Enrichment of genetically modified hematopoietic stem cells through epigenome editing

Abstract

Here, we developed an epigenome editing strategy to enrich base edited HSPCs by transiently downregulating CD33 expression. CD33 is a surface marker of the myeloid lineage, which is expressed on human HSCs with a high regenerative potential. With this approach, the transient repression will ensure CD33 downregulation only in the timeframe necessary to select and enrich base edited HSPCs. In particular, the present invention relates to a method for editing a population of eukaryotic cells comprising contacting the population of eukaryotic cells with an epigenome editing platform that comprises (a) one or more epigenome-editing enzyme(s), and (b) one or more guide RNA molecule(s) designed for guiding the epigenome-editing enzyme to a target sequence in the gene encoding for the CD33 so as to knock-down the expression of CD33.

Description

[0001] ENRICHMENT OF GENETICALLY MODIFIED HEMATOPOIETIC STEM CELLS

[0002] THROUGH EPIGENOME EDITING

[0003] FIELD OF THE INVENTION:

[0004] The present invention is in the field of medicine, in particular epigenome editing and haematology.

[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 / del étions (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 pathway19. 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 2. 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 BE 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 to. 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 regarding 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.

[0012] CD33 is a surface marker of the myeloid lineage, which is also highly expressed in malignant blasts of acute myeloid leukemia (AML) patients28 30. 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, in clinical trials (NCT03971799, NCT03927261), and experimental models of chimeric antigen receptor (CAR) T-cell immunotherapies30,32. 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)32. 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, to reduce the toxic side effect of the antibody.

[0013] SUMMARY OF THE INVENTION:

[0014] The present invention is defined by the claims. In particular, the present invention relates to a method of inhibiting the expression of CD33 in a population of eukaryotic cells through epigenome editing.

[0015] DETAILED DESCRIPTION OF THE INVENTION:

[0016] Main definitions:

[0017] As used herein, the terms “polypeptide”, “peptide” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by nonamino acids. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, pegylation, or any other manipulation, such as conjugation with a labelling component. As used herein the term “amino acid” includes natural and / or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics.

[0018] As used herein, the term “nucleic acid molecule” or “polynucleotide” refers to a DNA molecule (for example, but not limited to, a cDNA or genomic DNA). The nucleic acid molecule can be single-stranded or double-stranded. As used herein, the term "gene" refers to a fundamental unit of heredity that encompasses a sequence of nucleotides encoding a specific polypeptide or functional RNA molecule. A gene comprises not only the coding sequence responsible for producing the gene product but also the non-coding sequences that may play roles in regulating gene expression, such as introns and untranslated regions (UTRs). Additionally, a gene includes the promoter region, which is essential for initiating the transcription of the coding sequence.

[0019] As used herein, the term "encode", or "encoding" or "encoded" refers to a nucleic acid sequence that codes for a polypeptide sequence.

[0020] 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.

[0021] 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 reduction in the expression of a gene or its gene product(s).

[0022] 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.

[0023] 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.

[0024] As used herein, the term “stringent conditions” for hybridization refer to conditions under which a nucleic acid having complementarity to a target sequence predominantly hybridizes with the target sequence, and substantially does not hybridize to non-target sequences. Stringent conditions are generally sequence-dependent, and vary depending on a number of factors. In general, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes to its target sequence. Non-limiting examples of stringent conditions are described in detail in Tijssen (1993), Laboratory Techniques In Biochemistry And Molecular Biology-Hybridization With Nucleic Acid Probes Part I, Second Chapter “Overview of principles of hybridization and the strategy of nucleic acid probe assay”, Elsevier, N.Y.

[0025] As used herein, the term “hybridization” or “hybridizing” refers to a process where completely or partially complementary nucleic acid strands come together under specified hybridization conditions to form a double-stranded structure or region in which the two constituent strands are joined by hydrogen bonds. Although hydrogen bonds typically form between adenine and thymine or uracil (A and T or U) or cytosine and guanine (C and G), other base pairs may form (e.g., Adams et al., The Biochemistry of the Nucleic Acids, 11th ed., 1992).

[0026] As used herein, the term “fusion polypeptide” or “fusion protein” means a protein created by joining two or more polypeptide sequences together. The fusion polypeptides encompassed in this invention include translation products of a chimeric gene construct that joins the nucleic acid sequences encoding a first polypeptide, e.g., an RNA-binding domain, with the nucleic acid sequence encoding a second polypeptide, e.g., an effector domain, to form a single open-reading frame. In other words, a “fusion polypeptide” or “fusion protein” is a recombinant protein of two or more proteins which are joined by a peptide bond or via several peptides. The fusion protein may also comprise a peptide linker between the two domains. As used herein the term “wild type” is a term of the art understood by skilled persons and means the typical form of an organism, strain, gene or characteristic as it occurs in nature as distinguished from mutant or variant forms.

[0027] As used herein, the term “derived from” refers to a process whereby a first component (e.g., a first molecule), or information from that first component, is used to isolate, derive or make a different second component (e.g., a second molecule that is different from the first).

[0028] As used herein, the “percent identity” between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described below. The percent identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (Needleman, Saul B. & Wunsch, Christian D. (1970). "A general method applicable to the search for similarities in the amino acid sequence of two proteins". Journal of Molecular Biology. 48 (3): 443-53.). The percent identity between two nucleotide or amino acid sequences may also be determined using for example algorithms such as EMBOSS Needle (pair wise alignment; available at www.ebi.ac.uk). For example, EMBOSS Needle may be used with a BLOSUM62 matrix, a “gap open penalty” of 10, a “gap extend penalty” of 0.5, a false “end gap penalty”, an “end gap open penalty” of 10 and an “end gap extend penalty” of 0.5. In general, the “percent identity” is a function of the number of matching positions divided by the number of positions compared and multiplied by 100. For instance, if 6 out of 10 sequence positions are identical between the two compared sequences after alignment, then the identity is 60%. The % identity is typically determined over the whole length of the query sequence on which the analysis is performed. Two molecules having the same primary amino acid sequence or nucleic acid sequence are identical irrespective of any chemical and / or biological modification. According to the invention a first amino acid sequence having at least 90% of identity with a second amino acid sequence means that the first sequence has 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% of identity with the second amino acid sequence.

[0029] As used herein, the term “linker” refers to any means, entity or moiety used to join two or more entities. A linker can be a covalent linker or a non-covalent linker. Examples of covalent linkers include covalent bonds or a linker moiety covalently attached to one or more of the proteins or domains to be linked. The linker can also be a non-covalent bond, e.g., an organometallic bond through a metal center such as platinum atom. For covalent linkages, various functionalities can be used, such as amide groups, including carbonic acid derivatives, ethers, esters, including organic and inorganic esters, amino, urethane, urea and the like. To provide for linking, the domains can be modified by oxidation, hydroxylation, substitution, reduction etc. to provide a site for coupling. Methods for conjugation are well known by persons skilled in the art and are encompassed for use in the present invention. Linker moieties include, but are not limited to, chemical linker moieties, or for example a peptide linker moiety (a linker sequence). It will be appreciated that modification which do not significantly decrease the function of the RNA-binding domain and effector domain are preferred.

