Discernible protein variants of BCL-2 for use in cell therapy

Abstract

The present disclosure relates to the use of cells having discernible protein with engineered or naturally occurring mutation(s) but functional protein for use in therapy. The present invention also relates to the use of cells having discernible BCL-2 protein variants but functional protein for use in therapy, in particular adoptive cell therapy.

Description

[0001] DISCERNIBLE PROTEIN VARIANTS OF BCL-2 FOR USE IN CELLTHERAPY TECHNICAL FIELD The present disclosure relates to the use of cells having discernible protein with engineered or naturally occurring mutation(s) but functional protein for use in therapy. Thepresent invention also relates to the use of cells having discernible BCL-2 protein variantsbut functional protein for use in therapy, in particular adoptive cell therapy. BACKGROUND OF THE INVENTION Most targeted therapies are either small-molecule drugs or based on monoclonalantibodies. However, targeted therapy is often associated with severe unwanted sideeffects. Indeed, while cancer targeted therapy with small molecules has been successful intargeting and eradicating malignant cells expressing a specific antigen, it does often notdiscriminate between normal and malignant cells and thus induces destruction of thenormal hematopoietic system. Targeted therapies, which can be directed against receptortyrosine kinases or anti-apoptotic proteins eliminate all cells expressing the targetmolecule. However, most cancer antigens are shared with normal hematopoietic or othercells. Thus, to identify targets to kill diseased cells including tumors while avoiding damageto healthy cells is a major challenge for targeted therapies. In particular, in myeloid diseasesincluding myeloid malignancies such as myelodysplastic syndrome (MDS), acute myeloidleukemia (AML) or Chronic Myeloid Leukemia (CMML) cell surface and intracellularantigens such as CD117, CD33, BCL-2, or CD123 are shared with normal myeloidprogenitors and even hematopoietic stem cells. Therefore, targeted therapy targetingCD117, CD33, BCL-2 or CD123 antigen for MDS, AML or CMML can be associated withdepletion of normal hematopoietic cells in addition to malignant cells in patients (Gill S. I.Best practice & Research Clinical Hematology, 2019). As a consequence, targeted therapiesincluding small molecules, mAbs, T cell engagers or CAR T have in many cases been elusive, in part owing to the absence of truly disease-specific antigens (Gill S. I. Best practice & Research Clinical Hematology, 2019). The inventors in previous patent applications showed that a single amino acid differencein surface protein variants can be genetically engineered into hematopoietic cells to changethe antigenicity and be discriminated by specific and selective targeted drugs such asantibodies (WO2017 / 186718, WO2018 / 083071). Contrary to the approach where a proteinis removed (KO cells), the protein variants in these cells retain their normal expression andfunction and enable the target proteins with important non-redundant functions.BCL-2 is a single-pass membrane protein belonging to a family of apoptosis regulatingproteins and residing in the mitochondrial outer membrane through a C-terminalmembrane anchor. BCL-2 is expressed primarily in cells of the hematopoietic tissues suchas bone marrow, thymus, and spleen but is also found in endothelium and neurons. Morespecifically, BCL-2 is expressed in a variety of normal hematopoietic lineages such as B- andT-cells as well as in hematopoietic stem and progenitor cells (HSPC). The overall structureof BCL-2 consists of two central, predominately hydrophobic helices (helix 5 and 6) packedagainst four amphipathic α-helices, followed by the transmembrane domain.The BCL-2 family is typically classified into three groups, including pro-apoptotic initiators,pro-apoptotic effectors, and anti-apoptotic proteins. BCL-2 has anti-apoptotic activity andis required for the survival of mature lymphocytes, melanocyte stem cells and cells of thedeveloping kidney (Veis DJ et al., Cell 1993). Multiple anti-apoptotic proteins such as BCL-2, BCL-XL or MCL-1 can be co-expressed in many cell types and tissues. And like the pro-survival BCL-2 proteins, multiple pro-apoptotic initiators (e.g., BIM, BID, BIK, PUMA or BAD)and effectors (e.g., BAX, BAK or BOK) are found expressed in cells at the same time. Theapoptotic-promoting effects from the pro-apoptotic initiators and effectors are counteredby their direct interaction with the anti-apoptotic family members such as BCL-2 (reviewedin Adams MC, Front. Oncol. 2019).Conserved helical sequence regions, known as Bcl-2 homology (BH) motifs or domainsform the interaction sites of the BCL-2 family members and regulate intrinsic apoptosis.BCL2 binds to and inactivates BAX and other pro-apoptotic proteins such as BAK, therebyinhibiting apoptosis, ultimately preventing mitochondrial membrane permeabilization(reviewed in Campbell KJ and Tait SWG, Open Cell 2018; Tzifi F et al., Advances inHematology 2012). In more detail, BCL-2 interacts with pro-apoptotic members like BAXand BAK through their BH3 domains (Vela L et al., J Biol Chem 2013).The present disclosure aimed to identify amino acid residues of BCL-2 that can besubstituted in a manner such that a) the function of BCL-2 is not, or at least notsubstantially, altered, i.e. the variant of BCL-2 is functionally indistinguishable from the wildtype version of BCL-2, and b) a binding moiety, such as a small molecule, binds to the wildtype version of BCL-2, but shows a substantially decreased or no binding to the alteredversion of BCL-2, i.e. the variant of BCL-2 is distinguishable from the wild type version ofBCL-2 by way of binding / non-binding to the binding moiety.Most single amino acid substitution in any given target protein will only affect the bindingof a moiety, if the amino acid substitution is part of, or is close to, the binding site of themoiety, typically a pocket or groove. As also will be appreciated, single amino acidsubstitutions that do affect binding of a binding moiety to a target protein can also impactthe functionality of the target protein. It is therefore a highly sophisticated andunpredictable task to identify those amino acid substitutions that fulfill both requirements,i.e. that do affect binding of a moiety to a target protein, but at the same time do not, ornot substantially, affect the function of the target protein.Certain naturally occurring Bcl-2 resistance variants are known, such as G101V (NatCommun (2019) 10(1):2385) and D103Y (Acta Oncol (2021) 60(4):528-530).WO2023 / 133423 discloses the introduction of modified Bcl-2 RNA into HSCs to conferresistance to these cells. WO 2024 / 091959 generates venetoclax-resistant NK cells byknocking the Bcl-2 G101V variant into iPSCs. Increased resistance is only shown for G101V,but not for other Bcl-2 variants. The present disclosure also aimed to identify amino acid residues of BCL-2 that can besubstituted in a manner such that a) the expression of BCL-2 is not, or not substantially,altered, i.e. the level of BCL-2 expression is indistinguishable or substantiallyindistinguishable from the wild type version of BCL-2, and b) a binding moiety, such as a small molecule, that binds to the wild type version of BCL-2, but shows a substantially decreased or no binding to the altered version of BCL-2. While naturally occurring Bcl-2 variants, e.g. G101V and D103Y, only show 6.8 to 11.5 foldloss of venetoclax binding, certain variants disclosed herein (e.g. F104A, F104C, F104T,F104G, F104N) achieve a 100-200 fold loss of venetoclax binding, while retaining proteinintegrity and binding of known Bcl-2 ligands, such as BAK, BIM and BAX.Several BCL-2 targeted small molecule inhibitors are known in the art. BCL-2 is the targetof venetoclax (ABT-199), which is approved for the treatment of acute myeloid leukemia (AML) and chronic lymphocytic leukemia (CLL). Navitoclax (ABT-263), a related molecule and dual inhibitor of BCL-2 and BCL-XL is in clinical trials for various hematologicalmalignancies and solid tumors. AZD-4320 is also structurally related to venetoclax andnavitoclax. It is a dual inhibitor of Bcl2 and BCL-xL and in phase 1 / 2 clinical evaluation as adendrimer nanoparticle formulation, AZD-0466, in hematological malignancies as well asnon-Hodgkin's lymphoma (NHL). Lisaftoclax is also structurally related to venetoclax andinhibits both BCL-2 and BCL-XL. This inhibitor is in phase 2 clinical studies against CLL andsmall lymphocytic leukemia (SLL). Sonrotoclax, which is a structurally related inhibitor tovenetoclax targets BCL-2 and is in Phase 2 studies to treat CLL. S55746 is structurally not related to venetoclax and it is investigated in Phase 1 studies to treat patients suffering from CLL, NHL, and multiple myeloma. SUMMARY OF THE INVENTION One of the objectives of the present disclosure is to develop a safer method for thetreatment of malignancies, in particular cancer, hematological malignancies, and myeloiddiseases. The inventors thus sought variations of the protein BCL-2, which retain orsubstantially retain normal function and / or expression, and wherein a binding moiety, suchas an BLC-2 inhibitor, is capable of binding to the original, typically the wild-type, versionof the BCL-2 protein, but does not, or substantially weaker, bind to the variant BCL-2protein. This is achieved via single or multiple amino acid or nucleotide variations.In particular, the inventors identified variants of BCL-2, which change the binding of aspecific small-molecule inhibitor to BCL-2, while retaining its normal expression andfunction, such as the interaction with BIM, BAK and BAX, as well as its pro-survival and anti-apoptotic activity. This was achieved via a sophisticated campaign involving a rationaldesign approach, screening project and a comparison to naturally occurring polymorphisms. The present disclosure relates to a mammalian cell or a population of cells expressing afirst isoform of BCL-2 for use in a medical treatment in a patient in need thereof, saidpatient having cells expressing a second isoform of BCL-2, wherein said cell expressing said first isoform comprises genomic DNA with at least one polymorphism or genetically engineered allele, wherein said polymorphism or genetically engineered allele is not present in the genome of the patient having cells expressing said second isoform of BCL-2, and preferably wherein said first and second isoform are functional. Alternatively said firstisoform is generated via RNA editing. Preferably said function is the induction of apoptosis,the binding of BH3-only proteins (such as Bim and Bad) or the binding of pro-apoptotic proteins (such as Bax). The present disclosure also relates to a mammalian cell or a population of cells expressinga first isoform of BCL-2 for use in a medical treatment in a patient in need thereof, saidpatient having cells expressing a second isoform of BCL-2, wherein said cell expressing said first isoform comprises genomic DNA with at least one polymorphism or genetically engineered allele, wherein said polymorphism or genetically engineered allele is not present in the genome of the patient having cells expressing said second isoform of BCL-2 and preferably wherein said first and second isoform are expressed at the same level or substantially the same level. In a particular embodiment, the present disclosure relates to the mammalian cell orpopulation of cells, preferably hematopoietic stem cells for use in a medical treatment in a patient in need thereof wherein said medical treatment comprises: administering a therapeutically efficient amount of said cell or population of cells expressing said firstisoform of BCL-2 to said patient in need thereof, in combination with a therapeutically efficient amount of a depleting agent that binds specifically to said second isoform of BCL-2 to specifically deplete patient cells expressing said second isoform of BCL-2, preferably torestore normal hematopoiesis after targeted therapy in the treatment of hematopoieticdisease, and preferably in the treatment of malignant hematopoietic disease such as acutemyeloid leukemia (AML), myelodysplastic syndromes (MDS), chronic lymphocytic leukemia(CLL), mantle-cell lymphoma, multiple myeloma (MM), diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), marginal zone B-cell lymphoma, non-Hodgkin's lymphoma (NHL), T-cell prolymphocytic leukemia, Waldenstrom's macroglobulinaemia, chronic myelomonocytic leukemia (CML), precursor cell lymphoblastic leukaemia- lymphoma, B-cell lymphoma, blastic plasmacytoid dendritic cell neoplasm (BPDCN), or solidtumor malignancies such as non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC)prostate cancer, ovarian cancer, triple negative breast cancer (TNBC), colorectal cancer,head and neck cancer or breast cancer. In other embodiments the medical treatmentrelates to the restoration of the hematopoietic or the immune function in genetic diseasesof the hematopoietic or immune system, such as severe combined immunodeficiencysyndrome (SCID), sickle cell disease (SCD), beta-thalassemia, Fanconi anemia or Diamond-Blackfan anemia. In other embodiments the medical treatment relates to the restoration of the normalfunction in genetic diseases that are not originating in the hematopoietic and immune system but that can be treated by use of modified hematopoietic cells. In other embodiments the medical treatment relates to the restoration of the normalimmune function in autoimmune diseases, such as systemic lupus erythematosus (SLE),systemic sclerosis (SSc) or multiple sclerosis (MS).In another particular embodiment, the present disclosure relates to the mammalian cellor population of cells for use in a medical treatment in a patient in need thereof, whereinsaid medical treatment comprises: administering a therapeutically efficient amount of said cell or population of cells expressing said first isoform to said patient in need thereof incombination with a therapeutically efficient amount of a depleting agent that bindsspecifically to said first isoform to specifically deplete transferred cells expressing first isoform, preferably for use in adoptive cell transfer therapy, more preferably for the treatment of malignant hematopoietic disease such as acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), chronic lymphocytic leukemia (CLL), mantle-cell lymphoma, multiple myeloma (MM), diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), marginal zone B-cell lymphoma, non-Hodgkin's lymphoma (NHL), T-cell prolymphocytic leukemia, Waldenstrom's macroglobulinaemia, chronic myelomonocytic leukemia (CML), precursor cell lymphoblastic leukaemia-lymphoma, B-cell lymphoma,blastic plasmacytoid dendritic cell neoplasm (BPDCN) and other proliferative neoplasms orsolid tumor malignancies such as non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC) prostate cancer, ovarian cancer, triple negative breast cancer (TNBC), colorectal cancer, head and neck cancer or breast cancer again more preferably wherein said depleting agent is administered subsequently to said cell or population of cells expressing said first isoform of protein to avoid eventual severe side effects such as graft-versus-hostdisease due to the transplantation. In another aspect, the present disclosure relates to apharmaceutical composition comprising a mammalian cell, preferably a hematopoieticstem cell, a hematopoietic progenitor cell or an immune cell such as a lymphoid cell or a T-cell as described above and preferably a depleting agent and a pharmaceutically acceptablecarrier. The present disclosure also relates to a depleting agent for use in preventing or reducingthe risk of severe side effects in a patient having received a cell expressing a first isoform of BCL-2, wherein said patient’s native cells express a second isoform of BCL-2, and whereinsaid depleting agent binds specifically to said first isoform of BCL-2 and does not bind tosaid second isoform of BCL-2. In certain embodiments said depleting agents bindssubstantially weaker to said second isoform of BCL-2. The present disclosure also relates to a depleting agent for use in preventing or reducing the risk of severe side effects in a patient having received a cell expressing a first isoform of BCL-2, wherein said patient’s native cells express a second isoform of BCL-2, and whereinsaid depleting agent binds specifically to said first isoform of BCL-2 and does not bind tosaid second isoform of BCL-2, and wherein said first and second isoform are substantiallyfunctionally identical. In certain embodiments said depleting agent binds substantiallyweaker to said second isoform of BCL-2. The present disclosure also relates to a depleting agent for use in preventing or reducing the risk of severe side effects in a patient having received a cell expressing a first isoform of BCL-2, wherein said patient’s native cells express a second isoform of BCL-2, and whereinsaid depleting agent binds specifically to said first isoform of BCL-2 and does not bind tosaid second isoform of BCL-2, and wherein said first and second isoform are expressed at substantially the same level. In certain embodiments said depleting agent binds substantially weaker to said second isoform of BCL-2. The present disclosure also relates to a depleting agent for use in preventing or reducing the risk of severe side effects in a patient having received a cell expressing a first isoform of BCL-2, wherein said patient’s native cells express a second isoform of BCL-2, and whereinsaid depleting agent binds specifically to said first isoform of BCL-2 and does not bind tosaid second isoform of BCL-2, and wherein said first and second isoform bind to BAX.The present disclosure also relates to a depleting agent for use in preventing or reducing the risk of severe side effects in a patient having received a cell expressing a first isoform of BCL-2, wherein said patient’s native cells express a second isoform of BCL-2, and wherein said depleting agent binds specifically to said first isoform of BCL-2 and does not bind to said second isoform of BCL-2, and wherein said first and second isoform bind to BAK. The present disclosure also relates to a depleting agent for use in preventing or reducing the risk of severe side effects in a patient having received a cell expressing a first isoform of BCL-2, wherein said patient’s native