Life-Limiting Peripheral Organ Dysfunction in Feline Sandhoff Disease Emerges after Effective CNS Gene Therapy.

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.

Therapeutic benefit after intracranial gene therapy delivered during the symptomatic stage in a feline model of Sandhoff disease.

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.

PEA15 loss of function and defective cerebral development in the domestic cat.

Cerebral cortical size and organization are critical features of neurodevelopment and human evolution, for which genetic investigation in model organisms can provide insight into developmental mechanisms and the causes of cerebral malformations. However, some abnormalities in cerebral cortical proliferation and folding are challenging to study in laboratory mice due to the absence of gyri and sulci in rodents. We report an autosomal recessive allele in domestic cats associated with impaired cerebral cortical expansion and folding, giving rise to a smooth, lissencephalic brain, and that appears to be caused by homozygosity for a frameshift in PEA15 (phosphoprotein expressed in astrocytes-15). Notably, previous studies of a Pea15 targeted mutation in mice did not reveal structural brain abnormalities. Affected cats, however, present with a non-progressive hypermetric gait and tremors, develop dissociative behavioral defects and aggression with age, and exhibit profound malformation of the cerebrum, with a 45% average decrease in overall brain weight, and reduction or absence of the ectosylvian, sylvian and anterior cingulate gyrus. Histologically, the cerebral cortical layers are disorganized, there is substantial loss of white matter in tracts such as the corona radiata and internal capsule, but the cerebellum is relatively spared. RNA-seq and immunohistochemical analysis reveal astrocytosis. Fibroblasts cultured from affected cats exhibit increased TNFα-mediated apoptosis, and increased FGFb-induced proliferation, consistent with previous studies implicating PEA15 as an intracellular adapter protein, and suggesting an underlying pathophysiology in which increased death of neurons accompanied by increased proliferation of astrocytes gives rise to abnormal organization of neuronal layers and loss of white matter. Taken together, our work points to a new role for PEA15 in development of a complex cerebral cortex that is only apparent in gyrencephalic species.

Adeno-Associated Virus Gene Therapy in a Sheep Model of Tay-Sachs Disease.

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.

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

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

AAV-mediated gene delivery attenuates neuroinflammation in feline Sandhoff disease.

Sandhoff disease (SD) is a lysosomal storage disorder characterized by the absence of hydrolytic enzyme ß-N-acetylhexosaminidase (Hex), which results in storage of GM2 ganglioside in neurons and unremitting neurodegeneration. Neuron loss initially affects fine motor skills, but rapidly progresses to loss of all body faculties, a vegetative state, and death by five years of age in humans. A well-established feline model of SD allows characterization of the disease in a large animal model and provides a means to test the safety and efficacy of therapeutic interventions before initiating clinical trials. In this study, we demonstrate a robust central nervous system (CNS) inflammatory response in feline SD, primarily marked by expansion and activation of the microglial cell population. Quantification of major histocompatibility complex II (MHC-II) labeling revealed significant up-regulation throughout the CNS with areas rich in white matter most severely affected. Expression of the leukocyte chemokine macrophage inflammatory protein-1 alpha (MIP-1α) was also up-regulated in the brain. SD cats were treated with intracranial delivery of adeno-associated viral (AAV) vectors expressing feline Hex, with a study endpoint 16weeks post treatment. AAV-mediated gene delivery repressed the expansion and activation of microglia and normalized MHC-II and MIP-1α levels. These data reiterate the profound inflammatory response in SD and show that neuroinflammation is abrogated after AAV-mediated restoration of enzymatic activity.

Mucopolysaccharidosis-like phenotype in feline Sandhoff disease and partial correction after AAV gene therapy.

Sandhoff disease (SD) is a fatal neurodegenerative disease caused by a mutation in the enzyme ß-N-acetylhexosaminidase. Children with infantile onset SD develop seizures, loss of motor tone and swallowing problems, eventually reaching a vegetative state with death typically by 4years of age. Other symptoms include vertebral gibbus and cardiac abnormalities strikingly similar to those of the mucopolysaccharidoses. Isolated fibroblasts from SD patients have impaired catabolism of glycosaminoglycans (GAGs). To evaluate mucopolysaccharidosis-like features of the feline SD model, we utilized radiography, MRI, echocardiography, histopathology and GAG quantification of both central nervous system and peripheral tissues/fluids. The feline SD model exhibits cardiac valvular and structural abnormalities, skeletal changes and spinal cord compression that are consistent with accumulation of GAGs, but are much less prominent than the severe neurologic disease that defines the humane endpoint (4.5±0.5months). Sixteen weeks after intracranial AAV gene therapy, GAG storage was cleared in the SD cat cerebral cortex and liver, but not in the heart, lung, skeletal muscle, kidney, spleen, pancreas, small intestine, skin, or urine. GAG storage worsens with time and therefore may become a significant source of pathology in humans whose lives are substantially lengthened by gene therapy or other novel treatments for the primary, neurologic disease.

