UMMS Affiliation

Department of Pediatrics; Horae Gene Therapy Center; Vector Core; Department of Microbiology and Physiology Systems

Publication Date

2020-10-16

Document Type

Article

Disciplines

Genetics and Genomics | Microbiology | Therapeutics | Viruses

Abstract

A number of packaging systems are available for production of recombinant adeno-associated virus vectors (rAAVs). Among these, the use of a two-plasmid cotransfection system, in which Rep and Cap genes and Ad helper genes are on the same plasmid, has not been frequently employed for good manufacturing practices (GMP) production, even though it presents some practical advantages over the common three-plasmid (triple) transfection method. To confirm and expand the utility of the two-plasmid system, we generated GMP-compatible versions of this system and used those package reporter genes in multiple capsid variants in direct comparison with triple transfection. Vector yields, purity, and empty-to-full ratios were comparable between double and triple transfection methods for all capsid variants tested. We performed an in vivo side-by-side comparison of double and triple transfection vectors following both intravenous injection and intramuscular injection in mice. Expression and transduction were evaluated in muscle and liver 4 weeks after injection. Additional studies of bioactivity were conducted in vivo using packaged vectors carrying a variety of cargos, including the therapeutic transgene, microRNA, and single- or double-stranded vector. Results showed that cargos packaged using double transfection were equivalently bioactive to those packaged using a triple transfection system. In conclusion, these data suggest the utility of midrange (1E12-1E16) GMP-compatible packaging of adeno-associated virus (AAV) vectors for several AAV capsids.

Keywords

AAV, gene expression, gene therapy, gene transfer, plasmids, viral vectors

Rights and Permissions

Copyright Qiushi Tang et al., 2020; Published by Mary Ann Liebert, Inc. This Open Access article is distributed under the terms of the Creative Commons License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

DOI of Published Version

10.1089/biores.2020.0031

Source

Tang Q, Keeler AM, Zhang S, Su Q, Lyu Z, Cheng Y, Gao G, Flotte TR. Two-Plasmid Packaging System for Recombinant Adeno-Associated Virus. Biores Open Access. 2020 Oct 16;9(1):219-228. doi: 10.1089/biores.2020.0031. PMID: 33117614; PMCID: PMC7590824. Link to article on publisher's site

Journal/Book/Conference Title

BioResearch open access

Related Resources

Link to Article in PubMed

PubMed ID

33117614

Creative Commons License

Creative Commons Attribution 4.0 License
This work is licensed under a Creative Commons Attribution 4.0 License.

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