Methods, systems, and compositions for exogenous control of protein expression

Abstract

Described herein are methods, compositions, and systems for control of transgenes comprising contacting a cell expressing an RNA with a trigger oligonucleotide, wherein the RNA comprises a first nucleotide sequence comprising a conditional nucleotide sequence that comprises a region configured to hybridize to said trigger oligonucleotide, wherein said conditional nucleotide sequence comprises a stem-loop sequence comprising one or more editable codons.

Description

METHODS, SYSTEMS, AND COMPOSITIONS FOR EXOGENOUS CONTROL OF PROTEIN EXPRESSIONCROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 580,846, filed on September 6, 2023, which is incorporated by reference herein in its entirety.BACKGROUND

[0002] Control of transgene expression can be important for therapeutic applications.SUMMARY

[0003] In some aspects, the present disclosure provides for a method for expressing an output protein in a cell, comprising: contacting a cell expressing an RNA with a trigger oligonucleotide, wherein the RNA comprises: (i) a first nucleotide sequence comprising a conditional nucleotide sequence that comprises a region configured to hybridize to said trigger oligonucleotide, wherein said conditional nucleotide sequence comprises a stem-loop sequence comprising one or more editable codons, and (ii) a transgene encoding an output protein; wherein said trigger oligonucleotide is a non-natural oligonucleotide; and wherein said trigger oligonucleotide and said first nucleotide sequence are configured such that said trigger oligonucleotide: (a) selectively activates expression of said protein; or (b) selectively inhibits expression of said protein. In some embodiments, said cell expresses said RNA from a stable open reading frame (ORF). In some aspects, the present disclosure provides for a method for expressing an output protein in a cell, comprising: contacting a cell expressing an RNA from a stable open reading frame (ORF) with a trigger oligonucleotide, wherein the RNA comprises: (i) a first nucleotide sequence comprising a conditional nucleotide sequence that comprises a region configured to hybridize to said trigger oligonucleotide, wherein said conditional nucleotide sequence comprises a stem-loop sequence comprising one or more editable codons, and (ii) a transgene encoding an output protein; wherein said trigger oligonucleotide and said first nucleotide sequence are configured such that said trigger oligonucleotide: (a) selectively activates expression of said protein; or (b) selectively inhibits expression of said protein. In some embodiments, said trigger oligonucleotide is a non-natural oligonucleotide. In some embodiments, said trigger oligonucleotide comprises a synthetic oligonucleotide or an oligonucleotide with at least one non-natural linkage or non-natural base. In some embodiments, said trigger oligonucleotide comprises a non-natural sequence. In some embodiments, said trigger oligonucleotide is not a circular oligonucleotide.

[0004] The method any one of claims 1-7, wherein said stem-loop is not configured to bind a Cas protein. In some embodiments, said RNA is transcribed from a vector introduced to said cell prior to said contacting in said cell said RNA with said trigger oligonucleotide. In some embodiments, said RNA is transcribed from a genomic or episomal locus of said cell. In some embodiments, said trigger oligonucleotide is an in vitro transcribed (IVT) RNA or a synthetic RNA. In some embodiments, the method further comprises integrating a sequence encoding said RNA into a genomic locus. In some embodiments, expression of said output protein is selectively activated in said cell in the presence of said trigger RNA. In some embodiments, said one or more editable codons comprise: (i) 5'-UAG, 5'-UGA, or 5'-UAA; (ii) 5'-AUG-3', or 5'- AUA-3'. In some embodiments, expression of said output protein is selectively inhibited in said cell in the presence of said trigger RNA. In some embodiments, said one or more editable codons comprise: (i) 5'-AUG-3'; or (ii) a 5'-AUA-3' non-start codon. In some embodiments, said output protein comprises a therapeutic protein. In some embodiments, said region configured to hybridize to said trigger oligonucleotide comprises an amount of sequence complementarity sufficient to permit hybridization to said target RNA. In some embodiments, said region configured to hybridize to said trigger oligonucleotide comprises at least 60% complementarity to said trigger RNA. In some embodiments, said one or more editable codons further comprises a start codon. In some embodiments, said stem-loop sequence further comprises a Kozak sequence operably linked to said start codon. In some embodiments, said one or more editable codons further comprises a non-stop, non-start codon that is edited to become a start codon by said target cell. In some embodiments, said stem-loop sequence further comprises a Kozak sequence operably linked to said non-stop, non-start codon. In some embodiments, said nonstop, non-start codon further comprises 5'-AUA -3'. In some embodiments, said protein comprises a toxin, killing factor, a T-cell receptor, a chimeric antigen receptor, a fluorescent protein, a genomic modification protein, a transcription factor, an antigen, an antibody or a functional fragment thereof, a therapeutic protein, or an enzyme. In some embodiments, said cell expressing said RNA comprises an adenosine deaminase acting on RNA (ADAR) protein or a coding sequence encoding thereof. In some embodiments, said region configured to hybridize to said trigger oligonucleotide and said stem-loop sequence comprising one or more editable codons are non-overlapping. In some embodiments, said one or more editable codons are in a stem sequence of said stem-loop sequence. In some embodiments, said one or more editable codons comprise at least one base that is mismatched with a sequence within the stem-loop opposite said one or more editable codons. In some embodiments, an ORF encoding said protein is in frame with said one or more editable codons. In some embodiments, a stem of said stem-loop is at least 12 base pairs in length. In some embodiments, said trigger oligonucleotide: (i) comprises a sequence having 50% or less identity to a genomic sequence of said cell; (ii) does not comprise a stretch of bases with 90% or more identity to a genomic sequence of said cell, wherein said stretch is greater than about 30 bases in length. In some embodiments, said trigger oligonucleotide is or comprises at least 54 nucleotides in length. In some embodiments, said contacting comprises combining said cell expressing said RNA with a viral vector encoding said trigger oligonucleotide. In some embodiments, said trigger oligonucleotide is exogenous to said cell. In some embodiments, said trigger oligonucleotide comprises an antisense oligonucleotide (ASO). In some embodiments, said contacting comprises contacting said trigger oligonucleotide packaged in an LNP to said cell expressing said RNA. In some embodiments, said contacting comprises contacting a GalNAc-conjugated RNA to said cell expressing said RNA. In some embodiments, said contacting comprises contacting a trigger RNA comprising at least one nonnatural nucleotide to said cell expressing said RNA. In some embodiments, the method further comprises contacting said cell with a viral vector encoding said RNA prior to said contacting with said trigger RNA to produce said cell expressing said RNA. In some embodiments, the method further comprises contacting said cell with a donor oligonucleotide encoding said RNA to produce said cell expressing said RNA. In some embodiments, the method further comprises contacting said cell with a donor oligonucleotide encoding said RNA, a Cas protein, and a guide RNA compatible with said Cas protein to produce said cell expressing said RNA. In some embodiments, the method further comprises contacting said cell with a donor oligonucleotide encoding said RNA and a PiggyBac transposase, a Bxbl integrase, a PhiC or PhiC31 integrase, or a recombinase. In some embodiments, said donor oligonucleotide further comprises a homology-directed repair template. In some embodiments, said cell further comprises an open reading frame (ORF) encoding said RNA. In some embodiments, said cell further comprises a promoter operably linked to said open reading frame (ORF) encoding said RNA. In some embodiments, said cell further comprises a small-molecule or RNA-binding riboswitch operably linked to said sequence encoding said protein. In some embodiments, said promoter further comprises an inducible promoter. In some embodiments, said promoter comprises a CMV promoter, a CMV-tetO tetracycline responsive promoter, or a UAS promoter. In some embodiments, said cell comprises a T-cell or a precursor thereof, a T-reg cell or a precursor thereof, a natural killer T-cell or a precursor thereof, an NK-cell or a precursor thereof, a hepatocyte or a precursor thereof, a retinal pigment epithelial cell or a precursor thereof, a neuron or a precursor thereof, an epithelial cell or a precursor thereof, a retinal cell or a precursor thereof, a CHO cell, a HEK293 cell, a NSO murine myeloma cell, a PER.C6 cell, or an Sp2 / 0cell. In some embodiments, said cell comprises a T-cell, a T-reg cell, a natural killer T-cell, an NK-cell, or a precursor thereof. In some embodiments, said output protein further comprises a hormone, an antibody, a chimeric antigen receptor (CAR), a soluble receptor, an extracellular domain of a hormone receptor, an extracellular domain of a VEGF receptor, an interleukin, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11, caspase 12, caspase 13, p53 upregulated modulator of apoptosis (PUMA), Bakl, iCasp9, ricin toxin chain A, diphtheria toxin, IL-18, IL-23-alpha, IL-15, IL-12- alpha, IL-12-beta, an anti-IL-18 antibody, an anti-IL-lOR antibody, an anti-CSFIR antibody, tocilizumab, anakinra, IL-7, IL-2R-beta, CCL19, IL-36-gamma, IL-37, or FOXO1. In some embodiments, said protein further comprises an anti-tumor antibody or fragment thereof. In some embodiments, the method further comprises contacting said cell with a first viral vector encoding said RNA; further comprising contacting said cell with a second viral vector encoding said trigger RNA, wherein said first and said second viral vector comprise different viral serotypes with overlapping tissue tropism. In some embodiments, said first viral vector and said second viral vector comprise nonoverlapping selections from one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV8, or AAV9. In some embodiments, said stem-loop sequence comprises a sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or 100% sequence identity to any one of SEQ ID NOs: 5, 6, 7, 8, 17, 18, or 19, a stem-loop sequence of Table 1, or a reverse complement thereof. In some embodiments, said stem-loop sequence is configured to bind an ADAR protein.

[0005] In some aspects, the present disclosure provides for a system comprising: (a) a viral vector configured to transduce a cell or a donor oligonucleotide configured for expression in a cell, encoding an RNA comprising: (i) a first nucleotide sequence comprising a conditional nucleotide sequence that comprises a region configured to hybridize to said trigger oligonucleotide, wherein said conditional nucleotide sequence comprises a stem-loop sequence comprising one or more editable codons, and (ii) a transgene encoding an output protein; and (b) a trigger oligonucleotide, wherein said trigger oligonucleotide comprises a synthetic oligonucleotide or an oligonucleotide with at least one non-natural linkage or non-natural base; and wherein said trigger oligonucleotide and said first nucleotide sequence are configured such that said trigger oligonucleotide: (a) selectively activates expression of said protein; or (b) selectively inhibits expression of said protein. In some embodiments, said viral vector isconfigured to stably transduce a cell, or said donor oligonucleotide is configured for stable integration in a cell. In some aspects, the present disclosure provides for a system comprising: (a) a viral vector configured to stably transduce a cell or a donor oligonucleotide configured for stable expression in a cell, encoding an RNA comprising: (i) a first nucleotide sequence comprising a conditional nucleotide sequence that comprises a region configured to hybridize to said trigger oligonucleotide, wherein said conditional nucleotide sequence comprises a stem-loop sequence comprising one or more editable codons, and(ii) a transgene encoding an output protein; and (b) a trigger oligonucleotide; wherein said trigger oligonucleotide and said first nucleotide sequence are configured such that said trigger oligonucleotide: (a) selectively activates expression of said protein; or (b) selectively inhibits expression of said protein. In some aspects, the present disclosure provides for a system comprising: (a) a cell stably encoding an RNA, wherein said RNA comprises: (i) a first nucleotide sequence comprising a conditional nucleotide sequence that comprises a region configured to hybridize to said trigger oligonucleotide, wherein said conditional nucleotide sequence comprises a stem-loop sequence comprising one or more editable codons, and (ii) a transgene encoding an output protein; and (b) a trigger oligonucleotide; wherein said trigger oligonucleotide and said first nucleotide sequence are configured such that said trigger oligonucleotide: (a) selectively activates expression of said protein; or (b) selectively inhibits expression of said protein. In some embodiments, said trigger oligonucleotide is a non-natural oligonucleotide. In some embodiments, said trigger oligonucleotide comprises a synthetic oligonucleotide or an oligonucleotide with at least one non-natural linkage or non-natural base. In some embodiments, said trigger oligonucleotide comprises a synthetic oligonucleotide or an oligonucleotide with at least one non-natural linkage or non-natural base. In some embodiments, said trigger oligonucleotide comprises a non-natural sequence. In some embodiments, said trigger oligonucleotide is not a circular oligonucleotide. In some embodiments, said RNA is transcribed from a vector introduced to said cell prior to said contacting in said cell said RNA with said trigger oligonucleotide. In some embodiments, said RNA is transcribed from a genomic or episomal locus of said cell. In some embodiments, said trigger oligonucleotide is an in vitro transcribed (IVT) RNA or a synthetic RNA. In some embodiments, expression of said output protein is selectively activated in said cell in the presence of said trigger RNA. In some embodiments, said one or more editable codons comprise: (i) 5'-UAG, 5'-UGA, or 5'-UAA; (ii) 5'-AUG-3', or 5'-AUA-3'. In some embodiments, expression of said output protein is selectively inhibited in said cell in the presence of said trigger RNA. In some embodiments, said one or more editable codons comprise: (i) 5'-AUG-3'; or (ii) a 5'-AUA-3' non-start codon. In some embodiments, said outputprotein comprises a therapeutic protein. In some embodiments, said region configured to hybridize to said trigger oligonucleotide comprises an amount of sequence complementarity sufficient to permit hybridization to said target RNA. In some embodiments, said region configured to hybridize to said trigger oligonucleotide comprises at least 60% complementarity to said trigger RNA. In some embodiments, said one or more editable codons further comprises a start codon. In some embodiments, said one or more editable codons further comprises a nonstop, non-start codon that is edited to become a start codon by said target cell. In some embodiments, said non-stop, non-start codon further comprises 5'-AUA -3'. In some embodiments, said protein comprises a toxin, killing factor, a T-cell receptor, a chimeric antigen receptor, a fluorescent protein, a genomic modification protein, a transcription factor, an antigen, an antibody or a functional fragment thereof, a therapeutic protein, or an enzyme. In some embodiments, said cell encoding said RNA comprises an adenosine deaminase acting on RNA (ADAR) protein or a coding sequence encoding thereof. In some embodiments, an ORF encoding said protein is in frame with said one or more editable codons. In some embodiments, said trigger oligonucleotide: (i) comprises a sequence having 50% or less identity to a genomic sequence of said cell; (ii) does not comprise a stretch of bases with 90% or more identity to a genomic sequence of said cell, wherein said stretch is greater than about 30 bases in length. In some embodiments, said trigger oligonucleotide is at least 54 nucleotides in length. In some embodiments, said contacting comprises combining said cell expressing said RNA with a viral vector encoding said trigger oligonucleotide. In some embodiments, said trigger oligonucleotide is exogenous to said cell. In some embodiments, said trigger oligonucleotide comprises an antisense oligonucleotide (ASO). In some embodiments, said trigger oligonucleotide is encapsulated in an LNP. In some embodiments, said trigger oligonucleotide is GalNAc- conjugated. In some embodiments, said cell further comprises an open reading frame (ORF) encoding said RNA. In some embodiments, said cell further comprises a promoter operably linked to said open reading frame (ORF) encoding said RNA. In some embodiments, said cell further comprises a small-molecule or RNA-binding riboswitch operably linked to said sequence encoding said protein. In some embodiments, said promoter further comprises an inducible promoter. In some embodiments, said promoter comprises a CMV promoter, a CMV-tetO tetracycline responsive promoter, or a UAS promoter. In some embodiments, said cell comprises a T-cell or a precursor thereof, a T-reg cell or a precursor thereof, a natural killer T- cell or a precursor thereof, an NK-cell or a precursor thereof, a hepatocyte or a precursor thereof, a retinal pigment epithelial cell or a precursor thereof, a neuron or a precursor thereof, an epithelial cell or a precursor thereof, a retinal cell or a precursor thereof, a CHO cell, a HEK293cell, a NSO murine myeloma cell, a PER.C6 cell, or an Sp2 / 0 cell. In some embodiments, said output protein further comprises a hormone, an antibody, a chimeric antigen receptor (CAR), a soluble receptor, an extracellular domain of a hormone receptor, an extracellular domain of a VEGF receptor, an interleukin, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11, caspase 12, caspase 13, p53 upregulated modulator of apoptosis (PUMA), Bakl, iCasp9, ricin toxin chain A, diphtheria toxin, IL-18, IL- 23-alpha, IL-15, IL-12-alpha, IL-12-beta, an anti-IL-18 antibody, an anti-IL-lOR antibody, an anti-CSFIR antibody, tocilizumab, anakinra, IL-7, IL-2R-beta, CCL19, IL-36-gamma, IL-37, or FOXOL In some embodiments, said trigger oligonucleotide is encoded on a viral vector. In some embodiments, said viral vector comprises an AAV1, AAV2, AAV3, AAV4, AAV5, AAV8, or AAV9 vector. In some embodiments, said stem-loop sequence comprises a sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or 100% sequence identity to any one of SEQ ID NOs: 5, 6, 7, 8, 17, 18, or 19. In some embodiments, said stem-loop sequence comprises a sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or 100% sequence identity to a stem-loop sequence in Table 1, a reverse complement thereof, or an RNA cognate thereof. In some embodiments, said stem-loop is not configured to bind a Cas protein. In some embodiments, said stem-loop sequence further comprises a Kozak sequence operably linked to a start codon. In some embodiments, said stem-loop sequence further comprises a Kozak sequence operably linked to a non-stop, non-start codon. In some embodiments, said region configured to hybridize to said trigger oligonucleotide and said stemloop sequence comprising one or more editable codons are non-overlapping. In some embodiments, said one or more editable codons are in a stem sequence of said stem-loop sequence. In some embodiments, said one or more editable codons comprise at least one base that is mismatched with a sequence within the stem-loop opposite said one or more editable codons. In some embodiments, a stem of said stem-loop is at least 12 base pairs in length.

