A method for increasing fetal hemoglobin content by editing the +55 kb region of the erythroid-specific BCL11A enhancer.

Abstract

Reactivation of fetal hemoglobin remains a critical goal in the treatment of sickle cell disease and beta thalassemia patients. Several genome editing strategies have been developed with the goal of reactivating fetal gamma globin expression as a potential treatment for beta hemoglobinopathies. BCL11A is one of the major repressors of gamma globin. To avoid DSB-induced toxicity, the erythroid-specific BCL11A enhancer, specifically the ATF4 binding site, can be targeted using CBE and ABE-mediated base editing approaches to downregulate BCL11A expression and reactivate HbF. Here, we utilized CBE and ABE to analyze the ATF4 binding site in SCD HSPCs and identified a critical base conversion that induces changes in enhancer activity, BCL11A downregulation, and subsequently, HbF reactivation and sickle phenotype rescue.

Description

[Technical field]

[0001] Field of the invention: The present invention relates to the field of medicine, in particular to the field of hematology.

[0002] Background of the invention: Beta-hemoglobinopathies, beta-thalassemia, and sickle cell disease (SCD) are monogenic disorders caused by mutations in the beta-globin locus that affect the synthesis or structure of adult hemoglobin (Hb). Beta-thalassemia is caused by mutations in the beta-globin gene (HBB) locus that reduce (beta+) or eliminate (beta0) the production of beta-globin chains contained in the adult hemoglobin (HbA) tetramer, leading to precipitation of uncoupled alpha-globin chains, red blood cell death, and severe anemia (Taher et al., 2018). In SCD, an A>T mutation in the HBB gene causes a substitution of valine for glutamic acid at position 6 of the beta-globin chain (betaS), which is involved in the deoxygenation-induced polymerization of sickle hemoglobin (HbS). This primary event results in red blood cell (RBC) sickling, hemolysis, vaso-occlusive acute episodes, multi-organ damage, and is often associated with severely reduced life expectancy (Piel et al., 2017; Kata et al., 2018).

[0003] The only definitive cure for hemoglobinopathies is transplantation of allogeneic hematopoietic stem cells (HSCs) from HLA-matched donors, an option available for <30% of patients (Chandrakasan and Malik, 2014). Gene therapy approaches based on transplantation of autologous genetically modified HSCs have been investigated as a treatment option for patients lacking a matched donor (Cavazzana et al., 2017). Genome editing technologies have been exploited to develop therapeutic approaches for hemoglobinopathies based on direct gene correction. These approaches use designer nucleases, such as the CRISPR / Cas9 system, which induce DNA double-strand breaks (DSBs) via single guide RNAs (gRNAs) complementary to specific genomic targets (Cavazzana et al., 2017). DSBs can be repaired via homology-directed repair (HDR) by providing a donor DNA template containing the wild-type sequence. However, HDR-mediated gene correction is insufficiently efficient in HSCs.

[0004] Clinical history of beta-hemoglobinopathies indicates that the severity of both beta-thalassemia and SCD is mitigated by the synthesis of fetal gamma globin in adulthood (Forget, 1998). Fetal hemoglobin (HbF) compensates for HbA deficiency in beta-thalassemia, and gamma globin exerts a strong anti-sickling effect in SCD by replacing mutant sickle beta chains (Cavazzana et al., 2017). Notably, single nucleotide polymorphisms (SNPs) in BCL11A have been associated with elevated expression of HbF in adulthood (Canver et al., 2015). BCL11A is one of the major repressors of HBG1 / 2. Studies aimed at completely inactivating BCL11A have shown that it has deleterious effects on lymphoid development and critical HSC functions (Liu et al., 2003;Yu et al., 2012;Guda et al., 2015) as well as human RBC enucleation (Chang et al., 2017). Conversely, precise BCL11A downregulation through targeting its erythroid-specific enhancer can derepress γ-globin without adverse effects (Wu et al., 2019). Several genome editing strategies have been developed based on disrupting transregulatory elements via the generation of deletions or insertions in patients' hematopoietic stem / progenitor cells (HSPCs) and reactivating HbF expression in their erythroid progeny, with the goal of reactivating fetal γ-globin expression as a potential treatment for both β-thalassemia and SCD. CRISPR / Cas9 disruption of GATA1 and ATF4 activator binding sites (BS) within the erythroid-specific BCL11A enhancer (+58 kb and +55 kb regions) efficiently reactivates HbF expression in erythroid cell lines and primary HSPCs ( Wu et al., 2019 ; Huang et al., 2020 ).

[0005] It is noteworthy that HSCs are highly sensitive to DNA DSBs (Milyavsky et al., 2010) - especially in the case of multiple on-target or simultaneous on-target and off-target events. Even when highly specific gRNAs are used, Cas9 / gRNA treatment of human HSPCs induces a DNA damage response that can lead to apoptosis (Cromer et al., 2018). CRISPR / Cas9 can cause P53-dependent cytotoxicity and cell cycle arrest, resulting in the negative selection of cells with a functional P53 pathway (Haapaniemi et al., 2018). Furthermore, the generation of several on-target DSBs, simultaneous on-target and off-target DSBs, or even a single on-target DSB is associated with the risk of deletions, inversions, and translocations (Kosicki et al., 2018). Therefore, the development of novel, effective, and safe therapeutic strategies for β-hemoglobinopathies based on precise base editing rather than DSB-induced DNA repair has been a priority. The basic components of base editors are catalytically disabled Cas9 nuclease and deaminase; these ultimately produce CG to TA or AT to GC conversions (for CBE and ABE, respectively) (Rees and Liu, 2018). The base editing approach allows precise DNA repair in the virtual absence of DSBs, thus eliminating the risk of DSB-induced apoptosis, translocations and insertions or deletions of large portions of DNA. Importantly, base editing occurs in quiescent cells, suggesting that true HSCs can be genetically modified using this novel technology (Yeh et al., 2018), resulting in uniform and predictable base changes compared to the heterogeneous and unpredictable mutagenesis induced by NHEJ. Base editing has been utilized to correct mutations responsible for β-hemoglobinopathies (Antoniou et al., 2021; Newby et al., 2021). However, inducing HbF reactivation represents a universal approach that can be applied to all patients, regardless of the specific disease-causing mutation (Zeng et al., 2020; Antoniou et al., 2021).

[0006] Summary of the invention: The invention is defined by the claims. In particular, the invention relates to a method for increasing fetal hemoglobin content in eukaryotic cells and its use for the treatment of hemoglobinopathies.

[0007] Detailed description of the invention: To avoid DSB-induced toxicity, the erythroid-specific BCL11A enhancer, specifically the ATF4 binding site, can be targeted using a CBE- and ABE-mediated base editing approach to downregulate BCL11A expression and reactivate HbF. Here, we leveraged CBE and ABE to probe the ATF4 binding site in SCD HSPCs and identified a critical base conversion that induced changes in enhancer activity, BCL11A downregulation, and subsequently, HbF reactivation and sickling phenotype rescue.

[0008] Definition: As used herein, the term "beta-hemoglobinopathy" has its general meaning in the art and refers to any deficiency in the structure or function of any hemoglobin in an individual, including any mutation, such as a defect in the primary, secondary, tertiary or quaternary structure of hemoglobin caused by a deletion or substitution mutation in the coding region of the HBB gene, or a mutation or deletion in the promoter or enhancer of such a gene, that results in a reduction in the amount of hemoglobin produced compared to normal or standard conditions.

[0009] The term "sickle cell disease" as used herein has its general meaning in the art and refers to a group of autosomal recessive inherited blood disorders resulting from mutations in globin genes and characterized by red blood cells with abnormal, rigid, sickle-shaped morphology. They are defined by the presence of the βS globin gene encoding a β-globin chain variant in which glutamic acid is replaced by valine at amino acid position 6 of the peptide: incorporation of βS globin in the Hb tetramer (HbS, sickle Hb) leads to Hb polymerization and to the clinical phenotype. The term includes sickle cell anemia (HbSS), sickle hemoglobin C disease (HbSC), sickle beta plus thalassemia (HbS / β+), or sickle beta zero thalassemia (HbS / β0).

[0010] As used herein, the term "β thalassemia" refers to a hemoglobinopathy resulting from an altered ratio of β-like globin polypeptide chains to α globin polypeptide chains, resulting in underproduction of normal hemoglobin tetrameric protein and precipitation of free unpaired α globin chains.

[0011] As used herein, the term "hematopoietic stem cell" or "HSC" refers to blood cells that have the ability to self-renew and differentiate into precursors of blood cells. These progenitor cells are immature blood cells that cannot self-renew 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 self-renew and maintain a continuous production of hematopoietic stem cells that give rise to all mature blood cells throughout life. In some embodiments, hematopoietic progenitor or stem cells are isolated from peripheral blood cells.

[0012] As used herein, the term "peripheral blood cells" refers to the cellular components of blood that are found in the circulating pool of blood, including red blood cells, white blood cells, and platelets. In some embodiments, the eukaryotic cells are bone marrow-derived stem cells.

[0013] As used herein, the term "bone marrow-derived stem cells" refers to stem cells found in bone marrow. Stem cells may exist in bone marrow as adherent stromal cell types with pluripotent potential, or as cells expressing CD34 or CD45 cell surface proteins, which identify hematopoietic stem cells capable of differentiating into blood cells.

[0014] As used herein, the term "mobilization" or "stem cell mobilization" refers to a process that involves the mobilization of stem cells from their resident tissue or organ to peripheral blood after treatment with a mobilization 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 mobilization agent administered. Some mobilization agents act as agonists or antagonists that prevent the attachment of stem cells to cells or tissues of their microenvironment. Other mobilization agents induce the release of proteases that cleave adhesion molecules or support structures between stem cells and their attachment sites.

[0015] As used herein, the term "mobilizing agents" refers to a broad range of molecules that act to enhance the mobilization of stem cells from their resident tissues or organs, such as bone marrow (e.g., CD34+ stem cells) and spleen (e.g., Hox11+ stem cells), into peripheral blood. Mobilizing agents include chemotherapy drugs, such as cyclophosphamide and cisplatin; cytokines, and chemokines, such as granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), stem cell factor (SCF), Fms-related tyrosine kinase 3 (flt-3) ligand, stromal cell-derived factor 1 (SDF-1); agonists of chemokine (CC motif) receptor 1 (CCR1), such as chemokine (CC motif) ligand 3 (CCL3, also known as macrophage inflammatory protein-1α (Mip-1α)); chemokine (CXC motif) receptor 2 (CCR2) agonists, such as chemokine (CC motif) ligand 2 (CCL2, also known as macrophage inflammatory protein-1α (Mip-1α)); Mobilizing agents include agonists of CXCR1 and 2 (CXCR2), such as chemokine (CXC motif) ligand 2 (CXCL2) (also known as growth-related 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; very late antigen (VLA)-4 inhibitors; antagonists of CXCR4, such as TG-0054, plerixafor (also known as AMD3100), and AMD3465, or any combination of the aforementioned agents. Mobilizing agents increase the number of stem cells in peripheral blood, thus allowing a more available source of stem cells for use in transplantation, organ repair or regeneration, or disease treatment.

[0016] As used herein, the term "isolated cell" refers to a cell that has been removed from an organism in which it was originally found, or the progeny of such a cell. Optionally, the eukaryotic cell has been cultured in vitro, for example, in the presence of other cells. Optionally, the eukaryotic cell is subsequently introduced into a second organism, or reintroduced into the organism from which it (or the cell from which it was derived) was isolated. As used herein, the term "isolated population" in reference to an isolated population of cells refers to a population of cells that has been removed and separated from a mixed or heterogeneous population of cells. In some embodiments, the isolated population is a substantially pure population of cells, as compared to the heterogeneous population from which the cells are isolated or enriched.

[0017] As used herein, the term "alpha globin" or "beta globin" has its general meaning in the art and refers to the proteins encoded in humans by the HBA1 and HBA2 genes. The human alpha globin gene cluster, located on chromosome 16, spans approximately 30 kb and contains seven loci: 5'-zeta-pseudozeta-mu-pseudoalpha-1-alpha-2-alpha-1-theta-3'. The alpha-2 (HBA2) and alpha-1 (HBA1) coding sequences are identical. These genes differ slightly over the 5' untranslated region and introns, but they differ significantly over the 3' untranslated region. The ENSEMBL IDs (i.e., gene identifier numbers from the Ensembl Genome Browser database) for HBA1 and HBA2 are ENSG00000206172 and ENSG00000188536, respectively.

[0018] As used herein, the term "beta globin" or "β globin" has its common meaning in the art and refers to the globin protein which, together with alpha globin (HBA), constitutes the most common form of hemoglobin (Hb) in adults. Normal adult Hb is a heterotetramer consisting of two alpha chains and two beta chains. HBB is encoded by the HBB gene on human chromosome 11. It is 146 amino acids long and has a molecular weight of 15,867 Da.

