Engineered synthetic IRES with improved and robust translation in circular RNA

Abstract

Provided herein, in some aspects, are recombinant ribonucleic acid (RNA) molecules (e.g., linear mRNA, circular RNA) comprising engineered internal ribosome entry sites (IRES) elements and methods for their use in modulating gene expression in cells and subjects. Also provided herein are methods of engineering IRES elements for improved translational efficiency of RNA (e.g., linear mRNA, circular RNA).

Description

[0001] ENGINEERED SYNTHETIC IRES WITH IMPROVED AND ROBUST TRANSLATION IN CIRCULAR RNA

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 580,968 filed September 6, 2023, and entitled “Engineered Synthetic IRES with Improved and Robust Translation in Circular RNA,” the contents of which are hereby incorporated by reference herein in its entirety for all purposes.

[0004] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0005] The contents of the electronic sequence listing (M065670538WO00-SEQ-JRV.xml; Size: 218,368 bytes; and Date of Creation: August 20, 2024) is herein incorporated by reference in its entirety.

[0006] FIELD

[0007] This disclosure relates to recombinant circular ribonucleic acid (circRNA) molecules comprising engineered internal ribosome entry sites (IRES) elements and methods for their use in modulating gene expression in cells and subjects.

[0008] FEDERALLY SPONSORED RESEARCH

[0009] This invention was made with government support under HDTRA 1-22- 1-0032 awarded by the Defense Threat Reduction Agency. The government has certain rights in the invention.

[0010] BACKGROUND

[0011] Recombinant circular ribonucleic acids (circRNAs) are a class of non-coding RNAs that form covalently closed-loop structures without 5' caps or 3' poly(A) tails. Recently, circular RNAs have been identified as next-generation mRNA therapeutics due to their low immunogenicity, long half-lives and engineering potential. For example, Eukaryotic Toeholds (eToeholds) have been shown to enable RNA-responsive translational control (Zhao, Mao, et al. Nat Biotechnol. 2022 Apr;40(4):539-545. doi: 10.1038 / s41587-021- 01068-2. Epub 2021 Oct 28.) and small molecules can be used to induce translation in engineered circRNAs (Xiang et al. Nat Comms 2019). However, translational efficiency of circular RNA is often much lower within the first 24 hours after transfection or administration, as compared to linear mRNA translation. Methods for improving translational efficiency of RNA, especially circRNA, are highly desirable.

[0012] SUMMARY

[0013] There is a pressing need to achieve robust and high levels of expression of RNA constructs. Internal Ribosome Entry Sites (IRES) elements are sequences found in the 5' untranslated region (5' UTR) of viral and some cellular mRNA; IRES elements are a primary component for driving cap-independent protein translation in coding RNA. The inventors have found that selective modifications to IRES elements improve translational efficiency by as much as 114-fold, compared to expression mediated by wildtype EMCV IRES elements. In some embodiments, the engineered IRES elements described herein comprise domains having one or more insertion sequences (e.g., stem loops, spacer sequences, aptamers) which improve their translational efficiency. In some embodiments, the engineered IRES elements described herein comprise domains having one or more truncations which improve their translational efficiency. The engineered IRES elements described herein can be used, in some aspects, to mediate expression of circular RNA molecules and linear RNA molecules for therapeutic use or for expression of polypeptide for production. Furthermore, the engineered IRES described herein can be used, in some aspects, to mediate any capindependent translation. In some aspects, the engineered IRES elements described herein can be used for methods of mRNA and protein production via cell-free in vitro transcription and translation (IVT).

[0014] In some aspects, provided herein are engineered internal ribosome entry site (IRES) elements, comprising a plurality of domains, each domain comprising at least one stem loop, such that the IRES element comprises at least a 3’ terminal domain and a 5’ terminal domain, and optionally, one or more central domains between the 3’ terminal domain and the 5’ terminal domain; and an insertion sequence, wherein the insertion sequence is inserted into one of the plurality of domains. In some embodiments, the insertion sequence comprises a secondary structure. In some embodiments, the secondary structure comprises a stem loop. In some embodiments, the insertion sequence comprises an aptamer. In some embodiments, the aptamer is an eIF4G-binding aptamer (e.g., binds to eIF4G). In some embodiments, the insertion sequence comprises a spacer sequence. In some embodiments, the spacer sequence is less than lOObp in length. In some embodiments, the spacer sequence is 12-50bp in length. In some embodiments, the spacer sequence is about 32 bp in length. In some embodiments, the spacer sequence is 12-16bp in length. In some embodiments, the spacer sequence does not comprise a secondary structure.

[0015] In some embodiments, the engineered IRES element comprises two or more insertion sequences inserted into two or more of the plurality of domains. In some embodiments, the two or more insertion sequences are no less than 20bp apart. In some embodiments, at least one insertion sequence is complementary to at least one other insertion sequence. In some embodiments, an engineered IRES element provided herein comprises an insertion sequence inserted into the 3’ terminal domain. In some embodiments, an engineered IRES element provided herein comprises an insertion sequence inserted into the 5’ terminal domain.

[0016] In some embodiments, the engineered IRES element comprises at least one domain comprising a truncated stem loop. In some embodiments, the truncated stem loop is deleted.

[0017] In some aspects, provided herein are engineered internal ribosome entry site (IRES) elements, comprising a plurality of domains each comprising at least one stem loop, such that the IRES element comprises at least a 3’ terminal domain and a 5’ terminal domain, and optionally, one or more central domains between the 3’ terminal domain and the 5’ terminal domain; wherein at least one stem loop in the IRES element is a truncated stem loop. In some embodiments, the truncated stem loop is deleted. In some embodiments, the truncated stem loop is in the 3’ terminal domain. In some embodiments, the truncated stem loop is in the 5’ terminal domain. In some embodiments, the engineered IRES element comprises two or more truncated stem loops.

[0018] In some embodiments, the engineered IRES element further comprises an insertion sequence. In some embodiments, the insertion sequence comprises a secondary structure. In some embodiments, the insertion sequence comprises a stem loop. In some embodiments, the insertion sequence comprises an aptamer. In some embodiments, the aptamer binds to eIF4G.

[0019] In some embodiments, the engineered IRES element comprises an insertion sequence which is a spacer sequence. In some embodiments, the spacer sequence is less than lOObp in length. In some embodiments, the spacer sequence is 12-50bp in length. In some embodiments, the spacer sequence is about 32 bp in length. In some embodiments, the spacer sequence is 12-16bp in length. In some embodiments, the spacer sequence does not comprise a secondary structure.

[0020] In some embodiments, the engineered IRES element comprises two or more insertion sequences inserted into two or more domains. In some embodiments, the two or more insertion sequences are no less than 20bp apart. In some embodiments, at least one insertion sequence is complementary to at least one other insertion sequence. In some embodiments, the engineered IRES element comprises an insertion sequence inserted into the 3’ terminal domain. In some embodiments, the engineered IRES element comprises an insertion sequence inserted into the 5’ terminal domain.

[0021] In some embodiments, the engineered IRES element comprises a 3’ terminal domain and a 5’ terminal domain wherein both the 3’ terminal domain and the 5’ terminal domain comprise a truncated stem loop.

[0022] In some embodiments, an engineered IRES element described herein is modified from a wildtype IRES element. In some embodiments, the engineered IRES element is derived from a human genome. In some embodiments, the engineered IRES element is a short IRES. In some embodiments, the wildtype IRES element is an IRES element of Taura syndrome virus, Triatoma virus, Theiler's encephalomyelitis virus, simian Virus 40, Solenopsis invicta virus 1, Rhopalosiphum padi virus, Reticuloendotheliosis virus, human poliovirus 1, Plautia stall intestine virus, Kashmir bee virus, Human rhinovirus (iHRV), Homalodisca coagulata virus- 1, Human Immunodeficiency Virus type 1, Homalodisca coagulata virus- 1, Himetobi P virus, Hepatitis C virus, Hepatitis A virus, Hepatitis GB virus, foot and mouth disease virus, Human enterovirus 71 (EV71), Equine rhinitis virus, Ectropis obliqua picoma-like virus, Encephalomyocarditis virus (EMCV), Drosophila C Virus, Crucifer tobamo virus, Cricket paralysis virus, Bovine viral diarrhea virus 1, Black Queen Cell Virus, Aphid lethal paralysis virus, Avian encephalomyelitis virus, Acute bee paralysis virus, Hibiscus chlorotic ringspot virus, Classical swine fever virus, Human FGF2, Human SFTPA1, Human AML1 / RUNX1, Drosophila antennapedia, Human AQP4, Human AT1R, Human BAG-1, Human BCL2, Human BiP, Human c-IAPl, Human c-myc, Human eIF4G, Mouse NDST4L, Human LEF1, Mouse HIF1 alpha, Human n.myc, Mouse Gtx, Human p27kipl, Human PDGF2 / c-sis, Human p53, Human Pim-1, Mouse Rbm3, Drosophila reaper, Canine Scamper, Drosophila Ubx, Salivirus, Cosavirus, Parechovirus, Human UNR, Mouse UtrA, Human VEGF-A, Human XIAP, Drosophila hairless, S. cerevisiae TFIID, S. cerevisiae YAP1, Human c-src, Human FGF-1, Simian picomavirus, Turnip crinkle virus, Coxsackievirus B3 (CVB3), Coxsackievirus A (CVB1 / 2), or Poliovirus (PV). In some embodiments, the wildtype IRES element is EMCV, EV71, CVB3, iHRV, or PV. In some embodiments, the wildtype IRES element is EMCV. In some embodiments, the IRES element is EMCV-A7, EMCV- A3, or EMCV-26C.

[0023] Also provided herein, in some aspects, are circular ribonucleic acids (circRNA) comprising an engineered IRES element described herein. In some embodiments, a circRNA provided herein further comprises an open reading frame encoding a peptide, protein, or fragment thereof. In some embodiments, a circRNA provided herein further comprises an untranslated region (UTR). In some embodiments, a circRNA provided herein further comprises one or more intron regions or fragments thereof. In some embodiments, a circRNA provided herein comprises a frameshift mutation. In some embodiments, translation of a circRNA provided herein expresses an amount of a functional peptide, protein, or fragment thereof greater than the circRNA comprising a frameshift mutation absent the engineered IRES element.

[0024] Also provided herein, in some aspects, are linear ribonucleic acids (RNA) comprising an engineered IRES element described herein.

[0025] Also provided herein, in some aspects, are plasmids encoding an engineered IRES element described herein. In some embodiments, a plasmid provided herein comprises an enhancer sequence. In some embodiments, a plasmid provided herein comprises one or more intron regions or fragments thereof. In some embodiments, a plasmid provided herein comprises an N-terminal tag. In some embodiments, a plasmid provided herein comprises a C-terminal tag.

[0026] Also provided herein, in some aspects, are methods of expressing a polypeptide, the method comprising expressing a circRNA, a linear RNA, or a plasmid comprising an engineered IRES element described herein in a host cell in conditions under which the RNA is translated into a polypeptide.

[0027] Also provided herein, in some aspects, are methods of expressing a polypeptide, the method comprising expressing a circRNA, a linear RNA, or a plasmid comprising an engineered IRES element described herein in a cell-free composition in conditions under which the RNA is translated into a polypeptide.

[0028] BRIEF DESCRIPTION OF DRAWINGS

[0029] 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. The figures are illustrative only and are not required for enablement of the invention disclosed herein.

[0030] FIGs. 1A-1E show design and function of engineered enterovirus A71 (EV71) internal ribosome entry site (IRES) elements having various internal insertion sequences. FIG. 1A is a schematic of the predicted structure of an EV71 IRES element (MXFold2). Stem loops (SL) 1-7 are shown. FIG. IB shows sites for insertion of an insertion sequence in an EV71 IRES element (730 bases). FIG. 1C shows IRES -dependent mCherry expression in HEK293FT cells transfected with linear mRNAs having, from 5’ to 3’: a nucleic acid encoding GFP, stop codon, stem loops to block ribosome read-through, an EV71 IRES element with theophylline-binding aptamer insertions, and a nucleic acid encoding mCherry. Expression is shown for mRNA having: a wildtype EV71 IRES element (“EV71”), or an engineered EV71 IRES element having a theophylline-binding aptamer insertion at one of SL-1 (“VI”), SL-2 (“V2”), SL-3 (“V3”), SL-4 (“V4”), SL-5 (“V5”), SL-6 (“V6”), SL-7 (“V7”), SL-8 (“V8”), SL-9 (“V9”), SL-10 (“V10”), SL-11 (“Vll”), SL-12 (“V12”), SL-13 (“V13”), SL-14 (“V-14”), SL-15 (“V15”), SL-16 (“V16”), SL-17 (“V17”), or SL-18 (“V18”) in the presence of ImM theophylline (“Induced”) and in the absence of theophylline (“Uninduced”). FIG. ID shows IRES -dependent mCherry expression in HEK293FT cells transfected with linear mRNAs having, from 5’ to 3’: a nucleic acid encoding GFP, stop codon, stem loops to block ribosome read-through,, an EV71 IRES element with tetracyclinebinding aptamer insertions, and a nucleic acid encoding mCherry. Expression is shown for mRNA having: a wildtype EV71 IRES element (“EV71”) or an engineered EV71 IRES element having a tetracycline-binding aptamer at one of SL-1 to SI- 18 (as in FIG. 1C), in the presence of 200pM tetracycline (“Induced”) and in the absence of tetracycline (“Uninduced”). FIG. IE shows IRES-dependent mCherry expression in HEK293FT cells transfected with linear mRNAs having, from 5’ to 3’: a nucleic acid encoding GFP, stop codon, stem loops to block ribosome read-through, an EV71 IRES element with (6R,S)- folinic acid-binding aptamer insertions, , and a nucleic acid encoding mCherry. Expression is shown for mRNA having: a wildtype EV71 IRES element (“EV71”) or an engineered EV71 IRES element having a (6R,S)-folinic acid-binding aptamer insertion at one of SL-1 to SI- 18 (as in FIG. 1C), in the presence of ImM folinic acid (“Induced”) and in the absence of folinic acid (“Uninduced”).

[0031] FIGs. 2A-2B show IRES-dependent mCherry expression in HEK293FT cells transfected with linear mRNAs having, from 5’ to 3’: a nucleic acid encoding GFP, stop codon, stem loops to block ribosome read-through, a Coxsackievirus B3 (CVB3) IRES element or Poliovirus (PV) IRES element with theophylline-binding aptamer insertions, , and a nucleic acid encoding mCherry. FIG. 2A shows mCherry / GFP expression for mRNA having a wildtype CVB3 IRES element (“CVB3”) or an engineered CVB3 with a theophylline-binding aptamer insertion at one of SL-1 (“VI”), SL-2 (“V2”), SL-3 (“V3”), SL-4 (“V4”), SL-5 (“V5”), SL-6 (“V6”), SL-7 (“V7”), SL-8 (“V8”), SL-9 (“V9”), SL-10 (“V10”), SL-11 (“Vll”), SL-12 (“V12”), SL-13 (“V13”), SL-14 (“V-14”), SL-15 (“V15”), or SL-16 (“V16”), in the presence of ImM theophylline (“Induced”) and in the absence of theophylline (“Uninduced”). FIG. 2B shows mCherry / GFP expression for mRNA having a wildtype PV IRES element (“PV”) or an engineered PV with a theophylline-binding aptamer insertion at one of SL-1 (“VI”), SL-2 (“V2”), SL-3 (“V3”), SL-4 (“V4”), SL-5 (“V5”), SL-6 (“V6”), SL-7 (“V7”), SL-8 (“V8”), SL-9 (“V9”), SL-10 (“V10”), SL-11 (“Vll”), SL-12 (“V12”), SL-13 (“V13”), SL-14 (“V-14”), SL-15 (“V15”), or SL-16 (“V16”), in the presence of ImM theophylline (“Induced”) and in the absence of theophylline (“Uninduced”).

[0032] FIGs. 3A-3B show design and function of engineered EV71 internal ribosome entry site (IRES) elements having various terminal and internal insertion sequences. FIG. 3A is a schematic showing internal insertion sites and 5’ terminal (“Front”) and 3’ terminal (“End”) “edge” insertion sites for 12bp or 16bp complementary insertion sequences on an EV71 IRES element. FIG. 3B shows IRES-dependent mCherry expression in HEK293FT cells transfected with linear mRNAs having, from 5’ to 3’: a nucleic acid encoding GFP, stop codon, stem loops to block ribosome read-through, an EV71 IRES element with theophylline- binding aptamer insertions, , and a nucleic acid encoding mCherry. mCherry / GFP expression is shown for mRNA with a wildtype EV71 IRES element (“EV71”); an engineered EV71 IRES element having a theophylline-binding aptamer insertion at one of SL-1 (“VI”), SL-2 (“V2”), SL-3 (“V3”), SL-16 (“V16”), SL-17 (“V17”), or SL-18 (“V18”); or an engineered EV71 IRES element having a theophylline-binding aptamer insertion at one of SL-1 (“VI”), SL-2 (“V2”), SL-3 (“V3”), SL-16 (“V16”), SL-17 (“V17”), or SL-18 (“V18”), and one of: a 12bp complementary insertion sequence at the 5’ terminal (“Comp Front 12bp”), a 16bp complementary insertion sequence at the 5’ terminal (“Comp Front 16bp”), a 12bp complementary insertion sequence at the 3’ terminal (“Comp End 12bp”), a 16bp complementary insertion sequence at the 3’ terminal (“Comp End 16bp”). Expression is shown in the presence of ImM theophylline (“Induced”) and in the absence of theophylline (“Uninduced”).

[0033] FIGs. 4A-4C show design and function of engineered EV71 IRES elements with domain truncations. FIG. 4A is a schematic showing deletion of SL-1 and SL-7 of EV71 IRES. FIG. 4B is a schematic of the predicted structure of an EV71 IRES element (MXFold2) in which SL-1 and SL-7 are deleted. FIG. 4C shows IRES-dependent mCherry expression in HEK293FT cells transfected with linear mRNAs having, from 5’ to 3’: a nucleic acid encoding GFP, stop codon, stem loops to block ribosome read-through, an EV71 IRES element with stem loop deletions, a nucleic acid encoding a P2A peptide, and a nucleic acid encoding mCherry. mCherry / GFP expression is shown for mRNA having no IRES, (“No IRES”), having a wildtype EV71 IRES element (“WT”), or an engineered EV71 IRES element in which SL-1 has been deleted (“Al”); SL-2 has been deleted (“A2”); SL-3 has been deleted (“A3”); SL-4 has been deleted (“A4”); SL-5 has been deleted (“A5”); SL-6 has been deleted (“A6”); SL-6A (“SL-6 #1” in FIG. 4A) has been deleted (“A6A”); SL-6B (“SL-6 #2” in FIG. 4A) has been deleted (“A6B”); SL-7 has been deleted (“A7”); SL-1 and SL-2 have been deleted (“Al, 2”); SL-1 and SL-6 have been deleted (“Al, 6”); SL-1 and SL- 6A have been deleted (“A1,6A”); SL-1 and SL-6B have been deleted (“A1,6B”); SL-1 and SL-7 have been deleted (“Al, 7”); SL-2 and SL-6 have been deleted (“A2,6”); SL-2 and SL- 6A have been deleted (“A2,6A”); SL-2 and SL-6B have been deleted (“A2,6B”); SL-2 and SL-7 have been deleted (“A2,7”); SL-6 and SL-7 have been deleted (“A6,7”); SL-6A and SL-7 have been deleted (“A6A,7”); SL-6B and SL-7 have been deleted (“A6B,7”); SL-1, SL-2, and SL-6 have been deleted (“Al, 2, 6”); SL-1, SL-2, and SL-6A have been deleted (“A 1,2, 6 A”); SL-1, SL-2, and SL-6B have been deleted (“A1,2,6B”); SL-1, SL-2, and SL-7 have been deleted (“Al, 2, 7”); SL-2, SL-6, and SL-7 have been deleted (“A2,6,7”); SL-2, SL-6A, and SL-7 have been deleted (“A2,6A,7”); SL-2, SL-6B, and SL-7 have been deleted (“A2,6B,7”); SL-1, SL-2, SL-6, and SL-7 have been deleted (“Al, 2, 6, 7”); SL-1, SL-2, SL- 6A, and SL-7 have been deleted (“A1,2,6A,7”); or SL-1, SL-2, SL-6B, and SL-7 have been deleted (“A1,2,6B,7”).

