Improved multi-cistronic systems and their use

Abstract

Provided herein are optimized 2A ribosome skipping elements, polynucleotide sequences encoding such optimized 2A ribosome skipping elements, and multicistronic systems, constructs, vectors, and engineered cells that utilize them to express multiple polypeptides from a single transcript. Furthermore, pharmaceutical compositions comprising engineered vectors and / or cells for treating disorders are disclosed.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 511,459, filed on June 30, 2023, the entire disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0002] Sequence Listing This application includes an electronically - submitted sequence listing XML, which is incorporated herein by reference in its entirety. The XML file was created in XX month, 20XX, named XXXXXXX, and is XXX,XXX bytes in size.

Background Art

[0003] Multicistronic expression systems can generate multiple separate polypeptides from a single transcript using 2A ribosome - skipping elements. However, conventional 2A ribosome - skipping elements can result in incomplete separation of polypeptides due to inefficient ribosome skipping.

Summary of the Invention

[0004] Provided herein are optimized 2A ribosome - skipping elements and polynucleotide sequences encoding such optimized 2A ribosome - skipping elements.

[0005] In some embodiments, the 2A ribosome skipping element includes at least a functional portion of the P2A ribosome skipping element and at least a functional portion of the T2A ribosome skipping element. The 2A ribosome skipping element may include the P2A ribosome skipping element and the T2A ribosome skipping element, for example, a P2A-T2A sequence. In some embodiments, the 2A ribosome skipping element includes the amino acid sequence ATNFSLLKQAGDVEENPGPGSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 269).

[0006] Also provided herein are engineered polynucleotides comprising a first sequence encoding a first polypeptide and a second sequence encoding a second polypeptide, wherein the first and second sequences are operably linked via a linker polynucleotide sequence encoding a 2A ribosome skipping element, comprising at least a portion of a P2A element and a T2A element. In some embodiments, the engineered polynucleotide comprises a sequence oriented 5' to 3' according to the formula: P1-L-P2 In the formula, P1 comprises the first sequence, L comprises the linker polynucleotide sequence encoding the 2A ribosome skipping element, and P2 comprises the second sequence.

[0007] In some embodiments, the 2A ribosome skipping element includes the amino acid sequence ATNFSLLKQAGDVEENPGPGSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 269).

[0008] Also provided herein are vectors comprising the manipulated polynucleotides described herein, manipulated cells comprising the manipulated polynucleotides or vectors described herein, pharmaceutical compositions comprising the manipulated polynucleotides, vectors, or cells described herein, and methods for preparing or using such cells.

[0009] These and other features, aspects, and advantages of this disclosure will be better understood in relation to the following description and accompanying drawings. [Brief explanation of the drawing]

[0010] [Figure 1A] Figures 1A–1E show the results of assays to evaluate different 2A linkers between different payload components in a multi-cistronic system. [Figure 1B] Figures 1A–1E show the results of assays to evaluate different 2A linkers between different payload components in a multi-cistronic system. [Figure 1C] Figures 1A–1E show the results of assays to evaluate different 2A linkers between different payload components in a multi-cistronic system. [Figure 1D] Figures 1A–1E show the results of assays to evaluate different 2A linkers between different payload components in a multi-cistronic system. [Figure 1E] Figures 1A–1E show the results of assays to evaluate different 2A linkers between different payload components in a multi-cistronic system. [Modes for carrying out the invention]

[0011] This disclosure generally relates to modified degron polypeptides that have increased sensitivity to pomalidomide. Also provided are inducibly degradable proteins, inducible cell death systems, and activation condition-controlled polypeptides using the modified degron polypeptides described herein, as well as the methods and / or uses thereof.

[0012] Unless otherwise specified, terms used in the claims and specification are defined as set forth below.

[0013] The term "remission" refers to any therapeutically beneficial outcome in the treatment of a disease state, such as a cancerous disease state, including prevention, reduction of severity or progression, or remission.

[0014] The term "in situ" refers to a process that occurs in living cells that grow independently from a living organism, such as in tissue cultures.

[0015] The term "in vivo" refers to processes that occur within a living organism.

[0016] As used herein, the term “mammal” includes, but is not limited to, humans and non-humans, including humans, non-human primates, dogs, cats, mice, cattle, horses, and pigs.

[0017] The term "percent identity" in the context of sequences of two or more nucleic acids or polypeptides refers to two or more sequences or subsequences that, when compared and aligned for the greatest match, have a specified percentage of the same nucleotide or amino acid residue, as measured using one of the sequence comparison algorithms described herein (e.g., BLASTP and BLASTN, or other algorithms available to those skilled in the art) or by visual inspection. Depending on the application, percentage "identity" may exist across regions of the sequences being compared, for example, across functional domains, or alternatively, across the entire length of the two sequences being compared.

[0018] In sequence comparison, typically, one sequence acts as the reference sequence compared to the test sequence. When using a sequence comparison algorithm, the test sequence and the reference sequence are entered into the computer, the coordinates of subsequences are specified if necessary, and the program parameters of the sequence comparison algorithm are specified. The sequence comparison algorithm then calculates the sequence identity percentage of the test sequence to the reference sequence based on the specified program parameters.

[0019] Optimal alignment of arrays for comparison can be done, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by a search for the similarity method of Pearson & Lipman, Proc. Nat’l. Acad. Sci. USA 85:2444 (1988), by computerized execution of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis), or by visual inspection (generally see Ausubel et al., infra).

[0020] One example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, and software for performing BLAST analysis, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990), is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ).

[0021] The term "sufficient amount" means an amount sufficient to produce the desired effect, e.g., an amount sufficient to modulate protein aggregation in a cell.

[0022] The term "therapeutically effective amount" is an amount effective to relieve the symptoms of a disease. A therapeutically effective amount can be a "preventively effective amount" because prevention can be considered treatment.

[0023] As used herein and in the appended claims, it should be noted that the singular forms “a,” “an,” and “the” refer to multiple subjects unless the context explicitly indicates otherwise.

[0024] The use of common terms such as “first,” “second,” and “third” in the claims to modify claim elements does not in itself indicate any priority, priority, or order of one claim element over another, nor does it indicate the chronological order in which the actions of the method are performed, but is simply used as a label to distinguish one claim element having a particular name from another element having the same name (for the sake of using common terms). Similarly, the use of these terms herein does not in itself imply any necessary priority, priority, or order.

[0025] Multi-cistronic and multi-promoter systems In some embodiments, the manipulated nucleic acid is configured to produce multiple polypeptides. For example, the nucleic acid may be configured to produce 2 to 20 different polypeptides.In some embodiments, the manipulated nucleic acids are 2-20, 2-19, 2-18, 2-17, 2-16, 2-15, 2-14, 2-13, 2-12, 2-11, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-20, 3-19, 3-18, 3-17, 3-16, 3-15, 3-14, 3-13, 3-12, 3-11, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-20, 4-19, 4-18, 4-17, 4-16, 4-15, 4-14, 4-13, 4-12, 4-1 1, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-20, 5-19, 5-18, 5-17, 5-16, 5-15, 5-14, 5-13, 5-12, 5-11, 5-10, 5-9, 5-8, 5-7, 5-6, 6-20, 6-19, 6-18, 6-17, 6-16, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, 6-7, 7-20, 7-19, 7-18, 7-17, 7-16, 7-15, 7-14, 7-13, 7-12, 7-11, 7-10, 7-9, 7-8, 8-20 , 8~19, 8~18, 8~17, 8~16, 8~15, 8~14, 8~13, 8~12, 8~11, 8~10, 8~9, 9~20, 9~19, 9~18, 9~17, 9~16, 9~15, 9~14, 9~13, 9~12, 9~11, 9~10, 10~20, 10~19, 10~18, 10~17, 10~16, 10~15, 10~14, 10~13, 10~12, 10~11, 11~20, 11~19, 11~18, 11~17, 11~16, 11~15, 11~14, 11~13, 11~12, 12~20, 1 It is configured to produce polypeptides of 2-19, 12-18, 12-17, 12-16, 12-15, 12-14, 12-13, 13-20, 13-19, 13-18, 13-17, 13-16, 13-15, 13-14, 14-20, 14-19, 14-18, 14-17, 14-16, 14-15, 15-20, 15-19, 15-18, 15-17, 15-16, 16-20, 16-19, 16-18, 16-17, 17-20, 17-19, 17-18, 18-20, 18-19, or 19-20.In some embodiments, the engineered nucleic acid is configured to produce polypeptides 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, the engineered nucleic acid is configured to produce two polypeptides. In some embodiments, the engineered nucleic acid is configured to produce three polypeptides. In some embodiments, the engineered nucleic acid is configured to produce four polypeptides. In some embodiments, the engineered nucleic acid is configured to produce five polypeptides.

[0026] In some embodiments, the engineered nucleic acid can be multicistronic, meaning that two or more distinct polypeptides (e.g., multiple exogenous polynucleotides, or effector molecules, or engineered polypeptides as described herein) can be produced from a single transcript. In some embodiments, the engineered nucleic acid can be multicistronic through the use of various linkers, for example, a polynucleotide sequence encoding a first exogenous polynucleotide, or effector molecule, may be linked to a nucleotide sequence encoding a second exogenous polynucleotide, or effector molecule, for example, in a 5' to 3' orientation, such as first gene:linker:second gene.

[0027] Multi-cistronic expression systems can generate multiple separate polypeptides from a single transcript using 2A ribosome skipping elements. However, conventional 2A ribosome skipping elements can lead to incomplete polypeptide separation due to inefficient ribosome skipping. There is a need for optimized 2A ribosome skipping elements to improve the production of distinct polypeptides from a single transcript.

[0028] Provided herein are optimized 2A ribosome skipping elements and linker polynucleotide sequences encoding such optimized 2A ribosome skipping elements.

[0029] In some embodiments, the 2A ribosome skipping element includes at least a functional portion of the P2A ribosome skipping element and at least a functional portion of the T2A ribosome skipping element. The 2A ribosome skipping element may include the P2A ribosome skipping element and the T2A ribosome skipping element, for example, a P2A-T2A sequence. In some embodiments, the 2A ribosome skipping element includes the amino acid sequence ATNFSLLKQAGDVEENPGPGSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 269). In some embodiments, the P2A ribosome skipping element includes the amino acid sequence described in SEQ ID NO: 195. In some embodiments, the P2A ribosome skipping element includes the amino acid sequence described in SEQ ID NO: 271. In some embodiments, the T2A ribosome skipping element includes the amino acid sequence described in SEQ ID NO: 267. In some embodiments, the 2A ribosome skipping element includes the E2A G4S T2A(OPt2A) ribosome skipping element. In some embodiments, the E2A G4S T2A(OPt2A) ribosome skipping element comprises the amino acid sequence described in SEQ ID NO: 265. In some embodiments, the 2A ribosome skipping element comprises the P2A 3-T2A 2(Opt2A 2.0) ribosome skipping element. In some embodiments, the P2A 3-T2A 2(Opt2A 2.0) ribosome skipping element comprises the amino acid sequence described in SEQ ID NO: 269. The 2A ribosome skipping element enables the production of distinct polypeptides encoded by the first and second genes during translation.

[0030] In some embodiments, the linker is a cleavable linker polypeptide sequence, such as a furin cleavage site or a TEV cleavage site, or comprises such sequences, and following expression, the cleavable linker polypeptide is cleaved to produce distinct polypeptides encoded by the first and second genes. In some embodiments, the cleavable linker sequence includes a flexible linker sequence that further facilitates cleavage. In some embodiments, the flexible linker includes a Gly-Ser-Gly sequence.

[0031] In some embodiments, the linker polynucleotide sequence includes a polynucleotide sequence encoding an internal ribosome entry site (IRES), so that separate polypeptides encoded by the first and second genes are produced during translation. In some embodiments, the linker polynucleotide sequence includes a polynucleotide sequence encoding a splice receptor, such as a viral splice receptor.

[0032] In some embodiments, the linker includes a combination of linker sequences, such as a furin-2A linker, which can produce a distinct polypeptide through 2A ribosome skipping, followed by further cleavage of the furin site allowing for complete removal of the 2A residue. In some embodiments, the linker combination may include a furin sequence, a flexible linker, and a 2A linker. Thus, in some embodiments, the linker is a furin-Gly-Ser-Gly-2A fusion polypeptide. In some embodiments, the linker is a furin-Gly-Ser-Gly-T2A fusion polypeptide.

[0033] Generally, a multi-cistronic system used in accordance with this disclosure comprises any number or combination of linkers and can express any number of genes or portions thereof (for example, the engineered nucleic acid may encode the first, second, third, and fourth effector molecules described herein, each separated by a linker so as to produce separate polypeptides encoded by the first, second, and third effector molecules).

[0034] As used herein, “linker” refers to a polypeptide or multi-cistronic linker described herein that links a first polypeptide sequence with a second polypeptide sequence.

[0035] Manipulated nucleic acids and polypeptides This disclosure provides, in particular, engineered nucleic acids comprising one or more expression cassettes (e.g., one, two, three, four, five, or more expression cassettes), for example, any expression cassette described herein. In some embodiments, at least one expression cassette provided by this disclosure comprises a promoter and an exogenous polynucleotide sequence. In some embodiments, the promoter is operable and / or directly ligated to the exogenous polynucleotide sequence.

[0036] In some embodiments, one or more expression cassettes may be multicistronic, i.e., multiple distinct polypeptides (e.g., multiple exogenous polynucleotides, or effector molecules) may be produced from a single transcript. For example, a multicistronic expression cassette may encode both a first ACP and a second ACP, and for example, both may be expressed from a single expression cassette driven by a constitutive promoter. In another embodiment, a multicistronic expression cassette may encode both an effector molecule and an antigen-recognizing receptor, and for example, both may be expressed from a single expression cassette driven by an ACP-responsive promoter. Expression cassettes may also be multicistronic by the use of various linkers, for example, a polynucleotide sequence encoding a first target protein (the manipulated polypeptide described herein) may be linked to a nucleotide sequence encoding a second target protein (e.g., first gene:linker:second gene, 5' to 3' orientation). Multicistronic features and options are described herein, for example, in the section "Multicistronic and Multipromoter Systems".

[0037] In some embodiments, the engineered nucleic acid is selected from DNA, cDNA, RNA, mRNA, and naked plasmids (linear or circular). Also provided herein are expression vectors containing the engineered nucleic acid.

[0038] In some embodiments, the engineered nucleic acid further comprises an insulator. The insulator may be localized between a first expression cassette and a second expression cassette. The insulator is a cis-regulatory element having an enhancer-blocking or barrier function. Enhancer-blocker insulators block enhancers from acting on the promoter of a proximal gene. Barrier insulators prevent euchromatin silencing. An example of a preferred insulator of this disclosure is the A2 insulator described in Liu M, et al., Nat Biotechnol. 2015 Feb;33(2):198-203. Additional insulators are described in West et al., Genes & Dev, 002. 16: 271-288, both of which are incorporated in whole by reference. Other suitable examples of insulators include, but are not limited to, A1 insulators, CTCF insulators, Gypsy insulators, HS5 insulators, and β-globin locus insulators such as cHS4. In some embodiments, the insulator is an A2 insulator, an A1 insulator, a CTCF insulator, an HS5 insulator, a Gypsy insulator, a β-globin locus insulator, or a cHS4 insulator.

[0039] Exemplary polypeptides may include, but are not limited to, ligand-binding domains, cell death-inducing domains, regulated cell survival polypeptides, activation-conditionally regulated polypeptides, nucleic acid-binding domains, transcription effector domains, chimeric receptors, such as chimeric antigen receptors, and other effector molecules.

[0040] Ligand-binding domain In some embodiments of this disclosure, the engineered polypeptide includes a ligand-binding domain. In some embodiments, the ligand-binding domain interacts with a ligand, such as a homologous ligand. In some embodiments, the interaction between the ligand-binding domain and the ligand results in oligomerization, e.g., dimerization, of multiple ligand-binding domains. In some embodiments, the interaction between the ligand-binding domain and the ligand results in a functional association between multiple ligand-binding domains, thereby resulting in a biologically relevant output (e.g., increased or decreased gene expression, increased or decreased biological signaling, etc.). In some embodiments, the functional association between multiple ligand-binding domains is direct in that the multiple ligand-binding domains are in direct contact with each other. In some embodiments, the functional association between multiple ligand-binding domains is indirect in that the multiple ligand-binding domains are not in direct contact with each other (e.g., the functional association of the multiple ligand-binding domains may be facilitated by one or more intermediate agents or molecules, e.g., polypeptides, nucleic acids, etc.). In some embodiments, the functional association includes oligomerization, polymerization, and / or complex formation between two or more engineered polypeptides of this disclosure.

