Sustained transgene expression of IMID-responsive peptide-suicide protein fusion polypeptides and uses thereof

Abstract

Provided herein are targeting constructs for sustained transgene expression of inducible cell death systems that include degron mutants and cereblon mutants. Also provided are pharmaceutical compositions comprising the targeting constructs, and methods for use of the same. The methods of use include methods of inducing cell death in a cell, and methods of treating patients in need thereof.

Description

SUSTAINED TRANSGENE EXPRESSION OF IMID-RESPONSIVE PEPTIDE-SUICIDEPROTEIN FUSION POLYPEPTIDES AND USES THEREOF1. CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of priority under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 678,385, filed August 1, 2024. The disclosure of the prior application is considered part of and is herein incorporated by reference in the disclosure of this application in its entirety.2. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing XML which has been submitted electronically and is hereby incorporated by reference in its entirety. Said XML copy, created on Month XX, 20XX, is named XXXXXXX, and is XXX, XXX bytes in size.3. BACKGROUND

[0003] Currently available cell and gene therapy products can lack control of function, which can lead to safety concerns such as toxicity in subjects that receive the therapies. Thus, additional methods of controlling and regulating these therapies are needed.4. SUMMARY

[0004] Disclosed herein, in various embodiments, is a targeting construct comprising: (a) a first homology arm corresponding to a 5' target sequence comprising a first region of homology to a target genomic locus; (b) a nucleotide insert comprising a nucleotide sequence encoding a fusion polypeptide comprising: (i) a modified degron; and (ii) a caspase 9 domain or derivative or functional fragment thereof; (c) a second homology arm corresponding to a 3' target sequence comprising second region of homology to the target genomic locus, wherein the modified degron comprises an amino acid sequence corresponding to a SEQ ID NO: 1 or a variant thereof, optionally wherein the modified degron comprises a mutation selected from the group consisting of G30E and R27H, wherein the targeting construct is configured such that upon its recombination with the target genomic locus, the fusion polypeptide coding sequence is integrated into a STEL gene and becomes operably linked to the STEL gene regulatory element. In some embodiments, the targeting construct further comprises a modified cereblon (CRBN) and a second caspase 9 domain or derivative or functional fragment thereof, wherein the modified CRBN comprises an amino acid sequence corresponding to a SEQ ID NO: 4 or a variant thereof. In some embodiments, the modified degron comprises a mutation selected from the group consisting of 137 IN, Q325H, T359V, Q325R, and H397F.

[0005] Also disclosed herein, in various embodiments, is a targeting construct comprising: (a) a first homology arm corresponding to a 5' target sequence comprising a first region of homology to a target genomic locus; (b) a nucleotide insert comprising a nucleotide sequence encoding a fusion polypeptide comprising: (i) a modified cereblon (CRBN); and (ii) a caspase 9 domain or derivative or functional fragment thereof; (c) a second homology arm corresponding to a 3' target sequence comprising second region of homology to the target genomic locus, wherein the modified CRBN comprises an amino acid sequence corresponding to a SEQ ID NO: 4 or a variant thereof, optionally wherein the modified degron comprises a mutation selected from the group consisting of I371N, Q325H, T359V, Q325R, and H397F, wherein the targeting construct is configured such that upon its recombination with the target genomic locus, the fusion polypeptide coding sequence is integrated into a STEL gene and becomes operably linked to the STEL gene regulatory element. In some embodiments, the nucleotide insert further comprises a modified degron and a second caspase 9 domain or derivative or functional fragment thereof.

[0006] Also disclosed herein, in various embodiments, is a targeting construct comprising: (a) a first homology arm corresponding to a 5' target sequence comprising a first region of homology to a target genomic locus; (b) a nucleotide insert comprising a nucleotide sequence encoding a fusion polypeptide comprising: (i) a modified degron, wherein the modified degron comprises an amino acid sequence corresponding to a SEQ ID NO: 1 or a variant thereof, optionally wherein the modified degron or variant comprises a mutation selected from the group consisting of G30 or R27; (ii) a first caspase 9 domain or derivative or functional fragment thereof; (iii) a modified CRBN, wherein the modified CRBN comprises an amino acid sequence corresponding to a SEQ ID NO: 4 or a variant thereof, optionally wherein the modified degron comprises a mutation selected from the group consisting of 1371, Q325, T359, and H397; (iv) a second caspase 9 domain or derivative or functional fragment thereof; (c) a second homology arm corresponding to a 3' target sequence comprising second region of homology to the target genomic locus, wherein the targeting construct is configured such that upon its recombination with the target genomic locus, the fusion polypeptide coding sequence is integrated into a STEL gene and becomes operably linked to the STEL gene regulatory element.

[0007] Also disclosed herein, in various embodiments, is a targeting construct comprising: (a) a first homology arm corresponding to a 5' target sequence comprising a first region of homology to a target genomic locus; (b) a nucleotide insert comprising a nucleotide sequence encoding a fusion polypeptide comprising: (i) a modified degron; (ii) a first caspase 9 domain or derivative or functional fragment thereof; (iii) a modified cereblon; and (iv) a second caspase 9domain or derivative or functional fragment thereof; (c) a second homology arm corresponding to a 3' target sequence comprising second region of homology to the target genomic locus, wherein the modified degron comprises an amino acid sequence corresponding to a reference degron sequence (SEQ ID NO: 1) or a variant thereof, wherein the modified cereblon comprises an amino acid sequence corresponding to a reference cereblon sequence (SEQ ID NO: 4) or a variant thereof. In some embodiments, the modified degron comprises at least one amino acid substitution selected from the group consisting of G30E and R27H. In some embodiments, the modified cereblon comprises at least one amino acid substitution selected from the group consisting of 137 IN, Q325H, T359V, Q325R, and H397F. In some embodiments, the targeting construct is configured such that upon its recombination with the target genomic locus, the fusion polypeptide coding sequence is integrated into a STEL gene and becomes operably linked to the STEL gene regulatory element.

[0008] In some embodiments, the modified CRBN comprises one or more amino acid substitutions selected from the group consisting of 137 IN, Q325H, T359V, Q325R, and H397F relative to SEQ ID NO: 4. In some embodiments, the modified CRBN comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 5-8. In some embodiments, the modified CRBN comprises an 137 IN amino acid substitution relative to SEQ ID NO: 4. In some embodiments, the modified CRBN comprises Q325H, 137 IN, and T359V amino acid substitutions relative to SEQ ID NO: 4. In some embodiments, the modified CRBN comprises Q325R, 137 IN, H397F, and T359V amino acid substitutions relative to SEQ ID NO: 4.

[0009] In some embodiments, the modified degron comprises an amino acid substitution selected from the group consisting of G30E and R27H, relative to SEQ ID NO: 1. In some embodiments, the modified degron comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, the modified degron comprises a R27H amino acid substitution relative to SEQ ID NO: 1. In some embodiments, the modified degron comprises a G30E amino acid substitution relative to SEQ ID NO: 1. In some embodiments, the modified degron comprises a R27H amino acid substitution relative to SEQ ID NO: 1 and the modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the modified degron comprises a G30E amino acid substitution relative to SEQ ID NO: 1 and the modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the modified degroncomprises an amino acid sequence set forth in SEQ ID NO: 1 and the modified CRBN comprises an 137 IN amino acid substitution relative to SEQ ID NO: 4. In some embodiments, the modified degron comprises a G30E amino acid substitution relative to SEQ ID NO: 1 and the modified CRBN comprises an 137 IN amino acid substitution relative to SEQ ID NO: 4. In some embodiments, the modified degron comprises a R27H amino acid substitution relative to SEQ ID NO: 1 and the modified CRBN comprises Q325H, 137 IN, and T359V amino acid substitutions relative to SEQ ID NO: 4. In some embodiments, the modified degron comprises a R27H amino acid substitution relative to SEQ ID NO: 1 and the modified CRBN comprises Q325R, 137 IN, H397F, and T359V amino acid substitutions, relative to SEQ ID NO: 4.

[0010] In some embodiments, the modified degron is N-terminal to the caspase 9 domain or derivative or functional fragment thereof. In some embodiments, the modified degron is C- terminal to the caspase 9 domain or derivative or functional fragment thereof. In some embodiments, the modified CRBN is N-terminal to the caspase 9 domain or derivative or functional fragment thereof. In some embodiments, the modified CRBN is C-terminal to the caspase 9 domain or derivative or functional fragment thereof. In some embodiments, the fusion polypeptide comprises a linker between the modified degron and the caspase 9 domain or derivative or functional fragment thereof. In some embodiments, the fusion polypeptide comprises a linker between the modified CRBN and the caspase 9 domain or derivative or functional fragment thereof.

[0011] In some embodiments, the targeting construct is configured such that upon its recombination with the target genomic locus, the STEL gene is modified such to incorporate the fusion polypeptide coding sequence 3' to the STEL protein coding sequence. In some embodiments, the targeting construct is configured such that upon its recombination with the target genomic locus, the STEL gene is modified such to incorporate the fusion polypeptide coding sequence 5' to the STEL protein coding sequence.

[0012] In some embodiments, the nucleotide insert further comprises a nucleotide sequence encoding a separator sequence. In some embodiments, the targeting construct is configured such that upon recombination of the targeting construct with the target genomic locus, the separator sequence coding sequence is positioned between the coding sequence of the STEL protein and the fusion polypeptide coding sequence. In some embodiments, the separator sequence is an internal ribosome entry site (“IRES”). In some embodiments, the separator sequence is a selfcleaving peptide. In some embodiments, the self-cleaving peptide is a 2A peptide. In some embodiments, the self-cleaving peptide is T2A, P2A, E2A, F2A, PQR, Opt2A, or Opt2A_2.0. In some embodiments, the STEL gene encodes a polypeptide involved in one or more of:glycolysis, ribonucleopolypeptide complex formation, focal adhesion, cell-substrate adherens junction, cell-substrate junction, cell anchoring, extracellular exosome, extracellular vesicle, intracellular organelle, anchoring junction, RNA binding, nucleic acid binding (e.g., rRNA or mRNA binding), and polypeptide binding.

[0013] In some embodiments, the STEL gene encodes a ribosomal polypeptide. In some embodiments, the STEL gene is RPL13A, RPLPO, RPL10, RPL13, RPSJ8, RPL3, RPLP1, RPL15, RPL41, RPL11, RPL32, RPL18 A, RPL19, RPL28, RPL29, RPL9, RPL8, RPL6, RPL18, RPL7, RPL7A, RPL21, RPL37A, RPL 12, RPL5, RPL34, RPL35A, RPL30, RPL24, RPL39, RPL37, RPL14, RPL27A, RPLP2, RPL23A, RPL26, RPL36, RPL35, RPL23, RPL4, or RPL22. In some embodiments, the STEL gene encodes a ribosomal polypeptide small subunit (RPS). In some embodiments, the STEL gene is RPS2, RPS19, RPS14, RPS3A, RPS12, RPS3, RPS6, RPS23, RPS27A, RPS8, RPS4X, RPS7, RPS24, RPS27, RPS15A, RPS9, RPS28, RPS13, RPS A, RPS5, RPS 16, RPS25, RPS 15, RPS20, or RPS 11. In some embodiments, the STEL gene encodes a mitochondrial polypeptide. In some embodiments, the STEL gene is MT-CO1, MT-CO2, MT-ND4, MT-ND1, or MT-ND2. In some embodiments, the STEL gene encodes an actin polypeptide. In some embodiments, the STEL gene is ACTG1 or ACTB. In some embodiments, the STEL gene encodes a eukaryotic translation factor. In some embodiments, the STEL gene is EEF1A1, EEF2, or EIF1. In some embodiments, the STEL gene encodes a histone. In some embodiments, the STEL gene is H3F3A or H3F3B. In some embodiments, the STEL gene is FTL, FTH1, TPT1, IMSB10, GAPDH, PTMA, GNB2L1, NACA, YBX1, NPM1, FAU, UBA52, HSP90AB1, MYL6, SERF2, or SRP14. In some embodiments, the STEL gene is GAPDH. In some embodiments, the STEL gene is RPL 13 A. In some embodiments, the STEL gene is RPL7. In some embodiments, the STEL gene is RPLPO.

[0014] In some embodiments, the nucleotide insert further comprises a transgene. In some embodiments, the transgene is linked to the fusion polypeptide coding sequence. In some embodiments, the fusion polypeptide coding sequence and transgene are connected via a nucleotide sequence encoding a separator sequence. In some embodiments, the separator sequence is an internal ribosome entry site (“IRES”). In some embodiments, the separator sequence is a nucleotide sequence encoding a self-cleaving peptide (the “self-cleaving peptide coding sequence”). In some embodiments, the self-cleaving peptide is a 2A peptide. In some embodiments, the self-cleaving peptide is T2A, P2A, E2A, F2A, PQR, Opt2A, or Opt2A_2.0. In some embodiments, the transgene encodes a therapeutic polypeptide.

[0015] In some embodiments, the targeting construct comprises a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to a nucleic acid sequence set forth in SEQ ID NO: 19, 21, 23, 25, 27, or 29. In some embodiments, the targeting construct comprises a nucleic acid sequence set forth in SEQ ID NO: 19, 21, 23, 25, 27, or 29.

[0016] In some embodiments, the targeting construct encodes a polypeptide comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to an amino acid sequence set forth in SEQ ID NO: 18, 20, 22, 24, 26, or 28. In some embodiments, the targeting construct encodes a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 18, 20, 22, 24, 26, or 28.

[0017] Also disclosed herein, in various embodiments, is a system comprising: (a) the targeting construct disclosed herein; (b) a CRISPR-associated endonuclease (“Cas nuclease”) or a nucleic acid encoding a Cas nuclease; and (c) a guide RNA (“gRNA”) comprising a scaffold for binding the Cas polypeptide and a spacer sequence corresponding to the STEL gene, or a nucleic acid encoding the gRNA. In some embodiments, the guide RNA is a single guide RNA (“sgRNA”). In some embodiments, the system comprises the Cas nuclease and gRNA. In some embodiments, the system is in the form of a ribonucleoprotein particle (“RNP”).

[0018] Also disclosed herein, in various embodiments, is a method of producing a gene- edited target cell, comprising: (a) introducing the system disclosed herein into a target cell; and (b) culturing the target cell under conditions in which gene editing occurs, thereby producing gene-edited target cell. In some embodiments, the target cell is a stem cell, or a cell differentiated from a stem cell. In some embodiments, the target cell is a stem cell. In some embodiments, the stem cell is a human embryonic stem cell, an induced pluripotent stem cell (“iPSC”) or a cell differentiated therefrom. In some embodiments, the target cell is: (a) a regulatory T cell, a myeloid cell, a dendritic cell, a macrophage (e.g., an immunosuppressive macrophage), a myeloid progenitor cell, or a precursor or progenitor cell thereof; (b) a cell in the human nervous system, optionally selected from dopaminergic neuron, a microglial cell, an oligodendrocyte, an astrocyte, a cortical neuron, a spinal or oculomotor neuron, an enteric neuron, a Placode-derived cell, a Schwann cell, and a trigeminal or sensory neuron, or a precursor or progenitor cell thereof; (c) a cell in the human cardiovascular system, optionally selected from a cardiomyocyte, an endothelial cell, and a nodal cell, or a precursor or progenitor cell thereof; (d) a cell in the human metabolic system, optionally selected from a hepatocyte, a cholangiocyte, and a pancreatic beta cell, or a precursor or progenitor cell thereof, or (e) a cell in the human ocular system, optionally selected from a retinal pigment epithelial cell, aphotoreceptor cone cell, a photoreceptor rod cell, a bipolar cell, a ganglion cell, or a precursor or progenitor cell thereof. In some embodiments, the gene-edited target cell is of ectoderm lineage, optionally wherein the gene-edited target cell is a neuron. In some embodiments, the gene-edited target cell is of mesoderm lineage, optionally wherein the gene-edited target cell is a cardiomyocyte.

[0019] Also disclosed herein, in various embodiments, is a gene-edited target cell obtained or obtainable by the method provided herein.

[0020] Also disclosed herein, in various embodiments, is a gene-edited target cell comprising a STEL gene that comprises a nucleic acid encoding a fusion polypeptide under the transcriptional control of a STEL gene regulatory element, the fusion polypeptide comprising: (a) a modified CRBN; and (b) a caspase 9 domain or derivative or functional fragment thereof; wherein the modified CRBN comprises a mutation selected from the group consisting of 137 IN, Q325H, T359V, Q325R, and H397F.

[0021] Also disclosed herein, in various embodiments, is a gene-edited target cell comprising a STEL gene that comprises a nucleic acid encoding a fusion polypeptide under the transcriptional control of a STEL gene regulatory element, the fusion polypeptide comprising: (a) a modified degron; and (b) a caspase 9 domain or derivative or functional fragment thereof; wherein the modified degron comprises a mutation selected from the group consisting of G30E and R27H.

[0022] Also disclosed herein, in various embodiments, is a gene-edited target cell comprising a STEL gene that comprises a nucleic acid encoding a fusion polypeptide under the transcriptional control of a STEL gene regulatory element, the fusion polypeptide comprising: (a) a modified degron; (b) a caspase 9 domain or derivative or functional fragment thereof, wherein the modified degron comprises a mutation selected from the group consisting of G30E and R27H; (c) a modified CRBN, wherein the modified CRBN comprises a mutation selected from the group consisting of I371N, Q325H, T359V, Q325R, and H397F; (d) a caspase 9 domain or derivative or functional fragment thereof; wherein the modified CRBN comprises a mutation selected from the group consisting of 137 IN, Q325H, T359V, Q325R, and H397F; and wherein the modified degron comprises a mutation selected from the group consisting of G30E and R27H.

[0023] In some embodiments, the modified degron is as defined herein. In some embodiments, the modified CRBN is as defined herein. In some embodiments, the caspase 9 domain or derivative or functional fragment thereof is as defined herein. In some embodiments, the fusion polypeptide is as defined herein. In some embodiments, the STEL gene is configuredas defined herein. In some embodiments, the gene-edited target cell further comprises a transgene in the STEL gene. In some embodiments, the transgene is as defined herein. In some embodiments, the target cell is as defined herein.

[0024] Also disclosed herein, in various embodiments, is a pharmaceutical composition comprising the gene-edited target cell disclosed herein and a pharmaceutically acceptable carrier.

[0025] Also disclosed herein, in various embodiments, is a method of treating a patient in need thereof, comprising administering to the patient the gene-edited target cell or the pharmaceutical composition disclosed herein. In some embodiments, the method further comprises controlling the gene-edited target cell population in the patient by: (a) monitoring, optionally, the gene-edited target cell population in the patient; and / or (b) administering an inducer of the modified degron and / or modified cereblon if the patient experiences adverse events related to the gene-edited target cell population.

[0026] Also disclosed herein, in various embodiments, is a method of mitigating adverse events or a safety risk associated with cell therapy in the form of the gene-edited target cells or the pharmaceutical composition disclosed herein, the method comprising administering to a patient who received the gene-edited target cells or pharmaceutical composition an inducer of a degron or modified cereblon if the patient experiences adverse events or a safety risk related to the gene-edited target cells or pharmaceutical composition. In some embodiments, the inducer of the modified degron or modified cereblon is thalidomide, iberdomide, lenalidomide, or pomalidomide. In some embodiments, the inducer of the modified degron or modified cereblon is pomalidomide. In some embodiments, a combination of two or more inducers of the modified degron or modified cereblon is administered. In some embodiments, the combination has a synergistic effect and / or utilizes reduced dosing (e.g., reduced dosing amount and / or frequency) than would be required a single inducer of the modified degron or modified cereblon.5. BRIEF DESCRIPTION OF THE FIGURES

[0027] FIGS. 1A-1D are schematic diagrams of exemplary constructs of the present disclosure, comprising a nucleic acid sequence encoding a CRBN-suicide protein fusion polypeptide and a CRIMP-suicide protein fusion polypeptide and disclosed herein flanked by sustained transcription expression locus (STEL) homology arms or GAPDH homology arms.

[0028] FIG. 1A is an exemplary construct, which comprises from 5’ to 3’, a STEL left homology arm (LHA), a separator sequence (SS), a truncated Caspase 9 (truncCasp9), a cereblon domain (delCRBN), a second separator sequence (SS), a second truncCasp9, a CRIMP domain, and a STEL right homology arm (RHA). FIG. IB is another exemplary construct,which comprises from 5’ to 3’, a GAPDH left homology arm, a T2A sequence, a truncated Caspase 9 (truncCasp9), a cereblon domain (delCRBN), a second P2A sequence, a second truncCasp9, a CRIMP domain, and a GAPDH right homology arm. Modified degron peptides and Caspase-9 sequences in the constructs depicted in FIGS. 1A and IB are described in Sections 6.3.1 and 6.3.2, respectively. FIG. 1C shows an exemplary construct which comprises from 5’ to 3’, a STEL left homology arm (LHA), a separator sequence (SS), a truncated Caspase 9 (truncCasp9), a cereblon domain (delCRBN), a second separator sequence (SS), a second truncCasp9, a CRIMP domain, a third separator sequence, a transgene, and a STEL right homology arm (RHA). FIG. ID shows an exemplary construct which comprises from 5’ to 3’, a GAPDH left homology arm, a T2A sequence, a truncated Caspase 9 (truncCasp9), a cereblon domain (delCRBN), a second P2A sequence, a second truncCasp9, a CRIMP domain, an Opt2A sequences, a puromycin selection market, and a GAPDH right homology arm.

[0029] FIG. 2 shows a bright-field microscopy image of iPSCs following Nucleofection of Constructs 1-4 compared to Nucleofection of a control construct (pmaxGFP), no construct (RNP only), or cells only (No DNA / No RNP).

[0030] FIGS. 3A-3B is a graph depicting the results of a ddPCR analysis of the suicide switch encoded by Constructs 2 and 4 in the presence and absence of pomalidomide. FIG. 3A shows the fractional abundance of Construct 2 or Construct 4 in the absence or presence of a range of POM concentrations. FIG. 3B shows the fractional abundance of each sample expressed as percentage of each construct's respective untreated control sample.

[0031] FIG. 4 shows results from agarose gel electrophoresis showing DNA amplicons corresponding to left HA junction integration analysis (left side) and zygosity analysis (right side) using three different clonally derived cells lines, designated c49, c84, and c.189. Above the agarose photograph shows expected DNA amplicon size derived from designed primers.

[0032] FIG. 5 shows an experimental setup to assess the pomalidomide (POM)-response curve of the Construct 4 suicide switch integrated into clonally derived cell lines c.49, c.84, and c.189 that were generated as shown in FIG. 4.

[0033] FIGS. 6A-6D shows graphs depicting the results of the experiment shown in FIG. 5 to assess the POM dose-dependent responsiveness of the Construct 4 suicide switch. FIG. 6A shows the dose response curve of unedited iPSCs at the respective POM concentrations compared to a puromycin (puro) positive kill control. FIG. 6B shows the dose response curve of edited c.49 cells containing Construct 4 suicide switch exhibiting the homozygous genotype, compared to a puromycin (puro) positive kill control. FIG. 6C shows the dose response curve of edited c.84 cells containing Construct 4 suicide switch exhibiting the heterozygous genotype,compared to a puromycin (puro) positive kill control. FIG. 6D shows the dose response curve of edited c.189 cells containing a mistargeted Construct 4 suicide switch, compared to a puromycin (puro) positive kill control.

[0034] FIG. 7 are brightfield images of unedited iPSCs and edited cells (c.49, c.84, and c.189) expressing the Construct 4 suicide switch in the presence of either 0.01 nM or 4 nM pomalidomide at either 6 hours or 24 hours following pomalidomide exposure.

[0035] FIGS. 8A-8B shows a schematic of myeloid differentiation of hPSCs and the experimental workflow. FIG. 8A shows a schematic of myeloid differentiation. FIG. 8B shows an experimental plate layout to assess the POM dose-dependent responsiveness of the Construct 4 suicide switch in a cell line expressing the homozygous genotype (c.49) or the heterozygous genotype (c.84) after myeloid differentiation compared to unedited cells.

[0036] FIG. 9 shows a graph demonstrating the percentage cell death after induction of the Construct 4 suicide switch in myeloid cells that were differentiated from c.49 and c.84 edited iPSCs at the respective POM concentrations.

[0037] FIGS. 10A-10C show pomalidomide dose response curves of clonal edited iPSCs expressing Construct 4 (homozygous and heterozygous), a heterogenous pool of cells edited for Construct 5 after puromycin selection, or a heterogenous pool of cells edited for Construct 6 after puromycin selection, at the respective pomalidomide concentrations. FIG. 10A shows the dose response curve at 0.1 nM pomalidomide. FIG. 10B shows the dose response curve at 1 nM pomalidomide. FIG. 10C shows the dose response curve at 4 nM pomalidomide.

[0038] FIG. 11 shoes experimental overview of to assess the ability of 40mg / kg POM responsiveness of a suicide switch in vivo 4 days post-acute POM treatment (3 days).

[0039] FIG. 12 shows representative images of human cells (Ku80+) in the mouse hippocampus of WT (unedited) + vehicle (Veh), WT (Unedited) + POM, IMiD (edited) + Veh, and IMiD (edited) + POM).6. DETAILED DESCRIPTION6.1.Definitions

[0040] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. In case of conflict, the present specification, including definitions, will control. Generally, nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics,analytical chemistry, synthetic organic chemistry, medicinal and pharmaceutical chemistry, and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to manufacturer’s specifications, as commonly accomplished in the art or as described herein. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Throughout this specification and embodiments, the words “have” and “comprise,” or variations such as “has,” “having,” “comprises,” or “comprising,” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. All publications and other references mentioned herein are incorporated by reference in their entirety. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

[0041] Gene-Edited Target Cell: As used herein, the term “gene-edited target cell” refers to a cell engineered to express an IMiD responsive peptide-suicide protein fusion polypeptide of the disclosure from a STEL locus, e.g., via introduction of a targeting construct of the disclosure, or its descendants and progeny. A gene-edited target cell need not be of the same cell type as the cell into which the targeting construct was initially introduced. For example, the targeting construct may be introduced into a stem cell, such as an iPSC or a hESC, upon which the nucleotide sequence flanked by the homology arms of the targeting construct is integrated into the STEL locus of the stem cell. The stem cell can then be differentiated to produce a differentiated cell type, for example any of the cell types disclosed in Section 6.4. Both the stem cell and the differentiated cell are referred to herein as a “gene-edited target cell”. In addition to encoding an IMiD responsive peptide-suicide protein fusion polypeptide of the disclosure, the gene-edited target cell may include a transgene. In some embodiments, the transgene is introduced via the same targeting construct as the fusion polypeptide coding sequence and both the transgene and the fusion polypeptide are expressed from the same allele of the STEL gene (whether on a heterozygous or homozygous basis). In other embodiments, the fusion protein coding sequence and the transgene are positioned in different alleles of the STEL gene. In yet other embodiments, the fusion protein coding sequence is in a STEL gene and the transgene is in a different locus, whether a different STEL locus or a non-STEL locus.

[0042] iPSC: The term “induced pluripotent stem cell” or “iPSC” refers to a type of pluripotent stem cell artificially prepared from a non-pluripotent cell, such as an adult somatic cell, partially differentiated cell or terminally differentiated cell, such as a fibroblast, a cell of hematopoietic lineage, a myocyte, a neuron, an epidermal cell, or the like, by introducing orcontacting the cell with one or more reprogramming factors. iPSCs can be derived from multiple different cell types, including terminally differentiated cells. iPSCs have an embryonic stem (ES) cell-like morphology, growing as flat colonies with large nucleo-cytoplasmic ratios, defined borders and prominent nuclei. In addition, iPSCs express one or more key pluripotency markers known by one of ordinary skill in the art, including but not limited to Alkaline Phosphatase, SSEA3, SSEA4, Sox2, Oct3 / 4, Nanog, TRA160, TRA181, TDGF 1, Dnmt3b, Fox03, GDF3, Cyp26al, TERT, and zfp42.

[0043] Examples of methods of generating and characterizing iPSCs may be found in, for example, US Patent Publication Nos. US20090047263, US20090068742, US20090191159, US20090227032, US20090246875, and US20090304646, and PCT patent publications WO2013177133 and WO2022204567, the disclosures of which are incorporated herein by reference. Generally, to generate iPSCs, somatic cells are provided with reprogramming factors (e.g., Oct4, SOX2, KLF4, MYC, Nanog, Lin28, etc. known in the art to reprogram the somatic cells to become pluripotent stem cells.

[0044] Kill Switch: In the context of the present disclosure, the term “kill switch” refers to an inducible suicide gene or protein, for example a drug-inducible suicide gene or protein. Exemplary drug-inducible suicide proteins include the IMiD responsive peptide-suicide protein fusion polypeptide comprising the modified cereblon and / or the modified degron (e.g., CRIMP). The addition of appropriate drugs (e.g., IMiDs such as pomalidomide in the context of IMiD responsive peptide-suicide protein fusion polypeptides disclosed herein) triggers the suicide proteins and kills the cells that express them. These features allow clinicians to kill off exogenously administered cell therapies, for example if cytokine release syndrome (CRS) develops or the cells become tumorigenic.

[0045] Linker or Linker Sequence: The terms “linker” or “linker sequence” as used in reference to a fusion polypeptide, refers to a part that connects two or more domains, parts, or entities. In some embodiments, the linker may comprise an amino acid or a peptide. Generally, linkers have no specific biological activity other than to join or to preserve some minimum distance or other spatial relationship between the parts.

[0046] Nucleic Acid: The terms “nucleic acid” or “oligonucleotide” or “polynucleotide” as used herein means at least two nucleotides covalently linked together. The depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementary strand of a depicted single strand. Many variants of a nucleic acid may be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also encompasses substantially identical nucleic acids and complements thereof. A single strandprovides a probe that may hybridize to a target sequence under stringent hybridization conditions. Thus, a nucleic acid also encompasses a probe that hybridizes under stringent hybridization conditions.

[0047] Nucleic acids may be single stranded or double stranded, or may contain portions of both double stranded and single stranded sequence. The nucleic acid may be DNA, both genomic and cDNA, RNA, or a hybrid, where the nucleic acid may contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine. Nucleic acids may be obtained by chemical synthesis methods or by recombinant methods.

[0048] Operably linked: The term “operably linked” refers to a functional relationship between two or more peptide or polypeptide domains or nucleic acid (e.g., DNA) segments. In the context of transcriptional regulation, the term refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence. For example, a promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system.

[0049] Polypeptide, peptide, and protein: The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids.

[0050] Pluripotent: As used herein, the term “pluripotent” or “pluripotency” refers to the capacity of a cell to self-renew and to differentiate into cells of any of the three germ layers: endoderm, mesoderm, or ectoderm. “Pluripotent stem cells” or “PSCs” include, for example, embryonic stem cells derived from the inner cell mass of a blastocyst or derived by somatic cell nuclear transfer, and iPSCs derived from non-pluripotent cells.

[0051] STEL: The terms “sustained transgene expression locus” or “STEL” refer to a locus in the genome of a cell that enables persistent and stable expression of a transgene in that cell, e.g., through differentiation of the cell from one state or type to another state or type. STEL of the present disclosure include, without limitation, loci of robustly expressed endogenous genes, for instance certain housekeeping genes that are active in multiple cell types such as those involved in gene expression (e.g., transcription factors and histones), cellular metabolism (e.g., GAPDH and NADH dehydrogenase), or cellular structures (e.g., actin), or those that encode ribosomal proteins (e.g., large or small ribosomal subunits, such as RPL13A, RPLP0 and RPL7). Additional examples of STEL include those that form ribonucleoprotein complex, focal adhesion, cell-substrate adherens junction, cell-substrate junction, cell anchoring, extracellularexosome, extracellular vesicle, intracellular organelle, or anchoring junction. Some of the proteins are involved in RNA binding, nucleic acid binding (e.g., rRNA or mRNA binding), or protein binding.

[0052] STEL Protein, STEL Polypeptide: The terms “STEL protein” and “STEL polypeptide” are used interchangeably herein to refer to a polypeptide encoded by a STEL gene, or a polypeptide having at least 85% (e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) sequence identity thereto.

[0053] Target Cell: The term “target cell” refers to a host cell into which is introduced a targeting construct that following integration into the host cell genome results in the production of a recombinant nucleic acid encoding a modified degron peptide-suicide protein fusion polypeptide disclosed herein. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Such progeny need not be identical to the parent cell into which the expression vector or targeting construct was initially introduced but include counterparts and progeny of the cell which carry the expression cassette or into which the targeting construct has integrated, as well as cells differentiated therefrom. Such counterparts and progeny are still included within the scope of the term “target cell” as used herein.

[0054] Targeting Construct: The term “targeting construct” refers to a recombinant nucleic acid molecule that can specifically interact with a STEL locus and which may further comprise a nucleotide sequence encoding a modified degron peptide-suicide protein fusion polypeptide disclosed herein. Recombination of the targeting construct and the STEL locus leads to the modification of the STEL locus, e.g., to introduce an IMiD responsive peptide-suicide protein fusion polypeptide disclosed herein into the STEL locus. Typically, a targeting construct comprises homology arms that allow integration of the targeting construct into a particular STEL locus.

[0055] Transfection: The term “transfection” refers to the introduction of nucleic acid molecules, such as targeting constructs, into cells, e.g., into eukaryotic cells. In the context of the present disclosure, the term “transfection” encompasses any method known to the skilled person for introducing nucleic acid molecules into cells, e.g., into eukaryotic cells, such as into mammalian cells. Such methods encompass, for example, electroporation, nucleofection, lipofection, e.g., based on cationic lipids and / or liposomes, calcium phosphate precipitation, nanoparticle-based transfection, virus-based transfection, or transfection based on cationic polymers, such as DEAE-dextran or polyethylenimine.6.2.Targeting Constructs

[0056] The present disclosure provides targeting constructs comprising nucleotide sequences encoding an IMiD responsive peptide-suicide protein fusion polypeptides disclosed herein (“fusion polypeptide coding sequences”) flanked by homology arms that direct the integration of the fusion polypeptide coding sequences into a STEL locus in a target cell genome.

[0057] The targeting constructs may be in the form of vectors. The term “vector” as used herein refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.

[0058] A targeting construct in the form of a vector may be linearized or released from a circular vector prior to its introduction into a target cell.

[0059] Alternatively, a targeting construct may be synthesized in vitro, e.g., using a DNA polymerase such as T7 prior to its introduction into a target cell.

[0060] Exemplary STEL loci are disclosed in Section 6.2.1.

[0061] Suitable homology arms are disclosed in Section 6.2.2.

[0062] The targeting constructs may further comprise nucleotide sequences encoding separator sequences, as described in Section 6.2.3, that allow the fusion polypeptide coding sequences to be positioned under common transcriptional regulatory control with a STEL protein (e.g., a protein encoded by the endogenous STEL locus) but allows the separate translation of the fusion polypeptide and the STEL protein or the post-translational cleavage of the fusion polypeptide and the STEL protein. Targeting constructs may further comprise one or more ribonucleoprotein (RNP) cut sites that flank the outer sides of the homology arms. These RNP cut sites allow for the endonuclease to cleave the targeting constructs, e.g., to linearize a circular targeting construct. In some embodiments, the targeting construct comprises no RNP cut sites, one RNP cut site, or two RNP cut sites.

[0063] Target cells into which the targeting constructs can be introduced and methods of introducing the targeting constructs into the target cells are disclosed in Section 6.5.

[0064] In some embodiments, genome editing of a cell with any one of the targeting constructs disclosed herein results in insertion of more than one copies (e.g., 1, 2, 3, 4, or 5 or more copies) of any one of the nucleotide inserts disclosed here that comprise a nucleotide sequence encoding the fusion polypeptides disclosed herein.6.2.1. STEL Loci

[0065] Sustained Transgene Expression Loci (“STEL loci”) are genetic loci endogenous to a target cell that are expressed robustly and consistently at a sustained level across and between different cell types (for example, as a cell differentiates, such as from a PSC to PSC derived cell). For example, the expression level of the endogenous gene does not change (e.g., decrease) by more than 50%, more than 40%, more than 35%, more than 30%, more than 25%, more than 20%, more than 15%, more than 10%, or more than 5% over five or more, ten or more, or 15 or more passages or as the cell state changes (e.g., state of pluripotency and / or differentiation).

[0066] In some embodiments, a STEL locus is a gene that is associated with cellular metabolism, such as GAPDH, NADH dehydrogenase, and phosphoglycerate kinase 1 (PGKI).

[0067] In some embodiments, a STEL locus is a ribosomal protein gene or ribosomal protein gene locus, such as an RPL or RPS gene locus. Examples of RPL genes are RPL10, RPL13, RPS18, RPL3, RPLP1, RPL13A, RPL15, RPL41, RPL11, RPL32, RPL18A, RPL19, RPL28, RPL29, RPL9, RPL8, RPL6, RPL18, RPL7, RPL7A, RPL21, RPL37A, RPL12, RPL5, RPL34, RPL35A, RPL30, RPL24, RPL39, RPL37, RPL14, RPL27A, RPLP2, RPLP0, RPL23A, RPL26, RPL36, RPL35, RPL23, RPL4, and RPL22. Examples of RPS genes are RPS2, RPS19, RPS14, RPS3A, RPS12, RPS3, RPS6, RPS23, RPS27A, RPS8, RPS4X, RPS7, RPS24, RPS27, RPS15A, RPS9, RPS28, RPS13, RPSA, RPS5, RPS16, RPS25, RPS15, RPS20, and RPS / / .

[0068] In some embodiments, a STEL gene encodes a mitochondrial protein. Examples of such genes are MT-C01, MT-C02, MT-ND4, MT-ND1, and MT-ND2.

[0069] In some embodiments, a STEL gene encodes a cytoskeletal protein, such as actin. Examples of actin genes are ACTG1 zmA ACTB.

[0070] In some embodiments, a STEL gene encodes a eukaryotic translation elongation factor, such as EEF1A1 and EEF2, or a eukaryotic translation initiation factor, such as EIF1.

[0071] In some embodiments, a STEL gene encodes a histone, such as H3F3A or H3F3B.

[0072] In other embodiments, a STEL gene is selected from FTL, FTH1, TPT1, IMSB10,PTMA, GNB2L1, NACA, YBX1, NPM1, FAU, UBA52, HSP90AB1, MYL6, SERF2, and SRP14.

[0073] In some embodiments, an IMiD responsive peptide-suicide protein fusion polypeptide sequence, comprising a modified degron (i.e., CRIMP) protein sequence and / or a modified CRBN protein sequence disclosed herein is introduced into a STEL gene, so that theSTEL gene is modified to include the modified degron peptide-suicide protein fusion polypeptide, optionally separated from the coding sequence of the STEL protein via a separator sequence. In some embodiments, the STEL locus is the GAPDH gene. In some embodiments, a modified degron peptide-suicide protein fusion polypeptide sequence disclosed herein in introduced into a STEL gene, by generating a break in an exon of a STEL site that is required for cell survival, such that if proper integration of the modified degron peptide-suicide protein fusion polypeptide sequence does not occur, cells having such improper integration do not survive. Such screening of improper integration events may be performed in accordance with methods described in WO 2021 / 226151, which is incorporated herein by reference.

[0074] In some embodiments, a modified CRBN peptide-suicide protein fusion polypeptide sequence disclosed herein is introduced into a STEL gene, so that the STEL gene is modified to include the modified CRBN peptide-suicide protein fusion polypeptide, optionally separated from the coding sequence of the STEL protein via a separator sequence. In some embodiments, the STEL locus is the GAPDH gene. In some embodiments, a modified CRBN peptide-suicide protein fusion polypeptide sequence disclosed herein in introduced into a STEL gene, by generating a break in an exon of a STEL site that is required for cell survival, such that if proper integration of the modified CRBN peptide-suicide protein fusion polypeptide sequence does not occur, cells having such improper integration do not survive. Such screening of improper integration events may be performed in accordance with methods described in WO 2021 / 226151, which is incorporated herein by reference.

[0075] In some embodiments, a modified degron peptide-suicide protein fusion polypeptide sequence disclosed herein is introduced into a STEL gene, so that the STEL gene is modified to include the modified degron peptide-suicide protein fusion polypeptide, optionally separated from the coding sequence of the STEL protein via a separator sequence. In some embodiments, the STEL locus is the GAPDH gene. In some embodiments, a modified degron peptide-suicide protein fusion polypeptide sequence disclosed herein in introduced into a STEL gene, by generating a break in an exon of a STEL site that is required for cell survival, such that if proper integration of the modified degron peptide-suicide protein fusion polypeptide sequence does not occur, cells having such improper integration do not survive. Such screening of improper integration events may be performed in accordance with methods described in WO 2021 / 226151, which is incorporated herein by reference.6.2.2. Homology Arms

[0076] Targeting constructs that are intended for integration into a STEL locus of a target cell genome typically comprise a heterologous sequence that is not present in the target cellgenome, e.g., a nucleotide sequence encoding a modified degron peptide-suicide protein fusion polypeptide disclosed herein.

[0077] The targeting constructs typically include one or more regions that are homologous to regions of DNA within or near (e.g., flanking or adjoining) a STEL locus sequence. These homologous regions are referred to here as “homology arms.” For ease of reference, the homology arms are referred to herein as first and second (i.e., 5' and 3', upstream and downstream, or left and right) homology arms. This terminology relates to the relative position of the homology arms to the nucleic acid insert within the targeting construct. The first and second homology arms correspond to regions within or near (e.g., flanking or adjoining) a STEL locus sequence, which are referred to herein as “first region of homology” and “second region of homology,” respectively. The regions within or near (e.g., flanking or adjoining) a STEL locus sequence are sometimes referred to herein as “target” sequences.

[0078] The current disclosure provides a targeting construct comprising a first homology arm that corresponds to a first region of homology, a nucleic acid insert, and a second homology arm that corresponds to a second region of homology to a STEL locus.

[0079] A homology arm and a target sequence “correspond” or are “corresponding” to one another when the two regions share a sufficient level of sequence identity to one another to act as substrates for a homologous recombination reaction, whereby the homology arms are suitable for directing recombination of a nucleic acid insert with a desired target sequence to facilitate genomic integration and / or replacement of endogenous sequence.

[0080] The term “homology” includes DNA sequences that are either identical or share sequence identity to a corresponding sequence. The sequence identity between a given target sequence and the corresponding homology arm found in the exogenous donor nucleic acid can be any degree of sequence identity that allows for homologous recombination to occur. For example, the amount of sequence identity shared by the homology arm of the exogenous donor nucleic acid (or a fragment thereof) and the target sequence (or a fragment thereof) can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, such that the sequences undergo homologous recombination. Moreover, a corresponding region of homology between the homology arm and the corresponding target sequence can be of any length that is sufficient to promote homologous recombination. In some targeting vectors, the intended mutation in the target genomic locus is included in an insert nucleic acid flanked by the homology arms.

