Poly-tailed and poly-capped mrnas and uses thereof

Abstract

To provide a modified mRNA having increased stability and enhanced in vivo efficacy.SOLUTION: (A) tail containing one or more additional poly-A tails or 5' caps are provided, which may be generated by ligation of a nucleic acid to the 3' end or 5' end of the RNA, respectively. Also provided are compositions comprising one or more of the modified mRNAs and methods of using the compositions for therapeutic or agricultural applications.SELECTED DRAWING: Figure 1E

Description

[Technical Field]

[0001] Electronic Sequence Listing Reference The contents of the electronic sequence listing (B119570144WO00-SEQ-JQM.xml, size: 61,200 bytes, and creation date: January 18, 2023) are incorporated herein by reference in their entirety. [Background technology]

[0002] Messenger RNA (mRNA) technology is an emerging alternative to traditional small molecule, DNA, and protein therapeutics, as well as traditional vaccine approaches, due to its potent, programmable nature and the ability to rapidly produce mRNA with desired sequences. mRNA therapy is a rapidly developing field and has been used to express therapeutic proteins, ranging from revascularization factors (e.g., vascular endothelial growth factor A (VEGF-A), GATA-binding protein 4 (GATA4), myocyte enhancer factor 2C (MEF2C), T-box transcription factor 5 (TBX5), and myocardin (MYOCD)) to vaccines against COVID-19, influenza, and Zika viruses. Despite recent clinical success, mRNA therapy still faces challenges of instability, toxicity, short-term efficacy, and potential immunological responses. Increasing mRNA stability and enhancing their efficacy in vivo remains a key issue that must be resolved to enhance the feasibility of mRNA therapeutics in clinical applications. Summary of the Invention

[0003] Provided herein are modified mRNAs with modified poly-A tails and / or modified 5' caps that improve mRNA stability in cells and in in vitro translation systems, thereby enhancing protein production, as well as methods for making and using such modified mRNAs. Conventional mRNAs contain a poly-A tail containing approximately 100-250 adenosine nucleotides at their 3' ends. The poly-A tail can be degraded or removed by cellular exonucleases that remove the 3' nucleotides. Once exonucleases remove the poly-A tail and begin to remove nucleotides from the 3' untranslated region (UTR) and / or open reading frame (ORF), the mRNA cannot be translated into the encoded protein. mRNAs that are more resistant to 3' exonuclease activity are degraded more slowly, are more stable, and therefore have an increased half-life within cells; therefore, more protein can be produced from a given mRNA molecule. The poly(A) tail is also required for binding to poly(A)-binding protein (PABP), which enhances translation of mRNA molecules by binding to the eukaryotic initiation factor 4 complex (eIF4G). Conventional mRNAs also contain a 5' cap, which, like the poly(A) tail, protects the mRNA from 5' exonucleases and serves as a site for recruitment of the translation machinery. Modified nucleotides containing one or more structural modifications in the nucleobase, sugar, or phosphate linkages of mRNA can inhibit 3' and 5' exonuclease activity, making the mRNA more stable. However, the same structural modifications that inhibit exonucleases can also hinder the ability of enzymes to incorporate these modified nucleotides into mRNA, making it difficult to produce modified mRNA.

[0004] Nevertheless, alternative strategies for enhancing the stability of mRNA molecules without significantly impairing mRNA synthesis and / or translation have been demonstrated. Provided herein are strategies for enhancing mRNA stability by either ligating multiple polyA tails to the 3' end of an mRNA and / or ligating multiple 5' caps to the 5' end. One strategy involves ligating an oligonucleotide containing one or more nucleotides modified with a click chemistry handle to the 3' and / or 5' end of an existing mRNA molecule. Next, an additional oligonucleotide containing either a 3' polyA tail or a 5' cap is ligated to one or more modified nucleotides via a click chemistry reaction (see Figures 1C and 1G). Alternatively, the same result can be achieved by ligating a dendrimer-linked oligonucleotide to the 3' and / or 5' end of an existing mRNA molecule via a click chemistry reaction. The dendrimer, also modified with a click chemistry handle, is then ligated via click chemistry to additional oligonucleotides containing either a 3' polyA tail or a 5' cap (see Figures 1E and 1I). In addition to extending the lifespan of mRNA by protecting existing mRNA molecules from exonuclease activity, thereby reducing ORF degradation, these strategies can also directly enhance mRNA translation, as each additional polyA tail or 5' cap ligated to the mRNA can interact with the modified mRNA and recruit the translation machinery to it. These strategies can be employed with each other and with other techniques known to enhance mRNA stability, including, for example, the introduction of modified nucleotides at the 3' and / or 5' end of the mRNA, the introduction of modified nucleotides into the 3' and / or 5' untranslated region (UTR) of the mRNA, the introduction of exonuclease-resistant sequences into the mRNA, and / or cyclization, which involves ligating the ends of a linear mRNA to generate a circular mRNA.

[0005] Thus, the present disclosure provides, in some aspects, (i) an open reading frame (ORF) encoding a protein; (ii) a polyA region; (iii) a 5' cap region; The polyA region is 3' to the ORF and comprises 10 or more nucleotides, the 5' cap region is 5' to the ORF and comprises two or more polyA tails, and / or the 5' cap region comprises two or more 5' caps.

[0006] In some embodiments, the modified mRNA comprises a polyA region comprising two or more polyA tails and a 5' cap region comprising a 5' cap. In some embodiments, the modified mRNA comprises a polyA region comprising a polyA tail and a 5' cap region comprising two or more 5' caps. In some embodiments, the modified mRNA comprises a polyA region comprising two or more polyA tails and a 5' cap region comprising two or more 5' caps.

[0007] In some embodiments, the modified mRNA comprises a 5' untranslated region (5'UTR) and a 3' untranslated region (3'UTR), wherein the ORF is between the 5'UTR and the 3'UTR, the 3'UTR is between the ORF and the polyA region, and the 5'UTR is between the 5' cap region and the ORF.

[0008] In some embodiments, the modified mRNA is a linear mRNA, the 5' cap region is at the 5' end of the modified mRNA, and the polyA region is at the 3' end of the modified mRNA.

[0009] In some embodiments, the modified mRNA is a circular mRNA and the polyA region is between the 3' UTR and the 5' cap region.

[0010] In some embodiments, the polyA region comprises two or more polyA tails, and at least two instances of the polyA tails are covalently linked by a first linker.

[0011] In some embodiments, the poly A region is -(first poly A tail)-[(first linker)-(second poly A tail) n1 ] n2 wherein each instance of n1 is independently an integer between 1 and 20, inclusive; n2 is an integer between 2 and 10, inclusive; the first polyA tail is covalently attached to the 3'UTR; and the first linker is covalently attached to the first polyA tail and the second polyA tail.

[0012] In some embodiments, each instance of n1 is 1.

[0013] In some embodiments, at least one instance of the first linker is covalently attached to an internal nucleotide of the first poly-A tail.

[0014] In some embodiments, at least one instance of the first linker is covalently attached to the second poly-A tail at the 3' nucleotide of the second poly-A tail.

[0015] In some embodiments, at least one instance of the first linker comprises at least one moiety formed by reacting two orthogonal click chemistry handles.

[0016] In some embodiments, at least one moiety formed by reacting two orthogonal click chemistry handles has the formula: It is TIFF2026016516000002.tif13128.

[0017] In some embodiments, each instance of the first linker is independently a substituted or unsubstituted C 1-200 Alkylene, substituted or unsubstituted C 1-200 Alkenylene, substituted or unsubstituted C 1-200 Alkynylene, substituted or unsubstituted C 1-200 Heteroalkylene, substituted or unsubstituted C 1-200 Heteroalkenylene, substituted or unsubstituted C 1-200heteroalkynylene, substituted or unsubstituted carbocyclylene, substituted or unsubstituted heterocyclylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene, optionally substituted or unsubstituted C 1-200 Alkylene, substituted or unsubstituted C 1-200 Alkenylene, substituted or unsubstituted C 1-200 Alkynylene, substituted or unsubstituted C 1-200 Heteroalkylene, substituted or unsubstituted C 1-200 Heteroalkenylene, and substituted or unsubstituted C 1-200 One or more carbon atoms in the parent chain of each instance of heteroalkynylene is independently replaced with a substituted or unsubstituted carbocyclylene, substituted or unsubstituted heterocyclylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene.

[0018] In some embodiments, substituted or unsubstituted C 1-200 Alkylene, substituted or unsubstituted C 1-200 Alkenylene, substituted or unsubstituted C 1-200 Alkynylene, substituted or unsubstituted C 1-200 Heteroalkylene, substituted or unsubstituted C 1-200 Heteroalkenylene, and substituted or unsubstituted C 1-200 One or more carbon atoms in the parent chain of each instance of heteroalkynylene independently may be Replaced by TIFF2026016516000003.tif13128.

