Epitope engineering of the KIT cell surface receptor

Abstract

Genetically engineered cells, such as hematopoietic stem cells (e.g., HSPCs), with one or more gene-edited genes for cell surface proteins, and their therapeutic uses, either alone or in combination with immunotherapy targeting the cell surface protein(s).

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 437,326, filed January 5, 2023, and U.S. Provisional Patent Application No. 63 / 530,217, filed August 1, 2023, each of which is incorporated by reference herein in its entirety. [Background technology]

[0002] Allogeneic hematopoietic stem / progenitor cell (HSPC) transplantation (HSCT) is currently used, at least in clinical settings, to treat high-risk forms of acute leukemia or myelodysplastic syndromes, but it results in long-term relapse-free survival of only 15–20%. Furthermore, despite recent successes of immunotherapies, their application to acute myeloid leukemia (AML) is hindered by the lack of leukemia-restricted targets. The most suitable candidates often possess affinity for targets exhibited by both diseased cells and healthy HSPCs. Therefore, the use of such candidates in AML therapy can result in immunosuppression and life-threatening hematopoietic toxicity. Finally, anti-myeloid / stem cell CAR-T-induced toxicity limits the applicability of these particular immunotherapies as salvage therapy in the limited time frame before HSCT, which may be insufficient for disease eradication.

[0003] Multiple myeloma (MM) is the second most common hematologic malignancy in adults. Despite the approval of several new therapeutic agents that have extended patient survival, MM remains largely incurable. Similar to AML, the development of immunotherapies for MM (e.g., CD38-targeted CAR-T) is limited by the fact that many surface targets are also widely expressed on hematopoietic cells.

[0004] There is a need for effective therapeutic agents that target cells of interest, such as cancer cells or host-affected hematopoietic stem cells. Most advantageously, such therapeutic agents would cause minimal damage to normal cell populations. Summary of the Invention

[0005] The present disclosure generally relates to genetically engineered hematopoietic cells, such as hematopoietic stem cells, progenitor cells, or T cells, with one or more gene-edited genes for cell surface proteins and chimeric antigen receptors capable of targeting the same cell surface proteins. In certain embodiments, the genetically engineered cells are human hematopoietic stem cells (HSCs).

[0006] Also provided herein are genetically engineered HSPCs that contain a genetically engineered KIT gene (also referred to herein as cKIT or hcKIT).

[0007] In embodiments, the engineered KIT gene is engineered so that its encoded protein has reduced binding to a therapeutic anti-KIT antibody, where the therapeutic anti-KIT antibody is SR1, or has the same six CDRs as SR1, or is otherwise capable of competing with SR1 for the KIT binding site. In some embodiments, the engineered HSPCs contain at least one mutation (typically one or two mutations) in the engineered KIT gene that results in a polypeptide with a mutation at either D121, S123, or both D121 and S123. In some embodiments, the mutation at position D121 is D121L. In some embodiments, the mutation at position S123 is S123P.

[0008] In another embodiment, the engineered KIT gene is engineered so that its encoded protein has reduced binding to a therapeutic anti-KIT antibody, where the therapeutic anti-KIT antibody is anti-KIT clone 104D2, A3C6E2, or an antibody that has the same six CDRs as anti-KIT clone 104D2 or A3C6E2, or that can otherwise compete for the KIT binding site with anti-KIT clone 104D2 or A3C6E2. In some embodiments, the engineered HSPCs contain at least one mutation in the engineered KIT gene that results in a polypeptide with a mutation at R55.

[0009] Populations of such genetically engineered cells are also provided, as are compositions and kits containing such cells.

[0010] Cells can be genetically engineered using CRISPR systems.CRISPR systems include guide nucleic acids, particularly guide RNAs, and nucleases.CRISPR systems can be base editing systems that utilize simple guide RNAs, or prime editing systems that utilize prime editing guide RNAs and optionally nicking guide RNAs.Provided herein are suitable polynucleotides that function as guide RNAs for use in base editing systems, or as prime editing or nicking guide RNAs that function in prime editing systems.

[0011] In some embodiments of the CRISPR system used to generate the genetically modified gene, the nuclease is Streptococcus pyogenes Cas9 (SpCas9), Staphylococcus aureus (SaCas9), Lachnospiraceae bacterium Cas12a (LbCas12a), or Acidaminococcus sp. BV3L6 (AsCas12a). In some embodiments, the CRISPR system comprises an SpCas9 nuclease. In some embodiments, the nuclease is a catalytically impaired SpCas9 nuclease linked to a base editor enzyme. In some embodiments, the base editor enzyme is a nucleotide deaminase. In some embodiments, the nucleotide deaminase is a cytidine deaminase or an adenosine deaminase.

[0012] Also provided are methods for treating hematological conditions (e.g., multiple myeloma, acute leukemia, or myelodysplastic syndrome, or other lymphoid and myeloid malignancies) comprising administering to a human subject (a) a population of genetically engineered hematopoietic stem / progenitor cells described herein and (b) a therapeutically effective amount of at least one agent comprising an antibody binding domain or an antibody or antibody fragment comprising the antibody binding domain.

[0013] In some embodiments of the therapeutic methods using genetically engineered cells containing a genetically engineered KIT gene, the antibody is an anti-KIT antibody. In some of these methods, the agent comprises a CAR-T cell containing an anti-KIT binding domain. In such methods, the hematological condition is multiple myeloma, acute leukemia, or myelodysplastic syndrome, or other myeloid and lymphoid malignancies, as well as non-malignant conditions.

[0014] Also provided is a chimeric antigen receptor (CAR) comprising a polypeptide. In one embodiment, the polypeptide comprises (a) one or more epitope-binding fragments that bind to epitopes of one or more cell surface lineage-specific proteins, (b) a hinge domain, (c) a transmembrane domain, (d) a costimulatory domain, and (e) a cytoplasmic signaling domain, wherein one of the cell surface lineage-specific proteins is KIT.

[0015] Also provided herein are cells expressing any one of the CARs described herein. In some embodiments, the cells are immune cells. In some embodiments, the immune cells are T cells. Compositions and kits containing such cells are also provided.

[0016] Also provided herein are methods of treating a hematological condition (e.g., a hematological malignancy), particularly multiple myeloma, comprising administering to a human subject (a) a population of genetically engineered hematopoietic stem / progenitor cells or T cells described herein, and (b) cells expressing any one of the CARs described herein.

[0017] Polypeptides formed from the engineered genes described herein are also provided, as are nucleic acids encoding the polypeptides, vectors containing the nucleic acids, and cells containing the nucleic acids or vectors. The disclosure also provides methods of making the polypeptides, comprising culturing cells under conditions that allow expression of the polypeptide, and optionally isolating the polypeptide.

[0018] definition As used herein, the terms "identity" and "identical" refer to the sequence identity between two amino acid sequences or two nucleic acid sequences. The phrases "percent identity" and "percent identical," as well as simply "identity," refer to the percentage of sequence identity found in a comparison of two or more amino acid or nucleic acid sequences. Two or more sequences can be anywhere from 0 to 100% identical, or any value therebetween. Identity can be determined by comparing positions in each sequence, which can be aligned for purposes of comparison with a reference sequence. If a position in a compared sequence is occupied by the same nucleotide base or amino acid, the molecules are identical at that position. The degree of identity between amino acid sequences is a function of the number of identical amino acids at positions shared by the amino acid sequences. The degree of identity between nucleic acid sequences is a function of the number of identical or matching nucleotides at positions shared by the nucleic acid (i.e., polynucleotide) sequences.

[0019] Methods for aligning sequences for comparison are well known in the art. Typically, one sequence serves as a reference sequence, and the test sequence is compared by aligning the residues of two sequences (for example, a candidate polypeptide or polynucleotide and a reference polypeptide or polynucleotide of a specific sequence) to optimize the number of identical amino acids or nucleotides along the length of the sequences. Gaps in either or both sequences are allowed when aligning to optimize the number of identical amino acids, but the amino acids or nucleotides in each sequence must still remain in their proper order. The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. For example, pairwise comparison analysis of sequences can be performed using the BESTFIT algorithm in the GCG package (version 10.2, Madison, WI). Alternatively, sequences can be compared using the BLAST 2 search algorithm of the Blastp program, as described by Tatiana et al. (FEMS Microbiol. Lett., 174, 247-250 (1999)) and available on the National Center for Biotechnology Information (NCBI) website. Default values ​​for all BLAST2 search parameters can be used, including matrix=BLOSUM62, open gap penalty=11, extended gap penalty=1, gap x_dropoff=50, expectation=10, word size=3, and filter on.

[0020] As used herein, the term "epitope" refers to the amino acid sequence (linear or conformational) of a protein, such as a cell surface antigen, that is bound by a complementarity determining region (CDR) of an antibody.

[0021] As used herein, "subject," "individual," and "patient" are used interchangeably and refer to a human.

[0022] As used herein, the term "effective amount" can be used interchangeably with the term "therapeutically effective amount" and refers to an amount of a cytotoxic agent, genetically engineered cell population, or pharmaceutical composition (e.g., a composition comprising a cytotoxic agent and / or genetically engineered cells) that, upon administration to a subject in need thereof, is sufficient to produce a desired activity, e.g., delay the onset of, halt the progression of, or improve, relieve, reduce, alleviate, or alleviate at least one symptom of a disorder.

[0023] As used herein, terms such as "a," "an," and "the" are not intended to refer to only a single entity, but include general classes for which specific examples may be used for illustration. The terms "a," "an," and "the" are used interchangeably with the term "at least one." The phrases "at least one of" and "including at least one of" followed by a list refer to any one of the items in the list and any combination of two or more items in the list.

[0024] As used herein, the term "or" is generally used in its ordinary sense, including "and / or," unless the context clearly dictates otherwise. The term "and / or" means one or all of the listed elements or a combination of any two or more of the listed elements.

[0025] Unless otherwise indicated, all numbers expressing quantities of ingredients, molecular weights, and the like used in the specification and claims should be understood to be modified in all instances by the term "about." Accordingly, unless specifically indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits by applying ordinary rounding techniques.

[0026] Also herein, the recitation of numerical ranges by endpoints includes all numbers subsumed within that range, as well as the endpoints (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.), and any subranges (e.g., 1 to 5 includes 1 to 4, 1 to 3, 2 to 4, etc.).

[0027] In the foregoing description, for clarity, certain embodiments may be described in isolation. Throughout this specification, references to "one embodiment," "an embodiment," "certain embodiments," or "some embodiments" mean that a particular feature, structure, composition, or characteristic described in connection with an embodiment is included in at least one embodiment of the present disclosure. Thus, the appearance of such phrases in various places throughout this specification does not necessarily refer to the same embodiment of the present disclosure. Furthermore, particular features, structures, compositions, or characteristics may be combined in any suitable manner in one or more embodiments. Thus, features described in the context of one embodiment may be combined with features described in the context of a different embodiment, unless the features are necessarily mutually exclusive.

[0028] The polynucleotide sequences described herein are described using DNA or RNA. Complements, reverse sequences, and reverse complements of DNA and RNA sequences can be easily determined by those skilled in the art and are understood to be within the scope of the present disclosure. It is also understood that sequences disclosed herein as DNA sequences can be converted from DNA sequences to RNA sequences by replacing each thymidine nucleotide (T) with a uridine nucleotide (U). When describing an RNA (e.g., a guide RNA) using a DNA sequence, it is understood that the corresponding RNA sequence is a DNA sequence in which each thymidine nucleotide (T) is replaced with a uridine nucleotide (U). For example, a guide RNA with the DNA sequence GCGTATAG has an RNA sequence of GCGUAUAG.

[0029] Polynucleotide and / or polypeptide or protein sequences may include one or more forms of typographical emphasis (e.g., underlined text, bold text, italicized text). Typographical emphasis is understood to be non-limiting. Sequences presented with typographical emphasis include sequences presented without typographical emphasis. Typographical emphasis may or may not indicate modified nucleotide bases or bonds, modified sequences relative to the presented sequence, spacers, specific codons or codons, specific amino acids or amino acids, the location of features such as primer binding sites, mutation sites, retrotranscriptase templates, complementarity-determining regions, or any combination thereof. Additionally, polynucleotide sequences may be presented in uppercase, lowercase, or a combination thereof. While letters in polynucleotide sequences may be used to distinguish portions of the sequence, the case of letters is non-limiting. Unless otherwise specified, lowercase and uppercase letters indicate the identity of nucleobases.

[0030] The above summary is not intended to describe each disclosed embodiment or every implementation thereof. The following description more particularly exemplifies exemplary embodiments. In several places throughout this application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.

[0031] In several places throughout this application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list. It is understood that the specific examples, materials, amounts, and procedures are to be broadly construed in accordance with the scope and spirit of the invention as set forth herein.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the corresponding art. Methods and materials are described herein; other suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Other features and advantages of the present invention will become apparent from the following detailed description, drawings, and claims. [Brief explanation of the drawings]

[0033] [Figure 1A] FIG. 1 is a schematic diagram of the cKIT Sleeping Beauty expression construct. [Figure 1B] 1 shows testing of different anti-human cKIT antibodies for cross-reactivity with mouse cKIT protein. [Figure 1C]1 shows the generation of a human-mouse chimeric cKIT protein for individualizing the binding domain of an anti-human cKIT antibody that does not cross-react with the mouse orthologous protein. [Figure 2A] FIG. 1 is a schematic diagram showing that orthologous mutations were grouped into three clusters, cloned, and transduced into HEK-293T cells. [Figure 2B] Fluorescence-activated cell sorting (FACS) analysis showing that one of the three subgroups (mD2_Group1) was sufficient to avoid binding of the therapeutic antibody (SR1). [Figure 2C] Figures 2A and 2B show a repetition of the approach shown, which further narrowed the group of orthologous mutations. [Figure 3A] A group of four mutations (D121G, R122L, S123P, Y125F) in the mouse cKIT protein responsible for the lack of binding of the SR1 antibody is shown, along with FACS analysis compared to the same human epitope. [Figure 3B] A bar graph comparing various human cKIT (hcKIT) mutations is shown. Mutations were cloned into a Sleeping Beauty transposon plasmid in different combinations and as single point mutations. After transduction, HEK-293T cells expressing all different variants were examined in the same flow cytometry experiment. The bar plot shows the ratio of mean fluorescence intensity (MFI) of therapeutic and control antibodies normalized to the same MFI ratio in the hcKIT WT control. [Figure 3C] A bar graph showing MFI-normalized ratios for the same mutations as in Figure 3B is shown. Because the mouse hcKIT protein is not fully cross-reactive with the human ligand, cells expressing hcKIT variants were stained and tested with fluorescently conjugated stem cell factor (SCF) ligand. The S123P hcKIT protein has reduced affinity for the SR1 antibody, which maintains binding of the human SCF ligand. [Figure 4A]Schematic diagram of the cKIT library experiment: HEK-293T cells were electroporated with a low dose of plasmid to achieve a low copy number of plasmid per cell, and then cultured with puromycin to select for positively transduced cells. [Figure 4B] FACS plot of 293T cells after puromycin selection. Most cells expressed variants recognized by both anti-cKIT antibodies, but a rare subpopulation recognized only by the control antibody (SR1-) was selected and expanded in culture. [Figure 4C] Flow cytometry experiments of four candidate amino acid (aa) perturbations that emerged through deep sequencing analysis of SR1-negative expanded cell populations are shown. The figure shows FACS analysis using MFI for both control and therapeutic antibodies for each variant. Two variants, D121L and S123P (indicated by arrows), were efficiently expressed and recognized by the control antibody but not by the SR1 antibody. [Figure 5A] 1 shows FACS plots of two library-derived variants expressed by the Sleeping Beauty transposon system in BAF3 cells and compared with variants encoding hcKIT and mouse SR1 epitopes expressed in the same cell line. [Figure 5B] Figure 1 shows the dose-affinity curves of the D121L and S123P hcKIT variants for SR1 and SCF, both conjugated to ALEXA FLUOR 647. Both variants showed similar affinity for the conjugated SCF cytokine, yet did not bind to SR1 even at higher concentrations. [Figure 6A] The location of the T to C transition in the endogenous human cKIT locus where the S123P mutation can be induced using an adenine base editor genome editing approach is shown. The table (below) shows the three different guides designed to induce the depicted transition mutation: S123P_gRNA1, S123P_gRNA2, S123P_gRNA3. [Figure 6B]Figure 6A shows a FACS analysis of cells treated to induce adenine base editing of KIT using the three sgRNAs shown in Figure 6A. This experiment was performed on K562 cells overexpressing cKIT from the endogenous locus via promoter replacement. [Figure 6C] Figure 1 shows the genome editing efficiency of three different sgRNAs in K562 cKIT on expressing cell lines. Genome editing efficiency was determined using Sanger sequencing. [Figure 7A] Schematic of the CD34+ cell growth inhibition experiment. Mobilized peripheral blood-derived CD34+ HSPCs were thawed and electroporated after 2 days with adenine base editor (ABE) mRNA and sgRNA for the mutant S123P or AAVS1 control locus. After 3 days in culture, each group was plated with increasing concentrations of SR1 antibody in the presence of SCF cytokine (125 nanograms per milliliter (ng / mL)). [Figure 7B] 1 shows a plot showing the editing efficiency of the S123P mutation 3 days after electroporation of CD34+ cells. [Figure 7C] Plots of the absolute number of total live cells (left) and CD34+ cells (right) edited for cKIT S123P or at the AAVS1 control locus on day 7 of culture in the presence of different doses of SR1 antibody are shown. Counts are normalized to the median of untreated cells for each editing group (n=4 replicates for each concentration of antibody). [Figure 8A] A schematic diagram showing an in vitro selection experiment for CD34+ cells is shown. Mobilized peripheral blood-derived CD34+ HSPCs were thawed and electroporated after 2 days with ABE mRNA and sgRNA for mutant S123P and BCL11A enhancer gRNA, or at the AAVS1 control locus. After 3 days in culture, each group was stained with CELL TRACE yellow or CELL TRACE CSFE dye, as indicated, mixed at a 1:1 ratio, and plated in SR1 at concentrations ranging from 0 nM to 3300 nM in the presence of SCF cytokines (125 ng / mL). [Figure 8B]A bar graph of the relative portion of stained cells across different concentrations of SR1 antibody after 4 days of SR1 treatment is shown. [Figure 8C] Curves showing the mean fluorescence intensity (MFI) of FITC in CELL TRACE CFSE stained cells and PE in CELL TRACE yellow stained cells are shown (n=4 replicates for each concentration of antibody). [Figure 9A] A schematic diagram showing that orthologous mutations were grouped into three clusters, cloned, and transduced into 293T cells is shown. [Figure 9B] Shown is a FACS analysis showing that one of the three subgroups was sufficient to avoid binding of two anti-hcKIT clones, 104D2 and A3C6E2. [Figure 9C] Figures 9A and 9B show a repetition of the approach shown, which further narrowed the group of orthologous mutations. [Figure 10A] Shown is a FACS analysis of a group of four mutations (E53T, I54L, R56S, L57T) in the mouse cKIT protein responsible for the lack of binding of the 104D2 / A3C6E2 antibody compared to the same human epitope. [Figure 10B] 1 shows a bar graph comparing various cKIT mutations. Mutations were cloned and tested in different combinations and as single point mutations. In the same experiment, the bar plot shows the ratio of MFI of therapeutic and control antibodies normalized to the same ratio of MFI in the cKIT WT control. [Figure 11A] 1 shows FACS analysis of NIH 3T3 cells stably transduced with the wild-type human cKIT gene (hcKIT) and Sleeping Beauty transposons carrying three candidate variants: S123P, D121L, and S123P-D121L. [Figure 11B] Figure 1 shows the MFI of cell lines expressing different cKIT variants using ALEXA FLUOR 488-conjugated stem cell factor (SCF) ligand. [Figure 12A]Figure 12A is a schematic diagram showing the prime editing approach to introduce the D121L+S123P mutation into the K562 reporter cell line, which confers resistance to anti-cKIT SR1 antibody therapy. Additionally, Figure 12A shows a cartoon representation of the prime editing protein in complex with the double-stranded gene. [Figure 12B] FACS 3 days after electroporation of a set of nine epegRNAs is shown. RTT+10 and PBS 10 / 13 / 15 were identified as the best performing PEG guides and selected for further development. [Figure 13A] A bar plot showing the editing efficiency of epegRNAss with PBS lengths of 13, 14, and 15 is shown. [Figure 13B] Figure 1 shows a bar plot depicting the effect of PAM mutations and seed sequence perturbations after editing using guides with RTT / PBS of +10 and +13. The results indicate that modifying the PAM codon significantly reduces editing efficiency, while mutations in the seed sequence can be tolerated as they did not dramatically affect editing efficiency. [Figure 13C] A table listing potential codons encoding the D121L mutation with and without the seed mutation at guide lengths of +10 to 13 and +10 to 14 is shown. [Figure 14A] A schematic diagram showing the cKIT locus targeted for editing and the spacer region of the epegRNA used in the experiment is shown. [Figure 14B] A bar graph of the editing efficiency of three cKIT epegRNAs in combination with five nicking guide variants is shown. [Figure 14C] 1 shows a bar graph illustrating the percentage of edited and knockout (KO) cells as determined by FACS analysis. [Figure 14D] FACS analysis of cell populations is shown, highlighting the effect of experimental manipulations on editing efficiency. [Figure 15A] Enumerate the set of engineered scaffolds. [Figure 15B]A bar plot of the editing efficiency of different variants of epegRNA+10 / 14 is shown, each variant having a sequential deletion of 3' nucleotides in the scaffold portion of epegRNA. [Figure 15C] A schematic representation of the secondary structure of the optimized scaffold is shown. [Figure 15D] Representative FACS plots of the editing efficiency of 10 / 14 and optimized 10 / 14 scaffolds are shown. [Figure 16A] FIG. 1 shows a schematic of a modified scaffold with a 3″ nucleotide “C” deletion. [Figure 16B] A comparison of editing efficiency (measured by FACS at D3) between modified scaffolds and 10–14 epeg guides is shown. [Figure 17] 1 shows the sequencing results of the genomic region after prime editing according to Example 14. DETAILED DESCRIPTION OF THE INVENTION