[0081] In some embodiments, the first homology arm is between 50 to 250 nucleotides in length. In some embodiments, the first homology arm is between 50-2000 nucleotides in length. In some embodiments, the first homology arm is between 50-1500 nucleotides in length. In some embodiments, the first homology arm is between 50-1000 nucleotides in length. In some embodiments, the first homology arm is between 50-500 nucleotides in length. In some embodiments, the first homology arm is between 150 to 250 nucleotides in length. In some embodiments, the first homology arm is 2000 nucleotides or less in length. In some embodiments, the first homology arm is 1500 nucleotides or less in length. In some embodiments, the first homology arm is 1000 nucleotides or less in length. In some embodiments, the first homology arm is 700 nucleotides or less in length. In some embodiments, the first homology arm is 650 nucleotides or less in length. In some embodiments, the first homology arm is 600 nucleotides or less in length. In some embodiments, the first homology arm is 550 nucleotides or less in length. In some embodiments, the first homology arm is 500 nucleotides or less in length. In some embodiments, the first homology arm is 400 nucleotides or less in length. In some embodiments, the first homology arm is 300 nucleotides or less in length. In some embodiments, the first homology arm is 250 nucleotides or less in length. In some embodiments, the first homology arm is 200 nucleotides or less in length. In some embodiments, the first homology arm is 150 nucleotides or less in length. In some embodiments, the first homology arm is less than 100 nucleotides in length. In some embodiments, the first homology arm is 50 nucleotides in length or less. In some embodiments, the first homology arm is 250, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, or 20 nucleotides in length. In some embodiments, the first homology arm is at least 20 nucleotides in length. In some embodiments, the first homology arm is at least 40 nucleotides in length. In some embodiments, the first homology arm is at least 50 nucleotides in length. In some embodiments, the first homology arm is at least 70 nucleotides in length. In some embodiments, the first homology arm is at least 100 nucleotides in length. In some embodiments, the first homology arm is at least 200 nucleotides in length. In some embodiments, the first homology arm is at least 300 nucleotides in length. In some embodiments, the first homology arm is at least 400 nucleotides in length. In some embodiments, the first homology arm is at least 500 nucleotides in length. In some embodiments, the first homology arm is at least 600 nucleotides in length. In some embodiments, the first homology arm is at least 700 nucleotides in length. In some embodiments, the first homology arm is at least 1000 nucleotides in length. In some embodiments, the first homology arm is at least 1500 nucleotides in length. In someembodiments, the first homology arm is at least 2000 nucleotides in length. In some embodiments, the first homology arm is about 20 nucleotides in length. In some embodiments, the first homology arm is about 40 nucleotides in length. In some embodiments, the first homology arm is 250 nucleotides in length or less. In some embodiments, the first homology arm is about 100 nucleotides in length. In some embodiments, the first homology arm is about 200 nucleotides in length.

[0082] In some embodiments, the second homology arm is between 50 to 250 nucleotides in length. In some embodiments, the second homology arm is between 50-2000 nucleotides in length. In some embodiments, the second homology arm is between 50-1500 nucleotides in length. In some embodiments, the second homology arm is between 50-1000 nucleotides in length. In some embodiments, the second homology arm is between 50-500 nucleotides in length. In some embodiments, the second homology arm is between 150 to 250 nucleotides in length. In some embodiments, the second homology arm is 2000 nucleotides or less in length. In some embodiments, the second homology arm is 1500 nucleotides or less in length. In some embodiments, the second homology arm is 1000 nucleotides or less in length. In some embodiments, the second homology arm is 700 nucleotides or less in length. In some embodiments, the second homology arm is 650 nucleotides or less in length. In some embodiments, the second homology arm is 600 nucleotides or less in length. In some embodiments, the second homology arm is 550 nucleotides or less in length. In some embodiments, the second homology arm is 500 nucleotides or less in length. In some embodiments, the second homology arm is 400 nucleotides or less in length. In some embodiments, the second homology arm is 300 nucleotides or less in length. In some embodiments, the second homology arm is 200 nucleotides in length or less. In some embodiments, the second homology arm is 150 nucleotides in length or less. In some embodiments, the second homology arm is 100 nucleotides in length or less. In some embodiments, the second homology arm is 50 nucleotides in length or less. In some embodiments, the second homology arm is 250, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, or 20 nucleotides in length. In some embodiments, the second homology arm is at least 20 nucleotides in length. In some embodiments, the second homology arm is at least 40 nucleotides in length. In some embodiments, the second homology arm is at least 50 nucleotides in length. In some embodiments, the second homology arm is at least 70 nucleotides in length. In some embodiments, the second homology arm is at least 100 nucleotides in length. In some embodiments, the second homology arm is at least 200nucleotides in length. In some embodiments, the second homology arm is at least 300 nucleotides in length. In some embodiments, the second homology arm is at least 400 nucleotides in length. In some embodiments, the second homology arm is at least 500 nucleotides in length. In some embodiments, the second homology arm is at least 600 nucleotides in length. In some embodiments, the second homology arm is at least 700 nucleotides in length. In some embodiments, the second homology arm is at least 1000 nucleotides in length. In some embodiments, the second homology arm is at least 1500 nucleotides in length. In some embodiments, the second homology arm is at least 2000 nucleotides in length. In some embodiments, the second homology arm is about 20 nucleotides in length. In some embodiments, the second homology arm is about 40 nucleotides in length. In some embodiments, the second homology arm is 250 nucleotides in length or less. In some embodiments, the second homology arm is about 100 nucleotides in length. In some embodiments, the second homology arm is about 200 nucleotides in length.

[0083] The first and second homology arms can be of the same length or can differ in length. In some embodiments, the first and second homology arms are amplified to allow for the quantitative assessment of gene editing events, such as targeted integration, at a target nucleic acid. In some embodiments, the assessment of the gene editing events may rely on the amplification of both the 5' junction and 3' junction at the site of targeted integration by amplifying the whole or a part of the homology arm using a single pair of PCR primers in a single amplification reaction. Accordingly, although the length of the first and second homology arms may differ, the length of each homology arm should be capable of amplification (e.g., using PCR), as desired.

[0084] In some embodiments, the length of the first and second homology arms does not differ by more than 75 nucleotides. Thus, in some embodiments, when the first and second homology arms differ in length, the length difference between the homology arms is less than 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 nucleotides or base pairs. In some embodiments, the first and second homology arms differ in length by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75 nucleotides. In some embodiments, the length difference between the first and second homology arms is less than 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 base pairs. In some embodiments, the first and second homology arms differ in length by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75 base pairs.

[0085] Homology arms are capable of directing recombination of a nucleic acid insert within or near a desired target STEL gene to facilitate genomic integration and / or replacement of endogenous sequence, e.g., integrate a modified degron peptide-suicide protein fusion polypeptide sequence disclosed herein into a STEL gene. Regardless of the format used, a donor template can be designed to avoid undesirable sequences. In certain embodiments, one or both homology arms can be shortened to avoid overlap with certain sequence repeat elements, e.g., Alu repeats, LINE elements, etc.

[0086] In some embodiments, the homology arms are designed such that, following integration of the targeting construct, the modified degron peptide-suicide protein fusion polypeptide coding sequence is positioned 3' to the STEL polypeptide coding sequence. In other embodiments, the homology arms are designed such that, following integration of the targeting construct, the modified degron peptide-suicide protein fusion polypeptide coding sequence is positioned 5' to the STEL polypeptide coding sequence. Typically, the STEL polypeptide coding sequence and the modified degron peptide-suicide protein fusion polypeptide coding sequence are separated by a separator sequence, such as an IRES or a 2A peptide coding sequence.

[0087] GAPDH Left Homology Arm: TTGGTATCGTGGAAGGACTCATGGTATGAGAGCTGGGGAATGGGACTGAGGCTCCC ACCTTTCTCATCCAAGACTGGCTCCTCCCTGCCGGGGCTGCGTGCAACCCTGGGGTT GGGGGTTCTGGGGACTGGCTTTCCCATAATTTCCTTTCAAGGTGGGGAGGGAGGTA GAGGGGTGATGTGGGGAGTACGCTGCAGGGCCTCACTCCTTTTGCAGACCACAGTC CATGCCATCACTGCCACCCAGAAGACTGTGGATGGCCCCTCCGGGAAACTGTGGCG TGATGGCCGCGGGGCTCTCCAGAACATCATCCCTGCCTCTACTGGCGCTGCCAAGGC TGTGGGCAAGGTCATCCCTGAGCTGAACGGGAAGCTCACTGGCATGGCCTTCCGTG TCCCCACTGCCAACGTGTCAGTGGTGGACCTGACCTGCCGTCTAGAAAAACCTGCC AAATATGATGACATCAAGAAGGTGGTGAAGCAGGCGTCGGAGGGCCCCCTCAAGG GCATCCTGGGCTACACTGAGCACCAGGTGGTCTCCTCTGACTTCAACAGCGACACCC ACTCCTCCACCTTTGACGCTGGGGCTGGCATTGCCCTCAACGACCACTTTGTCAAGC TCATTTCCTGGTATGTGGCTGGGGCCAGAGACTGGCTCTTAAAAAGTGCAGGGTCTG GCGCCCTCTGGTGGCTGGCTCAGAAAAAGGGCCCTGACAACTCTTTACATCTTCTAG GTATGACAACGAATTTGGCTACAGCAACAGGGTGGTGGACCTCATGGCCCACATGG CCTCCAAGGAG (SEQ ID NO: 39)

[0088] GAPDH Right Homology Arm:GACCCCTGGACCACCAGCCAAAGCAAGAGCACAAGAGGAAGAGAGAGACCCTCACTGCTGGGGAGTCCCTGCCACACTCAGTCCCCCACCACACTGAATCTCCCCTCCTCACAGTTGCCATGTAGACCCCTTGAAGAGGGGAGGGGCCTAGGGAGCCGCACCTTGTCATGTACCATCAATAAAGTACCCTGTGCTCAACCAGTTACTTGTCCTGTCTTATTCTAGGGTCTGGGGCAGAGGGGAGGGAAGCTGGGCTTGTGTCAAGGTGAGACATTCTTGCTGGGGAGGGACCTGGTATGTTCTCCTCAGACTGAGGGTAGGGCCTCCAAACAGCCTTGCTTGCTTCGAGAACCATTTGCTTCCCGCTCAGACGTCTTGAGTGCTACAGGAAGCTGGCACCACTACTTCAGAGAACAAGGCCTTTTCCTCTCCTCGCTCCAGTCCTAGGCTATCTGCTGTTGGCCAAACATGGAAGAAGCTATTCTGTGGGCAGCCCCAGGGAGGCTGACAGGTGGAGGAAGTCAGGGCTCGCACTGGGCTCTGACGCTGACTGGTTAGTGGAGCTCAGCCTGGAGCTGAGCTGCAGCGGGCAATTCCAGCTTGGCCTCCGCAGCTGTGAGGTCTTGAGCACGTGCTCTATTGCTTTCTGTGCCCTCGTGTCTTATCTGAGGACATCGTGGCCAGCCCCTAAGGTCTTCAAGCAGGATTCATCTAGGTAAACCAAGTACCTAAAACCATGCCCAAGGCGGTAAGGACTATATAATGTTTAAAAATCGGTAAAAATGCCCACCTCGCATAGT (SEQ ID NO: 40)

[0089] RPL 13 A Left Homology Arm:TCTTAAGCCCCTCTCTTTCTCTAACAGAAAAAGCGGATGGTGGTTCCTGCTGCCCTCAAGGTCGTGCGTCTGAAGCCTACAAGAAAGGTGAGTCCCAGCTTACGCTGCACCATCTACTTGGGAGATTTCAGGCCTGCTGAGGGACCTGGGGACCTGGAGCCTGGCAGATGATGTCCTTATCTCACGATGGTCTGCGGATGTCCCTGTGGGAATGGCGACAATGCCAATGGCTTAGCTGATGCCAGGAGGCTTGGGTGGGTGCTTTTCTAACAGGCCTGCAGAGAACAGTTGCATTATGATATGCCCAGCTGTCAGTCACCTCCCAGCTCTCAACAGCTCCGGCTCTTCAGGGTGTGGGGGCTTAGATATCCTTACAACTTCATTTGTTCACCCCCCCCCCCCCCCCCCGCAGTTTGCCTATCTGGGGCGCCTGGCTCACGAGGTTGGCTGGAAGTACCAGGCAGTGACAGCCACCCTGGAGGAGAAGAGGAAAGAGAAAGCCAAGATCCACTACCGGAAGAAGAAACAGCTCATGGTGAGGCCAGGGGCTGGTGCTGAGGGGGGCATCTCACTCCTGGACAGGCCTGGCAGGTGCCTTGCTCACAGAGTACTCTTAACTGGCAAAGGACCAGCCGGGGTTGGGGTGGGATGCAGTCCATGTAATGAGGGCAATGCAACCCCTCCTGACCACCACCACCTGCACTTATTCTTGGCAGAGGCTACGCAAACAAGCCGAGAAGAACGTGGAGAAGAAAATTGACAAATACACAGAGGTCCTCAAGACCCACGGACTCCTGGTC (SEQ ID NO: 41)

[0090] RPL13A Right Homology Arm:GCCCAATAAAGACTGTTAATTCCTCATGCGTTGCCTGCCCTTCCTCCATTGTTGCCCTGGAATGTACGGGACCCAGGGGCAGCAGCAGTCCAGGTGCCACAGGCAGCCCTGGGACATAGGAAGCTGGGAGCAAGGAAAGGGTCTTAGTCACTGCCTCCCGAAGTTGCTTGAAAGCACTCGGAGAATTGTGCAGGTGTCATTTATCTATGACCAATAGGAAGAGCAACCAGTTACTATGAGTGAAAGGGAGCCAGAAGACTGATTGGAGGGCCCTATCTTGTGAGTGGGGCATCTGTTGGACTTTCCACCTGGTCATATACTCTGCAGCTGTTAGAATGTGCAAGCACTTGGGGACAGCATGAGCTTGCTGTTGTACACAGGGTATTTCTAGAAGCAGAAATAGACTGGGAAGATGCACAACCAAGGGGTTACAGGCATCGCCCATGCTCCTCACCTGTATTTTGTAATCAGAAATAAATTGCTTTTAAAGAAATCTGGCGTCTTTGCACTGTGTCTGCTGTGGAGGCAGGCCCCTGGCAAATGGGGGGTGAGGAGCTTGAAGAGGGTAGAATGGGCTGTGCTAATATACAGAATATATGTAACTTGCTATAAATTGAATGATCCTTTATAGACACCGTTTACAAACCAAAGACATAAAATGTGGCCAGCAGTGCC TGGTGCTTCCTAGTTAATGTAAAGCTGTCTCATTCTAATTCAGCTGCAAAGTATGGA CCCATGCCCTGCTGCCAGGCTGCTGTAGTCCCGGCGGTCTGTAGAGACTAGCATTTTGCAAATGATAA (SEQ ID NO: 42)

[0091] RPLPO Left Homology Arm:CTGAGCTGCCAACCTGGCAATTATTGTCTGCTAAGGGTTCTCTTTATTCACCCTTACTTGGACTTCCTTTCCTGTAGGGAATCTCACGTAAAATGAAATCTTCCCTCCCCCAGGGTGTCCGCAATGTTGCCAGTGTCTGTCTGCAGATTGGCTACCCAACTGTTGCATCAGTACCCCATTCTATCATCAACGGGTACAAACGAGTCCTGGCCTTGTCTGTGGAGACGGATTACACCTTCCCACTTGCTGAAAAGGTAAAAGGATCCCACCAGGACCACAGTGGGCCTGACTGTGACAAATTAGCAGGGTGATGTGGCCTTCTACCTTACTGCTTTTATAGTTGTATTTTATATAGCAGATAATTTTGTGAGGGGATATTTGAGAGGTTGGGAGGCAGGGAAGGCGTTTCTCACTTGAGAAATGACAAGAGACCCAAAGAGGGGGTTAATGGGCAAGAGCTGGGCCTTAGGAACCCTGCCTCACTAGGCCATACCCAAGCTGTCCTGCTTGGGCTGCTTCTGACAGGAAAGGCTTCACACGGACTTTGATATTGTTGGTCCTTAAACTCTACCAAGGCAGGAGGGTGGTGGGTAATAGAGGAGTGTGGATGACCATTTTGACCACTTCCCCCCTCCTTTCAGGTCAAGGCCTTCTTGGCTGATCCATCTGCCTTTGTGGCT GCTGCCCCTGTGGCTGCTGCCACCACAGCTGCTCCTGCTGCTGCTGCAGCCCCAGCT AAGGTTGAAGCCAAGGAAGAGTCGGAGGAGTCGGACGAGGATATGGGATTTGGTCTCTTTGAC (SEQ ID NO: 43)

[0092] RPLPO Right Homology Arm:TCACCAAAAAGCAACGAACTTAGCCAGTTTTATTTGCAAAACAAGGAAATAAATGCTTACTTCTTTAAAAAGTCTCTTGACTCTTAATTTTGTAATTTTTTTTCCTTTTTGACAC AGGGTCTGGCTGTTGCCCAGGCTGGAGTGTGGTGGTGTAATCATAACTCACTGCACCCTTGAACTCCTGGGATCAAGGGATCCTCGTATCTCAGCCTCCCAAGTAGCTGGGACT ACAGGCACACACCATGACACTCAGCTACTAATTTTTAAATTTTTTTTTTGTAGAGAT GTTGCACAAGCTGGTCTCAAATTCCTGGCCTCAAGGAATCCTGCCTCAGCCTCCCAA AGTGCTAGGATTACAGGCTTGAGCCACCATGTGCCTGGCCCTTAATTTTGAGGTTTA TAGTGCCATATGCTAGAAACGAAAGCCATGGTAAAACCAGAGCTTTGTATTTAGGT GTTGATGTTTGGGTATCTAAATGAAGCTACCAATCAAACATCCTATACAGTTTTCTA GACACAGTTGTAACTATTACACTAGAATTACTGTTTCTATGGCTGCTGCATACTTGG AGTAGGTTTAGTGTCAGCTGAGATAGGCACCTGGTGGATGCTGGGGCCAGTCCCCT AGAGTAAAGTTTTTCAAACTGGGTGGTGCTCCAACTCGGTGGTAACCAATTTATATT TTCGAGATAGTCTCAAATATATTTGAGACTGGGGTGCAGTGGCTTGGACTTGGCTCA CTGCAACCTCCGCCTCCTGGGTTCAAGTGATTCTCCTGCCTCAGCCTCCCAAGTAGC TGC (SEQ ID NO: 44)6.2.3. Separator Sequences

[0093] The targeting constructs and recombinant target cell genomes described herein can also comprise a separator sequence between STEL polypeptide coding sequence and the modified degron peptide-suicide protein fusion polypeptide coding sequence. Such separator sequences can allow separate expression of the STEL polypeptide and the modified degron peptide-suicide protein fusion polypeptide under common control of STEL gene regulatory elements.

[0094] In some embodiments, the separator sequence is an internal ribosome entry site (IRES), which allows the STEL polypeptide to be translated separately from the modified degron peptide-suicide protein fusion polypeptide.

[0095] In some embodiments, the separator sequence is a self-cleaving peptide, associated with ribosomal skipping during translation, in which the ribosomes skip the peptide bond between a C-terminal Gly and Pro, resulting in the production of two separate polypeptides, i.e., the STEL polypeptide and the modified CRBN peptide-suicide protein fusion polypeptide, the modified CRIMP peptide-suicide protein fusion polypeptide, and / or the IMiD-responsive peptide suicide protein fusion polypeptide. A self-cleaving peptide causes ribosomal skipping during translation. Examples of self-cleaving peptides are 2A peptides, which are viral derived peptides with a typical length of 18-22 amino acids. 2A peptides include T2A, P2A, E2A, F2A, PQR (Lo et al., 2015, Cell Reports 13:2634-2644), Opt2A, and Opt2A_2.0. By way of example, P2A is a peptide of 19 amino acids; after the cleavage, a few amino acid residues from the P2A are left on the upstream polypeptide and a proline is left at the beginning of the secondpolypeptide. 2A residues left on the STEL polypeptide and the modified degron peptide-suicide protein fusion polypeptide do not affect their functionality.

[0096] In some embodiments, a 2A peptide, is a P2A peptide. In some embodiments, a P2A peptide comprises an amino acid sequence as set forth in SEQ ID NO: 9. In some embodiments, a P2A peptide is encoded by a nucleic acid sequence as set forth in SEQ ID NO: 10.P2A amino acid sequence: ATNFSLLKQAGDVEENPGP (SEQ ID NO: 9)P2A nucleic acid sequence:GCGACGAATTTTAGTCTACTGAAACAAGCGGGAGACGTGGAGGAAAACCCT GGACCT (SEQ ID NO: 10)

[0097] In some embodiments, a 2A peptide comprises a T2A peptide. In some embodiments, a T2A peptide comprises an amino acid sequence as set forth in SEQ ID NO: 11. In some embodiments, a T2A peptide is encoded by a nucleic acid sequence as set forth in SEQ ID NO: 12.T2A amino acid sequence: EGRGSLLTCGDVEENPGP (SEQ ID NO: 11)T2A nucleic acid sequence:GAAGGGCGCGGGTCTCTCCTCACTTGTGGAGATGTTGAGGAAAATCCAGGAC CA (SEQ ID NO: 12)

[0098] In some embodiments, a 2A peptide comprises an E2A G4S T2A (Opt2A) peptide. In some embodiments, an E2A G4S T2A (Opt2A) peptide comprises an amino acid sequence as set forth in SEQ ID NO: 13. In some embodiments, an Opt2A peptide is encoded by a nucleic acid sequence as set forth in SEQ ID NO: 14.Opt2A amino acid sequence:QCTNYALLKLAGDVESNPGPGSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 13) Opt2A nucleic acid sequence: CAGTGCACAAATTATGCACTGCTGAAGCTCGCCGGGGATGTCGAGAGTAACC CAGGACCTGGAAGCGGAGAAGGTCGTGGTAGTCTACTAACGTGTGGTGATGT AGAAGAAAATCCTGGACCT (SEQ ID NO: 14)

[0099] In some embodiments, a 2A peptide comprises a P2A 3-T2A 2 (Opt2A_2.0) peptide. In some embodiments, a P2A 3-T2A 2 (Opt2A_2.0) peptide comprises an amino acid sequence as set forth in SEQ ID NO: 15. In some embodiments, an Opt2A_2.0 peptide is encoded by a nucleic acid sequence as set forth in SEQ ID NO: 16.Opt2A_2.0 amino acid sequence: (SEQ ID NO: 15)ATNFSLLKQAGDVEENPGPGSGEGRGSLLTCGDVEENPGPOpt2A_2.0 nucleic acid sequence: (SEQ ID NO: 16) GCGACGAATTTTAGTCTACTGAAACAAGCGGGAGACGTGGAGGAAAACCCT GGACCTGGAAGCGGAGAAGGTCGTGGTAGTCTACTAACGTGTGGTGATGTA GAAGAAAATCCTGGACCT6.3.IMiD-Responsive Suicide Protein Fusion Polypeptides

[0100] The IMiD-Responsive suicide protein fusion polypeptides disclosed herein allow for temporal control of activity of a suicide protein that is activated upon dimerization. The IMiD- Responsive peptide can allow reversible control over their activity by administrating or removing an inducing agent or ligand such as an IMiD or a variant / derivative of thalidomide thereof, for example, thalidomide, iberdomide, lenalidomide, and pomalidomide. The IMiD- Responsive suicide protein fusion polypeptide may comprise more than one ligand binding domain (e.g., a ligand domain in a provided degron, a provided CRIMP, or a provided CRBN). A given ligand that consistently binds to a given ligand binding domain can be referred to as a cognate ligand pair.

