Engineering selective resistance to targeted therapies

Abstract

The present disclosure provides modified stem cells, in particular hematopoietic stem cells engineered to express a variant of a B-cell lymphoma 2 (Bcl-2) family protein, wherein the variant confers resistance to a cytotoxic inhibitor of the Bcl-2 family protein. Also provided are methods and uses of the modified cells for therapeutic applications.

Description

[0001] TITLE OF THE INVENTION

[0002] ENGINEERING SELECTIVE RESISTANCE TO TARGETED THERAPIES

[0003] CROSS REFERENCE TO RELATED APPLICATIONS

[0004] The present application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 665,030 filed June 27, 2024, which is incorporated herein by reference in its entirety.

[0005] SEQUENCE LISTING

[0006] The XML file named “046483-7465WOl .xml” created on June 24, 2025, comprising 22,104 bytes, is hereby incorporated by reference in its entirety.

[0007] BACKGROUND OF THE INVENTION

[0008] Adoptive transfer of genetically engineered cells can be employed in the treatment of cancer, inherited diseases affecting the hematopoietic or immune system, and other conditions. The engrafted cells are susceptible to cytotoxic, immunosuppressive or other drugs. This precludes continuous administration of drugs that could help the engrafted cells to work. There is a need in the art for novel and effective compositions and methods for enhancing the therapeutic index of therapies with cytotoxic agents and engrafted cells. The present invention addresses this need.

[0009] SUMMARY OF THE INVENTION

[0010] In some aspects, the present invention provides a stem cell, wherein the cell is engineered to express a variant of a B-cell lymphoma 2 (Bcl-2) family protein, wherein the variant confers to the stem cell resistance to a cytotoxic inhibitor of the Bcl-2 family protein.

[0011] In other aspects, the present invention provides a pharmaceutical composition comprising a population of stem cells comprising the stem cell of any one of the embodiments described herein and a pharmaceutically acceptable carrier.

[0012] In other aspects, the present invention provides a method for treating a subject in need of a stem cell transplantation, the method comprising administering a therapeutically effective amount of a population of stem cells comprising the stem cell of any one of the embodiments described herein to the subject, thereby treating the subject in need of the stem cell transplantation.

[0013] In another aspect, the present invention provides a method for generating the stem cell of any one of the embodiments described herein, the method comprising engineering a stem cell to express the variant of the B-cell lymphoma 2 (Bcl-2) family protein, wherein the variant confers to the stem cell resistance to the cytotoxic inhibitor of the Bcl-2 family protein.

[0014] BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The following detailed description of specific embodiments of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, specific embodiments are shown in the drawings. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0016] FIGs. 1 A-1B show methods of engineering resistance to either antibody-derived therapeutics or small molecule drugs.

[0017] FIG. 2 shows engineering CAR-T cells to unlock new drug combinations with cellular immunotherapies. A single line of therapy of either CAR-T cells or chemotherapy is often insufficient to control tumor growth. However, both treatments cannot be administered simultaneously as the chemotherapy is cytotoxic, hindering the efficacy of CAR-T treatment. The installation of a mutation in CAR-T cells to confer resistance to the chemotherapy allows the two therapies to work together, controlling tumor growth more effectively than either alone. CAR-T cells can also survive repeat administrations of the chemotherapy in the case of maintenance therapy to prevent relapse or lymphodepletion.

[0018] FIGs. 3A-3B show engineered resistance for hematopoietic stem cell (HSC) graft protection. FIG. 3A depicts installing a drug-resistant mutation in donor hematopoietic stem cells (HSCs) prior to HSC transplant (HSCT) can increase the therapeutic index of chemotherapies or antibody-based therapies. After HSCT, administration of drug can selectively eliminate any remaining blood cancer cells or wild-type (WT) host hematopoietic cells in the patient while sparing the donor graft with engineered resistance. Editing donor HSCs effectively shifts the therapeutic index of the drug, making post-HSCT administration of that drug more tolerable for the patient. FIG. 3B examines epitope editing in both donor HSCs and chimeric antigen receptor (CAR) T cells prior to patient infusion serves two functions. First, cancer cells that have the WT target epitope remain susceptible to edited CAR T cells, preserving CAR T cell effector function. Second, both the epitope-edited donor graft and epitope-edited CAR T cells are protected from CAR T cell-mediated killing, preserving the graft and preventing fratricide. This approach increases the repertoire of targetable cell surface antigens in hematologic cancers.

[0019] FIG. 4 shows in vivo selection of edited cell population using a small molecule or antibody. Therapeutic edit and selection / resistance edit shown. Top panel shows installation of both edits with low efficiency. Bottom panel illustrates the concept of enriching the edited population by repeated dosing of the selection agent over time.

[0020] FIGs. 5A-5B show (FIG. 5 A) F104L is one of several mutations in the BCL-2 protein that can confer resistance to venetoclax; and (FIG. 5B) the role that BCL-2 plays in the intrinsic pathway of apoptosis (reproduced from Youle & Strasser, Nature Reviews Molecular Cell Biology 2008).

[0021] FIG. 6 shows a schematic of ex vivo editing to install the BCLF104Lmutation in HSCs which are transplanted into an AML patient. The patient is treated with venetoclax for a prolonged time after transplantation, which allows the survival of the donor HSCs while preventing AML relapse.

[0022] FIG. 7 shows a schematic of ex vivo installation of a selection edit (such as BCL2F104L) and a therapeutic edit (BCL11 A) in HSCs which are transplanted into a patient. The patient receives venetoclax which promotes the enrichment of therapeutic cells. In this instance, treatment with venetoclax commences before the conditioning chemotherapy and continues through administration of conditioning chemotherapy and for months afterwards. This will allow to reduce the dose of chemotherapy needed and thereby reduce toxicity.

[0023] FIG. 8 shows CD34+ cells can be efficiently edited to BCL2F104Lusing CRISPR base editing. The bar graph shows editing across 7 donors. Human CD34+ cells were electroporated with ABE8e mRNA and sgRNA for AAVS1 (WT cells) or F104L at a 1 :1 mass ratio. Editing efficiency was determined via Sanger sequencing. AAVS1 is a safe harbor locus that is used as a negative control.

[0024] FIG. 9 shows the enrichment of MOLM-13 cells with the BCL2F104Lmutation in the presence of venetoclax and DMSO over a 34 day period. MOLM-13 is an acute myeloid leukemia cell line that is sensitive to venetoclax (addition of venetoclax kills the cells). After installing the indicated BCL2 mutation at an efficiency of 50%, the cells were exposed to low (31.25nM) or moderate (lOOnM) concentrations of venetoclax. At the indicated time points, cells were harvested, DNA extracted, and analyzed by Sanger sequencing. Addition of venetoclax increased the frequency of the BCL2 mutation, supporting the hypothesis that cells carrying this mutation are resistant to venetoclax.

[0025] FIG. 10 is a schematic showing the F104L mutation in BCL-2 confers resistance to venetoclax.

[0026] FIG. 11 shows the schematic of how donated HSCs that have engineered resistance can be used to prevent relapse.

[0027] FIG. 12 shows the installation of F104L mutation in CD34+ cells.

[0028] FIG. 13 shows the CD34+ venetoclax titration setup.

[0029] FIG. 14 shows the percent of live CD34+ cells after a 5 day administration of venetoclax at varying concentrations.

[0030] FIG. 15 shows the percent of live CD34+ cells after a 5 day administration of ARAC at varying concentrations.

[0031] FIG. 16 examines the F104L MOLM-13 enrichment at varying concentrations of venetoclax.

[0032] FIG. 17 shows the CD34+ CFU assay over 14 days.

[0033] FIG. 18 shows HSC vs. myeloid sequencing of the F104L mutation.

[0034] FIG. 19 shows the CD33+ growth curve of the F104L mutation.

[0035] FIG. 20 examines the percent cytochrome C released after administration of varying concentrations of BIM.

[0036] FIG. 21 examines the percent cytochrome C released after administration of different compounds.

[0037] FIG. 22 shows a schematic of the NSG mouse study setup.

[0038] FIG. 23 shows the percent of CD45 cells in different cell types at 6 week-old, 8 week- old, 12 week-old, and 17 week-old mice.

[0039] FIG. 24 shows in vivo study sequencing pre- and post-injection.

[0040] FIG. 25 shows in vivo engraftment after post-injection.

[0041] FIG. 26 shows the enrichment of UCK2 mutants.

[0042] FIG. 27 shows a visual summary of the experimental design. FIG. 28 shows a CD34+ growth curve from two different donors.

[0043] FIG. 29 examines how mutations affect the abundance of the BCL2 protein.

[0044] FIG. 30 shows the enrichment of UCK2 mutants.

[0045] FIG. 31 shows the enrichment of BCL2 mutants.

[0046] DETAILED DESCRIPTION OF THE INVENTION

[0047] The following disclosure provides many different embodiments, or examples, for implementing different features of the provided subject matter. Specific examples of components and arrangements are described below to simplify the present disclosure. These are, of course, merely examples and are not intended to be limiting. For example, the formation of a first feature over or on a second feature in the description that follows may include embodiments in which the first and second features are formed in direct contact, and may also include embodiments in which additional features may be formed between the first and second features, such that the first and second features may not be in direct contact. In addition, the present disclosure may repeat reference numerals and / or letters in the various examples. This repetition is for the purpose of simplicity and clarity and does not in itself dictate a relationship between the various embodiments and / or configurations discussed.

[0048] Furthermore, the experiments described herein, unless otherwise indicated, use conventional molecular and cellular biological and immunological techniques within the skill of the art. Such techniques are well known to the skilled worker, and are explained fully in the literature. See, e.g., Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y. (1987-2008), including all supplements, Molecular Cloning: A Laboratory Manual (Fourth Edition) by MR Green and J. Sambrook and Harlow et al., Antibodies: A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor (2013, 2nd edition).

[0049] Definitions

[0050] Unless otherwise defined, scientific and technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art. In the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The use of “or” means “and / or” unless stated otherwise. The use of the term “including,” as well as other forms, such as “includes” and “included,” is not limiting.

[0051] Generally, nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein is well-known and commonly used in the art. The methods and techniques provided herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to manufacturer’s specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well- known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0052] That the disclosure may be more readily understood, select terms are defined below.

[0053] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0054] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0055] “Activation,” as used herein, refers to the state of a cell that has been sufficiently stimulated to induce detectable cellular proliferation. Activation can also be associated with induced cytokine production, differentiation of cells (i.e., stem cells), and detectable effector functions.

[0056] As used herein, the term “adenine base pairing” refers to a type of CRISPR application that converts an adenine nucleotide to an inosine nucleotide, resulting in an A to G nucleotide base change. ABEs do not display significant A to non-G conversion at target loci. As used herein, to “alleviate” a disease means reducing the severity of one or more symptoms of the disease.

[0057] The term “antigen” as used herein is defined as a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen.

[0058] Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an “antigen” as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid.

[0059] As used herein, the term “autologous” is meant to refer to any material derived from the same individual to which it is later to be re-introduced into the individual.

[0060] As used herein, the term “base editing” refers to a genome editing technique that fuses deaminase enzymes to RNA-guided nickases (such as Cas9) to install precise nucleotide changes in the genome without introducing double-strand breaks.

[0061] The term “chimeric antigen receptor” as used herein refers to a synthetic immune receptor combining an extracellular antigen-binding domain (derived from antibodies or receptor ligands) with intracellular T cell signalling domains (derived from CD3 and costimulatory proteins like CD28), enabling T cells to specifically recognize and attack cancer cells based on surface antigen expression.

[0062] A “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate. In contrast, a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health.

[0063] The term “downregulation” as used herein refers to the decrease or elimination of gene expression of one or more genes.

[0064] As used herein, the term “epitope” refers to the specific portion of an antigen that is recognized and bound by an antibody or T-cell receptor.

[0065] “Effective amount” or “therapeutically effective amount” are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological result or provides a therapeutic or prophylactic benefit. Such results may include, but are not limited to an amount that when administered to a mammal, causes a detectable level of immune suppression or tolerance compared to the immune response detected in the absence of the composition of the invention. The immune response can be readily assessed by a plethora of art-recognized methods. The skilled artisan would understand that the amount of the composition administered herein varies and can be readily determined based on a number of factors such as the disease or condition being treated, the age and health and physical condition of the mammal being treated, the severity of the disease, the particular compound being administered, and the like.

[0066] “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0067] As used herein “endogenous” refers to any material from or produced inside an organism, cell, tissue or system.

[0068] As used herein, the term “engraftmenf ’ refers to a process in which transplanted stem cells travel through the blood to the bone marrow, where they begin to make new white blood cells, red blood cells, and platelets. The terms “engraftment”, “hematopoietic stem cell transplantation", “HSCT”, “transplant”, and “transplantation” can be used interchangeably throughout the application.

[0069] The term “epitope” as used herein is defined as a small chemical molecule on an antigen that can elicit an immune response, inducing B and / or T cell responses. An antigen can have one or more epitopes. Most antigens have many epitopes; i.e., they are multivalent. In general, an epitope is roughly about 10 amino acids and / or sugars in size. Preferably, the epitope is about 4- 18 amino acids, more preferably about 5-16 amino acids, and even more most preferably 6-14 amino acids, more preferably about 7-12, and most preferably about 8-10 amino acids. One skilled in the art understands that generally the overall three-dimensional structure, rather than the specific linear sequence of the molecule, is the main criterion of antigenic specificity and therefore distinguishes one epitope from another. Based on the present disclosure, a peptide used in the present invention can be an epitope.

[0070] As used herein, the term “exogenous” refers to any material introduced from or produced outside an organism, cell, tissue or system.

[0071] The term “expand” as used herein refers to increasing in number, as in an increase in the number of pluripotent stem cells. In one embodiment, the pluripotent stem cells that are expanded ex vivo increase in number relative to the number originally present in the culture. In another embodiment, the pluripotent stem cells that are expanded ex vivo increase in number relative to other cell types in the culture. The term “ex vivo ” as used herein, refers to cells that have been removed from a living organism, (e.g., a human) and propagated outside the organism (e.g., in a culture dish, test tube, or bioreactor).

[0072] The term “expression” as used herein is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0073] “Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., Sendai viruses, lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide. The term “hematopoietic stem cell” or “HSC” as described herein refers to multipotent primitive cells that can develop into all types of blood cells, including myeloid-lineage and lymphoid-lineage cells. HSCs can be found in several organs, such as peripheral blood (PB), bone marrow (BM), and umbilical cord blood (UCB).

[0074] “Identity” as used herein refers to the subunit sequence identity between two polymeric molecules particularly between two amino acid molecules, such as, between two polypeptide molecules. When two amino acid sequences have the same residues at the same positions; e.g., if a position in each of two polypeptide molecules is occupied by an arginine, then they are identical at that position. The identity or extent to which two amino acid sequences have the same residues at the same positions in an alignment is often expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of matching or identical positions; e.g., if half (e g., five positions in a polymer ten amino acids in length) of the positions in two sequences are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 of 10), are matched or identical, the two amino acids sequences are 90% identical.

[0075] The term “immune response” as used herein is defined as a cellular response to an antigen that occurs when lymphocytes identify antigenic molecules as foreign and induce the formation of antibodies and / or activate lymphocytes to remove the antigen.

[0076] The term “immunosuppressive” is used herein to refer to reducing overall immune response.

[0077] “Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0078] By the term “modified” as used herein, is meant a changed state or structure of a molecule or cell of the invention. Molecules may be modified in many ways, including chemically, structurally, and functionally. Cells may be modified through the introduction of nucleic acids.

[0079] By the term “modulating,” as used herein, is meant mediating a detectable increase or decrease in the level of a response in a subject compared with the level of a response in the subject in the absence of a treatment or compound, and / or compared with the level of a response in an otherwise identical but untreated subject. The term encompasses perturbing and / or affecting a native signal or response thereby mediating a beneficial therapeutic response in a subject, preferably, a human.

[0080] In the context of the present invention, the following abbreviations for the commonly occurring nucleic acid bases are used. “A” refers to adenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine.

[0081] The term “oligonucleotide” typically refers to short polynucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, C, G), this also includes an RNA sequence (i.e., A, U, C, G) in which “U” replaces “T ”

[0082] An “overexpression” or “significantly higher level of expression” of the gene products refers to an expression level or copy number in a test sample that is greater than the standard error of the assay employed to assess the level of expression. In embodiments, the overexpression can be at least two, at least three, at least four, at least five, or at least ten or more times the expression level of the gene in a control sample or the average expression level of gene products in several control samples

[0083] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).

[0084] “Parenteral” administration of an immunogenic composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrastemal injection, or infusion techniques.

[0085] The term “polynucleotide” as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, “nucleic acid” and “polynucleotide” as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides” and which comprise one or more “nucleotide sequence(s)”. The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences (i.e., “nucleotide sequences”) which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR, and the like, and by synthetic means.

[0086] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[0087] As used herein, the term “pluripotent stem cell” or “PSCs” refers to a stem cell that has the potential to differentiate into any of the three germ layers: endoderm (gut, lungs and liver), mesoderm (muscle, skeleton, blood vascular, urogenital, dermis), or ectoderm (nervous, sensory, epidermis), but not into extra-embryonic tissues like the placenta or yolk sac. An “induced pluripotent stem cell”, “iPS”, or “iPSC” refers to a type of pluripotent stem cell artificially derived from a non-pluripotent cell, typically an adult somatic cell, by inducing a "forced" expression of certain genes and transcription factors. PSCs and iPSCs are used interchangeably herein to differentiate into HSCs.

[0088] The term “preconditioning” as used herein refers to the administration of chemotherapy and / or radiation therapy prior to hematopoietic stem cell transplantation to eliminate existing cancer cells, suppress the immune system to prevent rejection of the transplanted stem cells, and create space in the bone marrow for the donor stem cells to engraft.

[0089] As used herein, the term “prime editing” refers to a genome editing technology that allows researchers to directly rewrite genomic DNA sequences by combining a modified Cas9 protein with an engineered reverse transcriptase enzyme and an elongated prime editing gRNA allowing to install almost all possible genomic alterations. By the term “specifically binds,” as used herein with respect to an antibody, is meant an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. But, such cross-species reactivity does not itself alter the classification of an antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific. In some instances, the terms “specific binding” or “specifically binding,” can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody.

[0090] The term “subject” is intended to include living organisms in which an immune response can be elicited (e.g., mammals). A “subject” or “patient,” as used herein, may be a human or non-human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals, as well as simian and non-human primate mammals. Preferably, the subject is human.

[0091] A “target site” or “target sequence” refers to a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule may specifically bind under conditions sufficient for binding to occur. In some embodiments, a target sequence refers to a genomic nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule may specifically bind under conditions sufficient for binding to occur.

[0092] The term “therapeutic” as used herein means a treatment and / or prophylaxis. A therapeutic effect is obtained by suppression, remission, or eradication of a disease state.

