BIFUNCTIONAL VECTORS ALLOWING BCL11A SILENCING AND EXPRESSION OF AN ANTI-SICKLING HBB AND USES THEREOF FOR GENE THERAPY OF B-HEMOGLOBINOPATHIES

Abstract

The #β-hemoglobinopathies #β-thalassemia (BT) and sickle cell disease (SCD) are the most frequent genetic disorders worldwide. These diseases are caused by mutations causing reduced or abnormal synthesis of the β-globin chain of the adult hemoglobin (Hb) tetramer. Here, the inventors intend to improve HSC-based gene therapy for β-thalassemia and SCD by developing an innovative, highly infectious LV vector expressing a potent anti-sickling β-globin transgene and a second biological function either increasing fetal γ-globin expression (for β-thalassemia and SCD). More particularly, the inventors have designed a novel lentivirus (LV), which carry two different functions: βAS3 gene addition and gene silencing. This last strategy allows the re-expression of the fetal γ-globin genes (HBG1 and HBG2) and production of the endogenous fetal hemoglobin (HbF). Elevated levels of HbF and HbAS3 (Hb tetramer containing βAS3-globin) will benefit the β-hemoglobinopathy phenotype by increasing the total amount of β-like globin that will: (i) reduce the alpha precipitates and improve the alpha/non alpha ratio in β-thalassemia, and (ii) reduce the sickling in SCD. This combined strategy will improve the β-hemoglobinopathy phenotype at a lower vector copy number (VCN) per cell compared to a LV expressing the βAS3 alone.

Claims

1. A nucleic acid molecule having the sequence as set forth in SEQ ID NO:1 wherein a sequence encoding for an artificial microRNA (amiR) suitable for reducing the expression of BCL11A, is inserted i) between the nucleotide at position 85 and the nucleotide 86 at position in SEQ ID NO:1 and/or ii) between the nucleotide at position 146 and the nucleotide at position 147 in SEQ ID NO:1.

2. The nucleic acid molecule of claim 1 wherein the amiR comprises a shRNA that is embedded into a miRNA backbone.

3. The nucleic acid molecule of claim 2 wherein the miRNA backbone is derived from miR-142, miR-155, miR-181 and/or miR-223.

4. The nucleic acid molecule of claim 2 wherein the shRNA adopts a stem-loop structure wherein a stem region is formed by a guide strand and a passenger strand.

5. The nucleic acid molecule of claim 4 wherein the sequence encoding for the guide strand comprises the sequence as set forth in SEQ ID NO: 2.

6. The nucleic acid molecule of claim 4 wherein a loop segment is encoded by the sequence as set forth in SEQ ID NO:3.

7. The nucleic acid molecule of claim 2 wherein the sequence encoding for the shRNA comprises the sequence as set forth in SEQ ID NO:4.

8. The nucleic acid molecule of claim 1 wherein the sequence encoding for the amiR comprises the sequence as set forth in SEQ ID NO:5.

9. The nucleic acid molecule of claim 1 that has a sequence as set forth in SEQ ID NO:6 or SEQ ID NO:7.

10. A transgene encoding for an anti-sickling β-globin (HBB) wherein said transgene comprises the nucleic acid molecule of claim 1.

11. The transgene of claim 10 which comprises the sequence as set forth in SEQ ID NO:9 or SEQ ID NO:10.

12. The transgene of claim 10 which is placed under the transcriptional control of the HBB promoter and key regulatory elements from the 16-kb human β-locus control region (βLCR), wherein the key regulatory elements comprise the 2 DNase I hypersensitive sites HS2 and HS3.

13. A viral vector comprising the transgene of claim 10.

14. The viral vector of claim 13 which is a lentiviral vector.

15. A method of obtaining a population of host cells transduced with the transgene of claim 10, which comprises the step of transducing a population of host cells in vitro or ex vivo with the viral vector of claim 13.

16. The method of claim 15 wherein the host cell is selected from the group consisting of hematopoietic stem/progenitor cells, hematopoietic progenitor cells, hematopoietic stem cells (HSCs), pluripotent cells and induced pluripotent stem cells (iPS).

17. A method of treating a hemoglobinopathy in a subject in need thereof, comprising transplanting into the subject a therapeutically effective amount of the population of host cells obtained by the method of claim 16.

