In vitro NIH3T3 mouse embryonic fibroblast cell model does not predict AAV2 or AAVdj-mediated cell transformation

Area of Science:

• Biotechnology
• Gene Therapy
• Oncology

Background:

• Recombinant adeno-associated virus (rAAV) gene therapy carries a risk of insertional mutagenesis.
• This mutagenesis has been linked to hepatocellular carcinoma (HCC) development in rAAV-treated mice.

Purpose of the Study:

• To determine if in vitro cell transformation assays (CTA) can detect AAV2 or AAVdj-mediated cell transformation.
• To assess the predictive value of in vitro assays for in vivo rAAV-induced HCC.

Main Methods:

• Utilized NIH3T3 mouse cell lines for in vitro cell transformation assays.
• Employed CRISPR-Cas9 to create double-strand breaks at the Rian locus, enhancing AAV DNA integration.
• Tested rAAV vectors targeting the Rian locus and a previously identified HCC-inducing vector.

Main Results:

• No significant difference in transformation frequency was observed between AAV-transduced NIH3T3 cells and untreated control cells.
• In vitro cell transformation assays did not detect AAV-mediated cell transformation in NIH3T3 cells.

Conclusions:

• In vitro cell transformation assays may not be predictive of rAAV-induced hepatocellular carcinoma in mice.
• The study suggests that either the transformation rate is below spontaneous levels or in vitro assays lack predictive power for rAAV gene therapy risks.