Effective cell and gene therapy in a murine model of Gaucher disease

Abstract

Gaucher disease (GD) is a lysosomal storage disorder due to an inherited deficiency in the enzyme glucosylceramidase (GCase) that causes hepatosplenomegaly, cytopenias, and bone disease as key clinical symptoms. Previous mouse models with GCase deficiency have been lethal in the perinatal period or viable without displaying the clinical features of GD. We have generated viable mice with characteristic clinical symptoms of type 1 GD by conditionally deleting GCase exons 9-11 upon postnatal induction. Both transplantation of WT bone marrow (BM) and gene therapy through retroviral transduction of BM from GD mice prevented development of disease and corrected an already established GD phenotype. The gene therapy approach generated considerably higher GCase activity than transplantation of WT BM. Strikingly, both therapeutic modalities normalized glucosylceramide levels and practically no infiltration of Gaucher cells could be observed in BM, spleen, and liver, demonstrating correction at 5-6 months after treatment. The findings demonstrate the feasibility of gene therapy for type 1 GD in vivo. Our type 1 GD mice will serve as an excellent tool in the continued efforts toward development of safe and efficient cell and gene therapy for type 1 GD.

Fig. 1.

Induction of Cre causes excision of exons 9–11 of the GCase gene with concomitant enzyme deficiency and substrate accumulation in the BM, spleen, and liver. (A) Schematic view of the Cre-mediated deletion of GCase exons 9–11. PCR was used to screen for deletion (primers: P1 and P2). black bars, GCase exons; gray bars, metaxin exons; open triangles, loxP sites. (B) (Upper) PCR confirmed successful deletion of exons 9–11 in the BM, blood, liver, and spleen of GD mice. (Lower) PCR verified the presence of Cre in the GD mice. Bands were visualized by ethidium bromide. (C) The enzyme activity was practically absent (spleen) or severely reduced (BM and liver) in GD mice compared with WT (tissues from three to six individual GD mice; age, 6.5–12 months). Heterozygotes (hets) had approximately half of the enzyme activity of WT (tissues from three to six individual heterozygotes; age, 6.5–12 months). WT GCase activity was set to 100%, and activity in the GD mice and heterozygotes is presented as the percentage of WT. Error bars represent standard deviation. (D) BM, spleen, and liver from 12-month-old GD mice show a massive accumulation of GluCer (n = 3). WT and heterozygotes were pooled (depicted control, n = 4; age, 8–12 months), because they were indistinguishable from each other with respect to GluCer accumulation. Results are shown as nanomoles of GluCer per milligram of protein.

Fig. 2.

Infiltration of Gaucher cells in the BM, spleen, and liver after conditional deletion of GCase exons 9–11. Columns labeled “GD mouse” show specimens from 12-month-old GD mice with massive infiltration of GluCer-engorged multinucleated cells in BM, spleen, and liver. Columns labeled “control” show tissues from heterozygous and WT mice lacking Gaucher cells. Results were obtained through hematoxylin and eosin and periodic acid Schiff staining. (Scale bars: first and second columns, 1,000 μm; third and fourth columns, 50 μm.)

Fig. 3.

Induced GCase deficiency causes splenomegaly and microcytic anemia. (A) Spleen size increased over time, and the spleen from a representative 16-month-old GD mouse was ≈10-fold larger compared with control (heterozygotes and WT). White areas in the spleens show splenic infarcts. (B) GD mice had a significant reduction in hematocrit (Hct) (n = 5) (Upper Left), hemoglobin (Hgb) (n = 5) (Upper Right), and mean red blood cell volume (MCV) (n = 7) (Lower Right) compared with control (heterozygoyes and WT; n = 7–8); there was no significant difference in the number of red blood cells between GD mice (n = 7) and control mice (n = 8) (Lower Left). The findings are presented as box plots, where the thick black line represents median values, the box represents 50% of values, and the whiskers represent the range of values. ∗, P = 0,003 (Mann–Whitney U test); O1 and O2, outliers 1 and 2.

Fig. 4.

Gene therapy can correct the disease phenotype in the GD mice. (A) GC vector (GC) and GFP vector (GFP) design (details are given in Supporting Materials and Methods) (B) Experimental design (Supporting Materials and Methods). BM cells transduced through VCM incubation or in coculture with vector producer cell lines were transplanted into lethally irradiated recipient GD mice (4.5–6 months after GD induction). Results are presented individually for each mouse. (C) VCM transduction. The relative GCase activity in BM, spleen, and liver was increased in the GD mice treated with GC vector (mice 1 and 2) compared with tissues from GFP-treated GD mice (mice 3 and 4). (D) Coculture transduction. A robust increase in the relative GCase activity was demonstrated in BM, spleen, and liver in the GD mice treated with GC vector (mice 5–7) compared with tissues from GFP-treated GD mice (mice 8–10). The y axis is in log-scale. (C and D) WT GCase activity was set to 100%, and activity in individual GD mice (GC vector- or GFP vector-treated) is presented as the percentage of WT. (E) GC-treated GD mice (VCM transduction mice 1 and 2) had normal levels of GluCer in BM, spleen, and liver compared with GFP-treated mice (mice 3 and 4). Control levels (WT and heterozygous tissue, n = 5; age, 8–12 months) are included for comparison. (F) GC vector-treated GD mice (coculture transduction, mice 5–7) had normal levels of GluCer in BM, spleen, and liver compared with GFP vector-treated mice (mice 8–10). Control levels (WT and heterozygous tissue, n = 5; age, 8–12 months) are included for comparison. (E and F) Results are shown as nanomoles of GluCer per milligram of protein. Error bars represent standard deviation. (G) Gaucher cells were practically eliminated in BM, spleen, and liver 5 months after transplantation in mice treated with GluCer, whereas GFP-treated mice maintained the massive infiltration of Gaucher cells. The first and second columns are representative of the VCM incubation transduction, and the third and fourth columns are representative of the coculture transduction (hematoxylin and eosin and periodic acid Schiff staining). (Scale bars: 50 μm.)