US 20180353618A1 ( 19) United States (12 ) Patent Application Publication ( 10) Pub . No. : US 2018 /0353618 A1 Burkhardt et al. (43 ) Pub . Date : Dec . 13 , 2018

(54 ) HETEROLOGOUS UTR SEQUENCES FOR Publication Classification ENHANCED MRNA EXPRESSION (51 ) Int. Cl. A61K 48 /00 ( 2006 .01 ) (71 ) Applicant: Moderna TX , Inc ., Cambridge , MA C12N 15 / 10 (2006 .01 ) (US ) C12N 15 /67 ( 2006 .01 ) (72 ) Inventors : David H . Burkhardt , Somerville , MA A61K 31/ 7115 ( 2006 .01 ) (US ) ; Romesh R . Subramanian , A61K 31 /712 ( 2006 .01 ) A61K 31/ 7125 (2006 . 01) Framingham , MA (US ) ; Christian A61P 1/ 16 ( 2006 .01 ) Cobaugh , Newton Highlands , MA (US ) A61P 31/ 14 (2006 .01 ) (52 ) U . S . CI. (73 ) Assignee : Moderna TX , Inc. , Cambridge , MA CPC . .. . . A61K 48 /0058 ( 2013 .01 ) ; C12N 15 / 1086 (US ) ( 2013 . 01 ) ; A61K 48 / 0066 ( 2013 .01 ) ; C12N 15 /67 ( 2013 .01 ); A61P 31 / 14 ( 2018 . 01 ) ; A61K ( 21 ) Appl . No. : 15 / 781, 827 31/ 712 (2013 .01 ) ; A61K 31/ 7125 ( 2013 .01 ) ; A61P 1 / 16 ( 2018 .01 ) ; A61K 31/ 7115 ( 22 ) PCT Filed : Dec . 9 , 2016 ( 2013 .01 ) ( 86 ) PCT No. : PCT/ US2016 / 065796 (57 ) ABSTRACT $ 371 (c )( 1 ), mRNAs containing an exogenous open reading frame (ORF ) ( 2 ) Date : flanked by a 5 ' untranslated region (UTR ) and a 3 ' UTR is Jun . 6 , 2018 provided , wherein the 5 ' and 3 ' UTRs are derived from a naturally abundant mRNA in a tissue . Also provided are Related U . S . Application Data methods for identifying the 5' and 3' UTRs, and methods for (60 ) Provisional application No. 62 / 265 ,233 , filed on Dec. making and using the mRNAs . 9 , 2015 . Specification includes a Sequence Listing .

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FIG . 2 Patent Application Publication Dec. 13 , 2018 Sheet 3 of 7 US 2018 /0353618 A1

1 ,000 mRNA 800 median : 9h Protein

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FIG . 7 US 2018 /0353618 A1 Dec. 13 , 2018

HETEROLOGOUS UTR SEQUENCES FOR B -lymphocyte , a helper T- lymphocyte , a cytotoxic T- lym ENHANCED MRNA EXPRESSION phocyte , a regulatory T -lymphocyte , or a natural killer cell ), a macrophage , a monocyte, a dendritic cell , a neutrophil , an BACKGROUND eosinophil or a reticulocyte . The naturally abundant mRNA may have, for example , a tissue half - life of at least about 9 [ 0001] Messenger RNA (mRNA ) is frequently used as a hours , or from about 5 to about 60 hours . The naturally gene delivery molecule in the field of cancer immunotherapy abundant mRNA may be , for example , transcribed from HP, and stem cell- based biomedical research as an alternative to FGB , HPR , ALB , C3 , FGA , SERPINA1 or SERPINA3 plasmid DNA . As a direct source of gene products , mRNA gene . The 5 ' UTR may have , for example , the has several advantages including a lack of requirement for sequence of SEQ ID NOS : 1 - 6 , 9 or 10 . The exogenous ORF nuclear entry , which poses a significant barrier to DNA may be , for example , a reporter gene or a therapeutic gene . delivery, especially in non -dividing cells . mRNA also has a The therapeutic gene may be , for example , interferon alpha , negligible chance of integrating into the host genome, avoid TNF -related apoptosis - inducing ligand , vascular adhesion ing aberrant transcription and expression of oncogenes protein 1 , or hepatocyte growth factor. The mRNA may caused by insertional mutagenesis . include , for example , at least one non - natural or modified [ 0002] For a given gene, the untranslated gene regions nucleotide . The at least one non -natural or modified nucleo (UTRs ) , including the 5 ' and 3 ' UTRs , are regions involved tide may include, for example , at least one backbone modi in the regulation of expression . The 5' UTR is a regulatory fication , sugar modification or base modification . The at region of DNA situated at the 5 ' end of all protein coding least one non -natural or modified nucleotide may include , sequence that is transcribed into mRNA but not translated for example , at least one base modification . The at least one into protein . 5 ' UTRs may contain various regulatory ele base modification , for example , may be selected from the ments , e .g ., 5' cap structure , G - quadruplex structure (G4 ), group consisting of 2 -amino - 6 - chloropurine riboside stem -loop structure , and internal ribosome entry sites 5 ' - triphosphate , 2 - aminoadenosine 5 '- triphosphate , 2 - thio ( IRES ) , which play a major role in the control of translation 5 '- triphosphate , 2 - thiouridine 5 '- triphosphate , initiation . The 3 ' UTR , situated downstream of the protein 4 - thiouridine 5 '- triphosphate , 5 -aminoallylcytidine coding sequence , has been found to be involved in numerous 5 '- triphosphate , 5 -aminoallyluridine 5 -triphosphate , 5 -bro regulatory processes including transcript cleavage , stability mocytidine 5 '- triphosphate, 5 - bromouridine 5 '- triphosphate , and polyadenylation , translation , and mRNA localization . 5 - iodocytidine 5 '- triphosphate , 5 - iodouridine 5' - triphos The 3 ' UTR serves as a binding site for numerous regulatory phate , 5 -methylcytidine 5 '- triphosphate , 5 -methyluridine proteins and small non - coding RNAs, e . g ., microRNAs. 5 ' - triphosphate , 6 -azacytidine 5 ' - triphosphate , 6 - azauridine [ 0003 ] Current mRNA therapies typically rely on native or 5 - triphosphate , 6 - chloropurine riboside 5 '- triphosphate , standard UTR sequences that are often not tissue- specific 7 -deazaadenosine 5' - triphosphate , 7 - deazaguanosine and /or give low levels of protein expression . There is a need 5 '- triphosphate , 8 - azaadenosine 5 -triphosphate , 8 -azidoad to identify novelUTRs that can stabilize therapeutic mRNA enosine 5' - triphosphate , benzimidazole riboside 5' - triphos and increase protein synthesis in a tissue - specific manner. phate , N1- methyladenosine 5 '- triphosphate , N1- methyl 5 - triphosphate , N6 -methyladenosine SUMMARY 5 '- triphosphate , O?- methylguanosine 5 '- triphosphate , [ 0004 ] The present disclosure relates to methods of N ' -methyl - 5 '- triphosphate , puromycin improving expression from an mRNA in a tissue ( e . g . , liver, 5 ' - triphosphate and 5 ' - triphosphate. The at least or cells in vitro , such as stem cells , hepatocytes or lympho one non -natural or modified nucleotidemay , for example, be cytes ) , and heterologous untranslated region (UTR ) N '- methyl - pseudouridine 5 '- triphosphate . sequences for enhancing protein synthesis from therapeutic [0007 ] Described herein are methods of identifying a 5' mRNAs ( including those UTRs that act in a tissue- specific UTR and a 3 ' UTR for increasing protein synthesis in a manner ), methods of identifying the same, and methods of desired tissue or a cell derived from the desired tissue, using the same as therapeutic agents . Sequence elements comprising a ) isolating a plurality of 5' UTRs and a plurality provided herein include , for example, UTRs derived from of 3 ' UTRs from mRNAs that are naturally abundant in the mRNAs that are naturally abundant in a specific tissue ( e . g . , desired tissue; b ) generating a library of test constructs , each liver) , to which the therapeutic mRNA ' s expression is comprising a reporter ORF flanked by one of the plurality of targeted . 5 ' UTRs and /or one of the plurality of 3 ' UTRs; c ) providing [0005 ] UTR sequences of the present disclosure may , for a reference construct comprising the reporter ORF flanked example , increase protein synthesis by increasing both the by reference 5 ' and 3 ' UTRs ; d ) expressing each of the test time that the mRNA remains in translating polysomes (mes constructs and the reference construct in the tissue ; e ) sage stability ) and / or the rate at which ribosomes initiate measuring the protein expression of the reporter ORF from translation on the message (message translation efficiency ) . the each of the test constructs and the reference construct; f) Thus , UTR sequences of the present disclosure lead to selecting at least one test construct having a higher protein prolonged protein synthesis , enabling successful treatment expression from the reporter ORF than the reference con of conditions that require continuous protein expression in a struct; and g ) identifying the 5 ' UTR and the 3 ' UTR of the tissue -specific manner. selected test construct . In a particular embodiment, the 10006 ] Described herein are mRNAs comprising an exog desired tissue is liver. In a particular embodiment, an mRNA enous open reading frame (ORF ) flanked by a 5 ' UTR and comprising an identified 5 ' UTR or 3 ' UTR has a tissue a 3 ' UTR , wherein the 5 ' UTR and the 3 ' UTR are derived half- life of at least 4 hours . In a particular embodiment, the from a naturally abundant mRNA in a tissue . The tissue may reporter ORF is eGFP, adiponectin or Factor VII . be , for example , liver , a stem cell or lymphoid tissue . The [0008 ] Described herein are methods treating a liver con lymphoid tissue may be , for example , a lymphocyte ( e . g ., a dition , comprising administering to a subject in need thereof US 2018 / 0353618 A1 Dec. 13 , 2018