[0019] As used herein, the term "gamma globin" or "gamma globin" has its general meaning in the art and refers to the proteins encoded by the HBG1 and HBG2 genes in humans. The HBG1 and HBG2 genes are normally expressed in the fetal liver, spleen, and bone marrow. The two gamma globin chains, together with the two alpha globin chains, make up fetal hemoglobin (HbF), which is normally replaced by adult hemoglobin (HbA) the year after birth. The ENSEMBL IDs (i.e., gene identifier numbers from the Ensembl Genome Browser database) for HBG1 and HBG2 are ENSG00000213934 and ENSG00000196565, respectively.

[0020] As used herein, the term "transcriptional repressor" has its general meaning in the art and refers to a protein (transcription factor) that reduces gene transcription of a gene or set of genes. Most repressors are DNA-binding proteins that bind to enhancer or promoter proximal elements. According to the present disclosure, the transcriptional repressor is BCL11A.

[0021] As used herein, the term "BCL11A" has its general meaning in the art and refers to the gene encoding the BAF chromatin remodeling complex subunit BCL11A (gene ID: 53335). This term is also known as EVI9; CTIP1; DILOS; ZNF856; HBFQTL5; BCL11A-L; BCL11A-S; BCL11a-M; or BCL11A-XL. Five alternatively spliced ​​transcript variants of this gene have been reported, encoding different isoforms. 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 switch from gamma to beta globin expression during the fetal to adult erythropoietic transition (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).

[0022] As used herein, the term "transcriptional activator" has its general meaning in the art and refers to a protein that increases gene transcription of a gene or set of genes. Most activators are DNA-binding proteins that bind to enhancer or promoter-proximal elements. According to the present disclosure, the activator ATF4.

[0023] As used herein, the term "ATF4" has its general meaning in the art and refers to activating transcription factor 4 (tax-responsive enhancer element B67), a protein encoded in humans by the ATF4 gene (gene ID: 468). ATF4 is thus a transcriptional activator originally identified as a widely expressed mammalian DNA-binding protein capable of binding to the tax-responsive enhancer element in the LTR of HTLV-1. The encoded protein was also isolated and characterized as cAMP response element binding protein 2 (CREB-2). The protein encoded by this gene belongs to a family of DNA-binding proteins that includes the AP-1 family of transcription factors, cAMP response element binding protein (CREB) and CREB-like proteins. These transcription factors share a leucine zipper region involved in protein-protein interactions and are located at the C-terminus of a stretch of basic amino acids that functions as a DNA-binding domain. Two alternative transcripts encoding the same protein have been described. Two pseudogenes are located on the X chromosome at q28 in a region containing a large inverted duplication.

[0024] As used herein, the term "transcriptional activator binding site" refers to a site present on DNA whereby a transcriptional activator according to the present disclosure binds. According to the present invention, the base editing enzyme of the present invention edits the genomic sequence of a eukaryotic cell such that the activator can bind to the transcriptional activator binding site.

[0025] As used herein, the phrase "the +55 kb region of the erythroid-specific BCL11A enhancer" refers to the region having the nucleotide acid sequence depicted in Figure 1 and set forth in SEQ ID NO: 1. The "ATF4 binding site" extends from nucleotide position 21 to nucleotide position 31 in SEQ ID NO: 1 (i.e., TTGCATCATCC (SEQ ID NO: 45)). [ka]

[0026] As used herein, the term "expression" refers to the process by which a polynucleotide is transcribed from a DNA template (e.g., into an mRNA or other RNA transcript) and / or the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. The transcript and the encoded polypeptide may be collectively referred to as a "gene product." If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in eukaryotic cells. Any method known in the art may be used to measure the expression of a gene (e.g., HPLC analysis of proteins and RT-qPCR analysis of mRNA). Typically, such methods are described in the Examples.

[0027] As used herein, the phrase "increasing fetal hemoglobin content" refers to fetal hemoglobin being at least 5% higher in eukaryotic cells treated with a gene editing platform than in comparable eukaryotic cells, where a gene editing platform targeting an unrelated locus is present or where a gene editing platform is not present. In some embodiments, the percentage of fetal hemoglobin expression in the eukaryotic cells is at least 10% higher, at least 20% higher, at least 30% higher, at least 40% higher, at least 50% higher, at least 60% higher, at least 70% higher, at least 80% higher, at least 90% higher, at least 1-fold higher, at least 2-fold higher, at least 5-fold higher, at least 10-fold higher, at least 100-fold higher, at least 1000-fold higher, or more, than in the eukaryotic cells.

[0028] As used herein, the phrase "suppressing BCL11A expression" refers to the expression of BCL11A being at least 5% lower in a eukaryotic cell contacted with a gene editing platform of the present invention than in a comparable eukaryotic cell not contacted with the gene. In some embodiments, the percentage of BCL11A expression in a eukaryotic cell is at least 10% lower, at least 20% lower, at least 30% lower, at least 40% lower, at least 50% lower, at least 60% lower, at least 70% lower, at least 80% lower, at least 90% lower, at least 1-fold lower, at least 2-fold lower, at least 5-fold lower, at least 10-fold lower, at least 100-fold lower, at least 1000-fold lower, or less than that in a eukaryotic cell not contacted with a gene editing platform.

[0029] As used herein, the terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acids of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. The term also encompasses modified amino acid polymers; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, pegylation, or any other manipulation, such as conjugation with a labeling component. As used herein, the term "amino acid" includes natural and / or unnatural or synthetic amino acids, including both glycine D or L optical isomers, amino acid analogs and peptidomimetics.

[0030] As used herein, the term "nucleic acid molecule" or "polynucleotide" refers to a DNA molecule (such as, but not limited to, cDNA or genomic DNA). A nucleic acid molecule can be single-stranded or double-stranded.

[0031] As used herein, the term "isolated" when referring to a nucleic acid molecule or polypeptide means that the nucleic acid molecule or polypeptide is substantially free from at least one other component with which it is associated or found in nature.

[0032] As used herein, the term "complementarity" refers to the ability of a nucleic acid to form hydrogen bonds with another nucleic acid sequence, either by traditional Watson-Crick base pairing or other non-traditional types. Percent complementarity indicates the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 are 50%, 60%, 70%, 80%, 90%, 100% complementary). "Fully complementary" means that all contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in the second nucleic acid sequence. As used herein, "substantially complementary" refers to a degree of complementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions.

[0033] As used herein, the term "stringent conditions" for hybridization refers to conditions under which a nucleic acid having a complementarity to a target sequence hybridizes predominantly to the target sequence and does not substantially hybridize to non-target sequences. Stringent conditions are generally sequence-dependent and vary depending on many factors. In general, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes to its target sequence. Non-limiting examples of stringent conditions are described in detail in Tijssen (1993), Laboratory Techniques In Biochemistry And Molecular Biology-Hybridization With Nucleic Acid Probes Part I, Second Chapter "Overview of principles of hybridization and the strategy of nucleic acid probe assay", Elsevier, NY.

[0034] As used herein, the term "hybridization" or "hybridize" refers to the process by which fully or partially complementary nucleic acid strands come together under specific hybridization conditions to form a double-stranded structure or region in which the two constituent strands are linked by hydrogen bonds. Hydrogen bonds typically form between adenine and thymine or uracil (A and T or U) or cytosine and guanine (C and G), although other base pairs can form (e.g., Adams et al., The Biochemistry of the Nucleic Acids, 11th ed., 1992).

[0035] As used herein, the term "fusion polypeptide" or "fusion protein" refers to a protein made by linking two or more polypeptide sequences together. A fusion polypeptide encompassed by the present invention comprises the translation product of a chimeric gene construct that links a nucleic acid sequence encoding a first polypeptide, e.g., an RNA-binding domain, with a nucleic acid sequence encoding a second polypeptide, e.g., an effector domain, to form a single open reading frame. 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.

[0036] The term "wild type" as used herein is a term of the art understood by those of skill in the art and refers to the typical form or characteristics of an organism, strain or gene as it occurs in nature, as distinguished from mutant or variant forms.

[0037] As used herein, the term "derived from" refers to a process of using a first component (e.g., a first molecule), or information from that first component, to isolate, derive, or create a different second component (e.g., a second molecule that is different from the first).

[0038] As used herein, the "percent identity" between two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions x 100), taking into account the number of gaps, and the length of each gap, which must be introduced for optimal alignment of the two sequences. Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described below. Percent identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (Needleman, Saul B. & Wunsch, Christian D. (1970). "A general method applicable to the search for similarities in the amino acid sequence of two proteins". Journal of Molecular Biology. 48(3): 443-53.). Percent identity between two nucleotide or amino acid sequences can also be determined, for example, using algorithms such as EMBOSS Needle (pairwise alignment; available at www.ebi.ac.uk). For example, EMBOSS Needle may be used with the BLOSUM62 matrix, a "gap open penalty" of 10, a "gap extension penalty" of 0.5, a false "end gap penalty", an "end gap open penalty" of 10, and an "end gap extension penalty" of 0.5. In general, "percent identity" is a function of the number of matching positions divided by the number of positions compared, multiplied by 100. For example, if 6 of 10 sequence positions are identical between the two compared sequences after alignment, then the identity is 60%. Percent identity is typically determined over the entire length of the query sequence over which the analysis is performed. Two molecules that have the same primary amino acid or nucleic acid sequence are identical regardless of any chemical and / or biological modifications.According to the present invention, a first amino acid sequence having at least 90% identity with a second amino acid sequence means that the first sequence has 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% identity with the second amino acid sequence.

[0039] As used herein, the term "linker" refers to any means, entity, or moiety used to link two or more entities. A linker can be a covalent linker or a non-covalent linker. Examples of covalent linkers include covalent bonds or linker moieties that are covalently attached to one or more of the proteins or domains to be linked. A linker can also be a non-covalent bond, for example, an organometallic bond through a metal center, such as a platinum atom. For covalent linkages, functional groups can be used, including, for example, amide groups, carbonic acid derivatives, ethers, esters, including organic and inorganic esters, amino, urethane, urea, and the like. To provide linkage, the domains can be modified by oxidation, hydroxylation, substitution, reduction, etc. to provide sites for coupling. Methods for conjugation are well known to those skilled in the art and are encompassed for use in the present invention. Linker moieties include, but are not limited to, chemical linker moieties, or, for example, peptide linker moieties (linker sequences). It will be understood that modifications that do not significantly reduce the function of the RNA binding domain and the effector domain are preferred.

[0040] As used herein, "linked" refers to two or more entities to form one entity. Conjugates include both peptide-small molecule conjugates as well as peptide-protein / peptide conjugates.

[0041] As used herein, the term "base editing enzyme" refers to a fusion protein that includes a defective CRISPR / Cas nuclease linked to a deaminase polypeptide. This term is also known as a "base editor". Two classes of base editing enzymes - cytosine base editing enzymes (CBEs) and adenine base editing enzymes (ABEs) - can be used to generate single base pair edits without double-strand breaks. Typically, cytosine base editing enzymes are made by fusing a defective CRISPR / Cas nuclease to a deaminase.

[0042] As used herein, the term "deaminase" refers to an enzyme that catalyzes a deamination reaction. The term "deamination," as used herein, refers to the removal of an amine group from a molecule. In some embodiments, the deaminase is a cytidine deaminase, which catalyzes the hydrolytic deamination of cytidine or deoxycytidine to uracil or deoxyuracil, respectively. In some embodiments, the deaminase is an adenosine deaminase, which catalyzes the hydrolytic deamination of adenosine to inosine, which is processed by cells like guanosine, making an A to G (or T to C) change.

[0043] As used herein, the term "nuclease" includes proteins (ie, enzymes) that induce cleavage in a nucleic acid sequence, for example, a single- or double-stranded break in a double-stranded DNA sequence.

[0044] As used herein, the term "CRISPR / Cas nuclease" has its general meaning in the art and refers to a segment of prokaryotic DNA that contains clustered regularly interspaced short palindromic repeats (CRISPR) and associated nucleases encoded by Cas genes. In bacteria, CRISPR / Cas loci code for an RNA-guided adaptive immune system against mobile genetic elements (viruses, transposable elements, and conjugative plasmids). Three types of CRISPR systems have been identified. CRISPR clusters contain a spacer, a sequence complementary to the preceding mobile element. CRISPR clusters are transcribed and processed into mature CRISPR (Clustered Regularly Interspaced Short Palindromic repeats) RNA (crRNA). CRISPR / Cas nucleases Cas9 and Cpf1 belong to type II and type V CRISPR / Cas systems and have strong endonuclease activity to cleave target DNA. Cas9 is guided by a mature crRNA that contains a unique target sequence of about 20 nucleotides (called the spacer) and a transactivating small RNA (tracrRNA) that also serves as a guide for RNase III-assisted processing of the pre-crRNA. The crRNA:tracrRNA duplex directs Cas9 to the target DNA through complementary base pairing between the spacer on the crRNA and a complementary sequence on the target DNA (called the protospacer). Cas9 recognizes a trinucleotide (NGG for S. pyogenes Cas9) protospacer adjacent motif (PAM) to identify the cleavage site (the third or fourth nucleotide upstream from the PAM).

