Composition and method for autonomous RNA switches for translational control

Abstract

The present disclosure provides recombinant polypeptides containing a payload sequence and one or more 3'-untranslated region (3'-UTR) sequences downstream of and operably linked to the payload sequence. The one or more 3'-UTR sequences, or RNA transcripts thereof, respond to an intracellular signal by modulating the translation, transcription, and / or stability of the payload sequence or an RNA transcript thereof. The disclosure also provides DNA constructs, vectors, cells, and systems including the provided recombinant polypeptides, as well as methods for modifying cells and / or preventing or treating a disease by using the provided materials.

Description

COMPOSITION AND METHOD FOR AUTONOMOUS RNA SWITCHES FORTRANSLATIONAL CONTROLCROSS-REFERENCES TO RELATED APPLICATIONS

[0001] The present application claims priority from U.S. Provisional Application No. 63 / 623,679 filed January 22, 2024, the entire contents of which are incorporated herein by reference for all purposes.STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] This invention was made with Government support under contract DGE- 1656518 awarded by the National Science Foundation and under contract CA270609 awarded by the National Institutes of Health. The Government has certain rights in the invention.BACKGROUND

[0003] Messenger RNA (mRNA) therapies are useful for treating disease. Typically, therapeutic genes are delivered as mRNAs packaged m lipid nanoparticles (LNPs) and the mRNAs are translated into functional proteins in target tissues. Due to its adaptability, rapid production, and lack of immunogenicity or genotoxic effects, mRNA therapy has played a crucial role for developing antiviral vaccines during the COVID- 19 pandemic (Kim Y -K. 2022. Exp Mol Med 54:455-465). While there have been improvements in mRNA therapies related to vaccination, cancer treatment, and in vivo engineering of therapeutic immune cells, there is a lack in cell therapies that use nucleic acid switches for conditional gene expression.BRIEF SUMMARY

[0004] This summary provides a high-level overview of various aspects of the disclosure and introduces some of the concepts that are described and illustrated in the present document and the accompanying figures. The summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used in isolation to determine the scope ofthe claimed subject matter. Covered embodiments of the disclosure are defined by the claims. not this summary. The subject matter should be understood by reference to appropriate portions of the entire specification, any or all figures, and each claim. Some of the exemplary embodiments of the present disclosure are discussed below.

[0005] In one aspect, the disclosure provides a recombinant polynucleotide. The recombinant polynucleotide includes a payload sequence encoding a payload polypeptide that is functional in or secreted by a target cell. The recombinant polynucleotide further includes a 3'- untranslated region (3'-UTR) sequence downstream of and operably linked to the payload sequence. The 3'-UTR sequence is an engineered variant of a wild-type 3'-UTR sequence. The 3'-UTR sequence or an RNA transcript thereof responds to an intracellular signal of the target cell by modulating expression of the payload polypeptide by the target cell.

[0006] In another aspect, the disclosure provides another recombinant polynucleotide. The recombinant polynucleotide includes a payload sequence encoding a payload polypeptide that is functional in or secreted by a target cell. The recombinant polynucleotide further includes a first wild-type 3’-UTR sequence downstream of and operably linked to the payload sequence. Tire recombinant polynucleotide further includes a second wild-type 3'-UTR sequence downstream of and operably linked to the payload sequence. The first and second wiki-type 3'- UTR sequences or RNA transcripts thereof each independently respond to an intracellular signal of the target cell by modulating expression of the payload polypeptide by the target cell.

[0007] In another aspect, the disclosure provides another recombinant polynucleotide. The recombinant polynucleotide includes a payload sequence. The payload sequence or a payload RNA transcript thereof is functional in or secreted by a target cell. The recombinant polynucleotide further includes a 3'-UTR sequence downstream of and operably linked to the payload sequence. The 3 -UTR sequence is an engineered vanant of a wild -type 3'-UTR sequence. The 3'-UTR sequence or a 3'-UTR RNA transcript thereof responds to an intracellular signal of the target cell by modulating translation or stability of the payload sequence or the payload RNA transcript in the target cell.

[0008] In another aspect, the disclosure provides another recombinant polynucleotide. The recombinant polynucleotide includes a payload sequence. The payload sequence or a pay load RNA transcript thereof is functional in or secreted by a target cell. The recombinant polynucleotide further includes a first wild-type 3'-UTR sequence downstream of and operably linked to the payload sequence. The recombinant polynucleotide further includes a secondwild-type 3'-UTR sequence downstream of and operably linked to the payload sequence. The first and second wild-type 3'-UTR sequences or RNA transcripts thereof each independently respond to an intracellular signal of the target cell by modulating translation or stability of the payload sequence or the payload RNA transcript in the target, cell.

[0009] In another aspect, the disclosure provides a DNA construct. The DNA construct includes a recombinant polynucleotide as disclosed herein. The DNA construct further includes a payload promoter operably linked to the payload sequence.

[0010] In another aspect, the disclosure provides a vector. Tire vector includes a recombinant polynucleotide as disclosed herein, or a DNA construct as disclosed herein.

[0011] In another aspect, the disclosure provides a system. The system includes a recombinant polynucleotide as disclosed herein, a DNA construct as disclosed herein, or a vector as disclosed herein. The system further includes a chimeric antigen receptor (CAR) or a polynucleotide, DNA construct, or vector including a sequence encoding the CAR.

[0012] In another aspect, the disclosure provides a cell. The cell includes a recombinant polynucleotide as disclosed herein, a DNA construct as disclosed herein, or a vector as disclosed herein.

[0013] In another aspect, the disclosure provides a population of cells. The population of cells includes a cell as disclosed herein.

[0014] In another aspect, the disclosure provides a pharmaceutical composition. The pharmaceutical composition includes a pharmaceutically acceptable earner or a pharmaceutically acceptable excipient. Tire pharmaceutical composition further includes a recombinant polynucleotide as disclosed herein, a DNA construct as disclosed herein, a vector as disclosed herein, a system as disclosed herein, a cell as disclosed herein, or a population of cells as disclosed herein.

[0015] In another aspect, the disclosure provides a method of modify ing a cell. Hie method includes contacting the cell with a recombinant polynucleotide as disclosed herein, a DNA construct as disclosed herein, or a vector as disclosed herein; or introducing the recombinant polynucleotide, the DNA construct, or the vector into the cell.

[0016] In another aspect, the disclosure provides a method for preventing or treating a disease in a subject. The method includes administering to the subject an amount of arecombinant polynucleotide as disclosed herein, a DNA construct as disclosed herein, a vector as disclosed herein, a sy stem as disclosed herein, a cell as disclosed herein, a population of cells as disclosed herein, or a pharmaceutical composition as disclosed herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Unless otherwise specified, all error bars in the drawings indicate the mean and standard error of the mean (SEM) of the plotted data.|0018] FIGS. 1A-C show exemplary T cell activation-induced mRNA switches. FIGS. IA- B show T cell activation when exemplary mRNA switches are induced. The two exemplary constructs are an ‘"activation-induced expression” lentiviral construct and a “constant expression” control. FIG. 1C shows that primary human T cells transduced with an activation- induced mRNA switch only express green fluorescence protein (GFP) when activated / stimulated (grey histogram) and not at baseline (white histogram; without activation / stim ulation). Histograms depict GFP fluorescence (arbitrary units; a.u.) as measured by flow cytometry. Data are representative of at least three independent experiments performed with different blood donors.

[0019] FIGS. 2A-B show quantification of T cell activation-induced mRNA switches as measured by flow cytometry . FIG. 2A shows percent GFP+. FIG. 2B shows GFP MFI (median fluorescent intensity) in arbitrary units (a.u.).Wild-type human UTR sequence specified m x- axis. Each point represents an independent experiment performed with a different blood donor and is the mean of triplicate technical replicates. Ordinary one-way ANOVA was used with Sidak’s multiple comparisons test. Asterisks represent the following: *p <.05, **p <.01, ***p <.001, **»*p <.0001. Switch 3'UTRs are selected as having statistically significant change in either GFP MFI or percent GFP’ cells. IL4 3 'UTR is included as a negative UTR control. CONST = constant expression control: MOCK ~ untransduced primary human T ceils; black = baseline (unstimulated); white = stimulated.

[0020] FIGS. 3A-B show the impact of exemplary mRNA switches with truncated IFNG 3’- UTR on T cell activation. FIG. 3 A shows percent GFP+. FIG. 3B shows GFP MFI (a.u,). The full length, wild-type IFNG 3 -UTR was truncated from the distal end to generate 3'-UTRs with lengths specified on the x-axis. Data is representative for one donor. CONST = constantexpression control; MOCK = untransduced primary human T cells; WT::::wiki-type; black bars=:baseline (unstimulated); white bars = stimulated.[0021 j FIGS. 4A-B show the impact of exemplary' mRNA switches with truncated JL2 3'- UTR on T cell activation. FIG. 4A shows percent GFP+. FIG. 4B shows GFP MFI (a.u.). Truncation of wild-type 1L23'-UTR. The full length, wild-type 11.2 3'-UTR was truncated from the distal end to generate 3'-UTRs with lengths specified on the x-axis. Black bars represent expression at baseline (un stimulated) and white bars represent expression when stimulated. Data is representative for one donor. CONST' = constant expression control; MOCK = untransduced primary human T cells; WT = wild-type.

[0022] FIGS. 5A-D show the impact of exemplary mRNA switches w'itli truncated type TNF 3 -UTR on T cell activation. FIGS. 5A and 5C show percent GFP+. FIGS, 5B and 5D show GFP MFI (a.u.). In FIGS. 5A-B, the full length, wild-type TNF 3'-UTR was truncated from the distal end to generate 3'-UTRs with lengths specified on the x-axis. In FIGS. 5C-D, the proximal 600-bp 3'-UTR was then truncated from the proximal end to generate 3'-UTRs with lengths spanning [starting bp]-[ending bp], as specified on the x-axis. Black bars represent expression at baseline (unstimulated) and white bars represent expression when stimulated. Data is representative for one donor. CONST ~ constant expression control; MOCK = untransduced primary human T cells; WT = wi Id-type.

[0023] FIGS. 6A-C show the impact of deleting AU-rich elements (AREs) in the IFNG 3'- UTR on T cell activation. FIG. 6A shows the IFNG 3'-UTR (SEQ ID NO: 3) with AREs (in bold) within the IFNG 3'-UTR. The AREs from the 5' to the 3’ direction are A A RE 1, A ARE 2, A ARE 3, Zk ARE 4, A ARE 5, A ARE 6 and Zk ARE 7; single deletions of AREs. FIG. 6B shows percent GFP+. FIG. 6C shows GFP MFI (a.u.). Black bars represent expression at baseline (unstimulated) and white bars represent expression when stimulated. Data is representative for one donor. CONST = constant expression control; MOCK = untransduced primary human T cells; WT = wild-type.

[0024] FIGS. 7A-B show' exemplary synthetic arrays with a minimal IFNG mRNA switch(mIFNG). Synthetic mRNA switches were generated by arraying mIFNG directly downstream of another wild-type 3’-L!TR, as specified in tire x-axis. FIG. 7A shows percent GFP+. FIG. 7B shows GFP MFI (a.u.), Black bars represent expression at baseline (unstimulated) and white bars represent expression when stimulated. Data is representative for one donor. CONST = constant expression control.

[0025] FIGS. 8A-F show' percent GFP+ for exemplary synthetic arrays of four mRNA switches: IL1B, IL13, 1L6, and CSF2. Specific mRNA switches are denoted in the x-axis, where black bars represent expression at baseline (unstimulated) and white bars represent expression when stimulated. Data is representative of two independent experiments performed in different blood donors. CONST:::constant expression control.

[0026] FIGS. 9A-B show exemplary synthetic singlets and doublets of mRNA switches chosen from IL1B, CSF2, 1L13, and IL6. FIG. 9A shows percent GFP+. FIG. 9B shows GFP MFI (a.u.). Specific mRNA switches are denoted in the x-axis, where black bars represent expression at baseline (unstimulated) and white bars represent expression when stimulated. Data is representative of two independent experiments performed in different blood donors. CONST:::constant expression control.

[0027] FIGS. 10A-E show' data from exemplary antigen-dependent mRNA switches in CD 19-targeted T cells. As shown in FIG. 10A, activation-dependent mRNA switches confer antigen-dependent protein expression in CD19 CAR-T cells. FIGS. 10B and 10D show percent GFP+ measured after 24 h and 120 h of co-culture, respectively. FIGS. 10C and 10E show' GFP MFI (a.u.) measured after 24 h and 120 h of co-culture, respectively. Black = K562 CD19-. White = K562 CD19+. Data is representative for one donor. CONST ~ constant expression control; MOCK = primary human T cells without reporter transduction. Data is gated on CAR+ cells.

[0028] FIGS. 11A-D show positive feedback with exemplary antigen-dependent mRNA switches in CD! 9-targeted T cells. As shown in FIG. HA, primary human T cells are engineered through lentiviral transduction of either an “mRNA switch CAR” or a “constitutive CAR.” FIG. 1 IB shows tumor intensity for CD19- and CDI9+ tumors, FIG. 11C shows CAR- T cell intensity, and FIG. 11D compares tumor intensity for mRNA switch CAR and constitutive CAR. Data is representative for one donor.

[0029] FIGS. 12A-B show quantification of fold change of T cell activation-induced mRNA switches as measured by flow' cytometry with the stimulation protocol described in FIGS. 1 A- C. Wild-type human UTR sequence specified in x-axis. Each point represents an independent experiment performed with a different blood donor in technical triplicate.

[0030] FIGS. 13A-D show' data from exemplary antigen-dependent mRNA swatches in CD19-targeted T cells. The example of FIGS. 13A-D is similar to that of FIGS. 10B-E exceptthat primary human T cells were engineered with 19BBz CAR instead of 1928z CAR. Data is representative for one donor[0031 j FIGS. 14A-D show' positive feedback with antigen-dependent mRNA switches in R0R1 -targeted CAR T cells. Primary human T cells were engineered through MSGV1 retroviral transduction of either an “RNA switch CAR” or a “constitutive CAR”, as shown in the plasmid schematic of FIG. I4A and SEQ ID NOs: 72 and 73. The graphs of FIGS. 14B and 14C show' that, when co-cultured with GFP-positive Nalm6 leukemia tumor cells engineered with R0R1, mRNA switch CAR T cells increase CAR T raw intensity (FIG. 14B), and normalized intensity to initial time point (FIG. 14C), as measured by mKate2 expression (CAR signal). The graph of FIG. 14D shows that mRNA switch CAR T cells kill antigen-positive tumor cells with similar dynamics to constitutive CAR T cells, as measured by GFP fluorescence (tumor signal), normalized to initial time point. Cells were co-cultured at a 1: 1 ratio and analyzed on an Incucyte live-cell imaging platform. The shaded region indicates SEM. Data is representative of two independent experiments performed in different blood donorsDETAILED DESCRIPTIONA. GENERAL

[0032] The present disclosure provides compositions and methods related to using 3’ untranslated regions (3'-UTRs) of nucleic acids as switches for controlled gene expression. 3'-UTRs are generally known to regulate mRNA-based processes, such as mRNA localization, mRNA stability, and translation (Mayr, Christine. Cold Spring Harbor Perspectives in Biology vol. 11,10 a034728. 1 Oct. 2019, doi: 10.1101 / cshperspect.a034728).

[0033] 3'-UTRs contain specific cis-regulatory elements, such as AU -rich elements (AREs), which destabilize mRNA and are implicated in mRNA decay. Id. AREs were predominantly found in 3'-UTRs of a certain class of genes that encodes short-lived factors, including cytokines, lymphokines, growth factors, and oncogenes (Caput, D et al. Proceedings of the National Academy of Sciences of the United States of America vol. 83,6 (1986): 1670-4. doi: 10.1073 / pnas.83.6.1670). Intnguingly, the AREs were sometimes more conserved than the coding regions of these early response genes (Shaw, G, and R Kamen. Cell vol. 46,5 (1986): 659-67. doi:10.1016 / 0092-8674(86)90341-7). The AREs and related RNA-binding proteinsare thought to influence inflammation in disease (Palanisamy, V et al. Journal of Denial Research vol. 91,7 (2012): 651-8. doi: 10. 1177 / 0022034512437372).[0034 j Because ofmRNA’s transient nature, mRNA can be used for temporal control of gene expression. For example, T cells can be engineered with mRNA to drive T cell function, such as on-demand discrimination between protective and pathogenic inflammation. Current strategies for controlling mRNA expression in T cells remain limited to human-prescribed drug -responsive RNA ligands (Lee, Sang Kil, and George A Calin. Genome Medicine vol. 2,10 77. 15 Oct. 2010, doi: 10.1186 / gml98; Wong, Remus S et al. Nucleic Acids Research vol. 46,3 (2018): 1541-1552. dot: 10.1093 / nar / gkxl228). Drag-responsive switches allow for clinicians to control timing and release of therapeutic payload after administration. Exogenous control of therapeutic output and dosage requires prior knowledge of optimal timing and cell states that may not be available in complex clinical presentations. In contrast, the autonomous nucleic acid switches of the present disclosure capture endogenous biological complexity’ and native cellular sensing and regulation for synthetic control of user-defined genetic payloads.

