Haploinsufficiency of the Sin3/HDAC corepressor complex member SIN3B causes a syndromic intellectual disability/autism spectrum disorder.

Proteins involved in transcriptional regulation harbor a demonstrated enrichment of mutations in neurodevelopmental disorders. The Sin3 (Swi-independent 3)/histone deacetylase (HDAC) complex plays a central role in histone deacetylation and transcriptional repression. Among the two vertebrate paralogs encoding the Sin3 complex, SIN3A variants cause syndromic intellectual disability, but the clinical consequences of SIN3B haploinsufficiency in humans are uncharacterized. Here, we describe a syndrome hallmarked by intellectual disability, developmental delay, and dysmorphic facial features with variably penetrant autism spectrum disorder, congenital malformations, corpus callosum defects, and impaired growth caused by disruptive SIN3B variants. Using chromosomal microarray or exome sequencing, and through international data sharing efforts, we identified nine individuals with heterozygous SIN3B deletion or single-nucleotide variants. Five individuals harbor heterozygous deletions encompassing SIN3B that reside within a ∼230 kb minimal region of overlap on 19p13.11, two individuals have a rare nonsynonymous substitution, and two individuals have a single-nucleotide deletion that results in a frameshift and predicted premature termination codon. To test the relevance of SIN3B impairment to measurable aspects of the human phenotype, we disrupted the orthologous zebrafish locus by genome editing and transient suppression. The mutant and morphant larvae display altered craniofacial patterning, commissural axon defects, and reduced body length supportive of an essential role for Sin3 function in growth and patterning of anterior structures. To investigate further the molecular consequences of SIN3B variants, we quantified genome-wide enhancer and promoter activity states by using H3K27ac ChIP-seq. We show that, similar to SIN3A mutations, SIN3B disruption causes hyperacetylation of a subset of enhancers and promoters in peripheral blood mononuclear cells. Together, these data demonstrate that SIN3B haploinsufficiency leads to a hitherto unknown intellectual disability/autism syndrome, uncover a crucial role of SIN3B in the central nervous system, and define the epigenetic landscape associated with Sin3 complex impairment.

Assessing the quality and value of metabolic chart data for capturing core outcomes for pediatric medium-chain acyl-CoA dehydrogenase (MCAD) deficiency.

BACKGROUND: Generating rigorous evidence to inform care for rare diseases requires reliable, sustainable, and longitudinal measurement of priority outcomes. Having developed a core outcome set for pediatric medium-chain acyl-CoA dehydrogenase (MCAD) deficiency, we aimed to assess the feasibility of prospective measurement of these core outcomes during routine metabolic clinic visits. METHODS: We used existing cohort data abstracted from charts of 124 children diagnosed with MCAD deficiency who participated in a Canadian study which collected data from birth to a maximum of 11 years of age to investigate the frequency of clinic visits and quality of metabolic chart data for selected outcomes. We recorded all opportunities to collect outcomes from the medical chart as a function of visit rate to the metabolic clinic, by treatment centre and by child age. We applied a data quality framework to evaluate data based on completeness, conformance, and plausibility for four core MCAD outcomes: emergency department use, fasting time, metabolic decompensation, and death. RESULTS: The frequency of metabolic clinic visits decreased with increasing age, from a rate of 2.8 visits per child per year (95% confidence interval, 2.3-3.3) among infants 2 to 6 months, to 1.0 visit per child per year (95% confidence interval, 0.9-1.2) among those ≥ 5 years of age. Rates of emergency department visits followed anticipated trends by child age. Supplemental findings suggested that some emergency visits occur outside of the metabolic care treatment centre but are not captured. Recommended fasting times were updated relatively infrequently in patients' metabolic charts. Episodes of metabolic decompensation were identifiable but required an operational definition based on acute manifestations most commonly recorded in the metabolic chart. Deaths occurred rarely in these patients and quality of mortality data was not evaluated. CONCLUSIONS: Opportunities to record core outcomes at the metabolic clinic occur at least annually for children with MCAD deficiency. Methods to comprehensively capture emergency care received at outside institutions are needed. To reduce substantial heterogeneous recording of core outcome across treatment centres, improved documentation standards are required for recording of recommended fasting times and a consensus definition for metabolic decompensations needs to be developed and implemented.

