TREATMENT OF CHRONIC GRANULOMATOUS DISEASE

Abstract

The present invention relates to an isolated human hematopoietic stem cell or progenitor cell, transduced with a lentiviral vector which comprises a coding nucleic acid sequence encoding a functional variant of a polypeptide selected from gp9lphox, p22phox, p40phox, p47phox, p67phox and Rac2; under transcriptional control of a promoter sequence that comprises or essentially consists of the miR223 promoter sequence (SEQ ID NO 01).

Claims

1. An isolated human hematopoietic stem cell or myeloid progenitor cell, transduced with a lentiviral vector, said lentiviral vector comprising a coding nucleic acid sequence encoding a functional variant of a polypeptide selected from gp91phox and p47phox; under transcriptional control of a miR223 promoter sequence (SEQ ID NO 01).

2. The isolated human hematopoietic stem cell or myeloid progenitor cell according to claim 1, wherein said lentiviral vector comprises or essentially consists of a nucleic acid sequence characterized by SEQ ID NO 02.

3. The human hematopoietic stem cell or myeloid progenitor cell according to claim 1, wherein the cell is a CD34 positive hematopoietic stem cell.

4. A method for preparing a therapeutic cell preparation, comprising the steps of: a. providing a preparation of cells comprising hematopoietic stem cells, particularly a preparation of CD34 positive hematopoietic stem cells, isolated from a patient suffering from chronic granulomatous disease associated with a gene defect associated with a defective gene encoding a polypeptide selected from gp9lphox and p47phox; b. transducing said preparation of cells with a lentiviral vector comprising i. a coding nucleic acid sequence encoding a functional variant of a polypeptide selected from gp91phox, and p47phox; under transcriptional control of ii. a miR223 promoter sequence (SEQ ID NO 01).

5. A method of treating chronic granulomatous disease in a patient using the transduced human hematopoietic stem cell or myeloid progenitor cell of claim 1.

6. The method according to claim 5, wherein the cell is an autologous cell.

7. The human hematopoietic stem cell or myeloid progenitor cell according to claim 2, wherein the cell is a CD34 positive hematopoietic stem cell.

Description

DESCRIPTION OF THE FIGURES

[0060] FIGS. 1 and 2 show certain embodiments of the vector for use in the invention.

[0061] FIG. 3 shows that miR223 driven p47phox expression upon gammaretroviral gene therapy restores ROS production and E. coli killing in human granulocytes;

[0062] FIG. 4 shows restoration of ROS production in human macrophages of a p47phox-deficient CGD patient after transduction of patient-derived monocytes upon lentiviral transduction of monocytes;

[0063] FIG. 5 DNA methylation of the silencing sensitive SFFV enhancer/promoter (CpG island) as well as of a part of the p47phox transgene in human CD34+cells after four weeks in cell culture. CD34+cells were transduced gamma-retrovirally and cultured for four weeks. DNA was isolated treated with sodium bisulfite, amplified in a PCR reaction and sequences thereafter.

[0064] Every horizontal line represents a sequencing result in which the information content was reduced to CpG dinucleotides: empty circles=unmethylated CpGs; black circles=methylated CpGs.

[0065] FIG. 6 miR223 driven GFP expression in CD11b+cells upon transduction of human HSCs of a healthy donor with a lentiviral vector encoding green fluorescent protein (GFP) under control of the miR223 promoter or the SFFV promoter, followed by myeloid differentiation to CD11b+cells in liquid cell culture.

EXAMPLES

[0066] The present invention is particularly based on the surprising finding that use of the miR223 promoter in gene therapy vectors advantageously overcomes known problems of transactivation and/or epigenetic inactivation. This was particularly shown by LV-SIN gene therapy vector for autosomal-recessive p47phox-deficient CGD. Of note, lentiviral transduction of human HSCs with a lentiviral vector encoding p47phox under control of the miR223 promoter for p47phox CGD treatment was never shown before.

Vectors Utilized Pre-Clinically to Characterize Transgene Expression

[0067] Data confirming a highly myelospecific transgene expression were generated with gamma-retroviral vectors and lentiviral vectors. Examples for utilized gamma-retroviral vectors are shown in FIG. 1. The ΔLNGFR-2A-p47phox fusion construct results in two separate proteins (cytoplasmic p47phox, and ΔLNGFR on the cell surface) both derived from one mRNA (v).