[0035] Further, conditional switches that can regulate gene expression in response to intracellular signals for autonomous decision processing are rare. Recent work has focused on developing RNA-responsive translational control, where translation of a user-defined payload is modulated by the presence or absence of target RNAs, For example, eToeholds rely on engineered mRNA internal ribosome entry sites where inhibitory'- loops are disrupted when hybridized with target RNAs (Zhao, Evan M et al. Nature Biotechnology vol. 40,4 (2022): 539- 545. doi:10.1038 / s41587-021-01068-2). More recently, base editing by adenosine deaminases acting on RNAs (ADARs) on mismatched adenosines in double -stranded RNA structures has been engineered to enable sensor transcript expression upon detection of an RNA target of interest ((Jiang, Kaiyi et al. Nature Biotechnology vol. 41,5 (2023): 698-707. doi: 10.1038 / s41587-022-01534-5; Qian, Yongjun et al. Nature vol. 610,7933 (2022): 713-721. dot: 10.1038 / s41586-022-05280-1; Kaseniit, K Eerik et al. Nature Biotechnology’ vol. 41 ,4 (2023): 482-487. doi: 10.1038 / s41587-022-01493-x; Gayet, Raphael V et al. Nature Communications vol. 14,1 1339. 11 Mar. 2023, doi: 10.1038 / s41467-023-36851-z)). For both approaches, cell type and / or cell state specificity is conferred by defining specific RNA target transcripts. While a recent wealth of single cell and bulk RNA sequencing data has captured many molecular signatures of cell states and cell types, some cell types / states may not have unique transcript signatures or may be characterized by translational or post-translational signatures not captured by’ mRNA expression. In contrast, the autonomous nucleic acidswitches described herein are not limited by transcript specificity for cell state or type-specific translation.[0036 j The present disclosure also provides inducible nucleic acid switches for T cell activation. Unlike T ceil activation-inducible promoters, such as the nuclear factor of activated T cells (NF AT) promoter (Smole, Anze et al. Cancer Cell vol, 40,12 (2022): 1470-1487. e7. dor 10.1016 / j.ccell.2O22.1 1.006; Zhang, Ling et al. Clinical Cancer Research vol. 21 ,10 (2015): 2278-88. dot: 10.1 158 / 1078-0432.CCR-14-2085), nucleic acid switches can be directly applied to mRNA payloads, enabling clinical applications where avoiding genomic integration or transient therapeutic dose is necessary. Furthermore, engineering nucleic acid switches for other cell types or cell states may be more facile as relevant regulatory sequences are largely- contained within the transcript, while promoters often require more distal regulatory sequences (e.g., enhancers) for lull function.B. DEFINITIONS10037 ] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary7skill in the relevant art.

[0038] As used herein, the term “3 '-untranslated region” or “3'-UTR” refers to a portion of an RNA molecule, e.g., an mRNA molecule, where the portion follows a translation termination codon. As used herein, the tenn also refers to the sequence of a DNA molecule, where the DNA sequence is transcribed to form this RNA molecule portion. The 3'-UTR of the mRNA is thus transcribed from DNA but is not translated into protein.

[0039] As used herein, the tenn “AU-rich elements” (“AREs”) refers to motif sequences rich in adenosine and uridine, e.g., AUUUA motif sequences. AREs can be found in the 3'-UTR of some mRNAs. AREs are typically found in mRNAs that have a short half-life, and can have a capacity to provoke degradation of the mRNA.

[0040] As used herein, the term “intracellular signal” refers to a change in a biophysical state or composition of an intracellular environment, where one or more molecules or systems within the intracellular environment are responsive to the change. Typically, the change includes an increase or decrease in an intracellular concentration of one or more endogenous signaling molecules produced by the cell. An “intracellular signal” can be produced in response to the detection or binding of one or more exogenous molecules by the cell. The term “intracellularsignal” is used here to differentiate endogenous signaling molecules from exogenous molecules, such as chemical reagents, prescription drugs, and environmental contaminants.[0041 j As used herein, the terms “polynucleotide” and “nucleic acid” are used interchangeably to refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides. The terms include RNA, DNA, and synthetic forms and mixed polymers of the above. In particular embodiments, a nucleotide refers to a ribonucleotide, deoxynucleotide or a modified form or analog of either type of nucleotide. Deoxyribonucleic acids and ribonucleic acids thus include both naturally occurring molecules and synthetic analogues. The polynucleotides of the disclosure also encompass all forms of sequences including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures, and the like. A reference to a nucleic acid sequence encompasses its complement unless otherwise specified. Thus, a reference to a nucleic acid molecule having a particular sequence should be understood to encompass its complementary strand, with its complementary sequence. Reference to a “polynucleotide” or “nucleic acid” that encodes a polypeptide sequence also includes codon-optimized nucleic acids and nucleic acids that comprise alternative codons that encode the same polypeptide sequence.

[0042] As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably to refer to a polymer of amino acid residues. All three terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. As used herein, the terms encompass amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds.

[0043] As used herein, the terms “variant,” and “fragment,” refer to a polynucleotide related to a wild-type polynucleotide, for example, either by nucleic acid sequence, structure (e.g., secondary and / or tertiary), activity (e.g., enzymatic activity) and / or function. Variants and fragments of a polynucleotide can include one or more nucleic acid variations (e.g., mutations, insertions, and deletions), truncations, modifications, or combinations thereof compared to a wild-type polynucleotide. A variant or fragment can include at least 50%, e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% of the sequence, structure, activity, and / or function of the corresponding wild-type polynucleotide.[0044 [ As used herein, the term “operably linked” refers to a functional linkage between a first nucleic acid sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the first nucleic acid sequence afflicts the transcription, translation, localization, or stability of the second nucleic acid sequence.

[0045] As used herein, the term ‘’promoter” refers to nucleic acid regions or sequences located upstream and / or downstream from a start of transcription and which are involved in recognition and binding of RNA polymerase and other proteins to initiate transcription.

[0046] As used herein, the terms “percent identical,” “percent identity,” “sequence identity,” or equivalents used in the context of two polynucleotides or polypeptides refer to the level of identity between the sequences of the two polynucleotides or polypeptides when aligned, e.g., using a sequence alignment program or algorithm. For example, in certain embodiments, a particular polynucleotide sequence may have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity w'itli a reference sequence as determined by using standard methods, e.g., BLAST. For sequence comparisons, one sequence is typically used as a reference sequence, to which query sequences are compared. Sequence comparison algorithms are readily available to one of ordinary skill in the art for comparison of test and query sequences. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. Methods of alignment of sequences for comparison are well-known in tire art. 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. LISA 85:2.444 (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, WI), or by manual alignment and visual inspection. Percent sequence identity and sequence similarity can be determined using the BLAST and BLAST 2,0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively.Software for performing BLAST analyses is publicly available through the National Center forBiotechnology Information (NCBI) web site.[0047 j As used herein, the term “recombinant,” as used in the context of a polynucleotide described herein, refers to a polynucleotide created through deliberate human intervention bybringing together genetic material from multiple, i.e., two or more different, sources.

[0048] As used herein, the term “’specifically binds” refers to a molecule (e.g., a chimeric antigen receptor; CAR) that binds to a target with greater affinity, avidity, more readily, and / or with greater duration to that target in a sample than it binds to a non-target molecule. For example, the CD19-28z CAR specifically binds the CD 19 antigen with greater affinity, avidity, more readily, and / or with greater duration than to a non-target molecule. For example, in some embodiments, the CD19-28z CAR binds to the CD19 antigen with at. least 2-fold greater affinity than non-target compounds, e.g., at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-foki, at least 25-fold, at least 50-fold or greater affinity. For example, in some embodiments, the CD19-28z CAR specifically binds to the CD 19 antigen typically with at least a 2-fold greater affinity than to a non-CD 19-28z CAR target.

[0049] As used herein, the term “vector” refers to a vehicle for transferring a nucleic acid molecule into a cell. Vector forms include, for example, plasmids or viral vectors, as well polymer particles, lipid nanoparticles (LNPs), and virus particles.

[0050] As used herein, the term “plasmid” refers to a circular, double-stranded DNA containing one or more sequences of interest, for example, sequences encoding one or more particular proteins. In some embodiments, a plasmid can further include regulatory sequences or other genetic elements that are operatively linked to a sequence encoding a particular protein.

[0051] As used herein, the terms “lipid particle,” “lipid nanoparticle,” and “LNP” refer to a particle comprising a phospholipid and an ionizable lipid. A lipid particle may comprise additional lipid components, such as a sterol and / or a conjugated lipid, and may further comprise a nucleic acid, wherein the nucleic acid may be encapsulated within the particle.

[0052] As used herein, the terms “DNA construct” and “expression cassette” refer to a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of one or more particular nucleic acid sequencesin a host cell. A DNA construct can be a part of a vector. Typically, a DNA construct includes a nucleic acid sequence to be transcribed, where the sequence is operably linked to a promoter.[0053 j The term “introducing,” as used in the context of a polynucleotide described herein, refers to presenting a nucleic acid sequence to a host cell in such a manner that the sequence gains access to the interior of the cell.

[0054] As used herein, the term “pharmaceutically acceptable carrier” refers to an excipient or diluent in a pharmaceutical composition. The pharmaceutically acceptable carrier must be compatible with the other ingredients of the formulation and not deleterious to the recipient. The pharmaceutically acceptable carrier must provide adequate pharmaceutical stability to the active ingredient. The required nature of the carrier must be appropriate for use with the mode of administration. For example, for intravenous administration, an aqueous solution earner is generally used; for oral administration, a solid earner is preferred.

[0055] As used herein, the term “subject” refers to a vertebrate, and preferably to a mammal. Mammalian subjects for which the provided composition is suitable include, but are not limited to, mice, rats, simians, humans, farm animals, sport animals, and pets. In some embodiments, the subject is human. In some embodiments, the subject is male. In some embodiments, the subject is female. In some embodiments, the subject is an adult. In some embodiments, the subject is an adolescent. In some embodiments, the subject is a child. In some embodiments, the subject is above 10 years of age, e.g., above 20 years of age, above 30 years of age, above 40 years of age, above 50 years of age, above 60 years of age, above 70 years of age, or above 80 years of age. In some embodiments, the subject is less than 80 years of age, e.g., less than 70 years of age, less than 60 years of age, less than 50 years of age, less than 40 years of age, less than 30 years of age, less than 20 years of age, or less than 10 years of age.

[0056] As used herein, the term “administering” refers to oral administration, administration as a suppository, topical contact, parenteral, intravenous, intraperitoneal, intramuscular, intralesional, intranasal, subcutaneous, intrathecal, intracerebroventricular, intraparenchymal, subretinal, or intravitreal administration, or the implantation of a siow-rcicasc device e.g., a mini-osmotic pump, to the subject.

[0057] As used herein, the term “therapeutically effective amount” refers to an amount or dose of a compound, composition, or formulation that produces therapeutic effects for which it is administered. The exact amount or dose will depend on the purpose of the treatment, andwill be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: Tire Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0058] As used herein, the terms “treat” and “treatment” refer to both therapeutic treatment and prophylactic or preventive measures, wherein the object is to eliminate, slow' down, or prevent disease progression or manifestation as indicated by undesired physiological change or disorder. For purpose of this disclosure, the benefits of treatment include, but are not limited to, partial or complete alleviation of symptoms, delay of disease progression, amelioration of disease, improvement in patient outcomes (e.g., increase in patient survival), stabilization of a disease parameter (e.g., tumor size), and remission. These benefits may detectable or undetectable.

[0059] As used herein, the singular forms “a,” “an,” and “the” include both singular and plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a payload sequence” optionally includes a combination of two or more payload sequences, and the like.

[0060] As used herein, the term “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).[0061 j As used herein, the terms “including,” “comprising,” “’having,” “containing,” and variations thereof, are inclusive and open-ended and do not exclude additional, unrecited elements or method steps beyond those explicitly recited. As used herein, the phrase “consisting of’ is closed and excludes any element, step, or ingredient not explicitly specified. As used herein, the phrase “consisting essentially of” limits the scope of the described feature to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the disclosed feature.

[0062] The term “optional” or “optionally” means that the subsequent described event, circumstance or substituent may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0063] The terms “first,” “second,” “third,” and the like when used herein with reference to elements or properties, are simply to more clearly distinguish or identify multiple elements or properties, and are not intended to indicate an order or other serial or numerical limitation.

[0064] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0065] Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes, such as variations of + / - 10% or less, + / - 1-5% or less, + / - 1% or less, and + / - 0.1% or less from the specified value. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.C. RECOMBINANT POLYNUCLEOTIDES

[0066] Disclosed herein are recombinant polynucleotides for use as nucleic acid switches in cells. The polynucleotide may comprise DNA or RNA. In some embodiments, the polynucleotide comprises mRNA . In some embodiments, the polynucleotide is an mRNA switch. In general, a polynucleotide comprises at least (a) a 3 '-untranslated region (3'-UTR) sequence and (b) a payload sequence, and the 3'-UTR sequence regulates expression of the payload sequence.1. 3 '-Untranslated Regions (3f-UTRs)

[0067] 3'-UTRs regulate gene expression through the binding of RNA-binding proteins (Mayr, Christine. Annual Review of Genetics, vol. 51 : 171-194, 2017, doi: 10.1146 / annurev- genet- 120116-024704). RBPs bind to 3 -UTR cis-elements and mediate 3'-UTR functions through the recruitment of effector proteins. As RBPs interact with diverse effector proteins,each 3'-U TR regulatory element has the potential to carry out several different functions, depending on the cell type or cellular state. Hie cellular state also determines the RBPs that can access 3'-UTRs at a given moment. The composition of RBPs bound to a 3'-UTR at a given moment is dynamic and can change depending on the local environment, including through recognizing of an exogeneous molecule by the cell, e.g., the binding of an exogenous molecule by the cell. In some embodiments, the exogenous molecule is an antigen, e.g., a cancer-related antigen.

[0068] The 3’ untranslated region (3'-UTR) of a messenger RNA (mRNA) lies downstream of the mRNA’s translation termination codon. Thus, the 3'-UTR is transcribed from DNA but is not translated into protein. In polynucleotides of the present disclosure, the 3'-UTR lies downstream of a translation termination codon. In some embodiments, the 3'-UTRs of the polynucleotides can regulate mRNA-based processes, such as mRNA localization, mRNA stability, and translation. In some embodiments, the 3'-UTRs of the polynucleotides can establish 3 '-UTR -mediated protein-protein interactions, and thus can regulate diverse protein features, including protein complex formation or posttranslational modifications, as well as alter protein conformations.

[0069] In some embodiments, a provided polynucleotide is introduced into a target cell where the polynucleotide 3'-UTR sequence can respond to one or more intracellular signals produced by the target cell. In some embodiments, the polynucleotide 3'-UTR sequence responds to the intracellular signal(s) by modulating expression of the polynucleotide payload sequence. In some embodiments, the polynucleotide 3'-UTR sequence responds to the intracellular signal(s) by regulating transcription, localization, stability, and / or translation of the polynucleotide payload sequence. In some embodiments, an RNA transcript of the polynucleotide 3'-UTR sequence responds to one or more intracellular signals produced by the target cell. In some embodiments, the RN A transcript of the polynucleotide 3'-UTR sequence responds to one or more intracellular signals by modulating the translation of the polynucleotide pay load sequence.

[0070] In some embodiments, the provided polynucleotide 3'~UTR sequence can respond to one or more intracellular signals produced by the target cell, and the polynucleotide 3'-UTR sequence can mediate degradation of the polynucleotide RNA transcript itself. In some embodiments, an RNA transcript of the polynucleotide 3'-UTR sequence can respond to one or more intracellular signals produced by the target cell, and the RNA transcript of thepolynucleotide 3'-UTR sequence can mediate degradation of the polynucleotide RNA transcript itself.

[0071] Many 3 -UTR sequences may be used in a provided recombinant polynucleotide. In some embodiments, the 3'-UTR sequence is a wild-type 3'-UTR sequence. In some embodiments, the wild-type 3 -UTR sequence is naturally occurring in a target cell, re., endogenous to the target cell. In some embodiments, the 3'-UTR sequence is an engineered variant of a wild-type 3'-UTR sequence. Engineered variants of a 3'-UTR sequence include 3'- UTR sequences with deletions, insertions, truncations, point mutations, and / or frameshift mutations.

[0072] In some embodiments, the 3'-L!TR sequence of a provided recombinant polynucleotide comprises a cytokine 3'-UTR sequence, a cell surface receptor 3'-UTR sequence, or a variation thereof. In some embodiments, the 3'-UTR sequence comprises a sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity with a cytokine 3’-UTR sequence, a cell surface receptor 3'- UTR sequence. In some embodiments, the 3'-UTR sequence is a cytokine 3'-UTR sequence or a cell surface receptor 3'-UTR sequence.

[0073] In some embodiments, the 3'-UTR sequence of a provided polynucleotide comprises the 3'-UTR sequence of interferon gamma (IFNG), tumor necrosis factor (TNF), Cluster of Differentiation 69 (CD69), colony stimulating factor (CSF)-2, interleukin (IL)-2, IL-1B, IL- 10, IL-12, IL-13, IL-21 , IL-22, IL-4, IL-6, IL-17A, or any variation or combination thereof. In some embodiments, the 3’-UTR sequence comprises a sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity with an IFNG 3'-UTR sequence, an interleukin IL- IB 3'-UTR sequence, an IL-2 3'-UTR sequence, an IL-4 3'-UTR sequence, an IL-6 3'-UTR sequence, an IL-10 3'-UTR sequence, an IL-12 3'- UTR sequence, an IL- 13 3'-UTR sequence, an IL-17A 3'-UTR sequence, an IL-21 3'-UTR sequence, an IL-22 3'-UTR sequence, a tumor necrosis factor (TNF) 3'-UTR sequence, a CD69 3'-(JTR sequence, or a CSF-2 3'-UTR sequence. In some embodiments, the 3'-UTR sequence is an IFNG 3'-UTR sequence, an interleukin IL- IB 3'-UTR sequence, an IL-2 3'-UTRsequence, an IL-4 3'-UTR sequence, an IL-6 3'-UTR sequence, an IL- 10 3'-UTR sequence, anIL-12 3'-UTR sequence, an IL-13 3'-UTR sequence, an IL-17A 3'-UTR sequence, an IL-21 3’- UTR sequence, an IL-22 3'-UTR sequence, a tumor necrosis factor (INF) 3'-UTR sequence, a CD69 3'-UTR sequence, or a CSF-2 3'-UTR sequence.

