Novel Biomarkers of Human GM1 Gangliosidosis Reflect the Clinical Efficacy of Gene Therapy in a Feline Model

Abstract

GM1 gangliosidosis is a fatal neurodegenerative disease that affects individuals of all ages. Favorable outcomes using adeno-associated viral (AAV) gene therapy in GM1 mice and cats have prompted consideration of human clinical trials, yet there remains a paucity of objective biomarkers to track disease status. We developed a panel of biomarkers using blood, urine, cerebrospinal fluid (CSF), electrodiagnostics, 7 T MRI, and magnetic resonance spectroscopy in GM1 cats-either untreated or AAV treated for more than 5 years-and compared them to markers in human GM1 patients where possible. Significant alterations were noted in CSF and blood of GM1 humans and cats, with partial or full normalization after gene therapy in cats. Gene therapy improved the rhythmic slowing of electroencephalograms (EEGs) in GM1 cats, a phenomenon present also in GM1 patients, but nonetheless the epileptiform activity persisted. After gene therapy, MR-based analyses revealed remarkable preservation of brain architecture and correction of brain metabolites associated with microgliosis, neuroaxonal loss, and demyelination. Therapeutic benefit of AAV gene therapy in GM1 cats, many of which maintain near-normal function >5 years post-treatment, supports the strong consideration of human clinical trials, for which the biomarkers described herein will be essential for outcome assessment.

Keywords: AAV gene therapy; gangliosidosis; lysosomal storage disorders; neurodegeneration.

Figure 1

GM1+AAV Cat Survival and CSF Biomarkers in GM1 Cats and Humans (A) Kaplan-Meier curve showing survival of GM1 cats after AAV gene therapy. Ongoing GM1 cats treated by AAV1 or AAVrh8 display survival up to or beyond 60 months of age compared to a mean survival of 8.0 ± 0.6 months in untreated GM1 cats. (B) Aspartate aminotransferase (AST) was significantly elevated in untreated GM1 cats (n = 4; p = 0.01) compared to normal cats (n = 16; ages ranging from 4 months to 5 years). CSF was evaluated at 16 weeks in the GM1+AAVrh8 cohort (n = 4) and GM1+AAV1 cohort (n = 3). In long-term (LT) follow-up of animals with minimal clinical disease (2–3 years post-treatment; n = 5 per serotype), AST levels were significantly reduced compared to untreated levels (p = 0.01). When treated animals reached the clinical humane endpoint (n = 3 from AAV1 and n = 2 from AAVrh8), AST levels were intermediate to normal and untreated levels. (C) CSF AST levels were increased in two late-infantile GM1 patients compared to control patients who did not have lysosomal storage disease (n = 13). CSF AST was near normal levels in almost all juvenile patients (n = 14), except for one (GSL020) who, like the late-infantile patients, had accelerated neurologic decline at the time of testing. (D) LDH was significantly elevated in the untreated GM1 cat (n = 4) compared to normal (n = 16; p = 0.003), and gene therapy normalized LDH levels both at 16 weeks (AAVrh8, n = 4; AAV1, n = 3) and long term (n = 5 per group; p = 0.01). (E) LDH in CSF from late-infantile GM1 patients (n = 2) was significantly increased compared to the juvenile cohort (n = 14). Normal LDH values were not measured. *p < 0.05 and **p < 0.01 from normal control cats, ^ƚp < 0.05 and ^Ŧp < 0.01 from untreated GM1 cats at humane endpoint, ^⋄⋄p < 0.01 from juvenile GM1 patients.

