Enrichment of gene-modified hematopoietic stem cells by multiplex base editing

Abstract

Here, we developed a strategy to enrich for base-edited HSPCs by downregulating CD33 expression to achieve higher BE efficiency. Taking advantage of the fact that the BE system does not cause DSBs, allowing multiple edits without genomic rearrangements, we simultaneously targeted (i) the HBG promoter to insert HPFH and HPFH-like mutations that reactivate HbF, and (ii) the CD33 gene to downregulate the expression of the CD33 surface marker. We enriched for the HBG promoter-edited population by selecting CD33 knockout cells, eliminating unedited cells that typically outnumber edited cells during transplantation. This strategy will enable ex vivo selection of corrected HSCs before transplantation. Furthermore, we envision using this strategy to enrich for edited HSCs over unedited HSCs in vivo by administering a toxin-conjugated anti-CD33 monoclonal antibody. The latter would target not only pharmaceutical-derived unedited HSCs but also endogenous HSCs. Thus, it may be possible to reduce the need for a full conditioning regimen and avoid its side effects.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention is in the fields of medicine, particularly gene editing and hematology.

[0002] Background of the Invention Sickle cell disease (SCD) and beta-thalassemia are genetic disorders caused by mutations in the beta-globin locus. In SCD, a point mutation in the HBB gene causes sickle cell beta-globin. S -Globin chains are formed, which leads to polymerization of sickle hemoglobin (HbS), sickling of red blood cells (RBC), anemia and organ damage 1,2 In β-thalassemia, there is a partial or complete absence of β-globin chains (β + and β 0 ) leads to precipitation of unbound α-globin chains, apoptosis of erythroid precursors, ineffective erythropoiesis, and anemia 3~5 Gene therapy approaches based on autologous genetically modified hematopoietic stem cell (HSC) transplantation are being investigated as a treatment option for patients who do not have a donor suitable for allogeneic HSC transplantation. 6 .

[0003] The severity of both SCD and β-thalassemia is mitigated by genetic persistence of fetal hemoglobin (HbF) in adulthood (HPFH) 7 This persistence is due to mutations located 200 to 115 nucleotides upstream from the transcription start site of the identical HBG1 and HBG2 γ-globin promoters. The HPFH mutations may generate de novo DNA motifs recognized by transcriptional activators (e.g., KLF1). 8~10 or disruption of binding sites (BS) for transcriptional repressors (e.g., LRF and BCL11A) 11A CRISPR-Cas9 nuclease strategy has been used to disrupt the BS of LRF and BCL11A repressors by forming insertion / deletions (InDels) via non-homologous end joining (NHEJ), which mimics the HPFH mutation and reactivates HbF expression. 12~15 .

[0004] HSCs are highly sensitive to DNA double-strand breaks (DSBs), especially in the case of multiple on-target events or simultaneous on-target and off-target events. 16 Even when highly specific single-guide RNAs (sgRNAs) are used, Cas9-sgRNA treatment of human HSPCs triggers a DNA damage response (DDR) that can lead to apoptosis. 17,18 CRISPR-Cas9 induces p53-dependent cytotoxicity and cell cycle arrest, allowing the selection of cells with dysfunctional p53 pathways. 19 Furthermore, the occurrence of multiple on-target DSBs, simultaneous on-target and off-target DSBs, or even a single on-target DSB can result in genomic deletions, inversions or translocations, chromosome losses, and chromosome breakage. 20~23 Therefore, the development of novel, effective, and safe treatment strategies for β-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 Cas9 nickase and deaminase, and introduce C-to-T and A-to-G point mutations, respectively. 24 Importantly, base editors do not generate DNA DSBs or induce DDR and any of the resulting events. Thus, base editors enable the simultaneous editing of multiple targets, an approach that would result in genome reorganization in the case of the CRISPR / Cas9 nuclease system.

[0005] We recently reported the introduction of HPFH and HPFH-like mutations into the -200 region of the HBG1 / 2 promoter via adenine and cytidine ABE and CBE with minimal off-target activity. This resulted in therapeutic HbF levels and rescued pathological phenotypes in human RBCs differentiated from base-edited SCD or β-thalassemia hematopoietic stem / progenitor cells (HSPCs; most contain hematopoietic progenitors, with less than 1% HSCs) in vitro. However, xenotransplantation experiments (of human HSPCs in immunodeficient mice) demonstrated long-term BE in HSCs, but the efficiency was reduced compared to that of HSPCs. 25 BE may cause some toxicity to true HSCs or may be less efficient in this cell population compared to hematopoietic progenitor cells.

[0006] More generally, several limitations further hinder the widespread application of genome editing approaches for hematopoietic disorders: (i) efficiency in HSCs and (ii) in vivo competition with infused, unedited HSCs or endogenous HSCs that are not completely eliminated by the myeloablation procedure patients undergo. Genetic modification of HSCs can affect their properties. In particular, editing strategies are based on a 48-72 hour in vitro treatment that involves electroporation, introduction of the editing complex, genetic modification of the target region, and exposure to cytokines. In this context, unedited HSCs may have a survival advantage and superior stemness compared to edited cells.

[0007] Several parameters can be further optimized regarding base editing efficiency in HSCs and fitness of edited HSCs. Furthermore, enrichment of edited HSCs ex vivo or in vivo relative to non-edited HSCs can maximize engraftment rates of genetically modified cells. This can also mitigate conditioning regimens and their side effects. Such an approach could potentially improve the success of therapeutic strategies for diseases where modified cells offer little or no selective advantage, such as β-hemoglobinopathies. 26,27 .

[0008] CD33 is a surface marker of myeloid lineage and is also highly expressed on malignant blast cells in patients with acute myeloid leukemia (AML) 28~30 Gemtuzumab, a toxin-conjugated anti-CD33 monoclonal antibody, is approved for the treatment of AML. Of note, CD33 is also expressed on human HSCs, which have high regenerative potential. 31 One of the major side effects of gemtuzumab is bone marrow suppression. Therefore, despite its lack of expression specificity, CD33 has been used as a target in AML patients treated with gemtuzumab and in clinical trials (NCT03971799, NCT03927261) and experimental models of chimeric antigen receptor (CAR) T-cell immunotherapy. 32,33 Interestingly, to avoid myelosuppression, researchers transplanted CD33 knockout (KO) HSPCs in combination with CAR T cells, which then efficiently targeted leukemia-specific CD33-expressing blasts. Importantly, CD33 KO HSPCs remained functional and were capable of engraftment and differentiation in animal models (mouse and non-human primates). 32 Furthermore, an ongoing clinical trial (NCT04849910) is based on transplantation of CD33 KO HSPCs into AML patients at high risk of relapse, who will need to be treated with gemtuzumab after transplantation to reduce the toxic side effects of the antibody.

[0009] Summary of the Invention The invention is defined by the claims. In particular, the invention relates to:

[0010] Detailed Description of the Invention Here, we developed a strategy to enrich for base-edited HSPCs by downregulating CD33 expression, a surface marker of myeloid lineage that is expressed on human HSCs with high regenerative potential, to achieve higher BE efficiency. 31 Importantly, CD33 KO HSPCs remained functional and were capable of engraftment and differentiation in animal models (mouse and non-human primates). 32 Based on these findings, we investigated the BE system, which allows multiple editing without causing genome rearrangements, by utilizing its property of not causing DSBs. 32 In this study, we simultaneously targeted (i) the HBG promoter to introduce HPFH and HPFH-like mutations that reactivate HbF, and (ii) the CD33 gene to downregulate the expression of the CD33 surface marker. By selecting for CD33 KO cells, we enriched for the HBG promoter-edited population and eliminated unedited cells, which would normally predominate over edited cells during transplantation. This strategy would enable ex vivo selection of restored HSCs prior to transplantation. Furthermore, we envision using this strategy to enrich edited HSCs over unedited HSCs in vivo by administering a toxin-conjugated anti-CD33 monoclonal antibody. The latter would target not only pharmaceutical-derived unedited HSCs but also endogenous HSCs. This potentially reduces the need for a full conditioning regimen and avoids its side effects.

[0011] Key Definitions As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. The terms also encompass amino acid polymers that have been modified, e.g., by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, pegylation, or any other manipulation, such as conjugation with a labeling moiety. As used herein, the term "amino acid" includes natural and / or unnatural or synthetic amino acids, including glycine and both D- and L-optical isomers, as well as amino acid analogs and peptidomimetics.

[0012] As used herein, the term "nucleic acid molecule" or "polynucleotide" refers to a DNA molecule (e.g., but not limited to, cDNA or genomic DNA). A nucleic acid molecule can be single-stranded or double-stranded.

[0013] As used herein, the term "exon" refers to a defined portion of a protein-coding nucleic acid or a nucleic acid sequence that is represented in a mature RNA molecule after any portion of a preprocessed (or precursor) RNA has been spliced ​​out. The mature RNA molecule may be messenger RNA (mRNA) or a functional non-coding RNA, such as rRNA or tRNA.

[0014] As used herein, the term "intron" refers to a nucleic acid region (within a gene) that is not translated into protein. Introns are non-coding portions that are transcribed into precursor mRNA (pre-mRNA) and subsequently removed by splicing during formation of the mature RNA.

[0015] As used herein, the term "splice site" refers to a short conserved sequence at the 5' end (donor site) or 3' end (acceptor site) of an intron to which the spliceosome binds and catalyzes the splicing of the intron from the pre-mRNA.

[0016] As used herein, the terms "encode" or "encoding" or "encoded" refer to a nucleic acid sequence that codes for a polypeptide sequence.

[0017] As used herein, the term "expression" refers to the process by which a polynucleotide is transcribed from a DNA template (e.g., into mRNA or other RNA transcript) and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. The transcript and the encoded polypeptide may be collectively referred to as a "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 gene expression (e.g., HPLC analysis of proteins and RT-qPCR analysis of mRNA). Typically, such methods are described in the Examples.

[0018] As used herein, the terms "decrease," "reduced," "reduction," and "repress" are all generally used to mean a reduction in a statistically significant amount, e.g., a reduction of 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 100% (i.e., non-existent levels compared to a reference sample), or any reduction of 10-100% compared to a reference level. As used herein, the term "knockdown" as used herein refers to a reduction in the expression of a gene or its gene product.

[0019] As used herein, the terms "increased," "increase," or "enhance," or "activate" are all generally used to mean an increase in a statistically significant amount, for example, an increase of 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 100% as compared to a base line level, any increase between 10 and 100%, or at least about a 2-fold increase, or at least about a 3-fold increase, or at least about a 4-fold increase, or at least about a 5-fold increase, or at least about a 10-fold increase or any increase between 2-fold and 10-fold or more as compared to a base line level.

[0020] 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). That is, the expression level of a phenotypic marker corresponding to "+" is high or moderate (also referred to as "+ / -"). A phenotypic marker corresponding to "-" also refers to an expression level of the phenotypic marker being zero or less than 10% of the cell population expressing said phenotypic marker.

[0021] As used herein, the term "antibody" is used herein to refer to a molecule having useful antigen-binding specificity. Those skilled in the art will readily appreciate that this term may also cover polypeptides that are antibody fragments or derivatives but can exhibit the same or similar functionality. Such antibody fragments or derivatives are intended to be encompassed by the term antibody as used herein. "Antibody" or "antibody molecule" herein refers not only to whole immunoglobulin molecules but also to fragments thereof, such as Fab, F(ab'), Fv, and other fragments. Similarly, the term antibody includes genetically engineered derivatives of antibodies, such as single-chain Fv molecules (scFv) and domain antibodies (dAbs). The term "monoclonal antibody" is used herein to encompass any isolated Ab, e.g., a conventional monoclonal antibody hybridoma, but also to encompass any cell-produced and isolated monospecific antibody, e.g., a sample of identical human immunoglobulin expressed in a mammalian cell line. Suitable monoclonal antibodies having the reactivity described herein can be prepared by known techniques, such as those disclosed in "Monoclonal Antibodies; A manual of techniques", H. Zola (CRC Press, 1988) and "Monoclonal Hybridoma Antibodies: Techniques and Application", S.G.R. Hurrell (CRC Press, 1982).