[0019] In some embodiments, at least one example of the first linker has the formula: TIFF2026016516000004.tif11128, L 1 Each instance of is independently a substituted or unsubstituted C 1-199 Alkylene, or substituted or unsubstituted C 1-199 is heteroalkylene, and L 2 Each instance of is independently a substituted or unsubstituted C 1-199 Alkylene, or substituted or unsubstituted C 1-199It is heteroalkylene.

[0020] In some embodiments, at least one example of the first linker has the formula: It is TIFF2026016516000005.tif16130.

[0021] In some embodiments, each instance of n1 is independently an integer from 2 to 20, inclusive.

[0022] In some embodiments, at least one example of the first linker comprises a dendrimer. In certain embodiments, at least one example of the dendrimer is a first-generation dendrimer. In certain embodiments, at least one example of the dendrimer is a second-generation dendrimer. In certain embodiments, at least one example of the dendrimer is a third-generation dendrimer. In certain embodiments, at least one example of the dendrimer is a fourth-generation dendrimer. In certain embodiments, at least one example of the dendrimer comprises 2, 3, 4, 5, 6, 7, or 8 dendrons.

[0023] In certain embodiments, at least one example of a dendrimer is a poly-propyleneimine dendrimer, a poly-propyleneamine dendrimer, a poly(glutamic acid) dendrimer, a polymelamine dendrimer, a polyester dendrimer, or a PEGylated dendrimer. In some embodiments, at least one example of a dendrimer is a polyamidoamine (PAMAM) dendrimer.

[0024] In some embodiments, at least one example of a PAMAM dendrimer has the formula: TIFF2026016516000006.tif22128, wherein each instance of n3 is independently an integer from 1 to 10 (inclusive); each instance of n4 is independently an integer from 0 to 10 (inclusive); and R 2 Each instance of is independently hydrogen or TIFF2026016516000007.tif13128, but in R 2 At least three examples of TIFF2026016516000008.tif13128, R 1 Each example is a substituted or unsubstituted C 1-10 Alkylene, substituted or unsubstituted C 1-10 Alkenylene, substituted or unsubstituted C 1-10 Alkynylene, substituted or unsubstituted C 1-10 Heteroalkylene, substituted or unsubstituted C 1-10 Heteroalkenylene, or substituted or unsubstituted C 1-10 heteroalkynylene, optionally substituted or unsubstituted C 1-10 Alkylene, substituted or unsubstituted C 1-10 Alkenylene, substituted or unsubstituted C 1-10 Alkynylene, substituted or unsubstituted C 1-10 Heteroalkylene, substituted or unsubstituted C 1-10 Heteroalkenylene, and substituted or unsubstituted C 1-10 One or more carbon atoms in the parent chain of each instance of heteroalkynylene is independently replaced with a substituted or unsubstituted carbocyclylene, a substituted or unsubstituted heterocyclylene, a substituted or unsubstituted arylene, or a substituted or unsubstituted heteroarylene, and each instance of n5 is independently an integer from 0 to 10 (inclusive).

[0025] In some embodiments, at least one example of a PAMAM dendrimer is Includes TIFF2026016516000009.tif44128.

[0026] In some embodiments, substituted or unsubstituted C 1-10 Alkylene, substituted or unsubstituted C 1-10 Alkenylene, substituted or unsubstituted C 1-10 Alkynylene, substituted or unsubstituted C 1-10 Heteroalkylene, substituted or unsubstituted C 1-10 Heteroalkenylene, and substituted or unsubstituted C 1-10One or more carbon atoms in the parent chain of each instance of heteroalkynylene independently may be Replaced by TIFF2026016516000010.tif13128.

[0027] In some embodiments, at least one example of the first linker has the formula: It is TIFF2026016516000011.tif47128.

[0028] In some embodiments, at least one instance of the first linker is covalently attached to the 3' nucleotide of the first poly-A tail.

[0029] In some embodiments, at least one instance of the first linker is covalently attached to the 3' nucleotide of the second poly-A tail.

[0030] In some embodiments, the polyA region of the modified mRNA comprises one or more modified nucleotides.

[0031] In some embodiments, three or more of the last 10 nucleotides of at least one of the two or more poly-A tails are modified nucleotides and / or non-adenosine nucleotides.

[0032] In some embodiments, one or more modified nucleotides of the polyA region are modified adenosine nucleotides.

[0033] In some embodiments, one or more modified nucleotides of the polyA region are modified non-adenosine nucleotides.

[0034] In some embodiments, one or more modified nucleotides of the polyA region comprise a modified nucleobase.

[0035] In some embodiments, the modified nucleobases comprised by one or more modified nucleotides of the polyA region are xanthine, aliaminouracil, aliaminothymidine, hypoxanthine, digoxigenylated adenine, digoxigenylated cytosine, digoxigenylated guanine, digoxigenylated uracil, 6-chloropurine riboside, N6-methyladenine, methylpseudouracil, 2-thiocytosine, 2-thiouracil, 5-methyluracil, 4-thiothymidine, 4-thiouracil, 5,6-dihydro-5-methyluracil, 5,6-dihydrouracil, 5-[(3-indolyl)propionamido-N-allyl]uracil, 5-aminoallylcytosine, 5-aminoallyluracil, 5-bromouracil, 5-bromocytosine, 5-carboxycytosine, 5-carboxymethylesteruracil, 5-carboxyuracil, 5-fluorouracil, 5-formylcytosine, 5-formyluracil, 5-hydroxycytosine, 5-hydroxymethylcytosine, 5-hydroxymethyluracil, 5-hydroxyuracil, 5-iodocytosine, 5-iodouracil, 5-methoxycytosine, 5-methoxyuracil le, 5-methylcytosine, 5-methyluracil, 5-propargylaminocytosine, 5-propargylaminouracil, 5-propynylcytosine, 5-propynyluracil, 6-azacytosine, 6-azauracil, 6-chloropurine, 6-thioguanine, 7-deazaadenine, 7-deazaguanine, 7-deaza-7-propargylaminoadenine, 7-deaza-7-propargylaminoguanine, 8-azaadenine, 8-azidoadenine, 8-chloroadenine, 8-oxoadenine, 8-oxoguanine, araadenine, aracytosine, araguanine, Alauracil, biotin-16-7-deaza-7-propargylaminoguanine, biotin-16-aminoallylcytosine, biotin-16-aminoallyluracil, cyanine 3-5-propargylaminocytosine, cyanine 3-6-propargylaminouracil, cyanine 3-aminoallylcytosine, cyanine 3-aminoallyluracil, cyanine 5-6-propargylaminocytosine, cyanine 5-6-propargylaminouracil, cyanine 5-aminoallylcytosine, cyanine 5-aminoallyluracil, cyanine 7-aminoallyluracil,Dabsyl-5-3-aminoallyluracil, desthiobiotin-16-aminoallyl-uracil, desthiobiotin-6-aminoallylcytosine, isoguanine, N1-ethylpseudouracil, N1-methoxymethylpseudouracil, N1-methyladenine, N1-methylpseudouracil, N1-propylpseudouracil, N2-methylguanine, N4-biotin-OBEA-cytosine, N4-methyl Thiocytosine, N6-methyladenine, O6-methylguanine, pseudoisocytosine, pseudouracil, thienocytosine, thienoguanine, thienouracil, xanthosine, 3-deazaadenine, 2,6-diaminoadenine, 2,6-daminoguanine, 5-carboxamidouracil, 5-ethynyluracil, N6-isopentenyladenine (i6A), 2-methyl-thi o-N6-isopentenyladenine (ms2i6A), 2-methylthio-N6-methyladenine (ms2m6A), N6-(cis-hydroxyisopentenyl)adenine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenine (ms2io6A), N6-glycinylcarbamoyladenine (g6A), N6-threonylcarbamoyladenine (t6A), 2-methylthio-N 6-threonylcarbamoyladenine (ms2t6A), N6-methyl-N6-threonylcarbamoyladenine (m6t6A), N6-hydroxynorvalylcarbamoyladenine (hn6A), 2-methylthio-N6-hydroxynorvalylcarbamoyladenine (ms2hn6A), N6,N6-dimethyladenine (m62A), and N6-acetyladenine (ac6A).

[0036] In some embodiments, one or more modified nucleotides of the polyA region comprise a modified sugar.

[0037] In some embodiments, the modified sugars comprised by one or more modified nucleotides in the poly A region are 2'-thioribose, 2',3'-dideoxyribose, 2'-amino-2'-deoxyribose, 2'deoxyribose, 2'-azido-2'-deoxyribose, 2'-fluoro-2'-deoxyribose, 2'-O-methylribose, 2'-O-methyldeoxyribose, 3'-amino-2',3'-dideoxyribose, 3'-azido- The ribose is selected from the group consisting of 2',3'-dideoxyribose, 3'-deoxyribose, 3'-O-(2-nitrobenzyl)-2'-deoxyribose, 3'-O-methylribose, 5'-aminoribose, 5'-thioribose, 5-nitro-1-indolyl-2'-deoxyribose, 5'-biotin-ribose, 2'-O,4'-C-methylene linked, 2'-O,4'-C-amino linked, and 2'-O,4'-C-thio linked ribose.