[0074] In some embodiments, the 3'-UTR sequence of a provided recombinant polynucleotide comprises a sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity with SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, I I, 12, 13, 14, 15, or 16. In some embodiments, the 3 '-UTR sequence is the sequence as set forth in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, I I, 12, 13, 14, 15, or 16. a) Truncation of 3'-lJTRs

[0075] In some embodiments, the 3'-UTR sequence of a provided recombinant polynucleotide comprises a truncated vanant of a wild-type 3'-UTR sequence. The truncated 3'-UTR variant may comprise a 5'-end truncation or a 3 '-end truncation. In some embodiments, the truncated 3'-UTR variant retains the proximal end of the wikl-type 3'-UTR sequence. In some embodiments, the truncated 3'-UTR variant retains tire distal end of the wild-type 3’-UTR sequence. In some embodiments, the truncated 3'-IJTR variant lacks the proximal end of the wild-type 3'-UTR sequence. In some embodiments, the truncated 3'-UTR variant lacks the distal end of the wild-type 3'-L!TR sequence. In some embodiments, the truncated 3'-UTR variant lacks the proximal end of the wild-type 3'-UTR sequence and lacks the distal end of the wild-type 3'-UTR sequence.

[0076] In some embodiments, the truncated 3'-UTR vanant of a wild-type 3'-UTR has a length that is at least 5 base pairs (bp) shorter, at least 6 bp shorter, at least 7 bp shorter, at least 8 bp shorter, at least 9 bp shorter, at least 10 bp shorter, at least 11 bp shorter, at least 12 bp shorter, at least 13 bp shorter, at least 14 bp shorter, at least 15 bp shorter, at least 16 bp shorter, at least 17 bp shorter, at least 18 bp shorter, at least 19 bp shorter, at least 20 bp shorter, at least 21 bp shorter, at least 22 bp shorter, at least 23 bp shorter, at least 24 bp shorter, at least 25 bp shorter, at least 26 bp shorter, at least 27 bp shorter, at least 28 bp shorter, at least 29 bp shorter, at least 30 bp shorter, at least 31 bp shorter, at least 32 bp shorter, at least 33 bp shorter, at least 34 bp shorter, at least 35 bp shorter, at least 36 bp shorter, at least 37 bp shorter, at least 38 bp shorter, at least 39 bp shorter, at least 40 bp shorter, at least 41 bp shorter, at least 42 bp shorter.at least 43 bp shorter, at least 44 bp shorter, at least 45 bp shorter, at least 46 bp shorter, at least 47 bp shorter, at least 48 bp shorter, at least 49 bp shorter, at least 50 bp shorter, at least 51 bp shorter, at least 52 bp shorter, at least 53 bp shorter, at least 54 bp shorter, at least 55 bp shorter, at least 56 bp shorter, at least 57 bp shorter, at least 58 bp shorter, at least 59 bp shorter, at least 60 bp shorter, at least 61 bp shorter, at least 62 bp shorter, at least 63 bp shorter, at least 64 bp shorter, at least 65 bp shorter, at least 66 bp shorter, at least 67 bp shorter, at least 68 bp shorter, at least 69 bp shorter, or at least 70 bp shorter than that of the wsld-type 3'-UTR.

[0077] In some embodiments, the truncated 3'-UTR variant of a wild-type 3'-LTR has a length that is no greater than 100 bp, no greater than 110 bp, no greater than 120 bp, no greater than 130 bp, no greater than 140 bp, no greater than 150 bp, no greater than 160 bp, no greater than 170 bp, no greater than 180 bp, no greater than 190 bp, no greater than 200 bp, no greater than 210 bp, no greater than 220 bp, no greater than 230 bp, no greater than 240 bp, no greater than 250 bp, no greater than 260 bp, no greater than 270 bp, no greater than 280 bp, no greater than 290 bp, no greater than 300 bp, no greater than 310 bp, no greater than 320 bp, no greater than 330 bp, no greater than 340 bp, or no greater than 350 bp.

[0078] In some embodiments, the truncated 3'-UTR variant of a wild-type 3'-UTR has a length that is at least 5% shorter, at least 6% shorter, at least 7% shorter, at least 8% shorter, at least 9% shorter, at least 10% shorter, at least 1 1% shorter, at least 12% shorter, at least 13% shorter, at least 14% shorter, at least 15% shorter, at least 16% shorter, at least 17% shorter, at least 18% shorter, at least 19% shorter, at least 20% shorter, at least 21 % shorter, at least 22% shorter, at least 23% shorter, at least 24% shorter, at least 25% shorter, at least 26% shorter, at least 27% shorter, at least 28% shorter, at least 29% shorter, at least 30% shorter, at least 31% shorter, at least 32% shorter, at least 33% shorter, at least 34% shorter, at least 35% shorter, at least 36% shorter, at least 37% shorter, at least 38% shorter, at least 39% shorter, or at least 40% shorter than that of the wild-type 3'-UTR.

[0079] In some embodiments, the truncated 3'-UTR variant of a provided recombinant polynucleotide comprises a sequence with at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity with a wild-type 3'-UTR sequence. In some embodiments, the truncated 3'-UTR variant comprises a sequence with at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity with an IFNG 3'-UTR sequence, an interleukin IL- IB 3'- UTR sequence, an IL-2 3'-UTR sequence, an IL-4 3'-UTR sequence, an IL-6 3'-UTR sequence, an IL-10 3'-UTR sequence, an IL-12 3'-UTR sequence, an IL-13 3'- UTR sequence, an IL-17A 3'-UTR sequence, an IL-21 3'-UTR sequence, an IL-22 3 -UTR sequence, a tumor necrosis factor (TNF) 3'-UTR sequence, a CD69 3'-UTR sequence, or a CSF-2 3'-UTR sequence. In some embodiments, the truncated 3 -UTR variant comprises a sequence with at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity with the sequence as set forth in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16.

[0080] In some embodiments, the truncated 3'-UTR vanant of a provided recombinant polynucleotide comprises a sequence with at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity with SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, 2.4, 25, 2.6, 27, 28, 29, 30, 31 , 32, 33, 34, 35, or 36. In some embodiments, the truncated 3'-UTR variant is the sequence as set forth in SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36. b) Deletion or Mutation of AU-Rich Elements (AREs)

[0081] AU -rich elements (AREs) are motifs m 3'-UTRs that enable rapid mRNA decay (Mayr, Christine. Annual Review of Genetics, vol. 51 : 171-194, 2.017, doi: 10,1146 / annurev-genet-1201 16-024704). AREs can be found in the 3'-UTRs of genes whose expression requires tight regulation, such as cytokines, lymphokmes, growth factors, and oncogenes (Id. and Caput D, Beutler B, Hartog K, Thayer R, Brown-Shimer S, Cerami A, 1986. Proc Natl Acad Sci 83: 1670-1674). In some instances, the mRNA half-life of such genes is shorter than 30 minutes. The role of AREs in restricting protein expression is highlighted by the fact that their deletion is associated with cancer, chronic inflammation, and auto-immune disease (Barreau C, Paillard L, Osborne HB. 2005. Nucleic Acids Res. 33: 7138-50). Thus, in some embodiments, deletion of one or more AREs from a 3'-UTR sequence prevents rapid degradation of polynucleotide mRNA.[0082 j In some embodiments, the 3'-UTR of a provided recombinant polynucleotide is a variant of a wild-type 3'-UTR, where the variant lacks one, two, three, four, five, six, seven, eight, nine, ten, or more AREs of the wild-type 3'-UTR sequence. The variant can lack the one or more AREs as a result of a deletion of one or more AREs, and / or as a result of a mutation with one or more AREs. In some embodiments, the 3'-UTR lacks one, two, three, four, five, six, seven, eight, nine, ten, or more AREs of an IFNG 3'-DTR sequence, an interleukin IL- IB 3'-UTR sequence, an IL, -2 3'-UTR sequence, an IE-4 3'-UTR sequence, an IL, -6 3'-UTR sequence, an IL-10 3'-UTR sequence, an IL-12 3'-UTR sequence, an IL-13 3'-UTR sequence, an IL-17A 3'-UTR sequence, an IL-21 3'-UTR sequence, an IL-22 3'-UTR sequence, a tumor necrosis factor (TNF) 3'-UTR sequence, a CD69 3'-UTR sequence, or a CSF-2 3’-LTR sequence. In some embodiments, the 3'~UTR. comprises a sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity with SEQ ID NO: 37, 38, 39, 40, 41, 42, or 43. In some embodiments, the truncated 3'-UTR variant is SEQ ID NO: 37, 38, 39, 40, 41, 42, or 43.

[0083] In addition to or as an alternative to deletion and / or mutation of one or more AREs of a 3'-UTR, a provided recombinant polynucleotide can include deletion and / or mutation of other cis-regulatory elements. For example, a 3'-UTR of a provided recombinant polynucleotide can additionally or alternatively include deletion and / or mutation of one or more sequence-based cis-regulatory elements such as GU-rich elements (GREs). In some examples, a 3'-UTR of a provided recombinant polynucleotide can additionally or alternatively include deletion and / or mutation of one or more structure-based cis-regulatory elements such as pseudoknots and / or constitutive decay elements (CDEs).Payload Sequences

[0084] Many sequences are suitable for use as a payload sequence in the provided recombinant polynucleotides . In many embodiments, a target cell expresses a payload product encoded by the payload sequence in response to the state of the target cell. In some embodiments, the state of the target cell changes when the target cell recognizes an exogeneous molecule, e.g., binds to an exogenous molecule. In some embodiments, the target cell produces an intracellular signal when the target cell recognizes an exogeneous molecule, e.g., binds to an exogenous molecule. Thus, in some embodiments, the target cell is stimulated when it recognizes the exogeneous molecule, e.g., binds to the exogenous molecule, and the payload product is expressed from the polynucleotide. In some embodiments, the payload product is not expressed from the polynucleotide when the target cell does not recognize an exogeneous molecule, e.g., does not bind to an exogenous molecule (i.e., the target cell is unstimulated). In some embodiments, the exogenous molecule is an antigen, e.g., a cancer-related antigen.

[0085] In some embodiments, the payload product is the payload sequence itself, or an RNA transcript thereof. For example, the payload sequence can be a noncoding RNA sequence. In some embodiments, the payload product is a polypeptide encoded by the payload sequence. In some embodiments, the payload product is functional in the target cell. In some embodiments, the payload product is secreted by the target cell.

[0086] In some embodiments, the polynucleotide 3'-UTR sequence modulates expression of the payload product. In some embodiments, the 3'-UTR sequence regulates the transcription, localization, stability, and / or translation of the payload product. In some embodiments, the payload product is a long noncoding RNA. In some embodiments, the payload product is an mRNA. In some embodiments, the payload product is a polypeptide.

[0087] In some embodiments, expression of the payload product, e.g., the payload polypeptide or the payload polynucleotide, by the target cell is induced by the presence of one or more intracellular signals produced by the target cell. In some embodiments, the payload product, e.g., the payload polypeptide or the payload polynucleotide, is not expressed in the absence of tire intracellular signal(s). In some embodiments, expression of the payload product, e.g., the payload polypeptide or the payload polynucleotide, by the target cell in the absence of an intracellular signal is no greater than 5%, no greater than 10%, no greater than 15%, no greater than 20%, no greater than 25%, no greater than 30%, no greater than 35%, no greater than 40%, no greater than 45%, no greater than 50%, no greater than 60%, no greater than 65%of the expression of the payload product, e.g., the payload polypeptide or the payload polynucleotide, when the intracellular signal is present. Methods for detecting and measuring polypeptide or polynucleotide expression levels are known to one of ordinary skill in the art. Exemplary methods include Western blot, fluorescence microscopy, Northern blot, qPCR, high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC / MS), enzyme-linked immunosorbent assay (ELISA), protein immunoprecipitation, immunoelectrophoresis, and in situ hybridization. In some embodiments, and as described in the Examples below, the payload product, e.g., protein expression levels, are detected and / or measured using flow cytometry.

[0088] The recombinant polynucleotides of the present disclosure may be used to introduce a variety of gene products to a ceil, a population of cells, or a subject, such as a protein that may be used to treat a disease. In some embodiments, the recombinant polynucleotide comprises a payload sequence that encodes a chimeric antigen receptor (CAR) polypeptide. The CAR polypeptide can comprise at least (1) an extracellular target-binding domain, (2) a transmembrane domain, (3) a hinge domain, and (4) an intracellular signaling domain. In some embodiments, the CAR is introduced to an immune cell, e.g., the target cell is a T cell.

[0089] In some embodiments, the extracellular target-binding domain comprises a polypeptide that binds to a target of interest. For example, if the target of interest is a cancer- related antigen, the extracellular target-binding domain comprises a polypeptide that binds to that cancer-related antigen and / or a cell that expresses that cancer-related antigen on its surface.

[0090] Exemplary cancer-related antigens include CDla, CDlb, CDlc, CDld, CDle, CD2, CD3delta, CD3epsilon, CD3gamma, CD4, CDS, CD6, CD7, CD8alpha, CD8beta, CD9, CD10, CDS la, CDS lb, CD1 1c, CDwl2. CD13, CD14. CD15u, CD16a, CD16b, CDwl7, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31 , CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD44R, CD46, CD47R, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49L CD50, CD51, CD52, CD53. CD54, CD55, CD56, CD57, CD58, CD59, CD60a, CD60b, CD60c, CD61, CD62.E, CD62L, CD62P, CD63, CD64, CD65, CD65s, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD75, CD75s, CD77, CD79a. CD79b, CD80, CD8I, CD82, CD83, CD84, CD85, CD86, CD87, CD88, CD89, CD90, CD91, CD92, CDw 93, CD94, CD95, CD96, CD97, CD98, CD99, CD100, CDIOL CD102, CD103, CD104, CD105, CD106, CD107a, CD107b, CD108,CDI09, CD110, CD111, CD112, CDwl 13, CD114, CD115, CDI 16, CD117, CD118, CDwl 19, CD 120a, CD 120b, CD 12 la, CDwl21b, CD 122, CD 123, CD 124. CDwl25, CD 126, CD 127, CDwl28a, CDwl28b, CD129, CD130, CD131, CD132, CD133, CD134, CD135, CDwl36, CDwl37, CD138, CD139, CD140a, CD140b, CD141, CD142, CD143, CD144, CDw!45, CD146, CD147, CD148, CDwl49, CD150, CD151, CD152, CD153, CD154, CD155, CD156a, CD156b, CDwl56C, CD157, CD158, CD159a, CD159c, CD160, CD161, CD162, CD162R, CD163, CD164, CD165, CD166, CD 167a, CD 168, CD169, CD170, CD171, CD172a, CD172b, CD172g, CD173, CD174, CD175, CD175s, CD176, CD177, CD178, CD179a, CD179b, CD180, CD181, CD182, CD183. CD184, CD185, CDwl86, CD191, CD192, CD193, CD195, CD196, CD197, CDwl98, CDwl99, CDw!97, CD200, CD201, CD202b, CD203c, CD204, CD205, CD206, CD207, CD208, CD209, CDw210, CD212, CD213al, CD213a2, CDw217, CDw218a, CDw218b, CD220, CD221, CD222, CD223, CD224, CD225, CD226, CD227, CD228, CD229, CD230, CD231. CD232, CD233, CD234, CD235a, CD235b, CD235ab, CD236, CD236R, CD238, CD239, CD240CE, CD240D, CD240DCE, CD241, CD242, CD243, CD244, CD245, CD246, CD247, CD248, CD249, CD252 CD253, CD254, CD256, CD257, CD258, CD261, CD262, CD263, CD264, CD265, CD266, CD267 , CD268, CD269, CD271, CD272, CD273, CD274, CD275, CD276, CD277, CD278, CD279, CD280, CD281, CD282, CD283, CD284, CD289, CD292, CDw293, CD294, CD295, CD296, CD297, CD298, CD299, CD3()0a, CD300c, CD300e, CD301, CD302, CD303, CD304, CD305, CD306, CD307, CD309, CD312, CD314, CD315, CD316, CD317. CD318, CD319, CD320, CD321, CD322, CD324, CDw325, CD326, CDw327, CDw328, CDw329, CD331, CD332, CD333, CD334, CD335, CD336, CD337, CDw338, CD339, CD340, CD344, CD349, CD350, CD351, CD352, CD353, CD354, CD355, CD357, CD358, CD360, CD361, CD362 CD363, Pax-5, kappa, lambda, CD200, cytoplasmic kappa, cytoplasmic lambda, cripto, EGFR, TSH- R, DU., 4, CTLA4, CXCR4, Endoglin, EPC JAM, ERBB2, FCGR1, FOLR, GD2 ganglioside, G-28, GD3 idiotype, Heat shock proteins, FIERI, HER2, HLA-DR10, HLA-DRB, human chorionic gonadotropin, IGF1R, IL-2 receptor, IL-6R, Integrins (avp3, a501, a604, al 103, a505, av05), MAGE-1, MAGE-2, MAGE-3, MAGE 4, anti-transferrin receptor, p97, MS4A1, MUC1 or MUC1-KLH, MUC16 (CAI 25), CEA, gplOO, MARTI, MPG, MS4A1, Nucleolin, Neu oncogene product, P21, PLAP-like testicular alkaline phosphatase, PSMA, PSA, ROBO4, TAG 72, T cell transmembrane protein, TNFRSF10B, TNFRSF13B, TPBG, TRAIL-R1, VCAM-1, VEGF, VEGF-A, VEGF-2, Annexin Al, Nucleolin, ROBO4, Amino-peptidase N, A-Iike-4 (DLL4), B7-H3, WT1, MUC1, LMP2, HPV E6 E7, EGFRvIII, HER-2 / neu, Idiotype,MAGE A3, p53 nonmutant, NY-ESO-1, GD2, MelanA / MARTl, Ras mutant, p53 mutant, Proteinase3 (PR1), bcr-abl, Tyrosinase, Survivin, hTERT, Sarcoma translocation breakpoints, EphA2, PAP, ML-IAP, AFP, ERG (TMPRSS2 ETS fusion gene), NA 17, PAX3, ALK, Androgen receptor, C-yclin Bl, Polysialic acid, MYCN, RhoC, TRP-2, GD3, Fucosyl GM1, Mesothelin, PSCA, MAGE Al, sLe(a), CYP1B1, PLAC1, GM3, BORIS, Tn, GloboH, ETV6- AML, NY-BR-1, RGS5, SART3, STn, Carbonic anhydrase IX, PAX5, OY-TES1, Sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3, Legumain, Page4, VEGFR2, MAD-CT-1, FAP, PDGFR-P3, MAD-CT-2, and Fos-related antigen 1. In some embodiments, the extracellular target-binding domain binds to CD 19.[0091 j In some embodiments, the extracellular target-binding domain is derived from an antibody or a fragment thereof. In some embodiments, the extracellular target-binding domain is derived from a naturally occurring ligand for the target.

