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Author Notes:

Xinping Huang, MD, Department of Neurology, Emory University, Atlanta, GA 30322, United States, xhuang4@emory.edu.

We would like to thank Dr. Thomas Kukar for gifting the HEK293T cells and Dr. Nancy Ciliax for administrative support.

Subjects:

Research Funding:

This research project was supported by Emory Neuroscience NINDS Core Facility grant for the Viral Vector Core, P30NS055077.

Keywords:

  • Science & Technology
  • Life Sciences & Biomedicine
  • Biochemical Research Methods
  • Biotechnology & Applied Microbiology
  • Virology
  • Biochemistry & Molecular Biology
  • Recombinant virus
  • Optimization
  • AAV
  • AAV2-GFP
  • Polyethylenimine (PEI)
  • N/P ratio
  • RECOMBINANT ADENOASSOCIATED VIRUS
  • FLUORESCENCE CORRELATION SPECTROSCOPY
  • LONG-TERM CORRECTION
  • GENE-THERAPY
  • INFECTIOUS TITER
  • VECTORS
  • CELLS
  • POLYETHYLENIMINE
  • COMPLEXES
  • DELIVERY

AAV2 production with optimized N/P ratio and PEI-mediated transfection results in low toxicity and high titer for in vitro and in vivo applications

Tools:

Journal Title:

Journal of Virological Methods

Volume:

Volume 193, Number 2

Publisher:

, Pages 270-277

Type of Work:

Article | Post-print: After Peer Review

Abstract:

The adeno-associated virus (AAV) is one of the most useful viral vectors for gene delivery for both in vivo and in vitro applications. A variety of methods have been established to produce and characterize recombinant AAV (rAAV) vectors; however most methods are quite cumbersome and obtaining consistently high titer can be problematic. This protocol describes a triple-plasmid co-transfection approach with 25kDa linear polyethylenimine (PEI) in 293T cells for the production of AAV serotype 2. Seventy-two hours post-transfection, supernatant and cells were harvested and purified by a discontinuous iodixanol density gradient ultracentrifugation, then dialyzed and concentrated with an Amicon 15 100,000 MWCO concentration unit. To optimize the protocol for AAV2 production using PEI, various N/P ratios and DNA amounts were compared. We found that an N/P ratio of 40 coupled with 1.05μg DNA per ml of media (21μg DNA/15cm dish) was found to produce the highest yields for viral replication and assembly measured multiple ways. The infectious units, as determined by serial dilution, were between 1×108 and 2×109IU/ml. The genomic titer of the viral stock was determined by qPCR and ranged from 2×1012 to 6×1013VG/ml. These viral vectors showed high expression both in vivo within the brain and in vitro in cell culture. The use of linear 25kDa polyethylenamine PEI as a transfection reagent is a simple, more cost-effective, and stable means of high-throughput production of high-titer AAV serotype 2. The use of PEI also eliminates the need to change cell medium post-transfection, lowering cost and workload, while producing high-titer, efficacious AAV2 vectors for routine gene transfer.

Copyright information:

© 2013 Elsevier B.V.

This is an Open Access work distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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