Compositions and methods related to nucleic acid sensors

Abstract

The present disclosure provides compositions and methods related to nucleic acid sensors. In particular, the present disclosure provides nucleic acid molecules that target transcripts of gene fusions or chromosome fusions, activate downstream events (including the generation of detectable signals), and / or exert therapeutic functions. The junction sequence in the target nucleic acid comprises at least a portion of the gene fusion or chromosome fusion, and the junction sequence in the target nucleic acid may comprise at least a portion of the gene fusion or chromosome fusion associated with cancer.

Description

[Technical Field]

[0001] (Priority statement) This application claims priority to U.S. Provisional Application No. 63 / 381,234, filed October 27, 2022, and U.S. Provisional Application No. 63 / 498,367, filed April 26, 2023, the entire contents of each of which are incorporated herein by reference. (Sequence Listing) The text of the computer-readable sequence listing submitted with this application, entitled "41317_601_SequenceListing.xml," created on October 26, 2023, and having a file size of 262,458 bytes, is hereby incorporated by reference in its entirety. (government fund) This invention was made with government support under R01 HG990004 awarded by the National Institutes of Health. The United States Government has certain rights in this invention.

[0002] (Field) The present disclosure provides compositions and methods related to nucleic acid sensors. In particular, the present disclosure provides nucleic acid molecules that target chromosomal fusions, mutant genes, and / or viral transcripts to activate downstream therapeutic functions, thereby reducing or preventing the deleterious effects of the fusions, genes, or transcripts. [Background technology]

[0003] (background) Cancer cells possess unique genetic or transcriptomic alterations that distinguish them from normal cells. These unique genetic features include mutations, deletions, and fusions of endogenous genes or transcripts, as well as the presence of viral genomes or transcripts. While previous cancer treatment approaches used nonspecific chemotherapy or radiation therapy, more recent cancer drug development efforts have focused on targeting unique molecular pathways within cancer cells or programming the immune system to attack cancer cells. While powerful, these existing targeted approaches require significant time and expense to develop. Many cancers possess recurrent chromosomal translocations, each of which results in the fusion of two genes. Well-known examples include infantile AML (e.g., infantile AML with CBFA2T3:GLIS2 fusion), supratentorial ependymoma with ZFTA / RELA fusion (ST-EPN), and Ewing's sarcoma with EWS / FLI1 fusion (EWS). CBFA2T3:GLIS2-positive AML is one of the most aggressive forms of infantile AML, with very few survivors. ZFTA:RELA-positive ST-EPN remains without any effective chemotherapy or targeted therapy. Localized EWS with the EWS:FLI1 fusion has a fairly good prognosis, but metastatic or recurrent EWS is usually fatal. A problem with many fusion-positive cancers is the lack of established downstream targets of the gene fusion, despite years of intensive research to understand the biology of the gene fusion. In other cancers caused by viral infection, cancer cells express viral transcripts that are not present in uninfected cells. Many cancers have mutations in endogenous genes or other forms of genetic alterations that are unique to the cancer cells. Therefore, there is a need for a rapid, cost-effective method for developing precisely targeted cancer therapeutics that does not rely on extensive knowledge of the underlying molecular biology of the target cancer cells. Summary of the Invention [Means for solving the problem]

[0004] (Abstract) Embodiments of the present disclosure provide a single-stranded nucleic acid sensor molecule comprising: a target sensing region having a nucleic acid sequence substantially complementary to a target nucleic acid, the target sensing region comprising a TAG or TGA stop codon opposite a corresponding CAA, CTA, CGA, ACA, TCA, GCA, CCA, CCT, or CCC triplet in the target nucleic acid located on at least one side of a junction sequence in the target nucleic acid; and a response gene located downstream of the target sensing region, the response gene being expressed when the TAG or TGA stop codon is converted to a TGG codon by RNA-specific adenosine deaminase (ADAR)-mediated RNA editing upon binding of the sensor molecule to the target nucleic acid.

[0005] In some embodiments, the junction sequence of the target nucleic acid corresponds to at least a portion of a gene, transcript, or chromosomal fusion. In some embodiments, the junction sequence of the target nucleic acid comprises a CBFA2T3-GLIS2 fusion sequence, an EML4-ALK fusion sequence, a ZFTA-RELA fusion sequence, an EWSR1-FL1 fusion sequence, a CCNH-C5orf30 fusion sequence, a TMEM135-CCDC67 fusion sequence, an EVT6-NTRK3 fusion sequence, a TMPRSS2-ERG fusion sequence, a TRMT11-GRIK2 fusion sequence, or a PVT1-MYC fusion sequence. A target nucleic acid junction sequence that "comprises" a given fusion sequence does not require that the junction sequence include the entire fusion sequence, but rather only a portion of the fusion sequence (e.g., in some embodiments, the junction sequence of the target nucleic acid comprises at least a portion of the complete sequence encoding such a fusion protein). An exemplary CBFA2T3-GLIS2 fusion sequence is shown, for example, in SEQ ID NO: 2. In some embodiments, the junction sequence of the target nucleic acid comprises at least a portion of SEQ ID NO: 2. An exemplary EML4-ALK fusion sequence is set forth in SEQ ID NO: 74. In some embodiments, the junction sequence of the target nucleic acid comprises at least a portion of SEQ ID NO: 74. An exemplary ZFTA-RELA fusion sequence is set forth in SEQ ID NO: 75. In some embodiments, the junction sequence of the target nucleic acid comprises at least a portion of SEQ ID NO: 75. An exemplary EWSR1-FL1 fusion sequence is set forth in SEQ ID NO: 76. In some embodiments, the junction sequence of the target nucleic acid comprises at least a portion of SEQ ID NO: 76. An exemplary CCNH-C5orf30 fusion sequence is set forth in SEQ ID NO: 77. In some embodiments, the junction sequence of the target nucleic acid comprises at least a portion of SEQ ID NO: 77. An exemplary TMEM135-CCDC67 fusion sequence is set forth in SEQ ID NO: 78. In some embodiments, the junction sequence of the target nucleic acid comprises at least a portion of SEQ ID NO: 78. An exemplary ETV6-NTRK3 fusion sequence is set forth in SEQ ID NO: 79. In some embodiments, the junction sequence of the target nucleic acid comprises at least a portion of SEQ ID NO: 79. An exemplary TMPRSS2-ERG fusion sequence is set forth in SEQ ID NO: 80. In some embodiments, the junction sequence of the target nucleic acid comprises at least a portion of SEQ ID NO:80.An exemplary TRMT11-GRIK2 fusion sequence is set forth in SEQ ID NO: 81. In some embodiments, the junction sequence of the target nucleic acid comprises at least a portion of SEQ ID NO: 81. An exemplary PVT1-MYC fusion sequence is set forth in SEQ ID NO: 82. In some embodiments, the junction sequence of the target nucleic acid comprises at least a portion of SEQ ID NO: 82.

[0006] In some embodiments, the junction sequence comprises a TP53(R248Q) mutant transcript. An exemplary TP53(R248Q) mutant transcript is shown in SEQ ID NO: 83. In some embodiments, the junction sequence of the target nucleic acid comprises at least a portion of SEQ ID NO: 83.

[0007] In some embodiments, the junction sequence in the target nucleic acid comprises a CBFA2T3-GLIS2 fusion sequence and the target sensing region comprises the nucleic acid sequence set forth in SEQ ID NO: 3. The target sensing region may comprise additional nucleic acids relative to that set forth in SEQ ID NO: 3. In some embodiments, the junction sequence of the target nucleic acid comprises a CBFA2T3-GLIS2 fusion sequence and the target sensing region comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 4 or SEQ ID NO: 5. For example, in some embodiments, the target sensing region comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 4 or SEQ ID NO: 5.

[0008] In some embodiments, the junction sequence of the target nucleic acid comprises an EML4-ALK fusion sequence, and the target sensing region comprises the nucleic acid sequence set forth in SEQ ID NO: 28. The target sensing region may comprise additional nucleic acids to the nucleic acid set forth in SEQ ID NO: 28. In some embodiments, the junction sequence of the target nucleic acid comprises an EML4-ALK fusion sequence, and the target sensing region comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 20 or SEQ ID NO: 29. For example, in some embodiments, the target sensing region comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 20 or SEQ ID NO: 29.

[0009] In some embodiments, the junction sequence of the target nucleic acid comprises a ZFTA-RELA fusion sequence and the target sensing region comprises the nucleic acid sequence set forth in SEQ ID NO: 32. The target sensing region may comprise additional nucleic acids to that set forth in SEQ ID NO: 32. In some embodiments, the junction sequence of the target nucleic acid comprises a ZFTA-RELA fusion sequence and the target sensing region comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 31 or SEQ ID NO: 30. For example, in some embodiments, the target sensing region comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 31 or SEQ ID NO: 30.

[0010] In some embodiments, the junction sequence of the target nucleic acid comprises an EWSR1-FL1 fusion sequence, and the target sensing region comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 33. For example, in some embodiments, the target sensing region comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 33.

[0011] In some embodiments, the junction sequence in the target nucleic acid comprises a CCNH-C5orf30 fusion sequence, and the target sensing region comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 84. For example, in some embodiments, the target sensing region comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 84.

[0012] In some embodiments, the junction sequence in the target nucleic acid comprises a TMEM135-CCDC67 fusion sequence and the target sensing region comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 85. For example, in some embodiments, the target sensing region comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 85.

[0013] In some embodiments, the junction sequence in the target nucleic acid comprises an EVT6-NTRK3 fusion sequence, and the target sensing region comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 86. For example, in some embodiments, the target sensing region comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 86.

[0014] In some embodiments, the junction sequence in the target nucleic acid comprises a TMPRSS2-ERG fusion sequence, and the target sensing region comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 87. For example, in some embodiments, the target sensing region comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 87.

[0015] In some embodiments, the junction sequence in the target nucleic acid comprises a TRMT11-GRIK2 fusion sequence, and the target sensing region comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 88. For example, in some embodiments, the target sensing region comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 88.

[0016] In some embodiments, the junction sequence in the target nucleic acid comprises a PVT1-MYC fusion sequence, and the target sensing region comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 89. For example, in some embodiments, the target sensing region comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 89.

[0017] In some embodiments, the junction sequence in the target nucleic acid comprises a TP53(R248Q) mutant transcript. The junction sequence of a target nucleic acid comprising a given mutant transcript indicates that the junction sequence comprises at least a portion of the mutant transcript. In some embodiments, the junction sequence comprises a TP53(R248Q) mutant transcript, and the target sensing region comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO:90. For example, in some embodiments, the target sensing region comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:90.

[0018] In some embodiments, the junction sequence of the target nucleic acid comprises a viral transcript. A junction sequence of the target nucleic acid comprising a given viral transcript indicates that the junction sequence comprises at least a portion of the viral transcript. In some embodiments, the viral transcript is a transcript associated with cancer. In some embodiments, the viral transcript is an Epstein-Barr virus (EBV) transcript or a Kaposi's sarcoma-associated herpesvirus (KSHV) transcript. In some embodiments, the viral transcript is the Epstein-Barr virus transcript EBNA1, and the target-sensing region comprises a nucleic acid sequence having at least 80% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to SEQ ID NO: 34.

[0019] In some embodiments, the viral transcript is KSHV transcript ORF71 and the target sensing region comprises a nucleic acid sequence having at least 80% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to SEQ ID NO:35.

[0020] In some embodiments, the target sensing region is at least about 50 nucleotides in length, hi some embodiments, the target sensing region is from about 50 nucleotides to about 1000 nucleotides in length.

[0021] In some embodiments, the response gene encodes at least one of a reporter protein, a caspase, a prodrug converting enzyme, or an enzyme that catalyzes a specific function.

[0022] In some embodiments, the sensor molecule further comprises a control gene. The control gene is constitutively expressed. For example, in some embodiments, the control gene is a fluorescent protein.

[0023] In some embodiments, the sensor molecule comprises a linker region located upstream of the response gene but downstream of the TAG or TGA stop codon. In some embodiments, the linker region comprises an XTEN80 peptide. In some embodiments, the linker region comprises a 2A peptide.

[0024] In some embodiments, the sensor molecule comprises an RNA aptamer sequence capable of binding to its cognate binding protein. In some embodiments, the RNA aptamer comprises a sequence capable of binding to at least one of MS2, PP7, BoxB, or Pumilio. In some embodiments, the cognate binding protein is fused to an ADAR protein (including any mutant, derivative, or variant thereof). In some embodiments, the cognate binding protein is fused to a domain of an ADAR protein. In some embodiments, the cognate binding protein is fused to an ADAR mutant protein. Exemplary ADAR mutant proteins include ADARdd(E488Q) and ADARddm(C377F,E488Q). In some embodiments, the cognitive binding protein is MCP. In some embodiments, the sensor molecule comprises MCP-ADARdd(E488Q) or MCP-ADARddm(C377F,E488Q).

[0025] Embodiments of the present disclosure also include expression vectors that include DNA sequences corresponding to any of the RNA sensor molecules described herein.

[0026] In some embodiments, provided herein is a sensor molecule that does not require any exogenous ADAR to detect target sequence.In some embodiments, sensor molecule comprises gene encoding ADAR or ADAR fusion, and ADAR or ADAR fusion is constitutively expressed.In some embodiments, ADAR fusion comprises ADAR enzyme fused with homologous aptamer binding protein.In some embodiments, ADAR fusion comprises mutant ADAR protein.Exemplary ADAR mutant protein includes ADARdd(E488Q) and ADARddm(C377F,E488Q).In some embodiments, cognitive binding protein is MCP.In some embodiments, sensor molecule comprises MCP-ADARdd(E488Q) or MCP-ADARddm(C377F,E488Q). For example, in some embodiments, the ADAR enzyme (or mutant ADAR enzyme) is fused to a cognate aptamer-binding protein (e.g., MS2 coat protein (MCP), PP7 coat protein (PCP), lambda N protein, or Pumilio / PUF-HD domain) to form a single recombinant protein. In some embodiments, the sensor molecule further comprises an RNA aptamer sequence that recruits the cognate aptamer-binding protein-ADAR fusion.

[0027] In some embodiments, the sensor molecule is an RNA molecule. In some embodiments, the expression vector is selected from the group consisting of pCR8-mRuby2-P2A-ccdbCam-E2A-EGFP vector, pCR8-mRuby2-P2A-ccdbCam-E2A-EGFP-BsaI(agat) vector, pCR8-mRuby2-P2A-ccdbCam-E2A-EGFP-NxMS2 vector, pCR8-mRuby2-P2A-Sensor-E2A-EGFP vector, pCR8-mRuby2-P2A-Sensor-E2A-EGFP-NxMS2 vector, pmax-mRuby2-P2A-Sensor-XTEN80-EGFP-NxMS2 vector, and MCP -ADARdd(E488Q) vector;pmax-MCP-ADARdd(E488Q) vector;pmax-MCP-ADARdd(E488Q)-P2A-Sensor-XTEN80-NTR1.1-NxMS2 vector;pmax-MCP-ADARdd(E488Q)-P2A-Sensor -E2A-NTR1.1-NxMS2 vector;pmax-MCP-ADARddm(C377F,E488Q)-P2A-Sensor-XTEN80-NTR1.1-NxMS2 vector;pmax-MCP-ADARddm(C377F,E488Q)-P2A-Sensor-E2A-NTR1.1-NxMS2 vector;pmax-MCP-ADARdd(E488Q)-P2A-Sensor-XTEN80-DTA-NxMS2 vector;pmax-MCP-ADARdd(E488Q)-P2A-Sensor-E2A-DTA-NxMS2 vector;p max-MCP-ADARddm(C377F,E488Q)-P2A-Sensor-XTEN80-DTA-NxMS2 vector;pmax-MCP-ADARddm(C377F,E488Q)-P2A-Sensor-E2A-DTA-NxMS2 vector;p The vector is selected from the group consisting of max-MCP-ADARdd(E488Q)-P2A-Sensor-XTEN80-BAX-NxMS2 vector; pmax-MCP-ADARdd(E488Q)-P2A-Sensor-E2A-BAX-NxMS2 vector; pmax-MCP-ADARddm(C377F,E488Q)-P2A-Sensor-XTEN80-BAX-NxMS2 vector; and pmax-MCP-ADARddm(C377F,E488Q)-P2A-Sensor-E2A-BAX-NxMS2 vector.

[0028] Embodiments of the present disclosure also include cells comprising any of the RNA sensor molecules described herein or any of the vectors described herein.

[0029] Embodiments of the present disclosure also include kits that include any of the RNA sensor molecules described herein, any of the vectors described herein, and / or any of the cells described herein.

[0030] The embodiments of the present disclosure also include the method for treating the subject with cancer or the subject suspected of having cancer.According to these embodiments, the method comprises administering to the subject any of the RNA sensor molecules described herein, any of the vectors described herein, and / or any of the cells described herein; and treating the subject.In some embodiments, the cancer is caused by chromosomal translocation and / or gene fusion.

[0031] Embodiments of the present disclosure also include methods for detecting gene fusion transcripts in cells. According to these embodiments, the method includes transfecting a cell with any of the RNA sensor molecules described herein or any of the vectors described herein, and evaluating the cell for expression of a reporter protein. [Brief explanation of the drawings]

[0032] [Figure 1-1] 1A-1E: Representative schematic diagrams of a target gene fusion and its transcript (FIG. 1A), and a nucleic acid sensor designed to target the gene fusion transcript (FIG. 1B-1E), according to one embodiment of the present disclosure. [Figure 1-2] Same as above. [Figure 1-3] Same as above. [Figure 1-4] Same as above. [Figure 1-5] Same as above.

[0033] [Figure 2-1]Figures 2A-2E: Representative schematic diagrams of exemplary vector systems for facilitating the cloning and testing of various sensor sequences and sensor structures of the present disclosure. Figure 2A: pCR8-mRuby2-P2A-ccdbCam-E2A-EGFP vector. Figure 2B: pCR8-mRuby2-P2A-ccdbCam-E2A-EGFP-BsaI(agat) vector; and pCR8-mRuby2-P2A-ccdbCam-E2A-EGFP-NxMS2 vector (e.g., N can be any number). Figure 2C: pCR8-mRuby2-P2A-Sensor-E2A-EGFP vector (Gateway destination expression vector in Figure 2D); and pCR8-mRuby2-P2A-Sensor-E2A-EGFP-NxMS2 vector (Gateway destination expression vector in Figure 2E). [Figure 2-2] Same as above. [Figure 2-3] Same as above.