[0022] As used herein, the term "complementarity" refers to the ability of a nucleic acid to form hydrogen bonds with another nucleic acid sequence, either by traditional Watson-Crick base pairing or other non-traditional types. % complementarity indicates the percentage of residues in a nucleic acid molecule that 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 would be 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Fully complementary" means that all contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. As used herein, "substantially complementary" refers to a degree of complementarity of 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.

[0023] As used herein, the term "stringent conditions" for hybridization refers to conditions under which a nucleic acid complementary to a target sequence will hybridize preferentially to the target sequence and will not hybridize substantially to non-target sequences. Stringent conditions are generally sequence-dependent and vary depending on many factors. Generally, the longer the sequence, the higher the temperature at which that sequence will specifically hybridize 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, NY.

[0024] As used herein, the terms "hybridization" or "hybridizing" refer to the process by which fully or partially complementary nucleic acid strands come together under specific hybridization conditions to form a double-stranded structure or region in which the two constituent strands are joined by hydrogen bonds. Hydrogen bonds are typically formed between adenine and thymine or uracil (A and T or U) or cytosine and guanine (C and G), but can also be formed with other base pairs (e.g., Adams et al., The Biochemistry of the Nucleic Acids, 11th ed., 1992).

[0025] As used herein, the term "fusion polypeptide" or "fusion protein" refers to a protein formed by linking two or more polypeptide sequences together. Fusion polypeptides encompassed by the present invention include the translation product of a chimeric gene construct that links a nucleic acid sequence encoding a first polypeptide, e.g., an RNA-binding domain, with a nucleic acid sequence encoding a second polypeptide, e.g., an effector domain, to form a single open reading frame. That is, a "fusion polypeptide" or "fusion protein" is a recombinant protein of two or more proteins linked by a peptide bond or via multiple peptides. Fusion proteins may also include a peptide linker between the two domains.

[0026] As used herein, the term "wild-type" is a term of the art understood by those skilled in the art and refers to the typical form of an organism, strain, gene or characteristic occurring in nature, as distinguished from mutant or variant forms.

[0027] As used herein, the term "derived from" refers to a process used to isolate, derive, or prepare a first component (e.g., a first molecule) or a second component that differs in information from the first component (e.g., a second molecule that differs from the first molecule).

[0028] As used herein, the "% identity" between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences (i.e., % identity = number of identical positions / total number of positions × 100). Comparison of sequences and determination of the % identity between two sequences can be achieved using a mathematical algorithm, as described below. The % 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 % identity between two nucleotide or amino acid sequences can also be determined using algorithms such as EMBOSS Needle (pairwise alignment; available at www.ebi.ac.uk). For example, EMBOSS Needle can be used with the BLOSUM62 matrix, a "Gap Open Penalty" of 10, a "Gap Extend Penalty" of 0.5, an "End Gap Penalty" of false, an "End Gap Open Penalty" of 10, and an "End Gap Extend Penalty" of 0.5. Generally, "% identity" is a function of the number of matching positions divided by the number of compared positions multiplied by 100. For example, if 6 out of 10 sequence positions are identical between two compared sequences after alignment, the identity is 60%. % identity is typically determined over the entire length of the query sequence being analyzed. Two molecules with the same primary amino acid or nucleic acid sequence are identical, regardless of chemical and / or biological modifications.According to the present invention, a first amino acid sequence that has at least 90% identity with a second amino acid sequence means that the first amino acid sequence has 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% identity with the second amino acid sequence.

[0029] As used herein, the term "linker" refers to any means, entity, or moiety used to connect two or more entities. A linker may be a covalent linker or a non-covalent linker. Examples of covalent linkers include linker moieties that covalently attach to one or more proteins or domains to be covalently bonded or linked. A linker may also be a non-covalent bond, such as an organometallic bond via a metal center, e.g., a platinum atom. For covalent bonds, various functional groups can be used, such as amide groups including carbonic acid derivatives, ethers, esters including organic and inorganic esters, amino, urethane, urea, etc. To provide the bond, the domains can be modified by oxidation, hydroxylation, substitution, reduction, etc. to provide sites for coupling. Methods for conjugation are well known to those of skill 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, peptide linker moieties (linker sequences). It will be understood that modifications that do not significantly impair the function of the RNA-binding domain and the effector domain are preferred.

[0030] As used herein, "linked" as used herein refers to the attachment of two or more entities to form a single entity. Conjugates encompass both peptide-small molecule conjugates and peptide-protein / peptide conjugates.

[0031] As used herein, the terms "editing," "edit," "editing," or "edited" include methods of modifying the nucleic acid sequence of a polynucleotide (e.g., a naturally occurring wild-type nucleic acid sequence or a naturally occurring mutant nucleic acid sequence by introducing an alteration into a particular genomic target). A genomic target may include a chromosomal region, a coding polynucleotide (e.g., a gene), a promoter, a non-coding polynucleotide, or any nucleic acid sequence. Changes to the nucleic acid may include deletions, additions, and other changes to the nucleic acid sequence in the genome.

[0032] As used herein, the term "base editing enzyme" refers to a fusion protein comprising a defective CRISPR / Cas nuclease linked to a deaminase polypeptide. This term is also known as a "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-strand breaks. Typically, cytosine base editing enzymes are generated by fusing a defective CRISPR / Cas nuclease to a deaminase.

[0033] As used herein, the term "deaminase" refers to an enzyme that catalyzes a deamination reaction. As used herein, the term "deamination" refers to the removal of an amine group from a molecule. In some embodiments, the deaminase is a cytidine deaminase, which catalyzes the hydrolytic deamination of cytidine or deoxycytidine to uracil or deoxyuracil, respectively. In some embodiments, the deaminase is an adenosine deaminase, which catalyzes the hydrolytic deamination of adenosine to inosine. Inosine is treated similarly to guanosine in cells, resulting in a change from A to G (or T to C).

[0034] As used herein, the term "nuclease" includes proteins (ie, enzymes) that induce cleavage of nucleic acid sequences, for example, single- or double-strand breaks in double-stranded DNA sequences.

[0035] As used herein, the term "CRISPR / Cas nuclease" has its common meaning in the art and refers to a segment of prokaryotic DNA containing clustered regularly interspaced short palindromic repeats (CRISPR) and associated nucleases encoded by Cas genes. In bacteria, the CRISPR / Cas locus encodes an RNA-guided adaptive immune system against mobile genetic elements (viruses, transposable elements, and conjugative plasmids). Three types of CRISPR systems have been identified. CRISPR clusters contain a spacer, a sequence complementary to the preceding mobile element. CRISPR clusters are transcribed and processed into mature CRISPR (clustered regularly interspaced short palindromic repeats) RNA (crRNA). The CRISPR / Cas nucleases Cas9 and Cpf1 belong to type II and type V CRISPR / Cas systems and have potent endonuclease activity that cleaves target DNA. Cas9 is guided by a transactivating small RNA (tracrRNA) that also serves as a guide for RNase III-assisted processing of the mature crRNA and pre-crRNA, which contain a unique target sequence of approximately 20 nucleotides (called the spacer). The crRNA:tracrRNA duplex guides Cas9 to the target DNA through complementary base pairing between the spacer on the crRNA and a complementary sequence on the target DNA (called the protospacer). Cas9 recognizes a three-nucleotide (NGG in S. pyogenes Cas9) protospacer adjacent motif (PAM) to specify the cleavage site (the third or fourth nucleotide upstream from the PAM).

[0036] As used herein, the term "Cas9" or "Cas9 nuclease" refers to an RNA-guided nuclease comprising the Cas9 protein or a fragment thereof (e.g., a protein comprising the active or inactive DNA cleavage domain of Cas9 and / or the gRNA-binding domain of Cas9). Cas9 nuclease may also be referred to as casn1 nuclease or CRISPR (clustered regularly interspaced short palindromic repeats)-associated nuclease. CRISPR is an adaptive immune system that provides defense against mobile genetic elements (viruses, transposable elements, and conjugative plasmids). CRISPR clusters contain a spacer, a sequence complementary to the preceding mobile element, and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems, transcoded small RNAs (tracrRNAs), endogenous ribonuclease 3 (rnc), and the Cas9 protein are required for proper processing of the pre-crRNA. The tracrRNA serves as a guide for RNase 3-assisted processing of the pre-crRNA. Subsequently, Cas9 / crRNA / tracrRNA endonucleolytically cleaves linear or circular dsDNA targets complementary to the spacer. The target strand not complementary to the crRNA is first endonucleolytically cleaved and then 3'-5' exonucleolytically trimmed. In nature, DNA binding and cleavage typically require both a protein and both RNAs. However, a single guide RNA ("sgRNA" or simply "gNRA") can be engineered 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 are incorporated herein by reference. Cas9 recognizes a short motif (PAM or protospacer adjacent motif) in the CRISPR repeat sequence, which helps distinguish between self and non-self.The sequence and structure of Cas9 nuclease are well known to those skilled in the art (e.g., "Complete genome sequence of an M1 strain of Streptococcus pyogenes." Ferretti et al., JJ, McShan WM, AjdicD. J., Savic DJ, SavicG., Lyon K., Primeaux C., Sezate S., Suvorov AN, Kenton S., Lai HS, Lin SP, Qian "CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III." Deltcheva E., Chylinski K., Sharma CM, Gonzales See Jinek M., Chao Y., Pirzada ZA, Eckert MR, 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 JA, Charpentier E. Science 337:816-821 (2012), each of which is incorporated by reference in its entirety. 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 the present disclosure.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 Corynebacterium ulcerans (NCBI Ref:NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Ref: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Ref:NC_021284.1);Prevotellaintermedia(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 torquisI(NCBI Ref:NC_018721.1);Streptococcus refers to Cas9 derived from L. 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).

[0037] As used herein, the term "defective CRISPR / Cas nuclease" refers to a CRISPR / Cas nuclease that is missing at least one nuclease domain.

[0038] As used herein, the term "nickase" has its general meaning in the art and refers to an endonuclease that cleaves only one strand of a DNA duplex. Thus, the term "Cas9 nickase" typically refers to a nickase derived from a Cas9 protein by inactivating one nuclease domain of the Cas9 protein.

[0039] As used herein, the term "guide RNA molecule" generally refers to an RNA molecule (or a collection of RNA molecules) that can bind to a Cas9 protein and target the Cas9 protein to a specific location within a target DNA. A guide RNA may contain two segments: a DNA-targeting guide segment and a protein-binding segment. The DNA-targeting segment contains a nucleotide sequence that is complementary to (or capable of hybridizing under at least stringent conditions with) a target sequence. The protein-binding segment interacts with a CRISPR protein, e.g., Cas9 or a Cas9-associated polypeptide. These two segments may be located on the same RNA molecule or on two or more separate RNA molecules. When the two segments are present on separate RNA molecules, the molecule containing the DNA-targeting guide segment may be referred to as a CRISPR RNA (crRNA), while the molecule containing the protein-binding segment is referred to as a trans-activating RNA (tracrRNA).

[0040] As used herein, the term "target sequence" or "target" refers to a nucleic acid containing a target nucleic acid sequence. A target nucleic acid can be single-stranded or double-stranded, and is often double-stranded DNA. As used herein, "target nucleic acid sequence," "target sequence," or "target region" refers to a specific sequence or its complementary strand that is desired to bind using the CRISPR system disclosed herein. As used herein, the term "target nucleic acid strand" refers to the strand of the target nucleic acid that is subject to base pairing with the guide RNA disclosed herein. That is, the strand of the 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 that is not complementary to the guide sequence is referred to as the "non-complementary strand." In the case of a double-stranded target nucleic acid (e.g., DNA), as long as there is an appropriate PAM site, each strand can be a "target nucleic acid strand" used to design crRNAs and guide RNAs and practice the methods of the present invention.

