Modular RNA-based RNA sensors utilizing ADAR editing

Abstract

This disclosure provides methods, compositions, systems, and kits for expressing proteins in target cells.

Claims

[Claim 1] A method for expressing a protein in target cells, wherein the method is The method involves contacting the target cells with a sensor RNA or a vector encoding the sensor RNA, wherein the sensor RNA or the vector encoding the sensor RNA is (i) A first nucleotide sequence comprising (1) a nucleotide sequence containing a region that hybridizes to a target RNA, and (2) a stem-loop sequence containing one or more editable codons, (ii) a second nucleotide sequence encoding the output protein, Here, The target RNA is present in the target cell. a) The stem-loop sequence, the sensor RNA, or the region between the first nucleotide sequence and the second nucleotide sequence contains one or more stop codons outside the frame of the editable codon, or b) The stem-loop sequence includes a sequence that is at least 80% identical to a sequence selected from the group consisting of sequence number 10, sequence number 11, and sequence number 12, or c) The target RNA contains one or more base mismatches opposite to the nucleotide sequence containing the region that hybridizes to the target RNA, or d) The method wherein the region that hybridizes to the target RNA contains 25 nucleotides or more of the editable codon at the 5' or 3' position, and includes one or more mismatches opposite to those of the target RNA. [Claim 2] The method according to claim 1, wherein the stem-loop sequence, the sensor RNA, or the region between the first nucleotide sequence and the second nucleotide sequence includes one or more stop codons located outside the frame of the editable codon. [Claim 3] The method according to claim 1 or 2, wherein the editable codon is a stop codon, a start codon, or an AUA codon. [Claim 4] The method according to any one of claims 1 to 3, wherein the editable codon comprises one or more bases that mismatch with the stem-loop sequence opposite to the one or more editable codons. [Claim 5] The method according to any one of claims 1 to 4, wherein the target RNA includes one or more base mismatches opposite to the nucleotide sequence containing the region that hybridizes to the target RNA. [Claim 6] The method according to any one of claims 1 to 5, wherein the target RNA includes 10 or more base mismatches opposite to the nucleotide sequence containing the region that hybridizes to the target RNA. [Claim 7] The method according to any one of claims 1 to 6, wherein the region that hybridizes to the target RNA includes 25 nucleotides or more at the 5' or 3' of the editable codon, and contains one or more base mismatches opposite to those of the target RNA. [Claim 8] The method according to any one of claims 1 to 7, wherein the sensor RNA further comprises a region at 5' of the stem-loop sequence that hybridizes to the target RNA. [Claim 9] The method according to any one of claims 1 to 8, wherein the sensor RNA further comprises a region of the stem-loop sequence that hybridizes to the target RNA at 3'. [Claim 10] The method according to any one of claims 1 to 7, wherein the sensor RNA further comprises a region at 5' of the stem-loop sequence that hybridizes to the target RNA, and a region at 3' of the stem-loop sequence that hybridizes to the target RNA. [Claim 11] The method according to any one of claims 1 to 10, wherein the sensor RNA further comprises a 5' UTR of 5' relative to the first nucleotide sequence, or a 3' UTR of 3' relative to the second nucleotide sequence. [Claim 12] The 5'UTR or the 3'UTR is Hs PEG10 5' and 3'UTR, mmPEG10 5' and 3'UTR, HsPNMA1 5' and 3'UTR, mmPNMA1 5' and 3'UTR, HsPNMA3 5' and 3'UTR, mmPNMA3 5' and 3'UTR, HsMAOP1 5' and 3'UTR, mmMAOP1 5' and 3'UTR, HsPNMA5 5' and 3'UTR, mmPNMA5 5' and 3'UTR, HsRTL1 5' and 3'UTR, mmRTL1 5' and 3'UTR, HsZCCHC12 5' and 3'UTR, mmZCCHC12 The method according to claim 11, selected from the group consisting of 5' and 3'UTR, HsASPRV1 5' and 3'UTR, mmADPRV1 5' and 3'UTR, HsARC1 5' and 3'UTR, and mmARC1 5' and 3'UTR. [Claim 13] The method according to any one of claims 1 to 12, wherein the stem-loop sequence includes a sequence that is at least 80% identical to a sequence selected from the group consisting of sequence number 10, sequence number 11, and sequence number 12. [Claim 14] The method according to any one of claims 1 to 13, wherein the sensor RNA includes a cleavage domain or a 2A self-cleavage domain between the first nucleotide sequence and the second nucleotide sequence. [Claim 15] The method according to any one of claims 1 to 14, wherein the output protein is selected from fluorescent proteins, genome-modified proteins, transcription factors, toxic factors, toxins, antigens, T cell receptors, chimeric antigen receptors, therapeutic proteins, and enzymes. [Claim 16] The method according to any one of claims 1 to 15, wherein the target RNA is associated with a disease, condition, cell type, or tissue. [Claim 17] The method according to any one of claims 1 to 16, wherein the sensor RNA contains one or more pseudouridines, or the sensor nucleotide sequence does not contain pseudouridines. [Claim 18] The method according to any one of claims 1 to 16, wherein the contact with the target cells comprises bringing the target cells into contact with an adeno-associated virus (AAV) containing the sensor RNA, the sensor RNA being encoded in an AAV vector. [Claim 19] The method according to any one of claims 1 to 18, wherein the contact includes administering to a patient. [Claim 20] (i) a fourth nucleotide sequence comprising a second cleavage domain and preceding the first nucleotide sequence, (ii) The method according to any one of claims 1 to 19, further comprising a fifth nucleotide sequence comprising a nucleotide sequence encoding a marker protein and preceding the fourth nucleotide sequence. [Claim 21] The method according to any one of claims 1 to 20, further comprising contacting the target cells with an RNA-acting adenosine deaminase (ADAR) protein or its coding sequence. [Claim 22] The method according to any one of claims 1 to 21, further comprising assaying for the presence of the output protein. [Claim 23] The method according to claim 22, wherein the assay includes using microscopy, flow cytometry, immunoblotting, a plate reader, or a combination thereof. [Claim 24] The method according to any one of claims 1 to 22, wherein the target RNA includes intracellular mRNA. [Claim 25] The method according to claim 24, wherein the region that hybridizes to the target RNA includes the 5'UTR or 3'UTR of the intracellular mRNA.

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