Compositions and methods for treating anemias

Abstract

The present disclosure relates to compositions and methods of increasing levels of fetal hemoglobin (HbF) in cells. The present disclosure further relates to methods for treating patients suffering from blood cell diseases, including those associated with reduced amounts of functional adult hemoglobin (HbA), such as sickle cell disease and β-thalassemias

Description

[0001]VIA Patent Center 808955.000150 (OTT: 2024-511) Date of Deposit: May 14, 2025 COMPOSITIONS AND METHODS FOR TREATING ANEMIAS CROSS-REFERENCE TO RELATED APPLICATION The present application is related to and claims priority under 35 U.S.C. § 119(e) to U.S. provisional patent application No.63 / 647,903, entitled “Compositions and Methods for Treating Anemias,” filed May 15, 2024. The entire content of the aforementioned patent application is incorporated herein by this reference. FIELD OF THE INVENTION The present disclosure relates to targets, compositions and methods of inducing fetal hemoglobin (hemoglobin γ (HB γ) or HbF) expression in erythroid cells. The present disclosure further relates to methods for treating patients suffering from diseases associated with blood cell disorders, such as Sickle Cell Disease (SCD) or β-thalassemias, including those where elevated expression of HbF protein can compensate for a mutant or defective hemoglobin β (HBβ) gene, a mutant or defective HBβ protein, or changes in HBβ protein levels. SEQUENCE LISTING The instant application contains a Sequence Listing which has been filed electronically in eXtensible Markup Language format and is hereby incorporated by reference in its entirety. Said XML file, created on May 14, 2025, is named 808955_000150.xml and is 27,785 Bytes in size. BACKGROUND OF THE INVENTION Hemoglobin is the critical protein involved in oxygen transport throughout the body of vertebrates. It is found in red blood cells and consists of two α subunits and two β-like subunits. The composition of hemoglobin is developmentally regulated, and the human genome encodes multiple versions of these proteins, which are expressed during distinct stages of development (Blobel et al., Exp Hematol 2015; Stamatoyannopoulos G, Exp Hematol 2005). In general, fetal hemoglobin (HbF) is composed of two subunits of hemoglobin γ (HBγ) and two subunits of hemoglobin α (HBα) and adult hemoglobin (HbA) is composed of two subunits of hemoglobin β (HBβ) and two subunits of HBα. Thus, the β-like subunit utilized during the fetal stage of development (HBγ) switches to hemoglobin β (HBβ) after birth. 1 122236082.1 VIA Patent Center 808955.000150 (OTT: 2024-511) Date of Deposit: May 14, 2025 The developmental regulation of the egression of β-like subunits has been the focus of intense studies for decades (Li et al. Blood 2002). All five β-like subunits in humans reside on chromosome 11, where their genomic location corresponds to their temporal expression pattern. A distal cluster of enhancer elements, called the locus control region (LCR), coordinates the expression pattern at the β globin locus, where multiple transcription factors, including GATA1, GATA2, KLF1, KLF2, and MYB and TALI, bind at specific locations within the LCR at specific times in development. The five human β-like subunits are epsilon (HBE1; ε), gammaG (HBG2; γ), gammaA (HBG1, γ), delta (HBδ; δ) and beta (HBβ; β). The HBE1 gene is expressed during embryonic development, the HBG1 and HBG2 genes are expression during fetal development, and HBδ and HBβ genes are expressed in adults. The HBG1 and HBG2 genes encode identical proteins except for a single amino acid change at residue 136 (HBG1 = gly; HBG2 = ala). Red blood cell disorders like Sickle Cell Disease (SCD) and β-thalassemias are caused by alterations within the gene for the hemoglobin β (HBβ) subunit. SCD affects millions of people worldwide and is the most common inherited blood disorder in the United States (70,000-80,000 Americans). SCD has a high incidence in African Americans, where it is estimated to occur in 1 in 500 individuals. SCD is an autosomal recessive disease caused by single homozygous mutations in both copies of the HBβ gene (E6V) that result in a mutant hemoglobin protein called HbS (ghr.nlm.nih.gov / condition / sickle-cell-disease). Under deoxygenated conditions, the HbS protein polymerizes, which leads to abnormal red blood cell morphology. This abnormal morphology can lead to multiple pathologic symptoms including vaso-occlusion, pain crises, pulmonary hypertension, organ damage, and stroke. β-thalassemia is caused by mutations in the HBβ gene and results in reduced hemoglobin production (ghr.nlm.nih.gov / condition / beta-thalassemia). The mutations in the HBβ gene typically reduce the production of adult β-globin protein, which leads to low levels of adult hemoglobin, HbA. This leads to a shortage of red blood cells and a lack of oxygen distribution throughout the body. Patients with β-thalassemias can have weakness, fatigue and are at risk of developing abnormal blood clots. Thousands of infants are born with β-thalassemia each year, and symptoms are typically detected within the first two years of life. The identification of factors that regulate the expression of fetal hemoglobin could be useful targets for the treatment of SCD and β-thalassemias, since upregulation of fetal hemoglobin could compensate for mutant HbS protein in SCD or a lack of HbA in β-thalassemias. Because β- 2 122236082.1 VIA Patent Center 808955.000150 (OTT: 2024-511) Date of Deposit: May 14, 2025 like globin expression is developmentally regulated, with a reduction in the fetal ortholog (γ) occurring shortly after birth concomitantly with an increase in the adult ortholog (β), it has been postulated that maintaining expression of the anti-sickling γ ortholog may be of therapeutic benefit in both children and adults. A fetal ortholog of HBβ, hemoglobin γ (HBγ) can reverse disease- related pathophysiology in these disorders by also forming complexes with the required hemoglobin α subunit (Paikari and Sheehan, Br J Haematol 2018; Lethe and Bauer, Lancet 2016). Expression of the fetal hemoglobin protein can reverse the SCD pathophysiology through inhibiting HbS polymerization and morphologically defective red blood cells. Functionally, upregulation of either the HBG1 or HBG2 gene can compensate for mutant or defective adult HBβ. Based on clinical and preclinical studies, upregulation of hemoglobin γ (HBγ) is the proposedmechanism for compounds including Pomalidomide and Hydroxyurea and targets includingEHMT1 / EHMT2 and LSD 1 (Moutouh-de Parseval et al. J Clin Invest 2008; Letvin et al. NEJM 1984; Renneville et al. Blood 2015; Shi et al. Nature Med 2015). Given the severity and lack of effective treatments for blood cell disorders, such as Sickle Cell Disease (SCD) and β-thalassemias, including those where elevated expression of HbF protein could compensate for a mutant or defective hemoglobin β (HBβ) gene, there is clearly a need for new methods of treatment for these disorders, particularly for those capable of increasing HbF levels in a subject. BRIEF SUMMARY OF THE INVENTION The present disclosure is based, at least in part, on the identification of novel targets for modulation to achieve advantageous induction of fetal hemoglobin (hemoglobin γ (HBγ) or HbF) expression in erythroid cells. The present disclosure further relates to methods for treating patients suffering from diseases associated with blood cell disorders (β-hemoglobinopathies), such as Sickle Cell Disease (SCD) or β-thalassemias. In an aspect, the disclosure provides for a method of treating a hemoglobinopathy in a subject, the method including administering at least one agent capable of reducing the expression and / or activity of at least one human Spt-Ada-Gcn5-Acetyltransferase (hSAGA) complex protein in the subject, thereby treating the hemoglobinopathy in the subject. In one aspect, the disclosure provides for a method of inducing fetal hemoglobin in a subject, the method including administering at least one agent capable of reducing at least one Spt- 3 122236082.1 VIA Patent Center 808955.000150 (OTT: 2024-511) Date of Deposit: May 14, 2025 Ada-Gcn5-Acetyltransferase (hSAGA) complex protein level and / or function in the subject. In some embodiments, the subject has or is predisposed to having a hemoglobinopathy. In certain embodiments, the hSAGA complex protein is TADA2B, TADA3, TADA1, TADA2A, TAF5L, TAF6L, TAF9, TAF10, TAF12, KAT2A, KAT2B, SGF29, TATA1, SUPT7L, SUPT7H, SUPT20H, SUPT3H, TRRAP, USP22, ATXN7L3, ATXN7, ENY2, SF3B3, and / or SF3B5. Optionally, the hSAGA complex protein is TADA2B, TADA3, TAF5L, and / or TADA1. In some embodiments, the hSAGA complex protein is TADA2B, TADA3, TADA1, TADA2A, TAF5L, and / or SUPT20H. In still further embodiments, the hSAGA complex protein is TADA2B, TADA1, TAF5L, or SUPT20H. In some embodiments of the disclosure, the hemoglobinopathy to be treated is a beta- hemoglobinopathy. Optionally, the beta-hemoglobinopathy is a sickle cell disease or β- thalassemia. In some embodiments, the disclosure provides for an agent that reduces or eliminates expression of a gene, regulatory activity of a gene, or activity of an encoded protein or enzyme. In some embodiments, reduction or elimination of gene expression is a gene deletion and / or a missense gene mutation. In still other embodiments, the agent is an inhibitory RNA, siRNA, miRNA, shRNA, a small molecule inhibitor, and / or an antibody. In other embodiments, the agent comprises a CRISPR / Cas9 system comprising a guide RNA (gRNA). In some embodiments, the disclosure provides for gRNAs. In some embodiments, the gRNA is a TADA2B gRNA. Optionally, the gRNA is SEQ ID NO: 1 (GAAGAUAUGGCUGCCCACGU), SEQ ID NO: 2 (ACGUGAGCAUGUACAUCCAC), or SEQ ID NO: 3 (ACAGACCACACCUGUCCCAG). In some embodiments, the gRNA is a TADA1 gRNA. Optionally, the gRNA is SEQ ID NO: 4 (AACAGCUGAAACAGCCUCCU), SEQ ID NO: 5 (CCACUCGAGGCCAGCUUGAA), or SEQ ID NO: 6 (AUCCUUUGCCACAAAUUGCU). In some embodiments, the gRNA is a TAF5L gRNA. Optionally, the gRNA is SEQ ID NO: 7 (CAAGAUGAAUAUGUAAGGUG), SEQ ID NO: 8 (UGCUCUCCUGUAAGACCUCU), or SEQ ID NO: 9 (CCAAACCGUUGUUCUCACUG). In some embodiments, the gRNA is a SUPT20H gRNA. Optionally, the gRNA is SEQ ID NO: 10 (UCCAGUAACAUGAAAUCUCC), SEQ ID NO: 11 (CAGGUUAAUAUUUUUCAUUG), or SEQ ID NO: 12 (AACAUCCUUACCUGCAUUGU). In some embodiments, the disclosure provides for a composition for inducing fetal hemoglobin in a subject, the composition including a CRISPR / Cas9 system, wherein the 4 122236082.1 VIA Patent Center 808955.000150 (OTT: 2024-511) Date of Deposit: May 14, 2025 CRISPR / Cas9 system includes at least one guide RNA (gRNA) targeting at least one hSAGA complex protein gene. In some embodiments, composition target hSAGA complex protein gene is TADA2B, TADA3, TADA1, TADA2A, TAF5L, TAF6L, TAF9, TAF10, TAF12, KAT2A, KAT2B, SGF29, TATA1, SUPT7L, SUPT7H, SUPT20H, SUPT3H, TRRAP, USP22, ATXN7L3, ATXN7, ENY2, SF3B3, and / or SF3B5. Optionally, the hSAGA complex protein is TADA2B, TADA3, TAF5L, and / or TADA1. In some embodiments, the hSAGA complex protein is TADA2B, TADA3, TADA1, TADA2A, TAF5L, and / or SUPT20H. In still other embodiments, the hSAGA complex protein is TADA2B, TADA1, TAF5L, or SUPT20H. In some embodiments, the composition may be used for the treatment of a hemoglobinopathy in a subject. In some embodiments, the disclosure provides for a population of cells modified using the composition of the disclosure. Definitions Unless specifically stated or obvious from context, as used herein, the term "about" is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. "About" can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. In certain embodiments, the term "approximately" or "about" refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). Unless otherwise clear from context, all numerical values provided herein are modified by the term "about." Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms "a," "an," and "the" are understood to be singular or plural. Ranges can be expressed herein as from "about" one particular value, and / or to "about" another particular value. When such a range is expressed, another aspect includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," 5 122236082.1 VIA Patent Center 808955.000150 (OTT: 2024-511) Date of Deposit: May 14, 2025 it is understood that the particular value forms another aspect. It is further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as "about" that particular value in addition to the value itself. It is also understood that throughout the application, data are provided in a number of different formats and that this data represent endpoints and starting points and ranges for any combination of the data points. For example, if a particular data point "10" and a particular data point "15" are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed. Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 as well as all intervening decimal values between the aforementioned integers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to sub-ranges, "nested sub-ranges" that extend from either end point of the range are specifically contemplated. For example, a nested sub-range of an exemplary range of 1 to 50 may comprise 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction. The transitional term "comprising," which is synonymous with "including," "containing," or "characterized by," is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. By contrast, the transitional phrase "consisting of" excludes any element, step, or ingredient not specified in the claim. The transitional phrase "consisting essentially of" limits the scope of a claim to the specified materials or steps "and those that do not materially affect the basic and novel characteristic(s)" of the claimed invention. The embodiments set forth below and recited in the claims can be understood in view of the above definitions. Other features and advantages of the disclosure will be apparent from the following description of the preferred embodiments thereof, and from the claims. Unless otherwise defined, 6 122236082.1 VIA Patent Center 808955.000150 (OTT: 2024-511) Date of Deposit: May 14, 2025 all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. All published foreign patents and patent applications cited herein are incorporated herein by reference. All other published references, documents, manuscripts and scientific literature cited herein are incorporated herein by reference. In the case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. BRIEF DESCRIPTION OF THE DRAWINGS The following detailed description, given by way of example, but not intended to limit the disclosure solely to the specific embodiments described, may best be understood in conjunction with the accompanying drawings, in which: FIG. 1 shows an illustration of the human SAGA (hSAGA) complex. The functional modules of SAGA, such as the HAT module, the deubiquitinating (DUB) module, the core, the splicing module (SM), and the activator-binding module (AM) are indicated. FIG. 2 shows an illustration of the human ATAC complex with its four-subunit HAT module. FIG. 3 shows flow quantification data from intracellular gamma-globin staining in erythrocytes from two donors. A significant increase in percentage of erythrocytes that expressed fetal hemoglobin (% F-cells) was seen for three independent gRNAs (TADA2Bg1, TADA2Bg3, and TADA2Bg10), as compared to non-transformed cells (NT). FIG. 4 shows that a significant increase in gamma-globin mRNA (HBG1 / 2) and a concomitant reduction in beta-globin mRNA (HBβ) levels was observed in primary human erythroid cells for donor 1 treated with CRISPR / Cas9 and TADA2B-targeting guide RNA, measured using qPCR. FIG.5 shows a significant increase in gamma-globin mRNA (HBG1 / 2) and a concomitant reduction in beta-globin mRNA (HBβ) levels in primary human erythroid cells for donor 2 treated with CRISPR / Cas9 and TADA2B-targeting guide RNA, measured using qPCR. 7 122236082.1 VIA Patent Center 808955.000150 (OTT: 2024-511) Date of Deposit: May 14, 2025 FIG.6 shows an increase in fetal hemoglobin (HbF) in erythroid cells from donor 1 with CRISPR / Cas9 induced TADA2B deletions (TADA2Bg1, TADA2Bg3, and TADA2Bg10) as compared to non-transformed cells (NT), measured using HPLC. FIG.7 shows an increase in fetal hemoglobin (HbF) in erythroid cells from donor 2 with CRISPR / Cas9 induced TADA2B deletions (TADA2Bg1, TADA2Bg3, and TADA2Bg10) as compared to non-transformed cells (NT), measured using HPLC. FIG.8 shows absolute cell counts throughout differentiation by cell count analysis. A cell growth defect was not observed in cells with the TADA2B deletion. FIG. 9 shows absence of negative selection (percent indels) of edited cells compared to non-edited cells. FIG.10 shows flow cytometry analysis for CD49d and CD233 / BAND3 for donor 1. No appreciable differentiation defect of cells with TADA2B deletion was found. FIG.11 shows flow cytometry analysis for CD49d and CD233 / BAND3 for donor 2. No appreciable differentiation defect of cells with TADA2B deletion was found. FIG.12 shows an increase in F-cell formation of edited cells, as compared to non-edited cells, measured using fetal hemoglobin allophycocyanin conjugates (HbF-APC) along a forward scatter area (FSC-A) via flow cytometry. FIG.13 shows % fetal peak area data for the hSAGA complex, measured by HPLC. HPLC- based protein measurements of CRISPR-Cas9 edited CD34+ cells on day 14 of erythroid differentiation were performed. Results for NTC (Non targeting control), +ve control (positive control), TADA1, TADA2A, TADA2B and TADA3 are shown. FIG.14 shows % fetal peak area data for the hSAGA complex, measured by HPLC. HPLC- based protein measurements of CRISPR-Cas9 edited CD34+ cells on day 14 of erythroid differentiation were performed. Results for NTC (Non targeting control), +ve control (BCL11A), TAF5L, and SUPT20H are shown. FIG.15 shows a cryo-EM structure of the TADA2B binding site. FIG.16 shows compound 1, a candidate HAT inhibitor. FIG.17 shows compound 2, a candidate HAT inhibitor. FIG.18 shows compound 3, a candidate HAT inhibitor. FIG.19 shows a common pharmacophoric model. 8 122236082.1 VIA Patent Center 808955.000150 (OTT: 2024-511) Date of Deposit: May 14, 2025 DETAILED DESCRIPTION OF THE INVENTION The present disclosure relates, at least in part, to targets, compositions and methods newly identified as capable of increasing fetal hemoglobin (HbF) in erythroid cells, e.g., by increasing expression of hemoglobin γ (HBγ). Such increases in HbF can be achieved through upregulation of hemoglobin γ mRNA levels (e.g., HBG1 or HBG2) and / or upregulation of fetal hemoglobin protein (HBγ) levels, which result in an elevation in HbF. The targets, compositions and / or methods of the instant disclosure can be used alone or in combination with another agent that upregulates HbF, or that targets symptoms of SCD and / or β-thalassemia, including but not limited to vaso-occlusion and anemia. Abbreviations As used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural references unless the content clearly dictates otherwise. As used in this specification, the term "and / or" is used in this disclosure to either "and" or "or" unless indicated otherwise. Throughout this specification, unless the context requires otherwise, the word "comprise," or variations such as "comprises" or "comprising," will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers. As used in this application, the terms "about" and "approximately" are used as equivalents. Any numerals used in this application with or without about / approximately are meant to cover any normal fluctuations appreciated by one of ordinary skill in the relevant art. In certain embodiments, the term "approximately" or "about" refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). "Administration" refers herein to introducing an agent or composition into a subject or contacting an agent or composition with a cell and / or tissue. 