Induction of fetal haemoglobin using paired CAS9 nickases

Abstract

The invention relates to a kit for disrupting binding sites of repressor leukemia / lymphoma-related factor (LRF) of a promotor of a hemoglobin gamma (HbG) locus, compositions comprising said kit, and medical use thereof for treating and preventing β-hemoglobinopathy in a subject, wherein the kit comprising a CRISPR-Cas9 nickase and a pair of guide RNAs (gRNAs), wherein the pair of gRNAs binds to target sequences and results in a disruption of binding sites of repressor LRF in a promotor of HbG locus.

Description

INDUCTION OF FETAL HAEMOGLOBIN USING PAIRED CAS9 NICKASESTECHNICAL FIELD

[0001] The present disclosure relates generally to the field of biotechnology. More particularly, the present disclosure relates to the field of gene therapy.BACKGROUND

[0002] The human globin gene clusters on chromosome 11 controls the expression of p-subunit, which together with a-subunit, produces the function haemoglobin (Hit). Several P-subunit exists, including the predominantly fetal y chain (HBG gene) and the adult 5 (HBD gene; minor) and P (HBB gene; major) chains. Individuals that inherit mutations that disrupt p-chain, a condition known as p- hemoglobinopathies, fail to produce functional haemoglobin (adult HbA: α2p2).

[0003] Conventional methods of treating P-hemoglobinopathies which use allogenic hematopoietic cells transplantation are limited by the availability of compatible donors. Furthermore, other conventional methods of gene therapy using CRISPR / Cas9 for P-hemoglobinopathies face issues such as off-targel effects, which cause detrimental health issues to the subject. The only approved gene therapy product for P-hemoglobinopathies which is based on lentiviral transfer of P-chain (P-A'J 87ymutant) causes random insertional mutagenesis in hematopoietic stem cells.

[0004] Therefore, there is a need for a gene therapy for treating P-hemoglobinopathies that addresses some of the above-mentioned problems.SUMMARY

[0005] In one aspect, the present disclosure provides a kit comprising: a CRISPR-Cas9 nickase; a pair of guide RNAs (gRN A), wherein the pair of guide RN As binds to target sequences, wherein the binding of the guide RNAs to target sequences will result in a disruption of binding sites of repressor LRF in a promoter of a hemoglobin gamma (HbG) locus.

[0006] In another aspect, the present disclosure provides a method for modifying a hemoglobin gamma (HbG) locus in a eukaryotic cell, the method comprising introducing into the eukaryotic cell a kit comprising: a CRTSPR-Cas9 nickase; a pair of guide RNAs (gRNA), wherein the pair of guide RNAs binds to target sequences, wherein the binding will result in a disruption of binding sites of repressor LRF in a promoter of a hemoglobin gamma (HbG) locus.

[0007] In another aspect, the present disclosure provides a method of treating p-hemoglobinopathy in a subject, wherein the method comprises the steps of: a) isolating hematopoietic cells from the subject; b) editing the isolated hematopoietic cells obtained from step a) with a kit of the disclosure ex vivo', c) expanding the edited cells obtained from step b) ex vivo', and d) administering a therapeutically effective amount of the expanded cells obtained from step c) to a subject with a P-hemoglobinopathy.

[0008] In another aspect, the present disclosure provides a gene therapy for preventing [>- hemoglobinopathy in a subject, wherein the gene therapy comprises the steps of: a) isolating hematopoietic cells from the subject; b) editing the isolated hematopoietic cells obtained from step a) with a kit of the disclosure ex vivo; c) expanding the edited cells obtained from step b) ex vivo; and d) administering a therapeutically effective amount of the expanded cells obtained from step c) to a subject susceptible to P-hemoglobinopathy.

[0009] In another aspect, the present disclosure provides a composition comprising the kit of the disclosure.

[0010] In another aspect, the present disclosure provides a method of treating P-hemoglobinopathy in a subject, wherein the method comprises administering a therapeutically effective amount of the composition of the disclosure to a subject with a P-hemoglobinopathy.

[0011] In another aspect, the present disclosure provides a gene therapy for preventing P- hemoglobinopathy in a subject, wherein the method comprises administering the composition of the disclosure to a subject susceptible to P-hemoglobinopathy.BRIEF DESCRIPTION OF DRAWINGS

[0012] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which: Disrupting HBG promoters restore gamma haemoglobin expression.

[0013] Fig. la is a schematic diagram of the human beta-globin loci on chromosome 11. Expression of beta-globin genes (HBB, HBD, HBG1, HBG2, and HBE) are modulated by the locus control region and is highly coordinated with the stage of development. Normally the P- and 8-chains are expressed in adult when the expression of fetal Gy- and Ay-chains are repressed. The two HBG genes are highly homologous, in both promoter and the coding regions.

[0014] Fig. lb is a schematic diagram showing the identical promoter sequences of HBG1 and HBG2. The binding sites of the repressors BCL11A and LRF are underlined. Naturally occurring heredity persistence of fetal haemoglobin (HPFH) mutations that resulted in elevated HbF level are in bold. The position and orientation of the gRNAs used herein are illustrated.Examination of individual gRNAs, and in combinations.

[0015] Fig. 2a is a schematic showing the T7E1 assay results of individual gRNA on cord blood (CB) 34+cells. The gRNAs were combined with wildtype Cas9 nucleases (Cas9) or Cas9 nickase (Cas9n) to produce the ribonucleoprotein complex (RNP), before electroporation into cord blood (CB)34+cells. Genomic DNA was harvested 4 days post electroporation and the extent of editing was evaluated by T7E1 assay. The percentages of cleavage were shown below the lanes, which represented the extent of editing. gRNA pair combined with Cas9 nickase (Cas9n) showed higher editing than single gRNA combined with Cas9 nuclease (Cas9).

[0016] Fig. 2b is a schematic showing the T7E1 assay results of initial testing of gRNA Al with paired nickases on cord blood (CB) 34+cells. gRNA pairs with Cas9 nickase (Cas9n) showed a higher extent of editing than a single gRNA with Cas9 nuclease (Cas9) or with Cas9 nickase (Cas9n).

[0017] Fig. 2c is a schematic showing the T7E I assay results of comprehensive testing of all combinations with paired nickases on cord blood (CB) 34+cells.

[0018] Fig. 2d is a schematic showing the T7E1 assay results of the most efficient nickase pairs (A2 + S3 and A2 + S4) in adult CD34+cells under the same experimental conditions.Testing of paired nickases in adult hematopoietic cells.

[0019] Fig. 3a is a schematic showing the T7E1 assay results comparing the efficiency of Cas9 nickase and nuclease in inducing HbF rc-cxprcssion. The RNPs (Cas9n with A2 +S4, and Cas9 with A2) was electroporated into adult CD34+cells, and the editing efficiency was evaluated as before on day 4 postelectroporation. gRNA pair with Cas9 nickase (Cas9n) showed a higher efficiency in inducing HbF reexpression than a single gRNA with Cas9 nuclease (Cas9).

[0020] Fig. 3b is a flow plot showing the functional consequence of editing of HBG promoter. The edited cells were expanded and differentiated into erythrocytes for 17-20 days. The cells were then harvested for both RNA extraction (RT-PCR assay) and intracellular staining (flow analysis). The percentage of HbF expressing CD235+erythrocyte (F cells) was determined by flow cytometry, shown by the representative flow plots (cells in Q2, which were CD235+and HbF+)). The percentage of HbF expressing CD235+erythrocyte (F cells) is higher when edited by gRNA pair with Cas9 nickase (Cas9n) compared to a single gRNA with Cas9 nuclease (Cas9).

[0021] Fig. 3c is a schematic showing the T7E1 assay results of the effect of editing on adult CD34+hematopoietic cells. Results of T7E1 assay (for editing efficiency), flow cytometry (percentage of F cells) and RT-PCR (level of gamma transcript) are summarized (n = 3). gRNA pair with Cas9 nickase (Cas9n) showed higher editing efficiency, HbF expression, and level of gamma transcript than single gRNA with Cas9 nuclease (Cas9).Paired nickases induce HbF on hematopoietic cells from individuals with Thalassemia.

[0022] Fig. 4a is a schematic showing the T7EI assay results of the editing efficiency of selected gRNA pairs. Selected gRNAs and Cas9 nickases and nucleases were introduced into CD34+thalassemia cells. Experimental details arc the same as described for adult CD34+cells in Fig. 3. gRNA pairs with Cas9 nickase (Cas9n) showed editing efficiency than single gRNA with Cas9 nuclease (Cas9).

[0023] Fig. 4b is a representative flow plot for samples Thal_#l showing the induction of HbF upon editing. gRNA pairs with Cas9 nickase (Cas9n) showed higher induction of HbF than single gRNA with Cas9 nuclease (Cas9).

[0024] Fig. 4c is a schematic showing the T7E1 assay results of editing on individual thalassemia samples was shown, with exact data shown for each sample (n = 3). gRNA pairs with Cas9 nickase (Cas9n) showed higher editing efficiency, HbF expression, and level of gamma transcript than single gRNA with Cas9 nuclease (Cas9).Paired Cas9 nickases greatly reduced off -target editing of the Cas9 nucleases.

[0025] Fig. 5a is a schematic showing the targeted amplicon sequencing analysis of the specificity of Cas9 editing. The genomic DNA was extracted from the edited cells 4-6 days post-electroporation. A total of 54 specific targets, including the editing site on HBG promoter and the potential off-target sites (OFT), were included in the amplicon panel for targeted sequencing. Editing by Cas9 nuclease or nickase, presented by Indcls (inscrtions / dclctions) created through non-homologous end joining (NHEJ), were analysed for the targets. Only targets with indels above the level of control (Cas9 or Cas9n alone) are shown, for adult CD34+samples. gRNA pair with Cas9 nickase (Cas9n) showed lower off-target editing than a single gRNA with Cas9 nuclease (Cas9).

[0026] Fig. 5b is a schematic showing the targeted amplicon sequencing analysis of the specificity of Cas9 editing. Only largels with indels above the level of control (Cas9 or Cas9n alone) are shown, for thalassemia CD34+samples. gRNA pairs with Cas9 nickase (Cas9n) showed lower off-target editing than a single gRNA with wildtype Cas9 nuclease (Cas9).On target editing of HBG promoters resulted in deletion of the inter-promoters region.

[0027] Fig. 6a is a schematic showing the gRNA targeting regions on HBG1 and HBG2 gene, and the position of primer probes (half-arrows) for detection of deletion. PCR was performed to detect the presence of -1.5 kb fragment resulting from the deletion. Single gRNA with wildtype Cas9 were introduced as before but the harvested genomic DNA was used for the deletion specific PCR instead.

[0028] Fig. 6b is a schematic showing the PCR results for testing of individual gRNA as well as the most efficient combinations in cord blood CD34+cells. gRNA pair with Cas9 nickase (Cas9n) showed higher percentage of deletion of inter-promoters region than a single gRNA with Cas9 nuclease (Cas9).

[0029] Fig. 6c is a schematic showing the PCR results for testing of individual gRNA as well as the most efficient combinations in thalassemia samples. gRNA pairs with Cas9 nickase (Cas9n) showed higher editing efficiency than a single gRNA with Cas9 nuclease (Cas9).

[0030] Fig. 6d is a schematic showing the digital PCR results to quantify the deletion created by editing using paired or single gRNAs. The change in copy number for the inter-promoters region was normalized to that of the control region (CD44), for both adult 34+and thalassemia samples (n = 3 each). gRNA pair with Cas9 nickase (Cas9n) showed higher deletion of inter-promoters region than a single gRNA with Cas9 nuclease (Cas9).Paired Cas9 nickases approach demonstrated efficiency comparable to that of single gRNA mediated editing of BCL11A.

[0031] Fig. 7a is a schematic showing the T7E1 assay results of the editing efficiency of Casgevy gRNA and Cas9 nuclease (Cas9), and selected gRNA pairs with Cas9 nickase (Cas9n). Representative gel images of the T7E1 assay from two independent experiments are shown, where the estimated editing efficiency is shown for each lane. gRNA pairs as claimed herein with Cas9 nickase (Cas9n) showed higher editing efficiency than Casgevy gRNA with Cas9 nuclease (Cas9).

