Epitope engineering of KIT cell surface receptors

Abstract

Genetically engineered cells (e.g., HSPCs), such as hematopoietic stem cells, having one or more genetically edited cell surface protein genes and therapeutic uses thereof, alone or in combination with immunotherapies targeting the cell surface proteins.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 437,326, filed on January 5, 2023, and U.S. Provisional Patent Application No. 63 / 530,217, filed on August 1, 2023, each of which is incorporated herein by reference in its entirety. Background Art

[0003] Allogeneic hematopoietic stem / progenitor cell (HSPC) transplantation (HSCT) is currently used, at least in clinical practice, to treat high-risk forms of acute leukemia or myelodysplastic syndrome, but results in only 15-20% long-term relapse-free survival. In addition, despite the recent success of immunotherapy, its application in acute myeloid leukemia (AML) has been hampered by the absence of leukemia-restricted targets. The most suitable candidates typically have affinity for targets displayed by both diseased cells and healthy HSPCs. Therefore, the use of such candidates in AML therapy may result in immunosuppression and life-threatening hematopoietic toxicity. Ultimately, the toxicity induced by anti-myeloid / stem cell CAR-Ts limits the applicability of these specific immunotherapeutics to salvage therapy within a limited time window before HSCT, where they may be insufficient to eradicate the disease.

[0004] Multiple myeloma (MM) is the second most common hematologic malignancy in adults. Despite the approval of several new therapeutic agents that have prolonged patient survival, MM remains largely incurable. Similar to AML, the development of immunotherapies for MM, such as CD38-targeted CAR-T, is limited by the fact that many surface targets are also widely expressed on hematopoietic cells.

[0005] There is a need for effective therapeutic agents that target cells of interest, such as cancer cells or diseased hematopoietic stem cells of the host. Optimally, such therapeutic agents cause minimal damage to normal cell populations. Summary of the Invention

[0006] The present disclosure generally relates to genetically engineered hematopoietic cells such as hematopoietic stem cells, progenitor cells or T cells having one or more genetically edited cell surface protein genes, and chimeric antigen receptors capable of targeting the same cell surface proteins.In certain embodiments, the genetically engineered cells are human hematopoietic stem cells (HSC).

[0007] Also provided herein is a genetically engineered HSPC comprising a genetically engineered KIT gene (also referred to herein as cKIT or hcKIT).

[0008] In some embodiments, the genetically engineered KIT gene is engineered such that the protein it encodes has reduced binding to a therapeutic anti-KIT antibody, and the therapeutic anti-KIT antibody is SR1 or an antibody that has the same six CDRs as S1 or is otherwise capable of competing with SR1 for the KIT binding site. In some embodiments, the genetically engineered HSPCs comprise at least one mutation (typically one or two mutations) in the genetically engineered KIT gene, resulting in a polypeptide with a mutation at 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.

[0009] In another embodiment, the genetically engineered KIT gene is engineered such that the encoded protein has reduced binding to a therapeutic anti-KIT antibody, and the therapeutic anti-KIT antibody is anti-KIT clone 104D2, A3C6E2, or an antibody that shares six CDRs with anti-KIT clone 104D2 or A3C6E2 or is otherwise capable of competing with anti-KIT clone 104D2 or A3C6E2 for the KIT binding site. In some embodiments, the genetically engineered HSPCs comprise at least one mutation in the genetically engineered KIT gene that results in a polypeptide with a mutation at position R55.

[0010] Populations of such genetically engineered cells are also provided, as are compositions and kits containing such cells.

[0011] The cells can be genetically engineered using a CRISPR system. The CRISPR system comprises a guide nucleic acid, particularly a guide RNA, and a nuclease. The CRISPR system can be a base editing system utilizing a simple guide RNA or a lead editing system utilizing a lead editing guide RNA and an optional nicking guide RNA. Suitable polynucleotides are provided herein that function as guide RNAs for base editing systems or as lead editing or nicking guide RNAs that function in lead editing systems.

[0012] In some embodiments of the CRISPR system for forming a genetically modified gene, the nuclease is Streptococcus pyogenes Cas9 (SpCas9), Staphylococcus aureus (Staphylococcusaureus) (SaCas9), Lachnospiraceae bacterium Cas12a (LbCas12a), or Acidaminococcus BV3L6 (AsCas12a). In some embodiments, the CRISPR system comprises a SpCas9 nuclease. In some embodiments, the nuclease is a catalytically impaired SpCas9 nuclease connected 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.

[0013] Also provided are methods for treating hematological disorders (e.g., multiple myeloma, acute leukemia, or myelodysplastic syndrome or other lymphoid and myeloid malignancies), comprising administering to a human subject: (a) a genetically engineered hematopoietic stem / progenitor cell or T cell population as 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.

[0014] In some embodiments of the treatment methods using genetically engineered cells comprising a genetically engineered KIT gene, the antibody is an anti-KIT antibody. In some of these methods, the agent comprises a CAR-T cell comprising an anti-KIT binding domain. For such methods, the hematologic disorder is multiple myeloma, acute leukemia, or myelodysplastic syndrome or other myeloid and lymphoid malignancies, as well as non-malignant disorders.

[0015] Also provided are chimeric antigen receptors (CARs) comprising polypeptides. In one embodiment, the polypeptide comprises: (a) one or more epitope-binding fragments that bind to an epitope of one or more cell surface lineage-specific proteins, (b) a hinge domain, (c) a transmembrane domain, (d) a co-stimulatory domain, and (e) a cytoplasmic signaling domain, wherein one of the cell surface lineage-specific proteins is KIT.

[0016] Also provided herein are cells expressing any 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.

[0017] Also provided herein are methods for treating hematological disorders (e.g., hematological malignancies), particularly multiple myeloma, comprising administering to a human subject: (a) a genetically engineered hematopoietic stem / progenitor cell or T cell population as described herein; and (b) a cell expressing any one of the CARs described herein.

[0018] Also provided are polypeptides formed from the genetically engineered genes described herein, as well as nucleic acids encoding the polypeptides, vectors comprising the nucleic acids, and cells comprising the nucleic acids or vectors. The present disclosure also provides a method for preparing the polypeptides, wherein the method comprises culturing cells under conditions that allow expression of the polypeptides and optionally isolating the polypeptides.

[0019] definition

[0020] As used herein, the terms "identity" and "identical" are used to refer to the sequence identity between two amino acid sequences or two nucleic acid sequences. The phrases "percent identity" and "percent identical," and simply "identity," refer to the percentage of sequence identity found in comparing two or more amino acid sequences or nucleic acid sequences. The identity of two or more sequences can be anywhere between 0-100% or any value therebetween. Identity can be determined by comparing the positions in each sequence that can be aligned for the purpose of comparison with a reference sequence. When a position in the compared sequences is occupied by the same nucleotide base or amino acid, the molecules are identical at that position. The degree of identity of an amino acid sequence is based on the number of identical amino acids at the positions shared by the amino acid sequences. The degree of identity between nucleic acid sequences is based on the number of identical or matching nucleotides at the positions shared by the nucleic acid (i.e., polynucleotide) sequences.

[0021] The sequence alignment method for comparison is well known in the art.Usually, a sequence serves as a reference sequence, by comparing the residue of two sequences (for example, the reference polypeptide or polynucleotide of candidate polypeptide or polynucleotide and specific sequence), a test sequence is compared with the reference sequence to optimize the number of identical amino acids or nucleotides in its sequence length. In order to optimize the number of identical amino acids, when comparing, any sequence or two sequences are allowed to have gaps, but the amino acid in each sequence or nucleotides must keep their correct order. Then the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are compared. For example, the BESTFIT algorithm in the GCG bag (version 10.2, Madison WI) can be used to carry out paired comparative analysis of sequence. Alternatively, sequence can be compared using the Blastp program of the BLAST 2 search algorithm, as described in the people such as Tatiana (FEMS Microbiol.Lett., 174,247-250 (1999)), and can be obtained on the website of the National Center for Biotechnology Information (NCBI). Default values for all BLAST 2 search parameters may be used, including matrix = BLOSUM62; open gap penalty = 11, extension gap penalty = 1, gap x_dropoff = 50, expectation = 10, wordlength = 3, and filter on.

[0022] As used herein, the term "epitope" refers to an 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.

[0023] As used herein, "subject," "individual," and "patient" are used interchangeably and refer to a human.

[0024] As used herein, the term "effective amount" is used interchangeably with the term "therapeutically effective amount" and refers to the amount of a cytotoxic agent, a population of genetically engineered cells, or a pharmaceutical composition (e.g., a composition comprising a cytotoxic agent and / or genetically engineered cells) that is sufficient to produce the desired activity, such as delaying the manifestation, arresting the progression, or ameliorating, alleviating, reducing, lessening, or alleviating at least one symptom of a disorder, upon administration to a subject in need thereof.

[0025] As used herein, terms such as "a," "an," and "the" are not intended to refer to only a single entity, but rather include general categories that can be illustrated using specific examples. 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.

[0026] As used herein, unless the content clearly dictates otherwise, the term "or" is generally employed in its ordinary sense, including "and / or." The term "and / or" means one or all of the listed elements or a combination of any two or more of the listed elements.

[0027] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and the like used in the specification and claims should be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and claims are approximate and may vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit 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 and by applying ordinary rounding techniques.

[0028] Also herein, the recitations of numerical ranges by endpoints include 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.).

[0029] In the foregoing description, specific embodiments may be described separately for clarity. References throughout this specification to "one embodiment," "embodiment," "certain embodiments," or "some embodiments" mean that a particular characteristic, configuration, composition, or feature described in conjunction with the 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, in one or more embodiments, particular characteristics, configurations, compositions, or features may be combined in any suitable manner. Thus, characteristics described in the context of one embodiment may be combined with characteristics described in the context of a different embodiment, except where the characteristics are necessarily mutually exclusive.

[0030] Polynucleotide sequences as described herein are described using DNA or RNA. It should be understood that the complementary sequence, reverse sequence and reverse complementary sequence of DNA and RNA sequences can be easily determined by those skilled in the art, and are all within the scope of the present disclosure. It should also be understood that the sequence disclosed as DNA sequence herein can be converted from DNA sequence to RNA sequence by replacing each thymidine nucleotide (T) with uridine nucleotide (U). When using DNA sequence to describe RNA (e.g., guide RNA), it should be understood that the corresponding RNA sequence is a DNA sequence in which each thymidine nucleotide (T) is replaced with uridine nucleotide (U). For example, the guide RNA of the DNA sequence GCGTATAG has an RNA sequence of GCGUAUAG.

[0031] Polynucleotide and / or polypeptide or protein sequences may include one or more forms of typographical emphasis (e.g., underlined text, bold text, italic text). It should be understood that typographical emphasis is non-limiting. Sequences described with typographical emphasis include sequences not described with typographical emphasis. Typographical emphasis may or may not indicate modified nucleotide bases or linkages; modified sequences relative to indicated sequences; characteristics, such as the position of spacers, one or more specific codons, one or more specific amino acids, primer binding sites, mutation sites, reverse transcriptase templates, complementary determining regions, etc.; or any combination thereof. In addition, polynucleotide sequences may be displayed in uppercase letters, lowercase letters, or a combination thereof. Although the case of letters in a polynucleotide sequence can be used to distinguish parts of a sequence, the case of letters is non-limiting. Unless otherwise indicated, lowercase and uppercase letters indicate the identity of a nucleobase.

[0032] The foregoing summary is not intended to describe each disclosed embodiment or every implementation thereof. The following description more particularly illustrates illustrative embodiments. In several places throughout this application, guidance is provided through lists of examples, which can be used in various combinations. In each case, the enumerated list serves only as a representative group and should not be construed as an exclusive list.

[0033] In several places throughout this application, guidance is provided through lists of examples, which examples can be used in various combinations. In each case, the enumerated list serves only as a representative group and should not be construed as an exclusive list. It should be understood that the specific examples, materials, amounts, and procedures should be interpreted broadly, in accordance with the scope and spirit of the invention as set forth herein.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art. Methods and materials are described herein; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict, the present specification (including definitions) shall prevail. Other features and advantages of the present invention will be apparent from the following detailed description, drawings, and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1A is a schematic representation of the cKIT Sleeping Beauty expression construct.

[0036] Figure 1B Shown is a cross-reactivity test of different anti-human cKIT antibodies with mouse cKIT protein.

[0037] Figure 1C Shown is the generation of human-mouse chimeric cKIT proteins for personalization of binding domains of anti-human cKIT antibodies that do not cross-react with the mouse orthologous protein.

[0038] Figure 2A is a schematic diagram showing that orthologous mutations were divided into three clusters, cloned, and transduced in HEK-293T cells.

[0039] Figure 2B Figure 3 is a fluorescence activated cell sorting (FACS) analysis showing that one of the three subgroups (mD2_Group 1) is sufficient to avoid binding of the therapeutic antibody (SR1).

[0040] Figure 2C Shown Figure 2A and 2B The presented approach was repeated, which further narrowed the set of orthologous mutations.

[0041] Figure 3A Shown are FACS analyses showing four mutation groups (D121G; R122L; S123P; Y125F) of the mouse cKIT protein that result in lack of SR1 antibody binding and compared to the same human epitope.

[0042] Figure 3BA bar graph comparing various human cKIT (hcKIT) mutations is shown. The mutations were cloned into the Sleeping Beauty transposon plasmid in different combinations and as single point mutations. After transduction, HEK-293T cells expressing all the different variants were tested in the same flow cytometry experiment. The bar graph shows the ratio of the mean fluorescence intensity (MFI) of the therapeutic and control antibodies, which has been normalized to the ratio of the same MFI in the hcKIT WT control.

[0043] Figure 3C Shows the display with Figure 3B Bar graph of MFI-normalized ratios of the same mutations. Because murine cKIT protein does not fully cross-react with human ligands, 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, while maintaining binding to the human SCF ligand.

[0044] Figure 4A Schematic diagram of the cKIT library experiment is shown. 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 positively transduced cells.

[0045] Figure 4B Figure 2 shows FACS profiles of 293T cells after puromycin selection. The majority of cells expressed variants recognized by both anti-cKIT antibodies, while a rare subpopulation recognized only by a control antibody (SR1-) was sorted and expanded in culture.

[0046] Figure 4C Figure 1 shows flow cytometry experiments for perturbations of four candidate amino acids (aa) that emerged from deep sequencing analysis of an expanded SR1-negative cell population. The figure shows FACS analysis of control and therapeutic antibodies for each variant using MFI. Both variants, D121L and S123P (indicated by arrows), were highly expressed and recognized by the control antibody, but not by the SR1 antibody.

[0047] Figure 5A Shown are FACS plots of two library-derived variants expressed by the Sleeping Beauty transposon system in BAF3 cells and compared to hcKIT and a variant encoding the murine SR1 epitope expressed in the same cell line.

[0048] Figure 5B Shown are dose affinity curves for the D121L and S123P hcKIT variants for SR1 and SCF, both conjugated to ALEXA FLUOR 647. Both variants were unable to bind SR1 even at higher concentrations, but displayed similar affinity for the conjugated SCF cytokine.

[0049] Figure 6A The position of the T to C transition in the endogenous human cKIT locus is shown, and the conversion can be induced by the adenine base editor genome editing method S123P mutation. The table (bottom) shows three different guides: S123P_gRNA1; S123P_gRNA2; S123P_gRNA3, which are designed to induce the depicted conversion mutations.

[0050] Figure 6B Shows the use Figure 6A FACS analysis of cells treated with the three indicated sgRNAs to induce adenine base editing of KIT. The experiments were performed on K562 cells that overexpress cKIT from its endogenous locus by promoter replacement.

[0051] Figure 6C Figure 2 shows the genome editing efficiency of three different sgRNAs in the K562 cKIT overexpressing cell line. Genome editing efficiency was determined using Sanger sequencing.

[0052] Figure 7A Schematic diagram of CD34+ cell growth inhibition experiment is shown. Mobilized peripheral blood-derived CD34+ HSPCs were thawed and electroporated with adenine base editor (ABE) mRNA and sgRNA targeting mutant S123P or AAVS1 control locus 2 days later. After culturing for 3 days, each group was plated with increasing concentrations of SR1 antibody in the presence of SCF cytokine (125 nanograms / milliliter (ng / mL)).

[0053] Figure 7B Shown is a graph showing the editing efficiency of the S123P mutation on day 3 after electroporation of CD34+ cells.

[0054] Figure 7C Figure 2 shows the absolute counts of total live cells (left) and CD34+ cells (right) edited for cKIT S123P or in the AAVS1 control locus in the presence of different doses of SR1 antibody on day 7 of culture. Counts were normalized according to the median of untreated cells in each editing group (n = 4 replicates per antibody concentration).

[0055] Figure 8AA schematic diagram showing an in vitro selection experiment for CD34+ cells is shown. Mobilized peripheral blood-derived CD34+ HSPCs were thawed and electroporated 2 days later with ABE mRNA and sgRNAs for mutation S123P and BCL11A enhancer gRNA or sgRNA in the AAVS1 control locus. After 3 days of culture, each group was stained with CELL TRACE yellow or CELLTRACE CSFE dye as indicated, mixed at a 1:1 ratio, and plated with SR1 at concentrations between 0 nM and 3300 nM in the presence of SCF cytokine (125 ng / mL).