[0034] FIG. 5 shows IRES-dependent mCherry expression in HEK293FT cells transfected with linear mRNAs having, from 5’ to 3’: a nucleic acid encoding GFP, stop codon, stem loops to block ribosome read-through, a CVB3 IRES element with stem loop deletions, , and a nucleic acid encoding mCherry. mCherry / GFP expression is shown for mRNA having a wildtype EV71 IRES element (“WT”), or an engineered CVB3 IRES element in which SL-1 has been deleted (“Al”); SL-2 has been deleted (“A2”); SL-3 has been deleted (“A3”); SL-4 has been deleted (“A4”); SL-5 has been deleted (“A5”); SL-6 has been deleted (“A6”); SL-1 and SL-2 have been deleted (“Al, 2”); SL-1 and SL-6 have been deleted (“Al, 6”); SL-2 and SL-6 have been deleted (“A2,6”); or SL-1, SL-2, and SL-6 have been deleted (“A 1,2, 6”). FIGs. 6A-6B show IRES-dependent TagBFP expression in HEK293FT cells cotransfected with plasmids encoding TagBFP as transfection control and plasmids encoding circular mRNAs having, an CVB3 IRES element or an EV71 IRES element with stem loop deletions and / or theophylline-binding aptamer insertions, and a nucleic acid encoding GFP FIG. 6A GFP / TagBFP expression is shown for mRNA having a wildtype EV71 IRES element (“WT”), or having an engineered EV71 IRES element having a theophylline-binding aptamer insertion at the 5’ terminal (“5”’), at the 3’ terminal (“3”’), at the 5’ terminal and at the 3’ terminal (“5’+3”’); in which SL-1 has been deleted (“Al”); a theophylline-binding aptamer insertion at the 5’ terminal and in which SL-1 has been deleted (“Al+5”’); a theophylline-binding aptamer insertion at the 3’ terminal and in which SL-1 has been deleted (“Al+3”’); a theophylline-binding aptamer insertion at the 5’ terminal and at the 3’ terminal, and in which SL-1 has been deleted (“A 1+5 ’,3”’); in which SL-7 has been deleted (“A7”); a theophylline-binding aptamer insertion at the 5’ terminal and in which SL-7 has been deleted (“A7+5”’); a theophylline-binding aptamer insertion at the 3’ terminal and in which SL-7 has been deleted (“A7+3”’); a theophylline-binding aptamer insertion at the 5’ terminal and at the 3’ terminal, and in which SL-7 has been deleted (“A7+5’,3”’); in which SL-1 and SL-7 have been deleted (“A 1,7”); a theophylline-binding aptamer insertion at the 5’ terminal and in which SL-1 and SL-7 have been deleted (“Al, 7+5’”); or a theophylline-binding aptamer insertion at the 3’ terminal and in which SL-1 and SL-7 have been deleted (“Al, 7+3”’). Expression is shown in the presence of ImM theophylline (“Induced”) and in the absence of theophylline (“Uninduced”). FIG. 6B GFP / TagBFP expression is shown for mRNA having a wildtype CVB3 IRES element (“WT”) , or having an engineered CVB3 IRES element having a theophylline-binding aptamer insertion at the 5’ terminal (“5”’), at the 3’ terminal (“3”’), at the 5’ terminal and at the 3’ terminal (“5’+3”’); in which SL-1 has been deleted (“Al”); a theophylline-binding aptamer insertion at the 5’ terminal and in which SL-1 has been deleted (“Al+5”’); a theophylline-binding aptamer insertion at the 3’ terminal and in which SL-1 has been deleted (“Al+3”’); a theophylline-binding aptamer insertion at the 5’ terminal and at the 3’ terminal, and in which SL-1 has been deleted (“Al+5’, 3’”); in which SL-6 has been deleted (“A6”); a theophylline-binding aptamer insertion at the 5’ terminal and in which SL-6 has been deleted (“A6+5”’); a theophylline-binding aptamer insertion at the 3’ terminal and in which SL-6 has been deleted (“A6+3”’); a theophylline-binding aptamer insertion at the 5’ terminal and at the 3’ terminal, and in which SL-6 has been deleted (“A6+5’,3”’); in which SL-1 and SL-6 have been deleted (“A 1,6”); a theophylline-binding aptamer insertion at the 5’ terminal and in which SL-1 and SL-6 have been deleted (“A 1,6+5’”); a theophylline-binding aptamer insertion at the 3’ terminal and in which SL-1 and SL-6 have been deleted (“Al, 6+3”’). a theophylline-binding aptamer insertion at the 5’ terminal and at the 3’ terminal, and in which SL-1 and SL-6 have been deleted (“Al, 6+5’, 3”’). Expression is shown in the presence of ImM theophylline (“Induced”) and in the absence of theophylline (“Uninduced”).

[0035] FIG. 7 shows IRES-dependent TagBFP expression in HEK293FT cells cotransfected with plasmids encoding TagBFP as transfection control and plasmids encoding circular mRNAs having, an CVB3 IRES element or an EV71 IRES element with stem loop deletions and / or theophylline-binding aptamer insertions, and a nucleic acid encoding GFP GFP / TagBFP expression is shown for mRNA having: a wildtype PV IRES element (“PV”); a PV IRES element with a theophylline-binding aptamer insertion at the 5’ terminal (“PV 5’”); a PV IRES element with a theophylline-binding aptamer insertion at the 3’ terminal (“PV 3’”); a PV IRES element with a theophylline-binding aptamer insertion at the 5’ terminal and at the 3’ terminal (“PV 5 ’+3’”); a wildtype EMCV IRES element (“EMCV”); a EMCV IRES element with a theophylline-binding aptamer insertion at the 5’ terminal (“EMCV 5’”); a EMCV IRES element with a theophylline-binding aptamer insertion at the 3’ terminal (“EMCV 3’”); a EMCV IRES element with a theophylline-binding aptamer insertion at the 5’ terminal and at the 3’ terminal (“EMCV 5 ’+3’”); a wildtype EMCV- A3 IRES element (“EMCV- A3”); a EMCV-A3 IRES element with a theophylline-binding aptamer insertion at the 5’ terminal (“EMCV- A3 5’”); or a EMCV-A3 IRES element with a theophylline-binding aptamer insertion at the 3’ terminal (“EMCV-A3 3’”). Expression is shown in the presence of ImM theophylline (“Induced”) and in the absence of theophylline (“Uninduced”).

[0036] FIGs. 8A-8C show IRES-dependent TagBFP expression in HEK293FT cells cotransfected with plasmids encoding TagBFP as transfection control and plasmids encoding circular mRNAs having, an CVB3 IRES element or an EV71 IRES element with stem loop deletions and / or theophylline-binding aptamer insertions, and a nucleic acid encoding GFP. FIG. 8A shows GFP / TagBFP expression for mRNA having: a wildtype EV71 IRES element (“EV71”); an EV71 IRES element with a spacer sequence having minimal secondary structure (“NS”) insertion at the 5’ terminal (“5’ NS”); an EV71 IRES element with an NS insertion at the 3’ terminal (“3’ NS”); an EV71 IRES element with an eIF4G-binding aptamer insertion at the 5’ terminal (“5’ eIF4G”); an EV71 IRES element in which SL-1 has been deleted (“Al”); an EV71 IRES element with an NS insertion at the 5’ terminal and in which SL-1 has been deleted (“Al 5 ’NS”); an EV71 IRES element with an NS insertion at the 3’ terminal and in which SL-1 has been deleted (“Al 3 ’NS”); an EV71 IRES element with an eIF4G-binding aptamer insertion at the 5’ terminal and in which SL-1 has been deleted (“Al 5’eIF4G”); an EV71 IRES element with an eIF4G-binding aptamer insertion at the 3’ terminal and in which SL-1 has been deleted (“Al 3’ eIF4G”); an EV71 IRES element in which SL-1 has been deleted (“A7”); an EV71 IRES element with an NS insertion at the 5’ terminal and in which SL-7 has been deleted (“A7 5 ’NS”); an EV71 IRES element with an NS insertion at the 3’ terminal and in which SL-7 has been deleted (“A7 3’NS”); an EV71 IRES element with an eIF4G-binding aptamer insertion at the 5’ terminal and in which SL-7 has been deleted (“A7 5’eIF4G”); an EV71 IRES element with an eIF4G-binding aptamer insertion at the 3’ terminal and in which SL-7 has been deleted (“A7 3’ eIF4G”); an EV71 IRES element in which SL-1 and SL-7 have been deleted (“A 1,7”); an EV71 IRES element with an NS insertion at the 5’ terminal and in which SL-1 and SL-7 have been deleted (“A 1,7 5 ’NS”); an EV71 IRES element with an NS insertion at the 3’ terminal and in which SL-1 and SL-7 have been deleted (“Al, 7 3’NS”); an EV71 IRES element with an eIF4G-binding aptamer insertion at the 5’ terminal and in which SL-1 and SL-7 have been deleted (“A 1,7 5’eIF4G”); or an EV71 IRES element with an eIF4G-binding aptamer insertion at the 3’ terminal and in which SL-1 and SL-7 have been deleted (“Al, 7 3’ eIF4G”). FIG. 8B shows GFP / TagBFP expression for mRNA having: a wildtype CVB3 IRES element (“CVB3”); a CVB3 IRES element with a spacer sequence having minimal secondary structure (“NS”) insertion at the 5’ terminal (“5’ NS”); a CVB3 IRES element with an NS insertion at the 3’ terminal (“3’ NS”); a CVB3 IRES element with an eIF4G-binding aptamer insertion at the 5’ terminal (“5’ eIF4G”); a CVB3 IRES element in which SL-1 has been deleted (“Al”); a CVB3 IRES element with an NS insertion at the 5’ terminal and in which SL-1 has been deleted (“Al 5’NS”); a CVB3 IRES element with an NS insertion at the 3’ terminal and in which SL-1 has been deleted (“Al 3’NS”); a CVB3 IRES element with an eIF4G-binding aptamer insertion at the 5’ terminal and in which SL-1 has been deleted (“Al 5’eIF4G”); a CVB3 IRES element with an eIF4G-binding aptamer insertion at the 3’ terminal and in which SL-1 has been deleted (“Al 3’ eIF4G”); a CVB3 IRES element in which SL-1 has been deleted (“A7”); a CVB3 IRES element with an NS insertion at the 5’ terminal and in which SL-6 has been deleted (“A6 5’NS”); a CVB3 IRES element with an NS insertion at the 3’ terminal and in which SL-6 has been deleted (“A6 3’NS”); a CVB3 IRES element with an eIF4G-binding aptamer insertion at the 5’ terminal and in which SL-6 has been deleted (“A6 5’eIF4G”); a CVB3 IRES element with an eIF4G-binding aptamer insertion at the 3’ terminal and in which SL-6 has been deleted (“A6 3’ eIF4G”); a CVB3 IRES element in which SL-1 and SL-6 have been deleted (“Al, 6”); a CVB3 IRES element with an NS insertion at the 5’ terminal and in which SL-1 and SL-6 have been deleted (“A 1,6 5 ’NS”); a CVB3 IRES element with an NS insertion at the 3’ terminal and in which SL-1 and SL-6 have been deleted (“Al, 6 3’NS”); a CVB3 IRES element with an eIF4G-binding aptamer insertion at the 5’ terminal and in which SL-1 and SL-6 have been deleted (“Al, 6 5’eIF4G”); or a CVB3 IRES element with an eIE4G-binding aptamer insertion at the 3’ terminal and in which SL-1 and SL-6 have been deleted (“Al, 6 3’ eIE4G”). FIG. 8C shows GFP / TagBFP expression for mRNA having: a wildtype PV IRES element (“PV_WT”); a PV IRES element with a spacer sequence having minimal secondary structure (“NS”) insertion at the 5’ terminal (“PV 5’ NS”); a PV IRES element with an NS insertion at the 3’ terminal (“PV 3’ NS”); a PV IRES element with an eIF4G-binding aptamer insertion at the 5’ terminal (“PV 5’ eIF4G”); a wildtype EMCV IRES element (“EMCV_WT”); a EMCV IRES element with a spacer sequence having minimal secondary structure (“NS”) insertion at the 5’ terminal (“EMCV 5’ NS”); a EMCV IRES element with an NS insertion at the 3’ terminal (“EMCV 3’ NS”); a EMCV IRES element with an eIF4G-binding aptamer insertion at the 5’ terminal (“EMCV 5’ eIF4G”); a wildtype EMCV-A3 IRES element (“EMCV-A3 WT”); an EMCV-A3 IRES element with a spacer sequence having minimal secondary structure (“NS”) insertion at the 5’ terminal (“EMCV- A3 5’ NS”); an EMCV- A3 IRES element with an NS insertion at the 3’ terminal (“EMCV- A3 3’ NS”); or an EMCV IRES element with an eIF4G-binding aptamer insertion at the 5’ terminal (“EMCV- A3 5’ eIF4G”).

[0037] FIGs. 9A-9G show an initial IRES screen based on CVB3 and EMCV in circular RNA. FIG. 9A is a schematic showing a rational design for engineered IRES-based rolling circle expression of circular RNA (circRNA), comprising: a CMV promoter (PCMV), 3’ flanking circularization cassettes, a nucleic acid encoding a split fluorescent protein (FP: a C terminus portion of GFP (151-241aa) with no stop codon, a nucleic acid encoding a T2A peptide, an engineered IRES element, a nucleic acid encoding a P2A peptide, and an N terminus portion (l-150aa) of GFP), and the flanking 5’ end of the homology arm a poly-A tail. FIG. 9B is a schematic showing a rational design for engineered IRES-based nonrolling circle expression of circRNA, comprising: a CMV promoter (PCMV), 3’ flanking circularization cassettes, nucleic acid encoding a split fluorescent protein (FP: a C terminus portion of GFP (151-241 aa) with a stop codon, a nucleic acid encoding a T2A peptide, an engineered IRES element, a nucleic acid encoding a P2A peptide, and an N terminus portion (l-150aa) of GFP), and the flanking 5’ end of the homology arm a poly-A tail. FIG. 9C is a table showing the length and a summary of fold-change improvement of three IRES candidates according to the rolling circle design of FIG. 9A. FIG. 9D shows mNeonGreen expression in HEK293FT cells transfected with engineered EMCV IRES elements in nonrolling circRNA (as in FIG. 9B) or rolling circRNA (as in FIG. 9A) for various EMCV IRES element designs. Expression is provided as fold change over modified EMCV control or EMCV control. FIG. 9E (top) shows the predicted structure of an EMCV wildtype IRES and (bottom) predicted changes to the structure of altered domains in an EMCV-A7 variant (“IRESite_Id557 emcv” of FIG. 9D), which include deletion of the 5’ terminal domain and 3’ terminal domain, and the insertion of an eIF4G aptamer at the 3’ terminal. FIG. 9F shows mNeonGreen expression in HEK293FT cells transfected with engineered CVB3 IRES elements in non-rolling circRNA (as in FIG. 9B) or rolling circRNA (as in FIG. 9A) for various CVB3 IRES element designs. Expression is provided as fold change over control modified EMCV control or EMCV control. FIG. 9G (top) shows the predicted structure of a CVB3 wildtype IRES and (bottom) predicted changes to the structure of altered domains in an CVB3 variant (IRESite_Id598 CVB3 of FIG. 9F), which include deletion of the 3’ terminal domain, and the insertion of an eIF4G-binding aptamer at the 5’ terminal.

[0038] FIG. 10 is a schematic showing a rational design for eIF4G-binding aptamer insertions in IRES elements (CVB3-B1, EMCV- A3, EMCV-A7) in a non-rolling circRNA. A 39bp eIF4G-binding aptamer can be inserted every 20bp in an IRES element, which may be useful for recruiting eIF4G and improving protein translation.

[0039] FIGs. 11A-11D show effects of eIF4G-binding aptamer insertions in IRES elements according to the rationale of FIG. 10. FIG. 11A shows an example of an engineered EMCV- A7 IRES element according to the rationale of FIG. 10. 28 sites for potential eIF4G-binding insertions are shown (P1-P28), with each site 20bp apart. Corresponding fold-change improvement (FC) in expression of mNeonGreen in HEK293FT cells transfected with nonrolling circRNA having an EMCV-A7 IRES element with an eIF4G-binding aptamer insertion at each location is shown. FIG. 11B shows fold-change of IRES-dependent mNeonGreen expression in HEK293FT cells transfected with non-rolling circRNA encoding mNeonGreen and having a wildtype EMCV-A7 IRES element or an engineered EMCV-A7 IRES elements with an eIF4G-binding aptamer insertion at one of each position shown in FIG. 11A. FIG. 11C shows fold-change of mNeonGreen vs wildtype control for engineered CVB3-B1 IRES elements having an eIF4G-binding aptamer insertion at one of 32 potential insertion sites, with each site 20bp apart. FIG. 11D shows fold-change of mNeonGreen vs wildtype control for engineered EMCV-A3 IRES elements having an eIF4G-binding aptamer insertion at one of 10 potential insertion sites, with each site 20bp apart.

[0040] FIGs. 12A-12C show differing expression of mNeonGreen in HEK293FT cells transfected with non-rolling circRNA encoding mNeonGreen and having an engineered EMCV-A7 IRES element, CVB3-B1 IRES element, EMCV-A3 IRES element, or iHRV IRES element according to the rationale of FIG. 10. FIG. 12A shows fold-change mNeonGreen expression for non-rolling circRNA having engineered EMCV-A7 IRES elements (as in FIG. 11B) compared to non-rolling circRNA having wildtype EMCV-A7 IRES elements. FIG. 12B shows fold-change mNeonGreen expression for non-rolling circRNA having engineered CVB3-B1 IRES elements (as in FIG. 11C). FIG. 12C shows fold-change mNeonGreen expression for non-rolling circRNA having engineered EMC V- A3 IRES elements (as in FIG. 11D) or an iHRV (“Jxc”) IRES element.

[0041] FIGs. 13A-13B show a summary of GFP reads (mNeon expression) of naturally occurring and engineered IRES elements in non-rolling circular RNA subjected to frameshift mutations, normalized against a BFP control for the same without frameshift mutations. Rep 1 and Rep 2 denote two individual biological replicates. FIG. 13A shows GFP reads for BFP only, modified EMCV (25C), CVB3, and EMCV IRES elements. FIG. 13B shows GFP reads for EMCV, and control (NRC-Entry). Bins correspond to their transfection levels, at half-log intervals in the TagBFP-Pacific Blue channel.

