Silencing of B4Galnt1 Gene Prevents GM2 Accumulation in Tay-Sachs Cells

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Introduction
TSD is a subtype of rare, inherited metabolic disorders characterized by the accumulation of GM2 ganglioside particularly in the nervous system.Under normal circumstances, GM2 gangliosides are broken down by specific enzymes called β-hexosaminidase A in lysosomes [1].Mutations in the Hexa gene in humans cause impairment in the degradation of GM2 ganglioside and result in progressive GM2 accumulation, especially in the central nervous system (CNS).Patients show clinically similar phenotypes, and the severity of the disease depends on the residual enzyme activity.Progressive neurological deterioration, motor dysfunction, vision and hearing impairment, seizures, feeding difficulties, and hypotonia are among the symptoms [2].Hexa-/-mouse was previously generated as a model for TSD; however, it did not exhibit neuropathological symptoms that are observed in patients.Unlike humans in mice, a GA2-routed GM2 degradation pathway mediated by neuraminidase was suggested [3,4].Recently, the role of plasma membrane-associated neuraminidase Neu3 in glycolipid degradation in mice was investigated by generating Hexa-/-Neu3-/-double knock-out mouse model, and that model has been introduced as a novel valid murine model of TSD [5].The Hexa-/--Neu3-/-mice were healthy at birth but died at nearly 5 months of age due to progressive neurodegeneration with neuronal loss, Purkinje's cell depletion, and neuroinflammation.Neuroinflammation and neurological abnormalities such as slow movement, ataxia, and tremors were also reported in this mouse model [5,6].
Gangliosides are the predominant glycosphingolipids found in neuronal cell membranes.Their distinctive surface patterns are formed through highly synchronized biosynthesis and degradation processes within secretory compartments (ER and Golgi) and endolysosomal systems, respectively.The ganglioside biosynthesis pathway involves the synthesis of ceramide backbone, sequential addition of sugar residues, sialylation, and the formation of complex gangliosides [7,8].This dynamic process requires the action of several enzymes like glycosyltransferases (i.e., B4Galnt1, GM3S, and GD3S), hexosaminidases (i.e., HexA and HexB), and neuraminidases (Neu1, Neu3, and Neu4).The GM2/ GD2 synthase (also known as β-1,4-N-acetylgalactosaminyl transferase), encoded by the B4galnt1 gene, acts at a specific step in the pathway to create lactoseries gangliosides, which serve as intermediates for the generation of more complex gangliosides with additional sialic acid residues.The enzyme specifically catalyzes the transfer of N-acetylgalactosamine onto lactosylceramide, gangliosides GM3, and GD3, leading to the formation of sialic acid-containing gangliosides, GM2 and GD2 (Figure 1(a)) [9].
Gene therapy presents an appealing option for addressing lysosomal storage disorders (LSD) to reduce or reverse the accumulation of storage material in affected tissues due to its well-defined and broad range of therapeutic effectiveness [10,11].Various viral gene transfer vectors, like adenovirus, adeno-associated virus (AAV), lentivirus, and herpes virus, have been administered in vitro models or directly to the CNS of in vivo models of LSDs [10,12].Among viral delivery systems, AAV vectors possess several advantages, including robust in vivo expression of the therapeutic gene, minimal harmful effects, and the ability to activate the transgene in both actively dividing and resting cells [13].AAVmediated gene therapy has been used for the treatment of several LSDs such as mucopolysaccharidosis [14], Pompe's disease [15], Niemann-Pick's disease [16], GM1 gangliosidosis [17], and GM2 gangliosidosis [2,18].So far, researchers have employed various serotypes of AAV vectors and transgene cassettes to enhance brain and spinal cord transduction.Among these, AAV9 vectors demonstrated better transduction capabilities by their ability to cross the bloodbrain barrier and transduce both neurons and glia [19], which has led to therapeutic strategies targeting various CNS diseases through intravascular AAV9-mediated gene delivery [20][21][22].AAV9-mediated gene and/or substrate reduction therapy is efficacious for the treatment of LSDs [23,24] and neurodegenerative diseases used in clinical applications [25,26].
Multiple therapeutical strategies have been investigated like enzyme replacement therapy, pharmaceutical chaperone therapy, and hematopoietic stem cell replacement therapy for TSD; however, currently, there is no effective treatment available [27].In the present study, we aimed to apply AAV9-shRNA-mediated substrate reduction therapy to prevent GM2 ganglioside accumulation in TSD cell models for the first time, and we investigated the impact of in vitro downregulation of B4Galnt1 by using highly selective viral shRNA-mediated gene silencing.