cells express a second isoform of BCL-2, and whereinsaid depleting agent binds specifically to said first isoform of BCL-2 and does not bind tosaid second isoform of BCL-2, and wherein said first and second isoform are anti-apoptotic. The present disclosure also relates to a depleting agent for use in preventing or reducing the risk of severe side effects in a patient having received a cell expressing a first isoform of BCL-2, wherein said patient’s native cells express a second isoform of BCL-2, and wherein said depleting agent binds specifically to said first isoform of BCL-2 and does not bind to said second isoform of BCL-2, and wherein said first and second isoform inhibit BID, BIMand / or related activator BH3-only proteins.The present disclosure also relates to a depleting agent for use in preventing or reducing the risk of severe side effects in a patient having received a cell expressing a first isoform of BCL-2, wherein said patient’s native cells express a second isoform of BCL-2, and whereinsaid depleting agent binds specifically to said first isoform of BCL-2 and does not bind tosaid second isoform of BCL-2, and wherein said first and second isoform lead to the normal differentiation of hematopoietic cells. The present disclosure also relates to a depleting agent for use in preventing or reducing the risk of severe side effects in a patient having received a cell expressing a first isoform of BCL-2, wherein said patient’s native cells express a second isoform of BCL-2, and whereinsaid depleting agent binds specifically to said first isoform of BCL-2 and does not bind tosaid second isoform of BCL-2, wherein said polymorphic or genetically engineered allele ischaracterized by at least one substitution of an amino acid in position G101, D103 and / orF104 of SEQ ID NO: 1, preferably an amino acid at position F104 of SEQ ID NO: 1. In certainembodiments said depleting agents bind substantially weaker to said second isoform ofBCL-2. The present disclosure also relates to a depleting agent for use in preventing or reducing the risk of severe side effects in a patient having received a cell expressing a first isoformof BCL-2, wherein said patient’s native cells express a second isoform of BCL-2, and whereinsaid depleting agent binds specifically to said first isoform of BCL-2 and does not bind to said second isoform of BCL-2, wherein said polymorphic or genetically engineered allele ischaracterized by a substitution of the amino acid at position G101, wherein residue G101is substituted with I, L or V. The present disclosure also relates to a depleting agent for use in preventing or reducing the risk of severe side effects in a patient having received a cell expressing a first isoform of BCL-2, wherein said patient’s native cells express a second isoform of BCL-2, and wherein said depleting agent binds specifically to said first isoform of BCL-2 and does not bind to said second isoform of BCL-2, wherein said polymorphic or genetically engineered allele ischaracterized by a substitution of the amino acid at position D103, wherein residue D103is substituted with I, F or Y. The present disclosure also relates to a depleting agent for use in preventing or reducing the risk of severe side effects in a patient having received a cell expressing a first isoform of BCL-2, wherein said patient’s native cells express a second isoform of BCL-2, and wherein said depleting agent binds specifically to said first isoform of BCL-2 and does not bind to said second isoform of BCL-2, wherein said polymorphic or genetically engineered allele is characterized by a substitution of the amino acid at position F104, wherein residue F104 is substituted with A, C, G, I, L, T, S or V. The present disclosure also relates to a depleting agent for use in preventing or reducing the risk of severe side effects in a patient having received a cell expressing a first isoform of BCL-2, wherein said patient’s native cells express a second isoform of BCL-2, and wherein said depleting agent binds specifically to said first isoform of BCL-2 and does not bind to said second isoform of BCL-2, wherein said polymorphic or genetically engineered allele is characterized by a substitution of the amino acid at position F104, wherein residue F104 is substituted with A, V, C or T. The present disclosure also relates to a depleting agent for use in preventing or reducing the risk of severe side effects in a patient having received a cell expressing a first isoform of BCL-2, wherein said patient’s native cells express a second isoform of BCL-2, and wherein said depleting agent binds specifically to said first isoform of BCL-2 and does not bind to said second isoform of BCL-2, wherein said polymorphic or genetically engineered allele is characterized by a substitution of the amino acid at position F104, wherein residue F104 issubstituted with A or V.In certain embodiments, said first isoform of BCL-2 is obtained by in vivo or ex vivomodifying the nucleic acid sequence encoding said first isoform of BCL-2 by gene editing, preferably by introducing into a cell a gene editing enzyme capable of inducing site-specific mutations(s) within a target sequence encoding a protein region involved in the binding of agent comprising at least a first antigen-binding region. In certain embodiments, said depeleting agent is a BCL-2 inhibitor. Preferred BCL-2inhibitors include venetoclax (ABT-199), navitoclax (ABT-263), AZD-4320, AZD-0466,lisaftoclax (APG2575), S55746, sonrotoclax, ABT- 731, ABT-737, apogossypol, Ascenta’span-BCL-2 inhibitors, curcumin (and analogs thereof), G3139), HA14-1, NH-1, obatoclax,and any derivative or analog thereof. Most preferred is venetoclax.In alternative embodiments, said depleting agent is an agent comprising an antigen-binding region that binds specifically to said second isoform of BCL-2. In certain embodiments, the present disclosure relates to a pharmaceutical compositioncomprising a mammalian cell, preferably a hematopoietic stem cell or an immune cell such as T-cell, as described herein, and preferably a depleting agent as also defined herein, and a pharmaceutically acceptable carrier. In certain embodiments, the present disclosure relates to a depleting agent for use inpreventing or reducing the risk of severe side effects in a patient having received a cell expressing a first isoform of BCL-2, wherein said patient’s native cells express a second isoform of BCL-2, and wherein said depleting agent binds specifically to said first isoform of BCL-2 and does not bind or binds substantially weaker to said second isoform of BCL-2. In certain embodiments, the present disclosure relates to a BCL-2 inhibitor for use inselectively depleting the host cells in a patient in need thereof, wherein said patient’snative cells express a second isoform of BCL-2, wherein a mammalian cell or a populationof cells expressing a first isoform of BCL-2 is administered to said patient prior to, concomitant with or after said BCL-2 inhibitor, and wherein said BCL-2 inhibitor specifically binds to said second isoform of BCL-2, but not (or substantially weaker), to said first isoformof BCL-2. Preferably, said BCL-2 inhibitor binds to a polypeptide comprising the amino acidsequence of SEQ ID No.1, but not (or substantially weaker), to a polypeptide comprisingthe amino acid sequence of SEQ ID No. 1, said polypeptide comprises at least onesubstitution of an amino acid at position G101, D103 or F104. FIGURE LEGENDS CellTiter Glo Luminescent assay to analyze cell viability of Kasumi-1, MV4-11,MOLM-14, THP-1, MOLM-13 and OCI-AML2 cells treated with increasing concentrations of Venetoclax, Navitoclax or AZD4320 (from top to bottom) for 72h in culture. This method determines the number of viable cells in culture based on the quantitation of the ATP present, an indicator of metabolically active cells. Luminescence signal was detected in EnVision multimode plate reader and normalized to control DMSO-treated cells (100%). CellTiter Glo Luminescent assay to analyze cell viability of HCT-116, 14,Kasumi-1 and THP-1 cells treated with increasing concentrations of Venetoclax, Navitoclax, AZD4320, Lisaftoclax or S55746 for 72h in culture. This method determines the number of viable cells in culture based on the quantitation of the ATP present, an indicator ofmetabolically active cells. Luminescence signal was detected in EnVision multimode platereader and normalized to control DMSO-treated cells (100%). Enrichment of Bcl-2 variants by Venetoclax treatment. Using CRISPR / Cas9 HDR-based gene editing and the HDR-enhancer AZD6748 (0.4 uM), Kasumi-1 cells were engineered for the shown Bcl-2 variants. A) Knock in frequency by Sanger sequencing before and after Venetoclax (VX) treatment (1 μM Venetoclax for 72h). B) Relativeexpansion of Bcl-2 variants compared to Bcl-2 WT, EP control and Bcl-2 knockout (KO) bycell-titer Glo after Venetoclax treatment (10 μM Venetoclax for 72h), shielding variants and BCL2 KO were pretreated with 10nM Venetoclax for 72h. DETAILED DESCRIPTION OF THE INVENTION Targeted therapy is a promising therapy to treat cancer, genetic and autoimmunediseases. Immunodepleting agents such as small molecules, antibodies or engineeredimmune cells directed to tumor antigen are administered into a patient to target and killtumor cells. However, as tumor proteins are also expressed in normal cells includinghematopoietic cells, this strategy can induce severe side effects to the patients, e.g., byaltering hematopoiesis. To restore hematopoiesis in the patient, hematopoietic cells canbe subsequently transplanted into the patient. However, the uptake and activity of thedepleting agent not only on the diseased cells but also to the newly transplanted healthycells can limit the maximal tolerated dose or limit the use to treatment before transplantation of healthy cells. Alternatively, transplanted cells need to be resistant to saidimmunodepleting agent in order not to be targeted and eliminated by it. One approach istherefore to select cells resistant to said depleting agent used in targeted therapy whileretaining their function to restore normal hematopoiesis in the patient.The inventors developed a method to identify functional allelic variants in the geneticsequence encoding the protein region responsible for the binding and inhibition of aspecific depleting agent. Such variants can be naturally occurring polymorphisms, acquiredresistance mutations (e.g., in cancer patients upon treatment) and / or designed andengineered variants. Different isoforms of proteins can be selected or generated. Said firstisoform of a protein encoded by a nucleic acid with said polymorphism is not recognizedby a specific depleting agent. This variant allele particularly does not alter or does notsubstantially alter the function of the protein and / or is expressed at the same orsubstantially the same level. Thus, said depleting agent can be used to bind specifically tothe one isoform and not, or not substantially, the other isoform thereby depletingspecifically cells expressing one isoform. For example, if the depleting agent bindsspecifically to the second isoform, but not the first isoform, said depleting agent willspecifically deplete cells expressing said second isoform. In another embodiment, said firstisoform can be recognized by a second agent and thus this second agent can be used todeplete specifically cells expressing the first isoform, but not second isoform. The cellsexpressing the first isoform of the protein encoded by at least one variant allele isadvantageously used in medical treatment in a patient having cells expressing a secondisoform, in particular for depleting specifically transplanted or patient cells by using asecond or first agent respectively. It is impossible to predict, which mutation in a protein can be used in such an approach.First, the mutations need to lie within the binding groove of the protein that is accessiblefor the depleting agent. Second, the depleting agent needs to bind to this stretch or in thisgroove of the protein. Third, binding needs to be affected sufficiently enough so that thedepleting agent can discriminate the first isoform from the second isoform. Residual binding to the other isoform should be minimal or, better, be completely absent. Fourth,the mutation should not affect, or only marginally affect, the function of the protein. Themutated isoform should fulfill its biological function at least to an extent that is tolerablein a given therapeutic setting. Even in cases where (co-) crystal structures exist, only experimental testing can prove the usefulness of any given mutation. Depleting agent, binding moieties and small molecules The present disclosure relates to a binding moiety which binds specifically to one isoformof BCL-2 on a cell and does not bind or binds substantially weaker to another isoform of BCL-2. “Specific binding”, “specifically bind to” and “binds specifically to” includes binding witha monovalent affinity of about 10-5M (KD) or stronger. Preferably, binding is considered specific when the binding affinity is between 10-5M (KD) and 10-10M (KD), optionally between 10-7M (KD) and 10-10M (KD), in particular at least 10-10M (KD). The affinity can be determined by various methods well known from the one skilled in the art. These methods include, but are not limited to, surface plasmon resonance (SPR), biolayer interferometry (BLI), microscale thermophoresis (MST), fluorescence resonance energytransfer (FRET) or fluorescence polarization (FP) and Scatchard plot. Whether a bindingdomain specifically reacts with or binds to a target can be tested readily by, inter alia, comparing the reaction of said binding domain with a target protein or antigen with the reaction of said binding domain with proteins or antigens other than the target protein. As used herein, the term "drug-binding site" means the part of a polypeptide or proteinto which a therapeutically active molecule, such as an antibody, a binding moitety, a smallmolecule or a BCL-2 inhibitor, binds. For antibodies and related molecules this part on thetarget protein is often referred to as “epitope”. Drug-binding sites and epitopes can be divided into two categories, pockets or grooves, depending on the shape of the drug- binding site. The term "binding moiety" as used herein refers to any moiety that binds to a targetmolecule, preferably a proteinaceous molecule, such as a polypeptide or protein. In certainembodiments, the binding moiety binds to the active site of the target molecule. In itsbroadest sense, the binding moiety may be any commonly known molecules, including asmall (organic) molecule. The binding moiety may also be a polypeptide or a protein. Suchbinding moieties typically contain an antigen binding region and consist of one or morepolypeptide chains. Preferred binding moieties comprising an antigen binding region areantibodies, antibody fragments, antibody drug conjugates, or another variant of an antibody or scaffold. Exemplary antibody fragments and scaffolds include single domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR, igNAR, bis-scFv, camelid antibodies, ankyrins, centyrins, domain antibodies, lipocalins,small modular immuno- pharmaceuticals, maxybodies, Protein A and affilins. Particularpreferred binding moieties are small molecules” The terms "small molecule", “small organic molecule” or “SMOL” are usedinterchangeably herein and refer to organic compounds, either synthesized in thelaboratory or found in nature, having a molecular weight of less than 10,000 Daltons,optionally less than 5,000 Daltons, and optionally less than 1,500 Daltons. Preferably, a small molecule has a molecular weight less than 1,000 Daltons. Binding moieties that are use in the context of the present disclosure that specifically toone isoform of BCL-2 on a cell and that do not bind, or bind substantially weaker, to anotherisoform of BCL-2 may be used to deplete cells expressing the first or the second isoform ofthe BCL-2. In the context of the depletion of the first or the second isoform, the bindingmoieties are are also referred to as “depleting agent”.Preferred binding moieties of the present disclosure are small molecules that bind to thetarget protein investigated herein, i.e., BCL-2. Such molecules are referred to herein as“BCL-2 inhibitors”. Several small molecule BCL-2 inhibitors are known in the art, some of which are already approved drugs, while others are currently in clinical development. BCL- 2 is the target of venetoclax (ABT-199), which was developed by AbbVie and Genentech and is approved for the treatment of acute myeloid leukemia (AML) and chronic lymphocytic leukemia (CLL). It is in clinical trials for multiple other cancer indications, both liquid and solid tumors. Navitoclax (ABT-263), a related molecule and dual inhibitor of Bcl- 2 and Bcl-XL is developed by AbbVie and also in clinical trials for various hematologicalmalignancies and solid tumors. Also analogs of navitoclax are known (see US7390799).AZD-4320 is also structurally related to venetoclax and navitoclax. It is a dual inhibitor ofBcl2 and Bcl-xL and was in phase 1 / 2 clinical evaluation as a dendrimer nanoparticleformulation, AZD-0466, in hematological malignancies as well as on-Hodgkin's lymphoma(NHL). Lisaftoclax (APG2575), which targets Bcl-2 and Bcl-xl, is developed by AscentagePharma. It is in phase 2 clinical evaluation for CLL and other cancer indications. S55746 is studied in phase 1 clinical trials by Servier, Vernalis and Novartis for the treatment of CLL, NHL and multiple myeloma. It has high affinity for Bcl-2 and poor affinity for Bcl-xl. Sonrotoclax developed bei BeiGene binds Bcl-2 and initiates phase 3 clinical trials for thetreatment of CLL and received orphan drug status for multiple myeloma. Other BCL-2inhibitors include ABT- 731, ABT-737, apogossypol, Ascenta’s pan-BCL-2 inhibitors,curcumin (and analogs thereof), dual BCL- 2 / BCL-XL inhibitors (InfinityPharmaceuticals / Novartis Pharmaceuticals), Genasense (G3139), HA14-1 (and analogs thereof; see WO2008118802), NH-1 (Shenayng Pharmaceutical University), obatoclax (andanalogs thereof, see WO2004106328), S-001 (Gloria Pharmaceuticals) and TW seriescompounds (Univ. of Michigan). Particular preferred BLC-2 inhibitors include those in Table1. Table 1: Compund CASStructure numberVenetoclax 1257044-40-8Navitoclax 923564-51-6AZD-4320 1357576-48-7Lisaftoclax 2180923-05-9