AAV-mediated gene delivery in a feline model of Sandhoff disease corrects lysosomal storage in the central nervous system.

Sandhoff disease (SD) is an autosomal recessive neurodegenerative disease caused by a mutation in the gene for the ß-subunit of ß-N-acetylhexosaminidase (Hex), resulting in the inability to catabolize ganglioside GM2 within the lysosomes. SD presents with an accumulation of GM2 and its asialo derivative GA2, primarily in the central nervous system. Myelin-enriched glycolipids, cerebrosides and sulfatides, are also decreased in SD corresponding with dysmyelination. At present, no treatment exists for SD. Previous studies have shown the therapeutic benefit of adeno-associated virus (AAV) vector-mediated gene therapy in the treatment of SD in murine and feline models. In this study, we treated presymptomatic SD cats with AAVrh8 vectors expressing feline Hex in the thalamus combined with intracerebroventricular (Thal/ICV) injections. Treated animals showed clearly improved neurologic function and quality of life, manifested in part by prevention or attenuation of whole-body tremors characteristic of untreated animals. Hex activity was significantly elevated, whereas storage of GM2 and GA2 was significantly decreased in tissue samples taken from the cortex, cerebellum, thalamus, and cervical spinal cord. Treatment also increased levels of myelin-enriched cerebrosides and sulfatides in the cortex and thalamus. This study demonstrates the therapeutic potential of AAV for feline SD and suggests a similar potential for human SD patients.

Molecular cloning, sequencing, and distribution of feline GnRH receptor (GnRHR) and resequencing of canine GnRHR.

GnRH receptors play vital roles in mammalian reproduction via regulation of gonadotropin secretion, which is essential for gametogenesis and production of gonadal steroids. GnRH receptors for more than 20 mammalian species have been sequenced, including human, mouse, and dog. This study reports the molecular cloning and sequencing of GnRH receptor (GnRHR) cDNA from the pituitary gland of the domestic cat, an important species in biomedical research. Feline GnRHR cDNA is composed of 981 nucleotides and encodes a 327 amino acid protein. Unlike the majority of mammalian species sequenced so far, but similar to canine GnRHR, feline GnRHR protein lacks asparagine in position three of the extracellular domain of the protein. At the amino acid level, feline GnRHR exhibits 95.1% identity with canine, 93.8% with human, and 88.9% with mouse GnRHR. Comparative sequence analysis of GnRHRs for multiple mammalian species led to resequencing of canine GnRHR, which differed from that previously published by a single base change that translates to a different amino acid in position 193. This single base change was confirmed in dogs of multiple breeds. Reverse transcriptase PCR analysis of GnRHR messenger RNA in different tissues from four normal cats indicated the presence of amplicons of varying lengths, including full-length as well as shortened GnRHR amplicons, pointing to the existence of truncated GnRHR transcripts in the domestic cat. This study is the first insight into molecular composition and expression of feline GnRHR and promotes better understanding of receptor organization, and distribution in various tissues of this species.

Biomarkers for disease progression and AAV therapeutic efficacy in feline Sandhoff disease.

The GM2 gangliosidoses, Tay-Sachs disease (TSD) and Sandhoff disease (SD), are progressive neurodegenerative disorders that are caused by a mutation in the enzyme ß-N-acetylhexosaminidase (Hex). Due to the recent emergence of novel experimental treatments, biomarker development has become particularly relevant in GM2 gangliosidosis as an objective means to measure therapeutic efficacy. Here we describe blood, cerebrospinal fluid (CSF), magnetic resonance imaging (MRI), and electrodiagnostic methods for evaluating disease progression in the feline SD model and application of these approaches to assess AAV-mediated gene therapy. SD cats were treated by intracranial injections of the thalami combined with either the deep cerebellar nuclei or a single lateral ventricle using AAVrh8 vectors encoding feline Hex. Significantly altered in untreated SD cats, blood and CSF based biomarkers were largely normalized after AAV gene therapy. Also reduced after treatment were expansion of the lysosomal compartment in peripheral blood mononuclear cells and elevated activity of secondary lysosomal enzymes. MRI changes characteristic of the gangliosidoses were documented in SD cats and normalized after AAV gene therapy. The minimally invasive biomarkers reported herein should be useful to assess disease progression of untreated SD patients and those in future clinical trials.

