CHIRIACO, FARINELLI Et Al. Regulated Lentiviral Vectors for X-CGD

CHIRIACO, FARINELLI et al. Regulated lentiviral vectors for X-CGD

Supplementary Material

A. Vector production

VSV-pseudotyped third-generation LVs were produced by co-transfection of the transfer, packaging (pMD2.Lg/p.RRE and pRSV.Rev) and envelope constructs (pMD2.G) into 293T cells by a Ca3PO4transfection. Briefly, supernatants were collected, passed through a 0.22μm filter, and purified by ultracentrifugation Vector particles were measured by HIV-1 gag p24 antigen immunocapture (NEN Life Science Products, MA, USA). Expression titer was estimated by a quantitative real time PCR on 293T cells, previously infected with limited dilution of concentrated vector supernatant. Concentrated vector expression titer ranged from 0.15 – 1.5 x 1010transducing units 293T (TU)/ml for all vectors.

B. Patients

BM and PB was collected from four X-CGD patients (mutations in CYBB gene) on the occasion of diagnostic or therapeutic procedures. Patient’s parents gave informed consent following standard ethical procedure with approval of the Children’s Hospital Bambino Gesù Ethical Committee.

The following mutations were identified: patient 1, cDNA change: c.742dupA, protein change p.Ile248Asnfsx36, western blot analysis: X91° (unpublished data); patient 2, deletion encompassing the whole gene, western blot analysis: X91° (unpublished data); patient 3 and patient 4, large deletions of the gene previously described from Di Matteo G et al.3; patient 5, cDNA changes c1287delT and c1290delC,western blot analysis: X91° (published in Di Matteo G et al.3).

C. Culture and differentiation of human CD34+ cells

For the experiments using the gp91phox transgene: BM cells were grown in Cell Gro medium (CellGenix, Germany) supplemented with human stem cell factor (huSCF) at 300 ng/ml, human Flt3-ligand (hu Flt3L) at 300 ng/ml, human thrombopoietin (hu TPO) at 100 ng/ml; and human interleukin-3 (hu IL3) at 60 ng/ml (all of them from Peprotech, Germany) for 12/24 hours. After pre-stimulation, cells were transduced at MOI 100 for 12 hours in a retronectin-coated plate (final concentration 33ug/ml). Four days after transduction, cells were collected for surface staining and the remaining cells were plated with RPMI medium containing 10% FBS, glutamine, antibiotics and Granulocytes Colony-Stimulation Factor (G-CSF) (Peprotech, Germany) at 100 ng/ml to induce granulocytic differentiation.

For the experiments using the GFP reporter gene: CB- or BM-derived CD34+ HSPC were cultured in Stem Span medium (Stem Cell Technologies) supplemented with huSCF100 ng/ml, huFlt3L 100 ng/ml, huTPO50 ng/ml; and hu-IL650 ng/ml (all of them from Peprotech, Germany). The medium for cells kept in non-differentiating culture conditions for more than 2 days included Stem Span,huSCF50ng/ml, huFlt3L 50 ng/ml, huTPO50 ng/ml, hu-IL650 ng/ml and StemRegenin 1 (SR1) 1µM (kind gift from M. Cooke, Novartis). Myeloid differentiation culture was performed in IMDM + 10% FCS + G-CSF 100ng/ml (Myelostim, Chugai Sanofi Aventis, France) + SCF 50ng/ml for the first week of culture.

D. Ex-vivo gene therapy in X-CGD mice

Murine BM samples were harvested from the femurs and tibias of B6.129S6- Cybbtm1Din/J mice, Ly5.2 (The Jackson Laboratory, USA) and the Lin- progenitors were isolated using the Lineage Cell Depletion Kit (Miltenyi, Germany) following manufacturer’s instructions. Lin- cells were cultured in StemSpan serum-free medium for 12/24 hours (StemCell Technologies, Canada) supplemented with 1% l-glutamine, 1% penicillin/streptomycin and full cytokine cocktail 100 ng/ml murine SCF (mu SCF), 50 ng/ml mu TPO, 100 ng/ml hu Flt3L and 20 ng/ml hu IL-3 (Peprotech, Germany). After pre-stimulation, Lin- cells were transduced at MOI 100 for 16-20 hours with the different vectors.Six to eight weeks old B6.129S6- Cybbtm1Din/J mice, Ly5.2 recipient mice were lethally irradiated (900 RAD split into 2 fractions 20 minutes apart) and transplanted with 5 x 105LV-transduced Lin- cells via tail vein injection. Mice were bled 20 weeks after transplantation and leukocyte populations were stained and gated into granulocytes (Gr1+, CD11b+, CD48low), monocytes (Gr1low, CD11b+, CD48high), B cells (B220+, CD48+) and T cells (CD3+,CD48+). BM, spleen and thymus were collected at sacrifice and analysed by flow cytometry, DHR and PCR for vector transduced cells.

