Compositions and methods for HBB-editing in hspc

Abstract

The present invention generally relates to the field of genome engineering (gene editing), and more specifically to gene therapy for the treatment of Sickle Cell Disease. Described herewith are ex vivo methods for preparing a population of cells enriched in human HBB-gene-edited Hematopoietic Stem and Progenitor Cells (HSPCs) with enhanced engraftment capacity and therapeutic potential suitable for use in the treatment of sickle cell disease, means to carry out these methods, the population of HBB-gene edited HSPCs produced by these methods and the use thereof in the treatment of sickle cell disease.

Description

[0001] COMPOSITIONS AND METHODS FOR HBB-EDITING IN HSPC

[0002] FIELD OF THE INVENTION

[0003] The present invention generally relates to the field of genome engineering (gene editing) of Haematopoietic Stem and Progenitor Cells (HSPCs) to correct mutations in the / - / BB-gene associated with hemoglobinopathies such as sickle cell disease, and more specifically to an improved process of production of / 788-gene-edited HSPCs suitable for use in gene therapy. The present invention provides means and methods for producing genetically modified HSPCs in an amount and state particularly adapted for gene therapy. This process involves site-specific endonucleases, such as TALE-nucleases, that specifically target, ex vivo, a non-functional endogenous / - / BB-gene comprising at least one mutation causing a hemoglobinopathy, such as sickle cell disease, in a patient, thereby allowing the restoration of the normal cellular phenotype of the red blood cells derived from the edited HSPCs. Indeed, the invention provides, among others, a method for producing a population of cells enriched in HSPCs having at least one allele of their endogenous non-functional / - / BB-gene (i.e. encoding a non-functional Hemoglobin beta subunit) that has been corrected and virtually no, or only a minimal amount of, HSPCs having two P° alleles of the / - / BB-gene. Moreover, the population of cells enriched in / - / BB-gene edited HSPCs disclosed herewith is also enriched in long-term HSCs which have an improved engraftment ability.

[0004] Thus, the present invention provides means and methods to produce said / - / BB-gene edited HSPCs, populations of cells comprising said engineered HSPCs, pharmaceutical compositions comprising said engineered HSPCs or populations of cells, as well as their use in the treatment of a hemoglobinopathy such as sickle cell disease.

[0005] BACKGROUND OF THE INVENTION

[0006] Sickle cell disease (SCD) is one of the most common inherited diseases affecting millions of people worldwide and is characterized by mutations in the / - / BB-gene encoding the p subunit of hemoglobin (Hb). The underlying pathophysiology of SCD stems from a single point mutation (A>T) in exon 1 of the HBB-gene which results in the sickle Hb allele, ps, and the formation of the P-globin S subunit (Goldstein et al., 1963, J. Biol. Chem, 238: 2016-27). Homozygous inheritance of two mutant alleles leads to Hb tetramers that are comprised of two normal a-globin subunits and two abnormal ps-globin subunits, leading to the production of Hemoglobin S ("HbS”), characterized by a Glu6Val mutation in the p-globin polypeptide, in HbSS patients. The normal adult Hemoglobin is abbreviated “HbA”.

[0007] Current treatment for SCD largely relies on the symptomatic management of acute complications (e.g., blood transfusions and exchange transfusions for severe VOC and / or ACS, pain management, and preventative strategies for life-threatening infections (e.g., prophylactic penicillin and vaccination) and stroke (transcranial doppler screening, chronic transfusion therapy).

[0008] Hydroxyurea (HU) which induces the production of Fetal Hemoglobin (“HbF”) has been the mainstay of treatment since the late 1980s. The increase in HbF reduces HbS polymerization, and subsequent sickling. Despite significant data, there have been several barriers to its use including fear of side-effects like teratogenesis, effects on fertility and possible increased risk of malignancy.

[0009] In the past few years, novel therapies targeting alternative aspects of the SCD pathophysiology have become available for patients with SCD. The use of Endari (L-glutamine) is based on the hypothesis that sickle RBCs are more susceptible to oxidant stress than normal RBCs and that supplementation with L-glutamine leads to improved transport and utilization of glutamine in sickle cells and improved cellular defenses against oxidative stress. Other therapies focus on the prevention of adhesion between platelets, red cells, monocytes and neutrophils, an interaction which constitutes an integral component of the underlying pathophysiology. Adakveo (crizanlizumab) is a humanized monoclonal antibody that blocks the activity of P-selectin and thus the cells adhesion. Oxbryta (voxelotor) is a small molecule that binds HbS and increases the oxygen affinity thereby inhibiting polymerization and subsequent RBCs damage.

[0010] Despite the number of treatments for this disease, these therapies remain symptomatic. The only available cure for patients with SCD is a Stem Cell Transplant (SCT) which is typically reserved to patients with severe disease, and who have an H LA-matched related donor, given the morbidity and mortality often observed with SCT.

[0011] Several potential products based on gene therapies are currently investigated in clinical trials that aim to improve the disease state through increasing production of Fetal Hemoglobin (“HbF”) by silencing the BCL11A gene which encodes an important repressor of the y-globin gene necessary for production of HbF. For instance, the use of autologous CD34+cells IMP transduced with BCH-BB694 lentiviral vector encoding short hairpin RNA (SHRNA) targeting BCL11A mRNA has been described by Esrick et al. (2020, N. Engl. J. Med. 384(3): 205-215). Other similar approaches currently under clinical development include the use of a Lentiviral P-A-T87Q Globin vector to avoid the formation of sickle dimers in CD34+ stem cells (Lentiglobin (Zynteglo, Bluebird bio)) or the use of CRISPR-Cas9 technology to disrupt BCL11A gene (CRISPR therapeutics and Vertex) which induces an overexpression of HbF (Frangoul et al., 2020, N. Engl. J. Med, 384(3): 252-260).

[0012] Other nuclease-based gene therapy strategies consist in exploiting homology directed repair (HDR) to correct / - / BB-gene by CRISPR-Cas9 in the presence of a DNA repair template either based on Adeno Associated Virus 6 (AAV6) (Lattanzi et al., 2021 , Sci. Transl. Med. 13(598) ; Dever etal., 2016, 539(7629): 384-389) or single-stranded oligodeoxynucleotide (ssODN) (Magis et al., 2022, iScience. 25(6): 104374 ; Park et al., 2019, Nucleic Acids Res. 47(15): 7955-7972 ; DeWitt et al., 2016, Sci. Transl. Med. 8(360)). The direct and precise HBB correction represents an alternative approach with the potential advantage to promote the endogenous expression of HbA rather than a surrogate version (HbF) harboring different physicochemical properties and oxygen affinity and to decrease the level of HbS and its negative downstream effects.

[0013] WO2019185920 relates to allele specific TALE-nucleases and methods to operate allele specific gene repair by homologous recombination in primary cells.

[0014] However, while a nuclease-mediated gene correction strategy is appealing, it is not devoid of any challenges, including safety and efficacy concerns related to the method used to vectorize the DNA repair template, the nuclease and the editing protocol used. For instance, a substantial fraction of engineered HSPCs could end up harboring inactivated, rather than corrected, HBB alleles. Such gene editing outcome, consisting in HSPCs harboring biallelic inactivated HBB, could promote the development of pathological p-thalassemic phenotype and reduce the proportion of corrected therapeutic cells in the final product.

[0015] Therefore, new gene therapy approaches enabling to precisely correct the / - / BB-gene ex vivo and rescue HbA production, without compromising the safety and therapeutic efficiency of the edited HSPCs, in particular when scaled up to GMP manufacturing conditions, are greatly needed.

[0016] SUMMARY OF THE INVENTION

[0017] The present invention addresses these needs by providing an ex vivo method for preparing a population of cells enriched in human / 788-gene-edited Hematopoietic Stem and Progenitor Cells (HSPCs) with enhanced engraftment capacity and therapeutic potential suitable for use in gene therapy in the treatment of hemoglobinopathies including sickle cell disease, as well as means to carry out this method, the population of / - / BB-gene edited HSPCs produced by this method and the use thereof in gene therapy. Also provided herewith are means and methods related to an improved gene therapy approach to treat sickle cell disease, allowing the correction of HBB deficiency in HSPCs, notably in long-term repopulating HSCs. Particularly, the present invention provides means and methods for genetically modifying HSPCs involving TALE-nucleases that specifically target a non-functional endogenous / 788-gene comprising at least one allele having a mutation causing a hemoglobinopathy (such as the mutation (A>T) at position 20 of the coding region of exon 1 that is associated with sickle cell disease) and a polynucleotide repair template. As a result, engineered / 788-edited HSPCs are provided, comprising an exogenous sequence comprising a nucleic acid sequence encoding a functional p-globin subunit, wherein said exogenous sequence is integrated in said HSPCs’ genome into a non-functional endogenous HBB locus and thus replaces and corrects the non-functional / - / BB-gene, thereby restoring the normal cellular phenotype of the red blood cells derived from the edited HSPCs by enabling the expression of a functional p-globin subunit, and, thus, Hemoglobin.

[0018] The present invention can be summarized by the following items:

[0019] 1. An ex vivo method for preparing / - / BB-gene edited Hematopoietic Stem and Progenitor Cells (HSPCs) from a population of HSPCs comprising at least one endogenous HBB- allele having a mutation (A>T) or (A>C) at position 20 of the coding region of exon 1 of / - / BB-gene, wherein said mutation is associated with a hemoglobinopathy, said method comprising the steps of: i) Introducing, into a population of HSPCs having an allele of an endogenous / - / BB-gene having said mutation, a monomeric TALE-nuclease comprising a monomer binding the HBB sequence of SEQ ID NO: 17 or SEQ ID NO: 19, or a heterodimeric TALE-nuclease comprising a first monomer binding the HBB sequence of SEQ ID NO: 17 or SEQ ID NO: 19 and a second monomer binding the HBB sequence of SEQ ID NO: 18; ii) introducing into said population of HSPCs a non-viral single-stranded polynucleotide repair template comprising the nucleic acid sequence of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16; whereby a population of / - / BB-gene edited HSPCs is obtained, which comprises HSPCs having at least one allele of the / - / BB-gene corrected at position 20 of the coding region of exon 1 of / - / BB-gene.

[0020] 2. The / 788-edited HSPCs obtainable by the method of preparation disclosed herewith.

[0021] 3. Engineered / 788-edited HSPCs comprising, integrated at the / 788-locus in said HSPCs’ genome, a polynucleotide comprising the exogenous sequence of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16, thereby allowing the expression of a functional beta-globin subunit.

[0022] 4. A method of treatment of a hemoglobinopathy associated with a mutation in at least one endogenous / 788-allele at position 20 of the coding region of exon 1 of / 788-gene in a patient, comprising administering in said patient a population of / 788-gene edited HSPCs as described herewith, isolated / 788-gene edited HSPCs as described herewith, or a pharmaceutical composition comprising said / 788-gene edited HSPCs, preferably wherein said / 788-gene edited HSPCs derive from said patient.

[0023] 5. Some aspects of the invention are applicable to other genes than HBB, thus, another item relates more generally to an ex vivo method for preparing a population of cells enriched in viable gene-edited Hematopoietic Stem and Progenitor Cells (HSPCs) from a population of HSPCs comprising a gene having a deleterious mutation, said method comprising introducing, into a population of HSPCs having the deleterious mutation:

[0024] (i) a TALE-nuclease cleaving a specific target locus; and

[0025] (ii) a non-viral circular single-stranded polynucleotide repair template; wherein the edited cells have integrated the polynucleotide repair template at the targeted locus; whereby a population of cells is obtained which comprises at least 20% of edited cells and at least 70%, at least 80%, or at least 90%, of viable cells.

[0026] The invention can be further summarized by the following particular items:

[0027] 1. An ex vivo method for preparing / 788-gene edited HSPCs from a population of HSPCs comprising at least one endogenous / 788-allele having a mutation (A>T) at position 20 of the coding region of exon 1 of / 788-gene, wherein said mutation is associated with sickle cell disease, said method comprising the steps of: i) Introducing, into a population of HSPCs having an allele of an endogenous / 788-gene having said mutation, a mRNA encoding an heterodimeric TALE-nuclease comprising a first monomer comprising the amino acid sequence of SEQ ID NO: 54 and a second monomer comprising the amino acid sequence of SEQ ID NO: 55; ii) introducing into said population of HSPCs a non-viral single-stranded DNA repair template comprising the polynucleotide sequence SEQ ID NO: 16; iii) optionally, introducing into the cells a mRNA encoding at least one HDR-enhancer comprising the amino acid sequence SEQ ID NO: 61 and a mRNA encoding at least one viability-enhancer comprising the amino acid sequence SEQ ID NO: 63; whereby a population of HBB-gene edited HSPCs is obtained, which comprises HSPCs having at least one allele of the / - / BB-gene corrected at position 20 of the coding region of exon 1 of / 788-gene.

[0028] 2. The method according to above item 1 , wherein said repair template is a circular ssDNA.

[0029] 3. The method according to any one of above items 1 and 2, wherein the mRNA encoding said TALE-nuclease and said repair template are introduced by two electroporation steps.

[0030] 4. The method according to any one of above items 1 and 2, wherein the mRNA encoding said TALE-nuclease and said repair template are introduced by one electroporation step.

[0031] 5. A population of / 788-gene edited HSPCs obtainable by the method according to any one of items 1 to 4.

[0032] 6. A population of / 788-gene edited HSPCs, wherein a polynucleotide comprising the exogenous sequence of SEQ ID NO: 13 has been integrated at the / 788-locus in the genome of at least 10%, preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, of the cells.

[0033] 7. The population of / 788-gene edited HSPCs according to item 5 or 6, wherein the HDR frequency at the / 788-locus is at least about 20%, such as at least about 25%, at least 30%, or at least about 35%, and the frequency of Indels at the / 788-locus is lower than about 30%, such as lower than about 25%, lower than about 20%, or lower than about 15%.

[0034] 8. The population of / 788-gene edited HSPCs according to any one of items 5 to 7, comprising at least 70%, such as at least about 75%, at least about 80%, of viable cells.

[0035] 9. A method of treatment of sickle cell disease associated with a mutation (A>T) at position 20 of the coding region of exon 1 of / 788-gene in at least one endogenous / 788-allele in a human patient, said method comprising administering, in said patient, a population of / 788-gene edited HSPCs according to any one of items 5 to 8, wherein said HSPCs derive from said patient.

[0036] 10. A kit for ex vivo editing at least one endogenous / 788-allele in HSPCs from a patient suffering from sickle cell disease, comprising: i) an isolated mRNA encoding a heterodimeric TALE-nuclease comprising a first monomer comprising the amino acid sequence of SEQ ID NO: 54 and a second monomer comprising the amino acid sequence of SEQ ID NO: 55;

[0037] (ii) an isolated non-viral single-stranded DNA repair template comprising the polynucleotide sequence SEQ ID NO: 16.

[0038] BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1. A. Representative illustration of the binding sites, at the / 788-locus, of a TALEN described in WQ2019185920 (“prior art TALEN-T0”) and a heterodimeric TALEN described herewith (“NEW-TALEN”), and the correction templates. B. Gene editing of / - / BB-locus in HbSS patients’ HSPCs (N=2 donors). C: Plating efficiency obtained after CFU assay (N=2 donors). D: Frequency of Hemoglobin tetramers (HbF / HbA / HbA2 / HbS) detected in BFLI-E colonies obtained from of CFU assay (N=1 donor).

[0040] Figure 2. A. Representative illustration of viral (AAV) or non-viral (ssODN) editing protocols in HSPCs and experimental design. B. Representative illustration of TALE-Nuclease’s binding sites at the / 7BB-locus on either the wild-type sequence or on the HBB mutated (”HBB ps”) sequence, and / 788-gene correction template. C. Ratio of HDR / lndels frequencies obtained with the AAV- or ssODN- mediated editing protocol.

[0041] Figure 3. Frequency of homology directed repair (HDR, black) and insertion / deletion (Indels, grey) allelic events at / - / BB-locus measured at day 4 by ddPCR in PLX HSPCs edited with TALE- Nuclease coupled to ssODN (A) or AAV (B) in presence (+) or not (-) of HDR-Enh01. C. Frequencies of viable cells evaluated at day 4 by flow cytometry in ssODN (grey) or AAV (black) edited cells in R&D or GMP compatible conditions in presence (+) or not (-) of Via-Enh01 and in presence of HDR-Enh01. D. Frequency of homology directed repair (HDR, black) and insertion / deletion (Indels, grey) allelic events at / - / BB-locus edited with TALE-Nuclease coupled to ssODN or AAV in presence (+) or absence (-) of HDR-Enh01 and / or Via-Enh01 in GMP- compatible conditions. E. Ratio of HDR / indels frequencies obtained in ssODN (grey) or AAV (black) edited HSPCs in presence of HDR-Enh01 and in presence (+) or absence (-) of Via-Enh01 in GMP-compatible conditions.

[0042] Figure 4. A. Representative illustration and experimental design of xenotransplantation of HSPCs in NCG mice. B. Human chimerism assessed as human CD45+ cells in bone marrow (BM) by flow cytometry at 16-18 weeks after injection. Untreated (black filled in), Mock following 1 or 2 electroporation (EP) protocols (dark and light greys filled in), ssODN- (light grey) and AAV- (black) edited HSPCs groups are represented. C. Frequency of HDR alleles evaluated in gDNA from hCD45+ cells obtained from BM 16-18 weeks after injection (Output) or in gDNA from HSPCs before injection (Input). Black lines represent median values (n= 18 mice edited group from a total of 3 HSPCs donors). D. Frequency of HDR allelic events in hCD45+ cells obtained from BM at 16- 18 weeks after injection, erythroid (BFU-E) and myeloid (CFU-GM) colonies derived from engrafted hCD45+ cells for ssODN (left panel) or AAV (right panel) groups. Black lines connect paired samples. Figure 5. A. Representative illustration of gene correction protocol in HbSS (“HBB ps”) HSPCs and experimental design. B. Frequency of homology directed repair (HDR, black circles) and insertion / deletion (Indels, grey circles) allelic events at / - / BB-locus in PLX HSPCs edited with ssODN protocol (left plot) or AAV protocol (right plot) in presence (+) or absence (-) of HDR-Enh01 and Via-Enh01 . C. Plots representing the frequency of myeloid (CFLI-GM), Erythroid (BFLI-E) and multipotential (CFU-GEMM) colonies formed in methylcellulose comparing untreated (white), ssODN- (grey) and AAV- (black) edited HSPCs with optimized (circle) or unoptimized (square) protocols. D. Frequency of HDR allelic events in single BFLI-E colonies for genotype assessment (P= corrected allele, ps= sickle allele, p°= Indels allele). Groups containing at least one corrected allele are considered corrected, while groups with two alleles harboring indels are considered as collateral effect. Frequencies of both subgroups are shown.

[0043] Figure 6. A. Representative illustration of gene correction protocol in HbSS (“HBB bs”) HSPCs followed by erythroid differentiation and experimental design. B. RP-HPLC quantification of globin chains in erythroid cells derived from either unedited (Mock), treated with TALE-Nuclease only (TALEN only) or TALE-Nuclease and ssODN or AAV protocols (Corrected). C. In vitro sickling assay measuring the proportion of sickled RBCs under hypoxic conditions (0% O2). Plots represent percentage of sickle shape and normal shape RBCs derived from Mock control , ssODN- and AAV- edited cells.

[0044] Figure 7. A. Representative illustration and experimental design of xenotransplantation of HSPCs in NBSGW mice. B. Human chimerism assessed as hCD45+ cells in bone marrow (BM) by flow cytometry at 16-18 weeks after injection. Mock electroporated (black), ssODN-(grey) edited HSPCs groups are represented (ns= non-significant). C. Multilineage engraftment in bone marrow (BM) assessed by flow cytometry 16-18 weeks after injection to evaluate the frequency of Lymphoid, Granulocytes, Erythroid cells or HSPCs of Mock (black) or ssODN (grey) edited HSPCs groups. D. Frequency of HDR (left plot) or Indels (right plot) alleles evaluated in gDNA from hCD45+ cells obtained from BM 16-18 weeks after injection (Output) or in gDNA from HSPCs before injection (Input).

[0045] Figure 8. A. Representative illustration of Oligo Capture Assay (OCA) to identify on-site and candidate off-sites in T-cells (n=3 donors) transfected with mRNA encoding TALEN-HBBSS. B. OCA score obtained for the on-site (HBB), as well as the first off-site candidates, are indicated. The unique off-site identified (HBD) has a score above 5. C. Representative illustration of off-sites validation assay in HbSS patients’ HSPCs. D. Frequencies of indels obtained by high-throughput DNA sequencing of TALEN-HBBSSon-site and the identified HBD off-site in HbSS patients’ HPSCs.

[0046] Figure 9. Experimental strategy A. and diagram B. to detect deletion, inversion, and translocation events at the HBB-HBD locus after editing. C. Frequencies of deletions (upper panels), inversions (middle panels), translocations (lower panels) at the HBB-HBD locus assessed in mock treated- (Grey circles) or ssODN optimized protocol treated- (white circles) HbSS patient HSPCs at day 4 (D4), day 7 (D7) of culture or 16-18 weeks in hCD45+ bone marrow engrafted cells (Vivo).

[0047] Figure 10. A. Schematic representation of the gene editing strategy for targeted integration at the B2M locus. B. Schematic representation of the protocol. C. Frequency of targeted integration (left panel) and cell viability (right panel).

[0048] Figure 11. A. Schematic representation of the gene editing strategy using circular or linear ssDNA for HA tag insertion at the B2M locus. B. schematic representation of the protocol. C. Frequency of targeted integration (black bars) and indels (white bars), D. Cell viability, E. MX1 mRNA quantification by RTqPCR in the indicated conditions tested.

[0049] Figure 12. A. Schematic representation of the gene editing strategy using circular or linear ssDNA for targeted insertion at the B2M locus. B. schematic representation of the protocol. C. Frequency of targeted integration, D. Cell viability, E. Plating efficiency (CFU Assay), and F. MX1 mRNA quantification by RTqPCR or G. P21 mRNA quantification by RTqPCR, in the indicated conditions tested.

[0050] Figure 13. A. Schematic representation of the gene editing strategy using 1 or 2 electroporation steps. For the sake of readability, mRNA encoding Via-Enh01 and HDR-Enh01 , electroporated during the first transfection are not displayed in these two schematics. B. Frequency of targeted integration, C. Cell viability, D. Plating efficiency (CFU Assay), and E. MX1 mRNA quantification by RTqPCR or F. P21 mRNA quantification by RTqPCR, in the conditions indicated.

[0051] Figure 14. A. Schematic representation of HSPCs editing protocol using an mRNA-encoded TALEN targeting the B2M locus and CssDNAI or AAV as DNA donor templates to insert a reported gene (“CSR2”) (2.2 kb) via disruptive insertion. mRNAs encoding Via-Enh01 and HDR- Enh01 were also incorporated in the process. The timing is indicated in days (D0-D4). Edited HSPCs retrieved 4 days post thawing were characterized by flow cytometry to assess the level of knock-in (KI) of DNA donor templates and knock-out (KO) of B2M as well as their viability. Their differentiation capacity into erythroid and myeloid progenitors as well as their transcriptomics profile were assessed by colony forming unit (CFU) assay and CITE-seq, respectively. Edited HSPCs retrieved 4 days post thawing were injected in NCG mice to assess their ability to engraft, differentiate and keep their editing events, 16 weeks after injection onset. B. In vitro experimental results illustrating the frequency of cells harboring KI events, the ratio KI / KO, the viability, the differentiation and proliferation capacities of HSPCs either untreated, edited with TALEN only (TALEN), or edited with TALEN and CssDNA or AAV donor templates (CssDNA or AAV, respectively). Results from 3 donors (circle: donor 1 , square: donor 2, triangle: donor 3). C. In vivo experimental results illustrating the level of human CD45+ cells (hCD45) engraftment and of KI frequencies and KI / KO ratio determined either before mice injection (input), or in hCD45+ cells engrafted in the bone marrow (BM) of NCG mice, 16 weeks after cells injection onset (output). The product of the frequency of hCD45+ cells engraftment and frequency of KI is also shown to illustrate the overall efficiency of each protocol of HSPC editing. On each box plot, the central mark indicates the median, the bottom and top edges of the box indicate the interquartile range (IQR), and the whiskers represent the maximum and minimum data point. Each dot represents data obtained from one HSPCs donor. D. Frequency of HSC-enriched subpopulations (“HSCe”) within total cells, and frequency of KI and KO within the HSC-enriched subpopulations, respectively.

[0052] Detailed description

[0053] For the purpose of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with the usage of that word in any other document, including any document incorporated herein by reference, the definition set forth below shall always control for purposes of interpreting this specification and its associated claims unless a contrary meaning is clearly intended (for example in the document where the term is originally used). The use of “or” means “and / or” unless stated otherwise. As used in the specification and claims, the singular form “a,” “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes a plurality of cells, including mixtures thereof. The use of “comprise,” “comprises,” “comprising,” “include,” “includes,” and “including” are interchangeable and not intended to be limiting. Furthermore, where the description of one or more embodiments uses the term “comprising,” those skilled in the art would understand that, in some specific instances, the embodiment or embodiments can be alternatively described using the language “consisting essentially of” and / or “consisting of”. As used herein, the term “about” means plus or minus 10% of the numerical value of the number with which it is being used.

[0054] Where a numerical limit or range is stated herein, the endpoints are included. Also, all values and subranges within a numerical limit or range are specifically included as if explicitly written out.

[0055] All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, with suitable methods and materials being described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will prevail. Further, the materials, methods, and examples are illustrative only and are not intended to be limiting, unless otherwise specified.

[0056] The practice of the present invention will employ, unless otherwise indicated, techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, gene editing, and immunology, which belong to the knowledge of the skilled in the art. Such techniques are explained fully in the literature. See, for example, Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and son Inc, Library of Congress, USA); Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrook et al, 2001 , Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic Acid Hybridization (B. D. Harries & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the series, Methods In ENZYMOLOGY (J. Abelson and M. Simon, eds. -in-chief, Academic Press, Inc., New York), specifically, Vols.154 and 155 (Wu et al. eds.) and Vol. 185, "Gene Expression Technology" (D. Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes l-IV (D. M. Weir and C. C. Blackwell, eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

[0057] Unless specifically defined herein, all technical and scientific terms used have the same meaning as commonly understood by a skilled artisan in the fields of gene therapy, biochemistry, genetics, immunology, cancer, molecular biology, and gene editing. Definitions of common terms in molecular biology may be found, for example, in Benjamin Lewin, Genes VII, published by Oxford University Press, 2000 (ISBN 019879276X); Kendrew et al. (eds.); The Encyclopedia of Molecular Biology, published by Blackwell Publishers, 1994 (ISBN 0632021829); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by Wiley, John & Sons, Inc., 1995 (ISBN 0471186341).

[0058] As used herein, a "donor" is a human from which one or more cells are isolated prior to administration of the cells, or progeny thereof, into a recipient. The one or more cells may be, e.g. a population of HSPCs, hematopoietic stem cells and / or hematopoietic progenitor cells to be engineered, expanded, enriched, or maintained according to the methods described herewith prior to administration of the cells or the progeny thereof into a recipient. In the autologous setting contemplated herewith, a “donor” is the patient to be treated with the engineered cells.

[0059] As used herein, a "recipient" is a patient that receives a transplant, such as a transplant containing a population of engineered cells, e.g. HSPCs. The transplanted cells administered to a recipient may be, e.g. autologous, syngeneic, or allogeneic cells. The invention applies in particular to transplantation of autologous engineered cells.

[0060] The term "subject" or “patient” as used herein means a human suffering from a disease associated with a deleterious mutation in a gene. As a result of said deleterious mutation, the subject’s cells express a non-functional gene. More specifically, a subject is a human suffering from a hemoglobinopathy related to a mutation in the / - / BB-gene. Patients who are homozygous / 7BB-(A>T) / / 7BB-(A>T) (“HBB-SS”) and producing beta-globin S (ps), homozygous HBB- (A>C) / / 7BB-(A>C) (“HBB-CC”) and producing beta-globin C (Pc), or heterozygous HBB- (A>T) / / 7BB-(A>C) (“HBB-SC”) and producing beta-globin S and beta-globin C (pspc) may benefit from a treatment comprising administering the / - / BB-gene edited HSPCs described herewith.

[0061] "Expansion" in the context of cells refers to the increase in the number of a characteristic cell type, or cell types, from an initial cell population of cells, which may or may not be identical. The initial cells used for expansion may not be the same as the cells generated from expansion.

[0062] "Cell population" includes eukaryotic cells, such as mammalian, e.g. human, cells isolated from biological sources, for example, blood product or tissues. A cell population can derive from more than one cell.