[0038] In some embodiments, one or more modified nucleotides of the polyA region comprise a modified phosphate.

[0039] In some embodiments, the modified phosphates comprised by one or more modified nucleotides of the polyA region are selected from the group consisting of phosphorothioate (PS), thiophosphate, 5'-O-methylphosphonate, 3'-O-methylphosphonate, 5'-hydroxyphosphonate, hydroxyphosphate, phosphoroselenoate, selenophosphate, phosphoramidate, carbophosphonate, methylphosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidinium ring, triazole ring, boranophosphate (BP), methylphosphonate, and guanidinopropylphosphoramidate.

[0040] In some embodiments, the polyA region comprises 1 to 3, 3 to 5, 5 to 10, 10 to 15, 15 to 30, 30 to 50, 50 to 100, or 100 to 200 phosphorothioates.

[0041] In some embodiments, the polyA region comprises between 5 and 30 phosphorothioates.

[0042] In some embodiments, the polyA region comprises between 3 and 5, between 5 and 10, between 10 and 15, between 15 and 30, between 30 and 50, between 50 and 100, or between 100 and 200 deoxyribose sugars.

[0043] In some embodiments, the polyA region comprises between 5 and 30 deoxyribose sugars.

[0044] In some embodiments, the mRNA comprises a 3' terminal nucleotide, wherein the 3' terminal nucleotide is dideoxyadenosine, dideoxycytidine, dideoxyguanosine, dideoxythymidine, dideoxyuridine, or inverted deoxythymidine.

[0045] In some embodiments, the polyA region comprises between 25 and 500 nucleotides.

[0046] In some embodiments, the polyA region comprises between 50 and 100, 100 and 150, 150 and 200, 200 and 300, 300 and 400, or 400 and 500 nucleotides.

[0047] In some embodiments, the polyA region comprises 10 or more adenosine nucleotides.

[0048] In some embodiments, 25 to 100%, 30 to 100%, 40 to 100%, 50 to 100%, 60 to 100%, 70 to 100%, 80 to 100%, 90 to 100%, 95 to 100%, 96 to 100%, 97 to 100%, 98 to 100%, or 99 to 100% of the nucleotides in the polyA region are adenosine nucleotides.

[0049] In some embodiments, the first poly-A tail comprises 10-50 adenosine ribonucleotides, 1-10 modified uridine deoxyribonucleotides containing attachment points, and a 3'-terminal dideoxyribonucleotide or an inverted deoxyribonucleotide, and the second poly-A tail comprises 10-50 adenosine ribonucleotides.

[0050] In some embodiments, the first poly-A tail comprises the nucleotide sequence set forth as 5'-rArArArArArArArArArArArAdU(a1)rArArArArArArArArArArArArArAdU(a1)rArArArArArArArArArArArArAdU(a1)ddC-3' (SEQ ID NO: 1); at least one instance of the second poly-A tail comprises the nucleotide sequence set forth as 5'-rArArArArArArArArArArArArArArArArArArArArArArArArArA*rA*rA*rA*rA*rA*rA*(b1)-3' (SEQ ID NO: 2); and each instance of the first linker is independently TIFF2026016516000012.tif16144, wherein "rA" represents an adenosine ribonucleotide, "dU(a1)" represents a modified uridine deoxyribonucleotide, "ddC" represents a cytosine dideoxyribonucleotide, "*" represents a phosphorothioate bond, a1 in each instance of dU(a1) represents the point of attachment at the uridine of dU(a1), b1 in rA*(b1) represents the point of attachment at the * in rA*(b1), each instance of a1 is bonded to one instance of a2, and each instance of b1 is bonded to one instance of b2. In some embodiments, the modified uridine deoxyribonucleotide is 5-octadiynyl deoxyuridine.

[0051] In some embodiments, the first poly-A tail and the second poly-A tail comprise between 10 and 50 adenosine ribonucleotides and a 3'-terminal azide moiety.

[0052] In some embodiments, the first poly-A tail and the second poly-A tail comprise between 10 and 50 adenosine ribonucleotides and a 5'-terminal azide moiety.

[0053] In some embodiments, the first poly-A tail and the second poly-A tail comprise a nucleotide sequence shown as 5'-rArArArArArArArArArArArArArArArArArArArArArArArArA*rA*rA*rA*rA*rA* / AzideN / -3' (SEQ ID NO: 3), where "AzideN" represents the 3' azide moiety and "*" represents a phosphorothioate linkage.

[0054] In some embodiments, the first poly-A tail and the second poly-A tail comprise the nucleotide sequence shown as 5'-5 / AzideN / rArArArArArArArArArArArArArArArArArArArArArArArArA*rA*rA*rA*rA*rA-3' (SEQ ID NO: 27), where "AzideN" represents the 3' azide moiety and "*" represents a phosphorothioate linkage.

[0055] In some embodiments, the 5' cap region comprises two or more 5' caps, wherein at least two instances of the 5' caps are covalently linked by a second linker.

[0056] In some embodiments, the 5' cap region is -(first 5' cap)-[(second linker)-(second 5' cap) m1 ] m2 wherein each instance of m1 is independently an integer between 1 and 20, inclusive; m2 is an integer between 2 and 10, inclusive; the first 5' cap is covalently linked to the 5' UTR; and the second linker is covalently linked to the first 5' cap and the second 5' cap.

[0057] In some embodiments, each instance of m1 is 1.

[0058] In some embodiments, at least one instance of the second linker is covalently attached to an internal nucleotide of the first 5' cap.

[0059] In some embodiments, at least one instance of the second linker is covalently attached to the second 5' cap at the 3' nucleotide of the second 5' cap.

[0060] In some embodiments, at least one instance of the second linker comprises at least one moiety formed by reacting two orthogonal click chemistry handles.

[0061] In some embodiments, at least one moiety formed by reacting two orthogonal click chemistry handles has the formula: It is TIFF2026016516000013.tif13128.

[0062] In some embodiments, each instance of the second linker is independently a substituted or unsubstituted C 1-200 Alkylene, substituted or unsubstituted C 1-200 Alkenylene, substituted or unsubstituted C 1-200 Alkynylene, substituted or unsubstituted C 1-200 Heteroalkylene, substituted or unsubstituted C 1-200 Heteroalkenylene, substituted or unsubstituted C 1-200 heteroalkynylene, substituted or unsubstituted carbocyclylene, substituted or unsubstituted heterocyclylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene, optionally substituted or unsubstituted C 1-200 Alkylene, substituted or unsubstituted C 1-200 Alkenylene, substituted or unsubstituted C 1-200 Alkynylene, substituted or unsubstituted C 1-200 Heteroalkylene, substituted or unsubstituted C 1-200 Heteroalkenylene, and substituted or unsubstituted C 1-200 One or more carbon atoms in the parent chain of each instance of heteroalkynylene is independently replaced with a substituted or unsubstituted carbocyclylene, substituted or unsubstituted heterocyclylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene.

[0063] In some embodiments, substituted or unsubstituted C 1-200 Alkylene, substituted or unsubstituted C 1-200 Alkenylene, substituted or unsubstituted C 1-200 Alkynylene, substituted or unsubstituted C 1-200 Heteroalkylene, substituted or unsubstituted C 1-200 Heteroalkenylene, and substituted or unsubstituted C 1-200 One or more carbon atoms in the parent chain of each instance of heteroalkynylene independently may be Replaced by TIFF2026016516000014.tif13128.

[0064] In some embodiments, at least one example of the second linker has the formula: TIFF2026016516000015.tif11128, L 3 Each instance of is independently a substituted or unsubstituted C 1-199 Alkylene, or substituted or unsubstituted C 1-199 is heteroalkylene, and L 4 Each instance of is independently a substituted or unsubstituted C 1-199 Alkylene, or substituted or unsubstituted C 1-199 It is heteroalkylene.

[0065] In some embodiments, at least one example of the second linker has the formula: It is TIFF2026016516000016.tif16130.

[0066] In some embodiments, each instance of m1 is independently an integer from 2 to 20, inclusive.

[0067] In some embodiments, at least one example of the second linker comprises a dendrimer. In certain embodiments, at least one example of the dendrimer is a first-generation dendrimer. In certain embodiments, at least one example of the dendrimer is a second-generation dendrimer. In certain embodiments, at least one example of the dendrimer is a third-generation dendrimer. In certain embodiments, at least one example of the dendrimer is a fourth-generation dendrimer. In certain embodiments, at least one example of the dendrimer comprises 2, 3, 4, 5, 6, 7, or 8 dendrons.