[0006] In some aspects, the present disclosure provides for a system comprising: (a) a viral vector configured to encode an RNA and stably transduce a cell, or a donor DNA configured toencode an RNA and stably integrate in a cell, wherein said RNA comprises: (i) a first nucleotide sequence comprising a conditional nucleotide sequence that comprises a region configured to hybridize to said trigger oligonucleotide, wherein said region configured to hybridize to said trigger oligonucleotide comprises at least one editable codon, and (ii) a transgene encoding an output protein; wherein said trigger oligonucleotide and said first nucleotide sequence are configured such that said trigger oligonucleotide: (a) selectively activates expression of said protein; or (b) selectively inhibits expression of said protein; wherein: (i) said region configured to hybridize to said trigger oligonucleotide is outside of an open reading frame (ORF) encoding said output protein; or (ii) said editable codon is not a stop codon. In some aspects, the present disclosure provides for a system comprising; (a) a cell stably encoding an RNA, a viral vector configured to encode an RNA, or a donor DNA configured to encode an RNA, wherein said RNA comprises: (i) a first nucleotide sequence comprising a conditional nucleotide sequence that comprises a region configured to hybridize to said trigger oligonucleotide, wherein said region configured to hybridize to said trigger oligonucleotide comprises at least one editable codon, and (ii) a transgene encoding an output protein; wherein said trigger oligonucleotide and said first nucleotide sequence are configured such that said trigger oligonucleotide: (a) selectively activates expression of said protein; or (b) selectively inhibits expression of said protein; wherein: (i) said region configured to hybridize to said trigger oligonucleotide is outside of an open reading frame (ORF) encoding said output protein; or (ii) said editable codon is not a stop codon. In some embodiments, said trigger oligonucleotide comprises a synthetic oligonucleotide or an oligonucleotide with at least one non-natural linkage or non-natural base. In some embodiments, said trigger oligonucleotide comprises a non-natural sequence. In some embodiments, said trigger oligonucleotide is not a circular oligonucleotide. In some embodiments, said RNA is transcribed from a genomic or episomal locus of said cell. In some embodiments, said RNA is transcribed from a vector introduced to said cell prior to said contacting in said cell said RNA with said trigger oligonucleotide. In some embodiments, said trigger oligonucleotide is an in vitro transcribed (IVT) RNA or a synthetic RNA. In some embodiments, expression of said output protein is selectively activated in said cell in the presence of said trigger RNA. In some embodiments, said one or more editable codons comprise: (i) 5'-UAG, 5'-UGA, or 5'-UAA; (ii) 5'-AUG-3', or 5'-AUA-3'. In some embodiments, expression of said output protein is selectively inhibited in said cell in the presence of said trigger RNA. In some embodiments, said one or more editable codons comprise: (i) 5'-AUG-3'; or (ii) a 5'-AUA-3' non-start codon. In some embodiments, said region configured to hybridize to said trigger oligonucleotide comprises an amount of sequencecomplementarity sufficient to permit hybridization to said target RNA. In some embodiments, said region configured to hybridize to said trigger oligonucleotide comprises at least 60% complementarity to said trigger RNA. In some embodiments, said one or more editable codons further comprises a start codon. In some embodiments, said one or more editable codons further comprises a non-stop, non-start codon that is edited to become a start codon by said target cell. In some embodiments, said non-stop, non-start codon further comprises 5'-AUA -3'. In some embodiments, said protein comprises a toxin, killing factor, a T-cell receptor, a chimeric antigen receptor, a fluorescent protein, a genomic modification protein, a transcription factor, an antigen, an antibody or a functional fragment thereof, a therapeutic protein, or an enzyme. In some embodiments, said cell expressing said RNA comprises an adenosine deaminase acting on RNA (ADAR) protein or a coding sequence encoding thereof. In some embodiments, an ORF encoding said protein is in frame with said one or more editable codons. In some embodiments, said trigger oligonucleotide: (i) comprises a sequence having 50% or less identity to a genomic sequence of said cell; (ii) does not comprise a stretch of bases with 90% or more identity to a genomic sequence of said cell, wherein said stretch is greater than about 30 bases in length. In some embodiments, said trigger oligonucleotide is or comprises at least 54 nucleotides in length. In some embodiments, said trigger oligonucleotide is exogenous to said cell. In some embodiments, said trigger oligonucleotide comprises an antisense oligonucleotide (ASO). In some embodiments, said trigger oligonucleotide is encapsulated in an LNP. In some embodiments, said trigger oligonucleotide is GalNAc-conjugated. In some embodiments, said cell further comprises an open reading frame (ORF) encoding said RNA. In some embodiments, said cell further comprises a promoter operably linked to said open reading frame (ORF) encoding said RNA. In some embodiments, said cell further comprises a small-molecule or RNA-binding riboswitch operably linked to said sequence encoding said protein. In some embodiments, said promoter further comprises an inducible promoter. In some embodiments, said promoter comprises a CMV promoter, a CMV-tetO tetracycline responsive promoter, or a UAS promoter. In some embodiments, said cell comprises a T-cell or a precursor thereof, a T- reg cell or a precursor thereof, a natural killer T-cell or a precursor thereof, an NK-cell or a precursor thereof, a hepatocyte or a precursor thereof, a retinal pigment epithelial cell or a precursor thereof, a neuron or a precursor thereof, an epithelial cell or a precursor thereof, a retinal cell or a precursor thereof, a CHO cell, a HEK293 cell, a NSO murine myeloma cell, a PER.C6 cell, or an Sp2 / 0 cell. In some embodiments, said output protein further comprises a hormone, an antibody, a chimeric antigen receptor (CAR), a soluble receptor, an extracellular domain of a hormone receptor, an extracellular domain of a VEGF receptor, an interleukin,caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11, caspase 12, caspase 13, p53 upregulated modulator of apoptosis (PUMA), Bakl, iCasp9, ricin toxin chain A, diphtheria toxin, IL-18, IL-23-alpha, IL-15, IL-12- alpha, IL-12-beta, an anti-IL-18 antibody, an anti-IL-lOR antibody, an anti-CSFIR antibody, tocilizumab, anakinra, IL-7, IL-2R-beta, CCL19, IL-36-gamma, IL-37, or FOXOL In some embodiments, said protein further comprises an anti-tumor antibody or fragment thereof. In some embodiments, said trigger oligonucleotide is encoded on a viral vector. In some embodiments, said viral vector comprises an AAV1, AAV2, AAV3, AAV4, AAV5, AAV8, or AAV9 vector.

[0007] In some aspects, the present disclosure provides for a method of controlling expression of a protein in a cell, comprising contacting the components of any of the systems described herein to a cell, or utilizing the components of any of the systems described herein in vitro or in vivo. In some embodiments, the method comprises sequentially contacting the components of the system to said cell.

[0008] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure.Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.INCORPORATION BY REFERENCE

[0009] All publications, patents, patent applications, and accession numbers mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0011] FIG. 1 depicts a diagram illustrating how an exogenous “trigger” oligonucleotide can be used to modulate protein production (e.g. therapeutic protein production) in a cell stably transfected with a transgenic vector.

[0012] FIGs. 2A, 2B, 2C, 2D, 2E, 2F, and 2G depict the results of the experiment in Example 1, showing that cells expressing sensor constructs demonstrate an increased level of GFP+ (e.g. output protein-positive) cells upon transfection of either mRNA or plasmid DNA encoding mRNA corresponding to the matching trigger. Cells that were either (a) WT 293T cells; (b) 293T cells treated with the SensA lentiviral construct; or (c) 293T cells treated with the or SensB lentiviral construct were treated with: : (i) 500 ng IVT mRNA bearing a region complementary to the hybridization region in the RNA sensor (SEQ ID NO: 12) in complex with transfection agent (liposomal, Lipofectamine MessengerMax, Thermo Fisher); (ii) 500 ng IVT mRNA bearing a region not complementary to the hybridization region in the RNA sensor (SEQ ID NO: 10) in complex with transfection agent (liposomal, Lipofectamine MessengerMax, Thermo Fisher); (iii) 500ng plasmid DNA encoding a region complementary to the hybridization region in the RNA sensor (SEQ ID NO: 11) and not encoding ADAR in complex with jetOPTIMUS® (Polyplus); (iv) 500ng plasmid DNA encoding a region not complementary to the hybridization region in the RNA sensor (SEQ ID NO: 9) and not encoding ADAR in complex with jetOPTIMUS® (Polyplus); (v) 500ng plasmid DNA encoding a region complementary to the hybridization region in the RNA sensor (SEQ ID NO: 11) and further 5ng plasmid DNA encoding ADAR in complex with jetOPTIMUS® (Polyplus); and (vi) 500ng plasmid DNA encoding a region not complementary to the hybridization region in the RNA sensor (SEQ ID NO: 9) and further 5ng plasmid DNA encoding ADAR in complex with jetOPTIMUS® (Polyplus). The results of the experiment for each type of cell (WT 293T, SensA-treated, or SensB -treated) are shown in FIG. 2A (flow cytometry of GFP vs. mCherry fluorescence for condition (i)), FIG. 2B (flow cytometry of GFP vs. mCherry fluorescence for condition (ii)), FIG. 2C (flow cytometry of GFP vs. mCherry fluorescence for condition (iii)), FIG. 2D (flow cytometry of GFP vs. mCherry fluorescence for condition (iv)); FIG. 2E (flow cytometry of GFP vs. mCherry fluorescence for condition (v)); FIG. 2F (flow cytometry of GFP vs. mCherry fluorescence for condition (vi)); and FIG. 2G (quantitation of the flow cytometry results of FIGs. 2A, 2B, 2C, 2D, 2E, and 2F).

[0013] FIG. 3A depicts a schematic showing one example of a logic-gate approach according to embodiments described herein. In this approach, two transgenic cassettes can be integrated into a single host cell to form a signal amplifier: (i) a first transgenic cassette (right) containing a constitutive promoter (“Promoter”) operably linked to a first transgene (“Transgene 1”) operablylinked to an RNA sensor construct (“RADAR”) bearing a stop codon configured to be editable to a non-stop codon as described herein, and configured to be responsive to a trigger RNA; and (ii) a second transgenic cassette (left) containing a stimulus-inducible promoter (“Inducible Promoter”, e.g. a hormone, small molecule, cell-phase; or cell-identity responsive promoter) operably linked to a sequence encoding the trigger RNA (“trigger”) and an optional second transgene (“transgene 2”). In this configuration, the stimulus that induces the inducible promoter triggers the cell to produce a trigger RNA, which allows the RNA sensor construct operably linked to the first transgene to be edited and express the first transgene (“Therapeutic ON”).

[0014] FIG. 3B depicts a schematic showing one example of a logic-gate approach according to embodiments described herein. In this approach, a single transgenic cassette is used to produce an “AND” gate: the host cell comprises a stimulus-inducible promoter (“Inducible Promoter” e.g. a hormone, small molecule, cell-phase; or cell-identity responsive promoter) operably linked to a transgene (“Transgene 1”) operably linked to an RNA sensor construct (“RADAR”) bearing an stop codon configured to be editable to a non-stop codon as described herein, and configured to be responsive to a trigger RNA. In this configuration, both the stimulus that induces the inducible promoter (which produces the mRNA containing the RNA sensor construct) and the trigger RNA (which triggers RNA editing to allow expression of the transgene from the mRNA) are needed for the transgene to be expressed.

[0015] FIG. 4A depicts a schematic showing an example “default-OFF” embodiment of an RNA sensor construct as described herein. In this example, the RNA sensor construct operably linked to an ORF encoding an output protein is configured to be responsive to a trigger RNA but contains an editable stop codon. The presence of the trigger RNA induces editing of the stop codon to a non-stop codon allowing for expression of the downstream output protein.

[0016] FIG. 4B depicts a schematic showing an example “default-ON” embodiment of an RNA sensor construct as described herein. In this example, the RNA sensor construct operably linked to an ORF encoding an output protein is configured to be responsive to a trigger RNA but contains an editable start codon. The presence of the trigger RNA induces editing of the start codon to a non-start codon, thus inhibiting expression of the downstream output protein.

[0017] FIG. 5 depicts example designs for RNA sensor constructs that can be used with expression control systems described herein.DETAILED DESCRIPTIONOverview

[0018] Regulating transgene expression in a graded manner spatiotemporally in response to an exogenous signal is highly desirable, e.g. for gene therapy or cell manufacturing applications where amount or timing of transgene expression is important for cell differentiation or therapeutic effect. Additionally, fine-tuned control of otherwise integrated or constitutively expressing transgenes can improve their safety and efficacy.

[0019] Here we describe methods, compositions, and systems for control of transgenes (e.g. DNA- or RNA-based medicines or therapeutic genes) using a synthetic exogenous trigger in the form of an oligonucleotide to purposely provide inducible or logic-gated expression of transgene into protein.

[0020] These systems, compositions, and methods can be used to provide temporal / dynamic control of transgenes through the addition of a triggering oligonucleotide (e.g. trigger RNA) at a separate timepoint from the original introduction of a transgene encoding an output protein (e.g., through viral transduction , such as lentivirus or AAV). The oligonucleotide used here can be specifically designed to have no off-target effects to endogenous transcripts by using completely novel, non-natural sequences. Thus these methods, compositions, and systems can be used to induce or turn off transgene output protein expression (depending on whether the RNA sensing employs a stop codon, start codon, non-start codon, upstream open reading frame, or splice site) in a reversible, temporary, and dose-responsible manner, e.g. allowing for tuning the expression / modulating the dose of therapeutic transgenes in a dynamic manner by modulating the dose of the synthetic trigger oligonucleotide separately from the administered genetic dose of the transgene. This can be particularly useful in applications delivery efficiency of a transgene carrying a therapeutic is variable across patients and leads to variable responses, or when redosing is not possible (e.g., AAV).