[0041] As used herein, the term "ribonucleoprotein complex" or "ribonucleoprotein particle" refers to a complex or particle comprising a nucleoprotein and a ribonucleic acid. As provided herein, "nucleoprotein" refers to a protein capable of binding to nucleic acid (e.g., RNA, DNA). When a nucleoprotein binds to a ribonucleic acid, the nucleoprotein is referred to as a "ribonucleoprotein." The interaction between a ribonucleoprotein and a ribonucleic acid can be direct, e.g., by a covalent bond, or indirect, e.g., by a non-covalent bond (e.g., electrostatic interaction (e.g., ionic bond, hydrogen bond, halogen bond), van der Waals interaction (e.g., dipole-dipole, dipole-induced dipole, London dispersion), ring stacking (pi effect), hydrophobic interaction, etc.).

[0042] As used herein, the term "CD33" has its general meaning in the art and refers to the transmembrane receptor encoded by the CD33 gene. This 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 of CD33 is set forth as SEQ ID NO:1.

[0043] [ka]

[0044] As used herein, the term "mutation" has its general meaning in the art and refers to a substitution, deletion, or insertion. The term "substitution" means that a specific amino acid residue at a specific position is removed and another amino acid residue is inserted at the same position. The term "deletion" means that a specific amino acid residue is removed. The term "insertion" means that one or more amino acid residues are inserted before or after a specific amino acid residue. As used herein, the term "point mutation" refers to a substitution that replaces one of the nucleotides in a target polynucleotide.

[0045] As used herein, the term "mutagenesis" refers to the introduction of mutations into a polynucleotide sequence.

[0046] As used herein, the term "variant" refers to a first composition (e.g., a first molecule) relative to a second composition (e.g., a second molecule, also referred to as a "parent" molecule). A variant molecule may be derived from, isolated from, based on, or homologous to a parent molecule. A variant molecule may have full sequence identity with the original parent molecule, or alternatively, may have less than 100% sequence identity with the parent molecule. For example, a sequence variant may be a second sequence that is at least 50;51;52;53;54;55;56;57;58;59;60;61;62;63;64;65;66;67;68;69;70;71;72;73;74;75;76;77;78;79;80;81;82;83;84;85;86;87;88;89;90;91;92;93;94;95;96;97;98;99;100% sequence identical to the original sequence.

[0047] As used herein, the term "hematopoietic stem cell" or "HSC" refers to a blood cell that has the ability to self-renew and differentiate into blood cell precursors. These precursor cells are immature blood cells that cannot self-renew and must differentiate into mature blood cells. Hematopoietic stem and progenitor cells exhibit many phenotypes, such as Lin-CD34+CD38-CD90+CD45RA-, Lin-CD34+CD38-CD90-CD45RA-, Lin-CD34+CD38+IL-3aloCD45RA-, and Lin-CD34+CD38+CD10+ (Daley et al., Focus 18:62-67, 1996; Pimentel, E., Ed., Handbook of Growth Factors Vol. III: Hematopoietic Growth Factors and Cytokines, pp. 1-2, CRC Press, Boca Raton, Fla., 1994). Within the bone marrow microenvironment, stem cells self-renew throughout life, maintaining a continuous production of hematopoietic stem cells that give rise to all mature blood cells. In some embodiments, hematopoietic progenitor cells or hematopoietic stem cells are isolated from peripheral blood cells.

[0048] 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 the progeny 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 the cell (or the progeny of the cell) is reintroduced into the organism from which it was isolated. As used herein, the term "isolated population," with respect to an isolated cell population, as used herein, refers to a cell population that has been removed and separated from a mixed or heterogeneous cell population. In some embodiments, an isolated population is a substantially pure cell population compared to the heterogeneous population from which the cells are isolated or enriched.

[0049] As used herein, the phrase "substantially pure cell population" refers to a cell population that contains at least 90, 91, 92, 03, 94, 95, 96, 97, 98 or 99% of the desired cell type.

[0050] As used herein, the term "enrichment" includes any isolation or sorting process that increases the relative abundance of one or more desired cell types in a cell population.

[0051] As used herein, the terms "alpha globin" or "α-globin" have their common meaning in the art and refer to the proteins encoded in humans by the HBA1 and HBA2 genes. The human alpha globin gene cluster is located on chromosome 16, spans approximately 30 kb, and contains seven loci: 5'-zeta-pseudozeta-mu-pseudoalpha-1-alpha-2-alpha-1-theta-3'. The coding sequences for alpha-2 (HBA2) and alpha-1 (HBA1) are identical. These genes differ slightly in the 5' untranslated region and introns, but significantly in the 3' untranslated region. The ENSEMBL IDs (i.e., gene identifier numbers from the Ensembl genome browser database) for HBA1 and HBA2 are ENSG00000206172 and ENSG00000188536, respectively.

[0052] As used herein, the term "beta globin" or "β-globin" has its common meaning in the art and refers to the globin protein that, together with alpha globin (HB), constitutes the most common form of hemoglobin (Hb) in adult humans. Normal adult human Hb is a heterotetramer consisting of two α chains and two β chains. HBB is encoded by the HBB gene on human chromosome 11. HBB is 146 amino acids in length and has a molecular weight of 15,867 Da.

[0053] As used herein, the terms "gamma globin" or "γ-globin" have their common meaning in the art and refer to the proteins encoded in humans by the HBG1 and HBG2 genes. The HBG1 and HBG2 genes are normally expressed in the fetal liver, spleen, and bone marrow. Two γ-globin chains and two α-globin chains make up fetal hemoglobin (HbF), which is normally replaced by adult hemoglobin (HbA) in the year following birth. The ENSEMBL IDs (i.e., gene identifier numbers from the Ensembl genome browser database) for HBG1 and HBG2 are ENSG00000213934 and ENSG00000196565, respectively.

[0054] As used herein, the term "β-hemoglobinopathy" has its general meaning in the art and refers to any defect in the structure or function of any hemoglobin in an individual, including defects in the primary, secondary, tertiary, or quaternary structure of hemoglobin caused by any mutation, e.g., a deletion or substitution mutation in the coding region of the HBB gene, or a mutation or deletion in the promoter or enhancer of the gene that results in a reduced amount of hemoglobin produced compared to normal or standard conditions.

[0055] As used herein, the term "sickle cell disease" has its common meaning in the art and refers to a group of autosomal recessive blood disorders resulting from mutations in globin genes and characterized by red blood cells exhibiting an abnormal, rigid, sickle-like shape. The disease is defined by the presence of the βS-globin gene, which encodes a variant of the β-globin chain in which the amino acid glutamate at position 6 of the peptide is replaced by valine. Incorporation of βS-globin into the Hb tetramer (HbS, sickle Hb) leads to polymerization of Hb and results in the clinical phenotype. The term includes sickle cell anemia (HbSS), sickle hemoglobin C disease (HbSC), sickle beta-plus thalassemia (HbS / β+), or sickle beta-zero thalassemia (HbS / β0).

[0056] As used herein, the term "β-thalassemia" refers to a hemoglobinopathy in which an altered ratio of α-globin to β-like globin polypeptide chains results in a deficiency in the production of normal hemoglobin tetrameric protein and the precipitation of free, unpaired α-globin chains.

[0057] As used herein, the term "treatment" or "treating" refers to both prophylactic or preventive treatment and curative or disease-modifying treatment, including treatment of patients at risk of or suspected of having a disease and patients suffering from or diagnosed with a disease or condition, including suppression of clinical recurrence. Treatment may be administered to a subject with a medical disorder or who may ultimately acquire a disease to prevent, cure, delay the onset, reduce the severity, or ameliorate one or more symptoms of the disorder or recurrent disorder, or to extend the subject's survival beyond that expected in the absence of such treatment. A "therapeutic regimen" refers to a pattern of disease treatment, e.g., the dosing pattern used during treatment. 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 portion of a therapeutic regimen) used for the initial treatment of a disease. The general purpose of an induction regimen is to provide a high level of drug to the patient during the initial period of the treatment regimen. An induction regimen may utilize (in part or in whole) a "loading regimen." A "loading regimen" may involve administering a larger amount of drug than a physician would use during a maintenance regimen, administering a drug more frequently than a physician would use during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a treatment regimen (or portion of a treatment regimen) used to maintain a patient during disease treatment, e.g., to keep a patient in remission for an extended period of time (months or years). A maintenance regimen may utilize continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., discontinuation treatment, intermittent treatment, treatment upon relapse, or treatment upon reaching certain predetermined criteria (e.g., pain, disease symptoms, etc.)).

[0058] As used herein, the term "therapeutically effective amount" refers to a sufficient quantity of a cell population to treat a disease at a reasonable benefit / risk ratio applicable to any medical treatment. It will be understood that the total dosage of the gene-editing platform will be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend on various factors, including the patient's age, weight, general health, sex, and diet, the timing and route of administration, the duration of treatment, drugs used in conjunction with or concurrently with the cell population, and similar factors well known in the medical field. In some embodiments, the cells are formulated by first harvesting the cells from the culture medium and then washing and concentrating the cells in a medium and container system (a "pharmaceutically acceptable" carrier) suitable for administration of a therapeutically effective amount. A suitable infusion medium can be any isotonic medium formulation, typically normal saline, Normosol® (Abbott), or Plasma-Lyte® (Baxter), although 5% dextrose in water or lactated Ringer's solution can also be utilized. The infusion medium may be supplemented with human serum albumin. The therapeutically effective amount of cells in the composition will depend on the relative representation of cells with the desired specificity, the age and weight of the recipient, and the severity of the condition being targeted. This number of cells is approximately 10 3 pieces / kg, preferably 5 x 10 3 Low at 10 pieces / kg 7 pcs / kg, preferably 10 8 The number of cells can be as high as 100 cells / kg. The number of cells will depend on the intended end use of the composition and the type of cells contained therein. Typically, the minimum dose is 2 million cells per kg. Usually, 2 million to 20 million cells are injected into a subject. The desired purity can be achieved by incorporating a sorting step. For the applications provided herein, the cells generally have a volume of 1 liter or less, and may be 500 ml or less, or even 250 ml or 100 ml or less. A clinically relevant number of cells can be distributed over multiple infusions that cumulatively equal or exceed the desired total cell volume.

[0059] method A first object of the present invention relates to a method for editing a eukaryotic cell, comprising contacting the eukaryotic cell with a gene editing platform comprising (a) at least one base editing enzyme and (b) a plurality of guide RNA molecules designed to guide the base editing enzyme to a plurality of target sequences, wherein at least one guide RNA molecule is designed to guide the base editing enzyme to a target sequence in a gene encoding CD33, thereby knocking down expression of CD33.

[0060] eukaryotic cell In some embodiments, the eukaryotic cells are 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 cells are hematopoietic stem cells.

[0061] In some embodiments, hematopoietic progenitor or stem cells are isolated from peripheral blood cells. As used herein, the term "peripheral blood cells" refers to the cellular components of blood, including red blood cells, white blood cells, and platelets, which are found in the circulating pool of blood.

[0062] In some embodiments, the eukaryotic cells are bone marrow-derived stem cells. As used herein, the term "bone marrow-derived stem cells" refers to stem cells found in bone marrow. Stem cells may be present in bone marrow either as adherent stromal cell types with pluripotent potential or as cells expressing the CD34 or CD45 cell surface proteins, which identify hematopoietic stem cells capable of differentiating into blood cells.

[0063] Typically, eukaryotic cells arise from the mobilization of stem cells.

