Improved multicistronic systems and uses thereof

Abstract

Provided herein are optimized 2A ribosome skipping elements and polynucleotide sequences encoding such optimized 2A ribosome skipping elements, as well as multicistronic systems, constructs, vectors, and engineered cells utilizing the same for expressing multiple polypeptides from a single transcript.

Description

IMPROVED MULTICISTRONIC SYSTEMS AND USES THEREOFCROSS REFERENCE TO RELATED APPLICATIONS

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

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

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

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

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

[0006] Also provided herein is an engineered polynucleotide comprising a first sequence encoding for a first polypeptide and a second sequence encoding for 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. In some embodiments, the engineered polynucleotide comprises a sequence according to formula, oriented from 5’ to 3’:Pl - L - P2 wherein Pl 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 comprises the amino acid sequence ATNFSLLKQAGDVEENPGPGSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 269).

[0008] Also provided herein are vectors comprising engineered polynucleotides described herein, engineered cells comprising engineered polynucleotides or vectors described herein, pharmaceutical compositions comprising engineered polynucleotides, vectors, or cells described herein, and methods of preparing or using such cells.BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0009] These and other features, aspects, and advantages of the present disclosure will become better understood with regard to the following description, and accompanying drawings.

[0010] FIGs. 1A-1E show results of an assay to evaluate different 2A linkers between different pay load components in a multicistronic system.DETAILED DESCRIPTION

[0011] The present disclosure generally relates to modified degron polypeptides with increased sensitivity to pomalidomide. Also provided are inducibly degradable proteins, inducible cell death systems, and activation-conditional control polypeptides employing modified degron polypeptides and methods and / or use thereof as described herein.

[0012] Terms used in the claims and specification are defined as set forth below unless otherwise specified.

[0013] The term “ameliorating” refers to any therapeutically beneficial result in the treatment of a disease state, e.g., a cancer disease state, including prophylaxis, lessening in the severity or progression, remission, or cure thereof.

[0014] The term “zTz situ” refers to processes that occur in a living cell growing separate from a living organism, e.g., growing in tissue culture.

[0015] The term “zzz vivo” refers to processes that occur in a living organism.

[0016] The term “mammal” as used herein includes both humans and non-humans, and includes, but is not limited to humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.

[0017] The term “percent identity,” in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm e.g., any of those described herein (e.g., BLASTP and BLASTN or other algorithms available to persons of skill), or by visual inspection. Depending on the application, the percent “identity” can exist over aregion of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared.

[0018] For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

[0019] Optimal alignment of sequences for comparison can be conducted, e.g., 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 the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations 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 (see generally Ausubel et al., infra).

[0020] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses 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 a desired effect, e.g., an amount sufficient to modulate protein aggregation in a cell.

[0022] The term “therapeutically effective amount” is an amount that is effective to ameliorate a symptom of a disease. A therapeutically effective amount can be a “prophylactic ally effective amount” as prophylaxis can be considered therapy.

[0023] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0024] Use of ordinal terms such as “first”, “second”, “third”, etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements. Similarly, the use of these terms in the specification does not by itself connote any required priority, precedence, or order.Multicistronic and Multiple Promoter Systems

[0025] In some embodiments, engineered nucleic acids are configured to produce multiple polypeptides. For example, nucleic acids may be configured to produce 2-20 different polypeptides. In some embodiments, engineered nucleic acids are configured to produce 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, 4-11, 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, 12-19, 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 polypeptides. In some embodiments, engineered nucleic acids are configured to produce 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 polypeptides. In some embodiments, engineered nucleic acids are configured to produce two polypeptides. In some embodiments, engineered nucleic acids are configured to produce three polypeptides. In some embodiments, engineered nucleic acids are configured to produce four polypeptides. In some embodiments, engineered nucleic acids are configured to produce five polypeptides.

[0026] In some embodiments, a provided engineered nucleic acid can be multicistronic, i.e., more than one separate polypeptide (e.g., multiple exogenous polynucleotides or effector molecules, or engineered polypeptides as described herein) can be produced from a single transcript. In some embodiments, engineered nucleic acids can be multicistronic through the use of various linkers, e.g., a polynucleotide sequence encoding a first exogenous polynucleotide or effector molecule can be linked to a nucleotide sequence encoding a second exogenous polynucleotide or effector molecule, such as in a first gene: I inker: second gene 5’ to 3’ orientation.

[0027] Multicistronic expression systems can employ 2A ribosome skipping elements to generate multiple separated polypeptides from a single transcript. However, conventional 2A ribosome skipping elements can result in incomplete separation of polypeptides due to inefficient ribosome skipping. There is a need for optimized 2A ribosome skipping elements for improved production of separate 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, a 2A ribosome skipping element comprises at least a functional portion of a P2A ribosome skipping element and at least a functional portion of a T2A ribosome skipping element. The 2A ribosome skipping element may comprise P2A ribosome skipping element and a T2A ribosome skipping element, e.g., a P2A-T2A sequence. In some embodiments, the 2A ribosome skipping element comprises the amino acid sequence ATNFSLLKQAGDVEENPGPGSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 269). In some embodiments, a P2A ribosome skipping element comprises an amino acid sequence as set forth in SEQ ID NO: 195. In some embodiments, a P2A ribosome skipping element comprises an amino acid sequence as set forth in SEQ ID NO: 271. In some embodiments, a T2A ribosome skipping element comprises an amino acid sequence as set forth in SEQ ID NO: 267. In some embodiments, a 2A ribosome skipping element comprises an E2A G4S T2A (Opt2A) ribosome skipping element. In some embodiments, an E2A G4S T2A (Opt2A) ribosome skipping element comprises an amino acid sequence as set forth in SEQ ID NO: 265. In some embodiments, a 2A ribosome skipping element comprises a P2A 3-T2A 2 (Opt2A 2.0) ribosome skipping element. In some embodiments, a P2A 3-T2A 2 (Opt2A 2.0) ribosome skipping element comprises an amino acid sequence as set forth in SEQ ID NO: 269. 2A ribosome skipping elements allow production of separate polypeptides encoded by the first and second genes are produced during translation.

[0030] In some embodiments, a linker is or comprises a cleavable linker polypeptide sequence, such as a Furin cleavage site or a TEV cleavage site, wherein following expression the cleavable linker polypeptide is cleaved such that separate polypeptides encoded by the first and second genes are produced. In some embodiments, a cleavable linker sequence comprises a flexible linker sequence that further promotes cleavage. In some embodiments, a flexible linker comprises a Gly-Ser-Gly sequence).

[0031] In some embodiments, a linker polynucleotide sequence comprises a polynucleotide sequence encoding an Internal Ribosome Entry Site (IRES), such that separate polypeptides encoded by the first and second genes are produced during translation. In some embodiments, a linker polynucleotide sequence comprises a polynucleotide sequence encoding a splice acceptor, such as a viral splice acceptor.

[0032] In some embodiments, a linker comprises combination of linker sequences, such as a Furin-2A linker that can produce separate polypeptides through 2A ribosome skipping followed by further cleavage of the Furin site to allow for complete removal of 2A residues. In some embodiments, a combination of linkers can include a Furin sequence, a flexible linker, and 2Alinker. Accordingly, in some embodiments, the linker is a Furin-Gly-Ser-Gly-2A fusion polypeptide. In some embodiments, a linker is a Furin-Gly-Ser-Gly-T2A fusion polypeptide.

[0033] In general, a multicistronic system used in accordance with the present disclosure may comprise any number or combination of linkers, to express any number of genes or portions thereof (e.g., an engineered nucleic acid can encode a first, a second, a third, and a fourth effector molecule or engineered polypeptide as described herein, each separated by linkers such that separate polypeptides encoded by the first, second, and third effector molecules are produced).

[0034] ‘Linkers,” as used herein can refer to polypeptides that link a first polypeptide sequence and a second polypeptide sequence or the multicistronic linkers as described herein.Engineered Nucleic Acids and Polypeptides

[0035] The present disclosure provides for, among other things, engineered nucleic acids comprising one or more expression cassettes (e.g., one, two, three, four, five, or more expression cassettes), e.g., any expression cassette described herein. In some embodiments, at least one expression cassette, as provided by the present disclosure, comprises a promoter and an exogenous polynucleotide sequence. In some embodiments, a promoter is operably and / or directly linked to an exogenous polynucleotide sequence.

[0036] In some embodiments, one or more expression cassettes can be multicistronic, i.e., more than one separate polypeptide (e.g., multiple exogenous polynucleotides or effector molecules) can be produced from a single transcript. For example, a multicistronic expression cassette can encode both a first ACP and a second ACP, e.g., both expressed from a single expression cassette driven by a constitutive promoter. In another example, a multicistronic expression cassette can encode both an effector molecule and an antigen recognizing receptor, e.g., both expressed from a single expression cassette driven by an ACP-responsive promoter. Expression cassettes can be multicistronic through the use of various linkers, e.g., a polynucleotide sequence encoding a first protein of interest (e.g., an engineered polypeptide as described herein) can be linked to a nucleotide sequence encoding a second protein of interest, such as in a first gene: I inker: second gene 5’ to 3’ orientation. Multicistronic features and options are described herein, e.g., in the section “Multicistronic and Multiple Promoter Systems.”

[0037] In some embodiments, the engineered nucleic acid is selected from: a DNA, a cDNA, an RNA, an mRNA, and a naked plasmid (linear or circular). Also provided herein is an expression vector comprising the engineered nucleic acid.

[0038] In some embodiments, the engineered nucleic acid further comprises an insulator. The insulator can be localized between the first expression cassette and the second expression cassette. An insulator is a cis-regulatory element that has enhancer-blocking or barrier function.Enhancer-blocker insulators block enhancers from acting on the promoter of nearby genes. Barrier insulators prevent euchromatin silencing. An example of a suitable insulator of the present disclosure is the A2 insulator as 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 by reference in their entirety. Other examples of suitable insulators include, without limitation, an Al insulator, a CTCF insulator, a gypsy insulator, an HS5 insulator, and a P-globin locus insulator, such as cHS4. In some embodiments, the insulator is an A2 insulator, an Al insulator, a CTCF insulator, an HS5 insulator, a gypsy insulator, a P-globin locus insulator, or a cHS4 insulator.

[0039] Exemplary polypeptides can include, but are not limited to, ligand binding domains, cell death-inducing domains, regulatable cell survival polypeptides, activation-conditional control polypeptides, nucleic acid binding domains, transcriptional effector domains, chimeric recceptors, e.g., chimeric antigen receptors, and other effector molecules.Ligand Binding Domains

[0040] In some embodiments of the present disclosure, an engineered polypeptide comprises a ligand binding domain. In some embodiments, a ligand binding domain interacts with a ligand such as a cognate ligand. In some embodiments, interaction between a ligand binding domain and a ligand results in oligomerization, e.g., dimerization, of a plurality of ligand binding domains. In some embodiments, interaction between a ligand binding domain and a ligand results in a functional association between a plurality of ligand binding domains, whereby the functional association results in a biologically relevant output (e.g., increase or decrease in gene expression, increase or decrease in biological signaling, etc.). In some embodiments, a functional association between a plurality of ligand binding domains is direct, in that said plurality of ligand binding domains contact each other directly. In some embodiments, a functional association between a plurality of ligand binding domains is indirect, in that said plurality of ligand binding domains do not contact each other directly (e.g., functional association of said plurality of ligand binding domains may be facilitated by one or more intermediate agents or molecules, e.g., polypeptides, nucleic acids, etc.). In some embodiments, functional association includes oligomerization, multimerization, and / or complex formation between two or more engineered polypeptides of the present disclosure.

[0041] Exemplary ligand binding domains can include a domain, or functional fragment thereof, such as one or more of: an ABI domain, a PYL domain, a caffeine-binding singledomain antibody, a cannabidiol binding domain, a hormone-binding domain of estrogen receptor (ER) domain, heavy chain variable region (VH) of an anti-nicotine antibody, light chain variableregion (VL) of an anti-nicotine antibody, a progesterone receptor domain, an FKBP domain, and / or an FRB domain. Example sequences of such domains are shown in Table D.

[0042] A ligand binding domain can include a degron. The terms “degron” and “degron domain,” as used herein, refer to a protein or a part thereof that is important in regulation of protein degradation rates. Various degrons known in the art, including but not limited to short amino acid sequences, structural motifs, and exposed amino acids, can be used in various embodiments of the present disclosure. Degrons identified from a variety of organisms can be used. Degrons and degron pathways are generally known, see, e.g., Varshazsky A., PNAS 2019 Jan 8; 116(2):358-366, hereby incorporated by reference.

[0043] The term “degradation sequence” as used herein, refers to a sequence that promotes degradation of an attached protein through either the proteasome or autophagy-lysosome pathways. Degradation sequences known in the art can be used for various embodiments of the present disclosure. In some embodiments, a degradation sequence comprises a degron identified from an organism, or a modification thereof. In some embodiments, a degradation sequence is a polypeptide that destabilize a protein such that half-life of the protein is reduced at least twofold, when fused to the protein. Many different degradation sequences / signals (e.g., of the ubiquitin- proteasome system) are known in the art, any of which may be used as provided herein. A degradation sequence may be operably linked to a cell receptor, but need not be contiguous with it as long as the degradation sequence still functions to direct degradation of the cell receptor. In some embodiments, the degradation sequence induces rapid degradation of the cell receptor. For a discussion of degradation sequences and their function in protein degradation, see, e.g., Kanemaki et al. (2013) Pflugers Arch. 465(3):419-425, Erales et al. (2014) Biochim Biophys Acta 1843(1) :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(1 l):520-528, Meinnel et al. (2006) Biol. Chem. 387(7):839- 851, Kim et al. (2013) 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; herein incorporated by reference.

