Synthetic receptors for extracellular and intracellular input-output control with cytosolic splicing

Abstract

Provided herein are modular programmable synthetic receptors comprising a ligand binding domain, a kinase domain, and an RNase domain. Also provided herein are engineered synthetic RNAs recognized by synthetic receptors provided herein, as well as cells comprising the synthetic receptors and engineered synthetic RNAs, and methods of making and using the same.

Description

[0001] Attorney Docket No.: PTBI-010 / 01WO SYNTHETIC RECEPTORS FOR EXTRACELLULAR AND INTRACELLULAR INPUT¬ OUTPUT CONTROL WITH CYTOSOL IC SPLICING

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U. S. Provisional Application No. 63 / 728,368, filed December 5, 2024, the content of which is incorporated by reference in its entirety.

[0003] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0004] The content of the electronic sequence listing (PTBI 010 01WO SeqList ST26.xml; Size: 813,568 bytes; and Date of Creation: November 26, 2025) is herein incorporated by reference in its entirety.

[0005] FIELD

[0006] Described herein are chimeric proteins capable of detecting a wide range of extracellular and intracellular signals with a programmable output, as well as polynucleotides encoding the chimeric proteins. Also described herein are engineered RNA polynucleotides comprising cytosolic intron structures capable of being spliced by the chimeric proteins, as well as engineered cells comprising said synthetic proteins and / or engineered polynucleotides, and methods of using the same to modulate cellular responses.

[0007] BACKGROUND

[0008] Existing programmable synthetic receptors primarily depend on DNA-based, transcription factor-mediated signal transduction. RNA-based designs have been developed utilizing mainly ribozymes, aptamers, and adenosine deaminases.

[0009] SUMMARY

[0010] Programmable synthetic receptors typically rely on transcription factor-based regulation to transduce signals, which precludes mRNA-only applications and introduces other drawbacks, such as delayed response time, increased circuit complexity, and larger payload sizes for cellular delivery. Although some RNA-based platforms have been developed, none are capable of responding to user-defined extracellular signals. The inventors surprisingly found that modular programmable synthetic receptors of the present disclosure are capable of detecting intracellular or extracellular signals (e.g., biomarker

[0011] 1

[0012] 326657998 Attorney Docket No.: PTBI-010 / 01WO inputs) and triggering cellular responses (e.g., production of therapeutic proteins), while bypassing transcriptional regulation.

[0013] Aspects of this disclosure provide a chimeric protein comprising: (i) a ligand binding domain, wherein the ligand binding domain is not a ligand binding domain of an inositol - requiring enzyme 1 ( IRE1 ) protein; (ii) a kinase domain of an IRE1 protein or a kinase domain derived from an IRE1 protein; and (iii) an RNase domain of an IRE1 protein or an RNase domain derived from an IRE1 protein. In some embodiments, the chimeric protein comprises, N-terminally to C-terminally: (i), (ii), and (iii).

[0014] In some embodiments, the ligand binding domain is capable of dimerization, trimerization, or multimerization in the presence of the ligand. In some embodiments, the chimeric protein forms a dimer, a trimer, or a multimer, in the presence of the ligand. In some embodiments, the chimeric protein is a monomeric protein in the absence of the ligand.

[0015] In some embodiments, the chimeric protein is a plasma membrane protein or a cytosolic protein.

[0016] In some embodiments, the ligand comprises a small molecule, a peptide, a protein, a lipid, a carbohydrate, or any combination thereof. In some embodiments, the ligand is a cancer biomarker or a cancer-associated antigen. In some embodiments, the ligand is a cell¬ surface ligand or a soluble ligand.

[0017] In some embodiments, the kinase domain comprises a catalytic loop and an activation loop, wherein the catalytic loop comprises an HRD motif and / or wherein the activation loop comprises a DFG motif or a DMG motif. In some embodiments, the kinase domain comprises the amino acid sequence of X1LHX2LX3IX4HRDX5KPX6NX7LX8X9 (SEQ ID NO: 53), where Xi is H or Y; X2 is E or S; X3 is G, N, K, or R; X4 is V or I; X5 is L or I; Xe is Q or H; X7 is E or I; Xs is I or V; and X9 is S, A, or N. In some embodiments, the kinase domain comprises the amino acid sequence of X1X2X3X4SDX5GX6X7KX8X9X10 (SEQ ID NO: 54), where Xi is S, K, or R; X2 is A, I, or L, X3 is K, M, or L, X is L, I, or V; X5 is M or F; Xe is I or L; X7 is S or C; Xs is R or K; X9 is M or L; and X10 is S, A D, or E. In some embodiments, the kinase domain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of any one of SEQ ID NOs: 15-20.

[0018] In some embodiments, the RNase domain comprises one or more catalytic residues corresponding to Y60, N74, K75, and H78 relative to SEQ ID NO: 21. In some embodiments, the RNase domain comprises residues Y60, N74, K75, and 1178 relative to SEQ ID NO: 21. In some embodiments, the RNase domain comprises the amino acid sequence of

[0019] 2

[0020] 326657998 Attorney Docket No.: PTBI-010 / 01WO RX1YX2X3X4X5X6X7DLLRX8X9RNKX10X11HX12 (SEQ ID NO: 55), where X 1 is T, R, or K; X2 is K, T, S, or H; X3 is G, Y, P, or S; X4 is G, D, N, S, or E, X5 is S, R, or K; Xe is V, I, M, or L; X7 is R, L, or M; Xs is A or V; X9 is M, I, or L; X10 is K, L, or Y; Xu is H or N; and X12 is Y, H, or F. In some embodiments, the RNase domain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of any one of SEQ ID NOs: 21-26.

[0021] In some embodiments, the ligand binding domain lacks a dimerization domain.

[0022] In some embodiments, the ligand binding domain comprises an antibody. In some embodiments, the antibody is a single-chain variable fragment (sc-Fv) or a nanobody. In some embodiments, the antibody is a bispecific antibody (e.g., comprising two nanobodies targeting different antigen epitopes). In some embodiments, the antibody comprises 1, 2, or more than 2, immunoglobulin single variable domains. In some embodiments, the antibody comprises a first immunoglobulin single variable domain that binds to a first epitope of the ligand and a second immunoglobulin single variable domain that binds to a second epitope of the ligand.

[0023] In some embodiments, the ligand binding domain comprises a dimerization domain. In some embodiments, the ligand binding domain is an extracellular ligand binding domain. In some embodiments, the ligand binding domain comprises a ligand binding domain of a FK506-binding protein (FKBP), a ligand binding domain of a tumor necrosis factor (TNF) receptor superfamily protein, a ligand binding domain of an interleukin-1 receptor (IL-1R) protein, a ligand binding domain of an interleukin-1 receptor accessory protein (IL-lRAcP), or a combination thereof. In some embodiments, the ligand binding domain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of any one of SEQ ID NOs: 8- 14

[0024] In some embodiments, the chimeric protein further comprises a transmembrane domain, optionally wherein the chimeric protein comprises, N-terminally to C -terminally: the ligand binding domain; the transmembrane domain, the kinase domain; and the RNase domain. In some embodiments, the transmembrane domain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence of SEQ ID NO: 27.

[0025] In some embodiments, the IRE1 protein of (ii) and the IRE1 protein of (iii) are the same. In some embodiments, the IRE1 protein of (ii ) and / or the IRE1 protein of (iii) are not human IRE1 protein, not a mammalian IRE1 protein, or not a eukaryotic IRE1 protein.

[0026] 3

[0027] 326657998 Attorney Docket No.: PTBI-010 / 01WO In some embodiments, the RNase domain, or the chimeric protein, is capable of excising a cytosolic intron comprised in a stem-loop splicing hairpin of an RNA molecule. In some embodiments, the cytosolic intron or the stem-loop splicing hairpin is derived from an RNA molecule encoding a x-box binding protein 1 (XBP1).

[0028] In some embodiments, the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of any one of SEQ ID NOs: 28-43 or 56.

[0029] In some embodiments, this disclosure provides a polynucleotide comprising a sequence encoding a chimeric protein of the present disclosure. In some embodiments, the polynucleotide is an RNA. In some embodiments, the IRE1 protein of (ii) and / or the IRE1 protein of (iii) are not human IRE1 protein, not a mammalian IRE1 protein, or not a eukaryotic protein, but the sequence encoding the chimeric protein is codon optimized for human, mammalian, or eukaryotic expression.

[0030] In some aspects, this disclosure provides an engineered RNA polynucleotide comprising a sequence of a stem-loop splicing hairpin comprising a cytosolic intron, wherein the cytosolic intron is capable of being excised by an RNase domain of an inositol-requiring enzyme 1 (IRE1) protein. In some embodiments, the engineered RNA polynucleotide comprises a sequence encoding an initial start codon. In some embodiments, the initial start codon is 5’ to the sequence of the stem-loop splicing hairpin. In some embodiments, the engineered RNA polynucleotide does not encode a x-box binding protein 1 (XBP1). In some embodiments, the engineered RNA polynucleotide encodes a protein of interest that is not, or does not comprise, a x-box binding protein 1 (XBPI).

[0031] In some embodiments, the engineered RNA polynucleotide expresses the protein of interest after the cytosolic intron is excised, but does not express the protein of interest when the cytosolic intron is not excised. In some embodiments, the engineered RNA polynucleotide does not express the protein of interest after the cytosolic intron is excised, but expresses the protein of interest when the cytosolic intron is not excised. In some embodiments, after the cytosolic intron is excised, the engineered RNA polynucleotide expresses a protein of interest that is different from the protein of interest expressed by the engineered RNA polynucleotide when the cytosolic intron is not excised.

[0032] In some embodiments, at least a part of the nucleotide sequence encoding the protein of interest is out of frame with the initial start codon when the cytosolic intron is not excised. In some embodiments, the entire nucleotide sequence encoding the protein of interest is out of frame with the initial start codon when the cytosolic intron is not excised.

[0033] 4

[0034] 326657998 Attorney Docket No.: PTBI-010 / 01WO In some embodiments, the engineered RNA polynucleotide comprises a second start codon, wherein the second start codon is: (i) 3’ to the sequence of the initial start codon, (ii) 3’ to the cytosolic intron, and / or (iii) out of frame with the initial start codon.

[0035] In some embodiments, the nucleotide sequence encoding the protein of interest is in frame with the second start codon. In some embodiments, wherein the nucleotide sequence encoding the protein of interest comprises the second start codon as its first codon.

[0036] In some embodiments, the engineered RNA polynucleotide comprises a first stop codon, wherein the first stop codon is 3' to the sequence of the stem-loop splicing hairpin, and wherein the first stop codon is in frame with the initial start codon. In some embodiments, wherein the first stop codon is 5’ to the nucleotide sequence encoding the protein of interest.

[0037] In some embodiments, the engineered RNA polynucleotide comprises a sequence encoding a cleavage peptide. In some embodiments, the sequence encoding the cleavage peptide is 5’ to, and in frame with, the sequence encoding the protein of interest. In some embodiments, the sequence encoding the cleavage peptide is 3’ to the sequence encoding the second start codon, and wherein the sequence encoding the cleavage peptide is in frame with the sequence encoding the second start codon. In some embodiments, the cleavage peptide is a self-cleaving peptide. In some embodiments, the self-cleaving peptide is a viral 2A peptide.

[0038] In some aspects, this disclosure provides an engineered RNA polynucleotide comprising: (i) a sequence of a stem-loop splicing hairpin comprising a cytosolic intron, wherein the cytosolic intron is capable of excision by an RNase domain of an inositol- requiring enzyme 1 (IREl) protein; (ii) a sequence encoding an initial start codon, wherein the initial start codon is 5’ to the sequence of the stem-loop splicing hairpin; (iii) a sequence encoding a first stop codon, wherein the first, stop codon is 3’ to the sequence of the stem-loop splicing hairpin, and wherein the first stop codon is in frame with the initial start codon; (iv) a sequence encoding a second start codon, wherein the second start codon is 3’ to the sequence of the initial start codon, optionally 3’ to the cytosolic intron, and wherein the second start, codon is out of frame with the initial start codon, and (v) a sequence encoding a viral 2A peptide, wherein the sequence encoding the viral 2A peptide is 3’ to the sequence encoding the second start codon, and wherein the sequence encoding the viral 2A peptide is in frame with the sequence encoding the second start codon.

[0039] In some aspects, this disclosure provides an engineered RNA polynucleotide comprising: (i) a sequence of a stem-loop splicing hairpin comprising a cytosolic intron, wherein the stem-loop splicing hairpin comprises a first portion comprising a sequence

[0040] 5

[0041] 326657998 Attorney Docket No.: PTBI-010 / 01WO having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 57 and a second portion comprising a sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 58, wherein the first nucleotide sequence and the second nucleotide sequence are complementary; (ii) a sequence encoding an initial start codon, wherein the initial start codon is 5’ to the sequence of the stem-loop splicing hairpin; (iii) a sequence encoding a first stop codon, wherein the first stop codon is 3' to the sequence of the stem-loop splicing hairpin, and wherein the start codon is in frame with the initial start codon, and (iv) a sequence encoding a second start codon, wherein the second start codon is 3’ to the sequence of the initial start codon, optionally 3’ to the cytosolic intron, and wherein the second start codon is out of frame with the initial start codon; optionally wherein the engineered RNA polynucleotide further comprises: (v) a sequence encoding a viral 2 A peptide, wherein the sequence encoding the viral 2 A peptide is 3’ to the sequence encoding the second start codon, and wherein the sequence encoding the viral 2A peptide is in frame with the sequence encoding the second start codon.

[0042] In some embodiments, the stem-loop splicing hairpin comprises the nucleotide sequence of X1X2GCX3GX4X5X6T (SEQ ID NO: 73), wherein Xi is C or T, X2 is C, T, or A; X3 is C, T, or A; X4 is T, A, or C; X5 is A or G; and Xs is C, A, or G. In some embodiments, the nucleotide sequence comprises an IRE1 cleavage site.

[0043] In some embodiments, the stem-loop splicing hairpin comprises the nucleotide sequence of X1CX2GX3X4GX5X6X7 (SEQ ID NO: 74), where Xi is T or C; X2 is A, T, or C; X3 is C or A; X4 is T or A; Xs is absent, T, or A; Xt> is absent, C or T; and X~ is G or A. In some embodiments, the nucleotide sequence comprises an IRE1 cleavage site.

[0044] In some aspects, this disclosure provides an engineered RNA polynucleotide comprising: (i) a sequence of a stem-loop splicing hairpin comprising a cytosolic intron, wherein the stem-loop splicing hairpin comprises the nucleotide sequence of X1X2GCX3GX4X5X6T (SEQ ID NO: 73), wherein Xi is C or T; X ■ is C, T, or A, X 3 is C, T. or A; X4 is T, A, or C; X5 is A or G; and Xs is C, A, or G and / or the nucleotide sequence of X1CX2GX3X4GX5X6X7 (SEQ ID NO: 74), where Xi is T or C; X2 is A, T, or C; X3 is C or A; X4 is T or A; X? is absent, T, or A; Xs is absent, C or T; and X7 is G or A; (ii) a sequence encoding an initial start codon, wherein the initial start codon is 5’ to the sequence of the stem-loop splicing hairpin; (iii) a sequence encoding a first stop codon, wherein the first stop codon is 3’ to the sequence of the stem-loop splicing hairpin, and wherein the start codon is in

[0045] 6

[0046] 326657998 Attorney Docket No.: PTBI-010 / 01WO frame with the initial start codon; (iv) a sequence encoding a second start codon, wherein the second start, codon is 3’ to the sequence of the initial start codon, optionally 3’ to the cytosolic intron, and wherein the second start codon is out of frame with the initial start codon; and (v) a sequence encoding a viral 2A peptide, wherein the sequence encoding the viral 2A peptide is 3’ to the sequence encoding the second start codon, and wherein the sequence encoding the viral 2A peptide is in frame with the sequence encoding the second start codon.

[0047] In some embodiments, the stem-loop splicing hairpin comprises the structure, from 5’ to 3’: a 5’ stem 1 region, a 5’ stem 2 region, a 5’ loop region, a 3’ stem 2 region, a 5’ stem 3 region, a 3’ loop region, a 3’ stem 3 region, and a 3’ stem 1 region, wherein the RNA bases of the 5’ stem 1 region are complementary to the RNA bases of the 3’ stem 1 region, the RNA bases of the 5’ stem 2 region are complementary to the RNA bases of the 3’ stem 2 region, and the RNA bases of the 5’ stem 3 region are complementary to the RNA bases of the 3’ stem 3 region.

[0048] In some embodiments, the cytosolic intron comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of any one of SEQ ID NOs: 65-70. In some embodiments, the stem-loop splicing hairpin comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of any one of SEQ ID NOs: 59-64. In some embodiments, the stem-loop splicing hairpin comprises a first portion comprising a sequence having at least 90% identity to SEQ ID NO: 57 and a second portion comprising a sequence having at least 90% identity to SEQ ID NO: 58, wherein the first portion and the second portion are complementary''.

[0049] In some embodiments, the viral 2A peptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any one of SEQ ID NOs: 75-78.

[0050] In some embodiments, the engineered RNA polynucleotide does not encode either a XBP1 protein or a functional fragment thereof. In some embodiments, the engineered RNA polynucleotide does not encode a fusion protein comprising the XBP 1 protein or functional fragment thereof.

[0051] In some embodiments, the engineered RNA further comprises a nucleotide sequence encoding a protein of interest, wherein the nucleotide sequence encoding the protein of interest is in frame with the second start codon, optionally wherein the nucleotide sequence encoding the protein of interest comprises the second start codon as its first codon.

[0052] 7

[0053] 326657998 Attorney Docket No.: PTBI-010 / 01WO In some embodiments, the protein of interest is a therapeutic protein. In some embodiments, the therapeutic protein is an antibody (e.g., scFv), a Fc fusion protein, a hormone, a cytokine, a chemokine, a cytotoxin, an enzyme catalyzing a prodrug activation, a transcription factor, a transactivator, an immunomodulatory' protein, or a DNA-modifying enzyme.

[0054] In some embodiments, the engineered RNA polynucleotide is a non-coding RNA. In some embodiments, the engineered RNA polynucleotide is converted from a non-functional non-coding RNA to a functional non-coding RNA upon excision of the cytosolic intron. In some embodiments, the engineered RNA polynucleotide is converted from a functional non¬ coding RNA to a non-functional non-coding RNA upon excision of the cytosolic intron. In some embodiments, the function of the non-coding RNA is altered after the cytosolic intron is excised.

[0055] In some embodiments, the stem-loop splicing hairpin (including or excluding a cytosolic intron) is not derived from a human stem-loop splicing hairpin, a mammalian stem-loop splicing hairpin, or a eukaryotic stem-loop splicing hairpin.

[0056] In some aspects, this disclosure provides recombinant DNA polynucleotides encoding the engineered RNA polynucleotide of the disclosure.

[0057] In some embodiments, this disclosure provides a polynucleotide comprising, or a combination of polynucleotides collectively comprising, a sequence encoding a chimeric protein of the present disclosure and a sequence encoding an RNA comprising a sequence of a stem-loop splicing hairpin comprising a cytosolic intron, wherein the cytosolic intron is capable of excision by the RNase domain of the chimeric protein.

[0058] In some embodiments, the stem-loop splicing hairpin comprises the structure, from 5’ to 3:. a 5’ stem 1 region, a 5’ stem 2 region, a 5’ loop region, a 3’ stem 2 region, a 5’ stem 3 region, a 3’ loop region, a 3’ stem 3 region, and a 3’ stem 1 region, wherein the RNA bases of the 5’ stem 1 region are complementary / to the RNA bases of the 3’ stem 1 region, the RNA bases of the 5’ stem 2 region are complementary to the RNA bases of the 3’ stem 2 region, and the RNA bases of the 5’ stem 3 region are complementary to the RNA bases of the 3’ stem 3 region.

[0059] In some embodiments, the RNA further comprises: a sequence encoding an initial start codon, wherein the initial start codon is 5’ to the sequence of the stem-loop splicing hairpin; a sequence encoding a first stop codon, wherein the first stop codon is 3’ to the sequence of the stem-loop splicing hairpin, and wherein the first stop codon is in frame with the initial start codon: and a sequence encoding a second start codon, wherein the second start

[0060] 8

[0061] 326657998 Attorney Docket No.: PTBI-010 / 01WO codon is 3’ to the sequence initial start codon, optionally 3’ to the cytosolic intron, and wherein the second start codon is out of frame with the initial start codon.

[0062] In some embodiments, the RNA further comprises a nucleotide sequence encoding a protein of interest, wherein the nucleotide sequence encoding the protein of interest is in frame with the second start codon, optionally wherein the nucleotide sequence encoding the protein of interest comprises the second start codon as its first codon.

[0063] In some embodiments, the IRE1 protein and the stem-loop splicing hairpin are from different species.

[0064] In some embodiments, the RNase domain, or the chimeric protein, is capable of excising the cytosolic intron comprised in the stem-loop splicing hairpin.

[0065] In some embodiments, the RNA is an engineered RNA polynucleotide of the disclosure.

[0066] In some embodiments, a) the kinase domain of the chimeri c protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 19; and b) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 25; and optionally wherein: c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 63. In some embodiments, a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 19; and b) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 25; and optionally wherein: c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 64. In some embodiments, a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at. least. 97%, at least 98%, or at. least. 99% identity to the sequence of SEQ ID NO: 19; and b) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least

[0067] 9

[0068] 326657998 Attorney Docket No.: PTBI-010 / 01WO 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 25;

[0069] and optionally wherein: c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 60. In some embodiments, a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity' to the sequence of SEQ ID NO: 19; and b) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 25; and optionally wherein: c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 62. In some embodiments, the kinase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 19; and b) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 25, and optionally wherein: c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 61. In some embodiments, a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 20; and b) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 26; and optionally wherein: c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 63, In some embodiments, a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 20; and b) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 26; and optionally wherein: c) the stem-loop splicing hairpin comprises a nucleotide

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[0071] 326657998 Attorney Docket No.: PTBI-010 / 01WO sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 64. In some embodiments, a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 20; and b) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 26; and optionally wherein: c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 80%, at least. 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 60. In some embodiments, a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 20; and b) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 26; and optionally wherein: c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 80%, at. least. 85%, at least 90%, at least 95%, at. least. 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 62. In some embodiments, a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at. least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 20; and b) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at. least. 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 26; and optionally wherein: c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 80%, at. least. 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 61. In some embodiments, a) the kinase domain of the chimeric protein comprises an amino acid sequence having at. least. 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 16; and b) the RNase domain of the chimeric protein comprises an amino acid sequence having at. least. 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 22; and optionally wherein: c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 80%, at least 85%, at

[0072] 11

[0073] 326657998 Attorney Docket No.: PTBI-010 / 01WO least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 63. In some embodiments, the kinase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 16; and b) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 22; and optionally wherein: c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 64. In some embodiments, a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 16; and b) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 22; and optionally wherein: c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 60. In some embodiments, a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 18; and b) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 24; and optionally wherein: c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 62. In some embodiments, a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 17; and b) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 23; and optionally wherein: c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at

[0074] 12

[0075] 326657998 Attorney Docket No.: PTBI-010 / 01WO least 99% identity to the sequence of SEQ ID NO: 63. In some embodiments, a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity' to the sequence of SEQ ID NO: 17; and b) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 23; and optionally wherein: c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 64. In some embodiments, a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 17; and b) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 23; and optionally wherein: c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 60. In some embodiments, a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 17; and b) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 23; and optionally wherein: c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 62. In some embodiments, a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least. 98%, or at least 99% identity to the sequence of SEQ ID NO: 17; and b) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 23; and optionally wherein: c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 61.

[0076] 13

[0077] 326657998 Attorney Docket No.: PTBI-010 / 01WO In some embodiments, a single polynucleotide comprises the sequence encoding the chimeric protein and the sequence encoding the RNA comprising the stem-loop splicing hairpin. In some embodiments, the single polynucleotide is a DNA polynucleotide, optionally wherein the DNA polynucleotide is a DNA plasmid or a viral vector. In some embodiments, the single polynucleotide is an RNA polynucleotide.

[0078] In some embodiments, a first polynucleotide comprises the sequence encoding the chimeric protein and a second polynucleotide comprises the sequence encoding the RNA comprising the sequence of a stem-loop splicing hairpin. In some embodiments, the first polynucleotide is an RNA and / or the second polynucleotide is an RNA.

[0079] In some embodiments, this disclosure provides a therapeutic composition comprising a polynucleotide or combination of polynucleotides of the present disclosure and one or more pharmaceutically acceptable excipients.

[0080] In some embodiments, this disclosure provides a viral particle comprising a polynucleotide or combination of polynucleotides of the present disclosure, optionally wherein the viral particle is an adeno-associated (AAV) particle, an adenovirus particle, or a lentiviral particle.

[0081] In some embodiments, this disclosure provides a cell comprising a chimeric protein of the present disclosure. In some embodiments, this disclosure provides a cell comprising an engineered RNA polynucleotide of the present disclosure. In some embodiments, this disclosure provides a cell comprising a polynucleotide or combination of polynucleotides of the present disclosure.

[0082] In some embodiments, the chimeric protein is expressed in the cytosol of the cell. In some embodiments, the chimeric protein is expressed on the plasma membrane of the cell.

[0083] In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell.

[0084] In some embodiments, in the presence of saturating level of the ligand, the cell expresses the protein of interest, or the non-coding RNA, at a level that is at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 70- fold, at least 100-fold, at least 200-fold, or at least 300-fold, higher or lower than the level of the protein of interest, or the non-coding RNA, expressed by the cell in the absence of the ligand.

[0085] In some embodiments, the IREl protein of (i) and / or the IRE1 protein of (ii) are derived from a species different from the cell. In some embodiments, the stem-loop splicing hairpin is derived from a species different from the cell. In some embodiments, the species

[0086] 14

[0087] 326657998 Attorney Docket No.: PTBI-010 / 01WO which the IRE1 protein of (i) and / or the IRE1 protein of (ii) are derived from is different from the species which the stem-loop splicing hairpin is derived from.

[0088] In some embodiments, the RNase domain, or the chimeric protein, is not capable of excising an endogenous cytosolic intron of an RNA molecule encoding an endogenous XBP1 protein of the cell. In some embodiments, an endogenous IRE1 protein of the cell is not capable of excising the cytosolic intron of the engineered RNA polynucleotide.

[0089] In some embodiments, this disclosure provides a method of inducing expression of a protein of interest in a cell, the cell comprising: a plurality of chimeric proteins of the present disclosure, and an RNA polynucleotide comprising a sequence of a stem-loop splicing hairpin comprising a cytosolic intron, wherein the cytosolic intron is capable of excision by the RNase domain of the chimeric protein, the method comprising exposing the plurality of chimeric proteins to ligands corresponding to their ligand binding domains, thereby promoting activation of the kinase domains and RNase domains of the chimeric proteins and promoting excision of the cytosolic intron of the RNA polynucleotide. In some embodiments, the RNA polynucleotide comprises: (i) a sequence of a stem-loop splicing hairpin comprising a cytosolic intron, wherein the cytosolic intron is capable of excision by the RNase domains of the chimeric proteins; (ii) a sequence encoding an initial start codon, wherein the initial start codon is 5’ to the sequence of the stem-loop splicing hairpin; (iii) a sequence encoding a first stop codon, wherein the first stop codon is 3' to the sequence of the stem-loop splicing hairpin, and wherein the first stop codon is in frame with the initial start codon; (iv) a sequence encoding a second start codon, wherein the second start codon is the first start codon 3’ to the sequence of the stem-loop splicing hairpin, and wherein the second start codon is out of frame with the initial start codon; and (v) a sequence encoding the protein of interest, wherein the nucleotide sequence encoding the protein of interest is in frame with the second start codon, optionally wherein the nucleotide sequence encoding the protein of interest comprises the second start codon as its first codon.

[0090] In some embodiments, the present disclosure provides a method of altering expression of a protein of interest in a cell, the cell comprising: a plurality of chimeric proteins of the present disclosure, and an RNA polynucleotide comprising a sequence of a stem-loop splicing hairpin comprising a cytosolic intron, wherein the cytosolic intron is capable of excision by the RNase domain of the chimeric protein, the method comprising exposing the plurality of chimeric proteins to ligands corresponding to their ligand binding domains, thereby promoting activation of the kinase domains and RNase domains of the chimeric proteins and promoting excision of the cytosolic intron of the RNA polynucleotide. In some

[0091] 15

[0092] 326657998 Attorney Docket No.: PTBI-010 / 01WO embodiments, the RNA polynucleotide comprises: (i) a sequence of a stem-loop splicing hairpin comprising a cytosolic intron, wherein the cytosolic intron is capable of excision by the RNase domains of the chimeric proteins; (ii) a sequence encoding an initial start codon, wherein the initial start codon is 5’ to the sequence of the stem-loop splicing hairpin; (iii) a sequence encoding a first stop codon, wherein the first stop codon is 3’ to the sequence of the stem-loop splicing hairpin, and wherein the first stop codon is in frame with the initial start codon; (iv) a sequence encoding a second start codon, wherein the second start codon is the first start codon 3’ to the sequence of the stem-loop splicing hairpin, and wherein the second start codon is out of frame with the initial start codon; and (v) a sequence encoding the protein of interest, wherein the nucleotide sequence encoding the protein of interest is in frame with the second start codon, optionally wherein the nucleotide sequence encoding the protein of interest comprises the second start codon as its first codon.