[0030] As used herein, the “linked” as used herein refers to the attachment of two or more entities to form one entity. A conjugate encompasses both peptide-small molecule conjugates as well as peptide-protein / peptide conjugates.

[0031] As used herein, the term "editing", "edit", "edition", or "edited" refers to a method of altering a nucleic acid sequence of a polynucleotide (e.g., a naturally-occurring wild type nucleic acid sequence or a naturally-occurring mutated nucleic acid sequence by introducing a change to a specific genomic target; the genomic target may include a chromosomal region, a coding polynucleotide (e.g., a gene), a promotor, a non-coding polynucleotide, or any nucleic acid sequence. The changes to a nucleic acid may include deletion, addition and other changes to the nucleic acid sequence in the genome.

[0032] As used herein, the terms “epigenome editing” or “epigenome engineering” refer to a type of genetic engineering in which the epigenome is modified at specific sites using engineered molecules targeted to those sites (as opposed to whole-genome modifications). Whereas gene editing involves changing the actual DNA sequence itself, epigenetic editing involves modifying and presenting DNA sequences to proteins and other DNA binding factors that influence DNA function. By "editing” epigenomic features in this manner, researchers can determine the exact biological role of an epigenetic modification at the site in question. The engineered proteins used for epigenome editing are composed of a DNA binding domain that target specific sequences and an effector domain that modifies epigenomic features. Currently, three major groups of DNA binding proteins have been predominantly used for epigenome editing: Zinc finger proteins, Transcription Activator-Like Effectors (TALEs) and nuclease deficient Cas9 fusions (CRISPR).

[0033] As used herein, the term “epigenomic-modifying enzyme” or “epigenome-editing enzyme” refers to any engineered molecule designed to alter the epigenomic features at specific genomic sites. These modifications do not change the DNA sequence but influence how genes are expressed by modifying the accessibility of DNA to transcription factors and other DNA- binding proteins. Such enzymes typically consist of a DNA binding domain that targets specific DNA sequences and an effector domain that induces epigenetic changes. Typically, the epigenome-editing enzyme of the present invention comprises a transcriptional repressor domain.

[0034] As used herein, the term “transcriptional repressor domain” refers to a specific region in a protein that can bind to DNA and inhibit the transcription of one or more genes. This domain functions by recruiting other proteins that modify chromatin structure, leading to a repressive chromatin state and thus, silencing gene expression.

[0035] As used herein, the term “Kriippel-associated box” (KRAB) refers to category of transcriptional repression domains present in approximately 400 human zinc finger proteinbased transcription factors (KRAB zinc finger proteins). The KRAB domain typically consists of about 75 amino acid residues, while the minimal repression module is approximately 45 amino acid residues. Over 10 independently encoded KRAB domains have been shown to be effective repressors of transcription, suggesting this activity to be a common property of the domain. KRAB domains can be fused with dCas9 CRISPR tools to form even stronger repressors. The dCas9KRABrepressor is a highly specific epigenome editing tool that can be used in loss-of-function screens to study gene function and discover targets for drug development. The dCas9KRABhas exceptional specificity to target a particular enhancer, silence only the target genes of that enhancer, and create a repressive heterochromatin environment at that site. dCas9- KRAB can be used to screen for novel regulatory elements within the endogenous genomic context by silencing proximal or distal regulatory elements and corresponding gene targets. The specificity of dCas9-KRAB repressors allows it to be used for transcriptome-wide specificity for silencing endogenous genes. Epigenetic mechanisms for disruption at targeted locus such as histone methylation. The KRAB domain, a common heterochromatin-forming motif in naturally occurring zinc finger transcription factors, has been genetically linked to dCas9 to create an RNA-guided synthetic repressor, dCas9KRAB. The KRAB recruits heterochromatinforming factors: Kapl, HP1, SETDB1, NuRD. It induces H3K9 tri -methylation, histone deacetylation. KRAB-based synthetic repressors can effectively silence the expression of single genes and have been employed to repress oncogenes, inhibit viral replication, and treat dominant negative diseases.

[0036] As used herein, the term “base-editing enzyme” refers to fusion protein comprising a defective CRISPR / Cas nuclease linked to a deaminase polypeptide. The term is also known as “base-editor”. Two classes of base-editing enzymes— cytosine base-editing enzymes (CBEs) and adenine base-editing enzymes (ABEs)— can be used to generate single base pair edits without double stranded breaks. Typically, cytosine base-editing enzymes are created by fusing the defective CRISPR / Cas nuclease to a deaminase.

[0037] As used herein, the term “nuclease” includes a protein (i.e. an enzyme) that induces a break in a nucleic acid sequence, e.g., a single or a double strand break in a double-stranded DNA sequence.

[0038] As used herein, the term “CRISPR / Cas nuclease” has its general meaning in the art and refers to segments of prokaryotic DNA containing clustered regularly interspaced short palindromic repeats (CRISPR) and associated nucleases encoded by Cas genes. In bacteria the CRISPR / Cas loci encode RNA-guided adaptive immune systems against mobile genetic elements (viruses, transposable elements and conjugative plasmids). Three types of CRISPR systems have been identified. CRISPR clusters contain spacers, the sequences complementary to antecedent mobile elements. CRISPR clusters are transcribed and processed into mature CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) RNA (crRNA). The CRISPR / Cas nucleases Cas9 and Cpfl belong to the type II and type V CRISPR / Cas system and have strong endonuclease activity to cut target DNA. Cas9 is guided by a mature crRNA that contains about 20 nucleotides of unique target sequence (called spacer) and a transactivating small RNA (tracrRNA) that also serves as a guide for ribonuclease Ill-aided processing of pre-crRNA. The crRNA:tracrRNA duplex directs Cas9 to target DNA via complementary base pairing between the spacer on the crRNA and the complementary sequence (called protospacer) on the target DNA. Cas9 recognizes a trinucleotide (NGG for S. Pyogenes Cas9) protospacer adjacent motif (PAM) to specify the cut site (the 3rdor the 4thnucleotide upstream from PAM).