themRNA as described herein . In a particular embodiment , 5 -methyluridine 5' triphosphate , 6 -azacytidine 5' triphos the liver condition is selected from the group consisting of: phate , 6 -azauridine 5' triphosphate , 6 -chloropurine riboside acute hepatitis , chronic hepatitis , liver cirrhosis , cirrhosis , 5 '- triphosphate , 7 - deazaadenosine 5 ' triphosphate , 7 -deaza fatty liver , liver cancer , glycogen storage disease , progres guanosine 5 ' triphosphate , 8 - azaadenosine 5 ' triphosphate , sive familial intrahepatic cholestasis 1 (PFIC1 ) , progressive 8 - azidoadenosine 5 ' triphosphate , benzimidazole riboside 5 ' familial intrahepatic cholestasis 2 (PFIC2 ) , progressive triphosphate , NP-methyladenosine 5' triphosphate , Nl- meth familial intrahepatic cholestasis 3 (PFIC3 ), adenylosucci ylguanosine 5 ' triphosphate , N?- methyladenosine 5 ' triphos nate lyase deficiency ( ASLD ) , citrullinemia , arginase - 1 defi phate , Oº -methylguanosine 5 ' triphosphate , NP- methyl ciency , primary hyperoxaluria type 1 (PH1 ) , ornithine tran pseudouridine 5' triphosphate , puromycin 5' -triphosphate scarbamylase deficiency (OTCD ) , homocystinuria , and xanthosine 5 ' triphosphate . In a particular embodiment, pheylketonuria , glycogen storage disease type IV (GSDIV ), the at least one modified or non -naturally occurring nucleo galactose - 1 - phosphate uridylyltransferase deficiency ( type I tide is N ' -methyl - pseudouridine 5 - triphosphate . In a par (GALTI ) , type II (GALTII ) or type 3 (GALTIII ) ) , long chain ticular embodiment, the tissue is liver. In a particular 3 -hydroxyacyl - CoA dehydrogenase deficiency (LCHADD ), embodiment, the mRNA has a half - life of at least 40 hours . 3 -methylcrotonyl - CoA carboxylase deficiency (MCCC1 In a particular embodiment, the heterologous 5 ' UTR and /or deficiency ), methylmalonic aciduria (MMA ) , MMA and the heterologous 3 ' UTR is derived from a gene selected homocystinuria type C (MMACHC ), thrombotic thrombo from the group consisting of: HP, FGB , HPR , ALB , C3 , cytopenic purpura ( TTP ), hyperornithinemia -hyperammo FGA , Col1A , Col6A , SERPINA1 and SERPINA3. In a nemia -homocitrullinuria syndrome (HHH ), complement particular embodiment, the 5 ' UTR comprises a nucleotide component 2 deficiency ( C2D ) , F2 , protein S deficiency sequence selected from the group consisting of: SEQ ID ( caused by allelic variants of PROS1) , alpha - 1 antitrypsin NOS: 1 - 6 , 9 and 10 . In a particular embodiment, the ORF is deficiency (A1AT ), glutaric academia 1 (GA -1 ) , glutaric a reporter gene or a therapeutic gene of interest. In a academia 2 (GA -2 ) , cystinosis (CTNS ), tyrosinemia, tyro particular embodiment , the therapeutic gene is selected from sinemia type 3 ( cause by allelic variants of HPD ) , D -bifunc the group consisting of: interferon alpha , TNF - related apop tional protein deficiency (DBP ) , Fanconi- Bickel syndrome tosis - inducing ligand , vascular adhesion protein 1 , hepato ( FBS ) , pseudoxanthoma elasticum (PXE ) , primary biliary cyte growth factor, G6PC , ABCB11, ABCB4, ASL1, ASS , cirrhosis , Pompe disease , glycerol kinase deficiency (GKD ) , Arg1 , AGXT, OTC , CBS , PAH , GBE , GALE , HADH . proprionic acidemia (PA ) and Crigler -Najjar syndrome MCCC1 , MMA , ADAMTS13 , SLC25A15 , C2 , F2 , PROS1 , (CN1 ) . In a particular embodiment, the liver condition is SERPINAI, GALT1 , ETFA , GCDH , CTNS , FAH , TAT , hepatitis C . In a particular embodiment , the therapeutic gene HPD , HSD17b4 , SLC2A2, GALC , ABCC6 , AHSG , PDC is selected from the group consisting of: interferon alpha , E2 , GAA , ATP8B1 , MMACHC , GK , PCCA , PCCB and TNF- related apoptosis - inducing ligand , vascular adhesion UGT1A1. protein 1 , hepatocyte growth factor , G6PC , ABCB11 , ABCB4, ASL1, ASS , Arg1, AGXT, OTC , CBS , PAH ,GBE , BRIEF DESCRIPTION OF THE DRAWINGS GALE , HADH , MCCC1, MMA , ADAMTS13 , SLC25A15 , [0010 ] FIG . 1 shows degradation process of formulated C2, F2 , PROS1, SERPINAI , GALTI, ETFA , GCDH , mRNA after delivery into a cell. After formulated mRNA is CTNS , FAH , TAT, HPD , HSD17b4 , SLC2A2 , GALC , delivered and endocytosed into a cell , ribosomes bind to ABCC6 , AHSG , PDC - E2 , GAA , ATP8B1, MMACHC , GK , mRNA to form polysomal mRNA for active translation , PCCA , PCCB and UGT1A1. followed by mRNA degradation . [0009 ] Described herein are methods of improving expres [0011 ] FIG . 2 shows major paths for mRNA degradation . sion of a protein in a desired cell or desired tissue, com Nearly all major mRNA decay pathways characterized in prising providing an mRNA to said cell or tissue, wherein eukaryotes are initiated by deadenylation by deadenylases . the mRNA comprises an open reading frame (ORF ) that Deadenylation primarily leads to decapping at the 5 ' end encodes said protein , flanked by a heterologous 5 ' untrans with subsequent 5' to 3 ' exonucleolytic digestion of the RNA lated region (UTR ) and / or a heterologous 3 ' UTR , wherein body by 5 ' exonucleases ( 5 ' exo ). Alternatively , when the 5 ' the 5 ' UTR and / or the 3 ' UTR are derived from an mRNA to 3 ' decay pathway is compromised , after deadenylation , that is naturally abundant mRNA in the desired tissue or the mRNA body can be degraded from the 3 ' end by a large tissues from which the desired cell was derived . In a complex called an exosome. particular embodiment, the mRNA comprises at least one [0012 ] FIG . 3 shows a wide range of stabilities of endog modified or non -naturally occurring nucleotide . In a particu enous mRNAs. By increasing the upper limit of mRNA lar embodiment, the at least one modified or non -naturally half - life , the quantity of protein delivered may be dramati occurring nucleotide comprises at least one backbone modi cally increased . fication , sugar modification or base modification . In a par [0013 ] FIG . 4 shows construction of a combinatorial ticular embodiment, the at least one modified or non library of UTRs with a reporter as described herein . The naturally occurring nucleotide comprises at least one base method involves identifying mRNAs likely to be stable in modification . In a particular embodiment, the at least one liver, synthesizing UTRs of these mRNA sequences , and base modification is selected from the group consisting of: building combinatorial library of UTRs flanking a reporter 2 - amino -6 - chloropurine riboside 5 ' triphosphate , 2 -amino gene, e . g . , enhanced Green Fluorescent Protein ( eGFP ) or 5 ' triphosphate , 2 - thiocytidine 5 ' triphosphate , any other suitable reporter gene . 2 - thiouridine 5' triphosphate , 4 -thiouridine 5 ' triphosphate , [0014 ] FIG . 5 shows selection of UTRs based on liver 5 - aminoallylcytidine 5 ' triphosphate , 5 - aminoallyluridine 54 abundance ( contrasted with abundance in other tissues , e . g . , triphosphate , 5 - bromocytidine 5 ' triphosphate , 5 - bromouri heart ) . mRNAs for the genes Haptoglobin (HP ), Hapto dine 5 ' triphosphate , 5 - iodocytidine 5 ' triphosphate , 5 - io globin - Related Protein (HPR ) , Albumin ( ALB ) , Comple douridine 5 ' triphosphate , 5 -methylcytidine 5 ' triphosphate , ment Component 3 (C3 ), Alpha - 1 - antitrypsin (SERPINA1 ) , US 2018 /0353618 A1 Dec. 13 , 2018