[0056] Figure 8B Shown are bar graphs showing the relative fraction of cells stained in different concentrations of SR1 antibody after 4 days of SR1 treatment.

[0057] Figure 8C Shown are curves showing the mean fluorescence intensity (MFI) of FITC in CELL TRACE CFSE-stained cells and the MFI of PE in CELL TRACE Yellow-stained cells (n=4 replicates per antibody concentration).

[0058] Figure 9A A schematic diagram showing that orthologous mutations were grouped into three clusters, cloned, and transduced in 293T cells is shown.

[0059] Figure 9B Shown is a FACS analysis showing that one of the three subsets was sufficient to avoid binding of two anti-hcKIT clones 104D2 and A3C6E2.

[0060] Figure 9C Shown Figures 9A-9B The presented approach was repeated, which further narrowed the set of orthologous mutations.

[0061] Figure 10A Figure 2 is a FACS analysis of four mutation groups (E53T; I54L; R56S; L57T) of the mouse cKIT protein that result in lack of 104D2 / A3C6E2 antibody binding, as shown and compared to the same human epitope.

[0062] Figure 10B A bar graph comparing various cKIT mutations is shown. The mutations were cloned and tested in different combinations and as single point mutations. In the same experiment, the bar graph shows the ratio of the MFI of the therapeutic antibody and the control antibody, which has been normalized to the ratio of the same MFI in the cKIT WT control.

[0063] Figure 11AShown are FACS analyses of NIH 3T3 cells stably transduced with the Sleeping Beauty transposon carrying the wild-type human cKIT gene (hcKIT) and three candidate variants: S123P, D121L, and S123P-D121L.

[0064] Figure 11B Shown are the MFIs of cell lines expressing different cKIT variants using ALEXA FLUOR 488-conjugated stem cell factor (SCF) ligand.

[0065] Figure 12A Schematic diagram showing the lead editing approach to introduce D121L+S123P mutations into the K562 reporter cell line, thereby conferring resistance to anti-cKIT SR1 antibody therapy. Figure 12A A cartoon representation of a prime editing protein complexed with a double-stranded gene is shown.

[0066] Figure 12B Shown is FACS 3 days after electroporation of a pool of nine epegRNAs. RTT+10 and PBS10 / 13 / 15 were identified as the best performing peg guides and were selected for further development.

[0067] Figure 13A A bar graph showing the editing efficiency of epegRNAss with PBS lengths of 13, 14, and 15 is shown.

[0068] Figure 13B Shown are bar graphs showing the effects of post-editing PAM mutations and seed sequence perturbations 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 do not significantly affect editing efficiency.

[0069] Figure 13C A table listing possible codons encoding the D121L mutation with and without the seed mutation at guide lengths +10-13 and +10-14 is shown.

[0070] Figure 14A Shown is a schematic diagram illustrating the cKIT locus targeted for editing and the spacer region of the epegRNA used in the experiments.

[0071] Figure 14B Bar graph showing the editing efficiency of three cKIT epegRNAs in combination with five nick-guide variants.

[0072] Figure 14C Shown are bar graphs illustrating the percentages of edited and knockout (KO) cells determined by FACS analysis.

[0073] Figure 14D FACS analysis of cell populations is shown, and the effects of experimental manipulations on editing efficiency are highlighted.

[0074] Figure 15A The collection of manipulated scaffolds is listed.

[0075] Figure 15B Shown is a bar graph of the editing efficiency of different variants of epegRNA+10 / 14, each containing sequential deletions of 3' nucleotides from the epegRNA scaffold portion.

[0076] Figure 15C Schematic diagram showing the secondary structure of the optimized scaffold.

[0077] Figure 15D Representative FACS plots showing the editing efficiency of 10 / 14 and optimized 10 / 14 scaffolds.

[0078] Figure 16A Schematic diagram of a modified scaffold with a 3" nucleotide "C" deletion is shown.

[0079] Figure 16B Shown is a comparison of editing efficiency (measured by FACS at D3) between the modified scaffold and the 10-14epeg guide.

[0080] Figure 17 The sequencing results of the genomic region after prime editing according to Example 14 are shown. DETAILED DESCRIPTION

[0081] Identifying suitable proteins for targeted cancer therapy presents significant challenges. Many potential target proteins are present on the cell surface of cancer cells as well as on the cell surface of normal non-cancerous cells, and the target proteins may be involved in the development and / or survival of the subject. Many target proteins contribute to the functionality of such cells. Therefore, therapies targeting these proteins may cause harmful effects on the subject, such as significant toxicity and / or other side effects. In addition, resistance to chimeric antigen receptor T cell (CAR-T) therapy remains a challenge for treating hematopoietic malignancies such as acute myeloid leukemia (AML) and multiple myeloma (MM), as the disease escapes CAR-T therapy due to the conversion of cancer antigens on cancer cells. In addition, the identification and manipulation of appropriate stem cell markers can be used to improve the conditioning of bone marrow transplantation and expand its application to non-malignant diseases. Effective immune-based conditioning can be particularly useful in the autologous transplantation environment of gene therapy. It should be noted that in this environment, if a selective advantage is given, the therapeutic cell product can be enriched in vivo with immunotherapy (e.g., subsequently administering the same conditioning agent). In one aspect of the present disclosure, replacement of cancer cells by a modified normal cell population is performed using normal cells that have been manipulated such that the cells do not bind a cytotoxic agent.

[0082] Therefore, the present disclosure provides methods, cells, compositions and kits for solving at least the above problems. The methods, cells, compositions and kits described herein provide effective treatment for hematological disorders, particularly malignancies, and the treatment allows targeting to be present not only on cancer cells but also on cells that are crucial for the development and / or survival of the subject. In some cases, described herein are genetically engineered cells (e.g., HSPC or T cells) such as hematopoietic stem cells / progenitor cells (HSPC) with genetic editing in one or more genes encoding cell surface proteins (e.g., KIT); methods for producing such cells, for example, gene editors (CRISPR) methods guided by nucleotides using specific guide RNAs; methods for treating hematopoietic disorders, particularly malignancies, using engineered hematopoietic cells alone or in combination with one or more cytotoxic agents (e.g., CAR-T cells), wherein the one or more cytotoxic agents can target wild-type cell surface antigens but do not target those antigens encoded by the editing genes in engineered hematopoietic cells; and kits comprising engineered hematopoietic cells.

[0083] Genetically engineered cells (e.g., HSPCs)

[0084] In some embodiments, genetically engineered cells (e.g., HSPCs or T cells) have edited KIT genes. In some embodiments, one or more of these genes are mutated. In some cases, the mutated KIT gene includes a mutation or deletion of one or more non-essential epitopes to retain (all or part of) the biological activity of the KIT gene.

[0085] Hematopoietic stem / progenitor cells (HSPCs)

[0086] In some embodiments, hematopoietic cell as described herein is hematopoietic stem cell / progenitor cell.Hematopoietic stem cell / progenitor cell (HSPC) can produce myeloid progenitor cell and lymphoid progenitor cell, and the progenitor cell further produces myeloid cell (such as monocyte, macrophage, neutrophil, basophil, dendritic cell, erythrocyte, platelet etc.) and lymphocyte (such as T cell, B cell, NK cell) respectively.The feature of HSPC is the expression of cell surface marker CD34 (such as CD34+), which can be used for identifying and / or separating HSPC.

[0087] In some embodiments, HSPC is obtained from a human subject. In some embodiments, the human subject is a non-human primate, a rodent (e.g., a mouse or rat), a cow, a pig, a horse, or a domesticated animal. In some embodiments, HSPC is obtained from a human patient such as a human patient suffering from a hematopoietic malignancy. In some embodiments, HSPC is obtained from a healthy donor. In some such embodiments, HSPC is obtained from a donor who has never suffered from a hematopoietic malignancy. In some embodiments, HSPC is obtained from a subject to which genetically engineered HSPC is subsequently administered. HSPC administered from the same subject from which the cell is obtained is referred to as an autologous cell. HSPC obtained from a subject of a non-administered subject is referred to as an allogeneic cell. In the embodiment in which the cell is an allogeneic cell, the method can be modified to reduce the incidence of rejection. The method for reducing the incidence of rejection is standard and well known in the art.

[0088] HSPC can be obtained from any suitable source using conventional means known in the art. In some embodiments, HSPC is obtained from a sample of a subject (or donor), such as bone marrow, blood (e.g., peripheral blood mononuclear cells (PBMC)) and / or umbilical cord (i.e., cord blood cells). Generally speaking, bone marrow cells can be obtained from the iliac crest, femur, tibia, spine, rib or other medullary cavity of a subject (or donor). Bone marrow can be taken out from a patient and separated by various separation and washing procedures known in the art.

[0089] HSPCs are usually present in the bone marrow, but can be mobilized into the circulating blood by administering a mobilizing agent so that HSPCs can be harvested from the peripheral blood. In some embodiments, a mobilizing agent, such as granulocyte colony stimulating factor (G-CSF), is administered to the subject (or donor) from whom the HSPCs are obtained. The number of HSPCs collected after mobilization using a mobilizing agent is generally greater than the number of cells obtained without the use of a mobilizing agent.

[0090] In some embodiments, a sample is obtained from a subject (or donor) and then the desired cell type (e.g., CD34+, CD34+CD38-, CD133+, CD90+, CD49f+) is enriched. For example, PBMC and / or CD34+ hematopoietic cells can be isolated from blood. Cells can also be separated from other cells, for example, by separating and / or activating with antibodies that bind to epitopes on the cell surface of the desired cell type. Another method that can be used includes negative selection using antibodies against cell surface markers to selectively enrich for specific cell types without the need for receptor engagement to activate cells.

[0091] Mutated cell surface antigens

[0092] In some embodiments, the hematopoietic stem / progenitor cells (HSPCs) or T cells described herein may contain edited genes 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 binding to or no binding to a cytotoxic agent (e.g., an anti-KIT antibody) as described herein. The mutant may include one or more mutations in an epitope to which the cytotoxic agent binds (e.g., a nucleotide sequence encoding the epitope and an amino acid sequence of the epitope) such that binding to the cytotoxic agent is reduced or eliminated compared to a natural or wild-type cell surface protein counterpart. Such a mutant may preferably maintain a biological activity substantially similar to that of the wild-type counterpart.

[0093] As used herein, the term "reduction in conjunction with " refers to a combination reduction of at least 25%. In conjunction with level, it can refer to the combination of cytotoxic agent and hematopoietic stem cell, progenitor cell or T cell compared with wild-type (that is, non-engineered, non-mutated) protein or the combination of cytotoxic agent and cell surface protein. In some embodiments, in conjunction with reduction of 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, in conjunction with reduction, there is substantially no detectable combination in conventional determinations. As used herein, "no binding" refers to substantially no binding, e.g., no detectable binding or only baseline binding as determined by a conventional binding assay. Binding and reduced binding can be measured using quantitative fluorescence reduction, e.g., by performing fluorescence activated cell sorting (FACS) titration.

[0094] In some embodiments, 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 so that the cytotoxic agent does not bind to the mutated epitope or has reduced binding to the mutated epitope. Such a mutant may have a substantially reduced binding affinity for the cytotoxic agent (e.g., having a binding affinity that is at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% lower than its wild-type counterpart) or may have eliminated binding activity for the cytotoxic agent. In other cases, the mutant contains a deletion of a region comprising the epitope of interest. Such a region can be encoded by an exon. In some embodiments, the region is a domain of a cell surface protein of interest encoding an epitope. In one example, the variant only lacks the epitope. The length of the deleted region can be in the range of 3-60 amino acids, e.g., 5 to 50, 5 to 40, 10 to 30, 10 to 20, 5 to 10, etc.

[0095] 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 a cell surface antigen. In some embodiments, the cytotoxic agent binds to more than one epitope of a cell surface antigen, and the cell (e.g., HSPC) is manipulated such that each epitope is absent and / or unavailable for binding by the cytotoxic agent.

[0096] The mutation or deletion in the mutant of the cell surface antigen may be located within or around a non-essential epitope, such that the mutation or deletion does not substantially affect the biological activity of the protein.

[0097] In some embodiments, genetically engineered cells as described herein (such as HSPC) have one or more editing genes of cell surface antigens so that editing gene expression has the mutant cell surface antigen of the mutation in one or more non-essential epitopes." non-essential epitope" (or a fragment comprising such non-essential epitope) refers to a domain within a cell surface protein / antigen, wherein the mutation is substantially less likely to affect the biological activity of the cell surface protein. For example, when engineered cells (such as HSPC or T cells) comprise deletions or mutations of non-essential epitopes of cell surface antigens, such engineered cells can proliferate and / or undergo erythropoiesis and differentiate to a level similar to that of cells expressing wild-type cell surface antigens. It is well known for identifying and / or verifying the non-essential epitopes in cell surface antigens. In addition, the functional method for evaluating cell surface antigens and engineered cells is known in the art, and includes research on, for example, proliferation assays, differentiation assays, colony formation assays, expression analysis (for example, gene and / or protein), protein localization assays, intracellular signaling assays, functional assays, and humanized mouse models.

[0098] Preparation of genetically engineered cells (e.g., HSPCs or T cells)

[0099] Any one of the genetically engineered cells (such as HSPC or T cells) comprising the editing genes encoding one or more cell surface antigens can be prepared by conventional methods or the methods described herein. In some embodiments, genetic engineering is performed using genome editing. As used herein, "genome editing" refers to a method for modifying the genome (including any protein encoding or non-coding nucleotide sequence) of an organism to change target gene expression. In general, genome editing methods involve the use of endonucleases capable of cleaving the nucleic acid of the genome. For example, an endonuclease can cleave the nucleic acid sequence of the genome at the target nucleotide sequence. In some cases, genome editing methods involve the use of catalytic "dead" nucleases or nucleases that are nickases. The repair of double-strand breaks in the genome typically 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 adenine or cytidine deaminase domains with respect to base editors). Other functional domains include reverse transcriptases, RNA binding proteins, transcription factors, DNA repair mechanisms, lead editors, CRISPR-Cas activators or inhibitors, etc.

[0100] Genome editing methods are generally classified based on the type of endonuclease involved in producing double-strand breaks in the target nucleic acid. Types of genome editing methods include the use of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, and clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) protein systems. Modification (editing) can include the use of CRISPR / Cas systems such as CRISPR / Cas9 to delete or mutate epitopes of specific cell surface proteins.

[0101] CRISPR-Cas system

[0102] In some embodiments, genetically engineered HSPCs are genetically engineered using a CRISPR system. The CRISPR system comprises guide nucleic acids and nucleases. Cas nucleases can be easily programmed to cleave target DNA sequences for genome editing in various organisms. One class of these nucleases is called Cas9 protein or Cas9 nuclease, which forms a complex with two short RNAs: crRNA and trans-activating crRNA (tracrRNA). crRNA and tracrRNA typically hybridize to form a guide RNA (gRNA). The most commonly used Cas9 ortholog, Streptococcus pyogenes cas9 (SpCas9), uses a crRNA with a 20-nucleotide (nt) "spacer" region at its 5' end that is complementary to the opposite strand of the "protospacer" region of the target DNA site. Efficient cleavage includes SpCas9 recognizing the protospacer adjacent motif (PAM). The crRNA and tracrRNA sequences can be joined to form a single, approximately 100-nt single guide RNA (sgRNA, a gRNA) to guide the DNA cleavage activity of SpCas9. A Cas protein identified as Cpf1 (also known as Cas12a) 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 with a 23-nt protospacer at its 3' end that is complementary to the target DNA sequence.

[0103] In some embodiments, the Cas endonuclease is a Cas9 nuclease or a variant thereof, which cleaves both strands of the double-stranded DNA of the target nucleic acid, thereby generating blunt ends. In some embodiments, the Cas endonuclease is a Cpf1 nuclease or a variant thereof, which results in cleavage of both strands of the double-stranded DNA of the target nucleic acid, resulting in staggered ends of the nucleic acid.

[0104] CRISPR Cas9 system

[0105] 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) with a known (wild-type) sequence (see uniprot.org / uniprotkb / Q99ZW2 / entry ; Accession No. AAK33936.1), or a sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to the amino acid sequence of a wild-type SpCas9 endonuclease, e.g., differing by at most 5%, at most 10%, at most 15%, or at most 20% in residues that are substituted, e.g., 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 No. CCK74173.1), or having 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, for example, with at most 5%, at most 10%, at most 15%, or at most 20% difference, for example, in residues replaced with conservative mutations. In preferred embodiments, the endonuclease retains the desired activity of the parent, such as nuclease activity (unless the parent is a nickase or dead Cas9) and / or the ability to interact with the guide RNA and target DNA.

[0106] Herein, in the context of amino acid sequences, "conservative" mutations (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 acids belong. For example, it is well known in the field of protein biochemistry that amino acids belonging to groups of amino acids with a specific size or characteristic (such as charge, hydrophobicity, and hydrophilicity) can be substituted by another amino acid without changing the activity of the protein, particularly in regions of the protein that are not directly related to biological activity. For example, non-polar (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; or Gln for Asn or vice versa to maintain a free -NH. Similarly, biologically active analogs of polypeptides that contain deletions or additions of one or more consecutive or non-consecutive amino acids but do not eliminate the functional activity of the polypeptide are also contemplated.

