Base editing approaches for the treatment of β-hemoglobin disorders

Abstract

Clinical history of β-hemoglobinopathy indicates that its severity is mitigated by the synthesis of fetal γ-globin in adulthood, typically associated with mutations in genes in the HBB cluster, also known as hereditary hyperfetal hemoglobinemia (HPFH). The inventors have identified that most of the known HPFH mutations in the γ-globin promoter (C>T, G>A, T>C, or A>G) can be reproduced using CBE- and ABE-mediated base editing approaches. In particular, the inventors have designed gRNAs that, when combined with CBE or ABE, generate HPFH mutations and either disrupt binding sites for transcriptional repressors (sites −200 and −115) or generate de novo DNA motifs recognized by transcriptional activators (e.g., −198T>C, −175T>C, and −113A>G). It is noteworthy that a subset of gRNAs targeting the -200 and 115 regions are predicted to simultaneously generate the HPFH mutation and also create base changes other than the HPFH mutation within or surrounding the LRF or BCL11A binding site, which may further reduce LRF and BCL11A occupancy. Thus, the present invention relates to a base editing approach for the treatment of β-hemoglobinopathies.

Description

[Technical Field]

[0001] Field of invention: This invention relates to medicine, particularly to the field of hematology. [Background technology]

[0002] Background of the invention: Beta-hemoglobin disorders (beta-thalassemia and sickle cell disease (SCD)) are monogenic disorders caused by mutations in the beta-globin gene locus that affect the synthesis or structure of hemoglobin (Hb) in adults. Beta-thalassemia reduces the production of beta-globin chains that make up the adult hemoglobin (HbA) tetramer. + ) or disappearance (β 0 This condition is caused by a mutation in the β-globin gene (HBB) locus, which leads to the sedimentation of unbound α-globin chains, death of erythrocytes, and severe anemia. (1) In SCD, the A>T mutation in the HBB gene causes a substitution of glutamate at position 6 of the β-globin chain with valine (β S This is the cause of sickle cell hemoglobin (HbS) polymerization induced by deoxygenation. This initial event leads to sickle cell (RBC) formation, hemolysis, vascular occlusive crisis, and multi-organ failure, often accompanied by a severely shortened life expectancy. (2) .

[0003] The only definitive treatment for β-hemoglobin disorders is allogeneic hematopoietic stem cell (HSC) transplantation from an HLA-compatible donor, an option applicable to less than 30% of patients. (3) Gene therapy approaches based on the transplantation of a patient's own genetically modified HSCs are being studied as a treatment option for patients without a suitable donor. (4)Genome editing techniques based on direct gene modification have been utilized to develop therapeutic approaches for β-hemoglobinopathies. These approaches use designer nucleases such as the CRISPR / Cas9 system that induce double-stranded DNA breaks (DSBs) via single-guide RNAs (gRNAs) complementary to specific genomic targets. (4) .

[0004] The clinical course of β-hemoglobinopathies indicates that the severity of both β-thalassemia and SCD is typically alleviated by the synthesis of fetal γ-globin in adulthood, which is also known as the mutation of hereditary persistence of fetal hemoglobin (HPFH), and is typically associated with gene mutations (deletions or point mutations) within the HBB cluster. (5) . Fetal hemoglobin (HbF) compensates for the deficiency of adult hemoglobin in β-thalassemia, and γ-globin exhibits a potent anti-sickling effect in SCD by replacing the mutant sickle β-chain. (4) . In particular, mutations in the two identical promoters of the γ-globin genes (HBG1 and HBG2) either generate de novo DNA motifs recognized by transcriptional activators (TAL1, KLF1, and GATA1) (6、7、8) or disrupt the binding sites of transcriptional repressors (LRF and BCL11A). (9) . As potential therapeutic methods for both β-thalassemia and SCD, several genome editing strategies have been developed to reactivate the expression of fetal hemoglobin in erythroid progenitor cells of patients' hematopoietic stem / progenitor cells (HSPCs), based on disrupting cis-regulatory sequences via the occurrence of deletions or insertions, mimicking the mutations of hereditary persistence of fetal hemoglobin. (10) . Disruption of the binding sites of the repressors LRF and BCL11A in the γ-globin promoter by CRISPR / Cas9 efficiently reactivates the expression of fetal hemoglobin and remits the phenotype of SCD RBCs. (1)However, most deletions that disrupt inhibitory factor binding sites are caused by microhomology-mediated end junctions (MMEJs), which may not be effective in the quiescent HSC fraction of HSPC populations that are regrowing over long periods and consist mostly of proliferating progenitor cells. (11、12) .

[0005] HSCs are particularly problematic when multiple on-target events are present, or when on-target and off-target events occur simultaneously, as they can lead to DNA DSBs. (13) It is noteworthy that it is extremely sensitive to this. Even when using highly specific gRNAs, treatment of human HSPCs with Cas9 / gRNA can induce a DNA damage response, which can lead to apoptosis. (14) CRISPR / Cas9 can induce P53-dependent cytotoxicity and cell cycle arrest, resulting in negative selection of cells with a functional P53 pathway. (15) Furthermore, the occurrence of several on-target DSBs, simultaneous on-target and off-target DSBs, or even a single on-target DSB carries the risk of deletion, inversion, and translocation. (16) Therefore, the development of novel, effective, and safe therapeutic strategies for β-hemoglobin disorders based on precise base editing should take precedence over DNA repair induced by DSBs.

[0006] In recent years, it has been shown that cytosine and adenine base editing enzymes (CBEs and ABEs) based on the CRISPR system can cause pinpoint changes in DNA with little to no double-spindle transitions (DSBs). (17) The basic components of base editing enzymes are non-catalytic Cas9 nucleases and deaminases; these ultimately produce the conversion of CG to TA, or AT to GC (for CBE and ABE, respectively). (18)Base editing approaches enable precise DNA repair without substantial DSBs, thus eliminating the risk of apoptosis, translocation, and most DNA insertions or deletions induced by DSBs. Furthermore, base editing has a lower level of off-target activity than Cas9 nuclease (17) Importantly, base editing occurs in quiescent cells, which allows for the genetic modification of bona fide HSCs using this new technology (19) suggesting that uniform and predictable base changes occur compared to the heterogeneous and unpredictable mutagenesis induced by non-homologous end joining (NHEJ). SUMMARY OF THE INVENTION PROBLEMS TO BE SOLVED BY THE INVENTION

[0007] Invention summary: The present invention is defined by the claims. In particular, the present invention relates to a base editing approach for the treatment of β-hemoglobinopathies. MEANS FOR SOLVING THE PROBLEMS

[0008] Detailed description of the invention: The inventors identified that most of the mutations in the γ-globin promoter for known hereditary persistence of fetal hemoglobin (C>T, G>A, T>C, or A>G) can be recapitulated using base editing approaches mediated by CBE and ABE (Figure 1). Compared to CRISPR / Cas9 nuclease-based strategies, base editing approaches may enable the simultaneous targeting of multiple regions within the γ-globin promoter, e.g., disruption of mutations in hereditary persistence of fetal hemoglobin that disrupt both the -200 and -115 binding sites. Considering the independent roles of LRF and BCL11A in γ-globin repression, this may have an additive effect on the reactivation of fetal hemoglobin (20)In contrast, a Cas9 nuclease-based strategy targeting two different regions of the γ-globin promoter (e.g., -200 and -115) would likely trigger deletion of the intervention sequence, which could be detrimental to promoter activity. (9) Furthermore, base editing can be designed to create multiple novel binding sites for transcription activators (e.g., -198T>C, -175T>C, and -113A>G). (21) Importantly, this sequence will avoid a potential Cas9-mediated deletion in the region between the HBG1 and HBG2 promoters, which would result from simultaneous cleavage in the two γ-globin promoters, leading to a deletion of HBG2 expression. (22) The inventors previously showed that this type of deletion occurs in 10-15% of HUDEP-2 cells edited by Cas9 nuclease. (11) Importantly, the base editing approach does not rely on microhomology-mediated end-binding pathways and will avoid the toxicity induced by possible double-strand breaks (DSBs). Therefore, we designed gRNAs that, when combined with CBE or ABE, either induce hereditary hyperhemoglobinemia mutations, disrupting the binding sites of transcription repressors (-200 and -115 sites) or generating de novo DNA motifs recognized by transcription activators (e.g., -198T>C, -175T>C, and -113A>G) (Figure 1). It is noteworthy that subsets of gRNAs targeting the -200 and -115 regions are predicted to simultaneously induce hereditary hyperhemoglobinemia mutations and other base changes within or around the LRF and BCL11A binding sites, thereby further reducing the occupancy of LRF and BCL11A. (23) . [Brief explanation of the drawing]

[0009] [Figure 1]HPFH mutations within the HBG1 / 2 promoters at the β-globin locus. A diagram of the β-globin locus on chromosome 11, showing the HBG2 and HBG1 genes and their promoters. The sequences of the identical promoters for HBG2 and HBG1 (nucleotides -214 to -98 upstream of the HBG transcription start site) are shown below. Black arrows indicate HPFH mutations described in the HBG1 and / or HBG2 promoters. HPFH mutations resulting in the creation of novel binding sites for transcription activators (TAL1, KLF1, and GATA1) are located above the DNA sequence, while HPFH mutations resulting in the disruption of transcription repressors (LRF and BCL11A) are located below the DNA sequence. HPFH mutations that can be caused by base editing are highlighted with rectangles. Ovals indicate transcription activators and repressors. Target sequences of gRNAs designed for use with base editing enzymes are reported in the lower part of the diagram (highlighted with black arrows) and aligned with the DNA sequences to which they bind. The target base is highlighted in bold white, and the remaining protospacer sequences are highlighted in gray. PAMs (protospacer adjacency motifs) for each gRNA are reported in black at the end of the arrows. [Figure 2]Efficient base editing of the HBG1 / 2 promoter in K562 cells. (A-M) Base editing efficiency from AT to GC (AC, N) or CG to TA (DM) in Sanger-sequenced samples, calculated by EditR software. The base editing efficiency ratio was measured by subtracting the base conversion rate in the control, which was considered background noise. The target and enzyme used are indicated above each graph. Data are expressed as mean ± standard deviation (n=2-3 biologically independent experiments). (O) Frequency of insertions and deletions (indels) measured by TIDE analysis is reported for control and base-edited samples. (P) Frequency of 4.9kb deletions measured by droplet digital PCR is reported for control and base-edited samples, as well as for positive control (DNA extracted from K562 cells edited using standard Cas9 nuclease). (Q) Binding site conversion after base editing, as analyzed by DiffLogo (26). *Two different gRNAs (BCL11A_bs_1 and BCL11A_bs_2) were used in combination with the enzymes evoCDA1-BE4max-NG and evoFERNY-BE4max-NG (Graphs E and F). Similarly, two different gRNAs (LRF_bs_1 and LRF_bs_2) were used in combination with the CBE-SpRY enzyme (Graphs L and M). [Figure 3]Efficient base editing of the HBG1 / 2 promoter in HUDEP-2 cells. Base editing efficiency from AT to GC (AC) or CG to TA (DH) in Sanger-sequenced samples, calculated by EditR software. Base editing efficiency was calculated as described in the caption of Figure 2. The target and enzyme used are indicated above each graph. (I) The frequency of insertions and deletions (indels), measured by TIDE analysis, is reported for control and base-edited samples. (J) Transformation of binding sites after base editing, as analyzed by DiffLogo (26). *Two different gRNAs (BCL11A_bs_1 and BCL11A_bs_2) were used in combination with the enzymes evoCDA1-BE4max-NG and evoFERNY-BE4max-NG (Graphs E and F). [Figure 4] Deinhibition of fetal hemoglobin after the development of TAL1 and KLF1 binding sites in HUDEP-2 cells. (A) Frequency of fetal hemoglobin-expressing cells in high-glycophorin A (GPA) populations at day 0 (D0) and day 9 (D9) of erythroid differentiation (as determined by flow cytometry). Base editing efficiency is shown above each black bar graph (D0). (B) RT-qPCR analysis of β- and γ-globin mRNA levels at days 0, 6, and 9 of erythroid differentiation. β- and γ-globin mRNA expression was normalized to α-globin mRNA and expressed as a ratio to total β-like globin. (C) Expression of γ-(Gγ-+Aγ-) and β-globin chains measured by reverse-phase high-performance liquid chromatography (RP-HPLC). β-like globin chain expression was normalized to α-globin. (D) Analysis of HbF and HbA by cation exchange HPLC. The inventors calculated the ratio of each Hb type to the total Hb tetramer. (E~G) Flow cytometry analysis of the late erythrocyte marker GYPA (E) and the early erythrocyte markers CD36 (F) and CD71 (G) on days 0 and 9 of HUDEP-2 differentiation. [Figure 5]Reactivation of HbF upon disruption of the BCL11A binding site in HUDEP-2 cells. (A-C) Flow cytometry analysis of late erythrocyte marker GYPA (A) and early erythrocyte markers CD36 (B) and CD71 (C) at day 0 and day 9 of HUDEP-2 differentiation. (D) Frequency of HbF-expressing cells in the high-GPA population at day 0 (D0) and day 9 (D9) of erythroid differentiation (as determined by flow cytometry). Base editing efficiency is shown above each black bar graph (D0). (E) RT-qPCR analysis of β- and γ-globin mRNA levels at days 0, 6, and 9 of erythroid differentiation. β- and γ-globin mRNA expression was normalized to α-globin mRNA and expressed as a ratio to total β-like globin. (F) Expression of γ-(Gγ-+Aγ-) and β-globin chains as measured by RP-HPLC. (G) Analysis of HbF and HbA by cation exchange HPLC. The inventors calculated the ratio of each Hb type to the total Hb tetramer. [Figure 6]Increase in HbF upon disruption of LRF binding sites in HUDEP-2 cells and HSPCs. (A) Frequency of HbF-expressing cells in a high-GPA population of undifferentiated HUDEP-2 cells (as determined by flow cytometry). (B) HSPCs were transfected with two plasmids expressing AncBE4max_NAA and LRF_bs_1gRNA, respectively, to edit the LRF binding site. Base editing efficiency at the LRF binding site in HSPC samples subjected to Sanger sequencing, as calculated by EditR software. The base editing efficiency ratio was calculated as described in the caption for Figure 2. The frequency of insertions and deletions (indels), as measured by TIDE analysis, is reported for control and base-edited samples. (C) Transformation of the binding site in HSPCs after base editing (26), as analyzed by DiffLogo. (D) Quantification of HbF and HbA by cation exchange HPLC in bulk populations of control and HBG-edited erythroblast burst-forming cell (BFU-E) colonies. The inventors plotted the ratio of each Hb type to the total Hb tetramer. The control cells included samples transfected with either TE buffer, or with the AncBE4max_NAA plasmid alone, or with the AncBE4max_NAA plasmid and a gRNA sample targeting a site unrelated to the β-globin locus. [Figure 7] Base editing efficiency within the HBG1 / 2 promoter in K562 cells using enzymes with low RNA off-target activity. Base editing efficiency from AT to GC(AC) or CG to TA(D) in Sanger-sequenced samples, calculated by EditR software. The base editing efficiency ratios were calculated as described in the caption for Figure 2. The target and enzyme used are indicated above each graph. For each target, the inventors compared classical base editing enzymes with base editing enzymes having lower RNA off-target activity. [Figure 8]Testing of CBE disruption of LRF binding sites in SCD HSPC. Base editing efficiency from CG to TA(A and B) calculated by EditR software in samples subjected to Sanger sequencing. (C) Frequency of insertions and deletions (indels) measured by TIDE analysis is reported for base-edited samples. (D) RT-qPCR analysis of βS- and γ-globin mRNA levels at day 13 of erythroid differentiation. βS- and γ-globin mRNA expression was normalized to α-globin mRNA and expressed as a ratio to total β-like globin. (E) Expression of γ-(Gγ-+Aγ-) and βS-globin chains measured by RP-HPLC. Expression of β-like globin chains was normalized to α-globin. (F) Analysis of HbF and HbS by cation exchange HPLC. The inventors calculated the ratio of each Hb type to total Hb tetramers. (G) Frequency of HbF-expressing cells in the high glycophorin A (GPA) population on day 19 of erythroid differentiation (as determined by flow cytometry). (H) Frequency of non-sickle cells upon oxygen removal in sham-transfected control and base-edited samples. (DF and H) Below each graph, the base-editing efficiency (BE%), the ratio of HbF to total Hb tetramers (HbF%) as measured by cation exchange HPLC, and the frequency of HbF-expressing cells (F cells%) as determined by flow cytometry are shown for each sample. Number of donors n=1. [Figure 9](ABE) Testing of SCD HSPC to disrupt LRF binding sites or create KLF1 binding sites. (E) Base editing efficiency from AT to GC(A and D) calculated by EditR software in samples subjected to Sanger sequencing. (F) Frequency of insertions and deletions (indels) measured by TIDE analysis is reported for base-edited samples. (G) RT-qPCR analysis of βS- and γ-globin mRNA levels at day 13 of erythroid differentiation. Expression of βS- and γ-globin mRNA was normalized to α-globin mRNA and expressed as a ratio to total β-like globin. (H) Expression of γ-(Gγ-+Aγ-) and βS-globin chains measured by RP-HPLC. Expression of β-like globin chains was normalized to α-globin. (H) Analysis of HbF and HbS by cation exchange HPLC. The inventors calculated the ratio of each Hb type to total Hb tetramers. (I) Frequency of non-sickle cells during oxygen desorption in control samples (spuriously transfected and edited at an unrelated AAVS1 locus) and base-edited samples. (J) Frequency of HbF-expressing cells (as determined by flow cytometry) in the high glycophorin A (GPA) population at day 19 of erythroblast differentiation. (F-I) Below each graph, the base editing efficiency (BE%), the ratio of HbF to total Hb tetramers (HbF%) as measured by cation exchange HPLC, and the frequency of HbF-expressing cells (F cells%) as determined by flow cytometry are shown for each sample. Number of donors n=1. [Figure 10] RNA-mediated base editing at the HBG1 / 2 promoter in K562 cells and SCD HSPCs. (A-H) Base editing efficiency from AT to GC or CG to TA in K562 and HSPCs. Base editing efficiency in Sanger-sequenced K562 cell (A-E) and SCD HSPC (F-H) samples was calculated using EditR software. The enzyme and gRNA used are shown above each graph. [Modes for carrying out the invention]