Alpha - 1 -antichymotrypsin (SERPINA3 ) , Fibrinogen Alpha [ 0023 ] RNA is notoriously unstable . Rapid degradation of Chain ( FGA ) , and Fibrinogen Beta Chain ( FGB ) are natu mRNA presents a major challenge to current therapeutic rally highly abundant in liver as compared with their preva paradigms. Chronic mRNA dosing may be required to lence in heart . produce enough protein activity to adequately address the 10015 ) FIG . 6 shows that modified RNAs having hepato condition being treated . If a therapeutic mRNA is rapidly cyte 5 ' UTRs out- performed modified RNAs having syn degraded following delivery , the result will be only a brief thetic or non -hepatic UTRs. modRNAs having hepatocyte 5' pulse of protein production . An extremely frequent dosing UTRs derived from HP, FGB , HPR , ALB , C3 , FGA , SER schedule would be necessary to achieve continuous protein PINA1 and SERPINA3 genes produced more eGFP than expression from this rapidly degraded mRNA . But such a synthetic and non -hepatic (CollA and Col6A ) UTRs in schedule would pose significant toxicity , compliance and primary hepatocytes after transfection . efficacy challenges that may limit therapeutic efficacy . [ 0016 ] FIG . 7 shows that modified mRNA out -performed Reducing dose frequency , therefore , is desirable for improv native mRNAs for all constructs . eGFP synthesis was ing therapeutic efficacy. reduced for all mRNAs containing only natural , [0024 ] Endogenous mRNAs show a wide range of stabili as compared with modRNAs ( compare with FIG . 6 ) . ties . The most stable endogenous mRNAs have half- lives of from 40 to 60 hours (Schwanhausser , B . et al . , Nature , DETAILED DESCRIPTION 473 : 337 - 42 , 2011 ) , as indicated by the arrow in FIG . 3 . By [0017 ] Disclosed herein are methods of improving expres increasing the upper limit ofmRNA half - life , the quantity of sion from an mRNA , e . g . , in a tissue -specific manner ( e . g ., protein delivered may be dramatically increased . liver , or cells in vitro , such as stem cells or lymphocytes ) , [0025 ] RNA stability may also be increased in a tissue untranslated region (UTR ) sequences for enhancing protein specific manner . Every tissue comprises a set of core genes synthesis from mRNAs of interest , such as , for example , that are active in for each tissue (Ramsköld , D . et al. , PLOS therapeutic mRNAs, methods of identifying the same, and Comput. Biol ., 5 :e1000598 , 2009 ) . The tissue -specific methods of using the same as therapeutic agents . UTRs are mRNAs from such core genes accounts for the majority of described , for example , to increase translation and mRNA all RNA molecules ( transcriptome) in each tissue . For stability . Globin 5 ' - and 3 '- UTRs , for example, were used to example , the ten most abundantly expressed genes in mouse improve translation and mRNA stability of heterologous liver represent 25 % of all mRNAs in that tissue. These mRNA and of in vitro transcribed mRNA for immune observations suggest that enhanced RNA stability contrib therapy (Zhao , Y et al. , Cancer Res. , 70 :9053 -61 , 2010 ; utes to abundance of these mRNAs in liver. Kreiter , S . et al ., Cancer Res. , 70 : 9031- 40 , 2010 ) . The [0026 ] As noted above , UTR sequences can modulate 5 -UTR of tobacco etch virus ( TEV ) was also shown to mRNA stability through a variety ofmechanisms , including enhance translation of in vitro transcribed mRNA in mam mRNA binding proteins, miRNA , and secondary structures, malian cells and was used to express erythropoietin in mice . which directly inhibit nucleolytic degradation . Molecular [ 0018 ] It is to be understood that the disclosure is not engineering has, in some instances , led to a 100x improve limited to the particular embodiments of the present disclo ment in protein synthesis , a major component of which is sure described below , as variations of the particular embodi altering UTR sequences ( Thess, A . et al ., Mol. Ther ., ments may be made and still fall within the scope of the 23 : 1456 -64 , 2015 ). appended claims. It is also to be understood that the termi [0027 ] The present disclosure improves upon current tech nology employed is for the purpose of describing particular nology to enhance expression from an mRNA construct , embodiments , and is not intended to be limiting . e . g . , by decreasing the rate ofmRNA degradation to increase [0019 ] As used herein , the singular forms “ a , " " an " and both the duration and the magnitude of protein synthesis “ the ” include plural reference unless the context clearly produced from an mRNA dose , especially in a tissue dictates otherwise . Unless defined otherwise , all technical specific manner . The mRNA described herein can contain , and scientific terms used herein have the samemeaning as for example , a hybrid sequence , which may comprise an commonly understood to one of ordinary skill in the art to open reading frame (ORF ) for a target polypeptide of which this disclosure belongs . interest coupled (upstream of the target of interest ) to a [0020 ] The term “ functionally linked ” or “ operably heterologous , e . g. , a tissue -specific , UTR derived from linked ” means in this context the sequential and function another naturally occurring or engineered gene ( e . g . , a arrangement between a 5 ' UTR , open reading frame (ORF ) , human gene or an engineered UTR from a human gene ). The and 3 ' UTR according to the present disclosure, wherein at mRNA construct( s ) described herein can optionally com least the 5 ' UTR modulates translation of said ORF. prise , for example , a poly -adenosine region (poly - A tail ) [0021 ] The number of molecules of mRNA that can be downstream of the target of the ORF. delivered in a single therapeutic dose is limited by the [0028 ] The mRNA (s ) described herein can comprise natu toxicity of the delivery vehicle . For many diseases of rally occurring or chemically modified ribo interest, a high level of protein synthesis is required from , i . e . , modified mRNAs (modRNAs ) . modRNAS each dose of mRNA to achieve a therapeutic effect. In some can be prepared as described in , for example , U . S . Pat . No . cases , currently available technology is unable to safely 8 ,278 ,036 ( incorporated herein by reference ), to include one produce this high level of synthesis . Thus , achieving a or more pseudouridine residues , for example . and therapeutic effect requires reducing the dose volume. cytidine , for example , can be replaced with 2 -thiouridine [0022 ] Disclosed herein are compositions and methods for and /or 5 -methylcytidine to increase stability of the mRNA . increasing protein synthesis by increasing both the time that [0029 ] The described mRNA can comprise at least one the mRNA remains in translating polysomes (message sta structural or chemical modification . The that is bility ) and the rate at which ribosomes initiate translation on modified in the mRNA , for example , can be a uridine ( U ) , the message (message translation efficiency ). a cytidine ( C ) , an ( A ) , or ( G ) . The modified US 2018 /0353618 A1 Dec. 13 , 2018 nucleoside can be , for example , m®C ( 5 -methylcytidine ), uridine (mcm s2U ) , 5 - aminomethyl - 2 - thio uridine mA ( N -methyladenosine ), s ’ U ( 2 -thiouridien ), (nm s2U ), 5 -methylaminomethyl uridine (mnm U ), 5 -meth (pseudouridine ) or Um ( 2 - 0 -methyluridine ). Some exem ylaminomethyl- 2 - thio uridine (mnmºs2U ), 5 -methylamin plary chemical modifications of nucleosides in the mRNA omethyl- 2 - seleno uridine (mnm se U ) , 5 - carbamoylmethyl molecule further include , for example , pyridine - 4 -one ribo uridine ( ncmU) , 5 - carboxymethylaminomethyl uridine nucleoside, 5 -aza -uridine , 2 - thio - 5 - aza uridine , 2 - thiouri (cmnm U ), 5 -carboxymethylaminomethyl - 2 - thio uridine dine , 4 -thio pseudouridine, 2 -thio pseudouridine , 5 -hy ( cmnm s2U ) , 5 - propynyl uridine , 1 - propynyl pseudouri droxyuridine, 3 -methyluridine , 5 - carboxymethyl uridine , dine, 5- taurinomethyl uridine (tcm U ) , 1 -taurinomethyl 1 -carboxymethyl pseudouridine, 5 -propynyl uridine , 1 -pro pseudouridine, 5 - taurinomethyl - 2 - thio uridine ( TM?s2U ) , pynyl pseudouridine, 5 - taurinomethyluridine , 1 - taurinom 1 - taurinomethyl- 4 - thio pseudouridine, 5 -methyl uridine ethyl pseudouridine , 5 - taurinomethyl- 2 -thio uridine , 1 - tau ( m U , e. g ., having the deoxythymine ) , 1- methyl rinomethyl- 4 - thio uridine , 5 -methyl uridine , 1 -methyl pseudouridine ( m y ) , 5 -methyl - 2 - thio uridine (mºs2U ) , pseudouridine , 4 - thio - 1 -methyl pseudouridine, 2 -thio - 1 1 -methyl - 4 - thio pseudouridine ( m , s * y ) , 4 -thio - 1 -methyl methyl pseudouridine , 1 -methyl - 1 -deaza pseudouridine , pseudouridine , 3 -methyl pseudouridine (mºv ) , 2 -thio - 1 2 -thio - 1 -methyl - 1 -deaza pseudouridine , dihydrouridine , methyl pseudouridine, 1 -methyl - 1 -deaza pseudouridine , dihydropseudouridine, 2 - thio dihydrouridine, 2 - thio dihy 2 -thio - 1 -methyl - 1 -deaza pseudouridine , dihydrouridine ( D ) , dropseudouridine, 2 -methoxyuridine , 2 -methoxy -4 - thio uri dihydropseudouridine , 5 , 6 - dihydrouridine , 5 -methyl dihy dine , 4 -methoxy pseudouridine , 4 -methoxy - 2 - thio pseudou drouridine (m®D ), 2 -thio dihydrouridine , 2 - thio dihy ridine, 5 - aza cytidine , pseudoisocytidine , 3 -methyl cytidine , dropseudouridine , 2 -methoxy uridine , 2 -methoxy - 4 - thio uri N4 -acetylcytidine , 5 - formylcytidine, N4- methylcytidine , dine, 4 -methoxypseudouridine , 4 -methoxy - 2 - thio 5 -hydroxymethylcytidine , 1 -methyl pseudoisocytidine , pyr pseudouridine , N ' -methyl pseudouridine , 3 - ( 3 - amino - 3 - car rolo - cytidine , pyrrolo - pseudoisocytidine, 2 -thio cytidine , boxypropyl ) uridine (acpU ) , 1 -methyl - 3 - ( 3 - amino - 3 - car 2 - thio - 5 -methyl cytidine, 4 - thio pseudoisocytidine , 4 - thio boxypropyl) pseudouridine (acpy ) , 5 -( isopentenylaminom 1 -methyl pseudoisocytidine, 4 - thio - 1 -methyl - 1 - deaza ethyl ) uridine ( inm ” U ) , 5 - ( isopentenylaminomethyl ) - 2 - thio pseudoisocytidine, 1 -methyl - 1 - deaza pseudoisocytidine , uridine ( inms2U ) , . alpha - thio uridine, 2 - O -methyl uridine zebularine, 5- aza zebularine, 5 -methyl zebularine , 5 -aza - 2 (Um ) 5 ,2 -0 - dimethyl uridine (m®Um ) , 2 -O -methyl thio zebularine , 2 - thio zebularine , 2 -methoxy cytidine , pseudouridine (ym ), 2 -thio - 2 - O -methyl uridine ( s2Um ), 2 -methoxy - 5 -methyl cytidine, 4 -methoxy pseudoisocyti 5 -methoxycarbonylmethyl - 2 -0 -methyl uridine (mcm Um ), dine , 4 -methoxy - 1 -methyl pseudoisocytidine , 2 -aminopu 5 -carbamoylmethyl - 2' -O -methyl uridine (ncm Um ), 5 -car rine, 2 ,6 -diaminopurine , 7 -deaza adenine, 7 -deaza -8 - aza boxymethylaminomethyl- 2 - O -methyl uridine ( cmnm Um ), adenine , 7 -deaza - 2 - aminopurine, 7 - deaza - 8 - aza - 2 - aminopu 3 , 2 - 0 - dimethyl uridine ( m 'Um ) , 5 - ( isopentenylaminom rine, 7 - deaza - 2 ,6 - diaminopurine , 7 - deaza - 8 -aza - 2 , 6 - diamin ethyl) - 2 - O -methyl uridine ( inm Um ) , 1 -thio uridine , deoxy opurine, 1- methyladenosine , Nø -methyladenosine , Nø- iso , 2 '- F - ara uridine, 2 '- F uridine , 2 -OH - ara uridine , pentenyladenosine , Nº- ( cis -hydroxyisopentenyl ) adenosine , 5 -( 2 - carbomethoxyvinyl) uridine , and 5 -( 3 -( 1- E -prope 2 -methylthio - N? - ( cis -hydroxyisopentenyl ) adenosine , nylamino ) uridine . Nº -glycinylcarbamoyladenosine , N?- threonylcarbamoylad [0031 ] In some embodiments , the modified nucleobase is enosine, 2 -methylthio -N? - threonyl carbamoyladenosine, a modified including , for example , 5 - aza cytidine, Nº, N - dimethyladenosine , 7 -methyladenine , 2 -methylthio 6 -aza cytidine , pseudoisocytidine, 3 -methyl cytidine (m°C ), adenine , 2 -methoxy adenine, , l -methyl inosine , N4 -acetyl cytidine (act ) , 5 -formyl cytidine (fC ), N4- methyl wyosine, wybutosine , 7 -deaza guanosine, 7 - deaza - 8 -aza cytidine (m + C ), 5 -methyl cytidine (m°C ), 5 -halo cytidine guanosine , 6 -thio guanosine , 6 - thio - 7 - deaza guanosine , ( e . g ., 5 - iodo cytidine ) , 5 -hydroxymethyl cytidine (hmC ) , 6 - thio - 7 -deaza - 8 -aza guanosine, 7 -methyl guanosine , 1 -methyl pseudoisocytidine, pyrrolo -cytidine , pyrrolo 6 - thio - 7 -methyl guanosine , 7 -methylinosine , 6 -methoxy pseudoisocytidine, 2 - thio cytidine (s2C ), 2 -thio -5 -methyl guanosine , 1 -methylguanosine , N2- methylguanosine , cytidine , 4 - thio pseudoisocytidine , 4 - thio - 1 - methyl pseudoi N ,N2 - dimethylguanosine , 8 - oxo guanosine, 7 -methyl - 8 socytidine, 4 - thio - 1- methyl - 1 - deaza pseudoisocytidine , oxo guanosine , 1 -methyl - 6 - thio guanosine, N2-methyl - 6 1 -methyl - 1 - deaza pseudoisocytidine , zebularine, 5 -aza thio guanosine , and N ?, N2 - dimethyl - 6 -thio guanosine. In zebularine, 5 -methyl zebularine , 5 -aza - 2 - thio zebularine , another embodiment, the modifications are independently 2 - thio zebularine , 2 -methoxy cytidine , 2 -methoxy -5 -methyl selected from the group consisting of 5 -methylcytosine , cytidine , 4 -methoxy pseudoisocytidine , 4 -methoxy - 1 pseudouridine and 1 -methylpseudouridine . methyl pseudoisocytidine, lysidine (k²C ), alpha- thio cyti [0030 ] In some embodiments , the modified nucleobase in dine, 2 - O -methyl cytidine (Cm ) , 5 , 2 - 0 -dimethyl cytidine the mRNA molecule is a modified including, for (mCm ), N + - acetyl- 2 - O -methyl cytidine ( ac + Cm ), N4, 2 - 0 example , pseudouridine ( ) , pyridine - 4 - one , dimethyl cytidine ( m *Cm ), 5 - formyl - 2 '- O -methyl cytidine 5 - aza uridine , 6 - aza uridine , 2 - thio - 5 - aza uridine, 2 -thio (fCm ), N4, N4 , 2 '- O - trimethyl cytidine ( m * , Cm ), 1 - thio cyti uridine (s2U ) , 4 - thio uridine (s4U ) , 4 - thio pseudouridine , dine, 2' - F - ara cytidine, 2' - F cytidine , and 2 '- OH - ara cytidine . 2 - thio pseudouridine , 5 -hydroxy uridine (hoºU ) , 5 - amino [0032 ] In some embodiments , the modified nucleobase is allyl uridine , 5 -halo uridine ( e . g . , 5 - iodom uridine or a modified adenine including , for example , 2 - amino , 5 -bromo uridine ) , 3 -methyl uridine (mºU ), 5 -methoxy uri 2 ,6 -diamino purine, 2 - amino -6 - halo purine ( e . g . , 2 - amino dine (moºU ) , uridine 5 - oxyacetic acid ( cmo U ) , uridine 6 - chloro purine ), 6 -halo purine ( e . g ., 6 - chloro purine ) , 5 -oxyacetic acid methyl ester (mcmoPU ), 5 -carboxymethyl 2 -amino -6 -methyl purine, 8 -azido adenosine , 7 -deaza uridine (cmU ) , l - carboxymethyl pseudouridine , 5 -car adenine , 7 - deaza - 8 - aza adenine , 7 -deaza - 2 - amino purine , boxyhydroxymethyl uridine ( chm®U ) , 5 - carboxyhydroxym 7 -deaza - 8 -aza - 2 - amino purine, 7 -deaza - 2 , 6 -diamino purine , ethyl uridine methyl ester (mchmU ), 5 -methoxycarbonyl 7 -deaza - 8 -aza - 2 , 6 -diamino purine, 1 -methyl adenosine methyl uridine (mcm®U ) , 5 -methoxycarbonylmethyl - 2 - thio mA( ) , 2 -methyl adenine (m ’ A ), N?- methyl adenosine US 2018 /0353618 A1 Dec. 13 , 2018

