Abstract
Recombinant adeno-associated virus (rAAV), produced from a nonpathogenic parvovirus, has become an increasing popular vector for gene therapy applications in human clinical trials. However, transduction and transgene expression of rAAVs can differ across in vitro and ex vivo cellular transduction strategies. This study compared 11 rAAV serotypes, carrying one reporter transgene cassette containing a cytomegalovirus immediate-early enhancer (eCMV) and chicken beta actin (CBA) promoter driving the expression of an enhanced green-fluorescent protein (eGFP) gene, which was transduced into four different cell types: human iPSC, iPSC-derived RPE, iPSC-derived cortical, and dissociated embryonic day 18 rat cortical neurons. Each cell type was exposed to three multiplicity of infections (MOI: 1E4, 1E5, and 1E6 vg/cell). After 24, 48, 72, and 96 h posttransduction, GFP-expressing cells were examined and compared across dosage, time, and cell type. Retinal pigmented epithelium showed highest AAV-eGFP expression and iPSC cortical the lowest. At an MOI of 1E6 vg/cell, all serotypes show measurable levels of AAV-eGFP expression; moreover, AAV7m8 and AAV6 perform best across MOI and cell type. We conclude that serotype tropism is not only capsid dependent but also cell type plays a significant role in transgene expression dynamics.
Highlights
There is an excellent safety record with respect to use of recombinant adeno-associated virus (AAV) vectors in human clinical trials [1,2,3]
The generation of individualized cell types from pluripotent stem cells offers a tremendous tool for developing Recombinant adeno-associated virus (rAAV) therapeutics for ailing individuals; comparison of results in in vitro cell-based assays to in vivo or ex vivo models is necessary to ensure the relevance of gene augmentation strategies for ultimate human clinical trials
All human protocols were approved by the institutional review board (IRB) at the University of Pennsylvania, and each donor provided signed informed consent (IRB 808828). iPSC cultures were maintained in iPSC medium (Dulbecco’s modified essential medium/Ham’s F12 nutrient media; DMEM/F12 (50 : 50; Corning)) containing glutamine, penicillin/streptomycin (Invitrogen), 15% KnockOut serum replacement (KSR; Invitrogen), 1 × non-essential amino acids (NEAA; Invitrogen), 0.1 mM β-mercaptoethanol (2-ME; Invitrogen), and 5 ng/mL of basic fibroblast growth factor on 0.1% gelatin-coated dishes with irradiated mouse embryonic fibroblast
Summary
There is an excellent safety record with respect to use of recombinant adeno-associated virus (AAV) vectors in human clinical trials [1,2,3]. Since animal models are not always available for a given disease (or may have an irrelevant phenotype), recent trend for evaluating proof-of-concept of gene therapy in laboratory studies has focused on use of induced pluripotent stem cell(iPSC-) based cell models from affected individuals [6,7,8,9]. Such models can be used to study pathologic mechanisms associated with known gene mutations. The generation of individualized cell types from pluripotent stem cells offers a tremendous tool for developing rAAV therapeutics for ailing individuals; comparison of results in in vitro cell-based assays to in vivo or ex vivo models is necessary to ensure the relevance of gene augmentation strategies for ultimate human clinical trials
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