De Novo and Bi-allelic Pathogenic Variants in NARS1 Cause Neurodevelopmental Delay Due to Toxic Gain-of-Function and Partial Loss-of-Function Effects.

Aminoacyl-tRNA synthetases (ARSs) are ubiquitous, ancient enzymes that charge amino acids to cognate tRNA molecules, the essential first step of protein translation. Here, we describe 32 individuals from 21 families, presenting with microcephaly, neurodevelopmental delay, seizures, peripheral neuropathy, and ataxia, with de novo heterozygous and bi-allelic mutations in asparaginyl-tRNA synthetase (NARS1). We demonstrate a reduction in NARS1 mRNA expression as well as in NARS1 enzyme levels and activity in both individual fibroblasts and induced neural progenitor cells (iNPCs). Molecular modeling of the recessive c.1633C>T (p.Arg545Cys) variant shows weaker spatial positioning and tRNA selectivity. We conclude that de novo and bi-allelic mutations in NARS1 are a significant cause of neurodevelopmental disease, where the mechanism for de novo variants could be toxic gain-of-function and for recessive variants, partial loss-of-function.

Bridging clinical care and research in Ontario, Canada: Maximizing diagnoses from reanalysis of clinical exome sequencing data.

We examined the utility of clinical and research processes in the reanalysis of publicly-funded clinical exome sequencing data in Ontario, Canada. In partnership with eight sites, we recruited 287 families with suspected rare genetic diseases tested between 2014 and 2020. Data from seven laboratories was reanalyzed with the referring clinicians. Reanalysis of clinically relevant genes identified diagnoses in 4% (13/287); four were missed by clinical testing. Translational research methods, including analysis of novel candidate genes, identified candidates in 21% (61/287). Of these, 24 families have additional evidence through data sharing to support likely diagnoses (8% of cohort). This study indicates few diagnoses are missed by clinical laboratories, the incremental gain from reanalysis of clinically-relevant genes is modest, and the highest yield comes from validation of novel disease-gene associations. Future implementation of translational research methods, including continued reporting of compelling genes of uncertain significance by clinical laboratories, should be considered to maximize diagnoses.

Biochemical Correction of GM2 Ganglioside Accumulation in AB-Variant GM2 Gangliosidosis.

GM2 gangliosidosis is a group of genetic disorders that result in the accumulation of GM2 ganglioside (GM2) in brain cells, leading to progressive central nervous system (CNS) atrophy and premature death in patients. AB-variant GM2 gangliosidosis (ABGM2) arises from loss-of-function mutations in the GM2 activator protein (GM2AP), which is essential for the breakdown of GM2 in a key catabolic pathway required for CNS lipid homeostasis. In this study, we show that intrathecal delivery of self-complementary adeno-associated virus serotype-9 (scAAV9) harbouring a functional human GM2A transgene (scAAV9.hGM2A) can prevent GM2 accumulation in in GM2AP-deficient mice (Gm2a-/- mice). Additionally, scAAV9.hGM2A efficiently distributes to all tested regions of the CNS within 14 weeks post-injection and remains detectable for the lifespan of these animals (up to 104 weeks). Remarkably, GM2AP expression from the transgene scales with increasing doses of scAAV9.hGM2A (0.5, 1.0 and 2.0 × 1011 vector genomes (vg) per mouse), and this correlates with dose-dependent correction of GM2 accumulation in the brain. No severe adverse events were observed, and comorbidities in treated mice were comparable to those in disease-free cohorts. Lastly, all doses yielded corrective outcomes. These data indicate that scAAV9.hGM2A treatment is relatively non-toxic and tolerable, and biochemically corrects GM2 accumulation in the CNS-the main cause of morbidity and mortality in patients with ABGM2. Importantly, these results constitute proof-of-principle for treating ABGM2 with scAAV9.hGM2A by means of a single intrathecal administration and establish a foundation for future preclinical research.

Efficacy of Adeno-Associated Virus Serotype 9-Mediated Gene Therapy for AB-Variant GM2 Gangliosidosis.

