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1.
Ann Neurol ; 94(5): 969-986, 2023 11.
Article in English | MEDLINE | ID: mdl-37526361

ABSTRACT

OBJECTIVE: GM2 gangliosidosis is usually fatal by 5 years of age in its 2 major subtypes, Tay-Sachs and Sandhoff disease. First reported in 1881, GM2 gangliosidosis has no effective treatment today, and children succumb to the disease after a protracted neurodegenerative course and semi-vegetative state. This study seeks to further develop adeno-associated virus (AAV) gene therapy for human translation. METHODS: Cats with Sandhoff disease were treated by intracranial injection of vectors expressing feline ß-N-acetylhexosaminidase, the enzyme deficient in GM2 gangliosidosis. RESULTS: Hexosaminidase activity throughout the brain and spinal cord was above normal after treatment, with highest activities at the injection sites (thalamus and deep cerebellar nuclei). Ganglioside storage was reduced throughout the brain and spinal cord, with near complete clearance in many regions. While untreated cats with Sandhoff disease lived for 4.4 ± 0.6 months, AAV-treated cats lived to 19.1 ± 8.6 months, and 3 of 9 cats lived >21 months. Correction of the central nervous system was so effective that significant increases in lifespan led to the emergence of otherwise subclinical peripheral disease, including megacolon, enlarged stomach and urinary bladder, soft tissue spinal cord compression, and patellar luxation. Throughout the gastrointestinal tract, neurons of the myenteric and submucosal plexuses developed profound pathology, demonstrating that the enteric nervous system was inadequately treated. INTERPRETATION: The vector formulation in the current study effectively treats neuropathology in feline Sandhoff disease, but whole-body targeting will be an important consideration in next-generation approaches. ANN NEUROL 2023;94:969-986.


Subject(s)
Gangliosidoses, GM2 , Sandhoff Disease , Child , Animals , Cats , Humans , Sandhoff Disease/genetics , Sandhoff Disease/therapy , Sandhoff Disease/veterinary , Multiple Organ Failure/therapy , Genetic Vectors , Central Nervous System/pathology , Genetic Therapy
2.
Gene Ther ; 28(3-4): 142-154, 2021 04.
Article in English | MEDLINE | ID: mdl-32884151

ABSTRACT

Sandhoff disease (SD) is an autosomal recessive lysosomal storage disease caused by defects in the ß-subunit of ß-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.


Subject(s)
Sandhoff Disease , Animals , Cats , Dependovirus/genetics , Disease Models, Animal , Genetic Therapy , Genetic Vectors/genetics , Humans , Sandhoff Disease/genetics , Sandhoff Disease/therapy , beta-N-Acetylhexosaminidases/genetics
3.
Sci Rep ; 10(1): 11096, 2020 Jul 01.
Article in English | MEDLINE | ID: mdl-32606448

ABSTRACT

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

4.
Mol Ther ; 28(10): 2150-2160, 2020 10 07.
Article in English | MEDLINE | ID: mdl-32592687

ABSTRACT

The GM2 gangliosidoses, Tay-Sachs disease (TSD) and Sandhoff disease (SD), are fatal lysosomal storage disorders caused by mutations in the HEXA and HEXB genes, respectively. These mutations cause dysfunction of the lysosomal enzyme ß-N-acetylhexosaminidase A (HexA) and accumulation of GM2 ganglioside (GM2) with ensuing neurodegeneration, and death by 5 years of age. Until recently, the most successful therapy was achieved by intracranial co-delivery of monocistronic adeno-associated viral (AAV) vectors encoding Hex alpha and beta-subunits in animal models of SD. The blood-brain barrier crossing properties of AAV9 enables systemic gene therapy; however, the requirement of co-delivery of two monocistronic AAV vectors to overexpress the heterodimeric HexA protein has prevented the use of this approach. To address this need, we developed multiple AAV constructs encoding simultaneously HEXA and HEXB using AAV9 and AAV-PHP.B and tested their therapeutic efficacy in 4- to 6-week-old SD mice after systemic administration. Survival and biochemical outcomes revealed superiority of the AAV vector design using a bidirectional CBA promoter with equivalent dose-dependent outcomes for both capsids. AAV-treated mice performed normally in tests of motor function, CNS GM2 ganglioside levels were significantly reduced, and survival increased by >4-fold with some animals surviving past 2 years of age.


