Browsing by Subject "rAAV"

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    Adding recombinant AAVs to the cancer therapeutics mix
    (Elsevier, 2022-10-02) Mulcrone, Patrick L.; Herzog, Roland W.; Xiao, Weidong; Pediatrics, School of Medicine
    Gene therapy is a powerful biological tool that is reshaping therapeutic landscapes for several diseases. Researchers are using both non-viral and viral-based gene therapy methods with success in the lab and the clinic. In the cancer biology field, gene therapies are expanding treatment options and the possibility of favorable outcomes for patients. While cellular immunotherapies and oncolytic virotherapies have paved the way in cancer treatments based on genetic engineering, recombinant adeno-associated virus (rAAV), a viral-based module, is also emerging as a potential cancer therapeutic through its malleability, specificity, and broad application to common as well as rare tumor types, tumor microenvironments, and metastatic disease. A wide range of AAV serotypes, promoters, and transgenes have been successful at reducing tumor growth and burden in preclinical studies, suggesting more groundbreaking advances using rAAVs in cancer are on the horizon.
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    Cryptic resolution sites in the vector plasmid lead to the heterogeneities in the rAAV vectors
    (Wiley, 2023) Zhang, Junping; Chrzanowski, Matthew; Frabutt, Dylan A.; Lam, Anh K.; Mulcrone, Patrick L.; Li, Lei; Konkle, Barbara A.; Miao, Carol H.; Xiao, Weidong; Pediatrics, School of Medicine
    Recombinant adeno-associated virus (rAAV) vectors carry a cassette of interest retaining only the inverted terminal repeats (ITRs) from the wild-type virus. Conventional rAAV production primarily uses a vector plasmid as well as helper genes essential for AAV replication and packaging. Nevertheless, plasmid backbone related contaminants have been a major source of vector heterogeneity. The mechanism driving the contamination phenomenon has yet to be elucidated. Here we identified cryptic resolution sites in the plasmid backbone as a key source for producing snapback genomes, which leads to the increase of vector genome heterogeneity in encapsidated virions. By using a single ITR plasmid as a model molecule and mapping subgenomic particles, we found that there exist a few typical DNA break hotspots in the vector DNA plasmid backbone, for example, on the ampicillin DNA element, called aberrant rescue sites. DNA around these specific breakage sites may assume some typical secondary structures. Similar to normal AAV vectors, plasmid DNA with a single ITR was able to rescue and replicate efficiently. These subgenomic DNA species significantly compete for trans factors required for rAAV rescue, replication, and packaging. The replication of single ITR contaminants during AAV production is independent of size. Packaging of these species is greatly affected by its size. A single ITR and a cryptic resolution site in the plasmid work synergistically, likely causing a source of plasmid backbone contamination.