GM2 gangliosidoses are a group of neurodegenerative lysosomal storage disorders that are characterized by the accumulation of GM2 gangliosides (GM2), leading to rapid neurological decline and death. The hydrolysis of GM2 requires the specific synthesis, processing, and combination of products of three genes-HEXA, HEXB, and GM2A-within the cell's lysosomes. Mutations in these genes result in Tay-Sachs disease, Sandhoff disease, or AB-variant GM2 gangliosidosis (ABGM2), respectively. ABGM2, the rarest of the three types, is characterized by a mutation in the GM2A gene, which encodes the GM2 activator (GM2A) protein. Being a monogenic disease, gene therapy is a plausible and likely effective method of treatment for ABGM2. This study aimed at assessing the effects of administering a one-time intravenous treatment of single-stranded Adeno-associated virus serotype 9 (ssAAV9)-GM2A viral vector at a dose of 1 × 1014 vector genomes (vg) per kilogram per mouse in an ABGM2 mouse model (Gm2a-/-). ssAAV9-GM2A was administered at 1-day (neonatal) or 6-weeks of age (adult-stage). The results demonstrated that, in comparison to Gm2a-/- mice that received a vehicle injection, the treated mice had reduced GM2 accumulation within the central nervous system and had long-term persistence of vector genomes in the brain and liver. This proof-of-concept study is a step forward towards the development of a clinically therapeutic approach for the treatment of patients with ABGM2.

Glutaminase Deficiency Caused by Short Tandem Repeat Expansion in GLS.

We report an inborn error of metabolism caused by an expansion of a GCA-repeat tract in the 5' untranslated region of the gene encoding glutaminase (GLS) that was identified through detailed clinical and biochemical phenotyping, combined with whole-genome sequencing. The expansion was observed in three unrelated patients who presented with an early-onset delay in overall development, progressive ataxia, and elevated levels of glutamine. In addition to ataxia, one patient also showed cerebellar atrophy. The expansion was associated with a relative deficiency of GLS messenger RNA transcribed from the expanded allele, which probably resulted from repeat-mediated chromatin changes upstream of the GLS repeat. Our discovery underscores the importance of careful examination of regions of the genome that are typically excluded from or poorly captured by exome sequencing.

Haploinsufficiency of PRR12 causes a spectrum of neurodevelopmental, eye, and multisystem abnormalities.

PURPOSE: Proline Rich 12 (PRR12) is a gene of unknown function with suspected DNA-binding activity, expressed in developing mice and human brains. Predicted loss-of-function variants in this gene are extremely rare, indicating high intolerance of haploinsufficiency. METHODS: Three individuals with intellectual disability and iris anomalies and truncating de novo PRR12 variants were described previously. We add 21 individuals with similar PRR12 variants identified via matchmaking platforms, bringing the total number to 24. RESULTS: We observed 12 frameshift, 6 nonsense, 1 splice-site, and 2 missense variants and one patient with a gross deletion involving PRR12. Three individuals had additional genetic findings, possibly confounding the phenotype. All patients had developmental impairment. Variable structural eye defects were observed in 12/24 individuals (50%) including anophthalmia, microphthalmia, colobomas, optic nerve and iris abnormalities. Additional common features included hypotonia (61%), heart defects (52%), growth failure (54%), and kidney anomalies (35%). PrediXcan analysis showed that phecodes most strongly associated with reduced predicted PRR12 expression were enriched for eye- (7/30) and kidney- (4/30) phenotypes, such as wet macular degeneration and chronic kidney disease. CONCLUSION: These findings support PRR12 haploinsufficiency as a cause for a novel disorder with a wide clinical spectrum marked chiefly by neurodevelopmental and eye abnormalities.

Expanding the phenotype of ASXL3-related syndrome: A comprehensive description of 45 unpublished individuals with inherited and de novo pathogenic variants in ASXL3.

The study aimed at widening the clinical and genetic spectrum of ASXL3-related syndrome, a neurodevelopmental disorder, caused by truncating variants in the ASXL3 gene. In this international collaborative study, we have undertaken a detailed clinical and molecular analysis of 45 previously unpublished individuals with ASXL3-related syndrome, as well as a review of all previously published individuals. We have reviewed the rather limited functional characterization of pathogenic variants in ASXL3 and discuss current understanding of the consequences of the different ASXL3 variants. In this comprehensive analysis of ASXL3-related syndrome, we define its natural history and clinical evolution occurring with age. We report familial ASXL3 pathogenic variants, characterize the phenotype in mildly affected individuals and discuss nonpenetrance. We also discuss the role of missense variants in ASXL3. We delineate a variable but consistent phenotype. The most characteristic features are neurodevelopmental delay with consistently limited speech, significant neuro-behavioral issues, hypotonia, and feeding difficulties. Distinctive features include downslanting palpebral fissures, hypertelorism, tubular nose with a prominent nasal bridge, and low-hanging columella. The presented data will inform clinical management of individuals with ASXL3-related syndrome and improve interpretation of new ASXL3 sequence variants.

