Degradation determinant fusion proteins, methods of production and uses thereof

Abstract

The present disclosure relates, in part, to degradation determinant-based methods for controlling gene-edited therapeutic cells administered to a patient, as well as targeting constructs encoding degradation determinants that are suitable for generating gene-edited target cells that can be eliminated after administration to a patient.

Description

[0001] 1. CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 443,614, filed on February 6, 2023, and U.S. Provisional Application No. 63 / 503,640, filed on May 22, 2023, the contents of which are incorporated herein by reference in their entirety.

[0003] 2. Sequence Listing

[0004] This application contains a sequence listing that has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML sequence listing, created on January 22, 2024, is named BRT-003WO_SL.xml and is 120,402 bytes in size. Background Art

[0005] In some cases, cell therapy offers tremendous promise for the treatment of a variety of diseases and conditions by replacing or repairing defective or damaged cells or tissues with autologous or allogeneic cells. Pluripotent stem cells, including induced pluripotent stem cells (iPSCs), which can be differentiated into desired cell types and engineered to recombinantly express therapeutic polypeptides with desired therapeutic properties, are particularly useful for cell therapy.

[0006] However, due to the nature and complexity of these "living drugs", it would be advantageous to have the ability to remove the cell products after they are introduced into the patient in the event of an adverse event, or as a safety measure after the cell products have performed their function. Adverse events induced by therapeutic cells require rapid and near complete elimination of the therapeutic cells. For example, excessive on-target effects, such as by immunomodulatory cells, may lead to cytokine storms associated with tumor lysis syndrome (TLS), cytokine release syndrome (CRS), or macrophage activation syndrome (MAS). In addition, certain off-target effects may also occur, such as cells of the cell therapy becoming cancerous. Therefore, when the number of genetically engineered cells needs to be reduced or they need to be eliminated from the patient, their controlled destruction is required.

[0007] Therefore, there is great interest in developing methods that can eliminate therapeutic cells if they cause serious adverse events (SAEs) or become obsolete after treatment. Summary of the Invention

[0008] The present disclosure provides stable, reliable "suicide genes" that can be used to eliminate therapeutic cells in situations where they trigger or are likely to trigger a severe adverse event (SAE) or become obsolete after treatment.

[0009] In particular, the present disclosure provides compositions and methods for removing part or all of a transplanted cell therapy by engineering an inducible apoptosis mechanism into cells for therapy, wherein the inducible apoptosis mechanism is in the form of a fusion protein comprising an essential protein and a drug-inducible degron. The destruction of this fusion protein is activated by a drug (e.g., a clinically approved small molecule drug, such as an immunomodulatory imide drug (IMiD)).

[0010] Fusion proteins (sometimes referred to as "kill switches") are typically expressed from endogenous essential gene loci, thereby overcoming the shortcomings of other kill switches, such as transcriptional silencing or mutation of the transgene expressing the kill switch during cell differentiation (e.g., for the preparation of cell therapy products). Because the kill switches of the present disclosure comprise essential proteins, cell survival depends on the presence of the kill switch (and the lack of induction of degradation determinants). When cells expressing the fusion protein are exposed to appropriate conditions (e.g., drugs), degradation of the fusion protein can induce apoptosis and provide better control of the induced apoptosis because: 1) the essential gene cannot be transcriptionally silenced without cell death, and 2) the kill switch itself (i.e., in an induced state) cannot mutate without killing the cell.

[0011] In general, the fusion proteins of the present disclosure comprise an essential polypeptide and one or more degrons, optionally linked via one or more peptide linkers.Fusion proteins are further described in Section 6.2 and numbered Examples 1 to 56.

[0012] The present disclosure provides fusion proteins, which generally comprise an essential polypeptide or a fragment or derivative thereof. An essential polypeptide is a polypeptide encoded by an essential gene, and its null mutation is detrimental to the survival of the affected cell. Therefore, degradation of the essential polypeptide via an inducible degron can be used to regulate the survival state of the target cell. Exemplary essential polypeptides are described in Section 6.2.1 and numbered Examples 40 to 56.

[0013] The fusion proteins of the present disclosure further comprise one or more degrons. In a broad sense, a degron is a peptide sequence or protein element, such as a structural motif, a short amino acid sequence, etc., which regulates the degradation rate of a protein, for example, by targeting the protein for polyubiquitination and subsequent degradation via the proteasome. In some embodiments, the fusion protein comprises a drug-inducible degron, wherein the stability of the degron is controlled by the presence or absence of a small molecule that binds to the degron. Further details about degrons and exemplary degrons are described in Section 6.2.2 and numbered Examples 2 to 27 and 35 to 39.

[0014] The fusion proteins of the present disclosure may further comprise an optional linker sequence located between the degron sequence and the essential polypeptide sequence. In the case of a fusion protein having multiple degrons, the individual degrons may be linked to each other via an optional linker. Section 6.2.3 and numbered Examples 28 to 34 describe suitable optional linkers.

[0015] The disclosure provides targeting constructs, which are designed to produce in target cells the genomic sequence encoding the fusion protein under the control of expression control elements. Targeting constructs typically include homology arms to guide the integration of constructs into the expected genomic locus in the target cell genome, such as essential gene locus, wherein the sequence flanks encoding a degron and an optional joint have two homology arms targeting the essential gene locus so that the essential gene is modified to express a fusion protein comprising the essential polypeptide and the degron optionally separated via a joint. Targeting constructs are further described in chapters and sections 6.3 and subsection 6.3.1 and numbered embodiments 57 to 157. Chapters and sections 6.3.2 and 6.3.3 describe integration sites and homology arms of targeting constructs of the present disclosure, respectively.

[0016] The constructs and methods disclosed herein can also be used to generate target cells to express both (a) a fusion protein comprising an essential polypeptide and a degron and (b) a recombinant polypeptide. The recombinant polypeptide can be expressed by a transgene that can be introduced into the target cell via the same targeting construct or expression vector as the one comprising the degron encoding sequence. Section 6.4 and numbered Examples 235 to 258 further describe and provide exemplary transgenes.

[0017] The present disclosure further provides targeting constructs and recombinant target cell genomes that can comprise a separator sequence between the degron encoding sequence and the transgene to allow separate expression of polypeptides encoded by a single expression cassette. Exemplary separator sequences are described in Section 6.5.

[0018] The present disclosure also provides an expression vector encoding the fusion protein of the present disclosure, which generally comprises an expression cassette comprising a fusion polypeptide operably linked to a regulatory element such as a promoter and optionally a self-replication element. Further information and examples of expression vectors are described in Section 6.6 and numbered Examples 314 to 318.

[0019] The present disclosure further provides methods and systems for generating gene-edited target cells comprising a nucleotide sequence encoding a fusion protein of the present disclosure. Further information and examples of suitable methods and systems are described in Sections 6.8 and 6.9 and numbered Examples 158 to 177.

[0020] Examples of recombinant and gene-edited target cells (e.g., comprising a nucleotide sequence encoding a fusion protein of the disclosure) are disclosed, for example, in Section 6.7 and numbered Examples 178 to 325.

[0021] The present disclosure further provides a method for treating a patient with a cell therapy, the method comprising administering cells engineered to express a fusion protein comprising an essential protein and an inducible degradation determinant. If the subject experiences the side effects of cell therapy or is considered to be at risk of the side effects of cell therapy, the cells can be eliminated in whole or in part by the induction of the degradation determinant, for example, by administering an inducer of the degradation determinant to the subject. In certain embodiments, the degradation determinant is a drug-inducible (e.g., IMiD inducible) degradation determinant and the cells are eliminated by administering a drug (e.g., IMiD). Further information and examples about the method of the present disclosure are described in sections 6.11 and numbered embodiments 327 to 366. For cell therapy, cells can be formulated as pharmaceutical compositions, for example, as described in sections 6.10 and numbered embodiment 326.

[0022] Additional features, advantages, and applications of the fusion proteins, nucleic acids (targeting constructs, expression vectors), cells, and methods of the present disclosure are described in more detail below. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figures 1A to 1D is a cartoon representation of a degron-essential polypeptide fusion protein and its coding sequence. Figure 1A represents a fusion protein comprising a degron polypeptide (D) linked to the N-terminus of an essential polypeptide, optionally via a linker (shown as a line connecting the degron and essential polypeptide). Figure 1B represents a fusion protein comprising a degron polypeptide (D) linked to the C-terminus of an essential polypeptide, optionally via a linker (shown as a line connecting the degron and essential polypeptide). Figure 1C is a diagram of a nucleic acid comprising, from 5' to 3', the endogenous promoter of an essential gene, the transcription start site (indicated by an arrow), and the coding sequence of a degron, an optional linker, and an essential polypeptide, which, when expressed, produces Figure 1A The fusion proteins shown. Figure 1D is a diagram of a nucleic acid comprising, from 5' to 3', an endogenous promoter, a transcription start site (indicated by an arrow), and coding sequences for the necessary polypeptides, optional linkers, and degrons, which, when expressed, produces Figure 1B The fusion proteins shown.

[0024] Figures 2A to 2D is a schematic diagram of exemplary targeting constructs and vectors that can be used to produce or introduce nucleic acids encoding the disclosed fusion proteins in target cells. Figure 2ARepresents a targeting construct having a degron encoding sequence flanked by a first homology arm and a second homology arm, wherein the degron encoding sequence is linked on its 5' end to the first homology arm via an optional linker encoding sequence, wherein the homology arms are configured such that integration of the targeting construct into an essential gene via recombination of the homology arms with the target genome results in the production of a modified essential gene encoding an essential polypeptide fused at its C-terminus to the degron via an optional linker. Figure 2B Represents a targeting construct having a degron encoding sequence flanked by a first homology arm and a second homology arm, wherein the degron encoding sequence is linked on its 3' end to the second homology arm via an optional linker encoding sequence, wherein the homology arms are configured such that integration of the targeting construct into an essential gene via recombination of the homology arms with the target genome results in the production of a modified essential gene encoding an essential polypeptide fused at its N-terminus to the degron via an optional linker. Figure 2C Indicates something similar to Figure 2A The targeting construct is similar to the targeting construct shown in , but has two degron encoding sequences connected to each other by a linker instead of a single degron encoding sequence. Figure 2D Indicates something similar to Figure 2B , but with two degron encoding sequences linked to each other by a linker instead of a single degron encoding sequence. Figure 2C and 2D The targeting construct in has two degron encoding sequences, but the targeting construct of the present disclosure can include more than two degron encoding sequences. In some embodiments, the degron encoding sequences are connected to each other via a linker sequence.

[0025] Figures 3A to 3F is a diagram depicting the incorporation of a targeting construct at an essential gene locus. Figure 3A yes Figure 2A Schematic diagram of monoallelic incorporation of a targeting construct at an essential gene locus depicted in . Figure 3B Yes Figure 2A Schematic diagram of biallelic incorporation of targeting constructs at essential gene loci depicted in . Figure 3C is described as integrated into its genome Figure 2A Cartoon illustration of the mechanism of inducible degron-mediated degradation of essential polypeptides and apoptosis in cells harboring a targeting construct. Similar effects can be achieved by introducing extrachromosomal vectors and knocking out essential genes, for example, at one or both alleles. Figure 3D yes Figure 2B Schematic diagram of monoallelic incorporation of a targeting construct at an essential gene locus depicted in . Figure 3E Yes Figure 2BSchematic diagram of biallelic incorporation of targeting constructs at essential gene loci depicted in . Figure 3F is described as integrated into its genome Figure 2B Cartoon illustration of the mechanism of inducible degron-mediated degradation of essential polypeptides and apoptosis in cells harboring a targeting construct. Similar effects can be achieved by introducing extrachromosomal vectors and knocking out essential genes, for example, at one or both alleles.

[0026] Figures 4A to 4D is a schematic diagram of an exemplary targeting construct comprising a transgene in addition to a degron. Figure 4A A construct targeting the 3' end of an essential gene coding sequence is shown, comprising, from 5' to 3', a first homology arm to the essential gene, an optional linker coding sequence, a degron coding sequence, an IRES coding sequence, a transgene, and a second homology arm to the essential gene. The homology arms are configured such that integration of the targeting construct into the essential gene via recombination of the homology arms with the essential gene results in the production of a modified essential gene encoding an essential polypeptide fused at its C-terminus to the degron via an optional linker, followed by an IRES and a transgene. Figure 4B A construct targeting the 5' end of an essential gene coding sequence is shown, comprising, from 5' to 3', a first homology arm of the essential gene - a transgene - an IRES coding sequence - a degron coding sequence - an optional linker coding sequence - and a second homology arm of the essential gene. The homology arms are configured such that integration of the targeting construct into the essential gene via recombination of the homology arms with the essential gene results in the production of a modified essential gene encoding an essential polypeptide fused at its N-terminus to the degron via an optional linker, preceded by an IRES and a transgene. Figure 4C and 4D Shows similar Figure 4A and 4B , but with two sets of degron sequences. Figures 4A to 4D The degron coding sequence and the transgene are depicted separated by an IRES coding sequence, but they can also be separated by a sequence encoding a self-cleaving peptide (such as a 2A peptide) in frame with the degron and transgene sequences. Figure 4C and 4D The targeting construct in has two degron encoding sequences, but the targeting construct of the present disclosure can include more than two degron encoding sequences. In some embodiments, the degron encoding sequences are connected to each other via a linker sequence.

[0027] Figures 5A to 5D is a diagram depicting exemplary effects of incorporation of a targeting construct at a target genomic locus and incorporation of a targeting construct at an essential gene. Figure 5A yes Figure 4A Schematic diagram of monoallelic incorporation of a targeting construct at an essential gene locus depicted in . Figure 5B yes Figure 4A Schematic diagram of biallelic incorporation of targeting constructs at essential gene loci depicted in . Figure 5C yes Figure 4B Schematic diagram of monoallelic incorporation of a targeting construct at an essential gene locus depicted in . Figure 5D yes Figure 4B Schematic diagram of biallelic incorporation of targeting constructs at essential gene loci depicted in .

[0028] Figures 6A to 6E is a diagram depicting the incorporation of different targeting constructs at the first and second allele loci of an essential gene. Figure 6A is a schematic diagram of the genomic integration of a first targeting construct comprising an IRES and coding sequence for a transgene designed to be incorporated immediately 5' of the endogenous stop codon of the first allele of an essential gene, and a second targeting construct, e.g. Figure 2A As shown, a linker and coding sequence for a degron are included, which is designed to be incorporated immediately 5' of the endogenous stop codon of the second allele of the essential gene. Figure 6B is a schematic diagram of the genomic integration of a first targeting construct comprising the coding sequence for a transgene and an IRES designed to be incorporated immediately 3' of the ATG start codon of the second allele of an essential gene and a second targeting construct, e.g. Figure 2A As shown, a linker and coding sequence for a degron are included, which is designed to be incorporated immediately 5' of the endogenous stop codon of the second allele of the essential gene. Figure 6C is a schematic diagram of the genomic integration of a first targeting construct comprising an IRES and coding sequence for a transgene designed to be incorporated immediately 5' of the endogenous stop codon of the first allele of an essential gene, and a second targeting construct, e.g. Figure 2B As shown, it comprises an optional linker and the coding sequence of a degron designed for incorporation immediately 3' of the ATG start codon of the second allele of the essential gene. Figure 6Dis a schematic diagram of the genomic integration of a first targeting construct comprising a linker and coding sequences for a degron designed to be incorporated immediately 3' of the ATG start codon of the first allele of an essential gene; a second targeting construct comprising an IRES and coding sequences for a transgene designed to be incorporated immediately 5' of the endogenous stop codon of the first allele of an essential gene; and a third construct comprising a linker and coding sequences for a degron designed to be incorporated immediately 3' of the ATG start codon of the second allele of an essential gene. Figure 6E is a schematic diagram of the genomic integration of a first targeting construct comprising coding sequences for a transgene and an IRES designed to be incorporated immediately 3' of the ATG start codon of the second allele of an essential gene; a second targeting construct comprising coding sequences for a linker and a degron designed to be incorporated immediately 5' of the endogenous stop codon of the second allele of an essential gene; and a third construct comprising coding sequences for a linker and a degron designed to be incorporated immediately 3' of the ATG start codon of the second allele of an essential gene.

[0029] Figures 7A to 7L is a schematic diagram of an exemplary targeting construct configuration, wherein the essential gene is GAPDH ( Figures 7A to 7J ) or RPL13A( Figures 7K to 7L ). Thus, in each construct, the nucleic acid insert was flanked by GAPDH or RPL13A homology arms. Figure 7A The construct in (e.g., a construct comprising the nucleotide sequence of SEQ ID NO: 1) has a nucleic acid insert flanked by left and right C-terminal GAPDH homology arms, which is designed to integrate the nucleic acid insert at the 3' end of the GAPDH locus, wherein the nucleic acid insert has a linker, i.e., linker 1 (GGS), and a degron (SEQ ID NO: 3) in the N-terminal to C-terminal direction. Figure 7B The construct in has a nucleic acid insert flanked by left and right C-terminal GAPDH homology arms, wherein the nucleic acid insert has Linker 2 (SEQ ID NO: 23) and Degron (SEQ ID NO: 3) in the N-terminal to C-terminal direction. Figure 7C The construct in has a nucleic acid insert flanked by left and right C-terminal GAPDH homology arms, wherein the nucleic acid insert has linker 3 (SEQ ID NO: 103) and a degron (SEQ ID NO: 3) in the N-terminal to C-terminal direction. Figure 7DThe construct in (e.g., a construct comprising the nucleotide sequence of SEQ ID NO: 2) has a nucleic acid insert flanked by left and right C-terminal GAPDH homology arms, wherein the nucleic acid insert has linker 4 (SEQ ID NO: 15) and a super degron (SEQ ID NO: 4) in the N-terminal to C-terminal direction. Figure 7E The construct in (e.g., a construct comprising the nucleotide sequence of SEQ ID NO: 29) has a nucleic acid insert flanked by left and right N-terminal GAPDH homology arms, which is designed to integrate the nucleic acid insert at the 5' end of the GAPDH locus, wherein the nucleic acid insert has a degron (SEQ ID NO: 3) and a linker, i.e., linker 1 (GGS), in the N-terminal to C-terminal direction. Figure 7F The construct in (e.g., a construct comprising the nucleotide sequence of SEQ ID NO: 30) has a nucleic acid insert flanked by left and right N-terminal GAPDH homology arms, wherein the nucleic acid insert has a super degron (SEQ ID NO: 4) and a linker, i.e., Linker 4 (SEQ ID NO: 15), in the N-terminal to C-terminal direction. Figure 7G The construct in has a nucleic acid insert flanked by left and right C-terminal GAPDH homology arms, wherein the nucleic acid insert has linker 1 (GGS), a degron (SEQ ID NO: 3), an IRES, and GFP in the N-terminal to C-terminal direction. Figure 7H The construct in has a nucleic acid insert flanked by left and right C-terminal GAPDH homology arms, wherein the nucleic acid insert has Linker 2 (SEQ ID NO: 23), Degron (SEQ ID NO: 3), IRES, and GFP in the N-terminal to C-terminal direction. Figure 7I The construct in has a nucleic acid insert flanked by left and right C-terminal GAPDH homology arms, wherein the nucleic acid insert has Linker 3 (SEQ ID NO: 103), Degron (SEQ ID NO: 3), IRES, and GFP in the N-terminal to C-terminal direction. Figure 7J The construct in has a nucleic acid insert flanked by left and right C-terminal GAPDH homology arms, wherein the nucleic acid insert has Linker 4 (SEQ ID NO: 15), Super Degron (SEQ ID NO: 4), IRES, and GFP in the N-terminal to C-terminal direction. Figure 7K The construct in (e.g., a construct comprising the nucleotide sequence of SEQ ID NO: 14) has a nucleic acid insert flanked by left and right C-terminal RPL13A homology arms, which is designed to integrate the nucleic acid insert at the 3' end of the RPL13A locus, wherein the nucleic acid insert has a linker, i.e., linker 1 (GGS), and a degron (SEQ ID NO: 3) in the N-terminal to C-terminal direction. Figure 7LThe construct in (e.g., a construct comprising the nucleotide sequence of SEQ ID NO: 17) has a nucleic acid insert flanked by left and right C-terminal RPL13A homology arms, which is designed to integrate the nucleic acid insert at the 3' end of the RPL13A locus, wherein the nucleic acid insert has a linker, i.e., linker 4 (SEQ ID NO: 15), and a super degron (SEQ ID NO: 4) in the N-terminal to C-terminal direction.

[0030] Figures 8A to 8B Shown are the effects of 3 μM pomalidomide (POM) treatment on the survival of cells edited with a targeting construct comprising a GFP tag and a fusion protein in which a degron is linked to an essential gene. Figure 8A Shows the use of Figure 7G Fractions of naive iPSCs (GFP+ data points within the box) and unedited cells (data points outside the box) gene-edited with targeting constructs containing GFP and a degron are shown. Figure 8B Shown are the fractions of gene-edited and non-edited iPSCs after six days of POM treatment.

[0031] Figures 9A to 9C Shown are the effects of 1 μM POM treatment on the survival of gene-edited iPSCs over time. Figure 9A It is a graph showing the change in the number of viable cells / well at different time points, expressed as a percentage of the cells / well at the beginning (% of T0). Figure 9B Representative images of wells containing gene-edited iPSCs after 92 hours of POM treatment. Figure 9C Representative images of untreated control wells containing gene-edited iPSCs monitored for 92 hours.

[0032] Figures 10A to 10B Shown is the effect of linker length on the survival of iPSC pools gene-edited with targeting constructs in which a degron or superdegron is linked to the essential gene GAPDH following treatment with 3 μM POM. Figure 10A is a graph showing the percentage of GFP-positive cells quantified using flow cytometry. Figure 10B is a graph showing the percentage of cells normalized to untreated conditions quantified using amplicon sequencing.

[0033] Figures 11A to 11D We demonstrate activation of targeting constructs in homozygous gene-edited iPSCs, in which a degron or superdegron is linked to GAPDH. Figure 11A is a graph showing qPCR results assessing GAPDH expression after 3 μM POM treatment for different durations. Figure 11BWestern blot assessment of GAPDH protein levels in cells gene-edited with a GAPDH-linked degron. Lane 1: Molecular weight marker; Lane 2: Untreated and untransfected parental cells; Lane 3: Untransfected parental cells treated with 3 μM POM for 24 hours; Lane 4: Untreated clone A cells transfected with degron 1; Lane 5: Clone A cells treated with 3 μM POM for 24 hours; Lane 6: Untreated clone B cells transfected with degron 1; Lane 7: Clone B cells treated with 3 μM POM for 24 hours; Lane 8: Untreated clone C cells transfected with degron 1; Lane 9: Clone C cells treated with 3 μM POM for 24 hours. Figure 11C Western blot assessment of GAPDH protein levels in cells gene-edited with a superdegradant linked to GAPDH. Lane 1: molecular weight marker; Lane 2: untreated clone A cells transfected with the superdegradant; Lane 3: clone A cells treated with 3 μM POM for 24 hours. Figure 11D is a graph showing the growth kinetics of different iPSC lines gene-edited with targeting constructs containing a degron or super degron linked to GAPDH.

[0034] Figures 12A to 12C Shown is the effect of POM concentration on the survival of iPSCs gene-edited with targeting constructs containing a degron or super degron linked to GAPDH. Figure 12A Shown are representative images of unedited and gene-edited iPSCs after 5 days of 0.5 μM POM treatment. Figure 12B is a graph showing the difference in cell confluence over time, expressed as the percentage of cells / well at the start of gene editing of cells containing a targeting construct comprising a degron linked to GAPDH (% of T0), after treatment with various concentrations of POM ranging from 0.03125 to 10 μM. Figure 12C is a graph showing the difference in cell confluence expressed as the percentage of cells / well at the start (% of T0) of cells gene-edited with a targeting construct comprising a superdegrader linked to GAPDH after treatment with different concentrations of POM ranging from 0.03125 to 10 μM.

[0035] Figure 13 is a cartoon illustrating an assay that can be used to assess targeting construct activity in dopaminergic (DA) neurons differentiated from gene-edited iPSCs. See Kriks et al., 2011, Nature 480(7378):547-551 and U.S. Patent No. 10,711,243, which are incorporated herein by reference in their entirety.

[0036] Figure 14 Shown are representative images of parental DA neurons and two lines of gene-edited DA neurons after 5 days of 0.13 μM POM treatment.

[0037] Figures 15A to 15D Graph showing the effect of POM concentration on cell survival of DA neurons. Figure 15A is a graph showing the percentage of cell death in unedited parental DA neurons after 5-day treatment with different POM concentrations. Figure 15B is a graph showing the percentage of cell death in DA neurons derived from iPSCs gene-edited with the targeting construct clone B after treatment with POM concentrations ranging from 0.13 μM to 1 μM for 5 days. Figure 15C is a graph showing the percentage of cell death in DA neurons derived from iPSCs gene-edited with the targeting construct Clone C after treatment with POM concentrations ranging from 0.13 μM to 1 μM for 5 days. Figure 15D is a graph showing the percentage of cell death in DA neurons derived from iPSCs gene-edited with the targeting construct clone B after treatment with POM concentrations ranging from 10 nM to 100 nM for 5 days.

[0038] Figures 16A to 16C Shown are the effects of an extended range of POM concentrations (0.01 μM to 100 μM) on the survival of DA neurons differentiated from unedited or gene-edited iPSCs. Figure 16A is a graph showing the percentage of cell death in DA neurons differentiated from unedited parental iPSCs after treatment with different POM concentrations for 6 days. Figure 16B is a graph showing the percentage of cell death in DA neurons derived from iPSCs gene-edited with a targeting construct comprising a linker three amino acids in length (3-aa linker) and a degron (clone A) after treatment with POM concentrations ranging from 0.01 μM to 100 μM for 6 days. Figure 16C is a graph showing the same treatment in another gene-edited iPSC clone (clone C) containing the same targeting construct comprising a 3 aa linker and a degron.

[0039] Figure 17 Is a cartoon illustrating an assay that can be used to assess targeting construct activity in myeloid progenitor (MP) cells differentiated from gene-edited iPSCs. See Douvaras et al., Jun 6, 2017;8(6):1516-1524 and PCT Publication Nos. WO 2023 / 150089 A1 and WO 2017 / 152081 A1, which are incorporated herein by reference in their entirety.

[0040] Figures 18A to 18DShown are the effects of 1 μM POM on cell survival of myeloid progenitor (MP) cells differentiated from unedited or gene-edited iPSCs with targeting constructs containing a 3-aa linker and a degron. Figure 18A is a graph showing the percentage of cell death of MP cells differentiated from unedited parental iPSCs after treatment with or without POM. Figures 18B to 18D Figure 2 shows the changes in the expression of 1 μM POM for 108 h or without treatment in three different iPSC clones: Clone A ( Figure 18B ), Clone B( Figure 18C ) or clone C( Figure 18D ) of MP cells that were gene-edited with a targeting construct containing a 3aa linker and a degron. 6. Specific Implementation Methods

[0041] 6.1. Definitions

[0042] Unless otherwise defined herein, the scientific and technical terms used in conjunction with the present disclosure should have the meanings 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 the methods and materials described herein can also be used in the practice or testing of the present disclosure. In the event of a conflict, the present specification (including definitions) shall prevail. In general, the nomenclature and techniques used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, organic synthetic chemistry, medical 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 carried out according to the manufacturer's instructions, as commonly accomplished in the art or as described herein. In addition, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular. Throughout this specification and examples, the words "have" and "comprise" or variations such as "has," "having," "comprises," or "comprising" will be understood to imply the inclusion of the stated integer or group of integers, but not to exclude 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.

[0043] Cell therapy: As used herein, the term "cell therapy" refers to a therapy in which cellular material is administered to a patient. The cellular material can be whole, living cells. For example, T cells capable of fighting cancer cells via cell-mediated immunity can be injected during immunotherapy. Cell therapy is also known as cellular therapy or cytotherapy.

[0044] Coding sequence: As used herein, the term "coding sequence" refers to a nucleic acid (RNA or DNA molecule) comprising a nucleotide sequence that encodes a protein or portion thereof (e.g., an essential polypeptide, linker, or degron component of a fusion protein of the present disclosure). The coding sequence can be codon-optimized for expression in a cell of interest.

[0045] Complement: As used herein, the term "complement" or "complementary" means that a nucleic acid can form Watson-Crick (e.g., AT / U and CG) or Hoogsteen base pairing between nucleotides or nucleotide analogs in the nucleic acid molecule. "Complementarity" refers to a property shared between two nucleic acid sequences such that when they are aligned antiparallel to each other, the nucleotide bases at every position will be complementary.

[0046] Degron: The term "degron" refers to a peptide sequence, protein element, or portion of a protein that is involved in regulating the degradation rate of a protein. Degrons can include short amino acid sequences, structural motifs, and exposed amino acids (e.g., lysine or arginine). The stability of a fusion protein comprising an essential polypeptide and a degron sequence is controlled, at least in part, by the degron sequence. In some embodiments, suitable degrons are constitutive, such that the degron exerts its effect on protein stability independent of external factors (e.g., the degron is not drug-inducible, temperature-inducible, etc.), while in other embodiments, the degron is inducible (e.g., the degron can be turned on or off by drugs, light, or temperature changes, etc.). In some embodiments, the degron provides an essential polypeptide, such as an essential polypeptide (e.g., GAPDH), fused thereto with controlled stability. A fusion protein comprising a degron and an essential polypeptide can be maintained in an "on" (or stable) condition until the cells expressing the fusion protein are eliminated, at which point the degron is induced and the fusion protein becomes unstable and ultimately degrades, leading to cell death. In some embodiments, the degron is drug-inducible, for example by an IMiD.

[0047] Electroporation: The term "electroporation" refers to the use of transmembrane electric field pulses to induce microscopic pores in biological membranes. These pores, often called "electropores," allow the transfer of macromolecules, ions, and water from one side of the membrane to the other. Electroporation is often used to introduce drugs, DNA, or other molecules into cells. Electroporation is the basis of nucleofection, which combines the principles of electrophoresis with cell-type-specific reagents to transfer macromolecules directly into the nucleus of target cells.

[0048] Endonuclease: As used herein, the term "endonuclease" refers to an enzyme that cuts phosphodiester bonds within a nucleic acid chain. Nucleic acids can be double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), RNA, double-stranded hybrids of DNA and RNA, and synthetic DNA (e.g., containing bases other than A, C, G, and T). Endonucleases can cut nucleic acids symmetrically, leaving "flat" ends, or at positions that are not directly opposite, creating overhangs, which can be referred to as "sticky ends." Sometimes, for convenience, the term endonuclease is simply referred to as "nuclease."

[0049] Essential genes: The term "essential genes" refers to genes that are essential for the survival of a cell or organism. Invalid mutations in essential genes are harmful to the survival of affected cells. Some essential genes are cell type or lineage specific, such as tumor specific or neuron specific. Such lineage-specific essential genes are essential for the survival of that cell type or lineage, but not for the survival of other cell types or the entire organism in some cases (see, for example, Zhang et al., 2021, Translational Psychiatry. 11 (317)). Essential genes can also be STEL genes.

[0050] Essential protein, essential polypeptide: The terms "essential protein" and "essential polypeptide" are used interchangeably herein to refer to a polypeptide encoded by an essential 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.

[0051] Gene-edited target cells: As used herein, the term "gene-edited target cells" refers to cells engineered to express the fusion protein of the present disclosure (comprising essential polypeptide sequences and degradation determinants) via the introduction of a targeting construct of the present invention, or its progeny and descendants. Typically, the nucleotide sequence flanked by the homology arms of the targeting construct is integrated into the genome of the cell. The target cells of gene editing do not need to have the same cell type as the cells into which the targeting construct was initially introduced. For example, a targeting construct can be introduced into stem cells, such as iPSC or hESC, thereby integrating the nucleotide sequence flanked by the homology arms of the targeting construct into the genome of the stem cell. The stem cells can then be differentiated to produce differentiated cell types, such as any cell type disclosed in Section 6.7.1. Both stem cells and differentiated cells are referred to herein as "gene-edited target cells". In addition to encoding the fusion protein of the present invention, the target cells of gene editing can also include a transgene, for example, as described in Section 6.4. In some embodiments, the fusion protein coding sequence and the transgene are inserted into the same essential gene. In certain embodiments, the fusion protein coding sequence and transgenic are both located in the same allele of the essential gene (whether heterozygous or homozygous). In other embodiments, the fusion protein coding sequence and transgenic are located in different alleles of the essential gene. In other embodiments, the fusion protein coding sequence and transgenic are located in different loci. Alternatively, one or both of the fusion protein coding sequence and transgenic are expressed from an extrachromosomal expression vector, such as in cells where the corresponding essential gene is knocked out at one or two alleles. In some aspects, the target cell of gene editing has a single copy of the fusion protein coding sequence in an allele of the corresponding essential gene.