18. The nucleic acid molecule of claim 1, wherein the BCL11A is the BCL11A-XL isoform.

19. The method of claim 16 wherein the pluripotent cells are embryonic stem cells (ES).

Description

FIGURES

[0064] FIG. 1: Introduction of the modified shRNA #5 embedded in the miR-223 backbone in intron 2 of the βAS3 transgene. (A) The amiR is composed by a shRNA embedded in the miR-223 backbone (top panel). The sequence of the different amiR components is shown (bottom panel-(SEQ ID NO: 27)). (B) The shRNA #5 embedded in the miR-223 backbone (SEQ ID NO: 28). This amiR targets BCLL11A-XL RNA. (C) The amiR was inserted inside intron 2 of the βAS3 transgene between positions 85 and 86 (where a 593-bp region was deleted) or between positions 146 and 147 (βAS3-miR/int2_del and βAS3-miR/int2, respectively).

[0065] FIG. 2: The presence of the amiR does not affect gene transfer efficiency in HUDEP-2 cells. VCN/cell was measured by ddPCR in HUDEP-2 cells transduced with αAS3, βAS3-miR/int2_del or βAS3-miR/int2 LVs at MOI 1, 5, 10 and 15. After transduction, cells were grown for 14 days before measuring the VCN/cell.

[0066] FIG. 3: The amiR reduces BCL11A XL mRNA expression levels. BCL11A XL mRNA levels were measured by RT-qPCR in mock- and LV-transduced HUDEP-2 cells after 9 days of differentiation. mRNA levels were normalized to LMNB2 expression.

[0067] FIG. 4: βAS3 transgene expression is not affected by the insertion of the amiR in intron 2. βAS3 mRNA levels were measured by RT-qPCR in mock- and LV-transduced HUDEP-2 cells after 9 days of differentiation. βAS3 mRNA levels were normalized to HBA expression. We plotted βAS3 mRNA levels per VCN. No significant statistical difference was observed between the 3 LVs.

[0068] FIG. 5: Induction of HBG1 and 2 gene expression upon BCL11A-XL silencing. HBG1/2 mRNAs were measured by RT-qPCR in HUDEP-2 cells after 9 days of differentiation. HBG1/2 mRNA levels were normalized to HBA expression. We plotted HBG1/2 mRNA levels per VCN. No significant difference was observed between the AS3-miR/int2_del and βAS3-miR/int23 LVs. HBG1/2 mRNA levels were significantly higher in βAS3-miR/int2_del- and βAS3-miR/int23-transduced cells than in βAS3-transduced samples (One-way ANOVA test; *** P<0.001).

[0069] FIG. 6: HbF induction upon BCL11A-XL silencing. (A) Representative flow cytometry analysis of HbF expression in terminally differentiated CD235a.sup.high HUDEP-2 cells after 9 days of differentiation. (B) Graphs showing the percentage of HbF.sup.+ cells and the corresponding mean fluorescence intensities (MFI). (C) Graphs showing the β-like-globin/α-ratios, as determined by reverse-phase HPLC.

[0070] FIG. 7: Erythroid differentiation is not altered upon transduction of HD HSPCs with the BCL11A amiR-expressing LVs. Flow cytometry analysis of CD71 (A), CD36 (B) and CD235a (C) expression. We plotted the percentage of erythroid cells derived from HD CD34.sup.+ HSPCs expressing CD71, CD36 or CD235a. These erythroid surface markers were analyzed along the differentiation at day 6 (D6), day 13 (D13), day 16 (D16), and day 20 (D20). The expression of the early erythroid markers CD36 and CD71 decreased along the differentiation while the expression of the late erythroid marker CD235a increased. Erythroid differentiation was not impacted in samples transduced with the LVs containing the amiR BCL11A (βAS3-miR/int2 and βAS3-miR/int2_del) compared to control cells (mock-transduced cells (Mock), cells transduced with the LV containing either the βAS3 alone (βAS3), or the βAS3 and a non-targeting (nt) amiR (βAS3-miR #nt/int2 and βAS3-miR #nt/int2_del).

[0071] FIG. 8: Transduction of HD HSPCs with BCL11A amiR-expressing LVs does not impact the enucleation rate of RBCs derived from HD CD34.sup.+ HSPCs. (A, B). Flow cytometry analysis of DRAQ5.sup.+ nucleated and DRAQ5.sup.− enucleated RBCs-derived HD CD34.sup.+ HSPCs. We measured the percentage of enucleated RBCs along the differentiation at day 6 (D6), day 13 (D13), day 16 (D16) and day 20 (D20). Enucleated RBCs were detected from day 13 and their proportion increased to up to 90% at D20. Enucleation was not impacted in samples transduced with the LVs containing the amiR BCL11A (βAS3-miR/int2 and βAS3-miR/int2_del) compared to control cells (mock-transduced cells (Mock), cells transduced with the LV containing either the βAS3 alone (βAS3), or the βAS3 and a non-targeting (nt) amiR (βAS3-miR #nt/int2 and βAS3-miR #nt/int2_del)).