[0107] In general, the 3' or 5' side of the target nucleic acid is flanked by a protospacer adjacent motif (PAM) that can interact with the endonuclease and further participate in targeting the endonuclease activity to the target nucleic acid. It is generally believed that the PAM sequence flanking the target nucleic acid depends at least in part on the endonuclease and the source from which the endonuclease is derived. For example, for 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). For the Cas9 endonuclease derived from Staphylococcus aureus, the PAM sequence is NNGRRT (N is A, C, G or T; R is A or G).

[0108] Therefore, in some embodiments, the endonuclease is engineered / modified so that it can recognize one or more PAM sequences. In some embodiments, the endonuclease has been engineered / modified to recognize one or more PAM sequences that are different from the PAM sequences recognized by the unengineered / modified endonuclease. In some embodiments, the endonuclease can be modified so that it can recognize a PAM sequence that lacks guanine. In some embodiments, the endonuclease can be modified so that it can recognize a PAM sequence that includes "ACA", "AGC" or "AAA". In some embodiments, the endonuclease has been engineered / modified to reduce the off-target activity of the enzyme. In some embodiments, the nucleotide sequence encoding the endonuclease is modified to change the PAM recognition of the endonuclease. For example, the Cas endonuclease (e.g., SpCas9) has a mutation at one or more of the following positions: A61, L1111, D1135, S1136, G1218, E1219, N1317, A1322, R1333, R1335, T1337. See, e.g., International Patent Application Publication Nos. WO 2016 / 141224 and WO 2017 / 040348, U.S. Patent Application Publication No. 2021 / 0284978A1.

[0109] 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 does not have nuclease activity. For example, the mutation is: (i) D10A or D10N and / or (ii) H840A, H840N, or H840Y. In some embodiments, the catalytically impaired SpCas9 comprises a mutation at position D10A. In some embodiments, the catalytically impaired SpCas9 comprises a mutation D10N. In some embodiments, the catalytically impaired SpCas9 comprises a mutation at position K918. In one or more embodiments, the catalytically impaired SpCas9 comprises a mutation K918N.

[0110] 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 has been modified to inactivate one or more catalytic residues of the endonuclease. In some embodiments, the Cas9 endonuclease has been modified to inactivate one of the catalytic residues of the endonuclease, referred to as a "nickase" or "Cas9n." The Cas9 nickase endonuclease cleaves one DNA strand of the target nucleic acid.

[0111] 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.

[0112] CRISPR Cpf1 (Cas12a)

[0113] In some embodiments, the Cas endonuclease is a Cpf1 nuclease (also known as Cas12a) or a variant thereof. The Cpf1 endonuclease generally recognizes a PAM sequence located at the 5' end of the target nucleic acid. For the Cpf1 nuclease, the PAM sequence is TTTN (N is A, C, G, or T). In some embodiments, the host cell expresses a Cpf1 nuclease derived from a bacterium of the family Lachnospiraceae (LbCpf1), Acidinococcus (AsCpf1), or Francisella tularensis (FnCpf1). The wild-type sequences of each are known: Type V CRISPR-associated protein Cpf1 (Lachnospiraceae ND2006), GenBank accession number WP_051666128.1; Type V CRISPR-associated protein Cpf1 [Aminaminococcus BV3L6], NCBI reference sequence: WP_021736722.1; Type V CRISPR-associated protein Cpf1 (Franciscoides tularensis), GenBank accession number WP_003040289.1.

[0114] In some embodiments, the Cpf1 endonuclease is a wild-type version of the nuclease. In some embodiments, the Cpf1 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., having at most 5%, at most 10%, at most 15%, or at most 20% of the residues replaced, e.g., with 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 dead Cas9) and / or the ability to interact with the guide RNA and target DNA.

[0115] In some embodiments, the Cas12a endonuclease is a catalytically inactive variant, which may be referred to as dCas12a.

[0116] Cas endonuclease functional domain and CRISPR base editing system

[0117] Alternatively or in addition, Cas endonucleases (i.e., Cas9 or Cas12a) can be fused to another protein or a 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., wherein the transcriptional repression domain is a Kruppel-associated box (KRAB) domain, an ERF repressor domain (ERD), or an mSin3A interaction domain (SID); wherein the transcriptional silencer is heterochromatin protein 1 (HP1)). In some embodiments, the heterologous functional domain is an enzyme that modifies the DNA methylation state (e.g., DNA methyltransferase (DNMT) or TET protein (such as TET1)). In some embodiments, the heterologous functional domain is an enzyme that modifies a histone subunit (e.g., histone acetyltransferase (HAT), histone deacetylase (HDAC), histone methyltransferase (HMT), or histone demethylase). In some embodiments, the heterologous functional domain is a biological tether (for example, MS2, Csy4 or λN).In some embodiments, the heterologous functional domain is FokI.

[0118] In some embodiments, the heterologous functional domain and the 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 the apolipoprotein B mRNA-editing enzyme 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 deaminases disclosed in Neugebauer, Monica et al., Nat. Biotechnol. 1-13 (2022) and Nat. Biotechnol., 41, 673–685 (2023).

[0119] 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 1 (ADAR1), ADAR2, ADAR3 that acts on RNA; adenosine deaminase 1 (ADAT1), ADAT2, ADAT3 that acts on tRNA; and naturally occurring or engineered tRNA-specific adenosine deaminase (TadA). For example, ABE8e-TadA-8e. In some embodiments, the TadA adenosine deaminase domain comprises a V106W mutation.

[0120] In some embodiments, the endonuclease is a base editor. The base editor endonuclease generally includes a catalytically inactive Cas endonuclease fused to a base editor. For example, the endonuclease is SpCas9, which has a mutation at D10, E762, D839, H983, or D986; and / or has a mutation at H840 or N863 and is fused to a base editor, such as those mentioned above.

[0121] Base editors can be used for CRISPR base editing methods, which can install point mutations directly in cell DNA without inducing double-stranded DNA breaks. For example, cytosine base editors target specific loci by guide RNA, and convert cytosine into uracil, which is then converted into thymine by base excision repair, producing a change from C to T (or G to A on the opposite chain). Adenine base editors convert adenosine into inosine, which is processed like a cell processes guanosine, producing a change from A to G (or T to C). In general, base editing technology edits target nucleotides without producing double-strand breaks or relying on homology to guide repair. Such systems are commercially available (for example, at www.addgene.org) and are described in, for example, AC Komor et al., Nature, 533:420-424 (2016).

[0122] In some embodiments, the heterologous functional domain is an enzyme, domain, or peptide that inhibits or enhances an endogenous DNA repair or base excision repair (BER) pathway, e.g., uracil DNA glycosylase inhibitor (UGI), which inhibits uracil DNA glycosylase (UDG, also known as uracil N-glycosylase, or UNG)-mediated uracil excision to trigger BER; or a DNA end-binding protein, such as Gam from bacteriophage Mu.

[0123] In some cases, endonuclease (Cas9 or Cas12a) is fused to one or more of nuclear localization sequence, cell penetrating peptide sequence, affinity tag and / or fluorescent protein.For example, nuclear localization sequence is SV40 large T antigen nuclear localization sequence (PKKKRKV; SEQ ID NO:1), nuclear plasmid nuclear localization sequence (KRPAATKKAGQAKKKK; SEQ ID NO:2) or c-Myc nuclear localization sequence (PAAKRVKLD; SEQ ID NO:3).For example, nuclear localization sequence is fused to the N-terminus and / or C-terminus of Cas9 or Cas12a protein.In some embodiments, when heterologous functional domain is fused to the N-terminus and / or C-terminus of Cas9 or Cas12a protein, nuclear localization sequence is fused to the N-terminus and / or C-terminus of heterologous functional domain-Cas protein complex or is inserted between heterologous functional domain and Cas protein.

[0124] The sequences of exemplary Cas endonucleases are provided below:

[0125] SEQ ID NO:4-SpRY-ABE8e-V106W 3xNLS adenine base editor amino acid sequence:

[0126]

[0127] SEQ ID NO:5-SpRY-ABE8e 3xNLS adenine base editor amino acid sequence:

[0128]

[0129] SEQ ID NO:6-SpRY-evoAPOBEC1-BE4 3xNLS adenine base editor amino acid sequence:

[0130]

[0131] SEQ ID NO:7-SpRY-K918N-ABE8e-V106W 3xNLS adenine base editor amino acid sequence:

[0132]

[0133] SEQ ID NO:8-SpRY-ABE8e-V106W 3xNLS adenine base editor nucleotide sequence:

[0134]

[0135] SEQ ID NO:9-Nucleotide sequence of SpRY-evoAPOBEC1-BE4 3xNLS adenine base editor:

[0136]

[0137] SEQ ID NO: 10-SpRY-K918N-ABE8e-V106W 3xNLS adenine base editor nucleotide sequence:

[0138]

[0139] CRISPR guide RNA

[0140] The terms "gRNA," "guide RNA," and "CRISPR guide sequence" are used interchangeably throughout and refer to nucleic acids comprising sequences that determine the specificity of the Cas DNA binding protein of the CRISPR / Cas system. The gRNA hybridizes (e.g., is partially or fully complementary) to a target nucleic acid sequence in the genome of the host cell and promotes specific association or targeting of an RNA-guided nuclease (such as Cas9 or Cpf1) to the target sequence. The gRNA may be unimolecular (comprising a single RNA molecule and alternatively referred to as a chimeric or sgRNA) or may be modular (comprising more than one, and typically two, separate RNA molecules, such as CRISPR RNA (crRNA) and a transactivating CRISPR RNA (tracrRNA), which are typically associated with each other, e.g., by duplexes or hybridization). Thus, in some cases, gRNA refers collectively to crRNA and tracrRNA (e.g., when using Cas9 nuclease—in those cases, the guide RNA may be referred to as a single guide RNA, i.e., sgRNA). In other cases, gRNA refers only to crRNA (e.g., when using Cpf1 endonuclease).

[0141] Guide RNA, whether unimolecular or modular, comprises 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," "complementary region," "spacer," and collectively referred to as "crRNA." The length of the gRNA or portion thereof that hybridizes to the target nucleic acid may include 15-25 nucleotides, 18-22 nucleotides, or 19-21 nucleotides. In some embodiments, the length of 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 some embodiments, the length of the gRNA sequence that hybridizes to the target nucleic acid is 10-30 or 15-25 nucleotides. 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.

[0142] In addition to the targeting domain, gRNA generally (but not necessarily) includes multiple domains that can affect the formation or activity of the Cas9 / gRNA complex. For example, this includes one or more polyA segments (which can be recognized as termination signals by RNA polymerase) and two or more additional double-stranded regions that participate in nuclease activity in vivo but not necessarily in in vitro nuclease activity. Although this specification focuses on gRNAs used with Cas9, there are other RNA-guided nucleases that utilize gRNAs that are different from the gRNAs described herein in some aspects. The design of other gRNAs is further described in, for example, International Publication No. WO 2019 / 084168.

[0143] Those skilled in the art will appreciate that, although there may be structural differences between gRNAs from different prokaryotic species or between Cpf1 and Cas9 gRNAs, the principles of gRNA operation are generally consistent. Due to this consistency of operation, gRNAs can be broadly defined by their targeting domain sequence, and those skilled in the art will appreciate that a given targeting domain sequence can be incorporated into any suitable gRNA, including single-molecule or chimeric gRNAs or gRNAs comprising one or more chemical modifications and / or sequential modifications (substitutions, additional nucleotides, truncations, etc.). Therefore, for the economy presented in this disclosure, gRNAs can be described solely in terms of their targeting domain sequence.

[0144] Table 1 below provides exemplary guide crRNAs for editing the KIT gene. As is well known, the selection of gRNA sequences 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.

[0145] 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 unequal molar amounts. In some embodiments, two or more guide RNAs are provided in optimized amounts so that editing of each target occurs at an equal frequency. In some embodiments, two or more guide RNAs are provided in optimized amounts so that editing of each target occurs at an optimal frequency.

[0146] Provided herein is a polynucleotide suitable for use as a guide spacer sequence having 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 a sequence shown in Table 1 (SEQ ID NOs: 14-16). Such a polynucleotide is suitable for use as a crRNA segment in a guide RNA that forms a genetically modified KIT gene, resulting in a polypeptide having a mutation at position S123, respectively.

[0147] Table 1:

[0148]

[0149] Lead Editing System

[0150] Lead editing is a nucleic acid editing system that enables the installation of point mutations (i.e., base-to-base conversion), small insertions, or almost any combination of small deletions in the cellular DNA at the desired locus. It involves using lead editing to guide the targeting of the "lead editor" nucleoprotein to the target site in DNA. In some embodiments, the lead 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 lead editing guide is RNA (pegRNA, PEG RNA, or engineered pegRNA (ePEG RNA)), which guides the lead editor enzyme to the targeted locus and also encodes the required editing. PegRNA includes a scaffold (which is bound to the lead editor), a spacer sequence (which is complementary to the genomic site), and an extension arm (which includes two domains not typically included in other guide RNAs), a primer binding site (PBS), and a reverse transcriptase template (RTT).

[0151] As with typical gRNA, pegRNA guides nickase to the target site by homology with genomic DNA locus.During the lead editing, once the lead editor compounded with pegRNA is positioned to the genomic site, polymerase (such as reverse transcriptase (RT)) will use DNA synthesis template to synthesize the new DNA chain containing the required editing.Then the new DNA chain replaces the corresponding endogenous DNA chain at the genomic site, so that the desired, edited nucleotide sequence is installed in the genome at the editing site.Such systems are commercially available (for example, from Addgene, Cambridge, MA), and are described in, for example, U.S. Patent number 11,447,770 and International Publication No. WO 2022 / 067130 and AV Anzalone et al., Nature, 576:149-157 (2019) and JL Doman et al., Nature Protocols, 17, pp. 2431–2468 (2022).

[0152] There are several versions of the lead editing system. In the initial version, the PE1 system, pegRNA guides the Cas9 nickase to the target sequence, where it cuts the non-target strand and produces 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 chain replaces the unedited 5' flap, and the resulting heteroduplex is resolved by the cell's mismatch repair (MMR) system. Alternatively, the edited 3' flap can be excised, and the target sequence will remain unchanged, but will be available as a substrate for another round of lead editing.

[0153] In the subsequent version of the PE2 system, the reverse transcriptase portion of the lead editor enzyme contains five mutations (D200N, L603W, T330P, T306K, and W313F). This Cas9 nickase-pentamutant reverse transcriptase fusion enzyme has increased activity, enhanced binding between the template and PBS, increased processivity, and improved thermal stability.

[0154] In another version of the PE3 system, the PE2 Cas9 nickase-five mutant reverse transcriptase fusion enzyme is used together with PEG RNA plus an additional simple (e.g., excluding PBS or RTT) gRNA, which guides the Cas9 nickase to nick the unedited (opposite) chain at a nearby site. This additional gRNA can be referred to as a nick guide. The newly edited chain is then beneficial as a template for repair during heteroduplex resolution. In another version of the PE3b system, the gRNA includes a spacer that binds only to the edited chain, thereby guiding the nicking of the unedited chain only after editing occurs. The PE2, PE3, and PE3b systems all use a PE2 Cas9 nickase-five mutant reverse transcriptase fusion enzyme.

[0155] Prime editing guide RNA

[0156] The present disclosure provides polynucleotides forming lead editing guide RNA (referred to as PEG RNA, Peg RNA, peg RNA or pegRNA), which are suitable for, for example, lead editing CRISPR PE1, PE2 and PE3 systems for modifying the cKIT gene. In some embodiments, PEG RNA is an engineered PEG RNA (ePEG RNA). The ePEG RNA may include a specific 3' structural motif. In some embodiments, pegRNA RNA or ePEG RNA can be used for lead editing CRISPR PE1, PE2 or PE3 systems to mutate the cKIT gene so that the resulting polypeptide has the double mutation S123P and D121L described herein. These polynucleotides (Peg RNA or ePEG RNA) include a spacer complementary to the genomic site, a scaffold bound to the lead editor, a primer binding site (PBS), and a reverse transcriptase template (RTT). In some embodiments, PEG RNA includes a 3' structural motif. PEG RNA and ePEG RNA can be represented by the following formula:

[0157] 5'-spacer-scaffold-RTT / PBS-3' structural motif.

[0158] The spacer identifies the target nucleic acid site (i.e., is complementary to the genomic site). The length of the spacer segment may include 10-30 nucleotides, 15-25 nucleotides, 18-22 nucleotides, or 19-21 nucleotides. In some embodiments, the length of the spacer is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. 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 RNA or ePEG RNA of the present disclosure, the spacer has the 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.

[0159] 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 lead editor. The length of the scaffold segment may include 50-105 nucleotides. In the exemplary PEG RNA and ePEG RNA of the present disclosure, the scaffold has the following sequence:

[0160] GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 18); or

[0161] GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTG (SEQ ID NO: 19).