[0002] S55746 1448584-12-0 Sonrotoclax 2383086-06-2 BCL-2 BCL-2 (UniProt: P10415; also known as apoptosis regulator Bcl-2), is a single-passmembrane protein expressed in the outer mitochondrial membrane in most cells of thehematopoietic system in the bone marrow, thymus, and spleen but it is also found inendothelium and neurons. More specifically, BCL-2 is expressed in a variety of normalhematopoietic lineages such as B- and T-cells as well as in hematopoietic stem andprogenitor cells (HSPC). The overall structure of BCL-2 consists of two central, predominately hydrophobic helices (BH1-4) packed against four amphipathic α-helices (BH5 and 6), followed by the transmembrane domain. Human BCL-2 has the following amino acid sequence: MAHAGRTGYDNREIVMKYIHYKLSQRGYEWDAGDVGAAPPGAAPAPGIFSSQPGHTPHPAA SRDPVARTSPLQTPAAPGAAAGPALSPVPPVVHLTLRQAGDDFSRRYRRDFAEMSSQLHLT PFTARGRFATVVEELFRDGVNWGRIVAFFEFGGVMCVESVNREMSPLVDNIALWMTEYLNRHLHTWIQDNGGWDAFVELYGPSMRPLFDFSWLSLKTLLSLALVGACITLGAYLGHK (SEQID No.1) In a particular embodiment, said protein is BCL-2. In other embodiments said protein isBCL-2 comprising the amino acid sequence of SEQ ID No. 1. In other embodiments saidprotein is BCL-2 consisting of the amino acid sequence of SEQ ID No. 1.In certain embodiments the present disclosure relates to a mammalian cell or apopulation of cells expressing a first isoform of the protein BCL-2 for use in a medicaltreatment in a patient in need thereof, said patient having cells expressing a second isoform of said protein, wherein said cell expressing said first isoform comprises genomic DNA with at least one polymorphism or genetically engineered allele, wherein said polymorphism or genetically engineered allele is not present in the genome of the patient having cells expressing said second isoform of said protein and preferably wherein said first and second isoform are functional. In certain embodiments the present disclosure relates to a mammalian cell or apopulation of cells expressing a first isoform of the protein BCL-2 for use in a medicaltreatment in a patient in need thereof, said patient having cells expressing a second isoform of said protein, wherein said cell expressing said first isoform comprises genomic DNA with at least one polymorphism or genetically engineered allele, wherein said polymorphism or genetically engineered allele is not present in the genome of the patient having cells expressing said second isoform of said protein, wherein said first and second isoform are functional. In certain embodiments the present disclosure relates to a mammalian cell or apopulation of cells expressing a first isoform of the protein BCL-2 for use in a medicaltreatment in a patient in need thereof, said patient having cells expressing a second isoform of said protein, wherein said cell expressing said first isoform comprises genomic DNA with at least one polymorphism or genetically engineered allele, wherein said polymorphism or genetically engineered allele is not present in the genome of the patient having cells expressing said second isoform of said protein, wherein said first and second isoform are substantially functionally identical. Knowledge about the exact function of BCL-2 is still limited, although certain activities ofBCL-2 have been described. In certain embodiments the present disclosure related to a firstand a second isoform of BCL-2 wherein both isoforms are functional. In certainembodiments the present disclosure related to a first and a second isoform of BCL-2wherein both isoforms are functionally indistinguishable. In the present invention"functionally indistinguishable" refers to a first and a second isoform of BCL-2 that areequally capable of performing the same function within a cell without significant impairment. In other words, the first and the second isoform are functionally largelyindistinguishable. A slight functional impairment may be acceptable. In a preferredembodiment, said first isoform of BCL-2 remains functional and retains the capacity ofperforming the same function as the corresponding wildtype isoform within a cell withoutsignificant impairment. In certain embodiments, both isoforms of BCL-2 have that samefunction. In certain embodiments, both isoforms are functionally indistinguishable. Incertain embodiments, both isoforms are expressed at the same level or substantially thesame level. One function of BCL-2 is the induction of apoptosis. Therefore, in certain embodimentsthe present disclosure relates to a mammalian cell or a population of cells expressing a firstisoform of the protein BCL-2 for use in a medical treatment in a patient in need thereof,said patient having cells expressing a second isoform of said protein, wherein said cell expressing said first isoform comprises genomic DNA with at least one polymorphism or genetically engineered allele, wherein said polymorphism or genetically engineered allele is not present in the genome of the patient having cells expressing said second isoform of said protein and wherein said first and said second isoform induce apoptosis. One function of BCL-2 is the binding of so called BH3-only proteins, such as Bim and Bad.Therefore, in certain embodiments the present disclosure relates to a mammalian cell or a population of cells expressing a first isoform of the protein BCL-2 for use in a medical treatment in a patient in need thereof, said patient having cells expressing a second isoform of said protein, wherein said cell expressing said first isoform comprises genomic DNA with at least one polymorphism or genetically engineered allele, wherein said polymorphism or genetically engineered allele is not present in the genome of the patient having cells expressing said second isoform of said protein and wherein said first and said secondisoform binds BH3-only proteins, such as Bim and Bad.One function of BCL-2 is the binding of pro-apoptotic proteins, such as Bax. Therefore, in certain embodiments the present disclosure relates to a mammalian cell or a population of cells expressing a first isoform of the protein BCL-2 for use in a medical treatment in apatient in need thereof, said patient having cells expressing a second isoform of saidprotein, wherein said cell expressing said first isoform comprises genomic DNA with at least one polymorphism or genetically engineered allele, wherein said polymorphism or genetically engineered allele is not present in the genome of the patient having cells expressing said second isoform of said protein and wherein said first and said secondisoform bind pro-apoptotic proteins, such as Bax.In certain embodiments the present disclosure relates to a mammalian cell or apopulation of cells expressing a first isoform of the protein BCL-2 for use in a medicaltreatment in a patient in need thereof, said patient having cells expressing a second isoform of said protein, wherein said cell expressing said first isoform comprises genomic DNA with at least one polymorphism or genetically engineered allele, wherein said polymorphism or genetically engineered allele is not present in the genome of the patient having cells expressing said second isoform of said protein and wherein said first and said second isoform are expressed substantially to the same degree. In certain embodiments, said first and said second isoform are expressed to the same degree. In line with the present disclosure, it is also possible to combine additional variants orisoforms of BCL-2 within the methods and compositions of the present disclosure. Suchisoforms may for example include double mutants. Such isoforms may for example alsoinclude single and double mutants. The methods and compositions of the present disclosure may also be used in the depletion of myeloid cells in solid tumors in order to enhance tumor responses. The methods and compositions of the present disclosure may also be combined with cellscombinations, in particular, when said protein is BCL-2 with knock out of other targets, suchas CD117, CD123, CD33, DLL-1, CD45, CD47, CD7, CLEC12A, CD44, FLT3, CD300LF, EVI2B,TPO and combinations thereof. The methods and compositions of the present disclosure may also comprise cellsexpressing first isoform of BCL-2 and other protein variants such as CD117 variants, CD123variants, CD33 variants, DLL-1 variants, CD45 variants, CD47 variants, CD7 variants,CLEC12A (CD371) variants, CD44 variants, FLT3 (CD135) variants, CD300LF variants, EVI2Bvariants, TPO variants, CD52 variants and any combination thereof.The first isoform and the second isoform of BCL-2 may be polymorphic alleles. In certain embodiments, the first isoform and the second isoform of BCL-2 are naturally occurring polymorphic alleles. In alternative embodiments, the first isoform and the second isoform of BCL-2 are single nucleotide polymorphism (SNP) alleles. The first isoform and the second isoform of BCL-2 may be naturally occurring variants.Preferably, the first isoform and the second isoform of BCL-2 are naturally occurringpolymorphic alleles. Also preferably, the first isoform and the second isoform of BCL-2 are SNP’s (single nucleotide polymorphisms). The first isoform and the second isoform of BCL-2 may also be genetically engineered alleles. Preferably the first isoform and the second isoform of BCL-2 differ by one, two, three, four or five amino acids. Most preferably the first isoform and the second isoform of BCL-2 differ by one amino acid. Various methods can be used to determine the mutation that is to be introduced into BCL-2 to generate the second isoform. For example, mutations can be randomly inserted, followed by the functional and binding screening of the variants generated. Alternatively, mutations can be rationally designed, for example by analysis of the secondary or tertiary protein structure of BCL-2. The depleting agent comprises a chemical structure, which binds specifically to one isoform of BCL-2 on a cell and does not bind or binds substantially weaker to another isoform. In certain embodiments, the present disclosure relates to a binding agent which bindsspecifically to a second isoform of BCL-2 and does not bind a first isoform of BCL-2. In otherembodiments, the present disclosure also relates to a binding agent which bindsspecifically to the first isoform of BCL-2 and does not bind a second isoform of BCL-2. Incertain embodiments said binding agent binds substantially weaker to said second isoform of BCL-2. In certain embodiments, the present disclosure relates to a small molecule which bindsspecifically to a second isoform of BCL-2 and does not bind a first isoform of BCL-2. In otherembodiments, the present disclosure also relates to a small molecule which bindsspecifically to the first isoform of BCL-2 and does not bind a second isoform of BCL-2. Incertain embodiments said small molecule binds substantially weaker to said secondisoform of BCL-2. In certain embodiments, the present disclosure relates to a BCL-2 inhibitor which bindsspecifically to a second isoform of BCL-2 and does not bind a first isoform of BCL-2. In otherembodiments, the present disclosure also relates to a BCL-2 inhibitor which bindsspecifically to the first isoform of BCL-2 and does not bind a second isoform of BCL-2. Incertain embodiments, said BCL-2 inhibitor binds substantially weaker to said secondisoform of BCL-2. In certain embodiments, the present disclosure relates to an agent comprising a first antigen binding region which binds specifically to a second isoform of BCL-2 and does notbind a first isoform of BCL-2. In other embodiments, the present disclosure also relates toan agent comprising a second antigen binding region which binds specifically to the firstisoform of BCL-2 and does not bind a second isoform of BCL-2. In certain embodiments saidagent binds substantially weaker to said second isoform of BCL-2. In certain embodiments, the present disclosure relates to an antibody which bindsspecifically to a second isoform of BCL-2 and does not bind a first isoform of BCL-2. In otherembodiments, the present disclosure also relates to an antibody which binds specifically tothe first isoform of BCL-2 and does not bind a second isoform of BCL-2. In certainembodiments said antibody binds substantially weaker to said second isoform of BCL-2.In certain embodiments, the present disclosure relates to an antibody fragment, antibodydrug conjugate, or another variant of an antibody or scaffold which binds specifically to asecond isoform of BCL-2 and does not bind a first isoform of BCL-2. In other embodiments,the present disclosure also relates to an antibody fragment, antibody drug conjugate, oranother variant of an antibody or scaffold which binds specifically to the first isoform ofBCL-2 and does not bind a second isoform of BCL-2. In certain embodiments said anantibody fragment, antibody drug conjugate, or another variant of an antibody or scaffold binds substantially weaker to said second isoform of BCL-2. The first and the second isoform of BCL-2 may differ from each other by only one amino acid substitution. Said one amino acid difference between the first and the second isoform may also be the result of the presence of a single nucleotide polymorphism, such as a naturally occurring single nucleotide polymorphism. The first and the second isoform of BCL-2 may also differ from each other by more than one amino acid, such as by two, by three or by more than three amino acids. The first and the second isoform of BCL-2 may also differ from each other in that one of the isoforms has an insertion of one, of two, of three or of more than three amino acids compared to the other isoform. The first and the second isoform of BCL-2 may also differ from each other in that one of the isoforms has a deletion of one, of two, of three or of more than three amino acids compared to the other isoform. The two isoforms may also differ from each other by combinations of amino acid substitutions, insertions and / or deletions. In a preferred embodiment, said depleting agent is a small molecule. In another preferred embodiment, said depleting agent is a BCL-2 inhibitor. If the two isoforms of BCL-2 differ by more than one amino acid, then the amino acids changed may be adjacent to each other, i.e., direct neighboring amino acids, or they may be separated. As used herein, an agent which does not bind or binds substantially weaker to a particular isoform of BCL-2 includes an agent which is not able to bind to cells expressing saidparticular isoform. For experimental testing, said agent may be used to compete with aligand labelled with a fluorescent marker and the binding is measured indirectly by displacing the labeled ligand using fluorescence resonance energy transfer (FRET) orfluorescence polarization (FP). Alternatively, ligand binding may be directly detected usingtechnologies such as surface plasmon resonance (SPR) or microscale thermophoresis(MST). Ligand binding may also be assessed by measuring changes in the secondary structure or the stability of BCL-2 upon binding, by using, e.g., circular dichroism spectroscopy (CD) or thermal shift assays. Typically testing is done in cell lines expressing the recombinant target protein, i.e. BCL-2. The target protein may be expressed in its entirety. Alternatively, a truncated version may be used, wherein said truncated version ata minimum, needs to include the ligand binding domain or the regions containing the drug-binding site. To monitor the expression of the variant isoforms, Western Blot or quantitative immunoprecipitation can be performed, using antibodies which bind the different protein isoforms equally well, i.e., where the epitope is not affected by the introduction of the variants. As a non-binding control, cells are used that do not express the protein of interest. As a maximum binding control, cells that normally do not express the protein of interest are transfected with the wildtype isoform. Different cell lines have different expression levels, but the expression is controlled through endogenous control elements such as promoters. Such cell lines can also be used to study the mode-of-action of a depleting agent, the effective shielding against a different mode-of-action, to test cytotoxicity and shielding / resistance from cytotoxicity or to test the function of the engineered receptors. Western Blot, ELISA or FACS can be used to analyze phosphorylation of signaling molecules. Analysis of gene expression changes can serve to analyze gene expression compared to normal function. Cells can also be used to demonstrate the feasibility of editing a specific variant via different approaches, e.g., homology directed repair (HDR), base editing or prime editing. Binding of said agent can result in depletion of the cell expressing the first isoform of BCL- 2. Various mechanisms can lead to cell depletion. Small molecule inhibitors can induce cell depletion via induction of apoptosis, necroptosis and / or ferroptosis, inhibition of pro- survival signals and pathways, blockade of pro-proliferative proteins, targeting of proteins for degradation, or inhibition of immune-surveillance pathways. Proliferation assays constitute an industry standard to quantitate an agent’s potency to induce cell death as described in the experimental part. BCL-2 expressing cell lines are incubated in the presence of different concentrations of small-molecule inhibitors and viable cells are measured by, e.g., quantitation of ATP as an indicator of metabolically active cells using reagents like Cell-Titer Glo (Promega). Alternatively, the effect of BCL-2 small- molecule inhibitors on caspase activity or annexin V amounts can be measured. The depleting agent according to the present disclosure binds specifically to one isoformof BCL-2, thereby allowing the depletion of cells expressing said isoform.More preferably, in specific embodiments, said depleting agent according to the present disclosure does not bind or binds substantially weaker to a first isoform of BCL-2 but binds specifically to a second isoform of BCL-2 and allows the depletion of said cells expressing said second isoform of BCL-2, in particular in methods of use as disclosed herein. In particular, said depleting agent which does not bind or binds substantially weaker to a first isoform of BCL-2 but binds specifically to a second isoform of BCL-2 expressed in patient’s cell is used to deplete patient’s cells but not hematopoietic stem cells or their progeny expressing said first isoform of BCL-2 transplanted to restore hematopoiesis in said patient. In another specific embodiments, said depleting agent according to the present disclosure does not bind or binds substantially weaker to a second isoform of BCL-2 but binds specifically to a first isoform of BCL-2 and allows the depletion of cells expressing said first isoform of BCL-2, in particular in methods of use as disclosed herein. In particular, said depleting agent which does not bind or binds substantially weaker to a second isoform of BCL-2 but binds specifically to a first isoform of BCL-2 expressed in transplanted cells is used to deplete specifically transplanted cells to avoid eventual severe side effects such as graft- versus-host disease due to transplantation. Methods by which immune cells can be genetically modified to express a recombinant protein variant are well known in the art. A nucleic acid molecule encoding the antigenreceptor may be introduced into the cell in the form of, e.g., a vector, or any other suitablenucleic acid construct, or by inserting the nucleic acid molecule into the genome using genome editing technologies. Vectors, and their required components, are well known in the art. Nucleic acid molecules encoding antigen binding region can be generated using any method known in the art, e.g., molecular cloning using PCR. Antigen binding region sequences can be modified using commonly used methods, such as site-directed mutagenesis. Polymorphism of BCL-2 The cell expressing the first isoform of BCL-2 according to the present disclosurecomprises genomic DNA with at least one polymorphic allele in the nucleic acid encodingsaid BCL-2 isoform. In particular, said polymorphism induces at least one mutation involvedin the binding of a specific agent in comparison to said second isoform. Said polymorphism is preferably within a nucleic acid sequence encoding the proteinregion of BCL-2 involved in binding of the first binding moiety, preferably located in thedrug-binding region of BCL-2. More particularly, said polymorphism is within a nucleic acidsequence encoding at least one specific amino acid residue involved in binding of the first binding moiety. Said polymorphism can be a mutation such as a deletion, a substitution, aninsertion, or a combination thereof of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15 or 20nucleotides. In a particular embodiment, said polymorphism is a single nucleotide polymorphism. The term “isoform” refers to a variant of a protein which differs from another variant ofthe same protein by at least one amino acid difference. In the context of the present disclosure such difference may be a substitution of a single amino acid, but such differences may also be double, triple or multiple amino acid substitutions, or insertions or deletions. Also naturally occurring SNPs are isoforms. The difference in the sequence of the two isoforms may also be genetically introduced.Also, here the sequence difference is preferably within a nucleic acid sequence encodingthe BCL-2 region involved in binding of the first agent, preferably located in the extracellularportion of said protein, in