[0021] Alternatively or additionally, the trigger oligonucleotide (e.g. RNA) can be transcribed from an integrated cassette within the same cell as the transgene to create logic gated or amplified circuitry within the cell. Such embodiments may require no secondary exogenous dosing of the trigger oligonucleotide, but rather have the oligonucleotide transcription controlled by a cell-state specific or inducible promoter. Furthermore, differences in serotype tropism can be used to yield highly specific expression with non-specific vectors, e.g. by delivering the transgene with a constitutive promoter with one vector, and the trigger with a different vector with overlapping, but not identical tropism; in this way, the intersection of the tropism of the two vectors yields the final expression profile of the transgene.Definitions

[0022] Before describing exemplary embodiments in greater detail, the following definitions are set forth to illustrate and define the meaning and scope of the terms used in the description.

[0023] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, New York (1994), and Hale & Markham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, N.Y. (1991) provide one of skill with the general meaning of many of the terms used herein. Still, certain terms are defined below for the sake of clarity and ease of reference.

[0024] Certain ranges are presented herein with numerical values being preceded by the term "about." The term "about" is generally 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. 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.

[0025] In general, terms used herein are generally intended as “open” terms (e.g., the term “including” is to be interpreted as “including but not limited to,” the term “having” is to be interpreted as “having at least,” the term “includes” is to be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases need not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing solely one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” or “an” is to be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation is to be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, generally signifies at least two recitations, or two or more recitations). Furthermore, in those instances where a conventionanalogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art may understand the convention (e.g. , “ a system having at least one of A, B, and C” may include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B, and C together, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art may understand the convention (e.g., “ a system having at least one of A, B, or C” may include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B, and C together, etc.).

[0026] In general, as used herein, virtually any disjunctive word and / or phrase presenting two or more alternative terms, is to be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”

[0027] In addition, where features or aspects of the disclosure are described in terms of Markush groups herein, the disclosure is also generally intended to encompass any individual member or subgroup of members of the Markush group.

[0028] In general, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, a range generally includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

[0029] As used herein, the singular forms “a”, “an”, and “the” generally include plural referents unless the context clearly dictates otherwise. For example, the term “a RNA sensor” refers to one or more RNA sensors, e.g., a single RNA sensor and multiple RNA sensors. It is further noted that the claims can be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0030] Reference to an item in the singular is generally to be understood as including the plural and vice versa unless explicitly stated otherwise or clear from the text. Grammatical conjunctions are intended to express any and all disjunctive conjunctions and conjunctions of conjunctive clauses, sentences, words, and the like, unless otherwise stated or clear from the context. Thus, the term "or" is generally understood to be an inclusive “or” encompassing both alternatives.

[0031] The terms “oligonucleotide”, “polynucleotide” and “nucleic acid,” used interchangeably herein, generally refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxynucleotides. Thus, this term includes, but is not limited to, single-, double-, or multistranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer including purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The terms “oligonucleotide”, “polynucleotide”, and “nucleic acid” generally are understood to include, as applicable to the embodiment being described, single-stranded (such as sense or antisense) and double-stranded polynucleotides.

[0032] In some embodiments, an oligonucleotide as described herein comprises one or more non-natural linkages or one or more non-natural nucleotides. Non-natural nucleotides can include, but are not limited to, 2'-fluoro, 2'-O-mcthyl, 2'-O-mcthyl, 2'-O-methoxy-ethyl, 2'-O- methoxy-ethoxy, 5 '-methyl, SNA, hGNA, hhGNA, mGNA, TN A, h'GNA, locked nucleic acids (LNAs), GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA, 2'-mUNA, or an abasic nucleotide (e.g. DNA or RNA). Non-natural linkages can include, but are not limited to, phosphorothioate and methylphosphonate. In some embodiments, an oligonucleotide as described herein comprises a modified uracil. Example nucleobases and nucleosides having a modified uracil include pseudouridine ( ), pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza- uridine, 2-thio-5 -aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2- thio-pseudouridine, 5 -hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5- iodo-uridine or 5-bromo-uridine), 3-methyl-uridine (m3U), 5 -methoxy-uridine (mo5U), uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5 -carboxymethyluridine (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5- carboxyhydroxymethyl -uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 5-methylaminomethyl-2-thio-uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2- thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (rm5U), 1-taurinomethyl-pseudouridine, 5 -taurinomethyl-2 -thio-uridine (rm5s2U), 1- taurinomethyl-4-thio-pseudouridine, 5 -methyl -uridine (m5U, i.e., having the nucleobase deoxythymine), 1 -methylpseudouridine (ml\| / ), 5 -methyl-2 -thio-uridine (m5s2U), l-methyl-4- thio-pseudouridine (mls4\| / ), 4-thio-l-methyl-pseudouridine, 3-methyl-pseudouridine (m3\| / ), 2- thio- 1 -methyl-pseudouridine, 1 -methyl- 1 -deaza-pseudouridine, 2-thio- 1 -methyl- 1 -deaza- pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5 -methyldihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, Nl- methyl-pseudouridine (also known as 1 -methylpseudouridine (m h| / )), 3-(3-amino-3- carboxypropyl)uridine (acp3U), l-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3 \| / ), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm5s2U), a-thio-uridine, 2'-O-mcthyl-uridinc (Um), 5,2'-O-dimethyl-uridine (m5Um), 2'-O- methyl-pseudouridine (\| / m), 2-thio-2'-O-methyl-uridine (s2Um), 5-mcthoxycarbonylmcthyl-2'- O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm5Um), 5- carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm5Um), 3 ,2 '-O-di methyl-uri di ne (m3Um), 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm5Um), 1 -thio-uridine, deoxy thymidine, 2'-F-ara-uridine, 2'-F-uridinc, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, and 5-[3-(l-E-propenylamino)uridine. In some embodiments, an oligonucleotide as described herein comprises a modified cytosine. Example nucleobases and nucleosides having a modified cytosine include 5 -aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m3C), N4-acetyl-cytidine (ac4C), 5-formyl-cytidine (f5C), N4-methyl-cytidine (m4C), 5- methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5 -iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio- cytidine (s2C), 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio- 1-methyl- pseudoisocytidine, 4-thio- 1 -methyl- 1 -deaza-pseudoisocytidine, 1 -methyl- 1 -deaza- pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2- thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy- pseudoisocytidine, 4-methoxy- 1-methyl-pseudoisocytidine, lysidine (k2C), a-thio-cytidine, 2'-O- methyl-cytidine (Cm), 5,2'-O-dimethyl-cytidine (m5Cm), N4-acctyl-2'-O-mcthyl-cytidinc (ac4Cm), N4,2'-O-dimcthyl-cytidinc (m4Cm), 5-formyl-2'-O-mcthyl-cytidinc (f5Cm), N4,N4,2'- O-trimethyl-cytidine (m4 2Cm), 1 -thio-cytidine, 2'-F-aracytidinc, 2'-F-cytidinc, and 2'-OH- aracytidine.

[0033] In some cases, it is advantageous to reduce the immunogenicity of the sensor RNA. Methods of reducing the immunogenicity of RNAs have been documented by, for example,Starostina et al. (Vaccines (Basel). 2021 May 3;9(5):452) which is specifically incorporated by reference herein. In general, methods of reducing the immunogenicity of a sensor RNA can involve the incorporation of modified ribonucleic acids into the sensor RNA. Modified ribonucleic acids that find use in the present disclosure includes, without limitation, pyridin-4- one ribonucleoside, 5 -aza-uridine, 2-thio-5 -aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2- thio-pseudouridine, 5 -hydroxyuridine, 3 -methyluridine, 5 -carboxymethyluridine, 1 - carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5- taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5 -taurinomethyl -2-thiouridine, 1-tau- rinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-l -methylpseudouridine, 2 -thio- 1-methyl-pseudouridine, 1 -methyl- 1-deaza-pseudouridine, 2-thio-l- methyl-l-deaza-pseudouridine, dihydrouridine, dihydropseudouridine. 2-thio-dihydrouridine, 2- thiodihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thiouridine, 4-methoxy- pseudouridine, 4-methoxy-2-thio-pseudouridine, 5 -azacytidine, pseudoisocytidine, 3-methyl- cytidine, N4-acetylcytidine, 5 -formylcytidine, N4-methylcytidine, 5 -hydroxymethylcytidine, 1- methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5- methyl-cytidine, 4-thio-pseudo isocytidine, 4-thio-l -methyl-pseudoisocytidine, 4-thio-l -methyl - 1-deaza-pseudoisocytidine, 1 -methyl- 1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5- methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5- methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-l -methyl-pseudoisocytidine, 2- aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2- aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2, 6- diaminopurine, 1 -methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis- hydroxyisopentenyadenosine, 2-methylthio-N-6-(cis-hydroxyisopentenyl) adenosine, N6- glycinylcarbamoyladenosine, N6-threonyl-carbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6.N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, 2- methoxy-adenine, inosine, 1-me thyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7- deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza- guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy- guanosine, 1 -methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo- guanosine, 7-me thyl-8-oxo-guanosine, 1 -methyl -6-thio-guanosine, N2-methyl-6-thio -guano sine, N2,N2-dimethyl -6-thio-guanosine, methylcytosine, pseudouridine, methyladenosine, etc. In some embodiments, the methylcytosine is 5- methylcytosine. In some embodiments, the pseudouridine is N 1-methyl-pseudouridine. In some embodiments, the methyladenosine is a N6- methyladenosine. In some embodiments, the methyladenosine is a N1 -methyladenosine. In someembodiments, a portion of the nucleotides present in the sensor RNA are composed of modified ribonucleic acids. For instance, a portion of the uridines in the sensor RNA are replaced with pseudouridines. When the uridines of the sensor RNA are replaced with pseudouridines, a certain percentage of the uridines are replaced with pseudouridines. For instance, about 1-10%, about 10-20%, about 20-30%, about 30-40%, about 40-50%, about 50-60%, about 60-70%, about 70- 80%, 80-90% or greater than 90% of the uridines are replaced with pseudouridines. In an embodiment, 75% or less of the uridines in the sensor RNAs are replaced with pseudouridines. In an embodiment, the sensor sequence or parts of it do not have pseudouridines

[0034] As used herein, the term “PiggyBac transposase”, refers to the transposase isolated from the cabbage looper moth Trichop lusia ni, or the nucleic acid sequence encoding said transposase, which can transport sequences flanked by PiggyBac terminal repeats (TRs) to a TTAA target site in the genome. Example sequences for PiggyBac transposase and TRs are provided in Table 1.

[0035] As used herein, the term “piggyBac-like transposon” generally refers to a DNA sequence flanked by TR sites compatible with piggyBac transposase. In some embodiments, a “piggyBac- like transposon” generally encompasses any DNA fragments that could be excised by natural or artificial transposases and reinserted into a TTAA target site in the genome, causing a target-site duplication (TSD) that flanks the element. In some embodiments, the term “piggyBac-like transposon” generally refers to a transposon which comprises 5' and 3' terminal repeats from a piggyBac transposon. In some embodiments, these sequences are or are derived from the piggyBac sequences described in Table 1.

[0036] As used herein, the term “bxbl integrase” generally refers to an integrase capable of integrating a cargo between locations flanked by bxbl attB sites and locations flanked by a bxbl attP site. Example attB, attP, and integrase sequences can be found in Table 1. Use of Bxbl integrase can be performed according to e.g. Thompson et al. BMC Biotechnol. 2012 Mar 21:12:9. doi: 10.1186 / 1472-6750-12-9, which is incorporated by reference herein in its entirety.

[0037] As used herein, the term “phiC31 integrase” generally refers to an integrase capable of integrating a cargo between locations flanked by phiC31 attB sites and locations flanked by a phiC31 attP site. Example attB, attP, and integrase sequences can be found in Table 1. Use of PhiC31 integrase can be performed according to e.g. Thompson et al. BMC Biotechnol. 2010 Feb 23:10:17. doi: 10.1186 / 1472-6750-10-17, which is incorporated by reference herein in its entirety.

[0038] In certain embodiments, the phiC31 and Bxbl integrases may be introduced into the human cell by introducing a nucleic acid encoding the phiC31 and Bxbl integrases into the cell.The nucleic acid encoding the phiC31 and / or Bxbl integrases may be an mRNA or a circular DNA.

[0039] Some methods described herein involve the use of targeting endonucleases. A targeting endonuclease can comprise a DNA-binding domain and a nuclease domain. The DNA binding domain of the targeting endonuclease can be programmable, meaning that it can be designed or engineered to recognize and bind different DNA sequences. In some embodiments, the DNA binding can be mediated by interactions between the DNA binding domain of the targeting endonuclease and the target DNA. Thus, in some cases the DNA-binding domain can be programed to bind a DNA sequence of interest by protein engineering. In other embodiments, DNA-binding can be mediated by a guide RNA that interacts with the DNA-binding domain of the targeting endonuclease and the target DNA. In such instances, the DNA-binding domain can be targeted to a DNA sequence of interest by designing the appropriate guide RNA.

[0040] Suitable targeting endonucleases can include zinc finger nucleases, clustered regularly interspersed short palindromic repeats (CRISPR) / CRISPR- associated (Cas) (CRISPR / Cas) nuclease systems, or CRISPR / Cas nickase system,. The targeting endonuclease can comprise wild- type or naturally- occurring DNA-binding and / or nuclease domains, modified versions of naturally-occurring DNA-binding and / or nuclease domains, synthetic or artificial DNA-binding and / or nuclease domains, or combinations thereof.

[0041] In some embodiments, the targeting endonuclease can be a zinc finger nuclease (ZFN). A ZFN can comprise a DNA-binding zinc finger region and a nuclease domain. The zinc finger region can comprise from about two to seven zinc fingers, for example, about four to six zinc fingers, wherein each zinc finger binds three nucleotides, and wherein the zinc fingers can be linked together using suitable linker sequences. The zinc finger region can be engineered to recognize and bind to any DNA sequence. See, for example, Beerli et al. (2002) Nat. Biotechnol. 20:135-141 ; Pabo et al. (2001 ) Ann. Rev. Biochem. 70:313-340; Isalan et al. (2001 ) Nat. Biotechnol. 19:656-660; Segal et al. (2001 ) Curr. Opin. Biotechnol. 12:632-637; Choo et al. (2000) Curr. Opin. Struct. Biol. 10:41 1 -416; Zhang et al. (2000) J. Biol. Chem. 275(43):33850- 33860; Doyon et al. (2008) Nat. Biotechnol. 26:702-708; and Santiago et al. (2008) Proc. Natl. Acad. Sci. USA 105:5809-5814. Publicly available web-based tools for identifying potential target sites in DNA sequences as well as designing zinc finger binding domains are known in the art.

[0042] In some embodiments, the targeting endonuclease can be a RNA-guided CRISPR / Cas nuclease system, which introduces a double- stranded break in the DNA. The CRISPR / Cas nuclease system comprises a CRISPR / Cas nuclease and a guide RNA. The CRISPR / Casnuclease can be derived from a type I (e.g. IA, IB, IC, ID, IE, or IF), type II (e.g. IIA, MB, or IIC), type I II (i., IMA or 1MB), or type V CRISPR system, which are present in various bacteria and archaea. The CRISPR / Cas system can be from Streptococcus sp. (e.g., Streptococcus pyogenes), Campylobacter sp. (e.g., Campylobacter jejuni), Francisella sp. (e.g., Francisella novicida), Acaryochloris sp., Acetohalobium sp., Acidaminococcus sp., Acidithiobacillus sp., Alicyclobacillus sp., Allochromatium sp., Ammonifex sp., Anabaena sp., Arthrospira sp., Bacillus sp., Burkholderiales sp., Caldicelulosiruptor sp., Candidatus sp., Clostridium sp., Crocosphaera sp., Cyano thece sp., Exiguobacterium sp., Finegoldia sp., Ktedonobacter sp., Lactobacillus sp., Lyngbya sp., Marinobacter sp., Methanohalobium sp., Microscilla sp., Microcoleus sp., Microcystis sp., Natranaerobius sp., Neisseria sp., Nitrosococcus sp., Nocardiopsis sp., Nodularia sp., Nostoc sp., Oscillatoria sp., Polaromonas sp., Pelotomaculum sp., Pseudoalteromonas sp., Petrotoga sp., Prevotella sp., Staphylococcus sp., Streptomyces sp., Streptosporangium sp., Synechococcus sp., or Thermosipho sp. Non-limiting examples of suitable CRISPR nuclease include Cas proteins, Cpf proteins, Cmr proteins, Csa proteins, Csb proteins, Csc proteins, Cse proteins, Csf proteins, Csm proteins, Csn proteins, Csx proteins, Csy proteins, Csz proteins, and derivatives or variants thereof. In some embodiments, the CRISPR / Cas nuclease can be a type II Cas9 protein. The CRISPR / Cas nuclease can further comprise at least one nuclear localization signal or cell-penetrating domain. The CRISPR / Cas nuclease system can also comprise a guide RNA (gRNA). The guide RNA can interact with the CRISPR / Cas nuclease to guide it to a target site in the genomic sequence. The target site can be generic except that the sequence be bordered by a protospacer adjacent motif (PAM). For example, PAM sequences for Cas9 include 3'-NGG, 3'- NGGNG, 3'-NNAGAAW, and 3'-ACAY and PAM sequences for Cpfl include 5'-TTN (wherein N is defined as any nucleotide, W is defined as either A or T, and Y is defined an either C or T). Each gRNA can comprise a sequence that is complementary to the target sequence. The gRNA can also comprise a scaffold sequence that forms a stem loop structure and a single-stranded region. The scaffold region can be the same in every gRNA. In some embodiments, the gRNA can be a single molecule (i.e., sgRNA). In other embodiments, the gRNA can be two separate molecules (i.e., crRNA and tracrRNA). Example Cas sequences are provided in Table 1.