[0039] As used herein, the term “Cas9” or “Cas9 nuclease” refers to an RNA-guided nuclease comprising a Cas9 protein, or a fragment thereof (e.g., a protein comprising an active or inactive DNA cleavage domain of Cas9, and / or the gRNA binding domain of Cas9). A Cas9 nuclease is also referred to sometimes as a casnl nuclease or a CRISPR (clustered regularly interspaced short palindromic repeat)-associated nuclease. CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements and conjugative plasmids). CRISPR clusters contain spacers, sequences complementary to antecedent mobile elements, and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc) and a Cas9 protein. The tracrRNA serves as a guide for ribonuclease 3 -aided processing of pre- crRNA. Subsequently, Cas9 / crRNA / tracrRNA endonucleolytically cleaves linear or circular dsDNA target complementary to the spacer. The target strand not complementary to crRNA is first cut endonucleolytically, then trimmed 3Z-5Zexonucleolytically. In nature, DNA- binding and cleavage typically requires protein and both RNAs. However, single guide RNAs (“sgRNA”, or simply “gNRA”) can be engineered so as to incorporate aspects of both the crRNA and tracrRNA into a single RNA species. See, e.g., Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J. A., Charpentier E. Science 337:816-821(2012), the entire contents of which is hereby incorporated by reference. Cas9 recognizes a short motif in the CRISPR repeat sequences (the PAM or protospacer adjacent motif) to help distinguish self versus non-self. Cas9 nuclease sequences and structures are well known to those of skill in the art (see, e.g., “Complete genome sequence of an Ml strain of Streptococcus pyogenes.” Ferretti et al., J. J., McShan W. M., Ajdic D. J., Savic D. J., Savic G., Lyon K., Primeaux C., Sezate S., Suvorov A. N., Kenton S., Lai H. S., Lin S. P., Qian Y., Jia H. G, Najar F. Z., Ren Q., Zhu H., Song L., White J., Yuan X., Clifton S. W., Roe B. A., McLaughlin R. E., Proc. Natl. Acad. Sci. U.S.A. 98:4658-4663(2001); “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” Deltcheva E., Chylinski K., Sharma C. M., Gonzales K., Chao Y., Pirzada Z. A., Eckert M. R., Vogel J., Charpentier E., Nature 471:602-607(2011); and “A programmable dual- RNA-guided DNA endonuclease in adaptive bacterial immunity.” Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J. A., Charpentier E. Science 337:816-821(2012), the entire contents of each of which are incorporated herein by reference). Cas9 orthologs have been described in various species, including, but not limited to S. pyogenes and S. thermophilus. Additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on this disclosure, and such Cas9 nucleases and sequences include Cas9 sequences from the organisms and loci disclosed in Chylinski, Rhun, and Charpentier, “The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems” (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference. In some embodiments, the term “Cas9” refers to Cas9 from: Corynebacterium ulcérons (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheria. (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Ref: NC 021284.1); Prevotella intermedia (NCBI Ref: NC_017861.1); Spiroplasma taiwanense (NCBI Ref: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021314.1); Belliella baltica (NCBI Ref: NC_018010.1); Psychroflexus torquisl (NCBI Ref: NC 018721.1); Streptococcus thermophilus (NCBI Ref: YP 820832.1); Listeria innocua (NCBI Ref: NP_472073.1); Campylobacter jejuni (NCBI Ref: YP_002344900.1); or Neisseria, meningitidis (NCBI Ref: YP_002342100.1). As used herein, the term “defective CRISPR / Cas nuclease” refers to a CRISPR / Cas nuclease having lost at least one nuclease domain.

[0040] As used herein, the term “nickase” has its general meaning in the art and refers to an endonuclease which cleaves only a single strand of a DNA duplex. Accordingly, the term “Cas9 nickase” refers to a nickase derived from a Cas9 protein, typically by inactivating one nuclease domain of Cas9 protein.

[0041] As used herein, the term “CRISPR interference” (CRISPRi) refers to a genetic perturbation technique that allows for sequence-specific repression of gene expression in prokaryotic and eukaryotic cells. Based on the bacterial genetic immune system - CRISPR (clustered regularly interspaced short palindromic repeats) pathway. CRISPRi regulates gene expression primarily on the transcriptional level. The technology uses a catalytically dead Cas9 (usually denoted as dCas9) protein that lacks endonuclease activity to regulate genes in an RNA-guided manner. Targeting specificity is determined by complementary base-pairing of a single guide RNA (sgRNA) to the genomic locus.

[0042] As used herein, the term “guide RNA molecule” generally refers to an RNA molecule (or a group of RNA molecules collectively) that can bind to a Cas9 protein and target the Cas9 protein to a specific location within a target DNA. A guide RNA can comprise two segments: a DNA-targeting guide segment and a protein-binding segment. The DNA-targeting segment comprises a nucleotide sequence that is complementary to (or at least can hybridize to under stringent conditions) a target sequence. The protein-binding segment interacts with a CRISPR protein, such as a Cas9 or Cas9 related polypeptide. These two segments can be located in the same RNA molecule or in two or more separate RNA molecules. When the two segments are in separate RNA molecules, the molecule comprising the DNA-targeting guide segment is sometimes referred to as the CRISPR RNA (crRNA), while the molecule comprising the protein-binding segment is referred to as the trans-activating RNA (tracrRNA).

[0043] As used herein, the term “target sequence” or “target” refers to a nucleic acid containing a target nucleic acid sequence. A target nucleic acid may be single-stranded or double-stranded, and often is double-stranded DNA. A “target nucleic acid sequence,” “target sequence” or “target region” as used herein, means a specific sequence or the complement thereof that one wishes to bind to using the CRISPR system as disclosed herein. As used herein, the term “target nucleic acid strand” refers to a strand of a target nucleic acid that is subject to base-pairing with a guide RNA as disclosed herein. That is, the strand of a target nucleic acid that hybridizes with the crRNA and guide sequence is referred to as the “target nucleic acid strand.” The other strand of the target nucleic acid, which is not complementary to the guide sequence, is referred to as the “non-complementary strand.” In the case of double-stranded target nucleic acid (e.g., DNA), each strand can be a “target nucleic acid strand” to design crRNA and guide RNAs and used to practice the method of this invention as long as there is a suitable PAM site.

[0044] As used herein, the term “ribonucleoprotein complex,” or “ribonucleoprotein particle” refers to a complex or particle including a nucleoprotein and a ribonucleic acid. A “nucleoprotein” as provided herein refers to a protein capable of binding a nucleic acid (e.g., RNA, DNA). Where the nucleoprotein binds a ribonucleic acid, it is referred to as “ribonucleoprotein.” The interaction between the ribonucleoprotein and the ribonucleic acid may be direct, e.g., by covalent bond, or indirect, e.g., by non-covalent bond (e.g. electrostatic interactions (e.g. ionic bond, hydrogen bond, halogen bond), van der Waals interactions (e.g. dipole-dipole, dipole-induced dipole, London dispersion), ring stacking (pi effects), hydrophobic interactions and the like).

[0045] 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 SEQ ID NO:1.

[0046] 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

[0047] 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 progenitor cells display a number of phenotypes, such as Lin-CD34+CD38-CD90+CD45RA-, Lin-CD34+CD38-CD90-CD45RA-, Lin-

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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 have a P-hemoglobinopathy (e.g a sickle cell disease or a P-thalassemia).

[0053] 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.

[0054] 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).

[0055] 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.

[0056] 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.]).

[0057] 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 epigenome 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 treatmenteffective 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.

[0058] Methods for editing:

[0059] The first object of the present invention relates to a method for editing a population of eukaryotic cells comprising contacting the population of eukaryotic cells with an epigenome editing platform that comprises (a) one or more epigenome-editing enzyme(s), and (b) one or more guide RNA molecule(s) designed for guiding the epigenome-editing enzyme to a target sequences in the gene encoding for the CD33 so as to knock-down the expression of CD33.

[0060] Epigenome editing enzyme:

[0061] In some embodiments, the epigenome-editing enzyme of the present invention comprises a defective CRISPR / Cas nuclease. The sequence recognition mechanism is the same as for the non-defective CRISPR / Cas nuclease. Typically, the defective CRISPR / Cas nuclease of the invention comprises at least one RNA binding domain. The RNA binding domain interacts with a guide RNA molecule as defined hereinafter. However, the defective CRISPR / Cas nuclease of the invention is a modified version with no nuclease activity. Accordingly, the defective CRISPR / Cas nuclease specifically recognizes the guide RNA molecule and thus guides the epigenome-editing enzyme to its target DNA sequence.