[0010] Definition: As used herein, “β-hemoglobin disorder” has its general meaning in the art and refers to any abnormality of the structure or function of any hemoglobin in an individual, and includes any mutation such as deletion or substitution mutation in the coding region of the β-globin gene, or abnormalities of the primary, secondary, tertiary, or quaternary structure of hemoglobin caused by mutations or deletions in the promoter or enhancer of such gene that result in a decrease in hemoglobin production compared to a normal or standard condition.

[0011] As used herein, the term “sickle cell disease” has its general meaning in the art and refers to a group of autosomal recessive blood disorders characterized by abnormal, stiff, sickle-shaped red blood cells resulting from mutations in the globin gene. They are defined by the presence of a βS-globin gene encoding a β-globin chain variant in which the 6th amino acid of the peptide is replaced by valine: the incorporation of βS-globin (HbS, sickle hemoglobin) into the hemoglobin tetramer results in hemoglobin polymerization and clinical phenotype. The term includes sickle cell anemia (HbSS), sickle hemoglobin C disease (HbSC), sickle β-plus thalassemia (HbS / β+), or sickle β-zero thalassemia (HbS / β0).

[0012] As used herein, the term "β-thalassemia" refers to a hemoglobin disorder resulting from an altered ratio of α-globin polypeptide chains to β-like globin polypeptide chains, which leads to decreased production of normal hemoglobin tetramer protein and precipitation of unpaired free α-globin chains.

[0013] As used herein, the terms “hematopoietic stem cells” or “HSCs” refer to blood cells that have the ability to regenerate and differentiate into precursors of blood cells. These progenitor cells are immature blood cells that cannot regenerate and must differentiate into mature blood cells. Hematopoietic stem 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 regenerate and maintain the continuous production of hematopoietic stem cells, giving rise to all mature blood cells throughout a person's life. In some embodiments, hematopoietic progenitor cells or hematopoietic stem cells are isolated from peripheral blood cells.

[0014] As used herein, the term “peripheral blood cells” refers to the cellular components of blood, including red blood cells, white blood cells, and platelets, found in the circulating blood pool. In some embodiments, eukaryotic cells are bone marrow-derived stem cells.

[0015] As used herein, the term "bone marrow-derived stem cells" refers to stem cells found in the bone marrow. Stem cells may reside in the bone marrow either as pluripotent adherent stromal cells or as cells expressing the CD34 or CD45 cell surface proteins that identify hematopoietic stem cells capable of differentiating into blood cells.

[0016] As used herein, the terms “mobilization” or “stem cell mobilization” refer to the process involved in the replenishment of stem cells from residing tissues or organs into the peripheral blood after treatment with a mobilizing agent. This process mimics the enhanced physiological release of stem cells from tissues or organs in response to stress signals during injury and inflammation. The mechanism of the mobilization process depends on the type of mobilizing agent administered. Some mobilizing agents act as agonists or antagonists, preventing stem cells from attaching to cells or tissues in their microenvironment. Other mobilizing agents induce the release of proteases that cleave adhesion molecules or supporting structures between stem cells and their attachment sites.

[0017] As used herein, the term "mobilizing agent" refers to a substance obtained from tissue or organs where stem cells are present, such as bone marrow (e.g., CD34). +This refers to a wide variety of molecules that act to enhance the recruitment of stem cells and spleen (e.g., Hox11-positive stem cells) into peripheral blood. Recruiters 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, and stromal cell-derived factor 1 (SDF-1); agonists of chemokine (CC motif) receptor 1 (CCR1), such as chemokine (CC motif) ligand 3 (CCL3, also known as macrophage inflammatory protein-1α (Mip-1α)); and chemokine (CXC motif) receptor Examples include agonists of pharmacokinetic 1 (CXCR1) and 2 (CXCR2), such as chemokine (CXC motif) ligand 2 (CXCL2) (also known as proliferation-associated oncogene protein-β (Gro-β)), and CXCL8 (also known as interleukin-8 (IL-8)); agonists of CXCR4, such as CTCE-02142, and Met-SDF-1; late-stage antigen (VLA)-4 inhibitors; antagonists of CXCR4, such as TG-0054, prelixafor (also known as AMD3100), and AMD3465, or any combination of the aforementioned agents. Mobilizers increase the number of stem cells in peripheral blood, thereby providing a more readily available source of stem cells for use in transplantation, organ repair or regeneration, or disease treatment.

[0018] As used herein, the term “isolated cells” refers to cells that have been removed from an organism in which they were originally observed, or that are descendants of such cells. In some cases, eukaryotic cells are cultured in vitro, for example, in the presence of other cells. In some cases, eukaryotic cells are later introduced into a second organism, or they (or their descendants) are reintroduced into the organism from which they were isolated. As used herein, the term “isolated population” refers to a population of cells that have been removed or separated from a mixed or heterogeneous population of cells. In some embodiments, the isolated population is substantially purer than the heterogeneous population (from which cells were isolated or concentrated).

[0019] As used herein, the terms “gamma globin” or “γ-globin” have their general meanings in the art and refer to the proteins encoded in humans by the HBG1 and HBG2 genes. The HBG1 and HBG2 genes are typically expressed in the fetal liver, spleen, and bone marrow. Two γ-globin chains, together with two α-globin chains, constitute fetal hemoglobin (HbF), which is usually replaced by adult hemoglobin (HbA) in the year following birth (Higgs DR, Vickers MA, Wilkie AO, Pretorius IM, Jarman AP, Weatherall DJ (May 1989). "A review of the molecular genetics of the human alpha-globin gene cluster". Blood. 73 (5): 1081-104.). The ENSEMBL IDs (i.e., gene identification numbers in the Ensemble Genome Browser database) for HBG1 and HBG2 are ENSG00000213934 and ENSG00000196565, respectively.

[0020] As used herein, the expression “increases fetal hemoglobin content” means that fetal hemoglobin is at least 5% higher in eukaryotic cells treated with a DNA-targeting endonuclease than in equivalent eukaryotic cells in which an endonuclease targeting an unrelated locus is present or absent. In some embodiments, the ratio of fetal hemoglobin expression in the eukaryotic cells is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 1x, at least 2x, at least 5x, at least 10x, at least 100x, at least 1000x or more than that of eukaryotic cells. In some embodiments, the increase in fetal hemoglobin expression can be measured using any method known in the art, such as high-performance liquid chromatography analysis of fetal γ-globin protein and RT-qPCR analysis of fetal γ-globin mRNA. Typically, the method is described in the examples.

[0021] As used herein, the term “expression” refers to the process by which a polynucleotide is transcribed from a DNA template (for example, into mRNA or other RNA transcripts), and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. Transcripts and the polypeptides they encode may collectively be referred to as “gene products.” If the polynucleotide originates from genomic DNA, expression may include the process of splicing mRNA within a eukaryotic cell.

[0022] As used herein, the term “promoter” has its general meaning in the art and refers to a nucleic acid sequence required for the expression of a gene operably linked to a promoter sequence. The HBG1 and HBG2 promoters are identical up to -221 bp and include the nucleic acid sequence shown in SEQ ID NO: 1 and Figure 1. According to the present invention, the first nucleotide in SEQ ID NO: 1 represents a nucleotide located at position -210 upstream of the HBG transcription start site, and the last nucleotide in SEQ ID NO: 1 represents a nucleotide located at position -100 upstream of the HBG transcription start site. For example, -The nucleotide at position -201 in the HBG1 or HBG2 promoter represents the nucleotide at position 10 of SEQ ID NO: 1. -The nucleotide at position -200 in the HBG1 or HBG2 promoter represents the nucleotide at position 11 of SEQ ID NO: 1. -The nucleotide at position -198 in the HBG1 or HBG2 promoter represents the nucleotide at position 13 of SEQ ID NO: 1. -The nucleotide at position -197 within the HBG1 or HBG2 promoter represents the nucleotide at position 14 of SEQ ID NO: 1. -The nucleotide at position -196 in the HBG1 or HBG2 promoter represents the nucleotide at position 15 of SEQ ID NO: 1, and -The nucleotide at position -195 in the HBG1 or HBG2 promoter represents the nucleotide at position 16 of SEQ ID NO: 1. -The nucleotide at position -194 in the HBG1 or HBG2 promoter represents the nucleotide at position 17 of SEQ ID NO: 1. -The nucleotide at position -175 in the HBG1 or HBG2 promoter represents the nucleotide at position 36 of SEQ ID NO: 1. -The nucleotide at position -117 in the HBG1 or HBG2 promoter represents the nucleotide at position 94 of SEQ ID NO: 1. -The nucleotide at position -116 in the HBG1 or HBG2 promoter represents the nucleotide at position 95 of SEQ ID NO: 1. -The nucleotide at position -115 in the HBG1 or HBG2 promoter represents the nucleotide at position 96 of SEQ ID NO: 1. -The nucleotide at position -114 in the HBG1 or HBG2 promoter represents the nucleotide at position 97 of SEQ ID NO: 1. The nucleotide at position -113 in the -HBG1 or HBG2 promoter corresponds to the nucleotide at position 98 of SEQ ID NO: 1.

[0023] Sequence ID 1:> Sequence of the promoter of HBG1 or HBG2 [ka]

[0024] In this specification, the "-200 region" within the promoter of HBG1 or HBG2 refers to the region encompassing the nucleotides at positions -197, -196, and -195, and thus relates to the region from the nucleotide at position 11 (i.e., -200) to the nucleotide at position 21 (i.e., -190) in SEQ ID NO: 1, more preferably the region from the nucleotide at position 14 to the nucleotide at position 16 in SEQ ID NO: 1.

[0025] As used herein, the "-115 region" within the promoter of HBG1 or HBG2 refers to the region encompassing the nucleotides at positions -116, -115, -114, and -113, and therefore relates to the region from the nucleotide at position 95 (i.e., -115) to the nucleotide at position 98 (i.e., -113) in SEQ ID NO: 1.

[0026] As used herein, the term “activator” refers to a transcription activator, which is a protein (transcription factor) that increases the gene transcription of one gene or a set of genes. Most activators are DNA-binding proteins that bind to enhancer or promoter proximal sequences. According to this disclosure, the activator is selected from the group consisting of KL1, TAL1, and GATA1.

[0027] As used herein, the term "activator-binding site" refers to a site on DNA to which the activator-binding site described in this disclosure binds. According to the present invention, the base editing enzyme of the present invention edits the genome sequence of a eukaryotic cell, thereby enabling the activator to bind to its activator-binding site.

[0028] As used herein, the term "KLF1" has its general meaning in the art and refers to the Kruppel-like factor 1 protein. The term is also known as EKLF; EKLF / KLF1. KLF1 is a hematopoietic-specific transcription factor that induces high levels of expression of adult β-globin and other erythroid genes. The zinc finger protein binds to the DNA sequence found in the β-hemoglobin promoter.

[0029] As used herein, the term "TAL1" has its general meaning in the art and refers to TAL bHLH transcription factor 1, an erythroid differentiation-inducing factor. This term is also known as SCL; TCL5; tal-1; and bHLHa17.

[0030] As used herein, the term "GATA1" has its general meaning in the art and refers to GATA-binding protein 1. This term is also known as GF1; GF-1; NFE1; XLTT; ERYF1; NF-E1; XLANP; XLTDA; and GATA-1. GATA1 is a protein belonging to the GATA transcription factor family. This protein plays a crucial role in erythrocyte development by regulating the switching from fetal hemoglobin to adult hemoglobin.