[0041] Exemplary ligand-binding domains may include one or more of the following domains or functional fragments thereof: ABI domain, PYL domain, caffeine-binding single-domain antibody, cannabidiol-binding domain, hormone-binding domain of estrogen receptor (ER) domain, heavy chain variable region (VH) of anti-nicotine antibody, light chain variable region (VL) of anti-nicotine antibody, progesterone receptor domain, FKBP domain, and / or FRB domain. Examples of sequences of such domains are shown in Table D.

[0042] The ligand-binding domain may include degron. As used herein, the terms “degron” and “degron domain” refer to a protein or a portion thereof that is important in regulating the rate of proteolysis. Various embodiments of this disclosure may use various degrons known in the art, including but not limited to short amino acid sequences, structural motifs, and exposed amino acids. Degrons identified from various living organisms may be used. Degrons and degron pathways are generally known; see, for example, Varshazsky A., PNAS 2019 Jan 8;116(2):358-366, incorporated herein by reference.

[0043] As used herein, the term “degradation sequence” refers to a sequence that promotes the degradation of a bound protein by either the proteasome or the autophagy-lysosome pathway. Degradation sequences known in the art may be used in various embodiments of this disclosure. In some embodiments, the degradation sequence includes degrons identified from living organisms, or modifications thereof. In some embodiments, the degradation sequence is a polypeptide that destabilizes a protein such that, when fused to the protein, the protein’s half-life is reduced by at least twofold. Many different degradation sequences / signals (e.g., the ubiquitin-proteasome system) are known in the art, and any of these may be used as provided herein. The degradation sequence may be operably ligated to a cell receptor, but does not need to be adjacent to it, as long as the degradation sequence still functions to direct the degradation of the cell receptor. In some embodiments, the degradation sequence induces rapid degradation of the cell receptor. For discussions of degradation sequences in protein degradation and their functions, please refer to, for example, Kanemaki et al. (2013) Pflugers Arch. 465(3):4l9-425, Erales et al. (2014) Biochim Biophys Acta 1843(l):216-221, Schrader et al. (2009) Nat. Chem. Biol. 5(11):815-822, Ravid et al. (2008) Nat. Rev. Mol. Cell. Biol. 9(9):679-690, Tasaki et al. (2007) Trends Biochem Sci. 32(11):520-528, Meinnel et al. (2006) Biol. Chem. 387(7):839-851, and Kim et al. (2013), which are incorporated herein by reference. See Autophagy 9(7): 1100-1103, Varshavsky (2012) Methods Mol. Biol. 832: 1-11, and Fayadat et al. (2003) Mol Biol Cell. 14(3): 1268-1278.

[0044] In some embodiments, the degron, or degradation sequence, is selected from: HCV NS4 degron, PEST (two copies of human IκBα residues 277-307), GRR (human p105 residues 352-408), DRR (yeast Cdc34 residues 210-295), SNS (tandem repeat of SP2 and NB of influenza A or influenza B (SP2-NB-SP2), RPB (four copies of yeast RPB residues 1688-1702), SPmix (influenza A virus M2 protein) The tandem repeats of SP1 and SP2 proteins (SP2-SP1-SP2-SP1-SP2), NS2 (three copies of residues 79-93 of the influenza A virus NS protein), ODC (residues 106-142 of ornithine decarboxylase), Nek2A, mouse ODC (residues 422-461), mouse ODC_DA (residues 422-461 of mODC including D433A and D434A point mutations), APC / C degron, COP1 E3 ligase-binding degron motif, CRL4-Cdt2-binding PIP degron, actinphylline-binding degron, KEAP1-binding degron, KLHL2 and KLHL3-binding degron, MDM2-binding motif, N-degron, hydroxyproline modification in hypoxia signaling, plant hormone-dependent SCF-LRR-binding degron, SCF ubiquitin ligase-binding phosphodegron, plant hormone-dependent SCF-LRR-binding degron, DSGxxS phosphate-dependent degron, Siah-binding motif, SPOP SBC-docking motif, and PCNA-binding PIP box. In some embodiments, degrons include modifications / mutations that reduce ubiquitination of the wild-type protein, for example, the peptide sequence or domain from which the degron originates. Modifications / mutations that reduce ubiquitination may include substitutions or more lysine residues. Modifications / mutations that reduce ubiquitination may include substitutions of all lysine residues.

[0045] In some embodiments, degron contains a cerebron (CRBN) polypeptide substrate domain that can bind to CRBN in response to an immunomodulatory drug (IMiD), thereby promoting ubiquitin-mediated degradation of ACP. In some embodiments, the CRBN polypeptide substrate domain is selected from: IKZF1, IKZF3, CKla, ZFP91, GSPT1, MEIS2, GSS E4F1, ZN276, ZN517, ZN582, ZN653, ZN654, ZN692, ZN787, and ZN827, or fragments thereof capable of drug-inducible binding to CRBN. In some embodiments, the CRBN polypeptide substrate domain is a chimeric fusion product of a native CRBN polypeptide sequence. In some embodiments, the CRBN polypeptide substrate domain is an IKZF3 / ZFP91 / IKZF3 chimeric fusion product having the amino acid sequence FNVLMVHKRSHTGERPLQCEICGFTCRQKGNLLRHIKLHTGEKPFKCHLCNYACQRRDAL (SEQ ID NO: 93).

[0046] In some embodiments, degron includes degron having the amino acid sequence of SEQ ID NO: 131. Degron may include modified d913 degron, which includes amino acid substitutions to the amino acid sequence of SEQ ID NO: 131. In some embodiments, degron includes modified degron having the amino acid sequence of SEQ ID NO: 133. d913 degron may include modifications / mutations that reduce ubiquitination of unmodified d913 having the amino acid sequence of SEQ ID NO: 131. d913 degron may include, for example, substitution of one or more lysine residues in unmodified d913 having the amino acid sequence of SEQ ID NO: 131; for example, d913 degron may include substitution of all lysine residues in unmodified d913 having the amino acid sequence of SEQ ID NO: 131. d913 degron may, for example, substitute one or more lysine residues with arginine residues in unmodified d913 having the amino acid sequence of SEQ ID NO: 131. d913 degron can include, for example, substituting all lysine residues with arginine residues in an unmodified d913 having the amino acid sequence of SEQ ID NO: 131, such as a modified degron having the amino acid sequence of SEQ ID NO: 133.

[0047] In some embodiments, cerebron (CRBN) is a wild-type CRBN polypeptide, for example, the amino acid sequence of SEQ ID NO: 127. In some embodiments, CRBN is a modified CRBN polypeptide. Modified CRBN may include mutations that reduce ubiquitination compared to wild-type CRBN. Modified CRBN may include deletions of amino acids 194-247, which are the DDB1 interaction domain; for example, modified CRBN may include the amino acid sequence of SEQ ID NO: 129. In some embodiments, the ligand-binding domain includes the hormone-binding domain of the estrogen receptor (ER) domain, and the homologous ligand is tamoxifen or its metabolites. In some embodiments, the tamoxifen metabolites are selected from the group consisting of: 4-hydroxytamoxifen, N-desmethyltamoxifen, tamoxifen-N-oxide, and endoxifen.

[0048] In some embodiments, the ligand-binding domain comprises a progesterone receptor domain, and the homologous ligand is mifepristone or a derivative thereof.

[0049] In some embodiments, the ligand-binding domain includes an ABI domain or a PYL domain, and the homologous ligand is abscisic acid.

[0050] In some embodiments, the ligand-binding domain comprises a caffeine-binding single-domain antibody, and the homologous ligand is caffeine or a derivative thereof.

[0051] In some embodiments, the ligand-binding domain comprises a cannabidiol-binding domain, and the homologous ligand is cannabidiol or a plant cannabinoid. In some embodiments, the cannabidiol-binding domain comprises a single-domain antibody or a nanobody. In some embodiments, the cannabidiol-binding domain comprises an amino acid sequence selected from the group consisting of the sequences CA14, DB6, DB11, DB18, and DB21 in Table D.

[0052] In some embodiments, the ligand-binding domain comprises the hormone-binding domain of the estrogen receptor (ER) domain, and the homologous ligand is tamoxifen or its metabolites. In some embodiments, the tamoxifen metabolites are selected from the group consisting of: 4-hydroxytamoxifen, N-desmethyltamoxifen, tamoxifen-N-oxide, and endoxifen.

[0053] In some embodiments, the ligand-binding domain comprises the heavy chain variable region (VH) or the light chain variable region (VL) of an anti-nicotine antibody, and the homologous ligand is nicotine or a derivative thereof.

[0054] In some embodiments, the ligand-binding domain comprises a progesterone receptor domain, and the homologous ligand is mifepristone or a derivative thereof.

[0055] In some embodiments, the ligand-binding domain comprises an FKBP domain or an FRB domain, and the homologous ligand is rapamycin, AP1903, AP20187, FK1012, its derivatives, or analogs.

[0056] Exemplary ligand-binding domains are described in U.S. Patent Application Publication No. 20230011052, International Publication No. 2022109421, and International Publication No. 2022216823, which are incorporated herein by reference in their entirety.

[0057] Ligand-cognate ligand pair This disclosure identifies specific ligands useful for binding to ligand-binding domains, as discussed herein. In many embodiments of this disclosure, ligands can bind to specific ligand-binding domains. A given ligand that consistently binds to a given ligand-binding domain may be called a homologous ligand pair.

[0058] In some embodiments, the ligand is FK1012, its derivatives, or analogs thereof.

[0059] In some embodiments, the ligand is abscisic acid.

[0060] In some embodiments, the ligand is rapamycin, its derivatives, or analogs. In some embodiments, the ligand is tamoxifen or its metabolites. In some embodiments, the tamoxifen metabolites are selected from the group consisting of: 4-hydroxytamoxifen, N-desmethyltamoxifen, tamoxifen-N-oxide, and endoxifen.

[0061] In some embodiments, the ligand is caffeine or a derivative thereof.

[0062] In some embodiments, the ligand is nicotine or a derivative thereof.

[0063] In some embodiments, the ligand is cannabidiol, or a plant-derived cannabinoid.

[0064] In some embodiments, the ligand is mifepristone or a derivative thereof.

[0065] In some embodiments, the ligand is an IMiD. In some embodiments, the IMiD is an FDA-approved drug. In some embodiments, the IMiD is selected from the group consisting of thalidomide, lenalidomide, and pomalidomide.

[0066] Cell death induction domain In some embodiments, the manipulated polypeptides of the Disclosure include an inducible cell death polypeptide. The inducible cell death polypeptide may include one or more ligand-binding domains and at least one cell death-inducing domain.

[0067] Exemplary cell death-inducing domains, the following proteins, e.g., caspases (e.g., any one of caspases 1-11, e.g., caspase 3, caspase 6, caspase 7, caspase 8, caspase 9, etc.), diphtheria toxin fragment A (DTA), Bax, Bak, Bok, Bad, Bcl-Xs, Bik, Bcl-2 interacting protein 3 (BNIP3), Fas, Fas-related protein with death domain (FADD), tumor necrosis factor receptor 1-associated death domain protein (TRADD), TNF receptor (TNF-R), APAF-1, granzyme B, second mitochondrial-derived activator of caspase (SMAC), Omi, Bmf, Bid, Bim, Apot The p53 upregulatory factor (PUMA), Noxa, Blk, Hrk, cytochrome c, Arts, TNF-associated apoptosis-inducing ligand (TRAIL), herpes simplex virus thymidine kinase (HSV-TK), varicella-zoster virus thymidine kinase (VZV-TK), viral spike protein, carboxylesterase, cytosine deaminase, nitroreductase Fksb, carboxypeptidase G2, carboxypeptidase A, horseradish peroxidase, linamorase, liver cytochrome P450-2B1, and / or purine nucleoside phosphorylase, or variants thereof, or one or more functional fragments thereof. As used herein, variants may refer to variants, splice variants, conformations, isoforms, allele variants, species variants, and species homologs of biomolecules, such as polynucleotides or polypeptides. In some embodiments, the variant biomolecule is a native variant. In some embodiments, the variant biomolecule is an engineered variant. In some embodiments, the variant is a sequence-optimized variant, e.g., a polynucleotide sequence-optimized variant. In some embodiments, the term “variant” refers to a polynucleotide variant, e.g., a gene or regulatory element, that contains one or more different nucleotides compared to a reference (or “parent”) sequence. Thus, a variant polynucleotide sequence may contain at least one mutation, substitution, insertion, or deletion compared to each of its reference sequences.A “variant” may have at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity (e.g., nucleic acid sequence identity or amino acid sequence identity) compared to a reference sequence (e.g., a naturally occurring sequence, or a fragment or derivative thereof, or an engineered sequence). In some embodiments, depending on the context, a “variant” may refer to a “functional fragment.” A “functional fragment” of a biomolecule, e.g., a polynucleotide or polypeptide, may refer to a fragment of the reference biomolecule (i.e., shorter and / or smaller) that has the same or similar functional activity as the reference biomolecule. As long as the functional fragment achieves at least a portion of the activity of the reference biomolecule, the similar functional activity may be greater than, approximately equal to, or less than the functional activity of the reference biomolecule. If the reference biomolecule is a polypeptide, the polypeptide fragment is intended to retain at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more of the activity of the reference polypeptide in a preferred assay. For example, if the reference biomolecule is a polypeptide, the polypeptide fragment may be a polypeptide that has been cleaved or otherwise modified to be shorter and / or smaller than the reference polypeptide, yet is still intended to retain the functional activity of the reference polypeptide, for example, binding to a specific receptor; if the reference biomolecule is a polynucleotide, the polynucleotide fragment is intended to retain a portion of the same activity of the reference polynucleotide. For example, in the case of a protein-coding polynucleotide, the polynucleotide fragment is intended to encode a protein having at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more of the activity of the protein encoded by the reference polynucleotide in a preferred assay.For polynucleotides acting as regulatory elements (e.g., promoters or enhancers), the polynucleotide fragment is intended to have at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more of the activity of the reference polynucleotide in a preferred assay. Exemplary cell death-inducing domain sequences may be found in Table C-1.

[0068] Regulatory cell survival polypeptides In some embodiments, the manipulated polypeptides of the Disclosure include a regulatory cell survival polypeptide. The regulatory cell survival polypeptide may include at least one ligand-binding domain.

[0069] Exemplary cell survival polypeptides include one or more of the following: XIAP, Bcl-2, Bcl-Xl, Bcl-w, Bcl-2-related protein A1 (BCL2A1), Mcl-1, FLICE-like inhibitory protein (c-FLIP), and adenovirus E1B-19K protein. Cell survival polypeptides may include XIAP. For example, wild-type XIAP having amino acid sequence number 107 may be included. Cell survival polypeptides may include modified XIAP. Modified XIAP may include one or more amino acid substitutions with reference to sequence number 107.

[0070] Activated conditionally controlled polypeptide (ACP) In some embodiments, the manipulated polypeptide of the Disclosure comprises an ACP. In some embodiments, the manipulated polypeptide of the Disclosure may form an ACP in the presence of a ligand. In some embodiments, the ACP is or comprises a transcription modulator. In some embodiments, the ACP is or comprises a transcription repressor. In some embodiments, the ACP is or comprises a transcription activator. In some embodiments, the ACP is or comprises a transcription factor. In some embodiments, the ACP comprises a DNA-binding domain. In some embodiments, the ACP comprises a transcription effector domain. In some embodiments, the ACP comprises a DNA-binding domain and a transcription effector domain. In some embodiments, the transcription factor comprises a zinc finger-containing transcription factor. In some embodiments, the zinc finger-containing transcription factor may be a synthetic transcription factor. In some embodiments, the ACP DNA-binding domain comprises a DNA-binding zinc finger protein domain (ZF protein domain). In some embodiments, the DNA-binding domain comprises a tetracycline (or derivative thereof) repressor (TetR) domain. In some embodiments, the ACP may include one or more ligand-binding domains.