[0044] In some embodiments, the degron or degradation sequence is selected from: HCV NS4 degron, PEST (two copies of residues 277-307 of human IKBOI), GRR (residues 352-408 of human pl05), DRR (residues 210-295 of yeast Cdc34), SNS (tandem repeat of SP2 and NB (SP2-NB-SP2 of influenza A or influenza B), RPB (four copies of residues 1688-1702 of yeast RPB), SPmix (tandem repeat of SP1 and SP2 (SP2-SP1-SP2-SP1-SP2 of influenza A virus M2 protein), NS2 (three copies of residues 79-93 of 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), an APC / C degron,a C0P1 E3 ligase binding degron motif, a CRL4-Cdt2 binding PIP degron, an actinfilin-binding degron, a KEAP1 binding degron, a KLHL2 and KLHL3 binding degron, an MDM2 binding motif, an N-degron, a hydroxyproline modification in hypoxia signaling, a phytohormone- dependent SCF-LRR-binding degron, an SCF ubiquitin ligase binding phosphodegron, a phytohormone-dependent SCF-LRR-binding degron, a DSGxxS phospho-dependent degron, an Siah binding motif, an SPOP SBC docking motif, and a PCNA binding PIP box. In some embodiments, a degron includes modifications / mutations that reduce ubiquitination relative to wild-type protein, e.g., relative to a peptide sequence or domain the degron is derived from. Modifications / mutations that reduce ubiquitiation can include replacing or or more lysine residues. Modifications / mutations that reduce ubiquitiation can include replacing all lysine residues.

[0045] In some embodiments, the degron comprises a cereblon (CRBN) polypeptide substrate domain capable of binding CRBN in response to an immunomodulatory drug (IMiD) thereby promoting ubiquitin pathway-mediated degradation of the 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 a fragment thereof that is capable of drug-inducible binding of CRBN. In some embodiments, the CRBN polypeptide substrate domain is a chimeric fusion product of native CRBN polypeptide sequences. In some embodiments, the CRBN polypeptide substrate domain is a IKZF3 / ZFP91 / IKZF3 chimeric fusion product having the amino acid sequence of FNVLMVHKRSHTGERPLQCEICGFTCRQKGNLLRHIKLHTGEKPFKCHLCNYACQRRD AL (SEQ ID NO: 93).

[0046] In some embodiments, a degron includes a degron having the amino acid sequence of SEQ ID NO: 131. A degron can include a modified d913 degron, including amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 131. In some embodiments, a degron includes a modified degron having the amino acid sequence of SEQ ID NO: 133. A d913 degron can include modifications / mutations that reduce ubiquitination relative to unmodified d913 having the amino acid sequence of SEQ ID NO: 131. A d913 degron can include replacing one or more lysine residues, e.g., relative to unmodified d913 having the amino acid sequence of SEQ ID NO: 131, such as A d913 degron can include replacing all lysine residues, e.g., relative to unmodified d913 having the amino acid sequence of SEQ ID NO: 131. A d913 degron can include replacing one or more lysine residues with arginine residues, e.g., relative to unmodified d913 including the amino acid sequence of SEQ ID NO: 131. A d913 degron can include replacing all lysine residues with arginine residues, e.g., relative to unmodified d913 having theamino acid sequence of SEQ ID NO: 131, such as a modified degron including the amino acid sequence of SEQ ID NO: 133.

[0047] In some embodiments, cereblon (CRBN) is a wild-type CRBN polypeptide, e.g., the amino acid sequence of SEQ ID NO: 127. In some embodiments, CRBN is a modified CRBN polypeptide. A modified CRBN can include mutations that reduce ubiquitination relative to wild-type CRBN. A modified CRBN can include a deletion of amino acids 194-247, which is the DDB1 interacting domain, e.g., a modified CRBN including the amino acid sequence of SEQ ID NO: 129. In some aspects, a ligand binding domain comprises a hormone-binding domain of estrogen receptor (ER) domain and the cognate ligand is tamoxifen or a metabolite thereof. In some aspects, the tamoxifen metabolite is selected from the group consisting of: 4- hydroxytamoxifen, N-desmethyltamoxifen, tamoxifen, tamoxifen-N-oxide, and endoxifen.

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

[0049] In some aspects, a ligand binding domain comprises an ABI domain or a PYL domain and the cognate ligand is abscisic acid.

[0050] In some aspects, a ligand binding domain comprises a caffeine-binding singledomain antibody and the cognate ligand is caffeine or a derivative thereof.

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

[0052] In some aspects, a ligand binding domain comprises a hormone-binding domain of estrogen receptor (ER) domain and the cognate ligand is tamoxifen or a metabolite thereof. In some aspects, the tamoxifen metabolite is selected from the group consisting of: 4- hydroxytamoxifen, N-desmethyltamoxifen, tamoxifen-N-oxide, and endoxifen.

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

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

[0055] In some aspects, a ligand binding domain comprises an FKBP domain or an FRB domain and the cognate ligand is rapamycin, API 903, AP20187, FK1012, derivatives thereof, or analogs thereof.

[0056] Exemplary ligand binding domains are described in US20230011052, WO2022109421, and WO2022216823, which are hereby incorporated by reference in their entirety.Ligands and Cognate Ligand Pairs

[0057] The present disclosure identifies certain ligands that are useful for binding to ligand binding domains, as discussed herein. In many embodiments of the present disclosure, a ligand may bind to a particular ligand binding domain. A given ligand that consistently binds to a given ligand binding domain can be referred to as a cognate ligand pair.

[0058] In some aspects, the ligand is FK1012, a derivative thereof, or an analog thereof.

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

[0060] In some aspects, the ligand is rapamycin, a derivative thereof, or an analog thereof. In some aspects, the ligand is tamoxifen or a metabolite thereof. In some aspects, the tamoxifen metabolite is selected from the group consisting of: 4-hydroxytamoxifen, N- desmethyltamoxifen, tamoxifen-N-oxide, and endoxifen.

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

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

[0063] In some aspects, the ligand is a cannabidiol or a phytocannabinoid.

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

[0065] In some aspects, the ligand is an IMiD. In some aspects, the IMiD is an FDA- approved drug. In some aspects, the IMiD is selected from the group consisting of: thalidomide, lenalidomide, and pomalidomide.Cell Death-Inducing Domains

[0066] In some embodiments, an engineered polypeptide of the present disclosure comprises an inducible cell death polypeptide. Inducible cell death polypeptides can include one or more ligand binding domains and at least one cell death- inducing domain.

[0067] Exemplary cell death-inducing domains can be derived from a protein such as one or more of: a caspase (e.g., any one of caspases 1-11, such as caspase 3, caspase 6, caspase 7, caspase 8, caspase 9), Diphtheria toxin fragment A (DTA), Bax, Bak, Bok, Bad, Bcl-xS, Bak, Bik, Bcl-2-interacting protein 3 (BNIP3), Fas, Fas-associated protein with death domain (FADD), tumor necrosis factor receptor type 1 -associated death domain protein (TRADD), a TNF receptor (TNF-R), APAF-1, granzyme B, second mitochondria-derived activator of caspases (SMAC), Omi, Bmf, Bid, Bim, p53-upregulated modulator of apoptosis (PUMA), Noxa, Blk, Hrk, Cytochrome c, Arts, TNF-related apoptosis-inducing ligand (TRAIE), Herpes Simplex Virus thymidine kinase (HSV-TK), Varicella Zoster Virus thymidine kinase (VZV-TK), viral Spike protein, Carboxyl esterase, cytosine deaminase, nitroreductase Fksb, Carboxypeptidase G2, Carboxypeptidase A, Horseradish peroxidase, Linamarase, Hepatic cytochrome P450-2B1, Purine nucleoside phosphorylase, or variants or functional fragments thereof. A“variant” as used herein may refer to mutants, splice variants, conformations, isoforms, allelic variants, species variants and species homologs of a biomolecule, e.g., a polynucleotide or a polypeptide. In some embodiments, a variant biomolecule is a natural variant. In some embodiments, a variant biomolecule is an engineered variant. In some embodiments, a 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, comprising one or more different nucleotides as compared to a reference (or “parent”) sequence. Accordingly, variant polynucleotide sequences may comprise at least one mutation, substitution, insertion, or deletion as compared to their respective reference sequence. “Variants” may have a sequence identity (e.g., nucleic acid sequence identity or amino acid sequence identity) of 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%, as compared to a reference sequence (e.g., a naturally occurring sequence or fragment or derivative thereof, or an engineered sequence). In some embodiments, based on 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 a reference biomolecule (i.e., shorter and / or smaller) with the same or similar functional activity of the reference biomolecule. It is contemplated that a similar functional activity could be greater, about equal, or less than the functional activity of the reference biomolecule, as long as the functional fragment achieves at least a portion of the activity of the reference biomolecule. When the reference biomolecule is a polypeptide, it is contemplated that the polypeptide fragment retains 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 suitable assay. For example, when the reference biomolecule is a polypeptide, it is contemplated that the polypeptide fragment may be a polypeptide that has been cleaved or otherwise modified to be shorter and / or smaller than the reference polypeptide, but still retains functional activity of the reference polypeptide, such as binding to a particular receptor; when the reference biomolecule is a polynucleotide, it is contemplated that the polynucleotide fragment retains some of the same activity of the reference polynucleotide. For example, in the case of a polynucleotide encoding a protein, it is contemplated that the polynucleotide fragment encodes a protein having 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 suitable assay. In the case of a polynucleotide thatacts a regulatory element (e.g., a promoter or enhancer), it is contemplated that the polynucleotide fragment has 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 activity as reference polynucleotide in a suitable assay. Exemplary cell death-inducing domain sequences can be found in Table C-l.Regulatable Cell Survival Polypeptides

[0068] In some embodiments, an engineered polypeptide of the present disclosure comprises a regulatable cell survival polypeptide. A regulatable cell survival polypeptide can comprise at least one ligand binding domain.

[0069] Exemplary cell survival polypeptides include one or more of XIAP, Bcl-2, Bcl-xL, Bcl-w, Bcl-2-related protein Al (BCL2A1), Mcl-1, FLICE- like inhibitory protein (c-FLIP), and an adenoviral E1B-19K protein. A cell survival polypeptide can include XIAP. A cell survival polypeptide can include wild-type XIAP, e.g., having the amino acid sequence SEQ ID NO: 107. A cell survival polypeptide can include modified XIAP. A modified XIAP can include one or more amino acid substitutions with reference to SEQ ID NO: 107.Activation-Conditional Control Polypeptides (ACPs)

[0070] In some embodiments, an engineered polypeptide of the present disclosure comprises an ACP. In some embodiments, an engineered polypeptide of the present disclosure may form an ACP in the presence of a ligand. In some embodiments, the ACP is or comprises a transcriptional modulator. In some embodiments, the ACP is or comprises a transcriptional repressor. In some embodiments, the ACP is or comprises a transcriptional activator. In some embodiments, the ACP is or comprises a transcription factor. In some embodiments, an ACP comprises a DNA-binding domain. In some embodiments, an ACP comprises a transcriptional effector domain. In some embodiments, the ACP comprises a DNA-binding domain and a transcriptional effector domain. In some embodiments, the transcription factor includes 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, an ACP can include one or more ligand binding domains.Nucleic Acid Binding Domains

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

[0072] In some embodiments, an engineered polypeptide comprises at least one transcription factor. In some embodiments, a transcription factor comprises at least one nucleic acid-binding domain. In some embodiments, a transcription factor comprises at least one nucleic acid-binding domain and at least one transcriptional effector domain.

[0073] In some aspects, the nucleic acid-binding domain comprises a DNA-binding zinc finger protein domain (ZF protein domain). In some aspects, the ZF protein domain is modular in design and is composed of zinc finger arrays (ZFA). In some aspects, the transcriptional effector domain is selected from the group consisting of: a Herpes Simplex Virus Protein 16 (VP 16) activation domain; an activation domain comprising four tandem copies of VP 16, a VP64 activation domain; a p65 activation domain of NFKB; an Epstein-Barr virus R transactivator (Rta) activation domain; a tripartite activator comprising the VP64, the p65, and the Rta activation domains (VPR activation domain); a tripartite activator comprising the VP64, the p65, and the HSF1 activation domains (VPH activation domain); a histone acetyltransferase (HAT) core domain of the human ElA-associated protein p300 (p300 HAT core activation domain); a Kruppel associated box (KRAB) repression domain; a Repressor Element Silencing Transcription Factor (REST) repression domain; a WRPW motif of the hairy-related basic helixloop-helix repressor proteins, the motif is known as a WRPW repression domain; a DNA (cytosine-5)-methyltransferase 3B (DNMT3B) repression domain; an HP1 alpha chromoshadow repression domain, and variants or functional fragments thereof. In some embodiments, a transcriptional effector domain is a tripartite activator comprising the VP64, the p65, and the Rta activation domains (VPR activation domain), or a variant or functional fragments thereof. In some embodiments, a transcriptional effector domain comprises a p65 domain, or a variant or functional fragments thereof.

[0074] In some embodiments, the ZF protein domain is modular in design and is composed of zinc finger arrays (ZFA). A zinc finger array comprises multiple zinc finger protein motifs that are 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 can be directly adjacent to each other, or separated by a flexible linker sequence. In some embodiments, a ZFA is an array, string, or chain of ZF motifs arranged in tandem. A ZFA can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 zinc finger motifs. The ZFA can have from 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, a ZF protein domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more ZFAs. In some embodiments, a ZF domain comprises from 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 comprises one to ten ZFA(s). In some embodiments, the ZF protein domain comprises at least one ZFA. In some embodiments, the ZF protein domain comprises at least two ZFAs. In some embodiments, the ZF protein domain comprises at least three ZFAs. In some embodiments, the ZF protein domain comprises at least four ZFAs. In some embodiments, the ZF protein domain comprises at least five ZFAs. In some embodiments, the ZF protein domain comprises at least ten ZFAs.