[0068] Examples for utilized lentiviral vectors are shown in FIG. 2.

Prevention of Transactivation in HSCs by Limiting Expression to Myeloid Cells

Myelospecificity of miR223 Promoter Activity Shown in Animal Studies

[0069] Lineage-negative bone marrow cells were isolated from p47phox -/- mice and transduced with gamma-retroviral vectors encoding p47phox or the ΔLNGFR-2A-p47 fusion construct under miR223 promoter control (FIG. 1). Recipient p47 -/- mice were lethally irradiated, and re-infused intravenously with transduced bone marrow cells. Six weeks after re-infusion, p47phox expression and ROS production were restored in granulocytes, indicating successful engraftment of transduced bone marrow cells. (Brendel et al. (2013) Hum Gene Ther Methods. 24: 151-159).

[0070] In parallel, lineage negative bone marrow cells were isolated from gp91phox-/- mice and transduced with a LV-SIN vector encoding gp91phox under miR223 promoter control.

[0071] Recipient gp91phox -/- mice were lethally irradiated, and re-infused intravenously with transduced bone marrow cells. Transduced cells engrafted, leading to transgene expression and restoration of ROS production in peripheral granulocytes of these mice (Brendel et al. (2013) ibid.).

[0072] Analysis of transgene expression revealed an extraordinary lineage specificity conferred by the miR223 promoter in both animal experiments limiting transgene expression almost exclusively to myeloid cells (Brendel et al. (2013) ibid.).

Restoration of Phagocyte Function by miR223-Driven Transgene Expression in Primary Derived Patient Material

[0073] In above mentioned animal experiments, miR223 driven gp91phox expression (lentiviral vector) as well as miR223 driven p47phox expression (gamma-retroviral vector) both restored ROS production in phagocytes upon gene therapy treatment in the corresponding animal models.

[0074] Reconstitution of ROS production and reconstitution of E. coli killing was tested in human granulocytes after treatment of HSCs with a gamma-retroviral vector and differentiation of cells in cell culture. For detection of cytoplasmic p47phox transgene expression in living granulocytes, the inventors generated a gamma-retroviral vector encoding a fusion construct consisting of the ΔLNGFR surface marker, linked to p47phox via 2A sequence from food and mouth disease virus. This fusion construct leads to synthesis of two proteins from one mRNA transcript, in one translation process: the cytoplasmic p47phox protein and the ΔLNGFR surface marker. This co-expression allows for indirect detection of p47phox by ΔLNGFR surface staining (Wohlgensinger et al. (2010) Gene Ther. 17: 1193-1199). In the gamma-retroviral vector utilized, the inventors expressed this fusion construct either under control of the constitutively active SFFV promoter, or of the miR223 promoter.

[0075] The inventors transduced human HSC of a p47phox-deficient CGD patient with these gammretroviral vectors, followed by differentiation of the cells to granulocytes in cell culture.

[0076] Differentiation was confirmed by FACS measurement of granulocytic CD11b surface marker expression. Detection of ΔLNGFR surface marker indirectly indicated p47phox expression.

[0077] ROS production was detected by a dihydrorhodamine 123 oxidation (DHR) assay upon stimulation of cells with PMA or with E. coli. ROS production in ex vivo differentiated granulocytes was observed only in ΔLNGFR-positive cells (FIG. 3).

[0078] Differentiated (CD11b+) and ΔLNGFR-positive granulocytes, and differentiated and ΔLNGFR-negative (non-transduced) cells were isolated by FACS-sorting, and applied in an E. coli killing assay as described (Ott et al. (2006) Nat Med 12: 401-409). In this assay, successful E. coli killing results in a rise in OD420 nm. Importantly, using the gamma-retroviral SIN p47phox miR223 vector, p47phox transgene expression, ROS production and E. coli killing were restored to similar amounts as in a gamma-retroviral SIN p47phox vector in which transgene expression is driven by the strong constitutively active SFFV promoter (FIG. 3).

[0079] FIG. 3 shows that miR223-driven p47phox expression restores ROS production and E. coli killing in human granulocytes. Lineage-negative HSCs of a p47phox CGD patient were gamma-retrovirally transduced with the ΔLNGFR/p47phox co-expression construct under SFFV or miR223 promoter control, and differentiated to CD11b+granulocytes.