[0031] As used herein, the term “repressor” refers to a transcription repressor, which is a protein (transcription factor) that reduces the gene transcription of one gene or a set of genes. Most repressors are DNA-binding proteins that bind to enhancer or promoter proximal sequences. According to this disclosure, repressors are selected from the group consisting of BCL11A and LRF.

[0032] Therefore, the term “transcriptional repressor binding site” refers to a site on DNA to which a transcriptional repressor binds. In some embodiments, the base editing enzymes of the present invention edit the genome sequence of eukaryotic cells so that the transcriptional repressor cannot bind to its binding site. In some embodiments, the DNA-targeted endonucleases of the present invention will inhibit the binding of LRF or BCL11A to its binding site.

[0033] As used herein, the term "BCL11A" has its general meaning in the art and refers to the gene (gene identification number: 53335) encoding the BAF chromatin remodeling complex subunit BCL11A. This term is also known as EVI9;CTIP1;DILOS;ZNF856;HBFQTL5;BCL11A-L;BCL11A-S;BCL11a-M; or BCL11A-XL. Five alternatively spliced ​​transcriptional variants of this gene, encoding distinct isoforms, have been reported. This protein associates with the SWI / SNF complex, which regulates gene expression through chromatin remodeling. BCL11A is highly expressed in several hematopoietic lineages and plays a role in the transition from gamma-globin to beta-globin during fetal to adult development (Sankaran VJ et al. "Human fetal hemoglobin expression is regulated by the developmental stage-specific repressor BCL11A", Science Science. 2008 Dec 19;322(5909):1839-42).

[0034] As used herein, the term "LRF" has its general meaning in the art and refers to a transcriptional repressor, a leukemia / lymphoma-associated factor (LRF), encoded by the ZBTB7A gene. LRFs are ZBTB transcription factors that bind to DNA via a C-terminal C2H2 type zinc finger and possibly recruit transcriptional repressor complexes via their N-terminal BTB domain (Lee SU, Maeda T. Immunol. Rev. 2012;247:107-119).

[0035] As used herein, the terms “polypeptide,” “peptide,” and “protein” are synonymous and refer to amino acid polymers of any length. These polymers may be linear or branched, may contain modified amino acids, or may contain non-amino acids. The term also encompasses modified amino acid polymers; for example, those subjected to disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, PEGylation, or any other operation, such as conjugation with a labeling component. As used herein, the term “amino acid” includes natural and / or unnatural or synthetic amino acids, such as glycine and both D- and L-isomers, as well as amino acid analogs and peptide mimetic compounds.

[0036] As used herein, the terms “nucleic acid molecule” or “polynucleotide” refer to a DNA molecule (for example, but not limited to, cDNA or genomic DNA). A nucleic acid molecule may be single-stranded or double-stranded.

[0037] As used herein, the term “isolated” means, when referring to a nucleic acid molecule or polypeptide, that the nucleic acid molecule or polypeptide is substantially free from at least one other component that it associates with or is found together with in its natural state.

[0038] As used herein, the term “complementarity” refers to the ability of a nucleic acid to form hydrogen bonds (or groups thereof) with another nucleic acid sequence, either through traditional Watson-Crick base pairing or other non-traditional types. Complementarity indicates the ratio of residues within a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (for example, 5, 6, 7, 8, 9, and 10 out of 10 are 50%, 60%, 70%, 80%, 90%, and 100% complementary, respectively). “Fully complementary” means that all consecutive residues in a nucleic acid sequence will form hydrogen bonds with the same number of consecutive residues in the second nucleic acid sequence. As used herein, “substantially complementary” means 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 two nucleic acids that hybridize under stringent conditions.

[0039] As used herein, the term “stringent conditions” for hybridization refers to conditions under which a nucleic acid complementary to the target sequence hybridizes primarily to the target sequence and substantially not 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 the sequence specifically hybridizes to its target sequence. Non-restrictive examples of stringent conditions are detailed 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.

[0040] As used herein, the terms “hybridization” or “hybridized” refer to the process by which fully or partially complementary nucleic acid strands assemble under specific hybridization conditions to form a double-stranded structure or region in which the two constituent strands are connected 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 other base pairs may also be formed (e.g., Adams et al., The Biochemistry of the Nucleic Acids, 11th ed., 1992).

[0041] As used herein, the term “base editing enzyme” refers to a fusion protein containing an incomplete CRISPR / Cas nuclease linked to a deaminase polypeptide. The term is also known as a “base editor.” Two classes of base editing enzymes, namely cytosine base editing enzymes (CBEs) and adenine base editing enzymes (ABEs), can be used to edit single base pairs without double-strand breaks. Typically, cytosine base editing enzymes are produced by fusing an incomplete CRISPR / Cas nuclease to a cytidine deaminase such as APOBEC. These base editing enzymes are targeted by gRNA at specific loci, allowing them to convert cytidine to uridine within a small editing window near the PAM site. Uridine is then converted to thymidine through base break repair, producing a C-to-T change (or G-to-A on the opposite strand). Similarly, adenosine base editing enzymes are produced by condensing an incomplete CRISPR / Cas nuclease with adenosine deaminase. These base editing enzymes are engineered to convert adenosine to inosine, which is then processed by cells in the same way as guanosine, producing an A-to-G (or T-to-C) change.

[0042] As used herein, the terms “fusion polypeptide” or “fusion protein” mean a protein produced by linking two or more polypeptide sequences together. A fusion polypeptide as encompassed in this invention includes the translation product of a chimeric gene construct in which a nucleic acid sequence encoding a first polypeptide, e.g., an RNA-binding domain, is linked to a nucleic acid sequence encoding a second polypeptide, e.g., an effector domain, thereby forming a single open reading frame. In other words, a “fusion polypeptide” or “fusion protein” is a recombinant protein of two or more proteins linked by peptide bonds or via several peptides. A fusion protein may also include a peptide linker between the two domains.

[0043] As used herein, the term “linker” refers to any means, entity, or portion used to connect two or more entities. The linker may be a covalent linker or a non-covalent linker. An example of a covalent linker includes a covalent bond or linker moiety covalently attached to one or more proteins or domains to be linked. The linker may also be a non-covalent bond, such as an organometallic bond through a metal center, such as a platinum atom. For covalent bonding, various functional groups can be used, such as amide groups (including carboxylic acid derivatives), ethers, esters (including organic and inorganic esters), aminos, urethanes, ureas, etc. For linkage, domains may be modified by oxidation, hydroxylation, substitution, reduction, etc., to provide a site for coupling. Methods of conjugation are well known to those skilled in the art and are incorporated for use in the present invention. The linker moiety includes, but is not limited to, a chemical linker moiety or, for example, a peptide linker moiety (linker sequence). It will be understood that modifications that do not significantly reduce the function of the RNA-binding domain and effector domain are preferred.

[0044] As used herein, “conjugated” refers to two or more entities joining together to form a single entity. Conjugates encompass both peptide small molecule conjugates and peptide-protein / peptide conjugates.

[0045] As used herein, the term "nuclease" includes proteins (i.e., enzymes) that induce cleavage within a nucleic acid sequence, for example, single-strand or double-strand cleavage within a double-stranded DNA sequence.

[0046] As used herein, the term “CRISPR / Cas nuclease” has its general meaning in the art and refers to a segment of prokaryotic DNA containing clustered, regularly arranged short palindromic sequence repeats (CRISPR) and associated nucleases encoded by the Cas gene. In bacteria, the CRISPR / Cas locus encodes an RNA-induced adaptive immune system against mobile genetic elements (viruses, translocation factors, and conjugative plasmids). Three types of CRISPR systems have been identified. CRISPR clusters contain spacers, which are sequences complementary to the preceding mobile genetic elements. CRISPR clusters are transcribed and processed into mature CRISPR (clustered, regularly arranged short palindromic sequence repeat) RNA (crRNA). CRISPR / Cas nucleases Cas9 and Cpf1 belong to the type II and type V CRISPR / Cas systems and possess potent endonuclease activity for cleaving target DNA. Cas9 is induced by a trans-activated small RNA (tracrRNA) that acts as a guide for the processing of mature crRNA containing a specific target sequence (called a spacer) of approximately 20 nucleotides, and pre-crRNA assisted by ribonuclease III. The double strand of crRNA instructs Cas9 to target DNA via complementary base pairing between the spacer on the crRNA and a complementary sequence (called a protospacer) on the target DNA. Cas9 recognizes the protospacer-adjacent motif (PAM) of the trinucleotide (NGG in the case of Cas9 in Streptococcus pyogenes) and identifies the cleavage site (the nucleotide third or fourth upstream from the PAM).

[0047] As used herein, the terms “Cas9” or “Cas9 nuclease” refer to RNA-inducible nucleases containing the Cas9 protein or fragments thereof (e.g., proteins containing the active or inactive DNA cleavage domain and / or the gRNA-binding domain of Cas9). Cas9 nucleases are sometimes also referred to as cason1 nucleases or CRISPR (clustered, regularly arranged short palindromic sequence repeats)-associated nucleases. CRISPR is an adaptive immune system that provides defense against mobile genetic elements (viruses, translocation factors, and conjugative plasmids). CRISPR clusters contain spacers, which are sequences complementary to the preceding mobile element, and target the invading nucleic acid. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In the type II CRISPR system, the correct processing of precrRNA requires trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and the Cas9 protein. tracrRNA acts as a guide for the processing of precrRNA, assisted by ribonuclease 3. Subsequently, Cas9 / crRNA / tracrRNA cleaves a linear or circular double-stranded DNA target complementary to the spacer using an endonuclease. Target strands not complementary to the crRNA are first cleaved with an endonuclease and then shortened to 3'-5' with an exonuclease. In nature, DNA binding and cleavage typically require both proteins and RNAs. However, single guide RNAs ("sgRNAs," or simply "gRNAs") may be engineered to incorporate both aspects of crRNA and tracrRNA into a single RNA species. See, for example, Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E. Science 337:816-821 (2012), the entire content of which is incorporated herein by reference. Cas9 recognizes short motifs (PAM or protospacer-adjacent motifs) within CRISPR repeat sequences to help distinguish between self and non-self.Cas9 nuclease sequences and structures 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, Ajdic DJ, Savic DJ, Savic G., Lyon K., Primeaux C., Sezate S., Suvorov AN, Kenton S., Lai HS, Lin SP, Qian Y., Jia HG, Najar FZ, Ren Q., Zhu H., Song L., White J., Yuan X., Clifton SW, Roe BA, McLaughlin RE, Proc. Natl. Acad. Sci. USA 98:4658-4663(2001); “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” Deltcheva E., Chylinski K., Sharma CM, Gonzales K., Chao See 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), the entire contents of each of these are incorporated herein by reference). Orthologs of Cas9 have been described in various species, including but not limited to Streptococcus pyogenes and Streptococcus thermophilus (S. thermophilus).Further suitable Cas9 nucleases and sequences will become apparent to those skilled in the art based on this disclosure, and such Cas9 nucleases and sequences include Cas9 sequences derived from organisms and loci disclosed in Chylinski, Rhun, and Charpentier, “The tracrRNA and Cas9 families of type II CRISPR-Cas immune 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 reference numbers: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI reference numbers: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI reference number: NC_021284.1); Prevotella intermedia (NCBI reference number: NC_017861.1); Spiroplasma taiwanense (NCBI reference number: NC_021846.1); and Streptococcus inie. iniae) (NCBI reference number: NC_021314.1); Bellilla baltica (NCBI reference number: NC_018010.1); Psychroflexus torquis I (NCBI reference number: NC_018721.1); Streptococcus thermophilus (NCBI reference number: YP_820832.1); Listeria innocua (NCBI reference number: NP_472073.1); Campylobacter jejuni (NCBI reference number: YP_002344900.1); or Neisseria meningitidis (NCBI reference number: This refers to Cas9 derived from YP_002342100.1). Typically, Cas9 nucleases contain the amino acid sequence shown in Sequence ID No. 2.

[0048] Sequence ID 2: Cas9 sequence [ka]

[0049] As used herein, the term "incomplete CRISPR / Cas nuclease" refers to a CRISPR / Cas nuclease lacking at least one nuclease domain.

[0050] 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 double helix. Therefore, the term "Cas9 nickase" typically refers to a nickase derived from the Cas9 protein by inactivating one nuclease domain of the Cas9 protein.

[0051] As used herein, the term “deaminase” refers to an enzyme that catalyzes a deamination reaction. In some embodiments, the deaminase is a cytidine deaminase that catalyzes the hydrolytic deamination of cytidine or deoxycytidine to uracil or deoxyuracil, respectively. In some embodiments, the deaminase is an adenosine deaminase that catalyzes the hydrolytic deamination of adenosine to inosine, which is then processed by cells in the same way as guanosine to produce an A to G (or T to C) change.

[0052] As used herein, the term “guide RNA molecule” generally refers to an RNA molecule (or group of RNA molecules) that can bind to the Cas9 protein and target the Cas9 protein to a specific location within target DNA. Guide RNA may consist of 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 at least capable of hybridizing under stringent conditions to) the target sequence. The protein-binding segment interacts with a CRISPR protein, such as Cas9 or a Cas9-related polypeptide. These two segments may be located within the same RNA molecule or within two or more separate RNA molecules. When the two segments are in separate RNA molecules, the molecule containing the DNA targeting guide segment is sometimes referred to as CRISPR RNA (crRNA), while the molecule containing the protein-binding segment is referred to as transactivating RNA (tracrRNA).

[0053] As used herein, the terms “target nucleic acid” or “target” refer to a nucleic acid containing a target nucleic acid sequence. The target nucleic acid may be single-stranded or double-stranded, and is often double-stranded DNA. As used herein, “target nucleic acid sequence,” “target sequence,” or “target region” means a specific sequence or its complement that is to be ligated using a CRISPR system as disclosed herein.

[0054] As used herein, the term “target nucleic acid strand” refers to the target nucleic acid strand subjected to base pairing with a guide RNA as disclosed herein. That is, the target nucleic acid strand that hybridizes with crRNA and a 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), each strand can be a “target nucleic acid strand” for designing crRNA and a guide RNA, and can be used to carry out the method of the present invention, provided that a suitable PAM site is present.

[0055] As used herein, the term "non-nuclease DNA modifying enzyme" refers to an enzyme that is not a nuclease but can introduce some modification, such as a mutation, into a DNA molecule.

[0056] As used herein, the term "cytidine deaminase" refers to an enzyme that catalyzes the irreversible hydrolytic deamination of cytidine and deoxycytidine to uridine and deoxyuridine, respectively. As used herein, the term "deamination" refers to the removal of an amino group from a molecule.

[0057] In this specification, the term "AID," or "activation-induced cytidine deaminase," refers to an enzyme belonging to the APOBEC cytidine deaminase enzyme family. AIDs are expressed in activated B cells and are required to induce somatic hypermutations (Muramatsu et al., Cell, 102(5): 553-63 (2000); Revy et al., Cell, 102(5): 565-75 (2000); Yoshikawa et al., Science, 296(5575): 2033-6 (2002)) by creating point mutations in the underlying DNA that codes for antibody genes (Martin et al., Proc. Natl. Acad. Sci. USA., 99(19): 12304-12308 (2002) and Nature, 415(6873): (2002); Petersen-Mart et al., Nature, 418(6893): 99-103 (2002)) by creating point mutations in the underlying DNA that codes for antibody genes. AID is also an essential protein factor for class switch recombination and gene conversion (Muramatsu et al., Cell, 102(5): 553-63 (2000); Revy et al., Cell, 102(5): 565-75 (2000)).