[0093] In some embodiments, the method comprises delivering the polynucleotide or combination of polynucleotides of the disclosure to the cell.

[0094] In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell.

[0095] In some embodiments, the stem-loop splicing hairpin comprises the nucleotide sequence of X1X2GCX3GX4X5X6T (SEQ ID NO: 73), wherein Xi is C or T, X2 is C, T, or A; X3 is C, T, or A; X4 is T, A, or C; X5 is A or G; and Xe is C, A, or G. In some embodiments, the stem-loop splicing hairpin comprises the nucleotide sequence of X1CX2GX3X4GX5X6X7 (SEQ ID NO: 74), where Xi is T or C; X2 is A, T, or C; X3 is C or A; X4 is T or A; X5 is absent, T, or A; Xe. is absent, C or T; and X7 is G or A.

[0096] In some embodiments, the cytosolic intron comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least. 97%, at least 98%, or at least 99% identity to the sequence of any one of SEQ ID NOs: 65-70. In some embodiments, the stem-loop splicing hairpin comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of any one of SEQ ID NOs: 59-64.

[0097] In some embodiments, a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at. least. 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 19; and b) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 25; and optionally wherein: c) the stem-

[0098] 16

[0099] 326657998 Attorney Docket No.: PTBI-010 / 01WO loop splicing hairpin comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 63. In some embodiments, a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 19; and b) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 25; and optionally wherein: c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 64. In some embodiments, a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 19; and b) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 25; and optionally wherein: c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 60. In some embodiments, a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 19; and b) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least. 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 25; and optionally wherein: c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 80%, at. least. 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 62. In some embodiments, a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at. least. 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 19; and b) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 25; and optionally wherein: c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least

[0100] 17

[0101] 326657998 Attorney Docket No.: PTBI-010 / 01WO 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 61, In some embodiments, a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 20; and b) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 26; and optionally wherein: c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 63. In some embodiments, a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 20; and b) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 26; and optionally wherein: c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 64. In some embodiments, a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 20; and b) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 26; and optionally wherein: c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 60. In some embodiments, a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity' to the sequence of SEQ ID NO: 20; and b) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 26; and optionally wherein: c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least

[0102] 18

[0103] 326657998 Attorney Docket No.: PTBI-010 / 01WO 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 62. In some embodiments, a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 20; and b) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 26; and optionally wherein: c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 61. In some embodiments, a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 16; and b) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 22; and optionally wherein: c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 63. In some embodiments, a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 16; and b) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 22; and optionally wherein: c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 64. In some embodiments, a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 16; and b) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 22; and optionally wherein: c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to

[0104] 19

[0105] 326657998 Attorney Docket No.: PTBI-010 / 01WO the sequence of SEQ ID NO: 60. In some embodiments, a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 18; and b) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 24; and optionally wherein: c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 62. In some embodiments, a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least. 98%, or at least 99% identity' to the sequence of SEQ ID NO: 17; and b) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 23; and optionally wherein: c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 63. In some embodiments, a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 17; and b) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 23; and optionally wherein: c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 64. In some embodiments, a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 17; and b) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 23; and optionally wherein: c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 66. In some embodiments, a) the kinase

[0106] 20

[0107] 326657998 Attorney Docket No.: PTBI-010 / 01WO domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least. 98%, or at least 99% identity to the sequence of SEQ ID NO: 17; and b) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 23; and optionally wherein: c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 62. In some embodiments, a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 17; and b) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 23; and optionally wherein: c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence of SEQ ID NO: 61.

[0108] In some embodiments, this disclosure provides a method of treating a disease, comprising administering to a subject a composition comprising the chimeric protein, the engineered RN A polynucleotide, the polynucleotide or combination of poly nucleotides, the viral particle, or the cell, of the present disclosure. In some embodiments, the disease is an inflammatory disease. In some embodiments, the ligand is a biomarker of the inflammatory disease, and / or the protein of interest is a therapeutic protein for treating the inflammatory disease. In some embodiments, the disease is a cancer. In some embodiments, the ligand is a cancer-associated antigen or biomarker, and / or the protein of interest is a therapeutic protein for treating the cancer.

[0109] In some embodiments, this disclosure provides a method comprising delivering a composition comprising a polynucleotide or combination of polynucleotides of the present disclosure to a cell. In some embodiments, the cell is an ex vivo cell or an in vivo cell. In some embodiments, the cell is in a human.

[0110] In some embodiments, the polynucleotide or combination of polynucleotides are comprised within one or more vectors. In some embodiments, the one or more vectors are non-viral delivery vectors, optionally wherein the non-viral delivery vectors are lipid nanoparticles. In some embodiments, the one or more vectors are viral delivery vectors. In

[0111] 21

[0112] 326657998 Attorney Docket No.: PTBI-010 / 01WO some embodiments, the viral delivery vector is an adeno-associated virus (AAV) vector, an adenovirus vector, or a lentivirus vector.

[0113] BRIEF DESCRIPTION OF THE DRAWINGS

[0114] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. It is to be understood that the data illustrated in the drawings in no way limit the scope of the disclosure.

[0115] FIGs. 1 A-1B. Design of programmable RNA smsIRE receptors. FIG. 1 A depicts a schematic illustration of the endogenous IRE1 pathway in humans. IRE I is an ER-resident transmembrane receptor with luminal and cytosolic domains. Under stress conditions, misfolded proteins accumulate in the ER, leading to the dimerization of the luminal IRE1 domain and the activation of the cytosolic domain. This enables the RNase domain of IRE 1 to splice a 26-nucleotide cytosolic intron from unspliced XBP1 (uXBPl) mRNA, resulting in the production of spliced XBP1 (sXBPl), a key transcription factor in the unfolded protein response. The unspliced XBP1 (uXBPl) produces a 261-amino acid (aa) product, while the frameshift in the spliced XBP1 (sXBPl) results in a longer 376-aa product. FIG. IB shows engineering of the smsIRE system for programmable mRNA splicing. The platform may be delivered as an mRNA encoding the programmable receptor and the synthetic splicing target. The receptor consists of ligand-induced actuator domains derived from the kinase-RNase domains of IRE1, and a modular extracellular ligand binding domain (such as FKBP, nanobodies, IL-lR / IL-lRAcP) for detecting their cognate soluble ligands (such as B / B, surface EGFP (surEGFP), IL-lp, TNF-a). Upon ligand binding, the smsIRE receptor undergoes dimerization, activating the IRE1 RNase domain, which results in the splicing of the cytosolic intron from the synthetic target mRNA. In the depicted embodiment, this splicing leads to the proper translation of a gene of interest (GOI) downstream of a T2A selfcleaving peptide, ensuring the independent translation of the desired protein product from the spliced hairpin.

[0116] FIGs. 2A-2H. Reprogramming Cytosolic RNA Splicing Using Human-Derived IRE1 Domains. FIG. 2A depicts a schematic representation of an mRNA splicing reporter. The reporter consists of the XBP1 mRNA splicing region (nucleotides 410-633) inserted upstream of the out-of-frame coding sequence of the fluorescent protein mScarlet. Upon removal of the cytosolic intron by IRE1, the translation of mScarlet is restored, providing a fluorescent

[0117] 22

[0118] 326657998 Attorney Docket No.: PTBI-010 / 01WO readout of the receptor splicing activity. FIG. 2B shows the splicing reporter output in HEK293 cells treated with the ER-stress inducer tunicamycin. The bar chart shows the mScarlet normalized units (norm, u.) for biological triplicates, n=3. Individual data points are depicted, with error bars indicating the standard deviation. An unpaired two-tailed t-test was performed, and asterisks indicate p < 0.0001 (****). FIG. 2C is a semi -quantitative RT-PCR gel showing the unspliced (top, 375 base pairs) and spliced (bottom, 349 base pairs) forms of the splicing reporter in HEK293 cells treated with tunicamycin for 0 (control), 2, or 6 hours. FIG. 2D are fluorescence microscopy images of HEK293 or HeLa IRE1 knockout (KO) cells transfected with the mRNA splicing reporter and treated with tunicamycin to induce ER stress. Increased fluorescence indicates splicing activity of the IRE1 receptor. Scale bars represent 250 m. FIG. 2E shows the splicing reporter output in HeLa IRE1 KO cells upon addition of tunicamycin. The bar graph illustrates the normalized units of mScarlet (norm, u.) for three biological replicates (n 3 ) Individual data points are depicted, with error bars indicating the standard deviation. FIG. 2F show splicing reporter induction with the human IRE1 cytosolic fragment fused with constitutive self-assembly domains in HeLa IRE1 KO cells. The fluorescent mScarlet (norm, u.) signal increases with dimerization stoichiometry for biological duplicates (n=2). The dashed line indicates the output of a genetically encoded, constitutively spliced fluorescent reporter driven by the EFl alpha promoter. FIG. 2G is a schematic of the inducible heterodimerization system using FKBP and FRB domains fused to IRE1 cytosolic regions. The addition of the small molecule A-'C induces heterodimerization, activating the IRE1 splicing domains and inducing the production of mScarlet by the reporter. FIG. 2H is dose-response curve showing the normalized mScarlet fluorescence in response to varying concentrations of the heterodimerizer A. Each data point shown is the average of biological triplicates (n=3), with error bars indicating the standard deviation.

[0119] FIGs. 3A-3E. Development of smsIREl: an Orthologous IRE I -Hairpin Pair Insulated from Human IRE1 Activity. FIG, 3 A shows human IRE1 splicing activity on different orthologous XBPl-like hairpins in HEK293 cells activated with tunicamycin. The heatmap shows the loglO-transformed mScarlet normalized units (norm, u.) for biological triplicates, n = 3. The different hairpin species are denoted at the bottom (Homo sapiens, Neurospora crassa, Magnaporthe grisea, Arabidopsis thaliana, Saccharomyces cerevisiae, and Candida albicans), and bar charts on top depict the mean fold change relative to the human wild-type (WT) hairpin sequence, with error bars indicating the standard deviation. FIG. 3B shows a heatmap of the pairwise comparison matrix of insulation scores for various XBPl-like hairpins (bottom) and IRE 1 -like splicing domains (left) fused to the B / B homodimerized

[0120] 23

[0121] 326657998 Attorney Docket No.: PTBI-010 / 01WO FKBPF36V. Higher scores indicate better insulation from human 1RE1 activity. Data are the loglO-transformed insulation score mean for biological triplicates, n = 3. FIG. 3C shows mScarlet normalized units (norm, u.) of HEK293 cells expressing the top-scoring insulated pairs of the splicing reporter (A. ihaliana) and FKBPF36V-IRE1 domains M. grisea) or the human components. Each dot represents an individual data point of the biological triplicates (n = 3), with error bars indicating the standard deviation. ANOVA with Sidak’s correction was performed to compare the mean of each treated column with its respective control, and asterisks indicate p < 0.05 (*) or p < 0.0001 (****). FIG. 3D is a semi-quantitative RT-PCR gel showing the unspliced (top, 183 base pairs) and spliced (bottom, 160 base pairs) forms of the A. ihaliana hairpin in the presence of FKBPF36Vfused with the IRE1 domain of M. grisea. The appearance of the spliced band occurs upon FKBPF36Vhomodimerization with the B / B homodimerizer. FIG. 3E shows the Sanger sequencing alignment of the A. ihaliana spliced band with the predicted exon-exon boundary of the splicing reporter. The excision point of the 23 -nucleotide cytosolic intron (SEQ ID NO: 66) is shown with a vertical dashed line between the 5’ and the 3’ cytosolic exons (SEQ ID NO: 111).

[0122] FIGs. 4A-4O. smsIRE Receptors Enable Detection of Soluble and Surface Antigens with RNA-Exclusive Encoding. FIG. 4A is a schematic illustrating the production, delivery, and execution of the smsIRE receptor system with mRNA-only components. The PCR- linearized DNA of the M. grisea (Mg) IRE1 and the A. ihaliana splicing hairpin target, each containing a 120-nucleotide 3’ poly-A tail, are in vitro transcribed to mRNA with the T7 polymerase. Upon delivery, the IRE1 mRNA is translated into its protein form, ready for activation by the chosen ligand. Upon ligand-induced dimerization, the mRNA of the synthetic splicing target is spliced to its active form, allowing the translation of the output. FIGs. 4B-4C show Microscopy images (FIG. 4B) and flow cytometry quantification (FIG. 4C) of HEK293 cells transfected with FKBPF36V-smsIRE mRNAs and treated with B / B to induce mRNA hairpin splicing and mScarlet. production. Cells were co-transfected with control mRNA producing constitutive mCerulean for normalization. Scale bars represent 250 um. The bar chart, shows normalized mScarlet units (norm, u.) for biological triplicates (n = 3). Gray points denote individual values, with error bars indicating the standard deviation. An unpaired two-tailed t-test was performed, and asterisks indicate p < 0.0001 (****). FIGs. 4D-4E is a schematic showing the transmembrane FKBPF36V-smsIRE receptor (FIG. 4D) and its dose-response curve (FIG.4E). Upon mRNA delivery, the smsIRE receptor is translated and localized to the plasma membrane due to the CD4 signal peptide and the glycophorin A (GpA) transmembrane domain, where it is activated by ligand-induced homodimerization

[0123] 24

[0124] 326657998 Attorney Docket No.: PTBI-010 / 01WO resulting in activation of the synthetic splicing target. The dose-response curve shows the mScarlet fluorescence in normalized units (norm, u.) at various concentrations of the B / B homodimerizer. Each data point represents the average of biological triplicates (n = 3), with error bars indicating the standard deviation. FIG. 4F shows schematics showing the ligand- induced dimerization of the transmembrane smsIRE receptor upon presentation of plasma membrane-immobilized surface EGFP (surEGFP). Each nanobody subunit (Lagl6 and LaG2) binds to a distinct epitope on separate surEGFP monomers, indirectly causing smsIRE receptor dimerization. Schematics (FIGs. 4G, 4J, 4M), microscopy images (FIGs. 4H, 4K, 4N), and quantification (FIGs. 41, 4L, 40) of membrane sensing after mRNA delivery of Lagl6-LaG2-smsIRE components with different cell configurations: (FIGs. 4G, 4H, 41) adherentsurEGFP"adherentsmsIRE, (FIGs. 4J, 4K, 4L) suspensionsurEGFP-adherentsmsIRE, and (FIGs. 4M, 4N, 40) suspensionsurEGFP-suspensionsmsIRE. Scale bars represent 25 pm.

[0125] Histograms show mScarlet expression in smsIRE-transfected populations, with each overlay representing one of the biological triplicates (n = 3).

[0126] FIGs. 5 A- 5 J. smsIRE Receptors Detect Biologically Relevant Inflammatory Molecules in Cell Lines and Human Primary T-cells with RNA-Ex elusive Encoding. FIGs.

[0127] 5A and 5C are schematics illustrating the ligand-induced dimerization of the transmembrane smsIRE receptor after mRNA delivery. The smsIRE binds IL-1 P through the IL-1R and IL-IRAcP ectodomains or senses TNF-a via a VHH nanobody. Upon ligand binding, the IRE1 RNase domain is activated, leading to cytosolic splicing of the synthetic mRNA reporter and enabling mScarlet translation. FIGs. 5B and 5D show dose-response curves showing mScarlet normalized units (norm, u. ) in response to varying concentrations of IL-1 (FIG. 5B) or TNF-a (FIG. 5D). Each data point represents the average of biological triplicates (n = 3), with error bars indicating the standard deviation. FIG. 5E is a diagram depicting the differentiation of THP-1 monocytes into macrophages to release IL- 1. THP-1 cells are treated with 25 n. M PM A for 48 hours (FIG. 5H), rested for 24 hours, and then stimulated with 1 pg / mL LPS for 3 hours, followed by 5 mM ATP for 2 hours. FIG. 5F shows quantification of splicing reporter fluorescence after detection of IL- Ip-containing medium collected from differentiated THP-1 macrophages by HEK293 cells transfected with smsIRE mRNAs. FIG. 5G is a schematic illustrating the sensing of TNF-a release by LPS-stimulated THP-1 monocytes using co-cultured HEK293GFPcells expressing the smsIRE receptors. FIG.

[0128] 5H shows a bar chart quantification of mScarlet signal in the GFPpositlveHEK293 population, upon co-culture with THP-1 monocytes. FIGs. 51 and 5L are schematics illustrating the sensing mechanism and quantification of IL-1 P detection by human primary T-cells delivered

[0129] 25

[0130] 326657998 Attorney Docket No.: PTBI-010 / 01WO with smsIRE mRNAs. All bar charts show mScarlet normalized units (norm, u.) for biological triplicates (n = 3), with gray points denoting individual values, and error bars indicating the standard deviation. For each condition, an unpaired two-tailed t-test was performed, and asterisks indicate p < 0.0001 (****).

[0131] FIGs. 6A-6G. smsIRE Receptors Secrete the Anti-inflammatory IL-10 cytokine Following Inflammatory Detection in Cell Lines and Human Primary T-cells with mRNA-Exclusive Encoding. FIG. 6A depicts a design of the IL-10 synthetic mRNA splicing output. The mRNA encoding the IL- 10 cytokine is placed out-of-frame downstream of theM grisea splicing hairpin and a T2A self-cleaving peptide sequence. Upon cytosolic intron removal, the T2A ensures that the IL- 10 cytokine is translated into the secretory pathway with its native sequence. FIGs. 6B and 6C are schematics illustrating the sensing of IL- Ip released by differentiated THP-1 macrophages and ELISA quantification of IL-10 production by mRNA- delivered HEK293 cells. ANOVA with Dunnett correction was performed to compare each condition to the control column (n = 3). FIGs. 6D and 6E are schematics illustrating the sensing of TNF-a released by THP-1 monocytes and ELISA quantification of IL- 10 production by mRNA-delivered, co-cultured HEK293 cells. ANOVA with Dunnett correction was performed to compare each condition to the control column (n = 3). FIGs. 6F and 6G are schematics illustrating the delivery of smsIRE mRNAs into human primary T-cells. Detection of IL-ip through IL-1R and ILl-RAcP leads to the activation of the IRE1 RNase domains, causing the cytosolic splicing of the IL-10 synthetic mRNA and the subsequent extracellular release of the mature cytokine protein product, quantified by ELISA. An unpaired two-tailed t-test was used for statistical analysis (n = 6).. All bar charts show single data points in gray, with error bars representing the standard deviation and asterisks indicate p < 0.0001 (****).

[0132] FIGs. 7A-7C. Endogenous XBP1 mRNA Splicing in HEK293 Cells. FIGs. 7A is a RT-PCR gel showing the unspliced (uXBPl, 248 base pairs) and spliced (sXBPI, 222 base pairs) forms of XBP1 mRNA in HEK293 cells treated with tunicamycin for 24 hours. FIG.

[0133] 7B is a RT-PCR gel depicting uXBPl and sXBPI bands in HEK293 cells transfected with 500 ng of junk DNA compared to non-transfected cells. FIG. 7C shows quantification of band intensities from RT-PCR gels in (FIG. 7A) and (FIG. 7B). For each condition, the intensity of the sXBPI band was normalized to the sum of the intensities of the spliced (sXBPI) and unspliced (uXBPl) bands, representing the total XBP1 signal. The bar chart illustrates the mean spliced / total XBP1 ratio for biological duplicates (n = 2) for non-

[0134] 26

[0135] 326657998 Attorney Docket No.: PTBI-010 / 01WO transfected cells, biological replicates (n = 9) for transfected cells, and biological triplicates for tunicamycin-treated cells (n = 3). Error bars indicate the standard deviation.

[0136] FIGs. 8A-8B. Structural Predictions and Sequence Alignments of the Bifurcated Stem-Loop Splicing Hairpin of different XBP1 Orthologues. FIG. 8 A shows mF old predictions of the secondary structure of the splicing hairpin region from Homo sapiens (SEQ ID NO: 112), Arabidopsis thaliana (SEQ ID NO: 114), Candida albicans (SEQ ID NO: 117), Saccharomyces cerevisiae (SEQ ID NO: 113), Neurospora crassa (SEQ ID NO: 116), and Magnaporlhe grisea (SEQ ID NO: 115) used for insulation screening (see FIGs. 3A-3E). With the exception of the Saccharomyces cerevisiae sequence, in which the cytosolic intron was trimmed to reduce its length and to introduce a frameshift event after cytosolic splicing, all other sequences are endogenous. In the Homo sapiens XBP1 splicing hairpin, the conserved stem and loop structural features shared among all the orthologues are highlighted with different colored arrows, FIG. 8B is the sequence alignment of the XBP1 orthologues (Homo sapiens (SEQ ID NO: 112), Arabidopsis thaliana (SEQ ID NO: 114), Candida albicans (SEQ ID NO: 117), Saccharomyces cerevisiae (SEQ ID NO: 113), Neurospora crassa (SEQ ID NO: 116), and Magnaporlhe grisea (SEQ ID NO: 115)) shown in (FIG. 8A). The sequence regions predicted to fold into conserved structural features are displayed on top, with arrows colored analogous to (FIG. 8A). For each position, the color scheme shows compatible base pairs where hue denotes the number of different types (1 = red, 2 = yellow, 3 = green, 4 = cyan, 5 = blue, and 6 = purple) of compatible base pairs (C-G, G-C, A-U, U-A, G-U, or U-G), and saturation decreases with the number of incompatible base pairs, thus showing sequence conservation within the column. Below the alignment, the histogram shows the frequency of the most conserved base pair for each position.

[0137] FIGs. 9A-9D. AlphaFold3 Predictions and Sequence Alignments of IRE1 Orthologues. FIG. 9A shows the AlphaFold3 predictions of the kinase (top domain) - RNase (bottom domain) regions for the IRE1 species used in the insulation screening (see FIGs. 3 A- 3E), including Homo sapiens, Arabidopsis thaliana, Candida albicans, Saccharomyces cerevisiae, Neurospora crassa, and Magnaporlhe grisea. FIGs. 9B-9C. AlphaFold3 predictions of the interaction between the 5’ loop (FIG. 9B), and the 3’ loop (FIG. 9C) of the XBP1 splicing region with the human RNase dimer of IRE 1. The cleaved G and C bases are depicted. Amino acid residues of the RNase that interact with the loops are depicted as sticks, with the interactions highlighted by dashed lines. FIG. 9D shows the sequence alignment of the RNase region from the chosen species (SEQ ID NOs: 21-26 and 118). Asterisks identify positions predicted by AlphaFold3 in (FIG. 9B) and (FIG. 9C) to interact with the 5’ or 3’

[0138] 27

[0139] 326657998 Attorney Docket No.: PTBI-010 / 01WO XBP1 loop, with red denoting general interacting amino acids and green indicating previously identified catalytic amino acids. Consensus sequence (>65%) identity is shown below the alignment.. Above the alignment, the secondary structure of the human IRE1 protein is depicted with a (alpha helix) and q (310 helix) notation. Residues are colored according to their physicochemical properties: HKR (cyan), DE (red), STNQ (brown), AVLIM (pink), FYW (blue), PG (orange), and C (green).

[0140] FIGs. 10A-10C. Functional Characterization of Orthologous IRE1 - Hairpin Pairs. FIG. 10A shows the correlation between the splicing activity of endogenous IRE1 activated with 5 pg / mL of tunicamycm in HEK293 cells and the splicing activity of transfected FKBP'36V-JRElf”imanactivated with 500 nM of B / B homodimerizer in IRE1 HeLa KO cells used for the insulation screening. The plot shows mScarlet normalized units (norm, u.) obtained for each splicing reporter. The dashed line represents the curve fitting, with an R2 value of 0.9895 and a p < 0.0001. FIG. 10B shows the mScarlet normalized units (norm, u.) obtained from each splicing hairpin reporter species for each tested FKBPF36V-IREl ortholog. FIG. 10C shows the dynamic range of the second-best insulating pair composed of the Arabidopsis thaliana hairpin and the Neurospora crassa IRE1. Individual data points are shown in gray, with error bars indicating the standard deviation from biological triplicates (n = 3). An unpaired t-test was used for statistical analysis, and asterisks indicate p < 0.0001 (****).

[0141] FIGs. 11A-11C. IL- 1 P and TNF-a Production by THP-1 Cells with Sensing by HEK293 Cells. FIG. 11A shows the concentration of TNF-a and IL-ip (pg / mL) quantified by automated ELISA, produced by undifferentiated and unstimulated THP-1 cells, undifferentiated THP-1 cells treated overnight with 1 pg / mL of LPS, or macrophage- differentiated THP-1 cells stimulated with 1 y.g / mL LPS for 3 hours followed by 5 mM ATP for 2 hours. Individual data points are shown, with error bars denoting the standard deviation from at least 6 biological replicates per condition (n > 6). FIG. 1 IB shows the average mScarlet signal (n = 3) of HEK293 cells transfected with IL-1R- and IL1RAcP- smsIRE mRN A upon addition of macrophage-differentiated and stimulated THP-1 medium on the smsIRE receptor. The signal is overlaid on the dose-response curve calculated with the purified IL-ip, and the concentration of IL-ip in the THP-1 medium was determined by automated ELISA. FIG. 11C shows the average mScarlet signal (n = 3) of HEK293 cells transfected with anti- TNF-a VHH-smsIRE mRNA co-cultured overnight with LPS- stimulated THP-1 monocytes. The signal is overlaid on the dose-response curve calculated

[0142] 28

[0143] 326657998 Attorney Docket No.: PTBI-010 / 01WO with purified TNF-a, and the concentration of TNF-a in the co-culture medium was determined by automated ELISA.

[0144] FIGs. 12A-12F. IL-10 Production with varying splicing hairpin RNA amounts and baseline IL-10 expression in HEK293 cells and human primary T-cells. FIGs. 12A and 12C show IL-10 (pg / mL) released by HEK293 cells transfected with an 8x fold increase of the splicing hairpin mRNA for the IL- 1 p (FIG. 12A) or TNF-a (FIG. 12C) cytokine converter smsIRE. For FIG. 12A, IL-10 release was triggered either by the addition of macrophage- differentiated and stimulated THP-1 cell medium or by direct addition of purified IL- ip (10 ng / mL). For FIG. 12C, IL-10 release was induced by overnight co-culture with LPS-stimulated THP-1 monocytes or by direct addition of purified TNF-a (10 ng / mL). Data are presented as the mean of biological triplicates (n = 3), with gray dot representing individual values and vertical bars indicating the standard deviation. ANOVA with Dunnett correction was performed to compare each condition to the control column, and asterisks indicate p < 0.0001 (****). FIG 12B and 12D are graphs showing IL-10 (pg / mL) production by HEK293 cells in response to sensing IL-ip (10 ng / mL, FIG. 12B) or TNF-a (10 ng / mL, FIG. 12D), with two different transfection amounts of IL-10 hairpin mRNA. The expected linear relationship between IL- 10 production and mRNA quantity is indicated by the solid green or orange line, respectively. FIG. 12E shows endogenous IL-10 production levels by primary T-cells, either unstimulated or stimulated with IL-ip (n = 2), compared to IL- 10 production by the active IL- lb cytokine converter smsIRE receptor (n = 6). FIG. 12F shows endogenous IL- 10 production by non-transfected HEK293 cells in the presence of IL-ip or TNF-a.

[0145] FIG. 13 is a sequence alignment which compares IRE1 kinase domains of H. sapiens (SEQ ID NO: 15), A. thaliana (SEQ ID NO: 16), C. albicans (SEQ ID NO: 17), 5. cerevisiae (SEQ ID NO: 18), N. crassa (SEQ ID NO: 19), and grisea (SEQ ID NO: 20). The sequence alignment shows the consensus motif X1LHX2LX3IX4HRDX5KPX6NX7LX8X9 (SEQ ID NO: 53), where Xi is H or Y, X2 is E or S; X3 is G, N, K, or R; X is V or I; X5 is L or I; X& is Q or H; X7 is E or I; Xs is I or V; and X9 is S, A, or N at positions 158-177 and the consensus motif X1X2X3X4SDX5GX6X7KX8X9X10 (SEQ ID NO: 54), where Xi is S, K, or R; X2 is A, I, or L; X3 is K, M, or L; X4 is L, I, or V; X5 is M or F; X& is I or L; X7 is S or C; Xs is R or K; X9 is M or L; and X10 is S, A, D, or E at positions 201-215. The sequence alignment also shows the consensus motifs at 100 %, 90%, 80% and 70% (SEQ ID NOs: 119-122).