[0043] In conjunction with ZFN or Cas-based integration approaches, a donor oligonucleotide can be utilized to supply cargo designed to be integrated at a stable site in a host cell. In some cases the donor oligonucleotide can comprise from 5' to 3' a first homology arm comprising a sequence of at least 20 nucleotides of a first genomic site 5' to the target deoxyribonucleic acid sequence, a synthetic DNA sequence of at least 10 nucleotides, and a second homology armcomprising a sequence of at least 20 nucleotides of a second genomic site 3' to the target sequence. In some embodiments, the first or second homology arm can comprise a sequence of at least 40, 80, 120, 150, 200, 300, 500, or 1,000 nucleotides.

[0044] By "hybridize" it is generally meant that a nucleic acid (e.g. RNA, DNA) contains a sequence of nucleotides that enables it to non-covalently bind, e.g. form Watson-Crick base pairs or G / U base pairs, “to another nucleic acid in a sequence-specific, antiparallel, manner (e.g., a nucleic acid specifically binds to a complementary nucleic acid) under the appropriate in vitro or in vivo conditions of temperature and solution ionic strength. Standard Watson-Crick basepairing includes: adenine / adenosine) (A) pairing with thymidine / thymidine (T), A pairing with uracil / uridine (U), and guanine / guanosine) (G) pairing with cytosine / cytidine (C). Inosine (I) bases pair with cytosine / cytidine. In addition, for hybridization between two RNA molecules (e.g., dsRNA), and for hybridization of a DNA molecule with an RNA molecule (e.g., when a DNA splint oligo base pairs with an mRNA segment, etc.): G can also base pair with U. For example, G / U base-pairing is partially responsible for the degeneracy (e.g., redundancy) of the genetic code in the context of tRNA anti-codon base-pairing with codons in mRNA. Thus, in the context of this disclosure, a G (e.g., of a protein-binding segment (e.g., dsRNA duplex) of a guide RNA molecule; of a target nucleic acid (e.g., target DNA or RNA) base pairing with a sensor RNA) is considered complementary to both a U and to C. For example, when a G / U basepair can be made at a given nucleotide position of a protein-binding segment (e.g., dsRNA duplex) of a sensor RNA molecule, the position is not considered to be non-complementary, but is instead considered to be complementary. Pseudouridine (pseudo-U, ) is meant to stand for an isomer of uridine, where the uracil nucleobase is attached through a carbon-carbon linkage to the sugar. The pseudouridine is in some cases modified. In some cases the pseudouridine modification is methylation, e.g., at the N1 position, forming N1 -methylpseudouridine. Pseudouridine and its modifications base pair like uridine. When U is shown in a sequence, e.g. UAG, the U may be a pseudouridine. When a T is shown in a sequence, it is meant that the RNA encoded by that sequences contains a U, a pseudouridine, or a modified pseudouridine. The DNA that encodes a U or pseudouridine contains T in place of U or pseudouridine.“Mismatched” generally signifies refers to a base that is opposite a non-complementary base in an otherwise double-stranded structure (e.g., a C:A mismatch), or that a base is opposite no bases (e.g., a base is in a loop structure).

[0045] Hybridization generally requires that the two nucleic acids contain complementary sequences, although mismatches between bases are possible. The conditions appropriate for hybridization between two nucleic acids depend on the length of the nucleic acids and the degreeof complementarity. The greater the degree of complementarity between two nucleotide sequences, the greater the value of the melting temperature (Tm) for hybrids of nucleic acids having those sequences. In some cases, the length for a nucleic acid hybridizing to another nucleic acid is 8 nucleotides or more (e.g., 10 nucleotides or more, 12 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, 22 nucleotides or more, 25 nucleotides or more, or 30 nucleotides or more).

[0046] It is generally understood that the sequence of a polynucleotide need not be 100% complementary to that of its target nucleic acid to hybridize. Moreover, a polynucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure, a ‘bulge’, and the like). A polynucleotide can include 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence complementarity to a target region within the target nucleic acid sequence to which it will hybridize. For example, an antisense nucleic acid in which 18 of 20 nucleotides of the antisense compound are complementary to a target region (e.g. is capable of specific hybridization) would represent 90 percent complementarity. The remaining noncomplementary nucleotides may be clustered or interspersed with complementary nucleotides and need not be contiguous to each other or to complementary nucleotides. Percent complementarity between particular stretches of nucleic acid sequences within nucleic acids can be determined using any convenient method. Example methods include BLAST programs (basic local alignment search tools) and PowerBLAST programs (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656) or by using the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), e.g., using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489).

[0047] The terms "peptide," "polypeptide," and "protein" are generally used interchangeably herein, and refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.

[0048] The term “naturally-occurring” as used herein as applied to a nucleic acid, a protein, a cell, or an organism, generally refers to a nucleic acid, protein, cell, or organism that is found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by a human in the laboratory is naturally occurring.

[0049] The term “exogenous” as used herein as applied to a nucleic acid or a protein generally refers to a nucleic acid or protein that is not normally or naturally found in or produced by a given bacterium, organism, or cell in nature. As used herein, the term “endogenous nucleic acid” refers to a nucleic acid that is normally found in or produced by a given bacterium, organism, or cell in nature. An “endogenous nucleic acid” is also referred to as a “native nucleic acid” or a nucleic acid that is “native” to a given bacterium, organism, or cell. As used herein, the term “endogenous polypeptide” refers to a polypeptide that is normally found in or produced by a given bacterium, organism, or cell in nature.

[0050] “Recombinant,” as used herein, generally signifies that a particular nucleic acid or protein is the product of various combinations of cloning, restriction, or ligation steps resulting in a construct having a structural coding or non-coding sequence distinguishable from endogenous nucleic acids found in natural systems. Generally, DNA sequences encoding the structural coding sequence can be assembled from cDNA fragments and short oligonucleotide linkers, or from a series of synthetic oligonucleotides, to provide a synthetic nucleic acid which is capable of being expressed from a recombinant transcriptional unit contained in a cell or in a cell-free transcription and translation system. Such sequences can be provided in the form of an open reading frame uninterrupted by internal non-translated sequences, or introns, which can be present in eukaryotic genes. Genomic DNA containing the relevant sequences can also be used in the formation of a recombinant gene or transcriptional unit. Sequences of non-translated DNA may be present 5' or 3' from the open reading frame, where such sequences do not interfere with manipulation or expression of the coding regions, and may indeed act to modulate production of a predetermined product by various mechanisms.

[0051] Thus, e.g., the term “recombinant” nucleic acid or “recombinant” protein generally refers to one which is not naturally occurring, e.g., is made by the artificial combination of two otherwise separated segments of sequence through human intervention. This artificial combination is often accomplished by either chemical synthesis methods, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. Such a procedure can be done to replace a codon with a redundant codon encoding the same or a conservative amino acid, while introducing or removing a sequence recognition site. Alternatively, it is performed to join together nucleic acid segments of predetermined functions to generate a predetermined combination of functions. This artificial combination is often accomplished by either chemical synthesis methods, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques.

[0052] “Construct” or “vector” generally refers to a recombinant nucleic acid (in some cases recombinant DNA) which has been generated for the purpose of the expression or propagation of a nucleotide sequence(s) of interest, or is to be used in the construction of other recombinant nucleotide sequences.

[0053] In some examples, a vector is a minicircle, plasmid, nanoplasmid, yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), cosmid, phagemid, bacteriophage genome, or baculovirus genome. Suitable vectors also include vectors derived from bacteriophages or plant, invertebrate, or animal (including human) viruses such as CELiD vectors, doggybone DNA (dbDNA) vectors, closed-end linear duplex DNA vectors (e.g. wherein each end is covalently closed by chemical modification), adeno-associated viral vectors (e.g. AAV1, AAV2, AAV4, AAV5 for CNS or lung, AAV6 for lung or skeletal muscle, AAV7, AAV8, AAV9, or pseudotyped combinations thereof such as AAV2 / 5, AAV2 / 2, AAV-DJ, or AAV-DJ8), retroviral vectors (e.g. MLV or self-inactivating or SIN versions thereof, or pseudotyped versions thereof), herpesviral (e.g. HSV- or EBV-based), lentiviral vectors (e.g. HIV-, FIV-, or EIAV -based, or pseudotyped versions thereof), or adenoviral vectors (e.g. Ad5- based, including replication-deficient, replication-competent, or helper-dependent versions thereof).

[0054] The term “lipid” generally refers to a group of organic compounds that include, but are not limited to, esters of fatty acids and are characterized by being insoluble in water, but soluble in many organic solvents. They can be divided into at least three classes: (1) “simple lipids,” which can include fats and oils as well as waxes; (2) “compound lipids,” which can include phospholipids and glycolipids; and (3) “derived lipids” which can include steroids.

[0055] The term “lipid particle” generally includes a lipid formulation that can be used to deliver an active agent or therapeutic agent, such as a nucleic acid (e.g., an RNA as described herein or a vector encoding the RNA), to a target or predetermined site of interest (e.g., cell, tissue, organ, and the like). In some embodiments, the lipid particle of the invention is a lipid nanoparticle, which can be formed from a cationic lipid, a non-cationic lipid, and optionally a conjugated lipid that prevents aggregation of the particle. In other embodiments, the active agent or therapeutic agent, such as a nucleic acid, may be encapsulated in the lipid portion of the particle, thereby protecting it from enzymatic degradation.

[0056] As used herein, the term “LNP” generally refers to a lipid nanoparticle.An LNP generally represents a particle made from lipids (e.g., a cationic lipid, a non-cationic lipid, and optionally a conjugated lipid that prevents aggregation of the particle), wherein the nucleic acid (e.g., an RNA as described herein, or a or vector encoding the RNA)can be fully encapsulated within the lipid. In certain instances, LNP can be useful for systemic applications, as they can exhibit extended circulation lifetimes following intravenous (i.v.) injection, they can accumulate at distal sites (e.g., sites physically separated from the administration site), and they can mediate silencing of target gene expression at these distal sites. The nucleic acid may be complexed with a condensing agent and encapsulated within an LNP (see e.g. PCT Publication No. WO 00 / 03683, the disclosure of which is herein incorporated by reference in its entirety for all purposes). As described herein, any of the RNAs or vectors encoding the RNAs can be encapsulated within an LNP. When LNPs are utilized, the LNP formulation can comprise a documented LNP formulation, such as: (1) SM-102 (e.g. heptadecane-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate):DSPC (e.g. l,2-Distearoyl-sn-glycero-3-phosphocholine):Cholesterol:DMG-PEG 2000 (e.g. 1 ,2-dimyristoyl- rac-glycero-3-methoxypolyethylene glycol-2000) at 50:10:38.5:1.5 molar ratios; or (2) MC3 (e.g. (6Z,9Z,28Z,31Z)-Heptatriaconta-6,9,28,31-tetraen-19-yl 4- (dimethylamino)butanoate):DSPC:Chol:DMG-PEG 20002000 (e.g. 1 ,2-dimyristoyl-rac-glycero- 3 -methoxypolyethylene glycol-2000) at 50:10:38.5:1.5 molar ratios; or (3) ALC-0315 (e.g. ((4- Hydroxybutyl)azanediyl)bis(hexane-6,l-diyl)bis(2-hexyldecanoate)):DSPC DSPC (e.g. 1,2- Distearoyl-sn-glycero-3-phosphocholine):Cholesterol:ALC-0159 (e.g. PEG-N,N- ditetradecylacetamide) at 46.3:9.4:42.7:1.6 molar ratios.

[0057] As used herein, the term "IVT RNA" generally refers to a nucleic acid molecule encoding a polypeptide sequence to be expressed in a host that can be generated by in vitro transcription and is translatable in a mammalian (and preferably human) cell or subject to produce the polypeptide. In some embodiments, the IVT RNA can be non-coding, e.g. not encoding a polypeptide sequence translatable in a mammalian cell (e.g. not comprising a Kozak sequence, a start codon, a promoter, or an open reading frame). Generating the IVT RNA can be accomplished by any suitable technique (e.g. in vitro transcription in cell lysates from vectors encoding the IVT RNA). The transcribed IVT RNA molecule can be modified further posttranscription, e.g., by adding a cap or other functional group. IVT RNAs can comprise a modified ribonucleic acid to reduce immunogenicity (e.g. in place of some or all of a particular canonical nucleotide, such as uracil). Any of the RNAs described herein (e.g. sensor RNAs, trigger RNAs) can be IVT RNAs.

[0058] The term “transformation” or “transfection” generally refers to a permanent or transient genetic change induced in a cell following introduction of a nucleic acid (e.g., DNA or RNA exogenous to the cell). Genetic change (“modification”) can be accomplished either by incorporation of the new DNA into the genome of the host cell, or by transient or stablemaintenance of the new DNA as an episomal element. Where the cell is a eukaryotic cell, a permanent genetic change is generally achieved by introduction of the DNA into the genome of the cell. Suitable methods of genetic modification include viral infection, transfection, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate precipitation, direct micro injection, and the like. The choice of method is generally dependent on the type of cell being transformed and the circumstances under which the transformation is taking place (e.g., in vitro, ex vivo, or in vivo). A general discussion of these methods can be found in Ausubel et al, Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995.

[0059] "Operably linked" generally refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For instance, a promoter is operably linked to a nucleotide sequence (e.g., a protein coding sequence, e.g., a sequence encoding an mRNA; a non-protein coding sequence, e.g., a sequence encoding a Shh protein; and the like) if the promoter affects its transcription or expression.

[0060] The term “adenosine deaminase acting on RNA” or “ADAR” generally refers to an enzyme that catalyze the hydrolytic C6 deamination of adenosine (A) to produce inosine (I) in RNA substrates that are double stranded. ADARs preferentially edit double stranded RNAs at sites of mismatches where mismatches containing adenosines and cytosines are edited more efficiently than other mismatches. Editing by ADARs results in nucleotide substitution in RNA, because the purine I generated as the result of the deamination reaction is recognized as G instead of A, both by ribosomes during translational decoding of mRNA and by RNA-dependent polymerases during RNA replication. The term “ADAR” encompasses any documented type of ADAR such as AD ARI (ADAR) or ADAR2 (ADARB2).

