Base editing methods and compositions

Abstract

Enhancing fetal hemoglobin (HbF) production through targeted promoter editing using base editing technologies. An embodiment includes a composition for enhancing fetal hemoglobin (HbF) expression in mammalian cells, the composition comprising a base editing system comprising at least two guide RNAs comprising targeting sequences selected from SEQ ID NO: 1 to SEQ ID NO: 5. Specifically, it involves the design and use of single guide RNAs (sgRNAs) targeting key regulatory elements within the gamma-globin (HBG1 / 2) promoter. The method combines the use of adenosine base editors to introduce mutations, creating new binding sites for transcriptional activators like TAL1 and KLF1, and disrupting repressor binding sites such as BCL11A and ZBTB7A / LRF. This dual-targeting approach significantly increases HbF expression, offering a potential therapeutic strategy for treating beta-hemoglobinopathies like sickle cell disease and beta-thalassemia.

Description

[0001] BASE EDITING METHODS AND COMPOSITIONS

[0002] PRIORITY CLAIM

[0003] The instant patent application is related to and claims priority from the India provisional application entitled, “ENHANCED GENE REACTIVATION VIA TARGETED PROMOTER EDITING”, Application No.:202441072108, Filed: 24 September 2024, which is incorporated in its entirety herewith to the extent not inconsistent with the description herein.

[0004] TECHNICAL FIELD

[0005] The present disclosure relates to the field of gene editing techniques for treating betahemoglobinopathies, more particularly the application of base editing techniques to enhance fetal hemoglobin (HbF).

[0006] BACKGROUND

[0007] Beta-hemoglobinopathies are caused by mutations in the beta-globin gene and can be classified into two major groups: structural alterations (HbS-sickle cell anemia) and synthetic alterations (thalassemia). Current curative therapies are limited to allogeneic hematopoietic stem cell transplantation from HLA-matched sibling donors and pharmacologic agents such as hydroxyurea.

[0008] Hereditary Persistence of Fetal Hemoglobin (HPFH) is a benign genetic condition caused by single-point mutations in the gamma-globin (HBG1 and HBG2) promoter, leading to high HbF expression in adulthood. Individuals with HPFH who also inherit beta-hemoglobinopathies often experience milder symptoms due to elevated fetal hemoglobin (HbF) levels. Genome editing approaches have focused on mimicking the effects of HPFH mutations to increase HbF levels by either creating new binding sites for erythroid activators or disrupting repressor binding sites, thereby enhancing HbF expression and improving clinical outcomes for patients with sickle cell disease and beta-thalassemia.

[0009] Hemoglobin switching is clinically important, as reactivation of fetal y-globin in the adult stage is regarded as an effective treatment for severe P-globin gene disorders, such as P-thalassemia and sickle cell disease. Although the exact mechanisms governing the transition from fetal to adult hemoglobin expression are still under investigation, several studies have highlighted the involvement of crucial transcription factors and chromatin complexes. The interplay of these transcriptional modulators regulates the differential expression of the various genes in the P-globin cluster during development. Naturally occurring mutations associated with HPFH affect the

[0010] PCT Application 1 AD JU-013-PCT binding of transcriptional repressors or create de novo activator sites that drive fetal globin expression. The major globin activators that bind the fetal y-globin promoters (HBG1 / 2) include GATA1, KLF1, TALI, and NFY, and the major repressors are BCL11A andZBTB7A. A mutation at -175 creates a de novo site for the activator TALI, and mutations that disrupt binding sites for repressors map around -115, the BCL11 A binding motif, and -200, the ZBTB7A binding site.

[0011] CN115851706A is directed to a base editing system that employs the adenosine base editor variant ABE8e in combination with a sgRNA library to target the HBG promoter in human cells. This system upregulates HbF expression, offering a potential treatment for beta-hemoglobinopathies such as beta-thalassemia and sickle cell disease.

[0012] Han et al. is directed to the use of a transformer base editor (tBE) to disrupt transcription-factor- binding motifs at the HBG1 / 2 promoters. Disruption of the BCLllA-binding motif resulted in the highest y-globin expression while minimizing off-target mutations.

[0013] Psatha et al. is directed to the use of a helper-dependent adenoviral vector (HD-Ad5 / 35++) with nuclease activity to simultaneously target the erythroid enhancer of BCL11A and the HBG promoter. This approach reactivates HbF by recreating the -114 to -102 HPFH deletion, significantly enhancing HbF levels compared to single target strategies.