[0045] As used herein, the term "Cas9" or "Cas9 nuclease" refers to an RNA-guided nuclease that includes Cas9 protein, or a fragment thereof (e.g., a protein that includes an active or inactive DNA cleavage domain of Cas9, and / or a gRNA binding domain of Cas9). Cas9 nuclease is also sometimes referred to as casn1 nuclease or CRISPR (clustered regularly interspaced short palindromic repeats)-associated nuclease. CRISPR is an adaptive immune system that provides defense against mobile genetic elements (viruses, transposable elements, and conjugative plasmids). CRISPR clusters include a spacer, a sequence complementary to the preceding mobile element, and a target invading nucleic acid. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems, accurate processing of the pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and the Cas9 protein. The tracrRNA serves as a guide for RNase 3-assisted processing of the pre-crRNA. Cas9 / crRNA / tracrRNA then endonucleolytically cleaves linear or circular dsDNA targets that are complementary to the spacer. The target strand that is not complementary to the crRNA is first endonucleolytically cleaved and then 3'-5' exonucleolytically trimmed. In nature, DNA binding and cleavage typically requires a protein and both RNAs. However, single guide RNAs ("sgRNAs", or simply "gNRAs") can be engineered to incorporate aspects of both crRNA and tracrRNA into a single RNA species. See, e.g., Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E. Science 337:816-821 (2012), the entire contents of which are hereby incorporated by reference herein. Cas9 recognizes a short motif (the PAM or protospacer adjacent motif) in the CRISPR repeat sequence, which helps distinguish self from 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 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 which are incorporated herein by reference. Cas9 orthologs have been described in a variety of species, including, but not limited to, S. pyogenes and S. thermophilus.Additional suitable Cas9 nucleases and sequences will be apparent to one of skill in the art based on this disclosure, including Cas9 sequences from the organisms and loci disclosed in Chylinski, Rhun, and Charpentier, "The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems" (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference. In some embodiments, the term "Cas9" refers to Cas9 from: Corynebacterium ulcerans (NCBI References: NC_015683.1, NC_017317.1); Corynebacterium diphtheriae (NCBI References: NC_016782.1, NC_016786.1); Spiroplasma sylphydicola (NCBI Reference: NC_021284.1); Prevotella intermedia (NCBI Reference: NC_017861.1); Spiroplasma taiwanense (NCBI Reference: NC_021846.1); Streptococcus iniae (NCBI Reference: NC_021314.1); Berriella baltica (NCBI Reference: NC_018010.1); Psychroflexus talkis I ( torquis I (NCBI Reference: NC_018721.1); Streptococcus thermophilus (NCBI Reference: YP_820832.1); Listeria innocua (NCBI Reference: NP_472073.1); Campylobacter jejuni (NCBI Reference: YP_002344900.1); or Neisseria meningitidis (NCBI Reference: YP_002342100.1). Typically, the Cas9 nuclease comprises the amino acid sequence shown in SEQ ID NO:2. [ka]

[0046] As used herein, the term "defective CRISPR / Cas nuclease" refers to a CRISPR / Cas nuclease that is missing at least one nuclease domain.

[0047] As used herein, the term "nickases" has its general meaning in the art and refers to endonucleases that cleave only one strand of a DNA duplex. Thus, the term "Cas9 nickases" refers to nickases derived from Cas9 proteins, typically by inactivating one nuclease domain of the Cas9 protein.

[0048] As used herein, the term "guide RNA molecule" generally refers to an RNA molecule (or collectively a group of RNA molecules) that can bind to Cas9 protein and target Cas9 protein to a specific location in a target DNA. A guide RNA can include two segments: a DNA-targeting guide segment and a protein-binding segment. The DNA-targeting segment includes a nucleotide sequence that is complementary to (or can hybridize under at least stringent conditions to) a target sequence. The protein-binding segment interacts with a CRISPR protein, such as Cas9 or a Cas9-associated polypeptide. These two segments can be located in the same RNA molecule or in two or more separate RNA molecules. When the two segments are in separate RNA molecules, the molecule that includes the DNA-targeting guide segment is sometimes referred to as a CRISPR RNA (crRNA), while the molecule that includes the protein-binding segment is referred to as a trans-activating RNA (tracrRNA).

[0049] As used herein, the term "target nucleic acid" or "target" refers to a nucleic acid that contains a target nucleic acid sequence. A target nucleic acid can be single-stranded or double-stranded, but is often double-stranded DNA. As used herein, "target nucleic acid sequence", "target sequence" or "target region" refers to a specific sequence or its complement that one wishes to bind using the CRISPR system disclosed herein.

[0050] As used herein, the term "target nucleic acid strand" refers to the strand of the target nucleic acid that is subjected to base pairing with the guide RNA disclosed herein. That is, the strand of the target nucleic acid that hybridizes with the crRNA and the guide sequence is referred to as the "target nucleic acid strand". The other strand of the target nucleic acid is not complementary to the guide sequence and is referred to as the "non-complementary strand". In the case of a double-stranded target nucleic acid (e.g., DNA), each strand becomes a "target nucleic acid strand" for designing the crRNA and guide RNA and can be used to carry out the method of the present invention as long as there is a suitable PAM site.

[0051] As used herein, the term "ribonucleoprotein complex" or "ribonucleoprotein particle" refers to a complex or particle comprising a nucleoprotein and a ribonucleic acid. As provided herein, a "nucleoprotein" refers to a protein capable of binding to a nucleic acid (e.g., RNA, DNA). When a nucleoprotein binds to a ribonucleic acid, it is referred to as a "ribonucleoprotein". The interaction between a ribonucleoprotein and a ribonucleic acid can be direct, for example, by covalent bonding, or indirect, for example, by non-covalent bonding (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 effect), hydrophobic interactions, and the like).

[0052] As used herein, the term "mutation" has its general meaning in the art and refers to a substitution, deletion, or insertion. The term "substitution" means that a particular amino acid residue at a particular position is removed and another amino acid residue is inserted at the same position. The term "deletion" means that a particular amino acid residue is removed. The term "insertion" means that one or more amino acid residues are inserted before or after a particular amino acid residue.

[0053] As used herein, the term "mutagenesis" refers to the introduction of mutations into a polynucleotide sequence.

[0054] As used herein, the term "variant" refers to a first composition (e.g., a first molecule) relative to a second composition (e.g., a second molecule, also referred to as a "parent" molecule). A variant molecule can be derived from, isolated from, based on, or homologous to a parent molecule. A variant molecule can have complete sequence identity with the original parent molecule, or alternatively, can have less than 100% sequence identity with the parent molecule. For example, a sequence variant can 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 compared to the original sequence.

[0055] As used herein, the term "treatment" or "treating" refers to both prophylactic or preventative treatments as well as curative or disease-modifying treatments, including treatments of patients at risk of or suspected of having a disease, as well as patients diagnosed as being ill or suffering from a disease or medical condition, including suppression of clinical recurrence. Treatments may be administered to subjects who have a medical disorder or who may eventually acquire a disorder to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurrent disorder, or to extend the subject's survival beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant a pattern of treatment of a disease, e.g., a pattern of dosing used during treatment. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or a portion of a therapeutic regimen) used for the initial treatment of a disease. The general goal of an induction regimen is to provide high levels of drug to the patient during the initial period of the treatment regimen. The induction regimen may (partially or entirely) use a "loading regimen", which may involve administering a larger dose of the drug than the physician would use during a maintenance regimen, administering the drug more frequently than the physician administers during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a treatment regimen (or a portion of a treatment regimen) used to maintain a patient between disease treatments, for example, to keep a patient in remission for an extended period of time (months or years). A maintenance regimen may use continuous treatment (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent treatment (e.g., interrupted treatment, intermittent treatment, treatment upon relapse, or treatment upon achievement of certain pre-determined criteria (e.g., pain, disease manifestation, etc.)).

[0056] As used herein, the term "therapeutically effective amount" refers to a sufficient amount of a cell population to treat a disease at a reasonable benefit / risk ratio applicable to any medical treatment. It will be understood that all use compositions of the present invention will be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend on a variety of factors, including the age, weight, general health, sex and diet of the patient, time of administration, route of administration, duration of treatment, drugs used in combination or simultaneously with the population of cells, and similar factors well known in the medical field. In some embodiments, the cells are formulated by first harvesting the cells from their culture medium, then washing the cells and concentrating them in a therapeutically effective amount in an appropriate medium and container system (a "pharmaceutical acceptable" carrier) for administration. A suitable infusion medium can be any isotonic medium formulation, typically saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), but also 5% dextrose in water or lactated Ringer's can be utilized. The infusion medium can be supplemented with human serum albumin. The therapeutically effective amount of cells in the composition will depend on the relative representation of cells with the desired specificity, on the age and weight of the recipient, and on the severity of the target condition. The number of cells will be about 10 3 / kg, preferably 5 x 10 3 It can be as low as 10 pieces / kg; 7 pcs / kg, preferably 10 8 The number of cells may be as high as 10 ...

[0057] method: Therefore, a first object of the present invention is a method for increasing fetal hemoglobin content in a eukaryotic cell, comprising contacting a eukaryotic cell with a gene editing platform consisting of (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 +55 kb region of the erythroid-specific BCL11A enhancer, thereby editing and disrupting an ATF4 binding site in the region to suppress expression of BCL11A and subsequently increase expression of gamma globin.

[0058] In some embodiments, the eukaryotic cells are selected from the group consisting of hematopoietic progenitor cells, hematopoietic stem cells (HSC), pluripotent cells (i.e., embryonic stem cells (ES) and induced pluripotent stem cells (iPS)). Typically, the eukaryotic cells are derived from stem cell mobilization.

[0059] In some embodiments, the base editing enzyme of the present invention comprises defective CRISPR / Cas nuclease. The sequence recognition mechanism is the same as for non-defective CRISPR / Cas nuclease. Typically, the defective CRISPR / Cas nuclease of the present invention comprises at least one RNA binding domain. The RNA binding domain interacts with guide RNA molecule as defined later herein. However, the defective CRISPR / Cas nuclease of the present invention is a modified version without nuclease activity. Thus, the defective CRISPR / Cas nuclease specifically recognizes guide RNA molecule, thus guiding the base editing enzyme to its target DNA sequence.

[0060] In some embodiments, defective CRISPR / Cas nucleases can be modified to increase nucleic acid binding affinity and / or specificity, alter enzymatic activity, and / or change another property of the protein. In some embodiments, the nuclease domain of the protein can be modified, deleted, or inactivated. In some embodiments, the protein can be truncated to remove domains that are not essential for the function of the protein. In some embodiments, the protein is truncated or modified to optimize the activity of the RNA binding domain.

[0061] In some embodiments, the CRISPR / Cas nuclease is a mutant CRISPR / Cas nuclease, i.e., a protein with one or more point mutations, insertions, deletions, truncations, a fusion protein, or a combination thereof. In some embodiments, the mutant has RNA-guided DNA binding activity, but lacks one or both of its nuclease active sites. In some embodiments, the mutant comprises an amino acid sequence with at least 50% identity to the wild-type amino acid sequence of the CRISPR / Cas nuclease. Various CRISPR / Cas nucleases can be used in the present invention. Non-limiting examples of suitable CRISPR / CRISPR / Cas nucleases include Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9, Cas10, Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Csel (or CasA), Csel2 (or CasB), Csel3 (or CasC), Csel4 (or CasD), Csel5 (or CasD), Csel6 (or CasC), Csel7 (or CasC), Csel8 (or CasC), Csel9 (or CasC), Csel10 (or CasC), Csel11 (or CasC), Csel12 (or CasC), Csel13 (or CasC), Csel14 (or CasC), Csel15 (or CasC), Csel16 (or CasC), Csel17 (or CasC), Csel18 (or CasC), Csel19 (or CasC), Csel20 (or CasC), Csel21 (or CasC), Csel22 (or CasC), Csel23 (or CasC), Csel24 (or CasC), Csel25 (or CasC), Csel26 (or CasC), Csel27 (or CasC), Csel28 (or CasC), Csel29 ...9 (or CasC), Csel29 ( These include se3 (or CasE), Cse4 (or CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csz1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cu1966. See, e.g., WO2014144761 WO2014144592, WO2013176772, US20140273226, and US20140273233. the contents of which are incorporated herein by reference in their entirety.

