Biology of Subgenomic AAV Vector Particles

亚基因组 AAV 载体颗粒的生物学

基本信息

批准号：
10560550
负责人：
WEIDONG XIAO
金额：
$ 51.56万
依托单位：
INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-02-05 至 2027-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10560550
关键词：
Adenoviruses Affect Back Bioinformatics Biology Birth Blood Coagulation Disorders Cells Circulation Clinical Clinical Trials Clustered Regularly Interspaced Short Palindromic Repeats DNA DNA lesion Data Analyses Defect Dependovirus Development Double-Stranded RNA Elements F8 gene Factor VIII Gene Delivery Generations Genes Genome Goals Hemophilia A Hemorrhage Human Immune response Inherited Integration Host Factors Knowledge Lead Lesion Libraries Ligase Ligation Link Longitudinal Studies Malignant Neoplasms Mediating Methods Modeling Molecular Nonhomologous DNA End Joining Nucleotides Oncogenic Outcome Pathway interactions Phenotype Population Process Production Promoter Regions Proteins Quality Control Reaction Recombinant adeno-associated virus (rAAV)Research Risk Role Running Safety Southern Blotting System Viral Viral Genome Viral Packaging Virus Replication XRCC5 gene adeno-associated viral vector biological adaptation to stress canine model cost design enzyme replacement therapy gene therapy genotoxicity hazard male next generation novel particle promoter single molecule tool vector vector genome

项目摘要

PROJECT SUMMARY/ABSTRACT Hemophilia A is an X-linked bleeding disorder caused by hereditary defects in the F8 gene (encoding coagulation factor VIII, FVIII), which affects approximately 1 in 5000 male births worldwide. Clinically, hemophilia A is manifested as a severe bleeding phenotype that typically requires costly protein replacement therapy. The recent development of a gene therapy holds the promise of curing hemophilia A. Although the outcome of clinical trials using AAV vectors for hemophilia A has been encouraging, Adeno-associated virus (AAV)-mediated delivery of FVIII has not yet received regulatory approval. Major hurdles persist since there are concerns on gradually decreasing levels of FVIII in circulation as well as clonal hepatocellular expansion in long-term studies in a canine model, which raises concerns over genotoxicity or even cancer formation when using AAV vectors for human gene therapy. Current AAV-FVIII gene delivery strategies (AAV-F8) follow a standard design, in which a mini- promoter is used to drive F8 expression with two flanking ITRs. Nevertheless, clinical-grade vectors produced were reasonably homogenous based on Southern blot analysis of vector genomes. Our new studies however, using single molecule sequencing (SMRT), have uncovered a broad complexity of vector population that has been missed using conventional methods of analysis. One notable subgenomic vector particle contains the snapback genome (SBG), which may lead to dsRNA production in the host cells. For other SBGs having only the promoter sequences, they run the risk of promoting oncogenic readthrough, which may be a substantial safety concern. The central hypothesis of this project is that the combination of trans factors such as host cellular proteins, viral helper functions and cis elements such as vector nucleotide compositions lead to SGP formation. Therefore, a comprehensive mechanistic understanding of how these factors mediate SGP formation will be essential for the development of new a vector production platform with reduced/eliminated SGP particles and for the design of strategies to control and manage the potential hazards of SGP. To achieve these goals, we will pursue the following aims: 1): To define the roles of trans factors in subgenomic particle formation; 2) to define the role of vector genome composition in subgenomic particle formation; and 3) to develop strategies to reduce/eliminate subgenomic particle formation.

项目概要/摘要 A 型血友病是一种 X 连锁出血性疾病，由 F8 基因（编码凝血功能）的遗传性缺陷引起。因子 VIII (FVIII)，影响全球大约五千分之一的男性新生儿。临床上，A型血友病是表现为严重的出血表型，通常需要昂贵的蛋白质替代疗法。最近的基因疗法的发展有望治愈甲型血友病。尽管临床试验的结果使用 AAV 载体治疗 A 型血友病一直令人鼓舞，腺相关病毒 (AAV) 介导的递送 FVIII 尚未获得监管部门的批准。主要障碍依然存在，因为人们逐渐担心在犬科动物的长期研究中，循环中 FVIII 水平降低以及克隆性肝细胞扩张模型，这引起了对人类使用 AAV 载体时的遗传毒性甚至癌症形成的担忧基因疗法。目前的 AAV-FVIII 基因传递策略 (AAV-F8) 遵循标准设计，其中一个微型启动子用于驱动带有两个侧翼 ITR 的 F8 表达。尽管如此，产生了临床级载体根据载体基因组的 Southern blot 分析，它们具有相当的同质性。然而，我们的新研究，使用单分子测序（SMRT），揭示了载体群体的广泛复杂性使用传统的分析方法会被遗漏。一种著名的亚基因组载体粒子包含折回基因组 (SBG)，这可能导致宿主细胞中 dsRNA 的产生。对于其他 SBG 仅具有启动子序列，它们存在促进致癌性通读的风险，这可能是一个重大的安全问题。该项目的中心假设是反式因子（例如宿主细胞）的组合蛋白质、病毒辅助功能和顺式元件（例如载体核苷酸组成）导致 SGP 形成。因此，对这些因素如何介导 SGP 形成的全面机制理解将是对于开发减少/消除 SGP 颗粒的新载体生产平台以及设计控制和管理 SGP 潜在危害的策略。为了实现这些目标，我们将追求以下目标：1）：定义反式因子在亚基因组颗粒形成中的作用； 2）定义载体基因组组成在亚基因组颗粒形成中的作用； 3) 制定策略减少/消除亚基因组颗粒的形成。