A new genotype of hepatitis A virus causing transient liver enzyme elevations in Mauritius-origin laboratory-housed Macaca fascicularis.

Hepatitis A virus (HAV) infects humans and nonhuman primates, typically causing an acute self-limited illness. Three HAV genotypes have been described so far for humans, and three genotypes have been described for nonhuman primates. We observed transiently elevated liver enzymes in Mauritius-origin laboratory-housed macaques in Germany and were not able to demonstrate an etiology including HAV by serology and polymerase chain reaction (PCR). HAV is a rare pathogen in cynomolgus macaques, and since all employees were routinely vaccinated against HAV, it was not a part of the routine vaccination and screening program. A deep sequencing approach identified a new HAV genotype (referred to as Simian_HAV_Macaca/Germany/Mue-1/2022) in blood samples from affected animals. This HAV was demonstrated by reverse transcription PCR in blood and liver and by in situ hybridization in liver, gall bladder, and septal ducts. A commercial vaccine was used to protect animals from liver enzyme elevation. The newly identified simian HAV genotype demonstrates 80% nucleotide sequence identity to other simian and human HAV genotypes. There was deeper divergence between Simian_HAV_Macaca/Germany/Mue-1/2022 and other previously described HAVs, including both human and simian viruses. In situ hybridization indicated persistence in the biliary epithelium up to 3 months after liver enzymes were elevated. Vaccination using a commercial vaccine against human HAV prevented reoccurrence of liver enzyme elevations. Because available assays for HAV did not detect this new HAV genotype, knowledge of its existence may ameliorate potential significant epidemiological and research implications in laboratories globally.

Neurofilament Light Chain: A Translational Safety Biomarker for Drug-Induced Peripheral Neurotoxicity.

Branaplam is a splicing modulator previously under development as a therapeutic agent for Spinal Muscular Atrophy Type 1 and Huntington's disease. Branaplam increased the levels of survival motor neuron protein in preclinical studies and was well tolerated in early clinical studies; however, peripheral neurotoxicity was observed in a preclinical safety study in juvenile dogs. The aim of this study was to determine whether serum neurofilament light chain (NfL) concentrations in dogs could serve as a monitoring biomarker for branaplam-induced peripheral neurotoxicity. A 30-week time-course investigative study in dogs treated with vehicle control (negative control), neurotoxic pyridoxine (positive control), or branaplam was conducted to assess neuropathology, nerve morphometry, electrophysiological measurements, gene expression profiles, and correlation to NfL serum concentrations. In branaplam-treated animals, a mild to moderate nerve fiber degeneration was observed in peripheral nerves correlating with increased serum NfL concentrations, but there were no observed signs or changes in electrophysiological parameters. Dogs with pyridoxine-induced peripheral axonal degeneration displayed clinical signs and electrophysiological changes in addition to elevated serum NfL. This study suggests that NfL may be useful as an exploratory biomarker to assist in detecting and monitoring treatment-related peripheral nerve injury, with or without clinical signs, associated with administration of branaplam and other compounds bearing a neurotoxic risk.

Liver injury in cynomolgus monkeys following intravenous and intrathecal scAAV9 gene therapy delivery.

Hepatotoxicity associated with intravenous/intrathecal adeno-associated virus (AAV) gene therapy has been observed in preclinical species and patients. In nonhuman primates, hepatotoxicity following self-complementary AAV9 administration varies from asymptomatic transaminase elevation with minimal to mild microscopic changes to symptomatic elevations of liver function and thromboinflammatory markers with microscopic changes consistent with marked hepatocellular necrosis and deteriorating clinical condition. These transient acute liver injury marker elevations occur from 3-4 days post intravenous administration to ∼2 weeks post intrathecal administration. No transaminase elevation or microscopic changes were observed with intrathecal administration of empty capsids or a "promoterless genome" vector, suggesting that liver injury after cerebrospinal fluid dosing in nonhuman primates is driven by viral transduction and transgene expression. Co-administration of prednisolone after intravenous or intrathecal dosing did not prevent liver enzyme or microscopic changes despite a reduction of T lymphocyte infiltration in liver tissue. Similarly, co-administration of rituximab/everolimus with intrathecal dosing failed to block AAV-driven hepatotoxicity. Self-complementary AAV-induced acute liver injury appears to correlate with high hepatocellular vector load, macrophage activation, and type 1 interferon innate virus-sensing pathway responses. The current work characterizes key aspects pertaining to early AAV-driven hepatotoxicity in cynomolgus macaques, highlighting the usefulness of this nonclinical species in that context.