9 122236082.1 VIA Patent Center 808955.000150 (OTT: 2024-511) Date of Deposit: May 14, 2025 Methods and Compositions In one aspect, the present disclosure provides methods for increasing the amount of fetal hemoglobin (HbF) in a cell. In particular embodiments, the method comprises increasing expression of one or more components of HbF in a cell. In particular embodiments, the component of HbF is a hemoglobin γ (HBγ), e.g., human hemoglobin subunit gamma-1 (HBGl) or human hemoglobin subunit gamma-2 (HBG2). In particular embodiments, the component of fetal hemoglobin is a hemoglobin α (HBα), e.g., human hemoglobin subunit alpha-1 (HBA1) or human hemoglobin subunit alpha-2 (HBA2). In certain embodiments, expression of both HBγ and HBα is increaseed. In certain embodiments, the fetal hemoglobin comprises a human hemoglobin subunit gamma-1 (HBGl) having the protein sequence set forth in NCBI Reference Sequence: NP_000550.2 and shown below: MGHFTEEDKATITSLWGKVNVEDAGGETLGRLLVVYPWTQRFFDSFGNLSSASA IMGNPKVKAHGKKVLTSLGDATKHLDDLKGTFAQLSELHCDKLHVDPENFKLLGNVLV TVLAIHFGKEFTPEVQASWQKMVTAVASALSSRYH (SEQ ID NO: 13). (WO 2020 / 106876) In certain embodiments, the HBGl protein is encoded by the polynucleotide sequence set forth in NCBI Reference Sequence: NM_000559.2 and shown below: acactcgctt ctggaacgtc tgaggttatc aataagctcc tagtccagac gccatgggtc atttcacaga ggaggacaag gctactatca caagcctgtg gggcaaggtg aatgtggaag atgctggagg agaaaccctg ggaaggctcc tggttgtcta cccatggacc cagaggttct ttgacagctt tggcaacctg tcctctgcct ctgccatcat gggcaacccc aaagtcaagg cacatggcaa gaaggtgctg acttccttgg gagatgccac aaagcacctg gatgatctca agggcacctt tgcccagctg agtgaactgc actgtgacaa gctgcatgtg gatcctgaga acttcaagct cctgggaaat gtgctggtga ccgttttggc aatccatttc ggcaaagaat tcacccctga ggtgcaggct tcctggcaga agatggtgac tgcagtggcc agtgccctgt cctccagata ccactgagct cactgcccat gattcagagc tttcaaggat aggctttatt ctgcaagcaa tacaaataat aaatctattc tgctgagaga tcac (SEQ ID NO: 14) In certain embodiments, the fetal hemoglobin comprises a human hemoglobin subunit gamma-2 (HBG2) having the protein sequence set forth in NCBI Reference Sequence: NP_000175.1 and shown below: 10 122236082.1 VIA Patent Center 808955.000150 (OTT: 2024-511) Date of Deposit: May 14, 2025 MGHFTEEDKATITSLWGKVNVEDAGGETLGRLLVVYPWTQRFFDSFGNLSSASA IMGNPKVKAHGKKVLTSLGDAIKHLDDLKGTFAQLSELHCDKLHVDPENFKLLGNVLV TVLAIHFGKEFTPEVQASWQKMVTGVASALSSRYH (SEQ ID NO: 15). In certain embodiments, the HBG2 protein is encoded by the polynucleotide sequence set forth in NCBI Reference Sequence: NM_000184.2, NCBI Reference Sequence: NM_000184.3, or shown below: acactcgctt ctggaacgtc tgaggttatc aataagctcc tagtccagac gccatgggtc atttcacaga ggaggacaag gctactatca caagcctgtg gggcaaggtg aatgtggaag atgctggagg agaaaccctg ggaaggctcc tggttgtcta cccatggacc cagaggttct ttgacagctt tggcaacctg tcctctgcct ctgccatcat gggcaacccc aaagtcaagg cacatggcaa gaaggtgctg acttccttgg gagatgccat aaagcacctg gatgatctca agggcacctt tgcccagctg agtgaactgc actgtgacaa gctgcatgtg gatcctgaga acttcaagct cctgggaaat gtgctggtga ccgttttggc aatccatttc ggcaaagaat tcacccctga ggtgcaggct tcctggcaga agatggtgac tggagtggcc agtgccctgt cctccagata ccactgagct cactgcccat gatgcagagc tttcaaggat aggctttatt ctgcaagcaa tcaaataata aatctattct gctaagagat cac (SEQ ID NO: 16) acactcgctt ctggaacgtc tgaggttatc aataagctcc tagtccagac gccatgggtc atttcacaga ggaggacaag gctactatca caagcctgtg gggcaaggtg aatgtggaag atgctggagg agaaaccctg ggaaggctcc tggttgtcta cccatggacc cagaggttct ttgacagctt tggcaacctg tcctctgcct ctgccatcat gggcaacccc aaagtcaagg cacatggcaa gaaggtgctg acttccttgg gagatgccat aaagcacctg gatgatctca agggcacctt tgcccagctg agtgaactgc actgtgacaa gctgcatgtg gatcctgaga acttcaagct cctgggaaat gtgctggtga ccgttttggc aatccatttc ggcaaagaat tcacccctga ggtgcaggct tcctggcaga agatggtgac tggagtggcc agtgccctgt cctccagata ccactgagct cactgcccat gatgcagagc tttcaaggat aggctttatt ctgcaagcaa tcaaataata aatctattct gctaagagat cacaca (SEQ ID NO: 17) In certain embodiments, the fetal hemoglobin comprises a human hemoglobin subunit alpha-1 (HBA1) having the protein sequence set forth in NCBI Reference Sequence: NP_000549.1 and shown below: 11 122236082.1 VIA Patent Center 808955.000150 (OTT: 2024-511) Date of Deposit: May 14, 2025 mvlspadktn vkaawgkvga hageygaeal ermflsfptt ktyfphfdls hgsaqvkghg kkvadaltna vahvddmpna lsalsdlhah klrvdpvnfk llshcllvtl aahlpaeftp avhasldkfl asvstvltsk yr (SEQ ID NO: 18) In certain embodiments, the HBA1 protein is encoded by the polynucleotide sequence set forth in NCBI Reference Sequence: NM_000558.4, NCBI Reference Sequence: NM_000558.5, or shown below: cataaaccct ggcgcgctcg cggcccggca ctcttctggt ccccacagac tcagagagaa cccaccatgg tgctgtctcc tgccgacaag accaacgtca aggccgcctg gggtaaggtc ggcgcgcacg ctggcgagta tggtgcggag gccctggaga ggatgttcct gtccttcccc accaccaaga cctacttccc gcacttcgac ctgagccacg gctctgccca ggttaagggc cacggcaaga aggtggccga cgcgctgacc aacgccgtgg cgcacgtgga cgacatgccc aacgcgctgt ccgccctgag cgacctgcac gcgcacaagc ttcgggtgga cccggtcaac ttcaagctcc taagccactg cctgctggtg accctggccg cccacctccc cgccgagttc acccctgcgg tgcacgcctc cctggacaag ttcctggctt ctgtgagcac cgtgctgacc tccaaatacc gttaagctgg agcctcggtg gccatgcttc ttgccccttg ggcctccccc cagcccctcc tccccttcct gcacccgtac ccccgtggtc tttgaataaa gtctgagtgg gcggcaaaaa aaaaaaaaaa aaaaaaa (SEQ ID NO: 19) actcttctgg tccccacaga ctcagagaga acccaccatg gtgctgtctc ctgccgacaa gaccaacgtc aaggccgcct ggggtaaggt cggcgcgcac gctggcgagt atggtgcgga ggccctggag aggatgttcc tgtccttccc caccaccaag acctacttcc cgcacttcga cctgagccac ggctctgccc aggttaaggg ccacggcaag aaggtggccg acgcgctgac caacgccgtg gcgcacgtgg acgacatgcc caacgcgctg tccgccctga gcgacctgca cgcgcacaag cttcgggtgg acccggtcaa cttcaagctc ctaagccact gcctgctggt gaccctggcc gcccacctcc ccgccgagtt cacccctgcg gtgcacgcct ccctggacaa gttcctggct tctgtgagca ccgtgctgac ctccaaatac cgttaagctg gagcctcggt ggccatgctt cttgcccctt gggcctcccc ccagcccctc ctccccttcc tgcacccgta cccccgtggt ctttgaataa agtctgagtg ggcggca (SEQ ID NO: 20) 12 122236082.1 VIA Patent Center 808955.000150 (OTT: 2024-511) Date of Deposit: May 14, 2025 In certain embodiments, the fetal hemoglobin comprises a human hemoglobin subunit alpha-2 (HBA2) having the protein sequence set forth in NCBI Reference Sequence: NP_000508.1 and shown below: mvlspadktn vkaawgkvga hageygaeal ermflsfptt ktyfphfdls hgsaqvkghg kkvadaltna vahvddmpna lsalsdlhah klrvdpvnfk llshcllvtl aahlpaeftp avhasldkfl asvstvltsk yr (SEQ ID NO: 21) In certain embodiments, the HBA2 protein is encoded by the polynucleotide sequences set forth in NCBI Reference Sequence: NM_000517.4, NCBI Reference Sequence: NM_000517.6, or shown below: cataaaccct ggcgcgctcg cgggccggca ctcttctggt ccccacagac tcagagagaa cccaccatgg tgctgtctcc tgccgacaag accaacgtca aggccgcctg gggtaaggtc ggcgcgcacg ctggcgagta tggtgcggag gccctggaga ggatgttcct gtccttcccc accaccaaga cctacttccc gcacttcgac ctgagccacg gctctgccca ggttaagggc cacggcaaga aggtggccga cgcgctgacc aacgccgtgg cgcacgtgga cgacatgccc aacgcgctgt ccgccctgag cgacctgcac gcgcacaagc ttcgggtgga cccggtcaac ttcaagctcc taagccactg cctgctggtg accctggccg cccacctccc cgccgagttc acccctgcgg tgcacgcctc cctggacaag ttcctggctt ctgtgagcac cgtgctgacc tccaaatacc gttaagctgg agcctcggta gccgttcctc ctgcccgctg ggcctcccaa cgggccctcc tcccctcctt gcaccggccc ttcctggtct ttgaataaag tctgagtggg cagcaaaaaa aaaaaaaaaa aa (SEQ ID NO: 22) actcttctgg tccccacaga ctcagagaga acccaccatg gtgctgtctc ctgccgacaa gaccaacgtc aaggccgcct ggggtaaggt cggcgcgcac gctggcgagt atggtgcgga ggccctggag aggatgttcc tgtccttccc caccaccaag acctacttcc cgcacttcga cctgagccac ggctctgccc aggttaaggg ccacggcaag aaggtggccg acgcgctgac caacgccgtg gcgcacgtgg acgacatgcc caacgcgctg tccgccctga gcgacctgca cgcgcacaag cttcgggtgg acccggtcaa cttcaagctc ctaagccact gcctgctggt gaccctggcc gcccacctcc ccgccgagtt cacccctgcg gtgcacgcct ccctggacaa gttcctggct tctgtgagca ccgtgctgac ctccaaatac cgttaagctg gagcctcggt agccgttcct cctgcccgct gggcctccca acgggccctc ctcccctcct tgcaccggcc 13 122236082.1 VIA Patent Center 808955.000150 (OTT: 2024-511) Date of Deposit: May 14, 2025 cttcctggtc tttgaataaa gtctgagtgg gcagca (SEQ ID NO: 23) In certain embodiments, the fetal hemoglobin comprises two HBG1 and / or HBG2 proteins and two HBA1 and / or HBA2 proteins. The methods disclosed herein may be practiced in vitro or in vivo. The methods disclosed herein comprise contacting a cell with an inhibitor of a target gene, mRNA or protein (which may collectively be referred to as a "target") disclosed herein, wherein inhibition of the target results in an increased amount of fetal hemoglobin in the cell, e.g., an erythroid or red blood cell. In particular embodiments, inhibition of the target results in an increased amount of HBG 1 or HBG2 in the cell. In particular embodiments, an amount of the inhibitor effective to result in increased levels of Hbγ and / or HbF is used. In particular embodiments, the methods comprise contacting a tissue, organ or organism, e.g., a mammal, with the inhibitor. In certain embodiments, one or more inhibitors, each targeting the same or different targets, may be used. In certain embodiments, the targeted complex is human SAGA, sometimes described as STAGA, a chromatin-modifying complex that can deposit and remove post-translational modifications (PTMs) of histones, such as acetylation, in a dynamic manner. hSAGA contains 20 subunits, which are organized in functional modules, such as HAT, histone H2Bub1 deubiquitinase (DUB), activator binding (AM), splicing (SM), and core modules (FIG.1). hSAGA complex is a regulatory hub involved in gene regulation, chromatin modification, DNA damage repair and signaling. In one aspect, the present disclosure provides methods for increasing the amount of fetal hemoglobin (HbF) in a cell by inhibiting or modulating the hSAGA complex. In certain embodiments, the hSAGA complex comprises KAT2A / 2B, TADA2B, TADA3, SGF29, ATXN7L3, ENY2, USP22, ATXN7 / L1 / L2, SF3B5, SF3B3, TAF6L, TAF9 / 9B, TAF5L, SUPT7L, TAF10, SUPT3H, TADA1, TAF12, SUPT20H, and / or TRRAP. In certain embodiments, the targeted complex is human ATAC, a chromatin-modifying complex that can deposit and remove post-translational modifications (PTMs) of histones, such as acetylation, in a dynamic manner. ATAC contains 10 subunits, which includes a HAT module (FIG.2). The ATAC complex modifies Histone H3 and perhaps H4. 