Jagdeep S. Walia

Construction of a hybrid β-hexosaminidase subunit capable of forming stable homodimers that hydrolyze GM2 ganglioside in vivo

Tay-Sachs or Sandhoff disease result from mutations in either the evolutionarily related HEXA or HEXB genes encoding respectively, the α- or β-subunits of β-hexosaminidase A (HexA). Of the three Hex isozymes, only HexA can interact with its cofactor, the GM2 activator protein (GM2AP), and hydrolyze GM2 ganglioside. A major impediment to establishing gene or enzyme replacement therapy based on HexA is the need to synthesize both subunits. Thus, we combined the critical features of both α- and β-subunits into a single hybrid µ-subunit that contains the α-subunit active site, the stable β-subunit interface and unique areas in each subunit needed to interact with GM2AP. To facilitate intracellular analysis and the purification of the µ-homodimer (HexM), CRISPR-based genome editing was used to disrupt the HEXA and HEXB genes in a Human Embryonic Kidney 293 cell line stably expressing the µ-subunit. In association with GM2AP, HexM was shown to hydrolyze a fluorescent GM2 ganglioside derivative both in cellulo and in vitro. Gene transfer studies in both Tay-Sachs and Sandhoff mouse models demonstrated that HexM expression reduced brain GM2 ganglioside levels.

Systemic Gene Transfer of a Hexosaminidase Variant Using an scAAV9.47 Vector Corrects GM2 Gangliosidosis in Sandhoff Mice

GM2 gangliosidosis is a group of neurodegenerative diseases caused by β-hexosaminidase A (HexA) enzyme deficiency. There is currently no cure. HexA is composed of two similar, nonidentical subunits, α and β, which must interact with the GM2 activator protein (GM2AP), a substrate-specific cofactor, to hydrolyze GM2 ganglioside. Mutations in either subunit or the activator can result in the accumulation of GM2 ganglioside within neurons throughout the central nervous system. The resulting neuronal cell death induces the primary symptoms of the disease: motor impairment, seizures, and sensory impairments. This study assesses the long-term effects of gene transfer in a Sandhoff (β-subunit knockout) mouse model. The study utilized a modified human β-hexosaminidase α-subunit (μ-subunit) that contains critical sequences from the β-subunit that enables formation of a stable homodimer (HexM) and interaction with GM2AP to hydrolyze GM2 ganglioside. We investigated a self-complementary adeno-associated viral (scAAV) vector expressing HexM, through intravenous injections of the neonatal mice. We monitored one cohort for 8 weeks and another cohort long-term for survival benefit, behavioral, biochemical, and molecular analyses. Untreated Sandhoff disease (SD) control mice reached a humane endpoint at approximately 15 weeks, whereas treated mice had a median survival age of 40 weeks, an approximate 2.5-fold survival advantage. On behavioral tests, the treated mice outperformed their knockout age-matched controls and perform similarly to the heterozygous controls. Through the enzymatic and GM2 ganglioside analyses, we observed a significant decrease in the GM2 ganglioside level, even though the enzyme levels were not significantly increased. Molecular analyses revealed a global distribution of the vector between brain and spinal cord regions. In conclusion, the neonatal delivery of a novel viral vector expressing the human HexM enzyme is effective in ameliorating the SD mouse phenotype for long-term. Our data could have implications not only for treatment of SD but also for Tay-Sachs disease (α-subunit deficiency) and similar brain disorders.

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.

Efficacy of a Bicistronic Vector for Correction of Sandhoff Disease in a Mouse Model

GM2 gangliosidoses are a family of severe neurodegenerative disorders resulting from a deficiency in the β-hexosaminidase A enzyme. These disorders include Tay-Sachs disease and Sandhoff disease, caused by mutations in the HEXA gene and HEXB gene, respectively. The HEXA and HEXB genes are required to produce the α and β subunits of the β-hexosaminidase A enzyme, respectively. Using a Sandhoff disease mouse model, we tested for the first time the potential of a comparatively lower dose (2.04 × 1013 vg/kg) of systemically delivered single-stranded adeno-associated virus 9 expressing both human HEXB and human HEXA cDNA under the control of a single promoter with a P2A-linked bicistronic vector design to correct the neurological phenotype. A bicistronic design allows maximal overexpression and secretion of the Hex A enzyme. Neonatal mice were injected with either this ssAAV9-HexB-P2A-HexA vector or a vehicle solution via the superficial temporal vein. An increase in survival of 56% compared with vehicle-injected controls and biochemical analysis of the brain tissue and serum revealed an increase in enzyme activity and a decrease in brain GM2 ganglioside buildup. This is a proof-of-concept study showing the “correction efficacy” of a bicistronic AAV9 vector delivered intravenously for GM2 gangliosidoses. Further studies with higher doses are warranted.

