Degron fusion proteins and methods for their production and use

Abstract

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

Description

[Technical Field]

[0001] 1. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 443,614, filed February 6, 2023, and U.S. Provisional Patent Application No. 63 / 503,640, filed May 22, 2023, the contents of which are incorporated herein by reference in their entireties.

[0002] 2. Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format, and is incorporated herein 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. 3. [Background technology]

[0003] Cell therapy offers great promise for the treatment of various diseases and conditions, in some cases by replacing or repairing missing or damaged cells or tissues with autologous or allogeneic cells. Pluripotent stem cells, including induced pluripotent stem cells (iPSCs), are particularly useful for cell therapy, as they can differentiate into cell types of interest and can be engineered to recombinantly express therapeutic polypeptides with desired therapeutic properties.

[0004] However, due to the nature and complexity of these "living drugs," it would be advantageous to have the ability to remove cell products after their introduction into a patient in the event of an adverse event or as a safety measure after the cell product has fulfilled its function. Therapeutic cell-induced adverse events require rapid and nearly complete elimination of the therapeutic cells. For example, excessive on-target effects, such as those caused by immunoregulatory cells, can result in cytokine storms associated with tumor lysis syndrome (TLS), cytokine release syndrome (CRS), or macrophage activation syndrome (MAS). Furthermore, certain off-target effects, such as cell therapy cells becoming cancerous, can also occur. Therefore, if genetically engineered cells need to be reduced in number or removed from a patient's body, their controlled destruction is necessary.

[0005] As a result, there is great interest in developing methods that can eliminate therapeutic cells if they cause serious adverse events (SAEs) after treatment or become obsolete. 4. Summary of the Invention

[0006] The present disclosure provides stable, reliable "suicide genes" that can be used to eliminate therapeutic cells that cause or may cause a serious adverse event (SAE) after treatment or become obsolete.

[0007] In particular, the present disclosure provides compositions and methods useful for partially or completely eliminating transplanted cell therapy by engineering an inducible apoptotic machinery into therapeutic cells in the form of a fusion protein comprising an essential protein and a drug-inducible degron, the destruction of which is activated by a drug (e.g., a clinically approved small molecule drug such as an immunomodulatory imid drug (IMiD)).

[0008] Fusion proteins (sometimes referred to as "kill switches") are typically expressed from an endogenous essential gene locus, thereby overcoming the drawbacks of other kill switches, such as transcriptional silencing or mutation of the transgene expressing the kill switch during cell differentiation (e.g., to generate a cell therapy product). Because the kill switches of the present disclosure incorporate an essential protein, cell survival depends on the presence of the kill switch (and the lack of induction of a degron). When cells expressing the fusion protein are exposed to suitable conditions (e.g., a drug), degradation of the fusion protein can induce apoptosis and provide better control of induced apoptosis because: 1) the essential gene cannot be transcriptionally silenced without cell death; and 2) the kill switch itself (i.e., in its induced state) cannot be mutated without killing the cell.

[0009] In general, fusion proteins of the present disclosure comprise an essential polypeptide and one or more degrons, optionally connected via one or more peptide linkers. Fusion proteins are further described in Section 6.2 and numbered embodiments 1-56.

[0010] The present disclosure typically provides fusion proteins comprising an essential polypeptide or a fragment or derivative thereof. An essential polypeptide is a polypeptide encoded by an essential gene, a null mutation of which is detrimental to the survival of affected cells. Thus, degradation of an essential polypeptide via an inducible degron can be used to regulate the survival status of target cells. Exemplary essential polypeptides are described in Section 6.2.1 and numbered embodiments 40-56.

[0011] Fusion proteins of the present disclosure further comprise one or more degrons. Generally, a degron is a peptide sequence or protein element, e.g., a structural motif, a short amino acid sequence, or the like, that regulates the degradation rate of a protein, e.g., by targeting the protein for polyubiquitylation and subsequent degradation via the proteasome. In some embodiments, the fusion protein comprises a drug-inducible degron, whereby the stability of the degron is controlled by the presence or absence of a small molecule that binds to the degron. Further details regarding degrons and exemplary degrons are provided in Section 6.2.2 and numbered embodiments 2-27 and 35-39.

[0012] Fusion proteins of the present disclosure can further include an optional linker sequence between the degron sequence and the essential polypeptide sequence. In fusion proteins with multiple degrons, the individual degrons can be connected to each other via an optional linker. Section 6.2.3 and numbered embodiments 28-34 describe suitable optional linkers.

[0013] The present disclosure provides targeting constructs designed to generate genomic sequences in target cells that encode fusion proteins under the control of expression regulatory elements. The targeting constructs typically include homology arms that direct integration of the construct into a desired genomic locus, e.g., an essential gene locus, in the target cell genome, where a sequence encoding a degron and an optional linker flanks two homology arms that target the essential gene locus, such that the essential gene is modified to express a fusion protein comprising the essential polypeptide and the degron, optionally separated by a linker. Targeting constructs are further described in Section 6.3 and Subsection 6.3.1 and numbered embodiments 57-157. Sections 6.3.2 and 6.3.3 describe integration sites and homology arms, respectively, for targeting constructs of the present disclosure.

[0014] The constructs and methods of the present disclosure can also be used to generate target cells for expressing both (a) a fusion protein comprising an essential polypeptide and a degron and (b) a recombinant polypeptide. The recombinant polypeptide can be expressed from a transgene, and the transgene can be introduced into the target cell via the same targeting construct or expression vector as the targeting construct or expression vector comprising the degron-encoding sequence. Section 6.4 and numbered embodiments 235-258 further describe and provide exemplary transgenes.

[0015] The present disclosure further provides targeting constructs and recombinant target cell genomes that can include a separator sequence between the degron-encoding sequence and the transgene to allow for separate expression of polypeptides encoded by a single expression cassette. Exemplary separator sequences are described in Section 6.5.

[0016] The present disclosure also provides expression vectors encoding the fusion proteins of the present disclosure, which typically include an expression cassette comprising the fusion polypeptide operably linked to a regulatory element, such as a promoter, and optionally, an autonomously replicating element. Further information and examples regarding expression vectors are provided in Section 6.6 and numbered embodiments 314-318.

[0017] The present disclosure further provides methods and systems for producing gene-edited target cells comprising a nucleotide sequence encoding a fusion protein of the present disclosure. Further information and examples regarding suitable methods and systems are described in Sections 6.8 and 6.9, and in numbered embodiments 158-177.

[0018] For example, examples of recombinant and gene-edited target cells comprising nucleotide sequences encoding the fusion proteins of the present disclosure are disclosed, e.g., in Section 6.7 and numbered embodiments 178-325.

[0019] The present disclosure further provides methods of treating patients with cell therapy, including administering cells engineered to express a fusion protein comprising an essential protein and an inducible degron. If a subject experiences adverse effects of cell therapy or is considered at risk for adverse effects of cell therapy, the cells may be eliminated, in whole or in part, by induction of the degron, e.g., by administering an inducer of the degron to the subject. In some embodiments, the degron is a drug-inducible (e.g., IMiD-inducible) degron, and the cells are eliminated by administration of a drug (e.g., an IMiD). Further information and examples regarding the methods of the present disclosure are described in Section 6.11 and numbered embodiments 327-366. For cell therapy, the cells may be formulated as a pharmaceutical composition, e.g., as described in Section 6.10 and numbered embodiment 326.

[0020] Additional features, advantages, and uses of the fusion proteins, nucleic acids (targeting constructs, expression vectors), cells, and methods of the present disclosure are more particularly described below. 5. [Brief explanation of the drawings]

[0021] [Figure 1A]

[0023] Figure 1A shows an illustration of degron essential polypeptide fusion proteins and their coding sequences. Figure 1A shows 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]

[0023] Figure 1B shows an illustration of degron essential polypeptide fusion proteins and their coding sequences. Figure 1B shows 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]

[0033] Figure 1C is an illustration of a nucleic acid containing, from 5' to 3', the endogenous promoter of an essential gene, a transcription start site (represented by an arrow), and the coding sequence for a degron, an optional linker, and an essential polypeptide, which, upon expression, results in a fusion protein as shown in Figure 1A. [Figure 1D]

[0039] Figure 1D is an illustration of a nucleic acid containing, from 5' to 3', the endogenous promoter, transcription start site (represented by an arrow), and coding sequence for an essential polypeptide, an optional linker, and a degron, which, upon expression, results in a fusion protein as shown in Figure 1B. [Figure 2A]

[0023] Figure 2A is a schematic diagram of exemplary targeting constructs and vectors that can be used to generate or introduce into target cells a nucleic acid encoding a fusion protein of the present disclosure. Figure 2A depicts a targeting construct having a degron-encoding sequence flanked by first and second homology arms, where the degron-encoding sequence is connected at its 5' end to the first homology arm via an optional linker-encoding sequence, and the homology arms are configured such that integration of the targeting construct into an essential gene via recombination between the homology arms and 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 the optional linker. In some embodiments, the degron-encoding sequences are connected to each other via a linker sequence. [Figure 2B]

[0023] Figure 2B is a schematic diagram of exemplary targeting constructs and vectors that can be used to generate or introduce into target cells a nucleic acid encoding a fusion protein of the present disclosure. Figure 2B depicts a targeting construct having a degron-encoding sequence flanked by first and second homology arms, where the degron-encoding sequence is connected at its 3' end to the second homology arm via an optional linker-encoding sequence, and the homology arms are configured such that integration of the targeting construct into an essential gene via recombination between the homology arms and 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 the optional linker. In some embodiments, the degron-encoding sequences are connected to each other via a linker sequence. [Figure 2C]

[0023] Figure 2C is a schematic diagram of exemplary targeting constructs and vectors that can be used to generate or introduce into target cells a nucleic acid encoding a fusion protein of the present disclosure. Figure 2C depicts a targeting construct similar to the targeting construct shown in Figure 2A, but having two degron-encoding sequences connected to each other with a linker instead of a single degron-encoding sequence. While the targeting constructs in Figures 2C and 2D have two degron-encoding sequences, targeting constructs of the present disclosure can include three or more degron-encoding sequences. In some embodiments, the degron-encoding sequences are connected to each other via a linker sequence. [Figure 2D]

[0023] Figure 2D is a schematic diagram of exemplary targeting constructs and vectors that can be used to generate or introduce into target cells a nucleic acid encoding a fusion protein of the present disclosure. Figure 2D depicts a targeting construct similar to the targeting construct shown in Figure 2B, but having two degron-encoding sequences connected to each other with a linker instead of a single degron-encoding sequence. While the targeting constructs of Figures 2C and 2D have two degron-encoding sequences, targeting constructs of the present disclosure can include three or more degron-encoding sequences. In some embodiments, the degron-encoding sequences are connected to each other via a linker sequence. [Figure 3A]Figure 3A shows the integration of a targeting construct at an essential gene locus. Figure 3A is a schematic diagram of the monoallelic integration of the targeting construct shown in Figure 2A at an essential gene locus. A similar effect can be achieved by introducing an extrachromosomal vector to, for example, knock out an essential gene at one or both alleles. [Figure 3B] Figure 3B shows integration of a targeting construct at an essential gene locus. Figure 3B is a schematic diagram of biallelic integration of the targeting construct shown in Figure 2A at an essential gene locus. A similar effect can be achieved by introducing an extrachromosomal vector to, for example, knock out an essential gene at one or both alleles. [Figure 3C] Figure 3C shows integration of a targeting construct at an essential gene locus. Figure 3C is an illustration showing the mechanism of inducible degron-mediated degradation of essential polypeptides and apoptosis in cells whose genomes incorporate the targeting construct of Figure 2A. A similar effect can be achieved by introducing an extrachromosomal vector to knock out essential genes, for example, in one or both alleles. [Figure 3D] Figure 3D shows the integration of a targeting construct at an essential gene locus. Figure 3D is a schematic diagram of the single-allelic integration of the targeting construct shown in Figure 2B at an essential gene locus. A similar effect can be achieved by introducing an extrachromosomal vector to knock out an essential gene, for example, at one or both alleles. [Figure 3E] Figure 3E shows integration of a targeting construct at an essential locus. Figure 3E is a schematic diagram of biallelic integration of the targeting construct shown in Figure 2B at an essential locus. Figure 3F is an illustration showing the mechanism of inducible degron-mediated degradation of an essential polypeptide and apoptosis in cells whose genomes incorporate the targeting construct of Figure 2B. A similar effect can be achieved by introducing an extrachromosomal vector to knock out an essential gene, for example, at one or both alleles. [Figure 3F]Figure 3F shows integration of a targeting construct at an essential gene locus. Figure 3F is an illustration showing the mechanism of inducible degron-mediated degradation of essential polypeptides and apoptosis in cells whose genomes incorporate the targeting construct of Figure 2B. A similar effect can be achieved by introducing an extrachromosomal vector to knock out essential genes, for example, in one or both alleles. [Figure 4A] Schematic diagrams of exemplary targeting constructs containing a transgene in addition to a degron. Figure 4A shows a construct targeting the 3' end of an essential gene coding sequence, comprising, from 5' to 3', a first homology arm of the essential gene, an optional linker-encoding sequence, a degron-encoding sequence, an IRES-encoding 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 between the homology arms and the essential gene generates a modified essential gene encoding an essential polypeptide fused at its C-terminus to a degron via an optional linker, followed by an IRES and transgene. While Figures 4A-4D show the degron-encoding sequence and transgene separated by an IRES-encoding sequence, they may also be separated by a sequence encoding a self-cleaving peptide, such as a 2A peptide, in frame with the degron and transgene sequences. Furthermore, while the targeting constructs in Figures 4C and 4D have two degron-encoding sequences, targeting constructs of the present disclosure may include three or more degron-encoding sequences. In some embodiments, the degron coding sequences are connected to each other via a linker sequence. [Figure 4B]Schematic diagrams of exemplary targeting constructs containing a transgene in addition to a degron. Figure 4B shows a construct targeting the 5' end of an essential gene coding sequence, containing, from 5' to 3', a first homology arm of the essential gene, transgene, IRES coding sequence, degron coding sequence, 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 between the homology arms and the essential gene results in the generation of a modified essential gene encoding an essential polypeptide fused at its N-terminus to a degron via an optional linker, followed by an IRES and transgene. While Figures 4A-4D show the degron coding sequence and transgene separated by an IRES coding sequence, they may also be separated by a sequence encoding a self-cleaving peptide, such as a 2A peptide, in frame with the degron and transgene sequences. Furthermore, while the targeting constructs in Figures 4C and 4D have two degron coding sequences, targeting constructs of the present disclosure may include three or more degron coding sequences. In some embodiments, the degron coding sequences are connected to each other via a linker sequence. [Figure 4C]

[0039] Figures 4C and 4D are schematic diagrams of exemplary targeting constructs containing a transgene in addition to a degron. Figures 4C and 4D show targeting constructs similar to those in Figures 4A and 4B, respectively, but with two sets of degron sequences. While Figures 4A-4D show the degron coding sequence and transgene separated by an IRES coding sequence, they may also be separated by a sequence encoding a self-cleaving peptide, such as a 2A peptide, in frame with the degron and transgene sequences. Furthermore, while the targeting constructs in Figures 4C and 4D have two degron coding sequences, targeting constructs of the present disclosure can include three or more degron coding sequences. In some embodiments, the degron coding sequences are connected to each other via a linker sequence. [Figure 4D]

[0039] Figures 4C and 4D are schematic diagrams of exemplary targeting constructs containing a transgene in addition to a degron. Figures 4C and 4D show targeting constructs similar to those in Figures 4A and 4B, respectively, but with two sets of degron sequences. While Figures 4A-4D show the degron coding sequence and transgene separated by an IRES coding sequence, they may also be separated by a sequence encoding a self-cleaving peptide, such as a 2A peptide, in frame with the degron and transgene sequences. Furthermore, while the targeting constructs in Figures 4C and 4D have two degron coding sequences, targeting constructs of the present disclosure can include three or more degron coding sequences. In some embodiments, the degron coding sequences are connected to each other via a linker sequence. [Figure 5A] Figure 5A shows an exemplary effect of integration of a targeting construct at a target genomic locus and integration of the targeting construct at an essential gene. Figure 5A is a schematic diagram of monoallelic integration of the targeting construct shown in Figure 4A at an essential gene locus. [Figure 5B] Figure 5B shows an exemplary effect of integration of a targeting construct at a target genomic locus and integration of the targeting construct at an essential gene. Figure 5B is a schematic diagram of biallelic integration of the targeting construct shown in Figure 4A at an essential gene locus. [Figure 5C] Figure 5C shows an exemplary effect of integration of a targeting construct at a target genomic locus and integration of the targeting construct at an essential gene. Figure 5C is a schematic diagram of monoallelic integration of the targeting construct shown in Figure 4B at an essential gene locus. [Figure 5D] Figure 5D shows an exemplary effect of integration of a targeting construct at a target genomic locus and integration of the targeting construct at an essential gene. Figure 5D is a schematic diagram of biallelic integration of the targeting construct shown in Figure 4B at an essential gene locus. [Figure 6A]

[0033] Figure 6A shows the integration 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 transgene coding sequence designed to integrate immediately 5' of the endogenous stop codon of the first allele of an essential gene, and a second targeting construct comprising a linker and degron coding sequence designed to integrate immediately 5' of the endogenous stop codon of the second allele of the essential gene, for example, as shown in Figure 2A. [Figure 6B]

[0033] Figure 6B shows the integration of different targeting constructs at the first and second allele loci of an essential gene. Figure 6B is a schematic diagram of the genomic integration of a first targeting construct containing the coding sequence of a transgene and an IRES designed to be integrated immediately 3' of the ATG start codon of the second allele of an essential gene, and a second targeting construct containing the coding sequence of a linker and a degron designed to be integrated immediately 5' of the endogenous stop codon of the second allele of an essential gene, for example, as shown in Figure 2A. [Figure 6C]

[0033] Figure 6C shows the integration of different targeting constructs at the first and second allele loci of an essential gene. Figure 6C is a schematic diagram of the genomic integration of a first targeting construct containing an IRES and transgene coding sequence designed to integrate immediately 5' of the endogenous stop codon of the first allele of an essential gene, and a second targeting construct containing an optional linker and degron coding sequence designed to integrate immediately 3' of the ATG start codon of the second allele of the essential gene, for example, as shown in Figure 2B. [Figure 6D]6A and 6B show the integration of different targeting constructs at the first and second allele loci of an essential gene. Figure 6D is a schematic diagram of the genomic integration of a first targeting construct containing a linker and degron coding sequence designed to integrate immediately 3' of the ATG start codon of the first allele of the essential gene, a second targeting construct containing an IRES and transgene coding sequence designed to integrate immediately 5' of the endogenous stop codon of the first allele of the essential gene, and a third targeting construct containing a linker and degron coding sequence designed to integrate immediately 3' of the ATG start codon of the second allele of the essential gene. [Figure 6E]

[0033] Figure 6E shows the integration of different targeting constructs at the first and second allele loci of an essential gene. Figure 6E is a schematic diagram of the genomic integration of a first targeting construct containing coding sequences for a transgene and an IRES designed to integrate immediately 3' to the ATG start codon of the second allele of an essential gene, a second targeting construct containing coding sequences for a linker and a degron designed to integrate immediately 5' to the endogenous stop codon of the second allele of an essential gene, and a third construct containing coding sequences for a linker and a degron designed to integrate immediately 3' to the ATG start codon of the second allele of an essential gene. [Figure 7A] 7A-7J) or RPL13A (FIGS. 7K-7L). Thus, the nucleic acid insert is flanked by GAPDH or RPL13A homology arms in each construct. The construct in FIG. 7A (e.g., the construct containing the nucleotide sequence of SEQ ID NO: 1) has a nucleic acid insert flanked by left and right C-terminal GAPDH homology arms designed to integrate the nucleic acid insert at the 3' end of the GAPDH locus, whereby the nucleic acid insert contains, from the N- to C-terminus, a linker, i.e., linker 1 (GGS), and a degron (SEQ ID NO: 3). [Figure 7B]7A-7J) or RPL13A (FIGS. 7K-7L). Thus, the nucleic acid insert is flanked by GAPDH or RPL13A homology arms in each construct. The construct in FIG. 7B has a nucleic acid insert flanked by left and right C-terminal GAPDH homology arms, such that the nucleic acid insert contains, from the N- to C-terminus, linker 2 (SEQ ID NO: 23) and degron (SEQ ID NO: 3). [Figure 7C] 7A-7J) or RPL13A (FIGS. 7K-7L). Thus, the nucleic acid insert is flanked by GAPDH or RPL13A homology arms in each construct. The construct in FIG. 7C has a nucleic acid insert flanked by left and right C-terminal GAPDH homology arms, such that the nucleic acid insert contains, from N- to C-terminal, linker 3 (SEQ ID NO: 103) and a degron (SEQ ID NO: 3). [Figure 7D] 7A-7J) or RPL13A (FIGS. 7K-7L). Thus, the nucleic acid insert is flanked by GAPDH or RPL13A homology arms in each construct. The construct in FIG. 7D (e.g., the 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, such that the nucleic acid insert contains, from the N- to C-terminus, linker 4 (SEQ ID NO: 15) and superdegron (SEQ ID NO: 4). [Figure 7E]7A-7J) or RPL13A (FIGS. 7K-7L). Thus, the nucleic acid insert is flanked by GAPDH or RPL13A homology arms in each construct. The construct in FIG. 7E (e.g., the 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 designed to integrate the nucleic acid insert at the 5' end of the GAPDH locus, whereby the nucleic acid insert contains, from the N- to C-terminus, a degron (SEQ ID NO: 3) and a linker, i.e., linker1 (GGS). [Figure 7F] 7A-7J) or RPL13A (FIGS. 7K-7L). Thus, the nucleic acid insert is flanked by GAPDH or RPL13A homology arms in each construct. The construct in FIG. 7F (e.g., the 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, such that the nucleic acid insert contains, from the N- to C-terminus, a superdegron (SEQ ID NO: 4) and a linker, i.e., linker 4 (SEQ ID NO: 15). [Figure 7G] 7A-7J) or RPL13A (FIGS. 7K-7L). Thus, the nucleic acid insert is flanked by GAPDH or RPL13A homology arms in each construct. The construct in FIG. 7G has a nucleic acid insert flanked by left and right C-terminal GAPDH homology arms, such that the nucleic acid insert contains, from N to C-terminal, linker 1 (GGS), a degron (SEQ ID NO: 3), an IRES, and GFP. [Figure 7H]7A-7J) or RPL13A (FIGS. 7K-7L). Thus, the nucleic acid insert is flanked by GAPDH or RPL13A homology arms in each construct. The construct in FIG. 7H has a nucleic acid insert flanked by left and right C-terminal GAPDH homology arms, such that the nucleic acid insert contains, from N to C-terminal, linker 2 (SEQ ID NO: 23), a degron (SEQ ID NO: 3), an IRES, and GFP. [Figure 7I] 7A-7J) or RPL13A (FIGS. 7K-7L). Thus, the nucleic acid insert is flanked by GAPDH or RPL13A homology arms in each construct. The construct in FIG. 7I has a nucleic acid insert flanked by left and right C-terminal GAPDH homology arms, such that the nucleic acid insert contains, from N- to C-terminal, linker 3 (SEQ ID NO: 103), a degron (SEQ ID NO: 3), an IRES, and GFP. [Figure 7J] 7A-7L show schematic diagrams of exemplary targeting construct configurations, where the essential gene is GAPDH (FIGS. 7A-7J) or RPL13A (FIGS. 7K-7L). Thus, the nucleic acid insert is flanked by GAPDH or RPL13A homology arms in each construct. The construct in FIG. 7J has a nucleic acid insert flanked by left and right C-terminal GAPDH homology arms, such that the nucleic acid insert contains, from N to C-terminal, linker 4 (SEQ ID NO: 15), a superdegron (SEQ ID NO: 4), an IRES, and GFP. [Figure 7K]7A-7L show schematic diagrams of exemplary targeting construct configurations, where the essential gene is GAPDH (FIGS. 7A-7J) or RPL13A (FIGS. 7K-7L). Thus, the nucleic acid insert is flanked by GAPDH or RPL13A homology arms in each construct. The construct in FIG. 7K (e.g., the 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 designed to integrate the nucleic acid insert at the 3' end of the RPL13A locus, whereby the nucleic acid insert comprises, from the N- to C-terminus, a linker, i.e., linker 1 (GGS), and a degron (SEQ ID NO: 3). [Figure 7L] 7A-7J) or RPL13A (FIGS. 7K-7L). Thus, the nucleic acid insert is flanked by GAPDH or RPL13A homology arms in each construct. The construct in FIG. 7L (e.g., the 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 designed to integrate the nucleic acid insert at the 3' end of the RPL13A locus, whereby the nucleic acid insert contains, from the N- to C-terminus, a linker, i.e., linker 4 (SEQ ID NO: 15), and a superdegron (SEQ ID NO: 4). [Figure 8A] Figure 8A shows the effect of 3 μM pomalidomide (POM) treatment on the survival of cells edited with a targeting construct containing a GFP marker and a fusion protein in which the degron is linked to an essential gene. Figure 8A shows the fraction of untreated iPSCs gene-edited with a targeting construct containing GFP and a degron, as seen in Figure 7G (GFP+ data points within the box), and unedited cells (data points outside the box). [Figure 8B] Figure 8B shows the effect of 3 μM pomalidomide (POM) treatment on the survival of cells edited with a targeting construct containing a GFP marker and a fusion protein in which the degron is linked to an essential gene. Figure 8B shows the fraction of gene-edited and non-edited iPSCs after 6 days of POM treatment. [Figure 9A]Figure 9 shows the effect of 1 μM POM treatment on the survival of gene-edited iPSCs over time. Figure 9A is a graph showing the change in the number of viable cells / well at various time points, expressed as a percentage of starting cells / well (% of TO). [Figure 9B] Figure 9B shows the effect of 1 μM POM treatment on the survival of gene-edited iPSCs over time. Figure 9B is a representative image of a well containing gene-edited iPSCs after 92 hours of POM treatment. [Figure 9C] Figure 9C shows the effect of 1 μM POM treatment on the survival of gene-edited iPSCs over time. Figure 9C shows a representative image of untreated control wells containing gene-edited iPSCs monitored for 92 hours. [Figure 10A] Figure 10 shows the effect of linker length on the survival of iPSC pools gene-edited with targeting constructs, where the degron or super-degron is linked to the essential gene GAPDH, after treatment with 3 μM POM. Figure 10A is a graph showing the percentage of cells that are GFP-positive, as quantified using flow cytometry. [Figure 10B] Figure 10B shows the effect of linker length on the survival of iPSC pools gene-edited with targeting constructs, where the degron or superdegron is linked to the essential gene GAPDH, after treatment with 3 μM POM. Figure 10B is a graph showing the percentage of cells normalized to the untreated state, quantified using amplicon sequencing. [Figure 11A] Figure 11A shows qPCR results assessing GAPDH expression after different durations of 3 μM POM treatment, demonstrating activation of the targeting construct in homozygously gene-edited iPSCs, where the degron or superdegron is linked to GAPDH. [Figure 11B]This demonstrates activation of the targeting construct in homozygously gene-edited iPSCs, where the degron or super-degron is linked to GAPDH. Figure 11B shows Western blot analysis of GAPDH protein levels in cells gene-edited with a GAPDH-linked degron. Lane 1: molecular weight marker; Lane 2: untreated, 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] Activation of the targeting construct in homozygously gene-edited iPSCs was demonstrated, where the degron or superdegron was linked to GAPDH. Figure 11C shows Western blot analysis of GAPDH protein levels in cells gene-edited with a superdegron linked to GAPDH. Lane 1: molecular weight marker; Lane 2: untreated Clone A cells transfected with the superdegron; Lane 3: Clone A cells treated with 3 μM POM for 24 hours. [Figure 11D] Figure 11D demonstrates activation of targeting constructs in homozygously gene-edited iPSCs, where the degron or superdegron is linked to GAPDH. Figure 11D is a graph showing the growth rates of different iPSC lines gene-edited with targeting constructs containing a degron or superdegron linked to GAPDH. [Figure 12A] Figure 12 shows the effect of POM concentration on the survival of iPSCs gene-edited with targeting constructs containing a degron or superdegron linked to GAPDH. Figure 12A shows representative images of unedited and gene-edited iPSCs 5 days after 0.5 μM POM treatment. [Figure 12B]Figure 12B shows the effect of POM concentration on the survival of iPSCs gene-edited with a targeting construct containing a degron or superdegron linked to GAPDH. Figure 12B is a graph showing the difference in cell confluence over time, shown as the percentage of cells / well at the start of cells gene-edited with a targeting construct containing a degron linked to GAPDH, after treatment with different concentrations of POM ranging from 0.03125 to 10 μM. [Figure 12C] Figure 12C shows the effect of POM concentration on the survival of iPSCs gene-edited with a targeting construct containing a degron or superdegron linked to GAPDH. Figure 12C is a graph showing the difference in cell confluency, expressed as the percentage of cells / well at the start (% of TO), of cells gene-edited with a targeting construct containing a superdegron linked to GAPDH after treatment with different concentrations of POM ranging from 0.03125 to 10 μM. [Figure 13]