[0162] 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.

[0163] 3' structural motifs 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. WO 2022 / 067130. Particularly effective 3' structural motifs are tevopreQ1 motifs (JL Doman et al., Nature Protocols, 17, pp. 2431–2468 (2022)), which have the sequence CGCGGTTCTATCTAGTTACGCGTTAAACCAACTAGAATTT (SEQ ID NO: 20). In some embodiments, when PEG RNA includes tevopreQ1 motifs (SEQ ID NO: 20) or a sequence with a listed sequence identity to SEQ ID NO: 20, the PEG RNA is referred to as engineered PEG RNA (ePEG RNA). In some embodiments of the ePEG RNA disclosed herein, 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.

[0164] In some embodiments, PEG RNA may include a poly (T) sequence as a 3' structural motif. In RNA form, PEG RNA may include a poly (U) sequence as a 3' structural motif. The poly (T) or poly (U) sequence may include 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) core bases. In some embodiments, the poly (T) or poly (U) sequence includes 6 thymine or 6 uracil core bases. One or more nucleotides in the poly (T) sequence or poly (U) sequence may be modified. Examples of modifications include 2'-O-methylation of sugars (for RNA), phosphorothioate internucleoside linkages, or both.

[0165] The reverse transcriptase template (i.e., reverse transcriptase template or RTT) provides a DNA synthesis template containing the sequence to be edited. The length of the RTT segment may be 15-40 nucleotides or 20-30 nucleotides. In some embodiments, the length of the RTT is 20, 23, or 26 nucleotides. 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 RNA or ePEG RNA 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.

[0166] The primer binding site (PBS) is bound to the 3' flap. The length of the primer binding site segment can be 5-25 nucleotides or 10-15 nucleotides. In some embodiments, the length of the PBS segment is 10, 13, 14 or 15 nucleotides. 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.

[0167] In certain embodiments, the polynucleotides (PEG or ePEG RNA) of the present disclosure, suitable for modifying the cKIT gene, particularly providing the mutations S123P, D121L, or both, have the general structure listed above, wherein the RTT / PBS sequence comprises:

[0168] ccttgttn 1 n 2 n 3 n a n b n c n 4 n 5 n 6 n 7 n 8 n 9<h2 style=";text-align:left;direction:ltr">tatgggaaaga(RTT / PBS+7 / 10)(SEQ ID NO:23);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0169] <h2 style=";text-align:left;direction:ltr"> tttccttgttn<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 3 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> a <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> b <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> c <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 4 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 5 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 6 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 7 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 9 <h2 style=";text-align:left;direction:ltr"> tatgggaaaga(RTT / PBS+10 / 10)(SEQ ID NO:24);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0170] <h2 style=";text-align:left;direction:ltr"> gcttttccttgttn<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 3 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> a <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> b <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> c <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 4 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 5 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 6 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 7 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 9 <h2 style=";text-align:left;direction:ltr"> tatgggaaaga(RTT / PBS+13 / 10)(SEQ IDNO:25);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0171] <h2 style=";text-align:left;direction:ltr"> ccttgttn<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 3 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> a <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> b <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> c <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 4 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 5 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 6 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 7 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 9 <h2 style=";text-align:left;direction:ltr"> tatgggaaagaaga(RTT / PBS+7 / 13)(SEQ ID NO:26);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0172] <h2 style=";text-align:left;direction:ltr"> tttccttgttn<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 3 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> a <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> b <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> c <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 4 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 5 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 6 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 7 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 9<h2 style=";text-align:left;direction:ltr">tatgggaaagaaga(RTT / PBS+10 / 13)(SEQ IDNO:27);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0173] <h2 style=";text-align:left;direction:ltr"> gcttttccttgttn<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 3 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> a <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> b <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> c <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 4 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 5 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 6 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 7 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 9 <h2 style=";text-align:left;direction:ltr"> tatgggaaagaaga(RTT / PBS+13 / 13)(SEQ IDNO:28);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0174] <h2 style=";text-align:left;direction:ltr"> gcttttccttgttn<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 3 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> a <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> b <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> c <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 4 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 5 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 6 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 7 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 9 <h2 style=";text-align:left;direction:ltr"> tatgggaaagaagaca(RTT / PBS+7 / 15)(SEQID NO:29);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0175] <h2 style=";text-align:left;direction:ltr"> tttccttgttn<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 3 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> a <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> b <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> c <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 4 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 5 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 6 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 7 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 9 <h2 style=";text-align:left;direction:ltr"> tatgggaaagaagaca(RTT / PBS+10 / 15)(SEQ IDNO:30);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0176] <h2 style=";text-align:left;direction:ltr"> gcttttccttgttn<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 3 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> a <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> b <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> c <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 4 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 5 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 6 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 7 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 9tatgggaaagaagaca(RTT / PBS+13 / 15)(SEQID NO:31); or

[0177] 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 IDNO:32);

[0178] in:

[0179] n 1 n 2 n 3 represents a codon selected from ctg, tta, ttg, ctt, cta or ctc;

[0180] n 4 n 5 n 6 represents a codon selected from ccc, cct, cca, ccg, tcc, tct, tca, tcg, agt or agc;

[0181] n 7 n 8 n 9 represents a codon selected from ttg, tta, ctt, ctc, cta or ctg;

[0182] n a n b n c represents a codon selected from the group consisting of cgt, cgc, cga, cgg, aga or agg; or

[0183] Its combination.

[0184] In some embodiments, n a n b n c In some embodiments, n a n b n c It's agg.

[0185] In some embodiments, the polynucleotides (PEG or ePEG RNA) of the present disclosure comprise a spacer of the sequence gttgtcttctttcccataca (SEQ ID NO: 17) and an RTT / PBS sequence of any one of SEQ ID NO: 23 to SEQ ID NO: 32. a n b n c is cgc. 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.

[0186] In some embodiments, the polynucleotides (PEG or ePEG RNA) of the present disclosure comprise a spacer of the sequence cttctttcccatacaaggag SEQ ID NO: 100 and an RTT / PBS sequence of any one of SEQ ID NO: 23 to SEQ ID NO: 32. a n b n c is cgc. 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.

[0187] In certain embodiments, the polynucleotides (PEG or ePEG RNA) of the present disclosure, suitable for modifying the cKIT gene, particularly the gene providing the D121L mutation, have the general structure listed above, wherein the spacer is SEQ NO: 100, and the RTT / PBS sequence comprises:

[0188] ttccttgttn 1 n 2 n 3 n a n b n c tccttgtatgggaaag(RTT / PBS+9 / 14)(SEQ ID NO:101);

[0189] ttccttgttn 1 n 2 n 3 n a n b n c tccttgtatgggaaagaa(RTT / PBS+9 / 16)(SEQ ID NO:102);

[0190] in:

[0191] n 1 n 2 n 3 represents a codon selected from ctg, tta, ttg, ctt, cta or ctc;

[0192] n a n b n c represents a codon selected from the group consisting of cgt, cgc, cga, cgg, aga or agg; or

[0193] Its combination.

[0194] In some embodiments, the polynucleotide (PEG or ePEG RNA) of the present 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.

[0195] In some embodiments, the polynucleotide (PEG or ePEG RNA) of the present 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.

[0196] 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.

[0197] 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.

[0198] Examples of ePEG RNAs are shown below, wherein the spacer sequence is shown in italics, the scaffold (between the spacer sequence and the RTT / PBS sequence) and the 3' structural motif are shown in uppercase, and the PBS / RTT sequence is shown underlined (wherein the codon for the D121L mutation in the cKIT gene is shown in bold, the codon for the S123P mutation in the cKIT gene is shown in double underline, and the codon for the L124 mutation in the cKIT gene is shown in bold and double underline). The primer binding site (PBS) sequence and the reverse transcriptase template (RTT) sequence are of different lengths, wherein the RTT segment is identified by the number of bases after the last expected 3' edit.

[0199] [ePEG RTT / PBS+7 / 10](ePEG1)(SEQ ID NO:33):

[0200]

[0201] [ePEG RTT / PBS+10 / 10](ePEG4)(SEQ ID NO:34):

[0202]

[0203] [ePEG RTT / PBS+13 / 10](ePEG7)(SEQ ID NO:35):

[0204]

[0205] [ePEG RTT / PBS+7 / 13](ePEG2)(SEQ ID NO:36):

[0206]

[0207] [ePEG RTT / PBS+10 / 13](ePEG5)(SEQ ID NO:37):

[0208]

[0209] [ePEG RTT / PBS+13 / 13](ePEG8)(SEQ ID NO:38):

[0210]

[0211] [ePEG RTT / PBS+7 / 15](ePEG3)(SEQ ID NO:39):

[0212]

[0213] [ePEG RTT / PBS+10 / 15](ePEG6)(SEQ ID NO:40):

[0214]

[0215] [ePEG RTT / PBS+13 / 15](ePEG9)(SEQ ID NO:41):

[0216]

[0217] [ePEG RTT / PBS+10 / 14](SEQ ID NO:42):

[0218]

[0219] Scaffold optimized [ePEG RTT / PBS+10 / 14] (SEQ ID NO: 43):

[0220]

[0221] Scaffold optimized [ePEG RTT / PBS+10 / 13] (SEQ ID NO: 44):

[0222]

[0223] Scaffold optimized [ePEG RTT / PBS+10 / 15] (SEQ ID NO: 45):

[0224]

[0225] In some embodiments, the ePEG RNA of the present disclosure, suitable for modifying the cKIT gene, particularly providing the mutations S123P and D121L (preferably a double mutation), 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 ePEG RNA sequences of SEQ ID NOs: 33-45. In certain embodiments, a particularly effective ePEG RNA is [ePeg RTT / PBS+10 / 14] (SEQ ID NO: 42).

[0226] Examples of ePEG RNA and PEG RNA are shown below, with the spacer sequence shown in italics and the scaffold (between the spacer sequence and the RTT / PBS sequence) and 3' structural motif shown in uppercase. The primer binding site (PBS) sequence and the reverse transcriptase template (RTT) sequence are of varying lengths, with the RTT segment identified by the number of bases following the last expected 3' edit.

[0227] [PEG RTT / PBS+9 / 14](ePEG-D)(SEQ ID NO:103):

[0228] cttctttcccatacaaggagGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCccttgttctgaggtccttgtatgggaaagCGCGGTTCTATCTAGTTACGCGTTAAAACCAACTAGAATTT

[0229] [ePEG RTT / PBS+9 / 16](ePEG-E)(SEQ ID NO:104):

[0230] cttctttcccatacaaggagGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCtttccttgttctgaggccttgtatgggaaagaaCGCGGTTCTATCTAGTTACGCGTTAAAACCAACTAGAATTT ccttgtatgggaaaga

[0231] [PEG-D(+9-14)D121L R122R](SEQ ID NO:105)

[0232] cttctttcccatacaaggagGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCttccttgttCTGAGGtccttgtatgggaaagTTTTTT

[0233] In some embodiments, the PEG RNA or ePEG RNA of the present disclosure, suitable for modifying the cKIT gene, in particular providing a gene with mutations S123P, D121L, or both (preferably a double mutation), 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 of SEQ ID NOs: 103-105. In some embodiments, the PEG RNA is [PEG-D(+9-14)D121LR122R] (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.

[0234] CRISPR nick guide RNA

[0235] The present disclosure provides polynucleotides that function as nicking guide RNAs (ngRNAs) suitable for use in, for example, lead editing CRISPR PE3 systems. These polynucleotides include:

[0236] [nick guide 1] (SEQ ID NO: 46) tttgggccactagtcatgaa;

[0237] [nick guide 2] (SEQ ID NO: 47) gccattccaactactgattt;

[0238] [nick 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:

[0239] n1 n 2 n 3 represents a codon selected from ctg, tta, ttg, ctt, cta or ctc;

[0240] n 4 n 5 n 6 represents a codon selected from ccc, cct, cca, ccg, tcc, tct, tca, tcg, agt or agc;

[0241] n 7 n 8 n 9 represents a codon selected from ttg, tta, ctt, ctc, cta or ctg;

[0242] n a n b n c represents a codon selected from the group consisting of cgt, cgc, cga, cgg, aga or agg; or

[0243] its combination;

[0244] [nick guide 4] (SEQ ID NO: 49) gtgaccaattattccctcaa; and

[0245] [nick guide 5] (SEQ ID NO: 50) gaggtttattcctgacccca.

[0246] 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.

[0247] 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 nick guide is ttgttctgcgccccttgtat (SEQ ID NO: 51).

[0248] Genetically engineered cells (e.g., HSPCs)

[0249] 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 genetically engineered cells (e.g., HSPCs) as described herein, comprising editing genes for expressing one or more cell surface antigens in a mutant form.

[0250] The method for producing genetically engineered cells may involve providing cells and introducing components of the gene editing system guided by nucleotides into cells for genome editing. In some embodiments, a nucleic acid comprising a gRNA hybridized with a portion of a nucleotide sequence encoding a cell surface antigen or predicted to hybridize therewith is introduced into the cell. In some embodiments, gRNA is introduced into the cell on a vector. In some embodiments, Cas endonuclease is introduced into the cell. In some embodiments, Cas endonuclease is introduced into the cell as a nucleic acid encoding Cas endonuclease. In some embodiments, gRNA and the nucleotide sequence encoding Cas endonuclease are introduced into the cell on the same nucleic acid (e.g., the same vector). In some embodiments, Cas endonuclease is introduced into the cell in the form of a protein. In some embodiments, Cas endonuclease and gRNA are preformed in vitro and introduced into the cell as a ribonucleoprotein complex.

[0251] Genetically engineered cells expressing mutant KIT

[0252] 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).

[0253] In some embodiments, the methods described herein involve genetically engineering a mutant KIT gene in a HSPC population using a nucleotide-guided gene editing system. In some embodiments, the methods described herein involve genetically engineering KIT by mutating position S123 or D121 in a HSPC population. In some embodiments, the methods described herein involve genetically engineering a mutant KIT gene in a HSPC population using a nucleotide-guided gene editing system (including a guide sequence provided by any of the sequences provided herein).

[0254] 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.

[0255] In some embodiments, the genetically engineered HSPCs comprise a genetically engineered KIT gene, wherein the genetically engineered KIT gene is engineered such that the encoded protein has reduced binding to a therapeutic anti-KIT antibody (e.g., SR1 antibody). In some cases, the genetically engineered KIT gene encodes a protein having a mutation at position S123 (e.g., S123P). In some cases, the genetically engineered KIT gene encodes a protein having a mutation at position D121 (e.g., D121L). In some cases, the genetically engineered KIT gene encodes a protein having mutations at positions S123P and D121.