[0042] FIGs. 14 shows individual GFP reads (mNeon expression) corresponding to summaries shown in FIGs. 13A-13B.

[0043] DETAILED DESCRIPTION

[0044] Though recombinant ribonucleic acid (RNA) technologies and therapeutics are beginning to revolutionize medicine and life science research, linear RNAs face several challenges in stability. Heterologous messenger RNA (mRNA), which can be used to express pay loads in a host cell or subject, are also subject to endogenous degradation mechanisms. Once mRNA enters the cytoplasm, host cell enzymes deadenylate poly(A) tails at the 3’ tail of the mRNA and remove 5’ cap structures, exposing the remaining transcript to digestion by exonucleases. Because heterologous linear RNAs are quickly cleared from host cells, effective usage often requires high doses and / or frequent, repeated administration, which are often costly and burdensome. Recombinant circular RNAs (circRNA) provide an appealing alternative to linear RNA technologies due to their low immunogenicity, long half-lives and engineering potential. circRNA are a class of RNA that form covalently closed-loop structures and which can be found in nature. Indeed, thousands of endogenous circRNA have been identified and found to have complex roles in the regulation of endogenous gene expression (Qu et al. 2015, Cancer Lett, 365(2): 141-148). Though most naturally occurring circRNA are noncoding, circRNA containing internal ribosome entry site (IRES) elements have been shown to be able to initiate translation of proteins (Chen, C.Y. and Sarnow, P., 1995. Science, 268(5209): 415-417). Because circRNA lack 5' or 3' termini, they are resistant to the degradation mechanisms which target the terminals of linear RNA, making circRNA more stable and longer lasting in cells. However, despite these advantages, circRNA appear to lack the translational efficiency of mRNA. Translation inefficiency is also observed in linear RNAs with IRES elements (e.g., viral RNA), and is considered to be due, in some aspects, to the intricate and dynamic secondary and tertiary structures formed by IRESs (Koch et al., 2020. Nat Struct Mol Bio, 27: 1095-1104). Given the added complexity of its non-linear structure, the geometry of circRNA is thought to further interfere with IRES element structure, thereby hindering translational efficiency. Translational efficiency, as used herein, refers to the rate at which translation products (e.g., proteins) are generated from mRNA; translational efficiency is typically measured as protein per mRNA per hour.

[0045] Surprisingly, the inventors have identified methods for engineering IRES elements to improve translational efficiency of RNA and engineered IRES elements that provide improved translational efficiency of RNA. Disclosed herein are methods and compositions relating to engineered IRES elements. In some embodiments, the engineered IRES elements disclosed herein comprise certain placements of insertion sequences which improve translational efficiency of recombinant circRNA. In some embodiments, the engineered IRES elements disclosed herein comprise certain truncations which improve translational efficiency of recombinant circRNA. In some embodiments, the engineered IRES elements disclosed herein comprise certain placements of insertion sequences which improve translational efficiency of recombinant linear (e.g., mRNA). In some embodiments, the engineered IRES elements disclosed herein comprise certain truncations which improve translational efficiency of recombinant linear (e.g., mRNA). Recombinant Polynucleotides

[0046] Aspects of the disclosure relate to recombinant polynucleotides (e.g., linear RNA, circRNA) comprising engineered IRES elements disclosed herein. As used herein, the terms “polynucleotide,” “nucleic acid” and “oligonucleotide” are used interchangeably to refer to a polymer or oligomer of polynucleotide monomers (e.g., nucleotides). Polynucleotide monomers include, but are not limited to, deoxyribonucleotides and ribonucleotides. In some embodiments, polynucleotide monomers are naturally occurring. In some embodiments, polynucleotide monomers are synthetic. In some embodiments, polynucleotide monomers are chemically modified (e.g., methylated, acetylated, thiolated, ethylated).

[0047] In some embodiments, a polynucleotide is a ribonucleic acid (RNA) molecule. RNA molecules are typically single strands, but can form double-stranded polymers with other RNA molecules (e.g., by intermolecular base pairing) or internal double- stranded structures (e.g., by intramolecular base pairing). In some embodiments, RNA molecules are linear, such that they comprise a 5’ and 3’ terminus and do not form a closed structure. In some embodiments, a linear RNA molecule is a messenger RNA (mRNA) (e.g., mature mRNA, precursor mRNA). In some embodiments, a mRNA is a precursor mRNA, wherein the mRNA comprises introns and does not comprise a 5’ cap (e.g., Cap-0, Cap-1, Cap-2) and 3’ poly(A)denosine (poly(A)) tail. In some embodiments, an mRNA is a mature mRNA, wherein the mRNA does not comprise introns and comprises a 5’ cap (e.g., Cap-0, Cap-1, Cap-2) and a 3’ poly(A) tail. Typically, mature mRNA are generated from precursor mRNAs which are spliced, capped, and poly(A)denylated. Canonical splicing of an mRNA is catalyzed by a spliceosome, which binds to a donor site (5’ terminal of an intron) and an acceptor site (3’ terminal of an intron) on an intron. When the intron is excised, compatible exon terminals are spliced together. During capping, a series of enzymes add a cap, such as Cap-0 (7-methylguanosine), to the 5’ terminal of an mRNA molecule. Poly(A) tails, typically comprising about 200 adenine bases, are added to the 3’ terminal of an mRNA molecule by poly(A)denylate polymerase.

[0048] In some embodiments, RNA molecules are circRNA, such that they form a continuous, closed-loop structure. CircRNA do not comprise 5’ and 3’ termini, 5’ caps, or 3’ poly(A) tails. In some embodiments, circRNA are generated (e.g., circularized) from a precursor mRNA. Typically, circRNA are generated from linear RNA by back-splicing of a downstream 5’ donor sites to an upstream 3’ acceptor sites; accordingly, many circRNA molecules comprise a back-splice junction. Because the resulting circular molecule is covalently closed without accessible 5’ and 3’ termini, 5’ caps and 3’ poly(A) tails are absent in circRNA.

[0049] In some embodiments, a polynucleotide is a deoxyribonucleic acid (DNA) molecule. DNA molecules typically form double stranded polymers with other DNA molecules (e.g., by intermolecular base pairing), but can form single stranded structures (e.g., circDNA). In some embodiments, a DNA molecule can be transcribed to generate an RNA molecule (e.g., circRNA), such that the DNA can be said to encode the RNA molecule. In some embodiments, a DNA molecule encodes a non-coding RNA. In some embodiments, a DNA molecule encodes a payload, such that the DNA molecule can be transcribed to produce a coding RNA. In some embodiments, a DNA molecule is transcribed (e.g., expressed) in a cell or cell-free transcription system. Provided herein, in some aspects, are DNA molecules comprising sequences encoding RNA.

[0050] As used herein, the term “recombinant” refers to polynucleotides which comprise elements (e.g., sequences, higher order structures) or combinations of elements which are distinguishable from naturally occurring polynucleotides. The term “element,” as used herein, refers to discrete sequences or regions of sequences, including, but not limited to, IRES elements, coding sequences (e.g., gene or transgene), expression control sequences (e.g., promoters, enhancers, termination sequences), enzyme recognition sequences, and sequences having certain secondary structures (e.g., stem loops, bulges).

[0051] In some embodiments, recombinant polynucleotides comprise one or more elements isolated from one or more naturally occurring polynucleotides (e.g., genes). In some embodiments, recombinant polynucleotides comprise naturally occurring elements or combinations thereof, wherein the combination or arrangement of elements is not naturally occurring. In some embodiments, recombinant polynucleotides comprise engineered and / or synthetic elements. In some embodiments, recombinant polynucleotides are the product of cloning, restriction, polymerase chain reaction (PCR), ligation steps, or any combination thereof. The skilled artisan will understand that an individual element (e.g., expression control sequence) comprises a 5’ terminal and a 3’ terminal regardless of its localization in a linear or circular polynucleotide, such that an element in a circular polynucleotide retains an orientation and can be described accordingly.

[0052] In some embodiments, a recombinant polynucleotide comprises a coding sequence. A coding sequence (e.g., gene or transgene) is a sequence of nucleotides which can, under certain conditions, be transcribed and / or translated to generate a payload. In some embodiments, a coding sequence is transcribed and / or translated in a cell or cell-free translation system to generate a payload. As used herein, the term “payload” refers to one or more gene products of interest for delivery to or expression by an organism. A payload may be a functional nucleic acid, a peptide, or a protein or fragment thereof.

[0053] In some embodiments, a payload is a functional nucleic acid payload comprising a functional RNA. A “functional RNA,” as used herein, refers to an RNA which is not translated into a protein, but can knock down, knock in, increase, inhibit, or otherwise modulate gene expression. Non-limiting examples include short hairpin RNA (shRNA), microRNA (miRNA), artificial microRNA (amiRNA), small interfering RNA (siRNA), or circular RNA (circRNA).

[0054] In some embodiments, a payload is a peptide or protein or fragment thereof. The term “peptide,” as used herein, refers to a polymer of amino acids linked by peptide bonds. The length of a peptide may vary, but is typically less than 100 amino acids. Polymers of amino acids equal to or greater than 100 amino acids are referred to as “polypeptides”. In some embodiments, a polypeptide is a protein. Typically, a “protein” is a three-dimensional structure formed by the folding or binding of one or more polypeptides. Peptides and proteins or fragments thereof may be naturally occurring (e.g. products of translation of endogenous genes), or recombinantly or synthetically produced.

[0055] In some embodiments, a protein payload comprises a selectable marker, reporter or therapeutic protein. As used herein, a “selectable marker” refers to a peptide, protein or fragment thereof that can be used to screen cells by artificial selection. Non-limiting examples of selectable markers include antibiotic resistance proteins (e.g., ampicillin, puromycin) and negative selection markers (e.g., thymidine kinase). As used herein, a “reporter” is a peptide, protein or fragment thereof which alters the appearance of a cell such that cells can be visually or optically screened for presence or absence of the peptide or protein. Non-limiting examples of reporters include enzymes and fluorescent proteins. As used herein, a “therapeutic protein” refers to a protein which replaces or interferes with activity of deficient or absent endogenous proteins; augments activity of existing metabolic or synthetic pathways; provide a novel function or activity; or interferes with the activity of a pathogen or toxic molecule. Non-limiting examples of therapeutic proteins include membrane proteins, membrane-associated proteins, secreted proteins, intracellular proteins, immunomodulatory proteins, antigens, antibodies, or fragments thereof.

[0056] In some embodiments, a protein or fragment thereof comprises a protein tag (e.g., N- terminal tag, C-terminal tag). Non-limiting examples of protein tags include poly(His)tags, chitin binding protein (CBP), maltose binding protein (MBP), Strep-tag, and FLAG-tag. In some embodiments, a polynucleotide (e.g., RNA) comprises an untranslated region. An “untranslated region” (UTR), as used herein, refers to untranslated sequences on either side of one or more coding sequences in an RNA molecule. A UTR on the 5’ side of one or more coding sequences is referred to as a 5’ UTR. In some embodiments, a UTR is on the 3’ side of one or more coding sequences is referred to as a 3’ UTR. In some embodiments, a polynucleotide comprises a UTR derived from a virus. In some embodiments, a UTR comprises one or more expression control sequences. In some embodiments, a UTR comprises an engineered IRES element.

[0057] In some embodiments, a recombinant polynucleotide comprises a UTR comprising one or more expression control sequence. As used herein, an expression control sequence is any sequence which can regulate (e.g., promote, enhance, silence) expression or one or more coding sequences (e.g., genes, transgenes) to which it is operably coupled. In some embodiments, a polynucleotide comprises one or more expression control sequences operably linked to one or more coding sequences. As used herein, “operably linked” expression control sequences are those which are capable of effecting or modulating transcription of a coding sequence (e.g., have the coding sequence under transcriptional control). In some embodiments, an expression control sequence (e.g., promoter) is contiguous with a coding sequence to which it is operably linked. In some embodiments, an expression control sequence is not contiguous to a coding sequence to which it is operably linked, and effects or modulates expression of the coding sequence from a distance.

[0058] In some embodiments, an expression control sequence is a promoter. A “promoter” is a sequence in DNA which is recognized by the endogenous or introduced transcription machinery of a cell such that it directs the initiation of transcription of an RNA product from a coding sequence (e.g., gene or transgene). The function of a promoter may be further modulated by additional expression control sequences (i.e. enhancers, silencers, expression control sequences). Promoters may be native, constitutive, inducible, or tissue specific, for example, a promoter may be a cytomegalovirus (CMV) or spleen focus-forming virus (SFFV) promoter. A promoter can be understood to have a coding sequence “under transcriptional control” if it is in the correct location and orientation relative to the coding sequence such that it promotes transcription of the coding sequence. In some embodiments, an expression control sequence is a regulatory element which is not a promoter. Non-limiting examples of expression control sequences include transcription initiation sequences, termination sequences, translational efficiency sequences (e.g., Kozak sequences), and cleaving sequences, such as 2A-peptide (e.g., T2A, P2A) encoding sequences. In some embodiments, the RNA polynucleotide is a linear RNA comprising an engineered IRES element disclosed herein. In some embodiments, a linear RNA comprises an engineered IRES element disclosed herein and a coding sequence, wherein the engineered IRES element is operably linked to the coding sequence. In some embodiments, a linear RNA comprises an engineered IRES element operably linked to a coding sequence encoding a peptide, protein, or fragment thereof. In some embodiments, the linear RNA is a messenger RNA (mRNA).

[0059] In some embodiments, the RNA polynucleotide is a circRNA comprising an engineered IRES element disclosed herein. In some embodiments, a circRNA comprises an engineered IRES element disclosed herein and a coding sequence, wherein the engineered IRES element is operably linked to the coding sequence. In some embodiments, a circRNA comprises an engineered IRES element operably linked to a coding sequence encoding a peptide, protein, or fragment thereof.

[0060] Polynucleotides may be used to express a payload. In some embodiments, a host cell is by a polynucleotide (e.g., circRNA, linear RNA) under conditions in which the polynucleotide is introduced into the cell and a payload can be expressed by the host cell. In some embodiments, a payload encoded by a polynucleotide (e.g., circRNA, linear RNA) is expressed in a cell-free composition under conditions under which the payload can be translated.

[0061] Engineered. Internal Ribosome Entry Site (IRES) Elements

[0062] Disclosed herein are polynucleotides comprising engineered internal ribosome entry site (IRES) elements. As used herein, an “IRES element” refers to a cis RNA (e.g., mRNA, circRNA) sequence region which can promote cap-independent initiation of translation of the RNA. The terms “IRES element,” “IRES,” “IRES sequence,” and “IRES region” are used interchangeably herein. As used herein, “cap-independent” refers to a mechanism for initiation of translation of an RNA polynucleotide which does not require a 5’ cap (e.g., Capfl, Cap-1, Cap-2); cap -independent mechanisms for initiating translation are found in many viral RNA and in some eukaryotic mRNA. In contrast, “cap-dependent” mechanisms require a 5’ cap (e.g., Cap-0) which recruits a series of initiation factors and catalyzes the formation of a functional ribosome to initiate translation by a ribosome; cap-dependent mechanisms for initiating translation are found in most eukaryotic mRNA. RNA polynucleotides capable of cap-independent initiation of translation, such as those having an IRES element, bypass some mechanisms of cap-dependent translation. IRES elements can indirectly recruit a ribosomal subunit by binding to initiation factor which then recruits the ribosomal subunit, directly recruit a ribosomal subunit by binding to it directly. IRES elements may comprises a domain having a region of complementarity to a ribosomal RNA (rRNA). Provided herein are engineered IRES elements (e.g., modified from a naturally occurring IRES element) which may promote translation of RNA (e.g., circRNA).

[0063] Engineered IRES elements may be derived from naturally occurring (e.g., derived from a genome of a wildtype virus or eukaryote) or synthetic (e.g., artificial) IRES elements. Naturally occurring IRES elements are commonly found in 5’-UTRs of many viral mRNA and are more rarely observed in eukaryotic mRNA. In some embodiments, an engineered IRES element is derived from a naturally occurring IRES element, e.g., is modified from a naturally occurring IRES element. In some embodiments, an engineered IRES element is derived from a synthetic IRES element.

[0064] In some embodiments, an engineered IRES element is derived from a naturally occurring IRES element of a virus. Non-limiting examples of viral IRES elements include IRES elements of Taura syndrome virus, Triatoma virus, Theiler's encephalomyelitis virus, simian Virus 40, Solenopsis invicta virus 1, Rhopalosiphum padi virus, Reticuloendotheliosis virus, human poliovirus 1, Plautia stall intestine virus, Kashmir bee virus, Human rhinovirus (iHRV), Homalodisca coagulata virus- 1, Human Immunodeficiency Virus type 1, Homalodisca coagulata virus- 1, Himetobi P virus, Hepatitis C virus, Hepatitis A virus, Hepatitis GB virus, foot and mouth disease virus, Human enterovirus 71 (EV71), Equine rhinitis virus, Ectropis obliqua picoma-like virus, Encephalomyocarditis virus (EMCV), Drosophila C Virus, Crucifer tobamo virus, Cricket paralysis virus, Bovine viral diarrhea virus 1, Black Queen Cell Virus, Aphid lethal paralysis virus, Avian encephalomyelitis virus, Acute bee paralysis virus, Hibiscus chlorotic ringspot virus, Classical swine fever virus, Human FGF2, Human SFTPA1, Human AML1 / RUNX1, Drosophila antennapedia, Human AQP4, Human AT1R, Human BAG-1, Human BCL2, Human BiP, Human c-IAPl, Human c- myc, Human eIF4G, Mouse NDST4L, Human LEF1, Mouse HIF1 alpha, Human n.myc, Mouse Gtx, Human p27kipl, Human PDGF2 / c-sis, Human p53, Human Pim-1, Mouse Rbm3, Drosophila reaper, Canine Scamper, Drosophila Ubx, Salivirus, Cosavirus, Parechovirus, Human UNR, Mouse UtrA, Human VEGF-A, Human XIAP, Drosophila hairless, S. cerevisiae TFIID, S. cerevisiae YAP1, Human c-src, Human FGF-1, Simian picornavirus, Turnip crinkle virus, Coxsackievirus B3 (CVB3), Coxsackievirus A (CVB1 / 2), and Poliovirus (PV). In some embodiments, an engineered IRES element is modified from a naturally occurring (e.g., wildtype) IRES element, wherein the naturally occurring IRES element is EMCV, EV71, CVB3, iHRV, or PV. In some embodiments, an engineered IRES element is modified from a naturally occurring (e.g., wildtype) IRES element, wherein the naturally occurring IRES element is EMCV. In some embodiments, an engineered IRES element is modified from a naturally occurring (e.g., wildtype) IRES element, wherein the naturally occurring IRES element is EMCV-A7, EMCV-A3, or EMCV-26C. In some embodiments, an engineered IRES element is modified from a naturally occurring (e.g., wildtype) IRES element, wherein the naturally occurring IRES element is EMCV-A7. In some embodiments, a naturally occurring EMCV-A7 IRES element comprises the sequence set forth in SEQ ID NO: 114. In some embodiments, an engineered IRES element is modified from a naturally occurring (e.g., wildtype) IRES element, wherein the naturally occurring IRES element is EMCV- A3. In some embodiments, a naturally occurring EMCV- A3 IRES element comprises the sequence set forth in SEQ ID NO: 115. In some embodiments, an engineered IRES element is modified from a naturally occurring (e.g., wildtype) IRES element, wherein the naturally occurring IRES element is EMCV-26C. In some embodiments, a naturally occurring EMCV-26C IRES element comprises the sequence set forth in SEQ ID NO: 116. In some embodiments, an engineered IRES element is modified from a naturally occurring (e.g., wildtype) IRES element, wherein the naturally occurring IRES element is EV71. In some embodiments, a naturally occurring EV71 IRES element comprises the sequence set forth in SEQ ID NO: 117. In some embodiments, an engineered IRES element is modified from a naturally occurring (e.g., wildtype) IRES element, wherein the naturally occurring IRES element is CVB3. In some embodiments, a naturally occurring CVB3 IRES element comprises the sequence set forth in SEQ ID NO: 118. In some embodiments, an engineered IRES element is modified from a naturally occurring (e.g., wildtype) IRES element, wherein the naturally occurring IRES element is iHRV. In some embodiments, a naturally occurring iHRV IRES element comprises the sequence set forth in SEQ ID NO: 119. In some embodiments, an engineered IRES element is modified from a naturally occurring (e.g., wildtype) IRES element, wherein the naturally occurring IRES element is PV. In some embodiments, a naturally occurring PV IRES element comprises the sequence set forth in SEQ ID NO: 120.

