A Novel Vector Platform to Actualize T Cell Modification In Vivo

摘要 为了解决对改进的基因编辑递送系统的需求，我们提出构建嵌合的“AdAAV”。 载体由腺病毒（Ad）和与其衣壳缀合的多个腺相关病毒（AAV）组成， 面我们计划采用SpyTag/SpyCatcher技术将AAV缀合到Ad衣壳上。值得注意的 在这方面，Ads能够选择性地靶向某些组织（例如在huAd 5的情况下的肝脏），其具有高表达。 转导效率。此外，我们的小组已经能够设计Ad纤维，以促进组织特异性 靶向T细胞。此外，通过选择合适的AAV血清型，和/或通过衣壳工程改造， AAV也可以选择性地靶向所需的组织。因此，AdAAV可提供上级靶向 通过工程化的Ad纤维和AAV衣壳（其匹配以靶向相同的腺病毒载体）两者的组合作用， 组织类型）。作为概念验证，我们计划用AdAAV靶向T细胞。此外，由于AAVs携带 单链DNA，它们可以提供单链供体模板，这是已知的增强编辑 同源定向修复（HDR）的效率。在AdAAV内，Cas蛋白可由Ad AAV编码。 单链DNA模板可以包含在AAV的基因组内，而单链DNA模板可以包含在AAV的基因组内。凭借 用于HDR的单链供体模板的优点，以及由于供体模板的高拷贝数， 模板由与每个广告相关联的多个AAV携带，这种设计可能会大大增加编辑 效率最后，AdAAV将具有大的包装容量，因为它将由以下各项的总和组成： 广告的容量（已经很高了）和AAV的容量。基于这些因素，我们认为AdAAV 可能形成一种强大而通用的新的基因编辑疗法递送系统， 与现有方法相关的局限性。我们高度原创的AdAAV输送系统将大大提高 现有的CRISPR-Cas基因编辑疗法的多功能性，通过规避其应用的几个关键障碍， 更广泛的适用性。AdAAV的设计特征将潜在地使其成为一种理想的载体， 基因编辑交付的挑战，从而广泛增强了基于基因编辑的 治疗

ABSTRACT To address the need for improved gene editing delivery systems, we propose constructing a chimeric “AdAAV” vector consisting of an adenovirus (Ad) with multiple adeno-associated viruses (AAVs) conjugated to its capsid's surface. We plan to employ the SpyTag/SpyCatcher technology to conjugate AAVs onto the Ad capsid. Of note in this regard, Ads are able to selectively target certain tissues (such as the liver in the case of huAd5) with high transduction efficiencies. Furthermore, our group has been able to engineer Ad fibers to facilitate tissue-specific targeting of T cells. In addition, by choosing an appropriate AAV serotype, and/or through capsid engineering AAVs can also selectively target desired tissues as well. AdAAVs may therefore provide superior targeting through the combined effects of both engineered Ad fiber and AAV capsid (which are matched to target the same tissue type). As a proof-of-concept, we plan to target T cells with AdAAVs. In addition, because AAVs carry single-stranded DNA, they can provide single-stranded donor templates, which are known to enhance the editing efficiency of homology directed repair (HDR). Within an AdAAV, the Cas protein can be encoded by the Ad genome while the single-stranded DNA template can be embodied within the genome of the AAVs. By virtue of the advantages of single-stranded donor templates for HDR, and by virtue of the high copy number of donor templates carried by the multiple AAVs associated with each Ad, this design might substantially increase editing efficiency. Finally, the AdAAV would possess a large packaging capacity since it would consist of a sum of the Ad's capacity (which is already high) and the AAV's capacity. Due to these factors, we suggest that AdAAVs may form a powerful and versatile new delivery system for gene editing therapies which overcomes many of the limitations associated with existing approaches. Our highly original AdAAV delivery system will greatly enhance the versatility of existing CRISPR-Cas gene editing therapies by circumventing several key obstacles to their broader applicability. The design features of AdAAV will potentially make it an ideal vector by which to address the challenges of gene editing delivery and thereby broadly enhance the general feasibility of gene editing-based therapies.