Antisense oligonucleotide (ASO)-mediated down-regulation of CD33 to safely enrich for genetically modified cells

Abstract

The present invention relates to a recombinant antisense oligonucleotide that targets CD33 mRNA and to a method of preparing a substantially pure population of edited eukaryotic cells comprising the steps of i) editing a population of eukaryotic cells by the use of base editors, (ii) treating the same population with antisense oligonucleotides according to the invention to transiently downregulate CD33 and iii) enriching the population of edited eukaryotic cells that is negative for CD33.

Description

[0001] ANTISENSE OLIGONUCLEOTIDE (ASO)-MEDIATED DOWN-REGULATION OF

[0002] CD33 TO SAFELY ENRICH FOR GENETICALLY MODIFIED CELLS

[0003] FIELD OF THE INVENTION:

[0004] The present invention is in the field of medicine, in particular haematology and downregulation by antisense oligonucleotide of CD33.

[0005] BACKGROUND OF THE INVENTION:

[0006] SCD and P-thalassemia are genetic diseases caused by mutations in the P-globin locus. In SCD, a point mutation in the HBB gene leads to the formation of the sickle ps-globin chain, which causes the polymerization of sickle hemoglobin (HbS), red blood cell (RBC) sickling, anemia, and organ damage1,2. In P-thalassemia, the partial or total absence of P-globin chains (P+and P°, respectively) leads to the precipitation of noncoupled a-globin chains, apoptosis of erythroid precursors, ineffective erythropoiesis, and anemia3 5. Gene therapy approaches based on the transplantation of autologous, genetically modified hematopoietic stem cells (HSCs) have been investigated as a treatment option for patients lacking a compatible donor for allogeneic HSC transplantation6.

[0007] The severity of both SCD and P-thalassemia is lessened by the hereditary persistence of fetal hemoglobin (HbF) in adulthood (HPFH)7. This persistence is due to mutations located 200 to 115 nucleotides upstream of the transcription start sites of the identical HBG1 and HBG2 y- globin promoters. HPFH mutations either generate de novo DNA motifs recognized by transcriptional activators (e.g., KLF I )8 10or disrupt binding sites (BS) for transcriptional repressors (e.g., LRF and BCL11A)11. CRISPR-Cas9 nuclease strategies have been used to disrupt the LRF and BCL11A repressor BS via the non-homologous end-joining (NHEJ)- mediated generation of insertions / deletions (InDeis) that mimic HPFH mutations and reactivate HbF expression12 l 5.

[0008] HSCs are highly sensitive to DNA double-strand breaks (DSBs)16- especially in case of multiple on-target events or concomitant on-target and off-target events. Even when highly specific single guide RNAs (sgRNAs) are used, the Cas9-sgRNA treatment of human HSPCs induces a DNA damage response (DDR) that can lead to apoptosis17,18. CRISPR-Cas9 can cause p53-dependent cell toxicity and cell cycle arrest, resulting in the selection of cells with a dysfunctional p53 pathway1919. Furthermore, the generation of several on-target DSBs, simultaneous on-target and off-target DSBs, or even a single on-target DSB can lead to genomic deletions, inversions or translocations, chromosome loss, and chromothripsis20 23. Hence, the development of novel, efficacious, safe treatment strategies for P-hemoglobinopathies based on precise base editing (rather than DSB-induced DNA repair) is highly desirable. Cytidine and adenine base editors (CBEs and ABEs) are composed of a Cas9 nickase and a deaminase, and introduce C-to-T and A-to-G point mutations24, respectively. Importantly, base editors do not generate DNA DSBs or induce DDR and any consequent event. Hence, base editors allow the simultaneous editing of multiplex targets, an approach that would lead to genomic rearrangements in the case of the CRISPR / Cas9 nuclease system.

[0009] The inventors recently reported adenine and cytidine ABE- and CBE-mediated introduction of HPFH and HPFH-like mutations in the -200 region of the HBG1 / 2 promoters with minimal off-target activity. This led to therapeutic HbF levels in human RBCs differentiated from base edited SCD or P-thalassemia hematopoietic stem / progenitor cells (HSPCs; containing mostly hematopoietic progenitors and less than 1% of HSCs) and rescued the pathological phenotype in vitro. However, even though xenotransplantation experiments (of human HSPCs in immunodeficient mice) showed base editing in long-term HSCs, the efficiency was reduced compared to input HSPCs25. BEs might have induced some toxicity in bona fide HSCs or might be less efficient in in this cell population compared to hematopoietic progenitors.

[0010] More in general, several limitations still hamper the broad application of genome editing approaches for hematopoietic disorders: (i) the efficiency in HSCs and (ii) in vivo competition of the infused unedited HSCs or the endogenous HSCs that are not completely eliminated with the myeloablation treatments that patients undergo. Genetic modification of HSCs can potentially affect their properties. In particular, editing strategies are based on a 48- to 72-h in vitro treatment including the electroporation procedure, the introduction of the editing system complexes, the genetic modification of the target regions, and the exposure to cytokines. In this context non-edited HSCs might have a survival advantage and better sternness compared to edited cells.

[0011] Several parameters could be further optimized with regard to the base-editing efficiency in HSCs, and the fitness of the edited HSCs. Furthermore, enriching for ex vivo or in vivo edited versus non-edited HSCs could be exploited to maximize the engraftment of genetically modified cells. This could also allow the reduction of the conditioning regimen and its side effects. Such an approach could increase the success of therapeutic strategies for diseases with no or little selective advantage of corrected cells such as P-hemoglobinopathies26,27. CD33 is a surface marker of the myeloid lineage, which is also highly expressed in malignant blasts of acute myeloid leukemia (AML) patients28 31. Gemtuzumab, a toxin- conjugated anti-CD33 monoclonal antibody has been approved for treating AML. Of note, CD33 is also expressed on human HSCs with a high regenerative potential31and one of the main side effects of Gemtuzumab is myelosuppression. Therefore, even though it lacks expression specificity, CD33 has been used as a target in patients with AML that are treated with Gemtuzumab, and in clinical trials (NCT03971799, NCT03927261) and experimental models of chimeric antigen receptor (CAR) T-cell immunotherapies32 36. Interestingly, to avoid myelosuppression, researchers have combined transplantation of CD33 knock-out (KO) HSPCs with CAR T-cells efficiently targeting the from-now-on leukemia-specific CD33 -expressing blasts. Importantly, CD33 KO HSPCs remained functional and were able to engraft and differentiate in animal models (mice and non-human primates)30. Furthermore, an ongoing clinical trial (NCT04849910) is based on the transplantation of CD33 KO HSPCs in AML patients with high risk of relapse, who require Gemtuzumab treatment post-transplantation, so as to reduce the toxic side effect of the antibody.

[0012] SUMMARY OF THE INVENTION:

[0013] The present invention is defined by the claims. In particular, the present invention relates to recombinant antisense oligonucleotide that targets CD33 mRNA wherein the antisense oligonucleotide is selected from SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9.

[0014] DETAILED DESCRIPTION OF THE INVENTION:

[0015] The use of CRISPR base editors to simultaneously target the HBG promoters (to reactivate HbF production), and the CD33 gene (to downregulate the expression of this surface marker), could represent an intriguing approach to select and enrich edited HSPCs with reduced CD33 expression. This selection strategy would allow to obtain a pure population of HSPCs able to give rise to RBC re-expressing fetal hemoglobin. Nevertheless, the multiplex base editing can induce mutational risks due to the targeting of two different loci and therefore it can lead to genomic rearrangements like translocation, inversions or large deletions. Furthermore, even though CD33 knockout is reported not to affect HSC functions, engraftment capability and differentiation in animal models (mice and non-human primates)30, the CD33 protein is still an essential marker of myeloid cells. Finally, the permanent CD33 KO would prevent the use of Gemtuzumab in the case of AML. The use of antisense oligonucleotides (ASOs) to transiently downregulate CD33 appears safer than a permanent CD33 KO generated by the BE strategy. In fact, ASOs can modulate the splicing or induce mRNA degradation by a steric block of translation without the risk of generating genomic rearrangements.

[0016] Main definitions:

[0017] As used herein, the term "oligonucleotide" refers to a nucleic acid sequence, 3'-5' or 5'- 3' oriented, which may be single- or double-stranded. The oligonucleotide used in the context of the invention may in particular be DNA or RNA. The term also includes "oligonucleotide analog" which refers to an oligonucleotide having (i) a modified backbone structure, e.g., a backbone other than the standard phosphodiester linkage found in natural oligo- and polynucleotides, and (ii) optionally, modified sugar moieties, e.g., morpholino moieties rather than ribose or deoxyribose moieties. Oligonucleotide analogs support bases capable of hydrogen bonding by Watson-Crick base pairing to standard polynucleotide bases, where the analog backbone presents the bases in a manner to permit such hydrogen bonding in a sequencespecific fashion between the oligonucleotide analog molecule and bases in a standard polynucleotide {e.g., single-stranded RNA or single-stranded DNA). Particularly, analogs are those having a substantially uncharged, phosphorus containing backbone. A substantially uncharged, phosphorus containing backbone in an oligonucleotide analog is one in which a majority of the subunit linkages, e.g., between 50-100%, typically at least 60% to 100% or 75% or 80% of its linkages, are uncharged, and contain a single phosphorous atom. The term “oligonucleotide” also refers to an oligonucleotide sequence that is inverted relative to its normal orientation for transcription and so corresponds to an RNA or DNA sequence that is complementary to a target gene mRNA molecule expressed within the host cell (e.g., it can hybridize to the target gene mRNA molecule through Watson-Crick base pairing).

[0018] As used herein, the term “antisense oligonucleotide” or “ASO” refers to a single strand of DNA, RNA, or modified nucleic acids that is complementary to a chosen sequence. Antisense RNA can be used to prevent protein translation of certain mRNA strands by binding to them. Antisense DNA can be used to target a specific, complementary (coding or non-coding) RNA. In some embodiments, the antisense oligonucleotide of the present invention is an antisense RNA. In some embodiments, the antisense oligonucleotide of the present invention is an antisense DNA.