[0064] As used herein, the term "mobilization" or "stem cell mobilization" refers to the process by which stem cells are recruited from their tissue or organ into the peripheral blood after treatment with a mobilizing agent. This process mimics the physiological promotion of stem cell release from tissues or organs in response to stress signals during injury and inflammation. The mechanism of the mobilization process depends on the type of mobilizing agent administered. Some mobilizing agents act as agonists or antagonists that prevent stem cells from adhering to cells or tissues in their microenvironment. Other mobilizing agents induce the release of proteases that cleave adhesion molecules or support structures between stem cells and their attachment sites. As used herein, the term "mobilizing agent" refers to a wide range of molecules that promote the mobilization of stem cells from their tissues or organs, such as bone marrow (CD34+ stem cells) and spleen (e.g., Hox11+ stem cells), into the peripheral blood. Mobilizing agents include chemotherapeutic agents, such as cyclophosphamide and cisplatin; cytokines and chemokines, such as granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), stem cell factor (SCF), Fms-related tyrosine kinase 3 (flt-3) ligand, stromal cell-derived factor 1 (SDF-1); agonists of chemokine (CC motif) receptor 1 (CCR1), such as chemokine (CC motif) ligand 3 (CCL3, macrophage-derived factor 1); chemokine (C-X-C motif) receptor 1 (CXCR1) and 2 (CXCR2) agonists, such as chemokine (C-X-C motif) ligand 2 (CXCL2) (also known as growth-associated oncogene protein-β (Gro-β)) and CXCL8 (also known as interleukin-8 (IL-8)); CXCR4 agonists, such as CTCE-02142 and Met-SDF-1; Very Late Antigen (VLA)-4 inhibitors; CXCR4 antagonists, such as TG-0054, plerixafor (also known as AMD3100), and AMD3465, or any combination of the above agents. Mobilizing agents increase the number of stem cells in the peripheral blood, thereby making them more accessible.

[0065] Base editing enzymes In some embodiments, the base editing enzyme of the present invention comprises a defective CRISPR / Cas nuclease. The sequence recognition mechanism is the same as that of a non-defective CRISPR / Cas nuclease. Typically, the defective CRISPR / Cas nuclease of the present invention comprises at least one RNA-binding domain. The RNA-binding domain interacts with a guide RNA molecule, as defined below. On the other hand, the defective CRISPR / Cas nuclease of the present invention is a modified version that does not have nuclease activity. Therefore, the defective CRISPR / Cas nuclease specifically recognizes the guide RNA molecule, thereby guiding the base editing enzyme to the target DNA sequence.

[0066] In some embodiments, the truncated CRISPR / Cas nuclease can be modified to improve nucleic acid binding affinity and / or specificity, alter enzymatic activity, and / or alter another property of the protein. In some embodiments, the nuclease domain 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.

[0067] In some embodiments, the CRISPR / Cas nuclease comprises a mutant CRISPR / Cas nuclease, i.e., a protein with one or more point mutations, insertions, deletions, truncations, a fusion protein, or a combination thereof. In some embodiments, the mutant has 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 that is at least 50% identical to the wild-type amino acid sequence of the CRISPR / Cas nuclease. Various CRISPR / Cas nucleases can be used in the present invention. Non-limiting examples of suitable CRISPR / Cas nucleases include Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9, Cas10, Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Csel (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csz1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cu1966. See, e.g., WO 2014144761, 2014144592, 2013176772, US 20140273226, and 20140273233, the contents of which are incorporated by reference in their entireties.

[0068] In some embodiments, the CRISPR / Cas nuclease is derived from a Type II CRISPR-Cas system, hi some embodiments, the CRISPR / Cas nuclease is derived from a Cas9 protein. Cas9 proteins are found in Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus species, 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, Polaromonas species, Crocosphaera watsonii, Cyanothece species, Microcystis aeruginosa, Synechococcus species, Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter species, Nitrosococcushalophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc species, Arthrospira maxima, Arthrospiraplatensis, Arthrospira species, Lyngbya species, Microcoleus chthonoplastes, Oscillatoria species, Petrotoga mobilis, Thermosipho africanus or Acaryochlorismarina.

[0069] In some embodiments, the CRISPR / Cas nuclease is a mutant of a wild-type CRISPR / Cas nuclease (e.g., Cas9) or a fragment thereof. In some embodiments, the CRISPR / Cas nuclease is a mutant Cas9 protein from S. pyogenes.

[0070] Methods for generating a Cas9 protein (or a fragment thereof) with 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 which are incorporated herein by reference). For example, the DNA cleavage domain of Cas9 is known to contain two subdomains: an HNH nuclease subdomain and a RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, while the RuvC1 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)).

[0071] In some embodiments, the CRISPR / Cas nuclease of the invention is a nickase, particularly a Cas9 nickase, i.e., Cas9 from S. pyogenes, having one mutation selected from the group consisting of D10A and H840A. In some embodiments, the nickase of the invention comprises the amino acid sequence set forth in SEQ ID NO:2 or SEQ ID NO:3.

[0072] [ka]

[0073] [ka]

[0074] In some embodiments, Cas9 variants with mutations other than D10A or H840A are used. These variants result, for example, in nuclease-inactivated Cas9 (dCas9). Such mutations include, for example, other amino acid substitutions at D10 and H840 or other substitutions within the nuclease domain of Cas9 (e.g., substitutions in the HNH nuclease subdomain and / or the RuvC1 subdomain). In some embodiments, variants of dCas9 are provided that are at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to SEQ ID NO: 2 or 3. In some embodiments, variants of dCas9 are provided that have amino acid sequences that are about 5 amino acids, about 10 amino acids, about 15 amino acids, about 20 amino acids, about 25 amino acids, about 30 amino acids, about 40 amino acids, about 50 amino acids, about 75 amino acids, about 100 amino acids, or more, shorter or longer than SEQ ID NO:2 or 3.

[0075] According to the present invention, the second component of the base editing enzymes disclosed herein comprises a non-nuclease DNA-modifying enzyme that is a deaminase.

[0076] In some embodiments, the deaminase is an adenosine deaminase. In some embodiments, the deaminase is an ADAT family deaminase. In some embodiments, the adenosine deaminase mutant is a TadA deaminase. In some embodiments, the adenosine deaminase mutant is Staphylococcus aureus TadA, Bacillus subtilis TadA, Salmonella typhimurium TadA, Shewanella putrefaciens TadA, Haemophilus influenzae F3031 TadA, Caulobacter crescentus TadA, or Geobacter sulfurreducens TadA, or a fragment thereof. In some embodiments, the TadA deaminase is E. coli TadA deaminase (ecTadA). In some embodiments, the TadA deaminase is a truncated E. coli TadA deaminase. For example, a truncated ecTadA may lack one or more N-terminal amino acids relative to full-length ecTadA. In some embodiments, a truncated ecTadA may lack 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 N-terminal amino acid residues relative to full-length ecTadA. In some embodiments, a truncated ecTadA may lack 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 C-terminal amino acid residues relative to full-length ecTadA. In some embodiments, the TadA deaminase is a TadA deaminase. * 7.10. In some embodiments, the TadA deaminase is TadA *8 mutant. For example, deaminases are described in International PCT Application Nos. WO 2018 / 027078, 2017 / 070632, 2020 / 168132, and 2021 / 050571, each of which is incorporated herein by reference in its entirety. Also, Komor, AC, et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016); Gaudelli, NM, et al., “Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage” Nature 551, 464-471 (2017); Komor, AC, et al., “Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity” Science Advances 3:eaao4774 (2017) and Rees, HA, et al., “Base editing: precision chemistry on the genome and transcriptome of living cells.” Nat Rev Genet. 2018 Dec;19(12):770-788. doi: See also 10.1038 / s41576-018-0059-1, the contents of which are incorporated herein by reference in their entireties. An exemplary amino acid sequence for wild-type TadA (wt) adenosine deaminase is set forth as SEQ ID NO:4. In some embodiments, the amino acid sequence of the adenosine deaminase comprises at least 90% sequence identity with SEQ ID NO:4. In some embodiments, the amino acid sequence of the adenosine deaminase comprises a modification at position 82 numbered in SEQ ID NO:4.In some embodiments, the amino acid sequence of the adenosine deaminase comprises a V82S modification, where position 82 is as numbered in SEQ ID NO:4. In some embodiments, the amino acid sequence of the adenosine deaminase comprises a modification at position 166, as numbered in SEQ ID NO:4. In some embodiments, the amino acid sequence of the adenosine deaminase comprises a T166R modification, where position 166 is as numbered in SEQ ID NO:4. In some embodiments, the amino acid sequence of the adenosine deaminase comprises modifications at positions 82 and 166, as numbered in SEQ ID NO:4. In some embodiments, the amino acid sequence of the adenosine deaminase comprises a V82S and a T166R modification, where positions 82 and 166 are as numbered in SEQ ID NO:4. In some embodiments, the adenosine deaminase variant further comprises one or more of the following modifications: Y147T, Y147R, Q154S, Y123H, and Q154R. In some embodiments, the adenosine deaminase variant further comprises one or more of the following modifications: Y147T + Q154R; Y147T + Q154S; Y147R + Q154S; V82S + Q154S; V82S + Y147R; V82S + Q154R; V82S + Y123H; I76Y + V82S; V82S + Y123H + Y147T; V82S + Y123H + Y147R; V82S + Y123H In some embodiments, the adenosine deaminase mutant comprises a combination of modifications selected from the group consisting of: H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R and I76Y+V82S+Y123H+Y147R+Q154R. In some embodiments, the adenosine deaminase mutant is TadA. * 8.1, TadA * 8.2, TadA * 8.3, TadA * 8.4, TadA * 8.5, TadA * 8.6, TadA * 8.7, TadA * 8.8, TadA * 8.9, TadA *8.10, TadA * 8.11, TadA * 8.12, TadA * 8.13, TadA * 8.14, TadA * 8.15, TadA * 8.16, TadA * 8.17, TadA * 8.18, TadA * 8.19, TadA * 8.20, TadA * 8.21, TadA * 8.22, TadA * 8.23 or TadA * 8.24. In some embodiments, the adenosine deaminase is provided as a single (e.g., provided as a monomer) TadA mutant described above. In some embodiments, the adenosine deaminase is provided as a heterodimer of wild-type TadA (TadA(wt)) linked to a TadA mutant described above.

[0077] [ka]

[0078] In some embodiments, the deaminase is fused to the N-terminus of the defective CRISPR / Cas nuclease. In some embodiments, the deaminase is fused to the C-terminus of the defective CRISPR / Cas nuclease. In some embodiments, the defective CRISPR / Cas nuclease and the deaminase are fused via a linker. In some embodiments, the linker is a (GGGGS)n (SEQ ID NO: 5), (G)n, (EAAAK)n (SEQ ID NO: 6), (GGS)n, or SGSETPGTSESATPES (SEQ ID NO: 7) motif (see, e.g., Guilinger JP, Thompson DB, Liu DR). Further 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. No. 6,219,999, the entire contents of which are incorporated herein by reference.

[0079] In some embodiments, the fusion protein may include additional features. Other exemplary features that may be present are a localization sequence, such as a nuclear localization sequence (NLS), a cytoplasmic localization sequence, a transport sequence, such as a nuclear export sequence, or other localization sequence, and a sequence tag useful for solubilizing, purifying, or detecting the fusion protein. Suitable localization signal sequences and protein tag sequences are provided herein and include, but are not limited to, a biotin carboxylase carrier protein (BCCP) tag, a myc tag, a calmodulin tag, a FLAG tag, a hemagglutinin (HA) tag, a polyhistidine tag (also known as a histidine tag or His tag), a maltose-binding protein (MBP) tag, a nus tag, a glutathione-S-transferase (GST) tag, a green fluorescent protein (GFP) tag, a thioredoxin tag, an S tag, a soft tag (e.g., soft tag 1, soft tag 3), a strep tag, a biotin ligase tag, a FlAsH tag, a V5 tag, and an SBP tag. Further suitable features will be apparent to those skilled in the art.