[0076] An exemplary ZF protein domain is shown in the sequenceSRPGERPFQCRICMRNFSRRHGLDRHTRTHTGEKPFQCRICMRNFSDHSSLKRHLRTHT GSQKPFQCRICMRNFSVRHNLTRHLRTHTGEKPFQCRICMRNFSDHSNLSRHLKTHTGS QKPFQCRICMRNFSQRSSLVRHLRTHTGEKPFQCRICMRNFSESGHLKRHLRTHLRGS (SEQ ID NO: 57). In some embodiments, a ZF protein domain comprises the amino acid sequence of SEQ ID NO: 57.Transcriptional Effector Domains

[0077] The present disclosure provides for, in some embodiments, engineered polypeptides comprising at least one transcriptional effector domain. In some embodiments, an engineered polypeptide may comprise at least one ligand binding domain and at least one transcriptional effector domain.

[0078] In some embodiments, an inducible cell death polypeptide as provided herein can include at least one transcriptional effector domain. In some embodiments, an ACP as provided herein can include at least one transcriptional effector domain. In some embodiments, an inducible cell death polypeptide comprises at least one ligand binding domain and at least one transcriptional effector domain. In some embodiments, an ACP comprises at least one ligand binding domain and at least one transcriptional effector domain.

[0079] In someembodiments, a transcriptional effector domain comprises one or more of: a Herpes Simplex Virus Protein 16 (VP 16) activation domain; an activation domain comprising four tandem copies of VP 16, a VP64 activation domain; a p65 activation domain of NFKB; an Epstein-Barr virus R transactivator (Rta) activation domain; a tripartite activator comprising the VP64, the p65, and the Rta activation domains (VPR activation domain); a tripartite activatorcomprising the VP64, the p65, and the HSF1 activation domains (VPH activation domain); a histone acetyltransferase (HAT) core domain of the human El A- associated protein p300 (p300 HAT core activation domain); a Kriippel associated box (KRAB) repression domain; a Repressor Element Silencing Transcription Factor (REST) repression domain; a WRPW motif of the hairy-related basic helix-loop-helix repressor proteins, the motif is known as a WRPW repression domain; a DNA (cytosine-5)-methyltransferase 3B (DNMT3B) repression domain; and an HP1 alpha chromoshadow repression domain. In some embodiments, a transcriptional effector domain is a tripartite activator comprising the VP64, the p65, and the Rta activation domains (VPR activation domain). In some embodiments, a transcriptional effector domain comprises a p65 domain.

[0080] In some embodiments, the transcriptional effector domain comprises a transcriptional repressor domain. In some embodiments, the transcriptional repressor domain is selected from the group consisting of: a Kriippel associated box (KRAB) repression domain; a truncated Kriippel associated box (KRAB) repression domain; a Histone Deacetylase 4 (HDAC4) repressor domain; a Scleraxis (SCX) HLH domain, an Inhibitor of DNA binding 1 (ID1) HLH domain, a HECT domain and RCCl-like domain-containing protein 2 (HERC2) Cyt-b5 domain, a Twist-related protein 1 (TWST1) HLH domain, an Homeobox protein Nkx-2.2 (NKX22) homeodomain, an Inhibitor of DNA binding 1 (ID3) HLH domain, and a Twist-related protein 2 (TWST2) HLH domain, and EED repressor domain; a Repressor Element Silencing Transcription Factor (REST) repression domain; a WRPW motif of the hairy-related basic helixloop-helix repressor proteins, the motif is known as a WRPW repression domain; a DNA (cytosine-5)-methyltransferase 3B (DNMT3B) repression domain; and an HP1 alpha chromoshadow repression domain.In some embodiments, the transcriptional effector domain comprises a transcriptional activation domain. In some aspects, the transcriptional activation domain is selected from the group consisting of: a Herpes Simplex Virus Protein 16 (VP16) activation domain; an activation domain comprising four tandem copies of VP16; a VP64 activation domain; a p65 activation domain of NFKB; an Epstein-Barr virus R transactivator (Rta) activation domain; a tripartite activator comprising the VP64, the p65, and the Rta activation domains (VPR activation domain); a tripartite activator comprising the VP64, the p65, and the HSF1 activation domains (VPH activation domain); and a histone acetyltransferase (HAT) core domain of the human ElA-associated protein p300 (p300 HAT core activation domain). In some embodiments, a transcriptional activator domain is a tripartite activator comprising the VP64, the p65, and the Rta activation domains (VPR activation domain). In some embodiments, a transcriptional activator domain comprises a p65 domain. Transcriptional activation domains can also be referred to as transcriptional activator domains.

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

[0082] An engineered nucleic acid provided by the present disclosure can encode an effector domain, such as a transcriptional effector domain. In some embodiments a transcriptional effector domain comprises an effector domain (e.g., activator domain or repressor domain) of a transcription factor. Transcription factor effector domains are also known as transactivation domains, and act as scaffold domains for proteins such as transcription coregulators that act to activate or repress transcription of genes. Any suitable transcriptional effector domain can be used including, but not limited to, a Herpes Simplex Virus Protein 16 (VP 16) activation domain; an activation domain consisting of four tandem copies of VP 16, a VP64 activation domain; a p65 activation domain of NFKB; an Epstein-Barr virus R transactivator (Rta) activation domain; a tripartite activator comprising the VP64, the p65, and the Rta activation domains, the tripartite activator is known as a VPR activation domain; a histone acetyltransferase (HAT) core domain of the human ElA-associated protein p300, known as a p300 HAT core activation domain; a Kriippel associated box (KRAB) repression domain; a truncated Kriippel associated box (KRAB) repression domain; a Repressor Element Silencing Transcription Factor (REST) repression domain; a WRPW motif of the hairy-related basic helix-loop-helix repressor proteins, the motif is known as a WRPW repression domain; a DNA (cytosine-5)-methyltransferase 3B (DNMT3B) repression domain; and an HP1 alpha chromoshadow repression domain, or any combination thereof. In some embodiments, a transcriptional effector domain is a tripartite activator comprising the VP64, the p65, and the Rta activation domains (VPR activation domain). In some embodiments, a transcriptional effector domain comprises a p65 domain.

[0083] Exemplary transcriptional effector domain protein sequences are shown in Table 1. Exemplary transcriptional effector domain nucleotide sequences are shown in Table 2.Table 1. Transcriptional Effector Domain (Protein)Table 2. Transcriptional Effector Domain (Nucleotide)Chimeric receptors

[0084] In some embodiments, the chimeric receptor is a chimeric antigen receptor (CAR). In general, CARs are chimeric proteins that include an antigen-binding domain and polypeptide molecules that are heterologous to the antigen-binding domain, such as peptides heterologous to an antibody that an antigen-binding domain may be derived from. Polypeptide molecules that are heterologous to the antigen-binding domain can 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 combinations thereof.

[0085] In some embodiments, the chimeric antigen receptor is an activating chimeric antigen receptor (aCAR and also generally referred to as CAR unless otherwise specified). In some embodiments, binding of the chimeric antigen receptor to its cognate ligand is sufficient to induce activation of the immunoresponsive cell. In some embodiments, binding of the chimeric antigen receptor to its cognate ligand is sufficient to induce stimulation of the immunoresponsive cell. In some embodiments, activation of an immunoresponsive cell results in killing of target cells. In some embodiments, activation of an immunoresponsive cell results in cytokine or chemokine expression and / or secretion by the immunoresponsive cell. In some embodiments, stimulation of an immunoresponsive cell results in cytokine or chemokine expression and / or secretion by the immunoresponsive cell. In some embodiments, stimulation of an immunoresponsive cell induces differentiation of the immunoresponsive cell. In some embodiments, stimulation of an immunoresponsive cell induces proliferation of the immunoresponsive cell. In some embodiments, activation and / or stimulation of the immunoresponsive cell can be combinations of the above responses.

[0086] The number of ABDs in a binding molecule, such as the chimeric proteins described herein, defines the “valency” of the binding molecule. A binding molecule having a single ABD is “monovalent”. A binding molecule having a plurality of ABDs is said to be “multivalent”. A multivalent binding molecule having two ABDs is “bivalent.” A multivalent binding molecule having three ABDs is “trivalent.” A multivalent binding molecule having four ABDs is “tetravalent.” In various multivalent embodiments, all of the plurality of ABDs have the same recognition specificity and can be referred to as a “monospecific multivalent” binding molecule. In other multivalent embodiments, at least two of the plurality of ABDs have different recognition specificities. Such binding molecules are multivalent and “multispecific.” In multivalent embodiments in which the ABDs collectively have two recognition specificities, the binding molecule is “bispecific.” In multivalent embodiments in which the ABDs collectively have three recognition specificities, the binding molecule is “trispecific.” In multivalent embodiments in which the ABDs collectively have a plurality of recognition specificities fordifferent epitopes present on the same antigen, the binding molecule is “multiparatopic.” Multivalent embodiments in which the ABDs collectively recognize two epitopes on the same antigen are “biparatopic.”

[0087] In various multivalent embodiments, multivalency of the binding molecule improves the avidity of the binding molecule for a specific target. As described herein, “avidity” refers to the overall strength of interaction between two or more molecules, e.g. a multivalent binding molecule for a specific target, wherein the avidity is the cumulative strength of interaction provided by the affinities of multiple ABDs. Avidity can be measured by the same methods as those used to determine affinity, as described above. In certain embodiments, the avidity of a binding molecule for a specific target is such that the interaction is a specific binding interaction, wherein the avidity between two molecules has a KD value below 10-6M, 10-7M, 10-8M, 10-9M, or 10-10M. In certain embodiments, the avidity of a binding molecule for a specific target has a KD value such that the interaction is a specific binding interaction, wherein the one or more affinities of individual ABDs do not have has a KD value that qualifies as specifically binding their respective antigens or epitopes on their own. In certain embodiments, the avidity is the cumulative strength of interaction provided by the affinities of multiple ABDs for separate antigens on a shared specific target or complex, such as separate antigens found on an individual cell. In certain embodiments, the avidity is the cumulative strength of interaction provided by the affinities of multiple ABDs for separate epitopes on a shared individual antigen.

[0088] A CAR of the present disclosure may be a first, second, or third generation CAR. "First generation" CARs comprise a single intracellular signaling domain, generally derived from a T cell receptor chain. "First generation" CARs generally have the intracellular signaling domain from the CD3-zeta (CD3Q chain, which is the primary transmitter of signals from endogenous TCRs. "First generation" CARs can provide de novo antigen recognition and cause activation of both CD4+ and CD8+ T cells through their CD3(^ chain signaling domain in a single fusion molecule, independent of HLA-mediated antigen presentation. "Second generation" CARs add a second intracellular signaling domain from one of various costimulatory molecules (e.g., CD28, 4- IBB, ICOS, 0X40) to the cytoplasmic tail of the CAR to provide additional signals to the T cell. "Second generation" CARs provide both co-stimulation (e.g., CD28 or 4- IBB) and activation (CD3Q. Preclinical studies have indicated that "Second Generation" CARs can improve the anti-tumor activity of immunoresponsive cell, such as a T cell. "Third generation" CARs have multiple intracellular co-stimulation signaling domains (e.g., CD28 and 4- IBB) and an intracellular activation signaling domain (CD3Q.

[0089] In some embodiments, the chimeric antigen receptor is a chimeric inhibitory receptor (iCAR).

[0090] Binding of the extracellular antigen-binding domain of a CAR of the present disclosure can be determined by, for example, an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), FACS analysis, a bioassay (e.g., growth inhibition), bio-layer interferometry (e.g., Octet / FORTEBIO®), surface plasmon resonance (SPR) technology (e.g., Biacore®), or a Western Blot assay. Each of these assays generally detect the presence of protein- antibody complexes of particular interest by employing a labeled reagent (e.g., an antibody or scFv) specific for the complex of interest. For example, the scFv can be radioactively labeled and used in an RIA assay. The radioactive isotope can be detected by such means as the use of a y counter or a 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 for the extracellular antigen-binding domain and wherein the secondary antibody is labeled (e.g., radioactively or with a fluorescent marker).

[0091] Antigen-binding domains of the present disclosure can include any domain that binds to the antigen including, without limitation, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a bispecific antibody, a conjugated antibody, a human antibody, a humanized antibody, and a functional fragment thereof, including but not limited to a singledomain antibody (sdAb) such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain (VHH) of camelid derived nanobody, and to an alternative scaffold known in the art to function as antigen-binding domain, such as a recombinant fibronectin domain, a T cell receptor (TCR), a recombinant TCR with enhanced affinity, or a fragment thereof, e.g., single chain TCR, and the like. In some instances, it is beneficial for the antigen-binding domain to be derived from the same species in which the CAR will ultimately be used in.

[0092] In some embodiments, the extracellular antigen-binding domain comprises 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 comprises an antigen-binding fragment of an antibody.

[0093] In some embodiments, the extracellular antigen-binding domain comprises a F(ab) fragment. In certain embodiments, the extracellular antigen-binding domain comprises a F(ab') fragment.

[0094] In some embodiments, the extracellular antigen-binding domain comprises an scFv. In some embodiments, the extracellular antigen-binding domain comprises two single chain variable fragments (scFvs). 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 comprises a first scFv and a second scFv. In some embodiments, the first scFv and the second scFv bind 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 comprises a heavy chain variable domain (VH) and a light chain variable domain (VL).

[0095] In certain embodiments, the VH and VL are separated by a peptide linker. In certain embodiments, the peptide linker comprises any of the amino acid sequences shown in Table 6.

[0096] In certain embodiments, the scFv comprises the structure VH-L-VL or VL-L-VH, wherein 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 the one or more scFvs comprises the structure VH-L-VL or VL-L-VH, wherein VH is the heavy chain variable domain, L is the peptide linker, and VL is the light chain variable domain. When there are two or more scFv linked together, each scFv can be linked to the next scFv with a peptide linked. In some embodiments, each of the one or more scFvs is separated by a peptide linker.Table 6. Peptide Linkers

[0097] Suitable antigen recognizing receptors for use as an effector molecule recognize antigens that 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, Claudinl8.2, cMet, CSPG4, CTLA, DLK1, DLL3, DR5, EGFR, ENPP3, EpCAM, EphA2, Ephrin A4, ETBR, FGFR2, FGFR3, FRalpha, 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, MUCIC, NaPi2B, Nectin 4, NKG2D, NOTCH3, NY ESO 1, Ovarin, P-cadherin, pan- Erb2, PSCA, PSMA, PTK7, ROR1, S Aures, SCT, SLAMF7, SLITRK6, SSTR2, STEAP1, Survivin, TDGF1, TIM1, and WT1, or any combination thereof.