[0065] In some embodiments, an engineered IRES element is derived from a naturally occurring IRES element of a eukaryote (e.g., human). Observation of virus-like endogenous IRES elements in eukaryotes (e.g., humans), is infrequent; however, short “IRES-like” elements or “putative IRES” elements have been observed to mediate circRNA translation in eukaryotes (Fan et al., 2022, Nat Commun (13): 3751). The skilled artisan will understand that eukaryotic IRES elements described herein may include both typical IRES elements and “IRES-like” or “putative IRES” elements. Non-limiting examples of viral and human IRES sequences can be found on IRESbase (on the world wide web at reprod.njmu.edu.cn / cgi- bin / iresbase / index.php) or IRESite (on the world wide web at iresite.org / IRESite_web.php).

[0066] Typically, an IRES element comprises one or more domains. As used herein, a “domain” is a structurally or functionally distinct segment of an IRES element. Engineered IRES elements described herein comprise at least one domain. In some embodiments, an engineered IRES element comprises a single domain. In some embodiments, an IRES element comprises two or more domains, such that a first domain is a 5’ terminal domain (also referred to herein as a “front” domain) and second domain is a 3’ terminal domain (also referred to herein as an “end” domain). In some embodiments, an IRES element having a 5’ terminal domain and a 3’ terminal domain comprises one or more central domains (also referred to herein as “internal” domain) between the 5’ terminal domain and 3’ terminal domain. Typically, at least one domain in an IRES element comprises one or more higher order structures (e.g., secondary structure). A domain having a higher order structure may be located anywhere within the IRES element, so long as the IRES element retains its ability to initiate translation.

[0067] A “higher order structure,” as used herein, refers to any non-linear configuration of nucleotides in a polynucleotide or element thereof (e.g., IRES element), such as secondary structures. Secondary structures comprise any non-linear structure formed by base pair interactions (e.g., chemical pairing, repulsion) within the same polynucleotide. In some embodiments, a polynucleotide (e.g., RNA) or element thereof (e.g., IRES element) comprises one or more helices or “stems.” As used herein, a “helix” or “stem” refers to pairing of complementary nucleotides within a single polynucleotide (e.g., RNA molecule), such that the polynucleotide forms a double helical segment. In some embodiments, a polynucleotide (e.g., RNA) or element thereof (e.g., IRES element) comprises one or more loops. As used herein, a “loop” refers to a non-helical loop of unpaired nucleotides. A double helical segment may comprise a “bulge,” which is a loop formed by a plurality of non-complementary nucleotides on one strand of a double helical segment. A double helical strand may comprise an “internal loop,” which is a loop formed by separation of the double helical tract caused by a plurality of overlapping, non-complementary nucleotides on both strands. In some embodiments, a polynucleotide or element thereof comprises a stem loop. As used herein, a “stem loop” or “hairpin” comprises a helix (z.e., stem) and loop, wherein a polynucleotide strand folds back on itself. A stem loop may also comprise additional structural elements, for example, a junction, here referring to a multi-branch loop or point of connection between distinct helical sections of the same polynucleotide molecule (e.g., RNA strand). Non-limiting examples of secondary structures comprising a junction include a tetraloop (a junction connecting four helices) or clover leaf. In some embodiments, a polynucleotide (e.g., RNA) or element thereof (e.g., IRES element) having a higher order structure comprises a combination of one or more helix, loop, stem loop, or junction, for example, in a domain of an IRES element. Further descriptions of these and additional polynucleotide higher order structures are provided by Hendrix et al., 2005, Q Rev Biophys, 38(3): 221-243; Nowakowski and Tinoco, 1997, Semin Virol, 8(3): 153-165; Taylor and Sobczak, 2020, Int J Mol Sci, 21, 5161.

[0068] Engineered IRES elements may be of any length. In some embodiments, an engineered IRES element is a long IRES element, such that it consists of at least 100 nucleotides. In some embodiments, a naturally occurring IRES element is a short IRES element, such that it consists of fewer than 100 nucleotides. In some embodiments, an engineered IRES element is derived from a naturally occurring IRES element. In some embodiments, an engineered IRES element is derived from a naturally occurring or synthetic IRES element, in which one or more insertion sequences have been introduced to one or more domains. In some embodiments, an engineered IRES element is derived from a naturally occurring or synthetic IRES element, in which one or more domains has been deleted. In some embodiments, an engineered IRES element is between 50 to 1000 nucleotides in length. In some embodiments, an engineered IRES element is between about 50 to about 100, about 50 to about 200, about 50 to about 300, about 50 to about 400, about 50 to about 500, about 50 to about 600, about 50 to about 700, about 50 to about 800, about 50 to about 900, or about 50 to about 1000 nucleotides in length. In some embodiments, an engineered IRES element is between about 100 to about 200, about 150 to about 250, about 200 to about 300, about 250 to about 350, about 300 to about 400, about 350 to about 450, about 400 to about 500, about 450 to about 550, about 500 to about 600, about 550 to about 650, about 600 to about 700, about 650 to about 750, about 700 to about 800, about 750 to about 850, about 800 to about 900, about 850 to about 950, or about 900 to about 1000 nucleotides in length. In some embodiments, an engineered IRES element is between 400 to 900 nucleotides in length. In some embodiments, an engineered IRES element is between about 400 to about 600, about 450 to about 650, 400 to about 700, about 500 to about 700, about 550 to about 750, about 500 to about 800, about 600 to about 800, about 650 to about 850, about 600 to about 900, or about 700 to about 900 nucleotides in length. In some embodiments, an engineered IRES element is between 500 to 800 nucleotides in length. In some embodiments, an engineered IRES element is about 500, about 550, about 600, about 650, about 700, about 750, or about 800 nucleotides in length. As used herein, “about” means plus or minus 10% or less of the value indicated. For example, about 800 nucleotides in length means that the length can be between 720-880 nucleotides, including any value between 720-880.

[0069] The inventors have identified that insertions to domains of an IRES element may improve translational efficiency. Engineered IRES elements disclosed herein may comprise one or more insertion sequences. As used herein, an “insertion sequence” refers to a sequence inserted into a domain of an IRES element which maintains or improves the translational efficiency of the IRES. In some embodiments, an insertion sequence comprises a secondary structure. In some embodiments, an insertion sequence comprises a stem loop. In some embodiments, an insertion sequence comprises two or more stem loops. In some embodiments, an insertion sequence comprising a secondary structure is inserted into a 5’ terminal domain of an IRES element. In some embodiments, an insertion sequence comprising a secondary structure is inserted into a 3’ terminal domain of an IRES element. In some embodiments, an insertion sequence comprising a secondary structure is inserted into a central domain of an IRES element. In some embodiments, an insertion sequence comprising a secondary structure is inserted into a 5’ terminal domain and a 3’ terminal domain of an IRES element.

[0070] In some embodiments, an insertion sequence comprises an aptamer. Aptamers are short, single- stranded polynucleotide sequences which bind specific targets and typically comprise secondary structures. In some embodiments, aptamers bind one or more peptides or proteins or fragments thereof (e.g., RNA binding protein). In some embodiments, aptamers bind small molecules. In some embodiments, aptamers bind heavy metal ions. In some embodiments, aptamers bind toxins. In some embodiments, aptamers bind other polynucleotides (e.g., DNA, RNA, aptamers, miRNA). In some embodiments, an aptamer is a theophylline-binding aptamer. In some embodiments, an aptamer is a tetracycline -binding aptamer. In some embodiments, an aptamer is a (6R,S)-folinic acid-binding aptamer. In some embodiments, an aptamer is an eIF4G-binding aptamer. In some embodiments, an eIF4G binding aptamer comprises the sequence set forth in SEQ ID NO: 121. Additional exemplary aptamer sequences can be found in the UTexas Aptamer Database (sites . utexas . edu / ap tamerdatabase / ) . In some embodiments, an insertion sequence comprises a spacer sequence. As used herein, a “spacer sequence” is a short sequence (e.g., fewer than 100 nucleotides in length) having minimal secondary structure (e.g., a linear sequence). In some embodiments, a spacer sequence does not comprise a secondary structure, such that the spacer sequence is a linear sequence. Typically, a spacer sequence is 100 nucleotides or fewer in length. In some embodiments, a spacer sequence is 1 to 100 nucleotides long. In some embodiments, a spacer sequence is 12 to 50 nucleotides long. In some embodiments, a spacer sequence is 32 nucleotides long. In some embodiments, a spacer sequence is 12 nucleotides long. In some embodiments, a spacer sequence is 16 nucleotides long.

[0071] In some embodiments, an insertion sequence confers additional functions to an IRES element. In some embodiments, an insertion sequence comprises a binding site for a ligand. In some embodiments, an insertion sequence comprises an aptamer binding site. In some embodiments, higher order structures interact with cofactors and / or RNA binding proteins. In some embodiments, an insertion sequence comprises a binding site for one or more co-factors or RNA binding proteins. In some embodiments, an insertion sequence comprises a ribosomal binding site (RBS). In some embodiments, an insertion sequence comprises a ribozyme binding site. In some embodiments, an insertion sequence modifies or regulates RNA expression. In some embodiments, an insertion sequence comprises a binding site for one or more translational factors. In some embodiments, an insertion sequence comprises a binding site for miRNA (e.g., miRNA sponge). In some embodiments, an insertion sequence comprises a binding site for small molecules. In some embodiments, an insertion sequence comprises a cloverleaf structure. In some embodiments, an insertion sequence comprises an anti-codon loop. In some embodiments, an insertion sequence has one or more properties of a transfer RNA (trRNA), such that the insertion sequence is capable of recognizing (e.g., binding to) a codon in mRNA.

[0072] In some embodiments, two or more insertion sequences are inserted into one or more domains of an IRES element. In some embodiments, two or more complementary insertion sequences are inserted into two or more domains of an IRES element. A first insertion sequence can be understood to be complementary to a second insertion sequence if the first insertion sequence and second insertion sequence would be capable of binding (e.g., forming a double stranded sequence). For example, a first insertion sequence may have a region of complementarity having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementarity to a corresponding region of complementarity in the second insertion sequence. In some embodiments, a first insertion sequence is inserted into a first domain of an IRES element and a second insertion sequence, which is complementary to the first insertion sequence, is inserted into a second domain of an IRES element. In some embodiments, a first insertion sequence is inserted into a 5’ terminal domain of an IRES element and a second insertion sequence, which is complementary to the first insertion sequence, is inserted into a 3’ terminal domain of an IRES element.

[0073] Engineered IRES elements disclosed herein may comprise one or more truncated domains. As used herein, a “truncated” domain is any domain of an engineered IRES element in which the truncated domain is shortened in length relative to a corresponding domain of the IRES element from which the engineered IRES element is derived. In some embodiments, an engineered IRES element comprises a truncated domain in which one or more nucleotides have been deleted. In some embodiments, an engineered IRES element comprises a truncated domain in which a previously existing higher order structure (e.g., stem loop) has been shortened. In some embodiments, an engineered IRES element comprises a truncated domain in which a previously existing higher order structure (e.g., stem loop) has been deleted, such that the domain no longer comprises a higher order structure. In some embodiments, an engineered IRES element comprises a truncated domain in which all nucleotides have been deleted.

[0074] Insertion sequences may be inserted into any domain of an IRES element; similarly, any domain of an IRES element may be truncated.

[0075] Exemplary Engineered. IRES Elements

[0076] The inventors have identified several combinations of insertion sequences, truncations, and combinations thereof which improve translational efficiency of an engineered IRES element relative to wildtype (e.g., unmodified) IRES elements.

[0077] Provided herein, in some aspects, are engineered IRES elements comprising a single domain comprising one or more insertion sequences. In some embodiments, an engineered IRES element comprises a single domain comprising one or more insertion sequences, wherein one or more insertion sequence comprises a secondary structure (e.g., stem loop). In some embodiments, an engineered IRES element comprises a single domain comprising one or more insertion sequences, wherein one or more insertion sequence comprises an aptamer. In some embodiments, an engineered IRES element comprises a single domain comprising one or more insertion sequences, wherein the one or more insertion sequence comprises a spacer sequence. In some embodiments, an engineered IRES element comprises a single domain comprising one or more insertion sequences, wherein the one or more insertion sequence comprises a spacer sequence of 1 to 100 nucleotides in length. In some embodiments, an engineered IRES element comprises a single domain comprising one or more insertion sequences, wherein the one or more insertion sequence comprises a spacer sequence of 12 to 50 nucleotides in length. In some embodiments, an engineered IRES element comprises a single domain comprising one or more insertion sequences, wherein the one or more insertion sequence comprises a spacer sequence of 32 nucleotides in length. In some embodiments, an engineered IRES element comprises a single domain comprising one or more insertion sequences, wherein the one or more insertion sequence comprises a spacer sequence of 12 to 16 nucleotides in length. In some embodiments, an engineered IRES element comprises a single domain comprising one or more insertion sequences, wherein the one or more insertion sequence comprises a spacer sequence of 12 nucleotides in length. In some embodiments, an engineered IRES element comprises a single domain comprising one or more insertion sequences, wherein the one or more insertion sequence comprises a spacer sequence of 16 nucleotides in length.

[0078] Provided herein, in some aspects, are engineered IRES elements comprising two or more domains, such that a first domain is a 3’ terminal domain and a second domain is a 5’ terminal domain. In some embodiments, an engineered IRES element comprising a 3’ terminal domain and a 5’ terminal domain comprises one or more insertion sequences in the 5’ terminal domain. In some embodiments, an engineered IRES element comprising a 3’ terminal domain and a 5’ terminal domain comprises insertion of a secondary structure in the 5’ terminal domain. In some embodiments, an engineered IRES element comprising a 3’ terminal domain and a 5’ terminal domain comprises insertion of a stem loop in the 5’ terminal domain. In some embodiments, an engineered IRES element comprising a 3’ terminal domain and a 5’ terminal domain comprises insertion of an aptamer in the 5’ terminal domain. In some embodiments, an engineered IRES element comprising a 3’ terminal domain and a 5’ terminal domain comprises insertion of an eIF4G aptamer in the 5’ terminal domain. In some embodiments, an engineered IRES element comprising a 3’ terminal domain and a 5’ terminal domain comprises insertion of a spacer sequence in the 5’ terminal domain. In some embodiments, an engineered IRES element comprising a 3’ terminal domain and a 5’ terminal domain comprises insertion of a spacer sequence in the 5’ terminal domain, wherein the spacer sequence is 1 to 100 nucleotides in length. In some embodiments, an engineered IRES element comprising a 3’ terminal domain and a 5’ terminal domain comprises insertion of a spacer sequence in the 5’ terminal domain, wherein the spacer sequence is 12 to 50 nucleotides in length. In some embodiments, an engineered IRES element comprising a 3’ terminal domain and a 5’ terminal domain comprises insertion of a spacer sequence in the 5’ terminal domain, wherein the spacer sequence is 32 nucleotides in length. In some embodiments, an engineered IRES element comprising a 3’ terminal domain and a 5’ terminal domain comprises insertion of a spacer sequence in the 5’ terminal domain, wherein the spacer sequence is 12 to 16 nucleotides in length. In some embodiments, an engineered IRES element comprising a 3’ terminal domain and a 5’ terminal domain comprises insertion of a spacer sequence in the 5’ terminal domain, wherein the spacer sequence is 12 nucleotides in length. In some embodiments, an engineered IRES element comprising a 3’ terminal domain and a 5’ terminal domain comprises insertion of a spacer sequence in the 5’ terminal domain, wherein the spacer sequence is 16 nucleotides in length.

[0079] Provided herein, in some aspects, are engineered IRES elements comprising three or more domains, such that a first domain is a 3’ terminal domain, a second domain is a 5’ terminal domain, and a third or more domains are central domains positioned between the 3’ terminal domain and the 5’ terminal domain. In some embodiments, an engineered IRES element comprising a 3’ terminal domain, a 5’ terminal domain, and one or more central domains comprises one or more insertion sequences in the 5’ terminal domain. In some embodiments, an engineered IRES element comprising a 3’ terminal domain, a 5’ terminal domain, and one or more central domains comprises insertion of a secondary structure in the 5’ terminal domain. In some embodiments, an engineered IRES element comprising a 3’ terminal domain, a 5’ terminal domain, and one or more central domains comprises insertion of a stem loop in the 5’ terminal domain. In some embodiments, an engineered IRES element comprising a 3’ terminal domain, a 5’ terminal domain, and one or more central domains comprises insertion of an aptamer in the 5’ terminal domain. In some embodiments, an engineered IRES element comprising a 3’ terminal domain, a 5’ terminal domain, and one or more central domains comprises insertion of an eIF4G aptamer in the 5’ terminal domain. In some embodiments, an engineered IRES element comprising a 3’ terminal domain, a 5’ terminal domain, and one or more central domains comprises insertion of a spacer sequence in the 5’ terminal domain. In some embodiments, an engineered IRES element comprising a 3’ terminal domain, a 5’ terminal domain, and one or more central domains comprises insertion of a spacer sequence in the 5’ terminal domain, wherein the spacer sequence is 1 to 100 nucleotides in length. In some embodiments, an engineered IRES element comprising a 3’ terminal domain, a 5’ terminal domain, and one or more central domains comprises insertion of a spacer sequence in the 5’ terminal domain, wherein the spacer sequence is 12 to 50 nucleotides in length. In some embodiments, an engineered IRES element comprising a 3’ terminal domain, a 5’ terminal domain, and one or more central domains comprises insertion of a spacer sequence in the 5’ terminal domain, wherein the spacer sequence is 32 nucleotides in length. In some embodiments, an engineered IRES element comprising a 3’ terminal domain, a 5’ terminal domain, and one or more central domains comprises insertion of a spacer sequence in the 5’ terminal domain, wherein the spacer sequence is 12 to 16 nucleotides in length. In some embodiments, an engineered IRES element comprising a 3’ terminal domain, a 5’ terminal domain, and one or more central domains comprises insertion of a spacer sequence in the 5’ terminal domain, wherein the spacer sequence is 12 nucleotides in length. In some embodiments, an engineered IRES element comprising a 3’ terminal domain, a 5’ terminal domain, and one or more central domains comprises insertion of a spacer sequence in the 5’ terminal domain, wherein the spacer sequence is 16 nucleotides in length.