[0052] Guide RNA or gRNA: As used herein, the term "guide RNA" refers to a ribonucleic acid having a DNA targeting sequence (also referred to as a "spacer" or "DNA targeting segment") and a protein binding sequence (also referred to as a "protein binding segment"). The DNA targeting sequence has sufficient complementarity to the target DNA (e.g., genomic DNA) sequence to hybridize with the target DNA sequence and guide sequence-specific binding of the nucleic acid-targeting complex to the target DNA sequence. The DNA targeting sequence typically includes a "protospacer-like" sequence as described herein. The protein binding sequence interacts with a site-specific modifying enzyme (e.g., an endonuclease as described in Section 6.9.2). Site-specific cleavage of the target DNA occurs at a location determined by: (i) base pairing complementarity between the guide RNA and the target DNA; and (ii) a short motif in the target DNA (called a protospacer adjacent motif (PAM)). The protein binding segment of the guide RNA includes, in part, two complementary nucleotide stretches that hybridize to each other to form a double-stranded RNA duplex (dsRNA duplex). In some embodiments, the guide RNA is a single-stranded guide RNA (sgRNA).

[0053] IMiD: The term "IMiD" refers to immunomodulatory imide drugs and includes thalidomide and structural analogs of thalidomide that can act as immunomodulators. Examples of IMiDs include pomalidomide, thalidomide, lenalidomide, iberdomide, and avadomide.

[0054] iPSC: The term "induced pluripotent stem cell" or "iPSC" refers to a pluripotent stem cell artificially prepared from non-pluripotent cells (such as adult somatic cells, partially differentiated cells or terminally differentiated cells, such as fibroblasts, cells of the hematopoietic lineage, muscle cells, neurons, epidermal cells, etc.) by introducing or contacting one or more reprogramming factors into the cells. iPSC can be derived from a variety of different cell types, including terminally differentiated cells. iPSC has an embryonic stem (ES) cell-like morphology, grows as flat colonies, has a large nuclear-cytoplasmic ratio, clear boundaries and prominent nuclei. In addition, iPSC expresses one or more key pluripotency markers known to those 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.

[0055] The example of the method for generating and characterizing iPSC can be found in, for example, U.S. Patent Publication Nos. US20090047263, US20090068742, US20090191159, US20090227032, US20090246875 and US20090304646 and PCT Patent Publication Nos. WO2013177133 and WO2022204567, the disclosures of which are incorporated herein by reference. Typically, in order to generate iPSC, somatic cells are provided with reprogramming factors known in the art (e.g., Oct4, SOX2, KLF4, MYC, Nanog, Lin28, etc.) to reprogram somatic cells into pluripotent stem cells.

[0056] Knockout: The term "knockout" and related terms (e.g., "knockout (knocking out)") refer to the reduction / partial elimination of gene expression of one or more genes or the complete elimination of gene expression of one or more genes, whether from one or two alleles. In some embodiments, the term knockout refers to the partial elimination of gene expression at one or two alleles (e.g., a modification that results in at least 50%, at least 60%, or at least 70% reduction in gene expression in the absence of modification). In other embodiments, the term knockout refers to the complete elimination of gene expression at one or two alleles.

[0057] Linker or linker sequence: The term "linker" or "linker sequence" as used with respect to a fusion protein refers to a moiety that connects two or more domains, parts, or entities. In some embodiments, a linker may comprise an amino acid or a peptide. Generally, a linker has no specific biological activity other than connecting or maintaining some minimum distance or other spatial relationship between the parts.

[0058] Nucleic acid: As used herein, the terms "nucleic acid" or "oligonucleotide" or "polynucleotide" mean at least two nucleotides covalently linked together. The description of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also includes the complementary strand of the described single strand. Many variants of a nucleic acid can be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also includes substantially identical nucleic acids and their complements. A single strand provides a probe that can hybridize to a target sequence under stringent hybridization conditions. Thus, a nucleic acid also includes a probe that hybridizes under stringent hybridization conditions.

[0059] Nucleic acids can be single-stranded or double-stranded, or can contain portions of both double-stranded and single-stranded sequences. Nucleic acids can be DNA (both genomic and cDNA), RNA, or hybrids, wherein nucleic acids can contain a combination of deoxyribonucleotides and ribonucleotides, and a combination of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. Nucleic acids can be obtained by chemical synthesis or recombinant methods.

[0060] Nuclease: The terms "nuclease" and "endonuclease" are used interchangeably herein to refer to an enzyme having endonuclease catalytic activity for nucleic acid cleavage, as well as nuclease-inactive variants thereof.

[0061] Nucleofection: The term "nucleofection" refers to an electroporation-based transfection method that uses a combination of electrical parameters and cell type-specific reagents to transfer nucleic acids, proteins, or ribonucleoprotein complexes directly into the nucleus of target cells.

[0062] 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 transcription of the coding sequence in an appropriate host cell or other expression system.

[0063] 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, may include modified amino acids, and may be interrupted by non-amino acids.

[0064] Pluripotent: As used herein, the term "pluripotent" or "pluripotency" refers to the ability of a cell to self-renew and 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 by somatic cell nuclear transfer, as well as iPSCs derived from non-pluripotent cells.

[0065] Recombinant target cell: As used herein, the term "recombinant target cell" refers to a cell engineered to express a fusion protein of the present invention (comprising an essential polypeptide sequence and a degradation determinant), and includes the progeny and descendants of a target cell into which a targeting construct or expression vector of the present invention was initially introduced. Recombinant target cells do not need to be of the same cell type as the cell into which the targeting construct or expression vector was initially introduced. For example, the cell initially engineered to express the fusion protein of the present invention may be a stem cell, such as an iPSC or hESC. The stem cell may then be differentiated to produce a differentiated cell type, such as any cell type disclosed in Section 6.7.1. Both stem cells and differentiated cells are referred to herein as "recombinant target cells." In addition to encoding the fusion protein of the present invention, the recombinant target cell may also include a transgene, for example, as described in Section 6.4. In some embodiments, the fusion protein coding sequence and the transgene are located in the same gene, such as an essential gene encoding the essential polypeptide portion of the fusion polypeptide. In some embodiments, both the fusion protein coding sequence and the transgene are located in the same allele of the essential gene (whether heterozygous or homozygous). In other embodiments, the fusion protein coding sequence and the transgene are located in different alleles of the essential gene. Alternatively, one or both of the fusion protein coding sequence and the transgene are expressed from an extrachromosomal expression vector, such as in a target cell in which an essential gene is knocked out at one or two loci. In some aspects, the recombinant target cell has a single copy of the fusion protein coding sequence in one allele of the corresponding essential gene. In other aspects, the recombinant target cell has two or more copies of the fusion protein coding sequence, such as one or more copies at each allele of the corresponding essential gene.

[0066] Corresponding to: The term "corresponding to" as used herein with respect to the fusion proteins and essential genes of the present disclosure means that the fusion protein comprises the amino acid sequence of the essential protein encoded by the essential gene (or a fragment or variant of the essential protein).

[0067] Reprogramming factor, reprogramming protein: As used herein, the term "reprogramming factor" or "reprogramming protein" refers to a protein, peptide, functional fragment of a protein or peptide, or a small molecule that, when overexpressed or otherwise introduced into a cell alone or in combination with other proteins, peptides, functional fragments, or proteins or peptides or other small molecules, induces the cell to transition from one differentiated state to another differentiated state. In some embodiments, the reprogramming factor induces somatic cells to transition from a differentiated state to a pluripotent state. The reprogramming factors used herein may be human proteins or modified forms thereof that retain the desired biological effect.

[0068] Ribonucleoprotein (RNP) complex: "ribonucleoprotein complex" or "ribonucleoprotein particle" as provided herein refers to a complex or particle comprising a nucleoprotein and ribonucleic acid. "Nucleoprotein" as provided herein refers to a protein capable of binding to nucleic acids (e.g., RNA, DNA). When a nucleoprotein is bound to ribonucleic acid, it is referred to as a "ribonucleoprotein". The interaction between a ribonucleoprotein and ribonucleic acid can be direct, such as through a covalent bond, or indirect, such as through a non-covalent bond (e.g., electrostatic interaction (e.g., ionic bond, hydrogen bond, halogen bond), van der Waals interaction (e.g., dipole-dipole, dipole-induced dipole, London dispersion), ring stacking (π effect), hydrophobic interaction, etc.). In an embodiment, a ribonucleoprotein includes an RNA binding motif that is non-covalently bound to the ribonucleic acid. For example, a positively charged aromatic amino acid residue (e.g., lysine residue) in the RNA binding motif can form an electrostatic interaction with the negative nucleic acid phosphate backbone of the RNA, thereby forming a ribonucleoprotein complex. In some embodiments, any of the nucleases disclosed herein are in an RNP with a guide RNA.

[0069] STEL:Term " sustained transgenic expression locus " or " STEL " refers to the locus in the cell genome that can make transgene persistent and stably expressed in the cell.STEL of the present disclosure includes but is not limited to the locus of the endogenous gene of robust expression, for example, certain housekeeping genes with activity in a variety of cell types, such as those involved in gene expression (for example, transcription factors and histones), cell metabolism (for example, GAPDH) or cell structure (for example, actin), or encoding ribosomal proteins (for example, large or small ribosomal subunits, such as RPL13A, RPLP0 and RPL7). Other examples of STEL include those forming ribonucleoprotein complexes, focal adhesions, cell-matrix adhesion junctions, cell-matrix connections, cell anchoring, extracellular exosomes, extracellular vesicles, intracellular organelles or anchoring connections. Some proteins participate in RNA binding, nucleic acid binding (for example, rRNA or mRNA binding) or protein binding.STEL gene can also be an essential gene (sometimes referred to as "essential STEL gene" in this article).

[0070] 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.

[0071] Subject: The term "subject" or "patient" refers to an organism that undergoes the procedures and / or treatments of the present disclosure. Subjects can include humans and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, and reptiles. In preferred embodiments, the subject is a human.

[0072] Target cell: The term "target cell" refers to a host cell into which (i) an expression vector or (ii) a targeting construct is introduced, which, upon integration into the host cell genome, results in the production of a recombinant nucleic acid encoding a fusion protein comprising an essential polypeptide, a degron, and an optional linker. It should be understood that such terms refer not only to a specific subject cell, but also 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 cells carrying the expression cassette or having the targeting construct integrated therein, as well as cells differentiated therefrom. Such counterparts and progeny are still included within the scope of the term "target cell" as used herein.

[0073] Targeting construct: The term "targeting construct" refers to a recombinant nucleic acid molecule capable of specifically interacting with an essential gene locus. Recombination of the targeting construct and the target genomic locus results in modification of the essential gene, such as modifying the essential polypeptide to include a degron coding sequence and / or introducing a transgene into the essential gene locus. Typically, the targeting construct comprises homology arms that allow integration of the targeting construct into a specific genetic locus (e.g., an essential gene).

[0074] Transfection: The term "transfection" refers to the introduction of a nucleic acid molecule, such as a DNA or RNA (e.g., mRNA) molecule, into a cell, for example, into a eukaryotic cell. In the context of the present invention, the term "transfection" encompasses any method known to the skilled artisan for introducing a nucleic acid molecule into a cell, for example, into a eukaryotic cell, such as a mammalian cell. Such methods include, 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 a cationic polymer (such as DEAE-dextran or polyethyleneimine).

[0075] Vector: The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid connected thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop to which additional DNA segments can be connected. Another type of vector is a viral vector, in which additional DNA segments can be connected to the viral genome. Certain vectors can replicate autonomously in the host cell into which they are introduced (for example, bacterial vectors with bacterial replication origins and additional mammalian vectors). Other vectors (for example, non-additional mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, thereby replicating together with the host genome. In certain embodiments, the vector is a viral vector, such as a lentiviral vector, an adenoviral vector or an adeno-associated virus (AAV) vector. In addition, certain vectors can instruct the expression of the nucleotide sequence operably connected thereto. Such vectors are referred to as "expression vectors" herein.

[0076] Fusion Proteins

[0077] The present disclosure relates to fusion proteins comprising (i) an essential polypeptide and (ii) a degron, optionally connected via a peptide linker. In some embodiments, the degron is located at the N-terminus of the essential polypeptide. In other embodiments, the degron is located at the C-terminus of the essential polypeptide.

[0078] Nucleic acids encoding the disclosed fusion proteins can be introduced into target cells, for example, in the form of vectors or targeting constructs described in Section 6.6, which results in integration of the exogenous nucleotide sequence encoding the fusion protein into an essential gene in the genome of the target cell.

[0079] In some embodiments, nucleic acids encoding fusion proteins of the present disclosure can be produced in situ in a target cell by introducing a targeting construct, e.g., as described in Section 6.3, which recombines with an essential gene in the genome of the target cell to produce a modified essential gene encoding a fusion protein of the present disclosure comprising:

[0080] (i) essential polypeptides, as described in Section 6.2.1;

[0081] (ii) Degron, as described in Section 6.2.2;

[0082] (iii) and optionally a linker, as described in Section 6.2.3, which connects the essential polypeptide and the degron.

[0083] Thus, when a fusion protein is expressed due to integration of a targeting construct encoding a degron into an essential gene encoding an essential polypeptide, the essential gene is the essential gene for the targeting construct. When a fusion protein is expressed due to integration of a targeting construct encoding an essential polypeptide and a degron into a different locus than the essential gene encoding the essential polypeptide, the other locus is the essential gene for the targeting construct.

[0084] In some embodiments, the fusion protein is configured such that the degron is located at the N-terminus of the essential polypeptide, e.g., Figure 1A In other embodiments, the fusion protein is configured such that the degron is located at the C-terminus of the essential polypeptide, e.g., Figure 1B shown.

[0085] In some embodiments, the fusion protein comprises only one degron. In other embodiments, the fusion protein comprises two or more degrons. In some embodiments, the degrons are connected in series and separated by a linker. Figure 2A and Figure 2B After integration of the targeting construct shown in , a fusion protein containing only one degron is generated. Figure 2C and Figure 2D Following the targeting construct shown in , a fusion protein containing two degrons was generated.

[0086] In some embodiments, the fusion protein further comprises a self-cleaving peptide sequence (e.g., as described in Section 0) and a polypeptide encoded by a transgene (e.g., as described in Section 0). Such fusion proteins can be produced, for example, by incomplete processing of the self-cleaving peptide. Appropriate processing of a fusion protein having a self-cleaving peptide sequence at its N-terminus or C-terminus will produce a fusion protein comprising some amino acid residues of the self-cleaving peptide. The term "fusion protein of the present disclosure" encompasses fusion proteins comprising a suitably processed or incompletely processed self-cleaving peptide sequence (and, in some embodiments, a polypeptide sequence encoded by a transgene).

[0087] Therefore, in some embodiments, the fusion protein of the present disclosure lacks a self-cleaving peptide sequence. In other embodiments, the fusion protein of the present disclosure comprises a suitably processed self-cleaving peptide sequence (which can be a single amino acid residue). In further embodiments, the fusion protein of the present disclosure comprises an incompletely processed self-cleaving peptide sequence and optionally a transgenic encoded polypeptide sequence.

[0088] In some embodiments, the degron is an inducible degron. The fusion of an essential polypeptide with a degron allows for the control of its stability by induction of the degron. In the absence of an inducer, the degron is inactive and the essential polypeptide is stable. In the presence of an inducer, the degron is active, while the essential polypeptide is unstable, leading to its destruction. The destruction of the essential polypeptide is detrimental to cell survival. Therefore, the fusion proteins of the present disclosure can be used to control cell survival.

[0089] 6.2.1. Essential polypeptides

[0090] The fusion proteins disclosed herein generally comprise an essential polypeptide or a fragment or derivative thereof (for convenience, collectively referred to herein as an "essential protein" or "essential polypeptide"). In some embodiments, the essential polypeptide is a STEL polypeptide. In other embodiments, the essential polypeptide is a non-STEL polypeptide.

[0091] In certain embodiments, the essential polypeptide (whether STEL or non-STEL polypeptide) belongs to the category of essential polypeptide identified below.In other embodiments, the essential polypeptide (whether STEL or non-STEL polypeptide) does not come from the category of essential polypeptide identified below (e.g., the category is a conditional subject).

[0092] In addition, in some embodiments, the essential polypeptide (whether STEL or non-STEL polypeptide) is one of the essential polypeptides identified below. In other embodiments, the essential polypeptide (whether STEL or non-STEL polypeptide) is not one of the essential polypeptides identified below (e.g., specific essential polypeptides are the subject of a proviso).

[0093] In some embodiments, the essential polypeptide is encoded by a gene associated with cellular metabolism, such as GAPDH. In various embodiments, the essential polypeptide has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the GAPDH polypeptide of SEQ ID NO: 100.

[0094] In some embodiments, the essential polypeptide is a ribosomal polypeptide (RPL), such as a polypeptide encoded by an RPL gene. 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. In various embodiments, the essential polypeptide has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the RPL13A or RPLP0 polypeptide of SEQ ID NO: 101 or SEQ ID NO: 102, respectively.

[0095] In some embodiments, the essential polypeptide is a ribosomal polypeptide small subunit (RPS), such as a polypeptide encoded by an RPS gene. 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 RPS11.

[0096] In some embodiments, the essential polypeptide is a cytoskeletal protein, such as actin. Examples of actin encoding genes are ACTG1 and ACTB.

[0097] In some embodiments, the essential polypeptide is a eukaryotic translation elongation factor, such as EEF1A1 and EEF2, or a eukaryotic translation initiation factor, such as EIF1.

[0098] In some embodiments, the essential polypeptide is a histone, such as the histones encoded by genes H3F3A and H3F3B.

[0099] In other embodiments, the essential polypeptide is a STEL polypeptide selected from FTH1, TPT1, PTMA, GNB2L1, NACA, YBX1, NPM1, FAU, UBA52, HSP90AB1, MYL6, SERF2, and SRP14.

[0100] In other embodiments, the essential polypeptide is a non-STEL polypeptide, such as HDAC3, DNMT1, NADH dehydrogenase, and PGK1.

[0101] The fusion proteins of the present disclosure typically comprise a naturally occurring essential polypeptide sequence, such as when the fusion protein encoding sequence is constituted upon integration of the targeting construct into the locus of an essential gene.

[0102] Alternatively, the essential polypeptide sequence in the fusion protein can be a variant of the wild-type essential polypeptide sequence with substitutions, additions and deletions, for example, when expressed via an expression vector or from a target cell genome modified by a targeting construct encoding the complete fusion protein. Without being bound by theory, it is believed that recombinant expression of a fusion protein comprising an essential polypeptide and a degradation determinant can "poison" the natural cell essential polypeptide and cause it to be unstable when the degradation determinant is activated, leading to cell death even when the essential gene is intact. In various embodiments, the essential polypeptide sequence in the fusion protein has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% sequence identity to the wild-type amino acid sequence of the essential polypeptide.

[0103] 6.2.2. Degron

[0104] The fusion proteins described herein comprise one or more peptide sequences that can act as a "cut-off switch" or "kill switch" for eliminating target cells that have been engineered to express the fusion proteins of the present disclosure. Such "kill switch" peptide sequences are referred to herein as degrons.

[0105] In general, a degron is a peptide sequence or protein element that regulates the degradation rate of a protein, for example by targeting the protein for polyubiquitination and subsequent degradation via the proteasome. A degron can include short amino acid sequences, structural motifs, and exposed amino acids (e.g., lysine or arginine).

[0106] The stability of the degron is at least partially controlled by the degron sequence. In some embodiments, suitable degrons are constitutive, such that the degron exerts its effect on protein stability independent of experimental control (e.g., the degron is not drug-inducible, temperature-inducible, etc.). In some embodiments, the degron provides an essential polypeptide (e.g., GAPDH) fused thereto with controllable stability, such that the fusion protein can be "turned on" (e.g., stable) or "turned off" (e.g., unstable, degraded) depending on the desired conditions.

[0107] In some embodiments, the degron is a drug-inducible degron, wherein the presence or absence of a drug can switch the protein from an "off" (e.g., unstable) state to an "on" (e.g., stable) state, or vice versa. In some embodiments, the stability of the degron is controlled by the presence or absence of a small molecule that binds to the degron.

[0108] Examples of suitable degrons controlled by the presence or absence of small molecules include, but are not limited to, degrons controlled by immunomodulatory imide drugs (IMiDs, such as pomalidomide, thalidomide, lenalidomide, ibedomide, avalidomide, etc.), Shield-1, DHFR, and / or growth hormones. Other inducible degrons are temperature-sensitive degrons, light-inducible degrons, and degrons activated by expression of another protein (e.g., TEV protease). Non-limiting examples of suitable degrons are known in the art (e.g., Dohmen et al., Science, 1994. 263(5151):1273-1276; Schoeber et al., 2009, Am J Physiol Renal Physiol. 296(1):F204-11; Chu et al., 2008, Bioorg Med Chem Lett. 18(22):5941-4; Kanemaki, 2012, Pflugers Arch. Dec 28; Yang et al., 2012, Mol Cell. 48(4):487-8; Barbour et al., 2013, Biosci Rep. 33(1); and Greussing et al., 2012, J Vis Exp. (69), the contents of which are herein incorporated by reference in their entireties).

[0109] In some embodiments, the fusion protein comprises an inducible degron sequence fused to an essential polypeptide. In some embodiments, the degron is a zinc finger degron controllable by an IMiD (e.g., thalidomide, lenalidomide, pomalidomide, and / or its analogs). In some embodiments, the IMiD-sensitive degron is an engineered degron, such as a super degron, which has increased sensitivity to IMiDs and is capable of degrading the essential polypeptide more effectively than degradation achieved with a non-engineered degron.

[0110] Fusion of a degron sequence to a polypeptide sequence can be used to generate a polypeptide with an off switch. For example, fusion of an IMiD-sensitive degron to an essential polypeptide (e.g., GAPDH) generates a GAPDH-degron fusion protein, the expression of which can be turned off via targeted degradation of the GAPDH-degron fusion protein in the presence of an IMiD (such as pomalidomide). Degradation of a fusion protein comprising an essential polypeptide and a degron can lead to apoptosis in cells engineered to express the fusion protein.

[0111] In certain embodiments, the fusion protein of the present disclosure acts as a kill switch to eliminate cells engineered to express the fusion protein. In certain embodiments, the fusion protein of the present disclosure allows for the elimination of cells in vitro, for example, in the context of functional screening as disclosed in Natsume and Kanemaki, 2017, Annu Rev Genet.51:83-102, wherein it is possible to quickly control the expression of the protein of interest fused to the degradation determinant to determine whether the protein of interest is essential for cell viability. In other embodiments, the fusion protein of the present disclosure allows for the elimination of cells in vivo, for example, after gene therapy as disclosed in sections 6.11.

[0112] In some embodiments, the degron is an inducible degron.

[0113] In some embodiments, degrons are inducible by small molecules such as drugs.

[0114] In some embodiments, the degron can be induced by IMiD. Examples of IMiD-inducible degron sequences are disclosed in Koduri et al., 2019, Proc. Nat'l Acad. Sci. USA 116(7):2539–2544, WO 2021 / 188286A2, and WO 2019 / 089592 A1, the contents of each of which are incorporated herein in their entirety.

[0115] In some embodiments, the degron comprises or consists of the amino acid sequence: RPFQCNQCGASFTQKGNLLRHIKLH (SEQ ID NO: 3, from Koduri et al.), FNVLMVHKRSHTGERPLQCEICGFTCRQKGNLLRHIKLHTGEKPFKCHLCNYACQRRDAL (SEQ ID NO: 4, corresponding to SEQ ID NO: 42 of WO 2021 / 188286 A2), FNVLMVHKRSHTGERP (SEQ ID NO: 5, corresponding to SEQ ID NO: 97 of WO 2019 / 089592 A1), FNVLMVHRRSHTGERP (SEQ ID NO: 6, corresponding to SEQ ID NO: 100 of WO 2019 / 089592 A1), TGEKPFKCHLCNYACQRRDAL (SEQ ID NO: 7, corresponding to SEQ ID NO: 101 of WO 2019 / 089592 A1), NO:102), TGERPFRCHLCNYACQRRDAL (SEQ ID NO:8, corresponding to SEQ ID NO:103 of WO2019 / 089592A1), FQCNQCGASFT (SEQ ID NO:9, corresponding to SEQ ID NO:528 of WO2021 / 188286A2), FQCPICGLVIK (SEQ ID NO:10, corresponding to SEQ ID NO:529 of WO2021 / 188286A2), LQCEICGFTCR (SEQ ID NO:11, corresponding to SEQ ID NO:530 of WO2021 / 188286A2), LQCEICGYQCR (SEQ ID NO:12, corresponding to SEQ ID NO:531 of WO2021 / 188286A2) or LQCEVCGFQCR (SEQ ID NO:13, corresponding to SEQ ID NO:532 of WO2021 / 188286A2).

[0116] In some embodiments, the degron is a super degron. SEQ ID NO: 4 is an example of a super degron sequence.

[0117] In some embodiments, the degron is a SMASh (small molecule assisted shutdown) tag degron, which is a self-cleaving degron that can be stabilized after treatment with a small molecule (e.g., a drug such as asunaprevir). In the absence of a protease inhibitor, the SMASh tag will self-cleave, and the protein will be expressed at relatively normal levels. However, in the presence of a drug, the SMASh tag degron remains fused to the protein, inducing rapid degradation of all newly synthesized fusion proteins.

[0118] 6.2.3. Connectors

[0119] The fusion proteins of the present disclosure may comprise an optional linker sequence positioned between the essential polypeptide sequence and the degron sequence.

[0120] Suitable linkers for use in the methods of the present disclosure are well known to those skilled in the art and include peptide linkers. In particular embodiments, linkers are used to separate the essential polypeptide and the degron by a distance sufficient to ensure that the essential polypeptide retains its desired functional properties. In some embodiments, the peptide linker sequence adopts a flexible extended conformation and does not exhibit a tendency to form an ordered secondary structure.

[0121] Typical amino acids in flexible peptide linkers include Gly, Asn, and Ser. Thus, in certain embodiments, the linker includes a combination of one or more of Gly, Asn, and Ser amino acids. Other near-neutral amino acids, such as Thr and Ala, may also be used in the linker sequence. Exemplary linkers are disclosed in Maratea et al., 1985, Gene 40:39-46; Murphy et al., 1986, Proc. Nat'l. Acad. Sci. USA 83:8258-62; U.S. Patent No. 4,935,233; and U.S. Patent No. 4,751,180, the contents of which are incorporated herein in their entirety.

[0122] The peptide linker can be a repetition of an amino acid sequence or one or more amino acid sequences. In some embodiments, the sequence can be repeated 2 times. In some embodiments, the sequence can be repeated 3 times. In some embodiments, the sequence can be repeated 4 times. In some embodiments, the sequence can be repeated 5 times or more.

[0123] 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 a range of lengths defined by any pair of the foregoing 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, etc.).

[0124] In some embodiments, the linker is a "short" linker of up to 15 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length, or a range of lengths bounded by any pair of the foregoing values ​​(e.g., between 1 and 3 amino acids, between 1 and 12 amino acids in length, between 2 and 12 amino acids in length, between 1 and 10 amino acids in length, etc.).

[0125] Non-limiting examples of linker sequences are listed in Table 1 below.

[0126]

[0127] 6.3. Targeting Constructs

[0128] 6.3.1. Targeting constructs for fusion protein expression

[0129] The present disclosure provides a targeting construct designed to produce a genomic sequence in a target cell under the control of an expression control element, the genomic sequence encoding a fusion protein as described herein, e.g., an essential polypeptide, a degron, and optionally a linker sequence as described in Section 6.2.1. The targeting construct typically includes homology arms to direct the integration of the construct into the desired genomic locus in the genome of the target cell.

[0130] The targeting constructs of the present disclosure can include the entire coding sequence of the fusion protein for integration of the complete fusion protein coding sequence into a genomic locus in the genome of the target cell. Depending on the site of integration, the targeting construct can further include expression regulatory sequences, such as promoter sequences, or utilize expression regulatory sequences within the genome of the target cell at the intended integration site.

[0131] In certain embodiments, the targeting construct does not include the entire coding sequence of the fusion protein, but only includes the degron coding sequence and optional linker sequence except the homology arms. The homology arms may or may not include the essential polypeptide coding sequence, because the coding sequence of the entire fusion protein (continuous or with intron sequence) is formed when the targeting construct is integrated into the target cell genome. The homology arms can be designed to recombinant with essential genes and / or flanking sequences.

[0132] In some embodiments, the targeting constructs of the present disclosure comprise:

[0133] (i) a first homology arm as described in Section 6.3.3 corresponding to a region comprising an essential gene as described in Section 6.3.2

[0134] or a 5' target sequence in the first region homologous to its flanking sequence;

[0135] (ii) a nucleotide sequence encoding a degron (e.g., an inducible degron, such as a drug-inducible degron) as described in Section 6.2.2 ("degron encoding sequence"), and optionally a nucleotide sequence encoding a 5' or 3' linker to the degron encoding sequence;

[0136] (iii) a second homology arm as described in Section 6.3.3, which corresponds to a 3' target sequence comprising a second region homologous to the essential gene or its flanking sequence as described in Section 6.3.2;

[0137] Wherein the targeting construct is configured such that upon its recombination with the target genomic locus, the essential gene is modified to encode a fusion protein as described in Section 6.2, comprising the essential polypeptide, the degron, and optionally a linker.

[0138] In some aspects, the targeting construct further comprises a transgene as described in Section 6.4, e.g., located between the degron sequence and the second homology arm. In certain other aspects, the targeting construct of the present disclosure further comprises a spacer sequence as described in Section 6.5, wherein the fusion protein coding sequence and the transgene are linked via a spacer sequence (e.g., a nucleotide sequence encoding an internal ribosome entry site (IRES) or a self-cleaving peptide).

[0139] Exemplary configurations of targeting constructs lacking a transgene are presented in Figures 1 and 3.

[0140] An exemplary configuration of a targeting construct comprising a transgene is depicted in FIG4 , and an exemplary configuration of integration of a targeting construct into an essential gene locus is depicted in FIG4 . Figures 5A to 5D In. Figures 5A to 5DAs shown, the targeting construct can be integrated 5' to the essential protein coding sequence or 3' to the essential protein coding sequence, and cells heterozygous or homozygous for the essential gene modification can be selected.

[0141] As an alternative (or in addition) to incorporating a transgene into a targeting construct comprising a degron coding sequence, a first targeting construct comprising a degron coding sequence can be introduced into cells together with a second targeting construct lacking the degron but comprising the transgene. The targeting constructs can both target the same gene, and heterozygous cells can then be selected, e.g. Figure 6A 6F, or they can target different loci so that transgenic and the fusion protein comprising essential polypeptide and degron are expressed from different genes. In certain embodiments, the first targeting construct and the second targeting construct are both introduced into the STEL locus, as described in PCT application WO 2021 / 072329 A1. In certain embodiments, the first targeting construct and the second targeting construct are introduced into identical STEL locus, such as GAPDH locus. In other embodiments, the first targeting construct and the second targeting construct are introduced into different STEL loci, such as GAPDH locus and another STEL locus. In certain embodiments, the first targeting construct (that is, the targeting construct comprising the degron coding sequence) is introduced into the GAPDH locus, and the second targeting construct (that is, the targeting construct comprising transgenic) is introduced into different STEL loci. In other embodiments, the second targeting construct (that is, the targeting construct comprising transgenic) is introduced into the GAPDH locus, and the first targeting construct (that is, the targeting construct comprising the degron coding sequence) is introduced into different STEL loci. In yet other embodiments, a first targeting construct (i.e., a targeting construct comprising a degron encoding sequence) is introduced into an essential gene other than the STEL gene, and a second targeting construct (i.e., a targeting construct comprising a transgene) is introduced into the STEL locus.

[0142] The nucleic acid sequences of exemplary constructs are listed in Table 2.

[0143]

[0144]

[0145]

[0146]

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158] Methods for introducing targeting constructs into target cells are described in Sections 6.8 and 6.9.

