Velcro AAV Vector for tissue-specific delivery of genome editing reagents with enhanced cargo capacity

项目摘要 基于核酸酶的体细胞基因组编辑，包括使用CRISPR/Cas9和DNA碱基编辑器的方法 (BE)是一项变革性技术，有可能治愈许多人类疾病。然而，要翻译 随着基因组编辑广泛应用于临床，对更安全，更有效的技术的需求尚未得到满足 将基因组编辑机器递送到体内疾病相关的体细胞和组织中。虽然Adeno- 相关病毒（AAV）载体能够在体内递送具有高度编辑的CRISPR/Cas9系统。 尽管它们具有有限的包装能力，缺乏靶向细胞/组织的特异性，并且可以诱导 由于Cas9的持续表达导致的遗传毒性和免疫应答。此外，大多数非病毒方法 使用全身施用体内递送CRISPR/Cas9仍然无效。解决这些 面对挑战，我们开发了Velcro AAV平台-插入亮氨酸拉链（LZ）的AAV载体 策略性地在衣壳表面上，使得载体生产和转导效率最低 受影响然后LZ衔接子可用于蛋白质到衣壳上的模块化和通用连接， 例如细胞靶向纳米抗体或肽以及基因组编辑试剂。我们的核心假设是 Velcro AAV将提供改善的细胞靶向特异性和增加的包装能力， 从而影响转导效率，使得能够在体内进行更安全和更稳健的体细胞基因组编辑。在阶段 1（UG 3），将构建Velcro AAV载体，表征和优化用于基于纳米抗体的免疫原性。 内皮靶向（Aim 1a）和Cas9/BE蛋白附着（Aim 2a）。纳米抗体/核酸酶的作用 对病毒滴度和转导效率的附着进行定量。小鼠研究将在 目的1b、2b和2c测试Velcro AAV载体特异性靶向内皮的能力， 增加了用于体内基因编辑的包装能力。靶向特异性和基因编辑效率 Velcro AAV载体将在第2阶段（UH 3）通过Aim 3中的猪研究进一步确定。如果成功， 拟议的研究将产生一种新的输送平台技术的强有力的临床前证明， 提供特异性细胞/组织靶向、更大的货物容量和瞬时核酸酶活性， 高效的体细胞基因组编辑。

PROJECT SUMMARY Nuclease-based somatic genome editing, including approaches that use CRISPR/Cas9 and DNA Base Editor (BE), is a transformative technology that has the potential to cure many human diseases. However, to translate genome editing into widespread clinical use, there is an unmet need for safer and more effective technologies to deliver genome editing machinery into disease-relevant somatic cells and tissues in vivo. Although Adeno- Associated Viral (AAV) vectors are capable of delivering CRISPR/Cas9 systems in vivo with high editing efficiency, they have limited packaging capacity, lack the specificity in targeting cells/tissues, and can induce genotoxicity and immune responses due to persistent expression of Cas9. Further, most nonviral methods for in vivo delivery of CRISPR/Cas9 using systemic administration remain ineffective. To address these challenges, we have developed the Velcro AAV platform – AAV vectors with Leucine Zippers (LZ) inserted strategically onto the capsid surface such that vector production and transduction efficiencies are minimally impacted. The LZ adaptors can then be used for modular and versatile attachment of proteins onto the capsid, such as cell-targeting nanobodies or peptides as well as genome editing reagents. Our central hypothesis is that Velcro AAVs will provide improved cell-targeting specificity and increased packaging capacity without affecting transduction efficiency, enabling safer and more robust somatic genome editing in vivo. During Phase 1 (UG3), Velcro AAV vectors will be constructed, characterized and optimized for nanobody-based endothelium-targeting (Aim 1a) and Cas9/BE protein attachment (Aim 2a). The effects of nanobody/nuclease attachment on viral titers and transduction efficiency will be quantified. Mouse studies will be carried out in Aims 1b, 2b and 2c to test the ability of Velcro AAV vectors to specifically target the endothelium with increased packaging capacity for gene editing in vivo. The targeting specificity and gene editing efficiency of Velcro AAV vectors will be further determined in Phase 2 (UH3) through pig studies in Aim 3. If successful, the proposed studies will yield strong preclinical demonstration of a new delivery platform technology that can provide specific cell/tissue targeting, larger cargo capacity, and transient nuclease activity, enabling safe and efficient somatic genome editing in humans.