Department of Basic and Translational Sciences, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA, USA.
Department of Basic and Translational Sciences, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA, USA.
Biomaterials. 2023 Jul;298:122142. doi: 10.1016/j.biomaterials.2023.122142. Epub 2023 May 2.
Diabetes Mellitus is a silent epidemic affecting >500 million, which claimed 6.7 million lives in 2021, a projected increase of >670% in <20 years old in the next two decades but insulin is unaffordable for the large majority of the globe. Therefore, we engineered proinsulin in plant cells to facilitate oral delivery. Stability of the proinsulin gene and expression in subsequent generations, after removal of the antibiotic-resistance gene, was confirmed using PCR, Southern and western blots. Proinsulin expression was high (up to 12 mg/g DW or 47.5% of total leaf protein), stable up to one year after storage of freeze-dried plant cells at ambient temperature and met FDA regulatory requirements of uniformity, moisture content and bioburden. GM1 receptor binding, required for uptake via gut epithelial cells was confirmed by pentameric assembly of CTB-Proinsulin. IP insulin injections (without C peptide) in STZ mice rapidly decreased blood glucose level leading to transient hypoglycemia, followed by hepatic glucose compensation. On the other hand, other than the 15-min lag period of oral proinsulin (transit time required to reach the gut), the kinetics of blood sugar regulation of oral CTB-Proinsulin in STZ mice was very similar to naturally secreted insulin in healthy mice (both contain C-peptide), without rapid decrease or hypoglycemia. Elimination of expensive fermentation, purification and cold storage/transportation should reduce cost and increase other health benefits of plant fibers. The recent approval of plant cell delivery of therapeutic proteins by FDA and approval of CTB-ACE2 for phase I/II human clinical studies augur well for advancing oral proinsulin to the clinic.
糖尿病是一种无声的流行疾病，影响了超过 5 亿人，在 2021 年造成了 670 万人死亡，预计在未来 20 年内，20 岁以下的患者将增加超过 670%，但胰岛素对全球绝大多数人来说是负担不起的。因此，我们在植物细胞中构建了前胰岛素，以促进口服递送。通过 PCR、Southern 和 Western blot 证实了去除抗生素抗性基因后，前胰岛素基因的稳定性和后代的表达。前胰岛素的表达量很高（高达 12mg/gDW 或总叶蛋白的 47.5%），在冻干植物细胞在环境温度下储存一年后仍然稳定，并且符合 FDA 对均匀性、水分含量和生物负荷的监管要求。通过 CTB-前胰岛素的五聚体组装，证实了 GM1 受体结合对于通过肠道上皮细胞摄取是必需的。STZ 小鼠中 IP 胰岛素注射（不含 C 肽）可迅速降低血糖水平，导致短暂性低血糖，随后肝脏葡萄糖补偿。另一方面，除了口服前胰岛素（到达肠道所需的 15 分钟滞后时间）之外，STZ 小鼠口服 CTB-前胰岛素调节血糖的动力学与健康小鼠中天然分泌的胰岛素（两者都含有 C 肽）非常相似，没有快速下降或低血糖。消除昂贵的发酵、纯化和冷藏/运输应该降低成本，并增加植物纤维的其他健康益处。最近 FDA 批准了通过植物细胞递送治疗性蛋白质，以及 CTB-ACE2 用于 I/II 期人体临床试验，这为将口服前胰岛素推进临床应用提供了良好的前景。