[0023] Figure 1 is a cartoon depicting 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 by reference in their entireties. [Figure 14] Representative images of parental DA neurons and gene-edited DA neurons of two lines after 5 days of 0.13 μM POM treatment are shown. [Figure 15A] Figure 15A shows the effect of POM concentration on cell survival of DA neurons. Figure 15A is a graph showing the percentage of cell death of unedited parental DA neurons after treatment with different POM concentrations for 5 days. [Figure 15B] Figure 15B shows the effect of POM concentration on DA neuron cell survival. Figure 15B is a graph showing the percentage of cell death in DA neurons derived from iPSCs gene-edited with targeting construct clone B after 5 days of treatment with POM concentrations ranging from 0.13 μM to 1 μM. [Figure 15C]Figure 15C shows the effect of POM concentration on DA neuron cell survival. Figure 15C is a graph showing the percentage of cell death in DA neurons derived from iPSCs gene-edited with targeting construct clone C after 5 days of treatment with POM concentrations ranging from 0.13 μM to 1 μM. [Figure 15D] Figure 15D shows the effect of POM concentration on DA neuron cell survival. Figure 15D is a graph showing the cell death rate of DA neurons derived from iPSCs gene-edited with targeting construct clone B after 5 days of treatment with POM concentrations ranging from 10 nM to 100 nM. [Figure 16A] Figure 16 shows the effect of a wide 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] Figure 16B shows the effect of a wide range of POM concentrations (0.01 μM to 100 μM) on the survival of DA neurons differentiated from unedited or gene-edited iPSCs. Figure 16B shows the percentage of cell death in DA neurons derived from iPSCs gene-edited with a targeting construct containing a 3-amino acid linker (3-aa linker) and a degron (clone A) after 6 days of treatment with POM concentrations ranging from 0.01 μM to 100 μM. [Figure 16C] Figure 16C shows the effect of a wide range of POM concentrations (0.01 μM to 100 μM) on the survival of DA neurons differentiated from unedited or gene-edited iPSCs. Figure 16C shows the same treatment on another gene-edited iPSC clone containing the same targeting construct with a 3 aa linker and degron (clone C). [Figure 17]

[0023] Figure 1 is a cartoon depicting 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., 2017 Jun 6;8(6):1516-1524, and PCT Publication Nos. 2023 / 150089 A1 and 2017 / 152081 A1, which are incorporated by reference in their entireties. [Figure 18A] Figure 18 shows the effect of 1 μM POM on cell survival of myeloid progenitor (MP) cells differentiated from unedited iPSCs or gene-edited iPSCs containing a targeting construct containing a 3-aa linker and a degron. Figure 18A is a graph showing the percentage cell death of MP cells differentiated from unedited parental iPSCs after treatment with or without POM. [Figure 18B] Figure 18B shows the effect of 1 μM POM on cell survival of myeloid progenitor (MP) cells differentiated from unedited iPSCs or gene-edited iPSCs containing a targeting construct comprising a 3-aa linker and a degron. Figure 18B is a graph showing the percentage cell death of MP cells derived from three different iPSC clones (clone A) gene-edited with a targeting construct comprising a 3-aa linker and a degron after treatment with 1 μM POM for 10 hours or left untreated. [Figure 18C] Figure 18C shows the effect of 1 μM POM on cell survival of myeloid progenitor (MP) cells differentiated from unedited iPSCs or gene-edited iPSCs containing a targeting construct comprising a 3-aa linker and a degron. Figure 18C is a graph showing the percentage cell death of MP cells derived from three different iPSC clones (clone B) gene-edited with a targeting construct comprising a 3-aa linker and a degron after treatment with 1 μM POM for 10 hours or left untreated. [Figure 18D]Figure 18 shows the effect of 1 μM POM on cell survival of myeloid progenitor (MP) cells differentiated from unedited iPSCs or gene-edited iPSCs containing a targeting construct comprising a 3-aa linker and a degron. Figure 18D is a graph showing the percentage cell death of MP cells derived from three different iPSC clones (clone C) gene-edited with a targeting construct comprising a 3-aa linker and a degron after treatment with 1 μM POM for 108 hours or left untreated. 6. DETAILED DESCRIPTION OF THE INVENTION

[0022] 6.1.Definition Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by one of ordinary skill in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this disclosure. In case of conflict, the present specification, including definitions, will control. Generally, the nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, pharmaceutical and medicinal chemistry, and protein and nucleic acid chemistry, and hybridization described herein are those well known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly practiced in the art or as described herein. Furthermore, unless the context otherwise requires, singular forms include plural forms and plural forms include the singular form. Throughout this specification and the embodiments, the words "have" and "comprise," or variations such as "has," "having," "comprises," or "comprising," should be understood to mean the inclusion of the stated element or group of elements, but not the exclusion of any other element or group of elements. All publications and other references mentioned herein are incorporated by reference in their entirety. Although several documents are cited herein, this citation does not constitute an admission that any of these documents forms part of the widespread general knowledge in the art.

[0023] 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 may be intact, live cells. For example, T cells, which can fight cancer cells via cell-mediated immunity, may be injected during immunotherapy. Cell therapy is also referred to as cellular therapy or cytotherapy.

[0024] 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 a portion thereof (e.g., an essential polypeptide, linker, or degron component of a fusion protein of the present disclosure). The coding sequence may be codon-optimized for expression in a cell of interest.

[0025] 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 pairs between nucleotides or nucleotide analogs of a nucleic acid molecule. "Complementarity" refers to the property shared between two nucleic acid sequences such that, when they are aligned antiparallel to each other, the nucleotide bases at each position are complementary.

[0026] Degron: The term "degron" refers to a peptide sequence, protein element, or portion of a protein that is involved in regulating the rate of protein degradation. 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, a suitable degron is constitutive (e.g., the degron is not drug-inducible, temperature-inducible, etc.) such that the degron exerts its effect on protein stability independently of external factors, while in other embodiments, the degron is inducible (e.g., the degron can be turned on or off by drugs, light exposure, temperature changes, etc.). In some embodiments, a degron provides an essential polypeptide, such as an essential polypeptide (e.g., GAPDH) to which it is fused with controllable stability. A fusion protein comprising a degron and an essential polypeptide can be maintained in an "on" (or stable) state until the cells expressing the fusion protein are removed, at which time the degron is induced, causing the fusion protein to become unstable and ultimately degraded, resulting in cell killing. In some embodiments, the degron is drug-inducible, for example, by an IMiD.

[0027] Electroporation: The term "electroporation" refers to the use of transmembrane electric field pulses to induce microscopic pores in biological membranes. These pores, commonly called "electropores," allow macromolecules, ions, and water to pass from one side of the membrane to the other. Typically, electroporation has been used to introduce drugs, DNA, or other molecules into cells. Electroporation is the basis for nucleofection, which combines electrophoretic principles with cell-type-specific reagents to transfer macromolecules directly into the nucleus of target cells.

[0028] Endonuclease: As used herein, the term "endonuclease" refers to an enzyme that cleaves phosphodiester bonds in a nucleic acid chain. The nucleic acid 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 cleave nucleic acids symmetrically, leaving "blunt" ends, or may generate overhangs that are not directly opposed and may be called "sticky ends." Sometimes, for convenience, the term endonuclease is simply referred to as "nuclease."

[0029] Essential gene: The term "essential gene" refers to a gene that is essential for the survival of a cell or organism. Null mutations in essential genes are detrimental to the survival of affected cells. Some essential genes are cell type or lineage specific, e.g., tumor-specific or neuronal-specific. Such lineage-specific essential genes are required for the survival of that cell type or lineage, but not other cell types, or in some cases, the survival of the entire organism (see, e.g., Zhang et al., 2021, Translational Psychiatry. 11(317)). The essential gene may be a STEL gene.

[0030] 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.

[0031] Gene-edited target cell: As used herein, the term "gene-edited target cell" refers to a cell that has been engineered to express a fusion protein (including an essential polypeptide sequence and a degron) of the present disclosure through the introduction of a targeting construct of the present disclosure, or its progeny and descendants. Typically, the nucleotide sequences flanking the homology arms of the targeting construct are integrated into the genome of the cell. The gene-edited target cell need not be the same cell type as the cell into which the targeting construct was originally introduced. For example, the targeting construct can be introduced into a stem cell, such as an iPSC or hESC, where the nucleotide sequences flanking the homology arms of the targeting construct are integrated into the genome of the stem cell. The stem cell can then be differentiated to produce a differentiated cell type, such as any of the cell types 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 a fusion protein of the present disclosure, the gene-edited target cell can 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 some embodiments, both the fusion protein coding sequence and the transgene are located at the same allele of the essential gene (whether heterozygous or homozygous). In other embodiments, the fusion protein coding sequence and the transgene are located at different alleles of the essential gene. In yet other embodiments, the fusion protein coding sequence and the transgene are at different loci. Alternatively, one or both of the fusion protein coding sequence and the transgene are expressed from an extrachromosomal expression vector, for example, in a cell in which the corresponding essential gene is knocked out at one or both alleles. In certain aspects, the gene-edited target cell has a single copy of the fusion protein coding sequence at one allele of the corresponding essential gene.

[0032] 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 with the target DNA (e.g., genomic DNA) to hybridize with the target DNA sequence and direct sequence-specific binding of the nucleic acid-targeting complex to the target DNA sequence. The DNA-targeting sequence generally 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 both (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 partially comprises two complementary stretches of nucleotides that hybridize with each other to form a double-stranded RNA duplex (dsRNA duplex). In some embodiments, the guide RNA is a single-stranded guide RNA (sgRNA).

[0033] 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, iveldomide, and avadomide.

[0034] iPSC: The term "induced pluripotent stem cell" or "iPSC" refers to a type of pluripotent stem cell artificially prepared from non-pluripotent cells, such as adult somatic cells, partially differentiated cells, or terminally differentiated cells, such as fibroblasts, hematopoietic lineage cells, muscle cells, neurons, and epithelial cells, by introducing or contacting the cells with one or more reprogramming factors. iPSCs can be derived from several different cell types, including terminally differentiated cells. iPSCs have embryonic stem (ES) cell-like morphology and grow as flat colonies with a large nucleic acid-to-cytoplasm ratio, distinct borders, and prominent nuclei. In addition, iPSCs express one or more key pluripotency markers known to those skilled in the art, including, but not limited to, alkaline phosphatase, SSEA3, SSEA4, Sox2, Oct3 / 4, Nanog, TRA160, TRA181, TDGF1, Dnmt3b, FoxO3, GDF3, Cyp26al, TERT, and zfp42.

[0035] Examples of methods for generating and characterizing iPSCs can be found, for example, in U.S. Patent Publication Nos. 2009 / 0047263, 2009 / 0068742, 2009 / 01911159, 2009 / 0227032, 2009 / 024675, and 2009 / 0304646, and PCT Patent Publication Nos. 2013 / 177133 and 2022 / 204567, the disclosures of which are incorporated herein by reference. Generally, to generate iPSCs, somatic cells are provided with reprogramming factors known in the art (e.g., Oct4, SOX2, KLF4, MYC, Nanog, Lin28, etc.) to reprogram somatic cells to become pluripotent stem cells.

[0036] Knockout: The term "knockout" and related terms (e.g., "knockout") refer to both the partial ablation of gene expression of one or more genes, or the complete ablation of gene expression of one or more genes, whether from one or both alleles. In some embodiments, the term knockout refers to the partial ablation of gene expression at one or both alleles (e.g., a modification that results in at least a 50%, at least a 60%, or at least a 70% reduction in gene expression in the absence of the modification). In other embodiments, the term knockout refers to the complete ablation of gene expression at one or both alleles.

[0037] 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, moieties, 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 joining or maintaining some minimum distance or other spatial relationship between components.

[0038] Nucleic Acid: As used herein, the term "nucleic acid" or "oligonucleotide" or "polynucleotide" refers to at least two nucleotides covalently linked to each other. A depiction of a single strand also defines the sequence of the complementary strand. Thus, a nucleic acid also encompasses the complementary strand of a depicted single strand. Many variants of a nucleic acid can be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also encompasses 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 encompasses a probe that hybridizes under stringent hybridization conditions.

[0039] Nucleic acids can be single-stranded or double-stranded, or can contain portions of both double-stranded and single-stranded sequence. Nucleic acids can be DNA, RNA, or hybrids, both genomic and cDNA, where the nucleic acid can contain combinations of deoxyribonucleotides and ribonucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, and isoguanine. Nucleic acids can be obtained by chemical synthesis or by recombinant methods.

[0040] Nuclease: The terms "nuclease" and "endonuclease" are used interchangeably herein to refer to enzymes that have endonucleolytic catalytic activity for nucleic acid cleavage, as well as nuclease-inactivated variants thereof.

[0041] 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.

[0042] Operably linked: The term "operably linked" refers to a functional relationship between two or more peptide or polypeptide domains or nucleic acid (e.g., DNA) segments. In the context of transcriptional regulation, the term refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence. For example, a promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system.

[0043] 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 polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids.

[0044] Pluripotency: 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 from somatic cell nuclear transfer, and iPSCs derived from non-pluripotent cells.

[0045] Recombinant target cell: As used herein, the term "recombinant target cell" refers to a cell engineered to express a fusion protein (including an essential polypeptide sequence and a degron) of the present disclosure, including the progeny and successors of a target cell into which a targeting construct or expression vector of the present disclosure was originally introduced. A recombinant target cell need not be the same cell type as the cell into which a targeting construct or expression vector was originally introduced. For example, a cell originally engineered to express a fusion protein of the present disclosure may be a stem cell, such as an iPSC or hESC. The stem cell can then be differentiated to produce a differentiated cell type, e.g., any of the cell types disclosed in Section 6.7.1. Both stem cells and differentiated cells are referred to herein as "recombinant target cells." In addition to encoding a fusion protein of the present disclosure, a recombinant target cell can contain a transgene, e.g., as described in Section 6.4. In some embodiments, the fusion protein coding sequence and the transgene are located in the same gene, e.g., 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 at the same allele of the essential gene (whether on a heterozygous or homozygous basis). In other embodiments, the fusion protein coding sequence and the transgene are located at 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, e.g., in a target cell where the essential gene is knocked out at one or both alleles. In certain aspects, a recombinant target cell has a single copy of the fusion protein coding sequence at one allele of the corresponding essential gene. In other aspects, a recombinant target cell has two or more copies of the fusion protein coding sequence, e.g., one or more copies at each allele of the corresponding essential gene.

[0046] Corresponding: As used herein in reference to the fusion proteins and essential genes of the present disclosure, the term "corresponding" 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).

[0047] 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 other small molecule that, when overexpressed or otherwise introduced into a cell, alone or in combination with other proteins, peptides, functional fragments of proteins or peptides, or other small molecules, induces the cell to transition from one differentiation state to another. In some embodiments, the reprogramming factor induces somatic cells to transition from a differentiation state to a pluripotent state. As used herein, a reprogramming factor can be a human protein or a modified version thereof that retains the desired biological effect.

[0048] Ribonucleoprotein (RNP) complex: As provided herein, a "ribonucleoprotein complex" or "ribonucleoprotein particle" refers to a complex or particle comprising a nucleoprotein and a ribonucleic acid. As provided herein, a "nucleoprotein" refers to a protein capable of binding to nucleic acids (e.g., RNA, DNA). When a nucleoprotein binds to a ribonucleic acid, it is referred to as a "ribonucleoprotein." The interaction between a ribonucleoprotein and a ribonucleic acid can be direct, e.g., by a covalent bond, or indirect, e.g., by a non-covalent bond (e.g., electrostatic interactions (e.g., ionic bonds, hydrogen bonds, halogen bonds), van der Waals interactions (e.g., dipole-dipole, dipole-induced dipole, London dispersion), ring stacking (pi effect), hydrophobic interactions, etc.). In embodiments, a ribonucleoprotein comprises an RNA-binding motif non-covalently bound to a ribonucleic acid. For example, positively charged aromatic amino acid residues (e.g., lysine residues) in the RNA-binding motif can form electrostatic interactions with the negative nucleic acid phosphate backbone of RNA, thereby forming a ribonucleoprotein complex. In some embodiments, any one of the nucleases disclosed herein is in an RNP having a guide RNA.

[0049] STEL: The term "persistent transgene expression locus" or "STEL" refers to a locus in the genome of a cell that allows for sustained and stable expression of a transgene in that cell. STELs of the present disclosure include, but are not limited to, robustly expressed endogenous genes, such as those involved in gene expression (e.g., transcription factors and histones), cellular metabolism (e.g., GAPDH), or cellular structure (e.g., actin), or the loci of certain housekeeping genes that are active in multiple cell types, such as those encoding ribosomal proteins (e.g., large or small ribosomal subunits such as RPL13A, RPLP0, and RPL7). Additional examples of STELs include those that form ribonucleoprotein complexes, focal adhesions, cell-matrix adhesive junctions, cell-matrix junctions, cell anchorages, extracellular exosomes, extracellular vesicles, intracellular organelles, or anchorage junctions. Some of the proteins are involved in RNA binding, nucleic acid binding (e.g., rRNA or mRNA binding), or protein binding. The STEL gene may also be an essential gene (sometimes referred to herein as an "essential STEL gene").

[0050] 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.

[0051] Subject: The term "subject" or "patient" refers to an organism that undergoes a procedure and / or treatment of the present disclosure. Subjects can include human 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 a preferred embodiment, the subject is a human.

[0052] Target cell: The term "target cell" refers to a host cell into which (i) an expression vector or (ii) a targeting construct has been introduced, which, after integration into the host cell genome, results in the production of a recombinant nucleic acid encoding a fusion protein comprising the essential polypeptide, the degron, and an optional linker. It is understood that such terms are intended to refer not only to the particular 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 originally introduced, but include counterparts and progeny of the cell carrying the expression cassette or into which the targeting construct has been integrated, as well as cells differentiated therefrom. Such counterparts and progeny remain within the scope of the term "target cell" as used herein.

[0053] 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, e.g., to modify an essential polypeptide, include a degron-encoding sequence, and / or introduce a transgene into the essential locus. Typically, the targeting construct contains homology arms that allow integration of the targeting construct into a specific locus, e.g., an essential gene.

[0054] 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, e.g., a eukaryotic cell. In the context of the present invention, the term "transfection" encompasses any method known to those of skill in the art for introducing a nucleic acid molecule into a cell, e.g., a eukaryotic cell, such as a mammalian cell. Such methods include, for example, electroporation, nucleofection, lipofection based on, e.g., cationic lipids and / or liposomes, calcium phosphate precipitation, nanoparticle-based transfection, viral-based transfection, or transfection based on cationic polymers such as DEAE-dextran or polyethyleneimine.

[0055] Vector: The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, into which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of the host cell upon introduction into the host cell, and thereby are replicated along with the host genome. In some embodiments, the vector is a viral vector, e.g., a lentiviral vector, an adenoviral vector, or an adeno-associated viral (AAV) vector. Furthermore, certain vectors are capable of directing the expression of nucleotide sequences to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0056] Fusion Proteins 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 at the N-terminus of the essential polypeptide. In other embodiments, the degron is at the C-terminus of the essential polypeptide.

[0057] Nucleic acids encoding the fusion proteins of the present disclosure can be introduced into target cells, for example, in the form of a vector as described in Section 6.6, or in the form of a targeting construct that results in the integration of the exogenous nucleotide sequence encoding the fusion protein into an essential gene in the genome of the target cell.

[0058] In some embodiments, a nucleic acid encoding a fusion protein of the present disclosure can be purified in situ in a target cell by introducing a targeting construct, e.g., as described in Section 6.3, that recombines with an essential gene in the target cell genome to create a modified essential gene encoding a fusion protein of the present disclosure comprising: (i) an essential polypeptide as described in Section 6.2.1; (ii) a degron as described in Section 6.2.2; (iii) and an optional linker, as described in Section 6.2.3, connecting the essential polypeptide and the degron.

[0059] Thus, if a fusion protein is expressed as a result of integration of a targeting construct encoding a degron into an essential gene encoding an essential polypeptide, the essential gene is an essential gene of the targeting construct. If a fusion protein is expressed as a result of 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 an essential gene of the targeting construct.

[0060] In some embodiments, the fusion protein is configured such that the degron is at the N-terminus of the essential polypeptide, for example, as shown in Figure 1A. In other embodiments, the fusion protein is configured such that the degron is at the C-terminus of the essential polypeptide, for example, as shown in Figure 1B.

[0061] In some embodiments, the fusion protein contains only one degron. In other embodiments, the fusion protein contains two or more degrons. In some embodiments, the degrons are in tandem and separated by a linker. A fusion protein containing only one degron is produced after integration of the targeting construct shown in Figures 2A and 2B. A fusion protein containing two degrons is produced after integration of the targeting construct shown in Figures 2C and 2D.

[0062] In some embodiments, the fusion protein further comprises a self-cleaving peptide sequence, e.g., as described in Section 6.5, and a polypeptide encoded by a transgene, e.g., as described in Section 6.4. Such fusion proteins can be produced, for example, by incomplete processing of the self-cleaving peptide. Proper processing of a fusion protein having a self-cleaving peptide sequence at the N- or C-terminus results in a fusion protein comprising several amino acid residues of the self-cleaving peptide. Fusion proteins comprising a properly processed or incompletely processed self-cleaving peptide sequence (and, in some embodiments, a polypeptide sequence encoded by a transgene) are encompassed by the term "fusion proteins of the present disclosure."

[0063] Thus, in some embodiments, the fusion proteins of the present disclosure lack a self-cleaving peptide sequence. In other embodiments, the fusion proteins of the present disclosure comprise a properly processed self-cleaving peptide sequence (which may be a single amino acid residue). In further embodiments, the fusion proteins of the present disclosure comprise an incompletely processed self-cleaving peptide sequence and, optionally, a transgene-encoded polypeptide sequence.

[0064] In some embodiments, the degron is an inducible degron. Fusing an essential polypeptide to a degron allows its stability to be controlled by the 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 and the essential polypeptide is unstable, leading to its destruction. Destruction of the essential polypeptide is detrimental to cell survival. Thus, the fusion proteins of the present disclosure can be used to control cell survival.

[0065] 6.2.1 Essential Polypeptides Fusion proteins of the present disclosure typically include an essential polypeptide or a fragment or derivative thereof (all collectively referred to herein for convenience 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.

[0066] In some embodiments, the essential polypeptide (whether a STEL polypeptide or a non-STEL polypeptide) is from a category of essential polypeptides identified below. In other embodiments, the essential polypeptide (whether a STEL polypeptide or a non-STEL polypeptide) is not from a category of essential polypeptides identified below (e.g., that category is subject to a provisos).

[0067] Furthermore, in some embodiments, the essential polypeptide (whether a STEL polypeptide or a non-STEL polypeptide) is one of the essential polypeptides identified below. In other embodiments, the essential polypeptide (whether a STEL polypeptide or a non-STEL polypeptide) is not one of the essential polypeptides identified below (e.g., certain essential polypeptides are subject to provisos).

[0068] 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.

[0069] In some embodiments, the essential polypeptide is a ribosomal polypeptide (RPL), e.g., 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.

[0070] In some embodiments, the essential polypeptide is a ribosomal polypeptide small subunit (RPS), e.g., 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.

[0071] In some embodiments, the essential polypeptide is a cytoskeletal protein such as actin. Examples of actin-encoding genes are ACTG1 and ACTB.

[0072] 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.

[0073] In some embodiments, the essential polypeptide is a histone, for example, a histone encoded by genes H3F3A and H3F3B.

[0074] 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.

[0075] In other embodiments, the essential polypeptide is a non-STEL polypeptide, such as HDAC3, DNMT1, NADH dehydrogenase, and PGK1.

[0076] Fusion proteins of the present disclosure typically include a native essential polypeptide sequence, for example, when the fusion protein coding sequence is constructed upon integration of a targeting construct into an essential locus.

