Persistent expression of FLAG-tagged micro dystrophin in nonhuman primates following intramuscular and vascular delivery.
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.
Rodino-Klapac
Louise R
LR
Department of Pediatrics, The Ohio State University/Nationwide Children's Hospital, Columbus, Ohio 43205, USA.
Montgomery
Chrystal L
CL
Bremer
William G
WG
Shontz
Kimberly M
KM
Malik
Vinod
V
Davis
Nancy
N
Sprinkle
Spencer
S
Campbell
Katherine J
KJ
Sahenk
Zarife
Z
Clark
K Reed
KR
Walker
Christopher M
CM
Mendell
Jerry R
JR
Chicoine
Louis G
LG
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*
D016189:Dystrophin; D009842:Oligopeptides; D010455:Peptides; C085706:FLAG peptide