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. 2015 Mar 10;23(5):835–844. doi: 10.1038/mt.2015.16

Lentiviral Gene Therapy Using Cellular Promoters Cures Type 1 Gaucher Disease in Mice

Maria Dahl 1,2,2, Alexander Doyle 1,2,2, Karin Olsson 1,2, Jan-Eric Månsson 3, André R A Marques 4, Mina Mirzaian 4, Johannes M Aerts 4, Mats Ehinger 5, Michael Rothe 6, Ute Modlich 6, Axel Schambach 6, Stefan Karlsson 1,2,*
PMCID: PMC4427872  PMID: 25655314

Abstract

Gaucher disease is caused by an inherited deficiency of the enzyme glucosylceramidase. Due to the lack of a fully functional enzyme, there is progressive build-up of the lipid component glucosylceramide. Insufficient glucosylceramidase activity results in hepatosplenomegaly, cytopenias, and bone disease in patients. Gene therapy represents a future therapeutic option for patients unresponsive to enzyme replacement therapy and lacking a suitable bone marrow donor. By proof-of-principle experiments, we have previously demonstrated a reversal of symptoms in a murine disease model of type 1 Gaucher disease, using gammaretroviral vectors harboring strong viral promoters to drive glucosidase β-acid (GBA) gene expression. To investigate whether safer vectors can correct the enzyme deficiency, we utilized self-inactivating lentiviral vectors (SIN LVs) with the GBA gene under the control of human phosphoglycerate kinase (PGK) and CD68 promoter, respectively. Here, we report prevention of, as well as reversal of, manifest disease symptoms after lentiviral gene transfer. Glucosylceramidase activity above levels required for clearance of glucosylceramide from tissues resulted in reversal of splenomegaly, reduced Gaucher cell infiltration and a restoration of hematological parameters. These findings support the use of SIN-LVs with cellular promoters in future clinical gene therapy protocols for type 1 Gaucher disease.

Introduction

Insufficient activity of the enzyme glucosylceramidase (GCase) is the underlying cause of Gaucher disease (GD), the most prevalent of the lysosomal storage disorders (LSDs).1,2,3 This results in a severe reduction in glucosylceramide (GluCer) degradation and its subsequent accumulation, primarily in cells of mononuclear phagocyte origin.2 These cells turn into disease characteristic “Gaucher cells”, infiltrating tissues throughout the body and giving rise to a diverse set of symptoms. Clinical manifestations of GD normally begin with splenomegaly and hepatomegaly, anemia, and thrombocytopenia.2 Patients display a multitude of symptoms, ranging from those being entirely asymptomatic to those displaying severe childhood-onset disease.2 Classically, GD has been clinically divided into three subtypes, where patients of type 1 exhibit visceral symptoms, while types 2 and 3 also have an effect on the central nervous system. Type 1 is the most common form and is primarily a macrophage disorder without central nervous system involvement.2 Although expensive and noncurative, intravenous enzyme replacement therapy is the treatment of choice and alleviates peripheral symptoms in most patients.4,5,6,7,8 Allogeneic bone marrow transplantation (BMT) is the only curative treatment option, not without challenges such as donor availability and risks related to the transplantation procedure.9,10 For life long correction of type 1 Gaucher disease (GD1), infusion of ex vivo genetically corrected autologous hematopoietic stem and progenitor cells (HSPCs) into patients is an attractive clinical option, with recent success using this method in treating patients with X-linked severe combined immunodeficiency (SCID-X1), adenosine deaminase deficiency (ADA-SCID), and Wiskott-Aldrich syndrome.11,12,13 In a proof-of-principle study using a gammaretroviral vector with the viral promoter spleen focus–forming virus (SFFV), established disease symptoms were corrected in a conditional mouse model displaying the pathology and symptoms of type 1 GD.14 Since the strong viral long terminal repeats may cause insertional mutagenesis following vector integration15 and the SFFV promoter gives supraphysiological expression levels,16 this is not the optimal platform for development of clinical gene therapy. As self-inactivating (SIN) lentiviral vectors have an increased safety profile in comparison to gammaretroviral vectors, they are currently the vectors of choice in clinical trials.17,18 Previously, BMT experiments demonstrated that engraftment of less than 10% normal bone marrow (BM) cells, corresponding to a GCase activity of 10 nmol/mg protein/hour, was sufficient to reverse the pathology in BM and spleen of recipient GD1 mice.19 Here, we use genetic studies to demonstrate that as few as 6% functional macrophages are sufficient to prevent disease progression. These observations collectively suggest that lentiviral vectors containing physiological promoters may drive sufficient GBA expression for disease correction. In this study, we have evaluated the efficacy and safety of SIN lentiviral vectors harboring the human phosphoglycerate kinase (PGK) and the CD68 promoter, in an early and late intervention of GD1. The PGK promoter is ubiquitously expressed, giving physiological expression levels and described in several gene therapy studies.15,16,20 The CD68 promoter has been described as directing transgene expression to macrophage populations,21,22 with stable gene expression having been achieved in vivo from transplanted HSCs genetically corrected by a vector harboring the CD68 promoter.23 The CD68 gene is a member of the lysosomal/endosomal-associated membrane glycoprotein family and is highly expressed by human monocytes and tissue macrophages.22 Recently, a CD68-GFP transgenic reporter mouse was developed, exhibiting GFP expression in both monocytes and tissue-resident macrophage populations.24