E. Vpx-VLP production

To produce concentrated Vpx-incorporating viral-like particles (VLPs), 293T cells were co-transfected with a VSV-g expressing plasmid and the Simian Immunodeficiency Virus-derived packaging plasmid SIV3+, as previously described48.

F. Transduction of human monocyte-derived macrophages

Human blood monocytes were isolated from PBMC of 3X-CGD patients and 3healthy controls using the CD14 MicroBeads kit (Miltenyi, Germany). Isolated CD14+ cells were plated for 2 hours in RPMI medium (Sigma Aldrich, USA). After 2 hours of incubation at 37°C, cells were rinsed twice with RPMI medium to remove of unattached cells and then cultured in RPMI medium supplemented with 10% heat-inactivated FBS (Sigma Aldrich, USA), 4 mmol/l l-glutamine, antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin) and 100 ng/ml of human M-CSF (Miltenyi, Germany). After 7 days of culture Vpx-VLP was added and after 4-6 hours of incubation monocyte-derived macrophages were transduced with LVs PGK.GFP, MSP.GFP, PGK.gp91_126T(2) and MSP.gp91_126T(2) at different MOIs between 1 and 10. After 5 days cells were scraped from each well after a 5 minutes incubation with Trypsin-EDTA (Sigma Aldrich, USA) and analysed for gp91phox expression.

G. Flow cytometric analysis

For immunostaining, FITC labeled anti-human gp91phox (anti-flavocytochrome B558, clone 7D5) (MBL, Medical and Biological Laboratories Co., Japan) was added and the cells were incubated for 20 min at 4 °C, washed, and analyzed on a FACS Calibur or FACS Canto II(Becton-Dickinson, USA).

The progeny of human CD34+ cells were labeled with the following antibodies: anti-human gp91phox FITC, anti-CD11b APC, Pacific Blue or APC-Alexa 780 (BD Pharmingen or eBiosciences), anti-human CD34 PE, PE-Cy7 (BDPharmingen), anti-human CD38 APC or V450 (BD), anti-human CD90 APC or PE (BD), CD45RA Brilliant Violet (BD), CD10 APC (BD), anti-human CD45 APC-H7 (BD), anti-human CD16 PE, PE-Cy5 or PE-Cy7 (BD or DAKO), anti-human CD14 PE (BD), anti-human CD13 APC or PE (BD), anti-human CD33 (PE-Cy7), anti-human CD235a PE or APC (DAKO or BD), anti-CD19 V500 (BD), anti-human NGFR APC or PE (Miltenyi). Generally, cells were stained at a density of 10^7/ml for 20 min at 4 °C, washed, and analysed on an LSRII Fortessa flow cytometer or FACS Canto II (Becton-Dickinson). To perform the intracellular staining, cells were fixed and permeabilized with the Cytofix/Cytoperm kit (BD, USA), according tomanufacturer’s instructions, and then stained with PElabeled anti-human gp91phox(anti-flavocytochrome b558, clone 7D5) (MBL, Medical and Biological Laboratories Co., Japan).

Monocyte-derived macrophages were stained with FITC labeled anti-human gp91phox(anti-flavocytochrome B558, clone 7D5) (MBL, Medical and Biological Laboratories Co., Japan), anti-human CD14 PE (BD), anti-human CD206 (Miltenyi, Germany) and analysed on a FACS Canto II (Becton-Dickinson).Murine HSPCs and their progeny were stained with the following antibodies: anti-human gp91phox FITC (anti-flavocytochrome B558, clone 7D5) (MBL, Japan), anti-mouse Sca1 PE-Cy7, anti-mouse CD150 APC, anti-mouse cKit APC-eFluor 780, anti-mouse CD48 Pacific Blue, anti-mouse lineage biotin-antibody cocktail and anti-biotin PE (from BD Pharmingen, USA or eBioscience, USA).

