Cynthia J. Tifft

GM1 gangliosidosis is an inherited, progressively neurodegenerative lysosomal storage disorder with no approved therapy. We report 5-year safety and 3-year clinical, biochemical, and neuroimaging efficacy outcomes following expanded-access intravenous AAV9-GLB1 gene therapy in GT01, a 6-10-year-old female with juvenile-onset GM1 gangliosidosis. During participation in a natural history study before gene transfer, GT01 developed seizures, dysarthria, loss of ambulation, and progressive neurodegeneration. She received a single intravenous administration of AAV9-GLB1 at 1.5×10 ¹³ vector genomes per kilogram of body weight. Early improvements included the resolution of dysphagia, increased interactions with others, assisted ambulation, and gains in specific domains of adaptive functioning. She was seizure-free without anti-epileptic drugs 18 months post dosing with sustained seizure freedom at the study completion. Cerebrospinal fluid concentrations of GM1 ganglioside and H3N2b (a pentasaccharide biomarker indicative of biochemical improvement in this disorder) concentrations decreased by 50% and β-galactosidase reached normal activity. Brain, thalamic, and net fiber tract volume assessed by differential tractography increased and ventriculomegaly improved three years post-dosing. These results indicate that meaningful benefit can be achieved even in advanced neurologic disease.

Due to shared ß-hexosamindase A deficiencies and significant clinical overlap, the predominating subtypes of late-onset GM2 gangliosidosis, late-onset Tay-Sachs (LOTS) and late-onset Sandhoff disease (LOSD) have been considered essentially indistinguishable. However, growing evidence supports several distinctions between the two entities. We highlight these distinctions through the cross-sectional evaluation of 27 (21 LOTS and 6 LOSD) late-onset GM2 gangliosidosis participants. Study protocol included physical examinations, assessments of gait, balance, muscle strength, ataxia, and nerve conduction velocities, and brain magnetic resonance imaging. Lower limb weakness and later development of upper limb weakness was highly prevalent in both diseases. Accompanying gait disturbances, balance issues, and dysmetria were also prevalent in both cohorts. Strength evaluations showed weakness in both the LOTS and LOSD cohorts compared to controls primarily impacting extensor muscles. In contrast, BARS scores for dysarthria and oculomotor dysfunction were present and heterogenous in LOTS participants and absent in LOSD participants. Twenty-four percent of LOTS participants and none of the LOSD participants had a history of neuropsychiatric symptoms. Cerebellar volume including lobules V and VI were lower in LOTS compared to LOSD and normative data. However, severe length-dependent sensory neuropathy was present in all LOSD participants but not in LOTS participants. The finding of lower cerebellar volume in LOTS suggests the distinctive features of the LOTS phenotype are related to cerebellar dysfunction. However, the cause of the phenotypic differences between LOTS and LOSD remains a mystery, and the molecular and biochemical basis for the dichotomy requires further investigation.

GM1 gangliosidosis, caused by biallelic variants in GLB1, results from deficiency of lysosomal β-galactosidase, which degrades GM1 ganglioside. This fatal neurodegenerative disease currently has no effective therapy. In a phase 1-2, open-label, dose-escalation study, we assessed immunosuppression and a single intravenous infusion of adeno-associated virus serotype 9 (AAV9) encoding β-galactosidase in children with type II GM1 gangliosidosis with late-infantile or juvenile onset. The primary end point was safety. Secondary end points included changes from baseline in the cerebrospinal fluid (CSF) GM1 ganglioside concentration and β-galactosidase activity, clinical assessments (including the Clinical Global Impression-Improvement [CGI-I] score, assessed on a scale from 1 [very much improved] to 7 [very much worse]), and neuroimaging patterns. Nine participants were enrolled. Over a 3-year period, 124 adverse events occurred, 30 of which (8 gastrointestinal events, 21 laboratory abnormalities associated with inflammation, and 1 tachycardia event) were deemed by the investigator as being possibly, probably, or definitely related to the gene therapy. Five serious adverse events occurred, including vomiting that led to hospitalization in one participant, which was attributed to the gene therapy. Serum aspartate and alanine aminotransferase levels increased in all participants and returned to baseline levels by 18 months. In all participants, the CSF β-galactosidase level increased and CSF GM1 ganglioside level decreased. Expressive communication and gross motor scores appeared stable, but fine motor and receptive communication scores decreased. The median CGI-I score was 3 (indicating minimal improvement) at 2 years and 4 (indicating no change) at 3 years; in historical controls, scores have been shown to increase (indicating worsening) over time. Neuroimaging showed patterns consistent with reduced rates of cerebral atrophy and favorable changes in myelination as compared with baseline. In this study involving nine participants with type II GM1 gangliosidosis, a single infusion of AAV9 encoding β-galactosidase was associated with adverse events, including severe vomiting in one participant and elevated liver-enzyme levels in all participants. Secondary end-point results suggested improvements in biochemical markers and neuroimaging patterns and stable or reduced rates of developmental deterioration in some measures. (Funded by the National Human Genome Research Institute and others; ClinicalTrials.gov number, NCT03952637.).

