Feasibility and safety of systemic rAAV9-hNAGLU delivery for treating mucopolysaccharidosis IIIB: toxicology, biodistribution, and immunological assessments in primates

Abstract

No treatment is currently available for mucopolysaccharidosis (MPS) IIIB, a neuropathic lysosomal storage disease caused by autosomal recessive defect in α-N-acetylglucosaminidase (NAGLU). In anticipation of a clinical gene therapy treatment for MPS IIIB in humans, we tested the rAAV9-CMV-hNAGLU vector administration to cynomolgus monkeys (n=8) at 1E13 vg/kg or 2E13 vg/kg via intravenous injection. No adverse events or detectable toxicity occurred over a 6-month period. Gene delivery resulted in persistent global central nervous system and broad somatic transduction, with NAGLU activity detected at 2.9-12-fold above endogenous levels in somatic tissues and 1.3-3-fold above endogenous levels in the brain. Secreted rNAGLU was detected in serum. Low levels of preexisting anti-AAV9 antibodies (Abs) did not diminish vector transduction. Importantly, high-level preexisting anti-AAV9 Abs lead to reduced transduction in liver and other somatic tissues, but had no detectable impact on transgene expression in the brain. Enzyme-linked immunoabsorbent assay showed Ab responses to both AAV9 and rNAGLU in treated animals. Serum anti-hNAGLU Abs, but not anti-AAV9 Abs, correlated with the loss of circulating rNAGLU enzyme. However, serum Abs did not affect tissue rNAGLU activity levels. Weekly or monthly peripheral blood interferon-γ enzyme-linked immunospot assays detected a CD4(+) T-cell (Th-1) response to rNAGLU only at 4 weeks postinjection in one treated subject, without observable correlation to tissue transduction levels. The treatment did not result in detectable CTL responses to either AAV9 or rNAGLU. Our data demonstrate an effective and safe profile for systemic rAAV9-hNAGLU vector delivery in nonhuman primates, supporting its clinical potential in humans.

Figures

FIG. 1.

No abnormal changes in blood chemistry in NHP after a systemic rAAV9-hNAGLU vector delivery. NHP were treated with an IV injection of 1E13 vg/kg (a, Group 1, adult) or 2E13 vg/kg (b, Group 2, sexually immature) rAAV9-CMV-hNAGLU vector. Blood samples were assayed for blood chemistry panel before and after the vector delivery. AAV, adeno-associated virus; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate Aminotransferase; CK, creatine kinase; IV, intravenous; NHP, nonhuman primates; NT, nontreated controls; V, treated with vector only; V+P, treated with vector and immunosuppression.

FIG. 2.

No abnormal changes in hematology in NHP receiving a systemic rAAV9-hNAGLU vector delivery. (a) Group 1: 1E13 vg/kg group. (b) Group 2: 2E13 vg/kg group. Blood samples were assayed for hematology before and after the vector delivery. *Subject with high preexisting anti-AAV9 antibodies. RBC, red blood cell count; WBC, white blood cell count.

FIG. 3.

Effective CNS and widespread somatic transduction in NHP after a systemic rAAV9-hNAGLU gene delivery. (a) Tissue NAGLU activity. NAGLU activity is expressed as units/mg protein, 1 unit=release of 1 nmol 4MU/hr. Group 1: treated with 1E13 vg/kg; Group 2: treated with 2E13 vg/kg. (b) Immunofluorescence detection for rNAGLU using a polyclonal Ab against hNAGLU. Red fluorescence indicates NAGLU-positive cells/signals. Yellow arrows indicate neurons; blue arrows, blood vessels; yellow arrowheads, NAGLU-overexpressing hepatocytes; green arrowheads, NAGLU-overexpressing cardiomyocytes. *Subject with high preexisting anti-AAV9 antibodies. CNS, central nervous system; HRT, heart; LIV, liver; TH, thalamus (brain).

FIG. 4.

Serum and CSF NAGLU activity in NHP after rAAV9 vector delivery (a) Group 1: 1E13 vg/kg group. (b) Group 2: 2E13 vg/kg group. Serum and CSF samples were assayed for NAGLU activity at end point. NAGLU activity is expressed as units/ml. 1 unit NAGLU activity=formation of 4MU/hr. *Subject with high preexisting anti-AAV9 antibodies.

FIG. 5.

Systemic rAAV9 gene delivery-mediated robust antibody response against the vector. Serum samples were assayed weekly and/or monthly for antibodies against AAV9 vector by ELISA. (a) Group 1: 1E13 vg/kg group. (b) Group 2: 2E13 vg/kg group. *Subject with high preexisting anti-AAV9 antibodies. ELISA, enzyme-linked immunoabsorbent assay.

FIG. 6.

rAAV9-hNAGLU gene delivery-mediated antibody responses against rNAGLU and serum NAGLU activity levels. Group 1: 1E13 vg/kg group. Group 2: 2E13 vg/kg group. Serum samples were assayed weekly and/or monthly for antibodies against the full-length hNAGLU protein by ELISA. (a) Anti-hNAGLU IgG levels. (b) Correlation of anti-hNAGLU IgG levels to serum NAGLU activity. *Subject with high preexisting anti-AAV9 antibodies.