title: Large-Scale Production of Adeno-Associated Viral Vector Serotype-9 Carrying the Human Survival Motor Neuron Gene.
abstract: Recombinant 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 μg 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.
authors.affiliation: Department of Biotechnology, Ege University, Izmir, Turkey. afrash99@yahoo.com.|Young Researchers and Elites Club, North Tehran Branch, Islamic Azad University, Tehran, Iran. afrash99@yahoo.com.
authors.forename: Afrooz
authors.initials: A
authors.lastname: Rashnonejad
authors.affiliation: Medical School, Faculty of Medicine, Ege University, Izmir, Turkey. ghoamini@yahoo.com.
authors.forename: Gholamhossein Amini
authors.initials: GA
authors.lastname: Chermahini
authors.affiliation: Gene Therapy Center, University of Massachusetts Medical School, 368 Plantation St., AS6-2049, Worcester, MA, 01605, USA. shaoyong.li@umassmed.edu.
authors.forename: Shaoyong
authors.initials: S
authors.lastname: Li
authors.affiliation: Faculty of Medicine, Department of Medical Genetics, Ege University, Izmir, Turkey. f.ferda.ozkinay@ege.edu.tr.
authors.forename: Ferda
authors.initials: F
authors.lastname: Ozkinay
authors.affiliation: Gene Therapy Center, University of Massachusetts Medical School, 368 Plantation St., AS6-2049, Worcester, MA, 01605, USA. guangping.gao@umassmed.edu.
authors.forename: Guangping
authors.initials: G
authors.lastname: Gao
mesh_terms: D000229:Dependovirus / Q000235:genetics* / Q000254:growth & development; D004742:Enhancer Elements, Genetic; D005786:Gene Expression Regulation; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000096:biosynthesis / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D011401:Promoter Regions, Genetic / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid; D055532:SMN Complex Proteins / Q000235:genetics*; D014162:Transfection
chemical_list: D055532:SMN Complex Proteins
keywords: Adeno-associated viral vector; Electron microscopy; Gene therapy; Human embryonic kidney 293 cells; Spinal muscular atrophy (SMA); Survival Motor Neuron (SMN) gene; Transient transfection