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Assessing the biopotency of the rAAV9 vector In Vitro.
Pratikshya Adhikari1, Peter G Nichols1, Stephen M Vorobiov1
1Center for Molecular Medicine, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America.
Plos One
|February 26, 2026
Summary
A new cell-based potency assay using HuH-7 cells reliably measures the effectiveness of adeno-associated virus serotype 9 (AAV9) gene therapy vectors. This assay supports the quality and consistency of AAV9 gene therapy products.
Area of Science:
- Gene Therapy Vector Development
- Biotechnology and Bioprocessing
- Molecular and Cellular Biology
Background:
- Recombinant Adeno-associated Virus (AAV) gene therapy vectors require robust potency assays for clinical applications.
- AAV serotype 9 (AAV9) is a promising vector for neurogenetic diseases due to its neurotropism and blood-brain barrier penetration.
- Existing AAV9 potency assays face challenges with commonly used cell lines, necessitating alternative methods.
Purpose of the Study:
- To develop and validate a cell-based potency assay for self-complementary AAV9 vectors expressing human N-sulfoglucosamine sulfohydrolase (hSGSH).
- To evaluate the suitability of the human hepatoma (HuH-7) cell line for assessing AAV9 vector infection and transgene expression.
- To confirm the reliability, reproducibility, and stability of the developed AAV9 potency assay.
Main Methods:
- A cell-based potency assay was established using the HuH-7 cell line to evaluate infection by scAAV9-hSGSH vectors.
- Transgene expression (SGSH production) and vector genome copies were quantified in transduced HuH-7 cells.
- Correlation between vector dose and biological response (SGSH expression, genome copies) was analyzed; assay reproducibility and vector stability under long-term storage were assessed.
Main Results:
- HuH-7 cells reproducibly expressed the hSGSH transgene upon infection with scAAV9 vectors, leading to measurable SGSH production.
- SGSH expression and vector genome copies showed a strong linear correlation with the viral vector dose (R² = 0.71-0.95).
- The assay demonstrated high reliability and reproducibility, with intra-assay and inter-assay coefficients of variation below 20% and 25%, respectively; long-term storage (2.5 years at 2-4°C) did not impact vector biopotency.
Conclusions:
- A practical and reliable in vitro cell-based potency assay for rAAV9 vectors was successfully developed using HuH-7 cells.
- This assay effectively assesses AAV9 biopotency, providing a valuable alternative to animal models for quality control of gene therapy products.
- The findings support the functional quality and consistency of AAV9 gene therapy vectors, crucial for clinical translation.