[0093] The term “transfected” or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0094] The terms “treat”, “treatment” and “treating” refer to the reduction or amelioration of the progression, severity and / or duration of a proliferative disorder, or the amelioration of one or more symptoms (e.g., one or more discernible symptoms) of a proliferative disorder resulting from the administration of one or more therapies (e.g., one or more therapeutic agents such as a HSC of the invention). In specific embodiments, the terms “treat”, “treatment” and “treating” refer to the amelioration of at least one measurable physical parameter of a proliferative disorder, such as growth of a tumor, not necessarily discernible by the patient. In other embodiments the terms “treat”, “treatment” and “treating” -refer to the inhibition of the progression of a proliferative disorder, either physically by, e.g., stabilization of a discernible symptom, physiologically by, e g., stabilization of a physical parameter, or both. In other embodiments the terms “treat”, “treatment” and “treating” refer to the reduction or stabilization of tumor size or cancerous cell count. In some embodiments, “treatment” refers to an approach for obtaining a beneficial or a desired result including, but not limited to: a therapeutic benefit; or prevention of a condition, e.g., a side effect, e.g., an unwanted effect as described herein. In some embodiments, a therapeutic benefit is obtained by eradication or amelioration of the underlying disorder being treated. In some embodiments, a therapeutic benefit is obtained by reduction of, eradication, or amelioration of one or more of the symptoms, e.g., physiological symptoms, associated with the underlying disorder such that an improvement, e.g., change, is observed in the patient. In some embodiments, the patient can still be afflicted with the underlying disorder. In some embodiments, treatment comprises prevention of a condition, e.g., a side effect, e.g., an unwanted side effect from a therapy. Treatment or prevention of a condition or a side effect need not be a complete treatment or prevention of the condition or side effect.

[0095] An “underexpression” or “significantly lower level of expression” of products e.g., the markers set forth herein) refers to an expression level in a test sample that is greater than the standard error of the assay employed to assess expression, for example, at least 1.5, twice, at least three, at least four, at least five, or at least ten or more times less than the expression level of the gene in a control sample, or the average expression level of gene products in several control samples. "Variant" as the term is used herein, is a nucleic acid sequence or a peptide sequence that differs in sequence from a reference nucleic acid sequence or peptide sequence respectively, but retains essential properties of the reference molecule. Changes in the sequence of a nucleic acid variant may not alter the amino acid sequence of a peptide encoded by the reference nucleic acid, or may result in amino acid substitutions, additions, deletions, fusions and truncations. Changes in the sequence of peptide variants are typically limited or conservative, so that the sequences of the reference peptide and the variant are closely similar overall and, in many regions, identical. A variant and reference peptide may differ in amino acid sequence by one or more substitutions, additions, or deletions in any combination. A variant of a nucleic acid or peptide may be a naturally occurring such as an allelic variant, or may be a variant that is not known to occur naturally. Non-naturally occurring variants of nucleic acids and peptides may be made by mutagenesis techniques or by direct synthesis.

[0096] A “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, Sendai viral vectors, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.

[0097] The term “xenogeneic” refers to a graft derived from an animal of a different species.

[0098] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range. Engineering graft protection

[0099] The susceptibility of engrafted cells to certain therapies precludes continuous dosing of therapies. For example, venetoclax is routinely given to patients with leukemia; when administered after hematopoietic stem cell (HSC) transplantation, the drug is poorly tolerated since it suppresses growth of the engrafted donor stem cells. The susceptibility of engrafted cells to such therapies precludes continuous dosing and therefore increases the risk of relapse.

[0100] Engineering resistance to the conditioning agent in the donor graft could increase the therapeutic window of the conditioning agent and allow its use in post-transplant maintenance settings. In some aspects, the present invention provides a strategy to engineer hematopoietic stem cells (either allogeneic or autologous) to be resistant to venetoclax by the compositions and methods disclosed herein. This would allow continued administration of venetoclax even after transplant as it would only target unedited host cells. Conceptually, this approach increases the conditioning agent’s therapeutic index by allowing more prolonged exposure than would otherwise be tolerated.

[0101] In some aspects, the present invention provides a stem cell. The stem cell is engineered to express a variant of a B-cell lymphoma 2 (Bcl-2) family protein. The variant confers to the stem cell resistance to a cytotoxic inhibitor of the Bcl2 family protein.

[0102] In some embodiments, the stem cell is a pluripotent stem cell.

[0103] In other embodiments, the stem cell is a hematopoietic stem cell (HSC).

[0104] In some embodiments, the HSC is CD34+ HSC.

[0105] Sources of Stem Cells (e.g., HSCs or Progenitor Cells)

[0106] In some embodiment, prior to any expansion, a source of the cells is obtained from a subject. Non-limiting examples of subjects include humans, dogs, cats, mice, rats, non-human primates, swine and transgenic species thereof. Preferably, the subject is a human. The cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, cord blood, lymph node tissue, spleen tissue, umbilical cord, and tumors. In certain embodiments, a HSC or progenitor cell line available in the art, may be used. In certain embodiments, the cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll separation. In one embodiment, cells from the circulating blood of an individual are obtained by apheresis or leukapheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. The cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media, such as phosphate buffered saline (PBS) or wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations, for subsequent processing steps. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free, Mg-free PBS. Alternatively, the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media.

[0107] In another embodiment, the cells are isolated from peripheral blood by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient. Alternatively, the cells can be isolated from umbilical cord. In any event, a specific subpopulation of stem cells (e.g., HSC or progenitor cells) can be further isolated by positive or negative selection techniques.

[0108] The cord blood mononuclear cells so isolated can be depleted of cells expressing certain antigens, including, but not limited to, CD4, CD5, CD8, CD1 lb, CD14, CD19, CD24, CD45, CD56, and CD66b. Depletion of these cells can be accomplished using an isolated antibody, a biological sample comprising an antibody, such as ascites, an antibody bound to a physical support, and a cell bound antibody.

[0109] Enrichment of a cell population by negative selection can be accomplished using a combination of antibodies directed to surface markers unique to the negatively selected cells. A preferred method is cell sorting and / or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD34+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD4, CD5, CD8, CD14, CD20, CD1 lb, CD16, HLA-DR, and CD8.

[0110] For isolation of a desired population of cells by positive or negative selection, the concentration of cells and surface (e.g., particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly decrease the volume in which beads and cells are mixed together (i.e., increase the concentration of cells), to ensure maximum contact of cells and beads. For example, in one embodiment, a concentration of 2 billion cells / ml is used. In one embodiment, a concentration of 1 billion cells / ml is used. In a further embodiment, greater than 100 million cells / ml is used. In a further embodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / ml is used. In yet another embodiment, a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells / ml is used. In further embodiments, concentrations of 125 or 150 million cells / ml can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion.

[0111] The cells can also be frozen after the washing step, which does not require the monocyteremoval step. While not wishing to be bound by theory, the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population. After the washing step that removes plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, in a non-limiting example, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or other suitable cell freezing media. The cells are then frozen to -80°C at a rate of 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing may be used as well as uncontrolled freezing immediately at -20°C or in liquid nitrogen.

[0112] In one embodiment, the cell is obtained from cells selected from the group consisting of peripheral blood mononuclear cells, cord blood cells, bone marrow, lymph nodes, and a spleen. In another embodiment, the cell expresses the marker CD34.

[0113] Expansion of Stem Cells

[0114] The present invention includes a population of cells comprising the modified cells described herein. In one embodiment, the method for generating the modified cell described herein also includes expanding the cell or the modified cell. In one embodiment, the expansion is prior to the step of introducing any modification to the cell.

[0115] In some embodiments, the cells disclosed herein can be expanded by about 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, 200 fold, 300 fold, 400 fold, 500 fold, 600 fold, 700 fold, 800 fold, 900 fold, 1000 fold, 2000 fold, 3000 fold, 4000 fold, 5000 fold, 6000 fold, 7000 fold, 8000 fold, 9000 fold, 10,000 fold, 100,000 fold, 1,000,000 fold, 10,000,000 fold, or greater, and any and all whole or partial integers therebetween. In one embodiment, the cells are expanded in the range of about 20 fold to about 50 fold.

[0116] The cells can be incubated in cell medium in a culture apparatus for a period of time or until the cells reach high cell density for optimal passage before passing the cells to another culture apparatus. The culturing apparatus can be of any culture apparatus commonly used for culturing cells in vitro. The cell medium may be replaced during the culture of the cells at any time. Preferably, the cell medium is replaced about every 2 to 3 days. The cells are then harvested from the culture apparatus whereupon the cells can be used immediately or cryopreserved to be stored for use at a later time. In one embodiment, the invention includes cryopreserving the expanded cells. The cryopreserved cells are thawed prior to introducing nucleic acids into the cell.

[0117] In another embodiment, the method further comprises isolating the cell and expanding the cell. In another embodiment, the invention further comprises cryopreserving the cell prior to expansion. In yet another embodiment, the invention further comprises thawing the cryopreserved cell prior to introducing the nucleic acids.

[0118] The culturing step as described herein (contact with agents as described herein) can be very short, for example less than 24 hours such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours. The culturing step as described further herein (contact with agents as described herein) can be longer, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days.

[0119] Various terms are used to describe cells in culture. Cell culture refers generally to cells taken from a living organism and grown under controlled condition. A primary cell culture is a culture of cells, tissues or organs taken directly from an organism and before the first subculture. Cells are expanded in culture when they are placed in a growth medium under conditions that facilitate cell growth and / or division, resulting in a larger population of the cells. When cells are expanded in culture, the rate of cell proliferation is typically measured by the amount of time required for the cells to double in number, otherwise known as the doubling time.

[0120] Each round of subculturing is referred to as a passage. When cells are subcultured, they are referred to as having been passaged. A specific population of cells, or a cell line, is sometimes referred to or characterized by the number of times it has been passaged. For example, a cultured cell population that has been passaged ten times may be referred to as a P10 culture. The primary culture, i.e., the first culture following the isolation of cells from tissue, is designated PO. Following the first subculture, the cells are described as a secondary culture (Pl or passage 1). After the second subculture, the cells become a tertiary culture (P2 or passage 2), and so on. It will be understood by those of skill in the art that there may be many population doublings during the period of passaging; therefore the number of population doublings of a culture is greater than the passage number. The expansion of cells (i.e., the number of population doublings) during the period between passaging depends on many factors, including but is not limited to the seeding density, substrate, medium, and time between passaging.

[0121] In one embodiment, the cells may be cultured for several hours (about 3 hours) to about 14 days or any hourly integer value in between. Conditions appropriate for HSC or progenitor cell culture include an appropriate media (e.g., serum free human hematopoietic stem cell media (StemSpan™ SFEM II, Minimal Essential Media or RPMI Media 1640 or, X-vivo 15, (Lonza)) that may contain factors necessary for proliferation and viability, such as but not limited to, serum (e.g., fetal bovine or human serum), GM-CSF, insulin, IFN-gamma, interleukin-1 (IL-1), IL-3, IL-4, IL-6, IL-7, IL-10, IL-12, IL-15, SCF, TGF-beta, TNF-a and TPO, or any other additives for the growth of cells known to the skilled artisan. In one embodiment, the cell culture includes IL-3, IL-6, GM-CSF, SCF and TPO. Other additives for the growth of cells include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetyL cysteine and 2-mercaptoethanol. Media can include RPMI 1640, AIM-V, DMEM, MEM, a- MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and / or an amount of cytokine(s) sufficient for the growth and expansion of HSC or progenitor cells. Antibiotics, e.g., penicillin and streptomycin, are included only in experimental cultures, not in cultures of cells that are to be infused into a subject. The target cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37° C) and atmosphere (e.g., air plus 5% CO2).

[0122] The medium used to culture the cells may include an agent that can stimulate the modified cells to expand. The cell modified by the methods disclosed herein can be expanded approximately 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, 200 fold, 300 fold, 400 fold, 500 fold, 600 fold, 700 fold, 800 fold, 900 fold, 1000 fold, 2000 fold, 3000 fold, 4000 fold, 5000 fold, 6000 fold, 7000 fold, 8000 fold, 9000 fold, 10,000 fold, 100,000 fold, 1,000,000 fold, 10,000,000 fold, or greater. In one embodiment, the modified cell expands in the range of about 20 fold to about 50 fold, or more by culturing the modified cell.

[0123] Bcl-2 Family Proteins

[0124] In various embodiments, the cells of the present invention disclosed herein are engineered to express a variant of a B-cell lymphoma 2 (Bcl-2) family protein, wherein the variant confers to the stem cell resistance to a cytotoxic inhibitor of the Bcl-2 family protein.

[0125] The BCL-2 family of proteins comprises prosurvival members such as BCL-2, BCL-XL, BCL-W, MCL1, and BFL1, proapoptotic BH3-only proteins such as BIM and BAD, and the proapoptotic final effectors BAK and BAX. Bcl-2 family proteins are critical regulators of the mitochondrial apoptotic pathway.

[0126] In some embodiments, the Bcl-2 family protein is selected from the group consisting of Bcl-2, BCL-XL, BCL-W, MCL1, BFL1, BIM, BAD, BAK, and BAX.

[0127] In other embodiments, the Bcl-2 family protein is human Bcl-2.

[0128] Multiple isoforms of human Bcl-2 are known and are suitable for use in the invention, including the alpha and beta isoforms.

[0129] Human BCL2, isoform alpha, comprises the following amino acid sequence: MAHAGRTGYDNREIVMKYIHYKLSQRGYEWDAGDVGAAPPGAAPAPGIFSSQPGHTPH PAASRDPVARTSPLQTPAAPGAAAGPALSPVPPVVHLTLRQAGDDFSRRYRRDFAEMSS QLHLTPFTARGRFATVVEELFRDGVNWGRIVAFFEFGGVMCVESVNREMSPLVDNIAL WMTEYLNRHLHTWIQDNGGWDAFVELYGPSMRPLFDFSWLSLKTLLSLALVGACITLG AYLGHK (SEQ ID NO: 1).

[0130] Human BCL2, isoform alpha, comprises the following cDNA sequence: atggcgcacgctgggagaacggggtacgataaccgggagatagtgatgaagtacatccattataagctgtcgcagaggggctacgagtg ggatgcgggagatgtgggcgccgcgcccccgggggccgcccccgcaccgggcatcttctcctcccagcccgggcacacgccccatcc agccgcatcccgggacccggtcgccaggacctcgccgctgcagaccccggctgcccccggcgccgccgcggggcctgcgctcagcc cggtgccacctgtggtccacctgaccctccgccaggccggcgacgacttctcccgccgctaccgccgcgacttcgccgagatgtccagcc agctgcacctgacgcccttcaccgcgcggggacgctttgccacggtggtggaggagctcttcagggacggggtgaactgggggaggatt gtggccttctttgagttcggtggggtcatgtgtgtggagagcgtcaaccgggagatgtcgcccctggtggacaacatcgccctgtggatgac tgagtacctgaaccggcacctgcacacctggatccaggataacggaggctgggatgcctttgtggaactgtacggccccagcatgcggcc tctgtttgatttctcctggctgtctctgaagactctgctcagtttggccctggtgggagcttgcatcaccctgggtgcctatctgggccacaagtg a (SEQ ID NO: 2).

[0131] Human BCL2, isoform beta, comprises the following amino acid sequence: MAHAGRTGYDNREIVMKYIHYKLSQRGYEWDAGDVGAAPPGAAPAPGIFSSQPGHTPH PAASRDPVARTSPLQTPAAPGAAAGPALSPVPPVVHLTLRQAGDDFSRRYRRDFAEMSS QLHLTPFTARGRFATVVEELFRDGVNWGRIVAFFEFGGVMCVESVNREMSPLVDNIAL WMTEYLNRHLHTWIQDNGGWVGALGDVSLG (SEQ ID NO: 3).

[0132] Human BCL2, isoform beta, comprises the following cDNA sequence: atggcgcacgctgggagaacagggtacgataaccgggagatagtgatgaagtacatccattataagctgtcgcagaggggctacgagtg ggatgcgggagatgtgggcgccgcgcccccgggggccgcccccgcaccgggcatcttctcctcccagcccgggcacacgccccatcc agccgcatcccgggacccggtcgccaggacctcgccgctgcagaccccggctgcccccggcgccgccgcggggcctgcgctcagcc cggtgccacctgtggtccacctgaccctccgccaggccggcgacgacttctcccgccgctaccgccgcgacttcgccgagatgtccagcc agctgcacctgacgcccttcaccgcgcggggacgctttgccacggtggtggaggagctcttcagggacggggtgaactgggggaggatt gtggccttctttgagttcggtggggtcatgtgtgtggagagcgtcaaccgggagatgtcgcccctggtggacaacatcgccctgtggatgac tgagtacctgaaccggcacctgcacacctggatccaggataacggaggctgggtaggtgcacttggtgatgtgagtctgggc (SEQ ID NO: 4).

[0133] In some embodiments, the variant of Bcl-2 confers resistance to a cytotoxic inhibitor of the Bcl-2.

[0134] In some embodiments, the Bcl-2 is human Bcl-2 and the variant comprises an amino acid substitution selected from the group consisting of F104L, G101V, D103E, D103Y, F101C, F101L, V92L, T187I, A131V, S1O5F, D103G, D103N, A149T, V148A, L169P, and any combination thereof.

[0135] In some embodiments, the Bcl-2 is human BAX and the variant comprises a G179E mutation.

[0136] In some embodiments, the variant comprises the F104L substitution in Bcl-2.

[0137] In some embodiments, the F104L Bcl-2 comprises the following amino acid sequence: MAHAGRTGYDNREIVMKYIHYKLSQRGYEWDAGDVGAAPPGAAPAPGIFSSQPGHTPH PAASRDPVARTSPLQTPAAPGAAAGPALSPVPPVVHLTLRQAGDDLSRRYRRDFAEMSS QLHLTPFTARGRFATVVEELFRDGVNWGRIVAFFEFGGVMCVESVNREMSPLVDNIAL WMTEYLNRHLHTWIQDNGGWDAFVELYGPSMRPLFDFSWLSLKTLLSLALVGACITLG AYLGHK (SEQ ID NO: 5). In some embodiments, F104L Bcl-2 is encoded by a nucleic acid comprising the following nucleotide sequence: ATGGCCCATGCCGGAAGAACCGGCTACGACAATAGAGAGATCGTCATGAAGTACAT CCACTACAAGCTGTCCCAGAGGGGCTATGAGTGGGACGCCGGAGATGTGGGCGCTG CTCCTCCCGGAGCTGCCCCCGCCCCCGGAATTTTTTCCAGCCAGCCCGGCCATACCC CTCACCCCGCCGCCTCCAGAGATCCCGTGGCTAGAACCAGCCCTCTGCAAACCCCCG CCGCCCCCGGCGCCGCTGCTGGACCCGCCCTCAGCCCCGTGCCTCCCGTGGTGCACC TCACACTGAGGCAAGCCGGAGACGATCTGAGCAGAAGATATAGAAGGGACTTCGCC GAGATGAGCAGCCAGCTGCATCTGACCCCTTTCACAGCCAGAGGCAGATTTGCCAC CGTCGTGGAGGAGCTCTTCAGAGACGGCGTGAATTGGGGAAGAATCGTGGCCTTCTT CGAGTTCGGCGGCGTCATGTGCGTCGAGAGCGTGAATAGGGAGATGTCCCCCCTCGT GGACAACATCGCCCTCTGGATGACAGAGTATCTGAATAGACATCTGCACACATGGA TCCAAGACAACGGAGGCTGGGACGCCTTTGTGGAACTCTACGGCCCTAGCATGAGA CCTCTGTTCGACTTCAGCTGGCTGTCTCTGAAGACACTGCTGTCTCTGGCTCTGGTGG GAGCTTGCATTACACTGGGAGCCTATCTGGGACACAAG (SEQ ID NO: 6).