EHMT1 and EHMT2 genes encode human euchromatin histone lysine methyltransferase 1 and 2 (EHMT1 alias GLP; EHMT2 alias G9a) that form heteromeric GLP/G9a complexes with essential roles in epigenetic regulation of gene expression. While EHMT1 haploinsufficiency has been established as the cause of Kleefstra syndrome 1, the pathogenesis of G9a dysfunction in human disease remains largely unknown. We identified seven de novo EHMT2 variants in patients with clinical presentation, episignatures, histone modifications and transcriptomic profiles similar to those of Kleefstra syndrome 1. In vitro studies revealed that these variants encode for structurally stable G9a proteins that are catalytically incompetent due to aberrant interactions either with histone H3 tail or with S-adenosylmethionine. Heterozygous mice carrying a patient-derived variant exhibited growth retardation, facial/skull dysmorphia and aberrant behavior. Here we report pathogenic EHMT2 variants that likely exert dominant-negative effect on GLP/G9a complexes and thus genocopy the EHMT1 haploinsufficiency via a distinct molecular mechanism, defining an autosomal dominant EHMT2 -related Kleefstra syndrome.

AIM To define the natural history of diffusion tensor imaging (DTI) in Chediak-Higashi Disease (CHD) participants in relation to normative development. Methods Twenty-five DTI scans from 15 CHD participants were compared with 100 DTI scans from 100 neurotypical controls (NC). Comparisons were evaluated for DTI metrics including fractional anisotropy (FA), and mean, axial, and radial diffusivity (MD, AD, RD, respectively). Correlational tractography was also performed to identify group differences between CHD and NC. Results Pediatric CHD participants had DTI metrics similar to NC, however, progressive pathogenic increases in MD, AD, and RD were observed in CHD participants compared to NC in white matter pathways of the whole brain, corpus callosum, and cerebellum. Correlational fiber tractography identified fiber tracts with decreased FA, and increased MD, AD, and RD in CHD compared to NC. Interpretation Progressive deviations in DTI metrics highlight the progressive neurodegeneration of CHD. Aberrations in cerebellar white matter is reflective of the clinical neurologic phenotype of CHD including cerebellar dysfunction and cognitive decline. The corollary of progressive aberrations in DTI metrics and progressive clinical neurodegeneration suggest DTI may be a suitable neuroimaging marker for CHD. Future studies should evaluate functional magnetic resonance imaging and volumetric studies in this cohort.

Seventy-five unique variants in the KCNMA1 gene have been identified from individuals with neurological disorders. However, variant pathogenicity and evidence for disease causality are lacking in most cases. In this study, the KCNMA1 variants N999S and E656A (rs886039469 and rs149000684, respectively) were investigated from two individuals presenting with neurological disorders. N999S was previously shown to produce strong gain-of-function (GOF) changes in homomeric BK channel properties in vitro and is found as a heterozygous allele associated with epilepsy and paroxysmal dyskinesia in humans. Although its pathogenicity has been demonstrated in heterozygous animal models, the GOF classification for N999S has not been validated in a heterozygous patient-derived tissue. Conversely, the GOF pathogenicity for E656A is based solely on homomeric channels expressed in vitro and is inconclusive. For either variant, the properties of single heterozygous channels and allele expression is unknown. In this study, we profiled the wild-type and mutant KCNMA1 transcripts from primary human skin fibroblasts of heterozygous patients and unaffected controls and performed patch-clamp electrophysiology to characterize endogenous BK channel current properties. GOF gating was observed in single BK channel recordings from both channel types. Fibroblasts from the individual harboring the E656A variant showed decreases in the number of BK channels detected and E656A-containing transcripts compared to controls. These results show that single BK channels can be reliably detected in primary fibroblasts obtained from human skin biopsies, suggesting their utility for establishing variant pathogenicity, and reveal the BK channel expression and functional changes associated with two heterozygous patient genotypes.