[0080] A variety of base-editing enzymes are known in the art (see, e.g., Improving cytidine and adenine base-editing enzymes by expression optimization and ancestral reconstruction. Nat Biotechnol. 2018 May 29), typically including those listed in Table A.

[0081] Table A: Some exemplary base editing enzymes [Table 1]

[0082] Guide RNA molecules

[0013] A second component of the gene editing platform disclosed herein comprises a plurality of guide RNA molecules suitable for directing a base editing enzyme to a plurality of target sequences. In particular, the gene editing platform disclosed herein comprises i) at least one guide RNA molecule designed to direct a base editing enzyme to a target sequence of interest, and ii) at least one guide RNA molecule designed to direct the base editing enzyme to a target sequence in a gene encoding CD33, thereby knocking down expression of CD33. Thus, in some embodiments, the gene editing platform disclosed herein comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 20 guide RNA molecules.

[0083] In particular, the guide RNA molecule comprises a region that is complementary to and capable of hybridizing to a preselected target site of interest. In some embodiments, the guide sequence may comprise from about 10 nucleotides to more than about 25 nucleotides. For example, the region that base pairs between the guide sequence and the corresponding target site sequence may 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, e.g., 20 nucleotides in length.

[0084] Typically, software programs are used to identify candidate CRISPR target sequences on both strands of a DNA nucleic acid molecule containing an HBG gene based on the length of the desired guide sequence and the CRISPR motif sequence (PAM) for a designated CRISPR enzyme. One requirement for selecting an appropriate target nucleic acid is that the target nucleic acid have a 3' PAM site / sequence. Each target sequence and its corresponding PAM site / sequence are referred to herein as a Cas target site. Type II CRISPR systems are among the best-characterized and require only the Cas9 protein and a guide RNA complementary to the target sequence to affect target cleavage. For example, target sites for S. pyogenes Cas9, whose PAM sequence is NGG, can be identified by searching for 5'-Nx-NGG-3' on both the input sequence and the reverse complement of the input. Because multiple occurrences of a DNA target site in a genome can lead to nonspecific genome editing, after identifying all potential sites, the program filters sequences based on the number of times they appear in the associated reference genome. For CRISPR enzymes whose sequence specificity is determined by a "seed" sequence, e.g., 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 occurrence of the seed:PAM sequence in the relevant genome. The user may be able to select the length of the seed sequence. The user may also be able to specify the number of occurrences of the seed:PAM sequence in the genome for filtering purposes. The default is screening for unique sequences. The filtering level is altered by changing both the length of the seed sequence and the occurrence of the sequence in the genome. Additionally or alternatively, the program may provide a guide sequence complementary to the reported target sequence by providing the reverse complement of the identified target sequence. Further details of methods and algorithms for optimizing sequence selection can be found in US 61 / 836,080.No. 6,239,999, which is incorporated herein by reference.

[0085] In some embodiments, the gene editing platform of the invention targets the CD33 gene and comprises at least one guide RNA molecule designed to disrupt the start codon when coupled with a CBE, or to disrupt the splice donor site of exons 1-2 when coupled with either a CBE or an ABE, and / or to disrupt the splice acceptor site of exons 1-2 when coupled with an ABE.

[0086] Typically, the guide RNA molecule targets a nucleic acid sequence selected from Table 1.

[0087] In some embodiments, the gene editing platform comprises at least one guide RNA molecule that targets CD33 and that, when coupled to either a CBE or an ABE, disrupts the splice donor site of exons 1-2. In some embodiments, the guide RNA molecule targets the nucleic acid sequence set forth in SEQ ID NO: 13.

[0088] In some embodiments, the gene editing platform comprises (a) at least one adenine base editor (ABE); and (b) a plurality of guide RNA molecules designed to guide the base editing enzyme to a plurality of target sequences, wherein at least one guide RNA molecule is designed to direct the ABE to the target sequence set forth in SEQ ID NO: 13.

[0089] The guide RNA molecules of the present invention can be prepared 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 (200 mers or longer in length) using TC-RNA chemistry (see, e.g., U.S. Pat. No. 8,202,983) allows for the production of RNAs with specialized characteristics superior to those possible with the four basic ribonucleotides (A, C, G, and U). In particular, the RNA molecules of the present invention can be prepared by recombinant techniques using host cell systems or in vitro translation-transcription systems known in the art. Details of such systems and techniques can be found, for example, in WO 2014144761, WO 2014144592, WO 2013176772, U.S. Pat. No. 20140273226, and WO 20140273233, the contents of which are incorporated herein by reference in their entireties.

[0090] In some embodiments, the guide RNA molecule may contain one or more modifications. Such modifications may include the inclusion of at least one non-naturally occurring or modified nucleotide or its analog. Modified nucleotides may be modified at the ribose, phosphate, and / or base moiety. Modified nucleotides may include 2'-O-methyl, 2'-deoxy, or 2'-fluoro analogs. The nucleic acid backbone may be modified; for example, a phosphorothioate backbone may be used. Locked nucleic acids (LNA) or bridged nucleic acids (BNA) may also be used. Further examples of modified bases include, but are not limited to, 2-aminopurine, 5-bromo-uridine, pseudouridine, inosine, and 7-methylguanosine.

[0091] use In some embodiments, the gene editing platform is used to modify a target polynucleotide sequence of interest in a eukaryotic cell for any purpose. In some embodiments, the target polynucleotide sequence of interest in a eukaryotic cell is modified to generate a mutant cell, resulting in a genotype different from its original genotype. In some embodiments, the target polynucleotide sequence of interest in a eukaryotic cell is modified 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 a eukaryotic cell is modified to induce a genetic mutation (e.g., to disrupt the function of a gene or genetic element). In some embodiments, the modification may be a homozygous modification or a heterozygous modification. In some embodiments, the modification may be an insertion, a deletion, or a combination thereof. As will be understood by those skilled in the art, an insertion / deletion in the coding region of a genomic sequence will result in a frameshift mutation or a premature stop codon. In some embodiments, the modification may be a point mutation.

[0092] In some embodiments, the gene editing platform of the present invention is used to generate knockouts of target polynucleotide sequences. Knocking out selected polynucleotide sequences can be useful for a number of applications, for example, knocking out target polynucleotide sequences of interest in eukaryotic cell clones in vitro for research purposes and knocking out target polynucleotide sequences ex vivo to treat or prevent disorders associated with increased expression of the target polynucleotide sequence. As used herein, the term "knockout" includes deleting all or part of a target polynucleotide sequence in a manner that attenuates the function of the target polynucleotide sequence.

[0093] In some embodiments, the modification may change a 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. This correction includes, but is not limited to, inserting a nucleotide sequence that was deleted from the target polynucleotide sequence by deletion, deleting a nucleotide sequence from the target polynucleotide sequence by insertion mutation, and replacing an incorrect nucleotide sequence with a correct nucleotide sequence.

[0094] In some embodiments, the modification reduces or increases expression of a target polynucleotide sequence of interest.

[0095] In some embodiments, the gene editing platform is used to increase fetal hemoglobin content in eukaryotic cells.

[0096] In some embodiments, a gene editing platform is used to edit the HBG1 or HBG2 promoter and subsequently increase expression of γ-globin.

[0097] In some embodiments, the gene editing platform is suitable for introducing several mutations into the HBG1 or HBG2 promoter such that at least one transcription activator binding site is introduced into the HBG1 or HBG2 promoter. In some embodiments, the gene editing platform is particularly suitable for introducing new transcription activator binding sites for KLF1, TAL1, or GATA1. In some embodiments, the gene editing platform disclosed herein introduces a -198T>C mutation into the HBG1 or HBG2 promoter to newly allow binding of the KLF1 activator to the promoter. In some embodiments, the gene editing platform disclosed herein introduces a -175T>C mutation into the HBG1 or HBG2 promoter to newly allow binding of the TAL1 activator to the promoter. In some embodiments, the gene editing platform disclosed herein introduces a -113A>G mutation into the HBG1 or HBG2 promoter to newly allow binding of the GATA1 activator to the promoter. In some embodiments, the gene editing platforms disclosed herein are particularly suitable for editing the -200 region in the HBG1 or HBG2 promoter to disrupt a binding site for the LRF repressor. In some embodiments, the gene editing platforms disclosed herein introduce at least one mutation selected from the group consisting of -201C>T, -200C>T, -197C>T, -196C>T, -195C>T, and -194C>T in the HBG1 or HBG2 promoter to disrupt a binding site for the LRF repressor. In some embodiments, the gene editing disclosed herein is particularly suitable for editing the -115 region in the HBG1 or HBG2 promoter to disrupt a binding site for the BCL11A repressor.In some embodiments, the gene editing platform disclosed herein introduces at least one mutation selected from the group consisting of -114C>T, -113C>T, -115C>T, and -116C>T in the HBG1 or HBG2 promoter to disrupt a binding site for the BCL11A repressor.

[0098] In some embodiments, the gene editing platform is used to edit the +55 kb region of the erythroid-specific BCL11A enhancer, thereby disrupting the ATF4 binding site in said region to repress expression of BCL11A and subsequently increase expression of γ-globin.

[0099] In some embodiments, the gene editing platform is used to repress the expression of α-globin.

[0100] In some embodiments, the gene editing platform is used to edit the MCS-R2 region present in the locus control region of the HBA1 and HBA2 genes, thereby editing the MCS-R2 region and subsequently repressing α-globin expression in the eukaryotic cell. In some embodiments, the gene editing platform is suitable for introducing several mutations into the MCS-R2 region to disrupt at least one transcription activator binding site in the region. In some embodiments, the gene editing platform is particularly suitable for disrupting at least one transcription activator binding site for GATA1 or NF-E2 in the MCS-R2 region.

[0101] In some embodiments, the gene editing platform is used to restore normal expression of β-globin in eukaryotic cells that have a CD39(CAG>TAG) mutation. In some embodiments, the gene editing platform includes at least one guide RNA molecule to direct an adenine base editor to at least one target sequence that includes the CD39(CAG>TAG) mutation, thereby restoring production of β-globin in the eukaryotic cell.

[0102] In some embodiments, the gene editing platform is used to restore normal expression of β-globin in eukaryotic cells that have an IVS2-1(G>A) mutation. In some embodiments, the gene editing platform includes at least one guide RNA molecule to direct an adenine base editor to at least one target sequence that includes the IVS2-1(G>A) mutation, thereby restoring production of β-globin in the eukaryotic cell.

[0103] In some embodiments, the gene editing platform is used to restore normal expression of β-globin in eukaryotic cells that have an IVS1-110(G>A) mutation. In some embodiments, the gene editing platform includes at least one guide RNA molecule to direct an adenine base editor to at least one target sequence that includes the IVS1-110(G>A) mutation, thereby restoring production of β-globin in the eukaryotic cell.

[0104] vector In some embodiments, various components of the gene editing platform of the present invention are provided to eukaryotic cells through expression from one or more expression vectors. For example, nucleic acids encoding guide RNA molecules or base editing enzymes can be cloned into one or more vectors for their introduction into eukaryotic cells. The vectors are typically prokaryotic vectors, such as plasmids, shuttle vectors, or insect vectors, for storing or manipulating nucleic acids encoding the guide RNA molecules or base editing enzymes disclosed herein. Preferably, the nucleic acids are isolated and / or purified. Thus, the present invention provides recombinant constructs or vectors comprising sequences encoding one or more of the above-described guide RNA molecules or base editing enzymes. Examples of such constructs include vectors, such as plasmids or viral vectors, into which a nucleic acid sequence of the present invention has been inserted in a forward or reverse orientation. In some embodiments, the constructs further comprise regulatory sequences. "Regulatory sequences" include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include those directing constitutive expression of a nucleotide sequence and inducible regulatory sequences. The design of an expression vector may depend on factors such as the choice of eukaryotic cells to be transformed, transfected, or infected, the desired expression level, etc. Many suitable vectors and promoters are known to those skilled in the art and are commercially available. Cloning and expression vectors suitable for use in eukaryotic hosts are also described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press). Vectors may be capable of autonomous replication or integration into host DNA. Vectors may also contain sequences suitable for amplifying expression.In addition, expression vectors preferably contain 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 culture, or tetracycline or ampicillin resistance in E. coli. Any of the techniques known in the art can be used to introduce foreign nucleotide sequences into host cells, including 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 host cells.