[0014] Antoniou et al. is directed to base editing to generate multiple mutations within the -200 region of the HBG promoters, including creating a KLF1 activator binding site. This method reactivates HbF in patient-derived hematopoietic stem / progenitor cells, offering a safer alternative to Cas9- nuclease approaches by avoiding large genomic rearrangements and achieving higher y-globin levels.

[0015] Considering the limitations of existing therapies, there is a pressing need for gene editing strategies that can safely and efficiently introduce combinations of beneficial HPFH-like mutations in the HBG promoters to maximize HbF expression while avoiding large deletions and unintended genomic alterations that occur with nuclease-based methods.

[0016] Aspects of the present disclosure are directed to base editing methods and compositions.

[0017] SUMMARY

[0018] Aspects of the present disclosure are directed to base editing methods and compositions.

[0019] PCT Application 2 AD JU-013-PCT According to an aspect of the present disclosure, a method of enhancing fetal hemoglobin (HbF) expression in mammalian cells includes introducing into the mammalian cells a base editing system consisting at least a first guide RNA and a second guide RNA, where the first guide RNA and the second guide RNA comprise targeting sequences selected from SEQ ID NOs: 1 to 5 complementary to at least a first target region and a second target region within a gamma-globin (HBG1 / 2) promoter, and where the base editing system introduces base substitutions in the target regions to disrupt transcriptional repressor binding sites, create de novo activator sites, or a combination thereof, thereby altering transcriptional regulation and enhancing HbF production.

[0020] In an embodiment, the base substitutions are introduced sequentially at the first target region and second target region by delivering the corresponding guide RNAs in separate editing steps.

[0021] In yet another embodiment, the HBG1 / 2 promoter target region is located at least 250 base pairs upstream from the transcription start site (TSS) of the HBG1 / 2 gene.

[0022] In yet another embodiment, the base substitutions are located at positions selected from -200, - 175, -123, and -115 relative to the TSS.

[0023] In yet another embodiment, disrupting transcriptional repressor binding sites includes introducing base substitutions at positions -200 or -115 in the HBG1 / 2 promoter target region.

[0024] In yet another embodiment, the transcriptional repressor binding sites are binding sites for BCL11A or ZBTB7A / LRF.

[0025] In yet another embodiment, creating de novo activator sites includes introducing base substitutions at positions -123 or -175 in the HBG1 / 2 promoter target region.

[0026] In yet another embodiment, the de novo activator sites are binding sites for the globin activators KEF1 or TALI.

[0027] In yet another embodiment, the method includes introducing combinations of base substitutions to disrupt transcriptional repressor binding sites and create de novo activator sites in the HBG1 / 2 promoter target region.

[0028] PCT Application 3 AD JU-013-PCT In yet another embodiment, the mammalian cells are selected from HUDEP-2 cells, hematopoietic stem and progenitor cells (HSPCs), or induced pluripotent stem cells (iPSCs).

[0029] In yet another embodiment, enhancing HbF production is used for treating P-thalassemia or sickle cell disease.

[0030] In yet another embodiment, the base editing system is delivered to mammalian cells using a viral vector, a non-viral vector, electroporation, or by cell -penetrating peptide (CPP) delivery.

[0031] According to another aspect of the present disclosure, a composition for enhancing fetal hemoglobin (HbF) expression in mammalian cells includes a base editing system containing at least two guide RNAs with targeting sequences selected from SEQ ID NOs: 1 to 5, where the guide RNAs direct the base editor to at least two distinct target regions within the HBG1 or HBG2 promoter.

[0032] In an embodiment, the mammalian cells are selected from iPSCs or hematopoietic stem and progenitor cells (HSPCs).

[0033] According to another aspect of the present disclosure, a method for treating P-thalassemia or sickle cell disease in a subject includes editing HSPCs obtained from the subject using the method described herein to enhance HbF production, and administering the edited HSPCs back to the subject.

[0034] According to another aspect of the present disclosure, a pharmaceutical composition for treating P-thalassemia or sickle cell disease includes the edited mammalian cells obtained from the methods described herein and a pharmaceutically acceptable carrier.