[0159] In some embodiments, the target cell is modified to contain only one copy of a nucleotide sequence encoding a fusion protein comprising an essential polypeptide and a degron. For example, when integrating a targeting construct into the host cell genome, engineered cells are selected that contain an insertion of the targeting construct at only one allele of the essential gene. In some embodiments, a second allele is engineered to incorporate a transgene, for example, by introducing a second targeting construct comprising the transgene and a targeting construct comprising a degron coding sequence.

[0160] In other embodiments, the target cell is modified to include two copies of a nucleotide sequence encoding a fusion protein comprising an essential polypeptide and a degron. For example, when integrating the targeting construct into the host cell genome, engineered cells are selected that contain insertions of the targeting construct at both alleles of the essential gene.

[0161] Integration Sites

[0162] Targeting constructs intended to integrate into the genome of a target cell typically comprise heterologous sequences not present in the genome of the target cell, such as degron coding sequences or transgenes.

[0163] In certain embodiments, the degron sequence is introduced into the essential gene so that the essential gene is modified to express a fusion protein comprising the essential polypeptide and the degron optionally separated via a joint. It will be readily understood by those skilled in the art that the term "essential gene" is not limited to the STEL gene. However, due to the robust expression from the STEL gene locus, in certain embodiments, particularly when used for transgenic colocalization into the genome of the host cell, the essential gene introducing the degron also is a STEL gene.

[0164] In certain embodiments, the degron sequence is introduced into the essential gene (no matter it is STEL or non-STEL gene) of the functional category of the essential gene identified below. In other embodiments, the degron sequence is introduced into the essential gene (no matter it is STEL or non-STEL gene) of the classification (e.g., the classification is a conditional theme) that is not from the essential gene identified below.

[0165] In some embodiments, a degron sequence is introduced into an individual essential gene (whether STEL or non-STEL gene) selected from the individual essential genes identified below. In other embodiments, a degron sequence is introduced into an essential gene (whether STEL or non-STEL gene) that is not one of the essential genes identified below (e.g., specific essential genes are the subject of a proviso).

[0166] In some embodiments, the essential gene is the GAPDH gene.

[0167] In the case where the essential gene is the STEL gene, a transgene can also be introduced into the STEL locus so that the transgene can be expressed from a constitutively expressed locus. In some embodiments, a transgene is introduced into the GAPDH gene.

[0168] The degron encoding sequence and the transgene can be introduced into the same STEL gene, either via a single targeting construct, as shown in Figures 4 and 5, or via different targeting constructs, as shown in Figure 6. Target cells can be selected that are homozygous or heterozygous for both the degron encoding sequence and the transgene.

[0169] Targeting constructs generally include one or more regions homologous to the DNA region in or near (e.g., flanking or adjacent to) the target sequence. These homology regions are referred to herein as "homology arms." For ease of reference, homology arms are referred to herein as the first and second (i.e., 5' and 3', upstream and downstream, or left and right) homology arms. The term relates to the relative position of homology arms and nucleic acid inserts within the targeting construct. The first homology arm and the second homology arm correspond to the regions within the target genome locus, and are referred to herein as "the first homologous region" and "the second homologous region."

[0170] In some embodiments, the targeting construct comprises homology arms for targeted integration of a heterologous sequence into an essential gene locus, wherein integration of the heterologous sequence introduces a degron coding sequence in frame with the essential polypeptide coding sequence, directly linked, or linked via a linker sequence. Thus, after integration, the essential gene locus is engineered to express the fusion polypeptide of the present disclosure.

[0171] In some embodiments, essential genes are active in multiple cell types, such as genes involved in gene expression (e.g., transcription factors and / or histones), cellular metabolism (e.g., glyceraldehyde 3-phosphate dehydrogenase (GAPDH)), or cellular structure (e.g., actin), or encoding ribosomal proteins (e.g., large or small ribosomal subunits, such as RPL13A, RPLP0, and / or RPL7).

[0172] Other examples of essential genes include those involved in one or more of glycolysis, ribonucleoprotein complex formation, focal adhesions, cell-matrix adherens junctions, cell-matrix junctions, cell anchorage, extracellular exosomes, extracellular vesicles, intracellular organelles, or anchorage junctions. Some proteins are involved in RNA binding, nucleic acid binding (e.g., rRNA or mRNA binding), or protein binding.

[0173] In some embodiments, essential genes are robustly and consistently expressed in the pluripotent state and during differentiation (e.g., as examined by single-cell RNA sequencing (scRNAseq) analysis). For example, the expression level of an endogenous gene changes (e.g., decreases) by no more than 50%, no more than 40%, no more than 35%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, no more than 10%, or no more than 5% over 5 or more, 10 or more, or 15 or more passages, or as the cell state changes (e.g., state of pluripotency and / or differentiation).

[0174] In some embodiments, an essential gene is a gene associated with cellular metabolism, such as GAPDH.

[0175] In some embodiments, the essential gene is a ribosomal protein gene or a 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, RPL 12, 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 RPS11.

[0176] In some embodiments, the essential gene encodes a cytoskeletal protein, such as actin. Examples of actin genes are ACTG1 and ACTB.

[0177] In some embodiments, the essential genes encode eukaryotic translation elongation factors, such as EEF1A1 and EEF2, or eukaryotic translation initiation factors, such as EIF1.

[0178] In some embodiments, the essential genes encode histones, such as H3F3A and H3F3B.

[0179] In other embodiments, the essential gene is a STEL gene selected from the group consisting of FTH1, TPT1, PTMA, GNB2L1, NACA, YBX1, NPM1, FAU, UBA52, HSP90AB1, MYL6, SERF2, and SRP14.

[0180] In other embodiments, the essential gene is a non-STEL gene, such as HDAC3, DNMT1, NADH dehydrogenase, and PGK1.

[0181] 6.3.3. Homology arms

[0182] The present disclosure provides a targeting construct comprising a first homology arm corresponding to a first region of homology, a nucleic acid insert, and a second homology arm corresponding to a second region of homology.

[0183] The homology arms and target sequence are said to "correspond" to each other when the two regions share a sufficient level of sequence identity with each other to serve as substrates for a homologous recombination reaction, wherein the homology arms are suitable for directing recombination of the nucleic acid insert with the desired genomic locus to promote genomic integration and / or replacement of the endogenous sequence.

[0184] The term "homologous" refers to a DNA sequence that is identical or shares sequence identity with a corresponding sequence. The sequence identity between the corresponding homology arms found in a given target sequence and an exogenous donor nucleic acid can be the sequence identity of any degree that allows homologous recombination to occur. For example, the amount of the sequence identity shared by the homology arms of the exogenous donor nucleic acid (or its fragment) and the target sequence (or its fragment) 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, so that the sequence undergoes homologous recombination. In addition, the corresponding homology region between the homology arms and the corresponding target sequence can have any length that is enough to promote homologous recombination. In some targeting vectors, the desired mutation in the target genomic locus is included in an insert nucleic acid flanked by homology arms.

[0185] In some embodiments, the length of the first homology arm is between 50 and 250 nucleotides. In some embodiments, the length of the first homology arm is between 50 and 2000 nucleotides. In some embodiments, the length of the first homology arm is between 50 and 1500 nucleotides. In some embodiments, the length of the first homology arm is between 50 and 1000 nucleotides. In some embodiments, the length of the first homology arm is between 50 and 500 nucleotides. In some embodiments, the length of the first homology arm is between 150 and 250 nucleotides. In some embodiments, the length of the first homology arm is 2000 nucleotides or less. In some embodiments, the length of the first homology arm is 1500 nucleotides or less. In some embodiments, the length of the first homology arm is 1000 nucleotides or less. In some embodiments, the length of the first homology arm is 700 nucleotides or less. In some embodiments, the length of the first homology arm is 650 nucleotides or less. In some embodiments, the length of the first homology arm is 600 nucleotides or less. In some embodiments, the length of the first homology arm is 550 nucleotides or less. In some embodiments, the length of the first homology arm is 500 nucleotides or shorter. In some embodiments, the length of the first homology arm is 400 nucleotides or shorter. In some embodiments, the length of the first homology arm is 300 nucleotides or shorter. In some embodiments, the length of the first homology arm is 250 nucleotides or shorter. In some embodiments, the length of the first homology arm is 200 nucleotides or shorter. In some embodiments, the length of the first homology arm is 150 nucleotides or shorter. In some embodiments, the length of the first homology arm is less than 100 nucleotides. In some embodiments, the length of the first homology arm is 50 nucleotides or shorter. In some embodiments, the length of 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 some embodiments, the length of the first homology arm is at least 20 nucleotides. In some embodiments, the length of the first homology arm is at least 40 nucleotides. In some embodiments, the length of the first homology arm is at least 50 nucleotides. In some embodiments, the length of the first homology arm is at least 70 nucleotides. In some embodiments, the length of the first homology arm is at least 100 nucleotides. In some embodiments, the length of the first homology arm is at least 200 nucleotides. In some embodiments, the length of the first homology arm is at least 300 nucleotides. In some embodiments, the length of the first homology arm is at least 400 nucleotides.In some embodiments, the length of the first homology arm is at least 500 nucleotides. In some embodiments, the length of the first homology arm is at least 600 nucleotides. In some embodiments, the length of the first homology arm is at least 700 nucleotides. In some embodiments, the length of the first homology arm is at least 1000 nucleotides. In some embodiments, the length of the first homology arm is at least 1500 nucleotides. In some embodiments, the length of the first homology arm is at least 2000 nucleotides. In some embodiments, the length of the first homology arm is about 20 nucleotides. In some embodiments, the length of the first homology arm is about 40 nucleotides. In some embodiments, the length of the first homology arm is 250 nucleotides or shorter. In some embodiments, the length of the first homology arm is about 100 nucleotides. In some embodiments, the length of the first homology arm is about 200 nucleotides.

[0186] In some embodiments, the length of the second homology arm is between 50 and 250 nucleotides. In some embodiments, the length of the second homology arm is between 50 and 2000 nucleotides. In some embodiments, the length of the second homology arm is between 50 and 1500 nucleotides. In some embodiments, the length of the second homology arm is between 50 and 1000 nucleotides. In some embodiments, the length of the second homology arm is between 50 and 500 nucleotides. In some embodiments, the length of the second homology arm is between 150 and 250 nucleotides. In some embodiments, the length of the second homology arm is 2000 nucleotides or shorter. In some embodiments, the length of the second homology arm is 1500 nucleotides or shorter. In some embodiments, the length of the second homology arm is 1000 nucleotides or shorter. In some embodiments, the length of the second homology arm is 700 nucleotides or shorter. In some embodiments, the length of the second homology arm is 650 nucleotides or shorter. In some embodiments, the length of the second homology arm is 600 nucleotides or shorter. In some embodiments, the length of the second homology arm is 550 nucleotides or shorter. In some embodiments, the length of the second homology arm is 500 nucleotides or shorter. In some embodiments, the length of the second homology arm is 400 nucleotides or shorter. In some embodiments, the length of the second homology arm is 300 nucleotides or shorter. In some embodiments, the length of the second homology arm is 200 nucleotides or shorter. In some embodiments, the length of the second homology arm is 150 nucleotides or shorter. In some embodiments, the length of the second homology arm is 100 nucleotides or shorter. In some embodiments, the length of the second homology arm is 50 nucleotides or shorter. In some embodiments, the length of 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 some embodiments, the length of the second homology arm is at least 20 nucleotides. In some embodiments, the length of the second homology arm is at least 40 nucleotides. In some embodiments, the length of the second homology arm is at least 50 nucleotides. In some embodiments, the length of the second homology arm is at least 70 nucleotides. In some embodiments, the length of the second homology arm is at least 100 nucleotides. In certain embodiments, the length of the second homology arm is at least 200 nucleotides. In certain embodiments, the length of the second homology arm is at least 300 nucleotides. In certain embodiments, the length of the second homology arm is at least 400 nucleotides. In certain embodiments, the length of the second homology arm is at least 500 nucleotides.In some embodiments, the length of the second homology arm is at least 600 nucleotides. In some embodiments, the length of the second homology arm is at least 700 nucleotides. In some embodiments, the length of the second homology arm is at least 1000 nucleotides. In some embodiments, the length of the second homology arm is at least 1500 nucleotides. In some embodiments, the length of the second homology arm is at least 2000 nucleotides. In some embodiments, the length of the second homology arm is about 20 nucleotides. In some embodiments, the length of the second homology arm is about 40 nucleotides. In some embodiments, the length of the second homology arm is 250 nucleotides or shorter. In some embodiments, the length of the second homology arm is about 100 nucleotides. In some embodiments, the length of the second homology arm is about 200 nucleotides.

[0187] The first homology arm and the second homology arm can have the same length or can have different lengths.In certain embodiments, the first homology arm and the second homology arm are amplified to allow quantitative assessment of gene editing events at the target nucleic acid, such as targeted integration.In certain embodiments, the quantitative assessment of gene editing events can rely on amplifying all or part of the targeted integration site by using a single pair of PCR primers to amplify the homology arms in a single amplification reaction to amplify the 5' connection and the 3' connection. Therefore, although the length of the first homology arm and the second homology arm can be different, the length of each homology arm should be able to be amplified as needed (for example, using PCR). In addition, when amplifying the first homology arm and the second homology arm, it is necessary to react in a single PCR reaction the length difference of the first homology arm and the second homology arm, the length difference between the first homology arm and the second homology arm should allow the use of a single pair of PCR primers to carry out PCR amplification.

[0188] In some embodiments, the lengths of the first homology arm and the second homology arm differ by no more than 75 nucleotides. Thus, in some embodiments, when the first homology arm and the second homology arm are different lengths, 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, or 1 nucleotides or base pairs. 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 length. In some embodiments, the difference in length between the first homology arm and the second homology arm is less than 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 base pairs. 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,

[0189] Homology arms can guide the recombination of nucleic acid inserts with desired target genomic loci to promote genomic integration and / or replacement of endogenous sequences. Regardless of the format used, donor templates can be designed to avoid unwanted sequences. In certain embodiments, one or both homology arms can be shortened to avoid overlapping with certain sequence repeat elements (e.g., Alu repeats, LINE elements, etc.).

[0190] 6.4. Transgenic

[0191] In some embodiments, the constructs and methods of the present disclosure are designed to engineer target cells to express both (a) a fusion protein comprising an essential polypeptide and a degron and (b) a recombinant polypeptide. In some embodiments, the recombinant polypeptide is expressed by a transgene.

[0192] In some embodiments, the transgene is introduced into the target cell via the same targeting construct or expression vector as the one comprising the degron coding sequence. The transgene can be located 5' or 3' to the degron coding sequence. Illustrative examples of suitable targeting constructs comprising both a transgene and a degron coding sequence are shown in FIG4 .

[0193] In some embodiments, a transgene can be expressed from a different allele of an essential gene that has been modified to express a fusion protein comprising an essential polypeptide and a degron, e.g. Figure 6A and 6C In some embodiments, the essential gene is the STEL gene. In some embodiments, the transgene is located in a different gene / locus than the essential gene to which the degron is fused as described herein.

[0194] In certain embodiments, transgenic and fusion protein are expressed from different loci.In certain embodiments, different loci are all STEL loci, such as GAPDH loci and another STEL loci.In other embodiments, different loci are all non-STEL loci.In other embodiments again, one in loci is STEL (such as, GAPDH) loci, and another locus is non-STEL loci.

[0195] In yet further embodiments, the transgene is expressed from an expression vector and the fusion protein is expressed from a genomic locus (eg, a STEL locus).

[0196] In yet further embodiments, the fusion protein is expressed from an expression vector and the transgene is expressed from a genomic locus (eg, a STEL locus).

[0197] In some embodiments, the transgene encodes a reporter protein, such as a fluorescent protein (e.g., green fluorescent protein, red fluorescent protein, cyan fluorescent protein, yellow fluorescent protein, blue fluorescent protein, DsRed, mCherry, mKate2, and tdTomato) and an enzyme (e.g., luciferase and lacZ). Once the therapeutic cells are implanted in the patient, the reporter protein can help track the therapeutic cells.

[0198] In some embodiments, the transgene encodes a therapeutic molecule, such as a therapeutic nucleotide or a therapeutic polypeptide or protein.

[0199] In some embodiments, the therapeutic molecule encoded by the transgene is a therapeutic nucleotide, such as an oligonucleotide (e.g., a miRNA, a gapmer, a steric blocking ON, an antagomir, a small interfering RNA (siRNA), a microRNA mimic, a splice switch ON, or an aptamer).

[0200] In some embodiments, the therapeutic transgene is a miRNA or other small interfering nucleic acid that can regulate gene expression via RNA transcript cleavage / degradation or translational inhibition of mRNA. Non-limiting examples of miRNA genes or other small interfering nucleic acids that can be used as therapeutic transgenes include hsa-let-7a, hsa-let-7a*, hsa-let-7b, hsa-let-7b*, hsa-let-7c, hsa-let-7c*, hsa-let-7d, hsa-let-7d*, hsa-let-7e, hsa-let-7e*, hsa-let-7f, hsa-let-7f-1*, hsa-let-7f-2*, hsa-let-7g, hsa-let-7g*, hsa-let-7l, hsa-let-7l* ... , hsa-miR-1, hsa-miR-100, hsa-miR-100*, hsa-miR-101, hsa-miR-101*, hsa-miR-103, hsa-miR-105, hsa-miR-105*, hsa-miR-106a, hsa-mi R-106a*, hsa-miR-106b, hsa-miR-106b*, hsa-miR-107, hsa-miR-10a, hsa-miR-10a*, hsa-miR-10b, hsa-miR-10b*, hsa-miR-1178, hsa-miR -1179, hsa-miR-1180, hsa-miR-1181, hsa-miR-1182, hsa-miR-1183, hsa-miR-1184, hsa-miR-1185, hsa-miR-1197, hsa-miR-1200, hsa-mi R-1201, hsa-miR-1202, hsa-miR-1203, hsa-miR-1204, hsa-miR-1205, hsa-miR-1206, hsa-miR-1207-3p, hsa-miR-1207-5p, hsa-miR-1208, hsa-miR-122, hsa-miR-122*, hsa-miR-1224-3p, hsa-miR-1224-5p, hsa-miR-1225-3p, hsa-miR-1225-5p, hsa-miR-1226, hsa-miR-1226*, h sa-miR-1227, hsa-miR-1228, hsa-miR-1228*, hsa-miR-1229, hsa-miR-1231, hsa-miR-1233, hsa-miR-1234, hsa-miR-1236, hsa-miR-1237,hsa-miR-1238, hsa-miR-124, hsa-miR-124*, hsa-miR-1243, hsa-miR-1244, hsa-miR-1245, hsa-miR-1246, hsa-miR-1247, hsa-miR-1248, hsa-miR- 1249, hsa-miR-1250, hsa-miR-1251, hsa-miR-1252, hsa-miR-1253, hsa-miR-1254, hsa-miR-1255a, hsa-miR-1255b, hsa-miR-1256, hsa-miR-1257, h sa-miR-1258, hsa-miR-1259, hsa-miR-125a-3p, hsa-miR-125a-5p, hsa-miR-125b, hsa-miR-125b-1*, hsa-miR-125b-2*, hsa-miR-126, hsa-miR-12 6*, hsa-miR-1260, hsa-miR-1261, hsa-miR-1262, hsa-miR-1263, hsa-miR-1264, hsa-miR-1265, hsa-miR-1266, hsa-miR-1267, hsa-miR-1268, hsa-m iR-1269, hsa-miR-1270, hsa-miR-1271, hsa-miR-1272, hsa-miR-1273, hsa-miR-127-3p, hsa-miR-1274a, hsa-miR-1274b, hsa-miR-1275, hsa-miR- 127-5p、hsa-miR-1276、hsa-miR-1277、hsa-miR-1278、hsa-miR-1279、hsa-miR-128、hsa-miR-1280、hsa-miR-1281、hsa-miR-1282、hsa-miR-1283、hs a-miR-1284, hsa-miR-1285, hsa-miR-1286, hsa-miR-1287, hsa-miR-1288, hsa-miR-1289, hsa-miR-129*, hsa-miR-1290, hsa-miR-1291, hsa-miR-1 292, hsa-miR-1293, hsa-miR-129-3p, hsa-miR-1294, hsa-miR-1295, hsa-miR-129-5p, hsa-miR-1296, hsa-miR-1297, hsa-miR-1298, hsa-miR-1299hsa-miR-1300, hsa-miR-1301, hsa-miR-1302, hsa-miR-1303, hsa-miR-1304, hsa-miR-1305, hsa-miR-1306, hsa-miR-1307, hsa-miR-1308, hsa-miR- 130a, hsa-miR-130a*, hsa-miR-130b, hsa-miR-130b*, hsa-miR-132, hsa-miR-132*, hsa-miR-1321, hsa-miR-1322, hsa-miR-1323, hsa-miR-1324, hs a-miR-133a, hsa-miR-133b, hsa-miR-134, hsa-miR-135a, hsa-miR-135a*, hsa-miR-135b, hsa-miR-135b*, hsa-miR-136, hsa-miR-136*, hsa-miR-13 7, hsa-miR-138, hsa-miR-138-1*, hsa-miR-138-2*, hsa-miR-139-3p, hsa-miR-139-5p, hsa-miR-140-3p, hsa-miR-140-5p, hsa-miR-141, hsa-miR-1 41*, hsa-miR-142-3p, hsa-miR-142-5p, hsa-miR-143, hsa-miR-143*, hsa-miR-144, hsa-miR-144*, hsa-miR-145, hsa-miR-145*, hsa-miR-146a, hsa -miR-146a*、hsa-miR-146b-3p、hsa-miR-146b-5p、hsa-miR-147、hsa-miR-147b、hsa-miR-148a、hsa-miR-148a*、hsa-miR-148b、hsa-miR-148b*、hsa -miR-149, hsa-miR-149*, hsa-miR-150, hsa-miR-150*, hsa-miR-151-3p, hsa-miR-151-5p, hsa-miR-152, hsa-miR-153, hsa-miR-154, hsa-miR-154* hsa-miR-155, hsa-miR-155*, hsa-miR-15a, hsa-miR-15a*, hsa-miR-15b, hsa-miR-15b*, hsa-miR-16, hsa-miR-16-1*, hsa-miR-16-2*, hsa-miR-17hsa-miR-17*, hsa-miR-181a, hsa-miR-181a*, hsa-miR-181a-2*, hsa-miR-181b, hsa-miR-181c, hsa-miR-181c*, hsa-miR-181d, hsa-miR-182, hsa- miR-182*, hsa-miR-1825, hsa-miR-1826, hsa-miR-1827, hsa-miR-183, hsa-miR-183*, hsa-miR-184, hsa-miR-185, hsa-miR-185*, hsa-miR-186, hs a-miR-186*、hsa-miR-187、hsa-miR-187*、hsa-miR-188-3p、hsa-miR-188-5p、hsa-miR-18a、hsa-miR-18a*、hsa-miR-18b、hsa-miR-18b*、hsa-miR- 190, hsa-miR-190b, hsa-miR-191, hsa-miR-191*, hsa-miR-192, hsa-miR-192*, hsa-miR-193a-3p, hsa-miR-193a-5p, hsa-miR-193b, hsa-miR-193b *, hsa-miR-194, hsa-miR-194*, hsa-miR-195, hsa-miR-195*, hsa-miR-196a, hsa-miR-196a*, hsa-miR-196b, hsa-miR-197, hsa-miR-198, hsa-miR- 199a-3p、hsa-miR-199a-5p、hsa-miR-199b-5p、hsa-miR-19a、hsa-miR-19 a*、hsa-miR-19b、hsa-miR-19b-1*、hsa-miR-19b-2*、hsa-miR-200a、hsa- miR-200a*, hsa-miR-200b, hsa-miR-200b*, hsa-miR-200c, hsa-miR-200c*, hsa-miR-202, hsa-miR-202*, hsa-miR-203, hsa-miR-204, hsa-miR-205 、hsa-miR-206、hsa-miR-208a、hsa-miR-208b、hsa-miR-20a、hsa-miR-20a*、hsa-miR-20b、hsa-miR-20b*、hsa-miR-21、hsa-miR-21*、hsa-miR-210、hsa-miR-211, hsa-miR-212, hsa-miR-214, hsa-miR-214*, hsa-miR-215, hsa-miR-216a, hsa-miR-216b, hsa-miR-217, hsa-miR-218, hsa-miR-218-1 *, hsa-miR-218-2*, hsa-miR-219-1-3p, hsa-miR-219-2-3p, hsa-miR-219-5p, hsa-miR-22, hsa-miR-22*, hsa-miR-220a, hsa-miR-220b, hsa-miR-2 20c、hsa-miR-221、hsa-miR-221*、hsa-miR-222、hsa-miR-222*、hsa-miR-223、hsa-miR-223*、hsa-miR-224、hsa-miR-23a、hsa-miR-23a*、hsa-miR- 23b、hsa-miR-23b*、hsa-miR-24、hsa-miR-24-1*、hsa-miR-24-2*、hsa-miR-25、hsa-miR-25*、hsa-miR-26a、hsa-miR-26a-1*、hsa-miR-26a-2*、hsa- miR-26b, hsa-miR-26b*, hsa-miR-27a, hsa-miR-27a*, hsa-miR-27b, hsa-miR-27b*, hsa-miR-28-3p, hsa-miR-28-5p, hsa-miR-296-3p, hsa-miR-29 6-5p、hsa-miR-297、hsa-miR-298、hsa-miR-299-3p、hsa-miR-299-5p、hsa-miR-29a、hsa-miR-29a*、hsa-miR-29b、hsa-miR-296-1*、hsa-miR-296-2 *、hsa-miR-29c、hsa-miR-29c*、hsa-miR-300、hsa-miR-301a、hsa-miR-30 1b、hsa-miR-302a、hsa-miR-302a*、hsa-miR-302b、hsa-miR-302b*、hsa-m iR-302c、hsa-miR-302c*、hsa-miR-302d、hsa-miR-302d*、hsa-miR-302e、hsa-miR-302f、hsa-miR-30a、hsa-miR-30a*、hsa-miR-30b、hsa-miR-30b*、hsa-miR-30c, hsa-miR-30c-1*, hsa-miR-30c-2*, hsa-miR-30d, hsa-miR-30d*, hsa-miR-30e, hsa-miR-30e*, hsa-miR-31, hsa-miR-31*, hsa-miR-3 2, hsa-miR-32*, hsa-miR-320a, hsa-miR-320b, hsa-miR-320c, hsa-miR-320d, hsa-miR-323-3p, hsa-miR-323-5p, hsa-miR-324-3p, hsa-miR-324-5 p、hsa-miR-325、hsa-miR-326、hsa-miR-328、hsa-miR-329、hsa-miR-330-3p、hsa-miR-330-5p、hsa-miR-331-3p、hsa-miR-331-5p、hsa-miR-335、hs a-miR-335*, hsa-miR-337-3p, hsa-miR-337-5p, hsa-miR-338-3p, hsa-miR-338-5p, hsa-miR-339-3p, hsa-miR-339-5p, hsa-miR-33a, hsa-miR-33a *, hsa-miR-33b, hsa-miR-33b*, hsa-miR-340, hsa-miR-340*, hsa-miR-342-3p, hsa-miR-342-5p, hsa-miR-345, hsa-miR-346, hsa-miR-34a, hsa-mi R-34a*、hsa-miR-34b、hsa-miR-34b*、hsa-miR-34c-3p、hsa-miR-34c-5p、 hsa-miR-361-3p、hsa-miR-361-5p、hsa-miR-362-3p、hsa-miR-362-5p、hs a-miR-363, hsa-miR-363*, hsa-miR-365, hsa-miR-367, hsa-miR-367*, hsa-miR-369-3p, hsa-miR-369-5p, hsa-miR-370, hsa-miR-371-3p, hsa-miR -371-5p、hsa-miR-372、hsa-miR-373、hsa-miR-373*、hsa-miR-374a、hsa-miR-374a*、hsa-miR-374b、hsa-miR-374b*、hsa-miR-375、hsa-miR-376a、hsa-miR-376a*, hsa-miR-376b, hsa-miR-376c, hsa-miR-377, hsa-miR-377*, hsa-miR-378, hsa-miR-378*, hsa-miR-379, hsa-miR-379*, hsa-miR-3 80, hsa-miR-380*, hsa-miR-381, hsa-miR-382, hsa-miR-383, hsa-miR-384, hsa-miR-409-3p, hsa-miR-409-5p, hsa-miR-410, hsa-miR-411, hsa-miR -411*, hsa-miR-412, hsa-miR-421, hsa-miR-422a, hsa-miR-423-3p, hsa-miR-423-5p, hsa-miR-424, hsa-miR-424*, hsa-miR-425, hsa-miR-425*, hs a-miR-429, hsa-miR-431, hsa-miR-431*, hsa-miR-432, hsa-miR-432*, hsa-miR-433, hsa-miR-448, hsa-miR-449a, hsa-miR-449b, hsa-miR-450a, hs a-miR-450b-3p, hsa-miR-450b-5p, hsa-miR-451, hsa-miR-452, hsa-miR-452*, hsa-miR-453, hsa-miR-454, hsa-miR-454*, hsa-miR-455-3p, hsa-m iR-455-5p、hsa-miR-483-3p、hsa-miR-483-5p、hsa-miR-484、hsa-miR-485-3p、hsa-miR-485-5p、hsa-miR-486-3p、hsa-miR-486-5p、hsa-miR-487a、 hsa-miR-487b, hsa-miR-488, hsa-miR-488*, hsa-miR-489, hsa-miR-490-3p, hsa-miR-490-5p, hsa-miR-491-3p, hsa-miR-491-5p, hsa-miR-492, hsa -miR-493、hsa-miR-493*、hsa-miR-494、hsa-miR-495、hsa-miR-496、hsa-miR-497、hsa-miR-497*、hsa-miR-498、hsa-miR-499-3p、hsa-miR-499-5p、hsa-miR-500, hsa-miR-500*, hsa-miR-501-3p, hsa-miR-501-5p, hsa-miR-502-3p, hsa-miR-502-5p, hsa-miR-503, hsa-miR-504, hsa-miR-505, hsa -miR-505*, hsa-miR-506, hsa-miR-507, hsa-miR-508-3p, hsa-miR-508-5p, hsa-miR-509-3-5p, hsa-miR-509-3p, hsa-miR-509-5p, hsa-miR-510, hs a-miR-511, hsa-miR-512-3p, hsa-miR-512-5p, hsa-miR-513a-3p, hsa-miR-513a-5p, hsa-miR-513b, hsa-miR-513c, hsa-miR-514, hsa-miR-515-3p hsa-miR-515-5p, hsa-miR-516a-3p, hsa-miR-516a-5p, hsa-miR-516b, hsa-miR-517*, hsa-miR-517a, hsa-miR-517b, hsa-miR-517c, hsa-miR-518a -3p、hsa-miR-518a-5p、hsa-miR-518b、hsa-miR-518c、hsa-miR-518c*、hs a-miR-518d-3p、hsa-miR-518d-5p、hsa-miR-518e、hsa-miR-518e*、hsa-m iR-518f、hsa-miR-518f*、hsa-miR-519a、hsa-miR-519b-3p、hsa-miR-519c-3p、hsa-miR-519d、hsa-miR-519e、hsa-miR-519e*、hsa-miR-520a-3p、hs a-miR-520a-5p、hsa-miR-520b、hsa-miR-520c-3p、hsa-miR-520d-3p、hsa -miR-520d-5p、hsa-miR-520e、hsa-miR-520f、hsa-miR-520g、hsa-miR-520 h、hsa-miR-521、hsa-miR-522、hsa-miR-523、hsa-miR-524-3p、hsa-miR-524-5p、hsa-miR-525-3p、hsa-miR-525-5p、hsa-miR-526b、hsa-miR-526b*、hsa-miR-532-3p, hsa-miR-532-5p, hsa-miR-539, hsa-miR-541, hsa-miR-541*, hsa-miR-542-3p, hsa-miR-542-5p, hsa-miR-543, hsa-miR-544, hsa -miR-545、hsa-miR-545*、hsa-miR-548a-3p、hsa-miR-548a-5p、hsa-miR-548b-3p、hsa-miR-5486-5p、hsa-miR-548c-3p、hsa-miR-548c-5p、hsa-miR -548d-3p、hsa-miR-548d-5p、hsa-miR-548e、hsa-miR-548f、hsa-miR-548g、hsa-miR-548h、hsa-miR-548i、hsa-miR-548j、hsa-miR-548k、hsa-miR-5 481, hsa-miR-548m, hsa-miR-548n, hsa-miR-548o, hsa-miR-548p, hsa-miR-549, hsa-miR-550, hsa-miR-550*, hsa-miR-551a, hsa-miR-551b, hsa-mi R-551b*, hsa-miR-552, hsa-miR-553, hsa-miR-554, hsa-miR-555, hsa-miR-556-3p, hsa-miR-556-5p, hsa-miR-557, hsa-miR-558, hsa-miR-559, hsa -miR-561、hsa-miR-562、hsa-miR-563、hsa-miR-564、hsa-miR-566、hsa-miR-567、hsa-miR-568、hsa-miR-569、hsa-miR-570、hsa-miR-571、hsa-miR- 572, hsa-miR-573, hsa-miR-574-3p, hsa-miR-574-5p, hsa-miR-575, hsa-miR-576-3p, hsa-miR-576-5p, hsa-miR-577, hsa-miR-578, hsa-miR-579, h sa-miR-580, hsa-miR-581, hsa-miR-582-3p, hsa-miR-582-5p, hsa-miR-583, hsa-miR-584, hsa-miR-585, hsa-miR-586, hsa-miR-587, hsa-miR-588hsa-miR-589, hsa-miR-589*, hsa-miR-590-3p, hsa-miR-590-5p, hsa-miR-591, hsa-miR-592, hsa-miR-593, hsa-miR-593*, hsa-miR-595, hsa-miR -596, hsa-miR-597, hsa-miR-598, hsa-miR-599, hsa-miR-600, hsa-miR-601, hsa-miR-602, hsa-miR-603, hsa-miR-604, hsa-miR-605, hsa-miR-606 hsa-miR-607, hsa-miR-608, hsa-miR-609, hsa-miR-610, hsa-miR-611, hsa-miR-612, hsa-miR-613, hsa-miR-614, hsa-miR-615-3p, hsa-miR-615- 5p、hsa-miR-616、hsa-miR-616*、hsa-miR-617、hsa-miR-618、hsa-miR-619、hsa-miR-620、hsa-miR-621、hsa-miR-622、hsa-miR-623、hsa-miR-624、 hsa-miR-624*, hsa-miR-625, hsa-miR-625*, hsa-miR-626, hsa-miR-627, hsa-miR-628-3p, hsa-miR-628-5p, hsa-miR-629, hsa-miR-629*, hsa-miR-630, hsa-miR-631, hsa-miR-632, hsa-miR-633, hsa-miR-634, hsa-miR-635, hsa-miR-636, hsa-miR-637, hsa-miR-638, hsa-miR-639, hsa-miR-64 0, hsa-miR-641, hsa-miR-642, hsa-miR-643, hsa-miR-644, hsa-miR-645, hsa-miR-646, hsa-miR-647, hsa-miR-648, hsa-miR-649, hsa-miR-650, hs a-miR-651, hsa-miR-652, hsa-miR-653, hsa-miR-654-3p, hsa-miR-654-5p, hsa-miR-655, hsa-miR-656, hsa-miR-657, hsa-miR-658, hsa-miR-659hsa-miR-660, hsa-miR-661, hsa-miR-662, hsa-miR-663, hsa-miR-663b, hsa-miR-664, hsa-miR-664*, hsa-miR-665, hsa-miR-668, hsa-miR-671-3p hsa-miR-671-5p, hsa-miR-675, hsa-miR-7, hsa-miR-708, hsa-miR-708*, hsa-miR-7-1*, hsa-miR-7-2*, hsa-miR-720, hsa-miR-744, hsa-miR-744* hsa-miR-758, hsa-miR-760, hsa-miR-765, hsa-miR-766, hsa-miR-767-3p, hsa-miR-767-5p, hsa-miR-768-3p, hsa-miR-768-5p, hsa-miR-769-3p, hs a-miR-769-5p, hsa-miR-770-5p, hsa-miR-802, hsa-miR-873, hsa-miR-874, hsa-miR-875-3p, hsa-miR-875-5p, hsa-miR-876-3p, hsa-miR-876-5p, h sa-miR-877, hsa-miR-877*, hsa-miR-885-3p, hsa-miR-885-5p, hsa-miR-886-3p, hsa-miR-886-5p, hsa-miR-887, hsa-miR-888, hsa-miR-888*, hsa- miR-889, hsa-miR-890, hsa-miR-891a, hsa-miR-891b, hsa-miR-892a, hsa-miR-892b, hsa-miR-9, hsa-miR-9*, hsa-miR-920, hsa-miR-921, hsa-miR- 922, hsa-miR-923, hsa-miR-924, hsa-miR-92a, hsa-miR-92a-1*, hsa-miR-92a-2*, hsa-miR-92b, hsa-miR-92b*, hsa-miR-93, hsa-miR-93*, hsa-miR -933、hsa-miR-934、hsa-miR-935、hsa-miR-936、hsa-miR-937、hsa-miR-938、hsa-miR-939、hsa-miR-940、hsa-miR-941、hsa-miR-942、hsa-miR-943、hsa-miR-944, hsa-miR-95, hsa-miR-96, hsa-miR-96*, hsa-miR-98, hsa-miR-99a, hsa-miR-99a*, hsa-miR-99b and hsa-miR-99b*. ,