[0062] In some embodiments, the CRISPR / Cas nuclease is derived from a type II CRISPR-Cas system. In some embodiments, the CRISPR / Cas nuclease is derived from a Cas9 protein. Cas9 proteins are found in Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus spp., Nocardiopsis dassonevirei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenite, Bacillus spp., Bacillus subtilis ... Ireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacteria, Polaromonas naphthalenivorans, Polaromonas spp., Crocosphaera watsonii, Cyanoseis spp., Microcystis aeruginosa, Synechococcus spp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor vexii becscii), Candidatus desulfordis, Clostridium botulinum, Clostridium difficile, Finegordia magna, Natlanaerobius thermophilus, Perotomacrum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter spp., Nitrosococcus halophilus, Nitrosococcus watsonii, Schweitzer spp. The bacteria may be from the genus Alteromonas haloplanktis, Ctedonobacter racemifur, Methanohalobium ebestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc spp., Arthrospira maxima, Arthrospira platensis, Arthrospira spp., Lyngbya spp., Microcoleus ctnoplastes, Oscilatoria spp., Petrotoga mobilis, Thermosipho africanus, or Acaryochloris marina.

[0063] In some embodiments, the CRISPR / Cas nuclease is a mutant or fragment of a wild-type CRISPR / Cas nuclease, such as Cas9. In some embodiments, the CRISPR / Cas nuclease is a mutant Cas9 protein from S. pyogenes.

[0064] Methods for generating Cas9 proteins (or fragments thereof) with inactive DNA cleavage domains are known (see, e.g., Jinek et al., Science. 337:816-821(2012); Qi et al., "Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression"(2013)Cell. 28; 152(5):1173-83, the entire contents of each of which are incorporated herein by reference). For example, the DNA cleavage domain of Cas9 is known to contain two subdomains, the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, whereas the RuvC1 subdomain cleaves the non-complementary strand. Mutations within these subdomains can suppress the nuclease activity of Cas9. For example, the mutations D10A and H841A completely inactivate the nuclease activity of S. pyogenes Cas9 (Jinek et al., Science. 337:816-821(2012); Qi et al., Cell. 28; 152(5):1173 -83(2013).

[0065] In some embodiments, the CRISPR / Cas nuclease of the present invention is a nickase, more specifically a Cas9 nickase, i.e., Cas9 from S. pyogenes, having one mutation selected from the group consisting of D10A and H840A. In some embodiments, the nickase of the present invention comprises the amino acid sequence shown in SEQ ID NO:3 or SEQ ID NO:4. [ka] [ka]

[0066] In some embodiments, Cas9 variants with mutations other than D10A or H840A are used, resulting in, for example, a nuclease-inactivated Cas9 (dCas9). Such mutations include, by way of example, other amino acid substitutions at D10 and H840, or other substitutions within the nuclease domain of Cas9 (e.g., substitutions in the HNH nuclease subdomain and / or the RuvC1 subdomain). In some embodiments, variants of dCas9 are provided that are at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, or at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to SEQ ID NO: 2 or 3. In some embodiments, variants of dCas9 are provided that have an amino acid sequence that is shorter or longer than SEQ ID NO: 2 or 3 by about 5 amino acids, by about 10 amino acids, by about 15 amino acids, by about 20 amino acids, by about 25 amino acids, by about 30 amino acids, by about 40 amino acids, by about 50 amino acids, by about 75 amino acids, by about 100 amino acids or more.

[0067] In accordance with the present invention, the second component of the base editing enzymes disclosed herein comprises a non-nuclease DNA modifying enzyme that is a deaminase.

[0068] In some embodiments, the deaminase is a cytidine deaminase. In some embodiments, the deaminase is an apolipoprotein B mRNA editing complex (APOBEC) family deaminase. In some embodiments, the deaminase is an APOBEC1 family deaminase. In some embodiments, the deaminase is an activation-induced cytidine deaminase (AID). In some embodiments, the deaminase is an ACF1 / ASE deaminase.

[0069] In some embodiments, the deaminase is AID: activation-induced cytidine deaminase, APOBEC1: apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 1, APOBEC3A: apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3A, APOBEC3B: apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3B, APOBEC3C: apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3C, APOBEC3D: apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3D, APOBEC3F: apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3F, APOBEC3G: apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3G, APOBEC3H: apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3H, ADA: adenosine deaminase, ADAR1: adenosine deaminase acting on RNA 1, Dnmt1: DNA (cytosine-5-)-methyltransferase 1. Selected from the group consisting of Dnmt3a: DNA (cytosine-5-) methyltransferase 3 alpha, Dnmt3b: DNA (cytosine-5-) methyltransferase 3 beta, and Tet1: methylcytosine dioxygenase.

[0070] In some embodiments, the deaminase is derived from activation-induced cytidine deaminase (AID). AID is a cytidine deaminase that can catalyze the reaction of deamination of cytosine in the context of DNA or RNA. When delivered to the target site, AID changes the C base to a U base. In dividing cells, this can lead to a C to T point mutation. Alternatively, the C to U change triggers cellular DNA repair pathways, primarily the excision repair pathway, whereby the mismatched UG base pair is removed and replaced with a TA, AT, CG, or GC pair. As a result, a point mutation can be generated at the target CG site. In some embodiments, the DNA modifying enzyme is AID*Δ, an AID mutant with an ablated nuclear export signal (NES) and increased SHM activity (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).

[0071] In some embodiments, the deaminase consists of a variant of the amino acid sequence shown in SEQ ID NOs: 5-15. [ka] [ka] [ka] [ka] [ka] [ka]

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[0072] In some embodiments, the deaminase is an adenosine deaminase. In some embodiments, the deaminase is an ADAT family deaminase. In some embodiments, the adenosine deaminase variant is a TadA deaminase. In some embodiments, the adenosine deaminase variant is Staphylococcus aureus TadA, Bacillus subtilis TadA, Salmonella typhimurium TadA, Shewanella putrefaciens TadA, Haemophilus influenzae F3031 TadA, Caulobacter crescentus TadA, or Geobacter sulfurreducens TadA, or a fragment thereof. In some embodiments, the TadA deaminase is Escherichia coli TadA deaminase (ecTadA). In some embodiments, the TadA deaminase is a truncated E. coli TadA deaminase. For example, the truncated ecTadA may lack one or more N-terminal amino acids compared to full-length ecTadA. In some embodiments, the truncated ecTadA may lack 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 N-terminal amino acid residues compared to full-length ecTadA. In some embodiments, the truncated ecTadA may lack 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 C-terminal amino acid residues compared to full-length ecTadA. In some embodiments, the TadA deaminase is TadA*7.10. In some embodiments, the TadA deaminase is a TadA*8 variant. For example, deaminases are described in International PCT Applications WO2018 / 027078, WO2017 / 070632, WO / 2020 / 168132, and WO / 2021 / 050571, each of which is incorporated by reference in its entirety.Also, Komor, AC, et al., “Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016); Gaudelli, NM, et al., “Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage” Nature 551, 464-471(2017); Komor, AC, et al., “Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A base editors with higher efficiency and product purity” Science Advances 3:eaao4774(2017)), and Rees, HA, et al., “Base editing: precision chemistry on the genome and transcriptome of living cells.” Nat Rev Genet. 2018 Dec;19(12):770-788. doi: See, e.g., 10.1038 / s41576-018-0059-1, the entire contents of which are hereby incorporated by reference herein. An exemplary amino acid sequence for wild-type TadA (wt) adenosine deaminase is set forth as SEQ ID NO: 16. In some embodiments, the amino acid sequence of the adenosine deaminase comprises at least 90% sequence identity to SEQ ID NO: 16. In some embodiments, the amino acid sequence of the adenosine deaminase comprises a modification at position 82 numbered in SEQ ID NO: 16. In some embodiments, the amino acid sequence comprising the adenosine deaminase comprises a V82S modification, in which position 82 is as numbered in SEQ ID NO: 16.In some embodiments, the amino acid sequence of the adenosine deaminase comprises a modification at position 166 numbered in SEQ ID NO: 16. In some embodiments, the amino acid sequence of the adenosine deaminase comprises a T166R modification, where position 166 is as numbered in SEQ ID NO: 16. In some embodiments, the amino acid sequence of the adenosine deaminase comprises modifications at positions 82 and 166 numbered in SEQ ID NO: 16. In some embodiments, the amino acid sequence of the adenosine deaminase comprises a V82S and a T166R modification, where positions 82 and 166 are as numbered in SEQ ID NO: 16. In some embodiments, the adenosine deaminase variant further comprises one or more of the following modifications: Y147T, Y147R, Q154S, Y123H, and Q154R. In some embodiments, the adenosine deaminase variant comprises a combination of modifications selected from the group consisting of: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y123H+Y147R;V82S+Y123H+Q154R;Y147R+Q154R+Y123H;Y147R+Q154R+I76Y;Y147R+Q154R+T166R;Y123H+Y147R+Q154R+I76Y;V82S+Y123H+Y147R+Q154R;and I76Y+V82S+Y123H+Y147R+Q154R. In some embodiments, the adenosine deaminase variant is TadA*8.1, TadA*8.2, TadA*8.3, TadA*8.4, TadA*8.5, TadA*8.6, TadA*8.7, TadA*8.8, TadA*8.9, TadA*8.10, TadA*8.11, TadA*8.12, TadA*8.13, TadA*8.14, TadA*8.15, TadA*8.16, TadA*8.17, TadA*8.18, TadA*8.19, TadA*8.20, TadA*8.21, TadA*8.22, TadA*8.23, or TadA*8.24.In some embodiments, the adenosine deaminase is provided as a single (e.g., provided as a monomer) TadA variant, as described above. In some embodiments, the adenosine deaminase is provided as a heterodimer of wild-type TadA (TadA(wt)) linked to a TadA variant, as described above. [ka]

[0073] In some embodiments, the deaminase is fused to the N-terminus of the defective CRISPR / Cas nuclease. In some embodiments, the deaminase is fused to the C-terminus of the defective CRISPR / Cas nuclease. In some embodiments, the defective CRISPR / Cas nuclease and the deaminase are fused via a linker. In some embodiments, the linker comprises a (GGGGS)n (SEQ ID NO: 17), (G)n, (EAAAK)n (SEQ ID NO: 18), (GGS)n, SGSETPGTSESATPES (SEQ ID NO: 19) motif (see, e.g., Guilinger JP, Thompson DB, Liu DR. Additional suitable linker motifs and linker configurations will be apparent to one of skill in the art. In some embodiments, suitable linker motifs and configurations include those described in Chen et al., Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev. 2013; 65(10):1357-69, the entire contents of which are incorporated herein by reference.

[0074] In some embodiments, the fusion protein may include additional features. Other exemplary features that may be present are localization sequences, such as a nuclear localization sequence (NLS), a cytoplasmic localization sequence, a transport sequence, such as a nuclear transport sequence, or other localization sequences, as well as sequence tags that are useful for solubilizing, purifying, or detecting the fusion protein. Suitable localization signal sequences and protein tag sequences are provided herein and include, but are not limited to, biotin carboxylase carrier protein (BCCP) tags, myc tags, calmodulin tags, FLAG tags, hemagglutinin (HA) tags, polyhistidine tags (also referred to as histidine tags or His tags), maltose binding protein (MBP) tags, nus tags, glutathione S transferase (GST) tags, green fluorescent protein (GFP) tags, thioredoxin tags, S tags, Softag (e.g., Softag 1, Softag 3), strep tags, biotin ligase tags, FlAsH tags, V5 tags, and SBP tags. Additional suitable features will be apparent to those skilled in the art.

[0075] A variety of base-editing enzymes are known in the art (see, e.g., Improving cytidine and adenine base-editing enzymes by expression optimization and ancestral reconstruction. Nat Biotechnol. 2018 May 29), and typically include those listed in Table A. [Table 1] TIFF2024543966000018.tif152169 [ka] [ka] [ka] [ka] [ka] [ka]

[0076] The second component of the gene editing platform disclosed herein is composed of at least one guide RNA molecule suitable for guiding a base editing enzyme to at least one target sequence located in the +55 kb region of the erythroid-specific BCL11A enhancer. The guide RNA molecule of the present invention thus comprises a guide sequence for providing targeting specificity. It comprises a region that is complementary to and capable of hybridizing to a preselected target site of interest in the +55 kb region of the erythroid-specific BCL11A enhancer. According to the present disclosure, the guide RNA targets the ATF4 binding site and edits the site, thus disrupting the binding of ATF4 to the binding site.