particular, in the drug-binding region. More particularly, saidsequence difference is within a nucleic acid sequence encoding at least one specific amino acid residue involved in binding of the first agent. Said sequence difference can be amutation such as a deletion, a substitution, an insertion or a combination thereof of at least1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15 or 20 nucleotides. In a particular embodiment, said sequence difference is a single point mutation. The present disclosure provides polymorphisms in BCL-2, including in particularpolymorphisms including substitution of the residues G101, D103 or F104. Particularpreferred polymorphisms are a substitution of the residue F104.Preferred polymorphisms include substitutions of the residue G101. In certain preferredembodiments, residue G101 of BCL-2 is substituted with V, I or L. In other preferredembodiments, residue G101 of BCL-2 is substituted with I.Other preferred polymorphisms include substitutions of the residue D103. In certainpreferred embodiments, residue D103 of BCL-2 is substituted with I, Y, F, K or L. In otherpreferred embodiments, residue D103 of BCL-2 is substituted with F, I or K. In yet otherpreferred embodiments, residue D103 of BCL-2 is substituted with I. Other preferred polymorphisms include substitutions of the residue F104. In certainpreferred embodiments, residue F104 of BCL-2 is substituted with V, C, T, A, G, I, L or S. Inother preferred embodiments, residue F104 of BCL-2 is substituted with V, C, T or A. Inother preferred embodiments, residue F104 of BCL-2 is substituted with V or A.It will be appreciated that amino acid may be designated by the 3-letter code or the 1- letter code, which both are familiar to the skilled person.Table 2 shows the 20 natural occurring amino acids:Amino acid Three letter code One letter codealanine Ala Aarginine Arg Rasparagine Asn Naspartic acid Asp Dcysteine Cys Cglutamic acid Glu Eglutamine Gln Qglycine Gly Ghistidine His Hisoleucine Ile Ileucine Leu Llysine Lys Kmethionine Met Mphenylalanine Phe Fproline Pro Pserine Ser Sthreonine Thr Ttryptophan Trp Wtyrosine Tyr Yvaline Val VIn the experiments of the present disclosure certain variants of specific residues were identified. For practical reasons it is impossible to test any and all possible variants. It will however be understood that an identified variant may be substituted with a similar aminoacid residue. For example, an acidic amino acid can be replaced by another acidic aminoacid, since it can be expected to have the same effect. Likewise, a charged amino acid canbe replaced by another charged amino acid. As an example, T264E is expected to beequivalent to T264D, since both, D and E are acidic amino acids. In a particular embodiment, said cell according to the present disclosure is selected from a subject comprising native genomic DNA with at least one natural polymorphism allele, preferably single nucleotide polymorphism (SNP) in the nucleic acid encoding said isoform. In a particular embodiment, cells are selected from a subject that comprises native genomic DNA with at least one natural polymorphism allele, in particular SNP, in a nucleicacid sequence encoding BCL-2 region involved in anti-BCL-2 agent binding, preferablylocated in the extracellular portion of said protein, more preferably in a solvent-exposed secondary structure element. Certain naturally occurring SNPs are described in the literature. These naturally SNPs may be used within the spirit of the present disclosure with a respective binding agent which isable to discriminate such SNP from another isoform of BCL-2. A list of natural occurringSNPs of BCL-2 can be found in any respective database, such as gnomAD(https: / / gnomad.broadinstitute.org / ), dbSNP (https: / / www.ncbi.nlm.nih.gov / snp / =) orGeneCards (https: / / www.genecards.org / ). Gene editing In another particular embodiment, said cell expressing the first isoform of BCL-2 accordingto the present disclosure is obtained by gene editing, preferably by changing the sequence encoding said protein in the patient’s native genomic DNA. The cell can be genetically engineered by introducing into the cell a gene editing system to induce said polymorphism resulting in insertion, deletion and / or substitution of amino acids of the protein. Said gene editing modality targets a nucleic acid sequence, named herein target sequence encoding protein region involved in first agent binding as described above. In particular, when said protein is BCL-2, said gene editing modality targets a nucleicacid encoding at least one amino acid residue in position G101, D103 and / or F104 of SEQID NO: 1. Preferably amino acid residue G101 is substituted with V, L or I, and / or residueD103 is substituted with I, Y, F, K or L, and / or residue F104 is substituted with A, C, G, L, I,S, T or V. Gene editing enzymes may be sequence-specific nucleases, base editors, primeeditors or CRISPR-transposon based systems. The term “nuclease” refers to a wild type or variant enzyme capable of catalyzing the hydrolysis (cleavage) of phosphodiester bonds between nucleotides of a nucleic acid (DNA or RNA) molecule, preferably a DNA molecule. By "cleavage" is intended a double-strand break or a single-strand break event. The term “sequence-specific nuclease” refers to a nuclease which cleaves nucleic acid in a sequence-specific manner. Different types of site-specific nucleases can be used, such as Meganucleases, TAL-nucleases (TALEN), Zing-finger nucleases (ZFN), or RNA / DNA guided endonucleases like Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) / Cas system and Argonaute (Review in Li et al., Nature Signal transduction and targeted Therapy, 5, 2020; Guha et al., Computational and Structural Biotechnology Journal, 2017, 15, 146-160). According to the present disclosure, the nuclease generates a DNA cleavage within a target sequence, said target sequence encodes a protein region involved in first agent binding as described above. In particular embodiments, the inventors use the CRISPR system to induce a cleavage within a target sequence encoding protein region recognized by first agent as described above. By “target sequence”, it is intended targeting a part of the sequence encoding the regionon BCL-2 involved in first agent binding as described as described above and / or sequencesadjacent to said region on BCL-2 involved in first agent binding, in particular at least one(one or two) sequence of up to 50 nucleotides adjacent to said region on BCL-2 involved infirst agent binding, preferably 20, 15, 10, 9, 8, 7, 6 or 5 nucleotides adjacent to said agent binding site. The CRISPR system involves two or more components, a Cas protein (CRISPR-associated protein) and a guide RNA. The guide RNA can be a single guide RNA or a dual guide RNA. The Cas protein is a DNA endonuclease that uses a guide RNA sequence as a guide to recognize and generate double-strand cleavage in DNA that is complementary to the target sequence. Cas systems that generate single strand breaks require only one nuclease domain. Cas systems that generate double strand breaks require two nuclease domains.Cas protein may comprise two active cutting sites, such as HNH nuclease domain and RuvC-like nuclease domain. By Cas protein is also meant an engineered endonuclease, homologue or orthologue ofCas 9 which is capable of cleaving target nucleic acid sequence. In particular embodiments, Cas protein may induce a cleavage in the nucleic acid target sequence which cancorrespond to either a double-stranded break or a single- stranded break. Cas proteinvariant may be a Cas endonuclease that does not naturally exist in nature and that is obtained by protein engineering or by random mutagenesis. The Cas protein can be one type of the Cas proteins known in the art. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), SaCas9, Cas12, Cas12a (Cpf1), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl , Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Cmrl , Cmr3, Cmr4, Cmr5, Cnrr6, Csbl , Csb2, Csb3, Csxl7, CsxM, Csx lO, Cs l6, CsaX, Csx3, Cs l, Csxl5, Csfl, Csf2, CsO, Csf4, homologs, orthologs thereof, or modified versions thereof. Preferably Cas protein is Streptococcus pyogenes Cas 9 protein. Cas is contacted with a guide RNA (gRNA) designed to comprise a complementary sequence to the target sequence to specifically induce DNA cleavage within said target sequence, in particular according to the present disclosure a complementary sequence of a part of the target sequence encoding the protein region recognized by agent as described above. As used herein, a “guide RNA”, “gRNA”, “sgRNA” or “single guide RNA” refers to a nucleicacid that promotes the specific targeting or homing of a gRNA / Cas complex to a target nucleic acid. In particular, gRNA refers to RNA that comprises a transactivating crRNA (tracrRNA) and a crRNA. Preferably, said guide RNA corresponds to a crRNA and tracrRNA which can beused separately or fused together to generate a single guide RNA. The complementary sequence pairing with the target sequence recruits Cas to bind and cleave the DNA at the target sequence. According to the present disclosure, crRNA is engineered to comprise a complementary sequence to a part of a target sequence as described above encoding protein region recognized by agent, such that it is capable of targeting said region. In a preferred embodiment sgRNA is used to target the binding site of the said binding agent. In another preferred embodiment, the guide RNA contains chemically modifications known to the person skilled in the art. In a particular embodiment, the crRNA comprises a sequence of 5 to 50 nucleotides, preferably 15 to 30 nucleotides, more preferably 20 nucleotides which is complementary to the target sequence. As used herein, the terms "complementary sequence" refers tothe sequence part of a polynucleotide (e.g., part of crRNA or tracRNA) that can hybridize toanother part of polynucleotides under standard low stringent conditions. Preferentially, the sequences are complementary to each other pursuant to the complementaritybetween two nucleic acid strands relying on Watson-Crick base pairing between thestrands, i.e., the inherent base pairing between adenine and thymine (A-T) nucleotides andguanine and cytosine (G-C) nucleotides. Said gRNA can be designed by any methods known by one of skill in the art in view of the present disclosure. According to the present disclosure said target sequence encodes protein region on BCL-2 involved in first agent binding, preferably located in the extracellular portion of BCL-2,more preferably in an extracellular loop in comparison to said second isoform, again more preferably comprising amino acid residues involved in agent binding. In a preferred embodiment, when protein is BCL-2, said target sequence encodes a BCL-2region involved in binding of a first agent, such as anti-BCL-2 agent binding as disclosedabove. Preferably said target sequence encodes at least one residue in position G101, D103and / or F104 of SEQ ID NO: 1.The DNA strand break that is introduced by the nuclease according to the disclosure can result in mutation of the DNA at the cleavage site via non-homologous end joining (NHEJ) which often results in small insertions and / or deletions or replacement of the DNA surrounding the cleavage site via homology-directed repair (HDR). In a preferred embodiment, said polymorphism within nucleic acid encoding the isoformof BCL-2 is induced via HDR repair following the DNA cleavage and the introduction of anexogeneous nucleotide sequence, named herein HDR template. HDR template comprises a first and a second portion of sequence which are homologous to regions 5’ and 3’ of the target sequence, respectively and a middle sequence portion comprising polymorphism. Following cleavage of the target sequence, a homologous recombination event is achieved between the genome containing the target sequence and the HDR template and the genomic sequence containing the target sequence is replaced by the exogeneous sequence. Preferably, homologous sequences of at least 20 bp, preferably more than 30 bp, more preferably more than 50 bp and most preferably less than 200 bp are used. Homologous sequences may be dsDNA or ssDNA. Preferably the homologous sequences are ds DNA. Indeed, shared DNA homologies are located in regions flanking upstream and downstream the site of the break and the exogeneous sequence to be introduced should be locatedbetween the two arms. The flanking sequences may be symmetrical or asymmetrical. Bothstrands of the target nucleic acid, i.e., the plus strand or the minus strand, may be targeted.Optionally, a PAM sequence may be used, which may be silenced to improve HDR. In a preferred embodiment, the cell according to the present disclosure is genetically engineered by introducing into said cell said site-specific nuclease which targets thesequence encoding the region on BCL-2 recognized by said first agent as described aboveand a HDR template. In another particular embodiment, said gene editing enzyme is a DNA base editor as described in Komor et al., Nature 533, 420-424, and in Rees HA, Liu DR. Nat Rev Genet.2018;19: 770-788, or a prime editor as described in Anzalone et al. Nature, 2019, 576: 149-157, Matsoukas et al.,Front Genet. (2020) 11: 528, Chen et al. Cell (2021) 184: 5635-52,Koblan et al, Nat Biotechnol (2021) 39: 1414-25 and Kantor A. et al. Int. J. Mol. Sci. 2020, 21(6240). Base editor or prime editor can be used to introduce mutations at specific sites in the target sequence. According to the present disclosure, the base editor or prime editor generates a mutation within the target sequence by sequence-specific targeting of the sequence encoding theregion on BCL-2 involved in first agent binding.In particular, said base editor or prime editor are CRISPR base or prime editors. Said CRISPR base or prime editor may comprise as catalytically inactive sequence specific nuclease a dead Cas protein (dCas). It may also comprise Cas9 with a mutated nuclease domain. dCas refers to a modified Cas nuclease which lacks endonucleolytic activity. Nuclease activity can be inhibited or prevented in dCas proteins by one or more mutations and / or one or more deletions in the HNH and / or RuvC-like catalytic domains of the Cas protein. The resulting dCas protein lacks nuclease activity but bind to a guide RNA (gRNA)- DNA complex with high specificity and efficiency to specific target sequence. In a particular embodiment, said dCas may be a Cas nickase wherein one catalytic domain of the Cas is inhibited or prevented. Said base editor is complexed with a guide RNA (gRNA) designed to comprise a complementary sequence of the target nucleic acid sequence to specifically bind said target sequence as described above. Said gRNA can be designed by any methods known by one of skill in the art in view of the present disclosure. In a particular embodiment, said gRNA may target the sequenceencoding the region on BCL-2 recognized by said first agent as described above.As non-limiting examples said base editor is a nucleotide deaminase domain fused to a dead Cas protein, in particular Cas nickase. Said nucleotide deaminase may be an adenosine deaminase or cytidine deaminase. Said nucleotide deaminase may be natural or engineered deaminase. In a particular embodiment, said base editor may be as non-limiting examples selected from the group consisting of: BE1, BE2, BE3, BE4, HF-BE3, Sa-BE3, Sa-BE4, BE4-Gam, saBE4- Gam, YE1-BE3, EE-BE3, YE2-BE3, YEE-BE3, VQR-BE3, VRER-BE3, SaKKH-BE3, cas12a-BE, Target-AID, Target-AID-NG, xBE3, eA3A-BE3, A3A-BE3, BE-PLUS, TAM, CRIPS-X, ABE7.9, ABE7.10, ABE7.10* xABE, ABESa, ABEmax, ABE8e, VQR-ABE, VRER-ABE and SaKKH-ABE. Said prime editor consists of a fusion of a catalytically inactive sequence specific nuclease as described above, particularly a Cas nickase and a catalytically active engineered reverse transcriptase (RT) enzyme. Said fusion protein is used in combination with a prime editing guide RNA (pegRNA) which contains the complementary sequence to the target sequenceas described above, particularly when protein is BCL-2 comprises one of the sequencesdescribed herein and also an additional sequence comprising a sequence that binds to theprimer binding site region on the DNA. In a particular embodiment, said reverse transcriptase enzyme is a Maloney murine leukemia virus RT enzyme and variants thereof. Said prime editor may be as non-limiting examples selected from the group consisting of: PE1, PE2, PE3 and PE3b, or any of the prime editors described in Chen et al. Cell (2021) 184: 5635-52 or Koblan et al, Nat Biotechnol (2021) 39: 1414-25.Small molecule BCL-2 inhibitorsDue to the broad expression of BCL-2 in hematopoietic cells the administration of BCL-2inhibitors results in thrombocytopenia, neutropenia, anemia, and leukopenia. The presentdisclosure provides a solution of how to reduce these side effects by using depleting agentsthat bind to a first isoform, but not to a second isoform of BCL-2, or vice versa. In a particular embodiment, said depleting agent which binds to said second isoform ofBCL-2 and does not bind or binds substantially weaker to said first isoform of BCL-2 asdescribed above binds specifically to a drug binding site including the amino acids G101,D103 and / or F104 of SEQ ID NO: 1. In certain preferred embodiments said depleting agentsbinds specifically to a drug binding site including the amino acid F104 of SEQ ID NO: 1. Inother preferred embodiments said depleting agents binds specifically to a drug binding siteincluding the amino acid F104 of SEQ ID NO: 1. In certain embodiments, the BCL-2 inhibitor is selected from venetoclax (ABT-199),navitoclax (ABT-263), AZD-4320, AZD-0466, lisaftoclax (APG2575), S55746, sonrotoclax,ABT- 731, ABT-737, apogossypol, Ascenta’s pan-BCL-2 inhibitors, curcumin (and analogsthereof), G3139), HA14-1, NH-1, obatoclax, or any derivatives or analog thereof.In certain embodiments, the BCL-2 inhibitor is selected from Venetoclax (CAS # 1257044-40-8), Navitoclax (CAS # 923564-51-6), AZD-4320 (CAS# 1357576-48-7), Lisaftoclax(2180923-05-9), S55746 (1448584-12-0), Sonrotoclax (2383086-06-2), or a variant or an analog thereof. In certain embodiments, the BCL-2 inhibitor has the following structure: . In certain embodiments, the BCL-2 inhibitor has the following structure . In certain embodiments, the BCL-2 inhibitor has the following structure .In certain embodiments, the BCL-2 inhibitor has the following structure .In certain embodiments, the BCL-2 inhibitor has the following structure .In certain embodiments, the BCL-2 inhibitor has the following structure Natural and induced resistance variants of BCL-2 emerging after venetoclax treatment With the increased clinical use of venetoclax BCL-2 resistance variants have been emerging (reviewed in Tausch et al., Haematologica.2019; Liu et al., Front Oncol.2022). These arise usually through point mutations in BCL-2, which affect venetoclax binding but retain at least some level of binding of the natural interaction partners of BCL-2. The first and probably most prominent escape variant is G101V, which was discovered in CLLpatients at leukemia progression (Blombery P, Cancer Discov 2019). The G101V mutationreduces venetoclax binding affinity by more than 100-fold, and retains, e.g., BIM binding(Birkinshaw RW et al. Nat Commun 2019), thus still exerting its anti-apoptotic function.Following this discovery, other missense mutations were detected in patients, alsoresulting in some acquisition of venetoclax resistance. Examples are D103Y (Takacs F et al.,Acta Oncol 2021), F104I (Blombery P et al., Br J Haematol. 