A Novel Vector Strategy to Feasibilize Cellular Therapy for Heart Disease

摘要 最近，基于通过CAR-T技术对T细胞进行遗传重编程的细胞治疗的潜力已经被证实。 已经探索了心脏疾病，这种策略所体现的承诺已经赢得了流行的 和科学想象力。然而，在目前的临床CAR-T细胞治疗实践中，体外方法必须 这是限制更广泛地实施这一技术的主要时间和成本障碍。 因此，在患者体内原位产生CAR-T的稳健方法将极大地促进临床实践。 应用这种有前途的方法。为此，我们建议开发一种强大的基因递送系统， 具有多个T细胞嗜性腺相关病毒（AAV）的连接的“SAd. 临床上批准的T细胞嗜性猿猴（黑猩猩）腺病毒36（SAd）在其衣壳表面上。值得注意的是， captain工程化的SAd和AAV都能够选择性地靶向各种组织，包括具有高表达的T细胞。 因此，我们假设SAd.AAV可以通过以下途径提供上级靶向： 两种工程化病毒衣壳靶向相同组织或细胞上的不同受体的联合作用 类型.重要的是，携带单链敲入供体DNA模板的AAV已被广泛显示为 支持高编辑效率同源定向修复（HDR）。此外，强大但短暂的 通过SAd表达基因编辑器还提供了高度期望的“打了就跑”基因编辑减少潜力 与在实现目标编辑之后延长的编辑器表达相关联的不利影响。因此，我们认为， 由于每种病毒在基因编辑中的优势，SAd.AAV潜在地增加了组织靶向性， 这是一种新的CRISPR-Cas基因编辑疗法，具有特异性和效率，并提高了现有CRISPR-Cas基因编辑疗法的安全性。作为服务的证明 概念，在构建和表征第一个T细胞靶向的SAd.AAV后，我们计划实现高效的 用SAd.AAV基因编辑平台在体内产生心脏病特异性CAR-T细胞以敲入 体内T细胞中的可转换CARi转基因。体内生产的原理证明 基因编辑的可转换CARi-T细胞将为CARi-T干预的后续研究建立一个关键平台， 心脏病的小鼠模型。我们还预计，SAd.AAV可以迅速重新设计， 靶向与NHLBI相关的各种临床重要器官，用于基于基因编辑的治疗。

ABSTRACT Recently, the potential of cellular therapy based upon genetic reprogramming of T cells via CAR-T technology has been explored for diseases of the heart, and the promise embodied in this strategy has captured popular and scientific imagination. However, in current clinical CAR-T cell therapy practice, extracorporeal methods must be applied, which represent a major time and cost barrier restricting wider implementation of this technology. Thus, robust methods to generate CAR-T in situ, within the patients, would greatly facilitate the practical application of this promising approach. For this, we propose to develop a robust gene delivery system consisting of a conjoined “SAd.AAV” nano-vector with multiple T cell-tropic adeno-associated viruses (AAVs) conjugated to a clinically approved, T-cell tropic simian (chimpanzee) adenovirus 36 (SAd) on its capsid's surface. Of note, both capsid-engineered SAd and AAVs are able to selectively target various tissues including T cells with high transduction efficiencies, and we hypothesized that SAd.AAV may therefore provide superior targeting through the combined effects of both engineered viral capsids targeting different receptors on the same tissue or cell type. Importantly, AAVs carrying single-stranded knock-in donor DNA templates have been widely shown to support high editing efficiency of homology directed repair (HDR). Furthermore, the robust but transient expression of gene editor by SAd also provides highly desirable “hit-and-run” gene editing reducing potential adverse effects associated with prolonged editor expression after the on-target editing is achieved. Therefore, by virtue of the advantages of each virus in gene editing, SAd.AAV potentially increases the tissue targeting specificity and efficiency and improves safety of existing CRISPR-Cas gene editing therapies. As a proof of concept, after constructing and characterizing the first T cell-targeted SAd.AAV, we plan to achieve efficient production of the heart disease-specific CAR-T cells in vivo with an SAd.AAV gene editing platform to knock-in a switchable CARi transgene in the T cells in vivo. A proof-of-principle demonstration of in vivo production of gene-edited switchable CARi-T cells will establish a key platform for follow-on studies of CARi-T interventions in murine models of heart diseases. We furthermore anticipate that the SAd.AAV can be rapidly redesigned to target a wide variety of clinically important organs relevant to the NHLBI for gene editing-based therapies.