[0058] As used herein, the terms “ribonucleoprotein complex” or “ribonucleoprotein particle” refer to a complex or particle containing a nucleoprotein and ribonucleic acid. A “nucleoprotein” as provided herein refers to a protein capable of binding to nucleic acids (e.g., RNA, DNA). When a nucleoprotein binds to ribonucleic acid, it is referred to as a “ribonucleoprotein.” Interactions between ribonucleoproteins and ribonucleic acid may be direct, for example by covalent bonds, or indirect, for example by non-covalent bonds (e.g., electrostatic interactions (e.g., ionic bonds, hydrogen bonds, halogen bonds), van der Waals interactions (e.g., dipole-dipole, dipole-induced dipole, London dispersion), ring stacking (Pi action), hydrophobic interactions, etc.).

[0059] As used herein, the term “wild type” is a technical term understood by those skilled in the art and means a typical, naturally occurring form of an organism, strain, gene, or characteristic, distinct from a mutant or variant form.

[0060] As used herein, the term “mutation” has its general meaning in the art and refers to substitution, deletion, or insertion. “Substitution” means that a specific amino acid residue is removed at a particular location and another amino acid residue is inserted at the same location. “Deletion” means that a specific amino acid residue is removed. “Insertion” means that one or more amino acid residues are inserted before or after a particular amino acid residue.

[0061] As used herein, the term “mutagenicity” refers to the introduction of mutations into a polynucleotide sequence. In accordance with the present invention, mutations are introduced into a target DNA molecule encoding the variable domain of an antibody, thereby mimicking somatic hypermutation.

[0062] As used herein, the term “mutant” refers to a first composition (e.g., the first molecule) in relation to a second composition (e.g., the second molecule, also called the “parent” molecule). The mutant molecule may be derived from, isolated from, based on, or homologous to the parent molecule. The mutant molecule may have total sequence identity with the original parent molecule, or alternatively, have less than 100% sequence identity with the parent molecule. For example, a variant of a sequence 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% identical in sequence to the original sequence. Sequence identity rates are frequently measured in terms of the rate of identity (or similarity or homology); the higher the rate, the more similar the two sequences are. Sequence alignment methods for comparison are well known in the art. Various programs and alignment algorithms are used in Smith and Waterman, Adv. Appl. Math., 2:482, 1981; Needleman and Wunsch, J. Mol. Biol., 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85:2444, 1988; Higgins and Sharp, Gene, 73:237-244, 1988; Higgins and Sharp, CABIOS, 5:151-153, 1989; Corpet et al. Nuc. Acids Res., 16:10881-10890, 1988; Huang et al., Comp. Appls Biosci., 8:155-165, 1992; and Pearson et al., Meth. Mol. Biol. It is described in 24:307-31, 1994.Altschul et al., Nat. Genet., 6:119-129, 1994, present a detailed discussion of sequence alignment methods and homology calculations. For example, sequence comparison can be performed using the alignment tools ALIGN (Myers and Miller, CABIOS 4:11-17, 1989) or LFATA (Pearson and Lipman, 1988) (Internet Program® 1996, WR Pearson and the University of Virginia, fasta20u63 version 2.0u63, released December 1996). ALIGN compares entire sequences, while LFATA compares locally similar regions. These alignment tools and their respective instruction manuals are available, for example, on the NCSA website. Alternatively, for comparing amino acid sequences longer than approximately 30 amino acids, the Blast2 sequence function can be used with the default BLOSUM62 matrix set to default parameters (gap presence cost 11, and gap cost per residue 1). When aligning short peptides (shorter than approximately 30 amino acids), alignment should be performed using the Blast2 sequence function with the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalty). The BLAST sequence comparison system is available, for example, from the NCBI website; see also Altschul et al., J. Mol. Biol., 215:403-410, 1990; Gish. & States, Nature Genet., 3:266-272, 1993; Madden et al. Meth. Enzymol., 266:131-141, 1996; Altschul et al., Nucleic Acids Res., 25:3389-3402, 1997; and Zhang & Madden, Genome Res., 7:649-656, 1997.

[0063] As used herein, the term "derived from" refers to a process of isolating, deriving, or preparing a second component (e.g., a second molecule distinct from the first molecule) using a first component (e.g., a first molecule) or information from a first component.

[0064] As used herein, the term “therapeutic effective dose” means a sufficient number of cell populations to treat a disease with a reasonable benefit-risk ratio applicable to any medical treatment. It will be understood that the total daily dose of the composition of the present invention will be determined by the attending physician within reasonable medical judgment. The specific therapeutic effective dose level for any particular patient will depend on a wide variety of factors, including the patient’s age, weight, overall health, sex, and diet, time of administration, route of administration, duration of treatment, drugs used in combination with or concurrently with the cell population, and similar factors well known in the medical field. In some embodiments, cells are formulated by first collecting them from their culture medium, and then washing and concentrating the cells in a medium and container system (“pharmaceutically acceptable” carrier) suitable for administration at the therapeutic effective dose. Suitable infusion media may be any isotonic media formulation, typically ordinary saline, Normosol R (Abbott), or Plasmalite A (Baxter), but a 5% dextrose aqueous solution or Ringer’s lactate solution may also be used. The infusion medium may be supplemented with human serum albumin. The effective treatment amount of cells in the composition depends on the relative proportion of cells with the desired specificity, the recipient's age and weight, and the severity of the condition being targeted. The amount of these cells can range from as low as about 10 3 / kg, preferably 5 × 10 3 / kg; up to 10 7 / kg, preferably 10 8The amount may be per kg. The number of cells will depend on the final intended use of the composition, as well as the type of cells contained therein. Typically, the minimum dose is 2 million cells / kg. Usually, 2 million to 20 million cells are injected into a subject. The desired purity can be achieved by introducing a sorting process. For the applications provided herein, the cells are generally in 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 reasonable number of cells may be distributed over multiple injections, cumulatively equivalent to or exceeding the desired total amount of cells.

[0065] method: Therefore, the first object of the present invention relates to a method for increasing the fetal hemoglobin content in eukaryotic cells, comprising the steps of contacting eukaryotic cells with a gene editing platform comprising (a) at least one base editing enzyme and (b) at least one guide RNA molecule for guiding the base editing enzyme to at least one target sequence in the HBG1 or HBG2 promoter, thereby editing the promoter, and subsequently increasing the expression of gamma globulin in the eukaryotic cells.

[0066] In some embodiments, the gene editing platform is suitable for introducing several mutations into the HBG1 or HBG2 promoter, thereby introducing a binding site for at least one transcription activator into the promoter. In some embodiments, the gene editing platform is particularly suitable for introducing a novel binding site for a transcription activator for KLF1, TAL1, or GATA1.

[0067] In some embodiments, the gene editing platform disclosed herein introduces a -198T>C mutation into the HBG1 or HBG2 promoter, thereby enabling the KLF1 activator to bind to the promoter.

[0068] In some embodiments, the gene editing platform disclosed herein introduces a -175T>C mutation into the HBG1 or HBG2 promoter, thereby enabling the TAL1 activator to bind to the promoter.

[0069] In some embodiments, the gene editing platform disclosed herein introduces a -113A>G mutation into the HBG1 or HBG2 promoter, thereby enabling the GATA1 activator to bind to the promoter.

[0070] In some embodiments, the gene editing disclosed herein is particularly suitable for editing the -200 region within the HBG1 or HBG2 promoter, thereby disrupting the binding site for the LRF repressor. In some embodiments, the gene editing platform disclosed herein introduces at least one mutation selected from the group consisting of -201C>T, -200C>T, -197C>T, -196C>T, -195C>T, and -194C>T, thereby disrupting the binding site for the LRF repressor.

[0071] In some embodiments, the gene editing disclosed herein is particularly suitable for editing the -115 region within the HBG1 or HBG2 promoter, thereby disrupting the 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, thereby disrupting the binding site for the BCL11A repressor.

[0072] In some embodiments, eukaryotic cells are selected from a group consisting of hematopoietic progenitor cells, hematopoietic stem cells (HSCs), and pluripotent cells (i.e., embryonic stem cells (ES) and induced pluripotent stem cells (iPS)). Typically, eukaryotic cells arise from the recruitment of stem cells.

[0073] In some embodiments, the base editing enzyme of the present invention comprises an incomplete CRISPR / Cas nuclease. The sequence recognition mechanism is the same as that of the incomplete CRISPR / Cas nuclease. Typically, the incomplete 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 hereafter herein. However, the incomplete CRISPR / Cas nuclease of the present invention is a modified form that has no nuclease activity at all. Therefore, the incomplete CRISPR / Cas nuclease specifically recognizes the guide RNA molecule and thus guides the base editing enzyme to its target DNA sequence.

[0074] In some embodiments, an incomplete CRISPR / Cas nuclease may be modified to enhance its binding affinity and / or specificity to nucleic acids, to alter its enzymatic activity, and / or to change another property of the protein. In some embodiments, the nuclease domain of the protein may be modified, deleted, or inactivated. In some embodiments, the protein may be cleaved and shortened to remove a domain that is not essential for the protein's function. In some embodiments, the protein may be cleaved and shortened or modified to optimize the activity of its RNA-binding domain.

[0075] In some embodiments, the CRISPR / Cas nuclease consists of a mutant CRISPR / Cas nuclease, i.e., a protein, fusion protein, or combination thereof having one or more point mutations, insertions, deletions, or cleavage shortenings. In some embodiments, the mutant has RNA-induced DNA-binding activity but lacks one or both of its nuclease active sites. In some embodiments, the mutant contains an amino acid sequence having at least 50% identity with the wild-type amino acid sequence of the CRISPR / Cas nuclease. Various CRISPR / Cas nucleases can be used in the present invention. Unrestrictive examples of suitable CRISPR / 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, Cse1 (or CasA), Cse2 (or CasB), Examples include 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. For example, see International Publication No. 2014 / 144761, International Publication No. 2014 / 144592, International Publication No. 2013 / 176772, U.S. Patent Application Publication No. 2014 / 0273226, and U.S. Patent Application Publication No. 2014 / 0273233 (their entire contents are incorporated herein by reference).

[0076] In some embodiments, the CRISPR / Cas nuclease is derived from the type II CRISPR-Cas system. In some embodiments, the CRISPR / Cas nuclease is derived from the Cas9 protein. The Cas9 protein is found in, in particular, pyogenic streptococci, Streptococcus thermophilus, streptococci, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, and Exiguobacterium sibiricum. Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp.), Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus cardus Caldus), Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ctenobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp.), Arthrospira maxima, Arthrospira platensis, Arthrospira genus (Arthrospira sp.), Lyngbya genus (Lyngbya sp.)It may be derived from *Microcoleus chthonoplastes*, the genus *Oscillatoria*, *Petrotoga mobilis*, *Thermosipho africanus*, or *Acaryochloris marina*.

[0077] In some embodiments, the CRISPR / Cas nuclease is a mutant or fragment of a wild-type CRISPR / Cas nuclease (e.g., Cas9). In some embodiments, the CRISPR / Cas nuclease is a mutant Cas9 protein derived from Streptococcus pyogenes.

[0078] Methods for producing Cas9 proteins (or fragments thereof) with inactive DNA cleavage domains are known (see, for example, 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: the HNH nuclease subdomain and the 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 D10A and H841A mutations completely inactivate the Cas9 nuclease activity of Streptococcus pyogenes (Jinek et al., Science. 337:816-821(2012); Qi et al., Cell. 28; 152(5):1173-83 (2013)).

[0079] In some embodiments, the CRISPR / Cas nuclease of the present invention is a nickase, more specifically a Cas9 nickase, i.e., a Cas9 derived from Streptococcus pyogenes having one mutation selected from the group consisting of D10A and H840A. In some embodiments, the nickase of the present invention comprises an amino acid sequence such as that shown in SEQ ID NO: 3 or SEQ ID NO: 33.

[0080] Sequence ID 3> Sequence of the nCas9 protein of Streptococcus pyogenes with the D10A mutation [ka]

[0081] Sequence ID 33> Sequence of the nCas9 protein of Streptococcus pyogenes with the H840A mutation [ka]

[0082] In some embodiments, Cas9 mutants having mutations other than D10A or H840A are used, thereby obtaining, for example, Cas9 with an inactivated nuclease (dCas9). Such mutations include, for example, other amino acid substitutions in D10 and H840, or other substitutions within the nuclease domain of Cas9 (e.g., substitutions within the HNH nuclease domain and / or the RuvC1 subdomain). In some embodiments, mutants 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 SEQ ID NO: 3. In some embodiments, variants of dCas9 are provided that have an amino acid sequence that is 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 SEQ ID NO: 3.

[0083] According to the present invention, the second component of the base editing enzyme disclosed herein includes a deaminase, which is a DNA modifying enzyme that is not a nuclease.

[0084] In some embodiments, the deaminase is cytidine deaminase. In some embodiments, the deaminase is a deaminase of the apolipoprotein B mRNA editing complex (APOBEC) family. In some embodiments, the deaminase is a deaminase of the APOBEC1 family. In some embodiments, the deaminase is cytidine deaminase (AID) that is induced by activation. In some embodiments, the deaminase is ACF1 / ASE deaminase.

[0085] In some embodiments, the deaminase is: AID: Cytidine deaminase induced by activation, APOBEC1: mRNA editing enzyme for apolipoprotein B, catalytic polypeptide-like 1, APOBEC3A: mRNA editing enzyme for apolipoprotein B, catalytic polypeptide-like 3A, APOBEC3B: mRNA editing enzyme for apolipoprotein B, catalytic polypeptide-like 3B, APOBEC3C: mRNA editing enzyme for apolipoprotein B, catalytic polypeptide-like 3C, APOBEC3D: mRNA editing enzyme for apolipoprotein B, catalytic polypeptide-like 3D, APOBEC3F: mRNA editing enzyme for apolipoprotein B The following enzymes are selected from the group consisting of catalytic polypeptide-like enzyme 3F, APOBEC3G: mRNA editing enzyme for apolipoprotein B, catalytic polypeptide-like enzyme 3G, APOBEC3H: mRNA editing enzyme for apolipoprotein B, catalytic polypeptide-like enzyme 3H, ADA: adenosine deaminase, ADAR1: adenosine deaminase acting on RNA1, Dnmt1: DNA(cytosine-5-)-methyltransferase 1, Dnmt3a: DNA(cytosine-5-)-methyltransferase 3α, Dnmt3b: DNA(cytosine-5-)-methyltransferase 3β, and Tet1: methylcytosine dioxygenase.