[0019] As used herein, the term “exon” refers to a defined section of nucleic acid that encodes for a protein, or a nucleic acid sequence that is represented in the mature form of an RNA molecule after either portion of a pre-processed (or precursor) RNA have been removed by splicing. The mature RNA molecule can be a messenger RNA (mRNA) or a functional form of a non-coding RNA, such as rRNA or tRNA.

[0020] As used herein, the term "encode", or "encoding" or "encoded" refers to a nucleic acid sequence that codes for a polypeptide sequence.

[0021] As used herein, the term “expression” refers to the process by which a polynucleotide is transcribed from a DNA template (such as into and mRNA or other RNA transcript) and / or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides may be collectively referred to as “gene product”. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell. Any method known in the art can be used to measure the expression of the gene (e. g. HPLC analysis of protein and RT-qPCR analysis of mRNA.) Typically, said methods are described in the EXAMPLE.

[0022] As used herein, the term “inhibitor of expression” refers to a natural or synthetic compound that has a biological effect to inhibit or decrease the expression of a gene, e.g. the CD33 gene. It will be understood to those persons of skill in the relevant art that inhibiting expression of a gene, e.g. the CD33 gene, typically results in a decrease or even abolition of the gene product (protein, e.g. CD33 protein) in target cells or tissues, although various levels of inhibition may be achieved. Inhibiting or decreasing expression is typically referred to as knockdown

[0023] As used herein, the terms "decrease", "reduced", "reduction" "repress" are all used generally to mean a decrease by a statistically significant amount, for example, a decrease by at least 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (i.e. absent level as compared to a reference sample), or any decrease between 10-100% as compared to a reference level. As used herein, the term "knock-down" as used herein refers to a reduction in the expression of a gene or its gene product(s).

[0024] As used herein, the terms “expressing (positive or +)” and “not expressing (negative or -)” are well known in the art and refer to the expression level of a phenotypic marker of interest (e.g. CD33), in that the expression level of the phenotypic marker corresponding to “+” is high or intermediate, also referred as The phenotypic marker corresponding to is a null expression level of the phenotypic marker or also refers to less than 10 % of a cell population expressing the said phenotypic marker. As used herein, the term “complementarity” refers to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick base-pairing or other non-traditional types. A percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary). “Perfectly complementary” means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. “Substantially complementary” as used herein refers to a degree of complementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions.

[0025] As used herein, the term “CD33” has its general meaning in the art and refers to the transmembrane receptor encoded by the CD33 gene. The term is also known as Siglec-3 (sialic acid binding Ig-like lectin 3), SIGLEC3, SIGLEC-3, gp67, or p67). An exemplary amino acid sequence for CD33 is shown as SEP ID NO:1.

[0026] SEQ ID NO : 1 >sp | P20138 | CD33 HUMAN Myeloid cell surface antigen CD33 0S=Homo sapiens OX=9606 GN=CD33 PE=1 SV=2 MPLLLLLPLLWAGALAMDPNFWLQVQESVTVQEGLCVLVPCTFFHPI PYYDKNSPVHGYW FREGAI I SRDSPVATNKLDQEVQEETQGRFRLLGDPSRNNCSLSIVDARRRDNGSYFFRM ERGSTKYSYKSPQLSVHVTDLTHRPKILI PGTLEPGHSKNLTCSVSWACEQGTPPI FSWL SAAPTSLGPRTTHSSVLI ITPRPQDHGTNLTCQVKFAGAGVTTERTIQLNVTYVPQNPTT GI FPGDGSGKQETRAGWHGAIGGAGVTALLALCLCLI FFIVKTHRRKAARTAVGRNDTH PTTGSASPKHQKKSKLHGPTETSSCSGAAPTVEMDEELHYASLNFHGMNPSKDTSTEYSE VRTQ

[0027] As used herein, the term “hematopoietic stem cell” or “HSC” refers to blood cells that have the capacity to self-renew and to differentiate into precursors of blood cells. These precursor cells are immature blood cells that cannot self-renew and must differentiate into mature blood cells. Hematopoietic stem and progenitor cells display a number of phenotypes, such as Lin-CD34+CD38-CD90+CD45RA-, Lin-CD34+CD38-CD90-CD45RA-, Lin- CD34+CD38+IL-3aloCD45RA-, and Lin-CD34+CD38+CD 10+(Daley et al., Focus 18:62-67, 1996; Pimentel, E., Ed., Handbook of Growth Factors Vol. Ill: Hematopoietic Growth Factors and Cytokines, pp. 1-2, CRC Press, Boca Raton, Fla., 1994). Within the bone marrow microenvironment, the stem cells self-renew and maintain continuous production of hematopoietic stem cells that give rise to all mature blood cells throughout life. In some embodiments, the hematopoietic progenitor cells or hematopoietic stem cells are isolated form peripheral blood cells.

[0028] As used herein, the term "isolated cell" refers to a cell that has been removed from an organism in which it was originally found, or a descendant of such a cell. Optionally the eukaryotic cell has been cultured in vitro, e.g., in the presence of other cells. Optionally the eukaryotic cell is later introduced into a second organism or reintroduced into the organism from which it (or the cell from which it is descended) was isolated. As used herein, the term "isolated population" with respect to an isolated population of cells as used herein refers to a population of cells that has been removed and separated from a mixed or heterogeneous population of cells. In some embodiments, an isolated population is a substantially pure population of cells as compared to the heterogeneous population from which the cells were isolated or enriched.

[0029] As used herein, the expression “substantially pure population of cells” means a population of cells that contains at least 90, 91, 92, 03, 94, 95, 96, 97, 98, or 99% of the desired cell type.

[0030] As used herein, the term "enriching" includes any isolation or sorting process that increases the relative abundance of a desired cell type, or cell types, in a population of cells.

[0031] As used herein, the terms “subject” or “patient” denote a mammal, such as a rodent, a feline, a canine, and a primate. Particularly, the subject according to the invention is a human, a mouse or a rat. More particularly, the subject according to the invention has or is susceptible to suffer from a P-hemoglobinopathy.

[0032] As used herein, the term "P-hemoglobinopathy" has its general meaning in the art and refers to any defect in the structure or function of any hemoglobin of an individual, and includes defects in the primary, secondary, tertiary or quaternary structure of hemoglobin caused by any mutation, such as deletion mutations or substitution mutations in the coding regions of the HBB gene, or mutations in, or deletions of, the promoters or enhancers of such gene that cause a reduction in the amount of hemoglobin produced as compared to a normal or standard condition.

[0033] As used herein, the term "sickle cell disease" has its general meaning in the art and refers to a group of autosomal recessive genetic blood disorders, which results from mutations in a globin gene and which is characterized by red blood cells that assume an abnormal, rigid, sickle shape. They are defined by the presence of PS-globin gene coding for a P-globin chain variant in which glutamic acid is substituted by valine at amino acid position 6 of the peptide: incorporation of the PS-globin in the Hb tetramers (HbS, sickle Hb) leads to Hb polymerization and to a clinical phenotype. The term includes sickle cell anemia (HbSS), si ckl e-hemoglobin C disease (HbSC), sickle beta-plus- thalassaemia (HbS / p+), or sickle beta-zerothalassaemia (HbS / pO).

[0034] As used herein, the term "P-thalassemia" refers to a hemoglobinopathy that results from an altered ratio of a-globin to P-like globin polypeptide chains resulting in the underproduction of normal hemoglobin tetrameric proteins and the precipitation of free, unpaired a-globin chains.

[0035] As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).

[0036] As used herein, the term "therapeutically effective amount" is meant a sufficient amount of population of cells to treat the disease at a reasonable benefit / risk ratio applicable to any medical treatment. It will be understood that the total usage the gene editing platform will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the age, body weight, general health, sex and diet of the patient, the time of administration, route of administration, the duration of the treatment, drugs used in combination or coincidental with the population of cells, and like factors well known in the medical arts. In some embodiments, the cells are formulated by first harvesting them from their culture medium, and then washing and concentrating the cells in a medium and container system suitable for administration (a "pharmaceutically acceptable" carrier) in a treatment-effective amount. Suitable infusion medium can be any isotonic medium formulation, typically normal saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), but also 5% dextrose in water or Ringer's lactate can be utilized. The infusion medium can be supplemented with human serum albumin. A treatment-effective amount of cells in the composition is dependent on the relative representation of the cells with the desired specificity, on the age and weight of the recipient, and on the severity of the targeted condition. This number of cells can be as low as approximately 103 / kg, preferably 5xl03 / kg; and as high as 107 / kg, preferably 108 / kg. The number of cells will depend upon the ultimate use for which the composition is intended, as will the type of cells included therein. Typically, the minimal dose is 2 millions of cells per kg. Usually 2 to 20 millions of cells are injected in the subject. The desired purity can be achieved by introducing a sorting step. For uses provided herein, the cells are generally in a volume of a liter or less, can be 500 ml or less, even 250 ml or 100 ml or less. The clinically relevant number of cells can be apportioned into multiple infusions that cumulatively equal or exceed the desired total amount of cells.

[0037] Antisense oligonucleotides (ASOs) of the present invention

[0038] The first object of the present invention relates to isolated, synthetic or recombinant antisense oligonucleotide that targets CD33 mRNA.

[0039] According to the invention, the antisense oligonucleotide of the present invention thus targets an mRNA encoding CD33 gene product, and is capable of reducing the amount of CD33 expression in cells. Methods for determining whether an oligonucleotide is capable of reducing the amount of CD33 in cells are known to those skilled in the art. This may for example be done by analyzing CD33 RNA expression such as by RT-qPCR, in situ hybridization or CD33 protein expression such as by immunohistochemistry, Western blot, and by comparing CD33 protein expression or CD33 functional activity in the presence and in the absence of the antisense oligonucleotide to be tested.

[0040] In particular, the antisense oligonucleotide comprises a sequence that is at least partially complementary, particularly perfectly complementary, to a region of the sequence of said mRNA, said complementarity being sufficient to yield specific binding under intra-cellular conditions. As immediately apparent to the skilled in the art, by a sequence that is “perfectly complementary to” a second sequence is meant the reverse complement counterpart of the second sequence, either under the form of a DNA molecule or under the form of a RNA molecule. A sequence is “partially complementary to” a second sequence if there are one or more mismatches. In particular, the antisense oligonucleotide of the present invention is designed to be complementary to a sequence located to the exon 2 within the pre-mRNA molecule encoding for CD33.

