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. Author manuscript; available in PMC: 2025 May 12.
Published in final edited form as: J Gene Med. 2020 May 7;22(9):e3205. doi: 10.1002/jgm.3205

Improvement of motor and behavioral activity in Sandhoff mice transplanted with human CD34+ cells transduced with a HexA/HexB expressing lentiviral vector

Julie Beegle 1, Kyle Hendrix 1, Haley Maciel 1, Jan A Nolta 1, Joseph S Anderson 1
PMCID: PMC12067969  NIHMSID: NIHMS1599114  PMID: 32335981

Abstract

Background:

Tay-Sachs and Sandhoff disease are debilitating genetic diseases that affect the central nervous system leading to neurodegeneration through the accumulation of GM2 gangliosides. There are no cures for these diseases and treatments do not alleviate all symptoms. Hematopoietic stem cell gene therapy offers a promising treatment strategy to deliver wild type enzymes to affected cells. By genetically modifying HSC to express wild type HexA and HexB, systemic delivery of functional enzyme can be achieved.

Methods:

Primary human HSPC and Tay-Sachs affected cells were used to evaluate the functionality of the vector. An immunodeficient and humanized mouse model of Sandhoff disease was used to evaluate whether the HexA/HexB lentiviral vector transduced cells were able to improve the phenotypes associated with Sandhoff disease. An immunodeficient NOD-RAG1−/−IL2−/− (NRG) mouse model was used to evaluate whether the HexA/HexB vector transduced human CD34+ cells were able to engraft and undergo normal multi-lineage hematopoiesis.

Results:

HexA/HexB lentiviral vector transduced cells demonstrated strong expression of HexA and HexB and restored enzyme activity in Tay-Sachs affected cells. Upon transplantation into a humanized Sandhoff disease mouse model, improved motor and behavioral skills were observed. Decreased GM2-gangliosides were observed in the brains of HexA/HexB vector transduced cell transplanted mice. Increased peripheral blood levels of HexB was also observed in transplanted mice. Normal hematopoiesis in the peripheral blood and various lymphoid organs was also observed in transplanted NRG mice.

Conclusions:

These results highlight the potential use of stem cell gene therapy as a treatment strategy for Tay-Sachs and Sandhoff disease.

Keywords: stem cell – haemopoietic, gene – therapy, vector – viral

INTRODUCTION:

Tay-Sachs disease (TSD) and Sandhoff disease (SD) are lysosomal storage diseases (LSD) characterized by an accumulation of GM2-gangliosides due to a defective beta-hexosaminidase (Hex) enzyme [1–6]. HexA and HexB form a heterodimer which act to degrade GM2-gangliosides. Mutations in the HexA and HexB subunits define TSD and SD, respectively, leading to progressive and fatal neurodegeneration. TSD and SD are classified according to disease severity as infantile, juvenile, or adult-onset forms [1–6]. The infantile form is the most severe developing in the first months of life with symptoms beginning early along with developmental arrest. The remaining years of life consist of a decline to a semi-vegetative and unresponsive state with seizures, choking on oral secretions, blindness, deafness, screaming episodes and spasticity [1–6]. Late-onset GM2-gangliosidosis is the second most common form, but because early symptoms are common to other diseases, affected adults may be misdiagnosed for a long time. Symptoms include speech difficulties, muscle weakness, tremor and ataxia. Manic-depressive or psychotic episodes are present in about 30% of affected persons. The majority of late-onset patients are wheelchair-bound by age 30–40. The juvenile forms vary greatly in severity from case to case but all juvenile forms of GM2 gangliosidosis are fatal [1–6].

Currently, there is no cure or corrective therapy for TSD or SD and supportive care only marginally prolongs the lives of patients. Numerous therapeutic approaches have been developed to treat TSD and SD with some being evaluated in a clinical setting. These include enzyme replacement therapy, allogeneic HSC and blood transplants, adeno-associated virus delivery of HexA and HexB, substrate reduction therapy, gene editing using a CRISPR system, and chaperone therapy [7–19]. Enzyme replacement therapy is a straight-forward approach of injecting the wild-type HexA and/or HexB protein product, however, it does not readily enter the central nervous system (CNS) [7–9]. Allogeneic HSC and blood transplants have been utilized to systemically deliver HexA and HexB to patients through the blood system from donors who are unaffected by TSD or SD [10–12]. This approach has demonstrated some evidence of improvement in patients, however, side effects including rejection of the donor cells or graft-versus host disease could occur when using cells from another person. Gene therapy and gene editing approaches including adeno-associated virus (AAV) delivery of wild-type HexA and HexB and the addition of a HexM open reading frame via a CRISPR system are currently being evaluated as viable therapeutic options [13–15, 17]. AAV has demonstrated strong expression of delivered genes, however, large doses of AAV are required to be injected and do not readily enter the CNS if injected in a systemic manner. Substrate reduction therapy has been evaluated in a group of juvenile GM2 gangliosidosis patients but did not display improvement in clinical symptoms [16]. Chaperone therapy using pyrimethamine has also been in late onset TSD patients and displayed promising results in speech improvement in some patients, however, doses of pyrimethamine need to be tailored to each patient as increased doses caused TSD symptoms to increase in severity [18–19].

Autologous HSC gene therapy for TSD and SD offers a potential alternative approach by delivering wild-type HexA and HexB systemically, including in the CNS, while using a patient’s own blood cells. Transplantation of CD34+ HSC genetically engineered to overexpress wild-type HexA and HexB could potentially alleviate TSD and SD symptoms, as recently demonstrated with similar strategies for metachromatic leukodystrophy (MLD) and adrenoleukodystrophy (ALD) [20–23]. Cells derived from the vector transduced blood stem cells migrate into the brain and establish long-term residence as microglia [24]. Through cross correction, HexA and HexB proteins secreted by the vector transduced hematopoietic lineage cells can be taken up by affected neurons in the CNS via the mannose-6-phosphate receptor where they can degrade GM2-gangliosides. This would allow for an alleviation of symptoms and potentially halt the progression of these diseases. Lentiviral vectors are an effective delivery vehicle for the wild type enzymes due to their ability to transduce dividing and non-dividing cells, including HSC [25–27]. They also provide long-term expression of introduced genes due to their ability to integrate into target cells and are less prone to gene silencing in differentiated cells. This approach differs from the above mentioned therapies currently being evaluated for TSD and SD in that it would use a patient’s own cells, would deliver the actual enzymes being affected by the respective diseases, and would be performed ex vivo, alleviating the need to directly inject vector into the body.