[0032] Fig. 7b is a schematic showing the PCR results of the deletion of inter-promoters region of HBGs (HBG2) specific in thalassemia cells, by Casgcvy gRNA and Cas9 nuclease (Cas9), and selected gRNA pairs with Cas9 nickase (Cas9n). gRNA pairs as claimed herein with Cas9 nickase (Cas9n) showed higher deletion of inter-promoters regions than Casgevy gRNA with Cas9 nuclease (Cas9).

[0033] Fig. 7c is a schematic showing the induction of HbF upon editing by Casgevy gRNA and Cas9 nuclease (Cas9), and selected gRNA pairs with Cas9 nickase (Cas9n), as measured using flow cytometry. gRNA pairs with Cas9 nickase (Cas9n) showed higher induction of HbF than a single gRNA with Cas9 nuclease (Cas9).

[0034] Fig. 7d is a schematic showing the induction of HbF upon editing by Casgevy gRNA and Cas9 nuclease (Cas9). and selected gRNA pairs with Cas9 nickase (Cas9n), as measured by the level of gamma transcript encoding for HbF using RT-PCR. gRNA pairs with Cas9 nickase (Cas9n) showed higher induction of HbF than a single gRNA with Cas9 nuclease (Cas9), as measured by the level of gamma transcript encoding for HbF using RT-PCR.Mouse engraftment of edited human thalassemia CD34+ cells

[0035] Fig. 8a is a schematic showing the overall view of the mouse engraftment of edited human thalassemia CD34+ cells. The thalassemia CD34+ cells were edited in vitro as described herein by electroporation. NSG mice were transplanted with either control (Ctr; no Cas9 or guide RNA) or edited cells (with Cas9n + guide RNA A3 & S4) cells intravenously. Bone marrow (BM) cells were collected 16 weeks after transplantation for analysis. Data of one animal from each group is shown.

[0036] Fig. 8b is a schematic showing the human cell chimerism of the bone marrow cells, as presented by the percentage of human hCD45+ cells over the total of human hCD45+ and mouse mCD45+ cells.

[0037] Fig. 8c is a schematic showing the multilineage reconstitution of the engrafted human cells, as shown by the percentage of hCD19+ B cells, hCD33+ myeloid cells, hCD34+ progenitor cells, and hCD3+ T cells. The schematic shows that the engrafted human cells comprised mainly of hCD19+ B cells, some hCD33+ myeloid cells and hCD34+ progenitor cells, with minimal hCD3+ T cells.

[0038] Fig. 8d is a schematic showing the efficiency of editing of the cells before and after transplantation (recovered from bone marrow, and hCD34+ enriched), as indicated as “Input” and “BM34+ enriched” respectively. Representative gel images of T7E1 assay on fragments amplified from the HBG1 promoter are shown. The estimated editing efficiency are shown below the respective lanes.

[0039] Fig. 8e is a schematic showing the deletion of inter-HBGs promoter region as quantified by Taqman copy number variation assay. The copy number was normalized to that of control and set at 2.DEFINITIONS

[0040] As used herein, the terms “gene”, “locus”, and “gene locus”, refer to a region of nucleic acid on a eukaryotic chromosome which comprise, but are not limited to, the enhancer, silencer, promoter, 5’ untranslated region, open reading frame, 3’ untranslated region, and terminator.

[0041] As used herein, the term “target sequence” and “target site” are used interchangeably and refer to a section of a target molecule which is to be recognised using the gene editing method as disclosedherein. Such target(s) can be, but are not limited to DNA, RNA, single nucleotide polymorphism (SNP), microRNA (miRNA), genomic DNA, viral DNA, protein, post-translational modified proteins, cell surface receptors, metabolites, lipids, carbohydrates and small molecules.

[0042] As used herein, the term “nuclease” refers to an enzyme capable of cleaving phosphodiester bonds between nucleotides of nucleic acids. The cleaving of phosphodiester bonds between nucleotides of nucleic acids can occur on, but is not limited to, a DNA or RNA. The cleaving of phosphodicstcr bonds can occur on one or more sites on a DNA or RNA. These sites can comprise a nucleotide sequence which is complementary to the enzyme. The cleaving of phosphodiester bonds can also occur at sites which are not complementary to the enzyme. When the cleavage of phosphodiester bonds occurs on a DNA or RNA, the DNA or RNA may be single or double stranded. Thus, the cleavage of phosphodicstcr bonds can occur on a single strand of the DNA or RNA or on both strands of the DNA or RNA. Accordingly, the cleavage of phosphodiester bonds by a nuclease can be a single stranded break or a double stranded break. In one example, the cleavage by a nuclease is a double stranded break. The cleavage of phosphodiester bonds by a nuclease can occur at a phosphodiester bond within a DNA or RNA, or at a phosphodiester bond on an exposed end of a DNA or RNA. Examples of nucleases can be, but are not limited to, DNA exonucleases, DNA endonucleases, RNA exonucleases, RNA endonucleases, and CRISPR-associated nucleases. Examples of CRISPR-associated nucleases can be, but arc not limited to, Casl. Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9. CaslO. Casl2, Casl3, or Cas 14. In one example, the nuclease is Cas9.

[0043] As used herein, the term “nickase” refers to a nuclease which cleaves phosphodiester bonds between nucleotides of nucleic acids which can be, but are not limited to, on a single strand of a DNA or RNA. In one example, the cleavage by a nickase is a single stranded break. The cleaving of phosphodiester bonds can occur on one or more sites on a single strand of a DNA or RNA. These sites can comprise a nucleotide sequence which is complementary to the enzyme. The cleaving of phosphodicstcr bonds can also occur at sites which arc not complementary to the enzyme. Nickases can be derived from nucleases, where a nuclease is mutated to obtain a nickase. Examples of nucleases from which a nickase can be derived can be, but are not limited to, DNA exonucleases, DNA endonucleases, RNA exonucleases, RNA endonucleases, and CRISPR-associated nucleases. Examples of CRISPR- associated nucleases can be, but arc not limited to, Casl, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, CaslO, Casl2, Casl3, or Cas 14. In a specific example, the nickase is obtained from Cas9. Examples of mutations which can be performed to obtain a nickase will be known in the art. In a specific example, a nickase is obtained from a Cas9 nuclease via the D10A mutation.

[0044] As used herein, the terms “gRNA” and “guide RNA” arc used interchangeably and refer to a RNA strand that is complementary to a target sequence which can be, but is not limited to, on a DNA, or another RNA. Thus, in one example, the “gRNA” and “guide RNA” used herein can be, but are not limited to, a CRISPR RNA (crRNA), a trans- activating CRISPR RNA (tracrRNA), or a single guide RNA (sgRNA). In a further example, the “gRNA” and “guide RNA” used herein is a crRNA;tracrRNA.The size of the “gRNA” and “guide RNA” used herein can be, but is not limited to, 30 to 150 nucleotides. In a specific example, the size of the “gRNA’’ and “guide RNA” used herein is about 20 nucleotides, about 37 nucleotides, about 67 nucleotides, or about 100 nucleotides. The "gRNA” or “guide RNA” as used herein can recognise and bind to a target sequence on a DNA, or another RNA which the “gRNA” or “guide RNA” is complementary to. The “gRNA” or “guide RNA” as used herein can be bound to an enzyme, where the enzyme can be a nuclease or nickase. In one example, the enzyme is a nuclease. In another example, the enzyme is a nickase. In a further example, the enzyme is a Cas9 nuclease. Tn yet another example, the enzyme is a Cas9 nickase. Tn a specific example, the Cas9 nuclease comprises a sequence as defined in SEQ ID NO: 9. The Cas9 nickase comprises a D10 mutation relative to the sequence as defined in SEQ ID NO: 9. In a further example, the Cas9 nickase comprises a sequence as defined in SEQ ID NO: 10. The “gRNA” or “guide RNA” as used herein, when bound to an enzyme, can guide the enzyme to a target sequence which the “gRNA” or “guide RNA” recognises. Upon guidance of the enzyme to the target sequence, the enzyme can perform its enzymatic activity such as the cleavage of phosphodiester bonds if the enzyme is a nuclease or nickase.

[0045] As used herein, the term “mutant” refers to a molecule which comprises a structure different from its native form, where such structural differences are termed a “mutation”. As used herein, the term “native form” can be, but is not limited to, the wild-type form or sequence known in the art, a reference form or sequence, or any combinations thereof. As used herein, a “mutant”, can be, but is not limited to a mutant DNA, RNA, genomic DNA, messenger RNA (mRNA), or protein. “Mutations” in the context of a mutant DNA, RNA, genomic DNA, or messenger RNA (mRNA) can be but are not limited to, additions, deletions, substitutions, homologous recombination, inversions, frameshift, missense, fusions, nonsense mutations, or any combinations thereof. “Mutations” in the context of proteins can be a change in structure. Accordingly, due to the presence of mutations in a mutant, a mutant can have have properties different from its native form.

[0046] In the context of proteins such as enzymes, differences in properties can be, but arc not limited to, a change in enzymatic activity, and a change in the recognition of complementary binding sites. In one example, a mutant Cas9 nickase refers to a Cas9 nickase which was mutated relative to a reference Cas9 protein, which can be, but is not limited to, a Cas9 nuclease, or another Cas9 nickase. In one example, the Cas9 protein is a Cas9 nuclease. In another example, the Cas9 protein is a Cas9 nuclease comprising a sequence as defined in SEQ ID NO: 9. In another example, a mutant Cas9 nickase is a Cas9 nickase comprising a mutation relative to the sequence as defined in SEQ ID NO: 9. In a further example, a mutant Cas9 nickase is a Cas9 nickase comprising a D10A mutation relative to the sequence as defined in SEQ ID NO: 9. In a more specific example, a mutant Cas9 nickase comprises a sequence as defined in SEQ ID NO: 10. Thus, a mutant Cas9 nickase can have differences in enzymatic activity relative to a reference Cas9 protein which can be, but is not limited to, Cas9 nuclease, or another Cas9 nickase. Differences in enzymatic activity can be, but are not limited to, the resulting products from enzymatic digestion, the substrate recognised for enzymatic digestion, or the specificity to a substraterecognised for enzymatic reaction. In one example, a mutant Cas9 nickase generates a single stranded break on a nucleic acid sequence which can be, but is not limited to, a DNA or RNA. In another example, a mutant Cas9 nickase when paired with a guide RNA (gRNA) has lower off-target effects relative to a reference Cas9 nickase which can be, but is not limited to, a Cas9 nickase without the mutation.

[0047] In the context of nucleic sequences such as those located on a DNA or RNA, differences in properties, can be caused by, but arc not limited to, a change in structure, a change in the number of bases, a change in the number of nucleotides, and a change in the modifications present on the sequences. When such changes occur on a nucleic sequence located on a DNA or RNA, target sequences can be altered in ways such as, but are not limited to, a change in structure, a change in the number of bases, a change in the number of nucleotides, and a change in the modifications present on the target sequences. Accordingly, a change in nucleic sequence located on a DNA or RNA can result in the loss of sequences which are recognised and bound by complementary enzymes. The loss of such sequences results in complementary enzymes failing to bind at such sequences.

[0048] In the context of nucleic sequences located on a gene locus on a DNA, differences in properties can be caused by, but are not limited to, a change in structure, a change in the number of bases, a change in the number of nucleotides, and a change in the modifications present on the gene locus. The differences in properties can lead to changes in the gene locus which can be, but are not limited to, the loss of whole or part thereof or loss in function of the whole or part thereof of the gene locus, a fusion of a first gene locus to a second gene locus, or combinations thereof. The changes in the gene locus can subsequently lead to a change in gene expression which can be, but are not limited to, a complete or partial loss in gene expression, a decrease in gene expression, an increase in gene expression, or combinations thereof. Other changes in properties can result in, but are not limited to, the partial or complete loss of sequences which are recognised and bound by complementary enzymes, the partial or complete loss in a whole or part thereof of a gene, and subsequently the partial or complete loss of gene expression, or an alteration in gene expression.

