title: Persistent expression of FLAG-tagged micro dystrophin in nonhuman primates following intramuscular and vascular delivery.
abstract: Animal models for Duchenne muscular dystrophy (DMD) have species limitations related to assessing function, immune response, and distribution of micro- or mini-dystrophins. Nonhuman primates (NHPs) provide the ideal model to optimize vector delivery across a vascular barrier and provide accurate dose estimates for widespread transduction. To address vascular delivery and dosing in rhesus macaques, we have generated a fusion construct that encodes an eight amino-acid FLAG epitope at the C-terminus of micro-dystrophin to facilitate translational studies targeting DMD. Intramuscular (IM) injection of AAV8.MCK.micro-dys.FLAG in the tibialis anterior (TA) of macaques demonstrated robust gene expression, with muscle transduction (50-79%) persisting for up to 5 months. Success by IM injection was followed by targeted vascular delivery studies using a fluoroscopy-guided catheter threaded through the femoral artery. Three months after gene transfer, >80% of muscle fibers showed gene expression in the targeted muscle. No cellular immune response to AAV8 capsid, micro-dystrophin, or the FLAG tag was detected by interferon-gamma (IFN-gamma) enzyme-linked immunosorbent spot (ELISpot) at any time point with either route. In summary, an epitope-tagged micro-dystrophin cassette enhances the ability to evaluate site-specific localization and distribution of gene expression in the NHP in preparation for vascular delivery clinical trials.
authors.affiliation: Department of Pediatrics, The Ohio State University/Nationwide Children's Hospital, Columbus, Ohio 43205, USA.
authors.forename: Louise R
authors.initials: LR
authors.lastname: Rodino-Klapac
authors.forename: Chrystal L
authors.initials: CL
authors.lastname: Montgomery
authors.forename: William G
authors.initials: WG
authors.lastname: Bremer
authors.forename: Kimberly M
authors.initials: KM
authors.lastname: Shontz
authors.forename: Vinod
authors.initials: V
authors.lastname: Malik
authors.forename: Nancy
authors.initials: N
authors.lastname: Davis
authors.forename: Spencer
authors.initials: S
authors.lastname: Sprinkle
authors.forename: Katherine J
authors.initials: KJ
authors.lastname: Campbell
authors.forename: Zarife
authors.initials: Z
authors.lastname: Sahenk
authors.forename: K Reed
authors.initials: KR
authors.lastname: Clark
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Walker
authors.forename: Jerry R
authors.initials: JR
authors.lastname: Mendell
authors.forename: Louis G
authors.initials: LG
authors.lastname: Chicoine
mesh_terms: D000818:Animals; D015153:Blotting, Western; D000229:Dependovirus / Q000235:genetics; D016189:Dystrophin / Q000235:genetics / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007269:Injections, Intra-Arterial / Q000379:methods*; D007273:Injections, Intramuscular / Q000379:methods*; D008253:Macaca mulatta; D051379:Mice; D008810:Mice, Inbred C57BL; D009137:Muscular Dystrophy, Animal / Q000378:metabolism* / Q000628:therapy*; D009842:Oligopeptides; D010455:Peptides / Q000235:genetics / Q000378:metabolism*
chemical_list: D016189:Dystrophin; D009842:Oligopeptides; D010455:Peptides; C085706:FLAG peptide