[0138] Cytotoxic Inhibitors

[0139] In various embodiments, the variant expressed in the stem cell confers to the stem cell and its progeny resistance to a cytotoxic inhibitor of the Bcl-2 family protein.

[0140] For example, the BCL-2 inhibitor venetoclax, which is commonly used in the treatment of CLL and AML, is a cytotoxic inhibitor of the Bcl-2 family protein and may trigger apoptosis in cells. To circumvent this limitation, a venetocl ax-resistant mutation of the anti-apoptotic protein BCL-2 (F104L) can be expressed in cells.

[0141] In some embodiments, the cytotoxic inhibitor is selected from the group consisting of a small molecule and an inhibitory nucleic acid.

[0142] In other embodiments, the cytotoxic inhibitor is a small molecule.

[0143] In one embodiment, the cytotoxic inhibitor is a pro-apoptotic drug.

[0144] In some embodiments, the cytotoxic inhibitor is selected from the group consisting of venetoclax (ABT-199), navitoclax (ABT-263), ABT-737, sabutoclax (BL97C1), obatoclax (GX15-070,), TW-37, AT-101, HA14-1, RU486, BAM7, A-1331852, A-l 155463, BDA-366, UMI-77, and BIB 1-1. In other embodiments, the cytotoxic inhibitor is venetoclax.

[0145] Modifications

[0146] Genetically modifying stem cells (e.g., HSCs) with viral vectors or non-viral gene editing technologies can, for example, 1) replace missing or malfunctioning genes, 2) disrupt diseasecausing genes, and 3) correct genetic mutations. For example, genetically engineered HSCs can be infused into a patient, where they engraft and replace damaged / malfunctioning cells or produce therapeutic proteins.

[0147] In other embodiments, the stem cell is engineered to express the variant of the B-cell lymphoma 2 (Bcl-2) family protein, wherein the variant confers to the stem cell resistance to the cytotoxic inhibitor of the Bcl-2 family protein, and wherein the stem cell further comprises a genetic modification.

[0148] In one embodiment, the genetic modification replaces a missing or malfunctioning gene; disrupts a disease-causing gene; and / or corrects a genetic mutation.

[0149] In another embodiment, the genetic modification comprises a therapeutic edit of a target sequence associated with a disease or disorder.

[0150] In some embodiments, the disease or disorder comprises a cancer, a hemoglobinopathy, an inherited disease or disorder, or a metabolic disease or disorder.

[0151] In other embodiments, the disease or disorder comprises cancer.

[0152] In one embodiment, the disease or disorder comprises sickle-cell disease.

[0153] In some embodiments, the genetic modification comprises editing an enhancer of BCL11A gene.

[0154] In other embodiments, the cytotoxic inhibitor and genetic modification can correspond to other drug-drug resistance mutation pairs, not just for example Bcl-2 (e.g., Bcl-2 F104L mutation) and venetoclax. In some embodiments, the genetic modification is in a gene other than bcl-2 that confers resistance to a drug other than venetoclax, for application in a variety of therapeutic contexts including, but not limited to, sickle cell disease, a- or P-thalassemia, or fanconia anemia.

[0155] Pharmaceutical Compositions In other aspects, the present invention provides a pharmaceutical composition comprising a population of stem cells comprising a stem cell; and a pharmaceutically acceptable carrier; wherein stem cell is engineered to express a variant of a B-cell lymphoma 2 (Bcl-2) family protein, wherein the variant confers to the stem cell resistance to a cytotoxic inhibitor of the Bcl- 2 family protein.

[0156] In various embodiments, the stem cell, the Bcl-2 family protein, the variant, the cytotoxic inhibitor, the genetic modification, each is as described elsewhere herein.

[0157] In one embodiment, the stem cell is a HSC, the Bcl-2 family protein is a human bcl-2, the variant is a human bcl-2 comprising a F104L substitution therein, and the cytotoxic inhibitor is venetoclax. In other embodiments, the HSC further comprises a genetic modification of an enhancer of the BCL11A gene.

[0158] In some embodiments, the modified stem cells (e.g., HSCs) of the population make up at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more of the total cells in the population.

[0159] Pharmaceutical compositions and formulations generally include one or more optional pharmaceutically acceptable carrier or excipient. In some embodiments, the composition includes at least one additional therapeutic agent.

[0160] The term “pharmaceutical formulation” or “pharmaceutical composition” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative. In some aspects, the choice of carrier is determined in part by the particular cell and / or by the method of administration. Accordingly, there are a variety of suitable formulations. For example, the pharmaceutical composition can contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers are described, e.g., by Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG).

[0161] Buffering agents in some aspects are included in the compositions. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffering agents is used. The buffering agent or mixtures thereof are typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).

[0162] The formulations can include aqueous solutions. The formulation or composition may also contain more than one active ingredient useful for the particular indication, disease, or condition being treated with the cells, preferably those with activities complementary to the cells, where the respective activities do not adversely affect one another. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended. Thus, in some embodiments, the pharmaceutical composition further includes other pharmaceutically active agents or drugs, such as chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and / or vincristine. The pharmaceutical composition in some embodiments contains the cells in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount. Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects. The desired dosage can be delivered by a single bolus administration of the cells, by multiple bolus administrations of the cells, or by continuous infusion administration of the cells.

[0163] Formulations include those for intravenous and intraperitoneal. In some embodiments, the cell populations are administered parenterally. The term “parenteral,” as used herein, includes intravenous and intraperitoneal administration. In some embodiments, the cells are administered to the subject using peripheral systemic delivery by intravenous or intraperitoneal injection. Compositions in some embodiments are provided as sterile liquid preparations.

[0164] Sterile injectable solutions can be prepared by incorporating the cells in a solvent, such as in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, and / or colors, depending upon the route of administration and the preparation desired. Standard texts may in some aspects be consulted to prepare suitable preparations.

[0165] Various additives which enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, and sorbic acid. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0166] The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by fdtration through sterile filtration membranes.

[0167] Methods for Treatment

[0168] In some aspects, the present invention provides a method for treating a subject in need of a stem cell transplantation. The method comprises administering a therapeutically effective amount of a population of stem cells comprising a stem cell; wherein stem cell is engineered to express a variant of a B-cell lymphoma 2 (Bcl-2) family protein, wherein the variant confers to the stem cell resistance to a cytotoxic inhibitor of the Bcl-2 family protein, thereby treating the subject in need of the stem cell transplantation.

[0169] In various embodiments, the stem cell, the Bcl-2 family protein, the variant, the cytotoxic inhibitor, the genetic modification, each is as described elsewhere herein.

[0170] In one embodiment, the stem cell is a HSC, the Bcl-2 family protein is a human bcl-2, the variant is a human bcl-2 comprising a F104L substitution therein, and the cytotoxic inhibitor is venetoclax. In other embodiments, the HSC further comprises a genetic modification of an enhancer of the BCL11A gene.

[0171] In some embodiments, the subject is a human.

[0172] In some embodiments, the stem cell transplantation is hematopoietic stem cell transplantation.

[0173] In some embodiments, the subject has a disease or disorder.

[0174] In some embodiments, the disease or disorder comprises a cancer, a hemoglobinopathy, an inherited disease or disorder, or a metabolic disease or disorder

[0175] In some embodiments, the disease or disorder comprises chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), myelodysplastic syndrome (MDS), myeloproliferative neoplasms (MPN), juvenile myelomonocytic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, severe aplastic anemia, Fanconi's anemia, paroxysmal nocturnal hemoglobinuria (PNH), pure red cell aplasia, amegakaryocytosis / congenital thrombocytopenia, severe combined immunodeficiency syndrome (SCID), Wiskott-Aldrich syndrome, thalassemia (alpha, beta or other), sickle cell disease, Hurler's syndrome, adrenoleukodystrophy, metachromatic leukodystrophy, myelodysplasia, refractory anemia, chronic myelomonocytic leukemia, agnogenic myeloid metaplasia, or familial ery throphagocy ti c ly mphohi sti ocy tosi s .

[0176] In some embodiments, the disease or disorder comprises chronic lymphocytic leukemia (CLL) or acute myeloid leukemia (AML).

[0177] In other embodiments, the method further comprising administering a cytotoxic inhibitor to the subj ect.

[0178] In some embodiments, the cytotoxic inhibitor is administered to the subject prior to, simultaneously with, or after administering the population of stem cells. In some embodiments, the cytotoxic inhibitor is selected from the group consisting of a small molecule, an antibody, and an inhibitory nucleic acid.

[0179] In some embodiments, the cytotoxic inhibitor is a small molecule.

[0180] In some embodiments, the cytotoxic inhibitor is a pro-apoptotic drug.

[0181] In some embodiments, the cytotoxic inhibitor is selected from the group consisting of venetoclax (ABT-199), navitoclax (ABT-263), ABT-737, sabutoclax (BI-97C1), obatoclax (GX15-070,), TW-37, AT-101, HA14-1, RU486, BAM7, A-1331852, A-l 155463, BDA-366, UMI-77, BH3I-1, anti -metabolites, alkylating agents (e.g. busulfan, melphalan, cyclophosphamide, fludarabine, cytarabine) and any combination thereof.

[0182] In some embodiments, the cytotoxic inhibitor is venetoclax.

[0183] In other aspects, the invention includes a method of protecting a stem cell (e.g., hematopoietic stem or progenitor cell) from a therapy comprising a cytotoxic inhibitor (e.g., venetoclax) in a subject in need thereof, the method comprising administering a therapeutically effective amount of a population of stem cells comprising a stem cell; wherein stem cell is engineered to express a variant of a B-cell lymphoma 2 (Bcl-2) family protein, wherein the variant confers to the stem cell resistance to a cytotoxic inhibitor of the Bcl-2 family protein, thereby treating the subject in need of the stem cell transplantation.

[0184] In various embodiments, the stem cell, the Bcl-2 family protein, the variant, the cytotoxic inhibitor, the genetic modification, each is as described elsewhere herein.

[0185] In one embodiment, the stem cell is a HSC, the Bcl-2 family protein is a human bcl-2, the variant is a human bcl-2 comprising a F104L substitution therein, and the cytotoxic inhibitor is venetoclax. In other embodiments, the HSC further comprises a genetic modification of an enhancer of the BCL11A gene.

[0186] In another aspect, the invention includes a method for adoptive cell transfer therapy, the method comprising administering to a subject in need thereof an effective amount of the stem cell and a chimeric antigen receptor T cell (CAR T cell) therapy, wherein stem cell is engineered to express a variant of a B-cell lymphoma 2 (Bcl-2) family protein, wherein the variant confers to the stem cell resistance to a cytotoxic inhibitor of the Bcl-2 family protein, thereby treating the subject in need of the stem cell transplantation.

[0187] In various embodiments, the stem cell, the Bcl-2 family protein, the variant, the cytotoxic inhibitor, the genetic modification, each is as described elsewhere herein. In one embodiment, the stem cell is a HSC, the Bcl-2 family protein is a human bcl-2, the variant is a human bcl-2 comprising a F104L substitution therein, and the cytotoxic inhibitor is venetoclax. In other embodiments, the HSC further comprises a genetic modification of an enhancer of the BCL11A gene.

[0188] The modified cells described herein can be administered to a subject, preferably a mammal, even more preferably a human. In one embodiment, the modified cell differentiates into at least one blood cell type in the subject. In another embodiment, the modified cell is capable of self-renewal after administration into the subject.

[0189] In one embodiment, the condition is a cancer. Examples of various cancers include but are not limited to breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer and the like. In certain embodiments, the cancer is a leukemia, such as acute myeloid leukemia.

[0190] Further, in other embodiments, stem cells (e.g., the HSCs) of the invention can be administered to a subject, preferably a mammal, even more preferably a human, to suppress an immune reaction. The stem cells can be administered to suppress an immune reaction, such as those common to autoimmune diseases such as diabetes, psoriasis, rheumatoid arthritis, multiple sclerosis, GVHD, enhancing allograft tolerance induction, transplant rejection, and the like. In addition, the stem cells of the present invention can be used for the treatment of any condition in which a diminished or otherwise inhibited immune response, especially a cell-mediated immune response, is desirable to treat or alleviate the disease.

[0191] Further, in other embodiments, the stem cells e.g., the HSCs) can be administered to a subject, preferably a mammal, even more preferably a human, to treat a condition, such as an autoimmune disease. Examples of various autoimmune diseases include but are not limited to Examples of autoimmune disease include but are not limited to, Acquired Immunodeficiency Syndrome (AIDS, which is a viral disease with an autoimmune component), alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune lymphoproliferative syndrome (ALPS), autoimmune thrombocytopenic purpura (ATP), Behcet's disease, cardiomyopathy, celiac sprue-dermatitis hepetiformis; chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy (CIDP), cicatricial pemphigoid, cold agglutinin disease, CREST syndrome, Crohn's disease, Degos' disease, dermatomyositis-juvenile, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, Graves' disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA nephropathy, insulin-dependent diabetes mellitus, juvenile chronic arthritis (Still's disease), juvenile rheumatoid arthritis, mixed connective tissue disease, multiple sclerosis, myasthenia gravis, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomena, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma (progressive systemic sclerosis (PSS), also known as systemic sclerosis (SS)), Sjogren's syndrome, stiff-man syndrome, systemic lupus erythematosus, Takayasu arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vitiligo and Wegener's granulomatosis.

[0192] In other embodiments, the stem cells (e.g., the HSCs) described herein can also be modified and used to treat inflammatory disorders. Examples of inflammatory disorders include but are not limited to, chronic and acute inflammatory disorders. Examples of inflammatory disorders include Alzheimer's disease, asthma, atopic allergy, allergy, atherosclerosis, bronchial asthma, eczema, glomerulonephritis, graft vs. host disease, hemolytic anemias, osteoarthritis, sepsis, stroke, transplantation of tissue and organs, vasculitis, diabetic retinopathy and ventilator induced lung injury.

[0193] In another embodiment, the stem cells (e.g., the HSCs) described herein may be used for the manufacture of a medicament for the treatment of an immune response in a subject in need thereof.

[0194] Stem cells (e.g., the HSCs) of the invention can be administered in dosages and routes and at times to be determined in appropriate pre-clinical and clinical experimentation and trials. Cell compositions may be administered multiple times at dosages within these ranges. Administration of the cells of the invention may be combined with other methods useful to treat the desired disease or condition as determined by those of skill in the art.

[0195] The stem cells (e.g., the HSCs) of the invention to be administered may be autologous, allogeneic or xenogeneic with respect to the subject undergoing therapy. The administration of the stem cells (e.g., the HSCs) of the invention may be carried out in any convenient manner known to those of ordinary skill in the art. In some embodiments, the cells of the present invention may be administered to a subject by injection, transfusion, implantation, or transplantation. In some embodiments, compositions described herein may be administered to a patient transarterially, by intravenous (z.v.) injection, or intraperitoneally. In other embodiments, the cells of the invention are injected directly into a site of inflammation in the subject, a local disease site in the subject, a lymph node, an organ, a tumor, and the like.

[0196] Methods for administration of HSCs including, but not limited to, for transplantation therapy are known and may be used in connection with the provided methods and compositions. For example, HSC transplantation (HSCT) methods are described, e.g., in US Patent No.

[0197] 11,648,301; US Patent No. 11,904,016; US Patent Application Publication No. 2022 / 0153876; Giralt et al. (2009). Cancer Treat Res. 144: 1-21; and Khaddour et al. (2024). Treasure Island (FL): StatPearls Publishing. In some embodiments, the cell therapy, e.g., HSCT therapy is carried out by autologous transfer, in which the cells are isolated and / or otherwise prepared from the subject who is to receive the cell therapy, or from a sample derived from such a subject. Thus, in some aspects, the cells are derived from a subject, e.g., patient, in need of a treatment and the cells, following isolation and processing are administered to the same subject.

[0198] In some embodiments, the cell therapy, e.g., HSCT therapy, is carried out by allogeneic transfer, in which the cells are isolated and / or otherwise prepared from a subject other than a subject who is to receive or who ultimately receives the cell therapy, e.g., a first subject. In such embodiments, the cells then are administered to a different subject, e.g., a second subject, of the same species. In some embodiments, the first and second subjects are genetically identical. In some embodiments, the first and second subjects are genetically similar.

[0199] The precise amount of the compositions of the present invention to be administered can be determined by a physician with consideration of individual differences in age, weight, immune response, and condition of the patient (subject). In some embodiments, a pharmaceutical composition comprising the modified cells described herein may be administered at a dosage of 104to 109cells / kg body weight, preferably 1CP to 106cells / kg body weight, including all integer values within those ranges. Cell compositions may also be administered multiple times at these dosages. In some embodiments, the cells can be administered by using infusion techniques that are commonly known (see, e.g., Rosenberg et al., New Eng. I. of Med. 319: 1676, 1988). The optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient and adjusting the treatment accordingly.

[0200] In still further embodiments, the subject has been treated with a therapeutic agent targeting the disease or condition, e.g. the tumor, prior to administration of the cells or composition containing the cells. In some embodiments, the subject is refractory or non- responsive to the other therapeutic agent. In some embodiments, the subject has persistent or relapsed disease, e.g., following treatment with another therapeutic intervention, including chemotherapy, radiation, and / or CAR T cell therapy. In some embodiments, the administration effectively treats the subject despite the subject having become resistant to another therapy.

[0201] In some embodiments, the subject is responsive to the other therapeutic agent, and treatment with the therapeutic agent reduces disease burden. In some embodiments, the subject is initially responsive to the therapeutic agent, but exhibits a relapse of the disease or condition over time. In some embodiments, the subject has not relapsed. In some such embodiments, the subject is determined to be at risk for relapse, such as at a high risk of relapse, and thus the cells are administered prophylactically, e.g., to reduce the likelihood of or prevent relapse. In some aspects, the subject has not received prior treatment with another therapeutic agent.

[0202] In some embodiments, the subject has persistent or relapsed disease, e.g., following treatment with another therapeutic intervention, including chemotherapy, radiation, and / or CART cell therapy. In some embodiments, the administration effectively treats the subject despite the subject having become resistant to another therapy.

[0203] In some other embodiments, the cells of the present invention can be used for the treatment of any condition related to a cancer, especially a cell-mediated immune response against a tumor cell(s), where it is desirable to treat or alleviate the disease. In other embodiments, the types of cancers to be treated with the modified cells or pharmaceutical compositions of the invention include certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignancies e.g., sarcomas, carcinomas, and melanomas. In some exemplary embodiments, cancers include but are not limited to B-cell malignancies such as B- cell lymphomas and leukemias and the like, as well as colorectal cancer, breast cancer, ovarian cancer, renal cancer, non-small cell lung cancer, melanoma, lymphoma, and hepatocellular cancers. In certain embodiments, the cancer is a B-cell lymphoma. Exemplary embodiments of a B-cell lymphoma include, but are not limited to Burkitt lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), diffuse large B-cell lymphoma, follicular lymphoma, and mantle cell lymphoma. In some embodiments, the cancers can be non-solid tumors (such as hematological tumors) or solid tumors. Adult tumors / cancers and pediatric tumors / cancers are also included. In one embodiment, the cancer is a solid tumor or a hematological tumor. In certain embodiments, the cancer is a leukemia and / or a lymphoma. In certain embodiments, the cancer cells express CD19.