[0105] In some embodiments, various components of the gene editing platform of the present invention are provided to a cell population through the use of an RNA-encoding system. For example, a base editing system can be provided to a cell population through the use of chemically modified mRNA-encoding adenine or cytidine base editors in conjunction with modified guide RNAs, such as those described in Jiang, T., Henderson, JM, 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 systems encoding base editing enzymes (e.g., ABEs) are prepared by introducing various chemical modifications into both the mRNA encoding the base editing enzyme and the guide RNA. In particular, the modifications consist of uridine-depleted mRNAs modified with 5-methoxyuridine, where synonymous codons are introduced to delete as many uridines as possible without changing the coding sequence, and all remaining uridines can be replaced with 5-methoxyuridine. The optimized base editing system exhibits higher editing efficiency at some genomic sites compared to DNA-encoding systems. The modified mRNA and guide RNA can also be encapsulated in lipid nanoparticles (LNPs) to enable LNP-mediated delivery.

[0106] In some embodiments, various components of the gene editing platform of the present invention are provided to a cell population through the use of ribonucleoprotein (RNP) complexes. For example, a base editing enzyme can be pre-complexed with one or more guide RNA molecules to form a ribonucleoprotein (RNP) complex. In this manner, the RNP complex can be introduced into eukaryotic cells. The introduction of the RNP complex can be timed. Cells can be synchronized with other cells in the G1, S, and / or M phases of the cell cycle. RNP delivery avoids many of the pitfalls associated with mRNA, DNA, or viral delivery. Typically, an RNP complex is produced by simply mixing a protein (i.e., a base editing enzyme) with one or more guide RNA molecules in an appropriate buffer. This mixture is incubated at room temperature for 5-10 minutes before electroporation. Electroporation is a delivery technique in which an electric field is applied to one or more cells to increase the permeability of the cell membrane. In some embodiments, genome editing efficiency can be improved by adding a transfection enhancer oligonucleotide.

[0107] In some embodiments, multiple rounds of sequential transfection are performed to achieve a desired level of mutagenesis in the cells.

[0108] Concentration method A further object of the present invention relates to a method for producing a substantially pure population of edited eukaryotic cells, comprising the steps of: i) editing a population of eukaryotic cells by the editing method disclosed herein; and ii) enriching the population of edited eukaryotic cells that are negative for CD33.

[0109] According to the present invention, CD33 is indeed used as a negative marker to enrich for edited eukaryotic cells. Furthermore, the CD33 of edited cells - Selection leads to higher base editing efficiency and removes poorly edited cells that may compete with edited cells for engraftment in the bone marrow.

[0110] Enrichment of cells can be achieved by any means known to those skilled in the art. In some embodiments, methods for enriching cells include flow cytometry, cell sorting, magnetically activated cell sorting (e.g., as commercially used by Miltenyi Biotec MACS Technology or Dynal magnetic bead selection), antibody panning, and erythrocyte resetting. Other methods for enrichment are also contemplated by the present invention. According to the present invention, the method includes selecting cells that have reduced or substantially no expression of CD33 as a cell surface marker. For example, flow cytometry can be used to enrich for cells that are negative for CD33. Thus, FACS can be used in conjunction with the methods described herein to isolate and detect cell populations of the present invention. FACS typically involves the use of a flow cytometer capable of simultaneously exciting and detecting multiple fluorochromes, such as a BD Biosciences FACSCanto™ flow cytometer, used substantially according to the manufacturer's instructions. The cytometry system may include a cytometry sample fluidics subsystem, as described below. Additionally, the cytometry system includes a cytometer fluidically coupled to the cytometry sample fluidics subsystem. The systems of the present disclosure may include numerous additional components, such as data output devices (e.g., monitors, printers, and / or speakers), software (e.g., Flowjo, Laluza, etc.), data input devices (e.g., interface ports, mice, keyboards, etc.), fluid handling components, power supplies, etc. Typically, a cell population is contacted with a panel of antibodies specific for a particular phenotypic marker of interest (e.g., CD33). Typically, the antibodies are labeled with tags to facilitate isolation and detection of the cell population of interest. Suitable labels include fluorescent molecules, radioisotopes, nucleotide chromophores, enzymes, substrates, chemiluminescent moieties, magnetic particles, bioluminescent moieties, etc. Thus, 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 agents, e.g., antibodies, for use in the methods of the 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), PE-Cy5 conjugates (cyclom, R670, Tricolor, Quaternary Fluorescence Imaging), and the like. Fluorescent Red), PE-Cy7 conjugate, Red 613, PE-Texas Red, PerCP, PerCPeFluor710, PE-CF594, peridinin chlorphyll protein, TruRed (PerCP-Cy5.5 conjugate), FluorX, fluorescein isothiocyanate (FITC), BODIPY-FL, TRITC, X-rhodamine (XRITC), Lissamine rhodamine B, Texas Red, allophycocyanin (APC), APC-Cy7 conjugate, Alexa Fluor Fluor350, Alexa Fluor405, Alexa Fluor430, Alexa Fluor488, Alexa Fluor500, Alexa Fluor514, Alexa Fluor532, Alexa Fluor546, Alexa Fluor555, Alexa Fluor568, Alexa Fluor594, Alexa Fluor610, Alexa Fluor633, Alexa Fluor647, Alexa Fluor660, Alexa Fluor680, Alexa Fluor700, Alexa Fluor750, Alexa Fluor790, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, BV785, BV711, BV421, BV605, BV510, or BV650. The assay may include binding the antibody to a solid support. The solid surface may be a microtiter plate coated with the antibody. Alternatively, the solid surface can be a bead, e.g., an activated bead, a magnetically responsive bead, etc. The beads can be composed of a variety of materials, including (but not limited to) glass, plastic, polystyrene, and acrylic.In addition, the beads are preferably fluorescently labeled. In some embodiments, the fluorescent beads are those contained in TruCount™ tubes available from Becton Dickinson Biosciences (San Jose, California).

[0111] Treatment method A further object of the present invention relates to a method of treatment in a patient in need thereof, comprising transplanting a therapeutically effective amount of a population of edited eukaryotic cells (enriched for CD33- cells or not) obtained by the method disclosed herein.

[0112] In particular, the methods are particularly suited for treating β-hemoglobinopathy. In some embodiments, the β-hemoglobinopathy is sickle cell disease. In some embodiments, the β-hemoglobinopathy is β-thalassemia.

[0113] In some embodiments, the cell population is autologous to the subject, meaning that the cell population is derived from the same subject.

[0114] In some embodiments, the methods of treatment disclosed herein further comprise administering to the patient a therapeutically effective amount of an agent capable of depleting CD33-positive cells.

[0115] In some embodiments, the agent is an antibody that binds to CD33 and depletes CD33+ cells (ie, a "depleting antibody").

[0116] As used herein, the term "depletion" with respect to CD33+ cells refers to a measurable reduction in the number of CD33+ cells in a 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.

[0117] In some embodiments, the depleting antibodies mediate antibody-dependent cell-mediated cytotoxicity. As used herein, the term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a cell-mediated reaction in which nonspecific cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibodies on target cells and subsequently cause lysis of the target cells. In some embodiments, the depleting antibodies are IgG1 antibodies. In some embodiments, the depleting antibodies are IgG3 antibodies.

[0118] In some embodiments, the antibody suitable for depleting CD33+ cells is conjugated to a therapeutic moiety, ie, a drug.

[0119] In some embodiments, the therapeutic moiety may be, for example, a cytotoxin, a chemotherapeutic agent, a cytokine, an immunosuppressant, an immunostimulatory agent, a lytic peptide, or a radioisotope. Such conjugates are referred to herein as "antibody-drug conjugates" or "ADCs."

[0120] In some embodiments, the antibody suitable for depletion of CD33+ cells is conjugated to a cytotoxic moiety. Cytotoxic moieties include, for example, taxol; cytochalasin B; gramicidin D; ethidium bromide; emetine; mitomycin; etoposide; tenoposide; vincristine; vinblastine; colchicine; doxorubicin; daunorubicin; dihydroxyanthracin dione; tubulin inhibitors, for example, maytansine or an analog or derivative thereof; antimitotic agents, for example, monomethyl auristatin E or F or an analog or derivative thereof; dolastatin 10 or 15 or an analog thereof; irinotecan or an analog thereof; mitoxantrone; mithramycin; actinomycin D; 1-dehydrotestosterone; glucocorticoids; procaine; tetracaine; lidocaine; propranolol; puromycin; calicheamicin or an analog or derivative thereof; antimetabolites, for example, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine , fludarabine, 5-fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine, or cladribine; alkylating agents such as mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin; platinum derivatives such as cisplatin or carboplatin; duocarmycin A, duocarmycin SA, rachelmycin (CC-1065) or analogs or derivatives thereof; antibiotics such as dactinomycin, bleomycin, daunorubicin, doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC); pyrrolo[2,1-c][1,4]-benzodiazepines (PDB);Diphtheria toxin and related molecules, such as diphtheria A chain and its active fragments and hybrid molecules, ricin toxin, such as ricin A or deglycosylated ricin A chain toxin, cholera toxin, Shiga-like toxins, 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, allorin, saporin, modeccin, geranin, 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 inhibitors, curtin, crotin, sapaonaria officinalis inhibitors, gelonin, mitogelin, restrictocin, phenomycin, and enomycin toxins; ribonucleases (RNases); DNases I, Staphylococcal endotoxin A; pokeweed antiviral protein; diphtherin toxin; and Pseudomonas endotoxin.

[0121] In some embodiments, antibodies suitable for depleting CD33+ cells are conjugated to auristatin or its peptide analogs, derivatives, or prodrugs. Auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cytoplasmic division (Woyke et al. (2001) Antimicrob. Agents and Chemother. 45(12): 3580-3584), and have anticancer (US Pat. No. 5,663,149) and antifungal activities (Pettit et al., (1998) Antimicrob. Agents and Chemother. 42: 2961-2965). For example, auristatin E can be reacted with para-acetylbenzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other exemplary auristatin derivatives include AFP, MMAF (monomethyl auristatin F), and MMAE (monomethyl auristatin E). Suitable auristatins and auristatin analogs, derivatives, and prodrugs, as well as linkers suitable for conjugating auristatins to Abs, are described, for example, in U.S. Pat. Nos. 5,635,483, 5,780,588, and 6,214,345, and WO Nos. 02088172, 2004010957, 2005081711, 2005084390, 2006132670, 03026577, 200700860, 207011968, and 205082023.

[0122] In some embodiments, the antibody suitable for depletion of CD33+ cells is gemtuzumab ozogamicin, a recombinant humanized IgG4 kappa antibody conjugated to a calicheamicin derivative, a cytotoxic antitumor antibiotic. In particular, gemtuzumab comprises a light chain set forth in SEQ ID NO:8 and a heavy chain set forth in SEQ ID NO:9.

