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

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 for delivering wild-type enzymes to affected cells. By genetically modifying hematopoietic stem cells to express wild-type HexA and HexB, systemic delivery of functional enzyme can be achieved.

Methods: Primary human hematopoietic stem/progenitor cells 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 multilineage 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: gene; hemopoietic; stem cell; therapy; viral vector.

Figure 1.

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).

Figure 2.

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.

Figure 3.

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.

Figure 4.

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.

Figure 5.

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.

Figure 6.

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.

Figure 7.

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.

Figure 8.

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.

Figure 9.

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.