Hematopoietic stem cell gene therapy ameliorates CNS involvement in murine model of GM1-gangliosidosis

Tsunogai, Toshiki; Ohashi, Toya; Shimada, Yohta; Higuchi, Takashi; Kimura, A.; Watabe, Ayako M.; Kato, Fusao; Ida, Hiroyuki; Kobayashi, Hiroshi

doi:10.1016/j.omtm.2022.04.012

Cited by 7 publications

(3 citation statements)

References 51 publications

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“…However, various therapeutic approaches have been tested preclinically in small and large animal models of LSDs, some of which have led to the development of clinical trials for these diseases. Specifically, the β-Gal −/− mice have been extensively exploited to test the efficacy of various therapies, such as substrate deprivation therapy, as well as ex vivo and in vivo gene therapy [ 31 , 33 , 34 , 35 , 42 ]. In all instances, correction of the visceral organ pathology was accompanied by partial reversal of some of the neurodegenerative aspects of this disease, including neuronal cell death and neuroinflammation [ 18 ].…”

Section: Discussionmentioning

confidence: 99%

“…This favors the formation of these contact sites and facilitates the transfer of Ca 2+ from the ER to the mitochondria, ultimately evoking the mitochondria apoptotic pathway [ 30 ]. β-Gal −/− mice have also been exploited for testing various therapeutic modalities, including in vivo and ex vivo gene therapy, with promising improvement of the CNS pathology [ 31 , 33 , 34 , 35 ].…”

Section: Introductionmentioning

confidence: 99%

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Preclinical Enzyme Replacement Therapy with a Recombinant β-Galactosidase-Lectin Fusion for CNS Delivery and Treatment of GM1-Gangliosidosis

Weesner

Annunziata

Yang

et al. 2022

Cells

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GM1-gangliosidosis is a catastrophic, neurodegenerative lysosomal storage disease caused by a deficiency of lysosomal β-galactosidase (β-Gal). The primary substrate of the enzyme is GM1-ganglioside (GM1), a sialylated glycosphingolipid abundant in nervous tissue. Patients with GM1-gangliosidosis present with massive and progressive accumulation of GM1 in the central nervous system (CNS), which leads to mental and motor decline, progressive neurodegeneration, and early death. No therapy is currently available for this lysosomal storage disease. Here, we describe a proof-of-concept preclinical study toward the development of enzyme replacement therapy (ERT) for GM1-gangliosidosis using a recombinant murine β-Gal fused to the plant lectin subunit B of ricin (mβ-Gal:RTB). We show that long-term, bi-weekly systemic injection of mβ-Gal:RTB in the β-Gal−/− mouse model resulted in widespread internalization of the enzyme by cells of visceral organs, with consequent restoration of enzyme activity. Most importantly, β-Gal activity was detected in several brain regions. This was accompanied by a reduction of accumulated GM1, reversal of neuroinflammation, and decrease in the apoptotic marker caspase 3. These results indicate that the RTB lectin delivery module enhances both the CNS-biodistribution pattern and the therapeutic efficacy of the β-Gal ERT, with the potential to translate to a clinical setting for the treatment of GM1-gangliosidosis.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Preclinical Enzyme Replacement Therapy with a Recombinant β-Galactosidase-Lectin Fusion for CNS Delivery and Treatment of GM1-Gangliosidosis

Weesner

Annunziata

Yang

et al. 2022

Cells

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“…One of the important methods in delivering therapeutic components is choosing the right type of vector. According to the previous studies of researchers, it can be found that different viral vectors such as lentiviruses, 95,[102][103][104] retroviruses, 105 adenoviruses, 77 and AAVs can be used for gene therapy of GM1 gangliosidosis disease.…”

Section: Pros and Cons Of Therapeutic Vectors And Limitations Of Gene...mentioning

confidence: 99%

Gene therapy approaches for GM1 gangliosidosis: Focus on animal and cellular studies