[0072] FIG. 9: HBG genes are de-repressed in primary erythroid cells transduced with the BCL11A amiR-expressing LVs. HBG1 and HBG2 mRNA levels were measured by RT-qPCR in erythroid precursors derived from HD CD34.sup.+ HSPCs after 13 days of differentiation. HBG mRNA levels were normalized to HBA gene expression. We plotted HBG mRNA levels per VCN. HBG mRNA levels were higher in transduced cells with LVs containing the BCL11A amiR (βAS3-miR/int2 and βAS3-miR/int2_del) than in control cells transduced with LV containing the βAS3 alone (βAS3) or the βAS3 and a non-targeting (nt) amiR (βAS3-miR #nt/int2 and βAS3-miR #nt/int2_del).

[0073] FIG. 10: γ-globin induction in primary erythroid cells transduced with the BCL11A amiR-expressing LVs. (A) Western blot analysis of γ-globin expression in RBCs derived from HD CD34.sup.+ HSPCs after 16 days of differentiation. α-globin was used as the loading control. γ-globin expression was normalized to α-globin. (B) We plotted γ-globin chain expression levels per VCN and γ-globin chain fold-increase between control (βAS3-miR #nt) and BCL11A-miR transduced cells (βAS3-miR) for the LVs containing the BCL11A amiR in position int2 or int2_del.

[0074] γ-globin chain levels were higher in BCL11A amiR-transduced cells (βAS3-miR/int2 and βAS3-miR/int2_del) compared to control cells transduced with LV containing the βAS3 alone (βAS3) or the βAS3 and a non-targeting (nt) amiR (βAS3-miR #nt/int2 and βAS3-miR #nt/int2_del).

[0075] FIG. 11: Increased therapeutic globin levels in cells transduced with BCL11A amiR-expressing LVs. Graphs showing the β-like globin/α-globin ratios (A) and the (βAS3+γ)/VCN ratios (B) as measured by RP-HPLC and the percentage of hemoglobin tetramers (C) and the (HbF+HbAS3)/VCN ratios (D) as determined by cation exchange-HPLC (CE-HPLC). In graphs A and C, the VCN is indicated.

[0076] Globin chain and hemoglobin expression was assessed in RBCs derived from HD CD34.sup.+ HSPCs after 16 days of differentiation. γ-globin and HbF expression were higher in BCL11A amiR-transduced cells (βAS3-miR/int2 and βAS3-miR/int2_del) compared to mock-transduced cells (Mock) or cells transduced with LV expressing βAS3 and a non-targeting (nt) amiR (βAS3-miR #nt/int2). γ-globin de-repression coupled with βAS3 transgene expression leads to a 2-fold increase in therapeutic globins (βAS3+γ) and hemoglobin tetramers (HbF+HbAS3) per VCN. Fold-increase is indicated above the graphs.

Example: A Novel Lentiviral Vector for Gene Therapy of B-Hemoglobinopathies: Co-Expression of a Potent Anti-Sickling Transgene and a MicroRNA Downregulating BCL11A

[0077] Methods:

[0078] Lentiviral Vector Production and Titration

[0079] Third-generation LVs were produced by calcium phosphate transient transfection of HEK293T cells with the transfer vector (pCCL.βAS3, pCCL.βAS3-miR/int2_del or βAS3-miR/int2, pCCL.βAS3-miR #nt/int2_del or βAS3-miR #nt/int2), the packaging plasmid pHDMH gpm2 (encoding gag/pol), the Rev-encoding plasmid pBA Rev, and the vesicular stomatitis virus glycoprotein G (VSV-G) envelope-encoding plasmid pHDM-G. The viral infectious titer, expressed as transduction units per ml (TU/ml) was measured in HCT116 cells after transduction using serial vector dilutions. Three days after transduction, genomic DNA was extracted and the vector copy number (VCN) per cell was measured by qPCR. The VCN per cell was used to calculate the viral infectious titer.