[0086] In some embodiments, the deaminase is derived from cytidine deaminase (AID), which is induced by activation. AID is a cytidine deaminase capable of catalyzing the deamination of cytosine in the context of DNA or RNA. Upon reaching a target site, AID changes the cytidine base to a uridine base. In dividing cells, this can lead to a point mutation from cytosine to thymidine. Alternatively, the change from C to U may trigger an intracellular DNA repair pathway, primarily the cleavage repair pathway, which would remove the UG base pair mismatch and replace it with a TA pair, AT pair, CG pair, or GC pair. As a result, a point mutation would occur at the target CG site. In some embodiments, the DNA modifying enzyme is AID*Δ, which is an AID mutant with increased somatic hypermutation activity and denucleated nuclear export signaling (NES) (Hess GT, Fresard L, Han K, Lee CH, Li A, Cimprich KA, Montgomery SB, Bassik MC: Directed evolution using dCas9-targeted somatic hypermutation in mammalian cells. Nat Methods 2016, 13(12):1036-1042).

[0087] In some embodiments, the deaminase is an adenosine deaminase. In some embodiments, the deaminase is a deaminase of the ADAT family. For example, an adenosine deaminase of the ADAT family can be fused to a Cas9 protein, such as a nuclease-inactivating Cas9 domain, thereby producing a Cas9-ADAT fusion protein.

[0088] In some embodiments, the deaminase consists of amino acid sequence variants as shown in SEQ ID NOs: 4-14.

[0089] Sequence ID 4 Human AID: [ka]

[0090] Sequence ID 5: Human APOBEC-3G [ka]

[0091] Sequence ID 6: Human APOBEC-3F [ka]

[0092] Sequence ID 7 Human APOBEC-3B [ka]

[0093] Sequence ID 8 Human APOBEC-3C [ka]

[0094] Sequence ID 9: Human APOBEC-3A [ka]

[0095] Sequence ID No. 10 Human APOBEC-3H [ka]

[0096] Sequence ID 11: Human APOBEC-3D [ka]

[0097] Sequence ID 12: Human APOBEC-1 [ka]

[0098] Sequence ID 13: Human ADAT-2 [ka]

[0099] Sequence ID 14 Human ADAT-1 [ka]

[0100] In some embodiments, the deaminase is fused to the N-terminus of an incomplete CRISPR / Cas nuclease. In some embodiments, the deaminase is fused to the C-terminus of an incomplete CRISPR / Cas nuclease. In some embodiments, the incomplete CRISPR / Cas nuclease and the deaminase are fused via a linker. In some embodiments, the linker includes the motifs (GGGGS)n (SEQ ID NO: 3), (G)n, (EAAAK)n (SEQ ID NO: 4), (GGS)n, and SGSETPGTSESATPES (SEQ ID NO: 5) (see, for example, Guilinger JP, Thompson DB, and Liu DR). Additional suitable linker motifs and linker structures will be apparent to those skilled in the art. In some embodiments, suitable linker motifs and linker structures are those described in Chen et al., Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev. 2013; 65(10):1357-69 (the entire contents of which are incorporated herein by reference).

[0101] In some embodiments, the fusion protein may include additional features. Other exemplary features that may be present include localization sequences, such as nuclear localization sequences (NLS), cytoplasmic localization sequences, export sequences, such as nuclear export sequences, or other localization sequences, as well as sequence tags useful for solubilization, purification, or detection of the fusion protein. Suitable localization signal sequences and protein tag sequences are provided herein, including, but are not limited to, biotin carboxylase carrier protein (BCCP) tags, myc tags, calmodulin tags, FLAG tags, hemagglutinin (HA) tags, polyhistidine tags (also known as histidine tags or His tags), maltose-binding protein (MBP) tags, nus tags, glutathione-S-transferase (GST) tags, green fluorescent protein (GFP) tags, thioredoxin tags, S tags, Softag (e.g., Softag1, Softag3), streptavidin tags, biotin lineage tags, FIAsH tags, V5 tags, and streptavidin-binding peptide tags. Additional suitable features will be apparent to those skilled in the art.

[0102] Various base-editing enzymes are known in the art (see, for example, Improving cytidine and adenine base-editing enzymes by expression optimization and ancestral reconstruction. Nat Biotechnol. 2018 May 29), and these typically include those listed in Table A.

[0103] [Table 1]

[0104] The second component of the gene editing platform disclosed herein comprises at least one guide RNA molecule suitable for directing a base editing enzyme to at least one target sequence located within the HBG1 or HBG2 promoter. Therefore, the guide RNA molecule of the present invention includes a guide sequence for constituting targeting specificity. It includes a region that is complementary to and hybridizable to a pre-selected target site of interest within the HBG1 or HBG2 promoter.

[0105] In some embodiments, this guide sequence may contain approximately 10 to more than 25 nucleotides. For example, the base-pairing region between the guide sequence and the corresponding target site sequence may be approximately 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 approximately 17 to 20 nucleotides in length, for example, 20 nucleotides.

[0106] Typically, a software program is used to identify candidate CRISPR target sequences on both strands of a DNA nucleic acid molecule containing the HBG gene, based on the desired guide sequence length and the CRISPR motif sequence (PAM) for a specific CRISPR enzyme. One requirement for selecting a suitable target nucleic acid is that it has a 3' PAM site / sequence. Each target sequence and its corresponding PAM site / sequence are referred to herein as Cas targeting sites. One of the best-characterized systems, the Type II CRISPR system, requires only the Cas9 protein and a guide RNA complementary to the target sequence to cause target cleavage. For example, a target site for Cas9 derived from Streptococcus pyogenes with the PAM sequence NGG can be identified by searching for 5'-Nx-NGG-3' on both the input sequence and its reverse complement. Because the presence of multiple DNA target sites in the genome can lead to nonspecific genome editing, after identifying all possible sites, the program filters out sequences based on the number of occurrences in the relevant reference genome. For CRISPR enzymes where sequence specificity is determined by a “seed” sequence, such as 11-12 bp 5' from the PAM sequence, including the PAM sequence itself, the filtering step may be based on the seed sequence. Therefore, to avoid editing at additional genomic loci, the results are filtered based on the number of occurrences of the seed:PAM sequence in the relevant genome. The user may be allowed to select the length of the seed sequence. The user may also be allowed to identify the number of occurrences of the seed:PAM sequence in the genome for the purpose of passing through the filter. The default is to screen for specific sequences. The filter level changes with changes in both the length of the seed sequence and the number of occurrences of the sequence in the genome. The program may, additionally or alternatively, provide complementary guide sequences to the reported target sequences(s) by providing their reverse complements. Further details of methods and algorithms for optimizing sequence selection can be found in U.S. Patent Application No. 61 / 836,080, which is incorporated herein by reference.

[0107] In some embodiments, the base editing enzyme and the corresponding guide RNA molecule are selected according to Table B.

[0108] [Table 2]

[0109] 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 more in length) using terminal-continuing RNA (TC-RNA) chemical reactions (see, for example, U.S. Patent No. 8,202,983) makes it possible to produce RNAs with special characteristics superior to those made possible by RNAs consisting of the four basic ribonucleotides (A, C, G, and U). In particular, the RNA molecules of the present invention can be prepared using recombinant techniques with host cell systems or in vitro translation-transcription systems known in the art. Details of such systems and technologies can be found, for example, in International Publication Nos. 2014 / 144761, 2014 / 144592, 2013 / 176772, U.S. Patent Application Publication Nos. 2014 / 0273226 and U.S. Patent Application Publication Nos. 2014 / 0273233, the contents of which are incorporated herein by reference in their entirety.

[0110] In some embodiments, the guide RNA molecule may include one or more modifications. Such modifications may include the inclusion of at least one non-natural nucleotide, or a modified nucleotide, or an analog thereof. The modified nucleotide may be modified in the ribose, phosphate, and / or base moiety. The modified nucleotide may include a 2'-O-methyl analog, a 2'-deoxy analog, or a 2'-fluoro analog. The nucleic acid backbone may be modified, for example, a phosphorothioate backbone may be used. The use of locked nucleic acid (LNA) or cross-linked nucleic acid (BNA) may also be possible. Further examples of modified bases include, but are not limited to, 2-aminopurine, 5-bromouridine, pseudouridine, inosine, and 7-methylguanosine.

[0111] In some embodiments, multiple guide RNA molecules are designed to target multiple sequences within the HBG1 or HBG2 promoter. In some embodiments, the gene editing platform disclosed herein thus comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 20 guide RNA molecules, as disclosed herein.

[0112] In some embodiments, multiple base editing enzymes are designed to target multiple sequences within the HBG1 or HBG2 promoter, along with multiple guide RNA molecules. In some embodiments, the gene editing platform disclosed herein thus comprises two, three, or four base editing enzymes and two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, or twenty RNA molecules, as disclosed herein.

[0113] In some embodiments, various components of the gene editing platform of the present invention are delivered 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 introducing them into eukaryotic cells. The vectors are typically prokaryotic cell vectors, e.g., plasmids, or shuttle vectors or insertion vectors for the storage or manipulation of nucleic acids encoding the guide RNA molecules or base editing enzymes disclosed herein. Preferably, the nucleic acids are isolated and / or purified. Accordingly, the present invention provides recombinant constructs or vectors having the sequences encoding one or more of the above-mentioned guide RNA molecules or base editing enzymes. Examples of constructs include vectors, e.g., plasmids or viral vectors, in which the nucleic acid sequences of the present invention are inserted in the forward or reverse direction. In some embodiments, the constructs further include regulatory sequences. The "regulatory sequences" include promoters, enhancers, and other expression control sequences (e.g., polyadenylation signals). The regulatory sequences include nucleic acid sequences and sequences that instruct the constitutive expression of inducible regulatory sequences. The design of expression vectors may depend on factors such as the selection of eukaryotic cells to be transformed, transfected, or infected, and the desired expression level. 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 cell hosts are also described in Sambrook et al. (2001, Molecular Cloning: A Manual of Experiments, Cold Spring Harbor Press). Vectors can replicate autonomously or be incorporated into host DNA. Vectors may also contain sequences suitable for amplifying expression. Furthermore, expression vectors preferably contain one or more selection marker genes that result in phenotypic characteristics for the selection of transformed host cells, such as dihydrofolate reductase or neomycin resistance for eukaryotic cell cultures, or tetracycline or ampicillin resistance in E. coli. Any procedure known in the art for introducing exogenous nucleotide sequences into host cells may be used.Examples include the use of calcium phosphate transfection, polyblens, protoplast fusion, electroporation, nucleofection, liposomes, microinjection, naked DNA, plasmid vectors, viral vectors (both episomal and embedded), and any other well-known method for introducing cloned genomic DNA, cDNA, synthetic DNA, or other exogenous genetic material into host cells.

[0114] In some embodiments, various components of the gene editing platform of the present invention are provided to a cell population through the use of RNA-encoded systems. For example, a base editing system may be provided to a cell population through the use of an adenine or cytidine base editor encoded by chemically modified mRNA, together with a modified guide RNA, such as that 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, a base editing enzyme (e.g., ABE) system encoded by engineered RNA is prepared by introducing various chemical modifications into both mRNA encoding the base editing enzyme and the guide RNA. In particular, the modification consists of uridine-deleting mRNA modified with 5-methoxyuridine. Synonymous codons may be introduced to delete as much uridine as possible without altering the coding sequence, and all remaining uridine may be substituted with 5-methoxyuridine. The aforementioned optimized base editing system exhibits higher editing efficiency at several genomic sites compared to DNA-encoded systems. Modified mRNA and guide RNA can also be encapsulated in lipid nanoparticles (LNPs) to enable LNP-mediated delivery.

[0115] In some embodiments, various components of the gene editing platform of the present invention are delivered to a cell population through the use of ribonucleoprotein (RNP) complexes. For example, a base editing enzyme can form a ribonucleoprotein (RNP) complex by pre-complexing it with one or more guide RNA molecules. Thus, the RNP complex can be introduced into eukaryotic cells. The introduction of the RNA complex can be time-set. 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 delivery by mRNA, DNA, or viruses. Typically, the RNA complex is prepared simply by mixing a protein (i.e., a base editing enzyme) and one or more guide RNA molecules in a suitable buffer. This mixture is incubated at room temperature for 5-10 minutes before electroporation. Electroporation is a delivery technique that applies an electric field to one or more cells to increase the permeability of the cell membrane. In some embodiments, gene editing efficiency can be improved by the addition of transfection-enhancing oligonucleotides.

[0116] In some embodiments, multiple sequential transfections are performed to achieve a desired level of mutagenesis within the cell.

[0117] A further object of the present invention relates to a method for increasing fetal hemoglobin levels in a subject requiring such a method, the method comprising the step of transplanting a therapeutically effective amount of eukaryotic cell population obtained by the method described above.

[0118] In some embodiments, the cell population is self-identical to the subject, meaning that the cell population originates from the same subject.

[0119] In some embodiments, the subjects are diagnosed with hemoglobin disorders. Therefore, the method of the present invention is particularly suitable for the treatment of hemoglobin disorders.

[0120] In some embodiments, β-hemoglobin disorders are sickle cell disease.

[0121] In some embodiments, the hemoglobin disorder is β-thalassemia.

[0122] kit The present invention further provides a kit containing reagents for carrying out the above method, comprising all components of a gene editing platform as disclosed herein for carrying out mutagenesis. To achieve this objective, one or more reaction components, e.g., guide RNA molecules and nucleic acid molecules encoding a base editing enzyme for the method disclosed herein, may be supplied in the form of a kit for use. In some embodiments, the kit comprises one or more base editing enzymes and one or more guide RNA molecules. In some embodiments, the kit may comprise 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 maintained 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 the host cells, one or more reagents (e.g., probes or PCR primers) for detecting the expression of the guide RNA or base editing enzyme or for confirming the state of the target nucleic acid, and a buffer or culture medium for the reaction. The kit may also comprise one or more of the following components: supporting reagents, termination reagents, modification reagents, or digestion reagents, osmotic modifiers, and detection instruments. The components used may be provided in various forms. For example, the components (e.g., enzymes, RNA, probes and / or primers) may be suspended in an aqueous solution, or as freeze-dried or lyophilized powders, pellets, or beads. In the latter case, the components, when restored, form a complete mixture of components for use in the assay. The kits of the present invention may be provided at any suitable temperature. For example, in the storage of kits containing protein components or complexes thereof in liquid, they are preferably provided and maintained below 0°C, preferably -20°C or below, or otherwise in a frozen state. The kit may also include packaging materials for holding containers or combinations of containers. Typical packaging materials for such kits and systems include a solid matrix (e.g., glass, plastic, paper, foil, microparticles, etc.) that holds the reactive components or detection probes in one of a wide variety of structures (e.g., vials, microtiter plate wells, microarrays, etc.).The kit may further include instructions for use of the ingredients, recorded in a tangible form.

