BACKGROUND

Messenger RNA (mRNA) is frequently used as a gene delivery molecule in the field of cancer immunotherapy and stem cell-based biomedical research as an alternative to plasmid DNA. As a direct source of gene products, mRNA has several advantages including a lack of requirement for nuclear entry, which poses a significant barrier to DNA delivery, especially in non-dividing cells. mRNA also has a negligible chance of integrating into the host genome, avoiding aberrant transcription and expression of oncogenes caused by insertional mutagenesis.

For a given gene, the untranslated gene regions (UTRs), including the 5′ and 3′ UTRs, are regions involved in the regulation of expression. The 5′ UTR is a regulatory region of DNA situated at the 5′ end of all protein coding sequence that is transcribed into mRNA but not translated into protein. 5′ UTRs may contain various regulatory elements, e.g., 5′ cap structure, G-quadruplex structure (G4), stem-loop structure, and internal ribosome entry sites (IRES), which play a major role in the control of translation initiation. The 3′ UTR, situated downstream of the protein coding sequence, has been found to be involved in numerous regulatory processes including transcript cleavage, stability and polyadenylation, translation, and mRNA localization. The 3′ UTR serves as a binding site for numerous regulatory proteins and small non-coding RNAs, e.g., microRNAs.

Current mRNA therapies typically rely on native or standard UTR sequences that are often not tissue-specific and/or give low levels of protein expression. There is a need to identify novel UTRs that can stabilize therapeutic mRNA and increase protein synthesis in a tissue-specific manner.

SUMMARY

The present disclosure relates to methods of improving expression from an mRNA in a tissue (e.g., liver, or cells in vitro, such as stem cells, hepatocytes or lymphocytes), and heterologous untranslated region (UTR) sequences for enhancing protein synthesis from therapeutic mRNAs (including those UTRs that act in a tissue-specific manner), methods of identifying the same, and methods of using the same as therapeutic agents. Sequence elements provided herein include, for example, UTRs derived from mRNAs that are naturally abundant in a specific tissue (e.g., liver), to which the therapeutic mRNA's expression is targeted.

UTR sequences of the present disclosure may, for example, increase protein synthesis by increasing both the time that the mRNA remains in translating polysomes (message stability) and/or the rate at which ribosomes initiate translation on the message (message translation efficiency). Thus, UTR sequences of the present disclosure lead to prolonged protein synthesis, enabling successful treatment of conditions that require continuous protein expression in a tissue-specific manner.

Described herein are mRNAs comprising an exogenous open reading frame (ORF) flanked by a 5′ UTR and a 3′ UTR, wherein the 5′ UTR and the 3′ UTR are derived from a naturally abundant mRNA in a tissue. The tissue may be, for example, liver, a stem cell or lymphoid tissue. The lymphoid tissue may be, for example, a lymphocyte (e.g., a B-lymphocyte, a helper T-lymphocyte, a cytotoxic T-lymphocyte, a regulatory T-lymphocyte, or a natural killer cell), a macrophage, a monocyte, a dendritic cell, a neutrophil, an eosinophil or a reticulocyte. The naturally abundant mRNA may have, for example, a tissue half-life of at least about 9 hours, or from about 5 to about 60 hours. The naturally abundant mRNA may be, for example, transcribed from HP, FGB, HPR, ALB, C3, FGA, SERPINA1 or SERPINA3 gene. The 5′ UTR may have, for example, the nucleotide sequence of SEQ ID NOS: 1-6, 9 or 10. The exogenous ORF may be, for example, a reporter gene or a therapeutic gene. The therapeutic gene may be, for example, interferon alpha, TNF-related apoptosis-inducing ligand, vascular adhesion protein 1, or hepatocyte growth factor. The mRNA may include, for example, at least one non-natural or modified nucleotide. The at least one non-natural or modified nucleotide may include, for example, at least one backbone modification, sugar modification or base modification. The at least one non-natural or modified nucleotide may include, for example, at least one base modification. The at least one base modification, for example, may be selected from the group consisting of 2-amino-6-chloropurine riboside 5′-triphosphate, 2-aminoadenosine 5′-triphosphate, 2-thiocytidine 5′-triphosphate, 2-thiouridine 5′-triphosphate, 4-thiouridine 5′-triphosphate, 5-aminoallylcytidine 5′-triphosphate, 5-aminoallyluridine 5′-triphosphate, 5-bromocytidine 5′-triphosphate, 5-bromouridine 5′-triphosphate, 5-iodocytidine 5′-triphosphate, 5-iodouridine 5′-triphosphate, 5-methylcytidine 5′-triphosphate, 5-methyluridine 5′-triphosphate, 6-azacytidine 5′-triphosphate, 6-azauridine 5′-triphosphate, 6-chloropurine riboside 5′-triphosphate, 7-deazaadenosine 5′-triphosphate, 7-deazaguanosine 5′-triphosphate, 8-azaadenosine 5′-triphosphate, 8-azidoadenosine 5′-triphosphate, benzimidazole riboside 5′-triphosphate, N1-methyladenosine 5′-triphosphate, N1-methylguanosine 5′-triphosphate, N6-methyladenosine 5′-triphosphate, O6-methylguanosine 5′-triphosphate, N1-methyl-pseudouridine 5′-triphosphate, puromycin 5′-triphosphate and xanthosine 5′-triphosphate. The at least one non-natural or modified nucleotide may, for example, be N1-methyl-pseudouridine 5′-triphosphate.