[0061] As used herein “AD ARI” generally refers to an adenosine deaminase acting on RNA that catalyzes the hydrolytic C6 deamination of adenosine (A) to produce inosine (I) in RNA substrates that are double stranded. ADAR1 has 2 main isoforms, pl 50 and pl 10. The term “AD ARI” encompasses AD ARI from various species. Amino acid sequences of AD ARI from various species are publicly available. See, e.g., GenBank Accession Nos. NP 001102 (Homo sapiens AD ARI pl 50), NP 001180424.1 (Homo sapiens AD ARI pl 10), NP 001139768 (Mus musculus AD ARI pl 50), NP 001033676 (Mus musculus AD ARI pl 10). The term "AD ARI" as used herein also encompasses fragments, fusion proteins, and variants (e.g., variants having one or more amino acid substitutions, addition, deletions, or insertions) that retain AD ARI enzymatic activity.

[0062] As used herein “ADAR2” generally refers to an adenosine deaminase acting on RNA that catalyzes the hydrolytic C6 deamination of adenosine (A) to produce inosine (I) in RNAsubstrates that are double stranded. ADAR2 is exclusively localized to the nucleus. The term “ADAR2” encompasses ADAR2 from various species. Amino acid sequences of ADAR2 from various species are publicly available. See, e.g., GenBank Accession Nos. NP_056648.1 (Homo sapiens ADAR2), NP OO 1020008.1 (Mus musculus ADAR2), ACO52474.1 (Doryteuthis opalescens ADAR2). The term "ADAR2" as used herein also encompasses fragments, fusion proteins, and variants (e.g., variants having one or more amino acid substitutions, addition, deletions, or insertions) that retain ADAR2 enzymatic act.

[0063] The term “editable codon” as used herein generally refers to a 3 -nucleotide sequence that is editable by an ADAR protein or a derivative thereof. The codon may be a start codon, a stop codon or an AUA codon. The codon contains a sequence that contains an adenosine base. In general, in the methods disclosed herein, the editable codon is a start codon that is edited to become a non-start codon, a stop codon that is edited to become a non-stop codon, or a non-start codon (e.g., AUA) that is edited to become a start codon.

[0064] As used herein, the term "transgene" generally refers to a partly heterologous or entirely heterologous nucleic acid sequence that is not present or not fully expressed in the host cell (e.g. mammalian, primate, eukaryotic, or prokaryotic) into which it is introduced. This may include, for example, the case where the target gene is not properly expressed in a mammalian host cell and thus a corrected form of the target gene is introduced as a transgene. Therefore, in some embodiments, the transgene can be a potential therapeutic target gene. The transgene may be from another cell type, or from another species, or may be synthetically produced. Alternatively, the transgene may be obtained from a host cell that is functionally linked to a regulatory region different from that present in the native gene. Alternatively, the transgene may be an allele or variant of a gene present in the host cell. In some embodiments, a transgene can include one or more transcriptional regulatory sequences and any other nucleic acid, such as introns, that may be necessary for optimal expression of a selected nucleic acid.

[0065] The term “pharmaceutically acceptable carrier” is generally intended to denote any material, which is inert in the sense that it substantially does not have a therapeutic and / or prophylactic effect per se. Such an excipient is added with the purpose of making it possible to obtain a pharmaceutical composition having acceptable technical properties. Examples of suitable pharmaceutically acceptable carriers or diluents include, but are not limited to, ethanol, water, glycerol, propylene glycol, glycerin, diethylene glycol monoethylether, vitamin A and E oils, mineral oil, PPG2 myristyl propionate, magnesium carbonate, potassium phosphate, silicon dioxide, vegetable oils such as castor oil and derivatives thereof, plant gums, gelatin, animal oils, solketal, calcium, carbonate, dibasic calcium phosphate, tribasic calcium phosphate, calciumsulfate, microcrystalline cellulose, powdered cellulose, dextrans, dextrin, dextrose, fructose, kaolin, lactose, mannitol, sorbitol, starch, pre-gelatinized starch, sucrose, sugar etc.

[0066] In some embodiments, any of the compositions described herein can be administered intravenously, subcutaneously, intraventricularly, intrathecally, intracerebroventricularly, transdermally, intramuscularly, orally, by inhalation, nasally, rectally, intratumorally, or proxi- tumorally to a subject. Proxi-tumorally may denote administration to the tissue within proximity of a tumor, or administration into a region that would be predicted to be accessible to the tumor via the lymphatic system (e.g. an adjoining lymph node). Intratumoral or proxi-tumoral approaches may involve the use of additional imaging techniques such as e.g. endoscopic ultrasonography (see e.g. Shirley et al. Gastroenterol Res Pract. 2013; 2013: 207129) or via a brochioscope (see e.g. Rojas-Solanoet al. J Bronchology Interv Pulmonol. 2018 Jul; 25(3): 168— 17). In some embodiments, the composition is administered into at least one of the cervical, epitrochlear, supraclavicular, cervical, axillary, mediastinal, supratrochlear, mesenteric, inguinal, femoral, or popliteal lymph nodes. In some cases, lymph-node based administration may serve as a method of centralized local delivery to a tissue region.

[0067] Included in the current disclosure are variants of any of the polypeptides described herein with one or more conservative amino acid substitutions. Such conservative substitutions can be made in the amino acid sequence of a polypeptide without disrupting the three-dimensional structure or function of the polypeptide. Conservative substitutions can be accomplished by substituting amino acids with similar hydrophobicity, polarity, and R chain length for one another. Additionally or alternatively, by comparing aligned sequences of homologous proteins from different species, conservative substitutions can be identified by locating amino acid residues that have been mutated between species (e.g., non-conserved residues) without altering the basic functions of the encoded proteins. Such conservatively substituted variants may include variants with at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about65%, at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99%, or 100% sequence identity to any one of the polypeptide protein sequences described herein.

[0068] In some embodiments, such conservatively substituted variants are functional variants. Such functional variants can encompass sequences with substitutions such that the activity ofcritical active site residues of the endonuclease are not disrupted. In some embodiments, a functional variant of any of the proteins described herein lacks substitution of at least one conserved or functional residue.

[0069] Conservative substitution tables providing functionally similar amino acids are available from a variety of references (see, for e.g., Creighton, Proteins: Structures and Molecular Properties (W H Freeman & Co.; 2nd edition (December 1993), which is incorporated by reference in its entirety herein). The following eight groups each contain amino acids that are conservative substitutions for one another:

[0070] 1) Alanine (A), Glycine (G);

[0071] 2) Aspartic acid (D), Glutamic acid (E);

[0072] 3) Asparagine (N), Glutamine (Q);

[0073] 4) Arginine (R), Lysine (K);

[0074] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

[0075] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

[0076] 7) Serine (S), Threonine (T); and

[0077] 8) Cysteine (C), Methionine (M).

[0078] Percent identity or complementarity between particular stretches of nucleic acid sequences or within nucleic acids described herein can be determined using any convenient method. Example methods include BLAST programs (basic local alignment search tools) and PowerBLAST programs (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656) or by using the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), e.g., using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489).Example Embodiments

[0079] In some aspects, the present disclosure provides for a method for expressing an output protein in a cell, comprising: contacting a cell expressing an RNA with a trigger oligonucleotide, wherein the RNA comprises: (i) a first nucleotide sequence comprising a conditional nucleotide sequence that comprises a region configured to hybridize to said trigger oligonucleotide, wherein said conditional nucleotide sequence comprises a stem-loop sequence comprising one or more editable codons, and (ii) a transgene, a sequence, or an ORF encoding an output protein; wherein said trigger oligonucleotide is a non-natural oligonucleotide; and wherein said trigger oligonucleotide and said first nucleotide sequence are configured such that said triggeroligonucleotide: (a) selectively activates expression of said protein; or (b) selectively inhibits expression of said protein. In some embodiments, said trigger oligonucleotide and said first nucleotide sequence are configured such that said trigger oligonucleotide selectively activates expression of said protein. In some embodiments, said trigger oligonucleotide and said first nucleotide sequence are configured such that said trigger oligonucleotide selectively inhibits expression of said protein. In some embodiments, said cell expresses said RNA from a stable open reading frame (ORF). In some aspects, the present disclosure provides for a method for expressing an output protein in a cell, comprising: contacting a cell expressing an RNA from a stable open reading frame (ORF) with a trigger oligonucleotide, wherein the RNA comprises: (i) a first nucleotide sequence comprising a conditional nucleotide sequence that comprises a region configured to hybridize to said trigger oligonucleotide, wherein said conditional nucleotide sequence comprises a stem-loop sequence comprising one or more editable codons, and (ii) a transgene, a sequence, or an ORF encoding an output protein; wherein said trigger oligonucleotide and said first nucleotide sequence are configured such that said trigger oligonucleotide: (a) selectively activates expression of said protein; or (b) selectively inhibits expression of said protein. In some embodiments, said trigger oligonucleotide and said first nucleotide sequence are configured such that said trigger oligonucleotide selectively inhibits expression of said protein.

[0080] In some aspects, the present disclosure provides for a system comprising: (a) a viral vector configured to transduce a cell or a donor oligonucleotide configured for expression in a cell, encoding an RNA comprising: (i) a first nucleotide sequence comprising a conditional nucleotide sequence that comprises a region configured to hybridize to said trigger oligonucleotide, wherein said conditional nucleotide sequence comprises a stem-loop sequence comprising one or more editable codons, and (ii) a transgene, a sequence, or an ORF encoding an output protein; and (b) a trigger oligonucleotide, wherein said trigger oligonucleotide comprises a synthetic oligonucleotide or an oligonucleotide with at least one non-natural linkage or nonnatural base; and wherein said trigger oligonucleotide and said first nucleotide sequence are configured such that said trigger oligonucleotide: (a) selectively activates expression of said protein; or (b) selectively inhibits expression of said protein. In some embodiments, said trigger oligonucleotide and said first nucleotide sequence are configured such that said trigger oligonucleotide selectively activates expression of said protein. In some embodiments, said trigger oligonucleotide and said first nucleotide sequence are configured such that said trigger oligonucleotide selectively inhibits expression of said protein. In some embodiments, said viralvector is configured to stably transduce a cell, or said donor oligonucleotide is configured for stable integration in a cell.

[0081] In some aspects, the present disclosure provides for a system comprising: (a) a viral vector configured to stably transduce a cell or a donor oligonucleotide configured for stable expression in a cell, encoding an RNA comprising: (i) a first nucleotide sequence comprising a conditional nucleotide sequence that comprises a region configured to hybridize to said trigger oligonucleotide, wherein said conditional nucleotide sequence comprises a stem-loop sequence comprising one or more editable codons, and (ii) a transgene, a sequence, or an ORF encoding an output protein; and (b) a trigger oligonucleotide; wherein said trigger oligonucleotide and said first nucleotide sequence are configured such that said trigger oligonucleotide: (a) selectively activates expression of said protein; or (b) selectively inhibits expression of said protein. In some embodiments, said trigger oligonucleotide and said first nucleotide sequence are configured such that said trigger oligonucleotide selectively activates expression of said protein. In some embodiments, said trigger oligonucleotide and said first nucleotide sequence are configured such that said trigger oligonucleotide selectively inhibits expression of said protein.

[0082] In some aspects, the present disclosure provides for a system comprising: (a) a cell stably encoding an RNA, wherein said RNA comprises: (i) a first nucleotide sequence comprising a conditional nucleotide sequence that comprises a region configured to hybridize to said trigger oligonucleotide, wherein said conditional nucleotide sequence comprises a stem-loop sequence comprising one or more editable codons, and (ii) a transgene, a sequence, or an ORF encoding an output protein; and (b) a trigger oligonucleotide; wherein said trigger oligonucleotide and said first nucleotide sequence are configured such that said trigger oligonucleotide: (a) selectively activates expression of said protein; or (b) selectively inhibits expression of said protein. In some embodiments, said trigger oligonucleotide and said first nucleotide sequence are configured such that said trigger oligonucleotide selectively activates expression of said protein. In some embodiments, said trigger oligonucleotide and said first nucleotide sequence are configured such that said trigger oligonucleotide selectively inhibits expression of said protein. In some embodiments, said trigger oligonucleotide is a non-natural oligonucleotide. In some embodiments, said trigger oligonucleotide comprises a synthetic oligonucleotide or an oligonucleotide with at least one non-natural linkage or non-natural base.

[0083] In some aspects, the present disclosure provides for a system comprising; (a) a cell stably encoding an RNA, a viral vector configured to encode an RNA, or a donor DNA configured to encode an RNA, wherein said RNA comprises: (i) a first nucleotide sequence comprising a conditional nucleotide sequence that comprises a region configured to hybridize to said triggeroligonucleotide, wherein said region configured to hybridize to said trigger oligonucleotide comprises at least one editable codon, and (ii) a transgene, a sequence, or an ORF encoding an output protein; wherein said trigger oligonucleotide and said first nucleotide sequence are configured such that said trigger oligonucleotide: (a) selectively activates expression of said protein; or (b) selectively inhibits expression of said protein; wherein: (i) said region configured to hybridize to said trigger oligonucleotide is outside of an open reading frame (ORF) encoding said output protein; or (ii) said editable codon is not a stop codon. In some embodiments, said trigger oligonucleotide and said first nucleotide sequence are configured such that said trigger oligonucleotide selectively activates expression of said protein. In some embodiments, said trigger oligonucleotide and said first nucleotide sequence are configured such that said trigger oligonucleotide selectively inhibits expression of said protein. In some embodiments, said region configured to hybridize to said trigger oligonucleotide is outside of an open reading frame (ORF) encoding said output protein. In some embodiments, said editable codon is not a stop codon.

[0084] In some aspects, the present disclosure provides for a system comprising: (a) a viral vector configured to encode an RNA and stably transduce a cell, or a donor DNA configured to encode an RNA and stably integrate in a cell, wherein said RNA comprises: (i) a first nucleotide sequence comprising a conditional nucleotide sequence that comprises a region configured to hybridize to said trigger oligonucleotide, wherein said region configured to hybridize to said trigger oligonucleotide comprises at least one editable codon, and (ii) a transgene, a sequence, or an ORF encoding an output protein; wherein said trigger oligonucleotide and said first nucleotide sequence are configured such that said trigger oligonucleotide: (a) selectively activates expression of said protein; or (b) selectively inhibits expression of said protein; wherein: (i) said region configured to hybridize to said trigger oligonucleotide is outside of an open reading frame (ORF) encoding said output protein; or (ii) said editable codon is not a stop codon. In some embodiments, said trigger oligonucleotide and said first nucleotide sequence are configured such that said trigger oligonucleotide selectively activates expression of said protein. In some embodiments, said trigger oligonucleotide and said first nucleotide sequence are configured such that said trigger oligonucleotide selectively inhibits expression of said protein. In some embodiments, said region configured to hybridize to said trigger oligonucleotide is outside of an open reading frame (ORF) encoding said output protein. In some embodiments, said editable codon is not a stop codon.