[0080] In some embodiments, an engineered IRES element comprising a 3’ terminal domain, a 5’ terminal domain, and one or more central domains comprises one or more insertion sequences in the 3’ terminal domain. In some embodiments, an engineered IRES element comprising a 3’ terminal domain, a 5’ terminal domain, and one or more central domains comprises insertion of a secondary structure in the 3’ terminal domain. In some embodiments, an engineered IRES element comprising a 3’ terminal domain, a 5’ terminal domain, and one or more central domains comprises insertion of a stem loop in the 3’ terminal domain. In some embodiments, an engineered IRES element comprising a 3’ terminal domain, a 5’ terminal domain, and one or more central domains comprises insertion of an aptamer in the 3’ terminal domain. In some embodiments, an engineered IRES element comprising a 3’ terminal domain, a 5’ terminal domain, and one or more central domains comprises insertion of an eIF4G aptamer in the 3’ terminal domain. In some embodiments, an engineered IRES element comprising a 3’ terminal domain, a 5’ terminal domain, and one or more central domains comprises insertion of a spacer sequence in the 3’ terminal domain. In some embodiments, an engineered IRES element comprising a 3’ terminal domain, a 5’ terminal domain, and one or more central domains comprises insertion of a spacer sequence in the 3’ terminal domain, wherein the spacer sequence is 1 to 100 nucleotides in length. In some embodiments, an engineered IRES element comprising a 3’ terminal domain, a 5’ terminal domain, and one or more central domains comprises insertion of a spacer sequence in the 3’ terminal domain, wherein the spacer sequence is 12 to 50 nucleotides in length. In some embodiments, an engineered IRES element comprising a 3’ terminal domain, a 5’ terminal domain, and one or more central domains comprises insertion of a spacer sequence in the 3’ terminal domain, wherein the spacer sequence is 32 nucleotides in length. In some embodiments, an engineered IRES element comprising a 3’ terminal domain, a 5’ terminal domain, and one or more central domains comprises insertion of a spacer sequence in the 3’ terminal domain, wherein the spacer sequence is 12 to 16 nucleotides in length. In some embodiments, an engineered IRES element comprising a 3’ terminal domain, a 5’ terminal domain, and one or more central domains comprises insertion of a spacer sequence in the 3’ terminal domain, wherein the spacer sequence is 12 nucleotides in length. In some embodiments, an engineered IRES element comprising a 3’ terminal domain, a 5’ terminal domain, and one or more central domains comprises insertion of a spacer sequence in the 3’ terminal domain, wherein the spacer sequence is 16 nucleotides in length.

[0081] In some embodiments, an engineered IRES element comprising a 3’ terminal domain, a 5’ terminal domain, and one or more central domains comprises one or more insertion sequences in a central domain. In some embodiments, an engineered IRES element comprising a 3’ terminal domain, a 5’ terminal domain, and one or more central domains comprises insertion of a secondary structure in a central domain. In some embodiments, an engineered IRES element comprising a 3’ terminal domain, a 5’ terminal domain, and one or more central domains comprises insertion of a stem loop in a central domain. In some embodiments, an engineered IRES element comprising a 3’ terminal domain, a 5’ terminal domain, and one or more central domains comprises insertion of an aptamer in a central domain. In some embodiments, an engineered IRES element comprising a 3’ terminal domain, a 5’ terminal domain, and one or more central domains comprises insertion of an eIF4G aptamer in a central domain. In some embodiments, an engineered IRES element comprising a 3’ terminal domain, a 5’ terminal domain, and one or more central domains comprises insertion of a spacer sequence in a central domain. In some embodiments, an engineered IRES element comprising a 3’ terminal domain, a 5’ terminal domain, and one or more central domains comprises insertion of a spacer sequence in a central domain, wherein the spacer sequence is 1 to 100 nucleotides in length. In some embodiments, an engineered IRES element comprising a 3’ terminal domain, a 5’ terminal domain, and one or more central domains comprises insertion of a spacer sequence in a central domain, wherein the spacer sequence is 12 to 50 nucleotides in length. In some embodiments, an engineered IRES element comprising a 3’ terminal domain, a 5’ terminal domain, and one or more central domains comprises insertion of a spacer sequence in a central domain, wherein the spacer sequence is 32 nucleotides in length. In some embodiments, an engineered IRES element comprising a 3’ terminal domain, a 5’ terminal domain, and one or more central domains comprises insertion of a spacer sequence in a central domain, wherein the spacer sequence is 12 to 16 nucleotides in length. In some embodiments, an engineered IRES element comprising a 3’ terminal domain, a 5’ terminal domain, and one or more central domains comprises insertion of a spacer sequence in a central domain, wherein the spacer sequence is 12 nucleotides in length. In some embodiments, an engineered IRES element comprising a 3’ terminal domain, a 5’ terminal domain, and one or more central domains comprises insertion of a spacer sequence in a central domain, wherein the spacer sequence is 16 nucleotides in length.

[0082] Provided herein, in some aspects, are engineered IRES elements comprising a single domain comprising one or more truncations. In some embodiments, an engineered IRES element comprises a single domain comprising a truncation, wherein one or more secondary structures (e.g., stem loop) have been shortened. In some embodiments, an engineered IRES element comprises a single domain comprising a truncation, wherein one or more secondary structures (e.g., stem loop) have been deleted.

[0083] Provided herein, in some aspects, are engineered IRES elements comprising two or more domains, such that one domain is a 3’ terminal domain and another domain is a 5’ terminal domain. In some embodiments, an engineered IRES element comprising a 3’ terminal domain and a 5’ terminal domain comprises a truncation in the 5’ terminal domain. In some embodiments, an engineered IRES element comprising a 3’ terminal domain and a 5’ terminal domain comprises a truncation in the 5’ terminal domain, wherein one or more secondary structures (e.g., stem loop) have been shortened. In some embodiments, an engineered IRES element comprising a 3’ terminal domain and a 5’ terminal domain comprises a truncation in the 5’ terminal domain, wherein one or more secondary structures (e.g., stem loop) have been deleted.

[0084] In some embodiments, an engineered IRES element comprising a 3’ terminal domain and a 5’ terminal domain comprises a truncation in the 3’ terminal domain. In some embodiments, an engineered IRES element comprising a 3’ terminal domain and a 5’ terminal domain comprises a truncation in the 3’ terminal domain, wherein one or more secondary structures (e.g., stem loop) have been shortened. In some embodiments, an engineered IRES element comprising a 3’ terminal domain and a 5’ terminal domain comprises a truncation in the 3’ terminal domain, wherein one or more secondary structures (e.g., stem loop) have been deleted.

[0085] In some embodiments, an engineered IRES element comprising a 3’ terminal domain and a 5’ terminal domain comprises a truncation in the 5’ terminal domain and the 3’ terminal domain. In some embodiments, an engineered IRES element comprising a 3’ terminal domain and a 5’ terminal domain comprises a truncation in the 5’ terminal domain and the 3’ terminal domain, wherein one or more secondary structures (e.g., stem loop) in each domain have been shortened. In some embodiments, an engineered IRES element comprising a 3’ terminal domain and a 5’ terminal domain comprises a truncation in the 5’ terminal domain and the 3’ terminal domain, wherein one or more secondary structures (e.g., stem loop) in each domain have been deleted.

[0086] Provided herein, in some aspects, are engineered IRES elements comprising three or more domains, such that one domain is a 3’ terminal domain, another domain is a 5’ terminal domain, and one or more domains are central domains positioned between the 3’ terminal domain and the 5’ terminal domain. In some embodiments, an engineered IRES element comprising a 3’ terminal domain and a 5’ terminal domain comprises a truncation in the 5’ terminal domain and a central domain. In some embodiments, an engineered IRES element comprising a 3’ terminal domain and a 5’ terminal domain comprises a truncation in the 5’ terminal domain and a central domain, wherein one or more secondary structures (e.g., stem loop) in each domain have been shortened. In some embodiments, an engineered IRES element comprising a 3’ terminal domain and a 5’ terminal domain comprises a truncation in the 5’ terminal domain and a central domain, wherein one or more secondary structures (e.g., stem loop) in each domain have been deleted.

[0087] In some embodiments, an engineered IRES element comprising a 3’ terminal domain and a 5’ terminal domain comprises a truncation in in the 3’ terminal domain and a central domain. In some embodiments, an engineered IRES element comprising a 3’ terminal domain and a 5’ terminal domain comprises a truncation in in the 3’ terminal domain and a central domain, wherein one or more secondary structures (e.g., stem loop) in each domain have been shortened. In some embodiments, an engineered IRES element comprising a 3’ terminal domain and a 5’ terminal domain comprises a truncation in in the 3’ terminal domain and a central domain, wherein one or more secondary structures (e.g., stem loop) in each domain have been deleted.

[0088] Provided herein, in some aspects, are engineered IRES elements comprising a single domain comprising one or more truncations and one or more insertion sequences. In some embodiments, an engineered IRES element comprises a single domain comprising a truncation, wherein one or more secondary structures (e.g., stem loop) have been shortened, and one or more insertion sequences (e.g., stem loops, aptamers, spacer sequences) . In some embodiments, an engineered IRES element comprises a single domain comprising a truncation, wherein one or more secondary structures (e.g., stem loop) have been deleted, and one or more insertion sequences (e.g., stem loops, aptamers, spacer sequences) .

[0089] Provided herein, in some aspects, are engineered IRES elements comprising two or more domains, such that one domain is a 3’ terminal domain and another domain is a 5’ terminal domain. In some embodiments, an engineered IRES element comprising a 3’ terminal domain and a 5’ terminal domain comprises a truncation in the 5’ terminal domain and one or more insertion sequences (e.g., stem loops, aptamers, spacer sequences) in the 3’ terminal domain. In some embodiments, an engineered IRES element comprising a 3’ terminal domain and a 5’ terminal domain comprises a truncation in the 5’ terminal domain, wherein one or more secondary structures (e.g., stem loop) have been shortened, and one or more insertion sequences (e.g., stem loops, aptamers, spacer sequences) in the 3’ terminal domain. In some embodiments, an engineered IRES element comprising a 3’ terminal domain and a 5’ terminal domain comprises a truncation in the 5’ terminal domain, wherein one or more secondary structures (e.g., stem loop) have been deleted, and one or more insertion sequences (e.g., stem loops, aptamers, spacer sequences) in the 3’ terminal domain.

[0090] In some embodiments, an engineered IRES element comprising a 3’ terminal domain and a 5’ terminal domain comprises a truncation in the 3’ terminal domain, and one or more insertion sequences (e.g., stem loops, aptamers, spacer sequences) in the 5’ terminal domain. In some embodiments, an engineered IRES element comprising a 3’ terminal domain and a 5’ terminal domain comprises a truncation in the 3’ terminal domain, wherein one or more secondary structures (e.g., stem loop) have been shortened, and one or more insertion sequences (e.g., stem loops, aptamers, spacer sequences) in the 5’ terminal domain. In some embodiments, an engineered IRES element comprising a 3’ terminal domain and a 5’ terminal domain comprises a truncation in the 3’ terminal domain, wherein one or more secondary structures (e.g., stem loop) have been deleted, and one or more insertion sequences (e.g., stem loops, aptamers, spacer sequences) in the 5’ terminal domain.

[0091] Provided herein, in some aspects, are engineered IRES elements comprising three or more domains, such that one domain is a 3’ terminal domain, another domain is a 5’ terminal domain, and one or more domains are central domains positioned between the 3’ terminal domain and the 5’ terminal domain. In some embodiments, an engineered IRES element comprises a truncation in the 5’ terminal domain and one or more insertion sequences (e.g., stem loops, aptamers, spacer sequences) in a central domain. In some embodiments, an engineered IRES element comprises a truncation in the 5’ terminal domain, wherein one or more secondary structures (e.g., stem loop) in the 5’ terminal domain have been shortened, and one or more insertion sequences (e.g., stem loops, aptamers, spacer sequences) in a central domain. In some embodiments, an engineered IRES element comprises a truncation in the 5’ terminal domain, wherein one or more secondary structures (e.g., stem loop) in the 5’ terminal domain have been deleted, and one or more insertion sequences (e.g., stem loops, aptamers, spacer sequences) in a central domain.

[0092] In some embodiments, an engineered IRES element comprises a truncation in the 3’ terminal domain and one or more insertion sequences (e.g., stem loops, aptamers, spacer sequences) in a central domain. In some embodiments, an engineered IRES element comprises a truncation in the 3’ terminal domain, wherein one or more secondary structures (e.g., stem loop) in the 3’ terminal have been shortened, and one or more insertion sequences (e.g., stem loops, aptamers, spacer sequences) in a central domain. In some embodiments, an engineered IRES element comprises a truncation in the 3’ terminal domain, wherein one or more secondary structures (e.g., stem loop) in the 3’ terminal domain have been deleted, and one or more insertion sequences (e.g., stem loops, aptamers, spacer sequences) in a central domain.

[0093] In some embodiments, an engineered IRES element comprises a truncation in a central domain and one or more insertion sequences (e.g., stem loops, aptamers, spacer sequences) in the 5’ terminal domain. In some embodiments, an engineered IRES element comprises a truncation in a central domain, wherein one or more secondary structures (e.g., stem loop) in the central domain have been shortened, and one or more insertion sequences (e.g., stem loops, aptamers, spacer sequences) in the 5’ terminal domain. In some embodiments, an engineered IRES element comprises a truncation in a central domain, wherein one or more secondary structures (e.g., stem loop) in the central domain have been deleted, and one or more insertion sequences (e.g., stem loops, aptamers, spacer sequences) in the 5’ terminal domain.

[0094] In some embodiments, an engineered IRES element comprises a truncation in a central domain and one or more insertion sequences (e.g., stem loops, aptamers, spacer sequences) in the 3’ terminal domain. In some embodiments, an engineered IRES element comprises a truncation in a central domain, wherein one or more secondary structures (e.g., stem loop) in the central domain have been shortened, and one or more insertion sequences (e.g., stem loops, aptamers, spacer sequences) in the 3’ terminal domain. In some embodiments, an engineered IRES element comprises a truncation in a central domain, wherein one or more secondary structures (e.g., stem loop) in the central domain have been deleted, and one or more insertion sequences (e.g., stem loops, aptamers, spacer sequences) in the 3’ terminal domain.

[0095] Exemplary Engineered EV71 IRES Elements

[0096] In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises an insertion sequence. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises an insertion sequence at the 5’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises an insertion sequence at the 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises an insertion sequence at the 5’ terminal domain and the 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises an insertion sequence at a central domain. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises an insertion sequence at domain I. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises an insertion sequence at domain II. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises an insertion sequence at domain III. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises an insertion sequence at domain IV. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises an insertion sequence at domain V. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises an insertion sequence at domain VI. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises an insertion sequence at domain VII.

[0097] In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises an insertion sequence having a secondary structure (e.g., a stem loop insertion sequence). In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a stem loop insertion sequence. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a stem loop insertion sequence at the 5’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a stem loop insertion sequence at the 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a stem loop insertion sequence at the 5’ terminal domain and the 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a stem loop insertion sequence at a central domain. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a stem loop insertion sequence at domain I. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a stem loop insertion sequence at domain II. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a stem loop insertion sequence at domain III. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a stem loop insertion sequence at domain IV. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a stem loop insertion sequence at domain V. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a stem loop insertion sequence at domain VI. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises an insertion sequence at domain VII.

[0098] In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a spacer insertion sequence. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a spacer insertion sequence, wherein the spacer insertion sequence is 1 to 100 nucleotides in length. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a spacer insertion sequence, wherein the spacer insertion sequence is 12 to 50 nucleotides in length. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a spacer insertion sequence, wherein the spacer insertion sequence is 32 nucleotides in length. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a spacer insertion sequence, wherein the spacer insertion sequence is 12 to 16 nucleotides in length. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a spacer insertion sequence, wherein the spacer insertion sequence is 12 nucleotides in length. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a spacer insertion sequence, wherein the spacer insertion sequence is 16 nucleotides in length. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a spacer insertion sequence at the 5’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a spacer insertion sequence at the 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a spacer insertion sequence at the 5’ terminal domain and the 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a spacer insertion sequence at a central domain. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a spacer insertion sequence at domain I. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a spacer insertion sequence at domain II. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a spacer insertion sequence at domain III. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a spacer insertion sequence at domain IV. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a spacer insertion sequence at domain V. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a spacer insertion sequence at domain VI. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises an insertion sequence at domain VII.

[0099] In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises an aptamer insertion sequence. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a theophylline-binding aptamer insertion sequence. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a tetracycline-binding aptamer insertion sequence. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a (6R,S)-folinic acid-binding aptamer insertion sequence. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises an eIF4G-binding aptamer insertion sequence. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises an aptamer insertion sequence at the 5’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises an aptamer insertion sequence at the 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises an aptamer insertion sequence at the 5’ terminal domain and the 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises an aptamer insertion sequence at a central domain. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises an aptamer insertion sequence at domain I. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises an aptamer insertion sequence at domain II. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises an aptamer insertion sequence at domain III. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises an aptamer insertion sequence at domain IV. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises an aptamer insertion sequence at domain V. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises an aptamer insertion sequence at domain VI. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises an insertion sequence at domain VII.

[0100] In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated 5’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated 5’ terminal domain and a truncated 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated central domain. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain I. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain II. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain III. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain IV. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain V. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain VI. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain VII. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain I and a truncated domain II. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain I and a truncated domain VI. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain I and a truncated domain VII. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain II and a truncated domain VI. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain II and a truncated domain VII. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain VI and a truncated domain VII. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain I, a truncated domain II, and a truncated domain VI. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain I, a truncated domain II, and a truncated domain VII. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain I, a truncated domain II, a truncated domain VI, and a truncated domain VII.

[0101] In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated 5’ terminal domain, wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated 3’ terminal domain, wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated 5’ terminal domain, wherein one or more secondary structures (e.g., a stem loop) have been shortened, and a truncated 3’ terminal domain, wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated central domain, wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and a truncated domain I, wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain II, wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain III, wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain IV, wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain V, wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain VI, wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain VII, wherein one or more secondary structures (e.g., a stem loop) have been shortened.

[0102] In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain I and a truncated domain II, wherein one or more secondary structures (e.g., a stem loop) in each domain have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain I and a truncated domain VI, wherein one or more secondary structures (e.g., a stem loop) in each domain have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain I and a truncated domain VII, wherein one or more secondary structures (e.g., a stem loop) in each domain have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain II and a truncated domain VI, wherein one or more secondary structures (e.g., a stem loop) in each domain have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain II and a truncated domain VII, wherein one or more secondary structures (e.g., a stem loop) in each domain have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain VI and a truncated domain VII, wherein one or more secondary structures (e.g., a stem loop) in each domain have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain I, a truncated domain II, and a truncated domain VI, wherein one or more secondary structures (e.g., a stem loop) in each domain have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain I, a truncated domain II, and a truncated domain VII, wherein one or more secondary structures (e.g., a stem loop) in each domain have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain I, a truncated domain II, a truncated domain VI, and a truncated domain VII, wherein one or more secondary structures (e.g., a stem loop) in each domain have been shortened.