The findings reported here demonstrate that SIN lentiviral vectors harboring cellular promoters robustly reduce manifest GD1 symptoms in animals. This provides support for the development of a clinical gene therapy protocol for GD1 using these vectors.

Results

A low fraction of GCase producing macrophages prevents disease development in mice with induced deletion in the GBA genes

In an effort to generate an increased yield of GD1 mice for experimental purposes, disease progression was assessed in Mx1 Cre+ GBAflox/flox mice (hereafter referred to as flox/flox). These mice have two “floxed” alleles of the GBA gene, with exons 9–11 deleted after birth through induction of Cre recombination expression25 under the control of the Mx1 promoter, ensuring efficient recombination in hematopoietic cells.26,27 Six months postinduction, disease progression in these flox/flox mice was compared to Mx1 Cre+ GBAnull/flox mice (referred to as null/flox), originally described by Enquist et al.14 (Figure 1a). As expected, GCase activity was severely reduced in the bone marrow (BM), spleen, and liver of null/flox mice in comparison to WT (Figure 1b). A reduction in enzyme activity was also observed in the flox/flox mice, although these levels were significantly elevated in both BM and liver compared to null/flox mice (Figure 1b). Accumulation of GluCer was observed in the BM, liver, and spleen of null/flox mice, although strikingly no substrate accumulation was observed in the organs of flox/flox mice (Figure 1c), the residual GCase activity in induced flox/flox mice being sufficient to prevent accumulation of GluCer. To address whether the lack of disease progression in flox/flox mice was due to insufficient recombination efficiency of the GBA gene, PCR was performed on single hematopoietic colonies from BM seeded into methylcellulose media to assess recombination at the clonal level. A variation in excision efficiency of 25–94% was observed in flox/flox BM, while null/flox mice were consistent at 100% (Figure 1d; Supplementary Table S1). It was observed that complete recombination was necessary for disease progression since even in a flox/flox mouse with an excision efficiency of 94%, GCase activity was elevated 1.8-fold in BM and 2.9-fold in spleen compared to null/flox (Supplementary Table S1). Additionally, the null/flox mouse with 100% recombination had elevated GluCer levels, 44-fold in spleen and 4.5-fold in BM, compared to the flox/flox mouse (Supplementary Table S1). Consistent with previous findings from BMT experiments, where the GD1 phenotype was reversed after transplantation with a mixture of 10% WT and 90% GD1 BM cells, these results indicate that low numbers of functional macrophages can correct the disease phenotype in GD1 mice.19 To ensure the establishment of GD pathology in subsequent gene therapy experiments, we used only null/flox mice (henceforth referred to as GD1 mice).

Figure 1.

Figure 1

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A low percentage of normal macrophages rescues the Gaucher disease phenotype. (a) This panel demonstrates the experimental design. The recombination efficiency of the glucosidase β-acid (GBA) gene was determined by DNA polymerase chain reaction (PCR) of clonally derived individual hematopoietic colonies from GBAflox/flox and GBAnull/flox mice at 6 months of age, bulk hematopoietic cells were analyzed for levels of GCase and GluCer. (b) The relative GCase activity in bone marrow (BM), liver, and spleen was determined in WT, GBAflox/flox, and GBAnull/flox mice (n = 3 per group) and (c) substrate accumulation in the tissues (WT, n = 6; GBAflox/flox n = 6; GBAnull/flox, n = 4). (d) BM from GBAflox/flox and GBAnull/flox mice 6 months of age was harvested and colony forming unit-granulocyte macrophage (CFU-GM) assay performed. PCR targeting the excised region of GBA gene in individual colonies (minimum of 25 per group) was performed to determine recombination efficiency (n = 3 per group). *P < 0.05, **P < 0.01, Student t-test. Error bars represent mean + SEM.