[0077] 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 entire fusion protein. Without being bound by theory, it is believed that recombinant expression of a fusion protein comprising an essential polypeptide and a degron "poisons" the native cellular essential polypeptide, resulting in its destabilization when the degron is activated, leading to cell death even if 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.

[0078] 6.2.2.Degron The fusion proteins described herein contain one or more peptide sequences that can function as "shut-off switches" or "kill switches" to eliminate 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.

[0079] Generally, degrons are peptide sequences or protein elements that regulate the rate of protein degradation, for example, by targeting proteins for polyubiquitylation and subsequent degradation via the proteasome. Degrons can include short amino acid sequences, structural motifs, and exposed amino acids (e.g., lysine or arginine).

[0080] The stability of the degron is controlled, at least in part, by the degron sequence. In some embodiments, a suitable degron is constitutive, such that the degron exerts its effect on protein stability independent of experimental controls (e.g., the degron is not drug-inducible, temperature-inducible, etc.). In some embodiments, the degron provides the essential polypeptide (e.g., GAPDH) to which it is fused with controllable stability, such that the fusion protein can be turned "on" (e.g., stable) or "off" (e.g., unstable, degraded) depending on desired conditions.

[0081] In some embodiments, the degron is a drug-inducible degron, whereby 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.

[0082] Examples of suitable degrons regulated by the presence or absence of a small molecule include, but are not limited to, degrons regulated by immunomodulatory imide drugs (IMiDs, e.g., pomalidomide, thalidomide, lenalidomide, iverdomide, avadomide, etc.), Shield-1, DHFR, and / or auxin. Other inducible degrons are temperature-sensitive degrons, light-inducible degrons, and degrons activated by the expression of another protein, e.g., TEV protease. Non-limiting examples of suitable degrons are known in the art (see, 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 incorporated herein by reference in their entireties).

[0083] 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 that can be controlled by an IMiD, such as thalidomide, lenalidomide, pomalidomide, and / or analogs thereof. In some embodiments, the IMiD-sensitive degron is an engineered degron, e.g., a superdegron, that has increased susceptibility to an IMiD, allowing for more efficient degradation of the essential polypeptide compared to degradation achieved with a non-engineered degron.

[0084] Fusing a degron sequence to a polypeptide sequence can be used to produce a polypeptide with an off-switch. For example, fusing an IMiD-sensitive degron to an essential polypeptide such as GAPDH results in a GAPDH-degron fusion protein, the expression of which can be turned off in the presence of an IMiD such as pomalidomide through targeted degradation of the GAPDH-degron fusion protein. Degradation of the fusion protein containing the essential polypeptide and degron can result in apoptosis of cells engineered to express the fusion protein.

[0085] In some embodiments, the fusion proteins of the present disclosure function as a kill switch to eliminate cells engineered to express the fusion protein. In some embodiments, the fusion proteins of the present disclosure enable in vitro cell elimination, for example, in the context of functional screening as disclosed by Natsume and Kanemaki, 2017, Annu Rev Genet. 51:83-102, where rapid control of expression of a protein of interest fused to a degron is enabled to determine whether the protein of interest is essential for cell survival. In other embodiments, the fusion proteins of the present disclosure enable in vivo cell elimination, for example, following gene therapy as disclosed in Section 6.11.

[0086] In some embodiments, the degron is an inducible degron.

[0087] In some embodiments, the degron is inducible by a small molecule, e.g., a drug.

[0088] In some embodiments, the degron is inducible by an IMiD. Examples of IMiD-inducible degron sequences are disclosed in Koduri et al., 2019, Proc. Nat'l Acad. Sci. USA 116(7):2539-2544, WO2021 / 188286 A2, and WO2019 / 089592 A1, the contents of each of which are incorporated herein in their entirety.

[0089] In some embodiments, the degron comprises the amino acid sequence RPFQCNQCGASFTQKGNLLRHIKLH (SEQ ID NO: 3, Koduri et al.), FNVLMVHKRSHTGERPLQCEICGFTCRQKGNLLRHIKLHTGEKPFKCHLCNYACQRRDAL (SEQ ID NO: 4, corresponding to SEQ ID NO: 42 in WO2021 / 188286 A2), FNVLMVHKRSHTGERP (SEQ ID NO: 5, corresponding to SEQ ID NO: 97 in WO2019 / 089592 A1), FNVLMVHRRSHTGERP (SEQ ID NO: 6, corresponding to SEQ ID NO: 100 in WO2019 / 089592 A1), TGEKPFKCHLCNYACQRRDAL (SEQ ID NO: 7, corresponding to SEQ ID NO: 102 in WO2019 / 089592 A1), TGEKPFKCHLCNYACQRRDAL (SEQ ID NO: 8, corresponding to SEQ ID NO: 103 in WO2019 / 089592 A1), or TGERPFRCHLCNYACQRRDAL (SEQ ID NO: 94 in WO2019 / 089592 A1). A2), FQCNQCGASFT (SEQ ID NO: 9, corresponding to SEQ ID NO: 528 of WO2021 / 188286 A2), FQCPICGLVIK (SEQ ID NO: 10, corresponding to SEQ ID NO: 529 of WO2021 / 188286 A2), LQCEICGFTCR (SEQ ID NO: 11, corresponding to SEQ ID NO: 530 of WO2021 / 188286 A2), LQCEICGYQCR (SEQ ID NO: 12, corresponding to SEQ ID NO: 531 of WO2021 / 188286 A2), or LQCEVCGFQCR (SEQ ID NO: 13, corresponding to SEQ ID NO: 532 of WO2021 / 188286 A2).

[0090] In some embodiments, the degron is a superdegron. SEQ ID NO: 4 is an example of a superdegron sequence.

[0091] In some embodiments, the degron is a SMASh (small molecule-assisted shutoff) tag degron, which is a self-cleaving degron that can be stabilized by treatment with a small molecule, such as asunaprevir. In the absence of a protease inhibitor, the SMASh tag self-cleaves, and the protein is expressed at a relatively normal level. However, in the presence of a drug, the SMASh tag degron remains fused to the protein, causing all newly synthesized fusion proteins to be rapidly degraded.

[0092] Linker The fusion proteins of the present disclosure can include an optional linker sequence between the essential polypeptide sequence and the degron sequence.

[0093] 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 certain embodiments, a linker is used to separate the essential polypeptide and the degron by a distance sufficient to ensure that the essential polypeptide retains its required functional properties. In some embodiments, the peptide linker sequence adopts a flexible extended conformation and does not exhibit a tendency to develop an ordered secondary structure.

[0094] Typical amino acids in flexible peptide linkers include Gly, Asn, and Ser. Thus, in certain embodiments, the linker comprises one or more combinations 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. Pat. Nos. 4,935,233 and 4,751,180, the entire contents of which are incorporated herein by reference.

[0095] The peptide linker can be a single amino acid sequence or one or more repeats of an amino acid sequence. In some embodiments, the sequence can be used in two repeats. In some embodiments, the sequence can be used in three repeats. In some embodiments, the sequence can be used in four repeats. In some embodiments, the sequence can be used in five or more repeats.

[0096] In some embodiments, the peptide linker is 1 to 30 amino acids in length. In various aspects, the peptide linker is 1 to 3 amino acids in length, 3 to 8 amino acids in length, 3 to 10 amino acids in length, 5 to 15 amino acids in length, 11 to 20 amino acids in length, 15 to 25 amino acids in length, 21 to 30 amino acids in length, or a range of lengths bounded by any pair of the above values ​​(e.g., 3 to 15 amino acids in length, 8 to 20 amino acids in length, 25 to 30 amino acids in length, etc.).

[0097] 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 above values ​​(e.g., 1-3 amino acids, 1-12 amino acids in length, 2-12 amino acids in length, 1-10 amino acids in length, etc.).

[0098] Non-limiting examples of linker sequences are shown in Table 1 below. [Table 1]

[0099] 6.3. Targeting constructs 6.3.1. Targeting Constructs for Fusion Protein Expression The present disclosure provides targeting constructs designed to generate, under the control of expression regulatory elements, a genomic sequence in a target cell that encodes a fusion protein as described herein, e.g., an essential polypeptide as described in Section 6.2.1, a degron, and, optionally, a linker sequence. The targeting construct typically includes homology arms that direct integration of the construct into an intended genomic locus within the target cell genome.

[0100] The targeting constructs of the present disclosure can include the entire coding sequence of a fusion protein, for integration of the entire fusion protein coding sequence into a genomic locus in the genome of a target cell. Depending on the site of integration, the targeting construct can further include expression control sequences, such as a promoter sequence, or can utilize expression control sequences within the genome of the target cell at the intended integration site.

[0101] In some 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 in addition to the homology arms. The homology arms may or may not include essential polypeptide coding sequences, as the coding sequence of the entire fusion protein (contiguous or with intron sequences) is assembled upon integration of the targeting construct into the target cell genome. The homology arms can be designed to recombine with essential genes and / or flanking sequences.

[0102] In some embodiments, a targeting construct of the present disclosure comprises: (i) a first homology arm, as described in Section 6.3.3, corresponding to a 5' target sequence that includes a first region of homology to an essential gene or its adjacent sequence, as described in Section 6.3.2; (ii) a nucleotide sequence encoding a degron, e.g., an inducible degron, such as a drug-inducible degron ("degron-encoding sequence") as described in Section 6.2.2, and optionally, a linker 5' or 3' to the degron-encoding sequence; (iii) a second homology arm, as described in Section 6.3.3, corresponding to a 3' target sequence that includes a second region of homology to an essential gene or its adjacent sequence, as described in Section 6.3.2; Here, 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 an essential polypeptide, a degron, and optionally, a linker.

[0103] In certain embodiments, the targeting construct further comprises a transgene, e.g., as described in Section 6.4, between the degron sequence and the second arm of homology. In certain other embodiments, the targeting constructs of the present disclosure further comprise a separator sequence, e.g., as described in Section 6.5, wherein the fusion protein coding sequence and the transgene are connected via the separator sequence, e.g., a nucleotide sequence encoding an internal ribosome entry site (IRES) or a self-cleaving peptide.

[0104] Exemplary configurations of targeting constructs lacking a transgene are shown in FIGS.

[0105] Exemplary configurations of targeting constructs containing transgenes are shown in Figure 4, and exemplary configurations of targeting construct integration into essential gene loci are shown in Figures 5A-5D. As shown in Figures 5A-5D, the targeting construct can integrate 5' of the essential protein coding sequence or 3' of the essential protein coding sequence, allowing for selection of cells that are either heterozygous or homozygous for the essential gene modification.

[0106] Alternatively (or in addition) to incorporating a transgene into a targeting construct containing a degron-encoding sequence, a first targeting construct containing a degron-encoding sequence can be introduced into cells that lack a degron but have a second targeting construct containing a transgene. Both targeting constructs may target the same gene, followed by selection for heterozygous cells as shown in Figures 6A-6F, or they may target different loci such that the transgene and the fusion protein containing the essential polypeptide and degron are expressed from separate genes. In some embodiments, both the first and second targeting constructs are introduced into the STEL locus, as described in PCT Application No. 2021 / 072329A1. In some embodiments, the first and second targeting constructs are introduced into the same STEL locus, e.g., the GAPDH locus. In other embodiments, the first and second targeting constructs are introduced into different STEL loci, e.g., the GAPDH locus and another STEL locus. In some embodiments, a first targeting construct (i.e., a targeting construct comprising a degron coding sequence) is introduced into the GAPDH locus, and a second targeting construct (i.e., a targeting construct comprising a transgene) is introduced into a different STEL locus. In other embodiments, a second targeting construct (i.e., a targeting construct comprising a transgene) is introduced into the GAPDH locus, and a first targeting construct (i.e., a targeting construct comprising a degron coding sequence) is introduced into a different STEL locus. In yet other embodiments, a first targeting construct (i.e., a targeting construct comprising a degron coding sequence) is introduced into an essential gene that is not a STEL gene, and a second targeting construct (i.e., a targeting construct comprising a transgene) is introduced into the STEL locus.

[0107] Nucleic acid sequences, eg, constructs, are listed in Table 2. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10] [Table 2-11] [Table 2-12]

[0108] Methods for introducing targeting constructs into target cells are described in Sections 6.8 and 6.9.

[0109] In some embodiments, target cells are modified to contain only one copy of a nucleotide sequence encoding a fusion protein comprising an essential polypeptide and a degron. For example, when the targeting construct is integrated into the host cell genome, engineered cells containing the targeting construct insert in only one allele of the essential gene are selected. In some embodiments, the second allele is engineered to incorporate a transgene, for example, by introducing a second targeting construct containing the transgene and a targeting construct containing a degron-encoding sequence.

[0110] In other embodiments, the target cells are modified to contain two copies of a nucleotide sequence encoding a fusion protein comprising an essential polypeptide and a degron, e.g., when the targeting construct is integrated into the host cell genome, engineered cells containing targeting construct inserts into both alleles of the essential gene are selected.

[0111] 6.3.2. Integration Site Targeting constructs intended for integration into the target cell genome typically include a heterologous sequence, such as a degron-encoding sequence or a transgene, that is not present in the target cell genome.

[0112] In some embodiments, a degron sequence is introduced into an essential gene such that the essential gene is modified to express a fusion protein comprising the essential polypeptide and the degron, optionally separated via a linker. One skilled in the art will readily appreciate that the term "essential gene" is not limited to the STEL gene. However, for robust expression from the STEL locus, in some embodiments, the essential gene into which the degron is introduced is also a STEL gene, particularly when used to co-localize the transgene in the genome of the host cell.

[0113] In some embodiments, the degron sequence is introduced into an essential gene (whether a STEL gene or a non-STEL gene) of the functional categories of essential genes identified below. In other embodiments, the degron sequence is introduced into an essential gene (whether a STEL gene or a non-STEL gene) that is not from the categories of essential genes identified below (e.g., the categories are subject to provisos).

[0114] In some embodiments, the degron sequence is introduced into an individual essential gene (whether a STEL gene or a non-STEL gene) selected from the individual essential genes identified below. In other embodiments, the degron sequence is introduced into an essential gene (whether a STEL gene or a non-STEL gene) that is not one of the essential genes identified below (e.g., the particular essential gene is subject to provisos).

[0115] In some embodiments, the essential gene is the GAPDH gene.

[0116] If the essential gene is the STEL gene, the transgene may be introduced into the STEL locus so that the transgene can be expressed from the locus for sustained expression. In some embodiments, the transgene is introduced into the GAPDH gene.

[0117] The degron coding sequence and the transgene can be introduced into the same STEL gene, whether 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 coding sequence and the transgene.

[0118] A targeting construct typically contains one or more regions that are homologous to regions of DNA within or near (e.g., adjacent or neighboring) the target sequence. These homologous regions are referred to herein as "homology arms." For ease of reference, the homology arms are referred to herein as first and second (i.e., 5' and 3', upstream and downstream, or left and right) homology arms. This term refers to the relative position of the homology arms with respect to the nucleic acid insert within the targeting construct. The first and second homology arms correspond to regions within the target genomic locus, referred to herein as the "first homology region" and "second homology region," respectively.

[0119] In some embodiments, the targeting construct comprises homology arms that target integration of a heterologous sequence into an essential locus, whereby integration of the heterologous sequence introduces the degron coding sequence in-frame with the essential polypeptide coding sequence, connected directly or via a linker sequence, such that after integration, the essential locus is engineered to express a fusion polypeptide of the present disclosure.

[0120] 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 encode ribosomal proteins (e.g., large and small ribosomal subunits, such as RPL13A, RPLP0, and / or RPL7).

[0121] Further examples of essential genes include those involved in one or more of glycolysis, ribonucleoprotein complex formation, focal adhesions, cell-substrate adherens junctions, cell-substrate junctions, cell anchorage, extracellular exosomes, extracellular vesicles, intracellular organelles, or anchorage junctions. Some of the proteins are involved in RNA binding, nucleic acid binding (e.g., rRNA or mRNA binding), or protein binding.

[0122] In some embodiments, the essential gene is robustly and consistently expressed in the pluripotent state as well as during differentiation (e.g., as examined by single-cell RNA sequencing (scRNAseq) analysis). For example, the expression level of the endogenous gene does not change (e.g., decrease) by more than 50%, more than 40%, more than 35%, more than 30%, more than 25%, more than 20%, more than 15%, more than 10%, or more than 5% over 5 or more passages or as the cell state changes (e.g., pluripotency and / or differentiation state).

[0123] In some embodiments, the essential gene is a gene associated with cellular metabolism, such as GAPDH.

[0124] In some embodiments, the essential gene is a ribosomal protein gene or ribosomal protein locus, such as the RPL or RPS locus. Examples of RPL genes are RPL10, RPL13, RPS18, RPL3, RPLP1, RPL13A, RPL15, RPL41, RPL11, RPL32, RPL18A, RPL19, RPL28, RPL29, RPL9, RPL8, RPL6, RPL18, RPL7, RPL7A, RPL21, RPL37A, RPL12, RPL5, RPL34, RPL35A, RPL30, RPL24, RPL39, RPL37, RPL14, RPL27A, RPLP2, RPLP0, RPL23A, RPL26, RPL36, RPL35, RPL23, RPL4, and RPL22. Examples of RPS genes are RPS2, RPS19, RPS14, RPS3A, RPS12, RPS3, RPS6, RPS23, RPS27A, RPS8, RPS4X, RPS7, RPS24, RPS27, RPS15A, RPS9, RPS28, RPS13, RPSA, RPS5, RPS16, RPS25, RPS15, RPS20, and RPS11.

[0125] In some embodiments, the essential gene encodes a cytoskeletal protein, such as actin. Examples of actin genes are ACTG1 and ACTB.

[0126] In some embodiments, the essential genes encode eukaryotic translation elongation factors, such as EEF1A1 and EEF2, or eukaryotic translation initiation factors, such as EIF1.

[0127] In some embodiments, the essential genes encode histones such as H3F3A and H3F3B.

[0128] In other embodiments, the essential gene is a STEL gene selected from FTH1, TPT1, PTMA, GNB2L1, NACA, YBX1, NPM1, FAU, UBA52, HSP90AB1, MYL6, SERF2, and SRP14.

[0129] In other embodiments, the essential gene is a non-STEL gene, such as HDAC3, DNMT1, NADH dehydrogenase, and PGK1.

[0130] 6.3.3.Homology Arm The present disclosure provides a targeting construct comprising a first homology arm corresponding to a first homology region, a nucleic acid insert, and a second homology arm corresponding to a second homology region.

[0131] A homology arm and a target sequence "correspond" or "correspond" to one another if the two regions share a sufficient level of sequence identity with each other to act as substrates for a homologous recombination reaction, whereby the homology arm is suitable for directing recombination of the nucleic acid insert with a desired genomic locus to facilitate genomic integration and / or replacement of the endogenous sequence.

[0132] The term "homology" includes DNA sequences that are either identical to or share sequence identity with corresponding sequences. The sequence identity between a given target sequence and the corresponding homology arm found in the exogenous donor nucleic acid can be any degree of sequence identity that allows homologous recombination to occur. For example, the amount of sequence identity shared by the homology arm of the exogenous donor nucleic acid (or a fragment thereof) and the target sequence (or a fragment thereof) can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, resulting in the sequences undergoing homologous recombination. Furthermore, the corresponding homologous regions between the homology arms and the corresponding target sequence can be of any length sufficient to promote homologous recombination. In some targeting vectors, the intended mutation of the target locus is contained in the insert nucleic acid flanking the homology arms.

[0133] In some embodiments, the first homology arm is 50 to 250 nucleotides in length. In some embodiments, the first homology arm is 50 to 2000 nucleotides in length. In some embodiments, the first homology arm is 50 to 1500 nucleotides in length. In some embodiments, the first homology arm is 50 to 1000 nucleotides in length. In some embodiments, the first homology arm is 50 to 500 nucleotides in length. In some embodiments, the first homology arm is 150 to 250 nucleotides in length. In some embodiments, the first homology arm is 2000 nucleotides in length or less. In some embodiments, the first homology arm is 1500 nucleotides in length or less. In some embodiments, the first homology arm is 1000 nucleotides in length or less. In some embodiments, the first homology arm is 700 nucleotides in length or less. In some embodiments, the first homology arm is 650 nucleotides in length or less. In some embodiments, the first homology arm is 600 nucleotides or less in length. In some embodiments, the first homology arm is 550 nucleotides or less in length. In some embodiments, the first homology arm is 500 nucleotides or less in length. In some embodiments, the first homology arm is 400 nucleotides or less in length. In some embodiments, the first homology arm is 300 nucleotides or less in length. In some embodiments, the first homology arm is 250 nucleotides or less in length. In some embodiments, the first homology arm is 200 nucleotides or less in length. In some embodiments, the first homology arm is 150 nucleotides or less in length. In some embodiments, the first homology arm is less than 100 nucleotides in length. In some embodiments, the first homology arm is 50 nucleotides or less in length.In some embodiments, the first homology arm is 250, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, or 20 nucleotides in length. In some embodiments, the first homology arm is at least 20 nucleotides in length. In some embodiments, the first homology arm is at least 40 nucleotides in length. In some embodiments, the first homology arm is at least 50 nucleotides in length. In some embodiments, the first homology arm is at least 70 nucleotides in length. In some embodiments, the first homology arm is at least 100 nucleotides in length. In some embodiments, the first homology arm is at least 200 nucleotides in length. In some embodiments, the first homology arm is at least 300 nucleotides in length. In some embodiments, the first homology arm is at least 400 nucleotides in length. In some embodiments, the first homology arm is at least 500 nucleotides in length. In some embodiments, the first homology arm is at least 600 nucleotides in length. In some embodiments, the first homology arm is at least 700 nucleotides in length. In some embodiments, the first homology arm is at least 1000 nucleotides in length. In some embodiments, the first homology arm is at least 1500 nucleotides in length. In some embodiments, the first homology arm is at least 2000 nucleotides in length. In some embodiments, the first homology arm is about 20 nucleotides in length. In some embodiments, the first homology arm is about 40 nucleotides in length. In some embodiments, the first homology arm is 250 nucleotides or less in length. In some embodiments, the first homology arm is about 100 nucleotides in length. In some embodiments, the first homology arm is about 200 nucleotides in length.

[0134] In some embodiments, the second homology arm is 50 to 250 nucleotides in length. In some embodiments, the second homology arm is 50 to 2000 nucleotides in length. In some embodiments, the second homology arm is 50 to 1500 nucleotides in length. In some embodiments, the second homology arm is 50 to 1000 nucleotides in length. In some embodiments, the second homology arm is 50 to 500 nucleotides in length. In some embodiments, the second homology arm is 150 to 250 nucleotides in length. In some embodiments, the second homology arm is 2000 nucleotides in length or less. In some embodiments, the second homology arm is 1500 nucleotides in length or less. In some embodiments, the second homology arm is 1000 nucleotides in length or less. In some embodiments, the second homology arm is 700 nucleotides in length or less. In some embodiments, the second homology arm is 650 nucleotides in length or less. In some embodiments, the second homology arm is 600 nucleotides or less in length. In some embodiments, the second homology arm is 550 nucleotides or less in length. In some embodiments, the second homology arm is 500 nucleotides or less in length. In some embodiments, the second homology arm is 400 nucleotides or less in length. In some embodiments, the second homology arm is 300 nucleotides or less in length. In some embodiments, the second homology arm is 200 nucleotides or less in length. In some embodiments, the second homology arm is 150 nucleotides or less in length. In some embodiments, the second homology arm is 100 nucleotides or less in length. In some embodiments, the second homology arm is 50 nucleotides or less in length. In some embodiments, the second homology arm is 250, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, or 20 nucleotides in length.In some embodiments, the second homology arm is at least 20 nucleotides in length. In some embodiments, the second homology arm is at least 40 nucleotides in length. In some embodiments, the second homology arm is at least 50 nucleotides in length. In some embodiments, the second homology arm is at least 70 nucleotides in length. In some embodiments, the second homology arm is at least 100 nucleotides in length. In some embodiments, the second homology arm is at least 200 nucleotides in length. In some embodiments, the second homology arm is at least 300 nucleotides in length. In some embodiments, the second homology arm is at least 400 nucleotides in length. In some embodiments, the second homology arm is at least 500 nucleotides in length. In some embodiments, the second homology arm is at least 600 nucleotides in length. In some embodiments, the second homology arm is at least 700 nucleotides in length. In some embodiments, the second homology arm is at least 1000 nucleotides in length. In some embodiments, the second homology arm is at least 1500 nucleotides in length. In some embodiments, the second homology arm is at least 2000 nucleotides in length. In some embodiments, the second homology arm is about 20 nucleotides in length. In some embodiments, the second homology arm is about 40 nucleotides in length. In some embodiments, the second homology arm is 250 nucleotides in length or less. In some embodiments, the second homology arm is about 100 nucleotides in length. In some embodiments, the second homology arm is about 200 nucleotides in length.

[0135] The first and second homology arms may be the same length or may be different lengths. In some embodiments, the first and second homology arms are amplified to enable quantitative assessment of a gene editing event, such as targeted integration, in a target nucleic acid. In some embodiments, quantitative assessment of a gene editing event may rely on amplifying both the 5' junction and the 3' junction at the site of targeted integration by amplifying all or part of the homology arm using a single pair of PCR primers in a single amplification reaction. Thus, the lengths of the first and second homology arms may be different, but the length of each homology arm should be amplifiable (e.g., using PCR) as desired. Furthermore, when both the first and second homology arms are amplified, a difference in length between the first and second homology arms in a single PCR reaction is desirable, and the difference in length between the first and second homology arms should enable PCR amplification using a single pair of PCR primers.

[0136] In some embodiments, the lengths of the first and second homology arms do not differ by more than 75 nucleotides. Thus, in some embodiments, if the first and second homology arms differ in length, the difference in length between the homology arms is less than 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide or base pair. In some embodiments, the first and second homology arms differ in length by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75 nucleotides. In some embodiments, the difference in length between the first and second homology arms is less than 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 base pair. In some embodiments, the first and second homology arms differ in length by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75 base pairs.

[0137] The homology arms can direct recombination of the nucleic acid insert with a desired target genomic locus to facilitate genomic integration and / or replacement of endogenous sequences. Regardless of the format used, the donor template can be designed to avoid undesired sequences. In certain embodiments, one or both homology arms can be shortened to avoid overlap with certain sequence repeat elements, such as Alu repeats, LINE elements, etc.

[0138] 6.4.Transgene 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, which in some embodiments is expressed from a transgene.