[0098] In some embodiments, the antigen recognizing receptor recognizes an antigen selected from: 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, Claudinl8.2, cMet, CSPG4, CTLA, DLK1, DLL3, DR5, EGFR, ENPP3, EpCAM, EphA2, Ephrin A4, ETBR, FGFR2, FGFR3, FRalpha, 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, MUC1, MUC16, MUCIC, NaPi2B, Nectin 4, NKG2D, NOTCH3, NY ESO 1, Ovarin, P-cadherin, pan-Erb2, PSCA, PSMA, PTK7, ROR1, S Aures, SCT, SLAMF7, SLITRK6, SSTR2, STEAP1, Survivin, TDGF1, TIM1, and WTl.

[0099] Exemplary antigen-binding domain sequences are described in, e.g., U.S. Patent Application Pub. Nos. US20230011052 and US20210017277, International Patent Application Pub. Nos. W02022082059, WO2022115565, WO 2022236142, U.S. Patent Nos. 11,299,546; 9,409,994, which are hereby incorporated by reference in their entirety.

[0100] In some embodiments, a chimeric receptor is a T cell receptor (TCR).

[0101] In some embodiments, the multiple polypeptides can include a first chimeric receptor and a second chimeric receptor.Effector molecules

[0102] Any suitable effector molecule can be encoded by the engineered nucleic acid or expressed by the engineered cell. Suitable effector molecules can be grouped into therapeutic classes based on structure similarity, sequence similarity, or function. Effector molecule therapeutic classes include, but are not limited to, cytokines, chemokines, homing molecules, growth factors, co-activation molecules, tumor microenvironment modifiers, receptors, ligands, antibodies, polynucleotides, peptides, and enzymes.

[0103] In some embodiments, each effector molecule is independently selected from a therapeutic class, wherein the therapeutic class is selected from: a cytokine, a chemokine, a homing molecule, a growth factor, a co-activation molecule, a tumor microenvironment modifier a, a receptor, a ligand, an antibody, a polynucleotide, a peptide, and an enzyme.

[0104] Chemokines are small cytokines or signaling proteins secreted by cells that can induce directed chemotaxis in cells. Chemokines can be classified into four main subfamilies: CXC, CC, CX3C and XC, all of which exert biological effects by binding selectively to chemokine receptors located on the surface of target cells. Non-limiting examples of chemokines that may be encoded by the engineered nucleic acids of the present disclosure include: CCL21a, CXCL10, CXCL11, CXCL13, a CXCL10-CXCL11 fusion protein, CCL19, CXCL9, and XCL1, or any combination thereof. In some embodiments, the chemokine is selected from: CCL21a, CXCL10, CXCL11, CXCL13, a CXCL10-CXCL11 fusion protein, CCL19, CXCL9, and XCL1.

[0105] Non-limiting examples of cytokines that may be encoded by the engineered nucleic acids of the present disclosure include: ILl-beta, IL2, IL4, IL6, IL7, IL10, IL12, an IL12p70 fusion protein, IL15, IL17A, IL18, IL21, IL22, Type I interferons, Interferon-gamma, and TNF- alpha, or any combination thereof. In some embodiments, the cytokine is selected from: ILl- beta, IL2, IL4, IL6, IL7, IL10, IL12, an IL12p70 fusion protein, IL15, IL17A, IL18, IL21, IL22, Type I interferons, Interferon-gamma, and TNF-alpha.

[0106] Effector molecules can include homing molecules. “Homing,” refers to active navigation (migration) of a cell to a target site (e.g., a cell, tissue (e.g., tumor), or organ). A “homing molecule” refers to a molecule that directs cells to a target site. In some embodiments, a homing molecule functions to recognize and / or initiate interaction of an engineered cell to a target site. Non-limiting examples of homing molecules include CXCR1, CCR9, CXCR2, CXCR3, CXCR4, CCR2, CCR4, FPR2, VEGFR, IL6R, CXCR1, CSCR7, PDGFR, anti-integrin alpha4,beta7; anti-MAdCAM; CCR9; CXCR4; SDF1; MMP-2; CXCR1; CXCR7; CCR2; CCR4; and GPR15, or any combination thereof. In some embodiments, the homing molecule is selectedfrom: anti-integrin alpha4,beta7 ; anti-MAdCAM; CCR9; CXCR4; SDF1; MMP-2; CXCR1; CXCR7; CCR2; CCR4; and GPR15.

[0107] Suitable growth factors for use as an effector molecule include, but are not limited to, FLT3L and GM-CSF, or any combination thereof. In some embodiments, the growth factor is selected from: FLT3L and GM-CSF.

[0108] Suitable co-activation molecules for use as an effector molecule include, but are not limited to, c-Jun, 4-1BBL and CD40L, or any combination thereof. In some embodiments, the co-activation molecule is selected from: c-Jun, 4-1 BBL and CD40L.

[0109] A “tumor microenvironment” is the cellular environment in which a tumor exists, including surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, lymphocytes, signaling molecules and the extracellular matrix (ECM) (see, e.g., Pattabiraman, D.R. & Weinberg, R.A. Nature Reviews Drug Discovery 13, 497-512 (2014); Balkwill, F.R. 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 an effector molecule include, but are not limited to, adenosine deaminase, TGFbeta inhibitors, immune checkpoint inhibitors, VEGF inhibitors, and HPGE2, or any combination thereof. In some embodiments, the tumor microenvironment modifier is selected from: adenosine deaminase, TGFbeta inhibitors, immune checkpoint inhibitors, VEGF inhibitors, and HPGE2.

[0110] Effector molecules can include TGFbeta inhibitors. Suitable TGFbeta inhibitors for use as an effector molecule include, but are not limited to, an anti-TGFbeta peptide, an anti- TGFbeta antibody, a TGFb-TRAP, or combinations thereof. In some embodiments, the TGFbeta inhibitors are selected from: an anti-TGFbeta peptide, an anti-TGFbeta antibody, a TGFb- TRAP, and combinations thereof.

[0111] Effector molecules can include immune checkpoint inhibitors. Suitable immune checkpoint inhibitors for use as an effector molecule include, but are not limited to, anti-PD-1 antibodies, anti-PD-Ll 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-TREMl antibodies, and anti-TREM2 antibodies, or any combination thereof. In some embodiments, the immune checkpoint inhibitors are selected from: anti-PD-1 antibodies, anti-PD-Ll 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-TREMl antibodies, and anti-TREM2 antibodies.

[0112] Illustrative immune checkpoint inhibitors include pembrolizumab (anti-PD- 1 ; MK- 3475 / Keytruda® - Merck), nivolumamb (anti-PD- 1; Opdivo® - BMS), pidilizumab (anti-PD- 1 antibody; CT-011 - Teva / CureTech), AMP224 (anti-PD-1; NCI), avelumab (anti-PD-Ll; Bavencio® - Pfizer), durvalumab (anti-PD-Ll; MEDI4736 / Imfinzi® - Medimmune / AstraZeneca), atezolizumab (anti-PD-Ll; Tecentriq® - Roche / Genentech), BMS- 936559 (anti-PD-Ll - BMS), tremelimumab (anti-CTLA-4; Medimmune / AstraZeneca), ipilimumab (anti-CTLA-4; Yervoy ® - BMS), lirilumab (anti-KIR; BMS), monalizumab (anti- NKG2A; Innate Pharma / AstraZeneca).

[0113] Exemplary effector molecules also include VEGE inhibitors. Suitable VEGF inhibitors for use as an effector molecule include, but are not limited to, anti- VEGF antibodies, anti- VEGF peptides, or combinations thereof. In some embodiments, the VEGF inhibitors comprise anti- VEGF antibodies, anti- VEGF peptides, or combinations thereof.

[0114] In some embodiments, each effector molecule is a human-derived effector molecule.Promoters

[0115] In some embodiments, an engineered nucleic acid of the present disclosure comprises one or more promoters, e.g., in one or more expression cassettes. In some embodiments, an engineered nucleic acid of the present disclosure comprises a first expression cassette comprising a first promoter operably linked to an exogenous polynucleotide sequence. In some embodiments, an engineered nucleic acid of the present disclosure comprises a second expression cassette comprising a promoter operably linked to a second exogenous polynucleotide sequence encoding one or more effector molecules. In some embodiments, the first expression cassette and second expression cassette are each encoded by a separate engineered nucleic acid of the present disclosure. In some embodiments, the first expression cassette and the second expression cassette are encoded by the same engineered nucleic acid of the present disclosure.

[0116] In some embodiments, a promoter is an ACP-responsive promoter. In some embodiments, an ACP-responsive promoter of the present disclosure comprises an ACP-binding domain and a promoter sequence. In some embodiments, the ACP-responsive promoter is operable linked to a nucleotide sequence encoding an effector molecule (e.g., a protein of interest, e.g., any engineered polypeptide described herein).

[0117] A “promoter” refers to a control region of a nucleic acid sequence at which initiation and rate of transcription of the remainder of a nucleic acid sequence are controlled. In someembodiments, a promoter comprises sub-regions at which regulatory proteins or other molecules may bind, such as RNA polymerase, an ACP, and / or transcription factors. Promoters may be constitutive, inducible, repressible, tissue- specific or any combination thereof. In some embodiments, a promoter is a constitutive promoter. In some embodiments, a promoter is an inducible promoter. A promoter drives expression or drives transcription of the nucleic acid sequence that it regulates. Herein, a promoter is considered to be “operably linked” when it is in a correct functional location and orientation in relation to a nucleic acid sequence it regulates to control (“drive”) transcriptional initiation and / or expression of that sequence.

[0118] In some embodiments, a promoter may be one naturally associated with a particular gene or sequence, as may be obtained by isolating the 5’ non-coding sequences located upstream of the coding segment of said gene or sequence. Such a promoter can be referred to as “endogenous.” In some embodiments, a coding nucleic acid sequence may be positioned under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated 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 “naturally occurring” such as, for example, those that contain different elements of different transcriptional regulatory regions and / or mutations that alter expression through methods of genetic engineering. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and / or nucleic acid amplification technology, including polymerase chain reaction (PCR) (see, e.g., U.S. Pat. No. 4,683,202 and U.S. Pat. No. 5,928,906).

[0119] Promoters of an engineered nucleic acid of the present disclosure may be “inducible promoters,” which refer to promoters that are characterized by regulating (e.g., initiating or activating) transcriptional activity when in the presence of, influenced by or contacted by a signal. The signal may be endogenous or a normally exogenous condition (e.g., light), compound (e.g., chemical or non-chemical compound) or protein (e.g., an engineered polypeptide described herein, e.g., an ACP) that contacts an inducible promoter in such a way as to be active in regulating transcriptional activity from the inducible promoter. Activation of transcription may involve directly acting on a promoter to drive transcription or indirectly acting on a promoter by inactivation a repressor (e.g., an engineered polypeptide described herein) that is preventing the promoter from driving transcription. Conversely, deactivation of transcription may involve directly acting on a promoter to prevent transcription or indirectly acting on a promoter by activating a repressor that then acts on the promoter.

[0120] In some embodiments, a promoter is “responsive to” or “modulated by” a local tumor state (e.g., inflammation or hypoxia) or signal if in the presence of that state or signal, 1transcription from the promoter is activated, deactivated, increased, or decreased. In some embodiments, the promoter comprises a response element. A “response element” is a short sequence of DNA within a promoter region that binds specific molecules (e.g., transcription factors, or an engineered polypeptide as described herein, such as an ACP) that modulate (regulate) gene expression from the promoter. Response elements that may be used in accordance with the present disclosure include, without limitation, a phloretin-adjustable control element (PEACE), a zinc-finger DNA-binding domain (DBD), an interferon-gamma-activated sequence (GAS) (Decker, T. et al. J Interferon Cytokine Res. 1997 Mar; 17(3): 121-34, incorporated herein by reference), an interferon-stimulated response element (ISRE) (Han, K. J. et al. J Biol Chem. 2004 Apr 9;279(15): 15652-61, incorporated herein by reference), a NF- kappaB response element (Wang, V. et al. Cell Reports 2012; 2(4): 824-839, incorporated herein by reference), and a STAT3 response element (Zhang, D. et al. J of Biol Chem. 1996; 271: 9503-9509, incorporated herein by reference). Other response elements are encompassed herein. Response elements can also contain tandem repeats (e.g., consecutive repeats of the same nucleotide sequence encoding the response element) to generally increase sensitivity of the response element to its cognate binding molecule. Tandem repeats can be labeled 2X, 3X, 4X, 5X, etc. to denote the number of repeats present.

[0121] Non-limiting examples of responsive promoters (also referred to as “inducible promoters”) (e.g., TGF-beta responsive promoters) are listed in Table 3. Additionally, Table 3 provides exemplary promoter and transcription factor combinations that can be used in accordance with the present disclosure, as well as the effect of a select inducer molecule on said promoter and transcription factor combinations. For example, transcription factor response to an inducer is shown in the TF column (where A represents activation; DA represents deactivation; and DR represents derepression) and transgene transcription response to an inducer is shown in the T column (where B represents binding; D represents dissociation; and n.d. represents not determined) (see Homer, M. & Weber, W. FEBS Fetters 586 (2012) 20784-2096m, and references cited therein). Non-limiting examples of components that may be included in an inducible promoter (e.g., minimal promoters and responsive elements) are shown in Table 4.Table 3. Exemplary Inducible PromotersTable 4. Exemplary Components of Inducible Promoters

[0122] Other non-limiting examples of promoters include the cytomegalovirus (CMV) promoter, the elongation factor 1-alpha (EFla) promoter, the elongation factor (EFS) promoter, the MND promoter (a synthetic promoter that contains the U3 region of a modified MoMuLV LTR with myeloproliferative sarcoma virus enhancer), the phosphoglycerate kinase (PGK)promoter, the spleen focus-forming 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.Table 5. Exemplary Constitutive Promoters

[0123] In some embodiments, the promoter sequence is derived from a promoter selected from: minP, NFkB response element, CREB response element, NFAT response element, SRFresponse element 1, SRF response element 2, API response element, TCF-LEF response element promoter fusion, Hypoxia responsive element, SMAD binding element, STAT3 binding site, minCMV, YB_TATA, minTK, inducer molecule responsive promoters, and tandem repeats thereof.