[0034] [Figure 3-1]Figures 3A-3C: Representative schematic diagrams of four exemplary sensor sequences for detecting the CBFA2T3-GLIS2 fusion sequence, with lengths of 93 (CBFA2T3GLIS2_93_Sensor), 351 (CBFA2T3GLIS2_351_Sensor), and 495 (CBFA2T3GLIS2_495_Sensor), located approximately at the center of the fusion junction. A sensor consisting of four MS2 stem-loops inserted within the sensor region was also designed (CBFA2T3GLIS2_avidity5) (Figure 3A). Representative fluorescence detection data (using flow cytometry) in HEK293 cells transfected with the sensor response vector (pmax-mRuby2-P2A-Sensor-E2A-EGFP-9xMS2), the MCP-ADARdd(E488Q) vector (pmax-MCP-ADARdd(E488Q)), and either a control empty vector or vectors expressing the test fusion genes (pmax-CBFA2T3-GLIS2_FL or pmax-CBFA2T3-GLIS3_mini750) (Figure 3B). Representative fluorescence detection data (using flow cytometry) in HEK293 cells transfected with the sensor-responsive vector (pmax-mRuby2-P2A-Sensor-E2A-EGFP-9xMS2 or pmax-mRuby2-P2A-Sensor-XTEN80-EGFP-9xMS2), the MCP-ADARdd(E488Q) vector (pmax-MCP-ADARdd(E488Q)), and either a control empty vector or vectors expressing the test fusion genes (pmax-CBFA2T3-GLIS2_FL or pmax-CBFA2T3-GLIS3_mini750) (Figure 3C). The presence of the fusion transcript resulted in an increase in GFP signal not observed in cells transfected with the empty vector control, demonstrating specific detection of the fusion CBFA2T3-GLIS2 transcript in vivo in HEK293T cells. [Figure 3-2] Same as above. [Figure 3-3] Same as above.

[0035] [Figure 4]FIG. 4: A representative schematic diagram of a nucleic acid sensor-NTR designed to detect gene fusion transcripts, according to one embodiment of the present disclosure.

[0036] [Figure 5A] Figure 5A-5B: Figure 5A shows a schematic diagram of an exemplary sensor-NTR (CBFAZT3GLIS2_495_Sensor) with a length of 495, located approximately at the center of the fusion junction, for detecting the CBFA2T3-GLIS2 fusion sequence. The CBFAZT3GLIS2_495_Sensor is cloned upstream of an XTEN80 protein linker, an NTR1.1 coding sequence, and nine copies of the MS2 stem-loop (9xMS2). A schematic diagram of the pmax-MCP-ADARdd(E488Q) vector expressing MCP-ADARdd(E488Q), which can be recruited to 9xMS2. Figure 5B contains representative cell viability data (using the CellTiter-Glo 2.0 assay) in HEK293T cells transfected with the sensor-NTR vector (pmax-mRuby2-P2A-Sensor(CBFA2T3GLIS2_495)-XTEN80-NTR1.1-9xMS2), the MCP-ADARdd(E488Q) vector, and either a control empty vector, a combination of vectors expressing non-fusion components (pmax-CBFA2T3 and pmax-GLIS2), or a vector expressing the CBFA2T3-GLIS2 fusion gene (pmax-CBFA2T3-GLIS2_FL). The presence of the fusion transcript, but not the empty vector or non-fusion component transcript, resulted in a decrease in cell viability, demonstrating specific detection and elimination of the cell population expressing the CBFA2T3-GLIS2 transcript in vivo in HEK293T cells. [Figure 5B] Same as above.

[0037] [Figure 6A]Figure 6A-6B: Figure 6A includes a schematic diagram of an exemplary sensor-NTR (EML4ALK_501_Sensor) with a length of 501, located approximately at the center of the EML4-ALK fusion sequence, for detecting the fusion junction. The EML4ALK_501_Sensor is cloned upstream of an E2A peptide, an NTR1.1 coding sequence, and nine copies of the MS2 stem-loop (9xMS2). A schematic diagram of the pmax-MCP-ADARdd(E488Q) vector expressing MCP-ADARdd(E488Q), which can be recruited to 9xMS2.

[0038] [Figure 6B] Figure 6B contains representative cell viability data (using the CellTiter-Glo 2.0 assay) in HEK293T cells transfected with the sensor-NTR vector (pmax-mRuby2-P2A-Sensor(EML4ALK_501)-E2A-NTR1.1-9xMS2), the MCP-ADARdd(E488Q) vector, and either a control empty vector or a vector expressing the EML4-ALK fusion gene (pmax-EML4-ALK). The presence of the fusion transcript, but not the empty vector, resulted in a decrease in cell viability, demonstrating specific detection and elimination of the cell population expressing the EML4-ALK transcript in vivo in HEK293T.

[0039] [Figure 7] FIG. 7: Representative schematic of a nucleic acid ADAR-sensor-NTR designed to detect gene fusion transcripts, according to one embodiment of the present disclosure.

[0040] [Figure 8A]Figure 8A: A representative schematic diagram of an exemplary ADAR-sensor-NTR (CBFAZT3GLIS2_495_Sensor) construct with a length of 495, located approximately at the center of the fusion junction, for detecting the CBFA2T3-GLIS2 fusion sequence. The construct consists of the coding sequence for MCP-ADARdd(E488Q), a P2A peptide, CBFAZT3GLIS2_495_Sensor, an E2A peptide, an NTR1.1 coding sequence, and nine copies of the MS2 stem-loop (9xMS2). The 9xMS2 construct can recruit MCP-ADARdd(E488Q) expressed from the same RNA molecule. The central sequence shows the portion of the fusion transcript sequence spanning the junction, including the bolded and underlined CCA triplet (top, 3'→5'), and the corresponding portion of the sensor sequence, including the two bolded and underlined stop codons (bottom, 5'→3').

[0041] [Figure 8B] Figure 8B: Representative cell viability data (using the CellTiter-Glo 2.0 assay) in HEK293T cells transfected with the ADAR-Sensor-NTR vector (pmax-MCP_ADARdd(E488Q)-P2A-Sensor(CBFA2T3GLIS2_495)-E2A-NTR1.1-9xMS2) and either a control empty vector, a combination of vectors expressing non-fusion components (pmax-CBFA2T3 and pmax-GLIS2), or a vector expressing the CBFA2T3-GLIS2 fusion gene (pmax-CBFA2T3-GLIS2_FL). The presence of the fusion transcript, but not the empty vector or non-fusion component transcript, resulted in a decrease in cell viability, demonstrating specific detection and elimination of the cell population expressing the CBFA2T3-GLIS2 transcript in vivo in HEK293T.

[0042] [Figure 9A]Figure 9A: Representative schematic diagram of exemplary ADAR-sensor-NTR constructs with various sensor lengths (LS) for detecting the CBFA2T3-GLIS2 fusion sequence. Each construct consists of the coding sequence for MCP-ADARdd(E488Q), a P2A peptide, a CBFA2T3-GLIS2 sensor with a sensor length (LS) of 90, 150, or 495 nt, an E2A peptide, an NTR1.1 coding sequence, and nine copies of the MS2 stem-loop (9xMS2). The 9xMS2 can recruit MCP-ADARdd(E488Q) expressed from the same RNA molecule. The central sequence shows the portion of the fusion transcript sequence spanning the junction containing the bolded and underlined CCA triplet (top, 3'→5'), as well as the corresponding portion of the sensor sequence containing the two bolded and underlined sensor stop codons (bottom, 5'→3').

[0043] [Figure 9B] Figure 9B: Alignment of the target and 501-nt sensor sequences for the CBFA2T3-GLIS2 fusion. The alignment is shown in a group of three columns. The top column shows the target sequence in reverse (3' to 5'). The middle column shows number-coded flags for this alignment. The bottom column shows the sensor sequence in the 5' to 3' direction. The nucleotides are ordered to the codon encoded by the sensor. The 0-flag indicates that the target and sensor are aligned at that position. The 1-flag indicates the first nucleotide of the sensor stop codon, the 2-flag indicates the second nucleotide of the sensor stop codon, and the 3-flag indicates the third nucleotide of the sensor stop codon. The 4-flag indicates an insertion in the sensor sequence to preserve the frame between the sensor stop codons. The corresponding target position has a gap (-) for the sensor. The 5-flag and 6-flag indicate mutations to the sensor sequence to eliminate unwanted stop codons or unwanted start codons, respectively. The sensed stop codons are underlined. Right-pointing and left-pointing arrows indicate the start and end of the sensor sequence for the indicated sensor length (LS).

[0044] [Figure 9C] Figure 9C: Representative cell viability data (using the CellTiter-Glo 2.0 assay) for HEK293T cells transfected with ADAR-sensor-NTR vectors carrying the 90nt, 150nt, or 495nt sensor, and either a control empty vector or a vector expressing the CBFA2T3-GLIS2 fusion gene (pmax-CBFA2T3-GLIS2_FL). The ADAR mutant variants, single mutant MCP-ADARdd(E488Q) and double mutant MCP-ADARddm(C377F,E488Q), were included. The bar graph shows the normalized cell viability of CBFA2T3-GLIS2 fusion-positive cells compared to fusion-negative cells for ADAR-sensor-NTR constructs with the indicated sensor lengths and ADAR mutant variants. Increasing sensor length generally results in decreased normalized cell viability (higher cell elimination). Furthermore, the double mutant MCP-ADARddm(C377F,E488Q) induces a higher degree of cell ablation compared to the single mutant MCP-ADARdd(E488Q).

[0045] [Figure 10A] Figure 10A: Representative schematic diagram of exemplary ADAR-sensor-NTR constructs with various sensor lengths (LS) for detecting EML4-ALK fusion sequences. The constructs consist of the coding sequence for MCP-ADARdd(E488Q), a P2A peptide, an EML4-ALK sensor with a sensor length (LS) of 90 nt, 150 nt, or 501 nt, an E2A peptide, an NTR1.1 coding sequence, and nine copies of the MS2 stem-loop (9xMS2). The 9xMS2 can recruit MCP-ADARdd(E488Q) expressed from the same RNA molecule. The central sequence shows the portion of the fusion transcript sequence spanning the junction containing the bolded and underlined CCA triplet (top, 3'→5'), as well as the corresponding portion of the sensor sequence containing the two bolded and underlined sensor stop codons (bottom, 5'→3').

[0046] [Figure 10B] Figure 10B: Alignment of the target and 501-nt sensor sequences for the EML4-ALK fusion. The alignment is shown in a group of three columns. The top column shows the target sequence in reverse (3' to 5'). The middle column shows number-coded flags for this alignment. The bottom column shows the sensor sequence in the 5' to 3' direction. The nucleotides are ordered to the codon encoded by the sensor. The 0-flag indicates that the target and sensor are aligned at that position. The 1-flag indicates the first nucleotide of the sensor stop codon, the 2-flag indicates the second nucleotide of the sensor stop codon, and the 3-flag indicates the third nucleotide of the sensor stop codon. The 4-flag indicates an insertion in the sensor sequence to preserve the frame between the sensor stop codons. The corresponding target position has a gap (-) for the sensor. The 5-flag and 6-flag indicate mutations to the sensor sequence to eliminate unwanted stop codons or unwanted start codons, respectively. The 7-flag indicates a deletion in the sensor sequence to preserve the frame between the sensor stop codons. The sensor stop codons are underlined. The fusion junctions are indicated. Right and left arrows indicate the start and end of the sensor sequence for the indicated sensor length (LS).

[0047] [Figure 10C]Figure 10C: Representative cell viability data (using the CellTiter-Glo 2.0 assay) for HEK293T cells transfected with ADAR-sensor-NTR vectors containing the 90nt, 150nt, or 501nt sensor, and either a control empty vector or a vector expressing the EML4-ALK fusion gene (pmax-EML4-ALK). The ADAR mutant variants, single mutant MCP-ADARdd(E488Q) and double mutant MCP-ADARddm(C377F,E488Q), were included. The bar graph shows the normalized cell viability of EML4-ALK fusion-positive cells compared to fusion-negative cells for ADAR-sensor-NTR constructs containing the indicated sensor length and ADAR mutant variant. Increasing sensor length generally results in decreased normalized cell viability (higher cell elimination). Furthermore, the double mutant MCP-ADARddm(C377F,E488Q) induces a higher degree of cell ablation compared to the single mutant MCP-ADARdd(E488Q).

[0048] [Figure 11A] Figure 11A: Representative schematic diagram of exemplary ADAR-sensor-NTR constructs with various sensor lengths (LS) for detecting ZFTA-RELA fusion sequences. The constructs consist of the coding sequence for MCP-ADARdd(E488Q), a P2A peptide, a ZFTA-RELA sensor with a sensor length (LS) of 90 nt, 150 nt, or 501 nt, an E2A peptide, an NTR1.1 coding sequence, and nine copies of the MS2 stem-loop (9xMS2). The 9xMS2 can recruit MCP-ADARdd(E488Q) expressed from the same RNA molecule. The central sequence shows the portion of the fusion transcript sequence spanning the junction containing the bolded and underlined CCA triplet (top, 3'→5'), as well as the corresponding portion of the sensor sequence containing the two bolded and underlined sensor stop codons (bottom, 5'→3').

[0049] [Figure 11B]Figure 11B: Alignment of the target and 501-nt sensor sequences for the ZFTA-RELA fusion. The alignment is shown in a group of three columns. The top column shows the target sequence in reverse (3' to 5'). The middle column shows number-coded flags for this alignment. The bottom column shows the sensor sequence in the 5' to 3' direction. The nucleotides are ordered to the codon encoded by the sensor. The 0-flag indicates that the target and sensor are aligned at that position. The 1-flag indicates the first nucleotide of the sensor stop codon, the 2-flag indicates the second nucleotide of the sensor stop codon, and the 3-flag indicates the third nucleotide of the sensor stop codon. The 4-flag indicates an insertion in the sensor sequence to preserve the frame between the sensor stop codons. The corresponding target position has a gap (-) for the sensor. The 5-flag and 6-flag indicate mutations to the sensor sequence to eliminate unwanted stop codons or unwanted start codons, respectively. The 8-flag indicates a mutation to the sensor sequence to allow synthesis for cloning. The sensor stop codon is underlined. Right and left arrows indicate the start and end of the sensor sequence for the indicated sensor length (LS).

[0050] [Figure 11C] Figure 11C: Representative cell viability data (using CellTiter-Glo 2.0 assay) in HEK293T cells transfected with ADAR-sensor-NTR vectors carrying the 90nt, 150nt, or 501nt sensor, and either a control empty vector or a vector expressing the ZFTA-RELA fusion gene (pmax-ZFTA-RELA). The bar graph shows the normalized cell viability of ZFTA-RELA fusion-positive cells compared to fusion-negative cells for ADAR-sensor-NTR constructs with the indicated sensor lengths. The 501nt sensor is superior to the shorter 90nt and 150nt sensors in inducing a higher degree of cell elimination.

[0051] [Figure 11D] Figure 11D: A representative schematic diagram of a lentivirus carrying an exemplary ADAR-sensor-NTR construct for detecting endogenous ZFTA-RELA fusion transcripts expressed in BDX-1425EPN cancer cells and inducing cell elimination. The construct consists of the coding sequence for MCP-ADARdd(E488Q), a P2A peptide, a ZFTA-RELA sensor with a sensor length of 501 nt, an XTEN80 linker or E2A peptide, an NTR1.1 coding sequence, and nine copies of the MS2 stem-loop (9xMS2). The 9xMS2 can recruit MCP-ADARdd(E488Q) expressed from the same RNA molecule. The central sequence shows the portion of the fusion transcript sequence spanning the junction containing the bolded and underlined CCA triplet (top, 3'→5'), as well as the corresponding portion of the sensor sequence containing two bolded and underlined stop codons (bottom, 5'→3').

[0052] [Figure 11E] Figure 11E: Representative cell viability data (using CellTiter-Glo 2.0 assay) of untransduced BDX-1425EPN cells or BDX-1425EPN cells transduced with lentivirus carrying an ADAR-sensor-NTR vector with a 501nt sensor and either an XTEN80 peptide or an E2A peptide between the sensor and the NTR. Bar graphs show cell viability of untransduced cells (no Tdx), cells transduced with a lentiviral sensor with an XTEN80 peptide, or cells transduced with a lentiviral sensor with an E2A peptide.

[0053] [Figure 12A]Figure 12A: A representative schematic diagram of an exemplary ADAR-sensor-NTR (EWSR1_FLI1_501_Sensor) with a length of 501 for detecting the EWSR1-FLI1 fusion sequence. The construct consists of the coding sequence for MCP-ADARdd(E488Q), a P2A peptide, EWSR1_FLI1_501_Sensor, an E2A peptide, an NTR1.1 coding sequence, and nine copies of the MS2 stem-loop (9xMS2). The 9xMS2 can recruit MCP-ADARdd(E488Q) expressed from the same RNA molecule. The central sequence shows the portion of the fusion transcript sequence spanning the junction containing the bolded and underlined CCA triplet (top, 3'→5'), as well as the corresponding portion of the sensor sequence containing the two bolded and underlined stop codons (bottom, 5'→3'). <97nt> indicates 97 nucleotides not shown in the schematic.

[0054] [Figure 12B]Figure 12B: Alignment of target and sensor sequences for the EWSR1-FLI1 fusion. The alignment is shown in a group of three columns. The top column shows the target sequence in reverse (3' to 5'). The middle column shows number-coded flags for this alignment. The bottom column shows the sensor sequence in the 5' to 3' direction. The nucleotides are ordered to the codon encoded by the sensor. The 0-flag indicates that the target and sensor are aligned at that position. The 1-flag indicates the first nucleotide of the sensor stop codon, the 2-flag indicates the second nucleotide of the sensor stop codon, and the 3-flag indicates the third nucleotide of the sensor stop codon. The 4-flag indicates an insertion in the sensor sequence to preserve the frame between the sensor stop codons. The corresponding target position has a gap (-) for the sensor. The 5-flag and 6-flag indicate mutations to the sensor sequence to eliminate unwanted stop codons or unwanted start codons, respectively. The 7-flag indicates a deletion in the sensor sequence to preserve the frame between the sensor stop codons, which are underlined. The fusion junction is indicated.

[0055] [Figure 12C] Figure 12C: Representative cell viability data (using the CellTiter-Glo 2.0 assay) in HEK293T cells transfected with the ADAR-Sensor-NTR vector (pmax-MCP_ADARdd(E488Q)-P2A-Sensor(EWSR1FLI1_501)-E2A-NTR1.1-9xMS2) and either a control empty vector or a vector expressing the EWSR1-FLI1 fusion minigene (pmax-EWSR1-FLI1). The line graph shows the normalized cell viability of EWSR1-FLI1 fusion-positive cells compared with fusion-negative cells at the indicated days after drug addition for two runs of the experiment. The presence of the fusion transcript, but not the empty vector, resulted in a decrease in cell viability, demonstrating specific detection and elimination of the cell population expressing the EWSR1-FLI1 transcript in vivo in HEK293T.

[0056] [Figure 13] FIG. 13: A representative schematic diagram of a nucleic acid ADAR-sensor-DTA designed to detect gene fusion transcripts, according to one embodiment of the present disclosure.

[0057] [Figure 14A] Figure 14A: A representative schematic diagram of an exemplary ADAR-sensor-DTA (CBFA2T3GLIS2_495_Sensor) with a length of 495 for detecting the CBFA2T3-GLIS2 fusion sequence. The construct consists of the coding sequence for MCP-ADARdd(E488Q), a P2A peptide, CBFA2T3GLIS2_495_Sensor, an E2A peptide, a DTA coding sequence, and nine copies of the MS2 stem-loop (9xMS2). The 9xMS2 can recruit MCP-ADARdd(E488Q) expressed from the same RNA molecule. The central sequence shows the portion of the fusion transcript sequence spanning the junction containing the bolded and underlined CCA triplet (top, 3'→5') and the corresponding portion of the sensor sequence containing the two bolded and underlined stop codons (bottom, 5'→3').