[0034] Identifying proteins suitable for targeted cancer therapy is extremely challenging. Many potential target proteins are present on both the cell surface of cancer cells and normal, non-cancerous cells, and may be involved in the development and / or survival of a subject. Many target proteins contribute to the functionality of such cells. Therefore, treatments targeting these proteins may result in adverse effects in subjects, such as significant toxicity and / or other side effects. Furthermore, resistance to chimeric antigen receptor T cell (CAR-T) therapy remains a challenge in the treatment of hematopoietic malignancies such as acute myeloid leukemia (AML) and multiple myeloma (MM), due to evasion of CAR-T therapy due to switching of cancer antigens on cancer cells. Furthermore, the identification and manipulation of appropriate stem cell markers can be utilized to improve bone marrow transplant conditioning and extend its application to non-malignant diseases. Effective immune-based conditioning may be particularly useful in autologous transplant settings for gene therapy. In particular, in this setting, therapeutic cell products can be enriched in vivo with immunotherapy if they confer a selective advantage (e.g., by subsequent administration of the same conditioning agent). In one aspect of the present disclosure, replacement of cancer cells with a modified population of normal cells is performed using normal cells that have been engineered so that the cells do not bind to cytotoxic drugs.

[0035] Thus, the present disclosure provides methods, cells, compositions, and kits aimed at addressing at least the above-mentioned problems. The methods, cells, compositions, and kits described herein provide effective treatment for hematological conditions, particularly malignant tumors, and enable targeting of one or more cell surface proteins present not only on cancer cells but also on cells essential for their development and / or survival in a subject. In some cases, described herein are genetically engineered cells (e.g., hematopoietic stem / progenitor cells (HSPCs) or T cells) with gene editing in one or more genes encoding cell surface proteins, e.g., KIT; methods for producing same using, for example, a nucleotide-guided gene editor (CRISPR) approach with specific guide RNAs; methods for treating hematopoietic conditions, particularly malignant tumors, using the engineered hematopoietic cells alone or in combination with one or more cytotoxic agents (e.g., CAR-T cells) that target wild-type cell surface antigens but not those encoded by the edited genes in the engineered hematopoietic cells; and kits comprising the engineered hematopoietic cells.

[0036] Genetically engineered cells (e.g., HSPCs) In some embodiments, the genetically engineered cells (e.g., HSPCs or T cells) have an edited KIT gene. In some embodiments, one or more of these genes are mutated. In some cases, the mutated KIT gene contains mutations or deletions in one or more non-essential epitopes such that the biological activity of the KIT gene is retained (in whole or in part).

[0037] Hematopoietic stem / progenitor cells (HSPC) In some embodiments, the hematopoietic cells described herein are hematopoietic stem / progenitor cells. Hematopoietic stem / progenitor cells (HSPCs) can give rise to both myeloid and lymphoid progenitor cells, which further give rise to myeloid cells (e.g., monocytes, macrophages, neutrophils, basophils, dendritic cells, erythrocytes, platelets, etc.) and lymphoid cells (e.g., T cells, B cells, NK cells), respectively. HSPCs are characterized by expression of the cell surface marker CD34 (e.g., CD34+), which can be used to identify and / or isolate HSPCs.

[0038] In some embodiments, the HSPCs are obtained from a human subject. In some embodiments, the human subject is a non-human primate, rodent (e.g., mouse or rat), cow, pig, horse, or livestock. In some embodiments, the HSPCs are obtained from a human patient, such as a human patient suffering from a hematopoietic malignancy. In some embodiments, the HSPCs are obtained from a healthy donor. In some embodiments, the HSPCs are obtained from a donor not suffering from a hematopoietic malignancy. In some embodiments, the HSPCs are obtained from a subject to whom the genetically engineered HSPCs will subsequently be administered. HSPCs administered to the same subject from which the cells were obtained are referred to as autologous cells. HSPCs obtained from a subject other than the subject to whom the cells will be administered are referred to as allogeneic cells. In embodiments in which the cells are allogeneic cells, the method can be modified to reduce the incidence of rejection. Methods for reducing the incidence of rejection are standard and well known in the art.

[0039] HSPCs can be obtained from any suitable source using conventional means known in the art. In some embodiments, HSPCs are obtained from a sample from a subject (or donor), such as bone marrow, blood (e.g., peripheral blood mononuclear cells (PBMCs)), and / or umbilical cord (i.e., cord blood cells). Generally, bone marrow cells can be obtained from the iliac eminence, femur, tibia, spine, ribs, or other medullary cavity of a subject (or donor). Bone marrow can be harvested from a patient and isolated through various separation and purification procedures known in the art.

[0040] HSPCs are usually present in bone marrow, but can be mobilized into the circulating blood by administering a mobilization agent to collect HSPCs from peripheral blood. In some embodiments, the subject (or donor) from which HSPCs are obtained is administered a mobilization agent such as granulocyte colony-stimulating factor (G-CSF). The number of HSPCs collected after mobilization using a mobilization agent is typically greater than the number of cells obtained without using a mobilization agent.

[0041] In some embodiments, a sample is obtained from a subject (or donor) and then enriched for a desired cell type (e.g., CD34+, CD34+CD38-, CD133+, CD90+, CD49f+). For example, PBMCs and / or CD34+ hematopoietic cells can be isolated from blood. Cells can also be isolated from other cells, for example, by isolation and / or activation with an antibody that binds to an epitope on the cell surface of the desired cell type. Another method that can be used includes negative selection using antibodies against cell surface markers that selectively enrich for a particular cell type without activating the cells by receptor binding.

[0042] Mutant cell surface antigens In some embodiments, the hematopoietic stem / progenitor cells (HSPCs) or T cells described herein may contain an edited gene encoding one or more cell surface proteins of interest (e.g., KIT) in a mutated form (mutant or variant, used interchangeably herein). The mutant may have reduced or no binding to a cytotoxic agent (e.g., an anti-KIT antibody) described herein. The mutant may include one or more mutations in the epitope to which the cytotoxic agent binds (e.g., the nucleotide sequence encoding the epitope and the amino acid sequence of the epitope), such that binding to the cytotoxic agent is reduced or eliminated compared to the native or wild-type cell surface protein counterpart. Such variants may be preferred to maintain substantially similar biological activity as the wild-type counterpart.

[0043] As used herein, the term "reduced binding" refers to binding that is reduced by at least 25%. The level of binding can refer to the amount of binding of a cytotoxic agent to hematopoietic stem cells, progenitor cells, or T cells, or the amount of binding of a cytotoxic agent to a cell surface protein, when compared to the wild-type (i.e., unengineered, unmutated) protein. In some embodiments, binding is reduced by at least 25%, at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. In some embodiments, binding is reduced such that there is substantially no detectable binding in conventional assays. As used herein, "no binding" refers to substantially no binding, e.g., no detectable binding, or only baseline binding as determined in conventional binding assays. Binding and reduced binding can be measured using quantitative fluorescence reduction, e.g., by performing fluorescence-activated cell sorting (FACS) titration.

[0044] In some cases, the variant (mutant) contains one or more amino acid residue substitutions (e.g., 1, 2, 3, 4, 5, or more) within the epitope of interest, such that the cytotoxic agent does not bind to the mutated epitope or has reduced binding to the mutated epitope. Such variants may have substantially reduced binding affinity to the cytotoxic agent (e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% lower binding affinity than their wild-type counterparts) or may have abolished binding activity to the cytotoxic agent. In other cases, the variant contains a deletion of a region containing the epitope of interest. Such a region may be encoded by an exon. In some embodiments, the region is a domain of the cell surface protein of interest that encodes the epitope. In one example, the variant has only the epitope deleted. The length of the deleted region can range from 3 to 60 amino acids, for example, 5 to 50, 5 to 40, 10 to 30, 10 to 20, 5 to 10, etc.

[0045] In some embodiments, the cytotoxic agent binds to one or more (e.g., at least 2, at least 3, at least 4, at least 5, or more) epitopes of the cell surface antigen. In some embodiments, the cytotoxic agent binds to multiple epitopes of the cell surface antigen, and the cells (e.g., HSPCs) are engineered so that each of the epitopes is absent and / or unavailable for binding by the cytotoxic agent.

[0046] The mutation(s) or deletion(s) in the variant of the cell surface antigen may be within or surrounding a non-essential epitope such that the mutation(s) or deletion(s) do not substantially affect the biological activity of the protein.

[0047] In some embodiments, the genetically engineered cells (e.g., HSPCs) described herein have one or more edited genes for a cell surface antigen, such that the edited genes express a mutant cell surface antigen with mutations in one or more non-essential epitopes. A "non-essential epitope" (or a fragment containing it) refers to a domain within a cell surface protein / antigen, the mutation of which is unlikely to substantially affect the biological activity of the cell surface protein. For example, when engineered cells (e.g., HSPCs or T cells) contain deletions or mutations in non-essential epitopes of a cell surface antigen, such engineered cells can proliferate and / or undergo erythroid differentiation to a similar level as cells expressing the wild-type cell surface antigen. Methods for identifying and / or validating non-essential epitopes in cell surface antigens are well known. Furthermore, methods for assessing cell surface antigens and functionality of engineered cells are known in the art and include, for example, proliferation assays, differentiation assays, colony formation, expression analysis (e.g., gene and / or protein), protein localization assays, intracellular signaling assays, functional assays, and studies in humanized mouse models.

[0048] Preparation of genetically engineered cells (e.g., HSPCs or T cells) Any of the genetically engineered cells (e.g., HSPCs or T cells) encoding one or more cell surface antigens can be prepared by conventional methods or by the methods described herein. In some embodiments, the genetic engineering is performed using genome editing. As used herein, "genome editing" refers to a method of modifying a genome, including any protein-coding or non-coding nucleotide sequence of an organism, to alter the expression of a target gene. Generally, genome editing methods involve the use of endonucleases capable of cleaving nucleic acids in the genome. For example, endonucleases can cleave nucleic acid sequences in the genome at target nucleotide sequences. In some cases, genome editing methods involve the use of nucleases that are catalytically "dead" nucleases or nickases. Repair of double-strand breaks in the genome often introduces mutations and / or introduces exogenous nucleic acids into the target site. In some cases, genome editing methods involve the use of catalytically inactive or partially inactive endonucleases fused to functional domains, such as, in the case of base editors, an adenine or cytidine deaminase domain. Other functional domains include reverse transcriptases, RNA-binding proteins, transcription factors, DNA repair machinery, prime editors, CRISPR-Cas activators or repressors, etc.

[0049] Genome editing methods are generally classified based on the type of endonuclease involved in generating double-strand breaks in target nucleic acids. Genome editing methods include the use of zinc finger enzymes (ZFNs), transcription activator-like effector-based nucleases (TALENs), meganucleases, and clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) protein systems. Modifications (editing) can include deletion or mutation of epitopes of specific cell surface proteins using CRISPR / Cas systems, such as CRISPR / Cas9.

[0050] CRISPR-Cas system In some embodiments, engineered HSPCs are engineered using the CRISPR system. The CRISPR system includes a guide nucleic acid and a nuclease. Cas nucleases can be easily programmed to cleave target DNA sequences for genome editing in various organisms. One class of these nucleases, called Cas9 protein or Cas9 nuclease, forms a complex with two short RNAs: crRNA and transactivating crRNA (tracrRNA). The crRNA and tracrRNA typically hybridize to form a guide RNA (gRNA). The most commonly used Cas9 ortholog, S. pyogenes cas9 (SpCas9), uses a crRNA with a 20-nucleotide (nt) "spacer" region at its 5' end that is complementary to the strand opposite the "protospacer" region of the target DNA site. SpCas9 recognizes a protospacer adjacent motif (PAM) for efficient cleavage. The crRNA and tracrRNA sequences can be joined to form a single, approximately 100-nt, single guide RNA (sgRNA, a type of gRNA) that directs the DNA cleavage activity of SpCas9. A Cas protein called Cpf1 (also called Cas12a) has been identified that can also be programmed to cleave target DNA sequences. Unlike SpCas9, Cpf1 does not contain a tracrRNA sequence but instead uses a single 42-nt crRNA, which has 23 nt at its 3' end that is complementary to the protospacer of the target DNA sequence.

[0051] In some embodiments, the Cas endonuclease is a Cas9 nuclease or a variant thereof that cleaves both strands of double-stranded DNA of the target nucleic acid, resulting in blunt ends. In some embodiments, the Cas endonuclease is a Cpf1 nuclease or a variant thereof that cleaves both strands of double-stranded DNA of the target nucleic acid, resulting in shifting of the ends of the nucleic acid.

[0052] CRISPR-Cas9 system In some embodiments, the Cas endonuclease is a Cas9 enzyme or a variant thereof. In some embodiments, the Cas9 endonuclease is derived from Streptococcus pyogenes (SpCas9) having a known (wild-type) sequence (see uniprot.org / uniprotkb / Q99ZW2 / entry, accession number AAK33936.1) or has a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to the amino acid sequence of the wild-type SpCas9 endonuclease, e.g., with up to 5%, up to 10%, up to 15%, or up to 20% difference in residues replaced with conservative mutations. In some embodiments, the Cas9 endonuclease is derived from Staphylococcus aureus (SaCas9) having a known (wild-type) sequence (see uniprot.org / uniprotkb / J7RUA5 / entry, accession number CCK74173.1) or has a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to the amino acid sequence of wild-type SaCas9, e.g., with up to 5%, up to 10%, up to 15%, or up to 20% difference, e.g., of residues replaced with conservative mutations. In preferred embodiments, the endonuclease retains the desired activity of the parent, e.g., nuclease activity (unless the parent is a nickase or death Cas9), and / or the ability to interact with guide RNA and target DNA.

[0053] As used herein, in the context of amino acid sequences, "conservative" variations (i.e., conservative substitutions) of amino acids in endonucleases or other polypeptides described herein can be selected from other members of the class to which the amino acid belongs. For example, in the field of protein biochemistry, it is well known that an amino acid belonging to a group of amino acids having a particular size or property (such as charge, hydrophobicity, and hydrophilicity) can be substituted for another amino acid without altering the activity of the protein, particularly in regions of the protein not directly related to biological activity. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and tyrosine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, or glutamine. Positively charged (basic) amino acids include arginine, lysine, or histidine. Negatively charged (acidic) amino acids include aspartic acid or glutamic acid. Conservative substitutions include, for example, Lys for Arg or vice versa to maintain a positive charge, Glu for Asp or vice versa to maintain a negative charge, Ser for Thr or vice versa to maintain a free -OH, Gln for Asn or vice versa to maintain a free -NH2. Similarly, biologically active analogs of polypeptides containing one or more consecutive or non-consecutive amino acid deletions or additions that do not eliminate the functional activity of the polypeptide are also contemplated.

[0054] Generally, the target nucleic acid is flanked on the 3' or 5' side by a protospacer adjacent motif (PAM), which can interact with the endonuclease and further participate in targeting the endonuclease activity to the target nucleic acid. Generally, the PAM sequence flanking the target nucleic acid is believed to depend, at least in part, on the endonuclease and the source from which the endonuclease is derived. For example, in the case of the Cas9 endonuclease derived from Streptococcus pyogenes, the PAM sequence is NGG, although the PAM sequences NAG and NGA can be recognized with lower efficiency (N is A, C, G, or T). In the case of the Cas9 endonuclease derived from Staphylococcus aureus, the PAM sequence is NNGRRT (N is A, C, G, or T, and R is A or G).

[0055] Thus, in some embodiments, an endonuclease is engineered / modified to recognize one or more PAM sequences. In some embodiments, the endonuclease is engineered / modified to recognize one or more PAM sequences that are different from the PAM sequences that the endonuclease recognizes without engineering / modification. In some embodiments, the endonuclease may be modified to recognize PAM sequences that lack guanine. In some embodiments, the endonuclease may be modified to recognize PAM sequences that include "ACA," "AGC," or "AAA." In some embodiments, the endonuclease is engineered / modified to reduce the off-target activity of the enzyme. In some embodiments, the nucleotide sequence encoding the endonuclease is modified to alter the PAM recognition of the endonuclease. For example, a Cas endonuclease (e.g., SpCas9) has mutations at one or more of positions A61, L1111, D1135, S1136, G1218, E1219, N1317, A1322, R1333, R1335, and T1337. See, e.g., International Patent Application Publication Nos. WO2016 / 141224 and WO2017 / 040348, and U.S. Patent Application Publication No. 2021 / 0284978A1.

[0056] In some embodiments, the Cas9 endonuclease is a catalytically inactive (i.e., catalytically impaired) Cas9. For example, dCas9 contains mutations at catalytically active residues (D10, E762, D839, H983, or D986, and / or H840 or N863) and has no nuclease activity. For example, the mutations are (i) D10A or D10N and / or (ii) H840A, H840N, or H840Y. In some embodiments, the catalytically impaired SpCas9 contains a mutation at position D10A. In some embodiments, the catalytically impaired SpCas9 contains a mutation D10N. In some embodiments, the catalytically impaired SpCas9 contains a mutation at position K918. In one or more embodiments, the catalytically impaired SpCas9 contains a mutation K918N.

