In vivo CRISPR engineering of B cells to produce anti-HIV broadly neutralizing antibodies using novel nanoparticles

项目总结/摘要 艾滋病毒爆发至今已近半个世纪，但寻求治愈艾滋病毒的努力仍未完成。 抗逆转录病毒药物鸡尾酒可以抑制艾滋病毒感染，但由于副作用， 以及访问和遵守方面的限制。注射广泛中和抗体（bNAb）， 预防艾滋病毒反弹已经取得了一些成功，但需要定期重新注射多种抗体， 保持抑制和病毒逃逸。因此，费用和持续获取仍然是限制因素。遗传 已经提出了患者细胞的工程化以克服所有这些不足，并且可以构成 如果成功，一次性治疗具有终身治疗价值。在这个提案中，我们利用一本小说 贾斯汀·泰勒博士的实验室开发的一种方法，通过基因工程改造B细胞来表达bNAb 用于治疗人类免疫缺陷病毒（HIV）。这一战略已经被用于 改造B细胞以产生针对流感病毒、呼吸道合胞病毒 EB病毒和HIV [Moffett et al.，Science Immunology，2019]。虽然这种方法可以确保 保护性抗体的生产，基因工程过程需要10天的复杂的前处理， 体内生产，不能广泛分销。为了克服这些障碍，我们将增选一本小说， 詹妮弗·阿代尔博士实验室开发的合成纳米颗粒， 在单一的被动步骤中进行工程化[Shahbazi等人，Nature Materials，2019].我们表明，这 纳米粒子可以在不到一天的时间内组装起来，用于对未受刺激的原始人类进行基因工程改造。 血细胞，并且可以被修饰以在体内特异性地与靶血细胞类型相互作用。这里我们将 开发这种可扩展的纳米制剂作为疫苗样的体内递送系统， 在HIV感染的临床相关非人灵长类动物模型中使用多种bNAb的免疫。我们将 使用这些纳米颗粒直接对天然原代B细胞亚型进行基因工程改造， 造血干细胞和祖细胞，可以提供终身补充抗体- 产生B细胞。这项研究不仅将开发出一套独特的抗艾滋病毒工具， 公平分配基因编辑疗法的变革性进展。

PROJECT SUMMARY / ABSTRACT The quest for an HIV cure remains incomplete, nearly half a century since the onset of the epidemic. Antiretroviral drug cocktails can suppress HIV infection, but suffer in their success owing to side effects and limitations in access and compliance. Injection of broadly neutralizing antibodies (bNAbs) to prevent HIV rebound has had some success, but requires regular re-injection of multiple antibodies to maintain suppression and viral escape. Thus, cost and continued access remain limitations. Genetic engineering of patient cells has been proposed to overcome all of these shortfalls, and could constitute a one-time treatment with lifelong therapeutic value if successful. In this proposal, we leverage a novel approach developed by Dr. Justin Taylor’s laboratory to genetically engineer B cells to express bNAbs for the treatment of human immunodeficiency virus (HIV). This strategy has already been used to engineer B cells to produce antibodies protective against influenza virus, respiratory syncytial virus, Epstein-barr virus and HIV [Moffett et al., Science Immunology, 2019]. While this approach can ensure protective antibody production, the genetic engineering process required 10 days of complicated ex vivo manufacturing and is not broadly distributable. To overcome these barriers, we will co-opt a novel, synthetic nanoparticle that was developed in Dr. Jennifer Adair’s laboratory to deliver genetic engineering in a single, passive step [Shahbazi et al., Nature Materials, 2019]. We show that this nanoparticle can be assembled in less than a day to genetically engineer unstimulated, primary human blood cells and can be modified to specifically interact with target blood cell types in vivo. Here we will develop this scalable nanoformulation as a vaccine-like in vivo delivery system to direct humoral immunity with multiple bNAbs in a clinically-relevant nonhuman primate model of HIV infection. We will use these nanoparticles to directly genetically engineer native primary B cell subtypes, and hematopoietic stem and progenitor cells, which can provide lifelong replenishment of antibody- producing B cells. This research will not only develop a unique tool set against HIV but will provide transformative advances in equitable distribution of gene editing therapies.