[0101] For example, without being bound by theory, it is thought that in the absence of an inducing agent, the ligand binding domains, together with their suicide protein fusion partner, remain largely in monomeric form, such that they cannot induce apoptosis. In the presence of an IMiD (e.g., pomalidomide) or a variant / derivative thereof, however, it is believed that the ligand binding peptide(s) can then associate with the ligand, allowing the suicide proteins to also associate or dimerize and initiate apoptosis.

[0102] The modified degron (e.g., CRIMP) peptide-suicide protein fusion polypeptides disclosed herein comprise (1) (a) a modified degron peptide disclosed herein, (b) a suicide protein disclosed herein, and (c) an optional linker separating the modified degron peptide and suicide protein, and / or (2) (a) a modified cereblon (CRBN) peptide disclosed herein, (b) a suicide protein disclosed herein, and (c) an optional linker separating the modified degron peptide and suicide protein . In some embodiments, the modified degron peptide-suicide protein fusion polypeptide comprises a modified degron peptide N-terminal to the suicide protein. In some embodiments, the modified CRBN peptide-suicide protein fusion polypeptide comprises a modified CRBN peptide N-terminal to the suicide protein. In some embodiments, the modified degron peptide-suicide protein fusion polypeptide comprises a modified degron peptide C- terminal the suicide protein. In some embodiments, the modified CRBN peptide-suicide protein fusion polypeptide comprises a modified CRBN peptide C-terminal the suicide protein. In yet further embodiments, a modified degron peptide-suicide protein fusion polypeptide comprises a modified degron peptide both N-terminal and C-terminal to the suicide protein. In yet furtherembodiments, a modified CRBN peptide-suicide protein fusion polypeptide comprises a modified CRBN peptide both N-terminal and C-terminal to the suicide protein. The N- and C- terminal modified degron peptides or modified CRBN peptides can be the same or different. In each case, the suicide protein and the modified degron peptide sequence and / or the suicide protein and the modified CRBN peptide sequence may be separated by a linker sequence.

[0103] Modified degron peptides and modified CRBN peptides are set forth in Section 6.3.1 below.

[0104] Suicide proteins are set forth in Section 6.3.2 below.

[0105] Exemplary linker sequences are set forth in Section 6.3.4 below.6.3.1. The Modified Degron Peptide

[0106] Degrons are a protein or a portion thereof that is involved in the degradation of proteins. It can comprise a specific sequence of amino acids that tags a protein for degradation by ubiquitination by ubiquitin ligases. Ligands that bind to degrons, such as an IMiD or a variant / derivative of thalidomide thereof, for example, thalidomide, iberdomide, lenalidomide, and pomalidomide, have been found to induce degradation of some substrates by functionally associating with components of the E3 ubiquitin ligase complex, such as cereblon (CRBN). In this way, the association of CRBN and a modified degron through an IMiD ligand can bring together (i.e., dimerize) multiple suicide proteins to promote apoptosis.

[0107] Suicide protein activity can be regulated by fusing the suicide protein to a modified degron or modified CRBN. The modified degron peptide comprises an amino acid sequence corresponding to an unmodified degron peptide sequence (SEQ ID NO: 1), and comprises a R27H amino acid substitution or a G30E amino acid substitution.

[0108] Fusing a suicide protein to a modified degron peptide and / or a modified CRBN peptide is believed to maintain the suicide protein in monomeric form in the absence of a modified degron / CRBN inducer (e.g., an IMiD), such that the suicide protein cannot induce apoptosis. In the presence of an IMiD (e.g., pomalidomide), the modified degron / CBRN peptides, and consequently the suicide protein, can dimerize, activating the apoptosis-inducing capability of the suicide protein. This dimerization can be achieved by administering one or more IMiDs. Exemplary IMiDs include, but are not limited to, thalidomide, iberdomide, lenalidomide, and pomalidomide and variants or derivatives thereof.

[0109] It is to be understood that the additional amino acid substitutions to the modified degron peptide (CRIMP) disclosed herein are with reference to SEQ ID NO: 1.

[0110] In some embodiments, the modified degron polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1.

[0111] In some embodiments, the modified degron polypeptide (CRIMP) comprises a R27H amino acid substitution and comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 2.

[0112] In some embodiments, the modified degron polypeptide (CRIMP) comprises a G30E amino acid substitution and comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 3.

[0113] In some embodiments, the modified degron peptide (CRIMP) further comprises an N-terminal methionine residue.6.3.2. The Modified Cereblon (CRBN) Peptide

[0114] As described in 6.3, the IMiD-responsive suicide protein fusion polypeptide(s) can comprise a ligand binding domain comprising a CRBN domain or a modified CRBN domain. Degrons are a protein or a portion thereof that are involved in the degradation of proteins.Ligands that bind to degrons, such as an IMiD or a variant / derivative of thalidomide thereof, for example, thalidomide, iberdomide, lenalidomide, and pomalidomide, have been found to induce degradation of some substrates by functionally associating with components of the E3 ubiquitin ligase complex, such as cereblon (CRBN). In this way, the association of CRBN and a modified degron through an IMiD ligand can bring together (i.e., dimerize) multiple suicide proteins to promote apoptosis.

[0115] Suicide protein activity can be regulated by fusing the suicide protein to CRBN or a modified CRBN. The modified CRBN peptide comprises an amino acid sequence corresponding to a wild-type CRBN peptide sequence (SEQ ID NO: 4). In some embodiments, acereblon (CRBN), as used in accordance with the present disclosure, is a wild-type CRBN polypeptide, e.g., a CRBN comprising the amino acid sequence of SEQ ID NO: 4. In some embodiments, a CRBN is a modified CRBN. In some embodiments, a modified CRBN comprises mutations that reduce ubiquitination relative to wild-type CRBN. In some embodiments, the DDB1 interacting domain is deleted in a modified CBRN. In some embodiments, amino acids 194-247 are deleted in a modified CBRN, e.g., a modified CRBN comprising the amino acid sequence of SEQ ID NO: 5.

[0116] In some embodiments, the CRBN or modified CRBN further comprises an N- terminal methionine (M).6.3.3. The Suicide Protein

[0117] The IMiD-responsive suicide protein fusion polypeptide comprises a caspase 9 domain or derivative or functional fragment thereof (“suicide protein”).

[0118] The activity of the suicide protein is inducible by dimerization. Thus, in some embodiments, the suicide protein is inactive or has low / reduced activity in a monomeric form but has suicide protein activity in a dimeric form.

[0119] In some embodiments, the caspase 9 sequence is a truncated caspase 9 (“truncCasp9”) with its Caspase Activation and Recruitment Domain (CARD) motif removed. In the presence of an IMiD (e.g., pomalidomide and variants or derivatives thereof), the IMiD- responsive domains dimerize, which is believed to subsequently cause homodimerization of truncCasp9 and induction of apoptosis.

[0120] The dimerization of modified CRBN and modified degron peptides (i.e., IMiD- responsive suicide proteins), each fused to a truncCasp9 fusion polypeptide can be achieved by administering one or more IMiDs. Exemplary IMiDs include pomalidomide and / or variants and derivatives, such as thalidomide, iberdomide, lenalidomide, and pomalidomide. Without being bound by theory, it is believed that administering a combination of two or more IMiDs (such as a combination of pomalidomide and one or more of its metabolites or a combination of two or more pomalidomide variants / derivatives) can result in synergism, thereby lowering the minimally effective dosage of each agent included in the combination. Consequently, apoptosiscan be achieved by administration of pharmacologically relevant concentrations of each agent in a combination.

[0121] In some embodiments, dimerization of IMiD-responsive suicide proteins is achieved by administering only one IMiD, such as thalidomide, iberdomide, lenalidomide, and pomalidomide. In other embodiments, dimerization of IMiD-responsive suicide proteins is achieved by administering any combination of two or more IMiDs, such as thalidomide, iberdomide, lenalidomide, and pomalidomide, in any combination. In some embodiments, the dose of each agent administered in combination is less than the dose that would be administered to achieve dimerization using a single agent.

[0122] An exemplary truncCasp9 sequence is set forth below as SEQ ID NO: 17:

[0123] The derivative of the caspase 9 domain may comprise at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, at least 99% identity, or 100% identity to SEQ ID NO: 17.MDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMV EVKGDLTAKKMVLALLELARQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIV NIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLD AISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGI YKQMPGCFNFLRKKLFFKTS (SEQ ID NO: 17)6.3.4. The Linker

[0124] The modified CRBN peptide-suicide protein fusion polypeptide and / or the modified degron peptide-suicide protein fusion polypeptide of the disclosure may comprise an optional linker sequence between the modified CRBN peptide and the suicide protein sequence and / or the modified degron and the suicide protein sequence.

[0125] Suitable linkers for use in the fusion polypeptide of the present disclosure are well known to those of skill in the art and include peptide linkers. In particular embodiments, the linker is used to separate the modified CRBN peptide and the suicide protein sequence, and the modified degron peptide by a distance sufficient to ensure that the suicide protein retains its function. Preferred peptide linker sequences adopt a flexible extended conformation and do not exhibit a propensity for developing an ordered secondary structure.

[0126] Typical amino acids in flexible peptide linkers include Gly, Asn and Ser. Accordingly, in particular embodiments, the linker comprises a combination of one or more of Gly, Asn and Ser amino acids. Other near neutral amino acids, such as Thr and Ala, also may be used in the linker sequence. Exemplary linkers are disclosed in Maratea et a / ., 1985, Gene 40:39-46; Murphy etal., 1986, Proc. Nat’l. Acad. Sci. USA 83: 8258-62; U.S. Pat. No. 4,935,233; and U.S. Pat. No. 4,751,180, the contents of which are incorporated herein in their entireties.

[0127] Peptide linkers can be one amino acid sequence or repeats of one or more amino acid sequences. In some embodiments, a sequence can be used in repeats of 2. In some embodiments, a sequence can be used in repeats of 3. In some embodiments, a sequence can be used in repeats of 4. In some embodiments, a sequence can be used in repeats of 5 or more.

[0128] In some embodiments, the peptide linker is between 1 and 30 amino acids in length. In various aspects, the peptide linker is between 1 and 3 amino acids in length, between 3 and 8 amino acids in length, between 3 and 10 amino acids in length, between 5 and 15 amino acids in length, between 11 and 20 amino acids in length, between 15 and 25 amino acids in length, between 21 and 30 amino acids in length, or is a length range bounded by any pair of the forgoing values (e.g., between 3 and 15 amino acids in length, between 8 and 20 amino acids in length, between 25 and 30 amino acids in length, and so on and so forth).

[0129] Non-limiting examples of linker sequences are set forth in Table 4 below.6.4.Targeting Construct Sequences

[0130] In some embodiments, a targeting construct comprises one or more modified CRBN peptide sequences and one or more suicide proteins. In some embodiments, a targeting construct comprises 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or 8 or more distinct modified CRBN peptide sequences. In some embodiments, a targeting construct comprises 1 or more, 2 or more, 3 or more, 4 or more, or 5 or more copies of a single modified CRBN peptide sequence. In some embodiments, a targeting construct comprises 1 or more, 2 or more, 3 or more, 4 or more, 5 or more copies of distinct modified CRBN -Caspase9 constructs. In some embodiments, a targeting construct comprises 1 or more, 2 or more, 3 or more, 4 or more, 5 or more copies of distinct modified CRBN -truncCaspase9 constructs.

[0131] In some embodiments, a targeting construct comprises one or more modified degron (e.g., CRIMP) peptide sequences and one or more suicide proteins. In some embodiments, atargeting construct comprises 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or 8 or more distinct modified degron peptide sequences. In some embodiments, a targeting construct comprises 1 or more, 2 or more, 3 or more, 4 or more, or 5 or more copies of a single modified degron peptide sequence. In some embodiments, a targeting construct comprises 1 or more, 2 or more, 3 or more, 4 or more, 5 or more copies of distinct modified degron-Caspase9 constructs or modified CRBN-Caspase9 constructs. In some embodiments, a targeting construct comprises 1 or more, 2 or more, 3 or more, 4 or more, 5 or more copies of distinct modified degron -truncCaspase9 constructs.

[0132] In some embodiments, a targeting construct comprises a CRIMP sequence and a modified CRBN peptide sequence. In some embodiments, the modified degron polypeptide (CRIMP) comprises a R27H amino acid substitution relative to SEQ ID NO: 1 and the modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the modified degron polypeptide (CRIMP) comprises a G30E amino acid substitution relative to SEQ ID NO: 1 and the modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 4. In some embodiments, the modified degron polypeptide (CRIMP) comprises an amino acid sequence set forth in SEQ ID NO: 1 and the modified CRBN comprises an 137 IN amino acid substitution, relative to SEQ ID NO: 4. In some embodiments, the modified degron polypeptide (CRIMP) comprises a G30E amino acid substitution relative to SEQ ID NO: 1 and the modified CRBN comprises an 137 IN amino acid substitution, relative to SEQ ID NO: 4. In some embodiments, the modified degron polypeptide (CRIMP) comprises a R27H amino acid substitution relative to SEQ ID NO: 1 and the modified CRBN comprises Q325H, 137 IN, and T359V amino acid substitutions, relative to SEQ ID NO: 4. In some embodiments, the modified degron comprises a R27H amino acid substitution relative to SEQ ID NO: 1 and the modified CRBN comprises Q325R, 137 IN, H397F, and T359V amino acid substitutions, relative to SEQ ID NO: 4.

[0133] In some embodiments, a targeting construct comprises a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to a nucleic acid sequence set forth in SEQ ID NOs: 19, 21, 23, 25, 27, or 29.

[0134] In some embodiments, a targeting construct comprises a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to the nucleic acid sequence set forth in SEQ ID NO: 19. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 80% identical to the nucleic acidsequence set forth in SEQ ID NO: 19. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 85% identical to the nucleic acid sequence set forth in SEQ ID NO: 19. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 90% identical to the nucleic acid sequence set forth in SEQ ID NO: 19. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 91% identical to the nucleic acid sequence set forth in SEQ ID NO: 19. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 92% identical to the nucleic acid sequence set forth in SEQ ID NO: 19. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 93% identical to the nucleic acid sequence set forth in SEQ ID NO: 19. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 94% identical to the nucleic acid sequence set forth in SEQ ID NO: 19. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 95% identical to the nucleic acid sequence set forth in SEQ ID NO: 19. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 96% identical to the nucleic acid sequence set forth in SEQ ID NO: 19. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 97% identical to the nucleic acid sequence set forth in SEQ ID NO: 19. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 98% identical to the nucleic acid sequence set forth in SEQ ID NO: 19. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 99% identical to the nucleic acid sequence set forth in SEQ ID NO: 19. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 99.5% identical to the nucleic acid sequence set forth in SEQ ID NO: 19. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 99.9% identical to the nucleic acid sequence set forth in SEQ ID NO: 19. In some embodiments, a targeting construct comprises the nucleic acid sequence set forth in SEQ ID NO: 19.

[0135] In some embodiments, a targeting construct comprises a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to the nucleic acid sequence set forth in SEQ ID NO: 21. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 80% identical to the nucleic acid sequence set forth in SEQ ID NO: 21. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 85% identical to the nucleic acid sequence set forth in SEQ ID NO: 21. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 90% identical to the nucleic acid sequence set forth in SEQ ID NO: 21. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 91% identical to the nucleic acidsequence set forth in SEQ ID NO: 21. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 92% identical to the nucleic acid sequence set forth in SEQ ID NO: 21. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 93% identical to the nucleic acid sequence set forth in SEQ ID NO: 21. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 94% identical to the nucleic acid sequence set forth in SEQ ID NO: 21. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 95% identical to the nucleic acid sequence set forth in SEQ ID NO: 21. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 96% identical to the nucleic acid sequence set forth in SEQ ID NO: 21. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 97% identical to the nucleic acid sequence set forth in SEQ ID NO: 21. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 98% identical to the nucleic acid sequence set forth in SEQ ID NO: 21. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 99% identical to the nucleic acid sequence set forth in SEQ ID NO: 21. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 99.5% identical to the nucleic acid sequence set forth in SEQ ID NO: 21. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 99.9% identical to the nucleic acid sequence set forth in SEQ ID NO: 21. In some embodiments, a targeting construct comprises the nucleic acid sequence set forth in SEQ ID NO: 21.

[0136] In some embodiments, a targeting construct comprises a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to the nucleic acid sequence set forth in SEQ ID NO: 23. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 80% identical to the nucleic acid sequence set forth in SEQ ID NO: 23. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 85% identical to the nucleic acid sequence set forth in SEQ ID NO: 23. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 90% identical to the nucleic acid sequence set forth in SEQ ID NO: 23. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 91% identical to the nucleic acid sequence set forth in SEQ ID NO: 23. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 92% identical to the nucleic acid sequence set forth in SEQ ID NO: 23. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 93% identical to the nucleic acid sequence set forth in SEQ ID NO: 23. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 94% identical to the nucleic acidsequence set forth in SEQ ID NO: 23. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 95% identical to the nucleic acid sequence set forth in SEQ ID NO: 23. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 96% identical to the nucleic acid sequence set forth in SEQ ID NO: 23. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 97% identical to the nucleic acid sequence set forth in SEQ ID NO: 23. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 98% identical to the nucleic acid sequence set forth in SEQ ID NO: 23. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 99% identical to the nucleic acid sequence set forth in SEQ ID NO: 23. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 99.5% identical to the nucleic acid sequence set forth in SEQ ID NO: 23. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 99.9% identical to the nucleic acid sequence set forth in SEQ ID NO: 23. In some embodiments, a targeting construct comprises the nucleic acid sequence set forth in SEQ ID NO: 23.

[0137] In some embodiments, a targeting construct comprises a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to the nucleic acid sequence set forth in SEQ ID NO: 25. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 80% identical to the nucleic acid sequence set forth in SEQ ID NO: 25. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 85% identical to the nucleic acid sequence set forth in SEQ ID NO: 25. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 90% identical to the nucleic acid sequence set forth in SEQ ID NO: 25. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 91% identical to the nucleic acid sequence set forth in SEQ ID NO: 25. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 92% identical to the nucleic acid sequence set forth in SEQ ID NO: 25. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 93% identical to the nucleic acid sequence set forth in SEQ ID NO: 25. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 94% identical to the nucleic acid sequence set forth in SEQ ID NO: 25. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 95% identical to the nucleic acid sequence set forth in SEQ ID NO: 25. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 96% identical to the nucleic acid sequence set forth in SEQ ID NO: 25. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 97% identical to the nucleic acid31sequence set forth in SEQ ID NO: 25. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 98% identical to the nucleic acid sequence set forth in SEQ ID NO: 25. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 99% identical to the nucleic acid sequence set forth in SEQ ID NO: 25. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 99.5% identical to the nucleic acid sequence set forth in SEQ ID NO: 25. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 99.9% identical to the nucleic acid sequence set forth in SEQ ID NO: 25. In some embodiments, a targeting construct comprises the nucleic acid sequence set forth in SEQ ID NO: 25.

[0138] In some embodiments, a targeting construct comprises a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to the nucleic acid sequence set forth in SEQ ID NO: 27. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 80% identical to the nucleic acid sequence set forth in SEQ ID NO: 27. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 85% identical to the nucleic acid sequence set forth in SEQ ID NO: 27. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 90% identical to the nucleic acid sequence set forth in SEQ ID NO: 27. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 91% identical to the nucleic acid sequence set forth in SEQ ID NO: 27. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 92% identical to the nucleic acid sequence set forth in SEQ ID NO: 27. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 93% identical to the nucleic acid sequence set forth in SEQ ID NO: 27. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 94% identical to the nucleic acid sequence set forth in SEQ ID NO: 27. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 95% identical to the nucleic acid sequence set forth in SEQ ID NO: 27. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 96% identical to the nucleic acid sequence set forth in SEQ ID NO: 27. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 97% identical to the nucleic acid sequence set forth in SEQ ID NO: 27. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 98% identical to the nucleic acid sequence set forth in SEQ ID NO: 27. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 99% identical to the nucleic acid sequence set forth in SEQ ID NO: 27. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 99.5% identical to the nucleic acidsequence set forth in SEQ ID NO: 27. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 99.9% identical to the nucleic acid sequence set forth in SEQ ID NO: 27. In some embodiments, a targeting construct comprises the nucleic acid sequence set forth in SEQ ID NO: 27.