Mouse models for TSD and SD are available, however, the strain which has a deleted HexA gene does not display motor or behavioral symptoms of Tay-Sachs disease due to a work-around mechanism in the mice [28–29]. These mice only display minor GM2-ganglioside storage in the brain [28]. Therefore, the SD mouse model which has normal levels of HexA but lacks expression of HexB is more ideal for evaluating new therapeutic approaches as improvement of clinical symptoms can be evaluated [29]. SD mice begin showing symptoms around three months of age and die within the next 30–45 days. Symptoms include increased GM2-ganglioside accumulation in the CNS and decreased hind limb function and movement. No HexB expression is detected in the periphery or in the CNS [29]. These current models are immunocompetent strains and would reject any human cells transplanted. Therefore, to be able to evaluate the efficacy of our therapeutic approach using human CD34+ HSC, we have generated a new immunodeficient HexB knockout mouse model which is capable of accepting human CD34+ HSC for engraftment and multi-lineage hematopoiesis. This model was generated by crossing B6-RAG2−/−IL2−/− mice with B6-HexB−/− mice. Upon confirming their genotypes, progression of disease symptoms, and acceptance of human CD34+ HSC for engraftment, the new immunodeficient SD mouse model (BRG-SD) was used to evaluate the effectiveness of the HexA/HexB lentiviral vector transduced human CD34+ cells to improve the phenotypes associated with SD.

In our current study, we have demonstrated improved motor and behavioral activity in HexB deficient mice transplanted and engrafted with human CD34+ cells transduced with a HexA/HexB lentiviral vector. Overexpression and restoration of functional enzyme activity was observed in disease-specific cells and HSPC derived immune cells. Normal differentiation of human immune cells in the peripheral blood and lymphoid organs was also observed in NOD-RAG1−/−IL2rg−/− (NRG) mice upon transplantation of human CD34+ cells transduced with the HexA/HexB lentiviral vector [30]. This study highlights the potential use of this strategy to treat TSD and SD.

MATERIALS AND METHODS:

Lentiviral Vector Design and Production:

All lentiviral vectors were generated using a self-inactivating third-generation lentiviral vector backbone named CCLc-x (Figure 1a). To generate the HexA and HexB expressing lentiviral vectors, wild type human HexA (hHexA) and human HexB (hHexB) were PCR amplified from human peripheral blood mononuclear cell cDNA and cloned into pCR2.1 TOPO (ThermoFisher, Grand Island, NY). Clones were then confirmed by sequencing (Laragen, Culver City, CA). hHexA was then subcloned under the control of the MNDU3 promoter and hHexB was subcloned under the control of the phosphoglycerate kinase (PGK) promoter (named MAPB) (Figure 1c). A third expression cassette including an enhanced green fluorescent protein (EGFP) gene under the control of an elongation factor-1 alpha (EF1α) promoter was cloned downstream from the HexB gene in another version of the vector (named MAPBEE) (Figure 1d). This vector which contains the EGFP expression cassette was used to track and/or sort the transduced for various experiments. A control vector containing only an EGFP reporter gene was generated by cloning the EGFP gene under the control of the PGK promoter (EGFP) (Figure 1b). Experiments involving recombinant DNA were performed following the NIH guidelines.

Figure 1.

Figure 1.

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HexA/HexB lentiviral vectors: a) A self-inactivating lentiviral vector backbone, CCLc-x, was used to construct the HexA/HexB lentiviral vectors. b) A control EGFP alone vector which only contains an EGFP expression cassette was used as a control vector. c) Wild type human HexA and HexB were cloned under the control of MNDU3 and PGK promoters, respectively (MAPB). d) An EGFP gene under the control of an EF1α promoter was inserted downstream of the HexB gene (MAPBEE).

Lentiviral vectors were generated in human embryonic kidney (HEK)-293 cells by transfecting the cells with a 1:5:5 ratio of envelope vesicular stomatitis virus glycoprotein (VSVG), a packaging plasmid (Δ8.9) containing the vector capsid and reverse transcriptase genes, and one of the above mentioned transfer plasmids, either one of the HexA/HexB vectors (MAPB or MAPBEE) or the control EGFP vector. Forty-eight hours post-transfection, vector supernatants were collected and concentrated by ultrafiltration. Transducing unit titers of each vector were calculated by transducing HEK-293T cells and analyzing EGFP expression by flow cytometry forty-eight hours post-transduction for vectors containing the EGFP expression cassette. For the HexA/HexB vector that does not contain EGFP, total genomic DNA from the transduced cells was extracted using the Wizard Genomic DNA Isolation System (Promega, Madison, WI) and analyzed by quantitative PCR using Taqman Real-Time PCR Master Mix (ThermoFisher, Grand Island, NY) and a psi sequence primer and probe pair: psi probe 5’-AGCTCTCTCGACGCAGGACTCGGC=3’, psi forward 5’-ACCTGAAAGCGAAAGGGAAAC-3’, psi reverse 5’-CGCACCCATCTCTCTCCTTCT-3’. Flow cytometry for EGFP expression was performed using a Beckman Coulter Cytomics FC500 and CXP software.

TSD-affected cell culture and transduction:

Human fibroblasts and B cells obtained from individuals affected by TSD were purchased from Coriell Institute (Camden, NJ). Fibroblasts were cultured in Dulbecco’s minimum essential medium (DMEM) high glucose (GE Healthcare, Logan, UT) supplemented with 20% fetal bovine serum (FBS) and 1X each of penicillin-streptomycin and L-glutamine at 37oC with 5% CO2. Cells were left either nontransduced (NT), transduced at a multiplicity of infection (MOI) of 20 with the EGFP control vector, or transduced at an MOI of 20 with the MAPBEE vector and 8 mg/ml protamine sulfate for a minimum of 3 hours at 37oC. Forty-eight hours after transduction, cells were analyzed for EGFP expression by flow cytometry and expanded for use in subsequent Western blot and enzyme activity experiments. Flow cytometry was performed using a Beckman Coulter Cytomics FC500 and CXP software.