Clinical evaluation of R860Q semi-conservative amino acid substitution in CACNA1C gene in association with long QT syndrome

Article history: Received 26 November 2016 Accepted 31 March 2017 Available online 10 April 2017 she underwent insertion of an implantable cardioverter defibrillator for secondary prevention purpose. A genetic testing panel including sequencing and deletion/duplication analysis of 12 genes associated with LQTS was performed which revealed two variants of unknown significance – one in the CACNA1C gene and the other in the KCNE1 gene. We obtained a targeted 4-generation pedigree (Fig. 1), which revealed

363. Improvement of Sandhoff Phenotype Following Intravenous Injection of Adeno-Associated Viral Vector Expressing a Hexosaminidase Isoenzyme in Adult Sandhoff Mice: Preclinical Safety and Efficacy Study

GM2 gangliosidosis disorders stem from a Hexosaminidase A (HexA) isoenzyme deficiency. In humans, HexA is the sole enzyme able to catabolize GM2 ganglioside (GM2). The inability to effectively catabolize GM2 leads to neurodegeneration of the central nervous system. HexA is comprised of 2 subunits (α, β) and works with the GM2 activator protein (GM2AP). In the recent work by Tropak et al. (Mol Ther Met Clin Dev, in press), a hybrid subunit, named µ-subunit, was created (patent pending) by combining the stabilization and GM2AP binding sites of the β-subunit while conserving the catalytic properties of the α-subunit. The ‘µ’-subunit, coded by HEXM, can homodimerize and form a stable, functional enzyme, named HexM, which can interact with GM2AP to hydrolyse GM2. Previous work for successful correction of Sandhoff mice using AAV was only shown in neonatal mice (with immature blood-brain barrier (BBB)) and may not directly help in designing a human clinical trial. In the current study, we examined the efficacy/safety of IV injections of the scAAV9/HEXM vector at two doses in adult SD mice (with mature BBB). In addition, we also tested if an adjunct IV injection of mannitol provides any enhancement in efficacy. At 6 weeks old, the vector was injected via tail vein in cohorts of n=17 and n=15 SD mice at 2.5E+12 or 1.0E+13 vg/mouse, respectively. Another cohort of 16 mice received IV mannitol (3g/kg) prior to an IV injection of 2.5E+12 vg scAAV9/HEXM. Some mice from low dose group were euthanized at 16 weeks for direct analysis with untreated SD control mice, while the remainder were left until for terminal survival. Analysis of survival benefit, locomotor behaviour, biochemical and molecular parameters were performed. While untreated SD mice had a 16 week humane endpoint, 4 of 7 mice in higher dose group are now surviving past 56 weeks, 1 of 12 mice in the low dose cohort, and 4 of 9 mice in the mannitol cohort are surviving past 52 weeks. These increases in survival are all highly significant compared to the ~16 week humane endpoint of untreated SD mice. Behaviourally, there are no major significant differences in locomotion between the groups until after 15 weeks, when the adjunct mannitol group significantly outperforms the PBS group. Survival and behaviour monitoring, and the biochemical analyses for this study are ongoing. The preliminary results from this study show delayed onset of the SD phenotype with a single AAV9/HEXM injection and a significant benefit of a pre-injection of IV mannitol. This study is the first to show that an IV gene transfer using a scAAV/HEXM vector can provide survival and behavioural benefit in adult SD mice especially with adjunct use of mannitol. We propose that these results can advise the design of a human gene therapy trial for SD and the related Tay-Sachs disease.