[0080] ΔLNGFR/p47phox-expressing granulocytes were FACS sorted, and applied in an E. coli killing assay. A rise in OD420 nm indicates the restoration of E. coli killing.

[0081] Restoration of phagocyte function was also shown in primary derived p47phox CGD patient material. The lenti_miR223_p47_WPREmut6 vector (FIGS. 2 and 4) was used to transduce monocytes from a p47phox-deficient CGD patient. Transduced monocytes were differentiated to macrophages for seven days resulting in CD14+/CD206+macrophages. p47phox transgene expression was confirmed by intracellular FACS staining, and ROS production upon PMA stimulation was confirmed in a nitroblue teterazolium (NBT) reduction assay (FIG. 4).

[0082] FIG. 4 illustrates the restoration of ROS production in human macrophages of a p47phox-deficient CGD patient. Monocytes of a p47phox CGD patient were isolated from blood, transduced with the LV-SIN lenti_miR223_p47_WPREmut6 vector, and differentiated to macrophages. Differentiation to CD14+/CD206+macrophages and restoration of p47phox expression were confirmed by FACS (left). Restoration of ROS production upon PMA stimulation was confirmed by NBT assay. Yellow arrow: NBT-positive, i.e. ROS producing, macrophage, empty arrow: NBT-negative macrophage.

Prevention of Epigenetic Inactivation of Transgene Expression

Kinetics of Epigenetic Inactivation In Vivo and in Cell Culture

[0083] In the first temporarily successful clinical X-CGD gene therapy study, transgene expression was driven by the gammaretroviral promoter/enhancer within the viral LTR. Epigenetic inactivation of transgene expression started about one year after gene therapy, manifesting in an increasing discrepancy between gene marking and gene function within the 1.5 years thereafter (Siler et al. (2015) Current Gene Therapy 15: 416-427). The sequence of the promoter/enhancer within the LTR of the utilized gamma-retroviral vector corresponds to the SFFV promoter/enhancer utilized in various vectors as ubiquitously active promoter. This SFFV promoter was tested for its susceptibility to epigenetic modification in CGD animals three and six months after gene therapy treatment. Epigenetic SFFV promoter inactivation by DNA methylation in mice was already detectable after three months and increased thereafter over time (Zhang et al. (2010) Mol Ther 18: 1640-49).

[0084] Cells in a very primitive differentiation state like embryonic carcinoma cell lines (He et al. (2005) J Virol 79: 13497-508), embryonic stem cells (Liew et al. (2007) Stem Cells 25: 1521-28) and induced pluripotent stem cells (iPSCs) (Hotta & Ellis (2008) J Cell Biochem 105: 940-8) are known for their high DNA methylation activity. Epigenetic inactivation of the SFFV promoter within a lentiviral vector by P19 embryonic carcinoma cells is already detectable six to 12 days after transduction of the cells (Zhang et al. (2010) ibid.).

Novel Anti-Silencing Activity of the miR223 Promoter

[0085] The inventors tested the susceptibility of the miR223 promoter to DNA methylation in P19 cells and in a p47phox iPSC cell line.

[0086] Within a lentiviral vector (FIG. 2) an average 57.1% of CpGs within the miR223 promoter and more than 70% of CpGs within the SFFV promoter were methylated in P19 cells after 20 days (unpublished data). As P19 cells cannot be differentiated into the myeloid lineage (to phagocytes) and as the miR223 promoter is a myelospecific promoter, the inventors tested their lentiviral vector encoding p47phox under control of the miR223 promoter in p47phox CGD patient derived iPSCs (iPSC CGD1.1; Jiang et al. (2012) Stem Cells 30: 599-611) as these cells possess a high DNA methylation activity and can be propagated to phagocytes. iPSC CGD1.1 cells were transduced with a lentiviral vector encoding p47phox under control of the miR223 promoter (see Table 1 below) at multiplicity of infection 2 (MOI 2, i.e. twice as many viral particles as cells).

[0087] Starting day+13 post-transduction, iPSCs were propagated first to “embryoid bodies” and further to monocyte-releasing “monocyte factories”. First monocytes could then be harvested starting day+43 post transduction. Harvested monocytes were further differentiated to macrophages by seven days' incubation in medium supplemented with M-CSF.