Subject(s)
Dependovirus/genetics , Genetic Therapy , Genetic Vectors/genetics , Sandhoff Disease/therapy , Animals , Disease Management , Disease Models, Animal , G(M2) Ganglioside/metabolism , Gene Expression , Genetic Predisposition to Disease , Genetic Therapy/methods , Genetic Vectors/administration & dosage , Mice , Mutation , Sandhoff Disease/genetics , Tay-Sachs Disease/genetics , Tay-Sachs Disease/metabolism , Tay-Sachs Disease/therapy , Transgenes , beta-N-Acetylhexosaminidases/genetics , beta-N-Acetylhexosaminidases/metabolism
5.
Sci Rep ; 10(1): 3751, 2020 02 28.
Article in English | MEDLINE | ID: mdl-32111883

ABSTRACT

A major characteristic of Alzheimer's disease (AD) is the accumulation of misfolded amyloid-ß (Aß) peptide. Several studies linked AD with type 2 diabetes due to similarities between Aß and human amylin. This study investigates the effect of amylin and pramlintide on Aß pathogenesis and the predisposing molecular mechanism(s) behind the observed effects in TgSwDI mouse, a cerebral amyloid angiopathy (CAA) and AD model. Our findings showed that thirty days of intraperitoneal injection with amylin or pramlintide increased Aß burden in mice brains. Mechanistic studies revealed both peptides altered the amyloidogenic pathway and increased Aß production by modulating amyloid precursor protein (APP) and γ-secretase levels in lipid rafts. In addition, both peptides increased levels of B4GALNT1 enzyme and GM1 ganglioside, and only pramlintide increased the level of GM2 ganglioside. Increased levels of GM1 and GM2 gangliosides play an important role in regulating amyloidogenic pathway proteins in lipid rafts. Increased brain Aß burden by amylin and pramlintide was associated with synaptic loss, apoptosis, and microglia activation. In conclusion, our findings showed amylin or pramlintide increase Aß levels and related pathology in TgSwDI mice brains, and suggest that increased amylin levels or the therapeutic use of pramlintide could increase the risk of AD.


Subject(s)
Amyloid Precursor Protein Secretases/metabolism , Amyloid beta-Protein Precursor/metabolism , Islet Amyloid Polypeptide/pharmacology , Membrane Microdomains/metabolism , Protein Processing, Post-Translational , Alzheimer Disease/genetics , Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Amyloid Precursor Protein Secretases/genetics , Amyloid beta-Protein Precursor/genetics , Animals , Cerebral Amyloid Angiopathy/genetics , Cerebral Amyloid Angiopathy/metabolism , Cerebral Amyloid Angiopathy/pathology , G(M1) Ganglioside/genetics , G(M1) Ganglioside/metabolism , G(M2) Ganglioside/genetics , G(M2) Ganglioside/metabolism , Membrane Microdomains/genetics , Membrane Microdomains/pathology , Mice , Mice, Transgenic , N-Acetylgalactosaminyltransferases/genetics , N-Acetylgalactosaminyltransferases/metabolism
6.
Hum Gene Ther ; 29(3): 312-326, 2018 03.
Article in English | MEDLINE | ID: mdl-28922945

ABSTRACT

Tay-Sachs disease (TSD) is a fatal neurodegenerative disorder caused by a deficiency of the enzyme hexosaminidase A (HexA). TSD also occurs in sheep, the only experimental model of TSD that has clinical signs of disease. The natural history of sheep TSD was characterized using serial neurological evaluations, 7 Tesla magnetic resonance imaging, echocardiograms, electrodiagnostics, and cerebrospinal fluid biomarkers. Intracranial gene therapy was also tested using AAVrh8 monocistronic vectors encoding the α-subunit of Hex (TSD α) or a mixture of two vectors encoding both the α and ß subunits separately (TSD α + ß) injected at high (1.3 × 1013 vector genomes) or low (4.2 × 1012 vector genomes) dose. Delay of symptom onset and/or reduction of acquired symptoms were noted in all adeno-associated virus-treated sheep. Postmortem evaluation showed superior HexA and vector genome distribution in the brain of TSD α + ß sheep compared to TSD α sheep, but spinal cord distribution was low in all groups. Isozyme analysis showed superior HexA formation after treatment with both vectors (TSD α + ß), and ganglioside clearance was most widespread in the TSD α + ß high-dose sheep. Microglial activation and proliferation in TSD sheep-most prominent in the cerebrum-were attenuated after gene therapy. This report demonstrates therapeutic efficacy for TSD in the sheep brain, which is on the same order of magnitude as a child's brain.