[0139] In some embodiments, a targeting construct comprises a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to the nucleic acid sequence set forth in SEQ ID NO: 29. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 80% identical to the nucleic acid sequence set forth in SEQ ID NO: 29. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 85% identical to the nucleic acid sequence set forth in SEQ ID NO: 29. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 90% identical to the nucleic acid sequence set forth in SEQ ID NO: 29. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 91% identical to the nucleic acid sequence set forth in SEQ ID NO: 29. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 92% identical to the nucleic acid sequence set forth in SEQ ID NO: 29. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 93% identical to the nucleic acid sequence set forth in SEQ ID NO: 29. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 94% identical to the nucleic acid sequence set forth in SEQ ID NO: 29. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 95% identical to the nucleic acid sequence set forth in SEQ ID NO: 29. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 96% identical to the nucleic acid sequence set forth in SEQ ID NO: 29. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 97% identical to the nucleic acid sequence set forth in SEQ ID NO: 29. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 98% identical to the nucleic acid sequence set forth in SEQ ID NO: 29. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 99% identical to the nucleic acid sequence set forth in SEQ ID NO: 29. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 99.5% identical to the nucleic acid sequence set forth in SEQ ID NO: 29. In some embodiments, a targeting construct comprises a nucleic acid sequence at least 99.9% identical to the nucleic acid sequence set forth in SEQ ID NO: 29. In some embodiments, a targeting construct comprises the nucleic acid sequence set forth in SEQ ID NO: 29.

[0140] In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to an amino acid sequence set forth in SEQ ID NOs: 18, 20, 22, 24, 26, or 28.

[0141] In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to the amino acid sequence set forth in SEQ ID NO: 18. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 18. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 18. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 18. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 91% identical to the amino acid sequence set forth in SEQ ID NO: 18. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 92% identical to the amino acid sequence set forth in SEQ ID NO: 18. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 93% identical to the amino acid sequence set forth in SEQ ID NO: 18. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 94% identical to the amino acid sequence set forth in SEQ ID NO: 18. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 18. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 96% identical to the amino acid sequence set forth in SEQ ID NO: 18. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 97% identical to the amino acid sequence set forth in SEQ ID NO: 18. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 18. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 18. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence atleast 99.5% identical to the amino acid sequence set forth in SEQ ID NO: 18. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 99.9% identical to the amino acid sequence set forth in SEQ ID NO: 18. In some embodiments, a targeting construct encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 18.

[0142] In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to the amino acid sequence set forth in SEQ ID NO: 20. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 20. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 20. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 20. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 91% identical to the amino acid sequence set forth in SEQ ID NO: 20. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 92% identical to the amino acid sequence set forth in SEQ ID NO: 20. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 93% identical to the amino acid sequence set forth in SEQ ID NO: 20. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 94% identical to the amino acid sequence set forth in SEQ ID NO: 20. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 20. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 96% identical to the amino acid sequence set forth in SEQ ID NO: 20. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 97% identical to the amino acid sequence set forth in SEQ ID NO: 20. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 20. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 20. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence atleast 99.5% identical to the amino acid sequence set forth in SEQ ID NO: 20. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 99.9% identical to the amino acid sequence set forth in SEQ ID NO: 20. In some embodiments, a targeting construct encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 20.

[0143] In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to the amino acid sequence set forth in SEQ ID NO: 22. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 22. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 22. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 22. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 91% identical to the amino acid sequence set forth in SEQ ID NO: 22. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 92% identical to the amino acid sequence set forth in SEQ ID NO: 22. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 93% identical to the amino acid sequence set forth in SEQ ID NO: 22. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 94% identical to the amino acid sequence set forth in SEQ ID NO: 22. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 22. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 96% identical to the amino acid sequence set forth in SEQ ID NO: 22. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 97% identical to the amino acid sequence set forth in SEQ ID NO: 22. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 22. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 22. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence atleast 99.5% identical to the amino acid sequence set forth in SEQ ID NO: 22. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 99.9% identical to the amino acid sequence set forth in SEQ ID NO: 22. In some embodiments, a targeting construct encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 22.

[0144] In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to the amino acid sequence set forth in SEQ ID NO: 24. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 24. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 24. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 24. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 91% identical to the amino acid sequence set forth in SEQ ID NO: 24. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 92% identical to the amino acid sequence set forth in SEQ ID NO: 24. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 93% identical to the amino acid sequence set forth in SEQ ID NO: 24. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 94% identical to the amino acid sequence set forth in SEQ ID NO: 24. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 24. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 96% identical to the amino acid sequence set forth in SEQ ID NO: 24. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 97% identical to the amino acid sequence set forth in SEQ ID NO: 24. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 24. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 24. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence atleast 99.5% identical to the amino acid sequence set forth in SEQ ID NO: 24. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 99.9% identical to the amino acid sequence set forth in SEQ ID NO: 24. In some embodiments, a targeting construct encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 24.

[0145] In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to the amino acid sequence set forth in SEQ ID NO: 26. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 26. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 26. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 26. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 91% identical to the amino acid sequence set forth in SEQ ID NO: 26. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 92% identical to the amino acid sequence set forth in SEQ ID NO: 26. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 93% identical to the amino acid sequence set forth in SEQ ID NO: 26. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 94% identical to the amino acid sequence set forth in SEQ ID NO: 26. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 26. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 96% identical to the amino acid sequence set forth in SEQ ID NO: 26. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 97% identical to the amino acid sequence set forth in SEQ ID NO: 26. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 26. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 26. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence atleast 99.5% identical to the amino acid sequence set forth in SEQ ID NO: 26. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 99.9% identical to the amino acid sequence set forth in SEQ ID NO: 26. In some embodiments, a targeting construct encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 26.

[0146] In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to the amino acid sequence set forth in SEQ ID NO: 28. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 28. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 85% identical to the amino acid sequence set forth in SEQ ID NO: 28. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 28. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 91% identical to the amino acid sequence set forth in SEQ ID NO: 28. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 92% identical to the amino acid sequence set forth in SEQ ID NO: 28. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 93% identical to the amino acid sequence set forth in SEQ ID NO: 28. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 94% identical to the amino acid sequence set forth in SEQ ID NO: 28. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 28. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 96% identical to the amino acid sequence set forth in SEQ ID NO: 28. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 97% identical to the amino acid sequence set forth in SEQ ID NO: 28. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 98% identical to the amino acid sequence set forth in SEQ ID NO: 28. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 28. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence atleast 99.5% identical to the amino acid sequence set forth in SEQ ID NO: 28. In some embodiments, a targeting construct encodes a polypeptide comprising an amino acid sequence at least 99.9% identical to the amino acid sequence set forth in SEQ ID NO: 28. In some embodiments, a targeting construct encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 28.6.5.Target Cells and Methods of Editing

[0147] In some embodiments, a targeting construct is introduced into target cells or populations of target cells in order to produce engineered target cells in which an IMiD- responsive peptide-suicide protein fusion polypeptide coding sequence (i.e., a CRBN peptide sequence fused to a suicide protein sequence and / or a CRIMP peptide sequence fused to a suicide protein sequence) is integrated into and expressed from a STEL locus. In some embodiments, a targeting construct is introduced into target cells or populations of target cells in order to produce engineered target cells in which a CRIMP peptide sequence fused to a suicide protein sequence is integrated into and expressed from a first STEL locus and a CRBN peptide sequence fused to a suicide protein sequence is integrated into and expressed from a second STEL locus.

[0148] In some embodiments, the methods of the disclosure may be employed to express a IMiD-responsive peptide-suicide protein fusion polypeptide in mitotic or post-mitotic target cells in vivo and / or ex vivo and / or in vitro (e.g., to produce engineered target cells that can be reintroduced into an individual).

[0149] Any type of cell that may be of interest may be engineered to incorporate an IMiD- responsive peptide-suicide protein fusion polypeptide coding sequence into a STEL locus. In various embodiments, the target cell is a stem cell, e.g., a human embryonic stem cell (hESC), an induced pluripotent stem cell (iPSC), a germ cell; a somatic cell, e.g., a fibroblast, a hematopoietic cell, a neuron, a muscle cell, a bone cell, a hepatocyte, a pancreatic cell; an in vitro or in vivo embryonic cell of an embryo at any stage, e.g., a 1-cell, 2-cell, 4-cell, 8-cell, etc. stage zebrafish embryo; etc. . Cells may be from established cell lines, or they may be primary cells, where “primary cells”, “primary cell lines”, and “primary cultures” are used interchangeably herein to refer to cells and cells cultures that have been derived from a subject and allowed to grow in vitro for a limited number of passages, e.g., splittings, of the culture. For example, primary cultures include cultures that may have been passaged 0 times, 1 time, 2 times, 4 times, 5 times, 10 times, or 15 times, but not enough times to go through the crisis stage. Primary cell lines can be maintained for fewer than 10 passages in vitro. Target cells are, insome embodiments, unicellular organisms, or are grown in culture. Preferably, the target cells are of human origin.

[0150] In some embodiments, a cell engineered to incorporate an IMiD-responsive peptide- suicide protein fusion polypeptide coding sequence is a cell therapy modality. In some embodiments, the cell therapy modality is an autologous cell therapy modality. In some embodiments, the cell therapy modality is an allogeneic cell therapy modality.

[0151] If the cells are primary cells, such cells may be harvested from an individual by any suitable method. For example, leukocytes may be suitably harvested by apheresis, leukocytapheresis, density gradient separation, etc., while cells from tissues such as skin, muscle, bone marrow, spleen, liver, pancreas, lung, intestine, stomach, etc. are most suitably harvested by biopsy. An appropriate solution may be used for dispersion or suspension of the harvested cells. Such solution will generally be a balanced salt solution, e.g., normal saline, phosphate-buffered saline (PBS), Hank’s balanced salt solution, etc., suitably supplemented with fetal calf serum or other naturally occurring factors, in conjunction with an acceptable buffer at low concentration, e.g., from 5-25 mM. Suitable buffers include HEPES, phosphate buffers, lactate buffers, etc. The cells may be used immediately, or they may be stored, frozen, for long periods of time, being thawed and capable of being reused. In such cases, the cells will generally be frozen in 10% dimethyl sulfoxide (DMSO), 50% serum, 40% buffered medium, or some other such solution as is commonly used in the art to preserve cells at such freezing temperatures and thawed in a manner as commonly known in the art for thawing frozen cultured cells.6.5.1. Gene Editing Systems and Methods of Producing Cells

[0152] In some aspects, the present disclosure provides a method of producing a gene-edited target cell comprising introducing a targeting construct into the target cell and culturing the target cell under conditions that allow gene editing to occur. In some embodiments, a targeting construct is introduced into a target cell via nucleofection, as part of a gene editing system (e.g., a CRISPR / Cas-based gene editing system) designed to cleave target sequences within or near (e.g., flanking or adjoining) a STEL locus to facilitate homologous recombination of the targeting constructs of the target sequences. In some embodiments, the gene editing system comprises an endonuclease (e.g., a CRISPR-associated endonuclease or “Cas nuclease”), a guide RNA (e.g., single guide RNA or sgRNA), as well as the targeting construct. In some embodiments, the guide RNA is sgRNA. In some embodiments, the endonuclease is a Cas nuclease. In some embodiments, the gene editing system comprises a nucleic acid encoding the endonuclease. In some embodiments, the gene editing system is in the form of a compositionknown as a ribonucleoprotein or RNP complex. An RNP complex can be assembled by combining an endonuclease with a ribonucleic acid.

[0153] In some embodiments, the method comprises introducing a targeting construct configured to introduce a nucleotide sequence encoding a CRIMP peptide sequence fused to a suicide protein sequence and a nucleotide sequence encoding a CRBN peptide sequence fused to a suicide protein sequence into a STEL locus. In some embodiments, the method comprises introducing a first targeting construct configured to introduce a nucleotide sequence encoding a CRIMP peptide sequence fused to a suicide protein sequence into a first STEL locus and a second targeting construct configured to introduce a nucleotide sequence encoding a CRBN peptide sequence fused to a suicide protein sequence into a second STEL locus.6.5.2. Stem Cells

[0154] In some embodiments, the target cells that are engineered to express an IMiD- responsive peptide-suicide protein fusion polypeptide coding sequence from a STEL locus are stem cells, particularly pluripotent stem cells (PSCs) such as induced pluripotent stem cells (iPSCs) or human embryonic stem cells (hESCs), which are the starting point for the potential generation of large numbers of a specific cell type that can be delivered for regenerative medicine in patients with many different diseases. In some embodiments, the stem cells are human stem cells.

[0155] Following engineering to a PSC to express an IMiD-responsive peptide-suicide protein fusion polypeptide coding sequence, the PSC can be differentiated into a cell type of interest for cell therapy.

[0156] The PSCs, e.g., ESCs or recombinant PSCs can be differentiated into cells suitable for therapy, including the cells in the endoderm (e.g., lung, thyroid, or pancreatic cells, or progenitors thereof), ectoderm (e.g., skin, neuronal, or pigment cells, or progenitors thereof), and mesoderm (e.g., cardiac cells, skeletal muscle cells, red blood cells, smooth muscle cells, or progenitors thereof) lineages.

[0157] In some embodiments, the PSCs, e.g., ESCs or recombinant PSCs are differentiated into cardiovascular cells (cardiovascular cells (e.g., cardiomyocytes, pacemaker cell, cardiac fibroblasts, epicardial cells, endocardial cells, valvular interstitial cells, cardiac and vascular smooth muscle cells, cardiac and vascular endothelial cells, Purkinje fibers, or His-bundle cells).

[0158] In some embodiments, the PSCs, e.g., ESCs or recombinant PSCs are differentiated into cells in the endoderm (e.g., lung, thyroid, or pancreatic cells, or progenitors thereof), ectoderm (e.g., skin, neuronal, or pigment cells, or progenitors thereof) or mesoderm (e.g.,cardiac cells, skeletal muscle cells, red blood cells, smooth muscle cells, or progenitors thereof) lineages.

[0159] In some embodiments, a recombinant PSC of the disclosure is differentiated into a cardiac cell, or a precursor or progenitor cell thereof. In various embodiments, the cardiac cell is a cardiac progenitor cell or a mature or immature (atrial or ventricular) cardiomyocyte. In other embodiments, the cardiac cell is a cardiac endothelial cell or a nodal cell.

[0160] In some embodiments, a recombinant PSC of the disclosure is differentiated into a regulatory T cell, a myeloid cell, a dendritic cell, a macrophage (e.g., an immunosuppressive macrophage), a myeloid progenitor cell, or a precursor or progenitor cell thereof. Details on differentiation of PSCs into myeloid progenitor cells can be found in International (PCT) Publication Numbers WO 2023 / 150089 Al and WO 2017 / 152081 Al.

[0161] In some embodiments, a recombinant PSC of the disclosure is a neural cell or a precursor or progenitor cell thereof. In some embodiments, a neural cell is a microglia, a macroglia (e.g., oligodendrocytes, an astrocytes, a Schwann cells, or an enteric glia) and neurons, or neural stem and precursor cells or progenitor cells of any of the foregoing cells. In some embodiments, a recombinant PSC of the disclosure is differentiated into an oligodendrocyte progenitor cell or an oligodendrocyte.

[0162] In some embodiments, a recombinant PSC of the disclosure is differentiated into a neural lineage cell, for example a neural crest cells, an astrocyte, a dopaminergic neuron progenitor cell, a dopaminergic neuron cells, a midbrain dopaminergic neuron progenitor cell, a midbrain dopaminergic neuron, an authentic midbrain dopamine (DA) neuron, a dopaminergic neuron precursor cell, a floor plate midbrain progenitor cell, a floor plate midbrain DA neuron.

[0163] In some embodiments, a recombinant PSC of the disclosure is differentiated into a cell of the ocular system, such as a photoreceptor cell, a photoreceptor precursor cell, a retinal pigmented epithelium cell, a neural retinal cell, a neural retinal progenitor cell, or a precursor or progenitor cell thereof.

[0164] In further embodiments, a recombinant PSC of the disclosure is differentiated into a microglial cell or a microglial progenitor cell.

[0165] In further embodiments, a recombinant PSC of the disclosure is differentiated into a cell in the human metabolic system, optionally selected from a hepatocyte, a cholangiocyte, and a pancreatic beta cell, or a precursor or progenitor cell thereof.

[0166] In further embodiments, a recombinant PSC of the disclosure is differentiated into an enteric progenitor cell or an enteric cell.6.5.3. Differentiated Cells

[0167] In various embodiments, a cell at any stage of differentiation is engineered to express a IMiD responsive peptide-suicide protein fusion polypeptide. The differentiated cell may be derived from an engineered iPSC disclosed herein.

[0168] Exemplary differentiated cell types that can be engineered to express an IMiD- responsive peptide-suicide protein fusion polypeptide coding sequence include the cells in the endoderm (e.g., lung, thyroid, or pancreatic cells, or progenitors thereof), ectoderm (e.g., skin, neuronal, or pigment cells, or progenitors thereof) and mesoderm (e.g., cardiac cells, skeletal muscle cells, red blood cells, smooth muscle cells, or progenitors thereof) lineages.Alternatively, PSCs can be differentiated into cells in these lineages and then engineered with a targeting construct of the disclosure.

[0169] In some embodiments, a cardiac cell is engineered to express an IMiD-responsive peptide-suicide protein fusion polypeptide coding sequence. In some embodiments, the cardiac cell is a cardiac progenitor cell or a mature or immature (atrial or ventricular) cardiomyocyte. In other embodiments, the cardiac cell is a cardiac endothelial cell or a nodal cell.

[0170] Differentiated cell types can include cardiovascular cells (e.g., cardiomyocytes, pacemaker cell, cardiac fibroblasts, epicardial cells, endocardial cells, valvular interstitial cells, cardiac and vascular smooth muscle cells, cardiac and vascular endothelial cells, Purkinje fibers, or His-bundle cells).

[0171] In some embodiments, a human immune cell selected from a regulatory T cell, a myeloid cell, a dendritic cell, and / or a macrophage (e.g., an immunosuppressive macrophage), or a precursor or progenitor cell thereof is engineered to express an IMiD-responsive peptide- suicide protein fusion polypeptide coding sequence.

[0172] In some embodiments, a neural cell or a precursor or progenitor cell thereof, is engineered to express an IMiD-responsive peptide-suicide protein fusion polypeptide coding sequence. In some embodiments, a neural cell is a microglia, a macroglia (e.g., oligodendrocytes, an astrocytes, a Schwann cells, or an enteric glia) and neurons, or neural stem and precursor cells of any of the foregoing cells. In some embodiments, an oligodendrocyte progenitor cell or an oligodendrocyte is engineered to express an IMiD-responsive peptide- suicide protein fusion polypeptide coding sequence.

[0173] In some embodiments, a neural lineage cell is engineered to express an IMiD- responsive peptide-suicide protein fusion polypeptide coding sequence. In various embodiments, the neural lineage cell is a neural crest cell, an astrocyte, a dopaminergic neuron progenitor cell, a dopaminergic neuron cells, a midbrain dopaminergic neuron progenitor cell, a midbraindopaminergic neuron, an authentic midbrain dopamine (DA) neuron, a dopaminergic neuron precursor cell, a floor plate midbrain progenitor cell, a floor plate midbrain DA neuron.

[0174] In some embodiments, a cell of the ocular system or a precursor or progenitor cell thereof is engineered to express an IMiD-responsive peptide-suicide protein fusion polypeptide coding sequence. In various embodiments, the cell of the ocular system is a photoreceptor cell, a photoreceptor precursor cell, a retinal pigmented epithelium cell, a neural retinal cell, or a neural retinal progenitor cell.

[0175] In further embodiments, a microglial cell or a microglial progenitor cell is engineered to express an IMiD-responsive peptide-suicide protein fusion polypeptide coding sequence.

[0176] In further embodiments, a cell in the human metabolic system is engineered to express an IMiD-responsive peptide-suicide protein fusion polypeptide coding sequence. In various embodiments, the cell in the human metabolic system is optionally selected from a hepatocyte, a cholangiocyte, and a pancreatic beta cell or a precursor or progenitor cell thereof.

[0177] In further embodiments, an enteric progenitor cell or an enteric cell is engineered to express an IMiD-responsive peptide-suicide protein fusion polypeptide coding sequence.

[0178] In some embodiments of any of the aforementioned cells, the cell is a human cell.

[0179] Any of the foregoing differentiated cell types can be differentiated from PSCs prior to engineering them to express an IMiD-responsive peptide-suicide protein fusion polypeptide coding sequence.6.6.Methods of Administration

[0180] The present disclosure provides methods of using the therapeutic cells disclosed herein for treating a patient in need of cell therapy. The methods comprise administering to the patient a gene-edited target cell engineered to express an IMiD-responsive peptide-suicide protein fusion polypeptide coding sequence from a STEL locus, for example a gene-edited target cell as disclosed in Section 6.4 or any subsection thereof. In some embodiments, the gene- edited target cell is comprised in a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

[0181] The methods can further comprise controlling the gene-edited target cell population in the patient by (a) optionally monitoring the gene-edited target cell population in the patient; and / or (b) administering an inducer of a modified CRBN and / or modified degron (e.g., an IMiD, such as pomalidomide) if the patient experiences adverse events related to the gene-edited target cell population.

