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

项目总结 基于核酸酶的体细胞基因组编辑，包括使用CRISPR/Cas9和DNA Base Editor的方法 (BE)，是一种具有变革性的技术，有可能治愈许多人类疾病。然而，要翻译成 基因组编辑进入广泛的临床应用，对更安全、更有效的技术的需求尚未得到满足 将基因组编辑机制输送到体内与疾病相关的体细胞和组织中。尽管腺- 相关病毒(AAV)载体能够以高度编辑的方式在体内传递CRISPR/Cas9系统 效率，它们包装能力有限，缺乏靶向细胞/组织的特异性，并可诱导 持续表达Cas9的遗传毒性和免疫反应。此外，大多数非病毒治疗方法 采用全身给药的CRISPR/Cas9体内给药仍然无效。要解决这些问题 挑战，我们开发了魔术贴AAV平台-插入亮氨酸拉链(LZ)的AAV载体 战略性地转移到衣壳表面，以便将载体的生产和转导效率降至最低 受到了影响。然后LZ接头可用于将蛋白质模块化和多功能地附着到衣壳上， 如细胞靶向纳米体或多肽以及基因组编辑试剂。我们的中心假设是 尼龙搭扣AAV将提供更好的细胞靶向特异性和更大的包装能力，而不需要 影响转导效率，实现更安全和更强大的体内体细胞基因组编辑。在阶段中 1(UG3)，将构建、表征和优化基于纳米体的魔术贴AAV载体 内皮靶向(Aim 1a)和Cas9/BE蛋白结合(Aim 2a)。纳米体/核酸酶的作用 病毒滴度和转导效率的附着将被量化。老鼠研究将在#年进行。 目标1b、2b和2c测试魔术贴AAV载体针对内皮细胞的能力 增加了体内基因编辑的包装能力。靶向特异性和基因编辑效率 魔术贴AAV载体将在第二阶段(UH3)通过AIM 3中的猪研究进一步确定。如果成功， 拟议的研究将产生强有力的临床前演示，证明一种新的交付平台技术可以 提供特定的细胞/组织靶向、更大的货物容量和瞬时核酸酶活性，从而实现安全和 人类的高效体细胞基因组编辑。

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.