[0068] In certain embodiments, at least one example of a dendrimer is a poly-propyleneimine dendrimer, a poly-propyleneamine dendrimer, a poly(glutamic acid) dendrimer, a polymelamine dendrimer, a polyester dendrimer, or a PEGylated dendrimer. In some embodiments, at least one example of a dendrimer is a polyamidoamine (PAMAM) dendrimer.

[0069] In some embodiments, at least one example of a PAMAM dendrimer has the formula: TIFF2026016516000017.tif22128, wherein each instance of m3 is independently an integer from 1 to 10 (inclusive); each instance of m4 is independently an integer from 0 to 10 (inclusive); R 12 Each instance of is independently hydrogen or TIFF2026016516000018.tif13128, but in R 12 At least three examples of TIFF2026016516000019.tif13128, R 11 Each example is a substituted or unsubstituted C 1-10 Alkylene, substituted or unsubstituted C 1-10 Alkenylene, substituted or unsubstituted C 1-10 Alkynylene, substituted or unsubstituted C 1-10 Heteroalkylene, substituted or unsubstituted C 1-10Heteroalkenylene, or substituted or unsubstituted C 1-10 heteroalkynylene, optionally substituted or unsubstituted C 1-10 Alkylene, substituted or unsubstituted C 1-10 Alkenylene, substituted or unsubstituted C 1-10 Alkynylene, substituted or unsubstituted C 1-10 Heteroalkylene, substituted or unsubstituted C 1-10 Heteroalkenylene, and substituted or unsubstituted C 1-10 One or more carbon atoms in the parent chain of each instance of heteroalkynylene is independently replaced with a substituted or unsubstituted carbocyclylene, a substituted or unsubstituted heterocyclylene, a substituted or unsubstituted arylene, or a substituted or unsubstituted heteroarylene, and each instance of m5 is independently an integer of 0 to 10 (inclusive).

[0070] In some embodiments, at least one example of a PAMAM dendrimer is Includes TIFF2026016516000020.tif44128.

[0071] In some embodiments, substituted or unsubstituted C 1-10 Alkylene, substituted or unsubstituted C 1-10 Alkenylene, substituted or unsubstituted C 1-10 Alkynylene, substituted or unsubstituted C 1-10 Heteroalkylene, substituted or unsubstituted C 1-10 Heteroalkenylene, and substituted or unsubstituted C 1-10 One or more carbon atoms in the parent chain of each instance of heteroalkynylene independently may be Replaced by TIFF2026016516000021.tif13128.

[0072] In some embodiments, at least one example of the first linker has the formula: It is TIFF2026016516000022.tif49128.

[0073] In some embodiments, at least one instance of the second linker is covalently attached to the 3' nucleotide of the first 5' cap.

[0074] In some embodiments, at least one instance of the second linker is covalently attached to the 3' nucleotide of at least one instance of the second 5' cap.

[0075] In some embodiments, the 5' cap region of the modified mRNA comprises one or more modified nucleotides.

[0076] In some embodiments, one or more modified nucleotides in the 5' cap region comprise a modified nucleobase.

[0077] In some embodiments, the modified nucleobase is xanthine, allylaminouracil, allylaminothymidine, hypoxanthine, digoxigenylated adenine, digoxigenylated cytosine, digoxigenylated guanine, digoxigenylated uracil, 6-chloropurine riboside, N6-methyladenine, methylpseudouracil, 2-thiocytosine, 2-thiouracil, 5-methyluracil, 4-thiothymidine, 4-thiouracil, 5,6-dihydro-5-methyluracil, 5,6-dihydrouracil, 5-[(3-indolyl)propionamido-N-allyl ]uracil, 5-aminoallylcytosine, 5-aminoallyluracil, 5-bromouracil, 5-bromocytosine, 5-carboxycytosine, 5-carboxymethylesteruracil, 5-carboxyuracil, 5-fluorouracil, 5-formylcytosine, 5-formyluracil, 5-hydroxycytosine, 5-hydroxymethylcytosine, 5-hydroxymethyluracil, 5-hydroxyuracil, 5-iodocytosine, 5-iodouracil, 5-methoxycytosine, 5-methoxyuracil, 5-methylcytosine, 5-methyluracil, 5- Propargylaminocytosine, 5-propargylaminouracil, 5-propynylcytosine, 5-propynyluracil, 6-azacytosine, 6-azauracil, 6-chloropurine, 6-thioguanine, 7-deazaadenine, 7-deazaguanine, 7-deaza-7-propargylaminoadenine, 7-deaza-7-propargylaminoguanine, 8-azaadenine, 8-azidoadenine, 8-chloroadenine, 8-oxoadenine, 8-oxoguanine, araadenine, aracytosine, araguanine, arauracil, biotin-16-7-deaza-7 -propargylaminoguanine, biotin-16-aminoallylcytosine, biotin-16-aminoallyluracil, cyanine 3-5-propargylaminocytosine, cyanine 3-6-propargylaminouracil, cyanine 3-aminoallylcytosine, cyanine 3-aminoallyluracil, cyanine 5-6-propargylaminocytosine, cyanine 5-6-propargylaminouracil, cyanine 5-aminoallylcytosine, cyanine 5-aminoallyluracil, cyanine 7-aminoallyluracil, dabsyl-5-3-aminoallyluracil,Desthiobiotin-16-aminoallyl-uracil, desthiobiotin-6-aminoallylcytosine, isoguanine, N1-ethylpseudouracil, N1-methoxymethylpseudouracil, N1-methyladenine, N1-methylpseudouracil, N1-propylpseudouracil, N2-methylguanine, N4-biotin-OBEA-cytosine, N4-methylcytosine, N6-methyl Thioadenine, O6-methylguanine, pseudoisocytosine, pseudouracil, thienocytosine, thienoguanine, thienouracil, xanthosine, 3-deazaadenine, 2,6-diaminoadenine, 2,6-daminoguanine, 5-carboxamidouracil, 5-ethynyluracil, N6-isopentenyladenine (i6A), 2-methyl-thio-N6-isopentenyladenine (i6A) ms2i6A), 2-methylthio-N6-methyladenine (ms2m6A), N6-(cis-hydroxyisopentenyl)adenine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenine (ms2io6A), N6-glycinylcarbamoyladenine (g6A), N6-threonylcarbamoyladenine (t6A), 2-methylthio-N6-threonylcarbamoyladenine (t6A), N6,N6-dimethyladenine (m62A), N6-acetyladenine (ac6A), N6-hydroxynorvalylcarbamoyladenine (hn6A), 2-methylthio-N6-hydroxynorvalylcarbamoyladenine (ms2hn6A), N6,N6-dimethyladenine (m62A), and N6-acetyladenine (ac6A).

[0078] In some embodiments, one or more modified nucleotides in the 5' cap region comprise a modified sugar.

[0079] In some embodiments, the modified sugar comprised by one or more modified nucleotides in the 5' cap region is 2'-thioribose, 2',3'-dideoxyribose, 2'-amino-2'-deoxyribose, 2'deoxyribose, 2'-azido-2'-deoxyribose, 2'-fluoro-2'-deoxyribose, 2'-O-methylribose, 2'-O-methyldeoxyribose, 3'-amino-2',3'-dideoxyribose, 3'-azido 2',3'-dideoxyribose, 3'-deoxyribose, 3'-O-(2-nitrobenzyl)-2'-deoxyribose, 3'-O-methylribose, 5'-aminoribose, 5'-thioribose, 5-nitro-1-indolyl-2'-deoxyribose, 5'-biotin-ribose, 2'-O,4'-C-methylene-linked, 2'-O,4'-C-amino-linked, and 2'-O,4'-C-thio-linked ribose.

[0080] In some embodiments, one or more modified nucleotides in the 5' cap region comprise a modified phosphate.

[0081] In some embodiments, the modified phosphate comprised by one or more modified nucleotides in the 5' cap region is selected from the group consisting of phosphorothioate (PS), thiophosphate, 5'-O-methylphosphonate, 3'-O-methylphosphonate, 5'-hydroxyphosphonate, hydroxyphosphate, phosphoroselenoate, selenophosphate, phosphoramidate, carbophosphonate, methylphosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidinium ring, triazole ring, boranophosphate (BP), methylphosphonate, and guanidinopropylphosphoramidate.

[0082] In some embodiments, the 5' cap region comprises 1 to 3, 3 to 5, 5 to 10, 10 to 15, 15 to 30, 30 to 50, 50 to 100, or 100 to 200 phosphorothioates.

[0083] In some embodiments, the 5' cap region comprises between 5 and 30 phosphorothioates.

[0084] In some embodiments, the 5' cap region comprises between 3 and 5, between 5 and 10, between 10 and 15, between 15 and 30, between 30 and 50, between 50 and 100, or between 100 and 200 deoxyribose sugars.

[0085] In some embodiments, the 5' cap region comprises between 5 and 30 deoxyribose sugars.