[0041] In some embodiments, the antisense oligonucleotide of the present invention has a length from 15 to 25 nucleotides. In particular, the oligonucleotide of the present invention has a length of 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides.

[0042] In some embodiments, the antisense oligonucleotide of the invention comprises or consists of a sequence selected from Table 2 (see EXAMPLE).

[0043] Preferentially, the antisense oligonucleotide of the invention comprises or consists of the sequence as set forth in SEQ ID NO:7 or SEQ ID NO:4.

[0044] In some embodiments, the oligonucleotide of the present invention is further modified, particularly chemically modified, in order to increase the stability and / or therapeutic efficiency in vivo. The one skilled in the art can easily provide some modifications that will improve the efficacy of the oligonucleotide such as stabilizing modifications and modifications avoiding the RNase H activation in order to avoid degradation of the targeted transcript (C. Frank Bennett and Eric E. Swayze, RNA Targeting Therapeutics: Molecular Mechanisms of Antisense Oligonucleotides as a Therapeutic PlatformAnnu. Rev. Pharmacol. Toxicol. 2010.50:259-293; Juliano RL. The delivery of therapeutic oligonucleotides. Nucleic Acids Res. 2016 Aug 19;44(14):6518-48). In particular, the oligonucleotide used in the context of the invention may comprise modified nucleotides. Chemical modifications may occur at three different sites: (i) at phosphate groups, (ii) on the sugar moiety, and / or (iii) on the entire backbone structure of the oligonucleotide. Typically, chemical modifications include backbone modifications, heterocycle modifications, sugar modifications, and conjugation strategies.

[0045] For example the oligonucleotide may be selected from the group consisting of oligodeoxyribonucleotides, oligoribonucleotides, small regulatory RNAs (sRNAs), U7- or Ul- mediated ASOs or conjugate products thereof such as peptide-conjugated or nanoparticle- complexed ASOs, chemically modified oligonucleotide by backbone modifications such as morpholinos, phosphorodiamidate morpholino oligomers (Phosphorodiamidate morpholinos, PMO), peptide nucleic acid (PNA), phosphorothioate (PS) oligonucleotides, stereochemically pure phosphorothioate (PS) oligonucleotides, phosphoramidates modified oligonucleotides, thiophosphoramidate-modified oligonucleotides, and methylphosphonate modified oligonucleotides; chemically modified oligonucleotide by heterocycle modifications such as bicycle modified oligonucleotides, Bicyclic Nucleic Acid (BNA), tricycle modified oligonucleotides, tricyclo-DNA-antisense oligonucleotides (ASOs), nucleobase modifications such as 5-methyl substitution on pyrimidine nucleobases, 5-substituted pyrimidine analogues, 2-Thio-thymine modified oligonucleotides, and purine modified oligonucleotides; chemically modified oligonucleotide by sugar modifications such as Locked Nucleic Acid (LNA) oligonucleotides, 2 ’,4 ’-Methyleneoxy Bridged Nucleic Acid (BNA), ethylene-bridged nucleic acid (ENA), constrained ethyl (cEt) oligonucleotides, 2’ -Modified RNA, 2’- and 4’ -modified oligonucleotides such as 2’-0-Me RNA (2’-0Me), 2’-O-Methoxyethyl RNA (MOE), 2’ -Fluoro RNA (FRNA), and 4’-Thio-Modified DNA and RNA; chemically modified oligonucleotide by conjugation strategies such as N-acetyl galactosamine (GalNAc) oligonucleotide conjugates such as 5 ’-GalNAc and 3 ’-GalNAc ASO conjugates, lipid oligonucleotide conjugates, cell penetrating peptides (CPP) oligonucleotide conjugates, targeted oligonucleotide conjugates, antibody-oligonucleotide conjugates, polymer-oligonucleotide conjugate such as with PEGylation and targeting ligand; and chemical modifications and conjugation strategies described for example in Bennett and Swayze, 2010 (RNA targeting therapeutics: molecular mechanisms of antisense oligonucleotides as a therapeutic platform. Annu Rev Pharmacol Toxicol. 2010;50:259-93); Wan and Seth, 2016 (The Medicinal Chemistry of Therapeutic Oligonucleotides. J Med Chem. 2016 Nov 10;59(21):9645-9667); Juliano, 2016 (The delivery of therapeutic oligonucleotides. Nucleic Acids Res. 2016 Aug 19;44(14):6518-48); Lundin et al., 2015 (Oligonucleotide Therapies: The Past and the Present. Hum Gene Ther. 2015 Aug;26(8):475-85); and Prakash, 2011 (An overview of sugar-modified oligonucleotides for antisense therapeutics. Chem Biodivers. 2011 Sep;8(9): 1616-41). Indeed, for use in vivo, the oligonucleotide may be stabilized. A “stabilized” oligonucleotide refers to an oligonucleotide that is relatively resistant to in vivo degradation (e.g. via an exo- or endo-nuclease). Stabilization can be a function of length or secondary structure. In particular, oligonucleotide stabilization can be accomplished via phosphate backbone modifications, phosphodiester modifications, phosphorothioate (PS) backbone modifications, combinations of phosphodiester and phosphorothioate modifications, thiophosphoramidate modifications, 2' modifications (2'- O-Me, 2'-O-(2-methoxyethyl) (MOE) modifications and 2'-fluoro modifications), methylphosphonate, methylphosphorothioate, phosphorodithioate, p-ethoxy, and combinations thereof.

[0046] For example, the oligonucleotide may be employed as phosphorothioate derivatives (replacement of a non-bridging phosphoryl oxygen atom with a sulfur atom), which have increased resistance to nuclease digestion. 2 ’-methoxy ethyl (MOE) modification (such as the modified backbone commercialized by IONIS Pharmaceuticals) is also effective. Additionally or alternatively, the oligonucleotide of the present invention may comprise completely, partially or in combination, modified nucleotides which are derivatives with substitutions at the 2' position of the sugar, in particular with the following chemical modifications: O-m ethyl group (2'-0-Me) substitution, 2-methoxyethyl group (2'-0-M0E) substitution, fluoro group (2'- fluoro) substitution, chloro group (2'-Cl) substitution, bromo group (2'-Br) substitution, cyanide group (2'-CN) substitution, trifluoromethyl group (2'-CF3) substitution, OCF3 group (2'-OCF3) substitution, OCN group (2'-OCN) substitution, O-alkyl group (2'-O-alkyl) substitution, S-alkyl group (2'-S-alkyl) substitution, N-alkyl group (2'-N-akyl) substitution, O-alkenyl group (2'-O- alkenyl) substitution, S-alkenyl group (2'-S-alkenyl) substitution, N-alkenyl group (2'-N- alkenyl) substitution, SOCH3 group (2'-SOCH3) substitution, SO2CH3 group (2'-SO2CH3) substitution, ONO2 group (2'-ONO2) substitution, NO2 group (2'-NO2) substitution, N3 group (2'-N3) substitution and / or NH2 group (2'-NH2) substitution. Additionally or alternatively, the oligonucleotide of the present invention may comprise completely or partially modified nucleotides wherein the ribose moiety is used to produce locked nucleic acid (LNA), in which a covalent bridge is formed between the 2' oxygen and the 4' carbon of the ribose, fixing it in the 3'-endo configuration. These molecules are extremely stable in biological medium, able to activate RNase H such as when LNA are located to extremities (Gapmer) and form tight hybrids with complementary RNA and DNA.

[0047] In some embodiments, the oligonucleotide used in the context of the invention comprises modified nucleotides selected from the group consisting of LNA, 2’-0Me analogs, 2'-O-Met, 2'-O-(2 -methoxy ethyl) (MOE) oligomers, 2’ -phosphorothioate analogs, 2’ -fluoro analogs, 2’ -Cl analogs, 2’-Br analogs, 2’-CN analogs, 2’-CF3 analogs, 2’-OCF3 analogs, 2’- OCN analogs, 2’ -O-alkyl analogs, 2’ -S-alkyl analogs, 2’ -N-alkyl analogs, 2’ -O-alkenyl analogs, 2’ -S-alkenyl analogs, 2’ -N-alkenyl analogs, 2’-SOCH3 analogs, 2’-SO2CH3 analogs, 2’-ONO2 analogs, 2’-NO2 analogs, 2’-N3 analogs, 2’-NH2 analogs, tri cyclo (tc)-DNAs, U7 short nuclear (sn) RNAs, tricyclo-DNA-oligoantisense molecules and combinations thereof (U.S. Provisional Patent Application Serial No. 61 / 212,384 For: Tricyclo-DNA Antisense Oligonucleotides, Compositions and Methods for the Treatment of Disease, filed April 10, 2009, the complete contents of which is hereby incorporated by reference).

[0048] In a particular embodiment, the oligonucleotide according to the invention is a LNA oligonucleotide. As used herein, the term "LNA" (Locked Nucleic Acid) (or "LNA oligonucleotide") refers to an oligonucleotide containing one or more bicyclic, tricyclic or polycyclic nucleoside analogues also referred to as LNA nucleotides and LNA analogue nucleotides. LNA oligonucleotides, LNA nucleotides and LNA analogue nucleotides are generally described in International Publication No. WO 99 / 14226 and subsequent applications; International Publication Nos. WO 00 / 56746, WO 00 / 56748, WO 00 / 66604, WO 01 / 25248, WO 02 / 28875, WO 02 / 094250, WO 03 / 006475; U.S. Patent Nos. 6,043,060, 6268490, 6770748, 6639051, and U.S. Publication Nos. 2002 / 0125241, 2003 / 0105309, 2003 / 0125241, 2002 / 0147332, 2004 / 0244840 and 2005 / 0203042, all of which are incorporated herein by reference. LNA oligonucleotides and LNA analogue oligonucleotides are commercially available from, for example, Proligo LLC, 6200 Lookout Road, Boulder, CO 80301 USA. Other forms of oligonucleotides of the present invention are oligonucleotide sequences coupled to small nuclear RNA molecules such as U1 or U7 in combination with a viral transfer method based on, but not limited to, lentivirus or adeno-associated virus (Denti, MA, et al, 2008; Goyenvalle, A, et al, 2004).