[0077] In some embodiments, the guide sequence can comprise from about 10 nucleotides to more than about 25 nucleotides. For example, the region of base pairing between the guide sequence and the corresponding target site sequence can be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, or more than 25 nucleotides in length. In some embodiments, the guide sequence is about 17-20 nucleotides in length, such as 20 nucleotides.

[0078] Typically, a software program is used to identify candidate CRISPR target sequences on both strands of a DNA nucleic acid molecule based on the desired guide sequence length and the CRISPR motif sequence (PAM) for a particular 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 target sites. Type II CRISPR systems are one of the most well-characterized systems and only require the Cas 9 protein and a guide RNA complementary to the target sequence to affect target cleavage. For example, a target site for Cas9 from S. pyogenes with the PAM sequence NGG can be identified by searching for 5'-Nx-NGG-3' in the input sequence and in the reverse complement of the input. After identifying all potential sites, the program filters out sequences based on the number of times they appear in the associated reference genome, since multiple occurrences of DNA target sites in a genome can lead to non-specific genome editing. For CRISPR enzymes where sequence specificity is determined by a "seed" sequence, such as the 11-12 bp 5' from the PAM sequence, including the PAM sequence itself, the filtering step may be based on the seed sequence. Thus, to avoid editing at additional genomic loci, the results are filtered based on the number of occurrences of the seed:PAM sequence in the relevant genome. The user may be able to choose the length of the seed sequence. The user may also specify the number of occurrences of the seed:PAM sequence in the genome for purposes of passing the filter. The default is to screen for unique sequences. The filtering level is modified by varying 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 a sequence of guide sequences that are complementary to the reported target sequence by providing the reverse complement of the identified target sequence. Further details of methods and algorithms for optimizing sequence selection can be found in U.S. Patent Application No. 61 / 836,080; incorporated herein by reference.

[0079] In some embodiments, a gene editing platform comprising: a) a cytidine base editing enzyme, and b) and at least one guide RNA molecule suitable for introducing: - one C>T mutation in the first cytidine residue of SEQ ID NO: 45 (TTG C ATCATCC) and / or - one C>T mutation in the second cytidine residue of SEQ ID NO: 45 (TTGCAT C ATCC) and / or - one C>T mutation in the third cytidine residue of SEQ ID NO: 45 (TTGCATCAT C C) and / or - one C>T mutation in the fourth cytidine SEQ ID NO: 45 (TTGCATCATC C ).

[0080] In some embodiments, the gene editing platform comprises a) a cytidine base editing enzyme and b) at least one guide RNA molecule suitable for generating a +55CBE I editing profile of the ATF4 binding site (i.e., TTG T AT T AT TT (SEQ ID NO: 32)).

[0081] In some embodiments, the gene editing platform comprises a) a cytidine base editing enzyme and b) at least one guide RNA molecule suitable for generating a +55 CBE II editing profile at the ATF4 binding site (i.e., TTGCAT T AT TT (SEQ ID NO: 33)).

[0082] In some embodiments, a gene editing platform comprising: a) an adenine base editing enzyme, and b) and at least one guide RNA molecule suitable for introducing: - one A>G mutation at the first adenine residue in SEQ ID NO: 45 (TTGC A TCATCC) and / or - one A>G mutation in the second adenine residue of SEQ ID NO: 45 (TTGCATC ATCC).

[0083] In some embodiments, the gene editing platform comprises a) an adenine base editing enzyme and b) at least one guide RNA molecule (TTGCATC) suitable for generating a +55 ABE II profile at the ATF4 binding site. G TCC (SEQ ID NO: 35).

[0084] In some embodiments, the gene editing platform comprises a) an adenine base editing enzyme and b) at least one guide RNA molecule (TTGC G T.C. G TCC (SEQ ID NO: 46).

[0085] In some embodiments, the guide RNA targets a sequence selected from Table 1 (see Examples).

[0086] In some embodiments, the gene editing platform comprises a) a base editing enzyme that is ABE-SpRY or CBE-SpRY, and b) at least one guide RNA molecule that targets one of the sequences selected in Table 1 or Table 4.

[0087] In some embodiments, the gene editing platform comprises a) a base editing enzyme and b) at least one guide RNA molecule selected according to a combination described in Table 4.

[0088] The guide RNA molecules of the present invention can be produced 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 (200mer or longer) using TC-RNA chemistry (see, e.g., U.S. Pat. No. 8,202,983) allows for the production of RNAs with special features beyond those allowed by the four basic ribonucleotides (A, C, G, U). In particular, the RNA molecules of the present invention can be produced using recombinant techniques using host cell systems or in vitro translation-transcription systems known in the art. Details of such systems and techniques can be found, for example, in WO2014144761 WO2014144592, WO2013176772, US20140273226, and US20140273233, the contents of which are incorporated herein by reference in their entirety.

[0089] 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 or modified nucleotide, or an analog thereof. The modified nucleotide may be modified at the ribose, phosphate, and / or base moiety. The modified nucleotide may include 2'-O-methyl analogs, 2'-deoxy analogs, or 2'-fluoro analogs. The nucleic acid backbone may be modified, for example, a phosphorothioate backbone may be used. The use of locked nucleic acids (LNA) or bridged nucleic acids (BNA) may also be possible. Further examples of modified bases include, but are not limited to, 2-aminopurine, 5-bromouridine, pseudouridine, inosine, 7-methylguanosine.

[0090] In some embodiments, multiple guide RNA molecules are designed to target multiple sequences in the +55 kb region of the erythroid-specific BCL11A enhancer. 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.

[0091] In some embodiments, multiple base editing enzymes with multiple guide RNA molecules are designed to target multiple sequences in the +55 kb region of the erythroid-specific BCL11A enhancer. In some embodiments, the gene editing platform disclosed herein thus comprises 2, 3, or 4 base editing enzymes and 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 20 RNA molecules as disclosed herein.

[0092] In some embodiments, the different components of the gene editing platform of the invention are provided to eukaryotic cells through expression from one or more expression vectors. For example, nucleic acids encoding guide RNA molecules or base editing enzymes can be cloned into one or more vectors for their introduction into eukaryotic cells. The vectors are typically prokaryotic vectors, such as plasmids, or shuttle vectors, or insect vectors, for storage or manipulation of nucleic acids encoding guide RNA molecules or base editing enzymes disclosed herein. Preferably, the nucleic acids are isolated and / or purified. Thus, the invention provides recombinant constructs or vectors having sequences encoding one or more of the guide RNA molecules or base editing enzymes described above. Examples of constructs include vectors, such as plasmids or viral vectors, into which the nucleic acid sequences of the invention are inserted in forward or reverse orientation. In some embodiments, the construct further comprises a regulatory sequence. "Regulatory sequence" includes promoters, enhancers, and other expression control elements (e.g., polyadenylation signals).

[0093] Regulatory sequences include sequences that direct constitutive expression of a nucleotide sequence as well as inducible regulatory sequences. The design of an expression vector may depend on factors such as the choice of eukaryotic cells to be transformed, transfected, or infected, the desired level of expression, and the like. Many suitable vectors and promoters are known to those of skill in the art and are commercially available. Suitable cloning and expression vectors for use with eukaryotic hosts are also described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press). The vector may be capable of autonomous replication or integration into host DNA. The vector may also include appropriate sequences for amplifying expression. The expression vector also preferably includes one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or tetracycline or ampicillin resistance in, for example, E. coli. Any of the procedures known in the art for introducing foreign nucleotide sequences into a host cell may be used. Examples include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, nucleofection, liposomes, microinjection, naked DNA, plasmid vectors, viral vectors (both episomal and integrative), and other well-known methods for introducing cloned genomic DNA, cDNA, synthetic DNA, or other foreign genetic material into host cells.

[0094] In some embodiments, the different components of the gene editing platform of the present invention are provided to a population of cells through the use of RNA-encoded systems. For example, CBE-SpRY and ABE-SpRY are provided by protein-encoding mRNA sequences, such as SEQ ID NO: 17 and SEQ ID NO: 18, respectively. In particular, the sequences of the mRNAs are provided as SEQ ID NO: 30 and SEQ ID NO: 31. In particular, the base editing system can be provided to a population of cells through the use of chemically modified mRNA-encoded adenine or cytidine base editors together with modified guide RNAs, as described in Jiang, T., Henderson, JM, Coote, K. et al. Chemical modifications of adenine base editor mRNA and guide RNA expand its application scope. Nat Commun 11, 1979(2020). In particular, engineered RNA-encoded base editing enzyme (e.g., ABE) systems are prepared by introducing various chemical modifications into both the base editing enzyme-encoded mRNA and the guide RNA. In particular, the modification consists in a uridine-depleted mRNA modified with 5-methoxyuridine: synonymous codons may be introduced to deplete uridine as much as possible without modifying the coding sequence, and all remaining uridines are replaced with 5-methoxyuridine. The optimized base editing system shows higher editing efficiency at some genomic sites compared to DNA-encoded systems. It is also possible to encapsulate the modified mRNA and guide the RNA into lipid nanoparticles (LNPs) to enable lipid nanoparticle (LNP)-mediated delivery.

[0095] In some embodiments, the different components of the gene editing platform of the present invention are provided to a population of cells through the use of a ribonucleoprotein (RNP) complex. For example, a base editing enzyme can be pre-complexed with one or more guide RNA molecules to form a ribonucleoprotein (RNP) complex. The RNP complex can be introduced into a eukaryotic cell in this manner. The introduction of the RNP complex can be timed. The cell can be synchronized with other cells at the G1, S, and / or M phases of the cell cycle. RNP delivery avoids many of the pitfalls associated with mRNA, DNA, or viral delivery. Typically, an RNP complex is produced by simply mixing a protein (i.e., a base editing enzyme) and one or more guide RNA molecules in a suitable buffer. The mixture is incubated at room temperature for 5-10 minutes prior to electroporation. Electroporation is a delivery technique in which an electric field is applied to one or more cells to increase the permeability of the cell membrane. In some embodiments, genome editing efficiency can be improved by adding transfection enhancer oligonucleotides.

[0096] In some embodiments, multiple sequential transfections are performed to reach a desired level of mutagenesis in a cell.

[0097] A further object of the present invention is a method for treating a beta-hemoglobinopathy in a subject in need thereof, comprising transplanting a therapeutically effective amount of a population of eukaryotic cells obtained by the method described above.

[0098] In some embodiments, the population of cells is autologous to the subject, meaning that the population of cells is derived from the same subject.

[0099] In some embodiments, the beta-hemoglobinopathy is sickle cell disease.

[0100] In some embodiments, the beta-hemoglobinopathy is beta-thalassemia.

[0101] kit The present invention further provides a kit comprising reagents for carrying out the above-described methods, including all components of the gene editing platform disclosed herein for carrying out mutagenesis. To that end, one or more reaction components for the methods disclosed herein, such as guide RNA molecules and nucleic acid molecules encoding base editing enzymes, can be provided 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 can comprise one or more other reaction components. In some embodiments, suitable amounts of one or more reaction components are provided in one or more containers or carried 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 cell, one or more reagents for detecting the expression of the guide RNA or base editing enzyme or verifying the status of the target nucleic acid (e.g., probes or PCR primers), and buffers or culture media for the reaction. The kit may also comprise one or more of the following components: a support, a termination reagent, a modification reagent or a digestion reagent, an osmolyte, and an apparatus for detection. The components used can be provided in a variety of forms. For example, the components (e.g., enzymes, RNA, probes and / or primers) can be suspended in aqueous solution or as freeze-dried or lyophilized powders, pellets, or beads. In the latter case, the components, when reconstituted, form a complete mixture of components for use in the assay. The kits of the invention can be provided at any suitable temperature. For example, for storage of kits containing protein components or their complexes in liquid, they are preferably provided and maintained below 0°C, preferably below -20°C, or otherwise in a frozen state. The kits can also include packaging materials for holding a container or combination of containers.Typical packaging materials for such kits and systems include a solid matrix (e.g., glass, plastic, paper, foil, microparticles, and the like) that holds the reaction components or detection probes in various configurations (e.g., in vials, microtiter plate wells, microarrays, and the like). The kits may further include instructions recorded in tangible form for use of the components.