[0182] The present disclosure further provides methods of mitigating adverse events or a safety risk associated with cell therapy in the form of gene-edited target cells engineered toexpress an IMiD-responsive peptide-suicide protein fusion polypeptide coding sequence from a STEL locus, e.g., gene-edited target cell as disclosed in Section 6.4 or any subsection thereof, comprising administering to a patient who received the gene-edited target cells an inducer of a modified CRBN and / or modified degron (e.g., an IMiD, such as pomalidomide) if the patient experiences adverse events or a safety risk related to the gene-edited target cells or pharmaceutical composition.

[0183] In some embodiments, the inducer of the modified CRBN and / or modified degron is an IMiD, such as pomalidomide and its variants or derivatives.

[0184] In some aspects, the methods comprise administering a single inducer of the modified CRBN and / or modified degron. In some embodiments, the single inducer of the modified CRBN and / or modified degron is thalidomide, iberdomide, lenalidomide, and pomalidomide.Enumerated EmbodimentsEmbodiment 1 A targeting construct comprising: a. a first homology arm corresponding to a 5' target sequence comprising a first region of homology to a target genomic locus; b. a nucleotide insert comprising a nucleotide sequence encoding a fusion polypeptide comprising: i.a modified degron; and ii.a caspase 9 domain or derivative or functional fragment thereof; c. a second homology arm corresponding to a 3' target sequence comprising second region of homology to the target genomic locus, d. wherein the modified degron comprises an amino acid sequence corresponding to a SEQ ID NO: 1 or a variant thereof, optionally wherein the modified degron comprises a mutation selected from the group consisting of G30E and R27H, e. wherein the targeting construct is configured such that upon its recombination with the target genomic locus, the fusion polypeptide coding sequence is integrated into a STEL gene and becomes operably linked to the STEL gene regulatory element.Embodiment 2 The targeting construct of embodiment 1 , wherein the targeting construct further comprises a modified cereblon (CRBN) and a second caspase 9 domain or derivative or functional fragment thereof, wherein the modified CRBN comprises anamino acid sequence corresponding to a SEQ ID NO: 4 or a variant thereof, optionally wherein the modified degron comprises a mutation selected from the group consisting of I371N, Q325H, T359V, Q325R, and H397F.Embodiment 3 A targeting construct comprising: a. a first homology arm corresponding to a 5' target sequence comprising a first region of homology to a target genomic locus; b. a nucleotide insert comprising a nucleotide sequence encoding a fusion polypeptide comprising: i.a modified cereblon (CRBN); and ii.a caspase 9 domain or derivative or functional fragment thereof; c. a second homology arm corresponding to a 3' target sequence comprising second region of homology to the target genomic locus, d. wherein the modified CRBN comprises an amino acid sequence corresponding to a SEQ ID NO: 4 or a variant thereof, optionally wherein the modified degron comprises a mutation selected from the group consisting of 137 IN, Q325H, T359V, Q325R, and H397F, e. wherein the targeting construct is configured such that upon its recombination with the target genomic locus, the fusion polypeptide coding sequence is integrated into a STEL gene and becomes operably linked to the STEL gene regulatory element.Embodiment 4 The targeting construct of embodiment 3, wherein the nucleotide insert further comprises a modified degron and a second caspase 9 domain or derivative or functional fragment thereof.Embodiment 5 A targeting construct comprising: a. a first homology arm corresponding to a 5' target sequence comprising a first region of homology to a target genomic locus; b. a nucleotide insert comprising a nucleotide sequence encoding a fusion polypeptide comprising: i.a modified degron, wherein the modified degron comprises an amino acid sequence corresponding to a SEQ ID NO: 1 or a variant thereof, optionally wherein the modified degron or variant comprises a mutation selected from the group consisting of G30 or R27; ii.a first caspase 9 domain or derivative or functional fragment thereof;iii.a modified CRBN, wherein the modified CRBN comprises an amino acid sequence corresponding to a SEQ ID NO: 4 or a variant thereof, optionally wherein the modified degron comprises a mutation selected from the group consisting of 1371, Q325, T359, and H397; iv.a second caspase 9 domain or derivative or functional fragment thereof; c. a second homology arm corresponding to a 3' target sequence comprising second region of homology to the target genomic locus, d. wherein the targeting construct is configured such that upon its recombination with the target genomic locus, the fusion polypeptide coding sequence is integrated into a STEL gene and becomes operably linked to the STEL gene regulatory element.Embodiment 6 A targeting construct comprising: a. a first homology arm corresponding to a 5' target sequence comprising a first region of homology to a target genomic locus; b. a nucleotide insert comprising a nucleotide sequence encoding a fusion polypeptide comprising: i.a modified degron; ii.a first caspase 9 domain or derivative or functional fragment thereof; iii.a modified cereblon; and iv.a second caspase 9 domain or derivative or functional fragment thereof; c. a second homology arm corresponding to a 3' target sequence comprising second region of homology to the target genomic locus, d. wherein the modified degron comprises an amino acid sequence corresponding to a reference degron sequence (SEQ ID NO: 1) or a variant thereof, e. wherein the modified cereblon comprises an amino acid sequence corresponding to a reference cereblon sequence (SEQ ID NO: 4) or a variant thereof, f. optionally wherein the modified degron comprises at least one amino acid substitution selected from the group consisting of G30E and R27H, g. optionally wherein the modified cereblon comprises at least one amino acid substitution selected from the group consisting of I371N, Q325H, T359V, Q325R, and H397F,h. wherein the targeting construct is configured such that upon its recombination with the target genomic locus, the fusion polypeptide coding sequence is integrated into a STEL gene and becomes operably linked to the STEL gene regulatory element.Embodiment 7 The targeting construct of any one of embodiments 2-6, wherein the modified CRBN comprises one or more amino acid substitutions selected from the group consisting of 137 IN, Q325H, T359V, Q325R, and H397F relative to SEQ ID NO:4.Embodiment 8 The targeting construct of any one of embodiments 2-7, wherein the modified CRBN comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 5-8.Embodiment 9 The targeting construct of any one of embodiments 2-8, wherein the modified CRBN comprises an 137 IN amino acid substitution relative to SEQ ID NO:4.Embodiment 10 The targeting construct of any one of embodiments 2-8, wherein the modified CRBN comprises Q325H, 137 IN, and T359V amino acid substitutions relative to SEQ ID NO: 4.Embodiment 11 The targeting construct of any one of embodiments 2-8, wherein the modified CRBN comprises Q325R, I371N, H397F, and T359V amino acid substitutions relative to SEQ ID NO: 4.Embodiment 12 The targeting construct of any one of embodiments 1-11, wherein the modified degron comprises an amino acid substitution selected from the group consisting of G30E and R27H, relative to SEQ ID NO: 1.Embodiment 13 The targeting construct of any one of embodiments 1-12, wherein the modified degron comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3.Embodiment 14 The targeting construct of any one of embodiments 1-13, wherein the modified degron comprises a R27H amino acid substitution relative to SEQ ID NO:1.Embodiment 15 The targeting construct of any one of embodiments 1-13, wherein the modified degron comprises a G30E amino acid substitution relative to SEQ ID NO: 1.Embodiment 16 The targeting construct of any one of embodiments 1-6, wherein the modified degron comprises a R27H amino acid substitution relative to SEQ ID NO: 1 and the modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 4.Embodiment 17 The targeting construct of any one of embodiments 1-6, wherein the modified degron comprises a G30E amino acid substitution relative to SEQ ID NO: 1 and the modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 4.Embodiment 18 The targeting construct of any one of embodiments 1-6, wherein the modified degron comprises an amino acid sequence set forth in SEQ ID NO: 1 and the modified CRBN comprises an 137 IN amino acid substitution relative to SEQ ID NO: 4.Embodiment 19 The targeting construct of any one of embodiments 1-6, wherein the modified degron comprises a G30E amino acid substitution relative to SEQ ID NO: 1 and the modified CRBN comprises an 137 IN amino acid substitution relative to SEQ ID NO: 4.Embodiment 20 The targeting construct of any one of embodiments 1-6, wherein the modified degron comprises a R27H amino acid substitution relative to SEQ ID NO: 1 and the modified CRBN comprises Q325H, 137 IN, and T359V amino acid substitutions relative to SEQ ID NO: 4.Embodiment 21 The targeting construct of any one of embodiments 1-6, wherein the modified degron comprises a R27H amino acid substitution relative to SEQ ID NO: 1 and the modified CRBN comprises Q325R, 137 IN, H397F, and T359V amino acid substitutions, relative to SEQ ID NO: 4.Embodiment 22 The targeting construct of any one of embodiments 1-21, wherein the modified degron is N-terminal to the caspase 9 domain or derivative or functional fragment thereof.Embodiment 23 The targeting construct of any one of embodiments 1-21, wherein the modified degron is C-terminal to the caspase 9 domain or derivative or functional fragment thereof.Embodiment 24 The targeting construct of any one of embodiments 1-23, wherein the modified CRBN is N-terminal to the caspase 9 domain or derivative or functional fragment thereof.Embodiment 25 The targeting construct of any one of embodiments 1-23, wherein the modified CRBN is C-terminal to the caspase 9 domain or derivative or functional fragment thereof.Embodiment 26 The targeting construct of any one of embodiments 1-25, wherein the fusion polypeptide comprises a linker between the modified degron and the caspase 9 domain or derivative or functional fragment thereof.Embodiment 27 The targeting construct of any one of embodiments 1-25, wherein the fusion polypeptide comprises a linker between the modified CRBN and the caspase 9 domain or derivative or functional fragment thereof.Embodiment 28 The targeting construct of any one of embodiments 1-27, which is configured such that upon its recombination with the target genomic locus, the STEL gene is modified such to incorporate the fusion polypeptide coding sequence 3' to the STEL protein coding sequence.Embodiment 29 The targeting construct of any one of embodiments 1-27, which is configured such that upon its recombination with the target genomic locus, the STEL gene is modified such to incorporate the fusion polypeptide coding sequence 5' to the STEL protein coding sequence.Embodiment 30 The targeting construct of any one of embodiments 1-29, wherein the nucleotide insert further comprises a nucleotide sequence encoding a separator sequence.Embodiment 31 The targeting construct of embodiment 30, which is configured such that upon recombination of the targeting construct with the target genomic locus, the separator sequence coding sequence is positioned between the coding sequence of the STEL protein and the fusion polypeptide coding sequence.Embodiment 32 The targeting construct of embodiment 30 or 31 , wherein the separator sequence is an internal ribosome entry site (“IRES”).Embodiment 33 The targeting construct of embodiment 30 or 31 , wherein the separator sequence is a self-cleaving peptide.Embodiment 34 The targeting construct of embodiment 33, wherein the selfcleaving peptide is a 2A peptide.Embodiment 35 The targeting construct of embodiment 33 or 34, wherein the selfcleaving peptide is T2A, P2A, E2A, F2A, PQR, Opt2A, or Opt2A_2.0.Embodiment 36 The targeting construct of any one of embodiments 1-35, wherein the STEL gene encodes a polypeptide involved in one or more of: glycolysis, ribonucleopolypeptide complex formation, focal adhesion, cell-substrate adherens junction, cell-substrate junction, cell anchoring, extracellular exosome, extracellular vesicle, intracellular organelle, anchoring junction, RNA binding, nucleic acid binding (e.g., rRNA or mRNA binding), and polypeptide binding.Embodiment 37 The targeting construct of any one of embodiments 1-36, wherein the STEL gene encodes a ribosomal polypeptide.Embodiment 38 The targeting construct of embodiment 37, wherein the STEL gene is RPL13A, RPLPO, RPL10, RPL13, RPSJ8, RPL3, RPLP1, RPL15, RPL41, RPL11, RPL32, RPL18 A, RPL19, RPL28, RPL29, RPL9, RPL8, RPL6, RPL18, RPL7, RPL7A, RPL21, RPL37A, RPL 12, RPL5, RPL34, RPL35A, RPL30, RPL24, RPL39, RPL37, RPL14, RPL27A, RPLP2, RPL23A, RPL26, RPL36, RPL35, RPL23, RPL4, or RPL22.Embodiment 39 The targeting construct of any one of embodiments 1-37, wherein the STEL gene encodes a ribosomal polypeptide small subunit (RPS).Embodiment 40 The targeting construct of embodiment 39, wherein the STEL gene is RPS2, RPS19, RPS14, RPS3A, RPS12, RPS3, RPS6, RPS23, RPS27A, RPS8, RPS4X, RPS7, RPS24, RPS27, RPS15A, RPS9, RPS28, RPS13, RPSA, RPS5, RPS 16, RPS25, RPS15, RPS20, or RPS11.Embodiment 41 The targeting construct of any one of embodiments 1-36, wherein the STEL gene encodes a mitochondrial polypeptide.Embodiment 42 The targeting construct of embodiment 41, wherein the STEL gene is MT-CO1, MT-CO2, MT-ND4, MT-ND1, or MT-ND2.Embodiment 43 The targeting construct of any one of embodiments 1-36, wherein the STEL gene encodes an actin polypeptide.Embodiment 44 The targeting construct of embodiment 43, wherein the STEL gene is ACTG1 or ACTB.Embodiment 45 The targeting construct of any one of embodiments 1-36, wherein the STEL gene encodes a eukaryotic translation factor.Embodiment 46 The targeting construct of embodiment 45, wherein the STEL gene is EEF1A1, EEF2, or EIF1.Embodiment 47 The targeting construct of any one of embodiments 1-36, wherein the STEL gene encodes a histone.Embodiment 48 The targeting construct of embodiment 47, wherein the STEL gene is H3F3A or H3F3B.Embodiment 49 The targeting construct of any one of embodiments 1-36, wherein the STEL gene is FTL, FTH1, TPT1, IMSB10, GAPDH, PTMA, GNB2L1, NACA, YBX1, NPM1, FAU, UBA52, HSP90AB1, MYL6, SERF2, or SRP14.Embodiment 50 The targeting construct of embodiment 49, wherein the STEL gene is GAPDH.Embodiment 51 The targeting construct of embodiment 49, wherein the STEL gene is RPL13A.Embodiment 52 The targeting construct of embodiment 49, wherein the STEL gene is RPL7.Embodiment 53 The targeting construct of embodiment 49, wherein the STEL gene is RPLPO.Embodiment 54 The targeting construct of any one of embodiments 1-53, wherein the nucleotide insert further comprises a transgene.Embodiment 55 The targeting construct of embodiment 54, wherein the transgene is linked to the fusion polypeptide coding sequence.Embodiment 56 The targeting construct of embodiment 55, wherein the fusion polypeptide coding sequence and transgene are connected via a nucleotide sequence encoding a separator sequence.Embodiment 57 The targeting construct of embodiment 56, wherein the separator sequence is an internal ribosome entry site (“IRES”).Embodiment 58 The targeting construct of embodiment 56, wherein the separator sequence is a nucleotide sequence encoding a self-cleaving peptide (the “self-cleaving peptide coding sequence”).Embodiment 59 The targeting construct of embodiment 58, wherein the selfcleaving peptide is a 2A peptide.Embodiment 60 The targeting construct of embodiment 58 or 59, wherein the selfcleaving peptide is T2A, P2A, E2A, F2A, PQR, Opt2A, or Opt2A_2.0.Embodiment 61 The targeting construct of any one of embodiments 54-60, wherein the transgene encodes a therapeutic polypeptide.Embodiment 62 The targeting construct of any one of embodiments 1-61, wherein the targeting construct comprises a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to a nucleic acid sequence set forth in SEQ ID NO: 19, 21, 23, 25, 27, or 29.Embodiment 63 The targeting construct of embodiment 62, wherein the targeting construct comprises a nucleic acid sequence set forth in SEQ ID NO: 19, 21, 23, 25, 27, or 29.Embodiment 64 The targeting construct of any one of embodiments 1-62, wherein the targeting construct encodes a polypeptide comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to an amino acid sequence set forth in SEQ ID NO: 18, 20, 22, 24, 26, or 28.Embodiment 65 The targeting construct of embodiment 63, wherein the targeting construct encodes a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 18, 20, 22, 24, 26, or 28.Embodiment 66 A system comprising: a. the targeting construct of any one of embodiments 1-65;b. a CRISPR-associated endonuclease (“Cas nuclease”) or a nucleic acid encoding a Cas nuclease; and c. a guide RNA (“gRNA”) comprising a scaffold for binding the Cas polypeptide and a spacer sequence corresponding to the STEL gene, or a nucleic acid encoding the gRNA.Embodiment 67 The system of embodiment 66, wherein the guide RNA is a single guide RNA (“sgRNA”).Embodiment 68 The system of embodiment 66 or 67, which comprises the Cas nuclease and gRNA.Embodiment 69 The system of any one of embodiments 66-68, which is in the form of a ribonucleoprotein particle (“RNP”).Embodiment 70 A method of producing a gene-edited target cell, comprising: a. introducing the system of any one of embodiments 68-69 into a target cell; and b. culturing the target cell under conditions in which gene editing occurs, thereby producing gene-edited target cell.Embodiment 71 The method of embodiment 70, wherein the target cell is a stem cell, or a cell differentiated from a stem cell.Embodiment 72 The method of embodiment 70 or 71, wherein the target cell is a stem cell.Embodiment 73 The method of embodiment 72, wherein the stem cell is a human embryonic stem cell, an induced pluripotent stem cell (“iPSC”) or a cell differentiated therefrom.Embodiment 74 The method of embodiment 70 or 71, wherein the target cell is: a. a regulatory T cell, a myeloid cell, a dendritic cell, a macrophage (e.g., an immunosuppressive macrophage), a myeloid progenitor cell, or a precursor or progenitor cell thereof; b. a cell in the human nervous system, optionally selected from dopaminergic neuron, a microglial cell, an oligodendrocyte, an astrocyte, a cortical neuron, a spinal or oculomotor neuron, an enteric neuron, a Placode-derived cell, a Schwann cell, and a trigeminal or sensory neuron, or a precursor or progenitor cell thereof; c. a cell in the human cardiovascular system, optionally selected from a cardiomyocyte, an endothelial cell, and a nodal cell, or a precursor or progenitor cell thereof; d. a cell in the human metabolic system, optionally selected from a hepatocyte, a cholangiocyte, and a pancreatic beta cell, or a precursor or progenitor cell thereof, or e. a cell in the human ocular system, optionally selected from a retinal pigment epithelial cell, a photoreceptor cone cell, a photoreceptor rod cell, a bipolar cell, a ganglion cell, or a precursor or progenitor cell thereof.Embodiment 75 The method of any one of embodiments 70-74, wherein the gene- edited target cell is of ectoderm lineage, optionally wherein the gene-edited target cell is a neuron.Embodiment 76 The method of any one of embodiments 70-74, wherein the gene- edited target cell is of mesoderm lineage, optionally wherein the gene-edited target cell is a cardiomyocyte.Embodiment 77 A gene-edited target cell obtained or obtainable by the method of any one of embodiments 70-76.Embodiment 78 A gene-edited target cell comprising a STEL gene that comprises a nucleic acid encoding a fusion polypeptide under the transcriptional control of a STEL gene regulatory element, the fusion polypeptide comprising: a. a modified CRBN; and b. a caspase 9 domain or derivative or functional fragment thereof; wherein the modified CRBN comprises a mutation selected from the group consisting of I371N, Q325H, T359V, Q325R, and H397F.Embodiment 79 A gene-edited target cell comprising a STEL gene that comprises a nucleic acid encoding a fusion polypeptide under the transcriptional control of a STEL gene regulatory element, the fusion polypeptide comprising: a. a modified degron; and b. a caspase 9 domain or derivative or functional fragment thereof;wherein the modified degron comprises a mutation selected from the group consisting of G30E and R27H.Embodiment 80 A gene-edited target cell comprising a STEL gene that comprises a nucleic acid encoding a fusion polypeptide under the transcriptional control of a STEL gene regulatory element, the fusion polypeptide comprising: a. a modified degron; b. a caspase 9 domain or derivative or functional fragment thereof, wherein the modified degron comprises a mutation selected from the group consisting of G30E and R27H; c. a modified CRBN, wherein the modified CRBN comprises a mutation selected from the group consisting of I371N, Q325H, T359V, Q325R, and H397F; d. a caspase 9 domain or derivative or functional fragment thereof; wherein the modified CRBN comprises a mutation selected from the group consisting of 137 IN, Q325H, T359V, Q325R, and H397F; and wherein the modified degron comprises a mutation selected from the group consisting of G30E and R27H.Embodiment 81 The gene-edited target cell of embodiment 78 or 79, wherein the modified degron is as defined in any one of embodiments 1-59.Embodiment 82 The gene-edited target cell of any one of embodiments 78, 79, or 80, wherein the modified CRBN is as defined in any one of embodiments 2-59.Embodiment 83 The gene-edited target cell of any one of embodiments 78-82, wherein the caspase 9 domain or derivative or functional fragment thereof is as defined in any one of embodiments 1-65.Embodiment 84 The gene-edited target cell of any one of embodiments 78-83, wherein the fusion polypeptide is as defined in any one of embodiments 1-65.Embodiment 85 The gene-edited target cell of any one of embodiments 78-84, wherein the STEL gene is configured as defined in any one of embodiments 1-65.Embodiment 86 The gene-edited target cell of any one of embodiments 78-85, which further comprises a transgene in the STEL gene.Embodiment 87 The gene-edited target cell of embodiment 86, wherein the transgene is as defined in any one of embodiments 54-65.Embodiment 88 The gene-edited target cell of any one of embodiments 78-87, wherein the target cell is as defined in any one of embodiments 71-77.Embodiment 89 A pharmaceutical composition comprising the gene-edited target cell of any one of embodiments 78-88 and a pharmaceutically acceptable carrier.Embodiment 90 A method of treating a patient in need thereof, comprising administering to the patient the gene-edited target cell of any one of embodiments 78-88 or the pharmaceutical composition of embodiment 89.Embodiment 91 The method of embodiment 90, which further comprises controlling the gene-edited target cell population in the patient by: a. monitoring, optionally, the gene-edited target cell population in the patient; and / or b. administering an inducer of the modified degron and / or modified cereblon if the patient experiences adverse events related to the gene-edited target cell population.Embodiment 92 A method of mitigating adverse events or a safety risk associated with cell therapy in the form of the gene-edited target cells any one of embodiments 78- 88 or the pharmaceutical composition of embodiment 89, the method comprising administering to a patient who received the gene-edited target cells or pharmaceutical composition an inducer of a degron or modified cereblon if the patient experiences adverse events or a safety risk related to the gene-edited target cells or pharmaceutical composition.Embodiment 93 The method of embodiment 91 or 92, wherein the inducer of the modified degron or modified cereblon is thalidomide, iberdomide, lenalidomide, or pomalidomide.Embodiment 94 The method of embodiment 93, wherein the inducer of the modified degron or modified cereblon is pomalidomide.Embodiment 95 The method of any one of embodiments 91-94, wherein a combination of two or more inducers of the modified degron or modified cereblon is administered.Embodiment 96 The method of embodiment 95, wherein the combination has a synergistic effect and / or utilizes reduced dosing (e.g., reduced dosing amount and / orfrequency) than would be required a single inducer of the modified degron or modified cereblon.7. EXAMPLES7.1.Example 1: Design and Generation of IMiD-Responsive Suicide Switch Protein Constructs

[0185] Kill switch constructs of the disclosure were designed to trigger apoptosis in response to immunomodulatory imide drugs (IMiDs) such as pomalidomide that may be able to penetrate through the blood-brain barrier. However, implementation of such kill switches in pluripotent stem cell (PSC)-derived cell products can be difficult because expression of transgenes can be lost after differentiating a PSC to another cell type e.g., an immune cell such as a T cell or macrophage, a CNS cell such as a neuron, microglia, macroglia, or precursor thereof, or a cardiovascular cell. Thus, to implement such kill switches in pluripotent stem cell (PSC)-derived cell products, constructs were designed such that a single construct comprising a sequence that encodes a modified version of Cereblon (CRBN), termed del.CRBN, fused to a truncated Caspase 9 sequence, was transcriptionally linked to a sequence encoding a CRIMP domain fused to another truncated Caspase 9 sequence, and flanked by homology arms complementary to genomic sequences of a sustained transcription expression locus (STEL) (FIG. 1A). See WO2021072329A1, which is incorporated herein by reference in its entirety. The del.CRBN (e.g., SEQ ID NO: 5) is a modified version of CRBN that has the DDB1 domain removed to prevent its association with the E3 ubiquitin ligase complex. CRIMP (CRBN-IMiD binding partner) comprises a degron sequence derived from IKZF3 protein, and complexes with CRBN only in the presence of IMiD drugs such as pomalidomide. Caspase 9 is truncated by removal of its CARD domain, such that self cleavage is prevented. Addition of an IMiD allows complexing of del.CRBN and CRIMP and subsequent homodimerization of truncated Caspase 9 and induction of apoptosis.