[0204] In other embodiments, the disease or disorder is a hemoglobinopathy. Hemoglobinopathy is the medical term for a group of inherited blood disorders involving the hemoglobin, the protein of red blood cells. There are two main groups: abnormal structural hemoglobin variants caused by mutations in the hemoglobin genes, and the thalassemias, which are caused by an underproduction of otherwise normal hemoglobin molecules. In some exemplary embodiments, hemoglobinopathies include, but are not limited to hemoglobin C disease, hemoglobin S-C disease, sickle cell anemia, and thalassemias.

[0205] In some embodiments, the disease or disorder is an inherited disease or disorder, such as a genetic disorder. A genetic disorder is a disease caused in whole or in part by a change in the DNA sequence away from the normal sequence. Genetic disorders can be caused by a mutation in one gene (monogenic disorder), by mutations in multiple genes (multifactorial inheritance disorder), by a combination of gene mutations and environmental factors, or by damage to chromosomes (changes in the number or structure of entire chromosomes). In some exemplary embodiments, disorders include, but are not limited to achondroplasia, alpha-1 antitrypsin deficiency, antiphospholipid syndrome, attention deficit hyperactivity disorder, autism, autosomal dominant polycystic kidney disease, breast cancer, Charcot-Marie-Tooth disease, colon cancer, cri du chat syndrome, Crohn’s disease, cystic fibrosis, Dercum disease, down syndrome, Duane syndrome, Duchenne Muscular Dystrophy, factor V Leiden thrombophilia, familial hypercholesterolemia, graft versus host disease (GVHD), hemochromatosis, hemophilia, severe combined immunodeficiency, sickle cell disease, and thalassemia.

[0206] In some embodiments, the disease or disorder is a metabolic disease or disorder. Some metabolic disorders are inherited metabolic disorders, which are genetic conditions that result in metabolism problems. Most people with inherited metabolic disorders have a defective gene that results in an enzyme deficiency. In some exemplary embodiments, metabolic diseases or disorders include, but are not limited to Tay-Sachs disease, Hurler syndrome, phenylketonuria, Wilson disease, and hemochromatosis.

[0207] In some embodiments, the stem cells to be administered can be autologous, with respect to the subject undergoing therapy.

[0208] In some embodiments, the cells are administered at a desired dosage, which in some embodiments includes a desired dose or number of cells or cell type(s) and / or a desired ratio of cell types. Thus, the dosage of cells in some embodiments is based on a total number of cells (or number per kg body weight) and a desired ratio of the individual populations or sub-types. In some embodiments, the dosage of cells is based on a desired total number (or number per kg of body weight) of cells in the individual populations or of individual cell types. In some embodiments, the dosage is based on a combination of such features, such as a desired number of total cells, desired ratio, and desired total number of cells in the individual populations.

[0209] In some embodiments, the populations or sub-types of cells, such as PSCs and HSCs, are administered at a tolerated difference of a desired dose of total cells, such as a desired dose of HSCs. In some embodiments, the desired dose is a desired number of cells or a desired number of cells per unit of body weight of the subject to whom the cells are administered, e.g., cells / kg. In some embodiments, the desired dose is at or above a minimum number of cells or minimum number of cells per unit of body weight. In some embodiments, among the total cells, administered at the desired dose, the individual populations or sub-types are present at or near a desired output ratio, e.g., within a certain tolerated difference or error of such a ratio.

[0210] In some embodiments, the cells are administered at or within a tolerated difference of a desired dose of one or more of the individual populations or sub-types of cells, such as a desired dose of stem cells and / or a desired dose of HSCs. In some aspects, the desired dose is a desired number of cells of the sub-type or population, or a desired number of such cells per unit of body weight of the subject to whom the cells are administered, e.g., cells / kg. In some aspects, the desired dose is at or above a minimum number of cells of the population or subtype, or minimum number of cells of the population or sub-type per unit of body weight. Thus, in some embodiments, the dosage is based on a desired fixed dose of total cells and a desired ratio, and / or based on a desired fixed dose of one or more, e.g., each, of the individual sub-types or subpopulations. Thus, in some embodiments, the dosage is based on a desired fixed or minimum dose of HSCs, and / or is based on a desired fixed or minimum dose of HSCs. In certain embodiments, the cells, or individual populations of sub-types of cells, are administered to the subject at a range of about one million to about 100 billion cells, such as, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), such as about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), and in some cases about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells) or any value in between these ranges.

[0211] In some embodiments, the dose of total cells and / or dose of individual sub-populations of cells is within a range of between at or about IxlO5cells / kg to about IxlO11cells / kg 104and at or about 1011cells / kilograms (kg) body weight, such as between 105and 106cells / kg body weight, for example, at or about 1 x 103cells / kg, 1.5 x 105cells / kg, 2 x 105cells / kg, or 1 x 106cells / kg body weight. For example, in some embodiments, the cells are administered at, or within a certain range of error of, between at or about 104and at or about 109cells / kilograms (kg) body weight, such as between 105and 106cells / kg body weight, for example, at or about I x lO5cells / kg, 1.5 x 105T cells / kg, 2 x 105cells / kg, or 1 x 106cells / kg body weight. In other exemplary embodiments, a suitable dosage range of modified cells for use in a method of the present disclosure includes, without limitation, from about IxlO3cells / kg to about IxlO6cells / kg, from about IxlO6cells / kg to about IxlO7cells / kg, from about IxlO7cells / kg about IxlO8cells / kg, from about IxlO8cells / kg about IxlO9cells / kg, from about IxlO9cells / kg about IxlO10cells / kg, from about IxlO10cells / kg about IxlO11cells / kg. In an exemplary embodiment, a suitable dosage for use in a method of the present disclosure is about IxlO8cells / kg. In an exemplary embodiment, a suitable dosage for use in a method of the present disclosure is about IxlO7cells / kg. In other embodiments, a suitable dosage is from about IxlO7total cells to about 5xl07total cells. In some embodiments, a suitable dosage is from about IxlO8total cells to about 5xl08total cells. In some embodiments, a suitable dosage is from about 1.4xl07total cells to about 1 .IxlO9total cells. In an exemplary embodiment, a suitable dosage for use in a method of the present disclosure is about 7xl09total cells.

[0212] In some embodiments, the cells are administered at or within a certain range of error of between at or about 104and at or about 109cells / kilograms (kg) body weight, such as between 105and 106cells / kg body weight, for example, at or about 1 x 105cells / kg, 1.5 x 105cells / kg, 2 x 105cells / kg, or 1 x 106cells / kg body weight. In some embodiments, the cells are administered at or within a certain range of error of, greater than, and / or at least about 1 x 106, about 2.5 x 106, about 5 x 106, about 7.5 x 106, or about 9 x 106cells, and / or at least about 1 x 106, about 2.5 x 106, about 5 x 106, about 7.5 x 106, or about 9 x 106cells, and / or at least about l x 106, about 2.5 x 106, about 5 x 106, about 7.5 x 106, or about 9 x 106cells. In some embodiments, the cells are administered at or within a certain range of error of between about 108and 1012or between about IO10and 1011T cells, between about 108and 1012or between about IO10and 1011cells, and / or between about 108and 1012or between about IO10and 1011cells.

[0213] In some embodiments, the cells are administered at or within a tolerated range of a desired output ratio of multiple cell populations or sub-types, such as HSCs, PSCs, or sub-types. In some aspects, the desired ratio can be a specific ratio or can be a range of ratios, for example, in some embodiments, the desired ratio is between at or about 5: 1 and at or about 5: 1 (or greater than about 1 :5 and less than about 5: 1), or between at or about 1 :3 and at or about 3: 1 (or greater than about 1 :3 and less than about 3: 1), such as between at or about 2: 1 and at or about 1 :5 (or greater than about 1 : 5 and less than about 2: 1, such as at or about 5: 1, 4.5: 1, 4: 1, 3.5: 1, 3: 1, 2.5: 1, 2: 1, 1.9: 1, 1.8: 1, 1.7: 1, 1.6: 1, 1.5: 1, 1.4: 1, 1.3: 1, 1.2: 1, 1.1 : 1, 1 : 1, 1 : 1.1, 1: 1.2, 1 : 1.3, 1 : 1.4, 1 : 1.5, 1: 1.6, 1 : 1.7, 1 : 1.8, 1 : 1.9: 1 :2, 1 :2.5, 1 :3, 1 :3.5, 1 :4, 1 :4.5, or 1 :5. In some aspects, the tolerated difference is within about 1%, about 2%, about 3%, about 4% about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50% of the desired ratio, including any value in between these ranges.

[0214] In some embodiments, a dose of modified cells is administered to a subject in need thereof, in a single dose or multiple doses. In some embodiments, a dose of modified cells is administered in multiple doses, e.g., once a week or every 7 days, once every 2 weeks or every 14 days, once every 3 weeks or every 21 days, once every 4 weeks or every 28 days. In an exemplary embodiment, a single dose of modified cells is administered to a subject in need thereof. In an exemplary embodiment, a single dose of modified cells is administered to a subject in need thereof by rapid intravenous infusion.

[0215] In other embodiments, for the prevention or treatment of disease, the appropriate dosage can depend on the type of disease to be treated, the type of cells or recombinant receptors, the severity and course of the disease, whether the cells are administered for preventive or therapeutic purposes, previous therapy, the subject's clinical history and response to the cells, and the discretion of the attending physician. The compositions and cells are in some embodiments suitably administered to the subject at one time or over a series of treatments.

[0216] In some embodiments, the cells are administered as part of a combination treatment, such as simultaneously with or sequentially with, in any order, another therapeutic intervention, such as an antibody or engineered cell or receptor or agent, such as a cytotoxic or therapeutic agent. The cells in some embodiments are co-administered with one or more additional therapeutic agents or in connection with another therapeutic intervention, either simultaneously or sequentially in any order. In some contexts, the cells are co-administered with another therapy sufficiently close in time such that the cell populations enhance the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, the cells are administered prior to the one or more additional therapeutic agents. In some embodiments, the cells are administered after the one or more additional therapeutic agents. In some embodiments, the one or more additional agents includes a cytokine, such as IL-2, for example, to enhance persistence. In some embodiments, the methods comprise administration of a chemotherapeutic agent.

[0217] In certain embodiments, the modified cells of the invention can be administered to a subject in combination with an immune checkpoint antibody (e.g., an anti-PDl, anti-CTLA-4, or anti-PDLl antibody). For example, the modified cell can be administered in combination with an antibody or antibody fragment targeting, for example, PD-1 (programmed death 1 protein). In some exemplary embodiments, anti -PD-1 antibodies include, but are not limited to, pembrolizumab (KEYTRUDA®, formerly lambrolizumab, also known as MK-3475), and nivolumab (BMS-936558, MDX-1106, ONO-4538, OPDIVA®) or an antigen-binding fragment thereof. In certain embodiments, the modified cell may be administered in combination with an anti-PD-Ll antibody or antigen-binding fragment thereof. Examples of anti-PD-Ll antibodies include, but are not limited to, BMS-936559, MPDL3280A (TECENTRIQ®, Atezolizumab), and MED 14736 (Durvalumab, Imfinzi). In certain embodiments, the modified cell may be administered in combination with an anti-CTLA-4 antibody or antigen-binding fragment thereof. An example of an anti- CTLA-4 antibody includes, but is not limited to, Ipilimumab (trade name Yervoy). Other types of immune checkpoint modulators may also be used including, but not limited to, small molecules, siRNA, miRNA, and CRISPR systems. Immune checkpoint modulators may be administered before, after, or concurrently with the modified cell comprising the CAR. In certain embodiments, combination treatment comprising an immune checkpoint modulator may increase the therapeutic efficacy of a therapy comprising a modified cell of the present invention.

[0218] In certain embodiments, the subject is provided a secondary treatment. Secondary treatments include but are not limited to chemotherapy, radiation, surgery, and medications.

[0219] In some embodiments, a specific dosage regimen of the present disclosure includes a lymphodepletion step prior to the administration of the stem cells. In an exemplary embodiment, the lymphodepletion step includes administration of cyclophosphamide and / or fludarabine.

[0220] In some embodiments, the lymphodepletion step includes administration of cyclophosphamide at a dose of between about 200 mg / m2 / day and about 2000 mg / m2 / day (e.g., 200 mg / m2 / day, 300 mg / m2 / day, or 500 mg / m2 / day). In an exemplary embodiment, the dose of cyclophosphamide is about 300 mg / m2 / day. In some embodiments, the lymphodepletion step includes administration of fludarabine at a dose of between about 20 mg / m2 / day and about 900 mg / m2 / day (e.g., 20 mg / m2 / day, 25 mg / m2 / day, 30 mg / m2 / day, or 60 mg / m2 / day). In an exemplary embodiment, the dose of fludarabine is about 30 mg / m2 / day.

[0221] In some embodiment, the lymphodepletion step includes administration of cyclophosphamide at a dose of between about 200 mg / m2 / day and about 2000 mg / m2 / day (e.g., 200 mg / m2 / day, 300 mg / m2 / day, or 500 mg / m2 / day), and fludarabine at a dose of between about 20 mg / m2 / day and about 900 mg / m2 / day (e.g., 20 mg / m2 / day, 25 mg / m2 / day, 30 mg / m2 / day, or 60 mg / m2 / day). In an exemplary embodiment, the lymphodepletion step includes administration of cyclophosphamide at a dose of about 300 mg / m2 / day, and fludarabine at a dose of about 30 mg / m2 / day.

[0222] In an exemplary embodiment, the dosing of cyclophosphamide is 300 mg / m2 / day over three days, and the dosing of fludarabine is 30 mg / m2 / day over three days.

[0223] In an exemplary embodiment, for a subject having cancer, the subject receives lymphodepleting chemotherapy including 300 mg / m2of cyclophosphamide by intravenous infusion 3 days prior to administration of the stem cells. In an exemplary embodiment, for a subject having cancer, the subject receives lymphodepleting chemotherapy including 300 mg / m2of cyclophosphamide by intravenous infusion for 3 days prior to administration of the stem cells.

[0224] In an exemplary embodiment, for a subject having cancer, the subject receives lymphodepleting chemotherapy including fludarabine at a dose of between about 20 mg / m2 / day and about 900 mg / m2 / day (e.g., 20 mg / m2 / day, 25 mg / m2 / day, 30 mg / m2 / day, or 60 mg / m2 / day). In an exemplary embodiment, for a subject having cancer, the subject receives lymphodepleting chemotherapy including fludarabine at a dose of 30 mg / m2for 3 days.

[0225] In an exemplary embodiment, for a subject having cancer, the subject receives lymphodepleting chemotherapy including cyclophosphamide at a dose of between about 200 mg / m2 / day and about 2000 mg / m2 / day (e.g., 200 mg / m2 / day, 300 mg / m2 / day, or 500 mg / m2 / day), and fludarabine at a dose of between about 20 mg / m2 / day and about 900 mg / m2 / day (e.g., 20 mg / m2 / day, 25 mg / m2 / day, 30 mg / m2 / day, or 60 mg / m2 / day). In an exemplary embodiment, for a subject having cancer, the subject receives lymphodepleting chemotherapy including cyclophosphamide at a dose of about 300 mg / m2 / day, and fludarabine at a dose of 30 mg / m2for 3 days.

[0226] S el ecti on / Enri chm ent

[0227] In other aspects, the present invention provides a method for selecting for cells, the method comprising contacting a population of cells comprising the cells with venetoclax to enrich for the cells, wherein the cells are engineered to express a variant of a B-cell lymphoma 2 (Bcl-2) family protein, wherein the variant confers to cells resistance to a cytotoxic inhibitor of the Bcl-2 family protein.

[0228] In various embodiments, the stem cell, the Bcl-2 family protein, the variant, the cytotoxic inhibitor, the genetic modification, each is as described elsewhere herein.

[0229] In one embodiment, the stem cell is a HSC, the Bcl-2 family protein is a human bcl-2, the variant is a human bcl-2 comprising a F104L substitution therein, and the cytotoxic inhibitor is venetoclax.

[0230] In other embodiments, the stem cells (e.g., HSCs) further comprise a genetic modification. In some embodiments, the genetic modification replaces a missing or malfunctioning gene; disrupts a disease-causing gene; and / or corrects a genetic mutation.

[0231] In some embodiments, the genetic modification comprises a therapeutic edit of a target sequence associated with a disease or disorder.

[0232] In some embodiments, the disease or disorder comprises a cancer, a hemoglobinopathy, an inherited disease or disorder, or a metabolic disease or disorder.

[0233] In some embodiments, the disease or disorder comprises cancer.

[0234] In some embodiments, the disease or disorder comprises sickle-cell disease.

[0235] In some embodiments, the genetic modification comprises a modified enhancer of a BCL11A gene.

[0236] Methods for Generating Modified Cells In other aspects, the present invention also provides a method for generating a stem cell, wherein the cell is engineered to express a variant of a B-cell lymphoma 2 (Bcl-2) family protein, wherein the variant confers to the stem cell resistance to a cytotoxic inhibitor of the Bcl- 2 family protein.

[0237] In various embodiments, the stem cell, the Bcl-2 family protein, the variant, the cytotoxic inhibitor, the genetic modification, each is as described elsewhere herein.

[0238] In one embodiment, the stem cell is a HSC, the Bcl-2 family protein is a human bcl-2, the variant is a human bcl-2 comprising a F104L substitution therein, and the cytotoxic inhibitor is venetoclax. In other embodiments, the stem cells (e.g., HSCs) further comprise a genetic modification.

[0239] In some embodiments, the method comprises engineering a stem cell to express the variant of the B-cell lymphoma 2 (Bcl-2) family protein, wherein the variant confers to the stem cell resistance to the cytotoxic inhibitor of the Bcl-2 family protein. In one embodiment, the substitution is a F104L substitution in a gene for the human bcl-2. In another embodiment, the nucleic acid comprises a nucleotide sequence encoding variant, wherein the variant is the human Bcl-2 comprising the F104L substitution.

[0240] In other embodiments, the method further comprises introducing a nucleic acid capable of providing the genetic modification. In one embodiment, the invention may further comprise obtaining a stem cell from a subject in need of stem cell transplantation therapy. In another embodiment, the cell may be obtained from peripheral blood mononuclear cells, cord blood cells, bone marrow, lymph nodes, and / or a spleen. In other embodiments, cell may be CD34+.

[0241] In certain embodiments of the invention, the nucleic acid capable of expressing the variant or providing the genetic modification is a CRISPR system. The CRISPR system may comprise a Cas expression vector and a guide nucleic acid sequence specific for the endogenous gene(s)(s) and / or a Cas9 protein complexed with a guide nucleic acid sequence specific for the endogenous gene. The CRISPR system may comprise an inducible promoter. The stem cell (e.g., hematopoietic stem or progenitor cell) may be exposed to an agent that activates the inducible promoter in the Cas expression vector.

[0242] Certain embodiments of the invention further comprise expanding the cells. Expansion may be prior to the step of introducing the nucleic acid. The cells may be cryopreserved then thawed prior to introducing the nucleic acids. The nucleic acid may be introduced by transducing the cell, or transfecting the cell, or electroporating the cell.

[0243] CRISPR / Cas

[0244] Genome editing using programmable nucleases enables precise editing at specific genomic loci, which can be used to remove deleterious mutations or insert protective mutations. To date, there are three major classes of nucleases - zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered, regularly interspaced, short palindromic repeat (CRISPR)-associated nucleases. Of these, CRISPR-associated nucleases have proven to be markedly superior to the others in terms of the ease and simplicity of use.