Lentiviral gene therapy confers stable gene expression in transplanted animals and successfully prevents Gaucher disease progression in an early intervention procedure

Several lentiviral vectors with viral and cellular promoters were designed, driving expression of codon optimized GBA complementary DNA. Codon optimization generated an approximate twofold increase in GCase expression compared to noncodon optimized samples (Supplementary Figure S1). As a positive control, we used the SFFV promoter generating robust expression in all hematopoietic lineages. A vector containing the phosphoglycerate kinase (PGK) promoter was designed to represent a general housekeeping promoter, expected to express the GBA transgene in all hematopoietic lineages at lower levels than SFFV. Finally, we utilized the CD68 promoter to drive increased transgene expression in the macrophage population. As a negative control, we used a vector containing a noncoding DNA sequence driven by the PGK promoter (Figure 2). To assess the genetically corrected HSPCs in an early and late intervention of GD1, we designed two experimental approaches.

Figure 2.

Figure 2

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Schematic representation of the lentiviral vector design. The figure shows the vectors where the therapeutic vectors contain the codon optimized human glucosidase β-acid (GBA) cDNA driven by the CD68, PGK, and SFFV promoters, respectively. The negative control vector lacks the GBA gene and contains a spacer sequence downstream of the PGK promoter (PGK.Control). cPPT, central polypurine tract; RSV, Rous sarcoma virus enhancer/promoter; SD and SA, splice donor and splice acceptor sites, respectively; Ψ, packaging signal.

As previously reported by Enquist et al.,14 GD1 mice do not display manifest symptoms until 5 months postinduction. We addressed whether BMT of young mice (i.e., 10–12 weeks of age) with genetically corrected cells could prevent disease development. BM was harvested from GD1 donor mice where the disease had been induced as previously described14 prior to harvest. BM was lineage depleted to enrich for stem and progenitor cells which were transduced with the therapeutic SIN lentiviral vectors and transplanted into age matched lethally irradiated GD1 recipients (Figures 2 and 3a). An average engraftment of 28–53% of genetically corrected cells was observed 5 weeks post-transplant in peripheral blood (PB). Five months (20 weeks) posttransplant, the percentage of GFP+ cells in BM was measured at 6–48% suggesting reasonable gene transfer efficiency of long-term HSCs that generate hematopoiesis in the recipients. At 5 months posttransplant, the GCase activity was robustly increased in BM, spleen, and liver of recipient mice (Figure 3e–g) and GluCer levels significantly reduced compared to controls (Figure 3h–j). The enzyme activities in the livers of treated animals was less elevated than in BM and spleen. This may be expected as only 15% of the total number of liver cells are macrophages28 (Kupffer cells) (Figure 3g). Glucosylsphingosine (Lyso-GlyCer), a minor substrate for GCase, displayed a robust decrease in BM, spleen, and liver for all therapeutic vectors, while no differences in levels of a negative control lipid C18-Ceramide could be detected between samples (Supplementary Figure S2a–f). Hallmark features of GD1 include hepatosplenomegaly, microcytic anemia, and the presence of infiltrating disease characteristic Gaucher cells in the organs.2 A reversal of splenomegaly was observed posttransplant, with a significant decrease in the spleen to body mass ratio using all therapeutic vectors, whereas the PGK.Control vector had no therapeutic effect as expected (Figure 3d). Histopathological examination revealed that no Gaucher cells were detected in BM, spleen, and liver, whereas Gaucher cells had begun to infiltrate the tissues of untreated (PGK.Control) mice (Figure 4).

Figure 3.

Figure 3

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Efficient early intervention of Gaucher disease by lentiviral gene transfer. (a) Experimental design of early intervention study. (b) Transduction efficiencies at 5 weeks in peripheral blood (PGK.Control, n = 6; CD68.GBA, n = 6; PGK.GBA, n = 6; SFFV.GBA, n = 5). (c) Percentages of GFP-expressing cells in whole BM at 20 weeks post-transplant (PGK.Control, n = 8; CD68.GBA, n = 8; PGK.GBA, n = 8; SFFV.GBA, n = 7). (d) Spleen mass: body mass ratio was calculated for the GD1 transplanted mice relative to WT (WT, n = 6; PGK.Control, n = 8; CD68.GBA, n = 8; PGK.GBA, n = 8; SFFV.GBA, n = 6). (e–g) The GCase activity was determined in BM (e), spleen (f), and liver (g) for the different groups (WT, n = 6; PGK.Control, n = 8; CD68.GBA, n = 8; PGK.GBA, n = 8; SFFV.GBA, n = 7). (h–j) GluCer accumulation was measured in BM (h), spleen (i), and liver (j) (WT, n = 6; PGK.Control, n = 8; CD68.GBA, n = 8; PGK.GBA, n = 8; SFFV.GBA, n = 7). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 Mann–Whitney U-test. Error bars represent mean + SEM.