[0124] 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, hEFlaVl, hCAGG, hEFlaV2, hACTb, heIF4Al, hGAPDH, hGRP78, hGRP94, hHSP70, hKINb, and hUBIb.

[0125] In some embodiments, an ACP-responsive promoter is a synthetic promoter. In some embodiments, the ACP-responsive promoter comprises a minimal promoter. In some embodiments, the ACP-binding domain comprises one or more zinc finger binding sites. In some embodiments, an ACP-binding domain comprises 1, 2, 3, 4, 5 ,6, 7, 8, 9, 10, or more zinc finger binding sites. In some embodiments, the ACP-binding domain comprises one zinc finger binding site. In some embodiments, the ACP-binding domain comprises two zinc finger binding sites. In some embodiments, the ACP-binding domain comprises three zinc finger binding sites. In some embodiments, the ACP-binding domain comprises four zinc finger binding sites. In some embodiments, an ACP-binding domain comprises the polynucleotide sequence of: cgggtttcgtaacaatcgcatgaggattcgcaacgccttcGGCGTAGCCGATGTCGCGctcccgtctcagtaaaggtcGG CGTAGCCGATGTCGCGcaatcggactgccttcgtacGGCGTAGCCGATGTCGCGcgtatcagtcgcctcgg aacGGCGTAGCCGATGTCGCGcattcgtaagaggctcactctcccttacacggagtggataACTAGTTCTAGA GGGTATATAATGGGGGCCA (SEQ ID NO: 100).

[0126] In some embodiments, an ACP-responsive promoter comprises an enhancer that promotes transcription when an antigen recognizing receptor engages a cognate antigen, e.g., an antigen expressed on a target cell. Enhancers can include, but are not limited to, enhancers enriched in the ATAC-seq of activated T cells (Gate et al. Nat Genet. Author manuscript; available in PMC 2019 Jan 9; herein incorporated by reference for all purposes) or enhancers associated with upregulated genes in single-cell RNA seq data (Xhangolli et al. Genomics Proteomics Bioinformatics. 2019 Apr; 17(2): 129- 139. Doi: 10.1016 / j.gpb.2019.03.002; herein incorporated by reference for all purposes). In some embodiments, an enhancer is or comprises a synthetic enhancer. Synthetic enhancers can include multiple iterations of transcription factor binding sites. In some embodiments, a synethetic enhancer comprises one or more (e.g., one, two, three, four, five, or more) iterations of one or more distinct transcription factor binding sites. In some embodiments, a synthetic enhancer comprises four iterations of two distinct transcription factor binding sites in an aaaabbbb or abababab organization. Illustrative nonlimiting examples of genes from which enhancers can be derived include, but are not limited to,ATF2, ATF7, BACH1, BATF, Bcl-6, Blimp-1, BMH, CBFB, CREB1, CREM, CTCF, E2F1, EBF1, EGR1, ETV6, FOS, F0XA1, FOXA2, GATA3, HIF1A, IKZF1, IKZF2, IRF4, JUN, JUNB, JUND, Lefl, NFAT, NFIA, NFIB, NFKB, NR2F1, Nur77, PU.l, RELA, RUNX3, SCRT1, SCRT2, SP1, STAT4, STAT5A, T-Bet, Tcf7, ZBED1, ZNF143, or ZNF217.Post-Transcriptional Regulatory Elements

[0127] In some embodiments, an engineered nucleic acid of the present disclosure comprises a post-transcriptional regulatory element (PRE). In some embodiments, a PRE can enhance gene expression via enabling tertiary RNA structure stability and 3’ end formation. Non-limiting examples of PREs include the Hepatitis B virus PRE (HPRE) and the Woodchuck Hepatitis Virus PRE (WPRE). In some embodiments, the post-transcriptional regulatory element is a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE). In some embodiments, the WPRE comprises the alpha, beta, and gamma components of the WPRE element. In some embodiments, the WPRE comprises the alpha component of the WPRE element.Engineered Cells

[0128] Also provided herein are engineered cells, and methods of producing engineered cells, that comprise one or more engineered nucleic acids of the present disclosure. Accordingly, provided engineered cells, which typically contain one or more engineered nucleic acids as described herein, do not occur in nature. In some embodiments, engineered cells are isolated (e.g., an engineered cell may be substantially isolated from other cell types including nonengineered cells and / or other distinct engineered cells). In some embodiments, an engineered cell is isolated for a particular use (e.g., any use described herein).. In some embodiments, an engineered cell comprises one or more vectors that comprises one or more engineered nucleic acids as described. In some embodiments, an engineered cell comprises one or more loci within its genome that comprise one or more engineered nucleic acids as described herein. In some embodiments, engineered cells comprise at least one promoter operably linked to an exogenous polynucleotide sequence (e.g., a polynucleotide sequence encoding any engineered polypeptide as described herein).

[0129] An engineered cell of the present disclosure, in some embodiments, comprises an engineered nucleic acid integrated into the cell’s genome. In some embodiments, an engineered cell comprises an engineered nucleic acid capable of expression without integrating into the cell’s genome. In some embodiments, an engineered cell comprises a vector that comprises an engineered nucleic acid. In some embodiments, an engineered cell is contacted with anengineered nucleic acid (e.g., in the form of an mRNA) so that said engineered nucleic acid is expressed in the engineered cell.Engineered Cell Types

[0130] In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is a human cell. In some embodiments, an engineered cell or isolated engineered cell is a human primary cell. In some embodiments, an primary cell is a somatic cell. In some embodiments, a primary cell is a stem cell. In some embodiments, a primary cell is an induced pluripotent stem cell (iPSC). In some embodiments, an engineered cell is derived from a subject. In some embodiments, an engineered cell is allogeneic with reference to a subject.

[0131] An engineered cell of the present disclosure, in some embodiments, can be made using a cell isolated from a subject. In some embodiments, a subject is known to have, or is suspected of having, cancer. In some embodiments, an engineered cell is made using a cancer cell isolated from a subject. Cell isolation methods include, but are not limited to, sorting techniques based on cell-surface marker expression, such as FACS sorting, positive isolation techniques, and negative isolation, magnetic isolation, and combinations thereof. An engineered cell can be allogenic with reference to a subject being administered a treatment. Allogenic modified cells can be HLA-matched to a subject being administered a treatment. An engineered cell can be a cultured cell, such as an ex vivo cultured cell. An engineered cell can be an ex vivo cultured cell, such as a primary cell isolated from a subject. Cultured cell can be cultured with one or more cytokines.

[0132] In some embodiments, an engineered or isolated engineered cell of the present disclosure is selected from: a T cell (e.g., a CD8+ T cell, a CD4+ T cell, or a gamma-delta T cell), a cytotoxic T lymphocyte (CTL), a regulatory T cell, a Natural Killer T (NKT) cell, a Natural Killer (NK) cell, a B cell, a tumor-infiltrating lymphocyte (TIL), an innate lymphoid cell, a mast cell, an eosinophil, a basophil, a neutrophil, a myeloid cell, a macrophage (e.g., an Ml macrophage or an M2 macrophage), a monocyte, a dendritic cell, an erythrocyte, a platelet cell, a neuron, an oligodendrocyte, an astrocyte, a placode-derived cell, a Schwann cell, a cardiomyocyte, an endothelial cell, a nodal cell, a microglial cell, a hepatocyte, a cholangiocyte, a beta cell, a human embryonic stem cell (ESC), an ESC-derived cell, a pluripotent stem cell, a mesenchymal stromal cell (MSC), an induced pluripotent stem cell (iPSC), and an iPSC-derived cell.

[0133] In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is a T cell (e.g., a , a CD8+ T cell, a CD4+ T cell, or a gamma-delta T cell). In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is acytotoxic T lymphocyte (CTL). In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is a regulatory T cell. In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is a Natural Killer T (NKT) cell. In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is a Natural Killer (NK) cell. In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is a B cell. In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is a tumor-infiltrating lymphocyte (TIL). In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is an innate lymphoid cell. In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is a mast cell. In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is an eosinophil. In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is a basophil. In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is a neutrophil. In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is a myeloid cell. In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is a macrophage e.g., an Ml macrophage or an M2 macrophage). In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is a monocyte. In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is a dendritic cell. In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is an erythrocyte. In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is a platelet cell. In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is a neuron. In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is an oligodendrocyte. In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is an astrocyte. In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is a placode-derived cell. In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is a Schwann cell. In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is a cardiomyocyte. In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is an endothelial cell. In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is a nodal cell. In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is a microglial cell. In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is a hepatocyte. In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is a cholangiocyte. In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is a beta cell. In someembodiments, an engineered cell or isolated engineered cell of the present disclosure is a human embryonic stem cell (ESC). In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is an ESC-derived cell. In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is a pluripotent stem cell. In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is a mesenchymal stromal cell (MSC). In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is an induced pluripotent stem cell (iPSC). In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is an iPSC- derived cell. In some embodiments, an engineered cell is autologous. In some embodiments, an engineered cell is allogeneic. In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is a CD34+ cell, a CD3+ cell, a CD8+ cell, a CD 16+ cell, and / or a CD4+ cell.

[0134] In some embodiments, an engineered cell of the present disclosure is a cell therapy modality. In some embodiments, a cell therapy modality can express one or more therapeutic proteins. In some embodiments, a therapeutic protein is a chimeric antigen receptor (CAR) or a modified T cell receptor (TCR). In some embodiments, a cell therapy modality is a CAR-T cell or a CAR-NK cell. In some embodiments, a cell therapy modality is a CAR-T cell. In some embodiments, a cell therapy modality is a CAR-NK cell.

[0135] In some embodiments, an engineered cell of the present disclosure is a tumor cell. In some embodiments, an engineered cell of the present disclosure is selected from: an adenocarcinoma cell, a bladder tumor cell, a brain tumor cell, a breast tumor cell, a cervical tumor cell, a colorectal tumor cell, an esophageal tumor cell, a glioma cell, a kidney tumor cell, a liver tumor cell, a lung tumor cell, a melanoma cell, a mesothelioma cell, an ovarian tumor cell, a pancreatic tumor cell, a prostate tumor cell, a skin tumor cell, a thyroid tumor cell, and a uterine tumor cell.

[0136] In some embodiments, an engineered cell of the present disclosure is a bacterial cell. In some embodiments, an engineered cell of the present disclosure is selected from: Clostridium beijerinckii, Clostridium sporogenes, Clostridium novyi, Escherichia coli, Pseudomonas aeruginosa, Listeria monocytogenes, Salmonella typhimurium, and Salmonella choleraesuis.

[0137] Also provided herein are methods of culturing the engineered cells of the present disclosure. Provided engineered cells may be cultured using any suitable method. One skilled in the art will recognize that culturing conditions will depend on the particular engineered cell of interest. One skilled in the art will recognize that culturing conditions will depend on the specific downstream use of an engineered cell, for example, specific culturing conditions for subsequent administration of an engineered cell to a subject.Methods of Engineering Cells

[0138] Also provided herein are compositions and methods for engineering cells with any nucleic acid as described herein.

[0139] Provided engineered cells may be engineered through introduction (z.e., delivery) of one or more polynucleotides (e.g., engineered nucleic acids) of the present disclosure. Delivery methods include, but are not limited to, viral-mediated delivery, lipid-mediated transfection, nanoparticle delivery, electroporation, sonication, and cell membrane deformation by physical means. One skilled in the art will appreciate the choice of delivery method can depend on the specific cell type to be engineered.

[0140] In some embodiments, an engineered cell is transduced using an oncolytic virus. Examples of oncolytic viruses include, but are not limited to, an oncolytic herpes simplex virus, an oncolytic adenovirus, an oncolytic measles virus, an oncolytic influenza virus, an oncolytic Indiana vesiculovirus, an oncolytic Newcastle disease virus, an oncolytic vaccinia virus, an oncolytic poliovirus, an oncolytic myxoma virus, an oncolytic reovirus, an oncolytic mumps virus, an oncolytic Maraba virus, an oncolytic rabies virus, an oncolytic rotavirus, an oncolytic hepatitis virus, an oncolytic rubella virus, an oncolytic dengue virus, an oncolytic chikungunya virus, an oncolytic respiratory syncytial virus, an oncolytic lymphocytic choriomeningitis virus, an oncolytic morbillivirus, an oncolytic lentivirus, an oncolytic replicating retrovirus, an oncolytic rhabdovirus, an oncolytic Seneca Valley virus, an oncolytic sindbis virus, and any variant or derivative thereof. In some embodiments, an oncolytic virus is a recombinant oncolytic virus. In some embodiments, a recombinant oncolytic virus comprises an engineered nucleic acid as provided herein. In some embodiments, an oncolytic virus is a recombinant oncolytic virus comprising a first expression cassette and a second expression cassette. In some embodiments, an oncolytic virus further comprises a third expression cassette.