[0102] The present invention is further illustrated by the following figures and examples, which should not, however, be construed in any way as limiting the scope of the present invention. [Brief description of the drawings]

[0103] [Figure 1] Figure 1. sgRNA design for targeting the +55 kb region of the erythroid-specific BCL11A enhancer. A-B. Schematic diagram of a portion of the BCL11A gene on chromosome 2 depicting exons 2, 3, and 4 (Ex2, 3, and 4) and DNase I hypersensitive sites +62 kb, +58 kb, and +55 kb. The sequence of the +55 kb region of the erythroid-specific BCL11A enhancer is depicted. The ATF4 transcriptional activator BS is highlighted in bold. The target sequences of the base editing enzymes and the sgRNAs used are reported (highlighted with arrows) and are aligned with the DNA sequences to which they bind in the strand direction. The oval shapes indicate the transcriptional activator and ATF4. [Diagram 2]Figure 2. RNA-mediated base editing of the erythroid-specific BCL11A enhancer in SCD hPSC-derived erythroblasts. A. Experimental protocol used for base editing experiments in SCD HSPCs. Base editor mRNA and sgRNA were co-transfected in SCD HSPCs. Cells were differentiated into mature RBCs using a triphasic erythroid differentiation protocol or placed in methylcellulose-containing medium under conditions supporting erythroid (BFU-E) and granulomonocyte (CFU-GM) differentiation. B. CG to TA or AT to GC base editing efficiency, calculated by EditR software, in erythroblasts differentiated from SCD HSPCs that were edited in the +55 kb region and subjected to Sanger sequencing. Data are expressed as mean ± SEM (n=3 biologically independent experiments, 3 donors). CBE I and II profiles were generated by CBE-SpRY and ATF4_BS_1 and ATF4_BS_2 sgRNAs, respectively, and ABE I and II profiles were generated by ABE-SpRY and ATF4_BS_1 and ATF4_BS_2 sgRNAs, respectively. D. Frequency of InDels measured by TIDE analysis in control, base-edited and Cas9-edited samples subjected to Sanger sequencing. Data are expressed as mean ± SEM (n=3 biologically independent experiments, 3 donors). ****p≦0.0001 (ordinary one-way ANOVA). [Diagram 3]Figure 3. RNA-mediated base editing of the BCL11A enhancer in SCD HSPCs reactivates HbF in erythroid colonies without affecting progenitor cells. A. CG to TA or AT to GC base editing efficiency calculated by EditR software in BFU-E and CFU-GM pooled colonies subjected to Sanger sequencing. BFU-E and CFU-GM derived from SCD HSPCs edited in the +55 kb region. Data are expressed as mean ± SEM (n = 3 biologically independent experiments, 3 donors). +55: CBE I and II profiles were generated by CBE-SpRY, ABE I and II profiles were generated by ABE-SpRY. B-C. InDel frequency measured by TIDE analysis for control, base edited and Cas9 edited BFU-E (B) and CFU-GM (C) pooled samples subjected to Sanger sequencing. Data are expressed as mean ± SEM (n = 3 biologically independent experiments, 3 donors). *** p ≤ 0.001; **** p ≤ 0.0001 (ordinary one-way ANOVA). D. CFC frequency for control and edited samples. Data are expressed as mean ± SEM (n = 3 biologically independent experiments, 3 donors). E. Analysis of HbF and HbS by CE-HPLC in BFU-E. We calculated the percentage of each Hb type across the total Hb tetramer. Data are expressed as mean ± SEM (n = 3 biologically independent experiments, 3 donors). F. Expression of γ-globin chains measured by RP-HPLC in BFU-E. γ-globin expression was normalized to α-globin. Data are expressed as mean ± SEM (n = 3 biologically independent experiments, 3 donors). [Figure 4]Figure 4. Disruption of the erythroid-specific BCL11A enhancer does not affect the erythroid differentiation process of SCD HSPCs. A. Frequency of enucleated cells at days 6, 13, 16, and 19 of erythroid differentiation measured by flow cytometric analysis of DRAQ5 nuclear staining in control (untreated, or mock-transfected with TE buffer, or transfected with BE mRNA alone, or transfected with BE mRNA and sgRNA targeting the unrelated AAVS1 locus) and edited samples. Data are expressed as mean ± SEM (n = 3 biologically independent experiments, 3 donors). B-D. Frequency of CD71+ (B), CD36+ (C), and GPA+ (D) cells at days 6, 13, and 19 of erythroid differentiation measured by flow cytometric analysis. Data are expressed as mean ± SEM (n = 3 biologically independent experiments, 3 donors). E. Frequency of α4-integrin+, BAND3+, and α4-integrin+ / BAND3+ in 7AAD- / GPA+ cells at days 6, 13, and 19 of erythroid differentiation as determined by flow cytometric analysis. Data are expressed as mean ± SEM (n = 3 biologically independent experiments, 3 donors). [Diagram 5]Figure 5. Disruption of the erythroid-specific BCL11A enhancer reactivates HbF and ameliorates the sickling phenotype in RBCs derived from base-edited SCD HSPCs. A. Analysis of HbF and HbS by CE-HPLC in SCD patient RBCs. The percentage of each Hb type across the total Hb tetramers was calculated. Data are expressed as mean ± SEM (n = 3 biologically independent experiments, 3 donors). * p ≤ 0.05; *** p ≤ 0.001 (2-way ANOVA). B. RT-qPCR analysis of βS- and γ-globin mRNA levels in SCD patient erythroblasts at day 13 of erythroid differentiation. βS- and γ-globin mRNA expression was normalized to α-globin mRNA and expressed as the percentage of βS- + γ-globin mRNA. Data are expressed as mean ± SEM (n = 3 biologically independent experiments, 3 donors). **p≦0.01; ****p≦0.0001 (2-way ANOVA). C. Expression of γ-globin chain measured by RP-HPLC in SCD patient RBCs at the end of erythroid differentiation. γ-globin expression was normalized to α-globin. Data are expressed as mean ± SEM (n=3 biologically independent experiments, 3 donors). D. Frequency of HbF and HbS expressing cells in the GPA+ population for control and edited samples. Data are expressed as mean ± SEM (n=3 biologically independent experiments, 3 donors). E. Frequency of sickling cells upon O2 deprivation in control and edited samples. Data are expressed as mean ± SEM (n=2 biologically independent experiments, 2 donors). [Figure 6]Figure 6. Reactivation of HbF in single erythroid progenitors. A. HBG mRNA relative expression in single BFU-E colonies with different editing profiles and either mono- or bi-allelic (mono) or bi-allelic (bi) editing (Ctrl n=23; CBE I mono n=9; CBE I bi n=11; CBE II mono n=4; CBE II bi n=11; ABE I mono n=9; ABE I bi n=15; ABE II mono n=6; ABE II bi n=2; 1 donor). HBG mRNA expression was normalized to HBA1 / 2 mRNA and expressed as a percentage of total HBB+HBG mRNA. BFU-E derived from SCD HSPCs mock-transfected with TE buffer, transfected with BE mRNA alone, or transfected with BE mRNA and sgRNA targeting the irrelevant AAVS1 locus were used as negative controls (Ctrl). *p≦0.05; **p≦0.01; ***p≦0.001; ****p≦0.0001 (one-way ANOVA). B. Correlation between HBG mRNA relative expression and base editing efficiency in single BFU-E colonies with different editing profiles (Ctrl n=23; CBE I n=20; CBE II n=15; ABE I n=24; ABE II n=8; 1 donor). HBG mRNA expression was normalized to HBA1 / 2 mRNA and expressed as a percentage of total HBB+HBG mRNA. Base editing efficiency was calculated by EditR software in samples subjected to Sanger sequencing. BFU-E derived from SCD HSPCs mock-transfected with TE buffer, transfected with BE mRNA alone, or transfected with BE mRNA and sgRNA targeting an irrelevant AAVS1 locus were used as negative controls.ns p>0.05; *p≦0.05; ** p≦0.01; *** p≦0.001; **** p≦0.0001 (CBE I: R2=0.3989, Y=0.4685*X+29.98, p<0.0001 significance of non-zero slope; CBE II: R2=0.4072, Y=0.3532*X+31.75, p<0.0001 significance of non-zero slope; ABE I: R2=0.07206, Y=0.1431*X+31.24, p=0.0651 significance of non-zero slope; ABE II: R2=0.4710, Y=0.7134*X+30.87, p<0.0001 significance of non-zero slope; multiple t-test). [Figure 7] Figure 7. Disruption of the erythroid-specific BCL11A enhancer in SCD HSPC-derived cells using AncBE4max-OPT. CG to TA base editing efficiency calculated by EditR software in DNA samples obtained from cells cultured in HSPC medium and subjected to Sanger sequencing. SCD HSPCs were edited in the +55 kb region using either CBE-SpRY or AncBE4max-OPT. Data are expressed as single values ​​(n=1 donor). [Figure 8] Figure 8. Disruption of erythroid-specific BCL11A enhancer in SCD HSPC-derived cells with highly processive ABE. A. AT to GC base editing efficiency calculated by EditR software in SCD HSPC-derived cells (erythroid liquid culture, BFU-E colony, CFU-GM colony) subjected to Sanger sequencing. SCD HSPCs were edited in the +55 kb region with ABE8e. Data are expressed as mean ± SEM (n = 2 biologically independent experiments, 2 donors). B. Analysis of HbF and HbS by CE-HPLC in SCD-derived BFU-E. The percentage of each Hb type across the total Hb tetramer was calculated. Data are expressed as mean ± SEM (n = 2 biologically independent experiments, 2 donors). ****p ≤ 0.0001 (2-way ANOVA).

[0104] Working Example: method HSPC purification and culture We obtained human non-mobilized peripheral blood CD34+ HSPCs from SCD patients. Eligible SCD samples for research purposes were obtained from the "Hôpital Necker-Enfants malades" Hospital (Paris, France). Written informed consent was obtained from all adult subjects. All experiments were performed in accordance with the Declaration of Helsinki. The study was approved by the local Institutional Review Board (ref: DC 2014-2272, CPP Ile-de-France II "Hôpital Necker-Enfants malades"). HSPCs were purified by immunomagnetic selection using an AutoMACS (Miltenyi Biotec) after immunostaining with a CD34 MicroBead Kit (Miltenyi Biotec). 48 hours prior to transfection, CD34+ cells were thawed and cultured at 5 × 10 in “HSPC medium” containing StemSpan (STEMCELL Technologies) supplemented with penicillin / streptomycin (Gibco), 250 nM StemRegenin1 (STEMCELL Technologies), and the following recombinant human cytokines (PeproTech): human stem cell factor (SCF) (300 ng / ml), Flt-3L (300 ng / ml), thrombopoietin (TPO) (100 ng / ml), and interleukin-3 (IL-3) (60 ng / ml). 5 The cells were cultured at a concentration of 10 cells / ml.

[0105] Plasmids The plasmids used in this study were: pCAG-CBE4max-SpRY-P2A-EGFP(RTW5133)(Addgene #139999), pCMV-T7-SpRY-P2A-EGFP(RTW4830)(Addgene #139989). pCMV_AncBE4max_P2A_GFP(Addgene #112100), ABE8e (Addgene #138489). A DNA fragment containing two copies of the 3' untranslated region (UTR) of the HBB gene and a 96 adenine polyA sequence (3'UTR+polyA) was purchased from Genscript. Similarly, another DNA fragment containing the uridine-depleted coding sequence of pCAG-CBE4max-SpRY-P2A-EGFP was generated (CBE-SpRY_U-delp). The CBE-SpRY-OPT plasmid was generated by inserting the 3'UTR+polyA fragment into the pCAG-CBE4max-SpRY-P2A-EGFP (Addgene #140003) plasmid and replacing the CBE4max-SpRY coding sequence with the CBE-SpRY_U-delp fragment. The CBE-SpRY-OPT plasmid contains a T7 promoter followed by Gnt to allow efficient capping. The ABE-SpRY-OPT plasmid was generated by inserting the 3'UTR+polyA fragment into the pCMV-T7-SpRY-P2A-EGFP(RTW4830) (Addgene #139989) plasmid. The AncBE4max-OPT plasmid was generated by inserting a point mutation in the PAM identification domain of the Cas9 nickase of the CBE-SpRY-OPT plasmid, which allows recognition of the NGG PAM, and by inserting a point mutation in the deaminase domain of the CBE-SpRY-OPT plasmid, which allows reconstruction of the enzyme's ancestral form.

[0106] sgRNA design We manually designed sgRNAs targeting the +55 kb region of BCL11A (Table 1). To generate sgRNA expression plasmids, oligonucleotides were annealed to generate sgRNA protospacers, and the duplex was ligated into Bbs I-digested MA128 plasmid (provided by M. Amendola, Geneson, France). For RNA-mediated base editing, we used chemically modified synthetic sgRNAs with 2'-O-methyl analogs and 3'-phosphorothioate nonhydrolyzable bonds in the first three 5' and 3' nucleotides (Synthego). [Table 2]

[0107] In vitro transcription of mRNA 10 μg of plasmid expressing the base editor was digested overnight with 20 units of a restriction enzyme that cuts once just after the polyA tail. The linearized plasmid was purified using a PCR purification kit (QIAGEN #28106) and eluted in 30 μl of DNase / RNase-free water. 1 μg of linearized plasmid was used as template for the in vitro transcription reaction (MEGAscript, Ambion #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 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 final ratio of cap:GTP of 4:1, allowing efficient capping of mRNA. The incubation time for the in vitro reaction was reduced to 30 min. For constructs without the lack of a polyA tail in the plasmid (ABE8e), an additional step of polyadenylation was performed using the manufacturer's guidelines (PolyA Tailing Kit, Ambion). The mRNA was precipitated using lithium chloride and resuspended in TE buffer in a final volume allowing to achieve a concentration of >1 μg / μl. The quality of the mRNA was checked using a Bioanalyzer (Agilent).