[0049] Other forms of oligonucleotides of the present invention are peptide nucleic acids (PNA). In peptide nucleic acids, the deoxyribose backbone of oligonucleotides are replaced with a backbone more akin to a peptide than a sugar. Each subunit, or monomer, has a naturally occurring or non-naturally occurring base attached to this backbone. One such backbone is constructed of repeating units of N-(2-aminoethyl)glycine linked through amide bonds. Because of the radical deviation from the deoxyribose backbone, these compounds were named peptide nucleic acids (PNAs) (Dueholm et al., New J. Chem., 1997, 21, 19-31). PNA binds both DNA and RNA to form PNA / DNA or PNA / RNA duplexes. The resulting PNA / DNA or PNA / RNA duplexes are bound with greater affinity than corresponding DNA / DNA, DNA / RNA or RNA / RNA duplexes as determined by Tm's. This high thermal stability might be attributed to the lack of charge repulsion due to the neutral backbone in PNA. The neutral backbone of the PNA also results in the Tm's of PNATDNA(RNA) duplex being practically independent of the salt concentration. Thus, the PNA / DNA(RNA) duplex interaction offers a further advantage over DNA / DNA, DNA / RNA or RNA / RNA duplex interactions which are highly dependent on ionic strength. Homopyrimidine PNAs have been shown to bind complementary DNA or RNA in an anti-parallel orientation forming (PNA)2 / DNA(RNA) triplexes of high thermal stability (see, e.g., Egholm, et al., Science, 1991, 254, 1497; Egholm, et al., J. Am. Chem. Soc., 1992, 114, 1895; Egholm, et al., J. Am. Chem. Soc., 1992, 114, 9677). In addition to increased affinity, PNA has also been shown to bind to DNA or RNA with increased specificity. When a PNA / DNA duplex mismatch is melted relative to the DNA / DNA duplex there is seen an 8 to 20° C. drop in the Tm. This magnitude of a drop in Tm is not seen with the corresponding DNA / DNA duplex with a mismatch present. The binding of a PNA strand to a DNA or RNA strand can occur in one of two orientations. The orientation is said to be anti -parallel when the DNA or RNA strand in a 5' to 3' orientation binds to the complementary PNA strand such that the carboxyl end of the PNA is directed towards the 5' end of the DNA or RNA and amino end of the PNA is directed towards the 3' end of the DNA or RNA. In the parallel orientation the carboxyl end and amino end of the PNA are just the reverse with respect to the 5 '-3' direction of the DNA or RNA. A further advantage of PNA compared to oligonucleotides is that their polyamide backbones (having appropriate nucleobases or other side chain groups attached thereto) is not recognized by either nucleases or proteases and are not cleaved. As a result, PNAs are resistant to degradation by enzymes unlike nucleic acids and peptides. W092 / 20702 describes a peptide nucleic acid (PNA) compounds which bind complementary DNA and RNA more tightly than the corresponding DNA. PNA have shown strong binding affinity and specificity to complementary DNA (Egholm, M., et al., Chem. Soc., Chem. Commun., 1993, 800; Egholm, M., et.al., Nature, 1993, 365, 566; and Nielsen, P., et.al. Nucl. Acids Res., 1993, 21, 197). Furthermore, PNA's show nuclease resistance and stability in cell-extracts (Demidov, V. V., et al., Biochem. Pharmacol., 1994, 48, 1309-1313). Modifications of PNA include extended backbones (Hyrup, B., et.al. Chem. Soc., Chem. Commun., 1993, 518), extended linkers between the backbone and the nucleobase, reversal of the amida bond (Lagriffoul, P. H., et.al., Biomed. Chem. Lett., 1994, 4, 1081), and the use of a chiral backbone based on alanine (Dueholm, K. L, et.al., BioMed. Chem. Lett., 1994, 4, 1077). Peptide Nucleic Acids are described in U.S. Pat. No. 5,539,082 and U.S. Pat. No. 5,539,083. Peptide Nucleic Acids are further described in U.S. patent application No. 08 / 686,113.

[0050] Typically, the oligonucleotides of the present invention are obtained by conventional methods well known to those skilled in the art. For example, the oligonucleotide of the invention can be synthesized de novo using any of a number of procedures well known in the art. For example, the b-cyanoethyl phosphoramidite method (Beaucage et al., 1981); nucleoside H-phosphonate method (Garegg et al., 1986; Froehler et al., 1986, Garegg et al., 1986, Gaffney et al., 1988). These chemistries can be performed by a variety of automated nucleic acid synthesizers available in the market. These nucleic acids may be referred to as synthetic nucleic acids. Alternatively, oligonucleotide can be produced on a large scale in plasmids (see Sambrook, et al., 1989). Oligonucleotide can be prepared from existing nucleic acid sequences using known techniques, such as those employing restriction enzymes, exonucleases or endonucleases. Oligonucleotide prepared in this manner may be referred to as isolated nucleic acids.

[0051] The one skilled in the art can easily provide some approaches and modifications for enhancing the delivery and the efficacy of oligonucleotides such as chemical modification of the oligonucleotides, lipid- and polymer-based nanoparticles or nanocarriers, ligand- oligonucleotide conjugates by linking oligonucleotides to targeting agents such as carbohydrates, peptides, antibodies, aptamers, lipids or small molecules and small molecules that improve oligonucleotide delivery such as described in Juliano RL. The delivery of therapeutic oligonucleotides. Nucleic Acids Res. 2016 Aug 19;44(14):6518-48. Lipophilic conjugates and lipid conjugates include fatty acid-oligonucleotide conjugates; sterololigonucleotide conjugates and vitamin-oligonucleotide conjugates.

[0052] In some embodiments, the oligonucleotide of the present invention is modified by substitution at the 3’ or the 5’ end by a moiety comprising at least three saturated or unsaturated, particularly saturated, linear or branched, particularly linear, hydrocarbon chains comprising from 2 to 30 carbon atoms, particularly from 5 to 20 carbon atoms, more particularly from 10 to 18 carbon atoms as described in WO2014195432.

[0053] In some embodiments, the oligonucleotide of the present invention is modified by substitution at the 3’ or the 5’ end by a moiety comprising at least one ketal functional group, wherein the ketal carbon of said ketal functional group bears two saturated or unsaturated, particularly saturated, linear or branched, particularly linear, hydrocarbon chains comprising from 1 to 22 carbon atoms, particularly from 6 to 20 carbon atoms, in particular 10 to 19 carbon atoms, and even more particularly from 12 to 18 carbon atoms as described in WO2014195430.

[0054] In a particular embodiment, the oligonucleotide of the present invention is conjugated to a second molecule. Typically said second molecule is selected from the group consisting of aptamers, antibodies or polypeptides. For example, the oligonucleotide of the present invention may be conjugated to a cell penetrating peptide. Cell penetrating peptides are well known in the art and include for example the TAT peptide (Bechara C, Sagan S. Cell-penetrating peptides: 20 years later, where do we stand? FEBS Lett. 2013 Jun 19;587(12): 1693-702). In some embodiments, the oligonucleotide of the present invention is associated with a carrier or vehicle, e.g., liposomes or micelles, although other carriers could be used, as would be appreciated by one skilled in the art. Liposomes are vesicles made of a lipid bilayer having a structure similar to biological membranes. Such carriers are used to facilitate the cellular uptake or targeting of the oligonucleotide, or improve the oligonucleotide's pharmacokinetic or therapeutic properties. For example, the oligonucleotide of the present invention may also be administered encapsulated in liposomes, pharmaceutical compositions wherein the active ingredient is contained either dispersed or variously present in corpuscles consisting of aqueous concentric layers adherent to lipidic layers. The oligonucleotide, depending upon solubility, may be present both in the aqueous layer and in the lipidic layer, or in what is generally termed a liposomic suspension. The hydrophobic layer, generally but not exclusively, comprises phospholipids such as lecithin and sphingomyelin, steroids such as cholesterol, more or less ionic surfactants such as diacetylphosphate, stearylamine, or phosphatidic acid, or other materials of a hydrophobic nature. The diameters of the liposomes generally range from about 15 nm to about 5 microns. The use of liposomes as drug delivery vehicles offers several advantages. Liposomes increase intracellular stability, increase uptake efficiency and improve biological activity. Liposomes are hollow spherical vesicles composed of lipids arranged in a similar fashion as those lipids, which make up the cell membrane. They have an internal aqueous space for entrapping water soluble compounds and range in size from 0.05 to several microns in diameter. Several studies have shown that liposomes can deliver nucleic acids to cells and that the nucleic acids remain biologically active. For example, a liposome delivery vehicle originally designed as a research tool, such as Lipofectin, can deliver intact nucleic acid molecules to cells. Specific advantages of using liposomes include the following: they are nontoxic and biodegradable in composition; they display long circulation half-lives; and recognition molecules can be readily attached to their surface for targeting to tissues. Finally, cost-effective manufacture of liposome-based pharmaceuticals, either in a liquid suspension or lyophilized product, has demonstrated the viability of this technology as an acceptable drug delivery system.