(mºA ), 2- methylthio - N?- methyl adenosine (ms ? m?A ), adenine, 7 -deaza adenine, 3 - deaza adenine, pyrazolo [3 ,4 - d ] N “ - isopentenyl adenosine (iA ), 2 -methylthio - N “ - isopente , imidazo [ 1 , 5 - a ] 1 , 3 , 5 triazinones , 9 - deaza nyl adenosine (ms ? i?A ), Nº- ( cis -hydroxyisopentenyl ) , imidazo [ 4 , 5 - d ]pyrazines , thiazolo [ 4 , 5 - d ]pyrimi adenosine ( 10A) , 2 -methylthio - N? - ( cis -hydroxyisopente dines , pyrazine - 2 - ones , 1 , 2 , 4 - triazine , pyridazine ; and 1 , 3 , nyl) adenosine (ms ? io A ), Nº - glycinylcarbamoyl adenosine 5 - triazine . When the nucleotides are depicted using the ( g?A ) , Nø - threonylcarbamoyl adenosine ( tºA ) , Nø- methyl shorthand A , G , C , T or U , each letter refers to the repre Nº -threonylcarbamoyl adenosine (mºtºA ), 2 -methylthio sentative base and /or derivatives thereof, e .g . , A includes Nó- threonylcarbamoyl adenosine (ms - gºA ), Nº, N -dim adenine or adenine analogs, e . g ., 7 - deaza adenine) . ethyl adenosine (m®2A ) , Nó- hydroxynorvalylcarbamoyl [0035 ] Other modifications include , for example, those in adenosine ( hnºA ) , 2 -methylthio - N? - hydroxynorvalylcar U . S . Pat. No. 8 , 835 , 108 ; U . S . Patent Application Publication bamoyl adenosine (ms ? hn?A ), N -acetyl adenosine (acºA ), No . 20130156849 ; Tavernier, G . et al ., J . Control. Release , 7 -methyl adenine , 2 -methylthio adenine , 2 - methoxy 150 :238 -47 , 2011 ; Anderson , B . et al. , Nucleic Acids Res. , adenine , alpha - thio adenosine , 2 - O -methyl adenosine ( Am ) , 39 : 9329 - 38 , 2011 ; Kormann , M . et al ., Nat. Biotechnol. , N “, 2 - 0 - dimethyl adenosine (m?Am ) Nº, N “ ,2 '- O - trimethyl 29 : 154 - 7 , 2011 ; Karikó , K . et al. , Mol. Ther , 16 : 1833 - 40 , adenosine (mº , Am ), 1, 2 - 0 -dimethyl adenosine (m°Am ), 2008 ; Karikó, K . et al ., Immunity , 23 : 165 -75 , 2005 ; and 2 '- O -ribosyl adenosine (phosphate ) (Ar ( p ) ), 2 -amino -N Warren , L . et al. , Cell Stem Cell , 7 :618 - 30 , 2010 ; the entire methyl purine , 1 - thio adenosine , 8 - azido adenosine, 2 ' - F - ara contents of each of which is incorporated herein by refer adenosine , 2 '- F adenosine, 2 -OH -ara adenosine, and Nó ence . The modRNAs can be prepared , for example , by ( 19 - amino -pentaoxanonadecyl ) adenosine . incorporation of base -modified nucleotides, such as [0033 ] In some embodiments , the modified nucleobase is N ' -methyl - pseudouridine 5 '- triphosphate , the chemical a modified guanine including , for example , inosine (I ) , structure of which is shown below . 1 -methyl inosine ( m I) , wyosine ( img ) , methylwyosine (mimG ) , 4 - demethyl wyosine ( img - 14 ) , isowyosine ( imG2 ), wybutosine ( yW ), peroxywybutosine (02yW ) , hydroxywybutosine (?HyW ) , undermodified hydroxy wybutosine (?HyWy ) , 7 -deaza guanosine, queuosine ( Q ) , NH epoxyqueuosine (oQ ) , galactosyl queuosine ( galQ ) , manno syl queuosine (manQ ), 7 - cyano -7 -deaza guanosine (preQo ) , 7 - aminomethyl - 7 - deaza guanosine ( preQ?) , archaeosine ( G + ) , 7 -deaza - 8 - aza guanosine , 6 -thio guanosine , 6 -thio - 7 deaza guanosine , 6 - thio - 7 -deaza - 8 - aza guanosine, 7 -methyl guanosine ( m ' G ) , 6 -thio - 7 -methyl guanosine , 7 -methyl 4 Lit inosine , 6 -methoxy guanosine , 1- methyl guanosine (m 'G ), HO OH N2-methyl - guanosine (mºG ), N ?, N2 - dimethyl guanosine (m² , G ), N2, 7 - dimethyl guanosine (m2 , 7G ), N², N2, 7 - dim ethyl guanosine (m22 , 7G ), 8 - oxo guanosine , 7 -methyl - 8 - oxo [0036 ] Described herein are UTRs derived from , for guanosine, 1 -methio guanosine, N2- methyl - 6 - thio guanos example , human genes that are naturally abundant and ine , N ,N2 - dimethyl- 6 - thio guanosine , alpha- thio guanos well - translated in the tissue being targeted . Such UTRsmay ine , 2 - O -methyl guanosine (Gm ), N2 -methyl - 2 - O -methyl be used to determine whether altering the UTRs extends guanosine (m°Gm ) , N , N2- dimethyl - 2 - O -methyl guanosine mRNA half - life , thereby increasing protein production from ( m ? Gm ), 1 -methyl - 2 - 0 -methyl guanosine ( m Gm ), N2, 7 mRNAs, e . g ., modRNAs. dimethyl -2 -0 -methyl guanosine (m2 , 7Gm ), 2 - O -methyl [0037 ] Described herein are libraries of constructs with inosine ( 1m ), 1, 2 - O -dimethyl inosine (m ’ Im ), 2 - O - ribosyl UTRs, which may be screened to identify candidate UTRs guanosine (phosphate ) (Gr (p )) , 1- thio guanosine, O?- methyl that specifically increase mRNA stability in target tissues , guanosine, 2 - F - ara guanosine , and 2 '- F guanosine . such as liver , or cells in vitro , such as stem cells or [0034 ] The nucleobase of the nucleotide can be indepen lymphocytes . dently selected from a purine , a pyrimidine, a purine or [ 0038 ] A combinatorial library of constructs (including , pyrimidine analog . For example , the nucleobase can each be for example , a reporter gene ) can be used to evaluate the independently selected from adenine , cytosine , guanine , effects of heterologous 5 ' and 3 ' UTRs on mRNA stability uracil or . The nucleobase can also include , for (FIG . 4 ). Such a library can be constructed , for example , by example , naturally occurring and synthetic derivatives of a identifying mRNAs likely to be stable in a specific tissue , base , including , but not limited to , pyrazolo [ 3 , 4 - d ]pyrimi e . g . , liver , synthesizing UTRs of these mRNA sequences , dines , 5 -methylcytosine ( 5 -me - C ) , 5 -hydroxymethyl cyto and building combinatorial library of UTRs flanking a sine , , hypoxanthine, 2 - amino adenine , 6 -methyl reporter gene, e . g . , enhanced Green Fluorescent Protein and other alkyl derivatives of adenine and guanine , 2 -propyl ( eGFP ) or any other suitable reporter gene. and other alkyl derivatives of adenine and guanine , 2 - thio Identification of mRNAs Stable in Liver uracil, 2 - thio and 2 -thio cytosine, 5 -propynyl uracil 100391 UTRs were selected based on liver abundance and and cytosine , 6 - azo uracil , cytosine and thymine , pseudou stability measurements . As shown in FIG . 5 ( inset) , mRNAs racil , 4 - thio uracil, 8 -halo ( e . g . , 8 -bromo ) , 8 - amino , 8 - thiol, for the genes Haptoglobin (HP ) , Haptoglobin -Related Pro 8 - thioalkyl, 8 -hydroxyl and other 8 - substituted and tein (HPR ) , Albumin ( ALB ), Complement Component 3 , 5 -halo particularly 5 -bromo , 5 -trifluoromethyl and (C3 Alpha - 1 - antitrypsin (SERPINA1 Alpha - 1 - antichy other 5 -substituted and , 7 -methyl guanine motrypsin (SERPINA3 ) , Fibrinogen Alpha Chain ( FGA ) , and 7 -methyl adenine , 8 - aza guanine and 8 - aza adenine , and Fibrinogen Beta Chain ( FGB ) are naturally highly deaza guanine, 7 -deaza guanine , 3 - deaza guanine, deaza abundant in liver as compared with their prevalence in heart. US 2018 /0353618 A1 Dec. 13 , 2018