Sustained normalization of neurological disease after intracranial gene therapy in a feline model.

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.

High resolution MRI anatomy of the cat brain at 3 Tesla.

BACKGROUND: Feline models of neurologic diseases, such as lysosomal storage diseases, leukodystrophies, Parkinson's disease, stroke and NeuroAIDS, accurately recreate many aspects of human disease allowing for comparative study of neuropathology and the testing of novel therapeutics. Here we describe in vivo visualization of fine structures within the feline brain that were previously only visible post mortem. NEW METHOD: 3Tesla MR images were acquired using T1-weighted (T1w) 3D magnetization-prepared rapid gradient echo (MPRAGE) sequence (0.4mm isotropic resolution) and T2-weighted (T2w) turbo spin echo (TSE) images (0.3mm×0.3mm×1mm resolution). Anatomic structures were identified based on feline and canine histology. RESULTS: T2w high resolution MR images with detailed structural identification are provided in transverse, sagittal and dorsal planes. T1w MR images are provided electronically in three dimensions for unrestricted spatial evaluation. COMPARISON WITH EXISTING METHODS: Many areas of the feline brain previously unresolvable on MRI are clearly visible in three orientations, including the dentate, interpositus and fastigial cerebellar nuclei, cranial nerves, lateral geniculate nucleus, optic radiation, cochlea, caudal colliculus, temporal lobe, precuneus, spinocerebellar tract, vestibular nuclei, reticular formation, pyramids and rostral and middle cerebral arteries. Additionally, the feline brain is represented in three dimensions for the first time. CONCLUSIONS: These data establish normal appearance of detailed anatomical structures of the feline brain, which provide reference when evaluating neurologic disease or testing efficacy of novel therapeutics in animal models.

Therapeutic response in feline sandhoff disease despite immunity to intracranial gene therapy.

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.

Phage display allows identification of zona pellucida-binding peptides with species-specific properties: novel approach for development of contraceptive vaccines for wildlife.

Multiple phage-peptide constructs, where the peptides mimic sperm epitopes that bind to zona pellucida (ZP) proteins, were generated via selection from a phage display library using a novel approach. Selections were designed to allow for identification of ZP-binding phage clones with potential species-specific properties, an important feature for wildlife oral vaccines as the goal is to control overpopulation of a target species while not affecting non-target species' reproduction. Six phage-peptide antigens were injected intramuscularly into pigs and corresponding immune responses evaluated. Administration of the antigens into pigs stimulated production of anti-peptide antibodies, which were shown to act as anti-sperm antibodies. Potentially, such anti-sperm antibodies could interfere with sperm delivery or function in the male or female genital tract, leading to contraceptive effects. Staining of semen samples collected from different mammalian species, including pig, cat, dog, bull, and mouse, with anti-sera from pigs immunized with ZP-binding phage allowed identification of phage-peptide constructs with different levels of species specificity. Based on the intensity of the immune responses and specificity of these responses in different species, two of the antigens with fusion peptide sequences GEGGYGSHD and GQQGLNGDS were recognized as the most promising candidates for development of contraceptive vaccines for wild pigs.

Infective and inactivated filamentous phage as carriers for immunogenic peptides.

The focus of this study is on development of vaccines using filamentous phage as a delivery vector for immunogenic peptides. The use of phage as a carrier for immunogenic peptides provides significant benefits such as high immunogenicity, low production costs, and high stability of phage preparations. However, introduction of live recombinant phage into the environment might represent a potential ecological problem. This, for example, may occur when vaccines are used in oral or nasal formulations in field conditions for wild and feral animals. To address this issue, comparative studies of antigenic properties of live and inactivated (non-viable) phage were accomplished. Inactivated phage, if released, will not propagate and will degrade as any other protein. In these experiments, a model phage clone that was previously selected from a phage display library and shown to stimulate production of anti-sperm antibodies with contraceptive properties was used. Multiple methods of phage inactivation were tested, including drying, freezing, autoclaving, heating, and UV irradiation. Under studied conditions, heating at 76°C for 3h, UV irradiation, and autoclaving resulted in complete phage inactivation. Phage samples treated by heat and UV were characterized by spectrophotometry and electron microscopy. To test antigenicity, live and inactivated phage preparations were injected into mice and antibody responses assayed by ELISA. It was found that phage killed by heat causes little to no immune responses, probably due to destruction of phage particles. In contrast, UV-inactivated phage stimulated production of IgG serum antibodies at the levels comparable to live phage. Thus, vaccines formulated to include UV-inactivated filamentous phage might represent environmentally safe alternatives to live phage vaccines.

