Assessment of Safety and Biodistribution of AAVrh.10hCLN2 Following Intracisternal Administration in Nonhuman Primates for the Treatment of CLN2 Batten Disease.

CLN2 disease is a fatal, childhood autosomal recessive disorder caused by mutations in ceroid lipofuscinosis type 2 (CLN2) gene, encoding tripeptidyl peptidase 1 (TPP-1). Loss of TPP-1 activity leads to accumulation of storage material in lysosomes and resultant neuronal cell death with neurodegeneration. Genotype/phenotype comparisons suggest that the phenotype should be ameliorated with increase of TPP-1 levels to 5-10% of normal with wide central nervous system (CNS) distribution. Our previous clinical study showed that intraparenchymal (IPC) administration of AAVrh.10hCLN2, an adeno-associated vector serotype rh.10 encoding human CLN2, slowed, but did not stop disease progression, suggesting that this may be insufficient to distribute the therapy throughout the CNS (Sondhi 2020). In this study, we assessed whether the less invasive intracisternal delivery route would be safe and provide a wider distribution of TPP-1. A study was conducted in nonhuman primates (NHPs) with intracisternal delivery to cerebrospinal fluid (CSF) of AAVrh.10hCLN2 (5 × 1013 genome copies) or phosphate buffered saline (PBS). No abnormal behavior was noted. CNS magnetic resonance imaging and clinical chemistry data were all unremarkable. Histopathology of major organs had no abnormal finding attributable to the intervention or the vector, except that in one out of two animals treated with AAVrh.10hCLN2, dorsal root ganglia showed mild-to-moderate mononuclear cell infiltrates and neuronal degeneration. In contrast to our previous NHP study (Sondhi 2012) with IPC administration where TPP-1 activity was >2 × above controls in 30% of treated brains, in the two intracisternal treated NHPs, the TPP-1 activity was >2 × above controls in 50% and 41% of treated brains, and 52% and 84% of brain had >1,000 vector genomes/µg DNA, compared to 0% in the two PBS NHP. CSF TPP1 levels in treated animals were 43-62% of normal human levels. Collectively, these data indicate that AAVrh.10hCLN2 delivered by intracisternal route is safe and widely distributes TPP-1 in brain and CSF at levels that are potentially therapeutic. Clinical Trial Registration: NCT02893826, NCT04669535, NCT04273269, NCT03580083, NCT04408625, NCT04127578, and NCT04792944.

Identification of Safe and Effective Intravenous Dose of AAVrh.10hFXN to Treat the Cardiac Manifestations of Friedreich's Ataxia.

Friedreich's ataxia (FA) is a life-threatening autosomal recessive disorder characterized by neurological and cardiac dysfunction. Arrhythmias and heart failure are the main cause of premature death. From prior studies in murine models of FA, adeno-associated virus encoding the normal human frataxin gene (AAVrh.10hFXN) effectively treated the cardiac manifestations of the disease. However, the therapeutic dose window is limited by high level of human frataxin (hFXN) gene expression associated with toxicity. As a therapeutic goal, since FA heterozygotes have no clinical manifestations of FA, we estimated the level of frataxin (FXN) necessary to convert the heart of a homozygote to that of a heterozygote. In noncardiac cells, FA heterozygotes have 30-80% of normal FXN levels (17.7-47.2 ng/mg, average 32.5 ng/mg) and FA homozygotes 2-30% normal levels (1.2-17.7 ng/mg, average 9.4 ng/mg). Therefore, an AAV vector would need to augment endogenous in an FA homozygote by >8.3 ng/mg. To determine the required dose of AAVrh.10hFXN, we administered 1.8 × 1011, 5.7 × 1011, or 1.8 × 1012 gc/kg of AAVrh.10hFXN intravenously (IV) to muscle creatine kinase (mck)-Cre conditional knockout Fxn mice, a cardiac and skeletal FXN knockout model. The minimally effective dose was 5.7 × 1011 gc/kg, resulting in cardiac hFXN levels of 6.1 ± 4.2 ng/mg and a mild (p < 0.01 compared with phosphate-buffered saline controls) improvement in mortality. A dose of 1.8 × 1012 gc/kg resulted in cardiac hFXN levels of 33.7 ± 6.4 ng/mg, a significant improvement in ejection fraction and fractional shortening (p < 0.05, both comparisons) and a 21.5% improvement in mortality (p < 0.001). To determine if the significantly effective dose of 1.8 × 1012 gc/kg could achieve human FA heterozygote levels in a large animal, this dose was administered IV to nonhuman primates. After 12 weeks, the vector-expressed FXN in the heart was 17.8 ± 4.9 ng/mg, comparable to the target human levels. These data identify both minimally and significantly effective therapeutic doses that are clinically relevant for the treatment of the cardiac manifestations of FA.

Safety of Intravenous Administration of an AAV8 Vector Coding for an Oxidation-Resistant Human α1-Antitrypsin for the Treatment of α1-Antitrypsin Deficiency.

α1-antitrypsin (AAT) deficiency is a common autosomal recessive hereditary disorder, with a high risk for the development of early-onset panacinar emphysema. AAT, produced primarily in the liver, functions to protect the lung from neutrophil protease; with AAT deficiency, unimpeded neutrophil proteases destroy the lung parenchyma. AAT is susceptible to oxidative damage resulting in an inability to inhibit its target proteases, neutrophil elastase, and cathepsin G. The major sites of oxidative modification on the AAT molecule are methionine residues 351 and 358. We have previously demonstrated that an engineered variant of AAT that resists oxidation by modifying both protein surface methionines (M351V and M358L) provides antiprotease protection, despite oxidative stress. In mice, intravenous delivery of the modified AAT(AVL) variant by AAV serotype 8, AAV8hAAT(AVL), primarily to the liver resulted in long-term expression of an AAT that resists oxidative inactivation. In this study, we evaluated the safety of intravenous administration of AAV8hAAT(AVL) in a dose-escalating, blinded, placebo-controlled toxicology study in wild-type mice. The study assessed organ histology and clinical pathology findings of mice, intravenously administered AAV8hAAT(AVL) at three doses (5.0 × 1011, 5.0 × 1012, and 5.0 × 1013 genome copies [gc]/kg), compared to control mice injected intravenously with phosphate-buffered saline. As previously demonstrated, administration of AAV8hAAT(AVL) resulted in dose-dependent expression of high, potentially therapeutic, levels of serum human AAT protein that persist for at least 6 months. Antibodies against the AAV8 capsid were elicited as expected, but there was no antibody detected against the AAT(AVL) protein generated by the AAV8hAAT(AVL) vector. There was no morbidity or mortality observed in the study. The data demonstrate that intravenous administration of AAV8hAAT(AVL) is safe with no significant adverse effect attributed to AAV8hAAT(AVL) vector at any dose. This study demonstrates that AAV8hAAT(AVL) has a safety profile consistent with the requirements for proceeding to a clinical study.