TSD-affected B cells were cultured in Iscove’s modified Dulbecco’s medium (IMDM) (GE Healthcare, Logan, UT) supplemented with 20% fetal bovine serum (FBS) and 1X each of penicillin-streptomycin and L-glutamine at 37oC with 5% CO2. Cells were left either nontransduced (NT) or transduced at a multiplicity of infection (MOI) of 20 with the MAPB vector and 8 mg/ml protamine sulfate for a minimum of 3 hours at 37oC. Forty-eight hours after transduction, cells were analyzed for HexA/HexB activity.

Isolation and Transduction of human CD34+ HSPC:

Human CD34+ HSPC were isolated from umbilical cord blood obtained from the UC Davis Umbilical Cord Blood Collection Program by Ficoll-Paque (GE Healthcare, Logan UT) density gradient, and further purified by CD34 magnetic bead column separation (Miltenyi Biotec, Auburn, CA). Total CD34+ cells were cultured for forty-eight hours in XVIVO-10 media (Lonza, Anaheim, CA) supplemented with 50 ng/ml stem cell factor (SCF), thrombopoietin (TPO), and Flt-3 ligand (R&D Systems, Minneapolis, MN). After forty-eight hours post-isolation, the CD34+ cells were left either nontransduced (NT) or transduced with either the EGFP control vector, the MAPBEE vector, or the MAPB vector at an MOI of 20 and 8 mg/ml protamine sulfate for a minimum of 3 hours at 37oC. EGFP control and MAPBEE vector transduced CD34+ cells were then sorted based on EGFP expression for subsequent experiments.

Western Blot:

Total cell extracts were collected from cells using a Pierce RIPA buffer (ThermoFisher, Richardson, TX) supplemented with Halt Protease Inhibitor (ThermoFisher, Richardson, TX) and flash frozen. Total protein concentrations were determined using a BCA Protein Assay Kit (ThermoFisher, Richardson, TX) following the manufacturer’s protocol. Proteins were loaded into polyacrylamide gels and transferred onto polyvinylidene fluoride membranes. Membranes were then incubated with their respective primary antibodies: mouse anti-human HexA (R&D Systems, Minneapolis, MN) and mouse anti-human HexB (Novus Biologicals, Littleton, CO). Mouse anti-human actin (Sigma-Aldrich, St. Louis, MO) was used as an internal control. Goat anti-mouse horseradish peroxidase (HRP) conjugated secondary antibodies were then added. The blots were then developed using SuperSignal West Pico Chemiluminescent Substrate (ThermoFisher, Richardson, TX).

Hexosaminidase enzyme activity assay:

For the hexosaminidase enzyme assays, total cell extracts from the TSD-affected fibroblasts or B cells or the CD34+ cell derived macrophages were generated by lysing the cells in 0.01M phosphate-citrate buffer (pH 4.4) containing 0.1% Triton-X 100 with six consecutive freeze-thaw cycles. Extracts were then clarified by centrifugation, the supernatants were collected, Halt protease Inhibitor (ThermoFisher, Salt Lake City, UT) was added, and the extracts were snap frozen. Total protein concentrations were determined using a BCA Protein Assay Kit (ThermoFisher, Salt Lake City, UT) following the manufacturer’s protocol. Total HexA/B activity was determined by incubating 1μg of cell lysates with either 4-methylumbelliferyl N-acetyl-β-D-glucosaminide (MUG) or 4-Methylumbelliferyl-6-sulfo-N-acetyl-β-D-glucosaminide potassium salt (MUGS) (Millipore, Salt Lake City, UT) for 1 hour at 37oC in 0.01M phosphate citrate buffer pH 4.4. Enzyme activity was determined by measuring fluorescence at 360nm (excitation) and 450nm (emission). Experiments were performed in triplicate.

Colony-forming unit (CFU) assays:

CD34+ cells, either NT, fluorescently activated cell sorted (FACS) EGFP control vector transduced, FACS sorted MAPBEE vector transduced, or MAPB vector transduced cells (500 total cells) were cultured for 12 days in MethoCult medium (Stemcell Technologies, Vancouver, CA). Following the culture time period, the total burst forming unit-erythroid colonies (BFU-E), granulocyte/macrophage (GM) colonies, and granulocyte/erythrocyte/megakaryocyte/macrophage (GEMM) colonies were observed by microscopy and counted. Experiments were performed in triplicate.

Derivation of mature macrophages and analysis of phenotypic markers:

CFUs derived from the NT and vector transduced CD34+ cells were further derived into mature macrophages by plating the cells in six-well plates with DMEM supplemented with 10% FBS, 10 ng/ml macrophage colony stimulating factor (M-CSF) and 10 ng/ml of granulocyte-macrophage colony stimulating factor (GM-CSF) (R&D Systems, Minneapolis, MN) for 4 days with media changes every 2 days. Cells were observed by microscopy to identify macrophage morphology and analyzed by flow cytometry for expression of normal macrophage cell surface markers. Macrophages were stained with phycoerythrin (PE)-conjugated CD14, PE-conjugated HLA-DR, or PE-conjugated CD4 (BD Biosciences, San Jose, CA). Flow cytometry was performed using a Beckman Coulter Cytomics FC500 and CXP software. Experiments were performed in triplicate.