14 122236082.1 VIA Patent Center 808955.000150 (OTT: 2024-511) Date of Deposit: May 14, 2025 In one aspect, the present disclosure provides methods for increasing the amount of fetal hemoglobin (HbF) in a cell by inhibiting or modulating the ATAC complex. In certain embodiments the ATAC complex comprises SG29, TADA3, TADA2A, KAT2A / 2B, ZZZ3, YEATS2, NC2β, CSRP2BP, MBIP, and / or WDR5. In certain embodiments, the target gene, mRNA, or protein is KAT2A / GCN5 or Lysine Acetyltransferase 2A. KAT2A, or GCN5, is a histone acetyltransferase (HAT) that functions primarily as a transcriptional activator. It also functions as a repressor of NF-kappa-B by promoting ubiquitination of the NF-kappa-B subunit RELA in a HAT-independent manner In certain embodiments, the target gene, mRNA, or protein is KAT2B / PCAF or Lysine Acetyltransferase 2B or P300 / CBP-Associated Factor. CBP and p300 are large nuclear proteins that bind to many sequence-specific factors involved in cell growth and / or differentiation, including c-jun and the adenoviral oncoprotein E1A. The protein encoded by this gene associates with p300 / CBP. It has in vitro and in vivo binding activity with CBP and p300, and competes with E1A for binding sites in p300 / CBP. It has histone acetyl transferase activity with core histones and nucleosome core particles, indicating that this protein plays a direct role in transcriptional regulation. In certain embodiments, the target gene, mRNA, or protein is TADA2B or Transcriptional Adaptor 2B. TADA2B functions as a transcriptional adaptor protein that potentiates transcription through coordination of histone acetyltransferase (HAT) activity and by linking activation factors to basal transcriptional machinery. In certain embodiments, the target gene, mRNA, or protein is TADA3 or Transcriptional Adaptor 3. DNA-binding transcriptional activator proteins increase the rate of transcription by interacting with the transcriptional machinery bound to the basal promoter in conjunction with adaptor proteins, possibly by acetylation and destabilization of nucleosomes. The protein encoded by this gene is a transcriptional activator adaptor and a component of the histone acetyl transferase (HAT) coactivator complex which plays a crucial role in chromatin modulation and cell cycle progression. Along with the other components of the complex, this protein links transcriptional activators bound to specific promoters, to histone acetylation and the transcriptional machinery. In certain embodiments, the target gene, mRNA, or protein is TADA1 or Transcriptional Adaptor 1. TADA1L is a protein subunit of the human STAGA acetyltransferase complex, which is a chromatin-modifying multiprotein complex. 15 122236082.1 VIA Patent Center 808955.000150 (OTT: 2024-511) Date of Deposit: May 14, 2025 In certain embodiments, the target gene, mRNA, or protein is TADA2B or Transcriptional Adapter 2B. TADA2B is a protein subunit of the human STAGA acetyltransferase complex, which is a chromatin-modifying multiprotein complex. TADA2B functions as a transcriptional adaptor protein that potentiates transcription through coordination of histone acetyltransferase (HAT) activity and by linking activation factors to basal transcriptional machinery In certain embodiments, the target gene, mRNA, or protein is TAF5L or TATA-Box Binding Protein Associated Factor 5 Like. TAF5L is a protein subunit of the human STAGA acetyltransferase complex, which is a chromatin-modifying multiprotein complex, and is also a protein component of the human PCAF histone acetylase complex. The PCAF histone acetylase complex, which is composed of more than 20 polypeptides some of which are TAFs, is required for myogenic transcription and differentiation. In certain embodiments, the target gene, mRNA, or protein is SUPT20H or Suppressor of Ty 20 Homolog. SUPT20H is a protein subunit of the human STAGA acetyltransferase complex, which is a chromatin-modifying multiprotein complex. In certain embodiments, the disclosure provides methods of validating SAGA / ATAC components. The method may comprise HT validation using qPCR methods and RNA as the readout. Methods using qPCR have the advantage of being able to probe multiple components at once. However, such methods employing qPCR methods may be time consuming. The method may alternatively comprise individual validation using HPLC methods and proteins as the readout. Methods employing HPLC may be more reliable and less time consuming. In one embodiment, the present disclosure provides a method for increasing expression of a fetal hemoglobin (HbF) in a cell, comprising contacting a cell with an inhibitor of a target protein or protein complex that functions to regulate HbF expression. In some embodiments, the HbF comprises hemoglobin gamma and hemoglobin alpha. In some embodiments, the hemoglobin gamma comprises hemoglobin gamma G1 (HBG1) and / or or hemoglobin gamma G2 (HBG2). In particular embodiments, the target protein or protein complex exerts transcriptional control by determining what region(s) on chromatin are accessible to RNA polymerase, and can hence be transcribed to messenger RNA and subsequently to proteins such as hemoglobin γ (HBγ). Chromatin refers to the tightly compacted mixture of DNA and proteins, called histones, that form the chromosomes found in the cells of humans and other higher organisms. DNA is tightly wrapped around histone proteins, the architecture of which is further modulated by post-translational 16 122236082.1 VIA Patent Center 808955.000150 (OTT: 2024-511) Date of Deposit: May 14, 2025 modifications such as methylation or acetylation of the histone, actions which can effectively "silence" or repress the ability of the gene to undergo transcription to mRNA. Chromatin- modifying complexes exist that can deposit and remove these post-translational modifications in a dynamic manner, thereby controlling transcription. In one embodiment, the chromatin regulatory complexes are the transcriptional co-activator complexes ATAC (ADA-two-A-containing) and SAGA (Spt-Ada-GCN5-acetyltransferase), which can acetylate histones at distinct residues (Yayli, 2023). Acetylation is performed by Histone Acetyl Transferase (HAT) functions residing within the ATAC and SAGA complexes, that acetylate histone lysine residues. SAGA regulates gene expression by interacting with enhancer-bound activators, recruiting the transcriptional machinery and modifying promoter-proximal chromatin. Human SAGA is a 20-subunit, 1.4-Mda complex with five functional modules, a Histone Acetyl Transferse (HAT) module, a Splicing Module (SM), a Deubiquitinase Module (DUB), an Activator-binding Module (AM) and a Core Structural Module (FIG. 1) (Herbst, 2021, Yayli, 2023). The metazoan ATAC co-activator complex contains 10 well characterized subunits, out of which four subunits form the histone acetyltransferase (HAT) module. HAT domains more specifically acetylate lysine residues, thereby having lysine acetyltransferase (KAT) functions. The HAT module of human (h) ATAC contains the HAT enzyme KAT2A (also called GCN5) or KAT2B (also called PCAF) and the structural subunits SGF29, TADA3, and TADA2A. The six additional subunits of ATAC are YEATS2 and NC2β (also called DR1), which form a histone fold (HF) pair; ZZZ3; CSRP2BP (also called CSR2B, ATAC2, or KAT14); WDR5 (a WD40 repeat-containing protein); and MBIP (FIG.2). In mammals, three subunits of the ATAC HAT module, KAT2A / KAT2B, TADA3, and SGF29, are shared with the SAGA HAT module. The fourth and distinctive subunit of these related HAT modules is either TADA2A for the ATAC-specific HAT module or TADA2B for the SAGA- specific HAT subunit. Vertebrate ATAC and SAGA complexes harbor either KAT2A or KAT2B, which are mutually exclusive in their respective HAT modules. Composed of SF3B3 and SF3B5. Eukaryotic SAGA complexes preferentially acetylate histone H3 at lysine 9 and lysine 14 (H3K9 and H3K14) in the nucleus. In contrast, substrate specificities of the metazoan ATAC complexes are less well understood, but it has been suggested that ATAC acetylates both histone H3 and H4. In further embodiments, these transcriptional coactivator complexes are modulated to selectively derepress the expression of specific genes, most preferably the expression of gamma globin. Both the SAGA and ATAC complexes contain multiple enzymatic activities that could 17 122236082.1 VIA Patent Center 808955.000150 (OTT: 2024-511) Date of Deposit: May 14, 2025 provide unique targets for potential therapy development, including the HAT and DUB modules in SAGA and the two KATs in ATAC (Mustachio, 2020). The acetyltransferase domains of the KATs, the reader domains in GCN5 (bromodomain) and YEATS2 (the YEATS domain) and ubiquitin specific protease domain of USP22 all provide potential targets for inhibitor development. In further embodiments, inhibitor design is not limited to domains with enzymatic activity. For example, non-enzymatic domains within the SAGA and ATAC complexes can be targeted empirically via high-throughput screening, or by design, or by virtual screening, or by repression of gene expression via antisense oligonucleotides (ASOs), small inhibitory RNAs (siRNAs), or short hairpin RNAs (shRNAs) and the like, or other techniques available to those skilled in the art. For example, three-dimensional models based on available X-ray crystal structures, or homology models based on 3D structures of related proteins or domains, or virtual models created using the AlphaFold algorithm (Google DeepMind and Isomorphic Labs) followed by "hot spot" analyses (Clackson, 1995) can indicate regions or a cluster of residues on a protein that have a high propensity for ligand binding. Virtual screening is performed using, for example, molecular docking software whereby a virtual library of chemical compounds is interrogated to predict how well each compound binds to the target. The chemical library can be derived from various sources, such as commercial databases, compound libraries, or virtual chemical space generated through computational methods. Virtual docking is performed