Figure 2

CSF and Blood-Based Biomarkers of GM1 Gangliosidosis in Cats and Humans Blood-based biomarkers were measured at 5–12 weeks, 19–31 weeks, 50–80 weeks, and 100+ weeks in normal cats, GM1 cats, and GM1+AAVrh8- and GM1+AAV1-treated cats. Biomarkers were also tested in human patients. See the symbol legend at the bottom of the figure for cohort designations. (A) Blood AST levels were elevated in the untreated GM1 cat and normalized after AAV gene therapy. (B) AST abnormalities in CSF were also found in serum. *p < 0.05 and **p < 0.01 from CSF within a cohort. (C) Serum AST levels in each cohort of human GM1 patients (with trend lines shown). (D) Serum AST levels in individual human patients (left panel) or cats (right panel) over time are connected by solid lines, demonstrating that AST correlates with disease progression in any given GM1 cat or infantile patient. Utility of serum AST to chart disease progression in late-infantile patients is less clear (left panel). Other parameters altered in serum included calcium (E and F), creatinine (G and H), and albumin (I and J). Yellow horizontal lines indicate age-normalized levels in human patients. Cat numbers for serum: 5–12 week, 19–31 week, 50–80 week, and 100+ week cohorts, respectively, are as follows: normal, n = 16, 25, 11, and 15; GM1, n = 7, 18, 0, and 0; AAVrh8, n = 8, 12, 10, and 17; AAV1, n = 7, 7, 6, and 18. Cat numbers for CSF in (B): normal, n = 16; GM1, n = 4; GM1+AAV, n = 5 per cohort. Human numbers: infantile, n = 7; late infantile, n = 11; juvenile, n = 42. Statistics for all cat data except (B): *p < 0.05 and **p < 0.01 from normal control cats, ^ƚp < 0.05 and ^Ŧp < 0.01 from untreated GM1 cats at humane endpoint. Statistics for human data: **p < 0.01 from infantile, ^ƚp < 0.05 from late infantile, ^Ŧp < 0.01 from late infantile, ^⋄⋄p < 0.01 from juvenile GM1 patients.

Figure 3

EEG Tracings of GM1 Cats and Humans and the Effect of AAV Gene Therapy The late-infantile GM1 patient GSL013 exhibits rhythmic slowing of brain waves (A) compared to an age-matched normal control (B). Similarly, the GM1 cat exhibits rhythmic slowing (C) compared to normal controls (D). Between 4.5 and 5 years after AAV gene therapy, rhythmic slowing is largely ameliorated in treated cats (E and F). However, epileptiform activity is prominent in one AAV-treated cat (9-1356) just before endpoint (E). Representative images are shown from multiple EEGs: GM1 patient, n = 11; normal patient, n = 3; GM1 cat, n = 3; normal cat, n = 2; GM1+AAV1, n = 1; GM1+AAVrh8, n = 2.

Figure 4

Ultra-High-Field T2-Weighted MRI of GM1 Cats Normal cats have distinct regions of hypointense (dark) white matter and hyperintense (light) gray matter in the cortex or thalamus (A) and cerebellum (E), with DCN hypointensity especially prominent. Indistinct regions of white and gray matter (isointense) develop in untreated GM1 cats, perhaps most apparent in the DCN (F). Some regions of cortical white matter even become hyperintense to (lighter than; black arrow) gray matter, and cortical atrophy is noted by the increase in CSF (bright white; black arrowhead outlined in white) outlining the brain due to diminished gyri and enlarged sulci (B). Five years after gene therapy, gray and white matter intensities are largely restored in AAV-treated cats (C, D, G, and H) and thalamic hyperintensities (black arrowhead) are common (C and D), though not universal (i.e., not all treated cats had hyperintensities). Cat 8-1435 exhibits an area of hypointensity at the site of a thalamic hemorrhage during surgery (white arrowhead), from which the cat made a complete clinical recovery. In addition, bilateral hyperintense areas in the brainstem near the olivary nucleus are noted in one treated cat (8-1435; white arrow; H). Shown are representative images of several MRI scans: normal, n = 4; GM1, n = 6; GM1+AAVrh8, n = 3.

Figure 5

Single-Voxel Magnetic Resonance Spectroscopy of GM1 Cats MRS was performed in six voxels: thalamus (A), corona radiata (B), temporal cortex (C), parietal cortex (D), occipital cortex (E), and cerebellum (F) in normal cats at 4 months, 8 months, and 2–5 years (n = 4 per cohort); untreated GM1 cats at 4 months (n = 6) and at 8 months (n = 6); and GM1+AAVrh8 cats at 3–5 years (n = 3). Metabolites analyzed include the glial cell marker myoinositol (INS), neuronal marker N-acetylaspartate (NAA), demyelination indicators glycerophosphocholine and phosphocholine (GPC+PCh), NAA+N-acetylglutamate (NAA+NAAG), metabolism markers creatine and phosphocreatine (Cr+PCr), and glutamate and glutamine (Glu+Gln). *p < 0.05 and **p < 0.01 from normal control cats, ^ƚp < 0.05 from untreated GM1 cats at humane endpoint. Error bars are SD.