[0057] In some embodiments, the nucleotide sequence encoding the Cas9 endonuclease is further modified to alter the activity of the protein. In some embodiments, the Cas9 endonuclease is modified to inactivate one or more catalytic residues of the endonuclease. In some embodiments, the Cas9 endonuclease is modified to inactivate one of the catalytic residues of the endonuclease, referred to as "nickase" or "Cas9n." The Cas9 nickase endonuclease cleaves one DNA strand of the target nucleic acid.

[0058] In some embodiments, the catalytically impaired SpCas9 is NG-SpCas9 or SpRY-SpCas9. The endonuclease NG-SpCas9 nickase has the following mutations relative to wild-type SpCas0: D10A, L1111R, D1135V, G1218R, E1219F, A1322R, R1335V, and T1337R. The endonuclease SpRY-Cas9 nickase has the following mutations relative to wild-type SpCas9: D10A, A61R, L1111R, D1135L, S1136W, G1218K, E1219Q, N1317R, A1322R, R1333P, R1335Q, and T1337R.

[0059] CRISPR Cpf1 (Cas12a) In some embodiments, the Cas endonuclease is Cpf1 nuclease (also referred to as Cas12a) or a variant thereof. Cpf1 endonuclease generally recognizes a PAM sequence located at the 5' end of the target nucleic acid. For Cpf1 nuclease, the PAM sequence is TTTN (where N is A, C, G, or T). In some embodiments, the host cell expresses a Cpf1 nuclease from Lachnospiraceae bacterium (LbCpf1), Acidaminococcus sp. (AsCpf1), or Francisella tularensis (FnCpf1). The wild-type sequences of each are known: Type V CRISPR-associated protein Cpf1 (Lachnospiraceae bacterium ND2006), GenBank accession number WP_051666128.1; Type V CRISPR-associated protein Cpf1 [Acidaminococcus sp. BV3L6], NCBI reference sequence: WP_021736722.1; and Type V CRISPR-associated protein Cpf1 (Francisella tularensis), GenBank accession number WP_003040289.1.

[0060] In some embodiments, the Cpfl endonuclease is a wild-type version of a nuclease. In some embodiments, the Cpfl endonuclease is at least 80%, at least 85%, at least 90%, or at least 95% identical in amino acid sequence to the wild-type sequence, e.g., with up to 5%, up to 10%, up to 15%, or up to 20% of the residues replaced, e.g., by conservative mutations. In some embodiments, the endonuclease retains the desired activity of the parent, e.g., nuclease activity (unless the parent is a nickase or death Cas9), and / or the ability to interact with a guide RNA and target DNA.

[0061] In some embodiments, the Cas12a endonuclease is a catalytically inactive variant, which may be referred to as dCas12a.

[0062] Cas endonuclease functional domains and the CRISPR base editing system Alternatively, or in addition, a Cas endonuclease (i.e., Cas9 or Cas12a) can be fused to another protein or portion thereof, e.g., a heterologous functional domain. In some embodiments, the heterologous functional domain is a transcriptional activation domain (e.g., VP64 or NF-KB p65). In some embodiments, the heterologous functional domain is a transcriptional silencer or transcriptional repression domain (e.g., a transcriptional repression domain is a Kruppel-associated box (KRAB) domain, an ERF repressor domain (ERD), or an mSin3A-interacting domain (SID), and a transcriptional silencer is heterochromatin protein 1 (HP1)). In some embodiments, the heterologous functional domain is an enzyme that modifies the methylation state of DNA (e.g., a DNA methyltransferase (DNMT) or a TET protein (such as TET1)). In some embodiments, the heterologous functional domain is an enzyme that modifies a histone subunit (e.g., a histone acetyltransferase (HAT), a histone deacetylase (HDAC), a histone methyltransferase (HMT), or a histone demethylase). In some embodiments, the heterologous functional domain is a biological tether (e.g., MS2, Csy4, or lambda N). In some embodiments, the heterologous functional domain is FokI.

[0063] In some embodiments, the heterologous functional domain and endonuclease form a base editor. In some such embodiments, the heterologous functional domain can be a deaminase that modifies cytosine DNA bases, such as a cytidine deaminase from apolipoprotein B mRNA editing enzyme, the catalytic polypeptide-like (APOBEC) family of deaminases (including APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D / E, APOBEC3F, APOBEC3G, APOBEC3H, APOBEC4), activation-induced cytidine deaminase (AID), cytosine deaminase 1 (CDA1), and CDA2, and cytosine deaminase acting on tRNA (CDAT). Specific examples of base editors include evoAPOBEC1-BE4max, eA3A-BE5, EA-BE4max, or the deaminases disclosed in Neugebauer, Monica, et al., Nat. Biotechnol. 1-13 (2022) and Nat. Biotechnol., 41, 673-685 (2023).

[0064] In some embodiments, the heterologous functional domain is a deaminase that modifies adenosine DNA bases, for example, the deaminase is adenosine deaminase 1 (ADA1), ADA2, adenosine deaminase acting on RNA1 (ADAR1), ADAR2; adenosine deaminase acting on ADAR3; adenosine deaminase acting on tRNA1 (ADAT1), ADAT2, ADAT3; and naturally occurring or engineered tRNA-specific adenosine deaminase (TadA). For example, ABE8e-TadA-8e. In some embodiments, the TadA adenosine deaminase domain comprises the V106W mutation.

[0065] In some embodiments, the endonuclease is a base editor. The base editor endonuclease generally comprises a catalytically inactive Cas endonuclease fused to a base editor. For example, the endonuclease is SpCas9 with mutations at D10, E762, D839, H983, or D986, and / or H840 or N863, fused to a base editor, such as those described above.

[0066] Base editors can be used in CRISPR base editing methodologies, which can directly introduce point mutations into cellular DNA without inducing double-stranded DNA breaks. For example, cytosine base editors are targeted to specific gene loci by guide RNAs, converting cytidine to uridine, which is then converted to thymidine via base excision repair, resulting in a C to T change (or a G to A change on the opposite strand). Adenine base editors convert adenosine to inosine, which is treated like guanosine by cells, resulting in an A to G (or a T to C) change. In general, base editing techniques edit targeted nucleotides without causing double-stranded breaks or relying on homology-directed repair. Such systems are commercially available (e.g., www.addgene.org) and are described, for example, in A.C. Komor et al., Nature, 533:420-424 (2016).

[0067] In some embodiments, the heterologous functional domain is an enzyme, domain, or peptide that inhibits or enhances endogenous DNA repair or base excision repair (BER) pathways, e.g., uracil DNA glycosylase inhibitor (UGI), which inhibits uracil DNA glycosylase (UDG, also known as uracil N-glycosylase or UNG)-mediated excision of uracil to initiate BER, or a DNA end-binding protein such as Gam from bacteriophage Mu.

[0068] In some cases, the endonuclease (Cas9 or Cas12a) is fused to one or more of a nuclear localization sequence, a cell-penetrating peptide sequence, an affinity tag, and / or a fluorescent protein. For example, the nuclear localization sequence is the SV40 large T antigen nuclear localization sequence (PKKKRKV, SEQ ID NO: 1), the nucleoplasmin nuclear localization sequence (KRPAATKKAGQAKKKK, SEQ ID NO: 2), or the c-Myc nuclear localization sequence (PAAKRVKLD; SEQ ID NO: 3). For example, the nuclear localization sequence(s) are fused to the N-terminus and / or C-terminus of the Cas9 or Cas12a protein. In some embodiments, when a heterologous functional domain is fused to the N-terminus and / or C-terminus of the Cas9 or Cas12a protein, the nuclear localization sequence(s) are inserted at the N-terminus and / or C-terminus of the heterologous functional domain-Cas protein complex, or between the heterologous functional domain and the Cas protein.

[0069] Exemplary Cas endonuclease sequences are provided below: SEQ ID NO:4 - Amino acid sequence of SpRY-ABE8e-V106W 3×NLS adenine base editor: SEQ ID NO:5 - Amino acid sequence of SpRY-ABE8e 3×NLS adenine base editor: SEQ ID NO:6 - Amino acid sequence of SpRY-evoAPOBEC1-BE4 3×NLS adenine base editor: SEQ ID NO:7 - Amino acid sequence of SpRY-K918N-ABE8e-V106W 3xNLS adenine base editor: SEQ ID NO:8 - Nucleotide sequence of SpRY-ABE8e-V106W 3xNLS adenine base editor: SEQ ID NO:9 - Nucleotide sequence of SpRY-evoAPOBEC1-BE4 3xNLS adenine base editor: gagaatgtgatgctgctgacctctgacgcccccgagtataagccttgggccctggtcatccaggattctaacggcgagaataagatcaagatgctgagcggaggatccggaggatctggaggcagc accaacctgtctgacatcatcgagaaggagacaggcaagcagctggtcatccaggagagcatcctgatgctgcccgaagaagtcgaagaagtgatcggaaacaagcctgagagcgatatcctggtcc ataccgcctacgacgagagtaccgacgaaaatgtgatgctgctgacatccgacgccccagagtataagccctgggctctggtcatccaggattccaacggagagaacaaaatcaaaatgctgtctgg cggctcaaaaagaaccgccgacggcagcgaattcgagcccaagaagaagaggaaagtcggcagcggaagcaaaaggccggcggccacgaaaaaggccggccaggcaaaaaagaaaaagctcgagtaa SEQ ID NO:10 - SpRY-K918N-ABE8e-V106W 3xNLS adenine base editor nucleotide sequence:

[0070] CRISPR guide RNA The terms "gRNA," "guide RNA," and "CRISPR guide sequence" are used interchangeably throughout and refer to a nucleic acid comprising a sequence that determines the specificity of the Cas DNA-binding protein of a CRISPR / Cas system. A gRNA hybridizes (e.g., is either partially or fully complementary to) a target nucleic acid sequence within the genome of a host cell and facilitates the specific association or targeting of an RNA-guided nuclease, such as Cas9 or Cpfl, to the target sequence. A gRNA can be unimolecular (comprising a single RNA molecule, alternatively referred to as a chimera or sgRNA) or modular (comprising multiple, typically two separate RNA molecules, such as a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA), which are usually associated with each other, e.g., by duplexing or hybridization). Thus, in some cases, gRNA refers collectively to both crRNA and tracrRNA (e.g., when a Cas9 nuclease is used, in those cases the guide RNA may be referred to as a single guide RNA, i.e., sgRNA). In other cases, gRNA refers to only the crRNA (e.g., when Cpf1 endonuclease is used).

[0071] Guide RNAs, whether unimolecular or modular, contain a "targeting domain" that is fully or partially complementary to a target domain within a target sequence. Targeting domains are referred to by various names in the literature, including, but not limited to, "guide sequence," "complementarity region," "spacer," and generally, "crRNA." The gRNA, or portion thereof, that hybridizes to the target nucleic acid can be 15-25 nucleotides in length, 18-22 nucleotides in length, or 19-21 nucleotides in length. In some embodiments, the gRNA sequence that hybridizes to the target nucleic acid is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the gRNA sequence that hybridizes to the target nucleic acid is 10-30 nucleotides in length, or 15-25 nucleotides in length. In some embodiments, the gRNA sequence is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to the target nucleic acid.

[0072] In addition to the targeting domain, gRNAs typically (but not necessarily) contain multiple domains that can affect the formation or activity of the Cas9 / gRNA complex. This includes, for example, one or more polyA tracts that can be recognized by RNA polymerase as termination signals, and two or more additional double-stranded regions involved in nuclease activity in vivo, but not necessarily in vitro. While this description focuses on gRNAs for use with Cas9, there are other RNA-guided nucleases that utilize gRNAs that differ in some ways from those described herein. Other gRNA designs are further described, for example, in International Publication No. WO2019 / 084168.

[0073] Those skilled in the art will understand that while structural differences may exist between gRNAs derived from different prokaryotic species, or between Cpfl and Cas9 gRNAs, the principles by which gRNAs operate are generally consistent. Because of this consistency of operation, gRNAs may be broadly defined by their targeting domain sequence, and those skilled in the art will understand that a given targeting domain sequence can be incorporated into any suitable gRNA, including monomolecular or chimeric gRNAs, or gRNAs containing one or more chemical and / or sequential modifications (substitutions, additional nucleotides, truncations, etc.). Therefore, for economy of presentation in this disclosure, gRNAs will be described only in terms of their targeting domain sequence.

[0074] Exemplary guide crRNAs for editing the KIT gene are shown in Table 1 below. As is well known, the selection of a gRNA sequence can depend on factors such as the number of predicted on-target and / or off-target binding sites. In some embodiments, the gRNA sequence is selected to maximize potential on-target sites and minimize potential off-target sites.

[0075] In some embodiments, multiple gRNAs are introduced into cells. In some embodiments, two or more guide RNAs are transfected into cells in equimolar amounts. In some embodiments, two or more guide RNAs are provided in non-equimolar amounts. In some embodiments, two or more guide RNAs are provided in amounts that are optimized to ensure that editing of each target occurs at equal frequency. In some embodiments, two or more guide RNAs are provided in amounts that are optimized to ensure that editing of each target occurs at optimal frequency.

[0076] Provided herein are polynucleotides suitable for use as guide spacer sequences having sequences at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequences set forth in Table 1 (SEQ ID NOS: 14-16). Such polynucleotides are suitable for use as crRNA segments in guide RNAs forming genetically modified KIT genes that result in polypeptides with a mutation at position S123, respectively. [Table 1]

[0077] Prime Editing System Prime editing is a nucleic acid editing system that allows for the creation of virtually any combination of point mutations (i.e., base-to-base transversions), small insertions, or small deletions in cellular DNA at desired loci. It involves targeting a "prime editor" nucleoprotein to a target site within DNA using a prime editing guide. In some embodiments, the prime editor is a fusion enzyme in which Moloney murine leukemia virus reverse transcriptase (M-MLV RT) is fused to the C-terminus of Cas9 H840A nickase. The prime editing guide is an RNA (pegRNA, PEG RNA, or engineered pegRNA (ePEG RNA)) that directs the prime editor enzyme to the target locus and encodes the desired edits. The pegRNA contains a scaffold (that binds to the prime editor), a spacer sequence (complementary to the genomic site), and an extension arm containing two domains not typically found in other guide RNAs: a primer binding site (PBS) and a reverse transcriptase template (RTT).

[0078] Like a typical gRNA, the pegRNA directs the nickase to the target site by homology to the genomic DNA locus. During prime editing, once the prime editor complexed with the pegRNA is localized to the genomic site, a polymerase (e.g., reverse transcriptase (RT)) uses a DNA synthesis template to synthesize a new strand of DNA containing the desired edit. The new strand of DNA then replaces the corresponding endogenous DNA strand at the genomic site, thereby installing the desired edited nucleotide sequence into the genome at the edit site. Such systems are commercially available (e.g., from Addgene, Cambridge, MA) and are described, for example, in U.S. Pat. No. 11,447,770 and International Publication No. WO2022 / 067130, as well as AV Anzalone et al., Nature, 576:149-157 (2019), and JL Doman et al., Nature Protocols, 17, pages 2431-2468 (2022).

[0079] There are several versions of the prime editing system. In the early version, the PE1 system, a PEG RNA directs Cas9 nickase to the target sequence, where it nicks the non-target strand and generates a 3' flap. The 3' flap binds to the primer binding site (PBS) of the PEG RNA, and the desired edit is incorporated into the DNA by reverse transcription. The edited DNA strand replaces the unedited 5' flap, and the resulting heteroduplex is resolved by the cellular mismatch repair (MMR) system. Alternatively, the edited 3' flap can be excised, leaving the target sequence unchanged but available as a substrate for another round of prime editing.

[0080] In a subsequent version, in the PE2 system, the reverse transcriptase portion of the prime editor enzyme contained five mutations (D200N, L603W, T330P, T306K, and W313F). This Cas9 nickase-quintuple mutant reverse transcriptase fusion enzyme increased activity, enhanced binding between the template and PBS, increased processivity, and improved thermostability.

[0081] In another version, the PE3 system, a PE2 Cas9 nickase-quintuple mutant reverse transcriptase fusion enzyme is used with PEG RNA plus an additional simple (e.g., no PBS or RTT) gRNA, directing the Cas9 nickase to nick the unedited (opposite) strand at a nearby site. This additional gRNA can be referred to as a nicking guide. The newly edited strand is then preferred as a template for repair during heteroduplex dissociation. In yet another version, the PE3b system, the gRNA contains a spacer that binds only to the edited strand, thereby guiding the nicking of the unedited strand only after editing has occurred. The PE2, PE3, and PE3b systems all use a PE2 Cas9 nickase-quintuple mutant reverse transcriptase fusion enzyme.

[0082] Prime-editing guide RNA The present disclosure provides polynucleotides that form prime editing guide RNAs (referred to as PEG RNA, Peg RNA, peg RNA, or pegRNA) suitable for use in prime editing CRISPR PE1, PE2, and PE3 systems to modify, for example, the cKIT gene. In some embodiments, the PEG RNA is an engineered PEG RNA (ePEG RNA). The ePEG RNA may contain a specific 3' structural motif. In some embodiments, the pegRNA RNA or ePEG RNA may be used in a prime editing CRISPR PE1, PE2, or PE3 system to mutate the cKIT gene such that the produced polypeptide has the double mutations, S123P and D121L, described herein. These polynucleotides (Peg RNA or ePEG RNA) contain a spacer complementary to the genomic site, a scaffold that binds to the prime editor, a primer binding site (PBS), and a reverse transcriptase template (RTT). In some embodiments, the PEG RNA contains a 3' structural motif. PEG RNA and ePEG RNA can be represented by the following formula: 5'-spacer-scaffold-RTT / PBS-3' structural motif.

[0083] The spacer identifies the target nucleic acid site (i.e., is complementary to the genomic site). The spacer segment can comprise 10-30 nucleotides in length, 15-25 nucleotides in length, 18-22 nucleotides in length, or 19-21 nucleotides in length. In some embodiments, the spacer is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the spacer is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to the target nucleic acid. In exemplary PEG RNAs or ePEG RNAs of the present disclosure, the spacer has the following sequence: gttgtcttctttcccataca (SEQ ID NO: 17) or cttctttcccatacaaggag (SEQ ID NO: 100) In some embodiments of the PEG RNA or ePEG RNA of the present disclosure, the spacer has a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the spacer sequence of SEQ ID NO: 17 or SEQ ID NO: 100.

[0084] The scaffold (also referred to as the core or backbone) is at least partially responsible for holding the PEG RNA or ePEG RNA together and allowing it to interact with the prime editor. A scaffold segment can comprise 50 to 105 nucleotides in length. In exemplary PEG RNAs and ePEG RNAs of the present disclosure, the scaffold has the following sequence: GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 18), or GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTG (SEQ ID NO: 19).

[0085] In some embodiments of the PEG RNA or ePEG RNA of the present disclosure, the scaffold has a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the scaffold sequence of SEQ ID NO: 18 or 19.

[0086] The 3' structural motif can protect PEG RNA or ePEG RNA from degradation in cells. Exemplary 3' structural motifs that can be included in PEG RNA or ePEG RNA are described in International Publication No. WO2022 / 067130. A particularly effective 3' structural motif is the tevopreQ1 motif, which has the sequence CCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAATTT (SEQ ID NO: 20) (JL Doman et al., Nature Protocols, 17, pages 2431-2468 (2022)). In some embodiments, when PEG RNA includes the tevopreQ1 motif (SEQ ID NO: 20) or a sequence having the sequence identity listed in SEQ ID NO: 20, the PEG RNA is referred to as an engineered PEG RNA (ePEG RNA). In some embodiments of an ePEG RNA of the present disclosure, the 3' structural motif has a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the 3' structural motif sequence of SEQ ID NO: 20. In some embodiments, the PEG RNA does not comprise SEQ ID NO: 20 or a portion thereof.