iteratively, ranking virtual "hits" by docking score, and selectively enriching favorable features in subsequent rounds of screening. Filters can be applied to prioritize compounds with desirable drug-like properties, absence of toxicophores, and other factors. Once promising compounds are identified, selective synthesis and biological profiling in assays assessing potency, selectivity, metabolic properties and other characteristics are performed. A combination of virtual and "wet" chemistry affords lead structures for further optimization by those skilled in the art of medicinal chemistry. These techniques can be used to identify novel ligands that modulate transcriptional adaptors such as TADA1, TADA2A, TADA2B, or TADA3. In further embodiments, inhibitor design can be performed by scaffold evolution using shape or similarity screening. This involves comparing the three-dimensional shapes and chemical features of a reference compound (or compounds) against a database of compounds to identify those with similar structural and / or pharmacophoric features. Specifically, a reference compound 18 122236082.1 VIA Patent Center 808955.000150 (OTT: 2024-511) Date of Deposit: May 14, 2025 is converted into a 3D molecular representation in formats like .mol2 or .sdf, critical features required for binding can be defined, and then shape-based similarity methods (e.g., shape overlays, Gaussian volume overlap, or 3D similarity fingerprints) are used to compare the shape and spatial arrangement of pharmacophoric features between the reference compound and compounds in the database. This can be used to 'evolve' inhibitors of unrelated Histone Acetyl Transferases into new compounds with specifies for KAT2A / GCN5 or KAT2B / PCAF, or to evolve known bromodomain inhibitors into new compounds with specificity these targets. In some embodiments, the disclosure provides methods of reducing or eliminating expression of a gene, regulatory activity of a gene, or activity of an encoded protein or enzyme. The methods of reducing or eliminating expression of a gene, regulatory activity of a gene, or activity of an encoded protein or enzyme may comprise partially inactivating, fully inactivating, or deleting a gene or enzyme. The methods of reducing or eliminating expression of a gene, regulatory activity of a gene, or activity of an encoded protein or enzyme may comprise a knockout (KO) mutation that fully eliminates the expression or activity of a gene, protein, or enzyme. The methods of reducing or eliminating expression of a gene, regulatory activity of a gene, or activity of an encoded protein or enzyme may also comprise a knock-down that reduces, but does not entirely eliminate, the expression or activity of a gene, protein, or enzyme. The methods of reducing or eliminating expression of a gene, regulatory activity of a gene, or activity of an encoded protein or enzyme may comprise anything that reduces, prevents, or blocks the biosynthesis of a product produced by an enzyme. The methods of reducing or eliminating expression of a gene, regulatory activity of a gene, or activity of an encoded protein or enzyme may include, for example, a mutation in a gene encoding a protein or enzyme, a mutation in a genetic regulatory element involved in the expression of a gene encoding an enzyme, the introduction of a nucleic acid which produces a protein that reduces or inhibits the activity of an enzyme, or the introduction of a nucleic acid (e.g., antisense NAs, RNAi, TALEN, siRNA, CRISPR, or CRISPRi, noting that implementation of each of the preceding nucleic acid modalities / therapeutics is well-known in the art) or protein which inhibits the expression of a protein or enzyme. The disruption may be introduced using any method known in the art. For the purposes of the present disclosure, methods of reducing or eliminating expression of a gene, regulatory activity of a gene, or activity of an encoded protein or enzyme are laboratory-generated, not naturally occurring. 19 122236082.1 VIA Patent Center 808955.000150 (OTT: 2024-511) Date of Deposit: May 14, 2025 As used herein, the methods of reducing or eliminating expression of a gene, regulatory activity of a gene, or activity of an encoded protein or enzyme comprises the use of any agent that suppresses or inhibits expression of a target gene through transcription and translation. Any type of agent that specifically inhibits expression of a target gene may be included, and both inhibitors based on transcription regulation and post-transcriptional translation regulation, such as RNAi, are included. Examples of the agent may include but are not limited to an antisense nucleotide, siRNA (small interfering RNA), shRNA (small hairpin RNA), ribozyme, aptamer, anti-microRNA, microRNA mimic, nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), CRISPR-Cas9 systems (e.g., gRNA, sgRNA), polycistronic tRNA- gRNA systems, and self-ribozyme-flanked RNAs (Gao & Zhao 2014, Xie et al.2015, Zetsche et al. 2017), but are not limited thereto. In the present disclosure, the target gene is a member of a SAGA (Spt-Ada-Gcn5-Acetyltransferase) complex and / or a SAGA-like complex (e.g., PCAF complex, TFTC [TATA-binding-protein-free TAFII-containing complex], and / or STAGA [SPT3- TAFII31-GCN5L acetylase]). In other embodiments, the disease is a hemoglobinopathy, such as sickle cell disease, sickle cell trait, hemoglobin C disease, hemoglobin C trait, hemoglobin S / C disease, hemoglobin D disease, hemoglobin E disease, a thalassemia, a condition associated with hemoglobin with increased oxygen affinity, a condition associated with hemoglobin with decreased oxygen affinity, unstable hemoglobin disease, and / or methemoglobinemia. The methods of the disclosure have utility in the treatment of a subject having a hemoglobinopathy disease, such as sickle cell anemia or β-thalassemia. The methods of the disclosure, include, in some embodiments, a CRISPR / CasX:gRNA system. Embodiments of the CasX:gRNA systems employed in this disclosure, their functions, and their use in the editing of target nucleic acids in cells are described more fully elsewhere herein, with CRISPR / CasX methods more generally being widely known in the art. In some embodiments, the disclosure provides methods of modifying a STAGA complex protein gene in a cell, the method comprising introducing into the cell a CRISPR system. In some embodiments of the methods, the cells to be modified are autologous with respect to a subject to be administered said cell(s). In other embodiments, the cells to be modified are allogeneic with respect to a subject to be administered said cell(s). Thus, the compositions and methods described herein can be used to engineer a variety of cells in which mutations exist in the beta-globin gene 20 122236082.1 VIA Patent Center 808955.000150 (OTT: 2024-511) Date of Deposit: May 14, 2025 and are associated with disease, e.g., hemoglobinopathies, including sickle-cell disease and α- and β-thalassemias. This approach, therefore, can be used to modify cells for applications in a subject with a hemoglobinopathy-related disease such as, but not limited to, sickle-cell disease and alpha- and β-thalassemias. In another aspect, provided herein are populations of cells comprising a STAGA complex protein gene modified ex vivo by embodiments of any of the systems or methods described herein. In some embodiments, cells that have been genetically modified in this way may be administered to a subject for purposes such as gene therapy; e.g., in methods of treatment of a hemoglobinopathy-related disease, such as sickle cell disease or β-thalassemia where the administration results in an decreased expression of beta-globin and an increase of fetal hemoglobin (HbF) in the subject. In other embodiments, the disclosure provides compositions of modified cells for use as a medicament in the treatment of a hemoglobinopathy-related disease. In some embodiments, the disclosure provides methods of modifying a STAGA complex (or a human SAGA or hSAGA complex) protein gene in a population of cells by in vitro or ex vivo methods. The methods provide that the cells can be obtained from a subject using any number of techniques known to the skilled artisan; e.g., a biopsy of the marrow or by obtaining a sample of the peripheral blood. The desired cells may be separated from the remainder of the sample, washed to remove fluids and debris and, optionally, placed in an appropriate buffer or media for subsequent processing steps. The method may include one or more steps of i) introducing into the cells CasX:gRNA system components for editing of target nucleic acids; ii) introducing into the cells a nucleic acid or vector encoding CasX:gRNA system components to the cells; iii) expansion of the cells in an appropriate medium under conditions suitable for their propagation, and iv) cryopreservation of the cells for subsequent administration to the subject. Thus, the CasX:gRNA systems and methods described herein can be used to modify a variety of cells associated with the hemoglobinopathy to produce populations of cells in which the expression of a STAGA complex complex protein is reduced or eliminated and HbF is increased. This approach, therefore, could be used for methods of treatment in a subject with a hemoglobinopathy such as sickle cell anemia or β-thalassemia, amongst others. In some cases, the cells are contacted with a CasX and a gRNA wherein the gRNA is a guide RNA (gRNA). In other cases, the cells are contacted with a CasX and a gRNA wherein the gRNA is a chimera comprising DNA and RNA. 21 122236082.1 VIA Patent Center 808955.000150 (OTT: 2024-511) Date of Deposit: May 14, 2025 In one embodiment, the target nucleic acid of the cell is modified by contacting the cells with a CasX protein, a guide nucleic acid (gRNA) comprising a targeting sequence complementary to a STAGA complex complex protein nucleic acid, and a donor template wherein the donor template is inserted into or replaces a portion of the target nucleic acid sequence of the cell such that the STAGA complex complex protein is not expressed