The clinical utility of procainamide-induced late potentials on the signal averaged ECG.

BACKGROUND: Late potentials (LPs) identified on the signal averaged electrocardiogram (SAECG) are a marker for an increased risk of arrhythmias in Brugada syndrome (BrS). Procainamide is a sodium channel blocker used to diagnose BrS. The effects of Procainamide on the SAECG in those with BrS and the significance of Procainamide-induced LPs are unknown. METHODS: Procainamide provocation was performed for suspected BrS with 12-lead and SAECG pre- and post-infusion. Filtered QRS duration (fQRSd), duration of low amplitude signals <40 µV (LAS40) and root-mean-square voltage in the terminal 40 ms (RMS40) were determined. RESULTS: Data from 150 patients were included in the analysis (mean age 44.5 years, 109 males). Procainamide increased fQRSd (Pre 118.8 ± 10.5 ms, post 121.2 ± 10.2 ms, p < 0.001) and LAS40 (Pre 38.7 ± 9.8 ms, post 40.2 ± 10.5 ms, p = 0.005) and decreased RMS40 (Pre 24.6 ± 12 ms, post 22.8 ± 12 ms, p = 0.002). LPs were present in 68/150 (45%) at baseline. Fifteen patients with negative baseline SAECGs had LPs unmasked by Procainamide, but six patients had LPs at baseline that were no longer present following Procainamide. Comparing those with normal hearts (n = 48) to those with a final diagnosis of BrS (n = 38), Procainamide prolonged fQRSd to a greater extent in those with BrS. Comparing those with Procainamide-induced LPs to those with no LPs at any time did not highlight any aspect of phenotype and did not correlate with a history of ventricular arrhythmias. CONCLUSIONS: Procainamide influences the SAECG, provoking LPs in a small proportion of patients. However, there is no evidence that Procainamide-induced LPs provide additional diagnostic information or aid risk stratification.

A Comprehensive Review: Sphingolipid Metabolism and Implications of Disruption in Sphingolipid Homeostasis.

Sphingolipids are a specialized group of lipids essential to the composition of the plasma membrane of many cell types; however, they are primarily localized within the nervous system. The amphipathic properties of sphingolipids enable their participation in a variety of intricate metabolic pathways. Sphingoid bases are the building blocks for all sphingolipid derivatives, comprising a complex class of lipids. The biosynthesis and catabolism of these lipids play an integral role in small- and large-scale body functions, including participation in membrane domains and signalling; cell proliferation, death, migration, and invasiveness; inflammation; and central nervous system development. Recently, sphingolipids have become the focus of several fields of research in the medical and biological sciences, as these bioactive lipids have been identified as potent signalling and messenger molecules. Sphingolipids are now being exploited as therapeutic targets for several pathologies. Here we present a comprehensive review of the structure and metabolism of sphingolipids and their many functional roles within the cell. In addition, we highlight the role of sphingolipids in several pathologies, including inflammatory disease, cystic fibrosis, cancer, Alzheimer's and Parkinson's disease, and lysosomal storage disorders.

Investigating Immune Responses to the scAAV9-HEXM Gene Therapy Treatment in Tay-Sachs Disease and Sandhoff Disease Mouse Models.

GM2 gangliosidosis disorders are a group of neurodegenerative diseases that result from a functional deficiency of the enzyme ß-hexosaminidase A (HexA). HexA consists of an α- and ß-subunit; a deficiency in either subunit results in Tay-Sachs Disease (TSD) or Sandhoff Disease (SD), respectively. Viral vector gene transfer is viewed as a potential method of treating these diseases. A recently constructed isoenzyme to HexA, called HexM, has the ability to effectively catabolize GM2 gangliosides in vivo. Previous gene transfer studies have revealed that the scAAV9-HEXM treatment can improve survival in the murine SD model. However, it is speculated that this treatment could elicit an immune response to the carrier capsid and "non-self"-expressed transgene. This study was designed to assess the immunocompetence of TSD and SD mice, and test the immune response to the scAAV9-HEXM gene transfer. HexM vector-treated mice developed a significant anti-HexM T cell response and antibody response. This study confirms that TSD and SD mouse models are immunocompetent, and that gene transfer expression can create an immune response in these mice. These mouse models could be utilized for investigating methods of mitigating immune responses to gene transfer-expressed "non-self" proteins, and potentially improve treatment efficacy.