[0245] The CRISPR / Cas system is a facile and efficient system for inducing targeted genetic alterations. Target recognition by the Cas9 protein requires a ‘seed’ sequence within the guide RNA (gRN A) and a conserved di-nucleotide containing protospacer adjacent motif (PAM) sequence upstream of the gRNA-binding region. The Cas9 protein, under direction from the gRNA, binds to its target DNA sequence and cuts both strands of the DNA at a specific locus. This double-stranded DNA break is repaired by either non-homologous end joining (NHEJ) or homology-directed repair (HDR). NHEJ frequently causes small insertions or deletions (indels) at the breakage site that can lead to a frameshift mutation of the protein encoded by the gene. HDR utilizes a repair template that is copied into the gene, thus engineering specific mutations.

[0246] The CRISPR / CAS system can thereby be engineered to cleave virtually any DNA sequence by redesigning the gRNA in cell lines (such as 293T cells), primary cells, and stem and progenitor cells. In one aspect, the invention includes a modified hematopoietic stem or progenitor cell comprising a nucleic acid capable of expressing the variant or providing the genetic modification.

[0247] One example of a CRISPR / Cas system used to inhibit gene expression, CRISPRi, is described in U.S. Publication No.: 2014 / 0068797. CRISPRi induces permanent gene disruption that utilizes the RNA-guided Cas9 endonuclease to introduce DNA double stranded breaks which trigger error-prone repair pathways to result in frame shift mutations. A catalytically dead Cas9 lacks endonuclease activity. When coexpressed with a guide RNA, a DNA recognition complex is generated that specifically interferes with transcriptional elongation, RNA polymerase binding, or transcription factor binding. This CRISPRi system efficiently represses expression of targeted genes.

[0248] CRISPR / Cas gene disruption occurs when a guide nucleic acid sequence specific for a target gene and a Cas endonuclease are introduced into a cell and form a complex that enables the Cas endonuclease to introduce a double strand break at the target gene. The CRISPR / CAS system can also simultaneously target multiple genomic loci by co-expressing a single CAS9 protein with two or more gRNAs, making this system uniquely suited for multiple gene editing or synergisti c acti vation of target genes.

[0249] In some embodiments, the CRISPR system includes a Cas expression vector and a guide nucleic acid sequence specific for expressing the variant or providing the genetic modification. In another embodiment, the Cas expression vector induces expression of Cas9 endonuclease. Other endonucleases may also be used, including but not limited to, T7, Cas3, Cas8a, Cas8b, CaslOd, Csel, Csyl, Csn2, Cas4, CaslO, Csm2, Cmr5, Fokl, other nucleases known in the art, and any combination thereof.

[0250] In one embodiment, introducing the CRISPR system comprises introducing an inducible CRISPR system. The CRISPR system may be induced by exposing the modified stems cells (e.g., hematopoietic stem or progenitor cell) to an agent that activates an inducible promoter in the CRISPR system, such as the Cas expression vector. In such an embodiment, the Cas expression vector includes an inducible promoter, such as one that is inducible by exposure to an antibiotic (e.g., by tetracycline or a derivative of tetracycline, for example doxycycline).

[0251] However, it should be appreciated that other inducible promoters can be used. The inducing agent can be a selective condition (e.g., exposure to an agent, for example an antibiotic) that results in induction of the inducible promoter. This results in expression of the Cas expression vector.

[0252] The guide nucleic acid sequence is specific for a gene and targets that gene for Cas endonuclease-induced double strand breaks. The sequence of the guide nucleic acid sequence may be within a locus of the gene. In one embodiment, the guide nucleic acid sequence is at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more nucleotides in length.

[0253] The guide nucleic acid sequence includes a RNA sequence, a DNA sequence, a combination thereof (a RNA-DNA combination sequence), or a sequence with synthetic nucleotides. The guide nucleic acid sequence can be a single molecule or a double molecule. In one embodiment, the guide nucleic acid sequence comprises a single guide RNA.

[0254] In other embodiment, methods of introducing a nucleic acid into a stem cell (e.g., the hematopoietic stem or progenitor cell) include physical, biological and chemical methods. Physical methods for introducing a polynucleotide, such as RNA, into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. RNA can be introduced into target cells using commercially available methods which include electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, Mass.) or the Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendorf, Hamburg Germany). RNA can also be introduced into cells using cationic liposome mediated transfection using lipofection, using polymer encapsulation, using peptide mediated transfection, or using biolistic particle delivery systems such as “gene guns” (see, for example, Nishikawa, et al. Hum Gene Then, 12(8): 861 -70 (2001).

[0255] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362. Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e g., an artificial membrane vesicle).

[0256] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St. Louis, MO; dicetyl phosphate (“DCP”) can be obtained from K & K Laboratories (Plainview, NY); cholesterol (“Choi”) can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20°C. Chloroform is used as the only solvent since it is more readily evaporated than methanol. “Liposome” is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions that have different structures in solution than the normal vesicular structure are also encompassed. For example, the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules. Also contemplated are lipofectamine- nucleic acid complexes.

[0257] Regardless of the method used to introduce the nucleic acid into the cell, a variety of assays may be performed to confirm the presence of the nucleic acid in the cell. Such assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the invention. In some embodiments, the nucleic acid introduced into the cell comprises RNA. In another embodiment, at least one component of the CRISPR system comprises RNA. In yet another embodiment, the guide nucleic acid sequence is a RNA. In another embodiment, the RNA comprises in vitro transcribed RNA or synthetic RNA. The RNA is produced by in vitro transcription using a polymerase chain reaction (PCR)-generated template. DNA of interest from any source can be directly converted by PCR into a template for in vitro mRNA synthesis using appropriate primers and RNA polymerase. The source of the DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or any other appropriate source of DNA.

[0258] PCR can be used to generate a template for in vitro transcription of RNA which is then introduced into cells. Methods for performing PCR are well known in the art. Primers for use in PCR are designed to have regions that are substantially complementary to regions of the DNA to be used as a template for the PCR. Substantially complementary sequences are able to anneal or hybridize with the intended DNA target under annealing conditions used for PCR. The primers can be designed to be substantially complementary to any portion of the DNA template. For example, the primers can be designed to amplify the portion of a gene that is normally transcribed in cells (the open reading frame), including 5' and 3' UTRs. The primers can also be designed to amplify a portion of a gene that encodes a particular domain of interest. In one embodiment, the primers are designed to amplify the coding region of a human cDNA, including all or portions of the 5' and 3' UTRs. Primers useful for PCR are generated by synthetic methods that are well known in the art. “Forward primers” are primers that contain a region of nucleotides that are substantially complementary to nucleotides on the DNA template that are upstream of the DNA sequence that is to be amplified. “Upstream” is used herein to refer to a location 5, to the DNA sequence to be amplified relative to the coding strand. “Reverse primers” are primers that contain a region of nucleotides that are substantially complementary to a doublestranded DNA template that are downstream of the DNA sequence that is to be amplified. “Downstream” is used herein to refer to a location 3' to the DNA sequence to be amplified relative to the coding strand.

[0259] Chemical structures that have the ability to promote stability and / or translation efficiency of the RNA may also be used. The RNA preferably has 5' and 3' UTRs. In one embodiment, the 5' UTR is between zero and 3000 nucleotides in length. The length of 5' and 3' UTR sequences to be added to the coding region can be altered by different methods, including, but not limited to, designing primers for PCR that anneal to different regions of the UTRs. Using this approach, one of ordinary skill in the art can modify the 5' and 3' UTR lengths required to achieve optimal translation efficiency following transfection of the transcribed RNA.

[0260] The 5' and 3' UTRs can be the naturally occurring, endogenous 5' and 3' UTRs for the template. Alternatively, UTR sequences that are not endogenous for the template can be added by incorporating the UTR sequences into the forward and reverse primers or by any other modifications of the template. The use of UTR sequences that are not endogenous to the template can be useful for modifying the stability and / or translation efficiency of the RNA. For example, it is known that AU-rich elements in 3' UTR sequences can decrease the stability of RNA. Therefore, 3' UTRs can be selected or designed to increase the stability of the transcribed RNA based on properties of UTRs that are well known in the art.

[0261] In one embodiment, the 5' UTR can contain the Kozak sequence of the endogenous gene. Alternatively, when a 5' UTR that is not endogenous to the gene of interest is being added by PCR as described above, a consensus Kozak sequence can be redesigned by adding the 5' UTR sequence. Kozak sequences can increase the efficiency of translation of some RNA transcripts, but does not appear to be required for all RNAs to enable efficient translation. The requirement for Kozak sequences for many RNAs is known in the art. In other embodiments the 5' UTR can be derived from an RNA virus whose RNA genome is stable in cells. In other embodiments various nucleotide analogues can be used in the 3' or 5' UTR to impede exonuclease degradation of the RNA.

[0262] To enable synthesis of RNA from a DNA template without the need for gene cloning, a promoter of transcription should be attached to the DNA template upstream of the sequence to be transcribed. When a sequence that functions as a promoter for an RNA polymerase is added to the 5' end of the forward primer, the RNA polymerase promoter becomes incorporated into the PCR product upstream of the open reading frame that is to be transcribed. In one embodiment, the promoter is a T7 polymerase promoter, as described elsewhere herein. Other useful promoters include, but are not limited to, T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for T7, T3 and SP6 promoters are known in the art.

[0263] In one embodiment, the mRNA has both a cap on the 5' end and a 3' poly(A) tail which determine ribosome binding, initiation of translation and stability mRNA in the cell. On a circular DNA template, for instance, plasmid DNA, RNA polymerase produces a long concatameric product which is not suitable for expression in eukaryotic cells. The transcription of plasmid DNA linearized at the end of the 3' UTR results in normal sized mRNA which is not effective in eukaryotic transfection even if it is poly adenylated after transcription.

[0264] On a linear DNA template, phage T7 RNA polymerase can extend the 3' end of the transcript beyond the last base of the template (Schenborn and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003).

[0265] The conventional method of integration of polyA / T stretches into a DNA template is molecular cloning. However polyA / T sequence integrated into plasmid DNA can cause plasmid instability, which is why plasmid DNA templates obtained from bacterial cells are often highly contaminated with deletions and other aberrations. This makes cloning procedures not only laborious and time consuming but often not reliable. That is why a method which allows construction of DNA templates with polyA / T 3' stretch without cloning highly desirable.

[0266] The polyA / T segment of the transcriptional DNA template can be produced during PCR by using a reverse primer containing a polyT tail, such as 100T tail (size can be 50-5000 T), or after PCR by any other method, including, but not limited to, DNA ligation or in vitro recombination. Poly(A) tails also provide stability to RNAs and reduce their degradation. Generally, the length of a poly(A) tail positively correlates with the stability of the transcribed RNA. In one embodiment, the poly(A) tail is between 100 and 5000 adenosines.

[0267] Poly(A) tails of RNAs can be further extended following in vitro transcription with the use of a poly(A) polymerase, such as E. coli polyA polymerase (E-PAP). In one embodiment, increasing the length of a poly(A) tail from 100 nucleotides to between 300 and 400 nucleotides results in about a two-fold increase in the translation efficiency of the RNA. Additionally, the attachment of different chemical groups to the 3' end can increase mRNA stability. Such attachment can contain modified / artificial nucleotides, aptamers and other compounds. For example, ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerase. ATP analogs can further increase the stability of the RNA.

[0268] 5' caps also provide stability to RNA molecules. In a preferred embodiment, RNAs produced by the methods disclosed herein include a 5' cap. The 5' cap is provided using techniques known in the art and described herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444 (2001); Stepinski, et al., RNA, 7: 1468-95 (2001); Elango, et al., Biochim. Biophys. Res. Commun, 330:958-966 (2005)).

[0269] The RNAs produced by the methods disclosed herein can also contain an internal ribosome entry site (IRES) sequence. The IRES sequence may be any viral, chromosomal or artificially designed sequence which initiates cap-independent ribosome binding to mRNA and facilitates the initiation of translation. Any solutes suitable for cell electroporation, which can contain factors facilitating cellular permeability and viability such as sugars, peptides, lipids, proteins, antioxidants, and surfactants can be included.

[0270] The RNAs described herein may be introduced into the cell by a variety of methods known in the art. In some embodiments, the RNA is electroporated into the cells. In one embodiment, the CRISPR system comprises RNA that is electroporated into the cells. In yet another embodiment, the CRISPR system comprises at least one guide nucleic acid sequence that is an RNA and electroporated into the cells.

[0271] In other embodiments, the methods also provide the ability to control the level of expression over a wide range by changing, for example, the promoter or the amount of input RNA, making it possible to individually regulate the expression level.

[0272] In some embodiments, an RNA transgene can be delivered to a cell and expressed therein, as a minimal expressing cassette without the need for any additional viral sequences. Under these conditions, integration of the transgene into the host cell genome is unlikely. Cloning of cells is not necessary because of the efficiency of transfection of the RNA and its ability to uniformly modify the entire lymphocyte population.

[0273] Genetic modification of the cells with in vitro-transcribed RNA (IVT-RNA) makes use of two different strategies both of which have been successively tested in various animal models. Cells are transfected with in vitro-transcribed RNA by means of lipofection or electroporation. It is desirable to stabilize IVT-RNA using various modifications in order to achieve prolonged expression of transferred IVT-RNA.

[0274] Some IVT vectors are known in the literature which are utilized in a standardized manner as template for in vitro transcription and which have been genetically modified in such a way that stabilized RNA transcripts are produced. Currently protocols used in the art are based on a plasmid vector with the following structure: a 5' RNA polymerase promoter enabling RNA transcription, followed by a gene of interest which is flanked either 3' and / or 5' by untranslated regions (UTR), and a 3' polyadenyl cassette containing 50-70 A nucleotides. Prior to in vitro transcription, the circular plasmid is linearized downstream of the polyadenyl cassette by type II restriction enzymes (recognition sequence corresponds to cleavage site). The polyadenyl cassette thus corresponds to the later poly(A) sequence in the transcript. As a result of this procedure, some nucleotides remain as part of the enzyme cleavage site after linearization and extend or mask the poly(A) sequence at the 3' end. It is not clear, whether this nonphysiological overhang affects the amount of protein produced intracellularly from such a construct.

[0275] In another aspect, the RNA construct is delivered into the cells by electroporation. See, e.g., the formulations and methodology of electroporation of nucleic acid constructs into mammalian cells as taught in US 2004 / 0014645, US 2005 / 0052630A1, US 2005 / 0070841A1, US 2004 / 0059285A1, US 2004 / 0092907A1. The various parameters including electric field strength required for electroporation of any known cell type are generally known in the relevant research literature as well as numerous patents and applications in the field. See e g., U.S. Pat. No. 6,678,556, U.S. Pat. No. 7,171,264, and U.S. Pat. No. 7,173,116. Apparatus for therapeutic application of electroporation are available commercially, e.g., the MedPulser™ DNA Electroporation Therapy System (Inovio / Genetronics, San Diego, Calif), and are described in patents such as U.S. Pat. No. 6,567,694; U.S. Pat. No. 6,516,223, U.S. Pat. No. 5,993,434, U.S. Pat. No. 6,181,964, U.S. Pat. No. 6,241,701, and U.S. Pat. No. 6,233,482; electroporation may also be used for transfection of cells in vitro as described e.g. in US20070128708A1. Electroporation may also be utilized to deliver nucleic acids into cells in vitro. Accordingly, electroporation-mediated administration into cells of nucleic acids including expression constructs utilizing any of the many available devices and electroporation systems known to those of skill in the art presents an exciting new means for delivering an RNA of interest to a target cell.

[0276] It should be understood that the method and compositions that would be useful in the present invention are not limited to the particular formulations set forth in the examples. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the cells, expansion and culture methods, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention. The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, fourth edition (Sambrook, 2012); “Oligonucleotide Synthesis” (Gait, 1984); “Culture of Animal Cells” (Freshney, 2010); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1997); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Short Protocols in Molecular Biology” (Ausubel, 2002); “Polymerase Chain Reaction: Principles, Applications and Troubleshooting”, (Babar, 2011); “Current Protocols in Immunology” (Coligan, 2002). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.

[0277] EXAMPLES

[0278] The invention is now described with reference to the following Examples. These Examples are provided for the purpose of illustration only, and the invention is not limited to these Examples, but rather encompasses all variations that are evident as a result of the teachings provided herein.

[0279] Example 1 Venetoclax-Resistant HSCs

[0280] To prevent the cytotoxic effects of chemotherapy during engraftment, Human HSCs were engineered to be resistant to the cytotoxic effects of pro-apoptotic small molecule venetoclax. F104L is one of several substitutions in the BCL-2 protein that can confer resistance to venetoclax (FIG. 5 A). The schematic of ex vivo editing (FIG. 6) of F104L in HSCs which are transplanted into an AML patient. The patient receives venetoclax, which promotes the survival of the donor HSCs in order to prevent AML relapse. FIG. 7 shows a schematic of ex vivo installation of a selection edit (F104L) and a therapeutic edit (BCL11A) in HSCs which are transplanted into a patient. The patient receives venetoclax which promotes the enrichment of therapeutic cells. Guide RNA sequences for human BCL-2

[0281] Guide RNA sequences for human UCK2

[0282] Guide RNA sequences for mouse BCL-2

[0283] CD34+ cells can be efficiently edited to F104L through CRISPR base editing. The bar graph shows editing to F104L across 7 donors (FIG. 8). Cells were electroporated using the Lonza 4D-Nucleofector System program DZ100 with ABE8e mRNA and sgRNA for AAVS1 (WT cells) or Fl 04L at a 1 : 1 mass ratio. Editing efficiency was determined via Sanger sequencing. Once it was demonstrated that CD34+ could be efficiently edited to express F104L, it was then tested if the CD34+ F104L cells were resistant to venetoclax over a period of time. CD34+ cells were isolated from donor apheresis bags or cord blood bags. CD34+ cells were cultured in STEMCELL Technologies StemSpan SFEMII supplemented with 1% Penstrep and 100 ng / uL of cytokines SCF, TPO, IL-6, and FLT3-L. 24 hours after isolation and stimulation with cytokines, cells were electroporated with a 1 : 1 mass ratio of Cas9 mRNA and sgRNA. The cells were then left to rest for 5-6 days. After resting, DNA was extracted and prepared for Sanger sequencing to confirm installation of resistance mutation. The titration was set up in 96 well plates with 5eO5 cells / mL and venetoclax concentrations ranging from 0 to 8000 nM. Cells were exposed to venetoclax for 5 (FIG. 9) or 14 days (FIG. 11). Data with a non-linear regression for 5 days and 14 days is shown by FIGs. 10 and 13, respectively. For the 14 day titrations, 50uL of fresh media and venetoclax was added at day 7. To read out the plates, cells were prepared for flow cytometry and stained with APC Annexin V and PI. Live cells were determined to be negative for both Annexin V and PI. The percent of live cells per condition was normalized against the respective DMSO condition and graphed. Confirming that CD34+ F104L cells were resistant to venetoclax, it was then tested if CD34+F104L cells had similar colony formation to their wildtype counterparts. CD34+ were isolated from donor apheresis bags or cord blood bags. CD34+ cells were cultured in STEMCELL Technologies StemSpan SFEMII supplemented with 1% Penstrep and 100 ng / uL of cytokines SCF, TPO, IL-6, and FLT3-L. 24 hours after isolation and stimulation with cytokines, cells were electroporated with a 1 : 1 mass ratio of Cas9 mRNA and sgRNA. The cells were then left to rest for 24 hours. After resting, cells were cultured in STEMCELL Technologies MethoCult media and plated at a concentration of 500 cells / mL in technical duplicates. 14 days later, the plates were imaged and colonies were manually counted (FIG. 13). Data points represent the average colony count between the technical duplicates for each donor.