The two predominating subtypes of late-onset GM2 gangliosidosis are late-onset Tay-Sachs (LOTS) and late-onset Sandhoff disease (LOSD). Due to shared deficiencies of ß-hexosamindase A and significant clinical overlap, the two diseases have been considered indistinguishable. However, a growing body of evidence supports the notion of several distinctions between the two diseases. In this study, we highlight these distinctions through the cross-sectional evaluation of 27 late-onset GM2 gangliosidosis participants. Twenty-one participants with LOTS and 6 with LOSD were included in this study. We performed physical examinations alongside assessments for gait, balance, muscle strength, ataxia, nerve conduction velocities, and analyzed brain magnetic resonance imaging. Lower limb weakness (95% in LOTS, 100% in LOSD) and later development of upper limb weakness (90% in LOTS, 83% in LOSD) was highly prevalent in both cohorts. Accompanying gait disturbances, balance issues, and dysmetria (as assessed by the brief ataxia rating scale [BARS]) were also prevalent in both cohorts. Strength testing for the quadriceps and hamstrings demonstrated weakness in both cohorts, primarily impacting extensor muscles. Supratentorial gray and white matter volumes in both cohorts were similar to normative data. In contrast, BARS scores for dysarthria and oculomotor dysfunction were present and heterogenous in LOTS participants and absent in LOSD participants. 24% of LOTS participants and none of the LOSD participants had a history of neuropsychiatric symptoms. Cerebellar volume including lobules V and VI were lower in LOTS compared to LOSD and normative data. However, length dependent sensory neuropathy was present in all LOSD participants but absent in LOTS participants. Dysfunction of the posterior cerebellum (lobules VI, VII, and IX) has been shown to cause cerebellar cognitive affective syndrome (CCAS), that includes cognitive and behavioral disturbances. Furthermore, cerebellar dysfunction of lobules V and VI has been linked to dysarthric speech, and dysfunction of the posterior cerebellum has been linked to oculomotor symptoms. The finding of low cerebellar lobule volumes in LOTS, suggests the distinctive features of the LOTS phenotype are related to cerebellar dysfunction. However, the sensory symptoms unique to LOSD remains a mystery. The molecular and biochemical basis for the dichotomy between the LOTS and LOSD phenotypes requires further investigation.

Background: GM1 gangliosidosis, caused by biallelic variants in GLB1 , results from deficiency of lysosomal β-galactosidase, the enzyme primarily responsible for degradation of GM1 ganglioside. This progressive neurodegenerative disease is uniformly fatal with no approved therapies, but preclinical studies utilizing gene therapy have shown promising results. Methods: This phase 1-2 open label dose escalation study utilized a single intravenous administration of adeno-associated virus serotype 9 (AAV9) encoding β-galactosidase in the first nine enrolled Type II GM1 gangliosidosis participants. The primary endpoint was safety after 3 years; secondary and exploratory efficacy outcomes included cerebrospinal fluid (CSF) GM1 ganglioside and β-galactosidase, clinical assessments, and neuroimaging changes. Results: One serious adverse event was attributed to the vector, i.e., vomiting requiring rehospitalization for IV hydration. Serum aspartate and alanine aminotransferase levels increased following gene transfer but returned to baseline by 18 months. Per protocol analysis found stability in Vineland Expressive Communication and Gross Motor and significant declines in Fine Motor and Receptive Communication. Median CGI-Improvement scores at 2 and 3 years after gene transfer were “minimally improved” or “no change”. CSF β-galactosidase increased and GM1 ganglioside decreased in all participants. MRI showed improved myelination by differential tractography and declines in cerebral atrophy. MRS showed reduced loss of N -acetylaspartate+ N -acetylaspartyl glutamate (NAA) compared with historical controls. Conclusions: A single IV infusion of AAV9 encoding β-galactosidase was well-tolerated among the first nine Type II GM1 gangliosidosis participants. Secondary and exploratory outcomes suggested improvements in biochemical markers and neuroimaging and stabilized or reduced rates of developmental deterioration ( NCT03952637 ).

Late-onset Tay-Sachs (LOTS) disease is a lysosomal storage disorder most commonly caused by a point mutation (c.805G>A) in the HEXA gene encoding the α subunit of the lysosomal enzyme β-hexosaminidase A. LOTS manifests as a range of gradually worsening neurological symptoms beginning in young adulthood. Here, we explored the efficacy of an adenine base editor (ABE) programmed with an sgRNA to correct the HEXA c.805G>A mutation. Base editing in fibroblasts from a patient with LOTS successfully converted the pathogenic HEXA c.805A to G and partially restored β-hexosaminidase activity, with minimal genome-wide off-target editing. We generated a LOTS mouse model in which the mice exhibited decreased β-hexosaminidase activity, accumulation of GM2 ganglioside in the brain, progressive neurological manifestations, and reduced lifespan. Treatment of LOTS mice with the neurotropic virus AAV-PHP.eB carrying the ABE and an sgRNA targeting the LOTS point mutation partially corrected the c.805G>A mutation in the CNS, significantly increased brain β-hexosaminidase activity, and substantially reduced GM2 ganglioside accumulation in the brain. Moreover, the therapy delayed symptom onset and significantly extended median lifespan. These findings highlight the potential of base editing as an effective treatment for LOTS and its broader applicability to other lysosomal storage disorders.