[0080] HUDEP-2 Cell Culture, Differentiation and Transduction

[0081] HUDEP-2 cells (HUDEP-2) were cultured and differentiated as previously described (Antoniani et al., 2018; Canver et al., 2015; Kurita et al., 2013). HUDEP-2 cells were expanded in a basal medium composed of StemSpan SFEM (Stem Cell Technologies) supplemented with 10.sup.−6M dexamethasone (Sigma), 100 ng/ml human stem cell factor (hSCF) (Peprotech), 3 IU/ml erythropoietin (EPO) Eprex (Janssen-Cilag, France), 100 U/ml L-glutamine (Life Technologies), 2 mM penicillin/streptomycin and 1 μg/ml doxycycline (Sigma). HUDEP-2 cells were transduced at a cell concentration of 10.sup.6 cells/ml in basal medium supplemented with 4 ug/ml protamine sulfate (Choay). After 24 h, cells were washed and cultured in fresh basal medium. Cells were differentiated for 9 days in Iscove's Modified Dulbecco's Medium (IMDM) (Life Technologies) supplemented with 330 μg/ml holo-transferrin (Sigma), 10 μg/ml recombinant human insulin (Sigma), 2 IU/ml heparin (Sigma), 5% human AB serum (Eurobio AbCys), 3 IU/mL erythropoietin, 100 ng/mL human SCF, 1 μg/ml doxycycline, 100 U/ml L-glutamine, and 2 mM penicillin/streptomycin.

[0082] HSPC Purification and Transduction

[0083] Human adult HSPCs were obtained from healthy donors (HD). Written informed consent was obtained from all subjects. All experiments were performed in accordance with the Declaration of Helsinki. The study was approved by the regional investigational review board (reference, DC 2014-2272, CPP Ile-de-France II “Hôpital Necker-Enfants malades”). HSPCs were purified by immunomagnetic selection (Miltenyi Biotec) after immunostaining using the CD34 MicroBead Kit (Miltenyi Biotec).

[0084] CD34.sup.+ cells were thawed and cultured for 24 h at a concentration of 10.sup.6 cells/mL in pre-activation medium composed of X-VIVO 20 supplemented with penicillin/streptomycin (Gibco) and recombinant human cytokines: 300 ng/mL SCF, 300 ng/mL Flt-3 L, 100 ng/mL TPO, 20 ng/mL interleukin-3 (IL-3) (Peprotech) and 10 mM SR1 (StemCell). After pre-activation, cells (3.10.sup.6 cells/mL) were cultured in pre-activation medium supplemented with 10 μM PGE2 (Cayman Chemical) on RetroNectin coated plates (10 μg/cm2, Takara Bio) for at least 2 h. Cells (3.10.sup.6 cells/mL) were then transduced for 24 h on RetroNectin coated plates in the pre-activation medium supplemented with 10 μM PGE2, protamine sulfate (4 μg/mL, Protamine Choay) and Lentiboost (1 mg/ml, SirionBiotech).

[0085] In Vitro Erythroid Differentiation

[0086] Mature RBCs from mock- and LV-transduced CD34.sup.+ HSPCs were generated using a three-step protocol (Weber et al., 2018). Briefly, from day 0 to 6, cells were grown in a basal erythroid medium (BEM) supplemented with SCF, IL3, erythropoietin (EPO) (Eprex, Janssen-Cilag) and hydrocortisone (Sigma). From day 6 to 20, they were cultured on a layer of murine stromal MS-5 cells in BEM supplemented with EPO from day 6 to day 9 and without cytokines from day 9 to day 20. From day 13 to 20, human AB serum was added to the BEM.

[0087] Vector Copy Number Quantification by ddPCR

[0088] Genomic DNA was extracted from HUDEP-2 cells 14 days after transduction or from primary erythroid cells at day 13 of differentiation using the PureLink Genomic DNA Mini Kit (Invitrogen). DNA was digested using Dral restriction enzyme (NEB) at 37° C. for 30 min and then mixed with the ddPCR reaction mix composed of 2X ddPCR SuperMix for probes (no dUTP) (Bio-Rad), forward (for) and reverse (rev) primers (at a final concentration of 900 nM) and probes (at a final concentration of 250 nM). We used probes and primers specific for: (i) albumin (VIC-labeled ALB probe with a QSY quencher, 5′-CCTGTCATGCCCACACAAATCTCTCC-3′ (SEQ ID NO: 11); FOR ALB primer, 5′-GCTGTCATCTCTTGTGGGCTGT-3′(SEQ ID NO: 12); REV ALB primer, 5′ ACTCATGGGAGCTGCTGGTTC-3′ (SEQ ID NO: 13)), and for (ii) the LV (FAM-labeled LV probe with a MGB quencher, 5′-CGCACGGCAAGAGGCGAGG-3′ (SEQ ID NO: 14); FOR LV primer 5′-TCCCCCGCTTAATACTGACG-3′(SEQ ID NO: 15); REV LV primer 5′-CAGGACTCGGCTTGCTGAAG-3′ (SEQ ID NO: 16)). Droplets were generated using a QX200 droplet generator (Bio-Rad) with droplet generation oil for probes (Bio-Rad) onto a DG8 cartridge (Bio-Rad) and transferred on a semi-skirted 96 well plate (Eppendorf AG). After sealing with a pierce-able foil heat seal using a PX1 PCR plate sealer (Bio-Rad), the plate was loaded on a SimpliAmp Thermal Cycler (ThermoFisher Scientific) for PCR amplification using the following conditions: 95° C. for 10 min, followed by 40 cycles at 94° C. for 30 sec and 60° C. for 1 min, and by a final step at 98° C. for 10 min. The plate was analyzed using the QX200 droplet reader (Bio-Rad) (channel 1: FAM, channel 2: VIC) and analyzed using the QuantaSoft analysis software (Bio-Rad), which quantifies positive and negative droplets and calculate the starting DNA concentration using a Poisson algorithm. The VCN) per cell were calculated as (LV copies*2)/(albumin copies).