Described herein are methods of identifying a 5′ UTR and a 3′ UTR for increasing protein synthesis in a desired tissue or a cell derived from the desired tissue, comprising a) isolating a plurality of 5′ UTRs and a plurality of 3′ UTRs from mRNAs that are naturally abundant in the desired tissue; b) generating a library of test constructs, each comprising a reporter ORF flanked by one of the plurality of 5′ UTRs and/or one of the plurality of 3′ UTRs; c) providing a reference construct comprising the reporter ORF flanked by reference 5′ and 3′ UTRs; d) expressing each of the test constructs and the reference construct in the tissue; e) measuring the protein expression of the reporter ORF from the each of the test constructs and the reference construct; f) selecting at least one test construct having a higher protein expression from the reporter ORF than the reference construct; and g) identifying the 5′ UTR and the 3′ UTR of the selected test construct. In a particular embodiment, the desired tissue is liver. In a particular embodiment, an mRNA comprising an identified 5′ UTR or 3′ UTR has a tissue half-life of at least 4 hours. In a particular embodiment, the reporter ORF is eGFP, adiponectin or Factor VII.

Described herein are methods treating a liver condition, comprising administering to a subject in need thereof the mRNA as described herein. In a particular embodiment, the liver condition is selected from the group consisting of: acute hepatitis, chronic hepatitis, liver cirrhosis, cirrhosis, fatty liver, liver cancer, glycogen storage disease, progressive familial intrahepatic cholestasis 1 (PFIC1), progressive familial intrahepatic cholestasis 2 (PFIC2), progressive familial intrahepatic cholestasis 3 (PFIC3), adenylosuccinate lyase deficiency (ASLD), citrullinemia, arginase-1 deficiency, primary hyperoxaluria type 1 (PH1), ornithine transcarbamylase deficiency (OTCD), homocystinuria, pheylketonuria, glycogen storage disease type IV (GSDIV), galactose-1-phosphate uridylyltransferase deficiency (type I (GALTI), type II (GALTII) or type 3 (GALTIII)), long chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD), 3-methylcrotonyl-CoA carboxylase deficiency (MCCC1 deficiency), methylmalonic aciduria (MMA), MMA and homocystinuria type C (MMACHC), thrombotic thrombocytopenic purpura (TTP), hyperornithinemia-hyperammonemia-homocitrullinuria syndrome (HHH), complement component 2 deficiency (C2D), F2, protein S deficiency (caused by allelic variants of PROS1), alpha-1 antitrypsin deficiency (A1AT), glutaric academia 1 (GA-1), glutaric academia 2 (GA-2), cystinosis (CTNS), tyrosinemia, tyrosinemia type 3 (cause by allelic variants of HPD), D-bifunctional protein deficiency (DBP), Fanconi-Bickel syndrome (FBS), pseudoxanthoma elasticum (PXE), primary biliary cirrhosis, Pompe disease, glycerol kinase deficiency (GKD), proprionic acidemia (PA) and Crigler-Najjar syndrome (CN1). In a particular embodiment, the liver condition is hepatitis C. In a particular embodiment, the therapeutic gene is selected from the group consisting of: interferon alpha, TNF-related apoptosis-inducing ligand, vascular adhesion protein 1, hepatocyte growth factor, G6PC, ABCB11, ABCB4, ASL1, ASS, Arg1, AGXT, OTC, CBS, PAH, GBE, GALE, HADH, MCCC1, MMA, ADAMTS13, SLC25A15, C2, F2, PROS1, SERPINA1, GALT1, ETFA, GCDH, CTNS, FAH, TAT, HPD, HSD17b4, SLC2A2, GALC, ABCC6, AHSG, PDC-E2, GAA, ATP8B1, MMACHC, GK, PCCA, PCCB and UGT1A1.