[0103] In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated 5’ terminal domain, wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated 3’ terminal domain, wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated 5’ terminal domain, wherein one or more secondary structures (e.g., a stem loop) have been deleted, and a truncated 3’ terminal domain, wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated central domain, wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and a truncated domain I, wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain II, wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain III, wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain IV, wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain V, wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain VI, wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain VII, wherein one or more secondary structures (e.g., a stem loop) have been deleted.

[0104] In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain I and a truncated domain II, wherein one or more secondary structures (e.g., a stem loop) in each domain have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain I and a truncated domain VI, wherein one or more secondary structures (e.g., a stem loop) in each domain have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain I and a truncated domain VII, wherein one or more secondary structures (e.g., a stem loop) in each domain have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain II and a truncated domain VI, wherein one or more secondary structures (e.g., a stem loop) in each domain have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain II and a truncated domain VII, wherein one or more secondary structures (e.g., a stem loop) in each domain have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain VI and a truncated domain VII, wherein one or more secondary structures (e.g., a stem loop) in each domain have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain I, a truncated domain II, and a truncated domain VI, wherein one or more secondary structures (e.g., a stem loop) in each domain have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain I, a truncated domain II, and a truncated domain VII, wherein one or more secondary structures (e.g., a stem loop) in each domain have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain I, a truncated domain II, a truncated domain VI, and a truncated domain VII, wherein one or more secondary structures (e.g., a stem loop) in each domain have been deleted.

[0105] In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain I and an insertion sequence at the 5’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain I and an insertion sequence at the 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain I, an insertion sequence at the 5’ terminal domain, and an insertion sequence at the 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain VII and an insertion sequence at the 5’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain VII and an insertion sequence at the 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain VII, an insertion sequence at the 5’ terminal domain, and an insertion sequence at the 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain I, a truncated domain VII, and an insertion sequence at the 5’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype EV71 IRES element and comprises a truncated domain I, a truncated domain VII, and an insertion sequence at the 3’ terminal domain.

[0106] Exemplary Engineered CVB3 IRES Elements

[0107] In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises an insertion sequence. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises an insertion sequence at the 5’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises an insertion sequence at the 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises an insertion sequence at the 5’ terminal domain and the 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises an insertion sequence at a central domain. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises an insertion sequence at domain I. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises an insertion sequence at domain II. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises an insertion sequence at domain III. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises an insertion sequence at domain IV. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises an insertion sequence at domain V. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises an insertion sequence at domain VI.

[0108] In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises an insertion sequence having a secondary structure (e.g., a stem loop insertion sequence). In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a stem loop insertion sequence. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a stem loop insertion sequence at the 5’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a stem loop insertion sequence at the 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a stem loop insertion sequence at the 5’ terminal domain and the 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a stem loop insertion sequence at a central domain. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a stem loop insertion sequence at domain I. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a stem loop insertion sequence at domain II. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a stem loop insertion sequence at domain III. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a stem loop insertion sequence at domain IV. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a stem loop insertion sequence at domain V. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a stem loop insertion sequence at domain VI.

[0109] In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a spacer insertion sequence. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a spacer insertion sequence, wherein the spacer insertion sequence is 1 to 100 nucleotides in length. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a spacer insertion sequence, wherein the spacer insertion sequence is 12 to 50 nucleotides in length. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a spacer insertion sequence, wherein the spacer insertion sequence is 32 nucleotides in length. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a spacer insertion sequence, wherein the spacer insertion sequence is 12 to 16 nucleotides in length. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a spacer insertion sequence, wherein the spacer insertion sequence is 12 nucleotides in length. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a spacer insertion sequence, wherein the spacer insertion sequence is 16 nucleotides in length. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a spacer insertion sequence at the 5’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a spacer insertion sequence at the 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a spacer insertion sequence at the 5’ terminal domain and the 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a spacer insertion sequence at a central domain. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a spacer insertion sequence at domain I. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a spacer insertion sequence at domain II. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a spacer insertion sequence at domain III. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a spacer insertion sequence at domain IV. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a spacer insertion sequence at domain V. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a spacer insertion sequence at domain VI.

[0110] In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises an aptamer insertion sequence. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a theophylline-binding aptamer insertion sequence. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a tetracycline -binding aptamer insertion sequence. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a (6R,S)-folinic acid-binding aptamer insertion sequence. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises an eIF4G-binding aptamer insertion sequence. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises an aptamer insertion sequence at the 5’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises an aptamer insertion sequence at the 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises an aptamer insertion sequence at the 5’ terminal domain and the 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises an aptamer insertion sequence at a central domain. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises an aptamer insertion sequence at domain I. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises an aptamer insertion sequence at domain II. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises an aptamer insertion sequence at domain III. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises an aptamer insertion sequence at domain IV. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises an aptamer insertion sequence at domain V. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises an aptamer insertion sequence at domain VI.

[0111] In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a truncated domain. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a truncated 5’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a truncated 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a truncated 5’ terminal domain and a truncated 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a truncated central domain. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a truncated domain I. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a truncated domain II. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a truncated domain III. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a truncated domain IV. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a truncated domain V.

[0112] In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a truncated domain I and a truncated domain II. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a truncated domain I and a truncated domain VI. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a truncated domain II and a truncated domain VI. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a truncated domain I, a truncated domain II, and a truncated domain VI.

[0113] In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a truncated domain wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a truncated 5’ terminal domain, wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a truncated 3’ terminal domain, wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a truncated 5’ terminal domain, wherein one or more secondary structures (e.g., a stem loop) have been shortened, and a truncated 3’ terminal domain, wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a truncated central domain, wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and a truncated domain I, wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a truncated domain II, wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a truncated domain III, wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a truncated domain IV, wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a truncated domain V, wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a truncated domain VI, wherein one or more secondary structures (e.g., a stem loop) have been shortened.

[0114] In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a truncated domain I and a truncated domain II, wherein one or more secondary structures (e.g., a stem loop) in each domain have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a truncated domain I and a truncated domain VI, wherein one or more secondary structures (e.g., a stem loop) in each domain have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a truncated domain II and a truncated domain VI, wherein one or more secondary structures (e.g., a stem loop) in each domain have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a truncated domain I, a truncated domain II, and a truncated domain VI, wherein one or more secondary structures (e.g., a stem loop) in each domain have been shortened.

[0115] In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a truncated domain wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a truncated 5’ terminal domain, wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a truncated 3’ terminal domain, wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a truncated 5’ terminal domain, wherein one or more secondary structures (e.g., a stem loop) have been deleted, and a truncated 3’ terminal domain, wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a truncated central domain, wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and a truncated domain I, wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a truncated domain II, wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a truncated domain III, wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a truncated domain IV, wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a truncated domain V, wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a truncated domain VI, wherein one or more secondary structures (e.g., a stem loop) have been deleted.

[0116] In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a truncated domain I and a truncated domain II, wherein one or more secondary structures (e.g., a stem loop) in each domain have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a truncated domain I and a truncated domain VI, wherein one or more secondary structures (e.g., a stem loop) in each domain have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a truncated domain II and a truncated domain VI, wherein one or more secondary structures (e.g., a stem loop) in each domain have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a truncated domain I, a truncated domain II, and a truncated domain VI, wherein one or more secondary structures (e.g., a stem loop) in each domain have been deleted.

[0117] In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a truncated domain I and an insertion sequence at the 5’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a truncated domain I and an insertion sequence at the 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a truncated domain I, an insertion sequence at the 5’ terminal domain, and an insertion sequence at the 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a truncated domain VI and an insertion sequence at the 5’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a truncated domain VI and an insertion sequence at the 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a truncated domain VI, an insertion sequence at the 5’ terminal domain, and an insertion sequence at the 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a truncated domain I, a truncated domain VI, and an insertion sequence at the 5’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype CVB3 IRES element and comprises a truncated domain I, a truncated domain VI, and an insertion sequence at the 3’ terminal domain.

[0118] Exemplary Engineered PV IRES Elements

[0119] In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises an insertion sequence. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises an insertion sequence at the 5’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises an insertion sequence at the 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises an insertion sequence at the 5’ terminal domain and the 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises an insertion sequence at a central domain. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises an insertion sequence at domain I. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises an insertion sequence at domain II. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises an insertion sequence at domain III. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises an insertion sequence at domain IV. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises an insertion sequence at domain V. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises an insertion sequence at domain VI. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises an insertion sequence having a secondary structure (e.g., a stem loop insertion sequence). In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a stem loop insertion sequence. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a stem loop insertion sequence at the 5’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a stem loop insertion sequence at the 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a stem loop insertion sequence at the 5’ terminal domain and the 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a stem loop insertion sequence at a central domain. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a stem loop insertion sequence at domain I. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a stem loop insertion sequence at domain II. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a stem loop insertion sequence at domain III. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a stem loop insertion sequence at domain IV. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a stem loop insertion sequence at domain V. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a stem loop insertion sequence at domain VI.

[0120] In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a spacer insertion sequence. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a spacer insertion sequence, wherein the spacer insertion sequence is 1 to 100 nucleotides in length. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a spacer insertion sequence, wherein the spacer insertion sequence is 12 to 50 nucleotides in length. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a spacer insertion sequence, wherein the spacer insertion sequence is 32 nucleotides in length. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a spacer insertion sequence, wherein the spacer insertion sequence is 12 to 16 nucleotides in length. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a spacer insertion sequence, wherein the spacer insertion sequence is 12 nucleotides in length. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a spacer insertion sequence, wherein the spacer insertion sequence is 16 nucleotides in length. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a spacer insertion sequence at the 5’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a spacer insertion sequence at the 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a spacer insertion sequence at the 5’ terminal domain and the 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a spacer insertion sequence at a central domain. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a spacer insertion sequence at domain I. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a spacer insertion sequence at domain II. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a spacer insertion sequence at domain III. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a spacer insertion sequence at domain IV. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a spacer insertion sequence at domain V. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a spacer insertion sequence at domain VI.

[0121] In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises an aptamer insertion sequence. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a theophylline-binding aptamer insertion sequence. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a tetracycline-binding aptamer insertion sequence. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a (6R,S)-folinic acid-binding aptamer insertion sequence. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises an eIF4G-binding aptamer insertion sequence. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises an aptamer insertion sequence at the 5’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises an aptamer insertion sequence at the 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises an aptamer insertion sequence at the 5’ terminal domain and the 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises an aptamer insertion sequence at a central domain. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises an aptamer insertion sequence at domain I. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises an aptamer insertion sequence at domain II. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises an aptamer insertion sequence at domain III. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises an aptamer insertion sequence at domain IV. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises an aptamer insertion sequence at domain V. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises an aptamer insertion sequence at domain VI.

[0122] In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a truncated domain. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a truncated 5’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a truncated 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a truncated 5’ terminal domain and a truncated 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a truncated central domain. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a truncated domain I. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a truncated domain II. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a truncated domain III. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a truncated domain IV. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a truncated domain V. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a truncated domain VI.

[0123] In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a truncated domain wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a truncated 5’ terminal domain, wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a truncated 3’ terminal domain, wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a truncated 5’ terminal domain, wherein one or more secondary structures (e.g., a stem loop) have been shortened, and a truncated 3’ terminal domain, wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a truncated central domain, wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and a truncated domain I, wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a truncated domain II, wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a truncated domain III, wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a truncated domain IV, wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a truncated domain V, wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a truncated domain VI, wherein one or more secondary structures (e.g., a stem loop) have been shortened.

[0124] In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a truncated domain wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a truncated 5’ terminal domain, wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a truncated 3’ terminal domain, wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a truncated 5’ terminal domain, wherein one or more secondary structures (e.g., a stem loop) have been deleted, and a truncated 3’ terminal domain, wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a truncated central domain, wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and a truncated domain I, wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a truncated domain II, wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a truncated domain III, wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a truncated domain IV, wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a truncated domain V, wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype PV IRES element and comprises a truncated domain VI, wherein one or more secondary structures (e.g., a stem loop) have been deleted.

[0125] Exemplary Engineered iHRV IRES Elements

[0126] In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises an insertion sequence. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises an insertion sequence at the 5’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises an insertion sequence at the 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises an insertion sequence at the 5’ terminal domain and the 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises an insertion sequence at a central domain. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises an insertion sequence at domain I. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises an insertion sequence at domain II. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises an insertion sequence at domain III. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises an insertion sequence at domain IV. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises an insertion sequence at domain V. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises an insertion sequence at domain VI.

[0127] In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises an insertion sequence having a secondary structure (e.g., a stem loop insertion sequence). In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a stem loop insertion sequence. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a stem loop insertion sequence at the 5’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a stem loop insertion sequence at the 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a stem loop insertion sequence at the 5’ terminal domain and the 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a stem loop insertion sequence at a central domain. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a stem loop insertion sequence at domain I. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a stem loop insertion sequence at domain II. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a stem loop insertion sequence at domain III. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a stem loop insertion sequence at domain IV. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a stem loop insertion sequence at domain V. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a stem loop insertion sequence at domain VI.

[0128] In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a spacer insertion sequence. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a spacer insertion sequence, wherein the spacer insertion sequence is 1 to 100 nucleotides in length. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a spacer insertion sequence, wherein the spacer insertion sequence is 12 to 50 nucleotides in length. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a spacer insertion sequence, wherein the spacer insertion sequence is 32 nucleotides in length. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a spacer insertion sequence, wherein the spacer insertion sequence is 12 to 16 nucleotides in length. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a spacer insertion sequence, wherein the spacer insertion sequence is 12 nucleotides in length. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a spacer insertion sequence, wherein the spacer insertion sequence is 16 nucleotides in length. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a spacer insertion sequence at the 5’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a spacer insertion sequence at the 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a spacer insertion sequence at the 5’ terminal domain and the 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a spacer insertion sequence at a central domain. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a spacer insertion sequence at domain I. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a spacer insertion sequence at domain II. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a spacer insertion sequence at domain III. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a spacer insertion sequence at domain IV. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a spacer insertion sequence at domain V. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a spacer insertion sequence at domain VI.

[0129] In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises an aptamer insertion sequence. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a theophylline-binding aptamer insertion sequence. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a tetracycline -binding aptamer insertion sequence. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a (6R,S)-folinic acid-binding aptamer insertion sequence. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises an eIF4G-binding aptamer insertion sequence. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises an aptamer insertion sequence at the 5’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises an aptamer insertion sequence at the 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises an aptamer insertion sequence at the 5’ terminal domain and the 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises an aptamer insertion sequence at a central domain. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises an aptamer insertion sequence at domain I. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises an aptamer insertion sequence at domain II. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises an aptamer insertion sequence at domain III. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises an aptamer insertion sequence at domain IV. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises an aptamer insertion sequence at domain V. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises an aptamer insertion sequence at domain VI.

[0130] In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a truncated domain. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a truncated 5’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a truncated 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a truncated 5’ terminal domain and a truncated 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a truncated central domain. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a truncated domain I. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a truncated domain II. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a truncated domain III. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a truncated domain IV. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a truncated domain V. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a truncated domain VI.

[0131] In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a truncated domain wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a truncated 5’ terminal domain, wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a truncated 3’ terminal domain, wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a truncated 5’ terminal domain, wherein one or more secondary structures (e.g., a stem loop) have been shortened, and a truncated 3’ terminal domain, wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a truncated central domain, wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and a truncated domain I, wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a truncated domain II, wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a truncated domain III, wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a truncated domain IV, wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a truncated domain V, wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a truncated domain VI, wherein one or more secondary structures (e.g., a stem loop) have been shortened.

[0132] In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a truncated domain wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a truncated 5’ terminal domain, wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a truncated 3’ terminal domain, wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a truncated 5’ terminal domain, wherein one or more secondary structures (e.g., a stem loop) have been deleted, and a truncated 3’ terminal domain, wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a truncated central domain, wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and a truncated domain I, wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a truncated domain II, wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a truncated domain III, wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a truncated domain IV, wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a truncated domain V, wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype iHRV IRES element and comprises a truncated domain VI, wherein one or more secondary structures (e.g., a stem loop) have been deleted.

[0133] Exemplary Engineered EMCV IRES Elements

[0134] In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises an insertion sequence. In some embodiments, an engineered IRES element is derived from a wildtype EMCV- A3 IRES element and comprises an insertion sequence. In some embodiments, an engineered IRES element is derived from a wildtype EMCV-A7 IRES element and comprises an insertion sequence. In some embodiments, an engineered IRES element is derived from a wildtype EMCV-26C IRES element and comprises an insertion sequence. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises an insertion sequence at the 5’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises an insertion sequence at the 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises an insertion sequence at the 5’ terminal domain and the 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises an insertion sequence at a central domain. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises an insertion sequence at domain D. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises an insertion sequence at domain E. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises an insertion sequence at domain F. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises an insertion sequence at domain G. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises an insertion sequence at domain H. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises an insertion sequence at domain I. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises an insertion sequence at domain J. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises an insertion sequence at domain K. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises an insertion sequence at domain L. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises an insertion sequence at domain M. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises an insertion sequence at domain N.

[0135] In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises an insertion sequence having a secondary structure (e.g., a stem loop insertion sequence). In some embodiments, an engineered IRES element is derived from a wildtype EMCV- A3 IRES element and comprises an insertion sequence having a secondary structure (e.g., a stem loop insertion sequence). In some embodiments, an engineered IRES element is derived from a wildtype EMCV-A7 IRES element and comprises an insertion sequence having a secondary structure (e.g., a stem loop insertion sequence). In some embodiments, an engineered IRES element is derived from a wildtype EMCV-26C IRES element and comprises an insertion sequence having a secondary structure (e.g., a stem loop insertion sequence). In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a stem loop insertion sequence. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a stem loop insertion sequence at the 5’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a stem loop insertion sequence at the 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a stem loop insertion sequence at the 5’ terminal domain and the 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a stem loop insertion sequence at a central domain. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a stem loop insertion sequence at domain D. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a stem loop insertion sequence at domain E. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a stem loop insertion sequence at domain F. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a stem loop insertion sequence at domain G. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a stem loop insertion sequence at domain H. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a stem loop insertion sequence at domain I. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a stem loop insertion sequence at domain J. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a stem loop insertion sequence at domain K. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a stem loop insertion sequence at domain L. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a stem loop insertion sequence at domain M. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a stem loop insertion sequence at domain N.