[0201] In some embodiments, the transgenic protein or polypeptide is a therapeutic protein or polypeptide. The therapeutic protein or polypeptide can be introduced into a protein or peptide that is not present in the patient. The therapeutic protein or polypeptide can also replace proteins that are defective or abnormal (e.g., with mutations) in the patient, such as those associated with rare or orphan diseases. Examples of such rare diseases may include spinal muscular atrophy (SMA), Huntington's disease, Rett syndrome (e.g., methyl-CpG binding protein 2 (MeCP2); UniProtKB-P51608), amyotrophic lateral sclerosis (ALS), Duchenne muscular dystrophy, Friedreich's ataxia (e.g., ataxia), progranulin (PRGN) (associated with non-Alzheimer's brain degeneration, including frontotemporal dementia (FTD), progressive non-fluent aphasia (PNFA) and semantic dementia) etc.

[0202] Familial hypercholesterolemia, muscular dystrophy, mucopolysaccharidoses, cystic fibrosis, diabetes, and coagulation disorders can also be targeted with therapeutic proteins and peptides that replace absent, defective, or abnormal proteins in a patient's body. Non-limiting examples of therapeutic proteins and polypeptides that replace absent, defective, or abnormal proteins include insulin, growth hormone, coagulation factors, albumin, H-protein, T-protein, dystonia, neurofilament light chain (NEFL), and various enzymes that can be used in enzyme replacement therapy, such as lactase, lipase, amylase, adenosine deaminase, β-glucocerebrosidase, carbamoyl synthetase I, ornithine transcarbamylase (OTC), argininosuccinate synthetase, argininosuccinate lyase (ASL) for treating argininosuccinate lyase deficiency, arginase, fumaryl acetate hydrolase, phenylalanine hydroxylase, alpha-1 antitrypsin, rhesus alpha-fetoprotein (AFP), rhesus chorionic gonadotropin (CG), glucose-6-phosphatase, porphobilinogen deaminase, cystathionine β-synthase, branched-chain ketoacid decarboxylase, albumin, isovaleryl-CoA dehydrogenase, propionyl-CoA carboxylase, methyl Malonyl-CoA mutase, glutaryl-CoA dehydrogenase, β-glucosidase, pyruvate carboxylate, liver phosphorylase, phosphorylase kinase and glycine decarboxylase, α-L-iduronidase (IDUA), iduronate-2-sulfatase (IDS), sulfamidase, N-acetylgalactosamine-6-sulfate sulfatase (GALNS), arylsulfatase B, hyaluronidase, β-glucuronidase, phosphoenolpyruvate carboxykinase ( PEPCK), cyclin-dependent kinase-like 5 (CDKL5), galactosephosphate uridyltransferase, branched-chain α-ketoacid dehydrogenase, fumaryl acetoacetate hydrolase, methylmalonyl-CoA mutase, argininosuccinate synthetase, lecithin cholesterol acyltransferase, hypoxanthine guanine phosphoribosyltransferase, biotinylase, α-galactosidase A, hexosaminidase, ceramidase, asparagine glucosidase, and α-fucosidase

[0203] In some embodiments, therapeutic proteins or polypeptides can be used to enhance existing pathways. Some non-limiting examples of enhanced therapeutic proteins and polypeptides include peptide hormones for treating hormone deficiency or infertility, growth and differentiation factors, and proteins for treating hematopoietic defects, anemia caused by blood therapy, or myelodysplastic syndrome.

[0204] Non-limiting examples of hormones and growth differentiation factors that can be used as therapeutic proteins or polypeptides include glucagon, glucagon-like peptide-1 (GLP1), parathyroid hormone (PTH), growth hormone-releasing factor (GRF), follicle-stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiogenin, angiostatin, granulocyte colony-stimulating factor (GCSF), erythropoietin (EPO) (including, for example, human, canine or feline epo), connective tissue growth factor (CTGF), neutral trophic factors (including, for example, basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin growth factors I and II (IGF-I and IGF-II)), any of the transforming growth factor alpha superfamily (including TGFα, activin, inhibin) or bone morphogenetic proteins (BMPs) BMPs any one of 1-15, any one of the heregluin / neuregulin / ARIA / neu differentiation factor (NDF) family of growth factors, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophins NT-3 and NT-4 / 5, ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), neurotrophins, agrin, any one of the semaphorin / collapsin family, netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrin, noggin, sonic hedgehog, and tyrosine hydroxylase.

[0205] In some embodiments, therapeutic proteins or polypeptides can be used to provide new functions or activities to endogenous proteins, or to introduce non-endogenous proteins with new functions or activities. Some non-limiting examples are proteins and peptides for enzymatic degradation of macromolecules, such as papain, collagenase, hyaluronidase, botulinum toxin types A and B; and proteins and peptides for enzymatic degradation of small molecule metabolites, such as L-asparaginase, polyethylene glycol-asparaginase, and rasburicase. Other examples can include chimeric or hybrid polypeptides having non-naturally occurring amino acid sequences comprising insertions, deletions, or amino acid substitutions. For example, single-chain engineered immunoglobulins can be used in certain immunocompromised patients. Further examples of non-naturally occurring gene sequences can include antisense molecules and catalytic nucleic acids, such as ribozymes, which can be used to reduce overexpression of a target.

[0206] In certain embodiments, therapeutic nucleic acids, proteins or polypeptides can be used to interfere with molecules or organisms. Non-limiting examples include those used to treat infection or various forms of cancer, such as proteins and peptides produced only in hyperproliferative cells or at higher levels compared to normal cells, for example, polypeptides encoded by oncogenes myb, myc, fyn, and translocation genes bcr / abl, ras, src, P53, neu, trk and EGRF. The following is a non-limiting list of exemplary genes (e.g., oncogenes and tumor suppressor genes) known to be associated with cancer development that can be targeted by therapeutic transgenes: AARS, ABCB1, ABCC4, ABI2, ABL1, ABL2, ACK1, ACP2, ACY1, ADSL, AK1, AKR1C2, AKT1, ALB, ANPEP, ANXA5, ANXA7, AP2M1, APC, ARHGAP5, ARHGEF5, ARID4A, ASNS, ATF4, ATM, ATP5B, ATP5O, AXL, BAR D1, BAX, BCL2, BHLHB2, BLMH, BRAF, BRCA1, BRCA2, BTK, CANX, CAP1, CAPN1, CAPNS1, CAV1, CBFB, CBLB, CCL2, CCND1, CCND2, CCND3 , CCNE1, CCT5, CCYR61, CD24, CD44, CD59, CDC20, CDC25, CDC25A, CDC25B, CDC2L5, CDK10, CDK4, CDK5, CDK9, CDKL1, CDKN1A, CDKN 1B, CDKN1C, CDKN2A, CDKN2B, CDKN2D, CEBPG, CENPC1, CGRRF1, CHAF1A, CIB1, CKMT1, CLK1, CLK2, CLK3, CLNS1A, CLTC, COL1A1, CO L6A3, COX6C, COX7A2, CRAT, CRHR1, CSF1R, CSK, CSNK1G2, CTNNA1, CTNNB1, CTPS, CTSC, CTSD, CUL1, CYR61, DCC, DCN, DDX10, DEK, DHCR7, DHRS2, DHX8, DLG3, DVL1, DVL3, E2F1, E2F3, E2F5, EGFR, EGR1, EIF5, EPHA2, ERBB2, ERBB3, ERBB4, ERCC3, ETV1, ETV3, ETV 6. F2R, FASTK, FBN1, FBN2, FES, FGFR1, FGR, FKBP8, FN1, FOS, FOSL1, FOSL2, FOXG1A, FOXO1A, FRAP1, FRZB, FTL, FZD2, FZD5, FZD9,<h2 style=";text-align:left;direction:ltr">G22P1、GAS6、GCN5L2、GDF15、GNA13、GNAS、GNB2、GNB2L1、GPR39、GRB2、GSK3 A、GSPT1、GTF2I、HDAC1、HDGF、HMMR、HPRT1、HRB、HSPA4、HSPA5、HSPA8、HSPB 1、HSPH1、HYAL1、HYOU1、ICAM1、ID1、ID2、IDUA、IER3、IFITM1、IGF1R、IGF2R 、IGFBP3、IGFBP4、IGFBP5、IL1B、ILK、ING1、IRF3、ITGA3、ITGA6、ITGB4、JAK1 JARID1A, JUN, JUNB, JUND, K-ALPHA-1, KIT, KITLG, KLK10, KPNA2, KRAS2, KRT18, KRT2A, KRT9, LAMB1, LAMP2, LCK, LCN2, LEP, LITAF, LRPAP1, LTF, LYN, L ZTR1、MADH1、MAP2K2、MAP3K8、MAPK12、MAPK13、MAPKAPK3、MAPRE1、MARS、MA S1、MCC、MCM2、MCM4、MDM2、MDM4、MET、MGST1、MICB、MLLT3、MME、MMP1、MMP14、 MMP17、MMP2、MNDA、MSH2、MSH6、MT3、MYB、MYBL1、MYBL2、MYC、MYCL1、MYCN、M YD88、MYL9、MYLK、NEO1、NF1、NF2、NFKB1、NFKB2、NFSF7、NID、NINE、NMBR、NME 1、NME2、NME3、NOTCH1、NOTCH2、NOTCH4、NPM1、NQO1、NR1D1、NR2F1、NR2F6、N RAS、NRG1、NSEP1、OSM、PA2G4、PABPC1、PCNA、PCTK1、PCTK2、PCTK3、PDGFA、PD GFB、PDGFRA、PDPK1、PEA15、PFDN4、PFDN5、PGAM1、PHB、PIK3CA、PIK3CB、PIK 3CG、PIM1、PKM2、PKMYT1、PLK2、PPARD、PPARG、PPIH、PPP1CA、PPP2R5A、PRDX2 、PRDX4、PRKAR1A、PRKCBP1、PRNP、PRSS15、PSMA1、PTCH、PTEN、PTGS1、PTMA、 PTN、PTPRN、RAB5A、RAC1、RAD50、RAF1、RALBP1、RAP1A、RARA、RARB、RASGRF1、RB1, RBBP4, RBL2, REA, REL, RELA, RELB, RET, RFC2, RGS19, RHOA, RHOB, RHOC, RHOD, RIPK1, RPN2, RPS6KB1, RRM1, SARS, SELENBP1, SEMA3C, SEMA4D, SEPP1, SERPINH1, SFN, SFPQ, SFRS7, SHB, SHH, SIAH2, SIVA, SIVA TP53, SKI, SKIL, SLC16A1, SLC1A4, SLC20A1, SMO, Sphingomyelin phosphodiesterase 1 (SMPD1), SNAI2, SND1, SNRPB2, SOCS1, SOCS3, SOD1, SORT1, SPINT2, SPRY2, SRC, SRPX, STAT1, STAT2, STAT3, STAT5B, STC1, TAF1, TBL3, TBRG4, TCF1, TCF7L2, TFAP2C, TFDP1, TFDP2, TGFA, TGFB1, TGFBI, TGFBR2, TGFBR3, THBS1, TIE, TIMP1, TIMP3, TJP1, TK1, TLE1, TNF, TNFRSF10A, TNFRSF10B, TNFRSF1A, TNFRSF1B, TNFRSF6, TNFSF7, TNK1, TOB1, TP53, TP53BP2, TP5313, TP73, TPBG, TPT1, TRADD, TRAM1, TRRAP, TSG101, TUFM, TXNRD1, TYRO3, UBC, UBE2L6, UCHL1, USP7, VDAC1, VEGF, VHL, VIL2, WEE1, WNT1, WNT2, WNT2B, WNT3, WNT5A, WT1, XRCC1, YES1, YWHAB, YWHAZ, ZAP70 and ZNF9.

[0207] In some embodiments, the therapeutic transgene can be an apoptosis regulator. Non-limiting examples of apoptosis regulators include RPS27A, ABL1, AKT1, APAF1, BAD, BAG1, BAG3, BAG4, BAK1, BAX, BCL10, BCL2, BCL2A1, BCL2L1, BCL2L10, BCL2L11, BCL2L12, BCL2L13, BCL2L2, BCLAF1, BFAR, BID, BIK, NAIP, BIRC2, BIRC3, XIAP, BIRC5, BIR C6, BIRC7, BIRC8, BNIP1, BNIP2, BNIP3, BNIP3L, BOK, BRAF, CARD10, CARD11, NLRC4, CARD14, NOD2, NOD1, CARD6 , CARDS, CARDS, CASP1, CASP10, CASP14, CASP2, CASP3, CASP4, CASP5, CASP6, CASP7, CASP8, CASP9, CFLAR, CIDEA , CIDEB, CRADD, DAPK1, DAPK2, DFFA, DFFB, FADD, GADD45A, GDNF, HRK, IGF1R, LTA, LTBR, ​​MCL1, NOL3, PYCARD, RI PK1, RIPK2, TNF, TNFRSF10A, TNFRSF10B, TNFRSF10C, TNFRSF10D, TNFRSF11B, TNFRSF12A, TNFRSF14, TNFRSF19 , TNFRSF1A, TNFRSF1B, TNFRSF21, TNFRSF25, CD40, FAS, TNFRSF6B, CD27, TNFRSF9, TNFSF10, TNFSF14, TNFSF18 , CD40LG, FASLG, CD70, TNFSF8, TNFSF9, TP53, TP53BP2, TP73, TP63, TRADD, TRAF1, TRAF2, TRAF3, TRAF4 and TRAF5.

[0208] In some embodiments, therapeutic proteins or polypeptides can be used to deliver other compounds or proteins, such as radionuclides, cytotoxic drugs, or effector proteins, to target tissues or organs.

[0209] In some other embodiments, therapeutic proteins and polypeptides include those that can be used to treat individuals with autoimmune diseases and conditions by conferring a broad-based protective immune response against targets associated with autoimmunity, including cell receptors and cells that produce "self" directed antibodies. T cell-mediated autoimmune diseases include rheumatoid arthritis (RA), multiple sclerosis (MS), Sjögren's syndrome, sarcoidosis, insulin-dependent diabetes mellitus (IDDM), autoimmune thyroiditis, reactive arthritis, ankylosing spondylitis, scleroderma, polymyositis, dermatomyositis, psoriasis, vasculitis, Wegener's granulomatosis, Crohn's disease, and ulcerative colitis.

[0210] In some embodiments, the therapeutic protein is a receptor or a ligand for a receptor. Non-limiting examples of receptors include any of receptors for hormones, growth factors, cytokines, lymphokines, regulatory proteins, and immune system proteins, as well as cholesterol-regulated and / or lipid-regulated receptors, including low-density lipoprotein (LDL) receptors, high-density lipoprotein (HDL) receptors, very low-density lipoprotein (VLDL) receptors, and scavenger receptors. In some embodiments, the therapeutic protein is a member of the steroid hormone receptor superfamily, including glucocorticoid receptors and estrogen receptors, vitamin D receptors, and other nuclear receptors.

[0211] Therapeutic proteins and polypeptides also include complement regulatory proteins, such as complement regulatory proteins, membrane cofactor proteins (MCPs), decay accelerating factors (DAFs), CR1, CF2, CD59, and C1 esterase inhibitors (C1-INH). In some embodiments, the therapeutic protein can be a non-covalent binding agent other than mAb, Fc fusion protein, or polyclonal immunoglobulin (e.g., see Dimitrov, 2012, Table 4 of Methods Mol Biol. 899: 1–26, which is incorporated herein by reference).

[0212] In some embodiments, the transgene is a therapeutic protein that can be used to treat lysosomal storage diseases. In some embodiments, the therapeutic protein is a lysosomal enzyme, such as α-L-iduronidase, arylsulfatase A, β-glucocerebrosidase, acid sphingomyelinase, α-galactosidase, or β-galactosidase.

[0213] In certain embodiments, transgenic is a therapeutic protein that can be used to treat hemophilia or other inherited blood disorders. In certain embodiments, therapeutic polypeptides are factor VIII and factor IX. In certain embodiments, the therapeutic transgenic comprises the first 57 base pairs of the factor VIII heavy chain encoding a 10 amino acid signal sequence, and a human growth hormone (hGH) polyadenylation sequence. In alternative embodiments, the therapeutic transgenic further comprises A1 and A2 domains, and 5 amino acids from the N-terminus of the B domain and / or 85 amino acids from the C-terminus of the B domain, and A3, C1, and C2 domains. In yet other embodiments, nucleic acids encoding the factor VIII heavy and light chains are provided in a single minigene separated by 42 nucleic acids encoding the 14 amino acids of the B domain (see U.S. Patent No. 6,200,560).

[0214] In some embodiments, the therapeutic protein can be an immune system-related protein or polypeptide, such as an antibody, a Fab fragment, an immunoglobulin light chain, an immunoglobulin heavy chain, an Fc fusion protein, an immunoadhesin, an interferon, a lymphokine, an immunomodulator, e.g., a cytokine or cytokine receptor, or an interleukin or an interleukin receptor. Immune system modulating therapeutic proteins and polypeptides may include, but are not limited to, thrombopoietin (TPO), interleukins IL-1 to IL-36 (including, for example, human interleukins IL-1, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-6, IL-8, IL-10, IL-12, IL-11, IL-12, IL-13, IL-15, IL-18, IL-21, IL-23, IL-27, IL-31, IL-35), monocyte chemoattractant protein, leukemia inhibitory factor, granulocyte-macrophage colony stimulating factor, Fas ligand, tumor necrosis factor α and β, interferon α, β and γ, stem cell factor, flk-2 / flt3 ligand. Thus, in some embodiments, a transgene may comprise a nucleic acid encoding a proinflammatory or immunosuppressive agent. For example, in some embodiments, the transgene may comprise a nucleic acid encoding one of IL-1Ra, IL-1β, IL-6, IL-10, IL-12, IL-15, GM-CSF, IFN-α, IFN-β, IFN-γ, TNF-α, CCL2, CCL5, CXCL9, CXCL10, CXCL12, TGFβ, or CSF-1. Gene products produced by the immune system may also be used in the present invention. These include, but are not limited to, immunoglobulins IgG, IgM, IgA, IgD, and IgE, chimeric immunoglobulins, humanized antibodies, single-chain antibodies, T cell receptors, chimeric T cell receptors, single-chain T cell receptors, MHC class I and class II molecules, and engineered immunoglobulins and MHC molecules.

[0215] In some other embodiments, the immunomodulatory therapeutic protein is a human leukocyte antigen ("HLA") polypeptide, including but not limited to HLA-Ib class polypeptides. In some embodiments, the HLA polypeptide is an isoform of HLA-E, HLA-F, or HLA-G (e.g., HLA-G1, -G2, -G3, -G4, -G5, -G6, or -G7). Other suitable immunomodulatory polypeptides include but are not limited to CD47, PD-L1, CTLA-4, M-CSF, TGF-β1, IFN-γ, and various isoforms thereof.

[0216] In some embodiments, the transgene is a therapeutic polypeptide comprising an antibody or antigen-binding fragment thereof (eg, scFv).

[0217] In some embodiments, the therapeutic protein or polypeptide is a bone morphogenetic protein, an engineered protein scaffold, a serum protein, a globular protein, a defense protein, a membrane or membrane-bound protein, a channel (e.g., an ion exchange channel), a signaling protein, a regulatory protein, a transport protein, a sensory protein, a motor protein, a storage protein, a structural protein, or a thrombolytic protein.

[0218] In some embodiments, the therapeutic protein is a transcription factor, such as jun, fos, max, mad, serum response factor (SRF), AP-1, AP2, myb, MyoD and myogenin, ETS-box containing proteins, TFE3, E2F, ATF1, ATF2, ATF3, ATF4, ZF5, NFAT, CREB, HNF-4, C / EBP, SP1, CCAAT-box binding protein, interferon regulatory factor (IRF-1), Wilms' tumor protein, ETS binding protein, STAT and GATA-box binding protein, e.g., GATA-3, and the forkhead family of winged helix proteins.

[0219] In some embodiments, the transgene is a therapeutic polypeptide that binds to a pathogenic polypeptide, such as tau, α-synuclein, or β-amyloid polypeptide.

[0220] In some embodiments, the transgene is a therapeutic polypeptide that targets cancer cells. In some embodiments, the therapeutic polypeptide is a chimeric antigen receptor that binds a tumor-associated antigen, such as CD19 or CD20.

[0221] In some embodiments, the therapeutic polypeptide is a T cell receptor (TCR) or an antigen-binding fragment thereof, such as a recombinant TCR. In some embodiments, the recombinant TCR can bind to an antigen of interest, such as, but not limited to, an antigen selected from CD279, CD2, CD95, CD152, CD223, CD272, TIM3, KIR, A2aR, SIRPα, CD200, CD200R, CD300, LPA5, NY-ESO, PD1, PDL1, or MAGE-A3 / A6.

[0222] In some embodiments, the TCR or antigen-binding fragment thereof can bind to a viral antigen, such as from hepatitis A, hepatitis B, hepatitis C (HCV), human papillomavirus (HPV) (e.g., HPV-16 (such as HPV-16E6 or HPV-16E7), HPV-18, HPV-31, HPV-33, or HPV-35), Epstein-Barr virus (EBV), human herpesvirus 8 (HHV-8), human T-cell leukemia virus 1 (HTLV-1), human T-cell leukemia virus 2 (HTLV-2), or cytomegalovirus (CMV).

[0223] In some embodiments, the therapeutic protein or polypeptide can be a neutralizing antibody against a viral pathogen. Such antiviral antibodies may include anti-influenza antibodies against one or more of influenza A, influenza B, and influenza C. Other target pathogenic viruses include arenaviruses (including funin, machupo, and Lassa), filoviruses (including Marburg and Ebola), hantaviruses, picornaviruses (including rhinoviruses, echoviruses), coronaviruses, paramyxoviruses, measles virus, respiratory syncytial virus, enveloped viruses, coxsackieviruses, JC virus, parvovirus B19, parainfluenza virus, adenovirus, reovirus, variola virus (smallpox) and vaccinia virus (cowpox) of the poxviridae family, and varicella-zoster virus (pseudorabies). For example, in some embodiments, the therapeutic protein can be an anti-Ebola antibody (e.g., 2G4, 4G7, 13C6), an anti-influenza antibody (e.g., FI6, CR8033), or an anti-RSV antibody (e.g., palivizumab, motavizumab).

[0224] In some embodiments, the therapeutic protein can be a neutralizing antibody construct directed against a bacterial pathogen. In one embodiment, the neutralizing antibody construct is directed against the bacteria themselves. In another embodiment, the neutralizing antibody construct is directed against a toxin produced by bacteria, such as the pathogen of anthrax, a toxin produced by Bacillius anthracis.Examples of airborne bacterial pathogens include, e.g., Neisseria meningitidis (meningitis), Klebsiella pneumonia (pneumonia), Pseudomonas aeruginosa (pneumonia), Pseudomonas pseudomallei (pneumonia), Pseudomonas mallei (pneumonia), Acinetobacter (pneumonia), Moraxella catarrhalis, Moraxella lacunata, Alkaligenes, Cardiobacterium, Haemophilus influenzae (flu), Haemophilus parainfluenzae, Bordetella pertussis (whooping cough), Francisella tularensis (whooping cough), and Pseudomonas aeruginosa (pneumonia). tularensis (pneumonia / fever), Legionella pneumoniae (Legionnaires' disease), Chlamydia psittaci (pneumonia), Chlamydia pneumoniae (pneumonia), Mycobacterium tuberculosis (Tuberculosis (TB)), Mycobacterium kansasii (TB), Mycobacterium avium (pneumonia), Nocardia asteroides (pneumonia), Bacillus anthracis (anthrax), Staphylococcus aureus (pneumonia), Streptococcus pyogenes (scarlet fever), Streptococcus pneumoniae (pneumonia), Corynebacteria diphtheria (diphtheria), Mycoplasma pneumoniae (pneumonia).

[0225] The therapeutic protein may be an antibody to other infectious agents, such as parasites or fungi, including, for example, Aspergillus species, Absidia corymbifera, Rhixpus stolonifer, Mucor plumbeaus, Cryptococcus neoformans, Histoplasm capsulatum, Blastomyces dermatitidis, Coccidioides immitis, Penicillium species, Micropolyspora faeni, Thermoactinomyces vulgaris, Alternaria alternate, Cladosporium species, Helminthosporium, and Stachybotrys species.

[0226] In addition, in some embodiments, transgenic encoding cell lineage commitment factor.For example, in some embodiments, transgenic encoding gene product, the gene product promotes cell to more specialized cell type differentiation when expressed.For example, in some embodiments, transgenic encoding pedigree commitment factor, it promotes cell to fibroblast, hematopoietic cell, neuron, neuroglia, oligodendrocyte, myocyte, osteocyte, hepatocyte, pancreatic cell, bone marrow cell or bone marrow progenitor cell, microglia or microglial progenitor cell, T cell (for example, CD4+T cell, such as Treg) differentiation.For example, in some embodiments, transgenic includes CD4, CD25, ThPOK, FOXP3, CD45RA, CD62L, HELIOS, GITR, IKAROS, CTLA4, GATA3, TOX, ETS1, TCF7, LEF1, RORA, TNFR2, EOS, IRF5, SATB1, GATA1 or C-MYB.

[0227] 6.5. Delimited Sequences

[0228] The targeting constructs and recombinant target cell genomes described herein may also comprise a separator sequence between the degron encoding sequence and the transgene. Such separator sequences may allow for the separate expression of polypeptides encoded by a single expression cassette.

[0229] In some embodiments, the separator sequence is an internal ribosome entry site (IRES), which allows for separate translation of the transgene from a fusion protein comprising the sequences of the essential polypeptide and the degron.

[0230] In some embodiments, the separating sequence is a self-cleaving peptide, which is related to the ribosome skipping during translation, wherein the ribosome skips the peptide bond between the C-terminal Gly and Pro, resulting in the production of two separate polypeptides, i.e., a transgenic and a fusion protein comprising the sequence of the essential polypeptide and the degradation determinant. In a further embodiment, the polypeptide coding sequence comprising the fusion protein in the expression cassette can be separated by a translation skip sequence (i.e., the same frame coding sequence of the self-cleaving peptide) so that the translation of the mRNA transcript from the polycistronic box will produce a separate protein. The self-cleaving peptide can cause the ribosome to skip during translation. An example of a self-cleaving peptide is a 2A peptide, which is a virus-derived peptide with a typical length of 18 to 22 amino acids. The 2A peptide includes T2A, P2A, E2A, F2A, and PQR (Lo et al., 2015, Cell Reports 13: 2634-2644). For example, P2A is a 19-amino acid peptide; after cleavage, several amino acid residues from P2A remain on the upstream polypeptide, while proline remains at the start of the second polypeptide. It is believed that the 2A residues remaining on the fusion protein and the polypeptide encoded by the transgene will not affect their function.

[0231] 6.6. Expression Vectors

[0232] The present disclosure provides expression vectors encoding the fusion proteins of the present disclosure.The expression vectors generally comprise an expression cassette comprising a fusion polypeptide as described in Section 6.2 operably linked to regulatory elements such as a promoter and, optionally, a self-replicating element.

[0233] Without being bound by theory, it is believed that recombinant expression of a fusion protein comprising an essential polypeptide and a degron can "poison" native cellular proteins and cause instability when the degron is activated, leading to cell death even when the essential gene is intact and has not been modified by recombination with the targeting construct of the present disclosure. Thus, the fusion protein of the present disclosure can be expressed by an expression cassette that is not integrated into the essential gene. In some embodiments, the expression cassette is part of an extrachromosomal vector. In other embodiments, the expression cassette is integrated into the target cell genome without modifying the sequence of the native essential polypeptide.