[0256] An exemplary amino acid sequence of a genetically engineered KIT is provided below:

[0257] SEQ ID NO: 53 (KIT-S123P variant):

[0258] MRGARGAWDFLCVLLLLLRVQTGSSQPSVSPGESPPSIHPGKSDLIVRVGDEIRLLCTDPGFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPAKLFLVDRPLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIKSVKRAYHRCLHCSVDQEGKSVLSEKFILKVRPAFKAVPVVSVSKASYLLREGEEFTVTTCTIKDVSSSVYSTWKRENSQTKLQEKYNSWHHGDFNYERQATLTISSARVNDSGVFMCYANNTFGSANVTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPEHQQWIYMNRTFTDKWEDYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVNAAIAFNVYVNTKPEILTYDRLVNGMLQCVAAGFPEPTIDWYFCPGTEQRCSASVLPVDVQTLNSSGPPFGKLVVQSSIDSSAFKHNGT VECKAYNDVGKTSAYFNFAFKGNNKEQIHPHTLFTPLLIGFVIVAGMMCIIVMILTYKYLQKPMYEVQWKVVEEINGNNYVYIDPTQLPYDHKWEFPNRLSFGKTLGAGAFGKVVEATAYGLIKSDAAMTVAVKMLKPSAHLTEREALMSELKVLSYLGNHMIVNLLGACTIGGPTLVITEYCCYGDLNLNFLRKRDSFICSKQEDHAEAALYKNLLHSKSCSDSTNEYMDMKPGVSYVV PTKADKRRSVRIGSYIERDVTPAIMEDDELALDLEDLLSFSYQVAKGMAFLASKNCIHRDLAARNILLTHGRITKICDFGLARDIKNDSNYVVKGNARLPVKWMAPESIFNCVYTFESDVWSYGIFLWELFSLGSSPYPGMPVDSKFYKMIKEGFRMLSPEHAPAEMYDIMCWDADPLKRPTFKQIVQLIEKQISESTNHIYSNLANCSPNRQKPVVDHSVRINSVGSTASSSQPLLVHDDV

[0259] SEQ ID NO: 54 (KIT-D121L variant):

[0260] MRGARGAWDFLCVLLLLLRVQTGSSQPSVSPGESPPSIHPGKSDLIVRVGDEIRLLCTDPGFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPAKLFLVLRSLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIKSVKRAYHRCLHCSVDQEGKSVLSEKFILKVRPAFKAVPVVSVSKASYLLREGEEFTVTTCTIKDVSSSVYSTWKRENSQTKLQEKYNSWHHGDFNYERQATLTISSARVNDSGVFMCYANNTFGSANVTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPEHQQWIYMNRTFTDKWEDYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVNAAIAFNVYVNTKPEILTYDRLVNGMLQCVAAGFPEPTIDWYFCPGTEQRCSASVLPVDVQTLNSSGPPFGKLVVQSSIDSSAFKHNGT VECKAYNDVGKTSAYFNFAFKGNNKEQIHPHTLFTPLLIGFVIVAGMMCIIVMILTYKYLQKPMYEVQWKVVEEINGNNYVYIDPTQLPYDHKWEFPNRLSFGKTLGAGAFGKVVEATAYGLIKSDAAMTVAVKMLKPSAHLTEREALMSELKVLSYLGNHMIVNLLGACTIGGPTLVITEYCCYGDLNLNFLRKRDSFICSKQEDHAEAALYKNLLHSKSCSDSTNEYMDMKPGVSYVV PTKADKRRSVRIGSYIERDVTPAIMEDDELALDLEDLLSFSYQVAKGMAFLASKNCIHRDLAARNILLTHGRITKICDFGLARDIKNDSNYVVKGNARLPVKWMAPESIFNCVYTFESDVWSYGIFLWELFSLGSSPYPGMPVDSKFYKMIKEGFRMLSPEHAPAEMYDIMCWDADPLKRPTFKQIVQLIEKQISESTNHIYSNLANCSPNRQKPVVDHSVRINSVGSTASSSQPLLVHDDV

[0261] SEQ ID NO: 55 (KIT-D121L / S123P variant):

[0262] MRGARGAWDFLCVLLLLLRVQTGSSQPSVSPGESPPSIHPGKSDLIVRVGDEIRLLCTDPGFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPAKLFLVLRPLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIKSVKRAYHRCLHCSVDQEGKSVLSEKFILKVRPAFKAVPVVSVSKASYLLREGEEFTVTTCTIKDVSSSVYSTWKRENSQTKLQEKYNSWHHGDFNYERQATLTISSARVNDSGVFMCYANNTFGSANVTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPEHQQWIYMNRTFTDKWEDYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVNAAIAFNVYVNTKPEILTYDRLVNGMLQCVAAGFPEPTIDWYFCPGTEQRCSASVLPVDVQTLNSSGPPFGKLVVQSSIDSSAFKHNGT VECKAYNDVGKTSAYFNFAFKGNNKEQIHPHTLFTPLLIGFVIVAGMMCIIVMILTYKYLQKPMYEVQWKVVEEINGNNYVYIDPTQLPYDHKWEFPNRLSFGKTLGAGAFGKVVEATAYGLIKSDAAMTVAVKMLKPSAHLTEREALMSELKVLSYLGNHMIVNLLGACTIGGPTLVITEYCCYGDLNLNFLRKRDSFICSKQEDHAEAALYKNLLHSKSCSDSTNEYMDMKPGVSYVV PTKADKRRSVRIGSYIERDVTPAIMEDDELALDLEDLLSFSYQVAKGMAFLASKNCIHRDLAARNILLTHGRITKICDFGLARDIKNDSNYVVKGNARLPVKWMAPESIFNCVYTFESDVWSYGIFLWELFSLGSSPYPGMPVDSKFYKMIKEGFRMLSPEHAPAEMYDIMCWDADPLKRPTFKQIVQLIEKQISESTNHIYSNLANCSPNRQKPVVDHSVRINSVGSTASSSQPLLVHDDV

[0263] 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 sequence comprises a mutation at S123P and / or D121L, and wherein the polypeptide sequence has reduced binding to a therapeutic anti-KIT antibody (e.g., an SR1 antibody). Also provided herein are nucleic acids encoding the polypeptide sequences, vectors comprising the nucleic acids, cells comprising the nucleic acids or vectors, and methods of making the polypeptides, the methods comprising culturing the cells under conditions permissive for expression of the polypeptide and optionally isolating the polypeptide.

[0264] In some embodiments, the methods described herein involve genetically engineering a mutant KIT gene in a HSPC population using a nucleotide-guided gene editing system. In some embodiments, the methods described herein involve genetically engineering KIT by mutating position R55S in a HSPC population. In some embodiments, the methods described herein involve genetically engineering a mutant KIT gene in a HSPC population using a nucleotide-guided gene editing system.

[0265] In some embodiments, the genetically engineered HSPCs comprise a genetically engineered KIT gene, wherein the genetically 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 genetically engineered KIT gene encodes a protein having a mutation at position R55 (e.g., R55S).

[0266] SEQ ID NO: 56 (KIT-R55S variant):

[0267] MRGARGAWDFLCVLLLLLRVQTGSSQPSVSPGESPPSIHPGKSDLIVRVGDEISLLCTDPGFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPAKLFLVDRSLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIKSVKRAYHRCLHCSVDQEGKSVLSEKFILKVRPAFKAVPVVSVSKASYLLREGEEFTVTCTIKDVSSSVYSTWKRENSQTKLQEKYNSWHHGDFNYERQATLTISSARVNDSGVFMCYANNTFGSANVTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPEHQQWIYMNRTFTDKWEDYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVNAAIAFNVYVNTKPEILTYDRLVNGMLQCVAAGFPEPTIDWYFCPGTEQRCSASVLPVDVQTLNSSGPPFGKLVVQSSIDSSAFKHNGT VECKAYNDVGKTSAYFNFAFKGNNKEQIHPHTLFTPLLIGFVIVAGMMCIIVMILTYKYLQKPMYEVQWKVVEEINGNNYVYIDPTQLPYDHKWEFPNRLSFGKTLGAGAFGKVVEATAYGLIKSDAAMTVAVKMLKPSAHLTEREALMSELKVLSYLGNHMIVNLLGACTIGGPTLVITEYCCYGDLNLNFLRKRDSFICSKQEDHAEAALYKNLLHSKSCSDSTNEYMDMKPGVSYVV PTKADKRRSVRIGSYIERDVTPAIMEDDELALDLEDLLSFSYQVAKGMAFLASKNCIHRDLAARNILLTHGRITKICDFGLARDIKNDSNYVVKGNARLPVKWMAPESIFNCVYTFESDVWSYGIFLWELFSLGSSPYPGMPVDSKFYKMIKEGFRMLSPEHAPAEMYDIMCWDADPLKRPTFKQIVQLIEKQISESTNHIYSNLANCSPNRQKPVVDHSVRINSVGSTASSSQPLLVHDDV

[0268] 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 the sequence set forth in SEQ ID NO: 56, wherein the polypeptide sequence comprises a mutation at R55S, and wherein 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 vectors, and methods of making the polypeptides, the methods comprising culturing the cells under conditions permissive for expression of the polypeptide and optionally isolating the polypeptide.

[0269] Genetically engineered cells expressing multiplex systems (e.g., HSPCs)

[0270] In some embodiments, the cell surface protein KIT can be combined with other genetic engineering strategies, such as: i) editing of other epitopes on other target proteins; ii) other therapeutic base or lead editing methods (e.g., BCL11A erythroid enhancer); and iii) conventional gene therapy using integration vectors. For example, in some embodiments, this can be achieved by simultaneously transfecting two or more guide RNAs for different target surface proteins. In some embodiments, the two or more guide RNAs are provided sequentially or continuously, i.e., in two or more separate transfections.

[0271] Immunotherapeutic agents specific for cell surface antigens

[0272] Cytotoxic agents that target cells expressing cell surface antigens (e.g., cancer cells) can be used in conjunction 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 (e.g., target cancer cells) expressing specific cell surface antigens. Such cytotoxic agents may comprise protein binding fragments that bind to and target specific cell surface antigen epitopes.

[0273] Therapeutic antibodies / antibody-drug conjugates

[0274] Herein, genetically engineered genes are engineered so that the combination of their encoded proteins and therapeutic antibodies is reduced. In this context, "therapeutic" antibodies refer to antibodies that alleviate one or more existing symptoms or clinical signs relevant to an illness (such as a hematologic disorder). "Antibody" refers to a molecule containing at least one antigen binding site that immunospecifically binds to a specific antigen target of interest. Therefore, the term "antibody" includes but is not limited to full-length antibodies and / or their variants, fragments thereof, peptibodies and variants thereof, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (such as bispecific antibodies) formed by at least two complete antibodies, human antibodies, humanized antibodies, and mimics of the structure and / or function of antibodies or their specific fragments or parts (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 (sdAb), variable fragments (Fv), single chain variable fragments (scFv), or disulfide-linked Fv (sdFv); diabodies or bivalent diabodies; linear antibodies; single chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0275] In some embodiments, cytotoxic agents include therapeutic antibodies that can be conjugated to drugs (e.g., anticancer drugs) to form antibody-drug conjugates (ADCs). 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.

[0276] In some embodiments, the therapeutic anti-KIT antibody is the anti-KIT SR1 antibody or the 104D2 and A3C6E2 anti-KIT clones.

[0277] Suitable toxins or drugs for use in antibody-drug conjugates are well known in the art and will be apparent to one of ordinary skill in the art. See, for example, 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 linker or a non-cleavable linker) to attach the antibody and drug molecule. Examples of antibody-drug conjugates include, but are not limited to, brentuximab vedotin, glembatumumab vedotin / CDX-011, depatuxizumab 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 / RG7596 / DCDS4501A, and SGN-LIV1A. vedotin / ASG-22ME, AG-15ME, AGS67E, telisotuzumab vedotin / ABBV-399, ABBV-221, ABBV-085, GSK-2857916, tisotumab vedotin / HuMax-TF-ADC, HuMax-Axl-ADC, pinatuzumab vedotin / RG7593 / DCDT2980S, lifastuzumab vedotin / RG7599 / DNIB0600A, indusatumab vedotin / MLN-0264 / TAK-264, and vandortuzumab vedotin Vedotin) / RG7450 / DSTP3086S, Vedotin-sofituzumabvedotin / RG7458 / DMUC5754A, RG7600 / DMOT4039A, RG7336 / DEDN6526A, ME1547, PF-06263507 / ADC 5T4, trastuzumab emtansine / T-DM1, mirvetuximab soravtansine / IMGN853, coltuximab ravtansine / SAR3419, naratuximab emtansine / IMGN529, indatuximab ravtansine / BT-062, anetumab ravtansine / BAY 94-9343, SAR408701, SAR428926, AMG 224, PCA062, HKT288, LY3076226, SAR566658, lorvotuzumab mertansine / IMGN901, cantuzumab mertansine / SB-408075, cantuzumab ravtansine / IMGN242, laprituximab emtansine / IMGN289, IMGN388, bivatuzumab mertansine, AVE9633, BIIB015, MLN2704, AMG 172, AMG 595, LOP 628, vadastuximab talirine / SGN-CD33A, SGN-CD70A, SGN-CD19B, SGN-CD123A, SGN-CD352A, rovalpituzumab tesirine / SC16LD6.5, SC-002, SC-003, ADCT-301 / HuMax-TAC-PBD, ADCT-402, MEDI3726 / ADC-401, IMGN779, IMGN632, gemtuzumabozogamicin, inotuzumab ozogamicin / CMC-544, PF-06647263, CMD-193, CMB-401, trastuzumabduocarmazine / SYD985, BMS-936561 / MDX-1203, sacituzumab govitecan / IMMU-132, labetuzumab govitecan / IMMU-130, DS-8201a, U3-1402, milatuzumab doxorubicin / IMMU-110 / hLL1-DOX, BMS-986148, RC48-ADC / hertuzumab-vc-MMAE, PF-06647020, PF-06650808, PF-06664178 / RN927C, atinib-lupartumab amadotin / BAY1129980, aprutumab ixadotin / BAY1187982, ARX788, AGS62P1, XMT-1522, AbGn-107, MEDI4276, DSTA4637S / RG7861.

[0278] In some embodiments, the combination of antibody-drug conjugates and the epitope of cell surface protein induces the internalization of antibody-drug conjugates, and the drug (or toxin) can be released intracellularly. In some embodiments, the combination of antibody-drug conjugates and the epitope of cell surface protein induces the internalization of toxins or drugs, which allows toxins or drugs to kill cells (target cells) expressing cell surface proteins. In some embodiments, the combination of antibody-drug conjugates and the epitope of cell surface protein induces the internalization of toxins or drugs, which can regulate the activity of cells (target cells) expressing cell surface proteins. The type of toxins or drugs used in the antibody-drug conjugates described herein is not limited to any specific type.

[0279] In some embodiments, two or more (e.g., 2, 3, 4, 5 or more) epitopes of a cell surface antigen have been modified so that two or more (e.g., 2, 3, 4, 5 or more) different cytotoxic agents (e.g., two ADCs) can target the two or more epitopes. In some embodiments, the toxins carried by the ADCs can work synergistically to enhance efficacy (e.g., death of target cells). In some embodiments, two or more (e.g., 2, 3, 4, 5 or more) epitopes of a cell surface protein have been modified so that two or more (e.g., 2, 3, 4, 5 or more) different cytotoxic agents (e.g., two ADCs) can target the epitopes of the two or more cell surface antigens. In some embodiments, one or more (e.g., 1, 2, 3, 4, 5 or more) epitopes of a cell surface antigen have been modified, and one or more (e.g., 1, 2, 3, 4, 5 or more) epitopes of an additional cell surface protein have been modified, such that two or more (e.g., 2, 3, 4, 5 or more) different cytotoxic agents (e.g., two ADCs) are capable of targeting an epitope of a cell surface antigen and an epitope of an additional cell surface antigen. In some embodiments, targeting more than one cell surface antigen or one cell surface antigen and one or more additional cell surface proteins / antigens can reduce the recurrence of hematopoietic malignancies.

[0280] In some embodiments, the methods described herein involve administering an ADC targeting an epitope of a cell surface antigen that has mutated in a genetically engineered hematopoietic cell population. In some embodiments, the methods described herein involve administering an ADC targeting an epitope of a cell surface antigen that has mutated in a genetically engineered cell (e.g., HSPC) population and one or more additional cytotoxic agents that can target one or more additional cell surface proteins. In some embodiments, the agents can work synergistically to enhance efficacy by targeting more than one cell surface protein.

[0281] The ADCs described herein can be used as follow-up treatment for subjects who have undergone combination therapy as described herein.

[0282] In some embodiments, the methods described herein involve administering to a subject a population of genetically engineered cells lacking a non-essential epitope in a cell surface antigen (e.g., type 1 or type 2) and one or more immunotherapeutics (e.g., ADCs) targeting cells expressing the cell surface antigen. In any of the embodiments described herein, for example, if a hematopoietic malignancy recurs, one or more additional immunotherapeutics (e.g., targeting one or more additional epitopes and / or antigens) may be further administered to the subject.

[0283] Immune cells expressing chimeric antigen receptors (CARs)

[0284] In some embodiments, a cytotoxic agent that targets an epitope of a specific cell surface antigen as described herein is an immune cell expressing a chimeric antigen receptor (CAR) comprising an epitope-binding fragment (e.g., a single-chain antibody) that is capable of binding to an epitope of a cell surface protein (e.g., KIT).

[0285] As used herein, "chimeric antigen receptor" (CAR or abbreviated chimeric receptor) refers to a non-naturally occurring molecule that can be expressed on the surface of a host cell and includes a binding domain that provides CAR specificity (e.g., an epitope binding fragment that is bound to an epitope of a cell surface lineage-specific protein). Generally speaking, CAR includes at least two domains derived from different molecules.

[0286] Recognition of target cells (e.g., cancer cells) with specific protein epitopes on the cell surface by the epitope-binding fragment of the CAR transduces an activation signal to the signaling domain 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.

[0287] In some embodiments, immune cells express more than one CAR (e.g., 2, 3, 4, 5 or more), referred to as bispecific or multispecific immune cells. In some embodiments, immune cells express more than one CAR, at least one of which targets an epitope of a cell surface antigen. In some embodiments, immune cells express more than one CAR, each of which targets an epitope of a specific cell surface antigen. In some embodiments, immune cells express more than one CAR, 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 more than one cell surface protein or one cell surface protein and one or more additional cell surface proteins can reduce the recurrence of hematopoietic malignancies. In some embodiments, immune cells express CAR targeting more than one epitope (e.g., more than one epitope of an antigen or more than one epitope of an antigen), referred to as bispecific CAR.

[0288] In some embodiments, the cytotoxic agent targets an epitope of two or more lineage-specific cell surface proteins. In some embodiments, two or more CARs are expressed in the same immune cell, such as a bispecific chimeric receptor. 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 groups of cells express two or more different CARs. Two or more cells expressing different CARs can be administered or administered sequentially. In some embodiments, the cytotoxic agent targets an epitope of KIT. In some embodiments, CAR targeting KIT is expressed in the same immune cell (i.e., bispecific immune cell). 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 groups of cells express two or more different CARs. Two or more groups of cells expressing CAR targeting KIT can be administered simultaneously or sequentially.

[0289] In addition to the epitope-binding fragments described herein, the CAR may further comprise one or more of the following: a hinge domain (e.g., a CD28 hinge, an IgG4 hinge, or a CD8α hinge), a transmembrane domain (e.g., CD28™, CD8α™, 4-1BB™), a co-stimulatory domain (e.g., CD28z, 4-1BB, ICOS, OX40), a cytoplasmic signaling domain (e.g., CD3z), and combinations thereof.

