Safe and Sustained Expression of Human Iduronidase After Intrathecal Administration of Adeno-Associated Virus Serotype 9 in Infant Rhesus Monkeys

Abstract

Many neuropathic diseases cause early, irreversible neurologic deterioration, which warrants therapeutic intervention during the first months of life. In the case of mucopolysaccharidosis type I, a recessive lysosomal storage disorder that results from a deficiency of the lysosomal enzyme α-l-iduronidase (IDUA), one of the most promising treatment approaches is to restore enzyme expression through gene therapy. Specifically, administering pantropic adeno-associated virus (AAV) encoding IDUA into the cerebrospinal fluid (CSF) via suboccipital administration has demonstrated remarkable efficacy in large animals. Preclinical safety studies conducted in adult nonhuman primates supported a positive risk-benefit profile of the procedure while highlighting potential subclinical toxicity to primary sensory neurons located in the dorsal root ganglia (DRG). This study investigated the long-term performance of intrathecal cervical AAV serotype 9 gene transfer of human IDUA administered to 1-month-old rhesus monkeys (N = 4) with half of the animals tolerized to the human transgene at birth via systemic administration of an AAV serotype 8 vector expressing human IDUA from the liver. Sustained expression of the transgene for almost 4 years is reported in all animals. Transduced cells were primarily pyramidal neurons in the cortex and hippocampus, Purkinje cells in the cerebellum, lower motor neurons, and DRG neurons. Both tolerized and non-tolerized animals were robust and maintained transgene expression as measured by immunohistochemical analysis of brain tissue. However, the presence of antibodies in the non-tolerized animals led to a loss of measurable levels of secreted enzyme in the CSF. These results support the safety and efficiency of treating neonatal rhesus monkeys with AAV serotype 9 gene therapy delivered into the CSF.

Keywords: AAV9; MPS I; infant; intrathecal; rhesus monkey.

Figure 1.

Body-weight gain in rhesus monkeys after a single AAV9.hIDUA intrathecal injection at 4 weeks of age. Body weight was monitored regularly and compared to historical controls. Dashed lines represent the average body weight ±2 SD from historical controls. F, female; M, male; AAV9, adeno-associated virus serotype 9; hIDUA, human α-L-iduronidase; SD, standard deviation.

Figure 2.

Sustained increased CSF IDUA activity in tolerized rhesus monkeys after a single AAV9.hIDUA intrathecal (i.t.) injection at 4 weeks of age. (A) A 4-methylumbelliferone-based artificial substrate assay was used to measure IDUA activity in CSF samples from monkeys aged 4 weeks to 3 years and 9 months. Results are expressed as nanomoles of fluorescent product released per milliliter every 3 h (the detection limit was 0.1 IU/mL/3 h). The gray area between the dotted lines represents the average ±1 SD of IDUA activity obtained at baseline in the CSF of 26 rhesus monkeys, including the pre-vector samples from infants (N = 4) and juveniles/adults unrelated to the study (N = 22). (B) ELISA measuring anti-hIDUA antibody levels in CSF (dilution 1:20). (C) ELISA measuring anti-hIDUA antibody levels in serum (dilution 1:1,000). CSF, cerebrospinal fluid; ELISA, enzyme-linked immunosorbent assay.

Figure 3.

Long-term expression of hIDUA in neurons. Immunostaining of hIDUA 3 years and 8 months post AAV9.hIDUA i.t. injection. Representative stains from the frontal cortex (A and B), cerebellum (C and D), and cervical spinal cord (E and F) are shown. Scale bars: (A, B, E, and F) 100 μm; (C and D) 200 μm. (G) Quantification of hIDUA-positive neurons (% of total two neurons) in deep layers of the frontal cortex, hippocampus layers CA2-CA3, Purkinje cell layer in the cerebellum, cervical, thoracic, lumbar spinal cord anterior horns, cervical, thoracic, and lumbar DRG. Negative control = untreated rhesus monkey tissues stained with the same hIDUA antibody.

Figure 4.

Representative histology of DRG, spinal cord axons, and peripheral nerves. Microscopic images of central and peripheral nervous system tissues of rhesus monkeys administered AAV9 vector expressing human IDUA via intrathecal cervical injection at 4 weeks of age. There were no test article-related microscopic findings in either the tolerized or non-tolerized cohort. The images are representative of the central and peripheral nervous system tissues from each cohort. The cervical DRG (A and D), thoracic spinal cord (B and E), and sciatic nerve (C and F) were microscopically normal in all animals. Hematoxylin and eosin staining. Scale bars: (A, C, D, and F) 100 μm; (B and E) 200 μm.