[0085] The trigger oligonucleotide can have a variety of features, and be introduced to the cell in a variety of ways. In some embodiments, the trigger oligonucleotide is or can comprise RNA.In some embodiments, said trigger oligonucleotide can be a non-natural oligonucleotide. In some embodiments, said trigger oligonucleotide comprises a synthetic oligonucleotide or an oligonucleotide with at least one non-natural linkage or non-natural base. In some embodiments, said trigger oligonucleotide comprises a non-natural sequence. In some embodiments, said trigger oligonucleotide: (i) comprises a sequence having 50% or less identity to a genomic sequence of said cell or a natural genomic sequence; (ii) does not comprise a stretch of greater than 30 bases identical to a genomic sequence of said cell, or (iii) does not comprise a stretch of bases with 90% or more identity to a genomic sequence of said cell, wherein said stretch is greater than about 30 bases in length. In some embodiments, a non-natural sequence can comprise a sequence with about 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, or 1% or less sequence identity to a genomic sequence of said cell. In some embodiments, a non-natural sequence can comprise a sequence with about 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, or 1% or less sequence identity to natural sequence. In some embodiments, a non-natural sequence can have about 60 or fewer, about 55 or fewer, about 50 or fewer, about 45 or fewer, about 40 or fewer, about 35 or fewer, about 30 or fewer, about 25 or fewer, about 20 or fewer, about 15 or fewer, or about 10 or fewer consecutive bases identical to a genomic sequence of said cell. In some embodiments, said trigger nucleotide is or comprises at least about 30, at least about 35, at least about 40, at least about 45, at least about 46, at least about 47, at least about 48, at least about 49, at least about 50, at least about 51, at least about 52, at least about 53, at least about 54, at least about 55, at least about 56, at least about 57, at least about 58, at least about 59, at least about 60, at least about 61, at least about 62, at least about 63, at least about 64, at least about 65, at least about 66, at least about 67, at least about 68, at least about 69, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, at least about 100, at least about 110, at least about 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 190, or at least about 200 nucleotides in length, or any range between these values. In some embodiments, the trigger RNA is or comprises about 180 nucleotides to about 360 nucleotides in length. In some embodiments, the trigger RNA is or comprises at most 360 or at most 500 nucleotide in length. In some embodiments, said trigger oligonucleotide is a circular oligonucleotide (e.g. circular RNA). In some embodiments the said trigger oligonucleotide is not a circular oligonucleotide (e.g. not a circular RNA). In some embodiments, said trigger oligonucleotide is exogenous to said cell. In some embodiments, said trigger oligonucleotide comprises an antisense oligonucleotide (ASO). In some embodiments,said contacting comprises contacting said trigger RNA packaged in an LNP to said cell expressing said RNA. In some embodiments, said contacting comprises contacting a GalNAc- conjugated RNA to said cell expressing said RNA. In some embodiments, said contacting comprises contacting a trigger RNA comprising at least one non-natural nucleotide to said cell expressing said RNA.

[0086] The region configured to hybridize to said trigger oligonucleotide can be a variety of lengths. In some embodiments, the region is or comprises 8 nucleotides or more (e.g., 10 nucleotides or more, 12 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, 22 nucleotides or more, 25 nucleotides or more, 30 nucleotides or more, 35 nucleotides or more, 40 nucleotides or more, 45 nucleotides or more, 50 nucleotides or more, 55 nucleotides or more, 60 nucleotides or more, 65 nucleotides or more, 70 nucleotides or more, 75 nucleotides or more, 80 nucleotides or more, 85 nucleotides or more, 90 nucleotides or more, 95 nucleotides or more, or100 nucleotides or more, or any range between these values). In some embodiments, the region configured to hybridize to said trigger oligonucleotide is the same length as the trigger oligonucleotide. In some embodiments, said region configured to hybridize to said trigger oligonucleotide comprises an amount of sequence complementarity sufficient to permit hybridization to said target RNA. In some embodiments, said region configured to hybridize to said trigger oligonucleotide comprises at least 60% complementarity to said trigger RNA. In some embodiments, said region configured to hybridize to said trigger oligonucleotide comprises at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or 100% complementarity to the trigger RNA, or any range between these values. In some embodiments, said region configured to hybridize to said trigger oligonucleotide and said stemloop sequence comprising one or more editable codons are non-overlapping.

[0087] The cell can be a variety of types and comprise a variety of origins. In some embodiments, the target cell is a cell that is in a particular disease state. The cell may be in any disease state. In some embodiments, the target cell is a particular cell type. Non-limiting examples of candidate cell types include connective tissue elements such as fibroblast, skeletal tissue (bone and cartilage), skeletal, cardiac and smooth muscle, epithelial tissues (e.g., liver, lung, breast, skin, bladder and kidney), neural cells (glia and neurons), endocrine cells (adrenal, pituitary, pancreatic islet cells), bone marrow cells, melanocytes, and many different types ofhematopoietic cells. Suitable cells can also be cells representative of a specific body tissue from a subject. The types of body tissues include, but are not limited, to blood, muscle, nerve, brain, heart, lung, liver, pancreas, spleen, thymus, esophagus, stomach, intestine, kidney, testis, ovary, hair, skin, bone, breast, uterus, bladder, spinal cord and various kinds of body fluids. In some embodiments, said cell comprises a T-cell or a precursor thereof, a T-reg cell or a precursor thereof, a natural killer T-cell or a precursor thereof, an NK-cell or a precursor thereof, a hepatocyte or a precursor thereof, a retinal pigment epithelial cell or a precursor thereof, a neuron or a precursor thereof, an epithelial cell or a precursor thereof, a retinal cell or a precursor thereof, a CHO cell, a HEK293 cell, a NSO murine myeloma cell, a PER.C6 cell, or an Sp2 / 0 cell. In some embodiments, said cell comprises a T-cell, a T-reg cell, a natural killer T-cell, an NK-cell, or a precursor thereof. Cells suitable for use in a subject method include cells of a variety of subject hosts. Generally, such subject hosts are “mammals” or “mammalian”, where these terms are used broadly to describe organisms which are within the class mammalia, including the orders carnivore (e.g., dogs and cats), rodentia (e.g., mice, guinea pigs and rats), and primates (e.g., humans, chimpanzees and monkeys). In many aspects, the subject host will be a human. In certain embodiments, the subject host is a plant. In some embodiments, said cell comprises an adenosine deaminase acting on RNA (ADAR) protein or a coding sequence encoding thereof. In some embodiments, the cell further comprises a small-molecule (e.g. theophylline, tetracycline, or tocamuycin) or RNA-binding riboswitch operably linked to said sequence encoding said protein as described e.g. in Eriksson et al. Front Med (Lausanne). 2022 Dec 13:9:1052318. doi: 10.3389 / fined.2022.1052318. eCollection 2022, which is incorporated by reference herein in its entirety.

[0088] The editable codon can generally be a 3 -nucleotide sequence that is editable by an ADAR protein or a derivative thereof. The codon may be a start codon, a stop codon or an AUA codon. The codon contains a sequence that contains an adenosine base. In general, in the methods disclosed herein, the editable codon is a start codon that is edited to become a non-start codon, a stop codon that is edited to become a non-stop codon, or a non-start codon (e.g., AUA) that is edited to become a start codon. When the editable codon is within a stem-loop, the stem loop can comprise at least 1 nucleotide opposite the editable codon that is mispaired. When the editable codon is not within a stem-loop, the trigger oligonucleotide can comprise at least 1 nucleotide opposite the editable codon that is mispaired. In some embodiments, said one or more editable codons comprise: (i) 5'-UAG, 5'-UGA, or 5'-UAA; (ii) 5'-AUG-3', or 5'-AUA-3'. In some embodiments, said one or more editable codons comprise: (i) 5'-AUG-3'; or (ii) a SLAVAS' non-start codon. In some embodiments, said one or more editable codons are in a stemsequence of a stem-loop sequence. In some embodiments, said one or more editable codons comprise at least one base that is mismatched with a sequence opposite said one or more editable codons. In some embodiments, an ORF or transgene encoding said protein is in frame with said one or more editable codons.

[0089] The stem loop can comprise a variety of lengths and structures. In some embodiments, the stem-loop is configured to bind ADAR (e.g. AD ARI or ADAR2). In some embodiments, the stem-loop is not configured to bind a Cas enzyme. In some embodiments, said stem-loop sequence comprises a sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or 100% sequence identity to any one of SEQ ID NOs: 5, 6, 7, 8, 17, 18, or 19. In some embodiments, the stem-loop sequence comprises a sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or 100% sequence identity to any of the stemloops in Table 1, a reverse complement thereof, or an RNA cognate thereof. In some embodiments, said one or more editable codons further comprises a non-stop, non-start codon that is edited to become a start codon by said target cell. In some embodiments, a stem of said stem-loop is at least 12 base pairs in length. For example, the stem-loop may be 50 bp or less, 45 bp or less, 40 bp or less, 35 bp or less, 30 bp or less, 25 bp or less, or 20 bp or less. In some embodiments, the length of the stem-loop can be 18-50 bps. In some embodiments, said stemloop sequence further comprises a Kozak sequence operably linked to said non-stop, non-start codon. In some embodiments, said non-stop, non-start codon further comprises 5'-AUA -3'. In some embodiments, said one or more editable codons further comprises a start codon and said stem-loop sequence further comprises a Kozak sequence operably linked to said start codon. In some embodiments, said region configured to hybridize to said trigger oligonucleotide and said stem-loop sequence comprising one or more editable codons are non-overlapping.

[0090] The transgene or sequence encoding the output protein can be a partly heterologous or entirely heterologous nucleic acid sequence that is not present or not fully expressed in the host cell (e.g. mammalian, primate, eukaryotic, or prokaryotic) into which it is introduced. In some cases, the output protein may be from another cell type, or from another species, or may besynthetically produced. In some cases, the output protein comprises a sequence of a therapeutic protein. In some embodiments, said protein comprises a toxin, killing factor, a T-cell receptor, a chimeric antigen receptor, a fluorescent protein, a genomic modification protein, a transcription factor, an antigen, an antibody or a functional fragment thereof, a therapeutic protein, an enzyme, a hormone, soluble receptor, an extracellular domain of a hormone receptor, an extracellular domain of a VEGF receptor, an interleukin, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11, caspase 12, caspase 13, p53 upregulated modulator of apoptosis (PUMA), Bakl, iCasp9, ricin toxin chain A, diphtheria toxin, IL- 18, IL-23 -alpha, IL-15, IL-12-alpha, IL-12-beta, an anti-IL-18 antibody, an anti-IL-lOR antibody, an anti-CSFIR antibody, tocilizumab, anakinra, IL-7, IL-2R-beta, CCL19, IL-36- gamma, IL-37, or FOXO1, or any combination thereof. In some embodiments when said cell comprises a T-cell, a T-reg cell, a natural killer T-cell, or a precursor thereof, said output protein further comprises an interleukin, IL- 18, IL-23 -alpha, IL-15, IL-12-alpha, IL-12-beta, an anti-IL- 18 antibody, an anti-IL-lOR antibody, an anti-CSFIR antibody, tocilizumab, anakinra, IL-7, IL- 2R-beta, CCL19, IL-36-gamma, IL-37, FOXO1, or a chimeric antigen receptor, or any combination thereof. In some embodiments, said protein further comprises an anti-tumor antibody or fragment thereof. In some embodiments, said protein comprises any of the proteins described in Table 1. In some embodiments, an ORF or transgene encoding said protein is in frame with said one or more editable codons.

[0091] The RNA (e.g. the RNA encoding the first nucleotide sequence and / or the transgene) can be expressed by a variety of methods. In some embodiments, the RNA is expressed via a genomically integrated copy of a sequence encoding the RNA or is transcribed from a genomic locus of said cell. In some embodiments, the RNA is expressed via an episomal copy of a sequence encoding the RNA or is transcribed from an episomal locus of said cell. In some embodiments, said RNA is transcribed from a vector introduced to said cell prior to said contacting in said cell said RNA with said trigger oligonucleotide. Thus, in some cases, the cell is contacted with a viral vector to integrate a sequence encoding the RNA prior to contacting with the trigger oligonucleotide. In some embodiments, the method further comprises integrating a sequence encoding said RNA into a genomic locus. In some cases, the cell is contacted with a donor oligonucleotide encoding said RNA to produce said cell expressing said RNA. When a donor nucleotide is utilized, a variety of methods can be used to integrate the sequence from the donor oligonucleotide encoding the RNA to a genomic locus of the cell. In some embodiments where a donor oligonucleotide is utilized, a Cas protein, and a guide RNA (e.g. targeting a specific integration site) compatible with said Cas protein are contacted to saidcell produce said cell expressing said RNA. When Cas protein and guide RNA are used, the donor oligonucleotide can further comprise a homology-directed repair template. In some cases the donor oligonucleotide can comprise from 5' to 3' a first homology arm comprising a sequence of at least 20 nucleotides of a first genomic site 5' to the target deoxyribonucleic acid sequence, a synthetic DNA sequence of at least 10 nucleotides, and a second homology arm comprising a sequence of at least 20 nucleotides of a second genomic site 3' to the target sequence. In some embodiments, the first or second homology arm can comprise a sequence of at least 40, 80, 120, 150, 200, 300, 500, or 1,000 nucleotides. In some embodiments where a donor oligonucleotide is utilized, a PiggyBac transposase, a Bxbl integrase, a PhiC31 integrase, or a recombinase (e.g. a serine recombinase) are contacted to the cell alongside the donor oligonucleotide (e.g. wherein the donor oligonucleotide comprises flanking sequences compatible with the PiggyBac transposase, Bxbl integrase, recombinase, or serine recombinase). In some embodiments, said cell further comprises an promoter operably linked to said open reading frame (ORF) encoding said RNA. In some embodiments, said cell further comprises a small-molecule or RNA-binding riboswitch operably linked to said sequence encoding said protein. In some embodiments, said promoter further comprises an inducible promoter. In some embodiments, said promoter comprises a CMV promoter, an SFFV promoter, a CAG promoter, a PGK promoter, an EFla promoter, a JeT promoter, an A2UCOE promoter, a CMV-tetO tetracycline responsive promoter, or a UAS promoter. In some embodiments, the method further comprises contacting said cell with a first viral vector encoding said RNA; further comprising contacting said cell with a second viral vector encoding said trigger RNA, wherein said first and said second viral vector comprise different viral serotypes with overlapping tissue tropism. In some embodiments, said first viral vector and said second viral vector comprise nonoverlapping selections from one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV8, or AAV9.

[0092] In some aspects, the present disclosure provides for a method of controlling expression of a protein in a cell, comprising contacting the components any of the systems described herein to a cell or utilizing the components of any of the systems described herein in vitro or in vivo. In some embodiments, the method comprises sequentially contacting the components of the system to said cell. In some embodiments, the components of the system are contacted at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days or 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months apart.

[0093] In some aspects, the present disclosure provides for a nucleic acid molecule comprising or encoding any of the sequences, identified sub-elements, or any combination thereof described in Table 1, or a sequence having at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or 100% sequence identity to any of the sequences, identified sub-elements, or combinations thereof described in Table 1.Table 1: Sequences of Genes and Components Described HereinEXAMPLESExample 1. -Control of transgenes via exogenous RNA

[0094] It was hypothesized that exogenous RNAs (rather than endogenous or intracellular RNAs) could serve as a trigger for regulation of endogenous transgenes introduced by stable transfection methods. To test this, 3rdgeneration lentiviruses with puromycin selection cassetteswere constructed having an open reading frame (ORF) comprising an RNA sensor construct under a CMV promoter, and bearing mCherry in-frame upstream of the RNA sensor construct and GFP in-frame downstream of the RNA sensor construct. A first version (whole construct, SEQ ID NO: 1) of the lentivirus was constructed having an RNA sensor construct bearing hybridization regions to a trigger RNA on either side of a stem loop containing a stop codon (“SensA”, SEQ ID NO: 5), and a second version of the lentivirus (whole construct, SEQ ID NO: 3) was constructed having an RNA sensor construct bearing a stop codon in the middle of a single hybridization region to a trigger RNA (“SensB”, SEQ ID NO: 7). Additionally, a plasmid (functional sequences shown in SEQ ID NO: 11) encoding an exogenous trigger RNA in the 3'UTR of BFP was constructed. In vitro transcribed (IVT) mRNA (SEQ ID NO: 12) was prepared from the plasmid using T7 RNA polymerase (Areterna), RNA clean up (NEB Monarch RNA Clean Up kit). The “trigger” sequence in the trigger RNA was randomly generated and evaluated for similarity with the human genome, discarding sequences with any match as evaluated with BLAST.

[0095] To test exogenous regulation of SensA and SensB containing constructs, 293T cells were first transfected with both the SensA and SensB lentiviruses at a multiplicity of infection (MOI) of 10, following by puromycin selection at Ipg / mL for approximately 2 weeks starting 24-26 hours after lentivirus transduction.