[0139] In some embodiments, the transgene is introduced into the target cell via the same targeting construct or expression vector as the one containing the degron-encoding sequence. The transgene can be located 5' or 3' of the degron-encoding sequence. An exemplary embodiment of a suitable targeting construct containing both the transgene and the degron-encoding sequence is shown in Figure 4.

[0140] In some embodiments, the transgene may 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, as shown in Figures 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.

[0141] In some embodiments, the transgene and the fusion protein are expressed from separate loci. In some embodiments, the separate loci are both STEL loci, e.g., a GAPDH locus and another STEL locus. In other embodiments, the separate loci are both non-STEL loci. In still other embodiments, one of the loci is a STEL (e.g., GAPDH) locus and the other locus is a non-STEL locus.

[0142] In still further embodiments, the transgene is expressed from an expression vector and the fusion protein is expressed from a genomic locus (e.g., the STEL locus).

[0143] In still further embodiments, the fusion protein is expressed from an expression vector and the transgene is expressed from a genomic locus (e.g., the STEL locus).

[0144] 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). Reporter proteins can aid in tracking the therapeutic cells once they are implanted into a patient.

[0145] In some embodiments, the transgene encodes a therapeutic molecule, such as a therapeutic nucleotide or a therapeutic polypeptide or protein.

[0146] In some embodiments, the therapeutic molecule encoded by the transgene is a therapeutic nucleotide, e.g., an oligonucleotide (e.g., an miRNA, a gapmer, a steric block ON, an antagomir, a small interfering RNA (siRNA), a microRNA mimic, a splice-switching ON, or an aptamer).

[0147] In some embodiments, the therapeutic transgene is an miRNA or other small interfering nucleic acid that can regulate gene expression through cleavage / degradation of RNA transcripts or translational repression 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, and hsa-let-7g*. , hsa-let-71, hsa-let-71*, hsa-miR-1, hsa-miR-100, hsa-miR-100*, hsa-miR-101, hsa-miR-101*, hsa-miR-103, hsa-miR-105, hsa-mi R-105*, hsa-miR-106a, hsa-miR-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, h sa-miR-1197, hsa-miR-1200, hsa-miR-1201, hsa-miR-1202, hsa-miR-1203, hsa-miR-1204, hsa-miR-1205, hsa-miR-1206, hsa-miR-120 7-3p, 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*, hsa-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, hs a-miR-1255b、hsa-miR-1256、hsa-miR-1257、hsa-miR-1258、hsa-miR-125 9、hsa-miR-125a-3p、hsa-miR-125a-5p、hsa-miR-125b、hsa-miR-125b-1* hsa-miR-125b-2* hsa-miR-126 hsa-miR-126* hsa-miR-1260 hsa-miR-1261 hsa-miR-1262 hsa-miR-1263 hsa-miR-1264 hsa-miR-1265 hsa-m iR-1266, hsa-miR-1267, hsa-miR-1268, hsa-miR-1269, hsa-miR-1270, hsa-miR-1271, hsa-miR-1272, hsa-miR-1273, hsa-miR-127-3p, hsa-miR-12 74a, 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, hs a-miR-1281, hsa-miR-1282, hsa-miR-1283, hsa-miR-1284, hsa-miR-1285, hsa-miR-1286, hsa-miR-1287, hsa-miR-1288, hsa-miR-1289, hsa-miR-1 29*, hsa-miR-1290, hsa-miR-1291, hsa-miR-1292, hsa-miR-1293, hsa-miR-129-3p, hsa-miR-1294, hsa-miR-1295, hsa-miR-129-5p, hsa-miR-1296hsa-miR-1297, hsa-miR-1298, hsa-miR-1299, hsa-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, hs a-miR-1322, hsa-miR-1323, hsa-miR-1324, hsa-miR-133a, hsa-miR-133b, hsa-miR-134, hsa-miR-135a, hsa-miR-135a*, hsa-miR-135b, hsa-miR-13 5b*、hsa-miR-136、hsa-miR-136*、hsa-miR-137、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-141*, hsa-miR-142-3p, hsa-miR-142-5p, hsa-miR-143, hsa-miR-143*, hsa-miR-144, hsa-miR-144*, hs a-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-m iR-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-1 52, 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-16hsa-miR-16-1*, hsa-miR-16-2*, hsa-miR-17, hsa-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, h sa-miR-185, hsa-miR-185*, hsa-miR-186, hsa-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-19a*, 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-220c hsa-miR-221 hsa-miR-221* hsa-miR-222 hsa-miR-222* hsa-miR-223 hsa-miR-223* hsa-miR-2 24, 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-2 6a, 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, hs a-miR-28-5p, hsa-miR-296-3p, hsa-miR-296-5p, 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-301b、hsa-miR-302a、hsa-miR-3 02a*、hsa-miR-302b、hsa-miR-302b*、hsa-miR-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-32, 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-324-5p、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, hsa-miR-335*, hsa-miR-337-3p, hsa-miR-337-5p, hsa-miR-338-3p, hsa-miR-338-5p, hsa-miR-339-3p, hsa-miR-339-5 p、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-mi R-346, hsa-miR-34a, hsa-miR-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, hsa-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* hs a-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-379hsa-miR-379*, hsa-miR-380, 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*、hsa-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, hsa-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-miR-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, hs a-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, hs a-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-503hsa-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, h sa-miR-509-5p, hsa-miR-510, hsa-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-51 7b、hsa-miR-517c、hsa-miR-518a-3p、hsa-miR-518a-5p、hsa-miR-518b、h sa-miR-518c、hsa-miR-518c*、hsa-miR-518d-3p、hsa-miR-518d-5p、hsa-m iR-518e, hsa-miR-518e*, hsa-miR-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、hsa-miR-520a-5p、hsa-miR-520b、hsa-miR -520c-3p、hsa-miR-520d-3p、hsa-miR-520d-5p、hsa-miR-520e、hsa-miR-5 20f, hsa-miR-520g, hsa-miR-520h, 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-542-5phsa-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-mi R-548j, hsa-miR-548k, hsa-miR-5481, hsa-miR-548m, hsa-miR-548n, hsa-miR-548o, hsa-miR-548p, hsa-miR-549, hsa-miR-550, hsa-miR-550*, hs a-miR-551a, hsa-miR-551b, hsa-miR-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, hsa-miR-580, hsa-miR-581, hsa-miR-582-3p, hsa-miR-582-5p, hsa-miR-583, hsa-miR-584, hsa-miR-585, hs a-miR-586, hsa-miR-587, hsa-miR-588, hsa-miR-589, hsa-miR-589*, hsa-miR-590-3p, hsa-miR-590-5p, hsa-miR-591, hsa-miR-592, hsa-miR-593hsa-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-m iR-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, hs a-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-m iR-638, hsa-miR-639, hsa-miR-640, hsa-miR-641, hsa-miR-642, hsa-miR-643, hsa-miR-644, hsa-miR-645, hsa-miR-646, hsa-miR-647, hsa-miR-64 8, hsa-miR-649, hsa-miR-650, hsa-miR-651, hsa-miR-652, hsa-miR-653, hsa-miR-654-3p, hsa-miR-654-5p, hsa-miR-655, hsa-miR-656, hsa-miR-6 57, hsa-miR-658, hsa-miR-659, hsa-miR-660, hsa-miR-661, hsa-miR-662, hsa-miR-663, hsa-miR-663b, hsa-miR-664, hsa-miR-664*, hsa-miR-665hsa-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, hsa-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, hsa-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* are included.

[0148] In some embodiments, the transgene encodes a therapeutic protein or polypeptide. The therapeutic protein or polypeptide may introduce a protein or peptide not present in the patient. The therapeutic protein or polypeptide may also replace a protein that is deficient or abnormal (e.g., mutated) in the patient, for example, a patient associated with a rare or orphan disease. 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., frataxin), progranulin (PRGN) (associated with non-Alzheimer's brain degeneration, including frontal dementia (FTD), progressive non-fluent aphasia (PNFA), and semantic dementia), and the like.

[0149] Therapeutic proteins and polypeptides that replace absent, deficient, or abnormal proteins in patients may also target familial hypercholesterolemia, muscular dystrophy, mucopolysaccharidosis, cystic fibrosis, diabetes, and blood clotting disorders.Non-limiting examples of therapeutic proteins and polypeptides that replace absent, deficient, or abnormal proteins include insulin, growth hormone, clotting factors, albumin, H-protein, T-protein, dystonin, 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 I, and arginine transcarbamylase (ARS). argininosuccinate lyase (ASL) for the treatment of 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, cystathione beta synthase, branched-chain keto acid decarboxylase, albumin, isovaleryl-CoA dehydrogenase, propionyl-CoA calcitonin carboxylase, methylmalonyl-CoA mutase, glutaryl-CoA dehydrogenase, beta-glucosidase, pyruvate carboxylate, hepatic phosphorylase, phosphorylase kinase, and glycine decarboxylase, α-L-iduronidase (IDUA), iduronate-2-sulfatase (IDS), sulfamidase, N-acetylgalactosamine-6-sulfatase (GALNS), arylsulfatase B, hyaluronidase, beta-glucuronidase, phosphoenolpyruvate carboxykinase (P EPCK), cyclin-dependent kinase-like 5 (CDKL5), galactose-phosphate uridyltransferase, branched-chain α-keto acid dehydrogenase, fumarylacetoacetate hydrolase, methylmalonyl-CoA mutase, argininosuccinate synthetase, lecithin-cholesterol acyltransferase, hypoxanthine guanine phosphoribosyltransferase, biotinidase, α-galactosidase A, hexosaminidase, ceramidase, aspartylglucosaminidase, and α-fucosidase.

[0150] In some embodiments, therapeutic proteins or polypeptides can be used to enhance existing pathways. Some non-limiting examples of enhancing therapeutic proteins and polypeptides include peptide hormones used to treat hormone deficiencies or infertility, growth and differentiation factors, and proteins for treating hematopoietic deficiencies, hemotherapy-induced anemia, or myelodysplastic syndromes.

[0151] Non-limiting examples of hormones and growth and 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), angiopoietin, angiostatin, granulocyte colony-stimulating factor (GCSF), erythropoietin (EPO) (including, for example, human, canine, or feline EPO), connective tissue growth factor (CTGF), neutral factors, such as basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin growth factor I and II. Examples of such proteins include any one of the transforming growth factor α superfamily, including IGF-I and IGF-II, TGFα, activin, and inhibin, or any one of the bone morphogenetic proteins (BMPs) BMP1 to 15, any one of the heligulin / neuregulin / ARIA / neural 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), neurulin, agrin, any one of the semaphorin / collapsin family, netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrin, noggin, sonic hedgehog, and tyrosine hydroxylase.

[0152] In some embodiments, therapeutic proteins or polypeptides can be used to provide a new function or activity to an endogenous protein or to introduce a non-endogenous protein with a new function or activity. Some non-limiting examples are proteins and peptides used in the enzymatic degradation of macromolecules, such as papain, collagenase, hyaluronidase, and botulinum toxins types A and B, as well as proteins and peptides used in the enzymatic degradation of small molecule metabolites, such as L-asparaginase, PEG-asparaginase, and rasburicase. Other examples can include chimeric or hybrid polypeptides with non-naturally occurring amino acid sequences containing insertions, deletions, or amino acid substitutions. For example, single-chain engineered immunoglobulins can be useful in certain immunodeficient 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 targets.

[0153] In some embodiments, therapeutic nucleic acids, proteins, or polypeptides can be used to interfere with molecules or organisms. Non-limiting examples include proteins and peptides that are produced exclusively or at higher levels in hyperproliferative cells compared to normal cells, such as those used to treat infectious diseases or various forms of cancer, such as polypeptides encoded by the oncogenes myb, myc, and fyn, and the 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 suppressors) known to be associated with the development of cancer that can be targeted by therapeutic transgenes: AARS, ABCB1, ABCC4, ABI2, ABL1, ABL2, ACK1, ACP2, ACY1, ADSL, AK1, AKR1C2, AKT1, ALB, ANPEP, ANXA5, A NXA7, AP2M1, APC, ARHGAP5, ARHGEF5, ARID4A, ASNS, ATF4, ATM, ATP5B, ATP5O, AXL, BARD1, BAX, BCL2, BHL HB2, BLMH, BRAF, BRCA1, BRCA2, BTK, CANX, CAP1, CAPN1, CAPNS1, CAV1, CBFB, CBLB, CCL2, CCND1, CCND2, C CND3, CCNE1, CCT5, CCYR61, CD24, CD44, CD59, CDC20, CDC25, CDC25A, CDC25B, CDC2L5, CDK10, CDK4, CDK5 , CDK9, CDKL1, CDKN1A, CDKN1B, CDKN1C, CDKN2A, CDKN2B, CDKN2D, CEBPG, CENPC1, CGRRF1, CHAF1A, CIB1, CKMT1, CLK1, CLK2, CLK3, CLNS1A, CLTC, COL1A1, COL6A3, COX6C, COX7A2, CRAT, CRHR1, CSF1R, CSK, CSNK1 G2, CTNNA1, CTNNB1, CTPS, CTSC, CTSD, CUL1, CYR61, DCC, DCN, DDX10, DEK, DHCR7, DHRS2, DHX8, DLG3, DVL 1, DVL3, E2F1, E2F3, E2F5, EGFR, EGR1, EIF5, EPHA2, ERBB2, ERBB3, ERBB4, ERCC3, ETV1, ETV3, ETV6, F2R,FASTK、FBN1、FBN2、FES、FGFR1、FGR、FKBP8、FN1、FOS、FOSL1、FOSL2、FOXG1A 、FOXO1A、FRAP1、FRZB、FTL、FZD2、FZD5、FZD9、G22P1、GAS6、GCN5L2、GDF15、 GNA13、GNAS、GNB2、GNB2L1、GPR39、GRB2、GSK3A、GSPT1、GTF2I、HDAC1、HDGF 、HMMR、HPRT1、HRB、HSPA4、HSPA5、HSPA8、HSPB1、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、LA MP2、LCK、LCN2、LEP、LITAF、LRPAP1、LTF、LYN、LZTR1、MADH1、MAP2K2、MAP3K8 、MAPK12、MAPK13、MAPKAPK3、MAPRE1、MARS、MAS1、MCC、MCM2、MCM4、MDM2、MD M4、MET、MGST1、MICB、MLLT3、MME、MMP1、MMP14、MMP17、MMP2、MNDA、MSH2、MS H6、MT3、MYB、MYBL1、MYBL2、MYC、MYCL1、MYCN、MYD88、MYL9、MYLK、NEO1、NF1 、NF2、NFKB1、NFKB2、NFSF7、NID、NINE、NMBR、NME1、NME2、NME3、NOTCH1、NOTC H2、NOTCH4、NPM1、NQO1、NR1D1、NR2F1、NR2F6、NRAS、NRG1、NSEP1、OSM、PA2G 4、PABPC1、PCNA、PCTK1、PCTK2、PCTK3、PDGFA、PDGFB、PDGFRA、PDPK1、PEA15、 PFDN4、PFDN5、PGAM1、PHB、PIK3CA、PIK3CB、PIK3CG、PIM1、PKM2、PKMYT1、PL K2、PPARD、PPARG、PPIH、PPP1CA、PPP2R5A、PRDX2、PRDX4、PRKAR1A、PRKCBP1、PRNP, PRSS15, PSMA1, PTCH, PTEN, PTGS1, PTMA, PTN, PTPRN, RAB5A, RAC1, RAD50, RAF1, RALBP1, RAP1A, RAR B. RASGRF1, RB1, RBBP4, RBL2, REA, REL, RELA, RELB, RET, RFC2, RGS19, RHOA, RHOB, RHOC, RHOD, RIPK1, RPN2, RPS6 KB1, RRM1, SARS, SELENBP1, SEMA3C, SEMA4D, SEPP1, SERPINH1, SFN, SFPQ, SFRS7, SHB, SHH, SIAH2, SIVA, SIVA TP53 ) , SNAI2 , SND1 , SNRPB2 , SOCS1 , SOCS3 , SOD1 , SORT1 , SPINT2 , SPRY2 , SRC , SRPX , ST AT1, STAT2, STAT3, STAT5B, STC1, TAF1, TBL3, TBRG4, TCF1, TCF7L2, TFAP2C, TFDP 1, TFDP2, TGFA, TGFB1, TGFBI, TGFBR2, TGFBR3, THBS1, TIE, TIMP1, TIMP3, TJP1, T K1, TLE1, TNF, TNFRSF10A, TNFRSF10B, TNFRSF1A, TNFRSF1B, TNFRSF6, TNFSF7, TN K1, TOB1, TP53, TP53BP2, TP5313, TP73, TPBG, TPT1, TRADD, TRAM1, TRRAP, TSG101 TUFM, TXNRD1, TYRO3, UBC, UBE2L6, UCHL1, USP7, VDAC1, VEGF, VHL, VIL2, WEE1 NT1, WNT2, WNT2B, WNT3, WNT5A, WT1, XRCC1, YES1, YWHAB, YWHAZ, ZAP70, and ZNF9.

[0154] In some embodiments, the therapeutic transgene may 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, B IRC6, 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, RIPK1 , RIPK2, TNF, TNFRSF10A, TNFRSF10B, TNFRSF10C, TNFRSF10D, TNFRSF11B, TNFRSF12A, TNFRSF14, TNFRSF19, TNFR SF1A, 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.

[0155] 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.

[0156] In some other embodiments, therapeutic proteins and polypeptides include those that may be useful for treating individuals suffering from autoimmune diseases and disorders by conferring a broad protective immune response against targets associated with autoimmunity, including cellular receptors and cells that produce "self"-directed antibodies. T cell-mediated autoimmune diseases include rheumatoid arthritis (RA), multiple sclerosis (MS), Sjogren'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.

[0157] In some embodiments, the therapeutic protein is a receptor or a ligand for a receptor. Non-limiting examples of receptors include receptors for hormones, growth factors, cytokines, lymphokines, regulatory proteins, and immune system proteins, as well as receptors for cholesterol regulation and / or lipid regulation, including low-density lipoprotein (LDL) receptors, high-density lipoprotein (HDL) receptors, very-low-density lipoprotein (VLDL) receptors, and any one of scavenger receptors. In some embodiments, the therapeutic protein is a member of the steroid hormone receptor superfamily, which includes glucocorticoid receptors, estrogen receptors, vitamin D receptors, and other nuclear receptors.

[0158] 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 inhibitor (C1-INH). In some embodiments, the therapeutic protein can be a mAb, a non-covalent binder other than an Fc fusion protein, or a polyclonal immunoglobulin (see, e.g., Table 4 of Dimitrov, 2012, Methods Mol Biol. 899:1-26, incorporated herein by reference).

[0159] In some embodiments, the transgene is a therapeutic protein that can be used to treat a lysosomal storage disorder, hi some embodiments, the therapeutic protein is a lysosomal enzyme such as alpha-L-iduronidase, arylsulfatase A, beta-glucocerebrosidase, acid sphingomyelinase, alpha-galactosidase, or beta-galactosidase.

[0160] In some embodiments, the transgene is a therapeutic protein that can be used to treat hemophilia or other inherited blood disorders. In some embodiments, the therapeutic polypeptides are Factor VIII and Factor IX. In some embodiments, the therapeutic transgene 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 transgene further comprises the A1 and A2 domains, and 5 amino acids from the N-terminus of the B domain and / or the C-terminal 85 amino acids of the B domain, and the 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).

[0161] 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, such as a cytokine or cytokine receptor, or an interleukin or interleukin receptor. Immune system regulating therapeutic proteins and polypeptides include, but are not limited to, thrombopoietin (TPO), interleukins IL-1 through IL-36 (e.g., 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 chemotactic proteins, leukemia inhibitory factor, granulocyte-macrophage colony-stimulating factor, Fas ligand, tumor necrosis factors α and β, interferons α, β, and γ, stem cell factor, and flk-2 / flt3 ligand. Thus, in some embodiments, the transgene may comprise a nucleic acid encoding a pro-inflammatory or immunosuppressant 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 are also useful 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, class I and class II MHC molecules, and engineered immunoglobulins and MHC molecules.

[0162] In some other embodiments, the immunomodulatory therapeutic protein is a human leukocyte antigen ("HLA") polypeptide, including, but not limited to, an HLA class Ib polypeptide. 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-βI, IFN-γ, and their various isoforms.

[0163] In some embodiments, the transgene is a therapeutic polypeptide, including an antibody or antigen-binding fragment thereof, such as an scFv.

[0164] In some embodiments, the therapeutic protein or polypeptide is a bone morphogenetic protein, an engineered protein scaffold, a serum protein, a globular protein, a protective 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.

[0165] In some embodiments, the therapeutic protein is a transcription factor, e.g., 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 proteins, interferon regulatory factor (IRF-1), Wilms tumor protein, ETS binding proteins, STATs, and GATA box binding proteins, e.g., GATA-3, and the forkhead family of winged helix proteins.

[0166] In some embodiments, the transgene is a therapeutic polypeptide that binds to a pathogenic polypeptide, such as tau, alpha-synuclein, or beta-amyloid polypeptide.

[0167] In some embodiments, the transgene is a therapeutic polypeptide that targets cancer cells, hi some embodiments, the therapeutic polypeptide is a chimeric antigen receptor that binds to a tumor-associated antigen, such as CD19 or CD20.

[0168] In some embodiments, the therapeutic polypeptide is a T cell receptor (TCR) or an antigen-binding fragment thereof, e.g., a recombinant TCR. In some embodiments, the recombinant TCR is capable of binding to an antigen of interest, for example, but not limited to, an antigen selected from CD279, CD2, CD95, CD152, CD223, CD272, TIM3, KIR, A2aR, SIRPa, CD200, CD200R, CD300, LPA5, NY-ESO, PD1, PDL1, or MAGE-A3 / A6.

[0169] In some embodiments, the TCR or antigen-binding fragment thereof can bind to a viral antigen, for example, an antigen from hepatitis A, hepatitis B, hepatitis C (HCV), human papillomavirus (HPV) (e.g., HPV-16 (such as HPV-16 E6 or HPV-16 E7), 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).

[0170] In some embodiments, the therapeutic protein or polypeptide can be a neutralizing antibody against a viral pathogen. Such anti-viral antibodies can include anti-influenza antibodies against one or more of influenza A, influenza B, and influenza C. Other target pathogenic viruses include arenaviruses (including Junin, Machupo, and Lassa), filoviruses (including Marburg and Ebola), hantaviruses, picornoviridae (including rhinoviruses, echoviruses), coronaviruses, paramyxoviruses, morbilliviruses, respiratory syncytial viruses, togaviruses, coxsackieviruses, JC virus, parvovirus B19, parainfluenza, adenoviruses, reoviruses, and viruses from the poxvirus family, including varicella (smallpox)) and vaccinia (cowpox), and varicella varicella (pseudomeasles). 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.

[0171] In some embodiments, the therapeutic protein can be a neutralizing antibody construct against a bacterial pathogen. In one embodiment, the neutralizing antibody construct is against the bacterium itself. In another embodiment, the neutralizing antibody construct is against a toxin produced by a bacterium, such as the toxin produced by Bacillus anthracis, the causative agent of anthrax. Examples of airborne bacterial pathogens include, for example, 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 (influenza), Haemophilus parainfluenzae (influenza), parainfluenzae, Bordetella pertussis (whooping cough), Francisella 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, Staphylococcus aureusaureus (pneumonia), Streptococcus pyogenes (scarlet fever), Streptococcus pneumoniae (pneumonia), Corynebacteria diphtheria (diphtheria), and Mycoplasma pneumoniae (pneumonia).

[0172] The therapeutic protein may be, for example, an antibody against a parasitic or other fungal infectious agent, including Aspergillus spp., Absidiacorymbifera, Lyxspus stolonifera, Mucorporambeus, Cryptococcus neoformans, Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides immitis, Penicillium spp., Micropolisporaphaenii, Thermoactinomyces vulgaris, Alternaria alternata, Cladosporium spp., Helminthosporium, and Stachybotrys spp.

[0173] Further, in some embodiments, the transgene encodes a cell lineage commitment factor. For example, in some embodiments, the transgene encodes a gene product that, when expressed, promotes the differentiation of a cell into a more specialized cell type. For example, in some embodiments, the transgene encodes a lineage commitment factor that promotes the differentiation of a cell toward a fibroblast, a hematopoietic cell, a neuron, a glial cell, an oligodendrocyte, a myocyte, an osteocyte, a hepatocyte, a pancreatic cell, a myeloid cell or myeloid progenitor cell, a microglial cell or microglial progenitor cell, a T cell, e.g., a CD4+ T cell, e.g., a Treg, or the like. For example, in some embodiments, the transgene 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.

[0174] 6.5. Separation Array The targeting constructs and recombinant target cell genomes described herein can also include a separator sequence between the degron-encoding sequence and the transgene, which can allow for separate expression of polypeptides encoded by a single expression cassette.

[0175] In some embodiments, the separator sequence is an internal ribosome entry site (IRES) that allows the transgene to be translated separately from the fusion protein comprising the essential polypeptide and the degron sequence.

[0176] In some embodiments, the separator sequence is a self-cleaving peptide associated with ribosomal skipping during translation, causing the ribosome to skip the peptide bond between the C-terminal Gly and Pro, resulting in the production of two distinct polypeptides: a transgene and a fusion protein containing the essential polypeptide and a degron sequence. In further embodiments, the polypeptide coding sequences comprising the fusion protein within the expression cassette can be separated by a translation skip sequence (i.e., an in-frame coding sequence for a self-cleaving peptide), such that translation of the mRNA transcript from the polycistronic cassette results in distinct proteins. The self-cleaving peptide causes ribosomal skipping during translation. Examples of self-cleaving peptides are 2A peptides, which are virus-derived peptides typically 18-22 amino acids in length. 2A peptides include T2A, P2A, E2A, F2A, and PQR (Lo et al., 2015, Cell Reports 13:2634-2644). As an example, P2A is a 19 amino acid peptide; after cleavage, several amino acid residues from P2A are left on the upstream polypeptide and a proline is left at the beginning of the second polypeptide. The P2A residues left on the fusion protein and the polypeptide encoded by the transgene are not believed to affect their function.

[0177] 6.6. Expression Vectors The present disclosure provides expression vectors encoding the fusion proteins of the present disclosure. Expression vectors typically include an expression cassette comprising a fusion polypeptide as described in Section 6.2 operably linked to regulatory elements such as a promoter, and optionally, an autonomously replicating element.