[0141] In some embodiments, a virus as provided in accordance with the present disclosure is an oncolytic virus. In some embodiments, avirus is a recombinant virus that encodes one more transgenes encoding one or more effector molecules, such as any of the engineered nucleic acids described herein. In some embodiments, an engineered cell is engineered via transduction with an oncolytic virus.Viral-Mediated Delivery

[0142] Any suitable viral vector-based delivery platform can be used to make engineered cells of the present disclosure. In general, a viral vector-based delivery platform engineers a cell through introducing (i.e., delivering) a nucleic acid payload (e.g., any engineered nucleic acid as described herein) into a host cell. A viral vector-based delivery platform may compriseengineered or recombinant viruses to deliver engineered nucleic acids of the present disclosure to a cell.

[0143] A viral vector-based delivery platform can deliver one or more nucleic acid payloads, e.g., an engineered nucleic acid, gene, or transgene as described herein, within the same nucleic acid. In some embodiments, a viral vector-based delivery platform (e.g., a platform using a recombinant virus or an engineered virus) can deliver one or more transgenes, including, but not limited to, any engineered nucleic acid described herein that encodes one or more effector molecules. In some embodiments, one or more transgenes encoding one or more effector molecules can be configured to express the one or more effector molecules. A viral vector-based delivery platform can encode one or more genes in addition to the one or more transgenes (e.g., transgenes encoding the one or more effector molecules), such as viral genes needed for viral infectivity and / or viral production (e.g., capsid proteins, envelope proteins, viral polymerases, viral transcriptases, etc.), referred to as cis-acting elements or genes.

[0144] A viral vector-based delivery platform can comprise more than one viral vector, such as separate viral vectors encoding the engineered nucleic acids, genes, or transgenes described herein, and referred to as trans-acting elements or genes. For example, a helper-dependent viral vector-based delivery platform can provide additional genes needed for viral infectivity and / or viral production on one or more additional separate vectors in addition to the vector encoding the one or more effector molecules. One viral vector can deliver more than one engineered nucleic acids, such as one vector that delivers engineered nucleic acids that are configured to produce two or more effector molecules. More than one viral vector can deliver more than one engineered nucleic acids, such as more than one vector that delivers one or more engineered nucleic acid configured to produce one or more effector molecules. The number of viral vectors used can depend on the packaging capacity of the above mentioned viral vector-based vaccine platforms, and one skilled in the art can select the appropriate number of viral vectors.

[0145] In general, any of the viral vector-based systems can be used for the in vitro production of molecules, such as effector molecules, or used in vivo and ex vivo gene therapy procedures, e.g., for in vivo delivery of the engineered nucleic acids encoding one or more effector molecules. The selection of an appropriate viral vector-based system will depend on a variety of factors, such as cargo / payload size, immunogenicity of the viral system, target cell of interest, gene expression strength and timing, and other factors appreciated by one skilled in the art.

[0146] Viral vector-based delivery platforms can be RNA-based viruses or DNA-based viruses. Exemplary viral vector-based delivery platforms include, but are not limited to, a herpes simplex virus, a adenovirus, a measles virus, an influenza virus, a Indiana vesiculovirus, aNewcastle disease virus, a vaccinia virus, a poliovirus, a myxoma virus, a reovirus, a mumps virus, a Maraba virus, a rabies virus, a rotavirus, a hepatitis virus, a rubella virus, a dengue virus, a chikungunya virus, a respiratory syncytial virus, a lymphocytic choriomeningitis virus, a morbillivirus, a lentivirus, a replicating retrovirus, a rhabdovirus, a Seneca Valley virus, a sindbis virus, and any variant or derivative thereof. Other exemplary viral vector-based delivery platforms are described in the art, such as vaccinia, fowlpox, self-replicating alphavirus, marabavirus, adenovirus (See, e.g., Tatsis et al., Adenoviruses, Molecular Therapy (2004) 10, 616 — 629), or lentivirus, including but not limited to second, third or hybrid second / third generation lentivirus and recombinant lentivirus of any generation designed to target specific cell types or receptors (See, 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 containing the human ubiquitin C promoter, Nucl. Acids Res. (2015) 43 (1): 682-690, Zufferey et al., Self-Inactivating Lentivirus Vector for Safe and Efficient In vivo Gene Delivery, J. Virol. (1998) 72 (12): 9873- 9880).

[0147] The sequences may be preceded with one or more sequences targeting a subcellular compartment. Upon introduction (i.e. delivery) into a host cell, infected cells (i.e., an engineered cell) can express, and in some case secrete, the one or more effector molecules. Vaccinia vectors and methods useful in immunization protocols are described in, e.g., U.S. Pat. No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover et al. (Nature 351:456-460 (1991)). A wide variety of other vectors useful for the introduction (i.e., delivery) of engineered nucleic acids, e.g., Salmonella typhi vectors, and the like will be apparent to those skilled in the art from the description herein.

[0148] The viral vector-based delivery platforms can be a virus that targets a tumor cell, herein referred to as an oncolytic virus. Examples of oncolytic viruses include, but are not limited to, an oncolytic herpes simplex virus, an oncolytic adenovirus, an oncolytic measles virus, an oncolytic influenza virus, an oncolytic Indiana vesiculovirus, an oncolytic Newcastle disease virus, an oncolytic vaccinia virus, an oncolytic poliovirus, an oncolytic myxoma virus, an oncolytic reovirus, an oncolytic mumps virus, an oncolytic Maraba virus, an oncolytic rabies virus, an oncolytic rotavirus, an oncolytic hepatitis virus, an oncolytic rubella virus, an oncolytic dengue virus, an oncolytic chikungunya virus, an oncolytic respiratory syncytial virus, an oncolytic lymphocytic choriomeningitis virus, an oncolytic morbillivirus, an oncolytic lentivirus, an oncolytic replicating retrovirus, an oncolytic rhabdovirus, an oncolytic Seneca Valley virus, an oncolytic sindbis virus, and any variant or derivative thereof. Any of theoncolytic viruses described herein can be a recombinant oncolytic virus comprising one more transgenes (e.g., an engineered nucleic acid) encoding one or more effector molecules. The transgenes encoding the one or more effector molecules can be configured to express the one or more effector molecules.

[0149] In some embodiments, the virus is selected from: a lentivirus, a retrovirus, an oncolytic virus, an adenovirus, an adeno-associated virus (AAV), and a virus-like particle (VLP).

[0150] The viral vector-based delivery platform can be retrovirus-based. In general, retroviral vectors are comprised of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the one or more engineered nucleic acids (e.g., transgenes encoding the one or more effector molecules) into the target cell to provide permanent transgene expression. Retroviral-based delivery systems include, but are not limited to, those based upon murine leukemia, virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency vims (SIV), human immunodeficiency vims (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol. 66:2731-2739 (1992); Johann et ah, J. Virol. 66:1635-1640 (1992); Sommnerfelt et al., Virol. 176:58-59 (1990); Wilson et ah, J. Virol. 63:2374-2378 (1989); Miller et al, J, Virol. 65:2220-2224 (1991); PCT / US94 / 05700). Other retroviral systems include the Phoenix retrovirus system.

[0151] The viral vector-based delivery platform can be lentivirus-based. In general, lentiviral vectors are retroviral vectors that are able to transduce or infect non-dividing cells and typically produce high viral titers. Lentiviral-based delivery platforms can be HIV -based, such as ViraPower systems (ThermoFisher) or pLenti systems (Cell Biolabs). . Lentiviral-based delivery platforms can be SIV, or FIV-based. Other exemplary lentivirus-based delivery platforms are described in more detail in U.S. Pat. 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; 6,955,919, each herein incorporated by reference for all purposes.

[0152] The viral vector-based delivery platform can be adenovirus-based. In general, adenoviral based vectors are capable of very high transduction efficiency in many cell types, do not require cell division, achieve high titer and levels of expression, and can be produced in large quantities in a relatively simple system. In general, adenoviruses can be used for transient expression of a transgene within an infected cell since adenoviruses do not typically integrate into a host’s 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;WO 94 / 12649, WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984 and WO 95 / 00655, each herein incorporated by reference for all purposes. Other exemplary adenovirus-based delivery platforms are described in more detail in U.S. Pat. Nos. 5585362; 6,083,716, 7,371,570; 7,348,178; 7,323,177; 7,319,033; 7,318,919; and 7,306,793 and International Patent Application WO96 / 13597, each herein incorporated by reference for all purposes.

[0153] The viral vector-based delivery platform can be adeno-associated virus (AAV)-based. Adeno-associated virus (“AAV”) vectors may be used to transduce cells with engineered nucleic acids (e.g., any of the engineered nucleic acids described herein). AAV systems can be used for the in vitro production of effector molecules, or used in vivo and ex vivo gene therapy procedures, e.g., for in vivo delivery of the engineered nucleic acids encoding one or more effector molecules (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. 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; US 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 herein incorporated by reference for all purposes). Exemplary methods for constructing recombinant AAV vectors are described in more detail in U.S. Pat. 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 herein incorporated by reference for all purposes. In general, an AAV-based vector comprises a capsid protein having an amino acid sequence corresponding to any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV.RhlO, AAV11 and variants thereof.

[0154] The viral vector-based delivery platform can be a virus-like particle (VLP) platform. In general, VLPs are constructed by producing viral structural proteins and purifying resulting viral particles. Then, following purification, a cargo / payload (e.g., any of the engineered nucleic acids described herein) is encapsulated within the purified particle ex vivo. Accordingly, production of VLPs maintains separation of the nucleic acids encoding viral structural proteins and the nucleic acids 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 translation expression systems. The purified viral particles can be denatured and reformed in the presence of the desired cargo to produce VLPs using any suitable method. Production of VLPs are described in more detail in Seow et al. (Mol Ther. 2009 May; 17(5): 767-777), herein incorporated by reference for all purposes.

[0155] The viral vector-based delivery platform can be engineered to target (i.e., infect) a range of cells, target a narrow subset of cells, or target a specific cell. In general, the envelope protein chosen for the viral vector-based delivery platform will determine the viral tropism. The virus used in the viral vector-based delivery platform can be pseudotyped to target a specific cell of interest. The viral vector-based delivery platform can be pantropic and infect a range of cells. For example, pantropic viral vector-based delivery platforms can include the VSV-G envelope. The viral vector-based delivery platform can be amphotropic and infect mammalian cells. Accordingly, one skilled in the art can select the appropriate tropism, pseudotype, and / or envelope protein for targeting a desired cell type.Lipid Structure Delivery Systems

[0156] Engineered nucleic acids of the present disclosure (e.g., any of the engineered nucleic acids described herein) can be introduced into a cell using a lipid-mediated delivery system. In general, a lipid-mediated delivery system uses a structure composed of an outer lipid membrane enveloping an internal compartment. Examples of lipid-based structures include, but are not limited to, a lipid-based nanoparticle, a liposome, a micelle, an exosome, a vesicle, an extracellular vesicle, a cell, or a tissue. Lipid structure delivery systems can deliver a cargo / payload (e.g., any of the engineered nucleic acids described herein) in vitro, in vivo, or ex vivo.

[0157] A lipid-based nanoparticle can include, but is not limited to, a unilamellar liposome, a multilamellar liposome, and a lipid preparation. As used herein, a “liposome” is a generic term encompassing in vitro preparations of lipid vehicles formed by enclosing a desired cargo, e.g., an engineered nucleic acid, such as any of the engineered nucleic acids described herein, within a lipid shell or a lipid aggregate. Liposomes may be characterized as having vesicular structures with a bilayer membrane, generally comprising a phospholipid, and an inner medium that generally comprises an aqueous composition. Liposomes include, but are not limited to, emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. Liposomes can be unilamellar liposomes. Liposomes can be multilamellar liposomes. Liposomes can be multivesicular liposomes. Liposomes can be positively charged, negatively charged, or neutrally charged. In certain embodiments, the liposomes are neutral in charge. Liposomes can be formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of a desired purpose, e.g., criteria for in vivo delivery, such as liposome size, acid lability and stability of the liposomes in the blood stream. A variety of methods are available for preparing liposomes, asdescribed in, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng. 9; 467 (1980), U.S. Pat. Nos. 4,235,871, 4,501,728, 4,501,728, 4,837,028, and 5,019,369, each herein incorporated by reference for all purposes.

[0158] A multilamellar liposome is generated spontaneously when lipids comprising phospholipids are suspended in an excess of aqueous solution such that multiple lipid layers are separated by an aqueous medium. Water and dissolved solutes are entrapped in closed structures between the lipid bilayers following the lipid components undergoing self-rearrangement. A desired cargo (e.g., a polypeptide, a nucleic acid, a small molecule drug, an engineered nucleic acid, such as any of the engineered nucleic acids described herein, a viral vector, a viral-based delivery system, etc.) can be encapsulated in the aqueous interior of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the polypeptide / nucleic acid, interspersed within the lipid bilayer of a liposome, entrapped in a liposome, complexed with a liposome, or otherwise associated with the liposome such that it can be delivered to a target entity. Lipophilic molecules or molecules with lipophilic regions may also dissolve in or associate with the lipid bilayer.

[0159] A liposome used according to the present embodiments can be made using any suitable method. Preparations of liposomes are described in further detail in WO 2016 / 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 herein incorporated by reference for all purposes.

[0160] Liposomes can be cationic liposomes. Examples of cationic liposomes are described in more detail in U.S. Patent No. 5,962,016; 5,030,453; 6,680,068, U.S. Application 2004 / 0208921, and International Patent Applications W003 / 015757A1, WO04029213A2, and W002 / 100435A1, each hereby incorporated by reference in their entirety.

[0161] Lipid-mediated gene delivery methods are described, for instance, in WO 96 / 18372; WO 93 / 24640; Mannino & Gould-Fogerite, BioTechniques 6(7): 682-691 (1988); U.S. Pat. No. 5,279,833 Rose U.S. Pat. No. 5,279,833; W091 / 06309; and Feigner et al., Proc. Natl. Acad. Sci. USA 84: 7413-7414 (1987), each herein incorporated by reference for all purposes.

