1-specific TCR T cells in HLA-A∗02:01 patients with advanced soft tissue sarcoma.PubMed
Qiuzhong Pan, Desheng Weng, Jiayong Liu, et al.
Cell Rep Med. 2023 Aug 15;4(8):101133. doi: 10.1016/j.xcrm.2023.101133.
New York esophageal squamous cell carcinoma-1 (NY-ESO-1)-specific T cell receptor (TCR) T cell therapy is effective in tumors with NY-ESO-1 expression, but a safe and effective TCR-T cell therapeutic protocol remains to be improved. Here, we report a phase 1 investigational new drug clinical trial with TCR affinity-enhanced specific T cell therapy (TAEST16001) for targeting NY-ESO-1. Enrolled patients receive TAEST16001 cell infusion after dose-reduced lymphodepletion with cyclophosphamide (15 mg/kg/day × 3 days) combined with fludarabine (20 mg/m/day × 3 days), and the TCR-T cells are maintained with low doses of interleukin-2 injection post-adoptive transfer. Analysis of 12 patients treated with the regimen demonstrates no treatment-related serious adverse events. The overall response rate is 41.7%. The median progression-free survival is 7.2 months, and the median duration of response is 13.1 months. The protocol of TAEST16001 cells delivers a safe and highly effective treatment for patients with advanced soft tissue sarcoma (ClinicalTrials.gov: NCT04318964).
76Adoptive T-Cell Therapy in Sarcomas.PubMed
Monika Kucharczyk, Emine Hatipoglu, Robin L Jones, et al.
Curr Oncol Rep. 2025 Oct;27(10):1131-1143. doi: 10.1007/s11912-025-01706-x. Epub 2025 Aug 14.
PURPOSE OF REVIEW: To summarise and evaluate the latest adoptive T-cell therapies in sarcomas, focusing on therapeutic targets, efficacy, safety, and limitations. RECENT FINDINGS: An increasing number of clinical trials are investigating adoptive T-cell therapies in sarcomas, most targeting NY-ESO-1 and MAGE-A4 through engineered T-cell receptors (TCR-T). The FDA approval of afamitresgene autoleucel for advanced synovial sarcoma and the breakthrough designation of letetresgene autoleucel for myxoid/round cell liposarcoma signify a major turning point. Chimeric antigen receptor T strategies target mainly B7H3, GD2, FGFR4, and HER2, with innovations including dual antigen targeting and safety switches. Tumour infiltrating lymphocyte therapy, including lifileucel, is under investigation with checkpoint inhibitors or oncolytic agents to enhance efficacy and manage toxicity. Adoptive T-cell therapy demonstrates early promise in sarcomas, particularly TCR-T therapy. Challenges include HLA restriction, tumour heterogeneity, and manufacturing complexity. Future strategies involving novel antigens, multi-targeting, and combinatorial regimens could broaden patient eligibility and improve therapeutic outcomes.
77The Emerging World of TCR-T Cell Trials Against Cancer: A Systematic Review.PubMed
Jianxiang Zhang, Lingyu Wang
Technol Cancer Res Treat. 2019 Jan 1;18:1533033819831068. doi: 10.1177/1533033819831068.
T-cell receptor-engineered T-cell therapy and chimeric antigen receptor T-cell therapy are 2 types of adoptive T-cell therapy that genetically modify natural T cells to treat cancers. Although chimeric antigen receptor T-cell therapy has yielded remarkable efficacy for hematological malignancies of the B-cell lineages, most solid tumors fail to respond significantly to chimeric antigen receptor T cells. T-cell receptor-engineered T-cell therapy, on the other hand, has shown unprecedented promise in treating solid tumors and has attracted growing interest. In order to create an unbiased, comprehensive, and scientific report for this fast-moving field, we carefully analyzed all 84 clinical trials using T-cell receptor-engineered T-cell therapy and downloaded from ClinicalTrials.gov updated by June 11, 2018. Informative features and trends were observed in these clinical trials. The number of trials initiated each year is increasing as expected, but an interesting pattern is observed. NY-ESO-1, as the most targeted antigen type, is the target of 31 clinical trials; melanoma is the most targeted cancer type and is the target of 33 clinical trials. Novel antigens and underrepresented cancers remain to be targeted in future studies and clinical trials. Unlike chimeric antigen receptor T-cell therapy, only about 16% of the 84 clinical trials target against hematological malignancies, consistent with T-cell receptor-engineered T-cell therapy's high potential for solid tumors. Six pharma/biotech companies with novel T-cell receptor-engineered T-cell ideas and products were examined in this review. Multiple approaches have been utilized in these companies to increase the T-cell receptor's affinity and efficiency and to minimize cross-reactivity. The major challenges in the development of the T-cell receptor-engineered T-cell therapy due to tumor microenvironment were also discussed here.
78A phase 1 trial of NY-ESO-1-specific TCR-engineered T-cell therapy combined with a lymph node-targeting nanoparticulate peptide vaccine for the treatment of advanced soft tissue sarcoma.PubMed
Mikiya Ishihara, Yoshihiro Nishida, Shigehisa Kitano, et al.
Int J Cancer. 2023 Jun 15;152(12):2554-2566. doi: 10.1002/ijc.34453. Epub 2023 Feb 17.
The efficacy of immune checkpoint inhibitors is limited in refractory solid tumors. T-cell receptor gene-modified T (TCR-T)-cell therapy has attracted attention as a new immunotherapy for refractory cold tumors. We first investigated the preclinical efficacy and mode of action of TCR-T cells combined with the pullulan nanogel:long peptide antigen (LPA) vaccine in a mouse sarcoma model that is resistant to immune checkpoint inhibition. Without lymphodepletion, the pullulan nanogel:LPA vaccine markedly increased the number of TCR-T cells in the draining lymph node and tumor tissue. This change was associated with enhanced CXCR3 expression in TCR-T cells in the draining lymph node. In the phase 1 trial, autologous New York esophageal squamous cell carcinoma 1 (NY-ESO-1)-specific TCR-T cells were infused twice into HLA-matched patients with NY-ESO-1 soft tissue sarcoma (STS). The pullulan nanogel:LPA vaccine contains an epitope recognized by TCR-T cells, and it was subcutaneously injected 1 day before and 7 days after the infusion of TCR-T cells. Lymphodepletion was not performed. Three patients with refractory synovial sarcoma (SS) were treated. Two out of the three patients developed cytokine release syndrome (CRS) with low-to-moderate cytokine level elevation. We found obvious tumor shrinkage lasting for more than 2 years by tumor imaging and long-term persistence of TCR-T cells in one patient. In conclusion, NY-ESO-1-specific TCR-T-cell therapy plus vaccination with the pullulan nanogel carrying an LPA containing the NY-ESO-1 epitope without lymphodepletion is feasible and can induce promising long-lasting therapeutic effects in refractory SS (Registration ID: JMA-IIA00346).
79Human cancer-targeted immunity via transgenic hematopoietic stem cell progeny.PubMed
Theodore S Nowicki, Nataly Naser Al Deen, Cole W Peters, et al.
Nat Commun. 2025 Jul 1;16(1):5599. doi: 10.1038/s41467-025-60816-z.
Adoptive transfer of genetically engineered T cells expressing a tumor-antigen-specific transgenic T cell receptor (TCR) can result in clinical responses in a variety of malignancies. However, these responses are frequently short-lived, and patients typically relapse within several months. This phenomenon is largely due to poor persistence of the transgenic T cells, as well as a progressive loss of their functionality and terminal differentiation in vivo. This underscores the need for cell therapy approaches able to sustain the initial antitumor efficacy and lead to long-term antitumor efficacy. Herein, we report the use of tandem cell therapies involving autologous T cells and hematopoietic stem cells engineered to express the NY-ESO-1 TCR for the treatment of solid tumors in a first-in-human phase I clinical trial (NCT03240861). This therapy is shown to be safe, feasible, and leads to initial tumor regression activity. T cell progeny from the HSC progenitors is shown to provide circulating transgenic NY-ESO-1 TCR-T cells, which display tumor-antigen-specific antitumor functionality, without any evidence of anergy or exhaustion. These results demonstrate the utility of transgenic HSCs to generate a self-renewing source of tumor-specific cellular immunotherapy in human participants. Clinicaltrials.gov: NCT NCT03240861.
80CRISPR/Cas9-Mediated Genome Editing in Cancer Therapy.PubMed
Shuai Ding, Jinfeng Liu, Xin Han, et al.
Int J Mol Sci. 2023 Nov 15;24(22):16325. doi: 10.3390/ijms242216325.
The Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system, an RNA-based adaptive immune system found in bacteria and archaea, has catalyzed the development and application of a new generation of gene editing tools. Numerous studies have shown that this system can precisely target a wide range of human genes, including those associated with diseases such as cancer. In cancer research, the intricate genetic mutations in tumors have promoted extensive utilization of the CRISPR/Cas9 system due to its efficient and accurate gene editing capabilities. This includes improvements in Chimeric Antigen Receptor (CAR)-T-cell therapy, the establishment of tumor models, and gene and drug target screening. Such progress has propelled the investigation of cancer molecular mechanisms and the advancement of precision medicine. However, the therapeutic potential of genome editing remains underexplored, and lingering challenges could elevate the risk of additional genetic mutations. Here, we elucidate the fundamental principles of CRISPR/Cas9 gene editing and its practical applications in tumor research. We also briefly discuss the primary challenges faced by CRISPR technology and existing solutions, intending to enhance the efficacy of this gene editing therapy and shed light on the underlying mechanisms of tumors.
81TIM-3, LAG-3, or 2B4 gene disruptions increase the anti-tumor response of engineered T cells.PubMed
Beatrice Claudia Cianciotti, Zulma Irene Magnani, Alessia Ugolini, et al.
Front Immunol. 2024 Feb 29;15:1315283. doi: 10.3389/fimmu.2024.1315283. eCollection 2024.
BACKGROUND: In adoptive T cell therapy, the long term therapeutic benefits in patients treated with engineered tumor specific T cells are limited by the lack of long term persistence of the infused cellular products and by the immunosuppressive mechanisms active in the tumor microenvironment. Exhausted T cells infiltrating the tumor are characterized by loss of effector functions triggered by multiple inhibitory receptors (IRs). In patients, IR blockade reverts T cell exhaustion but has low selectivity, potentially unleashing autoreactive clones and resulting in clinical autoimmune side effects. Furthermore, loss of long term protective immunity in cell therapy has been ascribed to the effector memory phenotype of the infused cells. METHODS: We simultaneously redirected T cell specificity towards the NY-ESO-1 antigen via TCR gene editing (TCR) and permanently disrupted , or genes (IR) via CRISPR/Cas9 in a protocol to expand early differentiated long-living memory stem T cells. The effector functions of the TCR-IR and IR competent (TCR-IR) cells were tested in short-term co-culture assays and under a chronic stimulation setting . Finally, the therapeutic efficacy of the developed cellular products were evaluated in multiple myeloma xenograft models. RESULTS: We show that upon chronic stimulation, TCR-IR cells are superior to TCR-IR cells in resisting functional exhaustion through different mechanisms and efficiently eliminate cancer cells upon tumor re-challenge . Our data indicate that TIM-3 and 2B4-disruption preserve T-cell degranulation capacity, while LAG-3 disruption prevents the upregulation of additional inhibitory receptors in T cells. CONCLUSION: These results highlight that TIM-3, LAG-3, and 2B4 disruptions increase the therapeutic benefit of tumor specific cellular products and suggest distinct, non-redundant roles for IRs in anti-tumor responses.
82Transgenic mice for in vivo epigenome editing with CRISPR-based systems.PubMed
Matthew P Gemberling, Keith Siklenka, Erica Rodriguez, et al.
Nat Methods. 2021 Aug;18(8):965-974. doi: 10.1038/s41592-021-01207-2. Epub 2021 Aug 2.
CRISPR-Cas9 technologies have dramatically increased the ease of targeting DNA sequences in the genomes of living systems. The fusion of chromatin-modifying domains to nuclease-deactivated Cas9 (dCas9) has enabled targeted epigenome editing in both cultured cells and animal models. However, delivering large dCas9 fusion proteins to target cells and tissues is an obstacle to the widespread adoption of these tools for in vivo studies. Here, we describe the generation and characterization of two conditional transgenic mouse lines for epigenome editing, Rosa26:LSL-dCas9-p300 for gene activation and Rosa26:LSL-dCas9-KRAB for gene repression. By targeting the guide RNAs to transcriptional start sites or distal enhancer elements, we demonstrate regulation of target genes and corresponding changes to epigenetic states and downstream phenotypes in the brain and liver in vivo, and in T cells and fibroblasts ex vivo. These mouse lines are convenient and valuable tools for facile, temporally controlled, and tissue-restricted epigenome editing and manipulation of gene expression in vivo.
83Therapeutic in vivo delivery of gene editing agents.PubMed
Aditya Raguram, Samagya Banskota, David R Liu
Cell. 2022 Jul 21;185(15):2806-2827. doi: 10.1016/j.cell.2022.03.045. Epub 2022 Jul 6.
In vivo gene editing therapies offer the potential to treat the root causes of many genetic diseases. Realizing the promise of therapeutic in vivo gene editing requires the ability to safely and efficiently deliver gene editing agents to relevant organs and tissues in vivo. Here, we review current delivery technologies that have been used to enable therapeutic in vivo gene editing, including viral vectors, lipid nanoparticles, and virus-like particles. Since no single delivery modality is likely to be appropriate for every possible application, we compare the benefits and drawbacks of each method and highlight opportunities for future improvements.
84CRISPR-Cas9 genome editing using targeted lipid nanoparticles for cancer therapy.PubMed
Daniel Rosenblum, Anna Gutkin, Ranit Kedmi, et al.
Sci Adv. 2020 Nov 18;6(47). doi: 10.1126/sciadv.abc9450. Print 2020 Nov.
Harnessing CRISPR-Cas9 technology for cancer therapeutics has been hampered by low editing efficiency in tumors and potential toxicity of existing delivery systems. Here, we describe a safe and efficient lipid nanoparticle (LNP) for the delivery of Cas9 mRNA and sgRNAs that use a novel amino-ionizable lipid. A single intracerebral injection of CRISPR-LNPs against (sgPLK1-cLNPs) into aggressive orthotopic glioblastoma enabled up to ~70% gene editing in vivo, which caused tumor cell apoptosis, inhibited tumor growth by 50%, and improved survival by 30%. To reach disseminated tumors, cLNPs were also engineered for antibody-targeted delivery. Intraperitoneal injections of EGFR-targeted sgPLK1-cLNPs caused their selective uptake into disseminated ovarian tumors, enabled up to ~80% gene editing in vivo, inhibited tumor growth, and increased survival by 80%. The ability to disrupt gene expression in vivo in tumors opens new avenues for cancer treatment and research and potential applications for targeted gene editing of noncancerous tissues.
85Gene therapy clinical trials worldwide to 2023-an update.PubMed
Samantha L Ginn, Mawj Mandwie, Ian E Alexander, et al.
J Gene Med. 2024 Aug;26(8):e3721. doi: 10.1002/jgm.3721.
To date, 3,900 gene therapy clinical trials have been completed, are ongoing or have been approved worldwide. Our database brings together global information on gene therapy clinical activity from trial databases, official agency sources, published literature, conference presentations and posters kindly provided to us by individual investigators or trial sponsors. This review presents our analysis of clinical trials that, to the best of our knowledge, have been or are being performed worldwide. As of our March 2023 update, we have entries on 3,900 trials undertaken in 46 countries. We have analyzed the geographical distribution of trials, the disease indications (or other reasons) for trials, the proportions to which different vector types are used, and which genes have been transferred. Details of the analyses presented, and our searchable database are on The Journal of Gene Medicine Gene Therapy Clinical Trials Worldwide website at https://a873679.fmphost.com/fmi/webd/GTCT. We also provide an overview of the progress being made around the world, and discuss key trends since the previous review, namely the unprecedented increase in gene therapy clinical trial activity, including the implementation of genome editing technology with the potential to transform the field moving forward.
86CRISPR/Cas9 for cancer research and therapy.PubMed
Tianzuo Zhan, Niklas Rindtorff, Johannes Betge, et al.
Semin Cancer Biol. 2019 Apr;55:106-119. doi: 10.1016/j.semcancer.2018.04.001. Epub 2018 Apr 16.
CRISPR/Cas9 has become a powerful method for making changes to the genome of many organisms. First discovered in bacteria as part of an adaptive immune system, CRISPR/Cas9 and modified versions have found a widespread use to engineer genomes and to activate or to repress the expression of genes. As such, CRISPR/Cas9 promises to accelerate cancer research by providing an efficient technology to dissect mechanisms of tumorigenesis, identify targets for drug development, and possibly arm cells for cell-based therapies. Here, we review current applications of the CRISPR/Cas9 technology for cancer research and therapy. We describe novel Cas9 variants and how they are used in functional genomics to discover novel cancer-specific vulnerabilities. Furthermore, we highlight the impact of CRISPR/Cas9 in generating organoid and mouse models of cancer. Finally, we provide an overview of the first clinical trials that apply CRISPR/Cas9 as a therapeutic approach against cancer.
87Gene therapy: a double-edged sword with great powers.PubMed
Ran Tang, Zhigang Xu
Mol Cell Biochem. 2020 Nov;474(1-2):73-81. doi: 10.1007/s11010-020-03834-3. Epub 2020 Jul 21.
Gene therapy is the treatment of a disease through transferring genetic material into cells of the patients. In the recent several years, gene therapy has experienced rapid progress and achieved huge success. Over two dozens of gene therapies have been approved for clinical use by the drug regulatory agencies from different countries. However, concerns about its efficacy and safety have accompanied gene therapy since its birth. In the present manuscript, we first introduce various strategies employed in gene therapy, which includes ex vivo gene delivery v.s. in vivo gene delivery; gene addition v.s. genome editing; inherited disease v.s. acquired disease; and somatic gene therapy v.s. germline gene therapy. Then we discuss the clinical outcomes of some approved gene therapies. We finish our discussion with the safety issues related to gene therapy. We will see that with the technology improvement, somatic gene therapy has been proved to be efficient and safe enough for clinical practice. However, germline gene therapy has important efficiency and safety issues at present, and should not be put into clinical practice before these issues are solved.