[0055] In some embodiments, the oligonucleotide of the present invention is complexed with a complexing agent to increase cellular uptake of oligonucleotides. An example of a complexing agent includes cationic lipids. Cationic lipids can be used to deliver oligonucleotides to cells. The term “cationic lipid” includes lipids and synthetic lipids having both polar and non-polar domains and which are capable of being positively charged at or around physiological pH and which bind to polyanions, such as nucleic acids, and facilitate the delivery of nucleic acids into cells. In general cationic lipids include saturated and unsaturated alkyl and alicyclic ethers and esters of amines, amides, or derivatives thereof. Straight-chain and branched alkyl and alkenyl groups of cationic lipids can contain, e.g., from 1 to about 25 carbon atoms. Particularly, straight chain or branched alkyl or alkene groups have six or more carbon atoms. Alicyclic groups include cholesterol and other steroid groups. Cationic lipids can be prepared with a variety of counterions (anions) including, e.g., C1-, Br-, I-, F-, acetate, trifluoroacetate, sulfate, nitrite, and nitrate. Examples of cationic lipids include: polyethylenimine, polyamidoamine (PAMAM) starburst dendrimers, Lipofectin (a combination of DOTMA and DOPE), Lipofectase, Lipofectamine, DOPE, Cytofectin (Gilead Sciences, Foster City, Calif.), and Eufectins (JBL, San Luis Obispo, Calif.). Cationic liposomes may comprise the following: N-[l-(2,3- dioleoloxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA), N-[l-(2,3-dioleoloxy)- propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP), 3p-[N-(Nz,NZ- dimethylaminoethane)carbamoyl]cholesterol (DC-Chol), 2, 3, -dioleyloxy -N-

[0056] [2(sperminecarboxamido)ethyl]-N,N-dimethyl-l-propanaminium trifluoroacetate (DOSPA), l,2-dimyristyloxypropyl-3-dimethy-l-hydroxyethyl ammonium bromide; and dimethyldioctadecylammonium bromide (DDAB). The cationic lipid N-(l-(2,3- dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), for example, was found to increase 1000-fold the antisense effect of a phosphorothioate oligonucleotide. (Vlassov et al., 1994, Biochimica et Biophysica Acta 1197:95-108). Oligonucleotides can also be complexed with, e.g., poly(L-lysine) or avidin and lipids may, or may not, be included in this mixture (e.g., steryl-poly(L-lysine). Cationic lipids have been used in the art to deliver oligonucleotides to cells (see, e.g., U.S. Pat. Nos. 5,855,910; 5,851,548; 5,830,430; 5,780,053; 5,767,099; Lewis et al. 1996. Proc. Natl. Acad. Sci. USA 93:3176; Hope et al. 1998. Molecular Membrane Biology 15: 1). Other lipid compositions which can be used to facilitate uptake of the instant oligonucleotides can be used in connection with the claimed methods. In addition to those listed supra, other lipid compositions are also known in the art and include, e.g., those taught in U.S. Pat. No. 4,235,871; U.S. Pat. Nos. 4,501,728; 4,837,028; 4,737,323.

[0057] In some embodiments, the eukaryotic cells are genetically engineered so as to correct a particular gene deficit by expression a particular transgene of interest or by repressing the expression of a particular gene.

[0058] In some embodiments, the eukaryotic cells are genetically engineered HSCs. As used herein, the term “genetically engineered HSC” or “genetically modified HSC” refers to a cell or cells that have undergone gene editing so as to alter a target gene in the cell’s genome, or have been altered such that an exogenous gene or exogenous gene sequence is expressed in the cell.

[0059] Typically the eukaryotic cells, such as HSCs, are genetically engineered by any method well known in the art. One common method of genetic engineering is the use of CRISPR-Cas9 technology, which allows for the targeted modification of specific genes within the cell’s genome. This technique employs a guide RNA (gRNA) to direct the Cas9 enzyme to a precise location on the DNA strand, where it creates a double-strand break. The cell's natural repair mechanisms then come into play, either by homologous recombination, where a desired DNA sequence is inserted, or by non-homologous end joining, which can result in gene disruption. In some embodiments, the eukaryotic cells are genetically engineered by using base editing systems, including Cytosine Base Editors (CBEs) and Adenine Base Editors (ABEs). These systems allow for the precise conversion of one DNA base pair into another without causing double-strand breaks. CBEs convert cytosine (C) to thymine (T), while ABEs convert adenine (A) to guanine (G). This refined editing technique minimizes the risks associated with traditional CRISPR-Cas9 methods, such as unintended mutations or genomic instability. Base editing is particularly advantageous for correcting single-nucleotide mutations that cause genetic disorders, offering a more efficient and safer approach to gene therapy. Another approach involves the use of viral vectors, such as lentiviruses or retroviruses, to deliver exogenous genes into the cells. These vectors are engineered to carry the gene of interest and integrate it into the host genome. This method is particularly useful for introducing genes that encode therapeutic proteins or for correcting genetic deficiencies.

[0060] Methods of editing:

[0061] A further object of the present invention relates to a method of editing a population of eukaryotic cells comprising contacting the population of eukaryotic cells with: a gene editing platform that comprises (a) at least one base-editing enzyme, and (b) one or more guide RNA molecule(s) designed for guiding the base-editing enzyme(s) to one or more target sequence(s), and with one or more antisense oligonucleotide(s) of the present invention for transiently inhibiting the expression of CD33 in the population of eukaryotic cells.

[0062] In some embodiments, the eukaryotic cell is selected from the group consisting of hematopoietic progenitor cells, hematopoietic stem cells (HSCs), pluripotent cells (i.e. embryonic stem cells (ES) and induced pluripotent stem cells (iPS)). More preferably the eukaryotic cell is a hematopoietic stem cell. In some embodiments, the hematopoietic progenitor cells or hematopoietic stem cells are isolated form peripheral blood cells. As used herein, the term “peripheral blood cells” refer to the cellular components of blood, including red blood cells, white blood cells, and platelets, which are found within the circulating pool of blood.

[0063] In some embodiments, the eukaryotic cell is a bone marrow derived stem cell. As used herein the term “bone marrow-derived stem cells” refers to stem cells found in the bone marrow. Stem cells may reside in the bone marrow, either as an adherent stromal cell type that possess pluripotent capabilities, or as cells that express CD34 or CD45 cell-surface protein, which identifies hematopoietic stem cells able to differentiate into blood cells.

[0064] Typically, the eukaryotic cell results from a stem cell mobilization.

[0065] As used herein, the term “mobilization” or “stem cell mobilization” refers to a process involving the recruitment of stem cells from their tissue or organ of residence to peripheral blood following treatment with a mobilization agent. This process mimics the enhancement of the physiological release of stem cells from tissues or organs in response to stress signals during injury and inflammation. The mechanism of the mobilization process depends on the type of mobilization agent administered. Some mobilization agents act as agonists or antagonists that prevent the attachment of stem cells to cells or tissues of their microenvironment. Other mobilization agents induce the release of proteases that cleave the adhesion molecules or support structures between stem cells and their sites of attachment. As used herein, the term “mobilization agent” refers to a wide range of molecules that act to enhance the mobilization of stem cells from their tissue or organ of residence, e.g., bone marrow (e.g., CD34+ stem cells) and spleen (e.g., Hoxl l+ stem cells), into peripheral blood. Mobilization agents include chemotherapeutic drugs, e.g., cyclophosphamide and cisplatin; cytokines, and chemokines, e.g., granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colonystimulating factor (GM-CSF), stem cell factor (SCF), Fms-related tyrosine kinase 3 (flt-3) ligand, stromal cell-derived factor 1 (SDF-1); agonists of the chemokine (C — C motif) receptor 1 (CCR1), such as chemokine (C — C motif) ligand 3 (CCL3, also known as macrophage inflammatory protein-la (Mip-la)); agonists of the chemokine (C — X — C motif) receptor 1 (CXCR1) and 2 (CXCR2), such as chemokine (C — X — C motif) ligand 2 (CXCL2) (also known as growth-related oncogene protein-P (Gro-P)), and CXCL8 (also known as interleukin- 8 (IL-8)); agonists of CXCR4, such as CTCE-02142, and Met-SDF-1,; Very Late Antigen (VLA)-4 inhibitors; antagonists of CXCR4, such as TG-0054, plerixafor (also known as AMD3100), and AMD3465, or any combination of the previous agents. A mobilization agent increases the number of stem cells in peripheral blood, thus allowing for a more accessible source of stem cells.

[0066] To perform the knock-down of CD33 using antisense oligonucleotides (ASOs), the population of eukaryotic cells is contacted with the ASOs by employing one of several delivery methods. Electroporation is a common technique used to introduce ASOs into cells. This method involves applying an electrical field to the cells, which temporarily creates pores in the cell membrane, allowing the ASOs to enter. Another effective method for delivering ASOs is the use of lipid nanoparticles (LNPs). LNPs are tiny particles composed of lipids that can encapsulate the ASOs and facilitate their delivery into cells. These nanoparticles merge with the cell membrane, enabling the ASOs to be released into the cellular environment and subsequently taken up by the cells to exert their gene-knockdown effect.

[0067] In some embodiments, the gene editing platform is used to alter a target polynucleotide sequence of interest in the eukaryotic cell for any purpose. In some embodiments, the target polynucleotide sequence of interest in the eukaryotic cell is altered to generate a mutate cell, which results in a genotype that differs from its original genotype. In some embodiments, the target polynucleotide sequence of interest in the eukaryotic cell is altered to correct or repair a genetic mutation (e.g., to restore a normal phenotype to the cell). In some embodiments, the target polynucleotide sequence of interest in the eukaryotic cell is altered to induce a genetic mutation (e.g., to disrupt the function of a gene or genomic element). In some embodiments, the alteration may be a homozygous alteration or a heterozygous alternation. In some embodiments, the alteration may be an insertion, deletion, or the combination thereof. As will be appreciated by those skilled in the art, an insertion / deletion in a coding region of a genomic sequence will result in a frameshift mutation or a premature stop codon. In some embodiments, the alteration may be a point mutation.

[0068] In some embodiments, the gene editing platform of the present invention is used to generate a knock-out of a target polynucleotide sequence. The knocking out of a selected polynucleotide sequence can be useful for many applications, such as knocking out a target polynucleotide sequence of interest in the eukaryotic cell clone in vitro for research purposes; and knocking out a target polynucleotide sequence ex vivo for treating or preventing a disorder associated with increased expression of the target polynucleotide sequence. As used herein, the term "knock out" includes deleting all or a portion of the target polynucleotide sequence in a way that mutes the function of the target polynucleotide sequence.

[0069] In some embodiments, the alternation may result in a change of the target polynucleotide sequence of interest from an undesired sequence to a desired sequence. In some embodiments, the gene editing platform of the present invention is used to correct any type of mutation or error in a target polynucleotide sequence of interest, including but not limited to inserting a nucleotide sequence that is missing from a target polynucleotide sequence due to a deletion, deleting a nucleotide sequence from a target polynucleotide sequence due to an insertion mutation, and replacing an incorrect nucleotide sequence with a correct nucleotide sequence.