Synthesis of UTRS fibroblast cell line ( the only cell line in which globalmRNA stabilities have been tested -showing a half- life of ~ 40 - 50 [0040 ] 5' and 3' UTRs of each of HP , HPR , ALB , C3 , hours for each (Schwanhausser , B . et al. , Nature, 473 : 337 SERPINAI, SERPINA3 , FGA , and FGB genes were 42 , 2011 ) ; and an engineered , synthetic 5 ' UTR (SEQ ID obtained via PCR and then sequenced by methods known in NO : 11; Mandal, P. & Rossi, D ., Nat. Protoc. , 8: 568 -82 , the art . The 5 ' UTRs of these genes are shown in Table 1 . 2013 ). [0042 ] Twelve 3 'UTRs were used : eight from HP, FGB , TABLE 1 HPR , ALB , C3 , FGA , SERPINA1, SERPINA3 , two from SEQ collagens (Col1A and Col6A ), a synthetic 3' UTR (Mandal , ID P . & Rossi, D . , Nat. Protoc. , 8 : 568 - 82 , 2013 ) , and one from Gene NO 5 ' UTR Sequence a long non -coding RNA ( IncRNA ) , which is known to be HP 1 GGGAGAUGCCCCACAGCACUGCCCUUCCAGAGGCAAG stable and shown to stabilize other messages without ACCAACCAAG depending on a poly - A tail. [0043 ] eGFP , a fluorescent protein , was used as a reporter, FGB 2 GGGAGAUAUAUAUAGGAUUGAAGAUCUCUCAGUUAAG and the eGFP ORF was used in all constructs to test 5 ' and UCUACAUGAAAAGG 3 'UTRs . eGFP was used because it can be easily measured HPR 3 GGGACUGCCCUUCCAGAGGCAAGACCAACCAAG by fluorescence read -out . Other suitable reporters , such as, for example , adiponectin and Factor VII, for example , are ALB 4 GGGAGUAUAUUAGUGCUAAUUUCCCUCCGUUUGUCCU known in the art and may also be used . AGCUUUUCUCUUCUGUCAACCCCACA [0044 ] If any of the tested UTRs, i. e. , from HP, FGB , C3 5 GGGAGAUAAAAAGCCAGCUCCAGCAGGCGCUGCUCAC HPR , ALB , C3 , FGA , SERPINA1 and SERPINA3 genes , UCCUCCCCAUCCUCUCCCUCUGUCCCUCUGUCCCUCU for example , display greater stability and protein synthesis GACCCUGCACUGUCCCAGCACC than the UTRs from collagen (Col1A and Col6A ) and FGA 6 GGGAGCAAUCCUUUCUUUCAGCUGGAGUGCUCCUCA engineered UTRs , these UTRs may be functionally linked to GGAGCCAGCCCCACCCUUAGAAAAG a gene of interest, such as therapeutic gene , to test stability and synthesis of the gene of interest in , for example , liver, SERPINA1 7 GGGAUUCAUGAAAAUCCACUACUCCAGACAGACGGCU as compared with that in , for example , non - liver tissues . UUGGAAUCCACCAGCUACAUCCAGCUCCCUGAGGCAG Alternatively , stability and synthesis may be tested in any AGUUGAGA other organ , tissue or cell system , including but not limited Colla 8 GGGGUGUCCCAUAGUGUUUCCAAACUUGGAAAGGGC to stem cells or lymphocytes in vitro . GGGGGAGGGCGGGAGGAUGCGGAGGGCGGAGGUAU GCAGACAACGAGUCAGAGUUUCCCCUUGAAAGCCUCA [0045 ] All 132 possible combinations of the eleven 5 ' AAAGUGUCCACGUCCUCAAAAAGAAUGGAACCAAUUU UTRs and twelve 3 ' UTRs, plus eGFP reporter gene , were AAGAAGCCAGCCCCGUGGCCACGUCCCUUCCCCCAUU synthesized using natural nucleotides or modRNAs (which CGCUCCCUCCUCUGCGCCCCCGCAGGCUCCUCCCAG CUGUGGCUGCCCGGGCCCCCAGCCCCAGCCCUCCCA contain non - natural nucleotides, e . g ., NP- methyl - pseudouri UUGGUGGAGGCCCUUUUGGAGGCACCCUAGGGCCAG dine 5 ' triphosphate ) . GGAAACUUUUGCCGUAUAAAUAGGGCAGAUCCGGGCU [0046 ] To test which UTRs perform best , eGFP mRNAS UUAUUAUUUUAGCACCACGGCAGCAGGAGGUUUCGGC and eGFP modRNAs may be synthesized with variable UTR UAAGUUGGAGGUACUGGCCACGACUGCAUGCCCGCG CCCGCCAGGUGAUACCUCCGCCGGUGACCCAGGGGC sequences as a pool, mRNAs or modRNAs transfected UCUGCGACACAAGGAGUCUGCAUGUCUAAGUGCUAGA individually into primary hepatocytes, and eGFP production assessed at time points after transfection . Col6A 9 GGGGCUUACUCGGCGCCCGCGCCUCGGGCCGUCGGG AGCGGAGCCUCCUCGGGACCAGGACUUCAGGGCCAC Generation of Plasmid DNA AGGUGCUGCCAAG [0047 ] Plasmids containing the sequences for the UTR SERPINA3 10 GGGACAAUGACUCCUUUCGGUAAGUGCAGUGGAAGC library were synthesized and cloned into an in vitro tran UGUACACUGCCCAGGCAAAGCGUCCGGGCAGCGUAG scription plasmid (DNA018 ). All 23 UTRs ( eleven 5 ' UTRs GCGGGCGACUCAGAUCCCAGCCAGUGGACUUAGCCC and twelve 3 ' UTRs ) and the reporter ORF were synthesized CUGUUUGCUCCUCCGAUAACUGGGGUGACCUUGGUU AAUAUUCACCAGCAGCCUCCCCCGUUGCCCCUCUGGA and combinatorially cloned into DNA018 . Synthesis was UCCACUGCUUAAAUACGGACGAGGACAGGGCCCUGUC performed using Gibson cloning , and constructs were UCCUCAGCUUCAGGCACCACCACUGACCUGGGACAGU inserted into the plasmid template using Bsal, thus, the GAAUCGACA components were free of Bsal cloning sites . Engi 11 GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUA neered AGAGCCACC Generation of In Vitro Translated RNAs [ 0048 ] Plasmids were pooled by 3 ' UTR , generating 12 pools , each containing 11 plasmids with identical 3 ' UTRs Construction of Combinatorial Library of UTRs Flanking a and differing 5 ' UTRs . Each pool was then linearized with Reporter Gene EcoRI, producing single - stranded template . mRNAs and modRNAs were transcribed from each pool with 17 poly [0041 ] Eleven 5' UTRs were used : eight from HP , FGB , merase , using all natural nucleotides or nucleotides contain HPR , ALB , C3 , FGA , SERPINA1, and SERPINA3, (SEQ ing Nl-methyl - pseudouridine 5 ' triphosphate , respectively . ID NOS : 1 - 7 and 10 ), which are derived from natural mRNAs and modRNAs were capped during the in vitro mRNAs in human liver tissue ; two from collagens CollA transcription reaction using the anti - reverse cap analogue and Co16A SEQ ID NOS : 9 and 10 ), which were among the ( ARCA ) . In vitro translated RNAs were then purified using most stable mRNAs in a study performed in a human methods known in the art . US 2018 / 0353618 A1 Dec. 13 , 2018