[0092] Many suitable transmembrane domains may be used in accordance with a CAR of the present disclosure. In some embodiments, the transmembrane domain is derived from the transmembrane domain of the alpha, beta or zeta chain of the T-cell receptor, CD28, CD27, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. In some embodiments, the transmembrane domain is derived from the transmembrane region(s) of, KIRDS2, 0X40, CD2, CD27, LFA-1 (CD I la, CD 18), ICOS (CD278), 4-1 BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CDI60, CD19, IL2R beta, IL2R gamma, IL7R a, ITGAI, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49E ITGAD, CD1 Id, 1TGAE, ( D 103, ITGAL, CD! la, LFA-1, ITGAM, CD1 lb, ITGAX, CD1 1c, ITGB 1 , CD29, JTGB2, CD 18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CDI60 (BY55), PSGL1, CD100, (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3). BLAME, (SLAMF8), SELPLG (CD 162), LTBR, PAG / Cbp, NKG2D, or NKG2C. In some embodiments, the transmembrane domain is the native CD28 transmembrane domain or a variant thereof.

[0093] In some embodiments, tire CAR may comprise one or more hinge domains that covalently link the transmembrane domain to the extracellular target-binding domain. In some embodiments, the hinge domain is derived from a naturally occurring immunoglobulin hinge region, e.g., a human IgGl or a human IgG4, or a variant thereof. In some embodiments, the hinge domain is derived from the extracellular regions of membrane proteins, such as CD8alpha, CD4, CD28, PD1, CD 152, and CD7. In some embodiments, the hinge domain is the native CD28 hinge domain or a variant thereof.[0094 j In some embodiments, the CAR may comprise one or more intracellular signaling domains. In some embodiments, the intracellular signaling domain is derived from a polypeptide sequence of an immune cell co-stimulatory domain, activation domain, or cytoplasmic domain. In general, the intracellular signaling domain activates one or more native effector functions of the immune cell in which comprises the CAR. In some embodiments, the intracellular signaling domain increases CAR immune cell cytokine production. In some embodiments, the intracellular signaling domain increases immune cell replication. In some embodiments, the intracellular signaling domain prevents CAR immune cell exhaustion. In some embodiments, the intracellular signaling domain immune cell anti-tumor activity'. In some embodiments, the intracellular signaling domain increases survival of CAR immune cells.

[0095] In CARs that have more than one intracellular signaling domains, a primary signaling domain regulates primaiy activation of the CAR complex. The primaiy signaling domain can function in a stimulatory way or in an inhibitory way. A primaiy' intracellular signaling domain that acts in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs. Examples of IT AM containing primary intracellular signaling domains include those of CD3 zeta, common FcR gamma, Fc gamma Rlla, FcR beta (Fc Epsilon Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12. In some embodiments, a CAR comprises an intracellular signaling domain, e.g, a primaiy' signaling domain, of CD3 zeta.

[0096] In addition to the primary signaling domain, the CAR may also comprise one or more costimulatory signaling domains that are usefill in the context of the recombinant polynucleotide of the present disclosure. For example, the intracellular signaling domain of the CAR can comprise a CD3 zeta chain portion and one or more costimulatory signaling domains. Hie costimulatory signaling domain refers to a portion of the CAR comprising the intracellular domain of a costimulatoiy' molecule. A costimulatory’ molecule can be derived from a cell surface molecule other than an antigen receptor or its ligands that is required for an efficient response of lymphocytes to an antigen. Examples of such molecules include CD27, CD28, 4- 1BB (CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-I), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that binds to CD83, and the like.For example, CD27 costimulation has been demonstrated to enhance expansion, effector function, and survival of human CAR-T cells in vitro and augments human T cell persistence and antitumor activity in vivo (Song et al. Blood. 2012; 119(3):696-706). Further examples of such costimulatory molecules include CDS, ICAM-1 , GITR, BAFFR, HVEM (LIGIITR), SLAMF7, NKp80 (KLRFI), NKp44, NKp3(), NKp46, CD 160, CD 19, CD4, CD8alpha, CD8beta, 1L2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a. ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl Id, ITGAE, CD103, ITGAL, CDl la, LFA-l , ITGAM, CD1 lb, ITGAX, CDl 1c, ITGB I, CD29, ITGB2, CD 18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), NKG2.D, CEACAM1, CRTAM, Ly9 (CD229), CDl 60 (BY55), PSGL1, CDl 00 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM, (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, and CD 19a.

[0097] In some embodiments, the CAR is the CD19-28z CAR. that specifically binds the CD19 antigen. In some embodiments, the cells expressing the CD19-28z CAR, e.g., CDl 9-28z CAR-T cells, are capable of binding to CD19 or CD19-positive cancer cells. In some embodiments, the cells expressing the CD19-28z CAR, e.g., CD19-28z CAR-T cells, are capable of lysing CD19-positive cancer cells. In some embodiments, the CD 19-positive cancer cells are tumor cells.3. Sequence Combinations

[0098] Also provided herein are combinations and arrangements of sequences that may be found in a recombinant polynucleotide of the present disclosure. In some embodiments, the polynucleotide comprises (a) a 3'-UTR sequence and (b) a payload sequence. In some embodiments where the polynucleotide comprises only one 3'~UTR sequence, the polynucleotide is herein referred to as a “singlet.” In some embodiments, the 3'-UTR sequence is located downstream of the pay load sequence. In some embodiments, the 3'-UTR sequence is operably linked to the payload sequence, in some embodiments, the 3'-UTR sequence is adjacent to the payload sequence.

[0099] Hie use of more than one 3’-L!TR sequence in a recombinant polynucleotide may be useful in decreasing baseline (or “leaky”) expression of the polynucleotide payload when the target cell does not bind to an exogenous molecule (i ,e., the target cell is unstimulated). In some embodiments, the polynucleotide comprises two, three, four, five, or more 3'-UTR sequences; these polynucleotides are herein termed “synthetic arrays.” In some embodiments, one of thetwo, three, four, five, or more 3'-UTR sequences is adjacent to and / or downstream from the payload sequence. In some embodiments, one, some, or all of the two, three, four, five, or more 3'-UTR sequences comprise a wild-type 3'-UTR sequence. In some embodiments, one, some, or all of the tw'O, three, four, five, or more 3'-UTR sequences comprise an engineered variant of a wild-type 3'-UTR sequence. In some embodiments, one, some, or all of the two, three, four, five, or more 3'-UTR sequences are endogenous to the target cell. In some embodiments, one, some, or all of the two, three, four, five, or more 3'-UTR sequences have the same 3'-UTR sequence. In some embodiments, one, some, or all of the one, two, three, four, five, or more 3'- UTR sequences have different 3'-UTR sequences.

[0100] In some embodiments, each of the two, three, four, five, or more 3'-UTR sequences of a provided recombinant polynucleotide responds to an intracellular signal of the target cell. In some embodiments, each of the one, two, three, four, five, or more 3'-UTR sequences responds to the binding of an exogenous molecule by the target cell. In some embodiments, some or all of the two, three, four, five, or more 3'-UTR sequences respond to the same intrinsic signal or to the binding of the same exogenous molecule by the target cell. In some embodiments, some or all of the two, three, four, five, or more 3'-UTR sequences respond to different intrinsic signals or to the binding of different exogenous molecules by the target cell.

[0101] In some embodiments, the provided recombinant polynucleotide comprises two 3'- UTR sequences and the two 3'-UTR sequences are next to each other. In some embodiments where the polynucleotide comprises two 3'-UTR sequences, one of the two 3'-UTR sequences is adjacent to and / or downstream of the payload sequence. In some embodiments where the polynucleotide comprises two 3'~UTR sequences, one of the two 3'-UTR sequences is between the payload sequence and the other 3 ’-UTR sequence. In some embodiments, the order of sequences in the 5' to 3' direction of the polynucleotide is the payload sequence, a 3'-UTR sequence, and another 3’-UTR sequence. In many embodiments, one or both of the 3 ’-UTR sequences are operably linked to the payload sequence.

[0102] In some embodiments where the provided recombinant polynucleotide comprises two 3'-UTR sequences, each of the two 3'-UTR sequences comprises a wild-type 3'-UTR sequence. Tire two 3'-UTR sequences can be different or the same. In embodiments where the two 3'- UTR sequences are the same and are next to each other (i.e., adjacent), the recombinant nucleotide is herein referred to as a “synthetic doublet.” In some embodiments, one or both of the 3'-UTR sequences are endogenous to the target cell.

[0103] In some embodiments where the provided recombinant polynucleotide comprises two 3'-UTR sequences, one of the two 3'-UTR sequences comprises a wild-type 3'-UTR sequence while the other comprises an engineered variant of a wild-type 3'-UTR sequence. In some embodiments, the wild-type 3'-UTR sequence is endogenous to the target cell.

[0104] In some embodiments wdrere the provided recombinant polynucleotide comprises two 3’'-UTR sequences, each of the two 3'-UTR sequences comprises an engineered variant of a wild-type 3'-UTR sequence. The two 3'-UTR sequences can be different or the same.

[0105] In some embodiments where the provided recombinant polynucleotide comprises two 3'-UTR sequences and one of the two 3'-UTR sequences is adjacent to the payload sequence, the 3'-UTR sequence that is adjacent to the payload sequence comprises a cytokine 3'-UTR sequence, a cell surface receptor 3 -UTR sequence, or a variation thereof. In some embodiments, the 3'-UTR sequence that is adjacent to the payload sequence comprises a sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least. 94%, at. least. 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity with a cytokine 3'-UTR sequence, a cell surface receptor 3'- UTR sequence. In some embodiments, the 3'-UTR sequence that is adjacent to the payload sequence is a cytokine 3'-UTR sequence or a cell surface receptor 3'-UTR sequence.

[0106] In some embodiments where the provided recombinant polynucleotide comprises two 3'-UTR sequences and one of the two 3'-UTR sequences is adjacent to the payload sequence, the 3'-UTR sequence that is adjacent to the payload sequence comprises the 3'-UTR sequence of IFNG, TNF, CD69, CSF-2, interleukin IL-2, IL-1B, IL-10, IL-12, IL-13, IL-21, IL-22, IL- 4, IL-6, IL-17A, or any variation or combination thereof. In some embodiments, the 3’-UTR sequence that is adjacent to the payload sequence comprises a sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity with an IFNG 3 -UTR sequence, an interleukin IL,- IB 3'-UTR sequence, an IL-2 3'- UTR sequence, an IL-4 3'-UTR sequence, an IL-6 3'-UTR sequence, an IL-10 3'-UTR sequence, an IL- 123'-UTR sequence, an IL-13 3'-UTR sequence, an IL-17A 3'-UTR sequence, an IL-21 3'-UTR sequence, an IL-22 3'-UTR sequence, a tumor necrosis factor (TNF) 3'-UTR sequence, a. CD69 3'-UTR sequence, or a. CSF-2 3'-UTR sequence. In some embodiments, the3'-UTR sequence that is adjacent to the payload sequence is an IFNG 3'-UTR sequence, an interleukin IL- IB 3’-DTR sequence, an IL-2 3'-DTR sequence, an IL-4 3'-UTR sequence, an IL-6 3'-UTR sequence, an IL-10 3'-UTR sequence, an IL-12 3'-UTR sequence, an IL-13 3'- UTR sequence, an IL-17A 3'-UTR sequence, an IL-21 3'-UTR sequence, an IL-22 3'-UTR sequence, a tumor necrosis factor (TNF) 3'-UTR sequence, a CD69 3'-UTR sequence, or a CSF-2 3'-UTR sequence.

[0107] In some embodiments where the provided recombinant polynucleotide comprises two 3 -UTR sequences and one of the two 3'-UTR sequences is adjacent to the payload sequence, the 3'-UTR sequence that is adjacent to the payload sequence comprises a sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity with SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , or 43. In some embodiments, the 3’-UTR sequence that is adjacent to the payload sequence is the sequence as set forth in SEQ ID NO: 20, 21 , 22, 23, 24, 25, 26, 30, 31 , 34, 36, 37, or 41.

[0108] In some embodiments where the provided recombinant polynucleotide comprises a synthetic doublet, i .e., a polynucleotide comprising two adjacent 3'-UTR sequences, the sequence of the two adjacent 3'-UTRs comprises a sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity with SEQ ID NO: 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, or 68. In some embodiments where the polynucleotide comprises a synthetic doublet, the sequence of the two adjacent 3'-UTRs is as set forth in SEQ ID NO: 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, or 68.

[0109] In some embodiments where the provided recombinant polynucleotide comprises a synthetic array, i.e,, a polynucleotide comprising two or more 3'-UTR sequences, two of the two or more 3'-UTR sequences are next to each other (i.e., adjacent). In some embodiments where the polynucleotide comprises a synthetic array, the sequence of the two adjacent 3'-UTRs comprises a sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity with SEQ ID NO: 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, or 68. In some embodiments where the polynucleotide comprises a synthetic array, the sequence of the two adjacent 3'- UTRs is as set forth in SEQ ID NO: 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, or 68.D. DNA CONSTRUCTS, VECTORS, AND CELLS1. DNA Constructs and Vectors

[0110] Also provided herein are DNA constructs that each comprise a recombinant polynucleotide of the present disclosure, wherein each polynucleotide contains at least one 3'- UTR sequence and a polynucleotide payload sequence. The DNA construct may also comprise certain DNA elements that enable the generation, function, visualization, selection, and / or characterization of the construct. Exemplary DNA elements include a promoter, a reporter, an enhancer, a transcription terminator, a polyadenylation sequence, and a post-transcriptional regulatory element (PRE).

[0111] In some embodiments, the provided DNA construct comprises a payload promoter operably linked to the payload sequence. In some embodiments, the payload promoter is a constitutive promoter. Exemplary constitutive promoters include PGK, EFla core promoter (EF-S) SV40, CMV, UBC, EFl A, and CAGG. In some embodiments, the payload promoter is an inducible promoter. Exemplary' inducible promoters include chemically regulated promoters and physically regulated promoters. Chemically regulated promoters include, for example, alcohol-regulated promoters (e.g., an alcohol dehydrogenase (alcA) gene promoter), tetracycline-regulated promoters (e.g., a tetracycline-responsive promoter, a tetracycline operator sequence (tetO), a tet-On promoter, or a tet-Off promoter), steroid regulated promoters (e.g., a rat glucocorticoid receptor, a promoter of an estrogen receptor, or a promoter of an ecdysone receptor), or metal -regulated promoters (e.g., a metalloprotein promoter). Physically regulated promoters include, for example temperature-regulated promoters (e.g., a heat shock promoter) and light-regulated promoters (e.g., a light-inducible promoter or a light-repressible promoter).

[0112] In some embodiments, the DNA construct also comprises a reporter sequence encoding a reporter polypeptide that produces a detectable signal when the reporter polypeptideis expressed by the target cell. In some embodiments, the reporter functions as a switch reporter, providing an indication of the expression of the polynucleotide payload. In some embodiments, the reporter functions as a transduction promoter, providing an indication of transformation of a target cell with the DNA construct. In some embodiments, the DNA construct reporter sequence comprises a promoter that modulates expression of the reporter gene and is herein termed “reporter promoter.” In some embodiments, the reporter promoter is a constitutive promoter. In some embodiments, the reporter promoter is an inducible promoter. In some embodiments, the payload promoter and the reporter promoter are divergently oriented, i.e., oriented in the opposite direction, relative to one another. See, e.g., FIGS. 1A and 11A. In some embodiments, the reporter polypeptide is a fluorescent protein. In some embodiments, the fluorescent protein is a green fluorescent protein (GFP), enhanced GFP (eGFP), TurboGFP, red fluorescent protein (RFP), tdTomato, mCheny, yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), or blue fluorescent protein (BFP).

[0113] In some embodiments, the DNA construct also comprises a PRE. In some embodiments, the PRE is located adjacent to the reporter sequence. In some embodiments, the DNA construct also comprises a polyadenylation sequence.

[0114] In some embodiments, the DNA construct comprises a polyadenylation sequence. In some embodiments, the polyadenylation sequence is adjacent, to the distal end of the 3'-UTR sequence.

[0115] The polynucleotides and DNA constructs disclosed herein can be introduced into cells, e.g., particularly selected target cells, using appropriate vectors to produce engineered cells that can regulate expression of a polynucleotide payload sequence in response to the state of the ceil, or in response to the presence or absence of an exogenous molecule, e.g., an antigen. The polynucleotides, DNA constructs, and vectors can be prepared and introduced into target cells according to standard cloning techniques and methods for introducing nucleic acids into cells that are well-known to one of ordinary skill in the art, such as those disclosed in Molecular Cloning: A Laboratory Manual, 2ndedition (1989) (Sambrook, Fritsch, and Maniatis); Molecular Cloning: A Laboratory’ Manual, 4thedition (2012) (Green and Sambrook); Current Protocols in Molecular Biology (1987) (F.M. Ausubel et al. eds.).