Figure 4.

Figure 4

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Diminished Gaucher cell infiltration in treated animals. Representative histologic analysis of the BM, spleens, and livers from wild-type (WT), GD1 control vector, and therapeutic vector-treated animals 20 weeks posttransplant in the early intervention study. The untreated PGK.Control samples show samples from 6-month-old GD1 mice with infiltration of GluCer- laden cells in bone marrow (BM), spleen, and liver. Periodic acid Schiff staining. Scale bars: upper panel (BM samples); 20 μm, lower panels (spleen and liver samples); 100 μm.

In summary, a specific GCase activity was achieved in the therapeutic vector treated animals resulting in robust reduction of GluCer levels and subsequent prevention of Gaucher cell infiltration and splenomegaly development.

Manifest Gaucher symptoms can be successfully corrected by cellular and lysosomal promoters via lentiviral gene transfer

We next asked whether manifest GD1 symptoms could be corrected by a late intervention approach by transplanting mice that had been induced 5–8 months prior to treatment, exhibiting an established disease phenotype (Figures 2 and 5a). At 5 weeks posttransplant, engraftment of gene corrected cells was detected at 30–54% in PB, being maintained at 13–37% in BM at 20 weeks post transplant (Figure 5b–c). The average gene copy number in BM was estimated for five mice per vector and ranged between 2.2–4.1, with a general tendency of correlation between vector copy number and percentage GFP+ cells observed (Supplementary Figure S3). As expected, the SFFV.GBA vector-treated mice exhibited the highest GCase activity, at 9.5-fold of WT levels in BM and threefold of WT in the spleen (Figure 5e,f), while PGK.Control vector-treated animals displayed very low enzyme activity in all organs (Figure 5e–g). PGK.GBA and CD68.GBA vectors significantly increased the enzyme activity in BM to 65 and 42% of WT respectively (Figure 5e), with a significant increase in GCase activity also found in the spleens of PGK.GBA and CD68.GBA vector treated mice (Figure 5f). These increases in GCase activities reduced substrate levels in the BM, spleen, and liver using all therapeutic vectors, whereas mice treated with the PGK.Control vector continued to accumulate GluCer in these tissues (Figure 5h–j). Although there was variation mouse to mouse, a correlation between engraftment level of transduced cells and both increase in GCase activity and decrease of GluCer levels was evident for all therapeutic vectors (Supplementary Figure S4a,b). The CD68.GBA- and PGK.GBA-treated animals exhibited a reduction in spleen to body mass ratio, resulting in a spleen size close to WT (Figure 5d). An increase in several blood parameters, including mean red cell volume (MCV) and hemoglobin (Hgb), was observed in all therapeutic vector treatments compared to PGK.Control (Supplementary Figure S5). Mice treated with the CD68.GBA and PGK.GBA vectors exhibited dramatically reduced numbers of Gaucher cells, with only the odd Gaucher cell remaining upon histopathological examination (Supplementary Figure S6). Thus, modest increases in the levels of enzyme activity either by a cellular or lysosomal promoter effectively reverse pathophysiological symptoms in GD1 mice.

Figure 5.

Figure 5

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Robust correction of manifest GD1 symptoms by lentiviral gene transfer. (a) Experimental design of late intervention study. (b) Transduction efficiencies at 5 weeks in peripheral blood (PGK.Control, n = 11; CD68.GBA, n = 10; PGK.GBA, n = 6; SFFV.GBA, n = 9). (c) Percentages of GFP-expressing cells in whole BM at 20 weeks post-transplant (PGK.Control, n = 12; CD68.GBA, n = 10; PGK.GBA, n = 9; SFFV.GBA, n = 11). (d) Spleen mass: body mass ratio was calculated for the GD1 transplanted mice relative to WT (WT, n = 7; PGK.Control, n = 12; CD68.GBA, n = 10; PGK.GBA, n = 9; SFFV.GBA, n = 11). (e–g) The GCase activity was determined in BM (e), spleen (f), and liver (g) for the different groups (WT, n = 7; PGK.Control, n = 12; CD68.GBA, n = 10; PGK.GBA, n = 9; SFFV.GBA, n = 11). (h–j) GluCer accumulation was measured in BM (h), spleen (i), and liver (j) (WT, n = 7; PGK.Control, n = 12; CD68.GBA, n = 10; PGK.GBA, n = 9; SFFV.GBA, n = 11). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 Mann–Whitney U-test. Error bars represent mean + SEM.