or is expressed at a reduced level. In other cases, the CasX and gRNA are delivered to the cells of the population in a vector (embodiments of which are described herein), wherein the STAGA complex protein gene is modified such that the STAGA complex protein is not expressed or is expressed at a reduced level. In another aspect, the present disclosure relates to methods of treating a hemoglobinopathy- related disease or disorder in a subject, including but not limited to sickle-cell disease or β- thalassemia in which repression or elimination of expression of the STAGA complex protein by modifying the STAGA complex protein gene in target cells of the subject ameliorates the signs, symptoms, or effects of the disease or disorder, notwithstanding that the subject may still be afflicted with the underlying disease or disorder. A number of therapeutic strategies have been used to design the compositions for use in the methods of treatment of a subject with a hemoglobinopathy-related disease. In some embodiments, the method comprises administering to the subject having a hemoglobinopathy (e.g., sickle cell anemia or β-thalassemia) a therapeutically effective dose of a CRISPR nuclease and guide RNA disclosed herein. In some embodiments, the method of treatment comprises administering to the subject a therapeutically effective dose of: i) a CasX:gRNA system comprising a first CasX protein and a first gRNA with a targeting sequence complementary to the target nucleic acid; ii) the CasX:gRNA system comprising a first CasX protein and a first gRNA with a targeting sequence complementary to the target nucleic acid and a donor template; iii) a nucleic acid encoding the CasX:gRNA system of (i) or (ii); iv) a vector comprising the nucleic acid of (iii), which can be an AAV of any of the embodiments described herein; v) a XDP comprising the CasX:gRNA system of (i) or (ii); or vi) combinations of two or more of (i)-(v), wherein 1) the STAGA complex protein gene of the cells of the subject targeted by the first gRNA is modified (e.g., knocked-down or knocked-out) by the CasX protein and, optionally, the donor template; and 2) an increase in production of fetal hemoglobin (HbF) results in the subject. In some embodiments, the method of treating further comprises administering a second gRNA or a nucleic acid encoding the second gRNA, wherein the second gRNA has a targeting sequence 22 122236082.1 VIA Patent Center 808955.000150 (OTT: 2024-511) Date of Deposit: May 14, 2025 complementary to a different or overlapping portion of the target nucleic acid sequence compared to the first gRNA. In some embodiments, the present disclosure provides a CasX:gRNA composition, a nucleic acid encoding a CasX:gRNA composition, a vector comprising the nucleic acid, or a XDP comprising an RNP of the CasX:gRNA for use as a medicament for the treatment of a hemoglobinopathy, including sickle-cell disease or β-thalassemia. In some cases, the cells targeted for modification are selected from the group consisting of hematopoietic stem cells (HSC), hematopoietic progenitor cells (HPC), CD34+ cells, mesenchymal stem cells (MSC), induced pluripotent stem cells (iPSC), common myeloid progenitor cells, proerythroblast cells, and erythroblast cells. In an embodiment, the cells are erythrocytes. In some embodiments, the erythrocytes are erythroid cells differentiated from primary human stem and progenitor cells (HSPCs). In some embodiments, the subject to be treated is selected from the group consisting of rodent, dogs, cats, horse, mouse, rat, and non-human primate. In an embodiment, the subject is a mammal. In another embodiment, the subject is a human. In some embodiments the SAGA and / or SAGA-like complex is STAGA (SPT3-TAFII31- GCN5L acetylase complex). In further embodiments, the STAGA complex protein gene is TADA2B (also known as Transcriptional adaptor 2B, Transcriptional Adapter 2-beta, ADA2-Like Protein Beta, ADA2-Beta, ADA2B, MGC21874), TADA3 (also known as Transcriptional Adaptor 3, HADA3, ADA3, TADA3L, NGG1), TADA1 (also known as Transcriptional Adaptor 1, STAF42, TADA1L, HADA1, ADA1), TADA2A (also known as Transcriptional Adaptor 2ª, TADA2L, HADA2, ADA2A, ADA2), TAF5L, TAF6L, TAF9, TAF10, TAF12, KAT2A (also known as GCN5), KAT2B (also known as PCAF), SGF29, TATA1, SUPT7L, SUPT7H, SUPT20H, SUPT3H, TRRAP, USP22, ATXN7L3, ATXN7, ENY2, SF3B3, and / or SF3B5. In still further embodiments, the STAGA complex protein gene is TADA2B, TADA3, TADA1, and / or TADA2A. In some embodiments, the STAGA complex protein gene is TADA2B. In some embodiments, the STAGA complex protein is TADA2B (also known as Transcriptional adaptor 2B, Transcriptional Adapter 2-beta, ADA2-Like Protein Beta, ADA2- Beta, ADA2B, MGC21874), TADA3 (also known as Transcriptional Adaptor 3, HADA3, ADA3, TADA3L, NGG1), TADA1 (also known as Transcriptional Adaptor 1, STAF42, TADA1L, HADA1, ADA1), TADA2A (also known as Transcriptional Adaptor 2A, TADA2L, HADA2, 23 122236082.1 VIA Patent Center 808955.000150 (OTT: 2024-511) Date of Deposit: May 14, 2025 ADA2A, ADA2), TAF5L, TAF6L, TAF9, TAF10, TAF12, KAT2A (also known as GCN5), KAT2B (also known as PCAF), SGF29, TATA1, SUPT7L, SUPT7H, SUPT20H, SUPT3H, TRRAP, USP22, ATXN7L3, ATXN7, ENY2, SF3B3, and / or SF3B5. In still further embodiments, the STAGA complex protein is TADA2B, TADA3, TADA1, and / or TADA2A. In some embodiments, the STAGA complex protein is TADA2B. EXAMPLES Example 1: Deletion of TADA2B in human stem and progenitor cells To begin, three independent CRISPR / Cas9 guide RNAs (gRNAs) were prepared to target transcriptional adaptor 2B (TADA2B). These gRNAs were named TADA2Bg1, TADA2Bg2, and TADA2Bg3. Next, using the three independent gRNAs, TADA2B was deleted using CRISPR / Cas9 in human erythroid cells differentiated from primary human stem and progenitor cells (HSPCs), from two independent donors (Donor 1 and Donor 2). TADA2B deletion resulted in a significant increase in erythroid cells that expressed fetal- hemoglobin (F-cells), as evaluated and quantified by intracellular gamma-globin staining by flow cytometry (FIG. 3). This finding was validated in HSPCs obtained from 2 independent donors (FIG.3). Example 2: Significant increases in gamma-globin mRNA and decreases in beta-globin mRNA were observed when TADA2B was targeted Next, the results of Example 1 above were validated at the transcript level. It was found that TADA2B deletion resulted in a significant increase in gamma-globin mRNA levels, with a concomitant reduction in beta-globin mRNA levels, in primary human erythroid cells from 2 donors (FIG.4 and FIG.5). Using HPLC, a profound increase in fetal hemoglobin (HbF) resulting from TADA2B deletion was demonstrated (FIG.6 and FIG.7). Example 3: No cell growth defect was observed in TADA2B-deleted cells Notably, no cell growth defect was observed in cells having TADA2B deletions, as demonstrated by cell count analysis (FIG. 8). Absence of negative selection of edited compared to non-edited cells can be seen in FIG. 9. Furthermore, no appreciable differentiation defect of 24 122236082.1 VIA Patent Center 808955.000150 (OTT: 2024-511) Date of Deposit: May 14, 2025 TADA2B-deleted cells was detected by flow cytometry analysis for CD49d and BAND3 (also known as CD233) (FIG.10 and FIG.11). Example 4: Impact of TADA3, TAF5L, and TADA1 deletions TADA2B encodes a component of the STAGA complex. Other components of the STAGA complex were also deleted, and the impact on F-cell formation of these deletions was determined. Deletion of TADA3, TAF5L, and TADA1 each also resulted in a significant increase in F-cells, as compared to cells transduced with non-targeting sgRNA (FIG.12). Example 5: CD34+ culture and CRISPR / Cas9 gene editing Human Mobilized Peripheral Blood Primary CD34+ cells (StemCell Technologies) were expanded from thaw by seeding 100,000 viable cells / mL in a culture flask containing CD34+ Phase 1 Media comprised of IMDM, 100 ng / mL hSCF, 5 ng / mL IL-3, 3 IU / mL EPO, 250 ug / mL transferrin, 2.5% normal human serum, 1% pen / strep, 10 ng / mL heparin, 10 ug / mL insulin. On day 3 post-thaw cells were supplemented by adding an additional 1X culture volume of CD34+ Phase 1 Media. On day 7 post-thaw, cells were differentiated towards the erythroid lineage by complete medium exchange into CD34+ Phase 2 Media comprised of IMDM, 100 ng / mL hSCF, 3 IU / mL EPO, 250 ug / mL transferrin, 2.5% normal human serum, 1% pen / strep, 10 ng / mL heparin, 10 ug / mL insulin. Cell treatments were started at the time of changing to differentiation media. On culture day 7, CD34+ erythroid lineage cells were electroporated with an Amaxa 384- well HT Nucleofector System (Lonza) with the P3 primary cell kit according to the manufactures protocol. sgRNA and TrueCut Cas9 Protein V2 (Thermo Fisher) were complexed in a 2:1 molar ratio for 10 minutes at RT. Cells were washed 1x with PBS, resuspended in buffer P3 +Supplement and arrayed into a XL cuvette containing the RNP complexes at a density of 600,000 cells per reaction.100 µl of cells plus RNP complexes were transferred to the Lonza electroporation plate and electroporated using program DG-137. Pre-equilibrated media was added to the electroporation plate and cells recovered in the 37-degree incubator for 10 minutes before being added to 4 mL Phase 2 media for downstream assays (FIG.13 and FIG.14). 