88Gene therapy: advances, challenges and perspectives.PubMed
Giulliana Augusta Rangel Gonçalves, Raquel de Melo Alves Paiva
Einstein (Sao Paulo). 2017 Jul-Sep;15(3):369-375. doi: 10.1590/S1679-45082017RB4024.
The ability to make site-specific modifications to the human genome has been an objective in medicine since the recognition of the gene as the basic unit of heredity. Thus, gene therapy is understood as the ability of genetic improvement through the correction of altered (mutated) genes or site-specific modifications that target therapeutic treatment. This therapy became possible through the advances of genetics and bioengineering that enabled manipulating vectors for delivery of extrachromosomal material to target cells. One of the main focuses of this technique is the optimization of delivery vehicles (vectors) that are mostly plasmids, nanostructured or viruses. The viruses are more often investigated due to their excellence of invading cells and inserting their genetic material. However, there is great concern regarding exacerbated immune responses and genome manipulation, especially in germ line cells. In vivo studies in in somatic cell showed satisfactory results with approved protocols in clinical trials. These trials have been conducted in the United States, Europe, Australia and China. Recent biotechnological advances, such as induced pluripotent stem cells in patients with liver diseases, chimeric antigen receptor T-cell immunotherapy, and genomic editing by CRISPR/Cas9, are addressed in this review.
89Pluripotent Stem Cell-Based Cell Therapy-Promise and Challenges.PubMed
Shinya Yamanaka
Cell Stem Cell. 2020 Oct 1;27(4):523-531. doi: 10.1016/j.stem.2020.09.014.
Human pluripotent stem cells such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) provide unprecedented opportunities for cell therapies against intractable diseases and injuries. Both ESCs and iPSCs are already being used in clinical trials. However, we continue to encounter practical issues that limit their use, including their inherent properties of tumorigenicity, immunogenicity, and heterogeneity. Here, I review two decades of research aimed at overcoming these three difficulties.
90Pluripotent stem-cell-derived therapies in clinical trial: A 2025 update.PubMed
Agnete Kirkeby, Heather Main, Melissa Carpenter
Cell Stem Cell. 2025 Jan 2;32(1):10-37. doi: 10.1016/j.stem.2024.12.005.
Since the first derivation of human pluripotent stem cells (hPSCs) 27 years ago, technologies to control their differentiation and manufacturing have advanced immensely, enabling increasing numbers of clinical trials with hPSC-derived products. Here, we revew the landscape of interventional hPSC trials worldwide, highlighting available data on clinical safety and efficacy. As of December 2024, we identify 116 clinical trials with regulatory approval, testing 83 hPSC products. The majority of trials are targeting eye, central nervous system, and cancer. To date, more than 1,200 patients have been dosed with hPSC products, accumulating to >10 clinically administered cells, so far showing no generalizable safety concerns.
91Culturing human pluripotent stem cells for regenerative medicine.PubMed
Hiroki Ozawa, Takuya Matsumoto, Masato Nakagawa
Expert Opin Biol Ther. 2023 Jan-Jun;23(6):479-489. doi: 10.1080/14712598.2023.2225701. Epub 2023 Jun 23.
INTRODUCTION: The development of human pluripotent stem cell (hPSC) culture protocols has led to the establishment of directed differentiation induction methods, resulting in their application in regenerative medicine. Cell therapy products derived from hPSCs have been transplanted into patients, and promising results have been observed in some ongoing clinical trials. AREA COVERED: This review provides an overview of the challenges associated with the culture of hPSCs for clinical applications and the development of culture technologies designed to address these challenges. We also review future cell culture strategies for large-scale manufacturing to enhance patient access. EXPERT OPINION: Despite the great potential of hPSCs, difficulties such as safety, quality control, and cost management continue to pose obstacles to their product development and clinical translation. A substantial contribution of these issues lies in the cell culture process. Therefore, selecting the appropriate ancillary materials (AMs) and integrating effective culture methods in standard operating procedures (SOPs) from the early stages of clinical development are essential for success. Moreover, incorporating an automated scaling process is imperative to ensure the commercial feasibility of hPSC-based products. [Figure: see text]Human pluripotent stem cells (hPSCs) show great potential as a valuable resource for regenerative medicine. However, three significant obstacles must be overcome: safety, quality, and costs. Thankfully, recent progress in hPSC culture techniques has effectively tackled these challenges, opening up exciting possibilities for realizing hPSC-based regenerative medicine.
92Age-Related Macular Degeneration: Therapeutic Strategies Based on Stem Cells.PubMed
Isai Mathivanan, Enzmann Volker, Keerthana Thyagarajan, et al.
Stem Cell Rev Rep. 2025 Oct;21(7):1867-1882. doi: 10.1007/s12015-025-10917-5. Epub 2025 Jun 24.
Age-related macular degeneration (AMD) is the leading cause of irreversible vision loss among the elderly in developed countries, manifesting in two primary forms: neovascular (wet) AMD and non-neovascular (dry) AMD. Current treatments, such as anti-VEGF therapy, offer limited efficacy, particularly for dry AMD, highlighting the urgent need for alternative strategies. Advancements in contemporary treatment strategies for these eye conditions are a pressing medical concern. Stem cell-based therapies have emerged as a promising approach for retinal regeneration due to their capacity for self-renewal and differentiation into retinal cell types, including retinal pigment epithelial (RPE) cells and photoreceptors, two key cell populations damaged in AMD. Among the various sources, pluripotent stem cells such as human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) show significant potential in generating functional RPE cells and restoring retinal architecture and function. Preclinical and early clinical studies have demonstrated promising outcomes, including improved visual acuity and anatomical integration. However, challenges such as immune rejection, tumorigenicity, limited long-term integration, and ethical concerns continue to impede clinical translation. This review critically evaluates current stem cell-based therapeutic strategies for AMD, including advances in mesenchymal stem cells, retinal organoids, and combinatorial approaches with gene and nanotherapy. Furthermore, this review outlines the translational bottlenecks and future directions required to advance these therapies toward clinical application.
93Stem cells in clinical trials for treatment of retinal degeneration.PubMed
Henry Klassen
Expert Opin Biol Ther. 2016;16(1):7-14. doi: 10.1517/14712598.2016.1093110. Epub 2015 Sep 28.
INTRODUCTION: After decades of basic science research involving the testing of regenerative strategies in animal models of retinal degenerative diseases, a number of clinical trials are now underway, with additional trials set to begin shortly. These efforts will evaluate the safety and preliminary efficacy of cell-based products in the eyes of patients with a number of retinal conditions, notably including age-related macular degeneration, retinitis pigmentosa and Stargardt's disease. AREAS COVERED: This review considers the scientific work and early trials with fetal cells and tissues that set the stage for the current clinical investigatory work, as well the trials themselves, specifically those either now completed, underway or close to initiation. The cells of interest include retinal pigment epithelial cells derived from embryonic stem or induced pluripotent stem cells, undifferentiated neural or retinal progenitors or cells from the vascular/bone marrow compartment or umbilical cord tissue. EXPERT OPINION: Degenerative diseases of the retina represent a popular target for emerging cell-based therapeutics and initial data from early stage clinical trials suggest that short-term safety objectives can be met in at least some cases. The question of efficacy will require additional time and testing to be adequately resolved.
94Immunological aspects of RPE cell transplantation.PubMed
Sunao Sugita, Michiko Mandai, Hiroyuki Kamao, et al.
Prog Retin Eye Res. 2021 Sep;84:100950. doi: 10.1016/j.preteyeres.2021.100950. Epub 2021 Jan 19.
Retinal pigment epithelial (RPE) cells have several functions, including support of the neural retina and choroid in the eye and immunosuppression. Cultured human RPE cells directly suppress inflammatory immune cells. For instance, they directly suppress the activation of T cells in vitro. In contrast, transplanted allogeneic human RPE cells are rejected by bystander immune cells such as T cells in vivo. Recently, human embryonic stem cell-derived RPE cells have been used in several clinical trials, and human induced pluripotent stem cell (iPSC)-RPE cells have also been tested in our clinical study in patients with retinal degeneration. Major safety concerns after stem cell-based transplantation surgery include hyper-proliferation, tumorigenicity, or ectopic tissue formation, but these events have currently not been seen in any of these patients. However, if RPE cells are allogeneic, there are concerns about immune rejection issues that have been raised in previous clinical trials. We therefore performed a preclinical study of allogeneic iPSC-RPE cell transplantation in animal rejection models. We then conducted autogenic or allogeneic iPSC-RPE cell transplantation in clinical studies of patients with age-related macular degeneration. In this review, we focus on immunological studies of RPE cells, including iPSC-derived cells. iPSC-RPE cells have unique inflammatory (immunosuppressive and immunogenic) characteristics like primary cultured RPE cells. The purpose of this review is to summarize the current findings obtained from preclinical (basic research) and clinical studies in iPSC-RPE cell transplantation, especially the immunological aspects.
95The influence of femtosecond laser intrastromal lenticules on the characteristics and maturity in tissue-engineered stem cell-derived retinal pigment epithelium sheets.PubMed
Gu Jianing, Su Zhanyu, Wang Yini, et al.
Stem Cell Res Ther. 2025 Jun 20;16(1):316. doi: 10.1186/s13287-025-04463-7.
BACKGROUND: Recent advances in clinical trials have involved the transplantation of induced retinal pigment epithelium (iRPE) cells from stem cells in creating a functional monolayer that mimics the characteristics of natural adult RPE cells. One method of achieving this goal is through the use of tissue engineering. In this research, decellularised femtosecond laser intrastromal lenticules (dfLEN) were employed as a scaffold for cultivating a bioengineered iRPE monolayer sheet. METHODS: iRPE cells were obtained by differentiating induced pluripotent stem cells (iPSC). These cells were then seeded on decellularized FLI-lenticules (dfLEN). The functionality, characterization, and oxidative stress of iRPE cultured on dfLEN were compared with those cultured on plates (TCP) using various assays such as immunofluorescence (IF), Edu, CCK8, ELISA, DFCH-DA, and JC-1. Additionally, RNA-seq assays and electron microscope (SEM and TEM) were used to test the iRPE characteristic on engineered dfLEN. Finally, we evaluated the biocompatibility of iRPE-dfLEN sheets by transplanting them into the subretinal space of New Zealand white rabbits. RESULTS: The iRPE cells cultured on dfLEN exhibited morphology and physiology similar to that of native RPE tissue. The dfLEN not only increased the resistance capacity of iRPE cells but also improved their functional properties compared to TCP. In addition, our results indicate that dfLEN enhances the expression of genes associated with cilium assembly, resulting in notable improvements in ciliogenesis in iRPE cells. Finally, the dfLEN-iRPE sheets demonstrated favorable biocompatibility and some viability when transplanted into the subretinal space of rabbits for a period of 14 days. CONCLUSIONS: We determine that engineered RPE sheets using dfLEN scaffolds enhance RPE characteristics and functions, and suggest that dfLEN scaffolds promote cilium process maturation and polarization of iPSC-derived epithelial cells. Such a strategy for constructing iRPE sheets holds significant potential for advancing RPE cell therapy, disease models, and drug screening platforms.
96Gain of 20q11.21 in human pluripotent stem cells enhances differentiation to retinal pigment epithelium.PubMed
Loriana Vitillo, Fabiha Anjum, Zoe Hewitt, et al.
Stem Cell Res Ther. 2025 Feb 21;16(1):82. doi: 10.1186/s13287-025-04196-7.
BACKGROUND: Cell therapies based on human pluripotent stem cells (hPSCs) are in clinical trials with the aim of restoring vision in people with age-related macular degeneration. The final cell therapy product consists of retinal pigment epithelium (RPE) cells differentiated from hPSCs. However, hPSCs recurrently acquire genetic abnormalities that give them an advantage in culture with unknown effects to the clinically-relevant cell progeny. One of the most common genetic abnormalities in hPSCs is the sub-karyotype 20q11.21 copy number variant, known to carry oncogenes. Understanding the impact of this variant on RPE differentiation and its potential for malignant transformation is crucial for the development of safe and effective cell therapies. METHODS: We monitored the RPE differentiation efficiency of hPSCs with or without the 20q11.21 variant. We then phenotyped the purified RPE cells for functionality, purity and tumorigenicity potential. RESULTS: We observed that 20q11.21 clones exhibited an enhanced differentiation capacity, developing pigmented foci at a higher rate and yield compared to normal clones. Gene expression analysis confirmed the upregulation of key RPE markers in 20q11.21 clones. The enhanced differentiation capacity of 20q11.21 clones was found to be dependent on the activity of BCL-XL, located within the amplicon. Furthermore, we demonstrated that 20q11.21-containing RPE cells displayed a mature phenotype, maintained long-term stability, and exhibited no malignant transformation capacity in vitro. CONCLUSION: We demonstrated that gain of 20q11.21 enhances the speed and yield of RPE differentiation without compromising the phenotype of the derivatives. Finally, we discovered that 20q11.21-localised BCL-XL is important for RPE differentiation with potential non-canonical roles in retinal biology.
97Single-cell-pore-sized 3D printed scaffolds for retinal pigment epithelial cell therapy.PubMed
Hang Liu, Daniel Soo Lin Wong, Bhav Harshad Parikh, et al.
Acta Biomater. 2025 Nov;207:294-310. doi: 10.1016/j.actbio.2025.10.033. Epub 2025 Oct 17.
Cell therapy is one of the most promising methods to treat retinal degenerative diseases, and crucial to its success is optimizing biomaterials to facilitate the delivery of retinal pigment epithelial (RPE) cells. This study explores the application of single-cell-pore-sized 3D printed polycaprolactone (PCL) scaffolds for cultivating human embryonic stem cell-derived RPE cell sheets. It compares them with track-etched polyethylene terephthalate (PET) membranes, the commercial products used in clinical trials for RPE cell delivery. We engineered two types of scaffolds at the microscale to optimize cell culture conditions, specifically focusing on pore size and fiber spacing. Protein expression analysis demonstrated that one scaffold with a pore size of ∼10 µm facilitated superior cellular integrity and function. Functional assessments, including barrier integrity, permeability, and phagocytosis assays, indicated that this scaffold enhanced nutrient exchange and maintained effective RPE functions akin to PET membranes. In an in vivo study, color fundus, optical coherence tomography, immunohistochemistry, and electroretinography revealed that 3D printed scaffolds exhibited biocompatibility, stability, and minimal inflammatory responses in the subretinal space of porcine models for 2 months and rabbit models for 14 months, with no adverse impact on retinal structure or function over either period. The findings suggest that 3D-printed biodegradable scaffolds present a viable alternative for RPE cell delivery, potentially advancing therapies for retinal degenerative conditions. STATEMENT OF SIGNIFICANCE: Cell therapy shows great promise for treating eye diseases that lead to vision loss. A crucial aspect of this therapy is delivering specialized retinal pigment epithelial (RPE) cells effectively. Our research presents a 3D-printed scaffold made from polycaprolactone (PCL), designed to carry RPE cells derived from human stem cells and dissolve after placement in the eye. We tested this scaffold in rabbits and pigs to evaluate its surgical handling, cell delivery effectiveness, and safety for human application. Our results refine implant design, paving the way for safer and more effective treatments for retinal diseases. Overall, this research enhances the application of cell therapy with scaffolds and offers valuable insights for future medical practices.
98Pre-clinical safety and efficacy of human induced pluripotent stem cell-derived products for autologous cell therapy in Parkinson's disease.PubMed
Jeha Jeon, Young Cha, Yean Ju Hong, et al.
Cell Stem Cell. 2025 Mar 6;32(3):343-360.e7. doi: 10.1016/j.stem.2025.01.006. Epub 2025 Feb 13.
Human induced pluripotent stem cell (hiPSC)-derived midbrain dopaminergic cells (mDACs) represent a promising source for autologous cell therapy in Parkinson's disease (PD), but standardized regulatory criteria are essential for clinical translation. In this pre-clinical study, we generated multiple clinical-grade hiPSC lines from freshly biopsied fibroblasts of four sporadic PD patients using episomal reprogramming and differentiated them into mDACs using a refined 21-day protocol. Rigorous evaluations included whole-genome/exome sequencing, RNA sequencing, and in vivo studies, including a 39-week Good Laboratory Practice-compliant mouse safety study. While mDACs from all lines met safety criteria, mDACs from one patient failed to improve rodent behavioral outcomes, underscoring inter-individual variability. Importantly, in vitro assessments did not reliably predict in vivo efficacy, identifying dopaminergic fiber density as a key efficacy criterion. These findings support comprehensive quality control guidelines for autologous cell therapy and pave the way for a clinical trial with eight sporadic PD patients, scheduled to commence in 2025.
99Preclinical quality, safety, and efficacy of a human embryonic stem cell-derived product for the treatment of Parkinson's disease, STEM-PD.PubMed
Agnete Kirkeby, Jenny Nelander, Deirdre B Hoban, et al.
Cell Stem Cell. 2023 Oct 5;30(10):1299-1314.e9. doi: 10.1016/j.stem.2023.08.014.
Cell replacement therapies for Parkinson's disease (PD) based on transplantation of pluripotent stem cell-derived dopaminergic neurons are now entering clinical trials. Here, we present quality, safety, and efficacy data supporting the first-in-human STEM-PD phase I/IIa clinical trial along with the trial design. The STEM-PD product was manufactured under GMP and quality tested in vitro and in vivo to meet regulatory requirements. Importantly, no adverse effects were observed upon testing of the product in a 39-week rat GLP safety study for toxicity, tumorigenicity, and biodistribution, and a non-GLP efficacy study confirmed that the transplanted cells mediated full functional recovery in a pre-clinical rat model of PD. We further observed highly comparable efficacy results between two different GMP batches, verifying that the product can be serially manufactured. A fully in vivo-tested batch of STEM-PD is now being used in a clinical trial of 8 patients with moderate PD, initiated in 2022.
100Continuous human iPSC-macrophage mass production by suspension culture in stirred tank bioreactors.PubMed
Mania Ackermann, Anna Rafiei Hashtchin, Felix Manstein, et al.
Nat Protoc. 2022 Feb;17(2):513-539. doi: 10.1038/s41596-021-00654-7. Epub 2022 Jan 17.