[0123] The present invention will be further illustrated with reference to the following drawings and embodiments. However, these embodiments and drawings should not be construed as limiting the scope of the present invention in any way. [Examples]

[0124] Examples: method: Cell line culture K562 cells were maintained in RPMI1640 (Lonza), supplemented with glutamine-containing 10% fetal bovine serum (Lonza), 2 mM Hepes (Life Technologies), 100 nM sodium pyruvate (Life Technologies), and penicillin and streptomycin (Life Technologies). HUDEP-2 cells were treated with 1 μg / mL doxycycline (Sigma-Ace), 10 -6 The cells were cultured in StemSpan SFEM (StemCell Technologies) supplemented with M dexamethasone (Sigma-Ace), 100 ng / mL human stem cell factor (SCF) (Peprotec), 3 IU / mL erythropoietin (Necker Hospital Pharmacy), L-glutamine (Life Technologies), and penicillin / streptomycin. HUDEP-2 cells were differentiated over 9 days in Iskov-modified Dulbecco's medium (IMDM) (Life Technologies) supplemented with 330 μg / mL holo-transferrin (Sigma-Technologies), 10 μg / mL recombinant human insulin (Sigma-Technologies), 2 IU / mL heparin (Sigma-Technologies), 5% human type AB serum, 3 IU / mL erythropoietin, 100 ng / mL human stem cell factor, 1 μg / mL doxycycline, 1% L-glutamine, and 1% penicillin / streptomycin. Erythroid differentiation was monitored by flow cytometry analysis of CD36, CD71, and GYPA surface markers, as well as standard May-Grünwald-Giemsa staining.

[0125] Purification and cultivation of HSPC The inventors of this invention have identified human umbilical cord blood (CB) CD34 + HSPCs are obtained from healthy donors and from unmobilized human peripheral blood CD34 + HSPCs were obtained from SCD patients. Eligible cord blood samples for research purposes were customarily obtained from the cord blood bank at St. Louis Hospital (Paris, France). Eligible SCD samples for research purposes were customarily obtained from Necker Children's Hospital (Paris, France). Written informed consent was obtained from all adult subjects. All experiments were conducted in accordance with the Declaration of Helsinki. The study was approved by the Regional Review Board (Reference number: DC2014-2272, CPP Ile-de-France II, Necker Children's Hospital). HSPCs were purified by immunomagnetic selection using AutoMACS (Milteny Biotech) after immunostaining with a CD34 microbead kit. CD34 was used 48 hours prior to transfection. + cells (10 6 Cells (100 ng / ml) were thawed and cultured in "HSPC medium" containing StemSpan (Stem Cell Technologies) supplemented with penicillin / streptomycin (Gibco), 250 nM StemRegenin 1 (Stem Cell Technologies), and the following recombinant human cytokines (Peprotec): 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).

[0126] plasmid The plasmids used in this study were pCMV_ABEmax_P2A_GFP (Adgene 112101), pCMV_AncBE4max_P2A_GFP (Adgene 112100), pBT374 (Adgene 125615), pBT372 (Adgene 125613), pCMV-ABEmaxAW (Adgene 125647), and pMJ (Adgene). Examples include AncBE4max_NAA plasmid (Adgene, No. 42234), ABE8e (Adgene, No. 138489), pCMV-BE4max-NRCH (Adgene, No. 136920), pCAG-CBE4max-SpG-P2A-EGFP (RTW4552) (Adgene, No. 139998), and pCAG-CBE4max-SpRY-P2A-EGFP (RTW5133) (Adgene, No. 139999). The AncBE4max_NAA plasmid was created by substituting the PAM interaction domain of SpCas9n with one of the SmaCas9 molecules, while the AncBE4max_R33A / K34A plasmid was created by inserting the R33A and K34A mutations into the APOBEC1 domain of the AncBE4max plasmid (Adgene, No. 112094). A DNA fragment (3'UTR+PolyA) containing two copies of the 3' untranslated region (UTR) of the HBB gene and 96 adenine polyA sequences was purchased from GenScript (gene synthesis). Similarly, a DNA fragment containing the coding sequence of pCAG-CBE4max-SpRY-P2A-EGFP with a uridine deletion was constructed (CBE-SpRY_U-delp). The CBE-SpRY-OPT plasmid was constructed by inserting the 3'UTR+PolyA fragment into pCAG-CBE4max-SpRY-P2A-EGFP and replacing CBE4max-SpRY with the CBE-SpRY_U-delp fragment. Furthermore, the inventors replaced the CAG synthesis promoter of the CBE-SpRY plasmid with the T7 promoter. The ABE-SpRY-OPT plasmid was created by inserting a 3'UTR+ poly(A) fragment into pCMV-T7-ABEmax(7.10)-SpRY-P2A-EGFP(RTW5025) (Adgene, product code 140003).

[0127] γRNA design To construct the gRNA expression plasmid, oligonucleotides were annealed to create a gRNA protospacer, and the double-stranded gRNA was ligated to a Bbs I-digested MA128 plasmid (provided by M. Amendola of Genethon, France).

[0128] [Table 3]

[0129] mRNA transcription in vitro Plasmids expressing 10 μg of CBE-SpRY-OPT, ABE-SpRY-OPT, or ABE8e were digested overnight with 20 units of restriction enzyme, cleaved once immediately after the poly-A tail (AflII for CBE-SpRY, and ABE-SpRY and SapI for ABE8e). The chain-like plasmids were purified using a PCR purification kit (Qiagen, No. 28106) and eluted in 30 μl of DNase / RNase-free water. 1 μg of the chain-like plasmid was used as a template for the in vitro transcription reaction (MEGAscript, Ambion, No. AM1334). The in vitro transcription 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, N-7003) was used at a final concentration of 12.0 mM, resulting in a 4:1 cap:GTP final ratio that allowed for efficient mRNA capping. The incubation time for in vitro reactions was reduced to 30 minutes. After treatment with deoxynuclease I (MEGAscript, Ambion, AM1334), a poly-A tail was attached to the ABE8e mRNA according to the manufacturer's protocol (Poly-A tailing kit, Ambion, AM1350). The mRNA was precipitated using lithium chloride and resuspended in a final volume of TE buffer that allowed for concentrations of 1 μg / μl or higher. mRNA quality was confirmed using a bioanalyzer (Agilent).

[0130] Plasmid transfection K562 cells and HUDEP-2 cells (10 6Cells (individual cells / conditions) were transfected with a plasmid expressing 3.6 μg of a nucleotide editing enzyme and a plasmid containing 1.2 μg of gRNA. For nucleotide editing enzyme plasmids that did not express GFP, the inventors co-transfected them with a plasmid expressing 250 ng of GFPmax (Lonza). For K562 and HUDEP-2 cells, the inventors used the AMAXA cell line nucleofector kit V (VCA-1003) and the U-16 and L-29 programs (Nucleofector II), respectively. In K562 cells, transfection efficiency was evaluated by flow cytometry 18 hours after transfection using a Fortessa X20 (BD Biosciences) flow cytometer. Cells were maintained in culture medium for at least 3 days, and genomic DNA extraction was performed on day 3. GFP + HUDEP-2 cells were sorted 18 hours after transfection using an SH800 cell sorter (Sony Biotechnology), and the sorted cells were grown in culture medium. Genomic DNA extraction was performed 3 days after transfection. CD34 + HSPC(10 6 Cells (individual cells / conditions) were transfected with a plasmid expressing 3.6 μg of the enzyme and a plasmid containing 2.4 μg of gRNA. For base editing enzyme plasmids that do not express GFP, the inventors co-transfected them with 250 ng of GFPmax expression plasmid (Lonza). The inventors used the AMAXA Human CD34 Cell Nucleofector Kit (VPA-1003) and the U-08 program (Nucleofector II). 18 hours after transfection, GFP + CD34 +HSPCs were sorted using an SH800 cell sorter (Sony Biotechnology), seeded in cytokine-enriched HSPC medium (described above) at a concentration of 500,000 / mL for at least 6 days, and then either genomic DNA extraction was performed, or differentiation into mature RBCs was carried out using a three-phase erythroid differentiation protocol (Weber, Frati Science Advances 2020) for up to 20 days. Genomic DNA extraction was performed on day 6, total RNA extraction was performed on day 13, and functional analysis (flow cytometry, RP-HPLC, CE-HPLC, and sickle cell assay) to evaluate HbF expression was performed on day 19.

[0131] RNA transfection K562 cells (2 x 10 5 Cells (individual cells / conditions) were transfected at a final concentration of 1.5 μM with 2.0 μg of mRNA expressing a single base editor and synthetic gRNA purchased from Synthego containing chemical modifications (2'-O-methyl in the first three bases and the last base, and a 3' phosphorothioate bond between the first three bases and the last two bases). The inventors used the P3 Primary Cell 4D-Nucleofector X Kit S (Lonza) and the CA137 program (Nucleofector 4D). Cells transfected with TE buffer served as negative controls. RNA-transfected K562 cells were maintained in culture medium for at least 3 days before genomic DNA extraction and base editing analysis.

[0132] CD34 + HSPC (2×10 5Cells (100,000 cells / condition) were transfected at a final concentration of 4.6 μM with 3.0 μg of single-nucleotide editor expression mRNA and synthetic gRNA containing chemical modifications purchased from Synthego (2'-O-methyl in the first three bases and the last base, and a 3'-phosphorothioate bond between the first three bases and the last two bases). The inventors used the P3 Primary Cell 4D-Nucleofector X Kit S (Lonza) and the CA137 program (Nucleofector 4D). Cells transfected in TE buffer, or with base editor mRNA alone, or with base editor mRNA and gRNA targeting the AAVS1 locus served as negative controls. RNA-transfected HSPCs were seeded in HSPC medium (above) at a concentration of 500,000 cells / mL and cultured for at least 6 days before genomic DNA extraction and base editing analysis.

[0133] Evaluation of editing efficiency Genomic DNA was obtained 3 days after transfection for K562 cells and HUDEP2 cells, and CD34 + At HSPC, six days after transfection, DNA was extracted from control and edited cells using the PURE LINK Genomic DNA Mini-Kit (Life Technologies, Inc.) according to the manufacturer's instructions. To evaluate the efficiency of base editing at gRNA target sites, the inventors performed PCR, followed by Sanger sequencing and EditR analysis (EditR: a method for quantifying base editing from Sanger sequencing). (27) TIDE analysis (Tracking of InDels by Decomposition) was also performed to evaluate the ratio of insertions and deletions (indels) in the base-edited samples. (28) .

[0134] [Table 4]

[0135] The frequency of 4.9kb deletions was quantified by amplifying HBG1-HBG2 intervening region I or II, respectively, using digital droplet PCR (ddPCR) with either the EvaGreen mix or primer / probe mix (Bio-Rad). The short extension time (approximately 1 minute) allowed for PCR amplification of the genomic region containing the deletion. Control primers that anneal to the same chromosome (chromosome 11) or human albumin (hALB) (chromosome 4) were used as DNA loading controls.

[0136] [Table 5]

[0137] Flow cytometry analysis Differentiated HUDEP-2 cells were fixed and permeabilized using BD Cytofix / Cytoperm solution (BD Pharmingen), and stained with an antibody that recognizes HbF (anti-HbF antibody conjugated to allophycocyanin (APC), MHF05, Life Technologies). Flow cytometry analysis of CD36, CD71, and GYPA erythrocyte surface markers was performed using anti-CD36 antibody conjugated to V450 (561535, BD Horizon), anti-CD71 antibody conjugated to FITC (555536, BD Pharmingen), and anti-GYPA antibody conjugated to PE-Cy7 (563666, BD Pharmingen). Control cells and SCD RBCs differentiated from edited HSPCs were fixed with 0.05% glutaraldehyde, permeabilized with 0.1% Triton X-100, and stained with an antibody that recognizes HbF (anti-HbF antibody conjugated to FITC, clone 2D12 552829 BD). Flow cytometry analysis of CD36, CD71, GYPA, BAND3, and α4-integrin erythrocyte surface markers was performed using anti-CD36 antibody conjugated to V450 (561535, BD Horizon), anti-CD71 antibody conjugated to fluorescein isothiocyanate (FITC) (555536, BD Pharmingen), anti-GYPA antibody conjugated to phycoerythrin-cyanine 7 (PE-Cy7) (563666, BD Pharmingen), anti-BAND3 antibody conjugated to phycoerythrin (9439, IBGRL), and anti-CD49d antibody conjugated to allophycocyanin (559881, BD). Flow cytometry analysis of DRAQ5 (enucleation) and 7AAD (viability) was performed using anti-double-stranded DNA dyes (65-0880-96, Invitrogen, and 559925, BD, respectively). Flow cytometry analysis was performed using a Fortessa X20 (BD Biosciences) or a Galios flow cytometer. Data were analyzed using FlowJo (BD Biosciences) or KALUZA software.

[0138] Colony-forming cell (CFC) assay HSPC was mixed in methylcellulose-containing culture medium (GFH4435, Stem Cell Technologies) in a 1 x 10⁶ mixture. 3 Cells were seeded at a concentration of 10 cells / mL under conditions supporting erythroblast and granulocyte differentiation. Colonies of erythroblast burst-forming cells (BFU-E) and granulocyte-macrophage colony-forming cells (CFU-GM) were scored after 14 days. BFU-E and CFU-GM were randomly selected and collected as bulk populations (containing at least 30 colonies), and hemoglobin expression was evaluated by cation exchange-high-performance liquid chromatography (CE-HPLC).

[0139] RT-qPCR analysis of globin transcripts Total RNA was extracted from HUDEP-2 cells (days 0, 6, and 9 of differentiation) and erythroid cells differentiated from SCD HSPC (day 13) using the RNeasy Microkit (Qiagen) according to the manufacturer's instructions. Mature transcripts were reverse transcribed using the SuperScript First-Strand Synthesis System for RT-qPCR (Invitrogen) with oligo(dT) primers. RT-qPCR was performed using the iTaq Universal SYBR Green Master Mix (Bio-Rad) and the Viia7 Real-Time PCR System (Thermo Fisher Scientific).

[0140] [Table 6]

[0141] Analysis of globin chains by RP-HPLC RP-HPLC analysis was performed using a NexeraX2SIL-30AC chromatograph and LC solution software (Shimadzu). Globin chains were separated by HPLC using a 250 × 4.6 mm, 3.6 μm Aeris Widepore column (Phenomenex). The sample was eluted using a gradient mixture of solution A (water / acetonitrile / trifluoroacetic acid, 95:5:0.1) and solution B (water / acetonitrile / trifluoroacetic acid, 5:95:0.1). Absorbance was measured at 220 nm.

[0142] CE-HPLC analysis of hemoglobin tetramers Cation exchange HPLC analysis was performed using a NexeraX2 SIL-30AC chromatograph and LC Solution software (Shimadzu). Hemoglobin tetramers were separated by HPLC using two cation exchange columns (Polycat A, PolyLC, Columbia, Maryland). Samples were eluted using a gradient mixture of solution A (20 mM Bistris, 2 mM KCN, pH 6.5) and solution B (20 mM Bistris, 2 mM KCN, 250 mM NaCl, pH 6.8). Absorbance was measured at 415 nm.