[0290] 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 generally present between two domains of a protein and can allow for flexibility of the protein and movement of one or both domains relative to each other. Any amino acid sequence that provides such flexibility and movement of the epitope-binding fragment relative to the other domain of the chimeric receptor can be used. The hinge domain can contain about 10-200 amino acids, e.g., 15-150 amino acids, 20-100 amino acids, or 30-60 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.

[0291] In some embodiments, the hinge domain or at least a portion thereof is a hinge domain of a naturally occurring protein. In some embodiments, the hinge domain is of CD8α or CD28. In some embodiments, the hinge domain is a portion of the hinge domain of CD8α, such as a fragment containing at least 15 (e.g., 20, 25, 30, 35, or 40) consecutive amino acids of the hinge domain of CD8α or CD28.

[0292] The hinge domain of an antibody (such as an IgG, IgA, IgM, IgE or IgD antibody) is also suitable for use in chimeric receptors as described herein. In some embodiments, the hinge domain is a hinge domain connecting 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 an antibody and one or more constant regions of an antibody. In some embodiments, the hinge domain comprises the hinge domain of an antibody and the CH3 constant region of an antibody. In some embodiments, the hinge domain comprises the hinge domain of an antibody and the CH2 constant region and CH3 constant region of an 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 constant region and CH3 constant region of an IgG1 antibody. In some embodiments, the hinge region comprises the hinge region and CH3 constant region of an IgG1 antibody.

[0293] In some embodiments, CAR described herein may include one or more transmembrane domains, which may be in 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 suitable for CAR used herein can be obtained from naturally occurring proteins. Alternatively, the transmembrane domain can be a synthetic, non-naturally occurring protein segment, for example, a hydrophobic protein segment that is thermodynamically stable in a cell membrane.

[0294] The membrane spaning domain is based on the membrane spaning domain topology, including the number of times the membrane spaning domain passes through the membrane and the orientation of the protein for classification.For example, a single membrane spaning protein passes through the cell membrane once, and multiple membrane spaning proteins pass through the cell membrane at least twice (for example, 2, 3, 4, 5, 6, 7 times or more times). In some embodiments, the membrane spaning domain is a single membrane spaning domain. In some embodiments, the membrane spaning domain is a single membrane spaning domain that directs the N-terminus of the chimeric receptor to the extracellular side of the cell and the C-terminus of the chimeric receptor to the intracellular side of the cell. In some embodiments, the membrane spaning domain is obtained from a single membrane spaning protein. In some embodiments, the membrane spaning domain is CD28 or 4-1BB or CD8α.

[0295] In some embodiments, CAR described herein includes 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 effector function). The costimulatory signaling domain of the chimeric receptor described herein may 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).

[0296] In some embodiments, the CAR described herein comprises more than one (at least 2, at least 3, at least 4 or even more) costimulatory signaling domains. In some embodiments, the chimeric receptor comprises more than one costimulatory signaling domains obtained from different costimulatory proteins. In some embodiments, the chimeric receptor does not comprise a costimulatory signaling domain.

[0297] In general, in addition to the stimulation of antigen-specific signals, many immune cells also need costimulation to promote cell proliferation, differentiation and survival, and the effector functions of activated cells. The activation of the costimulatory signal transduction domain in host cells (such as immune cells) can induce cells to increase or reduce the production and secretion of cytokines, phagocytic properties, proliferation, differentiation, survival and / or cytotoxicity. The costimulatory signal transduction domain of any costimulatory protein can be applicable to CAR as described herein. The type of costimulatory signal transduction domain is based on factors such as immune cell types (for example, primary T cells, T cell lines, NK cell lines) and required immune effector functions (for example, cytotoxicity) such as expressing CAR to select. The example of the costimulatory signal transduction domain for CAR can be the cytoplasmic signal transduction domain of costimulatory protein, and the costimulatory protein includes but is not limited to CD27, CD28ζ (CD28z), 4-1BB, OX40, CD30, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3.

[0298] In some embodiments, the chimeric receptors described herein comprise one or more cytoplasmic signaling domains. Any cytoplasmic signaling domain can be used in the chimeric receptors described herein. Generally speaking, the cytoplasmic signaling domain transmits a signal, such as the interaction of an extracellular ligand binding domain with its ligand, to stimulate a cellular response, such as inducing an effector function (e.g., cytotoxicity) of the cell. In some embodiments, the cytoplasmic signaling domain is from CD3 zeta (CD3z).

[0299] In some embodiments, provided herein are CAR constructs targeting KIT or KIT plus other genes. The construct may further include at least one hinge domain (e.g., from CD28, CD8α, or an antibody), a transmembrane domain (e.g., from CD28), one or more costimulatory domains (one or more from CD28z), and a cytoplasmic signaling domain (e.g., from CD3z) or a combination thereof. In some instances, the methods described herein relate to administering to a subject a genetically engineered cell (e.g., HSPC) population (engineered to have mutant KIT or KIT plus other genes, such as those disclosed in International Publication No. WO 2023 / 159136) and / or expressing immune cells targeting KIT or KIT plus other genes, respectively, the CAR may further include at least one hinge domain (e.g., from CD28, CD8α, or an antibody), a transmembrane domain (e.g., from CD28), one or more costimulatory domains (one or more from CD28z), and a cytoplasmic signaling domain (e.g., from CD3z) or a combination thereof. In some embodiments, the immunotherapeutic product administered is a combination of immune cells expressing a single chimeric receptor targeting KIT.

[0300] Any of the CARs described herein can be prepared by conventional methods such as recombinant technology. The method for preparing a chimeric receptor herein involves generating a nucleic acid encoding a polypeptide comprising each of the domains of a chimeric receptor, the chimeric receptor comprising an epitope binding fragment and optionally a hinge domain, a transmembrane domain, at least one co-stimulatory signaling domain, and a cytoplasmic signaling domain. In some embodiments, the nucleic acids encoding the components of the chimeric receptor are linked together using recombinant technology.

[0301] In addition, any one of CAR can be expressed in immune cells and administered to human subjects by conventional methods. For example, T cells can be derived from T cells in the subject's own blood (autologous) or derived from T cells of another healthy donor (allogeneic). Once separated from the subject, these T cells are genetically engineered to express specific CARs, thereby programming them to target antigens present on the tumor surface. CAR-T cells are then infused into the subject by convention.

[0302] 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, wherein the CAR retains its ability to bind to KIT.

[0303] SEQ ID NO:67 anti-KIT SR1 CAR:

[0304] MLLLVTSLLLCELPHPAFLLIPQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGVIYSGNGDTSYNQKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARER DTRFGNWGQGTLVTVSSGSTSGSGKPGSSEGSTKGDIVMTQSPDSLAVSLGERATINCRASESVDIYGNSFMHWYQQKPGQPPKLLIYLASNLESGVPDRFSGSGSGTTDFTLTISSLQAEDVA VYYCQQNNEDPYTFGGGTKVEIKRAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRGGHSDYMNMTPRRPGPTRKHYQPY APPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0305] Methods of treating subjects

[0306] Genetically engineered cells (e.g., HSPCs) can be administered to a human subject in need of treatment, alone or in combination with one or more cytotoxic agents targeting one or more cell surface antigens as described herein. Since the cells have been genetically edited in the genes for one or more cell surface antigens, the cells and / or their progeny cells will express one or more cell surface antigens in a mutant form (e.g., but functional), such that they can escape targeting by cytotoxic agents.

[0307] Therefore, the present disclosure provides a method for treating a disease that generally affects the wild-type form of engineered cells, the method comprising administering (i) genetically engineered cells as described herein (e.g., HSPC) groups to a human subject in need, and optionally (ii) a cytotoxic agent targeting a cell surface antigen, the gene of which is genetically edited in the cell so that the cytotoxic agent does not target the wild-type form of engineered cells or its progeny cells. In the embodiment of administering (i) and (ii), the administration of (i) and (ii) can be performed simultaneously or in any order. In some embodiments, cytotoxic agent and / or cell can be mixed with a pharmaceutically acceptable carrier to form a pharmaceutical composition, which is also within the scope of the present disclosure.

[0308] In order to carry out 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 as described herein. In some embodiments, the subject is a human patient suffering from a hematopoietic malignancy.

[0309] As used herein, the term "effective amount" is used interchangeably with the term "therapeutically effective amount." As will be appreciated by those skilled in the art, the effective amount will vary depending on the particular condition being treated, the severity of the condition, individual patient parameters (including age, physical condition, size, sex, and weight), the duration of treatment, the nature of concurrent therapy (if any), the specific route of administration, and similar factors that are within the knowledge and expertise of the healthcare professional.

[0310] As described herein, the genetically engineered cells expressing chimeric receptors can be autologous to the subject, that is, the cells are obtained from a subject in need of treatment, manipulated so that the cells do not bind to cytotoxic agents, and then administered to the same subject. Compared to administering non-autologous cells, administering autologous cells to a subject can result in reduced rejection of host cells. For example, HSPCs are obtained from a biological sample of the subject, genetically engineered to the HSPCs, and administered to the same subject. In some cases, HSPCs are obtained from a biological sample, wherein the biological sample is CD34+ hematopoietic stem and progenitor cells derived from bone marrow cells, blood, umbilical cord blood cells, or mobilized peripheral blood.

[0311] Alternatively, the host cells are allogeneic cells, i.e., cells obtained from a first subject, genetically engineered, and then administered to a second subject that is different from the first subject but of the same species. For example, allogeneic immune cells can be derived from a human donor and administered to a human recipient that is different from the donor. In some embodiments, the genetically engineered cells have been further genetically engineered to reduce the host-versus-graft effect. For example, in some embodiments, immune cells and / or genetically engineered cells can be subjected to gene editing or gene silencing methods to reduce or eliminate the expression of one or more proteins involved in inducing a host immune response.

[0312] A typical amount of cells (ie, immune cells or genetically engineered cells of the disclosure) administered to a subject can be, for example, between 10 6 to 10 11 In some embodiments, less than 10 cells are administered to a subject. 6 In some embodiments, fewer than 10 cells are administered to a subject. 11 In some embodiments, one or more doses of cells include 10 6 cells to 10 11 cells, 10 7 cells to 10 10 cells, 10 8 cells to 10 9 cells, 10 6 cells to 10 8 cells, 10 7 cells to 10 9 cells, 10 7 cells to 10 10 cells, 10 7 cells to 10 11 cells, 10 8 cells to 10 10 cells, 10 8 cells to 10 11 cells, 10 9 cells to 10 10 cells, 10 9 cells to 10 11 cells or 10 10 cells to 10 11 cells.

[0313] In some embodiments, the methods described herein involve administering a population of genetically engineered cells (e.g., HSPCs) to a subject and administering one or more immunotherapeutic agents (e.g., cytotoxic agents). As will be appreciated by one of ordinary skill in the art, the immunotherapeutic agents may be of the same or different types (e.g., therapeutic antibodies, immune cell populations expressing chimeric antigen receptors, and / or antibody-drug conjugates).

[0314] In some embodiments, a cytotoxic agent (e.g., an immune cell expressing a CAR as described herein) comprising an epitope-binding fragment of an epitope binding to an epitope of a cell surface protein is administered prior to administering the genetically engineered cell. 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 longer before administering the genetically engineered cell.

[0315] Alternatively, in some embodiments, genetically engineered cells are administered before the cytotoxic agent (for example, expressing the immune cell of CAR as described herein) of the epitope-binding fragment of the epitope comprising the epitope binding fragment of the epitope of the 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 longer before administering the cytotoxic agent of the epitope binding fragment comprising the epitope binding fragment of the epitope of the cell surface protein.

[0316] In some embodiments, the cytotoxic agent targeting the cell surface protein and the genetically engineered cell (HSPC) group are administered substantially simultaneously. In some embodiments, the cytotoxic agent targeting the cell surface protein is administered and the patient is assessed for a period of time, and then the genetically engineered cell group is administered. In some embodiments, the genetically engineered cell group is administered and the patient is assessed for a period of time, and then the cytotoxic agent targeting the cell surface protein is administered.

[0317] Multiple administrations (e.g., dosages) of cytotoxic agents and / or genetically engineered cell populations are also within the scope of the present disclosure. In some embodiments, a cytotoxic agent and / or genetically engineered cell population is administered to a subject. In some embodiments, more than one (e.g., at least 2 times, at least 3 times, at least 4 times, at least 5 times or more) cytotoxic agent and / or genetically engineered cell population is administered to a subject. In some embodiments, a cytotoxic agent and / or genetically engineered cell population is administered to a subject at regular intervals (e.g., every six months).

[0318] Examples of routes of administration include intravenous, infusion, intradermal, subcutaneous, oral (eg, inhalation), transdermal (topical), transmucosal, and rectal administration.

[0319] Any of the methods described herein can be used to treat a hematological malignancy in a subject. As used herein, the term "treat" or "treatment" or "treating" or "to treat" refers to a therapeutic measure intended to alleviate a pathological condition or disorder, slow its progression, alleviate its symptoms, and / or stop its progression. Thus, a person in need of treatment includes those already suffering from the disorder. As used herein, treating cancer includes stabilizing cancer progression, slowing cancer progression, arresting cancer progression, reducing cancer size, or increasing overall survival in a subject diagnosed with cancer. Methods for assessing cancer progression are known in the art and include, for example, evaluating target lesions using imaging (e.g., X-rays, computed tomography, magnetic resonance imaging, caliper measurements, or positron emission tomography), cytology, or histology, or expression of tumor markers.

[0320] In some embodiments, the human subject suffers from a blood system disorder, 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 lymphoblastic leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, or chronic lymphoblastic leukemia and chronic lymphoblastic leukemia. Examples of blood system disorders other than hematopoietic malignancies include, but are not limited to, hemoglobinopathies, such as sickle cell disease, thalassemia, or primary immunodeficiency, such as SCID.

[0321] In some embodiments, cells involved in hematopoietic malignancies are resistant to conventional or standard therapeutic agents used to treat malignancies. For example, cells (e.g., cancer cells) may be resistant to chemotherapeutic agents and / or CAR-T cells used to treat malignancies.

[0322] In some instances, the hematopoietic malignancy includes high-risk acute myeloid leukemia (AML) or multiple myeloma.

[0323] Compositions and kits

[0324] Any immune cell expressing the chimeric receptor and / or genetically engineered cells (eg, HSPCs) described herein can be administered as a pharmaceutical composition in a pharmaceutically acceptable carrier.

[0325] The phrase "pharmaceutically acceptable" as used in conjunction with the compositions and / or cells of the present disclosure refers to that the molecular entities and other ingredients of such compositions are physiologically tolerable and generally do not produce adverse reactions when administered to humans. Preferably, as used herein, the term "pharmaceutically acceptable" means approved by federal regulatory agencies or state governments or listed for use in humans in the U.S. Pharmacopeia (US Pharmacopeia) or other generally recognized pharmacopeias. "Acceptable" means that the carrier is compatible with the active ingredients (e.g., nucleic acids, vectors, cells, or therapeutic antibodies) of the composition and does not negatively affect the subject to whom the composition is administered. Any pharmaceutical composition and / or cell used in the present method may comprise a pharmaceutically acceptable carrier, excipient, or stabilizer in the form of a lyophilized formulation or an aqueous solution.

[0326] Pharmaceutically acceptable carriers, including buffers, are well known in the art and may contain phosphates, citrates, 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 nonionic surfactants.

[0327] The medicine box for treating hematological disorders (such as hematopoietic malignancies) is also within the scope of the present disclosure. Such medicine box may include genetically engineered cells (such as HSPC), and optionally one or more cytotoxic agents targeting cell surface antigens, the genes of which are edited in hematopoietic cells. Such medicine box may include a container containing any one of the first pharmaceutical composition comprising genetically engineered cells (such as HSPC) as described herein, and optionally one or more additional containers containing a cytotoxic agent targeting a cell surface antigen as described herein (e.g., an immune cell expressing a chimeric receptor as described herein).

[0328] In some embodiments, the medicine box may include instructions for any one of the methods described herein. The included instructions may include a description of the use of genetically engineered cells (e.g., HSPCs), and optionally a description of one or more cytotoxic agents administered to a subject to achieve a desired activity in the subject. The medicine box may further include a description of whether the subject is suitable for treatment based on identifying whether the subject needs treatment. In some embodiments, the instructions include a description of administering genetically engineered cells (e.g., HSPCs) and optionally one or more cytotoxic agents to a subject in need of treatment.

[0329] Instructions for use of genetically engineered cells (e.g., HSPCs) and optionally cytotoxic agents as described herein generally include information on the dosage, dosing schedule, and route of administration for the intended treatment. The container may be a unit dose, bulk package (e.g., multi-dose package), or subunit dose. The instructions supplied in the kit of the present disclosure are typically written instructions on a label or package insert. The label or package insert indicates that the pharmaceutical composition is used to treat a disease or condition of a subject, delay its onset, and / or alleviate it.