The CD68 promoter generates increased transgene expression in monocytic cells and macrophages

To investigate the capacity of the CD68.GBA vector in targeting GBA expression to impaired macrophages, the relative expression levels of GFP within the GFP-positive lymphoid, myeloid, and progenitor compartments of the different test groups was determined in the BM of long-term reconstituted mice. For each therapeutic vector, the median fluorescence intensity of GFP in different cell compartments of the BM was normalized to that of the ubiquitously expressed PGK.Control vector. Median fluorescence intensity of GFP was determined in lymphoid cells (CD3+CD19+) as well as in granulocytes (Gr1+), monocytes (Mac1+), macrophages (Mac1+ F4/80+), and in the progenitor population (lineage negative, C-kit+ cells) (Figure 6; Supplementary Figure S7). As expected, the SFFV.GBA vector displayed high median fluorescence intensity levels in all cellular subsets (Figure 6) and exhibited 20-fold higher expression in the progenitor population compared to the cellular promoters (Supplementary Figure S7). Similar levels of relative GFP expression were detected in the lymphoid compartment when comparing the CD68.GBA and PGK.GBA vectors. A trend of elevated relative GFP expression was observed in mice treated with CD68.GBA in granulocytes. There was a 1.6-fold increase in relative GFP expression in monocytes and 1.7-fold in macrophages of CD68.GBA-treated mice compared to the PGK.GBA vector (Figure 6). Thus, elevated levels of GBA expression can be achieved in the macrophage cell compartment by utilizing the CD68.GBA vector, directing increased GCase expression to the target cell of Gaucher disease.

Figure 6.

Figure 6

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The CD68 promoter generates relatively high GFP transgene expression in myeloid cells and macrophages. The median fluorescence activity of GFP normalized to PGK.Control in BM macrophages (Mac1+ F4/80+), monocytes (Mac1+), granulocytes (Gr1+), and T-and B-cells (CD3+ CD19+) (PGK.Control, n = 12; CD68.GBA, n = 10; PGK.GBA, n = 9; SFFV.GBA, n = 11). Error bars represent mean + SEM.

Vector integration analysis

The average copy number of vectors per transduced cell ranged from approximately 2 to 4 where the average copy number of the therapeutic vectors with the cellular promoters ranged from 2.2 to 2.7 (Figure 7b). We analyzed the integration sites of five mice per group by conventional and restriction enzyme free (refree) linear amplification-mediated PCR (LAM-PCR) and subsequent 454 pyrosequencing (Figure 7a). After clustering a total of 107172 sequence reads for homology, we could align 598 individual sequences successfully to the murine genome. It is known that lentiviral vectors mostly integrate in coding regions without preference to insert close to the transcriptional start sites (TSS) of genes. These general tendencies were recapitulated by the vectors used in this study, as the majority of all hits (62.4%) were found inside of genes. We observed a significantly lower number of integrations in a ±10 kb window around the TSS compared to control studies using gammaretroviral vectors (Table 1). In case insertion sites of more abundant clones within a population would be sequenced more often, we analyzed the sequence read count distribution from the DNA samples of mice treated by the different vectors. In all analyzed samples the hematopoietic contribution was poly to oligo-clonal (Supplementary Table S2). We found no significant difference regarding the presence of high read clones (> 100 reads) or the polyclonal background (< 10 reads) between the different groups (Figure 7c,d). We further screened all neighboring genes of insertions with a read count above the 95% CI of the whole dataset for a possible role in cancer or hematologic malignancies (Supplementary Figure S8). In some cases, insertions with high read counts can indicate an increase in clonal abundance.29,30 It is, however, not a direct measurement of clonal contribution, due to known biases in the PCR-based technology.31 We further evaluated a possible overrepresentation of insertion sites in or near genes listed in cancer gene databases after in vivo selection (Supplementary Table S3). However, there was no statistical difference between the groups and no enrichment of insertion sites close to cancer-associated genes compared to a pretransplant sample of lentivirally transduced human CD34 cells (unpublished data, Manuel Grez and Julia Sürth). We identified common insertion sites (CIS) in the dataset with integration hotspots shared between the vector groups (Supplementary Table S4). None of the CIS identified in the mice of this study overlapped with those observed in previous clinical gene therapy trials.

Figure 7.

Figure 7

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Gene-corrected Gaucher cells exhibit a typical lentiviral insertion profile. (a) Integration analysis was performed on bone marrow (BM) from 10- to 13-month-old mice in the treatment study at 20 weeks posttransplantation. Five mice per vector group were analyzed. (b) Vector copy number was determined in BM. (c,d) The differences in the polyclonal background (<10 reads) and the amount of prominent insertions (>100 reads). Kruskal–Wallis analysis with Dunn's correction for multiple comparisons.

