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

Velcro AAV Vector 用于基因组编辑试剂的组织特异性递送，具有增强的负载能力

基本信息

批准号：
9810928
负责人：
Gang Bao
金额：
$ 77.64万
依托单位：
RICE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-01 至 2022-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9810928
关键词：
Address Affect Animals Atherosclerosis Blood Vessels CRISPR/Cas technology Capsid Cardiovascular Diseases Cell surface Cells Clinical DNA Disease Endothelial Cells Endothelium Engineering Enzymes Eye Family suidae Fluorescence Genes Goals Growth Guide RNA Harvest Human Immune response Immunohistochemistry In Vitro Inflammation Intravenous Leucine Zippers Ligands Liver Macular degeneration Methods Monitor Morbidity - disease rate Mus Orthologous Gene Other Genetics PECAM1 gene Pathology Peptides Phase Production Proteins Reagent Reporter Reporter Genes Retinal Serotyping Signal Transduction Solid Neoplasm Somatic Cell Specificity Staphylococcus aureus Streptococcus pyogenes Surface System Technology Testing Time Tissues Tomatoes Transgenic Mice Translating Tropism Variant Vascular Diseases Viral Viral Genome Viral Vector Western Blotting adeno-associated viral vector base cell type clinical translation combinatorial deep sequencing design experimental study genome editing genotoxicity human disease improved in vivo in vivo evaluation intravenous administration mortality nanobodies nuclease nucleic acid delivery pre-clinical promoter somatic cell gene editing transduction efficiency tumor growth vector

项目摘要

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.

项目概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。不过，要翻译一下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传递到体内与疾病相关的体细胞和组织中。虽然腺- 相关病毒 (AAV) 载体能够在体内提供具有高编辑能力的 CRISPR/Cas9 系统效率，它们的包装能力有限，缺乏靶向细胞/组织的特异性，并且可以诱导 Cas9 持续表达导致的遗传毒性和免疫反应。此外，大多数非病毒方法使用全身给药的 CRISPR/Cas9 体内递送仍然无效。为了解决这些面对挑战，我们开发了 Velcro AAV 平台 – 插入亮氨酸拉链 (LZ) 的 AAV 载体战略性地转移到衣壳表面，使载体生产和转导效率降至最低受到影响。然后，LZ 适配器可用于将蛋白质模块化且多功能地附着到衣壳上，例如细胞靶向纳米抗体或肽以及基因组编辑试剂。我们的中心假设是 Velcro AAV 将提供改进的细胞靶向特异性和增加的包装容量，而无需影响转导效率，从而实现更安全、更稳健的体内体细胞基因组编辑。阶段期间 1 (UG3)，Velcro AAV 载体将针对基于纳米抗体的构建、表征和优化内皮靶向（目标 1a）和 Cas9/BE 蛋白附着（目标 2a）。纳米抗体/核酸酶的作用病毒滴度和转导效率的附着将被量化。小鼠研究将在目标 1b、2b 和 2c 测试 Velcro AAV 载体特异性靶向内皮细胞的能力增加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 (UH3) 通过目标 3 中的猪研究进一步确定。如果成功，拟议的研究将对新的递送平台技术产生强有力的临床前演示，该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，从而实现安全和对人类进行有效的体细胞基因组编辑。