[0062] In some embodiments, the defective CRISPR / Cas nuclease can be modified to increase nucleic acid binding affinity and / or specificity, alter an enzymatic activity, and / or change another property of the protein. In some embodiments, the nuclease domains of the protein can be modified, deleted, or inactivated. In some embodiments, the protein can be truncated to remove domains that are not essential for the function of the protein. In some embodiments, the protein is truncated or modified to optimize the activity of the RNA binding domain.

[0063] In some embodiments, the CRISPR / Cas nuclease consists of a mutant CRISPR / Cas nuclease i.e. a protein having one or more point mutations, insertions, deletions, truncations, a fusion protein, or a combination thereof. In some embodiments, the mutant has the RNA-guided DNA binding activity, but lacks one or both of its nuclease active sites. In some embodiments, the mutant comprises an amino acid sequence having at least 50% of identity with the wild type amino acid sequence of the CRISPR / Cas nuclease. Various CRISPR / Cas nucleases can be used in this invention. Non-limiting examples of suitable CRISPR / CRISPR / Cas nucleases include Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9, CaslO, CaslOd, CasF, CasG, CasH, Csyl, Csy2, Csy3, Csel (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Cszl, Csxl5, Csfl, Csf2, Csfi, Csf4, and Cul966. See e.g., WO2014144761 WO2014144592, WO2013176772, US20140273226, and US20140273233, the contents of which are incorporated herein by reference in their entireties.

[0064] In some embodiments, the CRISPR / Cas nuclease is derived from a type II CRISPR-Cas system. In some embodiments, the CRISPR / Cas nuclease is derived from a Cas9 protein. The Cas9 protein can be from Streptococcus pyogenes, Streptococcus thermophilus, Staphylococcus aureus, Streptococcus sp., Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polar omonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vino sum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalter omonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, or Acaryochloris marina, inter alia.

[0065] Methods for generating a Cas9 protein (or a fragment thereof) having an inactive DNA cleavage domain are known (See, e.g., Jinek et al., Science. 337:816-821(2012); Qi et al., “Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression” (2013) Cell. 28; 152(5): 1173-83, the entire contents of each of which are incorporated herein by reference). For example, the DNA cleavage domain of Cas9 is known to include two subdomains, the HNH nuclease subdomain and the RuvCl subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, whereas the RuvCl subdomain cleaves the non-complementary strand. Mutations within these subdomains can silence the nuclease activity of Cas9. For example, the mutations D10A and H841A completely inactivate the nuclease activity of S. pyogenes Cas9 (Jinek et al., Science. 337:816-821(2012); Qi et al., Cell. 28; 152(5): 1173-83 (2013).

[0066] In some embodiments, the CRISPR / Cas nuclease of the present invention is nickase and more particularly a Cas9 nickase i.e. the Cas9 from S. pyogenes having one mutation selected from the group consisting of D10A and H840A.

[0067] In some embodiments, the Cas9 variants having mutations other than D10A or H840A are used, which e.g., result in nuclease inactivated Cas9 (dCas9). Such mutations, by way of example, include other amino acid substitutions at D10 and H840, or other substitutions within the nuclease domains of Cas9 (e.g., substitutions in the HNH nuclease subdomain and / or the RuvCl subdomain).

[0068] According to the present invention, the second component of the epigenome-editing enzyme disclosed herein comprises a transcriptional repressor domain. In some embodiments, the transcriptional repressor domain is a NuE domain, NcoR domain, SID domain or a SID4* domain. In some embodiments, the transcriptional repressor domain is a KRAB domain. The KRAB domain is a common heterochromatin-forming motif found in naturally occurring zinc finger transcription factors. The Kruppel-associated box (KRAB) domain recruits several heterochromatin-forming factors, including Kapl, HP1, SETDB1, and NuRD. These factors play a crucial role in inducing histone modifications such as H3K9 tri-methylation and histone deacetylation, contributing to the establishment of a repressive chromatin state. KRAB-based synthetic repressors are designed to effectively silence the expression of single genes, making them a valuable tool in epigenome editing. In this context, the epigenome-editing enzyme of the invention consists of a KRAB domain linked to a catalytically inactive Cas9 protein, commonly referred to as dCas9. This fusion protein, known as dCas9-KRAB, acts as an RNA- guided synthetic repressor.

[0069] In some embodiments, the transcriptional repressor domain is fused to the N-terminus of the defective CRISPR / Cas nuclease. In some embodiments, the transcriptional repressor domain is fused to the C-terminus of the defective CRISPR / Cas nuclease. In some embodiments, the defective CRISPR / Cas nuclease and the transcriptional repressor domain are fused via a linker. Suitable linker motifs and linker configurations will be apparent to those of skill in the art. In some embodiments, suitable linker motifs and configurations include those described in Chen et al., Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev. 2013; 65(10): 1357-69, the entire contents of which are incorporated herein by reference.

[0070] In some embodiments, the fusion protein may comprise additional features. Other exemplary features that may be present are localization sequences, such as nuclear localization sequences (NLS), cytoplasmic localization sequences, export sequences, such as nuclear export sequences, or other localization sequences, as well as sequence tags that are useful for solubilization, purification, or detection of the fusion proteins. Suitable localization signal sequences and sequences of protein tags are provided herein, and include, but are not limited to, biotin carboxylase carrier protein (BCCP) tags, myc-tags, calmodulin-tags, FLAG-tags, hemagglutinin (HA)-tags, polyhistidine tags, also referred to as histidine tags or His-tags, maltose binding protein (MBP)-tags, nus-tags, glutathione-S-transferase (GST)-tags, green fluorescent protein (GFP)-tags, thioredoxin-tags, S-tags, Softags (e.g., Softag 1, Softag 3), strep-tags, biotin ligase tags, FlAsH tags, V5 tags, and SBP-tags. Additional suitable features will be apparent to those of skill in the art.

[0071] In some embodiment, the epigenome-editing enzyme of the present invention is a Staphylococcus aureus Cas9-based repressor, namely SadCas9-KRAB (SaKRAB).

[0072] Guide RNA molecules: In particular, the guide RNA molecules include a region that is complementary and capable of hybridization to a pre-selected target site of interest (e.g. CD33). In some embodiment, this guide sequence can comprise from about 10 nucleotides to more than about 25 nucleotides. For example, the region of base pairing between the guide sequence and the corresponding target site sequence can be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, or more than 25 nucleotides in length. In some embodiments, the guide sequence is about 17-20 nucleotides in length, such as 20 nucleotides.