[0146] FIG. 14 is a sequence alignment which compares IRE1 RNase domains off / , sapiens (SEQ ID NO: 21), N. crassa (SEQ ID NO: 22), M. grisea (SEQ ID NO: 23), A. Ihaliana

[0147] 29

[0148] 326657998 Attorney Docket No.: PTBI-010 / 01WO (SEQ ID NO: 24), C. albicans (SEQ ID NO: 25), and 5. cerevisiae (SEQ ID NO: 26). The sequence alignment shows the consensus motif RX1YX2X3X4X5X6X7DLLRX8X9RNKX10X11HX12 (SEQ ID NO: 55), where Xiis T, R, or K; X2 is K, T, S, or H; X3 is G, Y, P, or S; X4is G, D, N, S, or E; X5is S, R, or K; X6is V, I, M, or L; X7is R, L, or M; X8is A or V; X9is M, I, or L, X10is K, L, or Y; X11is H or N; and X12is Y, H, or F at positions 62-83. The sequence alignment also shows the consensus motifs at 100 %, 90%, 80% and 70% (SEQ ID NOs: 123-126).

[0149] FIG. 15 shows a partial sequence alignment of stem-loop splicing hairpins from Homo sapiens (SEQ ID NO: 132), Arabidopsis ihaiiana (SEQ ID NO: 130), Candida albicans (SEQ ID NO: 131), Saccharomyces cerevisiae (SEQ ID NO: 127), Neurspora crassa (SEQ ID NO: 129), and Mgnaporthe grisea (SEQ ID NO: 128). Depicted is consensus motif X1AX2X3X4X5GCX6GX7XSX9TX10X11X12X13 (SEQ ID NO: 71), where Xi is A, C, or G; X2 is G or T, X3 is T or C; X4is C or T, Xs is A, T, or C; Xe is C, T, or A; X7 is T, A, or C; Xs is A or G; X9 is C, A, or G; X10 is T, G, or C; X11 is A or T; X12 is C, T, or G; and X13 is G, T, or A at positions 28-45 and consensus motif X1X2X3X4X5CX6GX7X8G (SEQ ID NO: 72), where Xi is A or C; X2 is C, T, or A; X3 is G, T, A, or C; X4 is G, A, or C; X5 is C or T, Xe is C, T, or A; 7 is A or C; and Xs is A or T at positions 59-69.

[0150] DETAILED DESCRIPTION

[0151] Most synthetic biology tools, such as programmable synthetic receptors, primarily rely on transcription factor-based regulation to transduce signals. For example, SynNotch23, MESA24, and Tango systems, involve fusing a transcription factor to the intracellular part of the receptor and releasing it through single-turnover proteolytic cleavage upon stimuli. Other strategies, such as GEMS27 and the artificial two-component system, employ an orthogonal transcription factor that is free-floating in the cytosol and activated through multiple-turnover receptor-mediated phosphorylation. This reliance on transcription factors not only precludes mRNA-only applications, but also introduces other drawbacks, such as delayed response times, increased circuit complexity and larger payload sizes for cellular delivery.

[0152] Although some RNA-based platforms have been developed, the field currently lacks an actuator capable of post-transcriptional response to user-defined extracellular signals. Classical RNA-acting RNAs, like ribozymes or aptamers, are inherently confined to the intracellular space and cannot interact with external cues. More recent protein-based RNA regulators, such as RNA-cleaving Cas proteins and orthogonal proteases are also canonically acting as repressors of protein synthesis. Finally, adenosine deaminases acting on RNA

[0153] 30

[0154] 326657998 Attorney Docket No.: PTBI-010 / 01WO systems, despite being capable of acting as “ON” switches, have been shown to sense only intracellular mRNA-inputs and still suffer from poor editing efficiency and potential off- target effects.

[0155] Described herein are modular programmable chimeric proteins capable of detecting extracellular and / or intracellular signals and triggering cellular responses through an engineered cytosolic RNA splicing mechanism following recognition of a specific hairpin sequence comprised in a synthetic mRNA. More specifically, modular chimeric proteins of the present disclosure are able to sense different signals and convert them through an engineered RNA splicing mechanism, bypassing transcriptional regulation and enabling a faster and more direct control on cellular response. The modular design of the chimeric proteins of the present disclosure allows for the substitution of various ligand-binding domains, enabling the system to be reprogrammed for diverse applications in therapeutics, diagnostics, and cell therapy. The modular chimeric proteins of the present disclosure are advantageous because signal transduction is capable of operating entirely at the RNA level, providing a faster and more efficient way to modulate cellular responses and making it easier to scale for various applications than currently available RNA-regulation platforms.

[0156] Additionally, modular chimeric proteins of the present disclosure are capable of amplifying extracellular and intracellular signals, compared to current approaches, since they operate as an enzymatic multi-turnover process, where one activated receptor can splice multiple synthetic mRNA target molecules, resulting in enhanced sensitivity compared to single¬ turnover systems. The inventors surprisingly found that modular chimeric proteins of the present disclosure can be implemented with a smaller genetic payload, making it advantageous for therapeutic applications (e.g., RNA-based therapies) and retains the ability to be encoded at the DNA level, enabling its usage in applications that require long-term expression or sustained activity over longer periods (e.g., gene therapy approaches with viral delivery),, while avoiding the complexity and time delay associated with transcriptional regulation. Further, the inventors surprisingly found that modular chimeric proteins of the present disclosure offer a faster and more efficient way to modulate cellular responses, with the advantage of signal transduction operating entirely at the RNA level, making it easier to scale for various applications.

[0157] In one aspect, the engineered cytosolic RNA splicing mechanisms described herein capitalize on the transduction mechanism of the inositol-requiring enzyme- 1 (IRE1) receptor. IREl is an evolutionarily conserved single-pass transmembrane protein that comprises I) an N-terminal, ER luminal domain that senses unfolded proteins and 2) a C -terminal

[0158] 31

[0159] 326657998 Attorney Docket No.: PTBI-010 / 01WO cytoplasmic region which initiates an unfolded protein response (UPR) via a serine / threonine kinase domain and an endoribonuclease (RNase) domain. In the endogenous UPR pathway, accumulation of misfolded proteins into the ER during stress conditions triggers the homodimerization of the IRE1 luminal domain, forcing the two cytosolic kinase domains to juxtapose in a transphosphorylation-competent orientation. This phosphate addition in the activation loop of the kinase region induces a conformational change that allows the two RNase subunits to form the active catalytic site which cleaves a species-specific consensus sequence present in tandem on a bifurcate stem loop hairpin of the x-box binding protein 1 (XBP1) mRNA. These dual cuts performed by the IRE1 RNase allow the subsequent rearrangement of the mRNA secondary' structure, which provokes ejection of the internal cytosolic intron fragment, followed by sealing of the remaining scar through a tRNA ligase.

[0160] Accordingly, this disclosure provides modular chimeric proteins termed synthetic RNA splicing IRE1 (smsIRE) that comprise (e.g., from N-terminal to C-terminal) a ligand binding domain, a kinase domain, and an RNase domain. smsIRE of the present disclosure operate through an orthogonal RNA splicing mechanism, in which the activated kinase- RNase domain recognizes a specific hairpin sequence contained in a synthetic RNA. Upon processing, the RNase domain cleaves the target RNA at two locations, excising the internal cytosolic intron sequence. The modular design of smsIRE allows for the substitution of various ligand binding domains, enabling the system to be reprogrammed for diverse applications (e.g., in therapeutics, diagnostics, cell therapy). Also provided herein are engineered synthetic RNA comprising hairpin sequences recognized by chimeric proteins provided herein.

[0161] I. Synthetic Proteins - Synthetic mRNA Splicing IREls (smsIREs) Aspects of this disclosure provide a chimeric protein comprising (i) a ligand binding domain; (ii) a kinase domain; and (iii) an RNase domain. In some embodiments, the chimeric protein comprises, N-terminally to C -terminally, the kinase domain of (ii) and the RNase domain of (iii). In some embodiments, the chimeric protein comprises the ligand binding domain of (i) N-terminal to the kinase domain of (ii) and the RNase domain of (iii). In some embodiments, the kinase domain of (ii) and the RNase domain of (iii) are, or are derived from, an IRE1 protein.

[0162] In some embodiments, the chimeric protein is a cell-surface protein. In some embodiments, the chimeric protein is a membrane-bound protein (e.g., plasma membrane

[0163] 32

[0164] 326657998 Attorney Docket No.: PTBI-010 / 01WO bound). In some embodiments, the chimeric protein is a transmembrane protein. In some embodiments, the chimeric protein is a plasma membrane protein.

[0165] In some embodiments, the chimeric protein is a soluble protein. In some embodiments, the chimeric protein is a cytosolic protein.

[0166] In contrast to currently available DNA-based receptor systems (which involve transcription factor-mediated signal transduction) and RNA-based solutions (such as ribozymes and aptamers, which are confined to intracellular space, cannot respond to extracellular signals, and lack modularity and compatibility with commonly used protein¬ based ligand binding domains), the chimeric proteins described herein have a modular design that allows for the substitution of various domains (e.g., ligand binding domain, kinase domain, RNase domain), enabling the system to be reprogrammed for diverse applications (e.g., therapeutics, diagnostics, cell therapy).

[0167] In some embodiments, the ligand binding domain is not a ligand binding domain of an inositol-requiring enzyme 1 (IRE1) protein. In some embodiments, the kinase domain is a kinase domain of an IRE1 protein. In some embodiments, the kinase domain is a kinase domain derived from an IRE I protein.

[0168] In some embodiments, the RNase domain is an RNase domain of an IRE1 protein. In some embodiments, the RNase domain is an RNase domain derived from an IREI protein.

[0169] An “IREI protein,” also referred to in the art as an Endoplasmic Reticulum To Nucleus Signaling 1 (ERN1) protein or a Serine / Threonine-Protein Kinase / Endoribonuclease, includes human Homo sapiens) IREI, the amino acid sequence of which may be found in, for example, NCBI Accession No. NP_001424.3. An IREI protein also includes Arabidopsis thaliana IREI, the amino acid sequence of which may be found in, for example, NCBI Accession No. NP 565419.1. An IREI protein also includes Candida albicans IREI, the amino acid sequence of which may be found in, for example, NCBI Accession No.

[0170] XP 717606.2. An IREI protein also includes Saccharomyces cerevisae IREI, the amino acid sequence of which may be found in, for example, NCBI Accession No. NP_011946.1. An IREI protein also includes Schizosaccharomyces pombe IREI, the amino acid sequence of which may be found in, for example, NP_013349.1. An IREI protein also includes Neurospora crassa IREI, the amino acid sequence of which may be found in, for example, NCBI Accession No. XP_959521.1. Other IREI proteins are known in the art and can be readily identified using sequence comparisons to these exemplary IREI proteins.

[0171] In some embodiments, the chimeric protein comprises a kinase domain of (ii) and a RNase domain of (iii) that are from, or derived from, the same IREI protein. In some

[0172] 33

[0173] 326657998 Attorney Docket No.: PTBI-010 / 01WO embodiments, the chimeric protein comprises a kinase domain of (ii) and a RNase domain of (iii) that are from, or derived from, different IREl protein. In some embodiments, the IREl protein(s) that the kinase domain and / or the RNase domain are derived from are not human IREl protein. In some embodiments, the IREl protein(s) that the kinase domain and / or the RNase domain are derived from are not an animal IREl protein.

[0174] In some embodiments, the chimeric protein, or its RNase domain (e.g., after phosphorylation ), iis capable of excising a cytosolic intron comprised in a stem-loop splicing hairpin of an RNA molecule. In some embodiments, the cytosolic intron or the stem-loop splicing hairpin is from, or derived from, an RNA molecule encoding a x-box binding protein 1 (XBP1).

[0175] Methods of measuring the function of a domain (e.g., a kinase domain or an RNase domain) are described herein and known to those having ordinary skill in the art.

[0176] a. Ligand binding domains

[0177] The chimeric proteins of the present disclosure comprise a ligand binding domain. A ligand binding domain is a region of a protein that is responsible for recognizing and binding to a cognate ligand (e.g., small molecule, cell-bound antigen, extracellular soluble protein), which induces conformational changes that activate or inhibit the protein’s function.

[0178] In some embodiments, the ligand binding domain is a ligand binding domain of an IREl. In some embodiments, the ligand binding domain is a ligand binding domain derived from IREl. The ligand binding domain of wild type IRE 1 is typically located in the luminal / extracellular region and resides in the ER lumen, where it detects the accumulation of misfolded or unfolded proteins within the ER and promotes the dimerization or oligomerization of IRE1 molecules, activating the kinase and RNase domains to regulate downstream signaling.

[0179] In some embodiments, the ligand binding domain is an animal (from kingdom animalia) IREl ligand binding domain. In some embodiments, the ligand binding domain is derived from an animal IREl ligand binding domain. In some embodiments, the animal IRE1 ligand binding domain is a Homo sapiens IRE I ligand binding domain, a Mus musculus IRE1 ligand binding domain, a Drosophila melanogaster IRE1 ligand binding domain, or a Caenorhabditis elegans IREl ligand binding domain.

[0180] In some embodiments, the ligand binding domain is a plant (from kingdom plantae) IREl ligand binding domain. In some embodiments, the ligand binding domain is derived from a plant IREl ligand binding domain. In some embodiments, the plant IREl ligand

[0181] 34

[0182] 326657998 Attorney Docket No.: PTBI-010 / 01WO binding domain is an Arabidopsis taliana IRE1 ligand binding domain, an Oryza sativa IRE1 ligand binding domain, or a Populus trichocarpa IRE I ligand binding domain.

[0183] In some embodiments, the ligand binding domain is a fungal IRE1 ligand binding domain. In some embodiments, the ligand binding domain is derived from a fungal IRE1 ligand binding domain. In some embodiments, the corresponding fungus is a plant- pathogenic fungus. In some embodiments, the fungal IRE1 ligand binding domain is a Saccharomyces cerevisiae IRE1 ligand biding domain, a Candida albicans IRE1 ligand binding domain, a Neurospora crassa IRE1 ligand binding domain, Magnaporthe grisea IRE1 ligand binding domain, an Schizosaccharomyces pombe IRE1 ligand binding domain, or an Aspergillus nidulans IRE1 ligand binding domain.

[0184] In some embodiments, the ligand binding domain comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least. 98%, or at least 99%) identity to any one of SEQ ID NOs: 1-7. In some embodiments, the ligand binding domain comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 1-7. In some embodiments, a polynucleotide of the disclosure comprising a sequence encoding the ligand binding domain comprising an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to any one of SEQ ID NOs: 1- 7. In some embodiments, a polynucleotide of the disclosure comprising a sequence encoding the ligand binding domain comprising or consisting of any one of SEQ ID NOs: 1-7.

[0185] In some embodiments, the ligand binding domain is not a ligand binding domain of an inositol-requiring enzyme 1 (IRE1 ) protein. In some embodiments, the ligand binding domain is not a H. sapiens IRE1 ligand binding domain (SEQ ID NO: 1). In some embodiments, the ligand binding domain is not an. thaliana IRE1 ligand binding domain (SEQ ID NO: 2). In some embodiments, the ligand binding domain is not a C. albicans IRE1 ligand binding domain (SEQ ID NO: 3). In some embodiments, the ligand binding domain is not a 5. cerevisiae 1RE1 ligand binding domain (SEQ ID NO: 4). In some embodiments, the ligand binding domain is not a > Y pombe IRE1 ligand binding domain (SEQ ID NO: 5). In some embodiments, the ligand binding domain is not a A7. crassa IRE1 ligand binding domain (SEQ ID NO: 6). In some embodiments, the ligand binding domain is not a grisea IREl ligand binding domain (SEQ ID NO: 7). In some embodiments, the ligand binding domain, or the chimeric protein, does not comprise or consist of a sequence having more than 15%, more than 20%, more than 25%, more than 30%, more than 35%, more than 40%, more than 50%,

[0186] 35

[0187] 326657998 Attorney Docket No.: PTBI-010 / 01WO more than 60%, more than 70%, or more than 80%, sequence identity to any one of SEQ ID NOs: 1-7.

[0188] In some embodiments, the ligand binding domain is capable of dimerization, trimerization, or multimerization. In some embodiments, the ligand binding domain is capable of forming a multimer comprising at least 4, 5, 6, 7, 8, 9, or 10 monomers. In some embodiments, binding to the ligand induces the dimerization, trimerization, or multimerization of the ligand binding domain. In some embodiments, the chimeric protein is a monomeric protein in the absence of the ligand — for example, when the chimeric protein is at physiologically relevant condition and concentration.

[0189] In some embodiments, the ligand binding domain lacks a dimerization, trimerization, or multimerization domain. In some embodiments, the ligand binding domains form a dimer, a trimer or a multimer upon binding to a ligand that is itself (or is capable of forming) a dimer, a trimer or a multimer. For example, a ligand may be an antibody that has at least two binding sites and is capable of simultaneously binding at least two ligand binding domains.

[0190] In some embodiments, the ligand comprises a small molecule, a peptide, a protein, an antibody, a lipid, a carbohydrate, or any combination thereof. In some embodiments, the ligand comprises a small molecule. In some embodiments, the ligand comprises a peptide. In some embodiments, the ligand comprises an antibody. In some embodiments, the ligand comprises a protein. In some embodiments, the ligand comprises a lipid. In some embodiments, the ligand comprises a carbohydrate.

[0191] In some embodiments, the ligand is a cancer biomarker or a cancer-associated antigen. Various cancer biomarkers and cancer-associated antigens are known in the art, see, for example, US2023 / 0028110, the content of which is incorporated by reference in its entirety.

[0192] In some embodiments, the ligand is a cell-surface ligand. In some embodiments, the ligand is a membrane-bound ligand (e.g., plasma membrane bound). In some embodiments, the ligand is a transmembrane ligand. In some embodiments, the ligand is a soluble ligand.

[0193] In some embodiments, the ligand binding domain binds peptides or protein ligands (e.g., tumor necrosis factor receptor ligand binding domain, interleukin-1 receptor ligand binding domain, interleukin-6 receptor ligand binding domain, toll-like receptor ligand binding domain, erythropoietin receptor ligand binding domain, growth hormone receptor ligand binding domain, prolactin receptor ligand binding domain, fibroblast growth factor receptor ligand binding domain, INF-α / β receptor ligand binding domain, IFN-γ receptor ligand binding domain, interleukin-2 receptor ligand binding domain). In some embodiments,

[0194] 36

[0195] 326657998 Attorney Docket No.: PTBI-010 / 01WO the ligand binding domain binds small molecules (e.g., FK506-binding protein ligand binding domain, cyclophilin ligand binding domain, G-protein coupled receptor ligand binding domain, nuclear receptor ligand binding domain, adenosine A2A receptor ligand binding domain, protein kinase inhibitor ligand binding domain, peroxisome proliferator-activated receptor ligand binding domain). In some embodiments, the ligand binding domain is a co¬ receptor (e.g., interleukin- 1 receptor accessory protein, glycoprotein 130, cytokine receptor¬ like factor 2, myeloid differentiation factor 2, cluster of differentiation 14).

[0196] In some embodiments, the ligand binding domain comprises a dimerization domain. A dimerization domain is a region of a protein that facilitates and stabilizes the interaction between two protein molecules, resulting in the formation of a dimer. In some embodiments, the dimerization domain is a leucine zipper domain, a SH2 domain, a SH3 domain, or a G- protein coupled receptor dimerization domain. In some embodiments, the dimerization domain is derived from a leucine zipper domain, a SH2 domain, a SH3 domain, or a G-protein coupled receptor dimerization domain. In some embodiments, the ligand binding domain lacks a dimerization domain.

[0197] In some embodiments, the ligand binding domain comprises an antibody. In some embodiments, the antibody is a monoclonal antibody, a chimeric antibody, a single-chain variable fragment (scFv), a nanobody, or a bi specific antibody. In some embodiments, the antibody comprises an immunoglobulin single variable domain. In some embodiments, the antibody comprises two immunoglobulin variable domains. In some embodiments, the antibody comprises more than two (e.g., 3, 4, or more than 4) immunoglobulin variable domains. In some embodiments, the antibody comprises a first immunoglobulin single variable domain that binds to a first epitope of a target protein and a second immunoglobulin single variable domain that binds to a second epitope of the target protein.

[0198] In some embodiments, the ligand binding domain comprises an extracellular ligand binding domain. An extracellular ligand binding domain is a region of a cell-surface protein (e.g., a cell surface receptor) located outside the cell that binds to extracellular ligands (e.g., peptides, proteins, small molecules, ions). In some embodiments, the ligand binding domain comprises a ligand binding domain of a receptor tyrosine kinase, a ligand binding domain of a G-protein coupled receptor, a ligand binding domain of a cytokine receptor, or a combination thereof. In some embodiments, the ligand binding domain comprises a ligand binding domain of a FK506-binding protein (FKBP), a ligand binding domain of a tumor necrosis factor (TNF) receptor superfamily protein, a ligand binding domain of an

[0199] 37

[0200] 326657998 Attorney Docket No.: PTBI-010 / 01 WO interleukin-1 receptor (1L-1R) protein, a ligand binding domain of an interleukin- 1 receptor accessory' protein (IL-lRAcP), or a combination thereof.

[0201] In some embodiments, the ligand binding domain comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96% at least 97%, at least 98%, or at least 99%) identity to any one of SEQ ID NO: 8-14. In some embodiments, the ligand binding domain comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 8-14.

[0202] Also described herein are polynucleotides comprising a sequence encoding a ligand binding domain (or a chimeric protein). In some embodiments, the ligand binding domain (or chimeric protein) comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96% at least 97%, at least 98%, or at least 99%) identity to any one of SEQ ID NO: 8-14. In some embodiments, the ligand binding domain (or a chimeric protein) comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 8-14.

[0203] Table 1. Exemplary ligand binding domains.

[0204] Description Amino Acid Sequence SEQ ID NO

[0205] H. sapiens MPARRLLLLLTLLLPGLGIFGSTSWFLPETLLFVSTLDGSLHAVSKRTGSIKW 1 1RE1 ligand TLKEDPVLQVPTHVEEPAFLPDPNDGSLYTLGSKNNEGLTKLPFTIPELVQASP binding CRSSDG1LYMGKKQDIWYVIDLLTGEKQQTLSSAFADSLCPSTSLLYLGRTEY domain T1TMYDTKTRELRWNATYFDYAASLPEDDVDYKMSHFVSNGDGLVVTVDSE SGDVLWIQNYASPVVAFYVWQREGLRKVMHINVAVETLRYLTFMSGEVGRI TKWKYPFPKETEAKSKLTPTLYVGKYSTSLYASPSMVHEGVAWPRGSTLPL LEGPQTDGVTIGDKGECVITPSTDVKFDPGLKSKNKLNY'LRNYWLLIGI-IHET PLSASTKMLERFPNNLPKHRENVIPADSEKKSFEEVINLVDQTSENAPTTVSR DVEEKPAII.'WAM-’E / WVDSMLIO)

[0206] A. thaliana MPPRCPFLRHLFFLLLLLSPWIMSPCGGAADDVTYPIVPSSPGRRSILQIRREPP 2 IRE1 ligand TEPNTKLVVDRDGKVFLKQQPKETPYWSFSTGSPMHSLYQAPANNNTENAT binding EITRPHIIVEYLNNSKAATTVDGYHNWTVQEFFRQKPLVTDDGVTLGSETTSA domain YLWGRSGRLIHVYKSTGDTKITNALVKPASTEDFVNEPLLIRRTDSKLEHFS KTTGKLVWNLTVSHFRAALLCDPVFNSGYDLGPKLQTGIYMPLLCGSQIDVR GPEIV1RVLHDQPMNVKMLPSPSLNHFESENSIMPFGKARESRKLQEQHKQKY TYLFGQ

[0207] C. albicans MHLFYFISYIISILLVSIIASRLERITTKYLSPQQQQQQQRRDQAYNPATPTNNI 3 IRE1 ligand YTSASLQPIAERSLDDWDLNNIILLSDINGSLHCVNRDDGNLIWSLPIDEPLVK binding IQSNIKDKSAAHNILWFVEPYQDGTLYYTTPKFGLNKLPTSIKDLVMESPFTLS domain GDDKIYTGTRKTSLYNINIHTGEIKSSFGNTDECPIPRSTLPPETAFNADDTIMI

[0208] GKTTYELSIHSKSNSDVAIWMNVTVSCVAVPNNIDNDLILQNQQSLDKIYFTPFH DRSLLAINKDIGTPIWISKLPGLPVSIFDVFSNSAKTNDYLLLPHPLKVLNDLQS NDINHQSMVFVNKTSSTSSNSQDHLNDQWVAMSFRNYPTLIKSAPISKYQEV LNKYYAGINTNTNNNNDKEFTWYIENFQIV’NNSNENIENLISGIHRVFELSPE NSYQPFERFHSPLDDIKRIGDGSGNDDKDEQSSAERTKNNGGAPDIIGGLKFPS RLSTLTSEILLIEPSKDNQLDQQSGNSHHNQDTSKLDNSNVTSS

[0209] S’, cerevisiae MRLLRRNMLVLTLLVCVFSSIISCSIPLSSRTSRRQ1VEDEVASTKKLNFNYGV 4

[0210]

[0211] IRE1 ligand DKNINSPIPAPRITTEGLPNAIKLSSYPTPNLLTADNRRANKKGRRAANSISVP

[0212] 38

[0213] 326657998 Attorney Docket No.: PTBI-010 / 01WO binding YLENRSLNELSLSDILIAAE> VEGGLHAVDRRNGfflIWSIEPENFQPLIEIQEPSR domain LETYETLIIEPFGDGNIYYFNAHQGLQKLPLSIRQLVSTSPLHLKTNIVVNDSG KIVEDEKVYTGSMR.’nMYTINKtl^NGEITSAFGPGSKNGYFGSQSVDCSPEEKIK LQEC EXAIIX IGK TIFELGIHSYTJGASY TWTYST AVQQNA i DVPi. Ai. QX TFSKD GMCIAPFRDKSLLASDLDFRIARWVSPTFPGIWGLFDVFNDLRTNENIIAT’HP FNTGDHESISSNn<\ATDQTSNLSWFALSSQNFPSL.\E. SAPISRYAASSDRWRVSS TFEDETLEKNAIAfGVHQIYlSrNTEYDffl^YENYEKTNSLDITHKYPPLlvnDSSVDT TOLHQNNEMNSLKEYMSPEDLEAYRKKIHEQISRELDEKNQNS

[0214] S. pombe MKSLKGRLLFLRKFVFFSLLILLFAHGASSSSSSFNYFDKRTNGKANNELALFS 5 IRE1 ligand PTADSPSSVDGVLELPSAENVFAESSLYNAFIVATVDGSLHSYDRITGQELWS binding LFTNANPGLSYTKDENSLLSSKFLSQSNFKSYNSTHGEFYSDSTLNISY'LSDDD domain TAAVFVEPIDGGILYAFM. QTGLVRLPIISIKDLVIIASPIRLLNNNVFVGSKNTT LFTEJVSNGDrVSQYPSGHRYTTHHSVriNLGTKREJSVPSGADSDLSFKDPSGK KLSESLDLLDDFNYQVTVSNKSFVDIARTEYTITIYSDSNVILDLVYIDWTPTK NEIMYESFHSSSFDSKLALSSYDSSLHIVDTHSKFIKQNIPLMSPAATVFDIVTL PIIKKKIDKSQTP / AKFPTSA'LLRQPIDTY LETWPQIARNKTEHA'Y TNFIIGNAW FAMSERirYPLVSLAPEASFLYYNGFYIPLNSIFGLHSLMATPKPFFALPGLPGYrDIPSYVESEGSTK. TI., PSIGKKPIPLLDPNPISSTP

[0215] A7, crassa MLRRPSGHVACRPEVLLAFAFLLVNLQWLQLVDAQPHHHARSPREDDDHAV 6 IRE1 ligand PNIATLSTLTGGRPQPVIEITRTHGRGKRTVVEIADGPKTESIRLSYHAQSDNT binding NNNNHADLISNNVRANALAPDSSVRAPLTQSNKVGSLGAELSQHQIARSLED domain WEVEDFVLLATVDGDLYATDRVRGEVRWHFKADHPMVETRHFRTSRSPVD EDFHEMDHWIWIVEPTRDGEIYIWRPSDSGPQLTKAfT'MTAlKQLVETYSGLH MNGIVYTGEKRTNMVTLNAATGAMIKNLGPRGVNINKVESERCLREYALGN SITSECSNRETITLGRTEYIVGVYRTDGSPIASLKYSEWAPNVLDGDLGQQHS A’lTOKRYriGKPDGTFFGFGLDRENGDRPLFSI-IQLTSPVARVFDYERRQWNP TGSSDTOLIILPQPLFPVNPAISRLRSAKVFVNFITEEGSWY. ALSGTQYTLILI-LY PP / UtFIOWPLESLNEDKLSE.'YLVGTFIDLPDWRIPKEINRL. VPLLDAPPPPAGD AyiTGNPTVPSSAGALPGLPAPDTATALPVQASD