Table 1. Number of insertions within 10 kb distance to the TSS.

graphic file with name mt201516t1.jpg

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Discussion

In this study, we show for the first time that ex vivo gene therapy of HSPCs by lentiviral vectors can correct a GD phenotype in a mouse model displaying clinical GD1 symptoms in both an early and a late intervention procedure. Our findings show that a GBA exon removal in excess of 94% of hematopoietic cells, observed in null/flox mice is necessary for the reduction in GCase activity below 10 nmol/mg protein/hour, required for the development of GD1 pathophysiology. In this study, we detect a robust decrease of GluCer content for all therapeutic vectors, even at low levels of engraftment. These findings, along with those described by Enquist et al.,19 indicate that modest increases in GCase activity or the presence of a low number of cells producing GCase (less than 6% of the hematopoietic cells) is sufficient for reversal of the GD1 phenotype. Collectively, these observations indicate that a relatively low level of gene transfer efficiency is sufficient in the development of gene therapy for Gaucher disease and that lentiviral vectors with internal cellular promoters can be applied successfully to treat patients with type 1 Gaucher disease.

Although HSPC gene therapy using gammaretroviral vectors has demonstrated clinical benefits in the treatment of primary immune deficiencies including X-linked severe combined immunodeficiency (SCID-X1), X-CGD and Wiskott-Aldrich syndrome, their use has been associated with the development of leukemia or myelodysplasia.32,33,34 This has been ascribed to the transactivation of proto-oncogenes (including LMO2 and MDS-EVI1) by the promoter sequences in the long terminal repeats of these vectors.35 Lentiviral vectors are replacing gammaretroviral vectors due to their increased safety profile,36 with recent clinical success in the treatment of X-linked adrenoleukodystrophy, β-thalassemia, Wiskott-Aldrich syndrome, and metachromatic leukodystrophy (MLD).11,12,13,37 The development of vectors incorporating codon optimized transgenes driven by endogenous and physiological promoters is removing the potentially genotoxic effects of very high expression levels, seen with viral promoters. Additionally, lentiviral vectors containing lineage-restricted promoters are being developed to drive correcting gene expression in disease affected cell populations, an example of this being the use of vectors containing the myeloid-specific gp91phox for the treatment of X-CGD.38,39 For the correction of developed GD1 in our model, we compared two SIN lentiviral vectors, both containing the codon optimized human GBA gene and a GFP reporter gene. The first vector contained the PGK promoter driving physiological levels of transgene expression; and the second vector containing the promoter for CD68, a lysosomal protein enriched in macrophages,40 previously described as effectively targeting transgene expression to macrophages.21,23,41

For effective treatment of GD, the long-term maintenance of gene corrected cells is essential. All vector-treated groups exhibited a consistent level of GFP expressing cells from initial transduction efficiencies to end point analysis at 20 weeks. The ability of the CD68 promoter to drive a relatively high transgene expression in the macrophage compartment was confirmed via flow cytometry, where relative GFP expression levels in BM was 1.6-fold in monocyte (Mac1+) and 1.7-fold in macrophage (Mac1+ F4/80+) populations compared to the PGK.GBA. Relative GFP expression levels within the lymphoid (CD3+ CD19+) compartment were similar in both CD68.GBA and PGK.GBA vector-treated mice, correlating with reports of CD68 expression in human PB lymphocytes.41 Gene therapy of HSPCs using either PGK.GBA or CD68.GBA vectors resulted in the reversal of the GD phenotype. GCase activity was increased in BM and spleen compared to PGK.Control-treated mice, with only a marginal increase observed in the liver, possibly due to the relatively low percentage of Kupffer cells found in this organ. An effective clearance of GluCer from the organs was observed as well as a drastic reduction in the numbers of infiltrating Gaucher cells. No infiltrating Gaucher cells were observed in the tissues of SFFV.GBA-treated mice, possibly due to the supraphysiological expression levels of GBA leading to increased kinetics of GluCer degradation in corrected macrophages. There was also a significant reversal in the splenomegaly observed in the treated mice compared to the PGK.Control mice. We conclude by LAM-PCR analyses that the vectors show no preference for insertions near the TSS, nor is there any overrepresentation of hits in cancer gene databases for any of the vectors. The SFFV promoter exhibits high transgene expression in progenitor cells and is thus unsuitable for use in a clinical vector.

A clinical gene therapy trial performed in three type 1 GD patients, infusing gammaretroviral vector gene corrected autologous CD34+ cells, resulted in only transient gene expression due to lack of myeloablation.42 For a successful treatment protocol for type 1 GD, it is likely that a reduced conditioning regime be applied to patients, as performed in Wiskott-Aldrich syndrome and ADA-SCID clinical trials,11,13 to allow for the engraftment of gene corrected HSCs.

