Persistent expression of FLAG-tagged micro dystrophin in nonhuman primates following intramuscular and vascular delivery

Parts used

Show 1 morepart→

Key findings

• The rAAV8 vector carrying MCK.micro-dystrophin.FLAG is built by cross-packaging, in which an AAV2 inverted terminal repeat–flanked genome is packaged into a non-AAV2 capsid supplied in trans. Production uses three-plasmid CaPO4 transfection of HEK293 cells with pAAV.MCK.microdys.FLAG, a rep2-cap8 modified AAV helper plasmid encoding cap serotype 8-like isolate rh.74, and an adenovirus type 5 helper plasmid (pAdhelper) expressing adenovirus E2A, E4 ORF6 and VA I/II RNA genes.
• An eight-amino-acid FLAG epitope (DYKDDDDK) fused in frame to the C-terminus of human micro-dystrophin (R4–R23/Δ71–78) distinguishes vector-derived micro-dystrophin from endogenous full-length dystrophin in rhesus macaque muscle, where the presence of an endogenous dystrophin gene otherwise precludes studying transgene distribution by immunohistochemistry. This was the first use of a FLAG epitope tag as a transgene marker to differentiate endogenous from vector-derived protein for assessing transgene distribution in a nonhuman primate.
• Micro-dystrophin carrying a C-terminal FLAG tag remains biopotent in dystrophic muscle. Injecting 10^11 vector genomes of rAAV8.MCK.micro-dys.FLAG into the tibialis anterior of 4-week-old mdx mice gives micro-dys.FLAG expression in 73.2 ± 10.4% of fibers at 4 weeks, detected with a FLAG-specific antibody and confirmed with the N-terminal dystrophin antibody Dys3, which recognizes human but not mouse dystrophin. Micro-dystrophin does not stain with the C-terminal dystrophin antibody Dys2.
• Micro-dystrophin.FLAG corrects a physiologic deficit in dystrophin-deficient muscle. Direct injection of 2 × 10^11 vg in 30 µl of rAAV8.MCK.micro-dys.FLAG into the left ventral quadrant of the diaphragm of 8-week-old mdx mice significantly protects diaphragm muscle strips from fatigue 8 weeks later under stimulation every second for 90 seconds (1 Hz amplitude, 80 ms duration, 130 ms frequency; two-way analysis of variance P < 0.001), without restoring function to C57/BL10 strain control levels.
• Micro-dystrophin.FLAG expression in rhesus macaque muscle persists for at least 5 months after a single intramuscular dose. Injecting 5 × 10^12 vg of rAAV8.MCK.micro-dys.FLAG as three ~333 µl depots 0.5 cm apart and 0.5 cm beneath the fascia in the tibialis anterior of AAV8 seronegative macaques transduces 50.1%, 79.1% and 62.3% of fibers at the injection site at 2 months and 37.6 ± 16.7%, 39 ± 5.4% and 54 ± 17.1% in peri-injection muscle at 5 months, the drop reflecting limited spread of vector from the target site. Vector genome copy number in treated muscle is 2.5 × 10^6 ± 2.4 × 10^6 vg/µg genomic DNA against 0.009 ± 0.009 vg/µg in the contralateral limb.
• FLAG-tagged micro-dystrophin localizes correctly to the sarcolemmal membrane in rhesus macaque muscle and colocalizes with endogenous full-length dystrophin, as shown by overlapping C-terminal dystrophin (Dys2) and anti-FLAG immunofluorescence. On western blot the N-terminal Dys3 antibody detects both the 427 kd endogenous dystrophin band and a 138 kd micro-dys.FLAG band in treated muscle, while the 138 kd band is absent from contralateral control muscle and carries no C-terminal Dys2 reactivity.
• Intramuscular rAAV8.MCK.micro-dys.FLAG in rhesus macaques elicits no transgene-directed T-cell response over 5 months. No micro-dys.FLAG IFN-γ ELISpot response rises above background in any animal at any time point against a positivity threshold of 50 spot-forming colonies per 10^6 peripheral blood mononuclear cells, and AAV8 capsid responses are limited to two transient, marginally positive values against capsid pool 3 — 82 spot-forming colonies per million PBMCs at day 14 in RQ6661 and 77 at day 42 in RQ6698. Anti-AAV8 capsid binding antibodies are nonetheless detectable in all animals as early as 2 weeks after gene transfer, confirming delivery of an immunogenic capsid dose.
• Vascular rAAV delivery can be confined to a single muscle group by catheterizing its feeding artery. Threading a fluoroscopy-guided 3-0 f catheter through the rhesus macaque femoral artery to the sural branch of the popliteal artery, which supplies the gastrocnemius, and infusing 2 × 10^12 vg/kg of rAAV8.MCK.micro-dys.FLAG in 2.5 ml/kg Tris buffered saline over 60 seconds between two tourniquets — one above the knee about 0.5 inches above the catheter tip and a second at the base of the gastrocnemius — with a 2.5 ml/kg saline preflush, a 10-minute dwell time and a 2.5 ml/kg postflush, targets expression to the gastrocnemius without noticeable edema or adverse effects.
• Pre-existing anti-AAV8 antibodies partially but not completely block muscle transduction by vascular rAAV8 delivery. Three months after isolated limb perfusion with 2 × 10^12 vg/kg rAAV8.MCK.micro-dys.FLAG, rhesus macaques without pre-existing AAV8 antibodies (01D299 and RQ6539) show 82% and 89% micro-dys.FLAG-positive fibers in the central gastrocnemius and 2.2 × 10^5 ± 1.7 × 10^5 vg/µg genomic DNA, while seropositive macaques (03E101 and 03E062) show 39% and 35% and 2.8 × 10^3 ± 1.3 × 10^3 vg/µg — about twofold lower expression, with gene transfer still achieved in both seropositive animals.
• rAAV8.MCK.micro-dys.FLAG delivered by isolated limb perfusion to rhesus macaque gastrocnemius is not immunogenic. No significant IFN-γ ELISpot response to AAV8 capsid or micro-dys.FLAG peptide pools appears in peripheral blood mononuclear cells through necropsy at 3 months, T cells isolated directly from necropsied muscle give no response even after clonal expansion and restimulation with capsid and micro-dys.FLAG peptide pools, and histology of expressing muscle shows no inflammation, necrosis or change in morphology.

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.