[0049 ] The method of modRNA generation may result in (MMA ) , MMA and homocystinuria type C (MMACHC ), several nucleotides being added to each end of the mRNA . thrombotic thrombocytopenic purpura ( TTP ) , hyperornith At the 5' end of each message , there may be found GGG . inemia -hyperammonemia -homocitrullinuria syndrome This is imposed by the T7 promoter being used for tran ( HHH ) , complement component 2 deficiency (C2D ) , F2, scription . At the 3 ' end of each message, there may be found protein S deficiency (caused by allelic variants of PROS1) , GAAUU following the poly - A tail . This may exist because alpha - 1 antitrypsin deficiency (A1AT ) , glutaric academia 1 (GA - 1 ) , glutaric academia 2 (GA - 2 ) , cystinosis (CTNS ) , EcoRI is used for template linearization . tyrosinemia , tyrosinemia type 3 ( cause by allelic variants of HPD ) , D -bifunctional protein deficiency (DBP ) , Fanconi Testing Protein Synthesis Bickel syndrome ( FBS ), pseudoxanthoma elasticum (PXE ) , [0050 ] modRNAs were generated from the construct of primary biliary cirrhosis , Pompe disease, glycerol kinase interest through in vitro transcription using just a single deficiency (GKD ) , proprionic acidemia (PA ) and Crigler plasmid as template , and eGFP synthesis was measured Najjar syndrome (CN , e . g ., CN1) . Protein expressed from following transfection in primary hepatocytes , using a fluo mRNAs comprising one or more heterologous UTRs can be rescence read -out . provided for treating liver condition (hepatitis C ) such as interferon alpha , TNF - related apoptosis - inducing ligand , EXAMPLE vascular adhesion protein 1 , hepatocyte growth factor, and the like may be provided . [0051 ] FIG . 6 shows that modRNAs having hepatocyte 5 ' [0055 ] Molecular targets for replacement therapies that UTRs derived from HP, FGB , HPR , ALB , C3, FGA , SER can include heterologous UTRs as described herein , include , PINA1 and SERPINA3 genes produced more eGFP than for example , gene products from the group consisting of: synthetic and non -hepatic (CollA and Col6A ) UTRs in interferon alpha , TNF -related apoptosis - inducing ligand , primary hepatocytes after transfection . These results suggest vascular adhesion protein 1 , hepatocyte growth factor, that hepatocyte 5 ' UTRs are liver - specific for RNA stability , G6PC , ABCB11, ABCB4 , ASL1, ASS , Arg1 , AGXT, OTC , protein synthesis , or both . All messages were transfected in CBS , PAH , GBE , GALE , HADH , MCCC1, MMA , triplicate in primary hepatocytes . Standard deviation is ADAMTS13 , SLC25A15 , C2 , F2 , PROS1, SERPINA1, ~ 30 % on all samples . GALT1, ETFA , GCDH , CTNS , FAH , TAT, HPD , HSD17b4, 10052 ]. On the other hand ,mRNAs containing only natural SLC2A2, GALC , ABCC6 , AHSG , PDC - E2 , GAA , nucleotides produced less eGFP than modRNAs, for all ATP8B1, MMACHC , GK , PCCA , PCCB and UGT1A1. constructs tested . As shown in FIG . 7 , eGFP synthesis was [0056 ] In addition to liver , the present disclosure may be reduced for all mRNAs containing only natural nucleotides , applied to other tissues and cells in which increased stability as compared with modRNAs (compare with FIG . 6 ) . These and /or translation efficiency of therapeutic mRNAs is results suggest that modified nucleotides increase mRNA desired . For example , to engineer lymphocytic cells or stem stability and/ or protein synthesis. Importantly, the liver cells for therapeutic purposes , stability of therapeutic specific effect of hepatocyte 5 ' UTRs persists because mRNAs after delivery may be increased by generating mRNAs containing only natural nucleotides and having mRNAs , including, for example , modRNAs, having UTRs , hepatocyte 5 ' UTRs still out- performed those having syn derived from mRNAs that are naturally abundant in lym thetic and non -hepatic ( CollA and Co16A ) UTRs. All mes phocytic cells or stem cells, linked to one or more thera sages were transfected in triplicate in primary hepatocytes . peutic ORFs. Standard deviation is ~ 30 % on all samples. [0057 ] The present disclosure differs from previous [0053 ] Thus, in addition to rendering modRNAs nonim mRNA therapy efforts , which have relied on standard UTR munogenic , the present disclosure shows the surprising sequences that are not tissue - specific , and give consistent results that the modified nucleoside pseudouridine can also but lower levels of protein expression . Thus, the present increase RNA stability and / or protein synthesis . disclosure provides a significant advantage over the art by [0054 ] The 5 ' and 3 ' UTRs may be functionally linked to enabling mRNA therapies for treatment of chronic condi an ORF , which ORF may be a therapeutic gene encoding tions that require sustained , high - level protein synthesis . protein for preventing or treating liver conditions such as , [0058 ] All references cited in this specification are herein for example , acute hepatitis , chronic hepatitis , liver cirrho incorporated by reference as though each reference was sis , cirrhosis , fatty liver , or liver cancer, and the like . specifically and individually indicated to be incorporated by Additional liver diseases or conditions that can be treated or reference . The citation of any reference is for its disclosure prevented by the compositions and methods described prior to the filing date and should not be construed as an herein include, for example , any liver- associated enzyme admission that the present disclosure is not entitled to deficiency such as, for example , glycogen storage disease , antedate such reference by virtue of prior disclosure . It will progressive familial intrahepatic cholestasis 1 (PFIC1 ) , pro - be understood that each of the elements described above , or gressive familial intrahepatic cholestasis 2 ( PFIC2 ) , progres two or more together may also find a useful application in sive familial intrahepatic cholestasis 3 (PFIC3 ) , adenylosuc other types of methods differing from the type described cinate lyase deficiency (ASLD ), citrullinemia , arginase - 1 above . Without further analysis , the foregoing will so fully deficiency, primary hyperoxaluria type 1 (PH1 ) , ornithine reveal the gist of the present disclosure that others can , by transcarbamylase deficiency (OTCD ) , homocystinuria , applying current knowledge , readily adapt it for various pheylketonuria , glycogen storage disease type IV (GSDIV ) , applications without omitting features that, from the stand galactose - 1 -phosphate uridylyltransferase deficiency ( galac point of prior art , fairly constitute essential characteristics of tosemia , e . g . , type I (GALTI ) , type II (GALTII ) or type 3 the generic or specific aspects of this disclosure set forth in (GALTIII )) , long chain 3 -hydroxyacyl - CoA dehydrogenase the appended claims. The foregoing embodiments are pre deficiency (LCHADD ) , 3 -methylcrotonyl - CoA carboxylase sented by way of example only ; the scope of the present deficiency MCCC1( deficiency ) , methylmalonic aciduria disclosure is to be limited only by the following claims. US 2018 /0353618 A1 Dec. 13 , 2018