[0058] [Figure 14B]Figure 14B: Representative cell viability data (using the CellTiter-Glo 2.0 assay) in HEK293T cells transfected with the ADAR-Sensor-DTA vector (pmax-MCP_ADARdd(E488Q)-P2A-Sensor(CBFA2T3GLIS2_495)-E2A-DTA-9xMS2) and either a control empty vector or a vector expressing the CBFA2T3-GLIS2 fusion gene (pmax-CBFA2T3-GLIS2_FL). The line graph shows the normalized cell viability of CBFA2T3-GLIS2 fusion-positive cells compared to fusion-negative cells at the indicated days after drug addition. The presence of the fusion transcript, but not the empty vector, resulted in a decrease in cell viability, demonstrating specific detection and elimination of the cell population expressing the CBFA2T3-GLIS2 transcript in vivo in HEK293T.

[0059] [Figure 15] FIG. 15: A representative schematic diagram of a nucleic acid ADAR-sensor-BAX designed to detect gene fusion transcripts, according to one embodiment of the present disclosure.

[0060] [Figure 16A] Figure 16A: A representative schematic diagram of an exemplary ADAR-sensor-BAX (CBFA2T3GLIS2_495_Sensor) construct with a length of 495 for detecting the CBFA2T3-GLIS2 fusion sequence. The construct consists of the coding sequence for MCP-ADARdd(E488Q), a P2A peptide, CBFA2T3GLIS2_495_Sensor, an E2A peptide or XTEN80 linker, a DTA coding sequence, and nine copies of the MS2 stem-loop (9xMS2). The 9xMS2 can recruit MCP-ADARdd(E488Q) expressed from the same RNA molecule. The central sequence shows the portion of the fusion transcript sequence spanning the junction containing the bolded and underlined CCA triplet (top, 3'→5'), as well as the corresponding portion of the sensor sequence containing the two bolded and underlined stop codons (bottom, 5'→3').

[0061] [Figure 16B] Figure 16B: Representative cell viability data (using the CellTiter-Glo 2.0 assay) in HEK293T cells transfected with the ADAR-sensor-BAX vector and either a control empty vector or a vector expressing the CBFA2T3-GLIS2 fusion gene (pmax-CBFA2T3-GLIS2_FL). The bar graph shows the normalized cell viability of CBFA2T3-GLIS2 fusion-positive cells compared to fusion-negative cells at day 5 after drug addition for the ADAR-sensor-BAX constructs with the indicated E2A or XTEN80 linkers.

[0062] [Figure 17] FIG. 17: Representative schematic of a nucleic acid ADAR-sensor-NTR designed to detect viral transcripts, according to one embodiment of the present disclosure.

[0063] [Figure 18A] Figure 18A: A representative schematic diagram of an exemplary ADAR-sensor-NTR (EBNA1_501_Sensor) with a sensor length of 501 for detecting Epstein-Barr virus (EBV)-EBNA1 transcripts. The construct consists of the coding sequence of MCP-ADARdd(E488Q), a P2A peptide, EBNA1_501_Sensor, an XTEN80 protein linker, an NTR1.1 coding sequence, and nine copies of the MS2 stem-loop (9xMS2). The 9xMS2 can recruit MCP-ADARdd(E488Q) expressed from the same RNA molecule. The central sequence shows the portion of the viral transcript sequence with the target CCA triplet in bold and underlined (top, 3'→5') and the corresponding portion of the sensor sequence with the sensor stop codon in bold and underlined (bottom, 5'→3').

[0064] [Figure 18B]Figure 18B: Alignment of target and sensor sequences for EBV-EBNA1. The alignment is shown in a group of three columns. The top column shows the target sequence in reverse (3' to 5'). The middle column shows number-coded flags for this alignment. The bottom column shows the sensor sequence in the 5' to 3' direction. The nucleotides are ordered to the codon encoded by the sensor. The 0-flag indicates that the target and sensor are aligned at that position. The 1-flag indicates the first nucleotide of the sensor stop codon, the 2-flag indicates the second nucleotide of the sensor stop codon, and the 3-flag indicates the third nucleotide of the sensor stop codon. The 5-flag and 6-flag indicate mutations to the sensor sequence to eliminate unwanted stop codons or unwanted start codons, respectively. The sensor stop codons are underlined.

[0065] [Figure 18C] Figure 18C: Representative cell viability data (using the CellTiter-Glo 2.0 assay) in HEK293T cells transfected with the ADAR-Sensor-NTR vector (pmax-MCP_ADARdd(E488Q)-P2A-Sensor(EBNA1_501)-XTEN80-NTR1.1-9xMS2) and either a control empty vector or a vector expressing the EBV-EBNA1 gene (pmax-EBNA1). The presence of viral transcripts, but not empty vector, resulted in a decrease in cell viability, demonstrating specific detection and elimination of the cell population expressing EBV-EBNA1 transcripts in vivo in HEK293T.

[0066] [Figure 19A]Figure 19A: A representative schematic diagram of an exemplary ADAR-sensor-NTR (KSHV_ORF71_501_Sensor) with a sensor length of 501 for detecting Kaposi's sarcoma-associated herpesvirus (KSHV)-ORF71 transcripts. The construct consists of the coding sequence of MCP-ADARdd(E488Q), a P2A peptide, KSHV_ORF71_501_Sensor, an XTEN80 protein linker, an NTR1.1 coding sequence, and nine copies of the MS2 stem-loop (9xMS2). The 9xMS2 can recruit MCP-ADARdd(E488Q) expressed from the same RNA molecule. The central sequence shows the portion of the viral transcript sequence with the target CCA triplet in bold and underlined (top, 3'→5') and the corresponding portion of the sensor sequence with the sensor stop codon in bold and underlined (bottom, 5'→3').

[0067] [Figure 19B] Figure 19B: Alignment of target and sensor sequences for KSHV-ORF71. The alignment is shown in a group of three columns. The top column shows the target sequence in reverse (3' to 5'). The middle column shows number-coded flags for this alignment. The bottom column shows the sensor sequence in the 5' to 3' direction. The nucleotides are ordered to the codon encoded by the sensor. The 0-flag indicates that the target and sensor are aligned at that position. The 1-flag indicates the first nucleotide of the sensor stop codon, the 2-flag indicates the second nucleotide, and the 3-flag indicates the third nucleotide. The 5-flag and 6-flag indicate mutations to the sensor sequence to eliminate an unwanted stop codon or an unwanted start codon, respectively. The sensor stop codon is underlined.

[0068] [Figure 19C]Figure 19C: Representative cell viability data (using the CellTiter-Glo 2.0 assay) in HEK293T cells transfected with the ADAR-Sensor-NTR vector (pmax-MCP_ADARdd(E488Q)-P2A-Sensor(KSHV_ORF71_501)-XTEN80-NTR1.1-9xMS2) and either a control empty vector or a vector expressing the KSHV-ORF71 gene (pOME0343_ORF71-tagged). The presence of viral transcripts, but not empty vector, resulted in a decrease in cell viability, demonstrating specific detection and elimination of the cell population expressing KSHV-ORF71 transcripts in vivo in HEK293T.

[0069] [Figure 20A] Figure 20A: A representative schematic diagram of an exemplary ADAR-sensor-NTR with two sensor stop codons surrounding the fusion junction. The construct consists of the coding sequence for MCP-ADARddm(C377F,E488Q), a P2A peptide, a sensor sequence, an E2A peptide, an NTR1.1 coding sequence, and nine copies of the MS2 stem-loop (9xMS2). The 9xMS2 can recruit MCP-ADARddm(C377F,E488Q) expressed from the same RNA molecule.

[0070] [Figure 20B]Figure 20B: Alignment of target and sensor sequences for CCNH-C5orf30 fusion. The alignment is shown in a group of three columns. The top column shows the target sequence in reverse (3' to 5'). The middle column shows number-coded flags for this alignment. The bottom column shows the sensor sequence in the 5' to 3' direction. The nucleotides are ordered to the codon encoded by the sensor. The 0-flag indicates that the target and sensor are aligned at that position. The 1-flag indicates the first nucleotide of the sensor stop codon, the 2-flag indicates the second nucleotide of the sensor stop codon, and the 3-flag indicates the third nucleotide of the sensor stop codon. The 4-flag indicates an insertion in the sensor sequence to preserve the frame between the sensor stop codons. The corresponding target position has a gap (-) for the sensor. The 5-flag and 6-flag indicate mutations to the sensor sequence to eliminate unwanted stop codons or unwanted start codons, respectively. The 7-flag indicates a deletion in the sensor sequence to preserve the frame between the sensor stop codons, which are underlined. The fusion junction is indicated.

[0071] [Figure 20C] Figure 20C: Representative cell viability data (using the CellTiter-Glo 2.0 assay) in HEK293T cells transfected with the ADAR-Sensor-NTR vector (pmax-MCP_ADARddm(C377F,E488Q)-P2A-Sensor(CCNH_C5orf30_Sensor_501)-E2A-NTR1.1-9xMS2) and either a control empty vector or a vector expressing the CCNH-C5orf30 minigene. The presence of the fusion transcript, but not the empty vector, resulted in a decrease in cell viability, demonstrating specific detection and elimination of the cell population expressing the CCNH-C5orf30 transcript in vivo in HEK293T.

[0072] [Figure 20D]Figure 20D: Alignment of target and sensor sequences for the TMEM135-CCDC67 fusion. The alignment is shown in a group of three columns. The top column shows the target sequence in reverse (3' to 5'). The middle column shows number-coded flags for this alignment. The bottom column shows the sensor sequence in the 5' to 3' direction. The nucleotides are ordered to the codon encoded by the sensor. The 0-flag indicates that the target and sensor are aligned at that position. The 1-flag indicates the first nucleotide of the sensor stop codon, the 2-flag indicates the second nucleotide of the sensor stop codon, and the 3-flag indicates the third nucleotide of the sensor stop codon. The 4-flag indicates an insertion in the sensor sequence to preserve the frame between the sensor stop codons. The corresponding target position has a gap (-) for the sensor. The 5-flag and 6-flag indicate mutations to the sensor sequence to eliminate unwanted stop codons or unwanted start codons, respectively. The 7-flag indicates a deletion in the sensor sequence to preserve the frame between the sensor stop codons, which are underlined. The fusion junction is indicated.

[0073] [Figure 20E] Figure 20E: Representative cell viability data (using the CellTiter-Glo 2.0 assay) in HEK293T cells transfected with the ADAR-Sensor-NTR vector (pmax-MCP_ADARddm(C377F,E488Q)-P2A-Sensor(TMEM135_CCDC67_Sensor_501)-E2A-NTR1.1-9xMS2) and either a control empty vector or a vector expressing the TMEM135-CCDC67 minigene. The presence of the fusion transcript, but not the empty vector, resulted in a decrease in cell viability, demonstrating specific detection and elimination of the cell population expressing the TMEM135-CCDC67 transcript in vivo in HEK293T.

[0074] [Figure 20F]Figure 20F: Alignment of target and sensor sequences for the ETV6-NTRK3 fusion. The alignment is shown in a group of three columns. The top column shows the target sequence in reverse (3' to 5'). The middle column shows number-coded flags for this alignment. The bottom column shows the sensor sequence in the 5' to 3' direction. The nucleotides are ordered to the codon encoded by the sensor. The 0-flag indicates that the target and sensor are aligned at that position. The 1-flag indicates the first nucleotide of the sensor stop codon, the 2-flag indicates the second nucleotide, and the 3-flag indicates the third nucleotide. The 4-flag indicates an insertion in the sensor sequence to preserve the frame between the sensor stop codons. The corresponding target position has a gap (-) for the sensor. The 5-flag and 6-flag indicate mutations to the sensor sequence to eliminate unwanted stop codons or unwanted start codons, respectively. The 7-flag indicates a deletion in the sensor sequence to preserve the frame between the sensor stop codons, which are underlined. The fusion junction is indicated.

[0075] [Figure 20G] Figure 20G: Representative microscopy images of HEK293T cells transfected with the ADAR-Sensor-NTR vector (pmax-MCP_ADARddm(C377F,E488Q)-P2A-Sensor(ETV6_NTRK3_Sensor_501)-E2A-NTR1.1-9xMS2) and either a control empty vector or a vector expressing the ETV6-NTRK3 minigene. The presence of the fusion transcript, but not the empty vector, resulted in a decrease in cell viability, demonstrating specific detection and elimination of the cell population expressing the ETV6-NTRK3 transcript in vivo in HEK293T.

[0076] [Figure 20H]Figure 20H: Alignment of target and sensor sequences for TMPRSS2-ERG fusion. The alignment is shown in a group of three columns. The top column shows the target sequence in reverse (3' to 5'). The middle column shows number-coded flags for this alignment. The bottom column shows the sensor sequence in the 5' to 3' direction. The nucleotides are ordered to the codon encoded by the sensor. The 0-flag indicates that the target and sensor are aligned at that position. The 1-flag indicates the first nucleotide of the sensor stop codon, the 2-flag indicates the second nucleotide, and the 3-flag indicates the third nucleotide. The 4-flag indicates an insertion in the sensor sequence to preserve the frame between the sensor stop codons. The corresponding target position has a gap (-) for the sensor. The 5-flag and 6-flag indicate mutations to the sensor sequence to eliminate unwanted stop codons or unwanted start codons, respectively. The 7-flag indicates a deletion in the sensor sequence to preserve the frame between the sensor stop codons, which are underlined. The fusion junction is indicated.

[0077] [Figure 20I] Figure 20I: Representative microscopy images of HEK293T cells transfected with the ADAR-Sensor-NTR vector (pmax-MCP_ADARddm(C377F,E488Q)-P2A-Sensor(TMPRSS2_ERG_Sensor_264)-E2A-NTR1.1-9xMS2) and either a control empty vector or a vector expressing the TMPRSS2-ERG minigene. The presence of the fusion transcript, but not the empty vector, resulted in a decrease in cell viability, demonstrating specific detection and elimination of the cell population expressing the TMPRSS2-ERG transcript in vivo in HEK293T.

[0078] [Figure 21A]Figure 21A: A representative schematic diagram of an exemplary ADAR-sensor-NTR with one sensory stop codon. The construct consists of the coding sequence of MCP-ADARddm(C377F,E488Q), a P2A peptide, a sensor sequence, an E2A peptide, an NTR1.1 coding sequence, and nine copies of the MS2 stem-loop (9xMS2). The 9xMS2 can recruit MCP-ADARddm(C377F,E488Q) expressed from the same RNA molecule.

[0079] [Figure 21B] Figure 21B: Alignment of target and sensor sequences for the TRMT11-GRIK2 fusion. The alignment is shown in a group of three columns. The top column shows the target sequence in reverse (3' to 5'). The middle column shows number-coded flags for this alignment. The bottom column shows the sensor sequence in the 5' to 3' direction. The nucleotides are ordered to the codon encoded by the sensor. The 0-flag indicates that the target and sensor are aligned at that position. The 1-flag indicates the first nucleotide of the sensor stop codon, the 2-flag indicates the second nucleotide of the sensor stop codon, and the 3-flag indicates the third nucleotide of the sensor stop codon. The 5-flag and 6-flag indicate mutations to the sensor sequence to eliminate unwanted stop codons or unwanted start codons, respectively. The sensor stop codon is underlined. The fusion junction is indicated.

[0080] [Figure 21C]Figure 21C: Representative cell viability data (using the CellTiter-Glo 2.0 assay) in HEK293T cells transfected with the ADAR-Sensor-NTR vector (pmax-MCP_ADARddm(C377F,E488Q)-P2A-Sensor(TRMT11_GRIK2ss_Sensor_201)-E2A-NTR1.1-9xMS2) and either a control empty vector or a vector expressing the TRMT11-GRIK2 minigene. The presence of the fusion transcript, but not the empty vector, resulted in a decrease in cell viability, demonstrating specific detection and elimination of the cell population expressing the TRMT11-GRIK2 transcript in vivo in HEK293T.

[0081] [Figure 21D] Figure 21D: Alignment of target and sensor sequences for PVT1-MYC fusion. The alignment is shown in a group of three columns. The top column shows the target sequence in reverse (3' to 5'). The middle column shows number-coded flags for this alignment. The bottom column shows the sensor sequence in the 5' to 3' direction. The nucleotides are ordered to the codon encoded by the sensor. The 0-flag indicates that the target and sensor are aligned at that position. The 1-flag indicates the first nucleotide of the sensor stop codon, the 2-flag indicates the second nucleotide of the sensor stop codon, and the 3-flag indicates the third nucleotide of the sensor stop codon. The 4-flag indicates an insertion in the sensor sequence to preserve the frame between the sensor stop codons. The corresponding target position has a gap (-) for the sensor. The 5-flag and 6-flag indicate mutations to the sensor sequence to eliminate unwanted stop codons or unwanted start codons, respectively. The 7-flag indicates a deletion in the sensor sequence to preserve the frame between the sensor stop codons, which are underlined. The fusion junction is indicated.

[0082] [Figure 21E]Figure 21E: Representative cell viability data (using the CellTiter-Glo 2.0 assay) in HEK293T cells transfected with the ADAR-Sensor-NTR vector (pmax-MCP_ADARddm(C377F,E488Q)-P2A-Sensor(PVT1_MYC_Sensor_498)-E2A-NTR1.1-9xMS2) and either a control empty vector or a vector expressing the PVT1-MYC minigene. The presence of the fusion transcript, but not the empty vector, resulted in a decrease in cell viability, demonstrating specific detection and elimination of the cell population expressing the PVT1-MYC transcript in vivo in HEK293T.

[0083] [Figure 22] FIG. 22: A representative schematic diagram of a nucleic acid ADAR-sensor-NTR designed to detect mutant transcripts, according to one embodiment of the present disclosure.

[0084] [Figure 23A] Figure 23A: A representative schematic diagram of an exemplary ADAR-sensor-NTR for the TP53(R248Q) mutant transcript. The construct consists of the coding sequence of MCP-ADARddm(C377F,E488Q), a P2A peptide, a sensor sequence, an XTEN80 linker peptide, an NTR1.1 coding sequence, and nine copies of the MS2 stem-loop (9xMS2). The 9xMS2 can recruit MCP-ADARddm(C377F,E488Q) expressed from the same RNA molecule.

[0085] [Figure 23B]Figure 23B: Alignment of target and sensor sequences for the TP53(R248Q) mutant transcript. The alignment is shown in a group of three columns. The top column shows the target sequence in reverse (3' to 5'). The middle column shows number-coded flags for this alignment. The bottom column shows the sensor sequence in the 5' to 3' direction. The nucleotides are ordered to the codon encoded by the sensor. The 0-flag indicates that the target and sensor are aligned at that position. The 1-flag indicates the first nucleotide of the sensor stop codon, the 2-flag indicates the second nucleotide of the sensor stop codon, and the 3-flag indicates the third nucleotide of the sensor stop codon. The 5-flag and 6-flag indicate mutations to the sensor sequence to eliminate unwanted stop codons or unwanted start codons, respectively. The sensor stop codons are underlined.