Macrophages derived from human induced pluripotent stem cells (iPSCs) have the potential to enable the development of cell-based therapies for numerous disease conditions. We here provide a detailed protocol for the mass production of iPSC-derived macrophages (iPSC-Mac) in scalable suspension culture on an orbital shaker or in stirred-tank bioreactors (STBRs). This strategy is straightforward, robust and characterized by the differentiation of primed iPSC aggregates into 'myeloid-cell-forming-complex' intermediates by means of a minimal cytokine cocktail. In contrast to the 'batch-like differentiation approaches' established for other iPSC-derived lineages, myeloid-cell-forming-complex-intermediates are stably maintained in suspension culture and continuously generate functional and highly pure iPSC-Mac. Employing a culture volume of 120 ml in the STBR platform, ~1-4 × 10 iPSC-Mac can be harvested at weekly intervals for several months. The STBR technology allows for real-time monitoring of crucial process parameters such as biomass, pH, dissolved oxygen, and nutrition levels; the system also promotes systematic process development, optimization and linear upscaling. The process duration, from the expansion of iPSC until the first iPSC-Mac harvest, is 28 d. Successful application of the protocol requires expertise in pluripotent stem cell culture, differentiation and analytical methods, such as flow cytometry. Fundamental know-how in biotechnology is also advantageous to run the process in the STBR platform. The continuous, scalable production of well-defined iPSC-Mac populations is highly relevant to various fields, ranging from developmental biology, immunology and cell therapies to industrial applications for drug safety and discovery.
101Mesenchymal and induced pluripotent stem cell-based therapeutics: a comparison.PubMed
Misha A Teale, Samuel Schneider, Dieter Eibl, et al.
Appl Microbiol Biotechnol. 2023 Jul;107(14):4429-4445. doi: 10.1007/s00253-023-12583-4. Epub 2023 May 29.
Stem cell-based cell therapeutics and especially those based on human mesenchymal stem cells (hMSCs) and induced pluripotent stem cells (hiPSCs) are said to have enormous developmental potential in the coming years. Their applications range from the treatment of orthopedic disorders and cardiovascular diseases to autoimmune diseases and even cancer. However, while more than 27 hMSC-derived therapeutics are currently commercially available, hiPSC-based therapeutics have yet to complete the regulatory approval process. Based on a review of the current commercially available hMSC-derived therapeutic products and upcoming hiPSC-derived products in phase 2 and 3, this paper compares the cell therapy manufacturing process between these two cell types. Moreover, the similarities as well as differences are highlighted and the resulting impact on the production process discussed. Here, emphasis is placed on (i) hMSC and hiPSC characteristics, safety, and ethical aspects, (ii) their morphology and process requirements, as well as (iii) their 2- and 3-dimensional cultivations in dependence of the applied culture medium and process mode. In doing so, also downstream processing aspects are covered and the role of single-use technology is discussed. KEY POINTS: • Mesenchymal and induced pluripotent stem cells exhibit distinct behaviors during cultivation • Single-use stirred bioreactor systems are preferred for the cultivation of both cell types • Future research should adapt and modify downstream processes to available single-use devices.
102Large-scale production of human pluripotent stem cell derived cardiomyocytes.PubMed
Henning Kempf, Birgit Andree, Robert Zweigerdt
Adv Drug Deliv Rev. 2016 Jan 15;96:18-30. doi: 10.1016/j.addr.2015.11.016. Epub 2015 Dec 3.
Regenerative medicine, including preclinical studies in large animal models and tissue engineering approaches as well as innovative assays for drug discovery, will require the constant supply of hPSC-derived cardiomyocytes and other functional progenies. Respective cell production processes must be robust, economically viable and ultimately GMP-compliant. Recent research has enabled transition of lab scale protocols for hPSC expansion and cardiomyogenic differentiation towards more controlled processing in industry-compatible culture platforms. Here, advanced strategies for the cultivation and differentiation of hPSCs will be reviewed by focusing on stirred bioreactor-based techniques for process upscaling. We will discuss how cardiomyocyte mass production might benefit from recent findings such as cell expansion at the cardiovascular progenitor state. Finally, remaining challenges will be highlighted, specifically regarding three dimensional (3D) hPSC suspension culture and critical safety issues ahead of clinical translation.
103Applications of Modern Cell Therapies: The Latest Data in Ophthalmology.PubMed
Ioannis Iliadis, Nadezhda A Pechnikova, Malamati Poimenidou, et al.
Life (Basel). 2025 Oct 16;15(10):1610. doi: 10.3390/life15101610.
Cell-based therapeutics are redefining interventions for vision loss by enabling tissue replacement, regeneration, and neuroprotection. This review surveys contemporary cellular strategies in ophthalmology through the lenses of therapeutic effectiveness, translational readiness, and governance. We profile principal sources-embryonic and induced pluripotent stem cells, mesenchymal stromal cells, retinal pigment epithelium, retinal progenitor and limbal stem cells-and enabling platforms including extracellular vesicles, encapsulated cell technology and biomaterial scaffolds. We synthesize clinical evidence across age-related macular degeneration, inherited retinal dystrophies, and corneal injury/limbal stem-cell deficiency, and highlight emerging applications for glaucoma and diabetic retinopathy. Delivery routes (subretinal, intravitreal, anterior segment) and graft formats (single cells, sheets/patches, organoids) are compared using standardized structural and functional endpoints. Persistent barriers include GMP-compliant derivation and release testing; differentiation fidelity, maturation, and potency; genomic stability and tumorigenicity risk; graft survival, synaptic integration, and immune rejection despite ocular immune privilege; the scarcity of validated biomarkers and harmonized outcome measures and ethical, regulatory, and health-economic constraints. Promising trajectories span off-the-shelf allogeneic products, patient-specific iPSC-derived grafts, organoid and 3D-bioprinted tissues, gene-plus-cell combinations, and cell-free extracellular-vesicle therapeutics. Overall, cell-based therapies remain investigational. With adequately powered trials, methodological harmonization, long-term surveillance, scalable xeno-free manufacturing, and equitable access frameworks, they may eventually become standards of care; at present, approvals are limited to specific products/indications and regions, and no cell therapy is the standard of care for retinal disease.
104Off-the-Shelf Chimeric Antigen Receptor Immune Cells from Human Pluripotent Stem Cells.PubMed
Handi Cao, Ryohichi Sugimura
Cancer Treat Res. 2022;183:255-274. doi: 10.1007/978-3-030-96376-7_9.
Autologous chimeric antigen receptor (CAR) T cells have expanded the scope and therapeutic potential of anti-cancer therapy. Nevertheless, autologous CAR-T therapy has been challenging due to labor some manufacturing processes for every patient, and the cost due to the complexity of the process. Moreover, T cell dysfunction results from the immunosuppressive tumor microenvironment in certain patients. Considering technical challenges in autologous donors, the development of safe and efficient allogeneic CAR-T therapy will address these issues. Since the advent of the generation of immune cells from pluripotent stem cells (PSCs), numerous studies focus on the off-the-shelf generation of CAR-immune cells derived from the universal donor PSCs, which simplifies the manufacturing process and standardizes CAR-T products. In this review, we will discuss advances in the generation of immune cells from PSCs, together with the potential and perspectives of CAR-T, CAR-macrophages, and CAR-natural killer (NK) cells in cancer treatment. The combination of PSC-derived immune cells and CAR engineering will pave the way for developing next-generation cancer immunotherapy.
105Targeted Disruption of the β2-Microglobulin Gene Minimizes the Immunogenicity of Human Embryonic Stem Cells.PubMed
Dachun Wang, Yuan Quan, Qing Yan, et al.
Stem Cells Transl Med. 2015 Oct;4(10):1234-45. doi: 10.5966/sctm.2015-0049. Epub 2015 Aug 18.
UNLABELLED: Human embryonic stem cells (hESCs) are a promising source of cells for tissue regeneration, yet histoincompatibility remains a major challenge to their clinical application. Because the human leukocyte antigen class I (HLA-I) molecules are the primary mediators of immune rejection, we hypothesized that cells derived from a hESC line lacking HLA-I expression could be transplanted without evoking a robust immune response from allogeneic recipients. In the present study, we used the replacement targeting strategy to delete exons 2 and 3 of β2-microglobulin on both gene alleles in hESCs. Because β2-microglobulin serves as the HLA-I light chain, disruption of the β2-microglobulin gene led to complete HLA-I deficiency on the cell surface of hESCs and their derivatives. Therefore, these cells were resistant to CD8+ T-cell-mediated destruction. Although interferon-γ (IFN-γ) treatment significantly induced β2-microglobulin expression, promoting CD8+ T cell-mediated killing of control hESCs and their derivatives, CD8+ T-cell-mediated cytotoxicity was barely observed with β2-microglobulin-null hESCs and their derivatives treated with IFN-γ. This genetic manipulation to disrupt HLA-I expression did not affect the self-renewal capacity, genomic stability, or pluripotency of hESCs. Despite being relatively sensitive to natural killer (NK) cell-mediated killing due to the lack of HLA-I expression, when transplanted into NK cell-depleted immunocompetent mice, β2-microglobulin-null hESCs developed into tumors resembling those derived from control hESCs in severe combined immunodeficiency mice. These results demonstrate that β2-microglobulin-null hESCs significantly reduce immunogenicity to CD8+ T cells and might provide a renewable source of cells for tissue regeneration without the need for HLA matching in the future. SIGNIFICANCE: This study reports the generation of a novel β2-microglobulin (B2M)-/- human embryonic stem cell (hESC) line. Differentiated mature cells from this line do not express cell surface human leukocyte antigen molecules even after interferon-γ stimulation and are resistant to alloreactive CD8+ T cells. Moreover, this B2M-/- hESC line contains no off-target integration or cleavage events, is devoid of stable B2M mRNA, exhibits a normal karyotype, and retains its self-renewal capacity, genomic stability, and pluripotency. Although B2M-/- hESC-derived cells are more susceptible to natural killer (NK) cells, murine transplantation studies have indicated that they are, overall, much less immunogenic than normal hESCs. Thus, these data show for the first time that, in vivo, the advantages provided by B2M-/- hESC-derived cells in avoiding CD8+ T-cell killing appear significantly greater than any disadvantage caused by increased susceptibility to NK cells.
106PD-L1 Promotes Immunological Tolerance and Enhances Visual Protection of hESC-RPE Grafts in Retinal Degeneration.PubMed
Bowen Li, Xue Zhang, Yajie Fang, et al.
Cell Prolif. 2025 Aug;58(8):e70007. doi: 10.1111/cpr.70007. Epub 2025 Feb 14.
Immune rejection is a major barrier to the successful human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) transplantation for age-related macular degeneration (AMD). Traditional strategies to mitigate immune rejection involve ablating major histocompatibility complex (MHC) molecules on hESC-RPE. An alternative approach is immune checkpoint overexpression, avoiding natural killer (NK) cell-mediated destruction due to MHC-I deficiency. Our study highlights the benefits of PD-L1 overexpression without requiring MHC gene deletion, which preserved the immunosuppressive functions of hESC-RPE on NK cells. In Vivo experiments in retinal degeneration models showed that PD-L1-expressing hESC-RPE grafts exhibited significantly higher survival, reduced apoptosis and enhanced visual protection. Single-cell transcriptomics revealed reduced immune activation and oxidative stress in PD-L1-overexpressing grafts. PD-L1's protective role was further evidenced by improved light transduction in host photoreceptors. These findings support PD-L1 overexpression as a promising strategy to improve the efficiency of hESC-RPE-based therapy for AMD.
107From Pluripotent Stem to CAR T Cells.PubMed
Cancer Discov. 2018 Jun;8(6):OF5. doi: 10.1158/2159-8290.CD-NB2018-057. Epub 2018 Apr 20.
Within the field of allogeneic chimeric antigen receptor T-cell therapy, La Jolla, CA-based Fate Therapeutics is exploring differentiation of induced pluripotent stem cells, rather than T cells harvested from donors, as their source material. Preclinical data on the company's off-the-shelf candidate, FT819, look promising so far.
108Application and current challenges of neural stem cells transplantation in clinical trials of spinal cord injury.PubMed
Wen-Jun Zhang, Jun Zhang
Int J Surg. 2025 Aug 1;111(8):5325-5336. doi: 10.1097/JS9.0000000000002548. Epub 2025 Jun 4.
Spinal cord injury (SCI) can lead to sensory, motor, and autonomic dysfunction, and even accompanied by persistent pathological pain, seriously affecting the psychosomatic health of patients. At present, there are no effective treatment methods. In recent years, cell transplantation replacement therapy has entered people's field of vision. Functional bioactive cells are transplanted into the host to exert the characteristics of biotherapy so as to repair the injured nerve and repair the function. Neural stem cells (NSCs) are a kind of pluripotent stem cells, which have been well applied in nerve injury repair and tissue engineering regeneration. In preclinical and animal models, NSCs transplantation can well repair SCI and repair part of the function. The therapeutic functions of NSCs include differentiation into neurons, promoting axonal regeneration and myelination, neuroprotection, immunoregulation, and improving the local inflammatory microenvironment of SCI, replacing injured neurons. it is beneficial to the repair and reconstruction of neural network after SCI. NSCs have also achieved some encouraging results in clinical trials of SCI. Transplantation of NSCs into patients with SCI is a safe and feasible treatment, which can partially restore the sensory and motor function of the patients, without obvious side effects. At present, NSCs are in the primary exploration stage in clinical trials, and there are many problems and challenges in their extensive development and application in clinical trials. Therefore, we comprehensively discussed the application of NSCs in clinical trials of SCI and the existing problems and challenges, so as to provide useful information for the application of NSCs in SCI in the future.
109Cell Therapies for Spinal Cord Injury: Trends and Challenges of Current Clinical Trials.PubMed
Richard D Bartlett, Sarah Burley, Mina Ip, et al.
Neurosurgery. 2020 Sep 15;87(4):E456-E472. doi: 10.1093/neuros/nyaa149.
Cell therapies have the potential to revolutionize the treatment of spinal cord injury. Basic research has progressed significantly in recent years, with a plethora of cell types now reaching early-phase human clinical trials, offering new strategies to repair the spinal cord. However, despite initial enthusiasm for preclinical and early-phase clinical trials, there has been a notable hiatus in the translation of cell therapies to routine clinical practice. Here, we review cell therapies that have reached clinical trials for spinal cord injury, providing a snapshot of all registered human trials and a summary of all published studies. Of registered trials, the majority have used autologous cells and approximately a third have been government funded, a third industry sponsored, and a third funded by university or healthcare systems. A total of 37 cell therapy trials have been published, primarily using stem cells, although a smaller number have used Schwann cells or olfactory ensheathing cells. Significant challenges remain for cell therapy trials in this area, including achieving stringent regulatory standards, ensuring appropriately powered efficacy trials, and establishing sustainable long-term funding. However, cell therapies hold great promise for human spinal cord repair and future trials must continue to capitalize on the exciting developments emerging from preclinical studies.
110Spinal Cord Injury and Related Clinical Trials.PubMed
Young-Hoon Kim, Kee-Yong Ha, Sang-Il Kim
Clin Orthop Surg. 2017 Mar;9(1):1-9. doi: 10.4055/cios.2017.9.1.1. Epub 2017 Feb 13.
Spinal cord injury (SCI) has been considered an incurable condition and it often causes devastating sequelae. In terms of the pathophysiology of SCI, reducing secondary damage is the key to its treatment. Various researches and clinical trials have been performed, and some of them showed promising results; however, there is still no gold standard treatment with sufficient evidence. Two therapeutic concepts for SCI are neuroprotective and neuroregenerative strategies. The neuroprotective strategy modulates the pathomechanism of SCI. The purpose of neuroprotective treatment is to minimize secondary damage following direct injury. The aim of neuroregenerative treatment is to enhance the endogenous regeneration process and to alter the intrinsic barrier. With advancement in biotechnology, cell therapy using cell transplantation is currently under investigation. This review discusses the pathophysiology of SCI and introduces the therapeutic candidates that have been developed so far.
111Translational Regenerative Therapies for Chronic Spinal Cord Injury.PubMed
Kyriakos Dalamagkas, Magdalini Tsintou, Amelia Seifalian, et al.
Int J Mol Sci. 2018 Jun 15;19(6):1776. doi: 10.3390/ijms19061776.
Spinal cord injury is a chronic and debilitating neurological condition that is currently being managed symptomatically with no real therapeutic strategies available. Even though there is no consensus on the best time to start interventions, the chronic phase is definitely the most stable target in order to determine whether a therapy can effectively restore neurological function. The advancements of nanoscience and stem cell technology, combined with the powerful, novel neuroimaging modalities that have arisen can now accelerate the path of promising novel therapeutic strategies from bench to bedside. Several types of stem cells have reached up to clinical trials phase II, including adult neural stem cells, human spinal cord stem cells, olfactory ensheathing cells, autologous Schwann cells, umbilical cord blood-derived mononuclear cells, adult mesenchymal cells, and autologous bone-marrow-derived stem cells. There also have been combinations of different molecular therapies; these have been either alone or combined with supportive scaffolds with nanostructures to facilitate favorable cell⁻material interactions. The results already show promise but it will take some coordinated actions in order to develop a proper step-by-step approach to solve impactful problems with neural repair.
112Neural stem cells derived from primitive mesenchymal stem cells reversed disease symptoms and promoted neurogenesis in an experimental autoimmune encephalomyelitis mouse model of multiple sclerosis.PubMed
Christina Brown, Christina McKee, Sophia Halassy, et al.
Stem Cell Res Ther. 2021 Sep 9;12(1):499. doi: 10.1186/s13287-021-02563-8.