[0089] RT-qPCR Analysis

[0090] RNA was extracted from HUDEP-2 cells after 9 days of differentiation or from primary erythroid cells at day 13 of differentiation using the RNeasy micro kit (QIAGEN). Reverse transcription of mRNA was performed using the SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen) with oligo(dT).sub.20 primers. qPCR was performed using the SYBR green detection system (BioRad). We used the following primers: βAS3 FOR, 5′-GCCACCACTTTCTGATAGGCAG-3′ (SEQ ID NO: 17); βAS3 REV, 5′-AAGGGCACCTTTGCCCAG-3′ (SEQ ID NO: 18); BCL11A-XL FOR, 5′-ATGCGAGCTGTGCAACTATG-3′ (SEQ ID NO: 19); BCL11A-XL REV, 5′-GTAAACGTCCTTCCCCACCT-3′ (SEQ ID NO: 20); HBG1/2 FOR, 5′ CCTGTCCTCTGCCTCTGCC-3′ (SEQ ID NO: 21); HBG1/2 REV, 5′-GGATTGCCAAAACGGTCAC-3′ (SEQ ID NO: 22); LMNB2 FOR, 5′-AGTTCACGCCCAAGTACATC-3′ (SEQ ID NO: 23); LMNB2 REV, 5′-CTTCACAGTCCTCATGGCC-3′(SEQ ID NO: 24); HBA FOR, 5′-CGGTCAACTTCAAGCTCCTAA-3′(SEQ ID NO: 25); HBA REV, 5′-ACAGAAGCCAGGAACTTGTC-3′(SEQ ID NO: 26). The samples were analyzed with the ViiA 7 Real-Time PCR System and software (Applied Biosystems).

[0091] Flow Cytometry

[0092] After nine days of differentiation, HUDEP-2 cells were stained with a monoclonal mouse anti-human CD235a antibody (clone GA-R2, BD Biosciences), then fixed and permeabilized with the fixation/permeabilization solution kit (BD Biosciences) and stained with a monoclonal mouse anti-human HbF antibody (clone HBF-1, ThermoFisher scientific). Cells were analyzed by flow cytometry using a BD LSRFortessa cell analyzer (BD Biosciences) and the Diva (BD Biosciences) and the FlowJo softwares.

[0093] In primary cell cultures, the expression of erythroid markers was monitored by flow cytometry using anti-CD36 (BD Horizon), anti-CD71 and anti-CD235a (BD PharMingen) antibodies and the proportion of enucleated RBCs was measured using the nuclear dye DRAQ5 (eBioscience). Flow cytometry analyses were performed using the Gallios analyzer and Kaluza software (Beckman-Coulter).

[0094] HPLC

[0095] HPLC analysis was performed using a NexeraX2 SIL-30AC chromatograph (Shimadzu) and the LC Solution software. Globin chains from differentiated HUDEP-2 cells (day 9) or from primary erythroid cells (day 16 of the in vitro erythroid differentiation) were separated by HPLC using a 250×4.6 mm, 3.6 μm Aeris Widepore column (Phenomenex). Samples were eluted with a gradient mixture of solution A (water/acetonitrile/trifluoroacetic acid, 95:5:0.1) and solution B (water/acetonitrile/trifluoroacetic acid, 5:95:0.1). The absorbance was measured at 220 nm.

[0096] Hemoglobin tetramers from mature RBCs (day 16 of the in vitro erythroid differentiation) were separated by CE-HPLC using a 2 cation-exchange column (PolyCAT A, PolyLC, Columbia). Samples were eluted with a gradient mixture of solution A (20 mM bis Tris, 2 mM KCN, pH, 6.5) and solution B (20 mM bis Tris, 2 mM KCN, 250 mM NaCl, pH, 6.8). The absorbance was measured at 415 nm.

