Biology of Subgenomic AAV Vector Particles

亚基因组 AAV 载体颗粒的生物学

• 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
• 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

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.

项目总结/摘要 血友病A是一种X连锁出血性疾病，由F8基因（编码凝血功能）的遗传缺陷引起 因子VIII，FVIII），其影响全世界约1/5000的男婴。临床上，血友病A是 表现为严重出血表型，通常需要昂贵的蛋白质替代疗法。近期 基因疗法的发展有望治愈血友病A。尽管临床试验的结果 使用AAV载体治疗血友病A一直令人鼓舞，腺相关病毒（AAV）介导的 FVIII尚未获得监管批准。主要障碍依然存在，因为人们担心， 犬长期研究中循环中FVIII水平降低以及克隆性肝细胞扩张 模型，这引起了人们对使用AAV载体用于人类时的遗传毒性甚至癌症形成的担忧。 基因治疗目前的AAV-FVIII基因递送策略（AAV-F8）遵循标准设计，其中微- 启动子用于驱动具有两个侧翼ITR的F8表达。尽管如此，临床级载体产生的 基于载体基因组的Southern印迹分析，它们是合理同源的。然而，我们的新研究， 使用单分子测序（SMRT），已经发现了载体群体的广泛复杂性， 使用传统的分析方法。一种值得注意的亚基因组载体颗粒含有 快速回复基因组（SBG），其可导致宿主细胞中的dsRNA产生。对于仅具有 启动子序列，它们冒着促进致癌通读的风险，这可能是一个实质性的 安全问题。该项目的中心假设是，宿主细胞等反式因子的组合， 蛋白质、病毒辅助功能和顺式元件如载体核苷酸组合物导致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 这对于开发具有减少/消除的SGP颗粒的新载体生产平台和用于 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 我们的目标是：1）：确定反式因子在亚基因组颗粒形成中的作用; 2）确定 载体基因组组成在亚基因组颗粒形成中的作用;和3）开发策略， 减少/消除亚基因组颗粒形成。