[0073] Typically, a software program is used to identify candidate CRISPR target sequences on both strands of the DNA nucleic acid molecule containing the HBG genes based on desired guide sequence length and a CRISPR motif sequence (PAM) for a specified CRISPR enzyme. One requirement for selecting a suitable target nucleic acid is that it has a 3' PAM site / sequence. Each target sequence and its corresponding PAM site / sequence are referred herein as a Cas- targeted site. Type II CRISPR system, one of the most well characterized systems, needs only Cas 9 protein and a guide RNA complementary to a target sequence to affect target cleavage. For example, target sites for Cas9 from S. pyogenes, with PAM sequences NGG, may be identified by searching for 5'-Nx-NGG-3' both on the input sequence and on the reversecomplement of the input. Since multiple occurrences in the genome of the DNA target site may lead to nonspecific genome editing, after identifying all potential sites, the program filters out sequences based on the number of times they appear in the relevant reference genome. For those CRISPR enzymes for which sequence specificity is determined by a “seed” sequence, such as the 11-12 bp 5' from the PAM sequence, including the PAM sequence itself, the filtering step may be based on the seed sequence. Thus, to avoid editing at additional genomic loci, results are filtered based on the number of occurrences of the seed:PAM sequence in the relevant genome. The user may be allowed to choose the length of the seed sequence. The user may also be allowed to specify the number of occurrences of the seed:PAM sequence in a genome for purposes of passing the filter. The default is to screen for unique sequences. Filtration level is altered by changing both the length of the seed sequence and the number of occurrences of the sequence in the genome. The program may in addition or alternatively provide the sequence of a guide sequence complementary to the reported target sequence(s) by providing the reverse complement of the identified target sequence(s). Further details of methods and algorithms to optimize sequence selection can be found in U.S. application Ser. No. 61 / 836,080; incorporated herein by reference.

[0074] Typically, the guide RNA molecule targets a nucleic acid sequence selected from Table In particular, the guide RNA molecule targets the nucleic acid sequence as set forth in SEQ ID

[0075] NO:2

[0076] In particular, the guide RNA molecule targets the nucleic acid sequence as set forth in SEQ ID NO:6

[0077] The guide RNA molecule of the present invention can be made by various methods known in the art including cell-based expression, in vitro transcription, and chemical synthesis. The ability to chemically synthesize relatively long RNAs (as long as 200 mers or more) using TC-RNA chemistry (see, e.g., U.S. Pat. No. 8,202,983) allows one to produce RNAs with special features that outperform those enabled by the basic four ribonucleotides (A, C, G and U). In particular, the RNA molecule of the present invention can be made with recombinant technology using a host cell system or an in vitro translation-transcription system known in the art. Details of such systems and technology can be found in e.g., WO2014144761 WO2014144592, WO2013176772, US20140273226, and US20140273233, the contents of which are incorporated herein by reference in their entireties.

[0078] In some embodiments, the guide RNA molecule may include one or more modifications. Such modifications may include inclusion of at least one non-naturally occurring nucleotide, or a modified nucleotide, or analogs thereof. Modified nucleotides may be modified at the ribose, phosphate, and / or base moiety. Modified nucleotides may include 2’-O-methyl analogs, 2’- deoxy analogs, or 2’ -fluoro analogs. The nucleic acid backbone may be modified, for example, a phosphorothioate backbone may be used. The use of locked nucleic acids (LNA) or bridged nucleic acids (BNA) may also be possible. Further examples of modified bases include, but are not limited to, 2-aminopurine, 5-bromo-uridine, pseudouridine, inosine, 7-methylguanosine.

[0079] The population o f eukaryotic cell:

[0080] 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.

[0081] 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. 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.

[0082] Typically, the eukaryotic cell results from a stem cell mobilization.

[0083] 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.

[0084] 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. 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.

[0085] In some embodiments, the eukaryotic cells, such as hematopoietic stem cells (HSCs), are genetically engineering 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 engineering 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.

[0086] Combination with gene editing platform:

[0087] In some embodiments, the method of the present invention further comprises the step of contacting the population of eukaryotic cell 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).

[0088] Typically, the epigenome editing performed with the epigenome editing enzyme and the gene editing performed with the base-editing platform are carried out concomitantly. Accordingly, a further object of the present invention relates to a method for editing a population of eukaryotic cells comprising contacting the population of eukaryotic cells with: an epigenome editing platform that comprises (a) one or more epigenome-editing enzyme(s), and (b) one or more guide RNA molecule(s) designed for guiding the epigenome-editing enzyme to a target sequence in the gene encoding for the CD33 so as to knock-down the expression of CD33 and, 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).

[0089] 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.

[0090] 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.

[0091] 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.

[0092] In some embodiments, the alteration results in reduced or increased expression of a target polynucleotide sequence of interest.

[0093] In some embodiments, the gene editing platform is used for increasing the fetal hemoglobin content in the eukaryotic cell.

[0094] In some embodiments, the method of the present invention further comprises the step of contacting the population of eukaryotic cell with a gene editing platform that comprises (a) at least one adenine-base editor (ABE), and (b) one or more guide RNA molecule(s) designed for guiding the ABE to the target sequence as set forth in SEQ ID NO:7.

[0095] Vectors:

[0096] In some embodiments, the different components of the editing platforms of the present invention are provided to the eukaryotic cell through expression from one or more expression vectors. For example, the nucleic acids encoding the guide RNA molecule or editing enzyme(s) can be cloned into one or more vectors for introducing them into the eukaryotic cell. The vectors are typically prokaryotic vectors, e.g., plasmids, or shuttle vectors, or insect vectors, for storage or manipulation of the nucleic acid encoding the guide RNA molecule or the editing enzyme herein disclosed. Preferably, the nucleic acids are isolated and / or purified. Thus, the present invention provides recombinant constructs or vectors having sequences encoding one or more of the guide RNA molecules or editing enzymes described above. Examples of the constructs include a vector, such as a plasmid or viral vector, into which a nucleic acid sequence of the invention has been inserted, in a forward or reverse orientation. In some embodiments, the construct further includes regulatory sequences. A “regulatory sequence” includes promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence, as well as inducible regulatory sequences. The design of the expression vector can depend on such factors as the choice of the eukaryotic cell to be transformed, transfected, or infected, the desired expression level, and the like. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available. Appropriate cloning and expression vectors for use with eukaryotic hosts are also described in e.g., Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press). The vector can be capable of autonomous replication or integration into a host DNA. The vector may also include appropriate sequences for amplifying expression. In addition, the expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell cultures, or such as tetracycline or ampicillin resistance in E. coli. Any of the procedures known in the art for introducing foreign nucleotide sequences into host cells may be used. Examples include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, nucleofection, liposomes, microinjection, naked DNA, plasmid vectors, viral vectors, both episomal and integrative, and any of the other well-known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell.

[0097] In some embodiments, the different components of the editing platform of the present invention are provided to the population of cells through the use of an RNA-encoded system. For instance, the editing system may be provided to the population of cells through the use of a chemically modified mRNA-encoded adenine or cytidine base editor together with modified guide RNA as described in Jiang, E, Henderson, J.M., Coote, K. et al. Chemical modifications of adenine base editor mRNA and guide RNA expand its application scope. Nat Commun 11, 1979 (2020). In particular, engineered RNA-encoded editing enzymes (e.g. ABE) system are prepared by introducing various chemical modifications to both mRNA that encoded the editing enzyme and guide RNA. In particular said modifications consist in uridine depleted mRNAs modified with 5-methoxyuridine: synonymous codons may be introduced to deplete uridines as much as possible without altering the coding sequence and replaced all the remaining uridines with 5-methoxyuridine. Said optimized base editing system exhibits higher editing efficiency at some genomic sites compared to DNA-encoded system. It is also possible to encapsulate the modified mRNA and guide RNA into lipid nanoparticle (LNP) for allowing lipid nanoparticle (LNP)-mediated delivery.