551. Enhancement of Gene Therapy Treatment for Sandhoff Disease Through Complimentary Drug Therapy

GM2 gangliosidoses is a group of neurodegenerative lysosomal storage disorders caused by deficiency in the β-hexosaminidase A (HexA) enzyme. HexA is a heterodimer composed of 2 subunits; α- (encoded by the HEXA gene) and β-hexosaminidase β (Encoded by the HEXB gene). Mutations in either gene may cause inactivity of HexA leading to either Tay-Sachs disease (TSD, HEXA mutation) or Sandhoff disease (SD, HEXB mutation) respectively. TSD and SD are clinically indistinguishable phenotypes principally affecting infants and young children that are fatal before the age of 4 years; there is currently no effective available treatment. A mouse model for SD has been developed and these mice reach a humane end point at 14-17 weeks of age. Gene therapy can be an important primary therapeutic strategy, but may fall short of a complete rescue. Neuroinflammation and neurodegeneration have been identified as hallmark pathological mechanisms in GM2 gangliosidosis and can be adjunctive therapeutic targets. Neuroanti-inflammatory/neuroprotective agents like non-steroidal anti-inflammatory drugs (NSAIDs), Histone deacetylase inhibitors and pharmacological chaperones have shown ameliorating effects in SD and similar disease models when used alone. Adeno associated virus (AAV) based expression of Hexosaminidase isoenzymes has been shown to increase survival of Sandhoff mice for long term. We tested the combined role of gene therapy and neuroanti-inflammatory/neuroprotective agents in SD mice. Our methods include injecting neonatal SD mice with a relatively low dose (2×1013 vg/kg) of a novel AAV9 vector expressing Hex A using both α and β subunits. A pilot study established the survival of these mice to be approximately 24 weeks, a 55% increase in life span over vehicle-injected controls. We observed a 47% increase in Hex A activity in the midbrain of treated mice as compared to the vehicle injected controls. Cohorts received treatment with neonatal gene therapy alone or in combination with indomethacin (a NSAID), pyrimethamine (a proven pharmacological chaperone for Hex A) and ITF2357 (a histone deactylase inhibitor) to elicit their combinational therapeutic potential. Each drug is administered daily via oral gavage starting the age of 6 weeks. All treatments have been completed and mice are being monitored for survival benefit and locomotor behaviour. Further analyses of enzyme activity, GM2 ganglioside levels and copy numbers will be done. The results of this study will establish a proof-of-concept for a novel combination gene therapy approach for treatment of GM2 gangliosidoses and similar disorders.

725. Assessment of CSF Route for Gene Delivery in Sandhoff Mice Using AAV9 Expressing an Hexosaminidase Isoenzyme

GM2 gangliosidoses are a group of neurodegenerative disorders, characterized by the malfunctioning Hexosaminidase A (HexA) enzyme. HexA is formed by heterodimerization of two subunits, α and β. Hex A, in interaction with GM2 activator protein (GM2AP), is the main isoenzyme able to hydrolyze GM2 gangliosides. HexA deficiencies result in Tay-Sachs (α-subunit deficiency) or Sandhoff disease (SD, β-subunit deficiency). In the recent work by Tropak et al. (Mol Ther Methods Clin Dev, in press), a hybrid human α-subunit, named “µ” and coded by HEXM, was created (patent pending) by incorporating the dimer stabilization and GM2AP binding sites of the β-subunit while maintaining the catalytic properties of the α-subunit. The µ-subunit is able to homodimerize to form a stable and functional enzyme, named HexM, which can interact with the human GM2AP and effectively hydrolyse GM2 gangliosides. Another advantage of this subunit (~1.6 kb) is that it can be packaged in a self-complementary adeno-associated virus (scAAV/HEXM). An intravenous route of scAAV administration has been shown to be successful, but brings with it translational issues including large scale viral preparation and relatively high vector uptake by liver. We tested the cerebrospinal fluid (CSF) route, as an alternative, for delivering scAAV9/HEXM. We injected 2.5E+11 vector genomes per mouse of scAAV9/HEXM via the cisterna magna at 6 weeks of age (n=13). Our controls include treatment with scAAV9/GFP (n=3) and vehicle (n=8). One additional cohort received an IV injection of 25% mannitol (2g/kg) post-AAV9 injection (n=10). We sacrificed part of the cohorts (n=4 each group) at 16 weeks of age (humane end-point of untreated SD mice) for tissue analysis. The remainder are being monitored for long-term survival. The parameters for analyses are survival benefit, locomotor behaviour, Hexosaminidase activity, GM2 ganglioside accumulation, and vector genome biodistribution. The preliminary results from this ongoing study show a significant survival advantage to the humane endpoint in HexM treated (average 28 weeks to date) as compared to negative controls (~16 weeks). The behaviour tests showed improved locomotor activity in HexM-treated mice as compared to negative controls. Results were similar when mannitol was administered in conjunction with scAAV9/HEXM. These preliminary results indicate that the intra-CSF route of administration of AAV9/HEXM is a tractable translatable approach for SD worth further exploration. Additional studies are focused on increased dosage and methods to improve distribution.