Subject(s)
Dependovirus , Genetic Therapy , Tay-Sachs Disease/therapy , beta-Hexosaminidase alpha Chain/biosynthesis , beta-Hexosaminidase beta Chain/biosynthesis , Animals , Brain/diagnostic imaging , Brain/enzymology , Disease Models, Animal , Echocardiography , Humans , Magnetic Resonance Imaging , Microglia/enzymology , Sheep , Tay-Sachs Disease/diagnostic imaging , Tay-Sachs Disease/enzymology , Tay-Sachs Disease/genetics , beta-Hexosaminidase alpha Chain/genetics , beta-Hexosaminidase beta Chain/genetics
7.
Sci Transl Med ; 6(231): 231ra48, 2014 Apr 09.
Article in English | MEDLINE | ID: mdl-24718858

ABSTRACT

Progressive debilitating neurological defects characterize feline G(M1) gangliosidosis, a lysosomal storage disease caused by deficiency of lysosomal ß-galactosidase. No effective therapy exists for affected children, who often die before age 5 years. An adeno-associated viral vector carrying the therapeutic gene was injected bilaterally into two brain targets (thalamus and deep cerebellar nuclei) of a feline model of G(M1) gangliosidosis. Gene therapy normalized ß-galactosidase activity and storage throughout the brain and spinal cord. The mean survival of 12 treated G(M1) animals was >38 months, compared to 8 months for untreated animals. Seven of the eight treated animals remaining alive demonstrated normalization of disease, with abrogation of many symptoms including gait deficits and postural imbalance. Sustained correction of the G(M1) gangliosidosis disease phenotype after limited intracranial targeting by gene therapy in a large animal model suggests that this approach may be useful for treating the human version of this lysosomal storage disorder.


Subject(s)
Brain/pathology , Genetic Therapy , Nervous System Diseases/therapy , Animals , Breeding , Cats , Dependovirus/metabolism , Disease Models, Animal , Disease Progression , Female , Humans , Lysosomes/enzymology , Magnetic Resonance Imaging , Male , Organ Specificity , Survival Analysis , beta-Galactosidase/genetics , beta-Galactosidase/therapeutic use
8.
Mol Ther ; 21(7): 1306-15, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23689599

ABSTRACT

Salutary responses to adeno-associated viral (AAV) gene therapy have been reported in the mouse model of Sandhoff disease (SD), a neurodegenerative lysosomal storage disease caused by deficiency of ß-N-acetylhexosaminidase (Hex). While untreated mice reach the humane endpoint by 4.1 months of age, mice treated by a single intracranial injection of vectors expressing human hexosaminidase may live a normal life span of 2 years. When treated with the same therapeutic vectors used in mice, two cats with SD lived to 7.0 and 8.2 months of age, compared with an untreated life span of 4.5 ± 0.5 months (n = 11). Because a pronounced humoral immune response to both the AAV1 vectors and human hexosaminidase was documented, feline cDNAs for the hexosaminidase α- and ß-subunits were cloned into AAVrh8 vectors. Cats treated with vectors expressing feline hexosaminidase produced enzymatic activity >75-fold normal at the brain injection site with little evidence of an immune infiltrate. Affected cats treated with feline-specific vectors by bilateral injection of the thalamus lived to 10.4 ± 3.7 months of age (n = 3), or 2.3 times as long as untreated cats. These studies support the therapeutic potential of AAV vectors for SD and underscore the importance of species-specific cDNAs for translational research.