BACKGROUND: Multiple sclerosis (MS) is an autoimmune inflammatory disease of the central nervous system (CNS). MS affects millions of people and causes a great economic and societal burden. There is no cure for MS. We used a novel approach to investigate the therapeutic potential of neural stem cells (NSCs) derived from human primitive mesenchymal stem cells (MSCs) in an experimental autoimmune encephalomyelitis (EAE) mouse model of MS. METHODS: MSCs were differentiated into NSCs, labeled with PKH26, and injected into the tail vein of EAE mice. Neurobehavioral changes in the mice assessed the effect of transplanted cells on the disease process. The animals were sacrificed two weeks following cell transplantation to collect blood, lymphatic, and CNS tissues for analysis. Transplanted cells were tracked in various tissues by flow cytometry. Immune infiltrates were determined and characterized by H&E and immunohistochemical staining, respectively. Levels of immune regulatory cells, Treg and Th17, were analyzed by flow cytometry. Myelination was determined by Luxol fast blue staining and immunostaining. In vivo fate of transplanted cells and expression of inflammation, astrogliosis, myelination, neural, neuroprotection, and neurogenesis markers were investigated by using immunohistochemical and qRT-PCR analysis. RESULTS: MSC-derived NSCs expressed specific neural markers, NESTIN, TUJ1, VIMENTIN, and PAX6. NSCs improved EAE symptoms more than MSCs when transplanted in EAE mice. Post-transplantation analyses also showed homing of MSCs and NSCs into the CNS with concomitant induction of an anti-inflammatory response, resulting in reducing immune infiltrates. NSCs also modulated Treg and Th17 cell levels in EAE mice comparable to healthy controls. Luxol fast blue staining showed significant improvement in myelination in treated mice. Further analysis showed that NSCs upregulated genes involved in myelination and neuroprotection but downregulated inflammatory and astrogliosis genes more significantly than MSCs. Importantly, NSCs differentiated into neural derivatives and promoted neurogenesis, possibly by modulating BDNF and FGF signaling pathways. CONCLUSIONS: NSC transplantation reversed the disease process by inducing an anti-inflammatory response and promoting myelination, neuroprotection, and neurogenesis in EAE disease animals. These promising results provide a basis for clinical studies to treat MS using NSCs derived from primitive MSCs.
113Pre-clinical study of induced pluripotent stem cell-derived dopaminergic progenitor cells for Parkinson's disease.PubMed
Daisuke Doi, Hiroaki Magotani, Tetsuhiro Kikuchi, et al.
Nat Commun. 2020 Jul 6;11(1):3369. doi: 10.1038/s41467-020-17165-w.
Induced pluripotent stem cell (iPSC)-derived dopaminergic (DA) neurons are an expected source for cell-based therapies for Parkinson's disease (PD). The regulatory criteria for the clinical application of these therapies, however, have not been established. Here we show the results of our pre-clinical study, in which we evaluate the safety and efficacy of dopaminergic progenitors (DAPs) derived from a clinical-grade human iPSC line. We confirm the characteristics of DAPs by in vitro analyses. We also verify that the DAP population include no residual undifferentiated iPSCs or early neural stem cells and have no genetic aberration in cancer-related genes. Furthermore, in vivo studies using immunodeficient mice reveal no tumorigenicity or toxicity of the cells. When the DAPs are transplanted into the striatum of 6-OHDA-lesioned rats, the animals show behavioral improvement. Based on these results, we started a clinical trial to treat PD patients in 2018.
114Exosomes derived from bone-marrow mesenchymal stem cells alleviate cognitive decline in AD-like mice by improving BDNF-related neuropathology.PubMed
Sen Liu, Min Fan, Jing-Xian Xu, et al.
J Neuroinflammation. 2022 Feb 7;19(1):35. doi: 10.1186/s12974-022-02393-2.
BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative disease characterized by a progressive decline in cognitive ability. Exosomes derived from bone-marrow mesenchymal stem cells (BMSC-exos) are extracellular vesicles that can execute the function of bone-marrow mesenchymal stem cells (BMSCs). Given the versatile therapeutic potential of BMSC and BMSC-exos, especially their neuroprotective effect, the aim of this study was to investigate the potential effect of BMSC-exos on AD-like behavioral dysfunction in mice and explore the possible molecular mechanism. METHODS: BMSC-exos were extracted from the supernatant of cultured mouse BMSCs, which were isolated from the femur and tibia of adult C57BL/6 mice, purified and sorted via flow cytometry, and cultured in vitro. BMSC-exos were identified via transmission electron microscopy, and typical marker proteins of exosomes were also detected via Western blot. A sporadic AD mouse model was established by intracerebroventricular injection of streptozotocin (STZ). Six weeks later, BMSC-exos were administered via lateral ventricle injection or caudal vein injection lasting five consecutive days, and the control mice were intracerebroventricularly administered an equal volume of solvent. Behavioral performance was observed via the open field test (OFT), elevated plus maze test (EPM), novel object recognition test (NOR), Y maze test (Y-maze), and tail suspension test (TST). The mRNA and protein expression levels of IL-1β, IL-6, and TNF-α in the hippocampus were measured via quantitative polymerase chain reaction (qPCR) and Western blot, respectively. Moreover, the protein expression of Aβ, BACE, IL-1β, IL-6, TNF-α, GFAP, p-Tau (Ser396), Tau5, synaptotagmin-1 (Syt-1), synapsin-1, and brain-derived neurotrophic factor (BDNF) in the hippocampus was detected using Western blot, and the expression of GFAP, IBA1, Aβ and DCX in the hippocampus was measured via immunofluorescence staining. RESULTS: Lateral ventricle administration, but not caudal vein injection of BMSC-exos improved AD-like behaviors in the STZ-injected mouse model, as indicated by the increased number of rearing, increased frequency to the central area, and increased duration and distance traveled in the central area in the OFT, and improved preference index of the novel object in the NOR. Moreover, the hyperactivation of microglia and astrocytes in the hippocampus of the model mice was inhibited after treatment with BMSC-exos via lateral ventricle administration, accompanied by the reduced expression of IL-1β, IL-6, TNF-α, Aβ and p-Tau and upregulated protein expression of synapse-related proteins and BDNF. Furthermore, the results of the Pearson test showed that the preference index of the novel object in the NOR was positively correlated with the hippocampal expression of BDNF, but negatively correlated with the expression of GFAP, IBA1, and IL-1β. Apart from a positive correlation between the hippocampal expression of BDNF and Syt-1, BDNF abundance was found to be negatively correlated with markers of glial activation and the expression of the inflammatory cytokines, Aβ, and p-Tau, which are characteristic neuropathological features of AD. CONCLUSIONS: Lateral ventricle administration, but not caudal vein injection of BMSC-exos, can improve AD-like behavioral performance in STZ-injected mice, the mechanism of which might be involved in the regulation of glial activation and its associated neuroinflammation and BDNF-related neuropathological changes in the hippocampus.
115The influence of BDNF on human umbilical cord blood stem/progenitor cells: implications for stem cell-based therapy of neurodegenerative disorders.PubMed
Edyta Paczkowska, Karolina Łuczkowska, Katarzyna Piecyk, et al.
Acta Neurobiol Exp (Wars). 2015;75(2):172-91. doi: 10.55782/ane-2015-2026.
Umbilical cord blood (UCB)-derived stem/progenitor cells (SPCs) have demonstrated the potential to improve neurologic function in different experimental models. SPCs can survive after transplantation in the neural microenvironment and indu ce neuroprotection, endogenous neurogenesis by secreting a broad repertoire of trophic and immunomodulatory cytokines. In this study, the influence of brain-derived neurotrophic factor (BDNF) pre-treatment was comprehensively evaluated in a UCB-derived lineage-negative (Lin-) SPC population. UCB-derived Lin- cells were evaluated with respect to the expression of (i) neuronal markers using immunofluorescence staining and (ii) specific (TrkB) receptors for BDNF using flow cytometry. Next, after BDNF pre-treatment, Lin- cells were extensively assessed with respect to apoptosis using Western blotting and proliferation via BrdU incorporation. Furthermore, NT-3 expression levels in Lin- cells using RQ PCR and antioxidative enzyme activities were assessed. We demonstrated neuronal markers as well as TrkB expression in Lin- cells and the activation of the TrkB receptor by BDNF. BDNF pre-treatment diminished apoptosis in Lin- cells and influenced the proliferation of these cells. We observed significant changes in antioxidants as well as in the increased expression of NT-3 in Lin- cells following BDNF exposure. Complex global miRNA and mRNA profiling analyses using microarray technology and GSEA revealed the differential regulation of genes involved in the proliferation, gene expression, biosynthetic processes, translation, and protein targeting. Our results support the hypothesis that pre-treatment of stem/progenitor cells could be beneficial and may be used as an auxiliary strategy for improving the properties of SPCs.
116Exploration of Cytokines That Impact the Therapeutic Efficacy of Mesenchymal Stem Cells in Alzheimer's Disease.PubMed
Herui Wang, Chonglin Zhong, Yi Mi, et al.
Bioengineering (Basel). 2025 Jun 12;12(6):646. doi: 10.3390/bioengineering12060646.
Current therapies for Alzheimer's disease (AD) includes acetylcholinesterase inhibitors, NMDA receptor antagonists, and amyloid beta (Aβ)/Tau-targeting drugs. While these drugs improve cognitive decline and target the pathological mechanisms, their outcomes still are still in debate. Mesenchymal stem cells (MSCs) offer a regenerative approach by modulating neuroinflammation and promoting neuroprotection. Although the paracrine of MSCs is efficient in various AD preclinical studies and the exosomes of MSCs have entered clinical trials, the key cytokines driving the efficacy remain unclear. Here, we evaluated human umbilical cord-derived MSCs (hUC-MSCs) and employed gene-silenced MSCs (siHGF-MSCs, siTNFR1-MSCs, siBDNF-MSCs) in APP/PS1 AD mice to investigate specific mechanisms. hUC-MSCs significantly reduced Aβ/Tau pathology and neuroinflammation, with cytokine-specific contributions: silencing HGF predominantly reduced Aβ/Tau clearance, although silencing TNFR1 or BDNF showed modest effects; silencing TNFR1 or BDNF more prominently weakened anti-neuroinflammation, while silencing HGF exerted a weaker influence. All three cytokines partially contributed to oxidative stress reduction and cognitive improvements. Our study highlights MSC-driven AD alleviation as a multifactorial strategy and reveals specific cytokines alleviating different aspects of AD pathology.
117Huc-MSCs-derived exosomes attenuate inflammatory pain by regulating microglia pyroptosis and autophagy via the miR-146a-5p/TRAF6 axis.PubMed
Tong Hua, Mei Yang, Honghao Song, et al.
J Nanobiotechnology. 2022 Jul 14;20(1):324. doi: 10.1186/s12951-022-01522-6.
BACKGROUND: Chronic inflammatory pain significantly reduces the quality of life and lacks effective interventions. In recent years, human umbilical cord mesenchymal stem cells (huc-MSCs)-derived exosomes have been used to relieve neuropathic pain and other inflammatory diseases as a promising cell-free therapeutic strategy. However, the therapeutic value of huc-MSCs-derived exosomes in complete Freund's adjuvant (CFA)-induced inflammatory pain remains to be confirmed. In this study, we investigated the therapeutic effect and related mechanisms of huc-MSCs-derived exosomes in a chronic inflammatory pain model. METHODS: C57BL/6J male mice were used to establish a CFA-induced inflammatory pain model, and huc-MSCs-derived exosomes were intrathecally injected for 4 consecutive days. BV2 microglia cells were stimulated with lipopolysaccharide (LPS) plus adenosine triphosphate (ATP) to investigate the effect of huc-MSCs-derived exosomes on pyroptosis and autophagy. Bioinformatic analysis and rescue experiments were used to demonstrate the role of miR-146a-5p/ TRAF6 in regulating pyroptosis and autophagy. Western blotting, RT-qPCR, small interfering RNA and Yo-Pro-1 dye staining were performed to investigate the related mechanisms. RESULTS: Huc-MSCs-derived exosomes alleviated mechanical allodynia and thermal hyperalgesia in CFA-induced inflammatory pain. Furthermore, huc-MSCs-derived exosomes attenuated neuroinflammation by increasing the expression of autophagy-related proteins (LC3-II and beclin1) and inhibiting the activation of NLRP3 inflammasomes in the spinal cord dorsal horn. In vitro, NLRP3 inflammasome components (NLRP3, caspase1-p20, ASC) and gasdermin D (GSDMD-F, GSDMD-N) were inhibited in BV2 cells pretreated with huc-MSCs-derived exosomes. Western blot and Yo-Pro-1 dye staining demonstrated that 3-MA, an autophagy inhibitor, weakened the protective effect of huc-MSCs-derived exosomes on BV2 cell pyroptosis. Importantly, huc-MSCs-derived exosomes transfected with miR-146a-5p mimic promoted autophagy and inhibited BV2 cell pyroptosis. TRAF6, as a target gene of miR-146a-5p, was knocked down via small-interfering RNA, which increased pyroptosis and inhibited autophagy. CONCLUSION: Huc-MSCs-derived exosomes attenuated inflammatory pain via miR-146a-5p/TRAF6, which increased the level of autophagy and inhibited pyroptosis.
118Human umbilical cord mesenchymal stem cell-derived exosomes attenuate neuroinflammation and oxidative stress through the NRF2/NF-κB/NLRP3 pathway.PubMed
Ji Che, Hui Wang, Jing Dong, et al.
CNS Neurosci Ther. 2024 Mar;30(3):e14454. doi: 10.1111/cns.14454. Epub 2023 Sep 12.
AIMS: We investigated whether human umbilical cord mesenchymal stem cell (hUC-MSC)-derived exosomes bear therapeutic potential against lipopolysaccharide (LPS)-induced neuroinflammation. METHODS: Exosomes were isolated from hUC-MSC supernatant by ultra-high-speed centrifugation and characterized by transmission electron microscopy and western blotting. Inflammatory responses were induced by LPS in BV-2 cells, primary microglial cultures, and C57BL/6J mice. H O was also used to induce inflammation and oxidative stress in BV-2 cells. The effects of hUC-MSC-derived exosomes on inflammatory cytokine expression, oxidative stress, and microglia polarization were studied by immunofluorescence and western blotting. RESULTS: Treatment with hUC-MSC-derived exosomes significantly decreased the LPS- or H O -induced oxidative stress and expression of pro-inflammatory cytokines (IL-6 and TNF-α) in vitro, while promoting an anti-inflammatory (classical M2) phenotype in an LPS-treated mouse model. Mechanistically, the exosomes increased the NRF2 levels and inhibited the LPS-induced NF-κB p65 phosphorylation and NLRP3 inflammasome activation. In contrast, the reactive oxygen species scavenger NAC and NF-κB inhibitor BAY 11-7082 also inhibited the LPS-induced NLRP3 inflammasome activation and switched to the classical M2 phenotype. Treatment with the NRF2 inhibitor ML385 abolished the anti-inflammatory and anti-oxidative effects of the exosomes. CONCLUSION: hUC-MSC-derived exosomes ameliorated LPS/H O -induced neuroinflammation and oxidative stress by inhibiting the microglial NRF2/NF-κB/NLRP3 signaling pathway.
119Mesenchymal stem cell-derived exosomes as a nanotherapeutic agent for amelioration of inflammation-induced astrocyte alterations in mice.PubMed
Panpan Xian, Yue Hei, Rui Wang, et al.
Theranostics. 2019 Aug 14;9(20):5956-5975. doi: 10.7150/thno.33872. eCollection 2019.
Mesenchymal stem cell-derived exosomes (MSC-Exo) have robust anti-inflammatory effects in the treatment of neurological diseases such as epilepsy, stroke, or traumatic brain injury. While astrocytes are thought to be mediators of these effects, their precise role remains poorly understood. To address this issue, we investigated the putative therapeutic effects and mechanism of MSC-Exo on inflammation-induced alterations in astrocytes. : Lipopolysaccharide (LPS)-stimulated hippocampal astrocytes in primary culture were treated with MSC-Exo, which were also administered in pilocarpine-induced status epilepticus (SE) mice. Exosomal integration, reactive astrogliosis, inflammatory responses, calcium signaling, and mitochondrial membrane potentials (MMP) were monitored. To experimentally probe the molecular mechanism of MSC-Exo actions on the inflammation-induced astrocytic activation, we inhibited the nuclear factor erythroid-derived 2, like 2 (Nrf2, a key mediator in neuroinflammation and oxidative stress) by sgRNA (in vitro) or ML385 (Nrf2 inhibitor) in vivo. : MSC-Exo were incorporated into hippocampal astrocytes as well as attenuated reactive astrogliosis and inflammatory responses in vitro and in vivo. Also, MSC-Exo ameliorated LPS-induced aberrant calcium signaling and mitochondrial dysfunction in culture, and SE-induced learning and memory impairments in mice. Furthermore, the putative therapeutic effects of MSC-Exo on inflammation-induced astrocytic activation (e.g., reduced reactive astrogliosis, NF-κB deactivation) were weakened by Nrf2 inhibition. : Our results show that MSC-Exo ameliorate inflammation-induced astrocyte alterations and that the Nrf2-NF-κB signaling pathway is involved in regulating astrocyte activation in mice. These data suggest the promising potential of MSC-Exo as a nanotherapeutic agent for the treatment of neurological diseases with hippocampal astrocyte alterations.
120Mesenchymal stem cells-derived extracellular vesicles ameliorate Alzheimer's disease in rat models via the microRNA-29c-3p/BACE1 axis and the Wnt/β-catenin pathway.PubMed
Sha Sha, Xueli Shen, Yunpeng Cao, et al.
Aging (Albany NY). 2021 Jun 4;13(11):15285-15306. doi: 10.18632/aging.203088.
Currently, Alzheimer's disease (AD) cannot be treated effectively. Mesenchymal stem cells (MSCs)-derived extracellular vesicles (EVs) (MSC-EVs) exhibit therapeutic effects on many diseases. This study investigated the mechanism of bone marrow MSC-EVs (BM-MSC-EVs) in a rat model of AD. The cognitive function, amyloid-β (Aβ) plaques, Aβ deposition areas and levels of Aβ1-42, Aβ decomposition-related factors (NEP and IDE), and inflammatory cytokines in BM-MSC-EVs-treated AD rats were measured. The effect of BM-MSC-EVs was studied in AD neuron model. microRNA (miR)-29c-3p and BACE1 expression, as well as levels of Wnt/β-catenin pathway-related factors in AD and EVs-treated AD models were detected. miR-29c-3p relationship with BACE1 was predicted and confirmed. miR-29c-3p and BACE1 were interfered to verify the mechanism of EVs in AD. The Wnt/β-catenin pathway inhibitor DKK1 was further added to EVs-treated AD neurons. BM-MSC-EVs showed therapeutic effects on AD rats and neurons. BM-MSC-EVs carried miR-29c-3p into AD neurons. miR-29c-3p targeted BACE1. Silencing miR-29c-3p in BM-MSCs reduced BM-MSC-EV therapeutic effect on AD, which was reversed after BACE1 knockdown. miR-29c-3p targeted BACE1 and activated the Wnt/β-catenin pathway, and the Wnt/β-catenin pathway inhibition impaired EV therapeutic effects on AD. We highlighted that BM-MSC-EVs delivered miR-29c-3p to neurons to inhibit BACE1 expression and activate the Wnt/β-catenin pathway, thereby playing a therapeutic role in AD. This study may provide a novel perspective for elucidating the mechanism of MSCs in the treatment of AD.