[0096] After lentivirus transduction and puromycin selection, cells that were either (a) WT 293 T cells; (b) 293T cells treated with the SensA lentiviral construct; or (c) 293T cells treated with the or SensB lentiviral construct were treated with: (i) 500 ng IVT mRNA bearing a region complementary to the hybridization region in the RNA sensor (SEQ ID NO: 12) in complex with transfection agent (liposomal, Lipofectamine MessengerMax, Thermo Fisher); (ii) 500 ng IVT mRNA bearing a region not complementary to the hybridization region in the RNA sensor (SEQ ID NO: 10) in complex with transfection agent (liposomal, Lipofectamine MessengerMax, Thermo Fisher); (iii) 500ng plasmid DNA encoding a region complementary to the hybridization region in the RNA sensor (SEQ ID NO: 11) and not encoding ADAR in complex with jetOPTIMUS® (Polyplus); (iv) 500ng plasmid DNA encoding a region not complementary to the hybridization region in the RNA sensor (SEQ ID NO: 9) and not encoding ADAR in complex with jetOPTIMUS® (Polyplus); (v) 500ng plasmid DNA encoding a region complementary to the hybridization region in the RNA sensor (SEQ ID NO: 11) and further 5ng plasmid DNA encoding ADAR in complex with jetOPTIMUS® (Polyplus); and (vi) 500ng plasmid DNA encoding a region not complementary to the hybridization region in the RNAsensor (SEQ ID NO: 9) and not further 5ng plasmid DNA encoding ADAR in complex with jetOPTIMUS® (Polyplus).

[0097] The results of the experiment for each type of cell (WT 293T, SensA-treated, or SensB- treated) are shown in FIG. 2A (flow cytometry of GFP vs. mCherry fluorescence for condition (i)), FIG. 2B (flow cytometry of GFP vs. mCherry fluorescence for condition (ii)), FIG. 2C (flow cytometry of GFP vs. mCherry fluorescence for condition (iii)), FIG. 2D (flow cytometry of GFP vs. mCherry fluorescence for condition (iv)); FIG. 2E (flow cytometry of GFP vs. mCherry fluorescence for condition (v)); FIG. 2F (flow cytometry of GFP vs. mCherry fluorescence for condition (vi)); and FIG. 2G (quantitation of the flow cytometry results of FIGs. 2A, 2B, 2C, 2D, 2E, and 2F). The results indicate stable 293T cells expressing sensor constructs demonstrate an increased level of GFP+ (e.g. output protein-positive) cells upon transfection of either mRNA (FIG. 2A and FIG. 2B), or plasmid DNA encoding mRNA (FIGs. 2C, 2D, 2E, and 2F) corresponding to the matching trigger (indicating that the trigger drove expression of the integrated output protein) when compared to a non-matching trigger control by flow analysis gated on the trigger or non-matching trigger transfected cells (indicating that the non-matching trigger did not drive expression of the integrated output protein). For the experiments in FIGs. 2A, 2B, 2C, 2D, 2E, and 2F BFP signal was used to gate on the presence of either matching or non-matching trigger in the cells so the analysis was restricted to the cells where transfection of all the desired elements was successful. Quantitation of the flow cytometry results (FIG. 2G) shows increased levels of GFP+ (e.g. output protein-positive) cells in the case that the trigger matches compared to the case when there is no matching trigger, and that the proportion of GFP+ cells is further increased in the presence of extra ADAR.Example 2.- (Prophetic) Control of transgenes via exogenous RNA in vivo

[0098] An AAV or another viral vector is constructed encoding a transgene under the control of an RNA sensor construct (e.g. a SensA or SensB construct, or any of the other RNA sensor constructs described herein) bearing an editable stop codon upstream from a transgene (“default off’) or an editable start codon upstream from a transgene (“default on”), which transgene can be a therapeutic transgene (e.g. Factor IX in the case of hemophilia B). An analogous negative control AAV is also generated not having the RNA sensor construct. A trigger sequence is randomly generated and evaluated for similarity with the human genome, discarding sequences with more than 30 matches as evaluated with BLAST. The trigger sequence is produced as an RNA (which can be a fully natural RNA, include non-natural nucleotides such as LNAs forstability, or be circular, or any combination of these). The trigger RNA can be in vitro transcribed or fully synthetic.

[0099] The AAV or another viral vector is administered to the subject(s). After a suitable amount of time to allow for stabilization (episomal stabilization in the case of AAV, integration in the case of integrating viral vectors) in the subject(s) (e.g. 2 weeks), expression of the transgene (e.g. Factor IX in the case of hemophilia B) is assessed by an ELISA assay for the transgene on a sample of peripheral blood to verify no transgene expression in the case of “default off’ designs or verify transgene expression in the case of “default on” designs.

[0100] After verifying transgene expression / no expression (e.g. 3 days later), an LNP formulation of the trigger RNA is administered to the subject(s). A negative control LNP bearing a marker RNA (e.g. luciferase, GFP, etc) is also administered to subject(s).

[0101] At various timepoints post-administration of the trigger LNP and negative control LNP, expression of the transgene is assessed in blood, liver, or other target organs of the subject(s), and transcriptome sequencing is performed on RNA from the subject(s) to verify editing of the mRNA encoded by the AAV.

[0102] Based on the observations post-administration of the LNPs, the dose of the trigger LNP can be adjusted to upregulate (in the case of “default off’ RNA sensor construct designs) or downregulate (in the case of “default on” RNA sensor designs) to a desired phenotypic range.

[0103] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A method for expressing an output protein in a cell, comprising: contacting a cell expressing an RNA with a trigger oligonucleotide, wherein the RNA comprises:(i) a first nucleotide sequence comprising a conditional nucleotide sequence that comprises a region configured to hybridize to said trigger oligonucleotide, wherein said conditional nucleotide sequence comprises a stem-loop sequence comprising one or more editable codons, and(ii) a transgene encoding an output protein; wherein said trigger oligonucleotide is a non-natural oligonucleotide; and wherein said trigger oligonucleotide and said first nucleotide sequence are configured such that said trigger oligonucleotide:(a) selectively activates expression of said protein; or(b) selectively inhibits expression of said protein.

2. The method of claim 1, wherein said cell expresses said RNA from a stable open reading frame (ORF).

3. A method for expressing an output protein in a cell, comprising: contacting a cell expressing an RNA from a stable open reading frame (ORF) with a trigger oligonucleotide, wherein the RNA comprises:(i) a first nucleotide sequence comprising a conditional nucleotide sequence that comprises a region configured to hybridize to said trigger oligonucleotide, wherein said conditional nucleotide sequence comprises a stem-loop sequence comprising one or more editable codons, and(ii) a transgene encoding an output protein; wherein said trigger oligonucleotide and said first nucleotide sequence are configured such that said trigger oligonucleotide:(a) selectively activates expression of said protein; or(b) selectively inhibits expression of said protein.

4. The method of claim 3, wherein said trigger oligonucleotide is a non-natural oligonucleotide.

5. The method of any one of claims 1-4, wherein said trigger oligonucleotide comprises a synthetic oligonucleotide or an oligonucleotide with at least one non-natural linkage or nonnatural base.

6. The method of any one of claims 1-5, wherein said trigger oligonucleotide comprises a non-natural sequence.

7. The method of any one of claims 1-6, wherein said trigger oligonucleotide is not a circular oligonucleotide.

8. The method any one of claims 1-7, wherein said stem-loop is not configured to bind a Cas protein.

9. The method of any one of claims 1-8, wherein said RNA is transcribed from a vector introduced to said cell prior to said contacting in said cell said RNA with said trigger oligonucleotide.

10. The method of any one of claims 1-9, wherein said RNA is transcribed from a genomic or episomal locus of said cell.

11. The method of any one of claims 1-10, wherein said trigger oligonucleotide is an in vitro transcribed (IVT) RNA or a synthetic RNA.

12. The method of any one of claims 1-11, further comprising integrating a sequence encoding said RNA into a genomic locus.

13. The method of any one of claims 1-12, wherein expression of said output protein is selectively activated in said cell in the presence of said trigger RNA.

14. The method of claim 13, wherein said one or more editable codons comprise: (i) 5'-UAG, 5'-UGA, or 5'-UAA; (ii) 5'- AUG-3', or 5'-AUA-3'.

15. The method of any one of claims 1-14, wherein expression of said output protein is selectively inhibited in said cell in the presence of said trigger RNA.

16. The method of claim 15, wherein said one or more editable codons comprise: (i) 5'-AUG- 3'; or (ii) a 5'-AUA-3' non-start codon.

17. The method of any one of claims 1-16, wherein said output protein comprises a therapeutic protein.

18. The method of any one of claims 1-17, wherein said region configured to hybridize to said trigger oligonucleotide comprises an amount of sequence complementarity sufficient to permit hybridization to said target RNA.

19. The method of any one of claims 1-18, wherein said region configured to hybridize to said trigger oligonucleotide comprises at least 60% complementarity to said trigger RNA.

20. The method of any one of claims 1-19, wherein said one or more editable codons further comprises a start codon.

21. The method of any one of claims 1-20, wherein said stem-loop sequence further comprises a Kozak sequence operably linked to said start codon.

22. The method of any one of claims 1-21, wherein said one or more editable codons further comprises a non-stop, non-start codon that is edited to become a start codon by said target cell.

23. The method of claim 22, wherein said stem-loop sequence further comprises a Kozak sequence operably linked to said non-stop, non-start codon.

24. The method of claim 22 or 23, wherein said non-stop, non-start codon further comprises 5'- AU A -y.

25. The method of any one of claims 1-24, wherein said protein comprises a toxin, killing factor, a T-cell receptor, a chimeric antigen receptor, a fluorescent protein, a genomic modification protein, a transcription factor, an antigen, an antibody or a functional fragment thereof, a therapeutic protein, or an enzyme.

26. The method of any one of claims 1-25, wherein said cell expressing said RNA comprises an adenosine deaminase acting on RNA (ADAR) protein or a coding sequence encoding thereof.

27. The method of any one of claims 1-26, wherein said region configured to hybridize to said trigger oligonucleotide and said stem-loop sequence comprising one or more editable codons are non-overlapping.

28. The method of any one of claims 1-27, wherein said one or more editable codons are in a stem sequence of said stem-loop sequence.

29. The method of any one of claims 1-28, wherein said one or more editable codons comprise at least one base that is mismatched with a sequence within the stem-loop opposite said one or more editable codons.

30. The method of any one of claims 1-29, wherein an ORF encoding said protein is in frame with said one or more editable codons.

31. The method of any one of claims 1-30, wherein a stem of said stem-loop is at least 12 base pairs in length.

32. The method of any one of claims 1-31, wherein said trigger oligonucleotide: (i) comprises a sequence having 50% or less identity to a genomic sequence of said cell; (ii) does not comprise a stretch of bases with 90% or more identity to a genomic sequence of said cell, wherein said stretch is greater than about 30 bases in length.

33. The method of any one of claims 1-32, wherein said trigger oligonucleotide is or comprises at least 54 nucleotides in length.

34. The method of any one of claims 1-33, wherein said contacting comprises combining said cell expressing said RNA with a viral vector encoding said trigger oligonucleotide.

35. The method of any one of claims 1-34, wherein said trigger oligonucleotide is exogenous to said cell.

36. The method of any one of claims 1-35, wherein said trigger oligonucleotide comprises an antisense oligonucleotide (ASO).

37. The method of any one of claims 1-36, wherein said contacting comprises contacting said trigger oligonucleotide packaged in an LNP to said cell expressing said RNA.

38. The method of any one of claims 1-37, wherein said contacting comprises contacting a GalNAc-conjugated RNA to said cell expressing said RNA.

39. The method of any one of claims 1-38, wherein said contacting comprises contacting a trigger RNA comprising at least one non-natural nucleotide to said cell expressing said RNA.

40. The method of any one of claims 1-39, further comprising contacting said cell with a viral vector encoding said RNA prior to said contacting with said trigger RNA to produce said cell expressing said RNA.

41. The method of any one of claims 1-39, further comprising contacting said cell with a donor oligonucleotide encoding said RNA to produce said cell expressing said RNA.

42. The method of any one of claims 1-39, further comprising contacting said cell with a donor oligonucleotide encoding said RNA, a Cas protein, and a guide RNA compatible with said Cas protein to produce said cell expressing said RNA.

43. The method of any one of claims 1-42, further comprising contacting said cell with a donor oligonucleotide encoding said RNA and a PiggyBac transposase, a Bxbl integrase, a PhiC or PhiC31 integrase, or a recombinase.

44. The method of any one of claims 41-43, wherein said donor oligonucleotide further comprises a homology-directed repair template.

45. The method of any one of claims 1-44, wherein said cell further comprises an open reading frame (ORF) encoding said RNA.

46. The method of claim 45, wherein said cell further comprises a promoter operably linked to said open reading frame (ORF) encoding said RNA.

47. The method of any one of claims 1-46, wherein said cell further comprises a smallmolecule or RNA-binding riboswitch operably linked to said sequence encoding said protein.

48. The method of any one of claims 46-47, wherein said promoter further comprises an inducible promoter.

49. The method of any one of claims 46-48, wherein said promoter comprises a CMV promoter, a CMV-tetO tetracycline responsive promoter, or a UAS promoter.

50. The method of any one of claims 1-49, wherein said cell comprises a T-cell or a precursor thereof, a T-reg cell or a precursor thereof, a natural killer T-cell or a precursor thereof, an NK-cell or a precursor thereof, a hepatocyte or a precursor thereof, a retinal pigment epithelial cell or a precursor thereof, a neuron or a precursor thereof, an epithelial cell or a precursor thereof, a retinal cell or a precursor thereof, a CHO cell, a HEK293 cell, a NSO murine myeloma cell, a PER.C6 cell, or an Sp2 / 0 cell.

51. The method of any one of claims 1-49, wherein said cell comprises a T-cell, a T-reg cell, a natural killer T-cell, an NK-cell, or a precursor thereof.

52. The method of any one of claims 1-51, wherein said output protein further comprises a hormone, an antibody, a chimeric antigen receptor (CAR), a soluble receptor, an extracellular domain of a hormone receptor, an extracellular domain of a VEGF receptor, an interleukin, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11, caspase 12, caspase 13, p53 upregulated modulator of apoptosis (PUMA), Bakl, iCasp9, ricin toxin chain A, diphtheria toxin, IL-18, IL-23-alpha, IL-15, IL-12- alpha, IL-12-beta, an anti-IL-18 antibody, an anti-IL-lOR antibody, an anti-CSFIR antibody, tocilizumab, anakinra, IL-7, IL-2R-beta, CCL19, IL-36-gamma, IL-37, or FOXO1.

53. The method of any one of claims 1-50, wherein said protein further comprises an antitumor antibody or fragment thereof.

54. The method of any one of claims 1-40 or 45-53, further comprising contacting said cell with a first viral vector encoding said RNA; further comprising contacting said cell with a second viral vector encoding said trigger RNA, wherein said first and said second viral vector comprise different viral serotypes with overlapping tissue tropism.

55. The method of claim 54, wherein said first viral vector and said second viral vector comprise nonoverlapping selections from one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV8, or AAV9.

56. The method of any one of claims 3-55, wherein said stem-loop sequence comprises a sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or 100% sequence identity to any one of SEQ ID NOs: 5, 6, 7, 8, 17, 18, or 19.

57. The method of any one of claims 3-56, wherein said stem-loop sequence is configured to bind an ADAR protein.

58. A system comprising:(a) a viral vector configured to transduce a cell or a donor oligonucleotide configured for expression in a cell, encoding an RNA comprising:(i) a first nucleotide sequence comprising a conditional nucleotide sequence that comprises a region configured to hybridize to said trigger oligonucleotide, wherein said conditional nucleotide sequence comprises a stem-loop sequence comprising one or more editable codons, and(ii) a transgene encoding an output protein; and(b) a trigger oligonucleotide, wherein said trigger oligonucleotide comprises a synthetic oligonucleotide or an oligonucleotide with at least one non-natural linkage or non-natural base; and wherein said trigger oligonucleotide and said first nucleotide sequence are configured such that said trigger oligonucleotide:(a) selectively activates expression of said protein; or(b) selectively inhibits expression of said protein.

59. The method of claim 56, wherein said viral vector is configured to stably transduce a cell, or said donor oligonucleotide is configured for stable integration in a cell.