Mobile element insertion detection in 89,874 clinical exomes.

PURPOSE: Exome sequencing (ES) is increasingly used for the diagnosis of rare genetic disease. However, some pathogenic sequence variants within the exome go undetected due to the technical difficulty of identifying them. Mobile element insertions (MEIs) are a known cause of genetic disease in humans but have been historically difficult to detect via ES and similar targeted sequencing methods. METHODS: We developed and applied a novel MEI detection method prospectively to samples received for clinical ES beginning in November 2017. Positive MEI findings were confirmed by an orthogonal method and reported back to the ordering provider. In this study, we examined 89,874 samples from 38,871 cases. RESULTS: Diagnostic MEIs were present in 0.03% (95% binomial test confidence interval: 0.02-0.06%) of all cases and account for 0.15% (95% binomial test confidence interval: 0.08-0.25%) of cases with a molecular diagnosis. One diagnostic MEI was a novel founder event. Most patients with pathogenic MEIs had prior genetic testing, three of whom had previous negative DNA sequencing analysis of the diagnostic gene. CONCLUSION: MEI detection from ES is a valuable diagnostic tool, reveals molecular findings that may be undetected by other sequencing assays, and increases diagnostic yield by 0.15%.

Liver transplantation for Gaucher disease presenting as neonatal cholestasis: Case report and literature review.

BACKGROUND: We present a rare case of neonatal cholestasis in a female infant with Gaucher Disease (GD), who received liver transplantation. We review the relevant literature on similar disease presentations. METHODS: A chart review of the index case was performed. PubMed and Medline databases were searched to identify other cases. RESULTS: A 4-day-old female was referred with conjugated hyperbilirubinemia. Physical examination revealed icterus with hepatosplenomegaly and normal neurologic examination. The diagnosis of GD was confirmed through liver biopsy, low glucocerebrosidase enzyme activity, and two pathogenic mutations in GBA gene. Despite early initiation of ERT, the patient had worsening of her liver failure and underwent a left lateral segment liver transplant from a living donor at 7 months of age. She experienced improvement of her liver enzymes and coagulation, but passed away at 8 months due to the late onset of neurologic involvement. Nine other cases of GD presenting with neonatal cholestasis have been reported. Forty-four percent (4/9) of cases received ERT and none were considered for transplant. Overall, the literature suggests a poor prognosis with death reported in 77% (7/9) cases. CONCLUSIONS: Neonatal presentation of GD represents a poor prognosis despite early initiation of treatment. Diagnosis remains a challenge as the presentation is rare and multiple tests such as BM biopsy, liver biopsy with both light and electron microscopy, enzymology, and genetic testing may need to be completed to reach a diagnosis. Neurological sequelae may manifest later making the decision to proceed with liver transplantation a difficult one.

Metabolism of Glycosphingolipids and Their Role in the Pathophysiology of Lysosomal Storage Disorders.

Glycosphingolipids (GSLs) are a specialized class of membrane lipids composed of a ceramide backbone and a carbohydrate-rich head group. GSLs populate lipid rafts of the cell membrane of eukaryotic cells, and serve important cellular functions including control of cell-cell signaling, signal transduction and cell recognition. Of the hundreds of unique GSL structures, anionic gangliosides are the most heavily implicated in the pathogenesis of lysosomal storage diseases (LSDs) such as Tay-Sachs and Sandhoff disease. Each LSD is characterized by the accumulation of GSLs in the lysosomes of neurons, which negatively interact with other intracellular molecules to culminate in cell death. In this review, we summarize the biosynthesis and degradation pathways of GSLs, discuss how aberrant GSL metabolism contributes to key features of LSD pathophysiology, draw parallels between LSDs and neurodegenerative proteinopathies such as Alzheimer's and Parkinson's disease and lastly, discuss possible therapies for patients.

VNUT and VMAT2 segregate within sympathetic varicosities and localize near preferred Cav2 isoforms in the rat tail artery.