[0116] In some embodiments, the polynucleotide or DNA construct is combined with a delivery vehicle, e.g., a plasmid or a iipid or polymer vector (including exosomes, liposomes, and lipid nanoparticles (LIMP)), that encapsulates, binds to, or otherwise is combined with thepolynucleotides or DNA construct. The delivery vehicle can improve the delivery efficiency. and thus, the functional efficacy, of the polynucleotide or DNA construct. In some embodiments, the vector is an LNP.|0117] In some embodiments, the polynucleotide or DMA construct is delivered to a cell via a virus or a viral vector. Examples of viruses and associated viral vectors (e.g., plasmids) include adeno-associated virus (AAV), adenovirus (e.g., a human adenovirus vector such as huAd5, huAd46; a chimpanzee adenovirus vector such as ChAdOxl, ChAd3; a rhesus macaque adenovirus vector such as RliAd54), flavivirus (e.g., a yellow fever (YF) virus vector), herpes simplex virus (HSV), lentivirus, measles virus, Newcastle disease virus (NDV), poxvirus (e.g., a vaccinia virus vector or a variola virus (W) vector), retrovirus, or vesicular stomatitis virus (VSV). In some embodiments, the vector or plasmid comprises the sequence of SEQ ID NO: 1, 2, 69, or 70.2. Cells

[0118] Also provided herein are cells that may be used as target cells tor polynucleotides,DNA constructs, vectors, or systems of the present disclosure. In some embodiments, the cell or population of the cells comprises a recombinant polynucleotide, a DNA construct, a vector , and / or a system of the present disclosure.

[0119] In some embodiments, the target cells are eukaryotic cells, such as mammalian cells, tor example, human cells. In some embodiments, the target cells are human immune cells, including but not limited to T cells (including primary T cells, naive T cells, CD4 T cells, CD8 T cells, stimulated T cells, cultured I' cells, immortalized T cells, helper I' cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, alpha / beta T cells, and gamma / delta T cells), B cells, natural killer (NK) cells, mast cells, macrophages, dendritic cells, monocytes, induced pluripotent stem cell, hematopoietic stem cell, myeloid progenitor cell, lymphoid progenitor cell, and myeloid-derived phagocytes. In some embodiments, the target cell is a T cell. In some embodiments, the target cell is a primary T cell.

[0120] In many embodiments, tire target cell is a cell that can express the polynucleotide payload. In some embodiments, the payload is a chimeric antigen receptor (CAR.) and the target cell is capable of expressing the CAR. In some embodiments, the target cell is an engineeredT cell that is capable of expressing the CAR. In some embodiments, the CAR is the CDI9-28zCAR and the target cell is an engineered T cell that is capable of expressing the CD19-28zCAR - in these embodiments, the target cell is termed the “CD19-28z CAR T cell.” In some embodiments, the CD 19-28z CAR T cell is capable of binding to CD 19 or CD 19-positive cells. e.g., CD19-positive cancer cells or CDl9-positive tumor cells. In some embodiments, the CD19-28z CAR T cell can lyse CD19-positive cells, e.g., CD19-positive cancer cells or CD19- positive tumor cells.

[0121] In some embodiments, target ceils are used to prevent or treat a disease in a patient. In some embodiments, the target cells are autologous cells, i.e., originally isolated from the patient who will receive the target cells. In some embodiments, the target cells are allogeneic celis, i .e., isolated from a subject who is different from the patient who will receive the target cells.E. PHARMACEUTICAL COMPOSITIONS

[0122] Also provided herein are pharmaceutical compositions for the modulating gene expression. Each pharmaceutical composition may comprise one or more recombinant polynucleotides, DNA constructs, vectors, systems, cells, or populations of cells of the present disclosure, and a pharmaceutically acceptable carrier or a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is used to modify a cell or a population of cells to comprise one or more recombinant polynucleotides, DNA constructs, vectors, and systems of the present disclosure. In some embodiments, the pharmaceutical composition is administered to a subject for therapeutic or prophylactic treatments.

[0123] In therapeutic applications, the pharmaceutical composition can be administered to a subject already suffering from a disease or condition, in an amount sufficient to cure or at least partially arrest the symptoms of the disease or condition, or to cure, heal, improve, or ameliorate the condition. In prophylactic applications, the pharmaceutical composition can be administered to a subject with a propensity or risk of exposure to conditions or diseases in order to prevent the occurrence of the condition or disease. The pharmaceutical composition can be administered to a subject before the onset of symptoms, or during or as soon as possible after the onset of the symptoms.

[0124] Hie pharmaceutical compositions described herein are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically or prophylactically effective. The quantity to be administered depends on a variety of factors including, e.g., the age, body weight, physical activity, and diet of the individual, the disease, disorder, or condition to be treated, and the stage or severity of the disease, disorder, orcondition. In certain embodiments, the size of the dose may also be determined by the existence, nature, and extent of any adverse side effects that accompany the administration of a therapeutic agent(s) in a particular individual. It should be understood, however, that the specific dose level and frequency of dosage for any particular patient may be varied and may depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, hereditary characteristics, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy.

[0125] In certain embodiments, the dose of the compound may take the form of solid, semisolid, lyophilized powder, or liquid dosage forms, such as, for example, tablets, pills, pellets, capsules, powders, solutions, suspensions, emulsions, suppositories, retention enemas, creams, ointments, lotions, gels, aerosols, foams, or the like, preferably in unit dosage forms suitable for simple admini stration of precise dosages.

[0126] As used herein, the term “unit dosage form” refers to physically discrete units suitable as unitan' dosages for humans and other mammals, each unit containing a predetermined quantity of a therapeutic or prophylactic agent calculated to produce the desired onset, tolerability, and / or therapeutic effects, in association with a suitable pharmaceutical excipient (e.g., an ampoule). In addition, more concentrated dosage forms may be prepared, from which the more dilute unit dosage forms may then be produced. The more concentrated dosage forms thus will contain substantially more than, e.g., at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, or more times the amount of the therapeutic compound.

[0127] Methods for preparing such dosage forms are known to those skilled in the art (see, e.g., Remington’s Pharmaceutical Sciences, supra). The dosage forms typically include a conventional pharmaceutical carrier or excipient and may additionally include other medicinal agents, carriers, adjuvants, diluents, tissue permeation enhancers, solubilizers, and the like. Appropriate excipients can be tailored to the particular dosage form and route of administration by methods well known in the art (see, e.g,, Remington’s Pharmaceutical Sciences, supra).

[0128] Administration of the pharmaceutical composition can be initiated within the first 48 hours of the onset of the symptoms, within the first 24 hours of the onset of the symptoms, within the first 6 hours of the onset of the symptoms, within 3 hours of the onset of the symptoms, or before the onset of any symptoms. The pharmaceutical composition can beadministered for a length of time necessary- for the treatment or the prevention of the disease, such as, for example, from about a few days to 2 weeks, and from about 1 month to about 3 months. The length of treatment can vary-’ for each subject. Administration of the pharmaceutical composition can be via any route that is suitable for the formulation of the composition. For example, a pharmaceutical composition for intravenous administration can be formulated as a liquid composition with appropriate properties that enable the handling and administration of the composition.

[0129] In some embodiments, the pharmaceutical composition includes a therapeutically effective amount of a pharmaceutically acceptable carrier or a pharmaceutically acceptable excipient. Examples of suitable excipients include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, saline, syrup, methylcellulose, ethylcellulose, hydroxypropylmethylcellulose, and polyacrylic acids such as Carbopols, e.g., Carbopol 941, Carbopol 980, Carbopol 981 , etc. In some embodiments, the pharmaceutical composition includes one or more of a diluent, adjuvant, or carrier in a formulation suitable for administration, e.g., administration to a human or other mammals. Suitable diluents, adjuvants, or carriers can include, for example, lipids, e.g., liposomes, e.g., liposome dendrimers; liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like; gum acacia; gelatin; starch paste; talc; keratm; colloidal silica; urea; and the like. Additional examples of suitable diluents include distilled water, buffered water, physiological saline, PBS, Ringer’s solution, dextrose solution, and Hank's solution. The pharmaceutical composition can also include additional substances to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, wetting agents, and detergents. In addition, auxiliary, thickening, lubricating, and coloring agents can alternatively or additionally be used. Pharmaceutical compositions can be formulated into preparations in solid, semisolid, liquid, or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols.

[0130] The pharmaceutical composition can also include any of a variety of stabilizing agents, such as an antioxidant for example. When the pharmaceutical composition includes a polypeptide, the polypeptide can be complexed with various well-known compounds that enhance the in vivo stability’ of the polypeptide, or otherwise enhance its pharmacologicalproperties (e.g., increase the half-life of the polypeptide, reduce its toxicity, and / or enhance solubility or uptake). Examples of such modifications or complexing agents include sulfate, gluconate, citrate, and phosphate. The nucleic acids or polypeptides of a composition can also be complexed wi th molecules that enhance their in vivo attributes. Such molecules include, for example, carbohydrates, polyamines, amino acids, other peptides, ions (e.g., sodium, potassium, calcium, magnesium, manganese), and lipids.F. METHODS OF USE

[0131] Disclosed herein are methods of using one or more recombinant polynucleotides, DNA constructs, vectors, systems, cells, populations of cells, or pharmaceutical compositions of the present disclosure.

[0132] In some embodiments, the recombinant polynucleotide, DNA construct, vector, or system of the present disclosure is used to modify a cell or a population of cells, thereby producing a target cell or a population of target cells that can regulate expression of a polynucleotide payload sequence m response to the presence or absence of an exogenous molecule. In some embodiments, the method comprises introducing the recombinant polynucleotide, DNA construct, or vector of the present disclosure into the cell or population of ceils. In some embodiments, the method comprises contacting the cell or population of cells with the recombinant polynucleotide, DNA construct, or vector of the present disclosure.

[0133] A recombinant polynucleotide, DNA construct, vector, system, cell, population of cells, or pharmaceutical composition of the present disclosure may be used to prevent or treat a disease in a subject, e.g., a human or a mammal subject. In some embodiments, the method comprises administering to the subject an amount of the recombinant polynucleotide, DNA construct, vector, system, cell, population of cells, or pharmaceutical composition of the present disclosure.

[0134] In some embodiments, the recombinant polynucleotide, DNA construct, vector, system, cell, population of cells, or pharmaceutical composition may be used for cancer treatment. In some embodiments, the recombinant polynucleotide, DNA construct, vector, system, cell, population of cells, or pharmaceutical composition facilitates cancer cell death or detection. In some embodiments, the cancer is chosen from biliary tract cancer, bladder cancer, brain cancer (e.g., glioblastomas, medulloblastomas), breast cancer, cervical cancer, choriocarcinoma, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer, leukemia (e.g., acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocyticleukemia, chronic myeloid leukemia), liver cancer, lymphoma (e.g., Hodgkin's disease, nonHodgkin lymphoma), lung cancer, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cancer (e.g,, renal cell adenocarcinoma, nephroblastoma), sarcoma (e.g., fibrosarcoma, leiomyosarcoma, liposarcoma, rhabdomyosarcoma, osteosarcoma), skin cancer (e.g., basal cell carcinoma, squamous cell carcinoma, melanoma), testicular cancer, and thyroid cancer.

[0135] In some embodiments, the cancer is a solid tumor. In some embodiments, the tumor is renal cell carcinoma, nasopharyngeal carcinoma, glioblastoma, melanoma, lung carcinoma, cervix carcinoma, breast carcinoma, ovarian carcinoma, mesothelioma, or a metastatic cancer. In some embodiments, the metastatic cancer is lung carcinoma, pancreatic carcinoma, or osteosarcoma.

[0136] In some embodiments, the cancer is a liquid tumor, e.g., a blood cancer such as a lymphoma or leukemia. In some embodiments, the leukemia is acute myelogenous leukemia (AML), B-cell acute lymphoblastic leukemia (B-ALL), chronic lymphocytic leukemia (CLL or B-CLL), hairy cell leukemia, B-cell prolymphocytic leukemia, Non-Hodgkin lymphoma or Non-Hodgkin’s lymphoma, Hodgkin lymphoma or Hodgkin’s lymphoma, multiple myeloma (MM), or T-cell leukemia. In some embodiments, the non-Hodgkin lymphoma is diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, follicle center lymphoma, mantle cell lymphoma, Burkitt lymphoma (BL), or Waldenstrom macroglobulinemia. In some embodiments, the T-cell leukemia is anaplastic large cell lymphoma, peripheral T cell lymphoma, cutaneous T cell lymphoma, extranodal natural killer (NK) / T-cell lymphoma, Epstein-Barr virus associated T cell lymphoma, or T-cell acute lymphoblastic leukemia,G. EXEMPLARY EMBODIMENTS

[0137] Tire following embodiments are contemplated. All combinations of features and embodiments are contemplated.

[0138] Embodiment 1 : A recombinant polynucleotide comprising a payload sequence encoding a payload polypeptide that is functional in or secreted by a target cell; and a 3'- untranslated region (3'-UTR) sequence downstream of and operably linked to the pay load sequence, the 3'-UTR sequence being an engineered variant of a wild-type 3'-UTR sequence; wherein the 3'-UTR sequence or an RNA transcript thereof responds to an intracellular signal of the target cell by modulating expression of the payload polypeptide by the target cell.

[0139] Embodiment 2: An embodiment of embodiment 1, wherein the target cell produces the intracellular signal when the target cell recognizes an antigen.

[0140] Embodiment 3: An embodiment of embodiment 1 or 2, wherein expression of the payload polypeptide by the target cell in the absence of the intracellular signal is less than 50% of expression of the payload polypeptide by the target cell in a presence of the intracellular signal.

[0141] Embodiment 4: An embodiment of any one of embodiments 1-3, wherein the wildtype 3'-UTR sequence is endogenous to the target cell.

[0142] Embodiment 5: An embodiment of any one of embodiments 1-4, wherein the wild- type 3'-UTR sequence is a cytokine 3 -UTR sequence.

[0143] Embodiment 6: An embodiment of any one of embodiments 1-5, wherein the wildtype 3'~UTR sequence is an interferon gamma (IF NG) 3'-UTR sequence, an interleukin (IL.)- 1B 3'-UTR sequence, an IL-2 3'-UTR sequence, an IL-4 3'-UTR sequence, an IL-6 3'-UTR sequence, an IL-10 3'-UTR sequence, an IL-12 3'-UTR sequence , an IL-13 3'-UTR sequence, an IL-17A 3’-UTR sequence, an IL-21 3'-UTR sequence, an IL-22 3'-UTR sequence, a tumor necrosis factor (INF) 3'-UTR sequence, a Cluster of Differentiation 69 (CD69) 3'-UTR sequence, a colony stimulating factor (CSF)-2 3'-UTR sequence, or a cytotoxic T-lymphocyte associated protein 4 (CTLA4) 3'-UTR sequence.

[0144] Embodiment 7: An embodiment of embodiment 6, wherein the wild-type 3'-UTR sequence has at least 85% sequence identity with SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 71.

[0145] Embodiment 8: An embodiment of any one of embodiments 1-7, wherein the 3'-UTR sequence is a truncated variant of the wild-type 3'-UTR sequence.

[0146] Embodiment 9: An embodiment of embodiment 8, wherein the truncated variant has at least 85% sequence identity with SEQ ID NO: 20, 21, 22, 23, 24, 25, 26, 30, 31, 34, or 36.

[0147] Embodiment 10: An embodiment of embodiment 8 or 9, wherein the truncated variant has a length at least 50 bp shorter than that of the wild-type 3'-UTR sequence.

[0148] Embodiment 11 : An embodiment of any one of embodiments 8-10, wherein the truncated variant has a length at least 20% shorter than that of the wild-type 3'-UTR sequence.

[0149] Embodiment 12: An embodiment of any one of embodiments 8-11, wherein the truncated variant has a length no greater than 250 bp.

[0150] Embodiment 13: An embodiment of any one of embodiments 8-12, wherein the truncated variant retains a proximal end of the wild-type 3'-UTR sequence.

[0151] Embodiment 14: An embodiment of any one of embodiments 8-12, wherein the truncated variant lacks a proximal end of the wild-type 3'-UTR sequence and a distal end of the wild-type 3'-UTR sequence.

[0152] Embodiment 15: An embodiment of any one of embodiments 1-14, wherein the engineered variant lacks one or more cis-regulatory elements of the wild-type 3'-LTR sequence.

[0153] Embodiment 16: An embodiment of embodiment 15, wherein the one or more cis- regulatory elem ents compri se one or more AU-rich elements (AREs) of the wild-type 3'-UTR sequence.

[0154] Embodiment 17: An embodiment of embodiment 16, wherein the engineered variant has at least 85% sequence identity with SEQ ID NO: 37 or 41.

[0155] Embodiment 18: An embodiment of any one of embodiments 1 -17, wherein the 3'- L7TR sequence is adjacent to the payload sequence.

[0156] Embodiment 19: An embodiment of any one of embodiments 1-17, wherein: the 3'- UTR sequence is a first 3'-UTR sequence; and the recombinant polynucleotide further comprises a second 3'-UTR sequence downstream of and operably linked to the payload sequence.

[0157] Embodiment 20: An embodiment of embodiment 19, wherein the second 3'-UTR sequence is between and adjacent to the payload sequence and the first 3'-UTR sequence.

[0158] Embodiment 21: An embodiment of embodiment 19, wherein the first 3'-sequence is between and adjacent to the payload sequence and the second 3 -UTR sequence.

[0159] Embodiment 22: An embodiment of any one of embodiments 19-21, wherein: the wiki-type 3'-UTR sequence is a first wild-type 3'-UTR sequence; and the second 3'-UTR sequence is a second wild-type 3 -UTR sequence, or is an engineered variant thereof.