[0216] M. grisea MLRRPSGHVACRPEVLLAFAFLLVNLQWLQLVDAQPHHHARSPREDDDHAV IRE1 ligand PNIATLSTLTGGRPQPX7TETPRTHGRGKRTVV7EIADGPKTESIRLSYHAQSDNT binding NNNNHADLISNNVRANALAPDSSXTIAPLTQSNKVGSLGAELSQHQIARSLED domain WEVEDFVLLATVDGDLYATDRVRGEVRWHFKADHPMVETRHFRTSRSPVD EDFHEMDHWIWIVEPTRDGEIYIWRPSDSGPQLTKAfT'MTAlKQLVETYSGLH MNGIVYTGEKRTNMVTLNAATGAMIKNLGPRGVNINKVESERCLREYALGN SITSECSNRETITLGRTEYIVGVYRTDGSPIASLKYSEWAPNVLDGDLGQQHS ATPDKRY1TGKPDGTFFGFGLDRENGDRPLFSHQLTSPVARVFDVLRRQWNP TGSSDTDL1ILPQPLFPVNPAISRLRSAKVFVNHTEEGSWYALSGTQYPLILHA PPAPIHKFWPLESLNEDKLSEALVGTHDLPDWRIPKEINRLVPLLDAPPPPAGD

[0217] AyiTGNPTVPSSAGALPGLPAPDTATALPVQASD

[0218] FKBP MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFML 8

[0219] GKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVE LLKLE FRB MVAILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKE 9

[0220] TSFNQAYGRDLMEAQEWCRKYAIKSGNVKDLLQAWDLYYHVFRRIS FKBPF36VMGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFML 10

[0221] GKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVE LLKLE IL1R MKVLLRLICFIALLISSLEADKCKEREEKIILVSSANEIDVRPCPLNPNEHKGTIT 11

[0222] WYKDDSKTPVSTEQASRIHQHKEKLWFVPAKVEDSGHYYCWRNSSYCLRI KISAKFVEbffiPM, CYNAQAIFKQKLPVAGDGGIWCPYMEFTKNENNELPKLQ WYKDCKPLLLDNTHFSGVKDRLWMNVAEKHRGNYTCHASYTYLGKQYPIT RVIEFm.£EN^PTRPVlVSPANETMEVDLGSQIQIJC]SrVTGQLSDIAY\X?KWN GSVIDEDDPVLGEDYYSVENPANKRRSTLITVLMSEIESRFYKHPFTCFAKNT HGIDAAY9QLIYTVTNFQK

[0223] ILlRac MTLLWCWSLYFYGILQSDASERCDDWGLDTMRQIQVFEDEPARIKCPLFEH 12

[0224] FLKF'KYSTAHSAGLTLIW YWTRQDRDLEEPINF ’RLPENRISKEKDVLWFRPTL LNDTGNYTCAtLRNTTYCSKVAFPLEVVQKDSCFNSPAIKLPVFIKLYIEYGIQR ITCPNVDGYYYSSVKPTITXVYMGCYKIQNFNNV1PEGMNLSFL1ALISNNGNYT

[0225]

[0226] CVVTYTENGRTFHLTRTLTVKVVGSPKNAVPPVIHSPNDHVVYEKEPGEELLI

[0227] 39

[0228] 326657998 Attorney Docket No.: PTBI-010 / 01WO PCTVYFSFLMDSRNEVVVWTIDGKKPDDITIDVTINESISHSRTEDETRTQILSIK KWSEDLKRSYVCHARSAKGEVAKAAKVKQKVPAPRYTVE

[0229] LaG16-LaG2 (eGFP binding) MTLLWCWSLYFYGILQSDASERCDDWGLDTMRQIQVFEDEPARIKCPLFEH 13 FLKFNYSTAHSAGLTLIWYWTRQDRDLEEPINFRLPENRISKEKDVLWFRPTL LNDTGNYTCMLRNTTYCSKVAFPLEVVQKDSCFNSPMKLPVHKLYIEYGIQR ITCPNVDGYFPSSVKPTITWYMGCYKIQNFNNVIPEGMNLSFLIALISNNGNYT CVVTYPENGRTFHLTRTLTVKVVGSPKNAVPPVIHSPNDHVVYEKEPGEELLI PCTVYFSFLMDSRNEVWWTIDGKKPDDITIDVTINESISHSRTEDETRTQILSIK KVTSEDLKRSYVCHARSAKGEVAKAAKVKQKVPAPRYTVE VHH2 (TNF binding) MAQVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWMYWVRQAPGKGLEW 14 VSEINTNGLITKYPDSVKGRFTISRDNAKNTLYLQMNSLKPEDTALYYCARSP

[0230]

[0231] SGFNRGQGTQVTVSS

[0232] b. Kinase domains

[0233] The chimeric proteins of the present disclosure comprise a kinase domain. A kinase domain is a region of a protein responsible for transferring a phosphate group (e.g., from ATP) to a specific substrate (e.g., protein, lipid, carbohydrate). In some embodiments, the kinase domain is a kinase domain of an inositol-requiring enzyme 1 (IRE I). In some embodiments, the kinase domain is a kinase domain derived from IREI.

[0234] The kinase domain of a wild type IREI protein is typically located in the cytoplasmic region of the protein and catalyzes the transfer of a phosphate group to itself upon dimerization, leading to conformational changes that activate the RNase domain of the protein.

[0235] The IREI kinase domains of the present disclosure comprise a catalytic loop, which catalyzes the addition of the phosphate to the target substrate, and an activation loop, which coordinate ions essential for stabilizing phosphate groups (e.g., from ATP).

[0236] In some embodiments, the catalytic loop comprises a HRD motif. In some embodiments, the catalytic loop is located in a consensus motif comprising the amino acid sequence of X1LHX2LX3IX4HRDX5KPX6NX7LX8X9 (SEQ ID NO: 53), where X1 is H or Y; X2 is E or S; X3 is G, N, K, or R; X4 is V or I; X5 is L or I; X6 is Q or H; X7 is E or I; X8 is I or V; and X9 is S, A, or N,

[0237] In some embodiments, the activation loop comprises a DFG conformation. In some embodiments, the activation loop comprises a DMG conformation. In some embodiments, the activation loop is located in a consensus motif comprising the amino acid sequence of X1X2X3X4SDX5GX6X7KX8X9X10 (SEQ ID NO: 54), where X1 is S, K, or R; X2 is A, I, or L; X3 is K, M, or L; X4 is L, I, or V; X5 is M or F; X6 is I or L; X7 is S or C; X8 is R or K; X9 is M or L; and X10 is S, A, D, or E.

[0238] In some embodiments, the kinase domain is an animal IREI kinase domain. In some embodiments, the kinase domain is derived from an animal IREI kinase domain. In some

[0239] 40

[0240] 326657998 Attorney Docket No.: PTBI-010 / 01WO embodiments, the animal IREl kinase domain is a Homo sapiens IREl kinase domain, aMus musculus IRE1 kinase domain, a Drosophila melanogaster IREl kinase domain, or a Caenorhabditis elegans IREl domain.

[0241] In some embodiments, the kinase domain is a plant IRE1 kinase domain. In some embodiments, the kinase domain is derived from a piant IREl kinase domain. In some embodiments, the plant IREl kinase domain is an Arabidopsis thaliana IRE1 kinase domain, an Oryza sativa IRE1 kinase domain, or a Populus trichocarpa IREl kinase domain.

[0242] In some embodiments, the kinase domain is a fungal IRE1 kinase domain. In some embodiments, the kinase domain is derived from a fungal IREl kinase domain. In some embodiments, the corresponding fungus is a plant-pathogenic fungus. In some embodiments, the fungal IREl kinase domain is a Saccharomyces cerevisiae IREl kinase domain, a Candida albicans IRE1 kinase domain, a Neurospora crassa IRE1 kinase domain, a Magnaporthe grisea IREl kinase domain, a Schizosaccharomyces pombe IRE1 kinase domain, or an Aspergillus nidulans IREl kinase domain.

[0243] In some embodiments, the IREl kinase domain is not derived from an animal IRE1 kinase domain. In some embodiments, the IRE1 kinase domain is not derived from a plant IREl kinase domain. In some embodiments, the IRE1 kinase domain is not derived from a fungal IREl kinase domain.

[0244] In some embodiments, the kinase domain comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to any one of SEQ ID NOs: 15-20. In some embodiments, the kinase domain comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to any one of SEQ ID NOs: 15-20, wherein the kinase domain comprises a catalytic loop and an activation loop. In some embodiments, the kinase domain comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to any one of SEQ ID NOs: 15-20, wherein the kinase domain comprises a catalytic loop and an activation loop, wherein the catalytic loop comprises an HRD motif and the activation loop comprises a DFG motif. In some embodiments, the kinase domain comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to any one of SEQ ID NOs: 15-20, wherein the kinase domain comprises a catalytic loop and an activation loop, wherein the catalytic loop comprises an HRD motif and the activation loop comprises a DMG motif.

[0245] 41

[0246] 326657998 Attorney Docket No.: PTBI-010 / 01WO In some embodiments, the kinase domain comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to any one of SEQ ID NOs: 15-20, wherein the kinase domain comprises a catalytic loop and an activation loop, wherein the catalytic loop comprises the amino acid sequence of SEQ ID NO: 53 and / or the activation loop comprises the amino acid sequence of SEQ ID NO: 54.

[0247] In some embodiments, the kinase domain comprises an amino acid sequence comprising a consensus sequence (consensus / 100%, consensu s / 90%, consensus / 80%, or consensus 70%) depicted in FIG. 13. In some embodiments, the kinase domain comprises an amino acid sequence comprising a consensus sequence of SEQ ID NO: 119. In some embodiments, the kinase domain comprises an amino acid sequence comprising a consensus sequence of SEQ ID NO: 120. In some embodiments, the kinase domain comprises an amino acid sequence comprising a consensus sequence of SEQ ID NO: 121. In some embodiments, the kinase domain comprises an amino acid sequence comprising a consensus sequence of SEQ ID NO: 122.

[0248] In some embodiments, the kinase domain comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 15-20.

[0249] Also described herein are polynucleotides comprising a sequence encoding a kinase domain (or a chimeric protein). In some embodiments, the kinase domain comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to any one of SEQ ID NOs: 15-20. In some embodiments, the kinase domain (or the chimeric protein) comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 15-20.

[0250] Table 2. Exemplary kinase domains. Highlighting HRD motif (bold) of the catalytic loop, which catalyzes the addition of the phosphate to the target substrate, and DFG / DMG motif of the activation loop (underlining), which enables the inactive / active conformational switching activity of the kinase.

[0251] Description Amino Acid Sequence SEQ ID NO

[0252] H. sapiens SWrVGKISFCPKDVLGHGAEGTTVYRGMFDNRDVAVKRILPECFSFADREVQ 15 IRE1 kinase LLRESDEHPNVIRYFCTEKDRQFQYIAIELCAATLQEYVEQKDFAHLGLEPITL domain LQQITSGLAffl, HSINIVHR»LKPHNnJSMPNAHGKIKAMISDFGLCKKLAVG RHSFSRRSGVPGTEGWIAPEMLSEDCKENPTYTVDIFSAGCVFYYVISEGSHPF GKSLQRQANILLGACSLDCLHPEKHEDVIARELIEKMIAMDPQKRPSAKHVL

[0253]

[0254] KHPFF

[0255] 42

[0256] 326657998 Attorney Docket No.: PTBI-010 / 01 WO >1. thaliana DGRKIGKLFLSSKEIAKGSNGTVVFEGIYEGRPVAVKRLVRSHHEVAFKEIQN 16 IRE1 kinase LIASDQFITNIIRWYGVEYDQDFVYLSLERCTCSLDDLIKSYLEFSMTKVLENN domain DSTEGVAAYKIQLDSLEGVKGNW^VGGHPSPLNnXLMRDIVCGIVHLHE LGIVHRDLKPQNVLISKDMTLSAKLSWGISKRMSRDMSSLGHLATGSGSSG WQAPEQIXQGRQIRAVDMFSLGCVIFYTITGCKHPFGDDLERDy^NIVKNKV DLFLVEHVPEASDLISRLLNPDPDLRPSATEVLLHPMF

[0257] C. albicans KKLQIENNLVISDKILGYGSHGTWFQGTFENRPVAVKRMLLDFYD1ANHEV 17 IRE1 kinase RLLQESDDHPNVVRYFCSQSSESEKFLYIALELCLCTLEDIIEKPQNMPNLCIP domain KRND1LYQLTSGLHYLHSLK1VHRD1KPQNILVAN1KKNGKRKNQITEIDETCE NNVRLLISDFGLCKKLENDQSSFRATTQNAASGTSGWRAPELLLNHDLWEIS ADSISSndSNSNSNGNGNGNGAINGSVSNSATSGKRLTKAIDIFSLGCVFYYIL TGGYI-IPFGDRYLREGNIIKGEYDLSLLMEKCPNDRYESIDLISKIISHDPSQRP NTGKILKHPLF

[0258] S. cerevisiae VVSEKILGYGSSGTVVFQGSFQGRPVAVKRMLIDFCDIALMEIKLLTESDDHP 18 IRE1 kinase NVdRYYCSETTDRFLYIALELCNLNLQDLVESKNVSDENLKLQKEYNPISLLR domain QIASGVAHLHSLKIIHRDLKPQNILVSTSSRFTADQQTGAENLRILISDFGLCK KLDSGQSSFRTNLNNPSGTSGWRAPELLEESNNLQCQV'ETEHSSSRHTWSSD SFYDPFTKRRLTRS1DIFSMGCVFYY1LSKGKHPFGDKYSRESN1IRGIFSLDEM KCLHDRSLIAEATOLISQWDHDPLKRPTAMKVLRHPLF

[0259] Y crassa PnXMGSLEVNQEQQLGIGSNGIIVFAGKWDGRDVAVKRMIArQFNEIASQE. T 19 IRFJ kinase KLLRESDDHPNVIRYFAQQQSAGFLYIALELCQASLADVIQRPSMFRELAQAG domain ERDMPGVT. YQVAKGLSHI. HSLRIVHRDLKPQNn, VNMGKDGRPRIIArSDFGL CKKLEGGQSSFG ATTAHAAGTTG WRAPELLLDDDGGPGPGATM' 1 F 1 DPGSS MHSASGTGSGYVGAGVT4VRRVTRAn)IFSLGLVFFY\TTKGHHPFDLGDRY MRESNIRKGKYT)LQLLEyT. GDYAHDAKDLIESMI, NSNPKKRPTAIG\7MAHPF F

[0260] M. grisea PIYKMGSLEWEDQQLGTGSNGTVVFAGKWGRDVAVKRMLIQFYDIASQE 20 IRE1 kinase TRLLRESDDHPNVIRYYAQQSRDAFLYIALELCQASLAEVIEKPAYFKNLAQ2A domain GEKDLPNVLYQITNGLSHLHSLRIVHRDLKPQNILVNMGKDGKPRLLVSDFG LCKKLEGGQSSFGATTAHAAGTTGWRAPELLLDDDARDNTATMVDASMSS AHSGSGSVQGSSDVPNRRATRAIDIFSLGLVFFYVLTKGSHPYDRGDRYMRE

[0261]

[0262] VNTRKGSFDLSRLEVTGDYAMEARDIVERMI^SFEPSERPTARDVMRHPFF

[0263] c. RNase domains

[0264] The chimeric proteins of the present disclosure comprise an RNase domain. An RNase domain is a region of a protein that has ribonuclease activity (e.g., capable of catalyzing the cleavage of RNA molecules). In some embodiments, the RNase domain is an RNase domain of an inositol-requiring enzyme 1 (IRE1). In some embodiments, the RNase domain is an RNase domain derived from IRE1.

[0265] The RNase domain of a wiki type IRE1 is typically responsible for cleaving specific mRNA molecules that encode transcription factors (e.g., x-box binding protein 1, XBP1) after activation by the kinase domain. In some embodiments, the RNase domain cleaves the mRNA in a non-canonical manner, leading to excision of a cytosolic intron and creation of a frameshift that results in the production of a functional transcription factor.

[0266] RNase domains of the present disclosure comprise one or more (e.g., 1, 2, 3, or 4) of the following conserved catalytic residues, relative to H. sapiens IRE1 RNase domain (SEQ ID NO: 21): Y60, N74, K75, H78. In some embodiments, the RNase domain comprises amino acids Y60, N74, K75, and H78, relative to SEQ ID NO: 21. In some embodiments,

[0267] 43

[0268] 326657998 Attorney Docket No.: PTBI-010 / 01WO RNase domains of the present disclosure comprise the consensus motif RX1YX2X3X4X5X6X7DLLRX8X9RNKX10X11HX12 (SEQ ID NO: 55), where Xiis T, R, or K; X2is K, T, S, or H; X3is G, Y, P, or S; X4is G, D, N, S, or E; X5 is S, R, or K; Xe is V, I, M, or L; X7 is R, L, or M; Xs is A or V; X9 is M, I, or L; X10 is K, L, or Y; Xu is H or N; and X12 is Y, H, or F.

[0269] In some embodiments, the RNase domain is an animal IREI RNase domain. In some embodiments, the RNase domain is derived from an animal IREI RNase domain. In some embodiments, the animal IREI RNase domain is a Homo sapiens IREI RNase domain, a Mas musculus IRE1 RNase domain, a Drosophila melanogaster IRE1 RNase domain, or a Caenorhabditis elegans IRE1 domain.

[0270] In some embodiments, the RNase domain is a plant IRE I RNase domain. In some embodiments, the RNase domain is derived from a plant IREi RNase domain. In some embodiments, the plant IREI RNase domain is an Arabidopsls thaliana IREI RNase domain, an Oryza saliva IREI RNase domain, or a Populus trichocarpa IREI RNase domain.

[0271] In some embodiments, the RNase domain is a fungal IREI RNase domain. In some embodiments, the RNase domain is derived from a fungal IREI RNase domain. In some embodiments, the corresponding fungus is a plant-pathogenic fungus. In some embodiments, the fungal IREI RNase domain is a Saccharomyces cerevisiae IREI RNase domain, a Candida albicans IREI RNase domain, a Neurospora crassa IREI RNase domain, a Magnaporthe grisea IREI RNase domain, a Schizosaccharomyces pombe IREI RNase domain, or an Aspergillus nidulans IREI RNase domain.

[0272] In some embodiments, the IREI RNase domain is not derived from an animal IREI RNase domain. In some embodiments, the IRE I RNase domain is not derived from a plant IREI RNase domain. In some embodiments, the IREI RNase domain is not derived from a fungal IRE i RNase domain.

[0273] In some embodiments, the RNase domain comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to any one of SEQ ID NOs: 21-26. In some embodiments, the RNase domain comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to any one of SEQ ID NOs: 21-26, wherein the RNase domain comprises one or more (e.g., 1, 2, 3, or 4) catalytic residues Y60, N74, K75, H78 relative to SEQ ID NO: 21. In some embodiments, the RNase domain comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at

[0274] 44

[0275] 326657998 Attorney Docket No.: PTBI-010 / 01WO least 96%, at least 97%, at least 98%, or at least 99%) identity to any one of SEQ ID NOs: 21-26, wherein the RNase domain comprises catalytic residues Y60, N74, K75, 1 78 relative to SEQ ID NO: 21. In some embodiments, the RNase domain comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to any one of SEQ ID NOs: 21-26, wherein the RNase domain comprises the amino acid sequence of RX1YX2X3X4X5X6X7DLLRX8X9RNKX10X11HX12 (SEQ ID NO: 55), where Xiis T, R, or K;

[0276] X2 is K, T, S, or H; X3 is G, Y, P, or S; X4is G, D, N, S, or E; X5 is S, R, or K; X6is V, I, M, or L; X7 is R, L, or M, Xs is A or V; X9 is M, I, or L, X10 is K, L, or Y; Xu is H or N; and X12 is Y, H, or F.

[0277] In some embodiments, the RNase domain comprises an amino acid sequence comprising a consensus sequence (consensus / 100%, consensus / 90%, consensus / 80%, or consensus 70%) depicted in FIG. 14. In some embodiments, the RNase domain comprises an amino acid sequence comprising a consensus sequence of SEQ ID NO: 123. In some embodiments, the RNase domain comprises an amino acid sequence comprising a consensus sequence of SEQ ID NO: 124. In some embodiments, the RNase domain comprises an amino acid sequence comprising a consensus sequence of SEQ ID NO: 125. In some embodiments, the RNase domain comprises an amino acid sequence comprising a consensus sequence of SEQ ID NO: 126. In some embodiments, the RNase domain comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 21-26.

[0278] Also described herein are polynucleotides comprising a sequence encoding an RNase domain (or a chimeric protein). In some embodiments, the RNase domain (or chimeric protein) comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to any one of SEQ ID NOs: 21-26. In some embodiments, the RNase domain (or chimeric protein) comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 21-26.

[0279] Table 3. Exemplary RNase domains. Highlighting conserved catalytic residues (bold). Description Amino Add Sequence SEQ ID NO

[0280] H. sapiens WSLEKQLQFFQDVSDRIEKESLDGP1VKQLERGGRAWKMDWRENITVPLQT 21 IRE 1' RNase DLRKFRTYKGGSVRDLLRAMRNKKHHYRELPAEVRETLGSLPDDFVCYFTS

[0281]

[0282] domain RFPHLLAHTYRAMELCSHERLFQPYYFHEPPEPQPPVIPDAL

[0283] 45

[0284] 326657998 Attorney Docket No.: PTBI-010 / 01WO >1. thaliana WNSEMRLSFLRDASDRVELENREzADSEILKAMESTAPVAIGGKWEKLEPWI 22 IRE1 RNase i NIGRYRRYKY DSIRDLLRV IRNKLNIfflRELPPEIQELVGT\ PI < H " DEYFAVRF domain PKLI.. IEAAT7V1SI.. I-ICREEEWRKYFKCDII

[0285] C. albicans WSFSKRLEFLLKVSDRFEIEKRDPPSPLLLKLEEHAKAVHNGNWHKLLNDDE 23 IRE1 RNase FMDNLGK YRK YSPEKLMDLLRAMRNKYHHY NDMPESLQLKMAPLPDGFY domain KYFNDKFPKLLMEIYYWEENFRNEHVFKEYY

[0286] S', cerevisiae WPKSKKLEHXKVSDRLEIENRDPPSALLMKFDAGSDFVIPSGDWTVKFDKTF 24 IRFJ RNase A4DNI.. ERYRKYIISSKIA4DI.. T., RAI, RNK\’FIHFAdDLPEDIAELMGPWDGFTOY domain FTKRFPNIJJGAATV'IIWENI. SDDQILREFLYS

[0287] AC crassa WSPRKRLNFLCDVSDHFEKEPRDPPSPALALLEDQSSCVITNGDFLKTLPREFV 25 IRE1 RNase ESLGKQRKYTGNRMLDLLRALRNKKNHYEDLTPQLRKMVGPLPEGYLGFFT domain TRFPNLLIKCWEVIADLELEESDRFKEYYEPAGL

[0288] M. grisea WSAKKRLAFLCDVSDHFEKEPRDPPSWPLQVLEEAAPDVITSGDFLRQLPREF 26 IRE1 RNase VDSLGKQRKYTGSRMLDLLRALRNKKNHYEDMPESLKKTVGPLPEGYLSF

[0289]

[0290] domain WTRRFDTLLINCWRIV’IDCGWDETDRFRDYYDLPGT

[0291] d. Transmembrane domains

[0292] In some embodiments, chimeric proteins of the present disclosure further comprise a transmembrane domain. In some embodiments, the transmembrane domain is located between (a) the ligand binding domain and (b) the kinase domain and the RNase domain. In some embodiments, chimeric proteins of the present disclosure comprise, from N-terminal to C -terminal: a ligand binding domain, a transmembrane domain, a kinase domain, and an RNase domain. A transmembrane domain is typically a hydrophobic region of a protein that spans the lipid bilayer of a cellular membrane (e.g., plasma membrane). In some embodiments, the transmembrane domain is a glycophorin A transmembrane domain. In some embodiments, the transmembrane domain comprising an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least. 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to TLIIFGVM AGVIGTILLISYGI (SEQ ID NO: 27). In some embodiments, the transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO: 27.

[0293] e. Exemplary chimeric proteins

[0294] In some embodiments, chimeric proteins of the present disclosure comprise an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at. least. 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to any one of SEQ ID NOs: 28-43 or 56. In some embodiments, chimeric proteins of the present disclosure comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 28-43 or 56.

[0295] Also described herein are polynucleotides comprising a sequence encoding a chimeric protein described herein. In some embodiments, the chimeric protein comprising an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least. 95%, at. least. 96%, at least 97%, at least 98%, or at least 99%) identity to any one of SEQ ID NOs:

[0296] 46

[0297] 326657998 Attorney Docket No.: PTBI-010 / 01 WO 28-43 or 56. In some embodiments, the chimeric protein comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 28-43 or 56.

[0298] Table 4. Exemplary chimeric protein sequences.