711. Intravenous Neonatal Gene Therapy Corrects GM2 Gangliosidosis in Sandhoff Mice for Long-Term, By Using AAV Viral Vector Expressing a New Hexosaminidase Variant

GM2 gangliosidosis is a group of neurodegenerative disorders, characterized by the malfunctioning Hexosaminidase A (HexA) enzyme, for which there is no treatment. HexA is composed of two similar, but non-identical subunits, the alpha and the beta, which must interact with the GM2 activator protein, a substrate-specific cofactor, to hydrolyze GM2. Mutation in either subunit (or the activator) results in the development of GM2 gangliosidosis. In these diseases, the malfunctioning protein is unable to play its role in cleaving GM2 ganglioside, whose accumulation within the neurons of the central nervous system is ultimately toxic. The resulting neuronal death induces the primary symptoms of the disease; motor impairment, seizures, and sensory impairments. The aim of this study is to observe the long-term in vivo effects of a novel treatment in a Sandhoff (beta deficient) mouse model. The treatment utilized a new Hex isoenzyme, Hex M, which functions as a homodimer in the treatment of GM2 gangliosidosis. The HexM subunit is a variant of the human Hex alpha subunit containing critical beta-components that allow it to form stable homodimers and interact with the GM2 activator protein to reduce substrate storage. Our methods include intravenous injections of the neonatal mice with a self-complementary vector (with a synthetic promoter) expressing HexM at day 0-1. We monitored one cohort for 8 weeks and another cohort long-term (>40 weeks) for biochemical, behavioural and molecular analyses. Through the enzymatic and GM2 ganglioside lipid analyses, we see that with a slight increase in enzyme activity, there is a significant increase in the clearance of GM2 gangliosides. On behavioural tests, the treated mice outperform their knockout age matched controls. While the untreated controls die before the age of 15 weeks, treated animals have survived to more than 40 weeks and are still being monitored. The molecular analyses reveal a uniform distribution of the vector between brain and spinal cord regions. In conclusion, the neonatal delivery of our newly synthesized viral vector expressing HexM to the Sandhoff mice provided long-term correction of the disease. This study will have implications not only for treatment of Sandhoff, but also Tay-Sachs disease (alpha deficiency).

712. Improved Reduction in GM2 Ganglioside Accumulation in Tay-Sachs Mice Using a New Hexosaminidase Variant

GM2 gangliosidosis is a family of three genetic neurodegenerative disorders caused by the accumulation of GM2 gangliosides (GM2). Two of these are due to the deficiency of one of 2 similar but non-identical subunits that comprise heterodimeric β-hexosaminidase A (HexA) which hydrolyzes GM2. Mutations in the α-subunit (encoded by HEXA) of the enzyme HexA lead to Tay-Sachs disease (TSD), wherein mutations in the β-subunit (encoded by HEXB) lead to Sandhoff disease (SD). In their acute infantile forms, both rapidly progress with fatal neurological deterioration during childhood. The most significant pathological feature of TSD and SD is GM2 accumulation in neurons. Since functional HexA is a heterodimer of the α- and β-subunits, the efficacy of overexpressing only the deficient subunit in a gene therapy approach is limited by the levels of the endogenous subunit. An effective approach to treat either TSD or SD would be to express the α- and β-subunits at equimolar ratios from the same vector, which for AAV has been limited by size restrictions.The present study used a new variant of the Hex α-subunit, containing critical sequences from the β-subunit that can form a stable homodimer (HexM) capable of hydrolyzing GM2. A self-complementary (sc) AAV genome was designed with a synthetic promoter to allow packaging of HEXM. To test the efficacy of HEXM compared to that of the unmodified HEXA, these were packaged into scAAV9 vectors and injected stereotaxically into 4 or 15 month old TSD mice along with an identical titer of scAAV9/GFP vector to track vector spread. The mice were euthanized after 4 weeks and brain sections were subjected to IHC analysis against GFP and GM2. The HexA-like activity was assessed by clearance of GM2 within the injected region, compared to the contralateral brain hemisphere. Qualitatively, a marked reduction of GM2 was apparent in the areas of highest GFP expression. The various Hex vectors showed a clear difference in their ability to degrade GM2. As predicted, human HEXM was more capable of clearing GM2 aggregates than human HEXA at either 4 or 15 months of age. Interestingly, mouse HEXA was more effective than human HEXA, indicating a species-incompatibility, presumably in the formation of a human α- and murine β-subunit heterodimer. This incompatibility further reinforces the utility of HexM to form a functional homodimer independent of the endogenous α- and β-subunit, which should be effective in treating both TSD and SD.In conclusion, modified HEXM can form a functional homodimer capable of clearing GM2 aggregates. It can be packaged in a scAAV9 vector, which is amenable for strategies directed at widespread CNS gene transfer. These technological advances overcome previous barriers and provide pivotal reagents to develop a translatable gene therapy for TSD and SD.