[0071] nucleic acid binding domain This disclosure provides, in some embodiments, engineered polypeptides comprising at least one nucleic acid-binding domain (e.g., a DNA-binding domain). In some embodiments, the engineered polypeptide comprises at least one ligand-binding domain and at least one nucleic acid-binding domain.

[0072] In some embodiments, the manipulated polypeptide comprises at least one transcription factor. In some embodiments, the transcription factor comprises at least one nucleic acid-binding domain. In some embodiments, the transcription factor comprises at least one nucleic acid-binding domain and at least one transcription effector domain.

[0073] In some embodiments, the nucleic acid binding domain includes a DNA-binding zinc finger protein domain (ZF protein domain). In some embodiments, the ZF protein domain is a modular design and consists of zinc finger sequences (ZFAs). In some embodiments, the transcription effector domain includes a herpes simplex virus protein 16 (VP16) activation domain; an activation domain containing four tandem copies of VP16; a VP64 activation domain; a p65 activation domain of NFκB; an Epstein-Barr virus R trans-activator (Rta) activation domain; a triple-activator containing VP64, p65, and Rta activation domains (VPR activation domain); a triple-activator containing VP64, p65, and HSF1 activation domains (VPH activation domain); and a histone acetyltransferase (HAT) core domain of human E1A-related protein p300 (p300). The transcription effector domain is selected from the group consisting of: HAT core activating domain; Kruppel association box (KRAB) repressor domain; repressor element silencing transcription factor (REST) ​​repressor domain; WRPW motif of hairy-associated basic helix-loop-helix repressor protein, known as the WRPW repressor domain; DNA (cytosine-5)-methyltransferase 3B (DNMT3B) repressor domain; and HP1 alpha-chromoshadow repressor domain, or variants thereof, or functional fragments. In some embodiments, the transcription effector domain is a triple activator comprising VP64, p65, and Rta activating domains (VPR activating domains), or variants thereof, or functional fragments. In some embodiments, the transcription effector domain comprises the p65 domain, or variants thereof, or functional fragments.

[0074] In some embodiments, the ZF protein domain is a modular design and consists of a zinc finger array (ZFA). The zinc finger sequence contains multiple zinc finger protein motifs linked together. Each zinc finger motif binds to a different nucleic acid motif. This results in a ZFA with specificity to any desired nucleic acid sequence. The ZF motifs may be directly adjacent to each other or separated by a flexible linker sequence. In some embodiments, the ZFA is a sequence, string, or chain of ZF motifs arranged in tandem. The ZFA may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 zinc finger motifs. ZFA may have 1-10, 1-15, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, or 5-15 zinc finger motifs.

[0075] In some embodiments, the ZF protein domain contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more ZFAs. In some embodiments, the ZF domain may contain 1-10, 1-15, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, or 5-15 ZFAs. In some embodiments, the ZF protein domain contains 1-10 ZFAs (or more). In some embodiments, the ZF protein domain contains at least one ZFA. In some embodiments, the ZF protein domain contains at least two ZFAs. In some embodiments, the ZF protein domain contains at least three ZFAs. In some embodiments, the ZF protein domain contains at least four ZFAs. In some embodiments, the ZF protein domain contains at least five ZFAs. In some embodiments, the ZF protein domain contains at least ten ZFAs.

[0076] An exemplary ZF protein domain is shown in the sequence SRPGERPFQCRICMRNFSRRHGLDRHTRTHTGEKPFQCRICMRNFSDHSSLKRHLRTHTGSQKPFQCRICMRNFSVRHNLTRHLRTHTGEKPFQCRICMRNFSDHSNLSRHLKTHTGSQKPFQCRICMRNFSQRSSLVRHLRTHTGEKPFQCRICMRNFSESGHLKRHLRTHLRGS (SEQ ID NO: 57). In some embodiments, the ZF protein domain includes the amino acid sequence of SEQ ID NO: 57.

[0077] Transcription effector domain This disclosure provides engineered polypeptides, in some embodiments, comprising at least one transcription effector domain. In some embodiments, the engineered polypeptide may comprise at least one ligand-binding domain and at least one transcription effector domain.

[0078] In some embodiments, the inducible cell death polypeptide provided herein may comprise at least one transcription effector domain. In some embodiments, the ACP provided herein may comprise at least one transcription effector domain. In some embodiments, the inducible cell death polypeptide may comprise at least one ligand-binding domain and at least one transcription effector domain. In some embodiments, the ACP may comprise at least one ligand-binding domain and at least one transcription effector domain.

[0079] In some embodiments, the transcription effector domain includes one or more of the following: herpes simplex virus protein 16 (VP16) activation domain; activation domains containing four tandem copies of VP16, VP64 activation domain; p65 activation domain of NFκB; Epstein-Barr virus R trans-activator (Rta) activation domain; triple activation factor containing VP64, p65, and Rta activation domains (VPR activation domain); tertiary activation factor containing VP64, p65, and HSF1 activation domains (VPH activation domain); histone acetyltransferase (HAT) core domain of human E1A-related protein p300 (p300 The transcription effector domains include: HAT core activating domain; Kruppel association box (KRAB) repressor domain; repressor element silencing transcription factor (REST) ​​repressor domain; WRPW motif of hairy-associated basic helix-loop-helix repressor protein, known as the WRPW repressor domain; DNA (cytosine-5)-methyltransferase 3B (DNMT3B) repressor domain; and HP1 alpha chromoshadow repressor domain. In some embodiments, the transcription effector domain is a triple activator comprising VP64, p65, and Rta activating domains (VPR activating domain). In some embodiments, the transcription effector domain comprises the p65 domain.

[0080] In some embodiments, the transcription effector domain includes a transcription repressor domain. In some embodiments, the transcription repressor domain includes a Kruppel-associated box (KRAB) repressor domain; a truncated Kruppel-associated box (KRAB) repressor domain; a histone deacetylase 4 (HDAC4) repressor domain; a Scleraxis (SCX) HLH domain; an inhibitor of the DNA-binding 1 (ID1) HLH domain; a HECT domain and an RCC1-like domain-containing protein 2 (HERC2) Cyt-b5 domain; a twist-associated protein 1 (TWST1) HLH domain; a homeobox protein Nkx-2.2 (NKX22) homeobox domain. The following are selected from the group consisting of a main, DNA-binding 1 (ID3) HLH domain inhibitor, twist-associated protein 2 (TWST2) HLH domain, and EED repressor domain; repressor element silencing transcription factor (REST) ​​repressor domain; WRPW motif of hairy-associated basic helix-loop helix repressor protein (the motif is known as the WRPW repressor domain); DNA (cytosine-5)-methyltransferase 3B (DNMT3B) repressor domain; and HP1 alpha-chromoshadow repressor domain. In some embodiments, the transcription effector domain includes a transcription activation domain. In some embodiments, the transcriptional activation domain is selected from the group consisting of: herpes simplex virus protein 16 (VP16) activation domain; activation domain containing four tandem copies of VP16; VP64 activation domain; p65 activation domain of NFκB; Epstein-Barr virus R trans-activator (Rta) activation domain; triple-activator containing VP64, p65, and Rta activation domains (VPR activation domain); triple-activator containing VP64, p65, and HSF1 activation domain (VPH activation domain); and histone acetyltransferase (HAT) core domain of human E1A-related protein p300 (p300 HAT core activation domain). In some embodiments, the transcriptional activation domain is a triple-activator containing VP64, p65, and Rta activation domains (VPR activation domain).In some embodiments, the transcriptional activation domain includes a p65 domain. The transcriptional activation domain may also be called a transcription activator domain.

[0081] In some embodiments, the engineered nucleic acid of the Disclosure comprises a polynucleotide encoding an inducible cell death polypeptide. In some embodiments, the engineered nucleic acid comprises a polynucleotide encoding an ACP. In some embodiments, the inducible cell death polypeptide provided herein may encode at least one transcription factor comprising at least one transcription effector domain. In some embodiments, the ACP provided herein comprises at least one transcription factor comprising at least one transcription effector domain. In some embodiments, the inducible cell death polypeptide provided herein may comprise at least one transcription factor comprising at least one nucleic acid binding domain and at least one transcription effector domain. In some embodiments, the ACP provided herein may comprise at least one transcription factor comprising at least one nucleic acid binding domain and at least one transcription effector domain. Furthermore, the ACP may comprise at least one ligand binding domain, as well as at least one transcription factor comprising at least one nucleic acid binding domain and at least one transcription effector domain.

[0082] The engineered nucleic acids provided by this disclosure may encode effector domains, such as transcription effector domains. In some embodiments, the transcription effector domain may include the effector domain of a transcription factor (e.g., an activator domain or a repressor domain). Transcription factor effector domains are also known as transactivation domains and act as scaffold domains for proteins, such as transcription coregulators, which act to activate or repress the transcription of genes. Any suitable transcription effector domains, but not limited to, include: the herpes simplex virus protein 16 (VP16) activation domain; the VP64 activation domain, which is an activation domain consisting of four tandem copies of VP16; the p65 activation domain of NFκB; the Epstein-Barr virus R trans-activator (Rta) activation domain; the triple activator containing the VP64, p65, and Rta activation domains (the triple activator is known as the VPR activation domain); and the histone acetyltransferase (HAT) core domain of human E1A-related protein p300 (p300HAT). The following domains may be used, including but not limited to: a core activating domain known as; a Kruppel association box (KRAB) repressor domain; a cleavage Kruppel association box (KRAB) repressor domain; a repressor element silencing transcription factor (REST) ​​repressor domain; the WRPW motif of the Hairy-associated basic helix-loop-helix repressor protein (this motif is known as a WRPW repressor domain); a DNA (cytosine-5)-methyltransferase 3B (DNMT3B) repressor domain; and an HP1 alpha-chromoshadow repressor domain, or any combination thereof. In some embodiments, the transcription effector domain is a triple activator comprising VP64, p65, and Rta activating domains (VPR activating domain). In some embodiments, the transcription effector domain comprises a p65 domain.

[0083] Exemplary transcription effector domain protein sequences are shown in Table 1. Exemplary transcription effector domain nucleotide sequences are shown in Table 2.

[0084] [Table 1]

[0085] [Table 2]

[0086] Chimeric receptor In some embodiments, the chimeric receptor is a chimeric antigen receptor (CAR). Generally, a CAR is a chimeric protein comprising an antigen-binding domain and a polypeptide molecule heterogeneous to the antigen-binding domain, such as a peptide heterogeneous to the antibody to which the antigen-binding domain can be induced. Polypeptide molecules heterogeneous to the antigen-binding domain may include, but are not limited to, a transmembrane domain, one or more intracellular signaling domains, a hinge domain, a spacer region, one or more peptide linkers, or a combination thereof.

[0087] In some embodiments, the chimeric antigen receptor is an activated chimeric antigen receptor (aCAR, and generally referred to as CAR unless otherwise specified). In some embodiments, binding of the chimeric antigen receptor to its homologous ligand is sufficient to induce activation of immune-responsive cells. In some embodiments, binding of the chimeric antigen receptor to its homologous ligand is sufficient to induce stimulation of immune-responsive cells. In some embodiments, activation of immune-responsive cells results in the killing of target cells. In some embodiments, activation of immune-responsive cells results in the expression and / or secretion of cytokines or chemokines by the immune-responsive cells. In some embodiments, stimulation of immune-responsive cells results in the expression and / or secretion of cytokines or chemokines by the immune-responsive cells. In some embodiments, stimulation of immune-responsive cells induces differentiation of immune-responsive cells. In some embodiments, stimulation of immune-responsive cells induces proliferation of immune-responsive cells. In some embodiments, activation and / or stimulation of immune-responsive cells may be a combination of the above responses.

[0088] The number of ABDs in a binding molecule, such as the chimeric proteins described herein, defines the "valence" of the binding molecule. A binding molecule having a single ABD is said to be "monovalent". A binding molecule having multiple ABDs is said to be "polyvalent". A polyvalent binding molecule having two ABDs is "divalent". A polyvalent binding molecule having three ABDs is "trivalent". A polyvalent binding molecule having four ABDs is "tetravalent". In various polyvalent embodiments, all of the multiple ABDs have the same recognition specificity and can be called a "monospecific polyvalent" binding molecule. In other polyvalent embodiments, at least two of the multiple ABDs have different recognition specificities. Such a binding molecule is polyvalent and "multispecific". In a polyvalent embodiment where the ABDs collectively have two recognition specificities, the binding molecule is "dualspecific". In a polyvalent embodiment where the ABDs collectively have three recognition specificities, the binding molecule is "triplespecific". In a polyvalent embodiment where ABD possesses multiple recognition specificities for different epitopes present on the same antigen, the binding molecule is a "multiple paratope." In a polyvalent embodiment where ABD recognizes two epitopes on the same antigen, it is a "double paratope."

[0089] In various polyvalent embodiments, polyvalent binding molecules improve the avidity of the binding molecule to a particular target. As used herein, “avidity” refers to the overall strength of the interaction between two or more molecules, e.g., polyvalent binding molecules to a particular target, and avidity is the cumulative strength of the interaction provided by the affinity of multiple ABDs. Avidity may be measured by the same method used to determine affinity, as described above. In a particular embodiment, the avidity of a binding molecule to a specific target is such that the interaction is a specific binding interaction, and the avidity between the two molecules is 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, or 10 -10It has a KD value less than M. In certain embodiments, the avidity of a binding molecule to a specific target has a KD value such that the interaction is a specific binding interaction, and one or more individual ABDs do not have a KD value such that they recognize the specific binding of each of their respective antigens or epitopes. In certain embodiments, avidity is the cumulative strength of the interaction resulting from the affinities of multiple ABDs to a shared specific target, such as distinct antigens found on individual cells, or to distinct antigens on a complex. In certain embodiments, avidity is the cumulative strength of the interaction resulting from the affinities of multiple ABDs to distinct epitopes on shared individual antigens.

[0090] The CARs of this disclosure may be first, second, or third-generation CARs. "First-generation" CARs generally contain a single intracellular signaling domain, typically derived from the T cell receptor chain. "First-generation" CARs generally have an intracellular signaling domain from the CD3-zeta (CD3ζ) chain, which is the primary signaling factor from the endogenous TCR. "First-generation" CARs provide de novo antigen recognition and can induce activation of both CD4+ and CD8+ T cells via the CD3ζ chain signaling domain within a single fusion molecule, independently of HLA-mediated antigen presentation. "Second-generation" CARs provide additional signaling to T cells by adding a second intracellular signaling domain from one of various co-stimulatory molecules (e.g., CD28, 4-1BB, ICOS, OX40) to the cytoplasmic tail of the CAR. "Second-generation" CARs provide both co-stimulation (e.g., CD28, or 4-1BB) and activation (CD3ζ). Preclinical studies have shown that "second-generation" CARs can improve the antitumor activity of immune-responsive cells, such as T cells. "Third-generation" CARs have multiple intracellular co-stimulatory signaling domains (e.g., CD28 and 4-1BB) and an intracellular activation signaling domain (CD3ζ).

[0091] In some embodiments, the chimeric antigen receptor is an inhibitory chimeric antigen receptor (iCAR).

[0092] The binding of the extracellular antigen-binding domain of the CARs of this disclosure may be determined, for example, by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition), biolayer interferometry (e.g., Octet / FORTEBIO®), surface plasmon resonance (SPR) technology (e.g., Biacore®), or Western blot assay. Each of these assays generally detects the presence of a particular protein-antibody complex of interest by using a labeling reagent (e.g., antibody, or scFv) specific to the complex of interest. For example, scFv may be radiolabeled and used in RIA assays. Radioisotopes may be detected by means such as the use of a γ counter or scintillation counter, or by autoradiography. In certain embodiments, the extracellular antigen-binding domain of the CAR is labeled with a fluorescent marker. Non-limiting examples of fluorescent markers include green fluorescent protein (GFP), blue fluorescent protein (e.g., EBFP, EBFP2, Azurite, and mKalamal), cyan fluorescent protein (e.g., ECFP, Cerulean, and CyPet), and yellow fluorescent protein (e.g., YFP, Citrine, Venus, and YPet). In certain embodiments, the extracellular antigen-binding domain of the CAR is labeled with a secondary antibody specific to the extracellular antigen-binding domain, and the secondary antibody is labeled (e.g., by radiation or with a fluorescent marker).