[0299] pMLS022 (CCdimer-ctoso& IREl’N

[0300] ^C-dimer GELAALKQELAALKKENAALKWEIAALKQGYY (SEQ ID NO: 44) Linker GGSGGTGGSGGT (SEQ ID NO: 45)

[0301] Homo sapiens IRE1 SEQ ID NO: 15 (see Table 2)

[0302] kinase domain

[0303] Homo sapiens IRE1 SEQ ID NO: 21 (see Table 3)

[0304] RNase domain

[0305] Full sequence MGEIAALKQEIAALKKENAALKWEIAALKQGYYGGSGGTGGSGGTSMHQQ QQI.. QHQQFQKELEKIQI. LQQQQQQITFHPPGDTAQDGELLDTSGPYSESSGT SSPSTSPRASNHSLCSGSSASKAGSSPSLEQDDGDEETSWIVGKISFCPKDVI. GHGAEGTiyA’RGMFDNRDVAWRILPECFSFADREVQLLRESDEI-IPN\TRAT CTEKDRQFQYIAIELCAATLQEYVEQKDFAHLGLEPITLLQQTTSGLAHLHSL NIWRDLKPHNILISWNAHGKIKAAflSDFGLCKKLAVGRHSFSRRSGWGTE GWIAPEMLSEDCKENPTYTVDIFSAGCVFYYVISEGSHPFGKSLQRQANILLG ACSLDCLHPEKHEDVTARELIEKMIAMDPQKRPSAKHVLKHPFFWSLEKQLQ FFQDVSDRIEKESLDGPIVKQLERGGRAVYTCMDWRENITVPLQTDLRKFRTY KGGSVRDLLRAMRNKKHHYRELPAEV'RETLGSLPDDFVCYFTSRFPHLLAH TYRAMELCSHERLFQPYYFHEPPEPQPPVTPDAL (SEQ ID NO: 28) pMLS023 (HexCoiLkla-cvtosuiiJREl115)

[0306] HexCoil AEAESALEYAQQALEKAQLALQAARQALKA (SEQ ID NO: 46)

[0307] Linker SEQ ID NO: 45

[0308] Homo sapiens IRE 1 SEQ ID NO: 15 (see Table 2)

[0309] kinase domain

[0310] Homo sapiens IRE 1 SEQ ID NO: 21 (see Table 3)

[0311] RNase domain

[0312] Full sequence MAEAESALEYAQQALEKAQLALQAARQALKAGGSGGTGGSGGTSMHQQQ QLQHQQFQKELEKIQLLQQQQQQLPFHPPGDTAQDGELLDTSGPYSESSGTS SPSTSPRASNHSLCSGSSASKAGSSPSLEQDDGDEETSWIVGKISFCPKDVLG HGAEGT1VYRGMFDNRDVAVKRILPECFSFADREVQLLRESDEHPNV1RYFC TEKDRQFQYIAIELCAATLQEYVEQKDFAHLGLEPITLLQQTTSGLAHLHSLN IVHRDLKPHNILISMPNAHGKIKAMISDFGLCKKLAVGRHSFSRRSGVPGTEG WLAPENRSEDCKENPIYTVDIFSAGCVFYYVISEGSIIPFGKSLQRQANILLGA CSLDCLHPEKHEDVIARELIEKMIAMDPQKRPSzXKHVLKHPFFWSLEKQLQF FQD VSDRIEKESLDGPIVKQLERGGRA VVKMD WRENITVPLQTDLRKFRTY KGGSVRDLLRAMRNiGkHI-IY " RELPAEVRETLGSLPDDFVCYFTSRI THLLAH TYR / YMELCSHERLFQPY YFHEPPEPQPPVTPDAL (SEQ ID NO: 29) pMLS025 (1301 -c rtosofclRE 1Hs)

[0313] 1301 KMEEL,1 KEEK ‘VAX LRANSV; {■. AKE. KAS AVFLGGV Hi 11 IT r l VPDADT VIK ELSFLKEMGAIIGAGTVTSVEQCRKAVESGAEFIVSPHLDEEISQFCKEKGVF AA.lPGYAnPTEIATkAAfKI. GHTII. KIEPGEWGPQFWAAKGPFPNV’KFWT GGVNT-DNVCEWFKAGVI^AVGVGSALVKGTPVEVAEKAKAFVEKIRGCTE (SEQ ID NO: 47)

[0314] Linker SEQ ID NO: 45

[0315] Homo sapiens IRE 1 SEQ ID NO: 15 (see Table 2)

[0316] kinase domain

[0317] Homo sapiens IRE1 SEQ ID NO: 21 (see Table 3)

[0318] RNase domain

[0319] Full sequence MKNffiFI. FKKHKIAAAVT. RANSV’EEAKKKAI. AWLGGVTIIJEITFTATDADTVT KELSFLKEMGAIIGAGTVTSVEQCRKAVESGAEFIVSPHLDEEISQFCKEKGV FYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPT GGWLDNVCEWFKAGVLAVGVGSALVKGTPVEVAEKAKAFV'EKIRGCTEG GSGGTGGSGGTSMHQQQQLQHQQFQKELEKIQLLQQQQQQLPFHPPGDTA

[0320]

[0321] QDGELLDTSGPYSESSGTSSPSTSPRASNHSLCSGSSASKAGSSPSLEQDDGD 47

[0322] 326657998 Attorney Docket No.: PTBI-010 / 01WO EETSVVTVGKISFCPKDVLGHGAEG'nVYRGMFDNRDVAVKRILPECFSFAD REVQLLRESDEIIPNVIRYFCTEKDRQFQYIAIELCAATLQEYVEQKDFM-ILG LEPITLLQQTTSGLAHIJISIINTATIRDLKPHNILISMPNAHGKIKAMISDFGLCK KLAVGRHSFSRRSGVPGTEGWIAPEMI> SEDCKENPTYTVDIFSAGCVFYYVI SEGSHPFGKSLQRQANH.. LGACSLDCLHPEKHEDVIARELIEKMIAMDPQKRP SAKHVI. KHPFFWSLEKQLQFFQDVSDRTEKESLDGPIVKQLERGGRAVVKM DWREMTWLQTOLRKFRTYKGGSVRDLLRAMRNKKHHYRELPAEVRETI. GSLPDDFVCYFTSRFPHLLAHTYRAN1ELCSHERLFQPYYFHEPPEPQPPVTPD AL (SEQ ID NO: 30)

[0323] pMLS062 (FKBP-cyto^ii JRE lHs)

[0324] FKBP SEQ ID NO: 8 (see Table 1)

[0325] Linker SEQ ID NO: 45

[0326] Homo sapiens IRE1 SEQ ID NO: 15 (see Table 2)

[0327] kinase domain

[0328] Homo sapiens IRE1 SEQ ID NO: 21 (see Table 3)

[0329] RNase domain

[0330] Full sequence MGVQV'ETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFML GKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPG1IPPHATLVFDVE LLKLEGGSGGTGGSGGTSMHQQQQLQHQQFQKELEKIQLLQQQQQQLPFHP PGDTAQDGELLDTSGPYSESSGTSSPSTSPRASNHSLCSGSSASKAGSSPSLEQ DDGDEETSVV1VGK1SFCPKDVLGHGAEGTIVYRGMFDNRDVAVKRILPECF SFADREVQLLRESDEHPNVIRYFC'IEKDRQFQYIAIELCAATLQEYVEQKDF AHLGLEPnTLQQTTSGLAHLHSLNIVHRDLKPHNILISMPNAHGKIKAMISD FGLCKKLAVGRHSFSRRSGVPGTEGWIAPEMLSEDCKENPTYTVDIFSAGCV FYYVISEGSHPFGKSLQRQANILLGACSLDCLHPEKHEDVIAREL1EKMIAMD PQKRPSAKHVLKHPFFWSLEKQLQFFQDVSDRIEKESLDGP1VKQLERGGRA VVKMDWREN1TVPLQTDLRKFRTYKGGSVRDLLRAMRNKKHHYRELPAEV RETLGSLPDDFVCYFTSRFPHLLAHTYRAMELCSHERLFQPYYFHEPPEPQPP VTPDAL (SEQ ID NO: 31)

[0331] pMLS063 (FRB-cjtosniicIRElHs)

[0332] FRB SEQ ID NO: 9 (see Table 1)

[0333] Linker SEQ ID NO: 45

[0334] Homo sapiens IRE1 SEQ ID NO: 15 (see Table 2)

[0335] kinase domain

[0336] Homo sapiens TRE 1 SEQ ID NO: 21 (see Table 3)

[0337] RNase domain

[0338] Full sequence WARA\TIEM^TffiGLEEASRLYFGERNVKGMFEVI. EPLHAMMERGPQTI> K ETSFNQAYGRDLMEAQEWCRKYR4KSGNVKDLLQAWLYYHVFRRISGGS GGTGGSGGTSMHQQQQLQHQQFQKELEKIQLLQQQQQQLPFHPPGDTAQD GELLDTSGPYSESSGTSSPSTSPRASNHSLCSGSSASKAGSSPSLEQDDGDEET SWIVGKISFCPKDVI. GHGAEGTIVYRGMFDNRDVAVKRR> PECFSFADREV QLLRESDEHPNVIRYFCTEKDRQFQYIAIELCAAn. QEYVEQKDFAHI> GLEPI TLLQQTTSGI, AI-IU-ISLNIWRDI. KPHNIUSNfPNAHGKIKAMISDFGI, CKKLA VGRHSFSRRSGVPGTEGWIAPEMLSEDCKENPTYTVDIFSAGCVFYYVISEGS HPFGKSLQR. QANR. LGACSLDCLHPEKHEDVIARELIEKMIAA4DPQKRPSAK HVLKHPFFWSLEKQLQFFQDVSDRIEKESLDGPIVKQLERGGRAVVKMDWR ENITVPLQTDLRKFRTYKGGSVRDLLRAMRNKKHHYRELPAEVRETLGSLP DDFVCYFTSRFPHLLAHTYRAMELCSHERLFQPYYFHEPPEPQPPVTPDAL

[0339] (SEQ ID NO: 32)

[0340] PMLS300 (FKBPF36V-cvt»s»iieIRElH8)

[0341] FKBPF36VSEQ ID NO: 10 (see Table 1)

[0342] Linker SEQ ID NO: 45

[0343] Homo sapiens TRE 1 SEQ ID NO: 15 (see Table 2)

[0344] kinase domain

[0345] Homo sapiens IRE1 SEQ ID NO: 21 (see Table 3)

[0346] RNase domain

[0347] Full sequence MGVQVEITSPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFM LGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDV ELLKLEGGSGGTGGSGGTSMHQQQQLQHQQFQKELEKIQLLQQQQQQLPFH PPGDTAQDGELLDTSGPYSESSGTSSPSTSPRASNHSLCSGSSASKAGSSPSLE

[0348]

[0349] QDDGDEETSWIVGKISFCPKDVLGHGAEGTIVYRGMFDNRDVAVKRILPEC 48

[0350] 326657998 Attorney Docket No.: PTBI-010 / 01WO FSFADREVQLLRESDEHPNVIRYFCTEKDRQFQYIAIELCAATLQEYVEQKDF

[0351] Alls. GO: PITLLQQTTSGLAHLIISLNIVIIRDLKTHNILISMPNAIIGKIKAMISD FGLCKKLAVGRHSFSRRSGVPGTEGWIAPEMI> SEDCKENPTYTVD1FSAGCV FYYVISEGSHPFGKSLQRQANnXGACSLDCLHPEKHEDVIARELIEKMIAMD PQKRPSAKHVIXHPFFWSLEKQLQFFQDVSDRIEKESLDGPIATCQLERGGRA VVXMDWREMTVPLQTOLRKFRTYKGGSVRDLLRAMRNKKHHYRELPAEV RE U.. GSLPDDFVCA TTSRFPHLLAFr TYRAN1ELCSHERLFQPYA THEPPEPQPP VTPDAL (SEQ ID NO: 33)

[0352] pMLS380 (FKBPF36V

[0353] FKBPF36VSEQ ID NO: 10 (see Table 1)

[0354] Linker SEQ ID NO: 45

[0355] M. grisea IRE1 SEQ ID NO: 20 (see Table 2)

[0356] kinase domain

[0357] M. grisea IRE1 SEQ ID NO: 26 (see Table 3)

[0358] RNase domain

[0359] Full sequence MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFM LGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDV ELLKLEGGSGGTGGSGGTSMHGSVSRDDDPPQASVSEIVDKAKQLGDAPRRI EPDLRTiroNVQDLTGPIYKMGSLEVNEDQQLGTGSNGTWFAGKWDGRDV AVKRMLIQFYDIASQETRLLRESDDHPNV1RYYAQQSRDAFLYIALELCQAS LAEVIEKPAYFKNLAQAGEKDLPNVLYQITOGLSHLHSLRIVHRDLKPQNIL VNMGKDGKPRLLVSDFGLCKKLEGGQS'SFGATTAHAAGITGWRAPELLLD DDARDNTATMVDASMSSAHSGSGSVQGSSDVPNRRATRAIDIFSLGLVFFY VLTKGSHPYDRGDRYMREVNIRKGSFDLSRLEVLGDYAMEARDIVERMLSF EPSERPTARDVMRHPFFWSAKKRLAFLCDVSDHFEKEPRDPPSWPLQVLEEA APDV1TSGDFLRQLPREFVDSLGKQRKYTGSRMLDLLRALRNKKNHYEDMP ESLKKTVGPLPEGYLSFWTRRFDTLLINCWRIVIDCGWDETDRFRDYYDLPG

[0360] T (SEQ ID NO: 34)

[0361] pMLS381 (FKBPF36V

[0362] FKBPH6VSEQ ID NO: 10 (see Table 1)

[0363] Linker SEQ ID NO: 45

[0364] A', crassa IRE1 SEQ ID NO: 19 (see Table 2)

[0365] kinase domain

[0366] A7. crassa IRE1 SEQ ID NO: 25 (see Table 3)

[0367] RNase domain

[0368] Full sequence MGVQVETISPGDGRTTYKRGQTCyATIYTGMLEDGKKVDSSRDRNKPFKFM LGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDV ELLKLEGGSGGTGGSGGTSNffiTTDAPAEWKPKRKAHRGRRGGIKHRKGP RNENTQSRDDEPPEPTVDEWKKAQEIGQQPKLEPDVITIPNGVDNy^SGPnX MGSLE. VNQEQQLGTGSNGTWTAGKWDGRDVA\TCRMLVQFNETASQETKLL RESDDHPNVIRYFAQQQSAGFI. YIALELCQASLADVIQRPSK1FRELAQAGER DNfPG\'T. YQ\7AKGLSHLHSLRI\TIRDLKPQNIL\7N]\ / IGKDGRPRILVSDFGLC KKI. EGGQSSFGATTAHAAGTTGVv'RAPEU. LDDDGGPGPGATMTFl'DPGSS MHSASGTGSGVWGAGVNVRRVTRAroiFSLGLVFFYVLTKGHHPFDLGDRY MRESNIRKGKYDLQLLE\T, GDYAHDAKDLIESML, NSNPKKRPTAIGVMAHP FFWSPRKRLNFLCDVSDHFEKEPRDPPSPALALLEDQSSCVITNGDFLKTLPR EFVESLGKQRKYTGNRMLDLLRALRNKKNHYEDLTPQLRKMVGPLPEGYL GFFTTRFPNLLIKCWEVIADLELEESDRFKEYYEPAGL (SEQ ID NO: 35) pMLS382 (FKBPF36V-TM»IRE1M®)

[0369] SP MCRAISLRRLLLLLLQLSQLLAVTQG (SEQ ID NO: 48)

[0370] FKBPF36VSEQ ID NO: 10 (see Table 1)

[0371] GpA SEQ ID NO: 27

[0372] Linker SEQ ID NO: 45

[0373] M. grisea IRE1 SEQ ID NO: 20 (see Table 2)

[0374] kinase domain

[0375] M. grisea IRE1 SEQ ID NO: 26 (see Table 3)

[0376] RNase domain

[0377] Full sequence MCRAISLRRLLLLLLQLSQLLAVTQGMGVQVETISPGDGRTFPKRGQTCVVH

[0378] YTGA1LEDGKKVDSSRDRNKPFKFA1LGKQEVIRGWEEGVAQA1SVGQRAKL

[0379]

[0380] TISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGSGGTGGSGGTGGSGGTG

[0381] 49

[0382] 326657998 Attorney Docket No.: PTBI-010 / 01WO GSGGSSTITLIIFGVMAGVIGTILLISYGISNRIGSVSRDDDPPQASVSEIVDKAK QLGD / yPRRIEPDLRTIIDNVQDLTGPIYKNIGSLEVNEDQQLGTGSNGTVVFA GKWDGRDVAVKRMUQFYDIASQETRLLRESDDHPNVIRYYAQQSRDAFI, Y TALELCQASLAEVIEKPAYFKNT. AQAGEKDLPNVI^YQITNGLSHI^HSLRI\7HR. DLKPQNTT. VNMGKDGKPRLLVSDFGLCKKLEGGQSSFGATTAHAAGTTGW RAPELLLDDDARDNTATMVDASMSSAHSGSGSVQGSSDVPNRRATRAIDIFS LGLATFYW. TKGSHPYDRGDRYAfREWIRKGSFDI. SRI. EyT. GDYAAfEARDI VERMI., SFEPSERPTARDVMRHPFFWSAKKRLAFI> CDVSDHFEKEPRDPPSWP LQVI, EEAAPDVITSGDFI> RQLPREFVDSLGKQRKYTGSR]Vn., DLLRALRNKK NHYEDMPESLKKTVGPLPEGYLSFWTRRFDTLLINCWRIVIDCGWDETDRFR DYYDLPGT (SEQ ID NO: 36)

[0383] pMLS394 (ILlRHs-TM»IRElMg)

[0384] IL1R SEQ ID NO: 11 (see Table 1)

[0385] GpA SEQ ID NO: 27

[0386] M. grisea IRE1 SEQ ID NO: 20 (see Table 2)

[0387] kinase domain

[0388] M grisea IRE1 SEQ ID NO: 26 (see Table 3)

[0389] RNase domain

[0390] Full sequence MKVLLRLICFIALLISSLEADKCKEREEKIILVSSANEIDVRPCPLNPNEHKGTI TWYKDDSKTPVSTEQASRIHQHKEKLWFVPAKVEDSGHYYCWRNSSYCL RIK1SAKFVENEPNLCYNAQA1FKQKLPVAGDGGLVCPYMEFFKNENNELPK LQWYKDCKPLLLDNIHFSGVKDRLIVMNVAEKHRGNYTCHASYTYLGKQY PITRVIEFITLEENKPTRPVIVSPANETMEVDLGSQIQLICNVTGQLSDIAYWK WNGSVIDEDDPVLGEDYYSVENPANKRRSTLITVLNISEIESRFYKHPFTCFA KNTHGIDAAY1QLIYPVTNFQKSTITLI1FGVMAGV1GTILL1SYG1SMHGSVSR DDDPPQASVSEIVDKAKQLGDAPRRIEPDLRTI1DNVQDLTGPIYKMGSLEVN EDQQLGTGSNGTWFAGKWDGRDVAVKRMLIQFYDIASQETRLLRESDDHP NVIRYYAQOSRDAF'LYIALELCQASLAEVIEKPAYFKNLAQAGEKDLPNVLY QITNGLSI-ILI-ISLRIVHRDLKPQNILVNMGKDGKPRLLVSDFGLCKKLEGGQS SFGATTAHAAGTTGWRAPELELDDDARDNTATMVDASMSSAHSGSGSVQG SSDVPNRRATRAIDIFSLGLVFFYVLTKGSHPYDRGDRYMREVNIRKGSFDLS RLEVLGDY / XNffiARDIVERlYlLSFEPSERPT / W)VNmiPFFWS / UG< RLAFLCD VSDHFEKEPRDPPSWPLQVLEEAAPDyTTSGDFLRQLPREFVDSLGKQRKYT GSRMLDLLRALRNKKNIIYEDMPESLKKTVGPLPEGYLSFWTRRFDTLLINC WIVIDCGWETDRFRDYYDLPGT (SEQ ID NO: 37) pMLS395 (IL1RACPH5-TM»IRE1MS)

[0391] IL1RAcP SEQ ID NO: 12 (see Table 1)

[0392] GpA SEQ ID NO: 27

[0393] M. grisea IRE1 SEQ ID NO: 20 (see Table 2.)

[0394] kinase domain

[0395] Ad. grisea IRE1 SEQ ID NO: 26 (see Table 3)

[0396] RNase domain

[0397] Full sequence MTI> LWCWSLYFYGn, QSDASERCDDWGLDTlVlRQIQVFEDEPARIK. CPLFE HFLKFNYSTAHSAGLnJWYWTRQDRDLEEPINFRLPENRISKEKDVT. AVFRP TLLNDTGNYTCMLRNITYCSKVAFPLEVVQKDSCFNSPMKLPVHKLYIEYGI QRITCPNVDGYFPSSVKPTITWYMGCYKIQNFNNVTPEGMNLSFLIALISNNG NYTCAATYPENGRTFHLTRTI.. T\7KVVGSPKNAVPPV1HSPNDHVVYEKEPG EELLIPCTWFSFLMDSRNEVWWTIDGKKPDD1TIDVTINESISHSRTEDETRT QILSIKKY'TSEDLKRSYVCHARSAKGEVAKAAKVKQKVPAPRYTVESTITLII FGVMAGVIGTILLISYGISMHGSVSRDDDPPQASVSEIVDKAKQLGDAPRRIE PDLRTIIDNVQDLTGPIYKMGSLEVNEDQQLGTGSNGTWFAGKWDGRDVA

[0398] V'KRMLIQFYDIASQETRLLRESDDHPNVIRYYAQQSRDAFLYIALELCQASL AE VIEKPA YFKNLAQAGEKDLPN VLYQITNGLSHLHSLR1VHRDLKPQNIL V NMGKDGKPRLLVSDFGLCKKLEGGQSSFGATTAHAAG1TGWRAPELLLDD DARDNTATMVDASMSSAHSGSGSVQGSSDVPNRRATRAIDIFSLGLVFFYVL TKGSHPYDRGDRYMREVNIRKGSFDLSRLEVLGDYAMEARDIVERMLSFEP SERPTARDVMRHPFFWSAKKRLAFLCDVSDHFEKEPRDPPSWPLQVLEEAA PDVITSGDFLRQLPREFVDSLGKQRKYTGSRMLDLLRALRNKKNHYEDMPE SLKKTVGPLPEGYLSFWTRRFDTLLINCWRIV1DCGWDETDRFRDYYDLPGT (SEQ ID NO: 38)

[0399]

[0400] pMLS420 (FKBP^'-cyt^iielREl^)

[0401] 50

[0402] 326657998 Attorney Docket No.: PTBI-010 / 01 WO FKBPF36VSEQ ID NO: 10 (see Table 1)

[0403] Linker SEQ ID NO: 45

[0404] A. thaliana IRE1 SEQ ID NO: 16 (see Table 2)

[0405] kinase domain

[0406] A. thaliana IRE1 SEQ ID NO: 22 (see Table 3)

[0407] RNase domain

[0408] Full sequence MGVQ\7ETISPGDGRTFPKRGQTCVVHYTGMT., EDGKKVDSSRDRNKPFKFM LGKQEVIRGWEEGVAQMSVGQRAKLTESPDYAYGATGHPGIIPPHATLVFDV ELLKLEGGSGGTGGSGGTSMHSSRGSDVSLKAGPSKKKKNRKSAKDTNRQS VPRGQDQFELIEGGQMLLGFNNFQSGATDGRKIGKLFLSSKEIAKGSNGTW FEGIYEGRPVAVKRLVRSHHEVAFKEIQNIJASDQHTNIIRWYGkTiYDQDFV YLSLERCTCSLDDLIKSYLEFSMTKVLENNDSTEGVAAYKIQLDSLEGVIKG NNFWKVGGHPSPLMLKLMRDIVCGIVHLHELGIVHRDLKPQNAT. ISKDMTL SAKLSDMGISKRMSRDMSSLGHLATGSGSSGWQAPEQLLQGRQTRAVDMF SLGCVIFYTITGCKHPFGDDLERDVN1VKNKVDLFLVEHVPEASDLISRLLNP DPDLRPSATEVLLHPMFWNSEMRLSFLRDASDRVELENREADSEILKAMEST APVAIGGKWDEKLEPVFITNIGRYRRYKYDSIRDLLRVIRNKLNHHRELPPEI QELVGTVPEGFDEYFAVRFPKLLIEVYRVISLHCREEEVFRKYFKCDIKSEQ ID NO: 39)

[0409] pMLS421 (FKBPF36V“Cjtt>st>iieIRElCa)

[0410] FKBPF36VSEQ ID NO: 10 (see Table 1)

[0411] linker SEQ ID NO: 45

[0412] C. albicans IRE1 SEQ ID NO: 17 (see Table 2)

[0413] kinase domain

[0414] C. albicans IRE1 SEQ ID NO: 23 (see Table 3)

[0415] RNase domain

[0416] Full sequence MGVQVET1SPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFM LGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDV ELLKLEGGSGGTGGSGGTSMHTTPKKKRKRGSRGGRRGGARKNNKQKTNG DEDQEDQQQNDESVVDEEIIPTKSLIPPSSLPAIKSRKKLQIENNLVISDKILGY GSHGTVVFQGTTENRPVAVKRNIL. LDFYDLANI-IEVRLLQESDDI-IPNVY'RYFC SQSSESEKFLYIALELCLCTLEDIIEKPQNMPNLCIPKRNDILYQLTSGLHYLH SLK1VIIRDIKPQNILVANIKKNGKRKNQITEIDETCENNVRLLISDFGLCKKLE NDQSSFRATTQNAASGTSGWRAPELLLNI-IDLWEISADSISSIHSNSNSNGNG NGNGA’ TNGSVSNSATSGKRLT 'KAIDIFSLGCVFY ) ILTGGY HP! ’GDRYLREG NIIKGEYDLSLLMEKC? PNDRYESIDLISKIISHDPSQRPN TGKILKHPLFWSFSK RLEI T. LKVSDRF EIEKRDPPSPLLLKLI lEIIAKAV ITNGNWI-IISLLNDDEFNIDN i. GKVRKVSFEK:.\! D!. LR \MR\KYHHYX!)\!m: Si. Qi. K\T\Pi. FDG!;YKYLV DKFPKIT. MERAA'AT. ENFRNEI-iy?FKETzY (SEQ ID NO: 40)

[0417] pMLS422 (FKBPF36V-cytosoiicIRElSc)

[0418] FKBPF36VSEQ ID NO: 10 (see Table 1)

[0419] Linker SEQ ID NO: 45

[0420] S', cerevisiae IRE I SEQ ID NO: 18 (see Table 2)

[0421] kinase domain

[0422] S. cerevisiae IRE1 SEQ ID NO: 24 (see Table 3)

[0423] RNase domain

[0424] Full sequence MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFM LGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDV ELLKLEGGSGGTGGSGGTSMHQRFKILPPLYVLLSKTGFMPEKETPIVESKSLN CPSSSENVTKPFDMKSGKQVVFEGAVNDGSLKSEKDNDDADEDDEKSLDLT TEKKKRKRGSRGGKKGRKSRIANIPNFEQSLKNLWSEKILGYGSSGTVVTQ GSFQGRPVAVTCRMLIDFCDIALMEIKLLTESDDHPNYTRYYCSETTDRFLYTA LELCNLNLQDLVESKNVSDENLKLQKEYNPISLLRQIASGVAHLHSLKIIHRD LKPQNILVSTSSRFTADQQTGAENLRILISDFGLCKKLDSGQSSFRTNLNNPSG TSGWRAPELLEESNNLQCQVETEHSSSRHTVVSSDSFYDPFTKRRLTRSIDIFS MGCVFYY1LSKGKHPFGDKYSRESNIIRGIFSLDEMKCLHDRSLIAEATDLISQ MIDHDPLKRPTAMKVLRHPLFWPKSKKLEFLLKVSDRLE1ENRDPPSALLMK FDAGSDFVIPSGDWTVKFDK'miDNLERYRKYHSSKLMDLLRALRNKYHH FWLPEDIAELMGPVPDGFYDYFTKRFPNLL1GVYMIVKENLSDDQ1LREFLY S (SEQ ID NO: 41)

[0425]

[0426] pMLS433 (LaG16-LaG2-TM»IRElM8)

[0427] 51

[0428] 326657998 Attorney Docket No.: PTBI-010 / 01WO SP MALPVTALLLPLALLLHAARP ( SEQ ID NO: 49)

[0429] Myc-tag EQKLISEEDL (SEQ ID NO: 50)

[0430] LaG16 MAQVQLVESGGRLVQAGDSLRLSCAASGRTFSTSAMA^TRQAPGREREFV AAIWTVGNTn., GDSVXGRFTISRDRAKNTVDLQMDNI, EPEDTAVYYCSAR SRGYVLSVLRSVDSYDYWGQGTQVTVS (SEQ ID NO: 51)

[0431] LaG2 MAQVQLVESGGGLVQAGGSLRLSCAASGRTFSNYAMGWFRQAPGKEREFV AA1SWTGVSTYYADSVKGRFTISRDNDKNTVYVQMNSLIPEDTAIYYCAAV RARSFSDTYSRVNEYDYWGQGTQVTV (SEQ ID NO: 52)

[0432] Linker SEQ ID NO: 45

[0433] AL grisea IREI SEQ ID NO: 20 (see Table 2)

[0434] kinase domain

[0435] AL grisea IRE I SEQ ID NO: 26 (see Table 3)

[0436] RNase domain

[0437] Full sequence MALPVTALLLPLALLLHAARPEQKLISEEDLMAQVQLVESGGRLVQAGDSL RLSCAASGRTFSTS / W1AWFRQAPGREREFVAAITWTVGNTILGDSVKGRFTI SRDRAKNTWLQMDNLEPEDTAVYYCSARSRGYVLSVLRSWSYDYWGQG TQVTVSGGGGSGGGGSGGGGSMAQVQLVESGGGLVQAGGSLRLSCAASGR TFSNYAMGWFRQAPGKEREFVAAISWTGVSTYYADSVKGRFTISRDNDKNT WVQMNSLIPEDTAIYYCAAVRARSFSDTYSRVNEYDYWGQGTQVTVGGG GSGGGGSGGGGSTGGSGGTGGSGGTGGSGGSSTGGSGGTGGSGGTSTITLIIF GVMAGVIGTILLISYGISMHGSVSRDDDPPQASVSEIVDKAKQLGDAPRRIEP DLRTUDNVQDLTGPIYKMGSLEVNEDQQLGTGSNGTWFAGKWDGRDVA VKRMI.. IQFYDI A SQETRLLRF. SDDHPNVIRYYAQQSRD AFLYI ALELCQASL AEATEKPATTKNLAQAGEKDLPNAT. YQITNGI. SHl. HSLRIWRDLKPQNILV NMGKDGKPRLLVSDFGLCKKLEGGQSSFGATTAHAAGTTGWRAPELLLDD DARDNTATMVDASMSSAHSGSGSVQGSSDVPNRRATRAIDIFSLGLVFFYVT TKGSHPYDRGDRYMREVNriRKGSFDLSRLEW. GDYAMEARDIWRMLSFEP SERPTARDANIRIIPFFWSAKKRLAFLCDVSDHFEKEPRDPPSWLQAA. EEAA PDA TTSGDFLRQLPREFA TJSLGKQRKY1TGSRNfl.. DLLRALRNKKNHYI iDMPE SLKKWGPLPEGYLSFWTRRFDTLLINCWRIVIDCGWDETDRFRDYYDLPGT

[0438] (SEQ ID NO: 42)

[0439] PMLS479 (VHI-I2-TM»IRE1MS)

[0440] SP SEQ ID NO: 49

[0441] VHH2 SEQ ID NO: 14 (see Table 1)

[0442] GpA SEQ ID NO: 27

[0443] Linker SEQ ID NO: 45

[0444] M. grisea IRE1 SEQ ID NO: 20 (see Table 2)

[0445] kinase domain

[0446] M. grisea IREI SEQ ID NO: 26 (see Table 3)

[0447] RNase domain

[0448] Full sequence MALPVTALLLPLALLLHAARPEQKLISEEDLMAQVQLVESGGGLVQPGGSL RLSCAASGFTFSNYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVKGRFT

[0449] 1SRDNAKNTLYLQMNSLKPEDTALYYCARSPSGFNRGQGTQVTVSSGGGGS GGGGSGGGGSIGGSGGTGGSGGTGGSGGTGGSGGTGGSGGTSTITLIIFGVM AGVIGTILL1SYGISMHGSVSRDDDPPQASVSEIVDKAKQLGDAPRRIEPDLRT IIDNVQDLTGPIYKMGSLEVNEDQQLGTGSNGTVVFAGKWDGRDVAVKRM LIQFYDIASQETRLLRESDDHPNVIRYYAQQSRDAFLYIALELCQASLAEV1E KPAYFKNLAQAGEKDLPNALYQnNGLSHLHSLRIATIRDLKPQNILANMGK DGKPRLLVSDFGLCKKLEGGQSSFGATTAHAAGTTGWRAPELLLDDDARD

[0450] NliAINIVDASMSSAHSGSGSVQGSSDVPNRRAIRAIDIFSLGLVFFYVLTKGS IIPY'DRGDRY'NIREVNIRKGSFDLSRLEALGDYAA'IE. ARDrVERA'ILSFEPSERPT ARDVMMTPFFWSAKKJlLAFLODVSDIlFEKEPRDPPSWPLQVLEEAAPDVn' SGDFLRQLPREFVDSLGKQRKYTGSRMLDLLRALRNKKNHYEDMPESLKKT VGPLPEGYL SFWTRRFDTLLINC WRIVIDCG WDETDRFRDYYDLPGT (SEQ ID NO: 43)

[0451] pMLS021 foiosoiiJREl^)

[0452] Homo sapiens IRE 1 SEQ ID NO: 15 (see Table 2)

[0453] kinase domain

[0454] Homo sapiens IREI SEQ ID NO: 21 (see Table 3)

[0455]

[0456] RNase domain

[0457] 52

[0458] 326657998 Attorney Docket No.: PTBI-010 / 01WO Full sequence MI-IQQQQLQHQQFQKELEKIQLLQQQQQQLPITIPPGDTAQDGELLDTSGPYS ESSGTSSPSTSPRASM-ISLCSGSSASKAGSSPSLEQDDGDEETSVVIVGKISFCP KDVTGHGAEGTIVYRGMFDNRDVAy^KRRPECFSFADREVQLLRESDEHPN VIRYFCTEKDRQFQYIAniLCAATL, QEYVEQKDFAHI. GLEPim., QQTTSGLA HI, HSLNIVHRDLKPHNn., ISMPNAHGKIKAMISDFGLCKKLAVGRHSFSRRSG VPGTEGWIAPEMI, SEDCKENPTYT\T)IFSAGC\TYYVISEGSHPFGKSLQRQ ANR. XGACSLDCLHPEKHEDVIARELIEKMIAMDPQKRPSAKHVT. XHPFFWS LEKQLQFFQDVSDRIEKESLDGPIVKQLERGGRA\WKMDWRENITVPLQTDL RKFRiyRGGSVRDIJ.. RAAfRNKKI-n-IYREI, PAEWETI.. GSL. PDDFVC VF1 SHL

[0459]

[0460] PHLLAHTYRAMELCSHERLFQPYYFHEPPEPQPPVTPDAL (SEQ ID NO: 56)

[0461] II. Synthetic RNAs

[0462] Aspects of the disclosure provide an engineered RNA polynucleotide comprising a sequence of a stem-loop splicing hairpin comprising a cytosolic intron. In some embodiments, the cytosolic intron is capable of being excised by an RNase domain of an inositol-requiring enzyme 1 (IRE1) protein. In some embodiments, the engineered RNA polynucleotide of the present disclosure comprises a hairpin sequence recognized by a chimeric protein provided herein, binding of which results in RNA cleavage and excision of a cytosolic intron.