[0063] As used herein, "nucleic acid" or "polynucleotides" refers to nucleotides and / or polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments generated by the polymerase chain reaction (PCR), and fragments generated by any of ligation, scission, endonuclease action, and exonuclease action. Nucleic acid molecules can be composed of monomers that are naturally occurring nucleotides (such as DNA and RNA), or analogs of naturally occurring nucleotides (e.g. enantiomeric forms of naturally-occurring nucleotides), or a combination of both. Modified nucleotides can have alterations in sugar moieties and / or in pyrimidine or purine base moieties. Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azido groups, or sugars can be functionalized as ethers or esters. Moreover, the entire sugar moiety can be replaced with sterically and electronically similar structures, such as aza-sugars and carbocyclic sugar analogs. Examples of modifications in a base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substitutes. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Nucleic acids can be either single-stranded or double-stranded. To improve stability of the nucleic acids, phosphorothioate bonds can be introduced between any of the last 3-5 nucleotides at the 5'- or 3'-end of an oligonucleotide to inhibit exonuclease degradation. For instance, singlestranded nucleic acids can have phosphorothioate modifications on the first two nucleotides situated at the 5’ and 3’ ends of the nucleic acid.

[0064] By "vector" is meant a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. A "vector" can include, but is not limited to, a viral vector, a plasmid, an oligonucleotide, a RNA vector or a linear or circular DNA or RNA molecule which may consist of a chromosomal, non-chromosomal, semisynthetic or synthetic nucleic acids. Examples of vectors are those capable of autonomous replication (episomal vector) and / or expression of nucleic acids to which they are linked (expression vectors). Large numbers of suitable vectors are known to those of skill in the art and commercially available. Viral vectors include retrovirus, adenovirus, parvovirus (e.g. adeno-associated viruses (AAV), coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g. influenza virus), rhabdovirus (e.g. rabies and vesicular stomatitis virus), paramyxovirus (e.g. measles and Sendai), positive strand RNA viruses such as picornavirus and alphavirus, and double-stranded DNA viruses including adenovirus, herpesvirus (e.g. Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g. vaccinia, fowlpox and canarypox). Other viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example. Examples of retroviruses include avian leukosis-sarcoma, mammalian C-type, B-type viruses, D type viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B. N. Fields, et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996). Examples of vectors also include mRNA and circular RNA. Circular RNA can be synthesized, for instance, via the synthesis of one or multiple precursor linear RNA, followed by RNA circularization mediated by chemical or enzymatical ligation (Petkovic and Muller, 2018, Methods Mol. Biol. 1724: 167-180). Other examples of vectors include single-stranded DNAs (e.g. single-stranded oligodeoxynucleotides) and, in particular, single-stranded circular DNA.

[0065] The terms "polypeptide," "peptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues. The term also applies to amino acid polymers in which one or more amino acids are chemical analogues or modified derivatives of corresponding naturally occurring amino acids.

[0066] "Sequence identity" refers to the identity between two nucleic acid molecules or polypeptides. It refers to the residues in the two sequences which are the same when the sequences are aligned for maximum correspondence. When a position in the compared sequence is occupied by the same base (or amino acid), then the molecules are identical at that position. A degree of identity between nucleic acid sequences (or amino acid sequences) is a function of the number of identical or matching nucleotides (or amino acids) at positions shared by the aligned nucleic acid sequences (or amino acid sequences). Various alignment algorithms and / or programs may be used to calculate the identity between two sequences, including FASTA, or BLAST which are available as a part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), and can be used with, e.g. default setting. For example, polypeptides having at least 70%, 80%, 85%, 90%, 95%, 98% or 99% identity to specific polypeptides described herein and exhibiting substantially the same functions, as well as polynucleotide encoding such polypeptides, are contemplated.

[0067] By “gene” is meant the basic unit of heredity, consisting of a segment of DNA arranged in a linear manner along a chromosome, which codes for a specific protein or segment of protein. A gene typically includes a promoter, a 5' untranslated region, one or more coding sequences (exons), optionally introns, a 3' untranslated region. The gene may further comprise a terminator, enhancers and / or silencers.

[0068] The “Hemoglobin subunit Beta gene” (“ / - / BB-gene”) encodes the beta-globin subunit of Hemoglobin. This term covers the human / - / BB-gene (NCBI Gene ID: NG_059281.1) encoding the human beta-globin subunit, also called Hemoglobin beta subunit. Human / - / BB-gene spans from position 5,225,464 to position 5,227,071 on chromosome 11 (Reference Genome: GRCh38 / hg38). The cDNA sequence of the wild-type human / - / BB-gene has the nucleotide sequence of SEQ ID NO: 4. The functional beta-globin subunit encoded by wild-type HBB-gene generally has the amino acid sequence SEQ ID NO: 51 (NCBI reference NP_000509.1). In the present disclosure, mutations altering the function of / - / BB-gene, and thus, associated with a hemoglobinopathy, are generally within exon 1 of the / - / BB-gene. Exon 1 of human wild-type HBB- gene generally comprises the coding region of nucleic acid sequence SEQ ID NO: 1. An example of mutations altering the function of / - / BB-gene includes the mutation (A>T) at position 20 relative to the coding region of exon 1 of the wild-type / - / BB-gene, leading to SEQ ID NO: 2. Said (A>T) mutation in exon 1 of / - / BB-gene results in a mutated cDNA sequence of SEQ ID NO: 5 causing a Glu6Val mutation in the beta-globin subunit (Ps), which in turn results in a mutated betaS-globin subunit of SEQ ID NO: 52 that is associated with sickle cell disease. This (A>T) mutation at position 20 of the coding region of exon 1 of / - / BB-gene corresponds to a mutation at position 5,227,002 on chromosome 11. Another example of mutations altering the function of / - / BB-gene is the mutation (A>C) at position 20 of the coding region of exon 1 of / - / BB-gene, leading to SEQ ID NO: 3. Said (A>C) mutation in exon 1 of / - / BB-gene results in a mutated cDNA sequence of SEQ ID NO: 6 causing a Glu6Lys mutation in the beta-globin subunit (Pc), which in turn results in a mutated betaC-globin subunit of SEQ ID NO: 53 that is associated with Hemoglobin C disease. This (A>C) mutation in exon 1 of / - / BB-gene corresponds to a mutation at position 5,227,002 on chromosome 11.

[0069] Patients who are homozygous HBB- / 3S / HBB-I3S, homozygous HBB-pc / HBB-pc, or heterozygous HBB- / 3S / HBB- / 3Cmay benefit from a treatment based on the administration of HBB- gene edited HSPCs as described herewith.

[0070] As used herein, the term “locus” is the specific physical location of a DNA sequence (e.g. of a gene, such as the / - / BB-gene) in a genome. The term “locus” can refer to the specific physical location of a rare-cutting endonuclease target sequence on a chromosome. Such a locus can comprise a target sequence that is recognized and / or cleaved by a sequence-specific endonuclease as described herewith. It is understood that the locus of interest can not only qualify a nucleic acid sequence that exists in the main body of genetic material (i.e. in a chromosome) of a cell but also a portion of genetic material that can exist independently of said main body of genetic material such as plasmids, episomes, virus, transposons or in organelles such as mitochondria as non-limiting examples.

[0071] By “DNA target”, “DNA target sequence”, “target DNA sequence”, “nucleic acid target sequence”, or “target sequence” it is intended a polynucleotide sequence that can be targeted and processed by a sequence-specific endonuclease as described herewith. These terms refer to a specific DNA location, preferably a genomic location in a cell, but also a portion of genetic material that can exist independently of the main body of genetic material such as plasmids, episomes, virus, transposons or in organelles such as mitochondria as non-limiting example. The target sequence is defined by the 5’ to 3’ sequence of one strand of said target. Generally, the target sequence is adjacent or in the proximity of the locus to be processed either upstream (5’ location) or downstream (3’ location). In a preferred embodiment, the target sequences and the proteins are designed in order to have said locus to be processed located between two such target sequences. Depending on the catalytic domains of the endonucleases, the target sequences may be distant from 5 to 50 bases (bp), preferably from 10 to 40 bp, more preferably from 15 to 30, even more preferably from 15 to 25 bp. These later distances define the spacer referred to in the description and the examples. It can also define the distance between the target sequence and the nucleic acid sequence being processed by the catalytic domain on the same molecule.

[0072] As used herein, “exogenous sequence” generally refers to any nucleic acid sequence that was not initially present at the selected locus. By opposition “endogenous sequence” means a cell genomic sequence initially present at a locus. An “exogenous sequence” is thus a foreign sequence introduced into the cell, and thus allows distinguishing engineered cells over sister cells that have not integrated this exogenous sequence at the locus. An exogenous polynucleotide generally has an exogenous sequence.

[0073] By “sequence-specific nuclease” it is meant any active molecule that has the ability to specifically recognize a selected polynucleotide sequence in a genomic locus, preferably of at least 9 bp, more preferably of at least 10 bp and even more preferably of at least 12 bp in length, and that catalyzes the breakage of the covalent backbone of a polynucleotide. Non-limiting examples of a “sequence-specific nuclease” according to the invention include reagents that have nickase or endonuclease activity. The sequence-specific nuclease can be a chimeric polypeptide comprising a DNA binding domain and another domain displaying catalytic activity. Such catalytic activity can be for instance an endonuclease cleaving both strands of the DNA to perform gene inactivation, or nickase or double nickase to preferentially perform gene insertion by creating cohesive ends to facilitate gene integration by homologous recombination, or to perform base editing as described in Komor et al. (2016) Nature 19;533(7603):420-4.

[0074] The term “endonuclease” generally refers to any wild-type or variant enzyme capable of catalyzing the hydrolysis (cleavage) of bonds between nucleic acids within a DNA or RNA molecule, preferably a DNA molecule. Endonucleases do not cleave the DNA or RNA molecule irrespective of its sequence but recognize and cleave the DNA or RNA molecule at specific polynucleotide sequences, further referred to as “target sequences” or “target sites”. Endonucleases can be classified as rare-cutting endonucleases when having typically a polynucleotide recognition site greater than 10 base pairs (bp) in length, more preferably of 14-55 bp. Rare-cutting endonucleases significantly increase homologous recombination by inducing DNA double-strand breaks (DSBs) at a defined locus thereby allowing gene repair or gene insertion therapies (Pingoud, A. and G. H. Silva (2007). Precision genome surgery. Nat. Biotechnol. 25(7): 743-4.). Endonucleases include nucleases comprising a DNA binding domain derived from an Artificial transcription-activator-like effector (TALE) fused to a catalytic domain (herewith called “TALE-nucleases”). Depending on the catalytic domain, a TALE-nuclease may be active as a monomer or as a heterodimer.

[0075] “TALE” stands for “Artificial transcription-activator-like effector”. TALEs form a special class of proteins that can bind DNA originally derived from the phytopathogenic bacterial genus Xanthomonas 15 (Kay S. et al. (2007) Science 318: 648-651). Artificial TALE proteins have emerged to be versatile and sequence specific gene tools offering flexible applications upon elucidation of a DNA recognition ‘code’, linking the amino-acid sequence of the TALE with its bound genomic DNA sequence (Moscou J.M. et al. (2009) Science. 326:1501). TALE binding is driven by a series of 33 to 35 amino-acid-long repeats that differ at essentially two positions, the so-called repeat variable dipeptide (RVD). Each base of one strand in the DNA target is contacted by a single repeat, with predictable specificity resulting from the linear arrangement of RVDs. The biochemical structure-function studies suggest that the amino acid present at position 13 uniquely identifies a nucleotide on the DNA target major groove (Deng et al. (2012) Science 335:720-723; Stella et al. (2013) Acta Crystallogr Sect. D. Bio. I Crystallogr. 69(9):1707-1716). This DNA-protein interaction unit is stabilized by the amino acid at position 12. For the creation of TALEs with variable precision and binding affinity, six conventional RVDs are generally used (NG, HD, Nl, NK, NH, and NN). HD and NG are associated with cytosine (C) and thymine (T) respectively. NN is a degenerate RVD showing binding affinity for both guanine (G) and adenine (A), but its specificity for guanine is reported to be stronger. RVD Nl binds with A and NK binds with G. It is worth noting that the binding affinity of TALE is influenced by the methylation status of the target DNA sequence (Streubel et al. (2012) Nat Biotechnol 30(7): 593-595). Methylated cytosine is not efficiently bound by the canonical RVDs. However, they can be accommodated by a certain degree of degeneracy in TALEs as described by Valton J, et al. (2012, J. Biol. Chem. 287(46): 38427-38432). This code was adopted to effectively engineer TALE DNA-binding scaffold specificity via modular assembly in order to form different associations of TALE proteins with various enzymatic domains, such 10 as transcriptional activators, repressors, base editors or nucleases with potential ability to act on genomic sequences (Voytas et al. (2011) Science. 333(6051): 1843-6).

[0076] The term "cleavage" when used in reference to nucleic acids refers to the breakage of the covalent backbone of a polynucleotide. Cleavage can be initiated by a variety of methods including, but not limited to, enzymatic or chemical hydrolysis of a phosphodiester bond. Both single-stranded cleavage and double-stranded cleavage are possible, and double-stranded cleavage can occur as a result of two distinct single-stranded cleavage events. Double stranded DNA, RNA, or DNA / RNA hybrid cleavage can result in the production of either blunt ends or staggered ends (also called sticky or cohesive ends).

[0077] “Hematopoietic Stem and / or Progenitor Cells (HSPCs)” refers to Hematopoietic Stem Cells (HSCs) and Hematopoietic Progenitor Cells (HPCs). Hematopoietic stem cells (HSCs) maintain and replenish all blood cell types in the bone marrow and respond to changing needs for blood cells in peripheral tissues. HSCs give rise to multipotent (produce most blood cell subsets), oligopotent (lymphoid or myeloid restricted), and unipotent Hematopoietic Progenitor Cells (HPCs). A subset of HPCs proliferates into a single set of mature blood cells.

[0078] By “hematopoietic stem cells” (“HSCs”), it is meant multipotent stem cells derived from the bone marrow that have the capacity to self-renew and the unique ability to differentiate into all of the different cell types and tissues of the myeloid or lymphoid cell lineages, including but not limited to, granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, erythrocytes), thrombocytes (e.g., megakaryoblasts, platelet producing megakaryocytes, platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts, and lymphocytes (e.g., NK cells, B-cells and T-cells). It is known in the art that such cells may or may not include CD34+ cells. CD34+ cells are immature cells that express the CD34 cell surface marker. In humans, CD34+ cells are believed to include a subpopulation of cells with the stem cell properties defined above, whereas in mice, HSCs are CD34-. In addition, HSC also refers to long term repopulating HSC (LT-HSC) and short-term repopulating HSC (ST- HSC). LT-HSC and ST-HSC are distinguished based on functional potential and on cell surface marker expression. For example, in some embodiments, human LT-HSCs are CD34+, CD38-, CD45RA-, CD90+, and CD133+, and ST-HSCs are CD34+, CD38-, CD45RA-, CD90-, and CD133-. In addition, ST-HSCs are less quiescent (i.e., more active) and more proliferative than LT-HSCs under homeostatic conditions. However, LT-HSCs have greater self-renewal potential (i.e., they survive throughout adulthood, and can be serially transplanted through successive recipients), whereas ST-HSCs have limited self-renewal (i.e., they survive for only a limited period of time, and do not possess serial transplantation potential). Any of these HSCs can be used in any of the methods described herein. In some embodiments, ST-HSC are useful because they are highly proliferative and thus, can more quickly give rise to differentiated progeny.

[0079] By “long term repopulating HSCs” or “LT-HSCs” it is meant a type of hematopoietic stem cells capable of maintaining self-renewal and multilineage differentiation potential throughout life. Phenotype markers characteristic for LT-HSCs include, but are not limited to, CD34+, CD38-, CD45RA-, CD90+, and CD133+.

[0080] By “primary cell” or “primary cells” it is meant cells taken directly from living tissue (e.g. biopsy material or blood sample) and established for growth in vitro for a limited amount of time, meaning that they can undergo a limited number of population doublings. Primary cells are opposed to continuous tumorigenic or artificially immortalized cell lines. Non-limiting examples of such cell lines are CHO-K1 cells; HEK293 cells; Caco2 cells; U2-OS cells; NIH 3T3 cells; NSO cells; SP2 cells; CHO-S cells; DG44 cells; K-562 cells, U-937 cells; MRC5 cells; IMR90 cells; Jurkat cells; HepG2 cells; HeLa cells; HT-1080 cells; HCT-116 cells; Hu-h7 cells; Huvec cells; Molt 4 cells.

[0081] By “originating from a patient” it is meant that a cell or cells, such as HSPCs, have been obtained from a patient’s tissue sample, e.g. from a patient suffering from a hemoglobinopathy associated with a mutation in / - / BB-gene. In general, cells are provided from patients through a variety of methods known in the art, as for instance by leukapheresis techniques as reviewed by Schwartz et al. (J. Clin. Apher., 2013, 28(3): 145-284). HSPCs can be taken from bone marrow, and more particularly from the pelvis, at the iliac crest, using a needle or syringe. Alternatively, HSPCs may be harvested from the circulating peripheral blood, while blood donors are injected with a HSPC mobilizing agent, such as granulocyte-colony stimulating factor (G-CSF) and / or plerixafor, or CXCL2, that induces cells to leave the bone marrow and circulate in the blood vessels. HSPCs may also be harvested from cord blood. HSPCs may also be obtained from induced pluripotent stem (iPS) cells derived from the patient.

[0082] As used herein, the terms "treat," "treatment," "treating," and the like, refer to obtaining a desired pharmacologic and / or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or may be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease. "Treatment," as used herein, covers any treatment of a disease in a mammal (e.g. a human), and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, / .e., arresting its development; and (c) relieving the disease, e.g. causing regression of the disease, e.g. to completely or partially remove symptoms of the disease.

[0083] As used herein, the term "pharmaceutical composition" refers to the active ingredient of interest in combination with a pharmaceutically acceptable carrier and / or excipient e.g. a carrier and / or excipient commonly used in the pharmaceutical industry. The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of mammals, such as human beings, without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0084] As used herein, the term "administering," refers to the placement of a compound, cell, or population of cells as disclosed herein into a subject by a method or route which results in at least partial delivery of the agent at a desired site. Pharmaceutical compositions comprising the compounds or cells disclosed herein can be administered by any appropriate route which results in an effective treatment in the patient. The patient who can be treated with the materials and methods disclosed herewith can be a mammal, including a human and a non-human primate.

[0085] An "effective amount" or "therapeutically effective amount" refers to that amount of a composition described herein which, when administered to a subject (e.g. human), is sufficient to aid in treating a disease. The amount of a composition that constitutes a "therapeutically effective amount" will vary depending on the cell preparations, the condition and its severity, the manner of administration, and the age of the subject to be treated, but can be determined routinely by one of ordinary skill in the art having regard to his own knowledge and to this disclosure. When referring to an individual active ingredient or composition, administered alone, a therapeutically effective dose refers to that ingredient or composition alone. When referring to a combination, a therapeutically effective dose refers to combined amounts of the active ingredients, compositions or both that result in the therapeutic effect, whether administered concurrently, simultaneously, or sequentially.

[0086] Disclosed herewith is an improved gene therapy approach to treat hemoglobinopathies, including sickle cell disease, related to a mutation in the / - / BB-gene, allowing the correction of HBB deficiency in HSPCs, notably in long-term repopulating HSCs. Particularly, the present invention provides means and methods for genetically modifying HSPCs involving sequence-specific nucleases, such as TALE-nucleases, that specifically target a non-functional endogenous HBB- gene comprising at least one allele having a mutation causing an hemoglobinopathy, such as sickle cell disease. As a result, engineered / - / BB-edited HSPCs are provided, which comprise an exogenous polynucleotide sequence comprising a nucleic acid sequence encoding a functional hemoglobin beta subunit that is integrated in said HSPCs’ genome into an originally non-functional endogenous / 788-gene, thereby restoring the normal cellular phenotype by enabling the expression of a functional Hemoglobin.

[0087] With “non-functional endogenous / 788-gene”, it is meant at least one allele of an endogenous / 788-gene, which contains one or more mutations causing a red blood cell disorder such as sickle cell disease and Hemoglobin beta C disease. In general, a non-functional endogenous / 788-gene refers to at least one allele of the / 788-gene that comprises the mutation (A>T) at position 20 of the coding region of exon 1 of the / 788-gene. A non-functional endogenous / 788-gene can also refer to at least one allele of the / 788-gene that comprises the mutation (A>C) at position 20 of the coding region of exon 1 of the / 788-gene. The nomenclature “(A>T)” or“(A>C)” indicates that the correct functional base (A) is replaced by a mutated non-functional (T or C) base.

[0088] Generally, once integrated in the endogenous / 788-locus, said exogenous sequence allows the expression of a functional Hemoglobin beta subunit, that can, in turn, form a functional adult Hemoglobin after association with a functional Hemoglobin alpha subunit.

[0089] The present invention aims at editing (i.e. correcting) mutations in the / 788-gene causing red blood cell disorders, also called hemoglobinopathies. In particular, the present invention aims at editing (i.e. correcting) the mutation (A>T) or (A>C) present at position 20 of the coding region of exon 1 of the endogenous / 788-gene in HSPCs, notably in HSPCs originating from a patient suffering from sickle cell disease or Hemoglobin peta C disease.

[0090] As shown in the examples, the present inventors have demonstrated that the combined use of the specific TALE-nucleases and polynucleotide repair templates described herewith allows an efficient and specific correction of / 788-mutations in HSPCs, with virtually no or limited off- target activity. They have made several independent surprising observations. For instance, they have surprisingly found that carrying out the / 788-gene editing in presence of a Homology Directed Repair-enhancer and / or viability-enhancer further improves the efficiency of production of the edited HSPCs in a state that is compatible with their possible use in the treatment of a hemoglobinopathy, e.g. in a state associated with an increased engraftment potential. Also surprising is the observation that the engraftment of / 788-edited HSPCs produced according to the methods described herewith, in GMP conditions, is higher when the repair template is a non- viral single-stranded DNA rather than an AAV. Still more surprising is that the correction of the / 788-mutations and engraftment of the / 788-edited HSPCs are further improved when the geneediting process uses a single-stranded DNA repair template in a circular form. 7. Means and methods for preparing HBB-gene edited Hematopoietic Stem and Progenitor Cells (HSPCs) with enhanced engraftment capacity and therapeutic potential

[0091] In one aspect, it is provided an ex vivo method for preparing HBB-gene edited Hematopoietic Stem and Progenitor Cells (HSPCs) from a population of HSPCs comprising at least one endogenous HBB-allele having a mutation (A>T) or (A>C) at position 20 of the coding region of exon 1 of / - / BB-gene, wherein said mutation is associated with a hemoglobinopathy, said method comprising the steps of: i) Introducing, into a population of HSPCs having an allele of an endogenous / - / BB-gene having said mutation, a monomeric TALE-nuclease comprising a monomer binding the HBB sequence of SEQ ID NO: 17 or SEQ ID NO: 19, or a heterodimeric TALE-nuclease comprising a first monomer binding the HBB sequence of SEQ ID NO: 17 or SEQ ID NO: 19 and a second monomer binding the HBB sequence of SEQ ID NO: 18; ii) introducing into said population of HSPCs a non-viral single-stranded polynucleotide repair template comprising the nucleic acid sequence of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16; whereby a population of / - / BB-gene edited HSPCs is obtained, which comprises HSPCs having at least one allele of the / - / BB-gene corrected at position 20 of the coding region of exon 1 of HBB-gene.

[0092] 1.1. HSPCs to be gene edited

[0093] The Hematopoietic Stem and Progenitor Cells (HSPCs) comprising a mutation in the HBB- gene to be edited according to the invention may be primary cells. Primary cells are generally used in cell therapy as they are deemed more functional and less tumorigenic. In general, primary HSPCs can be obtained from a patient suffering from a disorder associated with the expression of a non-functional gene in HSPCs, or the lack of expression of a functional gene in HSPCs, or in cells derived therefrom, through a variety of methods known in the art. For example, primary HSPCs can be taken from bone marrow, and more particularly from the pelvis, at the iliac crest, using a needle or syringe. Alternatively, HSPCs may be harvested from the circulating peripheral blood, while blood donors are injected with a HSPC mobilizing agent, such as chemokine (C-X-C motif) receptor 4 (CXCR4) antagonists, such as AMD3100 (also known as Plerixafor and MOZOBIL (Genzyme, Boston, Mass.)), granulocyte-colony stimulating factor (G-CSF), and chemokine (C-X-C motif) ligand 2 (CXCL2, also referred to as GROP), which induces cells to leave the bone marrow and circulate in the blood vessels. HSPCs may also be harvested from cord blood.

[0094] According to some embodiments, the HSPCs are mammalian HSPCs, and preferably human HSPCs.

[0095] More particularly, the HSPCs are human HSPCs from a patient suffering from a disorder associated with the expression of a non-functional / - / BB-gene in his / her HSPCs, or the lack of expression of a functional gene in his / her HSPCs, or in cells derived therefrom.

[0096] In some embodiments, at least one allele of said non-functional / - / BB-gene has a mutation (A>T) at position 20 of the coding region of exon 1 of the / - / BB-gene, said mutation being associated with sickle cell disease. According to some embodiments, the HSPCs are isolated from sickle cell disease patients having a HBB- / 3S / HBB- / 3Sgenotype (“HbSS patients”).

[0097] In some embodiments, at least one allele of said non-functional / - / BB-gene has a mutation (A>C) at position 20 of the coding region of exon 1 of the / - / BB-gene, said mutation being associated with Hemoglobin beta C disease. According to some embodiments, the HSPCs are isolated from patients suffering from Hemoglobin beta C disease, said patients having a HBB- / 3C / HBB-I3Cgenotype (“HbCC patients”).

[0098] According to other embodiments, the HSPCs are isolated from patients suffering from severe hemoglobinopathies associated with the mutation (A>T) in one allele and the mutation (A>C) in another allele of the non-functional endogenous / - / BB-gene, said patients having a HBB- / 3S / HBB- / 3Cgenotype (“HbSC patients”).

[0099] According to some embodiments, the HSPCs comprising at least one endogenous HBB- allele having a mutation associated with a hemoglobinopathy, which are edited according to the method of the invention, are G-CSF-mobilized or plerixafor-mobilized HSPCs from sickle cell disease patients presenting a ps / psgenotype (HbSS patients).

[0100] The HSPCs to be edited according to the method of the invention can be “fresh” cells, meaning that they have been freshly harvested from an individual as described above, without having been frozen and stored in liquid nitrogen before use. Alternatively, the HSPCs to be edited according to the method of the invention can have been frozen and stored in liquid nitrogen until use. Frozen HSPCs are thawed in appropriate conditions before being used in the method of the invention. Techniques to freeze (or “cryopreserve”) and thaw HSPCs without altering their viability are well known to the one skilled in the art. HSPCs can be cryopreserved in a medium containing fetal bovine serum (FBS) and 10% dimethyl sulfoxide (DMSO) in liquid nitrogen. 7.2. HBB-gene correction in the HSPCs

[0101] The HSPCs comprising at least one endogenous HBB-allele having a mutation (A>T) or (A>C) at position 20 of the coding region of exon 1 of HBB-gene, which are edited according to the methods described herewith, have their originally mutated / 788-gene corrected as a result of DNA Homology Directed Repair (HDR) mechanism induced by the combined actions of a TALE- nuclease (an engineered nuclease optimized to cleave the mutated HBB-gene) and a polynucleotide repair template.

[0102] 7.2.7. TALE-nuclease

[0103] TALE-nucleases used in the methods described herewith specifically target a nonfunctional endogenous gene in HSPCs, or cells derived thereof, wherein said non-functional gene comprises deleterious mutations and is associated with a disorder.

[0104] In particular, TALE-nucleases used in the methods described herewith specifically target a non-functional endogenous HBB-gene in HSPCs, or cells derived thereof, wherein said nonfunctional gene is associated with a hemoglobinopathy such as sickle cell disorder. After having been HBB-gene edited, the restoration of the normal cellular phenotype in the HSPCs or cells derived thereof is obtained.

[0105] Targeted (i.e. site-directed) integration to achieve gene repair is suitably done by using sequence-specific nucleases inducing DNA cleavage and exogenous polynucleotide donor templates bearing homology to the target site and comprising the corrective sequence. By “site directed integration”, it is meant that integration of the polynucleotide repair template occurs at the locus where DNA cleavage is induced by the sequence-specific nuclease. Thus, the target sequence that is recognized by the nuclease is preferably comprised in the endogenous HBB- gene having the mutation to be edited.

[0106] As used herewith “TALE-nuclease(s)” refers to engineered protein(s) resulting from the fusion of at least a DNA binding domain typically derived from Transcription Activator Like Effector proteins (TALE) with a catalytic domain having endonuclease activity (Boch et al., 2009, Science 326(5959): 1509-12; Moscou and Bogdanove, 2009, Science 326(5959): 1501). Such catalytic domain usually derives from enzymes, such as for instance I-Tevl, l-Crel, Onu-1 , ColE7, NucA and Fok-I. As used herewith a TALE-nuclease can be in a monomeric or dimeric form depending on the catalytic domain selected, as detailed for instance in WO2012138927.