[0160] Embodiment 23: An embodiment of embodiment 22, wherein the second wild-type 3'-UTR sequence is endogenous to the target cell.[0161 j Embodiment 24: An embodiment of embodiment 22 or 23, wherein the second wildtype 3'-UTR sequence is a cytokine 3'-UTR sequence.

[0162] Embodiment 25: An embodiment of embodiment 24, wherein the second wild-type 3'-UTR sequence is an IFNG 3'-UTR sequence, an IL-1B 3'-UTR sequence, an IL-2 3'-UTR sequence, an IL-4 3'-UTR sequence, an IL-6 3'-LTR sequence, an IL- 10 3’-UTR sequence, an IL-12 3'-UTR sequence , an IL-13 3'-UTR sequence , an IL-17A 3'-UTR sequence, an IL-21 3'-UTR sequence, an IL-22 3'-UTR sequence, a TNF 3'-UTR sequence, a CD69 3'-UTR sequence, a CSF-2 3'-UTR sequence, or a CTLA4 3'-UTR sequence.

[0163] Embodiment 26: An embodiment of embodiment 25, wherein the second wild-type 3'-UTR sequence has at least 85% sequence identity with SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 01- 71.

[0164] Embodiment 27: An embodiment of any one of embodiments 22-26, wherein the second wild-type 3'-UTR sequence is the same as the first wild-type 3'-UTR sequence.

[0165] Embodiment 28: An embodiment of any one of embodiments 19-27, wherein the second 3'-UTR sequence has at least 85% sequence identity with SEQ ID NO: 20, 21, 2.2, 23, 24, 25, 26, 30, 31, 34, 36, 37, or 41.

[0166] Embodiment 29: An embodiment of any one of embodiments 1-28, wdierein the target cell is a mammalian cell.

[0167] Embodiment 30: An embodiment of embodiment 2.9, wherein the mammalian cell is a human cell.

[0168] Embodiment 31 : An embodiment of any one of embodiments 1-30, wherein the target cell is an immune cell.

[0169] Embodiment 32: An embodiment of embodiment 31, wherein the immune cell is a T cell.

[0170] Embodiment 33: An embodiment of embodiment 32, wherein the T ceil is a primary T cell.

[0171] Embodiment 34: An embodiment of embodiment 32, wherein the T cell is a chimeric antigen receptor (CAR) T cell.

[0172] Embodiment 35: An embodiment of any one of embodiments 1-34, wherein the payload polypeptide is a CAR or a fragment thereof.

[0173] Embodiment 36: A recombinant polynucleotide comprising: a payload sequence encoding a payload polypeptide that is functional in or secreted by a target cell; a first wildtype 3’-UTR sequence downstream of and operably linked to the payload sequence; and a second wild-type 3'-L7TR sequence downstream of and operably linked to the payload sequence; wherein the first and second wild-type 3'-UTR sequences or RNA transcripts thereof each independently respond to an intracellular signal of the target cell by modulating expression of the payload polypeptide by the target cell.

[0174] Embodiment 37 : An embodiment of embodiment 36, w'herein the target ceil produces the intracellular signal when the target cell recognizes an antigen.

[0175] Embodiment 38: An embodiment of embodiment 36 or 37, wherein expression of the payload polypeptide by the target cell in the absence of tire intracellular signal is less than 50% of expression of the payload polypeptide by the target cell in a presence of the intracellular signal.

[0176] Embodiment 39: An embodiment of any one of embodiments 36-38, wherein at least one of the first wild-type 3'-UTR sequence and the second wild-type 3'-UTR sequence is endogenous to the target cell.

[0177] Embodiment 40: An embodiment of any one of embodiments 36-39, wherein at least one of the first wild-type 3'-UTR sequence and the second wild-type 3'-UTR sequence is a cytokine 3'-UTR sequence.

[0178] Embodiment 41 : An embodiment of embodiment 40, wherein at least one of the first wild-type 3 -UTR sequence and the second wild-type 3'-UTR sequence is an IFNG 3’-UTR sequence, an IL- IB 3 -UTR sequence, an IL-2 3'-UTR sequence, an IL-4 3'-UTR sequence, an IL-6 3'-UTR sequence, an IL- 10 3'-UTR sequence, an IL- 12 3'-UTR sequence , an IL- 13 3'- UTR sequence , an IL-17A 3'-UTR sequence, an IL-21 3'-UTR sequence, an IL-22 3'-UTR sequence, a TNF 3'-UTR sequence, a CD69 3'-UTR sequence, a CSF-2 3'-UTR sequence, or a CTLA4 3'-UTR sequence.

[0179] Embodiment 42: An embodiment of embodiment 41, wherein at least one of the first wild-type 3'-UTR sequence and the second wild-type 3'-UTR sequence has at least 85% sequence identity’ with SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, or 71.

[0180] Embodiment 43: An embodiment of any one of embodiments 36-42, wherein the second wild-type 3'-UTR sequence is the same as the first wild-type 3'-UTR sequence.

[0181] Embodiment 44: An embodiment of any one of embodiments 36-43, wherein the target cell is a mammalian cell.

[0182] Embodiment 45: An embodiment of embodiment 44, wherein the mammalian cell is a human cell.

[0183] Embodiment 46: An embodiment of any one of embodiments 36-45, wherein the target cell is an immune cell.

[0184] Embodiment 47: An embodiment of embodiment 46, wherein the immune cell is a T cell.

[0185] Embodiment 48: An embodiment of embodiment 47, wherein the T ceil is a primary T cell.

[0186] Embodiment 49: An embodiment of embodiment 48, wherein the T celi is a CAR T cell.

[0187] Embodiment 50: An embodiment of any one of embodiments 36-49, wherein the payload polypeptide is a CAR or a fragment thereof.

[0188] Embodiment 51 : A recombinant polynucleotide comprising: a payload sequence, wherein the payload sequence or a payload RNA transcript thereof is functional in or secreted by a target cell; and a 3'-UTR sequence downstream of and operably linked to the pay load sequence, the 3'-UTR sequence being an engineered variant of a wild-type 3'-UTR sequence; wherein the 3'-UTR sequence or a 3'-UTR RNA transcript thereof responds to an intracellular signal of the target cell by modulating translation or stability of the payload sequence or the payload RNA transcript in the target cell.

[0189] Embodiment 52: An embodiment of embodiment 51, wherein the target cell produces the intracellular signal when tire target cell recognizes an antigen.

[0190] Embodiment 53: An embodiment of embodiment 51 or 52, wherein the wild-type 3'-UTR sequence is endogenous to the target cell.[0191 j Embodiment 54: An embodiment of any one of embodiments 51 -53. wherein the wild-type 3'-UTR sequence is a cytokine 3'-UTR sequence.

[0192] Embodiment 55: An embodiment of any one of embodiments 51-54, wherein the wiid-type 3'-UTR sequence is an interferon gamma (IFNG) 3'-U TR sequence, an interleukin (IL)- IB 3'-UTR sequence, an IL-2 3'-UTR sequence, an IL-4 3'-UTR sequence, an IL-6 3’- UTR sequence, an IL-10 3'-UTR sequence, an IL-12 3 -UTR sequence , an IL-13 3'-UTR sequence, an IL-17A 3 '-UTR sequence, an IL-21 3 '-UTR sequence, an IL-22 3 '-UTR sequence, a tumor necrosis factor (TNF) 3'-L!TR sequence, a Cluster of Differentiation 69 (CD69) 3'- UTR sequence, a colony stimulating factor (CSF)-2 3 -UTR sequence, or a cytotoxic T- lymphocyte associated protein 4 (CTLA4) 3'-UTR sequence.

[0193] Embodiment 56: An embodiment of embodiment 55, wherein the wild-type 3'-UTR sequence has at least 85% sequence identity with SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 71.

[0194] Embodiment 57: An embodiment of any one of embodiments 51-56, wherein the 3'-UTR sequence is a truncated variant of the wild-type 3'-UTR sequence.

[0195] Embodiment 58: An embodiment of embodiment 57, wherein the truncated variant has at least 85% sequence identity with SEQ ID NO: 20, 21, 22, 23, 24, 25, 26, 30, 31, 34, or 36.

[0196] Embodiment 59: An embodiment of embodiment 57 or 58, wherein the truncated variant has a length at least 50 bp shorter than that of the wiid-type 3'-UTR sequence.

[0197] Embodiment 60: An embodiment of any one of embodiments 57-59, wherein the truncated variant has a length at least 20% shorter than that of the wild-type 3'-UTR sequence.

[0198] Embodiment 61: An embodiment of any one of embodiments 57-60, wherein the truncated variant has a length no greater than 250 bp.

[0199] Embodiment 62: An embodiment of any one of embodiments 57-61, wherein the truncated variant retains a proximal end of the wild-type 3'-UTR sequence.

[0200] Embodiment 63: An embodiment of any one of embodiments 57-62, wherein the truncated variant lacks a proximal end of the wild-type 3'-UTR sequence and a distal end of the wild-type 3'-UTR sequence.

[0201] Embodiment 64: An embodiment of any one of embodiments 51-63, wherein the engineered variant lacks one or more AU-rich elements (AREs) of the wild -type 3'-UTR sequence.

[0202] Embodiment 65 : An embodiment of embodiment 64, wherein the engineered variant has at least 85% sequence identity with SEQ ID NO: 37 or 41 .

[0203] Embodiment 66: An embodiment of any one of embodiments 51-65, wherein the 3'- UTR sequence is adjacent to the payload sequence.

[0204] Embodiment 67: An embodiment of any one of embodiments 51-65, wherein: the 3'- UTR sequence is a first 3'-UTR sequence; and the recombinant polynucleotide further comprises a second 3'-UTR sequence downstream of and operably linked to the payload sequence.

[0205] Embodiment 68: An embodiment of embodiment 67, wherein the second 3'-UTR sequence is between and adjacent to the payload sequence and the first 3'-UTR sequence.

[0206] Embodiment 69: An embodiment of embodiment 67, wherein the first 3'-sequence is between and adjacent to the payload sequence and the second 3'-UTR sequence.

[0207] Embodiment 70: An embodiment of any one of embodiments 67-69, wherein: the wild-type 3'-UTR sequence is a first wild-type 3'-UTR sequence; and the second 3'-UTR sequence is a second wild-type 3'-UTR sequence, or is an engineered variant thereof.

[0208] Embodiment 71: An embodiment of embodiment 70, wherein the second vrild-type 3'-UTR sequence is endogenous to the target cell.

[0209] Embodiment 72: An embodiment of embodiment 70 or 71 , wherein the second wildtype 3'-UTR sequence is a cytokine 3'-UTR sequence.

[0210] Embodiment 73: An embodiment of embodiment 72, wherein the second vrild-type 3 -UTR sequence is an IFNG 3'-UTR sequence, an IL- IB 3'-UTR sequence, an IL-2 3'-UTR sequence, an IL-4 3'-UTR sequence, an IL-6 3'-UTR sequence, an IL-10 3'-UTR sequence, an IL- 12 3'-UTR sequence , an IL-13 3'-UTR sequence , an IL-17A 3'-UTR sequence, an IL-21 3'-UTR sequence, an IL -22 3'-UTR sequence, a TNF 3'-UTR sequence, a CD69 3'-UTRsequence, a CSF-2 3'-UTR sequence, or a cytotoxic T-lymphocyte associated protein 4 (CTLA4) 3'-UTR sequence.

[0211] Embodiment 74: An embodiment of embodiment 73, wherein the second wild-type 3'-UTR sequence has at least 85% sequence identity with SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, or 71.

[0212] Embodiment 75: An embodiment of any one of embodiments 70-74, wherein the second wild-type 3'-UTR sequence is the same as tire first wild-type 3'-UTR sequence.

[0213] Embodiment 76: An embodiment of any one of embodiments 67-75, wherein the second 3'-UTR sequence has at least 85% sequence identity with SEQ ID NO: 20, 21, 22, 23, 24, 25, 26, 30, 31, 34, 36, 37, or 41.

[0214] Embodiment 77: An embodiment of any one of embodiments 51-76, wherein the target cell is a mammalian cell .

[0215] Embodiment 78: An embodiment of embodiment 77, wherein the mammalian cell is a human cell.

[0216] Embodiment 79: An embodiment of any one of embodiments 51-78, wherein the target cell is an immune cell.

[0217] Embodiment 80: An embodiment of embodiment 79, wherein the immune cell is a T cell.

[0218] Embodiment 81: An embodiment of embodiment 80, wherein the T cell is a primary T cell.

[0219] Embodiment 82: An embodiment of embodiment 80, wherein the T cell is a chimeric antigen receptor (CAR) T cell.

[0220] Embodiment 83: A recombinant polynucleotide comprising: a payload sequence, wherein the payload sequence or a payload RNA transcript thereof is functional in or secreted by a target cell; a first wild-type 3 -UTR sequence downstream of and operably linked to the payload sequence; and a second wild-type 3'-UTR sequence downstream of and operably- linked to the payload sequence; wherein the first and second wild-type 3'-UTR sequences or RNA transcripts thereof each independently respond to an intracellular signal of the target cell by modulating translation or stability of the payload sequence or the payload RNA transcript in the target cell.

[0221] Embodiment 84: An embodiment of embodiment 83, wherein the target cell produces the intracellular signal when the target cell recognizes an antigen.[0222 j Embodiment 85 : An embodiment of embodiment 83 or 84, wherein at least one of the first wild-type 3'-UTR sequence and the second wild-type 3'-UTR sequence is endogenous to the target cell.

[0223] Embodiment 86: An embodiment of any one of embodiments 83-85, wherein at least one of the first wild-type 3'-UTR sequence and the second wild-type 3'-UTR sequence is a cytokine 3'-UTR sequence.

[0224] Embodiment 87: An embodiment of embodiment 86, wherein at least one of the first wild-type 3 -UTR sequence and the second wild-type 3'-UTR sequence is an IFNG 3'-UTR sequence, an IL-1B 3'-UTR sequence, an IL-2 3'-UTR sequence, an IL-4 3'-UTR sequence, an IL-6 3'-UTR sequence, an IL-10 3'-UTR sequence, an IL-12 3'-UTR sequence , an IL-13 3'- UTR sequence , an IL.-17A 3'-UTR sequence, an IL-21 3'-UTR sequence, an IL-22 3'-UTR sequence, a TNF 3'-UTR sequence, a CD69 3'-UTR sequence, a CSF-2 3'-UTR sequence, or a CTLA4 3'-UTR sequence.

[0225] Embodiment 88: An embodiment of embodiment 87, wherein at least one of the first wild-type 3'-UTR sequence and the second wild-type 3'-UTR sequence has at least 85% sequence identity with SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, I I, 12, 13, 14, 15, 16, or 71.

[0226] Embodiment 89: An embodiment of any one of embodiments 83-88, wherein the second wild-type 3'-UTR sequence is the same as the first wikl-type 3'-UTR sequence.

[0227] Embodiment 90: An embodiment of any one of embodiments 83-89, wherein the target cell is a mammalian cell.

[0228] Embodiment 91 : An embodiment of embodiment 90, wherein the mammalian cell is a human cell.

[0229] Embodiment 92: An embodiment of any one of embodiments 83-91 , wherein the target cell is an immune cell.

[0230] Embodiment 93: An embodiment of embodiment 92, wherein the immune cell is a T cell.

[0231] Embodiment 94: An embodiment of embodiment 93, wherein the T cell is a primary’ T cell.

[0232] Embodiment 95: An embodiment of embodiments 94, wherein the T cell is a CAR T cell.

[0233] Embodiment 96: An embodiment of any one of embodiments 83-95, wherein the payload polypeptide is a CAR or a fragment thereof.

[0234] Embodiment 97: An embodiment of any one of embodiments 1-96, wherein the recombinant polynucleotide comprises RNA.

[0235] Embodiment 98: An embodiment of embodiment 97, wherein the recombinant polynucleotide comprises mRNA.

[0236] Embodiment 99: An embodiment of any one of embodiments 1-96, wherein the recombinant polynucleotide comprises DNA.

[0237] Embodiment 100: A DNA construct comprising: the recombinant polynucleotide of embodiment 99; and a payload promoter operably linked to the payload sequence.

[0238] Embodiment 101: An embodiment of embodiment 100, wherein the payload promoter is constitutive.

[0239] Embodiment 102: An embodiment of embodiment 100 or 101 , wherein the DNA construct further comprises a poly adenylation sequence.

[0240] Embodiment 103: An embodiment of embodiment 102, wherein the polyadenylation sequence is adjacent to the distal end of the 3'-UTR sequence.

[0241] Embodiment 1044: An embodiment of any one of embodiments 100-103, wherein the DNA construct further comprises a reporter sequence encoding a reporter polypeptide that produces a detectable signal when the reporter polypeptide is expressed by the target cell.

[0242] Embodiment 105: An embodiment of embodiment 104, wherein the DNA construct further comprises a reporter promoter operably linked to the reporter polypeptide.

[0243] Embodiment 106: An embodiment of embodiment 104 or 105, wherein the reporter promoter is constitutive.

[0244] Embodiment 107: An embodiment of any one of embodiments 104-106, wherein the payload promoter and the reporter promoter are divergently oriented relative to one another.

[0245] Embodiment 108: An embodiment of any one of embodiments 104-107, wherein the DNA construct further comprises a post-transcriptional regulatory element (PRE) adjacent to the reporter sequence,

[0246] Embodiment 109: A vector comprising the recombinant polynucleotide of any one of embodiments 1 -99 or the DNA construct of any one of embodiments 100-108.

[0247] Embodiment 110: An embodiment of embodiment 109, wherein the vector is a plasmid.

[0248] Embodiment 111: An embodiment of embodiment 109, wherein the vector is a virus.

[0249] Embodiment 112: An embodiment of embodiment 109, wherein the vector is a lipid particle.

