A Novel Vector Strategy to Feasibilize Cellular Therapy for Heart Disease

摘要 最近，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 已经被探索用于心脏疾病，这一战略中体现的承诺已经赢得了人们的欢迎 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 这是制约这项技术更广泛实施的主要时间和成本障碍。 因此，在患者体内原位生成CAR-T的稳健方法将极大地促进实用 这一前景看好的方法的应用。为此，我们建议开发一种健壮的基因递送系统，包括 连接多个嗜T细胞腺相关病毒(AAVs)的SAd.AAV纳米载体的构建 一种临床批准的嗜T细胞猿猴(黑猩猩)腺病毒36(SAD)，位于其衣壳表面。值得注意的是， 衣壳工程设计的SAD和AAVs都能够选择性地靶向各种组织，包括高水平的T细胞 转导效率，我们假设SAd.AAV因此可能通过 两种针对同一组织或细胞上不同受体的工程化病毒衣壳的联合作用 打字。重要的是，携带单链敲入供体DNA模板的AAVs已被广泛显示为 支持高编辑效率的同源定向修复(HDR)。此外，健壮但短暂的 通过SAD表达的基因编辑也提供了非常理想的降低潜力的“肇事逃逸”基因编辑 在实现目标编辑之后，与长时间的编辑者表达相关联的不利影响。因此， 由于每种病毒在基因编辑方面的优势，SAd.AAV潜在地增加了组织靶向性 提高了现有CRISPR-Cas基因编辑疗法的特异性和效率，并提高了安全性。作为证明 概念，在构建并鉴定了第一个T细胞靶向SAd.AAV之后，我们计划实现高效 用SAd.AAV基因编辑平台敲入体内生产心脏病特异性CAR-T细胞 体内T细胞内可切换的CARI转基因。活体生产的原则性论证 基因编辑的可切换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，以 针对与NHLBI相关的广泛的临床重要器官进行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.