[0107] As described herewith, the TALE DNA binding domain is preferably derived from a Transcription Activator like Effector (TALE), wherein sequence specificity is driven by a series of 33-35 amino acids repeats originating from Xanthomonas or Ralstonia bacterial proteins AvrBs3, PthXol , AvrHahl , PthA, Tallc as non-limiting examples. These repeats differ essentially by two amino acids positions generally located at positions 12 and 13, also referred to as Repeat Variable Diresidue (RVD), which confer specificity of interaction with a base pair. Each base pair in the DNA target is contacted by a single repeat, A TALE nucleic acid binding domain generally corresponds to an engineered core TALE scaffold comprising a plurality of TALE repeat sequences, each repeat comprising a RVD specific to each nucleotide’s base into the “target nucleotide sequence” formed by the TALE recognition site(s). Preferably, RVDs are associated in the TALE polypeptide binding domain so that the RVD “HD” is for recognizing C, “NG” for recognizing T, “Nl” for recognizing A, “NN” for recognizing G or A. In some cases, critical amino acids 12 and 13 can be mutated into other amino acid residues in order to modulate their specificity towards nucleotides A, T, C and G and in particular to enhance the specificity. A TALE nucleic acid binding domain usually comprises between 8 and 30 TALE repeat sequences. More preferably, the core scaffold comprises between 8 and 20 TALE repeat sequences; more preferably 15 TALE repeat sequences. It can also comprise an additional single truncated TALE repeat sequence made of 20 amino acids located at the C-terminus of said TALE binding domain (i.e. half- TALE repeat sequence).

[0108] TALE binding domains generally further comprise a N-terminal domain responsible for the requirement of a first thymine base (TO) on the targeted sequence and a C-terminal domain that comprises a nuclear localization signals (NLS). Preferred TALE-nucleases include a N-terminal domain of at least, or about, 140 amino acids and / or preferably a C-terminal domain that can form a linker between the TALE binding domain and the nuclease catalytic domain which generally comprises from 15 to 60 amino acids; more preferably of about, or at least, 40 amino acids.

[0109] N- and C-terminal domains of the TALE binding domain can also be adjusted or mutated to modulate cleavage activity and target specificity as described for instance in WO2023094435.

[0110] Heterodimeric TALE-nucleases using Fok-1 as nuclease domain have been widely described in the art as, for instance, by Mussolino et al. (Nucleic Acids Research, 2011 , 39(21 ):9283-9293). Such heterodimeric TALE-nuclease comprises two TALE-nuclease monomers designed to bind together the specified target sequence. The polynucleotide target sequence then preferably comprises a first binding sequence recognized by the first TALE- monomer (i.e. left TALEN), a “spacer sequence” which is cleaved by the nuclease Fok-1 , and the second binding sequence that is recognized by the second monomer (i.e. right TALEN). In general, the spacer sequence comprises from 10 to 30, preferably from 12 to 20. nucleotides.

[0111] On another hand, monomeric TALE-nucleases using, for instance, Tev-1 or an engineered homing endonuclease such as l-Onul as catalytic domain, also referred to as “Compact TALEN” and “Mega-TAL”, respectively, which have been described for instance by Beurdelay et al. (Nature comm., 2013, 4:1762) and Boissel et al. (Nucleic Acids Research, 2013, 42(4): 2591-2601), can bind and cleave their target sequence by using only one TALE binding domain. In general, the TALE nuclease binds a genomic sequence that has from 8 to 30, preferably from 9 to 20, more preferably from 10 to 15 nucleotides.

[0112] Due to their high specificity, TALE-nucleases have proven to be particularly appropriate for therapeutic applications, especially under heterodimeric forms - i.e. working by pairs with a “right” monomer (also referred to as “5”’ or “forward”) and left” monomer (also referred to as “3”’ or “reverse”) as reported for instance by Mussolino et al. (Nucleic Acids Research, 2014, 42(10): 6762-6773).

[0113] Thus, is provided herewith a TALE-Nuclease targeting a non-functional endogenous HBB- gene in HSPCs, wherein at least one allele of the / - / BB-gene has a mutation (A>T) or (A>C) at position 20 of the coding region of exon 1 of the / - / BB-gene. Said TALE-Nuclease can be monomeric or dimeric. When in dimeric form the TALE-Nuclease is generally heterodimeric.

[0114] Particularly, it is provided herewith a TALE-Nuclease targeting the polynucleotide sequence of SEQ ID NO: 7. In particular embodiments, said TALE-Nuclease cleaves the HBB- gene within the region of polynucleotide sequence SEQ ID NO: 8.

[0115] According to some embodiments, said TALE-nuclease is heterodimeric and comprises at least one monomer binding the HBB sequence of SEQ ID NO: 17 or SEQ ID NO: 19. In further embodiments, said TALE-nuclease is a heterodimer comprising at least one monomer having the RVD sequence HD-HD-NG-NN-NG-NN-NN-NI-NN-NI-NI-NN-NG-HD-NG-NG.

[0116] According to some embodiments, said TALE-nuclease is heterodimeric and comprises a first monomer binding the HBB sequence of SEQ ID NO: 17 or SEQ ID NO: 19 and a second monomer binding the HBB sequence of SEQ ID NO: 18. In a particular embodiment, said TALE-nuclease is a heterodimer comprising a monomer binding SEQ ID NO: 17 and a monomer binding SEQ ID NO: 18.

[0117] In a further particular embodiment, said TALE-nuclease is a heterodimer comprising a monomer having the RVD sequence HD-HD-NG-NN-NG-NN-NN-NI-NN-NI-NI-NN-NG-HD-NG- NG and a monomer having the RVD sequence NG-HD-NI-HD-HD-NG-NG-NN-HD-HD-HD-HD- NI-HD-NI-NG.

[0118] In some embodiments, said heterodimeric TALE-nuclease forms a 11-bp spacer on the target region.

[0119] In some further embodiments, said TALE-Nuclease is heterodimeric and comprises a first monomer comprising the amino acid sequence of SEQ ID NO: 54 or a variant thereof comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, sequence identity with SEQ ID NO: 54, and a second monomer comprising the amino acid sequence of SEQ ID NO: 55 or a variant thereof comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, sequence identity with SEQ ID NO: 55, wherein the heterodimer targets the polynucleotide sequence of SEQ ID NO: 7. In particular, said first monomer binds the sequence of SEQ ID NO: 17 and said second monomer binds the sequence of SEQ ID NO: 18.

[0120] According to some embodiments, said TALE-Nuclease heterodimer comprises a first monomer comprising the amino acid sequence of SEQ ID NO: 54 and a second monomer comprising the amino acid sequence SEQ ID NO: 55.

[0121] In said TALE-Nuclease heterodimer, the first and second monomers and variants thereof are designed to form a heterodimer binding to the target polynucleotide sequence SEQ ID NO: 7 and comprise the RVD sequence HD-HD-NG-NN-NG-NN-NN-NI-NN-NI-NI-NN-NG-HD-NG-NG and RVD sequence NG-HD-NI-HD-HD-NG-NG-NN-HD-HD-HD-HD-NI-HD-NI-NG, respectively. The cleavage occurs within the target sequence in exon 1 of the endogenous HBB locus.

[0122] Also provided herewith is a TALE-Nuclease that is a monomeric TALE-nuclease comprising a monomer binding the HBB sequence of SEQ ID NO: 17 or SEQ ID NO: 19.

[0123] In some embodiments, said monomeric TALE-Nuclease comprises a monomer having the RVD sequence HD-HD-NG-NN-NG-NN-NN-NI-NN-NI-NI-NN-NG-HD-NG-NG.

[0124] In some further embodiments, said monomeric TALE-Nuclease comprises a monomer comprising the amino acid sequence of SEQ ID NO: 54 or a variant thereof comprising an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, sequence identity with SEQ ID NO: 54, wherein said monomer binds the sequence of SEQ ID 17.

[0125] In a particular embodiment, said monomeric TALE-Nuclease comprises a monomer comprising the amino acid sequence of SEQ ID NO: 54.

[0126] In a still further embodiment, said monomeric TALE-Nuclease comprises a monomer comprising the amino acid sequence of SEQ ID NO: 54 fused to a catalytic domain derived from Tev-1 or an engineered homing endonuclease such as l-Onul.

[0127] Also provided are isolated nucleic acids encoding the TALE-nucleases as defined herewith.

[0128] In one embodiment, said isolated nucleic acid encodes a dimeric TALE-Nuclease comprising a first monomer comprising the amino acid sequence of SEQ ID NO: 54, or a variant thereof comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, sequence identity with SEQ ID NO: 54, comprising the RVD sequence HD-HD-NG-NN-NG-NN-NN-NI-NN-NI-NI-NN-NG-HD-NG-NG and binding the sequence of SEQ ID NO: 17, and a second monomer comprising the amino acid sequence of SEQ ID NO: 55, or a variant thereof comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, sequence identity with SEQ ID NO: 55, comprising the RVD sequence NG-HD-NI-HD-HD-NG-NG-NN-HD-HD-HD-HD-NI-HD-NI- NG and binding the sequence of SEQ ID NO: 18.

[0129] In some embodiments, is provided an isolated nucleic acid encoding a dimeric TALE- nuclease, wherein the two monomers of said dimeric TALE-Nuclease comprise the amino acid sequences SEQ ID NO: 54 and SEQ ID NO: 55, respectively.

[0130] Also provided are isolated nucleic acids comprising the nucleic acid sequence of SEQ ID NO: 21 and / or SEQ ID NO: 22.

[0131] In other embodiments, said isolated nucleic acid encodes a monomeric TALE-Nuclease comprising a monomer comprising the amino acid sequence of SEQ ID NO: 54, or a variant thereof comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, sequence identity with SEQ ID NO: 54, comprising the RVD sequence NG-HD-NI-HD-HD-NG-NG-NN-HD-HD-HD-HD-NI-HD-NI-NG and binding the sequence of SEQ ID NO: 17. In some embodiments, is provided an isolated nucleic acid encoding a monomeric TALE- nuclease, wherein the monomer of said monomeric TALE-Nuclease comprises the amino acid sequence SEQ ID NO: 54.

[0132] Also provided is an isolated nucleic acid comprising the nucleic acid sequence of SEQ ID NO: 21.

[0133] According to some embodiments, said isolated nucleic acid is a mRNA.

[0134] According to other embodiments, said isolated nucleic acid is a circular RNA.

[0135] According to one embodiment, said TALE-nuclease is introduced in the form of an isolated nucleic acid, by electroporation.

[0136] Also provided is the ex vivo use of the TALE-nuclease as described herewith, the isolated nucleic acid encoding same or the vector encoding same for / 788-gene editing HSPCs, notably HSPCs of a patient suffering from a disorder associated with the expression of a non-functional endogenous / - / BB-gene, or a lack of expression of a functional endogenous / - / BB-gene, in his / her HSPCs or cells derived thereof.

[0137] 1.2.2. Template for gene correction

[0138] Also provided herewith is an isolated nucleic acid comprising a polynucleotide repair template (also called herewith "donor template”) to be integrated in said HSPCs’ genome at the specific locus of the non-functional / - / BB-gene to be cleaved and edited by said TALE-nuclease.

[0139] Said polynucleotide repair template can be in the form of an isolated nucleic acid or vector comprising same.

[0140] Said polynucleotide repair template comprises an exogenous sequence bearing homology to the / 788-target site of the TALE-nuclease and comprising the corrective / 788-sequence.

[0141] The corrective / 788-sequence comprises a portion of the / 788-gene nucleic acid sequence comprising the corrected A base at the position and context (i.e. surrounding nucleotides) corresponding to the nucleotide of position 20 of / 788-exon 1.

[0142] In particular embodiments, to avoid that the TALE-nuclease cleaves the gene that has been edited (corrected), silent mutations are added in the sequence of the polynucleotide repair template, for instance about 3 to 10 silent mutations, such as 3, 4, 5, 6, 7, 8, 9 or 10 silent mutations. For instance, the polynucleotide repair template sequence comprises the corrected A base at the position corresponding to the original T or C mutation of the endogenous / 7BB-mutated allele and 4 silent mutations, wherein at least one, at least two, or at least three of the silent mutations are placed on the 5’-side and / or on the 3’-side of the corrected base. Preferably, said silent mutations are positioned in the DNA binding sites of the TALEN to avoid that the TALEN binds and cleaves the neo-inserted repair template.

[0143] A silent mutation corresponds to the change of a single nucleotide within a protein-coding portion of a gene that does not affect the amino acids sequence of the protein encoded by the gene.

[0144] In order to facilitate targeted (i.e. site-directed) integration of the exogenous sequence via homologous recombination, said exogenous sequence comprises a left (5’) and a right (3’) homology sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity with the left (5’) and right (3’) regions of the endogenous gene to be edited, respectively. Said left (5’) and right (3’) regions of said endogenous gene to be edited are portions (e.g. from 50 to 500 nucleotide-long portions) of said endogenous gene and are positioned at the left (5’) and right (3’) side of the nucleotide(s) to be corrected in the target endogenous locus. Said left (5’) and right (3’) homologous regions are generally from about 20 to about 500 nucleotide-long (such as from about 40 to about 500, from about 50 to about 400, from about 50 to about 350, from about 50 to about 325, from about 90 to about 310, from about 85, 86, 87, 88, 89, or 90 to about 295, 296, 297, 298, 299, 300, 301 , 302, 303, 304, or 305 nucleotide-long).

[0145] In some embodiments, said polynucleotide repair template comprises an exogenous sequence comprising the sequence of SEQ ID NO: 13.

[0146] In some embodiments, said polynucleotide repair template comprises an exogenous sequence comprising the sequence of SEQ ID NO: 13 flanked, at the 5’ (left) and 3’ (right), by a nucleic acid portion having homology with the target endogenous / 788-gene to be edited.

[0147] In the current application, the target region of the endogenous / 788-gene to be edited comprises the sequence SEQ ID NO: 7.

[0148] In particular embodiments, said polynucleotide repair template comprises an exogenous sequence of about 50 to about 1000 nucleotides (such as from about 60 to about 800, from about 100 to about 700, from about 100 to about 650, from about 180 to about 650, from about 195 to about 620 nucleotides) comprising the sequence of SEQ ID NO: 13, wherein the portions of said exogenous sequence that are at the 5’ (left) and 3’ (right) positions of SEQ ID NO: 13 have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity with the corresponding portions of the endogenous / - / BB-gene to be edited, respectively.

[0149] In particular embodiments, said polynucleotide repair template comprises an exogenous sequence comprising, from 5’ to 3’: (i) a nucleic acid sequence that is about 20 to 500 nucleotide- long (such as about 40 to 500, about 50 to 400, about 50 to 350, about 50 to 325, about 90 to 310, about 85, 86, 87, 88, 89, 90 to 295, 296, 297, 298, 299, 300, 301 , 302, 303, 304, 305 nucleotide- long) and has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity with the left (5’) regions of the endogenous / - / BB-gene to be edited, (ii) the nucleic acid sequence of SEQ ID NO: 13, and (iii) a nucleic acid sequence that is about 20 to 500 nucleotide-long (such as about 40 to 500, about 50 to 400, about 50 to 350, about 50 to 325, about 90 to 310, about 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100 to 295, 296, 297, 298, 299, 300, 301 , 302, 303, 304, 305, 306, 307, 308, 309, 310 nucleotide-long) and has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity with the right (3’) region of the endogenous / - / BB-gene to be edited.

[0150] In some embodiments, said polynucleotide repair template comprises the nucleic acid sequence of SEQ ID NO: 14.

[0151] In some further embodiments, said polynucleotide repair template comprises a nucleic acid sequence comprising, from 5’ to 3’: (i) a nucleic acid sequence that is about 20 to 500 nucleotide- long (such as about 40 to 500, about 50 to 400, about 50 to 350, about 50 to 325, about 90 to 310, about 85, 86, 87, 88, 89, 90 to 295, 296, 297, 298, 299, 300, 301 , 302, 303, 304, 305 nucleotide- long) and has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity with the left (5’) regions of the endogenous / - / BB-gene to be edited, (ii) the nucleic acid sequence of SEQ ID NO: 14, and (iii) a nucleic acid sequence that is about 20 to 500 nucleotide-long (such as about 40 to 500, about 50 to 400, about 50 to 350, about 50 to 325, about 90 to 310, about 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100 to 295, 296, 297, 298, 299, 300, 301 , 302, 303, 304, 305, 306, 307, 308, 309, 310 nucleotide-long) and has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity with the right (3’) region of the endogenous / - / BB-gene to be edited.

[0152] For instance, said polynucleotide repair template comprises an exogenous sequence comprising, from 5’ to 3’: (i) a nucleic acid sequence that is from about 80 to about 100 nucleotide- long, for example from about 85 to about 95 nucleotide-long (such as about 80, 81 , 82, 83, 84, 85, 86, 87, 88, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100 nucleotide-long) and has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity with the left (5’) regions of the endogenous / - / BB-gene to be edited, (ii) the nucleic acid sequence of SEQ ID NO: 13, and (iii) a nucleic acid sequence that is from about 80 to about 100 nucleotide-long, for example from about 85 to about 95 nucleotide-long (such as about 80, 81 , 82, 83, 84, 85, 86, 87, 88, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100 nucleotide-long) and has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity with the right (3’) region of the endogenous / - / BB-gene to be edited.

[0153] In some embodiments, said polynucleotide repair template comprises an exogenous sequence comprising the sequence of SEQ ID NO: 16.

[0154] For instance, said polynucleotide repair template comprises an exogenous sequence comprising, from 5’ to 3’: (i) a nucleic acid sequence that is about 300 nucleotide-long (such as about 295, 296, 297, 298, 299, 300, 301 , 302, 304, 305, 306, 307, 308, 309, 310 nucleotide-long) and has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity with the left (5’) regions of the endogenous / - / BB-gene to be edited, (ii) the nucleic acid sequence of SEQ ID NO: 13, and (iii) a nucleic acid sequence that is about 300 nucleotide-long (such as about 295, 296, 297, 298, 299, 300, 301 , 302, 304, 305, 306, 307, 308, 309, 310 nucleotide-long) and has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity with the right (3’) region of the endogenous / - / BB-gene to be edited.

[0155] In some embodiments, said polynucleotide repair template comprises an exogenous sequence comprising the sequence of SEQ ID NO: 15.

[0156] Considering that the sequence of SEQ ID NO: 13 itself is different from an endogenous / - / BB-gene sequence, SEQ ID NO: 13 can be qualified as an exogenous sequence according to the definition provided herewith. Thus, to be concise, the term “exogenous” may be omitted in the sentences referring to a “polynucleotide repair template comprising an exogenous sequence comprising the sequence of SEQ ID NO: 13” (or any sequence comprising SEQ ID NO: 13 such as SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16), which would have the same meaning as a sentence like “polynucleotide repair template comprising the nucleic acid sequence of SEQ ID NO: 13 (or any sequence comprising SEQ ID NO: 13 such as SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16)”.

[0157] A polynucleotide repair template can be a single-stranded DNA and can be introduced into a cell in linear or circular form. If introduced in linear form, the ends of the polynucleotide repair template sequence can be protected (for instance, from exonucleolytic degradation) by methods known to those of skill in the art. For example, one or more dideoxynucleotide residues are added to the 3' terminus of a linear molecule and / or self-complementary oligonucleotides are ligated to one or both ends. See, for example, Chang et al. (Proc. Natl. Acad. Sci. USA, 1987, 84:4959- 4963), Nehls et al. (Science, 1996, 272: 886-889). Additional methods for protecting exogenous polynucleotides from degradation include, but are not limited to, addition of terminal amino group(s) and the use of modified inter-nucleotide linkages such as, for example, phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyribose residues.

[0158] In some embodiments, said polynucleotide repair template is in the form of a singlestranded nucleic acid such as a single-stranded DNA.

[0159] As used herewith “single-stranded DNA” also covers single-stranded Oligodeoxynucleotide (“ssODN”). The ssODNs generally do not exceed about 200 nucleotides.

[0160] Advantageously, as demonstrated in the example section, in some further embodiments, said polynucleotide repair template is a non-viral single-stranded DNA.

[0161] Said polynucleotide repair template can be in a linear or circular form.

[0162] Advantageously, in some embodiments, said polynucleotide repair template is in a circular form.

[0163] In particular embodiments, said polynucleotide repair template is a non-viral ssDNA comprising the nucleic acid sequence of SEQ ID NO: 16.

[0164] In still further embodiments, said polynucleotide repair template is a non-viral ssDNA comprising the nucleic acid sequence of SEQ ID NO: 16, wherein said sequence comprises phosphorothioate modification on the first two bases situated at the 5’- and 3’- ends of the sequence.

[0165] In further particular embodiments, said polynucleotide repair template is a linear singlestranded DNA.

[0166] In further particular embodiments, said polynucleotide repair template is a non-viral linear single-stranded DNA comprising the nucleic acid sequence of SEQ ID NO: 16, and optionally comprises phosphorothioate modification on the first two bases situated at the 5’- and 3’- ends of the sequence.

[0167] In further particular embodiments, said polynucleotide repair template is a non-viral circular single-stranded DNA comprising the nucleic acid sequence of SEQ ID NO: 16.

[0168] 1.2.3. Culture and transfection conditions According to one embodiment, prior to introducing the / - / BB-specific TALE-nuclease in the HSPCs to be edited, the HSPCs are cultured for at least 1 day in a medium comprising at least one compound stimulating cell cycle.

[0169] According to one embodiment, when the method of the invention uses fresh HSPCs, the HSPCs are cultured in a medium comprising at least one compound stimulating cell cycle for about 1 day or 2 days.

[0170] According to another embodiment, when the method of the invention uses frozen HSPCs, the HSPCs are cultured in a medium comprising at least one compound stimulating cell cycle for about 2 days.

[0171] The compounds stimulating cell cycle to be added in the culture medium of HSPCs are well known by the skilled in the art.

[0172] According to one embodiment, the culture medium comprises one or more of the compounds selected from the group consisting of: Thrombopoietin (TPO), Fms-like tyrosine kinase 3 ligand (Flt-3L), Stem Cell Factor (SCF), Interleukin 3 (IL-3), hydrolyzed Polyvinyl Alcohol (PVA), LIM171 , SR1 , aminoquinoline, and dmPGE2. In particular, the culture medium comprises Thrombopoietin (TPO), Interleukin 3 (IL-3), and stem cell factor (SCF).

[0173] According to a particular embodiment, after thawing, HSPCs are cultured at a concentration of 0.3 x 106cells / mL in complete medium: StemSpan II (Stemcell, #09655), 1X CD34 expansion supplement (Stemcell, #02691) and 1X penicillin-streptomicyn (Gibco, #15140- 122) at 37°C, 5% CO2.

[0174] According to another particular embodiment, for culture of HSPCs in GMP-compatible conditions, HSPCs are cultured in GMP Stem Cell Growth Medium (SCGM, CellGenix, Freiburg, Germany) supplemented with human cytokines (Cellgenix GMP-grade), TPO (100 ng / mL), Flt3 (300 ng / mL), SCF (300 ng / mL) and IL-3 (60 ng / mL).

[0175] The terms “GMP-compatible conditions”, as used herewith, refer to conditions of “Good Manufacturing Practices” allowing large-scale and consistent production and controlled quality of edited HSPCs, which can then be administered as a medicinal product to a patient in need thereof. For instance, GMP Stem Cell Growth Medium is produced following all applicable GMP guidelines and allows for the safe use in accordance with USP Chapter <1043> and ISO 20399:2022. An appropriate GMP Stem Cell Growth Medium is a xeno-free medium used for the serum-free expansion of low numbers of isolated human hematopoietic stem and progenitor cells (HSCs / CD34+cells). A GMP-compatible culture medium can be a GMP Stem Cell Growth Medium (SCGM, CellGenix, Freiburg, Germany) supplemented with human cytokines (Cellgenix GMP- grade), TPO (100 ng / mL), Flt3 (300 ng / mL), SCF (300 ng / mL) and IL-3 (60 ng / mL).

[0176] In some embodiments, the cells are placed in a culture medium comprising a sternnessenhancer, such as Valproic Acid (VPA), for at least one day.

[0177] The TALE-nuclease and polynucleotide repair template, as described herewith, are introduced in the HSPCs to be edited via at least one transfection step such as an electroporation step.

[0178] Media appropriate for carrying out the transfection step(s) (e.g. electroporation) are well known to the skilled person and include commercially available media.

[0179] In one embodiment of the method for preparing / - / BB-gene edited HSPCs described herewith, step i) of introducing a TALE-nuclease in the HSPCs and / or step ii) of introducing a polynucleotide repair template in the HSPCs, comprise(s) supplementing the medium in which the HSPCs have been placed with at least one enhancer of Homology Directed Repair.

[0180] As defined herewith, enhancers of Homology Directed Repair (abbreviated “HDR- enhancers”) include reagents which are known in the art to favor a given Double Strand Break repair pathway in the cell. Double strand break induced by endonucleases can be repaired by different pathways managed by different key proteins. One objective of the present invention is to stimulate homologous recombination events as far as possible over non-homologous end-joining (NHEJ) pathways or other error prone repair pathways.

[0181] Said enhancer of Homology Directed Repair can be an inhibitor of a pro-NHEJ factor, an agonist of a pro-HDR factor, or an inhibitor of a MMEJ factor. Also described herewith is the combined use of an inhibitor of a pro-NHEJ factor and an inhibitor of a pro-HDR factor.

[0182] To this aim, appropriate repair pathway reagents can either inhibit NHEJ pathway, such as compounds like STL127705, NU7441 , KU-0060648, NU7026, M3812, E-822, SCR7, RS-1 , can act on cell cycle, such as Wortmanin, Aphidicolin, mimosin thymidine, Hydroxy urea (HU), Nocodazole, ABT-751 , XL413, or favor targeted integration by so far unknown mechanisms, such as L755507, Brefeldin and Resveratrol. Other preferred HDR-enhancers are inhibitors of Iig4, xrcc4, Ku70, Ku80, DNA-PKcs, which can be shRNA or siRNA transfected or expressed into the cell directed against Iig4, xrcc4, Ku70, Ku80, DNA-PKcs transcripts. In further embodiments, the process described herewith further comprises expressing into the cells a nucleic acid encoding Rad51 , Rad52, E4orf6 / 7, dominant-negative p53 mutant protein (GSE56), inhibitor of 53PB1 and / or dominant-negative 53BP1. Such polynucleotides encoding Rad51 , Rad52, E4orf6 / 7, dominant-negative p53 mutant protein (GSE56), inhibitor of 53PB1 and / or dominant-negative 53BP1 can be transfected in the same time as the gene editing reagents and / or the nucleic acid template [Canny M.D., et al. (2018) Inhibition of 53BP1 favors homology-dependent DNA repair and increases CRISPR-Cas9 genome-editing efficiency. Nat Biotechnol. 36(1): 95-102], [Paulsen B.S., et al. (2017) Ectopic expression of RAD52 and dn53BP1 improves homology-directed repair during CRISPR-Cas9 genome editing. Nat Biomed 1(11):878-888], [Schiroli, G. et al. (2019) Precise Gene Editing Preserves Hematopoietic Stem Cell Function following Transient p53- Mediated DNA Damage Response Cell Stem Cell 24:551-565],

[0183] In one embodiment, said enhancer of Homology Directed Repair is an inhibitor of the p53- binding protein 1 (53BP1). 53BP1 plays a key role in Double Strand Break (DSB) repair following the NHEJ pathway. Several inhibitors have been described in WO2017 / 132746, incorporated by reference.

[0184] Independently of the present application, the Applicant has observed that aminoquinoline compound(s) such as chloroquine, as well as its derivative hydroxychloroquine, increase homologous recombination in HSCs (WO2022112596, incorporated by reference herewith). These compounds are also encompassed by the term “enhancer of Homology Directed Repair” as used herewith.

[0185] In a further embodiment said enhancer of Homology Directed Repair is an inhibitor of the DNA-PKcs. DNA-PKcs is a member of the phosphatidylinositol-3 (PI-3) kinase-like kinase family (PIKK) and is a key kinase involved in NHEJ repair.

[0186] In some embodiments, the DNA-PKCs inhibitor useful herewith is selected among the group consisting of: a small molecule inhibitor Nu7441 (Leahy (2004) Bioorg Med Chem Lett 14:6083-6087), the PI 3-kinase inhibitor LY294002 (Izzard (1999) Cancer Res 59:2581-2586), 2- amino-chromen-4-ones (WO03 / 024949), 1 (2-hydroxy-4-morpholin-4-yl-phenyl)-ethanone

[0187] (Kashishian (2003) Mol Cancer Ther 2:1257-1264), SU11752 (Ismail (2004) Oncogene 23:873- 882), or any one of the small molecule inhibitors of DNA-PKcs described in US 9,592,232 ; US 7,402,607 ; US 6,893,821 ; US 2018 / 0194782, all incorporated by reference herewith.

[0188] In a further embodiment said enhancer of Homology Directed Repair is an inhibitor of a NHEJ enzyme, such as an inhibitor of Ku70 / 80, including CYREN (Arnoult (2017) Nature 549:548- 552), or an inhibitor of DNA Ligase IV, including Scr7 (Maruyama (2015) Nat Biotechnol 33:538- 542).

[0189] In another embodiment, said enhancer of Homology Directed Repair is an inhibitor of the MMEJ pathway (Sfeir (2015) 40:701-714). Thus, in another embodiment, said HDR-enhancer is an inhibitor of the DNA polymerase theta (Pol Q), an inhibitor of PARP including Veliparib and Olaparib, or an inhibitor of MRE11 including Mirin and derivatives (Shibata (2014) Molec Cell 53:7- 18).

