Browsing by Author "Frabutt, Dylan A."

Now showing 1 - 5 of 5
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    A novel class of self-complementary AAV vectors with multiple advantages based on cceAAV lacking mutant ITR
    (Elsevier, 2024-02-03) Zhang, Junping; Frabutt, Dylan A.; Chrzanowski, Matthew; Li, Ning; Miller, Lohra M.; Tian, Jiahe; Mulcrone, Patrick L.; Lam, Anh K.; Draper, Benjamin E.; Jarrold, Martin F.; Herzog, Roland W.; Xiao, Weidong; Pediatrics, School of Medicine
    Self-complementary AAV vectors (scAAV) use a mutant inverted terminal repeat (mITR) for efficient packaging of complementary stranded DNA, enabling rapid transgene expression. However, inefficient resolution at the mITR leads to the packaging of monomeric or subgenomic AAV genomes. These noncanonical particles reduce transgene expression and may affect the safety of gene transfer. To address these issues, we have developed a novel class of scAAV vectors called covalently closed-end double-stranded AAV (cceAAV) that eliminate the mITR resolution step during production. Instead of using a mutant ITR, we used a 56-bp recognition sequence of protelomerase (TelN) to covalently join the top and bottom strands, allowing the vector to be generated with just a single ITR. To produce cceAAV vectors, the vector plasmid is initially digested with TelN, purified, and then subjected to a standard triple-plasmid transfection protocol followed by traditional AAV vector purification procedures. Such cceAAV vectors demonstrate yields comparable to scAAV vectors. Notably, we observed enhanced transgene expression as compared to traditional scAAV vectors. The treatment of mice with hemophilia B with cceAAV-FIX resulted in significantly enhanced long-term FIX expression. The cceAAV vectors hold several advantages over scAAV vectors, potentially leading to the development of improved human gene therapy drugs.
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    Chemical Modifications of the Capsid for Redirecting and Improving the Efficacy of Adeno-Associated Virus Vectors
    (Mary Ann Liebert, 2021-12) Lam, Anh Kim; Frabutt, Dylan A.; Li, Lei; Xiao, Weidong; Pediatrics, School of Medicine
    Adeno-associated virus (AAV) vector-directed gene therapy is one of the most exciting modalities of biotechnology as more applications enter clinical stage. Although AAV vectors generally feature low toxicity, high stability, and long-lasting transgene expression, potential challenging issues of AAV include high vector dose, limited tissue tropism, and the host immune response and inflammation, which are all related to the capsid protein. To overcome these challenges, various strategies have been developed to engineer AAV capsids. Apart from widely employed genetic engineering of capsid protein, powerful and versatile chemical modification strategies are underexploited. This minireview summarizes recent advances and our perspectives for future direction in AAV capsid chemical modification to enhance its therapeutic use for gene therapy.
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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.
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    Genomic Designs of rAAVs Contribute to Pathological Changes in the Livers and Spleens of Mice
    (Wiley, 2022) Mulcrone, Patrick L.; Zhang, Junping; Pride, P. Melanie; Lam, Anh K.; Frabutt, Dylan A.; Ball-Kell, Susan M.; Xiao, Weidong; Pediatrics, School of Medicine
    Recombinant AAV (rAAV) gene therapy is being investigated as an effective therapy for several diseases including hemophilia B. Reports of liver tumor development in certain mouse models due to AAV treatment and genomic integration of the rAAV vector has raised concerns about the long-term safety and efficacy of this gene therapy. To investigate whether rAAV treatment causes cancer, we utilized two mouse models, inbred C57BL/6 and hemophilia B Balb/C mice (HemB), to test if injecting a high dose of various rAAV8 vectors containing or lacking hFIX transgene, a Poly-A sequence, or the CB or TTR promoter triggered liver fibrosis and/or cancer development over the course of the 6.5-month study. We observed no liver tumors in either mouse cohort regardless of rAAV treatment through ultrasound imaging, gross anatomical assessment at sacrifice, and histology. We did, however, detect differences in collagen deposition in C57BL/6 livers and HemB spleens of rAAV-injected mice. Pathology reports of the HemB mice revealed many pathological phenomena, including fibrosis and inflammation in the livers and spleens across different AAV-injected HemB mice. Mice from both cohorts injected with the TTR-hFIX vector demonstrated minimal adverse events. While not tumorigenic, high dose of rAAVs, especially those with incomplete genomes, can influence liver and spleen health negatively that could be problematic for cementing AAVs as a broad therapeutic option in the clinic.
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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.