[0234] The expression vector can be a viral genome, single-stranded RNA or DNA, or double-stranded DNA, such as a plasmid.

[0235] The expression vector may include other coding or non-coding elements. For example, the expression cassette may be delivered as part of a viral genome (e.g., in an AAV, adenovirus, Sendai virus, or lentiviral genome) that includes certain genomic backbone elements (e.g., in the case of an AAV genome, inverted terminal repeats).

[0236] In some embodiments, the expression vector is a circular plasmid that has not been linearized.

[0237] In some embodiments, the expression vector is a circular plasmid that has been linearized.

[0238] In certain embodiments, expression vector is a viral genome. The viral genome provides a rich source of vectors that can be used to effectively deliver exogenous nucleic acids to the genome of target cells (e.g., mammalian cells, such as human cells). The viral genome is a particularly useful vector for delivering exogenous nucleic acids, because the nucleic acids contained in such genomes are typically incorporated into the genome of target cells by general or specialized transduction. These processes occur as part of the natural viral replication cycle, and do not require the addition of proteins or reagents to induce gene integration. Examples of viral vectors include AAV, retroviruses, adenoviruses (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvoviruses (e.g., adeno-associated virus), coronaviruses, negative-strand RNA viruses such as orthomyxoviruses (e.g., influenza virus), rhabdoviruses (e.g., rabies and vesicular stomatitis virus), paramyxoviruses (e.g., measles virus and Sendai virus), positive-strand RNA viruses such as picornaviruses and alphaviruses, and double-stranded DNA viruses, including adenoviruses, herpesviruses (e.g., herpes simplex virus type 1 and type 2, Epstein-Barr virus, cytomegalovirus), and poxviruses (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox, and canarypox). Other viruses that can be used to deliver exogenous nucleic acids include, for example, Norwalk virus, envelope virus, flavivirus, reovirus, papovavirus, hepadnavirus, and hepatitis virus. Examples of retroviruses include: avian leukemia-sarcoma, mammalian C-type, B-type virus, D-type virus, HTLV-BLV group, lentivirus, foamy virus (Coffin, JM, Retroviridae: The viruses and their replication, in Fundamental Virology, 3rd edition, BN Fields et al., eds., Lippincott-Raven Publishers, Philadelphia, 1996). Other examples include murine leukemia virus, murine sarcoma virus, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T-cell leukemia virus, baboon endogenous virus, gibbon ape leukemia virus, Mason Fisher monkey virus, simian immunodeficiency virus, simian sarcoma virus, Rous sarcoma virus and lentivirus. Other examples of vectors are described, for example, in U.S. Patent No. 5,801,030.

[0239] In some embodiments, the expression vector is a recombinant adeno-associated viral vector ("rAAV vector"). The rAAV vector used in the present invention is a recombinant nucleic acid construct comprising (1) an expression cassette (e.g., a nucleic acid encoding a fusion protein comprising an essential polypeptide and a degradation determinant optionally connected via a linker) and (2) a viral nucleic acid that promotes the expression of the fusion protein. The viral nucleic acid can include those AAV sequences required for cis replication and packaging DNA (e.g., functional ITR) into viral particles. Useful rAAV vectors have one or more AAV WT genes that are completely or partially deleted, but retain functional flanking ITR sequences. AAV ITRs can be any serotype suitable for a particular application. Methods using rAAV vectors are described in, for example, Tal et al., 2000, J. Biomed. Sci. 7: 279-291, and Monahan and Samulski, 2000, Gene Delivery 7: 24-30. rAAV can be derived from any suitable serotype, including AAV1, 2, 3, 4, 5, 6, 7, 8, and 9.

[0240] In some embodiments, the expression cassette comprises a transgene in addition to the fusion protein encoding sequence, such as a transgene encoding a therapeutic polypeptide.

[0241] The transgene and fusion protein can be expressed from a common promoter. In some embodiments, the transgene is separated from the fusion protein coding sequence by a translational skip sequence (e.g., a co-frame coding sequence of a self-cleaving peptide) such that translation of the mRNA transcript from the polycistronic cassette will produce a separate polypeptide, i.e., a fusion protein comprising an essential polypeptide and a degron, and a polypeptide encoded by the transgene. Self-cleaving peptides are described in Section 6.5.

[0242] Alternatively, the expression cassette can be a polycistronic expression cassette, in which the fusion protein coding sequence and the transgene are separated by an internal ribosome entry site (IRES) in the mRNA.

[0243] Target cells

[0244] In some embodiments, a targeting construct or expression vector is introduced into a target cell or population of target cells. Methods for introducing proteins and nucleic acids into target cells are further described in Section 6.9.

[0245] The target cells and target cell populations of the present disclosure can be cells engineered to express a fusion protein comprising an essential polypeptide and a degron, and optionally a transgene. A cell population can comprise, for example, a population in which at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70% of the cells have been engineered to express a fusion protein comprising an essential polypeptide and a degron.

[0246] In some embodiments, the methods of the present disclosure can be used to express fusion proteins in mitotic or post-mitotic target cells in vivo and / or ex vivo and / or in vitro (e.g., to generate engineered target cells that can be reintroduced into an individual).

[0247] Any type of cell can be of interest (e.g., stem cells, e.g., human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs), germ cells; somatic cells, e.g., fibroblasts, hematopoietic cells, neurons, glial cells, oligodendrocytes, myocytes, bone cells, hepatocytes, pancreatic cells, bone marrow cells or bone marrow progenitor cells (e.g., primitive bone marrow progenitor cells), microglia or microglial progenitor cells, T cells (e.g., CD4+ T cells, such as Tregs); embryonic cells in vitro or in vivo at any stage of the embryo, e.g., zebrafish embryos at the 1-cell, 2-cell, 4-cell, 8-cell, etc. stage; etc.). The cells can be from an established cell line, or they can be primary cells, where "primary cells," "primary cell lines," and "primary cultures" are used interchangeably herein to refer to cells and cell cultures that are derived from a subject and allowed to grow in culture for a limited number of passages (e.g., divisions) in vitro. For example, primary cultures include cultures that may have been passaged 0, 1, 2, 4, 5, 10, or 15 times, but not enough times to have undergone a crisis phase. Primary cell lines can be maintained in vitro for fewer than 10 generations. In some embodiments, the target cells are unicellular organisms or are grown in culture. Preferably, the target cells are human. In some embodiments, in the context of cell therapy, the target cells are autologous cells. In some embodiments, in the context of cell therapy, the target cells are allogeneic cells.

[0248] If the cell is a primary cell, such cells can be harvested from an individual by any suitable method. For example, leukocytes can be suitably harvested by blood component separation, leukocyte removal, density gradient separation, etc., and cells from tissues such as skin, muscle, bone marrow, spleen, liver, pancreas, lung, intestine, stomach are most suitable for harvesting by biopsy. Suitable solutions can be used to disperse or suspend the harvested cells. This solution is typically a balanced salt solution, for example, normal saline, phosphate buffered saline (PBS), Hank's balanced salt solution, etc., suitably supplemented with fetal bovine serum or other naturally occurring factors, in combination with an acceptable buffer of low concentration (for example, 5-25mM). Suitable buffer includes HEPES, phosphate buffer, lactate buffer, etc. The cells can be used immediately, or they can be stored for a long time, frozen, thawed, and can be reused. In this case, cells are usually frozen in 10% dimethyl sulfoxide (DMSO), 50% serum, 40% buffered culture medium, or some other such solutions as are commonly used in this area to preserve cells at this freezing temperature, and thawed in a manner commonly known in the art for thawing frozen culture cells.

[0249] Methods for introducing heterologous nucleic acids into target cells, eg, engineering target cells to express a fusion protein of the disclosure and optionally a transgene, are disclosed in Sections 6.8 and 6.9.

[0250] In certain embodiments, target cells are engineered to incorporate a single copy of the coding sequence of a fusion protein encoding an essential polypeptide and a degron. In other embodiments, target cells are engineered to incorporate two copies of the coding sequence of a fusion protein encoding an essential polypeptide and a degron. In certain embodiments, each allele of the essential gene locus is modified to express a fusion protein comprising an essential polypeptide and a degron. In other embodiments, target cells are engineered to express multiple copies of the coding sequence of an encoding fusion protein, the fusion protein comprising an essential polypeptide and a degron, wherein two or more copies are integrated at each allele of the corresponding essential gene locus. As described in sections 6.2, in each of the aforementioned embodiments, the fusion protein can include one, two or more series-linked degrons. In certain embodiments, the degron is separated by a joint.

[0251] In certain embodiments, by the targeting construct being integrated into the single allele of essential gene, realize single copy.The degron coding sequence can be positioned at the 5' or 3' place of essential polypeptide coding sequence, makes fusion rotein can comprise degron at its N-terminal or C-terminal.Essential polypeptide and degron can be separated by joint.Target cell can further be engineered to express transgenic.Transgenic can be from the identical allele of the essential gene that has been modified to encode fusion rotein, from opposite allele, from another genomic locus completely or from extrachromosomal expression vector expression.In certain embodiments, degron coding sequence and transgenic both are introduced in the identical allele or the different allele of identical essential STEL gene, for example, introduce in the GAPDH locus.

[0252] In certain embodiments, two copies are realized by integrating the targeting construct into two alleles of the essential gene. The degron coding sequence can be located at the 5' or 3' position of the essential polypeptide coding sequence so that the fusion protein can comprise the degron at its N-terminal or C-terminal end. The essential polypeptide and the degron can be separated by a joint. The target cell can be further engineered to express transgenic. Transgenic can be expressed from the identical allele of the essential gene modified to encode the fusion protein, from another genomic locus or from an extrachromosomal expression vector. In certain embodiments, the degron coding sequence and transgenic are both introduced into the identical allele of the essential STEL gene, for example, by introducing the GAPDH locus.

[0253] In some embodiments, the target cells of the present disclosure comprise a single allele of an essential gene, a targeting construct comprising a degron (e.g., according to Figure 2A 、 2B , 2C or 2D) is integrated into the single allele. Optionally, the target cell comprises a transgene integrated into one allele or two alleles of different gene loci.

[0254] In some embodiments, the target cells of the present disclosure comprise two alleles of an essential gene, a targeting construct comprising a degron (e.g., according to Figure 2A 、 2B , 2C or 2D) is integrated into both alleles. Optionally, the target cell comprises a transgene integrated into one allele or both alleles of different gene loci.

[0255] In some embodiments, the target cells of the present disclosure comprise Figure 3A In some embodiments, the target cells of the present disclosure comprise two essential gene loci configured as shown. Figure 3BIn some embodiments, the target cells of the present disclosure comprise two essential gene loci configured as shown. Figure 3D In some embodiments, the target cells of the present disclosure comprise two essential gene loci configured as shown. Figure 3E Two essential gene loci configured as shown. In each of the foregoing embodiments, the target cell optionally comprises a transgene integrated into one allele or both alleles of different gene loci.

[0256] In some embodiments, the target cells of the present disclosure comprise one allele of an essential gene, a targeting construct comprising a degron and a transgene (e.g., according to Figure 4A 、 4B , 4C, or 4D) is integrated into the allele. In some embodiments, the target cells of the present disclosure comprise two alleles of an essential gene, a targeting construct comprising a degron and a transgene (e.g., according to Figure 4A 、 4B , 4C, or 4D) into both alleles.

[0257] In some embodiments, the target cells of the present disclosure comprise one allele of an essential gene, a targeting construct comprising a degron and a transgene (e.g., according to Figure 7A 、 7B , 7C, 7D, 7E, 7F, 7G, 7H, 7I, 7J, 7K, or 7L) into the allele. In some embodiments, the target cells of the present disclosure comprise two alleles of an essential gene, a targeting construct comprising a degron and a transgene (e.g., according to Figure 7A 、 7B , 7C, 7D, 7E, 7F, 7G, 7H, 7I, 7J, 7K or 7L) is integrated into both alleles.

[0258] In some embodiments, the target cells of the present disclosure comprise Figure 5A In some embodiments, the target cells of the present disclosure comprise two essential gene loci configured as shown. Figure 5B In some embodiments, the target cells of the present disclosure comprise two essential gene loci configured as shown. Figure 5C In some embodiments, the target cells of the present disclosure comprise two essential gene loci configured as shown. Figure 5D In some embodiments, the target cells of the present disclosure comprise two essential gene loci configured as shown. Figure 6A In some embodiments, the target cells of the present disclosure comprise two essential gene loci configured as shown. Figure 6BIn some embodiments, the target cells of the present disclosure comprise two essential gene loci configured as shown. Figure 6C In some embodiments, the target cells of the present disclosure comprise two essential gene loci configured as shown. Figure 6D Two essential gene loci in the configuration shown.

[0259] 6.7.1. Stem cells

[0260] In some embodiments, the target cells engineered to express the fusion proteins of the present disclosure and, optionally, a transgene, are stem cells, particularly pluripotent stem cells (PSCs), such as induced pluripotent stem cells (iPSCs) or human embryonic stem cells (hESCs), which are a starting point for potentially generating large quantities of specific cell types that can be delivered for regenerative medicine in patients with many different diseases.

[0261] Suitable methods for introducing heterologous nucleic acid into stem cells, eg, engineering stem cells to express a fusion protein of the disclosure and optionally a transgene, are disclosed in Sections 6.8 and 6.9.

[0262] After engineering PSCs to express the fusion proteins of the present disclosure and, optionally, a transgene, the PSCs can be differentiated into the cell type of interest for cell therapy.

[0263] Recombinant PSCs can be differentiated into cells suitable for therapy, including cells in the endoderm (e.g., lung, thyroid, or pancreatic cells, or their progenitors), ectoderm (e.g., skin, neuronal, or pigment cells, or their progenitors), and mesodermal (e.g., cardiac cells, skeletal muscle cells, erythrocytes, smooth muscle cells, or their progenitors or precursors) lineages.

[0264] In some embodiments, the recombinant PSCs are differentiated into cells of the endoderm (e.g., lung, thyroid, or pancreatic cells, or progenitors or precursors thereof), ectoderm (e.g., skin, neuron, or pigment cells, or progenitors or precursors thereof), or mesodermal (e.g., cardiac cells, skeletal muscle cells, red blood cells, smooth muscle cells, or progenitors or precursors thereof) lineages.

[0265] In some embodiments, the recombinant PSCs of the present disclosure are differentiated into cardiac cells. In various embodiments, the cardiac cells are cardiac progenitor cells or mature or immature (atrial or ventricular) cardiomyocytes. In other embodiments, the cardiac cells are cardiac endothelial cells or ganglion cells.

[0266] In some embodiments, the recombinant PSCs of the present disclosure are differentiated into adult immune cells, optionally selected from T cells, T cells expressing chimeric antigen receptors (CARs) or recombinant TCRs, regulatory T cells, myeloid cells, dendritic cells and / or macrophages (e.g., immunosuppressive macrophages) or their progenitors or precursors. In some embodiments, the recombinant PSCs of the present disclosure are differentiated into myeloid progenitor cells, for example, as described in WO 2023 / 150089 A1, the contents of which are incorporated herein by reference in their entirety.

[0267] In some embodiments, the recombinant PSCs of the present disclosure are differentiated into oligodendrocyte progenitor or precursor cells, or oligodendrocytes.

[0268] In some embodiments, the recombinant PSCs of the present disclosure are differentiated into cells of a neural lineage, e.g., neural crest cells, astrocytes, dopaminergic neuron progenitor cells, dopaminergic neuron cells, midbrain dopaminergic neuron progenitor cells, midbrain dopaminergic neurons, bona fide midbrain dopamine (DA) neurons, dopaminergic neuron precursor cells, floor plate midbrain progenitor cells, floor plate midbrain DA neurons, or progenitors or precursors thereof.

[0269] In some embodiments, recombinant PSCs of the present disclosure are differentiated into cells of the ocular system, such as photoreceptor cells, photoreceptor progenitor cells or precursor cells, retinal pigment epithelial cells or their progenitor cells or precursors, neural retinal cells or their progenitor cells or precursors. In other embodiments, unedited PSCs are differentiated into cells of the ocular system and then recombined with the targeting constructs of the present disclosure.

[0270] In further embodiments, the recombinant PSCs of the present disclosure are differentiated into microglia or microglial progenitor or precursor cells.

[0271] In further embodiments, the recombinant PSCs of the present disclosure are differentiated into cells in the human metabolic system, optionally selected from hepatocytes, bile duct cells, and pancreatic beta cells, or progenitors or precursors thereof.

[0272] In further embodiments, the recombinant PSCs of the present disclosure are differentiated into intestinal progenitor or precursor cells or enterocytes.

[0273] 6.7.2. Differentiated cells

[0274] In various embodiments, cells at any stage of differentiation are engineered to express a fusion protein of the disclosure and, optionally, a transgene.

[0275] Suitable methods for introducing heterologous nucleic acid into differentiated cells, eg, engineering differentiated cells to express a fusion protein of the disclosure and, optionally, a transgene, are disclosed in Sections 6.8 and 6.9.

[0276] Exemplary differentiated cell types that can be engineered to express the fusion proteins of the present disclosure include cells in the endoderm (e.g., lung, thyroid, or pancreatic cells, or progenitors thereof), ectoderm (e.g., skin, neurons, or pigment cells, or progenitors or precursors thereof), and mesoderm (e.g., cardiac cells, skeletal muscle cells, erythrocytes, smooth muscle cells, or progenitors or precursors thereof). Alternatively, PSCs can be differentiated into cells in these lineages and then recombined with the targeting constructs of the present disclosure.

[0277] In some embodiments, the cardiac cells are engineered to express the fusion proteins of the present disclosure. In some embodiments, the cardiac cells are cardiac progenitor cells or mature or immature (atrial or ventricular) cardiomyocytes. In other embodiments, the cardiac cells are cardiac endothelial cells or ganglion cells.

[0278] In some embodiments, human immune cells are engineered to express the fusion protein of the present disclosure. Human immune cells are optionally selected from T cells, T cells expressing chimeric antigen receptors (CAR) or recombinant TCR, regulatory T cells, bone marrow cells, dendritic cells and / or macrophages (e.g., immunosuppressive macrophages) or their progenitor cells or precursors. In some embodiments, bone marrow progenitor cells are engineered to express the fusion protein of the present disclosure after differentiation from PSC, for example, as described in WO 2023 / 150089A1, the contents of which are incorporated herein by reference in their entirety.

[0279] In some embodiments, oligodendrocyte progenitor or precursor cells or oligodendrocytes are engineered to express a fusion protein of the disclosure.

[0280] In some embodiments, the neural lineage cell is engineered to express a fusion protein of the present disclosure. In various embodiments, the neural lineage cell is a neural crest cell, an astrocyte, a dopaminergic neuron progenitor cell, a dopaminergic neuron cell, a midbrain dopaminergic neuron progenitor cell, a midbrain dopaminergic neuron, a bona fide midbrain dopamine (DA) neuron, a dopaminergic neuron precursor cell, a floor plate midbrain progenitor cell, a floor plate midbrain DA neuron, or a progenitor or precursor thereof.

[0281] In some embodiments, cells of the ocular system are engineered to express a fusion protein of the present disclosure. In various embodiments, the cells of the ocular system are photoreceptor cells, photoreceptor progenitor cells or precursor cells, retinal pigment epithelial cells or progenitor cells or precursors thereof, neural retinal cells or progenitor cells or precursors thereof.

[0282] In further embodiments, microglial cells or microglial progenitor or precursor cells are engineered to express a fusion protein of the disclosure.

[0283] In further embodiments, cells in the human metabolic system are engineered to express the fusion proteins of the present disclosure.In various embodiments, cells in the human metabolic system are optionally selected from hepatocytes, bile duct cells, and pancreatic beta cells, or progenitors or precursors thereof.

[0284] In further embodiments, intestinal progenitor or precursor cells or intestinal cells are engineered to express a fusion protein of the disclosure.

[0285] Any of the aforementioned differentiated cell types can be differentiated from PSCs before engineering them to express the fusion proteins of the disclosure.

[0286] 6.8. Methods for delivering exogenous nucleic acids to target cells

[0287] The targeting constructs and expression vectors of the present disclosure are delivered to target cells to produce recombinant target cells comprising a nucleic acid encoding a fusion protein comprising an essential polypeptide, a degron, and an optional linker. The nucleic acid can be integrated into the target cell genome, such as when a targeting construct is used, or maintained extrachromosomally, such as when an extrachromosomal expression vector is used.

[0288] Exemplary methods for introducing targeting constructs and expression vectors are described below.

[0289] Techniques that can be used to introduce nucleic acids (such as targeting constructs or expression vectors disclosed herein) into target cells are known in the art. For example, electroporation can be used to permeabilize mammalian cells (e.g., human target cells) by applying an electrostatic potential to the target cells. Mammalian cells (such as human cells) subjected to an external electric field in this way subsequently tend to take up exogenous nucleic acids. Electroporation of mammalian cells is described in detail in, for example, Chu et al., 1987, Nucleic Acids Research 15:131. A similar technique, Nucleofection, is also known in the art. TM Nucleofection utilizes an applied electric field to stimulate the uptake of exogenous nucleic acids into the nucleus of eukaryotic cells. TM The techniques and protocols that can be used to perform this technique are described in detail in, for example, Distler et al., 2005, Experimental Dermatology 14:315 and US 2010 / 03171 14.

[0290] Other techniques for transfection of target cells include the squeeze-perforation method. This technique induces rapid mechanical deformation of the cell to stimulate the uptake of exogenous DNA through membrane pores formed in response to applied stress. The advantage of this technique is that it does not require a vector to deliver nucleic acids to cells such as human target cells. Squeeze-perforation is described in detail, for example, in Sharei et al., 2013, Journal of Visualized Experiments 81:e50980.

[0291] Lipofection represents another technique that can be used to transfect target cells. The method involves loading nucleic acids into liposomes, which typically present cationic functional groups, such as quaternary amines or protonated amines, to the outside of the liposomes. Due to the anionic nature of the cell membrane, this promotes electrostatic interactions between the liposomes and the cells, which ultimately leads to the uptake of exogenous nucleic acids, for example, by direct fusion of the liposomes with the cell membrane or by endocytosis of the complex. Lipofection is described in detail in, for example, U.S. Patent No. 7,442,386. Similar techniques that utilize ionic interactions with the cell membrane to stimulate the uptake of exogenous nucleic acids include contacting the cells with cationic polymer-nucleic acid complexes. Exemplary cationic molecules that associate with nucleic acids to impart a positive charge that facilitates interaction with cell membranes are activated dendrimers (described, for example, in Dennig, 2003, Topics in Current Chemistry 228:227) and diethylaminoethyl (DEAE)-dextran, the use of which as transfection agents is described in detail, for example, in Gulick et al., 1997, Current Protocols in Molecular Biology 40:1:9.2:9.2.1. Magnetic beads are another tool that can be used to transfect target cells in a gentle and efficient manner, as this approach utilizes an applied magnetic field to direct the uptake of nucleic acids. This technology is described in detail, for example, in US 2010 / 0227406.

[0292] Another useful tool for inducing target cells to take up exogenous nucleic acids is laser transfection, a technique that involves exposing cells to electromagnetic radiation of a specific wavelength to gently permeabilize the cells and allow nucleic acids to penetrate the cell membrane. This technique is described in detail, for example, in Rhodes et al., 2007, Methods in Cell Biology 82:309.

[0293] Microvesicle represents another potential medium that can be used to introduce nucleic acid (such as targeting construct disclosed herein) into the genome of target cell.For example, the microvesicle induced by co-overexpression of glycoprotein VSV-G and, for example, genome modification protein (such as nuclease) can be used for effectively delivering protein into cell, which then catalyzes the site-specific cutting of endogenous nucleic acid sequence, so as to prepare the genome of cell, for covalent incorporation of target nucleic acid, such as gene or regulatory sequence. Such vesicle (also referred to as Gesicle) is described in detail in, for example, Quinn et al., 2015, Genetic Modification of Target Cells by Direct Delivery of ActiveProtein (summary) for the genetic modification of eukaryotic cells. See: Methylation changes in early embryonic genes in cancer (summary), see: Proceedings of the 18th Annual Meeting of the American Society of Gene and Cell Therapy, Abstract Number 122.

[0294] In addition to the above, a variety of tools that can be used for incorporating target genes into target cells (such as human cells) have also been developed. A type of such method that can be used for incorporating transgenic or polynucleotide encoding fusion proteins into target cells involves the use of transposons. Transposons are polynucleotides encoding transposases, and comprise polynucleotide sequences or target genes flanked by 5' and 3' excision sites. Once transposons are delivered into cells, the expression of the transposase gene begins and produces an active enzyme that cuts the target gene from the transposon. This activity is mediated by the site-specific recognition of the transposon excision site by transposase. In some cases, these excision sites can be terminal repeats or inverted terminal repeats. Once excised from the transposon, the target gene can be integrated into the genome of the mammalian cell by the similar excision site present in the nuclear genome by transposase catalysis. This enables the target gene to be inserted into the nuclear DNA of cutting at the complementary excision site, and subsequently the covalent connection of the phosphodiester bond connected to the DNA of the mammalian cell genome completes the incorporation process. In some cases, the transposon can be a retrotransposon, so that the gene encoding the essential gene is first transcribed into an RNA product and then reverse transcribed into DNA before being incorporated into the mammalian cell genome. Exemplary transposon systems are the piggybac transposon (described in detail in, for example, WO 2010 / 085699) and the sleeping beauty transposon (described in detail in, for example, US 2005 / 01 12764).

[0295] Other tools for integrating exogenous nucleic acids into the genome of target cells are based on gene editing (or genome editing) of nucleases, such as CRISPR / Cas systems, zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Exemplary CRISPR / Cas gene editing methods are disclosed in Section 6.9. The use of ZFN and TALEN in genome editing applications is described in, for example, Urnov et al., 2010, Nature Reviews Genetics 11: 636 and Joung et al., 2013, Nature Reviews Molecular Cell Biology 14: 49.

[0296] Other genome editing technologies that can be used to incorporate nucleic acids containing transgenes or encoding fusion proteins into the genome of target cells include the use of ARCUS, which can be rationally designed to site-specifically cleave genomic DNA. TM Meganucleases. Given that clear structure-activity relationships have been established for this class of enzymes, it is advantageous to use these enzymes to incorporate transgenic or fusion protein-encoding nucleic acids into the genome of mammalian cells. Single-chain meganucleases can be modified at certain amino acid positions to produce nucleases that selectively cleave DNA at desired locations, enabling site-specific incorporation of essential genes into the nuclear DNA of target cells. These single-chain nucleases have been extensively described, for example, in U.S. Patent Nos. 8,021,867 and 8,445,251.

[0297] 6.9. Exemplary Gene Editing Methods

[0298] 6.9.1. Endonuclease system

[0299] The targeting constructs of the present disclosure can be incorporated into target cells via an endonuclease system.

[0300] An endonuclease system may include:

[0301] (i) a targeting construct as described in Section 6.3;

[0302] (ii) an endonuclease or a nucleic acid encoding the endonuclease as described in Section 6.9.2;

[0303] (iii) A guide RNA or a nucleic acid encoding the guide RNA as described in Section 6.9.3.

[0304] In some embodiments, an endonuclease system comprises:

[0305] (i) a targeting construct as described in Section 6.3;

[0306] (ii) an endonuclease as described in Section 6.9.2;

[0307] (iii) a guide RNA as described in Section 6.9.3.

[0308] The endonuclease system can be delivered to target cells in the form of a ribonucleoprotein complex, as described in Section 0.

[0309] 6.9.2. Endonucleases

[0310] The targeting constructs of the present disclosure can be incorporated into a specific target genomic locus by promoting homologous recombination at DNA breaks generated by appropriate endonucleases.

[0311] In some embodiments, the endonuclease is a CRISPR-associated endonuclease, such as a Cas endonuclease selected from, but not limited to, a type II, type IV, or type V Cas protein.

[0312] In some embodiments, the endonuclease is a Cas protein, including but not limited to Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas12a (e.g., Cpf1), or Cas12b, a homolog thereof, or a modified form thereof, such as a truncated form or variant of a wild-type Cas protein having nuclease activity.

[0313] In some embodiments, the Cas endonuclease is a Cpf1 (Cas12a) endonuclease or a variant, derivative or fragment thereof, such as, for example, Cpf1 derived from Francisella neocidae U112 (FnCpf1), Acidaminococcus sp. BV3L6 (AsCpf1, including improved variants such as enAsCpf1), Lachnospiraceae bacterium ND2006 (LbCpf1), Lachnospiraceae bacterium MA2020 (Lb2Cpf1), Lachnospiraceae bacterium MC2017 (Lb3Cpf1), Moraxella sp. 237 (MbCpf1), or Prevotella peptoneolyticus (PdCpf1).

[0314] In some embodiments, the Cas endonuclease is a Cas9 protein or a variant, derivative, or fragment thereof. In some embodiments, the Cas9 protein is SaCas9, SpCas9, SpCas9n, Cas9-HF, Cas9-H840A, FokI-dCas9, or D10A nickase.

[0315] In some embodiments, the Cas endonuclease is a V-type RNA programmable nuclease, as disclosed in WO 2022 / 258753A1, the contents of which are incorporated herein by reference in their entirety.

[0316] In some embodiments, the Cas endonuclease is a MAD nuclease, such as a MAD7 nuclease, as disclosed in U.S. Patent 10,337,028, the contents of which are incorporated herein by reference in their entirety.

[0317] In some aspects, the targeting construct can be incorporated into the target genomic locus using a non-CRISPR endonuclease, including but not limited to a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease (ZFN) homing endonuclease, a sequence-specific endonuclease, or a meganuclease.

[0318] Non-limiting examples of suitable endonucleases are listed in Table 3.

[0319]

[0320]

[0321]

[0322]

[0323] 6.9.3.gRNA

[0324] In some embodiments, the systems, compositions, and methods described herein employ genome-targeting nucleic acids, such as RNA molecules, that can direct the activity of a Cas polypeptide to a specific target sequence within a target nucleic acid. Such RNA molecules are referred to herein as "guide RNA" or "gRNA."

[0325] The guide RNA has at least a spacer sequence and a CRISPR repeat sequence (this CRISPR repeat sequence is also referred to as a "tracr pairing sequence") that can hybridize with the target nucleic acid sequence of interest. In the type II system, the gRNA also has a second RNA called a tracrRNA sequence. In type II guide RNA (gRNA), the CRISPR repeat sequence and the tracrRNA sequence hybridize with each other to form a duplex. In type V guide RNA (gRNA), crRNA forms a duplex. In both systems, the duplex binds to the site-specific polypeptide so that the guide RNA and the site-directed polypeptide form a complex. The genome-targeting nucleic acid provides target specificity to the complex by virtue of its binding to the site-specific polypeptide. Therefore, the genome-targeting nucleic acid guides the activity of the site-specific polypeptide.

[0326] In some embodiments, the genome-targeting nucleic acid is a bimolecular guide RNA having two RNA strands. The first strand has an optional spacer extension sequence, a spacer sequence, and a minimal CRISPR repeat sequence in the 5' to 3' direction. The second strand has a minimal tracrRNA sequence (complementary to the minimal CRISPR repeat sequence), a 3' tracrRNA sequence, and an optional tracrRNA extension sequence.

[0327] In some embodiments, the guide RNA is a single guide RNA (sgRNA). The single molecule guide RNA (sgRNA) in the II type system has an optional spacer extension sequence, a spacer sequence, a minimal CRISPR repeat sequence, a single molecule guide linker, a minimal tracrRNA sequence, a 3'tracrRNA sequence, and an optional tracrRNA extension sequence in the 5' to 3' direction. The optional tracrRNA extension may have an element that provides additional functions (e.g., stability) for the guide RNA. The single molecule guide linker connects the minimal CRISPR repeat sequence and the minimal tracrRNA sequence to form a hairpin structure. The optional tracrRNA extension has one or more hairpins. The single molecule guide RNA (sgRNA) in the V type system has a minimal CRISPR repeat sequence and a spacer sequence in the 5' to 3' direction. Alternatively, the single molecule guide RNA (sgRNA) in the V type system has an optional tracr extension sequence, a tracrRNA sequence, a single molecule guide linker, a minimal CRISPR repeat sequence, a spacer sequence, and an optional spacer extension sequence in the 5' to 3' direction.