[0086] [Figure 23C] Figure 23C: Representative cell viability data (using the CellTiter-Glo 2.0 assay) of HEK293T cells transfected with the ADAR-Sensor-NTR vector (pmax-MCP_ADARddm(C377F,E488Q)-P2A-Sensor(TP53_R248Q_Sensor111)-E2A-NTR1.1-9xMS2) and either a plasmid expressing wild-type TP53 (pmax-TP53) or a plasmid expressing mutant TP53 (R248Q) (pmax-TP53(R248Q)). The presence of mutant TP53 (R248Q) transcripts, but not wild-type TP53 transcripts, resulted in decreased cell viability, demonstrating specific detection and elimination of the cell population expressing the TP53 (R248Q) mutant transcript in vivo in HEK293T. DETAILED DESCRIPTION OF THE INVENTION

[0087] (Detailed explanation) The present disclosure provides compositions and methods related to nucleic acid sensors. In particular, the present disclosure provides nucleic acid molecules that target transcripts of gene fusions (or viral transcripts, or mutant genes) to activate downstream therapeutic functions, thereby reducing or preventing the deleterious effects of the gene fusions (or viral transcripts, or mutant genes). In accordance with these embodiments, experiments were conducted to investigate the ability of nucleic acid sensors (e.g., RNA sensors) to target genomic abnormalities of gene fusions (or viral transcripts, or mutant genes) without prior knowledge of the function of the fusion genes (or viral transcripts, or mutant genes). As further described herein, the nucleic acid sensors of the present disclosure target specific gene fusions (or viral transcripts, or mutant genes) and, upon binding to the fusion sequence on an RNA transcript expressed from the fusion gene (or viral transcript, or mutant gene) in living cancer cells, trigger a programmed event (e.g., a cytotoxic event, expression of an immunostimulatory protein, or other therapeutic function). For example, to the extent that cancer cells express the fusion transcript (or viral transcript, or mutant gene), the RNA-sensor approach of the present disclosure can seek out these cancer cells and destroy them by triggering downstream apoptotic events or activation of a prodrug, or execution of other programmed therapeutic function. This platform technology combines the advantage of targeting only cancer cells that harbor the fusion (or viral transcript, or mutant gene) (specificity) with the savings of a great deal of time by eliminating the need to study the biology of the fusion before developing an appropriate therapeutic (rapid development). Furthermore, this platform technology can be easily reprogrammed to target many different types of cancers that harbor fusion genes and transcripts.

[0088] The section headings used in this section and throughout this disclosure are for organizational purposes only and are not intended to be limiting.

[0089] (1.Definition) Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. In case of conflict, the present specification (including definitions) will prevail. Preferred methods and materials are described below, but methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods and examples disclosed herein are merely illustrative and are not intended to be limiting.

[0090] As used herein, the terms "comprise(s)," "include(s)," "having," "has," "can," "contain(s)," and variations thereof are intended to be open-ended transitional phrases, terms, or phrases that do not exclude the possibility of additional actions or structures. The singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments that "comprise," "consist," and "consist essentially of" the embodiments or elements presented herein, whether expressly stated or not.

[0091] For the recitation of numerical ranges herein, each intervening number is expressly contemplated with the same precision. For example, for the range 6 to 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated.

[0092] As used herein, "correlated to" refers to compared to.

[0093] The term "single-stranded" oligonucleotide generally refers to an oligonucleotide comprising a series of covalently linked nucleotide residues.

[0094] The term "oligomer" or "oligonucleotide" includes RNA or DNA sequences of more than one nucleotide in either single-stranded or double-stranded form, and these terms particularly include short sequences (e.g., dimers and trimers) in either single-stranded or double-stranded form that may be intermediates in the production of specific binding oligonucleotides. "Modified" forms used in candidate pools contain at least one non-natural residue. "Oligonucleotide" or "oligomer" collectively refers to polydeoxyribonucleotides (containing 2'-deoxy-D-ribose or modified forms thereof) (e.g., DNA), polyribonucleotides (containing D-ribose or modified forms thereof) (e.g., RNA), and any other type of polynucleotide (which may be an N- or C-glycoside of a purine or pyrimidine base, or a modified purine or pyrimidine base, or an abasic nucleotide). "Oligonucleotide" or "oligomer" may also be used to describe artificial synthetic polymers similar to RNA and DNA, including but not limited to peptide nucleic acid (PNA) oligos. The term "RNA analog" or "RNA derivative" or "modified RNA" generally refers to polymeric molecules that, in addition to containing ribonucleosides as their units, also contain at least one of the following: 2'-deoxy, 2'-halo (including 2'-fluoro), 2'-amino (preferably unsubstituted or mono- or di-substituted), 2'-mono-halomethyl, 2'-di-halomethyl, or 2'-tri-halomethyl, 2'-O-alkyl, 2'-O-halo-substituted alkyl, 2'-alkyl, azido, phosphorothioate, sulfhydryl, methylphosphonate, fluorescein, rhodamine, pyrene, biotin, xanthine, hypoxanthine, 2,6-diaminopurine, 2-hydroxy-6-mercaptopurine, N1-methyl-pseudouridine-5'-triphosphate (N1meΨTP), and pyrimidine bases substituted with sulfur at the 6-position or halo or C at the 5-position. 1~5Alkyl-substituted pyrimidine bases, basic linkers, 3'-deoxy-adenosine, and other available "chain terminators" or "non-extendable" analogs at the 3' end of the RNA, or labels (e.g., 32 P, 33 P), etc. All of the above can be incorporated into RNA using standard synthesis techniques as disclosed herein.

[0095] The terms "binding activity" and "binding affinity" generally refer to the tendency of a ligand molecule to bind or not bind to a target. The energetics of these interactions are important in "binding activity" and "binding affinity" because they can include the definition of the concentrations of interacting partners, the rate at which these partners can associate, and the relative concentrations of bound and free molecules in solution.

[0096] "Sequence identity" refers to the degree to which two polymer sequences (e.g., peptides, polypeptides, nucleic acids, etc.) have the same sequential composition of monomer subunits. The term "sequence similarity" refers to the degree to which two polymer sequences (e.g., peptides, polypeptides, nucleic acids, etc.) have similar polymer sequences. For example, similar amino acids are those that share the same biophysical characteristics and can be grouped into families (e.g., acidic (e.g., aspartic acid, glutamic acid), basic (e.g., lysine, arginine, histidine), nonpolar (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine)). "Percent sequence identity" (or "percent sequence similarity") is calculated by: (1) comparing two optimally aligned sequences over a comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window); (2) determining the number of positions containing identical (or similar) monomers (e.g., the same amino acid is present in both sequences, similar amino acids are present in both sequences) to obtain the number of matched positions; (3) dividing the number of matched positions by the total number of positions in the comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window); and (4) multiplying the result by 100 to obtain the percent sequence identity or sequence similarity. For example, if peptide A and peptide B are both 20 amino acids long and have identical amino acids at all positions except one, peptide A and peptide B have 95% sequence identity. If the amino acids at non-identical positions share the same biophysical characteristics (e.g., both are acidic), peptide A and peptide B have 100% sequence similarity.As another example, if peptide C is 20 amino acids long and peptide D is 15 amino acids long, and 14 of the 15 amino acids in peptide D are identical to some amino acids in peptide C, then peptide C and peptide D have 70% sequence identity, but peptide D has 93.3% sequence identity over the optimal comparison window of peptide C. For purposes of calculating "percent sequence identity" (or "percent sequence similarity") herein, any gap in the aligned sequences is treated as a mismatch at that position.

[0097] 2. Compositions and Methods An embodiment of the present disclosure provides a single-stranded nucleic acid sensor molecule, comprising: a target sensing region having a nucleic acid sequence substantially complementary to a target nucleic acid, the target sensing region comprising a TAG or TGA stop codon opposite a corresponding CAA, CTA, CGA, ACA, TCA, GCA, CCA, CCT, or CCC triplet in the target nucleic acid located on at least one side of a junction sequence in the target nucleic acid; and a response gene located downstream of the target sensing region, the response gene being expressed when the TAG or TGA stop codon is converted to a TGG codon by RNA-specific adenosine deaminase (ADAR)-mediated gene editing upon binding of the sensor molecule to the target nucleic acid. In some embodiments, the sensor molecule itself constitutively expresses an ADAR or ADAR fusion, which enables ADAR-mediated gene editing upon binding of the sensor molecule to the target nucleic acid. Such a sensor is referred to herein as an "all-in-one" sensor.

[0098] In some embodiments, the sensor molecules provided herein are used to detect fusions (e.g., gene fusions, chromosome fusions). In some embodiments, the junction sequence of the target nucleic acid corresponds to the sequence spanning the junction between the components of the gene fusion or chromosome fusion. In other words, in some embodiments, the junction sequence is a sub-portion of the gene fusion or chromosome fusion sequence that includes the sequences on both ends of the fusion junction. In some embodiments, the sensor molecules provided herein are used to detect mutant or viral transcripts in which a fusion is not present. Although the junction sequence is often used herein in reference to a fusion, the term "junction sequence" does not require that a fusion be present in the target nucleic acid. In some embodiments, for example, when the target sequence is a mutant or viral transcript, the junction sequence of the target nucleic acid refers to a portion of the mutant or viral transcript.

[0099] This paper shows that this sensor molecule can be widely applied to various gene fusion or chromosome fusion targets.In some embodiments, this gene fusion or chromosome fusion is associated with cancer.For example, in some embodiments, this gene fusion or chromosome fusion is CBFA2T3-GLIS2 fusion sequence, EML4-ALK fusion sequence, ZFTA-RELA fusion sequence, EWSR1-FL1 fusion sequence, CCNH-C5orf30 fusion sequence, TMEM135-CCDC67 fusion sequence, EVT6-NTRK3 fusion sequence, TMPRSS2-ERG fusion sequence, TRMT11-GRIK2 fusion sequence or PVT1-MYC fusion sequence.

[0100] In some embodiments, the junction sequence of the target nucleic acid corresponds to a sequence spanning a portion of the TP53(R248Q) mutant transcript. In some embodiments, the junction sequence comprises at least a portion of a sequence corresponding to one of the chromosomal fusions or mutant transcripts listed above. Exemplary junction sequences and corresponding target sensing sequences are provided herein.

[0101] In some embodiments, the junction sequence of the target nucleic acid corresponds to a sequence spanning a portion of a viral transcript. In some embodiments, the viral transcript is a transcript associated with cancer. In some embodiments, the viral transcript is an Epstein-Barr virus (EBV) transcript or a Kaposi's sarcoma-associated herpesvirus (KSHV) transcript. In some embodiments, the viral transcript is the Epstein-Barr virus transcript EBNA1, and the target-sensing region comprises a nucleic acid sequence having at least 80% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to SEQ ID NO: 34.

[0102] In some embodiments, the viral transcript is KSHV transcript ORF71 and the target sensing region comprises a nucleic acid sequence having at least 80% sequence identity (e.g., at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) to SEQ ID NO:35.

[0103] In some embodiments, embodiments of the present disclosure activate a downstream event (eg, the production of a detectable signal corresponding to a target nucleic acid) or exert a therapeutic function.

[0104] In some embodiments, the target sensing region is at least about 50 nucleotides in length, hi some embodiments, the target sensing region is from about 50 nucleotides to about 1000 nucleotides in length.

[0105] In some embodiments, the response gene encodes at least one of a reporter protein, a caspase, a prodrug-converting enzyme, or an enzyme that catalyzes a specific reaction.

[0106] In some embodiments, the sensor molecule further comprises a control gene, wherein the control gene is constitutively expressed.

[0107] In some embodiments, the sensor molecule comprises a linker region located upstream of the response gene but downstream of the TAG or TGA stop codon.

[0108] In some embodiments, the sensor molecule comprises an RNA aptamer sequence, and the RNA aptamer sequence can be bound to its cognate binding protein.In some embodiments, the RNA aptamer comprises a sequence that can be bound to at least one of MS2, PP7, BoxB or Pumilio.In some embodiments, the cognate binding protein is fused to ADAR protein.

[0109] In some embodiments, the sensor molecule is an RNA molecule.

[0110] Embodiments of the present disclosure also include expression vectors that include DNA sequences corresponding to any of the RNA sensor molecules described herein.

[0111] In some embodiments, the sensor molecule is an RNA molecule. In some embodiments, the expression vector is selected from the group consisting of pCR8-mRuby2-P2A-ccdbCam-E2A-EGFP vector, pCR8-mRuby2-P2A-ccdbCam-E2A-EGFP-BsaI(agat) vector, pCR8-mRuby2-P2A-ccdbCam-E2A-EGFP-NxMS2 vector, pCR8-mRuby2-P2A-Sensor-E2A-EGFP vector, pCR8-mRuby2-P2A-Sensor-E2A-EGFP-NxMS2 vector, pmax-mRuby2-P2A-Sensor-XTEN80-EGFP-NxMS2 vector, and MCP -ADARdd(E488Q) vector;pmax-MCP-ADARdd(E488Q) vector;pmax-MCP-ADARdd(E488Q)-P2A-Sensor-XTEN80-NTR1.1-NxMS2 vector;pmax-MCP-ADARdd(E488Q)-P2A-Sensor -E2A-NTR1.1-NxMS2 vector;pmax-MCP-ADARddm(C377F,E488Q)-P2A-Sensor-XTEN80-NTR1.1-NxMS2 vector;pmax-MCP-ADARddm(C377F,E488Q)-P2A-Sensor-E2A-NTR1.1-NxMS2 vector;pmax-MCP-ADARdd(E488Q)-P2A-Sensor-XTEN80-DTA-NxMS2 vector;pmax-MCP-ADARdd(E488Q)-P2A-Sensor-E2A-DTA-NxMS2 vector;p max-MCP-ADARddm(C377F,E488Q)-P2A-Sensor-XTEN80-DTA-NxMS2 vector;pmax-MCP-ADARddm(C377F,E488Q)-P2A-Sensor-E2A-DTA-NxMS2 vector;p The vector is selected from the group consisting of max-MCP-ADARdd(E488Q)-P2A-Sensor-XTEN80-BAX-NxMS2 vector; pmax-MCP-ADARdd(E488Q)-P2A-Sensor-E2A-BAX-NxMS2 vector; pmax-MCP-ADARddm(C377F,E488Q)-P2A-Sensor-XTEN80-BAX-NxMS2 vector; and pmax-MCP-ADARddm(C377F,E488Q)-P2A-Sensor-E2A-BAX-NxMS2 vector.

[0112] Embodiments of the present disclosure also include cells comprising any of the RNA sensor molecules described herein or any of the vectors described herein.

[0113] Embodiments of the present disclosure also include kits that include any of the RNA sensor molecules described herein, any of the vectors described herein, and / or any of the cells described herein.

[0114]

[0010] Embodiments of the present disclosure also include methods of treating a subject with or suspected of having cancer. According to these embodiments, the method includes administering any of the RNA sensor molecules described herein, any of the vectors described herein, and / or any of the cells described herein to the subject; and treating the subject. In some embodiments, the cancer genome comprises a chromosomal translocation and / or a gene fusion. In some embodiments, the cancer cells comprise a viral genome and / or express viral transcripts. In some embodiments, the cancer cells comprise a mutation in an endogenous gene. The RNA sensor, vector, and / or cells can be administered to the subject by any suitable route, including parenteral routes (e.g., injection, such as intravenous injection, intramuscular injection, intraarterial injection, subcutaneous injection, etc.). In some embodiments, delivery of the sensor to the subject is achieved by use of a vector (e.g., a viral vector (e.g., AAV, virus-like particle)), nanoparticles (e.g., mRNA-lipid nanoparticles), or other suitable means.

[0115] Embodiments of the present disclosure also include methods for detecting gene fusion transcripts in cells. According to these embodiments, the method includes transfecting a cell with any of the RNA sensor molecules described herein or any of the vectors described herein, and evaluating the cell for expression of a reporter protein. [Example]

[0116] 3. Working Example It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods of the present disclosure described herein are readily applicable and discernible, and can be made using appropriate equivalents without departing from the scope of the present disclosure or from the aspects and embodiments disclosed herein. Now that the present disclosure has been described in detail, the same will be more clearly understood by reference to the following examples, which are intended only to illustrate certain aspects and embodiments of the present disclosure and should not be construed as limitations on the scope of the disclosure. The disclosures of all journal references, U.S. patents, and publications referenced herein are incorporated herein by reference in their entireties.

[0117] The present disclosure has multiple aspects, illustrated by the following non-limiting examples.

[0118] Example 1 (Design of Fusion RNA Sensor) Fusion genes arise from genome rearrangements and / or chromosomal deletions, placing two genes that are originally separated in the normal genome in close proximity, allowing the transcription machinery to transcribe a chimeric RNA transcript (Figure 1A). The chimeric transcript (fusion transcript) possesses a unique junction sequence as a result of the fusion, or joins together on the same nucleic acid molecule sequence derived from two originally separate genes. These unique features serve as sensing targets for our RNA sensor.

[0119] An exemplary RNA sensor of the present disclosure (e.g., FIGS. 1B-1E) can be composed, from 5' to 3', of: (i) a control sequence, optionally constitutively expressing a detectable gene product (e.g., red fluorescent protein (RFP)); (ii) a 2A peptide sequence, optionally allowing the downstream peptide to be separated from the upstream peptide; and (iii) a sensor sequence that is reverse-complementary to the target sequence and has one or more 5'-TAG-3' sensing triplets that oppose a 5'-CCA-3' triplet (or 5'-CAA-3', 5'-CTA-3', 5'-CGA-3', 5'-ACA-3', 5'-TCA-3', 5'-GCA-3', 5'-CCT-3', or 5'-CCC-3') on the target sequence, surrounding the junction sequence of the fusion transcript. (iv) optionally, a 2A peptide or protein linker sequence that either allows the downstream peptide to be separated from the upstream peptide or allows "spacing" of the downstream peptide from the upstream peptide; (v) a response gene (e.g., green fluorescent protein, a caspase, or a prodrug-converting enzyme) whose expression depends on binding to a target sequence having a 5'-CCA-3' triplet (or 5'-CAA-3', 5'-CTA-3', 5'-CGA-3', 5'-ACA-3', 5'-TCA-3', 5'-GCA-3', 5'-CCT-3', or 5'-CCC-3') and activation of the upstream sensor fragment when its 5'-TAG-3' stop codon is converted to a 5'-TGG-3' codon upon ADAR-mediated RNA editing.

[0120] In some embodiments, an RNA aptamer sequence (e.g., an MS2-binding site, a PP7-binding site, a BoxB-binding site, or a Pumilio-binding site) can be inserted upstream of the regulatory sequence, into the sensor sequence, or downstream of the response sequence, which can recruit a recombinant ADAR fused to a cognate aptamer-binding protein (e.g., an MS2 coat protein (MCP), a PP7 coat protein (PCP), a lambda N protein, or a Pumilio / PUF-HD domain).