[0123] [ka]

[0124] [ka]

[0125] kit The present invention further provides a kit comprising all components of the gene editing platform disclosed herein for performing mutagenesis and containing reagents for carrying out the above-described method. To this end, one or more of the reaction components for the methods disclosed herein, such as a guide RNA molecule and a nucleic acid molecule encoding a base editing enzyme, can be provided in the form of a kit for use. In some embodiments, the kit comprises: (a) at least one base editing enzyme and a polynucleotide encoding it; and (b) multiple guide RNA molecules designed to guide the base editing enzyme to multiple target sequences, wherein at least one guide RNA molecule is designed to guide the base editing enzyme to a target sequence in a gene encoding CD33, thereby knocking down CD33 expression. Specifically, the kit comprises: (a) at least one adenine base editor (ABE) and a polynucleotide encoding it; and (b) multiple guide RNA molecules designed to guide the base editing enzyme to multiple target sequences, wherein at least one guide RNA molecule is designed to guide the ABE to the target sequence set forth in SEQ ID NO: 13. In some embodiments, the kit may include one or more other reaction components. In some embodiments, appropriate amounts of one or more reaction components are provided in one or more containers or supported on a substrate. Examples of additional components of the kit include, but are not limited to, one or more host cells, one or more reagents for introducing an exogenous nucleotide sequence into a host cell, one or more reagents for detecting expression of a guide RNA or base-editing enzyme or for confirming the status of a target nucleic acid (e.g., a probe or PCR primer), and a buffer or culture medium for the reaction. The kit may also include one or more of the following components: a support, a termination reagent, a modification or digestion reagent, an osmolyte, and a detection instrument. The components used can be provided in various forms. For example, the components (e.g., enzyme, RNA, probe, and / or primer) can be suspended in aqueous solution or provided as freeze-dried or lyophilized powders, pellets, or beads.In the latter case, the components, upon reconstitution, form a complete mixture of components for use in the assay. The kits of the present invention can be provided at any suitable temperature. For example, for storage, kits containing protein components or complexes thereof in liquid are preferably provided and maintained at 0°C or below, preferably -20°C or below, or otherwise frozen. The kits may 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, etc.) that hold the reaction components or detection probes in any of a variety of configurations (e.g., in vials, in microtiter plate wells, in microarrays, etc.). The kits may further include tangible recorded instructions for use of the components.

[0126] The present invention will be further illustrated by the following figures and examples, which should not be construed as limiting the scope of the present invention in any way. [Brief explanation of the drawings]

[0127] drawing [Figure 1]Figure 1. CD33 knockout via base editing in K562 cells. A. Schematic diagram of CD33 on chromosome 19 (chr19). The gray box indicates exons 1 and 2, and the black box indicates the ATG start codon, the splice donor site (SD) of exons 1-2, and the splice acceptor site (SA) of exons 1-2. The black arrow indicates the sgRNA designed to target CD33 using the target protospacer; the ATG, SD, and SA targets are highlighted in bold. B. Experimental protocol used for base editing experiments using BE- and sgRNA expression plasmids in K562 cells. C. AT-to-GC and CG-to-TA base editing efficiencies calculated by EditR software for samples subjected to Sanger sequencing. Data are expressed as mean ± SEM (n = 3 biologically independent experiments). D. Frequency of CD33+ cells in control (mock-transfected samples with TE buffer) and edited samples calculated by flow cytometry 5 days after treatment. Base editing efficiency (BE%) is shown under each condition. Data are expressed as mean ± SEM (n = 3 biologically independent experiments). [Figure 2]Figure 2. CD33-K562 cell selection eliminates unedited cells. A. Experimental protocol used for multiplex base editing experiments using BE- and sgRNA expression plasmids in K562 cells. B. AT-to-GC and CG-to-TA base editing efficiencies in the HBG promoter calculated using EditR software for samples transfected with one (single) or two (double) sgRNAs and subjected to Sanger sequencing. CD33 base editing efficiency (CD33 BE%) is shown below each condition. Data are expressed as mean ± SEM (n = 3 biologically independent experiments). C. Frequency of CD33+ cells in control (mock-transfected samples with TE buffer) and edited samples calculated by flow cytometry 5 days after treatment. Data are expressed as mean ± SEM (n = 3 biologically independent experiments). *p ≤ 0.05; ****p ≤ 0.0001 (one-way ANOVA). D. AT-to-GC and CG-to-TA base editing efficiencies in the CD33 and HBG promoters calculated by EditR software for samples transfected with two sgRNAs, sorted for CD33 expression, and subjected to Sanger sequencing. Data are expressed as mean ± SEM (n = 3 biologically independent experiments). **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001 (two-way ANOVA). [Figure 3]Figure 3. CD33- cell sorting results in high BE efficiency in SCD HSPCs. A. Experimental protocol used for multiplex base editing experiments using BE mRNA and chemically modified sgRNA in SCD HSPCs. B-C. Frequency of CD34+ cells for control (mock sample transfected with TE buffer) and single- (ABE-KLF1 and CBE-LRF) or double- (ABE-KLF1-CD33 and CBE-LRF-CD33) edited samples calculated by flow cytometry 1 day (B) and 2 days (C) after treatment. Data are expressed as single values ​​(n = 1 donor). D–E. CD33+ cell frequencies for control (mock transfected with TE buffer) and single- (ABE-KLF1 and CBE-LRF) or double- (ABE-KLF1-CD33 and CBE-LRF-CD33)-edited samples, calculated by flow cytometry 1 day (D) and 2 days (E) after treatment. Data are expressed as single values ​​(n = 1 donor). F. CFC frequencies for control (mock transfected with TE buffer), single- (ABE-KLF1 and CBE-LRF) or double- (ABE-KLF1-CD33 and CBE-LRF-CD33)-edited samples, and double-edited samples sorted for CD33 expression [ABE-KLF1-CD33(CD33-) and CBE-LRF-CD33(CD33-)]. Data are expressed as single values ​​(n = 1 donor). G. AT-to-GC and CG-to-TA base editing efficiencies in the CD33 and HBG promoters calculated by EditR software for samples transfected with two sgRNAs, sorted for CD33 expression, and subjected to Sanger sequencing. Data are expressed as single values ​​(n=1 donor).

[0128] Example Materials and Methods Cell lineage culture Human erythroleukemia K562 cells were maintained in RPMI 1640 (Lonza) containing glutamine and supplemented with 10% fetal bovine serum (Lonza), 2 mM Hepes (Life Technologies), 100 nM sodium pyruvate (Life Technologies), and penicillin and streptomycin (Life Technologies).

[0129] HSPC purification and culture Human non-fixed peripheral blood CD34+ HSPCs were obtained from SCD patients. Eligible SCD samples for research purposes were obtained from the "Hôpital Necker-Enfants maladies" Hospital (Paris, France). Written informed consent was obtained from all adult subjects. All experiments were performed in accordance with the Declaration of Helsinki. This study was approved by the local Institutional Review Board (reference: DC 2014-2272, CPP Ile-de-France II "Hôpital Necker-Enfants maladies"). HSPCs were immunostained with a CD34 MicroBead Kit (Miltenyi Biotec) and then purified by immunomagnetic selection with a MACS column (Miltenyi Biotec). 24 hours before transfection, CD34 + Cells were thawed and cultured at 5 × 10 cells / well in "HSPC medium" containing StemSpan (STEMCELL Technologies) supplemented with penicillin / streptomycin (Gibco), 250 nM StemRegenin1 (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). 5 The cells were cultured at a concentration of 1000 cells / ml.

[0130] Plasmid The plasmids used in this study were pCMV_ABEmax_P2A_GFP (Addgene #112101), ABEmax-OPT [generated by deleting a uridine from the coding sequence of the pCMV_ABEmax_P2A_GFP (Addgene #112101) plasmid and adding a DNA fragment containing two copies of the 3' untranslated region (UTR) of the HBB gene and a 96-adenine poly(A) sequence], and CBE-SpRY-OPT2. 25 Includes:

[0131] sgRNA design We previously designed an sgRNA targeting the -200 region of the HBG1 / 2 promoter. 25 An sgRNA targeting the CD33 gene was manually designed (Table 1). To generate the sgRNA expression plasmid, oligonucleotides were annealed to generate an sgRNA protospacer, and the duplex was ligated into a BbsI-digested MA128 plasmid (provided by M. Amendola, Genethon, France). For RNA-mediated base editing, we used a chemically modified synthetic sgRNA (Synthego) with a 2'-O-methyl analog and a 3'-phosphorothioate nonhydrolyzable bond in the first three 5' and 3' nucleotides.

[0132] Table 1. sgRNA target sequences [Table 2]

[0133] In vitro transcription of mRNA Ten μg of BE expression plasmid was digested overnight with 20 units of a restriction enzyme that cleaves once after the polyA tail or after the stop codon for constructs with or without a polyA tail. The linearized plasmid was purified using a PCR purification kit (QIAGEN) and eluted in 30 μl of DNase / RNase-free water. 1 μg of linearized plasmid was used as a template for an in vitro transcription (ivt) reaction (MEGAscript, Ambion). The ivt protocol was modified as follows: GTP nucleotide solution was used at a final concentration of 3.0 mM instead of 7.5 mM, and the anti-reverse cap analog N7-methyl-3'-O-methyl-guanosine-5'-triphosphate-5'-guanosine (ARCA, Trilink) was used at a final concentration of 12.0 mM. As a result, the final Cap:GTP ratio was 4:1, allowing efficient capping of mRNA. The incubation time for the ivt reaction was shortened to 30 min. For constructs that did not already contain a poly-A tail in the plasmid, an additional step of polyadenylation was performed using the manufacturer's guidelines (Poly-A tailing kit, Ambion). The mRNA was precipitated using lithium chloride and resuspended in a final volume of TE buffer that allowed a concentration of >1 μg / μl to be achieved. The quality of the mRNA was assessed using a Bioanalyzer (Agilent). CBE-SpRY-OPT2 mRNA, containing 5-methoxy UTP and capped with a Cap1 analog, and subjected to silica membrane purification was purchased from Trilink.

[0134] Plasmid transfection K562 cells (10 6Cells (cells / condition) were transfected with 3.6 μg of a base editor expression plasmid and 1.2 μg of an sgRNA-containing plasmid or 2.4 μg of two sgRNA-containing plasmids. Cells transfected with TE buffer served as a negative control. The AMAXA Cell Line Nucleofector Kit V (VCA-1003) and the U-16 program (Nucleofector II) were used. 18 hours after transfection, transfection efficiency was assessed by flow cytometry using a Gallios (Beckman Coulter) flow cytometer.

[0135] RNA transfection 0.5 x 10 for each condition 5 pieces~2×10 5 pieces CD34 + HSPCs were transfected with 3.0 μg of enzyme-encoding mRNA and one or two synthetic sgRNAs at a final concentration of 1.15 μM each. The P3 Primary Cell 4D-Nucleofector X Kit S (Lonza) and CA137 program (Nucleofector 4D) were used. Cells were incubated at 37°C for 10 minutes, then transferred from the Nucleocuvette strip wells to a 96-well plate and 80 μl of prewarmed "HSPC medium" was added. Transfected cells were incubated at 37°C. Cells transfected with TE buffer served as a negative control.

[0136] Colony-forming cell (CFC) assay CD34 + HSPCs were seeded at a concentration of 500 cells / mL in methylcellulose-based medium (GFH4435, Stem Cell Technologies) under conditions that support differentiation into erythroid and granulocyte / monocytes. BFU-E and CFU-GM colonies were counted after 14 days. Colonies were randomly picked and collected as bulk populations (containing at least 25 colonies) to assess base editing efficiency.

[0137] Editing efficiency evaluation Base editing efficiency and insertion and deletion (InDel) frequencies were assessed in K562 cells 3 days post-transfection, in cells grown in HSPC medium 6 days post-transfection, and in BFU-E and CFU-GM 14 days post-seeding.