[0087] In some embodiments, the PEG RNA may contain a poly(T) sequence as a 3' structural motif. In the RNA form, the PEG RNA may contain a poly(U) sequence as a 3' structural motif. The poly(T) or poly(U) sequence may contain 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more thymine (T) or uracil (U) nucleobases. In some embodiments, the poly(T) or poly(U) sequence contains 6 thymine or 6 uracil nucleobases. One or more of the nucleotides in the poly(T) or poly(U) sequence may be modified. Examples of modifications include 2'-O-methylation of the sugar (for RNA), phosphorothioate internucleoside linkages, or both.

[0088] The reverse transcriptase template (i.e., retrotranscriptase template or RTT) provides a DNA synthesis template containing the sequence of the desired edit. The RTT segment can be 15-40 nucleotides or 20-30 nucleotides in length. In some embodiments, the RTT is 20, 23, or 26 nucleotides in length. In exemplary PEG RNAs or ePEG RNAs of the present disclosure, the RTT segment has the sequence tttccttgttctgcgccccttgt (SEQ ID NO: 21). In some embodiments of the PEG RNAs or ePEG RNAs of the present disclosure, the RTT segment has a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the RTT sequence of SEQ ID NO: 21.

[0089] A primer binding site (PBS) is attached to the 3' flap. The primer binding site segment can be 5-25 nucleotides in length or 10-15 nucleotides in length. In some embodiments, the PBS segment is 10, 13, 14, or 15 nucleotides in length. In exemplary PEG RNAs or ePEG RNAs of the present disclosure, the PBS segment has the sequence atgggaaagaagac (SEQ ID NO: 22). In some embodiments of the PEG RNAs or ePEG RNAs of the present disclosure, the PBS segment has a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the PBS sequence of SEQ ID NO: 22.

[0090] In certain embodiments, polynucleotides (PEG or ePEG RNA) of the disclosure suitable for modification of the cKIT gene, particularly those resulting in the S123P, D121L, or both mutations, have the general structure described above, wherein the RTT / PBS sequence is: ccttgttn 1 n 2 n 3 n a n b n c n 4 n 5 n 6 n 7 n 8 n 9 tatgggaaaga (RTT / PBS+7 / 10) (SEQ ID NO: 23): tttccttgttn 1 n 2 n 3 n a n b n c n 4 n 5 n 6 n 7 n 8 n 9 tatgggaaaga (RTT / PBS+10 / 10) (SEQ ID NO: 24), gcttttccttgttn 1 n2 n 3 n a n b n c n 4 n 5 n 6 n 7 n 8 n 9 tatgggaaaga (RTT / PBS+13 / 10) (SEQ ID NO: 25), ccttgttn 1 n 2 n 3 n a n b n c n 4 n 5 n 6 n 7 n 8 n 9 tatgggaaagaaga (RTT / PBS+7 / 13) (SEQ ID NO: 26), tttccttgttn 1 n 2 n 3 n a n b n c n 4 n 5 n 6 n 7 n 8 n 9 tatgggaaagaaga (RTT / PBS+10 / 13) (SEQ ID NO: 27), gcttttccttgttn 1 n 2 n 3 n a n b n c n 4 n 5 n 6 n 7 n 8 n 9 tatgggaaagaaga (RTT / PBS+13 / 13) (SEQ ID NO: 28), gcttttccttgttn 1 n 2 n 3 n a n b n c n 4 n5 n 6 n 7 n 8 n 9 tatgggaaagaagaca (RTT / PBS+7 / 15) (SEQ ID NO: 29), tttccttgttn 1 n 2 n 3 n a n b n c n 4 n 5 n 6 n 7 n 8 n 9 tatgggaaagaagaca (RTT / PBS+10 / 15) (SEQ ID NO: 30), gcttttccttgttn 1 n 2 n 3 n a n b n c n 4 n 5 n 6 n 7 n 8 n 9 tatgggaaagaagaca (RTT / PBS+13 / 15) (SEQ ID NO: 31), or tttccttgttn 1 n 2 n 3 n a n b n c n 4 n 5 n 6 n 7 n 8 n 9 tatgggaaagaagac (RTT / PBS+10 / 14) (SEQ ID NO: 32), where: n 1 n 2 n 3 represents a codon selected from ctg, tta, ttg, ctt, cta, or ctc, or n 4 n 5 n 6represents a codon selected from ccc, cct, cca, ccg, tcc, tct, tca, tcg, agt, or agc, or n 7 n 8 n 9 represents a codon selected from ttg, tta, ctt, ctc, cta, or ctg, or n a n b n c represents a codon selected from cgt, cgc, cga, cgg, aga, or agg, or It's a combination of those.

[0091] In some embodiments, n a n b n c In some embodiments, n a n b n c is agg.

[0092] In some embodiments, the polynucleotide (PEG or ePEG RNA) of the present disclosure comprises the sequence gttgtcttctttcccataca (SEQ ID NO: 17) and a spacer of the RTT / PBS sequence of any one of SEQ ID NOs: 23 to 32. a n b n c In some embodiments, n a n b n cis agg. In some embodiments, the polynucleotide (ePEG RNA) comprises a 3' structural motif comprising SEQ ID NO: 20 or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 20. In some embodiments, the polynucleotide (PEG RNA) comprises a 3' structural motif comprising a poly(T) or poly(U) sequence having 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more thymine or uracil nucleobases.

[0093] In some embodiments, the polynucleotide (PEG or ePEG RNA) of the present disclosure comprises the sequence cttctttcccatacaaggag (SEQ ID NO: 100) and a spacer of the RTT / PBS sequence of any one of SEQ ID NOs: 23 to 32. a n b n c In some embodiments, n a n b n c is agg. In some embodiments, the polynucleotide (ePEG RNA) comprises a 3' structural motif comprising SEQ ID NO: 20 or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 20. In some embodiments, the polynucleotide (PEG RNA) comprises a 3' structural motif comprising a poly(T) or poly(U) sequence having 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more thymine or uracil nucleobases.

[0094] In certain embodiments, polynucleotides (PEG or ePEG RNA) of the disclosure suitable for modification of the cKIT gene, particularly those resulting in the D121L mutation, have the general structure described above, wherein the spacer is SEQ ID NO: 100 and the RTT / PBS sequence comprises: ttccttgttn 1 n 2 n 3 n a n b n c tccttgtatgggaaag(RTT / PBS+9 / 14) (SEQ ID NO: 101), ttccttgttn 1 n 2 n 3 n a n b n c tccttgtatgggaaagaa(RTT / PBS+9 / 16) (SEQ ID NO: 102), where: n 1 n 2 n 3 represents a codon selected from ctg, tta, ttg, ctt, cta, or ctc, or n a n b n c represents a codon selected from cgt, cgc, cga, cgg, aga, or agg, or It's a combination of those.

[0095] In some embodiments, the polynucleotide (PEG or ePEG RNA) of the disclosure is an RTT / PBS sequence comprising SEQ ID NO: 101 or SEQ ID NO: 102, wherein n a n b n cis agg. In some such embodiments, the polynucleotide (ePEG RNA) comprises a 3' structural motif comprising SEQ ID NO: 20 or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 20. In other such embodiments, the polynucleotide (PEG RNA) comprises a 3' structural motif comprising a poly(T) or poly(U) sequence having 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more thymine or uracil nucleobases.

[0096] In some embodiments, the polynucleotide (PEG or ePEG RNA) of the disclosure is an RTT / PBS sequence comprising SEQ ID NO: 101 or SEQ ID NO: 102, wherein n a n b n c is cgc. In some such embodiments, the polynucleotide (ePEG RNA) comprises a 3' structural motif comprising SEQ ID NO: 20 or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 20. In other such embodiments, the polynucleotide (PEG RNA) comprises a 3' structural motif comprising a poly(T) or poly(U) sequence having 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more thymine or uracil nucleobases.

[0097] In some embodiments of the polynucleotides (PEG or ePEG RNA) of the present disclosure, the RTT / PBS segment has a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to at least one of the RTT / PBS sequences of SEQ ID NOs: 23-32).

[0098] In some embodiments of the polynucleotides (PEG or ePEG RNA) of the present disclosure, the RTT / PBS segment has a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to at least one of the RTT / PBS sequences of SEQ ID NOs: 101-102.

[0099] Examples of ePEG RNAs are shown below, with the spacer sequence in italics, the scaffold (between the spacer and the RTT / PBS sequence) and 3' structural motif in uppercase, and the PBS / RTT sequence underlined (the codon for the D121L mutant cKIT gene is in bold, the codon for the S123P mutant cKIT gene is double underlined, and the codon for the L124 mutant cKIT gene is bold and double underlined). The lengths of the primer binding site (PBS) sequence and the retrotranscriptase template (RTT) sequence were varied, and the RTT segments are identified by the number of bases after the last intended 3' edit. [ePEG RTT / PBS+7 / 10] (ePEG1) (SEQ ID NO: 33): [ka] [ePEG RTT / PBS+10 / 10] (ePEG4) (SEQ ID NO: 34): [ka] [ePEG RTT / PBS+13 / 10] (ePEG7) (SEQ ID NO: 35): [ka] [ePEG RTT / PBS+7 / 13](ePEG2) (SEQ ID NO: 36): [ka] [ePEG RTT / PBS+10 / 13] (ePEG5) (SEQ ID NO: 37): [ka] [ePEG RTT / PBS+13 / 13] (ePEG8) (SEQ ID NO: 38): [ka] [ePEG RTT / PBS+7 / 15] (ePEG3) (SEQ ID NO: 39): [ka] [ePEG RTT / PBS+10 / 15] (ePEG6) (SEQ ID NO: 40): [ka] [ePEG RTT / PBS+13 / 15](ePEG9) (SEQ ID NO: 41): [ka] [ePEG RTT / PBS+10 / 14] (SEQ ID NO: 42): [ka] Scaffold optimized [ePEG RTT / PBS+10 / 14] (SEQ ID NO: 43): [ka] Scaffold optimized [ePEG RTT / PBS+10 / 13] (SEQ ID NO: 44): [ka]

[0100] Scaffold optimized [ePEG RTT / PBS+10 / 15] (SEQ ID NO: 45): [ka]

[0101] In some embodiments, ePEG RNAs of the present disclosure suitable for modifying cKIT genes, particularly those providing the mutations S123P and D121L (preferably the double mutation), have a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to at least one of the ePEG RNA sequences set forth in SEQ ID NOs: 33-45. In one particular embodiment, a particularly effective ePEG RNA is [ePeg RTT / PBS+10 / 14] (SEQ ID NO: 42).

[0102] Examples of ePEG RNA and PEG RNA are shown below, with the spacer sequence in italics and the scaffold (between the spacer and the RTT / PBS sequence) and 3' structural motif in uppercase. The primer binding site (PBS) and retrotranscriptase template (RTT) sequences are of varying lengths, and RTT segments are distinguished by the number of bases after the last intended 3' edit. [PEG RTT / PBS+9 / 14] (ePEG-D) (SEQ ID NO: 103): cttctttcccatacaaggagGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCccttgttctgaggtccttgtatgggaaag CGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAATTT [ePEG RTT / PBS+9 / 16] (ePEG-E) (SEQ ID NO: 104): cttctttcccatacaaggagGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCtttccttgttctgagg ccttgtatgggaaagaaCGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAATTT ccttgtatgggaaaga [PEG-D(+9-14)D121L R122R] (SEQ ID NO: 105) cttctttcccatacaaggagGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCttccttgttCTGAGGtccttgtatgggaaagTTTTTT

[0103] In some embodiments, the PEG RNA or ePEG RNA of the present disclosure is suitable for modifying the cKIT gene, particularly those providing the mutation S123P, D121L, or both (preferably a double mutation), and has a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to at least one of the sequences set forth in SEQ ID NOs: 103-105. In some embodiments, the PEG RNA is [PEG-D(+9-14)D121L R122R] (SEQ ID NO: 105) or a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 105.

[0104] CRISPR nicking guide RNA The present disclosure provides polynucleotides that function as nicking guide RNAs (ng RNAs) suitable for use in, for example, a prime-editing CRISPR PE3 system. These polynucleotides include: [Nicking guide 1] (SEQ ID NO: 46) tttgggccactagtcatgaa, [Nicking guide 2] (SEQ ID NO: 47) gccattccaactactgattt, [Nicking guide 3] (SEQ ID NO: 48) ttgttn 1 n 2 n 3 n a n b n c n 4 n 5 n 6 n 7 n 8 n 9 tat, where, n 1 n 2 n 3 represents a codon selected from ctg, tta, ttg, ctt, cta, or ctc, or n 4 n 5 n 6 represents a codon selected from ccc, cct, cca, ccg, tcc, tct, tca, tcg, agt, or agc, or n 7 n 8 n 9 represents a codon selected from ttg, tta, ctt, ctc, cta, or ctg, or n a n b n c represents a codon selected from cgt, cgc, cga, cgg, aga, or agg, or It is a combination of these, [Nicking guide 4] (SEQ ID NO: 49) gtgaccaattattccctcaa, and [Nicking guide 5] (SEQ ID NO: 50) gaggtttattcctgacccca is included.

[0105] In some embodiments of the present disclosure, the polynucleotide (ngRNA) has a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to at least one of the ngRNAs of SEQ ID NOs: 46-50.

[0106] In some embodiments of the present disclosure, particularly useful ngRNAs have a sequence that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to ttgttctgcgccccttgtat (SEQ ID NO: 51). In some embodiments, the nicking guide is ttgttctgcgccccttgtat (SEQ ID NO: 51).

[0107] Genetically engineered cells (e.g., HSPCs) Provided herein are "genetically engineered cells." This refers to cells that contain polynucleotides that the cell does not naturally possess. Also provided herein are methods for producing the genetically engineered cells (e.g., HSPCs) described herein that contain edited genes to express one or more cell surface antigens in a mutant form.

[0108] Methods for producing a genetically engineered cell can include providing a cell and introducing into the cell cellular components of a nucleotide-guided gene editing system for genome editing. In some embodiments, a nucleic acid comprising a gRNA that hybridizes, or is predicted to hybridize, to a portion of a nucleotide sequence encoding a cell surface antigen is introduced into the cell. In some embodiments, the gRNA is introduced into the cell on a vector. In some embodiments, a Cas endonuclease is introduced into the cell. In some embodiments, the Cas endonuclease is introduced into the cell as a nucleic acid encoding the Cas endonuclease. In some embodiments, the gRNA and the nucleotide sequence encoding the Cas endonuclease are introduced into the cell on the same nucleic acid (e.g., the same vector). In some embodiments, the Cas endonuclease is introduced into the cell in the form of a protein. In some embodiments, the Cas endonuclease and gRNA are preformed in vitro and introduced into the cell as a ribonucleoprotein complex.

[0109] Genetically engineered cells expressing mutant KIT In some embodiments, the cell surface protein is KIT. The amino acid sequence of wild-type KIT is known (uniprot.org / uniprotkb / P10721 / entry) (Accession No. CAA29548.1).

[0110] In some embodiments, the methods described herein comprise engineering mutant KIT genes in a population of HSPCs using a nucleotide-guided gene editing system. In some embodiments, the methods described herein comprise engineering KIT by mutating positions S123 or D121 in a population of HSPCs. In some embodiments, the methods described herein comprise engineering mutant KIT genes in a population of HSPCs using a nucleotide-guided gene editing system, the guide sequence of which is provided by any one of the sequences provided herein.

[0111] In some embodiments, the methods described herein involve genetically engineering KIT by mutating positions S123 and / or D121 in a population of HSPCs using a nucleotide-guided gene editing system, such as a prime editing system.

[0112] In some embodiments, the engineered HSPCs comprise an engineered KIT gene, wherein the engineered KIT gene is engineered such that the encoded protein has reduced binding to a therapeutic anti-KIT antibody (e.g., an SR1 antibody). In some cases, the engineered KIT gene encodes a protein having a mutation at position S123 (e.g., S123P). In some cases, the engineered KIT gene encodes a protein having a mutation at position D121 (e.g., D121L). In some cases, the engineered KIT gene encodes a protein having mutations at positions S123P and D121.

[0113] An exemplary amino acid sequence of an engineered KIT is shown below: SEQ ID NO: 53 (KIT-S123P variant): MRGARGAWDFLCVLLLLLRVQTGSSQPSVSPGESPPSIHPGKSDLIVRVGDEIRLLCTDPGFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPAKLFLVDRPLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIKSVKRAYHRCLHCSVDQEGKSVLSEKFILKVRPAFKAVPVVSVSKASYLLREGEEFTVTTCTIKDVSSSVYSTWKRENSQTKLQEKYNSWHHGDFNYERQATLTISSARVNDSGVFMCYANNTFGSANVTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPEHQQWIYMNRTFTDKWEDYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVNAAIAFNVYVNTKPEILTYDRLVNGMLQCVAAGFPEPTIDWYFCPGTEQRCSASVLPVDVQTLNSSGPPFGKLVVQSSIDSSAFKHNGT VECKAYNDVGKTSAYFNFAFKGNNKEQIHPHTLFTPLLIGFVIVAGMMCIIVMILTYKYLQKPMYEVQWKVVEEINGNNYVYIDPTQLPYDHKWEFPNRLSFGKTLGAGAFGKVVEATAYGLIKSDAAMTVAVKMLKPSAHLTEREALMSELKVLSYLGNHMIVNLLGACTIGGPTLVITEYCCYGDLNLNFLRKRDSFICSKQEDHAEAALYKNLLHSKSCSDSTNEYMDMKPGVSYVV PTKADKRRSVRIGSYIERDVTPAIMEDDELALDLEDLLSFSYQVAKGMAFLASKNCIHRDLAARNILLTHGRITKICDFGLARDIKNDSNYVVKGNARLPVKWMAPESIFNCVYTFESDVWSYGIFLWELFSLGSSPYPGMPVDSKFYKMIKEGFRMLSPEHAPAEMYDIMCWDADPLKRPTFKQIVQLIEKQISESTNHIYSNLANCSPNRQKPVVDHSVRINSVGSTASSSQPLLVHDDV SEQ ID NO: 54 (KIT-D121L variant): MRGARGAWDFLCVLLLLLRVQTGSSQPSVSPGESPPSIHPGKSDLIVRVGDEIRLLCTDPGFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPAKLFLVLRSLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIKSVKRAYHRCLHCSVDQEGKSVLSEKFILKVRPAFKAVPVVSVSKASYLLREGEEFTVTTCTIKDVSSSVYSTWKRENSQTKLQEKYNSWHHGDFNYERQATLTISSARVNDSGVFMCYANNTFGSANVTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPEHQQWIYMNRTFTDKWEDYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVNAAIAFNVYVNTKPEILTYDRLVNGMLQCVAAGFPEPTIDWYFCPGTEQRCSASVLPVDVQTLNSSGPPFGKLVVQSSIDSSAFKHNGT VECKAYNDVGKTSAYFNFAFKGNNKEQIHPHTLFTPLLIGFVIVAGMMCIIVMILTYKYLQKPMYEVQWKVVEEINGNNYVYIDPTQLPYDHKWEFPNRLSFGKTLGAGAFGKVVEATAYGLIKSDAAMTVAVKMLKPSAHLTEREALMSELKVLSYLGNHMIVNLLGACTIGGPTLVITEYCCYGDLNLNFLRKRDSFICSKQEDHAEAALYKNLLHSKSCSDSTNEYMDMKPGVSYVV PTKADKRRSVRIGSYIERDVTPAIMEDDELALDLEDLLSFSYQVAKGMAFLASKNCIHRDLAARNILLTHGRITKICDFGLARDIKNDSNYVVKGNARLPVKWMAPESIFNCVYTFESDVWSYGIFLWELFSLGSSPYPGMPVDSKFYKMIKEGFRMLSPEHAPAEMYDIMCWDADPLKRPTFKQIVQLIEKQISESTNHIYSNLANCSPNRQKPVVDHSVRINSVGSTASSSQPLLVHDDV SEQ ID NO: 55 (KIT-D121L / S123P variant): MRGARGAWDFLCVLLLLLRVQTGSSQPSVSPGESPPSIHPGKSDLIVRVGDEIRLLCTDPGFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPAKLFLVLRPLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIKSVKRAYHRCLHCSVDQEGKSVLSEKFILKVRPAFKAVPVVSVSKASYLLREGEEFTVTTCTIKDVSSSVYSTWKRENSQTKLQEKYNSWHHGDFNYERQATLTISSARVNDSGVFMCYANNTFGSANVTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPEHQQWIYMNRTFTDKWEDYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVNAAIAFNVYVNTKPEILTYDRLVNGMLQCVAAGFPEPTIDWYFCPGTEQRCSASVLPVDVQTLNSSGPPFGKLVVQSSIDSSAFKHNGT VECKAYNDVGKTSAYFNFAFKGNNKEQIHPHTLFTPLLIGFVIVAGMMCIIVMILTYKYLQKPMYEVQWKVVEEINGNNYVYIDPTQLPYDHKWEFPNRLSFGKTLGAGAFGKVVEATAYGLIKSDAAMTVAVKMLKPSAHLTEREALMSELKVLSYLGNHMIVNLLGACTIGGPTLVITEYCCYGDLNLNFLRKRDSFICSKQEDHAEAALYKNLLHSKSCSDSTNEYMDMKPGVSYVV PTKADKRRSVRIGSYIERDVTPAIMEDDELALDLEDLLSFSYQVAKGMAFLASKNCIHRDLAARNILLTHGRITKICDFGLARDIKNDSNYVVKGNARLPVKWMAPESIFNCVYTFESDVWSYGIFLWELFSLGSSPYPGMPVDSKFYKMIKEGFRMLSPEHAPAEMYDIMCWDADPLKRPTFKQIVQLIEKQISESTNHIYSNLANCSPNRQKPVVDHSVRINSVGSTASSSQPLLVHDDV

[0114] In some embodiments, provided herein are polypeptide sequences that are at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to one or more of the sequences set forth in SEQ ID NOs: 53, 54, and 55, wherein the polypeptide sequences comprise mutations S123P and D121L, and the polypeptide sequences have reduced binding to therapeutic anti-KIT antibodies (e.g., SR1 antibodies). Also provided herein are nucleic acids encoding the polypeptide sequences, vectors comprising the nucleic acids, cells comprising the nucleic acids or the vectors, and methods of making the polypeptides, the methods comprising culturing cells under conditions that allow expression of the polypeptides and, optionally, isolating the polypeptides.