[0093] The antigen-binding domains of this disclosure may include, but are not limited to, antigens, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, bispecific antibodies, complex antibodies, human antibodies, and humanized antibodies; and functional fragments thereof, including, but not limited to, single-domain antibodies (sdAbs), such as heavy-chain variable domains (VH), light-chain variable domains (VL), and camel-derived nanobody variable domains (VHH); any domains to which the binding occurs; and any domains to which the binding occurs, which may bind to alternative scaffolds known in the art, such as recombinant fibronectin domains, T cell receptors (TCRs), recombinant TCRs with enhanced affinity, or fragments thereof, such as single-chain TCRs. In some cases, it is beneficial that the antigen-binding domains originate from the same species in which the CARs are ultimately used.

[0094] In some embodiments, the extracellular antigen-binding domain includes an antibody. In certain embodiments, the antibody is a human antibody. In certain embodiments, the antibody is a chimeric antibody. In some embodiments, the extracellular antigen-binding domain includes an antigen-binding fragment of the antibody.

[0095] In some embodiments, the extracellular antigen-binding domain includes the F(ab) fragment. In certain embodiments, the extracellular antigen-binding domain includes the F(ab') fragment.

[0096] In some embodiments, the extracellular antigen-binding domain includes scFv. In some embodiments, the extracellular antigen-binding domain includes two single-stranded variable fragments (scFv). In some embodiments, each of the two scFvs binds to a distinct epitope on the same antigen. In some embodiments, the extracellular antigen-binding domain includes a first scFv and a second scFv. In some embodiments, the first scFv and the second scFv bind to distinct epitopes on the same antigen. In certain embodiments, the scFv is a mammalian scFv. In certain embodiments, the scFv is a chimeric scFv. In certain embodiments, the scFv includes a heavy-chain variable domain (VH) and a light-chain variable domain (VL).

[0097] In certain embodiments, VH and VL are separated by a peptide linker. In certain embodiments, the peptide linker comprises one of the amino acid sequences shown in Table 6.

[0098] In certain embodiments, an scFv comprises the structure VH-L-VL or VL-L-VH, where VH is the heavy chain variable domain, L is the peptide linker, and VL is the light chain variable domain. In some embodiments, each of one or more scFvs comprises the structure VH-L-VL or VL-L-VH, where VH is the heavy chain variable domain, L is the peptide linker, and VL is the light chain variable domain. When two or more scFvs are linked together, each scFv may be linked to the next scFv to which a peptide is linked. In some embodiments, each of one or more scFvs is separated by a peptide linker.

[0099] [Table 3]

[0100] Suitable antigen-recognition receptors for use as effector molecules include, but are not limited to, 5T4, ADAM9, AFP, AXL, B7-H3, B7-H4, B7-H6, C4.4, CA6, Cadherin 3, Cadherin 6, CCR4, CD123, CD133, CD138, CD142, CD166, CD25, CD30, CD352, CD37, CD38, CD44, CD56, CD66e, CD70, CD71, CD74, CD79b, CD80, CEA, CEACAM5, Claudin 18.2, cMet, CSPG4, CTLA, DLK1, DLL3, DR5, EGFR, ENPP3, EpCAM, EphA2, EphrinA4, ETBR, FGFR2, FGFR3, FR alpha, FRb, GCC, GD2, GFRa4, gpA33, GPC3, gpNBM, GPRC5, HER2, IL-13R, IL-13Ra, IL-13Ra2, IL- 8, IL-15, IL1RAP, integrin aV, KIT, L1CAM, LAMP1, Lewis Y, LeY, LIV-1, LRRC, LY6E, MCSP, mesothelin (MSLN), MUC1, MUC16, MUC1C, NaPi2B, Nectin 4, NKG2D, NOTCH3, NY It recognizes antigens including ESO 1, ovalin, p-cadherin, pan-Erb2, PSCA, PSMA, PTK7, ROR1, S Aures, SCT, SLAMF7, SLITRK6, SSTR2, STEAP1, Survivin, TDGF1, TIM1, TROP2, and WT1, or combinations thereof.

[0101] In some embodiments, the antigen-recognizing receptor recognizes antigens selected from the following: 5T4, ADAM9, AFP, AXL, B7-H3, B7-H4, B7-H6, C4.4, CA6, Cadherin 3, Cadherin 6, CCR4, CD123, CD133, CD138, CD142, CD166, CD25, CD30, CD352, CD37, CD38, CD44, CD56, CD66e, CD70, CD71, CD74, CD79b, CD80, CEA, CEACAM5, Claudin 18.2, cMet, CSPG4, CTLA, DLK1, DLL3, D R5, EGFR, ENPP3, EpCAM, EphA2, EphrinA4, ETBR, FGFR2, FGFR3, FR alpha, FRb, GCC, GD2, GFRa4, gpA33, GPC3, gpNBM, GPRC5, HER2, IL-13R, IL-13Ra, IL-13Ra 2, IL-8, IL-15, IL1RAP, integrin aV, KIT, L1CAM, LAMP1, Lewis Y, LeY, LIV-1, LRRC, LY6E, MCSP, mesothelin, MUC1, MUC16, MUC1C, NaPi2B, Nectin 4, NKG2D, NOTCH3, NY ESO 1, Obalin, P-Cadherin, pan-Erb2, PSCA, PSMA, PTK7, ROR1, S Aures, SCT, SLAMF7, SLITRK6, SSTR2, STEAP1, Survivin, TDGF1, TIM1, TROP2, and WT1.

[0102] Exemplary antigen-binding domain sequences are described, for example, in U.S. Patent Publication No. 20230011052, U.S. Patent Publication No. 20210017277, International Publication No. 2022082059, International Publication No. 2022115565, International Publication No. 2022236142, U.S. Patents No. 11,299,546 and 9,409,994, which are incorporated herein by reference in their entirety.

[0103] In some embodiments, the chimeric receptor is a T cell receptor (TCR).

[0104] In some embodiments, the polypeptides may include a first chimeric receptor and a second chimeric receptor.

[0105] Effector molecule Any suitable effector molecule known in the art can be encoded by an engineered nucleic acid or expressed by an engineered cell. Suitable effector molecules can be grouped into therapeutic classes based on structural similarity, sequence similarity, or function. The therapeutic classes of effector molecules include, but are not limited to, cytokines, chemokines, homing molecules, growth factors, coactivating molecules, tumor microenvironment modifiers, receptors, ligands, antibodies, polynucleotides, peptides, and enzymes.

[0106] In some embodiments, each effector molecule is independently selected from a therapeutic class, where the therapeutic class is selected from: cytokines, chemokines, homing molecules, growth factors, coactivating molecules, tumor microenvironment modifiers, receptors, ligands, antibodies, polynucleotides, peptides, and enzymes.

[0107] Chemokines are small cytokines, or signaling proteins, secreted by cells that can induce directional chemotaxis within cells. Chemokines can be classified into four main subfamilies: CXC, CC, CX3C, and XC, all of which exert their biological effects by selectively binding to chemokine receptors located on the surface of target cells. Non-limiting examples of chemokines that may be encoded by engineered nucleic acids in this disclosure include: CCL21a, CXCL10, CXCL11, CXCL13, CXCL10-CXCL11 fusion proteins, CCL19, CXCL9, and XCL1, and any combination thereof. In some embodiments, the chemokines are selected from: CCL21a, CXCL10, CXCL11, CXCL13, CXCL10-CXCL11 fusion proteins, CCL19, CXCL9, and XCL1.

[0108] Non-limiting examples of cytokines that may be encoded by the manipulated nucleic acids of this disclosure include: IL1-beta, IL2, IL4, IL6, IL7, IL10, IL12, IL12p70 fusion protein, IL15, IL17A, IL18, IL21, IL22, type I interferon, interferon-gamma, and TNF-alpha, or any combination thereof. In some embodiments, the cytokines are selected from: IL1-beta, IL2, IL4, IL6, IL7, IL10, IL12, IL12p70 fusion protein, IL15, IL17A, IL18, IL21, IL22, type I interferon, interferon-gamma, and TNF-alpha.

[0109] Effector molecules may include homing molecules. "Homing" refers to the active navigation (migration) of a cell to a target site (e.g., a cell, a tissue (e.g., a tumor), or an organ). "Homing molecule" refers to a molecule that directs a cell toward a target site. In some embodiments, homing molecules function to recognize and / or initiate an interaction between the manipulated cell and the target site. Non-limiting examples of homing molecules include CXCR1, CCR9, CXCR2, CXCR3, CXCR4, CCR2, CCR4, FPR2, VEGFR, IL6R, CXCR1, CSCR7, PDGFR, anti-integrin alpha-4, beta-7; anti-MAdCAM; CCR9; CXCR4; SDFl; MMP-2; CXCR1; CXCR7; CCR2; CCR4; and GPR15, or any combination thereof. In some embodiments, the homing molecules are selected from: anti-integrin alpha 4, beta 7; anti-MAdCAM; CCR9; CXCR4; SDFl; MMP-2; CXCR1; CXCR7; CCR2; CCR4; and GPR15.

[0110] Suitable growth factors for use as effector molecules include, but are not limited to, FLT3L and GM-CSF, or any combination thereof. In some embodiments, the growth factors are selected from: FLT3L and GM-CSF.

[0111] Suitable co-activating molecules for use as effector molecules include, but are not limited to, c-Jun, 4-1BBL, and CD40L, or any combination thereof. In some embodiments, the co-activating molecules are selected from: c-Jun, 4-1BBL, and CD40L.

[0112] The "tumor microenvironment" is the cellular environment in which a tumor resides, including the surrounding blood vessels, immune cells, fibroblasts, myeloid-derived inflammatory cells, lymphocytes, signaling molecules, and the extracellular matrix (ECM) (see, for example, Pattabiraman, DR & Weinberg, RA Nature Reviews Drug Discovery 13, 497-512 (2014); Balkwill, FR et al. J Cell Sci 125, 5591-5596, 2012; and Li, H. et al. J Cell Biochem 101(4), 805-15, 2007). Suitable tumor microenvironment modifiers for use as effector molecules include, but are not limited to, adenosine deaminase, TGF-beta inhibitors, immune checkpoint inhibitors, VEGF inhibitors, and HPGE2, or any combination thereof. In some embodiments, tumor microenvironment modifiers are selected from: adenosine deaminase, TGF-beta inhibitors, immune checkpoint inhibitors, VEGF inhibitors, and HPGE2.

[0113] The effector molecule may include a TGF beta inhibitor. Suitable TGF beta inhibitors for use as an effector molecule include, but are not limited to, anti-TGF beta peptides, anti-TGF beta antibodies, TGFb-TRAP, or combinations thereof. In some embodiments, the TGF beta inhibitor is selected from: anti-TGF beta peptides, anti-TGF beta antibodies, TGFb-TRAP, and combinations thereof.

[0114] Effector molecules may include immune checkpoint inhibitors. Suitable immune checkpoint inhibitors for use as effector molecules include, but are not limited to, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-PD-L2 antibodies, anti-CTLA-4 antibodies, anti-LAG-3 antibodies, anti-TIM-3 antibodies, anti-TIGIT antibodies, anti-VISTA antibodies, anti-KIR antibodies, anti-B7-H3 antibodies, anti-B7-H4 antibodies, anti-HVEM antibodies, anti-BTLA antibodies, anti-GAL9 antibodies, anti-A2AR antibodies, anti-phosphatidylserine antibodies, anti-CD27 antibodies, anti-TNFa antibodies, anti-TREM1 antibodies, and anti-TREM2 antibodies, or any combination thereof. In some embodiments, the immune checkpoint inhibitor is selected from: anti-PD-1 antibody, anti-PD-L1 antibody, anti-PD-L2 antibody, anti-CTLA-4 antibody, anti-LAG-3 antibody, anti-TIM-3 antibody, anti-TIGIT antibody, anti-VISTA antibody, anti-KIR antibody, anti-B7-H3 antibody, anti-B7-H4 antibody, anti-HVEM antibody, anti-BTLA antibody, anti-GAL9 antibody, anti-A2AR antibody, anti-phosphatidylserine antibody, anti-CD27 antibody, anti-TNFa antibody, anti-TREM1 antibody, and anti-TREM2 antibody.

[0115] Examples of immune checkpoint inhibitors include pembrolizumab (anti-PD-1; MK-3475 / Keytruda®- Merck), nivolumab (anti-PD-1; Opdivo®- BMS), pidilizumab (anti-PD-1 antibody; CT-011- Teva / CureTech), AMP224 (anti-PD-1; NCI), avelumab (anti-PD-L1; Bavencio®- Pfizer), durvalumab (anti-PD-L1; MEDI4736 / Imfinzi®- Medimmune / AstraZeneca), atezolizumab (anti-PD-L1; Tecentriq®- Roche / Genentech), and BMS-936559 (anti-PD-L1 - This includes BMS, tremelimumab (anti-CTLA-4; Medimmune / AstraZeneca), ipilimumab (anti-CTLA-4; Yervoy®-BMS), lirirumab (anti-KIR; BMS), and monalizumab (anti-NKG2A; Innate Pharma / AstraZeneca).

[0116] Exemplary effector molecules also include VEGF inhibitors. Suitable VEGF inhibitors for use as effector molecules include, but are not limited to, anti-VEGF antibodies, anti-VEGF peptides, or combinations thereof. In some embodiments, the VEGF inhibitor includes anti-VEGF antibodies, anti-VEGF peptides, or combinations thereof.

[0117] In some embodiments, each effector molecule is a human-derived effector molecule.

[0118] promoter In some embodiments, the engineered nucleic acids of the Disclosure include, for example, one or more promoters in one or more expression cassettes. In some embodiments, the engineered nucleic acids of the Disclosure include a first expression cassette including a first promoter operably ligated to an exogenous polynucleotide sequence. In some embodiments, the engineered nucleic acids of the Disclosure include a second expression cassette including a promoter operably ligated to a second exogenous polynucleotide sequence encoding one or more effector molecules. In some embodiments, the first expression cassette and the second expression cassette are each encoded by separate engineered nucleic acids of the Disclosure. In some embodiments, the first expression cassette and the second expression cassette are encoded by the same engineered nucleic acid of the Disclosure.

[0119] In some embodiments, the promoter is an ACP-responsive promoter. In some embodiments, the ACP-responsive promoter of this disclosure comprises an ACP-binding domain and a promoter sequence. In some embodiments, the ACP-responsive promoter is operably ligated to a nucleotide sequence encoding an effector molecule (e.g., a protein of interest, e.g., any manipulated polypeptide described herein).

[0120] A “promoter” refers to a regulatory region of a nucleic acid sequence that controls the initiation and rate of the rest of the transcription of that nucleic acid sequence. In some embodiments, a promoter may include a small region to which regulatory proteins and other molecules can be bound, such as RNA polymerase, ACP, and / or transcription factors. A promoter may be constitutive, inductive, repressive, tissue-specific, or any combination thereof. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inductive promoter. A promoter drives expression or drives the transcription of the nucleic acid sequence it regulates. Hereinafter, a promoter is considered “operably ligated” when it is in the correct functional position and orientation relative to the nucleic acid sequence it regulates in order to control (“drive”) the transcription initiation and / or expression of its sequence.

[0121] In some embodiments, the promoter may be a promoter that is naturally associated with a gene or a particular sequence, such that it may be obtained by isolating a 5' non-coding sequence located upstream of the coding segment of the gene or sequence. Such a promoter may be referred to as “endogenous.” In some embodiments, the coding nucleic acid sequence may be under the control of a recombinant or heterologous promoter, which refers to a promoter that does not normally associate with the encoded sequence in its natural environment. Such promoters may include promoters of other genes; promoters isolated from any other cell; and synthetic promoters or enhancers that are not “native,” such as those containing different elements and / or mutations in different transcriptional regulatory regions, for example, by genetic engineering methods to modify expression. In addition to synthetically producing nucleic acid sequences of promoters and enhancers, sequences may be produced using nucleic acid amplification techniques, including recombinant cloning and / or polymerase chain reaction (PCR) (see, for example, U.S. Patents 4,683,202 and 5,928,906).