Neurofilament light chain and dorsal root ganglia injury after adeno-associated virus 9 gene therapy in nonhuman primates.

In nonhuman primates (NHPs), adeno-associated virus serotype 9 (AAV9) vectorized gene therapy can cause asymptomatic microscopic injury to dorsal root ganglia (DRG) and trigeminal ganglia (TG) somatosensory neurons, causing neurofilament light chain (NfL) to diffuse into cerebrospinal fluid (CSF) and blood. Data from 260 cynomolgus macaques administered vehicle or AAV9 vectors (intrathecally or intravenously) were analyzed to investigate NfL as a soluble biomarker for monitoring DRG/TG microscopic findings. The incidence of key DRG/TG findings with AAV9 vectors was 78% (maximum histopathology severity, moderate) at 2-12 weeks after the dose. When examined up to 52 weeks after the dose, the incidence was 42% (maximum histopathology severity, minimal). Terminal NfL concentrations in plasma, serum, and CSF correlated with microscopic severity. After 52 weeks, NfL returned to pre-dose baseline concentrations, correlating with microscopic findings of lesser incidence and/or severity compared with interim time points. Blood and CSF NfL concentrations correlated with asymptomatic DRG/TG injury, suggesting that monitoring serum and plasma concentrations is as useful for assessment as more invasive CSF sampling. Longitudinal assessment of NfL concentrations related to microscopic findings associated with AAV9 administration in NHPs indicates NfL could be a useful biomarker in nonclinical toxicity testing. Caution should be applied for any translation to humans.

Single-Dose Intrathecal Dorsal Root Ganglia Toxicity of Onasemnogene Abeparvovec in Cynomolgus Monkeys.

Intravenous onasemnogene abeparvovec is approved for the treatment of spinal muscular atrophy in children < 2 years. For later-onset patients, intrathecal onasemnogene abeparvovec may be advantageous over intravenous administration. Recently, microscopic dorsal root ganglion (DRG) changes were observed in nonhuman primates (NHPs) following intrathecal onasemnogene abeparvovec administration. To characterize these DRG findings, two NHP studies evaluating intrathecal onasemnogene abeparvovec administration were conducted: a 12-month study with a 6-week interim cohort and a 13-week study with a 2-week interim cohort. The latter investigated the potential impact of prednisolone or rituximab plus everolimus on DRG toxicity. An additional 6-month, single-dose, intravenous NHP study conducted in parallel evaluated onasemnogene abeparvovec safety (including DRG toxicity) with or without prednisolone coadministration. Intrathecal onasemnogene abeparvovec administration was well tolerated and not associated with clinical observations. Microscopic onasemnogene abeparvovec-related changes were observed in the DRG and trigeminal ganglion (TG) and included mononuclear cell inflammation and/or neuronal degeneration, which was colocalized with high vector transcript expression at 6 weeks postdose. Incidence and severity of DRG changes were generally decreased after 52 weeks compared with 6 weeks postdose. Other onasemnogene abeparvovec-related microscopic findings of axonal degeneration, mononuclear cell infiltrates and/or gliosis in the spinal cord, dorsal spinal nerve root/spinal nerves, and/or peripheral nerves were absent or found at decreased incidences and/or severities after 52 weeks. DRG and/or TG microscopic findings following intravenous onasemnogene abeparvovec dosing included minimal to slight neuronal degeneration and mononuclear cell inflammation at 6 weeks and 6 months postdose. Nervous system microscopic findings following intrathecal onasemnogene abeparvovec (≥1.2 × 1013 vg/animal) trended toward resolution after 52 weeks, supporting nonprogression of changes, including in the DRG. Onasemnogene abeparvovec-related DRG findings were not associated with electrophysiology changes and were not ameliorated by prednisolone or rituximab plus everolimus coadministration. The pathogenesis is possibly a consequence of increased vector genome transduction and/or transgene expression.

