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A - Viral Vector Development -> AAV Vectors – Preclinical and Proof-of-Concept Studies

1: In vivo Selection of Randomly Integrated rAAV Vectors

Type: Oral Abstract Session

Presentation Details
Session Title: AAV Gene Therapy in Large Animal Models
Location: Room 204
Start Time: 5/16/2022 10:15
End Time: 5/16/2022 10:30

Recombinant adeno associated viruses (rAAVs) are a powerful tool for gene delivery. While historically thought to be non-integrating, it is now known that rAAVs can randomly integrate into the genome at low frequencies. Compared to episomal expression, these integrated vectors have the advantage of persisting through cell divisions. However, these integration events are typically too infrequent to result in therapeutic levels of expression of the delivered transgene. Our lab has previously demonstrated a strategy to expand a small population of hepatocytes that have the desired gene edit to therapeutic levels. This strategy utilizes a drug selectable cassette consisting of a Cytochrome P450 reductase (Cypor) targeted shRNA. Cypor is an obligate cofactor of Cytochrome P450 enzymes and is necessary for the metabolism of the drug acetaminophen to its hepatotoxic byproduct. In the absence of Cypor, acetaminophen cannot be metabolized, and hepatocytes that have lost Cypor are protected against high doses of acetaminophen. Due to the regenerative nature of hepatocytes, these acetaminophen-protected cells can expand to therapeutic levels. When the selectable shRNA is linked in cis with a therapeutic transgene, this method can selectively expand hepatocytes that have a stably integrated transgene. We hypothesized that this selection method could be used to expand rare random rAAV integration events to therapeutic levels. We delivered rAAV containing a U6-driven shRNA targeting Cypor and a CAG promoter driving human Factor IX (hFIX) into wildtype neonatal and adult mice. To expand the population of hepatocytes with the integrated rAAV, the mice were placed on an acetaminophen containing diet. Blood hFIX concentration increased over time up to 50-fold, readily reaching therapeutic levels. This demonstrates the expansion of hepatocytes expressing integrated copies of transgene. Immunofluorescent staining for Cypor confirmed the clonal expansion of Cypor-negative hepatocytes to 20% of the liver. Thus, we conclude that the random integration of conventional rAAV gene therapy vectors in combination with a selection cassette and regimen can be utilized to expand small populations of hepatocytes bearing chromosomal rAAV integrations to therapeutic levels.

Amita Tiyaboonchai, Anne Vonada, Markus Grompe

Oregon Health and Science University, Portland, OR
 A. Tiyaboonchai: None.

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