[0088] Average gene therapy vector copy number per genome (VCN) was determined by qPCR on day+16 in iPSCs as well as on day+85 in harvested monocytes. p47phox expression was quantified by flow cytometry (FACS) analysis on day+6, +10, +16, +20 in iPSCs, on day+23 in CD133+stem cells within embryoid bodies, in CD38dim/CD34+cells in embryoid bodies on day+104, in CD14+cells on day+52, in CD15+cells on day+48, in CD206+cells on day+68. The percentage of p47phox-positive cells per vector copy number and the FACS derived mean fluorescent intensity (MFI) of p47phox-positive cells were determined to compare miR223 promoter activity in individual cell populations. p47phox expression analysis in various differentiation stages revealed that in the average on stem cell level (iPSCs, CD133+cells, CD34+cells) (n=6), miR223-driven p47phox expression was barely detectable, with the percentage of p47-positive cells per vector copy number of 0.67+/−0.33(Stdev). Upon myeloid differentiation, p47phox expression strongly increased, with a percentage of p47-positive cells per vector copy number of 5.26+/−0.66. This rise in the percentage of p47phox expressing cells ran in parallel with a strong increase in FACS signal intensity (MFI), strongly indicating an activation of the miR223 promoter upon differentiation from stem cell level to myeloid cells.

[0089] Gene therapy mediated restoration of p47phox production in granulocytes, monocytes and macrophages generated from transduced p47phox CGD iPSCs resulted in the restoration of ROS production as revealed in a Dihydrorhodamine 123 oxidation (DHR) assay and in a nitroblue tetrazolium reduction (NBT) assay.

[0090] In parallel with the analysis of miR223 promoter activity on various differentiation levels between iPSCs and myeloid cells (granulocytes, monocytes, macrophages), DNA methylation analysis of the miR223 promoter within the gene therapy vector and of the endogenous miR223 promoter gene within the genome of transduced cells was performed in iPSCs on day+20 post transduction, and in monocytes obtained from transduced iPSCs upon differentiation on day+85 post iPSC transduction. Analysis of iPSCs and iPSC-derived monocytes upon transduction with vector comprising p47phox under control of the silencing-sensitive SFFV promoter served as control.

[0091] This analysis (Table 1) revealed that, in parallel with P19 cells on stem cell level, the vast majority (>80%) of CpG dinucleotides of the miR223 promoter were methylated, within the gene therapy vector encoded miR223 promoter, as well as within the native endogenous miR223 gene promoter in the genome of the transduced cells. On a functional level, the methylated miR223 promoter was inactive, as was shown by p47phox expression analysis.

TABLE-US-00003 TABLE 1 DNA methylation analysis of the gene therapy vector encoded miR223 promoter and of the native miR223 gene promoter on iPSC (induced progenitor stem cell) level (day + 20) and in iPSC-derived monocytes day + 85 post iPSC transduction. A vector encoding the silencing-sensitive SFFV promoter served as control. Change in miR223 promoter DNA demethylation was statistically significant. Promoter DNA methylation analysis in iPSCs (day + 20) and iPSC-derived monocytes (day + 85) after transduction of iPSC with a lentiviral vector Endogenous % methylated Viral encoded miR223 gene CpGs +/− SEM miR223 promoter SFFV promoter In iPSCs 86.8% +/− 4.8 80.0 +/− 7.5 72.4 +/− 9.7 In monocytes   31.3 +/− 4.0 20.8 +/− 7.4 70.6 +/− 6.3 p-value (iPSC vs <0.001 <0.001 >0.05 monoctyes

[0092] Surprisingly the inventors found that the initially methylated miR223 promoter within the analysed gene therapy vector and the native genomic miR223 gene promoter both became demethylated upon myeloid differentiation (Table 1).

[0093] In parallel to a rise in promoter activity, the percentage of CpG dinucleotide methylation within the miR223 promoter dropped from 87% to 31%.

[0094] In the inventors' previous gamma-retroviral X-CGD (gp91phox-deficient form of CGD) clinical gene therapy trial, transgene expression was driven by a constitutively active (SFFV) promoter. A gradual increase in gene therapy vector encoded promoter methylation caused clinically a gradual decrease in therapeutic efficacy over time (Ott et al. (2006) Nat Med 12: 401-409; Siler et al. (2015) Curr Gene Ther 15: 416-427). In this trial, the constitutively active promoter became methylated in HSC, and resulted in a release of HSC from bone marrow with methylated promoter within the gene therapy vector, and therefore with diminished therapeutic transgene expression.

