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.

Malignant cells drive the generation of a desmoplastic and immunosuppressive tumor microenvironment. Cancer-associated stromal cells (CASCs) are a heterogeneous population that provides both negative and positive signals for tumor cell growth and metastasis. Fibroblast activation protein (FAP) is a marker of a major subset of CASCs in virtually all carcinomas. Clinically, FAP expression serves as an independent negative prognostic factor for multiple types of human malignancies. Prior studies established that depletion of FAP+ cells inhibits tumor growth by augmenting anti-tumor immunity. However, the potential for immune-independent effects on tumor growth have not been defined. Herein, we demonstrate that FAP+ CASCs are required for maintenance of the provisional tumor stroma since depletion of these cells, by adoptive transfer of FAP-targeted chimeric antigen receptor (CAR) T cells, reduced extracellular matrix proteins and glycosaminoglycans. Adoptive transfer of FAP-CAR T cells also decreased tumor vascular density and restrained growth of desmoplastic human lung cancer xenografts and syngeneic murine pancreatic cancers in an immune-independent fashion. Adoptive transfer of FAP-CAR T cells also restrained autochthonous pancreatic cancer growth. These data distinguish the function of FAP+ CASCs from other CASC subsets and provide support for further development of FAP+ stromal cell-targeted therapies for the treatment of solid tumors.

Improved delivery of adeno-associated virus (AAV) vectors to the CNS will greatly enhance their clinical utility. Selection of AAV9 variants in a mouse model led to the isolation of a capsid called PHP.B, which resulted in remarkable transduction of the CNS following intravenous infusion. However, we now show here that this enhanced CNS tropism is restricted to the model in which it was selected, i.e., a Cre transgenic mouse in a C57BL/6J background, and was not found in nonhuman primates or the other commonly used mouse strain BALB/cJ. We also report the potential for serious acute toxicity in NHP after systemic administration of high dose of AAV.

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.

Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer-related deaths worldwide, with an exceedingly low 5-year survival rate. PDAC tumors are characterized by an extensive desmoplastic stromal response and hypovascularity, suggesting that tumor hypoxia could regulate PDAC initiation and/or progression. Using a well-defined, autochthonous KrasG12D-driven murine model, as well as human tumors, we demonstrate that hypoxia and stabilization of hypoxia-inducible factor 1α (HIF1α), a principal mediator of hypoxic adaptation, emerge early during preinvasive stages of PDAC. Surprisingly, pancreas-specific Hif1α deletion drastically accelerated KrasG12D-driven pancreatic neoplasia, and was accompanied by significant increases in intrapancreatic B lymphocytes, featuring prominent influx of a rare “B1b” B cell subtype. Finally, treatment of HIF1α-deficient mice with B cell-depleting αCD20 monoclonal antibodies inhibited progression of pancreatic intraepithelial neoplasia (PanIN). Our data reveal a previously unrecognized role for B cells in promoting pancreatic tumorigenesis, and implicate HIF1α as a critical regulator of PDAC development.