[0330] The medicine boxes provided herein are all in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, soft packaging, etc. It is also contemplated that packaging for use in combination with specific devices (such as inhalers, nasal application devices, or infusion devices) is provided. The medicine box can have a sterile access port (e.g., the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). The container can also have a sterile access port. At least one active agent in the pharmaceutical composition is a chimeric receptor variant as described herein.

[0331] The kit may optionally provide additional components, such as buffer and interpretive information. Typically, the kit comprises a container and a label or package insert on or associated with the container. In some embodiments, the disclosure provides an article of manufacture comprising the contents of the kit described above.

[0332] Example

[0333] The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. With regard to mentioning specific materials, they are merely for illustrative purposes and are not intended to limit the present invention. Those skilled in the art may develop equivalent means or reactants without exercising creative ability and without departing from the scope of the present invention.

[0334] Part 1) hcKIT base editing for enhanced in vivo selection of cells for immune-based non-genotoxic bone marrow transplant conditioning, anti-cancer immunotherapy, and gene therapy.

[0335] Example 1: Chimeric orthologous KIT variants revealing a collection of mutants that avoid SR1 binding

[0336] To identify the specific amino acids that abolished SR1 clone binding, two transgenes expressing human or mouse cKIT constructs were individually cloned into the Sleeping Beauty transfer vector co-expressing BFP and puromycin resistance, as Figure 1A HEK-293T cells were electroporated in SF solution using the Lonza 4D-Nucleofector system with 500 nanograms (ng) of transfer vector and 500 ng of a plasmid expressing SB100x transposase. Cells were selected with puromycin (2 micrograms / ml (ug / mL)) and analyzed by flow cytometry using KIT 79D, AB55, SR1, and KIT 104D2 control antibodies. Figure 1B As shown, while all antibodies bound to the human cKIT protein, only clone Ab55 was able to bind to the mouse orthologous construct. In view of this finding, and in order to identify the domains bound by the different non-cross-reactive antibodies, a set of five chimeric constructs, each containing one of the murine domains in the human construct, were cloned and expressed in HEK-293T cells. Figure 1C As shown, for the 104D2 (mD1) and A3C6E2 clones, the set of mutations that abolished the binding of the tested antibodies was located in domain 1, while for the SR1 clone, the set of mutations was located in domain 2 (mD2).

[0337] Subsequently, all orthologous point mutations in mouse and human domain 2 were grouped and cloned into three different constructs, which were tested on HEK-293T cells (see Figure 2A and Figure 2B ). Figure 2A is a schematic diagram showing that orthologous mutations were divided into three clusters, cloned, and transduced in HEK-293T cells. Figure 2B Fluorescence activated cell sorting (FACS) analysis showed that one of the three subgroups (mD2_Group 1) was sufficient to avoid binding of the therapeutic antibody (SR1). Repeating this approach allowed the identification of a small set of four point mutations that abolished SR1 binding and likely represented the epitope recognized by the antibody (see Figure 2C ).

[0338] exist Figure 3AIn the present study, four mutation groups (D121G; R122L; S123P; Y125F) of the mouse cKIT protein that resulted in a lack of SR1 antibody binding were shown in FACS analysis and compared to the same human epitope. To identify a mutation suitable for a base editing strategy, all single orthologous point mutations and amino acids that could result in ABE / CBE (adenine base editing / cytosine base editing) editing at the same codon were tested in the same experiment. Figure 3B The bar graph shows the ratio of mean fluorescence intensity (MFI) of the therapeutic and control antibodies, normalized to the ratio of the same MFI in the human cKIT WT control. Because mouse cKIT does not fully cross-react with human ligands, the same mutations were stained and tested with fluorescently conjugated stem cell factor ligand (SCF). Interestingly, the S123P protein had reduced affinity for the SR1 antibody, while maintaining binding of the human SCF ligand ( Figure 3C ).

[0339] Example 2: Novel mutations targeting SR1 binding resistance via the D2 NNN library

[0340] To expand on the findings of Example 1, a comprehensive library approach was designed to further define alternative codons involved in SR1 binding. A degenerate library in which each codon within the KIT extracellular domain 4 consisted of a degenerate base (NNN) was cloned into a Sleeping Beauty transfer plasmid expressing human KIT cDNA, the mTagBFP reporter gene, and puromycin resistance ( Figure 4A HEK-293T cells were electroporated with the library plasmid and the 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 The library region was 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 base editing methods ( Figure 4C Interestingly, this assay re-identified S123P as a candidate point mutation that abolishes SR1 binding and also highlighted D121L as another possible candidate.

[0341] Example 3: Characterization of SR1-resistant cKIT variants and development of base editing strategies

[0342] The two identified variants were expressed by the Sleeping Beauty transposon system in BAF3 cells and compared with hcKIT and murine SR1 epitopes ( Figure 5AThe same cell line was used for dose affinity determination of SR1 or SCF conjugated to ALEXA FLUOR 647. Both variants were highly effective in avoiding SR1 binding even at higher concentrations, but showed similar affinity for the conjugated SCF cytokine ( Figure 5B To introduce the desired single codon change (T to C) at the cKIT locus with high efficiency and low toxicity ( Figure 6A ), without introducing double-stranded DNA breaks, CRISPR-Cas base editing was tested. A set of sgRNAs predicted to introduce S123P mutations in combination with adenine base editors were designed (SEQ ID NOs: 14-16, see Tables 1 and Figure 6A ). The CRISPR-Cas9 base editor ABE8e (TadA-8e V106W) was selected to develop the editing strategy and was further optimized by mutating the Cas9 nickase protein to relax PAM specificity, allowing editing in the absence of a 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 stated, base editing experiments were performed by electroporating reporter K562 cells overexpressing the FLT3 gene with 500 ng of base editor plasmid and 300 picomoles (pmol) or 360 pmol sgRNA (Integrated DNA Technologies, Coralville, IA). The cells were then cultured and samples for genomic DNA and flow cytometry analysis were harvested 72 h after editing ( Figure 6B and 6C ).

[0343] Example 4: HSC editing for SR1 resistance

[0344] To translate base editing programs into primary cells, suitable delivery methods for base editors need to be developed, as bacterial plasmid transfection has been reported to be toxic to stem cells.

[0345] The base editing protocol was transferred to human CD34+ HSPCs using base editor mRNA generated by in vitro transcription. Functional mRNA encoding adenine base editor (SpRY-ABE8e-V106W 3xNLS) was generated by in vitro transcription (IVT) using a MEGASCRIPT T7 transcription kit (AM1333, available from Invitrogen, Carlsbad, CA) or a T7 HISCRIBE kit (available from New England Biolabs, Ipswich, MA) and a custom plasmid (SEQ ID NO: 68) template, which encodes the base editor reading frame downstream to the T7 promoter sequence, a minimal 5'UTR, and downstream to two copies of HBB (hemoglobin B) 3'UTR and a polyA sequence (60-120 base pairs long). Co-transcriptional capping was achieved by replacing 80% of the GTP with a 3'-O-Me-m7G(5')ppp(5')G RNA cap analog (S1411, available from New England Biolabs). IVT reaction products were purified using an RNAESY mini kit (available from Qiagen, Venice, Netherlands) or MONARCH mRNA CLEANUP (T205L, available from New England Biolabs), quantified spectrophotometrically, and analyzed for quality control using an Agilent fragment analyzer (available from Agilent Technologies, Santa Clara, CA).

[0346] To confirm that S123P cKIT base editing is feasible for primary human CD34+ hematopoietic stem and progenitor cells, in vitro base editing and extended culture experiments were performed. Mobilized peripheral blood-derived CD34+ HSPCs were thawed and cultured at 0.5-0.75 million cells / 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), UM171 35 nM (Selleckhem, Houston, TX). 48 hours after thawing, 0.15-0.25 million 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 (ug) of base editor mRNA (SpRY-ABE8e-V106W) and sgRNA (Integrated DNA Technologies) 250-450 picomoles (pmol / uL) per 20 microliters of reaction. The cells were cultured in the above medium for 5-7 days. To test specific resistance to SR1 antibodies, S123P-edited or AAVS1-edited CD34+ cells were expanded in vitro for 3 days after editing and then co-cultured 4 times with several doses of SR1 under each condition.

[0347] Figure 7A Figure 2. Experimental layout and timeline for in vitro expansion of mobilized peripheral blood-derived CD34+ HSPCs and cKIT base editing. Cells were electroporated with 7.5 micrograms (ug) of SpRY-ABE8e-V106W3xNLS mRNA and 300 pmol of S123P_gRNA_3sgRNA or AAVS1 gRNA. Figure 7B The editing efficiency obtained by Sanger sequencing on day 3 after electroporation is shown. After 4 days of culture with SR1 antibody, cell counts were measured by flow cytometry and are shown in Figure 7C middle.

[0348] SEQ ID NO:68 - pmRNA plasmid for in vitro transcription of the SpRY_ABE8e_V106W adenine base editor, including 5'UTR, HBB 3'UTRx2, and a 120 bp long polyA tail:

[0349]

[0350] Example 5: HSC editing mediated in vitro selection for SR1

[0351] To establish that S123P cKIT edits human CD34+ hematopoietic stem cells and progenitor cells can be enriched in culture in the presence of SR1 antibodies, in vitro base editing and extended culture experiments were performed. Mobilized peripheral blood-derived CD34+ HSPCs were thawed and cultured at 0.5-0.75 million / mL in SFEMII (StemCell Technologies) medium supplemented with 1% penicillin / streptomycin, SCF 125ng / mL (Peprotech), FTL3L 125ng / mL (Peprotech), TPO 62.5ng / mL (Peprotech), Stemregenin-1 0.75 micromolar (uM) (StemCell technologies), UM171 35nM (Selleckhem). 48 hours after thawing, 0.15-0.25 million HSPCs were electroporated using the Lonza 4D-Nucleofector system in P3 electroporation solution (Lonza), supplemented with 2.5-7.5ug base editor mRNA (SpRY-ABE8e-V106W) and sgRNA (Integrated DNA Technologies) 250-450 pmoles per 20 microliters (uL) reaction. To test the specific enrichment of edited cells in the presence of SR1 antibody, CD34+ cells edited with S123P and BCL11A enhancer or AAVS1 were stained with CELL TRACE yellow and CFSE, respectively, mixed at a 50:50 ratio, and expanded in vitro for 4 days after editing, co-cultured 4 times with several doses of SR1 in each condition.

[0352] Figure 8A Figure 2 shows the experimental layout and timeline for the in vitro expansion culture and cKIT base editing of mobilized peripheral blood-derived CD34+ HSPCs. Cells were electroporated with 7.5 micrograms (ug) of SpRY-ABE8e-V106W3xNLS mRNA and 300 pmol of S123P_gRNA_3 and BCL11A+55sgRNA, BCL11A+58sgRNA, or AAVS1 gRNA. Figure 8BThe relative percentages of cells at different antibody concentrations, measured by flow cytometry, are shown, demonstrating the selective advantage of S123P-edited cells over AAVS1 control cells in the presence of the antibody. Measuring the MFI of FITC in CFSE cells and the MFI of PE in CELL TRACE yellow cells highlights the possible mechanism of enrichment. In the presence of the antibody, AAVS1 cells retained higher levels of the dye. Thus, SR1 inhibited the proliferation of control cells, but not S123P and BCL11A triple-edited cells. Flow cytometric analysis is shown in Figure 8C middle.

[0353] Example 6: Chimeric orthologous KIT variants revealing a collection of mutants that avoid 104D2 / A3C6E2 binding

[0354] To identify the specific amino acids that abolished the binding of the 104D2 / A3C6E2 clone, two transgenes expressing human or mouse cKIT constructs were individually cloned into the Sleeping Beauty transfer vector that co-expresses BFP and puromycin resistance, e.g. Figure 1A HEK-293T cells were electroporated in SF solution using the Lonza 4D-Nucleofector system with 500 nanograms (ng) of transfer vector and 500 ng of a plasmid expressing SB100x transposase. Cells were selected with puromycin (2 micrograms / ml (ug / mL)) and analyzed by flow cytometry using AB55, SR1, and KIT 104D2 control antibody staining. Figure 1B As shown, while all antibodies bound to the human cKIT protein, only clone Ab55 was able to bind to the mouse orthologous construct. In view of this finding, and in order to identify the domains bound by the different non-cross-reactive antibodies, a set of five chimeric constructs, each containing one of the murine domains in the human construct, were cloned and expressed in HEK-293T cells. Figure 1C As shown, for the 104D2 (mD1) and A3C6E2 clones, the set of mutations that abolished the binding of the tested antibodies was located in domain 1, while for the SR1 clone, the set of mutations was located in domain 2 (mD2).

[0355] Subsequently, all orthologous point mutations in mouse and human domain 1 were grouped and cloned into three different constructs, which were tested on HEK-293T cells (see Figure 9A and Figure 9B ). Figure 9A is a schematic diagram showing that orthologous mutations were divided into three clusters, cloned, and transduced in HEK-293T cells. Figure 9BFluorescence activated cell sorting (FACS) analysis showed that one of the three subgroups (mD1_Group 1) was sufficient to avoid binding of therapeutic antibodies (104D2 or A3C6E2). Repeating this approach allowed the identification of a small set of four point mutations that abolished binding of 104D2 or A3C6E2 and likely represented the epitope recognized by the antibodies (see Figure 9C ).

[0356] exist Figure 10A In Figure 2, four mutation groups (E53T; I54L; R55S; L57T) of the mouse cKIT protein that resulted in lack of binding of the 104D2 or A3C6E2 antibodies were shown in FACS analysis and compared to the same human epitope. Figure 10B The bar graph shows the ratio of mean fluorescence intensity (MFI) of therapeutic and control antibodies for each of these mutations, normalized to the ratio of the same MFI in the human cKIT WT control.

[0357] Part 2) - Double mutagenesis via prime editing

[0358] Example 7: Evaluation of human cKIT gene with double mutations

[0359] NIH 3T3 (fibroblast) cells were stably transduced with the Sleeping Beauty transposon carrying the wild-type human cKIT gene (hcKIT) and three candidate variants: S123P, D121L, and S123P-D121L. After puromycin-mediated selection of transduced cells, transgene expression was analyzed using fluorescence-activated cell sorting (FACS) staining with two antibodies: cKIT control antibody 104D2 clone and AF488-conjugated anti-hcKIT SR1 clone. Figure 11A FACS analysis of NIH3T3 cells stably transduced with the Sleeping Beauty transposon carrying the wild-type human cKIT gene (hcKIT) and three candidate variants: S123P, D121L, and S123P-D121L is shown. Notably, these findings revealed interesting differences in the antibody binding patterns between the various cKIT variants. It should be noted that the mutation S123P exhibited relative protection from binding of the therapeutic antibody compared to the wild-type, indicating that the epitope recognized by the antibody has been significantly altered. In contrast, the mutations D121L and D121L-S123P were observed to completely abolish binding of the therapeutic antibody in this assay, indicating that the epitope necessary for antibody recognition was significantly disrupted.

[0360] To evaluate the responses of cell lines expressing different cKIT variants, we used AF488-conjugated stem cell factor (SCF) ligand for staining. Following SCF staining, we performed comparative analysis by measuring the mean fluorescence intensity (MFI) of SCF and normalizing the MFI to that of an anti-cKIT control antibody. The control antibody was used to account for variations in transgene expression levels in different cell lines. Figure 11B Shown are the MFIs of cell lines expressing different cKIT variants using ALEXAFLUOR 488 conjugated to stem cell factor (SCF) ligand. Interestingly, the results revealed sigmoidal dose-response curves for SCF activity for all tested variants, including the wild-type (WT) cKIT control. Notably, the variants exhibited dose-response curves comparable to that of the cKIT WT control, indicating similar binding affinities to SCF. This observation suggests that the introduced mutations (variants S123P, D121L, and S123P-D121L) do not significantly alter the binding characteristics of cKIT to SCF.

[0361] Example 8: Prime Editing (PE2) Method and Evaluation of Various ePEG RNAs

[0362] Given the potential utility of the mutation D121L-S123P in conferring resistance to anti-cKIT SR1 therapy, the development of a lead editing approach was explored. To facilitate this exploration, a K562 reporter cell line was employed, which artificially expresses cKIT from its endogenous locus via promoter editing. Figure 12A A schematic diagram showing the lead editing approach for introducing D121L+S123P mutations into the K562 reporter cell line, conferring resistance to anti-cKIT SR1 antibody therapy, is shown, and a cartoon representation of the lead editing protein complexed with the double-stranded gene is shown. This strategy allows screening of various engineered lead editing guide RNAs (ePEG RNAs) to evaluate their effectiveness in inducing targeted mutations at cKIT exon 3. By employing this PE2 lead editing system, specific ePEG RNAs were identified that confer resistance to anti-cKIT therapy. To optimize the lead editing process, nine ePEG RNA sets were designed, varying the primer binding site (PBS) sequence and the length of the reverse transcriptase template (RTT), indicated as the number of bases after the last expected 3' edit (PBS10 / 13 / 15 and RTT+7 / +10 / +13). K562 cells were electroporated with PEmax plasmid and ePEG-expressing plasmid according to the protocol described by JL Doman et al., Nature Protocols, 17, pp. 2431–2468 (2022). Three days after electroporation, cells were analyzed by FACS ( Figure 12BAmong the tested PEG boots, RTT+10 and PBS10 / 13 / 15 were selected as the best performing PEG boots for further development.