[0143] Sickle disease assay At the end of erythroid differentiation, mature RBCs derived from SCD HSPCs were incubated under hypoxic conditions (0% oxygen), and the sickle-forming process was monitored in real time using a video microscope with an AxioObserver Z1 microscope (Zeiss) and a 40x objective lens, with images taken every 20 minutes for at least 60 minutes. Images of the same field of view were taken throughout all stages and processed in ImageJ to determine the ratio of non-sickle-forming RBCs per acquired field of view in the entire RBC population. More than 400 cells were counted per condition.

[0144] result: Efficient base editing in the HBG promoter creates novel binding sites for the transcription activators of TAL1 and KLF1. The -175T>C HPFH mutation has been shown to recruit the TAL1 transcription activator to the HBG promoter. (6) On the other hand, the -198T>C HPFH mutation recruits the KLF1 transcription activator. (7) The inventors used gRNAs (TAL1_bs_1 and KLF1_bs_1) (Figure 1) that can target the bases at positions -175 and -198 within the HBG promoter. The target bases for these gRNAs are at positions 3 and 7, respectively. Transfection of the K562 erythroleukemia cell line with the ABEmax_GFP plasmid and the TAL1_bs_1 or KLF1_bs_1 gRNA plasmid resulted in A>G conversion (T>C on the opposite strand) with efficiencies of 61.0% and 30.3%, respectively, in the bulk cell population (Figures 2A, 2B, and 2Q).

[0145] The formation of binding sites for TAL1 and KLF1 leads to the deinhibition of γ-globin. K562 cells primarily express γ-globin, and for this reason, they cannot be used as a model for measuring γ-globin derepression. Therefore, we used the HUDEP-2 adult erythroid progenitor cell line to evaluate γ-globin derepression after the creation of binding sites for TAL1 and KLF1 activators. Following plasmid transfection using either the ABEmax_GFP plasmid or the TAL1_bs_1 or KLF1_bs_1 gRNA plasmid, GFP +HUDEP2 cells were selected and amplified. Base editing efficiencies were 65% and 45% at position -175 and -198, respectively (Figures 3A, 3B, and 3J). Control cells and edited cells were differentiated into mature erythrocytes. Flow cytometry analysis of cells edited at position -175 and -198 revealed a high frequency of HbF-expressing cells (76.0% and 80.3% on day 0 of differentiation, and 85.0% and 88.1% on day 9 of differentiation), while in the control population transfected only with the ABEmax_GFP plasmid, HbF-expressing cells accounted for approximately 3.0% (Figure 4A). Therefore, we observed an increase in γ-globin transcript production and a parallel decrease in adult β-globin mRNA (Figure 4B). Reverse-phase high-performance liquid chromatography (RP-HPLC) confirmed a significant increase in γ-globin and a simultaneous decrease in β-globin production (Figure 4C). The generation of TAL1 and KLF1 binding sites resulted in high HbF levels, accounting for up to 53.5% and 30.0% of total Hb, respectively, as determined by cation exchange HPLC (CE-HPLC) (Figure 4D). Base editing of the HBG1 / 2 promoter did not alter the differentiation of erythroid cells, as evaluated by flow cytometry analysis of the erythroid markers GYPA, CD36, and CD71 (Figures 4E-4G).

[0146] Base editing in the -115 region of the HBG promoter disrupts the binding site for the BCL11A transcription repressor and simultaneously creates a binding site for the GATA1 transcription activator. HPFH mutations in the -115 region of the HBG promoter cause elevated HbF expression by disrupting the binding site for the BCL11A repressor (-114C>T) or by creating a binding site for the GATA1 activator (-113A>G). We designed gRNAs (BCL11A_bs_1 and BCL11A_bs_2) that can be used by either ABE or CBE to produce these HPFH mutations (-114C>T and -113A>G) and additional HPFH-like mutations (-116A>G and -115C>T) (Figure 1). The target bases correspond to the reference base editing window. In particular, the -116, -115, -114, and -113 bases are located at positions 5-8 and 4-7, respectively, in gRNAs BCL11A_bs_1 and BCL11A_bs_2. After transfection of the K562 cell line with the ABEmax_GFP and gRNA BCL11A_bs_1 plasmid, the inventors successfully obtained A>G conversion at positions -116 and -113 (40.7% and 34.3%, respectively) (Figures 2C and 2Q). Transfection of the same gRNA BCL11A_bs_1 plasmid with the AncBE4max_GFP plasmid resulted in C>T conversion at positions -115 and -114 (34.8% and 28.5%, respectively) (Figures 2D and 2Q). In an attempt to increase the enzymatic options for disrupting the binding site of the BCL11A transcription repressor, the inventors transfected the K562 cell line with two plasmids, evoCDA1-BE4max-NG or evoFERNY-BE4max-NG, which are two enzymes that recognize NG PAM, in combination with two gRNAs that target the BCL11A binding site (BCL11A_bs_1 and BCL11A_bs_2).All four different combinations yielded the same C>T conversion (-115C>T and -114C>T), with efficiencies ranging from 18% to 40.5% (evoCDA1-BE4max-NG - BCL11A_bs_1; 40.5% -115C>T and 31.5% -114C>T, evoCDA1-BE4max-NG - BCL11A_bs_2; 30.0% -115C>T and 22.0% -114C>T, evoFERNY-BE4max-NG - BCL11A_bs_1; 22.0% -115C>T and 21.5% -114C>T, and evoFERNY-BE4max-NG - BCL11A_bs_2; 18.0% -115C>T and 19.0% -114C>T) (Figures 2E, 2F, and 2Q).

[0147] NGG-free base editors can disrupt the binding site of LRF transcription repressors by editing multiple bases and creating HPFH and HPFH-like mutations. The -200 region of the HBG promoter contains various HPFH mutations associated with high γ-globin expression in adulthood. Most of these mutations derepress the HBG gene by disrupting its binding site, thereby reducing its ability to bind to the LRF transcription repressor. The LRF binding site contains eight cytosines, all of which can theoretically be targeted by base editing to be converted to thymine. As a result, numerous HPFH mutations and additional mutations that can induce HPFH-like phenotypes by impairing LRF binding ability can be created (Figure 1). The absence of a reference SpyCas9 NGG PAM close to the LRF binding site prompted us to create a base editing enzyme containing an NGG-free Cas9 variant that enables editing of this site. This Cas9 variant recognizes an NAA PAM, which is ideal for targeting the LRF binding site. This is because it allows for the design of a gRNA (LRF_bs_2) with target bases (eight cytosines) positioned at positions 2-11. After the exchange of the PAM-interacting domain, the resulting enzyme (called AncBE4max_NAA) was combined with the LRF_bs_2 gRNA plasmid and transfected into the K562 cell line as a plasmid. The inventors were able to modify seven of the eight cytosines at the LRF binding site with an efficiency of up to 37.0% (8.7% of -202C>T; 20.3% of -201C>T; 37.0% of -200C>T; 30.7% of -197C>T; 27.7% of -196C>T; 16.3% of -195C>T; 5.7% of -194C>T) (Figures 2G and 2Q). Using the evoCDA1-BE4max-NG or evoFERNY-BE4max-NG enzymes, one or more gRNAs (LRF_bs_1) were designed to target the LRF binding site (Figure 1). Using these combinations, the inventors were able to target six of the eight cytosines in the motif, which are located at positions 1-8.Transfection of the K562 cell line with the LRF_bs_1 gRNA plasmid and either the evoCDA1-BE4max-NG or evoFERNY-BE4max-NG enzyme resulted in 8.0-28.5% (8.0% of -201C>T; 22.5% of -200C>T; 28.0% of -197C>T) with the evoCDA1-BE4max-NG enzyme. The efficiencies observed were 7.5% for -196C>T, 28.5% for -195C>T, and 21.0% for -194C>T, while the evoFERNY-BE4max-NG enzyme showed efficiencies ranging from 15.0% to 32.5% (28.0% for -197C>T, 32.5% for -196C>T, 25.5% for -195C>T, and 15.0% for -194C>T) (Figures 2H, 2I, and 2Q). The same gRNA was tested using more efficient NGG-free base editors, such as CBE-NRCH, CBE-SpG, and CBE-SpRY. When combined with LRF_bs_1 gRNA, CBE-NRCH, CBE-SpG, and CBE-SpRY yielded C>T efficiencies of up to 50.3%, 43.7%, and 46.3%, respectively, during plasmid transfection into K562 cells (Figures 2J-L and 2Q). Given the PAM-free nature of CBE-SpRY, combining this base editor with LRF_bs_2 gRNA resulted in an efficiency of 58.0%, surpassing all the enzymes mentioned above. Importantly, thanks to its wide editing window, CBE-SpRY converted all cytosines in the -200 region (Figures 2M and 2Q). Finally, the ABE8e enzyme plasmid was cotransfected into K562 cells along with KLF1_bs_1 gRNA, resulting in A>G modifications of both -198 A:T bp and -199 A:T bp with an efficiency of up to 72.7%, and consequently disrupting the LRF binding site (Figures 2N and 2Q).

[0148] HbF is reactivated by disrupting the binding sites of BCL11A and LRF transcription repressors through base editing. Previously reported base-editing approaches to target the -115 and -200 regions of the HBG promoter by generating HPFH and HPFH-like mutations were tested in the HUDEP-2 cell line to evaluate γ-globin derepression after disruption of the BCL11A and LRF repressor binding sites, and / or after creation of the GATA1 transcription activator binding site. Plasmids expressing four different enzymes (ABEmax_GFP, AncBE4max_GFP, evoCDA1-BE4max-NG, and evoFERNY-BE4max-NG) for both regions were individually transfected into HUDEP-2 cells in combination with single gRNA expression plasmids (BCL11A_bs_1, BCL11A_bs_2, LRF_bs_1, and LRF_bs_2). For plasmids that do not express GFP (evoCDA1-BE4max-NG and evoFERNY-BE4max-NG), a small amount of GFPmax expression plasmid was co-transfected. After transfection, GFP + Cells were sorted using FACS, amplified in culture medium, and differentiated into erythrocytes.

[0149] Editing the BCL11A binding site using the ABEmax_GFP enzyme and BCL11A_bs_1 gRNA resulted in conversions to -116A>G and -113A>G at frequencies of 56.0% and 57.0%, respectively, disrupting the BCL11A binding site and simultaneously creating a GATA1 binding site (Figures 3C and 3J). Using the AncBE4max_GFP enzyme combined with BCL11A_bs_1 gRNA, the inventors successfully generated HPFH (38.0% -114C>T) and HPFH-like (47.0% -115C>T) mutations (Figures 3D and 3J). Base editing using NGG-free PAM enzymes (evoCDA1-BE4max-NG and evoFERNY-BE4max-NG) in combination with either BCL11A_bs_1 or BCL11A_bs_2 gRNA was effective only when evoCDA1-BE4max-NG was used. This resulted in -115C>T and -114C>T conversions (40.0% and 27.0%, respectively) when using BCL11A_bs_1 gRNA, and -115C>T and -114C>T conversions (24.0% and 15.0%, respectively) when using BCL11A_bs_2 gRNA (Figures 3E, 3F, and 3J). Base editing of the BCL11A binding site using the above enzyme was evaluated by flow cytometry analysis of the erythrocyte markers GYPA, CD36, and CD71, and did not affect erythrocyte differentiation (Figures 5A-5C). Flow cytometry analysis showed an increased frequency of HbF-expressing cells (up to 87.8%) (Figure 5D). RT-qPCR analysis confirmed the increased production of γ-globin transcript and the simultaneous decrease in β-globin transcript production, consistent with the flow cytometry data (Figure 5E). RP-HPLC and CE-HPLC analysis confirmed these data, showing that HbF accounted for up to 31.8% of total Hb (Figures 5F-5G).

[0150] By using evoCDA1-BE4max-NG and evoFERNY-BE4max-NG enzymes combined with LRF_bs_1 gRNA to target the binding site of LRF suppressors, the evoCDA1-BE4max-NG enzyme achieved a base editing efficiency of up to 24.0% (4.0% of -201C>T; 24.0% of -197C>T; 23.0% of -196C>T; 23.0% of -195C>T; 17.0% of -194C>T), and evoFERNY-B The E4max-NG enzyme yielded base editing efficiencies of up to 20.0% (20.0% of -197C>T; 20.0% of -196C>T; 5.0% of -195C>T; 1.0% of -194C>T) (Figures 3G, 3H, and 3J), and simultaneously, HbF-expressing cells increased to 12.9% (3.5% in unedited control cells) and 21.4% (6.7% in unedited control cells), respectively (Figure 6A). Subsequently, the inventors developed a method using CD34 derived from primary umbilical cord blood. + Hematopoietic stem / progenitor cells (HSPCs) were used. CD34 + Cells were transfected with AncBE4max_NAA, LRF_bs_1 gRNA, and GFPmax plasmids, and selected for GFP expression. Cells were maintained in liquid culture medium or seeded on semi-solid media that allow proliferation and differentiation of erythroblasts and granulocyte-monocyte progenitor cells (colony-forming unit assay). Six days after transfection, the inventors obtained a conversion efficiency of 19.0% to -200C>T in liquid culture medium (Figures 6B-6C). Fourteen days after transfection, cation exchange HPLC analysis was performed to detect hemoglobin tetramers in erythroblast burst-forming cell (BFU-E) colonies derived from erythroblast progenitor cells (Figure 6D). This analysis revealed an 11.02% increase in HbF levels (68.22% in control samples, 79.24% in edited samples) (Figure 6D). In short, these data demonstrate that the novel AncBE4max_NAA base editing enzyme can target the binding site of LRF transcription repressors in primary HSPCs, thereby increasing HbF expression in those erythroblast progenitor cells.

[0151] Indels and large deletions were almost undetectable in cells with edited bases. One of the safety issues that arises with the use of CRISPR / Cas9 nucleases is the creation of insertions and deletions (indels) in the genome after double-strand breaks occur. However, the inventors can overcome this problem by using a base editing system because the base editor contains inactivated Cas9 nuclease. The inventors wanted to confirm that the base editing enzymes they used did not create double-strand breaks in the genome. For this reason, the inventors amplified the target region by PCR and performed Sanger sequencing, followed by TIDE analysis, on the base-edited samples and the control K562 sample. (28) (Figures 2O, 3I, and 6B). For almost all samples, the inventors detected no indels at all, except for cells transfected with the ABE8e and KLF1_bs_1 gRNA plasmid and the evoCDA1-BE4max-NG and LRF_bs_2 gRNA plasmid, which showed average indel percentages of 18.4% and 22.0%, respectively (Figure 2O).

[0152] Another problem that arises when editing the β-globin locus using Cas9 nuclease is the simultaneous cleavage of the same HBG1 / 2 promoter, which results in the deletion of the intervening 4.9kb genomic region and the loss of the HBG2 gene. Therefore, we tested whether the 4.9kb deletion is present in base-edited K562 cells. According to the indel profiles of the base-edited samples (Figure 2O), we observed low-frequency 4.9kb deletions only in samples treated with ABEmax-, ABE8e-, and evoFERNY-BE4max-NG (4.9%, 4.9%, and 3.2%, respectively; Figure 2P). As a positive control for the 4.9kb deletion, we used DNA extracted from K562 cells edited with standard Cas9 nuclease (Figure 2P).

