Severe Toxicity in Nonhuman Primates and Piglets Following High-Dose Intravenous Administration of an Adeno-Associated Virus Vector Expressing Human SMN

Abstract

Neurotropic adeno-associated virus (AAV) serotypes such as AAV9 have been demonstrated to transduce spinal alpha motor neurons when administered intravenously (i.v.) at high doses. This observation led to the recent successful application of i.v. AAV9 delivery to treat infants with spinal muscular atrophy, an inherited deficiency of the survival of motor neuron (SMN) protein characterized by selective death of lower motor neurons. To evaluate the efficiency of motor neuron transduction with an AAV9 variant (AAVhu68) using this approach, three juvenile nonhuman primates (NHPs; aged 14 months) and three piglets (aged 7-30 days) were treated with an i.v. injection of an AAVhu68 vector carrying a human SMN transgene at a dose similar to that employed in the spinal muscular atrophy clinical trial. Administration of 2 × 10 genome copies per kilogram of body weight resulted in widespread transduction of spinal motor neurons in both species. However, severe toxicity occurred in both NHPs and piglets. All three NHPs exhibited marked transaminase elevations. In two NHPs, the transaminase elevations resolved without clinical sequelae, while one NHP developed acute liver failure and shock and was euthanized 4 days after vector injection. Degeneration of dorsal root ganglia sensory neurons was also observed, although NHPs exhibited no clinically apparent sensory deficits. There was no correlation between clinical findings and T-cell responses to the vector capsid or transgene product in NHPs. Piglets demonstrated no evidence of hepatic toxicity, but within 14 days of vector injection, all three animals exhibited proprioceptive deficits and ataxia, which profoundly impaired ambulation and necessitated euthanasia. These clinical findings correlated with more severe dorsal root ganglia sensory neuron lesions than those observed in NHPs. The liver and sensory neuron findings appear to be a direct consequence of AAV transduction independent of an immune response to the capsid or transgene product. The present results and those of another recent study utilizing a different AAV9 variant and transgene indicate that systemic and sensory neuron toxicity may be general properties of i.v. delivery of AAV vectors at high doses, irrespective of the capsid serotype or transgene. Preclinical and clinical studies involving high systemic doses of AAV vectors should include careful monitoring for similar toxicities.

Lessons learned from lung and liver in-vivo gene therapy: implications for the future

“…Another example, where results from rodent studies do not translate directly to studies with large animal models, is the recent observation that high doses (2e14 GC/kg) of rAAV9 vectors can lead to severe acute toxicity in piglets and non-human primates when delivered intravenously [91,92]. In this case, and different from the trial results reported by Manno et al [59], the toxicity was likely due to an acute, innate immune response (around 5 days after vector administration).…”

How will the field of gene therapy survive its success?

“…For example, a recent experiment in rhesus monkeys found that the high doses of AAV that may need to be delivered intravenously for some therapies could be prohibitively toxic. 52 In three out of three primates and three out of three piglets, the investigators found that the high doses of AAV used resulted in severe toxicity, including hepatocellular necrosis and axonopathies in both the central and peripheral nervous system of the monkeys, and neuronal degeneration in the dorsal root ganglia of the piglets. In commenting on this report, Flotte et al 53 noted that the doses used in the animals with these serious outcomes are nevertheless being used in human trials that so far have not encountered dose-limiting toxicity, and that the toxicity encountered in the primates and piglets could have been due to some unidentified contaminants in the viral preparation.…”

“…A second reason for caution is that the limitations of gene therapy vectors for treatments requiring distribution to the entire body, as some gene‐editing treatments might require, are still being learned and may not always be appreciated. For example, a recent experiment in rhesus monkeys found that the high doses of AAV that may need to be delivered intravenously for some therapies could be prohibitively toxic . In three out of three primates and three out of three piglets, the investigators found that the high doses of AAV used resulted in severe toxicity, including hepatocellular necrosis and axonopathies in both the central and peripheral nervous system of the monkeys, and neuronal degeneration in the dorsal root ganglia of the piglets.…”

Adeno‐associated virus‐based Alzheimer's disease mouse models and potential new therapeutic avenues

“…Remarkably, this treatment triggered elevated serum aminotransferase levels in only few patients and these were attenuated by prednisolone. In contrast, a separate study employing pigs and NHPs exposed to dose‐matched AAV9 expressing the same transgene and administered similarly showed massive vector and transgene‐related toxicity, motor neuron degeneration, and even fatalities (Hinderer et al, ). The discrepancy between large animal models and clinical observations is difficult to reconcile but may highlight the inherent limitations of animal models as predictors of outcome for human studies.…”