Large-Scale Production of Adeno-Associated Viral Vector Serotype-9 Carrying the Human Survival Motor Neuron Gene

WOS: 000368064600004PubMed ID: 26607476Recombinant AAV (rAAV) vectors are a suitable vector for gene therapy studies because of desired characteristics such as low immunogenicity, transfection of non-dividing and dividing cells, and long-term expression of the transgene. In this study, the large-scale production of single stranded (ss) and self-complementary (sc) AAV9 carrying the human survival motor neuron (SMN) gene (AAV9-SMN) suitable for in vivo gene therapy studies of SMA was described. SMN cDNA has been cloned into pAAV-CB6-PI and pAAVsc-CB6-PI with and without its specific UTRs, respectively. Both plasmids bear CMV enhancer/beta-actin (CB) promoter, CMV IE enhancer, and polyadenylation signal sequences. 2.5 mu g of constructed pAAV-CB6-PI-SMN and pAAVsc-CB6-PI-SMN cause to, respectively, 4.853- and 2.321-fold increases in SMN protein levels in transfected cells compared to untransfected cells. Ss and scAAV9-SMN vectors were also produced from these plasmids by transient transfection of HEK293 cells using CaCl2 solution. The silver staining and electron microscopy analysis demonstrated good quality of both isolated vectors, ssAAV9-SMN and scAAV9-SMN, with the titers of 2.00E+13 and 1.00E+13 GC/ml. The results of this study show that, the plasmid containing UTR elements causes to twice more SMN gene expression in transfected cells. The quality control results show that both produced ss and scAAV9-SMN are suitable for in vivo studies.University of Massachusetts Medical School; National Institutes of HealthUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [R01NS076991-01, 1R21DA031952-01A, 2P01HL059407, 1P01AI100263-01]; Will Foundation; Jacob's Cure; NTSAD Foundation; Canavan Foundation; National High Technology Research and Development Program ("863" Program) of ChinaNational High Technology Research and Development Program of China [2012AA020810]This work is supported by University of Massachusetts Medical School (an internal grant), National Institutes of Health (R01NS076991-01, 1R21DA031952-01A, 2P01HL059407, 1P01AI100263-01), the Will Foundation, Jacob's Cure, NTSAD Foundation, Canavan Foundation, and partial support from a grant from the National High Technology Research and Development Program ("863" Program) of China (2012AA020810). G.G. is a co-founder of Voyager Therapeutics and holds equity in the company. G.G. is an inventor on patents with potential royalties licensed to Voyager Therapeutics and other pharmaceutical companies