[0162] Exosomes are small membrane vesicles of endocytic origin that are released into the extracellular environment following fusion of multivesicular bodies with the plasma membrane. The size of exosomes ranges between 30 and 100 nm in diameter. Their surface consists of a lipid bilayer from the donor cell’s cell membrane, and they contain cytosol from the cell that produced the exosome, and exhibit membrane proteins from the parental cell on the surface. Any exosomes useful for the delivery of nucleic acids can be used in accordance with the presentdisclosure, e.g., the exosomes described in more detail in U.S. Pat. No. 9,889,210, herein incorporated by reference for all purposes.

[0163] As used herein, the term “extracellular vesicle” or “EV” refers to a cell-derived vesicle comprising a membrane that encloses an internal space. In general, extracellular vesicles comprise all membrane-bound vesicles that have a smaller diameter than the cell from which they are derived. Generally extracellular vesicles range in diameter from 20 nm to 1000 nm, and can comprise various macromolecular cargo either within the internal space, displayed on the external surface of the extracellular vesicle, and / or spanning the membrane. The cargo can comprise nucleic acids (e.g., any of the engineered nucleic acids described herein), proteins, carbohydrates, lipids, small molecules, and / or combinations thereof. By way of example and without limitation, extracellular vesicles include apoptotic bodies, fragments of cells, vesicles derived from cells by direct or indirect manipulation (e.g., by serial extrusion or treatment with alkaline solutions), vesiculated organelles, and vesicles produced by living cells (e.g., by direct plasma membrane budding or fusion of the late endosome with the plasma membrane). Extracellular vesicles can be derived from a living or dead organism, explanted tissues or organs, and / or cultured cells.

[0164] As used herein the term “exosome” refers to a cell-derived small (between 20-300 nm in diameter, more preferably 40-200 nm in diameter) vesicle comprising a membrane that encloses an internal space, and which is generated from the cell by direct plasma membrane budding or by fusion of the late endosome with the plasma membrane. The exosome comprises lipid or fatty acid and polypeptide and optionally comprises a payload (e.g., a therapeutic agent), a receiver (e.g., a targeting moiety), a polynucleotide (e.g., a nucleic acid, RNA, or DNA, such as any of the engineered nucleic acids described herein), a sugar (e.g., a simple sugar, polysaccharide, or glycan) or other molecules. The exosome can be derived from a producer cell, and isolated from the producer cell based on its size, density, biochemical parameters, or a combination thereof. An exosome is a species of extracellular vesicle. Generally, exosome production / biogenesis does not result in the destruction of the producer cell. Exosomes and preparation of exosomes are described in further detail in WO 2016 / 201323, which is hereby incorporated by reference in its entirety.

[0165] As used herein, the term “nanovesicle” (also referred to as a “microvesicle”) refers to a cell-derived small (between 20-250 nm in diameter, more preferably 30-150 nm in diameter) vesicle comprising a membrane that encloses an internal space, and which is generated from the cell by direct or indirect manipulation such that said nanovesicle would not be produced by said producer cell without said manipulation. In general, a nanovesicle is a sub-species of an extracellular vesicle. Appropriate manipulations of the producer cell include but are not limitedto serial extrusion, treatment with alkaline solutions, sonication, or combinations thereof. The production of nanovesicles may, in some instances, result in the destruction of said producer cell. Preferably, populations of nanovesicles are substantially free of vesicles that are derived from producer cells by way of direct budding from the plasma membrane or fusion of the late endosome with the plasma membrane. The nanovesicle comprises lipid or fatty acid and polypeptide, and optionally comprises a payload (e.g., a therapeutic agent), a receiver (e.g., a targeting moiety), a polynucleotide (e.g., a nucleic acid, RNA, or DNA, such as any of the engineered nucleic acids described herein), a sugar (e.g., a simple sugar, polysaccharide, or glycan) or other molecules. The nanovesicle, once it is derived from a producer cell according to said manipulation, may be isolated from the producer cell based on its size, density, biochemical parameters, or a combination thereof.

[0166] Lipid nanoparticles (LNPs), in general, are synthetic lipid structures that rely on the amphiphilic nature of lipids to form membranes and vesicle like structures (Riley 2017). In general, these vesicles deliver cargo / pay loads, such as any of the engineered nucleic acids or viral systems described herein, by absorbing into the membrane of target cells and releasing the cargo into the cytosol. Lipids used in LNP formation can be cationic, anionic, or neutral. The lipids can be synthetic or naturally derived, and in some instances biodegradable. Lipids can include fats, cholesterol, phospholipids, lipid conjugates including, but not limited to, polyethyleneglycol (PEG) conjugates (PEGylated lipids), waxes, oils, glycerides, and fat soluble vitamins. Lipid compositions generally include defined mixtures of materials, such as the cationic, neutral, anionic, and amphipathic lipids. In some instances, specific lipids are included to prevent LNP aggregation, prevent lipid oxidation, or provide functional chemical groups that facilitate attachment of additional moieties. Lipid composition can influence overall LNP size and stability. In an example, the lipid composition comprises dilinoleylmethyl- 4- dimethylaminobutyrate (MC3) or MC3-like molecules. MC3 and MC3-like lipid compositions can be formulated to include one or more other lipids, such as a PEG or PEG-conjugated lipid, a sterol, or neutral lipids. In addition, LNPs can be further engineered or functionalized to facilitate targeting of specific cell types. Another consideration in LNP design is the balance between targeting efficiency and cytotoxicity.

[0167] Micelles, in general, are spherical synthetic lipid structures that are formed using single-chain lipids, where the single-chain lipid’s hydrophilic head forms an outer layer or membrane and the single-chain lipid’ s hydrophobic tails form the micelle center. Micelles typically refer to lipid structures only containing a lipid mono-layer. Micelles are described in more detail in Quader et al. (Mol Ther. 2017 Jul 5; 25(7): 1501-1513), herein incorporated by reference for all purposes.

[0168] Nucleic-acid vectors, such as expression vectors, exposed directly to serum can have several undesirable consequences, including degradation of the nucleic acid by serum nucleases or off-target stimulation of the immune system by the free nucleic acids. Similarly, viral delivery systems exposed directly to serum can trigger an undesired immune response and / or neutralization of the viral delivery system. Therefore, encapsulation of an engineered nucleic acid and / or viral delivery system can be used to avoid degradation, while also avoiding potential off-target effects. In certain examples, an engineered nucleic acid and / or viral delivery system is fully encapsulated within the delivery vehicle, such as within the aqueous interior of an LNP. Encapsulation of an engineered nucleic acid and / or viral delivery system within an LNP can be carried out using any suitable method, such as microfluidic mixing and droplet generation carried out on a microfluidic droplet generating device. Such devices include, but are not limited to, standard T-junction devices or flow-focusing devices. In an example, the desired lipid formulation, such as MC3 or MC3-like containing compositions, is provided to the droplet generating device in parallel with an engineered nucleic acid or viral delivery system and any other desired agents, such that the delivery vector and desired agents are fully encapsulated within the interior of the MC3 or MC3-like based LNP. In an example, the droplet generating device can control the size range and size distribution of the LNPs produced. Lor example, the LNP can have a size ranging from 1 to 1000 nanometers in diameter, e.g., 1, 10, 50, 100, 500, or 1000 nanometers. Pollowing droplet generation, the delivery vehicles encapsulating the cargo / payload (e.g., an engineered nucleic acid and / or viral delivery system) can be further treated or engineered to prepare them for administration.Nanoparticle Delivery

[0169] Nanomaterials can be used to deliver engineered nucleic acids (e.g., any of the engineered nucleic acids described herein). Nanomaterial vehicles, importantly, can be made of non-immunogenic materials and generally avoid eliciting immunity to the delivery vector itself. These materials can include, but are not limited to, lipids (as previously described), inorganic nanomaterials, and other polymeric 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), herein incorporated by reference for all purposes.Genomic Editing Systems

[0170] A genomic editing systems can be used to engineer a host genome to encode an engineered nucleic acid, such as an engineered nucleic acid of the present disclosure. In general, a “genomic editing system” refers to any system for integrating an exogenous gene into a hostcell’s genome. Genomic editing systems include, but are not limited to, a transposon system, a nuclease genomic editing system, and a viral vector-based delivery platform.

[0171] A transposon system can be used to integrate an engineered nucleic acid, such as an engineered nucleic acid of the present disclosure, into a host genome. Transposons generally comprise terminal inverted repeats (TIR) that flank a cargo / payload nucleic acid and a transposase. The transposon system can provide the transposon in cis or in trans with the TIR- flanked cargo. A transposon system can be a retrotransposon system or a DNA transposon system. In general, transposon systems integrate a cargo / payload (e.g., an engineered nucleic acid) randomly into a host genome. Examples of transposon systems include systems using a transposon of the Tcl / mariner transposon superfamily, such as a Sleeping Beauty transposon system, described in more detail in Hudecek et al. (Crit Rev Biochem Mol Biol. 2017 Aug;52(4):355-380), and U.S. Patent Nos. 6,489,458, 6,613,752 and 7,985,739, each of which is herein incorporated by reference for all purposes. Another example of a transposon system includes a PiggyBac transposon system, described in more detail in U.S. Patent Nos. 6,218,185 and 6,962,810, each of which is herein incorporated by reference for all purposes.

[0172] A nuclease genomic editing system can be used to engineer a host genome to encode an engineered nucleic acid, such as an engineered nucleic acid of the present disclosure. Without wishing to be bound by theory, in general, the nuclease-mediated gene editing systems used to introduce an exogenous gene take advantage of a cell’s natural DNA repair mechanisms, particularly homologous recombination (HR) repair pathways. Briefly, following an insult to genomic DNA (typically a double-stranded break), a cell can resolve the insult by using another DNA source that has identical, or substantially identical, sequences at both its 5’ and 3’ ends as a template during DNA synthesis to repair the lesion. In a natural context, HDR can use the other chromosome present in a cell as a template. In gene editing systems, exogenous polynucleotides are introduced into the cell to be used as a homologous recombination template (HRT or HR template). In general, any additional exogenous sequence not originally found in the chromosome with the lesion that is included between the 5’ and 3’ complimentary ends within the HRT (e.g., a gene or a portion of a gene) can be incorporated (i.e., “integrated”) into the given genomic locus during templated HDR. Thus, a typical HR template for a given genomic locus has a nucleotide sequence identical to a first region of an endogenous genomic target locus, a nucleotide sequence identical to a 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, such as any of the engineered nucleic acids encoding one or more effector molecules).

[0173] In some examples, a HR template can be linear. Examples of linear HR templates include, but are not limited to, a linearized plasmid vector, a ssDNA, a synthesized DNA, and a PCR amplified DNA. In particular examples, a HR template can be circular, such as a plasmid. A circular template can include a supercoiled template.

[0174] The identical, or substantially identical, sequences found at the 5’ and 3’ ends of the HR template, with respect to the exogenous sequence to be introduced, are generally referred to as arms (HR arms). HR arms can be identical to regions of the endogenous genomic target locus (i.e., 100% identical). HR arms in some examples can be substantially identical to regions of the endogenous genomic target locus. While substantially identical HR arms can be used, it can be advantageous for HR arms to be identical as the efficiency of the HDR pathway may be impacted by HR arms having less than 100% identity.

[0175] Each HR arm, i.e., the 5’ and 3’ HR arms, can be the same size or different sizes. Each HR arm can each be greater than or equal to 50, 100, 200, 300, 400, or 500 bases in length. Although HR arms can, in general, be of any length, practical considerations, such as the impact of HR arm length and overall template size on overall editing efficiency, can also be taken into account. An HR arms can be identical, or substantially identical to, regions of an endogenous genomic target locus immediately adjacent to a cleavage site. Each HR arms can be identical to, or substantially identical to, regions of an endogenous genomic target locus immediately adjacent to a cleavage site. Each HR arms can be identical, or substantially identical to, regions of an endogenous genomic target locus within a certain distance of a cleavage site, such as 1 base-pair, less than or equal to 10 base-pairs, less than or equal to 50 base-pairs, or less than or equal to 100 base-pairs of each other.

[0176] A nuclease genomic editing system can use a variety of nucleases to cut a target genomic locus, including, but not limited to, a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) family nuclease or derivative thereof, a Transcription activatorlike effector nuclease (TALEN) or derivative thereof, a zinc-finger nuclease (ZFN) or derivative thereof, and a homing endonuclease (HE) or derivative thereof.

[0177] A CRISPR-mediated gene editing system can be used to engineer a host genome to encode an engineered nucleic acid, such as an engineered nucleic acid encoding one or more of the effector molecules described herein. CRISPR systems are described in more detail in M. Adli (“The CRISPR tool kit for genome editing and beyond” Nature Communications; volume 9 (2018), Article number: 1911), herein incorporated by reference for all that it teaches. In general, a CRISPR-mediated gene editing system comprises a CRIS PR-associated (Cas) nuclease and a RNA(s) that directs cleavage to a particular target sequence. An exemplary CRISPR-mediated gene editing system is the CRISPR / Cas9 systems comprised of a Cas9 nuclease and a RNA(s)that has a CRISPR RNA (crRNA) domain and a trans-activating CRISPR (tracrRNA) domain. The crRNA typically has two RNA domains: a guide RNA sequence (gRNA) that directs specificity through base-pair hybridization to a target sequence (“a defined nucleotide sequence”), e.g., a genomic sequence; and an RNA domain that hybridizes to a tracrRNA. A tracrRNA can interact with and thereby promote recruitment of a nuclease (e.g., Cas9) to a genomic locus. The crRNA and tracrRNA polynucleotides can be separate polynucleotides. The crRNA and tracrRNA polynucleotides can be a single polynucleotide, also referred to as a single guide RNA (sgRNA). While the Cas9 system is illustrated here, other CRISPR systems can be used, such as the Cpfl system. Nucleases can include derivatives thereof, such as Cas9 functional mutants, e.g., a Cas9 “nickase” mutant that in general mediates cleavage of only a single strand of a defined nucleotide sequence as opposed to a complete double- stranded break typically produced by Cas9 enzymes.