[0190] In a further embodiment, said enhancer of Homology Directed Repair is a stimulator of the HDR pathway, such as an agonist of a HDR factor like for instance RAD51. Thus, said HDR- enhancer can be an agonist of RaD51 , including including RS-1 (Jayathilaka (2008) PNAS 105:15848- 15853).

[0191] In one embodiment, the at least one enhancer of Homology Directed Repair is selected from the group consisting of an inhibitor of 53BP1 , a dominant negative mutant of 53BP1 , an inhibitor of P53, and an inhibitor of Non-Homology-End-Joining.

[0192] In a further embodiment, said inhibitor of 53BP1 is a polypeptide comprising an amino acid sequence having at least 80% identity with SEQ ID NO: 61.

[0193] In a further embodiment, said inhibitor of P53 is GSE56, such as a polypeptide comprising an amino acid sequence having at least 80% identity with SEQ ID NO: 62.

[0194] In some aspects of the method described herewith, said enhancer of Homology Directed Repair is introduced by electroporation of a RNA (e.g. mRNA) encoding said HDR-enhancer.

[0195] In some embodiments, said HDR-enhancer is fused to said TALEN and both are delivered as mRNA by electroporation.

[0196] In some embodiments, the population of HSPCs is transfected with at least 0.1 pg, at least 1 pg, or at least 2 pg, preferably at least 4 pg RNA of SEQ ID NO: 26 for about 1.106cells.

[0197] In a further embodiment of the method described herewith, about 1.108cells are transfected with at least 10 pg, preferably at least 100 pg, more preferably at least 200 pg, still more preferably at least 400 pg RNA of SEQ ID NO: 26.

[0198] In one embodiment of the method for preparing / - / BB-gene edited HSPCs described herewith, step i) of introducing a TALE-nuclease in the HSPCs and / or step ii) of introducing a polynucleotide repair template in the HSPCs, comprise(s) supplementing the medium in which the HSPCs have been placed with at least one viability-enhancer.

[0199] Said viability-enhancer can be an inhibitor of BAX / BAK selected from the group consisting of B-cell lymphoma-extra large (BCL-XL), B-cell lymphoma 2 (BCL-2), Myeloid cell leukemia 1 (MCL-1), BCL2 like 10 (BCL2L10, BCL-B or DIVA), BCL2 related protein A1 (BFL-1 or A1), BCL2 like 2 (BCL-W).

[0200] In one embodiment, the method described herewith comprises introducing into the HSPCs at least one viability-enhancer, such as BCL-XL.

[0201] In a particular embodiment, said viability-enhancer has an amino acid sequence comprising SEQ ID NO: 63.

[0202] In one embodiment of the method described herewith, said viability-enhancer is introduced by electroporation of a RNA (e.g. mRNA) encoding said viability-enhancer.

[0203] In some embodiments, said viability-enhancer is fused to said TALEN and both are delivered as mRNA by electroporation.

[0204] In a particular embodiment of the method described herewith, said viability-enhancer is introduced by electroporation of a RNA having a sequence comprising SEQ ID NO: 27.

[0205] In one embodiment of the method described herewith, the population of HSPCs is transfected with at least 0.1 pg, or at least 0.5 pg, preferably at least 1 pg of RNA (e.g. mRNA) encoding BCL-XL, for 1.106cells.

[0206] In a further embodiment, step i) of introducing a TALE-nuclease in the HSPCs and / or step ii) of introducing a polynucleotide repair template in the HSPCs, comprise(s) supplementing the medium with at least one enhancer of Homologous Recombination and at least one viabilityenhancer.

[0207] In a particular embodiment, said enhancer of Homology Directed Repair comprises the amino acid sequence of SEQ ID NO: 61 and said viability-enhancer comprises the amino acid sequence of SEQ ID NO: 63.

[0208] If not used immediately, the population of gene-edited cells following the process described herewith are resuspended in a cryomedium and stored frozen until use.

[0209] In some embodiments, the cells can be treated with the HDR-enhancer(s) and viabilityenhancers) after having introduced the polynucleotide repair template, preferably at the same time or before the TALE-nuclease is introduced or expressed in the cell. In general, the HDR- enhancer(s) and viability-enhancer(s) are added to the culture medium. Alternatively, the cells are transferred into a fresh medium comprising the HDR-enhancer(s) and viability-enhancer(s) after an electroporation step introducing the TALE-nuclease and / or the polynucleotide repair template. Electroporation steps that are used to transfect HSPCs are typically performed in closed chambers comprising parallel plate electrodes producing a pulse electric field between said parallel plate electrodes greater than 100 volts / cm and less than 5,000 volts / cm, substantially uniform throughout the treatment volume such as described in WO / 2004 / 083379, which is incorporated by reference, especially from page 23, line 25 to page 29, line 11. One such electroporation chamber preferably has a geometric factor (cm-1) defined by the quotient of the electrode gap squared (cm2) divided by the chamber volume (cm3), wherein the geometric factor is less than or equal to 0.1 cm-1, wherein the suspension of the cells and the sequence-specific reagent is in a medium which is adjusted such that the medium has conductivity in a range spanning 0.01 to 1.0 milliSiemens. In general, the suspension of cells undergoes one or more pulsed electric fields. With the method, the treatment volume of the suspension is scalable, and the time of treatment of the cells in the chamber is substantially uniform.

[0210] In some embodiments, the gene editing method is carried out with at least two electroporation steps. In some embodiments, the first electroporation step consists in introducing RNA encoding the TALE-nuclease, and the second electroporation step consists in introducing the polynucleotide repair template. A second electroporation step to introduce the polynucleotide repair template can be carried out, for instance, at least 5 hours to 20 hours, from 10 to 20 hours, from 12 to 18 hours, or from 15 to 17 hours, such as about 16 hours, after a first electroporation step to introduce the TALE-nuclease.

[0211] Thus, in some embodiments, the ex vivo method for preparing / - / BB-gene edited HSPCs comprises two electroporation steps, wherein the RNA encoding the TALE-nuclease is introduced in the cells in a first electroporation step and the polynucleotide repair template ssDNA is introduced in a second electroporation step.

[0212] In some embodiments, the RNA encoding the TALE-nuclease is introduced by electroporation from 24 to 72 hours, such as from 24 to 48 hours or from 36 to 72 hours, after thawing of frozen HSPCs.

[0213] In a further particular embodiment, the polynucleotide repair template ssDNA is electroporated from 10 to 20 hours or from 12 to 18 hours, or from 15 to 17 hours, such as about 16 hours, after the electroporation of the RNA encoding the TALE-nuclease.

[0214] The process described herewith can also be performed in one step, in which the TALE- nuclease and the polynucleotide repair template are concomitantly introduced in the cell. For instance, both the TALE-nuclease and the polynucleotide repair template can be introduced in the cell during the same delivery step (such as electroporation) as described for instance by Sather et al. ((2015) Science Translational Medicine 7(307):307).

[0215] Thus, in some embodiments of the ex vivo method for preparing HBB-gene edited HSPCs described herewith, the RNA encoding the TALE-nuclease and the polynucleotide repair template ssDNA are introduced in the cells in one electroporation step.

[0216] In a further particular embodiment, the ex vivo method for preparing HBB-gene edited HSPCs described herewith comprises introducing the RNA encoding the TALE-nuclease and the circular single-stranded DNA repair template in one electroporation step. In that embodiment, steps i) and ii) are carried out in one electroporation step.

[0217] In another embodiment of the method described herewith, the HDR-enhancer and / or viability-enhancer is / are electroporated concomitantly to the RNA encoding the TALE-nuclease.

[0218] In another alternative both the TALE-nuclease and the polynucleotide repair template may be transfected by using nanoparticles, such as silica based mesoporous particles as described for instance in WO2016124765. In such embodiments, the HDR-enhancer(s) and viabilityenhancers) can be directly introduced in the nanoparticles or in any transition culture medium used during or after transfection / transduction steps.

[0219] 2. Products

[0220] 2. 1. Gene edited HSPCs

[0221] It derives from the above, that the engineered HSPCs according to the invention may be primary cells.

[0222] According to some embodiments, the HSPCs are mammalian HSPCs, and preferably human HSPCs. More particularly, the HSPCs are human HSPCs from a patient suffering from a disorder associated with the expression of a non-functional gene in his / her HSPCs, or the lack of expression of a functional gene in his / her HSPCs, or in cells derived therefrom.

[0223] According to some embodiments, said non-functional gene is the HBB-gene comprising at least one allele having a mutation associated with a hemoglobinopathy. For instance, at least one HBB-allele has a mutation (A>T) or (A>C) at position 20 of the coding region of exon 1 of HBB- gene, wherein said mutation is associated with a hemoglobinopathy. In a particular embodiment, said non-functional gene is the / 7BB-gene, wherein at least one allele of the / - / BB-gene has a mutation (A>T) at position 20 of the coding region of / 788-exon 1 , said mutation being associated with sickle cell disease.

[0224] The gene edited HSPCs of the invention are / - / BB-gene edited HSPCs which have at least one allele of the / - / BB-gene corrected at the original (A>T) mutation.

[0225] In another embodiment, said non-functional gene is the / - / BB-gene, wherein at least one allele of the / - / BB-gene has a mutation (A>C) at position 20 of the coding region of / 788-exon 1 , said mutation being associated with hemoglobin beta C disease.

[0226] The gene edited HSPCs of the invention are / 788-edited HSPCs which have at least one allele of the / 788-gene corrected at the original (A>C) mutation.

[0227] According to some embodiments, the engineered HSPCs comprise long-term repopulating HSC (LT-HSC).

[0228] According to some embodiments, the engineered HSPCs comprise short-term repopulating HSC (ST-HSC).

[0229] According to some embodiments, the engineered HSPCs comprise at least CD34+cells.

[0230] According to some embodiments, the engineered HSPCs comprise at least CD34+, CD90+, and CD133+cells.

[0231] According to some embodiments, the engineered HSPCs comprise at least CD34+, CD38' , CD45RA; CD90+, and CD133+cells.

[0232] According to some embodiments, the engineered HSPCs comprise at least CD34+, CD38' , and CD45RA' cells.

[0233] According to some embodiments, the engineered HSPCs comprise at least CD34+, CD38' , CD45RA; CD90; and CD133’ cells.

[0234] Further provided herewith are engineered / 788-edited HSPCs as well as populations of / 788-edited HSPCs obtainable by any of the production methods disclosed herein.

[0235] An aspect relates to a population of / 788-gene edited HSPCs obtainable by the methods described herewith.

[0236] In some embodiments, said population of / 788-gene edited HSPCs comprises at least about 10% of engineered / 788-edited HSPCs as described herewith. According to some embodiments, at least 20%, such as at least 30% or at least 40%, of the total cells of the population of cells are engineered / - / BB-edited HSPCs as described herewith.

[0237] According to some embodiments, at least 50%, such as at least 60% or at least 70%, of the total cells of the population of cells are engineered / - / BB-edited HSPCs as described herewith.

[0238] According to some embodiments, at least 80%, such as at least 90% or at least 95%, of the total cells of the population of cells are engineered / - / BB-edited HSPCs as described herewith.

[0239] A particular aspect relates to a population of / 788-gene edited HSPCs, wherein a polynucleotide comprising the exogenous sequence SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16, has been integrated at the / 788-locus in the genome of at least 10%, preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, of the cells.

[0240] In a particular embodiment of the population of / 788-gene edited HSPCs, a polynucleotide comprising the exogenous sequence of SEQ ID NO: 13 has been integrated at the / 788-locus in the genome of at least 10%, preferably at least 20%, 30%, 40%, 50%, 60%, 70%, of the cells.

[0241] The population of cells according to the present invention may comprise long-term repopulating HSCs (LT-HSCs). According to some embodiments, at least 0.1%, at least 1 %, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, or at least 30%, of said LT-HSCs are engineered / 788-edited HSPCs as described herewith.

[0242] According to some embodiments, the population of / 788-gene edited HSPCs comprises at least 0.1%, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, or at least 30%, of cells having a [CD34+CD38' CD45RA'CD90+CD133+] phenotype.

[0243] According to some embodiments, the population of / 788-gene edited HSPCs comprises at least about 70%, such as at least about 80%, about 85%, or about 90%, of viable cells.

[0244] According to some embodiments, in the population of / 788-gene edited HSPCs, the HDR frequency at the / 788-locus is at least about 20% such as at least about 25%, at least about 30%, at least about 35%, or at least about 40%.

[0245] According to some embodiments, in the population of / 788-gene edited HSPCs, the Indel frequency at the / 788-locus is less than about 30% such as less than about 25%, less than about 20%, less than about 15%, or less than about 10%.

[0246] According to some embodiments, in the population of / 788-gene edited HSPCs, the HDR / indels ratio at the / 788-locus is equal to or higher than 1 , equal to or higher than 1.2, 1.4, 1.5, or 1.7, equal to or higher than about 2, equal to or higher than about 2.5, equal to or higher than about 3, equal to or higher than about 3.2, 3.5, 3.8, equal to or higher than about 4, 4.1 , or

[0247] 4.2.

[0248] Any standard method known to the skilled person can be used to determine HDR and Indels. For instance, HDR / lndels are determined by ddPCR, such as illustrated in the Example section.

[0249] According to some embodiments, the population of / 788-gene edited HSPCs comprises more than 0.5x108edited cells, preferably more than 1x108edited cells, more preferably more than 1.5x108edited cells, even more preferably more than 2x108edited cells.

[0250] A still other aspect relates to a population of HSPCs, wherein cells from said population comprise (i) RNA encoding a TALE-nuclease comprising a monomer comprising the amino acid sequence of SEQ ID NO: 54 and a monomer comprising the amino acid sequence of SEQ ID NO: 55, and (ii) a non-viral repair template ssDNA comprising the nucleic acid sequence SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.

[0251] In some embodiments, said population of HSPCs comprises cells comprising RNA and a repair template as specified above, and (i) a RNA (e.g. mRNA) encoding a HDR-enhancer, such as a RNA comprising the nucleic acid sequence SEQ ID NO: 26, and / or (ii) a RNA (e.g. mRNA) encoding a viability-enhancer, such as a RNA comprising the nucleic acid sequence SEQ ID NO: 27.

[0252] Another aspect relates to isolated / - / BB-gene edited HSPCs obtainable by the methods described herewith.

[0253] Another particular aspect concerns isolated / - / BB-gene edited HSPCs, wherein a polynucleotide comprising the exogenous sequence of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16, has been integrated at the / 788-locus in the genome of said HSPCs.

[0254] In a particular embodiment of the isolated / - / BB-gene edited HSPCs, a polynucleotide comprising the exogenous sequence of SEQ ID NO: 13 has been integrated at the / 788-locus in the genome of said HSPCs.

[0255] Other embodiments relate to isolated HSPCs comprising: (i) RNA encoding a TALE- nuclease comprising a monomer comprising the amino acid sequence of SEQ ID NO: 54 and a monomer comprising the amino acid sequence of SEQ ID NO: 55; and (ii) a non-viral polynucleotide repair template ssDNA comprising the nucleic acid sequence of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:15, or SEQ ID NO: 16. In further embodiments, the isolated HSPCs as described herewith comprise a RNA (e.g. mRNA) encoding an enhancer of Homology Directed Repair, such as a RNA comprising the nucleic acid sequence of SEQ ID NO: 26 and / or a RNA (e.g. mRNA) encoding a viability-enhancer, such as a RNA comprising the nucleic acid sequence of SEQ ID NO: 27.

[0256] 2.2. Pharmaceutical compositions

[0257] An aspect relates to a pharmaceutical composition comprising a population of / - / BB-gene edited HSPCs or isolated / - / BB-gene edited HSPC, as described herewith.

[0258] In some embodiments, said pharmaceutical composition further comprises a pharmaceutically acceptable excipient and / or carrier.

[0259] Suitable pharmaceutically acceptable excipients and carriers are well-known to the skilled person, and have been described in the literature, such as in Remington's Pharmaceutical Sciences, the Handbook of Pharmaceutical Additives or the Handbook of Pharmaceutical Excipients.

[0260] In some embodiments, it is provided a cryopreserved pharmaceutical composition comprising: (a) a viable composition of / - / BB-gene edited HSPCs; (b) an amount of cryopreservative sufficient for the cryopreservation of the HSPCs; and (c) a pharmaceutically acceptable carrier.

[0261] As used herein, "cryopreservation" refers to the preservation of cells by cooling to low subzero temperatures, such as (typically) 77 K or -196°C (the boiling point of liquid nitrogen). At these low temperatures, any biological activity, including the biochemical reactions that would lead to cell death, is effectively stopped.

[0262] “Cryomedium” refers to the medium used for the cryopreservation. Cryomedium comprises cryoprotective agents which are often used at sub-zero temperatures to preserve the cells from damage due to freezing at low temperatures or warming to room temperature. The injurious effects associated with freezing can be circumvented by (a) use of a cryoprotective agent, (b) control of the freezing rate, and (c) storage at a temperature sufficiently low to minimize degradative reactions. Cryoprotective agents which can be used include but are not limited to dimethyl sulfoxide (DMSO), glycerol, polyvinylpyrrolidine, polyethylene glycol, albumin, dextran, sucrose, ethylene glycol, i-erythritol, D-Sorbitol, D-mannitol, D-sorbitol, i-inositol, D-lactose, choline chloride, amino acids, methanol, acetamide, glycerol monoacetate, and inorganic salts. In a preferred embodiment, DMSO is used, a liquid which is nontoxic to cells in low concentration. Being a small molecule, DMSO freely permeates the cell and protects intracellular organelles by combining with water to modify its freezability and prevent damage from ice formation. Addition of plasma (e.g., to a concentration of 20-25%) can augment the protective effect of DMSO. After the addition of DMSO, cells should be kept at 0-4°C. until freezing, since DMSO concentrations of about 1% are toxic at temperatures above 4°C.

[0263] Also provided herewith is the pharmaceutical composition as described herewith for use in the treatment of a hemoglobinopathy associated with a mutation in / - / BB-gene, such as sickle cell disease.

[0264] Also provided herewith is the pharmaceutical composition as described herewith for use in hematopoietic stem cell transplantation.

[0265] 2.3. TALE-nucleases

[0266] Also provided herewith are the TALE-nucleases, either as monomers or dimers, as described above under point 1.2.1.

[0267] In some embodiments, the TALE-nuclease described herewith targets the polynucleotide sequence of SEQ ID NO: 7 and cleaves the / - / BB-gene within the region of polynucleotide sequence SEQ ID NO: 8.

[0268] In one embodiment is provided a monomeric TALE-nuclease comprising a monomer binding the HBB sequence of SEQ I D NO: 17 or SEQ I D NO: 19.

[0269] In a particular embodiment, said TALE-nuclease is a monomer binding the HBB sequence of SEQ ID NO: 17.

[0270] In a particular embodiment, said TALE-nuclease is a monomer comprising the RVD sequence HD-HD-NG-NN-NG-NN-NN-NI-NN-NI-NI-NN-NG-HD-NG-NG.

[0271] In a particular embodiment, said TALE-nuclease is a monomer comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% identity with SEQ ID NO: 54.

[0272] In another embodiment is provided a heterodimeric TALE-nuclease comprising a first monomer binding the HBB sequence of SEQ I D NO: 17 or SEQ I D NO: 19 and a second monomer binding the HBB sequence of SEQ ID NO: 18.

[0273] In a particular embodiment is provided a heterodimeric TALE-nuclease comprising a first monomer binding the HBB sequence of SEQ ID NO: 17 and a second monomer binding the HBB sequence of SEQ ID NO: 18. In a particular embodiment, said TALE-nuclease is a heterodimer comprising a monomer having the RVD sequence HD-HD-NG-NN-NG-NN-NN-NI-NN-NI-NI-NN-NG-HD-NG-NG and a monomer having the RVD sequence NG-HD-NI-HD-HD-NG-NG-NN-HD-HD-HD-HD-NI-HD-NI- NG.

[0274] In a particular embodiment, said TALE-nuclease is a heterodimer comprising a first monomer comprising the amino acid sequence of SEQ ID NO: 54, or a variant thereof comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 54, such as at least 90%, at least 95%, or at least 99% identity, and binding the sequence of SEQ ID NO: 17, and a second monomer comprising the amino acid sequence of SEQ ID NO: 55, or a variant thereof comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 55, such as at least 90%, at least 95%, or at least 99% identity, and binding the sequence of SEQ ID NO: 18.

[0275] In a particular embodiment, said TALE-nuclease is a heterodimer comprising a first monomer comprising the amino acid sequence of SEQ ID NO: 54 and a second monomer comprising the amino acid sequence of SEQ ID NO: 55.

[0276] 2.4. Polynucleotides and vectors

[0277] Also provided herewith are the polynucleotides encoding said TALE-nucleases and the polynucleotide repair template as described above under point 1.2.1 and 1.2.2.

[0278] Thus, also provided herewith is an isolated nucleic acid encoding the TALE-nuclease of the present invention.

[0279] According to some embodiments, said isolated nucleic acid encoding the TALE-nuclease is RNA. Said RNA encoding the TALE-nuclease described herewith can be linear mRNA or circular RNA.

[0280] According to some embodiments, said isolated nucleic acid encoding the TALE-nuclease is a circular RNA.

[0281] In one embodiment is provided an isolated polynucleotide that is a non-viral singlestranded polynucleotide repair template comprising the nucleic acid sequence of SEQ ID NO: 13, or comprising the nucleic acid sequence of SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16. In some embodiments, said polynucleotide repair template comprises an exogenous sequence comprising the sequence of SEQ ID NO: 13 flanked, on its 5’ (left) and 3’ (right) ends, by a nucleic acid portion having homology with the target endogenous / - / BB-gene to be edited.

[0282] In a particular embodiment, said polynucleotide is a non-viral single-stranded DNA (ssDNA) comprising SEQ ID NO: 16.

[0283] Said polynucleotide can be a linear single-stranded DNA.

[0284] Preferably, said polynucleotide is a circular single-stranded DNA.

[0285] In a further particular embodiment is provided an isolated polynucleotide that is a non-viral circular single-stranded DNA comprising SEQ ID NO: 16.

[0286] Also disclosed herewith are isolated polynucleotides encoding the HDR-enhancer and Viability-enhancers as described above under point 1.2.3.

[0287] Disclosed herewith is an isolated polynucleotide encoding an HDR-enhancer comprising the amino acid sequence of SEQ ID NO: 61.

[0288] In a particular disclosure, said polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 26.

[0289] Also disclosed herewith is a polynucleotide encoding an HDR-enhancer comprising the amino acid sequence of SEQ ID NO: 62.

[0290] Disclosed herewith is an isolated polynucleotide encoding a viability-enhancer of amino acid sequence of SEQ ID NO: 63.

[0291] A particular disclosure relates to an isolated polynucleotide having a sequence comprising SEQ ID NO: 27.

[0292] One aspect provides the ex vivo use of an isolated nucleic acid or vector as described herewith, comprising the exogenous sequence as defined herein, for / - / BB-gene editing HSPCs, notably HSPCs of a patient suffering from sickle cell disease.

[0293] Another aspect provides the ex vivo use of any of the TALE-nuclease defined herewith, the polynucleotide encoding same or the vector encoding same in combination with an isolated nucleic acid or vector as defined herewith, comprising the exogenous sequence as defined herein, for use in / - / BB-gene editing HSPCs, notably HSPCs of a patient suffering from sickle cell disease.

[0294] 2.5. Kits In a further aspect, it is disclosed a kit for carrying out the methods described herewith.

[0295] In one embodiment, provided is a kit comprising:

[0296] (i) at least one isolated polynucleotide or vector encoding a TALE-Nuclease heterodimer comprising a first monomer binding the HBB sequence of SEQ ID NO: 17 or SEQ ID NO: 19 and a second monomer binding the HBB sequence of SEQ ID NO: 18, and

[0297] (ii) at least one isolated non-viral single-stranded polynucleotide repair template comprising the nucleic acid sequence of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16, and,

[0298] (iii) optionally, at least one isolated polynucleotide or vector encoding an HDR- enhancer; and

[0299] (iv) optionally, at least one isolated polynucleotide or vector encoding a viabilityenhancer.

[0300] In a particular embodiment, the TALE-nuclease encoded by said polynucleotide or vector comprised in said kit is any TALE-Nuclease heterodimer described above under points 1.2.1.

[0301] For instance, said kit comprises an isolated polynucleotide or vector encoding a TALE- Nuclease heterodimer comprising the amino acid sequence of SEQ ID NO: 54 and SEQ ID NO: 55.

[0302] In a particular embodiment, the isolated polynucleotide repair template comprised in said kit is any one defined above under points 1.2.2.

[0303] For instance, said kit comprises a non-viral single-stranded polynucleotide repair template comprising the nucleic acid sequence of SEQ ID NO: 15.

[0304] For instance, said kit comprises a non-viral single-stranded polynucleotide repair template comprising the nucleic acid sequence of SEQ ID NO: 16.

[0305] In a particular embodiment, the HDR-enhancer comprised in said kit is selected from the group consisting of an inhibitor of 53BP1 , a dominant negative mutant of 53BP1 , an inhibitor of P53, and an inhibitor of Non-Homology-End-Joining, such as an enhancer of Homologous Recombination of SEQ ID NO: 61.

[0306] In a particular embodiment, the viability-enhancer comprised in said kit has the amino acid sequence of SEQ ID NO: 63.

[0307] Another aspect relates to the ex vivo use of the kit described herewith, for / - / BB-gene editing HSPCs, notably HSPCs from a patient suffering from a hemoglobinopathy, in particular sickle cell disease. In a particular embodiment, provided herewith is the ex vivo use of the kit described herewith for editing at least one endogenous / 788-allele comprising a mutation (A>T) or (A>C) at position 20 of the coding region of exon 1 of / 788-gene in the HSPCs of a patient suffering from a hemoglobinopathy associated with said mutation.

[0308] 3. Methods of treatment

[0309] The method for preparing / 788-gene edited HSPCs described herewith is preferably practiced ex vivo to obtain stably engineered / 788-edited HSPCs. The resulting engineered HSPCs can then be engrafted into a patient in need thereof for a long-term in vivo production of corrected cells that integrated the exogenous sequence described herein and, thus, express a functional hemoglobin.

[0310] Thus, also provided herewith is a method of treatment of a hemoglobinopathy associated with a mutation (A>T) or (A>C) in at least one endogenous / 788-allele at position 20 of the coding region of exon 1 of / 788-gene in a patient, comprising administering in said patient a population of / 788-gene edited HSPCs as described herewith, isolated / 788-gene edited HSPCs as described herewith, or a pharmaceutical composition as described herewith.

[0311] In one embodiment, is provided herewith a method for treating sickle cell disease in a patient, such as a human patient, wherein said patient has an endogenous / 788-allele having a mutation (A>T) at position 20 of the coding region of exon 1 of the / 788-gene, the method comprising administering engineered / 788-edited HSPCs as described herewith, a population of cells as described herewith, or a pharmaceutical composition as described herewith to said patient.

[0312] In another embodiment, is provided herewith a method for treating hemoglobin beta C disease in a patient, such as a human patient, wherein said patient has an endogenous HBB- allele having a mutation (A>C) at position 20 of the coding region of exon 1 of / 788-gene, the method comprising administering engineered / 788-edited HSPCs as described herewith, a population of cells as described herewith, or a pharmaceutical composition as described herewith to said patient.

[0313] Further provided is a method for hematopoietic stem cell transplantation in a patient, such as a human patient, having a mutation (A>T) or (A>C) in at least one endogenous / 788-allele at position 20 of the coding region of exon 1 of / 788-gene, the method comprising administering engineered / - / BB-edited HSPCs as described herewith, a population of cells as described herewith, or a pharmaceutical composition as described herewith to said patient.

[0314] Further provided is a method for hematopoietic stem cell transplantation in a patient, such as a human patient, having a mutation (A>T) in at least one endogenous / 788-allele at position 20 of the coding region of exon 1 of / 788-gene, the method comprising administering engineered / - / BB-edited HSPCs as described herewith, a population of cells as described herewith, or a pharmaceutical composition as described herewith to said patient.

[0315] One embodiment relates to a method of treatment of a hemoglobinopathy associated with a mutation (A>T) or (A>C) in at least one endogenous / 788-allele at position 20 of the coding region of exon 1 of / 788-gene in a patient, said method comprising administering / 788-gene edited HSPCs comprising a polynucleotide comprising the exogenous sequence SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16, integrated at the / 788-locus in the genome of said HSPCs, or a population of cells comprising said edited HSPCs, or a pharmaceutical composition comprising said edited HSPCs or said population of cells, to said patient.

[0316] In a particular embodiment, said method of treatment of a hemoglobinopathy comprises administering / 788-gene edited HSPCs comprising a polynucleotide comprising the exogenous sequence SEQ ID NO: 13 integrated at the / 788-locus in the genome of said HSPCs, or a population of cells comprising said edited HSPCs, or a pharmaceutical composition comprising said edited HSPCs or said population of cells, to said patient.

