AAV5 tropism for primate striated muscle and C5-12 promoter selectivity in primates were unclear. AAV5, AAV8, and AAV9 carrying C5-12-microdystrophin-FLAG were compared after one intravenous dose in monkeys. AAV8 reached ~20% of wild-type dystrophin at 1.18 × 10^14 VG/kg.
The synthetic C5-12 promoter drives microdystrophin-FLAG expression selectively in striated muscle after systemic AAV delivery in cynomolgus monkeys (Macaca fascicularis). After intravenous AAV5, AAV8, or AAV9 carrying a C5-12-microdystrophin-FLAG cassette, protein in liver, lung, and peripheral blood mononuclear cells stays at assay background (~1,096 a.u.), and only kidney (AAV5 and AAV9) and liver (AAV9) rise marginally above baseline. Liver carries the highest vector genome (VG) levels of any tissue yet yields only low mRNA, showing the promoter, not vector distribution, confines expression.
02
In cynomolgus monkey heart after systemic delivery of a C5-12-microdystrophin-FLAG cassette at 1.18 × 10^14 VG/kg, AAV5 and AAV8 give the highest microdystrophin-FLAG protein in the left and right ventricles, and AAV9 the lowest. AAV5 has poor tropism for striated muscle in rodents yet is comparable to AAV8 in the primate heart, so rodent tropism does not predict the serotype ranking in primate heart. Each of the three serotypes expresses more in the ventricles, septum and apex than in the atria.
03
AAV8-C5-12-microdystrophin-FLAG at 1.18 × 10^14 VG/kg expresses microdystrophin-FLAG at ~20% of wild-type cynomolgus monkey dystrophin by LC-MS, and the protein localizes to the sarcolemma of ~40% of skeletal muscle fibers and ~15% of left ventricular cardiomyocytes. AAV8 at 1.18 × 10^14 VG/kg gives broader expression than AAV9 at the higher 2 × 10^14 VG/kg dose by LC-MS and immunofluorescence.
04
After systemic AAV delivery of C5-12-microdystrophin-FLAG in cynomolgus monkeys, skeletal muscle yields ~10-fold more mRNA per microgram total RNA than heart at the same vector genome level (1–2 × 10^5 VG/μg genomic DNA), and skeletal muscle mRNA above ~1 × 10^6 copies/μg gives 3- to 4-fold more microdystrophin-FLAG protein than heart. Skeletal muscle (including diaphragm) therefore expresses higher levels than heart in this primate model.
05
In cynomolgus monkey heart after systemic AAV5, AAV8, or AAV9 delivery of C5-12-microdystrophin-FLAG, the atria carry the highest VG levels but produce minimal mRNA, no microdystrophin-FLAG protein in the left atria, and low protein in the right atria, while ventricles, septum, and apex express the most protein. This pattern is consistent with lower C5-12 promoter activity in atria, though uncleared vector genomes after only one month is an alternative explanation.
06
Plasma clearance of systemically delivered AAV in cynomolgus monkeys depends on serotype: AAV5 vector genomes are mostly cleared from blood within ~24 h of intravenous dosing, while AAV8 and AAV9 vector genomes persist beyond 48 h.
07
Neutralizing antibodies raised by a single systemic AAV5, AAV8, or AAV9 dose in cynomolgus monkeys are largely serotype-specific at days 15 and 29, with one AAV8-treated animal developing neutralizing antibodies to both AAV8 and AAV9. Monkeys neutralizing AAV8 and/or AAV9 before treatment but not AAV5 no longer neutralize AAV8 or AAV9 after AAV5 dosing.
08
A compact 4,412-bp AAV cassette within the ITRs carries the C5-12 promoter, a human microdystrophin (actin-binding domain, hinge 1, spectrin-like repeats 1–3, hinge 2, spectrin-like repeat 24, hinge 4, and cysteine-rich region) with a C-terminal FLAG tag, and a synthetic poly(A), with no 5′ or 3′ UTR. Packaged in AAV5, AAV8, or AAV9 capsids using the baculovirus expression vector system (BEVS), the same cassette transduces striated muscle in cynomolgus monkeys.
1 / of8
Abstract
Safely achieving therapeutic expression levels with adeno-associated virus (AAV) gene therapy is a significant challenge for treating the large muscle mass in humans. Non-human primates (NHPs) provide a more accurate assessment of the feasibility of achieving an effective and safe dose than rodents. Here, we compared a single systemic administration of AAV5, AAV8, or AAV9 in NHPs, each packaging the C5-12-microdystrophin-FLAG expression cassette. At 1 month post-dose, we compared tissue vector genomes, mRNA, and microdystrophin-FLAG protein levels by meso-scale discovery-enzyme-linked immunosorbent assay, liquid chromatography-mass spectrometry, and immunofluorescence. The C5-12 promoter was highly selective for heart and skeletal muscles, when compared to off-target tissues such as peripheral blood mononuclear cells, lung, liver, and kidney. AAV8 led to higher levels of microdystrophin-FLAG mRNA and protein in the cardiac ventricles and skeletal muscles when compared to AAV5 or AAV9. The AAV8-microdystrophin-FLAG led to ∼20% of wild-type NHP dystrophin protein expression levels and was located on the sarcolemma of ∼40% of skeletal muscles fibers and ∼15% of left ventricular cardiomyocytes. Hematology, serum chemistry, and pathology were unremarkable. Thus, a systemic dose of ∼1.18 × 1014 vector genomes/kg AAV8 is predicted to be safe and efficacious for treating Duchenne muscular dystrophy (DMD) but has significant room for improvement.
Choosing a selection results in a full page refresh.