[0178] In general, the components of a CRISPR system interact with each other to form a Ribonucleoprotein (RNP) complex to mediate sequence specific cleavage. In some CRISPR systems, each component can be separately produced and used to form the RNP complex. In some CRISPR systems, each component can be separately produced in vitro and contacted (i.e., “complexed”) with each other in vitro to form the RNP complex. The in vitro produced RNP can then be introduced (i.e., “delivered”) into a cell’s cytosol and / or nucleus, e.g., a T cell’s cytosol and / or nucleus. The in vitro produced RNP complexes can be delivered to a cell by a variety of means including, but not limited to, electroporation, lipid-mediated transfection, cell membrane deformation by physical means, lipid nanoparticles (LNP), virus like particles (VLP), and sonication. In a particular example, in vitro produced RNP complexes can be delivered to a cell using a Nucleofactor / Nucleofection® electroporation-based delivery system (Lonza®). Other electroporation systems include, but are not limited to, MaxCyte electroporation systems, Miltenyi CliniMACS electroporation systems, Neon electroporation systems, and BTX electroporation systems. CRISPR nucleases, e.g., Cas9, can be produced in vitro (i.e., synthesized and purified) using any suitable method. CRISPR system RNAs, e.g., an sgRNA, can be produced in vitro (i.e., synthesized and purified) using a variety of RNA production techniques, such as in vitro transcription or chemical synthesis.

[0179] An in vitro produced RNP complex can be complexed at different ratios of nuclease to gRNA. An in vitro produced RNP complex can be also be used at different amounts in a CRISPR-mediated editing system. For example, depending on the number of cells desired to be edited, the total RNP amount added can be adjusted, such as a reduction in the amount of RNP complex added when editing a large number of cells in a reaction.

[0180] In some CRISPR systems, each component (e.g., Cas9 and an sgRNA) can be separately encoded by a polynucleotide with each polynucleotide introduced into a cell together or separately. In some CRISPR systems, each component can be encoded by a single polynucleotide (i.e., a multi-promoter or multicistronic vector, see description of exemplary multicistronic systems below) and introduced into a cell. Following expression of each polynucleotide encoded CRISPR component within a cell (e.g., translation of a nuclease and transcription of CRISPR RNAs), an RNP complex can form within the cell and can then direct site-specific cleavage.

[0181] Some RNPs can be engineered to have moieties that promote delivery of the RNP into the nucleus. For example, a Cas9 nuclease can have a nuclear localization signal (NLS) domain such that if a Cas9 RNP complex is delivered into a cell’s cytosol or following translation of Cas9 and subsequent RNP formation, the NLS can promote further trafficking of a Cas9 RNP into the nucleus.

[0182] The engineered cells described herein can be engineered using non-viral methods, e.g., the nuclease and / or CRISPR mediated gene editing systems described herein can be delivered to a cell using non-viral methods. The engineered cells described herein can be engineered using viral methods, e.g., the nuclease and / or CRISPR mediated gene editing systems described herein can be delivered to a cell using viral methods such as adenoviral, retroviral, lentiviral, or any of the other viral-based delivery methods described herein.

[0183] In some CRISPR systems, more than one CRISPR composition can be provided such that each separately target the same gene or general genomic locus at more than target nucleotide sequence. For example, two separate CRISPR compositions can be provided to direct cleavage at two different target nucleotide sequences within a certain distance of each other. In some CRISPR systems, more than one CRISPR composition can be provided such that each separately target opposite strands of the same gene or general genomic locus. For example, two separate CRISPR “nickase” compositions can be provided to direct cleavage at the same gene or general genomic locus at opposite strands.

[0184] In general, the features of a CRISPR-mediated editing system described herein can apply to other nuclease-based genomic editing systems. TALEN is an engineered site-specific nuclease, which is composed of the DNA- binding domain of TALE (transcription activator-like effectors) and the catalytic domain of restriction endonuclease Fokl. By changing the amino acids present in the highly variable residue region of the monomers of the DNA binding domain, different artificial TALENs can be created to target various nucleotides sequences. The DNA binding domain subsequently directs the nuclease to the target sequences and creates a doublestranded break. TALEN-based systems are described in more detail in U.S. Ser. No. 12 / 965,590;U.S. Pat. No. 8,450,471; U.S. Pat. No. 8,440,431; U.S. Pat. No. 8,440,432; U.S. Pat. No. 10,172,880; and U.S. Ser. No. 13 / 738,381, all of which are incorporated by reference herein in their entirety. ZFN-based editing 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 Publication Nos. 2005 / 0064474; 2007 / 0218528; 2005 / 0267061, all incorporated herein by reference in their entireties for all purposes.Other Engineering Delivery Systems

[0185] Various additional means are available to a skilled artisan for introducing engineered nucleic acids (e.g., any of the engineered nucleic acids described herein) into a cell or other target recipient entity, such as any of the lipid structures described herein. A skilled artisan will understand which methods are suitable for introducing engineered nucleic acids into a cell or other biological system.

[0186] In some embodiments, electroporation is used to deliver polynucleotides (e.g., any engineered nucleic acid described herein) to recipient entities. Electroporation is a method of internalizing a cargo / payload into a target cell or entity’s interior compartment through applying an electrical field to transiently permeabilize the outer membrane or shell of the target cell or entity. In general, the method involves placing cells or target entities between two electrodes in a solution containing a cargo of interest (e.g., any of the engineered nucleic acids described herein). The lipid membrane of the cells is then disrupted, i.e., permeabilized, by applying a transient set voltage that allows the cargo to enter the interior of the entity, such as the cytoplasm of the cell. In the example of cells, at least some, if not a majority, of the cells remain viable. Cells and other entities can be electroporated in vitro, in vivo, or ex vivo. Electroporation conditions (e.g., number of cells, concentration of cargo, recovery conditions, voltage, time, capacitance, pulse type, pulse length, volume, cuvette length, electroporation solution composition, etc.) vary depending on several factors including, but not limited to, the type of cell or other recipient entity, the cargo to be delivered, the efficiency of internalization desired, and the viability desired. Optimization of such criteria are within the scope of those skilled in the art. A variety devices and protocols can be used for electroporation. Examples include, but are not limited to, Neon® Transfection System, MaxCyte® Flow Electroporation™, Lonza® Nucleofector™ systems, and Bio-Rad® electroporation systems.

[0187] Other means for introducing engineered nucleic acids (e.g., any of the engineered nucleic acids described herein) into a cell or other target recipient entity include, but are notlimited to, sonication, gene gun, hydrodynamic injection, and cell membrane deformation by physical means.

[0188] Compositions and methods for delivering engineered mRNAs in vivo, 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 herein incorporated by reference for all purposes.Methods of Use

[0189] Methods for treatment of diseases are also encompassed by this disclosure. Said methods include administering a therapeutically effective amount of an engineered nucleic acid, engineered cell, or isolated engineered cell as described herein. In some embodiments, provided herein are methods of treating a subject in need thereof, the method comprising administering a therapeutically effective dose of any of the engineered cells, isolated engineered cells, or compositions disclosed herein.

[0190] In some embodiments, the present disclosure provides methods of treating a subject with a disease or disorder by administering to said subject an engineered nucleic acid, an engineered polypeptide, or an engineered cell (e.g., an isolated engineered cell), as described herein. It will be understood by a skilled artisan that an engineered nucleic acid can be provided to a subject using any suitable delivery system, e.g., any delivery system described herein. In some embodiments, a subject is administered an engineered nucleic acid (or a delivery system that provides an engineered nucleic acid to said subject, e.g., an engineered cell) and an IMiD. In some embodiments, administration of an engineered nucleic acid and an IMiD is sequential. In some embodiments, administration of an engineered nucleic acid and an IMiD is simultaneous. In some embodiments, administration of an engineered nucleic acid and an IMiD is via different routes. In some embodiments, an IMiD is an FDA-approved drug. In some embodiments, an IMiD is thalidomide. In some embodiments, an IMiD is a variant or derivative of thalidomide. In some embodiments, an IMiD is selected from the group consisting of: thalidomide, iberdomide, lenalidomide, and pomalidomide. In some embodiments, an IMiD is pomalidomide.In vivo Expression

[0191] The methods provided herein also include methods of delivering a composition in vivo that is capable of producing an engineered cell as described herein, e.g., capable of delivering any of the engineered nucleic acids described herein to a cell in vivo. Such compositions include any of the viral-mediated delivery platforms, any of the lipid structure delivery systems, any of the nanoparticle delivery systems, any of the genomic editing systems,or any of the other engineering delivery systems described herein that are capable of engineering a cell in vivo.

[0192] The methods provided herein also include methods of delivering a composition in vivo capable of producing any of the effector molecules described herein. The methods provided herein also include delivering a composition in vivo capable of producing two or more of the effector molecules described herein. Compositions capable of in vivo production of effector molecules include, but are not limited to, any of the engineered nucleic acids described herein. Compositions capable of in vivo production of effector molecules can be a naked mRNA or a naked plasmid.Pharmaceutical Compositions

[0193] Engineered nucleic acids or engineered cells provided herein can be formulated in pharmaceutical compositions. These compositions can comprise, in addition to one or more of the engineered nucleic acids or engineered cells, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other suitable materials for pharmaceutical formulation. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material can depend on the route of administration, e.g., oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, or intraperitoneal routes.

[0194] Whether it is a cell, polypeptide, nucleic acid, small molecule or other pharmaceutically useful compound according to the present disclosure that is to be given to an individual, administration is preferably in a “therapeutically effective amount” or “prophylactic ally effective amount”(as the case can be, although prophylaxis can be considered therapy), this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of protein aggregation disease being treated. Prescription of treatment, e.g., decisions on dosage, etc., is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors. Examples of the techniques and protocols mentioned above can be found in Remington’s Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.

[0195] A composition can be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.Tables A, B, and C-lTable A| Cell death inducing proteinsTable BTable C-lEXAMPLES

[0196] Below are examples of specific embodiments for carrying out the present disclosure. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present disclosure in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

[0197] The practice of the present disclosure will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A.L. 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).Example 1: Evaluation of single vector multicistronic systems having different 2A linkers between payload components

[0198] In this example, the following single vector multicistronic constructs were tested in order to to test different 2A linkers between different pay loads.

[0199] SB12689-SB12692: designed to express a first polypeptide comprising a cereblon(CRBN) domain operably linked to a transcriptional activator (minVPR), and a second polypeptide comprising a degron operably linked to ZF10-1 DNA binding domain. For these constructs, the CRBN component was operably linked to the degron component either via 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 transcriptional ON switch.

[0200] SB 12703-SB 12706: same variables as SB 12689-SB 12692 but with GFP-T2A (T2A sequences shown in SEQ ID NOs: 267 and 268) preceding the ImiD transcriptional switch.

[0201] For these experiments, SB09667 was used as a control with wild-type first and second polypeptide components operably linked via a P2A linker.

[0202] Methods

[0203] On Day 0, U87MG + SB01066 (4xTFBS pMin: mCherry reporter) cells were plated at le5 cells / well in 12 well plates. On Day 1, cells were transduced with virus (le5 pg p24 of virus to each well). On Day 2, cells were treated with 0, 0.001, 0.01, 0.1, 1, 4, 10, and 100 nM Pomalidomide. On Day 4, cells were harvested and analyzed for mCherry expression by flow cytometry.

[0204] Delta %mCherry+ was calculated by subtraction of %mCherry+ cells in the ND condition from the %mCherry+ cells from the specified drug treated condition. To calculate fold activation of the mCherry reporter, gMFI of mCherry was quantified by flow and first subtracting the background mCherry gMFI (gMFI evaluated for no virus (NV) control) from all quantified mCherry gMFI values. Next, the background subtracted mCherry gMFI from the indicated drug treated condition was divided by the background subtracted mCherry gMFI evaluated for the ND condition of the same construct.

[0205] Results are shown in FIGS. 1A-1E.

[0206] As shown in FIG. 1A, constructs utilizing an Opt2A or Opt2A_2.0 linker in the IMiD transcriptional switch exhibited a reduction of basal reporter expression as compared to constructs comprising the P2A linker. Furthermore, results depicted in FIG. 1A suggest that both Opt2A and Opt2A_2.0 reduced maximum induced expression of reporter at lower concentrations of pomalidomide (4 nM and less). This effect was not as apparent at 10 nM and 100 nM pomalidomide doses. Results depicted in FIG. IB suggest that Opt2A and Opt2A_2.0 in the IMiD transcriptional switch result in larger gains in % mCherry positive cells during induction, while reducing background, as compared to P2A. These trends were also apparent in constructs with GFP-T2A preceding the IMiD transcriptional switch (see FIGs. 1C, ID, and IE).OTHER EMBODIMENTS

[0207] While the present disclosure has been particularly shown and described with reference to a preferred embodiment and various alternate embodiments, it will be understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the present disclosure and appended claims.

[0208] All references, issued patents and patent applications cited within the body of the instant specification are hereby incorporated by reference in their entirety, for all purposes.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. An engineered polynucleotide comprising a first sequence encoding for a first polypeptide and a second sequence encoding for 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. The engineered polynucleotide of claim 1, comprising a sequence according to formula, oriented from 5’ to 3’:Pl - L - P2 wherein Pl comprises the first sequence, L comprises the linker polynucleotide sequence encoding the 2A ribosome skipping element, and P2 comprises the second sequence.

3. The engineered polynucleotide of any one of the preceding claims, wherein the 2A ribosome skipping element comprises the amino acid sequence ATNFSLLKQAGDVEENPGPGSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 269).

4. A vector comprising the engineered polynucleotide of any one of the preceding claims.

5. An engineered cell comprising the engineered polynucleotide or vector of any one of the preceding claims.

6. A pharmaceutical composition comprising the engineered polynucleotide or vector of any one of the preceding claims and a pharmaceutically acceptable carrier.

7. A pharmaceutical composition comprising the engineered cell of claim 5 and a pharmaceutically acceptable carrier.

8. A method, comprising administering to a subject a pharmaceutical composition of any one of the preceding claims.

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

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