Therapeutic benefit after intracranial gene therapy delivered during the symptomatic stage in a feline model of Sandhoff disease

Victoria J McCurdy, Aime K Johnson, Heather L Gray-Edwards, Ashley N Randle, Allison M Bradbury, Nancy E Morrison, Misako Hwang, Henry J Baker, Nancy R Cox, Miguel Sena-Esteves, Douglas R Martin, Victoria J McCurdy, Aime K Johnson, Heather L Gray-Edwards, Ashley N Randle, Allison M Bradbury, Nancy E Morrison, Misako Hwang, Henry J Baker, Nancy R Cox, Miguel Sena-Esteves, Douglas R Martin

Abstract

Sandhoff disease (SD) is an autosomal recessive lysosomal storage disease caused by defects in the β-subunit of β-N-acetylhexosaminidase (Hex), the enzyme that catabolizes GM2 ganglioside. Hex deficiency causes neuronal storage of GM2 and related glycoconjugates, resulting in progressive neurodegeneration and death, typically in infancy. No effective treatment exists for human patients. Adeno-associated virus (AAV) gene therapy led to improved clinical outcome and survival of SD cats treated before the onset of disease symptoms. Most human patients are diagnosed after clinical disease onset, so it is imperative to test AAV-gene therapy in symptomatic SD cats to provide a realistic indication of therapeutic benefits that can be expected in humans. In this study, AAVrh8 vectors injected into the thalamus and deep cerebellar nuclei of symptomatic SD cats resulted in widespread central nervous system enzyme distribution, although a substantial burden of storage material remained. Cats treated in the early symptomatic phase showed delayed disease progression and a significant survival increase versus untreated cats. Treatment was less effective when administered later in the disease course, although therapeutic benefit was still possible. Results are encouraging for the treatment of human patients and provide support for the development AAV-gene therapy for human SD.

Trial registration: ClinicalTrials.gov NCT02716246 NCT03315182.

Conflict of interest statement

Conflict of Interest

The authors are beneficiaries of a licensing agreement with Axovant Gene Therapies (New York City) based partly on this technology. Drs. Sena-Esteves and Martin are shareholders in Lysogene (Neuilly-sur-Seine, France).