[0178] Without being bound by theory, it is believed that recombinant expression of a fusion protein comprising an essential polypeptide and a degron "poisons" the native cellular protein, causing its destabilization when the degron is activated, resulting in cell death even if the essential gene is intact and not modified by recombination with a targeting construct of the present disclosure. Thus, the fusion proteins 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.

[0179] The expression vector may be a viral genome, single-stranded RNA or DNA, or double-stranded DNA, such as a plasmid.

[0180] Expression vectors can include other coding or non-coding elements. For example, the expression cassette can be delivered as part of a viral genome (e.g., in an AAV, adenovirus, Sendai virus, or lentivirus genome) that includes certain genomic backbone elements (e.g., inverted terminal repeats in the case of the AAV genome).

[0181] In some embodiments, the expression vector is a circular plasmid that is not linearized.

[0182] In some embodiments, the expression vector is a circular plasmid that has been linearized.

[0183] In some embodiments, the expression vector is a viral genome. Viral genomes provide a rich source of vectors that can be used for efficient delivery of exogenous nucleic acids into the genome of target cells (e.g., mammalian cells, such as human cells). Viral genomes are particularly useful vectors for delivering exogenous nucleic acids because the nucleic acids contained within such genomes are typically integrated into the genome of target cells by generalized or specialized transduction. These processes occur as part of the natural viral replication cycle and do not require additional proteins or reagents to induce gene integration. Examples of viral vectors include negative-strand RNA viruses such as AAV, retroviruses, adenoviruses (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvoviruses (e.g., adeno-associated viruses), coronaviruses, orthomyxoviruses (e.g., influenza viruses), positive-strand RNA viruses such as rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and Sendai viruses), picornaviruses, and alphaviruses, as well as double-stranded DNA viruses including adenoviruses, herpesviruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxviruses (e.g., vaccinia, modified vaccinia Ankara (MVA), fowlpox, and canarypox). Other viruses useful for delivering exogenous nucleic acids include, for example, Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus, and hepatitis virus. Examples of retroviruses include avian leukosis sarcoma, mammalian type C, type B, and type D viruses, the HTLV-BLV group, lentiviruses, and spumaviruses (Coffin, JM, Retroviridae: The viruses and their replication, Fundamental Virology, Third 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 monkey leukemia virus, Mason-Pfizer 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.

[0184] In some embodiments, the expression vector is a recombinant adeno-associated virus vector ("rAAV vector"). rAAV vectors useful in the present invention are recombinant nucleic acid constructs that include (1) an expression cassette (e.g., a nucleic acid encoding a fusion protein comprising an essential polypeptide and a degron, optionally connected via a linker) and (2) viral nucleic acid that facilitates expression of the fusion protein. The viral nucleic acid may include AAV sequences required in cis for DNA replication and packaging into virions (e.g., functional ITRs). Useful rAAV vectors lack one or more AAV WT genes in whole or in part, but retain functional flanking ITR sequences. The AAV ITRs can be of any serotype suitable for a particular application. Methods for using rAAV vectors are described, for example, in Tal et al., 2000, J. Biomed. Sci. 7:279-291, and Monahan and Samulski, 2000, Gene Delivery 7:24-30. The rAAV can be derived from any suitable serotype, including AAV1, 2, 3, 4, 5, 6, 7, 8, and 9.

[0185] In some embodiments, the expression cassette includes a transgene in addition to the fusion protein coding sequence, for example, a transgene encoding a therapeutic polypeptide.

[0186] 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 translation skip sequence (e.g., an in-frame coding sequence for a self-cleaving peptide), such that translation of the mRNA transcript from the polycistronic cassette results in a fusion protein comprising separate polypeptides, i.e., the essential polypeptide, the degron, and the polypeptide encoded by the transgene. Self-cleaving peptides are described in Section 6.5.

[0187] Alternatively, the expression cassette can be a polycistronic expression cassette with the fusion protein coding sequence and the transgene separated by an internal ribosome entry site (IRES) within the mRNA.

[0188] 6.7.Target cells In some embodiments, the 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.

[0189] 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. Cell populations can include, for example, populations 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 are engineered to express a fusion protein comprising an essential polypeptide and a degron.

[0190] 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 produce engineered target cells that can be reintroduced into an individual).

[0191] Any type of cell may 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, muscle cells, bone cells, hepatocytes, pancreatic cells, myeloid cells or myeloid progenitor cells, e.g., primitive myeloid progenitor cells, microglial cells or microglial progenitor cells, T cells, e.g., CD4+ T cells, e.g., Tregs; in vitro or in vivo embryonic cells of any stage, e.g., zebrafish embryos at the 1-cell, 2-cell, 4-cell, 8-cell, etc. stage, etc.). The cells may be from an established cell line, or they may be primary cells; "primary cells," "primary cell lines," and "primary cultures" are used interchangeably herein to refer to cells and cell cultures derived from a subject and capable of being grown in vitro for a limited number of passages, e.g., division, in culture. For example, a primary culture includes a culture that may have been passaged 0, 1, 2, 4, 5, 10, or 15 times, but not enough times to pass through a crisis stage. A primary cell line may be maintained in vitro for fewer than 10 passages. The target cell, in some embodiments, is a single-cell organism or grown in culture. Preferably, the target cell is of human origin. In some embodiments, the target cell is an autologous cell in the context of cell therapy. In some embodiments, the target cell is an allogeneic cell in the context of cell therapy.

[0192] If the cells are primary cells, they can be collected from an individual by any suitable method. For example, white blood cells can be suitably collected by apheresis, leukopheresis, density gradient separation, etc., while cells from tissues such as skin, muscle, bone marrow, spleen, liver, pancreas, lung, intestine, and stomach are most suitably collected by biopsy. An appropriate solution can be used to disperse or suspend the collected cells. Such solutions are generally balanced salt solutions, such as saline, phosphate-buffered saline (PBS), Hank's balanced salt solution, and the like, suitably supplemented with fetal bovine serum or other natural factors, along with a low concentration, e.g., 5-25 mM, of an acceptable buffer. Suitable buffers include HEPES, phosphate buffer, lactate buffer, and the like. The cells can be used immediately, or they can be stored for extended periods, frozen, thawed, and reused. In such cases, the cells would typically be frozen in 10% dimethyl sulfoxide (DMSO), 50% serum, 40% buffered medium, or some other such solution commonly used in the art for preserving cells at such freezing temperatures, and thawed in a manner commonly known in the art for thawing frozen cultured cells.

[0193] Methods for introducing heterologous nucleic acid into target cells, for example, to engineer the target cells to express a fusion protein of the present disclosure and optionally a transgene, are disclosed in Sections 6.8 and 6.9.

[0194] In some embodiments, target cells are engineered to integrate a single copy of a coding sequence encoding a fusion protein comprising an essential polypeptide and a degron. In other embodiments, target cells are engineered to integrate two copies of a coding sequence encoding a fusion protein comprising an essential polypeptide and a degron. In some embodiments, each allele of an essential locus is modified to express a fusion protein comprising an essential polypeptide and a degron. In yet other embodiments, target cells are engineered to express multiple copies of a coding sequence encoding a fusion protein comprising an essential polypeptide and a degron, where two or more copies are integrated into each allele of a corresponding essential locus. As described in Section 6.2, in each of the foregoing embodiments, the fusion protein can include one, two, or more degrons in tandem. In some embodiments, the degrons are separated by a linker.

[0195] In some embodiments, single copy is achieved by integrating the targeting construct into a single allele of the essential gene. The degron-coding sequence may be located 5' or 3' to the essential polypeptide-coding sequence, allowing the fusion protein to contain the degron(s) at its N- or C-terminus. The essential polypeptide and degron(s) may be separated by a linker. The target cell may further be engineered to express the transgene. The transgenes may be expressed from the same allele of the essential gene modified to encode the fusion protein, from opposite alleles, together from separate genomic loci, or from an extrachromosomal expression vector. In some embodiments, the degron-coding sequence(s) and the transgene are both introduced into the same or different alleles of the same essential STEL gene, for example, the GAPDH locus.

[0196] In some embodiments, two copies are achieved by integrating a targeting construct into both alleles of the essential gene. The degron-coding sequence(s) can be located 5' or 3' to the essential polypeptide-coding sequence, such that the fusion protein can contain the degron(s) at its N- or C-terminus. The essential polypeptide and degron(s) can be separated by a linker. The target cell may be further engineered to express a transgene. The transgene can be expressed from the same allele of the essential gene that has been modified to encode the fusion protein, from a separate genomic locus, or from an extrachromosomal expression vector. In some embodiments, the degron-coding sequence(s) and the transgene are both introduced into the same allele of the essential STEL gene, for example, the GAPDH locus.

[0197] In some embodiments, the target cells of the present disclosure comprise a single allele of an essential gene into which a targeting construct comprising a degron has been integrated, e.g., a targeting construct according to any one of Figures 2A, 2B, 2C, or 2D. Optionally, the target cells comprise transgenes integrated into one or both alleles of different loci.

[0198] In some embodiments, a target cell of the present disclosure comprises two alleles of an essential gene into which a targeting construct comprising a degron, e.g., a targeting construct according to any one of Figures 2A, 2B, 2C, or 2D, has been integrated. Optionally, the target cell comprises a transgene integrated into one or both alleles at different loci.

[0199] In some embodiments, a target cell of the present disclosure comprises two essential loci configured as shown in Figure 3A. In some embodiments, a target cell of the present disclosure comprises two essential loci configured as shown in Figure 3B. In some embodiments, a target cell of the present disclosure comprises two essential loci configured as shown in Figure 3D. In some embodiments, a target cell of the present disclosure comprises two essential loci configured as shown in Figure 3E. In each of the foregoing embodiments, the target cell optionally comprises a transgene integrated into one allele or both alleles of different loci.

[0200] In some embodiments, the target cells of the present disclosure comprise one allele of an essential gene into which a targeting construct comprising a degron and a transgene, e.g., a targeting construct according to any one of Figures 4A, 4B, 4C, or 4D, has been integrated. In some embodiments, the target cells of the present disclosure comprise two alleles of an essential gene into which a targeting construct comprising a degron and a transgene, e.g., a targeting construct according to any one of Figures 4A, 4B, 4C, or 4D, has been integrated.

[0201] In some embodiments, the target cells of the present disclosure comprise one allele of an essential gene into which a targeting construct comprising a degron and a transgene, e.g., a targeting construct according to any one of Figures 7A, 7B, 7C, 7D, 7E, 7F, 7G, 7H, 7I, 7J, 7K, or 7L, has been integrated. In some embodiments, the target cells of the present disclosure comprise two alleles of an essential gene into which a targeting construct comprising a degron and a transgene, e.g., a targeting construct according to any one of Figures 7A, 7B, 7C, 7D, 7E, 7F, 7G, 7H, 7I, 7J, 7K, or 7L, has been integrated.

[0202] In some embodiments, a target cell of the present disclosure comprises two essential loci configured as shown in FIG. 5A. In some embodiments, a target cell of the present disclosure comprises two essential loci configured as shown in FIG. 5B. In some embodiments, a target cell of the present disclosure comprises two essential loci configured as shown in FIG. 5C. In some embodiments, a target cell of the present disclosure comprises two essential loci configured as shown in FIG. 5D. In some embodiments, a target cell of the present disclosure comprises two essential loci configured as shown in FIG. 6A. In some embodiments, a target cell of the present disclosure comprises two essential loci configured as shown in FIG. 6B. In some embodiments, a target cell of the present disclosure comprises two essential loci configured as shown in FIG. 6C. In some embodiments, a target cell of the present disclosure comprises two essential loci configured as shown in FIG. 6D.

[0203] 6.7.1. Stem cells In some embodiments, target cells engineered to express a fusion protein 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 the starting point for the potential generation of many specific cell types that can be delivered for regenerative medicine in patients with many different diseases.

[0204] Suitable methods for introducing heterologous nucleic acid into stem cells, for example, to engineer stem cells to express a fusion protein of the present disclosure, and optionally a transgene, are disclosed in Sections 6.8 and 6.9.

[0205] After engineering the PSCs to express the fusion proteins of the present disclosure, and optionally a transgene, the PSCs can be differentiated into cell types of interest for cell therapy.

[0206] Recombinant PSCs can be differentiated into cells suitable for therapy, including cells in the endodermal (e.g., lung, thyroid, or pancreatic cells, or precursors thereof), ectodermal (e.g., skin, neuron, or pigment cell, or precursors thereof), and mesodermal (e.g., cardiac, skeletal muscle, red blood cell, smooth muscle cell, or precursor or precursor thereof) lineages.

[0207] In some embodiments, the recombinant PSCs are differentiated into cells of the endoderm (e.g., lung, thyroid, or pancreatic cells, or precursors or precursors thereof), ectoderm (e.g., skin, neurons, or pigment cells, or precursors or precursors thereof), or mesoderm (e.g., cardiac cells, skeletal muscle cells, red blood cells, smooth muscle cells, or precursors or precursors thereof).

[0208] 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 nodal cells.

[0209] In some embodiments, recombinant PSCs of the present disclosure are differentiated into human immune cells, optionally selected from T cells, T cells expressing a chimeric antigen receptor (CAR) or a recombinant TCR, regulatory T cells, myeloid cells, dendritic cells, and / or macrophages (e.g., immunosuppressive macrophages), or precursors or progenitors thereof. In some embodiments, recombinant PSCs of the present disclosure are differentiated into myeloid progenitor cells, for example, as described in WO2023 / 150089A1, the contents of which are incorporated herein by reference in their entirety.

[0210] In some embodiments, recombinant PSCs of the present disclosure are differentiated into oligodendrocyte precursor or progenitor cells, or oligodendrocytes.

[0211] In some embodiments, the recombinant PSCs of the present disclosure are differentiated into neural lineage cells, such as 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 precursors or precursors thereof.

[0212] In some embodiments, recombinant PSCs of the present disclosure are differentiated into cells of the ocular system, such as photoreceptor cells, photoreceptor precursor or progenitor cells, retinal pigment epithelial cells or precursors or precursors thereof, neural retinal cells or precursors or precursors thereof. In other embodiments, unedited PSCs are differentiated into cells of the ocular system, which are then recombined with a targeting construct of the present disclosure.

[0213] In further embodiments, the recombinant PSCs of the present disclosure are differentiated into microglial cells or microglial precursor or progenitor cells.

[0214] In further embodiments, the recombinant PSCs of the present disclosure are optionally differentiated into cells in the human metabolic system selected from hepatocytes, cholangiocytes, and pancreatic beta cells, or precursors or progenitors thereof.

[0215] In further embodiments, the recombinant PSCs of the present disclosure are differentiated into enteric precursor or progenitor cells or enteric cells.

[0216] 6.7.2. Differentiated cells In various embodiments, cells at any stage of differentiation are engineered to express the fusion proteins of the present disclosure, and optionally a transgene.

[0217] Suitable methods for introducing heterologous nucleic acid into differentiated cells, for example, to engineer the differentiated cells to express a fusion protein of the present disclosure, and optionally a transgene, are disclosed in Sections 6.8 and 6.9.

[0218] Exemplary differentiated cell types that can be engineered to express the fusion proteins of the present disclosure include cells in the endodermal (e.g., lung, thyroid, or pancreatic cells, or precursors thereof), ectodermal (e.g., skin, neural, or pigment cell, or precursors or precursors thereof), and mesodermal (e.g., cardiac, skeletal muscle, red blood cell, smooth muscle cell, or precursors or precursors thereof) lineages. Alternatively, PSCs can be differentiated into cells in these lineages and then recombined with the targeting constructs of the present disclosure.

[0219] In some embodiments, cardiac cells are engineered to express a fusion protein 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 nodal cells.

[0220] In some embodiments, human immune cells are engineered to express a fusion protein of the present disclosure. The human immune cells are optionally selected from T cells, T cells expressing a chimeric antigen receptor (CAR) or a recombinant TCR, regulatory T cells, myeloid cells, dendritic cells, and / or macrophages (e.g., immunosuppressive macrophages), or precursors or progenitors thereof. In some embodiments, myeloid progenitor cells are engineered to express a fusion protein of the present disclosure following differentiation from PSCs, for example, as described in WO2023 / 150089A1, the entire contents of which are incorporated herein by reference.

[0221] In some embodiments, oligodendrocyte precursor or progenitor cells or oligodendrocytes are engineered to express a fusion protein of the present disclosure.

[0222] In some embodiments, neural lineage cells are engineered to express the fusion proteins of the present disclosure. In various embodiments, the neural lineage cells are 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 progenitor cells, floor plate midbrain progenitor cells, floor plate midbrain DA neurons, or precursors or precursors thereof.

[0223] 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 precursor or precursor cells, retinal pigment epithelial cells or precursors or precursors thereof, neural retinal cells or precursors or precursors thereof.

[0224] In a further embodiment, microglial cells or microglial precursor or progenitor cells are engineered to express a fusion protein of the present disclosure.

[0225] In further embodiments, cells in the human metabolic system are engineered to express the fusion proteins of the present disclosure. In various embodiments, the cells in the human metabolic system are optionally selected from hepatocytes, bile duct cells, and pancreatic beta cells, or precursors or precursors thereof.

[0226] In a further embodiment, the enteric precursor or progenitor cells or enteric cells are engineered to express a fusion protein of the present disclosure.

[0227] Any of the aforementioned differentiated cell types can be differentiated from PSCs before the PSCs are engineered to express the fusion proteins of the present disclosure.

[0228] 6.8. METHODS FOR DELIVERY OF EXOGENOUS NUCLEIC ACID TO TARGET CELLS The targeting constructs and expression vectors of the present disclosure are delivered to target cells, thereby generating recombinant target cells containing a nucleic acid encoding a fusion protein comprising the essential polypeptide, the degron, and an optional linker. The nucleic acid can be integrated into the target cell genome, for example, when a targeting construct is used, or can remain extrachromosomal, for example, when an extrachromosomal expression vector is used.

[0229] Exemplary methods for introducing targeting constructs and expression vectors are described below.

[0230] Techniques that can be used to introduce nucleic acids, such as the targeting constructs or expression vectors of the present disclosure, 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 manner are then predisposed to the uptake of exogenous nucleic acids. Electroporation of mammalian cells is described in detail, for example, in Chu et al., 1987, Nucleic Acids Research 15:131. A similar technique, Nucleofection™, utilizes an applied electric field to stimulate the uptake of exogenous nucleic acids into the nuclei of eukaryotic cells. Nucleofection™ and protocols useful for implementing this technique are described in detail, for example, in Distler et al., 2005, Experimental Dermatology 14:315, and US2010 / 03171 14.

[0231] Additional techniques useful for transfection of target cells include squeezeporation. This technique induces rapid mechanical deformation of cells to stimulate the uptake of exogenous DNA through membrane pores that form in response to applied stress. This technique is advantageous in that no vector is required to deliver nucleic acid to cells, such as human target cells. Squeezeporation is described in detail, for example, in Sharei et al., 2013, Journal of Visualized Experiments 81:e50980.

[0232] Lipofection represents another technique useful for transfecting target cells. This method involves loading nucleic acids into liposomes, which often present cationic functional groups, such as quaternary or protonated amines, toward the exterior of the liposome. This promotes electrostatic interactions between the liposome and the cell due to the anionic nature of the cell membrane, ultimately leading to the uptake of the exogenous nucleic acid, for example, by direct fusion of the liposome with the cell membrane or by endocytosis of the complex. Lipofection is described in detail, for example, in U.S. Pat. No. 7,442,386. A similar technique, which utilizes ionic interactions with the cell membrane to induce the uptake of exogenous nucleic acids, involves contacting cells with a cationic polymer-nucleic acid complex. Exemplary cationic molecules that associate with nucleic acids to impart a positive charge favorable for interaction with cell membranes are activated dendrimers (e.g., as described in Dennig, 2003, Topics in Current Chemistry 228:227) and diethylaminoethyl (DEAE)-dextran, whose use as transfection agents is described in detail, e.g., 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 they utilize an applied magnetic field to direct the uptake of nucleic acids. This technique is described in detail, e.g., in US 2010 / 0227406.

[0233] Another useful tool for inducing the uptake of exogenous nucleic acid by target cells is laser infection, which is a technique that involves exposing cells to electromagnetic radiation of a specific wavelength, so as to gradually permeabilize cells and allow nucleic acid to penetrate the cell membrane.This technique is described in detail, for example, in Rhodes et al., 2007, Methods in Cell Biology 82:309.

[0234] Microvesicles represent another potential vehicle that can be used to introduce nucleic acids, such as the targeting constructs disclosed herein, into the genome of target cells. For example, microvesicles induced by co-overexpression of the glycoprotein VSV-G with genome-modifying proteins, such as nucleases, can be used to efficiently deliver proteins that catalyze site-specific cleavage of endogenous nucleic acid sequences to cells, preparing the genome of the cell for covalent incorporation of a nucleic acid of interest, such as a gene or regulatory sequence. The use of such vesicles, also called gesicles, for genetic modification of eukaryotic cells is described in detail, for example, in Quinn et al., 2015, Genetic Modification of Target Cells by Direct Delivery of Active Protein (abstract), and in Methylation changes in early embryonic genes in cancer (abstract), in Proceedings of the 18th Annual Meeting of the American Society of Gene and Cell Therapy, Abstract No. 122.

[0235] In addition to the above, various tools have been developed that can be used to integrate a gene of interest into target cells, such as human cells. One such method that can be used to integrate a transgene or polynucleotide encoding a fusion protein into target cells involves the use of transposons. Transposons are polynucleotides that encode a transposase enzyme and contain a polynucleotide sequence or gene of interest flanked by 5' and 3' excision sites. Once the transposon is delivered to a cell, expression of the transposase gene is initiated, resulting in an active enzyme that excises the gene of interest from the transposon. This activity is mediated by site-specific recognition of the transposon excision site by the transposase. In some instances, these excision sites can be terminal repeats or inverted terminal repeats. Once excised from the transposon, the gene of interest can be integrated into the genome of a mammalian cell by transposase-catalyzed cleavage of a similar excision site present in the cell's nuclear genome. This allows the gene of interest to be inserted into the excised nuclear DNA at the complementary excision site, after which the integration process is completed by covalent ligation of a phosphodiester bond that joins the gene of interest to the DNA of the mammalian cell genome. In certain cases, the transposon may be a retrotransposon, such that genes encoding essential genes are first transcribed into RNA products and then reverse transcribed into DNA before being integrated into the mammalian cell genome. Exemplary transposon systems are the piggybac transposon (described in detail, for example, in WO2010 / 085699) and the sleeping beauty transposon (described in detail, for example, in US2005 / 0112764).

[0236] Other tools for integrating exogenous nucleic acids into the genome of a target cell are based on nuclease-based gene editing (or genome editing), such as the CRISPR / Cas system, zinc finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs). Exemplary CRISPR / Cas gene editing approaches are disclosed in Section 6.9. The use of ZFNs and TALENs in genome editing applications is described, for example, in Urnov et al., 2010, Nature Reviews Genetics 11:636, and Joung et al., 2013, Nature Reviews Molecular Cell Biology 14:49.

[0237] Additional genome editing techniques that can be used to integrate nucleic acids containing transgenes or encoding fusion proteins into the genome of target cells include the use of ARCUS™ meganucleases, which can be rationally designed to site-specifically cleave genomic DNA. The use of these enzymes to integrate transgenes or nucleic acids encoding fusion proteins into the genome of mammalian cells is advantageous given the defined structure-activity relationships established for such enzymes. Single-stranded meganucleases can be modified at specific amino acid positions to create nucleases that selectively cleave DNA at desired locations, enabling site-specific integration of essential genes into the nuclear DNA of target cells. These single-stranded nucleases are extensively described, for example, in U.S. Patent Nos. 8,021,867 and 8,445,251.

[0238] 6.9. Exemplary Gene Editing Approaches 6.9.1 Endonuclease Systems The targeting constructs of the present disclosure can be incorporated into target cells via an endonuclease system.

[0239] Endonuclease systems include: (i) a targeting construct as described in Section 6.3; (ii) an endonuclease enzyme, or a nucleic acid encoding an endonuclease enzyme, as described in Section 6.9.2; (iii) a guide RNA, or a nucleic acid encoding a guide RNA, as described in Section 6.9.3.

[0240] In some embodiments, the endonuclease system comprises: (i) a targeting construct as described in Section 6.3; (ii) an endonuclease enzyme, as described in Section 6.9.2; (iii) a guide RNA as described in Section 6.9.3.

[0241] The endonuclease system can be delivered to the target cell in the form of a ribonucleoprotein complex, as described in Section 6.9.4.

[0242] Endonucleases The targeting constructs of the present disclosure can be integrated into specific target genomic loci by promoting homologous recombination at DNA breaks generated by suitable endonucleases.

[0243] 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.

[0244] 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 version thereof, such as a truncated version or mutant of a wild-type Cas protein that has nuclease activity.

[0245] In some embodiments, the Cas endonuclease is a Cpf1 (Cas12a) endonuclease, or a variant, derivative, or fragment thereof, such as Francisella novicida U112 (FnCpf1), Acidaminococcus sp. BV3L6 (including improved forms such as AsCpf1, enAsCpf1), Lachnospiraceae bacterium ND2006 (LbCpf1), Lachnospiraceae bacterium MA2020 (Lb2Cpfl), Lachnospiraceae bacterium MC2017 (Lb3Cpfl), Moraxella boehmii, bovoculi237 (MbCpf1), or Cpf1 from Prevotella disiens (PdCpf1).

[0246] 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.

[0247] In some embodiments, the Cas endonuclease is a V-type RNA programmable nuclease as disclosed in WO2022 / 258753A1, the entire contents of which are incorporated herein by reference.

[0248] In some embodiments, the Cas endonuclease is a MAD nuclease, such as MAD7 nuclease, as disclosed in U.S. Patent No. 10,337,028, the entire contents of which are incorporated herein by reference.

[0249] In some embodiments, the targeting construct may be integrated 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.

[0250] Non-limiting examples of suitable endonucleases are listed in Table 3. [Table 3-1] [Table 3-2]

[0251] gRNA In some embodiments, the systems, compositions, and methods described herein use 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 RNAs" or "gRNAs."

[0252] The guide RNA has at least a spacer sequence capable of hybridizing to the target nucleic acid sequence of interest and a CRISPR repeat sequence (such a CRISPR repeat sequence is also called a "tracer mate sequence"). 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), the crRNA forms a duplex. In both systems, the duplex binds to a site-directed polypeptide such 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 association with the site-directed polypeptide. Thus, the genome-targeting nucleic acid directs the activity of the site-directed polypeptide.