[0328] The modification of guide RNA can be used to enhance the formation or stability of the CRISPR-Cas genome editing complex comprising guide RNA and Cas endonuclease. The modification of guide RNA can also or alternatively be used to enhance the initiation, stability or kinetics of interaction between the target sequence in genome editing complex and genome, which can be used for example to enhance targeting activity. The modification of guide RNA can also or alternatively be used to enhance specificity, for example, compared with the effect at other (off-target) sites, the relative rate of genome editing at target site. Modification can also or alternatively be used to increase the stability of guide RNA, for example, by increasing its resistance to degradation of ribonuclease (RNA enzyme) present in the cell, thereby causing its half-life in the cell to increase.

[0329] Table 4 lists exemplary Cpf1 guide RNA sequences targeting the essential gene GAPDH.

[0330]

[0331]

[0332] Table 5 lists exemplary Cas9 guide RNA sequences targeting the essential STEL gene GAPDH.

[0333]

[0334] Table 6 lists exemplary Cas9 guide RNA sequences targeting the essential STEL gene RPL13A.

[0335]

[0336] Table 7 lists exemplary Cas9 guide RNA sequences targeting the essential STEL gene RPLP0.

[0337]

[0338] Table 8 lists exemplary Cpf1 guide RNA sequences targeting the essential STEL gene RPLP0.

[0339]

[0340]

[0341] Table 9 lists exemplary Cas9 guide RNA sequences targeting the essential STEL gene RPL7.

[0342]

[0343] 6.9.4. Ribonucleoprotein (RNP) Complex

[0344] In some embodiments, the endonuclease is delivered to the target cell in a composition called a ribonucleoprotein or RNP complex. The RNP complex is assembled by binding the endonuclease to the ribonucleic acid.

[0345] In some embodiments, the ribonucleoprotein complex comprises a Cas endonuclease complexed with a suitable ribonucleic acid. In some embodiments, the ribonucleic acid is a gRNA or sgRNA, which is further described in Section 6.9.3.

[0346] One of the most common techniques for delivering RNPs is electroporation, which creates holes in the cell membrane, allowing RNPs to enter the cytoplasm. In addition, electroporation can be combined with cell type-specific reagents in a technique called nucleofection, which creates holes in the nuclear membrane, allowing DNA templates to enter. In some embodiments, the RNP complex is delivered to the target cell via nucleofection.

[0347] 6.9.5. Gene Editing Methods

[0348] In certain embodiments, the method of the present disclosure includes introducing a targeting construct into a target cell (or a target cell population). The targeting construct of the present disclosure can be incorporated into the target cell by an endonuclease system, wherein the endonuclease system can be introduced into the host or target cell by any of a variety of well-known methods and any known methods. For example, the endonuclease system of the present disclosure can be delivered to the target cell via one or more vectors encoding the endonuclease system or in the form of a ribonucleoprotein complex.

[0349] Suitable methods include, for example, viral or phage infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, nucleofection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran-mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle-mediated nucleic acid delivery (see, for example, Panyam et al., Adv Drug Deliv Rev. 2012 Sep 13. pii: 50169-409X(12)00283-9. doi: 10.1016 / j.addr.2012.09.023), etc., including but not limited to exosome delivery. Nucleic acids can also be delivered by non-viral delivery vehicles, including but not limited to nanoparticles, liposomes, ribonucleoproteins, positively charged peptides, small molecule RNA conjugates, aptamer-RNA chimeras, and RNA fusion protein complexes. Some exemplary non-viral delivery vehicles are described in Peer and Lieberman, 2011, Gene Therapy, 18: 1127-1133.

[0350] In some embodiments, the endonuclease system comprises a ribonucleoprotein complex (e.g., a Cas endonuclease and sgRNA), e.g., as described in Section 6.9.4, and can be delivered to the target cell by nucleofection, electroporation, or similar methods.

[0351] As an alternative to RNP delivery, the endonuclease system can be delivered to target cells in the form of a nucleic acid via a delivery vector, such as a viral delivery vector.

[0352] Suitable nucleic acids comprising nucleotide sequences encoding Cas endonucleases and / or guide RNAs include expression vectors. In some embodiments, the expression vector is a viral construct, such as a recombinant adeno-associated virus construct (see, e.g., U.S. Patent No. 7,078,387), a recombinant adenoviral construct, a recombinant lentiviral construct, a recombinant retroviral construct, or the like. Suitable expression vectors include, but are not limited to, viral vectors (e.g., vaccinia virus-based viral vectors; poliovirus; adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO 94 / 12649, WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984 and WO 95 / 00655); adeno-associated virus (see, e.g., Ali et al., Hum Gene Ther 9:81 86, 1998, Flannery et al., PNAS 94:6916 6921,1997; Bennett et al., Invest Opthalmol Vis Sci 38:2857 2863,1997; Jomary et al., Gene Ther 4:683-690,1997, Rolling et al., Hum Gene Ther 10:641 648,1999; Ali et al., Hum Mol Genet 5:591 594,1996; Srivastava et al., WO 93 / 09239, Samulski et al., J. Vir. (1989) 63:3822-3828; Mendelson et al., Viral. (1988) 166:154-165; and Flotte et al., PNAS (1993) 90:10613-10617); SV40; herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshi et al., PNAS 94:10319-23, 1997; Takahashi et al., J Virol 73:7812-7816, 1999); retroviral vectors (e.g., murine leukemia virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and the like.

[0353] In addition to encoding the endonuclease and guide RNA, the nucleic acid vector may further comprise a targeting construct of the present disclosure. Alternatively, the targeting construct may be introduced into the target cell on a separate nucleic acid molecule.

[0354] The target cell is then grown under conditions where gene editing is performed. Without being bound by theory, it is believed that the endonuclease, under the guidance of the guide RNA, cleaves the target cell genome, allowing the first homology arm and the second homology arm of the targeting construct to recombine with the target cell genome, which results in the nucleotide sequence flanked by the homology arms of the targeting construct being integrated into the genome of the target cell.

[0355] In some embodiments, the target cell (e.g., a cell comprising a target DNA locus targeted by a targeting construct) is in vitro. In some embodiments, the target cell is in vivo.

[0356] 6.10. Pharmaceutical Compositions

[0357] Also disclosed herein are pharmaceutical formulations and medicaments comprising recombinant cells engineered to express the fusion proteins of the disclosure and, optionally, a transgene, and a pharmaceutically acceptable excipient.

[0358] Suitable excipients include, but are not limited to, salts, diluents (e.g., Tris-HCl, acetates, phosphates), preservatives (e.g., thimerosal, benzyl alcohol, parabens), binders, fillers, solubilizers, disintegrants, adsorbents, solvents, pH regulators, antioxidants, anti-infectives, suspending agents, wetting agents, viscosity modifiers, tonicity agents, stabilizers and other components and combinations thereof. Suitable pharmaceutically acceptable excipients can be selected from materials that are generally recognized as safe (GRAS) and can be administered to an individual without causing adverse biological side effects or unwanted interactions. Suitable excipients and their formulations are described in Remington's Pharmaceutical Sciences, 16th edition, 1980, Mack Publishing Co. Additionally, such compositions can be complexed with polyethylene glycol (PEG), metal ions, or incorporated into polymeric compounds such as polyacetic acid, polyglycolic acid, hydrogels, or incorporated into liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroid cells. Suitable dosage forms for administration (eg, parenteral administration) include solutions, suspensions, and emulsions.

[0359] The components of the pharmaceutical preparation can be dissolved or suspended in a suitable solvent, such as, for example, water, Ringer's solution, phosphate buffered saline (PBS) or isotonic sodium chloride. The preparation can also be a sterile solution, suspension or emulsion in a non-toxic, parenterally acceptable diluent or solvent such as 1,3-butanediol.

[0360] In some cases, the formulation may include one or more tonicity agents to adjust the isotonic range of the formulation. Suitable tonicity agents are well known in the art and include glycerol, mannitol, sorbitol, sodium chloride and other electrolytes. In some cases, the formulation may be buffered with an effective amount of a buffer required to maintain a pH suitable for parenteral administration. Suitable buffers are well known to those skilled in the art, and some examples of useful buffers are acetate, borate, carbonate, citrate and phosphate buffers.

[0361] In some embodiments, the formulation can be distributed or packaged in liquid form, or alternatively as a solid, for example, obtained by lyophilizing a suitable liquid formulation, which can be reconstituted with a suitable carrier or diluent before administration. In some embodiments, the formulation can include a pharmaceutically effective amount of a guide RNA and a type II Cas protein sufficient to edit a gene in a cell. The pharmaceutical composition can be formulated for medical and / or veterinary use.

[0362] Treatment methods

[0363] The recombinant target cells and pharmaceutical compositions of the present disclosure can be introduced into an individual for therapeutic purposes. For example, the therapeutic cells of the present disclosure can be used to treat genetic diseases by transplanting cells expressing a functional transgene into an affected tissue or organ of a subject. The recombinant target cells can also be used to treat tissue injury, trauma, aging-related cell damage, or tissue or organ damage associated with exposure to certain environmental factors or other conditions by replacing dead, damaged, impaired, or dysfunctional cells in the affected tissue, organ, or body system.

[0364] The recombinant target cells can be autologous to the subject or allogeneic to the subject.

[0365] The recombinant target cells described herein can be provided in a pharmaceutical composition containing cells and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier can be a cell culture medium, which optionally does not contain any animal-derived components. For storage and transportation, cells can be frozen at <-70°C (e.g., on dry ice or in liquid nitrogen). Prior to use, the cells can be thawed and diluted in a sterile cell culture medium that supports the cell type of interest.

[0366] The recombinant target cells can be administered to the patient systemically (e.g., by intravenous injection or infusion) or locally (e.g., by direct injection into local tissues, such as the heart, brain, and damaged tissue). Various methods for administering cells to a patient's tissue or organ are known in the art, including but not limited to intracoronary administration, intramyocardial administration, transendocardial administration, or intracranial administration.

[0367] A therapeutically effective number of recombinant target cells is administered to a patient. As used herein, the term "therapeutically effective" refers to a number of cells or an amount of a pharmaceutical composition that, when administered to a human subject suffering from or susceptible to a disease, disorder, and / or condition, is sufficient to treat, prevent, and / or delay the onset or progression of symptoms of the disease, disorder, and / or condition. It will be understood by those of ordinary skill in the art that a therapeutically effective amount is typically administered via a dosing regimen comprising at least one unit dose. In some embodiments, at least 10 [units of] dose are administered to a subject at one time at one or more sites. 3 (For example, at least 10 4 , at least 10 5 , at least 10 6 , at least 10 7 , at least 10 8 , at least 10 9 , at least 10 10 , at least 10 11 or at least 10 12 ) cells. In some embodiments, 10 cells are administered to a subject at one time at one or more sites. 3 to 10 18 (For example, 10 3 to 10 4 , 10 3 to 10 5 , 10 3 to 10 6 , 10 3 to 10 7 , 10 3 to 10 8 , 10 3 to 10 9 , 10 3 to 10 10 , 10 3 to 10 11 , 10 3 to 10 12 , 10 6 to 10 7 , 10 6 to 10 8 , 10 6 to 10 9 , 10 6 to 10 10 , 10 6 to 10 11 , 10 6 to 10 12 , 10 9 to 10 10 , 10 9 to 10 11 , 10 9 to 10 12) cells. In some embodiments, more than 10 cells are administered to a subject at one time at one or more sites. 12 (For example, more than 10 12 , more than 10 13 , more than 10 14 , more than 10 15 , more than 10 16 , more than 10 17 , more than 10 18 or more) cells.

[0368] In some embodiments, a method of treating comprises selectively killing recombinant target cells that have been transplanted into a subject. Recombinant target cells can be selected by inducing a degron, such as by administering to the subject a drug (such as an IMiD) that activates the degron, thereby resulting in degradation of a fusion protein comprising the degron. Degradation of the fusion protein comprising the essential polypeptide leads to apoptosis and thus to selective killing of the cell.

[0369] For example, if an adverse event occurs, such as an adverse event caused by overexpression of a therapeutic polypeptide, selective killing of the target cells can be performed. If the therapeutic goal has been achieved and the recombinant target cells are no longer needed for therapy, selective killing of the recombinant target cells can also be performed. In addition, selective killing of the recombinant target cells can be used to completely eradicate the graft, such as when the graft causes serious side effects, such as cytokine storm, excessive (systemic) inflammation, tumor formation, graft-versus-host disease, organ damage, or other health problems of the subject.

[0370] Selective killing of target cells can be induced at any time after administration of the target cells to a subject, for example, from one hour to one year (or longer) after administration of the target cells.

[0371] In various embodiments, selective killing of target cells is induced in a subject one hour to one day after administration of the target cells, one day to one week after administration of the target cells, one week to two weeks after administration of the cells, two weeks to one month after administration of the target cells, one month to three months after administration of the target cells, three months to one year after administration of the target cells, or any time range bounded by any two of the foregoing embodiments (e.g., two weeks to three months after administration of the target cells).

[0372] Therefore, the present disclosure provides a method of treatment in which the subject has previously received a cell therapy of a recombinant target cell as described herein, the method comprising administering to the subject an effective amount of a degradation determinant inducer. In some embodiments, the subject has previously received a recombinant target cell engineered to express a fusion protein comprising an essential polypeptide and an IMiD inducible degradation determinant, and the method comprises administering to the subject an IMiD in an amount that effectively selectively kills the recombinant target cell. Exemplary IMiDs include but are not limited to pomalidomide, thalidomide, lenalidomide, ibedomide, and avadomide. In various embodiments, if the subject experiences a cytokine storm, excessive (systemic) inflammation, tumor formation, graft-versus-host disease, or another health problem caused by cell therapy, IMiDs are administered.

[0373] 7. Numbered Examples

[0374] Although various specific embodiments have been shown and described, it should be understood that various changes can be made without departing from the spirit and scope of the present disclosure. The present disclosure is illustrated by the numbered embodiments set forth below. Unless otherwise indicated, any concept, aspect, and / or feature of the embodiments described in the above detailed description applies mutatis mutandis to any of the following numbered embodiments.

[0375] 1. A fusion protein comprising:

[0376] (a) essential polypeptides;

[0377] (b) degron; and

[0378] (c) Optionally; a linker.

[0379] 2. The fusion protein of embodiment 1, wherein the degradation determinant is located at the N-terminus of the essential polypeptide.

[0380] 3. The fusion protein of embodiment 1, wherein the degradation determinant is located at the C-terminus of the essential polypeptide.

[0381] 4. The fusion protein of any one of embodiments 1 to 3, wherein the degron is an inducible degron.

[0382] 5. The fusion protein of embodiment 4, wherein the degron is drug-inducible, temperature-sensitive, light-inducible, or activated by a polypeptide (optionally wherein the polypeptide is TEV protease).

[0383] 6. The fusion protein of embodiment 5, wherein the degron is drug-inducible.

[0384] 7. The fusion protein of embodiment 6, wherein the drug is an immunomodulatory drug (IMiD).

[0385] 8. The fusion protein of embodiment 7, wherein the IMiD is lenalidomide, ibelidomide, thalidomide, avalidomide, or pomalidomide.

[0386] 9. The fusion protein of embodiment 8, wherein the IMiD is ibedomide.

[0387] 10. The fusion protein of embodiment 8, wherein the IMiD is avadomid.

[0388] 11. The fusion protein of embodiment 8, wherein the IMiD is thalidomide.

[0389] 12. The fusion protein of embodiment 8, wherein the IMiD is lenalidomide.

[0390] 13. The fusion protein of embodiment 8, wherein the IMiD is pomalidomide.

[0391] 14. The fusion protein of any one of embodiments 1 to 13, wherein the degron comprises or consists of the amino acid sequence: RPFQCNQCGASFTQKGNLLRHIKLH (SEQ ID NO: 3), FNVLMVHKRSHTGERPLQCEICGFTCRQKGNLLRHIKLHTGEKPFKCHLCNYACQRRDAL (SEQ ID NO: 4, corresponding to SEQ ID NO: 42 of WO 2021 / 188286 A2), FNVLMVHKRSHTGERP (SEQ ID NO: 5, corresponding to SEQ ID NO: 97 of WO 2019 / 089592 A1), FNVLMVHRRSHTGERP (SEQ ID NO: 6, corresponding to SEQ ID NO: 100 of WO 2019 / 089592 A1), TGEKPFKCHLCNYACQRRDAL (SEQ ID NO: 7, corresponding to SEQ ID NO: 103 of WO 2019 / 089592 A1), NO:102), TGERPFRCHLCNYACQRRDAL (SEQ ID NO:8, corresponding to SEQ ID NO:103 of WO2019 / 089592A1), FQCNQCGASFT (SEQ ID NO:9, corresponding to SEQ ID NO:528 of WO2021 / 188286A2), FQCPICGLVIK (SEQ ID NO:10, corresponding to SEQ ID NO:529 of WO2021 / 188286A2), LQCEICGFTCR (SEQ ID NO:11, corresponding to SEQ ID NO:530 of WO2021 / 188286A2), LQCEICGYQCR (SEQ ID NO:12, corresponding to SEQ ID NO:531 of WO2021 / 188286A2) or LQCEVCGFQCR (SEQ ID NO:13, corresponding to SEQ ID NO:532 of WO2021 / 188286A2).

[0392] 15. The fusion protein of embodiment 14, wherein the degron comprises or consists of the amino acid sequence of SEQ ID NO: 3.

[0393] 16. The fusion protein of embodiment 14, wherein the degron comprises or consists of the amino acid sequence of SEQ ID NO: 4.

[0394] 17. The fusion protein of embodiment 14, wherein the degron comprises or consists of the amino acid sequence of SEQ ID NO: 5.

[0395] 18. The fusion protein of embodiment 14, wherein the degron comprises or consists of the amino acid sequence of SEQ ID NO: 6.

[0396] 19. The fusion protein of embodiment 14, wherein the degron comprises or consists of the amino acid sequence of SEQ ID NO: 7.

[0397] 20. The fusion protein of embodiment 14, wherein the degron comprises or consists of the amino acid sequence of SEQ ID NO: 8.

[0398] 21. The fusion protein of embodiment 14, wherein the degron comprises or consists of the amino acid sequence of SEQ ID NO: 9.

[0399] 22. The fusion protein of embodiment 14, wherein the degron comprises or consists of the amino acid sequence of SEQ ID NO: 10.

[0400] 23. The fusion protein of embodiment 14, wherein the degron comprises or consists of the amino acid sequence of SEQ ID NO: 11.

[0401] 24. The fusion protein of embodiment 14, wherein the degron comprises or consists of the amino acid sequence of SEQ ID NO: 12.

[0402] 25. The fusion protein of embodiment 14, wherein the degron comprises or consists of the amino acid sequence of SEQ ID NO: 13.

[0403] 26. The fusion protein of any one of embodiments 1 to 25, wherein the degron is a super degron.

[0404] 27. The fusion protein of embodiment 6, wherein the degradation determinant is a SMASh (small molecule assisted shutoff) tag degradation determinant.

[0405] 28. The fusion protein of any one of embodiments 1 to 27, comprising a linker sequence between the essential polypeptide and the degron.

[0406] 29. The fusion protein of embodiment 27, wherein the linker is 1 to 30 amino acids in length.

[0407] 30. The fusion protein of embodiment 29, wherein the linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length.

[0408] 31. The fusion protein of embodiment 29 or embodiment 30, wherein the linker is between 1 and 12 amino acids in length, between 2 and 12 amino acids in length, or between 1 and 10 amino acids in length.

[0409] 32. The fusion protein of embodiment 29 or embodiment 30, wherein the linker is 3 to 10 amino acids in length.

[0410] 33. The fusion protein of embodiment 29 or embodiment 30, wherein the linker is 11 to 20 amino acids in length.

[0411] 34. The fusion protein of embodiment 29 or embodiment 30, wherein the linker is 21 to 30 amino acids in length.

[0412] 35. The fusion protein of any one of embodiments 1 to 34, comprising a plurality of degrons.

[0413] 36. The fusion protein of embodiment 35, wherein the fusion protein comprises two or more degradation determinants.

[0414] 37. The fusion protein of embodiment 36, wherein the degradation determinants are in tandem.

[0415] 38. The fusion protein of embodiment 37, wherein each pair of degrons is separated by a linker, optionally wherein (a) the linker is a linker described in Section 6.2.3 and / or (b) all of the linkers separating pairs of degrons are identical.

[0416] 39. The fusion protein of any one of embodiments 35 to 38, wherein the plurality of degrons are induced in the same manner, optionally wherein the plurality of degrons are identical.

[0417] 40. The fusion protein of any one of embodiments 1 to 39, wherein the essential polypeptide is:

[0418] (a) a STEL polypeptide; or

[0419] (b) Non-STEL polypeptides.

[0420] 41. The fusion protein of any one of embodiments 1 to 40, wherein the essential polypeptide participates in one or more of: glycolysis, ribonucleopolypeptide complex formation, focal adhesions, cell-matrix adherens junctions, cell-matrix junctions, cell anchorage, extracellular exosomes, extracellular vesicles, intracellular organelles, anchorage junctions, RNA binding, nucleic acid binding (e.g., rRNA or mRNA binding), and polypeptide binding.

[0421] 42. The fusion protein of any one of embodiments 1 to 41, wherein the essential polypeptide (a) is a ribosomal polypeptide or (b) is not a ribosomal polypeptide.

[0422] 43. The fusion protein of embodiment 42, wherein the essential polypeptide is a ribosomal polypeptide, optionally wherein the ribosomal polypeptide is RPL13A, RPLP0, 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.

[0423] 44. The fusion protein of any one of embodiments 1 to 41, wherein the essential polypeptide (a) is a ribosomal polypeptide small subunit (RPS) or (b) is not a ribosomal polypeptide small subunit (RPS).

[0424] 45. The fusion protein of embodiment 44, wherein the essential polypeptide is a ribosomal polypeptide small subunit (RPS), optionally wherein the ribosomal polypeptide small subunit (RPS) is RPS2, RPS19, RPS14, RPS3A, RPS12, RPS3, RPS6, RPS23, RPS27A, RPS8, RPS4X, RPS7, RPS24, RPS27, RPS15A, RPS9, RPS28, RPS13, RPSA, RPS5, RPS16, RPS25, RPS15, RPS20, or RPS11.

[0425] 46. ​​The fusion protein of any one of embodiments 1 to 41, wherein the essential polypeptide (a) is an actin polypeptide or (b) is not an actin polypeptide.

[0426] 47. The fusion protein of embodiment 46, wherein the essential polypeptide is an actin polypeptide, optionally wherein the actin polypeptide is ACTG1 or ACTB.

[0427] 48. The fusion protein of any one of embodiments 1 to 41, wherein the essential polypeptide (a) is a eukaryotic translation factor or (b) is not a eukaryotic translation factor.

[0428] 49. The fusion protein of embodiment 48, wherein the essential polypeptide is a eukaryotic translation factor, optionally wherein the eukaryotic translation factor is EEF1A1, EEF2, or EIF1.

[0429] 50. The fusion protein of any one of embodiments 1 to 41, wherein the essential polypeptide (a) is a histone or (b) is not a histone.

[0430] 51. The fusion protein of embodiment 50, wherein the essential polypeptide is a histone, optionally wherein the histone is H3F3A or H3F3B.

[0431] 52. The fusion protein of any one of embodiments 1 to 41, wherein the essential polypeptide is (a) selected from FTH1, TPT1, GAPDH, PTMA, GNB2L1, NACA, YBX1, NPM1, FAU, UBA52, HSP90AB1, MYL6, SERF2, SRP14, RPL13A, RPL7, or RPLP0 or (b) is not FTH1, TPT1, PTMA, GNB2L1, NACA, YBX1, NPM1, FAU, UBA52, HSP90AB1, MYL6, SERF2, or SRP14.

[0432] 53. The fusion protein of embodiment 52, wherein the essential polypeptide is GAPDH.

[0433] 54. The fusion protein of embodiment 52, wherein the essential polypeptide is RPL13A.

[0434] 55. The fusion protein of embodiment 52, wherein the essential polypeptide is RPL7.

[0435] 56. The fusion protein of embodiment 52, wherein the essential polypeptide is RPLP0.

[0436] 57. A targeting construct comprising:

[0437] (a) a first homology arm corresponding to a 5' target sequence comprising a first region homologous to an essential gene encoding an essential polypeptide in a target genomic locus;

[0438] (b) a nucleotide sequence encoding a degron ("degron encoding sequence");

[0439] (c) a second homology arm corresponding to a 3' target sequence comprising a second region homologous to the essential gene in the target genomic locus

[0440] wherein the targeting construct is configured such that upon recombination with the target genomic locus, the essential gene is modified to encode a fusion protein comprising the essential polypeptide and the degron, optionally wherein the fusion protein has one or more characteristics as defined in any one of Examples 1 to 56 or Section 6.2.

[0441] 58. The targeting construct of embodiment 57, wherein the targeting construct is configured such that upon recombination with the target genomic locus, the essential gene is modified to encode a fusion protein comprising the degradation determinant at the C-terminus of the essential polypeptide.

[0442] 59. The targeting construct of embodiment 57, wherein the targeting construct is configured such that upon recombination with the target genomic locus, the essential gene is modified to encode a fusion protein comprising the degradation determinant at the N-terminus of the essential polypeptide.

[0443] 60. The targeting construct of any one of embodiments 57 to 59, wherein the degron is an inducible degron.

[0444] 61. The targeting construct of embodiment 60, wherein the degron is drug-inducible, temperature-sensitive, light-inducible, or activated by a polypeptide (optionally wherein the polypeptide is a TEV protease).

[0445] 62. The targeting construct of embodiment 61, wherein the degradation determinant is drug-inducible.

[0446] 63. The targeting construct of embodiment 62, wherein the drug is an immunomodulatory drug (IMiD).

[0447] 64. The targeting construct of embodiment 63, wherein the IMiD is lenalidomide, ibedomide, thalidomide, avalidomide, or pomalidomide.

[0448] 65. The targeting construct of embodiment 63, wherein the IMiD is ibedomide.

[0449] 66. The targeting construct of embodiment 63, wherein the IMiD is avadomide.

[0450] 67. The targeting construct of embodiment 63, wherein the IMiD is thalidomide.

[0451] 68. The targeting construct of embodiment 63, wherein the IMiD is lenalidomide.

[0452] 69. The targeting construct of embodiment 63, wherein the IMiD is pomalidomide.

[0453] 70. The targeting construct of any one of embodiments 57 to 69, wherein the degron comprises or consists of the amino acid sequence: RPFQCNQCGASFTQKGNLLRHIKLH (SEQ ID NO: 3), FNVLMVHKRSHTGERPLQCEICGFTCRQKGNLLRHIKLHTGEKPFKCHLCNYACQRRDAL (SEQ ID NO: 4, corresponding to SEQ ID NO: 42 of WO 2021 / 188286 A2), FNVLMVHKRSHTGERP (SEQ ID NO: 5, corresponding to SEQ ID NO: 97 of WO 2019 / 089592 A1), FNVLMVHRRSHTGERP (SEQ ID NO: 6, corresponding to SEQ ID NO: 100 of WO 2019 / 089592 A1), TGEKPFKCHLCNYACQRRDAL (SEQ ID NO: 7, corresponding to SEQ ID NO: 101 of WO 2019 / 089592 A1), NO:102), TGERPFRCHLCNYACQRRDAL (SEQ ID NO:8, corresponding to SEQ ID NO:103 of WO2019 / 089592A1), FQCNQCGASFT (SEQ ID NO:9, corresponding to SEQ ID NO:528 of WO2021 / 188286A2), FQCPICGLVIK (SEQ ID NO:10, corresponding to SEQ ID NO:529 of WO2021 / 188286A2), LQCEICGFTCR (SEQ ID NO:11, corresponding to SEQ ID NO:530 of WO2021 / 188286A2), LQCEICGYQCR (SEQ ID NO:12, corresponding to SEQ ID NO:531 of WO2021 / 188286A2) or LQCEVCGFQCR (SEQ ID NO:13, corresponding to SEQ ID NO:532 of WO2021 / 188286A2).

[0454] 71. The targeting construct of embodiment 70, wherein the degradation determinant comprises or consists of the amino acid sequence of SEQ ID NO: 3.

[0455] 72. The targeting construct of embodiment 70, wherein the degradation determinant comprises or consists of the amino acid sequence of SEQ ID NO: 4.

[0456] 73. The targeting construct of embodiment 70, wherein the degradation determinant comprises or consists of the amino acid sequence of SEQ ID NO: 5.

[0457] 74. The targeting construct of embodiment 70, wherein the degradation determinant comprises or consists of the amino acid sequence of SEQ ID NO: 6.

[0458] 75. The targeting construct of embodiment 70, wherein the degradation determinant comprises or consists of the amino acid sequence of SEQ ID NO: 7.

[0459] 76. The targeting construct of embodiment 70, wherein the degradation determinant comprises or consists of the amino acid sequence of SEQ ID NO: 8.

[0460] 77. The targeting construct of embodiment 70, wherein the degradation determinant comprises or consists of the amino acid sequence of SEQ ID NO: 9.

[0461] 78. The targeting construct of embodiment 70, wherein the degradation determinant comprises or consists of the amino acid sequence of SEQ ID NO: 10.

[0462] 79. The targeting construct of embodiment 70, wherein the degradation determinant comprises or consists of the amino acid sequence of SEQ ID NO: 11.

[0463] 80. The targeting construct of embodiment 70, wherein the degron comprises or consists of the amino acid sequence of SEQ ID NO: 12.

[0464] 81. The targeting construct of embodiment 70, wherein the degradation determinant comprises or consists of the amino acid sequence of SEQ ID NO: 13.

[0465] 82. The targeting construct of any one of embodiments 57 to 81, wherein the degron is a super degron.

[0466] 83. The targeting construct of embodiment 62, wherein the degradation determinant is a SMASh (small molecule assisted shutoff) tag degradation determinant.

[0467] 84. The targeting construct of any one of embodiments 57 to 83, wherein the fusion protein comprises a linker sequence between the essential polypeptide and the degron.

[0468] 85. The targeting construct of embodiment 82, wherein the linker is 1 to 30 amino acids in length.

[0469] 86. The targeting construct of embodiment 85, wherein the linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length.

[0470] 87. The targeting construct of embodiment 85, wherein the linker is between 1 and 12 amino acids in length, between 2 and 12 amino acids in length, or between 1 and 10 amino acids in length.

[0471] 88. The targeting construct of embodiment 85, wherein the linker is 3 to 10 amino acids in length.

[0472] 89. The targeting construct of embodiment 85, wherein the linker is 11 to 20 amino acids in length.

[0473] 90. The targeting construct of embodiment 85, wherein the linker is 21 to 30 amino acids in length.

[0474] 91. The targeting construct of any one of embodiments 57 to 90, wherein the fusion protein comprises multiple degradation determinants.

[0475] 92. The targeting construct of embodiment 91, wherein the fusion protein comprises two or more degradation determinants.

[0476] 93. The targeting construct of embodiment 92, wherein the degradation determinants are in tandem.

[0477] 94. The targeting construct of embodiment 93, wherein each pair of degrons is separated by a linker, optionally wherein (a) the linker is a linker described in Section 6.2.3 and / or (b) all of the linkers separating pairs of degrons are identical.

[0478] 95. The targeting construct of any one of embodiments 91 to 94, wherein the plurality of degrons are induced in the same manner, optionally wherein the plurality of degrons are the same.

[0479] 96. The targeting construct of any one of embodiments 57 to 95, wherein the essential gene is:

[0480] (a) STEL gene; or

[0481] (b) Non-STEL genes.

[0482] 97. The targeting construct of any one of embodiments 57 to 96, wherein the essential gene encodes a polypeptide involved in one or more of: glycolysis, ribonucleopolypeptide complex formation, focal adhesions, cell-matrix adherens junctions, cell-matrix junctions, cell anchorage, extracellular exosomes, extracellular vesicles, intracellular organelles, anchorage junctions, RNA binding, nucleic acid binding (e.g., rRNA or mRNA binding), and polypeptide binding.

[0483] 98. The targeting construct of any one of embodiments 57 to 97, wherein the essential gene (a) encodes a ribosomal polypeptide or (b) does not encode a ribosomal polypeptide.

[0484] 99. The targeting construct of embodiment 98, wherein the essential gene encodes a ribosomal polypeptide, optionally wherein the ribosomal polypeptide is RPL13A, RPLP0, RPL10, RPL13, RPS18, 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.

[0485] 100. The targeting construct of any one of embodiments 57 to 98, wherein the essential gene (a) encodes a ribosomal polypeptide small subunit (RPS) or (b) does not encode a ribosomal polypeptide small subunit (RPS).