ZP-binding peptides identified via phage display stimulate production of sperm antibodies in dogs.

Zona pellucida (ZP) glycoproteins play a central role in sperm-oocyte binding and fertilization. Sperm protein sequences that are involved in sperm-ZP recognition and have an important role in fertilization represent attractive targets for development of contraceptive vaccines, yet are currently unknown. To identify peptide sequences that recognize and bind to ZP proteins, we developed a novel selection procedure from phage display libraries that utilizes intact oocytes surrounded by ZP proteins. The major advantage of this procedure is that ZP proteins remain in their native conformation unlike a selection protocol previously published that utilized solubilized ZP on artificial solid support. Several peptides of 7 and 12 amino acids with binding specificity to canine ZP proteins were identified. Four of them (LNSFLRS, SSWYRGA, YLPIYTIPSMVY, and NNQSPILKLSIH) plus a control ZP-binding peptide (YLPVGGLRRIGG) from the literature were synthesized and tested for antigenic properties in dogs. NNQSPILKLSIH peptide stimulated production of anti-peptide antibodies. These antibodies bind to the acrosomal region of the canine sperm cell, demonstrating ability to act as sperm antibodies. The identified ZP-binding peptides (mimicking sperm cell surface antigens) may be useful in the design of immunocontraceptive agents for dogs.

Neurodegenerative lysosomal storage disease in European Burmese cats with hexosaminidase beta-subunit deficiency.

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.

Neural stem/progenitor cells modulate immune responses by suppressing T lymphocytes with nitric oxide and prostaglandin E2.

We and others have reported that neural stem/progenitor cells (NSCs) may exert direct anti-inflammatory activity. This action has been attributed, in part, to T-cell suppression. However, how T-cells become suppressed by NSCs remains unresolved. In this study, we explored one of these mechanisms and challenged some previously advanced hypotheses regarding underlying NSC-mediated T-cell suppression. We employed an easily observable and manipulatable system in which activated and non-activated T-cells were co-cultured with a stable well-characterized clone of lacZ-expressing murine NSCs. As in previous reports, NSCs were found to inhibit T-cell proliferation. However, this inhibition by NSCs was not due to suppression of T cell activation or induction of apoptosis of T cells during the early activation stage. High levels of nitric oxide (NO) and prostaglandin E2 (PGE2) were induced in the T cells when co-cultured with NSCs. In addition, inducible NOS (iNOS) and microsomal type 1 PGES (mPGES-1) were readily detected in NSCs in co-culture with T-cells, but not at all in NSCs cultured alone or in activated T cells cultured with or without NSCs. This finding suggested that activated T cells induced NO and PGE2 production in the NSCs. Furthermore, T-cell proliferation inhibited by co-culture with the NSCs was significantly restored by inhibitors of NO and PGE2 production. Therefore, NSCs appear to suppress T-cells, at least in part, by NO and PGE2 production which, in turn, would account for the well-documented reduction of central nervous system immunopathology by transplanted NSCs.

Comparative analysis of brain lipids in mice, cats, and humans with Sandhoff disease.

Sandhoff disease (SD) is a glycosphingolipid (GSL) storage disease that arises from an autosomal recessive mutation in the gene for the beta-subunit of beta-Hexosaminidase A (Hexb gene), which catabolizes ganglioside GM2 within lysosomes. Accumulation of GM2 and asialo-GM2 (GA2) occurs primarily in the CNS, leading to neurodegeneration and brain dysfunction. We analyzed the total lipids in the brains of SD mice, cats, and humans. GM2 and GA2 were mostly undetectable in the normal mouse, cat, and human brain. The lipid abnormalities in the SD cat brain were generally intermediate to those observed in the SD mouse and the SD human brains. GM2 comprised 38, 67, and 87% of the total brain ganglioside distribution in the SD mice, cats, and humans, respectively. The ratio of GA2-GM2 was 0.93, 0.13, and 0.27 in the SD mice, cats, and humans, respectively, suggesting that the relative storage of GA2 is greater in the SD mouse than in the SD cat or human. Finally, the myelin-enriched lipids, cerebrosides and sulfatides, were significantly lower in the SD brains than in the control brains. This study is the first comparative analysis of brain lipids in mice, cats, and humans with SD and will be important for designing therapies for Sandhoff disease patients.

Generation and characterization of recombinant feline beta-galactosidase for preclinical enzyme replacement therapy studies in GM1 gangliosidosis.

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.