2019), F104L and A113G (LukasF et al., Blood 2020). Missense mutations also appear together with G101V, for exampleD103E, D103V, D103Y, A113G, R129L and V156D (Kotmayer L et al., Int J Mol Sci. 2023.Blombery P et al., Blood.2020). Similar mutations were also induced by cultivating venetoclax-sensitive cell lines in vitro in the presence of low-dose venetoclax (Tahir et al. BMC Cancer (2017) 17:399) or applyingcontinuous venetoclax exposure in vivo in a mouse lymphoma model (Fresquet et al., Blood2014). This led to the identification of F101C and F101L resistance mutations in mouse BCL-2m, which correspond to F104C and F104L in humans. Comprehensive tiling of BCL-2 usingbase editors resulted also in the enrichment of mutants with venetoclax resistance, suchas the naturally occurring resistance variants D103E, D103Y and F104L as well as newmutants D103G, D103N, S105F, S105F / R106C, V148A, V148I / A149T, L169P / V170A andR106del (Sangree AK et al., Nat Commun.2022).So far, two of the naturally emerging variants have been used to generate venetoclax resistant cells for therapy. For example, CRISPR-Cas9 technology was employed to knock-in the BCL2 G101V mutation in iPSCs. Homozygous knock-in IPCs were then differentiated,resulting in NK cells with a 94-fold increased resistance to venetoclax (Bernareggi D et al., Blood 2020). Along the same lines Lee et al developed venetoclax-resistant CD19 CAR T-cells, which are insensitive to venetoclax by overexpression of BCL-2 F104L (Lee YG et al.,Cancer Discov 2022).Interestingly, although many naturally emerging and induced BCL-2 resistance variants have been described, only few (such as G101V, G101A, F104C and F104L) have beenthoroughly investigated with respect to their venetoclax, BIM and BAX binding (BirkinshawRW et al., Nature Communications 2019). Any such naturally occurring variants of BCL-2 may be used in the context of the presentdisclosure. Additionally, also artificially induced resistance variants may be utilized. Suchvariants are rationally designed or identified via respective screening campaigns.Cells expressing a first isoform of BCL-2 The present disclosure relates to a mammalian cell, preferably a hematopoietic cell, or apopulation of cells expressing a first isoform of BCL-2 wherein said cell or population ofcells express a first isoform of BCL-2, wherein said first isoform is not recognized by a BCL-2 inhibitor.The present disclosure also relates to a mammalian cell, preferably a hematopoietic cell,or a population of cells expressing a first isoform of BCL-2 wherein said cell or populationof cells express a first isoform of BCL-2 comprising at least one polymorphic allele in thenucleic acid encoding said first isoform, and wherein said first isoform is not recognized bya BCL-2 inhibitor.The cell expressing said first isoform of BCL-2 which is not recognized by the BCL-2inhibitor as described herein may not necessarily comprise said polymorphism orgenetically engineered allele in the genomic DNA. In certain embodiements, said BCL-2 inhibitor is selected from venetoclax (ABT-199), navitoclax (ABT-263), AZD-4320, AZD-0466, lisaftoclax (APG2575), S55746, sonrotoclax,ABT- 731, ABT-737, apogossypol, Ascenta’s pan-BCL-2 inhibitors, curcumin (and analogsthereof), G3139), HA14-1, NH-1, obatoclax, or any derivative or analog thereof. In preferredembodiments, said BCL-2 inhibitor is venetoclax.Said first isoform of BCL-2 may also be transiently expressed in said cell by anymethodology known to the person skilled in the art. Said first isoform of BCL-2 may also be generated in said cell by in vivo editing by anyappropriate means known to the person skilled in the art. Said cell or population of cells are particularly useful in medical treatment in a patientexpressing a second isoform of BCL-2.In a particular embodiment, said cells (e.g. hematopoietic stem cell) encoding orexpressing said first isoform of BCL-2 not recognized by a depleting agent (e.g.hematopoietic cells) are particularly useful in medical treatment to restore normalhematopoiesis after targeted therapy, such as small molecule administration to a patientexpressing said second isoform, in particular wherein the treatment comprisesadministering a therapeutically efficient amount of said hematopoietic cells expressing saidfirst isoform of BCL-2 in combination with a therapeutically efficient amount of a depletingagent targeting said second isoform of BCL-2. In particular, said hematopoietic cells,preferably hematopoietic stem cells are administered subsequently to said depletingagent. In another particular embodiment, said hematopoietic cells, preferably hematopoietic stem cells can be administered before or concurrently to said depleting agent. In another particular embodiment, said cells expressing said first isoform of BCL-2specifically recognized by a depleting agent which does not bind or binds substantiallyweaker to a second isoform of BCL-2 are particularly useful in medical treatment in apatient expressing said second isoform of BCL-2, in particular to avoid severe side-effectrelated to transplanted cells carrying the first isoform (safety switch), wherein the treatment comprises administering a therapeutically efficient amount of a depleting agenttargeting said first isoform of BCL-2.As used herein, the term cell relates to mammalian cells, preferably human cells. In a particular embodiment, said cells are hematopoietic cells. Hematopoietic cellscomprise immune cells including lymphocytes, such as B cells and T cells, natural killer cells,myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, granulocytes, dendritic cells (DC) and plasmacytoid dendritic cells (pDCs). In certain embodiments, said immune cells are T cells. In another preferred embodiment,said immune cells are primary T cells. As used herein, the term “T cell” includes cells bearinga T cell receptor (TCR) or a cell derived from a T cell bearing a TCR. T-cells according to thedisclosure can be selected from the group consisting of inflammatory T-lymphocytes,cytotoxic T-lymphocytes, regulatory T-lymphocytes, memory T-lymphocytes, tumorinfiltrating lymphocytes or helper T- lymphocytes included both type 1 and 2 helper T cellsand Th17 helper cells. In another embodiment, said cell can be derived from the groupconsisting of CD4+ T- lymphocytes and CD8+ T-lymphocytes or non-classical T cells such asMR1 restricted T cells, MAIT cells, NKT cells, gamma delta T cells or innate-like T cells. T-cells can be obtained from a number of non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, T-cells can be obtained from a unit of blood collected from a subject usingany number of techniques known to the skilled person. Alternatively, T cells can bedifferentiated from iPS cells. In another preferred embodiment, said hematopoietic cells are hematopoietic stem cells. The stem cells can be adult stem cells, embryonic stem cells, more particularly non-human stem cells, cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells or hematopoietic stem cells. Representative human stem cells are CD34+cells. Hematopoietic stem cells can be differentiated from iPS cells or can be harvested from umbilical cord blood, from bone marrow or from mobilized or not mobilized peripheral blood. In certain embodiments, the cell is an allogeneic cell which refers to a cell derived from adonor that presents with an HLA genotype that is identical, similar or different to the HLAgenotype of the person receiving the cell. The donor may be a related or unrelated person.In certain embodiments, the cell is an autologous cell which refers to a cell derived fromthe same person that is receiving the cell. Said cells may originate from a healthy donor or from a patient, in particular from apatient diagnosed with cancer, genetic disease or an auto-immune disease or from apatient diagnosed with an infection. Hematopoietic cells can be extracted from blood, bonemarrow or derived from stem cells. HSC’s can for example be derived from iPS (inducedpluripotent stem cells. A person skilled in the art will choose the more appropriate cells according to the patient or subject to be transplanted. The disclosure further relates to a composition of cells or a population of cells for use in the therapy as disclosed herein. In certain embodiments, the present disclosure relates to a mammalian cell or apopulation of cells expressing a first isoform of BCL-2 for use in a medical treatment in apatient in need thereof, wherein said patient has cells expressing a second isoform of BCL-2, wherein said cell expressing said first isoform comprises genomic DNA with at least one polymorphism or genetically engineered allele, wherein said polymorphism or genetically engineered allele is not present in the genome of the patient having cells expressing said second isoform of BCL-2, and wherein said polymorphic or genetically engineered allele ischaracterized by at least one substitution of an amino acid in position G101, D103 and / orF104 of SEQ ID NO: 1. In certain preferred embodiments, said polymorphic or geneticallyengineered allele is characterized by at least one substitution of an amino acid in position F104 of SEQ ID NO: 1. In other preferred embodiments, said polymorphic or genetically engineered allele ischaracterized by at least one substitution of an amino acid in position F104 of SEQ ID NO:1.In vitro method for preparing cell first isoformThe cell expressing the first isoform of BCL-2 according to the present disclosure can begenetically engineered by introducing into said cell a nucleic acid construct (e.g., mRNA)encoding at least one gene editing enzyme or ribonucleoprotein complex comprising geneediting enzyme and / or HDR template as described above. Alternatively, the gene editingsystem is transduced into said cells via a viral system, such as an adenoviral system. Said cell can also be genetically engineered by further introducing into said cell a nucleic acid construct encoding a CAR as described above. In particular, said method is an ex vivo method performed on a culture of cells. The term “nucleic acid construct” as used herein refers to a nucleic acid molecule resulting from the use of recombinant DNA technology. A nucleic acid construct is a nucleicacid molecule, either single- or double-stranded, which has been modified to containsegments of nucleic acid sequences, which are combined and juxtaposed in a manner, which would not otherwise exist in nature. A nucleic acid construct usually is a “vector”,i.e., a nucleic acid molecule which is used to deliver exogenously created DNA into a hostcell. Preferably, the nucleic acid construct comprises said gene editing enzyme, HDR template and / or CAR, operably linked to one or more control sequences. Said control sequences may be a ubiquitous, tissue-specific or inducible promoter which is functional in cells of targetorgans (i.e., hematopoietic cell). Such sequences which are well-known in the art includein particular a promoter, and further regulatory sequences capable of further controlling the expression of a transgene, such as without limitation, enhancer, terminator, intron, silencer. The nucleic acid construct as described above may be contained in an expression vector. The vector may be an autonomously replicating vector, i.e., a vector that exists as an extra- chromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extra-chromosomal element, a mini-chromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one that, when introduced into the host cell, is integratedinto the genome and replicated together with the chromosome(s) into which it has beenintegrated. Examples of appropriate vectors include, but are not limited to, recombinant integrating or non-integrating viral vectors and vectors derived from recombinant bacteriophage DNA,plasmid DNA or cosmid DNA. Preferably, the vector is a recombinant integrating or non-integrating viral vector. Examples of recombinant viral vectors include, but not limited to, vectors derived from herpes virus, retroviruses, lentivirus, vaccinia viruses, adenoviruses, adeno-associated viruses or bovine papilloma virus. The present disclosure relates to a method for expressing a first isoform of a protein in acell by introducing into said cell a nucleic acid construct (e.g. mRNA) encoding the geneediting enzyme or ribonucleoprotein complex comprising gene editing enzyme and / or HDR template as described above. Said method may further comprise a step of introducing into said cell a nucleic acid construct encoding a CAR. Said method involves introducing gene editing enzyme such as Cas protein, base editor or prime editor and guide RNA (crRNA, tracrRNa, or fusion guide RNA or pegRNA) into a cell. In particular, said gene editing enzyme is CRISPR / Cas gene editing enzyme as described above. In a more particular embodiment, said gene editing enzyme is a site-specific nuclease, more preferably CRISPR / Cas nuclease comprising a guide RNA and Cas protein, wherein said guide RNA in combination with Cas protein cleaves and induces cleavage within said target sequence comprising a nucleic acid encoding protein region involved in agent binding as described above. Said Cas nuclease may be a high-fidelity Cas nuclease such as a high fidelity Cas9 nuclease. Said gene editing enzyme, preferably guide RNA and / or Cas protein, base editor or prime editor as described above may be synthesized in situ in the cell as a result of the introduction of nucleic acid construct, preferably expression vector encoding said gene editing enzyme such as guide RNA and / or Cas protein, base editor or prime editor as described above into the cell. Alternatively, said gene editing enzyme such as guide RNA and / or Cas protein, base editor or prime editor may be produced outside the cell and then introduced thereto. Said nucleic acid construct or expression vector can be introduced into cell by any methods known in the art and include, as non-limiting examples, stable transduction methods in which the nucleic acid construct or expression vector is integrated into the cell genome, transient transfection methods in which the nucleic acid construct or expression vector is not integrated into the genome of the cell and virus-mediated methods. For example, transient transformation methods include for example microinjection,electroporation, cell squeezing, particle bombardment or in vivo targeting approaches.In vivo editingThe cell expressing the first isoform of BCL-2 according to the present disclosure may alsobe edited in vivo. Various technologies exist that enable therapeutic in vivo gene editing,including viral vectors, lipid nanoparticles and virus-like particles (see for example Cell(2022) 185: 2806-27. The molecular machinery to convert BCL-2 into a first isoform of BCL-2 which is not recognized by the depleting agent can be accomplished by any of thesemethods. In certain embodiments, the present disclosure relates a pharmaceutical compositioncomprising molecular machinery capable of in vivo editing a gene and a depleting agent,wherein said molecular machinery capable of in vivo editing a gene comprises allcomponents required to introduce a point mutation of wild type BCL-2 in a target cell intoan isoform of BCL-2, and wherein said depleting agent binds to wild type BCL-2, but notto said isoform of BCL-2 for use in a medical treatment in a patient in need thereof.Pharmaceutical composition and therapeutic use In a further aspect, the present disclosure also provides a pharmaceutical compositioncomprising cells or a population of cells expressing a first isoform of BCL-2 as describedabove with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. In a particular embodiment, said cell expressing the first isoform of BCL-2 is ahematopoietic stem cell. In another embodiment, said cell expressing said first isoform of BCL-2 is an immune cell,preferably a T-cell, more preferably a primary T cell, bearing a chimeric antigen receptor (CAR). In another embodiment, said cell expressing said first isoform of BCL-2 is a hematopoietic cell, or an iPSC-derived hematopoietic cells such as a T-cell, B-cell, an NK cell, a megakaryocyte, an erythrocyte, an HSC or an HSPC. The pharmaceutical composition may further comprise a depleting agent comprising afirst or second chemical structure as described above.The pharmaceutical composition is formulated in a pharmaceutically acceptable carrier according to the route of administration. Preferably, the composition is formulated to beadministered by intravenous injection. Pharmaceutical compositions suitable for suchadministration may comprise the cells expressing first isoform as described above, incombination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions (e.g., balanced salt solution (BSS)), dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes or suspending or thickening agents.Optionally, the composition comprising cells expressing first isoform of BCL-2 may befrozen for storage at any temperature appropriate for storage of the cells. For example, the cells may be frozen at about −20° C, −80° C or any other appropriate temperature. Cryogenically frozen cells may be stored in appropriate containers and prepared for storage to reduce risk of cell damage and maximize the likelihood that the cells will survive thawing. Alternatively, the cells may also be maintained at room temperature of refrigerated, e.g., at about 4° C. The present disclosure relates to the cell or population of cells expressing a first isoformod BCL-2 as described above for use as a medicament, in particular for use in targetedtherapy such as adoptive cell transfer therapy in a patient.According to the present disclosure, said cell or population of cells (e.g., hematopoieticcells) expressing a first isoform of BCL-2 as described above, is used in a medical treatmentin a patient in need thereof, wherein said medical treatment comprises administering a therapeutically efficient amount of cell or population of cells expressing said first isoform of BCL-2, in combination with a therapeutically efficient amount of a depleting agent (e.g.a BCL-2 inhibitor) that binds specifically to the second isoform or first isoform of BCL-2 tospecifically depleting the patients or the transplanted cells, respectively.As used herein, the term “in combination” or “in combination therapy” means that two(or more) different treatments are delivered to the subject during the course of thesubject's affliction with the disorder, e.g., the two or more treatments are delivered after the subject has been diagnosed with the disorder and before the disorder has been cured or eliminated or treatment has ceased for other reasons. In some embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as “simultaneous” or “concurrent delivery”. In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. The delivery can be such that an effect of the first treatment delivered is still detectable when the second isdelivered. In one embodiment, a depleting agent that binds to a second isoform or a firstisoform of BCL-2 is administered at a dose and / or dosing schedule described herein, andthe cells expressing the first isoform are administered at a dose and / or a dosing scheduledescribed herein. In some embodiments, “in combination with,” is not intended to imply that the depleting agent targeting the second (e.g. CAR cells or antibody recognizing asecond isoform of BCL-2) or the first isoform of BCL-2 and compositions of cells expressingsaid first isoform of BCL-2, must be administered at the same time and / or formulated for delivery together, although these methods of delivery are within the scope of this disclosure. The depleting agent (e.g. CAR cells or antibody targeting a second isoform ofBCL-2) can be administered concurrently with, prior to or subsequent to a dose of thehematopoietic stem cells expressing the first isoform of BCL-2. In certain embodiments,each agent will be administered at a dose and / or on a time schedule determined for that particular agent. Adoptive cell transfer therapy according to the disclosure can be used to treat patients diagnosed with cancer, genetic disease, autoimmune disease, infectious disease, a diseaserequiring a hematopoietic stem cell transplantation (HSCT), the prevention of organrejection, the tumor conditioning regimen, tumor maintenance treatment, minimal residual disease, the prevention of relapse. The present disclosure also relates to the use of cells expressing a first isoform of BCL-2 as described above in the manufacture of a medicament for adoptive transfer cell therapy in a patient. As used herein, the term “subject”, or “patient” refers to an animal, preferably to a mammal in which an immune response can be elicited including human, pig, chimpanzee, dog, cat, cow, mouse, rabbit or rat. More preferably, the patient is a human, including adult, child and human at the prenatal stage. As used herein, the term “treatment”, “treat” or “treating” refers to any act intended toameliorate the health status of patients such as therapy, prevention, prophylaxis, andretardation of the disease. In certain embodiments, such term refers to the amelioration or eradication of a disease or symptoms associated with a disease. In other embodiments, this term refers to minimizing the spread or worsening of the disease resulting from the administration of one or more therapeutic agents to a subject with such a disease. Cancers that may be treated include tumors that are not vascularized, or not yet substantially vascularized, as well as vascularized tumors. The cancers may comprise non- solid tumors (such as hematological tumors, for example, leukemias and lymphomas including relapses and treatment-related tumors e.g. secondary malignancies after use of cytotoxic therapy and hematopoietic stem cell transplantation (HSCT)) or may comprise solid tumors. The term "autoimmune disease" as used herein is defined as a disorder that results from an autoimmune response. An autoimmune disease is the result of an inappropriate and excessive response to a self-antigen. Infectious disease is a disease caused by pathogenic microorganism such as bacteria, viruses, parasites or fungi. In particular embodiments, infections according to the disclosure occur in immunosuppressed patients, such as patients after HSCT or patients who received a solid organ transplantation. In a preferred embodiment, the present disclosure relates to a cell expressing first isoformof BCL-2 as described above for use in the treatment of a hematological cancer, preferablyleukemia or lymphoproliferative disorders. Said lymphoproliferative disorders can beselected from the group consisting of: malignant hematopoietic disease such as such as acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), chronic lymphocytic leukemia (CLL), mantle-cell lymphoma, multiple myeloma (MM), diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), marginal zone B-cell lymphoma, non- Hodgkin's lymphoma (NHL), T-cell prolymphocytic leukemia, Waldenstrom's macroglobulinaemia, chronic myelomonocytic leukemia (CML), precursor cell lymphoblastic leukaemia-lymphoma, B-cell lymphoma, blastic plasmacytoid dendritic cellneoplasm (BPDCN) and other proliferative neoplasms, preferably MDS or AML.In a preferred embodiment, the present disclosure relates to a cell expressing first isoformof BCL-2 as described above for use in the treatment of a solid cancer, and preferably in the treatment of