[0035] Several aspects of the present disclosure are described below with reference to examples for illustration. However, one skilled in the relevant art will recognize that the disclosure can be practiced without one or more of the specific details or with other methods, components, materials and so forth. In other instances, well known structures, materials, or operations are not shown in detail to avoid obscuring the features of the disclosure. Furthermore, the features / aspects described can be practiced in various combinations, though only some of the combinations are described herein for conciseness.

[0036] PCT Application 4 AD JU-013-PCT BRIEF DESCRIPTION OF DRAWINGS

[0037] Example embodiments of the present disclosure will be described with reference to the accompanying drawings briefly described below.

[0038] Figure 1A is a schematic diagram depicting the four different gene clusters, including the -115 (BCL11A) cluster, the -200 (LRF) cluster, the -175 (TAE1) cluster, and the -123 (KEF) cluster, along with the gRNAs targeting these clusters.

[0039] Figure IB is a graph depicting the total on-target editing at each cluster in individually transduced and dual transduced HUDEP-2 cells.

[0040] Figure 1C is a graph depicting the indel frequencies at the -115, -200, -175, and -123 clusters, as well as in dual editing combinations.

[0041] Figure ID is a graph depicting the frequency of deletions occurring across the different gene clusters and combinations of clusters in HUDEP-2 cells.

[0042] Figure IE is a graph depicting the levels of fetal hemoglobin (HbF) expression following targeting of the -115, -200, -175, and -123 clusters individually and in combination.

[0043] Figure 2A is a graph depicting the editing efficiency at the -175 and -123 clusters individually and in combination in healthy donor HSPCs as evaluated by Sanger sequencing.

[0044] Figure 2B is a graph depicting the frequency of large deletions (4.2 kb) at the HBG1 and HBG2 promoters in healthy donor HSPCs as quantified by qRT-PCR.

[0045] Figure 2C is a graph depicting the effect of editing the -175 and -123 clusters individually and in combination on HbF tetramer production in healthy donor HSPCs as determined by HPLC.

[0046] Figure 2D is a graph depicting the impact of editing the -175 and -123 clusters on erythroid differentiation in healthy donor HSPCs.

[0047] Figure 2E is a graph depicting the impact of editing the -175 and -123 clusters on enucleation in healthy donor HSPCs.

[0048] PCT Application 5 AD JU-013-PCT Figure 2F is a graph depicting the editing efficiency at the -175 and -123 clusters individually and in combination in HSPCs isolated from a P-thalassemia patient sample.

[0049] Figure 2G is a graph depicting the elevation of fetal hemoglobin following editing of the -175 and -123 clusters individually and in combination in HSPCs isolated from a P-thalassemia patient sample.

[0050] Figure 2H is a graph depicting the reduction of delta globin chain in dual-edited HSPCs isolated from a P-thalassemia patient sample.

[0051] Figure 21 is a graph depicting erythroid differentiation in dual-edited HSPCs isolated from a P- thalassemia patient sample.

[0052] Figure 2 J is a graph depicting enucleation in dual-edited HSPCs isolated from a P-thalassemia patient sample.

[0053] Figure 2K is a graph depicting reduced reactive oxygen species (ROS) levels in dual-edited HSPCs isolated from a P-thalassemia patient sample.

[0054] In the drawings, like reference numbers generally indicate identical, functionally similar, and / or structurally similar elements. The drawing in which an element first appears is indicated by the leftmost digit(s) in the corresponding reference number. In the drawings, like reference numbers generally indicate identical, functionally similar, and / or structurally similar elements. The drawing in which an element first appears is indicated by the leftmost digit(s) in the corresponding reference number.

[0055] DETAILED DESCRIPTION

[0056] For the purpose of promoting an understanding of the principles of the invention, reference will now be made to the embodiment illustrated in the drawings and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the illustrated system, and such further applications of the principles of the invention as illustrated therein being contemplated as would normally occur to one skilled in the art to which the invention relates.

[0057] It will be understood by those skilled in the art that the foregoing general description and the

[0058] PCT Application 6 AD JU-013-PCT following detailed description are exemplary and explanatory of the invention and are not intended to be restrictive thereof.

[0059] Reference throughout this specification to “an aspect”, “another aspect” or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, appearances of the phrase “in an embodiment”, “in another embodiment” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.