[0463] In some embodiments, the engineered RNA polynucleotide does not encode a x-box binding protein 1 (XBP1), or a functional fragment thereof. In some embodiments, the engineered RNA polynucleotide does not encode a fusion protein comprising the XBP1 protein or functional fragment thereof.

[0464] The term “stem-loop hairpin,” as used herein, refers to a secondary' RNA structure formed by single-stranded RNA that comprise a base-paired stem and one or more (e.g., 1, 2, or 3) loop sequences with unpaired RNA bases. For example, a stem-loop hairpin may comprise, from 5’ to 3’: a 5’ stem region, a loop region, and a 3’ stem region, wherein the RNA bases of the 5’ stem region are complementary to the RNA bases of the 3’ stem region. A stem-loop hairpin may comprise, for example, from 5’ to 3’: a 5’ stem 1 region, a 5’ stem 2 region, a 5’ loop region, a 3’ stem 2 region, a 5’ stem 3 region, a 3’ loop region, a 3’ stem 3 region, and a 3’ stem 1 region, wherein the RNA bases of the 5’ stem 1 region are complementary to the RNA bases of the 3’ stem 1 region, the RNA bases of the 5’ stem 2 region are complementary / to the RNA bases of the 3’ stem 2 region, and the RNA bases of the 5’ stem 3 region are complementary to the RNA bases of the 3’ stem 3 region. Exemplary / stem loop structures are provided in FIG. 8A.

[0465] The term “stem-loop splicing hairpin,” as used herein, refers to an RNA having a stem-loop hairpin structure comprising a cytosolic intron. The stem-loop splicing hairpin may comprise a 5’ IRE1 cleavage site and a 3’ IRE1 cleavage site flanking the cytosolic intron, which facilitate excision of the cytosolic intron and are recognized by a chimeric 53

[0466] 326657998 Attorney Docket No.: PTBI-010 / 01WO protein provided herein. Exemplary stem-loop splicing hairpins are provided in FIG. 8A and Table 5.

[0467] In some embodiments, the stem-loop splicing hairpin comprises the nucleotide sequence of X1X2GCX3GX4X5X6T (SEQ ID NO: 73), wherein Xi is C or T; X2 is C, T, or A; X3 is C, T, or A, X4 is T, A, or C; X5 is A or G; and Xe is C, A, or G, optionally wherein the nucleotide sequence comprises an IRE1 cleavage site. In some embodiments, the stem-loop splicing hairpin comprises the nucleotide sequence of X1CX2GX3X4GX5X6X7 (SEQ ID NO: 74), where Xi is T or C; X2 is A, T, or C; X3 is C or A; X4 is T or A; X5 is absent, T, or A; Xs is absent, C or T; and X7 is G or A, optionally wherein the nucleotide sequence comprises an 1RE1 cleavage site. In some embodiments, the stem-loop splicing hairpin comprises: (i) the nucleotide sequence of X1X2GCX3GX4X5X6T (SEQ ID NO: 73), wherein Xi is C or T; X2 is C, T, or A; X3 is C, T, or A; X4 is T, A, or C; Xs is A or G; and Xe> is C, A, or G, wherein the nucleotide sequence comprises a first IRE1 cleavage site; and (ii) the nucleotide sequence of X1CX2GX3X4GX5X6X7 (SEQ ID NO: 74), where Xi is T or C; X is A, T, or C; X is C or A; X4 is T or A; X5 is absent, T, or A; Xs is absent, C or T; and X7 is G or A, wherein the nucleotide sequence comprises a second IRE1 cleavage site.

[0468] In some embodiments, the first IRE1 cleavage site is 5’ to the second IRE1 cleavage site. In some embodiments, such a first IRE1 cleavage site is called a 5’ IRE1 cleavage site, and such a second IRE1 cleavage site is called a 3’ IRE1 cleavage site.

[0469] In some embodiments, the first and the second IRE1 cleavage sites are separated by about 10 to about 50 nucleotides. In some embodiments, the first and the second IRE1 cleavage sites are separated by about 10 to about 15, about 15 to about 20, about 20 to about 25, about 25 to about 30, about 30 to about 35, about 35 to about 40, about 40 to about 45, about 45 to about 50, about 10 to about 20, about 15 to about 25, about 20 to about 30, about.

[0470] 25 to about 35, about 30 to about 40, about 35 to about 45, about 40 to about 50, about 10 to about 25, about 15 to about 30, about.20 to about 35, about 25 to about. 40, about 30 to about 45, about 35 to about 50, about 10 to about 30, about 15 to about 35, about 20 to about 40, about 25 to about 45, about 30 to about 50, about 10 to about 35, about 15 to about 40, about.

[0471] 20 to about 45, about 25 to about 50, about 10 to about 40, about 15 to about 45, about 20 to about 50, about 10 to about 45, about 15 to about 50, or about 10 to about 50, nucleotides.

[0472] In some embodiments, a 5’ IRE1 cleavage site is comprised within the nucleotide sequence of X1X2GCX3GX4X5X6T (SEQ ID NO: 73), wherein Xi is C or T; X2 is C, T, or A; X3 is C, T, or A; X4 is T, A, or C; X? is A or G; and Xe is C, A, or G. In some embodiments, a 3’ IRE1 cleavage site is comprised within the nucleotide sequence of X1CX2GX3X4GX5X6X7

[0473] 54

[0474] 326657998 Attorney Docket No.: PTBI-010 / 01WO (SEQ ID NO: 74), where Xi is T or C; X is A, T, or C; Xs is C or A; X4 is T or A; X5 is absent, T, or A; Xe is absent, C or T, and X7 is G or A. In some embodiments, a 5’ IREl cleavage site is comprised within the nucleotide sequence of any one of SEQ ID NOs: 98, 100, 102, 104, 106, and 108 and the 3’ IREl cleavage site is comprised within the nucleotide sequence of any one of SEQ ID NOs: 99, 101, 103, 105, 107, and 109.

[0475] In some embodiments, a 5’ IREl cleavage site is comprised within the nucleotide sequence of SEQ ID NO: 98 and the 3’ IREl cleavage site is comprised within the nucleotide sequence of SEQ ID NO: 99. In some embodiments, a 5’ IREl cleavage site is comprised within the nucleotide sequence of SEQ ID NO: 100 and the 3’ IREl cleavage site is comprised within the nucleotide sequence of SEQ ID NO: 101. In some embodiments, a 5’ IRE1 cleavage site is comprised within the nucleotide sequence of SEQ ID NO: 102 and the 3’ IREl cleavage site is comprised within the nucleotide sequence of SEQ ID NO: 103. In some embodiments, a 5’ IREl cleavage site is comprised within the nucleotide sequence of SEQ ID NO: 104 and the 3’ IREl cleavage site is comprised within the nucleotide sequence of SEQ ID NO: 105. In some embodiments, a 5’ IREl cleavage site is comprised within the nucleotide sequence of SEQ ID NO: 106 and the 3’ IREl cleavage site is comprised within the nucleotide sequence of SEQ ID NO: 107. In some embodiments, a 5’ IREl cleavage site is comprised within the nucleotide sequence of SEQ ID NO: 108 and the 3’ IREl cleavage site is comprised within the nucleotide sequence of SEQ ID NO: 109.

[0476] In some embodiments, any one or more of the thymine bases (Ts) in any one of the RNA sequences provided herein (e.g., as provided in SEQ ID NOs: 73-74 and 98-109) may independently and optionally be uracil bases (Us).

[0477] The terms “cytosolic intron” or “unconventional intron,” as used herein, refers to an RNA sequence located between two IREl cleavage sites (e.g., between a 5’ IREl cleavage site and a 3’ IREl cleavage site). Unlike conventional introns, which comprise a 5’ splice site and a 3’ splice site that allow its removal via conventional splicing reactions by spliceosomes, cytosolic introns are excised by an IREl protein (or an RNase domain of a chimeric protein described herein). Exemplary cytosolic introns are provided in FIG. 8A and Table 5.

[0478] In some embodiments, the cytosolic intron comprises a nucleotide sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to any one of SEQ ID NO: 65-70. In some embodiments, the cytosolic intron comprises or consists of the nucleotide sequence of any one of SEQ ID NOs: 65-70. In some embodiments, any one or more of the thymine bases

[0479] 55

[0480] 326657998 Attorney Docket No.: PTBI-010 / 01WO (Ts) in any one of the cytosolic introns provided herein (e.g., as provided in SEQ D NOs: 65-70) may independently and optionally be uracil bases (Us).

[0481] In some embodiments, the RNA polynucleotide is an mRNA polynucleotide encoding a protein of interest. In some embodiments, excision of the cytosolic intron (e.g., by IREl cleavage at the 5’ IRE I cleavage site and the 3’ IREl cleavage site) results in restoration of the proper reading frame for translation of the protein of interest. In some embodiments, excision of the cytosolic intron results in the removal of the proper reading frame for translation of the protein of interest. In some embodiments, removal of the proper reading frame results in production of an alternative protein of interest.

[0482] In some embodiments, the RNA polynucleotide is a non-coding RNA. In some embodiments, excision of the cytosolic intron results in conversion of the RNA polynucleotide into a functional non-coding RNA. In some embodiments, excision of the cytosolic intron results in conversion of the RNA polynucleotide into a n on-function al non¬ coding RNA. In some embodiments, excision of the cytosolic intron results in conversion of the RNA polynucleotide into an alternative non-coding RNA.

[0483] In some embodiments, the RNA polynucleotide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 stem-loop splicing hairpins comprising a cytosolic intron.

[0484] In some embodiments, the stem-loop splicing hairpin, either including or excluding a cytosolic intron, is not derived from a human stem-loop splicing hairpin. In some embodiments, the stem-loop splicing hairpin, either including or excluding a cytosolic intron, is not derived from a mammalian stem-loop splicing hairpin. In some embodiments, the stem-loop splicing hairpin, either including or excluding a cytosolic intron, is not derived from a eukaryotic stem-loop splicing hairpin. In some embodiments, the cytosolic intron is not derived from a human stem-loop splicing hairpin. In some embodiments, the cytosolic intron is not derived from a mammalian stem-loop splicing hairpin. In some embodiments, the cytosolic intron is not derived from a eukaryotic stem-loop splicing hairpin.

[0485] In some embodiments, the stem-loop splicing hairpin, cytosolic intron, the 5’ cleavage site, and / or 3’ IREl cleavage site is not a human stem-loop splicing hairpin, cytosolic intron, 5’ IREl cleavage site, or 3’ IREl cleavage site. In some embodiments, the stem-loop splicing hairpin, cytosolic intron, the 5’ cleavage site, and / or 3’ IREl cleavage site is not derived from a human stem-loop splicing hairpin, cytosolic intron, 5’ IREl cleavage site, or 3’ IREl cleavage site.

[0486] In some embodiments, the stem-loop splicing hairpin, cytosolic intron, the 5’ cleavage site, and / or 3’ IREl cleavage site is not a mammalian stem-loop splicing hairpin, cytosolic

[0487] 56

[0488] 326657998 Attorney Docket No.: PTBI-010 / 01WO intron, 5’ IRE1 cleavage site, or 3’ IRE1 cleavage site. In some embodiments, the stem-loop splicing hairpin, cytosolic intron, the 5’ cleavage site, and / or 3’ IRE 1 cleavage site is not derived from a mammalian stem-loop splicing hairpin, cytosolic intron, 5’ IRE1 cleavage site, or 3’ IRE1 cleavage site.

[0489] In some embodiments, the stem-loop splicing hairpin, cytosolic intron, the 5’ cleavage site, and / or 3’ IRE1 cleavage site is a plant stem-loop splicing hairpin, cytosolic intron, 5’ IRE1 cleavage site, or 3’ IRE1 cleavage site. In some embodiments, the stem-loop splicing hairpin, cytosolic intron, the 5’ cleavage site, and / or 3’ IRE1 cleavage site is derived from a plant stem-loop splicing hairpin, cytosolic intron, 5’ IRE1 cleavage site, or 3’ IRE1 cleavage site.

[0490] In some embodiments, the stem-loop splicing hairpin, cytosolic intron, the 5’ cleavage site, and / or 3’ IRE1 cleavage site is a fungal stem-loop splicing hairpin, cytosolic intron, 5’ IRE1 cleavage site, or 3’ IRE1 cleavage site. In some embodiments, the stem-loop splicing hairpin, cytosolic intron, the 5’ cleavage site, and / or 3’ IRE1 cleavage site is derived from a fungal stem-loop splicing hairpin, cytosolic intron, 5’ IRE1 cleavage site, or 3’ IRE1 cleavage site. In some embodiments, the corresponding fungus is a plant-pathogenic fungus.

[0491] In some embodiments, the engineered RNA polynucleotide comprises (i) a sequence of a stem-loop splicing hairpin comprising a cytosolic intron, wherein the cytosolic intron is capable of being excised by an RNase domain of an inositol-requiring enzyme 1 (IRE1) protein; and (ii) a sequence encoding an initial start codon. In some embodiments, the initial start codon is 5’ to the sequence of the stem-loop splicing hairpin.

[0492] In some embodiments, the engineered RNA polynucleotide encodes a protein of interest that is not a x-box binding protein 1 (XBP1). In some embodiments, the engineered RNA polynucleotide expresses the protein of interest after the cytosolic intron is excised, but does not express the protein of interest when the cytosolic intron is not excised. In some embodiments, the engineered RNA polynucleotide does not express the protein of interest after the cytosolic intron is excised, but expresses the protein of interest when the cytosolic intron is not excised. In some embodiments, after the cytosolic intron is excised, the engineered RNA polynucleotide expresses a protein of interest that is different from the protein of interest expressed by the engineered RNA polynucleotide when the cytosolic intron is not excised.

[0493] In some embodiments, at least a part of the nucleotide sequence encoding the protein of interest is out of frame with the initial start, codon when the cytosolic intron is not excised.

[0494] 57

[0495] 326657998 Attorney Docket No.: PTBI-010 / 01WO In some embodiments, the entire nucleotide sequence encoding the protein of interest is out of frame with the initial start, codon when the cytosolic intron is not excised.

[0496] In some embodiments, the engineered RNA polynucleotide comprises a second start codon. In some embodiments, the second start codon is 3’ to the sequence of the initial start codon. In some embodiments, the second start codon is 3’ to the cytosolic intron. In some embodiments, the second start codon is out of frame with the initial start codon.

[0497] In some embodiments, the nucleotide sequence encoding the protein of interest is in frame with the second start codon. In some embodiments, the nucleotide sequence encoding the protein of interest comprises the second start codon as its first codon.

[0498] In some embodiments, the engineered RNA polynucleotide comprises a stop codon (e.g., a first, stop codon). In some embodiments, the stop codon is 3’ to the sequence of the stem-loop splicing hairpin. In some embodiments, the stop codon is in frame with the initial start codon. In some embodiments, the stop codon is 5’ to the nucleotide sequence encoding the protein of interest.

[0499] In some embodiments, the engineered RNA polynucleotide further comprises a second stop codon. In some embodiments, the nucleotide sequence encoding the protein of interest is in frame with the second stop codon. In some embodiments, the nucleotide sequence encoding the protein of interest comprises the second stop codon as its last codon.

[0500] In some embodiments, the protein of interest of the disclosure is a therapeutic protein. In some embodiments, the therapeutic protein of the disclosure is an antibody (e.g., scfv), a Fc fusion protein, a hormone, a cytokine, a chemokine, a cytotoxin, an enzyme catalyzing a prodrug activation, a transcription factor, a transactivator, an immunomodulatory protein, or a DNA-modifying enzyme.

[0501] In some embodiments, the engineered RNA polynucleotide is a non-coding RNA. In some embodiments, the engineered RNA polynucleotide is converted from a non-functional non-coding RNA to a functional non-coding RNA upon excision of the cy tosolic intron. In some embodiments, the engineered RNA polynucleotide is converted from a functional non¬ coding RNA to a non-functional non-coding RNA upon exci sion of the cytosolic intron. In some embodiments, the function of the non-coding RNA is altered after the cytosolic intron is excised.

[0502] In some embodiments, the RNA polynucleotide comprises: (i) a sequence of a stem-loop splicing hairpin comprising a cytosolic intron; (ii) a sequence encoding a first start codon; (iii) a sequence encoding a first stop codon that is in frame with the initial start codon; and (iv) a sequence encoding a second start codon, preferably wherein the second start codon

[0503] 58

[0504] 326657998 Attorney Docket No.: PTBI-010 / 01WO is out of frame with the first codon. In some embodiments, the second start codon is 5’ to the first start codon. In some embodiments, the second start codon is 3’ to the first start codon, optionally 3’ to the cytosolic intron.

[0505] In some embodiments, engineered RNA polynucleotides of the present disclosure comprise a sequence of a stem-loop splicing hairpin comprising a cytosolic intron. In some embodiments, the stem-loop splicing hairpin comprises a first portion comprising a sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of TACAATTCGAATGAT (SEQ ID NO: 57) and a second portion comprising a sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%>, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of ATCATTCGAATTGTA (SEQ ID NO: 58), wherein the first nucleotide sequence and the second nucleotide sequence are complementary. In some embodiments, the stem-loop splicing hairpin comprises a first portion comprising or consisting of the sequence of SEQ ID NO: 57 and a second portion comprising or consisting of the sequence of SEQ ID NO: 58, wherein the first nucleotide sequence and the second nucleotide sequence are complementary'. In some embodiments, any one or more of the thymine bases (Ts) in any one of the first portion (SEQ ID NO: 57) and / or second portion (SEQ ID NO: 58) may independently and optionally be uracil bases (Us).

[0506] In some embodiments, the stem-loop splicing hairpin comprises the nucleotide sequence of X]AX2X5X4X5GCX6GX7XsX9TXioXiiXi2Xi3 (SEQ ID NO: 71), where Xi is A, C, or G; X2 is G or T, X3 is T or C; Xr is C or T; X5 is A, T, or C; Xs is C, T, or A; X7 is T, A, or C; Xs is A or G; X9 is C, A, or G; X10 is T, G, or C; X11 is A or T; X12 is C, T, or G; and X13 is G, T, or A. In some embodiments, the stem loop splicing hairpin comprises the nucleotide sequence of X1X2X3X4X5CX6GX7X8G (SEQ ID NO: 72), wherein Xi is A or C; X2 is C, T, or A, X3 is G, T, A, or C; X is G, A, or C; X5 is C or T; Xe is C, T, or A; X7 is A or C; and Xs is A or T. In some embodiments, the stem-loop splicing hairpin comprises the nucleotide sequence of SEQ ID NO: 71 and the nucleotide sequence of SEQ ID NO: 72. In some embodiments, the stem-loop splicing hairpin comprises a nucleotide sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to any one of SEQ ID NOs: 59-64. In some embodiments, the stem-loop splicing hairpin comprises a nucleotide sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to any one of SEQ ID NOs: 59-64, wherein the stem-loop

[0507] 59

[0508] 326657998 Attorney Docket No.: PTBI-010 / 01WO splicing hairpin comprises the nucleotide sequence of SEQ ID NO: 71. In some embodiments, the stem-loop splicing hairpin comprises a nucleotide sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to any one of SEQ ID NOs: 59-64, wherein the stem-loop splicing hairpin comprises the nucleotide sequence of SEQ ID NO: 72. In some embodiments, the stem-loop splicing hairpin comprises a nucleotide sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to any one of SEQ ID NOs: 59-64, wherein the stem-loop splicing hairpin comprises the nucleotide sequence of SEQ ID NO: 71 and SEQ ID NO: 72, In some embodiments, any one or more of the thymine bases (Ts) in any one of the stem-loop splicing hairpins provided herein (e.g., as provided in SEQ ID NOs: 59-64) may independently and optionally be uracil bases (Us).

[0509] In some embodiments, the stem-loop splicing hairpin comprises a sequence comprising a consensus sequence (consensus / 100%, consensus / 90%, consensus / 80%, or consensus 70%) depicted in FIG. 15.

[0510] In some embodiments, the stem-loop splicing hairpin comprises or consists of the nucleotide sequence of any one of SEQ ID NOs: 59-64.

[0511] In some embodiments, any one or more of the thymine bases (Ts) in any one of the nucleotide sequences provided herein (e.g., the stem-loop splicing hairpins, cytosolic intron, and IRE1 cleavage site sequences provided in Table 5) may independently and optionally be uracil bases (Us).

[0512] In some embodiments, engineered RNA polynucleotides of the present disclosure comprise a sequence encoding an initial start codon, wherein the initial start codon is 5’ to the sequence of the stem-loop splicing hairpin. For example, as shown in FIG. 8A, a stern-loop splicing hairpin may comprise, from 5’ to 3’: a 5’ Stem 1, a 5’ Stem 2, a 5’ Loop, a 3’ Stem 2, a 5’ Stern 3, a 3’ Loop, a 3’ Stern 3, and a 3’ Stem 1, the initial start codon may be located in the 5’ Stem 1 region, the 5’ Stem 2 region, or 5’ to the 5’ Stem 1 region and the 5’ Stem 2 region.

[0513] In some embodiments, engineered RNA polynucleotides of the present disclosure comprise a sequence encoding a first stop codon, wherein the first stop codon is 3’ to the sequence of the stem-loop splicing hairpin. For example, as shown in FIG. 8A, a stem-loop splicing hairpin may comprise, from 5’ to 3’: a 5’ Stem 1, a 5’ Stem 2, a 5’ Loop, a 3’ Stem 2, a 5’ Stem 3, a 3’ Loop, a 3’ Stem 3, and a 3’ Stem 1, the first stop codon may be located in

[0514] 60

[0515] 326657998 Attorney Docket No.: PTBI-010 / 01WO the 3’ Stem 3 region, the 3’ Stem 1 region, or 3’ to the 3’ Stem 3 region and the 3’ Stem 1 region. In some embodiments, the first stop codon is in frame with the initial start codon.

[0516] As used herein, the term “in frame” describes codons that are aligned with a reading frame established by a reference codon (e.g., an initial start codon). In some embodiments, the reference codon establishes the first triplet of nucleotides for the reading frame. Thus, for example, where a first stop codon is in frame with the initial start codon, translation initiated at the initial start codon would terminate at the first stop codon.

[0517] In some embodiments, engineered RNA polynucleotides of the present disclosure comprise a sequence encoding a second start codon, wherein the second start, codon is 3’ to the sequence of the initial start codon. For example, shown in FIG. 8A, where a stem-loop splicing hairpin comprises, from 5’ to 3’: a S’ Stem I, a 5’ Stem 2, a 5’ Loop, a 3’ Stem 2, a 5’ Stem 3, a 3’ Loop, a 3’ Stem 3, and a 3’ Stem 1, and where the initial start codon is located in the 5’ Stem 1 region, the second start codon may be located 3’ to the initial start codon in the 5’ Stem 1 region, in the 5’ Stem 2 region, in the 5’ Loop, in the 3’ Loop, in the 3’ Stem 3 region, or in the 3’ Stem 1 region. In some embodiments, the second start codon is out of frame with the initial start codon.

[0518] The term “out of frame” describes codons that are not aligned with the reading frame established by the reference codon (e.g., the initial start codon). Codons that are out of frame are mistranslated, typically resulting in frameshift mutations such as missense and nonsense mutations. Thus, for example, where a second start codon is 3’ to the sequence of the initial start codon and out of frame with the initial start codon, the second start codon is not translated (or recognized) when translation is initiated at. the initial start codon.

[0519] In some embodiments, engineered RNA polynucleotides of the present disclosure comprise a nucleotide sequence encoding a protein of interest. In some embodiments, the nucleotide sequence encoding the protein of interest is in frame with the second start codon. Thus, in some embodiments, where the nucleotide sequence encoding the protein of interest is in frame with the second start codon, the protein of interest is not produced when translation is initiated at the initial start codon. In some embodiments, the nucleotide sequence encoding the protein of interest, comprises the second start codon as its first codon.

[0520] In some embodiments, engineered RNA polynucleotides of the present disclosure comprise a sequence encoding a cleavage peptide. In some embodiments, the sequence encoding the cleavage peptide is 3’ to the sequence encoding the second start codon. In some embodiments, the sequence encoding the cleavage peptide is in frame with the sequence encoding the second start codon. In some embodiments, the sequence encoding the cleavage

[0521] 61

[0522] 326657998 Attorney Docket No.: PTBI-010 / 01WO peptide is 5’ to the sequence encoding the protein of interest. In some embodiments, the sequence encoding the cleavage peptide is in frame with the sequence encoding the protein of interest. In some embodiments, the cleavage peptide is a self-cleaving peptide.

[0523] In some embodiments, the cleavage peptide comprises a protease cleavage site. In some embodiments, the protease cleavage site is an endoprotease, endopeptidase, and / or endoproteinase cleavage site. In some embodiments, the protease cleavage site comprises a chymosin cleavage site. In some embodiments, the protease cleavage site comprises a trypsin cleavage site. In some embodiments, the protease cleavage site comprises a furin cleavage site. Further descriptions of cleavage sites can be found, for example, in US2025 / 0011828, the content of which is incorporated by reference in its entirety.

[0524] In some embodiments, the cleavage peptide is a viral 2A peptide. A 2A peptide is an amino acid sequence found in viruses that causes a self-cleaving reaction during protein synthesis, allowing for the production of multiple distinct proteins from translation of a single mRNA. In some embodiments, the viral 2A peptide is F2A from foot-and-mouth disease virus, E2A from Equine rhinitis A virus, P2A from Porcine teschovirus-1, or T2A from Thosea asigna virus. In some embodiments, the viral 2A peptide comprises an amino acid sequence having at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at. least. 97%, at least 98%, or at. least. 99%) identity to EGRGSLLTCGDVEENPGP (SEQ ID NO: 75), ATNFSLLKQAGDVEENPGP (SEQ ID NO: 76), QCTN YALLKLAGDVESNPGP (SEQ ID NO: 77), or VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 78). In some embodiments, the viral 2A peptide comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 75-78.

[0525] Table 5. Exemplary stem-loop splicing hairpin, cytosolic intron, and IRE1 cleavage sequences. Nucleotides involved in IRE1 processing are highlighted (underlining), as well as the cytosolic intron (bold). While each of the sequences utilize thymine (T), it is understood that any one or more of the thymine (T) bases in any one of the stem-loop splicing hairpin, cytosolic intron, and IRE1 cleavage sequences may independently and optionally be uracil (U) bases (e.g., in the context of an RNA).