[0122] The promoters of the engineered nucleic acids in this disclosure may be “inducible promoters,” which are characterized by modulating (e.g., initiating or activating) transcriptional activity in the presence of, being affected by, or in contact with a signal. The signal may be an endogenous or usually exogenous state (e.g., light), a compound (e.g., a chemical or non-chemical compound), or a protein (e.g., an engineered polypeptide as described herein, e.g., ACP) that comes into contact with the inducible promoter in a manner that is active in modulating transcriptional activity from the inducible promoter. Activation of transcription may include acting directly on the promoter to drive transcription, or acting indirectly on the promoter by inactivating a repressor (e.g., an engineered polypeptide as described herein) that prevents the promoter from driving transcription. Conversely, inactivation of transcription may include acting directly on the promoter to prevent transcription, or acting indirectly on the promoter by subsequently activating a repressor that acts on the promoter.

[0123] In some embodiments, the promoter is “responsive” or “regulated by” a signal if, in the presence of a local tumor condition (e.g., inflammation or hypoxia), or in that condition or signal, transcription from the promoter is activated, inactivated, increased, or decreased. In some embodiments, the promoter includes a response element, which is a short sequence of DNA within the promoter region that binds to a specific molecule (e.g., a transcription factor, or one of the manipulated polypeptides described herein, e.g., ACP) that modulates (regulates) gene expression from the promoter. Response elements that may be used in accordance with this disclosure include, but are not limited to, the phloretin-adjustable regulatory element (PEACE), zinc finger DNA-binding domain (DBD), interferon-gamma activating sequence (GAS) (Decker, T. et al. J Interferon Cytokine Res. 1997 Mar;17(3):121-34, incorporated herein by reference), interferon-stimulated response element (ISRE) (Han, KJ et al. J Biol Chem. 2004 Apr 9;279(15):15652-61, incorporated herein by reference), NF-kappa B response element (Wang, V. et al. Cell Reports 2012;2(4): 824-839, incorporated herein by reference), and STAT3 response element (Zhang, D. et al. J of Biol Chem. 1996;271: This includes 9503-9509 (as incorporated herein by reference). Other response elements are also incorporated herein. Response elements may also include tandem repeats (e.g., consecutive repeats of the same nucleotide sequence encoding the response element) to generally increase the sensitivity of the response element to its homologous molecules. Tandem repeats may be labeled as 2×, 3×, 4×, 5×, etc., to indicate the number of repeats present.

[0124] Table 3 lists non-limiting examples of responsive promoters (also called inductive promoters) (e.g., TGF-beta responsive promoter). Furthermore, Table 3 provides exemplary promoter and transcription factor combinations that may be used in accordance with this disclosure, as well as the effects of selective inducing molecules on such promoter and transcription factor combinations. For example, the transcription factor response to an inducer is shown in the TF column (where A represents activation, DA represents inactivation, and DR represents derepression), and the transgene transcription response to an inducer is shown in the T column (where B represents binding, D represents dissociation, and nd represents undetermined) (see M. & Weber, W. FEBS Letters 586 (2012) 20784-2096m and the references cited therein). Table 4 shows non-limiting examples of components that may be included in an inductive promoter (e.g., minimal promoter and response element).

[0125] [Table 4-1]

[0126] [Table 4-2]

[0127] [Table 5]

[0128] Other non-limiting examples of promoters include the cytomegalovirus (CMV) promoter, the elongation factor 1-alpha (EF1a) promoter, the elongation factor (EFS) promoter, the MND promoter (a synthetic promoter containing the U3 region of a modified MoMuLV LTR with a myeloproliferative sarcoma virus enhancer), the phosphoglycerate kinase (PGK) promoter, the splenic fociforming virus (SFFV) promoter, the Simian virus 40 (SV40) promoter, and the ubiquitin C (UbC) promoter. In some embodiments, the promoter is a constitutive promoter. Exemplary constitutive promoters are shown in Table 5.

[0129] [Table 6-1]

[0130] [Table 6-2]

[0131] [Table 6-3]

[0132] [Table 6-4]

[0133] [Table 6-5]

[0134] In some embodiments, the promoter sequence is derived from a promoter selected from: minP, NFkB response element, CREB response element, NFAT response element, SRF response element 1, SRF response element 2, AP1 response element, TCF-LEF response element promoter fusion, hypoxia-responsive element, SMAD binding element, STAT3 binding site, minCMV, YB_TATA, minTK, inducible molecule-responsive promoter, and their tandem repeats.

[0135] In some embodiments, the first promoter is a constitutive promoter, an inducible promoter, or a synthetic promoter. In some embodiments, the constitutive promoter is selected from: CMV, EFS, SFFV, SV40, MND, PGK, UbC, hEF1aV1, hCAGG, hEF1aV2, hACTb, heIF4A1, hGAPDH, hGRP78, hGRP94, hHSP70, hKINb, and hUBIb.

[0136] In some embodiments, the ACP-responsive promoter is a synthetic promoter. In some embodiments, the ACP-responsive promoter includes a minimal promoter. In some embodiments, the ACP-binding domain includes one or more zinc finger binding sites. In some embodiments, the ACP-binding domain includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more zinc finger binding sites. In some embodiments, the ACP-binding domain includes one zinc finger binding site. In some embodiments, the ACP-binding domain includes two zinc finger binding sites. In some embodiments, the ACP-binding domain includes three zinc finger binding sites. In some embodiments, the ACP-binding domain includes four zinc finger binding sites. In some embodiments, the ACP-binding domain contains the polynucleotide sequence cgggtttcgtaacaatcgcatgaggattcgcaacgccttcGGCGTAGCCGATGTCGCGctcccgtctcagtaaaggtcGGCGTAGCCGATGTCGCGcaatcggactgccttcgtacGGCGTAGCCGATGTCGCGcgtatcagtcgcctcggaacGGCGTAGCCGATGTCGCGcattcgtaagaggctcactctcccttacacggagtggataACTAGTTCTAGAGGGTATATAATGGGGGCCA (SEQ ID NO: 100).

[0137] In some embodiments, the ACP-responsive promoter includes an enhancer that promotes transcription when the antigen-recognition receptor binds to a congeneral antigen, e.g., an antigen expressed on a target cell. The enhancer may include, but is not limited to, an enhancer enhanced in the ATAC sequence of activated T cells (referred herein by reference for all purposes, Gate et al. Nat Genet. Author manuscript; available in PMC 2019 Jan 9), or a cell-associated enhancer upregulated by single-cell RNA sequence data (referred herein by reference for all purposes, Xhangolli et al. Genomics Proteomics Bioinformatics. 2019 Apr;17(2):129-139. Doi: 10.1016 / j.gpb.2019.03.002). In some embodiments, the enhancer is or includes a synthetic enhancer. The synthetic enhancer may include, for example, multiple repeats of a transcription factor binding site. In some embodiments, the synthetic enhancer comprises one or more (e.g., one, two, three, four, five, or more) repeats of one or more distinct transcription factor binding sites. In some embodiments, the synthetic enhancer comprises four repeats of two distinct transcription factor binding sites in the configuration aaaabbbb or abababab. Exemplary, non-limiting examples of genes that can induce enhancers include, but are not limited to, ATF2, ATF7, BACH1, BATF, Bcl-6, Blimp-1, BMI1, CBFB, CREB1, CREM, CTCF, E2F1, EBF1, EGR1, ETV6, FOS, FOXA1, FOXA2, GATA3, HIF1A, IKZF1, IKZF2, IRF4, JUN, JUNB, JUND, Lef1, NFAT, NFIA, NFIB, NFKB, NR2F1, Nur77, PU.1, RELA, RUNX3, SCRT1, SCRT2, SP1, STAT4, STAT5A, T-Bet, Tcf7, ZBED1, ZNF143, or ZNF217.

[0138] Post-transfer regulatory elements In some embodiments, the manipulated nucleic acids of the Disclosure include post-transcriptional regulatory elements (PREs). In some embodiments, PREs can enhance gene expression by enabling tertiary RNA structure stability and 3' end formation. Non-limiting examples of PREs include hepatitis B virus PREs (HPREs) and woodchuck hepatitis virus PREs (WPREs). In some embodiments, the post-transcriptional regulatory element is a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). In some embodiments, the WPRE includes alpha, beta, and gamma components of the WPRE element. In some embodiments, the WPRE includes the alpha component of the WPRE element.

[0139] Manipulated cells Also provided herein are cells comprising one or more engineered nucleic acids of the Disclosure, and methods for producing such cells. Thus, the engineered cells provided typically comprise one or more engineered nucleic acids as described herein and do not occur naturally. In some embodiments, the engineered cells are isolated (e.g., engineered cells may be substantially isolated from other cell types comprising unengineered cells and / or other distinct engineered cells). In some embodiments, the engineered cells are isolated for a specific use (e.g., any use as described herein). In some embodiments, the engineered cells comprise one or more vectors comprising one or more engineered nucleic acids as described herein. In some embodiments, the engineered cells comprise one or more loci within their genome comprising one or more engineered nucleic acids as described herein. In some embodiments, the engineered cells comprise at least one promoter operably ligated to an exogenous polynucleotide sequence (e.g., a polynucleotide sequence encoding any engineered polypeptide as described herein).

[0140] In some embodiments, the engineered cells of the Disclosure may include engineered nucleic acids that are incorporated into the cell's genome. In some embodiments, the engineered cells include engineered nucleic acids that can be expressed without being incorporated into the cell's genome. In some embodiments, the engineered cells include a vector containing the engineered nucleic acids. In some embodiments, the engineered cells come into contact with the engineered nucleic acids (for example, in the form of mRNA), thereby causing the engineered nucleic acids to be expressed in the engineered cells.

[0141] Manipulated cell types In some embodiments, the manipulated cells or isolated cells of this disclosure are human cells. In some embodiments, the manipulated cells or isolated manipulated cells are human primary cells. In some embodiments, the primary cells are somatic cells. In some embodiments, the primary cells are stem cells. In some embodiments, the primary cells are induced pluripotent stem cells (iPSCs). In some embodiments, the manipulated cells are derived from a subject. In some embodiments, the manipulated cells are homogeneous with respect to a subject.

[0142] In some embodiments, the manipulated cells of this disclosure may be prepared using cells isolated from a subject. In some embodiments, the subject is known to have cancer or is suspected to have cancer. In some embodiments, the manipulated cells are prepared using cancer cells isolated from the subject. Methods of cell isolation are known to those skilled in the art and include, but are not limited to, positive isolation techniques such as sorting techniques based on cell surface marker expression, e.g., FACS sorting, and negative isolation, magnetic isolation, and combinations thereof. The manipulated cells may be allogeneic with respect to the subject to which the treatment is being administered. Allogeneic modified cells may be HLA-matched to the subject to which the treatment is being administered. The manipulated cells may be cultured cells, such as ex vivo cultured cells. The manipulated cells may be ex vivo cultured cells, such as primary cells isolated from a subject. Cultured cells may be cultured with one or more cytokines.

[0143] In some embodiments of the Disclosure, the manipulated or isolated cells are selected from: T cells (e.g., CD8+ T cells, CD4+ T cells, or gamma delta T cells), cytotoxic T lymphocytes (CTLs), regulatory T cells, natural killer T (NKT) cells, natural killer (NK) cells, B cells, tumor-infiltrating lymphocytes (TILs), innate lymphoid cells, mast cells, eosinophils, basophils, neutrophils, myeloid cells, macrophages (e.g., M1 macrophages, or M2 macrophages), monocytes, dendritic cells, erythrocytes, platelet cells, neurons, oligodendrocytes, astrocytes, placode-derived cells, Schwann cells, cardiomyocytes, endothelial cells, nodular cells, microglia, hepatocytes, cholangiocarcinomas, beta cells, human embryonic stem cells (ESCs), ESC-derived cells, pluripotent stem cells, mesenchymal stem cells (MSCs), induced pluripotent stem cells (iPSCs), and iPSC-derived cells.

[0144] In some embodiments, the manipulated or isolated cells of the Disclosure are T cells (e.g., CD8+ T cells, CD4+ T cells, or gamma delta T cells). In some embodiments, the manipulated or isolated cells of the Disclosure are cytotoxic T lymphocytes (CTLs). In some embodiments, the manipulated or isolated cells of the Disclosure are regulatory T cells. In some embodiments, the manipulated or isolated cells of the Disclosure are natural killer T (NTK) cells. In some embodiments, the manipulated or isolated cells of the Disclosure are natural killer (NK) cells. In some embodiments, the manipulated or isolated cells of the Disclosure are B cells. In some embodiments, the manipulated or isolated cells of the Disclosure are tumor-infiltrating lymphocytes (TILs). In some embodiments, the manipulated or isolated cells of the Disclosure are innate lymphoid cells. In some embodiments, the manipulated or isolated cells of the Disclosure are mast cells. In some embodiments, the manipulated or isolated cells of the Disclosure are eosinophils. In some embodiments, the manipulated or isolated cells of the Disclosure are basophils. In some embodiments, the manipulated or isolated cells of the Disclosure are neutrophils. In some embodiments, the manipulated or isolated cells of the Disclosure are myeloid cells. In some embodiments, the manipulated or isolated cells of the Disclosure are macrophages (e.g., M1 macrophages or M2 macrophages). In some embodiments, the manipulated or isolated cells of the Disclosure are monocytes. In some embodiments, the manipulated or isolated cells of the Disclosure are dendritic cells. In some embodiments, the manipulated or isolated cells of the Disclosure are erythrocytes. In some embodiments, the manipulated or isolated cells of the Disclosure are platelet cells. In some embodiments, the manipulated or isolated cells of the Disclosure are neurons. In some embodiments, the manipulated or isolated cells of the Disclosure are oligodendrocytes. In some embodiments, the manipulated or isolated cells of the Disclosure are astrocytes.In some embodiments, the manipulated or isolated cells of the Disclosure are Placode-derived cells. In some embodiments, the manipulated or isolated cells of the Disclosure are Schwann cells. In some embodiments, the manipulated or isolated cells of the Disclosure are cardiomyocytes. In some embodiments, the manipulated or isolated cells of the Disclosure are endothelial cells. In some embodiments, the manipulated or isolated cells of the Disclosure are nodular cells. In some embodiments, the manipulated or isolated cells of the Disclosure are microglia cells. In some embodiments, the manipulated or isolated cells of the Disclosure are hepatocytes. In some embodiments, the manipulated or isolated cells of the Disclosure are cholangiocarcinomas. In some embodiments, the manipulated or isolated cells of the Disclosure are beta cells. In some embodiments, the manipulated or isolated cells of the Disclosure are human embryonic stem cells (ESCs). In some embodiments, the manipulated or isolated cells of the Disclosure are ESC-derived cells. In some embodiments, the manipulated or isolated cells of the Disclosure are pluripotent stem cells. In some embodiments, the manipulated or isolated cells of the Disclosure are mesenchymal stem cells (MSCs). In some embodiments, the manipulated or isolated cells of the Disclosure are induced pluripotent stem cells (iPSCs). In some embodiments, the manipulated or isolated cells of the Disclosure are iPSC-derived cells. In some embodiments, the manipulated cells are autologous. In some embodiments, the manipulated cells are allogeneic. In some embodiments, the manipulated or isolated cells of the Disclosure are CD34+ cells, CD3+ cells, CD8+ cells, CD16+ cells, and / or CD4+ T cells.

[0145] In some embodiments, the manipulated cells of this disclosure are a cell therapy modality. In some embodiments, the cell therapy modality may express one or more therapeutic proteins. In some embodiments, the therapeutic proteins are chimeric antigen receptors (CARs) or T cell receptors (TCRs). In some embodiments, the cell therapy modality is CAR-T cells or CAR-NK cells. In some embodiments, the cell therapy modality is CAR-T cells. In some embodiments, the cell therapy modality is CAR-NK cells.

[0146] In some embodiments, the manipulated cells of the Disclosure are tumor cells. In some embodiments, the manipulated cells of the Disclosure are tumor cells selected from adenocarcinoma cells, bladder tumor cells, brain tumor cells, breast tumor cells, cervical tumor cells, colorectal tumor cells, esophageal tumor cells, glioma cells, kidney tumor cells, liver tumor cells, lung tumor cells, melanoma cells, mesothelioma cells, ovarian tumor cells, pancreatic tumor cells, prostate tumor cells, skin tumor cells, thyroid tumor cells, and uterine tumor cells.