Stability of the MAPB vector in CD34+ cell derived macrophages:

To determine if there were any rearrangements or deletions of the MAPB vector transgenes in transduced cells, PCR was performed. Total genomic DNA was extracted from NT, EGFP vector transduced, and MAPB vector transduced CD34+ cell derived macrophages using the Wizard Genomic DNA Isolation System (Promega, Madison, WI). PCR was performed using high fidelity Taq and primers corresponding to vector-specific sequences:

psi forward 5’-ACCTGAAAGCGAAAGGGAAAC-3’,

MNDU3 reverse 5’-GCGCCGAGTGAGGGGTTGTGGGC-3’,

MNDU3 forward 5’-CGCCCTCAGCAGTTTCTAGAG-3’,

HexA reverse 5’-GGTCTGTTCAAACTCCTGCTCACAGAAGCCTAC-3’,

HexA forward 5’-CTTCCGCTGTGAGTTGCTGAGGCG-3’,

HexB reverse 5’-CATGTTCTCATGGTTACAATATCCAGC-3’. The MAPB transfer plasmid used for vector production was used as a positive control. PCR products were visualized on a 1% agarose gel.

Functionality of MAPB vector transduced CD34+ cell derived macrophages:

Functional analyses of MAPB vector transduced macrophages were performed using a macrophage phagocytosis assay and a cytokine secretion detection assay. NT or MAPB vector transduced CD34+ cell derived macrophages were incubated overnight with 1ug/ml FITC-labeled E. coli bioparticles (ThermoFisher, Grand Island, NY). The next day, the cells were washed twice with PBS, stained with a PE-conjugated anti-human CD14 antibody (BD Biosciences, San Jose, CA) and analyzed by flow cytometry. To further evaluate macrophage functionality, the cells were incubated overnight with 1ug/ml of lipopolysaccharide. On days 1 and 2 post-stimulation, culture supernatants were sampled and analyzed for IL-1β, IL-6, IL-10, and TNF using a BD Cytokine Array detection kit (BD Biosciences, San Jose, CA) according to the manufacturer’s protocol. Experiments were performed in triplicate.

Open field analysis of engrafted humanized SD mice:

To evaluate whether the HexA/HexB vector transduced human CD34+ HSC improved the SD phenotypes in transplanted mice, an immunodeficient HexB knockout mouse model (BRG-SD) was used. This model was generated by crossing the SD HexB−/− mice with the immunodeficient B6-RAG2−/−IL2rg−/− (BRG) mice to allow for the engraftment of transplanted human CD34+ HSC [25].

NT or MAPB vector transduced human CD34+ HSC (500,000 cells) were transplanted intrahepatically into 2–5 day old BRG-SD pups (N=12 per cohort) sublethally irradiated with 100 rad. Ninety days post-transplant, mice were subjected to a 10-minute open field assay to evaluate total distance moved, total time moving, and total vertical movements on a 45 × 45 cm photocell-equipped Accuscan which automatically records and analyzes parameters. Open field analyses were performed until humane endpoints of either 20% loss of body weight or paralysis of one hind limb were reached. Nontransplanted HexB+/+ (BRG-wild type) and HexB−/− (BRG-mutant) mice were used as controls.

Periodic Acid Schiff (PAS) staining of GM2-ganliosides:

Upon completion of the open field analyses, mice were euthanized and their brains were formalin fixed followed by dehydration in 30% sucrose. Brains were snap frozen and then sectioned in 40μm sections. After slide mounting, brain sections were stained with PAS. Pictures were taken of the cerebellum, hippocampus, midbrain, and prefrontal cortex at 20x magnification. PAS stained GM2-gangliosides were then counted and compared between mouse cohorts.

HexB ELISA:

To evaluate the levels of HexB in the peripheral blood of MAPB vector transduced cell engrafted BRG-SD mice, ELISAs were performed on peripheral blood plasma. Upon completion of the open field analyses, mice were euthanized and peripheral blood was obtained, spun down, and the plasma was collected. HexB ELISAs were performed using a Human HexB Sandwich ELISA kit (LifeSpan Biosciences, Seattle, WA) according to the manufacturer’s protocol.

Transplantation of NOD-RAG1−/−IL2rg−/− Mice:

NOD-RAG1−/−IL2rg−/− (NRG) mice (stock number 007799) were obtained from The Jackson Laboratory (Sacramento, CA) [26]. Two to five-day old NRG mice (N=15 per cohort) were sublethally irradiated with 100 rads and transplanted intrahepatically with either EGFP control vector or MAPBEE vector transduced human CD34+ HSC (300,000 total cells). To determine engraftment levels, at 3 months post-transplant, mice were bled via tail vein and analyzed by flow cytometry with a PE-CY7-conjugated anti-human CD45 antibody. Flow cytometry was performed using a Beckman Coulter FC-500. Mice were used in accordance with institutional and IACUC guidelines.

Multilineage Hematopoiesis of Engrafted Human Immune Cells:

To evaluate the derivation of human immune cells in transplanted NRG mice, cells from the spleen, thymus, peripheral blood, and bone marrow were labeled with antibodies specific for human immune cell markers and analyzed by flow cytometry. T cells from the spleen, thymus, and peripheral blood were labeled with a Brilliant violet (BV) 421-conjugated CD3 antibody, an allophycocyanin (APC) H7-conjugated CD4 antibody, and an Alexa fluor (AF) 700-conjugated CD8 antibody (BD Biosciences, San Jose, CA). B cells from the spleen and bone marrow were labeled with a PE-CY7-conjugated CD45 antibody and a PE-conjugated CD19 antibody (BD Biosciences, San Jose, CA). Macrophages from the bone marrow were labeled with a PE-CY7-conjugated CD45 antibody and a PE-conjugated CD14 antibody (BD Biosciences). CD34+ cells from the bone marrow were labeled with a PE-CY7-conjugated CD45 antibody and a PE-conjugated CD34 antibody (BD Biosciences, San Jose, CA). Flow cytometry was performed using a BD Fortessa.

RESULTS

Enzyme expression and restoration of total Hex activity in TSD-affected fibroblasts and B cells following lentiviral vector transduction:

Individuals affected by TSD and SD have decreased or nonexistent activity of HexA or HexB, respectively, leading to the buildup of GM2-gangliosides. Therefore, to initially evaluate the functionality of the HexA/HexB lentiviral vectors, MAPBEE and MAPB, vector transduced TSD-affected fibroblasts and B cells were analyzed for enzyme expression and enzyme activity.