[0115] In some embodiments, the methods described herein comprise engineering a mutant KIT gene in a population of HSPCs using a nucleotide-guided gene editing system. In some embodiments, the methods described herein comprise engineering KIT by mutating the R55S position in a population of HSPCs. In some embodiments, the methods described herein comprise engineering a mutant KIT gene in a population of HSPCs using a nucleotide-guided gene editing system.

[0116] In some embodiments, the engineered HSPCs comprise an engineered KIT gene, where the engineered KIT gene is engineered such that the encoded protein has reduced binding to a therapeutic anti-KIT antibody (e.g., 104D2 or A3C6E2 anti-cKIT clone). In some cases, the engineered KIT gene encodes a protein with a mutation at position R55 (e.g., R55S). SEQ ID NO: 56 (KIT-R55S variant): MRGARGAWDFLCVLLLLLRVQTGSSQPSVSPGESPPSIHPGKSDLIVRVGDEISLLCTDPGFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPAKLFLVDRSLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIKSVKRAYHRCLHCSVDQEGKSVLSEKFILKVRPAFKAVPVVSVSKASYLLREGEEFTVTCTIKDVSSSVYSTWKRENSQTKLQEKYNSWHHGDFNYERQATLTISSARVNDSGVFMCYANNTFGSANVTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPEHQQWIYMNRTFTDKWEDYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVNAAIAFNVYVNTKPEILTYDRLVNGMLQCVAAGFPEPTIDWYFCPGTEQRCSASVLPVDVQTLNSSGPPFGKLVVQSSIDSSAFKHNGT VECKAYNDVGKTSAYFNFAFKGNNKEQIHPHTLFTPLLIGFVIVAGMMCIIVMILTYKYLQKPMYEVQWKVVEEINGNNYVYIDPTQLPYDHKWEFPNRLSFGKTLGAGAFGKVVEATAYGLIKSDAAMTVAVKMLKPSAHLTEREALMSELKVLSYLGNHMIVNLLGACTIGGPTLVITEYCCYGDLNLNFLRKRDSFICSKQEDHAEAALYKNLLHSKSCSDSTNEYMDMKPGVSYVV PTKADKRRSVRIGSYIERDVTPAIMEDDELALDLEDLLSFSYQVAKGMAFLASKNCIHRDLAARNILLTHGRITKICDFGLARDIKNDSNYVVKGNARLPVKWMAPESIFNCVYTFESDVWSYGIFLWELFSLGSSPYPGMPVDSKFYKMIKEGFRMLSPEHAPAEMYDIMCWDADPLKRPTFKQIVQLIEKQISESTNHIYSNLANCSPNRQKPVVDHSVRINSVGSTASSSQPLLVHDDV

[0117] In some embodiments, provided herein is a polypeptide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence set forth in SEQ ID NO: 56, wherein the polypeptide sequence comprises a mutation at R55S, and the polypeptide sequence has reduced binding to a therapeutic anti-KIT antibody (e.g., 104D2 or A3C6E2 anti-cKIT clone). Also provided herein are nucleic acids encoding the polypeptide sequences, vectors comprising the nucleic acids, cells comprising the nucleic acids or the vectors, and methods of making the polypeptides, the methods comprising culturing cells under conditions allowing expression of the polypeptide and, optionally, isolating the polypeptide.

[0118] Genetically engineered cells expressing multiple systems (e.g., HSPCs) In some embodiments, the cell surface protein KIT may be combined with other genetic engineering strategies, such as: i) other epitope editing for other target proteins; ii) other therapeutic base or prime editing approaches (e.g., BCL11A erythroid enhancer); and iii) traditional gene therapy using integrating vectors. For example, in some embodiments, this can be achieved by co-transfecting two or more guide RNAs for different target surface proteins with each other. In some embodiments, the two or more guide RNAs are provided sequentially or consecutively, i.e., in two or more separate transfections.

[0119] Immunotherapeutic agents specific to cell surface antigens Cytotoxic agents that target cells expressing a cell surface antigen (e.g., cancer cells) can be used in combination with genetically engineered cells (e.g., HSPCs) as described herein. As used herein, the term "cytotoxic agent" refers to any agent that can directly or indirectly induce cytotoxicity in target cells expressing a particular cell surface antigen (e.g., target cancer cells). Such cytotoxic agents can include protein-binding fragments that bind to and target epitopes of a particular cell surface antigen.

[0120] Therapeutic antibodies / antibody-drug conjugates As used herein, the engineered gene is engineered so that its encoded protein has reduced binding to a therapeutic antibody. In this context, a "therapeutic" antibody refers to an antibody that ameliorates one or more existing symptoms or clinical signs associated with a condition, such as a hematological condition. An "antibody" refers to a molecule containing at least one antigen-binding site that immunospecifically binds to a specific antigen target of interest. Thus, the term "antibody" includes, but is not limited to, full-length antibodies and / or variants thereof, fragments thereof, peptibodies and variants thereof, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, at least two intact antibodies, human antibodies, humanized antibodies, and multispecific antibodies (e.g., bispecific antibodies) formed from antibody mimetics that mimic the structure and / or function of an antibody or specified fragment or portion thereof, including single-chain antibodies and fragments thereof. Thus, as used herein, the term "antibody" encompasses antibody fragments capable of binding to a biological molecule (such as an antigen or receptor) or portion thereof, including, but not limited to, Fab, Fab' and F(ab')2, pFc', Fd, single domain antibodies (sdAbs), variable fragments (Fv), single-chain variable fragments (scFv) or disulfide-linked Fvs (sdFv), diabodies or bivalent diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0121] In some embodiments, the cytotoxic agent includes a therapeutic antibody, which can be conjugated to a drug (e.g., an anti-cancer drug) to form an antibody-drug conjugate (ADC). In some embodiments, the agent is an antibody-drug conjugate. In some embodiments, the antibody-drug conjugate comprises an epitope-binding fragment and a toxin or drug that induces cytotoxicity in target cells.

[0122] In some embodiments, the therapeutic anti-KIT antibody is the anti-KIT SR1 antibody or the 104D2 and A3C6E2 anti-KIT clones.

[0123] Toxins or drugs suitable for use in antibody-drug conjugates are well known in the art and will be apparent to those skilled in the art. See, e.g., Peters et al. Biosci. Rep. (2015) 35(4):e00225, Beck et al. Nature Reviews Drug Discovery (2017) 16:315-337, Marin-Acevedo et al. J. Hematol. Oncol. (2018) 11:8, Elgundi et al. Advanced Drug Delivery Reviews (2017) 122:2-19. In some embodiments, the antibody-drug conjugate may further comprise a linker (e.g., a peptide linker, such as a cleavable or non-cleavable linker) connecting the antibody and the drug molecule. Examples of antibody-drug conjugates include, but are not limited to, brentuximab vedotin, glentuximab vedotin / CDX-011, depatuximab mafodotin / ABT-414, PSMA ADC, polatuzumab vedotin / RG7596 / DCDS4501A, denintuzumab mafodotin / SGN-CD19A, AGS-16C3F, CDX-014, RG7841 / DLYE5953A, RG7882 / DMUC406A, RG7986 / DCDS0780A, SGN-LIV1A, enfortumab vedotin / ASG-22ME, AG-15ME, AGS67E, telisotuzumab vedotin / ABBV-399, ABBV-221, ABBV-085, GSK-2857916, tisotuzumab vedotin / HuMax-TF-ADC, HuMax-Axl-ADC, pinatuzumab vedotin dotin / RG7593 / DCDT2980S, rifastuzumab vedotin / RG7599 / DNIB0600A, indusatumab vedotin / MLN-0264 / TAK-264, bundutuzumab vedotin / RG7450 / DSTP3086S, sofituzumab vedotin / RG7458 / DMUC5754A, RG7600 / DMOT4039A, RG7336 / DEDN6526A, ME1547, PF-06263507 / ADC5T4, trastuzumab emtansine / T-DM1, mirvetuximab soravtansine / IMGN853, coltuximab ravtansine / SAR3419,Naratuximab emtansine / IMGN529, indatuximab ravtansine / BT-062, anetumab ravtansine / BAY94-9343, SAR408701, SAR428926, AMG224, PCA062, HKT288, LY3076226, SAR566658, lorvotuzumab mertansine / IMGN901, cantuzumab mertansine / SB-408075, cantuzumab ravtansine / IMGN242, laprituximab emtansine / IMGN289, IMGN388, bivatuzumab mertansine, AVE9633, BIIB015, MLN2704, AMG172, AMG595, LOP628, vadastuximab butarilin / SGN-CD33A, SGN-CD70A, SGN-CD19B, SGN-CD123A, SGN-CD352A, rovalpituzumab tesirin / SC16LD6.5, SC-002, SC-003, ADCT-301 / HuMax-TAC-PBD, ADCT-402, ME DI3726 / ADC-401, IMGN779, IMGN632, gemtuzumab ozogamicin, inotuzumab ozogamicin / CMC-544, PF-06647263, CMD-193, CMB-401, trastuzumab duocarmazine / SYD985, BMS-936561 / MDX-1203, sacituzumab govitecan / IMMU-132, labetuzumab govitecan / IMMU-130, DS-8201a, U3-1402, milatuzumab doxorubicin Examples of such drugs include rhetuzumab / IMMU-110 / hLL1-DOX, BMS-986148, RC48-ADC / hertuzumab-vc-MMAE, PF-06647020, PF-06650808, PF-06664178 / RN927C, rupartumab amadotin / BAY1129980, aprtuzumab ixadotin / BAY1187982, ARX788, AGS62P1, XMT-1522, AbGn-107, MEDI4276, and DSTA4637S / RG7861.

[0124] In some embodiments, binding of an antibody-drug conjugate to an epitope of a cell surface protein can induce internalization of the antibody-drug conjugate, releasing the drug (or toxin) intracellularly. In some embodiments, binding of an antibody-drug conjugate to an epitope of a cell surface protein can induce internalization of a toxin or drug, thereby allowing the toxin or drug to kill a cell expressing the cell surface protein (a target cell). In some embodiments, binding of an antibody-drug conjugate to an epitope of a cell surface protein can induce internalization of a toxin or drug, which can modulate the activity of a cell expressing the cell surface protein (a target cell). The type of toxin or drug used in the antibody-drug conjugates described herein is not limited to any particular type.

[0125] In some embodiments, two or more (e.g., two, three, four, five or more) epitopes of a cell surface antigen are modified, allowing two or more (e.g., two, three, four, five or more) different cytotoxic agents (e.g., two ADCs) to be targeted to the two or more epitopes. In some embodiments, toxins carried by the ADCs may act synergistically to enhance efficacy (e.g., target cell death). In some embodiments, two or more (e.g., two, three, four, five or more) epitopes of a cell surface protein are modified, allowing two or more (e.g., two, three, four, five or more) different cytotoxic agents (e.g., two ADCs) to target the two or more epitopes of a cell surface antigen. In some embodiments, one or more (e.g., 1, 2, 3, 4, 5, or more) epitopes of a cell surface antigen are modified, one or more (e.g., 1, 2, 3, 4, 5, or more) epitopes of an additional cell surface protein are modified, allowing two or more (e.g., 2, 3, 4, 5, or more) different cytotoxic agents (e.g., two ADCs) to be targeted to an epitope of the cell surface antigen and an epitope of the additional cell surface antigen. In some embodiments, targeting multiple cell surface antigens, or a cell surface antigen and one or more additional cell surface proteins / antigens, can reduce recurrence of hematopoietic malignancies.

[0126] In some embodiments, the methods described herein involve administering an ADC that targets an epitope of a cell surface antigen that is mutated in a population of genetically engineered hematopoietic cells. In some embodiments, the methods described herein involve administering an ADC that targets an epitope of a cell surface antigen that is mutated in a population of genetically engineered cells (e.g., HSPCs) and one or more additional cytotoxic agents that may target one or more additional cell surface proteins. In some embodiments, the agents may act synergistically to enhance efficacy by targeting multiple cell surface proteins.

[0127] The ADCs described herein may be used as a follow-on treatment for subjects who have received the combination therapy described herein.

[0128] In some embodiments, the methods described herein comprise administering to a subject a population of genetically engineered cells lacking a nonessential epitope in a cell surface antigen (e.g., Type 1 or Type 2) and one or more immunotherapeutic agents (e.g., ADCs) that target cells expressing the cell surface antigen. In any of the embodiments described herein, the subject can be further administered one or more additional immunotherapeutic agents (e.g., targeting one or more additional epitopes and / or antigens), e.g., if the hematopoietic malignancy recurs.

[0129] Immune cells expressing chimeric antigen receptors (CARs) In some embodiments, the cytotoxic agent that targets an epitope of a particular cell surface antigen described herein is an immune cell that expresses a chimeric receptor (CAR), where the CAR comprises an epitope-binding fragment (e.g., a single-chain antibody) that can bind to an epitope of a cell surface protein (e.g., KIT).

[0130] As used herein, a "chimeric antigen receptor" (CAR or simply chimeric receptor) refers to a molecule of non-natural origin that can be expressed on the surface of a host cell and contains a binding domain (e.g., an epitope-binding fragment that binds to an epitope of a cell surface lineage-specific protein) that provides the specificity of the CAR. Generally, a CAR contains at least two domains derived from different molecules.

[0131] Recognition of a target cell (e.g., a cancer cell) bearing a specific protein epitope on its cell surface by the epitope-binding fragment of the CAR transmits an activating signal to the signaling domain(s) of the CAR (e.g., a costimulatory signaling domain and / or a cytoplasmic signaling domain), which can activate effector functions in immune cells expressing the CAR.

[0132] In some embodiments, the immune cells express multiple CARs (e.g., two, three, four, five, or more), referred to as bispecific or multispecific immune cells. In some embodiments, the immune cells express multiple CARs, at least one of which targets an epitope of a cell surface antigen. In some embodiments, the immune cells express multiple CARs, each of which targets an epitope of a cell surface antigen. In some embodiments, the immune cells express multiple CARs, at least one of which targets an epitope of a cell surface antigen and at least one of which targets an epitope of an additional cell surface antigen. In some embodiments, targeting multiple cell surface proteins, or a cell surface protein and one or more additional cell surface proteins, can reduce the recurrence of hematopoietic malignancies. In some embodiments, the immune cells express a CAR that targets multiple epitopes (e.g., multiple epitopes of one antigen, or epitopes of multiple antigens), referred to as a bispecific CAR.

[0133] In some embodiments, epitopes of two or more lineage-specific cell surface proteins are targeted by a cytotoxic agent. In some embodiments, two or more CARs, e.g., bispecific chimeric receptors, are expressed on the same immune cells. Such cells can be used in any of the methods described herein. In some embodiments, cells expressing chimeric receptors are "pooled," i.e., two or more cell populations express two or more different CARs. Two or more cells expressing different CARs can be administered simultaneously or sequentially. In some embodiments, an epitope of KIT is targeted by a cytotoxic agent. In some embodiments, CARs targeting KIT are expressed on the same immune cells (i.e., bispecific immune cells). Such cells can be used in any of the methods described herein. In some embodiments, cells expressing chimeric receptors targeting KIT are "pooled," i.e., two or more cell populations express two or more different CARs. Two or more cell populations expressing CARs targeting KIT can be administered simultaneously or sequentially.

[0134] In addition to the epitope-binding fragments described herein, a CAR may further comprise one or more of the following: a hinge domain (e.g., a CD28 hinge, an IgG4 hinge, or a CD8 alpha hinge), a transmembrane domain (e.g., CD28 TM, CD8 alpha TM, 4-1BB TM), a costimulatory domain (e.g., CD28z, 4-1BB, ICOS, OX40), a cytoplasmic signaling domain (e.g., CD3z), and combinations thereof.

[0135] In some embodiments, a hinge domain may be located between the epitope-binding fragment and the transmembrane domain. A hinge domain is an amino acid segment typically found between two domains of a protein, allowing flexibility of the protein and movement of one or both of those domains relative to one another. Any amino acid sequence that provides such flexibility and movement of the epitope-binding fragment relative to another domain of the chimeric receptor may be used. The hinge domain may contain approximately 10 to 200 amino acids, e.g., 15 to 150 amino acids, 20 to 100 amino acids, or 30 to 60 amino acids. In some embodiments, the hinge domain can be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 amino acids in length.

[0136] In some embodiments, the hinge domain, or at least a portion thereof, is the hinge domain of a naturally occurring protein. In some embodiments, the transmembrane domain is derived from CD8alpha or CD28. In some embodiments, the hinge domain is a portion of the hinge domain of CD8alpha, e.g., a fragment of the hinge domain of CD8alpha or CD28 containing at least 15 (e.g., 20, 25, 30, 35, or 40) consecutive amino acids.