non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC) prostate cancer, ovarian cancer, triple negative breast cancer (TNBC), colorectal cancer, head and neck cancer or breast cancer. In a preferred embodiment, the present disclosure relates to a cell expressing first isoformof BCL-2 as described above for use in solid tumor malignancies, preferably such as non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC) prostate cancer, ovarian cancer, triple negative breast cancer (TNBC), colorectal cancer, head and neck cancer or breast cancer. In a preferred embodiment, the present disclosure relates to a cell expressing first isoformof BCL-2 as described above used to reduce side effects emerging through the depletion ofhematopoietic cells, such as neutropenia, anemia, thrombocytopenia and lymphopenia inpatients treated with a binding moiety that specifically binds to BCL-2, such as an BCL-2 inhibitor. In a preferred embodiment, the present disclosure relates to a cell expressing first isoformof BCL-2 as described above for the use to reduce side effects emerging through thedepletion of hematopoietic cells, such as neutropenia, anemia, thrombocytopenia and lymphopenia in cancer patients treated with a binding moiety that specifically binds to BCL- 2, such as an BCL-2 inhibitor. In another particular embodiment, said cell or population of cells (e.g., hematopoieticcells) expressing a first isoform of BCL-2 as described above can be used for the treatmentof autoimmune disease such as lupus, multiple sclerosis, scleroderma or systemic sclerosis.Method for depleting specifically patient cells and not transplanted cells According to the present disclosure, said cell or population of cells (e.g. hematopoieticcells) expressing a first isoform of BCL-2 as described above, is used in a medical treatmentin a patient in need thereof, wherein said medical treatment comprises administering atherapeutically efficient amount of said cells or population of cells expressing said firstisoform of BCL-2, in combination with a therapeutically efficient amount of a depletingagent (e.g. a small molecule inhibitor) that binds specifically to a second isoform of BCL-2.Indeed, during targeted therapy, a depleting agent, such as a small molecule directed toBCL-2, can be administered to a patient to target and kill tumoral cells. However, as tumorantigens are also expressed in normal hematopoietic cells, this strategy can induce severeside effects to the patients by altering hematopoiesis. To restore hematopoiesis in the patient, hematopoietic cells can be subsequently transplanted into the patient. However,these cells need to be resistant to said agent, i.e., the depleting agent for BCL-2 expressingcells, in order not to be targeted by it.Thus, alternatively, according to the present disclosure, the depleting agent specificallybinding to a second isoform of BCL-2 can be administered to ablate specifically patient cellsexpressing said second isoform of BCL-2 and not transplanted cells expressing said firstisoform of BCL-2. The selective depletion of patient cells, but not transplanted cells, allowsto reconstitute the patient with a healthy hematopoietic system which will no longer be depleted by immunodepleting agent. Thus, according to the present therapeutic use, the patients have a functional immune system rather than go through a prolonged phase of immunosuppression. The use of cells according to the present disclosure eliminatesinfections or prolonged or excessive bleeding (through thrombocytopenia) as majorcomplications of current cancer therapy.In another embodiment, the present disclosure relates to a method for adoptive cell transfer therapy, preferably for hematopoietic stem cell transplantation to restore normalhematopoiesis in a patient having cells expressing a second isoform of BCL-2 comprising:(i) administering an effective amount of a cell (e.g. hematopoietic stem cells)expressing a first isoform of BCL-2 wherein said cell expressing said first isoform of BCL-2comprises genomic DNA with at least one polymorphic allele, preferably single nucleotidepolymorphism (SNP) allele, or a genetically engineered allele in the nucleic acid encodingsaid first isoform and wherein said polymorphism is not present in the genome of thepatient having cells expressing said second isoform of BCL-2 or a pharmaceuticalcomposition thereof; and(ii) administering a therapeutically efficient amount of an that specifically binds to saidsecond isoform of BCL-2 and does not bind or binds substantially weaker to said firstisoform of BCL-2 to deplete specifically cells expressing said second isoform of BCL-2(patient’s cells).Said cells expressing the first isoform of BCL-2 or pharmaceutical compositions thereof areadministered to a subject in combination with (e.g., before, simultaneously or following)an agent specifically binding to the second isoform of BCL-2.In a preferred embodiment, the depleting agent (e.g., small molecule inhibitor targetinga second isoform of BCL-2 is administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes,45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96hours before), or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, 12 weeks, or 16 weeks after) a doseof the hematopoietic stem cells expressing a first isoform of said protein (e.g., a firstisoform of BCL-2). By a “therapeutically efficient amount” or “effective amount” is intended a number ofcells, in particular hematopoietic stem cells expressing the first isoform of BCL-2 asdescribed above administered to a subject that is sufficient to constitute a treatment as defined above, in particular restoration of normal hematopoiesis in a patient. The administration of the cell or pharmaceutical composition according to the present disclosure may be carried out in any convenient manner, including injection, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermal, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous or intralymphatic injection, or intraperitoneally. In anotherembodiment, the cells or pharmaceutical compositions of the present disclosure arepreferably administered by intravenous injection. The cells or pharmaceutical compositionsof the present disclosure may be injected directly into a tumor, lymph node, or site ofinfection. The administration of the cells or population of cells can consist of the administration of 104-109cells per kg body weight, preferably 105to 107cells / kg body weight, morepreferably 2x106-5x106 cells per kg body weight including all integer values of cell numberswithin those ranges. The dosage administrated will be dependent upon the age, health and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment andthe nature of the effect desired. The cells or population of cells can be administered in oneor more doses. Timing of administration is within the judgment of managing physician and depends on the clinical condition of the subject. The cells or population of cells may be obtained from any source, such as a blood bank or a donor. While individual needs vary, determination of optimal ranges of effective amounts of a given cell type for a particular disease or conditions within the skill of the art. In particular, the disclosure also relates to depleting agents that specifically bind BCL-2 asdisclosed herein, such as BCL-2 inhibitors, for use in selectively depleting the host cells in asubject in need thereof. Method for depleting specifically transplanted cells and not patient cells (safety switch). According to the present disclosure, said cell or population of cells (e.g. hematopoieticcells) expressing a first isoform of BCL-2 as described above, is used in a medical treatmentin a patient in need thereof, wherein said medical treatment comprises administering a therapeutically efficient amount of a cell or a population of cells expressing said firstisoform of BCL-2, in combination with a therapeutically efficient amount of a depletingagent (e.g. a small molecule inhibitor) that binds specifically to said first isoform BCL-2. The cell or population of cells, preferably immune cells expressing the first isoform of BCL-2 of the present disclosure is particularly used in adoptive transfer cell transfer therapy intoa patient. Said transplanted cell expressing said first isoform of BCL-2 can be furtherdepleted in patients by administering a therapeutically efficient amount of a depletingagent that specifically binds to the first isoform of BCL-2 particularly and does not bind orbinds substantially weaker to the second isoform of BCL-2 expressed by patient’s cells toavoid eventual severe side effects such as graft-versus-host disease due to thetransplantation. In this case, said agent comprising the depleting agent which bindsspecifically to said first isoform of BCL-2 (expressed by transplanted cell) is administered todeplete specifically transplanted cells and not patient cells. Selective depletion of thetransplanted cells constitutes an important safety feature by providing a “safety switch”. Graft-versus-host disease (GvHD) relates to a medical complication following the receipt of transplanted tissue from a genetically different person. Immune cells in the donated tissue (the graft) recognize the recipient (the host) as foreign. ln certain embodiments, the medical condition is graft-versus-host disease caused by hematopoietic stem celltransplantation or adoptive cell transfer therapy wherein immune cells are transferred intopatient. Said side effects can also occur when transplanted cells, particularly immune cells harboring a CAR have severe side effects such as cytokine release syndrome and / orneurotoxicity. In this case, the transplanted cells expressing the first isoform of BCL-2 canbe eliminated when said cells become malignant or cause any type of unwanted on-targetor off-target damage as a safety switch. The present disclosure relates to a method for adoptive cell transfer therapy in a patienthaving cells expressing a second isoform of BCL-2 comprising:(i) administering an effective amount of a cell expressing a first isoform of BCL-2wherein said cell expressing said first isoform of BCL-2 comprises genomic DNA with at leastone polymorphism allele, preferably single nucleotide polymorphism (SNP) allele, or agenetically engineered allele in the nucleic acid encoding said first isoform BCL-2 andwherein said polymorphism is not present in the genome of the patient having cellsexpressing said second isoform of BCL-2 or a pharmaceutical composition thereof; and(ii) administering a therapeutically efficient amount of a depleting agent that bindsspecifically to said first isoform of BCL-2 and does not bind or binds substantially weaker tosaid second isoform of BCL-2 to deplete specifically cells expressing said first isoform ofBCL-2. Said cells expressing the first isoform of BCL-2 or pharmaceutical compositions thereofare administered to a subject in combination with (e.g., before, simultaneously or following) an agent comprising a second antigen binding region as described above. In a preferred embodiment, the depleting agent (e.g. CAR cells or antibody targeting a second isoform of BCL-2) is administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours before), or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, 12 weeks, or 16 weeks after) a dose of the hematopoietic stem cells expressing a first isoform of BCL-2. The administration of the cells or pharmaceutical composition according to the presentdisclosure may be carried out in any convenient manner, including injection, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermal, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous or intralymphatic injection, or intraperitoneally. In anotherembodiment, the cells or pharmaceutical compositions of the present disclosure arepreferably administered by intravenous injection. The cells or pharmaceutical compositionsof the present disclosure may be injected directly into a tumor, lymph node, or site ofinfection. The administration of the cells or population of cells can consist of the administration of 104-109cells per kg body weight, preferably 105to 107cells / kg body weight including all integer values of cell numbers within those ranges. The dosage administrated will be dependent upon the age, health and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment and the nature of the effect desired. The cells or populationof cells can be administered in one or more doses. Timing of administration is within thejudgment of managing physician and depends on the clinical condition of the subject. The cells or population of cells may be obtained from any source, such as a blood bank or a donor. While individual needs vary, determination of optimal ranges of effective amountsof a given cell type for a particular disease or conditions within the skill of the art.Accordingly, in specific embodiments, the disclosure relates to a depleting agent (e.g. aBCL-2 inhibitor) for use in preventing or reducing the risk of severe side effects in a patienthaving received a cell expressing a first isoform of BCL-2 as described above, wherein saidpatient have native cells expressing a second isoform of BCL-2, and wherein said depletingagent comprising a depleting agent which binds specifically to said first isoform of BCL-2and does not bind or binds substantially weaker to said second isoform of BCL-2.In another aspect, the present disclosure relates to a kit for expressing a first isoform BCL-2 as describe above into a cell, said kit comprising a gene editing enzyme, such as guideRNA in combination with a Cas protein, base editor or prime editor, nucleic acid construct, expression vector as described above or isolated cell according to the present disclosure. EXAMPLESExample 1: Design and selection of BCL-2 variants by rational designSmall molecule BCL-2 inhibitors are very efficacious in eliminating cancer cells, but resultin severe adverse events such as neutropenia, thrombocytopenia, anemia and lymphopenia through the depletion of healthy hematopoietic cells. In order to identifyisoforms of BCL-2 retaining normal expression levels and functional activity, co-crystalstructures of human wildtype and variant BCL-2 with venetoclax (PDB numbers 6O0K,6O0L, 6O0M and 6O0P) as well as co-crystal structures of human BCL-2 with its naturalbinding partners such as Bax (2XA0) were analyzed rationally. Residues within 4 Angstromof venetoclax were considered for mutagenesis. Residues that are important for binding to the exemplary natural binding partner, Bax, were excluded as well as residues that couldhave a larger impact on BCL-2 structure upon mutagenesis. Residues A100, G101, D103,F104, Y108, V156 and Y202 were targeted due to their proximity to venetoclax and due tominor involvement in binding Bax or other binding partners (Nat Commun (2019) 10:2385).A100 was mutated to Val, G101 to Val, Leu, Ser, Thr, and Ile, D103 to Glu, Tyr, Ile, Val,Phe, Lys, Leu, F104 to Cys, Leu, Val, Ser, Ala, Ile, Thr, Gly, Asn, His, Y108 to Phe and Ser, V156 to Asn, Gln, Leu, Met and Y202 to Ala and Ser. Small molecule BCL-2 inhibitors used in the following examples are listed in Table 3. Table 3: Number Compound CAS number SourceRefSM #1 Venetoclax 1257044-40-8 MedChemExpessRefSM #2 Navitoclax 923564-51-6 Selleck chemicalRefSM #3 AZD-4320 1357576-48-7 MedChemExpessRefSM #4 Lisaftoclax 2180923-05-9 MedChemExpressRefSM #5 S55746 1448584-12-0 MedChemExpressRefSM #6 Sonrotoclax 2383086-06-2 TargetMolExample 2: Production of BCL-2 variants Wildtype BCL-2, as well as variant BCL-2 (amino acids 1-207 based on UniProt: P10415)comprising a C-terminal GST tag were expressed in E. coli BL21 in terrific broth (TB) mediumsupplemented with 0.25 mM Isopropyl β- d-1-thiogalactopyranoside (IPTG). The cells wereharvested after an 18-hour induction at 18 °C. The pellet was resuspended in lysis buffer(50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 5 mM MgCl2, 10% glycerol, 0.25mM TCEP, 1x PMSF / benzamidine, DNase) and sonicated at 30 seconds on / off for 5 rounds at 50% amplitude. Following sonication, the resuspended pellet was put through the homogenizer (EmulsiFlex C5) at 18,000 psi and clarified at 25,000xg for 30 minutes. The supernatant wasflash frozen and stored at -80°C until purification. The lysate was batch bound withGlutathione Agarose resin (MCLAB cat #GAB-200) for 3-4 hours. The resin was washed with 20 CV of binding buffer. The target protein was eluted with an elution buffer containing 25 mM glutathione. Eluted protein was pooled and concentrated for size exclusion chromatography. Fractions corresponding to non-aggregated target protein were pooled and concentrated. Concentrated samples were clarified via centrifugation and quantifiedusing OD280, MW and ^. All proteins could well be produced.Example 3: Biophysical properties of BCL-2 variants Low aggregation propensity and high melting temperature are characteristics of thestructural integrity and stability of biomolecules. Therefore, the soluble domains ofwildtype and variant BCL-2 were expressed and purified as described in Example 2. The recombinant purified BCL-2 extracellular domains were then tested in various biophysicalassays to assess protein integrity and stability versus the wildtype protein. Parameterswere compared to wildtype BCL-2 (and if possible, natural SNPs) to make sure that allproperties of the protein variants are as similar as possible as the wild-type BCL-2 protein.To test BCL-2 variants, BCL-2 wildtype and variants were diluted to 0.5mg / mL, combinedwith Sypro Orange (Sigma-Adrich, S5692) and exposed to a temperature gradient from 25°Cto 90°C utilizing a RT-PCR machine (Biorad, CFX Opus Real-Time PCR Systems). The fluorescence is monitored in function of the temperature First derivative curve providesthe melting temperature in degrees Celsius. GST tagged BCL-2 WT has one single meltingtemperature measured at 52.5°C. F104S, a natural SNP, has two melting events at 51°C and55°C so as G101L, F104G and F104N. Y202S and Y202A have one melting event which ismore than two degrees different from the WT. All the other variants have one meltingtemperature in a similar range to WT.Purity of all variants was assessed after capture and preparative Size Exclusion purificationby HPLC Size Exclusion Chromatography (AdvanceBio SEC 300A 2.7 um 4.6x300 mm, P / N: PL1580-5301). Protein peaks were monitored at 220 and 280 nm and the relative peakareas were measured in %. BCL-2 wildtype as well as all the tested variants have puritysuperior to 95%, except BCL-2 V156Q variant which has a purity of 35% indicating a low purity. Results are shown in Table 4. Table 4: purity (%) Variants SNPTM1 [°C] elution time TM post DSF 2 [°C] DSF preparative (min) SEC not BCL-2_WTapplicable 52.50 not applicable 96.5 8.8BCL-2_A100V yes 52.75 not applicable 96.7 8.78BCL-2_G101V no 53.25 not applicable 95.4 8.75BCL-2_G101I no 52.25 not applicable 98.2 8.75BCL-2_G101S no 52.00 not applicable not tested not testedBCL-2_G101T no 53.75 not applicable 99.6 8.76BCL-2_G101L no 47.00 52.5 97.4 8.74BCL-2_D103E no 51.00 not applicable 94.9 8.8BCL-2_D103Y yes 52.75 not applicable 96.9 8.74BCL-2_D103I no 51.75 not applicable 98.1 8.78BCL-2_D103V no 51.25 not applicable 98.3 8.78BCL-2_D103F no 51.25 not applicable 97.9 8.8BCL-2_D103K no 52.50 not applicable 99.1 8.77BCL-2_D103L no 51.75 not applicable 99 8.79BCL-2_F104C no 52.25 not applicable 99.9 8.79BCL-2_F104L no 51.75 not applicable 94.6 8.78BCL-2_F104V no 52.25 not applicable 99.9 8.76BCL-2_F104S yes 51.00 55 99.9 8.72BCL-2_F104A no 52.75 not applicable 98.3 8.73BCL-2_F104I no 51.75 not applicable 97.4 8.77BCL-2_F104T no 51.75 not applicable 99.8 8.75BCL-2_F104G no 48.00 52.5 98.5 8.7BCL-2_F104N no 50.25 54.5 97.9 8.7BCL-2_F104H no 52.75 not applicable 99.9 8.73BCL-2_Y108F no 53.75 not applicable 96.9 8.8BCL-2_Y108S no 53.25 not applicable 99.9 8.7BCL-2_V156N no 53.50 not applicable 95.8 8.85BCL-2_V156Q no 55.25 not applicable 35 8.77BCL-2_V156L no 53.00 not applicable 97.7 8.82BCL-2_V156M no 53.00 not applicable 95.5 8.8BCL-2_Y202A no 49.50 not applicable 95.3 8.9BCL-2_Y202S no 57.25 not applicable 95.4 8.9Example 4: Determination of KD of BAK binding to BCL-2 variantsIn order to determine the average KD of BAK binding to BCL-2 wildtype and variants, 4 nMrecombinant BCL-2 protein were incubated with serial dilutions of fluorescently labeledBAK peptide (fBAK; 3 to 500 nM) for 10 min. Anti GST-Tb cryptate HTRF reagent was addedand incubated for 1 h before measurement of the HTRF signal (excitation: 337 nm,emission: 492-8 nm and 520-8 nm). BAK binding KDs were calculated as the fBAK concentrations at 50% of the maximumsignal (Bmax). Results are shown in Table 5. About 1.5-fold peptide KD was then used asthe concentration for the determination of venetoclax IC50 in the following Exmaple.Table 5: Average KD of BAK binding to recombinant, human BCL-2 Average peptide Peptide for comp Protein name KD, nMtesting, nM CommentBcl-2 WT 56.6 100Bcl-2 A100V 21.4 35Bcl-2 G101V 104.8 125Bcl-2 G101I 107.3 120Bcl-2 G101S 58.8 85Bcl-2 G101T 42.6 60Bcl-2 G101L 146.3 150Bcl-2 D103Y 26.5 35Bcl-2 D103I 36.9 50Bcl-2 D103V 39.5 50Bcl-2 D103E 48.8 85Bcl-2 D103F 39.6 50Bcl-2 D103K 57.6 90Bcl-2 D103L 37.3 60Bcl-2 F104L 77.0 100Bcl-2 F104V 48.1 70Bcl-2 F104S 162.6 200Bcl-2 F104A 33.2 50Bcl-2 F104C 47.9 70Bcl-2 F104I 87.5 100Bcl-2 F104T 44.1 70Bcl-2 F104G 263.3 150Bcl-2 F104H very poor binding Could not be testedBcl-2 F104N 1200 200 low peptide affinityBcl-2 Y108F 63.2 100Bcl-2 Y108S 997.8 300 low peptide affinityBcl-2 V156N 108.6 150Bcl-2 V156Q 97.2 