Subject(s)
Cat Diseases/enzymology , Cat Diseases/therapy , Sandhoff Disease/enzymology , Sandhoff Disease/therapy , beta-N-Acetylhexosaminidases/metabolism , Animals , Cat Diseases/genetics , Cats , Dependovirus/genetics , Disease Models, Animal , Genetic Therapy/methods , Genetic Vectors/genetics , Sandhoff Disease/genetics , beta-N-Acetylhexosaminidases/genetics
9.
Mol Genet Metab ; 97(1): 53-9, 2009 May.
Article in English | MEDLINE | ID: mdl-19231264

ABSTRACT

GM2 gangliosidosis is a fatal, progressive neuronopathic lysosomal storage disease resulting from a deficiency of beta-N-acetylhexosaminidase (EC 3.2.1.52) activity. GM2 gangliosidosis occurs with varying degrees of severity in humans and in a variety of animals, including cats. In the current research, European Burmese cats presented with clinical neurological signs and histopathological features typical of a lysosomal storage disease. Thin layer chromatography revealed substantial storage of GM2 ganglioside in brain tissue of affected cats, and assays with a synthetic fluorogenic substrate confirmed the absence of hexosaminidase activity. When the hexosaminidase beta-subunit cDNA was sequenced from affected cats, a 91 base pair deletion constituting the entirety of exon 12 was documented. Subsequent sequencing of introns 11 and 12 revealed a 15 base pair deletion at the 3' end of intron 11 that included the preferred splice acceptor site, generating two minor transcripts from cryptic splice acceptor sites in affected Burmese cats. In the cerebral cortex of affected cats, hexosaminidase beta-subunit mRNA levels were approximately 1.5 times higher than normal (P<0.001), while beta-subunit protein levels were substantially reduced on Western blots.


Subject(s)
Cat Diseases/enzymology , Lysosomal Storage Diseases/veterinary , Nerve Degeneration/complications , Nerve Degeneration/enzymology , beta-Hexosaminidase beta Chain/metabolism , Animals , Base Sequence , Blotting, Western , Cats , Cerebral Cortex/enzymology , Cerebral Cortex/pathology , Chromatography, Thin Layer , DNA Mutational Analysis , Europe , Gangliosidoses, GM2/enzymology , Gangliosidoses, GM2/pathology , Lipids/analysis , Lysosomal Storage Diseases/complications , Lysosomal Storage Diseases/enzymology , Molecular Sequence Data , Myanmar
10.
Metab Brain Dis ; 23(2): 161-73, 2008 Jun.
Article in English | MEDLINE | ID: mdl-18421424

ABSTRACT

Lysosomal beta-galactosidase is required for the degradation of GM1 ganglioside and other glycolipids and glycoproteins with a terminal galactose moiety. Deficiency of this enzyme leads to the lysosomal storage disorder, GM1 gangliosidosis, marked by severe neurodegeneration resulting in premature death. As a step towards preclinical studies for enzyme replacement therapy in an animal model of GM1 gangliosidosis, a feline beta-galactosidase cDNA was cloned into a mammalian expression vector and subsequently expressed in Chinese hamster ovary (CHO-K1) cells. The enzyme secreted into culture medium exhibited specific activity on two synthetic substrates as well as on the native beta-galactosidase substrate, GM1 ganglioside. The enzyme was purified from transfected CHO-K1 cell culture medium by chromatography on PATG-agarose. The affinity-purified enzyme preparation consisted mainly of the protein with approximate molecular weight of 94 kDa and displayed immunoreactivity with antibodies raised against a 16-mer synthetic peptide corresponding to C-terminal amino acid sequence deduced from the feline beta-galactosidase cDNA.


Subject(s)
G(M1) Ganglioside/biosynthesis , Gangliosidosis, GM1/enzymology , Genetic Therapy/methods , Recombinant Proteins/isolation & purification , beta-Galactosidase/isolation & purification , Animals , Antibody Specificity/immunology , CHO Cells , Cats , Chromatography, Agarose , Cloning, Molecular/methods , Cricetinae , Cricetulus , Culture Media, Conditioned/chemistry , DNA, Complementary/genetics , Disease Models, Animal , G(M1) Ganglioside/genetics , Gangliosidosis, GM1/genetics , Gangliosidosis, GM1/therapy , Genetic Vectors/genetics , Molecular Weight , Protein Structure, Tertiary/physiology , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Transfection/methods , beta-Galactosidase/genetics , beta-Galactosidase/metabolism
11.
Mol Genet Metab ; 94(2): 212-21, 2008 Jun.
Article in English | MEDLINE | ID: mdl-18353697

