Rapid, Simple, and Versatile Manufacturing of Recombinant Adeno-Associated Viral Vectors at Scale
- 1 October 2010
- journal article
- research article
- Published by Mary Ann Liebert Inc in Human Gene Therapy
- Vol. 21 (10), 1259-1271
- https://doi.org/10.1089/hum.2010.055
Abstract
Adeno-associated viral (AAV) manufacturing at scale continues to hinder the application of AAV technology to gene therapy studies. Although scalable systems based on AAV–adenovirus, AAV–herpesvirus, and AAV–baculovirus hybrids hold promise for clinical applications, they require time-consuming generation of reagents and are not highly suited to intermediate-scale preclinical studies in large animals, in which several combinations of serotype and genome may need to be tested. We observed that during production of many AAV serotypes, large amounts of vector are found in the culture supernatant, a relatively pure source of vector in comparison with cell-derived material. Here we describe a high-yielding, recombinant AAV production process based on polyethylenimine (PEI)-mediated transfection of HEK293 cells and iodixanol gradient centrifugation of concentrated culture supernatant. The entire process can be completed in 1 week and the steps involved are universal for a number of different AAV serotypes. Process conditions have been optimized such that final purified yields are routinely greater than 1 × 1014 genome copies per run, with capsid protein purity exceeding 90%. Initial experiments with vectors produced by the new process demonstrate equivalent or better transduction both in vitro and in vivo when compared with small-scale, CsCl gradient-purified vectors. In addition, the iodixanol gradient purification process described effectively separates infectious particles from empty capsids, a desirable property for reducing toxicity and unwanted immune responses during preclinical studies.Keywords
This publication has 38 references indexed in Scilit:
- Efficient Serotype-Dependent Release of Functional Vector into the Culture Medium During Adeno-Associated Virus ManufacturingHuman Gene Therapy, 2010
- Systematic Evaluation of AAV Vectors for Liver directed Gene Transfer in Murine ModelsMolecular Therapy, 2010
- A Simplified Baculovirus-AAV Expression Vector System Coupled With One-step Affinity Purification Yields High-titer rAAV Stocks From Insect CellsMolecular Therapy, 2009
- Sustained transgene expression despite T lymphocyte responses in a clinical trial of rAAV1-AAT gene therapyProceedings of the National Academy of Sciences of the United States of America, 2009
- Large-Scale Adeno-Associated Viral Vector Production Using a Herpesvirus-Based System Enables Manufacturing for Clinical StudiesHuman Gene Therapy, 2009
- Producing Recombinant Adeno-Associated Virus in Foster Cells: Overcoming Production Limitations Using a Baculovirus–Insect Cell Expression StrategyHuman Gene Therapy, 2009
- Transient Transfection Methods for Clinical Adeno-Associated Viral Vector ProductionHuman Gene Therapy, 2009
- Safety and Efficacy of Gene Transfer for Leber's Congenital AmaurosisThe New England Journal of Medicine, 2008
- High-titer, serum-free production of adeno-associated virus vectors by polyethyleneimine-mediated plasmid transfection in mammalian suspension cellsBiotechnology Letters, 2007
- Recombinant adeno-associated virus purification using novel methods improves infectious titer and yieldGene Therapy, 1999