[0147] In some embodiments, the manipulated cells of the Disclosure are bacterial cells. In some embodiments, the manipulated cells of the Disclosure are selected from: Clostridium beijerinckii, Clostridium sporogenes, Clostridium novyi, Escherichia coli, Pseudomonas aeruginosa, Listeria monocytogenes, Salmonella typhimurium, and Salmonella choleraesuis.

[0148] Furthermore, provided herein are methods comprising culturing the manipulated cells of the Disclosure. The manipulated cells provided may be cultured using any preferred method. Those skilled in the art will recognize that the culture conditions depend on the specific manipulated cells of interest. Those skilled in the art will also recognize that the culture conditions depend on the specific downstream use of the manipulated cells, for example, the specific culture conditions for subsequent administration of the manipulated cells to an interest.

[0149] Methods for manipulating cells Also provided herein are compositions and methods for manipulating cells having any nucleic acid described herein.

[0150] The manipulated cells provided may be manipulated via the introduction (i.e., delivery) of one or more polynucleotides (e.g., manipulated nucleic acids) of the present disclosure. Delivery methods include, but are not limited to, virus-mediated delivery, lipid-mediated transfection, nanoparticle delivery, electroporation, sonication, and cell membrane deformation by physical means. Those skilled in the art will understand that the choice of delivery method may depend on the specific cell type being manipulated.

[0151] In some embodiments, the manipulated cells are transduced using oncolytic viruses. Examples of oncolytic viruses include, but are not limited to, oncolytic herpes simplex virus, oncolytic adenovirus, oncolytic measles virus, oncolytic influenza virus, oncolytic Indianabecyclovirus, oncolytic Newcastle disease virus, oncolytic vaccinia virus, oncolytic poliovirus, oncolytic myxoma virus, oncolytic reovirus, oncolytic mumps virus, oncolytic maraba virus, oncolytic rabies virus, oncolytic rotavirus, oncolytic hepatitis virus, oncolytic rubella virus, oncolytic dengue virus, oncolytic chikungunya virus, oncolytic respiratory syncytial virus, oncolytic lymphocytic choriomeningitis virus, oncolytic morbillivirus, oncolytic lentivirus, oncolytic replication retrovirus, oncolytic rhabdovirus, oncolytic seneca-valley virus, oncolytic sindobisvirus, and any variants or derivatives thereof. In some embodiments, the oncolytic virus is a recombinant oncolytic virus. In some embodiments, the recombinant oncolytic virus comprises an engineered nucleic acid provided herein. In some embodiments, the oncolytic virus is a recombinant oncolytic virus comprising a first expression cassette and a second expression cassette. In some embodiments, the oncolytic virus further comprises a third expression cassette.

[0152] In some embodiments, the viruses provided pursuant to this disclosure are oncolytic viruses. In some embodiments, the viruses are recombinant viruses encoding one or more transgenes that encode one or more effector molecules, such as any of the engineered nucleic acids described herein. In some embodiments, cells are engineered via transduction with an oncolytic virus.

[0153] Virus-borne delivery The engineered cells of this disclosure can be prepared using any suitable viral vector-based delivery platform. Generally, viral vector-based delivery platforms engineer cells by introducing (i.e., delivering) a nucleic acid payload (e.g., one of the engineered nucleic acids described herein) into the host cell. The viral vector-based delivery platform may include an engineered virus or a recombinant virus for delivering the engineered nucleic acids of this disclosure to the cell.

[0154] A viral vector-based delivery platform can deliver one or more nucleic acid payloads, e.g., engineered nucleic acids, genes, or transgenes described herein, within the same nucleic acid. In some embodiments, a viral vector-based delivery platform (e.g., a platform using recombinant viruses or engineered viruses) can deliver one or more transgenes, including, but not limited to, any of the engineered nucleic acids described herein that encode one or more effector molecules. In some embodiments, one or more transgenes encoding one or more effector molecules may be configured to express one or more effector molecules. In addition to one or more transgenes (e.g., transgenes encoding one or more effector molecules), a viral vector-based delivery platform may encode one or more genes called cis-acting elements, or cis-acting genes, e.g., viral genes necessary for viral infectivity and / or virus production (e.g., capsid proteins, envelope proteins, viral polymerases, viral transcriptases, etc.).

[0155] A viral vector-based delivery platform may include two or more viral vectors, such as separate viral vectors encoding engineered nucleic acids, genes, or transgenes, as described herein and referred to as transactive elements or genes. For example, a helper-dependent viral vector-based delivery platform may provide additional genes required for viral infectivity and / or viral production on one or more additional separate vectors, in addition to a vector encoding one or more effector molecules. One viral vector may deliver two or more engineered nucleic acids, such as one vector that delivers engineered nucleic acids configured to produce two or more effector molecules. One or more viral vectors may deliver one or more engineered nucleic acids, such as one or more vectors that deliver one or more engineered nucleic acids configured to produce one or more effector molecules. The number of viral vectors used may depend on the packaging capacity of the viral vector-based vaccine platform mentioned above, but those skilled in the art can select an appropriate number of viral vectors.

[0156] In general, any viral vector system can be used for the in vitro production of molecules such as effector molecules, or for in vivo and ex vivo gene therapy procedures to deliver, for example, engineered nucleic acids encoding one or more effector molecules in vitro. The selection of an appropriate viral vector-based system depends on various factors, such as cargo / payload size, immunogenicity of the viral system, target cells, intensity and timing of gene expression, and other factors understood by those skilled in the art.

[0157] Viral vector-based delivery platforms may be RNA-based viruses or DNA-based viruses. Exemplary viral vector-based delivery platforms include, but are not limited to, herpes simplex virus, adenovirus, measles virus, influenza virus, Indiana beshiclovirus, Newcastle disease virus, vaccinia virus, poliovirus, myxoma virus, reovirus, mumps virus, maraba virus, rabies virus, rotavirus, hepatitis virus, rubella virus, dengue virus, chikungunya virus, respiratory syncytial virus, lymphocytic choriomeningitis virus, morbillivirus, lentivirus, replication retrovirus, rhabdovirus, seneca-valley virus, sindobis virus, and any variants or derivatives thereof.Other exemplary viral vector-based delivery platforms have been described in the Art and include, but are not limited to, vaccinia, fowlpox, self-replicating alphavirus, marabavirus, adenovirus (see, e.g., Tatsis et al., Adenoviruses, Molecular Therapy (2004) 10, 616-629), or lentiviruses, including second, third, or hybrid second / third generation lentiviruses and recombinant lentiviruses of any generation designed to target specific cell types or receptors (e.g., Hu et al., Immunization Delivered by Lentiviral Vectors for Cancer and Infectious Diseases, Immunol Rev. (2011) 239(1): 45-61, Sakuma et al., Lentiviral vectors: basic to translational, Biochem J. (2012) 443(3):603-18, Cooper et al., Rescue of splicing-mediated intron loss maximizes expression in lentiviral vectors). See also: containing the human ubiquitin C promoter, Nucl. Acids Res. (2015) 43 (1): 682-690; and Zufferey et al., Self-Inactivating Lentivirus Vector for Safe and Efficient In vivo Gene Delivery, J. Virol. (1998) 72 (12): 9873-9880).

[0158] This sequence may be preceded by one or more sequences that target intracellular compartments. Upon introduction (i.e., delivery) into host cells, infected cells (i.e., manipulated cells) may express and, in some cases, secrete one or more effector molecules. Useful vaccinia vectors and methods in immunization protocols are described, for example, in U.S. Patent No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin). The BCG vector is described in Stover et al. (Nature 351:456-460 (1991)). A wide variety of other vectors useful for the introduction (i.e., delivery) of manipulated nucleic acids, such as Salmonella phytiphimurium vectors and similar, will be apparent to those skilled in the art from the description herein.

[0159] The viral vector-based delivery platform can be a virus that targets tumor cells, and is referred to herein as an oncolytic virus. Examples of oncolytic viruses include, but are not limited to, oncolytic herpes simplex virus, oncolytic adenovirus, oncolytic measles virus, oncolytic influenza virus, oncolytic Indianabecyclovirus, oncolytic Newcastle disease virus, oncolytic vaccinia virus, oncolytic poliovirus, oncolytic myxoma virus, oncolytic reovirus, oncolytic mumps virus, oncolytic maraba virus, oncolytic rabies virus, oncolytic rotavirus, oncolytic hepatitis virus, oncolytic rubella virus, oncolytic dengue virus, oncolytic chikungunya virus, oncolytic respiratory syncytial virus, oncolytic lymphocytic choriomeningitis virus, oncolytic morbillivirus, oncolytic lentivirus, oncolytic replication retrovirus, oncolytic rhabdovirus, oncolytic seneca-valley virus, oncolytic sindobisvirus, and any variants or derivatives thereof. Any of the oncolytic viruses described herein may be recombinant oncolytic viruses comprising a recombinant oncolytic virus (recommended oncolytic virus) that includes another transgene (e.g., an engineered nucleic acid) encoding one or more effector molecules. The transgene encoding one or more effector molecules may be configured to express one or more effector molecules.

[0160] In some embodiments, the virus is selected from lentiviruses, retroviruses, oncolytic viruses, adenoviruses, adeno-associated viruses (AAVs), and virus-like particles (VLPs).

[0161] Viral vector-based delivery platforms can be retroviral-based. Generally, retroviral vectors consist of cis-acting long terminal repeats with packaging ability for exogenous sequences up to 6–10 kb. The smallest cis-acting LTR is sufficient for vector replication and packaging, and is then used to incorporate one or more manipulated nucleic acids (e.g., a transgene encoding one or more effector molecules) into target cells to provide persistent transgene expression. Retroviral delivery systems include, but are not limited to, delivery systems based on mouse leukemia virus (MuLV), gibbon leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, for example, Buchscher et al., J. Virol. 66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992); Sommnerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol. 63:2374-2378 (1989); Miller et al, J, Virol. 65:2220-2224 (1991); PCT / US94 / 05700). Other retroviruses include the Phoenix retrovirus family.

[0162] Viral vector-based delivery platforms can be lentivirus-based. Generally, lentiviral vectors are retroviral vectors that can transduce or infect non-dividing cells and typically produce high viral titers. Lentivirus-based delivery platforms can be HIV-based, such as the ViraPower system (ThermoFisher) or the pLenti system (Cell Biolabs). Lentivirus-based delivery platforms can also be SIV or FIV-based. Other exemplary lentivirus-based delivery platforms are described in more detail in U.S. Patents Nos. 7,311,907; 7,262,049; 7,250,299; 7,226,780; 7,220,578; 7,211,247; 7,160,721; 7,078,031; 7,070,993; 7,056,699; and 6,955,919, each incorporated herein by reference for all purposes.

[0163] Viral vector-based delivery platforms can be adenovirus-based. Generally, adenovirus-based vectors allow for very high transduction efficiency in many cell types, do not require cell division, achieve high titers and expression levels, and can be produced in large quantities in relatively simple systems. Generally, adenoviruses can be used for transient expression of transgenes within infected cells because adenoviruses are typically not integrated into the host genome. Adenovirus-based delivery platforms are described in more detail in Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; International Publication Nos. 94 / 12649, 93 / 03769, 93 / 19191, 94 / 28938, 95 / 11984, and 95 / 00655, each incorporated herein by reference for all purposes. Other exemplary adenovirus-based delivery platforms are described in more detail in U.S. Patents Nos. 5,585,362; 6,083,716, 7,371,570; 7,348,178; 7,323,177; 7,319,033; 7,318,919; and 7,306,793, as well as in International Patent Application No. 96 / 13597, each incorporated herein by reference for all purposes.

[0164] Viral vector-based delivery platforms may be adeno-associated virus (AAV) based. Adeno-associated virus ("AAV") vectors, with engineered nucleic acids (e.g., any of the engineered nucleic acids described herein), may be used to transduce cells. The AAV system can be used for the in vitro production of effector molecules, or for in vivo delivery of engineered nucleic acids encoding one or more effector molecules in in vivo and ex vivo gene therapy procedures (e.g., West et al., Virology 160:38-47 (1987); U.S. Patent Nos. 4,797,368; 5,436,146; 6,632,670; 6,642,051; 7,078,387; 7,314,912; 6,498,244; 7,906,111; U.S. Patent Publications US 2003-0138772, US 2007 / 0036760, and US 2009 / 0197338; Gao, et al., J. Virol, 78(12):6381-6388 (June 2004); Gao, et al, Proc Natl Acad Sci USA, 100(10):6081-6086 (May 13, 2003); and international patent applications WO 2010 / 138263 and WO 93 / 24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994), each incorporated herein by reference for all purposes. Exemplary methods for constructing recombinant AAV vectors are described in more detail in U.S. Patent No. 5,173,414; Tratschin et ah, Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et ah, Mol. Cell, Biol. 4:2072-2081 (1984), Hermonat & Muzyczka, PNAS 81:64666470 (1984), and Samuiski et ah, J. Virol. 63:03822-3828 (1989), each of which is incorporated herein by reference for all purposes.Generally, AAV-based vectors contain a capsid protein having an amino acid sequence corresponding to any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV.Rh10, AAV11, or their variants.

[0165] Viral vector-based delivery platforms can be virus-like particle (VLP) platforms. Generally, VLPs are constructed by producing viral structural proteins and purifying the resulting viral particles. After purification, the cargo / payload (e.g., one of the engineered nucleic acids described herein) is then ex vivo encapsulated within the purified particles. Thus, VLP production maintains the separation of the nucleic acid encoding the viral structural protein and the nucleic acid encoding the cargo / payload. The viral structural proteins used in VLP production can be produced in a variety of expression systems, including mammalian, yeast, insect, bacterial, or in vivo translational expression systems. The purified viral particles can be denatured and reformed in the presence of the desired cargo using any preferred method to produce VLPs. VLP production is described in more detail in Seow et al. (Mol Ther. 2009 May;17(5):767-777), which is incorporated herein by reference for all purposes.

[0166] Viral vector-based delivery platforms can be manipulated to target (i.e., infect) a range of cells, a narrow subset of cells, or specific cells. Generally, the tropism of the virus will be determined by the envelope protein chosen for the viral vector-based delivery platform. The virus used in a viral vector-based delivery platform may be pseudotyped to target specific cells of interest. A viral vector-based delivery platform may be panaffinity, capable of infecting a range of cells. For example, a panaffinity viral vector-based delivery platform may contain the VSV-G envelope. A viral vector-based delivery platform may be amphoteric, capable of infecting mammalian cells. Therefore, those skilled in the art can select the appropriate tropism, pseudotype, and / or envelope protein to target the desired cell type.

[0167] Lipid structure delivery system The engineered nucleic acids described herein (e.g., any of the engineered nucleic acids described herein) may be introduced into cells using lipid-mediated delivery systems. Generally, lipid-mediated delivery systems use structures consisting of an outer lipid membrane enclosing an internal compartment. Examples of lipid-based structures include, but are not limited to, lipid-based nanoparticles, liposomes, micelles, exosomes, vesicles, extracellular vesicles, cells, or tissues. Lipid structure delivery systems may deliver cargo / payloads (e.g., any of the engineered nucleic acids described herein) in vitro, in vivo, or ex vivo.

[0168] Lipid-based nanoparticles may include, but are not limited to, monolayer liposomes, multilayer liposomes, and lipid preparations. As used herein, “liposome” is a general term encompassing in vitro preparations of lipid vehicles formed by encapsulating a desired cargo, e.g., engineered nucleic acids, e.g., any of the engineered nucleic acids described herein, within a lipid shell or lipid aggregate. Liposomes may generally be characterized as having a vesicle structure with a bilayer membrane containing phospholipids, and an internal medium generally containing an aqueous composition. Liposomes may include, but are not limited to, emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, layered layers, and the like. Liposomes may be monolayer liposomes. Liposomes may be multilayer liposomes. Liposomes may be multilocular liposomes. Liposomes may be positively charged, negatively charged, or neutrally charged. In certain embodiments, liposomes are neutrally charged. Liposomes can generally be formed from standard vesicle-forming lipids, including neutral and negatively charged phospholipids, as well as sterols, such as cholesterol. Lipid selection is generally guided by criteria for in vivo delivery, such as liposome size, acid instability, and liposome stability in the bloodstream, taking into account the desired purpose. A variety of methods are available for preparing liposomes, as described, for example, in Szokan et al., Ann. Rev. Biophys. Bioeng. 9;467 (1980), U.S. Patents No. 4,235,871, 4,501,728, 4,501,728, 4,837,028, and 5,019,369, each incorporated herein by reference for all purposes.