[0317] In a particular embodiment, said method of treatment of a hemoglobinopathy comprises administering / 788-gene edited HSPCs comprising a polynucleotide comprising the exogenous sequence SEQ ID NO: 15 integrated at the / 788-locus in the genome of said HSPCs, or a population of cells comprising said edited HSPCs, or a pharmaceutical composition comprising said edited HSPCs or said population of cells, to said patient.

[0318] In a particular embodiment, said method of treatment of a hemoglobinopathy comprises administering / 788-gene edited HSPCs comprising a polynucleotide comprising the exogenous sequence SEQ ID NO: 16 integrated at the / 788-locus in the genome of said HSPCs, or a population of cells comprising said edited HSPCs, or a pharmaceutical composition comprising said edited HSPCs or said population of cells, to said patient.

[0319] In another embodiment of the method of treatment as described herewith, the hemoglobinopathy is selected from the group consisting of sickle cell disease associated with a mutation (A>T) at position 20 of the coding region of exon 1 of HBB-gene, hemoglobin beta C disease associated with a mutation (A>C) at position 20 of the coding region of exon 1 of HBB- gene, and a severe hemoglobinopathy associated with mutations (A>T) and (A>C) at position 20 of the coding region of exon 1 of HBB-gene.

[0320] In an embodiment, is provided a method for treating sickle cell disease in a human patient having a mutation (A>T) in at least one endogenous HBB-a\\e\e at position 20 of the coding region of exon 1 of HBB-gene, said method comprising administering, in said patient, a population of HBB-gene edited HSPCs;

[0321] - wherein a polynucleotide comprising the exogenous sequence SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16, has been integrated at the / - / BB-locus in the genome of at least 10%, preferably at least 20%, 30%, 40%, 50%, 60 % of the cells of the population; and

[0322] - wherein, optionally, said population comprises at least 0.1%, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, or at least 30%, of cells having a [CD34+CD38-CD45RA-CD90+CD133+] phenotype; and

[0323] - wherein, optionally, the ratio of HDR / indels at the / - / BB-locus in the cell population is higher than 1 , higher than 1.2, 1.4, 1 .5, or 1.7, equal to or higher than about 2, equal to or higher than about 2.5, equal to or higher than about 3, equal to or higher than about 3.2, 3.5, 3.8, equal to or higher than about 4, 4.1 , or 4.2.

[0324] In a particular embodiment, the method for treating sickle cell disease in a human patient comprises administering in said patient a population of HBB-gene edited HSPCs,

[0325] - wherein a polynucleotide comprising the exogenous sequence of SEQ ID NO: 13 or SEQ ID NO: 16 has been integrated at the / - / BB-locus in the genome of at least 10%, preferably at least 20%, 30%, 40%, 50%, 60 % of the cells of the population; and

[0326] - wherein, optionally, said population comprises at least 0.1%, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, or at least 30%, of cells having a [CD34+CD38-CD45RA-CD90+CD133+] phenotype; and

[0327] - wherein, optionally, the ratio of HDR / indels in the HBB locus in the cell population is higher than 1 , higher than 1.2, 1.4, 1 .5, or 1.7, equal to or higher than about 2, equal to or higher than about 2.5, equal to or higher than about 3, equal to or higher than 3.2, 3.4, 3.5, 3.6, 3.8, equal to or higher than about 4, 4.1 or 4.2. Another particular embodiment relates to a method for treating hemoglobin beta C disease in a human patient having an endogenous / - / BB-gene having a mutation (A>C) at position 20 of the coding region of exon 1 of / - / BB-gene, said method comprising administering in said patient a population of / - / BB-gene edited HSPCs,

[0328] - wherein a polynucleotide comprising the exogenous sequence SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16, has been integrated at the / 788-locus in the genome of at least 10%, preferably at least 20%, 30%, 40%, 50%, 60 % of the cells of the population; and

[0329] - wherein, optionally, said population comprises at least 0.1%, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, or at least 30%, of cells having a [CD34+CD38-CD45RA-CD90+CD133+] phenotype; and

[0330] - wherein, optionally, the HDR / indels ratio in the / 788-locus in the cell population is higher than 1 , higher than 1.2, 1.4, 1 .5, or 1.7, equal to or higher than about 2, equal to or higher than about 2.5, equal to or higher than about 3, equal to or higher than 3.2, 3.4, 3.5, 3.6, 3.8, equal to or higher than about 4, 4.1 or 4.2.

[0331] In a particular embodiment, the method for treating hemoglobin beta C disease in a human patient comprises administering in said patient a population of / 788-gene edited HSPCs,

[0332] - wherein a polynucleotide comprising the exogenous sequence of SEQ ID NO: 13 or SEQ ID NO: 16 has been integrated at the / 788-locus in the genome of at least 10%, preferably at least 20%, 30%, 40%, 50%, 60 % of the cells of the population; and

[0333] - wherein, optionally, said population comprises at least 0.1%, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, or at least 30%, of cells having a [CD34+CD38-CD45RA-CD90+CD133+] phenotype; and

[0334] - wherein, optionally, the HDR / indels ratio at the / 788-locus in the cell population is higher than 1 , higher than 1.2, 1.4, 1 .5, or 1.7, equal to or higher than about 2, equal to or higher than about 2.5, equal to or higher than about 3, equal to or higher than 3.2, 3.4, 3.5, 3.6, 3.8, equal to or higher than about 4, 4.1 or 4.2.

[0335] Generally, the treatment of hemoglobinopathy (such as sickle cell disease and hemoglobin beta C disease) related to an / 788-gene mutation according to the invention can be ameliorating, curative or prophylactic.

[0336] The administration of the / 788-edited HSPCs or population of cells as described herewith may be carried out in any convenient manner, including injection, transfusion, implantation or transplantation. The engineered / 788-edited HSPCs or population of cells may be administered to a patient by intravenous injection. The administration of the edited HSPCs or population of cells as described herewith can consist in the administration of 104to 108 / - / BB-edited cells per patient’s kg body weight, preferably 105to 107cells / kg body weight including all integer values of cell numbers within those ranges, e.g. from about 1x106to about 10x106, including for instance from 1x106to 5x106 / - / BB-edited cells per kg body weight.

[0337] The administration of the edited HSPCs or population of cells as described herewith can consist in the administration of a flat dose of at least 1x106cells, more than 10x106cells, more than 100x106cells, more than 200x106cells, such as from 100x106to 500x106cells.

[0338] The engineered HSPCs or population of described herewith can be administrated in one or more doses. According to some embodiments, the therapeutic effective amount of cells is administrated as a single dose. According to some embodiments, the therapeutic effective amount of cells is administrated as more than one dose over a period time. Timing of administration is within the judgment of managing physician and depends on the clinical condition of the patient. The dosage administered will be dependent upon the age, health and weight of the patient receiving the treatment, the kind of concurrent treatment, if any, frequency of treatment and the nature of the effect desired.

[0339] Also provided herewith are engineered / 788-gene edited HSPCs as described herewith, a population of cells as described herewith or a pharmaceutical composition as described herewith for use in the treatment of a hemoglobinopathy associated with a mutation (A>T) or (A>C) in at least one endogenous / 788-allele at position 20 of the coding region of exon 1 of / 788-gene in a patient.

[0340] In one embodiment, said patient is homozygous for the (A>T) mutation at position 20 of the coding region of exon 1 of / 788-gene and suffers from sickle cell disease.

[0341] In another embodiment, said patient is homozygous for the (A>C) mutation at position 20 of the coding region of exon 1 of / 788-gene and suffers from Hemoglobin beta C disease.

[0342] In a still other embodiment, said patient is heterozygous for this position and carries the (A>T) and (A>C) mutations at position 20 of the coding region of exon 1 of the two alleles of the / 788-gene and suffers from a severe hemoglobinopathy.

[0343] Thus, also provided herewith are engineered / 788-edited HSPCs as described herewith, a population of cells as described herewith or a pharmaceutical composition as described herewith for use in the treatment of sickle cell disease in a patient, such as a human patient, having an endogenous / - / BB-gene having a mutation (A>T) at position 20 of the coding region of exon 1 of / 788-gene.

[0344] Also provided herewith are engineered / 788-edited HSPCs as described herewith, a population of cells as described herewith or a pharmaceutical composition as described herewith for use in the treatment of Hemoglobin beta C disease in a patient, such as a human patient, having an endogenous / 788-gene having a mutation (A>C) at position 20 of the coding region of exon 1 of / 788-gene.

[0345] Also provided herewith are engineered / 788-edited HSPCs as described herewith, a population of cells as described herewith or a pharmaceutical composition as described herewith for use in the treatment of severe hemoglobinopathy in a patient, such as a human patient, having the mutations (A>T) and (A>C) at position 20 of the coding region of exon 1 of / 788-gene of the two alleles of the endogenous / 788-gene.

[0346] In one embodiment are provided / 788-gene edited HSPCs comprising a polynucleotide comprising the exogenous sequence SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16 integrated at the / 788-locus in the genome of said HSPCs for use in the treatment of a hemoglobinopathy associated with at least one mutation at position 20 of the coding region of exon 1 of / 788-gene in a patient.

[0347] In one embodiment are provided / 788-gene edited HSPCs comprising a polynucleotide comprising the exogenous sequence SEQ ID NO: 13 integrated at the / 788-locus in the genome of said HSPCs for use in the treatment of a hemoglobinopathy associated with at least one mutation at position 20 of the coding region of exon 1 of / 788-gene in a patient.

[0348] In a particular embodiment are provided / 788-gene edited HSPCs comprising a polynucleotide comprising the exogenous sequence SEQ ID NO: 15 or SEQ ID NO: 16 integrated at the / 788-locus in the genome of said HSPCs for use in the treatment of a hemoglobinopathy associated with at least one mutation at position 20 of the coding region of exon 1 of / 788-gene in a patient.

[0349] In another particular embodiment are provided / 788-gene edited HSPCs comprising a polynucleotide comprising the exogenous sequence SEQ ID NO: 16 integrated at the / 788-locus in the genome of said HSPCs for use in the treatment of a hemoglobinopathy associated with at least one mutation at position 20 of the coding region of exon 1 of / 788-gene in a patient. Generally, for the uses and in the methods of treatments described herewith, the HBB- gene edited HSPCs derive from the patient to be treated. Thus, in some embodiments, the methods of treatment comprise administering autologous / - / BB-edited HSPCs.

[0350] 4. Improved method for editing HSPCs

[0351] Another aspect of the present invention relates to an ex vivo method for preparing a population of cells enriched in gene-edited HSPCs with enhanced engraftment capacity and therapeutic potential suitable for use in gene therapy.

[0352] In one aspect of the invention, the gene-editing comprises correcting, ex vivo, a deleterious mutation in an endogenous gene of HSPCs from a patient suffering from a disorder associated with said deleterious mutation or integrating an exogenous functional gene sequence (i.e. without the deleterious mutation) in the genome of said patient’s HSPCs without necessarily correcting the endogenous mutated allele, thereby producing edited HSPCs comprising a functional gene sequence in their genome.

[0353] Depending on the type, number, localizations, of the deleterious mutations, the skilled person is perfectly able to determine whether it is more appropriate to correct the endogenous mutated allele or to integrate an exogenous functional gene sequence. The skilled person is also perfectly able to determine whether the endogenous mutated allele needs to be inactivated (e.g. by deletion) or not when a corrected functional gene sequence is integrated in the HSPC’s genome.

[0354] After having been gene edited, the restoration of the normal cellular phenotype in the HSPCs or cells derived therefrom is obtained. The gene-edited HSPCs can then be administered to the patient and treat the patient suffering from a disorder associated with said deleterious mutation.

[0355] Thus, provided herewith is an ex vivo method for preparing a population of cells enriched in viable gene-edited Hematopoietic Stem and Progenitor Cells (HSPCs) from a population of HSPCs comprising a gene having a deleterious mutation, said method comprising introducing, into a population of HSPCs having the deleterious mutation:

[0356] (i) a TALE-nuclease cleaving a specific target locus; and

[0357] (ii) a non-viral circular single-stranded polynucleotide repair template; wherein the edited cells have integrated the polynucleotide repair template at the targeted locus; whereby a population of cells is obtained which comprises at least 20% of edited cells and at least 70%, at least 80%, or at least 90%, of viable cells.

[0358] Any standard method known to the skilled person can be used to determine viability, including Nucleocounter and flow cytometry as illustrated in the Example section.

[0359] In some embodiments, said population of cells enriched in viable edited HSPCs comprises long-term repopulating HSC (LT-HSC).

[0360] In some embodiments, said population of cells enriched in viable edited HSPCs comprises at least 0.1%, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, or at least 30%, of LT-HSCs.

[0361] According to some embodiments, said population of cells comprises short-term repopulating HSC (ST-HSC).

[0362] According to some embodiments, said population of cells comprises at least CD34+cells.

[0363] According to some embodiments, said population of cells comprises at least CD34+, CD90+, and CD133+cells.

[0364] According to some embodiments, said population of cells comprises at least CD34+, CD38' , CD45RA; CD90+, and CD133+cells.

[0365] According to some embodiments, said population of cells comprises at least CD34+, CD38' , and CD45RA' cells.

[0366] According to some embodiments, said population of cells comprises at least CD34+, CD38' , CD45RA; CD90; and CD133’ cells.

[0367] According to some embodiments, said population of cells comprises at least 0.1%, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, or at least 30%, of cells having a [CD34+CD38-CD45RA-CD90+CD133+] phenotype.

[0368] According to some embodiments, the ratio of HDR / indels at the targeted-locus in the cell population is equal to or higher than 1 , equal to or higher than 1.2, 1.4, 1.5, or 1.7, equal to or higher than about 2, equal to or higher than about 2.5, equal to or higher than about 3, equal to or higher than 3.2, 3.4, 3.5, 3.6, 3.8, equal to or higher than about 4, or 4.2.

[0369] In some embodiments, said TALE-nuclease cleaves the HSPCs’ genomic DNA at a targeted locus selected from: (i) a non-functional endogenous gene in HSPCs, wherein said non- functional gene is associated with a disorder in a patient having said HSPCs, and (ii) a genomic safe harbor locus.

[0370] As indicated elsewhere in the present application, targeted (i.e. site-directed) integration to achieve gene repair or integration of a functional gene sequence is suitably done by using sequence-specific nucleases (e.g. TALE-nucleases) inducing DNA cleavage and exogenous polynucleotide donor templates bearing homology to the target site and comprising the corrective or functional gene sequence. By “site directed integration”, it is meant that integration of the polynucleotide repair template occurs at the locus where DNA cleavage is induced by the sequence-specific nuclease.

[0371] TALE-nucleases useful in the ex vivo method described in this section can be monomeric or dimeric. In general, TALE-nucleases have the structure described elsewhere in this application.

[0372] In a particular embodiment, said TALE-nuclease is as described under sections 1.2.1 and 2.3 and cleaves at least one / 788-allele having a mutation (A>T) or (A>C) at position 20 of the coding region of exon 1 of / - / BB-gene.

[0373] In a particular embodiment, said TALE-nuclease targeting HBB is a heterodimer comprising a monomer having the RVD sequence HD-HD-NG-NN-NG-NN-NN-NI-NN-NI-NI-NN- NG-HD-NG-NG and a monomer having the RVD sequence NG-HD-NI-HD-HD-NG-NG-NN-HD- HD-HD-HD-NI-HD-NI-NG.

[0374] In a particular embodiment, said TALE-nuclease targeting HBB is a heterodimer comprising a first monomer comprising the amino acid sequence of SEQ ID NO: 54, or a variant thereof comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 54, such as at least 90%, at least 95%, or at least 99% identity, and binding the sequence of SEQ ID NO: 17, and a second monomer comprising the amino acid sequence of SEQ ID NO: 55, or a variant thereof comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 55, such as at least 90%, at least 95%, or at least 99% identity, and binding the sequence of SEQ ID NO: 18.

[0375] In a particular embodiment, said TALE-nuclease targeting HBB is a heterodimer comprising a first monomer comprising the amino acid sequence of SEQ ID NO: 54 and a second monomer comprising the amino acid sequence of SEQ ID NO: 55.

[0376] In another embodiment, said TALE-nuclease cleaves in a genomic safe harbor locus. Genomic safe harbors are regions of the genome that can maintain an exogenous gene expression without affecting the function of host cells. Ideally, insertion into a safe harbor locus should have no impact on the expression of other genes.

[0377] In particular embodiments, a genomic safe harbor locus is more than 150 kb, more than 200 kb, or more than 300 kb, away from a known oncogene, and more than 30 kb, or more than 40 kb, away from a known transcription start site; and has no overlap with coding mRNA.

[0378] In particular embodiments, a genomic safe harbor locus is: (a) located at a distance of more than about 50 kb from the 5' end of each gene of the genome; (b) located at a distance of more than about 300 kb from each cancer-related gene of the genome; (c) located outside each gene transcription unit of the genome; (d) located outside of each ultra-conserved region of the genome; (e) located outside of each non-coding RNA region of the genome; and (f) located at a distance more than about 300 kb from each microRNA (miRNA) of the genome.

[0379] Examples of safe harbor loci include AAVS1 (located in an intron of the PPP1 R12C gene region), CCR5 (encoding a protein involved in chemotaxis and serving as a co-receptor for HIV cellular entry in T cells), hROSA26, ROGI1 (on chromosome 1 (q31.3), coordinates 195,338,589- 195,818,588), ROGI2 (on chromosome 3 (p24.3), coordinates 22,720,711-22,761 ,389), GSH7 (on chromosome 7 (q35), coordinates 145,090,941-145,219,513), GSH8 (on chromosome 7 (q35), coordinates 145,320,384-145,525,881), GSH31 (on chromosome X (q21.31), coordinates 89,174,426-89,179,074). Other examples of genomic safe harbors include the blood-specific sites BLD_GSH_1 to BLD_GSH_19 identified in Table 1 of Dewan et al. (Genome Biology, 2022, 23: 199) and the brain-specific sites BRN_GSH_1 to BRN_GSH_5 identified in T able 2 of the same paper. Still other examples include the SH3 locus on human chromosome 6p25.1 , the SH4 locus on human chromosome 7q31.2, the SH6 locus on human chromosome 21q21 .1 , the SH12 locus on human chromosome 13q34, the SH13 locus on human chromosome 3p12.2, the SH19 locus on human chromosome 22, the SH20 locus on human chromosome 12q21.2, the SH21 locus on human chromosome 3p24.1 , the SH33 locus on human chromosome 6p12.2, the SH7 locus on human chromosome 2p16.1 and the SH8 locus on human chromosome 5.

[0380] Other examples include the genomic safe harbor sites represented by SEQ ID NOs. 1-194 provided in WO2018226762 (see also Table 3 of WO2018226762).

[0381] Other examples of genomic safe harbor loci include the loci of sequences SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 75, said sequence numbers corresponding to the sequences disclosed in US20220211761.

[0382] In one embodiment, the polynucleotide repair template comprises an exogenous sequence bearing homology to the gene-target site of the TALE-nuclease and comprising the corrective or functional gene-sequence. The polynucleotide repair template can comprise the full-length or a fragment of the functional gene sequence to be integrated in the cells’ genome.

[0383] In particular embodiments, to avoid that the TALE-nuclease cleaves the gene that has been edited (corrected), silent mutations are added in the sequence of the polynucleotide repair template, for instance about 3 to 10 silent mutations, such as 3, 4, 5, 6, 7, 8, 9 or 10 silent mutations. For instance, the polynucleotide repair template sequence comprises the corrected base at the position corresponding to the original mutation of the endogenous mutated gene and 4 silent mutations, wherein at least one, at least two, or at least three of the silent mutations are placed on the 5’-side and / or on the 3’-side of the corrected base. Preferably, said silent mutations are positioned in the DNA binding sites of the TALEN to avoid that the TALEN binds and cleaves the neo-inserted repair template.

[0384] A silent mutation corresponds to the change of a single nucleotide within a protein-coding portion of a gene that does not affect the amino acids sequence of the protein encoded by the gene.

[0385] In order to facilitate targeted (i.e. site-directed) integration of the exogenous sequence via homologous recombination, said exogenous sequence comprises a left (5’) and a right (3’) homology sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least 99% identity with the left (5’) and right (3’) regions of the endogenous gene to be edited, respectively. Said left (5’) and right (3’) regions of said endogenous gene to be edited are portions (e.g. from 50 to 500 nucleotide-long portions) of said endogenous gene and are positioned at the left (5’) and right (3’) side of the nucleotide(s) to be corrected in the target endogenous locus. Said left (5’) and right (3’) homologous regions are generally from about 20 to about 500 nucleotide-long (such as from about 40 to about 500, from about 50 to about 400, from about 50 to about 350, from about 50 to about 325, from about 90 to about 310, from about 85, 86, 87, 88, 89, or 90 to about 295, 296, 297, 298, 299, 300, 301 , 302, 303, 304, or 305 nucleotide-long).

[0386] In one embodiment, said ex vivo method uses the TALE-nuclease and repair templates for correcting the / - / BB-gene described above, in particular in sections 1.2.1 and 2.3, and 1.2.2 and 2.4, respectively. In a particular embodiment, said ex vivo method uses a TALE-nuclease targeting HBB comprising a first monomer comprising the amino acid sequence of SEQ ID NO: 54 and a second monomer comprising the amino acid sequence of SEQ ID NO: 55, and a non-viral circular singlestranded polynucleotide repair template comprising the nucleic acid sequence of SEQ ID NO: 16.

[0387] The ex vivo method described herewith is not limited to / - / BB-gene correction and can apply to other genes, in particular to genes associated with monogenic disorders such as those listed in Table 1. Table 1. Monogenic diseases and functional gene for their treatment.

[0388] Thus, in other embodiments, the polynucleotide repair template comprises the coding sequence of a gene selected from IDUA, IDS, ARSB, GUSB, ABCD1, GALC, ARSA, PSAP, GBA, FUCA 1, MAN2B1, AGA, ASAH1, HEXA, GAA, SMPD1, LIPA and CDKL5.

[0389] Thus, in some embodiments, the polynucleotide repair template comprises the coding sequence of any one of SEQ ID NOs: 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, and 98.

[0390] When inserting an exogenous gene sequence within a genomic safe harbor, the polynucleotide repair template will include a coding region and all regulatory elements required for its expression.

[0391] In some embodiments, the HSPCs to be edited are mammalian HSPCs, and preferably human HSPCs. More particularly, the HSPCs are human HSPCs from a patient suffering from a disorder associated with the expression of a non-functional gene in his / her HSPCs, or the lack of expression of a functional gene in his / her HSPCs, or in cells derived therefrom. Said nonfunctional gene comprises said deleterious mutation(s) and is the gene to be edited.

[0392] In some embodiments, the ex vivo methods described in this section use the culture and transfection conditions described in section 1.2.3.

[0393] In some embodiments of the ex vivo methods described in this section, the TALE-nuclease cleaving a specific target locus of step (i) is introduced by electroporation of a RNA (linear mRNA or circular RNA) encoding said TALE-nuclease.

[0394] In some embodiments of the ex vivo method described in this section the non-viral circular single-stranded polynucleotide repair template of step (ii) is a ssDNA introduced by electroporation.

[0395] In a particular embodiment of the ex vivo method described herewith the TALE-nuclease cleaving a specific target locus of step (i) and the non-viral circular single-stranded polynucleotide repair template of step (ii) are introduced via a single transfection (e.g. electroporation) step. In another aspect of the invention, the gene-editing comprises editing HSPCs from a healthy donor so as to prevent rejection of transplanted edited HPSCs by a recipient who is not the donor due to differences in the major histocompatibility complex.

[0396] The major histocompatibility complex (MHC) is a cell surface multi-component molecule found in all vertebrates that mediates interactions of leukocytes with other leukocytes or other cells. The MHC gene family is divided into three groups: class I, class II and class III. In humans, MHC is referred to as human leukocyte antigen (HLA). The HLA class I (HLA-I) protein is expressed on all nucleated cells and consists of an HLA class I heavy chain (or alpha chain) and P-2 microglobulin (B2M). HLA class I protein presents peptides on the cell surface to CD8+ cytotoxic T cells. Six HLA class I alpha chains have been identified to date, including three classical (HLA-A, HLA-B and HLA-C) and three non-classical (HLA-E, HLA-F and HLA-G) alpha chains. The specificity for peptide binding on the HLA class I molecule peptide binding cleft is determined by the alpha chain. Recognition by CD8+ T cells of the peptides presented by the HLA class I molecule mediates cellular immunity. The HLA class I protein from an allogeneic source constitutes in itself a foreign antigen in the context of transplantation. The recognition of non-self HLA class I protein is a major hurdle in using pluripotent cells for transplantation or replacement therapies. Inactivating B2M prevents the expression of HLA ABC at the surface of edited cells which will not be recognized by allogeneic host’s T-cells.

[0397] However, B2M KO edited cells can become the target of NK cells through a mechanism known as missing self-recognition. Therefore, in the context of B2M KO, it is preferable to edit the cells further to prevent such NK-based attack and depletion. One possibility is to endow edited cells with a NK-specific inhibitor such as the non-polymorphic and chimeric HLA-E or CD47, two complexes known to strongly inhibit NK cell-mediated recognition and depletion of HLA ABC KO edited cells.

[0398] Thus, an embodiment of the ex vivo method for preparing a population of cells enriched in gene-edited HSPCs with enhanced engraftment capacity and therapeutic potential suitable for use in gene therapy as described herewith, comprises inactivating the endogenous B2 / W-gene and, optionally, integrating HLA-E gene and / or CD47 gene.

[0399] Thus, provided herewith is an ex vivo method for preparing a population of cells enriched in viable gene-edited Hematopoietic Stem and Progenitor Cells (HSPCs) for allogeneic transplantation, said method comprising introducing, into said population of HSPCs from a healthy donor: (i) a TALE-nuclease cleaving a specific target locus such as B2M locus or a genomic safe harbor locus; and

[0400] (ii) a non-viral circular single-stranded polynucleotide repair template comprising a nucleic acid sequence encoding a NK inhibitor such as HLA-E and CD47; wherein the edited cells have integrated the polynucleotide repair template at the targeted locus; whereby a population of cells is obtained which comprises at least 20% of edited cells and at least 70%, at least 80%, or at least 90%, of viable cells.

[0401] According to some embodiments, the ratio of HDR / indels at the targeted-locus in the cell population obtained by said method is equal to or higher than 1 , equal to or higher than 1.2, 1.4, 1.5, or 1.7, equal to or higher than about 2, equal to or higher than about 2.5, equal to or higher than about 3, equal to or higher than 3.2, 3.4, 3.5, 3.6, 3.8, equal to or higher than about 4, or 4.2.

[0402] According to some embodiments, said population of cells comprises short-term repopulating HSC (ST-HSC).

[0403] According to some embodiments, said population of cells comprises at least CD34+cells.

[0404] According to some embodiments, said population of cells comprises at least CD34+, CD90+, and CD133+cells.

[0405] According to some embodiments, said population of cells comprises at least CD34+, CD38' , CD45RA; CD90+, and CD133+cells.

[0406] According to some embodiments, said population of cells comprises at least CD34+, CD38' , and CD45RA' cells.

[0407] According to some embodiments, said population of cells comprises at least CD34+, CD38' , CD45RA; CD90; and CD133’ cells.

[0408] According to some embodiments, said population of cells comprises at least 0.1%, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, or at least 30%, of cells having a [CD34+CD38-CD45RA-CD90+CD133+] phenotype.

[0409] In a particular embodiment, said TALE-nuclease is as described in Example 6 and cleaves the B2M locus (TALEN-B2M). In a particular embodiment, said TALE-nuclease cleaving the B2M locus comprises two monomers encoded by the nucleic acid sequences SEQ ID NO: 47 and SEQ ID NO: 48, respectively.

[0410] In further particular embodiments, said TALE-nuclease cleaving the B2M locus is a heterodimer comprising a monomer having the RVD sequence NG-NI-NN-HD-NG-NN-NG-NN- HD-NG-HD-NN-HD-NN-HD-NG and a monomer having the RVD sequence NN-NN-NI-NG-NI-NN- HD-HD-NG-HD-HD-NI-NN-NN-HD-NG.

[0411] In a particular embodiment, said TALE-Nuclease targeting B2M comprises a monomer of amino acid sequence SEQ ID NO: 59 and a monomer of amino acid sequence SEQ ID NO: 60.

[0412] The genomic safe harbor loci are as described above.

[0413] Some embodiments of this aspect use the culture and transfection conditions described in section 1.2.3.

[0414] In some embodiments of this aspect, the TALE-nuclease cleaving a specific target locus of step (i) is introduced by electroporation of a RNA (linear mRNA or circular RNA) encoding said TALE-nuclease.

[0415] In some embodiments of this aspect, the non-viral circular single-stranded polynucleotide repair template of step (ii) is a ssDNA introduced by electroporation.

[0416] In a particular embodiment of this aspect, the TALE-nuclease cleaving a specific target locus of step (i) and the non-viral circular single-stranded polynucleotide repair template of step (ii) are introduced via a single transfection (e.g. electroporation) step.

[0417] Also provided herewith is a kit containing any one or more of the elements disclosed in the ex vivo methods and compositions described in this entire section.