[0284] As a control, AML cancer cells were engineered with the F104L mutation and tested to see if they were sensitive to venetoclax treatment. MOLM-13 cells were cultured in RPMI media supplemented with 1% Penstrep, 10% FBS, and ImM HEPES. MOLM-13 cells were electroporated with a 1 : 1 mass ratio of Cas9 mRNA and sgRNA. The cells were then left to rest for 4 days before extracting DNA and performing Sanger sequencing to confirm installation of the F104L mutation. Sequencing prior to venetoclax administration showed 51% editing to F104L in MOLM-13 cells. The cells were then seeded at a concentration of 5e05 cells / mL in a 12 well plate and dosed with either venetoclax or DMSO. Every two days, the cells would be reseeded (5e05 cells / mL) and re-dosed with fresh media containing either venetoclax or DMSO. From days 0 to 11, cells were dosed with 31.25 nM of venetoclax or a comparable amount of DMSO. From days 11 to 20, the cells were dosed with 100 nM of venetoclax. On day 20, the enriched cells were cultured without any venetoclax and sequenced one (day 27) and two (day 34) weeks post-drug. Administration of DMSO did not affect the percent editing to F104L. Administration of 31 ,25nM of venetoclax was able to enrich the edited population up to 81%, and further administration of lOOnM of venetoclax enriched the edited population up to 90%. The enrichment was durable as the edited population remained 95% F104L even up to two weeks after being cultured without venetoclax (FIG. 14).

[0285] The results demonstrate that the mutation of b-cell lymphoma 2 (bcl-2) in HSCs provided better survival and engraftment while co-currently treating cancer using pro-apoptotic small molecules. These results further highlight the importance of improving HSC survival and engraftment by targeting the anti-apoptotic role of b cl- 1. Venetoclax (also known as ABT-199), is a potent inhibitor of bcl-2 that is an FDA-approved drug for both lymphoid and myeloid malignancies (Cang S, et al., 2015, Journal of Hematology & Oncology, 8(1): 1-8; Roberts AW, et al., 2016, New England Journal of Medicine, 374(4):311-22; and Seymour JF, et al., 2018, New England Journal of Medicine, 378(12): 1107-20).

[0286] Venetoclax can negatively affect HSCT therapy by inhibiting the anti-apoptotic function of bcl-2. The results indicate that higher doses of venetoclax required for targeting venetocl ax- resistant lymphomas limited the HSC’s long-term persistence by promoting apoptosis in HSCs, leading to a diminished combination effect. To overcome this venetoclax-induced apoptosis in HSCs, venetoclax-resistant HSCs were developed by overexpressing a bcl-2 variant that harbors a point mutation (F104L) at the key residue for the binding to venetoclax (Tahir SK, et al., 2017, BMC Cancer, 17(l):l-10). As demonstrated herein, overexpression of variant bcl2 (F104L) completely rescues HSCs from venetoclax-induced toxicity, thereby allowing the long-term synergistic effect between HSCs and venetoclax in combination. Additionally, the results indicate that bcl-2 overexpression significantly enhanced overall HSC transplantation activity by promoting long-term survival. Taken together, these data shows that genetic modulation in HSCs that confer a resistance to a potent pro-apoptotic drug (e.g., F104L Bcl-2 variant having resistance to venetoclax) is a promising strategy by achieving a surprising synergistic combination effect while significantly reducing the undesired bystander effects. In addition, expression of anti-apoptotic molecules (e g., Bcl-2) in HSCs promotes the long-term survival of HSCs leading to augmentation of overall HSCs’ transplantation activity.

[0287] Discussion

[0288] Cellular immunotherapies have made remarkable progress in the past ten years and matured into a potent therapeutic modality for combatting cancer. These therapies typically involve harvesting autologous immune effector cells (IEC) such as T or natural killer (NK) cells followed by ex vivo genetic modification using viral (or less commonly, non-viral) vectors to express CARs or transgenic TCRs targeting tumor antigens. Once re-infused into the patient, the engineered cells can recognize and eliminate cancer cells by mounting an anti-tumor immune response. The engineered cells must persist at therapeutically relevant levels and maintain effector function while avoiding off-tumor toxicities. However, with some notable exceptions, outside of B and plasma cell malignancies, current immune cell therapies often lack profound anti-tumor efficacy and do not persist past 28 days post infusion. While re-dosing cell therapies could address some of these issues, the requirement for conditioning chemotherapy prior to each cell infusion, an increased risk for on-target / off-tumor toxicities, their high manufacturing costs, and the atypical pharmacokinetic profile of these “living drugs” present significant challenges in translating this approach into clinical practice. One way to improve the current paradigm is by combining engineered IEC with other cancer drugs in a rational manner to achieve one or more of the following goals: (i) anti-cancer synergy (synthetic lethality), (ii) mitigation of toxicity which could secondarily improve the therapeutic index, or (iii) neutralization of counter- regulatory mechanisms that impede the full efficacy of IEC. However, such combinations typically only work when administered sequentially (pretreatment) or at sub-pharmacologic doses due to the effects of cytotoxic drugs on the immune effector cells themselves (with few exceptions). Furthermore, the additional agents should ideally already have regulatory approval, eliminating the need to test two experimental approaches concurrently. Therefore, the next generation of immune cell therapies should have a wider therapeutic window, be amenable to combination therapy with standard of care (SoC) drugs, and be readily available “off-the-shelf’. Concurrent with the rise of cancer immunotherapies, advancements in CRISPR / Cas gene editing technologies have transformed our ability to manipulate and control cellular behavior. Zinc-finger nucleases, transcription activator-like nucleases (TALENs), CRISPR / Cas nucleases and their derived technologies, base editing, and prime editing have greatly expanded the scope of possible genome modifications. This genome editing toolkit now allows for targeted gene insertion, deletion, base-pair conversions, or a combination of those. Amongst other applications for these genome editing technologies reviewed elsewhere, they allow for editing the molecular targets of targeted therapeutics to confer cells with drug resistance. Additionally, genome editing can remove cell surface proteins to facilitate cell therapies to evade al lorej ection. Since CAR / TCR-engineered cell therapies already undergo ex vivo manipulation, further genetic augmentation can be readily integrated into manufacturing processes. Recent data has shown that genetic engineering can shield cellular immunotherapies and hematopoietic stem cell transplants from the effects of targeted therapies, conditioning regimens, and enables their transplantation into allogeneic recipients with delayed immune rejection.

[0289] Discussed below are strategies to engineer into IEC resistance to chemotherapies, small molecule targeted therapies, antibody-based therapies (bispecific engagers, antibody-drug- conjugates, and chimeric antigen receptors), and immune rejection. Additional therapeutic applications for which engineered resistance can be harnessed to enhance the efficacy, safety, and accessibility of cellular immunotherapies. Lastly, the utility of engineering healthy hematopoietic stem cells (HSCs) is explored to confer resistance to antigen-directed immunotherapies and small molecule targeted therapy to expand the therapeutic index of SoC targeted cancer therapies and facilitate in vivo selection of genetically modified HSCs for non- malignant applications.

[0290] Principles of engineering resistance to targeted therapies

[0291] Engineering resistance to the effects of targeted drugs requires a detailed understanding about the drug’s mechanism of action and molecular target. Use of chemotherapy, small molecule inhibitors and antibody -based immunotherapies in cancer has uncovered many drug resistance mechanisms that could be exploited to purposefully endow cell therapies with resistance to the effects of a targeted drug, some of which are discussed below (FIG. 1 A-1B, Table 1). A key consideration must be the dose-limiting toxicity of a given drug beyond the engineered cell population, which would define the maximum tolerated dose regardless of the resistant engineered cells.

[0292] Engineering cellular resistance to small molecule inhibitors

[0293] One strategy to confer resistance to small molecule drugs is transgenic overexpression of a drug resistance gene. By exploiting well-characterized drug resistance mechanisms, such as efflux pumps, enzymatic drug metabolism, or DNA repair, transgenic overexpression enables the modulation and enhancement of the host's natural defense systems against targeted drugs. For example, overexpression of ATP -binding cassette (ABC) transporters, such as p-glycoprotein (p- / MDRl), confer resistance against chemotherapeutic agents like doxorubicin and paclitaxel by potentiating drug efflux. However, MDR1 overexpression via retroviral transduction confers resistance to many drugs (due to the wide variety of drugs that are substrates for MDR1) and has been associated with myeloproliferative syndrome in preclinical mouse models. This poses significant safety concerns in case of malignant transformation of multi-drug-resistant cells (Box 1). A different resistance strategy involves overexpression of DNA repair proteins such as methylguanine methyltransferase (MGMT) or tyrosyl-DNA phosphodiesterase (TDP-I) to protect cells from DNA alkylating agents or topoisomerase inhibitors. A third resistance strategy is overexpressing glutathione s-transferase (GST) enzymes in cells to enhance drug metabolism and confer resistance to drugs that are metabolized by GST-mediated detoxification pathways such as busulfan, a chemotherapeutic drug used as a backbone of myeloablative conditioning regimens. Lastly, the expression of protein variants that decrease or abrogate drug binding has been explored. For example, antifolate drugs like methotrexate inhibit dihydrofolate reductase (DHFR) by binding to the enzyme’s active site and thereby inhibiting nucleotide biosynthesis. Resistance to antifolate drugs in hematopoietic stem cells can be achieved by overexpressing DHFR variants that decrease binding of methotrexate while preserving enzymatic activity. Accordingly, hematopoietic stem cells transduced with methotrexate resistant DHFR variants are protected from methotrexate induced myeloablation.

[0294] While transgenic overexpression of drug resistance genes has been widely explored in preclinical models, the translation of this approach is limited by several factors. Furthermore, if the cell engineering platform is non-viral (i.e., recombinant CRISPR / Cas with chemically synthesized gRNA systems), adding a lentiviral transduction step to the manufacturing process to achieve selective resistance is unnecessarily complex and resource-intensive. In essence, the resistance mechanism should be compatible with the platform that is used for therapeutic intervention.

[0295] The advent of designer nucleases including CRISPR / Cas nucleases, base editors, or prime editors enables the precise introduction of nucleic acid changes at the endogenous loci. These technologies are therefore well suited to install resistance mutations. Many resistance mutations to cytotoxic drugs have already been characterized in patients who received chemotherapy or targeted therapy. Often, these mutations are benign and do not contribute to oncogenesis but become positively selected following therapy. For example, resistance mutations to tyrosine kinase inhibitors (TKIs), hormone receptor antagonists, or BH3 mimetics have been identified and studied extensively in the context of cancer therapy resistance. While these mutations impair therapeutic efficacy when the drugs are used in their original purpose (to treat cancer), one could harness the resistance mutations that emerge through Darwinian selection and purposefully insert them into therapeutic cell therapies as a drug selection system or to facilitate combination therapy, as discussed later in this manuscript.

[0296] Engineering resistance to antigen-directed therapies

[0297] Engineering cellular resistance to antibody-based therapeutics can be achieved through 1) deletion of the target antigen, 2) modification of the targeted epitope, and 3) steric blockade of antibody binding. Genetically deleting target antigen expression is the most straightforward and effective way to confer resistance to antibody therapeutics. In fact, antigen loss is a common resistance mechanism to antigen-directed cancer therapeutics. Target antigen expression can be removed from the therapeutic cell population using CRISPR / Cas9 nucleases to induce insertions and deletions (indels). Indel formation commonly results in premature stop codons and mRNA degradation via nonsense-mediated decay, thereby leading to loss of protein expression. In this approach, the gRNA should be designed to target early exons to avoid the expression of truncated cell surface receptors that antibodies could recognize, even if the receptor itself is not functional. Alternatively, protein expression can also be silenced by inducing stop codons or removing start codons via base editing or epigenetic silencing using epigenome editors.

[0298] To engineer resistance against antibody-based therapies targeting antigens whose expression is indispensable for normal cell function, the group and others have recently developed epitope-editing. The goal of epitope editing is to change a target protein’s antigenic determinant (i.e. the epitope) to prevent monoclonal antibody or CAR-T cell recognition while preserving protein expression and function. Importantly, the edited cells can still be recognized by antibody clones directed against the same molecule if they target a different epitope. This approach requires mapping the amino acid sequence on the target protein that is required for antibody binding and then changing these amino acids using either CRISPR / Cas9 knock-in of a DNA donor template or using base or prime editors to edit single amino acids in the epitope. Since base or prime editing is associated with a low rate of DSBs and indels, this can lead to a relatively homogenous population of epitope-edited cells. CRISPR / Cas HDR knock-in, on the other hand, is usually associated with a high frequency of indels and, thus, a protein knockout, which can be problematic if the loss of protein expression is of functional consequence.

[0299] Genetic deletion and epitope editing both require gene editing of the target antigen which may not be feasible for all targets without significant off-target effects. As an alternative, resistance can also be achieved by masking the targeted epitope in cis (FIGs. 1 A-1B).

[0300] There are several methods to engineer resistance to antibody-derived therapeutics such as antibody drug conjugates (ADC) or CAR-T cell therapy in hematopoietic cells while cancer cells remain susceptible. One strategy is to delete the targeted antigen entirely (FIG. 1 A). In cases where the antigen is not dispensable, the epitope may be edited to prevent recognition by ADCs or CAR-T cells while preserving protein function. Alternatively, the epitope may be masked by expressing the therapeutic antibody fragment on the engineered cell (FIG. 1A, curved receptor). The antibody fragment binds to the target protein (FIG. 1 A, diamond-shaped receptor) in cis, preventing binding of the target by ADCs or CAR-T cells in trans.

[0301] There are several methods to engineer resistance to small molecule drugs in hematopoietic cells (FIG. IB). First, overexpressing ATP-binding cassette (ABC) transporters can confer resistance to chemotherapeutic agents. Similarly, overexpressing DNA repair proteins can confer resistance to DNA alkylating agents (FIG. IB). However, both methods utilize viral platforms that are associated with malignant transformation, and neither method confers specific resistance. That is, engineered cells may be resistant to multiple drugs. Alternatively, a non-viral platform such as CRISPR-Cas nucleases can be utilized to install a mutation known to confer resistance to a specific drug.

[0302] Epitope masking occurs when cells that express the therapeutic antibody fragment on the surface prevent the binding of other antibodies, resulting in protection. If expressed at high levels under a strong, constitutive promoter, the antibody fragment can bind the target antigen on the same cell (in cis) to block the interaction between the target antigen and therapeutic antibody or CAR-T cells between cells (in trans). Epitope masking due to accidental CD 19 CAR transduction into a leukemia cell has been shown to confer a leukemia blast cell with resistance to anti-CD19 CAR-T cells. Although unintentional in this case report, this resistance mechanism can be harnessed to protect transduced cells from antibody or CAR-T cell-mediated destruction. Engineering resistance to enhance the efficacy and safety of cell therapies for cancer Facilitating combination therapies

[0303] While CAR-T cells have delivered practice-changing efficacy in certain hematologic malignancies, they appear to have limited efficacy as a monotherapy in chronic lymphocytic leukemia (CLL) and solid tumors. In recent years, several approaches to improve CAR T therapy for cancers by combining these engineered cell therapies with existing drugs to achieve additive or synergistic efficacy have been proposed. However, combining cell therapies with existing anti-cancer drugs has been challenging because most drugs used for cancer therapy are either cytotoxic to lymphocytes or inhibit their effector function at the necessary pharmacologic dose (FIG. 2). Therefore, most combination therapy approaches had to be administered prior to the cell infusion or at sub-pharmacologic doses. The goal of engineering resistance to such drugs in IEC would be to shift the half-maximal inhibitory / excitatory concentration (IC / EC50) toward higher concentrations. In practice, this could allow for maintenance therapies to be continuously administered even after CAR-T administration. One way to do this is to identify mutations that occur naturally or that arise from long-term exposure to a drug that confers resistance. These mutations can then be deliberately installed in CAR T cells to facilitate the administration of drugs that would otherwise be toxic or impair T cell function by other mechanisms. A crucial consideration when selecting resistance mutations is ensuring that they are not linked to oncogenic potential.

[0304] For example, the BCL-2 inhibitor venetoclax, which is commonly used in the treatment of CLL and AML, may trigger apoptosis in T cells. To circumvent this limitation, a venetoclax- resistant mutation of the anti-apoptotic protein BCL-2 (F104L) can be overexpressed alongside a CD19-directed CAR to enable the combination of CAR-T cells with venetoclax. However, BCL- 2 family proteins are commonly overexpressed in leukemias and lymphomas, posing considerable safety concerns of this approach. Similarly, CAR-T cells can be engineered to be resistant to purine or pyrimidine nucleoside analogs (PNAs) by genetically deleting deoxycytidine kinase (dCK), an enzyme that metabolizes PNA prodrugs into their active substances. The modification enabled CD 19 CAR-T cell expansion and anti -tumor activity in the presence of clinically relevant doses of the PNA clofarabine.

[0305] Since CAR T cells are living drugs, they are susceptible to exhaustion in cases of chronic exposure to excessive amounts of antigen. Functionally, T cell exhaustion is characterized by reduced proliferation, cytokine production, and cytotoxicity. Therefore, strategies to mitigate CAR-T cell exhaustion are needed to improve long-term therapy efficacy. One approach to mitigate CAR-T cell exhaustion is by transiently blocking T cell signaling with the tyrosine kinase inhibitor dasatinib. This allowed CAR-T cells to rest from chronic, antigen-dependent signaling and prevented exhaustion. However, while transient dasatinib exposure may delay or prevent CAR-T cell exhaustion, it also transiently (and reversibly) impairs their anti -turn or functions. Therefore, one would ideally have two distinct populations of CAR-T cells: one that is responsive to dasatinib and gets rested during dasatinib exposure, while the other one is engineered to resist dasatinib and thus maintains active anti-tumor immunity even in the presence of dasatinib. For example, engineering CAR-T cells to express mutations in ABL1 (V299, F317, or T315) would confer dasatinib resistance and allow them to provide ongoing anti-tumor immunity while unedited CAR-T cells are transiently resting to prevent their exhaustion.

[0306] A different approach to improve immunotherapy responses in solid tumors is combining engineered T cell therapies with additional immunotherapies that can recruit macrophages and NK cells, such as anti-CD47 blocking antibodies. However, blocking the CD47 / SIRPa axis not only augments the phagocytic activity of tumor-associated macrophages against the tumor cells but also leads to the clearance of the therapeutic CAR-T or TCR-T cell population. To overcome this challenge, Yamada-Hunter et al. engineered CAR-T and TCR-T cells to express a CD47 variant (47E) that engaged SIRPa and provided a “don’t-eat-me” signal to tumor-associated macrophages that was not blocked by aCD47 antibodies. Engineering CD47 provided the T cells with selective resistance to macrophage clearance, enhancing their anti-tumor efficacy in combination with anti-CD47 blocking antibodies.

[0307] Editing resistance has recently been explored to enable combination therapies between adipose-derived mesenchymal stem cells and oncolytic viruses to treat brain metastasis. An allogeneic dual stem cell platform activated dendritic and T cell-mediated immune responses in the brain of metastatic melanoma murine models. The tandem stem cell platform consists of 2 populations: 1) stem cells loaded with oncolytic herpes simplex virus (oHSV) and 2) stem cells with a nectin 1 (Nl) receptor knockout. The second group of cells was further engineered to release immunomodulators such as GM-CSF and scFvPD-1. Due to the Nl knockout, these stem cells are resistant to oHSV, making the codelivery of oncolytic virotherapy and adjuvant immunomodulators possible.