H. Functional assays

DHR assay.Human CD34+ in vitro differentiated oxidative burst was determined by Burst test kit following manufacturer’s instructions (OrpegenPharma, Germany). Briefly, cells were filled with 2 × 107unlabeled opsonized bacteria E. coli, 20 μL of substrate solution (negative control), 20 μLfMLP (peptide N-formyl-MetLeuPhe) as chemotactic low physiological stimulus (low control) and 20 μLphorbol 12-myristate 13-acetate (PMA), a strong non-receptor activator (high control). All samples were incubated for 10 min at 37.0 °C in a water bath, dihydrorhodamine (DHR) 123 as a fluorogenic substrate was added and incubated again in the same conditions. The oxidative burst occurred with the production of reactive oxygen substrates (ROS) (superoxide anion, hydrogen peroxide) in granulocytes stimulated in vitro. In ROS-stimulated granulocytes, nonfluorescent DHR 123 underwent conversion to fluorescent rhodamine (R) 123 registered in the flow cytometer FACS Canto II. Samples were washed (5 min, 250 r/min, 4 °C), and supernatant was discarded. Samples were washed again (washing solution), centrifuged (5 min, 250 r/min, 4 °C) and the supernatant was decanted. An amount of 200 μL of DNA staining solution (centrifuged and incubated for 10 min at 0 °C in a dark place) was added to discriminate and exclude aggregation artifacts of bacteria and/or cells in cytometric flow analysis.

For DHR test on mouse peripheral blood, 100 μl of lysed blood was incubated with APC labeled anti-mouse CD11b and CD48 (BD Pharmingen) in PBSgg (PBS with 0,1% of gelatine and 0,09% of D-glucose) containing 1000 U/ml of catalase (Sigma Aldrich, USA) and 2 μl of 1 mg/ml of DHR123 (Invitrogen, USA) – total reaction volume 250 μl. After 10 minutes of incubation at 37° C cells were split into two tubes, adding only to one of them 0,5 μl of PMA (1 mg/ml) (Sigma Aldrich, USA). Reactions were incubated for further 15 minutes at 37° C, stopped on ice and immediately analyzed on a flow cytometer.

Cytochrome c assay. Superoxide release was assessed by a cytochrome c reduction assay. For this assay 0.5x106primary monocytes were harvested, washed in prewarmed PBS (Euroclone, Italy) and then in prewarmed PBS with 7.5mM of Glucose. The pellet was then resuspended in PBS with 7.5mM Glucose and 1.5mM cytochrome c from horse heart (cat.no. C2867, Sigma), and splitted in 2 wells of a 96 wells plate. Then PMA was added and the absorbance of each well was measured at 550 nm every 10 seconds for 25 minutes.The released amount of superoxide was calculated using the Lambert-Beer’s law A=21.1 x c x d, where 21.1 was the absorption coefficient, c was the concentration in mmol/l, and d the light path of the well (on a 96 plate) in cm. The results were expressed as nanomoles of superoxide released per 106 cells per minute.

I. Analyses of vector copy number

Cells were cultured for 14 days after transduction in order to eliminate non- integrated vector forms. Genomic DNA was extracted by using QIAamp DNA Blood mini kit or micro kit (QIAGEN, Germany), according to manufacturer’s instructions. Vector copy numbers per genome (VCN) were quantified by quantitative Real-Time PCR (Q-PCR) using the following primers for HIV: HIVfwd: 5’-TACTGACGCTCTCGCACC-3’; HIV rev 5’-TCTCGACGCAGGACTCG-3’) and probe (FAM 5’- ATCTCTCTCCTTCTAGCCTC-3’) against the primer binding site (PBS) region of LV. Endogenous DNA amount was quantified by a primer/probe set against the human telomerase gene (Telofwd: 5’-GGCACACGTGGCTTTTCG-3’; Telo rev: 5’- GGTGAACCTCGTAAGTTTATGCAA-3’; TAMRA-Probe: 5’- TCAGGACGTCGAGTGGACACGGTG-3’) or against the murine β-actin gene (Act fwd: 5-AGAGGGAAATCGTGCGTGAC-3’; Act rev: 5’-CAATAGTGATGACCTGGCCGT-3’; probe: VIC 5’-CACTGCCGCATCCTCTTCCTCCC-3’). Copies per genome were calculated by the formula: (copies LV/copies endogenous DNA) / (n° of housekeeping gene copies in the standard curve). The standard curve was generated by using a CEM cell line (CEMA 301#25) stably carrying one vector integrant (MolMed, Italy). CEMA 301#25 and CEMA 301#37 were grown in RPMI medium (Sigma Aldrich, USA) supplemented with 10% heat-inactivated FBS (FBS, Sigma Aldrich, USA), 4 mmol/l l-glutamine and antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin).DNA isolated from CEMA 301#37 cell line (MolMed, Italy) carrying four copies of vector integrant was used as a positive control. All reactions were carried out in duplicate in an ABI Prism 7300HT (Applied Biosystems, USA).