A low percentage of GD patients treated with enzyme replacement therapy display prominent GD symptoms such as bone and pulmonary disease. We believe that gene therapy may help these patients and in addition reduce health care costs drastically by providing a curative solution. This study provides evidence of the effectiveness of ex vivo gene therapy for the treatment of type 1 GD, using cellular promoters in lentiviral vectors. This work takes steps toward the development of safe and efficient clinical grade vectors, for use in a clinical protocol for gene therapy in type 1 GD patients.

Materials and Methods

GD1 mice. The generation of the GD1 mouse model has been described previously.14 Screening of mice after polyinosinic-polycytidylic acid (pIpC; Sigma-Aldrich, St. Louis, MO)-induced exon deletion was done by polymerase chain reaction (PCR) using platinum taq polymerase (Invitrogen, Carlsbad, CA) and the following primers (5′-TAGAGTCCCTCCAGCTTCCCAG-3′; 5′-GTACGTTCATGGCATTGCTGTTCACT-3′; 5′-ATTCCAGCTGTCCCTCGTCTCC-3′). To verify the presence of the Cre gene, the following primers were used (5′-AATGCTTCTGTCCGTTTGCCGGTC-3′; 5′-GATCCGTCGCATGACCAGTGAAAC-3′). Mice were maintained in individually ventilated cages with ad libitum food and water in the animal facility at Lund University Biomedical Center. Breeding and experimental procedures were approved by the Committee for Animal Ethics in Malmö/Lund, Sweden.

Lentiviral vectors. SIN lentiviral vectors used in this study were generated from the pRRL-cPPT-hPGK-EGFP-WPRE vector, originally described by Dull et al.43 and modified by Schambach et al.44 Codon-optimized human GBA cDNA (sequence provided in Supplementary Table S5) was designed and inserted downstream of SFFV (SFFV.GBA), hPGK (PGK.GBA), and CD68 (CD68.GBA) promoters. The sequence for the CD68 promoter is provided in Supplementary Figure S9). Following the GBA cDNA, internal ribosomal entry site (IRES), eGFP, and improved post-transcriptional regulatory element (PRE*) were inserted to form the vectors. A similar vector, in which the GBA cDNA was replaced with an equally long noncoding spacer sequence, was used as a control (PGK.Control). VSV-G-pseudotyped vectors were produced by transient transfection of 293T cells at the Vector Unit at Lund University.

Tissue and cell preparations. Peripheral blood (PB) was collected from tail vein using EDTA-coated Microvette tubes (Sarstedt AG & Co, Nümbrecht, Germany). At end point, mice were weighed before sacrifice by CO2 and cervical dislocation and BM, liver, and spleen collected. For evaluation of substrate accumulation, tissue pieces were submerged in liquid nitrogen directly after removal from the mice. Tissue and harvested BM cells for enzyme activity analysis or flow cytometry analysis were kept in phosphate-buffered saline (2% FCS). For histopathology, tissue samples were fixed in 4% paraformaldehyde, paraffin-embedded, and sectioned.

Enzyme assay. Cells were lysed by three freeze-thaw cycles. Aliquots of the lysate were incubated for 40 minutes at 37 °C with or without 400 µmol/l Conduritol B Epoxide (Sigma-Aldrich). GCase activities were determined fluorometrically after 2 hours of incubation at 37 °C with 15 mmol/l 4-methylumbelliferyl-D-glucuronide trihydrate (Sigma-Aldrich).

Substrate accumulation. The glucosylceramide content was determined as previously described in samples from the late intervention study.45 Additionally, high-performance liquid chromatography was used to analyze glucosylceramide and C18-Ceramide in most of the early intervention study samples, while ultraperformance liquid chromatography mass spectrometry was utilized for measurement of Lyso-GlyCer (glucosylsphingosine).46,47

Hematological analysis and histopathological studies. Hematological analysis was performed using Sysmex XE-5000 automated cell counter. Fixed, sectioned tissue was stained with hematoxylin-eosin or periodic acid-Schiff for microscopic examination.