Upon transduction of the TSD-affected fibroblasts with the MAPBEE lentiviral vector, Western blots were performed with anti-HexA and anti-HexB antibodies to evaluate the overexpression of HexA and HexB. As displayed in Figure 2a, strong bands corresponding to HexA and HexB were observed in the MAPBEE vector transduced fibroblasts (lane 3) compared to NT (lane 1) and EGFP alone vector transduced (lane 2) cells. Actin was used as an internal control and was observed in all lanes.

Figure 2.

Figure 2.

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Functionality of HexA/HexB expressing lentiviral vectors in TSD-affected human fibroblasts and B cells: a) TSD-affected fibroblasts were left nontransduced (NT) or transduced with a control EGFP lentiviral vector (EGFP) or the HexA/HexB lentiviral vector (MAPBEE). Total cell extracts were then analyzed by Western blot for the expression of HexA and HexB. Actin was used as an internal control. Total cell extracts from the fibroblasts were analyzed for b) total Hex (MUG) and c) HexA (MUGS) activity. TSD-affected B cells were either left NT or transduced with the HexA/HexB lentiviral vector (MAPB). Total cell extracts from the B cells were analyzed for d) total Hex (MUG) and e) HexA (MUGS) activity. Asterisk indicates p values <0.01.

To evaluate whether HexA and HexB expressed in vector transduced fibroblasts were enzymatically active, 4-MU enzyme activity assays was performed. Protein extracts obtained from vector transduced fibroblasts were incubated with the MUG (total Hex activity) or MUGS (HexA activity) substrates and measured for fluorescence activity. As displayed in Figure 2b, significant MUG enzyme activity was observed in MAPBEE (p<0.001) vector transduced cells as compared to control NT and EGFP vector transduced cells. For the MAPBEE transduced fibroblasts, enzyme activity levels averaged 926 μM/hr/mg compared to NT cells which averaged 109 μM/hr/mg and EGFP control vector transduced cells which averaged 125 μM/hr/mg. As displayed in Figure 2c, significant MUGS activity was observed in MAPBEE (p<0.01) vector transduced cells as compared to control NT and EGFP vector transduced cells. For the MAPBEE transduced fibroblasts, enzyme activity levels averaged 7202 nM/hr/mg compared to NT cells which averaged 932 nM/hr/mg and EGFP control vector transduced cells which averaged 1745 nM/hr/mg. To evaluate whether HexA and HexB expressed in the vector transduced B cells were enzymatically active, the 4-MU enzyme activity assays was performed as described above. As displayed in Figure 2d, significant MUG enzyme activity was observed in MAPB (p<0.01) vector transduced cells as compared to control NT cells. For the MAPB transduced B cells, enzyme activity levels averaged 17.1 mM/hr/mg compared to NT cells which averaged 4.5 mM/hr/mg. As displayed in Figure 2e, significant MUGS enzyme activity was observed in MAPB (p<0.01) vector transduced cells as compared to control NT cells. For the MAPB transduced B cells, enzyme activity levels averaged 16.7 mM/hr/mg compared to NT cells which averaged 4.1 mM/hr/mg.

Transduction, differentiation, and HexA/HexB expression in HexA/HexB vector transduced human CD34+ HSPC:

Due to the overexpression of HexA and HexB in CD34+ HSPC by lentiviral transduction, it is possible that detrimental growth and differentiation of the cells could occur. To evaluate this, an HSPC CFU assay was performed. MAPBEE vector transduced CD34+ HSPC were FACS sorted based on EGFP expression and used for the CFU assay to specifically identify any effects caused by HexA and HexB overexpression. As displayed in Figure 3a, no significant differences (p>0.05) of all formed colonies, BFU-E, GM, and GEMM, were observed in the MAPBEE vector transduced cells as compared to control NT and EGFP vector transduced cells. CFUs were further differentiated, in vitro, into mature macrophages and evaluated by flow cytometry for normal macrophage markers. Cells transduced with the MAPBEE vector, on average, displayed normal levels of CD14 (99.3%), CD4 (98.9%), and HLADR (96.8%) as compared to control NT (CD14=99.9%, CD4=99.9%, and HLADR=99.7%) and EGFP alone vector transduced (CD14=99.7%, CD4=99.9%, and HLADR=99.6%) cells (data not shown).

Figure 3.

Figure 3.

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Differentiation of human CD34+ HSPC transduced with the HexA/HexB lentiviral vectors: a-b) Human CD34+ HSPC were left NT, transduced with the control EGFP lentiviral vector, transduced with the EGFP-containing HexA/HexB lentiviral vector (MAPBEE), or the HexA/HexB vector that does not contain EGFP (MAPB). Following transduction, EGFP+ cells were sorted by FACS. The NT and transduced cells were plated in methylcellulose media supplemented with cytokines. On day 12 post-plating, total BFU-E, CFU-GM, and CFU-GEMM colonies were counted. c) CFU colonies were further differentiated into macrophages in vitro which were analyzed by flow cytometry for various macrophage cell surface markers. Histograms presented are representations of data obtained.

To further evaluate the expression and enzyme activity of HexA and HexB in human CD34+ cell derived macrophages, the MAPB vector was utilized. Human CD34+ HSPC were left either nontransduced (NT) or transduced with the MAPB vector and a CFU assay was performed. As displayed in Figure 3b, no significant difference (p>0.5) in colony numbers were observed between the MAPB vector transduced and NT cell cultures. CFUs were further differentiated into mature macrophages in vitro and analyzed by flow cytometry for normal macrophage markers. MAPB vector transduced macrophages displayed comparable levels, on average, of CD14 (99.2%), CD4 (94.2%), and HLADR (74.6%) as compared to NT macrophages (CD4=99.5%, CD4=95.8%, and HLADR=74.6%) (Figure 3c). Representative histograms are displayed in the figure.