[0169] Multilayer liposomes are spontaneously formed when lipids containing phospholipids are suspended in an excess aqueous solution, such that multiple lipid layers are separated by an aqueous medium. The water and dissolved solute are encapsulated in a closed structure between the lipid bilayers after the self-reconstitution of the lipid components. The desired cargo (e.g., polypeptides, nucleic acids, small molecule drugs, engineered nucleic acids, such as any of the engineered nucleic acids described herein, viral vectors, virus-based delivery systems, etc.) can be encapsulated within the aqueous interior of the liposome, attached to the liposome via linking molecules that associate with both the liposome and the polypeptide / nucleic acid, scattered within the lipid bilayer of the liposome, encapsulated within the liposome, complexed with the liposome, or otherwise associated with the liposome, so that it can be delivered to the target entity. Lipophilic molecules, or molecules with lipophilic regions, can also dissolve in or associate with the lipid bilayer.

[0170] Liposomes used according to this embodiment may be prepared using any preferred method. The preparation of liposomes is described in more detail in WO2016 / 201323, International Applications PCT / US85 / 01161 and PCT / US89 / 05040, and U.S. Patents 4,728,578, 4,728,575, 4,737,323, 4,533,254, 4,162,282, 4,310,505 and 4,921,706, each incorporated herein by reference for all purposes.

[0171] Liposomes may be cationic liposomes. Examples of cationic liposomes are described in more detail in U.S. Patents 5,962,016; 5,030,453; 6,680,068, U.S. Patent Application 2004 / 0208921, and International Patent Applications WO03 / 015757A1, WO04029213A2, and WO02 / 100435A1, each incorporated herein by reference in whole.

[0172] Lipid-mediated gene delivery methods are described, for example, in WO96 / 18372, WO93 / 24640, Mannino & Gould-Fogerite, BioTechniques 6(7): 682-691 (1988); U.S. Patent No. 5,279,833, Rose Patent No. 5,279,833, International Publication No. 91 / 06309, and Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7414 (1987), each incorporated herein by reference for all purposes.

[0173] Exosomes are small membrane vesicles of endocytosis origin that are released into the extracellular environment after fusion of the polyendoplasmic reticulum with the plasma membrane. Exosomes range in size from 30 to 100 nm in diameter. Their surface consists of a lipid bilayer from the donor cell membrane, they contain cytosol from the exosome-producing cell, and exhibit membrane proteins from the parent cell on their surface. Exosomes useful for nucleic acid delivery may be used in this disclosure, for example, those described in more detail in U.S. Patent No. 9,889,210, which are incorporated herein by reference for all purposes.

[0174] As used herein, the terms “extracellular vesicle” or “EV” refer to a cell-derived vesicle containing a membrane that encloses an internal space. Generally, extracellular vesicles include all membrane-bound vesicles having a diameter smaller than the cell from which they originate. Generally, extracellular vesicles range in diameter from 20 nm to 1000 nm and may contain a variety of macromolecular cargoes, either within the internal space presented on the outer surface of the extracellular vesicle and / or across the membrane. Cargoes may include nucleic acids (e.g., any of the engineered nucleic acids described herein), proteins, carbohydrates, lipids, small molecules, and / or combinations thereof. As an example, but not limited to, extracellular vesicles include apoptotic bodies, cell fragments, vesicles derived from cells by direct or indirect manipulation (e.g., serial extrusion or treatment with alkaline solution), vesicled organelles, and vesicles produced by living cells (e.g., direct plasma membrane budding or fusion of late endosomes with the plasma membrane). Extracellular vesicles can originate from living or dead organisms, explanted tissues or organs, and / or cultured cells.

[0175] As used herein, the term “exosome” refers to a small, cell-derived vesicle (20–300 nm in diameter, more preferably between 40–200 nm in diameter) that includes a membrane surrounding its internal space and is produced from a cell by direct plasma membrane budding or by fusion of late endosomes with the plasma membrane. Exosomes contain lipids, or fatty acids and polypeptides, and optionally include a payload (e.g., a therapeutic agent), a receiver (e.g., a targeting portion), polynucleotides (e.g., nucleic acids, RNA, or DNA, such as any of the engineered nucleic acids described herein), sugars (e.g., monosaccharides, polysaccharides, or glycans), or other molecules. Exosomes originate from producing cells and can be isolated from producing cells based on their size, density, biochemical parameters, or combinations thereof. Exosomes are a type of extracellular vesicle. Generally, exosome production / biosynthesis does not result in the destruction of the producing cell. Exosomes and the preparation of exosomes are described in further detail in WO2016 / 201323, which is incorporated herein by reference in its entirety.

[0176] As used herein, the term “nanopes” (also referred to as “microvesicles”) refers to small, cell-derived vesicles (20–250 nm in diameter, more preferably between 30–150 nm in diameter) that include a membrane surrounding their internal space and are produced by a cell through direct or indirect manipulation, thereby preventing the nanovesicles from being produced by the producing cell without such manipulation. Generally, nanovesicles are a subspecies of extracellular vesicles. Appropriate manipulation of the producing cell includes, but is not limited to, serial extrusion, treatment with alkaline solutions, sonication, or a combination thereof. Nanovesicle production may, in some cases, result in the destruction of the producing cell. Preferably, the nanovesicle population is substantially free of vesicles originating from the producing cell by direct budding from the plasma membrane or fusion of late endosomes with the plasma membrane. The nanovesicles comprise lipids, or fatty acids and polypeptides, and optionally include a payload (e.g., a therapeutic agent), a receiver (e.g., a targeting moiety), polynucleotides (e.g., nucleic acids, RNA, or DNA, such as any of the manipulated nucleic acids described herein), sugars (e.g., monosaccharides, polysaccharides, or glycans), or other molecules. Once the nanovesicles are induced from the producing cells according to the aforementioned procedure, they can be isolated from the producing cells based on their size, density, biochemical parameters, or a combination thereof.

[0177] Lipid nanoparticles (LNPs) are generally synthetic lipid structures that form membranes and vesicle-like structures, relying on the amphiphilic nature of lipids (Riley 2017). These vesicles typically deliver cargo / payloads, such as the manipulated nucleic acids or viral systems described herein, by being absorbed into the membrane of target cells and releasing the cargo into the cytosol. Lipids used in LNP formation can be cationic, anionic, or neutral. Lipids can be synthetic or naturally occurring and, in some cases, biodegradable. Lipids can include fats, cholesterol, phospholipids, lipid conjugates (including, but not limited to, polyethylene glycol (PEG) conjugates (PEGylated lipids)), waxes, oils, glycerides, and fat-soluble vitamins. Lipid compositions generally include mixtures of defined materials, such as cationic lipids, neutral lipids, anionic lipids, and amphiphilic lipids. In some cases, specific lipids are included to provide functional chemical groups that prevent LNP aggregation, prevent lipid oxidation, or promote the attachment of additional moieties. The lipid composition can affect the overall LNP size and stability. In one example, the lipid composition comprises dilinoleylmethyl-4-dimethylaminobutyrate (MC3) or an MC3-like molecule. MC3 and MC3-like lipid compositions can be formulated to include one or more other lipids, such as PEG or PEG-conjugate lipids, sterols, or neutral lipids. LNPs can also be further manipulated or functionalized to facilitate targeting of specific cell types. Another consideration in LNP design is the balance between targeting efficiency and cytotoxicity.

[0178] A micelle is generally a spherical synthetic lipid structure formed using single-chain lipids, where the hydrophilic head of the single-chain lipid forms the outer layer or membrane, and the hydrophobic tail of the single-chain lipid forms the micelle center. Typically, a micelle refers to a lipid structure containing only a lipid monolayer. Micelles are described in detail by Quader et al. (Mol Ther. 2017 Jul 5;25(7):1501-1513), which are incorporated herein by reference for all purposes.

[0179] Nucleic acid vectors directly exposed to serum, such as expression vectors, can have several undesirable consequences, including degradation of the nucleic acid by serum nucleases or off-target stimulation of the immune system by free nucleic acids. Similarly, viral delivery systems directly exposed to serum can induce undesirable immune responses and / or neutralization of the viral delivery system. Therefore, encapsulation of engineered nucleic acids and / or viral delivery systems can be used to avoid degradation while also avoiding potential off-target effects. In certain embodiments, the engineered nucleic acids and / or viral delivery systems are completely encapsulated within a delivery vehicle, for example, within the aqueous interior of an LNP. Encapsulation of engineered nucleic acids and / or viral delivery systems within an LNP can be carried out using any preferred method, such as microfluidic mixing and droplet generation performed on a microfluidic droplet generation device. Such devices include, but are not limited to, standard T-junction devices or flow focusing devices. In one embodiment, a desired lipid formulation, such as MC3 or an MC3-like composition, is supplied to a droplet-generating device in parallel with a processed nucleic acid or viral delivery system and any other desired drug, such that the delivery vector and the desired drug are completely encapsulated within the MC3 or MC3-like LNP. In one embodiment, the droplet-generating device can control the size range and size distribution of the produced LNPs. For example, the LNPs may have sizes in the range of 1 to 1000 nanometers in diameter, e.g., 1, 10, 50, 100, 500, or 1000 nanometers. After droplet generation, the delivery vehicle (e.g., processed nucleic acid and / or viral delivery system) encapsulating the cargo / payload may be further processed or manipulated to prepare them for administration.

[0180] Nanoparticle delivery Nanomaterials can be used to deliver engineered nucleic acids (e.g., any of the engineered nucleic acids described herein). Importantly, the nanomaterial vehicle can be fabricated from non-immunogenic materials and generally avoid inducing immunity to its delivery vector itself. These materials may include, but are not limited to, lipids (as previously described), inorganic nanomaterials, and other polymer materials. Nanomaterial particles are described in more detail in Riley et al. (Recent Advances in Nanomaterials for Gene Delivery - A Review. Nanomaterials 2017, 7(5), 94), which is incorporated herein by reference for all purposes.

[0181] Genome editing systems Genome editing systems can be used to manipulate the host genome to encode an engineered nucleic acid, for example, the engineered nucleic acid described herein. Generally, “genome editing system” refers to any system for incorporating an exogenous gene into the genome of a host cell. Genome editing systems include, but are not limited to, transposon systems, nuclease genome editing systems, and viral vector-based delivery platforms.

[0182] Transposon systems can be used to incorporate engineered nucleic acids, such as those disclosed herein, into the host genome. Transposons typically consist of a cargo / payload nucleic acid and a terminal inversion repeat (TIR) ​​adjacent to the transposase. Transposon systems can provide transposons in cis or trans orientation, accompanied by a TIR-adjacent cargo. Transposon systems can be retrotransposon systems or DNA transposon systems. Generally, transposon systems randomly incorporate the cargo / payload (e.g., engineered nucleic acid) into the host genome. Examples of transposon systems include systems using transposons from the Tc1 / mariner transposon superfamily, such as the Sleeping Beauty transposon system, which are described in more detail in Hudecek et al. (Crit Rev Biochem Mol Biol. 2017 Aug;52(4):355-380) and U.S. Patents 6,489,458, 6,613,752, and 7,985,739, each of which is incorporated herein by reference for all purposes. Another example of a transposon system is the PiggyBac transposon system, which is described in more detail in U.S. Patents 6,218,185 and 6,962,810, each of which is incorporated herein by reference for all purposes.

[0183] Nuclease-mediated gene editing systems can be used to manipulate the host genome to encode an engineered nucleic acid, for example, the engineered nucleic acid described herein. While we do not wish to be constrained by theory, nuclease-mediated gene editing systems, generally used to introduce exogenous genes, utilize the cell's innate DNA repair mechanisms, particularly the homologous recombination (HR) repair pathway. Simply put, following damage to genomic DNA (typically a double-strand break), the cell may resolve the damage by using another DNA source having identical or substantially identical sequences at both its 5' and 3' ends as a template during DNA synthesis to repair the damage. In natural contexts, HDRs may use other chromosomes present in the cell as templates. In gene editing systems, an exogenous polynucleotide is introduced into the cell and used as a homologous recombination template (HRT, or HR template). In general, any additional exogenous sequences not originally found in the chromosome, involving damage between the 5' and 3' complementary ends of an HRT (e.g., a gene or a portion of a gene), can be incorporated (i.e., "integrated") into a given genomic locus during a templated HDR. Thus, a typical HR template for a given genomic locus has a nucleotide sequence identical to the first region of the endogenous genomic target locus, a nucleotide sequence identical to the second region of the endogenous genomic target locus, and a nucleotide sequence encoding a cargo / payload nucleic acid (e.g., any of the engineered nucleic acids described herein, e.g., any of the engineered nucleic acids encoding one or more effector molecules).

[0184] In some embodiments, the HR template may be linear. Examples of linear HR templates include, but are not limited to, linear plasmid vectors, ssDNA, synthetic DNA, and PCR-amplified DNA. In certain embodiments, the HR template may be circular, for example, a plasmid. Circular templates may include supercoiled templates.

[0185] Identical or substantially identical sequences found at the 5' and 3' ends of the HR template are generally referred to as arms (HR arms) with respect to the introduced exogenous sequence. HR arms can be identical (i.e., 100% identical) to the region of the endogenous genomic target locus. In some embodiments, HR arms can be substantially identical to the region of the endogenous genomic target locus. While substantially identical HR arms can be used, it may be advantageous for HR arms to be identical, as the efficiency of the HDR pathway may be affected by HR arms with less than 100% identity.

[0186] Each HR arm, i.e., the 5' and 3' HR arms, may be the same size or different sizes. Each HR arm may be longer than or equal to 50, 100, 200, 300, 400, or 500 bases. While HR arms can generally be of any length, practical considerations such as the effect of HR arm length on overall editing efficiency and the overall template size may also be taken into account. HR arms may be identical or substantially identical to a region of an endogenous genomic target locus directly adjacent to a cleavage site. Each HR arm may be identical or substantially identical to a region of an endogenous genomic target locus directly adjacent to a cleavage site. Each HR arm may be identical or substantially identical to a region of an endogenous genomic target locus within a specific distance from a cleavage site, e.g., 1 base pair, 10 base pairs or less, 50 base pairs or less, or 100 base pairs or less.

[0187] Nuclease genome editing systems can cleave target genomic loci using a variety of nucleases, including, but not limited to, clustered, regularly spaced, short-interval palindromic repeat (CRISPR) family nucleases or derivatives thereof, transcriptional activator-like effector nucleases (TALENs) or derivatives thereof, zinc finger nucleases (ZFNs) or derivatives thereof, and homing endonucleases (HEs) or derivatives thereof.

[0188] CRISPR-mediated gene editing systems can be used to manipulate the host genome to encode manipulated nucleic acids, such as those encoding one or more of the effector molecules described herein. The CRISPR system is described in more detail in M. Adli ("The CRISPR tool kit for genome editing and beyond," Nature Communications; volume 9 (2018), Article number: 1911), all of which are incorporated herein by reference. Generally, a CRISPR-mediated gene editing system comprises a CRISPR-associated (Cas) nuclease and RNA that directs cleavage to a specific target sequence. An exemplary CRISPR-mediated gene editing system is the CRISPR / Cas9 system, which comprises a Cas9 nuclease and RNA having a CRISPR RNA (crRNA) domain and a transactivating CRISPR (tracrRNA) domain. crRNA typically has two RNA domains: a guide RNA (gRNA) that directs specificity to a target sequence ("defined nucleotide sequence"), such as a genomic sequence, through base-pair hybridization, and an RNA domain that hybridizes to tracrRNA. TracrRNA can interact with nucleases (e.g., Cas9) to facilitate recruitment to genomic loci. crRNA and tracrRNA polynucleotides can be distinct polynucleotides. crRNA and tracrRNA polynucleotides may also be single polynucleotides and are referred to as single guide RNAs (sgRNAs). While the Cas9 system is shown herein, other CRISPR systems can be used, such as the Cpf1 system. Nucleases may include their derivatives, such as Cas9 functional mutants, for example, Cas9 "nickase" mutants that generally mediate single-strand breaks of a defined nucleotide sequence, in contrast to the complete double-strand breaks typically produced by the Cas9 enzyme.

