A Novel Vector Platform to Actualize T Cell Modification In Vivo

摘要 为了满足对改进的基因编辑传递系统的需求，我们建议构建嵌合的“AdAAV” 由多个腺相关病毒(AAV)与其衣壳连接的腺病毒(Ad)组成的载体 浮出水面。我们计划使用SpyTag/SpyCatcher技术将AAV连接到广告衣壳上。值得注意的是 在这一点上，ADS能够选择性地靶向某些组织(例如在huAd5的情况下是肝脏)。 转导效率。此外，我们的团队已经能够通过改造Ad纤维来促进组织特异性 靶向T细胞。此外，通过选择适当的AAV血清型，和/或通过衣壳工程 AAVs还可以选择性地靶向所需的组织。因此，AdAAV可以提供更好的靶向 通过工程化的Ad纤维和AAV衣壳(它们匹配成相同的靶标)的组合作用 组织类型)。作为概念验证，我们计划用AdAAVs靶向T细胞。此外，由于AAVs携带 单链DNA，他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中，Cas蛋白可以由Ad 而单链DNA模板可以包含在AAVs的基因组中。凭借以下优势 HDR的单链供体模板的优点，以及供体拷贝数高的优点 由与每个广告相关联的多个AAV携带的模板，这种设计可能会大大增加编辑 效率。最后，AdAAV将拥有很大的包装容量，因为它将由 AD的容量(已经很高)和AAV的容量。鉴于这些因素，我们建议AdAAVs 可能会形成一种强大和多功能的新的基因编辑疗法的输送系统，它克服了许多 与现有方法相关的限制。我们独创的AdAAV传送系统将大大增强 现有CRISPR-Cas基因编辑疗法的多功能性，绕过了其 更广泛的适用性。AdAAV的设计特性可能使其成为一种理想的解决方案 基因编辑交付的挑战，从而广泛增强了基于基因编辑的一般可行性 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.