[0098] In some embodiments, the different components of the editing platform of the present invention are provided to the population of cells through the use of ribonucleoprotein (RNP) complexes. For instance, the editing enzyme can be pre-complexed with one or more guide RNA molecules to form a ribonucleoprotein (RNP) complex. The RNP complex can thus be introduced into the eukaryotic cell. Introduction of the RNP complex can be timed. The cell can be synchronized with other cells at Gl, S, and / or M phases of the cell cycle. RNP delivery avoids many of the pitfalls associated with mRNA, DNA, or viral delivery. Typically, the RNP complex is produced simply by mixing the proteins (i.e. the epigenome-editing enzyme) and one or more guide RNA molecules in an appropriate buffer. This mixture is incubated for 5-10 min at room temperature before electroporation. Electroporation is a delivery technique in which an electrical field is applied to one or more cells in order to increase the permeability of the cell membrane. In some embodiments, the editing efficiency can be improved by adding a transfection enhancer oligonucleotide.

[0099] Methods of enrichment:

[0100] 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.

[0101] In some embodiment, the present invention relates to a population of edited eukaryotic cells obtainable by the method of the present invention.

[0102] 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.

[0103] 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 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).

[0104] In some embodiments, cell enrichment is performed with an agent that is capable of depleting CD33 positive cells.

[0105] In some embodiments, the agent is an antibody that binds to CD33 and depletes CD33+ cells (i.e. a “depleting antibody”).

[0106] 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. 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.

[0107] In some embodiments, the antibody suitable for depletion of CD33+ cells is conjugated to a therapeutic moiety, i.e. a drug.

[0108] 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".

[0109] 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.

[0110] 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.

[0111] 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.

[0112] Methods of therapy:

[0113] 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.

[0114] 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.

[0115] In some embodiments, the population of cell is autologous to the subject, meaning the population of cells is derived from the same subject. In some embodiments, the method of therapy of the present invention further comprises administering the patient with an agent that is capable of depleting CD33 positive cells, such as an antibody that binds to CD33 and depletes CD33+ cells as described above.

[0116] Kits

[0117] In a further embodiment, the present invention relates to a kit comprises (a) at least epigenome-editing enzyme, and (b) one or more RNA molecule(s) designed for guiding the epigenome-editing enzyme(s) to a target sequence in the gene encoding for CD33 so as to knock-down the expression of CD33, optionally (c) one or more base-editing enzyme(s), optionally (d) one or more guide RNA molecule(s) designed for guiding the base-editing enzyme(s) to one or more target sequence(s) and optionally (e) an agent that is capable of depleting CD33 positive cells.

[0118] 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 epigenome-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, microparticles 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. 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.

[0119] FIGURES:

[0120] Figure 1. KRAB-mediated downregulation of CD33 in the HEL cell line. (A) Schematic representation of the CD33 locus. Boxes represent exons. Lines represent sgRNAs (g6; g6-15nt; g3). We reported the sequence of the protospacers. The potential nucleotides that can be converted are highlighted in bold. (B) Number of off-target sites in silico predicted using CRISPOR35. We classified the off-targets sites based on the number of mismatches present in the protospacer. Percentage of CD33+ (C) and (D) CD33+ / GFP+ HEL cells measured by flow cytometry. Cells were transfected with plasmids expressing editors and sgRNAs and collected 5 days after the treatment. Control samples were transfected with TE buffer. Data are expressed as mean ± SD of n=3 biologically independent experiments, ns = not significant (Two=way ANOVA with Tukey’s multiple comparison test).

[0121] Figure 2. Simultaneous CD33 downregulation and HBG base editing. (A) Schematic representation of the HBG locus. Boxes represent exons. The line represents the sgRNA (gl2). We reported the sequence of the protospacers. The potential nucleotides that can be converted are highlighted in bold. (B) Possible combinations of Cas9-derived editors (ABE or KRAB) with sgRNAs targeting CD33 (g6 / g6-15nt) or HBG (gl2). (C) Editing efficiency at the CD33 region 5 days after transfection (reported as A-T to G-C conversion) measured by EditR analysis of Sanger sequencing chromatograms. Cells were transfected with ABE or ABE+KRAB together with g6 or g6-15nt alone or in combination with gl2. Control samples were transfected with TE buffer. (D) Editing efficiency at the HBG region of cells treated as in (C). (E) Percentage of CD33+ / GFP+ cells treated as in (C) measured by flow cytometry 5 days after the treatment. (F) Fold-change of CD 33 expression relative to GAPDH measured by RT- qPCR. All data are expressed as mean ± SD of n=3 biologically independent experiments, ns = not significant (Two=way ANOVA with Tukey’s multiple comparison test).

[0122] Figure 3. Independent CD33 downregulation and HBG base editing. (A) Expected combinations of SaCas9-KRAB (SaKRAB) with sgRNAs targeting CD33 and ABE8.13m (ABE) targeting HBG. (B) Schematic representation of the CD33 locus. Boxes represent exons. Lines represent sgRNAs (g7, g8, g9). We reported the sequence of the protospacers. (C) Percentage of CD33+ / GFP+ HEL cells measured by flow cytometry. Cells were transfected with plasmids expressing editors and sgRNAs and collected 5 days after the treatment. Control samples were transfected with KRAB enzyme only. (D) Editing efficiency at the HBG region in cells treated with gl2 alone or in combination with g9. Data are expressed as mean ± SD of n=3 biologically independent experiments, ns = not significant (Two=way ANOVA with Tukey’s multiple comparison test).

[0123] Figure 4. KRAB-mediated downregulation of CD33 in HSPCs. (A) Viability of HSPCs based on side scatter (SSC) and forward scatter (FSC) measurement assessed by flow cytometry 2 days after electroporation with sgRNAs and editor-encoding mRNA. Control samples were transfected with TE buffer. (B) Percentage of CD33+ HSPCs measured by flow cytometry 2 days after electroporation. (C) Fold-change of CD 33 expression relative to GAPDH measured by RT-qPCR. (D) Editing efficiency at the HBG region of cells treated as in (A). (E) Fold-change of HBG expression relative to HBA, measured by RT-qPCR. All data are expressed as mean ± SD of n=2 or 3 biologically independent experiments. Experiments performed in SCD HSPCs are reported as circles and squares; experiments performed in HD HSPCs are reported as triangles.

[0124] EXAMPLE:

[0125] Introduction:

[0126] 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)32, 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. A safer approach is represented by a transient downregulation of CD33 and by avoiding the use of DNA-cleaving enzymes at the CD33 region to limit genomic alterations. The CRISPR-Cas system, soon after its description as a genome editor, was also adapted to work as a targeted gene regulation tool. The method, termed CRISPR interference (CRISPRi), was developed by fusing a catalytically inactive dead Cas9 (dCas9) with domains of negative transcriptional regulation33. The Krüppel- Associated Box (KRAB) domain coming from the human zinc-finger protein Koxl is the most used repressor, making the KRAB-dCas9 fusion a powerful platform for transient gene silencing. The epigenetic modification (i.e. deposition of tri-methylation on histone H3 lysine 9) is indeed not maintained during time and gene expression returns to baseline as soon as the complex is not present in the cell anymore34. Furthermore, the addition of DNMT3A and DNMT3B DNA methyltransferases allow a permanent silencing (Cappelluti MA, Mollica Poeta V, Valsoni S, Quarato P, Merlin S, Merelli I, Lombardo A.Nature. 2024 Mar;627(8003):416-423. doi: 10.1038 / s41586-024-07087-8. Epub 2024 Feb 28.PMID: 38418872).