[0121] Binding of the sensor fragment to the target sequence generates a C:A (or A:A, G:A) mismatch in the context of the mostly double-stranded RNA, presenting it as a substrate for RNA editing by endogenous or exogenously supplied ADARs. The RNA editing converts the A in the sensor RNA to an inosine, which is read as a G by the translational machinery. Because the edited A is in the context of a designed in-frame stop codon upstream of the response coding sequence, the A-to-I (A-to-G) conversion changes the stop codon to a 5'-TGG-3' codon encoding tryptophan, allowing the downstream response gene to be translated. The response gene may encode a fluorescent protein (e.g., GFP) to provide fluorescence upon detection of the target transcript. Alternatively, the response gene may encode a cell death protein (e.g., caspase), in which case apoptotic cell death is triggered upon target detection. Alternatively, the response gene encodes a prodrug-converting enzyme (e.g., bacterial nitroreductase (NTR)) that is expressed upon target detection, sensitizing the host cell and possibly nearby cells to the prodrug (e.g., CB1945 or MTZ). Alternatively, the response gene encodes an immunostimulatory or immunoattractive protein that attracts or recruits immune cells to the cellular microenvironment to elicit a local immune response and / or elimination of the target and nearby cells.

[0122] Example 2 (Cloning of vectors for fusion RNA sensors) To facilitate cloning and testing of various sensor sequences and structures, a vector system was created (Figure 2). For example, mRuby2 (control sequence), P2A, an AscI restriction site, ccdB-Cm (登録商標)The negative-positive selection cassette, FseI restriction site, E2A, and EGFP (response sequence) were assembled in 5'→3' order by multiple PCR reactions and cloned into the pCR8 / GW / TOPO Gateway donor vector by the TEDA ligation-free cloning method to generate the pCR8-mRuby2-P2A-ccdbCam-E2A-EGFP vector (Figure 2A). (登録商標) The negative-positive selection cassette, FseI restriction site, E2A, EGFP (response element), and BsaI restriction site capable of generating a 5'-agat-3' overhang were assembled in 5'→3' order by multiple PCR reactions and cloned into the pCR8 / GW / TOPO Gateway donor vector by the TEDA ligation-free cloning method to generate the pCR8-mRuby2-P2A-ccdbCam-E2A-EGFP-BsaI(agat) vector, which was then digested with BsaI enzyme, terminally dephosphorylated by Quick CIP, and ligated with an annealed double-stranded polyoligonucleotide encoding the MS2 stem-loop sequence to generate the pCR8-mRuby2-P2A-ccdbCam-E2A-EGFP-NxMS2 vector, which was then screened for various numbers of MS2 stem-loops (e.g., N = 9) (Figure 2B).

[0123] To insert the sensor sequence, the pCR8-mRuby2-P2A-ccdbCam-E2A-EGFP vector or the pCR8-mRuby2-P2A-ccdbCam-E2A-EGFP-NxMS2 vector was transfected with the ccdb-Cam vector. (登録商標)The selection cassette was double-digested with AscI and FseI restriction enzymes at the sites flanking it, and the intervening sequence was replaced by the sensor sequence provided by PCR or as a synthesized double-stranded DNA fragment via the TEDA method, resulting in the pCR8-mRuby2-P2A-Sensor-E2A-EGFP or pCR8-mRuby2-P2A-Sensor-E2A-EGFP-NxMS2 vectors (Figure 2C), which could then be shuttled into Gateway destination expression vectors (Figures 2D and 2E) to generate expression vectors for the sensor-responsive genes.

[0124] Example 3 (In vivo detection of CBFA2T3-GLIS2 fusion transcripts). Four sensor sequences were designed to detect the CBFA2T3-GLIS2 fusion sequence: 93 (CBFA2T3GLIS2_93_Sensor), 351 (CBFA2T3GLIS2_351_Sensor), and 495 (CBFA2T3GLIS2_495_Sensor), positioned approximately at the center of the fusion junction. A sensor consisting of four MS2 stem-loops inserted within the sensor region was also designed (CBFA2T3GLIS2_avidity5). Because the two sensor stop codons were not in frame with each other (the distance between them was not a multiple of three), an extra G was added to ensure that the two sensor stop codons were in frame with each other and with the upstream and downstream sequences (Figure 3A, sensor sequences, italicized G). All in-frame stops in the sensor region that were not part of the RNA editing substrate were mutated to non-stop codons (TAG → TGG, TAA → TAC, TGA → TGG), and all in-frame start codons (ATG) in the sensor region were mutated to ATC. A sensor-responsive expression vector was constructed according to the workflow in Example 2 (pmax-mRuby2-P2A-Sensor-E2A-EGFP-9xMS2). Minigenes (375-bp sequences on either side of the fusion transcript) and full-length CBFA2T3-GLIS were cloned into this pmax expression vector (pmax-CBFA2T3-GLIS2_FL or pmax-CBFA2T3-GLIS3_mini750) to serve as test fusion genes. An expression vector was constructed for the MS2 coat protein hyperactive ADAR (E488Q) fusion (pmax-MCP-ADARdd(E488Q)).The sensor response vector (pmax-mRuby2-P2A-Sensor-XTEN80-EGFP-9xMS2), the MCP-ADARdd(E488Q) vector (pmax-MCP-ADARdd(E488Q)), and either a control empty vector or vectors expressing the test fusion genes (pmax-CBFA2T3-GLIS2_FL or pmax-CBFA2T3-GLIS3_mini750) were transfected into HEK293T cells and analyzed for fluorescence by flow cytometry 48 h posttransfection (Figure 3B). The presence of the fusion transcript resulted in an increase in GFP signal that was not observed in cells transfected with the empty vector control, demonstrating specific detection of the fusion CBFA2T3-GLIS2 transcript in HEK293T cells in vivo. The reproducibility of the CBFA2T3-GLIS2_495 sensor probe with an E2A linker (N = 4) is also shown (Figure 3C).

[0125] Example 4 (Design of a fusion RNA sensor-NTR (nitroreductase) response). The chimeric transcript (fusion transcript) resulting from the fusion gene possesses a unique junction sequence that can serve as a target for detection (Figure 4). An RNA sensor-NTR response can be constructed to express nitroreductase (NTR) upon fusion transcript detection. The NTR expressed as a result of fusion transcript detection converts a prodrug (e.g., CB1954 (tretazicar)) into a cytotoxic agent that can cause cell death and diffuse to nearby cells, causing nearby cell death (also known as the bystander effect). Alternatively, MTZ (metronidazole) can be used to cause cell death in cells expressing the fusion transcript without the bystander effect.

[0126] One exemplary RNA sensor-NTR response of the present disclosure (e.g., FIG. 4 ) is composed, from 5′ to 3′, of: (i) a control sequence, optionally constitutively expressing a detectable gene product (e.g., red fluorescent protein (RFP)); (ii) a 2A peptide sequence, optionally allowing the downstream peptide to be separated from the upstream peptide; (iii) a sensor sequence reverse-complementary to the target sequence, having one or more 5′-TAG-3′ sensing triplets opposing a 5′-CCA-3′ triplet (or 5′-CAA-3′, 5′-CTA-3′, 5′-CGA-3′, 5′-ACA-3′, 5′-TCA-3′, 5′-GCA-3′, 5′-CCT-3′, or 5′-CCC-3′) on the target sequence, surrounding the junction sequence of the fusion transcript; (iv) a downstream (v) a nitroreductase (NTR) gene, whose expression depends on binding to a target sequence having a 5'-CCA-3' triplet (or 5'-CAA-3', 5'-CTA-3', 5'-CGA-3', 5'-ACA-3', 5'-TCA-3', 5'-GCA-3', 5'-CCT-3', or 5'-CCC-3') and activation of an upstream sensor fragment when its 5'-TAG-3' stop codon is converted to a 5'-TGG-3' codon upon ADAR-mediated RNA editing; and (vi) optionally, a set of binding sites (e.g., an MS2 stem-loop that can recruit an MS2 coat protein (MCP)-ADAR fusion).

[0127] Example 5 (In vivo detection of CBFA2T3-GLIS2 fusion transcripts by sensor-NTR to induce cell death in the presence of the CB1945 prodrug). The CBFA2T3GLIS2_495_Sensor was reprogrammed to express nitroreductase (NTR) upon detection of CBFA2T3-GLIS2 fusion transcripts by replacing its EGFP-responsive gene with the NTR1.1 coding sequence to generate pmax-mRuby2-P2A-Sensor(CBFA2T3GLIS2_495)-XTEN80-NTR1.1-9xMS2. The sensor region contains two sensory stop codons, followed by an XTEN80 protein linker, which is then followed by the NTR1.1 coding sequence and nine copies of the MS2 stem-loop (9xMS2). The 9xMS2 can recruit MCP-ADARdd(E488Q). Binding of the sensor region to the fusion transcript (detection) triggers editing of the two TAG stop codons to TGG codons, allowing the downstream NTR to be translated. The translated NTR can convert a prodrug (e.g., CB1954) into a cytotoxic drug, achieving cell ablation (Figure 5A). We cotransfected the sensor construct with pmax-MCP-ADARdd(E488Q) (expressing the MCP-ADARdd(E488Q) protein), empty vector (EV), pmax-CBFA2T3-GLIS2_FL (expressing the fusion transcript), or a combination of the non-fusion components pmax-CBFA2T3 and pmax-GLIS2 into HEK293T cells, and added the CB1954 prodrug 24 hours after transfection (Figure 5B). Cell viability was measured using the CellTiter-Glo 2.0 assay, and luminescent quantification of ATP indicated the presence of metabolically active cells 7 days after prodrug addition. The presence of the fusion transcript, but not the empty vector control or the non-fusion component combination, resulted in increased cell death in the presence of the CB1954 prodrug, indicating specific elimination of cells expressing the fusion CBFA2T3-GLIS2 transcript in vivo (Figure 5B).

[0128] Example 6 (In vivo detection of EML4-ALK fusion transcripts by sensor-NTR to induce cell death in the presence of the CB1945 prodrug). The EML4ALK_501_Sensor was reprogrammed to express nitroreductase (NTR) upon detection of the CBFA2T3-GLIS2 fusion transcript by replacing its EGFP-responsive gene with the NTR1.1 coding sequence to generate pmax-mRuby2-P2A-Sensor(EML4ALK_501)-E2A-NTR1.1-9xMS2. The sensor region contains two sensory stop codons, followed by an E2A peptide, the NTR1.1 coding sequence, and nine copies of the MS2 stem-loop (9xMS2). The 9xMS2 can recruit MCP-ADARdd(E488Q). Binding of the sensor region to the fusion transcript induces editing of the two TAG stop codons to TGG codons, allowing the downstream NTR to be translated. The translated NTR can convert a prodrug (e.g., CB1954) into a cytotoxic drug, achieving cell ablation (Figure 6A). We cotransfected HEK293T cells with the sensor construct, pmax-MCP-ADARdd(E488Q) (expressing the MCP-ADARdd(E488Q) protein), and either an empty vector (EV) or pmax-EML4-ALK(mini) (expressing a 750 fragment encompassing the EML4-ALK fusion junction), and added the CB1954 prodrug 24 hours after transfection (Figure 6B). Cell viability was measured using the CellTiter-Glo 2.0 assay, and luminescent quantification of ATP indicated the presence of metabolically active cells 7 days after prodrug addition. The presence of the fusion transcript, but not the empty vector control, resulted in increased cell death in the presence of the CB1954 prodrug, indicating specific elimination of cells expressing the fusion EML4-ALK transcript in vivo ( Fig. 6B ).

[0129] Example 7 (Design of an all-in-one ADAR-sensor-NTR (nitroreductase) construct). The chimeric transcript (fusion transcript) resulting from the fusion gene possesses a unique junction sequence that can serve as a target for detection (Figure 7). An all-in-one ADAR-sensor-NTR construct can be constructed to constitutively express a fusion protein containing an ADAR enzyme and an RNA sensor fragment linked to a nitroreductase (NTR) coding sequence that is translated upon fusion transcript detection. The NTR expressed as a result of fusion transcript detection converts a prodrug (e.g., CB1954 (tretazicar)) into a cytotoxic drug that can cause cell death and diffuse to nearby cells, resulting in the death of nearby cells (also known as the bystander effect). Alternatively, MTZ (metronidazole) can be used to cause cell death in cells expressing the fusion transcript without the bystander effect.

[0130] One exemplary ADAR-sensor-NTR construct of the present disclosure (e.g., FIG. 7 ) consists, from 5′ to 3′, of: (i) a coding sequence for an ADAR fusion protein (e.g., a fusion of an ADAR deaminase domain with an MS2 coat protein (MCP)); (ii) optionally, a 2A peptide sequence that allows the downstream peptide to be separated from the upstream peptide; (iii) a sensor sequence that is reverse-complementary to the target sequence and has one or more 5′-TAG-3′ sensing triplets that oppose a 5′-CCA-3′ (or 5′-CAA-3′, 5′-CTA-3′, 5′-CGA-3′, 5′-ACA-3′, 5′-TCA-3′, 5′-GCA-3′, 5′-CCT-3′, or 5′-CCC-3′) triplet on the target sequence, surrounding the junction sequence of the fusion transcript; and (iv) optionally, (v) a nitroreductase (NTR) gene, whose expression depends on binding to a target sequence having a 5'-CCA-3' triplet (or 5'-CAA-3', 5'-CTA-3', 5'-CGA-3', 5'-ACA-3', 5'-TCA-3', 5'-GCA-3', 5'-CCT-3', or 5'-CCC-3') and activation of an upstream sensor fragment when its 5'-TAG-3' stop codon is converted to a 5'-TGG-3' codon upon ADAR-mediated RNA editing; and (vi) optionally, a set of binding sites (e.g., an MS2 stem-loop that can recruit an MS2 coat protein (MCP)-ADAR fusion).

[0131] Example 8 (In vivo detection of CBFA2T3-GLIS2 fusion transcripts by all-in-one ADAR-sensor-NTR to induce cell death in the presence of the CB1945 prodrug.) pmax-MCP-ADAR-P2A-Sensor(CBFA2T3GLIS2_495)-E2A-NTR1.1-9xMS2 was constructed to express an RNA molecule containing the coding sequence for the ADAR deaminase domain fused to MCP (MCP-ADARdd(E488Q)), followed by a sequence encoding a P2A peptide, followed by a sensor region complementary to the target CBFA2T3-GLIS2 fusion transcript and two sensor stop codons surrounding the fusion junction, followed by the E2A peptide coding sequence and the NTR1.1 coding sequence, followed by nine copies of the MS2 stem-loop (9xMS2). The 9xMS2 can recruit MCP-ADARdd(E488Q) expressed from the same RNA molecule. Binding of the sensor domain to the fusion transcript induces editing of the two TAG stop codons to TGG codons, allowing the downstream NTR to be translated. The translated NTR can convert a prodrug (e.g., CB1954) into a cytotoxic drug, achieving cell ablation (Figure 8A). The all-in-one ADAR-sensor-NTR construct was cotransfected into HEK293T cells with either empty vector (EV), pmax-CBFA2T3-GLIS2_FL (expressing the fusion transcript), or a combination of the non-fusion components pmax-CBFA2T3 and pmax-GLIS2. The CB1954 prodrug was added 24 h posttransfection (Figure 8B). Cell viability was measured using the CellTiter-Glo 2.0 assay, and luminescent quantification of ATP indicated the presence of metabolically active cells 5 days after prodrug addition. The presence of the fusion transcript, but not the empty vector control or the non-fusion component combination, resulted in increased cell death in the presence of the CB1954 prodrug, indicating specific elimination of cells expressing the fusion CBFA2T3-GLIS2 transcript in vivo (Figure 8B).

[0132] Example 9 (In vivo detection of CBFA2T3-GLIS2 fusion transcripts by all-in-one ADAR-sensor-NTR constructs with various sensor lengths and ADAR mutants to induce cell death in the presence of the CB1945 prodrug). Various sensor lengths (L) complementary to the CBFA2T3-GLIS2 fusion transcripts were used. S To test the effect of sensor length on cell elimination efficacy, we constructed an ADAR-sensor-NTR construct with the C377F mutation (i.e., MCP-ADARddm(C377F,E488Q)) (Figure 9A-B). During cloning of the ADAR-sensor-NTR construct, we recovered an ADAR mutant with an additional C377F mutation (i.e., MCP-ADARddm(C377F,E488Q)), which was therefore included in the analysis. We cotransfected these all-in-one ADAR-sensor-NTR constructs with either empty vector (EV) or pmax-CBFA2T3-GLIS2_FL into HEK293T cells, and added the CB1954 prodrug 24 h after transfection (Figure 9C). Five days after prodrug addition, cell viability of samples expressing the CBFA2T3-GLIS2 fusion (fusion+) was measured using the CellTiter-Glo 2.0 assay to quantify metabolically active cells and normalized to cell viability of samples receiving the empty vector control (fusion-) (Figure 9C). S We observed a general increase in cell elimination activity (a decrease in normalized cell viability) with the addition of the MCP-ADARddm(C377F,E488Q) double mutant construct compared to the MCP-ADARdd(E488Q) single mutant construct (Figure 9C).

[0133] Example 10 (In vivo detection of EML4-ALK fusion transcripts by all-in-one ADAR-sensor-NTR constructs with various sensor lengths and ADAR mutants to induce cell death in the presence of CB1945 prodrug). Various sensor lengths (L SWe constructed ADAR-sensor-NTR constructs carrying single or double mutant ADARs with ALK (Figure 10A-B) to test the effect of sensor length on cell elimination efficacy. We co-transfected these all-in-one ADAR-sensor-NTR constructs with empty vector (EV) or pmax-EML4-ALK into HEK293T cells and added the CB1954 prodrug 24 hours after transfection (Figure 10C). Five days after prodrug addition, cell viability of samples expressing the EML4-ALK fusion (fusion+) was measured by quantifying metabolically active cells using the CellTiter-Glo 2.0 assay and normalized to the cell viability of samples receiving the empty vector control (fusion-) (Figure 10C). We observed that increasing sensor length L S We observed a general increase in cell elimination activity (a decrease in normalized cell viability) with the addition of the MCP-ADARddm(C377F,E488Q) double mutant construct compared to the MCP-ADARdd(E488Q) single mutant construct (Figure 10C).