[0250] Embodiment 113: A system comprising: the recombinant polynucleotide of any one of embodiments 1-99, the DNA construct of any one of embodiments 100-108, or the vector of any one of embodiments 109-1 12; and a CAR or a polynucleotide, DNA construct, or vector including a sequence encoding the CAR.

[0251] Embodiment 1 14: A cell comprising the recombinant polynucleotide of any one of embodiments 1 -99, the DNA construct of any one of embodiments 100-108, or the vector of any one of embodiments 109-112, wherein the cell is the target cell.

[0252] Embodiment 115: An embodiment of embodiment 114, wherein the cell further comprises a CAR or a polynucleotide, DNA construct, or vector including a sequence encoding the CAR.

[0253] Embodiment 116: An embodiment of embodiment 1 14 or 115, wherein the cell is in a mammal .

[0254] Embodiment 1 17: A population of cells comprising the cell of embodiment 1 14 or 1 15.

[0255] Embodiment 1 18: A pharmaceutical composition comprising: a pharmaceutically acceptable earner or a pharmaceutically acceptable excipient; and the recombinant polynucleotide of any one of embodiments 1-99, the DNA construct of any one of embodiments 100-108, the vector of any one of embodiments 109-112, the sy stem of embodiment 113, the cell of embodiment 114 or 1 15, or the population of cells of embodiment 117.

[0256] Embodiment 119: A method of modifying a cell, the method comprising contacting the cell with the recombinant polynucleotide of any one of embodiments 1-99, the DMA construct of any one of embodiments 100-108, or the vector of any one of embodiments 109- 112; or introducing the recombinant polynucleotide, the DNA construct, or the vector into the cell; wherein the cell is the target cell.

[0257] Embodiment 120: An embodiment of embodiment 119, wherein the cell is in a mammal.[0258 j Embodiment 121: A method for preventing or treating a disease in a subject, the method comprising administering to the subject an amount of the recombinant polynucleotide of any one of embodiments 1-99, the DM A construct of any one of embodiments 100-108, the vector of any one of embodiments 109-112, the system of embodiment 113, the cell of embodiment 114 or 1 15, the population of cells of embodiment 117, or the pharmaceutical composition of embodiment 1 18.

[0259] Embodiment 122: An embodiment of any one of embodiments 121, wherein the disease comprises a cancer.

[0260] Embodiment 123 : An embodiment of embodiment 122, wherein the cancer comprises a solid tumor.

[0261] Embodiment 124 : An embodiment of embodiment 122, wherein the cancer comprises a liquid tumor.EXAMPLES

[0262] The present disclosure will be better understood in view of the following non-limiting examples. The following examples are intended for illustrative purposes only and do not limit in any way the scope of the present invention.Overview,

[0263] Regulators' sequences found in the UTR of many endogenous cytokine transcripts play a critical role in rapid, stimuli-responsive transcript stability and translation. The followingExamples demonstrate the design and use of UTR sequences as nucleic acid switches.Example 1. T Cell Activation with 3 '-UTR Compositions

[0264] In this Example, a dual-color lentiviral reporter system was designed to quantitatively capture post-transcriptional regulation in primary human T cells on the single cell level (FIGS. 1A-B). Primary human T cells (FIG. IB) are transduced with “activation-induced expression” lentiviral constructs (FIG. 1 A) or a “constant expression” control that consist of a switch reporter (TurboGFP) and a transduction reporter (BFP) driven by constitutive promoters human PGK and the EFla core promoter (EF-S) (FIG. 1A), respectively. The activation-induced construct contained a 3 '-untranslated region (UTR) directly after the stop codon of GFP while constant expression const met did not (FIG. 1 A).

[0265] As shown in FIG. 1C, primary human T cells transduced with an activation-induced mRNA switch only expressed GFP when activated / stimulated (grey histogram) and not at baseline (white histogram; unstimulated). Histograms depict GFP expression as measured by flow' cytometry after stimulation on day 10 with Human T- Activator CD3 / CD28 Dynabeads (Gibco) at a 1: 1 bead-to-cell ratio for 24 hours. Cells were gated for BFP expression and analyzed for GFP expression.

[0266] Next, mRNA switches were designed using various endogenous 3 '-UTR sequences and tested for T cell activation. The 3 '-UTR sequences were the 3'-UTR sequences of IFNG. IL2, TNF, CD69, CSF2, IL1B, ILIO, III 2. JL13, IL21, IL22, JL4. JL6, IL17A and CTLA4. The dual-color lentiviral reporter system discussed above was used to test the mRNA switches. T cell activation was measured by flow cytometry' with the same stimulation protocol as described above. The cells were gated for BFP expression and analyzed tor GFP expression. As shown in FIGS. 2A-B and 12A-B, the 3'-UTR sequences confer T cell activation-inducible translation of genetic pay loads.Example 2. Truncated 3rUTR Sequences in mRNA Retain the Ability to Activate T Cells

[0267] In this Example, 3 '-UTR sequences were truncated and tested for ability to modulate switch dynamics of T cell activation-inducible expression of a reporter protein (GFP).

[0268] Primary' human T cells were engineered with the truncated 3'-UTR sequences discussed in this Example and restimulated on day 10 for 24 hours by 1 ug / mL plate-bound aCD3 and 1 ug / mL soluble aCD28 (Biolegend). Cells were then measured by flow' cytometry-. Cells are gated for BFP expression and analyzed for GFP expression.

[0269] First, the full length, wild-type IFNG 3'-UTR was truncated from the distal end to generate 3 ’-UTRs with lengths from 50 to 500 base pairs, as specified on the x-axis of FIGS. 3A-B. Retaining the proximal 200 nt ofthe IFNG 3'-UTR was sufficient to recapitulate mRNA switching as seen in both the percentage of GFP positive cells (FIG. 3A) as well as the median GFP intensity' (FIG. 3B).

[0270] Next, the full length, wild-type IL2 3'-UTR was truncated from the distal end to generate 3 '-UTRs with lengths from 50 to 250 base pairs, as specified on the x-axis of FIGS. 4A-B. Retaining the proximal 200 nt of the IL2 3'-UTR was sufficient to recapitulate mRNA switching as seen in both the percentage of GFP positive cells (FIG. 4A) as well as the median GFP intensity (FIG. 4B).

[0271] Next, full length, wild-type 1 NF3'-U TR was truncated from the distal end to generate 3 ’-UTRs with lengths from 200 to 600 base pairs, as specified on the x-axis of FIGS. 5A-B. The proximal 600 nt recapitulated mRNA switching as seen in both percentage of GFP positive cells (FIG. 5A) as well as the median GFP intensity (FIG. 5B) The proximal 600 nt 3'-UTR was then truncated from the proximal end to generate 3 '-UTRs with lengths spanning [starting nt] / [ending nt], as specified on the x-axis of FIGS. 5C-D. A 3'-UTR starting from base 400 and ending at base 600 of the INF 3'-UTR was sufficient to recapitulate mRNA switching.

[0272] Taken together, the results demonstrate that truncated UTRs may be used in areas where genetic payload size limits apply.Example 3. Deletion of Cis-Regulatory Elements in the 3f-UTR Can Modulate Gene Expression

[0273] Ihis Example demonstrates that mRNA switching behavior can be controlled by modifying cis-regulatory elements in the 3'-UTR.

[0274] AU-rich elements (AREs) are one of the most common determinants of RNA stability in mammalian cells. The IFNG 3 -UTR (SEQ ID NO: 3) contains several predicted AREs. In FIG. 6A, the AREs are numbered sequentially from ARE! to ARE7 in the 5' to 3' direction in wikl-type IFNG 3'-UTR and are indicated with bold letters. To investigate the role of AREs in T cell activation-dependent expression, 3 '-UTRs with deletions of each ARE were generated and their sequences are as set forth in SEQ ID NOS: 37-43.

[0275] Primary human T cells were engineered with these ARE-deleted 3'-UTR sequences and were measured by flow cytometry as described above. Cells were gated for BFP expression and analyzed for GFP expression ,

[0276] Deletion of AREs 2 through 4 resulted in leakier switches in comparison to the wildtype switch, while ARE 1 and ARE 5 did not seem to affect switching as seen in both percentage of GFP positive cells (FIG. 6B) and median GFP intensity (FIG. 6C). Tire results demonstrate that deletion of regulatory sequences within mRNA switches can modulate both the baseline (unstimulated) and stimulated genetic payload expression.Example 4, Synthetic Arrays of mRNA Switches Improve Fold Activation

[0277] This Example demonstrates that synthetic arrays of mRNA switches can improve fold activation of mRNA switches while dampening absolute signal (FIGS. 7A-B, 8A-F, and 9A- B). Primary human I' cells were engineered with the mRNA synthetic arrays discussed in this Example, The cells were measured by flow cytometry as discussed above. Cells were gated for BFP expression and analyzed for GFP expression.

[0278] First, synthetic arrays were designed with a minimal IFNG mRNA switch. As discussed in Example 2 above, the minimal IFNG mRNA switch (mIFNG) was identified as the proximal 200 bp of the IFNG 3'-UTR. Synthetic mRNA switches were generated by arraying mIFNG directly downstream of another wild-type 3'-UTR, as specified in the x-axis of FIGS. 7A-B. Synthetic mRNA switches were functional and generally showed improved fold activation and lower leakiness as seen in both percentage of GFP positive cells (FIG. 7A) as well as median GFP intensity (FIG. 7B).

[0279] Next, synthetic arrays were designed with four mRNA switches. Expanding on improving and / or modulating mRNA switches with synthetic arrays, four mRNA switches, using IL1B, CSF2, IL13, and 1L6 3'-UTRs, with varied switching behavior were selected and arrayed m both A-B and B-A positions (12 total). Specific mRNA switches are denoted in the x-axis of FIGS. 8A-F. mRNA switches generally did not seem to display array positiondependent behavior, except for 1L1B and IL13, where array position resulted in either leaky (1L1 B-IL1.?) or not leaky (ILi ' 3-IL1B} behavior (FIGS. 8A-F).

[0280] Next, synthetic doublets of mRNA switches were tested. IL1B, CSF2, 1L13, and IL63'-UTR were also used to generate synthetic doublets and compared to singlet mRNA switches (i ,e,, A-A vs A). Specific mRNA switches are denoted in the x-axis of FIGS. 9A-B. In general,doubling the 3'-UTRin the mRNA switch decreased baseline (unstimulated) leaky expression. improving mRNA switch fold activation (FIGS. 9A-B).Example 5. mRNA Switches for CAR Expression

[0281] This Example discusses applying mRNA switches in chimeric antigen receptor (CAR) T therapy. CD19-28z and CD19-BBz CAR-T cells, i.e., CAR-T cells that are capable of binding to CD 19 and lysing CD 19-positive tumor cells, were used. The CAR-T cells were engineered to express the GFP reporter protein under the control of the mRNA switch: the mRNA switch can be activated with the CAR-T cells were co-cultured with antigen-positive tumor cells, i.e., K562 leukemia cells (K562 CD19+), and tested for GFP expression (FIG. 10A).

[0282] Primary human T cells were engineered with mRNA switch reporter plasmids(specified in the x-axis of FIGS. 10B-E and 13A-D) and the CD19-28z or CD19-BBz CAR through lentiviral transduction. CAR-T cells were co-cultured with CD19+or CD19‘ K562 leukemiatumor cells in a 1 : 1 ratio for 24 hours. CAR-T cells were measured by flow cytometry. CAR-T cells were gated for CAR and BFP expression and analyzed for GFP expression.

[0283] As shown in FIGS. 10B-E and 13A-D, GFP was expressed when the CAR-T cells were co-cultured with K562 CD 19+ cells. Reporter protein expression showed no significant difference when co-cultured with antigen-negative tumor cells compared to baseline (unstimulated), The results demonstrate the use of mRNA switches in expressing tumorspecific antigens, which may enhance target recognition specificity at the site of disease. mRNA switch dynamics here were consistent with previous characterization in an antigenindependent stimulation system (FIG. 2A).|0284] Next, mRNA switches were tested for the ability to directly control CAR expression. The mRNA switch CAR containing 1FNG 3’-UTR and the constitutive CAR constructs were as shown in FIG. 1 1 A. Additional mRNA switch CAR and constitutive C AR constructs were prepared according to the schematics of FIG. 14A to test other 3'-UTR options. Primary human T cells were engineered using lentiviral transduction with either an “mRNA switch CAR” or a “constitutive CAR.” The cancer cells were GFP -positive Nalm6 leukemia tumor cells. Tumor cells were engineered to express GFP. The generated CAR-T cells were co-cultured with the Nalm6 cells at a 1: 1 ratio, then analyzed for tumor killing using in vitro Incucyte live cell microscopy.

[0285] The mRNA switch CAR-T cells decreased tumor intensity in CD 19+, antigenpositive, tumor cells as measured by GFP expression (FIG. 1 IB), and showed increased CAR- T cell intensity as measured by mCherry expression (FIG. 1 1C, with the shaded region indicating the SEM) or mKate2 expression (FIGS. 14B and 14C, with the shaded region indicating SEM). The graph of FIG. 1 ID shows mRNA switch CAR-T cells killing tumor cells more slowly than the constitutive CAR-T cells, and tire graph of FIG. 14D shows mRNA switch CAR T cells killing antigen -positive tumor cells with similar dynamics to constitutive CAR T cells. The results demonstrate that activation-dependent mRNA switches confer antigendependent protein expression in CD 19 CAR-T cells.[0286 j It is appreciated that certain features of the disclosure, which are, for clarity', described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombmation. All combinations of the embodiments pertaining to the disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every- such sub- combination was individually and explicitly disclosed herein.

[0287] Although the foregoing disclosure has been described in some detail by way of illustration and example for purpose of clarity of understanding, one of skill in the art will appreciate that certain changes and modifications within the spirit and scope of the disclosure may be practiced, e.g., within the scope of the appended claims. It should also be understood that aspects of the disclosure and portions of various recited embodiments and features can be combined or interchanged either in whole or in part. In the foregoing descriptions of the various embodiments, those embodiments which refer to another embodiment may be appropriately combined with other embodiments as will be appreciated by one of skill in the art. Furthermore, those of ordinary skill in the art will appreciate that the foregoing description is by way of example only, and is not intended to limit the disclosure. In addition, each reference provided herein is incorporated by reference in its entirety for all purposes to the same extent as if each reference was individually incorporated by reference.

Claims

WHAT IS CLAIMED IS:

1. A recombinant polynucleotide comprising: a payload sequence encoding a payload polypeptide that is functional in or secreted by a target cell: and a 3 ’-untranslated region (3'-UTR) sequence downstream of and operably linked to the payload sequence, the 3'-UTR sequence being an engineered variant of a wild-type 3'- UTR sequence; wherein the 3'-UTR sequence or an RNA transcript thereof responds to an intracellular signal of the target cell by modulating expression of the payload polypeptide by the target cell.

2. The recombinant polynucleotide of claim 1 , wherein the target cell produces the intracellular signal when the target cell recognizes an antigen.

3. Tire recombinant polynucleotide of claim 1 , wherein expression of the payload polypeptide by the target cell in the absence of the intracellular signal is less than 50% of expression of the payload polypeptide by the target cell in a presence of the intracellular signal.

4. The recombinant polynucleotide of claim 1 , wherein the wild-type 3'- UTR sequence is endogenous to the target cell.

5. The recombinant polynucleotide of claim 1, wherein the wild-type 3'- UTR sequence is a cytokine 3'-UTR sequence.

6. Tire recombinant polynucleotide of claim 1, wherein the wild-type 3'- UTR sequence is an interferon gamma (IFNG) 3'-UTR sequence, an interleukin (IL)- IB 3’- UTR sequence, an IL-2 3'-UTR sequence, an IL, -4 3'-UTR sequence, an IL-6 3 -UTR sequence, an IL- 10 3'-UTR sequence, an IL- 12 3'-UTR sequence , an IL-13 3'-UTR sequence, an IL-17A 3 -UTR sequence, an IL-21 3'-UTR sequence, an IL-22 3 -UTR sequence, a tumor necrosis factor (TNF) 3'-UTR sequence, a Cluster of Differentiation 69 (CD69) 3'-L!TR sequence, a colony stimulating factor (CSF)-2 3'-UTR sequence, or a cytotoxic T-lymphocyte associated protein 4 (CTLA4) 3'-UTR sequence.

7. The recombinant polynucleotide of claim 6, wherein the wild-type 3'- UTR sequence has at least 85% sequence identity with SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 71.

8. The recombinant polynucleotide of claim 1, wherein the 3'~UTR sequence is a truncated variant of the wild-type 3'~UTR. sequence.

9. The recombinant polynucleotide of claim 8, wherein the truncated variant has at least 85% sequence identity with SEQ ID NO: 20, 21 , 22, 23, 24, 25, 26, 30, 31 , 34, or 36.

10. The recombinant polynucleotide of claim 8, wherein the truncated variant has a length at least 50 bp shorter than that of the wikl-type 3'-UTR sequence.

11. Tire recombinant polynucleotide of claim 8, wherein the truncated variant has a length at least 20% shorter than that of the wild-type 3'-UTR sequence.

12. The recombinant polynucleotide of claim 8, wherein the truncated variant has a length no greater than 250 bp.

13. The recombinant polynucleotide of claim 8, wherein tire truncated variant retains a proximal end of the wild-type 3'-UTR sequence.

14. lire recombinant polynucleotide of claim 8, wherein the truncated variant lacks a proximal end of the wild-type 3'~UTR. sequence and a distal end of the wildtype 3'-UTR sequence.

15. The recombinant polynucleotide of claim 1, wherein the engineered variant lacks one or more cis-regulatory elements of the wild-type 3'-UTR sequence.

16. Tire recombinant polynucleotide of claim 15, wherein the one or more cis-regulatory elements comprise one or more AU-rich elements (AREs) of the wild -type 3'- UTR sequence.