25 122236082.1 VIA Patent Center 808955.000150 (OTT: 2024-511) Date of Deposit: May 14, 2025 Example 6: Hemoglobin HPLC Hemoglobin tetramers were analyzed using HPLC analysis following a protocol derived from Grevet et al. Science 2018. Briefly, approximately 1 million cells were lysed in 100 µL of water with 30 µL of lysate used per injection. Species were monitored with UV absorbance at 410 nm. Retention times for hemoglobin species was optimized using the hemoglobin FASC control (PerkinElmer) as isotype controls for HbF, HbA, HbS and HbC. Hemolysates were cleared by centrifugation and analyzed for identity and levels of hemoglobin variants (HbF and HbA) by cation-exchange HPLC with a weak cation-exchange column (Poly CAT A: 35 mm x 4.6 mm, Poly LC, Inc., Columbia, MD). Hemoglobin isotype peaks were eluted with a linear gradient of phase B from 0% to 80% at A410nm (Mobile Phase A: 20 mM Bis-Tris, 2 mM KCN, pH 6.95; Phase B:20 mM Bis-Tris, 2 mM KCN, 0.2 M sodium chloride, pH 6.55). The abundance of HbF and HbA was quantified by calculating the area under the curve for each species in the samples. The abundance of HbF was plotted compared to the NTC (Non-Targeting Control). CRISPR knockdown results for TADA1, TADA2A, TADA2B, and TADA3 are shown in FIG. 13, and CRISPR knockdown results for TAF5L and SUPT20H are shown in FIG.14. Example 7: Virtual Screening of TADA2B, TADA1, and TADA3 The recently published structure of 8H7G from Zhang, Y. et al. Cryo-EM structure of human SAGA transcriptional coactivator complex. Cell Discov.8, 125 (2022), and 7KTR Herbst, D. A. et al. Structure of the human SAGA coactivator complex. Nat. Struct. Mol. Biol.28, 989– 996 (2021) has enabled identification of TADA2B binding site (FIG.15). By usage of structure prediction tools and modeling against other organisms (6CW2, 6CW3) pockets for disruption of the TADA2B binding with the human SAGA complex may be identified. The site selected for virtual screening consists of a pocket formed optionally by residues, including but not limited to: H467, R468, G469, P470, G487, E488, D489, Q490, R491, E505, R507, G508, H509, T510, D511, N512, I513, K24, G27, S104, V105, R106, Q107, K108, F109, D110, H111, R112. In another site, the residues that form the pocket could be selected from the associated spliceosome U2 snRNP factors SF3B3 and SF3B5: S15, F16, A17, I18, H19, G20, N21, F22, Q28, R63, S64, L65, M66, A67, F68, R69, L70, T71, G72, D76, G118, Q119, F120, L121, A122, V123, D124, P125, K126, G127, H169, V170, V171, G172, V173, D174, F234, 26 122236082.1 VIA Patent Center 808955.000150 (OTT: 2024-511) Date of Deposit: May 14, 2025 L235, T237, P239, G240, V288, C289, S290, A291, T292, K294, A336, A337, A338, M339, C340, V341, K343, E352, M44, L50, K61, A62, R65. The method of screening consists of A) Receiving the protein structure in a digital format. B) preparation of the protein structure using the recommended procedure by the software provider of either Schrödinger Glide, Chemical Computing Group MOE, or Biopharmics Surflex Suite. C) virtually screening a digital library of small molecule structures consisting of approximately 7,457,000 compounds (which can be obtained in 4-6 weeks of ordering from suppliers such as ChemSpace, WuXi, or Enamine) against the site and predicting the binding affinity of each molecule. D) The library is rank-ordered by predicted binding affinity. E) A subset of molecules is selected for optimal binding based on favorable interactions with the binding site and F) final filtering for diversity, predicted ADME properties, and PAINS alerts. The resulting list of compounds is purchased and procured from suppliers and delivered for in-house testing. In a similar manner, the binding site for TADA1 is targeted by using a similar technique on residues (but not limited to) L138, C139, S140, H141, T142, M144, L145, P146, T147, R148, G149, Q150, L151, E152, G153, R154, M155, I156, V157, T158, A159, Y160, E161, H162, V167, V172, V175, V176, V179, L85, Q86, I87, A88, D89, D90, F91, I92, E93, S94, V96, T97, C100, E112, R527, D537, S538, R539, N541, R542 In a similar manner, the binding site for TADA3 is targeted by selecting residues near the unassigned density identified by Zhang et al, which consisted of residues proximal to the Yeast Ada3. To conduct a ligand based virtual screen (LBVS) and identify compounds that could be potential HAT inhibitors, a computer-implemented method is used to identify pharmacophoric features relating to possible binding of compounds 1, 2, and 3 (FIG. 16, FIG. 17, FIG. 18, respectively). The compounds were used either in isolation or in combination to identify common pharmacophoric elements and shared electrostatic shape. The method consisted of: A) Receiving the target ligands in a digital format. B) Identifying the 3D shape of the ligands and their projected pharmacophore features and electrostatic surface. C) Generating a model consisting of a subset of the features. D) Obtaining and preparing a digital library of small molecules. E) Ranking the library for fit against the target ligand(s), and F) Selecting which to include (but not limited to) filtering for PAINS, ADME and diversity properties. An example of an image comprising a common pharmacophoric model is shown in FIG.19. 27 122236082.1 VIA Patent Center 808955.000150 (OTT: 2024-511) Date of Deposit: May 14, 2025 Example 8: Ligand-Based Screening based on shape or similarity An aspect of the disclosure is ligand-based screening using shape or similarity to find novel inhibitors of the Histone Acetyl Transferase (HAT Domains) KAT2A / GCN5 and KAT2B / PCAF. The method may employ the following compounds: 28 122236082.1 VIA Patent Center 808955.000150 (OTT: 2024-511) Date of Deposit: May 14, 2025 In an aspect of the can be used for scaffold hopping. Scaffold hopping is the identification of isofunctional molecular structures with chemically completely different core structures. It is a subset of bioisosteric replacement where the core motif (pharmacophore; see Example 7 above) is replaced. Important interaction potentials of the molecule are usually maintained. In another aspect of the disclosure, IC50measurements for inhibitors using PCAF AlphaLisa Binding are used. His / Flag epitope tagged PCAF719-832 bromodomain is cloned, expressed and purified to homogeneity. PCAF bromodomain binding and inhibition of the compounds disclosed herein are assessed by monitoring the engagement of biotinylated small molecule ligand (known to bind the PCAF bromodomain) with the target using the AlphaLisa technology (Perkin-Elmer). Specifically, in a 384 well ProxiPlate PCAF bromodomain (225 nM final) is combined with the biotinylated small molecule ligand (6 nM final) in 50 mM HEPES (pH 7.5), 75 mM NaCl, 1 mM TCEP, 0.01% (w / v) BSA, and 0.008% (w / v) Brij-35 either in the presence of DMSO (final 0.2% DMSO) or compound dilution series in DMSO. After 15 minute incubation at room temperature AlphaLisa streptavidin acceptor beads and AlphaLisa anti- histidine done beads are added to a final concentration of 12.5 μg / mL each. After 90 minutes of 29 122236082.1 VIA Patent Center 808955.000150 (OTT: 2024-511) Date of Deposit: May 14, 2025 equilibration, plates are read on an Envision instrument and IC50s are calculated using a four parameter non-linear curve fit. In summary, to one skilled in the art, both structure-based and ligand-based computational techniques are used to identify candidate molecules that can disrupt the human SAGA and ATAC complexes, thereby increasing the transcription of gamma globin and consequently increasing HbF levels. All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the disclosure pertains. All references cited in this disclosure are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety individually. One skilled in the art would readily appreciate that the present disclosure is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The methods and compositions described herein as presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the disclosure. Changes therein and other uses will occur to those skilled in the art, which are encompassed within the spirit of the disclosure, are defined by the scope of the claims. In addition, where features or aspects of the disclosure are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group or other group. The use of the terms "a" and "an" and "the" and similar referents in the context of describing the disclosure (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open- ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or 30 122236082.1 VIA Patent Center 808955.000150 (OTT: 2024-511) Date of Deposit: May 14, 2025 exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure. Embodiments of this disclosure are described herein, including the best mode known to the inventors for carrying out the disclosed invention. Variations of those embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The disclosure illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations that are not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising", "consisting essentially of", and "consisting of" may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that 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 disclosure provides preferred embodiments, optional features, modification and variation of the concepts 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 disclosure as defined by the description and the appended claims. It will be readily apparent to one skilled in the art that varying substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention. Thus, such additional embodiments are within the scope of the present disclosure and the following claims. The present disclosure teaches one skilled in the art to test various combinations and / or substitutions of chemical modifications described herein toward generating conjugates possessing improved contrast, diagnostic and / or imaging activity. Therefore, the specific embodiments described herein are not limiting and one skilled in the art can readily appreciate that specific combinations of the modifications described herein can be tested without undue experimentation toward identifying conjugates possessing improved contrast, diagnostic and / or imaging activity. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the disclosure to be practiced otherwise than as specifically described herein. 31 122236082.1 VIA Patent Center 808955.000150 (OTT: 2024-511) Date of Deposit: May 14, 2025 Accordingly, this disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the disclosure unless otherwise indicated herein or otherwise clearly contradicted by context. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. Such equivalents are intended to be encompassed by the following claims. 32 122236082.1