[0086] In some embodiments, the mRNA comprises a 3' terminal nucleotide, wherein the 3' terminal nucleotide is dideoxyadenosine, dideoxycytidine, dideoxyguanosine, dideoxythymidine, dideoxyuridine, or inverted deoxythymidine.

[0087] In some embodiments, the 5' cap region comprises between 25 and 500 nucleotides.

[0088] In some embodiments, the 5' cap region comprises between 50 and 100, 100 and 150, 150 and 200, 200 and 300, 300 and 400, or 400 and 500 nucleotides.

[0089] In some embodiments, the 5' cap region comprises 1 to 3, 3 to 5, 5 to 7, or 7 to 10 5' caps.

[0090] In some embodiments, the 5' cap region comprises one or more 7-methylguanylic acid caps containing a 5'-5' triphosphate linkage.

[0091] In some embodiments, the 5' cap region comprises one or more 2,2,7-trimethylguanosine caps containing a 5'-5' triphosphate linkage.

[0092] In some embodiments, the 5' cap region comprises one or more modified 5' caps.

[0093] In some embodiments, the one or more modified 5' caps are locked nucleic acid (LNA) modified caps, 5' caps including a 5'-5' triphosphate linkage modified by 5'-phosphorothiolate, or 5' caps including a 5'-5' tetraphosphate linkage.

[0094] In some embodiments, the 5' cap region comprises one or more 5' caps comprising one or more modified nucleotides.

[0095] In some embodiments, one or more of the modified nucleotides comprised by the 5' cap are 2'-O-methylated nucleotides.

[0096] In some embodiments, the first 5' cap comprises 10 to 50 ribonucleotides, 1 to 10 modified uridine deoxyribonucleotides comprising an attachment point, and a 5' cap, and the second 5' cap comprises 10 to 50 ribonucleotides and a 5' cap.

[0097] In some embodiments, the first 5' cap comprises a nucleotide sequence set forth as 5'- / Cap / rGrGrGrArArAdU(c1)rArArGrArGrArGrArArArGrArArGrArGdU(c1)rArArGrArArGrArArAdU(c1)rA-3' (SEQ ID NO:4), the second 5' cap comprises a nucleotide sequence set forth as 5'- / Cap / rGrGrGrArGrArCrTrGrCrCrArCrCrA*rA*rA*rA*rA*rA*(d1)-3' (SEQ ID NO:5), and each instance of the second linker is independently TIFF2026016516000023.tif16144, in which "rA" represents an adenosine ribonucleotide, "rT" represents a thymidine ribonucleotide, "rC" represents a cytidine ribonucleotide, "rG" represents a guanosine ribonucleotide, "dU(c1)" represents a modified uridine deoxyribonucleotide, "dU(c1)" represents a modified uridine deoxyribonucleotide, "*" represents a phosphorothioate bond, "Cap" represents a 5' cap, c1 in each example of dU(c1) represents the point of attachment at the uridine of dU(c1), d1 in rA*(d1) represents the point of attachment at the * of rA*(d1), each example of c1 is bonded to an example of c2, and each example of d1 is bonded to an example of d2. In some embodiments, the modified uridine deoxynucleotide is 5-octadiynyl deoxyuridine.

[0098] In some embodiments, the first 5' cap comprises 5 to 10 ribonucleotides and a 5' azide moiety, and the second 5' cap comprises 10 to 50 ribonucleotides, a 3' azide moiety, and a 5' cap.

[0099] In some embodiments, the first 5' cap comprises a nucleotide sequence designated as 5'- / 5AzideN / rArArArArA-3' and the second 5' cap comprises a nucleotide sequence designated as 5'- / Cap / rGrGrGrArGrArCrTrGrCrCrArCrCrA*rA*rA*rA*rA*rA* / 3AzideN / -3' (SEQ ID NO: 6), where "rA" represents an adenosine ribonucleotide, "rT" represents a thymidine ribonucleotide, "rC" represents a cytidine ribonucleotide, "rG" represents a guanosine ribonucleotide, "dU(c1)" represents a modified uridine deoxyribonucleotide, "5AzideN" represents a 5' azido moiety, "3AzideN" represents a 3' azido moiety, "*" represents a phosphorothioate linkage, and "Cap" represents the 5' cap.

[0100] In some aspects, the present disclosure provides a method of producing any one of the modified mRNAs described herein, comprising: ligating a first RNA comprising an open reading frame (ORF) encoding a protein to a tailing nucleic acid comprising two or more poly-A tails and / or a capping nucleic acid comprising two or more 5' caps in the presence of an RNA ligase, whereby the RNA ligase forms a covalent bond between the 3' nucleotide of the first RNA and the 5' nucleotide of the tailing nucleic acid, and / or a covalent bond between the 5' nucleotide of the first RNA and the 3' nucleotide of the capping nucleic acid, to produce the modified mRNA.

[0101] In some embodiments, the modified mRNA comprises a 5' untranslated region (5'UTR) and a 3' untranslated region (3'UTR), wherein the ORF is between the 5'UTR and the 3'UTR, the 3'UTR is between the ORF and the ligated tailing nucleic acid, and the 5'UTR is between the ORF and the ligated capping nucleic acid.

[0102] In some embodiments, the tailing nucleic acid comprises a first polyA tail and one or more second polyA tails, wherein the first polyA tail is covalently linked to the 3'UTR.

[0103] In some aspects, the present disclosure provides modified mRNA produced by the methods of producing modified mRNA provided herein.

[0104] In some aspects, the present disclosure provides a delivery agent comprising any one of the modified mRNAs provided herein, wherein the delivery agent comprises a lipid, peptide, protein, antibody, carbohydrate, nanoparticle, or microparticle.

[0105] In some embodiments, the nanoparticles or microparticles are lipid nanoparticles or microparticles, polymer nanoparticles or microparticles, protein nanoparticles or microparticles, or solid nanoparticles or microparticles.

[0106] In some aspects, the present disclosure provides a cell comprising any one of the modified mRNAs provided herein.

[0107] In some embodiments, the cell is a mammalian cell.

[0108] In some aspects, the present disclosure provides a composition comprising any one of the modified mRNAs provided herein, a delivery agent provided herein, or a cell provided herein.

[0109] In some embodiments, the composition further comprises an additional agent. In some embodiments, the additional agent is an agent that has a therapeutic effect when administered to a subject. In some embodiments, the additional agent is a nucleotide, nucleic acid, amino acid, peptide, protein, small molecule, aptamer, lipid, or carbohydrate. In some embodiments, the additional agent is an shRNA, siRNA, or ASO. In some embodiments, the additional agent is an antigen or an adjuvant.

[0110] In some embodiments, the composition is a pharmaceutical composition, and the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0111] In some aspects, the present disclosure provides methods comprising introducing any one of the modified mRNA provided herein, the delivery agent provided herein, or the composition provided herein into a cell.

[0112] In some aspects, the present disclosure provides methods that include introducing into a subject any one of the modified mRNA provided herein, the delivery agent provided herein, the cell provided herein, or the composition provided herein.

[0113] In some aspects, the present disclosure provides a method of preventing a disease in a subject in need thereof, the method comprising introducing into the subject an effective amount of any one of the modified mRNA provided herein, the delivery agent provided herein, the cell provided herein, or the composition provided herein, wherein the open reading frame of the modified mRNA encodes a protein.

[0114] In some aspects, the present disclosure provides a method of treating a disease in a subject in need thereof, the method comprising introducing into the subject an effective amount of any one of the modified mRNA provided herein, the delivery agent provided herein, the cell provided herein, or the composition provided herein, wherein the open reading frame of the modified mRNA encodes a protein.

[0115] In some embodiments, the protein is a protein antigen or fragment thereof from a disease-causing cell or virus.

[0116] In some embodiments, the protein is a protein that is expressed in the subject at a level that is lower than the level of the reference value.

[0117] In some aspects, the present disclosure provides a method of replacing an enzyme in a subject, the method comprising introducing into a subject any one of a modified mRNA provided herein, a delivery agent provided herein, a cell provided herein, or a composition provided herein, wherein the open reading frame of the modified mRNA encodes the enzyme.

[0118] In some embodiments, the subject is a human.

[0119] In some aspects, the present disclosure provides any one of the modified mRNA provided herein, the delivery agent provided herein, the cell provided herein, or the composition provided herein for use in treating a disease in a subject in need thereof.

[0120] In some aspects, the present disclosure provides a kit comprising a first RNA and a tailing nucleic acid and / or a capping nucleic acid of the methods for producing any one of the modified mRNAs provided herein.

[0121] In some embodiments, the kit further comprises an RNA ligase.

