Naeimi Kararoudi Meisam, Alsudayri Alfahdah, Hill Cynthia L, Elmas Ezgi, Sezgin Yasemin, Thakkar Aarohi, Hester Mark E, Malleske Daniel T, Lee Dean A, Neal Matthew L, Perry Mark R, Harvilchuck Jill A, Reynolds Susan D
Nationwide Children's Hospital, Columbus, OH, United States.
Molecular, Cellular, and Developmental Biology Graduate Program, The Ohio State University, Columbus, OH, United States.
Front Genome Ed. 2022 Feb 7;4:781531. doi: 10.3389/fgeed.2022.781531. eCollection 2022.
Respiratory system damage is the primary cause of mortality in individuals who are exposed to vesicating agents including sulfur mustard (SM). Despite these devastating health complications, there are no fielded therapeutics that are specific for such injuries. Previous studies reported that SM inhalation depleted the tracheobronchial airway epithelial stem cell (TSC) pool and supported the hypothesis, TSC replacement will restore airway epithelial integrity and improve health outcomes for SM-exposed individuals. TSC express Major Histocompatibility Complex (MHC-I) transplantation antigens which increases the chance that allogeneic TSC will be rejected by the patient's immune system. However, previous studies reported that Beta-2 microglobulin (B2M) knockout cells lacked cell surface MHC-I and suggested that B2M knockout TSC would be tolerated as an allogeneic graft. This study used a Cas9 ribonucleoprotein (RNP) to generate B2M-knockout TSC, which are termed Universal Donor Stem Cells (UDSC). Whole genome sequencing identified few off-target modifications and demonstrated the specificity of the RNP approach. Functional assays demonstrated that UDSC retained their ability to self-renew and undergo multilineage differentiation. A preclinical model of SM inhalation was used to test UDSC efficacy and identify any treatment-associated adverse events. Adult male Sprague-Dawley rats were administered an inhaled dose of 0.8 mg/kg SM vapor which is the inhaled LD on day 28 post-challenge. On recovery day 2, vehicle or allogeneic Fisher rat UDSC were delivered intravenously ( = 30/group). Clinical parameters were recorded daily, and planned euthanasia occurred on post-challenge days 7, 14, and 28. The vehicle and UDSC treatment groups exhibited similar outcomes including survival and a lack of adverse events. These studies establish a baseline which can be used to further develop UDSC as a treatment for SM-induced airway disease.
呼吸系统损伤是接触包括硫芥（SM）在内的起疱剂的个体死亡的主要原因。尽管存在这些严重的健康并发症，但目前尚无针对此类损伤的实用治疗方法。先前的研究报告称，吸入SM会耗尽气管支气管气道上皮干细胞（TSC）库，并支持这样一种假说，即TSC替代将恢复气道上皮完整性并改善SM暴露个体的健康结果。TSC表达主要组织相容性复合体（MHC-I）移植抗原，这增加了异体TSC被患者免疫系统排斥的可能性。然而，先前的研究报告称，β2微球蛋白（B2M）基因敲除细胞缺乏细胞表面MHC-I，并表明B2M基因敲除的TSC作为异体移植物将被耐受。本研究使用Cas9核糖核蛋白（RNP）来生成B2M基因敲除的TSC，即通用供体干细胞（UDSC）。全基因组测序确定了很少的脱靶修饰，并证明了RNP方法的特异性。功能测定表明，UDSC保留了自我更新和进行多谱系分化的能力。使用SM吸入的临床前模型来测试UDSC的疗效，并确定任何与治疗相关的不良事件。成年雄性Sprague-Dawley大鼠吸入0.8 mg/kg SM蒸气剂量，这是攻击后第28天的吸入致死剂量。在恢复第2天，将赋形剂或异体Fisher大鼠UDSC静脉注射（每组n = 30）。每天记录临床参数，并在攻击后第7、14和28天进行计划安乐死。赋形剂和UDSC治疗组表现出相似的结果，包括存活率和无不良事件。这些研究建立了一个基线，可用于进一步开发UDSC作为治疗SM诱导的气道疾病的方法。