[0070] In some embodiments, the alteration results in reduced or increased expression of a target polynucleotide sequence of interest.

[0071] In some embodiments, the gene editing platform is used for increasing the fetal hemoglobin content in the eukaryotic cell.

[0072] Methods of enrichment:

[0073] A further object of the present invention relates to a method of preparing a substantially pure population of edited eukaryotic cells comprising the steps of i) editing a population of eukaryotic cells by the editing method of the present invention and ii) enriching the population of edited eukaryotic cells that is negative for CD33.

[0074] In some embodiment, the present invention relates to a population of edited eukaryotic cells obtainable by the method of the present invention.

[0075] According to the present invention, CD33 is indeed used as a negative marker for enriching the edited eukaryotic cells. Furthermore, CD33 selection of the edited cells leads to higher base-editing efficiencies and eliminates poorly edited cells that could outcompete edited cells in engrafting in the bone marrow.

[0076] Cell enrichment can be accomplished by any means known to one of ordinary skill in the art. In some embodiments, the method of enriching cells comprises flow cytometry, cell sorting, magnetic activated cell sorting (for example as commercially used in Miltenyi Biotec MACS Technology or Dynal magnetic bead selection), antibody panning and red-cell resetting. Other methods for enrichment are also contemplated by the present invention. According to the present invention, the method comprises selecting the cells that have reduced expression, or do not substantially express CD33 as a cell surface marker. For example, flow cytometry may be used to enrich for cells that negative for CD33. Accordingly, FACS can be used with the methods described herein to isolate and detect the population of cells of the present invention. FACS typically involves using a flow cytometer capable of simultaneous excitation and detection of multiple fluorophores, such as a BD Biosciences FACSCanto™ flow cytometer, used substantially according to the manufacturer's instructions. The cytometric systems may include a cytometric sample fluidic subsystem, as described below. In addition, the cytometric systems include a cytometer fluidically coupled to the cytometric sample fluidic subsystem. Systems of the present disclosure may include a number of additional components, such as data output devices, e.g., monitors, printers, and / or speakers, softwares (e.g. (Flowjo, Laluza.... ), data input devices, e.g., interface ports, a mouse, a keyboard, etc., fluid handling components, power sources, etc. Typically, the population of cells is contacted with a panel of antibodies specific for the specific phenotypic markers of interest (CD33). Typically, the antibodies are labelled with a tag to facilitate the isolation and detection of population of cells of the interest. Suitable labels include fluorescent molecules, radioisotopes, nucleotide chromophores, enzymes, substrates, chemiluminescent moieties, magnetic particles, bioluminescent moieties, and the like. As such, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Non-limiting examples of fluorescent labels or tags for labeling the agents such as antibodies for use in the methods of invention include Hydroxycoumarin, Succinimidyl ester, Aminocoumarin, Succinimidyl ester, Methoxycoumarin, Succinimidyl ester, Cascade Blue, Hydrazide, Pacific Blue, Maleimide, Pacific Orange, Lucifer yellow, NBD, NBD-X, R-Phycoerythrin (PE), a PE-Cy5 conjugate (Cychrome, R670, Tri-Color, Quantum Red), a PE-Cy7 conjugate, Red 613, PE-Texas Red, PerCP, PerCPeFluor 710, PE-CF594, Peri dinin chlorphyll protein, TruRed (PerCP-Cy5.5 conjugate), FluorX, Fluoresceinisothyocyanate (FITC), BODIPY-FL, TRITC, X-Rhodamine (XRITC), Lissamine Rhodamine B, Texas Red, Allophycocyanin (APC), an APC-Cy7 conjugate, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, BV 785, BV711, BV421, BV605, BV510 or BV650. The aforementioned assays may involve the binding of the antibodies to a solid support. The solid surface could be a microtitration plate coated with the antibodies. Alternatively, the solid surfaces may be beads, such as activated beads, magnetically responsive beads. Beads may be made of different materials, including but not limited to glass, plastic, polystyrene, and acrylic. In addition, the beads are preferably fluorescently labelled. In some embodiments, fluorescent beads are those contained in TruCount(TM) tubes, available from Becton Dickinson Biosciences, (San Jose, California).

[0077] In some embodiments, cell enrichment is performed with an agent that is capable of depleting CD33 positive cells. In some embodiments, the agent is an antibody that binds to CD33 and depletes CD33+ cells (i.e. a “depleting antibody”).

[0078] As used herein, the term “depletion” with respect to CD33+ cells, refers to a measurable decrease in the number of CD33+ cells in the subject. The reduction can be at least about 10%, e.g., at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more. In some embodiments, the depleting antibody binds to CD33.

[0079] In some embodiments, the depleting antibody mediates antibody-dependent cell- mediated cytotoxicity. As used herein the term “antibody-dependent cell-mediated cytotoxicity” or ‘ ADCC” refer to a cell-mediated reaction in which non-specific cytotoxic cells (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cell. In some embodiments, the depleting antibody is an IgGl antibody. In some embodiments, the depleting antibody is an IgG3 antibody.

[0080] In some embodiments, the antibody suitable for depletion of CD33+ cells is conjugated to a therapeutic moiety, i.e. a drug.

[0081] In some embodiments, the therapeutic moiety can be, e.g., a cytotoxin, a chemotherapeutic agent, a cytokine, an immunosuppressant, an immune stimulator, a lytic peptide, or a radioisotope. Such conjugates are referred to herein as an "antibody-drug conjugates" or "ADCs".

[0082] In some embodiments, the antibody suitable for depletion of CD33+ cells is conjugated to a cytotoxic moiety. The cytotoxic moiety may, for example, be selected from the group consisting of taxol; cytochalasin B; gramicidin D; ethidium bromide; emetine; mitomycin; etoposide; tenoposide; vincristine; vinblastine; colchicin; doxorubicin; daunorubicin; dihydroxy anthracin dione; a tubulin- inhibitor such as maytansine or an analog or derivative thereof; an antimitotic agent such as monomethyl auristatin E or F or an analog or derivative thereof; dolastatin 10 or 15 or an analogue thereof; irinotecan or an analogue thereof; mitoxantrone; mithramycin; actinomycin D; 1 -dehydrotestosterone; a glucocorticoid; procaine; tetracaine; lidocaine; propranolol; puromycin; calicheamicin or an analog or derivative thereof; an antimetabolite such as methotrexate, 6 mercaptopurine, 6 thioguanine, cytarabine, fludarabin, 5 fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine, or cladribine; an alkylating agent such as mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C; a platinum derivative such as cisplatin or carboplatin; duocarmycin A, duocarmycin SA, rachelmycin (CC-1065), or an analog or derivative thereof; an antibiotic such as dactinomycin, bleomycin, daunorubicin, doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC)); pyrrolo[2,l-c][l,4]-benzodiazepines (PDB); diphtheria toxin and related molecules such as diphtheria A chain and active fragments thereof and hybrid molecules, ricin toxin such as ricin A or a deglycosylated ricin A chain toxin, cholera toxin, a Shiga-like toxin such as SLT I, SLT II, SLT IIV, LT toxin, C3 toxin, Shiga toxin, pertussis toxin, tetanus toxin, soybean Bowman- Birk protease inhibitor, Pseudomonas exotoxin, alorin, saporin, modeccin, gelanin, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacca americana proteins such as PAPI, PAPII, and PAP-S, momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, and enomycin toxins; ribonuclease (RNase); DNase I, Staphylococcal enterotoxin A; pokeweed antiviral protein; diphtherin toxin; and Pseudomonas endotoxin.

[0083] In some embodiments, the antibody suitable for depletion of CD33+ cells is conjugated to an auristatin or a peptide analog, derivative or prodrug thereof. Auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis and nuclear and cellular division (Woyke et al (2001) Antimicrob. Agents and Chemother. 45(12): 3580-3584) and have anticancer (US5663149) and antifungal activity (Pettit et al., (1998) Antimicrob. Agents and Chemother. 42: 2961-2965. For example, auristatin E can be reacted with para-acetyl benzoic acid or benzoyl valeric acid to produce AEB and AEVB, respectively. Other typical auristatin derivatives include AFP, MMAF (monomethyl auristatin F), and MMAE (monomethyl auristatin E). Suitable auristatins and auristatin analogs, derivatives and prodrugs, as well as suitable linkers for conjugation of auristatins to Abs, are described in, e.g., U.S. Patent Nos. 5,635,483, 5,780,588 and 6,214,345 and in International patent application publications W002088172, W02004010957, W02005081711, W02005084390, W02006132670, WO03026577, W0200700860, W0207011968 and W0205082023.

[0084] In some embodiments, the antibody suitable for depletion of CD33+ cells is Gemtuzumab ozogamicin that is a recombinant humanized IgG4 kappa antibody, which is conjugated with calicheamicin derivative, a cytotoxic antitumor antibiotic.

[0085] Methods of therapy:

[0086] A further object of the present invention relates to a method of therapy in a subject in need thereof, the method comprising transplanting a therapeutically effective amount of a population of edited eukaryotic cells (enriched or not for CD33- cells) obtained by the methods of the present invention. In particular, the subject of the present invention suffers from a P-hemoglobinopathy. In some embodiments, the P-hemoglobinopathy is a sickle cell disease. In some embodiments, the P-hemoglobinopathy is a P-thalassemia.

[0087] In some embodiments, the population of cell is autologous to the subject, meaning the population of cells is derived from the same subject.

[0088] Kits:

[0089] In a further embodiment, the present invention relates to a kit comprises (a) at least one base-editing enzyme, and (b) one or more RNA molecule(s) designed for guiding the baseediting enzyme(s) to one or more target sequence(s), (c) one or more antisense oligonucleotide(s) according to the present invention and optionally (d) an agent that is capable of depleting CD33 positive cells.