[0049] It should also be understood that the absence of a specific mention of the terms “mutant” or “mutated” does not necessarily imply that the succeeding described protein or nucleic acid sequence is non-mutated. The text should be read in the context of the entirety of the disclosure.

[0050] As used herein, the term “disruption” when used in the context of a gene or part thereof, refers to a loss of a whole or part thereof of the gene, which can occur due to mutations that result from, but are not limited to, additions, deletions, substitutions, homologous recombination, inversions, frameshift, missense, fusions, nonsense mutations, or any combinations thereof. The loss of the whole or part thereof of the gene can result in a mutant gene. A mutant gene can have changes in properties such as, but are not limited to, a change in structure, a change in the number of bases, a change in the number of nucleotides, and a change in the modifications present on the sequences. Such changes in properties can result in, but are not limited to, the partial or complete loss of sequences which are recognised and bound by complementary enzymes, the partial or complete loss in a whole or part thereof of a gene, andsubsequently the partial or complete loss of gene expression, or an alteration in gene expression. Accordingly, the expression of sequences located downstream or upstream of the disrupted gene can also be altered. In one example, a human beta-globin gene locus is disrupted. In another example, a beta-globin gene loci is disrupted, where a beta-globin gene loci can be, but is not limited to, hemoglobin gamma 1 (HBG1) gene, hemoglobin gamma 2 (HBG2) gene, hemoglobin 8 (HBD) gene, hemoglobin [> (HBB) gene, or combinations thereof. In another example, the promoter of a beta-globin gene loci can be disrupted, which can be, but is not limited to, the promoter of a hemoglobin gamma 1 (HBGI ) gene, hemoglobin gamma 2 (HBG2) gene, hemoglobin 8 (HBD) gene, hemoglobin [1 (HBB) gene, or combinations thereof. In a specific example, the hemoglobin gamma 2 (HBG2) gene is disrupted. In a more specific example, the hemoglobin gamma 2 (HbG2) gene gene is lost. In another example, the loss of the hemoglobin gamma 2 (HBG2) gene resulted from the fusion of the hemoglobin gamma 1 (HBGI) gene to the promoter of the hemoglobin gamma 2 (HBG2) gene.

[0051] As used herein, the term “unmutated” has the same meaning as the term “non-mutated” and when used in the context of a nucleic acid, refers to the absence of mutations in the nucleic acid sequence relative to a reference nucleic acid sequence. When used in the context of a protein, the terms “unmutated” and “non-mutated” refer to the absence of differences in the protein’s corresponding nucleic acid sequence relative to a reference protein’s corresponding nucleic acid sequence, such that the protein is folded into the native structure of the protein, and not a mutant form. Thus, for example, an unmutated Cas9 nuclease refers to a Cas9 nuclease without mutations in the nucleic acid sequence and can refer to a wild-type Cas9 nuclease. In another example, an unmutated Cas9 nuclease can serve as a reference protein to which a mutant is compared, and refers to a Cas9 nuclease which lacks the mutations of the mutant. In a further example, an unmutated Cas9 nuclease can refer to a Cas9 nuclease which comprises a nucleic acid sequence that does not deviate from a nucleic acid sequence defined by a SEQ ID NO. When used in the context of a gene, the terms “unmutated” and “non-mutated” refer to the absence of differences in the nucleic acid sequence relative to a reference nucleic acid sequence, which can be, for example, a wild-type nucleic acid sequence. Thus, for example, an unmutated human beta-globin gene locus refers to a human beta-globin gene locus without mutations in the nucleic acid sequence. In another example, an unmutated human beta-globin gene locus can serve as a reference nucleic acid sequence to which a mutant is compared. Accordingly, in another example, an unmutated human beta-globin gene locus can be understood to refer to a human beta-globin gene locus which comprises the intact hemoglobin gamma 1 (HBGI) gene, hemoglobin gamma 2 (HBG2) gene, hemoglobin 3 (HBD) gene, and hemoglobin [> (HBB) gene. It should also be understood that the absence of a specific mention of the terms “unmutated” or “non-mutated” docs not necessarily imply that the succeeding described protein or nucleic acid sequence is a mutant. The text should be read in the context of the entirety of the disclosure.DETAILED DESCRIPTION

[0052] The human globin gene clusters on chromosome 11 controls the expression of p-subunit, which together with a-subunit, produces the functional haemoglobin (Hb). Several P-subunit exists, including the predominantly fetal y chain (HBG gene) and the adult 8 (HBD gene; minor) and p (HBB gene; major) chains (Fig. l a). Expression of specific [3-like globin chains during development follows the same order in which they are genetically arranged, a process known as “globin switching’’. The g-chain (HBE) which is expressed during the embryonic gestation is replaced by the two y-chains (Gy and Ay, from HBG2 and HBG1 respectively) during fetal development. The y chains are then gradually downregulated after birth, whereas 8 (HBD; minor) and P (HBB; major) chains become the predominant forms thereafter ( Fig. la).

[0053] Individuals who inherit mutations that disrupt p-chain, a condition known as p- hemoglobinopathies (Mendelian disorders such as thalassemia and sickle cell disease), start to develop symptoms few months after birth, due to a failure in an increased production of normal adult haemoglobin (HbA; c / ’po with a reciprocal reduction of fetal haemoglobin (HbF; a-y^). Such individuals develop a condition known as p-hemoglobinopathies, (such as thalassemia and sickle cell disease), and fail to produce functional haemoglobin (adult HbA: ct2f>2). Approximately 300,000- 400,000 children with P-hemoglobinopathies are born annually.

[0054] Rare individuals inherit a benign condition known as heredity persistence of fetal haemoglobin (HPFH), at which the silencing of HBG genes is retarded by mutations of the cis-rcgulatory elements, leading to persistently high production of y-chains and fetal haemoglobin (HbF; cx2y2). The reexpression of HbF alleviated the clinical severity of P-hemoglobinopathies by rescuing the defective HbA. The clinical severity of P-hemoglobinopathies can be ameliorated by naturally occurring mutations in the HBG promoter, namely hereditary persistence of fetal haemoglobin (HPFH), which revert haemoglobin synthesis from the defective adult P-globin to functional fetal y-globin well into adulthood.

[0055] Conventional methods of treating P-hcmoglobinopathics using allogenic hematopoietic cells transplantation are limited by the availability of compatible donors or transplant-related toxicities. Furthermore, emerging methods of gene therapy with CRISPR / Cas9 still face issues such as off-target effects, which cause detrimental health issues to the subject. The only approved gene therapy product for P-hcmoglobinopathics which is based on lentiviral transfer of P-chain (p-A T87Qmutant) causes random insertional mutagenesis in hematopoietic stem cells.

[0056] Therefore, there is a need for a gene therapy for treating P-hemoglobinopathies with minimal off-target effect.

[0057] Several gene therapy approaches had been adopted for P-hcmoglobinopathics. A direct way is to express a functional or anti-sickling copy of P-chain in hemopoietic cells. As disclosed herein, the present disclosure adopts the use of heredity persistence of fetal haemoglobin (HPFH) for disrupting control elements of the HbF gene to induce HbF production. Noticeably, many HPFH mutations are single nucleotide substitutions on HBG promoters that disrupt the binding sites of repressors LRF orBCL11A ( Fig. lb), which are potent inducers of HbF (up to 40% of Hb in the HPFH individuals). Current gene therapy methods which induce HPFH mutations by small insertions and deletions (indels) using CR1SPR / Cas9 nucleases can only result in HbF induction to a small extent.

[0058] The canonical CR1SPR / Cas9 nuclease (Cas9), which induces double strand break (DSB) and small indels on target DNA, can be converted into a nickase (Cas9n) that generates single strand nick instead. A pair of Cas9 nickases, when placed on opposite strands at a distance apart, can generate DSB and longer indels (tens to hundreds bps) to cover more heredity persistence of fetal haemoglobin (HPFH) mutation sites simultaneously. As discussed herein, paired Cas9 nickases (the D I 0A mutant) was used to target the cluster of heredity persistence of fetal haemoglobin (HPFH) mutations around the LRF binding site on HBG promoters.Paired Cas9 nickases are more efficient and highly specific as compared to single Cas9 nuclease for HBG promoters editing

[0059] While Cas9 nuclease generates double stranded DNA breaks to specific sites when guided by a guide RNA (gRNA), the mutant Cas9 nickase (Cas9n) generates single stranded DNA breaks. When used with a pair of guide RNA (gRNA) with proper spacing and orientation, the Cas9n (D10A mutant) can generate double strand DNA breaks with high specificity. Pairs of gRNAs that couple with Cas9n (gRNA + Cas9n) were designed (Table 4), demonstrating that these combinations can specifically gene- edit the HBG promoters. The gene editing results in the induction of HbF in hematopoietic progenitors obtained both from healthy donors (Fig. 3) and from individuals with thalassemia (Fig. 4). Successful editing showed deletion of inter-promoter regions (Fig. 6).

[0060] Thus, as disclosed herein, it is shown that paired Cas9 nickases are more efficient than the respective Cas9 nucleases in editing and causes better HbF induction.

[0061] Accordingly, disclosed herein is a kit or system comprising: a) a CRISPR-Cas9 nickase; and a pair of guide RNAs (gRNAs). In one example, the CRISPR-Cas9 nickase is a mutant of a CRISPR- Cas9 nuclease, where the sequence of a CRISPR-Cas9 nuclease is known in the art. In another example, the CRISPR-Cas9 nuclease comprises a sequence as defined in SEQ ID NO: 9. In another example, the CRTSPR-Cas9 nickase comprises a D I OA mutation relative to a CRTSPR-Cas9 nuclease comprising a sequence as defined in SEQ ID NO: 9. In a more specific example, the CRISPR-Cas9 nickase comprises a sequence as defined in SEQ ID NO: 10.

[0062] CRISPR-Cas9 nickases can be paired with guide RNAs (gRNAs) to modify the genome of a cell. Depending on the sequence of the guide RNAs (gRNAs), the location of binding on a genome, and subsequently the location of modification on a genome, will differ. The location of binding on a genome by a guide RNA (gRNA) is referred to as a “target sequence"’ as defined herein. In one example, the guide RNAs (gRNAs) as used herein binds to a target sequence located 300 nucleotides upstream or downstream relative to a LRF binding site in the promoter of the hemoglobin gamma ( HbG) locus. Tn another example, the hemoglobin gamma (HbG) locus is a mammalian or human hemoglobin gamma(HbG) locus. In yet another example, the LRF binding site in the promoter of the hemoglobin gamma (HbG) locus comprises a sequence as defined in SEQ ID NO: 83.100631 Thus, in a further example, the target sequence is located 300 nucleotides upstream or downstream relative to a LRF binding site in the promoter of the hemoglobin gamma (HbG) locus, where the LRF binding site comprises a sequence as defined in SEQ ID NO: 83. In a more specific example, the target sequence of the guide RNAs (gRNAs) as used herein comprises a sequence as defined in any one of SEQ ID NOs: 1 to 8.

[0064] As described herein, while the canonical CRTSPR / Cas9 nuclease (Cas9) induces double strand break (DSB), a Cas9 nickase (Cas9n) generates single strand nicks. Thus, to generate double strand DNA breaks, a Cas9 nickase can be paired with a pair of guide RNAs (gRNAs). In one example, a pair of guide RNAs (gRNAs) refers to a first guide RNA (gRNA) that binds to a first target sequence and a second guide RNA (gRNA) that binds to a second target sequence. In a further example, a pair of guide RNAs (gRNAs) refers to a first guide RNA (gRNA) that binds to a first target sequence located on a sense strand and a second guide RNA (gRNA) that that binds to a second target sequence located on an antisense strand. In another example, a pair of guide RNAs (gRNAs) as used herein refers to a pair of guide RNAs (gRNAs) that bind to target sequences as defined in any one of SEQ ID NOs: 1 to 8, or any combinations thereof. In another example, the pair of guide RNA (gRNA) binds to the target sequences in opposite directions.

