Browsing by Author "Firrman, Jenni"

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    Satellite Subgenomic Particles Are Key Regulators of Adeno-Associated Virus Life Cycle
    (MDPI, 2021-06-21) Zhang, Junping; Yu, Xiangping; Guo, Ping; Firrman, Jenni; Pouchnik, Derek; Diao, Yong; Samulski, Richard Jude; Xiao, Weidong; Pediatrics, School of Medicine
    Historically, adeno-associated virus (AAV)-defective interfering particles (DI) were known as abnormal virions arising from natural replication and encapsidation errors. Through single virion genome analysis, we revealed that a major category of DI particles contains a double-stranded DNA genome in a "snapback" configuration. The 5'- snapback genomes (SBGs) include the P5 promoters and partial rep gene sequences. The 3'-SBGs contains the capsid region. The molecular configuration of 5'-SBGs theoretically may allow double-stranded RNA transcription in their dimer configuration. Our studies demonstrated that 5-SBG regulated AAV rep expression and improved AAV packaging. In contrast, 3'-SBGs at its dimer configuration increased levels of cap protein. The generation and accumulation of 5'-SBGs and 3'-SBGs appears to be coordinated to balance the viral gene expression level. Therefore, the functions of 5'-SBGs and 3'-SBGs may help maximize the yield of AAV progenies. We postulate that AAV virus population behaved as a colony and utilizes its subgenomic particles to overcome the size limit of a viral genome and encodes additional essential functions.
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    Subgenomic particles in rAAV vectors result from DNA lesion/break and non-homologous end joining of vector genomes
    (Elsevier, 2022-08-24) Zhang, Junping; Guo, Ping; Yu, Xiangping; Frabutt, Dylan A.; Lam, Anh K.; Mulcrone, Patrick L.; Chrzanowski, Matthew; Firrman, Jenni; Pouchnik, Derek; Sang, Nianli; Diao, Yong; Herzog, Roland W.; Xiao, Weidong; Pediatrics, School of Medicine
    Recombinant adeno-associated virus (rAAV) vectors have been developed for therapeutic treatment of genetic diseases. Current rAAV vectors administered to affected individuals often contain vector DNA-related contaminants. Here we present a thorough molecular analysis of the configuration of non-standard AAV genomes generated during rAAV production using single-molecule sequencing. In addition to the sub-vector genomic-size particles containing incomplete AAV genomes, our results showed that rAAV preparations were contaminated with multiple categories of subgenomic particles with a snapback genome (SBG) configuration or a vector genome with deletions. Through CRISPR and nuclease-based modeling in tissue culture cells, we identified that a potential mechanism leading to formation of non-canonical genome particles occurred through non-homologous end joining of fragmented vector genomes caused by genome lesions or DNA breaks present in the host cells. The results of this study advance our understanding of AAV vectors and provide new clues for improving vector efficiency and safety profiles for use in human gene therapy.