As a next step in evaluating the stability of the MAPB vector in transduced cells, PCR was performed on total genomic DNA obtained from MAPB vector transduced CD34+ cell derived macrophages. Primers specific for the MAPB vector were used to evaluate whether any deletions or rearrangements of the expression cassettes had occurred during transduction. As displayed in Figure 4a, similar PCR bands were observed with the positive control MAPB plasmid (+) and the MAPB vector transduced cell genomic DNA (MAPB). No bands were observed with genomic DNA extracted from NT macrophages. A schematic of the MAPB vector and where the PCR primers bind is displayed in Figure 4a. A band corresponding to the psi forward and MNDU3 reverse PCR reaction is observed in Figure 4a1 in the EGFP lane due to the presence of both the psi signal and the MNDU3 promoter in the EGFP alone vector.

Figure 4.

Figure 4.

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Stability and functionality of a HexA/HexB lentiviral vector in human CD34+ HSPC derived macrophages: Macrophages were generated in vitro from NT or MAPB HexA/HexB lentiviral vector transduced human CD34+ HSPC. a) Total genomic DNA was extracted from macrophages and analyzed by PCR with vector specific primers. Primer binding sites and relative PCR products are displayed with the vector schematic below the PCR bands. b) Total cell extracts from the macrophages were analyzed by Western blots for the expression of HexA and HexB. Actin was used as an internal control. Total cell extracts from the macrophages were also analyzed for c) total Hex (MUG) and d) HexA (MUGS) activity. Asterisk indicates p<0.05.

To further evaluate expression of both HexA and HexB in MAPB vector transduced CD34+ cell derived macrophages, Western blots were performed. As displayed in Figure 4b, overexpression of both HexA and HexB was demonstrated in MAPB vector transduced as compared to NT macrophages. HexA and HexB expression was detected in NT macrophages since the CD34+ cells used in the assay were from a healthy individual.

A 4-MU enzyme activity assays were performed to evaluate the levels of MUG and MUGS enzyme activity in MAPB vector transduced macrophages. As displayed in Figures 4c and 4d, significant increases (p=0.016) and (p=0.0094) in MUG and MUGS enzyme activity, respectively, were observed in MAPB macrophages compared to NT macrophages due to lentiviral vector overexpression. Enzyme activity was observed in the NT macrophages as these were obtained from healthy donors.

Taken together, these results demonstrated the functionality of the HexA/HexB lentiviral vectors through overexpression of the enzymes and restoration of enzyme activity in TSD-affected cells. The overexpression of these enzymes in human CD34+ cells also did not affect their expansion or differentiation into phenotypically normal end stage macrophages.

Normal functionality of MAPB vector transduced CD34+ cell derived macrophages:

Along with normal expansion, differentiation, and phenotype, end stage immune cells derived from MAPB vector transduced CD34+ HSPC also need to function properly. Therefore, to evaluate normal functions of the vector transduced macrophages, a phagocytosis and cytokine secretion assay were performed. FITC-labeled E. coli particles were added to the NT and MAPB macrophage cultures followed by analysis by flow cytometry. As displayed in Figure 5a, similar levels of E. coli positive and CD14 positive cells, on average, were observed in the MAPB vector transduced macrophages (84.5%) compared to the NT macrophages (84.7%). Representative dot plots are displayed in the figure.

Figure 5.

Figure 5.

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Phagocytosis and cytokine secretion of HexA/HexB lentiviral vector transduced macrophages: Human CD34+ HSPC were left NT or transduced with the MAPB HexA/HexB lentiviral vector. Cells were then derived into mature macrophages. a) FITC-labelled E. coli bioparticles were added to the macrophage cultures and analyzed the following day by flow cytometry for FITC and with a PE-conjugated CD14 antibody. b) Macrophages were also stimulated with LPS. On day 1 (black bars) and day 2 (grey bars) post-stimulation, culture supernatants were analyzed for the cytokines IL-1β, IL-6, IL-10, and TNF. Flow cytometry plots displayed are representative of the data obtained.

To further evaluate the functionality of the MAPB macrophages, LPS was added to the cultures to stimulate the cells. On days 1 and 2 post-stimulation, culture supernatant samples were taken and analyzed by flow cytometry using a BD cytokine array. As displayed in Figure 5b, similar levels of the cytokines IL-1β, IL-6, IL-10, and TNF were observed in MAPB macrophages on days 1 and 2 post-stimulation as compared to cultures of NT macrophages. These results demonstrated that overexpression of HexA and HexB in CD34+ cell derived macrophages did not have an effect on macrophage phagocytosis or cytokine secretion upon antigen stimulation.

Improved motor function and decreased GM2-gangliosides in MAPB cell engrafted BRG-SD mice:

For new therapies to proceed toward human clinical trials, relevant animal studies assessing the in vivo efficacy of the therapeutic candidate need to be performed. Therefore, to evaluate the ability of the HexA/HexB lentiviral vectors to improve SD phenotypes, MAPB vector transduced human CD34+ HSC were transplanted into immunodeficient BRG-SD mice. Successfully engrafted mice were then subjected to an open field assay to assess total distance traveled, total time moving, and total vertical movements for a 10-minute period. This analysis was performed twice weekly from 90 days post-transplant until humane endpoints were reached and the mice were required to be euthanized. As displayed in Figure 6a, a significant increase (p=0.01, p<0.001, and p=0.01) in total distance moved was observed in mice transplanted with MAPB vector transduced human CD34+ HSC on weeks 17, 18, and 19 post-transplant, respectively. This was in comparison to nontransplanted HexB−/− (MUT) mice. As displayed in Figure 6b, a significant increase (p=0.007 and p=0.04) in total time moving was observed in mice transplanted with MAPB vector transduced human CD34+ HSC on weeks 18 and 19 post-transplant, respectively. This was in comparison to nontransplanted HexB−/− (MUT) mice. An increase in total time moving was observed on week 17, however, it was not significant (p=0.13). As displayed in Figure 6c, a significant increase (p=0.05, p=0.03, and p=0.01) in total vertical movements was observed in mice transplanted with MAPB vector transduced human CD34+ HSC on weeks 17, 18, and 19 post-transplant, respectively. This was in comparison to nontransplanted HexB−/− (MUT) mice. HexB−/− (MUT) mice transplanted with nontransduced (NT) human CD34+ HSC were used as cell transplant controls and displayed similar phenotypic deficiencies as nontransplanted HexB−/− (MUT) mice. Wild-type (WT) HexB+/+ mice were also used as controls for normal mouse phenotypes.