[0253] In some embodiments, the genome-targeting nucleic acid is a dual-molecule guide RNA having two RNA strands. The first strand comprises, from 5' to 3', an optional spacer extension sequence, a spacer sequence, and a minimal CRISPR repeat sequence. The second strand comprises a minimal tracrRNA sequence (complementary to the minimal CRISPR repeat sequence), a 3' tracrRNA sequence, and an optional tracrRNA extension sequence.

[0254] In some embodiments, the guide RNA is a single guide RNA (sgRNA). In a type II system, the single guide RNA (sgRNA) comprises, from 5' to 3', an optional spacer extension sequence, a spacer sequence, a minimal CRISPR repeat sequence, a single guide linker, a minimal tracrRNA sequence, a 3' tracrRNA sequence, and an optional tracrRNA extension sequence. The optional tracrRNA extension may comprise elements that contribute additional functionality (e.g., stability) to the guide RNA. The single guide linker links the minimal CRISPR repeat sequence and the minimal tracrRNA sequence to form a hairpin structure. The optional tracrRNA extension comprises one or more hairpins. In a type V system, the single guide RNA (sgRNA) comprises, from 5' to 3', a minimal CRISPR repeat sequence and a spacer sequence. Or alternatively, the single guide RNA (sgRNA) in a Type V system has, in the 5' to 3' direction, an optional tracr extension sequence, a tracrRNA sequence, a single guide linker, a minimal CRISPR repeat sequence, a spacer sequence, and an optional spacer extension sequence.

[0255] Modifications of guide RNAs can be used to enhance the formation or stability of a CRISPR-Cas genome editing complex containing a guide RNA and a Cas endonuclease. Modifications of guide RNAs can also, or alternatively, be used to enhance the initiation, stability, or kinetics of the interaction between a genome editing complex and a target sequence in a genome, which can be used, for example, to enhance on-target activity. Modifications of guide RNAs can also, or alternatively, be used to enhance specificity, e.g., the relative rate of genome editing at on-target sites compared to effects at other (off-target) sites. Modifications can also, or alternatively, be used to increase the stability of guide RNAs, for example, by increasing their resistance to degradation by ribonucleases (RNases) present in cells, thereby increasing their half-life within the cell.

[0256] Exemplary Cpf1 guide RNA sequences targeting the essential gene GAPDH are listed in Table 4. [Table 4]

[0257] Exemplary Cas9 guide RNA sequences targeting the essential STEL gene GAPDH are listed in Table 5. [Table 5]

[0258] Exemplary Cas9 guide RNA sequences targeting the essential STEL gene RPL13A are listed in Table 6. [Table 6]

[0259] Exemplary Cas9 guide RNA sequences targeting the essential STEL gene RPLP0 are listed in Table 7. [Table 7]

[0260] Exemplary Cpf1 guide RNA sequences targeting the essential STEL gene RPLP0 are listed in Table 8. [Table 8]

[0261] Exemplary Cas9 guide RNA sequences targeting the essential STEL gene RPL7 are listed in Table 9. [Table 9]

[0262] 6.9.4. Ribonucleoprotein (RNP) complexes In some embodiments, the endonuclease is delivered to the target cell in a composition known as a ribonucleoprotein or RNP complex, which is assembled by combining the endonuclease with a ribonucleic acid.

[0263] In some embodiments, the ribonucleoprotein complex comprises a Cas endonuclease complexed with a suitable ribonucleic acid, which in some embodiments is a gRNA or sgRNA, which are further described in Section 6.9.3.

[0264] One of the most common techniques for RNP delivery is electroporation, which creates pores in the cell membrane, allowing RNP to enter the cytoplasm.In addition, electroporation can be combined with cell-type specific reagents in a technique known as nucleofection, which creates pores in the nuclear membrane, allowing DNA templates to enter.In some embodiments, RNP complexes are delivered to target cells via nucleofection.

[0265] 6.9.5. Gene editing methods In some embodiments, the methods of the present disclosure include introducing a targeting construct into a target cell (or a population of target cells). The targeting construct of the present disclosure can be incorporated into the target cell with an endonuclease system, where the endonuclease system can be introduced into the host or target cell by any of a variety of 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.

[0266] Suitable methods include, for example, exosome delivery, but are not limited to, viral or bacteriophage 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, e.g., Panyam et al., Adv Drug Deliv Rev. 2012 Sep 13. pii:50169-409X(12)00283-9. doi:10.1016 / j.addr.2012.09.023), and the like. Nucleic acids can also be delivered by non-viral delivery vehicles, including but not limited to nanoparticles, liposomes, ribonucleoproteins, positively charged peptides, small 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.

[0267] In some embodiments, the endonuclease system comprises a ribonucleoprotein complex (e.g., a Cas endonuclease and an sgRNA), e.g., as described in Section 6.9.4, and can be delivered to the target cell via nucleofection, electroporation, or similar methods.

[0268] As an alternative to RNP delivery, the endonuclease system can be delivered into target cells in nucleic acid form via a delivery vector, for example, a viral delivery vector.

[0269] Suitable nucleic acids comprising a nucleotide sequence encoding a Cas endonuclease and / or a guide RNA include expression vectors, which in some embodiments are viral constructs, such as recombinant adeno-associated viral constructs (see, e.g., U.S. Patent No. 7,078,387), recombinant adenoviral constructs, recombinant lentiviral constructs, recombinant retroviral constructs, etc. Suitable expression vectors include, but are not limited to, viral vectors (e.g., vaccinia virus; poliovirus; adenovirus-based viral vectors (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 5:10881097, 1999; WO94 / 12649; WO93 / 03769; WO93 / 19191; WO94 / 28938; WO95 / 11984; and WO95 / 00655); adeno-associated viruses (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 in WO93 / 09239, Samulski et al., J. Vir. (1989) 63:3822-3828, Mendelson et al., Viral. (1988) 166:154-165, and Flotte et al. 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., vectors derived from murine leukemia virus, spleen necrosis virus, and retroviruses such as Ruth sarcoma virus, Harvey sarcoma virus, avian leukosis virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus).

[0270] 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.

[0271] The target cell is then grown under conditions that allow gene editing to occur. Without being bound by theory, it is believed that the endonuclease cleaves the target cell genome as guided by the guide RNA, allowing the first and second homology arms of the targeting construct to recombine with the target cell genome, resulting in the nucleotide sequences flanking the homology arms of the targeting construct being integrated into the genome of the target cell.

[0272] In some embodiments, the target cell (e.g., a cell containing the target DNA locus targeted by the targeting construct) is in vitro. In some embodiments, the target cell is in vivo.

[0273] Pharmaceutical Compositions Also disclosed herein are pharmaceutical formulations and medicaments comprising recombinant cells engineered to express the fusion proteins of the present disclosure, and optionally a transgene, together with a pharmaceutically acceptable excipient.

[0274] Suitable excipients include, but are not limited to, salts, diluents (e.g., Tris-HCl, acetate, phosphate), preservatives (e.g., thimerosal, benzyl alcohol, parabens), binders, fillers, solubilizers, disintegrants, adsorbents, solvents, pH adjusters, antioxidants, anti-infective agents, suspending agents, wetting agents, viscosity adjusters, tonicity agents, stabilizers, and other ingredients and combinations thereof. Suitable pharmaceutically acceptable excipients can be selected from materials generally recognized as safe (GRAS) and can be administered to individuals without causing undesirable biological side effects or undesirable interactions. Suitable excipients and their formulations are described in Remington's Pharmaceutical Sciences, 16th ed. 1980, Mack Publishing Co. In addition, such compositions can be complexed with polyethylene glycol (PEG), metal ions, or incorporated into polymeric compounds such as polyacetal acid, polyglycolic acid, hydrogels, or incorporated into liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroblasts. Suitable dosage forms for administration, e.g., parenteral administration, include solutions, suspensions, and emulsions.

[0275] The components of the pharmaceutical formulation 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 formulation can also be a sterile solution, suspension, or emulsion in a non-toxic, parenterally acceptable diluent or solvent, such as 1,3-butanediol.

[0276] In some cases, the formulation can contain one or more tonicity agents to adjust the isotonic range of the formulation.Suitable tonicity agents are well known in the art, and include glycerin, mannitol, sorbitol, sodium chloride, and other electrolytes.In some cases, the formulation can be buffered with an effective amount of buffer solution necessary to maintain a pH suitable for parenteral administration.Suitable buffer solutions are well known to those skilled in the art, and some examples of useful buffer solutions are acetate, borate, carbonate, citrate, and phosphate buffer solutions.

[0277] In some embodiments, the formulations can be distributed or packaged in liquid form, or alternatively, as a solid, e.g., obtained by lyophilization of a suitable liquid formulation that can be reconstituted with a suitable carrier or diluent prior to administration. In some embodiments, the formulations can include a pharmaceutically effective amount of guide RNA and type II Cas protein sufficient to edit a gene in a cell. Pharmaceutical compositions can be formulated for medical and / or veterinary use.

[0278] 6.11. Treatment Methods The recombinant target cells and pharmaceutical compositions of the present disclosure can be introduced into an individual for treatment. For example, the therapeutic cells of the present disclosure can be used to treat a genetic disease by transplanting cells expressing a functional transgene into the 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, injured, damaged, or dysfunctional cells in the affected tissue, organ, or body system.

[0279] The recombinant target cells may be autologous to the subject or allogeneic to the subject.

[0280] The recombinant target cells described herein may be provided in a pharmaceutical composition containing the cells and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be a cell culture medium, optionally free of any animal-derived components. For storage and transportation, the cells may be stored frozen below -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.

[0281] Recombinant target cells can be administered to a patient systemically (e.g., via intravenous injection or infusion) or locally (e.g., via direct injection into local tissues, such as the heart, brain, and the site of damaged tissue). A variety of methods are known in the art for administering cells to tissues or organs of a patient, including, but not limited to, intracoronary, intramyocardial, intraendocardial, or intracranial administration.

[0282] 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 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 a symptom(s) of the disease, disorder, and / or condition. It will be understood by those skilled 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 3 (e.g., 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 are administered to a subject at one or more sites at a time. In some embodiments, 10 3 ~10 18 (e.g., 10 3~10 4 , 10 3 ~10 5 , 10 3 ~10 6 , 10 3 ~10 7 , 10 3 ~10 8 , 10 3 ~10 9 , 10 3 ~10 10 , 10 3 ~10 11 , 10 3 ~10 12 , 10 6 ~10 7 , 10 6 ~10 8 , 10 6 ~10 9 , 10 6 ~10 10 , 10 6 ~10 11 , 10 6 ~10 12 , 10 9 ~10 10 , 10 9 ~10 11 , 10 9 ~10 12 ) cells are administered to a subject at one or more sites at a time. In some embodiments, 10 12 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 More than one) of the cells are administered to the subject at one time at one or more sites.

[0283] In some embodiments, the method of treatment involves selective killing of recombinant target cells transplanted into a subject. The recombinant target cells can be selected, for example, by administering to the subject a drug, such as an IMiD, that activates a degron, resulting in degradation of the fusion protein containing the degron. Degradation of the fusion protein containing the essential polypeptide triggers apoptosis, thus selectively killing the cells.

[0284] Selective killing of target cells can be performed when an adverse event occurs, such as an adverse event due to overexpression of a therapeutic polypeptide. Selective killing of recombinant target cells can also be performed when the therapeutic goal is achieved and the recombinant target cells are no longer needed for treatment. Furthermore, selective killing of recombinant target cells can be used to completely eradicate a graft, such as when the graft causes severe side effects in the subject, such as cytokine storm, excessive (systemic) inflammation, tumor formation, graft-versus-host disease, organ damage, or other health problems.

[0285] Selective killing of target cells can be induced any time after administration of the target cells to a subject, for example, from 1 hour to 1 year (or more) after administration of the target cells.

[0286] In various embodiments, selective killing of the target cells is induced in the subject 1 hour to 1 day after administration of the target cells, 1 day to 1 week after administration of the target cells, 1 week to 2 weeks after administration of the cells, 2 weeks to 1 month after administration of the target cells, 1 month to 3 months after administration of the target cells, 3 months to 1 year after administration of the target cells, or any time range bounded by two of the foregoing embodiments (e.g., 2 weeks to 3 months after administration of the target cells).

[0287] Accordingly, the present disclosure provides methods of treatment for a subject who has previously received cell therapy using recombinant target cells as described herein, comprising administering to the subject an effective amount of a degron inducer. In some embodiments, the subject previously received recombinant target cells engineered to express a fusion protein comprising an essential polypeptide and an IMiD-inducible degron, and the method comprises administering to the subject an amount of an IMiD effective to selectively kill the recombinant target cells. Exemplary IMiDs include, but are not limited to, pomalidomide, thalidomide, lenalidomide, iveldomide, and avadomide. In various embodiments, the IMiD is administered when the subject experiences cytokine storm, excessive (systemic) inflammation, tumor formation, graft-versus-host disease, or another health problem caused by cell therapy.