121Exosomes derived from hypoxia-preconditioned mesenchymal stromal cells ameliorate cognitive decline by rescuing synaptic dysfunction and regulating inflammatory responses in APP/PS1 mice.PubMed
Guo-Hong Cui, Jing Wu, Fang-Fang Mou, et al.
FASEB J. 2018 Feb;32(2):654-668. doi: 10.1096/fj.201700600R. Epub 2018 Jan 4.
Administration of exosomes derived from mesenchymal stromal cells (MSCs) could improve some neurologic conditions by transferring functional biomolecules to recipient cells. Furthermore, exosomes from hypoxic progenitor cells exerted better therapeutic effects in organ injury through specific cargoes. However, there are no related reports about whether exosomes derived from MSCs or hypoxia-preconditioned MSCs (PC-MSCs) could prevent memory deficits in Alzheimer disease (AD). In this study, the exosomes derived from MSCs or PC-MSCs were systemically administered to transgenic APP/PS1 mice. The expression of miR-21 in MSCs was significantly increased after hypoxic treatment. Injection of exosomes from normoxic MSCs could rescue cognition and memory impairment according to results of the Morris water maze test, reduced plaque deposition, and Aβ levels in the brain; could decrease the activation of astrocytes and microglia; could down-regulate proinflammatory cytokines (TNF-α and IL-1β); and could up-regulate anti-inflammatory cytokines (IL-4 and -10) in AD mice, as well as reduce the activation of signal transducer and activator of transcription 3 (STAT3) and NF-κB. Compared to the group administered exosomes from normoxic MSCs, in the group administered exosomes from PC-MSCs, learning and memory capabilities were significantly improved; the plaque deposition and Aβ levels were lower, and expression of growth-associated protein 43, synapsin 1, and IL-10 was increased; and the levels of glial fibrillary acidic protein, ionized calcium-binding adaptor molecule 1, TNF-α, IL-1β, and activation of STAT3 and NF-κB were sharply decreased. More importantly, exosomes from PC-MSCs effectively increased the level of miR-21 in the brain of AD mice. Additionally, replenishment of miR-21 restored the cognitive deficits in APP/PS1 mice and prevented pathologic features. Taken together, these findings suggest that exosomes from PC-MSCs could improve the learning and memory capabilities of APP/PS1 mice, and that the underlying mechanism may lie in the restoration of synaptic dysfunction and regulation of inflammatory responses through regulation of miR-21.-Cui, G.-H., Wu, J., Mou, F.-F., Xie, W.-H., Wang, F.-B., Wang, Q.-L., Fang, J., Xu, Y.-W., Dong, Y.-R., Liu, J.-R., Guo, H.-D. Exosomes derived from hypoxia-preconditioned mesenchymal stromal cells ameliorate cognitive decline by rescuing synaptic dysfunction and regulating inflammatory responses in APP/PS1 mice.
122Bone marrow mesenchymal stem cells-derived exosomes reduce Aβ deposition and improve cognitive function recovery in mice with Alzheimer's disease by activating sphingosine kinase/sphingosine-1-phosphate signaling pathway.PubMed
Xinhui Wang, Guojie Yang
Cell Biol Int. 2021 Apr;45(4):775-784. doi: 10.1002/cbin.11522. Epub 2021 Jan 7.
Exosomes are associated with the development and progression of Alzheimer's disease (AD), although the impact of these extracellular vesicles in brain pathological condition remains incompletely understood. Therefore, this study aimed to investigate the role and mechanism of exosomes signaling in AD. Double transgenic APP/PS1 mice were injected with bone marrow mesenchymal stem cells (BM-MSCs)-derived exosomes or combined with SKI-Ⅱ (sphingosine kinase [SphK] inhibitor) or VPC23019 (sphingosine-1-phosphate [S1P] 1 receptor blocker). We observed the spatial learning and memory ability of mice, and assessed the levels of amyloid and proteins. We found that exosomes improved spatial learning and memory ability of APP/PS1 mice, and enhanced the expression of SphK1 and S1P1. Moreover, exosomes inhibited the levels of amyloid and enhanced the expression of NeuN in cortex and hippocampus of APP/PS1 mice. Exosomes repressed the levels of Aβ1-40, Aβ1-42, BACE1, and PS1, and promoted the expression of neprilysin in APP/PS1 mice. The influence conferred by exosomes was abolished by SKI-Ⅱ or VPC23019. In conclusion, our article confirms that BM-MSCs-derived exosomes reduce Aβ deposition and promote cognitive function recovery in AD mice by activating SphK/S1P signaling pathway. Thus, our data suggest that S1P/SphK-containing exosomes should be explored as potential AD cure.
123Exosomes Derived from Human Amniotic Fluid Mesenchymal Stem Cells Preserve Microglia and Neuron Cells from Aβ.PubMed
Manuela Zavatti, Martina Gatti, Francesca Beretti, et al.
Int J Mol Sci. 2022 Apr 29;23(9):4967. doi: 10.3390/ijms23094967.
BACKGROUND: Neuroinflammation is involved in neuronal cell death that occurs in neurodegenerative diseases such as Alzheimer's disease (AD). Microglia play important roles in regulating the brain amyloid beta (Aβ) levels, so immunomodulatory properties exerted by mesenchymal stem cells may be exploited to treat this pathology. The evidence suggests that the mechanism of action of human amniotic fluid stem cells (hAFSCs) is through their secretome, which includes exosomes (exo). METHODS: We examined the effect of exosomes derived from human amniotic fluid stem cells (hAFSCs-exo) on activated BV-2 microglia cells by lipopolysaccharide (LPS) as a neuroinflammation model. To investigate the exo effect on the interplay between AD neurons and microglia, SH-SY5Y neuroblastoma cells treated with Aβ were exposed to a conditioned medium (CM) obtained from activated BV-2 or co-culture systems. RESULTS: We found that the upregulation of the markers of pro-inflammatory microglia was prevented when exposed to hAFSC-exo whereas the markers of the anti-inflammatory macrophage phenotype were not affected. Interestingly, the hAFSC-exo pretreatment significantly inhibited the oxidative stress rise and apoptosis occurring in the neurons in presence of both microglia and Aβ. CONCLUSION: We demonstrated that hAFSC-exo mitigated an inflammatory injury caused by microglia and significantly recovered the neurotoxicity, suggesting that hAFSC-exo may be a potential therapeutic agent for inflammation-related neurological conditions, including AD.
124Clinical safety and efficacy of allogenic human adipose mesenchymal stromal cells-derived exosomes in patients with mild to moderate Alzheimer's disease: a phase I/II clinical trial.PubMed
Xinyi Xie, Qingxiang Song, Chengxiang Dai, et al.
Gen Psychiatr. 2023 Oct 11;36(5):e101143. doi: 10.1136/gpsych-2023-101143. eCollection 2023.
BACKGROUND: There have been no effective treatments for slowing or reversing Alzheimer's disease (AD) until now. Growing preclinical evidence, including this study, suggests that mesenchymal stem cells-secreted exosomes (MSCs-Exos) have the potential to cure AD. AIMS: The first three-arm, drug-intervention, phase I/II clinical trial was conducted to explore the safety and efficacy of allogenic human adipose MSCs-Exos (ahaMSCs-Exos) in patients with mild to moderate AD. METHODS: The eligible subjects were assigned to one of three dosage groups, intranasally administrated with ahaMSCs-Exos two times per week for 12 weeks, and underwent follow-up visits at weeks 16, 24, 36 and 48. RESULTS: No adverse events were reported. In the medium-dose arm, Alzheimer's Disease Assessment Scale-Cognitive section (ADAS-cog) scores decreased by 2.33 (1.19) and the basic version of Montreal Cognitive Assessment scores increased by 2.38 (0.58) at week 12 compared with baseline levels, indicating improved cognitive function. Moreover, the ADAS-cog scores in the medium-dose arm decreased continuously by 3.98 points until week 36. There were no significant differences in altered amyloid or tau deposition among the three arms, but hippocampal volume shrank less in the medium-dose arm to some extent. CONCLUSIONS: Intranasal administration of ahaMSCs-Exos was safe and well tolerated, and a dose of at least 4×10 particles could be selected for further clinical trials. TRIAL REGISTRATION NUMBER: NCT04388982.
125Mesenchymal stem cell-derived exosome: a promising alternative in the therapy of Alzheimer's disease.PubMed
Mengtian Guo, Zhenyu Yin, Fanglian Chen, et al.
Alzheimers Res Ther. 2020 Sep 14;12(1):109. doi: 10.1186/s13195-020-00670-x.
Alzheimer's disease (AD) has been a devastating public health with the development of global aging. Approaches for reducing the current AD epidemic are becoming a primary focus of human healthcare due to the lack of achieved lasting and complete remission strategies to treat AD with the characteristics of heterogeneity and complexity. Exosomes, which is the new emerging approach to intercellular communication, provide novel perspective on identified therapeutic strategies of AD. Mesenchymal stem cell-derived exosomes (MSC-exos) are emerging to be an appealing therapeutic tool for AD, with the donor-derived properties and the characteristics of minimal immunogenicity, effortless storage, nature delivery vehicles, and low risks of tumor formation based on the previous researches. In this review, we elaborate the mechanism of MSC-exos in the treatment of AD and discuss limitations in the clinical application.
126Mesenchymal Stem Cell-Derived Exosomes as a Neuroregeneration Treatment for Alzheimer's Disease.PubMed
Siddharth Shah, Hadeel M Mansour, Tania M Aguilar, et al.
Biomedicines. 2024 Sep 17;12(9):2113. doi: 10.3390/biomedicines12092113.
BACKGROUND: Alzheimer's disease (AD) is the most prevalent kind of dementia and is a long-term degenerative disease. Pathologically, it is defined by the development of extracellular amyloid-β plaques and intracellular neurofibrillary tangles made up of hyperphosphorylated tau protein. This causes neuronal death, particularly in the hippocampus and cortex. Mesenchymal stem cell (MSC)-derived exosomes have been identified as possibly therapeutic and have promise for Alzheimer's disease due to their regenerative characteristics. METHODS: A systematic retrieval of information was performed on PubMed. A total of 60 articles were found in a search on mesenchymal stem cells, exosomes, and Alzheimer's disease. A total of 16 ongoing clinical trials were searched and added from clinicaltrials.gov. We added 23 supporting articles to help provide information for certain sections. In total, we included 99 articles in this manuscript: 50 are review articles, 13 are preclinical studies, 16 are clinical studies, 16 are ongoing clinical trials, and 4 are observational studies. Appropriate studies were isolated, and important information from each of them was understood and entered into a database from which the information was used in this article. The clinical trials on mesenchymal stem cell exosomes for Alzheimer's disease were searched on clinicaltrials.gov. RESULTS: Several experimental investigations have shown that MSC-Exo improves cognitive impairment in rats. In this review paper, we summarized existing understanding regarding the molecular and cellular pathways behind MSC-Exo-based cognitive function restoration, with a focus on MSC-Exo's therapeutic potential in the treatment of Alzheimer's disease. CONCLUSION: AD is a significant health issue in our culture and is linked to several important neuropathological characteristics. Exosomes generated from stem cells, such as mesenchymal stem cells (MSCs) or neural stem cells (NSCs), have been examined more and more in a variety of AD models, indicating that they may be viable therapeutic agents for the treatment of diverse disorders. Exosome yields may be increased, and their therapeutic efficacy can be improved using a range of tailored techniques and culture conditions. It is necessary to provide standardized guidelines for exosome manufacture to carry out excellent preclinical and clinical research.
127Mesenchymal stem cell-derived extracellular vesicles: A novel promising neuroprotective agent for Alzheimer's disease.PubMed
Jun Chang, Zihang Feng, Yujiao Li, et al.
Biochem Pharmacol. 2024 Apr;222:116064. doi: 10.1016/j.bcp.2024.116064. Epub 2024 Feb 17.
Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive loss of neurons in the brain. However, there are no effective drugs for AD. Mesenchymal stem cell-derived extracellular vesicles (MSCs-EVs), as a new mediator of intercellular communication, are associated with low immunogenicity, low risk of tumor formation, and good safety profile. Therefore, MSCs-EVs may be a safe and attractive cell-free nanotherapeutics, offering a new perspective for AD treatment. Although preclinical studies have demonstrated that MSCs-EVs have significant neuroprotective effects, the underlying mechanism is unclear. This study aimed to: outline the diagnostic and delivery roles of MSCs-EVs for AD treatment; summarize the optimal sources and delivery methods of MSCs-EVs; provide a comprehensive review on the neuroprotective mechanisms of MSCs-EVs; explore how to enhance the neuroprotective effects of MSCs-EVs; and discuss the limitations and potential of their translation to the clinic. Therefore, this study may provide a more precise theoretical reference and practical basis for clinical research of MSCs-EVs.
128Exosomes for the diagnosis and treatment of dementia.PubMed
Hyeon Su Joo, Ha Yeong Jeon, Eun Be Hong, et al.
Curr Opin Psychiatry. 2023 Mar 1;36(2):119-125. doi: 10.1097/YCO.0000000000000842. Epub 2023 Jan 11.
PURPOSE OF REVIEW: Dementia is a syndrome with several possible pathologies. To date, definitive methods for diagnosis and treatment of sub-types of dementia have not been established. Emerging evidence suggests that exosomes can provide important information for the diagnosis and treatment of several subtypes of dementia. This article reviews recent studies on the application of exosomes in dementia. RECENT FINDINGS: Exosomes are involved in the pathogenesis of Alzheimer's disease (AD) and Parkinson's disease (PD) through transporting toxic proteins such as amyloid beta (Aβ), tau, and α-synuclein. Exosomal microRNAs (miR) and proteins reflect the disease state, and therefore, exosomes can be used as diagnostic markers for diseases such as AD, PD, Huntington's disease (HD), vascular dementia (VaD), dementia with Lewy bodies (DLB), and frontotemporal dementia (FTD). Mesenchymal stem cell (MSC)-derived exosomes have been shown to ameliorate disease pathology, and improve cognitive function in AD, PD, and VAD. SUMMARY: Recent studies have shown that exosomes could be novel diagnostic agents for dementia because they contain molecules that could be potential biomarker candidates indicative of the type and stage of dementia. Therapeutic application of exosomes in dementia has revealed that exosomes only, or exosomes loaded with an active pharmaceutical ingredient (API), ameliorate disease phenotype of dementia. Further work is needed to exploit this potential.
129Stem Cell-Derived Exosomes as Nanotherapeutics for Autoimmune and Neurodegenerative Disorders.PubMed
Milad Riazifar, M Rezaa Mohammadi, Egest J Pone, et al.
ACS Nano. 2019 Jun 25;13(6):6670-6688. doi: 10.1021/acsnano.9b01004. Epub 2019 May 29.
To dissect therapeutic mechanisms of transplanted stem cells and develop exosome-based nanotherapeutics in treating autoimmune and neurodegenerative diseases, we assessed the effect of exosomes secreted from human mesenchymal stem cells (MSCs) in treating multiple sclerosis using an experimental autoimmune encephalomyelitis (EAE) mouse model. We found that intravenous administration of exosomes produced by MSCs stimulated by IFNγ (IFNγ-Exo) (i) reduced the mean clinical score of EAE mice compared to PBS control, (ii) reduced demyelination, (iii) decreased neuroinflammation, and (iv) upregulated the number of CD4+CD25+FOXP3+ regulatory T cells (Tregs) within the spinal cords of EAE mice. Co-culture of IFNγ-Exo with activated peripheral blood mononuclear cells (PBMCs) cells in vitro reduced PBMC proliferation and levels of pro-inflammatory Th1 and Th17 cytokines including IL-6, IL-12p70, IL-17AF, and IL-22 yet increased levels of immunosuppressive cytokine indoleamine 2,3-dioxygenase. IFNγ-Exo could also induce Tregs in vitro in a murine splenocyte culture, likely mediated by a third-party accessory cell type. Further, IFNγ-Exo characterization by deep RNA sequencing suggested that IFNγ-Exo contains anti-inflammatory RNAs, where their inactivation partially hindered the exosomes potential to induce Tregs. Furthermore, we found that IFNγ-Exo harbors multiple anti-inflammatory and neuroprotective proteins. These results not only shed light on stem cell therapeutic mechanisms but also provide evidence that MSC-derived exosomes can potentially serve as cell-free therapies in creating a tolerogenic immune response to treat autoimmune and central nervous system disorders.
130CAR T cell therapy for patients with solid tumours: key lessons to learn and unlearn.PubMed
Steven M Albelda
Nat Rev Clin Oncol. 2024 Jan;21(1):47-66. doi: 10.1038/s41571-023-00832-4. Epub 2023 Oct 30.
Chimeric antigen receptor (CAR) T cells have been approved for use in patients with B cell malignancies or relapsed and/or refractory multiple myeloma, yet efficacy against most solid tumours remains elusive. The limited imaging and biopsy data from clinical trials in this setting continues to hinder understanding, necessitating a reliance on imperfect preclinical models. In this Perspective, I re-evaluate current data and suggest potential pathways towards greater success, drawing lessons from the few successful trials testing CAR T cells in patients with solid tumours and the clinical experience with tumour-infiltrating lymphocytes. The most promising approaches include the use of pluripotent stem cells, co-targeting multiple mechanisms of immune evasion, employing multiple co-stimulatory domains, and CAR ligand-targeting vaccines. An alternative strategy focused on administering multiple doses of short-lived CAR T cells in an attempt to pre-empt exhaustion and maintain a functional effector pool should also be considered.