K. Calculation of comparative transgene expression levels

Optimization of miRT sequences (studies shown in Fig. 1 and Supplementary Fig. S2): we employed bidirectional miRNA reporter vectors (BdLVs). The following parameters were measured for each sample and cell subpopulation by flow cytometry, with a gate set on the NGFR+ (transduced) population: arithmetic mean fluorescence intensity (MFI) of GFP and NGFR in cells transduced with the miRNA reporter (GFPBdLV.miRTandNGFRBdLV.miRT, respectively), arithmetic MFI of GFP and NGFR in cells transduced with the control BdLV (GFPBdLV.CtrlandNGFRBdLV.Ctrl, respectively). The MFI fold change with respect to the control vector (Fig. 1b,d) was calculated as follows: MFI relative to PGK = (NGFRBdLV.Ctrl x GFPBdLV.miRT) / (NGFRBdLV.miRTxGFPBdLV.Ctrl).

Comparison of PGK.GFP with MSP.GFP (studies shown in Fig.2):

Transduction levels with the PGK.GFP LV and MSP.GFP.LV were estimated by vector copy number analysis and the percentage of GFP+ myeloid cells (see Fig. 1). Relative MFI was calculated as the arithmetic GFP mean fluorescence intensity of the GFP+ cells from the MSP divided by the corresponding MFI of the PGK transduced cells.

Comparison of MSP.GFP with MSP.GFP.126T and PGK.GFP (studies shown in Fig.2):

Transduction levels with the PGK.GFP LV, MSP.GFP.LV and MSP.GFP.126T LV were carefully matched and found to be comparable. Since GFP expression fell below the detectable level in MSP.GFP.126T LV transduced HSPC, and transduced cells could thus not reliably be distinguished from untransduced cells, we estimated the GFP MFI originating from LV transduced cells. The following parameters were acquired by flow cytometry: The arithmetic MFI of GFP of the total cells with the immune phenotype characterizing a specific subpopulation after transduction with one of the LVs (without distinguishing between transduced and untransduced cells): GFPsubpop x (all); The background GFP MFI of untransduced cells: GFPUT; The percentage of GFP+ cells measured in the most differentiated myeloid subpopulation (CD34-CD38+ cells in serum free culture; CD13+CD11b+CD16+ cells in granulocytic differentiation culture; CD13+CD14+CD16int cells in methylcellulose culture) after HSPC transduction with the single LVs: %GFPdiff. It is assumed that the differentiated cells are generated from transduced CD34+ HSPC, and that transduction efficiency in the differentiated cells reflects the one in CD34+ HSPC. The MFI originating from vector transduced cells was estimated as follows: MFIest = (GFPsubpop x (all)- GFPUTx (1- %GFPdiff/100)) / %GFPdiffx100. The correctness of this MFI estimation was validated by comparing the MFIest with the MFI of the GFP+ cells for the PGK.GFP vector where transduced cells could be unambiguously identified, showing excellent concordance between the 2 values in all subpopulations (data not shown).

Comparison of MSP.gp91with MSP.gp91_126T(2) and PGK.gp91 (studies shown in Fig.3b):

Since gp91phox expression fell below the detectable level in regulated LVs transduced HSPC, and transduced cells could thus not reliably be distinguished from untransduced cells, we estimated the gp91phox MFI originating from LV transduced cells. The following parameters were acquired by flow cytometry: The geometric MFI of gp91phox of the total cells with the immune phenotype characterizing a specific subpopulation after transduction with one of the LVs (without distinguishing between transduced and untransduced cells): gp91phoxsubpop x (all); The background gp91phox MFI of untransduced cells: gp91phoxUT; The percentage of gp91phox+ cells measured in the most differentiated myeloid subpopulation (CD11b+ cells) after HSPC transduction with the single LVs: %gp91phoxdiff. It is assumed that the differentiated cells are generated from transduced CD34+ HSPC, and that transduction efficiency in the differentiated cells reflects the one in CD34+ HSPC. The MFI originating from vector transduced cells was estimated as follows: MFIest = (gp91phoxsubpop x (all)- gp91phoxUTx (1- %gp91phoxdiff/100)) / %gp91phoxdiffx100. The correctness of this MFI estimation was validated by comparing the MFIest with the MFI of the gp91phox+ cells for the PGK.gp91 vector where transduced cells could be unambiguously identified, showing excellent concordance between the 2 values in all subpopulations (data not shown). The graphs of Fig. 3b shows the MFIest of the vectors divided by the MFIofgp91phox UT.

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