[0308] Expanding the repertoire of targetable antigens

[0309] Although CAR T cells efficiently kill target cells, toxicities often arise from on- target / off-tumor immune attack. Identifying cell surface antigens that are specific to cancer cells has proven challenging, since cancer-causing mutations occur in proteins that are only expressed in the cytoplasm and therefore not accessible to antibody-based therapies. Therefore, the field has heavily relied on targeting a limited number of lineage antigens such as CD 19, CD20, CD22, or BCMA, where on-target / off-tumor toxicities against healthy antigen-bearing cells are clinically tolerable. Similarly, CAR-T cells targeting T cell lineage antigens are susceptible to on-target / off-tumor toxicities against themselves, phenomena referred to as fratricide and suicide.

[0310] One method to prevent fratricide and suicide amongst CAR T cells is knocking out antigens targeted by CAR-T cells. For example, fratricide-resistant CAR-T cells targeting CD2, CD5, or CD7 were developed by knocking out the respective target antigen in either a single or double knockout setting (for dual-specific CARs). Knocking out the target gene from T cells enables their expansion after turnover of the wt protein and, in the case of CD5 deletion, even enhanced CAR-T cell efficacy. Another approach to prevent CAR-T cell fratricide without genetically deleting the target antigen is by preventing its surface trafficking. This can be achieved by expressing antibody fragments that are coupled to intracellular retention motifs. However, although antigen deletion or intracellular retention prevents CAR-T cell fratricide, these strategies do not spare endogenous healthy T cells in the patient, leading to profound immunodeficiency.

[0311] To prevent the antigen-dependent on-target / off-tumor toxicity against indispensable cell types of the hematopoietic system, one can combine CAR-T cells with engineered healthy donor HSPCs from which the antigen target was removed using CRISPR / Cas9. The absence of the target antigen renders the engineered HSPCs and all their progeny cells resistant to the antigen- directed immunotherapy (i.e., CAR-T cells, ADCs, and bispecific engagers), leaving only the patient-derived hematopoietic cells, including their cancer cells, highly susceptible to the drug. In the case of bone marrow malignancies, it is crucial to utilize healthy donor hematopoietic stem and progenitor cells (HSPCs) instead of autologous HSPCs to avoid introducing resistance into a malignant cell clone. For example, genetic deletion of CD33 (myeloid cell marker) or CD7 (T- and NK-cell marker) renders the engineered HSPCs and all their progeny cells resistant to anti- CD33 or anti-CD7 CAR-T cells and ADCs, leaving only the patient-derived hematopoietic cells, including their cancer cells, highly susceptible to the drug. This dual therapy approach allows regenerating the respective cell types that are otherwise permanently depleted. However, this approach relies on targeting (and genetically deleting) antigens dispensable for normal hematopoietic function. Although deletion of CD33 or CD7 is safe, these antigens may be exceptions rather than the rule for this approach. Importantly, if the antigen is dispensable for normal hematopoietic cells, it is likely to also be dispensable for cancer cells, thereby facilitating antigen-negative relapse.

[0312] Therefore, recent work has adapted this concept for targeting indispensable antigens (FIG. 3B). Epitope editing enabled targeting the otherwise undruggable pan-leukocyte antigen CD45 for a universal blood cancer therapy. A single amino acid substitution at the target epitope installed via base editing rendered cells resistant to anti-CD45 CAR-T cells and bispecific T cell engagers, allowing for the manufacturing of fratricide-resistant CART45 cells and the regeneration of hematopoiesis from epitope-edited hematopoietic stem cell transplant. Notably, CD45 deletion led to impairments in hematopoiesis and CAR-T cell function, demonstrating that targeting CD45 requires epitope editing, rather than conventional antigen deletion, to unlock the potential of anti-CD45 immunotherapy. Similar results were also reported for other antigens of functional relevance, such as the cytokine receptors CD117 or CD123. There, epitope editing also efficiently abrogated antibody binding while maintaining responses to their respective receptor ligands SCF and IL3 to facilitate targeting of CD117 and CD123 on cancer cells without depleting CD117 and CD 123 -expressing healthy HSPCs and myeloid cells. Engineering graft protection to enable post-transplant maintenance therapy

[0313] Preconditioning is essential in preparing patients for immune cell therapies and autologous or allogeneic stem cell transplants to reduce tumor burden and create a favorable environment to support cell engraftment. However, chemotherapeutic conditioning often comes with high toxicity levels and can lead to long-term complications, including organ damage, infertility, and secondary malignancies. To enable the broader application of cell and gene therapies, non-genotoxic conditioning approaches using antibodies targeting CD117 / c-kit or CD45 are being explored as a promising alternative. Non-genotoxic conditioning agents allow for safer, targeted depletion of the hosts healthy leukocytes and leukemia cells to facilitate the engraftment of donor HSPCs. They also provide an opportunity to engineer the transplanted cells with selective resistance to the conditioning agents to expand their utility in post-transplant maintenance therapies. Engineered resistance enables two critical clinical applications: graft protection in hematologic malignancies and in vivo selection in the setting of genetic diseases (FIG. 3A).

[0314] Conditioning chemotherapy is given to the patients in the days leading up to hematopoietic stem cell transplantation to reduce tumor burden. However, it must then be stopped as it would otherwise eliminate the engrafted donor stem cells. The susceptibility of engrafted cells to the conditioning agent precludes continuous dosing and leaves a risk of relapse if the conditioning chemotherapy does not successfully eliminate all cancer cells, particularly in autologous stem cell transplantation, where conditioning chemotherapy is the primary driver of tumor elimination. Engineering resistance to the conditioning agent in the donor graft could increase the therapeutic window of the conditioning agent and allow its use in post-transplant maintenance settings. For example, even targeted conditioning agents like anti-CDl 17 / c-kit or anti-CD45 antibodies must be stopped at the time of donor stem cell infusion as they cannot distinguish between antigen-positive host or donor-derived cells. Engineering hematopoietic stem cells (either allogeneic or autologous) prior to (re)infusion to be resistant to these antibodies or conceivably also to small molecules by the means described above would allow continued administration even after transplant as it would only target unedited host cells. Conceptually, this approach increases the conditioning agent’s therapeutic index by allowing more prolonged exposure than would otherwise be tolerated.

[0315] Protection and in vivo selection of gene-modified hematopoietic stem cell

[0316] Hematopoietic stem cell transplantation can cure some non-malignant diseases of the hematopoietic system, such as hemoglobinopathies or inborn errors of immunity. For example, allogeneic stem cell transplantation is the standard treatment for patients born with severe combined immunodeficiencies to replace their genetically defect immune system with a healthy, donor-derived one. However, with the advent of gene editing technologies, these inborn errors of immunity can conceivably now be treated with autologous stem cell transplants in which the genetic error has been corrected, thereby eliminating the risks and toxicity associated with allogeneic HSCT.

[0317] Genetically modifying autologous hematopoietic stem cells with viral vectors or non- viral gene editing technologies can 1) replace missing or malfunctioning genes, 2) disrupt disease-causing genes, and 3) correct genetic mutations. The genetically engineered HSCs are then infused into the patient, where they engraft and replace damaged / malfunctioning cells or produce therapeutic proteins. Alternatively, in vivo gene insertion / editing strategies are also being explored to circumvent the labor and cost-intensive nature of personalized ex vivo manufacturing. In any case, the re-infused or in vivo edited cells must persist long-term at therapeutic levels to achieve clinical benefits. However, autologous, or in vivo edited HSCs often lack a survival or proliferative advantage. Therefore, strategies to selectively enrich the therapeutic cell populations after gene editing / insertion have been investigated to increase the safety and efficacy of hematopoietic stem cell gene therapies.

[0318] The need for positively selecting the population of genetically corrected autologous HSCs was first recognized due to the low efficiency of ex vivo viral gene transfer and gene editing in hematopoietic stem cells (HSCs) during the early days of gene therapy, necessitating the enrichment of the transduced / edited cells for therapeutic efficacy. However, even with improvements in viral gene transfer technologies and the advent of highly efficient gene editing technologies such as CRISPR / Cas9, in vivo selection remains a crucial feature to solve the manufacturing bottleneck of edited HSC therapies. In vivo gene editing or transduction technologies aim to reduce manufacturing costs and time to meet the growing demand for therapeutic genome editing. Although the technologies for in vivo delivery of gene editing reagents such as engineered viruses, virus-like particles, and lipid nanoparticles appear promising and are rapidly improving, they are not as efficient as ex vivo editing and, in many cases, unlikely to be efficient enough to reach therapeutically relevant levels of modified cells. Therefore, additional modifications that allow for the safe and controlled positive selection of modified cells will help to broaden the scope of treatable conditions. For example, lipid nanoparticles can be encapsulated with editor mRNA (Cas9, base editor, prime editor) and two gRNAs: one for executing the “therapeutic edit” and the other for executing the “resistance edit” to a particular selection agent (i.e., a targeted drug). After administering the selection agent, the genetically corrected cells harboring the resistance mutation would be positively enriched (FIG. 4).

[0319] A therapeutic cell population can be enriched in vivo by installing both a selection edit and a therapeutic edit (FIG. 4). In vivo gene editing via delivery of editor mRNA and two gRNAs with lipid nanoparticles (LNPs) or virus-like particles (VLPs) may result in low- efficiency editing and a heterogeneous mixture of cells with both edits, cells with the selection edit only (FIG. 4), cells with the therapeutic edit only, or WT cells that failed to be edited (FIG. 4). Only the first two cell populations will be resistant to the selection agent. The edited cells can be enriched in vivo through repeat administration of the selection agent, bringing them up to frequency sufficient for therapeutic benefit . One caveat to this approach is that cells carrying the selection edit only will also be enriched, which can hinder the therapeutic efficacy of the engineered cell product. Additionally, the efficiency of successful double-gene editing will depend on the editing platform chosen.

[0320] The choice of selection agent and engineered resistance pair will depend on the platform employed to correct the underlying genetic disease. Generally, small molecules are an attractive class of selection agents because 1) there is a large chemical diversity of small molecules with a clinical track record and regulatory approvals, 2) their molecular targets are often well- characterized, 3) they have predictable pharmacokinetic profiles, and 4) resistance mechanisms are often known (due to the emergence of resistance mutations in cancer). However, a significant hurdle to translating small molecule-based drug selection systems is that their molecular targets are often proteins / pathways expressed / active in many cell types. Therefore, small molecule selection systems can have unintended toxicities to cells outside the hematopoietic system. Monoclonal antibodies (mAbs) are also attractive selection agents due to their high specificity for their target antigen (and thus the target cell lineage) and their effectiveness in depleting cells harboring the targeted antigen. Alternatively, cells can be engineered to be susceptible to antibodies by introducing an antigen normally not expressed in the target cell population. Engineered susceptibility has increasingly been used as a safety feature for cell therapies in the case of oncogenic transformation. However, the selection antigen should be absent from other important host cell lineages or ideally non-existent in humans to avoid toxicity from the depletion agent. While a non-human or entirely orthogonal antibody-antigen pair can be developed as a selection antigen, this approach requires an in-depth assessment of immunogenicity resulting from the introduction of xenogeneic proteins presented on the cell surface. Either way, stable expression of the selection marker is most compatible with viral gene therapy platforms in which the marker can be integrated into the existing vector for the therapeutic transgene. For non-viral gene editing platforms (i.e., CRISPR / Cas-based systems), however, adding viral vectors to the manufacturing process may be unnecessarily complex and expensive. However, a selection maker can be expressed transiently by inducing the expression of an otherwise silent reporter gene using CRISPRa and epigenome modifying enzyme to match the platform used for therapeutic intervention.

[0321] Increasing cell therapy accessibility through engineered immune evasion

[0322] Currently, hematopoietic stem cell transplants, CAR T cell therapy, and other cell therapies rely on autologous or HLA-matched donor cell material to avoid host immune rejection. This highly personalized approach increases the time, investments, and infrastructure needed to develop cell therapies, raising concerns about access in low-middle-income countries. Therefore, a primary goal of the field is to develop universal, off-the-shelf, allogeneic cell and gene therapies. Overcoming the host immune barrier to achieve sufficient engraftment and prevent rejection of transplanted cells often requires the lifelong use of immunosuppressive drugs. However, long-term systemic immunosuppression has been associated with an increased risk of morbidities and negatively impacts immune effector cell efficacy. Without immunosuppression however, cell therapies are readily rejected by the host immune system, therefore failing to persist long enough to produce a durable response. Engineering therapeutic cells to evade both the adaptive and innate immune responses without broad immunosuppression is crucial to making successful allogeneic therapies.

[0323] Cells engineered to remove HLA proteins and overexpress CD47 are considered “hypoimmune”. Hypoimmune cells are distinct from allogeneic cells, which refer to cells from a donor unrelated to the recipient and may still have immune recognition molecules such as MHC class I and II. Eliminating these molecules can be achieved by disrupting B2M and CIITA and protects cells from T cell-mediated immune rejection. However, removing these proteins primes cells for NK-mediated destruction via the “missing self’ response. This response occurs when cells have little to no MHC class I expression, and inhibitory receptors on NK cells, such as CD94 / NKG2A, can no longer bind. This problem can be overcome by overexpressing CD47, also known as the “do not eat me” signal. Interaction between CD47 and SIRP-a inhibits phagocytosis by myeloid cells as well as NK activation, and overexpression of CD47 appears to effectively prevent innate immune responses. Accordingly, B2M~ / ~ CIITA'^ CD47+hypoimmune CD 19 CAR T cells can persist in vivo and maintain tumor-killing efficiency. Compared to allogeneic CD 19 CAR T cells, hypoimmune CD 19 CAR T cells achieved superior killing while reliably evading both adaptive and innate immune responses. Alternatively, deletion of the adhesion ligands CD54 and CD58 in CAR-T cells has also been shown to reduce their susceptibility to NK cell-mediated immune rejection without affecting their effector function.

[0324] Rather than directly deleting surface proteins that play a role in alloreactivity, an alternative approach to creating allogeneic CAR-T cells is by making them resistant to lymphodepletion regimens or immunosuppressants. For instance, resistance to lymphodepletion with nucleoside analogues (such as clofarabine) or anti-CD52 antibodies (i.e. alemtuzumab) can be achieved by genetically deleting deoxycytidine kinase or CD52, respectively. Similarly, resistance to immunosuppressive agents such as steroids (dexamethasone) or calcineurin inhibitors (Cyclosporin A) can be accomplished by disrupting the glucocorticoid receptor or overexpressing a mutated calcineurin subunit A. Engineering resistance to lymphodepletion or immunosuppressive agents allows allogeneic CAR-T cells to maintain their anti-tumor efficacy while the lymphodepletion or immunosuppressive agents prevent their allorej ection. However, systemic immunosuppression also limits the engagement of the EICs with the patients’ endogenous immune system, potentially impairing their overall anti-tumor efficacy.

[0325] Although recent advancements in gene-editing technology have made incorporating multiple edits in primary T cells feasible, extensive ex vivo culturing required for such cell manipulation can lead to the exhaustion and loss of function of therapeutic T cells. Therefore, engineering immune evasion in induced pluripotent stem cells (iPSCs) has been explored to create a universal donor cell source for immunotherapies that can yield antigen-specific T cells sufficient for many doses. Again, a knockout of B2M and CIITA enabled the iPSCs to escape elimination by CD8 and CD4 T cells. To evade killing by NK cells, B2M / CIITA-K0 cells were transduced with a single-chain-trimer HLA-E molecule (scHLA-E). However, the forced expression of HLA-E was insufficient to protect iPSCs from NKG2A NK cells. Further knocking out PVR, a ligand of NK cell-activating receptor DNAM-1, allowed cells to evade this subset of NK cells. Hypoimmune iPSCs transduced with an anti-CD19 CAR inhibited tumor growth of two CD 19+ leukemia and lymphoma cells as effectively as wild-type iPS-T cells expressing anti-CD19 CAR.

[0326] Allogeneic therapeutic cells may also be cleared by antibody-mediated killing. The generation of graft-specific antibodies drives antibody-mediated rejection despite continued systemic immunosuppression. Antibody generation against CAR T cells has been observed, impairing the efficacy of allogeneic therapies. Overexpression of CD64 has been shown to protect human thyroid epithelial cells, pancreatic beta cells, and CD 19 CAR T cells from both antibody- and complement-mediated killing. Importantly, the killing capacity of the CAR T cells is not hindered by CD64 overexpression. Combining CD64 overexpression with the B2M~ / ~ CIITA '~ CD47+hypoimmune phenotype in transgenic iPSC-derived endothelial cells conferred immunity to both IgG antibody -mediated and cellular immune killing in vitro and humanized mice.

[0327] Another major obstacle to allogeneic cell therapies is the development of graft-versus- host-disease (GvHD). To mitigate this in CAR T cells, the endogenous T cell receptor (TCR) can be eliminated by disrupting the TRAC locus. Therefore, a truly allogeneic CAR-T cell therapy will require at least three or more gene knockouts (B2M, CIITA, CD54, CD58, PVR) and gene transfer (CAR / TCR, CD47, scHLA-E, CD64). Although CRISPR / Cas9 nucleases can effectively disrupt three genes even in a clinical manufacturing setting, multiplex nuclease editing increases the risk for chromosomal abnormalities resulting from multiple double-stranded breaks (DSBs) within the cells. One strategy to circumvent this is using CRISPR base editing to disrupt multiple genes without causing DSBs. The feasibility of this approach has been demonstrated by silencing B2M, TRAC, and PDCD1 in CD 19 CAR T cells. Triple KO CD 19 CAR T cells demonstrated the ability to kill both CD19pos / PD-Llposand CD 19pos / PD-Llnegtarget cells while reducing the incidence of DSBs, chromosomal rearrangements, and other genotoxicities.

[0328] Engineering healthy cells to withstand the effects of a drug, rather than modifying the drug itself, represents a paradigm shift to widen the therapeutic index. Most mechanisms to install selective resistance were discovered through reverse translational research in which resistance mechanisms that emerged after patients were treated with the respective agents were characterized. Harnessing known resistance mechanisms is “convenient” because the functional implications are well characterized. However, these are likely just the tip of the iceberg. Advancements in precision gene editing enable the field to not simply rely on naturally occurring resistance mechanisms but also imagine novel ways to endow cell therapies with selective resistance. To this extent, epitope editing has been developed to orthogonalize surface antigens to be resistant to antibody-based therapies while maintaining their essential functions.

[0329] Table 1. Method of engineering resistance to small molecule inhibitors or antibody -based immunotherapies.

[0330] Example 2

[0331] FIG. 28 shows CD34+ were isolated from donor apheresis bags or cord blood bags.