[0418] In particular embodiments, a kit includes a TALE-nuclease and an isolated non-viral circular single-stranded polynucleotide repair template as disclosed herewith. Elements may be provided individually or in combination, and may be provided in any suitable container, such as a vial, a bottle, a bag or a tube. In some embodiments, the kit includes instructions in one or more languages, for example in more than one language.

[0419] Another aspect relates to the gene-edited HSPCs obtained by the ex vivo methods described in this section, a pharmaceutical composition comprising the same, and the use thereof in gene therapy or for transplantation in a patient in need thereof.

[0420] The above written description of the invention provides a manner and process of making and using it such that any person skilled in this art is enabled to make and use the same, this enablement being provided in particular for the subject matter of the appended claims, which make up a part of the original description. Having generally described this invention, a further understanding can be obtained by reference to certain specific examples, which are provided herein for purposes of illustration only, and are not intended to limit the scope of the claimed invention.

[0421] Specific embodiments include the following items:

[0422] 1. An ex vivo method for preparing / - / BB-gene edited Hematopoietic Stem and Progenitor Cells (HSPCs) from a population of HSPCs comprising at least one endogenous HBB-allele having a mutation (A>T) or (A>C) at position 20 of the coding region of exon 1 of / - / BB-gene, wherein said mutation is associated with a hemoglobinopathy, said method comprising the steps of: i) introducing, into a population of HSPCs having an allele of an endogenous / - / BB-gene having said mutation, a monomeric TALE-nuclease comprising a monomer binding the HBB sequence of SEQ ID NO: 17 or SEQ ID NO: 19, or a heterodimeric TALE-nuclease comprising a first monomer binding the HBB sequence of SEQ I D NO: 17 or SEQ I D NO: 19 and a second monomer binding the HBB sequence of SEQ ID NO: 18; ii) introducing into said population of HSPCs a non-viral single-stranded polynucleotide repair template comprising the nucleic acid sequence of SEQ ID NO: 13, or comprising the nucleic acid sequence of SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16; whereby a population of / - / BB-gene edited HSPCs is obtained, which comprises HSPCs having at least one allele of the / - / BB-gene corrected at position 20 of the coding region of exon 1 of / - / BB-gene.

[0423] 2. The method according to item 1 , wherein the population of HSPCs having said mutation (A>T) is obtained from a patient suffering from sickle cell disease; or wherein the population of HSPCs having said mutation (A>C) is obtained from a patient suffering from hemoglobin beta C disease.

[0424] 3. The method according to any one of items 1 to 2, wherein said mutation is the mutation (A>T) at position 20 of the coding region of exon 1 of the / - / BB-gene and the population of HSPCs is obtained from a patient suffering from sickle cell disease.

[0425] 4. The method according to any one of claims 1 to 3, wherein said TALE-nuclease targets the polynucleotide sequence of SEQ ID NO: 7 and cleaves the / - / BB-gene within the region of polynucleotide sequence SEQ ID NO: 8. 5. The method according to any one of items 1 to 4, wherein said TALE-nuclease is a monomer comprising the RVD sequence HD-HD-NG-NN-NG-NN-NN-NI-NN-NI-NI-NN-NG-HD-NG-NG.

[0426] 6. The method according to item 5, wherein said TALE-nuclease is a monomer comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% identity with SEQ ID NO: 54.

[0427] 7. The method according to any one of items 1 to 4, wherein said TALE-nuclease is a heterodimer comprising a monomer binding SEQ ID NO: 17 and a monomer binding SEQ ID NO: 18.

[0428] 8. The method according to item 7, wherein said TALE-nuclease is a heterodimer comprising a monomer having the RVD sequence HD-HD-NG-NN-NG-NN-NN-NI-NN-NI-NI-NN-NG-HD- NG-NG and a monomer having the RVD sequence NG-HD-NI-HD-HD-NG-NG-NN-HD-HD- HD-HD-NI-HD-NI-NG.

[0429] 9. The method according to item 8, wherein said TALE-nuclease is a heterodimer comprising a monomer comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% identity with SEQ ID NO: 54 and a monomer comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% identity with SEQ ID NO: 55.

[0430] 10. The method according to any one of items 1 to 9, wherein said TALE-nuclease is introduced by electroporation of a RNA encoding said TALE-nuclease.

[0431] 11. The method according to item 10, wherein said RNA is linear mRNA or circular RNA.

[0432] 12. The method according to any one of items 10 to 11 , wherein the RNA encoding the TALE- nuclease is introduced by electroporation from 24 to 72 hours, such as from 24 to 48 hours or from 36 to 72 hours, after thawing of frozen HSPCs.

[0433] 13. The method according to any one of items 1 to 12, comprising introducing into said cells at least one enhancer of Homology Directed Repair (“HDR-enhancer”) selected from the group consisting of an inhibitor of 53BP1 , a dominant negative mutant of 53BP1 , an inhibitor of P53, and an inhibitor of Non-Homology-End-Joining.

[0434] 14. The method according to item 13, wherein said HDR-enhancer is a polypeptide comprising an amino acid sequence having at least 80% identity with SEQ ID NO: 61 , such as at least 90%, 95%, 98%, 99% identity. 15. The method according to item 14, wherein said HDR-enhancer is a polypeptide comprising an amino acid sequence having at least 80% identity with SEQ ID NO: 62, such as at least 90%, 95%, 98%, 99% identity.

[0435] 16. The method according to any one of items 13 to 15, wherein said HDR-enhancer is introduced by electroporation of a RNA encoding said HDR-enhancer.

[0436] 17. The method according to item 16, wherein said RNA encoding said HDR-enhancer is linear mRNA or circular RNA.

[0437] 18. The method according to item 16 or 17, wherein the cells are transfected with at least 0.1 pg, at least 1 pg, or at least 2 pg, preferably at least 4 pg RNA of SEQ ID NO: 26 for about 1.106 cells.

[0438] 19. The method according to any one of items 1 to 18, comprising introducing into said cells at least one viability-enhancer.

[0439] 20. The method according to item 19, wherein said viability-enhancer is an inhibitor of BAX / BAK selected from the group consisting of B-cell lymphoma-extra large (BCL-XL), B-cell lymphoma 2 (BCL-2), Myeloid cell leukemia 1 (MCL-1), BCL2 like 10 (BCL2L10, BCL-B or DIVA), BCL2 related protein A1 (BFL-1 or A1), BCL2 like 2 (BCL-W).

[0440] 21. The method according to item 19 or 20, wherein said viability-enhancer is B-cell lymphoma- extra-large (BCL-XL).

[0441] 22. The method according to any one of items 19 to 21 , wherein said viability-enhancer is a polypeptide comprising the amino acid sequence of SEQ ID NO: 63.

[0442] 23. The method according to any one of items 19 to 22, wherein said viability-enhancer is introduced by electroporation of a RNA encoding said viability-enhancer.

[0443] 24. The method according to item 23, wherein said RNA encoding said viability-enhancer is linear mRNA or circular RNA.

[0444] 25. The method according to item 24, wherein the cells are transfected with at least 0.1 pg, or at least 0.5 pg, preferably at least 1 pg of RNA encoding said viability-enhancer, for 1.106cells total. 26. The method according to any one of items 13 to 25, wherein said HDR-enhancer and / or said viability-enhancer is / are electroporated concomitantly to the RNA encoding the TALE- nuclease.

[0445] 27. The method according to any one of items 1 to 26, comprising placing the cells in a culture medium comprising a sternness-enhancer, such as Valproic Acid (VPA), for at least one day.

[0446] 28. The method according to any one of items 1 to 27, comprising, prior to step i), a step of culturing the HSPCs in a culture medium comprising at least one compound stimulating cell cycle, for at least about 1 day.

[0447] 29. The method according to any one of items 1 to 28, wherein the HSPCs are cultured, prior to step i), in a culture medium comprising one or more of: Thrombopoietin (TPO), Fms-like tyrosine kinase 3 ligand (Flt-3L), Stem Cell Factor (SCF), Interleukin 3 (IL-3), hydrolyzed Polyvinyl Alcohol (PVA), LIM171 , SR1 , aminoquinoline, and dmPGE2.

[0448] 30. The method according to item 29, wherein said culture medium comprises Thrombopoietin (TPO), Interleukin 3 (IL-3), and stem cell factor (SCF).

[0449] 31 . The method according to any one of items 1 to 30, wherein said repair template is a singlestranded DNA (’’ssDNA”).

[0450] 32. The method according to item 31 , wherein said ssDNA comprises the polynucleotide sequence SEQ ID NO: 16.

[0451] 33. The method of any one of items 1 to 32, wherein said repair template is a circular singlestranded DNA (“CssDNA”).

[0452] 34. The method according to any one of items 1 to 33, comprising two electroporation steps.

[0453] 35. The method according to any one of items 1 to 34, comprising introducing the RNA encoding the TALE-nuclease in a first electroporation step and introducing the polynucleotide repair template in a second electroporation step.

[0454] 36. The method according to any one of items 31 to 35, wherein the ssDNA is electroporated from 10 to 20 hours or from 12 to 18 hours, or from 15 to 17 hours, such as about 16 hours, after the electroporation of the RNA encoding the TALE-nuclease. 37. The method according to item 33, wherein step i) of introducing the RNA encoding the TALE- nuclease and step ii) of introducing the circular single-stranded DNA are carried out in one electroporation step.

[0455] 38. The method according to any one of items 1 to 37, wherein, after step i), the HSPCs are incubated at 30°C, overnight, then cultured at 37°C.

[0456] 39. The method according to any one of items 1 to 38, further comprising recovering the population of / 788-gene edited HSPCs in a cryomedium and storing frozen until use, optionally without purification step before conditioning as a pharmaceutical composition comprising said HBB- gene edited HSPCs.

[0457] 40. A population of / - / BB-gene edited HSPCs obtainable by the method according to any one of items 1 to 39.

[0458] 41 . The population of / - / BB-gene edited HSPCs according to item 40, wherein a polynucleotide comprising the exogenous sequence SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16, has been integrated at the / 788-locus in the genome of at least 10%, preferably at least 20%, 30%, 40%, 50%, 60 % of the cells.

[0459] 42. The population of / - / BB-gene edited HSPCs according to item 40 or 41 , wherein a polynucleotide comprising the exogenous sequence of SEQ ID NO: 13 has been integrated at the / 788-locus in the genome of at least 10%, preferably at least 20%, 30%, 40%, 50%, 60,%, 70%, 80%, of the cells.

[0460] 43. The population of / 788-gene edited HSPCs according to any one of items 40 to 42, wherein said population comprises at least at least 0.1%, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, or at least 30%, of cells having a [CD34+CD38-CD45RA- CD90+CD133+] phenotype.

[0461] 44. The population of / 788-gene edited HSPCs according to any one of items 40 to 43, wherein the HDR / indels ratio at the / 788-locus is equal to or higher than 1 , equal to or higher than 1.2, 1 .4, 1.5, or 1 .7, equal to or higher than about 2, equal to or higher than about 2.5, equal to or higher than about 3, equal to or higher than about 3.2, 3.5, 3.8, equal to or higher than about 4, 4.1 , or 4.2. 45. The population of HBB-gene edited HSPCs according to any one of items 40 to 44, wherein the number of edited cells is more than 0.5x108, preferably more than 1x108cells, more preferably more than 1.5x108cells, even more preferably more than 2x108cells.

[0462] 46. Isolated / - / BB-gene edited HSPCs obtainable by the method according to any one of items 1 to 39.

[0463] 47. Isolated / - / BB-gene edited HSPCs, wherein a polynucleotide comprising the exogenous sequence of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16, has been integrated at the / 788-locus in the genome of said HSPCs.

[0464] 48. A cryopreserved composition comprising a population of / - / BB-gene edited HSPCs according to any one of items 40 to 45 or comprising isolated / - / BB-gene edited HSPCs according to any one of items 46 to 47.

[0465] 49. A pharmaceutical composition comprising a population of / - / BB-gene edited HSPCs according to any one of items 40 to 45, isolated / - / BB-gene edited HSPCs according to any one of items 46 to 47, or a cryopreserved composition according to item 48; and a pharmaceutically acceptable excipient and / or carrier.

[0466] 50. The population of / - / BB-gene edited HSPCs according to any one of items 40 to 45, for use in the treatment of a hemoglobinopathy such as sickle cell disease.

[0467] 51 . The isolated / - / BB-gene edited HSPCs according to any one of items 46 to 47, for use in the treatment of a hemoglobinopathy such as sickle cell disease.

[0468] 52. The pharmaceutical composition according to item 49, for use in the treatment of a hemoglobinopathy such as sickle cell disease.

[0469] 53. A TALE-Nuclease selected from:

[0470] - a monomeric TALE-nuclease comprising a monomer binding the HBB sequence of SEQ ID NO: 17 or SEQ ID NO: 19;

[0471] - a heterodimeric TALE-nuclease comprising a first monomer binding the HBB sequence of SEQ ID NO: 17 or SEQ ID NO: 19 and a second monomer binding the HBB sequence of SEQ ID NO: 18.

[0472] 54. The TALE-Nuclease according to item 53, wherein said heterodimeric TALE-nuclease comprises a monomer having the RVD sequence HD-HD-NG-NN-NG-NN-NN-NI-NN-NI-NI- NN-NG-HD-NG-NG and a monomer having the RVD sequence NG-HD-NI-HD-HD-NG-NG- NN-HD-HD-HD-HD-NI-HD-NI-NG.

[0473] 55. The TALE-Nuclease heterodimer according to any one of items 53 to 54, comprising a first monomer comprising the amino acid sequence of SEQ ID NO: 54, or a variant thereof comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO:

[0474] 54, such as at least 90%, at least 95%, or at least 99% identity, and binding the sequence of SEQ ID NO: 17, and a second monomer comprising the amino acid sequence of SEQ ID NO:

[0475] 55, or a variant thereof comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 55, such as at least 90%, at least 95%, or at least 99% identity, and binding the sequence of SEQ ID NO: 18.

[0476] 56. The TALE-Nuclease heterodimer according to any one of items 53 to 55, comprising a first monomer comprising the amino acid sequence of SEQ ID NO: 54 and a second monomer comprising the amino acid sequence of SEQ ID NO: 55.

[0477] 57. An isolated polynucleotide or vector encoding a TALE-Nuclease heterodimer according to any one of items 53 to 56.

[0478] 58. The isolated polynucleotide according to item 57, which is a RNA such as mRNA.

[0479] 59. An isolated polynucleotide, wherein said polynucleotide is a non-viral single-stranded polynucleotide comprising the sequence of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16.

[0480] 60. The isolated polynucleotide according to item 59, which is a single-stranded DNA (ssDNA) comprising SEQ ID NO: 16.

[0481] 61. The isolated polynucleotide according to item 60, which is a circular ssDNA.

[0482] 62. A kit comprising:

[0483] (i) at least one isolated polynucleotide or vector encoding a TALE-Nuclease heterodimer comprising a first monomer binding the HBB sequence of SEQ ID NO: 17 or SEQ ID NO: 19 and a second monomer binding the HBB sequence of SEQ ID NO: 18, and

[0484] (ii) at least one isolated non-viral single-stranded polynucleotide repair template comprising the nucleic acid sequence of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16, and,

[0485] (iii) optionally, at least one isolated polynucleotide or vector encoding an HDR-enhancer; and (iv) optionally, at least one isolated polynucleotide or vector encoding a viability-enhancer.

[0486] 63. The kit according to item 62, wherein the TALE-nuclease encoded by said polynucleotide or vector is a TALE-Nuclease heterodimer according to any one of items 53 to 56.

[0487] 64. The kit according to item 62 or 63, wherein the TALE-nuclease encoded by said polynucleotide or vector is a TALE-Nuclease comprising a first monomer comprising the amino acid sequence of SEQ ID NO: 54 and a second monomer comprising the amino acid sequence of SEQ ID NO: 55.

[0488] 65. The kit according to any one of items 62 or 64, wherein said isolated polynucleotide repair template is the polynucleotide defined in any one of items 59 to 61.

[0489] 66. The kit according to any one of items 62 to 65, wherein said isolated polynucleotide repair template comprises the nucleic acid sequence of SEQ ID NO: 15 or SEQ ID NO: 16.

[0490] 67. The kit according to any one of items 62 to 66, wherein said HDR-enhancer is selected from the group consisting of an inhibitor of 53BP1 , a dominant negative mutant of 53BP1 , an inhibitor of P53, and an inhibitor of Non-Homology-End-Joining, such as an HDR-enhancer of SEQ ID NO: 61.

[0491] 68. The kit according to any one of items 62 to 67, wherein said viability-enhancer comprises the amino acid sequence of SEQ ID NO: 63.

[0492] 69. Ex vivo use of the kit according to any one of items 62 to 68 for editing at least one endogenous / 788-allele comprising a mutation (A>T) or (A>0) at position 20 of the coding region of exon 1 of / - / BB-gene in the HSPCs of a patient suffering from a hemoglobinopathy associated with said mutation.

[0493] 70. A method of treatment of a hemoglobinopathy associated with a mutation (A>T) or (A>C) in at least one endogenous / 788-allele at position 20 of the coding region of exon 1 of / - / BB-gene in a patient, comprising administering in said patient a population of / - / BB-gene edited HSPCs according to any one of items 40 to 45, isolated / - / BB-gene edited HSPCs according to any one of items 46 to 47, or a pharmaceutical composition according to item 49.

[0494] 71. The method of treatment according to item 70, wherein said hemoglobinopathy is sickle cell disease and wherein said mutation is the mutation (A>T) at position 20 of the coding region of exon 1 of the / - / BB-gene. 72. The method of treatment according to item 70 or 71 , wherein the population of / 788-gene edited HSPCs are prepared from HSPCs from the patient to be treated.

[0495] 73. A method of treatment of sickle cell disease associated with a mutation (A>T) in at least one endogenous / 788-allele at position 20 of the coding region of exon 1 of / - / BB-gene in a human patient, said method comprising administering, in said patient, a population of / - / BB-gene edited HSPCs, wherein a polynucleotide comprising the exogenous sequence of SEQ ID NO: 13 or SEQ ID NO: 16 has been integrated at the / 788-locus in the genome of at least 10%, preferably at least 20%, 30%, 40%, 50%, 60 % of the cells of the population; and wherein, optionally, said population comprises at least 0.1%, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, or at least 30%, of cells having a [CD34+CD38-CD45RA-CD90+CD133+] phenotype; and wherein, optionally, the ratio of HDR / indels in the / 788-locus in the cell population is equal to or higher than 1 , equal to or higher than 1.2, 1.4, 1.5, or 1.7, equal to or higher than about 2, equal to or higher than about 2.5, equal to or higher than about 3, equal to or higher than 3.2, 3.4, 3.5, 3.6, 3.8, equal to or higher than about 4, or 4.2.

[0496] Other specific embodiments include the following items:

[0497] 1. An ex vivo method for preparing a population of cells enriched in viable gene-edited Hematopoietic Stem and Progenitor Cells (HSPCs) from a population of HSPCs comprising a gene having a deleterious mutation, said method comprising introducing, into a population of HSPCs having the deleterious mutation:

[0498] (i) a TALE-nuclease cleaving a specific target locus; and

[0499] (ii) a non-viral circular single-stranded polynucleotide repair template comprising a corrective or functional gene sequence without the deleterious mutation; wherein the edited cells have integrated the polynucleotide repair template at the targeted locus; whereby a population of cells is obtained which comprises at least 20% of edited cells and at least 70%, at least 80%, or at least 90%, of viable cells.

[0500] 2. The ex vivo method according to item 1 , wherein said population of cells enriched in viable edited HSPCs comprises long-term repopulating HSC (LT-HSC). 3. The ex vivo method according to item 1 or 2, wherein said population of cells comprises at least 0.1%, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, or at least 30%, of cells having a [CD34+CD38-CD45RA-CD90+CD133+] phenotype.

[0501] 4. The ex vivo method according to any one of items 1 to 3, wherein said TALE-nuclease cleaves the HSPCs’ genomic DNA at a targeted locus selected from: (i) a non-functional endogenous gene in HSPCs, wherein said non-functional gene is associated with a disorder in a patient having said HSPCs, (ii) a genomic safe harbor locus, and (iii) B2 / W-gene.

[0502] 5. The ex vivo method according to any one of items 1 to 4, wherein said TALE-nuclease cleaves the HBB locus.

[0503] 6. The ex vivo method according to any one of items 1 to 5, wherein said repair template comprises the nucleic acid sequence of SEQ ID NO: 16.

[0504] 7. The ex vivo method according to any one of items 1 to 6, wherein said TALE-nuclease targeting HBB comprises a first monomer comprising the amino acid sequence of SEQ ID NO: 54 and a second monomer comprising the amino acid sequence of SEQ ID NO: 55, and said repair template comprises the nucleic acid sequence of SEQ ID NO: 16.

[0505] 8. The ex vivo method according to any one of items 1 to 7, wherein said TALE-nuclease cleaves at a genomic safe harbor locus.

[0506] 9. The ex vivo method according to any one of items 1 to 8, wherein said genomic safe harbor locus is selected from the group consisting of AAVS1 , CCR5, hROSA26, ROGI1 , and ROGI2.

[0507] 10. The ex vivo method according to any one of items 1 to 9, wherein said repair template comprises the coding sequence of a gene selected from IDUA, IDS, ARSB, GUSB, ABCD1, GALC, ARSA, PSAP, GBA, FUCA 1, MAN2B1, AGA, ASAH1, HEXA, GAA, SMPD1, LIPA and CDKL5.

[0508] 11 . The ex vivo method according to any one of items 1 to 10, wherein the mRNA encoding said TALE-nuclease and said polynucleotide repair template are introduced via a single transfection step.

[0509] 12. A population of cells enriched in viable gene-edited HSPCs obtainable by the method according to any one of items 1 to 11. The population of cells according to item 12, wherein said population of cells comprises longterm repopulating HSC (LT-HSC). The population of cells according to item 12 or 13, wherein said population of cells comprises at least 0.1%, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, or at least 30%, of cells having a [CD34+CD38-CD45RA-CD90+CD133+] phenotype. A pharmaceutical composition comprising the population of cells according to any one of items 12 to 14. A method of treatment of a disorder associated with a deleterious mutation in HSPCs comprising administering the population of cells according to any one of items 12 to 14 or the pharmaceutical composition according to item 15 to a patient in need thereof. The method according to item 16, wherein the gene-edited HSPCs derive from the patient’s HSPCs. An ex vivo method for preparing a population of cells enriched in viable gene-edited Hematopoietic Stem and Progenitor Cells (HSPCs) for allogeneic transplantation, said method comprising introducing, into a population of HSPCs from a healthy donor:

[0510] (i) a TALE-nuclease cleaving a specific target locus selected from B2M locus and a genomic safe harbor locus; and

[0511] (ii) a non-viral circular single-stranded polynucleotide repair template comprising a nucleic acid sequence encoding HLA-E and / or CD47; wherein the edited cells have integrated the polynucleotide repair template at the targeted locus; whereby a population of cells is obtained which comprises at least 20% of edited cells and at least 70%, at least 80%, or at least 90%, of viable cells. The ex vivo method according to item 18, wherein said population of cells enriched in viable edited HSPCs comprises long-term repopulating HSC (LT-HSC). The ex vivo method according to item 18 or 19, wherein said population of cells comprises at least 0.1%, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, or at least 30%, of cells having a [CD34+CD38-CD45RA-CD90+CD133+] phenotype. The ex vivo method according to any one of items 18 to 20, wherein said genomic safe harbor locus is selected from the group consisting of AAVS1 , CCR5, hROSA26, ROGI1 and ROGI2. 22. The ex vivo method according to any one of items 18 to 21 , wherein the mRNA encoding said TALE-nuclease and said polynucleotide repair template are introduced via a single transfection step.

[0512] 23. A population of cells enriched in viable gene-edited HSPCs obtainable by the method according to any one of items 18 to 22.

[0513] 24. The population of cells according to item 23, wherein said population of cells comprises longterm repopulating HSC (LT-HSC).

[0514] 25. A pharmaceutical composition comprising the population of cells according to any one of items 23 to 24.

[0515] 26. The use of the population of cells according to any one of items 23 to 24 or the pharmaceutical composition according to item 25 for allogeneic transplantation to a patient in need thereof.

[0516] 27. A kit for preparing a population of cells enriched in viable gene-edited Hematopoietic Stem and Progenitor Cells (HSPCs) comprising a TALE-nuclease cleaving a specific target locus; and a non-viral circular single-stranded polynucleotide repair template as described in any one of the above items.

[0517] EXAMPLES

[0518] 1 : Material and methods

[0519] TALE-Nucleases and HDR (Figure 2B)

[0520] Plasmids of the TALE-Nucleases targeting either wild-type (WT) HBB locus (TALEN surrogate, also called TALEN-HBBpp, comprising monomers of SEQ ID NO: 56 and SEQ ID NO: 55) or mutated HBB locus (HBBSS) (TALEN-HBBSScomprising monomers of SEQ ID NO: 54 and SEQ ID NO: 55), containing a T7 promoter and a polyA sequence, were produced and linearized for mRNA in vitro transcription. TALE-Nucleases mRNAs were produced by TriLink.

[0521] The TALE-Nuclease targeting the mutated HBBss locus described in WO2019185920 having the left arm binding on the sequence starting with the TO corresponding to the E6V mutation was used as a comparison (herewith called “TALEN-TO”). TALEN-TO is a heterodimer comprising a monomer comprising the amino acid sequence SEQ ID NO: 57 and a monomer comprising the amino acid sequence SEQ ID NO: 58. The left-monomer comprises the RVD sequence NN-NN- NI-NN-NI-NI-NN-NG-HD-NG-NN-HD-HD-NN-NG-NG and the right-monomer comprises the RVD sequence HD-HD-NI-HD-NN-NG-NG-HD-NI-HD-HD-NG-NG-NN-HD-NG. The two monomers of TALEN-TO were encoded by nucleic acids of SEQ ID NO: 24 and SEQ ID NO: 25, respectively.

[0522] For viral mediated HDR repair, a DNA matrix comprising SEQ ID NO: 13 flanked by about 300 nt left and right homology arm sequences was designed, forming a sequence of SEQ ID NO: 15, and carried by an AAV6 vector.

[0523] For non-viral mediated repair, a DNA donor template sequence comprising SEQ ID NO: 13 flanked by about 90 nt left and right homology arm sequences was designed, forming a sequence of SEQ ID NO: 16 in the form of a single-stranded oligonucleotide (ssODN). This 197 nt long single-stranded oligonucleotide was sourced from Integrated DNA Technology (IDT) and contained phosphorothioate modification on the first two bases situated at the 5’ and 3’ ends of the sequence. This ssODN donor template was used in its linear form in Examples 2 to 5, and in its linear or circular form in Example 7.

[0524] CD34+ HSPCs sourcing

[0525] Frozen CD34+ HSPCs purified from healthy donor G-CSF- and Plerixafor-mobilized peripheral blood were purchased from AllCells (Almeda) or Hemacare (Los Angeles). CD34+ HSPCs derived from HbSS patients were recovered from erythrocytapheresis bags provided by Hopital Necker-Enfants malades (Paris). Written informed consent was obtained from all adult subjects.

[0526] CD34+ cells isolation

[0527] Isolation of mononuclear cells was performed on erythrocytapheresis bags by diluting 2 times the red blood cell suspension with a medium containing PBS (Gibco, #70011044), 2% heat inactivated FBS (Gibco, #10082147) and 1 mM EDTA (Invitrogen, #15575020). Diluted red blood cells suspension was then distributed into Sepmate tubes (StemCell #85450) containing 15 mL of density gradient medium (StemCell, #07861). Tubes were centrifuged 10 minutes at 12000 g, the supernatant was poured in a new 50 mL tube, completed up to 45 mL of medium containing PBS, FBS and EDTA and centrifuged 7 min at 300 g. All pellets were pooled together to perform CD34+ cells isolation using CD34 progenitor kit according to manufacturer’s recommendations (Miltenyi #130-046-703). HbSS CD34+ cells were cryopreserved in medium containing FBS and 10% DMSO (Sigma, #D2438) in liquid nitrogen.

[0528] CD34+ HSPCs culture

[0529] After thawing, CD34+ HSPCs from either healthy donors (“HD”) and HbSS patients (“HbSS”) were cultured at a concentration of 0.3 x 106cells / mL in complete medium: StemSpan II (Stemcell, #09655), 1X CD34 expansion supplement (Stemcell, #02691) and 1X penicillin- streptomicyn (Gibco, #15140-122) at 37°C, 5% CO2.

[0530] For GMP-compatible conditions, cells were cultured in GMP Stem Cell Growth Medium (SCGM, CellGenix, Freiburg, Germany) supplemented with human cytokines (Cellgenix GMP- grade), TPO (100 ng / mL), Flt3 (300 ng / mL), SCF (300 ng / mL) and IL-3 (60 ng / mL).

[0531] HSPCs were assessed for viability by Nucleocounter or by the expression of CD34 and viability marker by flow cytometry, 2 days after gene editing. The flow cytometry staining was performed in PBS, 0.5% BSA and 2 mM EDTA with a CD34 VioBlue 1 / 50 (Miltenyi, # 130-124- 459) antibody and a fixable viability marker e780 1 / 1000 (eBioscience, #65-0865-18).