[0137] Hinge domains of antibodies, such as IgG, IgA, IgM, IgE, or IgD antibodies, are also compatible for use in the chimeric receptors described herein. In some embodiments, the hinge domain is the hinge domain joining the constant domains CH1 and CH2 of an antibody. In some embodiments, the hinge domain is of an antibody and comprises the hinge domain of the antibody and one or more constant regions of the antibody. In some embodiments, the hinge domain comprises the hinge domain of an antibody and the CH3 constant region of the antibody. In some embodiments, the hinge domain comprises the hinge domain of an antibody and the CH2 and CH3 constant regions of the antibody. In some embodiments, the antibody is an IgG, IgA, IgM, IgE, or IgD antibody. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the hinge region comprises the hinge region and the CH2 and CH3 constant regions of an IgG1 antibody. In some embodiments, the hinge region comprises the hinge region and CH3 constant region of an IgG1 antibody.

[0138] In some embodiments, the CAR described herein can comprise one or more transmembrane domain(s), which can be any form known in the art.As used herein, "transmembrane domain" refers to any protein structure that is thermodynamically stable in a cell membrane, preferably a eukaryotic cell membrane.The transmembrane domain that is suitable for use in the CAR used herein can be obtained from naturally occurring proteins.Alternatively, it can be a synthetic non-naturally occurring protein segment, for example, a hydrophobic protein segment that is thermodynamically stable in a cell membrane.

[0139] Transmembrane domains are classified based on transmembrane domain topology, including the number of times the transmembrane domain passes through the membrane and the orientation of the protein. For example, a single-pass membrane protein traverses the cell membrane once, while a multi-pass membrane protein traverses the cell membrane at least twice (e.g., two, three, four, five, six, seven, or more times). In some embodiments, the transmembrane domain is a single-pass transmembrane domain. In some embodiments, the transmembrane domain is a single-pass transmembrane domain that orients the N-terminus of the chimeric receptor toward the extracellular side of the cell and the C-terminus of the chimeric receptor toward the intracellular side of the cell. In some embodiments, the transmembrane domain is derived from a single-pass membrane protein. In some embodiments, the transmembrane domain is derived from CD28 or 4-1BB or CD8alpha.

[0140] In some embodiments, the CAR described herein comprises one or more costimulatory signaling domains. As used herein, the term "costimulatory signaling domain" refers to at least a portion of a protein that mediates intracellular signal transduction to induce an immune response, such as an effector function. The costimulatory signaling domain of the chimeric receptor described herein can be a cytoplasmic signaling domain from a costimulatory protein that transduces signals and regulates responses mediated by immune cells, such as T cells, NK cells, macrophages, neutrophils, or eosinophils.

[0141] In some embodiments, the CARs described herein comprise multiple (at least two, at least three, at least four, or more) costimulatory signaling domains. In some embodiments, the chimeric receptor comprises multiple costimulatory signaling domains obtained from different costimulatory proteins. In some embodiments, the chimeric receptor does not comprise a costimulatory signaling domain.

[0142] Many immune effector cells require costimulation in addition to antigen-specific signal stimulation to promote cell proliferation, differentiation, and survival and activate cellular effector functions. Activation of a costimulatory signaling domain in a host cell (e.g., an immune cell) can induce the cell to increase or decrease cytokine production and secretion, phagocytosis, proliferation, differentiation, survival, and / or cytotoxicity. The costimulatory signaling domain of any costimulatory protein may be compatible for use in the CARs described herein. The type(s) of costimulatory signaling domain(s) are selected based on factors such as the type of immune cell in which the CAR will be expressed (e.g., primary T cells, T cell lines, NK cell lines) and the desired immune effector function (e.g., cytotoxicity). Examples of costimulatory signaling domains for use in CARs may be cytoplasmic signaling domains of costimulatory proteins, including, but not limited to, CD27, CD28 zeta (CD28z), 4-1BB, OX40, CD30, ICOS, CD2, CD7, LIGHT, NKG2C, and B7-H3.

[0143] In some embodiments, the chimeric receptors described herein comprise one or more cytoplasmic signaling domain(s). Any cytoplasmic signaling domain may be used in the chimeric receptors described herein. Generally, the cytoplasmic signaling domain relays a signal, such as the interaction of an extracellular ligand-binding domain with its ligand, to stimulate a cellular response, such as inducing a cellular effector function (e.g., cytotoxicity). In some embodiments, the cytoplasmic signaling domain is derived from CD3zeta (CD3z).

[0144] In some embodiments, provided herein are CAR constructs that target KIT or other gene(s) in addition to KIT. The constructs further comprise at least a hinge domain (e.g., from CD28, CD8alpha, or an antibody), a transmembrane domain (e.g., from CD28), one or more costimulatory domains (e.g., from one or more of CD28z), a cytoplasmic signaling domain (e.g., from CD3z), or a combination thereof. In some examples, the methods described herein comprise administering to a subject a population of genetically engineered cells (e.g., HSPCs) (engineered to have mutant KIT or other gene(s) in addition to KIT, such as those disclosed in International Publication No. WO 2023 / 159136), and / or immune cells expressing CARs that target KIT or other gene(s) in addition to KIT, respectively (which may further comprise at least a hinge domain (e.g., from CD28, CD8 alpha, or an antibody), a transmembrane domain (e.g., from CD28), one or more costimulatory domains (from one or more of CD28z), and a cytoplasmic signaling domain (e.g., from CD3z), or a combination thereof). In some embodiments, the administered immunotherapy product is a combination of immune cells expressing individual chimeric receptors that target KIT.

[0145] Any of the CARs described herein can be prepared by conventional methods, such as recombinant techniques. The method for preparing a chimeric receptor herein includes generating nucleic acids encoding polypeptides containing each of the domains of the chimeric receptor, including an epitope-binding fragment and, optionally, a hinge domain, a transmembrane domain, at least one costimulatory signaling domain, and a cytoplasmic signaling domain. In some embodiments, nucleic acids encoding the components of the chimeric receptor are linked using recombinant techniques.

[0146] Additionally, any CAR can be expressed in immune cells and administered to human subjects by conventional methods. For example, T cells can be derived from the T cells in the subject's own blood (autologous) or from the T cells of another healthy donor (allogeneic). Once isolated from the subject, these T cells are genetically engineered to express a specific CAR and are programmed to target antigens present on the surface of tumors. Then, CAR-T cells are routinely infused into the subject.

[0147] In some embodiments, the CAR is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 67, and the CAR retains the ability to bind to KIT. Anti-KIT SR1 CAR of SEQ ID NO: 67: MLLLVTSLLLCELPHPAFLLIPQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGVIYSGNGDTSYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARER DTRFGNWGQGTLVTVSSGSTSGSGKPGSSEGSTKGDIVMTQSPDSLAVSLGERATINCRASESVDIYGNSFMHWYQQKPGQPPKLLIYLASNLESGVPDRFSGSGSGTDFTLTISSLQAEDVA VYYCQQNNEDPYTFGGGTKVEIKRAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRGGHSDYMNMTPRRPGPTRKHYQPY APPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0148] Target treatment method The genetically engineered cells (e.g., HSPCs) can be administered to a human subject in need of treatment either alone or in combination with one or more cytotoxic agents that target one or more cell surface antigens described herein. Because the cells have been genetically edited in the genes for one or more cell surface antigens, the cells and / or their progeny will express the one or more cell surface antigens in a mutant (e.g., functional) form such that they can avoid targeting by the cytotoxic agent.

[0149] Thus, the present disclosure provides methods for treating conditions that typically affect the wild-type morphology of engineered cells, the methods comprising administering to a human subject in need thereof (i) a population of genetically engineered cells (e.g., HSPCs) described herein and, optionally, (ii) a cytotoxic agent that targets a cell surface antigen, the gene of which has been gene-edited within the cells such that the cytotoxic agent does not target the wild-type morphology of the engineered cells or their progeny. In embodiments in which both (i) and (ii) are administered, administration of (i) and (ii) may be simultaneous or in any order. In some embodiments, the cytotoxic agent and / or cells may be mixed with a pharmaceutically acceptable carrier to form a pharmaceutical composition, which is also within the scope of the present disclosure.

[0150] To practice the methods described herein, an effective amount of genetically engineered cells (e.g., HSPCs) can be administered to a human subject in need of treatment. Optionally, the genetically engineered cells can be used in conjunction with a cytotoxic agent described herein. In some embodiments, the subject is a human patient with a hematopoietic malignancy.

[0151] As used herein, the term "effective amount" can be used interchangeably with the term "therapeutically effective amount." An effective amount, as recognized by one of ordinary skill in the art, will depend on the particular condition being treated, the severity of the condition, the age, health, size, sex, and weight, duration of treatment, the nature of concurrent therapy (if any), the particular route of administration, and similar factors within the knowledge and expertise of a medical professional.

[0152] As described herein, genetically engineered cells expressing chimeric receptors can be autologous to a subject; that is, the cells are obtained from a subject in need of treatment, engineered to prevent the cells from binding to cytotoxic drugs, and then administered to the same subject. Administration of autologous cells to a subject can result in reduced host cell rejection compared to administration of non-autologous cells. For example, HSPCs are obtained from a biological sample from the subject, the HSPCs are genetically engineered, and the genetically engineered HSPCs are administered to the same subject. In some cases, the HSPCs are obtained from a biological sample, where the biological sample is bone marrow cells, blood, umbilical cord blood cells, or mobilized peripheral blood-derived CD34+ hematopoietic stem and progenitor cells.

[0153] Alternatively, the host cells are allogeneic cells. That is, the cells are obtained from a first subject, genetically engineered, and then administered to a second subject of the same species as the first subject. For example, allogeneic immune cells can be derived from a human donor and administered to a human recipient different from the donor. In some embodiments, the engineered cells are further engineered to reduce the host-versus-graft effect. For example, in some embodiments, immune cells and / or engineered cells can be subjected to gene editing or silencing methods to reduce or eliminate the expression of one or more proteins involved in inducing a host immune response.

[0154] A typical amount of cells (i.e., immune cells or genetically engineered cells) administered to a subject is, for example, about 10 6 ~10 11 In some embodiments, the range is 106 It may be desirable to administer fewer than 10 cells to a subject. 11 It may be desirable to administer more than 10 cells to a subject. In some embodiments, one or more doses of cells comprise more than 10 6 cells ~ 10 11 cells, 10 7 cells ~ 10 10 cells, 10 8 cells ~ 10 9 cells, 10 6 cells ~ 10 8 cells, 10 7 cells ~ 10 9 cells, 10 7 cells ~ 10 10 cells, 10 7 cells ~ 10 11 cells, 10 8 cells ~ 10 10 cells, 10 8 cells ~ 10 11 cells, 10 9 cells ~ 10 10 cells, 10 9 cells ~ 10 11 cells, or 10 10 cells ~ 10 11 Contains cells.

[0155] In some embodiments, the methods described herein involve administering to a subject a population of genetically engineered cells (e.g., HSPCs) and one or more immunotherapeutic agents (e.g., cytotoxic agents). As will be appreciated by one of skill in the art, the immunotherapeutic agents can be of the same type or different types (e.g., therapeutic antibodies, populations of immune cells expressing chimeric antigen receptor(s), and / or antibody-drug conjugates).

[0156] In some embodiments, the cytotoxic agent comprising an epitope-binding fragment that binds to an epitope of a cell surface protein (e.g., an immune cell expressing a CAR described herein) is administered prior to administration of the engineered cells, which can be at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, at least 3 months, at least 4 months, at least 5 months, at least 6 months or more prior to administration of the engineered cells.

[0157] Alternatively, in some embodiments, the engineered cells are administered prior to the administration of a cytotoxic agent (e.g., an immune cell expressing a CAR described herein) comprising an epitope-binding fragment that binds to an epitope of a cell surface protein. This can be at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, at least 3 months, at least 4 months, at least 5 months, at least 6 months, or more prior to the administration of the cytotoxic agent comprising an epitope-binding fragment that binds to an epitope of a cell surface protein.

[0158] In some embodiments, the cytotoxic agent that targets a cell surface protein and the population of genetically engineered cells (HSPCs) are administered substantially simultaneously. In some embodiments, the cytotoxic agent that targets a cell surface protein is administered, the patient is evaluated for a period of time, and then the population of genetically engineered cells is administered. In some embodiments, the population of genetically engineered cells is administered, the patient is evaluated for a period of time, and then the cytotoxic agent that targets a cell surface protein is administered.

[0159] Also within the scope of the present disclosure are multiple administrations (e.g., doses) of cytotoxic agents and / or populations of genetically engineered cells. In some embodiments, the cytotoxic agents and / or populations of genetically engineered cells are administered to the subject once. In some embodiments, the cytotoxic agents and / or populations of genetically engineered cells are administered to the subject multiple times (e.g., at least two times, at least three times, at least four times, at least five times, or more). In some embodiments, the cytotoxic agents and / or populations of genetically engineered cells are administered to the subject at regular intervals, for example, every six months.

[0160] Examples of routes of administration include intravenous, infusion, intradermal, subcutaneous, oral (eg, inhalation), transdermal (topical), transmucosal, and rectal administration.

[0161] Any of the methods described herein can be for the treatment of a hematological malignancy in a subject. As used herein, the term "treat" or "treatment" or "treating" or "to treat" refers to a therapeutic measure aimed at alleviating, slowing the progression of, alleviating symptoms of, and / or halting the progression of a pathological condition or disorder. Thus, a person in need of treatment includes a person already with the disorder. As used herein, treating cancer includes stabilizing the progression of cancer, slowing the progression of cancer, stopping the progression of cancer, reducing the size of cancer, or extending the overall survival of a subject diagnosed with cancer. Methods for assessing cancer progression are known in the art and include, for example, evaluation of target lesions using imaging (e.g., X-ray, computed tomography scan, magnetic resonance imaging, caliper measurement, or positron emission tomography scan), cytology or histology, or expression of tumor marker(s).

[0162] In some embodiments, the human subject has a hematologic condition, such as a hematopoietic malignancy. As used herein, a hematopoietic malignancy refers to a malignant abnormality involving hematopoietic cells (e.g., blood cells, including progenitor cells and stem cells). Examples of hematopoietic malignancies include, but are not limited to, Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, or multiple myeloma. Exemplary leukemias include, but are not limited to, acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia or chronic lymphoblastic leukemia, and chronic lymphocytic leukemia. Examples of hematologic conditions other than hematopoietic malignancies include, but are not limited to, hemoglobin disorders, such as sickle cell disease, thalassemia, or primary immunodeficiencies, such as SCID.

[0163] In some embodiments, the cells involved in the hematopoietic malignancy are resistant to conventional or standard therapeutic agents used to treat the malignancy. For example, the cells (e.g., cancer cells) may be resistant to chemotherapeutic agents and / or CAR-T cells used to treat the malignancy.

[0164] In some cases, the hematopoietic malignancy includes high-risk acute myeloid leukemia (AML) or multiple myeloma.

[0165] Compositions and Kits Any of the immune cells expressing the chimeric receptors and / or genetically engineered cells (e.g., HSPCs) described herein can be administered as a pharmaceutical composition in a pharmaceutically acceptable carrier or excipient.

[0166] The phrase "pharmaceutically acceptable," when used in connection with compositions and / or cells of the present disclosure, refers to molecular entities and other components of such compositions that are physiologically tolerable and do not normally produce adverse reactions when administered to humans. Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopeia or other generally recognized pharmacopeia for use in humans. "Acceptable" means that the carrier is compatible with the active ingredients of the composition (e.g., nucleic acids, vectors, cells, or therapeutic antibodies) and does not adversely affect the subject to whom the composition(s) are administered. Any of the pharmaceutical compositions and / or cells used in the present methods may include pharmaceutically acceptable carriers, excipients, or stabilizers, whether in the form of a lyophilized formulation or an aqueous solution.

[0167] Pharmaceutically acceptable carriers, including buffers, are well known in the art and can include phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives; low molecular weight polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; amino acids; hydrophobic polymers; monosaccharides; disaccharides; and other carbohydrates; metal complexes; and / or non-ionic surfactants.

[0168] Kits for use in treating hematological conditions (e.g., hematopoietic malignancies) are also within the scope of the present disclosure. Such kits may include genetically engineered cells (e.g., HSPCs) and, optionally, one or more cytotoxic agents that target cell surface antigens whose genes have been edited in the hematopoietic cells. Such kits may include a container containing a first pharmaceutical composition comprising any of the genetically engineered cells (e.g., HSPCs) described herein, and, optionally, one or more additional containers containing one or more cytotoxic agents that target cell surface antigens also described herein (e.g., immune cells expressing a chimeric receptor described herein).

[0169] In some embodiments, the kit may include instructions for use in any of the methods described herein. The included instructions may include instructions for administering the genetically engineered cells (e.g., HSPCs) and, optionally, instructions for administering one or more cytotoxic agents to a subject to achieve an intended activity in the subject. The kit may further include instructions for selecting a suitable subject for treatment based on identifying whether the subject is in need of treatment. In some embodiments, the instructions include instructions for administering the genetically engineered cells (e.g., HSPCs) and, optionally, one or more cytotoxic agents to a subject in need of treatment.

[0170] The instructions associated with the use of the genetically engineered cells (e.g., HSPCs) and optionally cytotoxic agents described herein generally include information regarding the dosage, dosing regimen, and route of administration for the intended treatment. Containers may be unit doses, bulk packages (e.g., multi-dose packages), or subunit doses. Instructions provided with kits of the present disclosure are typically instructions found on a label or package insert. The label or package insert indicates that the pharmaceutical composition is used for treating, delaying the onset of, and / or alleviating a disease or disorder in a subject.

[0171] The kit provided herein is suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), etc. Packages for use in combination with specific devices, such as inhalers, nasal administration devices, or infusion devices, are also contemplated. The container holds or contains the formulation and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic injection needle). The container may also have a sterile access port. At least one active agent contained in the pharmaceutical composition is the chimeric receptor variant described herein.

[0172] Kits may optionally provide additional components such as buffers and interpretive information. Typically, kits include a container and a label or package insert(s) on or associated with the container. In some embodiments, the disclosure provides an article of manufacture comprising the contents of the kit described above. [Example]

[0173] The following specific examples are for the purpose of illustrating the invention and should not be construed as limiting the scope of the claims. To the extent that specific materials are mentioned, they are for illustrative purposes only and are not intended to limit the invention. Those skilled in the art will be able to readily develop equivalent means or reactants that do not depart from the scope of the invention using ordinary inventive skills.

[0174] Part 1) hcKIT base editing for enhanced immune-based non-genotoxic bone marrow transplant conditioning, anti-cancer immunotherapy, and in vivo selection of gene therapy cells. Example 1: Chimeric orthologous KIT variants revealed a series of mutations that circumvented SR1 binding To identify specific amino acids for suppressing SR1 clonal binding, two transgenes expressing human or mouse cKIT constructs were individually cloned into a Sleeping Beauty transfer vector coexpressing BFP and puromycin resistance, as shown in the schematic diagram in Figure 1 A. HEK-293T cells were electroporated using the Lonza 4D-Nucleofector system in SF solution containing 500 nanograms (ng) of transfer vector and 500 ng of a plasmid expressing SB100x transposase. Cells were selected with puromycin (2 micrograms per milliliter (µg / mL)), stained with KIT 79D, AB55, SR1, and KIT 104D2 control antibodies, and analyzed by flow cytometry. As shown in Figure 1B, all antibodies bound to the human cKIT protein, but only clone Ab55 was able to bind to the mouse ortholog construct. Considering this observation and to identify the domains bound by the different, non-cross-reactive antibodies, a set of five chimeric constructs, each containing one of the mouse domains in the human construct, was cloned and expressed in HEK-293T cells. As shown in Figure 1C, the set of mutations that abolished binding of the test antibodies was in domain 1 for the 104D2 (mD1) and A3C6E2 clones, but in domain 2 (mD2) for the SR1 clone.

