Intracellular translation of circular RNA
A circular mRNA molecule possessing features resembling native mammalian mRNA demonstrates improved translation, while retaining the properties of an extremely long half-life inside cells. This circular mRNA is functional inside mammalian cells, being able to compete against native cellular mRNAs for the eukaryotic translation initiation machinery. The invention possesses additional RNA elements compared to a previous invention containing only an IRES element for successful in vitro or in vivo translation.
1. A vector for making circular mRNA, said vector comprising the following elements operably connected to each other and arranged in the following sequence:
a) an RNA polymerase promoter,
b) a self circularizing intron 5′ slice junction,
c) an IRES,
d) an optional 5′ UTR,
e) a multiple cloning insertion site for inserting an ORF into said vector,
f) a 3′ UTR,
g) an optional polyA tract,
h) a self circularizing intron 3′ slice junction, and
i) an optional RNA polymerase terminator,
said vector allowing production of a circular mRNA that is able to compete against native cellular mRNAs for eukaryotic translation initiation machinery and that is translatable inside eukaryotic cells.
2. The vector of claim 1 , wherein the RNA polymerase promoter and terminator are from a T7 virus, T6 virus, SP6 virus, T3 virus, or T4 virus.
3. The vector of claim 1 , wherein the 3′ UTR is from human beta globin, human alpha globin xenopus beta globin, xenopus alpha globin, human prolactin, human GAP-43, human eEF1a1, human Tau, human TNF alpha, dengue virus, hantavirus small mRNA, bunyanavirus small mRNA, turnip yellow mosaic virus, hepatitis C virus, rubella virus, tobacco mosaic virus, human IL-8, human actin, human GAPDH, human tubulin, hibiscus chlorotic rinsgpot virus, woodchuck hepatitis virus post translationally regulated element, sindbis virus, turnip crinkle virus, tobacco etch virus or Venezuelan equine encephalitis virus.
4. The vector of claim 1 , wherein the 5′ UTR is from human beta globin, Xenopus laevis beta globin, human alpha globin, Xenopus laevis alpha globin, rubella virus, tobacco mosaic virus, mouse Gtx, dengue virus, heat shock protein 70 kDa protein 1A, tobacco alcohol dehydrogenase, tobacco etch virus, turnip crinkle virus, or adenovirus tripartite leader.
5. The vector of claim 1 , wherein the polyA track is at least 30 nucleotides long.
6. The vector of claim 1 , wherein the polyA track is at least 60 nucleotides long.
7. The vector of claim 1 , wherein the IRES is from 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 stali intestine virus, Kashmir bee virus, Human rhinovirus 2, Homalodisca coagulata virus-1, Human Immunodeficiency Virus type 1, Himetobi P virus, Hepatitis C virus, Hepatitis A virus, Hepatitis GB virus, foot and mouth disease virus, Human enterovirus 71, Equine rhinitis virus, Ectropis obliqua picorna-like virus, Encephalomyocarditis virus, Drosophila C Virus, Human coxsackievirus B3, Crucifer tobamovirus, 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-IAP1, Human c-myc, Human eIF4G, Mouse NDST4L, Human LEF1, Mouse HIF1 alpha, Human N-myc, Mouse Gtx, Human p27kip1, Human PDGF2/c-sis, Human p53, Human Pim-1, Mouse Rbm3, Drosophila reaper, Canine Scamper, Drosophila Ubx, 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, or an aptamer to eIF4G.
8. The vector of claim 1 , further including an RNA sequence that binds eIF4E when transcribed into the circular mRNA.
9. The vector of claim 8 , wherein the RNA sequence binding to eIF4E is from Mouse histone H4, Human cyclin D1, Pea enation mosaic virus RNA2, Panicum Mosaic Virus, or an RNA aptamer to eIF4E.
10. The vector of claim 1 , wherein the IRES is combined with a second IRES facilitating additional initiation factor recruitment, ribosome subunit binding, ribosome shunting, ribosome base pairing, or ribosome translocation.
11. The vector of claim 1 , wherein said self-circularizing catalytic intron is a Group I intron or Group II intron.
12. The vector of claim 1 , further comprising a nuclear transport element selected from Mason Pfizer Monkey Virus Constitutive Transport Element (CTE), 4E-SE element, woodchuck hepatitis virus post regulatory element, hepatitis B virus post regulatory element, or HIV rev response element.
13. The vector of claim 1 , said IRES comprising SEQ ID NO. 3.
14. A method of making circular mRNA, said method comprising adding ribonucleotide triphosphates, inorganic pyrophosphatase, RNase inhibitor, and an RNA polymerase to the vector of claim 1 in appropriate reaction buffer, transcribing RNA from said vector, and allowing self-circularization of said transcribed RNA to produce circular mRNA.
15. The method of claim 14 , said ribonucleotides including modified ribonucleotides m5C, m5U, m6A, s2U, Ψ, or 2′-O-methyl-U.
16. A method of making circular mRNA, said method comprising transfecting the vector of claim 1 and a phage polymerase or nucleic acid encoding a phage polymerase into a eukaryotic cell, allowing for transcription of said vector inside the cell to produce transcribed RNA, and allowing self-circularization of said transcribed RNA to produce circular mRNA.
17. A method of making circular mRNA, said method comprising transfecting the vector of claim 1 into a eukaryotic cell, wherein said vector is transcribed by a host cell RNA polymerase.