131CAR-T cell therapy for triple-negative breast cancer and other solid tumors: preclinical and clinical progress.PubMed
Chiara Corti, Konstantinos Venetis, Elham Sajjadi, et al.
Expert Opin Investig Drugs. 2022 Jun;31(6):593-605. doi: 10.1080/13543784.2022.2054326. Epub 2022 Mar 24.
INTRODUCTION: Most breast cancer-related deaths arise from triple-negative breast cancer (TNBC). Molecular heterogeneity, aggressiveness and the lack of effective therapies are major hurdles to therapeutic progress. Chimeric antigen receptor (CAR)-T cells have emerged as a promising immunotherapeutic strategy in TNBC. This approach combines the antigen specificity of an antibody with the effector function of T cells. AREAS COVERED: This review examines the opportunities provided by CAR-T cell therapies in solid tumors. Emerging targets, ongoing clinical trials, and prospective clinical implications in TNBC are considered later. An emphasis is placed on the key challenges and possible solutions for this therapeutic approach. EXPERT OPINION: A challenge for CAR-T cell therapy is the selection of the optimal targets to minimize on-target/off-tumor toxicity. Tumor escape via antigen loss and intrinsic heterogeneity is a further hurdle. TROP2, GD2, ROR1, MUC1 and EpCAM are promising targets. Persistence and trafficking to tumor cells may be enhanced by the implementation of CARs with a chemokine receptor and/or constitutively activated interleukin receptors. Fourth-generation CARs (TRUCKs) may redirect T-cells for universal cytokine-mediated killing. Combinatorial approaches and the application of CARs to other immune cells could revert the suppressive immune environment that characterizes solid neoplasms.
132Shaping Polarization Of Tumor-Associated Macrophages In Cancer Immunotherapy.PubMed
Jing Gao, Yuanzheng Liang, Liang Wang
Front Immunol. 2022 Jun 30;13:888713. doi: 10.3389/fimmu.2022.888713. eCollection 2022.
Different stimuli can polarize macrophages into two basic types, M1 and M2. Tumor-associated macrophages (TAMs) in the tumor microenvironment (TME) are composed of heterogeneous subpopulations, which include the M1 anti-tumor and M2 pro-tumor phenotypes. TAMs predominantly play a M2-like tumor-promoting role in the TME and regulate various malignant effects, such as angiogenesis, immune suppression, and tumor metastasis; hence, TAMs have emerged as a hot topic of research in cancer therapy. This review focuses on three main aspects of TAMs. First, we summarize macrophage polarization along with the effects on the TME. Second, recent advances and challenges in cancer treatment and the role of M2-like TAMs in immune checkpoint blockade and CAR-T cell therapy are emphasized. Finally, factors, such as signaling pathways, associated with TAM polarization and potential strategies for targeting TAM repolarization to the M1 pro-inflammatory phenotype for cancer therapy are discussed.
133CAR-T Cell Therapy in Hematological Malignancies: Current Opportunities and Challenges.PubMed
Xiaomin Zhang, Lingling Zhu, Hui Zhang, et al.
Front Immunol. 2022 Jun 10;13:927153. doi: 10.3389/fimmu.2022.927153. eCollection 2022.
Chimeric antigen receptor T (CAR-T) cell therapy represents a major breakthrough in cancer treatment, and it has achieved unprecedented success in hematological malignancies, especially in relapsed/refractory (R/R) B cell malignancies. At present, CD19 and BCMA are the most common targets in CAR-T cell therapy, and numerous novel therapeutic targets are being explored. However, the adverse events related to CAR-T cell therapy might be serious or even life-threatening, such as cytokine release syndrome (CRS), CAR-T-cell-related encephalopathy syndrome (CRES), infections, cytopenia, and CRS-related coagulopathy. In addition, due to antigen escape, the limited CAR-T cell persistence, and immunosuppressive tumor microenvironment, a considerable proportion of patients relapse after CAR-T cell therapy. Thus, in this review, we focus on the progress and challenges of CAR-T cell therapy in hematological malignancies, such as attractive therapeutic targets, CAR-T related toxicities, and resistance to CAR-T cell therapy, and provide some practical recommendations.
134Exhaustion-associated cholesterol deficiency dampens the cytotoxic arm of antitumor immunity.PubMed
Chengsong Yan, Lin Zheng, Shutan Jiang, et al.
Cancer Cell. 2023 Jul 10;41(7):1276-1293.e11. doi: 10.1016/j.ccell.2023.04.016. Epub 2023 May 26.
The concept of targeting cholesterol metabolism to treat cancer has been widely tested in clinics, but the benefits are modest, calling for a complete understanding of cholesterol metabolism in intratumoral cells. We analyze the cholesterol atlas in the tumor microenvironment and find that intratumoral T cells have cholesterol deficiency, while immunosuppressive myeloid cells and tumor cells display cholesterol abundance. Low cholesterol levels inhibit T cell proliferation and cause autophagy-mediated apoptosis, particularly for cytotoxic T cells. In the tumor microenvironment, oxysterols mediate reciprocal alterations in the LXR and SREBP2 pathways to cause cholesterol deficiency of T cells, subsequently leading to aberrant metabolic and signaling pathways that drive T cell exhaustion/dysfunction. LXRβ depletion in chimeric antigen receptor T (CAR-T) cells leads to improved antitumor function against solid tumors. Since T cell cholesterol metabolism and oxysterols are generally linked to other diseases, the new mechanism and cholesterol-normalization strategy might have potential applications elsewhere.
135EZH1/EZH2 inhibition enhances adoptive T cell immunotherapy against multiple cancer models.PubMed
Patrizia Porazzi, Siena Nason, Ziqi Yang, et al.
Cancer Cell. 2025 Mar 10;43(3):537-551.e7. doi: 10.1016/j.ccell.2025.01.013. Epub 2025 Feb 20.
Tumor resistance to chimeric antigen receptor T cell (CAR-T) and, in general, to adoptive cell immunotherapies (ACTs) is a major challenge in the clinic. We hypothesized that inhibiting the tumor drivers' methyltransferases EZH2 and EZH1 could enhance ACT by rewiring cancer cells to a more immunogenic state. In human B cell lymphoma, EZH2 inhibition (tazemetostat) improved the efficacy of anti-CD19 CAR-T by enhancing activation, expansion, and tumor infiltration. Mechanistically, tazemetostat-treated tumors showed upregulation of genes related to adhesion, B cell activation, and inflammatory responses, and increased avidity to CAR-T. Furthermore, tazemetostat improved CAR- and TCR-engineered T cell efficacy in multiple liquid (myeloma and acute myeloid leukemia) and solid (sarcoma, ovarian, and prostate) cancers. Lastly, combined EZH1/EZH2 inhibition (valemetostat) further boosted CAR-T efficacy and expansion in multiple cancers. This study shows that EZH1/2 inhibition reprograms tumors to a more immunogenic state and potentiates ACT in preclinical models of both liquid and solid cancers.
136Utilizing chemokines in cancer immunotherapy.PubMed
Florian Märkl, Duc Huynh, Stefan Endres, et al.
Trends Cancer. 2022 Aug;8(8):670-682. doi: 10.1016/j.trecan.2022.04.001. Epub 2022 Apr 29.
Immune checkpoint inhibition and chimeric antigen receptor (CAR) T cell therapy have demonstrated stunning clinical efficacy in many cancer types. However, most patients do not respond to immunotherapies or relapse after an initial response, stressing the need for improved strategies. Chemokines, as mediators of immune cell trafficking, play an important role in the composition of the tumor microenvironment and exert both pro- and antitumorigenic functions. Here, chemokines may represent valuable prognostic biomarkers of response to immunotherapy and a strategy to improve immunotherapies. In this review, the pleiotropic functions of chemokines in the tumor microenvironment (TME) and strategies of utilizing chemokines or chemokine antagonism in immunotherapy are discussed. The review highlights preclinical and clinical studies that apply or target chemokines in monotherapy or in combination therapies.
137Current understanding and management of CAR T cell-associated toxicities.PubMed
Jennifer N Brudno, James N Kochenderfer
Nat Rev Clin Oncol. 2024 Jul;21(7):501-521. doi: 10.1038/s41571-024-00903-0. Epub 2024 May 20.
Chimeric antigen receptor (CAR) T cell therapy has revolutionized the treatment of several haematological malignancies and is being investigated in patients with various solid tumours. Characteristic CAR T cell-associated toxicities such as cytokine-release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) are now well-recognized, and improved supportive care and management with immunosuppressive agents has made CAR T cell therapy safer and more feasible than it was when the first regulatory approvals of such treatments were granted in 2017. The increasing clinical experience with these therapies has also improved recognition of previously less well-defined toxicities, including movement disorders, immune effector cell-associated haematotoxicity (ICAHT) and immune effector cell-associated haemophagocytic lymphohistiocytosis-like syndrome (IEC-HS), as well as the substantial risk of infection in patients with persistent CAR T cell-induced B cell aplasia and hypogammaglobulinaemia. A more diverse selection of immunosuppressive and supportive-care pharmacotherapies is now being utilized for toxicity management, yet no universal algorithm for their application exists. As CAR T cell products targeting new antigens are developed, additional toxicities involving damage to non-malignant tissues expressing the target antigen are a potential hurdle. Continued prospective evaluation of toxicity management strategies and the design of less-toxic CAR T cell products are both crucial for ongoing success in this field. In this Review, we discuss the evolving understanding and clinical management of CAR T cell-associated toxicities.
138Cytokine release syndrome and associated neurotoxicity in cancer immunotherapy.PubMed
Emma C Morris, Sattva S Neelapu, Theodoros Giavridis, et al.
Nat Rev Immunol. 2022 Feb;22(2):85-96. doi: 10.1038/s41577-021-00547-6. Epub 2021 May 17.
A paradigm shift has recently occurred in the field of cancer therapeutics. Traditional anticancer agents, such as chemotherapy, radiotherapy and small-molecule drugs targeting specific signalling pathways, have been joined by cellular immunotherapies based on T cell engineering. The rapid adoption of novel, patient-specific cellular therapies builds on scientific developments in tumour immunology, genetic engineering and cell manufacturing, best illustrated by the curative potential of chimeric antigen receptor (CAR) T cell therapy targeting CD19-expressing malignancies. However, the clinical benefit observed in many patients may come at a cost. In up to one-third of patients, significant toxicities occur that are directly associated with the induction of powerful immune effector responses. The most frequently observed immune-mediated toxicities are cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome. This Review discusses our current understanding of their pathophysiology and clinical features, as well as the development of novel therapeutics for their prevention and/or management.
139ASTCT Consensus Grading for Cytokine Release Syndrome and Neurologic Toxicity Associated with Immune Effector Cells.PubMed
Daniel W Lee, Bianca D Santomasso, Frederick L Locke, et al.
Biol Blood Marrow Transplant. 2019 Apr;25(4):625-638. doi: 10.1016/j.bbmt.2018.12.758. Epub 2018 Dec 25.
Chimeric antigen receptor (CAR) T cell therapy is rapidly emerging as one of the most promising therapies for hematologic malignancies. Two CAR T products were recently approved in the United States and Europe for the treatment ofpatients up to age 25years with relapsed or refractory B cell acute lymphoblastic leukemia and/or adults with large B cell lymphoma. Many more CAR T products, as well as other immunotherapies, including various immune cell- and bi-specific antibody-based approaches that function by activation of immune effector cells, are in clinical development for both hematologic and solid tumor malignancies. These therapies are associated with unique toxicities of cytokine release syndrome (CRS) and neurologic toxicity. The assessment and grading of these toxicities vary considerably across clinical trials and across institutions, making it difficult to compare the safety of different products and hindering the ability to develop optimal strategies for management of these toxicities. Moreover, some aspects of these grading systems can be challenging to implement across centers. Therefore, in an effort to harmonize the definitions and grading systems for CRS and neurotoxicity, experts from all aspects of the field met on June 20 and 21, 2018, at a meeting supported by the American Society for Transplantation and Cellular Therapy (ASTCT; formerly American Society for Blood and Marrow Transplantation, ASBMT) in Arlington, VA. Here we report the consensus recommendations of that group and propose new definitions and grading for CRS and neurotoxicity that are objective, easy to apply, and ultimately more accurately categorize the severity of these toxicities. The goal is to provide a uniform consensus grading system for CRS and neurotoxicity associated with immune effector cell therapies, for use across clinical trials and in the postapproval clinical setting.
140Cytokine Release Syndrome with Chimeric Antigen Receptor T Cell Therapy.PubMed
Noelle Frey, David Porter
Biol Blood Marrow Transplant. 2019 Apr;25(4):e123-e127. doi: 10.1016/j.bbmt.2018.12.756. Epub 2018 Dec 23.
Chimeric antigen receptor (CAR)-modified T cells (CAR-Ts) targeting CD19 have resulted in unprecedented durable remissions for patients with relapsed and refractory B cell malignancies. Cytokine release syndrome (CRS), resulting from rapid immune activation induced by CAR-Ts, is the most significant treatment-related toxicity. CRS initially manifests with fever and can progress to life-threatening capillary leak with hypoxia and hypotension. The clinical signs of CRS correlate with T cell activation and high levels of cytokines including IL-6. Tocilizumab, an anti-IL-6 receptor antagonist, is the standard for CRS management, but optimal timing of administration is unclear. The development of a supportive infrastructure by treatment centers is important to maintain safe administration as access expands. Collaborative efforts are underway to harmonize the definition and grading of CRS to allow for better interpretation of toxicities across CAR-T products and clinical trials and allow for informed management algorithms.
141Cytokine Release Syndrome Biology and Management.PubMed
Dustin A Cobb, Daniel W Lee
Cancer J. 2021;27(2):119-125. doi: 10.1097/PPO.0000000000000515.
The successful application of chimeric antigen receptor (CAR) T cells for the treatment of relapsed and refractory B-cell malignancies has ushered in a new frontier for the immunotherapy of cancer. Despite its successes, CAR T-cell therapy presents several challenges. Cytokine release syndrome (CRS) triggered by robust and exponential CAR T-cell expansion is the most common adverse effect and may be severe or life-threatening. Although modulation of the interleukin 6 axis was appreciated early on as a means to manage CRS, the exact underlying mechanisms leading to severe CRS remain to be elucidated. What is clear is that severe CRS involves recruitment of the broader immune system into a hyperinflammatory and unregulated state. Myeloid-derived cells appear to play a critical role in this regard and are at the center of active investigation. In this article, we will focus on important elements of CRS, the clinical manifestations, underlying biology, and management strategies including grading, supportive care, and treatment via immunosuppression.
142Prevention and management of adverse events during treatment with bispecific antibodies and CAR T cells in multiple myeloma: a consensus report of the European Myeloma Network.PubMed
Heinz Ludwig, Evangelos Terpos, Niels van de Donk, et al.
Lancet Oncol. 2023 Jun;24(6):e255-e269. doi: 10.1016/S1470-2045(23)00159-6.
T-cell redirecting bispecific antibodies (BsAbs) and chimeric antigen receptor T cells (CAR T cells) have revolutionised multiple myeloma therapy, but adverse events such as cytokine release syndrome, immune effector cell-associated neurotoxicity syndrome (ICANS), cytopenias, hypogammaglobulinaemia, and infections are common. This Policy Review presents a consensus from the European Myeloma Network on the prevention and management of these adverse events. Recommended measures include premedication, frequent assessing for symptoms and severity of cytokine release syndrome, step-up dosing for several BsAbs and some CAR T-cell therapies; corticosteroids; and tocilizumab in the case of cytokine release syndrome. Other anti-IL-6 drugs, high-dose corticosteroids, and anakinra might be considered in refractory cases. ICANS often arises concomitantly with cytokine release syndrome. Glucocorticosteroids in increasing doses are recommended if needed, as well as anakinra if the response is inadequate, and anticonvulsants if convulsions occur. Preventive measures against infections include antiviral and antibacterial drugs and administration of immunoglobulins. Treatment of infections and other complications is also addressed.
143How I treat refractory CRS and ICANS after CAR T-cell therapy.PubMed
Michael D Jain, Melody Smith, Nirali N Shah
Blood. 2023 May 18;141(20):2430-2442. doi: 10.1182/blood.2022017414.
The clinical use of chimeric antigen receptor (CAR) T-cell therapy is growing rapidly because of the expanding indications for standard-of-care treatment and the development of new investigational products. The establishment of consensus diagnostic criteria for cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS), alongside the steady use of both tocilizumab and corticosteroids for treatment, have been essential in facilitating the widespread use. Preemptive interventions to prevent more severe toxicities have improved safety, facilitating CAR T-cell therapy in medically frail populations and in those at high risk of severe CRS/ICANS. Nonetheless, the development of persistent or progressive CRS and ICANS remains problematic because it impairs patient outcomes and is challenging to treat. In this case-based discussion, we highlight a series of cases of CRS and/or ICANS refractory to front-line interventions. We discuss our approach to managing refractory toxicities that persist or progress beyond initial tocilizumab or corticosteroid administration, delineate risk factors for severe toxicities, highlight the emerging use of anakinra, and review mitigation strategies and supportive care measures to improve outcomes in patients who develop these refractory toxicities.
144Engineering sonogenetic EchoBack-CAR T cells.PubMed
Longwei Liu, Peixiang He, Yuxuan Wang, et al.
Cell. 2025 May 15;188(10):2621-2636.e20. doi: 10.1016/j.cell.2025.02.035. Epub 2025 Apr 2.
Chimeric antigen receptor (CAR) T cell therapy for solid tumors encounters challenges such as on-target off-tumor toxicity, exhaustion, and limited T cell persistence. Here, we engineer sonogenetic EchoBack-CAR T cells using an ultrasensitive heat-shock promoter screened from a library and integrated with a positive feedback loop from CAR signaling, enabling long-lasting CAR expression upon focused-ultrasound (FUS) stimulation. EchoBack-hGD2CAR T cells, targeting disialoganglioside GD2, exhibited potent cytotoxicity and persistence in 3D glioblastoma (GBM) models. In mice, EchoBack-hGD2CAR T cells suppressed GBM without off-tumor toxicity and outperformed their constitutive counterparts. Single-cell RNA sequencing revealed enhanced cytotoxicity and reduced exhaustion in EchoBack-CAR T cells compared with the standard CAR T cells. This EchoBack design was further adapted to target prostate-specific membrane antigen (EchoBack-PSMACAR) for prostate cancer treatment, demonstrating long-lasting tumor suppression with minimal off-tumor toxicity. Thus, the sonogenetic EchoBack-CAR T cells can serve as a versatile, efficient, and safe strategy for solid tumor treatment.