[0288] 7. Numbered Embodiments While various specific embodiments have been illustrated and described, it will be understood that various changes can be made without departing from the spirit and scope of the present disclosure(s). The present disclosure is exemplified by the numbered embodiments set forth below. Unless otherwise specified, any concept, aspect, and / or feature of any of the embodiments described in the above detailed description applies mutatis mutandis to any of the numbered embodiments below. 1. (a) an essential polypeptide; (b) degron and (c) optionally, a linker. 2. The fusion protein of embodiment 1, wherein the degron is N-terminal to the essential polypeptide. 3. The fusion protein of embodiment 1, wherein the degron is C-terminal to the essential polypeptide. 4. The fusion protein of any one of embodiments 1 to 3, wherein the degron is an inducible degron. 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). 6. The fusion protein of embodiment 5, wherein the degron is drug-inducible. 7. The fusion protein of embodiment 6, wherein the drug is an immunomodulatory drug (IMiD). 8. The fusion protein of embodiment 7, wherein the IMiD is lenalidomide, iberdomide, thalidomide, avadomide, or pomalidomide. 9. The fusion protein of embodiment 8, wherein the IMiD is iberdomide. 10. The fusion protein of embodiment 8, wherein the IMiD is avadomide. 11. The fusion protein of embodiment 8, wherein the IMiD is thalidomide. 12. The fusion protein of embodiment 8, wherein the IMiD is lenalidomide. 13. The fusion protein of embodiment 8, wherein the IMiD is pomalidomide. 14. The degron has the amino acid sequence RPFQCNQCGASFTQKGNLLRHIKLH (SEQ ID NO: 3), FNVLMVHKRSHTGERPLQCEICGFTCRQKGNLLRHIKLHTGEKPFKCHLCNYACQRRDAL (SEQ ID NO: 4, corresponding to SEQ ID NO: 42 in WO2021 / 188286A2), FNVLMVHKRSHTGERP (SEQ ID NO: 5, corresponding to SEQ ID NO: 97 in WO2019 / 089592A1), FNVLMVHRRSHTGERP (SEQ ID NO: 6, corresponding to SEQ ID NO: 100 in WO2019 / 089592A1), TGEKPFKCHLCNYACQRRDAL (SEQ ID NO: 7, corresponding to SEQ ID NO: 102 in WO2019 / 089592A1), TGEKPFKCHLCNYACQRRDAL (SEQ ID NO: 7, corresponding to SEQ ID NO: 102 in WO2019 / 089592A1), TGEKPFKCHLCNYACQRR 14. The fusion protein according to any one of embodiments 1 to 13, comprising or consisting of DAL (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). 15. The fusion protein of embodiment 14, wherein the degron comprises or consists of the amino acid sequence of SEQ ID NO: 3. 16. The fusion protein of embodiment 14, wherein the degron comprises or consists of the amino acid sequence of SEQ ID NO: 4. 17. The fusion protein of embodiment 14, wherein the degron comprises or consists of the amino acid sequence of SEQ ID NO: 5. 18. The fusion protein of embodiment 14, wherein the degron comprises or consists of the amino acid sequence of SEQ ID NO: 6. 19. The fusion protein of embodiment 14, wherein the degron comprises or consists of the amino acid sequence of SEQ ID NO: 7. 20. The fusion protein of embodiment 14, wherein the degron comprises or consists of the amino acid sequence of SEQ ID NO: 8. 21. The fusion protein of embodiment 14, wherein the degron comprises or consists of the amino acid sequence of SEQ ID NO: 9. 22. The fusion protein of embodiment 14, wherein the degron comprises or consists of the amino acid sequence of SEQ ID NO: 10. 23. The fusion protein of embodiment 14, wherein the degron comprises or consists of the amino acid sequence of SEQ ID NO: 11. 24. The fusion protein of embodiment 14, wherein the degron comprises or consists of the amino acid sequence of SEQ ID NO: 12. 25. The fusion protein of embodiment 14, wherein the degron comprises or consists of the amino acid sequence of SEQ ID NO: 13. 26. The fusion protein of any one of embodiments 1 to 25, wherein the degron is a superdegron. 27. The fusion protein of embodiment 6, wherein the degron is a SMASh (small molecule assisted shutoff) tagged degron. 28. The fusion protein of any one of embodiments 1 to 27, comprising a linker sequence between the essential polypeptide and the degron. 29. The fusion protein of embodiment 27, wherein the linker is 1 to 30 amino acids in length. 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. 31. The fusion protein of embodiment 29 or 30, wherein the linker is 1 to 12 amino acids in length, 2 to 12 amino acids in length, or 1 to 10 amino acids in length. 32. The fusion protein of embodiment 29 or 30, wherein the linker is 3 to 10 amino acids in length. 33. The fusion protein of embodiment 29 or 30, wherein the linker is 11 to 20 amino acids in length. 34. The fusion protein of embodiment 29 or 30, wherein the linker is 21 to 30 amino acids in length. 35. The fusion protein of any one of embodiments 1 to 34, comprising multiple degrons. 36. The fusion protein of embodiment 35, wherein the fusion protein comprises two or more degrons. 37. The fusion protein of embodiment 36, wherein the degron is tandem. 38. The fusion protein of embodiment 37, wherein each pair of degrons is separated by a linker, and optionally (a) the linker is a linker described in Section 6.2.3, and / or (b) all linkers separating the pairs of degrons are the same. 39. The fusion protein of any one of embodiments 35-38, wherein the multiple degrons are similarly inducible, and optionally the multiple degrons are the same. 40. An essential polypeptide is (a) a STEL polypeptide, or (b) a non-STEL polypeptide. 41. The fusion protein of any one of embodiments 1 to 40, wherein the essential polypeptide is involved in one or more of glycolysis, ribonucleopolypeptide complex formation, focal adhesion, cell-substrate adhesive junctions, cell-substrate 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. 42. The fusion protein of any one of embodiments 1-41, wherein the essential polypeptide is (a) a ribosomal polypeptide or (b) a non-ribosomal polypeptide. 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, RPL18A, 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. 44. The fusion protein of any one of embodiments 1 to 41, wherein the essential polypeptide is (a) a ribosomal polypeptide small subunit (RPS) or (b) is not a ribosomal polypeptide small subunit (RPS). 45. The fusion protein of embodiment 44, wherein the essential polypeptide is a small ribosomal polypeptide subunit (RPS), and optionally the small ribosomal polypeptide subunit (RPS) is RPS2, RPS19, RPS14, RPS3A, RPS12, RPS3, RPS6, RPS23, RPS27A, RPS8, RPS4X, RPS7, RPS24, RPS27, RPS15A, RPS9, RPS28, RPS13, RPSA, RPS5, RPS 16, RPS25, RPS15, RPS20, or RPS11. 46. ​​The fusion protein of any one of embodiments 1-41, wherein the essential polypeptide is (a) an actin polypeptide, or (b) is not an actin polypeptide. 47. The fusion protein of embodiment 46, wherein the essential polypeptide is an actin polypeptide, and optionally the actin polypeptide is ACTG1 or ACTB. 48. The fusion protein of any one of embodiments 1-41, wherein the essential polypeptide is (a) a eukaryotic translation factor, or (b) is not a eukaryotic translation factor. 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. 50. The fusion protein of any one of embodiments 1-41, wherein the essential polypeptide is (a) a histone or (b) is not a histone. 51. The fusion protein of embodiment 50, wherein the essential polypeptide is a histone, optionally wherein the histone is H3F3A or H3F3B. 52. The fusion protein of any one of embodiments 1-41, wherein the essential polypeptide is selected from (a) 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. 53. The fusion protein of embodiment 52, wherein the essential polypeptide is GAPDH. 54. The fusion protein of embodiment 52, wherein the essential polypeptide is RPL13A. 55. The fusion protein of embodiment 52, wherein the essential polypeptide is RPL7. 56. The fusion protein of embodiment 52, wherein the essential polypeptide is RPLPO. 57. A targeting construct comprising: (a) a first homology arm corresponding to a 5' target sequence comprising a first region of homology to an essential gene encoding an essential polypeptide in a target genomic locus; (b) a nucleotide sequence encoding a degron (the "degron-coding sequence"); and (c) a second homology arm corresponding to the 3' target sequence, the second homology arm comprising a second region of homology to an essential gene in the target genomic locus. 57. A targeting construct 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, and optionally, the fusion protein has one or more features as defined in any one of embodiments 1-56 or Section 6.2. 58. The targeting construct of embodiment 57, wherein the targeting construct is configured such that, upon recombination with the target locus, the essential gene is modified to encode a fusion protein comprising a degron at the C-terminus of the essential polypeptide. 59. The targeting construct of embodiment 57, wherein the targeting construct is configured such that, upon recombination with the target locus, the essential gene is modified to encode a fusion protein comprising a degron at the N-terminus of the essential polypeptide. 60. The targeting construct of any one of embodiments 57-59, wherein the degron is an inducible degron. 61. The targeting construct of embodiment 60, wherein the degron is drug-inducible, temperature-sensitive, light-inducible, or polypeptide-activated (optionally, the polypeptide is TEV protease). 62. The targeting construct of embodiment 61, wherein the degron is drug-inducible. 63. The targeting construct of embodiment 62, wherein the drug is an immunomodulatory drug (IMiD). 64. The targeting construct of embodiment 63, wherein the IMiD is lenalidomide, iberdomide, thalidomide, avadomide, or pomalidomide. 65. The targeting construct of embodiment 63, wherein the IMiD is iberdomide. 66. The targeting construct of embodiment 63, wherein the IMiD is avadomide. 67. The targeting construct of embodiment 63, wherein the IMiD is thalidomide. 68. The targeting construct of embodiment 63, wherein the IMiD is lenalidomide. 69. The targeting construct of embodiment 63, wherein the IMiD is pomalidomide. 70. The degron has the amino acid sequence RPFQCNQCGASFTQKGNLLRHIKLH (SEQ ID NO: 3), FNVLMVHKRSHTGERPLQCEICGFTCRQKGNLLRHIKLHTGEKPFKCHLCNYACQRRDAL (SEQ ID NO: 4, corresponding to SEQ ID NO: 42 in WO2021 / 188286A2), FNVLMVHKRSHTGERP (SEQ ID NO: 5, corresponding to SEQ ID NO: 97 in WO2019 / 089592A1), FNVLMVHRRSHTGERP (SEQ ID NO: 6, corresponding to SEQ ID NO: 100 in WO2019 / 089592A1), TGEKPFKCHLCNYACQRRDAL (SEQ ID NO: 7, corresponding to SEQ ID NO: 102 in WO2019 / 089592A1), TGEKPFKCHLCNYACQRRDAL (SEQ ID NO: 7, corresponding to SEQ ID NO: 102 in WO2019 / 089592A1), TGEKPFKCHLCNYACQRR 69. The targeting construct of any one of embodiments 57 to 69, comprising or consisting of: DAL (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). 71. The targeting construct of embodiment 70, wherein the degron comprises or consists of the amino acid sequence of SEQ ID NO: 3. 72. The targeting construct of embodiment 70, wherein the degron comprises or consists of the amino acid sequence of SEQ ID NO: 4. 73. The targeting construct of embodiment 70, wherein the degron comprises or consists of the amino acid sequence of SEQ ID NO: 5. 74. The targeting construct of embodiment 70, wherein the degron comprises or consists of the amino acid sequence of SEQ ID NO: 6. 75. The targeting construct of embodiment 70, wherein the degron comprises or consists of the amino acid sequence of SEQ ID NO: 7. 76. The targeting construct of embodiment 70, wherein the degron comprises or consists of the amino acid sequence of SEQ ID NO: 8. 77. The targeting construct of embodiment 70, wherein the degron comprises or consists of the amino acid sequence of SEQ ID NO: 9. 78. The targeting construct of embodiment 70, wherein the degron comprises or consists of the amino acid sequence of SEQ ID NO: 10. 79. The targeting construct of embodiment 70, wherein the degron comprises or consists of the amino acid sequence of SEQ ID NO: 11. 80. The targeting construct of embodiment 70, wherein the degron comprises or consists of the amino acid sequence of SEQ ID NO: 12. 81. The targeting construct of embodiment 70, wherein the degron comprises or consists of the amino acid sequence of SEQ ID NO: 13. 82. The targeting construct of any one of embodiments 57-81, wherein the degron is a superdegron. 83. The targeting construct of embodiment 62, wherein the degron is a SMASh (small molecule assisted shutoff) tagged degron. 84. A targeting construct according to any one of embodiments 57 to 83, wherein the fusion protein comprises a linker sequence between the essential polypeptide and the degron. 85. The targeting construct of embodiment 82, wherein the linker is 1 to 30 amino acids in length. 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. 87. The targeting construct of embodiment 85, wherein the linker is 1 to 12 amino acids in length, 2 to 12 amino acids in length, or 1 to 10 amino acids in length. 88. The targeting construct of embodiment 85, wherein the linker is 3 to 10 amino acids in length. 89. The targeting construct of embodiment 85, wherein the linker is 11 to 20 amino acids in length. 90. The targeting construct of embodiment 85, wherein the linker is 21 to 30 amino acids in length. 91. A targeting construct according to any one of embodiments 57 to 90, wherein the fusion protein comprises multiple degrons. 92. The targeting construct of embodiment 91, wherein the fusion protein comprises two or more degrons. 93. The targeting construct of embodiment 92, wherein the degron is tandem. 94. The targeting construct of embodiment 93, wherein each pair of degrons is separated by a linker, and optionally (a) the linker is a linker described in Section 6.2.3, and / or (b) all linkers separating the pairs of degrons are the same. 95. A targeting construct according to any one of embodiments 91-94, wherein the multiple degrons are similarly inducible, and optionally, the multiple degrons are the same. 96. Essential genes are (a) the STEL gene, or (b) A targeting construct described in any one of embodiments 57 to 95, which is a non-STEL gene. 97. A targeting construct described in any one of embodiments 57 to 96, wherein the essential genes are involved in one or more of glycolysis, ribonucleopolypeptide complex formation, focal adhesion, cell-substrate adhesive junctions, cell-substrate 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. 98. A targeting construct described in any one of embodiments 57 to 97, wherein the essential gene (a) encodes a ribosomal polypeptide, or (b) does not encode a ribosomal polypeptide. 99. The targeting construct of embodiment 98, wherein the essential gene encodes a ribosomal polypeptide, and optionally the ribosomal polypeptide is RPL13A, RPLP0, RPL10, RPL13, RPSJ8, RPL3, RPLP1, 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, RPL23A, RPL26, RPL36, RPL35, RPL23, RPL4, or RPL22. 100. A targeting construct described in 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). 101. The targeting construct of embodiment 100, wherein the essential gene encodes a small ribosomal polypeptide subunit (RPS), and optionally the small ribosomal polypeptide subunit (RPS) is RPS2, RPS19, RPS14, RPS3A, RPS12, RPS3, RPS6, RPS23, RPS27A, RPS8, RPS4X, RPS7, RPS24, RPS27, RPS15A, RPS9, RPS28, RPS13, RPSA, RPS5, RPS 16, RPS25, RPS15, RPS20, or RPS11. 102. A targeting construct according to any one of embodiments 57-97, wherein the essential gene (a) encodes an actin polypeptide, or (b) does not encode an actin polypeptide. 103. The targeting construct of embodiment 102, wherein the essential gene encodes an actin polypeptide, and optionally, the actin polypeptide is ACTG1 or ACTB. 104. A targeting construct according to 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. 105. The targeting construct of embodiment 104, wherein the essential gene encodes a eukaryotic translation factor, and optionally, the eukaryotic translation factor is EEF1A1, EEF2, or EIF1. 106. A targeting construct according to any one of embodiments 57-97, wherein the essential gene (a) encodes a histone, or (b) does not encode a histone. 107. The targeting construct of embodiment 106, wherein the essential gene encodes a histone, and optionally the histone is H3F3A or H3F3B. 108. A targeting construct according to any one of embodiments 57 to 97, wherein the essential gene is selected from (a) 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. 109. The targeting construct of embodiment 108, wherein the essential gene is GAPDH. 110. The targeting construct of embodiment 108, wherein the essential gene is RPL13A. 111. The targeting construct of embodiment 108, wherein the essential gene is RPL7. 112. The targeting construct of embodiment 108, wherein the essential gene is RPLP0. 113. A targeting construct according to 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. 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. 115. A targeting construct according to any one of embodiments 57 to 114, wherein the difference in length between the first homology arm and the second homology arm, if present, is less than 75 nucleotides. 116. The targeting construct of embodiment 115, wherein the difference in length between the first homology arm and the second homology arm, if present, is less than 50 nucleotides. 117. The targeting construct of any one of embodiments 57 to 116, wherein the targeting construct further comprises a transgene between the degron coding sequence and the second homology arm. 118. The targeting construct of embodiment 113, wherein the transgene is linked to a nucleotide sequence encoding a fusion protein ("fusion protein coding sequence"). 119. The targeting construct of embodiment 118, wherein the fusion protein coding sequence and the transgene are connected via a nucleotide sequence encoding an internal ribosome entry site ("IRES"). 120. The targeting construct of embodiment 118, wherein the fusion protein coding sequence and the transgene are connected in frame via a nucleotide sequence encoding a self-cleaving peptide ("self-cleaving peptide coding sequence"). 121. The targeting construct of embodiment 120, wherein the self-cleaving peptide is a 2A peptide. 122. A targeting construct according to embodiment 120 or 121, wherein the self-cleaving peptide is T2A, P2A, E2A, F2A, or PQR. 123. The introduced gene is (a) encoding a therapeutic polypeptide; and / or (b) A targeting construct described in any one of embodiments 57 to 122, which is a transgene described in Section 6.4. 124. The targeting construct of embodiment 123, wherein the therapeutic polypeptide is a lysosomal enzyme. 125. The targeting construct of embodiment 124, wherein the lysosomal enzyme is alpha-L-iduronidase, arylsulfatase A, beta-glucocerebrosidase, acid sphingomyelinase, alpha-galactosidase, or beta-galactosidase. 126. The targeting construct of embodiment 123, wherein the therapeutic polypeptide is a polypeptide the deficiency of which is associated with hemophilia. 127. The targeting construct of embodiment 126, wherein the therapeutic polypeptide is factor VIII or factor IX. 128. The targeting construct of embodiment 123, wherein the therapeutic polypeptide is an immunomodulatory polypeptide. 129. The targeting construct of embodiment 128, wherein the immunomodulatory polypeptide is a human leukocyte antigen ("HLA") polypeptide. 130. The targeting construct of embodiment 129, wherein the HLA polypeptide is an HLA class Ib polypeptide. 131. The targeting construct of embodiment 129 or 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). 132. The targeting construct of embodiment 128, wherein the immunomodulatory polypeptide is a cytokine. 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. 134. The targeting construct of embodiment 123, wherein the therapeutic polypeptide is or comprises an antibody or an antigen-binding fragment thereof. 135. The targeting construct of embodiment 134, wherein the therapeutic polypeptide binds to the pathogenic polypeptide. 136. The targeting construct of embodiment 135, wherein the pathogenic polypeptide is a tau, alpha-synuclein, or beta-amyloid polypeptide. 137. The targeting construct of embodiment 134, wherein the therapeutic polypeptide binds to cancer cells. 138. The targeting construct of embodiment 137, wherein the therapeutic polypeptide is a chimeric antigen receptor. 139. A targeting construct according to embodiment 137 or 138, wherein the therapeutic polypeptide binds to a tumor-associated antigen. 140. The targeting construct of embodiment 139, wherein the tumor-associated antigen is CD19 or CD20. 141. A targeting construct according to any one of embodiments 57 to 140, which is a vector. 142. The targeting construct of embodiment 141, wherein the vector is a viral vector. 143. The targeting construct of embodiment 142, wherein the viral vector is an AAV vector, a retroviral vector, or a lentiviral vector. 144. The targeting construct of embodiment 141, wherein the vector is a DNA vector. 145. The targeting construct of embodiment 141, wherein the vector is an RNA vector. 146. A targeting construct according to any of embodiments 57 to 145, comprising a nucleotide sequence configured as shown in FIG. 7A, and optionally, the nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 1. 147. A targeting construct according to any of embodiments 57 to 145, comprising a nucleotide sequence configured as shown in Figure 7B. 148. A targeting construct according to any of embodiments 57 to 145, comprising a nucleotide sequence configured as shown in Figure 7C. 149. A targeting construct according to any of embodiments 57 to 145, comprising a nucleotide sequence configured as shown in Figure 7D, and optionally, the nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 2. 150. A targeting construct described in any of embodiments 57 to 145, comprising a nucleotide sequence configured as shown in Figure 7E, and optionally, the nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 29. 151. A targeting construct described in any of embodiments 57 to 145, comprising a nucleotide sequence configured as shown in Figure 7F, and optionally, the nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 30. 152. A targeting construct according to any of embodiments 57 to 145, comprising a nucleotide sequence configured as shown in Figure 7G. 153. A targeting construct according to any of embodiments 57 to 145, comprising a nucleotide sequence configured as shown in Figure 7H. 154. A targeting construct according to any of embodiments 57 to 145, comprising a nucleotide sequence configured as shown in Figure 7I. 155. A targeting construct according to any of embodiments 57 to 145, comprising a nucleotide sequence configured as shown in Figure 7J. 156. A targeting construct described in any of embodiments 57 to 145, comprising a nucleotide sequence configured as shown in Figure 7K, and optionally, the nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 14. 157. A targeting construct described in any of embodiments 57 to 145, comprising a nucleotide sequence configured as shown in Figure 7L, and optionally, the nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 17. 158. (a) a targeting construct according to any one of embodiments 57 to 157; and (b) a CRISPR-associated endonuclease ("Cas polypeptide") or a nucleic acid encoding a Cas polypeptide; (c) a guide RNA ("gRNA"), or a nucleic acid encoding the gRNA, comprising a scaffold for binding to a Cas polypeptide and a spacer sequence corresponding to an essential gene. 159. The system of embodiment 158, wherein the guide RNA is a single guide RNA ("sgRNA"). 160. The system according to embodiment 158 ​​or 159, comprising a Cas polypeptide and a gRNA. 161. The system according to any one of embodiments 158-160, which is in the form of a ribonucleoprotein particle ("RNP"). 162. A method for producing a gene-edited target cell, comprising: (a) introducing the system according to any one of embodiments 158 to 161 into target cells, optionally the target cells being as defined in section 6.7; (b) culturing the target cells under conditions where gene editing occurs, thereby producing gene-edited target cells. 163. The method of embodiment 162, wherein the target cells are stem cells or cells differentiated from stem cells. 164. The method of embodiment 162 or 163, wherein the target cells are stem cells. 165. The method of embodiment 164, wherein the stem cells are human embryonic stem cells, induced pluripotent stem cells ("iPSCs"), or cells differentiated therefrom. 166. The target cells are (a) human immune cells, optionally selected from T cells, T cells expressing a chimeric antigen receptor (CAR) or a recombinant TCR, regulatory T cells, myeloid cells, dendritic cells, and macrophages (e.g., immunosuppressive macrophages); (b) a cell in the human nervous system, optionally selected from a dopaminergic neuron, a microglial cell, an oligodendrocyte, an astrocyte, a cortical neuron, a spinal cord or oculomotor neuron, an enteric neuron, a placode-derived cell, a Schwann cell, and a trigeminal or sensory neuron; (c) cells within the human cardiovascular system, optionally selected from cardiomyocytes, endothelial cells, and nodal cells; (d) cells in a human metabolic system, 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 epithelial cell, a photoreceptor cone cell, a photoreceptor rod cell, a bipolar cell, or a ganglion cell; or (f) A method according to any one of embodiments 162 to 164, wherein the cell is a precursor or progenitor of any one of the aforementioned cells. 167. The method of any one of embodiments 162 to 166, wherein the gene-edited target cell is of the ectodermal lineage, and optionally, the gene-edited target cell is a neuron or a precursor or progenitor thereof. 168. The method of any one of embodiments 162-166, wherein the gene-edited target cell is of the mesodermal lineage, and optionally, the gene-edited target cell is a cardiomyocyte or a precursor or progenitor thereof. 169. The method of any one of embodiments 162-166, wherein the gene-edited target cell is of the endodermal lineage, and optionally, the gene-edited target cell is a lung cell, a thyroid cell, or a pancreatic cell, or a precursor or progenitor thereof. 170. The method of any one of embodiments 162 to 166, wherein the gene-edited target cell is a cardiac cell, a cardiac progenitor cell, or a mature or immature (atrial or ventricular) cardiomyocyte, a cardiac endothelial cell, a lymph node cell, or a precursor or progenitor thereof. 171. The method of any one of embodiments 162 to 166, wherein the gene-edited target cell is a T cell, a CAR-T cell, a recombinant TCR-expressing T cell, a regulatory T cell, a myeloid cell, a dendritic cell, and / or a macrophage, or a precursor or progenitor thereof. 172. The method of any one of embodiments 162 to 166, wherein the gene-edited target cells are oligodendrocytes or their precursors or precursors. 173. The method of any one of embodiments 162-166, wherein the gene-edited target cell is a neural crest cell, an astrocyte, a dopaminergic neuron, or a precursor or precursor thereof. 174. The method of any one of embodiments 162 to 166, wherein the gene-edited target cell is a photoreceptor cell, a retinal pigment epithelial cell, a neural retinal cell, or a precursor or progenitor thereof. 175. The method of any one of embodiments 162 to 166, wherein the gene-edited target cells are microglial cells or microglial precursor or progenitor cells. 176. The method of any one of embodiments 162 to 166, wherein the gene-edited target cells are hepatocytes, cholangiocytes, and pancreatic beta cells, or precursors or precursors thereof. 177. The method of any one of embodiments 162 to 166, wherein the gene-edited target cells are enteric cells, or enteric progenitor or precursor cells. 178. A gene-edited target cell obtained or obtainable by a method according to any one of embodiments 162 to 177. 179. (a) degron and (b) a gene-edited target cell comprising an essential gene encoding a fusion protein comprising an essential polypeptide, wherein the essential gene is encoded by an essential gene as defined in any one of embodiments 96 to 112. 180. The gene-edited target cell of embodiment 179, wherein the degron is at the C-terminus of the essential polypeptide. 181. The gene-edited target cell of embodiment 179, wherein the degron is at the N-terminus of the essential polypeptide. 182. The gene-edited target cell of any one of embodiments 179 to 181, wherein the degron is an inducible degron. 183. The gene-edited target cell of embodiment 182, wherein the degron is drug-inducible, temperature-sensitive, light-inducible, or polypeptide-activated (optionally, the polypeptide is TEV protease). 184. The gene-edited target cell of embodiment 183, wherein the degron is drug-inducible. 185. The gene-edited target cell of embodiment 184, wherein the drug is an immunomodulatory drug (IMiD). 186. The gene-edited target cell of embodiment 185, wherein the IMiD is lenalidomide, iberdomide, thalidomide, avadomide, or pomalidomide. 187. The gene-edited target cell of embodiment 185, wherein the IMiD is iberdomide. 188. The gene-edited target cell of embodiment 185, wherein the IMiD is avadomide. 189. The gene-edited target cell of embodiment 185, wherein the IMiD is thalidomide. 190. The gene-edited target cell of embodiment 185, wherein the IMiD is lenalidomide. 191. The gene-edited target cell of embodiment 185, wherein the IMiD is pomalidomide. 192. The degron is selected from the group consisting of the amino acid sequences RPFQCNQCGASFTQKGNLLRHIKLH (SEQ ID NO: 3), FNVLMVHKRSHTGERPLQCEICGFTCRQKGNLLRHIKLHTGEKPFKCHLCNYACQRRDAL (SEQ ID NO: 4, corresponding to SEQ ID NO: 42 in WO2021 / 188286A2), FNVLMVHKRSHTGERP (SEQ ID NO: 5, corresponding to SEQ ID NO: 97 in WO2019 / 089592A1), FNVLMVHRRSHTGERP (SEQ ID NO: 6, corresponding to SEQ ID NO: 100 in WO2019 / 089592A1), TGEKPFKCHLCNYACQRRDAL (SEQ ID NO: 7, corresponding to SEQ ID NO: 102 in WO2019 / 089592A1), TGEKPFKCHLCNYACQRRDAL ( 192. The gene-edited target cell of any one of embodiments 179 to 191, comprising or consisting of: SEQ ID NO: 8, corresponding to SEQ ID NO: 103 in WO2019 / 089592A1), FQCNQCGASFT (SEQ ID NO: 9, corresponding to SEQ ID NO: 528 in WO2021 / 188286A2), FQCPICGLVIK (SEQ ID NO: 10, corresponding to SEQ ID NO: 529 in WO2021 / 188286A2), LQCEICGFTCR (SEQ ID NO: 11, corresponding to SEQ ID NO: 530 in WO2021 / 188286A2), LQCEICGYQCR (SEQ ID NO: 12, corresponding to SEQ ID NO: 531 in WO2021 / 188286A2), or LQCEVCGFQCR (SEQ ID NO: 13, corresponding to SEQ ID NO: 532 in WO2021 / 188286A2). 193. The gene-edited target cell of claim 192, wherein the degron comprises or consists of the amino acid sequence of SEQ ID NO:3. 194. The gene-edited target cell of claim 192, wherein the degron comprises or consists of the amino acid sequence of SEQ ID NO:4. 195. The gene-edited target cell of claim 192, wherein the degron comprises or consists of the amino acid sequence of SEQ ID NO:5. 196. The gene-edited target cell of claim 192, wherein the degron comprises or consists of the amino acid sequence of SEQ ID NO:6. 197. The gene-edited target cell of claim 192, wherein the degron comprises or consists of the amino acid sequence of SEQ ID NO:7. 198. The gene-edited target cell of claim 192, wherein the degron comprises or consists of the amino acid sequence of SEQ ID NO:8. 199. The gene-edited target cell of claim 192, wherein the degron comprises or consists of the amino acid sequence of SEQ ID NO:9. 200. The gene-edited target cell of claim 192, wherein the degron comprises or consists of the amino acid sequence of SEQ ID NO: 10. 201. The gene-edited target cell of claim 192, wherein the degron comprises or consists of the amino acid sequence of SEQ ID NO: 11. 202. The gene-edited target cell of claim 192, wherein the degron comprises or consists of the amino acid sequence of SEQ ID NO: 12. 203. The gene-edited target cell of claim 192, wherein the degron comprises or consists of the amino acid sequence of SEQ ID NO: 13. 204. The gene-edited target cell of any one of embodiments 179 to 203, wherein the degron is a superdegron. 205. The gene-edited target cell of embodiment 184, wherein the degron is a SMASh (small molecule-assisted shutoff) tagged degron. 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 degron. 207. The gene-edited target cell of embodiment 205, wherein the linker is 1 to 30 amino acids in length. 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. 209. The gene-edited target cell of embodiment 207 or 208, wherein the linker is 1 to 12 amino acids in length, 2 to 12 amino acids in length, or 1 to 10 amino acids in length. 210. The gene-edited target cell of embodiment 207 or 208, wherein the linker is 3 to 10 amino acids in length. 211. The gene-edited target cell of embodiment 207 or 208, wherein the linker is 11 to 20 amino acids in length. 212. The gene-edited target cell of embodiment 207 or 208, wherein the linker is 21 to 30 amino acids in length. 213. A gene-edited target cell according to any one of embodiments 179 to 212, wherein the fusion protein comprises multiple degrons. 214. The gene-edited target cell of embodiment 213, wherein the fusion protein comprises two or more degrons. 215. The gene-edited target cell of embodiment 214, wherein the degron is tandem. 216. The gene-edited target cell of embodiment 215, wherein each pair of degrons is separated by a linker, and optionally (a) the linker is a linker described in Section 6.2.3, and / or (b) all linkers separating the pairs of degrons are the same. 217. A gene-edited target cell according to any one of embodiments 213 to 216, wherein the multiple degrons are similarly inducible, and optionally the multiple degrons are the same. 218. Essential genes are (a) the STEL gene, or (b) A gene-edited target cell described in any one of embodiments 179 to 217, wherein the gene is a non-STEL gene. 219. The gene-edited target cell of any one of embodiments 179 to 218, wherein the essential genes are involved in one or more of glycolysis, ribonucleopolypeptide complex formation, focal adhesion, cell-substrate adhesive junctions, cell-substrate 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. 220. A gene-edited target cell according to any one of embodiments 179 to 219, wherein the essential gene (a) encodes a ribosomal polypeptide, or (b) does not encode a ribosomal polypeptide. 221. The gene-edited target cell of embodiment 220, wherein the essential gene encodes a ribosomal polypeptide, and optionally the ribosomal polypeptide is RPL13A, RPLP0, RPL10, RPL13, RPSJ8, RPL3, RPLP1, 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, RPL23A, RPL26, RPL36, RPL35, RPL23, RPL4, or RPL22. 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). 223. The gene-edited target cell of embodiment 222, wherein the essential gene encodes a small ribosomal polypeptide subunit (RPS), and optionally the small ribosomal polypeptide 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. 224. A gene-edited target cell according to any one of embodiments 179 to 219, wherein the essential gene (a) encodes an actin polypeptide, or (b) does not encode an actin polypeptide. 225. The gene-edited target cell of embodiment 224, wherein the essential gene encodes an actin polypeptide, and optionally the actin polypeptide is ACTG1 or ACTB. 226. A gene-edited target cell according to 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. 227. The gene-edited target cell of embodiment 226, wherein the essential gene encodes a eukaryotic translation factor, and optionally the eukaryotic translation factor is EEF1A1, EEF2, or EIF1. 228. A gene-edited target cell according to any one of embodiments 179 to 219, wherein the essential gene (a) encodes a histone, or (b) does not encode a histone. 229. The gene-edited target cell of embodiment 228, wherein the essential gene encodes a histone, and optionally the histone is H3F3A or H3F3B. 230. The gene-edited target cell of any one of embodiments 179-219, wherein the essential gene is selected from (a) 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. 231. The gene-edited target cell of embodiment 230, wherein the essential gene is GAPDH. 232. The gene-edited target cell of embodiment 230, wherein the essential gene is RPL13A. 233. The gene-edited target cell of embodiment 230, wherein the essential gene is RPL7. 234. The gene-edited target cell of embodiment 230, wherein the essential gene is RPLP0. 235. A gene-edited target cell according to any one of embodiments 179 to 234, further comprising a transgene. 236. The gene-edited target cell of embodiment 235, wherein the transgene is linked to a nucleotide sequence encoding a fusion protein ("fusion protein coding sequence") via a nucleotide sequence encoding an internal ribosome entry site ("IRES"). 237. The gene-edited target cell of embodiment 235, wherein the transgene is linked to a nucleotide sequence encoding a fusion protein ("fusion protein coding sequence") via a nucleotide sequence encoding a self-cleaving peptide ("self-cleaving peptide coding sequence"). 238. The gene-edited target cell of embodiment 237, wherein the self-cleaving peptide is a 2A peptide. 239. The gene-edited target cell of embodiment 237 or 238, wherein the self-cleaving peptide is T2A, P2A, E2A, F2A, or PQR. 240.The introduced gene is (a) encoding a therapeutic polypeptide; and / or (b) A gene-edited target cell described in any one of embodiments 179 to 239, wherein the introduced gene is described in section 6.4. 241. The gene-edited target cell of embodiment 240, wherein the therapeutic polypeptide is a lysosomal enzyme. 242. The gene-edited target cell of embodiment 241, wherein the lysosomal enzyme is alpha-L-iduronidase, arylsulfatase A, beta-glucocerebrosidase, acid sphingomyelinase, alpha-galactosidase, or beta-galactosidase. 243. The gene-edited target cell of embodiment 240, wherein the therapeutic polypeptide is a polypeptide the deficiency of which is associated with hemophilia. 244. The gene-edited target cell of embodiment 243, wherein the therapeutic polypeptide is factor VIII or factor IX. 245. The gene-edited target cell of embodiment 240, wherein the therapeutic polypeptide is an immunomodulatory polypeptide. 246. The gene-edited target cell of embodiment 245, wherein the immunomodulatory polypeptide is a human leukocyte antigen ("HLA") polypeptide. 247. The gene-edited target cell of embodiment 246, wherein the HLA polypeptide is an HLA class Ib polypeptide. 248. The gene-edited target cell of embodiment 246 or 247, 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). 249. The gene-edited target cell of embodiment 245, wherein the immunomodulatory polypeptide is a cytokine or cytokine receptor. 250. The immunomodulatory polypeptide is a cytokine, and the cytokine is (a) 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; (b) The gene-edited target cell of embodiment 245 or 249, which is 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. 251. The gene-edited target cell of embodiment 245 or 249, wherein the immunomodulatory polypeptide is IL-1Ra. 252. The gene-edited target cell of embodiment 240, wherein the therapeutic polypeptide is or comprises an antibody or an antigen-binding fragment thereof. 253. The gene-edited target cell of embodiment 252, wherein the therapeutic polypeptide binds to the pathogenic polypeptide. 254. The gene-edited target cell of embodiment 253, wherein the pathogenic polypeptide is tau, alpha-synuclein, or beta-amyloid polypeptide. 255. The gene-edited target cell of embodiment 240, wherein the therapeutic polypeptide binds to the cancer cell. 256. The gene-edited target cell of embodiment 255, wherein the therapeutic polypeptide is a chimeric antigen receptor. 257. A gene-edited target cell according to embodiment 255 or 256, wherein the therapeutic polypeptide binds to a tumor-associated antigen. 258. The gene-edited target cell of embodiment 257, wherein the tumor-associated antigen is CD19 or CD20. 259. A recombinant cell engineered to express a fusion protein described in any one of embodiments 1 to 56, wherein optionally the recombinant cell is a gene-edited target cell described in any one of embodiments 178 to 258. 260. The recombinant cell of embodiment 259, which is obtainable by integration of the targeting construct shown in Figure 2A into a single allele of the essential locus. 261. The recombinant cell of embodiment 259, which is obtainable by integration of the targeting construct shown in Figure 2A into both alleles of the essential locus. 262. The recombinant cell of embodiment 259, which is obtainable by integration of the targeting construct shown in Figure 2B into a single allele of an essential locus. 263. The recombinant cell of embodiment 259, which is obtainable by integration of the targeting construct shown in Figure 2B into both alleles of the essential locus. 264. The recombinant cell of embodiment 259, which is obtainable by integration of the targeting construct shown in Figure 2C into a single allele of the essential locus. 265. The recombinant cell of embodiment 259, which is obtainable by integration of the targeting construct shown in Figure 2C into both alleles of the essential locus. 266. The recombinant cell of embodiment 259, which is obtainable by integration of the targeting construct shown in Figure 2D into a single allele of an essential locus. 267. The recombinant cell of embodiment 259, which is obtainable by integration of the targeting construct shown in Figure 2D into both alleles of the essential locus. 268. The recombinant cell of embodiment 259, which is obtainable by integration of the targeting construct shown in Figure 4A into a single allele of an essential locus. 269. The recombinant cell of embodiment 259, obtained by integrating the targeting construct shown in Figure 4A into both alleles of the essential locus. 270. The recombinant cell of embodiment 259, which is obtainable by integration of the targeting construct shown in Figure 4B into a single allele of the essential locus. 271. The recombinant cell of embodiment 259, which is obtainable by integration of the targeting construct shown in Figure 4B into both alleles of the essential locus. 272. The recombinant cell of embodiment 259, which is obtainable by integration of the targeting construct shown in Figure 4C into a single allele of the essential locus. 273. The recombinant cell of embodiment 259, obtained by integrating the targeting construct shown in Figure 4C into both alleles of the essential locus. 274. The recombinant cell of embodiment 259, which is obtainable by integration of the targeting construct shown in Figure 4D into a single allele of an essential locus. 275. The recombinant cell of embodiment 259, obtained by integrating the targeting construct shown in Figure 4D into both alleles of the essential locus. 276. The recombinant cell of embodiment 259, which is obtainable by integration of a targeting construct shown in Figure 7A, for example a targeting construct comprising the nucleotide sequence of SEQ ID NO: 1, into a single allele of an essential locus. 277. The recombinant cell of embodiment 259, which is obtainable by integration of a targeting construct shown in Figure 7A, for example a targeting construct comprising the nucleotide sequence of SEQ ID NO: 1, into both alleles of an essential locus. 278. The recombinant cell of embodiment 259, which is obtainable by integration of the targeting construct shown in Figure 7B into a single allele of the essential locus. 279. The recombinant cell of embodiment 259, which is obtainable by integration of the targeting construct shown in Figure 7B into both alleles of the essential locus. 280. The recombinant cell of embodiment 259, which is obtainable by integration of the targeting construct shown in Figure 7C into a single allele of the essential locus. 281. The recombinant cell of embodiment 259, which is obtainable by integration of the targeting construct shown in Figure 7C into both alleles of the essential locus. 282. The recombinant cell of embodiment 259, which is obtainable by integration of a targeting construct shown in Figure 7D, for example a targeting construct comprising the nucleotide sequence of SEQ ID NO: 2, into a single allele of an essential locus. 283. The recombinant cell of embodiment 259, which is obtainable by integration of a targeting construct shown in Figure 7D, for example a targeting construct comprising the nucleotide sequence of SEQ ID NO: 2, into both alleles of an essential locus. 284. The recombinant cell of embodiment 259, which is obtainable by integration of a targeting construct shown in Figure 7E, for example a targeting construct comprising the nucleotide sequence of SEQ ID NO: 29, into a single allele of an essential locus. 285. The recombinant cell of embodiment 259, which is obtainable by integration of a targeting construct shown in Figure 7E, for example a targeting construct comprising the nucleotide sequence of SEQ ID NO: 29, into both alleles of an essential locus. 286. The recombinant cell of embodiment 259, which is obtainable by integration of a targeting construct shown in Figure 7F, for example a targeting construct comprising the nucleotide sequence of SEQ ID NO: 30, into a single allele of an essential locus. 287. The recombinant cell of embodiment 259, which is obtainable by integration of a targeting construct shown in Figure 7F, for example a targeting construct comprising the nucleotide sequence of SEQ ID NO: 30, into both alleles of an essential locus. 288. The recombinant cell of embodiment 259, which is obtainable by integration of the targeting construct shown in Figure 7G into a single allele of an essential locus. 289. The recombinant cell of embodiment 259, which is obtainable by integration of the targeting construct shown in Figure 7G into both alleles of the essential locus. 290. The recombinant cell of embodiment 259, which is obtainable by integration of the targeting construct shown in Figure 7H into a single allele of the essential locus. 291. The recombinant cell of embodiment 259, which is obtainable by integration of the targeting construct shown in Figure 7H into both alleles of the essential locus. 292. The recombinant cell of embodiment 259, which is obtainable by integration of the targeting construct shown in Figure 7I into a single allele of an essential locus. 293. The recombinant cell of embodiment 259, which is obtainable by integration of the targeting construct shown in Figure 7I into both alleles of the essential locus. 294. The recombinant cell of embodiment 259, which is obtainable by integration of the targeting construct shown in Figure 7J into a single allele of an essential locus. 295. The recombinant cell of embodiment 259, which is obtainable by integration of the targeting construct shown in Figure 7J into both alleles of the essential locus. 296. The recombinant cell of embodiment 259, which is obtainable by integration of a targeting construct shown in Figure 7K, for example a targeting construct comprising the nucleotide sequence of SEQ ID NO: 14, into a single allele of an essential locus. 297. The recombinant cell of embodiment 259, which is obtainable by integration of a targeting construct shown in Figure 7K, for example a targeting construct comprising the nucleotide sequence of SEQ ID NO: 14, into both alleles of an essential locus. 298. The recombinant cell of embodiment 259, which is obtainable by integration of a targeting construct shown in Figure 7L, for example a targeting construct comprising the nucleotide sequence of SEQ ID NO: 17, into a single allele of an essential locus. 299. The recombinant cell of embodiment 259, which is obtainable by integration of a targeting construct shown in Figure 7L, for example a targeting construct comprising the nucleotide sequence of SEQ ID NO: 17, into both alleles of an essential locus. 300. The recombinant cell of embodiment 259, having an engineered essential gene having the configuration shown in Figure 3A. 301. The recombinant cell of embodiment 259, having an engineered essential gene having the configuration shown in Figure 3B. 302. The recombinant cell of embodiment 259, having an engineered essential gene having the configuration shown in Figure 3D. 303. The recombinant cell of embodiment 259, having an engineered essential gene having the configuration shown in Figure 3E. 304. The recombinant cell of embodiment 259, having an engineered essential gene having the configuration shown in Figure 5A. 305. The recombinant cell of embodiment 259, having an engineered essential gene having the configuration shown in Figure 5B. 306. The recombinant cell of embodiment 259, having an engineered essential gene having the configuration shown in Figure 5C. 307. The recombinant cell of embodiment 259, having an engineered essential gene having the configuration shown in Figure 5D. 308. The recombinant cell of embodiment 259, having an engineered essential gene having the configuration shown in Figure 6A. 309. The recombinant cell of embodiment 259, having an engineered essential gene having the configuration shown in Figure 6B. 310. The recombinant cell of embodiment 259, having an engineered essential gene having the configuration shown in Figure 6C. 311. The recombinant cell of embodiment 259, having an engineered essential gene having the configuration shown in Figure 6D. 312. The recombinant cell of embodiment 259, having an engineered essential gene having the configuration shown in Figure 6E. 313. The recombinant cell of any one of embodiments 259-312, further engineered to express a transgene from a locus other than an essential gene. 314. A recombinant cell according to embodiment 259, comprising an expression vector, comprising a nucleotide sequence encoding the fusion protein according to any one of embodiments 1 to 56. 315. The recombinant cell of embodiment 314, wherein the vector is a viral vector. 316. The recombinant cell of embodiment 315, wherein the viral vector is an AAV vector, a retroviral vector, or a lentiviral vector. 317. The recombinant cell of embodiment 314, wherein the vector is a DNA vector. 318. The recombinant cell of embodiment 314, wherein the vector is an RNA vector. 319. A recombinant cell according to any one of embodiments 314 to 318, wherein the corresponding essential gene is knocked out in a single allele. 320. A recombinant cell according to any one of embodiments 314 to 318, wherein the corresponding essential gene is knocked out in both alleles. 321. A recombinant cell according to any one of embodiments 314 to 320, which has been engineered to express a transgene. 322. The recombinant cell of embodiment 321, wherein the transgene is not expressed from the corresponding essential locus. 323. The recombinant cell of embodiment 321, wherein the transgene is expressed from the corresponding essential locus. 324. The recombinant cell of any one of embodiments 321 to 323, wherein the transgene is as defined in section 6.4. 325. The recombinant cell of any one of embodiments 259 to 324, wherein the cell is as defined in section 6.7. 326. A pharmaceutical composition comprising a gene-edited target cell according to any one of embodiments 178 to 258 or a recombinant cell according to any one of embodiments 259 to 325, and a pharmaceutically acceptable excipient. 327. Use of a gene-edited target cell according to any one of embodiments 178-258, or a recombinant cell according to any one of embodiments 259-325, for the manufacture of a medicament for treating a patient in need thereof, wherein optionally the gene-edited target cell or recombinant cell is (a) autologous to the patient, or (b) allogeneic to the patient. 328. The gene-edited target cell of any one of embodiments 178-258, the recombinant cell of any one of embodiments 259-325, or the pharmaceutical composition of embodiment 326, for use in treating a patient in need of treatment, wherein optionally the gene-edited target cell or recombinant cell is (a) autologous to the patient, or (b) allogeneic to the patient, or the pharmaceutical composition comprises cells that are (a) autologous to the patient, or (b) allogeneic to the patient. 329. A method of treating a subject with cell therapy, comprising administering to a subject in need thereof a gene-edited target cell of any one of embodiments 178-258, a recombinant cell of any one of embodiments 259-325, or a pharmaceutical composition of embodiment 326, optionally wherein the gene-edited target cell or recombinant cell is (a) autologous to the patient, or (b) allogeneic to the patient, or wherein the pharmaceutical composition comprises cells that are (a) autologous to the patient, or (b) allogeneic to the patient. 330. The method of embodiment 329, further comprising administering to the subject an inducer of degron. 331. The method of embodiment 330, wherein the subject is experiencing adverse effects of the cell therapy. 332. The method of embodiment 330, wherein the subject is at risk of developing adverse effects of the cell therapy. 333. The method of embodiment 331 or 332, wherein the adverse effect is cytokine storm or cytokine release syndrome (CRS). 334. The method of embodiment 331 or 332, wherein the adverse effect is excessive (systemic) inflammation. 335. The method of embodiment 331 or 332, wherein the adverse effect is tumor formation. 336. The method of embodiment 331 or 332, wherein the adverse effect is tumor lysis syndrome. 337. The method of embodiment 331 or 332, wherein the adverse effects include abnormally functioning neurons and / or dyskinesias. 338. The method of embodiment 331 or 332, wherein the adverse effects include abnormally functioning cardiomyocytes and / or cardiac arrhythmias. 339. The method of embodiment 331 or 332, wherein the adverse effect is macrophage activation syndrome (MAS). 340. The method of embodiment 331 or 332, wherein the adverse effect is graft-versus-host disease. 341. The method of any one of embodiments 330-340, wherein the degron is a drug-inducible degron. 342. The method of embodiment 341, wherein the drug is an immunomodulatory drug (IMiD). 343. The method of embodiment 342, wherein the IMiD is lenalidomide, iberdomide, thalidomide, avadomide, or pomalidomide. 344. The method of embodiment 342, wherein the IMiD is iberdomide. 345. The method of embodiment 342, wherein the IMiD is avadomide. 346. The method of embodiment 342, wherein the IMiD is thalidomide. 347. The method of embodiment 342, wherein the IMiD is lenalidomide. 348. The method of embodiment 342, wherein the IMiD is pomalidomide. 349. A method for reducing or eliminating engineered cells in a subject who has previously received cell therapy with gene-edited target cells described in any one of embodiments 178-258, recombinant cells described in any one of embodiments 259-325, or the pharmaceutical composition of embodiment 326, comprising administering to the subject an inducer of a degron, and optionally, the cell therapy comprises cells that are (a) autologous to the subject, or (b) allogeneic to the subject. 350. The method of embodiment 349, wherein the subject has experienced or is at risk for adverse effects of cell therapy. 351. The method of embodiment 350, wherein the adverse effect is cytokine storm or cytokine release syndrome (CRS). 352. The method of embodiment 350, wherein the adverse effect is excessive (systemic) inflammation. 353. The method of embodiment 350, wherein the adverse effect is tumor formation. 354. The method of embodiment 350, wherein the adverse effect is tumor lysis syndrome. 355. The method of embodiment 350, wherein the adverse effects include abnormally functioning neurons and / or dyskinesias. 356. The method of embodiment 350, wherein the adverse effects include abnormally functioning cardiomyocytes and / or cardiac arrhythmias. 357. The method of embodiment 350, wherein the adverse effect is macrophage activation syndrome (MAS). 358. The method of embodiment 350, wherein the adverse effect is graft-versus-host disease. 359. The method of any one of embodiments 349-351, wherein the degron is a drug-inducible degron. 360. The method of embodiment 359, wherein the drug is an immunomodulatory drug (IMiD). 361. The method of embodiment 360, wherein the IMiD is lenalidomide, iberdomide, thalidomide, avadomide, or pomalidomide. 362. The method of embodiment 360, wherein the IMiD is iberdomide. 363. The method of embodiment 360, wherein the IMiD is avadomide. 364. The method of embodiment 360, wherein the IMiD is thalidomide. 365. The method of embodiment 360, wherein the IMiD is lenalidomide. 366. The method of embodiment 360, wherein the IMiD is pomalidomide. [Example]