[0189] Generally, components of a CRISPR system interact with each other to form ribonucleoprotein (RNP) complexes, which mediate sequence-specific cleavage. In some CRISPR systems, each component can be produced separately and used to form the RNP complex. In some CRISPR systems, each component can be produced separately in vitro and brought into contact with each other in vitro (i.e., "complexed") to form the RNP complex. The RNP produced in vitro can then be introduced (i.e., "delivered") into the cytosol and / or nucleus of a cell, for example, the cytosol and / or nucleus of a T cell. The RNP complex produced in vitro can be delivered to cells by a variety of means, but are not limited to electroporation, lipid-mediated transfection, physical cell membrane deformation, lipid nanoparticles (LNPs), virus-like particles (VLPs), and sonication. In certain cases, RNP complexes produced in vitro can be delivered to cells using a Nucleofactor / Nucleofection® electroporation-based delivery system (Lonza®). Other electroporation systems include, but are not limited to, the MaxCyte electroporation system, the Miltenyi CliniMACS electroporation system, the Neon electroporation system, and the BTX electroporation system. CRISPR nucleases, such as Cas9, can be produced in vitro (i.e., synthesized and purified) using any preferred method. CRISPR RNAs, such as sgRNA, can be produced in vitro (i.e., synthesized and purified) using various RNA production techniques known to those skilled in the art, such as in vitro transcription or chemical synthesis.

[0190] RNP complexes produced in vitro can be complexed with nucleases to gRNAs in different ratios. RNP complexes produced in vitro can also be used in different amounts in CRISPR-mediated editing systems. For example, the total amount of RNP added can be adjusted depending on the number of cells to be edited, such as reducing the amount of RNP complex added when editing a large number of cells in the reaction.

[0191] In some CRISPR systems, each component (e.g., Cas9 and sgRNA) can be individually encoded by a polynucleotide, and each polynucleotide can be introduced into the cell together or individually. In some CRISPR systems, each component can be encoded by a single polynucleotide (i.e., a multi-promoter or multi-cistronic vector; see the description of exemplary multi-cistronic systems below) and introduced into the cell. Following the expression of the CRISPR component encoded by each polynucleotide in the cell (e.g., translation of the nuclease and transcription of the CRISPR RNA), an RNP complex can be formed in the cell and then directed for site-specific cleavage.

[0192] Some RNPs can be engineered to have a portion that facilitates the delivery of RNPs into the nucleus. For example, Cas9 nucleases may have a nuclear localization signaling (NLS) domain, and if the Cas9 RNP complex is delivered into the cell cytosol, or after Cas9 translation and subsequent RNP formation, the NLS can facilitate further transport of Cas9 RNPs into the nucleus.

[0193] The manipulated cells described herein can be manipulated using nonviral methods, for example, the nucleases and / or CRISPR-mediated gene editing systems described herein can be delivered to the cells using nonviral methods. The manipulated cells described herein can be manipulated using viral methods, for example, the nucleases and / or CRISPR-mediated gene editing systems described herein can be delivered to the cells using any of the viral methods, such as adenovirus, retrovirus, lentivirus, or other virus-based delivery methods described herein.

[0194] Some CRISPR systems can provide two or more CRISPR compositions, each independently targeting the same gene or common genomic locus with multiple target nucleotide sequences. For example, two separate CRISPR compositions can be provided and directed to cleave at two different target nucleotide sequences within a specific distance from each other. Some CRISPR systems can provide two or more CRISPR compositions, each independently targeting the reverse strand of the same gene or common genomic locus. For example, two separate CRISPR "nickase" compositions can be provided and directed to cleave at the same gene or common genomic locus in the reverse strand.

[0195] In general, the characteristics of the CRISPR-mediated editing systems described herein can be applied to other nuclease-based genome editing systems. A TALEN is an engineered site-specific nuclease, which consists of a DNA-binding domain of a TALE (transmission activator-like effector) and a catalytic domain of the restriction endonuclease Fokl. Different artificial TALENs can be generated by altering the amino acids present in the highly variable residue region of the monomer of the DNA-binding domain, thereby targeting various nucleotide sequences. The DNA-binding domain then directs the nuclease to the target sequence, creating a double-strand break. TALEN-based systems are described in more detail in U.S. Patent Nos. 12 / 965,590; 8,450,471; 8,440,431; 8,440,432; 10,172,880; and 13 / 738,381, all of which are incorporated herein by reference in their entirety. The ZFN-based editorial systems are described in more detail in U.S. Patent Nos. 6,453,242; 6,534,261; 6,599,692; 6,503,717; 6,689,558; 7,030,215; 6,794,136; 7,067,317; 7,262,054; 7,070,934; 7,361,635; 7,253,273; and U.S. Patent Publications 2005 / 0064474; 2007 / 0218528; and 2005 / 0267061, all of which are incorporated herein by reference in their entirety for all purposes.

[0196] Other operational delivery systems A variety of additional means are available to those skilled in the art for introducing engineered nucleic acids (e.g., any of the engineered nucleic acids described herein) into cells or other target recipient entities, such as any of the lipid structures described herein. Those skilled in the art will understand which method is suitable for introducing engineered nucleic acids into cells or other biological systems.

[0197] In some embodiments, electroporation is used to deliver polynucleotides (e.g., any engineered nucleic acids described herein) to a recipient entity. Electroporation is a method of internalizing cargo / payload into the internal compartment of a target cell or entity by applying an electric field to transiently permeate the outer membrane or shell of the target cell or entity. Generally, the method involves placing the cell or target entity between two electrodes in a solution containing the cargo of interest (e.g., any of the engineered nucleic acids described herein). The lipid membrane of the cell is then broken, i.e., made permeable, by applying a transient set voltage, thereby allowing the cargo to enter the interior of the entity, such as the cytoplasm of the cell. In the case of cells, at least some, but not the majority, of the cell remain viable. Cells and other entities can be electroporated in vitro, in vivo, or ex vivo. Electroporation conditions (e.g., cell number, cargo concentration, recovery conditions, voltage, time, capacitance, pulse type, pulse length, volume, cuvette length, electroporation solution composition, etc.) vary depending on several factors, including, but are not limited to, the type of cells or other recipient entities, the cargo delivered, the desired internalization efficiency, and the desired viability. Optimization of such criteria is within the scope of the art of the art. Various devices and protocols can be used for electroporation. Examples, but are not limited to, the Neon® transfection system, MaxCyte® Flow Electroporation®, Lonza® Nucleofector® system, and Bio-Rad® electroporation system.

[0198] Other means for introducing the manipulated nucleic acid (e.g., any of the manipulated nucleic acids described herein) into cells or other target recipient entities include, but are not limited to, sonication, gene guns, hydrodynamic injection, and cell membrane deformation by physical means.

[0199] Compositions and methods for in vivo delivery of manipulated mRNA, such as naked plasmids or mRNA, are described in detail in Kowalski et al. (Mol Ther. 2019 Apr 10;27(4):710-728) and Kaczmarek et al. (Genome Med. 2017;9: 60), each of which is incorporated herein by reference for all purposes.

[0200] How to use Methods for treating diseases are also encompassed by this disclosure. Such methods include administering a therapeutically effective dose of engineered nucleic acids, engineered cells, or isolated cells, as described herein. In some embodiments, what is provided herein is a method for treating a subject in need thereof, and the method includes administering a therapeutically effective dose of any of the engineered cells, isolated cells, or compositions disclosed herein.

[0201] In some embodiments, this disclosure provides a method for treating a subject having a disease or disorder by administering to the subject an engineered nucleic acid, engineered polypeptide, or engineered cell (e.g., isolated engineered cell) as described herein. Those skilled in the art will understand that the engineered nucleic acid may be delivered to the subject using any suitable delivery system, e.g., any delivery system described herein. In some embodiments, the subject is administered the engineered nucleic acid (or a delivery system that delivers the engineered nucleic acid to the subject, e.g., engineered cell), and IMiD. In some embodiments, the administration of the engineered nucleic acid and IMiD is sequential. In some embodiments, the administration of the engineered nucleic acid and IMiD is simultaneous. In some embodiments, the administration of the engineered nucleic acid and IMiD is via different routes. In some embodiments, IMiD is an FDA-approved drug. In some embodiments, IMiD is thalidomide. In some embodiments, IMiD is a variant or derivative of thalidomide. In some embodiments, IMiD is selected from the group consisting of thalidomide, iverdide, lenalidomide, and pomalidomide. In some embodiments, IMiD is pomalidomide.

[0202] In vivo expression The methods provided herein also include delivering in vivo a composition capable of producing the manipulated cells described herein, for example, capable of delivering any of the manipulated nucleic acids described herein to cells in vivo. Such a composition includes any of the virus-mediated delivery platforms, any of the lipid structure delivery systems, any of the nanoparticle delivery systems, any of the genome editing systems, or any of the other manipulated delivery systems described herein that are capable of manipulating cells in vivo.

[0203] The methods provided herein also include delivering in vivo a composition capable of producing any of the effector molecules described herein. The methods provided herein also include delivering in vivo a composition capable of producing two or more of the effector molecules described herein. The compositions capable of in vivo production of effector molecules include, but are not limited to, any of the manipulated nucleic acids described herein. The compositions capable of in vivo production of effector molecules may be naked mRNA or naked plasmids.

[0204] Pharmaceutical composition The engineered nucleic acids or engineered cells provided herein may be formulated into pharmaceutical compositions. These compositions may contain, in addition to one or more engineered nucleic acids or engineered cells, pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other materials suitable for pharmaceutical formulation. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The exact properties of the carrier or other materials may depend on the route of administration, e.g., oral, intravenous, cutaneous, or subcutaneous, nasal, intramuscular, or intraperitoneal routes.

[0205] Regardless of whether what is administered to an individual is a cell, polypeptide, nucleic acid, small molecule, or other pharmaceutically useful compound as disclosed herein, the administration is preferably a “therapeutic effective dose” or a “preventive effective dose” (in some cases, prevention may be considered treatment), which is sufficient to demonstrate benefit to the individual. The actual amount administered, as well as the rate of administration and the course of time, will depend on the nature and severity of the protein aggregation disorder being treated. The prescription of treatment, e.g., the determination of the dosage, is the responsibility of the general practitioner and other physicians, typically taking into account the disorder to be treated, the individual patient’s condition, the site of delivery, the method of administration, and other factors known to the practitioner. Examples of the above techniques and protocols can be found in Remington’s Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.

[0206] Depending on the condition to be treated, the composition may be administered alone, in combination with other treatments, concurrently, or sequentially.

[0207] [Table 7]

[0208] [Table 8]

[0209] [Table 9-1]

[0210] [Table 9-2]

[0211] [Table 9-3]

[0212] [Table 9-4]

[0213] [Table 9-5]

[0214] [Table 9-6]

[0215] [Table 9-7]

[0216] [Table 9-8]

[0217] [Table 9-9] [Examples]

[0218] The following are examples of specific embodiments for carrying out the present disclosure. These examples are provided for illustrative purposes only and are not intended to limit the scope of the present disclosure in any way. While efforts have been made to ensure accuracy regarding the numbers used (e.g., quantity, temperature, etc.), some degree of experimental error and deviation should naturally be tolerated.

[0219] In implementing this disclosure, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology will be used within the scope of the said technology. Such methods are fully described in the literature. See, for example, TECreighton, Proteins: Structures and Molecular Properties (WH Freeman and Company, 1993); AL Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3rd Ed. (Plenum Press) Vols A and B (1992).

[0220] Example 1: Evaluation of a single-vector multi-cistronic system having different 2A linkers between payload components In this embodiment, the following single-vector multi-cistronic constructs were tested to test different 2A linkers between different payloads.

[0221] SB12689-SB12692: Designed to express a first polypeptide containing a cereblon (CRBN) domain operably linked to a transcription activator (minVPR), and a second polypeptide containing degron operably linked to a ZF10-1 DNA-binding domain. For these constructs, the CRBN component was operably linked to the degron component via an E2A-G4S-T2A ("Opt2A") linker (SEQ ID NOs. 265 and 266) or a P2A_3-T2A_2 (Opt2A_2.0) linker (SEQ ID NOs. 269 and 270). The first and second polypeptides dimerize in the presence of an IMiD small molecule inducer to act as an inducible transcription on-switch.

[0222] SB12703-SB12706: This is the same variant as SB12689-SB12692, but GFP-T2A (the T2A sequence shown in SEQ ID NOs. 267 and 268) precedes the ImiD transcription switch.

[0223] For these experiments, SB09667 was used as a control having wild-type primary and secondary polypeptide components operably linked via a P2A linker.

[0224] method On day 0, U87MG+SB01066 (4×TFBS pMin:mCherry reporter) cells were seeded at a rate of 1e5 cells / well in a 12-well plate. On day 1, the cells were transduced with virus (1e5 pg p24 of the virus in each well). On day 2, the cells were treated with 0, 0.001, 0.01, 0.1, 1, 4, 10, and 100 nM pomalidomide. On day 4, the cells were harvested and mCherry expression was analyzed by flow cytometry.

[0225] Delta %mCherry+ was calculated by subtracting the %mCherry+ cells under ND conditions from the %mCherry+ cells from the specified drug treatment conditions. To calculate the activation factor of the mCherry reporter, the gMFI of mCherry was quantified by flow rate. First, the background mCherry gMFI (gMFI was evaluated for virus-free (NV) controls) was subtracted from all quantified mCherry gMFI values. Next, the background-subtracted mCherry gMFI from the indicated drug treatment conditions was divided by the background-subtracted mCherry gMFI evaluated for the same construct under ND conditions.

[0226] The results are shown in Figures 1A to 1E.

[0227] As shown in Figure 1A, in the IMiD transcription switch, constructs utilizing the Opt2A or Opt2A_2.0 linker showed reduced basal reporter expression compared to constructs containing the P2A linker. Furthermore, the results shown in Figure 1A suggest that both Opt2A and Opt2A_2.0 reduced maximal reporter induction expression at lower concentrations of pomalidomide (≤4 nM). This effect was not evident at 10 nM and 100 nM pomalidomide doses. The results shown in Figure 1B suggest that in the IMiD transcription switch, Opt2A and Opt2A_2.0, compared to P2A, resulted in a greater increase in mCherry-positive cell percentage during induction while reducing background. These trends were also evident in constructs containing GFP-T2A preceding the IMiD transcription switch (Figures 1C, 1D, and 1E).

[0228] Other Embodiments While this disclosure is specifically shown and described with reference to preferred embodiments and various alternative embodiments, it will be understood by those skilled in the art that various variations in form and detail can be made therein without departing from the spirit and scope of this disclosure and the appended claims.

[0229] All references, published patents, and patent applications cited herein are incorporated herein by reference in their entirety for all purposes.

Claims

1. An engineered polynucleotide comprising a first sequence encoding a first polypeptide and a second sequence encoding a second polypeptide, wherein the first sequence and the second sequence are operably linked via a linker polynucleotide sequence encoding a 2A ribosome skipping element comprising at least a portion of a P2A element and a T2A element.

2. Includes an array oriented from 5' to 3', following the formula below: P1-L-P2 The manipulated polynucleotide according to claim 1, wherein P1 comprises a first sequence, L comprises a linker polynucleotide sequence encoding a 2A ribosome skipping element, and P2 comprises a second sequence:

3. The manipulated polynucleotide according to any one of the prior claims, wherein the 2A ribosome skipping element comprises the amino acid sequence ATNFSLLKQAGDVEENPGPGGSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 269).

4. A vector comprising an manipulated polynucleotide as described in any one of the prior claims.

5. An engineered cell comprising an engineered polynucleotide or vector as described in any one of the prior claims.

6. A pharmaceutical composition comprising an engineered polynucleotide or vector as described in any one of the prior claims, and a pharmaceutically acceptable carrier.

7. A pharmaceutical composition comprising the manipulated cells described in claim 5 and a pharmaceutically acceptable carrier.

8. A method comprising administering a pharmaceutical composition described in any one of the prior claims to a subject.

9. A method for producing an engineered cell, comprising introducing an engineered polynucleotide or vector according to any one of the prior claims into a cell.

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