Assessing the biopotency of the rAAV9 vector In Vitro
Pratikshya Adhikari, Peter G. Nichols, Stephen M. Vorobiov, Tierra A. Bobo, Haiyan Fu
Abstract
The potency assay is critical to ensure the effectiveness and consistency of recombinant Adeno-associated Virus (AAV) gene therapy vectors, especially clinical-grade products. AAV serotype 9 (AAV9), known for its neurotropic properties and ability to cross the blood-brain barrier, has been a favored vector for targeting neurogenetic diseases. However, assessing AAV9 biopotency has been challenging due to the insusceptibility of the commonly used cell lines to AAV9.
Introduction
Mucopolysaccharidosis type IIIA (MPS IIIA), also known as Sanfilippo syndrome type A, is one of the neuropathic lysosomal storage disorders (LSDs) that result from a deficiency in enzymes responsible for catalyzing Glycosaminoglycans (GAG) [1]. MPS IIIA is an autosomal recessive disorder arising from mutations in the gene encoding N-sulfoglucosamine sulfohydrolase (SGSH).
Materials and method
rAAV viral vector
The testing article, scAAV9-mCMV-hSGSH viral vector was developed for treating MPS IIIA and previously characterized and tested in a MPS IIIA mouse model [13]. The viral vector genome consists of AAV2 terminal repeats, a truncated 228-bp mCMV promoter, the human SGSH (hSGSH) coding sequence cDNA, and a Simian Virus 40 (SV40) polyadenylation signal. The viral vector was produced at the UNC Vector Core, in compliance with the Current Good Manufacturing Practice (cGMP) enforced by the FDA.
Results
A cell-based in vitro potency assay for rAAV9 gene therapy vector
We hereby evaluated the potency of the rAAV9 vector using a cell-based system. The testing articles were different batches of lot LAV139 which is a cGMP product of scAAV9-based gene replacement vector, scAAV9-mCMV-hSGSH, consisting of a mini-CMV promoter that drives the expression of codon-optimized hSGSH [13].
Discussion
Due to the limited infectivity of AAV9 to fibroblast cells cultures in vitro [17], the use of cell-based potency assays as a measure of therapeutic efficiency poses a significant challenge for the translation of AAV9 gene therapy vectors for treating human diseases. In this study, we utilized a HuH-7 cell-based in vitro potency assay enabling rapid and effective potency testing of an AAV9 gene therapy vector.
Acknowledgments
We would like to thank Dr. Douglas M. McCarty for proofreading and editing assistance.
Citation: Adhikari P, Nichols PG, Vorobiov SM, Bobo TA, Fu H (2026) Assessing the biopotency of the rAAV9 vector In Vitro. PLoS One 21(2): e0341451. https://doi.org/10.1371/journal.pone.0341451
Editor: Chen Ling, Fudan University, CHINA
Received: November 26, 2024; Accepted: January 7, 2026; Published: February 26, 2026
Copyright: © 2026 Adhikari et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: The study is supported by the donations from the families and friends of children with Sanfilippo syndrome (MPS III) through Aislinn’s Wish Foundation and Abby Grace Foundation, received by HF. PA, TB and HF were also funded by a research grant from RTW Foundation. HF was also funded by SBIR grants from NIH (R44AI172670, R44NS135667). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The corresponding author, Haiyan Fu, is a faculty at UNC-CH and also the founder and president of NeuroGT, Inc. Other authors have no conflict of interest to disclose.