[0065] When pairing a Cas9 nickase with a pair of guide RNAs (gRNAs) as described herein, guide RNAs (gRNAs) can be selected with target sequences that do not overlap. As used herein, the term “do not overlap’’ when used in the context of the target sequences of individual guide RNAs (gRNAs) in a pair of selected guide RNAs (gRNAs) would thus exclude, for example, a combination of SEQ ID NO: 2 and SEQ ID NO: 5, or SEQ ID NO: 4 and SEQ ID NO: 5, but not a combination of, for example, SEQ ID NO: 2 and SEQ ID NO: 7, SEQ ID NO: 2 and SEQ ID NO: 8, or SEQ ID NO: 3 and SEQ ID NO: 8. The term “do not overlap” as used herein, when used in the context of the target sequences of guide RNAs (gRNAs), can be understood to mean that a first guide RNA (gRNA) will not bind to the same region as a second guide RNA (gRNA). Another way to define the term “do not overlap” as used herein can mean that the binding of a first guide RNA (gRNA) to a target sequence will not prevent the binding of a second guide RNA (gRNA), because the target sequence of the first guide RNA (gRNA) and second guide RNA (gRNA) are on different locations or have different sequences.

[0066] Accordingly, in one example, the target sequences of the individual guide RNAs (gRNAs) in a pair of selected guide RNAs (gRNAs) do not overlap and can be, but are not limited to, a combination of SEQ ID NO: 1 and SEQ ID NO: 5, SEQ ID NO: 1 and SEQ ID NO: 6, SEQ ID NO: 1 and SEQ ID NO: 7, SEQ ID NO: 1 and SEQ ID NO: 8, SEQ ID NO: 2 and SEQ ID NO: 6, SEQ ID NO: 2 and SEQ ID NO: 7, SEQ ID NO: 2 and SEQ ID NO: 8, SEQ ID NO: 3 and SEQ ID NO: 6, SEQ ID NO: 3 and SEQ ID NO: 7, SEQ ID NO: 3 and SEQ ID NO: 8, SEQ ID NO: 4 and SEQ ID NO: 7, SEQ ID NO: 4 and SEQ ID NO: 8.

[0067] In a more specific example, the target sequences of the individual guide RNAs (gRNAs) in a pair of selected guide RNAs (gRNAs) do not overlap and can be, but arc not limited to, a combination of SEQ ID NO: 2 and SEQ ID NO: 6, SEQ ID NO: 2 and SEQ ID NO: 7, SEQ ID NO: 2 and SEQ ID NO: 8, SEQ ID NO: 3 and SEQ ID NO: 6, SEQ ID NO: 3 and SEQ ID NO: 7, SEQ ID NO: 3 and SEQ ID NO: 8, SEQ ID NO: 4 and SEQ ID NO: 7, or SEQ ID NO: 4 and SEQ ID NO: 8. In a more specific example, the target sequences of the individual guide RNAs (gRNAs) in a pair of selected guide RNAs (gRNAs) do not overlap and can be, but are not limited to, a combination of SEQ ID NO: 2 and SEQ ID NO: 8, SEQ ID NO: 3 and SEQ ID NO: 8, or SEQ ID NO: 4 and SEQ ID NO: 8.

[0068] By pairing a Cas9 nickase with a pair of guide RNAs (gRNAs) as described herein, double strand breaks can be induced in a target sequence or region. In one example, the target sequence or region is a gene or part thereof. In a further example, target sequence or region is a promoter of the gene.

[0069] The induction of double stranded breaks in a gene or part thereof can result in a mutation of the gene or part thereof. As defined herein, the mutation of the gene or part thereof can result in a change in the structure of a gene, the expression of the gene, or any combinations thereof.

[0070] Accordingly, as described herein, a Cas9 nickase can be paired with a pair of guide RNAs (gRNAs) as described herein to induce double strand breaks in a target sequence or region. In one example, the induction of double stranded breaks can be on the sense and antisense strand. In a further example, the induction of double stranded breaks can be on the sense and antisense strand of the betaglobin gene loci.

[0071] In another example, the beta-globin gene loci can be, but is not limited to, hemoglobin gamma1 (HBG1), hemoglobin gamma 2 (HBG2), hemoglobin 8 (HBD), hemoglobin |i (HBB), or combinations thereof. Tn a further example, the target sequence or region is in the hemoglobin gamma I (HBG I ) gene, hemoglobin gamma 2 (HBG2) gene, or any combinations thereof. In another example, the target sequence or region is in the promoter of the hemoglobin gamma 1 (HBG1) gene, hemoglobin gamma2 (HBG2) gene, or any combinations thereof.

[0072] Tn another example, the target sequence or region is the LRF binding site in the promoter of the hemoglobin gamma (HBG) locus. In a further example, the target sequence or region is the LRF binding site in the promoter of the hemoglobin gamma 1 (HBG1) gene, hemoglobin gamma 2 (HBG2) gene, or any combinations thereof.

[0073] Accordingly, when the target sequence or region of the guide RNAs (gRNAs) as described herein is the LRF binding site in the promoter of the hemoglobin gamma (HBG) locus, the induction of double stranded breaks can disrupt the LRF binding site of the promoter of the hemoglobin gamma (HBG) locus.

[0074] The disruption of the LRF binding site can result in structural changes in the hemoglobin gamma 1 (HBG 1) gene, hemoglobin gamma 2 (HBG2) gene, or both the hemoglobin gamma 1 (HBG1) and hemoglobin gamma 2 (HBG2) gene. Structural changes can be, but are not limited to. the partial orcomplete loss of the promoter of the hemoglobin gamma 1 (HBGl) gene and the promoter of the hemoglobin gamma 2 (HBG2) gene, the fusion of the promoter of the hemoglobin gamma 1 (HBGl) gene to the promoter of the hemoglobin gamma 2 (HBG2) gene, the loss of the hemoglobin gamma 2 (HbG2) gene, or any combinations thereof.

[0075] As a result of the disruption of the LRF binding site, expression of the hemoglobin gamma 1 (HBGl) gene, hemoglobin gamma 2 (HBG2) gene, or any combinations thereof, can be altered. In one example, expression of the hemoglobin gamma 1 (HBGl) gene is induced. In a further example, induction of the hemoglobin gamma I (HBG I ) gene results in an increase in the level of y- globin.

[0076] Thus, disclosed herein is a kit comprising: a) a CRISPR-Cas9 nickase; and a pair of guide RNAs (gRNA), wherein the pair of guide RNAs binds to target sequences, wherein the binding will result in a disruption of binding sites of repressor LRF in a promotor of a hemoglobin gamma (HBG) locus.Pairs of gRNA + Cas9n having high editing efficiency on HBG promoters were identified

[0077] Pairs of guide RNAs (gRNAs) for testing with nickase or nuclease were identified (Table 4). The paired nickases are more efficient than the respective nucleases in editing and inducing HbF reexpression, as demonstrated in hematopoietic progenitors obtained from both healthy donors (Fig. 3) and thalassemia patients (Fig. 4). gRNA + Cas9n pairs are more efficient than the respective gRNA + Cas9 in editing and inducing HbF (Fig. 2 to 4) and show deletion of inter-promoter regions upon successful on-target editing (Fig. 6). In particular, the pair A2+S4 shows the best efficiency, followed by the pairs A3+S4 and A4+S4.

[0078] Accordingly, in one example, disclosed herein is a gene editing method with low off-target effect using paired Cas9 nickases. Described herein is also a gene editing method to induce HbF expression by targeting the cluster of heredity persistence of fetal haemoglobin (HPFH) mutations around the LRF binding site on HBG promoters.

[0079] The method as described herein can be performed to modify the genome of a eukaryotic cell. In one example, the eukaryotic cell is a hcmotopoictic stem cell. In a further example, the hcmotopoictic stem cell is a CD34+ or a CD133+ cell.

[0080] The modifying of a genome of a eukaryotic cell can occur in, for example, the hemoglobin gamma (HBG) locus. In a further example, the modifying occurs in a promotor of a hemoglobin gamma (HBG) locus. In a more specific example, the modifying occurs in the binding sites of repressor LRF located in a promotor of a hemoglobin gamma (HBG) locus. Accordingly, described herein is a method for modifying a hemoglobin gamma (HBG) locus in a eukaryotic cell.

[0081] As described herein, the modifying of a hemoglobin gamma (HBG) locus of a eukaryotic cell can be performed using a kit or system as described herein. In one example, the kit or system as used in the method described herein comprises: a) a CR1SPR-Cas9 nickase; and a pair of guide RNAs (gRNAs).

[0082] In one example, the CRISPR-Cas9 nickase is a mutant of a CRISPR-Cas9 nuclease, where the sequence of a CRISPR-Cas9 nuclease is known in the art. In another example, the CRISPR-Cas9nuclease comprises a sequence as defined in SEQ ID NO: 9. In another example, the CRISPR-Cas9 nickase comprises a D10A mutation relative to a CRISPR-Cas9 nuclease comprising a sequence as defined in SEQ ID NO: 9. In a more specific example, the CRISPR-Cas9 nickase comprises a sequence as defined in SEQ ID NO: 10.

[0083] In another example, the guide RNAs (gRNAs) as used in the method described herein binds to a target sequence located 300 nucleotides upstream or downstream relative to a LRF binding site in the promoter of the hemoglobin gamma (HBG) locus. In a further example, the LRF binding site in the promoter of the hemoglobin gamma (HBG) locus comprises a sequence as defined in SEQ ID NO: 83. In a more specific example, the guide RNAs (gRNAs) as used herein binds to target sequences as defined in any one of SEQ ID NOs: 1 to 8.

[0084] In a further example, the guide RNAs (gRNAs) as used in the method described herein can be a pair of guide RNAs (gRNAs) to induce a double stranded break. As used herein, in one example, paired guide RNAs (gRNAs) refer to a first guide RNA (gRNA) that comprises a target sequence located on a sense strand and a second guide RNA (gRNA) that comprises a target sequence located on an antisense strand. In another example, the pair of guide RNA (gRNA) binds to the target sequences in opposite directions. In another example, the paired guide RNAs (gRNAs) as used herein refers to a pair of guide RNAs (gRNAs) that bind to target sequences as defined in any one of SEQ ID NOs: 1 to 8.

[0085] As used herein, the term “do not overlap” when used in the context of the target sequences of individual guide RNAs (gRNAs) in a pair of selected guide RNAs (gRNAs) would thus exclude, for example, a combination of SEQ ID NO: 2 and SEQ ID NO: 5, or SEQ ID NO: 4 and SEQ ID NO: 5, but not a combination of, for example, SEQ ID NO: 2 and SEQ ID NO: 7, SEQ ID NO: 2 and SEQ ID NO: 8, or SEQ ID NO: 3 and SEQ ID NO: 8. The term “do not overlap” as used herein, when used in the context of the target sequences of guide RNAs (gRNAs), can be understood to mean that a first guide RNA (gRNA) will not bind to the same region as a second guide RNA (gRNA). Another way to define the term “do not overlap” as used herein can mean that the binding of a first guide RNA (gRNA) to a target sequence will not prevent the binding of a second guide RNA (gRNA), because the target sequence of the first guide RNA (gRNA) and second guide RNA (gRNA) are on different locations or have different sequences.

[0086] Accordingly, in a specific example, the target sequences of the individual guide RNAs (gRNAs) in a pair of selected guide RNAs (gRNAs) used in the method described herein do not overlap and can be, but is not limited to, a combination of SEQ ID NO: 1 and SEQ ID NO: 5, SEQ ID NO: 1 and SEQ ID NO: 6, SEQ ID NO: 1 and SEQ ID NO: 7, SEQ ID NO: 1 and SEQ ID NO: 8, SEQ ID NO: 2 and SEQ ID NO: 6, SEQ ID NO: 2 and SEQ ID NO: 7, SEQ ID NO: 2 and SEQ ID NO: 8, SEQ ID NO: 3 and SEQ ID NO: 6, SEQ ID NO: 3 and SEQ ID NO: 7, SEQ ID NO: 3 and SEQ ID NO: 8, SEQ ID NO: 4 and SEQ ID NO: 7, SEQ ID NO: 4 and SEQ ID NO: 8.