[0095] Utilizing the myelospecific miR223 promoter as internal promoter within a gene therapy vector, the surprisingly observed methylation pattern indicates that the promoter is inactive and methylated at stem cell level. Only upon differentiation, the miR223 promoter is activated and demethylated. This strongly indicates that this promoter is able to provide long-term efficacy.

[0096] As there is a continuous flux of myeloid cells derived from HSC that are released from bone marrow, in these newly produced cells the miR223 promoter actively became demethylated prior to release from bone marrow.

Kinetics of Promoter DNA Methylation

[0097] In a cell culture experiment, the inventors introduced the SFFV promoter in human CD34+bone marrow cells by gamma-retroviral transduction, cultured the cells for four weeks and quantified DNA methylation thereafter. The inventors were unable to detect significant amounts of DNA methylation in human CD34+cells after four weeks in cell culture (FIG. 5).

[0098] Silencing of the SFFV promoter in vivo in mouse bone marrow was reported to become detectable three months after gene therapy, and to progress further in the following three months (Zhang et al. (2010) ibid.).

[0099] Retroviral vectors are reported to be immediately silenced in pluripotent stem cells including early preimplantation embryos, embryonic stem (ES) cells and embryonic carcinoma (EC) cells (Pannell D, Ellis J. (2001) Rev Med Virol 11: 205-217). The embryonic carcinoma cell line P19 has been intensively utilized to test the susceptibility of promoters within gene therapy vectors to silencing (Knight et al. (2012) J Virol 86: 9088-95; Brendel et al. (2012) Gene Therapy 19: 1018-29; Zhang, Santilli & Thrasher (2017) Sci Rep. 7: 10213; Zhang et al. (2010) ibid.). Silencing of the SFFV promoter is consistently reported in this model to be finished already after 12 to 17 days.

[0100] As P19 cells can't be differentiated, this model is not compatible to test tissue specific promoters. Meanwhile it is known that retroviral expression is silenced in induced pluripotent stem cells (iPSCs) (Maherali et al. (2007) Cell Stem Cell 1: 55-70; Okita, Ichisaka & Yamanaka (2007) Nature 448: 313-317; Wernig et al. (2007) Nature 448: 318-324), which have the capacity to be differentiated in cell culture into any cell type. iPSCs were generated from CGD patient material. It was shown that these iPSCs have the same genetic defect as was found in the patient material. The cells can be differentiated to mature monocytes and macrophages, and in contrast to monocytes and macrophages from non-CGD iPSCs, the CGD iPSC derived monocytes and macrophages show no production of reactive oxygen species (ROS), as is true for monocytes and macrophages from CGD patients (Jiang et al. (2012) Stem Cells 30:599-611).

[0101] Upon retroviral introduction of the SFFV promoter into CGD iPSCs, the SFFV promoter, which was silenced in vivo in human bone marrow in the clinical X-CGD trial starting after 10 months, in mouse bone marrow after 3 to 6 months, and which was silenced in P19 cells after 12 to 17 days, was silenced in CGD iPSCs within 20 days (Hänseler et al. (2018) Matters; doi.: 10.19185/matters.201805000005). Upon differentiation of SFFV promoter harboring CGD iPSCs, the SFFV promoter driven transgene expression was strongly diminished in terminally differentiated monocytes and macrophages. This shows that 1) iPSCs have a strong promoter silencing activity, 2) the SFFV promoter, once being silenced remains silenced upon cell differentiation from iPSCs to monocytes (Hänseler et al. (2018) Matters; doi.: 10.19185/matters.201805000005). iPSCs had been used for the development of systems, which prevent the silencing of ubiquitously active promoters like the SFFV promoter. These anti-silencing systems mainly based on the use of insulators (Sanchez-Hernandez et al. (2018) Mol Ther Nucleic Acids 13: 16-28) or on the use of the ubiquitous chromatin opening element (UCOE) (Pfaff et al. (2013) Stem Cells 31: 488-99; Ackermann et al. (2014) Biomaterials 35: 1531-42; Hoffmann et al. (2017) Gene Ther. 24: 298-307).