[0127] Here, we developed an epigenome editing strategy to enrich base edited HSPCs in order to achieve higher BE efficiencies by transiently downregulating CD33 expression. CD33 is a surface marker of the myeloid lineage, which is expressed on human HSCs with a high regenerative potential. In their approach, this transient repression will ensure CD33 downregulation only in the timeframe necessary to select and enrich base edited HSPCs.

[0128] In particular, we simultaneously targeted: (i) the HBG promoters, to insert HPFH and HPFH-like mutations that reactivate HbF, and (ii) the CD33 gene, to downregulate the expression of the CD33 surface marker. By selecting CD33 negative cells, we enriched for populations edited at the HBG promoters and eliminated unedited cells that normally outcompete edited cells during transplantation.

[0129] This epigenome editing strategy will allow the ex vivo selection of corrected HSCs prior to transplantation.

[0130] Material and Methods

[0131] Plasmids

[0132] Plasmids used in this study include KRAB-dCas9 (Addgene #112195), SadCas9 KRAB (Addgene #188504), ABE8.13m-OPT [generated by uridine depletion of the coding sequence of the ABE8.13m (Addgene #136296) plasmid and by addition of a DNA fragment containing two copies of the 3’ untranslated region (UTR) of the HBB gene and a poly-A sequence of 96 adenines]. The sgRNA targeting the -200 region of the HBG promoter (gl2 - (CTTGGGGGCCCCTTCCCCAC - SEQ ID NO:7)) was previously generated25, while sgRNAs targeting the CD33 gene were manually designed (Table 1). To generate the sgRNA expression plasmid, oligonucleotides were annealed to create the sgRNA spacer and the duplexes were ligated into the Bbsl-digested MA128 plasmid (provided by M. Amendola, Genethon, France). The plasmids for SaCas9 sgRNAs were generated with the same strategy but ligated into the Bsal-digested pVAX plasmid (provided by A. Cereseto, CIBIO UniTN, Italy).

[0133] Table 1. List of sgRNA spacer sequences.

[0134] Synthetic sgRNA

[0135] For HSPCs RNA-mediated 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). mRNA in vitro transcription (ivt)

[0136] Ten micrograms of editor-expressing plasmids were digested overnight with 20 units of a restriction enzyme that cleaves once after the stop codon. The linearized plasmids were purified using the PCR purification kit (QIAGEN) and were eluted in 30 pl of DNase / RNase- free water. One microgram of linearized plasmid was used as template for the ivt reaction (MEGAscript, Ambion). The ivt protocol was modified as follows. The GTP nucleotide solution was used at a final concentration of 3.0 mM instead of 7.5 mM and the anti -reverse cap analog A7-methyl-3'-O-methyl-guanosine-5'-triphosphate-5'-guanosine (ARCA, Trilink) was used at a final concentration of 12.0 mM resulting in a final ratio of Cap:GTP of 4:1 that allows efficient capping of the mRNA. The incubation time for the ivt reaction was reduced to 30 min. The polyadenylation step was performed following manufacturer’s guidelines (PolyA tailing kit, Ambion). mRNA was precipitated using lithium chloride and resuspended in TE buffer in a final volume that allowed us to achieve a concentration of >1 pg / pl. The mRNA quality was evaluated using Tapestation 2200 (Agilent).

[0137] HEL cell culture and transfection Human erythroleukemia (HEL) cell line was maintained in RPMI 1640 (Lonza) medium 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). For the experiments 106cells / condition were transfected with 3.6 pg of ABE or KRAB-expressing plasmid and 1.2 pg of sgRNA-containing plasmid. For combinations a total of 7.2 for editors and 2.4 pg for sgRNAs was used. An additional plasmid expressing GFP was transfected to assess the nucleofection efficiency. Cells were nucleofected with the AMAXA Cell Line Nucleofector Kit V (VCA-1003) and the X-005 program (Nucleofector II). 24h after nucleofection, transfection efficiency was evaluated by flow cytometry, using the Novocyte (Agilent) flow cytometer.

[0138] HSPC purification and culture

[0139] Adult human non-mobilized peripheral blood CD34+HSPCs from SCD patients or bone marrow CD34+HSPCs from healthy donors were obtained from the “Hôpital 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-2024-6899, CPP Ile-de-France II “Hôpital 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 5 * 105cells / ml in StemSpan (STEMCELL Technologies) supplemented with penicillin / streptomycin (Gibco), 250 nM StemRegenin (STEMCELL Technologies), 38 nM UM171 (STEMCELL Technologies), and the following recombinant human cytokines (PeproTech): human stem cell factor (SCF) (300 ng / ml), Flt-3L (300 ng / ml), thrombopoietin (TPO) (100 ng / ml), and interleukin-3 (IL-3) (60 ng / ml).

[0140] RNA transfection

[0141] 0.1 x 105to 2 * 105CD34+HSPCs per condition were transfected with 3 pg of each enzyme-encoding mRNA and a single or a mix of synthetic sgRNAs (3 pg each). The P3 Primary Cell 4D-Nucleofector X Kit S (Lonza) was used with the CAI 37 program (Nucleofector 4D). Cells transfected with TE buffer served as negative controls.

[0142] Flow cytometry Flow cytometry analysis of CD33 surface marker for HEL cells was performed using APC-conjugated anti-CD33 (551378, BD). Acquisitions were performed using Novocyte (Agilent) flow cytometer and data were analyzed using FlowJo (BD Biosciences) software.

[0143] Evaluation of editing efficiency

[0144] The base editing efficiency was evaluated in HEL cells 5 days post-transfection or in HSPCs 6 days post-electroporation. Briefly, genomic DNA was extracted from control and edited cells using PURE LINK Genomic DNA Mini kit (Life Technologies) or Quick- DNA / RNA Miniprep (ZYMO Research) following manufacturer’s instructions. Target sites where PCR amplified and following Sanger sequencing, the chromatograms were analyzed with the EditR tool (EditR: A Method to Quantify Base Editing from Sanger Sequencing)38.

[0145] Table 2. Primers used to detect base-editing events.

[0146] Quantitative RT-PCR

[0147] Total RNA was extracted from HEL cells 5 days post-transfection using RNeasy micro kit (QIAGEN) or from HSPCs 2 days post-electroporation using Quick-DNA / RNA Miniprep (ZYMO Research). Mature transcripts were reverse transcribed using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) with random primers 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).

[0148] Table 3. Primers used to detect CD33 transcripts by qPCR.