[0087] In a more specific example, the target sequences of the individual guide RNAs (gRNAs) in a pair of selected guide RNAs (gRNAs) used in the method described herein do not overlap and can be, but is not limited to, a combination of SEQ ID NO: 2 and SEQ ID NO: 6, SEQ ID NO: 2 and SEQ ID NO: 7, SEQ ID NO: 2 and SEQ ID NO: 8, SEQ ID NO: 3 and SEQ ID NO: 6, SEQ ID NO: 3 and SEQ ID NO: 7, SEQ ID NO: 3 and SEQ ID NO: 8, SEQ ID NO: 4 and SEQ ID NO: 7, or SEQ ID NO: 4 and SEQ ID NO: 8. In a more specific example, the target sequences of the individual guide RNAs (gRNAs) in a pair of selected guide RNAs (gRNAs) do not overlap and can be, but is not limited to, a combination of SEQ ID NO: 2 and SEQ ID NO: 8, SEQ ID NO: 3 and SEQ ID NO: 8, or SEQ ID NO: 4 and SEQ ID NO: 8.

[0088] In another example, guide RNAs (gRNAs) with target sequences that overlap can be selected.

[0089] Accordingly, the method as described herein can modify the genome of a eukaryotic cell such as, for example, a hemotopoietic stem cell, a CD34+, or a CD133+ cell. The modification of the genome of a eukaryotic cell such as, for example, a hemotopoietic stem cell, a CD34+, or a CD133+ cell, can result in the modifying of a genome of, for example, the hemoglobin gamma (HBG) locus.

[0090] In a further example, the modification of the hemoglobin gamma (HBG) locus can be, but is not limited to, the promoter of the hemoglobin gamma 1 (HBG1) gene, hemoglobin gamma 2 (HBG2) gene, or any combinations thereof. In a specific example, the modification of the hemoglobin gamma (HBG) locus can be at the LRF binding site of the promoter of the hemoglobin gamma (HBG) locus.

[0091] Modification of the LRF binding site of the promoter of the hemoglobin gamma (HBG) locus can result in the disruption of the LRF binding site. Disruption of the LRF binding site can result in structural changes in the hemoglobin gamma (HBG) locus where the structural changes can be, but are not limited to, the partial or complete loss of the promoter of the hemoglobin gamma 1 (HBGl) gene and the promoter of the hemoglobin gamma 2 (HBG2) gene, the fusion of the promoter of the hemoglobin gamma 1 (HBGl) gene to the promoter of the hemoglobin gamma 2 (HBG2) gene, the loss of the hemoglobin gamma 2 (HBG2) gene, or any combinations thereof, in a hemotopoietic stem cell, a CD34+, or a CD133+ cell.

[0092] Structural changes in the hemoglobin gamma (HbG) locus can alter gene expression, in a hemotopoietic stem cell, a CD34+, or a CD 133+ cell. One example of an alteration in gene expression can be the induction of the expression of the hemoglobin gamma 1 (HBGl) gene in a hemotopoietic stem cell, a CD34+, or a CD133+ cell. In a further example, induction of the hemoglobin gamma 1 (HbGl) gene results in an increase in the level of y- globin in a hemotopoietic stem cell, a CD34+, or a CD133+ cell.

[0093] Thus, described herein is a method for modifying a hemoglobin gamma (HBG) locus in a eukaryotic cell, the method comprising introducing into the eukaryotic cell a kit comprising: a) a CRISPR-Cas9 nickase; and b) a pair of guide RNAs (gRNA), wherein the pair of guide RNAs binds to target sequences, wherein the binding will result in a disruption of binding sites of repressor LRF in a promotor of a hemoglobin gamma (HbG) locus.

[0094] The intrinsic off-target activity of the nuclease was eliminated by the paired nickases in the primary cells (Fig. 5). Mechanistically, editing of the HBG1 and HBG2 promoters simultaneously leads to the deletion of HBG2 containing inter-promoter region (Fig. 6). Compared with Cas9 nuclease, the Cas9 nickases reduces off-target mutagenesis in primary hematopoietic cells (Fig. 5 and 6). The pair gRNAs + Cas9n approach is more specific than the respective single gRNA + Cas9 (wildtype), minimizing potential off-target editing (Fig. 5). The low off-target effect of the gRNAs + Cas9n claimed herein allows for specificity in editing, particularly in hematopoietic cells for inducing HbF expression. Accordingly, the paired gRNAs with Cas9 nickase (Cas9n) claimed herein is suitable for modifying hematopoietic cells for inducing HbF expression due to the low off-target effects relative to single gRNA with Cas9 nuclease (Cas9) (Fig. 5). The low off-target effect is further illustrated in Fig. 6, which shows the successful deletion of inter-promoter regions upon editing by the paired gRNAs with Cas9 nickase (Cas9n) claimed herein.

[0095] When used in a subject in need thereof, the kit or system as described herein can cause disruption of the binding sites of repressor LRF with minal off-target effect. In one example, disruption of the binding sites of repressor LRF can result in the partial loss of the promoter of a hemoglobin gamma 1 (HBG1) gene and the promoter of a hemoglobin gamma 2 (HBG2) gene. In another example, disruption of the binding sites of repressor LRF can result in the fusion of the hemoglobin gamma 1 (HBGl) gene to the promoter of a hemoglobin gamma 2 (HBG2) gene. In a further example, fusion of the hemoglobin gamma 1 (HBGl) gene to the promoter of a hemoglobin gamma 2 (HBG2) gene can result in the loss of the promoter of the hemoglobin gamma 2 (HBG2) gene.

[0096] Thus, as described herein, disruption of the binding sites of repressor LRF induces expression of the hemoglobin gamma 1 (HBGl) gene. The expression of hemoglobin gamma 1 (HBGl) gene can thus result in an increase in an increase in a ' / -chain in a subject in need thereof.

[0097] Accordingly, described herein is a composition comprising the kit or system as described herein. In one example, the composition as described herein comprises a) a CRISPR-Cas9 nickase; and a pair of guide RNAs (gRNA), wherein the pair of guide RNAs binds to target sequences, wherein the binding will result in a disruption ofbinding sites of repressor LRF in a promotor of a hemoglobin gamma (HbG) locus.

[0098] The low off-target effect of the kit or system as described herein allows for the kit or system as described herein lo be formulated as composition to treat P-hemoglobinopathy. Thus, in one example, disclosed herein is a composition comprising the kit or system or system as described herein and a pharmaceutically acceptable excipient. Suitable excipients are well-known by a skilled person of the art and can be, but arc not limited to, polymers, lactose, sucrose, or sodium starch glycolatc. The composition can be in a form suitable for administration which can be, but is not limited to, a liquid composition, a solid composition, or any other forms known in the art. The liquid composition can be, but is not limited to, a suspension, a formulation, an emulsion, a cream, or any other liquid compositionsknown in the art. The solid composition can be, but is not limited to, a lyophilisate, a spray dried powders, a freeze dried powder, or any other solid compositions known in the art.[00991 Accordingly, in one example, the compositions as disclosed herein can be used in a method of treating a disease. In one example, the disease is P-hemoglobinopathy. In another example, the |3- hemoglobinopathy can be, but is not limited to, p-thalassemia, and sickle cell disease.

[0100] Described herein is thus a method of treating P-hemoglobinopathy in a subject, wherein the method comprises administering a (pharmaceutically or therapeutically effective amount of the) composition of the disclosure to a subject with a p-hemoglobinopathy.

[0101] Cells edited with the kit, system, or method as described herein are capable of persisting, engrafting and rcpopulating in a subject upon administration to said subject (Fig. 8). Therefore, cells edited with the kit, system, or method as described herein can also be used to treat P-hemoglobinopathy. Accordingly, also described herein is a method of treating P-hemoglobinopathy in a subject, wherein the method comprises the steps of a) isolating hematopoietic cells from the subject; b) editing the isolated hematopoietic cells obtained from step a with a kit of the disclosure ex vivo', c) expanding the edited cells obtained from step b) ex vivo', and d) administering a therapeutically effective amount of the expanded cells obtained from step c) to a subject with a P-hemoglobinopathy. Suitable modes of administration are well-known by a person skilled in the art. Thus, in one example, methods of administration can be, but is not limited to, subcutaneous, intravenous, intramuscular, infusion techniques, topically, orally, rectally, buccally, and vaginally. In a specific example, the mode of administration is transplantation.

[0102] Also disclosed herein is a composition for use in treating P-hemoglobinopathy. In one example, described herein is a use of a composition as described herein in the manufacture or preparation of a medicament for treating P-hemoglobinopathy. In another example, described herein is a composition for use in therapy or as a medicine.

[0103] A comparison of the editing efficiency of kit or system described herein shows that HbF induction using the kit or system described herein is higher than editing by conventional methods such as Casgevy (Fig. 7). Editing by paired nickases of the kit or system described herein generated higher levels of HbF+CD235a+ erythrocytes in hematopoietic cells from both a normal adult donor and a Thalassemia donor, compared to when editing was performed with Casgevy (Fig. 7c and 7d).

[0104] Accordingly, in one example, described herein is a gene therapy to induce HbF which demonstrates low off-target effect and higher editing compared to conventional methods. Also described herein is a gene therapy for preventing P-hemoglobinopathy in a subject, wherein the gene therapy comprises administering the composition of the disclosure to a subject susceptible to p- hemoglobinopathy .

[0105] Cells edited with the kit, system, or method as described herein are capable of persisting, engrafting and repopulating in a subject upon administration to said subject (Fig. 8). Therefore, cells edited with the kit, system, or method as described herein can also be used as a gene therapy forpreventing [i-hcmoglobinopathy. Thus, also described herein is a gene therapy for preventing [>- hemoglobinopathy in a subject, wherein the gene therapy comprises the steps of a) isolating hematopoietic cells from the subject; b) editing the isolated hematopoietic cells obtained from step a with a kit of the disclosure ex vivir, c) expanding the edited cells obtained from step b) ex vivir, and d) administering a therapeutically effective amount of the expanded cells obtained from step c) to a subject susceptible to P-hemoglobinopathy. In a further example, administration induces expression of the hemoglobin gamma 1 (HBGl) gene. In yet another example, administration increases y- globin expressing cells. In another example, the therapeutically effective amount of the expanded cells is administered by transplantation. As used herein, the prevention of P-hemoglobinopathy can be, but is not limited to, the prevention of the onset of [i-hcmoglobi nopathy or a symptom of P-hemoglobinopathy in a subject in need thereof or in a subject predisposed towards P-hemoglobinopathy.

[0106] A subject suitable for receiving the method of treatment as described herein can be, but is not limited to, a subject suffering from P-hemoglobinopathy, P-thalassemia, or sickle cell disease. In a further example, administration induces expression of the hemoglobin gamma 1 (HbGl) gene. In yet another example, administration increases y- globin expressing cells. In a further example, a subject administered with a composition of the disclosure can be induced to express the hemoglobin gamma 1 (HBG1) gene to treat fl-hemoglobinopathy, p-lhalasscmia, or sickle cell disease by increasing y- globin expressing cells.

[0107] In another example, a subject suitable for receiving the gene therapy as described herein can be, but is not limited to, a subject susceptible to p-hemoglobinopathy, P-thalassemia, or sickle cell disease. As used herein, a subject susceptible to hemoglobinopathy, P-thalassemia, or sickle cell disease can be, but is not limited to, a subject who have genetic mutations that disrupt P-chain. Such genetic mutations are known in the art.

[0108] Accordingly, in another example, a subject susceptible to P-hemoglobinopathy, P-thalassemia, or sickle cell disease, who is administered a composition of the disclosure can be induced to express the hemoglobin gamma 1 (HBG1) gene to prevent the onset of hemoglobinopathy, P-thalassemia. or sickle cell disease, or a symptom of hemoglobinopathy, p-thalassemia, or sickle cell disease. In yet another example, a subject administered a composition of the disclosure can be induced to increase y- globin expressing cells. In a further example, a subject administered a composition of the disclosure can be induced lo express lhe hemoglobin gamma 1 (HBG1) gene lo increase y- globin expressing cells to prevent the onset of hemoglobinopathy, P-thalassemia, or sickle cell disease, or a symptom of hemoglobinopathy, P-thalassemia, or sickle cell disease.

