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.

Beagle dogs are a key nonrodent species in nonclinical safety evaluation of new biomedical products. The Society of Toxicologic Pathology (STP) has published “best practices” recommendations for nervous system sampling in nonrodents during general toxicity studies ( Toxicol Pathol 41[7]: 1028-1048, 2013), but their adaptation to the Beagle dog has not been defined specifically. Here we provide 2 trimming schemes suitable for evaluating the unique neuroanatomic features of the dog brain in nonclinical toxicity studies. The first scheme is intended for general toxicity studies (Tier 1) to screen test articles with unknown or no anticipated neurotoxic potential; this plan using at least 7 coronal hemisections matches the STP “best practices” recommendations. The second trimming scheme for neurotoxicity studies (Tier 2) uses up to 14 coronal levels to investigate test articles where the brain is a suspected or known target organ. Collection of spinal cord, ganglia (somatic and autonomic), and nerves for dogs during nonclinical studies should follow published STP “best practices” recommendations for sampling the central ( Toxicol Pathol 41[7]: 1028-1048, 2013) and peripheral ( Toxicol Pathol 46[4]: 372-402, 2018) nervous systems. This technical guide also demonstrates the locations and approaches to collecting uncommonly sampled peripheral nervous system sites.

Axonal dystrophy (AD) is a common age-related neurohistological finding in vertebrates that can be congenital or induced by xenobiotics, vitamin E deficiency, or trauma/compression. To understand the incidence and location of AD as a background finding in Beagle dogs used in routine toxicity studies, we examined central nervous system (CNS) and selected peripheral nervous system (PNS) tissues in twenty 18- to 24-month-old and ten 4- to 5-year-old control males and females. Both sexes were equally affected. The cuneate, gracile, and cochlear nuclei and the cerebellar white matter (rostral vermis) were the most common locations for AD. Incidence of AD increased with age in the cuneate nucleus, cerebellar white matter (rostral vermis), trigeminal nuclei/tracts, and lumbar spinal cord. Axonal dystrophy in the CNS was not accompanied by neuronal degeneration/necrosis, nerve fiber degeneration, and/or glial reaction. Axonal dystrophy was not observed in the PNS (sciatic nerve, vagus nerve branches, or gastrointestinal mural autonomic plexuses).

Background CBA/J mice are standard experimental animals in auditory studies, and age-related changes in auditory pathways are well documented. However, changes in locomotion-related brain regions have not been systematically explored. Results We showed an increase in immunoreactivity for glial fibrillary acidic protein (GFAP) in the cerebellar molecular layer associated with Purkinje cells in mice at 24 weeks of age but not in the younger mice. Increased GFAP immunoreactivity appeared in the form of clusters and distributed multifocally consistent with hyperplasia of astrocytes that were occasionally associated with Purkinje cell degeneration. Three out of 12 animals at 16 and 24 weeks of age exhibited pre-convulsive clinical signs. Two of these 3 animals also showed increased GFAP immunoreactivity in the cerebellum. Rotarod behavioral assessments indicated decreased performance at 24 weeks of age. Conclusions These results suggest minimal to mild reactive astrocytosis likely associated with Purkinje cell degeneration in the cerebellum at 24 weeks of age in CBA/J mice. These findings should be taken into consideration prior to using this mouse strain for studying neuroinflammation or aging.

The use of Wistar Han (WH) rats in regulatory toxicology studies, including juvenile animal studies, is increasing. The current study was performed to characterize femur development in neonatal and juvenile WH rats. Beginning on postnatal day (PND) 4 through PND 50, groups of WH rats (n = 5/sex) were evaluated for the development and ossification of the femur by Alizarin Red S staining, micro-computed tomography (micro-CT) imaging, microscopic examination, and Collagen X immunostaining. The focus of these evaluations was limited to the proximal femur including the head, neck, greater trochanter, lesser trochanter, and shaft. Through the multiple methods utilized, it was determined that the femoral neck is the first structure of the proximal femur to ossify, which occurs between PND 15 and 20. The femoral head and greater trochanter begin to separate on PND 15 with separation completing on PND 25 (both still composed of cartilage). Evidence of mineralization and ossification of these structures is apparent at PND 25, with a progressive increase with age, reaching adult-like morphology by PND 40. This work has provided a thorough characterization of postnatal femur development in WH rats to help inform bone and femur effects in juvenile toxicity studies as well as the design of investigative studies.