Figures

Figure 1.
Figure 1.
Survival and clinical progression of post-symptomatic AAV-treated SD cats and untreated SD cats. (a) Age of symptom onset is shown for untreated SD cats (mean ± s.d, n = 14). The scale is based on gait defects, which ultimately define humane endpoint, with initial deficits at 2.1 months old. However, disease onset begins at 1.3 months old with subtle tremors of the head/tail that progress to overt whole-body tremors at 2.4 months old. Neurologic humane endpoint is reached at a score of 3. (b) Kaplan-Meier survival curves for untreated SD cats (solid black line, n = 14) and SD cats treated post-symptomatically. Survival of the EPS cohort is significantly increased compared to untreated (P = 0.0010, dashed black line, n = 4) and compared to the LPS cohort (P = 0.048). Survival of the LPS cohort is not significantly increased compared to untreated (P < 0.28, solid grey line, n = 3). Normal controls (not shown) remained alive and healthy throughout the study. (c, d) Composite clinical scores are shown for untreated SD cats (n = 14) while EPS (c) and LPS (d) treated cats are depicted separately. Open symbols denote the presence of subtle tremors, and checkered symbols denote the presence of overt whole body tremors, which were absent in all EPS treated cats, but present in all LPS treated cats. Scores were assigned using two separate, independent readouts, as described in materials and methods. Normal controls (not shown) scored a 10 on the clinical rating scale and had no tremors for the duration of the study.
Figure 2.
Figure 2.
Neurological exam performance of post-symptomatic AAV-treated SD cats versus untreated SD cats. Cats were given a neurological examination twice monthly and deficits were recorded. * Onset of deficit was significantly delayed compared to untreated SD cats (P ≤ 0.011); ▼, onset of deficit was significantly earlier compared to the EPS cohort (P ≤ 0.026). In the LPS cohort, pelvic hopping deficits and pelvic wheelbarrowing deficits were already apparent at the time of treatment for cat 7-866 and all deficits except thoracic wheelbarrowing were apparent at the time of treatment for cat 7-801. The first deficit to appear in untreated SD cats is pelvic hopping at 2.2 ± 0.7 months, and the last deficit to appear is thoracic wheelbarrowing at 3.0 ± 0.7 months. Abbreviations: P = pelvic limb; T = thoracic limb; EPT = extensor postural thrust; LPS = late post-symptomatic; EPS = early post-symptomatic. Data is expressed as mean ± s.d. No deficits were recorded in normal control cats.
Figure 3.
Figure 3.
MRI evaluation of late post-symptomatic AAV-treated SD cats and untreated controls. T2-weighted MR images (3 Tesla) were taken at the level of the caudate nucleus (a, c and e) and DCN (b, d and f). Cortical white matter is hypointense to (darker than) gray matter in normal cats but hyperintense to (lighter than) gray matter in untreated SD cats. Also, the DCN area is hypointense to surrounding gray matter in normal cats (outlined black arrowhead in panel b) but becomes hyperintense with disease progression in untreated SD cats. In LPS cat 11-994 at humane endpoint, hypointensity of cortical white to gray matter is improved compared to untreated but is not normal. However, the DCN area has turned hyperintense to gray matter, similar to untreated SD cats. At the time of imaging cat 11-994 was on a clinical rating score of 3 (Figure 1a). There were no appreciable differences between a normal cat brain at 6 months old and at older ages. Brain atrophy, indicated by the amount of CSF (bright white area), in cat 11-994 was comparable to the untreated SD cat.
Figure 4.
Figure 4.
Therapeutic enzyme distribution in the CNS of post-symptomatic AAV-treated SD cats. Tissues from post-symptomatic AAV-treated SD cats were collected at humane endpoint. (a) Shown are injection sites (white circles) and 0.6 cm coronal blocks of the brain (A-H) and spinal cord (I-O) as depicted in a previous study. Brain blocks were halved, and the right half was assayed for enzyme activity. Lysosomal Hex activity (red) was visualized throughout the brain (b) and spinal cord (c) of early post-symptomatic AAV-treated SD cat 11-831 (SD + AAV) at 18.9 months old. Similarly, Hex activity was visualized throughout the brain (d) and spinal cord (e) of late post-symptomatic AAV-treated SD cat 11-994 (SD + AAV) at 11.5 months old. Representative control sections are shown from untreated normal cats along with untreated SD cats, which express ≤0.02 fold normal HexA activity in the brain and spinal cord. Corresponding HexA activity against MUGS substrate is shown below each block as fold normal level (fold N). Each block from treated and untreated SD cats is normalized to the corresponding block from age-matched normal controls (the mean value from 5 normal cats). HexA specific activity in normal control cats ranged from 28.1 ± 7.5 to 57.4 ± 5.7 nmol 4MU/mg protein/hr in the brain and from 6.7 ± 0.8 to 17.5 ± 3.2 nmol 4MU/mg/hr in the spinal cord.
Figure 5.
Figure 5.
Storage in the CNS of post-symptomatic AAV-treated SD cats. (a) Storage in untreated SD cats is visualized by dark PAS staining in gray matter. In treated brains, ganglioside storage was present in Hex deficient areas (see corresponding naphthol staining for the same cat, 11-831, in Figures 4b and c), such as the temporal lobe (blocks d and e) and cervical spinal cord (block k). (b) Sample sites for sialic acid quantitation (circles) in brain (A-H) and spinal cord (K and O; half of each block was used). (c) Sialic acid levels were measured in untreated SD cats (n = 4) and in the EPS (n = 4) and LPS (n = 3) cohorts for comparison to normal cats (n = 4 for each cohort). **, all samples from untreated cats were significantly higher than normal (P = 0.015 for each block) in the cerebrum (2.6 – 4.9 fold normal), brainstem and cerebellum (2.0 - 3.8 fold normal) and spinal cord (3.2 - 3.8 fold normal); *, samples from treated cats that were not significantly higher than normal. Samples without this symbol were significantly higher than normal (P ≤ 0.030 for each block); ▼, samples from treated cats that were significantly lower than untreated (P ≤ 0.030); ▲, sample from treated cats that was significantly higher than untreated (P = 0.015); ●, sample that was significantly lower in the LPS cohort versus the EPS cohort (P = 0.037). Abbreviations: EPS = early post-symptomatic; LPS = late post-symptomatic; tx = treatment. Data is expressed as mean ± s.d. Samples were analyzed in duplicate and the average value is reported.
Figure 6.
Figure 6.
Normalization of lysosomal mannosidase activity in the CNS and liver of post-symptomatic AAV-treated SD cats. Cats from the EPS (n = 4, black bars) and LPS (n = 3, striped bars) cohorts were euthanized at humane endpoint and lysosomal mannosidase activity was compared to untreated SD cats (n = 5, gray bars) and normal cats (n = 5) in the brain (A-H), spinal cord (I-O), and liver (Liv). CNS sample lettering corresponds to Figure 4a. *, all samples from untreated SD cats were significantly higher than normal (A-H, I-O, and liver,P = 0.0061 for each block); **, samples B-F, H, I-O, and liver from the LPS cohort remained significantly higher than normal (P ≤ 0.037 for each block); ***, all CNS samples from the EPS cohort remained significantly higher than normal (P ≤ 0.019 for each block), but liver was not significantly higher than normal; ▼, samples from AAV-treated SD cats that were significantly lower than untreated (P ≤ 0.037); ●, samples that were significantly higher in the LPS cohort versus the EPS cohort (P ≤ 0.040). Data is expressed as mean ± s.d. Samples were analyzed in duplicate and the average value is reported.

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