[0109] As used herein, the terms “increase” and “decrease” refer to the relative alteration of a chosen trait or characteristic in a subset of a population in comparison to the same trait or characteristic as present in the whole population. An increase thus indicates a change on a positive scale, whereas a decrease indicates a change on a negative scale. The term “change”, as used herein, also refers to the difference between a chosen trait or characteristic of an isolated population subset in comparison to thesame trait or characteristic in the population as a whole. However, this term is without valuation of the difference scon.

[0110] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0111] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0112] Other embodiments are within the following claims and non- limiting examples.EXAMPLESPaired Cas9 nickases are more efficient than Cas9 nuclease in editing HBG promoters

[0113] The sequences of HBG1 and HBG2 promoters are identical from position -223 to the transcription start site + 1 . The region around the LRF binding site contains multiple heredity persistence of fetal haemoglobin (HPFH) mutations, including one (-196 C > T) that leads to high level (-40%) of Ay expression. This cluster of heredity persistence of fetal haemoglobin (HPFH) mutations can be targeted at once by paired nickases, possibly resulted in more prominent y-chains re-expression. Over the region, 4 pairs of gRNAs were identified, which are in opposite orientation (anti-sense A l to A4; and sense SI to S4) (Fig. IB; table 1). Pairing of an anti-sense and a sense gRNAs with Cas9 nickases in a PAM (protospaccr-adjaccnt motif)-out configuration, can generate efficient DSB with various lengths.

[0114] The efficiency of individual gRNAs with Cas9 nuclease in cord blood (CB) CD34+cells was tested. The gRNA was combined with the Cas9 protein (nuclease or nickase) to produce the ribonuclcoprotcin (RNP) complex, and was electroporated into the cells. In all experiments, the same amount of gRNAs and Cas9 protein was used for electroporation (i.e. 2.5 pg of Cas9 protein and 4 pg of gRNA, or 2 pg each for pair of gRNAs) so that the editing efficiency of nuclease and nickase can be directly compared. Genomic DNA was extracted 4 days post-electroporation, and the percentage of on- target editing was estimated by T7 endonuclease (T7E1) mismatch assay using primers specific toHBG1 promoter. All gRNAs demonstrated a level of editing with Cas9 nuclease, with S4 (54 %) being the most efficient gRNAs (Fig. 2A). Two other gRNAs were also shown to result in efficient editing (A2, 32%; A3; 48%). Both HBG1- and HBG2-specific primers gave similar result on the T7E1 assay (data not shown).

[0115] Next, gRNAs were tested with the nickase. The gRNA Al was able to edit substantially when paired with another gRNA and the nickases (12-22%; Fig. 2B). suggesting the nicking approach worked in primary hematopoietic cells. All combinations were then tested using the paired nickases. Surprisingly, most combinations worked quite well, with highest editing efficiency up to -85 - 86% for the A2 + S4 and A3 + S4 pairs (A2 / A3 / A4 + S2 / S3, ranged from 62 to 81%; summarized in table 4) (Fig. 2C). Notably, the paired nickases were more efficient than the respective nuclease alone (c.g. A2 + S4 pah' gave -86%; A2 and S4 alone gave -32% and -54 % respectively). In adult bone marrow (AD) CD34+ cells, the combinations A2 + S4 and A3 + S4 also demonstrated much higher editing efficiency (76% and 69% respectively) than that of the nuclease alone (37%, A2; Fig. 2D).Editing by paired nickases induce stronger HbF re-expression in hematopoietic progenitors

[0116] Although the paired nickases approach worked well in gene editing of cord blood (CB) CD34+cells, these cord blood cells have yet begun “globin switching” and, upon erythrocyte differentiation, expresses almost exclusively HbF (>95% F cells) even without editing. The consequence of editing in adult bone marrow (AD) CD34+cells was examined for the most efficient nickases pair A2 + S4 and the respective nuclease A2 for comparison. The edited adult bone marrow (AD) CD34+cells were differentiated into erythrocytes following the 3-phases expansion protocol. The erythrocytes were harvested at 17-20 day for quantification of y-chain production by flow cytometery and real-thrte PCR. The editing efficiency of paired nickases A2 + S4 was higher than that of nuclease A2 (71 + 4.8 % versus 3 I + 6.3 %; Fig. 3 A). In line with higher editing efficiency, the paired nickases also outperformed the nuclease by generating more F cells (44.3 ± 5.6 % versus 27 ± 5.7 %; Fig. 3B and Fig. 3C) and increasing production of y-chain transcripts (ration of y / a chains, 0.33 ± 0.07 % versus 0.22 ± 0.09 %; Fig. 3C). The substantial background level of F cells (-21% for the Cas9 and Cas9n controls) is likely intrinsic to the artificial culture condition of the assay. Thus, it was demonstrated that comparing to nuclease, the paired nickases was more efficient in HbF induction in normal hematopoietic cells.The paired nickases approach work efficiently on (1-thalassemia cells

[0117] Upon confirmation of the high efficiency of paired nickases in normal CD34+ cells, it was examined whether the same approach will work for those with defective P-chain. CD34+cells were collected from three individuals with (3-thalassemia having the following genotypes: Thal_#l (IVS II- 654 OT; CD 41 / 42 -CTTT, (3 0 / [3 +), Thal_#2 (41 / 42 -CTTT; Codon 43 G to T) and Thal_#3 (homozygous 41 / 42 -CTTT). Besides the previously examined pair of A2 + S4, the A3 / A4 + S4 pairs were also included for evaluation in P-thalassemia cells. The editing efficient was higher with paired nickases (A2 + S4, 60.7 ± 2.7%; A3 + S4, 64 ± 1%; A4 + S4, 59.7 ± 7.2%) than with the respective nucleases (A2, 36 ± 5.2%; A3, 46 ± 4%; A4, 41 ± 3%) (Fig. 4A and 4C). Again, all the paired nickasesin general produced more F cells (A2 + S4, 68.3 ± 3.9%; A3 + S4, 69.7 ± 9.1%; A4 + S4, 70.4 ± 2.8%) than that of the respective nucleases (A2, 63.9 ± 6.1%; A3, 56.7 ± 10.2%; A4, 53.2 ± 8.1%), and produced more y-chain transcript (ration of y / a chains, A2 + S4, 1.5 ± 0.9%; A4 + S4, 1.3 ± 0.7%; A2, I + 0.6%) (Fig. 40). When compared with the adult bone marrow (AD) cells, the ^-thalassemia cells have a much higher background level of F cells (thalassemia versus adult bone marrow (AD) cells, 51.8 ± 9.1 % versus 21.3 ± 5.2 %) and y-chain expression (ratio of y / ot chains for thalassemia versus adult bone marrow (AD) cells, 0.95 + 0.55 % versus 0.12 + 0.04 %) in the artificial ex vivo culture system, with higher variability between samples (shown by individual data points in Fig. 4C). For every sample, the paired nickases resulted in higher editing efficiency and HbF / y-chain production than the respective nucleases.Paired nickases minimize off-targel mutagenesis created by nuclease

[0118] The paired nickases as disclosed herein are efficient for editing the HBG promoters. Furthermore, the editing specificity of the paired nickases as disclosed herein was determined in comparison to that of nuclease. Targeted amplicon deep sequencing was employed to analyse the target site on HBG promoters, as well as a panel of 54 potential off-target sites (OFT) for the gRNAs A2, A3 and S4 (Table 5). These 54 off-target sites included off-target sites for gRNA A2 and A3 (23 and 2 sites respectively), and the predicted OFTs of gRNA S4 (29 sites). Targeted amplicon sequencing was carried out on the adult bone marrow (AD) and ^-thalassemia CD34+samples (3 each) to determine the percentage of non -homologo us end joining (NHEJ) mediated indels that resulted from Cas9 editing. Consistent with the result of T7E1 assay, the on-target editing by the paired nickase A2 + S4 (47.6 ± 4.4%) was higher than that of nuclease A2 (27.8 ± 0.8%) on adult bone marrow (AD) cells (Fig. 5A) and on the thalassemia cells (paired nickase A2 + S4, 61.6 + 6.5%; nuclease A2, 44.8 + 11.7%) (Fig. 5B). Other nickase pairs (A3 + S4) and nucleases (A3, S4) were also included for some experiment, which generated on-target editing as well (Fig. 5B).

[0119] For the off-target sites, no significant indels were found for the no gRNAs controls (above the background level of -0.1-0.2%, variable between OFTs). Also, an increase of indels for most of the OFTs was not found, except for the three gRNA A2 OFTs (OFT- 13, OFT-25 and OFT-40). For the adult bone marrow (AD) CD 34+ samples, indels were found exclusively over these sites for editing by A2 and nuclease (OFT-25, 0.4 ± 0.06%; OFT-40, 0.68 ± 0.1%; OFT-13, 0.31 ± 0.04%) but not for editing by the paired nickases A2 + S4 (all al background level of -0.1-0.2%, same as level of control) (Fig. 5 A). Consistently for the P-thalassemia cells, indels on these three sites were found only for editing by A2 and nuclease (OFT-25, 14.7 ± 7.2%; OFT-40, 1.25 ± 0.5%; OFT-13, 0.94 ± 0.4%; Fig. 5B). The paired nickases were able to eliminate off-target caused by the nuclease, demonstrating the greater specificity and enhanced efficiency of a paired nickase editing approach.Editing of HBG promoters lead to deletion of inter-HBGs region

[0120] The nearly identical HBG1 and HBG2 promoters can be recognized and edited by Cas9 RNPs simultaneously, as suggested by the results of the T7E1 assay as disclosed herein. The two closelydistanced DSBs in a highly homologous context can result in the loss of intervening HBG2 gene and promotor regions, fusing the HBG1 gene to the promoter of HBG2, thus resulting in the upregulation of HBG1 expression. To demonstrate the potential loss of intervening genome region, two different assays were employed (Fig. 6A). The first assay is a standard PCR reaction that aim to amplifies a unique deletion-specific ~ 1.5 kb fragment. And second, a digital PCR assay that quantifies the absolute copy number (CN) of the intcr-HBG region, with reference to another control region on the same arm of chromosome 11. On the cord blood (CB) CD34+cells, the detection of the deletion-specific fragments by PCR in all samples edited by nuclease Cas9 RNPs was achieved, regardless of the gRNAs used and the editing efficiency (Fig. 6B; the same samples for T7E1 assay of Fig. 2A). The same deletion-specific fragments were also detected for the Cas9 RNPs (paired nuclease A2 + S4 and A4 + S4; nuclease A2 and S4) treated P-thalassemia CD34+cells (Fig. 6C; the same samples for T7E1 assay of Fig. 4A). After screening by the semi-quantitative standard PCR assay, the extent of intervening HBG2 deletion on the Cas9 edited adult bone marrow (AD) and p-thalassemia CD34+cells was confirmed and quantified (Fig. 6D). When the copy number (CN) of the inter-HBG region of the no gRNAs controls was set to 2, the copy number (CN) was reduced significantly for cells edited by nuclease A2 (adult bone marrow (AD), 1.46 ± 0.07; P-thalassemia, 1.1 ± 0.13) and reduced further for the cells edited by paired nickases A2 + S4 (AD, 1.05 ± 0.01; P-thalassemia, 0.85 ± 0.1), confirming the deletion of inter-HBG region proportionally upon editing of the HBG promoters. Taken together, the above assays confirmed that the loss of HBG2 gene was common to all hematopoietic cells edited by Cas9 nuclease or paired nickases, and suggested that most of the y-chain expression in edited cells were mechanistically derived from desuppressed HBG1 rather than HBG2. r00121]The Cas9 mutant, the D10A nickase, was tested herein on its ability to reactivate y-chain expression by disrupting the binding sites of repressor LRF on HBG promoters. PAM-out Cas9 nickase pairs was shown herein to be more efficient and specific than the respective Cas9 nucleases in editing the HBG promoters, leading to greater HbF rc-cxprcssion in adult and P-thalassemia hematopoietic cells.