Described herein are methods of improving expression of a protein in a desired cell or desired tissue, comprising providing an mRNA to said cell or tissue, wherein the mRNA comprises an open reading frame (ORF) that encodes said protein, flanked by a heterologous 5′ untranslated region (UTR) and/or a heterologous 3′ UTR, wherein the 5′ UTR and/or the 3′ UTR are derived from an mRNA that is naturally abundant mRNA in the desired tissue or tissues from which the desired cell was derived. In a particular embodiment, the mRNA comprises at least one modified or non-naturally occurring nucleotide. In a particular embodiment, the at least one modified or non-naturally occurring nucleotide comprises at least one backbone modification, sugar modification or base modification. In a particular embodiment, the at least one modified or non-naturally occurring nucleotide comprises at least one base modification. In a particular embodiment, the at least one base modification is selected from the group consisting of: 2-amino-6-chloropurine riboside 5′ triphosphate, 2-aminoadenosine 5′ triphosphate, 2-thiocytidine 5′ triphosphate, 2-thiouridine 5′ triphosphate, 4-thiouridine 5′ triphosphate, 5-aminoallylcytidine 5′ triphosphate, 5-aminoallyluridine 5′ triphosphate, 5-bromocytidine 5′ triphosphate, 5-bromouridine 5′ triphosphate, 5-iodocytidine 5′ triphosphate, 5-iodouridine 5′ triphosphate, 5-methylcytidine 5′ triphosphate, 5-methyluridine 5′ triphosphate, 6-azacytidine 5′ triphosphate, 6-azauridine 5′ triphosphate, 6-chloropurine riboside 5′-triphosphate, 7-deazaadenosine 5′ triphosphate, 7-deazaguanosine 5′ triphosphate, 8-azaadenosine 5′ triphosphate, 8-azidoadenosine 5′ triphosphate, benzimidazole riboside 5′ triphosphate, N1-methyladenosine 5′ triphosphate, N1-methylguanosine 5′ triphosphate, N6-methyladenosine 5′ triphosphate, O6-methylguanosine 5′ triphosphate, N1-methyl-pseudouridine 5′ triphosphate, puromycin 5′-triphosphate and xanthosine 5′ triphosphate. In a particular embodiment, the at least one modified or non-naturally occurring nucleotide is N1-methyl-pseudouridine 5-triphosphate. In a particular embodiment, the tissue is liver. In a particular embodiment, the mRNA has a half-life of at least 40 hours. In a particular embodiment, the heterologous 5′ UTR and/or the heterologous 3′ UTR is derived from a gene selected from the group consisting of: HP, FGB, HPR, ALB, C3, FGA, Col1A, Col6A, SERPINA1 and SERPINA3. In a particular embodiment, the 5′ UTR comprises a nucleotide sequence selected from the group consisting of: SEQ ID NOS: 1-6, 9 and 10. In a particular embodiment, the ORF is a reporter gene or a therapeutic gene of interest. In a particular embodiment, the therapeutic gene is selected from the group consisting of: interferon alpha, TNF-related apoptosis-inducing ligand, vascular adhesion protein 1, hepatocyte growth factor, G6PC, ABCB11, ABCB4, ASL1, ASS, Arg1, AGXT, OTC, CBS, PAH, GBE, GALE, HADH, MCCC1, MMA, ADAMTS13, SLC25A15, C2, F2, PROS1, SERPINA1, GALT1, ETFA, GCDH, CTNS, FAH, TAT, HPD, HSD17b4, SLC2A2, GALC, ABCC6, AHSG, PDC-E2, GAA, ATP8B1, MMACHC, GK, PCCA, PCCB and UGT1A1.