Claims

VIA Patent Center 808955.000150 (OTT: 2024-511) Date of Deposit: May 14, 2025 Claims We Claim:

1. A method of treating a hemoglobinopathy in a subject, the method comprising administering at least one agent capable of reducing the expression and / or activity of at least one human Spt-Ada-Gcn5-Acetyltransferase (hSAGA) complex protein in the subject, thereby treating the hemoglobinopathy in the subject.

2. The method of claim 1, wherein the hSAGA complex protein is selected from the group consisting of TADA2B, TADA3, TADA1, TADA2A, TAF5L, TAF6L, TAF9, TAF10, TAF12, KAT2A, KAT2B, SGF29, TATA1, SUPT7L, SUPT7H, SUPT20H, SUPT3H, TRRAP, USP22, ATXN7L3, ATXN7, ENY2, SF3B3, and SF3B5, optionally wherein the hSAGA complex protein is TADA2B, TADA3, TAF5L, and / or TADA1.

3. The method of claim 1 or claim 2, wherein the hSAGA complex protein is selected from the group consisting of TADA2B, TADA3, TADA1, TADA2A, TAF5L, and SUPT20H.

4. The method of any one of the preceding claims, wherein the hSAGA complex protein is TADA2B, TADA1, TAF5L, or SUPT20H.

5. The method of any one of the preceding claims, wherein the hemoglobinopathy is a beta- hemoglobinopathy, optionally wherein the beta-hemoglobinopathy is a sickle cell disease or β- thalassemia.

6. The method of claim 1, wherein the agent reduces or eliminates expression of a gene, regulatory activity of a gene, or activity of an encoded protein or enzyme.

7. The method of claim 6, wherein the reduction or elimination of gene expression is the result of a gene deletion and / or a missense gene mutation.

8. The method of claim 6, wherein the agent is an inhibitory RNA, siRNA, miRNA, shRNA, a small molecule inhibitor, and / or an antibody. 33 122236082.1VIA Patent Center 808955.000150 (OTT: 2024-511) Date of Deposit: May 14, 2025 9. The method of claim 6, wherein the agent comprises a CRISPR / Cas9 system comprising a guide RNA (gRNA).

10. The method of claim 9, wherein the gRNA is a TADA2B gRNA, optionally wherein the gRNA comprises SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO:

3.

11. The method of claim 9, wherein the gRNA is a TADA1 gRNA, optionally wherein the gRNA comprises SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO:

6.

12. The method of claim 9, wherein the gRNA is a TAF5L gRNA, optionally wherein the gRNA comprises SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO:

9.

13. The method of claim 9, wherein the gRNA is a SUPT20H gRNA, optionally wherein the gRNA comprises SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO:

12.

14. A method of inducing fetal hemoglobin in a subject, the method comprising administering at least one agent capable of reducing at least one human Spt-Ada-Gcn5-Acetyltransferase (hSAGA) complex protein level and / or function in the subject, thereby inducing fetal hemoglobin in the subject.

15. The method of claim 14, wherein the subject has or is predisposed to having a hemoglobinopathy.

16. The method of claim 14, wherein the hSAGA complex protein is selected from the group consisting of TADA2B, TADA3, TADA1, TADA2A, TAF5L, TAF6L, TAF9, TAF10, TAF12, KAT2A, KAT2B, SGF29, TATA1, SUPT7L, SUPT7H, SUPT20H, SUPT3H, TRRAP, USP22, ATXN7L3, ATXN7, ENY2, SF3B3, and SF3B5, optionally wherein the hSAGA complex protein is TADA2B, TADA3, TAF5L, and / or TADA1. 34 122236082.1VIA Patent Center 808955.000150 (OTT: 2024-511) Date of Deposit: May 14, 2025 17. The method of claim 14, wherein the hSAGA complex protein is TADA2B, TADA3, TADA1, TADA2A, TAF5L, and / or SUPT20H.

18. The method of claim 14, wherein the hSAGA complex protein is TADA2B, TADA1, TAF5L, or SUPT20H.

19. The method of claim 15, wherein the hemoglobinopathy is a beta-hemoglobinopathy, optionally wherein the beta-hemoglobinopathy is a sickle cell disease or β-thalassemia.

20. The method of claim 14, wherein the at least one agent reduces or eliminates expression of a gene, regulatory activity of a gene, or activity of an encoded protein or enzyme.

21. The method of claim 20, wherein reduction or elimination of gene expression is the result of a gene deletion and / or a missense gene mutation.

22. The method of claim 14, wherein the at least one agent is an inhibitory RNA, siRNA, miRNA, shRNA, a small molecule inhibitor, and / or an antibody.

23. The method of claim 14, wherein the at least one agent comprises a CRISPR / Cas9 system comprising a guide RNA (gRNA).

24. The method of claim 23, wherein the gRNA is a TADA2B gRNA, optionally wherein the gRNA comprises SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO:

3.

25. The method of claim 23, wherein the gRNA is a TADA1 gRNA, optionally wherein the gRNA comprises SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO:

6.

26. The method of claim 23, wherein the gRNA is a TAF5L gRNA, optionally wherein the gRNA comprises SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO:

9. 35 122236082.1VIA Patent Center 808955.000150 (OTT: 2024-511) Date of Deposit: May 14, 2025 27. The method of claim 23, wherein the gRNA is a SUPT20H gRNA, optionally wherein the gRNA comprises SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO:

12.

28. A composition for inducing expression of fetal hemoglobin in a subject, the composition comprising a CRISPR / Cas9 system, wherein the CRISPR / Cas9 system comprises at least one guide RNA (gRNA) targeting at least one hSAGA complex protein gene.

29. The composition of claim 28, wherein the hSAGA complex protein gene is selected from the group consisting of TADA2B, TADA3, TADA1, TADA2A, TAF5L, TAF6L, TAF9, TAF10, TAF12, KAT2A, KAT2B, SGF29, TATA1, SUPT7L, SUPT7H, SUPT20H, SUPT3H, TRRAP, USP22, ATXN7L3, ATXN7, ENY2, SF3B3, and SF3B5, optionally wherein the hSAGA complex protein is TADA2B, TADA3, TAF5L, and / or TADA1.

30. The composition of claim 28, wherein the hSAGA complex protein is TADA2B, TADA3, TADA1, TADA2A, TAF5L, and / or SUPT20H.

31. The composition of claim 28, wherein the hSAGA complex protein is TADA2B, TADA1, TAF5L, or SUPT20H.

32. The composition of any one of claims 28-31 for the treatment of a hemoglobinopathy in a subject.

33. A population of cells modified using the composition of any one of claims 28-31. 36 122236082.1

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