[0122] Heredity persistence of fetal haemoglobin (HPFH) comprises of diverse mutations, including deletion forms that could span from a few to hundreds kb that often cause the loss of HBB gene, and non-dclction form that only affect single base in the promoters. Mechanistically, it is clear that the betalike goblin gene promotors (HBB, HBD and HBG1 / 2) are competing for the upstream locus control region (LCR), the master controller for expression of this gene cluster. As such, disrupting either the active HBB promoter or the repressed HBG promoters can favour the expression of HBG genes. Alternatively, the paired nickase approach, as demonstrated herein, mediates even higher editing efficiency and specificity as compared to the single gRNA nuclease.

[0123] Since development, CRIPSR / Cas9 nuclease is the most common choice for editing experiments, owing to its great versatility and accessibility. However, Cas9 nuclease is prone to off- targeting cleavages, which represents a major drawback for clinical application. Cas9 can tolerate somemismatch over 5’ end of the target sequence, creating source of off target editing. It was shown herein that the paired nickases can remarkably reduce the off-target effect of nuclease.100124] Double nicking by paired nickase is not widely employed for editing experiment because of the “presumed” low efficiency. Tn addition, to plan for paired nickases experiment, one must identify the rightly aligned gRNA pairs (i.e. in PAM-out) and evaluate the efficient of these different pairs, which present extra hurdles. The findings as shown herein on primary hematopoietic cells demonstrate that paired nickase can outmatch Cas9 nuclease on both editing efficient and specificity.

[0125] Simultaneous editing of HBG promoters leads to deletion of HBG2 containing inter-promoter region, fusing the promoter of HBG2 to that of HBG1 (Fig. 6). The same deletion was observed previously with Cas9-mcdiatcd editing of LCF or BCL11 A binding sites. In fact, the highly homologous HBG promoters are subjected to natural gene rearrangements that produced a number of novel y-chain variants in human, including duplications, triplication and even quadruplication of y-globin genes of various Gy and Ay combinations. One of these rearrangements, resembling the editing-induced HBG2 deletion, is a 5 kb deletion over the HBG promoter that fuses the first and second exons of HBG2 to third exon of HBG1, generating unique Gy- Ay hybrid chain. Individuals carrying this variant are normal phenotypically, except having reduced Gy to Ay ratio. Considering the absence of noticeable deleterious effect on erythrocytes differentiation and the marked increase in HbF expression and F cells with our gene-editing approach, it shows that re -expression of HBG1 alone can sufficiently correct f>- hemoglobinopathies.

[0126] Cas9 nuclease produces blunt DSB that predominantly repaired by non-homologous end joining (NHEJ); in contrast, the PAM-out paired D10A nickases produce 5’- overhangs that favours HDR. It appears that since Cas9 nuclease and nickases create different type of DNA lesions particular lesion can favour the formation of HBG2 deletion. Nonetheless the HBG2 deletion is found in cells edited either by nuclease and nickase in this study, indicating the deletion is not driven by the difference in DNA repair mechanism. As such, the difference in the extent of HBG2 deletion probably reflects the difference in editing efficiency. The results shown herein show that, due to high sequence homology, targeting any element on the HBG promoters generates the specific HGB2 deletion. The deletion of the entire HBG2 containing inter-promoter region in turn resulted in upregulation of HBG 1 expression and F cells.Paired Cas9 nickases approach demonstrated efficiency comparable to that of single gRNA mediated editing of BCL11A.

[0127] The single gRNA used by Casgevy, which comprises a sequence as defined in SEQ ID NO: 84, was combined with Cas9, and electroporated into the adult normal and or thalassemia CD34+cells. The editing efficiency was evaluated by T7E1 assay (Fig. 7a), using primers flanking the edited sites, where the primer comprise sequences as defined in SEQ ID NOs: 85 and 86. Editing by paired Cas9n was performed as described herein.

[0128] Deletion of inter-promoters region of HBGs (HBG2) specific for thalassemia cells, as edited by Casgcvy gRNA and Cas9 nuclease, and paired gRNAs with Cas9 nickase, was detected by PCR assay (Fig. 7b) as described herein.

[0129] Edited cells as obtained in Fig. 7a were expanded and differentiated in vitro into erythrocytes for 17 days, and the percentage of F cells (i.e. Fetal haemoglobin (HbF) expressing erythrocytes) was analysed by flow cytometry (Fig. 7c), while the level of gamma transcript which encodes for HbF was detected by RT-PCR as shown in Fig. 7d.

[0130] Fig. 7 summarises the comparison between the approach as described herein, which employs the use of paired nickases targeting the promoters of HBG (A2 / S4, A3 / S4 and A4 / S4) and that of Casgcvy, which employs the use of a nuclease targeting the enhancer of BCL11A and a gRNA 1617.

[0131] Fig. 7 shows that editing by paired nickases in hematopoietic cells from a normal adult donor generated 33.5% to 34.4 % of HbF+CD235a+ erythrocytes. In contrast, editing by Cas9 nuclease with a single gRNA 1617 from Casgevy generated 22.8% of HbF+CD235a+ erythrocytes upon in vitro differentiation, while the proportion of HbF+CD235a+ erythrocytes in unedited cells from a normal adult donor is 17.8%.

[0132] When tested on hematopoietic cells from a Thalassemia donor, which has a basal level of 60.1% of HbF+CD235a+ erythrocytes, editing by paired nickases increased the level of HbF+CD235a+ crythrocytcs to 73.5% to 75.2%, while editing by Cas9 nuclease with a gRNA 1617 from Casgcvy resulted in 71.8% of HbF+CD235a+ erythrocytes.

[0133] In summary, the present disclosure illustrates the feasibility of achieving efficient and specific editing of the HBG promoter by deploying paired Cas9 nickases. Potential non-specific editing brought by Cas9 nuclease can be eliminated, rendering a much safer editing approach for clinical translation and the treatment of [(-hemoglobinopathies.Mouse engraftment of edited human thalassemia CD34+ cells

[0134] To assess the impact of our editing approach on rcpopulating hematopoietic cells, human Thalassemia CD34+ cells edited as described herein were engrafted into immunodeficient NSG mice. Human cells were recovered from the mice 16 weeks post-engraftment. Fig. 8a shows the overall view of the animal experiment. The thalassemia CD34+ cells were edited in vitro as before by electroporation. NSG mice were transplanted with cither control (Ctr; no Cas9 or guide RNA) or edited cells (with Cas9n + guide A3 & S4) cells intravenously. Bone marrow (BM) cells were collected 16 weeks after transplantation for analysis. Data of one animal from each group was shown for illustration purpose.

[0135] Fig. 8b shows the human cell chimerism of the bone marrow cells, presented by the percentage of human hCD45+ cells over the total of human hCD45+ and mouse mCD45+ cells.

[0136] Fig. 8c shows the multilineage reconstitution of the engrafted human cells, composed mainly of hCD19+ B cells, some hCD33+ myeloid cells and hCD34+ progenitor cells, with minimal hCD3+ T cells.

[0137] Fig. 8d shows the efficiency of editing of the cells before (input) and after transplantation (recovered from bone marrow, and hCD34+ enriched). Representative gel images of the T7E1 assay on fragment amplified from HBG1 promoter. The estimated editing efficiency was shown below the respective lanes.

[0138] Fig. 8e shows the deletion of inter-HBGs promoter region as quantified by Taqman copy number variation assay. The copy number was normalized to that of control and set at 2.

[0139] Overall it is shown herein that the Cas9n edited CD34+ Thalassemia cells are capable of persisting, engrafting and repopulating immunodeficient mice. The efficient of editing in the BM recovered cells was lower than that of the pre-transplanted (input) cells.EXPERIMENTAL SECTIONStudy approval, cell processing and culture

[0140] The study was approved by Singhealth centralised institutional review board (CIRB 2019 / 2866; CIRB 2021 / 2541) and IRB of HKU. All donors provided inform consents. Cord bloods (CB) were obtained from Singapore Cord Blood Bank. Adult hemopoietic cells were obtained from KKH. Thalassemia samples were obtained from KKH and HKU. Mononuclear cells were separated by Ficoll- paque (cat. 17544203, GE Healthcare) and CD34+cells were enriched by magnetic microbeads (cat. 130-046-702, Miltenyi Biotec). The CD34+cells were cultured in completed StemPro-34 medium (with nutrient supplement, glutamine and Pen-Strep) (cat. 10639011, ThermoFisher) supplemented with 300 ng / ml SCF, 300 ng / ml FLT3L, 100 ng / ml TPO and 60 ng / ml IL-3 (cal. 300-07, 300-19, 300-18 and 200-03, all from Peprotech) for 48 hours before electroporation. Sequence of the primers used are summarized in Table 3.Guide RNA, Cas9 RNP and electroporation

[0141] Guide RNAs (gRNAs) were designed using GPP sgRNA Designer (and currently updated to CRISPick, https: / / portals.broadinstitute.org / gppx / crispick / public) and are summarized in Table 1. The gRNA was produced by hybridization of crRNA;tracrRNA duplex by mixing equal molarity of crRNA and tracrRNA, heated up to 94°C for 2mins, and cooled to 60°C for Imin, and further cooled to room temperature. The gRNA was then reconstituted to 2 pg / pl in duplex buffer (all from Integrated DNA Technologies, IDT). The Cas9 ribonucleoproteins (Cas9 RNP) complex was formed by mixing 2.5 pg of cither the nuclease Cas9 (Alt-R S.p. Cas9 Nuclease V3, cat. 1081059, IDT) or the nickase Cas9n (Alt-R S.p. Cas9 D10A nickase, cal 1081063, IDT) wilh 4 pg of gRNAs in 6 id of buffer R for 15 mins at room temperature, in the present of 1 pg / ul electroporation enhancer (cat. 1075916, IDT). The target cells were washed thrice with PBS (without Ca2+ / Mg2+), and resuspended in the desired volume of buffer R. Typically 0.1-0.2xl06cells were resuspended in 6 pl of buffer R. combined with 6 pl of Cas9 RNP complex, and electroporated using the 10 pl Neon tips with the pre-set program no. 24 of the Neon system (1600V, 10ms pulse, 3 pulses) (all from ThermoFisher, cat. MPK5000). Electroporated cells were released immediately to pre-warmed complete StemPro 34 medium (Phase 1 medium forerythrocyte expansion) for recovery and continuous culture. For 1-2 xlO6starting cells, the reagents were scaled up and 100 pl Neon® tips were used instead.T7E1 endonuclease mismatch cleavage assayThree phases serum-free expansion of erythrocyte

[0143] The 3 phases erythrocyte expansion culture was performed as described previously (Lee, de Vasconcellos et al. 2013). The StemPro-34 complete medium was used throughout, with specific supplements added each phase. The phase 1 medium (day 2 to 7 for the electroporated cells) was supplemented with 50 ng / mL SCF, 50 ng / mL FLT3 Ligand and 10 ng / mL IL-3. Phase 2 medium (day 7 to day 14) w as supplemented with 4 U / mL of erythropoietin (EPO) (cat. 100-64, Peprotech), 10 ng / mL SCF, 10 pg / mL insulin (cat. I9278-5ML, Sigma-Aldrich), 3 U / mL of heparin (cat. H3149-250KU, Sigma-Aldrich), and 0.8 mg / mL of holo Transferrin (cat. T0665, Sigma-Aldrich). Phase 3 medium (day 14 to day 21) was supplemented with 4 U / mL EPO, 3 pM RU486 (cat. M8046, Sigma-Aldrich), 10 pg / mL insulin, 3 U / mL heparin, and 0.8 mg / mL holo Transferrin. At the end of phase I expansion, some cells were collected for genomic DNA extraction. The expanded erythrocytes were then harvested for flow cytometry and RNA extraction between day 17 and day 21.Real-time quantitative PCR