125Bcl-2 V156L 264 200 low peptide affinityBcl-2 V156M 102.6 125Bcl-2 Y202A very poor binding Could not be testedBcl-2 Y202S very poor binding Could not be testedBCL-2 variants F104H, F104N, Y202A and Y202S showed very poor binding of BAK peptideand therefore no KD could be measured. Other variants such as Y108S and V156L exhibiteda greater than 3-fold reduction in KD, with F104S having 2.7-fold loss of BAK binding. Allother variants retained most of their BAK binding capacity. Example 5: Determination of IC50 of venetoclax binding to BCL-2 variants In order to determine the average IC50 of venetoclax to BCL-2 wildtype and variants, 4nM recombinant BCL-2 protein was incubated with venetoclax (serial titration of 1E-6 to5E-11 M in DMSO) at variable peptide concentrations (1E-6 to 5E-11 M; ca.1.5-fold peptideKD; see above) for 15 min. The fixed concentrations of fBAK peptide were added (seecolumn 3 in Table 5). After 10 min incubation, anti-GST-Tb cryptate HTRF reagent wasadded and incubated for 1 hour before measurement of the HTRF signal (excitation: 337nm, emission: 492-8 nm and 520-8 nm). Each measurement was performed twice withduplicates. Venetoclax IC50 values were determined as venetoclax concentrations at 50%of the maximum signal. Results are shown in Table 6 (average of two replicates).Table 6: Average IC50 of venetoclax binding to recombinant, human BCL-2 Peptide conc.av Loss of venetoclax Protein name KD to fBak for IC50 eragebinding compared[nM] determinationvenetoclax IC50[M to WT, fold [nM] ] Bcl-2 wildtype 56.6 100 1.87E-09 1.0Bcl-2 A100V 21.4 35 3.98E-09 2.1Bcl-2 G101V 104.8 125 1.27E-08 6.8Bcl-2 G101I 107.3 120 1.01E-08 5.4Bcl-2 G101S 58.8 85 2.24E-09 1.2Bcl-2 G101T 42.6 60 2.66E-09 1.4Bcl-2 G101L 146.3 150 2.06E-08 11.0Bcl-2 D103Y 26.5 35 2.16E-08 11.5Bcl-2 D103I 36.9 50 1.39E-08 7.4Bcl-2 D103V 39.5 50 6.38E-09 3.4Bcl-2 D103E 48.8 85 5.76E-09 3.1Bcl-2 D103F 39.6 50 1.89E-08 10.1Bcl-2 D103K 57.6 90 1.45E-08 7.7Bcl-2 D103L 37.3 60 2.86E-08 15.3Bcl-2 F104L 77.0 100 1.06E-08 5.7Bcl-2 F104V 48.1 70 1.66E-07 88.7Bcl-2 F104S 162.6 200 2.65E-07 141.6Bcl-2 F104A 33.2 50 3.75E-07 200.7Bcl-2 F104C 47.9 70 3.85E-07 205.9Bcl-2 F104T 44.1 70 2.79E-07 149.5Bcl-2 F104G 263.3 150 2.04E-07 109.3Bcl-2 F104H could not be tested due to low peptide affinityBcl-2 F104N 1200 200 1.89E-07 101.2Bcl-2 Y108F 63.16 100 2.23E-09 1.2Bcl-2 Y108S 997.8 300* 1.75E-08 9.3Bcl-2 V156N 108.6 150 4.57E-09 2.4Bcl-2 V156Q 97.2 125 3.97E-09 2.1Bcl-2 V156L 264 200 1.80E-09 1.0Bcl-2 V156M 102.6 125 1.81E-09 1.0Bcl-2 Y202A could not be tested due to low peptide affinityBcl-2 Y202S could not be tested due to low peptide affinityOf all tested BCL-2 variants, only G101V, G101I, G101L, D103I, D103Y, D103F, D103K,D103L, F104L, F104V, F104S, F104A, F104C, F104I, F104T, F104G, F104N and Y108S showeda more than 5-fold reduction of venetoclax binding, with G101I and F104L having ca 5- to6-fold reduced IC50 and F104V, F104A, F104C, F104T ranging from 75- to 210-fold affinityreduction. Except for G101L, F104S, F104G, F104N, and Y108S all of these variants boundthe BAK peptide within 2-fold of the wildtype BCL-2 KD.Example 6: Determination of KD between BCL-2 variants to BAX, BIM and small moleculeinhibitors by SPRDirect binding of BCL-2 wildtype and variants to BAX, BIM in peptide formats (Amino acidsequences are shown in Table 7) and to RefSM#1-6 was measured with a Biacore S200 system at25 °C using 1x HBS EP+ (Cytiva, PN: BR100669) with or without 1mM TCEP in single cycle kinetic mode. GST tagged BCL-2 WT and variants were captured using GST capture kit (Cytiva, PNBR100223) onto CM4 or CM5 sensor chip (Cytiva, PN: 29104988, 29104989) using followingconditions: 5µL / min, 20ug / mL, 30-180s injection. After washing with Biacore running buffer, BAX,BIM and RefSM#1-6 were injected at 50 μl / min for 1 min at 4 consecutive increasing concentrations,from 0.78 to 50nM for BIM, from 3.1 to 200nM for BAX and from 1 to 1000nM for small moleculeinhibitor. Dissociation was monitored for 15 minutes to 20 minutes injecting running buffer at50µl / min. Sensor chip was regenerated using 2min injection of 10mM glycine-HCl pH 1.7. Bindingkinetics were analysed using BIAevaluation software with solvent and blank correction. Results areshown in Table 8 (standard deviation in brackets).Table 7: Peptide Amino acid tag-sequenceBAX biot-Ahx-ADASTKKLSECLKRIGDELDSNMELQRMIAA (SEQ ID No.2)BIM biot-Ahx-DMRPEIWIAQELRRIGDEFNAYYARR (SEQ ID No.3)Table 8: Protein KD to BIM (nM) KD to BAX (nM)WT 0.22 12.45G101V 0.33 15.3 (4.38)G101I 0.30 19.2G101S 0.20 8.5G101T 0.18 9.2G101L 0.11 not testedD103E 0.15 6.9D103Y 0.20 26.3D103V 0.15 12D103F 0.51 not testedD103K 0.26 not testedD103L 0.09 not testedF104L 0.08 15.1 (2.40)F104V 0.17 9.85 (4.60)F104S 1.99 65.5 (2.12)F104A 0.47 22.5 (19.09)F104G 1.56 not testedF104I 0.05 52F104T 0.14 not testedF104H 2.04 not testedF104N 1.43 not testedAmong all the elected variants only D103F, D103L, F104L, F104S, F104G, F104I, F104H,F104N had an affinity that to BIM and / or BAX increased or decreased by two-fold or morecompared to wildtype. Mutations G101V, G101I, G101S, G101T G101L, D103E, D103Y,D103V, D103K, F104V, F104A, F104T and Y202S had a minor impact on the binding to BIMand BAX compared to wildtype. Table 9 summarizes the variants that significantly reduce (>75-fold) venetoclax bindingwhile retaining BAK binding within a factor of 2-fold and BIM and BAX binding within afactor of 5-fold. Table 9:Residue Mutation NaturalLoss of BAK Bax BIM binding resistance venetoclax binding binding (KD, x-fold vs. mutation binding (KD, x- (KD, x- wt) (IC50, x- fold vs. fold vs. fold vs. wt) wt) Wt) A no 201x 0.6 1.8 2.1 C yes 206x 0.8 n.a. n.a F104 V no 89x 0.9 0.8 0.8 T no 150x 0.8 4.2 0.6Example 7: Expression of recombinant wildtype and variant BCL-2 and cytotoxicity assayin human cell lines KASUMI-1, MOLM-13, MOLM-14, MV4-11, THP-1 and OCI-AML2 human cell lines were seeded at 10,000 cells per well in clear, sterile 96-well microtiter plates and incubated for1 hr at 37°C, 5% C02. Venetoclax, navitoclax and AZD4320 (10 mM stock solutions in DMSO)were added to the cells at concentrations between 2 nM and 10 uM. After 72 hrs incubation at 37°C, 5% C0250 µL CellTiter-Glo2.0 was added to each well and luminescence wasmeasured after 10 min at RT. The resulting proliferation curves and IC50 values are shownin Figure 1 and Table 10. Table 10 summarizes the half-maximal inhibitory concentrations(IC50) in µM calculated for each cell line and BCL-inhibitor . MV4-11 and KASUMI-1 cell lineshad the highest sensitivities to all three BCL-2 small molecule inhibitors and are thereforeparticularly well suited for functional proliferation and apoptosis assays. THP-1 has a highsensitivity to AZD4320. Table 10:Figure 2 shows results of the titration of cell lines to various BCL-2 inhibitors. Shown areresults of a CellTiter Glo Luminescent assay that analyzes cell viability of HCT-116, Molm-14, Kasumi-1 and THP-1 cells treated with increasing concentrations of Venetoclax, Navitoclax, AZD4320, Lisaftoclax or S55746 for 72h in culture. Luminescence signal was detected in EnVision multimode plate reader and normalized to control DMSO-treated cells (100%). Another cell line sensitive to venetoclax treatment is the cell line HCT-116 (Zeng C et al.,Experimental Cell Research 414, 2022). Venetoclax sensitive HCT-116 and THP-1 cell linesare also available as BCL-2 knock-out derivatives. Wildtype and variant BCL-2 were clonedinto standard plasmid and recombinantly expressed in BCL-2- / - HCT-116 or THP-1 cells.BCL-2 expression was confirmed by Western blot. Venetoclax, navitoclax, sonrotoclax,lisaftoclax, S55746, and AZD4320 will be titrated on these cells and viable cells will bequantified after 72 hours incubation using CellTiter-Glo2.0. Example 8: Generation of BCL-2 variants by gene editing in human cancer cell lines Certain BCL-2 variants were generated via base editing. Each base editor complex comprised a single guide RNA with a target site-specific spacer sequence (sgRNAs; Synthego; Science (2020) 368: 290-6) combined with either ABE8e(TadA-8e V106W)-Spry (PAM 5’-NNN-3’; Trilink;) or ABE8e(TadA-8e V106W)-NG (PAM 5’-NGN-3’; Trilink; Science (2020) 38: 290-6) base editor mRNA. SgRNAs were designed to overlap each target aminoacid and enable local A-to-G transitions via base editing that would thereby change thewild-type amino acid to a variant of interest. Certain BCL-2 variants were generated via HDR-mediated editing. A ribonucleoprotein (RNP) complex was employed, comprising an sgRNA with a target site-specific spacer (Synthego) as well as SpyFi Cas9 (PAM 5’-NGG-3’; Aldevron), to generate a double-stranded break near the target codon. The RNP was combined with a single-stranded oligodeoxynucleotide (ssODN) repair template that introduces the edit, yielding the respective amino acid variant through homology-derived repair of double-stranded break site. RNPs were also employed to generate a double-stranded break at exon 2 which, without providing a repair template, resulted in error-prone NHEJ-mediated repair and knock-out of BCL-2 expression. The variants generated via base editing and the sgRNA sequences and base editor format used are described in Table 11. The variants generated via HDR-mediated editing and the sgRNA sequences and ssODN sequences are described in Table 12. Table 11: Codon Target Residue (SEQ amino Strand sgRNA sequence 5'-3' (SEQ ID PAM (SEQ ID acid ID No.) ID No.) No.) sg165 + GAAGTCGTCGCCGGCCTGGC (4) GGA (5)sg159F104 GAA S / L / P+ GCGGCGGGAGAAGTCGTCGC (6) CGG (7)(12)sg166 + GAGAAGTCGTCGCCGGCCTG (8) GCG (9)sg167 + GGAGAAGTCGTCGCCGGCCT (10) GGC (11)Table 12: Codon Target Residue (SEQ ID aminosgRNA ID StrandssODN sequence 5'-3' (SEQ ID No.) acid No.) CTGCGCTCAGCCCGGTGCCACCT GTGGTCCACCTGACCCTCCGCCA sg159 +GGCAGTAGACGACTTCTCCCGCC GCTACCGCCGCGACTTCGCCGAG ATGTCCAGCCAGCTGCACCT (13) G101 GGC (11) VAGGTGCAGCTGGCTGGACATCTC GGCGAAGTCGCGGCGGTAGCGG sg160 +CGGGAGAAGTCGTCTACTGCCTG GCGGAGGGTCAGGTGGACCACA GGTGGCACCGGGCTGAGCGCAG (14) AGGTGCAGCTGGCTGGACATCTC GGCGAAGTCGCGGCGGTAGCGG F104 TTC (15) A sg159 +CGGGATGCATCATCGCCGGCCTG GCGGAGGGTCAGGTGGACCACA GGTGGCACCGGGCTGAGCGCAG (16) AGGTGCAGCTGGCTGGACATCTC GGCGAAGTCGCGGCGGTAGCGG sg160 CGGGATGCATCATCACCTGCCTG GCGGAGGGTCAGGTGGACCACA GGTGGCACCGGGCTGAGCGCAG (17) AGGTGCAGCTGGCTGGACATCTC GGCGAAGTCGCGGCGGTAGCGG sg159 CGGGAAAGATCATCGCCGGCCTG GCGGAGGGTCAGGTGGACCACA GGTGGCACCGGGCTGAGCGCAGF104 TTC (15) L(18) AGGTGCAGCTGGCTGGACATCTC GGCGAAGTCGCGGCGGTAGCGG sg160 CGGGAAAGATCATCACCTGCCTG GCGGAGGGTCAGGTGGACCACA GGTGGCACCGGGCTGAGCGCAG (19) AGGTGCAGCTGGCTGGACATCTC GGCGAAGTCGCGGCGGTAGCGG sg159 CGGGACGTATCATCGCCGGCCTG GCGGAGGGTCAGGTGGACCACA GGTGGCACCGGGCTGAGCGCAGF104 TTC (15) T(20) AGGTGCAGCTGGCTGGACATCTC GGCGAAGTCGCGGCGGTAGCGG sg160 CGGGACGTATCATCACCTGCCTG GCGGAGGGTCAGGTGGACCACA GGTGGCACCGGGCTGAGCGCAG (21) AGGTGCAGCTGGCTGGACATCTC GGCGAAGTCGCGGCGGTAGCGG sg159 CGGGATACATCATCGCCGGCCTG GCGGAGGGTCAGGTGGACCACA GGTGGCACCGGGCTGAGCGCAGF104 TTC (15) V(22) AGGTGCAGCTGGCTGGACATCTC GGCGAAGTCGCGGCGGTAGCGG sg160 CGGGATACATCATCACCTGCCTG GCGGAGGGTCAGGTGGACCACA GGTGGCACCGGGCTGAGCGCAG (23)Gene editing was either performed with a Lonza 4D Nucleofector or Thermo Fisher Neon.For the Lonza 4D Nucleofector, gene editing reagents were mixed with MV4-11 and Kasumi-1 cells and nucleofection buffer (SG; Lonza) and the cell suspension mix was then electroporated with the DK-100 program. Immediately after electroporation, pre-warmed medium was added directly in the receptacles and incubated for 20 minutes at 37°C for cells to recover optimally. For the Thermo Fisher Neon, gene editing reagents were mixed with Kasumi-1 cells and nucleofection buffer R. The electroporation was performed in100 µl tips with the following conditions: 1350 V, 35 ms, 1 pulse. Immediately afterelectroporation, cells were transferred into pre-warmed medium. Electroporated conditions were cultured in the presence of AZD7648 (0.4 µM) to increase HDR efficiency. AZD7648 was washed out 48 hours after electroporation and medium without AZD7648 was added. The medium was renewed every 48 hours. 5 days post-electroporation, bulk cells of each condition were spun down for 5 min at 500 RCF. Pellets were resuspended in 200 µl PBS and gDNA was isolated with column-based purification kits such as DNeasy Blood and Tissue kit (Quiagen). gDNA of each condition was subjected to PCR reaction (1) with primers Fwd-1 and Rev-1 to amplify across BCL-2. PCR product 1 was then either column-purified and sequenced by Sanger sequencing (Microsynth) using primer Fwd-1 or further amplified towards Next-generation sequencing (NGS, Miniseq, Illumina) in PCR reaction (2) with primers with manufacturer’s Nextera primers (Illumina) (see Table 13). Table 13: Primer Sequence (5’-3’) (SEQ ID No.) Target Amplicon SizeFwd-1GTGCCGGTTCAGGTACTCAG (24) BCL-2 426 bpp186 Rev-1ACAGGGTACGATAACCGGGA (25)p189 Fwd-2 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNNBCL-2 279 bpp207NNNTCCCTGAAGAGCTCCTCCAC (26) Rev-2 GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGN p200NNNNGCATCTTCTCCTCCCAGCC (27)Editing efficiency and purity are quantified from Sanger sequencing reads using EditR(CRISPR J. (2018) 1:239-250), or from Mini-seq FastQ files using CRISPRESSO (Clement et al (2019). Nat Biotech). Starting from day 7 - 13 post-electroporation depending on the grows of cells, cells werecultured in the presence of Venetoclax (0.1 – 10 µM) to enrich for edited cells. Cell viabilityand proliferation was assessed on day 3 of Venetoclax treatment. Up to three rounds of Venetoclax selection were conducted with indicated concentrations. After the last selection, gDNA was isolated as described above and the editing efficiency and purity was quantified from Sanger sequencing and Mini-seq FastQ files (Figure 3).HSPCs expressing BCL-2 variants exhibit a significant decrease in sensitivity to the BCL-2 inhibitors compared to wildtype cells. All investigated variants are amenable to editing. Experimental conditions may need tobe optimized, which is a matter of routine experimentation. Example 9: Introduction of BCL-2 variants by gene editing renders human cell lines resistant to killing by BCL-2 small molecule inhibitors To assess the sensitivity of MV411 or Kasumi-1 cells expressing the BCL-2 variants to thedifferent BCL-2 small molecule inhibitors, cell viability is measured in CTG and Incucyteassays. Briefly, for CTG measurements 3.000 cells per well are seeded in clear, sterile 384- well microtiter plates and incubated for 1 hr at 37°C, 5% C02. Venetoclax, navitoclax,lisaftoclax, AZD4320, sonrotoclax, and S55746 are added via Tecan D300eCONTROL to thecells at concentrations between 0.05 nM and 10 uM. After 72 hrs incubation at 37°C, 5%C0250 µL CTG is added to each well and luminescence is measured after 15-30 min at RT.For Incucyte analysis, 10,000 cells are seeded per well in 96-well plates flat bottom in thepresence of Cytotox Red dye at 1 / 4000 dilution. Venetoclax, navitoclax, lisaftoclax,AZD4320, sonrotoclax and S55746 are manually titrated on the cells at concentrationsbetween 0.05 nM and 10 µM. 4 images of 20x resolution are taken every 4 hours for a totalof 4 days.Example 10: Generation of BCL-2 variants by gene editing in primary CD34-positivehuman cells hCD34 HSPCs were enriched from healthy donor mobilized leukapheresis product (CytoCare) via the LP-34 process on the CliniMACS Prodigy (Miltenyi). hCD34+ HSPCs were thawed and resuspended in pre-warmed HSPC complete medium (StemSpan SFEM II (StemCell #09655) + 100 IU / mL hSCF (Cellgenix #1018-050) + 67 IU / mL hFlt3-Ligand (Cellgenix #1015-1000) + 5000 IU / mL hTPO (Cellgenix #1017-1000) + 132 IU / mL hIL3 (Cellgenix #1002-050)) and incubated as static culture in Vuelife 72-C (Cellgenix) for two days pre-electroporation. Certain BCL-2 variants were generated via base editing as described in Example 8. Certain BCL-2 variants were generated via HDR-mediated editing as described in Example 8. The variants generated via base editing and the sgRNA sequences and base editor format used are described in Table 11. The variants generated via HDR-mediated editing and the sgRNA sequences and ssODN sequences are described in Table 12. HSPCs were resuspended in Maxcyte electroporation buffer to a final viable cell density of 60 e6 / mL and mixed with 5 uM sgRNA and 0.5 ug ABE mRNA per condition in a final volume of 50 µL. The suspension was then pipetted into R50x3 cuvettes (Maxcyte) and electroporated using the GTx nucleofector (Maxcyte) via the HSC2 or HSC3 program. Immediately after electroporation, 950µL of pre-warmed complete stem cell medium was added directly in the R50x3 cuvettes and they were incubated for 20 minutes at 37°C for cells to recover. Edited cells were then resuspended in complete Stemspan medium to a cell density of 0.5 e6 / mL and incubated as static culture for 3 days. Five days post- electroporation, gDNA from bulk cells is isolated and PCR amplification and Sanger sequencing or NGS are performed on the target region (see Example 8). Editing results are then quantified as described in Example 8.Example 11: Introduction of BCL-2 variants by gene editing renders human CD34+ cellsresistant to killing by BCL-2 small molecule inhibitors Human CD34+ cells are gene edited as described above using base editing, prime editing or HDR to introduce the relevant variants. The edited CD34+ cells are used as either bulk cells or sorted to enrich edited cells carrying the respective BCL-2 variant. Approximately 103 to 104 engineered human CD34+ cells are seeded in 96 well plates in100 µl of medium supplemented with cytokines hSCF, hFLT3L, hTPO, hIL-3, hIL-6, hGM-CSF,hEPO, hIL-2, hIL-7 and hIL-11 to promote expansion and differentiation. Cells are incubated for 3-5 days at 37°C with serial dilutions of Venetoclax, navitoclax, lisaftoclax, AZD4320 andS55746 at concentrations ranging between 0.05 nM and 10 uM. At the end of theincubation period, 100µl of CTG (Promega Cat Nr: G9241) is added to each well andluminescence is measured after 15-30 min incubation at RT. Non-targeted control (NTC)edited CD34+ cells expressing wildtype BCL-2 cells serve as positive control for maximum killing.Alternatively, edited bulk CD34+ cells are mixed with Cytotox Red and treated with the BCL-2 small molecule inhibitors as described above for Incucyte analysis. Briefly, 10,000 cellsare seeded per well in 96-well plates flat bottom in the presence of Cytotox Red dye at1 / 4000 dilution. Venetoclax, navitoclax, lisaftoclax, AZD4320, sonrotoclax and S55746 are manually titrated on the cells at concentrations ranging between 0.05 nM and 10 uM. 4images of 20x resolution are taken every 4 hours for a total of 4 days. In both assays, non-targeted control (NTC) edited CD34+ cells expressing wildtype BCL-2 cells serve as positivecontrol for maximum killing. HSPCs expressing BCL-2 variants exhibit a significant decreasein sensitivity to the BCL-2 inhibitors compared to wildtype cells. Example 12: In vivo experiment Human CD34+ HSPCs are isolated from immobilized leukopaks (CytoCare) via the LP-34process on the CliniMacs Prodigy (Miltenyi). The human CD34+ HSPCs are edited to introduce BCL-2 variants as described above. Electroporation-only / mock-edited HSPCs areused as wildtype control cells. Two days after editing ca.0.5 - 1 mio HSPCs are transplanted each into immunodeficient mice (e.g., strain NBSGW (NOD.Cg-Kit W-41J Tyr + Prkdc scid Il2rg tm1Wjl / ThomJ), NSG (NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ), NSG-SGM3 (NOD.Cg- Prkdcscid Il2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav / MloySzJ) or NSG-SGM3-IL-15 (NOD.Cg- Prkdcscid Il2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav Tg(IL15)1Sz / J). Ca 14-20 weeks afterengraftment venetoclax, navitoclax, lisaftoclax, AZD4320, sonrotoclax and / or S55746 isadministered to the mice. Control groups of mice receive the vehicle. Depletion andshielding of BCL-2 wildtype and variant human cells is monitored in blood, bone marrowand spleen using flow cytometry and next generation sequencing. The differentiation ofBCL-2-edited HSPCs into lymphocytes will be assessed by quantifying human immune cellsubsets in the mice.Results show that venetoclax, navitoclax, lisaftoclax, AZD4320, sonrotoclax and / orS55746 deplete unedited (or wild-type) cells, while edited cells are shielded and enrichedin mice. Edited HSPCs show comparable immune cell development to WT HSPCs.Example 13: Venetoclax depletes leukemic cells, while sparing variant BCL-2 expressinghematopoietic cells BCL-2 variant CD34+ HSPCs or WT HSPCs are transplanted into immunodeficient mousestrains as described in example 12.8-15 weeks after HSPC engraftment mice additionally receive leukemia cells, e.g. a leukemia cell line or patient derived leukemia cells. Tumor growth is monitored by flow cytometry of blood samples and / or by total body bioluminescence imaging. Venetoclax, navitoclax, lisaftoclax, AZD4320, sonrotoclax and / or S55746 are administered to mice upon stable tumor engraftment. Control groups of mice receive the vehicle. Animals experiencing continuous tumor growth will be euthanized based on clinical scoring criteria. The spleen, blood and bone marrow will be analyzed for human tumor cells and non-tumor cells by flow cytometry. Tumor free animals will be terminated after a reasonable time without detectable tumor cells. Results show that venetoclax, navitoclax, lisaftoclax, AZD4320, sonrotoclax and / or S55746 deplete tumor cells and enhances survival of the mice, while edited cells are shielded from depletion and drive normal haematopoiesis.