[0149] Results

[0150] CRISPRi efficiently downregulates CD33 in the HEL cell line To transiently downregulate CD33 and avoid the cleavage of two genomic sites (HBG and CD33), we decided to exploit CRISPRi, specifically KRAB-SpdCas9, in combination with a sgRNA (g6) binding to the promoter region of the CD33 gene (Figure 1A). In parallel, we used an sgRNA (g3 - (CTGCTGTGGGCAGGTGAGTG - SEQ ID NO: 14)) that coupled with an adenine base editor (ABE), downregulate CD33 by targeting one of its splice donor sites. (Figure 1A). Compared to g3, g6 showed less in .s / 7 / œ-predicted off-target sites (Figure IB). Moreover, we designed a 15-nucleotide version of g6 (g6-15nt), since shorter sgRNAs proved to be still capable of modulating gene expression with CRISPRa / CRISPRi, but they lose their nuclease activity when combined with Cas9 nuclease36. We evaluated the efficiency of KRAB- SpdCas9 (KRAB) to downregulate CD33 expression in the HEL cell line. We transfected the cells with plasmids coding for the different sgRNAs and KRAB. We used ABE with g3 as a positive control for CD33 downregulation. An additional plasmid expressing GFP was used to control the transfection efficiency. We observed more than 60% downregulation of CD33 expression by flow cytometry with both KRAB-g6 and KRAB-g6-15nt (Figure 1C). No statistical difference was observed between normal and short sgRNA. We also measured the percentage of CD33 positive cells among the GFP positive cells reaching even higher downregulation levels (-90% of CD33-negative cells) (Figure ID).

[0151] Simultaneous base editing of HBG and KRAB-mediated downregulation of CD33

[0152] To parallelly obtain CD33 downregulation and base editing of the HBG promoter, we transfected HEL cells with plasmids encoding g6 or g6-15nt (targeting CD33 gl2 (targeting the HBG region) (Figure 2 A), and plasmids expressing ABE and KRAB. Since both editors are based on SpCas9, the two sgRNAs can complex with one or the other editor generating 4 possible ribonucleoproteins (RNP) (Figure 2B). We therefore asked whether the ABE- g6 / ABE-g6-15nt combinations could lead to undesired base editing in the CD33 or if KRAB- gl2 could lead to a decreased accessibility and in turn editing efficiency at the HBG locus. First, we measured by Sanger sequencing the editing efficiency at the CD33 region targeted by g6. We observed no A-to-G conversion in cells transfected with g6 / g6-15nt either with ABE alone or with ABE and KRAB together (Figure 2C). Next, we evaluated the editing at the HBG locus, observing no significant decrease between cells treated with ABE-gl2 only and samples transfected with ABE in combination with KRAB+g6 or KRAB+g6-15nt (Figure 2D). Finally, flow cytometry showed almost 80% reduction in the frequency of CD33+ cells (amongst the GFP+ populations) with the combination of KRAB+ABE g6+gl2 (Figure 2E). Unexpectedly, the KRAB+ABE g6-15nt+gl2 combination containing the shorter sgRNA was not able to downregulate CD33, suggesting a preference of the KRAB towards the canonical 20 nucleotide sgRNA. The results were further confirmed at the RNA level by RT-qPCR (Figure 2F).

[0153] Independent CD33 downregulation and HBG base editing

[0154] The capability of SpCas9 to complex with both CD33 and HBG sgRNAs could lead to unwanted effects such as CD33 base editing and HBG downregulation. To exclude this possibility, we decided to improve the strategy by uncoupling CD33 downregulation from HBG base editing. We exploited a Staphylococcus aureus Cas9-based repressor, namely SadCas9- KRAB (SaKRAB)37, in order to have a different Cas9 ortholog not able to bind to the sgRNA targeting the HBG promoters (Figure 3A). We designed 3 sgRNAs in the CD33 promoter region and we tested them in the HEL cell line (Figure 3B). The combination of SaKRAB with g9 resulted in almost 75% reduction of CD33+ cells (amongst the GFP+ population) (Figure 3C). In addition, we combined SaKRAB+g9 and ABE+gl2 observing similar levels of CD33 downregulation and no significant decrease in editing efficiency at the HBG promoters (Figure 3C, 3D)

[0155] CRISPRi efficiently downregulates CD33 in HSPCs

[0156] The strategies using KRAB or SaKRAB were tested in HSPCs from a healthy individual or a patient with SCD. The editing components were delivered as RNA and CD33 expression was evaluated 2 days after the electroporation. Viability was similar in HSPCs treated with ABE or KRAB editors and control samples electroporated only with TE buffer (Figure 4A). Both KRAB-g6 and SaKRAB-g9 alone led to a decrease of CD33+ HSPCs, reaching more than 50% reduction as observed with the base editing strategy (ABE-g3+gl2; Figure 4B). In parallel, we transfected ABE-gl2 in combination with KRAB-g6 or SaKRAB-g9. KRAB-g6 combined with ABE-gl2 was less effective than KRAB-g6 alone in reducing the frequency of CD33+ HSPCs suggesting a competition between KRAB and ABE editors to complex with the two sgRNAs (Figure 4B). On the contrary, samples treated with SaKRAB+g9 or SaKRAB- g9+ABE-gl2 were equally potent in decreasing the proportion of CD33+ cells (Figure 4B). The mRNA expression analysis by RT-qPCR confirmed the results observed by flow cytometry (Figure 4C). Finally, we observed a similar editing efficiency at the HBG locus with a modest reduction in cells treated with KRAB-g6 / ABE-gl2, confirming the potential competition between KRAB and ABE editors (Figure 4P). Editing of the HBG promoters resulted in a comparable increase in HBG transcripts across all the conditions treated with ABE-gl2 (Figure 4E). REFERENCES:

[0157] 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.

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Claims

42 -CLAIMS1. A method for editing a population of eukaryotic cells comprising contacting the population of eukaryotic cells with an epigenome editing platform that comprises (a) one or more epigenome-editing enzyme(s), and (b) one or more guide RNA molecule(s) designed for guiding the epigenome-editing enzyme to a target sequence in the gene encoding for the CD33 so as to knock-down the expression of CD33.

2. The method of claim 1 wherein the epigenome enzyme consists of a defective CRISPR / Cas nuclease that is fused to a transcriptional repressor domain.

3. The method of claim 2 wherein the transcriptional repressor domain is a KRAB domain.

4. The method according to claim 1 wherein the guide RNA molecule targets the nucleic acid sequence as set forth in SEQ ID NO:2 or SEQ ID NO:6.

5. The method according to claim 1 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)).

6. The method according to any one of claims 1 to 5 that comprises contacting the population of eukaryotic cells with:- the epigenome editing platform that comprises (a) one or more epigenomeediting enzyme(s), and (b) one or more guide RNA molecule(s) designed for guiding the epigenome-editing enzyme to a target sequence in the gene encoding for the CD33 so as to knock-down the expression of CD33 and, 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).

7. The method according to any one of claims 1 to 6 that further comprises the step that consists of enriching the population of edited eukaryotic cells that is negative for CD33.43 -8. The method of claim 7 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.

9. A population of edited eukaryotic cells obtainable by the method according to any one of claims 1 to 8.

10. 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 9.

11. The method according to claim 10 wherein the subject suffers from a 0- hemoglobinopathy.

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