Quantitative PCR Analysis for Viral Copy Number
Detection.Transfected HEK293T cells were resuspended in lysis buffer (150 mM NaCl, 50 mM Tris HCl, and pH 8.5).After freezing and thawing three times, they were centrifuged at 1,167 × g for 15 min at 4 °C.rAAV9-mB4Galnt1-shRNA virus particles were collected in the supernatant.Viral media and cell supernatant were treated with DNase I (Life Technologies) to remove any residual DNA contaminants not present within the intact virus particles.DNase I was inactivated through the addition of 0.5 mM EDTA and incubation at 65 °C for 10 min.Viral DNA was uncoated through digestion with proteinase K (Sigma-Aldrich) through incubation at 50 °C for 60 min.The AAV virus capsids were disassembled, and the AAV vector genome was released into the solution.Proteinase K was then inactivated by incubation at 95 °C for 20 min.
The number of viral genomes in each preparation was determined through quantitative real-time PCR with Light-Cycler 480 SYBR Green I Master Mix (Roche Life Science, Swiss) generating an amplicon from within the CMV promoter upstream of the mCherry transgene cassette: CMV forward (5 ′ -TCATATGCCAAGTACGCCCC-3 ′ ) and CMV reverse (5 ′ -CCCGTGAGTCAAACCGCTAT-3 ′ ).Samples were run on a LightCycler® 96 Instrument (Roche Life Science, Swiss).The conditions of PCR were as follows: 1 cycle of 10 min at 95 °C and 45 cycles of 20 s at 95 °C, 15 s at 61 °C, 3 Advances in Cell and Gene Therapy and 22 s at 72 °C.The values were read after each cycle.Samples were compared against a standard curve of AAV plasmid diluted from 1 × 10 3 to 1 × 10 7 copies per milliliter.The vector genomes per milliliter were calculated via quantitative real-time PCR.

Cell Lines and Transduction Assay. Mouse fibroblasts
were obtained from a skin biopsy of wild-type and Hexa-/-Neu3-/-mice at the age of 5 months.Human control and Tay-Sachs patient fibroblasts, obtained from forearm skin with a punch biopsy, were kindly donated by Dr. Nur Arslan, Child Neurology Department, Dokuz Eylül University.Mouse neuroglia cell (NG) cell line was generated from 100 mg brain tissue of 5-month-old wild-type and Hexa-/-Neu3-/-mice by using an adult brain dissociation kit according to manufacturer's instructions (Miltenyi Biotec, 130-107-677).After cell debris and red blood cell removal were performed, neurons were isolated by a neuron isolation kit according to the manufacturer's instructions (Miltenyi Biotec,.Primary cell lines were cultured in DMEM high glucose (Gibco) supplemented with 20% FBS (Gibco) and 1% (vol/vol) penicillin/streptomycin (Gibco) in a humidified incubator with 5% CO 2 at 37 °C.Primary cell lines were infected with human papillomavirus (HPV) type 16 E6E7 (HPV16E6E7) retrovirus (ATCC® CRL-2203™), and uninfected primary cells were eliminated with 50 μg/ ml of G418 (Sigma-Aldrich) for 14 days.The immortalized TSD neuroglia cells (Ng-125) obtained from the Tay-Sachs fetus were obtained [28].TSD neuroglia cells producing active HEXA enzyme throughout cDNA clone of the α subunit of Hex A (pCMV-Hex α) were used as control of human neuroglia cells [29].Immortalized cell lines were cultured in DMEM high glucose supplemented with 10% FBS and 1% (vol/vol) penicillin/streptomycin in a 37 °C, 5% CO 2 incubator.Immortalized cell lines were seeded at 40-60% confluence.Cells were fed DMEM high glucose supplemented with 10% FBS with prior infection, media changed to 2% FBS, and cells were exposed to AAV9-B4Galnt1-shRNA for 1 hour.After that, a complete medium was added and incubation continued for up to 72 hours.All infections were performed with the same number of vg (6 0 × 10 9 ) for 3 6 × 10 5 cells.The transduction efficiency of each cell was measured based on mCherry signal, and the % of infected cells is presented in Figure S1.
2.4.Quantitative PCR Analysis.Total RNA was extracted from skin fibroblast and neuroglia cells derived from humans and mice by using TRIzol Reagent (Geneaid).RNA was then reverse transcribed into cDNA using iScript cDNA Synthesis Kit (BioRad).50 ng RNA was used in each qRT-PCR reaction using iTaq Universal SYBR Green Supermix (BioRad) according to the manufacturer's instructions.The relative quantity of endogenous B4Galnt1 transcript in the cells had received the AAV9-B4Galnt1-shRNA compared with the cells that had no AAV9-B4Galnt1-shRNA.Primers in Table 1 were used in the qRT-PCR analysis.Each sample was run in triplicate, and relative concentration was calculated using the ddCt values normalized to endogenous GAPDH transcript.