[0090] This invention further provides kits containing reagents for performing the abovedescribed methods. In some embodiments, the kit can include one or more other reaction components. In some embodiments, an appropriate amount of one or more reaction components is provided in one or more containers or held on a substrate. Examples of additional components of the kits include, but are not limited to, one or more host cells, one or more reagents for introducing foreign nucleotide sequences into host cells, one or more reagents (e.g., probes or PCR primers) for detecting expression of the guide RNA or editing enzymes or verifying the target nucleic acid's status, and buffers or culture media for the reactions. The kit may also include one or more of the following components: supports, terminating, modifying or digestion reagents, osmolytes, and an apparatus for detection. The components used can be provided in a variety of forms. For example, the components (e.g., enzymes, RNAs, probes and / or primers) can be suspended in an aqueous solution or as a freeze-dried or lyophilized powder, pellet, or bead. In the latter case, the components, when reconstituted, form a complete mixture of components for use in an assay. The kits of the invention can be provided at any suitable temperature. For example, for storage of kits containing protein components or complexes thereof in a liquid, it is preferred that they are provided and maintained below 0° C., preferably at or below -20° C., or otherwise in a frozen state. The kits can also include packaging materials for holding the container or combination of containers. Typical packaging materials for such kits and systems include solid matrices (e.g., glass, plastic, paper, foil, micro-particles and the like) that hold the reaction components or detection probes in any of a variety of configurations (e.g., in a vial, microtiter plate well, microarray, and the like). The kits may further include instructions recorded in a tangible form for use of the components.

[0091] The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

[0092] FIGURES:

[0093] Figure 1. Downregulation of CD33 in the HEL cell line using ASO. (A) Viability of HEL cells based on side scatter (SSC) and forward scatter (FSC) measurement assessed by flow cytometry 1 day, 2 days, or 6 days after transfection. WT, wild-type untreated cells. TE, mock samples transfected with TE buffer. Cells were electroporated with ABE alone (ABE only), ABE and gRNA3 (ABE g3) or 5 different ASOs (ASO1, ASO2, ASO3, ASO4, ASO5; 220 pmol each). Data are expressed as mean ± SD of n=3 biologically independent experiments. (B) Frequency of CD33+HEL cells assessed by flow cytometry 1, 2, 6 days after electroporation. (C) CD33 mean fluorescence intensity (MFI) in HEL cells assessed by flow cytometry 1, 2, 6 days after electroporation. All data are expressed as mean ± SD n=3 biologically independent experiments. (D) Representative flow cytometry plots of CD33 MFI in HEL cells assessed by flow cytometry 2 days after electroporation. (E) Fold-change of CD33 expression (relative to GAPDH measured by RT-qPCR in HEL cells 2 days after transfection. Expression was normalized to the TE control. Data are expressed as mean ± SD of n=2 biologically independent experiments.

[0094] Figure 2. Downregulation of CD33 in HSPCs. (A) Viability of HSPCs based on SSC and FSC) measurement assessed by flow cytometry 1 day, 2 days or 6 days after transfection. TE, mock samples transfected with TE buffer. ASO CTR, cells transfected with a scramble non-targeting ASO. Cells were transfected with ABE and gRNA3 (ABE g3) or ASO 5 (220 or 330 pmol). (B) Frequency of CD33+HSPCs assessed by flow cytometry 2 days after electroporation. (C) Representative flow cytometry plots of CD33 expression 2 days after electroporation. All data are expressed as mean ± SD of n=3 biologically independent experiments.

[0095] Figure 3. Gemtuzumab ozogamicin efficiently targets CD33+HSPCs. (A) Frequency of CD33+HSPCs treated with several concentrations of gemtuzumab ozogamicin (GO; 5, 10 or 20 ng / ml). MFI was evaluated by flow cytometry 24 h after treatment. (B-C) CD33 MFI of HSPCs treated with several concentrations of GO (5, 10 or 20 ng / ml). MFI was evaluated by flow cytometry 24 h after treatment. Material & Methods

[0096] HEL cell line culture

[0097] Human acute erythroid leukemia HEL cells were maintained in RPMI 1640 (Lonza) containing glutamine and supplemented with 10% fetal bovine serum (Lonza), 2 mM Hepes (Life Technologies), 100 nM sodium pyruvate (Life Technologies), and penicillin and streptomycin (Life Technologies).

[0098] HSPC purification and culture

[0099] We obtained human non-mobilized peripheral blood CD34+ HSPCs from SCD patients. SCD samples eligible for research purposes were obtained from the “Hopital Necker-Enfants malades” Hospital (Paris, France). Written informed consent was obtained from all adult subjects. All experiments were performed in accordance with the Declaration of Helsinki. The study was approved by the regional investigational review board (reference: DC 2022-5364, CPP Ile-de-France II “Hopital Necker-Enfants malades”). HSPCs were purified by immunomagnetic selection with MACS columns (Miltenyi Biotec) after immunostaining with the CD34 MicroBead Kit (Miltenyi Biotec). Twenty-four hours before transfection, CD34+cells were thawed and cultured at a concentration of 5xl05cells / ml in the “HSPC medium” containing StemSpan (STEMCELL Technologies) supplemented with penicillin / streptomycin (Gibco), 250 nM StemRegeninl (STEMCELL Technologies), and the following recombinant human cytokines (PeproTech): human stem cell factor (SCF) (300 ng / ml), Flt3-L (300 ng / ml), thrombopoietin (TPO) (100 ng / ml), and interleukin-3 (IL-3) (20 ng / ml). sgRNA design

[0100] We manually designed the sgRNA targeting the CD33 gene (Table 1). For RNA- mediated base editing we used chemically modified synthetic sgRNAs harboring 2'-O-methyl analogs and 3'-phosphorothioate nonhydrolyzable linkages at the first three 5' and 3' nucleotides (Synthego).

[0101] Table 1. sgRNA target sequences. Antisense oligonucleotides (ASOs)

[0102] To enhance skipping of exon 2 in the CD 33 transcripts, we first investigated the presence of a putative Splicing Regulatory Element (SRE) into the exon sequence. To accomplish this, we used RBPmap and SpliceAid to analyze CD33 exon 2, predicting the binding affinities of various RNA-binding proteins along the exon sequence. Subsequently, we employed the eSkip-Finder resources to assess the potential exon skipping efficiency of ASOs targeting CD33 exon 2. We applied the CD33 exon 2 sequence, along with 200 base pairs of flanking sequences, and configured it to account for 2'-O-methoxyethyl (2’MOE) modifications. Based on information about RNA-binding protein (RBP) binding sites and ASO efficiency, we retained five ASOs (Table 2). These ASOs were forecasted to exhibit efficiencies ranging from 79.7% to 48.5%. ASOs were produced by IDT with the following modifications: (i) 2’MOE and (ii) phosphorothioate to enhance their stability37.

[0103] Table 2. Antisense oligonucleotides (ASOs) sequences

[0104] RNA transfection

[0105] 2xl05to 7xl05HEL or CD34+HSPCs per condition were transfected with ASOs or 3.0 pg of the enzyme encoding mRNA, and 3.2 pg of the synthetic sgRNA. We used the P3 Primary Cell 4D-Nucleofector X Kit S (Lonza) and the CA137 program (Nucleofector 4D). Cells were incubated at 37°C for 10 minutes before being transferred from the nucleocuvette strip well to a 96-well plate after the addition of pre-warmed “HSPC medium” per well. Cells were incubated at 37°C post-transfection. Cells transfected with TE buffer or WT cells served as negative controls.

[0106] Flow cytometry analysis

[0107] Flow cytometry analysis of CD33 surface marker for HEL cells and CD34+HSPCs were performed using the APC-conjugated anti-CD33 antibody (551378, BD). Flow cytometry analyses were performed using the Novocyte (Agilent Technologies) flow cytometer. Data were analyzed using Flow Jo (BD Biosciences) software.

[0108] RT-qPCR

[0109] 2xl05cells were harvested for total RNA isolation at day 2 post-transfection of HEL cells. RNA was isolated using the RNeasy Micro Kit (Qiagen). Mature transcripts were reverse transcribed using SuperScript III First-Strand Kit (Invitrogen) with oligodT following manufacturer’s instructions. RT-qPCR was performed using the iTaq universal SYBR Green master mix (BioRad) and the BioRad CFX384 Real-Time System (BioRad). Primers are listed in Table 3.

[0110] Table 3. Primers used to detect CD33 transcripts by RT-qPCR.

[0111] Targeting of CD33+ HSPCs with Gemtuzumah ozogamicin (GO)

[0112] HSPCs were cultured during 48h in the HSPC medium and then treated with Gemtuzumah ozogamicin (GO), an antibody-drug conjugate (MedChemExpress) for 24 h. This antibody is directed against CD33 and conjugated to the cytotoxic drug calicheamicin.

[0113] Results

[0114] ASO-mediated CD33 downregulation in HEL cells

[0115] To transiently downregulate CD33, 5 ASOs were designed to target CD33 (ASO1, ASO2, ASO3, ASO4 and ASO5). ASOs were transfected in the HEL cell line using a dose of 220 pmol. The effects of the ASOs were compared with that of a base editing approach inactivating CD33 using adenine base editor (ABE) and a gRNA targeting CD33 (g3). We measured cell viability (based on the morphology: SSC / FSC) and CD33 expression by flow cytometry at day 1 (DI), day 2 (D2) and day 6 (D6) after transfection (Figure 1). At DI, we observed a modest decrease in cell viability in samples treated with ASO2, ASO4 and ASO5 in comparison with control cells transfected only with the TE buffer (Figure 1A). However, at D2 and D6, the percentage of live cells in all samples treated with ASOs was similar to that measured in the control cells (Figure 1A). CD33 expression was decreased 2 days after treatment with ASO2, 4, and 6 as evaluated by flow cytometry (Figures 1B-1D) and by RT- qPCR (Figure IE). At D6, CD33 expression returned to normal levels in ASO-treated samples (Figure IB). On the contrary, in base edited cells, CD33 down-regulation persisted over time. These results show a transient downregulation of CD33 using ASOs compared to base editing. For CD 33 targeting in HSPC, we selected ASO5 as it showed the highest CD33 downregulation efficiency at day 2 in HEL cells (Figures 1B-1E).