[0526] Description Sequence SEQ ID NO Homo sapiens stem-loop GGCCTTGTAGTTGAGAACCAGGAGTTAAGACAGCGC 59

[0527] splicing hairpin TTGGGGATGGATGCCCTGGTTGCTGAAGAGGAGGCG GAAGCCAAGGGGAATGAAGTGAGGCCAGTGGCCGG GTCTGCTGAGTCCGCAGCACTCAGACTACGTGCAC CTCTGCAGCAGGTGCAGGCCCAGTTGTCACCCCTCC AGAACATCTCCCCTTGGATTCTGGCGGTATTGACTCT

[0528]

[0529] TCAGATTCAGAGTCTGATATCCTGTTGGGCATTC

[0530] 62

[0531] 326657998 Attorney Docket No.: PTBI-010 / 01WO Homo sapiens cytosolic CAGCACTCAGACTACGTGCACCTCTG 65 intron

[0532] Arabidopsis thaliana AATACTACCATGATGTCGAAGCAGGAGTCTGCTGTG 60 stem-loop splicing hairpin CTCTTGTTGGAATCCCTGCTGTTGGGTTCCCTGCTT TGGCTTCTGGGAGTAAA

[0533] Arabidopsis thalian CTGTGCTCTTGTTGGAATCCCTG 66 cytosolic intron

[0534] Candia albicans stem- TCCTCATCATCGTTAATTTCATCTTCCGGTCCAACTA 61 loop splicing hairpin ATCATTCTATAGCTGATTTAGCAGCAATCAGTCTAG CCAGCAGAGCCAATAGAATCATTTCAGTTGGACTTT GA TGG i H T IT TCGA1T ’1’1 AGGA

[0535] Candida albicans CAGCAATCAGTCTAGCCAG 67 cytosolic intron

[0536] Saccharomyces cerevisiae CATGACAATTGGCGTAAGTCAGCCGTACTTACGCC 62 stem-loop splicing hairpin AACCAGCTTGTACGGCCCGAAGCGCCGTCAGGTTT G

[0537] Saccharomyces cerevisiae CCGTACTTACGCCAACCAGCTTGTACGGCCCG 68 cytosolic intron

[0538] Neurospora crassa stem- GCGGATGCTACCTCCACTGCGACTGTGGACACTTCC 63 loop splicing hairpin CCCGATGCGACACAACATCCTGCTGTGGTGTTGTG TGATGACCTGCAGTGTCGGTCGGCGGAAGTGCCAC GCTCCAAGTGCCTGGCAGTGTCCCA

[0539] Neurospora crassa CTGTGGTGTTGTGTGATGACCTG 69 cytosolic intron

[0540] Magnaporthe grisea GAAGCGAAGACCTCGGCTGACCTGACACAACATCCT 64 stem-loop splicing hairpin GCCGAGATGTTGTGCACCGACCTGCAGTGTCAGTC AGCCAAGGTCCCGCAAGC

[0541] Magnaporthe grisea CCGAGATGTTGTGCACCGACCTG 70 cytosolic intron

[0542] Consensus Sequence XtXjGCXjGXiXjXgT, wherein X] is C or T; X2is C, T, or A; 73 Comprising 5’ IRE1 X; is C, T, or A; X« is T, A, or C; X$ is A or G; and X6is C. cleavage site A. or G

[0543] Consensus Sequence X1CX2GX3X4GX5X6X7, where X, is T or C; X2is A, T, or C: 74 Comprising 3’ IRE1 X3 is C or A; X4is T or A; X5is absent, T, or A; Xsis absent, cleavage site C or T; and X7is G or A

[0544] Homo sapiens 5 ’ IRE 1 CCGCAGCACT 98 cleavage sequence

[0545] Homo sapiens 3’ IRE1 TCTGCAGCA 99 cleavage sequence

[0546] Arabidopsis thaliana 5’ CTGCTGTGCT 100 IRE1 cleavage sequence

[0547] Arabidopsis thalian 3 ’ CCTGCTGTTG 101 IRE1 cleavage sequence

[0548] Candia albicans 5’ IRE1 TAGCAGCAAT 102 cleavage sequence

[0549] Candida albicans 3’ IRE1 CCAGCAGAG 103 cleavage sequence

[0550] Saccharomyces cerevisiae CAGCCGTACT 104 5' IRE1 cleavage

[0551] sequence

[0552] Saccharomyces cerevisi ae CCCGAAGCG 105 3’ IRE1 cleavage

[0553] sequence

[0554] Neurospora crassa stem- CTGCTGTGGT 106 s’ IRF.1 cleavage

[0555]

[0556] sequence

[0557] 63

[0558] 326657998 Attorney Docket No.: PTBI-010 / 01WO Neumspora crassa 3 ’ CCTGCAGTG 107

[0559] IREl cleavage sequence

[0560] Magnaporthe grisea 5’ CTGCCGAGAT 108

[0561] IREl cleavage sequence

[0562] Magnaporthe grisea 3 ’ CCTGCAGTG 109

[0563]

[0564] IREl cleavage sequence

[0565] III. Synthetic Circuits

[0566] Aspects of this disclosure provide a polynucleotide comprising a sequence encoding a chimeric protein of the disclosure. Aspects of this disclosure provide a polynucleotide comprising or encoding an engineered RNA polynucleotide of the disclosure.. Aspects of this disclosure provide a polynucleotide comprising a sequence encoding, or a combination of polynucleotides collectively comprising: a sequence encoding a chimeric protein (one or more) described herein; and a sequence encoding an RNA polynucleotide (one or more) described herein. In some embodiments, the polynucleotide is or comprises a DNA. In some embodiments, the polynucleotide is or comprises an RNA(e.g., a mRNA). In some embodiments, the RNA molecule of the disclosure is a modified RNA.

[0567] In some embodiments, the IRE1 protein(s) and the stem-loop splicing hairpin related to the polynucleotide or combination of polynucleotides of the disclosure are derived from the same species. In some embodiments, the IRE1 protein(s) and the stem-loop splicing hairpin related to the polynucleotide or combination of polynucleotides of the disclosure are derived from different species. In some embodiments, the IREl protein(s) related to the chimeric protein kinase and / or RNase domain, and the stem-loop splicing hairpin of the engineered RNA polynucleotide in the cell are derived from a non-human species. In some embodiments, the IREl protein(s) related to the chimeric protein kinase and / or RNase domain are derived from an animal species and the stem-loop splicing hairpin is derived from a plant species. In some embodiments, the IRE1 protein(s) related to the chimeric protein kinase and / or RNase domain are derived from an animal species and the stem-loop splicing hairpin is derived from a fungal species. In some embodiments, the 1RE1 protein(s) related to the chimeric protein kinase and / or RNase domain are derived from a plant species and the stem-loop splicing hairpin is derived from an animal species. In some embodiments, the IREl protein(s) related to the chimeric protein kinase and / or RNase domain are derived from a plant species and the stem-loop splicing hairpin is derived from a fungal species. In some embodiments, the IRE1 protein(s) related to the chimeric protein kinase and / or RNase domain are derived from a fungal species and the stem-loop splicing hairpin is derived from an animal species. In some embodiments, the IREl protein(s) related to the chimeric protein kinase and / or RNase domain are derived from a fungal species and the stem-loop splicing 64

[0568] 326657998 Attorney Docket No.: PTBI-010 / 01WO hairpin is derived from a plant species. In some embodiments, the RNase domain, or the chimeric protein, is capable of excising the cytosolic intron comprised in the stem-loop splicing hairpin (even though in some cases they are derived from different species).

[0569] In some embodiments, the sequence encoding the chimeric protein is codon optimized for human expression. In some embodiments, the sequence encoding the chimeric protein is codon optimized for mammalian expression. In some embodiments, the sequence encoding the chimeric protein is codon optimized for eukaryotic expression. In some embodiments, the kinase domain and / or the RNase domain of the chimeric protein is not derived from a human IRE1 protein, a mammalian IRE1 protein, or a eukaryotic IRE1 protein.

[0570] In some embodiments, the RNA polynucleotide comprises a sequence encoding an initial start codon, a sequence encoding a first stop codon, and a sequence encoding a second start codon. In some embodiments, the initial start codon is 5’ to the sequence of the stem-loop splicing hairpin. In some embodiments, the first stop codon is 3’ to the sequence of the stem-loop splicing hairpin. In some embodiments, the first stop codon is in frame with the initial start codon. In some embodiments, the second start codon is the first start codon 3’ to the sequence of the stem-loop splicing hairpin. In some embodiments, the second start codon is out of frame with the initial start codon.

[0571] In some embodiments, the RNA polynucleotide comprises a nucleotide sequence encoding a protein of interest. In some embodiments, the nucleotide sequence encoding the protein of interest is in frame with the second start codon. In some embodiments, the nucleotide sequence encoding the protein of interest comprises the second start codon as its first codon.

[0572] In some embodiments, the RNA polynucleotide is an engineered RNA polynucleotide described herein.

[0573] In some embodiments, the RNase domain of the chimeric protein is, or is derived from, a plant IRE1 RNase domain, whereas the stem-loop splicing hairpin, cytosolic intron, the 5’ cleavage site and / or the 3’ cleavage site is, or is derived from, a fungal stem-loop splicing hairpin, cytosolic intron, 5’ cleavage site and / or 3’ cleavage site. In some embodiments, the corresponding fungus is a plant-pathogenic fungus. In some embodiments, the kinase domain of the chimeric protein is, or is derived from, the kinase domain of the corresponding plant IRE1 protein.

[0574] In some embodiments, the RNase domain of the chimeric protein is, or is derived from, a fungal IRE1 RNase domain, whereas the stem-loop splicing hairpin, cytosolic intron, the 5’ cleavage site and / or the 3’ cleavage site is, or is derived from, a plant stem-loop

[0575] 65

[0576] 326657998 Attorney Docket No.: PTBI-010 / 01WO splicing hairpin, cytosolic intron, 5’ cleavage site and / or 3’ cleavage site. In some embodiments, the corresponding fungus is a plant-pathogenic fungus. In some embodiments, the kinase domain of the chimeric protein is, or is derived from, the kinase domain of the corresponding fungal IRE1 protein.

[0577] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 15 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 21. In some embodiments, the RNA. polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 59. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 15 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 21. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 59.

[0578] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 15 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 21. In some embodiments, the RNA polynucleotide comprises a. stem-loop splicing hairpin comprising a. nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 60. In some embodiments, the polynucleotide or combination of

[0579] 66

[0580] 326657998 Attorney Docket No.: PTBI-010 / 01WO polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 15 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 21. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 60.

[0581] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 15 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 21. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 61. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 15 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 21. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 61.

[0582] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 15 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 21. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least

[0583] 67

[0584] 326657998 Attorney Docket No.: PTBI-010 / 01WO 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 62. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 15 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 21. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 62.

[0585] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 15 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 21. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 63. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 15 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 21. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 63,

[0586] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 15 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 21. In some

[0587] 68

[0588] 326657998 Attorney Docket No.: PTBI-010 / 01WO embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 64. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 15 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 21. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 64.

[0589] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 15 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 22. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 59. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 15 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 22. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 59.

[0590] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 15 and an RNase domain comprising an amino acid sequence having at least

[0591] 69

[0592] 326657998 Attorney Docket No.: PTBI-010 / 01WO 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 22. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 60. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 15 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 22. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 60.

[0593] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 15 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 22. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 61. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 15 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 22. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 61.

[0594] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at

[0595] 70

[0596] 326657998 Attorney Docket No.: PTBI-010 / 01WO least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 15 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 22. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 62. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 15 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 22. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 62.

[0597] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 15 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 22. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 63, In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 15 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 22. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 63.

[0598] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding

[0599] 71

[0600] 326657998 Attorney Docket No.: PTBI-010 / 01WO an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 15 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 22. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 64. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 15 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 22. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 64.

[0601] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 15 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 23. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 59. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 15 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 23. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 59.

[0602] 72

[0603] 326657998 Attorney Docket No.: PTBI-010 / 01WO In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 15 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 23. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 60. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 15 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 23. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 60.

[0604] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 15 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 23. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 61. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 15 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 23. In

[0605] 326657998 Attorney Docket No.: PTBI-010 / 01WO some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 61.

[0606] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 15 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 23. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 6. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 15 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 23. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 62.

[0607] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 15 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 23. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 63. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase

[0608] 326657998 Attorney Docket No.: PTBI-010 / 01WO domain comprising or consisting of the amino acid sequence of SEQ ID NO: 15 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 23. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 63.

[0609] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 15 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 23. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 64. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 15 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 23. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 64.

[0610] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least. 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 15 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 24. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 59. In some embodiments, the polynucleotide or combination of

[0611] 75

[0612] 326657998 Attorney Docket No.: PTBI-010 / 01WO polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 15 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 24. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 59.

[0613] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 15 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 24. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 60. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 15 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 24. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 60.

[0614] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 15 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 24. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least

[0615] 76

[0616] 326657998 Attorney Docket No.: PTBI-010 / 01WO 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 61. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 15 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 24. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 61.

[0617] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 15 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 24. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 62. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 15 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 24. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 62,

[0618] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 15 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 24. In some

[0619] 326657998 Attorney Docket No.: PTBI-010 / 01WO embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 63. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 15 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 24. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 63.

[0620] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 15 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 24. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 64. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 15 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 24. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 64.

[0621] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 15 and an RNase domain comprising an amino acid sequence having at least

[0622] 78

[0623] 326657998 Attorney Docket No.: PTBI-010 / 01WO 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 25. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 59. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 15 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 25. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 59.

[0624] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 15 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 25. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 60. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 15 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 25. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 60.

[0625] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at

[0626] 79

[0627] 326657998 Attorney Docket No.: PTBI-010 / 01WO least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 15 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 25. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 61. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 15 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 25. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 61.

[0628] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 15 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 25. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 62, In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 15 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 25. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 62.

[0629] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding

[0630] 80

[0631] 326657998 Attorney Docket No.: PTBI-010 / 01WO an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 15 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 25. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 63. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 15 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 25. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 63.

[0632] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 15 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 25. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 64. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 15 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 25. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 64.

[0633] 81

[0634] 326657998 Attorney Docket No.: PTBI-010 / 01WO In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 15 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 26. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 59. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 15 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 26. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 59.

[0635] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 15 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 26. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 60. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 15 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 26. In

[0636] 82

[0637] 326657998 Attorney Docket No.: PTBI-010 / 01WO some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 60.

[0638] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 15 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 26. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 61. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 15 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 26. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 61.

[0639] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 15 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 26. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 62. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase

[0640] 83

[0641] 326657998 Attorney Docket No.: PTBI-010 / 01WO domain comprising or consisting of the amino acid sequence of SEQ ID NO: 15 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 26. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 62.

[0642] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 15 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 26. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 63. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 15 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 26. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 63.

[0643] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least. 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 15 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 26. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 64. In some embodiments, the polynucleotide or combination of

[0644] 84

[0645] 326657998 Attorney Docket No.: PTBI-010 / 01WO polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 15 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 26. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 64.

[0646] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 16 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 22. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 60. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 16 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 22. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 60.

[0647] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 16 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 22. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least

[0648] 85

[0649] 326657998 Attorney Docket No.: PTBI-010 / 01WO 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 61. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 16 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 22. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 61.

[0650] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 16 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 22. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 62. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 16 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 22. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 62,

[0651] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 16 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 22. In some

[0652] 86

[0653] 326657998 Attorney Docket No.: PTBI-010 / 01WO embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 63. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 16 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 22. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 63.

[0654] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 16 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 22. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 64. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 16 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 22. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 64.

[0655] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 16 and an RNase domain comprising an amino acid sequence having at least

[0656] 87

[0657] 326657998 Attorney Docket No.: PTBI-010 / 01WO 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 22. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 59. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 16 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 22. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 59.

[0658] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 16 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 21. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 59. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 16 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 21. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 59.

[0659] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at

[0660] 88

[0661] 326657998 Attorney Docket No.: PTBI-010 / 01WO least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 16 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 21. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 60. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 16 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 21. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 60.

[0662] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 16 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 21. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 61, In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 16 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 21. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 61.

[0663] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding

[0664] 89

[0665] 326657998 Attorney Docket No.: PTBI-010 / 01WO an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 16 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 21. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 62. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 16 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 21. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 62.

[0666] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 16 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 21. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 63. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 16 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 21. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 63.

[0667] 90

[0668] 326657998 Attorney Docket No.: PTBI-010 / 01WO In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 16 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 21. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 64. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 16 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 21. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 64.

[0669] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 16 and an RNase domain comprising an amino acid sequence having at least.

[0670] 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 23. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 59. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 16 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 23. In

[0671] 91

[0672] 326657998 Attorney Docket No.: PTBI-010 / 01WO some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 59.

[0673] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 16 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 23. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 60. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 16 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 23. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 60.

[0674] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 16 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 23. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 61. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase

[0675] 92

[0676] 326657998 Attorney Docket No.: PTBI-010 / 01WO domain comprising or consisting of the amino acid sequence of SEQ ID NO: 16 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 23. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 61.

[0677] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 16 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 23. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 62. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 16 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 23. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 62.

[0678] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least. 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 16 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 23. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 63. In some embodiments, the polynucleotide or combination of

[0679] 93

[0680] 326657998 Attorney Docket No.: PTBI-010 / 01WO polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 16 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 23. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 63.

[0681] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 16 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 23. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 64. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 16 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 23. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 64.

[0682] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 16 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 24. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least

[0683] 94

[0684] 326657998 Attorney Docket No.: PTBI-010 / 01WO 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 59. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 16 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 24. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 59.

[0685] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 16 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 24. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 60. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 16 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 24. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 60,

[0686] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 16 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 24. In some

[0687] 95

[0688] 326657998 Attorney Docket No.: PTBI-010 / 01WO embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 61. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 16 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 24. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 61.

[0689] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 16 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 24. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 62. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 16 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 24. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 62.

[0690] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 16 and an RNase domain comprising an amino acid sequence having at least

[0691] 96

[0692] 326657998 Attorney Docket No.: PTBI-010 / 01WO 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 24. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 63. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 16 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 24. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 63.

[0693] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 16 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 24. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 64. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 16 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 24. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 64.

[0694] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at

[0695] 97

[0696] 326657998 Attorney Docket No.: PTBI-010 / 01WO least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 16 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 25. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 59. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 16 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 25. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 59.

[0697] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 16 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 25. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 60, In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 16 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 25. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 60.

[0698] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding

[0699] 98

[0700] 326657998 Attorney Docket No.: PTBI-010 / 01WO an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 16 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 25. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 61. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 16 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 25. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 61.

[0701] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 16 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 25. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 62. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 16 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 25. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 62.

[0702] 99

[0703] 326657998 Attorney Docket No.: PTBI-010 / 01WO In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 16 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 25. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 63. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 16 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 25. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 63.

[0704] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 16 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 25. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 64. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 16 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 25. In

[0705] 100

[0706] 326657998 Attorney Docket No.: PTBI-010 / 01WO some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 64.

[0707] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 16 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 26. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 59. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 16 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 26. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 59.

[0708] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 16 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 26. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 60. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase

[0709] 101

[0710] 326657998 Attorney Docket No.: PTBI-010 / 01WO domain comprising or consisting of the amino acid sequence of SEQ ID NO: 16 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 26. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 60.

[0711] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 16 and an RNase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 26. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 61. In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 16 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 26. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 61.

[0712] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least. 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 16 and an RNase domain comprising an amino acid sequence having at least.

[0713] 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 26. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising a nucleotide sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity to the sequence of SEQ ID NO: 62. In some embodiments, the polynucleotide or combination of

[0714] 102

[0715] 326657998 Attorney Docket No.: PTBI-010 / 01WO polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 16 and an RNase domain comprising or consisting of the amino acid sequence of SEQ ID NO: 26. In some embodiments, the RNA polynucleotide comprises a stem-loop splicing hairpin comprising or consisting of the nucleotide sequence of SEQ ID NO: 62.

[0716] In some embodiments, the polynucleotide or combination of polynucleotides comprise a sequence encoding a chimeric protein described herein and a sequence encoding an RNA polynucleotide, wherein the chimeric protein comprises a kinase domain comprising an amino acid sequence having at least 80% (e.g., at least 80% at least 85%, at least 90%, at l...

Claims

Attorney Docket No.: PTBI-010 / 01 WOCLAIMSWhat is cla med is:

1. A chimeric protein comprising,(i) a ligand binding domain, wherein the ligand binding domain is not a ligand binding domain of an inositol-requiring enzyme 1 (IRE1) protein;(ii) a kinase domain of an IR. E1 protein or a kinase domain derived from an IRE1 protein; and(iii) an RNase domain of an IREl protein or an RNase domain derived from an IREi protein;optionally, the chimeric protein comprises N-terminally to C-terminally: (i), (ii), and (iii).

2. The chimeric protein of claim 1, wherein the ligand binding domain is capable of dimerization, trimerization, or multimerization in the presence of the ligand.

3. The chimeric protein of any one of claims 1-2, wherein the ligand comprises a small molecule, a peptide, a protein, a lipid, a carbohydrate, or any combination thereof.

4. The chimeric protein of claim 3, wherein the ligand is a small molecule.

5. The chimeric protein of any one of claims 1-4, wherein the ligand is a cancer biomarker or a cancer-associated antigen.

6. The chimeric protein of any one of claims 1-5, wherein the ligand is a cell-surface ligand or a soluble ligand.

7. The chimeric protein of any one of claims 1-6, wherein the chimeric protein forms a dimer, a trimer, or a multimer, in the presence of the ligand.

8. The chimeric protein of any one of claims 1-7, wherein the chimeric protein is a monomeric protein in the absence of the ligand.256326657998Attorney Docket No.: PTBI-010 / 01 WO9. The chimeric protein of any one of claims 1-8, wherein the chimeric protein is a plasma membrane protein or a cytosolic protein.

10. The chimeric protein of any one of claims 1-9, wherein the kinase domain comprises a catalytic loop and an activation loop, wherein the catalytic loop comprises an HRD motif and / or wherein the activation loop comprises a DFG motif or a DMG motif.

11. The chimeric protein of any one of claims 1-10, wherein the kinase domain comprises the amino acid sequence of X1LHX2LX3IX4HRDX5KPX6NX7LX8X9 (SEQ ID NO: 53), where Xi is H or Y; X2 is E or S; X is G, N, K, or R; X4 is V or I; Xs is L or I; Xe is Q or H; X7 is E or I; Xs is I or V; and X9 is S, A, or N.

12. The chimeric protein of any one of claims 1-11, wherein the kinase domain comprises the amino acid sequence of X1X2X3X4SDX5GX6X7KX8X9X10 (SEQ ID NO: 54), where Xi is S, K, or R; X2 is A, I, or L; X3 is K, M, or L; X4 is L, I, or V; X5 is M or F; XG is I or L; X7 is S or C, Xs is R or K; X9 is M or L, and X10 is S, A D, or E.

13. The chimeric protein of any one of claims 1-12, wherein the kinase domain comprises an amino acid sequence having at least 90% identity to the sequence of any one of SEQ ID NOs: 15-20.

14. The chimeric protein of any one of claims 1-13, wherein the RNase domain comprises one or more catalytic residues corresponding to Y60, N74, K75, and H78 relative to SEQ ID NO: 21.

15. The chimeric protein of any one of claims 1-14, wherein the RNase domain comprises residues Y60, N74, K75, and H78 relative to SEQ ID NO: 21.

16. The chimeric protein of any one of claims 1-15, wherein the RNase domain comprises the amino acid sequence of RX1YX2X3X4X5X6X7DLLRX8X9RNKX10X11HX12 (SEQ ID NO: 55), where Xi is T, R, or K; X2 is K, T, S, or H; X3 is G, Y, P, or S; X4is G, D, N, S, or E; X5is S, R, or K; XG is V, I, M, or L; X7 is R, L, or M; Xs is A or V; X9 is M, I, or L; Xio is K, L, or Y; Xu is H or N; and X12 is Y, H, or F.257326657998Attorney Docket No.: PTBI-010 / 01 WO17. The chimeric protein of any one of claims 1-16, wherein the RNase domain comprises an amino acid sequence having at least 90% identity to the sequence of any one of SEQ ID NOs: 21-26.

18. The chimeric protein of any one of claims 1-17, wherein the ligand binding domain lacks a dimerization domain.

19. The chimeric protein of any one of claims 1-18, wherein the ligand binding domain comprises an antibody.

20. The chimeric protein of claim 19, wherein the antibody is a single-chain variable fragment (scFv) or a nanobody.

21. The chimeric protein of claim 19, wherein the antibody is a bispecific antibody.

22. The chimeric protein of any one of claims 19-21, wherein the antibody comprises 1, 2, or more than 2, immunoglobulin single variable domains.

23. The chimeric protein of any one of claims 19-22, wherein the antibody comprises a first immunoglobulin single variable domain that binds to a first epitope of the ligand and a second immunoglobulin single variable domain that binds to a second epitope of the ligand.

24. The chimeric protein of any one of claims 1-17, wherein the ligand binding domain comprises a dimerization domain.

25. The chimeric protein of any one of claims 1-17 and 24, wherein the ligand binding domain comprises a ligand binding domain of a FK506-binding protein (FKBP), a ligand binding domain of a tumor necrosis factor (TNF) receptor superfamily protein, a ligand binding domain of an interleukin-1 receptor (IL-1R) protein, a ligand binding domain of an interleukin- 1 receptor accessory protein (IL-lRAcP), or a combination thereof.

26. The chimeric protein of any one of claims 1-25, wherein the ligand binding domain is an extracellular ligand binding domain.258326657998Attorney Docket No.: PTBI-010 / 01 WO27. The chimeric protein of any one of claims 1 -26, wherein the ligand binding domain comprises an amino acid sequence having at least 90% identity to the sequence of any one of SEQ ID NOs: 8-14.

28. The chimeric protein of any one of claims 1-27, wherein the chimeric protein further comprises a transmembrane domain, optionally wherein the chimeric protein comprises, N- terminally to C-terminally: the ligand binding domain; the transmembrane domain; the kinase domain; and the RNase domain.

29. The chimeric protein of claim 28, wherein the transmembrane domain comprises an amino acid sequence having at least 90% identity to the amino acid sequence of SEQ ID NO: 27.

30. The chimeric protein of any one of claims 1-29, wherein the IRE1 protein of (ii) and the IRE1 protein of (iii) are the same.

31. The chimeric protein of any one of claims 1-30, wherein the IRE1 protein of (ii) and / or the IRE1 protein of (iii) are not human IRE1 protein, not a mammalian IRE1 protein, or not a eukaryotic IRE1 protein.

32. The chimeric protein of any one of claims 1-31, wherein the RNase domain, or the chimeric protein, iis capable of excising a cytosolic intron comprised in a stem-loop splicing hairpin of an RNA molecule, optionally, the cytosolic intron or the stem-loop splicing hairpin is derived from an RNA molecule encoding a x-box binding protein 1 (XBP1).

33. The chimeric protein of any one of claims 1-32, wherein the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of any one of SEQ ID NOs: 28-43 or 56.

34. A polynucleotide comprising a sequence encoding the chimeric protein of any one of claims 1-33.

35. The polynucleotide of claim 34, wherein the polynucleotide is an RNA.259326657998Attorney Docket No.: PTBI-010 / 01 WO36. The polynucleotide of claim 34 or 35, wherein the IRE1 protein of (ii) and / or the IREl protein of (iii) are not human IREl protein, not a mammalian IREl protein, or not a eukaryotic protein, but the sequence encoding the chimeric protein is codon optimized for human, mammalian, or eukaryotic expression.

37. An engineered RNA polynucleotide comprising a sequence of a stem-loop splicing hairpin comprising a cytosolic intron, wherein the cytosolic intron is capable of being excised by an RNase domain of an inositol-requiring enzyme 1 (IREl) protein,optionally, the engineered RNA polynucleotide comprises a sequence encoding an initial start codon, optionally, the initial start codon is 5’ to the sequence of the stem-loop splicing hairpin;optionally, the engineered RNA polynucleotide does not encode a x-box binding protein 1 (XBP1).

38. The engineered RNA polynucleotide of claim 37, wherein the engineered RNA polynucleotide encodes a protein of interest that is not, or does not comprise, a x-box binding protein 1 (XBP1).

39. The engineered RNA polynucleotide of claim 38, wherein:(i) the engineered RNA polynucleotide expresses the protein of interest after the cytosolic intron is excised, but does not express the protein of interest when the cytosolic intron is not excised;(ii) the engineered RNA polynucleotide does not express the protein of interest after the cytosolic intron is excised, but expresses the protein of interest when the cytosolic intron is not excised; or(iii) after the cytosolic intron is excised, the engineered RNA polynucleotide expresses a protein of interest that is different from the protein of interest expressed by the engineered RNA polynucleotide when the cytosolic intron is not excised.