[0060] The terms “comprises”, “comprising”, or any other variations thereof, are intended to cover a nonexclusive inclusion, such that a process or method that comprises a list of steps does not include only those steps but may include other steps not expressly listed or inherent to such process or method. Similarly, one or more compositions or elements or structures or components preceded by “comprises... a” does not, without more constraints, preclude the existence of other compositions or elements or other structures or other components or additional compositions or additional elements or additional structures or additional components.

[0061] Unless otherwise defined, 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 invention belongs. The compositions, methods, and examples provided herein are illustrative only and not intended to be limiting.

[0062] Embodiments of the present disclosure will be described below in detail with reference to the accompanying drawings.

[0063] I. Cluster Design

[0064] In an aspect, sgRNAs were designed to specifically target critical regions within the gamma-globin promoter in order to reactivate HbF expression in adult cells. The gRNAs for targeting the HBG1 and HBG2 promoter regions were designed using SnapGene and Benchling. The primary purpose of designing these sgRNAs is to disrupt the binding sites of transcriptional repressors or to introduce new binding sites for transcriptional activators, ultimately leading to increased levels of HbF. This approach aims to mimic naturally occurring mutations found in individuals with HPFH, a benign condition where elevated HbF levels alleviate the symptoms of beta-hemoglobinopathies such as sickle cell disease and beta-thalassemia. The design of specific single -guide RNAs (sgRNAs) was centered on targeting four distinct clusters within the gamma-globin promoter

[0065] PCT Application 7 AD JU-013-PCT region: the -115 cluster, -123 cluster, -175 cluster, and -200 cluster.

[0066] Fig. 1A shows a pictorial representation of the four different gene clusters: -115 (BCL11 A) cluster, -200 (LRF cluster); -175 (TALI) cluster, and -123 (KLF) cluster and the gRNAs targeting these clusters. These clusters were selected based on their established roles in the regulation of fetal hemoglobin (HbF) expression during development and their involvement in the repression of HbF in adult cells. For instance, the -115 Cluster contains binding sites for BCL11A, a key repressor of HbF expression. Targeting this cluster is crucial for disrupting the interaction between BCL11A and the gamma-globin promoter, which could lead to increased HbF levels. The -123 Cluster is hypothesized to involve binding sites for transcription factors like KLF1, which activate HbF expression. Targeting this cluster could create binding sites, potentially modulating HbF levels. The -175 Cluster is significant due to the presence of potential binding sites for the TALI transcription factor, which is known to act as a strong activator of erythroid-specific genes. By targeting the -175 cluster, the goal is to enhance the binding of TALI, thereby promoting HbF expression. The introduction of TALI binding sites at this location could synergize with the disruption of repressor binding sites, further boosting HbF levels. The -200 Cluster contains binding sites for ZBTB7A / LRF, another repressor of HbF expression. Similar to the -115 cluster, targeting this region is aimed at disrupting the repressive effects of transcription factor- ZBTB7A / LRF, leading to HbF reactivation. The choice of this cluster is based on the understanding that removing multiple repressive or incorporating the activator binding site influences can significantly enhance HbF production.

[0067] The present disclosure is not limited to the four specific clusters (-115, -123, -175, and -200) described herein. It will be understood by those skilled in the relevant art that the disclosure is not limited to the four specific clusters described herein, and encompasses the targeting of additional clusters or regulatory regions within the gamma-globin gene promoter and associated regulatory elements.

[0068] In an embodiment, the total on-target editing at each cluster in individually transduced and dual transduced HUDEP-2 cells ranged between 20-90% based on the target base in the editing window (Figure IB). Despite the use of base editors, unintended insertions and deletions occurred, with greater frequency when two sgRNAs were introduced. The -200 cluster was characterized by the highest indel percentages, followed by the -123 and -115 clusters, while the -175 cluster exhibited negligible indel frequency (Figure 1C). Editing with sgRNAs targeting -115 and -123 resulted in -15-40% indel frequency, which affected the BCL11A binding site favorably, but also extended

[0069] PCT Application 8 AD JU-013-PCT to affect the creation of a possible KLF1 binding site at -123, resulting in reactivation of HbF due to only a single transcription factor binding modulation rather than two. Targeting either the HBG1 or HBG2 locus resulted in editing at both promoters due to sequence similarity, increasing the probability of concurrent nicking and leading to deletions of HBG2 or, in rare cases, inversions (Figure ID).