[0116] ASO5 mediated CD33 downregulation in SCD HSPCs

[0117] Two different doses of ASO5 were tested in SCD HSPCs (220 and 330 pmol). No toxicity was observed over time on HSPCs treated with ABE or ASOs even using the higher dose (Figure 2A). The frequency of CD33+HSPCs decreases in the presence of ASO5 at a dose of 220 pmol (Figure 2B-2C). At day 2, we observed 50% of CD33+ HSPCs treated ASO5 at a dose of 220 pmol in comparison with 80% in the control conditions (Figure 2B). An even higher downregulation was observed by using 330 pmol reaching 30% of CD33+ HSPCs (Figure 2A-2C). To conclude, the CD33 downregulation at D2 is the highest when using ASO5 at a dose of 330 pmol with no effect on cell viability.

[0118] Selection of CD 33' SCD HSPCs

[0119] In order to select only the edited cells based on the low / absent CD33 expression on HSPCs, we will use gemtuzumab ozogamicin (GO) a drug that allows to kill of the remaining unedited CD33+positive cells. To this aim, we tested several doses of GO on HSPCs (5, 10 and 20 ng / ml) to determine the best-performing concentration to use to kill the non-edited HSPCs (i.e., CD33 positive HSPCs). After 48 h of culture in a medium supplemented with HSPC- supporting cytokines, 75% of HSPCs express the CD33 (Figure 3A). The addition of GO led to a progressive decrease of CD33 expression, which was positively correlated with the dose (Figures 3A-3C). In particular, a dose at 20 ng / ml was the most efficient, leading to only 22% of HSPCs expressing the CD33 after treatment (Figures 3A-3C). In conclusion, GO could be used in combination with an ASO-base editing dual strategy in HSPCs to select only the edited HSPCs in vitro before reinfusion or in vivo directly in the patient. Hence, this strategy will improve the outcome of HSC-based gene therapy. REFERENCES:

[0120] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

[0121] 1. Kato, G. J. et al. Sickle cell disease. Nat Rev Dis Primers 4, (2018).

[0122] 2. Piel, F. B., Steinberg, M. H. & Rees, D. C. Sickle Cell Disease. N Engl JMed 376, 1561— 1573 (2017).

[0123] 3. Weatherall, D. J. Phenotype-genotype relationships in monogenic disease: lessons from the thalassaemias. Nat Rev Genet 2, 245-255 (2001).

[0124] 4. Cappellini, M. D., Porter, J. B., Viprakasit, V. & Taher, A. T. A paradigm shift on beta- thalassaemia treatment: How will we manage this old disease with new therapies? Blood Rev 32, 300-311 (2018).

[0125] 5. Taher, A. T., Weatherall, D. J. & Cappellini, M. D. Thalassaemia. Lancet 391, 155-167 (2018).

[0126] 6. Cavazzana, M., Antoniani, C. & Miccio, A. Gene Therapy for P-Hemoglobinopathies. Mol Ther 25, 1142-1154 (2017).

[0127] 7. Forget, B. G. Molecular basis of hereditary persistence of fetal hemoglobin. Ann N Y Acad Sci 850, 38-44 (1998).

[0128] 8. Wienert, B. et al Editing the genome to introduce a beneficial naturally occurring mutation associated with increased fetal globin. Nat Commun 6, (2015).

[0129] 9. Wienert, B. et al. KLF1 drives the expression of fetal hemoglobin in British HPFH. toot / 130, 803-807 (2017).

[0130] 10. Martyn, G. E. et al A natural regulatory mutation in the proximal promoter elevates fetal globin expression by creating a de novo GATA1 site. Blood 133, 852-856 (2019).

[0131] 11. Martyn, G. E., Quinlan, K. G. R. & Crossley, M. The regulation of human globin promoters by CCAAT box elements and the recruitment of NF-Y. Biochim Biophys Acta Gene RegulMech 1860, 525-536 (2017).

[0132] 12. Weber, L. et al Editing a y-globin repressor binding site restores fetal hemoglobin synthesis and corrects the sickle cell disease phenotype. Sci Adv 6, (2020).

[0133] 13. Wang, L. et al. Reactivation of y-globin expression through Cas9 or base editor to treat P-hemoglobinopathies. Cell Res 30, 276-278 (2020).

[0134] 14. Li, C. et al. In vivo HSPC gene therapy with base editors allows for efficient reactivation of fetal y-globin in P-YAC mice. Blood Adv 5, 1122-1135 (2021). Mayuranathan, T. et al. Adenosine Base Editing of y-Globin Promoters Induces Fetal Hemoglobin and Inhibit Erythroid Sickling. Blood 136, 21-22 (2020). Milyavsky, M. et al. A distinctive DNA damage response in human hematopoietic stem cells reveals an apoptosis-independent role for p53 in self-renewal. Cell Stem Cell 7, 186-197 (2010). Cromer, M. K. et al. Global Transcriptional Response to CRISPR / Cas9-AAV6-Based Genome Editing in CD34+ Hematopoietic Stem and Progenitor Cells. Mol Ther 26, 2431-2442 (2018). Schiroli, G. et al. Precise Gene Editing Preserves Hematopoietic Stem Cell Function following Transient p53-Mediated DNA Damage Response. Cell Stem Cell 24, 551- 565. e8 (2019). Haapaniemi, E., Botla, S., Persson, J., Schmierer, B. & Taipale, J. CRISPR-Cas9 genome editing induces a p53-mediated DNA damage response. Nat Med 24, 927-930 (2018). Kosicki, M., Tomberg, K. & Bradley, A. Repair of double-strand breaks induced by CRISPR-Cas9 leads to large deletions and complex rearrangements. Nat Biotechnol 36, 765-771 (2018). Boutin, J. et al. CRISPR-Cas9 globin editing can induce megabase-scale copy -neutral losses of heterozygosity in hematopoietic cells. Nat Commun 12, (2021). Leibowitz, M. L. et al. Chromothripsis as an on-target consequence of CRISPR-Cas9 genome editing. Nat Genet 53, 895-905 (2021). Turchiano, G. et al. Quantitative evaluation of chromosomal rearrangements in gene- edited human stem cells by CAST-Seq. Cell Stem Cell 28, 1136-1147. e5 (2021). Koblan, L. W. et al. Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction. Nat Biotechnol 36, 843-848 (2018). Antoniou, P. et al. Base-editing-mediated dissection of a y-globin cis-regulatory element for the therapeutic reactivation of fetal hemoglobin expression. Nat Commun 13, (2022). Marktel, S. et al. Intrabone hematopoietic stem cell gene therapy for adult and pediatric patients affected by transfusion-dependent B-thalassemia. Nat Med 25, 234-241 (2019). Magrin, E. et al. Long-term outcomes of lentiviral gene therapy for the P- hemoglobinopathies: the HGB-205 trial. Nat Med 28, 81-88 (2022). Laszlo, G. S., Estey, E. H. & Walter, R. B. The past and future of CD33 as therapeutic target in acute myeloid leukemia. Blood Rev 28, 143-153 (2014). Laszlo, G. S. et al. Expression and functional characterization of CD33 transcript variants in human acute myeloid leukemia. Oncotarget 7, 43281-43294 (2016). Kim, M. Y. et al. Genetic Inactivation of CD33 in Hematopoietic Stem Cells to Enable CAR T Cell Immunotherapy for Acute Myeloid Leukemia. Cell 173, 1439-1453. el9 (2018). Knapp, D. J. H. F. et al Single-cell analysis identifies a CD33 + subset of human cord blood cells with high regenerative potential. Nat Cell Biol 20, 710-720 (2018). Sailman, D. A. et al. Phase 1 / lb Safety Study of Prgn-3006 Ultracar-T in Patients with Relapsed or Refractory CD33-Positive Acute Myeloid Leukemia and Higher Risk Myelodysplastic Syndromes. Blood 140, 10313-10315 (2022). Awada, H. et al. Gemtuzumab ozogamicin plus standard induction hemotherapy improves outcomes of newly diagnosed intermediate cytogenetic risk acute myeloid leukemia. Blood Cancer J 13, (2023). Qin, H. et al. Systematic preclinical evaluation of CD33-directed chimeric antigen receptor T cell immunotherapy for acute myeloid leukemia defines optimized construct design. J Immunother Cancer 9, (2021). Appelbaum, F. R. & Bernstein, I. D. Gemtuzumab ozogamicin for acute myeloid leukemia. Blood 130, 2373-2376 (2017). Baron, J. & Wang, E. S. Gemtuzumab ozogamicin for the treatment of acute myeloid leukemia. Expert Rev Clin Pharmacol 11, 549-559 (2018). Sheng, L., Rigo, F., Frank Bennett, C., Krainer, A. R. & Hua, Y. Comparison of the efficacy of MOE and PMO modifications of systemic antisense oligonucleotides in a severe SMA mouse model. Nucleic Acids Res 48, 2853-2865 (2020).

Claims

34 -CLAIMS:

1. An antisense oligonucleotide suitable for knock-downing the expression of CD33 in an eukaryotic cell that comprises or consists of the sequence as set forth in SEQ ID NO:7, SEQ ID NO:4, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:6.

2. The antisense oligonucleotide according to claim 1 that comprises 2’ -MOE and / or phosphorothioate modifications.

3. A method of editing a population of eukaryotic cells comprising contacting the population of eukaryotic cells with: a gene editing platform that comprises (a) at least one base-editing enzyme, and (b) one or more guide RNA molecule(s) designed for guiding the base-editing enzyme(s) to one or more target sequence(s), and with one or more antisense oligonucleotide(s) according to claim 1 or 2 for transiently inhibiting the expression of CD33 in the population of eukaryotic cells.

4. The method according to claim 3 wherein the eukaryotic cell is selected from the group consisting of hematopoietic progenitor cells, hematopoietic stem cells (HSCs), pluripotent cells (i.e. embryonic stem cells (ES) and induced pluripotent stem cells (iPS)).

5. The method according to claim 3 or 4 that further comprises the step that consists of enriching the population of edited eukaryotic cells that is negative for CD33.

6. The method of claim 5 wherein cell enrichment is performed with an agent that is capable of depleting CD33 positive cells, such as an antibody that binds to CD33 and depletes CD33+ cells.

7. A population of edited eukaryotic cells obtainable by the method according to any one of claims 1 to 6.

8. A method of therapy in a subject in need thereof, the method comprising transplanting a therapeutically effective amount of the population of edited eukaryotic cells of claim 7.35 -9. The method according to claim 8 wherein the subject suffers from a P- hemoglobinopathy.

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