[0144] RNA was extracted from the RBC using the Rneasy extraction kit (cat. 74004, Qiagen). The cDNA was synthesised using Superscript VILO cDNA kit (cat. 11754050, Thermo). The real-time PCR reaction was setup by mixing the cDNA with 2x iQ SYBR Green Supcrmix (cat. 1708880, Bio-rad) and 0.2pM final of both forward and reverse primers, at 25 pl per well in the CFX96 Real-Time PCR Detection System. The condition of PCR reaction was: association at 95°C for 3mins, followed by 40 cycles of 95°C lOsec, annealing at 63 C for 30sec and extension at 72 'C for 20sec.Intracellular staining and flow cytometry

[0145] Intracellular staining of RBC was performed based on the protocol from Inside Stain Kit (130- 090-477, Miltneyi Biotec). Briefly, about 0.1 x l O6RBCs were fixed for 20 mins with 0.5 ml of Inside Fix. Cells were then washed once with autoMCAS rinsing buffer (cat. 130-091-222, Miltenyi Biotec), followed by 0.5 ml of Inside Perm. Afterward the cells were resuspended in 90 pl of Inside Perm andco-stained with anti-CD235 (1: 100; cat. 349106, Biolegend) and anti-HbF (1: 10; cat. 130-108-241, Miltcnyi Biotcc) antibodies in a total volume of 100 Jtl for 10 mins in dark. The cells were then washed once with 0.5 ml of Inside Perm, and resuspend in 0.25 ml of autoMACS buffer for flow acquisition on BD LSRI1. The percentage of HbF expressing erythrocyte (F cells) was analysed by Flowjo (BD biosciences).CRISPR target amplicon sequencing

[0146] The amplification primers for amplicon panel were designed and produced by the rhAmpSeq CRISPR analysis system (IDT). The amplicon panel was designed to include the targeted locus (the HBG promoter 5249974 to 5250057, hg38) and a total of 54 validated / predicted off-target loci (of the guides A2, A3 and S4; table 5). Amplicon libraries were constructed using 10-50 ng of genomic DNA per sample and were amplified using rhAmpSeq CRISPR library kit (cat. 10007317), following the instructions on protocol. The libraries were then indexed, pooled, and cleaned up by AMPure XP beads before sending for sequencing on the NovaSeq platform (Novogene), aiming for coverage of lOOOOx per target. The sequencing data was analysed on the cloud-based rhAmpSeq CRISPR analysis platform (BlueBee, IDT).Detection of inter-HBG region deletion

[0147] The deletion of inter-HBG region was screened by PCR and further quantified by digital PCR. For PCR: the PCR was performed using the GoTaq PCR master mix, under the same condition used for T7E1 assay. The amplified product was then resolve on 1% gel agarose. For digital PCR: digital PCR was performed on QIAcuity One (Qiagen). Primer pahs targeting the inter-HBG region (upstream of the HBG1 promoter) or the control region (targeting CD44 on chr.l l, same arm of HBG genes) (CNV assay; cat. PCH111-0175806A, Qiagen) were used to quantify the exact copy number. Each reaction consisted of the genomic DNA, 4 pl of 3x evagreen mix (cat. 2501 I I Qiagen) and 0.8 pM primers in a total volume of 12 pl, was loaded onto a well on the 8.5K 24 wells plate (cat. 250011, Qiagen). The quantification reactions were run in duplicate or triplicate using the program: activation at 95°C for 2 mins, followed by 40 cycles of denaturation at 95°C for 15 sec, annealing at 60°C for 15 sec and extension at 72"C for 15 sec, and with final extension at 40"C for 5 mins. Data analysis was done on QIAcuity Software Suite.Mouse engraftment of edited human thalassemia CD34+ cells

[0148] The thalassemia CD34+ cells were edited in vitro as described herein by electroporation. NSG mice were transplanted with either control (Ctr; no Cas9 or guide RNA) or edited cells (with Cas9n + guide A3 & S4) cells intravenously. Bone marrow (BM) cells were collected 16 weeks after transplantation for analysis.Statistical analysis

[0149] Data were reported as mean + standard error of the mean (SEM). Statistical significance was determined by two tailed Student t test.Table 1: gRNA sequences of the present disclosureTable 2: Nuclease and nickase sequences of the present disclosureTable 4: Editing efficiency of gRNA pairsTable 5: Targeted sites on HBG promotersTable 6.1: Sequences of HbG locus and promotersTable 6.2: Sequences of LRF binding sites in HbG locusTable 7: Casgevy gRNA and primers for evaluating editing efficiency by Casgevy gRNA

Claims

CLAIMS1. A kit comprising: a) a CRISPR-Cas9 nickase; and b) a pair of guide RNAs (gRNA), wherein the pair of guide RNAs binds to target sequences, wherein the binding will result in a disruption of binding sites of repressor LRF in a promotor of a hemoglobin gamma (HbG) locus.

2. The kit of claim 1, wherein the CRISPR-Cas9 nickase is a mutant nickase (Cas9n) comprising a D10A mutation relative to the Cas9 sequence of SEQ ID NO: 9.

3. The kit of any one of claims 1 to 2, wherein the target sequence of the guide RNA (gRNA) is located 300 nucleotides upstream or downstream relative to a LRF binding site in the promoter of the hemoglobin gamma (HbG) locus.

4. The kit of any one of claims 1 to 3, wherein the target sequence of the guide RNA (gRNA) comprises a pair of sequences selected from the group consisting of: SEQ ID NOs: 1 to 8.

5. The kit of any one of claims 1 to 4, wherein the target sequences of each pair of guide RNA (gRNA) do not overlap.

6. The kit of claim 5, wherein the target sequence of the guide RNA (gRNA) comprises a pair of sequences selected from the group consisting of: SEQ ID NO: 2 and 8, SEQ ID NO: 3 and 8, and SEQ ID NO: 4 and 8.

7. The kit of any one of claims 1 to 6, wherein the disruption of the binding sites of repressor LRF of the target sequence of the hemoglobin gamma (HbG) promoter results in the partial loss of the promoter of a hemoglobin gamma 1 (HbGl) gene and the promoter of a hemoglobin gamma 2 (HbG2) gene.

8. The kit of any one of claims 1 to 7, wherein the disruption of the binding sites of repressor LRF further results in the loss of a hemoglobin gamma 2 (HbG2) gene.

9. The kit of any one of claims 7 to 8, wherein the disruption of the binding sites of repressor LRF further results in a fusion of the promoter of the hemoglobin gamma 1 (HbGl) gene to the promoter of the hemoglobin gamma 2 (HbG2) gene.

10. The kit of any one of claims 1 to 9, wherein the disruption of the binding sites of repressor LRF induces expression of the hemoglobin gamma 1 (HbGl) gene.I I . The kit of any one of claims I to 10, wherein the pair of guide RNA (gRNA) bi nds to the target sequences in opposite directions.

12. The kit of any one of claims 1 to 11, wherein the hemoglobin gamma (HbG) locus is a mammalian or human hemoglobin gamma (HbG) locus.

13. A method for modifying a hemoglobin gamma (HbG) locus in a eukaryotic cell, the method comprising introducing into the eukaryotic cell a kit comprising: a) a CRISPR-Cas9 nickase; and b) a pair of guide RNAs (gRNA), wherein the pair of guide RNAs binds to target sequences, wherein the binding will result in a disruption of binding sites of repressor LRF in a promotor of a hemoglobin gamma (HbG) locus.

14. The method of claim 13, wherein the CRISPR-Cas9 nickase is a mutant nickase (Cas9n) comprising a D10A mutation relative to the Cas9 sequence of SEQ ID NO: 9.

15. The method of any one of claims 13 to 14, wherein the target sequence of the guide RNA (gRNA) is located 300 nucleotides upstream or downstream relative to a LRF binding site in the promoter of the hemoglobin gamma (HbG) locus.

16. The method of any one of claims 13 to 15, wherein the target sequence of the guide RNA (gRNA) comprises a pair of sequences selected from the group consisting of: SEQ ID NOs: 1 to 8.

17. The method of any one of claims 13 to 16, wherein the target sequences of each pair of guide RNA (gRNA) do not overlap.

18. The method of claim 17, wherein the target sequence of the guide RNA (gRNA) comprises a pair of sequences selected from the group consisting of: SEQ ID NO: 2 and 8, SEQ ID NO: 3 and 8, and SEQ ID NO: 4 and 8.

19. The method of claim 13 to 18, wherein the disruption of the binding sites of repressor LRF results in the partial loss of the promoter of a hemoglobin gamma 1 (HbGl ) gene and the promoter of a hemoglobin gamma 2 (HbG2) gene.

20. The method of any one of claims 13 to 19, wherein the disruption of the binding sites of repressor LRF further results in the loss of a hemoglobin gamma 2 (HbG2) gene.21 . The method of any one of claims 19 to 20, wherein the disruption of the binding sites of repressor LRF further results in a fusion of the promoter of the hemoglobin gamma 1 (HbGl) gene to the promoter of the hemoglobin gamma 2 (HbG2) gene.

22. The method of any one of claims 13 to 21 , wherein the disruption of the binding sites of repressor LRF induces expression of the hemoglobin gamma 1 (HbGl) gene.

23. The method of any one of claims 13 to 22, wherein the pair of guide RNA (gRNA) binds to the target sequences in opposite directions.

24. The method of any one of claims 13 to 23. wherein the hemoglobin gamma (HbG) locus is a mammalian or human hemoglobin gamma (HbG) locus.

25. The method of any one of claims 13 to 24, wherein the eukaryotic cell is a hemotopoietic stem cell.

26. The method of claim 25, wherein the hemotopoietic stem cell is a CD34+ or a CD133+ cell.

27. A method of treating P-hemoglobinopathy in a subject, wherein the method comprises the steps of: a) isolating hematopoietic cells from the subject; b) editing the isolated hematopoietic cells obtained from step a) with a kit of any one of claims 1 to 12 ex vivo; c) expanding the edited cells obtained from step b) ex vivo; and d) administering a therapeutically effective amount of the expanded cells obtained from step c) to a subject with a P-hcmoglobinopathy.

28. The method of claim 27, wherein the administration induces expression of the hemoglobin gamma 1 (HbGl) gene.

29. The method of any one of claims 27 to 28 wherein the administration increases y- globin expressing cells.

30. The method of any one of claims 27 to 29, wherein the P-hemoglobinopathy is selected from the group consisting of: P-thalasscmia, and sickle cell disease.3 I . The method of any one of claims 27 to 30, wherein the expanded cells obtained from step c) are administered to a subject with a P-hemoglobinopathy by transplantation.

32. A gene therapy for preventing P-hemoglobinopathy in a subject, wherein the gene therapy comprises the steps of: a) isolating hematopoietic cells from the subject; b) editing the isolated hematopoietic cells obtained from step a) with a kit of any one of claims 1 to 12 ex vivo; c) expanding the edited cells obtained from step b) ex vivo', and d) administering a therapeutically effective amount of the expanded cells obtained from step c) to a subject susceptible to P-hemoglobinopathy.

33. The gene therapy of claim 32, wherein the administration induces expression of the hemoglobin gamma 1 (HbGl) gene.

34. The gene therapy of any one of claims 32 to 33, wherein the administration increases y- globin expressing cells.

35. The gene therapy of any one of claims 32 to 34, wherein the P-hemoglobinopathy is selected from the group consisting of: p-thalassemia, and sickle cell disease.

36. The gene therapy of any one of claims 32 to 35, wherein the expanded cells obtained from step c) are administered to a subject with a P-hemoglobinopathy by transplantation.

37. A composition comprising the kit of any one of claims 1 to 12.

38. A method of treating P-hemoglobinopathy in a subject, wherein the method comprises administering a therapeutically effective amount of the composition of claim 37 to a subject with P-hemoglobinopathy.

39. The method of claim 38, wherein the administration induces expression of the hemoglobin gamma 1 (HbGl ) gene.

40. The method of any one of claims 38 to 39 wherein the administration increases y- globin expressing cells.41 . The method of any one of claims 38 to 40, wherein the fl-hemoglobinopathy is selected from the group consisting of: fl-thalassemia, and sickle cell disease.

42. A gene therapy for preventing P-hemoglobinopathy in a subject, wherein the gene therapy comprises administering a therapeutically effective amount of the composition of claim 37 to a subject susceptible to P-hemoglobinopathy.

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