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.
The administration of adeno-associated virus (AAV) vectors to nonhuman primates (NHP) via the blood or cerebrospinal fluid (CSF) can lead to dorsal root ganglion (DRG) pathology. The pathology is minimal to moderate in most cases; clinically silent in affected animals; and characterized by mononuclear cell infiltrates, neuronal degeneration, and secondary axonopathy of central and peripheral axons on histopathological analysis. We aggregated data from 33 nonclinical studies in 256 NHP and performed a meta-analysis of the severity of DRG pathology to compare different routes of administration, dose, time course, study conduct, age of the animals, sex, capsid, promoter, capsid purification method, and transgene. DRG pathology was observed in 83% of NHP that were administered AAV through the CSF, and 32% of NHP that received an intravenous (IV) injection. We show that dose and age at injection significantly affected the severity whereas sex had no impact. DRG pathology was minimal at acute time points (i.e., <14 days), similar from one to 5 months post-injection, and was less severe after 6 months. Vector purification method had no impact, and all capsids and promoters that we tested resulted in some DRG pathology. The data presented here from five different capsids, five different promoters, and 20 different transgenes suggest that DRG pathology is almost universal after AAV gene therapy in nonclinical studies using NHP. None of the animals receiving a therapeutic transgene displayed any clinical signs. Incorporation of sensitive techniques such as nerve-conduction velocity testing can show alterations in a minority of animals that correlate with the severity of peripheral nerve axonopathy. Monitoring sensory neuropathies in human central nervous system and high-dose IV clinical studies seems prudent to determine the functional consequences of DRG pathology.
The SARS-CoV-2 pandemic has affected more than 185 million people worldwide resulting in over 4 million deaths. To contain the pandemic, there is a continued need for safe vaccines that provide durable protection at low and scalable doses and are easily delivered. Here, AAVCOVID-1, an adeno-associated viral (AAV), Spike gene-based vaccine candidate demonstrates potent immunogenicity in mouse and nonhuman primates following a single injection and confers complete protection from SARS-CoV-2 challenge in macaques. Peak neutralizing antibody titers are sustained at 1 year and complemented by functional memory T-cell responses. The AAVCOVID vector has no relevant pre-existing immunity in humans, does not elicit cross-reactivity to common AAVs used in gene therapy, and its persistence and expression wanes following injection. The single, low dose requirement, high yield manufacturability, and 1-month stability for storage at room-temperature may make this technology well-suited to support effective immunization campaigns for emerging pathogens on a global scale.
SARS-CoV-2 variants have emerged with enhanced pathogenicity and transmissibility, and escape from pre-existing immunity, suggesting first-generation vaccines and monoclonal antibodies may now be less effective. Here we present an approach for preventing clinical sequelae and the spread of SARS-CoV-2 variants. First, we affinity matured an angiotensin-converting enzyme 2 (ACE2) decoy protein, achieving 1000-fold binding improvements that extend across a wide range of SARS-CoV-2 variants and distantly related, ACE2-dependent coronaviruses. Next, we demonstrated the expression of this decoy in proximal airway when delivered via intranasal administration of an AAV vector. This intervention significantly diminished clinical and pathologic consequences of SARS-CoV-2 challenge in a mouse model and achieved therapeutic levels of decoy expression at the surface of proximal airways when delivered intranasally to nonhuman primates. Importantly, this long-lasting, passive protection approach is applicable in vulnerable populations such as the elderly and immune-compromised that do not respond well to traditional vaccination. This approach could be useful in combating COVID-19 surges caused by SARS-CoV-2 variants and should be considered as a countermeasure to future pandemics caused by one of the many pre-emergent, ACE2-dependent CoVs that are poised for zoonosis.
Since its recent reformulation, alfaxalone has gained popularity as an injectable veterinary anesthetic, including promising studies demonstrating the use of alfaxalone-xylazine for anesthesia in mice. Here we sought to expand these studies by testing additional dose ranges, elaborating on physiologic monitoring, testing sex-and strain-associated differences, and evaluating efficacy during actual surgical conditions. C57BL/6J mice showed significant sex-associated differences in anesthetic sensitivity, with males requiring higher doses of alfaxalone (80-120 mg/kg IP alfaxalone with 10 mg/kg IP xylazine) than females (40-80 mg/kg IP alfaxalone with 10 mg/kg IP xylazine) to achieve a surgical plane of anesthesia. In addition, female outbred CD1 mice were less sensitive to alfaxalone than female inbred C57BL/6J mice. When used during actual surgery, alfaxalonexylazine administered intraperitoneally provided adequate anesthesia for a model of orthopedic surgery, whereas the same anesthetic regimen during laparotomy resulted in unacceptably high mortality; survival during laparotomy increased when drugs were administered subcutaneously. These results indicate that alfaxalone-xylazine may be a viable option for injectable surgical anesthesia in mice, although strain-and sex-associated differences and alternative routes of administration should be considered when optimizing the anesthetic regimen for specific experimental conditions.
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