Figure 6.

Figure 6.

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Open field analyses and GM2-ganglioside quantification of HexA/HexB lentiviral vector transduced cell engrafted BRG-SD mice: Human CD34+ HSC were left either NT or transduced with the MAPB HexA/HexB lentiviral vector (MAPB). Cells were transplanted intrahepatically into 2–5 day old BRG-SD (HexB−/−) mice. An open field assay evaluating a) total distance moved, b) total time moving, and c) total vertical movements was performed for 20 weeks post-transplant. Nontransplanted wild type (WT) and HexB−/− mutant (MUT) BRG-SD mice were used as controls. Single asterisks indicate p values <0.05. Double asterisks indicate prolonged survival of MAPB vector transduced cell engrafted mice compared to NT cell engrafted and nontransplanted MUT mice.

To further assess the ability of the HexA/HexB lentiviral vectors in vivo efficacy of the engrafted MAPB vector transduced CD34+ HSC, PAS staining was performed on brain sections from the open field BRG-SD mice post-euthanization. Aggregates were stained and counted from the cerebellum (CB), prefrontal cortex (PFC), midbrain (MB), and the hippocampus (HC). As displayed in Figure 7a, significant decreases (p<0.001) in the numbers of GM2-ganglioside aggregates were observed in mice transplanted with MAPB vector transduced CD34+ HSC (MAPB). This was in comparison to nontransplanted HexB−/− (MUT) mice. HexB−/− (MUT) mice transplanted with nontransduced (NT) human CD34+ HSC were used as cell transplant controls and displayed similar levels of GM2-gangliosides as nontransplanted HexB−/− (MUT) mice. Wild-type (WT) HexB+/+ mice were also used as controls for normal levels of GM2-gangliosides. Representative histology pictures from the prefrontal cortex (Figure 7b) and the midbrain (Figure 7c) are displayed.

Figure 7.

Figure 7.

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Detection of GM2-gangliosides by PAS staining: Following the open field assays, brain sections from the mice were analyzed in the cerebellum (CB), prefrontal cortex (PFC), midbrain (MB), and hippocampus (HC) for GM2-ganglioside aggregates by PAS staining. a) Aggregates were counted and compared between cohorts, wild-type (WT), mutant (MUT), nontransduced (NT), and MAPB. Representative histology pictures are presented from the b) prefrontal cortex and c) midbrain. Arrows indicate PAS stained GM2-ganglioside aggregates. Asterisks indicate p values <0.05.

Together, this data demonstrated that the overexpression of HexA and HexB in BRG-SD mice improves the motor functions of engrafted mice and decreases the accumulation of GM2-gangliosides in the brains of HexB−/− mice.

Overexpression of HexB in the peripheral blood of MAPB cell engrafted mice:

Due to the presence of MAPB vector transduced cells in the hematopoietic system of engrafted BRG-SD mice, the levels of HexB should be notably increased compared to nontransplanted mice as SD mice do not normally express HexB. Therefore, to assess the levels of HexB in the periphery of the transplanted HexB−/− mice, a HexB enzyme linked immunosorbent assay (ELISA) was performed. As displayed in Figure 8, a significant increase (p<0.001) in HexB enzyme was observed in the plasma of BRG-SD mice engrafted with the MAPB vector transduced CD34+ HSC as compared to nontransplanted mutant HexB−/− mice. A significant increase (p=0.01) in HexB enzyme was also observed in MAPB engrafted mice compared to WT HexB+/+ mice.

Figure 8.

Figure 8.

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HexB enzyme expression in the peripheral blood of HexA/HexB lentiviral vector transduced cell engrafted BRG-SD mice: Plasma from the peripheral blood of mice, wild type (WT), HexB−/− mutant (MUT), nontransduced (NT) CD34+ cell engrafted, and MAPB HexA/HexB lentiviral vector transduced cell engrafted mice, obtained from the open field analyses was evaluated using a HexB sandwich ELISA. Asterisk indicates p<0.001.

Multi-lineage hematopoiesis of HexA/HexB vector transduced human CD34+ HSC:

To evaluate the ability of HexA/HexB lentiviral vector transduced human CD34+ HSC to undergo normal development once engrafted in vivo, multi-lineage hematopoiesis was evaluated in an NRG immunodeficient mouse model. This model allows for the engraftment of human CD34+ HSC and the development of mature human cells including T cells, B cells, and macrophages in the peripheral blood and lymphoid organs including the spleen, thymus, and bone marrow. T cell analysis was performed on the blood, spleen, and thymus. B cell analysis was performed on the spleen and bone marrow. Macrophage and human CD34+ cell analyses were performed on the bone marrow. Cells were initially gated on EGFP and then analyzed for the human cell markers to specifically identify any adverse effects occurring in the HexA/HexB gene modified cells. As displayed in Figure 9, similar levels of all cell types analyzed in all lymphoid tissues was observed in MAPBEE vector transduced cell engrafted NRG mice as compared to EGFP alone vector transduced cell engrafted mice. No significant differences (p=0.36, p=0.85, p=0.73, and p=1.0) were observed in the spleen for CD3+, CD4+, CD8+, and CD4+/CD8+ T cells, respectively (Figure 9a). No significant differences (p= 0.27, p=0.47, p=0.69, and p=0.67) were observed in the thymus for CD3+, CD4+, CD8+, and CD4+/CD8+ T cells, respectively (Figure 9b). No significant differences (p=0.43, p=0.44, p=0.38, and p=0.34) were observed in the peripheral blood for CD3+, CD4+, CD8+, and CD4+/CD8+ T cells, respectively, (Figure 9c).

Figure 9.

Figure 9.