[0102] In summary, iPSCs are a relevant model for the analysis of gene therapy vector methylation, but this model had never been applied to analyze the susceptibility of tissue specific promoters to silencing.

Analysis of the Tissue Specific miR223 Promoter for its Susceptibility to Silencing

[0103] For the analysis of susceptibility to silencing of the miR223 promoter, the inventors utilized iPSCs, which were developed from a patient suffering from p47phox-deficient CGD (p47phox CGD iPSCs) and which were able to differentiate into monocytes and macrophages showing the CGD phenotype (Jiang et al. (2012) Stem Cells 30:599-611). p47phox CGD iPSCs were lentivirally transduced (day+1) with the inventors' gene therapy vector (UZH1p47CGD), or with a lentiviral vector encoding p47phox under control of constitutively active and silencing resistant version of the SFFV promoter (Lenti UCOE 1662fwd SFFV p47). Transduced iPSC were differentiated first to embryoid bodies (EB), then further to monocytes and macrophages. Average vector copy number per cell (VCN) was determined by qPCR in iPSC on day+16, and in monocytes on day+85 after transduction. Transgene expression was monitored in CD133-positive stem cells in embryoid bodies on day +23, in CD38dim/CD34+cells within embryoid bodies (which are cells comparable to HSC) on day+104, in monocytes on day+52, and in macrophages on day+68 after transduction. p47phox expression was detected by FACS analysis upon intracellular staining of p47phox protein in combination with CD34, CD38, CD14 and CD206 surface staining. To correct for the differences in transduction efficiencies, percentages of p47phox-positive cells were divided by the VCN.

p47phox Transgene Expression

[0104] In iPSC and in the iPSC derived homolog of HSC, i.e. the CD.sub.38dim/CD.sub.34hig.sup.h cells within embryoid bodies (from now on in this context termed “HSCs”), the miR223 promoter within the inventors gene therapy vector UZH1p47CGD resulted in markedly weaker p47phox transgene expression, as measured by signal intensity, and a low percentage of p47phox-positive cells per vector copy number. In contrast, in differentiated monocytes and macrophages, signal intensity and percentage of p47phox-positive cells per vector copy number was high in transduced cells (see following Table 2).

TABLE-US-00004 TABLE 2 shows the p47phox transgene expression in iPSCs and iPSC-derived cell populations characterized by percentage of p47phox-positive cells per vector copy number (CVN) and by flow cytometry-derived mean fluorescent signal intensity (MFI). Human p47phox-negative iPSCs were lenti-virally transduced and propagated via embryoid bodies to monocytes and to macrophages. Average vector copy number (VCN) was determined in iPSC, and in iPSC derived monocytes. Transgene expression was monitored in CD133+ stem cells, in HSC-like CD34+ cells within embryoid bodies, in monocytes, and in macrophages. p47phox expression was detected by FACS analysis upon intracellular staining. Percentages of p47phox-positive cells were normalized for transduction efficiencies by calculation of the percentage of p47-positive cells per VCN. miR223-driven p47phox expression showed a marginal expression in CD133+ and in CD34+ cells and a rise in expression upon differentiation as revealed by percent of p47phox-positive cells as well as in MFI. in iPSC- in iPSC- in iPSC- in iPSC- Induction of p47phox derived derived derived derived expression upon CD133+ HSC-like CD14+ CD206+ differentiation stem cells CD34+ cells monocytes macrophages % p47 +/VCN SFFV 14.7 3.4 6 4.6 miR223 0.6 0.2 5.5 5.7 MFI of p47 + SFFV 0.96 3.56 1.56 1.8 cells miR223 0.57 1.93 2.39 3.75 CGD control 0.48 3.09 1.12 1.22 fold induction SFFV 2 1.15 1.39 1.48 of MFI rel. to miR223 1.19 0.62 2.13 3.07 contr.

[0105] Importantly, the miR223 promoter driven p47phox expression led to a low background signal (percent positive cells/VCN) as well as expression strength (MFI) in stem cells. The low background signal in stem cells indicates an excellent safety profile of the UZH1p47CGD gene therapy vector.

