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

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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.

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Acknowledgments

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.

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Authors and Affiliations

  1. Department of Biotechnology, Ege University, Izmir, Turkey

    Afrooz Rashnonejad

  2. Young Researchers and Elites Club, North Tehran Branch, Islamic Azad University, Tehran, Iran

    Afrooz Rashnonejad

  3. Medical School, Faculty of Medicine, Ege University, Izmir, Turkey

    Gholamhossein Amini Chermahini

  4. Gene Therapy Center, University of Massachusetts Medical School, 368 Plantation St., AS6-2049, Worcester, MA, 01605, USA

    Shaoyong Li & Guangping Gao

  5. Faculty of Medicine, Department of Medical Genetics, Ege University, Izmir, Turkey

    Ferda Ozkinay

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  1. Afrooz Rashnonejad
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  2. Gholamhossein Amini Chermahini
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  3. Shaoyong Li
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  4. Ferda Ozkinay
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  5. Guangping Gao
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Correspondence to Guangping Gao.

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Research did not involve Human Participants and/or Animal.

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We confirm that this work is original and has not been published elsewhere nor is it currently under consideration for publication elsewhere. The manuscript has been seen and approved by all authors. We hereby assign the copyright of the enclosed manuscript to “Molecular Biotechnology” Journal.

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Supplementary material 1 (DOCX 19 kb)

12033_2015_9899_MOESM2_ESM.tif

Supplementary Fig. 1. The blast result of SMN cDNA in pCMV6-XL5-SMN and SMN reference sequence (GenBank Accession No. NM_000344.3). The protein sequence of SMN protein encoded by the gene is also demonstrated. The nucleotides which are shown with red color are the nucleotides with silent mutation (single nucleotide polymorphisms or SNPs), causing no changes in coded amino acids. (TIFF 5151 kb)

12033_2015_9899_MOESM3_ESM.tif

Supplementary Fig. 2. The sequencing analysis of pAAVsc-CB6-PI-SMN showing the correct insertion of SMN cDNA into plasmid backbone. (TIFF 952 kb)

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Rashnonejad, A., Chermahini, G.A., Li, S. et al. Large-Scale Production of Adeno-Associated Viral Vector Serotype-9 Carrying the Human Survival Motor Neuron Gene. Mol Biotechnol 58, 30–36 (2016). https://doi.org/10.1007/s12033-015-9899-5

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  • DOI: https://doi.org/10.1007/s12033-015-9899-5

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