[0136] In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a spacer insertion sequence. In some embodiments, an engineered IRES element is derived from a wildtype EMCV- A3 IRES element and comprises a spacer insertion sequence. In some embodiments, an engineered IRES element is derived from a wildtype EMCV-A7 IRES element and comprises a spacer insertion sequence. In some embodiments, an engineered IRES element is derived from a wildtype EMCV-26C IRES element and comprises a spacer insertion sequence. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a spacer insertion sequence, wherein the spacer insertion sequence is 1 to 100 nucleotides in length. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a spacer insertion sequence, wherein the spacer insertion sequence is 12 to 50 nucleotides in length. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a spacer insertion sequence, wherein the spacer insertion sequence is 32 nucleotides in length. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a spacer insertion sequence, wherein the spacer insertion sequence is 12 to 16 nucleotides in length. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a spacer insertion sequence, wherein the spacer insertion sequence is 12 nucleotides in length. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a spacer insertion sequence, wherein the spacer insertion sequence is 16 nucleotides in length. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a spacer insertion sequence at the 5’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a spacer insertion sequence at the 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a spacer insertion sequence at the 5’ terminal domain and the 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a spacer insertion sequence at a central domain. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a spacer insertion sequence at domain D. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a spacer insertion sequence at domain E. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a spacer insertion sequence at domain F. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a spacer insertion sequence at domain G. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a spacer insertion sequence at domain H. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a spacer insertion sequence at domain I. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a spacer insertion sequence at domain J. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a spacer insertion sequence at domain K. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a spacer insertion sequence at domain L. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a spacer insertion sequence at domain M. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a spacer insertion sequence at domain N.

[0137] In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises an aptamer insertion sequence. In some embodiments, an engineered IRES element is derived from a wildtype EMCV- A3 IRES element and comprises an aptamer insertion sequence. In some embodiments, an engineered IRES element is derived from a wildtype EMCV-A7 IRES element and comprises an aptamer insertion sequence. In some embodiments, an engineered IRES element is derived from a wildtype EMCV-26C IRES element and comprises an aptamer insertion sequence. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a theophylline-binding aptamer insertion sequence. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a tetracycline -binding aptamer insertion sequence. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a (6R,S)-folinic acid-binding aptamer insertion sequence. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises an eIF4G-binding aptamer insertion sequence. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises an aptamer insertion sequence at the 5’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises an aptamer insertion sequence at the 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises an aptamer insertion sequence at the 5’ terminal domain and the 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises an aptamer insertion sequence at a central domain. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises an aptamer insertion sequence at domain D. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises an aptamer insertion sequence at domain E. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises an aptamer insertion sequence at domain F. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises an aptamer insertion sequence at domain G. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises an aptamer insertion sequence at domain H. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises an aptamer insertion sequence at domain I. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises an aptamer insertion sequence at domain J. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises an aptamer insertion sequence at domain K. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises an aptamer insertion sequence at domain L. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises an aptamer insertion sequence at domain M. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises an aptamer insertion sequence at domain N.

[0138] In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a truncated domain. In some embodiments, an engineered IRES element is derived from a wildtype EMCV-A3 IRES element and comprises a truncated domain. In some embodiments, an engineered IRES element is derived from a wildtype EMCV-A7 IRES element and comprises a truncated domain. In some embodiments, an engineered IRES element is derived from a wildtype EMCV-26C IRES element and comprises a truncated domain. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a truncated 5’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a truncated 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a truncated 5’ terminal domain and a truncated 3’ terminal domain. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a truncated central domain. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a truncated domain D. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a truncated domain E. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a truncated domain F. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a truncated domain G. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a truncated domain H. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a truncated domain I. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a truncated domain J. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a truncated domain K. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a truncated domain L. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a truncated domain M. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a truncated domain N.

[0139] In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a truncated domain wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype EMCV- A3 IRES element and comprises a truncated domain wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype EMCV-A7 IRES element and comprises a truncated domain wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype EMCV-26C IRES element and comprises a truncated domain wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a truncated 5’ terminal domain, wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a truncated 3’ terminal domain, wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a truncated 5’ terminal domain, wherein one or more secondary structures (e.g., a stem loop) have been shortened, and a truncated 3’ terminal domain, wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a truncated central domain. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a truncated domain D, wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a truncated domain E, wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a truncated domain F, wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a truncated domain G, wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a truncated domain H, wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a truncated domain I, wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a truncated domain J, wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a truncated domain K, wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a truncated domain L, wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a truncated domain M, wherein one or more secondary structures (e.g., a stem loop) have been shortened. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a truncated domain N, wherein one or more secondary structures (e.g., a stem loop) have been shortened.

[0140] In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a truncated domain wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype EMCV- A3 IRES element and comprises a truncated domain wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype EMCV-A7 IRES element and comprises a truncated domain wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype EMCV-26C IRES element and comprises a truncated domain wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a truncated 5’ terminal domain, wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a truncated 3’ terminal domain, wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a truncated 5’ terminal domain, wherein one or more secondary structures (e.g., a stem loop) have been deleted, and a truncated 3’ terminal domain, wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a truncated central domain. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a truncated domain D, wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a truncated domain E, wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a truncated domain F, wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a truncated domain G, wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a truncated domain H, wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a truncated domain I, wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a truncated domain J, wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a truncated domain K, wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a truncated domain L, wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a truncated domain M, wherein one or more secondary structures (e.g., a stem loop) have been deleted. In some embodiments, an engineered IRES element is derived from a wildtype EMCV IRES element and comprises a truncated domain N, wherein one or more secondary structures (e.g., a stem loop) have been deleted.

[0141] Vectors

[0142] Aspects of the present disclosure relate to the insertion of heterologous polynucleotides into a host cell through the use of a vector. A “vector” is a vehicle for the transfer of a heterologous polynucleotide from a first environment into a second environment. A “host cell” is any cell which receives or is capable of receiving a heterologous polynucleotide of interest. In some embodiments, a host cell is a eukaryotic cell. In some embodiments, a host cell is a mammalian cell. In some embodiments, a host cell is a human cell. Host cells may be contacted by one or more vectors to introduce a heterologous polynucleotide into the cytoplasm. In some embodiments, host cells integrate inserted polynucleotides into their genome. In some embodiments, host cells transcribe or translate inserted polynucleotides. In some embodiments, host cells replicate inserted polynucleotides. In some embodiments, a vector is a plasmid. Plasmids are small extrachromosomal nucleic acid which can replicate autonomously; typically, plasmids comprise an origin of replication and at least one gene. Plasmids may exist naturally or be constructed synthetically with methods known by one skilled in the art. A plasmid may be delivered into a cell through a transfection. The term “transfection” refers to the process by which cells uptake foreign DNA in the absence of viral vectors. A cell has been “transfected” when exogenous DNA has entered the cytoplasm. Non-limiting examples of transfection include electroporation, heat shock, liposomes, nanoparticles, microinjection, sonoporation, photoporation, magnetofection, hydroporation, biolistics, continuous infusion and impalefection. Transfection can be used to introduce one or more plasmids into a host cell.

[0143] In some embodiments, a vector is a viral particle. A viral particle is a virion, comprising nucleic acids (e.g., viral genome) within a capsid. Capsids are the three- dimensional protein shells of a virus, which enclose a viral genome. In some embodiments, viral particles are enveloped. “Enveloped viruses” are viral particles which are encapsulated in a lipid membrane acquired from a host cell membrane. Viral envelopes may comprise one or more viral glycoproteins, which direct interactions of the virion with a host cell membrane, and may reduce viral immunogenicity. In some embodiments, viral particles comprise a viral genome or components thereof. In addition to components of a viral genome, viral particles used as vectors comprise a heterologous polynucleotide (e.g., DNA, RNA) of interest. Non-limiting examples of viral vectors include adeno-associated virus (AAV) vectors (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12), adenovirus vectors (e.g., HAdV2, HAdV5, Ad4, Ad5, Ad7, Ad26, ChAdOxl), baculovirus vectors, retrovirus vectors (e.g., gammaretrovirus, lentivirus), and herpesvirus vectors.

[0144] Vectors disclosed herein may be used to express a payload. In some embodiments, a host cell is contacted (e.g., transfected, transduced) by a vector (e.g., plasmid, virus) under conditions in which the vector is introduced into the cell and a payload can be expressed by the host cell. In some embodiments, a payload encoded by a vector (e.g., plasmid) is expressed in a cell-free composition under conditions under which the payload can be translated.

[0145] Exemplary Recombinant Polynucleotides

[0146] Provided herein, in some aspects, are circRNA comprising an engineered IRES element. In some embodiments, a circRNA comprises an engineered IRES element operably coupled to a coding sequence. In some embodiments, a circRNA disclosed herein comprises an engineered IRES element which is in-frame relative to the coding sequence, such that the engineered IRES element is positioned in the same reading frame as the encoded protein. In some embodiments, a circRNA comprising an in-frame engineered IRES element and a stop codon can be translated. In some embodiments, a circRNA comprising an in-frame engineered IRES element and lacking a stop-codon can be translated infinitely (e.g., rolling circle translation).

[0147] In some embodiments, a circRNA comprises an engineered IRES element is out-offrame relative to the coding sequence, such that the engineered IRES element is positioned outside of the reading frame of the encoded protein. In some embodiments, an engineered IRES element comprises a frameshift mutation relative to a non-modified (e.g., naturally occurring) IRES element. In some embodiments, an out-of-frame engineered IRES element cannot be translated. In some embodiments, a circRNA comprises an out-of-frame engineered IRES element which is still capable of being translated to express a payload. In some embodiments, translation of a circRNA comprising an out-of-frame engineered IRES element expresses a payload, wherein expression of the payload is greater than that expressed by a circRNA comprising a frameshift mutation absent the engineered IRES element.

[0148] In some embodiments, a circRNA comprising an engineered IRES element comprises a UTR. In some embodiments, a circRNA comprises an engineered IRES element comprised in a UTR (e.g., viral UTR). In some embodiments, a circRNA comprising an engineered IRES element comprises a UTR comprising one or more expression control sequences.

[0149] In some embodiments, a circRNA comprising an engineered IRES element comprises a promoter. In some embodiments, a circRNA comprising an engineered IRES element comprises an enhancer sequence.

[0150] In some embodiments, a circRNA comprising an engineered IRES element comprises an intron region or fragment thereof.

[0151] Provided herein, in some aspects, are linear RNA comprising an engineered IRES element disclosed herein. In some embodiments, a linear RNA comprises an engineered IRES element disclosed herein and a coding sequence, wherein the engineered IRES element is operably linked to the coding sequence.

[0152] In some embodiments, a linear RNA comprises a UTR. In some embodiments, a linear RNA comprises a UTR comprising an engineered IRES element.

[0153] Described herein, in some aspects, are DNA sequences which encode circRNA. In some embodiments, DNA sequences provided herein encode circRNA comprising an engineered IRES element disclosed herein. In some embodiments, DNA sequences provided herein encode circRNA comprising an IRES element disclosed herein and a coding sequence, wherein the IRES element is operably linked to the coding sequence. In some embodiments, a DNA sequence encoding a circRNA is a plasmid. In some embodiments, a DNA sequence encoding a circRNA is a plasmid comprising a sequence encoding an N-terminal tag. In some embodiments, a DNA sequence encoding a circRNA is a plasmid comprising a sequence encoding a C-terminal tag.

[0154] Exemplary DNA sequences encoding RNA comprising engineered IRES elements are set forth in SEQ ID NOs: 122-126. The skilled artisan will understand that any one or more of thymine bases (T) in any one of the polynucleotides provided herein may optionally be uracil bases (U), and / or any one or more U may optionally be a T.

[0155] EMBODIMENTS

[0156] Embodiment 1. An engineered internal ribosome entry site (IRES) element, comprising: a plurality of domains, each domain comprising at least one stem loop, such that the IRES element comprises at least a 3’ terminal domain and a 5’ terminal domain, and optionally, one or more central domains between the 3’ terminal domain and the 5’ terminal domain; and an insertion sequence, wherein the insertion sequence is inserted into one of the plurality of domains.

[0157] Embodiment 2. The engineered IRES element of embodiment 1, wherein the insertion sequence comprises a secondary structure.

[0158] Embodiment 3. The engineered IRES element of embodiment 2, wherein the secondary structure comprises a stem loop.

[0159] Embodiment 4. The engineered IRES element of embodiment 1 or 2, wherein the insertion sequence comprises an aptamer.

[0160] Embodiment 5. The engineered IRES element of embodiment 3, wherein the aptamer binds to eIF4G.

[0161] Embodiment 6. The engineered IRES element of embodiment 1, wherein the insertion sequence comprises a spacer sequence.

[0162] Embodiment 7. The engineered IRES element of embodiment 6, wherein the spacer sequence is 12-16bp in length.

[0163] Embodiment 8. The engineered IRES element of embodiment 6 or 7, wherein the spacer sequence does not comprise a secondary structure.

[0164] Embodiment 9. The engineered IRES element of any one of embodiments 1 to 8, comprising two or more insertion sequences inserted into two or more of the plurality of domains. Embodiment 10. The engineered IRES element of embodiment 9, wherein the two or more insertion sequences are no less than 20bp apart.

[0165] Embodiment 11. The engineered IRES element of embodiment 9, wherein at least one insertion sequence is complementary to at least one other insertion sequence.

[0166] Embodiment 12. The engineered IRES element of any one of embodiments 1 to 11, wherein an insertion sequence is inserted into the 3’ terminal domain.

[0167] Embodiment 13. The engineered IRES element of any one of embodiments 1 to 12, wherein an insertion sequence is inserted into the 5’ terminal domain.

[0168] Embodiment 14. The engineered IRES element of any one of embodiments 1 to 13, wherein at least one of the domains comprises a truncated stem loop.

[0169] Embodiment 15. The engineered IRES element of embodiment 14, wherein the truncated stem loop is deleted.

[0170] Embodiment 16. An engineered internal ribosome entry site (IRES) element, comprising: a plurality of domains each comprising at least one stem loop, such that the IRES element comprises at least a 3’ terminal domain and a 5’ terminal domain, and optionally, one or more central domains between the 3’ terminal domain and the 5’ terminal domain; wherein at least one stem loop in the IRES element is a truncated stem loop.

[0171] Embodiment 17. The engineered IRES element of embodiment 16, wherein the truncated stem loop is deleted.

[0172] Embodiment 18. The engineered IRES element of embodiment 16 or 17, wherein the truncated stem loop is in the 3’ terminal domain.

[0173] Embodiment 19. The engineered IRES element of any one of embodiments 16 to 18, wherein the truncated stem loop is in the 5’ terminal domain. Embodiment 20. The engineered IRES element of any one of embodiments 16 to 19, wherein two or more stem loops are truncated.

[0174] Embodiment 21. The engineered IRES element of any one of embodiments 16 to 20, further comprising an insertion sequence.

[0175] Embodiment 22. The engineered IRES element of embodiment 21, wherein the insertion sequence comprises a secondary structure.

[0176] Embodiment 23. The engineered IRES element of embodiment 21 or 22, wherein the secondary structure comprises a stem loop.

[0177] Embodiment 24. The engineered IRES element of embodiment 23, wherein the insertion sequence comprises an aptamer.

[0178] Embodiment 25. The engineered IRES element of embodiment 24, wherein the aptamer binds to eIF4G.

[0179] Embodiment 26. The engineered IRES element of embodiment 21, wherein the insertion sequence comprises a spacer sequence.

[0180] Embodiment 27. The engineered IRES element of embodiment 26, wherein the spacer sequence is less than lOObp in length.

[0181] Embodiment 28. The engineered IRES element of embodiment 27, wherein the spacer sequence is 12-50bp in length.

[0182] Embodiment 29. The engineered IRES element of embodiment 27 or 28, wherein the spacer sequence is about 32 bp in length.

[0183] Embodiment 30. The engineered IRES element of any one of embodiments 26-29, wherein the spacer sequence does not comprise a secondary structure. Embodiment 31. The engineered IRES element of any one of embodiments 16 to 30, comprising two or more insertion sequences inserted into two or more domains.

[0184] Embodiment 32. The engineered IRES element of embodiment 31, wherein the two or more insertion sequences are no less than 20bp apart.

[0185] Embodiment 33. The engineered IRES element of embodiment 31 or 32, wherein at least one insertion sequence is complementary to at least one other insertion sequence.

[0186] Embodiment 34. The engineered IRES element of any one of embodiments 16 to 33, wherein an insertion sequence is inserted into the 3’ terminal domain.

[0187] Embodiment 35. The engineered IRES element of any one of embodiments 16 to 34, wherein an insertion sequence is inserted into the 5’ terminal domain.

[0188] Embodiment 36. The engineered IRES element of any one of embodiments 16 to 35, wherein the 3’ terminal domain and the 5’ terminal domain both comprise a truncated stem loop.

[0189] Embodiment 37. The engineered IRES element of any one of embodiments 1 to 36, wherein the engineered IRES element is modified from a wildtype IRES element.

[0190] Embodiment 38. The engineered IRES element of any one of embodiments 1 to 37, wherein the engineered IRES element is derived from a human genome.

[0191] Embodiment 39. The engineered IRES element of embodiment 38, wherein the engineered IRES element is a short IRES.

[0192] Embodiment 40. The engineered IRES element of embodiment 37, wherein the wildtype IRES element is an IRES element of Taura syndrome virus, Triatoma virus, Theiler's encephalomyelitis virus, simian Virus 40, Solenopsis invicta virus 1, Rhopalosiphum padi virus, Reticuloendotheliosis virus, human poliovirus 1, Plautia stall intestine virus, Kashmir bee virus, Human rhinovirus (iHRV), Homalodisca coagulata virus- 1, Human Immunodeficiency Virus type 1, Homalodisca coagulata virus- 1, Himetobi P virus, Hepatitis C virus, Hepatitis A virus, Hepatitis GB virus, foot and mouth disease virus, Human enterovirus 71 (EV71), Equine rhinitis virus, Ectropis obliqua picoma-like virus, Encephalomyocarditis virus (EMCV), Drosophila C Virus, Crucifer tobamo virus, Cricket paralysis virus, Bovine viral diarrhea virus 1, Black Queen Cell Virus, Aphid lethal paralysis virus, Avian encephalomyelitis virus, Acute bee paralysis virus, Hibiscus chlorotic ringspot virus, Classical swine fever virus, Human FGF2, Human SFTPA1, Human AML1 / RUNX1, Drosophila antennapedia, Human AQP4, Human AT1R, Human BAG-1, Human BCL2, Human BiP, Human c-IAPl, Human c-myc, Human eIF4G, Mouse NDST4L, Human LEF1, Mouse HIF1 alpha, Human n.myc, Mouse Gtx, Human p27kipl, Human PDGF2 / c-sis, Human p53, Human Pim-1, Mouse Rbm3, Drosophila reaper, Canine Scamper, Drosophila Ubx, Salivirus, Cosavirus, Parechovirus, Human UNR, Mouse UtrA, Human VEGF-A, Human XIAP, Drosophila hairless, S. cerevisiae TFIID, S. cerevisiae YAP1, Human c-src, Human FGF-1, Simian picomavirus, Turnip crinkle virus, Coxsackievirus B3 (CVB3), Coxsackievirus A (CVB1 / 2), or Poliovirus (PV).

[0193] Embodiment 41. The engineered IRES element of embodiment 37 or 40, wherein the wildtype IRES element is EMCV, EV71, CVB3, iHRV, or PV.

[0194] Embodiment 42. The engineered IRES element of embodiment 37, 40, or 41, wherein the wildtype IRES element is EMCV.

[0195] Embodiment 43. The engineered IRES element of embodiment 42, wherein the IRES element is EMCV-A7, EMCV- A3, or EMCV-26C.

[0196] Embodiment 44. A circular ribonucleic acid (circRNA) comprising an engineered IRES element of any one of embodiments 1 to 43.

[0197] Embodiment 45. The circRNA of embodiment 44, further comprising an open reading frame encoding a peptide, protein, or fragment thereof.