[0138] Genomic DNA was extracted from control and edited cells using the PURE LINK Genomic DNA Mini kit (Life Technologies) or Quick-DNA / RNA Miniprep (ZYMO Research) according to the manufacturer's instructions. To assess the efficiency of base editing at the sgRNA target site, PCR was followed by Sanger sequencing and EditR analysis (EditR: a method for quantifying base editing from Sanger sequencing). 33 Tracking of InDels by Decomposition (TIDE) analysis was also performed to assess the proportion of InDels in the base-edited samples. 34 .

[0139] Table 2. Primers used to detect base editing and InDel events [Table 3]

[0140] Flow cytometry analysis K562 cells and CD34 + Flow cytometry analysis of the CD33 surface marker for HSPCs was performed using APC-conjugated anti-CD33 (551378, BD). Flow cytometry analysis was performed using a Gallios (Beckman Coulter) flow cytometer. Data were analyzed using FlowJo (BD Biosciences) software.

[0141] Fluorescence-activated cell sorting K562 cells were transfected as described above and 5 × 10 5 Cells were seeded at a concentration of 1000 cells / ml. Five days after transfection, cells were stained with APC-conjugated anti-CD33 (551378, BD) and sorted using an SH800 (Sony). Sorted and unsorted populations were collected for DNA extraction. CD34 + HSPCs were transfected 1 day after thawing as described above and plated at 5 x 10 in HSPC medium. 5 Two days after transfection, cells were stained with APC-conjugated anti-CD33 (551378, BD) and PE-conjugated anti-CD34 (550761, BD) and sorted using an SH800 (Sony). Sorted and unsorted populations were cultured in "HSPC medium" at a concentration of 5 × 10 cells / ml. 5 Cells were cultured at a concentration of 1000 / ml for 6 days and then either harvested for DNA extraction or plated for CFC assays.

[0142] result CD33 knockout via base editing in K562 cells The BE system allows for the generation of gene knockouts (KOs) through the introduction of point mutations. CBE mediates gene KO via the insertion of a stop codon, while both CBE and ABE alter gene expression through the disruption of either the ATG start codon or the splice / acceptor donor site. We designed sgRNAs targeting CD33 (CD33-1, CD33-2, and CD33-3) that mediate CD33 KO when coupled with CBE or ABE. When CD33-1 sgRNA is coupled with CBE, it disrupts the start codon; when CD33-2 sgRNA is coupled with either CBE or ABE, it disrupts the splice donor site of exons 1-2; and when CD33-3 sgRNA is coupled with ABE, it disrupts the splice acceptor site of exons 1-2 (Figure 1A). Transfection of K562 hematopoietic cells with a plasmid encoding CBE or ABE (CBE-SpRY-OPT2 or ABEmax) and a plasmid encoding sgRNA (Figure 1B) resulted in various BE efficiencies ranging from 7.0 to 80.0% (Figure 1C). Flow cytometry analysis demonstrated downregulation of CD33 at the protein level (29.6% and 17.4 to 5.8% in control and edited samples, respectively; Figure 1D). CD33-2 sgRNA and ABE were the most suitable combination for editing and knockout of CD33. Overall, these data demonstrate that BE efficiently downregulates CD33 expression in hematopoietic cell lineages.

[0143] CD33 - K562 cell selection removes non-edited cells BE does not cause DSBs, allowing for safe multiple editing without causing genome rearrangements. 32To assess the possibility of enriching for HBG promoter-edited cells through selection of CD33- cells, we performed multiplex BE simultaneously targeting the HBG promoter and CD33 gene (Figure 2A). Two BE systems targeting the HBG promoter that efficiently reactivate HbF were used: LRF_bs_2 sgRNA coupled with CBE-SpRY-OPT2 to disrupt the LRF repressor BS, and KLF1_bs_1 sgRNA coupled with ABEmax to generate the KLF1 activator BS. 25 To target CD33, we selected the CD33-2 sgRNA, which, when coupled with ABEmax, yielded the highest BE efficiency (80.0%) and potent CD33 downregulation (Figures 1C and 1D). The same sgRNA coupled with CBE-SpRY-OPT2 was selected because it resulted in CD33 downregulation despite its low BE efficiency (Figures 1C and 1D). K562 cells were transfected with a plasmid encoding either CBE or ABE (CBE-SpRY-OPT2 or ABEmax) and either an sgRNA-encoding plasmid targeting the HBG promoter (single editing) or two sgRNA-encoding plasmids targeting the HBG promoter and the CD33 gene, respectively (double editing; Figure 2A). No statistically significant difference in HBG editing efficiency was observed between the single-editing and double-editing conditions (Figure 2B). When CD33 was targeted, a strong downregulation of CD33 was observed in ABE-treated samples, and a mild decrease in CD33 expression was observed in CBE-treated samples (Fig. 2C). - population and CD33 + As expected, the frequency of the edited CD33 allele was significantly higher than that of the CD33 - Interestingly, CD33 + Compared with the CD33 population, - In the CD33 population, higher BE efficiency at the HBG promoter was observed (Fig. 2D). -This demonstrates that cell sorting removes defective editing cells.

[0144] CD33 - Cell sorting leads to higher BE efficiency in SCD HSPCs We applied the ABE or CBE dual editing strategy to HSPCs obtained from a single SCD patient. We used a clinically relevant protocol to deliver the BE system to SCD HSPCs based on RNA transfection (mRNA encoding a base editor and a chemically modified synthetic sgRNA; Figure 3A). 25 SCD HSPCs were transfected with mRNA encoding ABE or CBE (ABEmax or CBE-SpRY-OPT2) and either an sgRNA targeting the HBG promoter (single editing) or two sgRNAs targeting the HBG promoter and the CD33 gene (double editing; Figure 3A). At 1 and 2 days post-transfection, CD33 and CD34 expression was assessed in the control and edited populations. At both time points, most cells expressed CD34 + (Figures 3B and 3C). Edited cells showed a decrease in CD33 expression (Figures 3D and 3E). Two days after transfection, CD33 - population and CD33 + Populations were sorted. Sorted and unsorted populations were either expanded in liquid culture ("HSPC medium") or seeded in semi-solid medium that allows for erythroid and granulocytic / monocytic differentiation of single progenitor cells (CFC assay; Figure 3A). CFC assays revealed differences in CD33 expression between single-edited and double-edited cells, and between the bulk population and CD33 - We found no difference in progenitor cell survival between the selected and unselected populations (Figure 3F). Through RNA delivery, CBE was observed to be similar to that previously observed. 25 , was overall more efficient in HSPCs than in K562 (Figure 3G). As expected, CD33 - Compared with the CD33 population, +In this population, no or low BE in CD33 was observed. Importantly, for both the ABE and CBE strategies, CD33 - In the population, the bulk population or CD33 + Higher BE efficiency was detected at the HBG promoter than in the SCD HSPC population (Figure 3G). Collectively, these results suggest that multiple editing of the HBG promoter and CD33, as well as CD33 expression in SCD HSPCs, are involved in the differentiation of SCD HSPCs. - Combining BE with selection results in higher BE efficiency and eliminates poorly edited cells that may compete with edited cells for engraftment in the bone marrow.

[0145] References Throughout this application, various references are made to describe the state of the art to which this invention pertains, the disclosures of which are incorporated by reference into this disclosure.

[0146] [Table 4] TIFF2026506554000011.tif242169 TIFF2026506554000012.tif154169

Claims

1. 1. A method for editing a eukaryotic cell, comprising: contacting the eukaryotic cell with a gene editing platform comprising: (a) at least one base editing enzyme; and (b) a plurality of guide RNA molecules designed to guide the base editing enzyme to a plurality of target sequences; wherein at least one guide RNA molecule is designed to guide the base editing enzyme to a target sequence in a gene encoding CD33, thereby knocking down expression of CD33. method.

2. 2. The method of claim 1, wherein the eukaryotic cells are 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)).

3. The method of claim 1 or 2, wherein the base editing enzyme is a cytosine base editing enzyme (CBE) or an adenine base editing enzyme (ABE).

4. 4. The method of any one of claims 1 to 3, wherein the gene editing platform comprises i) at least one guide RNA molecule designed to guide the base editing enzyme to a desired target sequence, and ii) at least one guide RNA molecule designed to guide the base editing enzyme to a target sequence in a gene encoding CD33, thereby knocking down expression of CD33.

5. 5. The method of any one of claims 1 to 4, wherein the gene editing platform targets the CD33 gene and comprises at least one guide RNA molecule designed such that i) when coupled with a CBE, the start codon is destroyed, or ii) when coupled with either a CBE or an ABE, the splice donor site of exons 1-2 is destroyed, and / or iii) when coupled with an ABE, the splice acceptor site of exons 1-2 is destroyed.

6. 6. The method of any one of claims 1 to 5, wherein the guide RNA molecule targets a nucleic acid sequence selected from Table 1.

7. 7. The method of any one of claims 1 to 6, wherein the gene editing platform comprises at least one guide RNA molecule that targets CD33 and that, when coupled to either a CBE or an ABE, disrupts the splice donor site of exon 1-2.

8. The method of claim 7, wherein the guide RNA molecule targets the nucleic acid sequence set forth in SEQ ID NO:

13.

9. The method of any one of claims 1 to 8, wherein the gene editing platform comprises (a) at least one adenine base editor (ABE) and (b) a plurality of guide RNA molecules designed to guide the base editing enzyme to a plurality of target sequences, wherein at least one guide RNA molecule is designed to guide the ABE to the target sequence set forth in SEQ ID NO:

13.

10. 10. The method of any one of claims 1 to 9, wherein the gene editing platform is used to increase fetal hemoglobin content in eukaryotic cells.

11. 11. The method of claim 10, wherein a gene editing platform is used to edit the HBG1 or HBG2 promoter and subsequently increase expression of γ-globin.

12. The method of any one of claims 1 to 9, wherein the gene editing platform is used to repress the expression of α-globin.

13. 10. The method of any one of claims 1 to 9, wherein the gene editing platform is used to restore normal expression of β-globin in a eukaryotic cell.

14. 14. A population of edited eukaryotic cells obtainable by the method of any one of claims 1 to 13.

15. 1. A method for producing a substantially pure population of edited eukaryotic cells, comprising: i) editing a population of eukaryotic cells by the method of any one of claims 1 to 13; ii) Enriching the population of edited eukaryotic cells that are negative for CD33 and

16. 16. A substantially pure population of edited eukaryotic cells obtainable by the method of claim 15.

17. transplanting a therapeutically effective amount of the edited eukaryotic cell population of claim 14 or 16. A method of treatment in a patient in need thereof.

18. 18. The method of claim 17, wherein the patient is suffering from a β-hemoglobinopathy.

19. 19. The method of claim 17 or 18, further comprising administering to the patient a therapeutically effective amount of an agent capable of depleting CD33-positive cells.

20. The method of claim 19, wherein the agent is an antibody that binds to CD33 and depletes CD33+ cells.

21. 21. The method of claim 20, wherein the antibody suitable for depletion of CD33+ cells is conjugated to a cytotoxic moiety.

22. 21. The method of claim 20, wherein the antibody suitable for depleting CD33+ cells is gemtuzumab ozogamicin.

23. (a) at least one base editing enzyme or a polynucleotide encoding the same; and (b) a plurality of guide RNA molecules designed to guide the base editing enzyme to a plurality of target sequences; wherein at least one guide RNA molecule is designed to guide the base editing enzyme to a target sequence in a gene encoding CD33, thereby knocking down expression of CD33. kit.

24. 24. The kit of claim 23, comprising: (a) at least one adenine base editor (ABE) or a polynucleotide encoding same; and (b) a plurality of guide RNA molecules designed to guide the base-editing enzyme to a plurality of target sequences, wherein at least one guide RNA molecule is designed to guide the ABE to the target sequence set forth in SEQ ID NO: 13.

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