[0486] 101. The targeting construct of embodiment 100, wherein the essential gene encodes a ribosomal polypeptide small subunit (RPS), optionally wherein the ribosomal polypeptide small subunit (RPS) is RPS2, RPS19, RPS14, RPS3A, RPS12, RPS3, RPS6, RPS23, RPS27A, RPS8, RPS4X, RPS7, RPS24, RPS27, RPS15A, RPS9, RPS28, RPS13, RPSA, RPS5, RPS16, RPS25, RPS15, RPS20, or RPS11.

[0487] 102. The targeting construct of any one of embodiments 57 to 97, wherein the essential gene (a) encodes an actin polypeptide or (b) does not encode an actin polypeptide.

[0488] 103. The targeting construct of embodiment 102, wherein the essential gene encodes an actin polypeptide, optionally wherein the actin polypeptide is ACTG1 or ACTB.

[0489] 104. The targeting construct of any one of embodiments 57 to 97, wherein the essential gene (a) encodes a eukaryotic translation factor or (b) does not encode a eukaryotic translation factor.

[0490] 105. The targeting construct of embodiment 104, wherein the essential gene encodes a eukaryotic translation factor, optionally wherein the eukaryotic translation factor is EEF1A1, EEF2, or EIF1.

[0491] 106. The targeting construct of any one of embodiments 57 to 97, wherein the essential gene (a) encodes a histone or (b) does not encode a histone.

[0492] 107. The targeting construct of embodiment 106, wherein the essential gene encodes a histone, optionally wherein the histone is H3F3A or H3F3B.

[0493] 108. The targeting construct of any one of embodiments 57 to 97, wherein the essential gene is (a) selected from FTH1, TPT1, GAPDH, PTMA, GNB2L1, NACA, YBX1, NPM1, FAU, UBA52, HSP90AB1, MYL6, SERF2, SRP14, RPL13A, RPL7, or RPLP0 or (b) is not FTH1, TPT1, PTMA, GNB2L1, NACA, YBX1, NPM1, FAU, UBA52, HSP90AB1, MYL6, SERF2, or SRP14.

[0494] 109. The targeting construct of embodiment 108, wherein the essential gene is GAPDH.

[0495] 110. The targeting construct of embodiment 108, wherein the essential gene is RPL13A.

[0496] 111. The targeting construct of embodiment 108, wherein the essential gene is RPL7.

[0497] 112. The targeting construct of embodiment 108, wherein the essential gene is RPLP0.

[0498] 113. The targeting construct of any one of embodiments 57 to 112, wherein the first homology arm and the second homology arm are each 500 to 1,500 nucleotides in length.

[0499] 114. The targeting construct of embodiment 113, wherein the first homology arm and the second homology arm are each 600 to 1200 nucleotides in length or 700 to 1000 nucleotides in length.

[0500] 115. The targeting construct of any one of embodiments 57 to 114, wherein the difference in length between the first homology arm and the second homology arm, if any, is less than 75 nucleotides.

[0501] 116. The targeting construct of embodiment 115, wherein the difference in length between the first homology arm and the second homology arm, if any, is less than 50 nucleotides.

[0502] 117. The targeting construct of any one of embodiments 57 to 116, wherein the targeting construct further comprises a transgene positioned between the degron encoding sequence and the second homology arm.

[0503] 118. The targeting construct of embodiment 113, wherein the transgene is linked to a nucleotide sequence encoding the fusion protein ("fusion protein encoding sequence").

[0504] 119. The targeting construct of embodiment 118, wherein the fusion protein coding sequence and the transgene are linked via a nucleotide sequence encoding an internal ribosome entry site ("IRES").

[0505] 120. The targeting construct of embodiment 118, wherein the fusion protein coding sequence and the transgene are linked in frame via a nucleotide sequence encoding a self-cleaving peptide ("self-cleaving peptide coding sequence").

[0506] 121. The targeting construct of embodiment 120, wherein the self-cleaving peptide is a 2A peptide.

[0507] 122. The targeting construct of embodiment 120 or embodiment 121, wherein the self-cleaving peptide is T2A, P2A, E2A, F2A, or PQR.

[0508] 123. The targeting construct of any one of embodiments 57 to 122, wherein the transgene

[0509] (a) encodes a therapeutic polypeptide; and / or

[0510] (b) is a transgene as described in Section 6.4.

[0511] 124. The targeting construct of embodiment 123, wherein the therapeutic polypeptide is a lysosomal enzyme.

[0512] 125. The targeting construct of embodiment 124, wherein the lysosomal enzyme is α-L-iduronidase, arylsulfatase A, β-glucocerebrosidase, acid sphingomyelinase, α-galactosidase, or β-galactosidase.

[0513] 126. The targeting construct of embodiment 123, wherein the therapeutic polypeptide is a polypeptide whose deficiency is associated with hemophilia.

[0514] 127. The targeting construct of embodiment 126, wherein the therapeutic polypeptide is Factor VIII or Factor IX.

[0515] 128. The targeting construct of embodiment 123, wherein the therapeutic polypeptide is an immunomodulatory polypeptide.

[0516] 129. The targeting construct of embodiment 128, wherein the immunomodulatory polypeptide is a human leukocyte antigen ("HLA") polypeptide.

[0517] 130. The targeting construct of embodiment 129, wherein the HLA polypeptide is an HLA class Ib polypeptide.

[0518] 131. The targeting construct of embodiment 129 or embodiment 130, wherein the HLA polypeptide is an isoform of HLA-E, HLA-F, or HLA-G (e.g., HLA-G1, -G2, -G3, -G4, -G5, -G6, or -G7).

[0519] 132. The targeting construct of embodiment 128, wherein the immunomodulatory polypeptide is a cytokine.

[0520] 133. The targeting construct of embodiment 132, wherein the cytokine is IL-1, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-6, IL-8, IL-10, IL-12, IL-11, IL-12, IL-13, IL-15, IL-18, IL-21, IL-23, IL-27, IL-31 or IL-35.

[0521] 134. The targeting construct of embodiment 123, wherein the therapeutic polypeptide is or comprises an antibody or an antigen-binding fragment thereof.

[0522] 135. The targeting construct of embodiment 134, wherein the therapeutic polypeptide binds to a pathogenic polypeptide.

[0523] 136. The targeting construct of embodiment 135, wherein the pathogenic polypeptide is tau, α-synuclein, or β-amyloid polypeptide.

[0524] 137. The targeting construct of embodiment 134, wherein the therapeutic polypeptide binds to cancer cells.

[0525] 138. The targeting construct of embodiment 137, wherein the therapeutic polypeptide is a chimeric antigen receptor.

[0526] 139. The targeting construct of embodiment 137 or embodiment 138, wherein the therapeutic polypeptide binds to a tumor-associated antigen.

[0527] 140. The targeting construct of embodiment 139, wherein the tumor-associated antigen is CD19 or CD20.

[0528] 141. The targeting construct of any one of embodiments 57 to 140, which is a vector.

[0529] 142. The targeting construct of embodiment 141, wherein the vector is a viral vector.

[0530] 143. The targeting construct of embodiment 142, wherein the viral vector is an AAV vector, a retroviral vector, or a lentiviral vector.

[0531] 144. The targeting construct of embodiment 141, wherein the vector is a DNA vector.

[0532] 145. The targeting construct of embodiment 141, wherein the vector is an RNA vector.

[0533] 146. The targeting construct of any one of embodiments 57 to 145, comprising Figure 7A A nucleotide sequence configured as shown, optionally wherein the nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 1.

[0534] 147. The targeting construct of any one of embodiments 57 to 145, comprising Figure 7B Nucleotide sequences for the configurations shown.

[0535] 148. The targeting construct of any one of embodiments 57 to 145, comprising Figure 7C Nucleotide sequences for the configurations shown.

[0536] 149. The targeting construct of any one of embodiments 57 to 145, comprising Figure 7D A nucleotide sequence configured as shown, optionally wherein the nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 2.

[0537] 150. The targeting construct of any one of embodiments 57 to 145, comprising Figure 7E A nucleotide sequence configured as shown, optionally wherein the nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 29.

[0538] 151. The targeting construct of any one of embodiments 57 to 145, comprising Figure 7F A nucleotide sequence configured as shown, optionally wherein the nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 30.

[0539] 152. The targeting construct of any one of embodiments 57 to 145, comprising Figure 7G Nucleotide sequences for the configurations shown.

[0540] 153. The targeting construct of any one of embodiments 57 to 145, comprising Figure 7H Nucleotide sequences for the configurations shown.

[0541] 154. The targeting construct of any one of embodiments 57 to 145, comprising Figure 7I Nucleotide sequences for the configurations shown.

[0542] 155. The targeting construct of any one of embodiments 57 to 145, comprising Figure 7J Nucleotide sequences for the configurations shown.

[0543] 156. The targeting construct of any one of embodiments 57 to 145, comprising Figure 7K A nucleotide sequence configured as shown, optionally wherein the nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 14.

[0544] 157. The targeting construct of any one of embodiments 57 to 145, comprising Figure 7L A nucleotide sequence configured as shown, optionally wherein the nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 17.

[0545] 158. A system comprising:

[0546] (a) the targeting construct of any one of embodiments 57 to 157;

[0547] (b) a CRISPR-associated endonuclease ("Cas polypeptide") or a nucleic acid encoding a Cas polypeptide; and

[0548] (c) a guide RNA ("gRNA") or a nucleic acid encoding the gRNA, wherein the guide RNA comprises a scaffold for binding to the Cas polypeptide and a spacer sequence corresponding to the essential gene.

[0549] 159. The system of embodiment 158, wherein the guide RNA is a single guide RNA ("sgRNA").

[0550] 160. The system of embodiment 158 ​​or embodiment 159, comprising the Cas polypeptide and gRNA.

[0551] 161. The system of any one of embodiments 158 to 160, which is in the form of a ribonucleoprotein particle ("RNP").

[0552] 162. A method for producing a gene-edited target cell, the method comprising:

[0553] (a) introducing the system of any one of Embodiments 158 to 161 into a target cell, optionally wherein the target cell is as defined in Section 6.7; and

[0554] (b) culturing the target cell under conditions whereby gene editing occurs, thereby producing a gene-edited target cell.

[0555] 163. The method of embodiment 162, wherein the target cell is a stem cell or a cell differentiated from a stem cell.

[0556] 164. The method of embodiment 162 or embodiment 163, wherein the target cell is a stem cell.

[0557] 165. The method of embodiment 164, wherein the stem cell is a human embryonic stem cell, an induced pluripotent stem cell ("iPSC"), or a cell differentiated therefrom.

[0558] 166. The method of any one of embodiments 162 to 164, wherein the target cell is:

[0559] (a) a human immune cell, optionally selected from a T cell, a T cell expressing a chimeric antigen receptor (CAR) or a recombinant TCR, a regulatory T cell, a myeloid cell, a dendritic cell, and a macrophage (e.g., an immunosuppressive macrophage);

[0560] (b) cells in the human nervous system, optionally selected from dopaminergic neurons, microglia, oligodendrocytes, astrocytes, cortical neurons, spinal or oculomotor neurons, enteric neurons, placode-derived cells, Schwann cells, and trigeminal or sensory neurons;

[0561] (c) cells in the human cardiovascular system, optionally selected from the group consisting of cardiomyocytes, endothelial cells and ganglion cells;

[0562] (d) cells in the human metabolic system, which are optionally selected from hepatocytes, bile duct cells and pancreatic beta cells,

[0563] (e) a cell in the human ocular system, optionally selected from a retinal pigment epithelium cell, a photoreceptor cone cell, a photoreceptor rod cell, a bipolar cell or a ganglion cell, or

[0564] (f) A progenitor cell or precursor of any of the aforementioned cells.

[0565] 167. The method of any one of embodiments 162 to 166, wherein the target cell for gene editing is of ectodermal lineage, optionally wherein the target cell for gene editing is a neuron or a progenitor or precursor thereof.

[0566] 168. The method of any one of embodiments 162 to 166, wherein the target cell for gene editing is of mesodermal lineage, optionally wherein the target cell for gene editing is a cardiomyocyte or a progenitor or precursor thereof.

[0567] 169. The method of any one of embodiments 162 to 166, wherein the gene-edited target cell is of endoderm lineage, optionally wherein the gene-edited cell is a lung cell, a thyroid cell, or a pancreatic cell, or a progenitor or precursor thereof.

[0568] 170. A method according to any one of embodiments 162 to 166, wherein the target cell for gene editing is a cardiac cell, a cardiac progenitor cell or a mature or immature (atrial or ventricular) cardiomyocyte, a cardiac endothelial cell, a ganglion cell or a progenitor or precursor thereof.

[0569] 171. A method according to any one of embodiments 162 to 166, wherein the gene-edited target cells are T cells, CAR-T cells, T cells expressing recombinant TCRs, regulatory T cells, bone marrow cells, dendritic cells and / or macrophages or their progenitors or precursors.

[0570] 172. The method of any one of embodiments 162 to 166, wherein the target cell for gene editing is an oligodendrocyte or a progenitor or precursor thereof.

[0571] 173. The method of any one of embodiments 162 to 166, wherein the target cell for gene editing is a neural crest cell, an astrocyte, a dopaminergic neuron, or a progenitor or precursor thereof.

[0572] 174. The method of any one of embodiments 162 to 166, wherein the target cell for gene editing is a photoreceptor cell, a retinal pigment epithelial cell, a neural retinal cell, or a progenitor or precursor thereof.

[0573] 175. The method of any one of embodiments 162 to 166, wherein the target cell for gene editing is a microglia or a microglial progenitor or precursor cell.

[0574] 176. A method according to any one of embodiments 162 to 166, wherein the target cells for gene editing are hepatocytes, bile duct cells and pancreatic beta cells or their progenitors or precursors.

[0575] 177. The method of any one of embodiments 162 to 166, wherein the target cell for gene editing is an intestinal cell or an intestinal progenitor or precursor cell.

[0576] 178. A gene-edited target cell obtained or obtainable by the method of any one of embodiments 162 to 177.

[0577] 179. A gene-edited target cell comprising an essential gene encoding a fusion protein comprising:

[0578] (a) Degron;

[0579] (b) an essential polypeptide encoded by an essential gene as defined in any one of embodiments 96 to 112.

[0580] 180. The gene-edited target cell of embodiment 179, wherein the degradation determinant is located at the C-terminus of the essential polypeptide.

[0581] 181. The gene-edited target cell of embodiment 179, wherein the degradation determinant is located at the N-terminus of the essential polypeptide.

[0582] 182. The gene-edited target cell of any one of embodiments 179 to 181, wherein the degradation determinant is an inducible degradation determinant.

[0583] 183. The gene-edited target cell of embodiment 182, wherein the degradation determinant is drug-inducible, temperature-sensitive, light-inducible, or activated by a polypeptide (optionally wherein the polypeptide is a TEV protease).

[0584] 184. The gene-edited target cell of embodiment 183, wherein the degradation determinant is drug-inducible.

[0585] 185. The gene-edited target cell of embodiment 184, wherein the drug is an immunomodulatory drug (IMiD).

[0586] 186. The gene-edited target cell of embodiment 185, wherein the IMiD is lenalidomide, ibedomide, thalidomide, avalidomide or pomalidomide.

[0587] 187. The gene-edited target cell of embodiment 185, wherein the IMiD is ibedomide.

[0588] 188. The gene-edited target cell of embodiment 185, wherein the IMiD is avadomide.

[0589] 189. The gene-edited target cell of embodiment 185, wherein the IMiD is thalidomide.

[0590] 190. The gene-edited target cell of embodiment 185, wherein the IMiD is lenalidomide.

[0591] 191. The gene-edited target cell of embodiment 185, wherein the IMiD is pomalidomide.

[0592] 192. The gene-edited target cell according to any one of embodiments 179 to 191, wherein the degradation determinant comprises or consists of the following amino acid sequence: RPFQCNQCGASFTQKGNLLRHIKLH (SEQ ID NO: 3), FNVLMVHKRSHTGERPLQCEICGFTCRQKGNLLRHIKLHTGEKPFKCHLCNYACQRRDAL (SEQ ID NO: 4, corresponding to SEQ ID NO: 42 of WO 2021 / 188286A2), FNVLMVHKRSHTGERP (SEQ ID NO: 5, corresponding to SEQ ID NO: 97 of WO 2019 / 089592A1), FNVLMVHRRSHTGERP (SEQ ID NO: 6, corresponding to SEQ ID NO: 100 of WO 2019 / 089592 A1), TGEKPFKCHLCNYACQRRDAL (SEQ ID NO: 7, corresponding to SEQ ID NO: 10 of WO 2019 / 089592 A1), NO: 102), TGERPFRCHLCNYACQRRDAL (SEQ ID NO: 8, corresponding to SEQ ID NO: 103 of WO 2019 / 089592 A1), FQCNQCGASFT (SEQ ID NO: 9, corresponding to SEQ ID NO: 528 of WO 2021 / 188286 A2), FQCPICGLVIK (SEQ ID NO: 10, corresponding to SEQ ID NO: 529 of WO 2021 / 188286 A2), LQCEICGFTCR (SEQ ID NO: 11, corresponding to SEQ ID NO: 530 of WO 2021 / 188286 A2), LQCEICGYQCR (SEQ ID NO: 12, corresponding to SEQ ID NO: 531 of WO 2021 / 188286 A2) or LQCEVCGFQCR (SEQ ID NO: 13, corresponding to SEQ ID NO: 532 of WO 2021 / 188286 A2). NO:532).

[0593] 193. The gene-edited target cell of claim 192, wherein the degradation determinant comprises or consists of the amino acid sequence of SEQ ID NO: 3.

[0594] 194. The gene-edited target cell of claim 192, wherein the degradation determinant comprises or consists of the amino acid sequence of SEQ ID NO: 4.

[0595] 195. The gene-edited target cell of claim 192, wherein the degradation determinant comprises or consists of the amino acid sequence of SEQ ID NO: 5.

[0596] 196. The gene-edited target cell of claim 192, wherein the degradation determinant comprises or consists of the amino acid sequence of SEQ ID NO: 6.

[0597] 197. The gene-edited target cell of claim 192, wherein the degradation determinant comprises or consists of the amino acid sequence of SEQ ID NO: 7.

[0598] 198. The gene-edited target cell of claim 192, wherein the degradation determinant comprises or consists of the amino acid sequence of SEQ ID NO: 8.

[0599] 199. The gene-edited target cell of claim 192, wherein the degradation determinant comprises or consists of the amino acid sequence of SEQ ID NO: 9.

[0600] 200. The gene-edited target cell of claim 192, wherein the degradation determinant comprises or consists of the amino acid sequence of SEQ ID NO: 10.

[0601] 201. The gene-edited target cell of claim 192, wherein the degradation determinant comprises or consists of the amino acid sequence of SEQ ID NO: 11.

[0602] 202. The gene-edited target cell of claim 192, wherein the degradation determinant comprises or consists of the amino acid sequence of SEQ ID NO: 12.

[0603] 203. The gene-edited target cell of claim 192, wherein the degradation determinant comprises or consists of the amino acid sequence of SEQ ID NO: 13.

[0604] 204. The gene-edited target cell of any one of embodiments 179 to 203, wherein the degradation determinant is a super degradation determinant.

[0605] 205. The gene-edited target cell of embodiment 184, wherein the degradation determinant is a SMASh (small molecule assisted shutoff) tag degradation determinant.

[0606] 206. The gene-edited target cell of any one of embodiments 179 to 205, wherein the fusion protein comprises a linker sequence between the essential polypeptide and the degradation determinant.

[0607] 207. The gene-edited target cell of embodiment 205, wherein the linker is 1 to 30 amino acids in length.

[0608] 208. The gene-edited target cell of embodiment 207, wherein the linker is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length.

[0609] 209. The gene-edited target cell of embodiment 207 or embodiment 208, wherein the linker is between 1 and 12 amino acids in length, between 2 and 12 amino acids in length, or between 1 and 10 amino acids in length.

[0610] 210. The gene-edited target cell of embodiment 207 or embodiment 208, wherein the linker is 3 to 10 amino acids in length.

[0611] 211. The gene-edited target cell of embodiment 207 or embodiment 208, wherein the linker is 11 to 20 amino acids in length.

[0612] 212. The gene-edited target cell of embodiment 207 or embodiment 208, wherein the linker is 21 to 30 amino acids in length.

[0613] 213. The gene-edited target cell of any one of embodiments 179 to 212, wherein the fusion protein comprises multiple degradation determinants.

[0614] 214. The gene-edited target cell of embodiment 213, wherein the fusion protein comprises two or more degradation determinants.

[0615] 215. The gene-edited target cell of embodiment 214, wherein the degradation determinants are in tandem.

[0616] 216. The gene-edited target cell of embodiment 215, wherein each pair of degradation determinants is separated by a linker, optionally wherein (a) the linker is a linker described in Section 6.2.3 and / or (b) all of the linkers separating the degradation determinant pairs are the same.

[0617] 217. The gene-edited target cell of any one of embodiments 213 to 216, wherein the multiple degradation determinants are induced in the same manner, optionally wherein the multiple degradation determinants are the same.

[0618] 218. The gene-edited target cell of any one of embodiments 179 to 217, wherein the essential gene is:

[0619] (a) STEL gene; or

[0620] (b) Non-STEL genes.

[0621] 219. The gene-edited target cell of any one of embodiments 179 to 218, wherein the essential gene encodes a polypeptide involved in one or more of: glycolysis, ribonucleopolypeptide complex formation, focal adhesions, cell-matrix adherens junctions, cell-matrix junctions, cell anchorage, extracellular exosomes, extracellular vesicles, intracellular organelles, anchorage junctions, RNA binding, nucleic acid binding (e.g., rRNA or mRNA binding), and polypeptide binding.

[0622] 220. The gene-edited target cell of any one of embodiments 179 to 219, wherein the essential gene (a) encodes a ribosomal polypeptide or (b) does not encode a ribosomal polypeptide.

[0623] 221. The gene-edited target cell of embodiment 220, wherein the essential gene encodes a ribosomal polypeptide, optionally wherein the ribosomal polypeptide is RPL13A, RPLP0, 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.

[0624] 222. The gene-edited target cell of any one of embodiments 179 to 219, wherein the essential gene (a) encodes a ribosomal polypeptide small subunit (RPS) or (b) does not encode a ribosomal polypeptide small subunit (RPS).

[0625] 223. The gene-edited target cell of embodiment 222, wherein the essential gene encodes a ribosomal polypeptide small subunit (RPS), optionally wherein the ribosomal polypeptide small subunit (RPS) is RPS2, RPS19, RPS14, RPS3A, RPS12, RPS3, RPS6, RPS23, RPS27A, RPS8, RPS4X, RPS7, RPS24, RPS27, RPS15A, RPS9, RPS28, RPS13, RPSA, RPS5, RPS16, RPS25, RPS15, RPS20 or RPS11.

[0626] 224. The gene-edited target cell of any one of embodiments 179 to 219, wherein the essential gene (a) encodes an actin polypeptide or (b) does not encode an actin polypeptide.

[0627] 225. The gene-edited target cell of embodiment 224, wherein the essential gene encodes an actin polypeptide, optionally wherein the actin polypeptide is ACTG1 or ACTB.

[0628] 226. The gene-edited target cell of any one of embodiments 179 to 219, wherein the essential gene (a) encodes a eukaryotic translation factor or (b) does not encode a eukaryotic translation factor.

[0629] 227. The gene-edited target cell of embodiment 226, wherein the essential gene encodes a eukaryotic translation factor, optionally wherein the eukaryotic translation factor is EEF1A1, EEF2 or EIF1.

[0630] 228. The gene-edited target cell of any one of embodiments 179 to 219, wherein the essential gene (a) encodes a histone or (b) does not encode a histone.

[0631] 229. The gene-edited target cell of embodiment 228, wherein the essential gene encodes a histone, optionally wherein the histone is H3F3A or H3F3B.

[0632] 230. The gene-edited target cell of any one of embodiments 179 to 219, wherein the essential gene is (a) selected from FTH1, TPT1, GAPDH, PTMA, GNB2L1, NACA, YBX1, NPM1, FAU, UBA52, HSP90AB1, MYL6, SERF2, SRP14, RPL13A, RPL7, or RPLP0 or (b) is not FTH1, TPT1, PTMA, GNB2L1, NACA, YBX1, NPM1, FAU, UBA52, HSP90AB1, MYL6, SERF2, or SRP14.

[0633] 231. The gene-edited target cell of embodiment 230, wherein the essential gene is GAPDH.

[0634] 232. The gene-edited target cell of embodiment 230, wherein the essential gene is RPL13A.

[0635] 233. The gene-edited target cell of embodiment 230, wherein the essential gene is RPL7.

[0636] 234. The gene-edited target cell of embodiment 230, wherein the essential gene is RPLP0.

[0637] 235. The gene-edited target cell of any one of embodiments 179 to 234, further comprising a transgene.

[0638] 236. A gene-edited target cell according to embodiment 235, wherein the transgene is linked to the nucleotide sequence encoding the fusion protein ("fusion protein coding sequence") via a nucleotide sequence encoding an internal ribosome entry site ("IRES").

[0639] 237. The gene-edited target cell of embodiment 235, wherein the transgene is linked to the nucleotide sequence encoding the fusion protein ("fusion protein encoding sequence") via a nucleotide sequence encoding a self-cleaving peptide ("self-cleaving peptide encoding sequence").

[0640] 238. The gene-edited target cell of embodiment 237, wherein the self-cleaving peptide is a 2A peptide.

[0641] 239. The gene-edited target cell of embodiment 237 or 238, wherein the self-cleaving peptide is T2A, P2A, E2A, F2A or PQR.

[0642] 240. The gene-edited target cell of any one of embodiments 179 to 239, wherein the transgene (a) encodes a therapeutic polypeptide; and / or

[0643] (b) is a transgene as described in Section 6.4.

[0644] 241. The gene-edited target cell of embodiment 240, wherein the therapeutic polypeptide is a lysosomal enzyme.

[0645] 242. The gene-edited target cell of embodiment 241, wherein the lysosomal enzyme is α-L-iduronidase, arylsulfatase A, β-glucocerebrosidase, acid sphingomyelinase, α-galactosidase or β-galactosidase.

[0646] 243. The gene-edited target cell of embodiment 240, wherein the therapeutic polypeptide is a polypeptide whose defect is associated with hemophilia.

[0647] 244. The gene-edited target cell of embodiment 243, wherein the therapeutic polypeptide is factor VIII or factor IX.

[0648] 245. The gene-edited target cell of embodiment 240, wherein the therapeutic polypeptide is an immunomodulatory polypeptide.

[0649] 246. The gene-edited target cell of embodiment 245, wherein the immunomodulatory polypeptide is a human leukocyte antigen ("HLA") polypeptide.

[0650] 247. The gene-edited target cell of embodiment 246, wherein the HLA polypeptide is an HLA-Ib class polypeptide.

[0651] 248. The gene-edited target cell of Example 246 or Example 247, wherein the HLA polypeptide is an isotype of HLA-E, HLA-F, or HLA-G (e.g., HLA-G1, -G2, -G3, -G4, -G5, -G6, or -G7).

[0652] 249. The gene-edited target cell of embodiment 245, wherein the immunomodulatory polypeptide is a cytokine or a cytokine receptor.

[0653] 250. The gene-edited target cell of embodiment 245 or embodiment 249, wherein the immunomodulatory polypeptide is a cytokine, and the cytokine is:

[0654] or

[0655] (b) IL-1β, IL-6, IL-10, IL-12, IL-15, GM-CSF, IFN-α, IFN-β, IFN-γ, TNF-α, CCL2, CCL5, CXCL9, CXCL10, CXCL12, TGFβ, or CSF-1.

[0656] 251. The gene-edited target cell of embodiment 245 or embodiment 249, wherein the immunomodulatory polypeptide is IL-1Ra.

[0657] 252. The gene-edited target cell of embodiment 240, wherein the therapeutic polypeptide is or comprises an antibody or an antigen-binding fragment thereof.

[0658] 253. The gene-edited target cell of embodiment 252, wherein the therapeutic polypeptide is bound to a pathogenic polypeptide.

[0659] 254. The gene-edited target cell of embodiment 253, wherein the pathogenic polypeptide is tau, α-synuclein, or β-amyloid polypeptide.

[0660] 255. The gene-edited target cell of embodiment 240, wherein the therapeutic polypeptide binds to cancer cells.

[0661] 256. The gene-edited target cell of embodiment 255, wherein the therapeutic polypeptide is a chimeric antigen receptor.

[0662] 257. The gene-edited target cell of embodiment 255 or embodiment 256, wherein the therapeutic polypeptide binds to a tumor-associated antigen.

[0663] 258. The gene-edited target cell of embodiment 257, wherein the tumor-associated antigen is CD19 or CD20.

[0664] 259. A recombinant cell engineered to express the fusion protein of any one of embodiments 1 to 56, optionally wherein the recombinant cell is a target cell for gene editing of any one of embodiments 178 to 258.

[0665] 260. The recombinant cell according to embodiment 259, which can be Figure 2A This is achieved by integrating the targeting construct depicted in into a single allele of an essential gene locus.

[0666] 261. The recombinant cell according to embodiment 259, which can be Figure 2A This is achieved by integrating the targeting construct depicted in Figure 2 into both alleles of the essential gene locus.

[0667] 262. The recombinant cell according to embodiment 259, which can be Figure 2B This is achieved by integrating the targeting construct depicted in into a single allele of an essential gene locus.

[0668] 263. The recombinant cell according to embodiment 259, which can be Figure 2B This is achieved by integrating the targeting construct depicted in Figure 2 into both alleles of the essential gene locus.

[0669] 264. The recombinant cell according to embodiment 259, which can be Figure 2C This is achieved by integrating the targeting construct depicted in into a single allele of an essential gene locus.

[0670] 265. The recombinant cell according to embodiment 259, which can be Figure 2C This is achieved by integrating the targeting construct depicted in Figure 2 into both alleles of the essential gene locus.

[0671] 266. The recombinant cell according to embodiment 259, which can be Figure 2D This is achieved by integrating the targeting construct depicted in into a single allele of an essential gene locus.

[0672] 267. The recombinant cell according to embodiment 259, which can be Figure 2D This is achieved by integrating the targeting construct depicted in Figure 2 into both alleles of the essential gene locus.

[0673] 268. The recombinant cell according to embodiment 259, which can be Figure 4A This is achieved by integrating the targeting construct depicted in into a single allele of an essential gene locus.

[0674] 269. The recombinant cell according to embodiment 259, which can be Figure 4A This is achieved by integrating the targeting construct depicted in Figure 2 into both alleles of the essential gene locus.

[0675] 270. The recombinant cell according to embodiment 259, which can be Figure 4B This is achieved by integrating the targeting construct depicted in into a single allele of an essential gene locus.

[0676] 271. The recombinant cell according to embodiment 259, which can be Figure 4B This is achieved by integrating the targeting construct depicted in Figure 2 into both alleles of the essential gene locus.

[0677] 272. The recombinant cell according to embodiment 259, which can be Figure 4C This is achieved by integrating the targeting construct depicted in into a single allele of an essential gene locus.

[0678] 273. The recombinant cell according to embodiment 259, which can be Figure 4C This is achieved by integrating the targeting construct depicted in Figure 2 into both alleles of the essential gene locus.

[0679] 274. The recombinant cell according to embodiment 259, which can be Figure 4D This is achieved by integrating the targeting construct depicted in into a single allele of an essential gene locus.

[0680] 275. The recombinant cell according to embodiment 259, which can be Figure 4D This is achieved by integrating the targeting construct depicted in Figure 2 into both alleles of the essential gene locus.

[0681] 276. The recombinant cell according to embodiment 259, which can be Figure 7AThe targeting construct depicted in , for example, comprising the nucleotide sequence of SEQ ID NO: 1, is integrated into a single allele of an essential gene locus.

[0682] 277. The recombinant cell according to embodiment 259, which can be Figure 7A The targeting construct depicted in , for example, comprising the nucleotide sequence of SEQ ID NO: 1, is integrated into both alleles of an essential gene locus.

[0683] 278. The recombinant cell according to embodiment 259, which can be Figure 7B This is achieved by integrating the targeting construct depicted in into a single allele of an essential gene locus.

[0684] 279. The recombinant cell according to embodiment 259, which can be Figure 7B This is achieved by integrating the targeting construct depicted in Figure 2 into both alleles of the essential gene locus.