[0070] In contrast to previous studies, ABE8e editing at the -115 cluster introduced a mutation at -112, disrupting the BCL11A site but not creating a site for GATA1, whereas ABE7.10 created a GATA1 binding site and yielded higher HbF levels (Figure IE).

[0071] In an embodiment, sgRNAs targeting genomic safe-harbor loci such as AAVS1 and CCR5 were also used. These control sgRNAs validate the editing system and delivery methodology. Representative sequences include:

[0072] 1. AAVS1: 5'-ggggccactagggacaggat-3'

[0073] 2. CCR5: 5'-aaaatataatctttaagata-3'

[0074] These safe-harbor sgRNAs were employed in parallel to confirm editing specificity at non-y- globin loci.

[0075] II. Vector Design and Delivery

[0076] In another aspect, the designed sgRNAs against these gene clusters may be synthesized and cloned into a lentiviral backbone vector. The vector is engineered to carry a fluorescent reporter gene such as GFP (Green Fluorescent Protein) or RFP (Red Fluorescent Protein), under a strong promoter such as EFla. The use of these fluorescent markers enables the monitoring of transduction efficiency and facilitates the selection of successfully transduced cells. The sgRNA sequences are inserted into the lentiviral vectors using standard molecular cloning techniques. Specifically, the vectors are linearized using restriction enzymes compatible with the sgRNA overhangs, and the sgRNAs are ligated into the vectors. Following ligation, the recombinant plasmids are transformed into competent E. coli cells, which are then plated on selective media. Positive clones are identified by colony PCR, and the accuracy of the inserted sgRNA sequence is confirmed through Sanger sequencing. Once the sgRNA-containing plasmids are confirmed, they are used to produce lentivirus in HEK293T cells. This step involves co-transfecting the HEK293T cells with the sgRNA-expressing plasmid and a set of helper plasmids necessary for viral packaging and envelope formation.

[0077] In an embodiment, the base editing system may be introduced into mammalian cells using non-

[0078] PCT Application 9 AD JU-013-PCT viral vectors (e.g., lipid nanoparticles, plasmids), electroporation of nucleic acids or ribonucleoprotein complexes, or cell-penetrating peptide (CPP) delivery.

[0079] III. Example Implementation of Editing in Erythroid Progenitor Cells

[0080] In an embodiment, Human umbilical cord blood-derived erythroid progenitor (HUDEP) cell lines are transduced with the target guide RNA for base editing. These cells are genetically modified to express adenosine deaminase, which supports their proliferation and differentiation in culture. It further comprises a nucleotide sequence encoding a base editing protein or peptide. Prior to transduction, HUDEP-2 cells are cultured in erythroid expansion media to maintain their progenitor state and ensure optimal transduction efficiency. Initially, as shown in Fig 2A, HUDEP- 2 cells were exposed to lentivirus-carrying sgRNAs that target the individual gene clusters within the HBG1 / 2 promoter (-115, -123, -175, -200). Each of these lentiviruses expresses one reporter gene, such as GFP. Following transduction, the cells are incubated for 48 to 72 hours, after which GFP expression is monitored using flow cytometry. The GFP-positive cells, which indicate successful transduction and sgRNA expression, are expanded in culture for an additional 3-5 days. This expansion period allows sufficient time for the base editor, ABE, to introduce the desired adenine-to-guanine (A-to-G) conversions at the targeted loci within the promoter region.

[0081] After the initial base editing, the GFP-positive HUDEP-2 cells undergo a second round of transduction with a new set of lentiviruses that carry sgRNAs targeting different combinations of the four clusters and express a different reporter gene, such as RFP. This dual-reporter system allows for the differentiation between cells that have undergone only the first transduction (GFP- positive) and those that have undergone both transductions (GFP and RFP-positive). This approach allows for the assessment of combinatorial effects on HbF reactivation, such as disrupting a repressor site while simultaneously creating an activator binding site. Following the second transduction, the cells are again expanded for 5 days to allow the ABE8e enzyme sufficient time to introduce the second round of base edits. The presence of both GFP and RFP in the cells is monitored using flow cytometry, and double-positive cells (expressing both GFP and RFP) are isolated for further analysis.

