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：
10231050
负责人：
Gang Bao
金额：
$ 76.25万
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 2023-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10231050
关键词：
Address Affect Animals Atherosclerosis Blood Vessels CRISPR/Cas technology Capsid Cardiovascular Diseases Cell surface Cells Clinical Cre driver 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 Retina 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.

项目摘要基于核酸酶的体细胞基因组编辑，包括使用CRISPR/CAS9和DNA基础编辑器的方法（BE）是一种具有变革性的技术，具有治愈许多人类疾病的潜力。但是，要翻译基因组编辑为宽度临床使用，对更安全，更有效的技术的需求未满足在体内将基因组编辑机械输送到与疾病相关的体细胞和组织中。虽然adeno- 相关病毒（AAV）向量能够在体内传递CRISPR/CAS9系统，并具有高编辑效率，它们的包装能力有限，缺乏靶向细胞/组织的特异性，并且可以诱导由于cas9的持续表达，遗传毒性和免疫复杂。此外，大多数非病毒方法使用系统给药在体内交付CRISPR/CAS9仍然无效。解决这些挑战，我们已经开发了Velcro AAV平台 - 带有亮氨酸拉链（LZ）的AAV矢量从策略性地进入衣壳表面，使矢量产生和翻译效率最低受影响。然后，LZ适配器可用于蛋白质的模块化和多功能附件，例如靶向细胞的纳米化或肽以及基因组编辑试剂。我们的中心假设是魔术贴AAV将提供改进的细胞靶向特异性，并提高包装能力，而无需影响转移效率，使体内更安全，更健壮的体细胞基因组编辑。在阶段 1（ug3），将构建，对基于纳米的魔术贴，特征和优化内皮靶向（AIM 1A）和CAS9/BE蛋白质附着（AIM 2A）。纳米/核酸酶的作用将量化对病毒滴度和转移效率的附件。小鼠研究将在目标1b，2b和2c测试魔术贴量矢量专门针对内皮的能力体内基因编辑的包装能力增加。靶向特异性和基因编辑效率 Velcro AAV载体将通过AIM 3中的Pig研究进一步确定。拟议的研究将对新的交付平台技术产生强烈的临床前证明，该技术可以提供特定的细胞/组织靶向，较大的货物容量和瞬时核酸酶活性，使得安全和在人类中有效的体细胞基因组编辑。

项目成果

期刊论文数量（7）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Identification and Validation of CRISPR/Cas9 Off-Target Activity in Hematopoietic Stem and Progenitor Cells.

造血干细胞和祖细胞中 CRISPR/Cas9 脱靶活性的鉴定和验证。

DOI：
10.1007/978-1-0716-1979-7_19
发表时间：
2022
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
0
作者：
Park,SoHyun;Lee,CiaranM;Bao,Gang
通讯作者：
Bao,Gang

Site-Specific Post-translational Surface Modification of Adeno-Associated Virus Vectors Using Leucine Zippers.

DOI：
10.1021/acssynbio.9b00341
发表时间：
2020-02
期刊：
ACS synthetic biology
影响因子：
4.7
作者：
Nicole N. Thadani;Joanna Yang;Buhle Moyo;Ciaran M. Lee;Maria Y. Chen;Gang Bao;J. Suh
通讯作者：
Nicole N. Thadani;Joanna Yang;Buhle Moyo;Ciaran M. Lee;Maria Y. Chen;Gang Bao;J. Suh

Magnetic Forces Enable Control of Biological Processes In Vivo.

磁力能够控制体内的生物过程。