Best practices for veterinary toxicologic clinical pathology, with emphasis on the pharmaceutical and biotechnology industries.

The purpose of this paper by the Regulatory Affairs Committee (RAC) of the American Society for Veterinary Clinical Pathology (ASVCP) is to review the current regulatory guidances (eg, guidelines) and published recommendations for best practices in veterinary toxicologic clinical pathology, particularly in the pharmaceutical and biotechnology industries, and to utilize the combined experience of ASVCP RAC to provide updated recommendations. Discussion points include (1) instrumentation, validation, and sample collection, (2) routine laboratory variables, (3) cytologic laboratory variables, (4) data interpretation and reporting (including peer review, reference intervals and statistics), and (5) roles and responsibilities of clinical pathologists and laboratory personnel. Revision and improvement of current practices should be in alignment with evolving regulatory guidance documents, new technology, and expanding understanding and utility of clinical pathology. These recommendations provide a contemporary guide for the refinement of veterinary toxicologic clinical pathology best practices.

Anatomic, hematologic, and biochemical features of C57BL/6NCrl mice maintained on chronic oral corticosterone.

Metabolic syndrome is a condition that typically includes central obesity, insulin resistance, glucose intolerance, dyslipidemia, and hypertension. Disruption of the hypothalamic-pituitary-adrenal axis, a regulator of corticosterone secretion, occurs in some cases of metabolic syndrome and obesity, and Cushing hypercortisolemia is associated with obesity and metabolic disorders. We therefore assessed anatomic and clinical pathology in C57BL/6NCrl mice to evaluate the effects of chronic corticosterone in the drinking water at doses of 25, 50, and 100 µg/mL for 25 d. Treated mice developed obesity, glucose intolerance, electrolyte aberrations, and dyslipidemia that were dose-dependent and most severe in the 100-mu;g/mL treatment group. To evaluate return to normal function, additional C57BL/6NCrl mice received corticosterone-free water for 2 wk after the 25-d treatment period. According to results of gross examination, mice appeared to recover within days of exogenous corticosterone withdrawal; however, adrenal gland vacuolation and protein, lipid, and electrolyte abnormalities persisted. Together, these findings support chronic corticosterone exposure through the drinking water as a potentially useful, noninvasive method to induce some features of metabolic syndrome.

Drug-induced hemolytic anemia and thrombocytopenia associated with alterations of cell membrane lipids and acanthocyte formation.

CXCR3 is a chemokine receptor, upregulated upon activation of T cells and expressed on nearly 100% of T cells in sites of inflammation. SCH 900875 is a selective CXCR3 receptor antagonist. Thrombocytopenia and severe hemolytic anemia with acanthocytosis occurred in rats at doses of 75, 100, and 150 mg/kg/day. Massively enlarged spleens corresponded histologically to extramedullary hematopoiesis, macrophages, and hemosiderin pigment and sinus congestion. Phagocytosed erythrocytes and platelets were within splenic macrophages. IgG and/or IgM were not detected on erythrocyte and platelet membranes. Ex vivo increased osmotic fragility of RBCs was observed. Lipid analysis of the RBC membrane revealed modifications in phosphatidylcholine, overall cholesterol, and/or sphingomyelin. Platelets exhibited slender filiform processes on their plasma membranes, analogous to those of acanthocytes. The presence of similar morphological abnormalities in acanthocytes and platelets suggests that possibly similar alterations in the lipid composition of the plasma membrane have taken place in both cell types. This phenotype correlated with alterations in plasma lipids (hypercholesterolemia and low triglycerides) that occurred after SCH 900875 administration, although other factors cannot be excluded. The increased cell destruction was considered triggered by alterations in the lipid profile of the plasma membranes of erythrocytes and platelets, as reflected morphologically.