[0082] In an embodiment, HUDEP-2 cells expressing ABE8e and transduced with sgRNAs targeting each cluster. The editing efficiency varied depending on the locus, with the -175 cluster producing the highest levels of HbF expression, followed by the -123 cluster, while the repressor clusters -115 and -200 yielded comparatively lower levels of HbF induction (Figure IE). All gRNAs targeting the HBG1 / 2 promoters elevated HbF regardless of single or dual editing. However, differences

[0083] PCT Application 10 AD JU-013-PCT were observed when HbF levels were quantified by HPLC, revealing the relative strength of each locus (Figure IE).

[0084] Base editors are innovative tools derived from the CRISPR / Cas9 system, enabling precise transition mutations in DNA without causing double- stranded breaks (DSBs). Different types of base editors include cytosine base editors (CBEs), which convert C to T, and the recently developed ABE8e, which offers enhanced editing efficiency and processivity over its predecessors. Adenosine base editors (ABEs) are designed to convert adenine to inosine, which is read as guanine during DNA replication, repair, and transcription, effectively resulting in an A-to- G edit. ABE8e's improvements make it especially suitable for applications requiring extensive or complex base editing, such as installing naturally occurring HPFH mutations in the HBG1 and HBG2 promoters. This variant’s superior performance in precision and efficiency expands the potential for targeted gene therapies and genetic research.

[0085] In an embodiment, the base editing protein or peptide is an adenine base editor (ABE). The ABE may be selected from the group consisting of ABE7.10, ABE8e, and an ABE including SpRY. More specifically, the base editor is an ABE8 base editor selected from the group consisting of: ABE8.1, ABE8.2, ABE8.3, ABE8.4, ABE8.5, ABE8.6, ABE8.7, ABE8.8, ABE8.9, ABE8.10, ABE8.11, ABE8,12, and ABE8.13.

[0086] In an embodiment, ABE7.10 and ABE8e effectively disrupt the BCL11A binding site at -115, ABE8e introduces a mutation at -112 and does not create a GATA1 binding site, whereas ABE7.10 produces a GATA1 site and achieves higher HbF induction (Figure 2E).

[0087] IV. Editing Efficiency and Outcome

[0088] DNA may be isolated from these transduced cells and amplified with primers specific to the HBG1 / 2 promoter regions. After confirming successful amplification, the PCR products may undergo Sanger sequencing. The sequencing data may be analyzed using tools such as Inference of CRISPR Edits (ICE) and EditR to determine the presence of indels and to assess base editing efficiency.

[0089] The total editing at each cluster in individually transduced and dual transduced HUDEP-2 cells ranged between 20- 90% based on the target base in the editing window (Figure IB). Indel frequencies varied depending on the targeted clusters and combinations (Figure 1C). The -200 cluster exhibited the highest indel frequency, followed by the -123 and -115 clusters, while the -

[0090] PCT Application 11 AD JU-013-PCT 175 cluster exhibited negligible indels. Dual targeting of -115 and -123 resulted in substantial indels, impacting both disruption of the BCL11A binding site and the creation of the KLF1 site (Figure 1C). In addition, simultaneous editing increased the risk of deletions at the HBG2 promoter, or in rare cases inversions, due to homologous nicking across both HBG1 and HBG2 (Figure ID).

[0091] V. Combinatorial Effects of Editing

[0092] In one embodiment, the BCL11A binding site (-115 gene cluster) alone and in combination with the ZBTB7A / LRF (-200 gene cluster) site were targeted. Disrupting both BCL11A at -115 gene cluster and ZBTB7A / LRF at -200 gene cluster resulted in higher HbF levels compared to targeting them individually. However, this combined approach yielded a maximum 39-44% increase in HbF, indicating additional regulatory interactions in the y-globin promoter.

[0093] In another embodiment, the effects of introducing a TALI binding site at the -175 cluster while disrupting the BCL11A site at the -115 cluster were examined. TALI is a transcriptional activator, and its binding could potentially counteract the repressive effects of BCL11A. This combination was compared with other strategies, such as introducing a KLF1 binding site at the -123 cluster alongside BCL11A disruption. The results showed that the combination of TALI site introduction and BCL11A disruption (59-62%) was more effective in increasing HbF levels than combinations involving the KLF1 site (47-58%). This suggests that TALI has a stronger activating potential, making it a more promising target for HbF reactivation.