ATP and norepinephrine (NE) are coreleased from peripheral sympathetic nerve terminals. Whether they are stored in the same vesicles has been debated for decades. Preferential dependence of NE or ATP release on Ca2+ influx through specific voltage-gated Ca2+ channel (Cav2) isoforms suggests that NE and ATP are stored in separate vesicle pools, but simultaneous imaging of NE and ATP containing vesicles within single varicosities has not been reported. We conducted an immunohistochemical study of vesicular monoamine transporter 2 (VMAT2/SLC18A2) and vesicular nucleotide translocase (VNUT/SLC17A9) as markers of vesicles containing NE and ATP in sympathetic nerves of the rat tail artery. A large fraction of varicosities exhibited neighboring, rather than overlapping, VNUT and VMAT2 fluorescent puncta. VMAT2, but not VNUT, colocalized with synaptotagmin 1. Cav2.1, Cav2.2, and Cav2.3 are expressed in nerves in the tunica adventitia. VMAT2 preferentially localized adjacent to Cav2.2 and Cav2.3 rather than Cav2.1. VNUT preferentially localized adjacent to Cav2.3 > Cav2.2 >> Cav2.1. With the use of wire myography, inhibition of field-stimulated vasoconstriction with the Cav2.3 blocker SNX-482 (0.25 µM) mimicked the effects of the P2X inhibitor suramin (100 µM) rather than the α-adrenergic inhibitor phentolamine (10 µM). Variable sensitivity to SNX-482 and suramin between animals closely correlated with Cav2.3 staining. We concluded that a majority of ATP and NE stores localize to separate vesicle pools that use different synaptotagmin isoforms and that localize near different Cav2 isoforms to mediate vesicle release. Cav2.3 appears to play a previously unrecognized role in mediating ATP release in the rat tail artery. NEW & NOTEWORTHY Immunofluorescence imaging of vesicular nucleotide translocase and vesicular monoamine transporter 2 in rat tail arteries revealed that ATP and norepinephrine, classical cotransmitters, localize to well-segregated vesicle pools. Furthermore, vesicular nucleotide translocase and vesicular monoamine transporter 2 exhibit preferential localization with specific Cav2 isoforms. These novel observations address long-standing debates regarding the mechanism(s) of sympathetic neurotransmitter corelease.

Clinical spectrum of KIAA2022 pathogenic variants in males: Case report of two boys with KIAA2022 pathogenic variants and review of the literature.

KIAA2022 is an X-linked intellectual disability (XLID) syndrome affecting males more severely than females. Few males with KIAA2022 variants and XLID have been reported. We present a clinical report of two unrelated males, with two nonsense KIAA2022 pathogenic variants, with profound intellectual disabilities, limited language development, strikingly similar autistic behavior, delay in motor milestones, and postnatal growth restriction. Patient 1, 19-years-old, has long ears, deeply set eyes with keratoconus, strabismus, a narrow forehead, anteverted nares, café-au-lait spots, macroglossia, thick vermilion of the upper and lower lips, and prognathism. He has gastroesophageal reflux, constipation with delayed rectosigmoid colonic transit time, difficulty regulating temperature, several musculoskeletal issues, and a history of one grand mal seizure. Patient 2, 10-years-old, has mild dysmorphic features, therapy resistant vomiting with diminished motility of the stomach, mild constipation, cortical visual impairment with intermittent strabismus, axial hypotonia, difficulty regulating temperature, and cutaneous mastocytosis. Genetic testing identified KIAA2022 variant c.652C > T(p.Arg218*) in Patient 1, and a novel nonsense de novo variant c.2707G > T(p.Glu903*) in Patient 2. We also summarized features of all reported males with KIAA2022 variants to date. This report not only adds knowledge of a novel pathogenic variant to the KIAA2022 variant database, but also likely extends the spectrum by describing novel dysmorphic features and medical conditions including macroglossia, café-au-lait spots, keratoconus, severe cutaneous mastocytosis, and motility problems of the GI tract, which may help physicians involved in the care of patients with this syndrome. Lastly, we describe the power of social media in bringing families with rare medical conditions together.

Long-term correction of Sandhoff disease following intravenous delivery of rAAV9 to mouse neonates.