[0198] Embodiment 46. The circRNA of embodiment 44 or 45, further comprising an untranslated region (UTR). Embodiment 47. The circRNA of any one of embodiments 44 to 46, further comprising one or more intron regions or fragments thereof.

[0199] Embodiment 48. The circRNA of any one of embodiments 44 to 47, wherein the circRNA comprises a frameshift mutation.

[0200] Embodiment 49. The circRNA of embodiment 48, wherein translation of the circRNA expresses an amount of a functional peptide, protein, or fragment thereof greater than the circRNA comprising a frameshift mutation absent the engineered IRES element.

[0201] Embodiment 50. A linear ribonucleic acid (RNA) comprising the engineered IRES element of any one of embodiments 1 to 43.

[0202] Embodiment 51. A plasmid encoding the circRNA of any one of embodiments 44 to 49.

[0203] Embodiment 52. The plasmid of embodiment 51, further comprising an enhancer sequence.

[0204] Embodiment 53. The plasmid of embodiment 51 or 52, further comprising one or more intron regions or fragments thereof.

[0205] Embodiment 54. The plasmid of any one of embodiments 51 to 53, further comprising an N-terminal tag.

[0206] Embodiment 55. The plasmid of any one of embodiments 51 to 54, further comprising a

[0207] C-terminal tag.

[0208] Embodiment 56. A method of expressing a polypeptide, the method comprising expressing the circRNA of any one of embodiments 44 to 49, the linear RNA of embodiment 50, or the plasmid of any one of embodiments 51 to 55 in a host cell in conditions under which the RNA is translated into a polypeptide.

[0209] Embodiment 57. A method of expressing a polypeptide, the method comprising expressing the circRNA of any one of embodiments 44 to 49, the linear RNA of embodiment 50, or the plasmid of any one of embodiments 51 to 55 in a cell-free composition in conditions under which the RNA is translated into a polypeptide.

[0210] EXAMPLES

[0211] Example 1: Insertion of aptamers to EV71 IRES elements enhance translation in linear RNA

[0212] Several enterovirus A71 (EV71) internal ribosome entry site (IRES) elements having various aptamer insertion sequences were designed. Wildtype EV71 (FIG. 1A) was engineered to include theophylline-binding aptamers, tetracycline-binding aptamers, or (6R,S)-folinic acid-binding aptamers within 7 domains or “stem loops” (SL1-SL-7); 18 insertion sequence sites for the aptamers were identified (FIG. IB; VI -VI 8). Engineered EV71 IRES sequences were ordered as double strand DNA gene blocks from IDT or Twist.

[0213] To differentiate cap-dependent RNA expression from cap-independent (IRES- dependent) RNA expression, linear RNA bicistronic vectors were prepared from the gene blocks, using Gibson Assembly or Golden Gate Assembly method. Linear RNAs consisted of, from 5’ to 3’: a nucleic acid encoding GFP, a stop codon, stem loops to block ribosome read- through, an EV71 IRES element with aptamer insertions, and a nucleic acid encoding mCherry. HEK293FT cells were transfected with the linear RNAs, and, if induced, treated with a corresponding ligand (ImM theophylline, 200pM tetracycline, or ImM folinic acid). Exemplary mRNA sequences corresponding to tested EV71 IRES elements are shown in Table 1.

[0214] Table 1. Exemplary Engineered EV71 IRES Elements with Aptamer Insertions

[0215] Cells were harvested 2 days post-transfection and expression of GFP and mCherry was analyzed via flowcytometry. Transfection control-positive cells (GFP+) were gated for analysis, and the median mCherry fluorescence was normalized by the median GFP fluorescence. As shown in FIGs. 1C-1E, application of aptamer binding ligands to “induce” additional expression did not alter IRES-mediated expression. However, insertion of the tested aptamers at terminal domains (e.g., V1-V3 in SL-1, V17-18 in SL-7 in FIG. IE) and adjacent domains (e.g., V4-V5 in SL-2 and V16 in SL-6 of FIG. IE, respectively) was generally found to increase IRES-mediated expression. Insertion of tested aptamers at some central domains (e.g., SL-4 in FIG. IE) was also found to increase IRES-mediated expression.

[0216] To determine if these effects were specific to EV71 IRES elements, similar mRNAs were designed and prepared with engineered Coxsackievirus B3 (CVB3) IRES elements and Poliovirus (PV) IRES elements having theophylline-binding aptamer insertions. As shown in FIGs. 2A-2B, application of aptamer binding ligands again did not alter IRES-mediated expression, regardless of IRES element used. However, increased IRES-mediated expression by insertion of aptamers in terminal domains was confirmed in both engineered CVB3 IRES elements and engineered PV IRES elements, suggesting that insertion sequence at terminal domains of IRES elements may improve IRES-mediated expression generally.

[0217] Example 2: Insertion of complementary spacer sequences alters IRES-mediated expression in linear RNA

[0218] As terminal insertion sequences were found to increase activity of engineered IRES elements (e.g., as shown in Example 1), additional IRES elements were designed to include theophylline-binding aptamers at VI, V2, V3, V16, V17, or V18 (as in Example 1) or to include 12bp or 16bp complementary insertion sequences on the 5’ terminal (“Front”) and / or 3’ terminal (“End”) of each element (as shown in FIG. 3A). Engineered IRES sequences were designed, prepared, and tested as in Example 1. HEK293FT cells were transfected with mRNA having, from 5’ to 3’: a nucleic acid encoding GFP, stop codon, stem loops to block ribosome read-through, an EV71 IRES element with theophylline-binding aptamer insertions, and a nucleic acid encoding mCherry. Exemplary mRNA sequences corresponding to tested EV71 IRES elements are shown in Table 2 below. Table 2. Exemplary Engineered EV71 IRES Elements with Complementary Sequence Insertions

[0219] Cells were harvested 2 days post-transfection and expression of GFP and mCherry was analyzed via flowcytometry. Transfection control-positive cells (GFP+) were gated for analysis, and the median mCherry fluorescence was normalized by the median GFP fluorescence. As shown in FIG. 3B, insertion of complementary sequences at the 5’ terminal (“Front”) and 3’ terminal (“End”) of each IRES element at some, but not all, positions was found to increase EV71 IRES-mediated expression at similar levels to theophylline-binding aptamers insertions in terminal domains. However, 16bp insertions on the 3’ terminal of IRES elements generally impaired EV71-IRES mediated expression, and in some instances, decreased expression by as much as 7-fold.

[0220] Example 3: Truncations of stem loops in terminal domains improves EV71 translation in linear RNA Given the variable effect of terminal insertion sequences in engineered IRES elements (e.g., as shown in Example 2), additional IRES elements were designed to have truncated terminal domains (e.g., deletion of SL-1, SL-7 in FIG. 4A); an exemplary predicted structure of EV71 having deleted SL-1 and SL-7 is shown in FIG. 4B. Engineered IRES sequences were designed, prepared, and tested as in Examples 1 and 2. HEK293FT cells were transfected with mRNA having, from 5’ to 3’: a nucleic acid encoding GFP, stop codon, stem loops to block ribosome read-through, an EV71 IRES element or CVB3 IRES element with stem loop deletions, a nucleic acid encoding a P2A peptide, and a nucleic acid encoding mCherry. Exemplary mRNA sequences corresponding to tested EV71 IRES elements and CVB3 IRES elements are shown in Table 3 below.

[0221] Cells were harvested 2 days post-transfection and expression of GFP and mCherry was analyzed via flowcytometry. Transfection control-positive cells (GFP+) were gated for analysis, and the median mCherry fluorescence was normalized by the median GFP fluorescence. As shown in FIG. 4C, truncations of EV71 IRES domains (e.g., deletion of stem loops) had variable effects, with central domain truncations (e.g., SL-2 through SL-5) generally reducing expression, however, deletions of SL-1, SL-7, and both SL-1 and SL-7 improved IRES-mediated expression by 5-fold. The deleterious effects of central domain truncations was also found in similarly prepared CVB3 IRES elements.

[0222] Example 4: Engineered EV71 and CVB3 IRES elements enhance IRES-mediated expression of circRNA

[0223] In this example, modifications to IRES elements were tested for their effects on circRNA expression. Several IRES elements (EV71, CVB3, and EMCV) were engineered to include: theophylline-binding aptamers, eIF4G-binding aptamers, or spacer sequence with minimal secondary structure (“NS”) at 5’ terminals or 3’ terminals; and / or truncation of terminal domains. Engineered EV71 IRES sequences were ordered as double strand DNA gene blocks from IDT or Twist. To differentiate cap-dependent RNA expression from capindependent (IRES-dependent) RNA expression, circular RNA split GFP vectors were prepared and cloned using Gibson Assembly or Golden Gate Assembly method, where RNA circularization was driven by Alu repeats-promoted back splicing in the intron regions, the transcript of a nucleic acid encoding GFP was assembled upon circularization, and the nucleic acid encoding GFP was operably linked to the engineered IRES. HEK293FT cells were cotransfected with plasmids encoding TagBFP as transfection control and plasmids encoding the circular mRNAs, and contacted with ligands for corresponding aptamers, if applicable. Exemplary circRNA sequences corresponding to tested EV71 IRES elements, CVB3 IRES elements, and EMCV IRES elements are shown in Table 4 below.

[0224] Table 4. Exemplary Engineered IRES Elements with Domain Truncations and Insertions

[0225] Cells were harvested 2 days post-transfection and expression of GFP and TagBFP was analyzed via flowcytometry. Insertion of theophylline-binding aptamers improved IRES- mediated expression at some positions- for example, engineered EV71 IRES elements having 3’ theophylline-binding aptamer insertions generally expressed GFP at higher levels than wildtype; this effect was maintained or increased (by as much as 3x) when combined with certain domain truncations (e.g., Al, A7, Al, 7), as shown in FIG. 6A. Similarly, FIG. 6B shows that engineered CVB3 IRES elements benefit from aptamer insertions at the 5’ terminal and 3’ terminal, and from truncation of 5’ terminal domains (e.g., Al). FIG. 7 and FIG. 8A-8C demonstrate that engineered EMCV, EV71, and CVB3 IRES elements all generally benefit from NS insertions at the 3’ terminal. These results are consistent with findings in linear mRNAs (e.g., Examples 1-3). Furthermore, unlike in linear mRNAs, application of theophylline to engineered IRES elements having theophylline-binding aptamers generally improved expression (FIGs. 6A- 6B, FIG. 7); however, increased expression effects were found even in uninduced conditions, suggesting that sequence insertion alone may be useful for generally improving IRES-mediated expression of both linear mRNA and circRNA.

[0226] Example 5: Engineered IRES elements in rolling / non-rolling circle circRNA

[0227] In this example, an initial IRES screen based on engineered CVB3 and EMCV IRES elements in circRNA was performed. Plasmids encoding circRNA comprising engineered CVB3 IRES elements and engineered EMCV IRES elements were designed for both rolling circle expression and non-rolling circle expression. Non-rolling circRNA (as shown in FIG. 9 A) included: a CMV promoter (PCMV), 3’ flanking circularization cassettes, a nucleic acid encoding a split fluorescent protein (FP: a C terminus portion of GFP (151-241aa) with no stop codon, a nucleic acid encoding a T2A peptide, an engineered IRES element, a nucleic acid encoding a P2A peptide, and an N terminus portion (l-150aa) of GFP), and the flanking 5’ end of the homology arm a poly-A tail. Rolling circRNA (as shown in FIG. 9B) included: a CMV promoter (PCMV), 3’ flanking circularization cassettes, a nucleic acid encoding a split fluorescent protein (FP: a C terminus portion of GFP (151-241aa) with a stop codon, a nucleic acid encoding a T2A peptide, an engineered IRES element, a nucleic acid encoding a P2A peptide, and an N terminus portion (l-150aa) of GFP), and the flanking 5’ end of the homology arm a poly-A tail. Exemplary engineered IRES elements and corresponding sequences are provided in Table 5 below.

[0228] Table 5. EMCV and CVB3 IRES candidates search based on sequence and structure lomology.

[0229]

[0230] For non-rolling circRNA, a wildtype EMCV IRES element was used as a control. For rolling circRNA, a “Modified EMCV” control was used as a control, in which stop codons in a wildtype EMCV IRES element were functionally removed; modified EMCV control IRES elements included the following mutations relative to wildtype EMCV IRES element (e.g., corresponding to SEQ ID NO: 114-116): C27T, C211G, C302A, C477T, A517 A, and C498T.

[0231] HEK293FT cells were transfected with the circRNA and GFP expression (mNeonGreen) was determined. A summary of fold-change improvements of three IRES candidates according to the rolling circle circRNA design are shown in FIG. 9C. As shown in FIG. 9D, engineered EMCV IRES elements were especially effective in improving translation of GFP in non-rolling circRNA. The predicted structure of an exemplary engineered EMCV-A7 variant (“IRESite_Id557 emcv” of FIG. 9D and SEQ ID NO: 67) is shown in FIG. 9E. This engineered EMCV IRES includes deletion of the 5’ terminal domain and 3’ terminal domain, and the insertion of an eIF4G-binding aptamer at the 3’ terminal.

[0232] A similar experiment was subsequently performed using engineered CVB3 IRES elements, prepared according to the same non-rolling circRNA or rolling circRNA designs of FIG. 9A and FIG. 9B, respectively. Similarly to results shown for engineered EMCV IRES elements, engineered CVB3 IRES elements were especially effective in in improving translation of GFP in non-rolling circRNA, as shown in FIG. 9F. The predicted structure of an exemplary engineered (IRESite_Id598 CVB3 of FIG. 9F and SEQ ID NO: 73) is shown in FIG. 9G. This engineered CVB3 IRES includes deletion of the 3’ terminal domain, and the insertion of an eIF4G-binding aptamer at the 5’ terminal.

[0233] Example 6: eIF4G aptamer insertions at 20 nucleotide intervals improve translational efficiency of CVB3 and EMCV IRES elements In this example, engineered IRES elements were designed to include eIF4G-binding aptamer insertions in IRES elements (CVB3-B1, EMCV-A3, EMCV-A7) in a non-rolling circRNA. As shown in FIG. 10, 39bp eIF4G-binding aptamer (SEQ ID NO: 121) can be inserted every 20bp in an IRES element, which may be useful for recruiting eIF4G and improving protein translation. An exemplary engineered EMCV-A7 IRES element according to FIG. 10 is shown in FIG. 11 A.

[0234] Cells were transfected with non-rolling circle circRNA as described in Example 5. As shown in FIG. 11B, eIF4G-binding aptamer insertions into EMCV-A7 improved mNeonGreen expression, with insertions at sites 1, 14-17 and site 28 improving expression the most. Similar experiments were performed with engineered CVB3-B1 and EMCV-A3 (FIG. 11C, 11D).

[0235] Exemplary engineered IRES elements with eIF4G-binding aptamer insertions and corresponding sequences are provided in Table 6 below.

[0236] Table 6. eIF4G-binding aptamer insertion of EMCV and CVB3 candidates

[0237] Example 7: An IRES Toolbox, showing engineered IRES elements with different translational strength

[0238] In this example, HEK293FT cells were transfected with non-rolling circRNA encoding mNeonGreen and having an engineered EMCV-A7 IRES element, CVB3-B1 IRES element, EMCV- A3 IRES element, or iHRV IRES element according to the rationale of FIG. 10. As shown in FIGs. 12A-12C (fold-change) engineered IRES elements can increase expression of payloads in non-rolling circRNA at differing levels, which may be useful for a variety of applications.

[0239] Exemplary engineered IRES elements shown in FIGs. 12A-12C and corresponding sequences are provided in Table 7 below.

[0240] Table 7. HRV IRES and optimized synthetic IRES candidates

[0241] Example 8: Engineered IRES elements exhibit robust expression against frame changes Because non-rolling circRNA are susceptible to deleterious effects of frameshift mutations, each naturally derived IRES and synthetic IRES was subjected to a frameshift mutation. For instance, CVB3-WT, synthetic IRES 25C, synthetic IRES 26C were subjected to shift the start site by 1 bp, which is thought to shift the TSS start site accordingly. If the F.c remained relatively unchanged compared to corresponding non-frameshifted engineered IRES elements, then it can be concluded that the engineered IRES is more resistant and insulated in any given genetic context.

[0242] HEK294FT cells were transfected as described in Example 5, and GFP expression was determined. Surprisingly, as shown in FIG. 13A-13B, though some engineered IRES elements were susceptible to variation (e.g., Zen01-EMCV-26C+lF), other engineered IRES elements (e.g., ZcnO I -25C, an engineered EMCV IRES element) demonstrated enhanced robustness against frame shift mutation (F.c 2.20 - 2.28 between +1F and - IF). Individual GFP expression for exemplary engineered IRES elements are shown in FIG. 14.

[0243] Exemplary engineered IRES elements shown in FIGs. 13A-13B and corresponding sequences are provided in Table 8 below.

[0244] Table 8. Frame shifted mutant engineered IRES elements used for FIG. 13A-13B

[0245] Plasmid backbones used for FIGs. 13A-13B are provided in Table 9 below.

[0246] Table 9. Frame shifted mutant plasmid backbones used for FIG. 13 A- 13B

Claims

CLAIMSWhat is claimed is:

1. An engineered internal ribosome entry site (IRES) element, comprising: a plurality of domains each comprising at least one stem loop, such that the IRES element comprises at least a 3’ terminal domain and a 5’ terminal domain, and optionally, one or more central domains between the 3’ terminal domain and the 5’ terminal domain; wherein at least one stem loop in the IRES element is a truncated stem loop.

2. The engineered IRES element of claim 1, comprising a truncated stem loop in the 5’ terminal domain and / or the 3’ terminal domain of the IRES element.

3. The engineered IRES element of claim 1, further comprising an insertion sequence.

4. The engineered IRES element of claim 3, wherein the insertion sequence comprises a secondary structure, an aptamer, or a spacer sequence, optionally wherein the spacer sequence does not comprise a secondary structure.

5. The engineered IRES element of claim 1, comprising two or more insertion sequences inserted into two or more of the plurality of domains.

6. The engineered IRES element of claim 1, wherein the engineered IRES element is modified from a wildtype IRES element, derived from a human genome, or is a short IRES.

7. A circular ribonucleic acid (circRNA) comprising the engineered IRES element of claim 1 and a nucleic acid encoding a peptide, protein, or fragment thereof, optionally wherein the circRNA comprises a frameshift mutation, further optionally wherein translation of the circRNA expresses an amount of a functional peptide, protein, or fragment thereof greater than the circRNA comprising a frameshift mutation absent the engineered IRES element.

8. A linear ribonucleic acid (RNA) comprising the engineered IRES element of claim 1 and a nucleic acid encoding a peptide, protein, or fragment thereof.

9. A method of expressing a peptide, protein, or fragment thereof, the method comprising: contacting an isolated cell with a ribonucleic acid (RNA) comprising the engineered IRES element of claim 1 in conditions under which the RNA is translated into the peptide, protein, or fragment thereof.

10. An engineered internal ribosome entry site (IRES) element, comprising: a plurality of domains each comprising at least one stem loop, such that the IRES element comprises at least a 3’ terminal domain and a 5’ terminal domain, and optionally, one or more central domains between the 3’ terminal domain and the 5’ terminal domain; and an insertion sequence, wherein the insertion sequence is inserted into one of the plurality of domains.

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