[0153] By using enzymes with low RNA off-target activity, it is possible to target the binding sites of HBG repressors and activators. Base editing enzymes can cause off-target editing of intracellular RNA, mostly independent of gRNA. Mutations in the base editing enzyme deaminase can minimize RNA off-target editing. In adenine base editors, these mutations include E59A within the TadA domain, and TadA * The V106W mutation is within the domain, and enzymes with these mutations are called ABEmaxAW. (29) The mutations in the cytosine base editor are R33A and K34A within the APOBEC1 domain. (30) By inserting these mutations into the AncBE4max enzyme, the inventors created the AncBE4max_R33A / K34A base editing enzyme. The inventors' objective was to determine whether these enzymes, which exhibit low RNA off-target editing, could be used to create binding sites for TAL1 and KLF1 activators, or to disrupt the binding site for the BCL11A repressor. Transfection of K562 cells with the ABEmaxAW plasmid and TAL1_bs_1 or KLF1_bs_1 gRNA plasmids resulted in -175T>C (A>G relative to the opposite strand) and T>C (A>G relative to the opposite strand) conversions at frequencies of 25.0% and 17.0%, respectively (Figures 7A and 7B). Targeting the BCL11A binding site with ABEmaxAW in combination with BCL11A_bs_1 gRNA resulted in 36.0% -116A>G and 33.0% A>G conversions (Figure 7C), while targeting the same site with the same gRNA and the AncBE4max_R33A / K34A enzyme resulted in -115C>T and -114C>T conversions at frequencies of 29.0% and 24.0%, respectively (Figure 7D). In short, these data demonstrate that the binding sites of transcription activators or repressors within the HBG promoter can be efficiently targeted by using these safer forms of base editing enzymes.

[0154] In SCD HSPC, disruption of the LRF repressor binding site within the HBG promoter by CBE leads to HbF reactivation, rescuing the sickle phenotype. To demonstrate the effectiveness of our base-editing approach as a therapeutic strategy for treating SCD, we transfected unrecruited primary human adult SCD HSPCs with plasmids expressing base editors and plasmids expressing gRNAs. In particular, plasmids expressing CBE enzymes (CBE-NRCH, CBE-SpG-GFP, CBE-SpRY-GFP) were individually transfected in combination with single gRNA expression plasmids (LRF_bs_1, LRF_bs_2). To enrich the edited cells, we used plasmids expressing base editor-GFP fusions (CBE-SpG-GFP, CBE-SpRY-GFP), or we co-transfected plasmids expressing base editors (CBE-NRCH) and plasmids expressing GFPmax. GFP differentiated towards the erythrocyte lineage. 高い Cells were sorted by FACS using a three-phase protocol.

[0155] Base editing efficiency was measured in erythroblasts at the end of the first phase of erythroid differentiation (day 6). Samples treated with CBE and LRF_bs_1 gRNA, which convert four cytosines (LRF 4C) at the LRF binding site, showed editing efficiencies of approximately 22.4% (26.8%, 23.8%, and 16.5% using CBE-NRCH, CBE-SpG, and CBE-SpRY, respectively) (Figure 8A). In samples treated with CBE-SpRY and LRF_bs_2, all cytosines at the LRF binding site were converted to thymidine with an efficiency of up to 25.5% (Figure 8B). TIDE analysis of the base-edited samples confirmed the absence of indels (Figure 8C).

[0156] Subsequently, the inventors differentiated a bulk population of edited erythroblasts into mature RBCs and evaluated the recovery of HbF expression and sickle cell phenotype. Erythroid differentiation was measured along the differentiation process by flow cytometry analysis of late (CD36, CD71, and α4-integrin) and early (GYPA and BAND3) erythroid markers and was similar between the control and CBE-treated samples (data not shown). Enucleation rates were similar across the groups at various time points during differentiation, reaching over 90% in all samples by the end of the final phase (data not shown). CBE-treated LRF 4C and LRF 8C samples showed reactivation of fetal hemoglobin at both mRNA and protein levels when measured by RT-qPCR, RP-HPLC (Figures 8D and 8E), and CE-HPLC (22.3% and 9.1% HbF in edited and control samples, respectively, Figure 8F). Flow cytometry analysis revealed high frequencies of HbF-expressing RBCs (42.9%, 64.3%, and 70.0% in the control, LRF 4C, and LRF 8C samples, respectively; Figure 8G). To evaluate the effect of HbF reactivation on the sickle phenotype, we incubated mature RBCs under hypoxic conditions to induce HbS polymerization. Interestingly, editing of 4 or 8 cytosines in the LRF binding site relieved the sickle phenotype (23.2%, 52.5%, and 58.4% of non-sickle cells in the control, LRF 4C, and LRF 8C samples, respectively) (Figure 8H). Overall, these data demonstrate that base editing of the HBG1 / 2 promoter by CBE can lead to HbF reactivation and rescue the sickle phenotype in RBCs differentiated from HSPCs of SCD patients.

[0157] In SCD HSPC, ABE disrupts the binding site of the LRF inhibitor in the HBG promoter, or generates a KLF1 activator, leading to HbF activation and rescuing the sickle phenotype. ABE can also be used to disrupt the binding site of LRF transcription repressors or to create a binding site for KLF1 transcription activators. To demonstrate the therapeutic potential of ABE, the inventors performed the same set of experiments for CBE as described in the previous paragraphs in unmobilized primary human adult SCD HSPCs. More specifically, plasmids expressing ABEmax-GFP or ABE8e were individually transfected in combination with plasmids expressing a single gRNA (KLF1_bs_1 gRNA or AAVS1 gRNA targeting unrelated AAVS1 loci; Weber et al., Sc. Advances, 2020). To enhance the edited cells, the inventors used plasmids expressing the ABEmax-GFP fusion protein, or co-transfected with ABE8e-expressing plasmids and GFPmax-expressing plasmids. After transfection, GFP 中間 and GFP 高い Cells were sorted using FACS to obtain a diverse population of cells with varying editing efficiencies. The sorted cells were then fully differentiated into mature RBCs using a three-phase protocol.

[0158] Base editing efficiency was measured in erythroblasts at the end of the first phase of erythroid differentiation (day 6). Samples treated with ABEmax (which generates a KLF1 binding site, KLF1) and ABE8e (which converts two thymidines at the LRF binding site; LRF 2T) showed GFP 中間 and GFP 高い In the bulk population, efficiencies ranged from 41.0% to 52.3% and 56.5% to 76.0%, respectively (Figures 9A-9D). TIDE analysis of the base-edited samples confirmed the absence of indels in cells treated with ABEmax (Figure 9E), while moderate indel frequencies of 7.9% and 14.8% were observed in GFP. 中間 and GFP 高い These were detected in samples treated with ABE8e (Figure 9E).

[0159] HbF expression and recovery of sickle cell phenotype were evaluated by differentiating a bulk population from edited erythroblasts to mature RBCs. Erythroid differentiation was measured along the differentiation process by flow cytometry analysis of late (CD36, CD71, and α4-integrin) and early (GYPA and BAND3) erythroid markers and was similar between control and ABE-treated samples (data not shown). Enucleation rates were similar across groups at various time points during differentiation, reaching over 90% in all samples by the end of the final phase (data not shown). ABE-treated samples with either a KLF1 binding site or an LRF 2T profile expressed high HbF levels as measured by CE-HPLC (66.3% and 62.6%, respectively) (Figure 9H). These results were confirmed at the mRNA and single globin chain levels by RT-qPCR and RP-HPLC (Figures 9F and 9G). Flow cytometry analysis revealed RBCs expressing HbF at high frequencies (60.4%, ≥94.2%, and ≥81.4% in the control, ABEmax-treated, and ABE8e-treated samples, respectively) (Figure 9J). Sickling assays were performed on the control and edited samples. Corrected cells with high frequencies were observed in the ABEmax-treated and ABE8e-treated samples (14.7%, 75.7%, and 60.6% non-sickling cells in the control, ABEmax-treated, and ABE8e-treated samples, respectively) (Figure 9I). Overall, this study demonstrates that reactivation of fetal hemoglobin occurs by either disrupting the LRF inhibitory binding site or creating a KLF1 activator binding site within the -200 region of the HBG1 / 2 promoter using ABE, thereby rescuing the sickle phenotype in RBCs differentiated from HSPCs in SCD patients.

[0160] Efficient RNA-mediated editing of the HBG1 / 2 promoter in K562 cells and SCD HSPC To establish a clinically valid method for delivering a base editing system to primary HSPCs and achieve high editing efficiency with minimal toxicity, the inventors optimized a protocol based on the transfection of base editor-encoding mRNA and modified synthetic gRNA. First, the inventors optimized plasmids encoding CBE-SpRY and ABE-SpRY for in vitro transcription and mRNA generation. In particular, the inventors incorporated two copies of the 3' untranslated region (UTR) of the HBB gene (which has been shown to extend the mRNA half-life and improve protein levels). 31~33 ) and by inserting the polyA sequence after the 3'UTR, mRNA in the CBE-SpRY and ABE-SpRY constructs 34 It was further stabilized.

[0161] Next, the inventors performed in vitro mRNA transcription using plasmids of CBE-SpRY-OPT, ABE-SpRY-OPT, and ABE8e. In K562 cells, high base editing efficiency was obtained by transfection of CBE-SpRY, ABE-SpRY, and ABE8e mRNA together with modified synthetic gRNAs of LRF_bs_2, KLF1_bs_1, or BCL11A_bs_1, demonstrating that the inventors have produced fully functional mRNAs. In particular, transfection of CBE-SpRY mRNA in combination with LRF_bs_2 or BCL11A_bs_1 gRNA resulted in C>T conversions of 87.0% and 83.0%, respectively (Figures 10A and 10B). Similarly, transfection with ABE-SpRY mRNA and KLF1_bs_ or BCL11A_bs_1gRNA resulted in A>G conversions of 55.0% and 39.0%, respectively. Finally, co-transfection with ABE8e mRNA and BCL11A_bs_1gRNA yielded a base editing efficiency of 88.0% (Figures 10C-10E).

[0162] CBE-SpRY and ABE8e mRNA were also transfected into SCD HSPC in combination with chemically modified single gRNAs. CBE-SpRY mRNA combined with LRF_bs_1 or LRF_bs_2 gRNA resulted in 51.0% and 61.0% C>T conversion, respectively, while ABE8e mRNA combined with KLF1_bs_1 gRNA resulted in 75.0% A>G conversion (Figures 10F-10H). These results demonstrate that RNA-mediated delivery of base editors enables efficient targeting of the HBG1 / 2 promoter in SCD HSPC.

[0163] References: Throughout this application, various references describe the latest technology in the art to which the present invention pertains. The disclosures of these references are incorporated herein by reference.

[0164] [Table 7] TIFF0007856581000023.tif248165 TIFF0007856581000024.tif154165

Claims

1. A method for increasing the fetal hemoglobin content in isolated eukaryotic cells, comprising the steps of: contacting isolated eukaryotic cells with a gene editing platform comprising (a) at least one base editing enzyme and (b) at least one guide RNA molecule for guiding the base editing enzyme to at least one target sequence in the HBG1 or HBG2 promoter, thereby editing the promoter, and subsequently increasing the expression of gamma globulin in the eukaryotic cells; The gene editing platform introduces a -198T>C mutation into the HBG1 or HBG2 promoter, thereby enabling the KLF1 activator to bind to the promoter; or The gene editing is used to edit the -200 region within the HBG1 or HBG2 promoter, thereby disrupting the binding site to the LRF suppressor, and, The base editing enzyme and the corresponding guide RNA molecule are as follows: The base editing enzyme is ABEmax, and the corresponding guide RNA molecule is the sequence shown in Sequence ID No. 49; The base editing enzyme is CBE-NRCH, and the corresponding guide RNA molecule is the sequence shown in Sequence ID No. 52; The base editing enzyme is CBE-SpG, and the corresponding guide RNA molecule is the sequence shown in Sequence ID No. 52; The base editing enzyme is CBE-SpRY, and the corresponding guide RNA molecule is the sequence shown in Sequence ID No. 52; and The base editing enzyme is CBE-SpRY, and the corresponding guide RNA molecule is the sequence shown in Sequence ID No. 53; A method selected from the group consisting of the following.

2. The method according to claim 1, wherein the gene editing platform introduces at least one mutation selected from the group consisting of -201C>T, -200C>T, -197C>T, -196C>T, -195C>T, and -194C>T into the HBG1 or HBG2 promoter, thereby disrupting the binding site to the LRF suppressor.

3. The method according to claim 1, wherein the eukaryotic cells are selected from the group consisting of hematopoietic progenitor cells, hematopoietic stem cells (HSCs), and pluripotent cells (i.e., embryonic stem cells (ES) and induced pluripotent stem cells (iPS)).

4. The method according to claim 1, wherein multiple guide RNA molecules are designed to target multiple sequences within the HBG1 or HBG2 promoter.

5. The method according to claim 1, wherein multiple base editing enzymes, along with multiple guide RNA molecules, are designed to target multiple sequences within the HBG1 or HBG2 promoter.

6. A pharmaceutical composition for increasing fetal hemoglobin levels in a subject who requires it, comprising a therapeutically effective amount of a population of eukaryotic cells obtained by the method described in any one of claims 1 to 5.

7. The method according to claim 1, wherein the eukaryotic cells are derived from a subject diagnosed with a hemoglobin disorder such as sickle cell disease or β-thalassemia.

8. A kit for increasing the fetal hemoglobin content in eukaryotic cells, comprising a gene editing platform comprising (a) at least one base editing enzyme and (b) at least one guide RNA molecule for guiding the base editing enzyme to at least one target sequence within the HBG1 or HBG2 promoter, The gene editing platform introduces a -198T>C mutation into the HBG1 or HBG2 promoter, thereby enabling the KLF1 activator to bind to the promoter; or The gene editing is used to edit the -200 region within the HBG1 or HBG2 promoter, thereby disrupting the binding site to the LRF suppressor, and, The base editing enzyme and the corresponding guide RNA molecule are as follows: The base editing enzyme is ABEmax, and the corresponding guide RNA molecule is the sequence shown in Sequence ID No. 49; The base editing enzyme is CBE-NRCH, and the corresponding guide RNA molecule is the sequence shown in Sequence ID No. 52; The base editing enzyme is CBE-SpG, and the corresponding guide RNA molecule is the sequence shown in Sequence ID No. 52; The base editing enzyme is CBE-SpRY, and the corresponding guide RNA molecule is the sequence shown in Sequence ID No. 52; and The base editing enzyme is CBE-SpRY, and the corresponding guide RNA molecule is the sequence shown in Sequence ID No. 53; A kit selected from the group consisting of the following.

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