145Immune Effector Cell-Associated Hemophagocytic Lymphohistiocytosis-Like Syndrome.PubMed
Melissa R Hines, Tristan E Knight, Kevin O McNerney, et al.
Transplant Cell Ther. 2023 Jul;29(7):438.e1-438.e16. doi: 10.1016/j.jtct.2023.03.006. Epub 2023 Mar 9.
T cell-mediated hyperinflammatory responses, such as cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS), are now well-established toxicities of chimeric antigen receptor (CAR) T cell therapy. As the field of CAR T cells advances, however, there is increasing recognition that hemophagocytic lymphohistiocytosis (HLH)-like toxicities following CAR T cell infusion are occurring broadly across patient populations and CAR T cell constructs. Importantly, these HLH-like toxicities are often not as directly associated with CRS and/or its severity as initially described. This emergent toxicity, however ill-defined, is associated with life-threatening complications, creating an urgent need for improved identification and optimal management. With the goal of improving patient outcomes and formulating a framework to characterize and study this HLH-like syndrome, we established an American Society for Transplantation and Cellular Therapy panel composed of experts in primary and secondary HLH, pediatric and adult HLH, infectious disease, rheumatology and hematology, oncology, and cellular therapy. Through this effort, we provide an overview of the underlying biology of classical primary and secondary HLH, explore its relationship with similar manifestations following CAR T cell infusions, and propose the term "immune effector cell-associated HLH-like syndrome (IEC-HS)" to describe this emergent toxicity. We also delineate a framework for identifying IEC-HS and put forward a grading schema that can be used to assess severity and facilitate cross-trial comparisons. Additionally, given the critical need to optimize outcomes for patients experiencing IEC-HS, we provide insight into potential treatment approaches and strategies to optimize supportive care and delineate alternate etiologies that should be considered in a patient presenting with IEC-HS. By collectively defining IEC-HS as a hyperinflammatory toxicity, we can now embark on further study of the pathophysiology underlying this toxicity profile and make strides toward a more comprehensive assessment and treatment approach.
146Affordable Pricing of CRISPR Treatments is a Pressing Ethical Imperative.PubMed
Jon Rueda, Íñigo de Miguel Beriain, Lluis Montoliu
CRISPR J. 2024 Oct;7(5):220-226. doi: 10.1089/crispr.2024.0042. Epub 2024 Oct 10.
Casgevy, the world's first approved CRISPR-based cell therapy, has been priced at $2.2 million per patient. Although this hefty price tag was widely anticipated, the extremely high cost of this and other cell and gene therapies poses a major ethical issue in terms of equitable access and global health. In this Perspective, we argue that lowering the prices of future CRISPR therapies is an urgent ethical imperative. Although we focus on Casgevy as a case study, much of our analysis can be extrapolated to the controversies over affordable access to other gene and cell therapies. First, we explain why this first-of-its-kind CRISPR therapy might be so expensive. We then analyze the ethical issues of equity and global health of early CRISPR treatments. Next, we discuss potential solutions to lower the prices of CRISPR gene therapies. We conclude that the approval of CRISPR transforms our obligations of justice and compels us to bring future gene therapies to the maximum possible number of patients with serious genetic diseases at affordable prices.
147A roadmap for affordable genetic medicines.PubMed
Melinda Kliegman, Manar Zaghlula, Susan Abrahamson, et al.
Nature. 2024 Oct;634(8033):307-314. doi: 10.1038/s41586-024-07800-7. Epub 2024 Jul 17.
Twenty genetic therapies have been approved by the US Food and Drug Administration to date, a number that now includes the first CRISPR genome-editing therapy for sickle cell disease-CASGEVY (exagamglogene autotemcel, Vertex Pharmaceuticals). This extraordinary milestone is widely celebrated owing to the promise for future genome-editing treatments of previously intractable genetic disorders and cancers. At the same time, such genetic therapies are the most expensive drugs on the market, with list prices exceeding US$4 million per patient. Although all approved cell and gene therapies trace their origins to academic or government research institutions, reliance on for-profit pharmaceutical companies for subsequent development and commercialization results in prices that prioritize recouping investments, paying for candidate product failures and meeting investor and shareholder expectations. To increase affordability and access, sustainable discovery-to-market alternatives are needed that address system-wide deficiencies. Here we present recommendations of a multidisciplinary task force assembled to chart such a path. We describe a pricing structure that, once implemented, could reduce per-patient cost tenfold and propose a business model that distributes responsibilities while leveraging diverse funding sources. We also outline how academic licensing provisions, manufacturing innovation and supportive regulations can reduce cost and enable broader patient treatment.
148Beyond the traditional distinctions of genome editing: evaluating a vulnerability framework.PubMed
Ioanna Angelioudaki, Ana Ruxandra Badea, Martina Bodo, et al.
Front Genome Ed. 2024 Oct 16;6:1426228. doi: 10.3389/fgeed.2024.1426228. eCollection 2024.
Over 40 years ago, the 1982 Splicing Life report outlined the two distinctions that have orientated much of the normative and legal landscape of genetic intervention or genome editing since - that of somatic versus germline (or heritable interventions) and medical versus non-medical (or enhancement) applications. During this time, these distinctions have been used to ethically prioritize some areas of research and potential application, such as somatic treatments, while considering others for prohibition, such as germline enhancements. Nevertheless, somatic interventions may also be done for controversial enhancement purposes while some germline interventions may be done with greater justification (e.g., the enhancement of athletic ability versus the avoidance of Tay-Sachs disease). Even with new somatic treatments that are generally lauded, exemplified with the case of Casgevy, many issues still arise - such as cost and access, particularly salient on a global level. The concerns over a dystopian future of genetic haves and have nots, as a result of enhancement and/or germline interventions, that perhaps may happen, should not distract us from a greater attention to what is happening in the here and now. In this paper, we will highlight the limits of the two distinctions in terms of moving from questions of "should a technology be used" to "how should a technology be used." We argue that an additional focus on vulnerability and marginalization can be useful to support the attempt to better prioritize which interventions should be permitted or prohibited. We show how this can better dovetail with calls for effective (global) governance and reasonable consensus by focusing on the most urgent issues and developing policy accordingly, while leaving aside more abstract issues for further discussion.
149Development of a closed-system process for clinical-scale generation of DCs: evaluation of two monocyte-enrichment methods and two culture containers.PubMed
E C C Wong, S M Lee, K Hines, et al.
Cytotherapy. 2002;4(1):65-76. doi: 10.1080/146532402317251545.
BACKGROUND: Clinical immunotherapy trials using DCs depend on large-scale methods for DC generation that fulfil current good manufacturing practice requirements. Our goal was to develop data on two variables, monocyte-enrichment method and culture container, which could be used to design a closed-system process for ex vivo generation of immature DCs. METHODS: Mononuclear cells were collected by leukapheresis and enriched for monocytes by either counterflow centrifugal elutriation, or immunomagnetic selection using Isolex, an automated closed-system device. Monocytes were cultured for 7 days in serum-free medium with GM-CSF and IL-4, using either plastic flasks or gas-permeable Stericell bags. Monocytes and cultured DCs were evaluated for yield, flow cytometric phenotype, and in vitro function in MLR, and autologous recall responses to tetanus toxoid and influenza virus. RESULTS: Enriched monocyte products from elutriation and immunomagnetic selection were equivalent in yield and purity, and were capable of generating immature DCs in either flasks or bags. DCs from all four culture conditions were equivalent in yield, phenotype, and in vitro function. Mean DC yield was 67-80% per seeding monocyte, and 11-13% per starting mononuclear cell (MNC). A leukapheresis product containing 5 x 10(9) MNCs processed by this method could therefore yield approximately 5 x 10(8) immature DCs. DISCUSSION: In this manufacturing process, the Isolex system was equivalent to elutriation, and Stericell bags were equivalent to flasks. Together, the Isolex system and Stericell bags can be incorporated into a closed-system process to generate immature DCs.
150Closed system generation of dendritic cells from a single blood volume for clinical application in immunotherapy.PubMed
M Elias, J van Zanten, G A P Hospers, et al.
J Clin Apher. 2005 Dec;20(4):197-207. doi: 10.1002/jca.20054.
Dendritic cells (DC) used for clinical trials should be processed on a large scale conforming to current good manufacturing practice (cGMP) guidelines. The aim of this study was to develop a protocol for clinical grade generation of immature DC in a closed-system. Aphereses were performed with the Cobe Spectra continuous flow cell separator and material was derived from one volume of blood processed. Optimisation of a 3-phase collection autoPBSC technique significantly improved the quality of the initial mononuclear cell (MNC) product. Monocytes were then enriched from MNC by immunomagnetic depletion of CD19+ B cells and CD2+ T cells and partial depletion of NK cells using the Isolex 300I Magnetic cell selector. The quality of the initial mononuclear cell product was found to determine the outcome of monocyte enrichment. Enriched monocytes were cultured in Opticyte gas-permeable containers using CellGro serum-free medium supplemented with GM-CSF and IL-4 to generate immature DC. A seeding concentration of 1 x 10(6) was found optimal in terms of DC phenotype expression, monocyte percentage in culture, and cell viability. The differentiation pattern favours day 7 for harvest of immature DC. DC recovery, viability, as well as phenotype expression after cryopreservation of immature DC was considered in this study. DC were induced to maturation and evaluated in FACS analysis for phenotype expression and proliferation assays. Mature DC were able to generate an allogeneic T-cell response as well as an anti-CMV response as detected by proliferation assays. These data indicate that the described large-scale GMP-compatible system results in the generation of stable DC derived from one volume of blood processed, which are qualitatively and quantitatively sufficient for clinical application in immunotherapeutic protocols.
151Generation of immature autologous clinical grade dendritic cells for vaccination of cancer patients.PubMed
M Toungouz, C Quinet, E Thille, et al.
Cytotherapy. 1999;1(6):447-53. doi: 10.1080/0032472031000141304.
BACKGROUND: Dendritic cell (DC)-based vaccine is a promising approach for cancer therapy. Pioneer trials have been conducted using DC generated in research conditions. There is now a need for generating DC in clinical grade conditions, including the use of closed systems, avoidance of FCS and respect of good manufacturing practices (GMP). METHODS: DC were generated from 84 leukapheresis products of 27 cancer patients enrolled in two Phase I/II trials of vaccination of either MAGE+tumors (n = 24) or prostate cancer (n = 3). Monocytes were seeded in culture bags in a serum-free medium supplemented with IL-4 and GM-CSF. After a 7 day culture, DC were collected and most were pulsed with various MAGE-derived peptides. RESULTS: After a short leukapheresis (mean time: 66 min; mean processed blood: 5 L), a mean of 6 x 10(9) WBC were collected, from which 2.25 x 10(9) were seeded. The culture procedure yielded a large number of DC (mean: 62 x 10(6) DC) harboring the expected phenotype of immature DC (CD1a(+) CD14(-) HLA-DR(+) CD80(+) CD86(+) CD83(-)). This phenotype was not altered by peptide loading. These DC, either fresh or thawed, were functionally effective invitro. Their s.c. and i.v. injections were devoid of any short-term side effect and associated with the induction of immune responses in the patients. DISCUSSION: Large numbers of functional immature clinical grade DC can be generated in a closed system from leukapheresis products in cancer patients. These results provide the basis for large-scale studies of cancer immunotherapy under improved safety conditions.
152Innovative Payment Models for Sickle-Cell Disease Gene Therapies in Medicaid: Leveraging Real-World Data and Insights from CMMI's Gene Therapy Access Model.PubMed
Antal Zemplenyi, Jim Leonard, Garth C Wright, et al.
Pharmacoeconomics. 2025 May;43(5):583-594. doi: 10.1007/s40273-025-01474-3. Epub 2025 Feb 21.
OBJECTIVE: This study aims to evaluate the financial implications of implementing various payment models, including outcome-based agreements (OBAs), volume-based rebates, and guaranteed rebates, for the newly approved gene therapies, exagamglogene autotemcel (exa-cel) and lovotibeglogene autotemcel (lovo-cel), in the treatment of sickle-cell disease (SCD) from the perspective of Colorado Medicaid. The analysis specifically examines the cost of standard of care (SoC) for severe SCD, the impact of different eligibility criteria based on vaso-occlusive events (VOEs), and the potential financial impacts associated with rebate structures. METHODS: Data from the Colorado Department of Health Care Policy & Financing (HCPF) database was used to estimate the annual costs for Medicaid-enrolled patients with severe SCD from 2018 to 2023. Patients were selected based on various eligibility criteria, including the number of VOEs, acute chest syndrome events, and stroke diagnoses. Three-state Markov models (SCD, stable, and dead) were constructed to compare the costs of SoC and gene therapies. The durability of gene therapy effectiveness and the financial impact of OBAs, volume-based rebates, and guaranteed rebates were evaluated over a 6-year contract period, with scenarios reflecting different VOE criteria and treatment durability. RESULTS: The average annual SoC cost for severe SCD patients (N = 138) was US$45,941 (SD US$59,653), with higher costs associated with more frequent VOEs. Gene therapies exa-cel and lovo-cel, with one-off list prices of US$2.2 million and US$3.1 million, respectively, exhibited high upfront costs, resulting in a negative cumulative balance averaging - US$2.11 million for exa-cel and - US$3.00 million for lovo-cel per patient over 6 years compared with SoC. Outcome-based rebates could potentially save Medicaid approximately US$260K (uncertainty interval 88K-772K) per patient on average for exa-cel and US$367K (uncertainty interval 122K-1111K) for lovo-cel after they pay the full up-front cost. Volume-based and guaranteed rebates also offered potential savings but varied in impact based on contract duration and effectiveness of gene therapy. CONCLUSIONS: The study highlights critical considerations for Medicaid in negotiating OBAs for SCD gene therapies. Achieving budget neutrality over 6 years is unlikely due to low SoC costs. However, payment models can enhance value-based spending by linking high therapy costs and potential rebates to the health gains these treatments may offer. OBAs offer offsets contingent on therapy effectiveness durability and contract terms (such as length and price), while varying eligibility criteria impact budgets and outcomes. Medicaid real-world data is crucial for navigating complexities in defining eligible populations and structuring OBAs.
153Addressing the Value of Gene Therapy and Enhancing Patient Access to Transformative Treatments.PubMed
Rachel Salzman, Francesca Cook, Timothy Hunt, et al.
Mol Ther. 2018 Dec 5;26(12):2717-2726. doi: 10.1016/j.ymthe.2018.10.017. Epub 2018 Oct 30.
Although high upfront costs for the high value of gene therapy have resulted in concerns about sufficient reimbursement to allow patient access to these therapies, the significant benefits of gene therapies will not be realized unless patients have access to them. Stakeholders are discussing these issues, and the payment models being developed for the newly approved gene therapies provide an early indication of the flexibility that will be needed from treatment manufacturers, payers, and policy makers to optimize patient access. Maximizing patient access to effective gene therapies is one integral part of the overall mission of the American Society of Gene and Cell Therapy, along with maximizing the quality of therapies and minimizing their costs.
154Enhancing pediatric access to cell and gene therapies.PubMed
Crystal L Mackall, Catherine M Bollard, Nancy Goodman, et al.
Nat Med. 2024 Jul;30(7):1836-1846. doi: 10.1038/s41591-024-03035-1. Epub 2024 Jun 17.
Increasing numbers of cell and gene therapies (CGTs) are emerging to treat and cure pediatric diseases. However, small market sizes limit the potential return on investment within the traditional biopharmaceutical drug development model, leading to a market failure. In this Perspective, we discuss major factors contributing to this failure, including high manufacturing costs, regulatory challenges, and licensing practices that do not incorporate pediatric development milestones, as well as potential solutions. We propose the creation of a new entity, the Pediatric Advanced Medicines Biotech, to lead late-stage development and commercialize pediatric CGTs outside the traditional biopharmaceutical model in the United States-where organized efforts to solve this problem have been lacking. The Pediatric Advanced Medicines Biotech would partner with the academic ecosystem, manufacture products in academic good manufacturing practice facilities and work closely with regulatory bodies, to ferry CGTs across the drug development 'valley of death' and, ultimately, increase access to lifesaving treatments for children in need.
155Developments in Stem Cell Therapy by Utilizing Artificial Intelligence.PubMed
Priyanshi Goyal, Rishabha Malviya
Curr Pharm Des. 2023;29(28):2223-2228. doi: 10.2174/0113816128266696230926094423.
Undifferentiated cells, known as stem cells, have the capacity to self-renew and specialise in a variety of distinct mature and functional cell types while still retaining their original identity. For their significance in regenerative medicine, stem cells' deep potential has been widely examined, and this work has established the groundwork for cell-based therapeutics. With the potential to repair and restore specific organs or tissue injuries or chronic disease situations when the body's regenerative process is insufficient to heal, regenerative medicine is quickly advancing in healthcare. The most recent developments in stem cell-based treatments for regenerative medicine are covered in this review, focusing on mesenchymal stem cell-based treatments because these cells have received the most clinical research. The most recent applications of artificial intelligence algorithms in stem cell-based therapeutics are presented, along with their limitations and potential future applications.
156Harnessing the potential of CAR-T cell therapy: progress, challenges, and future directions in hematological and solid tumor treatments.PubMed
Gunjan Dagar, Ashna Gupta, Tariq Masoodi, et al.
J Transl Med. 2023 Jul 7;21(1):449. doi: 10.1186/s12967-023-04292-3.