[0134] Example 11 (In vivo detection of ZFTA-RELA fusion transcripts by all-in-one ADAR-sensor-NTR constructs with various sensor lengths to induce cell death in the presence of CB1945 prodrug). Various sensor lengths (L) complementary to ZFTA-RELA fusion transcripts were used. SWe constructed ADAR-sensor-NTR constructs with either the empty vector (EV) or pmax-ZFTA-RELA to test the effect of sensor length on cell elimination efficacy (Figure 11A-B). We co-transfected these all-in-one ADAR-sensor-NTR constructs with either the empty vector (EV) or pmax-ZFTA-RELA into HEK293T cells and added the CB1954 prodrug 24 hours after transfection (Figure 11C). Five days after prodrug addition, cell viability of samples expressing the ZFTA-RELA fusion (fusion+) was measured by quantifying metabolically active cells using the CellTiter-Glo 2.0 assay and normalized to cell viability of samples receiving the empty vector control (fusion-) (Figure 11C). We used 90 or 150 L S Compared with the sensor length L of 501 S We observed increased cell ablation activity (a decrease in normalized cell viability) in the presence of α- and β-actin (Figure 11C). We further tested the sensor in ablation of BDX-1425EPN cancer cells derived from ST-EPN-RELA tumors expressing an endogenous ZFTA-RELA fusion transcript (Figure 11D). We engineered a lentiviral vector carrying an all-in-one ADAR-sensor-NTR for ZFTA-RELA. We produced viral particles by cotransfecting Lenti-X 293T cells with a mixture of pLP1, pLP2, and VSV-G and a doxycycline-inducible lentiviral vector carrying the ADAR-sensor-NTR. Virus was collected from the Lenti-X 293T supernatant, filtered through a 45 μM PES filter, and concentrated to 1:100 using a Lenti-X Concentrator. The day before transduction, BXD-1425EPN cells were cultured in 24-well plates at 1.0 × 10 5Cells were seeded at a density of 10,000 cells / well. Prior to transduction, the medium was replaced with medium (0.5 mL / well) containing 10 μg / mL polybrene. 25 μL of virus was added to each well and incubated overnight. 24 hours after transduction, the medium was replaced. Two days after transduction, medium containing 2 μg / mL puromycin was added to each well. Antibiotic selection was continued for 10 days before seeding for cell removal experiments. Non-transduced (control) BXD-1425EPN cells and transduced BXD-1425EPN cells were seeded at a density of approximately 10,000 cells / well into wells of a 96-well plate in medium containing doxycycline (100 ng / mL). The CB1954 prodrug was added 24 hours after seeding, and 5 days after prodrug addition, cell viability of the samples was measured by quantifying metabolically active cells using the CellTiter-Glo 2.0 assay. BDX-1425EPN cells transduced with the all-in-one ADAR-sensor-NTR targeting ZFTA-RELA exhibited significant cell death in the presence of the CB1954 prodrug compared with untransduced cells, demonstrating the effectiveness of the all-in-one ADAR-sensor-NTR in eliminating cancer cells expressing the endogenous fusion transcript (Figure 11E).

[0135] Example 12 In vivo detection of EWSR1-FLI1 fusion transcripts using an all-in-one ADAR-sensor-NTR construct to induce cell death in the presence of the CB1945 prodrug. An ADAR-sensor-NTR construct carrying a 501-nt sensor complementary to the EWSR1-FLI1 fusion transcript was constructed to eliminate cells expressing the EWSR1-FLI1 fusion transcript (Figure 12A-B). We cotransfected the all-in-one ADAR-sensor-NTR construct with either empty vector (EV) or pmax-EWSR1-FL1 into HEK293T cells and added the CB1945 prodrug 24 hours after transfection (Figure 12C). Two and five days after prodrug addition, cell viability of samples expressing the EWSR1-FLI1 fusion (fusion+) was measured using the CellTiter-Glo 2.0 assay to quantify metabolically active cells and normalized to cell viability of samples receiving the empty vector control (fusion-) (Figure 12C). The presence of the fusion transcript, but not the empty vector control, resulted in increased cell death in the presence of the prodrug on day 2, with more pronounced cell death on day 5, indicating specific elimination of cells expressing the EWSR1-FLI1 fusion transcript in vivo (Figure 12C).

[0136] Example 13 (Sensor-DTA or ADAR-sensor-DTA (diphtheria toxin fragment A) design). The chimeric transcript (fusion transcript) resulting from the fusion gene possesses a unique junction sequence that can serve as a target for detection ( FIG. 13 ). Sensor-DTA or ADAR-sensor-DTA constructs can be constructed to optionally express a fusion protein containing an ADAR enzyme or fluorescent marker protein and an RNA sensor fragment linked to a diphtheria toxin fragment A (DTA) coding sequence that is translated upon fusion transcript detection. The DTA expressed as a result of fusion transcript detection results in cytotoxicity and target cell death.

[0137] One exemplary sensor-DTA construct or ADAR-sensor-DTA construct of the present disclosure (e.g., FIG. 13 ) is composed, from 5′ to 3′, of: (i) optionally, a coding sequence for an ADAR fusion protein (e.g., a fusion of an ADAR deaminase domain with an MS2 coat protein (MCP) or a fluorescent marker protein); (ii) optionally, a 2A peptide sequence that allows the downstream peptide to be separated from the upstream peptide; (iii) a sensor sequence that is reverse-complementary to a target sequence and has one or more 5′-TAG-3′ sensing triplets that oppose a 5′-CCA-3′ (or 5′-CAA-3′, 5′-CTA-3′, 5′-CGA-3′, 5′-ACA-3′, 5′-TCA-3′, 5′-GCA-3′, 5′-CCT-3′, or 5′-CCC-3′) triplet on the target sequence, surrounding the junction sequence of the fusion transcript. (iv) optionally, a 2A peptide or protein linker sequence that either allows the downstream peptide to be separated from the upstream peptide or allows "spacing" of the downstream peptide from the upstream peptide; (v) a diphtheria toxin fragment A (DTA) gene, whose expression depends on binding to a target sequence having a 5'-CCA-3' (or 5'-CAA-3', 5'-CTA-3', 5'-CGA-3', 5'-ACA-3', 5'-TCA-3', 5'-GCA-3', 5'-CCT-3', or 5'-CCC-3') triplet and activation of an upstream sensor fragment when its 5'-TAG-3' stop codon is converted to a 5'-TGG-3' codon upon ADAR-mediated RNA editing; and (vi) optionally, a group of binding sites (e.g., an MS2 stem-loop that can recruit an MS2 coat protein (MCP)-ADAR fusion).

[0138] Example 14 (In vivo detection of CBFA2T3-GLIS2 fusion transcripts by all-in-one ADAR-Sensor-DTA for inducing cytotoxicity and targeted cell death). pmax-MCP-ADAR-P2A-Sensor(CBFA2T3GLIS2_495)-E2A-DTA.1-9xMS2 was constructed to express an RNA molecule containing the coding sequence for an ADAR deaminase domain fused to MCP (MCP-ADARdd(E488Q)), followed by a sequence encoding a P2A peptide, followed by a sensor region complementary to the target CBFA2T3-GLIS2 fusion transcript and two sensor stop codons surrounding the fusion junction, followed by the coding sequence for the E2A peptide and the DTA, followed by nine copies of an MS2 stem-loop (9xMS2). The 9xMS2 can recruit MCP-ADARdd(E488Q) expressed from the same RNA molecule. Binding of the sensor domain to the fusion transcript induces editing of the two TAG stop codons to TGG codons, allowing the downstream DTA to be translated. Target-dependent expression of the DTA is cytotoxic, inducing target cell death (Figure 14A). The all-in-one ADAR-sensor-DTA construct was cotransfected with either empty vector (EV) or pmax-CBFA2T3-GLIS2_FL into HEK293T cells (Figure 14B). Cell viability was measured using the CellTiter-Glo 2.0 assay at 2 and 5 days posttransfection, and luminescent quantification of ATP indicated the presence of metabolically active cells. The presence of the fusion transcript, but not the empty vector control, resulted in increased cell death in transfected cells at day 2, which became more pronounced at day 5, indicating specific elimination of cells expressing the fusion CBFA2T3-GLIS2 transcript in vivo (Figure 14B).

[0139] Example 15 (Design of Sensor-BAX or ADAR-Sensor-BAX (BCL2-associated X, apoptosis regulator)). The chimeric transcript (fusion transcript) resulting from the fusion gene possesses a unique junction sequence that can serve as a target for detection (Figure 15). Sensor-BAX or ADAR-Sensor-BAX constructs can be constructed to optionally express a fusion protein containing an ADAR enzyme or fluorescent marker protein and an RNA sensor fragment linked to the coding sequence of the apoptosis regulator BCL2-associated X (BAX), which is translated upon detection of the fusion transcript. The expressed BAX protein induces or promotes apoptosis in target cells.

[0140] One exemplary sensor-BAX or ADAR-sensor-BAX construct of the present disclosure (e.g., FIG. 15 ) consists, from 5′ to 3′, of: (i) optionally, a coding sequence for an ADAR fusion protein (e.g., a fusion of an ADAR deaminase domain with an MS2 coat protein (MCP) or a fluorescent marker protein); (ii) optionally, a 2A peptide sequence that allows the downstream peptide to be separated from the upstream peptide; and (iii) a sensor sequence that is reverse-complementary to the target sequence, comprising one or more 5′-TAG-3′ sensing triplets opposite a 5′-CCA-3′ (or 5′-CAA-3′, 5′-CTA-3′, 5′-CGA-3′, 5′-ACA-3′, 5′-TCA-3′, 5′-GCA-3′, 5′-CCT-3′, or 5′-CCC-3′) triplet on the target sequence, surrounding the junction sequence of the fusion transcript. (iv) optionally, a 2A peptide or protein linker sequence that either allows the downstream peptide to be separated from the upstream peptide or allows "spacing" of the downstream peptide from the upstream peptide; (v) the BCL2-associated X (BAX) gene, whose expression depends on binding to a target sequence having a 5'-CCA-3' (or 5'-CAA-3', 5'-CTA-3', 5'-CGA-3', 5'-ACA-3', 5'-TCA-3', 5'-GCA-3', 5'-CCT-3', or 5'-CCC-3') triplet and activation of the upstream sensor fragment when its 5'-TAG-3' stop codon is converted to a 5'-TGG-3' codon upon ADAR-mediated RNA editing; and (vi) optionally, a group of binding sites (e.g., an MS2 stem-loop that can recruit an MS2 coat protein (MCP)-ADAR fusion).

[0141] Example 16 In vivo detection of CBFA2T3-GLIS2 fusion transcripts by all-in-one ADAR-sensor-BAX to induce apoptosis in target cells. pmax-MCP-ADAR-P2A-Sensor(CBFA2T3GLIS2_495)-E2A-BAX-9xMS2 and pmax-MCP-ADAR-P2A-Sensor(CBFA2T3GLIS2_495)-XTEN80-BAX-9xMS2 were constructed to express RNA molecules containing the coding sequence for the ADAR deaminase domain fused to MCP (MCP-ADARdd(E488Q)), followed by a sequence encoding a P2A peptide, followed by a sensor region complementary to the target CBFA2T3-GLIS2 fusion transcript and two sensor stop codons surrounding the fusion junction, followed by the coding sequence for the E2A peptide (or XTEN80, respectively) and BAX, followed by nine copies of the MS2 stem-loop (9xMS2). The 9xMS2 can recruit MCP-ADARdd(E488Q) expressed from the same RNA molecule. Binding of the sensor domain to the fusion transcript triggers editing of the two TAG stop codons to TGG codons, allowing downstream BAX to be translated. Target-dependent expression of BAX induces apoptosis in target cells (Figure 16A). The all-in-one ADAR-sensor-BAX construct was cotransfected with either empty vector (EV) or pmax-CBFA2T3-GLIS2_FL into HEK293T cells (Figure 16B). Two days after transfection, cell viability was measured by quantifying metabolically active cells using the CellTiter-Glo 2.0 assay. The presence of the fusion transcript, but not the empty vector control, resulted in increased cell death in transfected cells, indicating specific elimination of cells expressing the fusion CBFA2T3-GLIS2 transcript in vivo (Figure 16B).

[0142] Example 17 (Design of Sensor-NTR or ADAR-Sensor-NTR for Viral Transcripts) Virus-infected cells and some cancer cells express viral transcripts that are not present in uninfected or normal cells. These viral transcripts serve as unique fingerprints for these cells (Figure 17). Sensor-NTR or ADAR-sensor-NTR constructs can be constructed to optionally express a fusion protein containing an ADAR enzyme or fluorescent marker protein and an RNA sensor fragment linked to the coding sequence of nitroreductase (NTR), which is translated upon detection of the fusion transcript. The NTR expressed as a result of viral transcript detection converts a prodrug (e.g., CB1954 (Tretazicar)) into a cytotoxic agent that can cause cell death and diffuse to nearby cells, causing nearby cell death (also known as the bystander effect). Alternatively, MTZ (metronidazole) can be used to cause cell death in cells expressing the fusion transcript without the bystander effect.

[0143] One exemplary sensor-NTR construct or ADAR-sensor-NTR construct of the present disclosure (e.g., FIG. 17) is composed, from 5' to 3', of: (i) optionally, a coding sequence for an ADAR fusion protein (e.g., a fusion of an ADAR deaminase domain with an MS2 coat protein (MCP) or a fluorescent protein); (ii) optionally, a 2A peptide sequence that allows the downstream peptide to be separated from the upstream peptide; (iii) a sensor sequence that is reverse-complementary to a target viral sequence and has one or more 5'-TAG-3' sensing triplets opposite a 5'-CCA-3' (or 5'-CAA-3', 5'-CTA-3', 5'-CGA-3', 5'-ACA-3', 5'-TCA-3', 5'-GCA-3', 5'-CCT-3', or 5'-CCC-3') triplet on the target sequence on the target viral transcript. (iv) an optional 2A peptide or protein linker sequence that either allows the downstream peptide to be separated from the upstream peptide or allows "spacing" of the downstream peptide from the upstream peptide; (v) a nitroreductase (NTR) gene, whose expression depends on binding to a target sequence having a 5'-CCA-3' (or 5'-CAA-3', 5'-CTA-3', 5'-CGA-3', 5'-ACA-3', 5'-TCA-3', 5'-GCA-3', 5'-CCT-3', or 5'-CCC-3') triplet and activation of an upstream sensor fragment when its 5'-TAG-3' stop codon is converted to a 5'-TGG-3' codon during ADAR-mediated RNA editing; and (vi) an optional set of binding sites (e.g., an MS2 stem-loop that can recruit an MS2 coat protein (MCP)-ADAR fusion).

[0144] Example 18 (In vivo detection of Epstein-Barr virus (EBV)-EBNA1 transcripts by an all-in-one ADAR-sensor-NTR to induce cell death in the presence of the CB1945 prodrug). Epstein-Barr virus (EBV) is present in nasopharyngeal carcinoma, some gastric cancers, and some lymphomas. The virus remains in a latent cycle and expresses latent genes (e.g., EBNA1). We constructed the plasmid pmax-MCP-ADAR-P2A-Sensor(EBNA1_501)-XTEN80-NTR1.1-9xMS2 to express an RNA molecule containing the coding sequence for an ADAR deaminase domain fused to MCP (MCP-ADARdd(E488Q)), followed by a P2A peptide coding sequence, followed by a sensor region complementary to the target Epstein-Barr virus (EBV)-EBNA1 transcript with a sensor stop codon complementary to the target CCA triplet on the EBNA1 transcript, followed by the coding sequence for the XTEN80 peptide and the coding sequence for NTR1.1, followed by nine copies of the MS2 stem-loop (9xMS2) that can recruit MCP-ADARdd(E488Q) expressed from the same RNA molecule. Binding of the sensor region to the viral transcript induces editing of the TAG stop codon to a TGG codon, allowing the downstream NTR to be translated. The translated NTR can convert a prodrug (e.g., CB1954) into a cytotoxic drug, achieving cell ablation (Figures 18A-B). We cotransfected the all-in-one ADAR-sensor-NTR construct with either an empty vector (EV) or pmax-EBNA1 (expressing the EBV-EBNA1 transcript) into HEK293T cells and added the CB1954 prodrug 24 hours after transfection (Figure 18C). Seven days after prodrug addition, cell viability was measured by quantifying metabolically active cells using the CellTiter-Glo 2.0 assay. The presence of EBV-EBNA1 transcripts, but not empty vector controls, resulted in increased cell death in the presence of the CB1954 prodrug, indicating specific elimination of cells expressing EBV-EBNA1 transcripts in vivo (Fig. 18C).

[0145] Example 19 (In vivo detection of Kaposi's sarcoma-associated herpesvirus (KSHV)-ORF71 transcripts by an all-in-one ADAR-sensor-NTR to induce cell death in the presence of the CB1945 prodrug). Kaposi's sarcoma-associated herpesvirus (KSHV) is present in some sarcomas. During latency, the virus expresses latent genes (e.g., ORF71). The plasmid pmax-MCP-ADAR-P2A-Sensor(KSHV_ORF71_501)-XTEN80-NTR1.1-9xMS2 was constructed to express an RNA molecule containing the coding sequence for an ADAR deaminase domain fused to MCP (MCP-ADARdd(E488Q)), followed by a P2A peptide coding sequence, followed by a sensor region complementary to a target Kaposi's sarcoma-associated herpesvirus (KSHV)-ORF71 transcript, followed by a sensor stop codon complementary to a target CCA triplet on the KSHV-ORF71 transcript, followed by the coding sequence for the XTEN80 peptide and NTR1.1, followed by nine copies of the MS2 stem-loop (9xMS2) that can recruit MCP-ADARdd(E488Q) expressed from the same RNA molecule. Binding of the sensor region to the viral transcript induces editing of the TAG stop codon to a TGG codon, allowing the downstream NTR to be translated. The translated NTR can convert a prodrug (e.g., CB1954) into a cytotoxic drug, achieving cell ablation (Figures 19A-19B). The all-in-one ADAR-sensor-NTR construct was cotransfected with either an empty vector (EV) or pOME0343_ORF71 tagged (expressing KSHV-ORF71 transcript) into HEK293T cells, and the CB1954 prodrug was added 24 hours after transfection (Figure 19C). Seven days after prodrug addition, cell viability was measured by quantifying metabolically active cells using the CellTiter-Glo 2.0 assay. The presence of KSHV-ORF71 transcripts, but not empty vector controls, resulted in increased cell death in the presence of the CB1954 prodrug, indicating specific elimination of cells expressing KSHV-ORF71 transcripts in vivo (Fig. 19C ).

[0146] Example 20 (Exemplary software for designing sensor sequences) A ​​python program was created to facilitate the design of sensor sequences. This program accepts a target sequence in uppercase and its core sensing nucleotide (e.g., the middle cytosine (C) in a CCA triplet) in lowercase (i.e., CcA). The program first creates the reverse complement of the target sequence as the initial sensor sequence. Then, the nucleotide opposite the sensing nucleotide marked with a lowercase letter in the target is converted to adenosine (A), allowing ADAR to convert the adenosine (A) to inosine upon target binding. For sensing triplets other than CCA, the immediately upstream nucleotide is converted to T and the immediately downstream nucleotide is converted to G, respectively, to create a sensing stop codon (TAG). Then, for sensors with more than one sensing triplet, if the two sensing stop codons are not in frame (i.e., the number of nucleotides between the sensing stop codons is not a multiple of three), frame-correcting nucleotides are added approximately midway between the sensing stop codons on that sensor. The frame-correcting nucleotides ensure that the sensing stop codons are in frame with each other. Alternatively, an option (enabled by the -deletePlus1 flag) is available to delete nucleotides between the sensing stop codons to ensure that the stop codons are in frame. The sensor sequence is then scanned for stop codons that are not involved in sensing, and the unwanted stop codons are converted as follows: TAA → TAc, TAG → TgG, TGA → TGg. The sensor sequence is also scanned for unwanted start codons after the first sensing stop codon, converting them from ATG to AgG. Alternatively, an option (enabled by (-removeAllATG)) is available to convert all ATGs to AgGs. The program then outputs the target sequence, the sensor sequence, and the target-sensor alignment.To further facilitate bulk design efforts across databases of fusion transcripts, we created another Python program (BulkSensorRNADesign.py includes CODE LISTING as TXT or FIG?) to scan fusion transcript databases and automate the design of sensor sequences across tens of thousands of fusion sequences. The default setting for the program is to design sensors with two sensitive stop codons, but an option (--singleSTOP) is available to instruct the program to design sensors with one sensitive stop codon. The sensitive triplet defaults to CCA, but an option (--allowedTriplets) is available to include other triplets that can be used by ADAR (e.g., the nine triplets: CAA, CTA, CGA, ACA, TCA, GCA, CCA, CCT, or CCC). Furthermore, the program allows users to specify the minimum distance (--minDist) and maximum distance (--maxDist) between sensory stop codons, the padding size (--padding), which is the number of nucleotides before and after the sensory stop codons, and the number of sensors to design per target (--numOfSensorsPerTarget). The program receives annotations of fusion transcripts from FusionGDB (e.g., ccsm.uth.edu / FusionGDB / tables / TCGA_ChiTaRS_combined_fusion_ORF_analyzed_gencode_h19v19_In-frame_100k_check_cds_seq.txt). The program scans each fusion transcript in the database to identify the sensory triplets upstream and downstream of the fusion junction (breakpoint). The program then generates target design sequences, minimizing the distance between sensed stop codons and formatting them according to the requirements of SensorRNADesigner.py (i.e., sensed nucleotides in lower case within the context of upper case sequences).It calls functions in SensorRNADesigner.py to write the input target design sequence, sensor sequence, and target-sensor alignment for each design to a file. If more than one sensor design is requested, it outputs the next sensor design with the next shortest sensor stop codon distance, and so on, until it reaches the number of sensors to be designed or exhausts all possible designs according to its parameters.