Lysosomal Accumulation Is Reduced in TSD Mouse
Fibroblast and Neuroglia Cells.Here, we examined the effects of AAV9-m-shRNA1 transduction on the lysosomal accumulation of GM2 ganglioside in the TSD mouse fibroblast and neuroglia.In untreated conditions, significantly increased GM2 (Figures 4(a    We further displayed AAV9-h-shRNA1 transduction in the TSD neuroglia cell line.Immunofluorescence staining of the GM2 and LAMP1 demonstrated that the knockdown of B4GALNT1 by AAV9-h-shRNA1 significantly reduced the lysosomal accumulation of the GM2 ganglioside ( * * P < 0 001) (Figure 7(d)) and a lysosomal marker of LAMP1 ( * P < 0 05) in TSD patient neuroglia cell (Figure 7(h)).

Discussion
The Tay-Sachs disease is a neurodegenerative lysosomal storage disorder resulting from genetic mutations in the HEXA gene leading to the accumulation of GM2 ganglioside TSD Human Fibroblast TSD Human Neuroglia  Relative expression levels of (b, g) B3GALT4, (c, h) HEXB, (d, i) GM3S, and (e, j) GD3S in untransduced and AAV9-h-shRNA1transduced TSD human fibroblast and neuroglia, respectively (n = 3; * P < 0 05, * * P < 0 01, and * * * P < 0 005).Representative fluorescent images showed (a-c) GM2 ganglioside and (e-g) lysosome levels in the untreated and AAV9-transduced cells stained with anti-GM2 (green) and anti-Lamp1 (green) antibodies following 72 hours post-transduction.The total intensity of fluorescence was quantified using Fiji imaging software.Graphical representations were indicated for (d) GM2 and (h) Lamp1 intensity per cell (n = 3; * * P < 0 01, * * * P < 0 005, and * * * * P < 0 001; scale bar represents 100 μm).Advances in Cell and Gene Therapy in the CNS.There is no effective treatment for patients with Tay-Sachs yet.Enzyme replacement therapy, stem cell transplantation, substrate reduction therapy, or gene therapy comprises the main treatment strategies for many lysosomal storage disorders [31].Some of them provide or enhance the activity of the missing enzyme to re-establish substrate catabolism; however, others have no impact on the correction of neuropathology for diseases like Tay-Sachs because of the blood-brain barrier.Viral vector-based gene therapies are one of the novel treatment strategies for lysosomal storage disorders to provide the activity of the missing enzyme and re-establish substrate metabolism in the CNS [32].A bunch of studies have shown the potential for AAV treatment in the management of gangliosidosis models in vitro and in vivo [17,[33][34][35].In the present study, we suggest a new experimental treatment strategy for the Tay-Sachs disease using AAV-mediated RNAi technology targeting the human and mouse B4Galnt1 gene which catalyzes GM2 biosynthesis.