60. A system comprising:(a) a viral vector configured to stably transduce a cell or a donor oligonucleotide configured for stable expression in a cell, encoding an RNA comprising:(i) a first nucleotide sequence comprising a conditional nucleotide sequence that comprises a region configured to hybridize to said trigger oligonucleotide, wherein said conditional nucleotide sequence comprises a stem-loop sequence comprising one or more editable codons, and(ii) a transgene encoding an output protein; and(b) a trigger oligonucleotide; wherein said trigger oligonucleotide and said first nucleotide sequence are configured such that said trigger oligonucleotide:(a) selectively activates expression of said protein; or(b) selectively inhibits expression of said protein.

61. A system comprising:(a) a cell stably encoding an RNA, wherein said RNA comprises:(i) a first nucleotide sequence comprising a conditional nucleotide sequence that comprises a region configured to hybridize to said trigger oligonucleotide, wherein said conditional nucleotide sequence comprises a stem-loop sequence comprising one or more editable codons, and(ii) a transgene encoding an output protein; and(b) a trigger oligonucleotide; wherein said trigger oligonucleotide and said first nucleotide sequence are configured such that said trigger oligonucleotide:(a) selectively activates expression of said protein; or(b) selectively inhibits expression of said protein.

62. The system of any one of claims 58-61, wherein said trigger oligonucleotide is a nonnatural oligonucleotide.

63. The system of any one of claims 58-62, wherein said trigger oligonucleotide comprises a synthetic oligonucleotide or an oligonucleotide with at least one non-natural linkage or nonnatural base.

64. The system of any one of claims 58-63, wherein said trigger oligonucleotide comprises a synthetic oligonucleotide or an oligonucleotide with at least one non-natural linkage or nonnatural base.

65. The system of any one of claims 58-64, wherein said trigger oligonucleotide comprises a non-natural sequence.

66. The system of any one of claims 58-65, wherein said trigger oligonucleotide is not a circular oligonucleotide.

67. The system of any one of claims 61-66, wherein said RNA is transcribed from a vector introduced to said cell prior to said contacting in said cell said RNA with said trigger oligonucleotide.

68. The system of any one of claims 61-67, wherein said RNA is transcribed from a genomic or episomal locus of said cell.

69. The system of any one of claims 58-68, wherein said trigger oligonucleotide is an in vitro transcribed (IVT) RNA or a synthetic RNA.

70. The system of any one of claims 61-69, wherein expression of said output protein is selectively activated in said cell in the presence of said trigger RNA.

71. The system of any one of claims 58-70, wherein said one or more editable codons comprise: (i) 5'-UAG, 5'-UGA, or 5'-UAA; (ii) 5'-AUG-3', or 5'-AUA-3'.

72. The system of any one of claims 61-71, wherein expression of said output protein is selectively inhibited in said cell in the presence of said trigger RNA.

73. The system of any one of claims 58-72, wherein said one or more editable codons comprise: (i) 5'-AUG-3'; or (ii) a 5'-AUA-3' non-start codon.

74. The system of any one of claims 58-73, wherein said output protein comprises a therapeutic protein.

75. The system of any one of claims 58-74, wherein said region configured to hybridize to said trigger oligonucleotide comprises an amount of sequence complementarity sufficient to permit hybridization to said target RNA.

76. The system of any one of claims 58-75, wherein said region configured to hybridize to said trigger oligonucleotide comprises at least 60% complementarity to said trigger RNA.

77. The system of any one of claims 58-76, wherein said one or more editable codons further comprises a start codon.

78. The system of any one of claims 58-77, wherein said one or more editable codons further comprises a non-stop, non-start codon that is edited to become a start codon by said target cell.

79. The system of any one of claims 58-78, wherein said non-stop, non-start codon further comprises 5'- AU A -3'.

80. The system of any one of claims 58-79, wherein said protein comprises a toxin, killing factor, a T-cell receptor, a chimeric antigen receptor, a fluorescent protein, a genomic modification protein, a transcription factor, an antigen, an antibody or a functional fragment thereof, a therapeutic protein, or an enzyme.

81. The system of any one of claims 61-80, wherein said cell encoding said RNA comprises an adenosine deaminase acting on RNA (ADAR) protein or a coding sequence encoding thereof.

82. The system of any one of claims 58-81, wherein an ORF encoding said protein is in frame with said one or more editable codons.

83. The system of any one of claims 58-82, wherein said trigger oligonucleotide: (i) comprises a sequence having 50% or less identity to a genomic sequence of said cell; (ii) does not comprise a stretch of bases with 90% or more identity to a genomic sequence of said cell, wherein said stretch is greater than about 30 bases in length.

84. The system of any one of claims 58-83, wherein said trigger oligonucleotide is at least 54 nucleotides in length.

85. The system of any one of claims 58-84, wherein said contacting comprises combining said cell expressing said RNA with a viral vector encoding said trigger oligonucleotide.

86. The system of any one of claims 58-85, wherein said trigger oligonucleotide is exogenous to said cell.

87. The system of any one of claims 58-86, wherein said trigger oligonucleotide comprises an antisense oligonucleotide (ASO).

88. The system of any one of claims 58-87, wherein said trigger oligonucleotide is encapsulated in an LNP.

89. The system of any one of claims 58-88, wherein said trigger oligonucleotide is GalNAc- conjugated.

90. The system of any one of claims 61-89, wherein said cell further comprises an open reading frame (ORF) encoding said RNA.

91. The system of claim 90, wherein said cell further comprises a promoter operably linked to said open reading frame (ORF) encoding said RNA.

92. The system of any one of claims 61-91, wherein said cell further comprises a smallmolecule or RNA-binding riboswitch operably linked to said sequence encoding said protein.

93. The system of any one of claims 58-92, wherein said promoter further comprises an inducible promoter.

94. The system of any one of claims 58-93, wherein said promoter comprises a CMV promoter, a CMV-tetO tetracycline responsive promoter, or a UAS promoter.

95. The system of any one of claims 61-94, wherein said cell comprises a T-cell or a precursor thereof, a T-reg cell or a precursor thereof, a natural killer T-cell or a precursor thereof, an NK-cell or a precursor thereof, a hepatocyte or a precursor thereof, a retinal pigment epithelial cell or a precursor thereof, a neuron or a precursor thereof, an epithelial cell or a precursor thereof, a retinal cell or a precursor thereof, a CHO cell, a HEK293 cell, a NSO murine myeloma cell, a PER.C6 cell, or an Sp2 / 0 cell.

96. The system of any one of claims 58-95, wherein said output protein further comprises a hormone, an antibody, a chimeric antigen receptor (CAR), a soluble receptor, an extracellular domain of a hormone receptor, an extracellular domain of a VEGF receptor, an interleukin, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11, caspase 12, caspase 13, p53 upregulated modulator of apoptosis (PUMA), Bakl, iCasp9, ricin toxin chain A, diphtheria toxin, IL-18, IL-23-alpha, IL-15, IL-12- alpha, IL-12-beta, an anti-IL-18 antibody, an anti-IL-lOR antibody, an anti-CSFIR antibody, tocilizumab, anakinra, IL-7, IL-2R-beta, CCL19, IL-36-gamma, IL-37, or FOXO1.

97. The system of any one of claims 61-96, wherein said trigger oligonucleotide is encoded on a viral vector.

98. The system of claim 97, wherein said viral vector comprises an AAV 1 , AAV2, AAV3, AAV4, AAV5, AAV8, or AAV9 vector.

99. The system of any one of claims 61-98, wherein said stem-loop sequence comprises a sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or 100% sequence identity to any one of SEQ ID NOs: 5, 6, 7, 8, 17, 18, or 19.

100. The system of any one of claims 61-98, wherein said stem-loop sequence comprises a sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or 100% sequence identity to a stem-loop sequence in Table 1.

101. The system of any one of claims 61-100, wherein said stem-loop is not configured to bind a Cas protein.

102. The system of any one of claims 61-101, wherein said stem-loop sequence further comprises a Kozak sequence operably linked to a start codon.

103. The system of any one of claims 61-102, wherein said stem-loop sequence further comprises a Kozak sequence operably linked to a non-stop, non-start codon.

104. The system of any one of claims 61-103, wherein said region configured to hybridize to said trigger oligonucleotide and said stem-loop sequence comprising one or more editable codons are non-overlapping.

105. The system of any one of claims 61-104, wherein said one or more editable codons are in a stem sequence of said stem-loop sequence.

106. The system of any one of claims 61-105, wherein said one or more editable codons comprise at least one base that is mismatched with a sequence within the stem-loop opposite said one or more editable codons.

107. The system of any one of claims 61-106, wherein a stem of said stem-loop is at least 12 base pairs in length.

108. A system comprising:(a) a viral vector configured to encode an RNA and stably transduce a cell, or a donor DNA configured to encode an RNA and stably integrate in a cell, wherein said RNA comprises:(i) a first nucleotide sequence comprising a conditional nucleotide sequence that comprises a region configured to hybridize to said trigger oligonucleotide, wherein said region configured to hybridize to said trigger oligonucleotide comprises at least one editable codon, and(ii) a transgene encoding an output protein; wherein said trigger oligonucleotide and said first nucleotide sequence are configured such that said trigger oligonucleotide:(a) selectively activates expression of said protein; or(b) selectively inhibits expression of said protein; wherein: (i) said region configured to hybridize to said trigger oligonucleotide is outside of an open reading frame (ORF) encoding said output protein; or (ii) said editable codon is not a stop codon.

109. A system comprising;(a) a cell stably encoding an RNA, a viral vector configured to encode an RNA, or a donor DNA configured to encode an RNA, wherein said RNA comprises:(i) a first nucleotide sequence comprising a conditional nucleotide sequence that comprises a region configured to hybridize to said trigger oligonucleotide, wherein said region configured to hybridize to said trigger oligonucleotide comprises at least one editable codon, and(ii) a transgene encoding an output protein; wherein said trigger oligonucleotide and said first nucleotide sequence are configured such that said trigger oligonucleotide:(a) selectively activates expression of said protein; or(b) selectively inhibits expression of said protein; wherein: (i) said region configured to hybridize to said trigger oligonucleotide is outside of an open reading frame (ORF) encoding said output protein; or (ii) said editable codon is not a stop codon.

110. The system of any one of claims 108-109, wherein said trigger oligonucleotide comprises a synthetic oligonucleotide or an oligonucleotide with at least one non-natural linkage or nonnatural base.

111. The system of any one of claims 108-110, wherein said trigger oligonucleotide comprises a non-natural sequence.

112. The system of any one of claims 108-111, wherein said trigger oligonucleotide is not a circular oligonucleotide.

113. The system of any one of claims 108-112, wherein said RNA is transcribed from a genomic or episomal locus of said cell.

114. The system of any one of claims 109-113, wherein said RNA is transcribed from a vector introduced to said cell prior to said contacting in said cell said RNA with said trigger oligonucleotide.

115. The system of any one of claims 108-114, wherein said trigger oligonucleotide is an in vitro transcribed (IVT) RNA or a synthetic RNA.

116. The system of any one of claims 109-115, wherein expression of said output protein is selectively activated in said cell in the presence of said trigger RNA.

117. The system of any one of claims 108-116, wherein said one or more editable codons comprise: (i) 5'-UAG, 5'-UGA, or 5'-UAA; (ii) 5'-AUG-3', or 5'-AUA-3'.

118. The system of any one of claims 109-117, wherein expression of said output protein is selectively inhibited in said cell in the presence of said trigger RNA.

119. The system of any one of claims 108-118, wherein said one or more editable codons comprise: (i) 5'-AUG-3'; or (ii) a 5'-AUA-3' non-start codon.

120. The system of any one of claims 108-119, wherein said region configured to hybridize to said trigger oligonucleotide comprises an amount of sequence complementarity sufficient to permit hybridization to said target RNA.

121. The system of any one of claims 108-120, wherein said region configured to hybridize to said trigger oligonucleotide comprises at least 60% complementarity to said trigger RNA.

122. The system of any one of claims 108-121, wherein said one or more editable codons further comprises a start codon.

123. The system of any one of claims 108-122, wherein said one or more editable codons further comprises a non-stop, non-start codon that is edited to become a start codon by said target cell.

124. The system of any one of claims 108-123, wherein said non-stop, non-start codon further comprises 5'- AU A -3'.

125. The system of any one of claims 108-124, wherein said protein comprises a toxin, killing factor, a T-cell receptor, a chimeric antigen receptor, a fluorescent protein, a genomic modification protein, a transcription factor, an antigen, an antibody or a functional fragment thereof, a therapeutic protein, or an enzyme.

126. The system of any one of claims 109-125, wherein said cell expressing said RNA comprises an adenosine deaminase acting on RNA (ADAR) protein or a coding sequence encoding thereof.

127. The system of any one of claims 108-126, wherein an ORF encoding said protein is in frame with said one or more editable codons.

128. The system of any one of claims 108-127, wherein said trigger oligonucleotide: (i) comprises a sequence having 50% or less identity to a genomic sequence of said cell; (ii) does not comprise a stretch of bases with 90% or more identity to a genomic sequence of said cell, wherein said stretch is greater than about 30 bases in length.

129. The system of any one of claims 108-128, wherein said trigger oligonucleotide is or comprises at least 54 nucleotides in length.

130. The system of any one of claims 109-129, wherein said trigger oligonucleotide is exogenous to said cell.

131. The system of any one of claims 108-130, wherein said trigger oligonucleotide comprises an antisense oligonucleotide (ASO).

132. The system of any one of claims 108-131, wherein said trigger oligonucleotide is encapsulated in an LNP.

133. The system of any one of claims 108-132, wherein said trigger oligonucleotide is GalNAc-conjugated.

134. The system of any one of claims 109-132, wherein said cell further comprises an open reading frame (ORF) encoding said RNA.

135. The system of any one of claims 109-134, wherein said cell further comprises a promoter operably linked to said open reading frame (ORF) encoding said RNA.

136. The system of any one of claims 109-135, wherein said cell further comprises a smallmolecule or RNA-binding riboswitch operably linked to said sequence encoding said protein.

137. The system of any one of claims 108-136, wherein said promoter further comprises an inducible promoter.

138. The system of any one of claims 108-137, wherein said promoter comprises a CMV promoter, a CMV-tetO tetracycline responsive promoter, or a UAS promoter.

139. The system of any one of claims 109-138, wherein said cell comprises a T-cell or a precursor thereof, a T-reg cell or a precursor thereof, a natural killer T-cell or a precursor thereof, an NK-cell or a precursor thereof, a hepatocyte or a precursor thereof, a retinal pigment epithelial cell or a precursor thereof, a neuron or a precursor thereof, an epithelial cell or aprecursor thereof, a retinal cell or a precursor thereof, a CHO cell, a HEK293 cell, a NSO murine myeloma cell, a PER.C6 cell, or an Sp2 / 0 cell.

140. The system of any one of claims 108-139, wherein said output protein further comprises a hormone, an antibody, a chimeric antigen receptor (CAR), a soluble receptor, an extracellular domain of a hormone receptor, an extracellular domain of a VEGF receptor, an interleukin, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11, caspase 12, caspase 13, p53 upregulated modulator of apoptosis (PUMA), Bakl, iCasp9, ricin toxin chain A, diphtheria toxin, IL-18, IL-23-alpha, IL-15, IL-12- alpha, IL-12-beta, an anti-IL-18 antibody, an anti-IL-lOR antibody, an anti-CSFIR antibody, tocilizumab, anakinra, IL-7, IL-2R-beta, CCL19, IL-36-gamma, IL-37, or FOXO1.

141. The system of any one of claims 108-140, wherein said protein further comprises an antitumor antibody or fragment thereof.

142. The system of any one of claims 109-141, wherein said trigger oligonucleotide is encoded on a viral vector.

143. The system of claim 142, wherein said viral vector comprises an AAV1, AAV2, AAV3, AAV4, AAV5, AAV8, or AAV9 vector.

144. A method of controlling expression of a protein in a cell, comprising contacting the components of the system of any one of claims 58-143 to a cell.

145. The method of claim 144, comprising sequentially contacting the components of the system to said cell.

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