[0094] In yet another embodiment, the impact of creating dual activator sites at TALI (at the -175 cluster) and KLF1 (at the -123 cluster) — simultaneously within the HBG1 / 2 promoter regions was determined. The goal was to assess whether the combined presence of these activators could synergistically enhance HbF levels. This dual activation strategy led to a significant reactivation of HbF, achieving levels up to 69-78% in cells expressing ABE8e or its engineered variant, dABE8e. This suggests that activating multiple sites within the promoter can effectively disrupt repressor binding and facilitate the action of other transcriptional activators like GATA1 and NFY.

[0095] In yet another embodiment, TALI and KLF1 binding sites were sequentially introduced and its effect on steric hindrance was studied. This approach aimed to explore the effects of creating multiple activator sites in a specific sequence and how this might affect the binding of repressors like ZBTB7A / LRF and BCL11A. The results indicated that the sequential introduction of TALI and KLF1 sites led to steric hindrance, which impaired the binding of the repressors. This

[0096] PCT Application 12 AD JU-013-PCT disruption in repressor binding was associated with elevated HbF levels, with the sequence of introducing TALI first proving to be particularly effective.

[0097] In yet another embodiment, targeting the BCL11A site (-115 cluster) and introducing the TALI binding site (-175 cluster) efficiently increased HbF levels, surpassing the combination with the - 123 cluster that potentially creates a KLF1 binding site (Figure IE). Disrupting both repressors at -115 (BCL11A) and -200 (ZBTB7A / LRF) resulted in higher HbF levels than targeting them individually, reaching a maximum -50% increase (Figure IE). The order of sequential transduction was also important: introducing a TALI binding site first, followed by BCL11A disruption, was more effective than introducing a KLF1 binding site before BCL11A disruption (Figure 3B). Creation of dual activator sites by targeting the -175 and -123 clusters reactivated the maximum levels of HbF, reaching -60% by HPLC quantification (Figure IE). This was attributed to the binding of both TALI and KLF1, which also interfered with ZBTB7A / LRF and BCL11A binding due to steric hindrance, thereby supporting the recruitment of other activators such as GAT Al and NFY. Altogether, these results demonstrate that elevation of HbF levels is more pronounced when the promoter region becomes accessible to activators, in contrast to removal of repressors alone.

[0098] VI. Editing in Hematopoietic Stem and Progenitor Cells (HSPCs)

[0099] In an embodiment, the cells may be selected from CD34+ hematopoietic stem and progenitor cells (CD34+ HSPCs), cells within the erythroid lineage, and endothelial cells, but not limited to these. Cells may be sourced from bone marrow, peripheral blood, mobilized peripheral blood, cord blood, induced pluripotent stem cells (iPSCs) or patient-derived iPSCs. Furthermore, the differentiated cells derived from these progenitor or stem cells, particularly within the erythroid lineage, are characterized by an increased expression of gamma-globin protein compared to equivalent cells from the same subject that lack synthetic allele modifications. This exploration of various cell types offers potential for broader therapeutic applications in gene editing and regenerative medicine.

[0100] The present disclosure includes a composition for gene editing to increase HbF levels, comprising mammalian cells, such as HSPCs, or HUDEP-2 cells that have been edited using this base editing approach.

[0101] In an embodiment, sequential editing at the -175 and -123 clusters was further evaluated in HSPCs derived from healthy donor PBMNCs, as this combination was shown to reactivate the highest

[0102] PCT Application 13 AD JU-013-PCT levels of HbF. Unlike the efficient editing observed in HUDEP-2 cells with lentiviral delivery, low editing efficiency was initially observed at the -175 cluster. To overcome this, NG-Cas9 ABE8e and a gRNA recognizing an NG PAM were employed, resulting in improved editing at -175 and approximately 70% editing efficiency at the -123 cluster (Figure 2A). Minimal or no insertions or deletions were detected at the target sites, and large deletions typically observed with editing of the homologous HBG1 / 2 promoters were absent (Figure 2B). The differentiation profile and enucleation potential of the edited HSPCs were comparable to controls, indicating that the editing did not impair erythroid development (Figure 2C, 2D). Sequential editing at -175 and -123 induced the highest levels of HbF reactivation in differentiated erythroid cells, as quantified by HPEC (Figure 2E). These results demonstrate that base editing at these regulatory regions exhibits high HbF-inducing potential while maintaining normal hematopoietic differentiation.