[0186] Four constructs were designed comprising the IMiD-responsive Caspase 9 Suicide Switch and different versions of del.CRBN and CRIMP (Constructs 1-4, Table 1). The resulting four kill switch fusion polypeptide encoding sequence were inserted between the left and right homology arms targeting the GAPDH STEL site. A T2A peptide sequence linked one monomer of truncated Caspase 9 (iCasp9) and a del.CRBN variant to the GAPDH coding sequence, while a P2A peptide sequence linked the other monomer of iCasp9 and a CRIMP variant to the preceding sequences (FIG. IB; Constructs 1-4, Table 2).

[0187] Another set of constructs were designed to include a transgene sequence after a separator sequence downstream to the IMiD-responsive Caspase 9 Suicide Switch protein fusion polypeptide (FIG. 1C), wherein the transgene might be a reporter gene or a therapeutic gene. Hence two additional constructs (FIG. ID; Constructs 5-6, Table 2) were generated using the IMiD-responsive Caspase 9 Suicide Switch protein fusion polypeptides by inclusion of an optimized 2A peptide linker sequence (Opt2A) and a sequence encoding the puromycin resistance gene (Puro).

[0188] For all constructs listed on Table 2 (SEQ ID NOs:l-6), the sequences of the flanking left and right homology arms for targeting the GAPDH locus are shown below as SEQ ID NOs:30 and 31, respectively.TTGGTATCGTGGAAGGACTCATGGTATGAGAGCTGGGGAATGGGACTGAGGCTCCCACCTTTCTCATCCAAGACTGGCTCCTCCCTGCCGGGGCTGCGTGCAACCCTGGGGTTGGGGGTTCTGGGGACTGGCTTTCCCATAATTTCCTTTCAAGGTGGGGAGGGAGGTAGAGGGGTGATGTGGGGAGTACGCTGCAGGGCCTCACTCCTTTTGCAGACCACAGTCCATGCCATCACTGCCACCCAGAAGACTGTGGATGGCCCCTCCGGGAAACTGTGGCGTGATGGCCGCGGGGCTCTCCAGAACATCATCCCTGCCTCTACTGGCGCTGCCAAGGCTGTGGGCAAGGTCATCCCTGAGCTGAACGGGAAGCTCACTGGCATGGCCTTCCGTGTCCCCACTGCCAACGTGTCAGTGGTGGACCTGACCTGCCGTCTAGAAAAACCTGCCAAATATGATGACATCAAGAAGGTGGTGAAGCAGGCGTCGGAGGGCCCCCTCAAGGGCATCCTGGGCTACACTGAGCACCAGGTGGTCTCCTCTGACTTCAACAGCGACACCCACTCCTCCACCTTTGACGCTGGGGCTGGCATTGCCCTCAACGACCACTTTGTCAAGCTCATTTCCTGGTATGTGGCTGGGGCCAGAGACTGGCTCTTAAAAAGTGCAGGGTCTGGCGCCCTCTGGTGGCTGGCTCAGAAAAAGGGCCCTGACAACTCTTTACATCTTCTAGGTATGACAACGAATTTGGCTACAGCAACAGGGTGGTGGACCTCATGGCCCACATGGCCTCCAAGGAG(SEQ ID NO:30)GACCCCTGGACCACCAGCCAAAGCAAGAGCACAAGAGGAAGAGAGAGACCCTCACTGCTGGGGAGTCCCTGCCACACTCAGTCCCCCACCACACTGAATCTCCCCTCCTCACAGTTGCCATGTAGACCCCTTGAAGAGGGGAGGGGCCTAGGGAGCCGCACCTTGTCATGTACCATCAATAAAGTACCCTGTGCTCAACCAGTTACTTGTCCTGTCTTATTCTAGGGTCTGGGGCAGAGGGGAGGGAAGCTGGGCTTGTGTCAAGGTGAGACATTCTTGCTGGGGAGGGACCTGGTATGTTCTCCTCAGACTGAGGGTAGGGCCTCCAAACAGCCTTGCTTGCTTCGAGAACCATTTGCTTCCCGCTCAGACGTCTTGAGTGCTACAGGAAGCTGGCACCACTACTTCAGAGAACAAGGCCTTTTCCTCTCCTCGCTCCAGTCCTAGGCTATCTGCTGTTGGCCAAACATGGAAGAAGCTATTCTGTGGGCAGCCCCAGGGAGGCTGACAGGTGGAGGAAGTCAGGGCTCGCACTGGGCTCTGACGCTGACTGGTTAGTGGAGCTCAGCCTGGAGCTGAGCTGCAGCGGGCAATTCCAGCTTGGCCTCCGCAGCTGTGAGGTCTTGAGCACGTGCTCTATTGCTTTCTGTGCCCTCGTGTCTTATCTGAGGACATCGTGGCCAGCCCCTAAGGTCTTCAAGCAGGATTCATCTAGGTAAACCAAGTACCTAAAACCATGCCCAAGGCGGTAAGGACTATATAATGTTTAAAAATCGGTAAAAATGCCCACCTCGCATAGT(SEQ ID N0:31)

[0189] The sequences, SEQ ID Nos: 30 and 31 are positioned 3’ or 5’ respectively of GAPDH flanking / cut sites which are listed below as SEQ ID NO: 32 and SEQ ID NO: 33, respectively, and incorporate an exemplary gRNA sequence (SEQ ID NO.34) and its associated Casl2a / Cpfl PAM motif that targets the GAPDH endogenous coding sequence.TTTCATCTTCTAGGTATGACAACGATTACA (SEQ ID NO:32) TGTAATCGTTGTCATACCTAGAAGATGAAA (SEQ ID NO:33) ATCTTCTAGGTATGACAACGA - GAPDH Cpfl gRNA-1 (SEQ ID NO:34)

[0190] The GAPDH flanking or cut sites allows the nucleic acid to be cut by Casl2a / Cpfl endonuclease at the sequence flanking the left homology arm (L HA) towards its 5 ’ end and the sequence flanking the right homology arm (R HA) towards its 3 ’ end. The GAPDH Cpfl gRNA cut site is designed to be within exon 9 of the GAPDH gene, the last coding exon. Having a gRNA cut site targeting to an exon of GAPDH allows for enrichment and selection of cells with proper targeted integration, because cells with improper integration result in cell death (see WO2021226151).7.2.Example 2: Pooled Screening for Targeted Integration of IMiD-Responsive Suicide Switch Protein Constructs at the GAPDH locus

[0191] IMiD-responsive Caspase 9 Suicide Switch protein fusion polypeptide encoding constructs were delivered to human iPSCs via Nucleofection with a GAPDH targeting ribonucleoprotein (RNP) complex comprising of Cpfl endonuclease and a single guide RNA (sgRNA) targeting the coding sequence of the GAPDH gene. The sgRNA comprised a sequence 5' ATCTTCTAGGTATGACAACGA 3’ (SEQ ID NO:34). Briefly, iPSCs were cultured in Essential 8 (E8) medium and maintained at 37°C at 5% CO2 level between passages. Passages were done at 75 to 80% confluency. On the day of Nucleofection, iPSCs were thawed andimmediately Nucleofected in Lonza P3 Primary Cell Nucleofection buffer. 2.5 pg of each plasmid construct (Constructs 1-4, SEQ ID NOs:19, 21, 23, and 25) was Nucleofected into iPSCs using GAPDH Cpfl RNP with LONZA 4D Nucleofector. The Nucleofected cells were then plated at 50,000 cells / cm2per well and grown in E8 medium (plus Rock inhibitor Y-27632 from Tocris) for 72 to 96 hours. Knock-in pools were treated with 0, 0.01, 0.1, and IpM POM starting at 7d post-Nucleofection and treated for a total of 2d until 9d post-Nucleofection. Following POM treatment, each treatment condition for each construct was screened for targeted integration using ddPCR, as well as conventional PCR.

[0192] One day post-Nucleofection, significant iPSC toxicity was associated with Nucleofection of 2.5pg of Construct 1 (SEQ ID NO: 19) and Construct 3 (SEQ ID NO:23) (FIG. 2). As such, targeted integration efficiency was only determined for iPSC pools that were Nucleofected with 2.5pg of Construct 2 (SEQ ID NO:21) and Construct 4 (SEQ ID NO:25).

[0193] Junction GAPDH-T2A ddPCR analysis revealed that in the absence of adding the IMiD Suicide Switch activator, pomalidomide (POM), fusion polypeptide sequence integration was 0.6% for Construct 2 and 2.1% for Construct 4 (FIG. 3A) in pools of iPSCs transfected with either construct. The integration rate, as measured by fractional abundance of targeted cells, dropped in all instances where pomalidomide was added to the transfected iPSC pools to as low as 0.17% for Construct 2 and 0.1% for Construct 4 at IpM POM, the highest concentration tested. When quantified as a percentage of cell depletion of untreated cells, cells dropped to 28% for Construct 2-edited iPSCs and 6% for Construct 4-edited iPSCs after the addition of IpM POM (FIG. 3B). These results indicate that POM treatment reduced the amount of cells that carried integration of the IMiD kill-switch fusion sequences, Constructs 2 and 4, at the GAPDH locus.7.3.Example: 3: Screening of Clones for Targeted Integration of IMiD-Responsive Suicide Switch Protein Constructs at the GAPDH locus

[0194] Individual clones successfully transfected with Construct 4 (SEQ ID NO:25, Table 2) were screened using PCR via assessment for integration at the 5’ junction, as well as the zygosity of the integrated sequence. An agarose gel photograph (FIG. 4) displaying the results of PCR assessment showed a positive ~900 bp band after performing a 5’ Junction PCR with a forward primer of sequence 5’-TCCATGACAACTTTGGTATCG-3’ (SEQ ID NO: 35) binding outside the GAPDH left homology arm and a reverse primer of sequence 5’- TGGGCCAGGATTCTCC-3’ (SEQ ID NO: 36) binding within the T2A peptide sequence in 3 different clonally derived cell lines (c.49, c.84 and c.189). A positive control line (+) comprising a construct that contains the same GAPDH L HA sequence linked to a T2A sequence was alsopositive for the -900 bp band, while wildtype (WT) unedited cells were not. These results indicate correct integration of the IMiD-responsive suicide protein Construct 4 at the 5’ junction of integration. On the same agarose gel photograph (FIG.4H) a positive -4000 bp band was observed after performing a Zygosity PCR with a forward primer of sequence 5’- TGGACCTGACCTGCCGTCTA-3’ (SEQ ID NO: 37) binding internally within the GAPDH left homology arm and a reverse primer of sequence 5’- CCCCAGACCCTAGAATAAGACAGG-3’ (SEQ ID NO: 38) binding internally within the GAPDH right homology arm in clone 49, clone 84, and the positive control line, but was not detected in clone 189 and the wildtype unedited PSC line. These results suggest that clone 189 was incorrectly targeted as only the smaller -600 bp band representing a Wildtype Allele was detected. Clone 84 was both positive for the Targeted Allele band and the Wildtype Allele band indicating a heterozygously-edited Construct 4 clone, while Clone 49 was only positive for the Targeted Allele indicating a homozygously-edited Construct 4 clone.7.4.Example 4: Pomalidomide-induced Apoptosis in Clones Edited with IMiD- Responsive Suicide Switch Protein Constructs at the GAPDH locus

[0195] To determine whether IMiD-responsive suicide switch protein fusion polypeptides of the disclosure were able to induce apoptosis, a wildtype unedited iPSC line, an iPSC clonal cell line (c.49) comprising homozygous integration of Construct 4 (SEQ ID NO:25, Table 2), an iPSC clonal cell line (c.84) comprising heterozygous integration of Construct 4 (SEQ ID NO:25, Table 2), and an iPSC clonal cell line (c.189) that was mistargeted (see FIG. 4) were plated at a concentration of 12,000 cells per well of a 96-well plate and treated with treated with various concentrations of pomalidomide (0, 0.01, 0.1, 4, 10, lOOnM). Incucyte NucLight Rapid NIR Reagent (Cat# 4804, Sartorius) was added at a titration of 1:750, 24 hours after cells were plated. NucLight Rapid NIR Reagent is a nuclear dye which stains all cells and emits in the near infrared channel. Staining with this dye followed by image analysis using the onboard Incucyte Cell by Cell image analysis modules quantifies the percentage of live cells in each image. Cells were imaged on a Sartorius Incucyte SX5 Live-Cell Analysis Instrument, with images scanned every 3 hours for 24 hours. Puromycin, at 2pg / mL, was added as a positive control for cell death since cells are not resistant to puromycin. The experimental plate layout is depicted in FIG. 5. After plotting data from Incucyte scans (FIG. 6A-6D), a reduction in the percentage of live cells was not observed after the addition of pomalidomide (at any concentration) to the wildtype unedited iPSC line (FIG. 6A) and the c.189 mistargeted clonal iPSC line (FIG. 6D). In contrast, a reduction in the percentage of live cells comparable to the puromycin positive control was observed after the addition of pomalidomide from as low as 0.1 nM up to lOOnM for both thehomozygously-edited Construct 4 clonal iPSC line (c.49; FIG. 6B) and the heterozygously- edited Construct 4 clonal iPSC line (c.84; FIG. 6C). The addition of O.OlnM pomalidomide did not induce a reduction in cell number. Brightfield images comparing the cell lines at the lowest dose of pomalidomide tested (O.OlnM) and a higher dose of pomalidomide (4nM) (FIG. 7) highlight differences in sensitivities between cell lines, whereby no cell lines were sensitive to O.OlnM pomalidomide and only the Construct 4 correctly targeted homozygously- and heterozygously-edited cell lines were sensitive to pomalidomide at 4nM with most cell death already observed from 6 hours, and near complete death observed after 24 hours pomalidomide treatment. Overall, these data indicate that correct integration of Construct 4, either monoallelic or biallelic, allows induction of apoptosis by pomalidomide in edited iPSC clones.7.5.Example 5: Evaluation of Pomalidomide-induced Apoptotic Efficacy in PSC- Derived Myeloid Progenitor Cells Edited with IMiD-Responsive Suicide Switch Protein Constructs

[0196] To validate conservation of kill switch function after the differentiation of PSCs,PSCs engineered to incorporate the IMiD-responsive suicide switch protein fusion polypeptide Construct 4 (SEQ ID NO:25, Table 2) were differentiated to myeloid progenitor cells, according to the experimental workflow schematic depicted in FIG. 8A. A wildtype unedited iPSC line, an iPSC clonal cell line (c.49) comprising homozygous integration of Construct 4 (SEQ ID NO:25, Table 2), and an iPSC clonal cell line (c.84) comprising heterozygous integration of Construct 4 (SEQ ID NO:25, Table 2) were differentiated into myeloid progenitor cells through the addition of various growth factors and small molecules. Differentiated cells were plated at lx 105cells / cm2in an ultra-low attachment cell culture plate for further myeloid cell maturation. After 7 days in maturation medium, cells were collected and frozen down. For testing of IMiD- Responsive Suicide Switch activation, myeloid cells were plated at 1.5x 105cells / cm2in a 96- well tissue culture plate with a 1:750 dilution of ViaStain AOPI Staining Solution (Nexcelom) in X-VIVO 15 Medium with lOng / mL GM-CSF. The kill switch was induced with 0, 1, 4, 10 or lOOnM of pomalidomide over 5 days and images were collected every 8 hours on the Incucyte SX5 (Sartorius). Cells were stained with AO / PI dye, with dead cells uptaking Propidium Iodide and utilized to quantitate the percentage cell death. The experimental plate layout is depicted in FIG. 8B. After plotting data from Incucyte scans, FIG. 9 shows kill switch activity as measured by percentage cell death in myeloid progenitor cells following differentiation of unedited PSCs, and homozygously- or heterozygously-edited Construct 4 PSCs, at the indicated pomalidomide treatment conditions. Some pomalidomide-induced death was observed in edited myeloid progenitor cells from a dose of 4nM up, with the homozygously-edited clone demonstratingslightly higher sensitivity than the heterozygously-edited clone. No cell death was observed after addition of InM pomalidomide to edited myeloid progenitors. In all instances, unedited myeloid progenitors did not demonstrate pomalidomide-induced apoptosis. Taken together the results indicate some pomalidomide-induced cell death in myeloid progenitor cells engineered with IMiD-responsive suicide switch protein fusion polypeptide Construct 4.7.6.Example 6: Evaluation of Pomalidomide-induced Apoptosis in iPSC Pools Edited with IMiD-Responsive Suicide Switch Protein Constructs after Puromycin Selection

[0197] Two additional constructs were designed comprising the IMiD-responsive Caspase 9 Suicide Switch whereby the del.CRBN component was further mutated to improve switch dimerization and activation (Constructs 5 and 6, Table 1). Similar to the Constructs 1-4 previously described, the two additional kill switch fusion polypeptide encoding sequences were inserted between the left and right homology arms targeting the GAPDH STEL site. A T2A peptide sequence linked one monomer of truncated Caspase 9 (iCasp9) and a del.CRBN variant to the GAPDH coding sequence, while a P2A peptide sequence linked the other monomer of iCasp9 and a CRIMP variant to the preceding sequences. Additionally, the two constructs were generated with the inclusion of an optimized 2A peptide linker sequence (Opt2A) and a sequence encoding the puromycin resistance gene (Puro) to enable puromycin-based selection of correctly targeted constructs (FIG. ID; Constructs 5 and 6, Table 2).

[0198] IMiD-responsive Caspase 9 Suicide Switch protein fusion polypeptide encoding constructs were delivered to human iPSCs via Nucleofection with a GAPDH targeting ribonucleoprotein (RNP) complex comprising of Cpfl endonuclease and a single guide RNA (sgRNA) targeting the coding sequence of the GAPDH gene, 3.8pg of each plasmid construct (Constructs 5 and 6, SEQ ID NOs:5-6) was Nucleofected into iPSCs using GAPDH Cpfl RNP with LONZA 4D Nucleofector. After 8d in culture following Nucleofection, 0.25 pg / mL puromycin was added to iPSCs for 6d.