[0097] Western Blot

[0098] RBCs from day 16 of the in vitro erythroid differentiation, were lysed for 30 min at 4° C. using a lysis buffer containing: 10 mM Tris, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% SDS, 0.1% Na-deoxicholate, 140 mM NaCl (Sigma-Aldrich) and protease inhibitor cocktail (Roche-Diagnostics). Cell lysates were sonicated twice (50% amplitude, 10 sec per cycle, pulse 9 sec on/1 sec off) and underwent 3 cycles of freezing/thawing (3 min at −80° C./3 min at 37° C.). After centrifugation, the supernatant was collected and protein concentration was measured using the Pierce™ BCA Protein Assay Kit (ThermoScientific). After electrophoresis and protein transfer, γ- and α-globins were detected using the antibodies sc-21756 and sc-31110 (SantaCruz), respectively. The bands corresponding to γ- and α-globins were quantified using the Chemidoc and the Image lab Software (BioRad).

[0099] Results

[0100] Production of a Bifunctional LV for Gene Addition and Silencing

[0101] To re-express the HBG genes, we used an artificial microRNA (amiR) targeting BCL11A, described by Guda et al. (Guda et al., 2015) and Brendel et al. (Brendel et al., 2016). Briefly, this amiR is composed of the shRNA #5mod embedded in the miR-223 backbone (FIGS. 1A and 1B). This amiR targets the extra-large BCL11A isoform (BCL11A XL) responsible for HBG silencing (Liu et al., 2018; Trakarnsanga et al., 2014; Zhu et al., 2012). This strategy will avoid the potential side effects due to the silencing of other BCL11A isoforms. More precisely, the guide strand of this amiR targets the 3′ end of the coding sequence of BCL11A-XL mRNA (FIG. 1B). As in Guda's and Brendel's studies, we used the miR-223 backbone that has been extensively optimized to improve miRNA processing and reduce off-target binding by stringent strand selection (Amendola et al., 2009; Brendel et al., 2016; Guda et al., 2015).

[0102] Guda et al. (Guda et al., 2015) and Brendel et al. (Brendel et al., 2016) developed lentiviral vectors expressing an amiR targeting BCL11A to de-repress HBG. Compared to their studies, our approach is based on HBG de-repression through an amiR targeting BCL11A and the concomitant expression of the βAS3 transgene. This combined strategy will be more effective in providing therapeutic hemoglobin levels for both β-thalassemia and SCD.

[0103] Since amiR can be expressed using Pol II promoters (Amendola et al., 2009), we inserted our amiR in the second intron of the βAS3 transgene to express it under the control of the HBB promoter and 2 potent enhancers derived from the HBB locus control region (βAS3 LV; Weber et al., 2018), thus reducing potential amiR toxicity by limiting its expression to the erythroid lineage. Compared to the wild type intron of the HBB gene, βAS3 intron 2 carries a 593-bp deletion removing a region from 85 and 679 downstream of HBB exon 2. The total length of intron 2 is 257 nucleotides. The last 60 nucleotides of HBB intron 2 (which are retained in the βAS3 intron 2, nucleotides 198 to 257) are required for efficient 3′-end formation (Michael Antoniou et al., 1998).

[0104] To avoid negative effects on βAS3 RNA expression and processing (e.g. splicing and 3′end formation), we inserted the amiR between positions 85 and 86 or between 146 and 147 of the βAS3 intron 2 (βAS3-miR/int2_del and βAS3-miR/int2) because these regions are apparently not involved in RNA expression and splicing and far enough from the last 60 nucleotides to preserve 3′-end formation (FIG. 1C).

[0105] We generated 2 βAS3 LV-derived LVs containing the amiR in these two alternative positions (βAS3-miR/int2_del and βAS3-miR/int2). These LVs were tested in a human erythroid progenitor cell line (HUDEP-2; Kurita et al., 2013) and primary hematopoietic stem/progenitor cells (HSPCs) with the goal of achieving efficient BCL11A silencing without affecting βAS3 expression.

[0106] The Insertion of an amiR in βAS3 LV does not Affect Gene Transfer Efficiency

[0107] To assess the potential impact of the amiR on gene transfer efficiency, HUDEP-2 cells were transduced at increasing multiplicities of infection (MOI) with the different LV constructs: βAS3-miR/int2_del, βAS3-miR/int2 and the original LV containing only the βAS3 transgene (βAS3). Genomic DNA was extracted to measure the VCN per cell by ddPCR. Neither the insertion of the amiR, nor its position in intron 2 affected gene transfer efficiency (FIG. 2).

[0108] Bifunctional LVs Allow BCL11A-XL Silencing and βAS3 Transgene Expression

[0109] Mock- and LV-transduced HUDEP-2 cells were terminally differentiated into mature erythroblasts. We measured BCL11A-XL expression in mock- and LV-transduced HUDEP-2 cells. BCL11A-XL mRNA expression decreased in HUDEP-2 cells transduced with LVs containing the amiR (βAS3-miR/int2_del or βAS3-miR/int2) compared with control cells (mock-transduced or transduced with βAS3 LV) (FIG. 3). These results demonstrated that the amiR is expressed in the frame of the βAS3-expressing LVs and is able to reduce BCL11A-XL expression.