Advances in Cell and Gene Therapy
Substrate reduction therapy (SRT) acts upstream of the enzymatic defect, decreasing storage by silencing its biosynthetic pathway.β-1,4-N-Acetylgalactosaminyl transferase coded by the B4Galnt1 gene is a key enzyme in the ganglioside biosynthesis pathway involving the conversion of GM3 to GM2 [9].In our study, we selected the B4Galnt1 gene as a target for substrate reduction therapy to decrease GM2 biosynthesis and eventually its accumulation in cell models of the Tay-Sachs disease.Deficiency of the B4GALNT1 enzyme in mice leads to the complete absence of major brain gangliosides; instead, the less complex gangliosides GM3 and GD3 are found in higher concentrations since they are synthesized before the block in the biosynthetic pathway.Interestingly, despite the lack of complex ganglioside biosynthesis, the overall levels of glycosphingolipids (GSLs) in the B4Galnt1 KO mice remain comparable to those in normal mice due to the compensatory increase in the upstream synthesis of simple GSLs [36].Gangliosides play multifaceted roles especially during the neurodevelopmental stage, contributing to the important processes that shape the developing nervous system [37].Their involvement in neuronal migration, axon guidance, dendritic arborization, synaptogenesis, and myelination highlights their significance in ensuring proper neuronal development and function [38].Simple gangliosides, such as GM3, are prevalent during early neurodevelopmental stages and are involved in fundamental processes like cell proliferation, migration, and differentiation.As neurodevelopment progresses, there is a shift towards expressing more complex gangliosides, like GD1a, GD1b, and GT1b.These complex gangliosides are enriched in mature neurons and are intricately involved in synaptic plasticity, neurotransmitter release, and signal transduction [39].The balance between simple and complex gangliosides is tightly regulated throughout neurodevelopment, ensuring proper neuronal maturation and function.Dysregulation of ganglioside metabolism or expression has been implicated in various neurodevelopmental disorders, including intellectual disabilities, developmental delay, and neurological disorders like hereditary spastic paraplegia (HSP) [40].In humans, mutations in the B4GALNT1 gene have been shown to cause HSP which is a genetically heterogeneous group of neurological disorders characterized clinically by slow progressive lower limb spasticity and the axonal degeneration in the corticospinal tracts [41].It has been reported that mutations on B4GALNT1 that cause loss of function of the enzyme lead to HSP with an extreme phenotype, with very limited exceptions that reduced enzyme activity (M4 mutant) causes a mild phenotype of HSP disease.Additionally, an 85% reduction of the B4GALNT1 enzyme activity is caused by the read through of the nonsense mutation at codon 228 of the B4GALNT1 gene which alleviates the clinical pathology of HSP patients [42].We suggest that silencing the B4GALNT1 gene at the postnatal stage in TSD patients may prevent the neurodevelopmental abnormalities observed in HSP patients with B4GALNT1 deficiency.Here, we first aimed to determine the effectiveness of silencing B4GALNT1 by AAV-mediated-shRNA treatment in vitro and address the basis for future studies in vivo to treat the TSD mouse model we generated.
In the present study, we first prepared the shRNAs with AAV9 which is an efficient natural AAV serotype bypassing the BBB and efficiently targeting cells of the CNS [19,43].In our in vitro study, therefore, we transduced both mouse and human TSD fibroblast and neuroglia with rAAV9-B4Galnt1-shRNAs and determined m-shRNA1 and h-shRNA1 as the most efficient siRNAs in mouse and human cells, respectively.
We demonstrated that although silencing of B4galnt1 with m-shRNA1 and h-shRNA1 displayed an increasing pattern for B3GALT4 gene expression, it was statistically not significant.We speculate that this situation might be related to accumulated GM2 ganglioside amounts in each cell type where B3GALT4 catalyzes GM2 to GM1 conversion; however, this hypothesis requires further investigation.
Previously, it has been demonstrated that B4GALNT1 deficiency leads to increased synthesis of GM3 and GD3 gangliosides in human fibroblast and mouse models, as B4GALNT1 is at the downstream of GM3 synthesis in the ganglioside synthesis pathway [44,45].Consistently, our data showed significantly increased levels of GM3S expression in all cell models except mouse fibroblast.Elevated levels of GD3S expression in all cell models studied also suggest a higher rate of GM3 biosynthesis when B4GALNT1 is silenced.Additionally, significantly increased HexB gene expression levels in mouse fibroblast and human neuroglia might be related to the compensation mechanisms due to GSL alterations under the same conditions.Our immunocytochemical data using anti-GM2 and anti-LAMP1 also displayed silencing of the B4GALNT1 gene enabling the depletion of accumulated GM2 and lysosomal mass in both human and mouse TSD cell models.
Overall, we suggest a new strategy for the treatment of TSD with the proof concept of study using AAV-mediated RNAi technology targeting the B4GALNT1 gene at the upstream of the enzymatic defect in TSD pathology.We demonstrated that AAV9-B4Galnt1-shRNA transduction could effectively suppress B4GALNT1 expression and reverse lysosomal storage of GM2 ganglioside in both mouse and human TSD cell models.Therefore, we address that

Figure 1 :
Figure 1: Ganglioside biosynthesis and degradation pathway in Hexa-/-Neu3-/-TSD model.The dotted lines indicate the knock-out genes and proposed silencing of the B4Galnt1 gene by shRNA treatment (a).Schematic presentation of self-complementary recombinant AAV vector including shRNA silencing cassettes controlled by U6 promoter and mCherry reporter gene regulated by CMV promoter (b).Location of the target regions of the shRNA(s) for mouse and human B4Galnt1 gene (c).

Table 1 :
Primer sequences for the qRT-PCR analysis.