[0199] To determine whether IMiD-responsive suicide switches protein fusion polypeptides of the disclosure were able to induce apoptosis, a puromycin-selected targeted pool comprising Construct 5 (SEQ ID NO:27, Table 2) and a puromycin-selected targeted pool comprising Construct 6 (SEQ ID NO:29, Table 2) were compared to a wildtype unedited iPSC line, an iPSC clonal cell line (c.49) comprising homozygous integration of Construct 4 (SEQ ID NO:25, Table 2), and an iPSC clonal cell line (c.84) comprising heterozygous integration of Construct 4 (SEQ ID NO:25, Table 2). Briefly, 40,000 cells were plated for each described condition per well of a 24-well plate and treated with treated with either 0.1 nM, 1 nM, or 4nM pomalidomide. Cells were imaged on a Sartorius Incucyte SX5 Live-Cell Analysis Instrument, with imagesscanned every 2 hours for 24 hours. After plotting data from Incucyte scans (FIG. 10A-10C), a reduction in the percentage of live cells was not observed after the addition of pomalidomide (at any concentration) to the wildtype unedited iPSC line. Both pools of Construct 5 or Construct 6 edited iPSC pools demonstrated comparable pomalidomide-induced apoptosis to the homozygously-edited and heterozygously-edited Construct 4 iPSC clonal lines at the InM (FIG. 10B) and 4nM (FIG. IOC) pomalidomide conditions, but displayed increased reduction of cell number after O.lnM pomalidomide treatment (FIG. 10A). Taken together, the results indicate that IMiD-responsive suicide switch protein fusion polypeptide Construct 5 and Construct 6 display greater sensitivity than Construct 4 to pomalidomide-induced apoptosis in iPSCs.7.7.Example 7: Evaluation of Apoptotic Efficacy in PSC-Derived Myeloid Progenitor Cells Edited with IMiD-Responsive Suicide Switch Protein Constructs

[0200] To validate conservation of kill switch function after PSCs are differentiated to myeloid progenitor cells, clonal PSCs engineered to incorporate IMiD-responsive suicide switch protein fusion polypeptides Construct 5 (SEQ ID NO:27, Table 2) and Construct 6 (SEQ ID NO:29, Table 2) are differentiated to myeloid progenitor cells. Thereafter PSC-derived myeloid progenitor cells are treated with pomalidomide ranging from 0.1 to lOOnM before assessment of apoptosis. It is found that treatment of PSC-derived myeloid progenitor cells with pomalidomide results in apoptosis.7.8.Example 8: In Vivo Evaluation of Apoptotic Efficacy in PSC-Derived MyeloidProgenitor Cells Edited with an IMiD-Responsive Suicide Switch Protein Construct

[0201] A clonally derived PSC line engineered to incorporate IMiD-responsive suicide switch protein fusion polypeptide Construct 5 (SEQ ID NO:27, Table 2) was differentiated to myeloid progenitor cells and 210,000 cells / hemisphere were transplanted into the hippocampus of NSG QUAD (strain #028657) male mice. Briefly, two weeks prior to cell transplantation, mice were placed on 600mg / kg PLX3397 chow (AIN-76A with 600mg PLX3397 / kg diet) to ablate endogenous mouse microglia. On day 14 (24hrs) before surgery, animals were switched to control chow for the remainder of the experiment. On day 15 animals received bilateral cell transplantation of either WT (unedited) or IMiD suicide switch (KS MG, edited) into the hippocampus. Following cell transplantation, animals were left untouched for 4 weeks. On day 44 animals were split into vehicle or pomalidomide (POM; 40mg / kg) treatments and dosed via gavage for 3 consecutive days. Four days post the last drug treatment; animals were euthanizedvia cardiac perfusion and brain was collected for histological assessment of human cells (Ku80+) (FIG. 11).

[0202] After brains were collected, they were sectioned at 20 pm and stained for Ku80, a human cell marker. Here, we found that following 35 days in vivo, WT (unedited) cells fully engraft into the hippocampus (FIG. 12; top left quadrant). Furthermore, treatment with POM does not affect cell survival, indicating that an unedited cell line will be unresponsive to drug treatment (FIG. 12; top right quadrant). IMiD (edited) cells also fully engraft in the mouse (FIG. 12; bottom left quadrant) but when exposed to 3 days of POM (40mg / kg), a large reduction of Ku80+ human cells were observed from a representative image compared to a representative image of Vehicle-treated edited cells (FIG. 12; bottom right quadrant; 191 vs 3641 cells). Represented cell counts are further depicted in Table 3.

[0203] Taken together, these results indicate that myeloid progenitor cells engineered with IMiD-responsive suicide switch protein fusion polypeptide Construct 5 display some pomalidomide-induced cell death following in vivo transplantation. For further removal of human myeloid progenitor cells engineered with an IMiD-responsive suicide switch, a longer drug dosing regimen to ablate the cells are to be undertaken.Table 3

[0204] While the present disclosure has been particularly shown and described with reference to a preferred embodiment and various alternate embodiments, it will be understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the present disclosure and appended claims.

[0205] All references, issued patents and patent applications cited within the body of the instant specification are hereby incorporated by reference in their entirety, for all purposes.

Claims

1. WHAT IS CLAIMED IS1. A targeting construct comprising:(a) a first homology arm corresponding to a 5' target sequence comprising a first region of homology to a target genomic locus;(b) a nucleotide insert comprising a nucleotide sequence encoding a fusion polypeptide comprising:(i) a modified degron; and(ii) a caspase 9 domain or derivative or functional fragment thereof;(c) a second homology arm corresponding to a 3' target sequence comprising second region of homology to the target genomic locus, wherein the modified degron comprises an amino acid sequence corresponding to a SEQ ID NO: 1 or a variant thereof, optionally wherein the modified degron comprises a mutation selected from the group consisting of G30E and R27H, wherein the targeting construct is configured such that upon its recombination with the target genomic locus, the fusion polypeptide coding sequence is integrated into a STEL gene and becomes operably linked to the STEL gene regulatory element.

2. The targeting construct of claim 1 , wherein the targeting construct further comprises a modified cereblon (CRBN) and a second caspase 9 domain or derivative or functional fragment thereof, wherein the modified CRBN comprises an amino acid sequence corresponding to a SEQ ID NO: 4 or a variant thereof, optionally wherein the modified degron comprises a mutation selected from the group consisting of 137 IN, Q325H, T359V, Q325R, and H397F.

3. A targeting construct comprising:(a) a first homology arm corresponding to a 5' target sequence comprising a first region of homology to a target genomic locus;(b) a nucleotide insert comprising a nucleotide sequence encoding a fusion polypeptide comprising:(i) a modified cereblon (CRBN); and(ii) a caspase 9 domain or derivative or functional fragment thereof;(c) a second homology arm corresponding to a 3' target sequence comprising second region of homology to the target genomic locus,wherein the modified CRBN comprises an amino acid sequence corresponding to a SEQ ID NO: 4 or a variant thereof, optionally wherein the modified degron comprises a mutation selected from the group consisting of I371N, Q325H, T359V, Q325R, and H397F, wherein the targeting construct is configured such that upon its recombination with the target genomic locus, the fusion polypeptide coding sequence is integrated into a STEL gene and becomes operably linked to the STEL gene regulatory element.

4. The targeting construct of claim 3, wherein the nucleotide insert further comprises a modified degron and a second caspase 9 domain or derivative or functional fragment thereof.

5. A targeting construct comprising:(a) a first homology arm corresponding to a 5' target sequence comprising a first region of homology to a target genomic locus;(b) a nucleotide insert comprising a nucleotide sequence encoding a fusion polypeptide comprising:(i) a modified degron, wherein the modified degron comprises an amino acid sequence corresponding to a SEQ ID NO: 1 or a variant thereof, optionally wherein the modified degron or variant comprises a mutation selected from the group consisting of G30 or R27;(ii) a first caspase 9 domain or derivative or functional fragment thereof;(iii) a modified CRBN, wherein the modified CRBN comprises an amino acid sequence corresponding to a SEQ ID NO: 4 or a variant thereof, optionally wherein the modified degron comprises a mutation selected from the group consisting of 1371, Q325, T359, and H397;(iv) a second caspase 9 domain or derivative or functional fragment thereof;(c) a second homology arm corresponding to a 3' target sequence comprising second region of homology to the target genomic locus, wherein the targeting construct is configured such that upon its recombination with the target genomic locus, the fusion polypeptide coding sequence is integrated into a STEL gene and becomes operably linked to the STEL gene regulatory element.

6. A targeting construct comprising:(a) a first homology arm corresponding to a 5' target sequence comprising a first region of homology to a target genomic locus;(b) a nucleotide insert comprising a nucleotide sequence encoding a fusion polypeptide comprising:(i) a modified degron;(ii) a first caspase 9 domain or derivative or functional fragment thereof;(iii) a modified cereblon; and(iv) a second caspase 9 domain or derivative or functional fragment thereof;(c) a second homology arm corresponding to a 3' target sequence comprising second region of homology to the target genomic locus, wherein the modified degron comprises an amino acid sequence corresponding to a reference degron sequence (SEQ ID NO: 1) or a variant thereof, wherein the modified cereblon comprises an amino acid sequence corresponding to a reference cereblon sequence (SEQ ID NO: 4) or a variant thereof, optionally wherein the modified degron comprises at least one amino acid substitution selected from the group consisting of G30E and R27H, optionally wherein the modified cereblon comprises at least one amino acid substitution selected from the group consisting of 137 IN, Q325H, T359V, Q325R, and H397F, wherein the targeting construct is configured such that upon its recombination with the target genomic locus, the fusion polypeptide coding sequence is integrated into a STEL gene and becomes operably linked to the STEL gene regulatory element.

7. The targeting construct of any one of claims 2-6, wherein the modified CRBN comprises one or more amino acid substitutions selected from the group consisting of 137 IN, Q325H, T359V, Q325R, and H397F relative to SEQ ID NO: 4.

8. The targeting construct of any one of claims 2-7, wherein the modified CRBN comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 5-8, optionally wherein the modified CRBN comprises an 137 IN amino acid substitution relative to SEQ ID NO: 4, optionally wherein the modified CRBN comprises Q325H, I371N, and T359V amino acid substitutions relative to SEQ ID NO: 4, optionallywhere wherein the modified CRBN comprises Q325R, 137 IN, H397F, and T359V amino acid substitutions relative to SEQ ID NO: 4.

9. The targeting construct of any one of claims 1-8, wherein the modified degron comprises an amino acid substitution selected from the group consisting of G30E and R27H, relative to SEQ ID NO: 1, optionally wherein the modified degron comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3, optionally wherein the modified degron comprises a R27H amino acid substitution relative to SEQ ID NO: 1, optionally wherein the modified degron comprises a G30E amino acid substitution relative to SEQ ID NO: 1, optionally wherein the modified degron comprises a R27H amino acid substitution relative to SEQ ID NO: 1 and the modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 4, optionally wherein the modified degron comprises a G30E amino acid substitution relative to SEQ ID NO: 1 and the modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 4, optionally wherein the modified degron comprises an amino acid sequence set forth in SEQ ID NO: 1 and the modified CRBN comprises an 137 IN amino acid substitution relative to SEQ ID NO: 4, optionally wherein the modified degron comprises a G30E amino acid substitution relative to SEQ ID NO: 1 and the modified CRBN comprises an 137 IN amino acid substitution relative to SEQ ID NO: 4, optionally wherein the modified degron comprises a R27H amino acid substitution relative to SEQ ID NO: 1 and the modified CRBN comprises Q325H,137 IN, and T359V amino acid substitutions relative to SEQ ID NO: 4, optionally wherein the modified degron comprises a R27H amino acid substitution relative to SEQ ID NO: 1 and the modified CRBN comprises Q325R, 137 IN, H397F, and T359V amino acid substitutions, relative to SEQ ID NO: 4.

10. The targeting construct of any one of claims 1-9, wherein the modified degron and / or the modified CRBN is N-terminal or C-terminal to the caspase 9 domain or derivative or functional fragment thereof.

11. The targeting construct of any one of claims 1-10, wherein the fusion polypeptide comprises a linker between the modified degron and the caspase 9 domain or derivative or functional fragment thereof and / or wherein the fusion polypeptide comprises a linker between the modified CRBN and the caspase 9 domain or derivative or functional fragment thereof.

12. The targeting construct of any one of claims 1-11, which is configured such that upon its recombination with the target genomic locus, the STEL gene is modified such to incorporate the fusion polypeptide coding sequence 3' or 5’ to the STEL protein coding sequence.

13. The targeting construct of any one of claims 1-12, wherein the nucleotide insert further comprises a nucleotide sequence encoding a separator sequence, optionally wherein the targeting construct is configured such that upon recombination of the targeting construct with the target genomic locus, the separator sequence coding sequence is positioned between the coding sequence of the STEL protein and the fusion polypeptide coding sequence, optionally wherein the separator sequence is an internal ribosome entry site (“IRES”) or a self-cleaving peptide, optionally wherein the self-cleaving peptide is a 2A peptide, optionally wherein the self-cleaving peptide is T2A, P2A, E2A, F2A, PQR, Opt2A, or Opt2A_2.0.

14. The targeting construct of any one of claims 1-13, wherein the STEL gene encodes a polypeptide involved in one or more of: glycolysis, ribonucleopolypeptide complex formation, focal adhesion, cell-substrate adherens junction, cell-substrate junction, cell anchoring, extracellular exosome, extracellular vesicle, intracellular organelle, anchoring junction, RNA binding, nucleic acid binding (e.g., rRNA or mRNA binding), and polypeptide binding, optionally wherein:(a) the STEL gene encodes a ribosomal polypeptide, optionally wherein the STEL gene is RPL13A, RPLPO, RPL10, RPL13, RPSJ8, RPL3, RPLP1, RPL15, RPL41, RPL11, RPL32, RPL18 A, RPL19, RPL28, RPL29, RPL9, RPL8, RPL6, RPL18, RPL7, RPL7A, RPL21, RPL37A, RPL 12, RPL5, RPL34, RPL35A, RPL30, RPL24, RPL39, RPL37, RPL14, RPL27A, RPLP2, RPL23A, RPL26, RPL36, RPL35, RPL23, RPL4, or RPL22;(b) the STEL gene encodes a ribosomal polypeptide small subunit (RPS), optionally wherein the STEL gene is RPS2, RPS 19, RPS 14, RPS3A, RPS 12, RPS3, RPS6, RPS23, RPS27A, RPS8, RPS4X, RPS7, RPS24, RPS27, RPS15A, RPS9, RPS28, RPS13, RPSA, RPS5, RPS 16, RPS25, RPS15, RPS20, or RPSl l;(c) the STEL gene encodes a mitochondrial polypeptide, optionally wherein the STEL gene is MT-C01, MT-C02, MT-ND4, MT-ND1, or MT-ND2;(d) the STEL gene encodes an actin polypeptide, optionally wherein the STEL gene is ACTG1 or ACTB;(e) the STEL gene encodes a eukaryotic translation factor, optionally where the STEL gene is EEF1A1, EEF2, or EIF1;(f) the STEL gene encodes a histone, wherein the STEL gene is H3F3A orH3F3B;(g) the STEL gene is FTL, FTH1, TPT1, IMSB10, GAPDH, PTMA, GNB2L1, NACA, YBX1, NPM1, FAU, UBA52, HSP90AB1, MYL6, SERF2, or SRP14, optionally wherein the STEL gene is GAPDH, optionally wherein the STEL gene is RPL13A, optionally wherein the STEL gene is RPL7, optionally wherein the STEL gene is RPLPO.

15. The targeting construct of any one of claims 1-14, wherein the nucleotide insert further comprises a transgene, optionally wherein the transgene is linked to the fusion polypeptide coding sequence, optionally wherein the fusion polypeptide coding sequence and transgene are connected via a nucleotide sequence encoding a separator sequence, optionally wherein the separator sequence is an internal ribosome entry site (“IRES”), optionally wherein the separator sequence is a nucleotide sequence encoding a self-cleaving peptide (the “selfcleaving peptide coding sequence”), optionally wherein the the self-cleaving peptide is a 2A peptide, optionally wherein the self-cleaving peptide is T2A, P2A, E2A, F2A, PQR, Opt2A, or Opt2A_2.0, optionally wherein the transgene encodes a therapeutic polypeptide.

16. The targeting construct of any one of claims 1-15, wherein(a) the targeting construct comprises a nucleic acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to a nucleic acid sequence set forth in SEQ ID NO: 19, 21, 23, 25, 27, or 29, optionally wherein the targeting construct comprises a nucleic acid sequence set forth in SEQ ID NO: 19, 21, 23, 25,27, or 29; or(b) the targeting construct encodes a polypeptide comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to an amino acid sequence set forth in SEQ ID NO: 18, 20, 22, 24, 26, or28, optionally wherein the targeting construct encodes a polypeptide comprising an amino acid sequence set forth in SEQ ID NO: 18, 20, 22, 24, 26, or 28.

17. A system comprising:(a) the targeting construct of any one of claims 1-16;(b) a CRISPR-associated endonuclease (“Cas nuclease”) or a nucleic acid encoding a Cas nuclease; and(c) a guide RNA (“gRNA”) comprising a scaffold for binding the Cas polypeptide and a spacer sequence corresponding to the STEL gene, or a nucleic acid encoding the gRNA, optionally wherein the guide RNA is a single guide RNA (“sgRNA”), optionally wherein the system comprises the Cas nuclease and gRNA, optionally wherein the system is in the form of a ribonucleoprotein particle (“RNP”).

18. A method of producing a gene-edited target cell, comprising:(a) introducing the system of claim 17 into a target cell; and(b) culturing the target cell under conditions in which gene editing occurs, thereby producing gene-edited target cell.

19. The method of claim 18, wherein the target cell is(a) a stem cell, or a cell differentiated from a stem cell, optionally wherein the stem cell is a human embryonic stem cell, an induced pluripotent stem cell (“iPSC”) or a cell differentiated therefrom;(b) a regulatory T cell, a myeloid cell, a dendritic cell, a macrophage (e.g., an immunosuppressive macrophage), a myeloid progenitor cell, or a precursor or progenitor cell thereof;(c) a cell in the human nervous system, optionally selected from dopaminergic neuron, a microglial cell, an oligodendrocyte, an astrocyte, a cortical neuron, a spinal or oculomotor neuron, an enteric neuron, a Placode-derived cell, a Schwann cell, and a trigeminal or sensory neuron, or a precursor or progenitor cell thereof;(d) a cell in the human cardiovascular system, optionally selected from a cardiomyocyte, an endothelial cell, and a nodal cell, or a precursor or progenitor cell thereof;(e) a cell in the human metabolic system, optionally selected from a hepatocyte, a cholangiocyte, and a pancreatic beta cell, or a precursor or progenitor cell thereof, or(f) a cell in the human ocular system, optionally selected from a retinal pigment epithelial cell, a photoreceptor cone cell, a photoreceptor rod cell, a bipolar cell, a ganglion cell, or a precursor or progenitor cell thereof; optionally wherein the gene-edited target cell is of (a) ectoderm lineage, optionally wherein the gene-edited target cell is a neuron, or of (b) mesoderm lineage, optionally wherein the gene-edited target cell is a cardiomyocyte.

20. A gene-edited target cell obtained or obtainable by the method of any one of claims 18-19.

21. A gene-edited target cell comprising a STEL gene that comprises a nucleic acid encoding a fusion polypeptide under the transcriptional control of a STEL gene regulatory element, wherein(a) the fusion polypeptide comprises:(i) a modified CRBN, wherein the modified CRBN comprises a mutation selected from the group consisting of I371N, Q325H, T359V, Q325R, and H397F; and(ii) a caspase 9 domain or derivative or functional fragment thereof; or(b) the fusion polypeptide comprises:(i) a modified degron, wherein the modified degron comprises a mutation selected from the group consisting of G30E and R27H; and(ii) a caspase 9 domain or derivative or functional fragment thereof; or(c) the fusion polypeptide comprises:(i) a modified degron, wherein the modified CRBN comprises a mutation selected from the group consisting of I371N, Q325H, T359V, Q325R, and H397F; and wherein the modified degron comprises a mutation selected from the group consisting of G30E and R27H;(ii) a caspase 9 domain or derivative or functional fragment thereof, wherein the modified degron comprises a mutation selected from the group consisting of G30E and R27H; or(iii) a modified CRBN, wherein the modified CRBN comprises a mutation selected from the group consisting of I371N, Q325H, T359V, Q325R, and H397F;(iv) a caspase 9 domain or derivative or functional fragment thereof22. The gene-edited target cell of claim 21, wherein(a) the modified degron is as defined in any one of claims 1-16, optionally wherein the modified CRBN is as defined in any one of claims 2-16, and / or(b) the caspase 9 domain or derivative or functional fragment thereof is as defined in any one of claims 1-17; and / or(c) the fusion polypeptide is as defined in any one of claims 1-17; and / or(d) the STEL gene is configured as defined in any one of claims 1-17; optionally wherein a transgene is in the STEL gene and / or(e) the transgene is as defined in any one of claims 15-16, and / or(f) the target cell is as defined in any one of claims 20-21.

23. A pharmaceutical composition comprising the gene-edited target cell of claim 22 and a pharmaceutically acceptable carrier.

24. A method of treating a patient in need thereof, comprising administering to the patient the gene-edited target cell of claim 22 or the pharmaceutical composition of claim 23, optionally wherein the method further comprises controlling the gene-edited target cell population in the patient by:(a) monitoring, optionally, the gene-edited target cell population in the patient; and / or(b) administering an inducer of the modified degron and / or modified cereblon if the patient experiences adverse events related to the gene-edited target cell population.

25. A method of mitigating adverse events or a safety risk associated with cell therapy in the form of the gene-edited target cells of claim 22 or the pharmaceutical composition of claim 23, the method comprising administering to a patient who received the gene-edited target cells or pharmaceutical composition an inducer of a degron or modified cereblon if the patient experiences adverse events or a safety risk related to the gene-edited target cells or pharmaceutical composition.

26. The method of claim 24 or 25, wherein the inducer of the modified degron or modified cereblon is thalidomide, iberdomide, lenalidomide, or pomalidomide, optionally wherein the inducer of the modified degron or modified cereblon is pomalidomide, optionally wherein a combination of two or more inducers of the modified degron or modified cereblon is administered, optionally wherein the combination has a synergistic effect and / or utilizes reduced dosing (e.g., reduced dosing amount and / or frequency) than would be required a single inducer of the modified degron or modified cereblon.

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