[0289] 8. Working Example 8.1. Example 1: Design and Generation of Targeting Constructs The targeting construct was designed to contain a linker and degron sequence flanking the GAPDH homology arms, as shown in Figures 7A-7D. An additional targeting construct was designed to contain a linker, degron sequence, IRES sequence, and GFP transgene sequence flanking the GAPDH homology arms, as shown in Figures 7G-7J. The homology arms were designed to allow integration of the construct at the endogenous GAPDH locus immediately 5' to the GAPDH endogenous stop codon. The desired sequence was sent to GenScript (Piscataway, NJ) for de novo gene synthesis via on-site oligo design, oligo synthesis, and gene assembly. The amplified fragment was ligated into the pUC57-Kan cloning vector, transformed into bacteria, and the resulting plasmid containing the targeted construct was isolated.

[0290] 8.2. Example 2: Selective Targeting of Gene-Edited iPSCs The functionality of the kill switch was tested in iPSCs using the targeting construct shown in Figure 7G, which contains a linker flanking GAPDH homology arms, a degron sequence, an IRES, and a GFP transgene sequence. On the day of nucleofection, iPSCs were harvested and resuspended in Lonza P3 primary cell nucleofection buffer. Ribonucleoprotein (RNP) was complexed with sgRNA using a 1:2 ratio of protein:sgRNA (IDT). Nucleofection of the complexed RNP and targeting construct into resuspended iPSCs was achieved using a LONZA 4D Nucleofector. Nucleofected cells were then plated and assessed for targeting events.

[0291] Heterogeneous mixtures of unedited cells (GFP-negative) and cells edited with the construct shown in Figure 7G, which contains a degron fused to GAPDH as well as 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 had no effect on unedited cells (Figure 8A) but depleted the population of edited GFP-positive cells (Figure 8B), suggesting that inducible degradation of the degron-fused GAPDH protein killed the gene-edited cells.

[0292] In the next set of evaluations, clones biallelically modified with a degron fused to GAPDH and IRES-GFP (Figure 7G) were treated with 1 μM POM for 92 hours and imaged using an Incucyte™ microscope. The percentage of viable cells per well was quantified at several time points using Incucyte software and normalized for the number of viable cells per well at the first imaging time point. As shown in Figure 9A, the cell death rate of gene-edited cells exceeded their proliferation rate approximately 24 hours after the addition of POM. Furthermore, gene-edited cells were completely eliminated 92 hours after the addition of POM (Figures 9A and 9B). Untreated gene-edited cells proliferated normally over the same period (Figure 9C).

[0293] 8.3. Example 3: Effect of targeting construct linker length on survival of gene-edited iPSCs 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 flanking GAPDH homology arms, a degron or superdegron sequence, an IRES sequence, and a GFP transgene sequence, as shown in Figures 7G-7J, where the linker lengths were 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 contain a linker flanking GAPDH homology arms and a degron or superdegron sequence, as shown in Figures 7A-7D. For both sets, the homology arms were designed to allow integration of the construct at the endogenous GAPDH locus immediately 5' of the GAPDH endogenous stop codon. iPSCs were transfected individually with each construct.

[0294] The pool of iPSCs transfected with the first set of targeting constructs, potentially containing both monoallelic and biallelic gene-edited iPSCs, was assessed using flow cytometry, 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 containing the superdegron had the lowest percentage of GFP-positive cells, whereas approximately one-third to one-half of untreated cells transfected with one of the degron-containing targeting constructs were GFP-positive. However, treatment with POM only depleted GFP-positive cells when transfected with the targeting construct containing the degron with the shortest linker or the targeting construct containing the superdegron (Figure 10A).

[0295] Next, we evaluated iPSCs transfected with a second set of targeting constructs, where amplicon depletion was assessed after POM treatment compared to untreated cells. Consistent with the results of 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 superdegron. Taken together, the linker length and degron type of the targeting construct may be important factors in causing cell death in gene-edited cells.

[0296] 8.4. Example 4: Activation of targeting constructs in gene-edited iPSCs To determine whether gene editing of iPSCs with a targeting construct whose homology arms allow integration at the endogenous GAPDH locus affects GAPDH expression, three clones biallelically modified with a targeting construct containing a 3 aa linker, GGS, and a degron (shown in Figure 7G) and one clone biallelically modified with a targeting construct containing a superdegron (shown in Figure 7J) were treated with 3 μM POM for up to 3 days.

[0297] No significant differences in GAPDH expression were detected by qPCR between untreated and POM-treated gene-edited cells, all of which were similar to expression in the untransfected parental lines (Figure 11A). Assessment of protein expression by Western blot revealed that GAPDH protein was completely depleted after one day of treatment with 3 μM POM in the same iPSC clones (Figures 11B and 11C). Together, these results suggest that none of the targeting constructs altered GAPDH mRNA levels but enabled POM-induced degradation of GAPDH protein.

[0298] Next, we seeded unedited parental cells and gene-edited iPSCs at equal densities and tracked confluency over time using an Incucyte imager to assess the effect of gene editing with the targeting construct on growth rate. The results revealed that all three clones gene-edited with the degron-containing targeting construct grew at rates comparable to those of the unedited parental cells, whereas iPSCs gene-edited with the super-degron-containing targeting construct grew at a slower rate (Figure 11C).

[0299] 8.5. Example 5: Effect of POM Concentration on Gene-Edited iPSC Death The same gene-edited iPSC clones in Example 4 were treated with 0.5 μM POM for 5 days to determine whether there were any differences in viability. This POM treatment resulted in the complete killing of all gene-edited iPSCs but did not affect the growth of the unedited parental cells ( FIG. 12A ).

[0300] To determine the optimal POM concentration for inducing apoptosis, the confluency of gene-edited iPSC clones treated with different concentrations of POM ranging from 0.03125 μM to 10 μM was monitored for 5 days using an Incucyte system. Results revealed that 0.25 μM POM was able to induce 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). This concentration was even lower for iPSCs gene-edited with a targeting construct containing a superdegron, as the lowest POM concentration evaluated (0.03125 μM) was associated with apoptosis in all cells.

[0301] 8.6. Example 6: Activation of targeting constructs in dopaminergic neurons differentiated from gene-edited iPSCs. To determine the applicability of degron-containing targeting constructs to differentiated cells, unedited parental iPSCs and gene-edited iPSC clones B and C were differentiated into dopaminergic (DA) neurons, as shown in Figure 13. The 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 entireties. After plating down cells in the presence and absence of various concentrations of POM, cell death was assessed post-thaw. Annexin V dye was used to quantitate cell death in an Incucyte imager over a 5-day period. No differences in neuronal death were detected in DA neurons derived from unedited parental iPSCs (Figures 14 and 15A). All POM concentrations evaluated achieved complete death of all neurons differentiated from both clone B and clone C iPSCs by day 5 (Figures 14 and 15B-15C). Further evaluation with DA neurons derived from clone B revealed complete killing of these cells even at 10 nM POM (Figure 15C), the lowest concentration evaluated. Collectively, these results suggest that DA neurons differentiated from iPSCs gene-edited with a degron-containing targeting construct exhibit nanomolar sensitivity to POM.

[0302] 8.7. Example 7: Extended Testing of POM Concentrations for Activation of Targeting Constructs in Dopaminergic Neurons Differentiated from Gene-Edited iPSCs Extending the work shown in Example 6, a wider range of POM concentrations was tested in differentiated DA neurons from the unedited parent line and two clones biallelically modified with a targeting construct containing a linker that is three amino acids long (3-aa linker) and a degron: gene-edited clone A or gene-edited clone C. Cells were differentiated from iPSCs into DA neurons using the same protocol as shown in Figure 13. DA neurons were plated and stained with Annexin V dye, and cell death was measured using an Incucyte imager over a 144-hour period.

[0303] As shown in Figure 16A, no differences in cell death were observed in DA neurons derived from unedited parental iPSCs at any concentration of POM from the range tested (0.01 μM to 100 μM). DA neurons derived from clone A or clone C reached nearly complete cell death by 144 hours with as little as 0.1 μM POM (Figures 16B and 16C). These results indicate that POM has no effect on unedited neurons but can promote apoptosis in neurons modified to contain degron tags on essential genes.

[0304] 8.8. Example 8: Activation of essential gene-targeting constructs in myeloid progenitor cells differentiated from gene-edited iPSCs To expand the applicability of degron-containing constructs to additional differentiated cell types, unedited parental iPSCs containing targeting constructs as shown in Figure 7G, as well as biallelically edited iPSC clones A, B, and C, were differentiated into myeloid progenitor (MP) cells as shown in Figure 17. The differentiation protocol was adapted from Douvaras et al., 2017 Jun 6;8(6):1516-1524, and PCT Publication Nos. 2023 / 150089A1 and 2017 / 152081A1, which are incorporated herein by reference in their entireties. After plating down MP cells in the presence and absence of POM, cell death was assessed post-thaw. Cell death was quantified over a 108-hour period in an Incucyte Imager using acridine orange / propidium iodide (AO / PI) dye.

[0305] Analysis revealed that all four lines survived relatively well in the absence of POM, and the unedited parent line did not exhibit increased cell death in the presence of POM. All three gene-edited clones died in response to POM over the treatment period (Figures 18A-18D). These results suggest that myeloid precursors differentiated from iPSCs gene-edited with a targeting construct containing a degron exhibit sensitivity to POM.

[0306] 8.9. Example 9. Targeting of 3AA-degron and Superdegron Constructs at the RPL13A Locus and Activation of Targeting Constructs in Gene-Edited iPSCs and Differentiated Dopaminergic Neurons and Myeloid Progenitor Cells To demonstrate the functionality of the degron-based kill switch when linked to another essential gene, a targeting construct was designed to contain a linker and degron sequence 3 amino acids long (Linker 1; GGS) flanking the RPL13A homology arm, as shown in Figure 7K and SEQ ID NO: 14. A second targeting construct was designed to contain a linker and superdegron sequence 10 amino acids long (Linker 4; SEQ ID NO: 15) flanking the RPL13A homology arm, as shown in Figure 7L and SEQ ID NO: 17. Both targeting constructs were transfected into iPSCs, and amplicon depletion after POM treatment relative to untreated cells was assessed. Clonal lines generated from these transfected pools were then assessed to determine the optimal POM concentration required to induce apoptosis in iPSCs, as well as in dopaminergic neurons and myeloid progenitor cells differentiated from these gene-edited iPSCs.

[0307] 9. Sequence Listing Exemplary sequences of the present disclosure are provided below in Table 10 ("SEQ" refers to sequence number). [Table 10] [Table 10-2] [Table 10-3] [Table 10-4] [Table 10-5] [Table 10-6] [Table 10-7] [Table 10-8] [Table 10-9] [Table 10-10] [Table 10-11] [Table 10-12] [Table 10-13] [Table 10-14] [Table 10-15] [Table 10-16]

[0308] 10. Incorporation by Reference All publications, patents, patent applications, and other documents cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document was individually indicated to be incorporated by reference for all purposes. In the event of any conflict between the teachings of this specification and one or more of the references incorporated into this disclosure, the teachings of this specification are intended.

Claims

1. (a) an essential polypeptide; and (b) a degron; (c) optionally, a linker.

2. The fusion protein of claim 1 , wherein the degron is C-terminal to the essential polypeptide.

3. The fusion protein of claim 1 or 2, wherein the degron is an inducible degron.

4. 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 degron has the amino acid sequence RPFQCNQCGASFTQKGNLLRHIKLH (SEQ ID NO: 3), FNVLMVHKRSHTGERPLQCEICGFFTCRQKGNLLRHIKLHTGEKPFKCHLCNYACQRRDAL (SEQ ID NO: 4), FNVLMVHKRSHTGERP (SEQ ID NO: 5), FNVLMVHRRSHTGERP (SEQ ID NO: 6), TGEKPFKCHLCNYACQR 6. The fusion protein of any one of claims 1 to 5, comprising or consisting of: RDAL (SEQ ID NO: 7), TGERPFRCHLCNYACQRRDAL (SEQ ID NO: 8), FQCNQCGASFT (SEQ ID NO: 9), FQCPICGLVIK (SEQ ID NO: 10), LQCEICGFFTCR (SEQ ID NO: 11), LQCEICGYQCR (SEQ ID NO: 12), or LQCEVCGFQCR (SEQ ID NO: 13).

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

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

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

10. The fusion protein according to any one of claims 1 to 9, wherein the essential polypeptide is a STEL polypeptide, and optionally 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 of homology to an essential gene encoding an essential polypeptide in a target genomic locus; (b) a nucleotide sequence encoding a degron (the "degron-coding sequence"); and (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; 11. A targeting construct 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. 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, if present, the difference in length between the first homology arm and the second homology arm is less than 75 nucleotides.

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

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

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

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

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

19. 1. A method of producing a gene-edited target cell, comprising: (a) introducing into a target cell a system according to any one of claims 16 to 18; (b) culturing the target cell under conditions in which gene editing is effected, thereby producing a gene-edited target cell.

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

21. 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 target cells (a) human immune cells, optionally selected from T cells, T cells expressing a chimeric antigen receptor (CAR) or a recombinant TCR, regulatory T cells, myeloid cells, dendritic cells, and macrophages (e.g., immunosuppressive macrophages); (b) a cell in the human nervous system, optionally selected from a dopaminergic neuron, a microglial cell, an oligodendrocyte, an astrocyte, a cortical neuron, a spinal cord or oculomotor neuron, an enteric neuron, a placode-derived cell, a Schwann cell, and a trigeminal or sensory neuron; (c) cells within the human cardiovascular system, optionally selected from cardiomyocytes, endothelial cells, and nodal cells; (d) cells within the human metabolic system, 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 epithelial cell, a photoreceptor cone cell, a photoreceptor rod cell, a bipolar cell, or a ganglion cell; or 21. The method of claim 19 or 20, wherein the cell is (f) a precursor or progenitor of any one of the aforementioned cells.

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

24. (a) a degron; (b) an essential gene encoding a fusion protein comprising an essential polypeptide, the essential gene being encoded by the essential gene defined in claim 10. A gene-edited target cell.

25. 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 gene-edited target cell of claim 23 or 24.

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

27. 26. 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. 28. A pharmaceutical composition comprising a gene-edited target cell of claim 23 or 24, a recombinant cell of any one of claims 25 to 27, and a pharmaceutically acceptable excipient.

29. 28. Use of a gene-edited target cell of Claim 23 or 24, or a recombinant cell of any one of Claims 25-27, 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. 29. The gene-edited target cell of claim 23 or 24, the recombinant cell of any one of claims 25-27, or the pharmaceutical composition of claim 28, for use in treating a patient in need of treatment, wherein optionally the gene-edited target cell or the recombinant cell is (a) autologous to the patient, or (b) allogeneic to the patient, or the pharmaceutical composition comprises cells that are (a) autologous to the patient, or (b) allogeneic to the patient.

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

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

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

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

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

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