[0147] Example 21 (In vivo detection of CCNH-C5orf30 fusion transcripts, TMEM135-CCDC67 fusion transcripts, EVT6-NTRK3 fusion transcripts, and TMPRSS2-ERG fusion transcripts by all-in-one ADAR-sensor-NTR to induce cell death in the presence of the CB1945 prodrug.) RNA sensors were designed to target CCNH-C5orf30 fusion transcripts, TMEM135-CCDC67 fusion transcripts, ETV6-NTRK3 fusion transcripts, and TMPRSS2-ERG fusion transcripts. These RNA sensors contain the coding sequence for a double mutant ADAR deaminase domain fused to MCP (MCP-ADARddm(C377F,E488Q)), followed by the coding sequence for a P2A peptide, followed by a sensor region complementary to the target fusion transcript and two sensor stop codons surrounding the fusion junction, followed by the coding sequence for an E2A peptide and an NTR1.1 coding sequence, followed by nine copies of an MS2 stem-loop (9xMS2). The 9xMS2 can recruit MCP-ADARddm(C377F,E488Q) expressed from the same RNA molecule. Binding of the sensor region to the fusion transcript induces editing of the two TAG stop codons to TGG codons, allowing the downstream NTR to be translated. The translated NTR can convert a prodrug (e.g., CB1954) into a cytotoxic drug, achieving cell ablation (Figure 20A). For each fusion target, the all-in-one ADAR-Sensor-NTR construct was co-transfected with either an empty vector (EV) or a fusion minigene into HEK293T cells. 24 hours after transfection, the CB1954 prodrug was added. Cell viability was then measured 7 days after drug addition using the CellTiterGlo assay or microscopy (Figure 20A). CCNH_C5orf30_Sensor_501-loaded ADAR-Sensor-NTR (Figure 20B) was able to specifically eliminate cells in the presence of the CCNH-C5orf30 fusion transcript (Figure 20C).ADAR-Sensor-NTR equipped with TMEM135_CCDC67_Sensor_501 (Figure 20D) was able to specifically eliminate cells in the presence of TMEM135-CCDC67 fusion transcripts (Figure 20E). ADAR-Sensor-NTR equipped with EVT6_NTRK3_Sensor_501 (Figure 20F) was able to specifically eliminate cells in the presence of EVT6-NTRK3 fusion transcripts (Figure 20G). ADAR-Sensor-NTR equipped with TMPRSS2_ERG_Sensor_264 (Figure 20H) was able to specifically eliminate cells in the presence of TMPRSS2-ERG fusion transcripts (Figure 20I).

[0148] Example 22 (In vivo detection of TRMT11-GRIK2 and PVT1-MYC fusion transcripts by an all-in-one ADAR-sensor-NTR to induce cell death in the presence of the CB1945 prodrug). To target TRMT11-GRIK2 and PVT1-MYC fusion transcripts, we designed RNA sensors. These RNA sensors contain the coding sequence of a double mutant ADAR deaminase domain fused to MCP (MCP-ADARddm(C377F,E488Q)), followed by the coding sequence of a P2A peptide, followed by a sensor region complementary to the target fusion transcript and a single sensor stop codon proximal to the fusion junction, followed by the coding sequence of an E2A peptide and an NTR1.1 coding sequence, followed by nine copies of the MS2 stem-loop (9xMS2). The 9xMS2 can recruit the MCP- MCP-ADARddm (C377F, E488Q) expressed from the same RNA molecule. Binding of the sensor region to the fusion transcript induces editing of the TAG stop codon to a TGG codon, allowing the downstream NTR to be translated. The translated NTR can convert a prodrug (e.g., CB1954) into a cytotoxic drug, achieving cell ablation (Figure 21A). For each fusion target, the all-in-one ADAR-sensor-NTR construct was co-transfected with either an empty vector (EV) or a fusion minigene into HEK293T cells. 24 hours after transfection, CB1954 prodrug was added. Cell viability was then measured 7 days after drug addition using the CellTiterGlo assay (Figure 21A). ADAR-Sensor-NTR carrying TRMT11_GRIK2ss_Sensor_201 (Figure 21B) was able to specifically eliminate cells containing TRMT11-GRIK2 fusion transcripts (Figure 21C), and ADAR-Sensor-NTR carrying PVT1_MYC_Sensor_498 (Figure 21D) was able to specifically eliminate cells containing PVT1-MYC fusion transcripts (Figure 21E).

[0149] Example 23 (Design of Sensor-NTR or ADAR-Sensor-NTR for Mutant Transcripts). Somatic mutations can lead to diseases such as cancer. These mutant transcripts serve as unique fingerprints for these cells (Figure 22). Sensor-NTR or ADAR-Sensor-NTR constructs can be constructed to optionally express a fusion protein containing an ADAR enzyme or fluorescent marker protein and an RNA sensor fragment linked to the coding sequence of nitroreductase (NTR), which is translated upon mutant transcript detection. The NTR expressed as a result of fusion transcript detection converts a prodrug (e.g., CB1954 (Tretazicar)) into a cytotoxic agent that can cause cell death and diffuse to nearby cells, causing nearby cell death (also known as the bystander effect). Alternatively, MTZ (metronidazole) can be used to cause cell death in cells expressing the fusion transcript without the bystander effect.

[0150] One exemplary sensor-NTR construct or ADAR-sensor-NTR construct of the present disclosure (e.g., FIG. 22) is composed, from 5' to 3', of: (i) optionally, a coding sequence for an ADAR fusion protein (e.g., a fusion of an ADAR deaminase domain with an MS2 coat protein (MCP) or a fluorescent protein); (ii) optionally, a 2A peptide sequence that allows the downstream peptide to be separated from the upstream peptide; (iii) a sensor sequence that is reverse-complementary to the target sequence, having one or more 5'-TAG-3' sensing triplets opposite a 5'-CCA-3' (or 5'-CAA-3', 5'-CTA-3', 5'-CGA-3', 5'-ACA-3', 5'-TCA-3', 5'-GCA-3', 5'-CCT-3', or 5'-CCC-3') triplet on the target sequence on the target viral transcript. (iv) optionally, a 2A peptide or protein linker sequence that either allows the downstream peptide to be separated from the upstream peptide or allows "spacing" of the downstream peptide from the upstream peptide; (v) a nitroreductase (NTR) gene, whose expression depends on binding to a target sequence having a 5'-CCA-3' (or 5'-CAA-3', 5'-CTA-3', 5'-CGA-3', 5'-ACA-3', 5'-TCA-3', 5'-GCA-3', 5'-CCT-3', or 5'-CCC-3') triplet and activation of an upstream sensor fragment when its 5'-TAG-3' stop codon is converted to a 5'-TGG-3' codon upon ADAR-mediated RNA editing; and (vi) optionally, a group of binding sites (e.g., an MS2 stem-loop that can recruit an MS2 coat protein (MCP)-ADAR fusion).

[0151] Example 24 (In vivo detection of TP53(R248Q) mutant transcripts using an all-in-one ADAR-sensor-NTR to induce cell death in the presence of the CB1945 prodrug.) To detect TP53(R248Q) mutant transcripts, which carry a CCA triplet not present in wild-type TP53, we designed an RNA sensor. The RNA sensor contains the coding sequence for a double mutant ADAR deaminase domain fused to an MCP (MCP-ADARddm(C377F,E488Q)), followed by the coding sequence for a P2A peptide, followed by a sensor region complementary to the target TP53(R248Q) transcript and a sensor stop codon opposite the mutant-specific CCA triplet, followed by the coding sequence for an XTEN80 linker peptide and NTR1.1, followed by nine copies of the MS2 stem-loop (9xMS2). The 9xMS2 can recruit MCP-MCP-ADARddm(C377F,E488Q) expressed from the same RNA molecule. Binding of the sensor region to the TP53(R248Q) mutant transcript induces editing of the TAG stop codon to a TGG codon, allowing the downstream NTR to be translated. The translated NTR can convert a prodrug (e.g., CB1954) into a cytotoxic drug, achieving cell ablation (Figure 23A). The all-in-one ADAR-sensor-NTR construct was cotransfected into HEK293T cells with either an empty vector (EV), a plasmid expressing wild-type TP53, or a plasmid expressing mutant TP53(R248Q). The CB1954 prodrug was added 24 hours after transfection, and cell viability was then measured 7 days after drug addition using the CellTiterGlo assay (Figure 23A). ADAR-sensor-NTR carrying TP53_R248Q_Sensor111 (FIG. 23B) was able to specifically eliminate cells in the presence of TP53(R248Q) mutant transcripts but not wild-type TP53 transcripts (FIG. 23C).

[0152] (4. Array) Various embodiments of the present disclosure refer to one or more of the nucleic acid and amino acid sequences provided below.

[0153] CBFA2T3GLIS2_495_Sensor (sensor stop codon is lowercase and underlined, mismatch with target is lowercase): [ka]

[0154] CBFA2T3GLIS2_avidity5_Sensor (sensor stop codon lowercase and underlined, MS2 stem-loop lowercase and italicized, mismatch with target lowercase): [ka]

[0155] MS2SL (uppercase MS2 stem loop, lowercase spacer, agat: cloning overhang): agatggccAACATGAGGATCACCCATGTCTGCAGggcc (SEQ ID NO: 7)

[0156] P2A amino acid sequence: GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 13)

[0157] E2A amino acid sequence: GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 14)

[0158] XTEN80 amino acid sequence: [ka]

[0159] MCP-ADARdd(E488Q) amino acid sequence: [ka]

[0160] MCP-NES-ADARdd(E488Q) amino acid sequence: [ka]

[0161] >NTR1.1 amino acid sequence: [ka]

[0162] > mRuby2-P2A-Sensor(CBFA2T3GLIS2_495)-XTEN80-NTR1.1-9xMS2: [ka] [ka]

[0163] >EML4ALK_501_Sensor (sensor stop codon is lowercase and underlined, mismatch with target is lowercase, [del] is deletion relative to target): [ka]

[0164] > mRuby2-P2A-Sensor(EML4ALK_501)-E2A-NTR1.1-9xMS2: [ka] [ka]

[0165] >MCP-ADARddm(E488Q) amino acid sequence on the all-in-one vector [ka]

[0166] >MCP-ADARddm(C377F,E488Q) amino acid sequence (All-in-one) [ka]

[0167] >DTA amino acid sequence [ka]

[0168] >BAX amino acid sequence [ka]

[0169] >ZFTARELA_501_Sensor (sensor stop codon is lowercase and underlined, mismatch with target is lowercase, [c] is insertion of cytosine relative to target): [ka]

[0170] >EWSR1FLI1_501_Sensor (sensor stop codon is lowercase and underlined, mismatch with target is lowercase): [ka]

[0171] >EBNA1_501_Sensor (sensor stop codon is lowercase and underlined, mismatch with target is lowercase): [ka]

[0172] >KSHV_ORF71_501_Sensor (sensor stop codon is lowercase and underlined, mismatch with target is lowercase): [ka]

[0173] >MCP-ADARdd(E488Q)-Sensor(CBFA2T3GLIS2_495)-E2A-NTR1.1-9xMS2

change

change

[0174] >MCP-ADARddm(C377F,E488Q)-Sensor(CBFA2T3GLIS2_495)-E2A-NTR1.1-9xMS2

change

change

[0175] >MCP-ADARdd(E488Q)-Sensor(EML4ALK_501)-E2A-NTR1.1-9xMS2

change

change

[0176] >MCP-ADARdd(E488Q)-Sensor(ZFTARELA_501)-E2A-NTR1.1-9xMS2

change

change

change

[0177] >MCP-ADARdd(E488Q)-Sensor(EWSR1FLI1_501)-E2A-NTR1.1-9xMS2

change

change

change

[0178] >MCP-ADARddm(C377F,E488Q)-Sensor(CBFA2T3GLIS2_495)-E2A-DTA-9xMS2

change

change

change

[0179] >MCP-ADARdd(E488Q)-Sensor(CBFA2T3GLIS2_495)-E2A-BAX-9xMS2

change

change

change

[0180] >MCP-ADARdd(E488Q)-Sensor(CBFA2T3GLIS2_495)-XTEN80-BAX-9xMS2

change

change

change

[0181] >MCP-ADARdd(E488Q)-Sensor(EBNA1_501)-XTEN80-NTR1.1-9xMS2

change

change

change

[0182] >MCP-ADARdd(E488Q)-Sensor(KSHV_ORF71_501)-XTEN80-NTR1.1-9xMS2

change

change

change

[0183] MCP_ADARddm(C377F,E488Q)-P2A-Sensor(CCNH_C5orf30_Sensor_501)-E2A-NTR1.1-9xMS2

change

change

change

[0184] >MCP_ADARddm(C377F,E488Q)-P2A-Sensor(TMEM135_CCDC67_Sensor_501)-E2A-NTR1.1-9xMS2

Chem.

Chem.

Chem.

[0185] >MCP_ADARddm(C377F,E488Q)-P2A-Sensor(EVT6_NTRK3_Sensor_501)-E2A-NTR1.1-9xMS@

Chem.

Chem.

Chem.

[0186] >MCP_ADARddm(C377F,E488Q)-P2A-Sensor(TMPRSS2_ERG_Sensor_264)-E2A-NTR1.1-9xMS@

Chem.

Chem.

Chem.

[0187] >MCP_ADARddm(C377F,E488Q)-P2A-Sensor(TRMT1l_GRIK2ss_Sensor_201)-E2A-NTR1.1-9xMS@

Chem.

[0188] >MCP_ADARddm(C377F,E488Q)-P2A-Sensor(PVT1_MYC_Sensor_498)-E2A-NTR1.1-9xMS2 [ka] [ka]

[0189] >MCP_ADARddm(C377F,E488Q)-P2A-Sensor(TP53_R248Q_Sensor111)-XTEN80-NTR1.1-9xMS2 [ka] [ka] [ka]

[0190] >CCNH_C5orf30_Sensor_501 (sensor stop codon is lowercase and underlined, target mismatch / insertion / deletion is lowercase): [ka]

[0191] >TMEM135_CCDC67_Sensor_501 (sensor stop codon is lowercase and underlined, target mismatch / insertion / deletion is lowercase): [ka]

[0192] >EVT6_NTRK3_sensor_501 [ka]

[0193] >TMPRSS2_ERG_sensor_264 [ka]

[0194] >TRMT11_GRIK2ss_Sensor_201 [ka]

[0195] >PVT1_MYC_sensor_498 [ka]