[0175] We then classified all orthologous point mutations between mouse and human domain 2, cloned them into three different constructs, and tested them in HEK-293T cells (see Figures 2A and 2B). Figure 2A is a schematic diagram showing that the orthologous mutations were classified into three clusters, cloned, and transduced into HEK-293T cells. Figure 2B is a fluorescence-activated cell sorting (FACS) analysis showing that one of the three subgroups (mD2_Group1) was sufficient to avoid binding of a therapeutic antibody (SR1). Repeating this approach allowed us to identify a small set of four point mutations that were able to abolish SR1 binding and likely represent the epitope recognized by the antibody (see Figure 2C).

[0176] In Figure 3A, a group of four mutations (D121G, R122L, S123P, Y125F) in the mouse cKIT protein responsible for the lack of binding of the SR1 antibody is shown and compared to the same human epitope in a FACS analysis. To identify a single mutation suitable for the base editing strategy, all single orthologous point mutations and amino acids that can occur in ABE / CBE (adenine base editing / cytosine base editing) editing at the same codon were tested in the same experiment. The bar plot in Figure 3B shows the mean fluorescence intensity (MFI) ratios of the therapeutic and control antibodies normalized to the same MFI ratios in the human cKIT WT control. Because mouse cKIT is not completely cross-reactive with human ligands, the same mutations were stained and tested with fluorescently conjugated stem cell factor ligand (SCF). Interestingly, the S123P protein has reduced affinity for the SR1 antibody, maintaining binding of the human SCF ligand (Figure 3C).

[0177] Example 2: Novel mutations of SR1 binding resistance via the D2 NNN library To expand on the findings in Example 1, we designed a comprehensive library approach to further define the alternative codons involved in SR1 binding. A degenerate library composed of degenerate bases (NNN) for each codon within the KIT extracellular domain 4 was cloned into a Sleeping Beauty transfer plasmid expressing human KIT cDNA, an mTagBFP reporter, and puromycin resistance (Figure 4A). HEK-293T cells were electroporated with the library plasmid and pSB100X transposase plasmid to allow stable integration of the transgene. After puromycin selection, cells were FACS-sorted and expanded in culture to obtain a single positive population (Figure 4B). Library regions were PCR-amplified and sequenced by next-generation sequencing. Deep sequencing analysis of SR1-negative cells highlighted four candidate amino acids that were cloned, expressed, and tested along with amino acids that could be inserted at the same codon using a base editing approach (Figure 4C). Interestingly, this assay re-identified S123P as a candidate point mutation for abolishing SR1 binding and also highlighted D121L as another possible candidate.

[0178] Example 3: Characterization of SR1-resistant cKIT variants and development of base editing strategies The two identified variants were expressed using the Sleeping Beauty transposon system in BAF3 cells and compared to the hcKIT and mouse SR1 epitopes (Figure 5A). The same cell line was used for dose-affinity assays of either SR1 or SCF conjugated with ALEXA FLUOR 647. Both variants showed similar affinity for the conjugated SCF cytokine while efficiently avoiding SR1 binding, even at higher concentrations (Figure 5B). CRISPR-Cas base editing was tested in the cKIT locus to introduce the desired single codon change (T to C) with high efficiency and low toxicity without introducing double-stranded DNA breaks (Figure 6A). A panel of sgRNAs (SEQ ID NOS: 14-16, see Table 1 and Figure 6A) predicted to introduce the S123P mutation in combination with an adenine base editor was designed. The CRISPR-Cas9 base editor ABE8e (TadA-8e V106W) was selected for development of the editing strategy and further optimized by mutating the Cas9 nickase protein to relax PAM specificity to enable editing in the absence of the conventional NGG PAM. To this end, a SpRY-Cas9 variant of the base editor was cloned. To further increase efficiency, a third nuclear localization site (NLS) was fused to the C-terminal portion of the protein. Unless otherwise noted, base editing experiments were performed by electroporation of reporter K562 cells overexpressing the FLT3 gene with 500 ng of base editor plasmid and either 300 picomoles (pmol) or 360 pmol of sgRNA (Integrated DNA Technologies, Coralville, IA). Cells were then cultured, and samples for genomic DNA and flow cytometry analysis were harvested 72 hours after editing (Figures 6B and 6C).

[0179] Example 4: Editing HSCs for SR1 resistance To translate base editing procedures into primary cells, a suitable delivery method for base editors needs to be developed, as bacterial plasmid transfection is reported to be toxic to stem cells.

[0180] The base editing protocol was translated into human CD34+ HSPCs using base editor mRNA produced by in vitro transcription. Functional mRNA encoding the adenine base editor (SpRY-ABE8e-V106W 3xNLS) was produced by in vitro transcription (IVT) using the MEGASCRIPT T7 Transcription Kit (AM1333, available from Invitrogen, Carlsbad, CA) or the T7 HISCRIBE Kit (available from New England Biolabs, Ipswich, MA) and a custom plasmid (SEQ ID NO: 68) template (encoding the base editor reading frame downstream of a T7 promoter sequence, a minimal 5' UTR, and upstream of two copies of the HBB (hemoglobin B) 3' UTR and poly(A) sequence (60–120 base pairs in length). Cotranscriptional capping was achieved by replacing 80% of the GTP with the 3'-O-Me-m7G(5')ppp(5')G RNA cap analog (S1411, available from New England Biolabs). IVT reaction products were purified using either the RNAESY Mini Kit (available from Qiagen, Venlo, Netherlands) or MONARCH mRNA CLEANUP (T205L, available from New England Biolabs), quantified spectrophotometrically, and analyzed by an Agilent Fragment Analyzer (available from Agilent Technologies, Santa Clara, CA) for quality control.

[0181] To confirm the feasibility of S123P base editing in primary human CD34+ hematopoietic stem and progenitor cells, we performed in vitro base editing and expansion experiments. Mobilized peripheral blood-derived CD34+ HSPCs were thawed and cultured at 500,000–750,000 cells per mL in SFEMII medium (available from StemCell Technologies, Vancouver, Canada) supplemented with 1% penicillin / streptomycin, SCF 125 ng / mL (available from Peprotech, Cranbury, NJ), FTL3L 125 ng / mL (Peprotech), TPO 62.5 ng / mL (Peprotech), stemregenin-1 0.75 micromolar (uM) (StemCell Technologies), and UM171 35 nM (Selleckhem, Houston, TX). Forty-eight hours after thawing, 150,000–250,000 HSPCs were electroporated using the Lonza 4D-Nucleofector system in P3 electroporation solution (available from Lonza, Basel, Switzerland) supplemented with 2.5–7.5 micrograms (µg) of base-edited mRNA (SpRY-ABE8e-V106W) and 250–450 picomoles (pmol / µL) of sgRNA (Integrated DNA Technologies) per 20-microliter reaction. Cells were cultured for 5–7 days in the aforementioned medium. To test for specific resistance to the SR1 antibody, S123P-edited or AAVS1-edited CD34+ cells were expanded in vitro for 3 days after editing and then co-cultured with several doses of SR1, four times per condition.

[0182] Figure 7A shows the experimental layout and timeline for in vitro expansion and cKIT base editing of mobilized peripheral blood-derived CD34+ HSPCs. Cells were electroporated with 7.5 micrograms (µg) of SpRY-ABE8e-V106W 3xNLS mRNA and 300 pmol of S123P_gRNA_3 sgRNA or AAVS1 gRNA. Figure 7B shows the editing efficiency by Sanger sequencing 3 days after electroporation. After 4 days of culture with SR1 antibody, cell number was measured by flow cytometry and is shown in Figure 7C. pmRNA plasmid for in vitro transcription of SpRY_ABE8e_V106W adenine base editor, containing SEQ ID NO:68-5'UTR, HBB 3'UTR x 2, and a 120 bp long polyA tail:

[0183] Example 5: HSC editing for SR1-mediated in vitro selection To confirm that S123P cKIT can edit human CD34+ hematopoietic stem cells and progenitor cells can be enriched in culture in the presence of the SR1 antibody, we performed in vitro base editing and expansion experiments. Mobilized peripheral blood-derived CD34+ HSPCs were thawed and cultured at 500,000–750,000 / mL in SFEMII (StemCell Technologies) medium supplemented with 1% penicillin / streptomycin, SCF (125 ng / mL) (Peprotech), FTL3L (125 ng / mL) (Peprotech), TPO (62.5 ng / mL) (Peprotech), Stemregenin-1 (0.75 μM) (StemCell Technologies), and UM171 (35 nM) (Selleckhem). Forty-eight hours after thawing, 150,000–250,000 HSPCs were electroporated using the Lonza 4D-Nucleofector system in P3 electroporation solution (Lonza) supplemented with 2.5–7.5 μg of base-edited mRNA (SpRY-ABE8e-V106W) and 250–450 pmol of sgRNA (Integrated DNA Technologies) per 20 μL reaction. To test for specific enrichment of edited cells in the presence of SR1 antibody, S123P and BCL11A enhancer-edited or AAVS1-edited CD34+ cells were stained with CELL TRACE yellow and CFSE, respectively, mixed 50:50, expanded in vitro for 4 days after editing, and co-cultured with several doses of SR1 in four replicates per condition.

[0184] Figure 8A shows the experimental layout and timeline for in vitro expansion and cKIT base editing of mobilized peripheral blood-derived CD34+ HSPCs. Cells were electroporated with 7.5 micrograms (µg) of SpRY-ABE8e-V106W 3xNLS mRNA and 300 pmol of S123P_gRNA_3 and BCL11A +55 and +58 sgRNAs or AAVS1 gRNA. Figure 8B shows the relative percentage of cells measured by flow cytometry across different concentrations of antibody, demonstrating a selective advantage of S123P-edited cells over AAVS1 control cells in the presence of antibody. Measuring the MFI of FITC in CFSE cells and PE in CELL TRACE yellow cells can highlight possible mechanisms of enrichment. AAVS1 cells retain higher levels of dye in the presence of antibody. Thus, SR1 inhibits the proliferation of control cells but not S123P and BCL11A triple-edited cells. Flow cytometry analysis is displayed in Figure 8C.

[0185] Example 6: Chimeric orthologous KIT variants revealed a series of mutations that circumvented 104D2 / A3C6E2 binding To identify specific amino acids for abolishing 104D2 / A3C6E2 clonal binding, two transgenes expressing human or mouse cKIT constructs were individually cloned into a Sleeping Beauty transfer vector coexpressing BFP and puromycin resistance, as shown in the schematic diagram in Fig. 1 A. HEK-293T cells were electroporated using the Lonza 4D-Nucleofector system in SF solution containing 500 nanograms (ng) of transfer vector and 500 ng of a plasmid expressing SB100x transposase. Cells were selected with puromycin (2 micrograms per milliliter (µg / mL)), stained with AB55, SR1, and KIT 104D2 control antibodies, and analyzed by flow cytometry. As shown in Figure 1B, all antibodies bound to the human cKIT protein, but only clone Ab55 was able to bind to the mouse ortholog construct. Considering this observation and to identify the domains bound by the different, non-cross-reactive antibodies, a set of five chimeric constructs, each containing one of the mouse domains in the human construct, was cloned and expressed in HEK-293T cells. As shown in Figure 1C, the set of mutations that abolished binding of the test antibodies was in domain 1 for the 104D2 (mD1) and A3C6E2 clones, but in domain 2 (mD2) for the SR1 clone.

[0186] We then classified all orthologous point mutations between mouse and human domain 1 and cloned them into three different constructs, which were then tested in HEK-293T cells (see Figures 9A and 9B). Figure 9A is a schematic diagram showing that orthologous mutations were classified into three clusters, cloned, and transduced into HEK-293T cells. Figure 9B is a fluorescence-activated cell sorting (FACS) analysis showing that one of the three subgroups (mD1_Group1) was sufficient to avoid binding of therapeutic antibodies (104D2 or A3C6E2). Repeating this approach allowed us to identify a small set of four point mutations that were able to abolish binding of 104D2 or A3C6E2 and likely represent epitopes recognized by the antibodies (see Figure 9C).

[0187] In Figure 10A, a group of four mutations (E53T, I54L, R55S, L57T) in the mouse cKIT protein responsible for the lack of binding of the 104D2 or A3C6E2 antibodies are shown and compared to the same human epitope in FACS analysis. The bar plot in Figure 10B shows the ratio of mean fluorescence intensity (MFI) of the therapeutic and control antibodies for each of these mutations, normalized to the same MFI ratio in the human cKIT WT control.

[0188] Part 2) - Prime-edited double mutation Example 7: Evaluation of the human cKIT gene with a double mutation NIH 3T3 (fibroblast) cells were stably transduced with the wild-type human cKIT gene (hcKIT) and the Sleeping Beauty transposon harboring three candidate variants: S123P, D121L, and S123P-D121L. Following puromycin-mediated selection of transduced cells, transgene expression analysis was performed using fluorescence-activated cell sorting (FACS) staining with two antibodies: the cKIT control antibody 104D2 clone and the AF488-conjugated anti-hcKIT SR1 clone. Figure 11A shows FACS analysis of NIH 3T3 cells stably transduced with the wild-type human cKIT gene (hcKIT) and the Sleeping Beauty transposon harboring three candidate variants: S123P, D121L, and S123P-D121L. Notably, these findings revealed interesting differences in antibody binding patterns between the various cKIT variants. Notably, the mutation S123P exhibited relative protection against therapeutic antibody binding compared to the wild-type, indicating a clear change in the epitope recognized by the antibody. In contrast, the mutations D121L and D121L-S123P were observed to completely abolish therapeutic antibody binding in this assay, suggesting significant disruption of an epitope essential for antibody recognition.

[0189] To evaluate the responses of cell lines expressing different cKIT variants, staining was performed using AF488-conjugated stem cell factor (SCF) ligand. After SCF staining, comparative analysis was performed by measuring the mean fluorescence intensity (MFI) of SCF normalized to the MFI of an anti-cKIT control antibody. The control antibody was used to account for variations in transgene expression levels across different cell lines. Figure 11B shows the MFI of cell lines expressing different cKIT variants using ALEXA FLUOR 488-conjugated stem cell factor (SCF) ligand. Interestingly, the results revealed a sigmoidal dose-response curve for SCF activity across all variants tested, including the wild-type (WT) cKIT control. Notably, the variants exhibited dose-response curves comparable to those of the cKIT WT control, indicating similar binding affinities for SCF. This observation suggests that the introduced mutations (variants S123P, D121L, and S123P-D121L) did not significantly alter the binding properties of cKIT to SCF.

[0190] Example 8: Prime Editing (PE2) Approach and Evaluation of Various ePEG RNAs Given the potential utility of the D121L-S123P mutation in conferring resistance to anti-cKIT SR1 therapy, we explored the development of a prime-editing approach. To facilitate this investigation, we used a K562 reporter cell line that artificially expresses cKIT from its endogenous locus via promoter editing. Figure 12A is a schematic diagram illustrating the prime-editing approach for introducing the D121L+S123P mutation into the K562 reporter cell line, conferring resistance to anti-cKIT SR1 antibody therapy, and shows a cartoon representation of the prime-editing protein in complex with the double-stranded gene. This strategy enabled the screening of various engineered prime-editing guide RNAs (ePEG RNAs) to evaluate their efficacy in inducing targeted mutations in cKIT exon 3. Using this PE2 prime-editing system, we identified specific ePEG RNAs capable of conferring resistance to anti-cKIT therapy. To optimize the prime editing process, a set of nine ePEG RNAs was designed, varying the primer binding site (PBS) sequence and the length of the retrotranscriptase template (RTT), expressed as the number of bases after the final intended 3' edit (PBS 10 / 13 / 15 and RTT +7 / +10 / +13). K562 cells were electroporated with the PEmax plasmid and ePEG expression plasmid according to the protocol described in J.L. Doman et al., Nature Protocols, 17, pages 2431-2468 (2022). Three days after electroporation, cells were analyzed by FACS (Figure 12B). Among the tested PEG guides, RTT +10 and PBS 10 / 13 / 15 were selected as the top-performing PEG guides for further development.

[0191] The sequences of the nine ePEG RNAs are shown below, with the spacer sequence in italics, the scaffold and tevopreQ1 (JL Doman et al., Nature Protocols, 17, pages 2431-2468 (2022)) sequences in uppercase, and the RTT / PBS sequence underlined (the codon for the D121L mutation is bold, the codon for the S123P mutation is double underlined, and the codon for the L124 mutation is double underlined and bold). [ePEG RTT / PBS+7 / 10] (ePEG1) (SEQ ID NO: 33): [ka] [ePEG RTT / PBS+10 / 10] (ePEG4) (SEQ ID NO: 34): [ka] [ePEG RTT / PBS+13 / 10] (ePEG7) (SEQ ID NO: 35): [ka] [ePEG RTT / PBS+7 / 13](ePEG2) (SEQ ID NO: 36): [ka] [ePEG RTT / PBS+10 / 13] (ePEG5) (SEQ ID NO: 37): [ka] [ePEG RTT / PBS+13 / 13] (ePEG8) (SEQ ID NO: 38): [ka] [ePEG RTT / PBS+7 / 15] (ePEG3) (SEQ ID NO: 39): [ka] [ePEG RTT / PBS+10 / 15] (ePEG6) (SEQ ID NO: 40): [ka] [ePEG RTT / PBS+13 / 15](ePEG9) (SEQ ID NO: 41): [ka]

[0192] Example 9: Optimization of PBS length for ePEG RNA To further refine the PBS sequence length of the ePEG RNA, intermediate values ​​of 13 to 15, specifically 14, were tested, and three selected ePEGs were reevaluated in subsequent experiments: [ePeg RTT / PBS+10 / 13] (ePEG5) (SEQ ID NO: 37), [ePeg RTT / PBS+10 / 15] (ePEG6) (SEQ ID NO: 40), and the following SEQ ID NO: 42. [ePEG RTT / PBS+10 / 14] (SEQ ID NO: 42): [ka]

[0193] Editing efficiency was determined by identifying the % of SR1-negative cells by FACS analysis 3 days after electroporation. The results, shown in Figure 13A, which is a bar plot showing the editing efficiency of ePEG RNAs with PBS lengths of 13, 14, and 15, demonstrate that ePEG with a PBS length of 14 exhibited the highest efficiency in editing the K562 reporter cell line.

[0194] Example 10: Evaluation of PAM mutations and seed sequence perturbations after editing To explore the post-editing effects of PAM mutations and seed sequence perturbations, experiments were conducted using ePEG RNAs with RTT / PBS of +10 and +13. Different codons for amino acid S123P were tested to encode the PAM sequence on the opposite (non-coding) strand. Modifications to the codon for the amino acid at position L124 were tested to perturb the seed sequence. Additionally, the combined effect of both perturbations was examined. As shown in Figure 13B and the bar plot in Table A (below), the results indicate that modifying the PAM codon significantly reduces editing efficiency. Mutations in the seed sequence may be tolerated, as they did not dramatically affect editing efficiency. These findings suggest that although post-editing PAM mutations negatively affect editing efficiency, seed sequence perturbations may still be tolerated and may be of use in future applications. [Table 2]

[0195] To explore perturbations of mutation D121, all possible codons encoding L amino acids were tested with and without seed mutations at guide lengths of +10 to +13 and +10 to +14. See the table in Figure 13C. This analysis led to the development of several ePEG RNAs that demonstrate the potential to manipulate hematopoietic stem cells (HSCs). These findings highlight the potential of utilizing these ePEG RNAs for targeted modification in HSCs.

