Biology of Subgenomic AAV Vector Particles

亚基因组 AAV 载体颗粒的生物学

• 批准号: 10333190
• 负责人: WEIDONG XIAO
• 金额: $ 52.11万
• 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/10333190
• 关键词: Adenoviruses; Affect; Bioinformatics; Biology; Birth; Blood Circulation; Blood Coagulation Disorders; Cells; 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; base; 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/文摘