[0108] RNA transfection 2 x 10 per condition 5CD34+ HSPCs were transfected with 3.0 μg of enzyme-encoding mRNA and synthetic sgRNA at a final concentration of 2.3 μM, respectively. We used the P3 Primary Cell 4D-Nucleofector X Kit S (Lonza) and CA137 program (Nucleofector 4D). Non-transfected cells or cells transfected with TE buffer, or with only the enzyme-encoding mRNA, or with the enzyme-encoding mRNA and sgRNA targeting the AAVS1 locus served as negative controls.

[0109] Ribonucleoprotein (RNP) transfection The RNP complex was assembled at room temperature using 90 μM Cas9-GFP protein and 180 μM synthetic sgRNA (Cas9:sgRNA ratio 1:2). CD34+ HSPCs (2 × 10 5 Cells (100 cells / condition) were transfected with RNP complexes using the P3 Primary Cell 4D-Nucleofector X Kit S (Lonza) and the CA137 program (Nucleofector 4D) in the presence of a transfection enhancer (IDT). Non-transfected cells or cells transfected with TE buffer, or with only the enzyme-encoding mRNA, or with the enzyme-encoding mRNA and sgRNA targeting the AAVS1 locus served as negative controls.

[0110] HSPC differentiation Transfected CD34+ HSPCs were differentiated into mature RBCs using a triphasic erythroid differentiation protocol as previously described (Giarratana et al., 2005; Weber et al., 2020). During the first phase (days 0 to 6), cells were supplemented with 100 ng / ml recombinant human SCF (PeproTech), 5 ng / ml recombinant human IL-3 (PeproTech), 3 IU / ml EPO Eprex (Janssen-Cilag), and 10 -6 Cells were cultured in basal erythroid medium supplemented with 3 IU / ml EPO Eprex (Sigma). During the second phase (days 6 to 9), cells were co-cultured with MS-5 stromal cells in basal erythroid medium supplemented with 3 IU / ml EPO Eprex (Janssen-Cilag). During the third phase (days 9 to 20), cells were co-cultured with stromal MS-5 cells in cytokine-free erythroid basal medium. Erythroid differentiation was monitored by flow cytometric analysis of CD36, CD71, GYPA, BAND3, and α4-integrin erythroid surface markers, as well as of enucleated cells using DRAQ5 double-stranded DNA dye. Viable cells were identified using 7AAD.

[0111] Colony forming cell (CFC) assay CD34+ HSPCs were cultured at 1 × 10 in methylcellulose-based medium (GFH4435, Stem Cell Technologies) under conditions that support erythroid and granulo-monocytic differentiation. 3 Cells were seeded at a concentration of 1000 cells / mL. BFU-E and CFU-GM colonies were counted after 14 days. Colonies were randomly picked and collected as bulk populations (containing at least 25 colonies) to assess base editing efficiency, globin expression by RT-qPCR and RP-HPLC, and hemoglobin expression by CE-HPLC. BFU-E were randomly picked and collected as single colonies to assess base editing efficiency and globin expression by RT-qPCR.

[0112] Evaluation of editing efficiency Base editing efficiency and InDel frequency were evaluated in HSPC-derived erythroid cells at the end of the first stage of differentiation, and in BFU-E and CFU-GM 14 days after seeding. Genomic DNA was extracted from control and edited cells using PURE LINK Genomic DNA Mini kit (LifeTechnologies) or Quick-DNA / RNA Miniprep (ZYMO Research) according to the manufacturer's instructions. To evaluate base editing efficiency at the sgRNA target site, we performed PCR followed by Sanger sequencing and EditR analysis (Kluesner et al., 2018). TIDE analysis (tracking InDels by degradation) was also performed to evaluate the percentage of InDels in edited samples (Brinkman et al., 2014). [Table 3]

[0113] RT-qPCR Total RNA was extracted from SCD HSPCs differentiated to the erythroid lineage (day 13) using the RNeasy micro kit (QIAGEN) and from BFU-E pools using Quick-DNA / RNA Miniprep (ZYMO Research). RNA was treated with DNase using the DNase I kit (Invitrogen) 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 iTaq Universal SYBR Green Master Mix (Biorad) and the Viia7 Real-Time PCR System (ThermoFisher Scientific) or the CFX384 Touch Real-Time PCR Detection System (Biorad). [Table 4]

[0114] Flow cytometry analysis HSPC-derived erythroid cells were fixed with 0.05% cold glutaraldehyde and permeabilized with 0.1% TRITON X-100. After fixation and permeabilization, cells were stained with an antibody recognizing the GYPA erythrocyte surface marker (PE-Cy7 conjugated anti-GYPA antibody, 563666, BD Pharmingen) and either an antibody recognizing HbF (FITC conjugated anti-HbF antibody, clone 2D12 552829 BD) or an antibody recognizing HbS (anti-HbS antibody, H04181601, BioMedomics), followed by staining with a secondary antibody recognizing rabbit IgG (BV421 conjugated anti-rabbit IgG, 565014, BD). Flow cytometric analysis of CD36, CD71, GYPA, BAND3, and α4-integrin erythrocyte surface markers was performed using V450-conjugated anti-CD36 (561535, BD Horizon), FITC-conjugated anti-CD71 (555536, BD Pharmingen), PE-Cy7-conjugated anti-GYPA (563666, BD Pharmingen), PE-conjugated anti-BAND3 (9439, IBGRL), and APC-conjugated anti-CD49d (559881, BD). Flow cytometric analysis of enucleated or viable cells was performed using double-stranded DNA dyes (DRAQ5, 65-0880-96, Invitrogen and 7AAD, 559925, BD, respectively). Flow cytometric analysis was performed using a Fortessa X20 (BD Biosciences) or Gallios (Beckman Coulter) flow cytometer. Data was analyzed using FlowJo (BD Biosciences) software.

[0115] RP-HPLC analysis of globin chains RP-HPLC analysis was performed using a NexeraX2 SIL-30AC chromatograph and LC Solution software (Shimadzu). Globin chains were separated by HPLC using a 250 x 4.6 mm, 3.6 μm Aeris Widepore column (Phenomenex). Samples were eluted with 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.

[0116] CE-HPLC analysis of hemoglobin tetramer 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, MD). Samples were eluted with 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.

[0117] Sickling assay HSPC-derived mature RBCs obtained at the end of erythroid differentiation were incubated under graded hypoxic conditions (20% O2 for 20 min; 10% O2 for 20 min; 5% O2 for 20 min; 0% O2 for 60-80 min) and time-lapse analysis of sickling was performed in real time by video microscopy. Images were acquired every 20 min using an AxioObserver Z1 microscope (Zeiss) and a 40x objective. Throughout the time course, images were acquired and then processed using ImageJ to determine the percentage of non-sickled erythrocytes per acquired field in the total RBC population. More than 400 cells were counted per condition.

[0118] result RNA-mediated base editing in SCD HSPCs disrupts the +55 kb region of the erythroid-specific BCL11A enhancer To disrupt the BCL11A erythroid enhancer, we targeted the ATF4 BS in the +55 kb region (Figure 1). We primarily utilized CBE-SpRY and ABE-SpRY enzymes, taking into account their PAM-less nature, which allows us to design sgRNAs by placing base editing windows within different strands and positions of the ATF4 BS, thus enabling different types of conversion (Figure 1). To establish a clinically relevant method to introduce base editing systems in primary HSPCs and achieve high editing efficiency combined with minimal toxicity, we optimized a protocol based on transfection of mRNAs encoding base editors and synthetic modified sgRNAs. First, we optimized plasmids encoding CBE-SpRY and ABE-SpRY for in vitro transcription and mRNA production. In particular, we inserted two copies of the 3' untranslated region (UTR) of the HBB gene, which has been shown to increase the half-life of mRNA and improve protein levels (Ross and Sullivan, 1985; Kariko et al., 1999; Holtkamp et al., 2006), and a polyA sequence after the 3'UTR to further stabilize the mRNA (Gallie, 1991) in the CBE-SpRY and ABE-SpRY constructs (SEQ ID NO: 30 and SEQ ID NO: 31). The optimized plasmids CBE-SpRY-OPT and ABE-SpRY-OPT were used for in vitro transcription and mRNA production. [ka] TIFF2024543966000029.tif172169 [ka] TIFF2024543966000031.tif249169 TIFF2024543966000032.tif70169

[0119] In vitro transcribed CBE-SpRY-OPT or ABE-SpRY-OPT mRNA was transfected into SCD HSPCs (three non-mobilized donors) in combination with chemically modified sgRNA (Figure 2A). Transfected SCD HSPCs were differentiated toward the erythroid lineage (Figure 2A). CBE-SpRY in conjunction with ATF4_bs_1 or ATF4_bs_2 sgRNA led to disruption of the ATF4 BS with an efficiency of 47.0% ± 7.0 [mean ± standard error of the mean (SEM)] and 46.7% ± 3.8, respectively, and editing profiles hereafter named +55 CBE I or +55 CBE II, respectively, of TTGTATTATTT (SEQ ID NO: 32) and TTGCATTATTT (SEQ ID NO: 33), respectively; Figure 2B). ABE-SpRY in conjunction with ATF4_bs_1 or ATF4_bs_2 sgRNA led to disruption of the ATF4 BS with efficiencies of 63.7% ± 2.2 and 24.3% ± 2.7, and editing profiles hereafter termed +55 ABE I or +55 ABE II, respectively (TTGCGTCATCC (SEQ ID NO: 34) and TTGCATCGTCC (SEQ ID NO: 35), respectively; Figure 2B). In parallel, to compare base editing and Cas9 nuclease strategies, we transfected an RNP complex containing Cas9 nuclease and ATF4_bs_3 sgRNA (Huang et al., 2020) and disrupted the ATF4 BS in the +55 kb region of the erythroid-specific BCL11A enhancer (hereafter named +55 Cas9) with an efficiency of 86.8% ± 1.6 (Figure 2C). TIDE analysis confirmed the DSB-free nature of the base editor, since we did not detect InDels in the base-edited samples (Figure 2C). In conclusion, we were able to efficiently target the ATF4 BS at the +55 kb BCL11A enhancer region without causing DSBs in SCD HSPCs by targeting alternative bases.

[0120] RNA-mediated base editing of the BCL11A enhancer in SCD HSPCs reactivates HbF in erythroid colonies without affecting progenitor cell viability and differentiation Transfected SCD HSPCs were subjected to CFC assays (Figure 2A). While base editing efficiencies in BFU-E and CFU-GM pool colonies were similar to those observed in liquid erythroid cultures for sgRNAs targeting the +55 kb region, editing efficiencies tended to be lower and more variable in CFU-GM compared to BFU-E pools (Figure 3A). InDels were absent in base-edited samples as measured by TIDE analysis (Figures 3B and C). In Cas9 nuclease-treated samples, Indel efficiencies in BFU-E were similar to those observed in erythroid bulk and higher than in CFU-GM (Figures 3B and C). Neither RNA transfection of SCD HSPCs nor editing by itself affected erythroid or non-erythroid progenitors as shown by CFC assays. Indeed, no significant differences were observed between control and base-edited samples in terms of the number of erythroid BFU-E and granulomonocytic CFU-GM colonies (Figure 4D). These results demonstrate that our therapeutic strategy does not affect the viability of progenitor cells and their differentiation towards erythroid and granulomonocytic lineages.

[0121] The first insight into the therapeutic potential of our strategy was the reactivation of HbF in erythroid BFU-E pools upon RNA-mediated base editing of the erythroid-specific BCL11A enhancer. Notably, CE-HPLC analysis showed that base-edited samples expressed intermediate HbF levels between the control (22.1% × 7.4 in untreated samples) and the +55 Cas9 samples (49.7% × 2.2) (Figure 3E). There was a trend for higher HbF levels in +55 CBE samples compared to +55 ABE samples (41.9% ± 4.6, 39.1% ± 3.1, 35.3% ± 7.1, and 34.5% ± 10.8 for +55 CBE I, II, ABE I, and II profiles, respectively) (Figure 3E). These results were confirmed by RP-HPLC detecting single globin chains (Figure 3F). Overall, these data indicate that base editing-mediated disruption of the +55 kb erythroid-specific BCL11A enhancer reactivates the γ-globin gene in erythroid BFU-E colonies derived from base edited SCD HSPCs, with variable levels of expression based on the type of modification.