[0685] 280. The recombinant cell according to embodiment 259, which can be Figure 7C This is achieved by integrating the targeting construct depicted in into a single allele of an essential gene locus.

[0686] 281. The recombinant cell according to embodiment 259, which can be Figure 7C This is achieved by integrating the targeting construct depicted in Figure 2 into both alleles of the essential gene locus.

[0687] 282. The recombinant cell according to embodiment 259, which can be Figure 7D The targeting construct depicted in , for example, comprising the nucleotide sequence of SEQ ID NO: 2, is integrated into a single allele of an essential gene locus.

[0688] 283. The recombinant cell according to embodiment 259, which can be Figure 7D The targeting construct depicted in , for example, comprising the nucleotide sequence of SEQ ID NO: 2, is integrated into both alleles of the essential gene locus.

[0689] 284. The recombinant cell according to embodiment 259, which can be Figure 7E The targeting construct depicted in , for example, comprising the nucleotide sequence of SEQ ID NO: 29, is integrated into a single allele of an essential gene locus.

[0690] 285. The recombinant cell according to embodiment 259, which can be Figure 7EThe targeting construct depicted in , for example, comprising the nucleotide sequence of SEQ ID NO: 29, is integrated into both alleles of the essential gene locus.

[0691] 286. The recombinant cell according to embodiment 259, which can be Figure 7F The targeting construct depicted in , for example, comprising the nucleotide sequence of SEQ ID NO: 30, is integrated into a single allele of an essential gene locus.

[0692] 287. The recombinant cell according to embodiment 259, which can be Figure 7F The targeting construct depicted in , for example, comprising the nucleotide sequence of SEQ ID NO: 30, is integrated into both alleles of the essential gene locus.

[0693] 288. The recombinant cell according to embodiment 259, which can be Figure 7G This is achieved by integrating the targeting construct depicted in into a single allele of an essential gene locus.

[0694] 289. The recombinant cell according to embodiment 259, which can be Figure 7G This is achieved by integrating the targeting construct depicted in Figure 2 into both alleles of the essential gene locus.

[0695] 290. The recombinant cell according to embodiment 259, which can be Figure 7H This is achieved by integrating the targeting construct depicted in into a single allele of an essential gene locus.

[0696] 291. The recombinant cell according to embodiment 259, which can be Figure 7H This is achieved by integrating the targeting construct depicted in Figure 2 into both alleles of the essential gene locus.

[0697] 292. The recombinant cell according to embodiment 259, which can be Figure 7I This is achieved by integrating the targeting construct depicted in into a single allele of an essential gene locus.

[0698] 293. The recombinant cell according to embodiment 259, which can be Figure 7I This is achieved by integrating the targeting construct depicted in Figure 2 into both alleles of the essential gene locus.

[0699] 294. The recombinant cell according to embodiment 259, which can be Figure 7J This is achieved by integrating the targeting construct depicted in into a single allele of an essential gene locus.

[0700] 295. The recombinant cell according to embodiment 259, which can be Figure 7J This is achieved by integrating the targeting construct depicted in Figure 2 into both alleles of the essential gene locus.

[0701] 296. The recombinant cell according to embodiment 259, which can be Figure 7K The targeting construct depicted in , for example, comprising the nucleotide sequence of SEQ ID NO: 14, is integrated into a single allele of an essential gene locus.

[0702] 297. The recombinant cell according to embodiment 259, which can be Figure 7K The targeting construct depicted in , for example, comprising the nucleotide sequence of SEQ ID NO: 14, is integrated into both alleles of the essential gene locus.

[0703] 298. The recombinant cell according to embodiment 259, which can be Figure 7L The targeting construct depicted in , for example, comprising the nucleotide sequence of SEQ ID NO: 17, is integrated into a single allele of an essential gene locus.

[0704] 299. The recombinant cell according to embodiment 259, which can be Figure 7L The targeting construct depicted in , for example, comprising the nucleotide sequence of SEQ ID NO: 17, is integrated into both alleles of an essential gene locus.

[0705] 300. The recombinant cell of embodiment 259, comprising an engineered essential gene having Figure 3A The configuration depicted in .

[0706] 301. The recombinant cell of embodiment 259, comprising an engineered essential gene having Figure 3B The configuration depicted in .

[0707] 302. The recombinant cell of embodiment 259, comprising an engineered essential gene having Figure 3D The configuration depicted in .

[0708] 303. The recombinant cell of embodiment 259, comprising an engineered essential gene having Figure 3E The configuration depicted in .

[0709] 304. The recombinant cell of embodiment 259, comprising an engineered essential gene having Figure 5A The configuration depicted in .

[0710] 305. The recombinant cell of embodiment 259, comprising an engineered essential gene having Figure 5B The configuration depicted in .

[0711] 306. The recombinant cell of embodiment 259, comprising an engineered essential gene having Figure 5C The configuration depicted in .

[0712] 307. The recombinant cell of embodiment 259, comprising an engineered essential gene having Figure 5D The configuration depicted in .

[0713] 308. The recombinant cell of embodiment 259, comprising an engineered essential gene having Figure 6A The configuration depicted in .

[0714] 309. The recombinant cell of embodiment 259, comprising an engineered essential gene having Figure 6B The configuration depicted in .

[0715] 310. The recombinant cell of embodiment 259, comprising an engineered essential gene having Figure 6C The configuration depicted in .

[0716] 311. The recombinant cell of embodiment 259, comprising an engineered essential gene having Figure 6D The configuration depicted in .

[0717] 312. The recombinant cell of embodiment 259, comprising an engineered essential gene having Figure 6E The configuration depicted in .

[0718] 313. The recombinant cell of any one of embodiments 259 to 312, further engineered to express a transgene from a locus other than the essential gene.

[0719] 314. The recombinant cell of embodiment 259, comprising an expression vector comprising a nucleotide sequence encoding the fusion protein of any one of embodiments 1 to 56.

[0720] 315. The recombinant cell of embodiment 314, wherein the vector is a viral vector.

[0721] 316. The recombinant cell of embodiment 315, wherein the viral vector is an AAV vector, a retroviral vector, or a lentiviral vector.

[0722] 317. The recombinant cell of embodiment 314, wherein the vector is a DNA vector.

[0723] 318. The recombinant cell of embodiment 314, wherein the vector is an RNA vector.

[0724] 319. The recombinant cell of any one of embodiments 314 to 318, wherein the corresponding essential gene is knocked out at a single allele.

[0725] 320. The recombinant cell of any one of embodiments 314 to 318, wherein the corresponding essential gene is knocked out at both alleles.

[0726] 321. The recombinant cell of any one of embodiments 314 to 320, which is also engineered to express a transgene.

[0727] 322. The recombinant cell of embodiment 321, wherein said transgene is not expressed from a corresponding essential gene locus.

[0728] 323. The recombinant cell of embodiment 321, wherein said transgene is expressed from a corresponding essential gene locus.

[0729] 324. The recombinant cell of any one of embodiments 321 to 323, wherein the transgene is as defined in Section 6.4.

[0730] 325. The recombinant cell of any one of embodiments 259 to 324, wherein the cell is as defined in Section 6.7.

[0731] 326. A pharmaceutical composition comprising the gene-edited target cell of any one of embodiments 178 to 258 or the recombinant cell of any one of embodiments 259 to 325 and a pharmaceutically acceptable excipient.

[0732] 327. Use of the gene-edited target cell of any one of embodiments 178 to 258 or the recombinant cell of any one of embodiments 259 to 325 for the manufacture of a medicament for treating a patient in need thereof, optionally wherein the gene-edited target cell or the recombinant cell is (a) autologous to the patient or (b) allogeneic to the patient.

[0733] 328. The gene-edited target cell of any one of embodiments 178 to 258, the recombinant cell of any one of embodiments 259 to 325, or the pharmaceutical composition of embodiment 326, for use in treating a patient in need thereof, optionally wherein the gene-edited target cell or the recombinant cell is (a) autologous to the patient or (b) allogeneic to the patient, or the medicament comprises a cell that is (a) autologous to the patient or (b) allogeneic to the patient.

[0734] 329. A method of treating a subject with cell therapy, the method comprising administering to a subject in need thereof the gene-edited target cell of any one of embodiments 178 to 258, the recombinant cell of any one of embodiments 259 to 325, or the pharmaceutical composition of embodiment 326, optionally wherein the gene-edited target cell or the recombinant cell is (a) autologous to the patient or (b) allogeneic to the patient, or the pharmaceutical comprises a cell that is (a) autologous to the patient or (b) allogeneic to the patient.

[0735] 330. The method of embodiment 329, further comprising administering to said subject an inducer of said degron.

[0736] 331. The method of embodiment 330, wherein the subject has developed side effects from the cell therapy.

[0737] 332. The method of embodiment 330, wherein the subject is at risk for developing side effects of the cell therapy.

[0738] 333. The method of embodiment 331 or embodiment 332, wherein the side effect is cytokine storm or cytokine release syndrome (CRS).

[0739] 334. The method of embodiment 331 or embodiment 332, wherein the side effect is excessive (systemic) inflammation.

[0740] 335. The method of embodiment 331 or embodiment 332, wherein the side effect is tumor formation.

[0741] 336. The method of embodiment 331 or embodiment 332, wherein the side effect is tumor lysis syndrome.

[0742] 337. The method of embodiment 331 or embodiment 332, wherein the side effects include dysfunctional neurons and / or movement disorders.

[0743] 338. The method of embodiment 331 or embodiment 332, wherein the side effects include dysfunctional cardiomyocytes and / or arrhythmias.

[0744] 339. The method of embodiment 331 or embodiment 332, wherein the side effect is macrophage activation syndrome (MAS).

[0745] 340. The method of embodiment 331 or embodiment 332, wherein the side effect is graft-versus-host disease.

[0746] 341. The method of any one of embodiments 330 to 340, wherein said degron is a drug-inducible degron.

[0747] 342. The method of embodiment 341, wherein the drug is an immunomodulatory drug (IMiD).

[0748] 343. The method of embodiment 342, wherein the IMiD is lenalidomide, ibedomide, thalidomide, avalidomide, or pomalidomide.

[0749] 344. The method of embodiment 342, wherein the IMiD is ibelidomide.

[0750] 345. The method of embodiment 342, wherein the IMiD is avadomid.

[0751] 346. The method of embodiment 342, wherein the IMiD is thalidomide.

[0752] 347. The method of embodiment 342, wherein the IMiD is lenalidomide.

[0753] 348. The method of embodiment 342, wherein the IMiD is pomalidomide.

[0754] 349. A method of reducing engineered cells in a subject or eliminating engineered cells from the subject, wherein the subject previously received cell therapy with the gene-edited target cells of any one of embodiments 178 to 258, the recombinant cells of any one of embodiments 259 to 325, or the pharmaceutical composition of embodiment 326, the method comprising administering to the subject an inducer of the degradation determinant, optionally wherein the cell therapy comprises or consists of cells that are: (a) autologous to the subject or (b) allogeneic to the subject.

[0755] 350. The method of embodiment 349, wherein the subject experiences or is at risk of a side effect of the cell therapy.

[0756] 351. The method of embodiment 350, wherein the side effect is cytokine storm or cytokine release syndrome (CRS).

[0757] 352. The method of embodiment 350, wherein the side effect is excessive (systemic) inflammation.

[0758] 353. The method of embodiment 350, wherein the side effect is tumor formation.

[0759] 354. The method of embodiment 350, wherein the side effect is tumor lysis syndrome.

[0760] 355. The method of embodiment 350, wherein the side effects include dysfunctional neurons and / or movement disorders.

[0761] 356. The method of embodiment 350, wherein the side effects include dysfunctional cardiomyocytes and / or arrhythmias.

[0762] 357. The method of embodiment 350, wherein the side effect is macrophage activation syndrome (MAS).

[0763] 358. The method of embodiment 350, wherein the side effect is graft-versus-host disease.

[0764] 359. The method of any one of embodiments 349 to 351, wherein said degron is a drug-inducible degron.

[0765] 360. The method of embodiment 359, wherein the drug is an immunomodulatory drug (IMiD).

[0766] 361. The method of embodiment 360, wherein the IMiD is lenalidomide, ibedomide, thalidomide, avalidomide, or pomalidomide.

[0767] 362. The method of embodiment 360, wherein the IMiD is ibelidomide.

[0768] 363. The method of embodiment 360, wherein the IMiD is avadomid.

[0769] 364. The method of embodiment 360, wherein the IMiD is thalidomide.

[0770] 365. The method of embodiment 360, wherein the IMiD is lenalidomide.

[0771] 366. The method of embodiment 360, wherein the IMiD is pomalidomide.

[0772] 8. Examples

[0773] 8.1. Example 1: Design and Generation of Targeting Constructs

[0774] The targeting construct was designed to contain a linker and a degron sequence flanked by GAPDH homology arms, e.g. Figures 7A to 7D Additional targeting constructs were designed to contain a linker, a degron sequence, an IRES sequence, and a GFP transgene sequence flanked by GAPDH homology arms, as shown in FIG. Figures 7G to 7J As shown in . Homology arms were designed to enable integration of the construct into the endogenous GAPDH locus, immediately 5' of the endogenous stop codon of GAPDH. The desired sequence was sent to GenScript (Piscataway, NJ) for de novo gene synthesis via on-site oligonucleotide design, oligonucleotide synthesis, and gene assembly. The amplified fragment was ligated into the pUC57-Kan cloning vector, transformed into bacteria, and the resulting plasmid containing the targeting construct was isolated.

[0775] 8.2. Example 2: Selective Targeting of Gene-Edited iPSCs

[0776] use Figure 7G The targeting construct described in [ 15 ] tested the function of the kill switch in iPSC, and the targeting construct contained a linker, a degradation determinant sequence, an IRES, and a GFP transgenic sequence flanked by GAPDH homology arms. On the day of nuclear transfection, iPSC was harvested and resuspended in Lonza P3 primary cell nuclear transfection buffer. A 1:2 ratio of protein:sgRNA (IDT) was used to compound ribonucleoprotein (RNP) with sgRNA. The composite RNP was nuclear transfected into resuspended iPSC together with the targeting construct using LONZA 4D Nucleofector. The nuclear transfected cells were then plated and the targeting event was assessed.

[0777] Unedited cells (GFP negative) and Figure 7G A heterogeneous mixture of cells edited with the construct depicted in (containing the degron fused to GAPDH and GFP (GFP positive)) were treated with 3 μM pomalidomide (POM) for 6 days or fed with complete medium without POM (untreated). Treatment of cells with POM did not affect the expression of unedited cells ( Figure 8A ), but depletion of the edited GFP-positive cell population ( Figure 8B), indicating that inducible degradation of degron-fused GAPDH protein kills gene-edited cells.

[0778] In the next set of assessments, if Figure 7G As shown, clones modified bi-allelically with a degron fused to GAPDH and IRES-GFP were treated with 1 μM POM for 92 hours and imaged using the Incucyte. The percentage of viable cells / well was quantified at multiple time points using the Incucyte software and normalized to the number of viable cells / well at the first image time point. Figure 9A As shown, approximately 24 hours after the addition of POM, the cell death rate of gene-edited cells exceeded their proliferation rate. In addition, 92 hours after the addition of POM, the gene-edited cells were completely eliminated ( Figure 9A and 9B Untreated gene-edited cells proliferated normally for the same duration ( Figure 9C ).

[0779] 8.3. Example 3: Effect of Targeting Construct Linker Length on Survival of Gene-Edited iPSCs

[0780] Two sets of targeting constructs were generated as described in Section 8.1. The first set of targeting constructs was designed to contain a linker, a degron or super degron sequence, an IRES sequence, and a GFP transgene sequence flanked by GAPDH homology arms, such as Figures 7G to 7J , wherein the linker lengths are 3 aa (linker 1, GGS), 15 aa (linker 2, SEQ ID NO: 23), 27 aa (linker 3, SEQ ID NO: 103), and 10 aa (linker 4, SEQ ID NO: 15). The second set of targeting constructs was designed to include a linker and a degron or super degron sequence flanked by GAPDH homology arms, as shown in Figures 7A to 7D For both groups, homology arms were designed to enable integration of the construct into the endogenous GAPDH locus, immediately 5' of the endogenous stop codon of GAPDH. iPSCs were transfected individually with each construct.

[0781] Flow cytometry was used to evaluate the iPSC pools transfected with the first set of targeting constructs, which may include both monoallelic and bi-allelic edited iPSCs, where depletion of GFP signal was assessed after 4 days of treatment with 3 μM POM. Among the gene-edited iPSCs, those transfected with the targeting construct comprising the super degron had the lowest percentage of GFP-positive cells, while approximately 1 / 3 to 1 / 2 of the untreated cells transfected with one of the targeting constructs comprising the degron were GFP-positive. However, depletion of GFP-positive cells with POM treatment was only observed when transfected with the targeting construct comprising the degron or the targeting construct comprising the super degron with the shortest linker. Figure 10A ).

[0782] Next, iPSCs transfected with a second set of targeting constructs were evaluated, where amplicon depletion was assessed relative to untreated cells after POM treatment. Consistent with the results from the first set, targeting constructs containing a 3aa linker and a degron were associated with amplicon depletion. Similar levels of depletion were observed in cells transfected with targeting constructs containing a super degron. In summary, targeting construct linker length and degron type may be key factors in triggering cell death in gene-edited cells.

[0783] 8.4. Example 4: Activation of Targeting Constructs in Gene-Edited iPSCs

[0784] To determine whether gene editing of iPSCs with a targeting construct whose homology arms enabled integration into the endogenous GAPDH locus affected GAPDH expression, three clones (e.g., Figure 7G ) and one clone that was biallelicly modified with a targeting construct containing a super degron (as shown in Figure 7J ) lasts up to 3 days.

[0785] No significant differences in GAPDH expression were detected between untreated and POM-treated gene-edited cells by qPCR, all of which were similar to expression in the untransfected parental line ( Figure 11A Assessment of protein expression by Western blotting showed that GAPDH protein was completely depleted in the same iPSC clones after one day of treatment with 3 μM POM ( Figure 11B and 11C Taken together, these results indicate that none of the targeting constructs altered GAPDH mRNA levels but were able to induce POM-induced GAPDH protein degradation.

[0786] Next, unedited parental cells and gene-edited iPSCs were seeded at equal density and the confluence was tracked over time using an Incucyte imager to assess the effect of gene editing with the targeting construct on growth kinetics. The results showed that all three clones gene-edited with the targeting construct containing the degron grew at a rate comparable to that of the unedited parental cells, while iPSCs gene-edited with the targeting construct containing the super degron grew at a slower rate ( Figure 11C ).

[0787] 8.5. Example 5: Effect of POM Concentration on Death of Gene-Edited iPSCs

[0788] The same gene-edited iPSC clones from Example 4 were treated with 0.5 μM POM for 5 days to determine if there were any differences in survival rates. This POM treatment resulted in complete killing of all gene-edited iPSCs but did not affect the growth of unedited parental cells ( Figure 12A ).

[0789] To determine the optimal POM concentration for inducing apoptosis, the confluence of gene-edited iPSC clones treated with different concentrations of POM ranging from 0.03125 μM to 10 μM was monitored using Incucyte for 5 days. The results showed that 0.25 μM POM was able to trigger apoptosis (not shown), and 0.5 μM POM was sufficient to achieve complete killing of iPSCs gene-edited with a targeting construct containing a degron ( Figure 12B For iPSC cells gene-edited with a targeting construct containing a superdegrader, this concentration was even lower, as the lowest POM concentration evaluated (0.03125 μM) was associated with apoptosis in all cells.

[0790] 8.6. Example 6: Activation of Targeted Constructs in Dopaminergic Neurons Differentiated from Gene-Edited iPSCs.

[0791] To determine the applicability of the targeting constructs containing the degron to differentiated cells, unedited parental iPSCs as well as gene-edited iPSC clones B and C were differentiated into dopaminergic (DA) neurons, as shown in Figure 5. Figure 13The differentiation protocol was adapted from Kriks et al., 2011, Nature 480(7378):547-551 and U.S. Patent No. 10,711,243, which are incorporated herein by reference in their entirety. Cells were plated in the presence and absence of different concentrations of POM and assessed for post-thaw cell death. Cell death was quantified using Annexin V dye in an Incucyte imager over a 5-day period. No differences in neuronal death were detected in DA neurons derived from unedited parental iPSCs ( Figure 14 and 15A All POM concentrations evaluated achieved complete death of all neurons differentiated from both clone B and clone CiPSCs at day 5 ( Figure 14 and 15B Further evaluation of DA neurons derived from clone B showed that these cells were completely killed even with 10 nM POM, the lowest concentration evaluated ( Figure 15C Together, these results demonstrate that DA neurons differentiated from iPSCs gene-edited with a targeting construct containing a degron display nanomolar sensitivity to POM.

[0792] 8.7. Example 7: Extended Testing of POM Concentrations for Activating Targeting Constructs in Dopaminergic Neurons Differentiated from Gene-Edited iPSCs

[0793] Based on the work presented in Example 6, a wider range of POM concentrations was tested in differentiated DA neurons from the unedited parental line, as well as two clones that were bi-allelically modified with a targeting construct containing a linker three amino acids in length (3-aa linker) and a degron: gene-edited clone A or gene-edited clone C. Figure 13 Cells were differentiated from iPSCs into DA neurons using the same protocol as shown in . DA neurons were plated and stained with Annexin V dye, and cell death was measured over 144 hours using an Incucyte imager.

[0794] No differences in cell death were observed in DA neurons derived from unedited parental iPSCs at any concentration of POM from the tested range (0.01 μM to 100 μM). Figure 16A DA neurons derived from clone A or clone C reached almost complete cell death within 144 hours after exposure to as little as 0.1 μM POM ( Figure 16B and 16C These results suggest that POM does not affect unedited neurons but is able to promote apoptosis in neurons that have been modified to contain degron tags on essential genes.

[0795] 8.8. Example 8: Activation of Essential Gene Targeting Constructs in Myeloid Progenitor Cells Differentiated from Gene-Edited iPSCs

[0796] To extend the applicability of the degron-containing constructs to additional differentiated cell types, unedited parental iPSCs and those containing Figure 7G Bi-allelic edited iPSC clones A, B, and C with the targeting constructs shown in FIG were differentiated into Figure 17 Myeloid progenitor (MP) cells were differentiated as shown. The differentiation protocol was adapted from Douvaras et al., June 6, 2017;8(6):1516-1524 and PCT Publication Nos. WO 2023 / 150089 A1 and WO 2017 / 152081 A1, which are incorporated herein by reference in their entireties. MP cells were plated in the presence and absence of POM and cell death was assessed after thawing. Cell death was quantified in an Incucyte imager over a 108-hour period using acridine orange / propidium iodide (AO / PI) dye.

[0797] The analysis showed that all four cell lines survived reasonably well in the absence of POM, and the unedited parental cell line showed no increase in cell death in the presence of POM. All three gene-edited clones died in response to POM within the duration of treatment ( Figures 18A to 18D These results indicate that myeloid progenitor cells differentiated from iPSCs gene-edited with a targeting construct containing a degron exhibit sensitivity to POM.

[0798] 8.9. Example 9.3 Targeting of AA-degradon and superdegradon constructs at the RPL13A locus and activation of targeting constructs in gene-edited iPSCs and differentiated dopaminergic neurons and bone marrow progenitor cells

[0799] To demonstrate the functionality of the degron-based kill switch when linked to another essential gene, a targeting construct containing a linker of three amino acids in length (Linker 1; GGS) and the degron sequence flanked by RPL13A homology arms was designed, e.g. Figure 7K and SEQ ID NO: 14, and designed a second targeting construct comprising a linker (Linker 4; SEQ ID NO: 15) having a length of 10 amino acids and a super degron sequence flanked by RPL13A homology arms, such as Figure 7Land SEQ ID NO: 17. The two targeting constructs were transfected into iPSCs and amplicon depletion was assessed after POM treatment relative to untreated cells. Clonal lines generated from these transfected pools were then assessed to determine the optimal POM concentration required to induce apoptosis in iPSCs and dopaminergic neurons and myeloid progenitor cells differentiated from these gene-edited iPSCs.

[0800] 9. Sequence Listing

[0801] Exemplary sequences of the present disclosure are provided below in Table 10 (wherein "SEQ" refers to SEQ ID NO).

[0802]

[0803]

[0804]

[0805]

[0806]

[0807]

[0808]

[0809]

[0810]

[0811]

[0812]

[0813]

[0814]

[0815]

[0816]

[0817]

[0818]

[0819]

[0820]

[0821]

[0822]

[0823]

[0824] 10. Incorporated by Reference

[0825] All publications, patents, patent applications, and other documents cited in this application are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent application, or other document were individually indicated to be incorporated by reference herein for all purposes. In the event of any inconsistency between the teachings of one or more incorporated references and the present disclosure, the teachings of the present specification control.

Claims

1. A fusion protein comprising: (a) essential polypeptides; (b) degron; and (c) Optionally; a linker.

2. The fusion protein of claim 1, wherein the degradation determinant is located at the C-terminus of the essential polypeptide.

3. The fusion protein according to any one of claims 1 to 2, wherein the degron is an inducible degron. The fusion protein of claim 3 , wherein the degron is a drug-inducible degron.

5. The fusion protein of claim 4, wherein the drug is an immunomodulatory drug (IMiD).

6. The fusion protein according to any one of claims 1 to 5, wherein the degron comprises or consists of the following amino acid sequence: RPFQCNQCGASFTQKGNLLRHIKLH(SEQ ID NO:3), FNVLMVHKRSHTGERPLQCEICGFTCRQKGNLLRHIKLHTGEKPFKCHLCNYA CQRRDAL(SEQ ID NO:4), FNVLMVHKRSHTGERP(SEQ ID NO:5), FNVLMVHRRSHTGERP(SEQ ID NO:6), TGEKPFKCHLCNYACQRRDAL (SEQ ID NO:7), TGERPFRCHLCNYACQRRDAL (SEQ ID NO:8), FQCNQCGASFT (SEQ ID NO:9), FQCPICGLVIK (SEQ ID NO:10), LQCEICGFTCR (SEQ ID NO:11), LQCEICGYQCR (SEQ ID NO:12) or LQCEVCGFQCR (SEQ ID NO:13).

7. The fusion protein according to any one of claims 1 to 6, wherein the degron is a super degron.

8. The fusion protein according to any one of claims 1 to 7, comprising a linker sequence located between the essential polypeptide and the degron.

9. The fusion protein according to any one of claims 1 to 8, comprising two or more degrons, optionally wherein the degrons are in tandem.

10. The fusion protein according to any one of claims 1 to 9, wherein the essential polypeptide is a STEL polypeptide, optionally wherein the STEL polypeptide is GAPDH.

11. A targeting construct comprising: (a) a first homology arm corresponding to a 5' target sequence comprising a first region homologous to an essential gene encoding an essential polypeptide in a target genomic locus; (b) a nucleotide sequence encoding a degron ("degron encoding sequence"); (c) a second homology arm corresponding to a 3' target sequence comprising a second region of homology to the essential gene in the target genomic locus. wherein the targeting construct is configured such that upon recombination with the target genomic locus, the essential gene is modified to encode a fusion protein comprising the essential polypeptide and the degron, optionally wherein the fusion protein has one or more characteristics as defined in any one of claims 1 to 10.

12. The targeting construct of claim 11, wherein the first homology arm and the second homology arm are each 500 to 1,500 nucleotides in length, and / or wherein the difference in length between the first homology arm and the second homology arm, if any, is less than 75 nucleotides.

13. The targeting construct of claim 11 or claim 12, wherein the targeting construct further comprises a transgene located between the degron encoding sequence and the second homology arm.

14. The targeting construct of claim 13, wherein the transgene is linked to a nucleotide sequence encoding the fusion protein ("fusion protein encoding sequence").

15. The targeting construct according to any one of claims 11 to 14, which is a vector, optionally wherein the vector is a DNA vector or an RNA vector.

16. A system comprising: (a) a targeting construct according to any one of claims 11 to 15; (b) a CRISPR-associated endonuclease ("Cas polypeptide") or a nucleic acid encoding a Cas polypeptide; and (c) a guide RNA ("gRNA") or a nucleic acid encoding the gRNA, the guide RNA comprising a scaffold for binding to the Cas polypeptide and a spacer sequence corresponding to the essential gene.

17. The system of claim 16, wherein the guide RNA is a single guide RNA ("sgRNA").

18. The system of claim 16 or claim 17, which is in the form of a ribonucleoprotein particle ("RNP").

19. A method for producing a gene-edited target cell, the method comprising: (a) introducing the system according to any one of claims 16 to 18 into a target cell; as well as (b) culturing the target cell under conditions whereby gene editing occurs, thereby producing a gene-edited target cell.

20. The method of claim 19, wherein the target cell is a stem cell or a cell differentiated from a stem cell.

21. The method of claim 20, wherein the cell is (a) a human embryonic stem cell, (b) an induced pluripotent stem cell ("iPSC"), or (c) a cell differentiated from (a) or (b).

22. The method according to any one of claims 19 to 20, wherein the target cell is: (a) a human immune cell, optionally selected from a T cell, a T cell expressing a chimeric antigen receptor (CAR) or a recombinant TCR, a regulatory T cell, a myeloid cell, a dendritic cell, and a macrophage (e.g., an immunosuppressive macrophage); (b) cells in the human nervous system, optionally selected from dopaminergic neurons, microglia, oligodendrocytes, astrocytes, cortical neurons, spinal or oculomotor neurons, enteric neurons, placode-derived cells, Schwann cells, and trigeminal or sensory neurons; (c) cells in the human cardiovascular system, optionally selected from the group consisting of cardiomyocytes, endothelial cells and ganglion cells; (d) cells in the human metabolic system, which are optionally selected from hepatocytes, bile duct cells and pancreatic beta cells, (e) a cell in the human ocular system, optionally selected from a retinal pigment epithelium, a photoreceptor cone cell, a photoreceptor rod cell, a bipolar cell or a ganglion cell, or (f) A progenitor cell or precursor of any of the aforementioned cells.

23. A gene-edited target cell obtained or obtainable by the method according to any one of claims 19 to 22.

24. A gene-edited target cell comprising an essential gene encoding a fusion protein comprising: (a) Degron; (b) an essential polypeptide encoded by an essential gene as defined in claim 10.

25. A recombinant cell engineered to express the fusion protein of any one of claims 1 to 10, optionally wherein the recombinant cell is a target cell for gene editing according to any one of claim 23 or claim 24.

26. The recombinant cell of claim 25, further engineered to express a transgene from a locus other than the essential gene.

27. The recombinant cell according to claim 25, comprising an expression vector comprising a nucleotide sequence encoding the fusion protein according to any one of claims 1 to 10.

28. A pharmaceutical composition comprising the gene-edited target cell according to claim 23 or claim 24, the recombinant cell according to any one of claims 25 to 27, and a pharmaceutically acceptable excipient.

29. Use of the gene-edited target cell of claim 23 or claim 24 or the recombinant cell of any one of claims 25 to 27 for the manufacture of a medicament for treating a patient in need thereof, optionally wherein the gene-edited target cell or the recombinant cell is (a) autologous to the patient or (b) allogeneic to the patient.

30. The gene-edited target cell of claim 23 or claim 24, the recombinant cell of any one of claims 25 to 27, or the pharmaceutical composition of claim 28, for use in treating a patient in need thereof, optionally wherein the gene-edited target cell or the recombinant cell is (a) autologous to the patient or (b) allogeneic to the patient, or the medicament comprises a cell that is (a) autologous to the patient or (b) allogeneic to the patient.

31. A method for treating a subject with cell therapy, the method comprising administering to a subject in need thereof a gene-edited target cell according to claim 23 or claim 24, a recombinant cell according to any one of claims 25 to 27, or a pharmaceutical composition according to claim 28, optionally wherein the gene-edited target cell or the recombinant cell is (a) autologous to the patient or (b) allogeneic to the patient, or the drug comprises a cell that is (a) autologous to the patient or (b) allogeneic to the patient.

32. The method of claim 31 , further comprising administering to the subject an inducer of the degron.

33. The method of claim 31 or claim 32, wherein the degron is a drug-inducible degron.

34. The method of claim 33, wherein the drug is an immunomodulatory drug (IMiD).

35. A method of reducing engineered cells in a subject or eliminating engineered cells from the subject, the subject having previously received cell therapy with a gene-edited target cell according to claim 23 or claim 24, a recombinant cell according to any one of claims 25 to 27, or a pharmaceutical composition according to claim 28, the method comprising administering to the subject an inducer of the degradation determinant.

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