G(M2) gangliosidoses are severe neurodegenerative disorders resulting from a deficiency in ß-hexosaminidase A activity and lacking effective therapies. Using a Sandhoff disease (SD) mouse model (Hexb(-/-)) of the G(M2) gangliosidoses, we tested the potential of systemically delivered adeno-associated virus 9 (AAV9) expressing Hexb cDNA to correct the neurological phenotype. Neonatal or adult SD and normal mice were intravenously injected with AAV9-HexB or -LacZ and monitored for serum ß-hexosaminidase activity, motor function, and survival. Brain G(M2) ganglioside, ß-hexosaminidase activity, and inflammation were assessed at experimental week 43, or an earlier humane end point. SD mice injected with AAV9-LacZ died by 17 weeks of age, whereas all neonatal AAV9-HexB-treated SD mice survived until 43 weeks (P < 0.0001) with only three exhibiting neurological dysfunction. SD mice treated as adults with AAV9-HexB died between 17 and 35 weeks. Neonatal SD-HexB-treated mice had a significant increase in brain ß-hexosaminidase activity, and a reduction in G(M2) ganglioside storage and neuroinflammation compared to adult SD-HexB- and SD-LacZ-treated groups. However, at 43 weeks, 8 of 10 neonatal-HexB injected control and SD mice exhibited liver or lung tumors. This study demonstrates the potential for long-term correction of SD and other G(M2) gangliosidoses through early rAAV9 based systemic gene therapy.

Intrathecal delivery of a bicistronic AAV9 vector expressing ß-hexosaminidase A corrects Sandhoff disease in a murine model: A dosage study.

The pathological accumulation of GM2 ganglioside associated with Tay-Sachs disease (TSD) and Sandhoff disease (SD) occurs in individuals who possess mutant forms of the heterodimer ß-hexosaminidase A (Hex A) because of mutation of the HEXA and HEXB genes, respectively. With a lack of approved therapies, patients experience rapid neurological decline resulting in early death. A novel bicistronic vector carrying both HEXA and HEXB previously demonstrated promising results in mouse models of SD following neonatal intravenous administration, including significant reduction in GM2 accumulation, increased levels of Hex A, and a 2-fold extension of survival. The aim of the present study was to identify an optimal dose of the bicistronic vector in 6-week-old SD mice by an intrathecal route of administration along with transient immunosuppression, to inform possible clinical translation. Three doses of the bicistronic vector were tested: 2.5e11, 1.25e11, and 0.625e11 vector genomes per mouse. The highest dose provided the greatest increase in biochemical and behavioral parameters, such that treated mice lived to a median age of 56 weeks (>3 times the lifespan of the SD controls). These results have direct implications in deciding a human equivalent dose for TSD/SD and have informed the approval of a clinical trial application (NCT04798235).

Characterization of a phenotypically severe animal model for human AB-Variant GM2 gangliosidosis.

AB-Variant GM2 gangliosidosis (ABGM2) is a rare and lethal genetic disorder caused by mutations in the GM2A gene that lead to fatal accumulation of GM2 gangliosides (GM2) in neurons of the central nervous system (CNS). GM2A encodes a transport protein known as GM2 activator (GM2A) protein, which is essential for degrading GM2 into their GM3 form. ABGM2 presents in infantile-, juvenile-, and adult-onset forms; of the three, the infantile-onset is the most prominent, and by far the most severe, as evidenced by high levels of GM2 accumulation, widespread neurodegeneration, and death by the age of 4. Gm2a-/- mice are commonly used as a model of ABGM2. These mice are characterized by phenotypes most representative of predicted adult-onset form of ABGM2, which include moderate GM2 accumulation and mild neurological defects. This mild phenotype has been attributed to compensation by alternative GM2 degradation pathways mediated by sialidase, neuraminidase 3 (NEU3), a pathway that is more prominent in mice than humans. To assess the extent to which NEU3 contributes to GM2 degradation, we generated double knock-out (Gm2a-/-Neu3-/-) mice. Compellingly, these mice present with a clinical phenotype resembling that of a more severe ABGM2, including ataxia, reduced mobility and coordination, weight loss, poor body scores, and lethality by 6-7 months. Furthermore, these phenotypes correlate with a dramatic increase in GM2 accumulation in the CNS compared to levels observed in either Gm2a-/- or Neu3-/- mice. Taken together, these studies, for the first-time, confirm that the mild neurological phenotype of Gm2a-/- mice is due to compensatory activity on GM2 catabolism through an alternate breakdown pathway involving NEU3. These studies support the use of double knockout mice as a novel and highly relevant model for pre-clinical drug studies in a more severe form of ABGM2.