High-dose systemic adeno-associated virus vector administration causes liver and sinusoidal endothelial cell injury.

We analyzed retrospective data from toxicology studies involving administration of high doses of adeno-associated virus expressing different therapeutic transgenes to 21 cynomolgus and 15 rhesus macaques. We also conducted prospective studies to investigate acute toxicity following high-dose systemic administration of enhanced green fluorescent protein-expressing adeno-associated virus to 10 rhesus macaques. Toxicity was characterized by transaminitis, thrombocytopenia, and alternative complement pathway activation that peaked on post-administration day 3. Although most animals recovered, some developed ascites, generalized edema, hyperbilirubinemia, and/or coagulopathy that prompted unscheduled euthanasia. Study endpoint livers from animals that recovered and from unscheduled necropsies of those that succumbed to toxicity were analyzed via hypothesis-driven histopathology and unbiased single-nucleus RNA sequencing. All liver cell types expressed high transgene transcript levels at early unscheduled timepoints that subsequently decreased. Thrombocytopenia coincided with sinusoidal platelet microthrombi and sinusoidal endothelial injury identified via immunohistology and single-nucleus RNA sequencing. Acute toxicity, sinusoidal injury, and liver platelet sequestration were similarly observed with therapeutic transgenes and enhanced green fluorescent protein at doses ≥1 × 1014 GC/kg, suggesting it was the consequence of high-dose systemic adeno-associated virus administration, not green fluorescent protein toxicity. These findings highlight a potential toxic effect of high-dose intravenous adeno-associated virus on nonhuman primate liver microvasculature.

Adeno-associated virus-mediated expression of human butyrylcholinesterase to treat organophosphate poisoning.

Rare diseases defined by genetic mutations are classic targets for gene therapy. More recently, researchers expanded the use of gene therapy in non-clinical studies to infectious diseases through the delivery of vectorized antibodies to well-defined antigens. Here, we further extend the utility of gene therapy beyond the "accepted" indications to include organophosphate poisoning. There are no approved preventives for the multi-organ damage resulting from acute or chronic exposure to organophosphates. We show that a single intramuscular injection of adeno-associated virus vector produces peak expression (~0.5 mg/ml) of active human butyrylcholinesterase (hBChE) in mice serum within 3-4 weeks post-treatment. This expression is sustained for up to 140 days post-injection with no silencing. Sustained expression of hBChE provided dose-dependent protection against VX in male and female mice despite detectable antibodies to hBChE in some mice, thereby demonstrating that expression of hBChE in vivo in mouse muscle is an effective prophylactic against organophosphate poisoning.

AAV8-antiVEGFfab Ocular Gene Transfer for Neovascular Age-Related Macular Degeneration.

Sustained suppression of VEGF is needed in many patients with neovascular age-related macular degeneration (NVAMD), and gene transfer of a VEGF-neutralizing protein is a promising approach to achieve it. Initial clinical trials testing this approach have shown encouraging signals, but evidence of robust transgene expression and consistent antiangiogenic and antipermeability activity has been lacking. In this study, we demonstrate expression of an anti-human VEGF antibody fragment (antiVEGFfab) after subretinal injection of AAV8-antiVEGFfab. In transgenic mice expressing human VEGF in retina (rho/VEGF mice), a model of type 3 choroidal neovascularization (NV), eyes injected with ≥1 × 107 gene copies (GC) of AAV8-antiVEGFfab had significantly less mean area of NV than null vector-injected eyes. A dose-dependent response was observed with modest reduction of NV with ≤3 × 107, >50% reduction with ≥1 × 108 GC and almost complete elimination of NV with 3 × 109 or 1 × 1010 GC. In Tet/opsin/VEGF mice, in which doxycycline-induced high expression of VEGF leads to severe vascular leakage and exudative retinal detachment (RD), reduction of total RD by 70%-80% occurred with 3 × 109 or 1 × 1010 GC of AAV8-antiVEGFfab, an effect that was sustained for at least a month. These data strongly support initiating clinical trials testing subretinal injection of AAV8-antiVEGFfab in patients with NVAMD.