Hosseini,

Fallahi,

Tabei

et al. 2023

Cell Biochemistry & Function

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One of the most important inherited metabolic disorders is GM1 gangliosidosis, which is a progressive neurological disorder. The main cause of this disease is a genetic defect in the enzyme β‐galactosidase due to a mutation in the glb1 gene. Lack of this enzyme in cells (especially neurons) leads to the accumulation of ganglioside substrate in nerve tissues, followed by three clinical forms of GM1 disease (neonatal, juvenile, and adult variants). Genetically, many mutations occur in the exons of the glb1 gene, such as exons 2, 6, 15, and 16, so the most common ones reported in scientific studies include missense/nonsense mutations. Therefore, many studies have examined the genotype‐phenotype relationships of this disease and subsequently using gene therapy techniques have been able to reduce the complications of the disease and alleviate the signs and symptoms of the disease. In this regard, the present article reviews the general features of GM1 gangliosidosis and its mutations, as well as gene therapy studies and animal and human models of the disease.

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Creation of an in vitro model of GM1 gangliosidosis by CRISPR/Cas9 knocking‐out the GLB1 gene in SH‐SY5Y human neuronal cell line

Hosseini,

Fallahi,

Aligholi

et al. 2024

Cell Biochemistry & Function

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GM1 gangliosidosis is one type of hereditary error of metabolism that occurs due to the absence or reduction of β‐galactosidase enzyme content in the lysosome of cells, including neurons. In vitro, the use of neural cell lines could facilitate the study of this disease. By creating a cell model of GM1 gangliosidosis on the SH‐SY5Y human nerve cell line, it is possible to understand the main role of this enzyme in breaking down lipid substrate and other pathophysiologic phenomena this disease. To knock‐out the human GLB1 gene, guides targeting exons 14 and 16 of the GLB1 gene were designed using the CRISPOR and CHOP‐CHOP websites, and high‐efficiency guides were selected for cloning in the PX458 vector. After confirming the cloning, the vectors were transformed into DH5α bacteria and then the target vector was extracted and transfected into human nerve cells (SH‐SY5Y cell line) by electroporation. After 48 h, GFP+ cells were sorted using the FACS technique and homozygous (compound heterozygous) single cells were isolated using the serial dilution method and sequencing was done to confirm them. Finally, gap PCR tests, X‐gal and Periodic acid‐Schiff (PAS) staining, and qPCR were used to confirm the knock‐out of the human GLB1 gene. Additionally, RNA sequencing data analysis from existing data of the Gene Expression Omnibus (GEO) was used to find the correlation of GLB1 with other genes, and then the top correlated genes were tested for further evaluation of knock‐out effects. The nonviral introduction of two guides targeting exons 14 and 16 of the GLB1 gene into SH‐SY5Y cells led to the deletion of a large fragment with a size of 4.62 kb. In contrast to the non‐transfected cell, X‐gal staining resulted in no blue color in GLB1 gene knock‐out cells indicating the absence of β‐galactosidase enzyme activity in these cells. Real‐time PCR (qPCR) results confirmed the RNA‐Seq analysis outcomes on the GEO data set and following the GLB1 gene knock‐out, the expression of its downstream genes, NEU1 and CTSA, has been decreased. It has been also shown that the downregulation of GLB1‐NEU1‐CTSA complex gene was involved in suppressed proliferation and invasion ability of knock‐out cells. This study proved that using dual guide RNA can be used as a simple and efficient tool for targeting the GLB1 gene in nerve cells and the knockout SH‐SY5Y cells can be used as a model investigation of basic and therapeutic surveys for GM1 gangliosidosis disease.

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Hematopoietic stem cell gene therapy ameliorates CNS involvement in murine model of GM1-gangliosidosis

Cited by 7 publications

References 51 publications

Preclinical Enzyme Replacement Therapy with a Recombinant β-Galactosidase-Lectin Fusion for CNS Delivery and Treatment of GM1-Gangliosidosis

Preclinical Enzyme Replacement Therapy with a Recombinant β-Galactosidase-Lectin Fusion for CNS Delivery and Treatment of GM1-Gangliosidosis

Gene therapy approaches for GM1 gangliosidosis: Focus on animal and cellular studies

Creation of an in vitro model of GM1 gangliosidosis by CRISPR/Cas9 knocking‐out the GLB1 gene in SH‐SY5Y human neuronal cell line

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