ABSTRACT

G(M1) gangliosidosis is an inherited, fatal neurodegenerative disease caused by deficiency of lysosomal beta-d-galactosidase (EC 3.2.1.23) and consequent storage of undegraded G(M1) ganglioside. To characterize the genetic mutation responsible for feline G(M1) gangliosidosis, the normal sequence of feline beta-galactosidase cDNA first was defined. The feline beta-galactosidase open reading frame is 2010 base pairs, producing a protein of 669 amino acids. The putative signal sequence consists of amino acids 1-24 of the beta-galactosidase precursor protein, which contains seven potential N-linked glycosylation sites, as in the human protein. Overall sequence homology between feline and human beta-galactosidase is 74% for the open reading frame and 82% for the amino acid sequence. After normal beta-galactosidase was sequenced, the mutation responsible for feline G(M1) gangliosidosis was defined as a G to C substitution at position 1448 of the open reading frame, resulting in an amino acid substitution at arginine 483, known to cause G(M1) gangliosidosis in humans. Feline beta-galactosidase messenger RNA levels were normal in cerebral cortex, as determined by quantitative RT-PCR assays. Although enzymatic activity is severely reduced by the mutation, a full-length feline beta-galactosidase cDNA restored activity in transfected G(M1) fibroblasts to 18-times normal. beta-Galactosidase protein levels in G(M1) tissues were normal on Western blots, but immunofluorescence analysis demonstrated that the majority of mutant beta-galactosidase protein did not reach the lysosome. Additionally, G(M1) cat fibroblasts demonstrated increased expression of glucose-related protein 78/BiP and protein disulfide isomerase, suggesting that the unfolded protein response plays a role in pathogenesis of feline G(M1) gangliosidosis.


Subject(s)
Cat Diseases/genetics , Gangliosidosis, GM1/genetics , Gangliosidosis, GM1/veterinary , Mutation, Missense , beta-Galactosidase/genetics , Amino Acid Substitution , Animals , Cat Diseases/enzymology , Cats , Cell Line , Cells, Cultured , Cloning, Molecular , Endoplasmic Reticulum Chaperone BiP , Fibroblasts/enzymology , Gangliosidosis, GM1/enzymology , Heat-Shock Proteins/metabolism , Humans , Molecular Chaperones/metabolism , Molecular Sequence Data , Open Reading Frames , Protein Disulfide-Isomerases/metabolism , Protein Transport , beta-Galactosidase/analysis , beta-Galactosidase/metabolism
12.
Vet Immunol Immunopathol ; 109(1-2): 161-6, 2006 Jan 15.
Article in English | MEDLINE | ID: mdl-16169600

ABSTRACT

The mechanisms that initiate the pathophysiologic changes in the digital laminae in equine laminitis are poorly understood. Due to the fact that (1) the horse at risk of laminitis has many similarities clinically to the human sepsis patient and (2) our recent finding of marked laminar proinflammatory cytokine expression at the developmental time point of the black walnut extract (BWE) model of laminitis, we tested the possibility that, similar to organ damage in human sepsis, leukocyte emigration is an early event in laminitis. Using immunoperoxidase methods with an anti-equine CD13 monoclonal antibody that recognizes neutrophils and monocytes, we discovered that, whereas the dermal microvasculature of the skin commonly has a marginal pool of leukocytes, the normal laminar dermal microvasculature has minimal to no perivascular leukocytes. However, increases in leukocyte numbers occurred around the dermal vasculature of both the laminae and the skin in the majority of BWE-treated horses in the developmental stage and at the onset of clinical signs of lameness in the BWE model. These findings indicate that, similar to organ failure in human sepsis, leukocyte emigration is likely to play a significant role in initiating numerous pathophysiologic mechanisms that lead to the development of laminitis.


Subject(s)
Cell Movement/immunology , Foot Diseases/veterinary , Horse Diseases/immunology , Leukocytes, Mononuclear/immunology , Skin Diseases/veterinary , Animals , CD13 Antigens/immunology , Disease Models, Animal , Foot Diseases/immunology , Horses , Immunohistochemistry/veterinary , Leukocyte Count/veterinary , Skin Diseases/immunology
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