Lentivector transduction improves outcomes over transplantation of human HSCs alone in NOD/SCID/Fabry mice

Abstract

Fabry disease is a lysosomal storage disorder caused by a deficiency of α-galactosidase A (α-gal A) activity that results in progressive globotriaosylceramide (Gb(3)) deposition. We created a fully congenic nonobese diabetic (NOD)/severe combined immunodeficiency (SCID)/Fabry murine line to facilitate the in vivo assessment of human cell-directed therapies for Fabry disease. This pure line was generated after 11 generations of backcrosses and was found, as expected, to have a reduced immune compartment and background α-gal A activity. Next, we transplanted normal human CD34(+) cells transduced with a control (lentiviral vector-enhanced green fluorescent protein (LV-eGFP)) or a therapeutic bicistronic LV (LV-α-gal A/internal ribosome entry site (IRES)/hCD25). While both experimental groups showed similar engraftment levels, only the therapeutic group displayed a significant increase in plasma α-gal A activity. Gb(3) quantification at 12 weeks revealed metabolic correction in the spleen, lung, and liver for both groups. Importantly, only in the therapeutically-transduced cohort was a significant Gb(3) reduction found in the heart and kidney, key target organs for the amelioration of Fabry disease in humans.

Figure 1

Phenotypic characterization of the novel NOD/SCID/Fabry xenograft model. Eleven generations of backcrossings were performed to generate a fully congenic NOD/SCID/Fabry line. At each generation different phenotypic studies were performed. (a) Representative flow cytometry analysis of the percentage of T and B cells in peripheral blood. Different F₁₁-individuals were compared with the parental mouse lines. (b) Plasma α-gal A enzyme activity was analyzed for the new mouse line and also compared to an earlier Fabry mouse model. (c) Specific enzyme activity in different organs. (d) Gb₃ quantification by HPLC. Data represent mean ± SD from five individuals (F₁₁) per group. *P < 0.001 compared to wild-type control groups. α-gal A, α-galactosidase A; BL6, wild-type; Fabry, traditional Fabry mice; FITC, fluorescein isothiocyanate; Gb₃, globotriaosylceramide; HPLC, high-performance liquid chromatography; NOD, nonobese diabetic; NSF, NOD/SCID/Fabry; NS, NOD/SCID; SCID, severe combined immunodeficiency.

Figure 2

Lentivector schema. Schema of the lentivectors used in the present study. Control vector, LV-eGFP, and therapeutic vector, LV-α-gal A/IRES/hCD25. α-gal A, α-galactosidase A; cPPT, central polypurine tract; EF1-α, elongation factor 1-α promoter; eGFP, enhanced green fluorescent protein; IRES, internal ribosome entry site; LTR, long terminal repeat; RRE, Rev responsive element; SA, 3′ splice acceptor site; SD, 5′ splice donor site; SIN, self-inactivating LTR; WPRE, woodchuck hepatitis virus post-transcriptional regulatory element; ψ, human immunodeficiency virus packaging signal.

Figure 3

Phenotypic characterization of LV-transduced human CD34⁺ progenitor cells. (a) Representative flow cytometry analysis of the normal human CD34⁺ cells used in the in vivo experiment. (b) Percentage of eGFP or hCD25⁺ cells at 4 days after transduction of the progenitor cells. A representative flow cytometry analysis is shown. (c) Representative clonogenic assay evaluated at 14 days after transduction. (d) Progenitor enzymatic activity evaluated 14 days after transduction. Data are mean ± SD (n = 3). *P < 0.01 compared to NT or LV-eGFP control cells. α-gal A, α-galactosidase A; BFU-E, burst-forming unit-erythroid; CFU-GM, colony-forming unit-granulocyte and macrophage; CFU-GEMM, colony-forming unit-granulocyte, erythrocyte, macrophage, and megakaryocyte; eGFP, enhanced green fluorescent protein; FSC-H, forward scatter; IRES, internal ribosome entry site; LV, lentiviral vector; NT, nontransduced cells.

Figure 4

High level of engraftment of human HSCs in NOD/SCID/Fabry mice. Transduced progenitor cells (8 × 10⁵) were injected intravenously into sublethally irradiated NOD/SCID/Fabry mice. (a) Representative flow cytometry analysis of the percentage of hCD45⁺ cells in peripheral blood at different time points. (b) Percentage of hCD45⁺ cells in peripheral blood analyzed by flow cytometry at different time points. (c) Representative flow cytometry analysis for LV-eGFP positive cells in circulation. (d) Representative dot-plot analysis of human cell engraftment from bone marrow samples 12 weeks after transplantation. (e) Bone marrow engraftment of human cells in all mice evaluated 12 weeks after transplant. (f) Representative hCD45⁺/eGFP⁺ cell engraftment analysis from pooled bone marrow samples. (g) Percentage of hCD34⁺/eGFP⁺ cells in bone marrow pools. Each open circle or black square represents an individual mouse. Two independent experiments were performed. α-gal A, α-galactosidase A; APC, allophycocyanin; eGFP, enhanced green fluorescent protein; FSC-H, forward scatter; HSC, hematopoietic stem cells; IRES, internal ribosome entry site; LV, lentiviral vector; NOD, nonobese diabetic; NSF, NOD/SCID/Fabry; NS, NOD/SCID; SCID, severe combined immunodeficiency; SSC-H, side scatter.

Figure 5

α-Gal A activity augmentation and organ Gb₃ reduction in the LV-α-gal A/IRES/hCD25 transplanted mice. (a) Plasma α-gal A enzyme activity was evaluated at 4, 6, 8, 10, and 12 weeks after transplantation. Data were normalized to nontransplanted NOD/SCID/Fabry mice enzyme levels and represent mean ± SEM (n = 16). *P < 0.05, **P < 0.01, and ***P < 0.001 compared to the LV-eGFP control group. (b) Bone marrow, spleen, and liver α-gal A enzyme activity at killing. Data represent mean ± SEM (n = 16). *P < 0.01 compared to nontransplanted group and ^#P < 0.05 versus eGFP group. (c) HPLC-based Gb₃ quantification in heart, kidney, liver, lung, and spleen at 12 weeks after transplantation. Data represent mean ± SEM (n = 16). *P < 0.01 compared to nontransplanted group and ^&P < 0.01 or ^#P < 0.05 versus the LV-eGFP group. (d) Representative flow cytometry analyses of engineered cell tissue engraftment. α-gal A, α-galactosidase A; eGFP, enhanced green fluorescent protein; Gb₃, globotriaosylceramide; IRES, internal ribosome entry site; LV, lentiviral vector; NOD, nonobese diabetic; NSF, NOD/SCID/Fabry; NS, NOD/SCID; SCID, severe combined immunodeficiency.