[0122] In some aspects, the present disclosure provides a kit comprising a pharmaceutical composition provided herein, a device for administering the pharmaceutical composition to a subject, and instructions for administering the pharmaceutical composition to a subject. [The present invention 1001] (i) an open reading frame (ORF) encoding a protein; (ii) a polyA region; (iii) a 5' cap region; Including, the poly A region is 3' to the ORF and comprises 10 or more nucleotides, and the 5' cap region is 5' to the ORF; the polyA region comprises two or more polyA tails and / or the 5' cap region comprises two or more 5' caps; Modified mRNA. [The present invention 1002] 1001. A modified mRNA of the present invention, comprising a polyA region comprising two or more polyA tails, and a 5' cap region comprising one 5' cap. [The present invention 1003] 1001. A modified mRNA of the present invention, comprising a polyA region comprising one polyA tail and a 5' cap region comprising two or more 5' caps. [The present invention 1004] 1001. A modified mRNA of the present invention, comprising a polyA region comprising two or more polyA tails, and a 5' cap region comprising two or more 5' caps. [The present invention 1005] The modified mRNA of any of claims 1001 to 1004, wherein the modified mRNA comprises a 5' untranslated region (5'UTR) and a 3' untranslated region (3'UTR), the ORF is located between the 5'UTR and the 3'UTR, the 3'UTR is located between the ORF and the polyA region, and the 5'UTR is located between the 5' cap region and the ORF. [The present invention 1006] The modified mRNA of any of claims 1001 to 1005, wherein the modified mRNA is a linear mRNA, the 5' cap region is at the 5' end of the modified mRNA, and the poly A region is at the 3' end of the modified mRNA. [The present invention 1007] The modified mRNA of the present invention 1005 or 1006, wherein the modified mRNA is a circular mRNA and the poly A region is between the 3' UTR and the 5' cap region. [The present invention 1008] The modified mRNA of any of claims 1001 to 1007, wherein the polyA region comprises two or more polyA tails, and at least two instances of the polyA tails are covalently linked by a first linker. [The present invention 1009] The poly A region is -(first poly A tail)-[(first linker)-(second poly A tail) n1 ] n2 Including, each instance of n1 is independently an integer from 1 to 20, inclusive; n2 is an integer between 2 and 10, inclusive; the first poly A tail is covalently linked to the 3' UTR; the first linker is covalently attached to the first polyA tail and the second polyA tail; The modified mRNA of the present invention 1008. [The present invention 1010] 1009. A modified mRNA of the present invention, wherein each instance of n1 is 1. [The present invention 1011] 10. The modified mRNA of the present invention 1010, wherein at least one instance of said first linker is covalently linked to an internal nucleotide of said first polyA tail. [The present invention 1012] The modified mRNA of claim 1010 or 1011, wherein at least one instance of the first linker is covalently linked to the second polyA tail at the 3' nucleotide of the second polyA tail. [The present invention 1013] The modified mRNA of any of claims 1010 to 1012, wherein at least one example of the first linker comprises at least one moiety formed by reacting two orthogonal click chemistry handles. [The present invention 1014] At least one moiety formed by reacting two orthogonal click chemistry handles has the formula: The modified mRNA of the present invention 1013 is that of TIFF2026016516000024.tif13128. [The present invention 1015] Each instance of the first linker is independently a substituted or unsubstituted C 1-200 Alkylene, substituted or unsubstituted C 1-200 Alkenylene, substituted or unsubstituted C 1-200 Alkynylene, substituted or unsubstituted C 1-200 Heteroalkylene, substituted or unsubstituted C 1-200 Heteroalkenylene, substituted or unsubstituted C 1-200 heteroalkynylene, substituted or unsubstituted carbocyclylene, substituted or unsubstituted heterocyclylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene; Optionally, the substituted or unsubstituted C 1-200 Alkylene, substituted or unsubstituted C 1-200 Alkenylene, substituted or unsubstituted C 1-200 Alkynylene, substituted or unsubstituted C 1-200 Heteroalkylene, substituted or unsubstituted C 1-200 Heteroalkenylene, and substituted or unsubstituted C 1-200one or more carbon atoms in the parent chain of each instance of heteroalkynylene are independently replaced with a substituted or unsubstituted carbocyclylene, a substituted or unsubstituted heterocyclylene, a substituted or unsubstituted arylene, or a substituted or unsubstituted heteroarylene; A modified mRNA according to any one of 1010 to 1014 of the present invention. [The present invention 1016] The substituted or unsubstituted C 1-200 Alkylene, substituted or unsubstituted C 1-200 Alkenylene, substituted or unsubstituted C 1-200 Alkynylene, substituted or unsubstituted C 1-200 Heteroalkylene, substituted or unsubstituted C 1-200 Heteroalkenylene, and substituted or unsubstituted C 1-200 One or more carbon atoms in the parent chain of each instance of heteroalkynylene independently Any of modified mRNAs 1010 to 1015 of the present invention, replaced by TIFF2026016516000025.tif13128. [The present invention 1017] At least one example of the first linker has the formula: It is TIFF2026016516000026.tif11128, L 1 Each instance of is independently a substituted or unsubstituted C 1-199 Alkylene, or substituted or unsubstituted C 1-199 is heteroalkylene, L 2 Each instance of is independently a substituted or unsubstituted C 1-199 Alkylene, or substituted or unsubstituted C 1-199 is heteroalkylene, The modified mRNA of any one of 1010 to 1016 of the present invention. [The present invention 1018] At least one example of the first linker has the formula: Any of modified mRNAs 1010 to 1017 of the present invention, which is of TIFF2026016516000027.tif16130. [The present invention 1019] 1009. The modified mRNA of the present invention, wherein each instance of n1 is independently an integer between 2 and 20 (inclusive). [The present invention 1020] The modified mRNA of the present invention 1019, wherein at least one example of the first linker comprises a dendrimer. [The present invention 1021] The modified mRNA of the present invention 1020, wherein at least one example of the dendrimer is a polyamidoamine (PAMAM) dendrimer. [The present invention 1022] At least one example of the PAMAM dendrimer has the formula: TIFF2026016516000028.tif22128, wherein: each instance of n3 is independently an integer from 1 to 10, inclusive; each instance of n4 is independently an integer from 0 to 10, inclusive; R 2 each instance of is independently hydrogen or TIFF2026016516000029.tif13128, but R 2 At least three examples of TIFF2026016516000030.tif13128, R 1 Each instance of 1-10 Alkylene, substituted or unsubstituted C 1-10 Alkenylene, substituted or unsubstituted C 1-10 Alkynylene, substituted or unsubstituted C 1-10 Heteroalkylene, substituted or unsubstituted C 1-10 Heteroalkenylene, or substituted or unsubstituted C 1-10 heteroalkynylene, Optionally, the substituted or unsubstituted C 1-10 Alkylene, substituted or unsubstituted C 1-10 Alkenylene, substituted or unsubstituted C 1-10 Alkynylene, substituted or unsubstituted C 1-10 Heteroalkylene, substituted or unsubstituted C 1-10 Heteroalkenylene, and substituted or unsubstituted C1-10 one or more carbon atoms in the parent chain of each instance of heteroalkynylene are independently replaced with a substituted or unsubstituted carbocyclylene, a substituted or unsubstituted heterocyclylene, a substituted or unsubstituted arylene, or a substituted or unsubstituted heteroarylene; each instance of n5 is independently an integer from 0 to 10, inclusive; The modified mRNA of the present invention. [The present invention 1023] At least one example of the PAMAM dendrimer is Modified mRNA of the present invention 1022, including TIFF2026016516000031.tif55128. [The present invention 1024] Substituted or unsubstituted C 1-10 Alkylene, substituted or unsubstituted C 1-10 Alkenylene, substituted or unsubstituted C 1-10 Alkynylene, substituted or unsubstituted C 1-10 Heteroalkylene, substituted or unsubstituted C 1-10 Heteroalkenylene, and substituted or unsubstituted C 1-10 One or more carbon atoms in the parent chain of each instance of heteroalkynylene independently The modified mRNA of the present invention 1021, which is replaced by TIFF2026016516000032.tif12128. [The present invention 1025] At least one example of the first linker has the formula: The modified mRNA of the present invention 1024, which is of TIFF2026016516000033.tif62157. [The