[0110] We then compared βAS3 transgene expression in HUDEP-2 cells transduced with βAS3-miR/int2_del, βAS3-miR/int2 and βAS3 LV. βAS3 transgene was expressed at similar levels for each LV (FIG. 4). Neither the insertion of the amiR nor its position in intron 2 affected βAS3 transgene expression.

[0111] amiR-Mediated BCL11A-XL Down-Regulation Induces HbF Re-Expression in HUDEP-2

[0112] To evaluate if BCL11A-XL silencing is associated with HBG re-activation, we measured HBG mRNA expression levels in terminally differentiated HUDEP-2. HBG expression was substantially higher in mature erythroblasts transduced with amiR-expressing LVs than in cells transduced with the βAS3 LV or in mock-transduced cells (FIG. 5). These results shows that amiR-mediated BCL11A-XL silencing leads to HBG gene re-activation.

[0113] HbF expression was analyzed by flow cytometry in mock- and LV-transduced differentiated HUDEP-2 cells. Both the percentage of HbF populations and HbF content (measured as mean fluorescence intensity) were increased in samples transduced with LVs expressing the miR targeting BCL11A (FIGS. 6A, 6B and 6C). Reverse-phase HPLC analysis of single globin chains showed increased γ-globin expression upon BCL11A-XL silencing: overall the total amount of therapeutic β-like globin chains (γ+βAS3 globins) was higher in cells transduced with amiR-expressing LVs than in βAS3-transduced cells. Importantly, we observed a decrease in the levels of the endogenous adult β-globin (β.sup.A) chains, which could further counteract RBC sickling in SCD.

[0114] Bifunctional LVs Induce HbF Re-Expression in Primary Erythroid Cells

[0115] We transduced primary adult hematopoietic stem/progenitor cells (HSPCs) derived from healthy donors (HD) with bifunctional LVs harboring the amiR against BCL11A-XL. We introduced two new control LVs containing a non-targeting (nt) in the two different positions in intron 2 of the βAS3 transgene (βAS3-miR #nt/int2 and βAS3-miR #nt/int2_del). Mock- and transduced HSPCs were terminally differentiated into mature RBCs. Flow cytometry analysis of erythroid markers showed that erythroid differentiation was not altered upon HSPC transduction with bifunctional LVs (FIGS. 7A, 7B and 7C). Similarly, the proportion of enucleated RBCs along the differentiation was comparable between control and transduced samples with no impairment of enucleation upon expression of the amiR targeting BCLIIA-XL (FIGS. 8A and 8B).

[0116] To evaluate the potential therapeutic effect of this strategy, we measured HBG mRNA expression in mock- and LV-transduced erythroid cells derived from HSPCs. HBG genes were de-repressed in cells transduced with LVs containing the amiR (βAS3-miR/int2_del or βAS3-miR/int2) compared to control cells (transduced with βAS3- or βAS3-miR #nt-LVs). Notably, we observed a 7.5-fold increase in HBG mRNA expression per VCN in cells transduced with the LV harboring the BCL11A-XL amiR in the int2 position (βAS3-miR/int2) (FIG. 9). De-repression of HBG1/2 genes was confirmed by Western Blot analysis: a 4.4-fold increase of γ-globin expression was observed in cells transduced with βAS3-miR/int2 compared to control cells transduced with βAS3-miR #nt/int2 (FIGS. 10A and 10B). γ-globin de-repression coupled with βAS3 expression resulted in a 2-fold increase in the total amount of therapeutic β-like globins and hemoglobins per VCN in RBCs derived from βAS3-miR-LV-compared to βAS-miR #ntLV-transduced HSPCs (FIGS. 11A, 11B, 11C and 11D).

CONCLUSION

[0117] Overall, these results show that LVs expressing a βAS3 transgene and an amiR targeting BCL11A-XL could induce high-level of therapeutic globins. This combined strategy will likely be more effective than a classical gene addition approach to β-hemoglobinopathies.