[0196] Example 11: Development of PE3 and PE3b CRISPR systems To develop the PE3 system, we added simple guide RNAs to direct Cas9 nickase to nick the unedited DNA strand at nearby sites. Five nicking guides were designed. These ng RNAs included: [Nicking guide 1] (SEQ ID NO: 46) tttgggccactagtcatgaa, [Nicking guide 2] (SEQ ID NO: 47) gccattccaactactgattt, [Nicking guide 3] (SEQ ID NO: 115) ttgttn 1 n 2 n 3 cgcn 4 n 5 n 6 n 7 n 8 n 9 tat, where, n 1 n 2 n 3 represents a codon selected from ctg, tta, ttg, ctt, or ctc, or n 4 n 5 n 6 represents a codon selected from ccc, cct, cca, or ccg, or n 7 n 8 n 9 represents a codon selected from ttg, tta, ctt, ctc, or ctg, or ttgttctgcgccccttgtat (SEQ ID NO: 51), and combinations thereof. [Nicking guide 4] (SEQ ID NO: 49) gtgaccaattattccctcaa, and [Nicking guide 5] (SEQ ID NO: 50) gaggtttattcctgacccca is included.

[0197] Nicking guides 1 and 2 were positioned 5' to the intended edit, one complementary to the edited strand after prime editing (PE3b system), and nicking guides 4 and 5 were positioned 3' to the edit. See Figure 14A for a schematic showing the cKIT locus targeted for editing.

[0198] Figure 14B is a graph showing editing efficiency on day 3 (D3). Evaluation of editing efficiency was performed using FACS analysis, focusing on three previously characterized cKIT ePEG RNAs (ePEG2, ePEG4, and ePEG5) in combination with five nicking guide variants. Among the nicking guides (ng) tested, i.e., guides 3, 4, and 5, the data show that ng3 exhibits the highest efficacy in terms of editing efficiency. Notably, ng3 is also used as part of the PE3b strategy, which targets the flap region encoding the mutation after prime editing. This strategic use of ng3 aims to minimize the occurrence of double-strand breaks within the bulk population of edited cells.

[0199] A more comprehensive characterization of the combination of nicking guide 3 (ng3) and ePEG+10 / 14 was performed. Cells were electroporated with increasing doses of both the PEmax and ePEG plasmids. The bar plot in Figure 14C shows the percentage of edited and knockout (KO) cells as determined by FACS analysis on day 3 (D3).

[0200] The FACS plot in Figure 14D illustrates the results obtained under the highest dose conditions. This plot provides a visual representation of the cell population and highlights the effect of experimental manipulations on editing efficiency. Together, Figures 14C and 14D provide a comprehensive assessment of the impact of ng3 and ePEG+10 / 14 on editing efficiency and KO cells. These findings strongly support the notion that combining cKIT ePEG RNA with ng3 results in improved editing efficiency. Furthermore, the application of ng3 in the PE3b strategy highlights its effectiveness in reducing the occurrence of double-strand breaks, thereby enhancing the overall accuracy and efficacy of the editing process.

[0201] Example 12: Engineering the Scaffold of 10 / 14 ePEG RNA During large-scale ePEG RNA screening, it was discovered that scaffold engineering can enhance the editing efficiency of these ePEG RNAs. To further explore this, a set of engineered scaffolds was generated using ePEG RTT / PBS+10 / 14 (SEQ ID NO: 42) (also referred to as "10 / 14 ePEG RNA"), as shown in the schematic diagram in Figure 15A (only a portion of each modified ePEG sequence is shown).

[0202] The K562 reporter cell line was electroporated with different variants of ePEG RTT / PBS+10 / 14 (SEQ ID NO: 42), each with a sequential deletion of 3' nucleotides in the scaffold portion of the ePEG RNA. The bar plot in Figure 15B shows the editing efficiency on day 3 as measured by FACS analysis. Deletion of the last scaffold nucleotide appears to improve the efficiency of this guide. Figure 15C shows a schematic diagram of the secondary structure of the optimized scaffold (one 3' nucleotide deletion). Figure 15D shows a representative FACS plot of the editing efficiency of ePEG RTT / PBS+10 / 14 (SEQ ID NO: 42) and ePEG RTT / PBS+10 / 14 with the optimized scaffold (SEQ ID NO: 43). Scaffold optimized [ePEG RTT / PBS+10 / 14] (SEQ ID NO: 43): [ka]

[0203] Example 13: Engineering additional ePEG RNA scaffolds Figure 16A shows a schematic of the modified scaffold utilized in this study. The modified scaffold was strategically engineered to contain a deletion of its final 3' nucleotide, specifically the base "C." This targeted modification alters the sequence at its 3' end and was therefore investigated to have significant implications for the functionality of the scaffold. In Figure 16B, the efficiency of the editing process on the modified scaffold was evaluated at day 3 (D3) after treatment. Editing efficiency measurements were performed using FACS analysis. The results were then compared to those obtained with the 10 / 14 ePEG RNA, which was identified as a highly promising candidate for editing. This analysis highlights the impact of the deletion of the 3' nucleotide "C" on the editing process and provides valuable insight into the performance of the modified scaffold as a potential tool for genetic engineering. Scaffold optimized [ePEG RTT / PBS+10 / 13] (SEQ ID NO: 44): [ka] Scaffold optimized [ePEG RTT / PBS+10 / 15] (SEQ ID NO: 45): [ka]

[0204] Example 14: HSC prime editing for the cKIT D121L mutation To test whether the prime-editing strategy could induce mutations in human CD34+ hematopoietic stem and progenitor cells, we performed an in vitro prime-editing experiment. Mobilized peripheral blood-derived CD34+ HSPCs were thawed and cultured at 500,000–750,000 / mL in StemCell SFEMII medium supplemented with 1% penicillin / streptomycin, SCF (125 ng / mL, Peprotech), FTL3L (125 ng / mL, Peprotech), TPO (62.5 ng / mL, Peprotech), Stemregenin-1 (0.75 μM, StemCell Technologies), and UM171 (35 nM, StemCell). Twenty-four hours after thawing, 150,000–250,000 HSPCs were electroporated using the Lonza 4D-Nucleofector System in P3 electroporation solution (Lonza) supplemented with PEmax mRNA, using 2,000 ng of mRNA along with 200 pmol of synthetic epegRNA (sequence shown below) and 100 pmol of nicked sgRNA. Subsequently, electroporated HSPCs were split into two wells and cultured for 72 hours. Genomic DNA was harvested 3 days after nucleofection. For c-Kit PEGRNA-D (sequence shown below), the specific mutation introduced was D121L / R122R, and prime editing (PE) efficiency was quantified by Sanger sequencing of the relevant genomic region (Figure 17). [PEG-D(+9-14)D121L R122R] SEQ ID NO: 105 cttctttcccatacaaggagGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCttccttgttCTGAGGtccttgtatgggaaagTTTTTT

Claims

1. A genetically engineered hematopoietic stem / progenitor cell (HSPC) comprising a genetically engineered KIT gene, wherein the genetically engineered KIT gene is engineered such that its encoded protein has reduced binding to a therapeutic anti-KIT antibody, and the therapeutic anti-KIT antibody is SR1 or has the same six CDRs as SR1, or is otherwise an antibody capable of competing with SR1 for the KIT binding site.

2. 2. The genetically engineered HSPC of claim 1, wherein at least one mutation in the genetically engineered KIT gene results in a polypeptide having a mutation at D121, S123, or both D121 and S123.

3. 3. The genetically engineered HSPC of claim 2, wherein the mutation at position D121 is D121L.

4. 3. The genetically engineered HSPC of claim 2, wherein the mutation at position S123 is S123P.

5. 4. The genetically engineered HSPC of any one of claims 1 to 3, wherein the genetically engineered KIT gene encodes a polypeptide comprising the amino acid sequence of any one of SEQ ID NOs: 53, 54, or 55, or a polypeptide that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of any one of SEQ ID NOs: 53, 54, or 55.

6. 1. A genetically engineered hematopoietic stem cell (HSPC) comprising a genetically engineered KIT gene, wherein the genetically engineered KIT gene has been engineered such that its encoded protein has reduced binding to a therapeutic anti-KIT antibody, wherein the therapeutic anti-KIT antibody is anti-KIT clone 104D2, A3C6E2, or an antibody that has the same six CDRs as anti-KIT clone 104D2 or A3C6E2, or is otherwise capable of competing with anti-KIT clone 104D2 or A3C6E2 for the KIT binding site.

7. 7. The genetically engineered HSPC of claim 6, wherein at least one mutation in the genetically engineered KIT gene results in a polypeptide having a mutation at R55.

8. The genetically engineered HSPC of claim 7, wherein the mutation at position R55S is

9. 9. The genetically engineered HSPC of any of claims 6 to 8, wherein the genetically engineered KIT gene encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:56, or a polypeptide that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO:

56.

10. The genetically engineered HSPC of any one of claims 1 to 9, wherein the genetically engineered HSPC is genetically engineered using a CRISPR system comprising a guide nucleic acid and a nuclease.

11. A population of genetically engineered hematopoietic stem / progenitor cells (HSPCs), comprising the genetically engineered HSPCs of any one of claims 1 to 10.

12. 12. A pharmaceutical composition comprising the population of genetically engineered hematopoietic stem / progenitor cells of claim 11 and a pharmaceutically acceptable carrier.

13. 12. A kit comprising the population of genetically engineered hematopoietic stem / progenitor cells of claim 11 and, optionally, one or more cytotoxic agents that target cell surface antigens whose genes have been edited in the hematopoietic stem / progenitor cells.

14. 1. A method of treating a hematological condition, the method comprising administering to a human subject: (a) a population of genetically engineered hematopoietic stem / progenitor cells according to claim 11; and (b) administering a therapeutically effective amount of at least one agent comprising an anti-KIT antibody binding domain or an antibody or antibody fragment comprising an anti-KIT binding domain.

15. 15. The method of claim 14, wherein the hematological condition is acute myeloid leukemia (AML).

16. 12. A method for improving bone marrow transplant conditioning, the method comprising administering to a human subject the population of genetically engineered hematopoietic stem / progenitor cells of claim 11.

17. A chimeric antigen receptor (CAR) comprising a polypeptide, the polypeptide comprising: (a) one or more epitope-binding fragments that bind to epitopes of one or more cell surface lineage-specific proteins; (b) a hinge domain; (c) a transmembrane domain; (d) a costimulatory domain, and (e) comprises a cytoplasmic signaling domain; The chimeric antigen receptor (CAR), wherein one of the cell surface lineage-specific proteins is KIT.

18. A cell expressing the CAR described in claim 17.

19. The cell of claim 18 , wherein the cell is an immune cell.

20. 1. A method of treating a hematological malignancy, the method comprising administering to a human subject: (a) a population of genetically engineered hematopoietic stem / progenitor cells; and (b) administering the cells of claim 18 or 19.

21. 21. The method of claim 20, wherein the hematological malignancy comprises multiple myeloma.

22. 20. A pharmaceutical composition comprising the cells of claim 18 or 19 and a pharmaceutically acceptable carrier.

23. 20. A kit comprising the cells of claim 18 or 19 and, optionally, one or more cytotoxic agents that target cell surface antigens whose genes have been edited in the hematopoietic stem / progenitor cells.

24. A polypeptide comprising an amino acid sequence that is at least 80% identical to the sequence set forth in SEQ ID NO: 56, wherein the polypeptide comprises a mutation at R55S, and the polypeptide has reduced binding to a therapeutic anti-KIT antibody.

25. A polypeptide comprising an amino acid sequence that is at least 80% identical to the sequence set forth in any one of SEQ ID NOs: 53, 45, or 55, wherein the polypeptide comprises a mutation in S123P, DL121, or both, and the polypeptide has reduced binding to a therapeutic anti-KIT antibody.

26. A nucleic acid encoding the polypeptide of claim 24 or 25.

27. A vector comprising the nucleic acid of claim 26.

28. 28. A cell comprising the nucleic acid of claim 26 or the vector of claim 27.

29. 30. A method of producing a polypeptide, comprising culturing the cell of claim 28 under conditions that allow expression of the polypeptide, and optionally isolating the polypeptide.

30. A polynucleotide comprising a segment having a crRNA sequence that is at least 75% identical to one or more of the sequences shown in the table below. Table 1

31. 31. The polynucleotide of claim 30, comprising a segment having one or more crRNA sequences of SEQ ID NOs: 14-16.

32. A polynucleotide having the formula: 5'-spacer-scaffold-RTT / PBS-3' structural motif, During the ceremony, the spacer identifies a target nucleic acid site and comprises 10 to 30 nucleotides; the scaffold binds to a prime editor of a CRISPR system; the 3' structural motif protects the polynucleotide from degradation within the cell; the RTT / PBS segment has a sequence at least 75% identical to one or more of the following sequences: ccttgttn １ n ２ n ３ n ａ n ｂ n ｃ n ４ n ５ n ６ n ７ n ８ n ９ tatgggaaaga (RTT / PBS+7 / 10) (SEQ ID NO: 23), tttccttgttn １ n ２ n ３ n ａ n ｂ n ｃ n ４ n ５ n ６ n ７ n ８ n ９ tatgggaaaga (RTT / PBS+10 / 10) (SEQ ID NO: 24), gcttttccttgttn １ n ２ n ３ n ａ n ｂ n ｃ n ４ n ５ n ６ n ７ n ８ n ９ tatgggaaaga (RTT / PBS+13 / 10) (SEQ ID NO: 25), ccttgttn １ n ２ n ３ n ａ n ｂ n ｃ n ４ n ５ n ６ n ７ n ８ n ９ tatgggaaagaaga (RTT / PBS+7 / 13) (SEQ ID NO: 26), tttccttgttn １ n ２ n ３ n ａ n ｂ n ｃ n ４ n ５ n ６ n ７ n ８ n ９ tatgggaaagaaga (RTT / PBS+10 / 13) (SEQ ID NO: 27), gcttttccttgttn １ n ２ n ３ n ａ n ｂ n ｃ n ４ n ５ n ６ n ７ n ８ n ９ tatgggaaagaaga (RTT / PBS+13 / 13) (SEQ ID NO: 28), gcttttccttgttn １ n ２ n ３ n ａ n ｂ n ｃ n ４ n ５ n ６ n ７ n ８ n ９ tatgggaaagaagaca (RTT / PBS+7 / 15) (SEQ ID NO: 29), tttccttgttn １ n ２ n ３ n ａ n ｂ n ｃ n ４ n ５ n ６ n ７ n ８ n ９ tatgggaaagaagaca (RTT / PBS+10 / 15) (SEQ ID NO: 30), gcttttccttgttn １ n ２ n ３ n ａ n ｂ n ｃ n ４ n ５ n ６ n ７ n ８ n ９ tatgggaaagaagaca (RTT / PBS+13 / 15) (SEQ ID NO: 31), or tttccttgttn １ n ２ n ３ n ａ n ｂ n ｃ n ４ n ５ n ６ n ７ n ８ n ９ tatgggaaagaagac (RTT / PBS+10 / 14) (SEQ ID NO: 32), where: n １ n ２ n ３ represents a codon selected from ctg, tta, ttg, ctt, cta, or ctc; n ４ n ５ n ６ represents a codon selected from ccc, cct, cca, ccg, tcc, tct, tca, tcg, agt, or agc; n ７ n ８ n ９ represents a codon selected from ttg, tta, ctt, ctc, cta, or ctg; n ａ n ｂ n ｃ represents a codon selected from cgt, cgc, cga, cgg, aga, or agg, or The polynucleotide is a combination thereof.

33. n ａ n ｂ n ｃ The polynucleotide of claim 32, wherein is agg.

34. n ａ n ｂ n ｃ The polynucleotide of claim 32, wherein is cgc.

35. 35. The polynucleotide of any one of claims 32 to 34, wherein the spacer comprises gttgtcttctttcccataca (SEQ ID NO: 17).

36. 36. The polynucleotide of any one of claims 32 to 35, wherein the spacer comprises cttctttcccatacaaggag (SEQ ID NO: 100).

37. 37. The polynucleotide of any one of claims 32 to 36, wherein the 3' structural motif comprises SEQ ID NO:20 or a sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

20.

38. 37. The polynucleotide of any one of claims 32 to 36, wherein the 3' structural motif comprises a poly(T) or poly(U) sequence having 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more thymine or uracil nucleobases.

39. the RTT / PBS segment has a sequence at least 75% identical to one or more of the following sequences: ttccttgttn １ n ２ n ３ n ａ n ｂ n ｃ tccttgtatgggaaag (RTT / PBS+9 / 14) (SEQ ID NO: 101), ttccttgttn １ n ２ n ３ n ａ n ｂ n ｃｔ ccttgtatgggaaagaa(RTT / PBS+9 / 16) (SEQ ID NO: 102), where: n １ n ２ n ３ represents a codon selected from ctg, tta, ttg, ctt, cta, or ctc; n ａ n ｂ n ｃ represents a codon selected from cgt, cgc, cga, cgg, aga, or agg, or The polynucleotide according to any one of claims 32 to 38, which is a combination thereof.

40. A polynucleotide having the formula: 5'-spacer-scaffold-RTT / PBS-3' structural motif, During the ceremony, the scaffold binds to a prime editor of a CRISPR system; the 3' structural motif protects the polynucleotide from degradation within the cell; the RTT / PBS segment comprises a primer binding site (PBS) and a reverse transcriptase template; The polynucleotide wherein the spacer has a sequence that is at least 75% identical to the sequence gttgtcttctttcccataca (SEQ ID NO: 17).

41. A polynucleotide having the formula: 5'-spacer-scaffold-RTT / PBS-3' structural motif, During the ceremony, the scaffold binds to a prime editor of a CRISPR system; the 3' structural motif protects the polynucleotide from degradation within the cell; the RTT / PBS segment comprises a primer binding site (PBS) and a reverse transcriptase template; The polynucleotide wherein the spacer has a sequence that is at least 75% identical to the sequence cttctttcccatacaaggag (SEQ ID NO: 100).

42. A polynucleotide having a sequence that is at least 75% identical to the ePEG RNA sequences of SEQ ID NOs: 33-50 or 103-105.

43. The polynucleotide of claim 42, which is [ePeg RTT / PBS+10 / 14] (SEQ ID NO: 47).

44. PEG-D(+9-14)D121L R122R (SEQ ID NO: 105).

45. The following array: [Nicking guide 1] (SEQ ID NO: 46) tttgggccactagtcatgaa, [Nicking guide 2] (SEQ ID NO: 47) gccattccaactactgattt, [Nicking guide 3] (SEQ ID NO: 48) ttgttn １ n ２ n ３ n ａ n ｂ n ｃ n ４ n ５ n ６ n ７ n ８ n ９ tat (where n １ n ２ n ３ represents a codon selected from ctg, tta, ttg, ctt, cta, or ctc; n ４ n ５ n ６ represents a codon selected from ccc, cct, cca, ccg, tcc, tct, tca, tcg, agt, or agc; n ７ n ８ n ９ represents a codon selected from ttg, tta, ctt, ctc, cta, or ctg; n ａ n ｂ n ｃ represents a codon selected from cgt, cgc, cga, cgg, aga, or agg, or a combination thereof); [Nicking guide 4] (SEQ ID NO: 49) gtgaccaattattccctcaa, or [Nicking guide 5] (SEQ ID NO: 50) gagggttattcctgacccca. A polynucleotide having a sequence that is at least 50% identical to one or more of the following:

46. A polynucleotide having a sequence that is at least 50% identical to SEQ ID NO: 51 (ttgttctgcgccccttgtat).

47. 47. The polynucleotide of claim 46 having the sequence ttgttctgcgccccttgtat (SEQ ID NO:51).

Images