[0106] Human HSCs of a healthy donor were transduced with a lentiviral vector encoding GFP under control of the SFFV or the miR223 promoter, followed by differentiation to CD11b-positive myeloid cells in liquid cell culture. Expression of GFP was analyzed in human HSC-derived CD11b-positive cells by FACS analysis. Detection of GFP-positive human HSC-derived myeloid cells (FIG. 6, unpublished data) was in line with vector copy number (CVN) of 0.05 and shows transgene expression activity of the miR223 promoter in human HSC-derived myeloid cells as required for CGD gene therapy treatment.

[0107] Reconstitution of ROS production: ROS production was monitored in monocytes and macrophages by DHR oxidation and by NBT reduction assay upon stimulation with PMA (monocytes) or with PMA+fMLP (macrophages).

[0108] In the DHR assay, the non-fluorescent dihydrorhodamin turns fluorescent upon oxidation by ROS. Fluorescence signal intensities were detected by FACS analysis. Correspondingly, in FACS analysis a shift to the right indicates restoration of ROS production. In the NBT assay, the formation of dark blue formazan precipitates indicates ROS production.

[0109] Monocytes/macrophages from untransduced CGD iPSCs served as negative controls. As positive control served monocytes/macrophages obtained from CGD iPSCs, which were transduced with a vector encoding p47phox under control of a ubiquitously active and silencing resistant version of the SFFV promoter (Lenti UCOE1662fwd SFFV p47). miR223 driven p47phox expression by the inventors' gene therapy vector UZH1p47CGD resulted in reconstitution of ROS production and therefore in the correction of the CGD phenotype, as visualized in both the DHR assay and in the NBT assay.

[0110] Silencing (DNA methylation) analysis in the iPSC model: The inventors utilized p47-/- iPSCs to test for methylation of their gene therapy vector (UZH1p47CGD) and fortransgene expression activity upon iPSC differentiation to phagocytes. p47phox-negative iPSC were lenti-virally transduced and propagated to monocytes. DNA was isolated from transduced iPSC (day+20) and from iPSC-derived monocytes (d+97). For DNA methylation analysis, isolated DNA was exposed to sodium bisulfite, which converts only unmethylated cytosine to uracil. Subsequent PCR amplification converts uracil to thymidine.

[0111] Comparison of the original sequence with the sequence after bisulfite conversion reveals DNA methylation, i.e. in all methylated sequences a cytosine is detected by sequencing, whereas in unmethylated sequences thymidine is detected.

[0112] DNA methylation analysis was conducted in iPSCs and iPSC-derived monocytes upon transduction with the gene therapy vector (UZH1p47CGD) encoding p47phox under miR223 control. Methylation analysis covered the whole miR223 promoter sequence, as well as a part of the p47phox transgene. In addition, the inventors analyzed DNA methylation of the native miR223 gene promoter sequence of the iPSC genome.

[0113] Importantly, in iPSC both the virally encoded miR223 promoter, as well as the native miR223 promoter, were almost completely methylated within 20 days. Accordingly, in CD133+iPSCs and in iPSC-derived CD34+CD38- stem cells, p47phox transgene expression was extremely low (see Table 1 above).

[0114] Neither on iPSC level nor in monocytes, the degree of methylation between gene therapy vector encoded miR223 promoter sequence and native miR223 promoters, did not differ significantly. Surprisingly and in contrast to results obtained with the SFFV promoter (Hanseler et al. (2018) Matters; doi.: 10.19185/matters.201805000005), the inventors found that upon differentiation from iPSCs to terminally differentiated cells, methylation of both the viral and the native miR223 promoter dropped significantly, indicating active demethylation upon differentiation (Table 1) and thus activation (Table 2). Extrapolating to the long term in vivo situation, this finding indicates long term activity of the miR223 promoter, as being silenced in dormant HSC, the miR223 promoter is expected to become active upon HSC activation and differentiation. As mentioned earlier, UCOE based systems were developed, which can prevent the silencing of the SFFV, or of the Chim promoter (Zhang, Santilli & Thrasher (2017) Sci Rep 7: 10213). But the UCOE system embedded into a retroviral gene therapy vector harbors the risk of altering the DNA methylation of DNA in proximity to the retroviral integration site, also in HSC. This activity might in HSC activate oncogenes close to the integration site of the gene therapy vector in all consequence. In contrast to the UCOE system, the miR223 promoter within the vector UZH1p47CGD adapted to the surrounding methylation pattern in stem cells and only upon differentiation became demethylated, which is a significant safety feature.