BM cell purification, transduction, and transplantation. Femur, tibia, and iliac were harvested from donor mice (GD1 mice) and BM flushed using a 23G needle and syringe in phosphate-buffered saline (ThermoScientific, Logan, UT) (2% fetal calf serum). Cells were passed through a 40 µmol/l filter (Fischer Scientific) and lineage-marker depleted (lin-) cells were isolated by magnetic sorting, using lineage-specific antibodies (CD5, CD45R (B220), CD11b, Anti-Gr-1 (Ly-6G/C), 7-4 and Ter-119; Mitenyi Biotech, Bergisch Gladbach, Germany). Prior to transduction, lin- BM cells were prestimulated for 18 hours in StemSpan serum-free expansion medium (SFEM) (Stemcell technologies, Grenoble, France), containing 10 ng/ml murine stem cell factor (SCF) (Peprotech, Rocky Hill, NJ), 20 ng/ml murine thrombopoietin (TPO) (Peprotech, Rocky Hill, NJ), 20 ng/ml murine IGF2 (RnD Systems, Abingdon, UK), 10 ng/ml human FGF-1 (Peprotech), 2% penicillin/streptomycin, 2 mmol/l glutamine (Life Technologies, Paisley, UK), plated in wells precoated with 10 µg/cm2 Retronectin (TaKaRa, Otsu, Japan). Cells were transduced at a multiplicity of infection of 30 and incubated for 24 hours (37 °C, 5% CO2). Recipient mice (GD mice) were lethally irradiated (900 cGy) 4 hours before transplantation and transduced cells transplanted by tail vein injection.

Flow cytometry. The transduction efficiency (percentage GFP) was analyzed in BM cells before transplantation and in peripheral blood at 4–5 weeks posttransplantation. BM cells were analyzed at end point for engraftment and lineage distribution with the following fluorochrome-conjugated monoclonal antibodies; PE-conjugated CD19 (BD Biosciences, Franklin Lakes, NJ), PE-conjugated CD3 (BioLegend), Qdot605-conjugated streptavidin (Life Technologies) Gr1-biotin (BioLegend), PeCy7-conjugated Mac1 (BD Biosciences), APC-conjugated F4/80 (eBioscience), APC eFlour 780-conjugated C-kit (CD117) (eBioscience). Dead cells were detected with 7-aminoactinomycin D (7AAD; Sigma-Aldrich). All analyses were done on a FACSCanto (BD Biosciences). Results were analyzed using FlowJo software (Tree Star Software, San Carlos, CA).

Vector copy number and insertional mutagenesis analysis. LAM-PCR and restriction enzyme-free LAM-PCR (Re-free LAM-PCR) were performed on whole BM genomic DNA from samples of all vector-treated groups48,49 with slight modifications to the published protocol. For LAM-PCR, samples were digested with MluCI, MseI, and HinP1I (all NEB) in separate reactions and joint for nested PCR steps. During Re-free LAM-PCR, we did not use a blocking oligo but digested the internal control amplicons with SacI (NEB) like in the conventional LAM-PCR protocol. Barcoding, sequencing, and bioinformatic analysis using custom Perl scripts, HISAP, and MAVRIC were performed as previously described.50

SUPPLEMENTARY MATERIAL Table S1. Increased recombination efficiency results in more severe GD1 progression. Table S2. Poly to oligo-clonal hematopoietic contribution. Table S3. Hits in Cancer Gene Databases. Table S4. Common Insertion Site (CIS) analysis. Table S5. Sequence of codon optimized human GBA. Figure S1. Codon optimization of vectors increases GCase activity. Figure S2. Robust reduction of glucosylsphingosine in treated animals. Figure S3. GFP percentage in BM correlates with vector copy number (VCN). Figure S4. Increased GCase activity and reduced levels of GluCer accumulation correlates with engraftment levels of genetically corrected cells. Figure S5. Improved hematological parameters for treated animals. Figure S6. Robust reversal of Gaucher cell infiltration in spleens of treated animals. Figure S7. The SFFV promoter generates 20-fold higher transgene expression in progenitor cells. Figure S8. Read Count Distribution. Figure S9. The CD68 promoter sequence.

Acknowledgments

The authors thank Johan Richter, Ilana Moscatelli, Carmen Florens-Bjurström and Niels-Bjarne Woods for advice, Reinhard Hämmerle for vector analysis, Manuel Grez and Julia Sürth for help regarding insertion site analysis, Britt-Marie Rynmark for technical assistance with lipid analyses, Emma Rörby and Matilda Billing for technical assistance and Beata Lindqvist at the Vector Unit at Lund University for virus production. This work was supported by a Clinical Research Award from Scania University Hospital (ALF), the Swedish Research Council Linnaeus (Hemato-Linné grant), STEMTHERAPY, an infrastructure grant from The Swedish Research Council, the Swedish Cancer Foundation (Cancerfonden), the Swedish Cancer Society (S.K.), the Swedish Children's Cancer Society (G.K. and S.K.), a project grant from the Swedish Medical Research Council (S.K.), and the Royal Swedish Academy of Sciences (Tobias Prize) financed by the Tobias Foundation (S.K.). The authors declare no conflict of interest.

Supplementary Material

Supplementary Figures and Tables
Click here for additional data file. (1.4MB, pdf)

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