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 × 10¹⁴ 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.

Keywords: adeno-associated virus; axonopathy; gene therapy; hepatic toxicity; liver failure; shock.

Figure 1.

Acute transaminase elevations following intravenous (i.v.) administration of an adeno-associated virus (AAV) vector expressing human SMN to nonhuman primates (NHPs). (A) Study design, (B) serum alanine aminotransferase (ALT), (C) serum aspartate aminotransferase (AST), (D) serum alkaline phosphatase, and (E) serum total bilirubin. Unscheduled laboratory assessments were performed for all animals on study day 5 after animal 16C176 developed acute liver failure requiring euthanasia. AST was not performed on study day 5 for animals 16C116 and 16C215. Dashed lines indicate laboratory reference range.

Figure 2.

Liver histopathologic findings in juvenile NHPs treated with i.v. AAVhu68 expressing human SMN. Animal 16C176 required euthanasia on study day 4 and had massive acute hepatocellular necrosis (A) with sinusoidal fibrin deposition (B, arrowheads) and acute fibrin thrombi (C, arrow) in portal veins. Immunohistochemistry (IHC) for fibrinogen in animal 16C176 revealed prominent periportal sinusoidal fibrin deposition (D, arrowheads; fibrinogen IHC). The remaining two primates (E, 16C215; F 16C116) were clinically normal throughout the study, and both had similar findings in the liver consisting of single hepatocellular necrosis (arrows) predominantly surrounding portal areas with mild mononuclear cell infiltrates. Animal 16C215 (E) also had foci of hepatocellular regeneration (circle). Staining: hematoxylin and eosin; scale bar = 10 μm (A and C), 50 μm (B), and 100 μm (D–F).

Figure 3.

Representative central and peripheral nervous system histopathologic findings in juvenile NHPs treated with i.v. AAVhu68 expressing human SMN 28 days post injection, as depicted in images from animal 16C116. Both animals had an axonopathy (arrowheads) of the dorsal white matter tracts of the spinal cord (A). The dorsal axonopathy was typically bilateral and characterized by dilated myelin sheaths with and without myelomacrophages, consistent with axonal degeneration. The dorsal root ganglia (DRG) of the spinal cord (B) had minimal to mild neuronal cell body degeneration characterized by central chromatolysis (circle), satellitosis, and mononuclear cell infiltrates that surrounded and invaded neuronal cell bodies (neuronophagia; arrows). Mononuclear cell infiltrates were predominantly composed of CD3-positive T cells (C, arrowheads) with fewer CD20-positive B cells (D, arrowheads; CD3/CD20 IHC). A similar axonopathy (arrows) was observed in the peripheral nerves of the hind limb (sciatic nerve, E) and forelimb (median nerve, F). Animal 16C176 that was euthanized on study day 4 had no nervous system findings. Staining: hematoxylin and eosin; scale bar = 200 μm (A), 100 μm (B–F).

Figure 4.

Vector biodistribution in rhesus macaques. Rhesus macaques treated with i.v. AAVhu68 expressing human SMN were euthanized on study day 28, except for animal 16C176, which was euthanized on study day 4. Vector genomes were detected in tissue DNA samples by quantitative polymerase chain reaction (PCR). Values are expressed as vector genome copies (GC) per host diploid genome. Data are shown for four liver lobes (caudate, left, middle, and right).

Figure 5.

SMN expression in rhesus macaques. Human SMN RNA was detected by in situ hybridization (ISH) in the liver (A). The liver was stained with control probes for albumin (B) and green fluorescent protein (C). SMN-expressing cells were identified by ISH in the spinal cord (D). Motor neurons were identified by ChAT ISH (E). Rare patches of transduced neurons were detected by SMN ISH in the brain (F, DAPI nuclear stain). The percentage of ChAT+ motor neurons transduced at each level of spinal cord was quantified (G). Error bars = standard error of the mean.

Figure 6.

Representative histopathologic findings of piglets treated with i.v. AAVhu68 expressing human SMN at 7 and 30 days of age. Piglets in both groups had an axonopathy of the dorsal white matter tracts of the spinal cord (A), as depicted in piglet B. The dorsal axonopathy was bilateral and characterized by dilated myelin sheaths with and without myelomacrophages, consistent with axonal degeneration (arrowheads). The DRG of the spinal cord (B) exhibited varying degrees of neuronal cell body degeneration characterized by central chromatolysis (circles), satellitosis, and mononuclear cell infiltrates that surrounded and invaded neuronal cell bodies (neuronophagia; arrows), as depicted in piglet B. A similar axonopathy (arrows) was observed to varying degrees in the peripheral nerves of the hind limb (sciatic nerve, C) and forelimb (median nerve, D) in the majority of piglets, as depicted in piglet A. Staining: hematoxylin and eosin; scale bar = 200 μm (A), 100 μm (B–D).

Figure 7.

Vector biodistribution in piglets. Newborn piglets treated with i.v. AAVhu68 expressing human SMN were euthanized 13–14 days after injection. Vector genomes were detected in tissue DNA samples by quantitative PCR. Values are expressed as vector genome copies (GC) per host diploid genome. Data are shown for four liver lobes (caudate, left, middle, and right).

Figure 8.

SMN expression in the spinal cord of piglets. Human SMN RNA was detected by ISH in cervical (A), thoracic (B), and lumbar (C) spinal cord segments. Motor neurons were identified by ChAT ISH in corresponding sections (D–F). Representative images are shown.