[0122] Disruption of the erythroid-specific BCL11A enhancer reactivates HbF in RBCs derived from base-edited SCD HSPCs Erythroid BFU-E colonies have a high level of HbF background and contain only a small proportion of fully differentiated erythroid cells, thus not allowing us to pinpoint the most robust editing profile in terms of HbF reactivation in enucleated RBCs. Therefore, we evaluated the HbF reactivation achieved in mature RBCs derived from fully differentiated control and transfected SCD HSPCs (Figure 2A).

[0123] First, we evaluated the effect of our strategy on the erythroid differentiation process to assess the safety of these therapeutic strategies. To this end, we measured the frequency of enucleated cells (DRAQ5 cells) along erythroid differentiation. In all samples, we observed an increase in the enucleation rate along differentiation, with mature enucleated erythrocytes reaching approximately 60% of the total cells at the end of differentiation. Importantly, no significant differences in the enucleation rate were found between control and treated samples (Figure 4A), indicating that RNA-mediated base editing of the BCL11A erythroid-specific enhancer does not affect the enucleation process. To better evaluate the impact of our procedure on SCD HSPC erythroid differentiation, we evaluated the expression of erythroid surface markers (CD36, CD71, GPA, BAND3, and α4-integrin) along erythroid differentiation by flow cytometry. Along the line of differentiation, we observed similar increases in the frequency of cells expressing GPA and band 3, as well as similar decreases in the frequency of cells expressing CD36, CD71, and α4-integrin in control and edited samples (Figure 4B-E). In summary, by targeting the ATF4 BS at the erythroid-specific BCL11A enhancer, erythroid differentiation of SCD HSPCs was not impaired.

[0124] We next assessed the levels of HbF reactivation in RBCs derived from control and edited HSPCs. CE-HPLC analysis revealed HbF reactivation in all samples (Figure 5A). CBE-treated samples showed higher HbF levels (24.5% ± 7.2 and 23.6 ± 6.4 for CBE I and II profiles, respectively) compared to ABE-treated samples (16.5% ± 5.0 and 15.6% ± 4.1 for ABE I and II profiles, respectively) (Figure 5A). Notably, cells harboring the ABE I profile showed modest HbF levels despite high base editing efficiency, suggesting that the targeted base is not essential for ATF4 binding. Conversely, cells harboring the ABE II profile reactivated HbF despite low base editing efficiency, indicating that the converted base is critical for ATF4 binding. Disruption of the ATF4 BS with either CBE or Cas9 led to similar levels of HbF (29.3% ± 6.7 for Cas9-treated samples), even though the editing efficiency in the Cas9 nuclease-treated samples was two-fold higher than in the base-edited samples (Figure 5A). These results were confirmed by RT-qPCR and RP-HPLC at the mRNA and single globin chain levels (Figures 5B and C). Flow cytometry analysis confirmed these data, with +55 CBE I, II, and ABE I samples showing a high percentage of F cells (56.9% ± 13.6, 55.2% ± 11.9, and 54.0% ± 13.4, respectively), approaching the value observed in the Cas9 nuclease-treated samples (62.3% ± 10.3) (Figure 5D). The frequency of HbS-expressing cells, however, was similar between the control and all edited samples (Figure 5D). Finally, sickling assays were performed on control and edited samples. A high frequency of corrected cells was observed for the base-edited samples (up to 56.1% ± 25.5) (Figure 5E).

[0125] Overall, these data indicate that base editing-mediated disruption of the ATF4 activator BS in the +55 kb region leads to HbF reactivation and ameliorated pathological sickling phenotype. Interestingly, different bases or base combinations have distinct roles in the binding of these TFs, some of which are more critical, thus representing potent base editing targets for downregulating BCL11A and reactivating HbF.

[0126] HbF reactivation in single erythroid progenitor cells Transfected SCD HSPCs were subjected to CFC assay (Figure 2A). To accurately compare the efficacy of the different editing approaches, we measured γ-globin expression at the clonal level in BFU-E. We observed significant γ-globin reactivation in colonies with biallelic editing profiles of the CBE I and CBE II groups, while for colonies of the same groups with monoallelic editing, there was a trend for higher γ-globin expression compared to the control (Figure 6A). Interestingly, the ABE II profile allowed significantly higher γ-globin expression not only in colonies with biallelic editing, but also in colonies with monoallelic editing, highlighting the ability of a single A>G mutation (at position 8 of the ATF4 binding site) to induce high HbF levels (Figure 6A). Conversely, the ABE I profile did not show any significant γ-globin reactivation even in colonies with a different A>G mutation (at position 6 of the ATF4 binding site) and with biallelic editing (Figure 6A). Similarly, we observed a positive correlation between base editing efficiency and gamma globin expression in the CBE I, CBE II, and ABE II groups (Figure 6B). The generation of the ABE II profile was the most robust event in terms of gamma globin reactivation (Figure 6B). While the CBE I and CBE II profiles showed similar gamma globin levels, ABE I did not show significant gamma globin reactivation (Figure 6B). Overall, these data demonstrate the ability of different editing profiles to reactivate HbF through the disruption of the ATF4 transcriptional activator BS in the +55 kb region of the erythroid-specific BCL11A enhancer.

[0127] Disruption of the erythroid-specific BCL11A enhancer in SCD HSPCs using a more precise and highly processive base editor In an effort to generate the best performance profile (CBE I and ABE II) in a more efficient and accurate manner, we utilized a BE enzyme that recognizes the NGG PAM. This minimizes potential off-target effects thanks to PAM recognition compared to the nearly PAMless SpRY-based enzyme used in previous experiments. In this frame, we used the AncBE4max enzyme that recognizes the NGG PAM. We first optimized the AncBE4max plasmid for in vitro transcription and mRNA production by also adding two copies of the 3'UTR of HBB and a polyA sequence after the 3'UTR. The optimized plasmid AncBE4max-OPT was used for in vitro transcription and mRNA production. In vitro transcribed AncBE4max-OPT or CBE-SpRY-OPT mRNA was transfected in SCD HSPCs (one non-mobilized donor) in combination with chemically modified ATF4_bs_1 sgRNA (Figure 2A). Transfected SCD HSPCs were cultured in "HSPC medium." Both enzymes yielded similar levels of C>T conversion, demonstrating that we can also recapitulate the CBE I profile with a more precise base editor (FIG. 7).

[0128] In parallel, we used the NGG-recognizing ABE8e enzyme to generate ABE II profiles in a more efficient and accurate manner. We transfected SCD HSPCs (two non-mobilized donors) with ABE8e mRNA in combination with chemically modified ATF4_bs_2 sgRNA (Figure 2A). Transfected SCD HSPCs were differentiated toward erythroid lineage or subjected to CFC assay (Figure 2A). We observed high base conversion rates in both erythroid liquid cultures and BFU-E or CFU-GM colonies (up to about 90%; Figure 8A). Notably, we were not able to accurately generate ABE II profiles (one single A>G mutation at position 8 of the ATF4 binding site), but thanks to the high processivity of the enzyme, we generated a new editing profile, named ABE III, which has two A>G mutations at positions 6 and 8 (Figure 8A). The generation of the ABE III profile was highly efficiently associated with very high HbF in erythroid BFU-E colonies, due to the high level of BE efficiency obtained using ABE8e (Figures 8A and B).

[0129] Notably, alternative sgRNAs were designed to precisely recapitulate the ABE II profile with one single A>G mutation at position 8 of the ATF4 binding site with high efficiency (Table 4). [Table 5]

[0130] In conclusion, we were able to efficiently target the ATF4 BS at the +55 kb BCL11A enhancer region by using a more precise and highly efficient BE enzyme in SCD HSPCs.

[0131] References: Throughout this application, various references describe the state of the art to which this invention pertains, the disclosures of which are hereby incorporated by reference into this disclosure.

Table 6

Claims

1. An in vitro method for increasing fetal hemoglobin content in a eukaryotic cell, comprising contacting the eukaryotic cell with a gene editing platform consisting of (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 +55 kb region of an erythroid-specific BCL11A enhancer, thereby editing and disrupting an ATF4 binding site in the region to suppress expression of BCL11A and subsequently increase expression of gamma globin.

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

3. 2. The method of Claim 1, wherein the base editing enzyme comprises a nickase, more specifically a Cas9 nickase.

4. 4. The method of claim 3, wherein the nickase comprises the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO:

4.

5. 2. The method of claim 1, wherein the base editing enzyme is cytidine deaminase or adenosine deaminase.

6. 2. The method of claim 1, wherein the base editing enzyme is selected from the group consisting of ABE-SpRY (SEQ ID NO: 18), CBE-SpRY (SEQ ID NO: 17), ABE8e (SEQ ID NO: 37), ABE8e-SpRY (SEQ ID NO: 38), ABE8e-NRCH (SEQ ID NO: 39), and NG-ABE8e (SEQ ID NO: 40).

7. 2. The method of claim 1, wherein the guide RNA targets the ATF4 binding site and edits the site, thus disrupting binding of ATF4 to the binding site.

8. the gene editing platform comprising: a) a cytidine base editing enzyme; and b): - one C>T mutation in the first cytidine residue of SEQ ID NO: 45 (TTGCATCATCC), and / or - one C>T mutation in the second cytidine residue of SEQ ID NO: 45 (TTGCATCATCC), and / or - one C>T mutation at the third cytidine residue of SEQ ID NO: 45 (TTGCATCATCC), and / or - one C>T mutation at the fourth cytidine residue of SEQ ID NO: 45 (TTGCATCATCC) at least one guide RNA molecule suitable for introducing The method of claim 1 , comprising:

9. 9. The method of Claim 8, wherein the gene editing platform comprises: a) a cytidine base editing enzyme; and b) at least one guide RNA molecule (i.e., TTGTATTATTT (SEQ ID NO: 32)) suitable for generating a +55 CBE I editing profile at the ATF4 binding site.

10. 9. The method of Claim 8, wherein the gene editing platform comprises: a) a cytidine base editing enzyme; and b) at least one guide RNA molecule (i.e., TTGCATTATTT (SEQ ID NO: 33)) suitable for generating a +55 CBE II editing profile at the ATF4 binding site.

11. the gene editing platform comprising: a) an adenine base editing enzyme; and b): - one A>G mutation at the first adenine residue of SEQ ID NO: 45 (TTGCATCATCC), and / or - one A>G mutation at the second adenine residue of SEQ ID NO: 45 (TTGCATCATCC) at least one guide RNA molecule suitable for introducing The method of claim 1 , comprising:

12. 12. The method of Claim 11, wherein the gene editing platform comprises: a) an adenine base-editing enzyme; and b) at least one guide RNA molecule (TTGCATCGTCC (SEQ ID NO: 35)) suitable for generating a +55 ABE II profile at the ATF4 binding site.

13. 12. The method of Claim 11, wherein the gene editing platform comprises: a) an adenine base editing enzyme; and b) at least one guide RNA molecule (TTGCGTCGTCC (SEQ ID NO: 46)) suitable for generating a +55 ABE III profile at the ATF4 binding site.

14. 2. The method of claim 1, wherein the guide RNA targets a sequence selected from SEQ ID NO: 19 to SEQ ID NO:

21.

15. 2. The method of claim 1, wherein the gene editing platform comprises: a) a base editing enzyme that is ABE-SpRY or CBE-SpRY; ​​and b) at least one guide RNA molecule that targets one sequence selected from SEQ ID NOs: 19 to 21 and SEQ ID NOs: 41 to 44.

16. 2. The method of claim 1, wherein the gene editing platform comprises a) a base editing enzyme and b) at least one guide RNA molecule selected from SEQ ID NOs: 41 to 44.

17. 2. The method of claim 1, wherein the gene editing platform comprises multiple guide RNA molecules designed to target multiple sequences in the +55 kb region of the erythroid-specific BCL11A enhancer.

18. 10. The method of Claim 1, wherein different components of the gene editing platform are provided to the population of cells through the use of an RNA encoding system.

19. A method for producing a pharmaceutical composition for increasing fetal hemoglobin levels, wherein the pharmaceutical composition comprises a population of eukaryotic cells obtained by a method described in any one of claims 1 to 18.

20. A method for producing a pharmaceutical composition for treating beta-hemoglobinopathy, wherein the pharmaceutical composition comprises a population of eukaryotic cells obtained by a method described in any one of claims 1 to 18.

21. 21. The method of claim 20, wherein the beta-hemoglobinopathy is sickle cell disease or beta-thalassemia.

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