Two-Plasmid Packaging System for Recombinant Adeno-Associated Virus
Keeler, Allison M.
Flotte, Terence R.
UMass Chan AffiliationsDepartment of Microbiology and Physiology Systems
Horae Gene Therapy Center
Department of Pediatrics
Document TypeJournal Article
Genetics and Genomics
MetadataShow full item record
AbstractA 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.
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