REFERENCES

[0118] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure. [0119] Amendola M, Passerini L, Pucci F, Gentner B, Bacchetta R, Naldini L. 2009. Regulated and Multiple miRNA and siRNA Delivery Into Primary Cells by a Lentiviral Platform. Mol Ther J Am Soc Gene Ther 17:1039-1052. doi:10.1038/mt.2009.48 [0120] Brendel C, Guda S, Renella R, Bauer D E, Canver M C, Kim Y-J, Heeney M M, Klatt D, Fogel J, Milsom M D, Orkin S H, Gregory R I, Williams D A. 2016. Lineage-specific BCL11A knockdown circumvents toxicities and reverses sickle phenotype. J Clin Invest 126:3868-3878. doi:10.1172/JCI87885 [0121] Guda S, Brendel C, Renella R, Du P, Bauer D E, Canver M C, Grenier J K, Grimson A W, Kamran S C, Thornton J, de Boer H, Root D E, Milsom M D, Orkin S H, Gregory R I, Williams D A. 2015. miRNA-embedded shRNAs for Lineage-specific BCL11A Knockdown and Hemoglobin F Induction. Mol Ther 23:1465-1474. doi:10.1038/mt.2015.113 [0122] Kurita R, Suda N, Sudo K, Miharada K, Hiroyama T, Miyoshi H, Tani K, Nakamura Y. 2013. Establishment of Immortalized Human Erythroid Progenitor Cell Lines Able to Produce Enucleated Red Blood Cells. PLOS ONE 8:e59890. doi:10.1371/journal.pone.0059890 [0123] Liu N, Hargreaves V V, Zhu Q, Kurland J V, Hong J, Kim W, Sher F, Macias-Trevino C, Rogers J M, Kurita R, Nakamura Y, Yuan G-C, Bauer D E, Xu J, Bulyk M L, Orkin S H. 2018. Direct Promoter Repression by BCL11A Controls the Fetal to Adult Hemoglobin Switch. Cell 173:430-442.e17. doi:10.1016/j.ce11.2018.03.016 [0124] Marktel S, Scaramuzza S, Cicalese M P, Giglio F, Galimberti S, Lidonnici M R, Calbi V, Assanelli A, Bernardo M E, Rossi C, Calabria A, Milani R, Gattillo S, Benedicenti F, Spinozzi G, Aprile A, Bergami A, Casiraghi M, Consiglieri G, Masera N, D'Angelo E, Mirra N, Origa R, Tartaglione I, Perrotta S, Winter R, Coppola M, Viarengo G, Santoleri L, Graziadei G, Gabaldo M, Valsecchi M G, Montini E, Naldini L, Cappellini M D, Ciceri F, Aiuti A, Ferrari G. 2019. Intrabone hematopoietic stem cell gene therapy for adult and pediatric patients affected by transfusion-dependent β-thalassemia. Nat Med 25:234-241. doi:10.1038/s41591-018-0301-6 [0125] Miccio A, Cesari R, Lotti F, Rossi C, Sanvito F, Ponzoni M, Routledge S J E, Chow C-M, Antoniou M N, Ferrari G. 2008. In vivo selection of genetically modified erythroblastic progenitors leads to long-term correction of β-thalassemia. Proc Natl Acad Sci 105:10547-10552. doi:10.1073/pnas.0711666105 [0126] Miccio A, Poletti V, Tiboni F, Rossi C, Antonelli A, Mavilio F, Ferrari G. 2011. The GATA1-H52 enhancer allows persistent and position-independent expression of a β-globin transgene. PloS One 6:e27955. doi:10.1371/journal.pone.0027955 [0127] Michael Antoniou, Geraghty F, Hurst J, Grosveld F. 1998. Efficient 3′-end formation of human β-globin mRNA in vivo requires sequences within the last intron but occurs independently of the splicing reaction 9. [0128] Trakarnsanga K, Wilson M C, Lau W, Singleton B K, Parsons S F, Sakuntanaga P, Kurita R, Nakamura Y, Anstee D J, Frayne J. 2014. Induction of adult levels of β-globin in human erythroid cells that intrinsically express embryonic or fetal globin by transduction with KLF1 and BCL11A-XL. Haematologica 99:1677-1685. doi:10.3324/haematol.2014.110155 [0129] Weber L, Poletti V, Magrin E, Antoniani C, Martin S, Bayard C, Sadek H, Felix T, Meneghini V, Antoniou M N, El-Nemer W, Mavilio F, Cavazzana M, Andre-Schmutz I, Miccio A. 2018. An Optimized Lentiviral Vector Efficiently Corrects the Human Sickle Cell Disease Phenotype. Mol Ther Methods Clin Dev 10:268-280. doi:10.1016/j.omtm.2018.07.012 [0130] Zhu X, Wang Y, Pi W, Liu H, Wickrema A, Tuan D. 2012. NF-Y recruits both transcription activator and repressor to modulate tissue- and developmental stage-specific expression of human γ-globin gene. PloS One 7:e47175. doi:10.1371/journal.pone.0047175