40. The engineered RNA polynucleotide of any one of claims 37-39, wherein at least a part of the nucleotide sequence encoding the protein of interest is out. of frame with the initial start codon when the cytosolic intron is not excised; optionally, the entire nucleotide sequence260326657998Attorney Docket No.: PTBI-010 / 01 WOencoding the protein of interest is out of frame with the initial start codon when the cytosolic intron is not excised,41. The engineered RNA polynucleotide of any one of claims 37-40, comprising a second start codon, wherein the second start codon is:(i) 3’ to the sequence of the initial start codon,(ii) 3’ to the cytosolic intron, and / or(iii) out of frame with the initial start codon.

42. The engineered RNA polynucleotide of claim 41, wherein the nucleotide sequence encoding the protein of interest is in frame with the second start codon, optionally wherein the nucleotide sequence encoding the protein of interest comprises the second start codon as its first codon.

43. The engineered RNA polynucleotide of any one of claims 37-42, comprising a first stop codon, wherein the first stop codon is 3’ to the sequence of the stem-loop splicing hairpin, and wherein the first stop codon is in frame with the initial start codon; optionally, wherein the first stop codon is 5’ to the nucleotide sequence encoding the protein of interest.

44. The engineered RNA polynucleotide of any one of claims 37-43, comprising a sequence encoding a cleavage peptide; optionally, the sequence encoding the cleavage peptide is 5’ to, and in frame with, the sequence encoding the protein of interest.

45. The engineered RNA polynucleotide of claim 44, wherein the sequence encoding the cleavage peptide is 3’ to the sequence encoding the second start codon, and wherein the sequence encoding the cleavage peptide is in frame with the sequence encoding the second start codon.

46. The engineered RNA polynucleotide of claim 44 or 45, wherein the cleavage peptide is a self-cleaving peptide; optionally, the self-cleaving peptide is a viral 2A peptide.

47. An engineered RNA polynucleotide comprising:261326657998Attorney Docket No.: PTBI-010 / 01 WO(i) a sequence of a stem-loop splicing hairpin comprising a cytosolic intron, wherein the cytosolic intron is capable of excision by an RNase domain of an inositol- requiring enzyme 1 (IRE1) protein;(ii) a sequence encoding an initial start codon, wherein the initial start codon is 5’ to the sequence of the stem-loop splicing hairpin;(iii) a sequence encoding a first stop codon, wherein the first stop codon is 3' to the sequence of the stem-loop splicing hairpin, and wherein the first stop codon is in frame with the initial start codon;(iv) a sequence encoding a second start codon, wherein the second start, codon is 3’ to the sequence of the initial start codon, optionally 3’ to the cytosolic intron, and wherein the second start, codon is out of frame with the initial start codon; and(v) a sequence encoding a viral 2A peptide, wherein the sequence encoding the viral 2 A peptide is 3’ to the sequence encoding the second start, codon, and wherein the sequence encoding the viral 2A peptide is in frame with the sequence encoding the second start codon.

48. An engineered RNA polynucleotide comprising:(i) a sequence of a stem-loop splicing hairpin comprising a cytosolic intron, wherein the stem-loop splicing hairpin comprises a first portion comprising a sequence having at least 90% identity to the sequence of SEQ ID NO: 57 and a second portion comprising a sequence having at least 90% identity to the sequence of SEQ ID NO: 58, wherein the first nucleotide sequence and the second nucleotide sequence are complementary;(ii) a sequence encoding an initial start codon, wherein the initial start, codon is 5’ to the sequence of the stem-loop splicing hairpin;(iii) a sequence encoding a first stop codon, wherein the first stop codon is 3’ to the sequence of the stem-loop splicing hairpin, and wherein the start codon is in frame with the initial start codon; and(iv) a sequence encoding a second start codon, wherein the second start codon is 3’ to the sequence of the initial start codon, optionally 3’ to the cytosolic intron, and wherein the second start codon is out of frame with the initial start codon;optionally wherein the engineered RNA polynucleotide further comprises:(v) a sequence encoding a viral 2A peptide, wherein the sequence encoding the viral 2A peptide is 3’ to the sequence encoding the second start codon, and wherein the262326657998Attorney Docket No.: PTBI-010 / 01 WOsequence encoding the viral 2A peptide is in frame with the sequence encoding the second start codon.

49. The engineered RNA polynucleotide of any one of claims 37-48, wherein the stem-loop splicing hairpin comprises the nucleotide sequence of X1X2GCX3GX4X5X6T (SEQ ID NO: 73), wherein Xi is C or T; X2 is C, T, or A; X3 is C, T, or A; X4 is T, A, or C; X5 is A or G; and Xe is C, A, or G; optionally, wherein the nucleotide sequence comprises an IRE1 cleavage site.

50. The engineered RNA polynucleotide of any one of claims 37-49, wherein the stem-loop splicing hairpin comprises the nucleotide sequence of X1CX2GX3X4GX5X6X7 (SEQ ID NO: 74), where Xi is T or C; X2 is A, T, or C; X3 is C or A; X4 is T or A; X5 is absent, T, or A; Xe is absent, C or T; and X7 is G or A; optionally, wherein the nucleotide sequence comprises an IRE1 cleavage site.

51. An engineered RNA polynucleotide comprising:(i) a sequence of a stem-loop splicing hairpin comprising a cytosolic intron, wherein the stem-loop splicing hairpin comprises the nucleotide sequence of X1X2GCX3GX4X5XT (SEQ ID NO: 73), wherein Xi is C or T; X2is C, T, or A; X? is C, T, or A; X is T, A, or C, X5 is A or G; and Xe is C, A, or G and / or the nucleotide sequence of X1CX2GX3X4GX5X6X7 (SEQ ID NO: 74), where Xi is T or C; X2 is A, T, or C; X3 is C or A; Xr is T or A; X? is absent, T, or A; Xs is absent, C or T; and X7 is G or A;(ii) a sequence encoding an initial start codon, wherein the initial start codon is 5’ to the sequence of the stem-loop splicing hairpin;(iii) a sequence encoding a first stop codon, wherein the first stop codon is 3’ to the sequence of the stern-loop splicing hairpin, and wherein the start codon is in frame with the initial start codon;(iv) a sequence encoding a second start codon, wherein the second start codon is 3’ to the sequence of the initial start codon, optionally 3’ to the cytosolic intron, and wherein the second start codon is out of frame with the initial start codon; and(v) a sequence encoding a viral 2A peptide, w'herein the sequence encoding the viral 2A peptide is 3’ to the sequence encoding the second start codon, and wherein the sequence encoding the viral 2A peptide is in frame with the sequence encoding the second start codon.263326657998Attorney Docket No.: PTBI-010 / 01 WO52. The engineered RNA polynucleotide of any one of claims 37-51, wherein the stem-loop splicing hairpin comprises the structure, from 5’ to 3’: a 5’ stem 1 region, a 5’ stem 2 region, a 5’ loop region, a 3’ stem 2 region, a 5’ stem 3 region, a 3’ loop region, a 3’ stem 3 region, and a 3’ stem 1 region, wherein the RNA bases of the 5’ stem 1 region are complementary to the RNA bases of the 3’ stem 1 region, the RNA bases of the 5’ stem 2 region are complementary to the RNA bases of the 3’ stem 2 region, and the RNA bases of the 5’ stem 3 region are complementary to the RNA bases of the 3' stem 3 region.

53. The engineered RNA polynucleotide of any one of claims 37-52, wherein the cytosolic intron comprises a nucleotide sequence having at least 90% identity to the sequence of any one of SEQ ID NOs: 65-70.

54. The engineered RNA polynucleotide of any one of claims 37-53, wherein the stem-loop splicing hairpin comprises a nucleotide sequence having at least 90% identity to the sequence of any one of SEQ ID NOs: 59-64.

55. The engineered RNA polynucleotide of any one of claims 37-54, wherein the stem-loop splicing hairpin comprises a first portion comprising a sequence having at least 90% identity to SEQ ID NO: 57 and a second portion comprising a sequence having at least 90% identity to SEQ ID NO: 58, wherein the first portion and the second portion are complementary.

56. The engineered RNA polynucleotide of any one of claims 46-55, wherein the viral 2A peptide comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 75-78.

57. The engineered RNA polynucleotide of any one of claims 37-56, wherein the engineered RNA polynucleotide does not encode either a XBP1 protein or a functional fragment thereof, or a fusion protein comprising the XBP1 protein or functional fragment thereof.

58. The engineered RNA polynucleotide of any one of claims 37-57, wherein the engineered RNA further comprises a nucleotide sequence encoding a protein of interest,264326657998Attorney Docket No.: PTBI-010 / 01 WOwherein the nucleotide sequence encoding the protein of interest is in frame with the second start codon, optionally wherein the nucleotide sequence encoding the protein of interest comprises the second start codon as its first codon,59. The engineered RNA polynucleotide of any one of claims 37-58, wherein the protein of interest is a therapeutic protein; optionally, the therapeutic protein is an antibody (e.g., scFv), a Fc fusion protein, a hormone, a cytokine, a chemokine, a cytotoxin, an enzyme catalyzing a prodrug activation, a transcription factor, a transactivator, an immunomodulatory protein, or a DNA-modi tying enzyme.

60. The engineered RNA polynucleotide of any one of claims 37, 49-50, 52-55, and 57, wherein the engineered RNA polynucleotide is a non-coding RNA, and wherein:(i) the engineered RNA polynucleotide is converted from a non-functional non¬ coding RNA to a functional non-coding RNA upon excision of the cytosolic intron;(ii) the engineered RNA polynucleotide is converted from a functional non-coding RNA to a non-functional non-coding RNA upon excision of the cytosolic intron, or(iii) the function of the non-coding RNA is altered after the cytosolic intron is excised.

61. The engineered RNA polynucleotide of any one of claims 37-60, wherein the stem-loop splicing hairpin (including or excluding a cytosolic intron) is not derived from a human stern-loop splicing hairpin, a mammalian stem-loop splicing hairpin, or a eukaryotic stem-loop splicing hairpin.

62. A recombinant DNA polynucleotide encoding the engineered RNA polynucleotide of any one of claims 37-61.

63. A polynucleotide comprising, or a combination of polynucleotides collectively comprising, a sequence encoding the chimeric protein of any one of claims 1-33 and a sequence encoding an RNA comprising a sequence of a stem-loop splicing hairpin comprising a cytosolic intron, wherein the cytosolic intron is capable of excision by the RNase domain of the chimeric protein;265326657998Attorney Docket No.: PTBI-010 / 01 WOoptionally, wherein the RNA is the engineered RNA polynucleotide of any one of claims 37-61.

64. The polynucleotide or combination of polynucleotides of claim 63, wherein the stem-loop splicing hairpin comprises the structure, from 5’ to 3’: a 5’ stem 1 region, a 5’ stem 2 region, a 5’ loop region, a 3’ stem 2 region, a 5’ stem 3 region, a 3’ loop region, a 3’ stem 3 region, and a 3’ stem 1 region, wherein the RNA bases of the 5’ stem 1 region are complementary to the RNA bases of the 3’ stem 1 region, the RNA bases of the 5’ stem 2 region are complementary to the RNA bases of the 3’ stem 2 region, and the RNA bases of the 5’ stem 3 region are complementary to the RNA bases of the 3’ stem 3 region.

65. The polynucleotide or combination of polynucleotides of claim 63 or claim 64, wherein the RNA further comprises:a sequence encoding an initial start codon, wherein the initial start codon is 5’ to the sequence of the stem-loop splicing hairpin;a sequence encoding a first stop codon, wherein the first stop codon is 3’ to the sequence of the stem-loop splicing hairpin, and wherein the first stop codon is in frame with the initial start codon; anda sequence encoding a second start codon, wherein the second start codon is 3’ to the sequence initial start codon, optionally 3’ to the cytosolic intron, and wherein the second start codon is out of frame with the initial start codon.

66. The polynucleotide or combination of polynucleotides of claim 65, wherein the RNA further comprises a nucleotide sequence encoding a protein of interest, wherein the nucleotide sequence encoding the protein of interest is in frame with the second start codon, optionally wherein the nucleotide sequence encoding the protein of interest comprises the second start codon as its first codon.

67. The polynucleotide or combination of polynucleotides of any one of claims 63-66, wherein the IRE1 protein and the stem-loop splicing hairpin are from different species.

68. The polynucleotide or combination of polynucleotides of any one of claims 63-67, wherein the RNase domain, or the chimeric protein, is capable of excising the cytosolic intron comprised in the stem-loop splicing hairpin.266326657998Attorney Docket No.: PTBI-010 / 01 WO69. The polynucleotide or combination of polynucleotides of any one of claims 63-68, wherein the RNA is an RNA polynucleotide according to any one of claims 37-61.

70. The polynucleotide or combination of polynucleotides of any one of claims 63-69, wherein:a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 19; andb) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 25;and optionally wherein:c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 90% identity to the sequence of SEQ ID NO: 63.

71. The polynucleotide or combination of polynucleotides of any one of claims 63-69, wherein:a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 19; andb) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 25;and optionally wherein:c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 90% identity to the sequence of SEQ ID NO: 64.

72. The polynucleotide or combination of polynucleotides of any one of claims 63-69, wherein:a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 19; andb) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 25;and optionally wherein:c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 90% identity to the sequence of SEQ ID NO: 60.267326657998Attorney Docket No.: PTBI-010 / 01 WO73. The polynucleotide or combination of polynucleotides of any one of claims 63-69, wherein:a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 19; andb) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 25;and optionally wherein:c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 90% identity to the sequence of SEQ ID NO: 62.

74. The polynucleotide or combination of polynucleotides of any one of claims 63-69, wherein:a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 19; andb) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 25;and optionally wherein:c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 90% identity to the sequence of SEQ ID NO: 61.

75. The polynucleotide or combination of polynucleotides of any one of claims 63-69, wherein:a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 20; andb) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 26;and optionally wherein:c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 90% identity to the sequence of SEQ ID NO: 63.

76. The polynucleotide or combination of polynucleotides of any one of claims 63-69, wherein:a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 20; and268326657998Attorney Docket No.: PTBI-010 / 01 WOb) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 26;and optionally wherein:c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 90% identity to the sequence of SEQ ID NO: 64.

77. The polynucleotide or combination of polynucleotides of any one of claims 63-69, wherein:a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 20; andb) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 26;and optionally wherein:c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 90% identity to the sequence of SEQ ID NO: 60.

78. The polynucleotide or combination of polynucleotides of any one of claims 63-69, wherein:a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 20; andb) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 26;and optionally wherein:c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 90% identity to the sequence of SEQ ID NO: 62.

79. The polynucleotide or combination of polynucleotides of any one of claims 63-69, wherein:a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 20; andb) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 26;and optionally wherein:269326657998Attorney Docket No.: PTBI-010 / 01 WOc) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 90% identity to the sequence of SEQ ID NO: 61.

80. The polynucleotide or combination of polynucleotides of any one of claims 63-69, wherein:a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 16; andb) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 22;and optionally wherein:c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 90% identity to the sequence of SEQ ID NO: 63.

81. The polynucleotide or combination of polynucleotides of any one of claims 63-69, wherein:a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 16; andb) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 22;and optionally wherein:c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 90% identity to the sequence of SEQ ID NO: 64.

82. The polynucleotide or combination of polynucleotides of any one of claims 63-69, wherein:a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 16; andb) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 22;and optionally wherein:c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 90% identity to the sequence of SEQ ID NO: 60.270326657998Attorney Docket No.: PTBI-010 / 01 WO83. The polynucleotide or combination of polynucleotides of any one of claims 63-69, wherein:a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 18; andb) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 24;and optionally wherein:c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 90% identity to the sequence of SEQ ID NO: 62.

84. The polynucleotide or combination of polynucleotides of any one of claims 63-69, wherein:a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 17; andb) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 23;and optionally wherein:c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 90% identity to the sequence of SEQ ID NO: 63.

85. The polynucleotide or combination of polynucleotides of any one of claims 63-69, wherein:a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 17; andb) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 23;and optionally wherein:c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 90% identity to the sequence of SEQ ID NO: 64.

86. The polynucleotide or combination of polynucleotides of any one of claims 63-69, wherein:a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 17; and271326657998Attorney Docket No.: PTBI-010 / 01 WOb) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 23;and optionally wherein:c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 90% identity to the sequence of SEQ ID NO: 60.

87. The polynucleotide or combination of polynucleotides of any one of claims 63-69, wherein:a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 17; andb) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 23;and optionally wherein:c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 90% identity to the sequence of SEQ ID NO: 62.

88. The polynucleotide or combination of polynucleotides of any one of claims 63-69, wherein:a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 17; andb) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 23;and optionally wherein:c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 90% identity to the sequence of SEQ ID NO: 61.

89. The polynucleotide or combination of polynucleotides of any one of claims 63-88, wherein a single polynucleotide comprises the sequence encoding the chimeric protein and the sequence encoding the RNA comprising the stem-loop splicing hairpin.

90. The polynucleotide or combination of polynucleotides of claim 89, wherein the single polynucleotide is a DNA polynucleotide, optionally wherein the DNA polynucleotide is a DNA plasmid or a viral vector.272326657998Attorney Docket No.: PTBI-010 / 01 WO91. The polynucleotide or combination of polynucleotides of claim 89, wherein the single polynucleotide is an RNA polynucleotide.

92. The polynucleotide or combination of polynucleotides of any one of claims 63-88, wherein a first polynucleotide comprises the sequence encoding the chimeric protein and a second polynucleotide comprises the sequence encoding the RNA comprising the sequence of a stem-loop splicing hairpin.

93. The polynucleotide or combination of polynucleotides of claim 92, wherein the first polynucleotide is an RNA and / or the second polynucleotide is an RNA.

94. A therapeutic composition comprising the polynucleotide or combination of polynucleotides of any one of claims 63-93 and one or more pharmaceutically acceptable excipients.

95. A viral particle comprising the polynucleotide or combination of polynucleotides of any one of claims 63-93, optionally wherein the viral particle is an adeno-associated (AAV) particle, an adenovirus particle, or a lentiviral particle.

96. A cell comprising the chimeric protein of any one of claims 1-33.

97. A cell comprising the engineered RNA polynucleotide of any one of claims 37-61.

98. A cell comprising the polynucleotide or combination of polynucleotides of any one of claims 63-69.

99. The cell of any one of claims 96-98, wherein the chimeric protein is expressed in the cytosol of the cell.

100. The cell of any one of claims 96-98, wherein the chimeric protein is expressed on the plasma membrane of the cell.

101. The cell of any one of claims 96-100, wherein the cell is a eukaryotic cell.273326657998Attorney Docket No.: PTBI-010 / 01 WO102. The cell of any one of claims 96-101, wherein the cell is a mammalian cell.

103. The cell of any one of claims 97-102, wherein, in the presence of saturating level of the ligand, the cell expresses the protein of interest, or the non-coding RNA, at a level that is at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50- fold, at least 70-fold, at least 100-fold, at least 200-fold, or at least 300-fold, higher or lower than the level of the protein of interest, or the non-coding RNA, expressed by the cell in the absence of the ligand.

104. The cell of any one of claims 96-103, wherein:(a) the IRE1 protein of (i) and / or the IRE1 protein of (ii) are derived from a species different from the cell;(b) the stem-loop splicing hairpin is derived from a species different from the cell, and / or(c) the species which the IRE1 protein of (i) and / or the IRE1 protein of (ii) are derived from is different from the species which the stem-loop splicing hairpin is derived from.

105. The cell of any one of claims 96-104, wherein:(a) the RNase domain, or the chimeric protein, is not capable of excising an endogenous cytosolic intron of an RNA molecule encoding an endogenous XBP1 protein of the cell; and / or(b) an endogenous IRE1 protein of the cell is not capable of excising the cytosolic- intron of the engineered RNA polynucleotide.

106. A method of inducing expression of a protein of interest in a cell, the cell comprising: a plurality of chimeric proteins according to any one of claims 1-33; and an RNA polynucleotide comprising a sequence of a stem-loop splicing hairpin comprising a cytosolic intron, wherein the cytosolic intron is capable of excision by the RNase domain of the chimeric protein, the method comprising exposing the plurality of chimeric proteins to ligands corresponding to their ligand binding domains, thereby promoting activation of the kinase domains and RNase domains of the chimeric proteins and promoting excision of the cytosolic intron of the RNA polynucleotide.

107. The method of claim 106, wherein the RNA polynucleotide comprises:274326657998Attorney Docket No.: PTBI-010 / 01 WO(i) a sequence of a stem-loop splicing hairpin comprising a cytosolic intron, wherein the cytosolic intron is capable of excision by the RNase domains of the chimeric proteins;(ii) a sequence encoding an initial start codon, wherein the initial start codon is 5’ to the sequence of the stem-loop splicing hairpin;(iii) a sequence encoding a first stop codon, wherein the first stop codon is 3' to the sequence of the stem-loop splicing hairpin, and wherein the first stop codon is in frame with the initial start codon;(iv) a sequence encoding a second start codon, wherein the second start, codon is the first start codon 3’ to the sequence of the stem-loop splicing hairpin, and wherein the second start, codon is out of frame with the initial start codon; and(v) a sequence encoding the protein of interest, wherein the nucleotide sequence encoding the protein of interest is in frame with the second start codon, optionally wherein the nucleotide sequence encoding the protein of interest comprises the second start codon as its first codon.

108. A method of altering expression of a protein of interest in a cell, the cell comprising: a plurality of chimeric proteins according to any one of claims 1-33; and an RNA polynucleotide comprising a sequence of a stem-loop splicing hairpin comprising a cytosolic intron, wherein the cytosolic intron is capable of excision by the RNase domain of the chimeric protein, the method comprising exposing the plurality of chimeric proteins to ligands corresponding to their ligand binding domains, thereby promoting activation of the kinase domains and RNase domains of the chimeric proteins and promoting excision of the cytosolic intron of the RNA polynucleotide.

109. The method of claim 108, wherein the RNA polynucleotide comprises:(i) a sequence of a stem-loop splicing hairpin comprising a cytosolic intron, wherein the cytosolic intron is capable of excision by the RNase domains of the chimeric proteins;(ii) a sequence encoding an initial start codon, wherein the initial start codon is 5’ to the sequence of the stem-loop splicing hairpin;(iii) a sequence encoding a first stop codon, wherein the first stop codon is 3' to the sequence of the stem-loop splicing hairpin, and wherein the first, stop codon is in frame with the initial start codon;275326657998Attorney Docket No.: PTBI-010 / 01 WO(iv) a sequence encoding a second start codon, wherein the second start codon is the first start codon 3’ to the sequence of the stem-loop splicing hairpin, and wherein the second start codon is out of frame with the initial start codon; and(v) a sequence encoding the protein of interest, wherein the nucleotide sequence encoding the protein of interest is in frame with the second start codon, optionally wherein the nucleotide sequence encoding the protein of interest comprises the second start codon as its first codon.

110. The method of any one of claims 106-109, comprising delivering the polynucleotide or combination of polynucleotides of any one of claims 63-93 to the cell.

111. The method of any one of claims 106-110, wherein the cell is a eukaryotic cell.

112. The method of any one of claims 106-111, wherein the cell is a mammalian cell.

113. The method of any one of claims 106-112, wherein the stem-loop splicing hairpin comprises the nucleotide sequence of X1X2GCX3GX4X5X6T (SEQ ID NO: 73), wherein X1is C or T; X2is C, T, or A; X3is C, T, or A; X4is T, A, or C; X5is A or G; and X6is C, A, or G.

114. The method of any one of claims 106-113, wherein the stem-loop splicing hairpin comprises the nucleotide sequence of X1CX2GX3X4GX5X6X7(SEQ ID NO: 74), where X1is T or C; X2is A, T, or C; X3is C or A; X4is T or A; X5is absent, T, or A; X6is absent, C or T, and X7is G or A.

115. The method of any one of claims 106-114, wherein the cytosolic intron comprises a nucleotide sequence having at least 90% identity to the sequence of any one of SEQ ID NOs: 65-70,116. The method of any one of claims 106-115, wherein the stem-loop splicing hairpin comprises a nucleotide sequence having at least 90% identity to the sequence of any one of SEQ ID NOs: 59-64,117. The method of any one of claims 106-116, wherein:276326657998Attorney Docket No.: PTBI-010 / 01 WOa) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 19; andb) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 25;and optionally wherein:c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 90% identity to the sequence of SEQ ID NO: 63.

118. The method of any one of claims 106- 116, wherein:a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 19; andb) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 25;and optionally wherein:c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 90% identity to the sequence of SEQ ID NO: 64.

119. The method of any one of claims 106-116, wherein:a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 19; andb) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 25;and optionally wherein:c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 90% identity to the sequence of SEQ ID NO: 60.

120. The method of any one of claims 106-116, wherein:a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 19; andb) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 25;and optionally wherein:c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 90% identity to the sequence of SEQ ID NO: 62.277326657998Attorney Docket No.: PTBI-010 / 01 WO121. The method of any one of claims 106- 116, wherein:a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 19; andb) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 25;and optionally wherein:c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 90% identity to the sequence of SEQ ID NO: 61.

122. The method of any one of claims 106-116, wherein:a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 20; andb) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 26;and optionally wherein:c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 90% identity to the sequence of SEQ ID NO: 63.

123. The method of any one of claims 106-116, wherein:a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 20; andb) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 26;and optionally wherein:c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 90% identity to the sequence of SEQ ID NO: 64.

124. The method of any one of claims 106-116, wherein:a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 20; andb) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 26;and optionally wherein:278326657998Attorney Docket No.: PTBI-010 / 01 WOc) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 90% identity to the sequence of SEQ ID NO: 60.

125. The method of any one of claims 106-116, wherein:a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 20; andb) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 26;and optionally wherein:c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 90% identity to the sequence of SEQ ID NO: 62.

126. The method of any one of claims 106-116, wherein:a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 20; andb) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 26;and optionally wherein:c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 90% identity to the sequence of SEQ ID NO: 61.

127. The method of any one of claims 106-116, wherein:a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 16; andb) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 22;and optionally wherein:c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 90% identity to the sequence of SEQ ID NO: 63.

128. The method of any one of claims 106-116, wherein:a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 16; and279326657998Attorney Docket No.: PTBI-010 / 01 WOb) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 22;and optionally wherein:c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 90% identity to the sequence of SEQ ID NO: 64.

129. The method of any one of claims 106-116, wherein:a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 16; andb) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 22;and optionally wherein:c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 90% identity to the sequence of SEQ ID NO: 60.

130. The method of any one of claims 106-116, wherein:a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 18; andb) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 24;and optionally wherein:c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 90% identity to the sequence of SEQ ID NO: 62.

131. The method of any one of claims 106-116, wherein:a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 17; andb) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO:23;and optionally wherein:c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 90% identity to the sequence of SEQ ID NO: 63.

132. The method of any one of claims 106-116, wherein:280326657998Attorney Docket No.: PTBI-010 / 01 WOa) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 17; andb) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 23;and optionally wherein:c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 90% identity to the sequence of SEQ ID NO: 64.

133. The method of any one of claims 106-116, wherein:a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 17; andb) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 23;and optionally wherein:c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 90% identity to the sequence of SEQ ID NO: 66.

134. The method of any one of claims 106-116, wherein:a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 17; andb) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 23;and optionally wherein:c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 90% identity to the sequence of SEQ ID NO: 62.

135. The method of any one of claims 106-116, wherein:a) the kinase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 17; andb) the RNase domain of the chimeric protein comprises an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 23;and optionally wherein:c) the stem-loop splicing hairpin comprises a nucleotide sequence having at least 90% identity to the sequence of SEQ ID NO: 61.281326657998Attorney Docket No.: PTBI-010 / 01 WO136. The method of any one of claims 106-135, wherein the cell is according to any one of claims 96-105.

137. A method of treating a disease, comprising administering to a subject a composition comprising the chimeric protein of any one of claims 1-33, the engineered RNA polynucleotide of any one of claims 37-61, the polynucleotide or combination of polynucleotides of any one of claims 34-36 and 62-93, the viral particle of claim 95, or the cell of any one of claims 96-105; optionally wherein the disease is an inflammatory disease or a cancer.

138. The method of claim 137, wherein:(a) the ligand is a biomarker of the inflammatory disease, and / or the protein of interest is a therapeutic protein for treating the inflammatory disease; or(b) the ligand is a cancer-associated antigen or biomarker, and / or the protein of interest is a therapeutic protein for treating the cancer.

139. A method comprising delivering a composition comprising the polynucleotide or combination of polynucleotides of any one of claims 34-36 and 62-93 to a cell.

140. The method of claim 139, wherein the cell is an ex vivo cell or an in vivo cell; optionally, w'herein the cell is in a human.

141. The method of any one of claims 137-140, wherein the polynucleotide or combination of polynucleotides are comprised within one or more vectors.

142. The method of claim 141, wherein the one or more vectors are non-viral delivery vectors, optionally wherein the non-viral delivery vectors are lipid nanoparticles.

143. The method of claim 141, wherein the one or more vectors are viral delivery vectors.

144. The method of claim 143, wherein the viral delivery vector is an adeno-associated virus (AAV) vector, an adenovirus vector, or a lentivirus vector.282326657998

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