[0363] The sequences of the nine ePEG RNAs are shown below, with the spacer sequences in italics, the scaffold and tevopreQ1 (JL Doman et al., Nature Protocols, 17, pp. 2431–2468 (2022)) sequences in uppercase, and the RTT / PBS sequence underlined (the D121L mutated codon is in bold, the S123P mutated codon is double underlined, and the L124 mutated codon is double underlined and bold).

[0364] [ePEG RTT / PBS+7 / 10](ePEG1)(SEQ ID NO:33):

[0365]

[0366] [ePEG RTT / PBS+10 / 10](ePEG4)(SEQ ID NO:34):

[0367]

[0368] [ePEG RTT / PBS+13 / 10](ePEG7)(SEQ ID NO:35):

[0369]

[0370] [ePEG RTT / PBS+7 / 13](ePEG2)(SEQ ID NO:36):

[0371]

[0372] [ePEG RTT / PBS+10 / 13](ePEG5)(SEQ ID NO:37):

[0373]

[0374] [ePEG RTT / PBS+13 / 13](ePEG8)(SEQ ID NO:38):

[0375]

[0376] [ePEG RTT / PBS+7 / 15](ePEG3)(SEQ ID NO:39):

[0377]

[0378] [ePEG RTT / PBS+10 / 15](ePEG6)(SEQ ID NO:40):

[0379]

[0380] [ePEG RTT / PBS+13 / 15](ePEG9)(SEQ ID NO:41):

[0381]

[0382] Example 9: Optimizing the PBS length of ePEG RNA

[0383] To further refine the PBS sequence length of the ePEG RNA, intermediate values between 13 and 15, specifically 14, were tested, and three selected ePEGs were re-evaluated in subsequent experiments. Specifically, [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 were evaluated.

[0384] [ePEG RTT / PBS+10 / 14](SEQ ID NO:42):

[0385]

[0386] The editing efficiency was determined by FACS analysis on day 3 after electroporation to identify the % SR1 negative cells. The results are shown in Figure 13A , which is a bar graph showing the RNA editing efficiency of ePEG with PBS lengths of 13, 14, and 15, demonstrating that ePEG with a PBS length of 14 exhibits the highest efficiency in editing the K562 reporter cell line.

[0387] Example 10: Evaluation of post-editing PAM mutations and seed sequence perturbations

[0388] To explore the effects of post-editing PAM mutations and seed sequence perturbations, experiments were performed using ePEGRNAs with RTT / PBS of +10 and 13. Different codons were tested for the amino acid S123P, as it encodes the PAM sequence on the opposite (non-coding) strand. Modification of the codon for the amino acid at position L124 was tested to perturb the seed sequence. In addition, the combined effect of the two perturbations was also examined. Figure 13BAs shown in the bar graph and Table A (below), the results indicate that modifying the PAM codon significantly reduced editing efficiency. Mutations in the seed sequence can be tolerated because they do not significantly affect editing efficiency. These findings suggest that although post-editing PAM mutations have a negative impact on editing efficiency, perturbations in the seed sequence can still be tolerated and can potentially be used in future applications.

[0389] Table A

[0390]

[0391]

[0392] To explore the perturbation of mutation D121, all possible codons encoding L amino acids were tested with guide lengths of +10-13 and +10-14, with and without the seed mutation. Figure 13C Table in. This analysis led to the development of several ePEG RNAs that showed potential for manipulating hematopoietic stem cells (HSCs). These findings highlight the possibility of using these ePEG RNAs for targeted modification of HSCs.

[0393] Example 11: Development of PE3 and PE3b CRISPR Systems

[0394] To develop the PE3 system, five nicking guides were designed, which adds simple guide RNAs to direct the Cas9 nickase to nick the unedited DNA strand at a nearby site.

[0395] These ng RNAs include:

[0396] [nick guide 1] (SEQ ID NO: 46) tttgggccactagtcatgaa;

[0397] [nick guide 2] (SEQ ID NO: 47) gccattccaactactgattt;

[0398] [nick 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:

[0399] n 1 n 2 n3 represents a codon selected from ctg, tta, ttg, ctt or ctc;

[0400] n 4 n 5 n 6 represents a codon selected from ccc, cct, cca or ccg;

[0401] n 7 n 8 n 9 represents a codon selected from ttg, tta, ctt, ctc or ctg; or

[0402] combinations thereof, such as ttgttctgcgccccttgtat (SEQ ID NO: 51);

[0403] [nick guide 4] (SEQ ID NO: 49) gtgaccaattattccctcaa; and

[0404] [nick guide 5] (SEQ ID NO: 50) gaggtttattcctgacccca.

[0405] Nick guides 1 and 2 are located 5' of the intended edit, one of the nick guides is complementary to the edited strand following the prime edit (PE3b system), and nick guides 4 and 5 are located 3' of the edit. Figure 14A , which is a schematic diagram illustrating the cKIT locus for targeted editing.

[0406] Figure 14B is a graph illustrating editing efficiency on day 3 (D3). FACS analysis was used to evaluate editing efficiency, focusing on three previously characterized cKIT ePEG RNAs (ePEG2, ePEG4, and ePEG5) and five nick guide variants. Of the nick guides (ng) tested, i.e., guides 3, 4, and 5, the data indicated that ng3 exhibited the highest effectiveness in terms of editing efficiency. It should be noted that ng3 was also used as part of the PE3b strategy, which targets the flap region encoding mutations after lead editing. This strategic use of ng3 is intended to minimize the occurrence of double-strand breaks in a large number of edited cell populations.

[0407] The combination of nick-guide 3 (ng3) and ePEG+10 / 14 was more fully characterized. Cells were electroporated with increasing doses of PEmax and ePEG plasmids. Figure 14C The bar graph illustrates the percentage of edited cells and knockout (KO) cells determined by FACS analysis on day 3 (D3).

[0408] Figure 14DThe FACS graph in Figure 3 specifically shows the results obtained at the highest dose. This graph provides a visual representation of the cell population and highlights the impact of experimental manipulations on editing efficiency. In summary, Figure 14C and 14D A comprehensive assessment of the effects of ng3 and ePEG+10 / 14 on editing efficiency and KO cells is provided. These findings strongly support the notion that combining cKIT ePEG RNA with ng3 leads to improved editing efficiency. Furthermore, the use of ng3 in the PE3b strategy highlights its efficacy in reducing the occurrence of double-strand breaks, thereby enhancing the overall precision and efficiency of the editing process.

[0409] Example 12: Manipulation of 10 / 14 ePEG RNA Scaffolds

[0410] During a broad ePEG RNA screening, it was found that scaffold manipulation could enhance the editing efficiency of these ePEG RNAs. To further explore this, a collection of manipulated scaffolds was generated using ePEG RTT / PBS+10 / 14 (SEQ ID NO: 42) (also referred to as "10 / 14 ePEG RNA"). Figure 15A Schematic diagram depicted in (only a portion of each modified ePEG sequence is shown).

[0411] The K562 reporter cell line was electroporated with different variants of ePEG RTT / PBS+10 / 14 (SEQ ID NO: 42), each containing sequential deletions of 3' nucleotides from the scaffold portion of the ePEG RNA. Figure 15B The bar graph shows editing efficiency measured by FACS analysis on day 3. Deletion of the last scaffold nucleotide appears to improve the efficiency of this guide. Figure 15C Schematic diagram showing the secondary structure of the optimized scaffold (one 3' nucleotide is missing). Figure 15D Representative FACS plots showing editing efficiency of ePEG RTT / PBS+10 / 14 (SEQ ID NO:42) and scaffold-optimized ePEG RTT / PBS+10 / 14 (SEQ ID NO:43) are shown.

[0412] Scaffold optimized [ePEG RTT / PBS+10 / 14] (SEQ ID NO: 43):

[0413]

[0414] Example 13: Manipulation of additional ePEG RNA scaffolds

[0415] Figure 16AA schematic representation of the modified scaffold utilized in this study is presented. The modified scaffold was strategically engineered to include a deletion of its last 3' nucleotide, specifically the base "C". This targeted modification was found to have a significant impact on the functionality of the scaffold as it altered the sequence at the 3' end of the scaffold. Figure 16B In this study, the efficiency of the editing process of the modified scaffolds was assessed on day 3 (D3) after treatment. FACS analysis was used to measure the editing efficiency. The results were then compared with those obtained from 10 / 14ePEG RNA, which was identified as a very promising candidate for editing. The analysis highlighted the impact of the 3' nucleotide "C" deletion on the editing process and provided valuable insights into the performance of the modified scaffolds as potential tools for genetic manipulation.

[0416] Scaffold optimized [ePEG RTT / PBS+10 / 13] (SEQ ID NO: 44):

[0417]

[0418] Scaffold optimized [ePEG RTT / PBS+10 / 15] (SEQ ID NO: 45):

[0419]

[0420] Example 14: HSC lead editing targeting the cKIT D121L mutation

[0421] To test whether the lead editing strategy can induce mutations in human CD34+ hematopoietic stem and progenitor cells, an in vitro lead editing experiment was performed. Mobilized peripheral blood-derived CD34+ HSPCs were thawed and cultured at 0.5-0.75 million / mL in StemCell SFEMII medium supplemented with 1% penicillin / streptomycin, SCF 125ng / mL (Peprotech), FTL3L 125ng / mL (Peprotech), TPO 62.5ng / mL (Peprotech), Stemregenin-10.75 micromolar (uM) (StemCell technologies), UM171 35nM (Selleckhem). 24 hours after thawing, 0.15-0.25 million HSPCs were electroporated using the Lonza 4D-Nucleofector system in P3 electroporation solution (Lonza), and the solution was supplemented with PEmax mRNA, 2000ng of mRNA and 200pmol of synthetic epegRNA (sequence shown below) and 100pmol of cut sgRNA. Subsequently, the electroporated HSPCs were divided into two wells and cultured for 72 hours. Genomic DNA was harvested 3 days after nuclear transfection. For c-Kit pegRNA-D (sequence as follows), the specific mutation introduced was D121L / R122R, and the efficiency of lead editing (PE) was quantified by Sanger sequencing of the relevant genomic region. Figure 17 .

[0422] [PEG-D(+9-14)D121L R122R]SEQ ID NO:105

[0423] 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 the encoded protein has reduced binding to a therapeutic anti-KIT antibody, and wherein the therapeutic anti-KIT antibody is SR1 or an antibody that has the same six CDRs as SR1 or is otherwise capable of competing with SR1 for the KIT binding site.

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

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

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

5. 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. A genetically engineered hematopoietic stem cell (HSPC) comprising a genetically engineered KIT gene, wherein the genetically engineered KIT gene is engineered such that the encoded protein has reduced binding to a therapeutic anti-KIT antibody, and 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. The genetically engineered HSPC of claim 6, wherein at least one mutation in the genetically engineered KIT gene results in a polypeptide with a mutation at R55.

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

9. The genetically engineered HSPC of any one 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 genetically engineered hematopoietic stem / progenitor cell (HSPC) population comprising the genetically engineered HSPC according to any one of claims 1 to 10.

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

13. A kit comprising the genetically engineered hematopoietic stem / progenitor cell population of claim 11, and optionally one or more cytotoxic agents targeting a cell surface antigen whose gene is edited in the hematopoietic stem / progenitor cell.

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

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

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

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

18. A cell expressing the CAR of claim 17.

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

20. 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) The cell according to claim 18 or 19.

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

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

23. A kit comprising the cell of claim 18 or 19, and optionally one or more cytotoxic agents targeting a cell surface antigen, the gene of which is edited in the hematopoietic stem / progenitor cell.

24. A polypeptide comprising an amino acid sequence that is at least 80% identical to the sequence shown in SEQ ID NO: 56, wherein the polypeptide comprises a mutation at R55S, and wherein 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 at S123P, DL121, or both, and wherein the polypeptide has reduced binding to a therapeutic anti-KIT antibody.

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

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

29. A method of producing a polypeptide, the method comprising culturing the cell of claim 28 under conditions allowing 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 following table:

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

32. A polynucleotide having the formula: 5'-spacer-scaffold-RTT / PBS-3' structural motif, in: The spacer region recognizes the target nucleic acid site and comprises 10-30 nucleotides; The scaffold binds to a lead editor in a CRISPR system; The 3' structural motif protects the polynucleotide from degradation in the cell; and The RTT / PBS segment has a sequence that is at least 75% identical to one or more of the following sequences: <h2 style=";text-align:left;direction:ltr">ccttgttn<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 3 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> a <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> b <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> c <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 4 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 5 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 6 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 7 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 9 <h2 style=";text-align:left;direction:ltr"> tatgggaaaga(RTT / PBS+7 / 10)(SEQ ID NO:23); <h2 style=";text-align:left;direction:ltr">tttccttgttn<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 3 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> a <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> b <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> c <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 4 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 5 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 6 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 7 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 9 <h2 style=";text-align:left;direction:ltr"> tatgggaaaga(RTT / PBS+10 / 10)(SEQ ID NO:24); <h2 style=";text-align:left;direction:ltr">gcttttccttgttn<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 3 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> a <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> b <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> c <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 4 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 5 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 6 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 7 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 9 <h2 style=";text-align:left;direction:ltr"> tatgggaaaga(RTT / PBS+13 / 10)(SEQ ID NO:25); <h2 style=";text-align:left;direction:ltr">ccttgttn<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 3 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> a <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> b <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> c <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 4 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 5 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 6 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 7 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 9 <h2 style=";text-align:left;direction:ltr"> tatgggaaagaaga(RTT / PBS+7 / 13)(SEQ ID NO:26); <h2 style=";text-align:left;direction:ltr">tttccttgttn<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 3 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> a <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> b <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> c <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 4 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 5 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 6 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 7 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 9 <h2 style=";text-align:left;direction:ltr"> tatgggaaagaaga(RTT / PBS+10 / 13)(SEQ ID NO:27); <h2 style=";text-align:left;direction:ltr">gcttttccttgttn<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 3 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> a <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> b <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> c <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 4 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 5 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 6 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 7 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 9 <h2 style=";text-align:left;direction:ltr"> tatgggaaagaaga(RTT / PBS+13 / 13)(SEQ ID NO:28); <h2 style=";text-align:left;direction:ltr">gcttttccttgttn<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 3 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> a <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> b <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> c <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 4 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 5 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 6 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 7 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 9 <h2 style=";text-align:left;direction:ltr"> tatgggaaagaagaca(RTT / PBS+7 / 15)(SEQ IDNO:29); <h2 style=";text-align:left;direction:ltr">tttccttgttn<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 3 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> a <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> b <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> c <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 4 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 5 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 6 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 7 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 9 <h2 style=";text-align:left;direction:ltr"> 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 IDNO:31); or <h2 style=";text-align:left;direction:ltr">tttccttgttn<h2 style=";text-align:left;direction:ltr"> 1 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 2 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 3 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> a <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> b <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> c <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 4 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 5 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 6 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 7 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 8 <h2 style=";text-align:left;direction:ltr"> n<h2 style=";text-align:left;direction:ltr"> 9 <h2 style=";text-align:left;direction:ltr"> tatgggaaagaagac(RTT / PBS+10 / 14)(SEQ ID NO:32); in: 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 the group consisting of cgt, cgc, cga, cgg, aga or agg; or Its combination.

33. The polynucleotide of claim 32, wherein n a n b n c It's agg.

34. The polynucleotide of claim 32, wherein n a n b n c It's cgc.

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

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

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. 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 polynucleotide of any one of claims 32 to 38, wherein the RTT / PBS segment has a sequence that is 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); in: 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 the group consisting of cgt, cgc, cga, cgg, aga or agg; or Its combination.

40. A polynucleotide having the formula: 5'-spacer-scaffold-RTT / PBS-3' structural motif, in: The scaffold binds to a lead editor in a CRISPR system; The 3' structural motif protects the polynucleotide from degradation in the cell; and The RTT / PBS segment comprises a primer binding site (PBS) and a reverse transcriptase template; and 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, in: The scaffold binds to a lead editor in a CRISPR system; The 3' structural motif protects the polynucleotide from degradation in the cell; and The RTT / PBS segment comprises a primer binding site (PBS) and a reverse transcriptase template; and 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 at least 75% identical to the ePEG RNA sequence of SEQ ID NOs: 33-50 or SEQ ID NOs: 103-105.

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

44. The polynucleotide of claim 42, which is PEG-D(+9-14)D121L R122R] (SEQ ID NO: 105).

45. A polynucleotide having a sequence at least 50% identical to one or more of the following sequences: [nick guide 1] (SEQ ID NO: 46) tttgggccactagtcatgaa; [nick guide 2] (SEQ ID NO: 47) gccattccaactactgattt; [nick 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 the group consisting of cgt, cgc, cga, cgg, aga, or agg; or a combination thereof; [nick guide 4] (SEQ ID NO: 49) gtgaccaattattccctcaa; or [nick guide 5] (SEQ ID NO: 50) gaggtttattcctgacccca.

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

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

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