17. lire recombinant polynucleotide of claim 16, wherein the engineered variant has at. least 85% sequence identity' with SEQ ID NO: 37 or 41 .

18. The recombinant polynucleotide of claim 1 , wherein the 3'-UTR sequence is adjacent to the payload sequence.

19. The recombinant polynucleotide of claim 1, wherein: the 3'-UTR sequence is a first 3'-UTR sequence: and the recombinant polynucleotide further comprises a second 3'-UTR sequence downstream of and operably linked to the payload sequence.

20. Tire recombinant polynucleotide of claim 19, wherein the second 3'- UTR sequence is between and adjacent to the payload sequence and the first 3 -UTR sequence.

21. The recombinant polynucleotide of claim 19, wherein the first 3'- sequence is between and adjacent to the payload sequence and the second 3'-UTR sequence.

22. Tire recombinant polynucleotide of claim 19, wherein: the wild-type 3’-UTR sequence is a first wild-type 3'-UTR sequence; and the second 3'-UTR sequence is a second wild-type 3'-UTR sequence, or is an engineered variant thereof.

23. The recombinant polynucleotide of claim 22, wherein the second wildtype 3'-UTR sequence is endogenous to the target cell.

24. The recombinant polynucleotide of claim 22, wherein the second wildtype 3'-UTR sequence is a cytokine 3'-UTR sequence.

25. Tire recombinant polynucleotide of claim 24, wherein the second wildtype 3'-UTR sequence is an IFNG 3'-UTR sequence, an IL- IB 3'-UTR sequence, an IL-2 3’- UTR sequence, an IL-4 3'-UTR sequence, an IL, -6 3'-UTR sequence, an IL, -10 3'-UTR sequence, an IL-12 3'-UTR sequence , an IL-13 3'-UTR sequence , an IL-17A 3'-UTR sequence, an IL-21 3'-UTR sequence, an IL-22 3'-UTR sequence, a TNF 3'-UTR sequence, a CD69 3'-UTR sequence, a CSF-2 3'-UTR sequence, or a CTLA4 3'-UTR sequence.

26. The recombinant polynucleotide of claim 25, wherein the second wildtype 3'-UTR sequence has at least 85% sequence identity with SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, or 71.

27. The recombinant polynucleotide of claim 22, wherein the second wild- type 3'-UTR sequence is the same as the first wild-type 3'-UTR sequence.

28. The recombinant polynucleotide of claim 19, wherein the second 3'- UTR sequence has at least 85% sequence identity with SEQ ID NO: 20, 21, 22, 23, 24, 25, 26, 30, 31 , 34, 36, 37, or 41.

29. The recombinant polynucleotide of claim 1, wherein the target cell is a mammalian cell.

30. The recombinant polynucleotide of claim 29, wherein the mammalian cell is a human cell.

31. The recombinant polynucleotide of claim 1 , wherein the target cell is an immune cell.

32. The recombinant polynucleotide of claim 31 , wherein the immune cell is a T cell.

33. Tire recombinant polynucleotide of claim 32, wherein the T cell is a primary T cell.

34. The recombinant polynucleotide of claim 32, wherein the T cell is a chimeric antigen receptor (CAR) T cell.

35. The recombinant polynucleotide of claim 1, wherein the payload polypeptide is a CAR or a fragment thereof.

36. A recombinant polynucleotide comprising: a payload sequence encoding a payload polypeptide that is functional in or secreted by a target cell; a first wild-type 3'-UTR sequence downstream of and operably linked to the payload sequence; and a second w ild-ty pe 3'-UTR sequence downstream of and operably linked to the payload sequence;wherein the first and second wild-type 3'-UTR sequences or RNA transcripts thereof each independently respond to an intracellular signal of the target cell by modulating expression of the payload polypeptide by the target cell.

37. The recombinant polynucleotide of claim 36, wherein the target cell produces the intracellular signal when the target cell recognizes an antigen.

38. The recombinant polynucleotide of claim 36, wherein expression of the payload polypeptide by the target cell in the absence of the intracellular signal is less than 50% of expression of the payload polypeptide by the target cell in a presence of the intracellular signal.

39. The recombinant polynucleotide of claim 36, wherein at least one of the first wild-type 3'-UTR sequence and the second wild-type 3'-UTR sequence is endogenous to the target cell.

40. The recombinant polynucleotide of claim 36, wherein at least one of the first wild-type 3 -UTR sequence and the second wild-type 3'-UTR sequence is a cytokine 3'- UTR sequence.41 . Tire recombinant polynucleotide of claim 40, wherein at least one of the first wild-type 3’-UTR sequence and the second wild-type 3'-UTR sequence is an IFNG 3’- UTR sequence, an IL- IB 3'-UTR sequence, an IL-2. 3'-UTR sequence, an IL-4 3'-UTR sequence, an IL-6 3'-UTR sequence, an IL-10 3'-UTR sequence, an IL-12 3'-UTR sequence , an IL-13 3'-UTR sequence , an IL-17A 3'-UTR sequence, an IL-21 3 -UTR sequence, an IL- 22. 3'-UTR sequence, a INF 3'-UTR sequence, a CD69 3'-UTR sequence, a CSF-2 3'-UTR sequence, or a CTL.A4 3'-UTR sequence.

42. The recombinant polynucleotide of claim 41, wherein at least one of the first wild-type 3'-UTR sequence and the second wild-type 3'-UTR sequence has at least 85% sequence identity with SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, or 71.

43. Tire recombinant polynucleotide of claim 36, wherein the second wildtype 3'-UTR sequence is the same as the first wild-type 3'-UTR sequence.

44. The recombinant polynucleotide of claim 36, wherein the target cell is a mammalian cell.

45. The recombinant polynucleotide of claim 44, wherein the mammalian cell is a human cell.

46. The recombinant polynucleotide of claim 36, wherein the target cell is an immune cell.

47. The recombinant polynucleotide of claim 46, wherein the immune cell is a T cell ,48. The recombinant polynucleotide of claim 47, wherein the T cell is a primary T cell.

49. ITe recombinant polynucleotide of claim 48, wherein the T cell is a CAR T cell.

50. The recombinant polynucleotide of claim 36, wherein the payload polypeptide is a CAR or a fragment thereof.51 . A recombinant polynucleotide comprising: a payload sequence, wherein the payload sequence or a payload RNA transcript thereof is functional in or secreted by a target cell; and a 3'-UTR sequence downstream of and operably linked to the payload sequence, the 3'-UTR sequence being an engineered variant of a wild-type 3'-UTR sequence; wherein the 3'-UTR sequence or a 3'-UTR RN A transcript thereof responds to an intracellular signal of the target cell by modulating translation or stability of the payload sequence or the payload RNA transcript in the target cell.

52. The recombinant polynucleotide of claim 51, wherein the target cell produces the intracellular signal when the target cell recognizes an antigen.

53. The recombinant polynucleotide of claim 51 , wherein the wild-type 3'- UTR sequence is endogenous to the target cell.

54. The recombinant polynucleotide of claim 51, wherein the wild-type 3'- UTR sequence is a cytokine 3'-UTR sequence.

55. Tire recombinant polynucleotide of claim 51 , wherein the wild-type 3'- UTR sequence is an interferon gamma (IFNG) 3'-UTR sequence, an interleukin (IL)- IB 3'-UTR sequence, an IL-2 3'-UTR sequence, an IL-4 3'-UTR sequence, an IL-6 3'-UTR sequence, an IL-10 3'-UTR sequence, an IL-12 3'-UTR sequence , an IL-13 3'-UTR sequence, an IL-17A 3 -UTR sequence, an IL-21 3'-UTR sequence, an IL-22 3 '-UTR sequence, a tumor necrosis factor (TNF) 3'-UTR sequence, a Cluster of Differentiation 69 (CD69) 3'-UTR sequence, a colony stimulating factor (CSF)-2 3'-UTR sequence, or a cytotoxic T-lymphocyte associated protein 4 (CTLA4) 3 ’-UTR sequence.

56. The recombinant polynucleotide of claim 55, wherein the wild-type 3'- UTR sequence has at least 85% sequence identity with SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, or 71.

57. The recombinant polynucleotide of claim 51, wherein the 3'-UTR sequence is a truncated variant of the wild-type 3 -UTR sequence.

58. The recombinant polynucleotide of claim 57, wherein the truncated variant has at least 85% sequence identity with SEQ ID NO: 20, 21 , 22, 23, 24, 25, 26, 30, 31 ,34, or 36.

59. The recombinant polynucleotide of claim 57, wherein the truncated variant has a length at least 50 bp shorter than that of the wild-type 3 -UTR sequence.

60. The recombinant polynucleotide of claim 57, wherein the truncated variant has a length at least 20% shorter than that of the wild-type 3'-UTR sequence.

61. Tire recombinant polynucleotide of claim 57, wherein the truncated variant has a length no greater than 250 bp.

62. The recombinant polynucleotide of claim 57, wherein the truncated variant retains a proximal end of the wild-type 3'-UTR sequence.

63. The recombinant polynucleotide of claim 57, wherein the truncated variant lacks a proximal end of the wild-type 3'-UTR sequence and a distal end of the wildtype 3'-UTR sequence.

64. The recombinant polynucleotide of claim 51, wherein the engineered variant lacks one or more AU -rich elements (AREs) of the wdld-type 3 ’-UTR sequence.

65. The recombinant polynucleotide of claim 64, wherein the engineered variant has at least 85% sequence identity with SEQ ID NO: 37 or 41.

66. The recombinant polynucleotide of claim 51, wherein the 3'-UTR sequence is adjacent to the payload sequence.

67. The recombinant polynucleotide of claim 51 , wherein: the 3'-UTR sequence is a first 3'-UTR sequence; and the recombinant polynucleotide further comprises a second 3'-UTR sequence downstream of and operably linked to the payload sequence.

68. The recombinant polynucleotide of claim 67, wherein the second 3'- UTR sequence is between and adjacent to the pay load sequence and the first 3 -UTR sequence.

69. Tire recombinant polynucleotide of claim 67, wherein the first 3'- sequence is between and adjacent to the payload sequence and the second 3'-UTR sequence.

70. The recombinant polynucleotide of claim 67, wherein: the wild-type 3'-UTR sequence is a first wild-type 3’-UTR sequence; and the second 3'-UTR sequence is a second wild-type 3'-UTR sequence, or is an engineered variant thereof.

71. The recombinant polynucleotide of claim 70, wherein the second wildtype 3'-UTR sequence is endogenous to the target cell.

72. Tire recombinant polynucleotide of claim 70, wherein the second wildtype 3'-UTR sequence is a cytokine 3'-UTR sequence.

73. The recombinant polynucleotide of claim 72, wherein the second wildtype 3’-UTR sequence is an IFNG 3'-UTR sequence, an IL- IB 3'-UTR sequence, an IL-2 3'- UTR sequence, an IL-4 3'-UTR sequence, an IL-6 3'-UTR sequence, an IL- 10 3'-UTR sequence, an IL- 12 3'-UTR sequence , an IL- 13 3'-UTR sequence , an IL-17A 3'-UTR sequence, an IL-21 3'-UTR sequence, an IL-22 3'-UTR sequence, a INF 3'-UTR sequence, a CD69 3'-UTR sequence, a CSF-2 3'-UTR sequence, or a cytotoxic T-lymphocyte associated protein 4 (CTLA4) 3'-UTR sequence.

74. The recombinant polynucleotide of claim 73, wherein the second wildtype 3'-UTR sequence has at least 85% sequence identity with SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 71.

75. The recombinant polynucleotide of claim 70, wherein the second wildtype 3'-UTR sequence is the same as the first wild-type 3'-UTR sequence.

76. The recombinant polynucleotide of claim 67, wherein the second 3'- UTR sequence has at least 85% sequence identity with SEQ ID NO: 20, 21 , 22, 23, 24, 25, 26, 30, 31, 34, 36, 37, or 41.

77. The recombinant polynucleotide of claim 51, w herein the target cell is a mammalian cell.

78. Hie recombinant polynucleotide of claim 77, wherein the mammalian cell is a human cell.

79. The recombinant polynucleotide of claim 51, wherein the target cell is an immune cell.

80. The recombinant polynucleotide of claim 79, wherein the immune cell is a T cell.

81. lire recombinant polynucleotide of claim 80, wherein the T cell is a primary T cell.

82. The recombinant polynucleotide of claim 80, wherein the T cell is a chimeric antigen receptor (CAR) T cell.

83. A recombinant polynucleotide comprising: a payload sequence, wherein the payload sequence or a payload RNA transcript thereof is functional in or secreted by a target cell; a first wild-type 3'-UTR sequence downstream of and operably linked to the payload sequence; and a second wild-type 3'-UTR sequence downstream of and operably linked to the pay load sequence;wherein the first and second wild-type 3'-UTR sequences or RNA transcripts thereof each independently respond to an intracellular signal of the target cell by modulating translation or stability of the payload sequence or the payload RNA transcript in the target cell.

84. The recombinant polynucleotide of claim 83, wherein the target cell produces the intracellular signal when the target cell recognizes an antigen.

85. The recombinant polynucleotide of claim 83 , wherein at least one of the first wild-type 3'-UTR sequence and the second wild-type 3'-UTR sequence is endogenous to the target cell.

86. The recombinant polynucleotide of claim 83, wherein at least one of the first wild-type 3'-UTR sequence and the second wild-type 3'-UTR sequence is a cytokine 3'- UTR sequence.

87. The recombinant polynucleotide of claim 86, wherein at least one of the first wild-type 3'-UTR sequence and the second wild-type 3'-UTR sequence is an IFNG 3'- UTR sequence, an IL- IB 3'-UTR sequence, an IL-2 3'-UTR sequence, an IL-4 3'-UTR sequence, an IL-6 3'-UTR sequence, an IL-10 3'-UTR sequence, an IL-12 3'-UTR sequence , an IL- 13 3'-UTR sequence , an IL-17A 3’-UTR sequence, an IL-21 3 -UTR sequence, an IL- 22 3'-UTR sequence, a TNF 3'-UTR sequence, a CD69 3'-UTR sequence, a CSF-2 3'-UTR sequence, or a CTLA4 3'-IJTR sequence.

88. The recombinant polynucleotide of claim 87, wherein at least one of the first wild-type 3'-UTR sequence and the second wild-type 3'-UTR sequence has at least 85% sequence identity with SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 71.

89. The recombinant polynucleotide of claim 83, wherein the second wildtype 3'-UTR sequence is the same as the first wild-type 3'-UTR sequence.

90. lire recombinant polynucleotide of claim 83, wherein tire target cell is a mammalian cell.

91. The recombinant polynucleotide of claim 90, wherein the mammalian cell is a human cell.

92. The recombinant polynucleotide of claim 83, wherein the target cell is an immune cell.

93. The recombinant polynucleotide of claim 92, wherein the immune cell is a T cell.

94. The recombinant polynucleotide of claim 93, wherein the T cell is a primary T cell.

95. The recombinant polynucleotide of claim 94, wherein the T cell is a CAR T cell.

96. ITe recombinant polynucleotide of claim 83, wherein the payload polypeptide is a CAR or a fragment thereof.

97. The recombinant polynucleotide of claim 1, wherein the recombinant polynucleotide comprises RNA.

98. The recombinant polynucleotide of claim 97, wherein the recombinant polynucleotide comprises mRNA.

99. The recombinant polynucleotide of claim 1 , wherein the recombinant polynucleotide comprises DNA.

100. A DNA construct comprising: the recombinant polynucleotide of claim 99; and a payload promoter operably linked to the payload sequence.

101. The DNA construct of claim 100, wherein the pay load promoter is constitutive.

102. The DNA construct of claim 100, wherein the DNA construct further comprises a polyadenylation sequence.

103. The DNA construct of claim 102, wherein the polyadenyl ati on sequence is adjacent to the distal end of the 3'-UTR sequence.

104. The DNA construct of claim 100, wherein the DNA construct further comprises a reporter sequence encoding a reporter polypeptide that produces a detectable signal when the reporter polypeptide is expressed by the target cell.

105. The DNA construct of claim 104, wherein the DNA construct further comprises a reporter promoter operably linked to the reporter polypeptide.

106. The DNA construct of claim 104, wherein the reporter promoter is constitutive.

107. The DNA construct of claim 104, wherein the payload promoter and the reporter promoter are divergently oriented relative to one another.

108. The DNA construct of claim 104, wherein the DN A construct further comprises a post-transcriptional regulatory element (PRE) adjacent to the reporter sequence.

109. A vector comprising the recombinant polynucleotide of claim 1 or the DNA construct of claim 100.

110. Tire vector of claim 109, wherein the vector is a plasmid.1 11. The vector of claim 109, wherein the vector is a virus.

112. The vector of claim 109, wherein the vector is a lipid particle.

113. A system comprising: the recombinant polynucleotide of claim 1 ; and a CAR or a polynucleotide, DNA construct, or vector including a sequence encoding the CAR.

114. A cell comprising tire recombinant polynucleotide of claim 1, wherein the cell is the target cell.

115. The cell of claim 114, wherein the cell further comprises a CAR or a polynucleotide, DN A construct, or vector including a sequence encoding the CAR.

116. The cell of claim 114, wherein the cell is in a mammal.

117. A population of cells comprising the cell of claim 114.1 18, A pharmaceutical composition comprising: a pharmaceutically acceptable carrier or a pharmaceutically acceptable excipient; and the recombinant polynucleotide of claim 1.119, A method of modifying a cell, the method comprising contacting the cell with the recombinant polynucleotide of claim 1, wherein the cell is the target cell.120, Tire method of claim 119, wherein the cell is in a mammal.121 , A method for preventing or treating a disease in a subject, the method comprising administering to the subject an amount of the recombinant polynucleotide claim 1.122, The method of claim 121, wherein the disease comprises a cancer.123 , The method of claim 122, wherein the cancer comprises a solid tumor.

124. The method of claim 122, wherein the cancer comprises a liquid tumor.

Images