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Engraftment and multilineage hematopoiesis of HexA/HexB lentiviral vector transduced human CD34+ HSC in NRG mice: Human CD34+ HSC were transduced with either the EGFP control (EGFP) or HexA/HexB (MAPBEE) lentiviral vector. Cells were transplanted intrahepatically into 2–5 day old NRG mice. 16 weeks post-transplant, mice were euthanized and human EGFP+ T cells were analyzed for CD3, CD4, and CD8 expression in the a) spleen, b) thymus, and c) peripheral blood. d) Human EGFP+ B cells were analyzed in the spleen. e) Human EGFP+ B cells, macrophages, and CD34+ cells were analyzed in the bone marrow.

B cell development was also found to be similar in the spleen and bone marrow of NRG mice engrafted with the MAPBEE vector transduced human CD34+ HSC. No significant difference (p=0.56) was observed in the levels of CD45+/CD19+ cells in the spleens of MAPBEE vector transduced cell engrafted mice as compared to EGFP control vector transduced cell engrafted mice (Figure 9d). No significant difference (p=0.74) was observed in the levels of CD45+/CD19+ cells in the bone marrow of MAPBEE vector transduced cell engrafted mice as compared to EGFP control vector transduced cell engrafted mice (Figure 9e). Macrophage and CD34+ cell engraftment levels were similar in the bone marrow of MAPBEE and EGFP control vector transduced mice. No significant differences (p=0.69 and p=0.49) were observed in the levels of CD45+/CD14+ and CD45+/CD34+ cells, respectively, in the bone marrow of engrafted mice (Figure 9e).

These results demonstrated that HexA/HexB expressing vector transduced human CD34+ HSC were able to successfully engraft NRG mice and undergo normal multilineage hematopoiesis in the peripheral blood and various lymphoid organs.

DISCUSSION

Currently, there is no cure for TS or SD and current treatments only marginally alleviate symptoms. New therapies that have the potential of delivering wild type and functional enzymes to affected cells for the life of a patient need to be developed. HSC gene therapy for TSD and SD has the potential to achieve this goal by genetically modifying a patient’s own HSC to overexpress HexA and HexB. This has been achieved using a similar approach for MLD and ALD [20–23]. Therapeutic cells can migrate to the central nervous system and reside as microglia delivering functional enzyme to affected neurons [24]. Through a process called cross-correction, HexA and HexB proteins secreted by the vector transduced cells can be taken up by neurons via the mannose-6-phosphate receptor where they can degrade GM2-gangliosides. In our current study, we evaluated the use of this cross-correction strategy to express HexA and HexB via lentiviral vector transduction of human CD34+ HSC for the treatment of TSD and SD.

High tittered lentiviral vectors were generated which upon transduction of TSD-affected fibroblasts, restored enzyme activity due to the overexpression of HexA and HexB. This was the first step in demonstrating that wild type HexA and HexB transgenes introduced by a lentiviral vector were capable of expressing relevant enzyme levels. Safety and functionality were also observed in primary human cells of the hematopoietic lineage upon transduction with the HexA/HexB lentiviral vector. This was demonstrated in a CFU assay and the derivation of mature macrophages, in vitro, where normal proliferation and differentiation of the cells had occurred with a lack of toxicity due to the overexpression of HexA and HexB. Enzyme activity in HSPC derived macrophages was also demonstrated in cells transduced with the HexA/HexB lentiviral vector. and was significantly higher than NT macrophages obtained from a healthy donor. This result highlights the ability of HexA/HexB gene modified cells to provide higher efficacy, through higher expression levels of HexA and HexB, than nontransduced allogeneic cells [10–12].

In order to translate findings to a clinical setting, relevant animal models are required to effectively evaluate the safety and efficacy off potential new therapies. Therefore, we have generated an humanized version of the SD HexB−/− mouse model by crossing these mice with BRG immunodeficient mice. This cross allows for the transplantation, engraftment, and multi-lineage hematopoiesis of human CD34+ HSC. These mice retain the phenotypes of normal HexB−/− mice as neurological symptoms still occurred around 90 days of age followed by an average lifespan of 120 days [28]. Upon transplantation of BRG-SD mice with HexA/HexB vector transduced human CD34+ HSC, mice were then monitored via an open field assay for total distance moved, total time moving, and total vertical movements. During the second half of the open field assay, between weeks 17 and 20 post-transplant, a significant improvement in motor skills was observed in mice transplanted with the HexA/HexB expressing lentiviral vector as compared to nontransplanted mutant HexB−/− mice. A significant decrease in the accumulation of GM2-gangliosidie aggregates in multiple parts of the brains of HexA/HexB lentiviral vector transduced cell engrafted mice was also observed upon PAS staining of brain sections. This decrease in GM2-gangliosides can be correlated to the improved motor function as significantly higher levels of HexB were observed in the peripheral blood plasma of HexA/HexB lentiviral vector transduced cell engrafted mice.

In vivo safety as measured by successful engraftment and multilineage hematopoiesis of HexA/HexB lentiviral vector transduced human CD34+ HSC was observed in transplanted NRG mice. No difference in T cell, B cell, macrophage, or CD34+ cell engraftment or differentiation was observed in the peripheral blood, spleen, thymus, and bone marrow. The long-term engraftment of these cells highlighted that the overexpression of HexA and HexB did not affect their engraftment, proliferation, or differentiation.

Taken together, this HSC gene therapy approach for TSD and SD may provide a treatment alternative for patients affected with these incurable diseases. HSC gene therapy based on lentiviral vector insertion of a wild type copy of HexA and HexB offers a promising strategy to halt or even revert disease progression in treated patients. Furthermore, our results demonstrate that an HSC gene therapy approach may provide significantly better efficacy in a clinical setting than nontransduced allogeneic cells due to constitutively higher levels of systemic enzyme expression.

ACKNOWLEDGEMENTS:

We would like to thank the following for their support and funding for this research. The Cure Tay-Sachs Foundation and the California Institute for Regenerative Medicine grant #TRAN1-08519 and EDUC2-08397. The project described was supported by the National Center for Advancing Translational Sciences, National Institutes of Health, through grant number UL1 TR001860 and linked award KL2 TR001859. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

Footnotes

CONFLICTS OF INTEREST: The authors declare no conflicts of interest.

DATA AVAILABILITY STATEMENT:

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

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Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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