Claims

CLAIMS1. A mammalian cell or a population of cells expressing a first isoform of BCL-2 for use in amedical treatment in a patient in need thereof, said patient having cells expressing a second isoform of BCL-2, wherein said cell expressing said first isoform comprises a polymorphism or geneticallyengineered allele characterized by at least one substitution of an amino acid at positionG101, D103 or F104 of SEQ ID NO: 1 that is not present in the genome of the patienthaving cells expressing said second isoform of BCL-2.

2. The mammalian cell or population of cells for use according to claim 1, wherein said first and second isoform are substantially functionally identical and / or are expressed at thesame level or substantially the same level, preferably wherein said function is the induction of apoptosis, the binding of BH3-only proteins (such as Bim and Bad) or thebinding of pro-apoptotic proteins (such as Bax).

3. The mammalian cell or population of cells for use according to any one of the precedingclaims, wherein said residue G101 is substituted with V, I or L, and / or residue D103 issubstituted with I, Y, F, K or L, and / or residue F104 is substituted with A, V, C, T, G, I, Lor S.

4. The mammalian cell or population of cells for use according to claim 3, wherein said residue F104 is substituted with A, V, C or T, preferably A or V.

5. The mammalian cell or population of cells for use according to any one of the precedingclaims, wherein said first isoform of BCL-2 is obtained by in vivo or ex vivo modifyingthe nucleic acid sequence encoding said first isoform of BCL-2 by gene editing,preferably by introducing into a cell a gene editing enzyme capable of inducing site-specific mutations(s) within a target sequence encoding a protein region involved in the binding of agent comprising at least a first antigen-binding region.

6. The mammalian cell or population of cells, preferably hematopoietic stem cells for useaccording to any one of the preceding claims wherein said medical treatment comprisesadministering a therapeutically efficient amount of said cell or population of cells expressing said first isoform of BCL-2 to said patient in need thereof, in combinationwith a therapeutically efficient amount of a depleting agent that binds specifically to said second isoform of BCL-2 to specifically deplete patient cells expressing said secondisoform of BCL-2, preferably to restore normal haematopoiesis after targeted therapy in the treatment of hematopoietic disease, and preferably in the treatment of malignant hematopoietic disease such as acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (PMF), blastic plasmacytoid dendritic cell neoplasm (BPDCN) and other myeloproliferative neoplasms.

7. The mammalian cell or population of cells, preferably hematopoietic stem cells for use according to any one of the preceding claims wherein said medical treatment comprisesadministering a therapeutically efficient amount of said cell or population of cells expressing said first isoform of BCL-2 to said patient in need thereof, in combination with a therapeutically efficient amount of a depleting agent that binds specifically to said second isoform of BCL-2 to specifically deplete patient cells expressing said second isoform of BCL-2, preferably to restore normal haematopoiesis after targeted therapy in the treatment of a solid cancer, and preferably in the treatment of non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC) prostate cancer, ovarian cancer, triple negative breast cancer (TNBC), colorectal cancer, head and neck cancer or breast cancer.

8. The mammalian cell or population of cells for use according to claim 6 or 7, wherein saiddepleting agent is a small molecule, preferably a BCL-2 inhibitor.

9. The mammalian cell or population of cells for use according to claim 8, wherein said BCL-2 inhibitor is selected from venetoclax (ABT-199), navitoclax (ABT-263), AZD-4320, AZD-0466, lisaftoclax (APG2575), S55746, sonrotoclax, ABT- 731, ABT-737, apogossypol,Ascenta’s pan-BCL-2 inhibitors, curcumin (and analogs thereof), G3139), HA14-1, NH-1,obatoclax, or any derivative or analog thereof.

10. The mammalian cell or population of cells for use according to claim 6 or 7, whereinsaid depleting agent comprises an antigen-binding region that binds specifically to said second isoform of BCL-2.

11. A pharmaceutical composition comprising a mammalian cell, preferably a hematopoietic stem cell or an immune cell such as T-cell, as defined in any one of claims1 to 10, and preferably a depleting agent as defined in any one of claims 6 to 10, and apharmaceutically acceptable carrier.

12. A depleting agent for use in preventing or reducing the risk of severe side effects in a patient having received a cell expressing a first isoform of BCL-2, wherein said patient’s native cells express a second isoform of BCL-2, and wherein said depleting agent binds specifically to said first isoform of BCL-2 and does not bind or binds substantially weakerto said second isoform of BCL-2.

13. A BCL-2 inhibitor for use in selectively depleting the host cells in a patient in needthereof, wherein said patient’s native cells express a second isoform of BCL-2, whereina mammalian cell or a population of cells expressing a first isoform of BCL-2 isadministered to said patient prior to, concomitant with or after said BCL-2 inhibitor,and wherein said BCL-2 inhibitor specifically binds to said second isoform of BCL-2, butnot (or substantially weaker), to said first isoform of BCL-2.

14. A BCL-2 inhibitor for use according to claim 13, wherein said BCL-2 inhibitor binds to apolypeptide comprising the amino acid sequence of SEQ ID No. 1, but not (or substantially weaker), to a polypeptide comprising the amino acid sequence of SEQ ID No. 1, said polypeptide comprises at least one substitution of an amino acid at positionG101, D103 or F104.

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