[0196] >TP53_R248Q_sensor111 [ka] (5. Clauses) The present invention is further described by the following clauses: Clause 1: A single-stranded nucleic acid sensor molecule, comprising: a target sensing region having a nucleic acid sequence substantially complementary to a target nucleic acid, the target sensing region comprising a TAG or TGA stop codon opposite a corresponding CAA, CTA, CGA, ACA, TCA, GCA, CCA, CCT, or CCC triplet in the target nucleic acid located on at least one side of a junction sequence in the target nucleic acid; and a response gene located downstream of the target sensing region, the response gene being expressed when the TAG or TGA stop codon is converted to a TGG codon by RNA-specific adenosine deaminase (ADAR)-mediated gene editing upon binding of the sensor molecule to the target nucleic acid. A single-stranded nucleic acid sensor molecule comprising: Clause 2: The sensor of clause 1, wherein the target sensing region comprises a TAG or TGA stop codon opposite a corresponding CCA (or 5'-CAA-3', 5'-CTA-3', 5'-CGA-3', 5'-ACA-3', 5'-TCA-3', 5'-GCA-3', 5'-CCT-3' or 5'-CCC-3') triplet in the target nucleic acid. Clause 3: The sensor molecule of clause 1 or clause 2, wherein the junction sequence in the target nucleic acid corresponds to at least a portion of a gene or chromosomal fusion. Clause 4: The sensor molecule of Clause 3, wherein the junction sequence in the target nucleic acid comprises at least a portion of a gene or chromosomal fusion associated with cancer. Clause 5: The sensor molecule of any one of clauses 1 to 4, wherein the junction sequence in the target nucleic acid comprises a CBFA2T3-GLIS2 fusion sequence, an EML4-ALK fusion sequence, a ZFTA-RELA fusion sequence, an EWSR1-FL1 fusion sequence, a CCNH-C5orf30 fusion sequence, a TMEM135-CCDC67 fusion sequence, an EVT6-NTRK3 fusion sequence, a TMPRSS2-ERG fusion sequence, a TRMT11-GRIK2 fusion sequence, or a PVT1-MYC fusion sequence. Clause 6: The sensor molecule of clause 1 or clause 2, wherein the junction sequence in the target nucleic acid comprises a TP53(R248Q) mutant transcript. Clause 7: The sensor molecule of clause 5, wherein the junction sequence in the target nucleic acid comprises a CBFA2T3-GLIS2 fusion sequence and the target sensing region comprises the nucleic acid sequence shown in SEQ ID NO:3. Clause 8: The sensor molecule of clause 5, wherein the junction sequence in the target nucleic acid comprises a CBFA2T3-GLIS2 fusion sequence and the target sensing region comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 4 or SEQ ID NO: 5. Clause 9: The sensor molecule of clause 5, wherein the junction sequence in the target nucleic acid comprises an EML4-ALK fusion sequence and the target sensing region comprises the amino acid sequence shown in SEQ ID NO:28. Clause 10: The sensor molecule of clause 5, wherein the junction sequence in the target nucleic acid comprises an EML4-ALK fusion sequence and the target sensing region comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 20 or SEQ ID NO: 29. Clause 11: The sensor molecule of clause 5, wherein the junction sequence in the target nucleic acid comprises a ZFTA-RELA fusion sequence and the target sensing region comprises the amino acid sequence shown in SEQ ID NO: 32. Clause 12: The sensor molecule of clause 5, wherein the junction sequence in the target nucleic acid comprises a ZFTA-RELA fusion sequence and the target sensing region comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 31 or SEQ ID NO: 30. Clause 13: The sensor molecule of clause 5, wherein the junction sequence of the target nucleic acid comprises an EWSR1-FL1 fusion sequence and the target sensing region comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 33. Clause 14: The sensor molecule of clause 5, wherein the junction sequence in the target nucleic acid comprises a CCNH-C5orf30 fusion sequence and the target sensing region comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 84. Clause 15: The sensor molecule of clause 5, wherein the junction sequence in the target nucleic acid comprises a TMEM135-CCDC67 fusion sequence and the target sensing region comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 85. Clause 16: The sensor molecule of clause 5, wherein the junction sequence in the target nucleic acid comprises an EVT6-NTRK3 fusion sequence and the target sensing region comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 86. Clause 17: The sensor molecule of clause 5, wherein the junction sequence in the target nucleic acid comprises a TMPRSS2-ERG fusion sequence and the target sensing region comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 87. Clause 18: The sensor molecule of clause 5, wherein the junction sequence in the target nucleic acid comprises a TRMT11-GRIK2 fusion sequence and the target sensing region comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 88. Clause 19: The sensor molecule of clause 5, wherein the junction sequence in the target nucleic acid comprises a PVT1-MYC fusion sequence and the target sensing region comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 89. Clause 20: The sensor molecule of clause 5, wherein the junction sequence in the target nucleic acid comprises a TP53(R248Q) mutant transcript and the target sensing region comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:90. Clause 21: The sensor molecule of clause 1 or clause 2, wherein the junction sequence of the target nucleic acid corresponds to a viral transcript. Clause 22: The sensor molecule of clause 21, wherein the viral transcript is an Epstein-Barr virus (EBV) transcript or a Kaposi's sarcoma-associated herpesvirus (KSHV) transcript. Clause 23: The sensor molecule of clause 21, wherein the viral transcript is the Epstein-Barr virus transcript EBNA1 and the target sensing region comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:34. Clause 24: The sensor molecule of clause 21, wherein the viral transcript is KSHV transcript ORF71 and the target sensing region comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:35. Clause 25: The sensor molecule of any one of clauses 1 to 24, wherein said target sensing region is at least about 50 nucleotides in length. Clause 26: The sensor molecule of Clause 25, wherein the target sensing region is from about 50 nucleotides to about 1000 nucleotides in length. Clause 27: The sensor molecule of any one of clauses 1 to 26, wherein the response gene encodes at least one of a reporter protein, a caspase, a prodrug converting enzyme, or an enzyme that catalyzes another reaction. Clause 28: The sensor molecule of clause 27, wherein said response gene encodes nitroreductase (NTR), diphtheria toxin fragment A (DTA), or BCL2-associated X (BAX). Clause 29: The sensor molecule of any one of clauses 1 to 28, further comprising a control gene. Clause 30: The sensor molecule of Clause 29, wherein said control gene is constitutively expressed. Clause 31: The sensor of clause 20 or clause 30, wherein the control gene encodes a fluorescent protein. Clause 32: The sensor molecule of any one of clauses 1 to 31, comprising a linker region located upstream of said response gene but downstream of said TAG or TGA stop codon. Clause 33: The sensor molecule of Clause 32, wherein said linker region comprises a 2A peptide or an XTEN80 linker. Clause 34: The sensor molecule of Clause 33, wherein the linker region comprises SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15. Clause 35: A sensor molecule according to any one of clauses 1 to 34, comprising an RNA aptamer sequence capable of binding to its cognate binding protein. Clause 36: The sensor molecule of Clause 35, wherein said RNA aptamer sequence comprises a sequence capable of binding to at least one of MS2, PP7, BoxB, or Pumilio. Clause 37: The sensor molecule of clause 36 or clause 37, wherein said cognate binding protein is fused to an ADAR protein. Clause 38: The sensor molecule of any one of clauses 1 to 37, further comprising a gene encoding an ADAR or an ADAR fusion, wherein said ADAR or ADAR fusion is constitutively expressed. Clause 39: The sensor molecule of clause 38, wherein said ADAR fusion comprises an ADAR enzyme fused to a cognate aptamer-binding protein. Clause 40: The sensor molecule of clause 39, further comprising an RNA aptamer sequence that recruits said cognate aptamer-binding protein upon expression of said ADAR fusion. Clause 41: The sensor molecule of any one of clauses 1 to 40, which is an RNA molecule. Clause 42: An expression vector comprising a DNA sequence corresponding to any of the sensor molecules of any one of clauses 1 to 41. Article 43: (a) pCR8-mRuby2-P2A-ccdbCam-E2A-EGFP vector; (b)pCR8-mRuby2-P2A-ccdbCam-E2A-EGFP-BsaI(agate)ベクター; (c)pCR8-mRuby2-P2A-ccdbCam-E2A-EGFP-NxMS2ベクター; (d)pCR8-mRuby2-P2A-Sensor-E2A-EGFPベクター; (e)pCR8-mRuby2-P2A-Sensor-E2A-EGFP-NxMS2ベクター; (f)pmax-mRuby2-P2A-Sensor-XTEN80-EGFP-NxMS2ベクター; (g)MCP-ADARdd(E488Q)ベクター; (h)pmax-MCP-ADARdd(E488Q)ベクター; (i)pmax-MCP-ADARdd(E488Q)-P2A-Sensor-XTEN80-NTR1.1-NxMS2ベクター; (j)pmax-MCP-ADARdd(E488Q)-P2A-Sensor-E2A-NTR1.1-NxMS2ベクター; (k)pmax-MCP-ADARddm(C377F,E488Q)-P2A-Sensor-XTEN80-NTR1.1-NxMS2ベクター; (l)pmax-MCP-ADARddm(C377F,E488Q)-P2A-Sensor-E2A-NTR1.1-NxMS2ベクター; (m)pmax-MCP-ADARdd(E488Q)-P2A-Sensor-XTEN80-DTA-NxMS2ベクター; (n)pmax-MCP-ADARdd(E488Q)-P2A-Sensor-E2A-DTA-NxMS2ベクター; (o)pmax-MCP-ADARddm(C377F,E488Q)-P2A-Sensor-XTEN80-DTA-NxMS2ベクター; (p)pmax-MCP-ADARddm(C377F,E488Q)-P2A-Sensor-E2A-DTA-NxMS2ベクター; (q)pmax-MCP-ADARdd(E488Q)-P2A-Sensor-XTEN80-BAX-NxMS2 vector; (r)pmax-MCP-ADARdd(E488Q)-P2A-Sensor-E2A-BAX-NxMS2 vector; (s) pmax-MCP-ADARddm(C377F,E488Q)-P2A-Sensor-XTEN80-BAX-NxMS2 vector; and (t)pmax-MCP-ADARddm(C377F,E488Q)-P2A-Sensor-E2A-BAX-NxMS2 vector 43. The expression vector of clause 42, selected from the group consisting of: Clause 44: A cell comprising any of the sensor molecules of any one of clauses 1 to 41 or any of the vectors of clauses 42 or 43. Article 45: any of the sensor molecules according to any one of clauses 1 to 41; any of the vectors according to clause 42 or 43, and / or The cell according to clause 44 Includes a kit. Clause 46: A method of treating a subject having or suspected of having cancer, the method comprising administering to the subject any of the sensor molecules of any one of clauses 1 to 41, any of the vectors of clauses 42 or 43, and / or a cell of clause 44. Clause 47: The method of Clause 46, further comprising administering a prodrug to said subject, wherein said sensor molecule converts said prodrug into a cytotoxic agent, thereby treating cancer in said subject. Clause 48: The method of clause 46 or clause 47, wherein said cancer comprises a chromosomal translocation and / or a gene fusion. Clause 49: The method of clause 46 or clause 47, wherein said cancer contains a viral genome or expresses a viral transcript. Clause 50: The method of clause 46 or clause 47, wherein said cancer contains one or more genetic mutations or expresses one or more mutant gene transcripts. Clause 51: A method for detecting a transcript in a cell, comprising: transfecting a cell with any of the sensor molecules of any one of clauses 1 to 41 or any of the vectors of clauses 42 or 43; and assessing the cells for expression of a reporter protein. A method comprising:

[0197] It is understood that the above detailed description and accompanying examples are merely illustrative and should not be taken as limitations on the scope of the present disclosure, which is defined solely by the appended claims and equivalents thereof.

[0198] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including but not limited to, changes and modifications related to the chemical structures, substitutions, derivatives, intermediates, synthesis, compositions, formulations, or methods of use of the disclosure, can be made without departing from the spirit and scope of the disclosure.

Claims

1. A single-stranded nucleic acid sensor molecule, a target sensing region having a nucleic acid sequence substantially complementary to a target nucleic acid, the target sensing region comprising a TAG or TGA stop codon opposite a corresponding CAA, CTA, CGA, ACA, TCA, GCA, CCA, CCT, or CCC triplet in the target nucleic acid located on at least one side of a junction sequence in the target nucleic acid; and a response gene located downstream of the target sensing region, the response gene being expressed when the TAG or TGA stop codon is converted to a TGG codon by RNA-specific adenosine deaminase (ADAR)-mediated gene editing upon binding of the sensor molecule to the target nucleic acid. A single-stranded nucleic acid sensor molecule comprising:

2. 2. The sensor of claim 1, wherein the target sensing region comprises a TAG or TGA stop codon opposite a corresponding CCA triplet in the target nucleic acid.

3. The sensor molecule of claim 1 , wherein the junction sequence in the target nucleic acid corresponds to at least a portion of a gene or a chromosomal fusion.

4. The sensor molecule of claim 3 , wherein the junction sequence in the target nucleic acid comprises at least a portion of a gene or chromosomal fusion associated with cancer.

5. 2. The sensor molecule of claim 1, wherein the junction sequence in the target nucleic acid comprises a CBFA2T3-GLIS2 fusion sequence, an EML4-ALK fusion sequence, a ZFTA-RELA fusion sequence, an EWSR1-FL1 fusion sequence, a CCNH-C5orf30 fusion sequence, a TMEM135-CCDC67 fusion sequence, an EVT6-NTRK3 fusion sequence, a TMPRSS2-ERG fusion sequence, a TRMT11-GRIK2 fusion sequence, or a PVT1-MYC fusion sequence.

6. The sensor molecule of claim 1 , wherein the junction sequence in the target nucleic acid comprises a TP53(R248Q) mutant transcript.

7. 6. The sensor molecule of claim 5, wherein the junction sequence in the target nucleic acid comprises a CBFA2T3-GLIS2 fusion sequence and the target sensing region comprises the nucleic acid sequence shown in SEQ ID NO:

3.

8. 6. The sensor molecule of claim 5, wherein the junction sequence in the target nucleic acid comprises a CBFA2T3-GLIS2 fusion sequence, and the target sensing region comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO:4 or SEQ ID NO:

5.

9. 6. The sensor molecule of claim 5, wherein the junction sequence in the target nucleic acid comprises an EML4-ALK fusion sequence and the target sensing region comprises the nucleic acid set forth in SEQ ID NO:

28.

10. 6. The sensor molecule of claim 5, wherein the junction sequence in the target nucleic acid comprises an EML4-ALK fusion sequence and the target sensing region comprises a nucleic acid having at least 80% sequence identity to SEQ ID NO:20 or SEQ ID NO:

29.

11. 6. The sensor molecule of claim 5, wherein the junction sequence in the target nucleic acid comprises a ZFTA-RELA fusion sequence and the target sensing region comprises the nucleic acid sequence shown in SEQ ID NO:

32.

12. 6. The sensor molecule of claim 5, wherein the junction sequence in the target nucleic acid comprises a ZFTA-RELA fusion sequence and the target sensing region comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO:31 or SEQ ID NO:

30.

13. 6. The sensor molecule of claim 5, wherein the junction sequence of the target nucleic acid comprises a EWSR1-FL1 fusion sequence and the target sensing region comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO:

33.

14. 6. The sensor molecule of claim 5, wherein the junction sequence in the target nucleic acid comprises a CCNH-C5orf30 fusion sequence and the target sensing region comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO:

84.

15. 6. The sensor molecule of claim 5, wherein the junction sequence in the target nucleic acid comprises a TMEM135-CCDC67 fusion sequence and the target sensing region comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO:

85.

16. 6. The sensor molecule of claim 5, wherein the junction sequence in the target nucleic acid comprises an EVT6-NTRK3 fusion sequence and the target sensing region comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO:

86.

17. 6. The sensor molecule of claim 5, wherein the junction sequence in the target nucleic acid comprises a TMPRSS2-ERG fusion sequence and the target sensing region comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO:

87.

18. 6. The sensor molecule of claim 5, wherein the junction sequence in the target nucleic acid comprises a TRMT11-GRIK2 fusion sequence and the target sensing region comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO:

88.

19. 6. The sensor molecule of claim 5, wherein the junction sequence in the target nucleic acid comprises a PVT1-MYC fusion sequence and the target sensing region comprises a nucleic acid having at least 80% sequence identity to SEQ ID NO:

89.

20. 6. The sensor molecule of claim 5, wherein the junction sequence in the target nucleic acid comprises a TP53(R248Q) mutant transcript and the target sensing region comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO:

90.

21. The sensor molecule of claim 1 , wherein the junction sequence of the target nucleic acid corresponds to a viral transcript.

22. 22. The sensor molecule of claim 21, wherein the viral transcript is an Epstein-Barr virus (EBV) transcript or a Kaposi's sarcoma-associated herpesvirus (KSHV) transcript.

23. 22. The sensor molecule of claim 21, wherein the viral transcript is the Epstein-Barr virus transcript EBNA1 and the target sensing region comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO:

34.

24. 22. The sensor molecule of claim 21, wherein the viral transcript is KSHV transcript ORF71 and the target sensing region comprises a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO:

35.

25. The sensor molecule of claim 1 , wherein the target sensing region is at least about 50 nucleotides in length.

26. The sensor molecule of claim 1, wherein the target sensing region is from about 50 nucleotides to about 1000 nucleotides in length.

27. The sensor molecule of claim 1 , wherein the response gene encodes at least one of a reporter protein, a caspase, a prodrug-converting enzyme, or an enzyme that catalyzes another reaction.

28. 28. The sensor molecule of claim 27, wherein the response gene encodes nitroreductase (NTR), diphtheria toxin fragment A (DTA), or BCL2-associated X (BAX).

29. The sensor molecule of claim 1 , further comprising a control gene.

30. 30. The sensor molecule of claim 29, wherein the control gene is constitutively expressed.

31. 30. The sensor molecule of claim 29, wherein the control gene encodes a fluorescent protein.

32. The sensor molecule of claim 1 , comprising a linker region located upstream of the response gene but downstream of the TAG or TGA stop codon.

33. 33. The sensor molecule of claim 32, wherein the linker region comprises a 2A peptide or an XTEN80 peptide.

34. The sensor molecule of claim 33, wherein the linker region comprises SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO:

15.

35. 10. The sensor molecule of claim 1, comprising an RNA aptamer sequence capable of binding to its cognate binding protein.

36. 36. The sensor molecule of claim 35, wherein the RNA aptamer sequence comprises a sequence capable of binding to at least one of MS2, PP7, BoxB, or Pumilio.

37. 36. The sensor molecule of claim 35, wherein the cognate binding protein is fused to an ADAR protein.

38. The sensor molecule of claim 1 , further comprising a gene encoding an ADAR or an ADAR fusion, wherein the ADAR or ADAR fusion is constitutively expressed.

39. 39. The sensor molecule of claim 38, wherein the ADAR fusion comprises an ADAR enzyme fused to a cognate aptamer-binding protein.

40. 40. The sensor molecule of claim 39, further comprising an RNA aptamer sequence that recruits the cognate aptamer-binding protein upon expression of the ADAR fusion.

41. The sensor molecule of claim 1 which is an RNA molecule.

42. An expression vector comprising a DNA sequence corresponding to the sensor molecule of claim 1.

43. (a) pCR8-mRuby2-P2A-ccdbCam-E2A-EGFP vector; (b) pCR8-mRuby2-P2A-ccdbCam-E2A-EGFP-BsaI (agat) vector; (c) pCR8-mRuby2-P2A-ccdbCam-E2A-EGFP-NxMS2 vector; (d) pCR8-mRuby2-P2A-Sensor-E2A-EGFP vector; (e) pCR8-mRuby2-P2A-Sensor-E2A-EGFP-NxMS2 vector; (f) pmax-mRuby2-P2A-Sensor-XTEN80-EGFP-NxMS2 vector; (g) MCP-ADARdd(E488Q) vector; (h) pmax-MCP-ADARdd(E488Q) vector; (i) pmax-MCP-ADARdd(E488Q)-P2A-Sensor-XTEN80-NTR1.1-NxMS2 vector; (j) pmax-MCP-ADARdd(E488Q)-P2A-Sensor-E2A-NTR1.1-NxMS2 vector; (k) pmax-MCP-ADARddm (C377F, E488Q)-P2A-Sensor-XTEN80-NTR1.1-NxMS2 vector; (l) pmax-MCP-ADARddm (C377F, E488Q)-P2A-Sensor-E2A-NTR1.1-NxMS2 vector; (m) pmax-MCP-ADARdd(E488Q)-P2A-Sensor-XTEN80-DTA-NxMS2 vector; (n) pmax-MCP-ADARdd(E488Q)-P2A-Sensor-E2A-DTA-NxMS2 vector; (o) pmax-MCP-ADARddm (C377F, E488Q)-P2A-Sensor-XTEN80-DTA-NxMS2 vector; (p) pmax-MCP-ADARddm (C377F, E488Q)-P2A-Sensor-E2A-DTA-NxMS2 vector; (q) pmax-MCP-ADARdd(E488Q)-P2A-Sensor-XTEN80-BAX-NxMS2 vector; (r)pmax-MCP-ADARdd(E488Q)-P2A-Sensor-E2A-BAX-NxMS2 vector; (s) pmax-MCP-ADARddm(C377F,E488Q)-P2A-Sensor-XTEN80-BAX-NxMS2 vector; and (t)pmax-MCP-ADARddm(C377F,E488Q)-P2A-Sensor-E2A-BAX-NxMS2 vector 40. The expression vector of claim 39, selected from the group consisting of:

44. A cell comprising either the sensor molecule of claim 1 or the vector of claim 42.

45. The sensor molecule of claim 1 ; A vector according to claim 42, and / or The cell of claim 44. Includes a kit.

46. A method for treating a subject having or suspected of having cancer, comprising administering to the subject a sensor molecule described in claim 42, a vector described in claim 42, and / or a cell described in claim 44.

47. 47. The method of claim 46, further comprising administering a prodrug to the subject, wherein the sensor molecule converts the prodrug into a cytotoxic agent, thereby treating cancer in the subject.

48. 47. The method of claim 46, wherein the cancer comprises a chromosomal translocation and / or a gene fusion.

49. 47. The method of claim 46, wherein the cancer contains a viral genome or expresses a viral transcript.

50. 47. The method of claim 46, wherein the cancer contains one or more genetic mutations or expresses one or more mutant gene transcripts.

51. 1. A method for detecting a gene fusion transcript in a cell, comprising: transfecting a cell with the sensor molecule of claim 1 or the vector of claim 42; and assessing the cells for expression of a reporter protein. A method comprising:

Images