Biodistribution and Tolerability of AAV-PHP.B-CBh-SMN1 in Wistar Han Rats and Cynomolgus Macaques Reveal Different Toxicologic Profiles

Abstract

Recombinant adeno-associated viruses (AAVs) have emerged as promising vectors for human gene therapy, but some variants have induced severe toxicity in Rhesus monkeys and piglets following high-dose intravenous (IV) administration. To characterize biodistribution, transduction, and toxicity among common preclinical species, an AAV9 neurotropic variant expressing the survival motor neuron 1 ( SMN1 ) transgene (AAV-PHP.B-CBh- SMN1 ) was administered by IV bolus injection to Wistar Han rats and cynomolgus monkeys at doses of 2 × 10 13 , 5 × 10 13 , or 1 × 10 14 vg/kg. A dose-dependent degeneration/necrosis of neurons without clinical manifestations occurred in dorsal root ganglia (DRGs) and sympathetic thoracic ganglia in rats, while liver injury was not observed in rats. In monkeys, one male at 5 × 10 13 vg/kg was found dead on day 4. Clinical pathology data on days 3 and/or 4 at all doses suggested liver dysfunction and coagulation disorders, which led to study termination. Histologic evaluation of the liver in monkeys showed hepatocyte degeneration and necrosis without inflammatory cell infiltrates or intravascular thrombi, suggesting that hepatocyte injury is a direct effect of the vector following hepatocyte transduction. In situ hybridization demonstrated a dose-dependent expression of SMN1 transgene mRNA in the cytoplasm and DNA in the nucleus of periportal to panlobular hepatocytes, while quantitative polymerase chain reaction confirmed the dose-dependent presence of SMN1 transgene mRNA and DNA in monkeys. Monkeys produced a much greater amount of transgene mRNA compared with rats. In DRGs, neuronal degeneration/necrosis and accompanying findings were observed in monkeys as early as 4 days after test article administration. The present results show sensory neuron toxicity following IV delivery of AAV vectors at high doses with an early onset in Macaca fascicularis and after 1 month in rats, and suggest adding the autonomic system in the watch list for preclinical and clinical studies. Our data also suggest that the rat may be useful for evaluating the potential DRG toxicity of AAV vectors, while acute hepatic toxicity associated with coagulation disorders appears to be highly species-dependent.

Circulating neurofilament light chain as a promising biomarker of AAV-induced dorsal root ganglia toxicity in nonclinical toxicology species

“…Mild to moderate mononuclear cell infiltration was observed in monkeys exhibiting neuron/nerve fiber degeneration. In a similar monkey study, minimal focal to multifocal mononuclear cell infiltrate and reactive satellite glial cells were observed around necrotic neurons (shrunken neurons/neuronophagy) as early as day 5 post treatment at ≥5 × 10 13 vg/kg, 12 suggesting inflammation may directly contribute to the mechanism of toxicity in this species. In contrast, the minimal to mild mononuclear cell infiltration and glial cell hypertrophy observed in rats was likely a secondary response initiated to resolve the damaged neurons.…”

“…The DRG and spinal cord lesions from this rat study and a similar cynomolgus monkey study have been comprehensively characterized in a previous publication, which includes regional differences in histologic findings and immunohistochemical profiling of mononuclear cell infiltrates. 12 …”

“… 6 , 7 , 8 In clinical practice, anti-capsid inflammatory responses have been shown to be suppressed or resolved by co-administering AAV vectors with corticosteroids, 9 while rapamycin is reported to prevent humoral and cell-mediated responses in preclinical models. 10 , 11 Hepatic necrosis and coagulopathy have been observed in non-human primates (NHPs) treated with high doses of AAV, 12 , 13 while acute liver failure has been reported in two infants following treatment with an AAV vector containing the survival motor neuron 1 ( SMN1 ) gene (onasemnogene abeparvovec). 14 Moreover, neonatal mice are reported to develop hepatocellular carcinoma following AAV integration at the RNA imprinted and accumulated in nucleus ( Rian ) locus.…”

“…Following AAV administration, NHPs and rodents exhibit a high incidence of microscopic DRG lesions, although clinical sensory deficits are rarely observed. 5 , 12 , 13 , 17 , 18 , 19 , 20 , 21 These dose-dependent lesions are characterized by neuronal degeneration and/or necrosis, nerve fiber degeneration, and mononuclear cell infiltration. In one study, AAV-treated piglets exhibited severe DRG lesions accompanied by neurological deficits such as ataxia and impairments in conscious proprioception.…”

“…Comparable to these DRG findings, neuronal degeneration/necrosis and nerve fiber degeneration have also been observed in trigeminal and autonomic ganglia from AAV-treated rats and cynomolgus monkeys, indicating various ganglia throughout the body are affected. 12 In humans, sensory deficits (meningoradiculitis) were observed in one of two familial amyotrophic lateral sclerosis (ALS) patients following an intrathecal infusion of AAV containing anti-superoxide dismutase 1 ( SOD1 ) microRNA. 23 Beyond this single report, the human relevance of AAV-induced ganglia toxicity is currently unknown.…”