[0332] CD34+ cells were cultured in StemSpan SFEMII supplemented with 1% Penstrep and 2.5 ng / mL of cytokines TPO and IL-6, and 5ng / mL FLT3-L and SCF. 24 hours after isolation, cells were electroporated with a 1 : 1 mass ratio of Cas9 mRNA and sgRNA. The cells were then left to rest for 5-6 days. After resting, DNA was extracted and prepared for Sanger sequencing to confirm installation of resistance mutation. Cells were counted at various timepoints for 7 days to track cell proliferation. FIG. 29 shows total T cells were isolated from a healthy donor via apheresis. T cells were activated with CD3 / CD28 Dynabeads for 48 hours and cultured in X-VIVO media supplemented with 10% Human ab serum, 1% Penstrep, and 1% Glutamax. After 48 hours, T cells were debeaded with a magnet and electroporated with a 1 : 1 mass ratio of Cas9 mRNA and sgRNA. The cells were then left to rest for 5 days. After resting, DNA was extracted and prepared for Sanger sequencing to confirm installation of resistance mutations or knockout (KO). That same day, cells were fixed and permeabilized for intracellular staining of BCL2, BCL-xL, and MCL-1. Cells were stained overnight and analyzed via flow cytometry the next day. The geometric mean fluorescent intensity (MFI) was calculated for each sample. In comparison to the WT control (AAVS1), the F104L, L137P, and BCL2 KO cells had a reduced MFI for the BCL2 stain, suggesting that all three mutations reduces the abundance of the BCL2 protein. Although this result was expected for the BCL2 KO group, F104L and L137P were not expected to alter BCL2 protein abundance. In contrast, the MFI for BCL-xL and MCL- 1 were found to be similar between all four groups, suggesting that the mutations only alter BCL2 protein expression.

[0333] FIG. 30 shows MOLM-13 cells were cultured in RPMI media supplemented with 1% Penstrep, 10% FBS, and ImM HEPES. MOLM-13 cells were electroporated with a 1 : 1 mass ratio of Cas9 mRNA and sgRNA. The cells were then left to rest for 5 days before extracting DNA and performing Sanger sequencing to confirm installation of the two UCK2 mutations. Sequencing prior to azacitidine (AZA) administration is shown at Day 0. The cells were then seeded at a concentration of 5e05 cells / mL in a 12 well plate and dosed with luM of AZA or DMSO. Every two days, the cells would be re-seeded (5e05 cells / mL) and re-dosed with fresh media containing luM of AZA. After 7 days of culture, DNA was extracted and Sanger sequenced to show enrichment of the desired mutations.

[0334] FIG. 31 shows MOLM-13 cells were cultured in RPMI media supplemented with 1% Penstrep, 10% FBS, and ImM HEPES. MOLM-13 cells were electroporated with a 1 : 1 mass ratio of Cas9 mRNA and sgRNA. The cells were then left to rest for 5 days before extracting DNA and performing Sanger sequencing to confirm installation of the three BCL2 mutations. Sequencing prior to venetoclax (VEN) administration is shown at Day 0. The cells were then seeded at a concentration of 5e05 cells / mL in a 12 well plate and dosed with 31 ,25nM of VEN or DMSO. Every two days, the cells would be re-seeded (5e05 cells / mL) and re-dosed with fresh media containing luM of AZA. After 7 days of culture, DNA was extracted and Sanger sequenced to show enrichment of the desired mutations.

[0335] Other Embodiments

[0336] The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof. Enumerated Embodiments

[0337] The following enumerated embodiments are provided, the numbering of which is not to be construed as designating levels of importance:

[0338] Embodiment 1 provides a stem cell, wherein the cell is engineered to express a variant of a B-cell lymphoma 2 (Bcl-2) family protein, wherein the variant confers to the stem cell resistance to a cytotoxic inhibitor of the Bcl-2 family protein.

[0339] Embodiment 2 provides the stem cell of Embodiment 1, wherein the cell is a pluripotent stem cell.

[0340] Embodiment 3 provides the stem cell of Embodiment 1, wherein the stem cell is a hematopoietic stem cell (HSC).

[0341] Embodiment 4 provides the stem cell of any one of Embodiments 1-3, wherein the Bcl-2 family protein is selected from the group consisting of Bcl-2, BCL-XL, BCL-W, MCL1, BFL1, BIM, BAD, BAK, and BAX.

[0342] Embodiment 5 provides the stem cell of any one of Embodiments 1-4, wherein the Bcl-2 family protein is human Bcl-2.

[0343] Embodiment 6 provides the stem cell of any one of Embodiments 1-5, wherein the Bcl-2 family protein is human Bcl-2 and the variant comprises a substitution selected from the group consisting ofF104L, G101V, D103E, D103Y, F101C, F101L, V92L, T187I, A131V, S105F, D103G, D103N, A149T, V148A, L169P, and any combination thereof.

[0344] Embodiment 7 provides the stem cell of any one of Embodiments 1-6, wherein the variant comprises F104L Bcl-2.

[0345] Embodiment 8 provides the stem cell of any one of Embodiments 1-7, wherein the cytotoxic inhibitor is selected from the group consisting of a small molecule and an inhibitory nucleic acid.

[0346] Embodiment 9 provides the stem cell of any one of Embodiments 1-8, wherein the cytotoxic inhibitor is a small molecule.

[0347] Embodiment 10 provides the stem cell of any one of Embodiments 1-9, wherein the cytotoxic inhibitor is a pro-apoptotic drug.

[0348] Embodiment 11 provides the stem cell of any one of Embodiments 1-10, wherein the cytotoxic inhibitor is selected from the group consisting of venetoclax (ABT-199), navitoclax (ABT-263), ABT-737, sabutoclax (BI-97C1), obatoclax (GX15-070,), TW-37, AT-101, HA14-1, RU486, BAM7, A-1331852, A-l 155463, BDA-366, UMI-77, BH3I-1, anti-metabolites, alkylating agents (e.g. busulfan, melphalan, cyclophosphamide, fludarabine, cytarabine), and any combination thereof.

[0349] Embodiment 12 provides the stem cell of any one of Embodiments 1-11, wherein the cytotoxic inhibitor is venetoclax.

[0350] Embodiment 13 provides the stem cell of any one of Embodiments 1-12, further comprising a genetic modification.

[0351] Embodiment 14 provides the stem cell of Embodiment 13, wherein the genetic modification replaces a missing or malfunctioning gene; disrupts a disease-causing gene; and / or corrects a genetic mutation.

[0352] Embodiment 15 provides the stem cell of Embodiment 13, wherein the genetic modification comprises a therapeutic edit of a target sequence associated with a disease or disorder.

[0353] Embodiment 16 provides the stem cell of Embodiment 15, wherein the disease or disorder comprises a cancer, a hemoglobinopathy, an inherited disease or disorder, or a metabolic disease or disorder.

[0354] Embodiment 17 provides the stem cell of Embodiment 16, wherein the disease or disorder comprises cancer.

[0355] Embodiment 18 provides the stem cell of Embodiment 16, wherein the disease or disorder comprises sickle-cell disease.

[0356] Embodiment 19 provides the stem cell of any one of claims 13-18, wherein the genetic modification comprises editing an enhancer of BCL11A gene.

[0357] Embodiment 20 provides a pharmaceutical composition comprising a population of stem cells comprising the stem cell of any one of Embodiments 1-19 and a pharmaceutically acceptable carrier.

[0358] Embodiment 21 provides a method for treating a subject in need of a stem cell transplantation, the method comprising administering a therapeutically effective amount of a population of stem cells comprising the stem cell of any one of Embodiments 1-19 to the subject, thereby treating the subject in need of the stem cell transplantation.

[0359] Embodiment 22 provides the method of Embodiment 21, wherein the subject is a human. Embodiment 23 provides the method of Embodiments 21 or 22, wherein the stem cell transplantation is hematopoietic stem cell transplantation.

[0360] Embodiment 24 provides the method of any one of Embodiments 21-23, wherein the subject has a disease or disorder.

[0361] Embodiment 25 provides the method of Embodiment 24, wherein the disease or disorder comprises a cancer, a hemoglobinopathy, an inherited disease or disorder, or a metabolic disease or disorder.

[0362] Embodiment 26 provides the method of Embodiment 24, wherein the disease or disorder comprises chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), juvenile myelomonocytic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, severe aplastic anemia, Fanconi's anemia, paroxysmal nocturnal hemoglobinuria (PNH), pure red cell aplasia, amegakaryocytosis / congenital thrombocytopenia, severe combined immunodeficiency syndrome (SCID), Wiskott-Aldrich syndrome, betathalassemia major, sickle cell disease, Hurler's syndrome, adrenoleukodystrophy, metachromatic leukodystrophy, myelodysplasia, refractory anemia, chronic myelomonocytic leukemia, agnogenic myeloid metaplasia, or familial erythrophagocytic lymphohistiocytosis.

[0363] Embodiment 27 provides the method of Embodiment 23, wherein the disease or disorder comprises chronic lymphocytic leukemia (CLL) or acute myeloid leukemia (AML).

[0364] Embodiment 28 provides the method of any one of Embodiments 21-27, further comprising administering a cytotoxic inhibitor to the subject.

[0365] Embodiment 29 provides the method of Embodiment 28, wherein the cytotoxic inhibitor is administered to the subject prior to, simultaneously with, or after administering the population of stem cells.

[0366] Embodiment 30 provides the method of Embodiment 28 or 29, wherein the cytotoxic inhibitor is selected from the group consisting of a small molecule and an inhibitory nucleic acid.

[0367] Embodiment 31 provides the method of any one of Embodiments 28-30, wherein the cytotoxic inhibitor is a small molecule.

[0368] Embodiment 32 provides the method of any one of Embodiments 28-31, wherein the cytotoxic inhibitor is a pro-apoptotic drug. Embodiment 33 provides the method of any one of Embodiments 28-32, wherein the cytotoxic inhibitor is selected from the group consisting of venetoclax (ABT- 199), navitoclax (ABT-263), ABT-737, sabutoclax (BI-97C1), obatoclax (GX15-070,), TW-37, AT-101, HA14-1, RU486, BAM7, A-1331852, A-l 155463, BDA-366, UMI-77, BH3I-1, anti-metabolites, alkylating agents (e.g. busulfan, melphalan, cyclophosphamide, fludarabine, cytarabine), and any combination thereof.

[0369] Embodiment 34 provides the method of any one of Embodiments 28-33, wherein the cytotoxic inhibitor is venetoclax.

[0370] Embodiment 35 provides a method for selecting for cells, the method comprising contacting a population of cells comprising the cells with venetoclax to enrich for the cells, wherein the cells are engineered to express a variant of a B-cell lymphoma 2 (Bcl-2) family protein, wherein the variant confers to cells resistance to a cytotoxic inhibitor of the Bcl-2 family protein.

[0371] Embodiment 36 provides the method of Embodiment 35, wherein the cells comprise a genetic modification.

[0372] Embodiment 37 provides the method of Embodiment 36, wherein the genetic modification replaces a missing or malfunctioning gene; disrupts a disease-causing gene; and / or corrects a genetic mutation.

[0373] Embodiment 38 provides the method of Embodiment 36 or 37, wherein the genetic modification comprises a therapeutic edit of a target sequence associated with a disease or disorder.

[0374] Embodiment 39 provides the method of Embodiment 38, wherein the disease or disorder comprises a cancer, a hemoglobinopathy, an inherited disease or disorder, or a metabolic disease or disorder.

[0375] Embodiment 40 provides the method of Embodiment 38, wherein the disease or disorder comprises cancer.

[0376] Embodiment 41 provides the method of Embodiment 38, wherein the disease or disorder comprises sickle-cell disease.

[0377] Embodiment 42 provides the method of any one of Embodiments 35-41, wherein the genetic modification is of an enhancer of a BCL11A gene. Embodiment 43 provides a method for generating the stem cell of any one of claims 1-19, the method comprising engineering a stem cell to express the variant of the B-cell lymphoma 2 (Bcl-2) family protein, wherein the variant confers to the stem cell resistance to the cytotoxic inhibitor of the Bcl-2 family protein. Embodiment 44 provides the method of Embodiment 43, further comprising introducing a genetic modification to the stem cell.

[0378] Embodiment 45 provides the method of Embodiment 43 or 44, wherein the engineering comprises introducing into the stem cell (i) a substitution in a gene to express the variant and / or (ii) a nucleic acid capable of expressing the variant having the substitution. Embodiment 46 provides the method of any one of Embodiments 43-45, wherein the substitution is a F104L substitution in a gene for the human bcl-2.

[0379] The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

[0380] -11-

Claims

CLAIMS1. A stem cell, wherein the cell is engineered to express a variant of a B-cell lymphoma 2 (Bcl-2) family protein, wherein the variant confers to the stem cell resistance to a cytotoxic inhibitor of the Bcl-2 family protein.

2. The stem cell of claim 1, wherein the cell is a pluripotent stem cell.

3. The stem cell of claim 1, wherein the stem cell is a hematopoietic stem cell (HSC).

4. The stem cell of any one of claims 1-3, wherein the Bcl-2 family protein is selected from the group consisting of Bcl-2, BCL-XL, BCL-W, MCL1, BFL1, BIM, BAD, BAK, and BAX5. The stem cell of any one of claims 1-4, wherein the Bcl-2 family protein is human Bcl-2.

6. The stem cell of any one of claims 1-5, wherein the Bcl-2 family protein is human Bcl-2 and the variant comprises a substitution selected from the group consisting of F104L, GIO IV, D103E, D103Y, F101C, F101L, V92L, T187I, Al 3 IV, S105F, D103G, D103N, A149T, V148A, L169P, and any combination thereof.

7. The stem cell of any one of claims 1-6, wherein the variant comprises F104L Bcl-2.

8. The stem cell of any one of claims 1-7, wherein the cytotoxic inhibitor is selected from the group consisting of a small molecule and an inhibitory nucleic acid.

9. The stem cell of any one of claims 1-8, wherein the cytotoxic inhibitor is a small molecule.

10. The stem cell of any one of claims 1-9, wherein the cytotoxic inhibitor is a pro-apoptotic drug.

11. The stem cell of any one of claims 1-10, wherein the cytotoxic inhibitor is selected from the group consisting of venetoclax (ABT-199), navitoclax (ABT-263), ABT-737, sabutoclax (BI-97C1), obatoclax (GX15-070,), TW-37, AT-101, HA14-1, RU486, BAM7, A-1331852, A-l 155463, BDA-366, UMI-77, BH3I-1, anti-metabolites, alkylating agents (e.g. busulfan, melphalan, cyclophosphamide, fludarabine, cytarabine), and any combination thereof.

12. The stem cell of any one of claims 1-11, wherein the cytotoxic inhibitor is venetoclax.

13. The stem cell of claims 1-12, further comprising a genetic modification.

14. The stem cell of claim 13, wherein the genetic modification replaces a missing or malfunctioning gene; disrupts a disease-causing gene; and / or corrects a genetic mutation.

15. The stem cell of claim 13, wherein the genetic modification comprises a therapeutic edit of a target sequence associated with a disease or disorder.

16. The stem cell of claim 15, wherein the disease or disorder comprises a cancer, a hemoglobinopathy, an inherited disease or disorder, or a metabolic disease or disorder.

17. The stem cell of claim 16, wherein the disease or disorder comprises cancer.

18. The stem cell of claim 16, wherein the disease or disorder comprises sickle-cell disease.

19. The stem cell of any one of claims 13-18, wherein the genetic modification comprises editing an enhancer of BCL11A gene.

20. A pharmaceutical composition comprising a population of stem cells comprising the stem cell of any one of claims 1-19 and a pharmaceutically acceptable carrier.

21. A method for treating a subject in need of a stem cell transplantation, the method comprising administering a therapeutically effective amount of a population of stem cells comprising the stem cell of any one of claims 1-19 to the subject, thereby treating the subject in need of the stem cell transplantation.

22. The method of claim 21, wherein the subject is a human.

23. The method of claim 21 or 22, wherein the stem cell transplantation is hematopoietic stem cell transplantation.

24. The method of any one of claims 21-23, wherein the subject has a disease or disorder.

25. The method of claim 24, wherein the disease or disorder comprises a cancer, a hemoglobinopathy, an inherited disease or disorder, or a metabolic disease or disorder.

26. The method of claim 24, wherein the disease or disorder comprises chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), juvenile myelomonocytic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, severe aplastic anemia, Fanconi's anemia, paroxysmal nocturnal hemoglobinuria (PNH), pure red cell aplasia, amegakaryocytosis / congenital thrombocytopenia, severe combined immunodeficiency syndrome (SCID), Wiskott- Aldrich syndrome, beta-thalassemia major, sickle cell disease, Hurler's syndrome, adrenoleukodystrophy, metachromatic leukodystrophy, myelodysplasia, refractory anemia, chronic myelomonocytic leukemia, agnogenic myeloid metaplasia, or familial ery throphagocy ti c ly mphohi sti ocy tosi s .

27. The method of claim 23, wherein the disease or disorder comprises chronic lymphocytic leukemia (CLL) or acute myeloid leukemia (AML).-SO-28. The method of any one of claims 21 -27, further comprising administering a cytotoxic inhibitor to the subject.

29. The method of claim 28, wherein the cytotoxic inhibitor is administered to the subject prior to, simultaneously with, or after administering the population of stem cells.

30. The method of claim 28 or 29, wherein the cytotoxic inhibitor is selected from the group consisting of a small molecule and an inhibitory nucleic acid.

31. The method of any one of claims 28-30, wherein the cytotoxic inhibitor is a small molecule.

32. The method of any one of claims 28-31, wherein the cytotoxic inhibitor is a pro-apoptotic drug.

33. The method of any one of claims 28-32, wherein the cytotoxic inhibitor is selected from the group consisting of venetoclax (ABT-199), navitoclax (ABT-263), ABT-737, sabutoclax (BI-97C1), obatoclax (GX15-070,), TW-37, AT-101, HA14-1, RU486, BAM7, A-1331852, A-l 155463, BDA-366, UMI-77, BH3I-1, anti-metabolites, alkylating agents (e.g. busulfan, melphalan, cyclophosphamide, fludarabine, cytarabine), and any combination thereof.

34. The method of any one of claims 28-33, wherein the cytotoxic inhibitor is venetoclax.

35. A method for selecting for cells, the method comprising contacting a population of cells comprising the cells with venetoclax to enrich for the cells, wherein the cells are engineered to express a variant of a B-cell lymphoma 2 (Bcl-2) family protein, wherein the variant confers to cells resistance to a cytotoxic inhibitor of the Bcl-2 family protein.

36. The method of claim 35, wherein the cells comprise a genetic modification.

37. The method of claim 36, wherein the genetic modification replaces a missing or malfunctioning gene; disrupts a disease-causing gene; and / or corrects a genetic mutation.

38. The method of claim 36 or 37, wherein the genetic modification comprises a therapeutic edit of a target sequence associated with a disease or disorder.

39. The method of claim 38, wherein the disease or disorder comprises a cancer, a hemoglobinopathy, an inherited disease or disorder, or a metabolic disease or disorder.

40. The method of claim 38, wherein the disease or disorder comprises cancer.

41. The method of claim 38, wherein the disease or disorder comprises sickle-cell disease.

42. The method of any one of claims 35-41, wherein the genetic modification is of an enhancer of a BCL11A gene.

43. A method for generating the stem cell of any one of claims 1-19, the method comprising engineering a stem cell to express the variant of the B-cell lymphoma 2 (Bcl-2) family protein, wherein the variant confers to the stem cell resistance to the cytotoxic inhibitor of the Bcl-2 family protein.

44. The method of claim 43 further comprising introducing a genetic modification to the stem cell.

45. The method of claim 43 or 44, wherein the engineering comprises introducing into the stem cell (i) a substitution in a gene to express the variant and / or (ii) a nucleic acid capable of expressing the variant having the substitution.

46. The method of any one of claims 43-45, wherein the substitution is a F104L substitution in a gene for the human bcl-2.

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