SEQUENCE LISTING < 160 > NUMBER OF SEQ ID NOS : 11 < 210 > SEQ ID NO 1 < 211 > LENGTH : 47 < 212 > TYPE : RNA < 213 > ORGANISM : Homo sapiens < 400 > SEQUENCE : 1 gggagaugccccacagcacu gcccuuccag aggcaagaccaaccaag 47

< 210 > SEQ ID NO 2 < 211 > LENGTH : 51 < 212 > TYPE : RNA < 213 > ORGANISM : Homo sapiens < 400 > SEQUENCE : 2 gggagauaua uauaggauug aagaucucuc aguua agucu acaugaaaag g 51

< 210 > SEQ ID NO 3 < 211 > LENGTH : 33 < 212 > TYPE : RNA < 213 > ORGANISM : Homo sapiens < 400 > SEQUENCE : 3 gggacugccc uuccagaggc aagaccaacc aag 33

< 210 > SEQ ID NO 4 < 211 > LENGTH : 63 < 212 > TYPE : RNA < 213 > ORGANISM : Homo sapiens < 400 > SEQUENCE : 4 gggaguauau uagugcuaau uucccuccgu uuguccuagc uuuucucuuc ugucaacccc aca

< 210 > SEQ ID NO 5 < 211 > LENGTH : 96 < 212 > TYPE : RNA < 213 > ORGANISM : Homo sapiens < 400 > SEQUENCE : 5 gggagauaaa aagccagcuc cagcaggcgc ugcucacucc uccccauccu cucccucuqu cccucugucc cucugacccu gcacuguccc agcacc

< 210 > SEQ ID NO 6 < 211 > LENGTH : 61 < 212 > TYPE : RNA < 213 > ORGANISM : Homo sapiens < 400 > SEQUENCE : 6 gggagcaauc cuuucuuuca gcuggagugc uccucaggag ccagccccac ccuuagaaaa

< 210 > SEQ ID NO 7 < 211 > LENGTH : 82 < 212 > TYPE : RNA < 213 > ORGANISM : Homo sapiens US 2018 /0353618 A1 Dec. 13 , 2018

- continued

< 400 > SEQUENCE : 7 gggauucaug aaaauccacu acuccagaca gacggcuuug gaauccacca gcuacaucca gcucccugag gcagaguuga ga

< 210 > SEQ ID NO 8 < 211 > LENGTH : 474 < 212 > TYPE : RNA < 213 > ORGANISM : Homo sapiens < 400 > SEQUENCE : 8 gggguguccc auaguguuuc caaacuugga aagggcgggg gagggcggga ggaugcggag 60 ggcggaggua ugcagacaac gagucagagu uuccccuuga aagccucaaa aguguccacg 120 uccucaaaaa gaauggaacc aauuuaagaa gccagccccg uggccacguc ccuuccccca 180 uucgcucccu ccucugcgcc cccgcaggcu ccucccagcu guggcugccc gggcccccag 240 ccccagcccu cccauuggug gaggcccuuu uggaggcacc cuagggccag ggaaacuuuu 300 gccguauaaa uagggcagau ccgggcuuua uuauuuuagc accacggcag caggagguuu 360 cggcuaaguu ggagguacug gccacgacug caugcccgcg cccgccaggu gauaccuccg 420 ccggugaccc aggggcucug cgacacaagg agucugcaug ucuaagugcu agac 474

< 210 > SEQ ID NO 9 < 211 > LENGTH : 85 < 212 > TYPE : RNA < 213 > ORGANISM : Homo sapiens < 400 > SEQUENCE : 9 ggggcuuacu cggcgcccgc gccucgggcc gucgggagcg gagccuccuc gggaccagga 60 cuucagggcc acaggugcug ccaag 85

V< 210 > SEQ ID NO 10 V< 211 > LENGTH : 264 V< 212 > TYPE : RNA < 213 > ORGANISM : Homo sapiens < 400 > SEQUENCE : 10 gggacaauga cuccuuucgg uaagugcagu ggaagcugua cacugcccag gcaaagcguc 60 cgggcagcgu aggcgggcga cucagauccc agccagugga cuuagccccu guuugcuccu 120 ccgauaacug gggugaccuu gguuaauauu caccagcago cucccccguu gccccucugg 180 auccacugcu uaaauacgga cgaggacagg gcccugucuc cucagcuuca ggcaccacca 240 cugaccuggg acagugaauc gaca 264

< 210 > SEO ID NO 11 < 211 > LENGTH : 47 < 212 > TYPE : RNA < 213 > ORGANISM : artificial 20 > FEATURE : < 223 > OTHER INFORMATION : engineered 5 ' UTR < 400 > SEQUENCE : 11 gggaaauaag agagaaaaga agaguaagaa gaaauauaag agccacc 47 US 2018 /0353618 A1 Dec. 13 , 2018