[0532] Aliquots of HSPCs from healthy donors were frozen 2 days after gene editing for xenotransplantation experiments and for scRNAseq analysis.

[0533] Transfection and transduction of HSPCs (Figure 2A)

[0534] Two days after thawing, the cells were washed twice in BTXpress buffer and resuspended at a final concentration of 10 x 106cells / mL in the same solution. The cellular suspension (1 x 106cells) was mixed with 15 pg mRNA encoding each of the left and right TALE-Nuclease arms in the presence or absence of 4 pg and 1 pg mRNAs encoding HDR-Enh01 (SEQ ID NO: 26) and / or Via-Enh01 (SEQ ID NO: 27), respectively, in a final volume of 100 pl. The cellular suspension was transfected in cuvettes of 0.4 cm width using BTX AgilePulse technology. The electroporation program consisted of two 0.1 mS pulses at 1000 V / cm followed by four 0.2 mS pulses at 130 V / cm. Immediately after electroporation, HSPCs were transferred to a new plate containing prewarmed medium at the concentration of 2x106 / ml and placed 15 minutes at 37°C.

[0535] For transduction experiments, 15 minutes after TALE-Nuclease electroporation, HSPCs were cultured in the presence or absence of AAV particles (MOI = 7.5 x 105vg / cells) containing the HDR donor template, also called herewith “HBB gene repair matrix” or “repair template”, at the concentration of 2x106cells / ml and incubated 15 minutes at 37°C. HSPCs were then incubated at

[0536] 30°C overnight.

[0537] For ssODN transfection experiments, HSPCs were kept in culture at 30°C and 16 hours after TALE-Nuclease electroporation, a second electroporation was performed in the same conditions described above, using 1x106cells in presence or absence of 1000 pmol of ssODN in a final volume of 100 pl. HSPCs were electroporated following the same conditions and program described above. After ssODN transfection, HSPCs were seeded at the concentration of 2x106cells / ml and incubated at 30°C.

[0538] The following day, cells were seeded at a density of 0.3x106cells / mL in complete medium and cultured at 37°C in the presence of 5% CO2. GMP-compatible conditions were performed with the following modifications: cells were washed and resuspended in an in-house made GMP-compliant electroporation buffer for both mRNA and DNA electroporation. 500 pmol ssODN were used for electroporation.

[0539] Evaluation of editing efficiency and adverse events by ddPCR of edited HSPCs in cell culture, HSPCs-derived erythrocytes and bone marrow samples

[0540] Genomic DNA (gDNA) was extracted with Qiagen kit DNeasy Blood and Tissue Kit (Qiagen, #69506) or QiAmp DNA Micro kit (Qiagen, #56304) according to manufacturer’s instructions. BFLI-E clones were lyzed using DNA Extract All Reagents kit (Termo Scientific, # 4403319) according to manufacturer’s instructions. gDNA quantification is performed using NanodropOne device (ThermoFisher).

[0541] Assessment of editing rates was performed by digital droplet PCR (ddPCR) on 50 ng of purified gDNA or lysate (for CFU) using a triplex assay allowing the quantification of edited, nonedited and NHEJ-mediated indels events. This triplex assay was designed with an in / out PCR and three probes: one probe detecting edited sequences, one drop-off probe situated at the TALEN cutting site identifying indels events and one probe for reference (Miyaoka et al., Methods Mol. Biol. 2018; 1768:349). Assessment of adverse events was performed based on protocol described by Long et al. (Mol. Ther. 2018; 26(2): 468-479) using 100 ng of purified gDNA. All primers and probes sequences are shown in Table 2. ddPCR was performed following Biorad protocol. Genomic DNA was combined with 1 pl EcoRI restriction enzyme (ThermoScientific, #FD0274), 10 pM each of target primer and FAM probe mix, 10 pM each of reference primer and HEX probe mix, 1 x ddPCR Supermix probe without dllTP (Bio-Rad, UK) and nuclease-free water in a final 20pl volume.

[0542] Table 2. Primers and sequences

[0543] Identification and detection of candidate off-site targeting by oligo capture assay and high- throughput DNA sequencing

[0544] Oligo capture assay (OCA) was used to assess the specificity of TALE-Nuclease targeting HBBss of SEQ ID NO: 7. Briefly, primary T cells derived from HbSS-patients suffering from sickle cell disease were co-transfected with mRNA encoding TALEN-HBBSS(SEQ ID NO: 21 and SEQ ID NO: 22) and over-hanging double-stranded oligodeoxynucleotide (dsODN) and expanded for 6 days. Genomic DNA was recovered, sheared, end-repaired / A-tailed, processed, and analyzed by high-throughput DNA sequencing. The resulting sequences were mapped onto the human genome (GRCh38) to identify potential off-site candidates. The frequency of insertion and deletion events (indels) generated at potential off-site candidates were then quantitatively assessed using high-throughput DNA sequencing of candidate off-site-specific PCR amplicons obtained from HSPCs edited with TALEN-HBBSSin absence of dsODN.

[0545] Deep sequencing / amplicon sequencing for on- and off- target detection

[0546] 100 ng of genomic DNA was used per reaction in a 50 pL reaction with Phusion High- Fidelity PCR Master Mix (NEB). The PCR conditions were set to 1 cycle of 30 s at 98°C; 30 cycles of 10 s at 98°C, 30s at 60°C, 30 s at 72°C; 1 cycle of 5 min at 72°C; hold at 4°C. The PCR product was then purified with Omega NGS beads (1 : 1.2 ratio) and eluted into 30 pL of 10 mM Tris buffer pH7.4. The second PCR which incorporates NGS indices was then performed on the purified products from the first PCR. 15 pL of the first PCR products were set in a 50 pL reaction with Phusion High-Fidelity PCR Master Mix (NEB). The PCR conditions were set to 1 cycle of 30 s at 98°C; 8 cycles of 10 s at 98°C, 30 s at 62°C, 30 s at 72°C; 1 cycle of 5 min at 72°C; hold at 4°C. Purified PCR products were sequenced on MiSeq (Illumina) on a 2 x 250 nano V2 cartridge. Colony forming unit (CFU) assays

[0547] CD34+ HSPCs recovered two days after electroporation or human CD45+ recovered from mice bone marrow 16 weeks after in vivo injection, were plated in methylcellulose (Stemcell, #04435) for Colony Forming Unit (CFU). 200-500 cells (CD34+) or 200,000-300,000 (hCD45+) were resuspended in 100 pL of Stemspan II and transferred to an aliquot of 1 mL of methylcellulose, mixed and plated in a Smartdish well (Stemcell, #27371). Cells were cultured 12- 14 days in methylcellulose according to manufacturer’s instructions.

[0548] At the end of the culture, colonies were automatically counted by Stemvision (StemCell) to assess plating efficiency (number of colony counted at day 14 / number of cells plated at day 0). Bulk and single BFU-E colonies were picked at day 14 and lysed to obtain gDNA for evaluation of editing efficiency.

[0549] HPLC

[0550] HPLC experiments were performed on CFU derived BFLI-E bulk (~25 colonies) and HSPCs-derived erythroid cells (400,000 cells). Hemoglobin tetramers and monomers were quantified by Cation Exchange (CE) HPLC and globin chains by Reverse Phase (RP) HPLC, respectively as previously described (Weber et al. Sci. Adv. 2020; 6(7):1-15).

[0551] In vitro differentiation of CD34+ HSPCs into

[0552] Two days after TALE-Nuclease mRNA electroporation, HSPCs were plated at the concentration of 0.5 x 105cells / ml to start the erythroid lineage differentiation following Giarratana’s protocol (Giarratana et al. Nat. Biotechnol. 2005; 23(1):69-74). First, cells were plated for 6 days in a serum free medium containing SCF at 100 ng / mL (CellGenix, #001418-050), IL3 at 5 ng / mL (CellGenix, #001402-050), EPO at 3 UI / mL (Stemcell, #78007) and Hydrocortisone at 10 pM (Sigma, #H0888-1G). For the second step of differentiation, HSPCs were co-cultured on a MS5 cells layer in a medium containing EPO at 3 UI / mL for 3 days. Cells were kept in MS5 coculture and 10% of human AB serum-containing medium (Biowest, # S4190-100) for differentiation for a total of 21 days.

[0553] To assess the quality of erythroid differentiation, HSPCs derived erythrocytes were stained for flow cytometry analysis at different time points during differentiation process, using the following antibodies: CD36 V450 1 / 20 (BD, #561535), CD71 FITC 1 / 50 (BD, #555536), CD233 PE 1 / 50 (IBGRL, #9439), CD235a PECy7 1 / 100 (BD, #563666), CD49d APC 1 / 20 (BD, #559881), viability dye 7AAD 1 / 100 (BD, #559925), and Draq5 1 / 500 (eBioscience, #65-0880-96). Sickling assay

[0554] 21 days after the beginning of erythroid differentiation, HSPCs-derived erythroid cells were exposed to an oxygen-deprived atmosphere (0% O2), and the time course of sickling was monitored in real time by video microscopy. Images were captured every 20 min for at least 80 min using an AxioObserver Z1 microscope (Zeiss) and a 40x objective. Images of the same fields were taken throughout all stages and processed with Imaged to determine the percentage of non- sickled Red Blood Cells (RBC) among the total RBC population. 500 to 1 ,000 cells were counted per condition.

[0555] Transplantation of CD34+ HSPCs into NSG or NBSGW mice

[0556] Frozen aliquots of mobilised PB CD34+ cells from healthy donors obtained 2 days after editing were sent to TRANSCURE bioServices (Archamps, France) for xenotransplantation in NOD Prkdcfm26Cd52 / / 2rgem26Cd22 / NjuCrl (NCG) mice. Pre-transplant conditioning was based on Busulfan (Sigma, #B1170000) according to TRANSCURE bioServices (Archamps, France) protocol. Non-mobilized CD34+ cells derived from HbSS patients were transplanted fresh 2 days after editing in NOD.Cg-Kitw-41JTyr +PrkdcscidH2rgtm1Wjl / ThornJ (NBSGW) mice. Busulfan (Sigma, #B1170000) conditioning was performed at a dose of 15 mg / kg body weight through an intra peritoneal injection one day prior HSPCs transplantation.

[0557] Edited and non-edited control HSPCs were transplanted via tail vein (NCG) or retro-orbital (NBSGW) injection at 0.25 or 0.7x106cells for NCG mice and 5x105cells for NBSGW mice xenotransplantation.

[0558] For all xenotransplantation studies, 16-18 weeks after transplantation, mice were sacrificed and peripheral blood, bone marrow, spleen and thymus were recovered. 1x106cells from each organ were harvested and chimerism was assessed by flow cytometry using the following antibodies mouse CD45 Vioblue 1 / 50 (Miltenyi, #130-110-664), human CD45 APCVio770 1 / 50 (Miltenyi, #130-110-635) and viability dye 7AAD 1 / 200 (BD, #559925).

[0559] Human CD45+ cells from bone marrow were sorted using human CD45 Microbeads (Miltenyi, #130-045-801) and following manufacturer’s recommendations and gDNA was extracted to assess gene editing stability after engraftment. For NCG xenotransplantation an aliquot of sorted human CD45+ cells were plated for CFU assay.

[0560] All procedures and housing concerning NCG xenotransplantations were performed at TransCure bioServices (Archamps, France) and have been reviewed and approved by the local ethics committee (CELEAG). Multilineage engraftment

[0561] Cells recovered from mice bone marrow 16-18 weeks post injection, were assessed for multilineage engraftment in the lymphoid compartment using CD19 FITC 1 / 100 (Beckman Coulter, # A07768) and CD3 APC 1 / 50 (Miltenyi, # 130-113-135) antibodies ; granulocyte compartment using CD15 PE 1 / 50 (Beckman Coulter, # IM1954U), CD14 PECy7 1 / 50 (BD, # 562698) and CD11b APC 1 / 100 (BD, #553312) antibodies and erythroid compartment using CD36 FITC 1 / 50 (BD, # 555454), CD235a PE 1 / 50 (Invitrogen, # 12-9987-82) and CD71 APC (BD, #551374) antibodies.

[0562] Cells were incubated in a buffer containing PBS, 0.5% BSA, 2mM EDTA and antibodies 15 minutes at room temperature then were washed two times in PBS 1X. Cells were resuspended in PBS, 2% FBS and were read on flow cytometer MacsQuant 10 (Miltenyi).

[0563] Statistical Analysis

[0564] Comparisons of numerical variables between two groups were evaluated either with Mann-Whitney Wilcoxon’s paired test or Fisher’s exact test as specified in the figure legends. One-way ANOVA with Tukey’s multiple comparisons test or Kruskal-Wallis followed by Dunn’s multiple comparisons test was used in case of three or more groups. Significance threshold was set to 0.01 : *=0.01 ; **=0.001 ; ***=0.0001 ; “ns” means not significant. All statistical analyses were performed using GraphPad Prism v.9.4 (GraphPad).

[0565] Example 2: TALE-Nuclease choice

[0566] To select the most suitable TALE-Nuclease for HBB- / 3s gene correction, a side-by-side comparison of the editing efficiency of a TALEN described in the prior art WO2019185920 (herewith called “TALEN-T0”) and a new TALEN (described herewith and called “TALEN-HBB” or “TALEN-HBBss”) was performed in HSPCs derived from two different patients suffering from sickle cell disease.

[0567] TALEN-T0 is composed of a left monomer and a right monomer comprising the amino acid seguences SEQ ID NO: 57 and SEQ ID NO: 58, respectively. The mRNAs encoding TALEN-T0 comprised the amino acid seguences SEQ ID NO: 24 and SEQ ID NO: 25, respectively.

[0568] TALEN-HBBss is composed of a left monomer and a right monomer comprising the amino acid seguences SEQ ID NO: 54 and SEQ ID NO: 55, respectively. The mRNAs encoding TALEN- HBBss comprised the amino acid seguences SEQ ID NO: 21 and SEQ ID NO: 22, respectively.

[0569] The binding sites of TALEN-T0 and TALEN-HBB are represented in Figure 1A.

[0570] Targeted integration was performed using the same AAV repair matrix. Using a series of multiple read out, TALEN-HBB elicited a higher freguency of HDR (HBB- / 3s gene correction) than TALEN-TO and a similar frequency of Indels (HBB- / 3s gene inactivation) (Figure 1 B). In addition, none of the TALE-Nucleases treatment impacted the plating efficiency of edited HSPCs compared to unedited HSPCs (Figure 1C).

[0571] In comparison to TALEN-TO, the higher gene correction activity of TALEN-HBB is related to a higher frequency of HbA re-expression in BFLI-E colonies (Figure 1D). Overall, our results showed that TALEN-HBB elicits a higher propensity to rescue expression of HbA compared to TALEN-TO.

[0572] TALEN-HBB was thus selected as the TALEN-candidate for further evaluation.

[0573] Example 3: Optimization of HBB correction process in Healthy Donor’s Cells

[0574] In order to develop a process that is suitable for / - / BB-gene correction in view of a gene therapy approach to restore functional hemoglobin production in sickle cell disease (SCD) patients (also called herewith “HbSS patients”), two sets of TALE-Nucleases were produced, namely: a TALE-Nuclease comprising a left-monomer binding SEQ ID NO: 20 and a right- monomer binding SEQ ID NO: 18 for targeting the WT / 788-locus (“TALEN-HBBpp”) in healthy subjects. The mRNAs encoding TALEN-HBBpp comprised the amino acid sequences SEQ ID NO: 23 and SEQ ID NO: 22. TALEN-HBBpp is composed of a left monomer and a right monomer comprising the amino acid sequences SEQ ID NO: 56 and SEQ ID NO: 55, respectively; and a TALE-Nuclease comprising a left-monomer binding SEQ ID NO: 17 and a right- monomer binding SEQ ID NO: 18 for targeting the mutated / 788-locus in HbSS patients (“TALEN-HBBss”). The mRNAs encoding TALEN-HBBSScomprised the amino acid sequences SEQ ID NO: 21 and SEQ ID NO: 22. TALEN-HBBSSis composed of a left monomer and a right monomer comprising the amino acid sequences SEQ ID NO: 54 and SEQ ID NO: 55, respectively.

[0575] TALEN-HBBpp was first used in HSPCs mobilized from healthy donors (HD) to set up and optimize the TALEN-mediated gene editing protocol and select the most suitable DNA template delivery method to repair HBB via homologous directed repair (HDR). Viral and non-viral strategies based on either AAV or ssODN were tested. Both forms of DNA repair templates comprise SEQ ID NO: 13 to enable HBB gene edition and avoid TALEN-mediated cleavage of the edited sequence (Figure 2B). The first tested viral and non-viral gene editing protocols with mRNA TALEN electroporation followed either by AAV transduction or by ssODN second electroporation (see Example 1) led to 40% indels induction, and up to 38.5% and 30.23% of HBB gene correction using AAV and ssODN, respectively, leading to a ratio of HDR vs Indels events close to 1. The first optimization step carried out herewith aimed at reducing the level of indels in the HBB coding gene to mitigate the risk of generating p-thalassemic cells. To this aim, TALEN-HBBpp was co-transfected with a mRNA encoding an HDR-enhancer of amino acid sequence SEQ ID NO: 61 , named HDR-Enh01. A significant increase in HDR in both ssODN and AAV protocols comprising HDR-Enh01 compared to the protocols without HDR-Enh01 (34.3% vs 22.6% and 42.2% vs 33.3% in average, respectively) was observed. This effect was associated to a significant reduction of indels which decreased from 38.7% down to 19.2% with the ssODN protocol and from 32.4% to 16.8% with the AAV protocol (Figure 3A-B).

[0576] This protocol was then tested in GMP-compatible conditions (GMP-compliant culture medium and electroporation buffer). Unexpectedly, a drop in cell viability was observed when using either AAV or ssODN (71.6% and 39.6% viable cells, respectively) in GMP-compatible conditions compared to standard R&D conditions (Figure 3C). Thus, the gene editing process was further optimized by delivering together with TALEN-HBBpp and HDR-Enh01 , an mRNA encoding an antiapoptotic protein of amino acid sequence SEQ ID NO: 63, named Via-Enh01. The addition of Via-Enh01 led to a significant increase in HSPCs viability compared to HDR-Enh01 alone in GMP-compatible conditions (82.3% vs 39.6% and 91.4% vs 71.6% viable cells in ssODN and AAV, respectively) (Figure 3C). Surprisingly, when using ssODN protocol, the addition of Via- Enh01 contributed to a further increase in HDR frequency in the GMP-compatible conditions (Figure 3D, left panel). This could not be explained by the impact on cell survival since ViaEnhOI had no effect on Indels events frequency (Figure 3D right panel). This optimized protocol, carried out with GMP compatible material, elicited an HDR / lndels ratio of about 2 and 3 with the ssODN and AAV protocol, respectively.

[0577] The ability of the viral and non-viral DNA delivery strategies to edit long-term repopulating hematopoietic stem cells (LT-HSCs) was tested by using plerixafor-mobilized (PLX) HSPCs from three healthy donors as a clinically relevant source of CD34+ cells. The LT-HSCs edited with the optimized protocol were assessed for engraftment and hematopoietic reconstitution in Busulfan conditioned immunodeficient NCG mice (Figure 4A). Compared to AAV, ssODN-mediated editing led to higher levels of engraftment in bone marrow (BM) 16 weeks after injection (2.2% vs 58.1% median hCD45+ chimerism, respectively) (Figure 4B). Interestingly, neither one nor two electroporation steps impacted LT-HSCs as the mean engraftment levels were comparable between untreated and electroporated controls; likewise, ssODN engraftment was similar to the control groups (Figure 4B). To assess allelic editing in engrafted LT-HSCs, ddPCR on genomic DNA extracted from hCD45+ cells sorted from bone marrow (BM) was performed. Notably, ssODN significantly exceeded AAV, reaching a median HDR allelic frequency of 30.5% vs 9%, respectively (Figure 4C). Higher HDR allelic frequency for ssODN compared to AAV was also observed in CFU derived from engrafted CD34+ cells with no major differences observed among BFU-E and CFU-GM (Figure 4D).

[0578] Overall, these data show that non-viral mediated DNA delivery outperformed viral- mediated editing both in term of engraftment and HDR efficiency in clinically relevant cells.

[0579] Example 4: HBB correction in HbSS patients’ cells

[0580] To assess whether the optimized editing protocol described above would efficiently correct sickle-cell HSPCs, purified HSPCs from nine non-mobilized homozygous sickle patients (“nmHbSS”) were assessed for gene editing efficiencies and reproducibility (Figure 5A).

[0581] With the optimized editing protocol, 44.3% (ssODN) and 53.2% (AAV) average HBB correction was achieved while maintaining low level of HBB indels (21.4% and 17.3% with ssODN and AAV, respectively) in all patients’ cells tested (Figure 5B). These data also confirmed the advantage of the optimized protocol compared to its standard counterpart lacking HDR-Enh01 and Via-Enh01 (with standard protocol: 29.6% and 40.3% average HDR with ssODN and AAV, respectively, and 40.3% and 33.4% average indels with ssODN and AAV, respectively) (Figure 5B). The optimized protocol with ssODN led to higher CFU or BFU platting efficiency compared to the optimized protocol with AAV, but none of these protocols led to lineage skewing since all the 3 lineages (BFU-E, CFU-GEMM and CFU-GM) were present at the same proportion / frequency (Figure 5C). Our optimized protocol significantly reduced the proportion of HSPC-derived BFU-E clones harboring bi-allelic indels (less than 10% of “collateral effect” in all clones with either AAV or ssODN editing) and enabled reaching high levels of corrected BFU-E clones (58% and 75% in ssODN and AAV, respectively) (Figure 5D). Overall, the optimized gene editing process led to efficient and reproducible HBB correction in HbSS HSPCs, elicited therapeutically relevant fraction of corrected clones, while mitigated bi-allelic HBB inactivation.

[0582] To verify the therapeutic potential of edited HbSS HSPCs, these edited cells were differentiated into fully mature red blood cells (RBCs) and hemoglobin expression was assessed as well as the sickling properties of edited samples (Figure 6A). As producing bi-allelic indels in the HBB gene might generate p-thalassemic cells in treated patients, a control corresponding to the TALE-Nuclease alone (“TALEN only control”) was performed. Reverse Phase-HPLC, and Cation Exchange-HPLC measurements showed clinically relevant HbA expression in all corrected RBC groups (51% and 52% with ssODN and AAV, respectively), with a substantial reduction of HbS expression compared to Mock control (25% and 23%, respectively, vs 94%) (Figure 6B). Most importantly, the a- / non-a-globin ratio measured in corrected RBCs was comparable to “Mock EP control”, but significantly different from the “TALEN only control” that led to reactivation of HbF, high proportion of HbF+ cells and reduced level of the total p-like globins (Figure 6B). All the corrected RBCs showed significantly lower frequencies of sickle cells relative to “Mock EP control” as assessed by sickling assay (96.2% vs 47.1% and 45.2%), confirming that HbA expression was sufficient to inhibit Hb polymerization (Figure 6C).

[0583] Taken together, these data demonstrate the therapeutic potential of the optimized gene editing protocol that promotes high level of phenotypic correction and mitigates the generation of beta-thalassemic phenotype in fully mature RBCs.

[0584] The ability of corrected nmHbSS HSPCs with the ssODN optimized process to engraft long term and reconstitute hematopoiesis was tested in immunodeficient NBSGW mouse model (Figure 7A) in comparison to a mock double electroporated control. BM human chimerism and alleles correction frequencies were evaluated 16 to 18 weeks after transplantation. Overall, corrected HbSS HSPCs showed robust engraftment capacity with a median human chimerism comparable to the mock-electroporated sample control (30.5% vs 16% median, respectively) (Figure 7B). The human graft was multilineage and consisted of mostly lymphoid cells (CD19+ and CD3+) and myeloid cells (CD14+, CD15+ and CD11 b+) (Figure 7C). The cells engrafted in BM showed a relevant percentage of corrected alleles (median of 21 %) and no sign of indels enrichment (median of 13.4%) (Figure 7D-E).

[0585] Overall, these data confirmed the ability of the optimized gene editing protocol to produce corrected long-term repopulating hematopoietic stem cells derived from HbSS patients, validating its therapeutic potential.

[0586] Example 5: TALE-Nuclease specificity assessment

[0587] To examine the potential off-target cleavage at the highest dose of TALEN-HBBSS, an unbiased genome wide approach named Oligo Capture Assay (OCA) was used. Primary T-cells from HbSS patients were used to perform this procedure and predict the potential off-target cleavage for TALEN-HBBss (Figure 8A). OCA results enabled us to identify the on-site (in HBB gene) and 192 putative off-sites that were confidently mapped on the human genome (Figure 8B). The putative off-target sites identified were further investigated quantitatively on HSPCs from 2 HbSS donors engineered by our process (Figure 8C). This analysis revealed only one off-target site, located in the HBD gene. TALEN-HBBss cleavage activity at this off-site in HbSS patients’ HSPCs was found to be very low compared to the on-site cleavage activity (54.2% indels versus 1.1% at the HBB on-site and the HBD off-site, respectively) (Figure 8D).

[0588] Because TALEN-HBBss catalyzes DNA double stranded break, genetic rearrangements, including translocation, deletion, and inversion, could occur between the two cleavage sites. The frequency of such rearrangements was assessed by ddPCR using gDNA from edited HSPCs derived from HD and HbSS patients (Figure 9A-B). TALEN-HBBss cleavage activity could promote formation of deletions, inversions, and translocations between HBB and HBD with mean frequencies of 2.7% of deletion, 0.4% of inversion and 1.3% of translocation in HbSS cells (Figure 9C). The frequency of those genomic rearrangements was found to significantly decrease from culture samples to those obtained from BM of mice, 16 weeks after edited HSPC infusion onset (Figure 9C), suggesting a negative selection of adverse events in long-term repopulating cells.

[0589] Example 6: Gene editing optimization in HSPCs using single-stranded DNA repair template

[0590] 6.1. Material and methods

[0591] TALE-Nucleases and ssDNA HDR repair templates

[0592] Plasmids comprising the nucleic acid sequences of SEQ ID NO: 47 and SEQ ID NO: 48 encoding the two monomers of a B2M-targeting TALE-Nuclease and containing a T7 promoter and a polyA sequence were linearized for mRNA in vitro transcription. TALE-Nucleases mRNAs were produced by TriLink.

[0593] For HDR repair, several ssDNA matrices were used, which comprised either SEQ ID NO: 49 or SEQ ID NO: 50, either in a linear or a circular form.

[0594] CD34+ HSPC culture

[0595] CD34+ HSPC culture conditions were identical to previous protocol (see example 1) except that HSPC pellets were recovered from culture 1 day after ssDNA matrix electroporation for RNA extraction and evaluation of editing efficiency, MX1 and P21 levels of transcription.

[0596] CD34+ HSPC transfection

[0597] Two days after thawing, the cells were washed twice in BTX buffer and resuspended at a final concentration of 10 x 106cells / mL in the same solution. The cellular suspension (1 x 106cells) was mixed with 15 pg mRNA encoding each of the TALEN-B2M arms (SEQ ID NO: 59 and SEQ ID NO: 60) in the presence of 4 pg mRNA encoding HDR-Enh01 (SEQ ID NO: 61) and 1 pg of mRNA encoding Via-Enh01 (SEQ ID NO: 63), in a final volume of 100 pl. The cellular suspension was transfected using BTX AgilePulse technology. The electroporation program consisted of two 0.1 ms pulses at 1000 V / cm followed by four 0.2 ms pulses at 130 V / cm. Immediately after electroporation, the HSPCs were transferred to a new plate containing prewarmed medium at a concentration of 2x106 / mL and incubated for 15 minutes at 37°C. For ssDNA transfection (with ssDNA HLAE of SEQ ID NO: 49 or with ssDNA HA tag of SEQ ID NO: 50), HSPCs were kept in culture at 30°C for 16 hours after mRNAs electroporation, and a second electroporation was performed using AMAXA 2B device, program U008 and CD34 electroporation buffer, using 1x106cells in presence of 0.2 pM ssDNA (final concentration) in a final volume of 100 pl. After ssDNA transfection, HSPCs were seeded at a concentration of 2x106cells / mL and incubated at 30°C overnight.

[0598] For AAV transduction of HLAE (SEQ ID NO: 49), 15 min after TALEN electroporation, HSPCs were cultured in the presence or absence of AAV particles (MOI = 350 viral genome / cell) containing the HLAE at a concentration of 2 * 106cells / mL and incubated for 15 min at 37°C. HSPCs were then incubated at 30°C overnight.

[0599] The following day, cells were seeded at a density of 0.3x106cells / mL in complete medium and cultured at 37 °C in the presence of 5% CO2before being analyzed for viability, editing efficiency, transcriptomics and in vivo engraftment capacity. Viability and editing efficiency were analyzed by flow cytometry, 7 days after HSPCs thawing. Aliquots of 500 000 edited HSPCs obtained 4 days after thawing, were analyzed by CITE-Seq and aliquots of the same edited HSPCs were injected into NCG mice for in vivo engraftment assessment.