Traditional cancer treatments use nonspecific drugs and monoclonal antibodies to target tumor cells. Chimeric antigen receptor (CAR)-T cell therapy, however, leverages the immune system's T-cells to recognize and attack tumor cells. T-cells are isolated from patients and modified to target tumor-associated antigens. CAR-T therapy has achieved FDA approval for treating blood cancers like B-cell acute lymphoblastic leukemia, large B-cell lymphoma, and multiple myeloma by targeting CD-19 and B-cell maturation antigens. Bi-specific chimeric antigen receptors may contribute to mitigating tumor antigen escape, but their efficacy could be limited in cases where certain tumor cells do not express the targeted antigens. Despite success in blood cancers, CAR-T technology faces challenges in solid tumors, including lack of reliable tumor-associated antigens, hypoxic cores, immunosuppressive tumor environments, enhanced reactive oxygen species, and decreased T-cell infiltration. To overcome these challenges, current research aims to identify reliable tumor-associated antigens and develop cost-effective, tumor microenvironment-specific CAR-T cells. This review covers the evolution of CAR-T therapy against various tumors, including hematological and solid tumors, highlights challenges faced by CAR-T cell therapy, and suggests strategies to overcome these obstacles, such as utilizing single-cell RNA sequencing and artificial intelligence to optimize clinical-grade CAR-T cells.
157Bioprinting functional hepatocyte organoids derived from human chemically induced pluripotent stem cells to treat liver failure.PubMed
Guangya Li, Jianyu He, Jihang Shi, et al.
Gut. 2025 Jun 6;74(7):1150-1164. doi: 10.1136/gutjnl-2024-333885.
BACKGROUND: To treat liver failure, three-dimensional (3D) bioprinting is a promising technology used to construct hepatic tissue models. However, current research on bioprinting of hepatic tissue models primarily relies on conventional single-cell-based bioprinting, where individual functional hepatocytes are dispersed and isolated within hydrogels, leading to insufficient treatment outcomes due to inadequate cell functionality. OBJECTIVE: Here, we aim to bioprint a hepatic tissue model using functional hepatocyte organoids (HOs) and evaluate its liver-specific functions and . DESIGN: Human chemically induced pluripotent stem cells (hCiPSCs) were used as a robust and non-genome-integrative cell source to produce highly viable and functional HOs (hCiPSC-HOs). An oxygen-permeable microwell device was used to enhance oxygen supply, ensuring high cell viability and promoting hCiPSC-HOs maturation. To maintain the long-term biofunction of hCiPSC-HOs, spheroid-based bioprinting was employed to construct hepatic tissue models (3DP-HOs). 3DP-HOs were intraperitoneally implanted in mice with liver failure. RESULTS: 3DP-HOs demonstrated enhanced cell viability when compared with a model fabricated using single-cell-based bioprinting and exhibited gene profiles closely resembling hCiPSC-HOs while maintaining liver-specific functionality. Moreover, 3DP-HOs implantation significantly improved survival in mice with CCl-induced acute-on-chronic liver failure and also mice with liver failure. 3DP-HOs significantly reduced liver injury, inflammation and fibrosis indices while promoting liver regeneration and biofunction expression. CONCLUSION: Our bioprinted hepatic tissue model exhibits remarkable therapeutic efficacy for liver failure and holds great potential for clinical research in the field of liver regenerative medicine.
158Three-dimensional bioprinted hepatorganoids prolong survival of mice with liver failure.PubMed
Huayu Yang, Lejia Sun, Yuan Pang, et al.
Gut. 2021 Mar;70(3):567-574. doi: 10.1136/gutjnl-2019-319960. Epub 2020 May 20.
OBJECTIVE: Shortage of organ donors, a critical challenge for treatment of end-stage organ failure, has motivated the development of alternative strategies to generate organs in vitro. Here, we aim to describe the hepatorganoids, which is a liver tissue model generated by three-dimensional (3D) bioprinting of HepaRG cells and investigate its liver functions in vitro and in vivo. DESIGN: 3D bioprinted hepatorganoids (3DP-HOs) were constructed using HepaRG cells and bioink, according to specific 3D printing procedures. Liver functions of 3DP-HOs were detected after 7 days of differentiation in vitro, which were later transplanted into Fah-deficient mice. The in vivo liver functions of 3DP-HOs were evaluated by survival time and liver damage of mice, human liver function markers and human-specific debrisoquine metabolite production. RESULTS: 3DP-HOs broadly acquired liver functions, such as ALBUMIN secretion, drug metabolism and glycogen storage after 7 days of differentiation. After transplantation into abdominal cavity of mouse model of liver injury, 3DP-HOs further matured and displayed increased synthesis of liver-specific proteins. Particularly, the mice acquired human-specific drug metabolism activities. Functional vascular systems were also formed in transplanted 3DP-HOs, further enhancing the material transport and liver functions of 3DP-HOs. Most importantly, transplantation of 3DP-HOs significantly improved the survival of mice. CONCLUSIONS: Our results demonstrated a comprehensive proof of principle, which indicated that 3DP-HO model of liver tissues possessed in vivo hepatic functions and alleviated liver failure after transplantation, suggesting that 3D bioprinting could be used to generate human liver tissues as the alternative transplantation donors for treatment of liver diseases.
159Induced pluripotent stem cells in disease modelling and drug discovery.PubMed
R Grant Rowe, George Q Daley
Nat Rev Genet. 2019 Jul;20(7):377-388. doi: 10.1038/s41576-019-0100-z.
The derivation of induced pluripotent stem cells (iPSCs) over a decade ago sparked widespread enthusiasm for the development of new models of human disease, enhanced platforms for drug discovery and more widespread use of autologous cell-based therapy. Early studies using directed differentiation of iPSCs frequently uncovered cell-level phenotypes in monogenic diseases, but translation to tissue-level and organ-level diseases has required development of more complex, 3D, multicellular systems. Organoids and human-rodent chimaeras more accurately mirror the diverse cellular ecosystems of complex tissues and are being applied to iPSC disease models to recapitulate the pathobiology of a broad spectrum of human maladies, including infectious diseases, genetic disorders and cancer.
160iPSC-Derived Liver Organoids: A Journey from Drug Screening, to Disease Modeling, Arriving to Regenerative Medicine.PubMed
Cristina Olgasi, Alessia Cucci, Antonia Follenzi
Int J Mol Sci. 2020 Aug 27;21(17):6215. doi: 10.3390/ijms21176215.
Liver transplantation is the most common treatment for patients suffering from liver failure that is caused by congenital diseases, infectious agents, and environmental factors. Despite a high rate of patient survival following transplantation, organ availability remains the key limiting factor. As such, research has focused on the transplantation of different cell types that are capable of repopulating and restoring liver function. The best cellular mix capable of engrafting and proliferating over the long-term, as well as the optimal immunosuppression regimens, remain to be clearly well-defined. Hence, alternative strategies in the field of regenerative medicine have been explored. Since the discovery of induced pluripotent stem cells (iPSC) that have the potential of differentiating into a broad spectrum of cell types, many studies have reported the achievement of iPSCs differentiation into liver cells, such as hepatocytes, cholangiocytes, endothelial cells, and Kupffer cells. In parallel, an increasing interest in the study of self-assemble or matrix-guided three-dimensional (3D) organoids have paved the way for functional bioartificial livers. In this review, we will focus on the recent breakthroughs in the development of iPSCs-based liver organoids and the major drawbacks and challenges that need to be overcome for the development of future applications.
162Organoids from adult liver and pancreas: Stem cell biology and biomedical utility.PubMed
Christopher J Hindley, Lucía Cordero-Espinoza, Meritxell Huch
Dev Biol. 2016 Dec 15;420(2):251-261. doi: 10.1016/j.ydbio.2016.06.039. Epub 2016 Jun 27.
The liver and pancreas are critical organs maintaining whole body metabolism. Historically, the expansion of adult-derived cells from these organs in vitro has proven challenging and this in turn has hampered studies of liver and pancreas stem cell biology, as well as being a roadblock to disease modelling and cell replacement therapies for pathologies in these organs. Recently, defined culture conditions have been described which allow the in vitro culture and manipulation of adult-derived liver and pancreatic material. Here we review these systems and assess their physiological relevance, as well as their potential utility in biomedicine.
163RNA Delivery to Hematopoietic Stem and Progenitor Cells Targeted Lipid Nanoparticles.PubMed
Dennis Shi, Sho Toyonaga, Daniel G Anderson
Nano Lett. 2023 Apr 12;23(7):2938-2944. doi: 10.1021/acs.nanolett.3c00304. Epub 2023 Mar 29.
Ex vivo autologous hematopoietic stem cell (HSC) gene therapy has provided new therapies for the treatment of hematological disorders. However, these therapies have several limitations owing to the manufacturing complexities and toxicity resulting from required conditioning regimens. Here, we developed a c-kit (CD117) antibody-targeted lipid nanoparticle (LNP) that, following a single intravenous injection, can deliver RNA (both siRNA and mRNA) to HSCs in rodents. This targeted delivery system does not require stem cell harvest, culture, or mobilization of HSCs to facilitate delivery. We also show that delivery of Cre recombinase mRNA at a dose of 1 mg kg can facilitate gene editing to almost all (∼90%) hematopoietic stem and progenitor cells (HSPCs) , and edited cells retain their stemness and functionality to generate high levels of edited mature immune cells.
164Research advance in lipid nanoparticle-mRNA delivery system and its application in CAR-T cell therapy.PubMed
Baixin Ye, Yongxian Hu, Mingming Zhang, et al.
Zhejiang Da Xue Xue Bao Yi Xue Ban. 2022 Apr 25;51(2):185-191. doi: 10.3724/zdxbyxb-2022-0047.
Chimeric antigen receptor (CAR) T cell therapy has shown significant efficacy for hematological malignancies, however, it needs to be further optimized. Recently, the lipid nanoparticle (LNP)-mRNA delivery system as a nonviral gene transfer vector has gained rapid progress in CAR-T cell therapy. The claudin-6 (CLDN6) mRNA is delivered to antigen presenting cells (APCs) through LNP system, thereby enhancing the function of CLDN6 CAR-T cells for the clearance of solid tumor cells. For treatment of acute cardiac injury, the fibroblast activation protein (FAP) CAR mRNA can be delivered to T cells through LNP system for the production of FAP CAR-T cells, thereby blocking the process of myocardial fibrosis. The LNP-mRNA delivery system has advantages including having no integration in host genome, inexpensiveness, low toxicity and modifiability; on the other hand, it has certain disadvantages such as limited cell persistence caused by transient protein expression and limitations in preparation techniques. This article reviews the research advance in LNP-mRNA delivery system and its application in CAR-T cell therapy.
165Lipid nanoparticle-mediated mRNA delivery to CD34 cells in rhesus monkeys.PubMed
Hyejin Kim, Ryan Zenhausern, Kara Gentry, et al.
Nat Biotechnol. 2025 Nov;43(11):1813-1820. doi: 10.1038/s41587-024-02470-2. Epub 2024 Nov 22.
Transplantation of ex vivo engineered hematopoietic stem cells (HSCs) can lead to robust clinical responses but carries risks of adverse events from bone marrow mobilization, chemotherapy conditioning and other factors. HSCs have been modified in vivo using lipid nanoparticles (LNPs) decorated with targeting moieties, which increases manufacturing complexity. Here we screen 105 LNPs without targeting ligands for effective homing to the bone marrow in mouse. We report an LNP named LNP that delivers mRNA to murine HSCs in vivo, primary human HSCs ex vivo and CD34 cells in rhesus monkeys (Macaca mulatta) in vivo at doses of 0.25 and 0.4 mg kg. Without mobilization and conditioning, LNP can mediate delivery of mRNA to HSCs and their progenitor cells (HSPCs), as well as to the liver in rhesus monkeys, without serum cytokine activation. These data support the hypothesis that in vivo delivery to HSCs and HSPCs in nonhuman primates is feasible without targeting ligands.
166Enhancing regenerative approaches with nanoparticles.PubMed
Sabine van Rijt, Pamela Habibovic
J R Soc Interface. 2017 Apr;14(129). doi: 10.1098/rsif.2017.0093.
In this review, we discuss recent developments in the field of nanoparticles and their use in tissue regeneration approaches. Owing to their unique chemical properties and flexibility in design, nanoparticles can be used as drug delivery systems, to create novel features within materials or as bioimaging agents, or indeed these properties can be combined to create smart multifunctional structures. This review aims to provide an overview of this research field where the focus will be on nanoparticle-based strategies to stimulate bone regeneration; however, the same principles can be applied for other tissue and organ regeneration strategies. In the first section, nanoparticle-based methods for the delivery of drugs, growth factors and genetic material to promote tissue regeneration are discussed. The second section deals with the addition of nanoparticles to materials to create nanocomposites. Such materials can improve several material properties, including mechanical stability, biocompatibility and biological activity. The third section will deal with the emergence of a relatively new field of research using nanoparticles in advanced cell imaging and stem cell tracking approaches. As the development of nanoparticles continues, incorporation of this technology in the field of regenerative medicine will ultimately lead to new tools that can diagnose, track and stimulate the growth of new tissues and organs.
167In vivo manufacture and manipulation of CAR-T cells for better druggability.PubMed
Rui Hou, Xiaoxue Zhang, Xu Wang, et al.
Cancer Metastasis Rev. 2024 Sep;43(3):1075-1093. doi: 10.1007/s10555-024-10185-8. Epub 2024 Apr 9.
The current CAR-T cell therapy products have been hampered in their druggability due to the personalized preparation required, unclear pharmacokinetic characteristics, and unpredictable adverse reactions. Enabling standardized manufacturing and having clear efficacy and pharmacokinetic characteristics are prerequisites for ensuring the effective practicality of CAR-T cell therapy drugs. This review provides a broad overview of the different approaches for controlling behaviors of CAR-T cells in vivo. The utilization of genetically modified vectors enables in vivo production of CAR-T cells, thereby abbreviating or skipping the lengthy in vitro expansion process. By equipping CAR-T cells with intricately designed control elements, using molecule switches or small-molecule inhibitors, the control of CAR-T cell activity can be achieved. Moreover, the on-off control of CAR-T cell activity would yield potential gains in phenotypic remodeling. These methods provide beneficial references for the future development of safe, controllable, convenient, and suitable for standardized production of CAR-T cell therapy products.
168Mesenchymal stem cell-derived extracellular vesicles attenuate ferroptosis in aged hepatic ischemia/reperfusion injury by transferring miR-1275.PubMed
Yihang Gong, Qiang You, Xiaofeng Yuan, et al.
Redox Biol. 2025 Apr;81:103556. doi: 10.1016/j.redox.2025.103556. Epub 2025 Feb 18.
With an aging global population, the proportion of aged donor livers in graft pools is steadily increasing. Compared to young livers, aged livers exhibit heightened susceptibility to hepatic ischemia/reperfusion injury (HIRI), which significantly limits their utilisation in liver transplantation (LT) and exacerbates organ shortages. Our previous study demonstrated that ferroptosis is a pivotal trigger for HIRI vulnerability in aged livers. However, effective clinical strategies for the inhibition of ferroptosis remain elusive. Utilizing an aged mouse HIRI model, primary hepatocytes, and human liver organoids, this study provides hitherto undocumented evidence that mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) effectively alleviate HIRI in aged livers by inhibiting ferroptosis. Mechanistically, miR-1275, which was significantly enriched within MSC-EVs, was transferred to hepatocytes. Subsequently, miR-1275 downregulated the expression of SLC39A14, a crucial iron transporter that is upregulated in aged livers and plays a pivotal role in promoting ferroptosis. Furthermore, we found a negative correlation between SLC39A14 levels and prognosis of aged donor liver recipients using clinical LT samples. Silencing miR-1275 in MSC-EVs or modulating SLC39A14 levels in aged livers reversed MSC-EV-mediated mitigation of ferroptosis. Collectively, these findings revealed the novel therapeutic potential of MSC-EVs in attenuating aged HIRI, suggesting a promising treatment for improving prognosis and preventing serious complications in recipients of aged liver grafts during LT.
169Magnetic Nanoparticles and Methylprednisolone Based Physico-Chemical Bifunctional Neural Stem Cells Delivery System for Spinal Cord Injury Repair.PubMed
Wencan Zhang, Mingshan Liu, Jie Ren, et al.
Adv Sci (Weinh). 2024 Jun;11(21):e2308993. doi: 10.1002/advs.202308993. Epub 2024 Mar 22.
Neural stem cells (NSCs) transplantation is an attractive and promising treatment strategy for spinal cord injury (SCI). Various pathological processes including the severe inflammatory cascade and difficulty in stable proliferation and differentiation of NSCs limit its application and translation. Here, a novel physico-chemical bifunctional neural stem cells delivery system containing magnetic nanoparticles (MNPs and methylprednisolone (MP) is designed to repair SCI, the former regulates NSCs differentiation through magnetic mechanical stimulation in the chronic phase, while the latter alleviates inflammatory response in the acute phase. The delivery system releases MP to promote microglial M2 polarization, inhibit M1 polarization, and reduce neuronal apoptosis. Meanwhile, NSCs tend to differentiate into functional neurons with magnetic mechanical stimulation generated by MNPs in the static magnetic field, which is related to the activation of the PI3K/AKT/mTOR pathway. SCI mice achieve better functional recovery after receiving NSCs transplantation via physico-chemical bifunctional delivery system, which has milder inflammation, higher number of M2 microglia, more functional neurons, and axonal regeneration. Together, this bifunctional NSCs delivery system combined physical mechanical stimulation and chemical drug therapy is demonstrated to be effective, which provides new treatment insights into clinical transformation of SCI repair.
170New Developments in Imaging Cell-Based Therapy.PubMed
Olivier Martinez, Jane Sosabowski, John Maher, et al.
J Nucl Med. 2019 Jun;60(6):730-735. doi: 10.2967/jnumed.118.213348. Epub 2019 Apr 12.
Cancer immunotherapy is now established as a central therapeutic pillar in hematologic oncology. Cell-based therapies, with or without genetic modification ex vivo, have reached the clinic as the standard of care in limited indications and remain the subject of intense preclinical and translational development. Expanding on this, related therapeutic approaches are in development for solid-tumor and nonmalignant indications, broadening the scope of this technology. It has long been recognized that in vivo tracking of infused cellular therapies would provide unique opportunities to optimize their efficacy and aid in the assessment and management of toxicity. Recently, we have witnessed the introduction of novel tracers for passive labeling of cell products and advances in the introduction and use of reporter genes to enable longitudinal imaging. This review highlights the key developments over the last 5 y.