What is claimed is : f ) selecting at least one test construct having a higher 1 . An mRNA , comprising an open reading frame (ORF ) protein expression from the reporter ORF than the flanked by a heterologous 5 ' untranslated region (UTR ) reference construct ; and and/ or a heterologous 3 'UTR , wherein the 5 'UTR and /or the g ) identifying the 5 ' UTR and the 3 ' UTR of the selected 3 ' UTR are derived from mRNA that is naturally abundant test construct. in a tissue of interest . 2 . The mRNA of claim 1 , wherein the tissue is liver. 13 . The method of claim 12 , wherein the desired tissue is 3 . The mRNA of claim 2 , wherein the mRNA has a tissue liver. half - life of at least 4 hours. 14 . The method of claim 13 , wherein an mRNA compris 4 . The mRNA of claim 3 , wherein the heterologous 5 ing an identified 5 ' UTR or 3 ' UTR has a tissue half - life of UTR and /or the heterologous 3 ' UTR is derived from a gene at least 4 hours . selected from the group consisting of: HP, FGB , HPR , ALB , 15 . The method of claim 12, wherein the reporter ORF is C3 , FGA , Col1A , Col6A , SERPINA1 and SERPINA3 . eGFP , adiponectin or Factor VII . 5 . The mRNA of claim 1 , wherein the heterologous 5 ' 16 . A method of treating a liver condition , comprising UTR comprises a sequence selected from the group con administering to a subject in need thereof the mRNA of sisting of: SEQ ID NOs: 1 - 6 , 9 or 10 . claim 2 . 6 . The mRNA of claim 5 , wherein the ORF is a reporter 17 . The method of claim 16 , wherein the liver condition gene or a therapeutic gene of interest. is selected from the group consisting of: acute hepatitis , 7 . The mRNA of claim 6 , wherein the mRNA comprises chronic hepatitis , liver cirrhosis , cirrhosis , fatty liver, liver at least one modified or non - naturally occurring nucleotide . cancer, glycogen storage disease , progressive familial intra 8 . The mRNA of claim 7 , wherein the at least one hepatic cholestasis 1 (PFIC1 ) , progressive familial intrahe modified or non -naturally occurring nucleotide comprises at patic cholestasis 2 (PFIC2 ), progressive familial intrahepatic least one backbone modification , sugar modification or base cholestasis 3 (PFIC3 ) , adenylosuccinate lyase deficiency modification . (ASLD ), citrullinemia , arginase- 1 deficiency , primary 9 . The mRNA of claim 8 , wherein the at least one hyperoxaluria type 1 (PH1 ) , ornithine transcarbamylase modified or non -naturally occurring nucleotide comprises deficiency (OTCD ) , homocystinuria , pheylketonuria , glyco the at least one base modification . gen storage disease type IV (GSDIV ) , galactose - 1 - phos 10 . The mRNA of claim 9 , wherein the at least one base phate uridylyltransferase deficiency (type I (GALTI ) , type II modification is selected from the group consisting of: (GALTII ) or type 3 (GALTIII ) ), long chain 3 -hydroxyacyl 2 - amino -6 - chloropurine riboside 5 ' triphosphate , 2 -amino CoA dehydrogenase deficiency (LCHADD ) , 3 -methylcroto adenosine 5 ' triphosphate , 2 -thiocytidine 5 ' triphosphate , nyl- CoA carboxylase deficiency (MCCC1 deficiency ) , 2 - thiouridine 5 ' triphosphate , 4 -thiouridine 5' triphosphate , methylmalonic aciduria (MMA ), MMA and homocystinuria 5 - aminoallylcytidine 5 ' triphosphate , 5 - aminoallyluridine 5 type C (MMACHC ), thrombotic thrombocytopenic purpura triphosphate , 5 - bromocytidine 5 ' triphosphate , 5 -bromouri ( TTP ) , hyperornithinemia -hyperammonemia - homocitrul dine 5 ' triphosphate, 5 - iodocytidine 5 ' triphosphate , 5 - io linuria syndrome (HHH ) , complement component 2 defi douridine 5 ' triphosphate , 5 -methylcytidine 5 ' triphosphate , ciency (C2D ) , F2 , protein S deficiency ( caused by allelic 5 -methyluridine 5' triphosphate , 6 - azacytidine 5 ' triphos variants of PROS1 ) , alpha - 1 antitrypsin deficiency ( A1AT ) , phate , 6 -azauridine 5 ' triphosphate , 6 -chloropurine riboside glutaric academia 1 (GA - 1 ) , glutaric academia 2 (GA - 2 ) , 5 ' - triphosphate , 7 - deazaadenosine 5 ' triphosphate , 7 - deaza cystinosis (CTNS ) , tyrosinemia , tyrosinemia type 3 ( cause guanosine 5 ' triphosphate , 8 - azaadenosine 5 ' triphosphate , by allelic variants of HPD ), D -bifunctional protein defi 8 -azidoadenosine 5 ' triphosphate , benzimidazole riboside 5 ' ciency (DBP ) , Fanconi- Bickel syndrome (FBS ) , pseudox triphosphate , NP- methyladenosine 5' triphosphate , NP-meth anthoma elasticum (PXE ), primary biliary cirrhosis , Pompe ylguanosine 5 ' triphosphate , Nø -methyladenosine 5 ' triphos disease , glycerol kinase deficiency (GKD ), proprionic aci phate , O ' -methylguanosine 5 ' triphosphate , NP-methyl demia (PA ) and Crigler - Najjar syndrome ( CN1) . pseudouridine 5 ' triphosphate , puromycin 5 '- triphosphate 18 . The method of claim 16 , wherein the liver condition and xanthosine 5 ' triphosphate . is hepatitis C . 11 . The mRNA of claim 10, wherein the at least one 19 . The method of claim 18 , wherein the therapeutic gene modified or non - naturally occurring nucleotide is is selected from the group consisting of : interferon alpha , N ' -methyl - pseudouridine 5 ' triphosphate . TNF -related apoptosis - inducing ligand , vascular adhesion 12 . A method of identifying a 5 ' UTR and a 3 ' UTR for protein 1, hepatocyte growth factor, G6PC , ABCB11, increasing protein synthesis in a desired tissue or a cell ABCB4, ASL1, ASS , Arg1, AGXT, OTC , CBS, PAH , GBE , derived from the desired tissue , comprising : GALE , HADH , MCCC1, MMA, ADAMTS13 , SLC25A15 , a ) isolating a plurality of 5 ' UTRs and a plurality of 3 ' C2 , F2 , PROSI , SERPINAI, GALT1 , ETFA , GCDH , UTRs from mRNAs that are naturally abundant in the CTNS , FAH , TAT, HPD , HSD17b4 , SLC2A2 , GALC , desired tissue ; ABCC6 , AHSG , PDC -E2 ,GAA , ATP8B1, MMACHC , GK , b ) generating a library of test constructs , each comprising PCCA , PCCB and UGT1A1. a reporter ORF flanked by one of the plurality of 5 20 . A method of improving expression of a protein in a UTRs and / or one of the plurality of 3 ' UTRs; desired cell or desired tissue , comprising providing an c ) providing a reference construct comprising the reporter mRNA to said cell or tissue , wherein the mRNA comprises ORF flanked by reference 5 ' and 3 ' UTRs ; an open reading frame (ORF ) that encodes said protein , d ) expressing each of the test constructs and the reference flanked by a heterologous 5 ' untranslated region (UTR ) construct in the tissue; and /or a heterologous 3 ' UTR , wherein the 5 ' UTR and / or the e ) measuring the protein expression of the reporter ORF 3 'UTR are derived from an mRNA that is naturally abundant from the each of the test constructs and the reference mRNA in the desired tissue or tissues from which the desired construct ; cell was derived . US 2018 /0353618 A1 Dec. 13 , 2018

21 . The method of claim 20 , wherein the mRNA com pseudouridine 5 ' triphosphate , puromycin 5 '- triphosphate prises at least one modified or non -naturally occurring and xanthosine 5 ' triphosphate . nucleotide . 25 . The method of claim 24 , wherein the at least one modified or non -naturally occurring nucleotide is 22 . The method of claim 21, wherein the at least one N '- methyl - pseudouridine 5 - triphosphate . modified or non -naturally occurring nucleotide comprises at 26 . The method of claim 20 , wherein the tissue is liver. least one backbone modification , sugar modification or base 27 . The method of claim 20 , wherein the mRNA has a modification . half -life of at least 40 hours . 23 . The method of claim 22 , wherein the at least one 28 . The method of claim 20 , wherein the heterologous 5 modified or non -naturally occurring nucleotide comprises at UTR and / or the heterologous 3 ' UTR is derived from a gene least one base modification . selected from the group consisting of: HP, FGB , HPR , ALB , 24 . The method of claim 23 , wherein the at least one base C3 , FGA , Col1 A , Col6A , SERPINA1 and SERPINA3. modification is selected from the group consisting of: 29 . The method of claim 20 , wherein the 5 ' UTR com 2 -amino - 6 - chloropurine riboside 5 ' triphosphate , 2 - amino prises a nucleotide sequence selected from the group con adenosine 5 ' triphosphate , 2 -thiocytidine 5 ' triphosphate , sisting of: SEQ ID NOS : 1 -6 , 9 and 10 . 2 - thiouridine 5' triphosphate , 4 -thiouridine 5 ' triphosphate , 30 . Themethod of claim 20 , wherein the ORF is a reporter 5 -aminoallylcytidine 5 ' triphosphate, 5 -aminoallyluridine 5 ' gene or a therapeutic gene of interest. triphosphate , 5 - bromocytidine 54 triphosphate , 5 - bromouri 31 . The method of claim 20 , wherein the therapeutic gene dine 5 triphosphate , 5 - iodocytidine 5 ' triphosphate , 5 - io is selected from the group consisting of: interferon alpha , douridine 5 ' triphosphate , 5 -methylcytidine 5 ' triphosphate , TNF -related apoptosis - inducing ligand , vascular adhesion 5 -methyluridine 5 ' triphosphate , 6 - azacytidine 5 ' triphos protein 1, hepatocyte growth factor, G6PC , ABCB11, phate , 6 - azauridine 5 ' triphosphate , 6 - chloropurine riboside ABCB4 , ASL1, ASS , Arg1, AGXT, OTC , CBS, PAH , GBE , 5 -triphosphate , 7 -deazaadenosine 5 ' triphosphate , 7 - deaza GALE, HADH , MCCC1, MMA, ADAMTS13 , SLC25A15 , guanosine 5 ' triphosphate , 8 - azaadenosine 5 ' triphosphate , C2 , F2 , PROS1, SERPINA1, GALT1, ETFA , GCDH , 8 - azidoadenosine 5 ' triphosphate , benzimidazole riboside 5 CTNS , FAH , TAT, HPD , HSD17b4 , SLC2A2, GALC , triphosphate , NP- methyladenosine 5' triphosphate , NP-meth ABCC6 , AHSG , PDC - E2, GAA , ATP8B1, MMACHC , GK , ylguanosine 5 ' triphosphate , Nø -methyladenosine 5 ' triphos PCCA , PCCB and UGT1A1. phate , O®- methylguanosine 5 ' triphosphate , Nl- methyl * * * * *