[0600] Edited CD34+ HSPC

[0601] For flow cytometry analysis, CD34+ HSPCs recovered 4 days post linear or circular ssDNA electroporation were recovered and stained with Annexin V (BD, # 563973), E-fluor 780 viability dye (eBioscience # 65-0865-18), anti HLA ABC (miltenyi, # 130-120-141) or anti-B2M (biolegend, # 395712), anti-tag antibody (biolegend, # 901509) or anti HLAE (miltenyi , # 130-117-551) and anti-CD34 (Invitrogen, # 25-0349-42). Stained samples were then analysed by flow cytometry to determine the frequency of HA tag or HLAE insertion within viable, CD34 positive, HLA ABC (or B2M) positive or negative populations, respectively.

[0602] For colony forming units (CFU) assay, CD34+ HSPCs recovered two days after electroporation were plated in methylcellulose (Stemcell, #04435) for CFU assays. A total of 200- 800 cells (CD34+) were resuspended in 100 pL of Stemspan II and transferred to an aliquot of 1 mL of methylcellulose, mixed, and plated in a Smartdish well (Stemcell, #27371). Cells were cultured for 12-14 days in methylcellulose according to the manufacturer’s instructions. At the end of the culture, colonies were automatically counted using a Stemvision (StemCell) automated colony counter to assess plating efficiency (number of colonies counted at day 14 I number of cells plated at day 0). Bulk colonies were picked at day 14 and analysed by flow cytometry using the protocol described above, to evaluate gene insertion efficiency. For RTqPCR assay, CD34+ HSPCs recovered 1 day after ssDNA electroporation were pelleted, RNA was extracted using RNeasy® Plus Micro Kit (Qiagen, # 74034) and analysed for P21 and MX1 transcripts level using First Strand cDNA Synthesis Protocols (NEB, # E6560), SsoAdvanced™ Universal Probes Supermix (Biorad, # 1725281) and a set of oligonucleotides / probes specific for MX1 , P21 and for HPRT reference transcripts (Thermofisher, TaqManGene ExpressionAssay MX1 , #Hs00895608_m1 , TaqManGene ExpressionAssay CDKN1A, # Hs00355782_m1 and TaqManGene ExpressionAssay HPRT1 , # Hs02800695_m1 , respectively). CFX96 Touch Real time system (Bio-Rad) was used to perform the qPCR.

[0603] For CITE-Seq analysis, single cell droplets were generated using Chromium apparatus (1 OX Genomics), libraries were constructed using Chromium Next GEM Single Cel I5' Reagent Kits v2 (Dual Index), barcodes were implemented with TotalSeq™-B or -C with 10x Feature Barcoding Technology and barcoded libraries were sequenced using NovaSeq X plus 10B - 100 cycles (Illumina)

[0604] The in vivo engraftment capacity was assessed using Busulfan preconditioned NCG mice. NCG mice were injected i.v. with 0.75E6 edited cells. Mice were then sacrificed 16 weeks post injection onset. Their bone marrow was then analysed by flow cytometry to determine the level of hCD45+ cells engraftment (ratio hCD45+ / mCD45+ cells) and the frequency of gene insertion in CD45+ cells.

[0605] 6.2. Results

[0606] To evaluate the efficiency of TALEN-mediated non-viral gene targeting at the B2M locus in HSPCs, we designed a linear ssDNA matrix (“LssDNAI” of about 2.2 kb total length (SEQ ID NO: 49) encompassing the HLAE trimeric construct flanked by 5’ and 3’ arms (300 bp) homologous to the B2M locus (Figure 10A). This repair matrix was designed to insert, in a disruptive manner, the HLAE construct in frame with the B2M locus. Insertion of this construct would thus lead to HLA ABC(-) HSPCs expressing HLAE.

[0607] Our results show that linear ssDNA could be inserted at the B2M locus in a targeted fashion by the TALEN-B2M with an efficiency of gene knock-in (KI) reaching a mean of 15% (up to 30%, Figure 10C left panel). In this particular experimental setting, edited cells showed viability around 70% (Figure 10C, right panel).

[0608] Circular ssDNA compared to linear ssDNA Circular single strand DNA was compared to linear single strand DNA with a repair template allowing insertion of an HA Tag at the B2M locus (Figure 11A). Surprisingly, the side-by- side comparison performed in HSPCs showed that circularization of an about 0.7 kb HA tag matrix specific for B2M targeted tagging (“CssDNA2” of SEQ ID NO: 50) markedly increased the KI efficiency in comparison to its linear counterpart (i.e. of identical nucleic acid sequence) named “LssDNA2”. Six and 45 % of KI were obtained when using LssDNA2 compared to circular CssDNA2, respectively (Figure 11 C, left panel). Interestingly, this increase of KI was correlated to a slightly higher viability (Figure 11C, middle panel), a lower B2M knock-out (KO) (Figure 11C, left panel), and a decrease in the inflammation pattern (Figure 11C, right panel).

[0609] Altogether, these results demonstrate that circular ssDNA matrix enables an unexpected and important improved double-strand-break-induced targeted integration.

[0610] To confirm this proof of concept with different matrix lengths, a longer (about 2.2kb vs about 0.7kb) circular ssDNA (“CssDNAI”) encoding a HLAE trimeric construct was compared to its linear ssDNA counterpart (“LssDNAI”) of identical nucleic acid sequence (SEQ ID NO: 49) (Figure 12A). Our side-by-side comparison performed in HSPCs showed that circularization of the HLAE construct specific for B2M locus markedly increased the KI efficiency compared to its linear counterpart (13% and 24 % of KI obtained for LssDNAI and CssDNAI , respectively, Figure 12C). As shown above, this increase of KI observed with CssDNAI compared to LssDNAI was correlated to a higher viability (Figure 12D). In addition, our results show that switching from the linear to the circular ssDNA format leads to an increase of CFU plating efficiency (Figure 12E) and a decrease of MX1 and P21 markers (Figure 12F and Figure 12G, respectively), commonly associated to inflammation and cell fitness, respectively.

[0611] Altogether, our data, obtained for short single strand DNA (0.7 kb) and long single strand DNA (2.2 kb), show that circularization of ssDNA matrix enables higher DSB-mediated gene integration. Results obtained on HSPC inflammation (MX1 RTqPCR read out), HSPC fitness (P21 RTqPCR read out) and HSPC differentiation (CFU assay) suggest that circularization may improve DNA construct stability, prevent its degradation by endogenous DNA sensing pathway actors and mitigate P53 pathway-dependent cell death.

[0612] We thus confirm that circularizing ssDNA construct appears to be a relevant feature to markedly improve DSB induced gene insertion process in HSPCs. Circular ssDNA in a single-electroporation step process compared to a two- electroporation-steps process

[0613] To evaluate if a co-electroporation of TALEN, HDR-Enh01 , Via-Enh01 and CssDNA elicited a higher gene insertion outcome than a standard gene editing process involving two separated electroporation steps (i.e. TALEN, HDR-Enh01 and Via-Enh01 in a first electroporation step, and CssDNA in a second electroporation step), we performed a side-by-side experiment to compare the two processes. For that purpose, we used the CssDNA2 (SEQ ID NO: 50) designed to insert a HA Tag at the B2M locus in HSPCs.

[0614] Unexpectedly, our data first show that both processes elicited similar efficiency of HA Tag insertion at the B2M locus (49% versus 41 % for 2 electroporation steps and for 1 electroporation step, respectively) and cell viability (89.3% versus 87.4%, respectively). Interestingly, the level of inflammation and cell death markers (MX1 and P21 , respectively) were higher in the 2- electroporation-steps process than in the 1 -electroporation-step process, indicating that the latter triggers less cellular damage than the former. Finally, both processes elicited similar plating efficiency (Figure 13).

[0615] Altogether, our data unexpectedly show that transfection of TALEN, HDR-Enh01 , Via- Enh01 and CssDNA in a single-electroporation-step process produced edited cells with a similar efficiency of gene insertion than a standard 2-electroporation-steps process and has the advantage of mitigating inflammation and cell death signal commonly observed in the standard 2- electroporation-steps process.

[0616] Circular ssDNA compared to viral vector

[0617] To evaluate the efficiency of TALEN-mediated CssDNA-mediated gene targeting at the B2M locus in HSPCs and compare it to an AAV-mediated gene targeting approach, we designed a circular ssDNA matrix (CssDNAI , 2.2 kb total length, (SEQ ID NO: 49) encompassing the HLAE trimeric construct flanked by 5’ and 3’ arms (300 bp) homologous to the B2M locus. This matrix was designed to insert, in a disruptive manner, the HLAE construct in frame with the B2M locus. Insertion of this construct was expected to lead to HLA ABC(-) HSPCs expressing HLAE.

[0618] Using the protocol described in Figure 14A and in the Material and Methods section above, our in vitro results show that CssDNAI could be inserted at the B2M locus in a targeted fashion using the B2M TALEN (SEQ ID NO: 59 and 60). Our results showed an efficiency of gene knock- in (KI) reaching a mean of 30% (up to 49%) and a viability above 80%. Similar results were obtained with the AAV-mediated gene targeting approach (Figure 14B). Indeed, both CssDNA and AAV editing processes elicited similar KI frequency (30.7% ± 2.1 % and 26.1% ± 5.4%, mean ± SD, respectively), KI / KO ratio (0.61 ± 0.08 and 0.53 ± 0.14, mean ± SD, respectively), and cell viability (81.0% ± 1.0% and 82.5% ± 4.3%, mean ± SD, respectively) using 3 HSPC donors. Edited HSPCs were able to differentiate in vitro as observed by CFU assay.

[0619] The same cells were injected in NCG mice to assess their ability to engraft in the bone marrow and maintain their editing events. Unexpectedly, while both processes led to similar editing efficiencies (KI input) and viability in vitro, CssDNA-edited cells showed a higher bone marrow engraftment and a higher editing level (KI output) than AAV-edited HSPCs (Figure 14C). Both processes elicited similar levels of HSPC differentiation into Lymphocyte T and B, myeloid cells, monocyte, macrophages and neutrophiles with respect to untreated cells (data not shown).

[0620] These results were correlated to a higher frequency of HSC-enriched cell population and a higher frequency of HSC harboring gene insertion event in the CssDNA-edited cells compared to the AAV-edited HSPCs (Figure 14D). Indeed, we found a 3-fold higher KI frequency in hCD45 cells engrafted in animal injected with CssDNA-edited HSPCs compared to those injected with AAV-edited HSPCs.

[0621] Altogether, our results illustrate that editing HSPCs with TALEN and CssDNA leads to a higher level of potentially therapeutically relevant HSPCs engraftment in murine model.

[0622] To determine whether the advantageous effect of CssDNA and TALEN gene editing observed at the B2M locus was also observed at other loci in HSPCs, we designed 3 additional CssDNA donor templates (~3kb) specific for AAVS1 , CD11 B and S100A9 loci, which are three therapeutically relevant loci, demonstrated earlier to allow pan-lineage or myeloid-specific expression of therapeutic transgenes. Using the afore-mentioned gene editing protocol, loci- specific TALEN and CssDNA, we showed that AAVS1 , CD11 B and S100A9 loci were efficiently modified, reaching similar gene insertion frequency and viability than the ones obtained with TALENB2M and CssDNAI (34% ± 9.6%, 26% ± 6.6% and 39% ± 10.5%, mean KI frequency ± SD, respectively). This additional dataset indicates that CssDNA could be used to promote efficient gene insertion at multiple loci in HSPCs.

[0623] Example 7. Talen-mediated non viral HBB-aene correction in HSPCs usinq circular sinqle strand DNA The above example shows that an about 0.7 kb and an about 2.2 kb circular ssDNA matrices (CssDNA2 and CssDNAI , respectively) were more efficiently inserted at the B2M locus in HSPCs compared to their linear counterparts.

[0624] We sought to further confirm this proof of concept using a shorter (about 0.2 kb) circular ssODN encompassing 5 point-mutations specific for the HBB locus. This approach could be highly valuable to improve the efficiency of TALEN-mediated / - / BB-gene correction in the context of sickle cell anemia ex vivo gene therapy.

[0625] The side-by-side comparison of targeted insertion of a circular or linear ssODN of SEQ ID NO: 16 performed in HSPCs showed that circularization of the ssODN construct specific for HBB locus markedly increased the KI efficiency in comparison to its linear counterpart. Similarly to what has been demonstrated above for the B2M locus, this increase of KI was correlated with a higher viability of edited HSPCs.

Claims

CLAIMS1. An ex vivo method for preparing HBB-gene edited Hematopoietic Stem and Progenitor Cells (HSPCs) from a population of HSPCs comprising at least one endogenous / 788-allele having a mutation (A>T) or (A>C) at position 20 of the coding region of exon 1 of / 788-gene, wherein said mutation is associated with a hemoglobinopathy, said method comprising the steps of: iii) Introducing, into a population of HSPCs having an allele of an endogenous / 788-gene having said mutation, a monomeric TALE-nuclease comprising a monomer binding the HBB sequence of SEQ ID NO: 17 or SEQ ID NO: 19, or a heterodimeric TALE-nuclease comprising a first monomer binding the HBB sequence of SEQ ID NO: 17 or SEQ ID NO: 19 and a second monomer binding the HBB sequence of SEQ ID NO: 18; iv) introducing into said population of HSPCs a non-viral single-stranded polynucleotide repair template comprising the nucleic acid sequence of SEQ I D NO: 13, or comprising the nucleic acid sequence of SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16; whereby a population of / 788-gene edited HSPCs is obtained, which comprises HSPCs having at least one allele of the / 788-gene corrected at position 20 of the coding region of exon 1 of / 788-gene.

2. The method according to claim 1 , wherein the population of HSPCs having said mutation (A>T) is obtained from a patient suffering from sickle cell disease; or wherein the population of HSPCs having said mutation (A>C) is obtained from a patient suffering from hemoglobin beta C disease.

3. The method according to any one of claims 1 to 2, wherein said mutation is the mutation (A>T) at position 20 of the coding region of exon 1 of the / 788-gene and the population of HSPCs is obtained from a patient suffering from sickle cell disease.

4. The method according to any one of claims 1 to 3, wherein said TALE-nuclease targets the polynucleotide sequence of SEQ ID NO: 7 and cleaves the / 788-gene within the region of polynucleotide sequence SEQ ID NO: 8.

5. The method according to any one of claims 1 to 4, wherein said TALE-nuclease is a monomer comprising the RVD sequence HD-HD-NG-NN-NG-NN-NN-NI-NN-NI-NI-NN-NG-HD-NG-NG.

6. The method according to claim 5, wherein said TALE-nuclease is a monomer comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% identity with SEQ ID NO: 54.

7. The method according to any one of claims 1 to 4, wherein said TALE-nuclease is a heterodimer comprising a monomer binding SEQ ID NO: 17 and a monomer binding SEQ ID NO: 18.

8. The method according to claim 7, wherein said TALE-nuclease is a heterodimer comprising a monomer having the RVD sequence HD-HD-NG-NN-NG-NN-NN-NI-NN-NI-NI-NN-NG-HD-NG- NG and a monomer having the RVD sequence NG-HD-NI-HD-HD-NG-NG-NN-HD-HD-HD-HD- NI-HD-NI-NG.

9. The method according to claim 8, wherein said TALE-nuclease is a heterodimer comprising a monomer comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% identity with SEQ ID NO: 54 and a monomer comprising an amino acid sequence having at least 90%, at least 95%, at least 98%, or at least 99% identity with SEQ ID NO: 55.

10. The method according to any one of claims 1 to 9, wherein said TALE-nuclease is introduced by electroporation of a RNA encoding said TALE-nuclease.

11. The method according to claim 10, wherein said RNA is linear mRNA or circular RNA.

12. The method according to any one of claims 10 to 11 , wherein the RNA encoding the TALE- nuclease is introduced by electroporation from 24 to 72 hours after thawing of frozen HSPCs.

13. The method according to any one of claims 1 to 12, comprising introducing into said cells at least one enhancer of Homology Directed Repair (“HDR-enhancer”) selected from the group consisting of an inhibitor of 53BP1 , a dominant negative mutant of 53BP1 , an inhibitor of P53, and an inhibitor of Non-Homology-End-Joining.

14. The method according to claim 13, wherein said HDR-enhancer is a polypeptide comprising an amino acid sequence having at least 80% identity with SEQ ID NO: 61 , such as at least 90%, 95%, 98%, 99% identity.

15. The method according to claim 14, wherein said HDR-enhancer is a polypeptide comprising an amino acid sequence having at least 80% identity with SEQ ID NO: 62, such as at least 90%, 95%, 98%, 99% identity.

16. The method according to any one of claims 13 to 15, wherein said HDR-enhancer is introduced by electroporation of a RNA encoding said HDR-enhancer.

17. The method according to claim 16, wherein said RNA encoding said HDR-enhancer is linear mRNA or circular RNA.

18. The method according to claim 16 or 17, wherein the cells are transfected with at least 0.1 pg, at least 1 pg, or at least 2 pg, preferably at least 4 pg RNA of SEQ ID NO: 26 for about 1.106cells.

19. The method according to any one of claims 1 to 18, comprising introducing into said cells at least one viability-enhancer.

20. The method according to claim 19, wherein said viability-enhancer is an inhibitor of BAX / BAK selected from the group consisting of B-cell lymphoma-extra large (BCL-XL), B-cell lymphoma 2 (BCL-2), Myeloid cell leukemia 1 (MCL-1), BCL2 like 10 (BCL2L10, BCL-B or DIVA), BCL2 related protein A1 (BFL-1 or A1), BCL2 like 2 (BCL-W).

21. The method according to claim 19 or 20, wherein said viability-enhancer is B-cell lymphoma- extra-large (BCL-XL).

22. The method according to any one of claims 19 to 21 , wherein said viability-enhancer is a polypeptide comprising the amino acid sequence of SEQ ID NO: 63.

23. The method according to any one of claims 19 to 22, wherein said viability-enhancer is introduced by electroporation of a RNA encoding said viability-enhancer.

24. The method according to claim 23, wherein said RNA encoding said viability-enhancer is linear mRNA or circular RNA.

25. The method according to claim 24, wherein the cells are transfected with at least 0.1 pg, or at least 0.5 pg, preferably at least 1 pg of RNA encoding said viability-enhancer, for 1.106cells total.

26. The method according to any one of claims 13 to 25, wherein said HDR-enhancer and / or said viability-enhancer is / are electroporated concomitantly to the RNA encoding the TALE-nuclease.

27. The method according to any one of claims 1 to 26, comprising placing the cells in a culture medium comprising a sternness-enhancer, such as Valproic Acid (VPA), for at least one day.

28. The method according to any one of claims 1 to 27, comprising, prior to step i), a step of culturing the HSPCs in a culture medium comprising at least one compound stimulating cell cycle, for at least about 1 day.

29. The method according to any one of claims 1 to 28, wherein the HSPCs are cultured, prior to step i), in a culture medium comprising one or more of: Thrombopoietin (TPO), Fms-like tyrosine kinase 3 ligand (Flt-3L), Stem Cell Factor (SCF), Interleukin 3 (IL-3), hydrolyzed Polyvinyl Alcohol (P A), LIM171 , SR1 , aminoquinoline, and dmPGE2.

30. The method according to claim 29, wherein said culture medium comprises Thrombopoietin (TPO), Interleukin 3 (IL-3), and stem cell factor (SCF).

31. The method according to any one of claims 1 to 30, wherein said repair template is a singlestranded DNA C’ssDNA”).

32. The method according to claim 31 , wherein said ssDNA comprises the polynucleotide sequence SEQ ID NO: 16.

33. The method of any one of claims 1 to 32, wherein said repair template is a circular singlestranded DNA (“CssDNA”).

34. The method according to any one of claims 1 to 33, comprising two electroporation steps.

35. The method according to any one of claims 1 to 34, comprising introducing the RNA encoding the TALE-nuclease in a first electroporation step and introducing the polynucleotide repair template in a second electroporation step.

36. The method according to any one of claims 31 to 35, wherein the ssDNA is electroporated from 10 to 20 hours or from 12 to 18 hours, or from 15 to 17 hours, such as about 16 hours, after the electroporation of the RNA encoding the TALE-nuclease.

37. The method according to claim 33, wherein step i) of introducing the RNA encoding the TALE- nuclease and step ii) of introducing the circular single-stranded DNA are carried out in one electroporation step.

38. The method according to any one of claims 1 to 37, wherein, after step i), the HSPCs are incubated at 30°C, overnight, then cultured at 37°C.

39. The method according to any one of claims 1 to 38, further comprising recovering the population of / 788-gene edited HSPCs in a cryomedium and storing frozen until use, optionally without purification step before conditioning as a pharmaceutical composition comprising said / - / BB-gene edited HSPCs.

40. A population of H BB-gene edited HSPCs obtainable by the method according to any one of claims 1 to 39.41 . The population of / - / BB-gene edited HSPCs according to claim 40, wherein a polynucleotide comprising the exogenous sequence SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16, has been integrated at the / 788-locus in the genome of at least 10%, preferably at least 20%, 30%, 40%, 50%, 60 % of the cells.

42. The population of / - / BB-gene edited HSPCs according to claim 40 or 41 , wherein a polynucleotide comprising the exogenous sequence of SEQ ID NO: 13 has been integrated at the / 788-locus in the genome of at least 10%, preferably at least 20%, 30%, 40%, 50%, 60,%, 70%, 80%, of the cells.

43. The population of / 788-gene edited HSPCs according to any one of claims 40 to 42, wherein said population comprises at least at least 0.1%, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, or at least 30%, of cells having a [CD34+CD38-CD45RA- CD90+CD133+] phenotype.

44. The population of HBB-gene edited HSPCs according to any one of claims 40 to 43, wherein the HDR / indels ratio at the / - / BB-locus is equal to or higher than 1 , equal to or higher than 1 .2, 1.4, 1.5, or 1.7, equal to or higher than about 2, equal to or higher than about 2.5, equal to or higher than about 3, equal to or higher than about 3.2, 3.5, 3.8, equal to or higher than about 4, 4.1 , or 4.2.

45. The population of / 788-gene edited HSPCs according to any one of claims 40 to 44, wherein the number of edited cells is more than 0.5x108, preferably more than 1x108cells, more preferably more than 1.5x108cells, even more preferably more than 2x108cells.

46. Isolated / 788-gene edited HSPCs obtainable by the method according to any one of claims 1 to 39.

47. Isolated / 788-gene edited HSPCs, wherein a polynucleotide comprising the exogenous sequence of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16, has been integrated at the HBB-locus in the genome of said HSPCs.

48. A cryopreserved composition comprising a population of / 788-gene edited HSPCs according to any one of claims 40 to 45 or comprising isolated / 788-gene edited HSPCs according to any one of claims 46 to 47.

49. A pharmaceutical composition comprising a population of / 788-gene edited HSPCs according to any one of claims 40 to 45, isolated / 788-gene edited HSPCs according to any one of claims 46 to 47, or a cryopreserved composition according to claim 48; and a pharmaceutically acceptable excipient and / or carrier.

50. The population of / 788-gene edited HSPCs according to any one of claims 40 to 45, for use in the treatment of a hemoglobinopathy such as sickle cell disease.51 . The isolated / 788-gene edited HSPCs according to any one of claims 46 to 47, for use in the treatment of a hemoglobinopathy such as sickle cell disease.

52. The pharmaceutical composition according to claim 49, for use in the treatment of a hemoglobinopathy such as sickle cell disease.

53. A TALE-Nuclease selected from:- a monomeric TALE-nuclease comprising a monomer binding the HBB sequence of SEQ ID NO: 17 or SEQ ID NO: 19;- a heterodimeric TALE-nuclease comprising a first monomer binding the HBB sequence of SEQ ID NO: 17 or SEQ ID NO: 19 and a second monomer binding the HBB sequence of SEQ ID NO: 18.

54. The TALE-Nuclease according to claim 53, wherein said heterodimeric TALE-nuclease comprises a monomer having the RVD sequence HD-HD-NG-NN-NG-NN-NN-NI-NN-NI-NI-NN- NG-HD-NG-NG and a monomer having the RVD sequence NG-HD-NI-HD-HD-NG-NG-NN-HD- HD-HD-HD-NI-HD-NI-NG.

55. The TALE-Nuclease heterodimer according to any one of claims 53 to 54, comprising a first monomer comprising the amino acid sequence of SEQ ID NO: 54, or a variant thereof comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 54, such as at least 90%, at least 95%, or at least 99% identity, and binding the sequence of SEQ ID NO: 17, and a second monomer comprising the amino acid sequence of SEQ ID NO: 55, or a variant thereof comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 55, such as at least 90%, at least 95%, or at least 99% identity, and binding the sequence of SEQ ID NO: 18.

56. The TALE-Nuclease heterodimer according to any one of claims 53 to 55, comprising a first monomer comprising the amino acid sequence of SEQ ID NO: 54 and a second monomer comprising the amino acid sequence of SEQ ID NO: 55.

57. An isolated polynucleotide or vector encoding a TALE-Nuclease heterodimer according to any one of claims 53 to 56.

58. The isolated polynucleotide according to claim 57, which is a RNA such as mRNA.

59. An isolated polynucleotide, wherein said polynucleotide is a non-viral single-stranded polynucleotide comprising the sequence of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16.

60. The isolated polynucleotide according to claim 59, which is a single-stranded DNA (ssDNA) comprising SEQ ID NO: 16.

61. The isolated polynucleotide according to claim 60, which is a circular ssDNA.

62. A kit comprising:(i) at least one isolated polynucleotide or vector encoding a TALE-Nuclease heterodimer comprising a first monomer binding the HBB sequence of SEQ ID NO: 17 or SEQ ID NO: 19 and a second monomer binding the HBB sequence of SEQ ID NO: 18, and(ii) at least one isolated non-viral single-stranded polynucleotide repair template comprising the nucleic acid sequence of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16, and,(iii) optionally, at least one isolated polynucleotide or vector encoding an HDR- enhancer; and(iv) optionally, at least one isolated polynucleotide or vector encoding a viabilityenhancer.

63. The kit according to claim 62, wherein the TALE-nuclease encoded by said polynucleotide or vector is a TALE-Nuclease heterodimer according to any one of claims 53 to 56.

64. The kit according to claim 62 or 63, wherein the TALE-nuclease encoded by said polynucleotide or vector is a TALE-Nuclease comprising a first monomer comprising the amino acid sequence of SEQ ID NO: 54 and a second monomer comprising the amino acid sequence of SEQ ID NO: 55.

65. The kit according to any one of claims 62 or 64, wherein said isolated polynucleotide repair template is the polynucleotide defined in any one of claims 59 to 61 .

66. The kit according to any one of claims 62 to 65, wherein said isolated polynucleotide repair template comprises the nucleic acid sequence of SEQ ID NO: 15 or SEQ ID NO: 16.

67. The kit according to any one of claims 62 to 66, wherein said HDR-enhancer is selected from the group consisting of an inhibitor of 53BP1 , a dominant negative mutant of 53BP1 , an inhibitorof P53, and an inhibitor of Non-Homology-End-Joining, such as an HDR-enhancer of SEQ ID NO: 61.

68. The kit according to any one of claims 62 to 67, wherein said viability-enhancer comprises the amino acid sequence of SEQ ID NO: 63.

69. Ex vivo use of the kit according to any one of claims 62 to 68 for editing at least one endogenous / 788-allele comprising a mutation (A>T) or (A>C) at position 20 of the coding region of exon 1 of / 788-gene in the HSPCs of a patient suffering from a hemoglobinopathy associated with said mutation.

70. A method of treatment of a hemoglobinopathy associated with a mutation (A>T) or (A>C) in at least one endogenous / 788-allele at position 20 of the coding region of exon 1 of / 788-gene in a patient, comprising administering in said patient a population of / 788-gene edited HSPCs according to any one of claims 40 to 45, isolated / 788-gene edited HSPCs according to any one of claims 46 to 47, or a pharmaceutical composition according to claim 49.

71. The method of treatment according to claim 70, wherein said hemoglobinopathy is sickle cell disease and wherein said mutation is the mutation (A>T) at position 20 of the coding region of exon 1 of the / 788-gene.

72. The method of treatment according to claim 70 or 71 , wherein the population of / 788-gene edited HSPCs are prepared from HSPCs from the patient to be treated.

73. A method of treatment of sickle cell disease associated with a mutation (A>T) in at least one endogenous / 788-allele at position 20 of the coding region of exon 1 of / 788-gene in a human patient, said method comprising administering, in said patient, a population of / 788-gene edited HSPCs,- wherein a polynucleotide comprising the exogenous sequence of SEQ ID NO: 13 or SEQ ID NO: 16 has been integrated at the / 788-locus in the genome of at least 10%, preferably at least 20%, 30%, 40%, 50%, 60 % of the cells of the population; and- wherein, optionally, said population comprises at least 0.1%, at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, or at least 30%, of cells having a [CD34+CD38-CD45RA-CD90+CD133+] phenotype; and- wherein, optionally, the ratio of HDR / indels in the / - / BB-locus in the cell population is equal to or higher than 1, equal to or higher than 1.2, 1.4, 1.5, or 1.7, equal to or higher than about 2, equal to or higher than about 2.5, equal to or higher than about 3, equal to or higher than 3.2, 3.4, 3.5, 3.6, 3.8, equal to or higher than about 4, or 4.2.

Images