[0103] HSPCs isolated from a P-thalassemia patient sample were subjected to editing at the -175 and - 123 clusters. Editing efficiency was consistent with that seen in healthy donor HSPCs (Figure 2F), and dual editing led to robust elevation of fetal hemoglobin (Figure 2G). In these patient-derived cells, HbF reactivation was accompanied by a reduction in delta-globin chain levels (Figure 2H), decreased reactive oxygen species (ROS) levels (Figure 2K), and preservation of erythroid differentiation (Figure 21) and enucleation (Figure 2J). These effects were more pronounced relative to control cells, indicating that dual editing may enhance erythroid maturation while simultaneously reducing oxidative stress in P-thalassemia cells.

[0104] The below table showcases the different combinations of sgRNAs used in this study.

[0105] Table 1: Different combinations of sgRNAs used in the study

[0106] PCT Application 14 AD JU-013-PCT

Claims

CLAIMS:

1. A method of enhancing fetal hemoglobin (HbF) expression in mammalian cells, the method comprising: introducing into the mammalian cells, a base editing system comprising at least a first guide RNA and a second guide RNA, wherein the first guide RNA and the second guide RNA comprise targeting sequences selected from SEQ ID NOS: 1-5, complementary to at least a first target region and a second target region within a gamma-globin (HBG1 / 2) promoter; and wherein the base editing system introduces base substitutions in the target regions thereby disrupting transcriptional repressor binding sites, creating de novo activator sites, or a combination thereof, to alter transcriptional regulation and enhance HbF production.

2. The method of claim 1, wherein the base substitutions are introduced sequentially at the first target region and second target region, by delivering the corresponding guide RNA in separate editing steps.

3. The method of claim 1, wherein the base substitutions are introduced combinatorially at both the first target region and second target region by simultaneous delivery of the corresponding guide RNAs.

4. The method of claim 1, wherein the HBG1 / 2 promoter target region is located at least 250 base pairs upstream from the transcription start site (TSS) of the HBG1 / 2 gene.

5. The method of claim 1, wherein the base substitutions are located at specific positions in the HBG1 / 2 promoter target region selected from the group consisting of -200, -175, -123, and -115 relative to the TSS.

6. The method of claim 1, wherein disrupting transcriptional repressor binding sites comprises introducing base substitutions at positions -200 or -115 in the HBG1 / 2 promoter target region.

7. The method of claim 6, wherein transcriptional repressor binding sites are binding sites for BCL11 A or ZBTB7A / LRF.

8. The method of claim 1, wherein creating de novo activator sites comprises introducing base substitutions at positions -123 or -175 in the HBG1 / 2 promoter target region.PCT Application 15 AD JU-013-PCT9. The method of claim 8, wherein de novo activator sites are binding sites for the globin activator KLF1 or TALI.

10. The method of claim 1, wherein the method further comprises introducing combinations of the base substitutions to disrupt transcriptional repressor binding sites and create de novo activator sites in the HBG1 / 2 promoter target region.

11. The method of claim 1, wherein the mammalian cells are selected from HUDEP-2, HSPCs, iPSCs, or hematopoietic stem and progenitor cell (HSPC).

12. The method of claim 1, wherein enhancing HbF production is used for treating P- thalassemia or sickle cell disease.

13. The method of claim 1, wherein the base editing system is delivered to the mammalian cells using a viral vector, a non-viral vector, electroporation, or by cell-penetrating peptide (CPP) delivery.

14. A composition for enhancing fetal hemoglobin (HbF) expression in mammalian cells, the composition comprising: a base editing system comprising at least two guide RNAs comprising targeting sequences selected from SEQ ID NO: 1 to SEQ ID NO: 5, wherein the guide RNAs are capable of directing the base editor to at least two distinct target regions within the HBG1 / 2 promoter.

15. The composition of claim 14, wherein mammalian cells are selected from iPSCs or hematopoietic progenitors a hematopoietic stem and progenitor cell (HSPC).

16. A pharmaceutical composition comprising the edited mammalian cells of claim 14 and a pharmaceutically acceptable carrier.

17. A method for treating -thalassemia or sickle cell disease in a subject, the method comprising: editing HSPCs obtained from the subject using the method of claim 1 to enhance HbF production, and administering the edited HSPCs back to the subject.PCT Application 16 AD JU-013-PCT

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