present invention 1026] 1026. The modified mRNA of any of claims 1008 to 1025, wherein at least one instance of said first linker is covalently bound to the 3' nucleotide of said first polyA tail. [The present invention 1027] 1027. The modified mRNA of any of claims 1008 to 1026, wherein at least one instance of said first linker is covalently attached to the 3' nucleotide of said second poly-A tail. [The present invention 1028] The modified mRNA of any one of claims 1001 to 1027, wherein the poly A region of the modified mRNA comprises one or more modified nucleotides. [The present invention 1029] 1028. The modified mRNA of the present invention, wherein three or more of the last ten nucleotides of at least one of said two or more poly-A tails are modified nucleotides and / or non-adenosine nucleotides. [The present invention 1030] The modified mRNA of the present invention 1028 or 1029, wherein one or more modified nucleotides in the polyA region are modified adenosine nucleotides. [The present invention 1031] The modified mRNA of any of 1028 to 1030, wherein one or more modified nucleotides in the poly A region are modified non-adenosine nucleotides. [The present invention 1032] The modified mRNA of any one of 1028 to 1031, wherein one or more modified nucleotides in the polyA region comprise a modified nucleic acid base. [The present invention 1033] The modified nucleobase may be xanthine, aliaminouracil, aliaminothymidine, hypoxanthine, digoxigenylated adenine, digoxigenylated cytosine, digoxigenylated guanine, digoxigenylated uracil, 6-chloropurine riboside, N6-methyladenine, methylpseudouracil, 2-thiocytosine, 2-thiouracil, 5-methyluracil, 4-thiothymidine, 4-thiouracil, 5,6-dihydro-5-methyluracil, 5,6-dihydrouracil, 5-[(3-indolyl)propionamido-N-allyl]uracil, 5-aminoaliphatic acid ... cytosine, 5-aminoallyluracil, 5-bromouracil, 5-bromocytosine, 5-carboxycytosine, 5-carboxymethylesteruracil, 5-carboxyuracil, 5-fluorouracil, 5-formylcytosine, 5-formyluracil, 5-hydroxycytosine, 5-hydroxymethylcytosine, 5-hydroxymethyluracil, 5-hydroxyuracil, 5-iodocytosine, 5-iodouracil, 5-methoxycytosine, 5-methoxyuracil, 5-methylcytosine, 5-methyluracil, 5-propargylaminocytosine, 5-propargylaminocytosine, azacytosine, 5-propynyluracil, 6-azacytosine, 6-azauracil, 6-chloropurine, 6-thioguanine, 7-deazaadenine, 7-deazaguanine, 7-deaza-7-propargylaminoadenine, 7-deaza-7-propargylaminoguanine, 8-azaadenine, 8-azidoadenine, 8-chloroadenine, 8-oxoadenine, 8-oxoguanine, araadenine, aracytosine, araguanine, arauracil, biotin-16, 7-deaza-7-propargylaminoguanine, biotin- 16-aminoallylcytosine, biotin-16-aminoallyluracil, cyanine 3-5-propargylaminocytosine, cyanine 3-6-propargylaminouracil, cyanine 3-aminoallylcytosine, cyanine 3-aminoallyluracil, cyanine 5-6-propargylaminocytosine, cyanine 5-6-propargylaminouracil, cyanine 5-aminoallylcytosine, cyanine 5-aminoallyluracil, cyanine 7-aminoallyluracil, dabsyl-5-3-aminoallyluracil, desthiobiotin-16-aminoallyl-uracil,Desthiobiotin-6-aminoallylcytosine, isoguanine, N1-ethylpseudouracil, N1-methoxymethylpseudouracil, N1-methyladenine, N1-methylpseudouracil, N1-propylpseudouracil, N2-methylguanine, N4-biotin-OBEA-cytosine, N4-methylcytosine, N6-methyladenine, O6-methylguanine, pseudoisocytosine Thienocytosine, pseudouracil, thienocytosine, thienoguanine, thienouracil, xanthosine, 3-deazaadenine, 2,6-diaminoadenine, 2,6-daminoguanine, 5-carboxamidouracil, 5-ethynyluracil, N6-isopentenyladenine (i6A), 2-methyl-thio-N6-isopentenyladenine (ms2i6A), 2-methyl- 2-methylthio-N6-methyladenine (ms2m6A), N6-(cis-hydroxyisopentenyl)adenine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenine (ms2io6A), N6-glycinylcarbamoyladenine (g6A), N6-threonylcarbamoyladenine (t6A), 2-methylthio-N6-threonylcarbamoyladenine (ms2t 6A), N6-methyl-N6-threonylcarbamoyladenine (m6t6A), N6-hydroxynorvalylcarbamoyladenine (hn6A), 2-methylthio-N6-hydroxynorvalylcarbamoyladenine (ms2hn6A), N6,N6-dimethyladenine (m62A), and N6-acetyladenine (ac6A). [The present invention 1034] The modified mRNA of any of 1028 to 1033, wherein one or more modified nucleotides comprises a modified sugar. [This invention 1035] The modified sugar may be 2'-thioribose, 2',3'-dideoxyribose, 2'-amino-2'-deoxyribose, 2'deoxyribose, 2'-azido-2'-deoxyribose, 2'-fluoro-2'-deoxyribose, 2'-O-methylribose, 2'-O-methyldeoxyribose, 3'-amino-2',3'-dideoxyribose, 3'-azido-2',3'-dideoxyribose, 3'-deoxyribose 2'-O,4'-C-amino-linked ribose, 2'-O,4'-C-amino-linked ribose, and 2'-O,4'-C-thio-linked ribose. [The present invention 1036] The modified mRNA of any of 1028 to 1035, wherein one or more modified nucleotides comprises a modified phosphate. [This invention 1037] 1036. The modified mRNA of the present invention, wherein the modified phosphate is selected from the group consisting of phosphorothioate (PS), thiophosphate, 5'-O-methylphosphonate, 3'-O-methylphosphonate, 5'-hydroxyphosphonate, hydroxyphosphate, phosphoroselenoate, selenophosphate, phosphoramidate, carbophosphonate, methylphosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidinium ring, triazole ring, boranophosphate (BP), methylphosphonate, and guanidinopropylphosphoramidate. [The present invention 1038] The modified mRNA of the present invention 1036 or 1037, wherein the poly A region contains 1 to 3, 3 to 5, 5 to 10, 10 to 15, 15 to 30, 30 to 50, 50 to 100, or 100 to 200 phosphorothioates. [This invention 1039] The modified mRNA of the present invention 1038, wherein the poly A region contains 5 to 30 phosphorothioates. [The present invention 1040] The modified mRNA of any one of claims 1028 to 1039, wherein the poly A region comprises 3 to 5, 5 to 10, 10 to 15, 15 to 30, 30 to 50, 50 to 100, or 100 to 200 deoxyribose sugars. [The present invention 1041] The modified mRNA of the present invention 1040, wherein the poly A region contains 5 to 30 deoxyribose sugars. [The present invention 1042] The modified mRNA of any one of claims 1028 to 1041, wherein the mRNA comprises a 3'-terminal nucleotide, and the 3'-terminal nucleotide is dideoxyadenosine, dideoxycytidine, dideoxyguanosine, dideoxythymidine, dideoxyuridine, or inverted deoxythymidine. [This invention 1043] The modified mRNA of any one of claims 1001 to 1042, wherein the poly A region comprises 25 to 500 nucleotides. [This invention 1044] 1043. The modified mRNA of the present invention, wherein the poly A region comprises 50 to 100, 100 to 150, 150 to 200, 200 to 300, 300 to 400, or 400 to 500 nucleotides. [This invention 1045] The modified mRNA of any one of claims 1001 to 1044, wherein the poly A region contains 10 or more adenosine nucleotides. [The present invention 1046] The modified mRNA of any of claims 1001 to 1045, wherein 25 to 100%, 30 to 100%, 40 to 100%, 50 to 100%, 60 to 100%, 70 to 100%, 80 to 100%, 90 to 100%, 95 to 100%, 96 to 100%, 97 to 100%, 98 to 100%, or 99 to 100% of the nucleotides in the polyA region are adenosine nucleotides. [This invention 1047] The modified mRNA of any of claims 1008 to 1046, wherein the first poly-A tail comprises 10 to 50 adenosine ribonucleotides, 1 to 10 modified uridine deoxyribonucleotides containing a binding site, and a 3'-terminal dideoxyribonucleotide or an inverted deoxyribonucleotide, and the second poly-A tail comprises 10 to 50 adenosine ribonucleotides. [This invention 1048] the first poly A tail comprises: 5'-rArArArArArArAdU(a1)rArArArArArArArArArArArAdU(a1)rArArArArArArArArArArArArAdU(a1)ddC-3' (SEQ ID NO: 1) comprising the nucleotide sequence shown as At least one example of the second polyA tail is 5'-rArArArArArArArArArArArArArArArArArArArArArArArA*rA*rA*rA*rA*rA*(b1)-3' (SEQ ID NO: 2) comprising the nucleotide sequence shown as each instance of said first linker independently TIFF2026016516000034.tif16148, "rA" represents adenosine ribonucleotide; "dU(a1)" represents a modified uridine deoxyribonucleotide; "ddC" represents cytosine dideoxyribonucleotide; "*" represents a phosphorothioate bond, a1 in each example of dU(a1) represents the point of attachment to the uridine of said dU(a1); b1 of rA*(b1) represents the attachment point at * in rA*(b1), each instance of a1 is connected to an instance of a2, each instance of b1 is connected to an instance of b2; The modified mRNA of the present invention 1047. [This invention 1049] The modified mRNA of the present invention 1047 or 1048, wherein the modified uridine deoxynucleotide is 5-octadiynyl deoxyuridine. [The present invention 1050] The modified mRNA of any of claims 1008 to 1046, wherein the first poly-A tail and the second poly-A tail comprise 10 to 50 adenosine ribonucleotides and a 3'-terminal azide moiety. [This invention 1051] the first polyA tail and the second polyA tail are 5'-rArArArArArArArArArArArArArArArArArArArArArArArA*rA*rA*rA*rA*rA* / AzideN / -3' (SEQ ID NO: 3) comprising the nucleotide sequence shown as wherein "rA" represents an adenosine ribonucleotide, "AzideN" represents a 3' azide moiety, and "*" represents a phosphorothioate bond. The modified mRNA of the present invention. [This invention 1052] The modified mRNA of any of claims 1008 to 1046, wherein the first poly-A tail and the second poly-A tail comprise 10 to 50 adenosine ribonucleotides and a 5'-terminal azide moiety. [This invention 1053] the first polyA tail and the second polyA tail are 5'- / AzideN / rArArArArArArArArArArArArArArArArArArArArArArArA*rA*rA*rA*rA*rA-3' (SEQ ID NO: 27) comprising the nucleotide sequence shown as wherein "rA" represents an adenosine ribonucleotide, "AzideN" represents a 3' azide moiety, and "*" represents a phosphorothioate bond. The modified mRNA of the present invention 1052. [This invention 1054] A modified mRNA of any of claims 1001 to 1053, wherein the 5' cap region comprises two or more 5' caps, and at least two instances of the 5' caps are covalently linked by a second linker. [This invention 1055] the 5' cap region is -(first 5' cap)-[(second linker)-(second 5' cap) m1 ] m2 Including, each instance of m1 is independently an integer from 1 to 20, inclusive; m2 is an integer between 2 and 10, inclusive; the first 5' cap is covalently linked to the 5' UTR; the second linker is covalently attached to the first 5' cap and the second 5' cap. The modified mRNA of the present invention 1054. [The present invention 1056] 1055. A modified mRNA of the present invention, wherein each instance of m1 is 1. [This invention 1057] 1056. The modified mRNA of the present invention, wherein at least one instance of said second linker is covalently attached to an internal nucleotide of said first 5' cap. [This invention 1058] The modified mRNA of claim 1056 or 1057, wherein at least one example of the second linker is covalently linked to the second 5' cap at the 3' nucleotide of the second 5' cap. [This invention 1059] The modified mRNA of any of claims 1056 to 1058, wherein at least one example of the second linker comprises at least one moiety formed by reacting two orthogonal click chemistry handles. [The present invention 1060] At least one moiety formed by reacting two orthogonal click chemistry handles has the formula: The modified mRNA of the present invention 1059, which is of TIFF2026016516000035.tif13128. [The present invention 1061] Each instance of the second linker is independently a substituted or unsubstituted C 1-200 Alkylene, substituted or unsubstituted C 1-200 Alkenylene, substituted or unsubstituted C 1-200 Alkynylene, substituted or unsubstituted C 1-200 Heteroalkylene, substituted or unsubstituted C 1-200 Heteroalkenylene, substituted or unsubstituted C 1-200 heteroalkynylene, substituted or unsubstituted carbocyclylene, substituted or unsubstituted heterocyclylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene; Optionally, the substituted or unsubstituted C 1-200 Alkylene, substituted or unsubstituted C 1-200 Alkenylene, substituted or unsubstituted C 1-200 Alkynylene, substituted or unsubstituted C 1-200 Heteroalkylene, substituted or unsubstituted C 1-200 Heteroalkenylene, and substituted or unsubstituted C 1-200 one or more carbon atoms in the parent chain of each instance of heteroalkynylene are independently replaced with a substituted or unsubstituted carbocyclylene, a substituted or unsubstituted heterocyclylene, a substituted or unsubstituted arylene, or a substituted or unsubstituted heteroarylene; A modified mRNA according to any one of 1056 to 1060 of the present invention. [The present invention 1062] The substituted or unsubstituted C 1-200 Alkylene, substituted or unsubstituted C 1-200 Alkenylene, substituted or unsubstituted C 1-200 Alkynylene, substituted or unsubstituted C 1-200 Heteroalkylene, substituted or unsubstituted C 1-200 Heteroalkenylene, and substituted or unsubstituted C 1-200 One or more carbon atoms in the parent chain of each instance of heteroalkynylene independently A modified mRNA of any one of 1049 to 1054 of the present invention, which is replaced by TIFF2026016516000036.tif13128. [The present invention 1063] At least one example of the second linker has the formula: It is TIFF2026016516000037.tif11128, L 3 Each instance of is independently a substituted or unsubstituted C 1-199 Alkylene, or substituted or unsubstituted C 1-199 is heteroalkylene, L 4 Each instance of is independently a substituted or unsubstituted C 1-199 Alkylene, or substituted or unsubstituted C 1-199 is heteroalkylene, A modified mRNA according to any one of 1056 to 1062 of the present invention. [The present invention 1064] At least one example of the second linker has the formula: A modified mRNA of any one of 1056 to 1062 of the present invention, which is of TIFF2026016516000038.tif16132. [This invention 1065] 1056. The modified mRNA of the present invention, wherein each instance of m1 is independently an integer between 2 and 20 (inclusive). [The present invention 1066] The modified mRNA of the present invention 1065, wherein at least one example of the second linker comprises a dendrimer. [This invention 1067] The modified mRNA of the present invention 1066, wherein at least one example of the dendrimer is a polyamidoamine (PAMAM) dendrimer. [The present invention 1068] At least one example of the PAMAM dendrimer has the formula: TIFF2026016516000039.tif22128, wherein: each instance of m3 is independently an integer from 1 to 10, inclusive; each instance of m4 is independently an integer from 0 to 10, inclusive; R 12 each instance of is independently hydrogen or TIFF2026016516000040.tif13128, but R12 At least three examples of TIFF2026016516000041.tif13128, R 11 Each instance of 1-10 Alkylene, substituted or unsubstituted C 1-10 Alkenylene, substituted or unsubstituted C 1-10 Alkynylene, substituted or unsubstituted C 1-10 Heteroalkylene, substituted or unsubstituted C 1-10 Heteroalkenylene, or substituted or unsubstituted C 1-10 heteroalkynylene, Optionally, the substituted or unsubstituted C 1-10 Alkylene, substituted or unsubstituted C 1-10 Alkenylene, substituted or unsubstituted C 1-10 Alkynylene, substituted or unsubstituted C 1-10 Heteroalkylene, substituted or unsubstituted C 1-10 Heteroalkenylene, and substituted or unsubstituted C 1-10 one or more carbon atoms in the parent chain of each instance of heteroalkynylene are independently replaced with a substituted or unsubstituted carbocyclylene, a substituted or unsubstituted heterocyclylene, a substituted or unsubstituted arylene, or a substituted or unsubstituted heteroarylene; each instance of m5 is independently an integer from 0 to 10, inclusive; The modified mRNA of the present invention 1067. [This invention 1069] At least one example of the PAMAM dendrimer is The modified mRNA of the present invention 1067 or 1068, including TIFF2026016516000042.tif55128. [The present invention 1070] The substituted or unsubstituted C 1-10 Alkylene, substituted or unsubstituted C 1-10 Alkenylene, substituted or unsubstituted C 1-10 Alkynylene, substituted or unsubstituted C 1-10 Heteroalkylene, substituted or unsubstituted C 1-10Heteroalkenylene, and substituted or unsubstituted C 1-10 One or more carbon atoms in the parent chain of each instance of heteroalkynylene independently The modified mRNA of the present invention 1068, which is replaced by TIFF2026016516000043.tif13128. [This invention 1071] At least one example of the first linker has the formula: The modified mRNA of the present invention 1068, which is of TIFF2026016516000044.tif56148. [This invention 1072] 1072. The modified mRNA of any one of claims 1050 to 1071, wherein at least one instance of the second linker is covalently linked to the 5' or 3' nucleotide of the first 5' cap. [This invention 1073] The modified mRNA of any of claims 1050 to 1072, wherein at least one example of the second linker is covalently linked to the 5' or 3' nucleotide of at least one example of the second 5' cap. [This invention 1074] The modified mRNA of any of claims 1050 to 1073, wherein the 5' cap region of the modified mRNA comprises one or more modified nucleotides. [This invention 1075] 1074. The modified mRNA of the present invention, wherein one or more modified nucleotides comprises a modified nucleobase. [This invention 1076] The modified nucleobase may be xanthine, aliaminouracil, aliaminothymidine, hypoxanthine, digoxigenylated adenine, digoxigenylated cytosine, digoxigenylated guanine, digoxigenylated uracil, 6-chloropurine riboside, N6-methyladenine, methylpseudouracil, 2-thiocytosine, 2-thiouracil, 5-methyluracil, 4-thiothymidine, 4-thiouracil, 5,6-dihydro-5-methyluracil, 5,6-dihydrouracil, 5-[(3-indolyl)propionamido-N-allyl]uracil, 5-aminoaliphatic acid ... cytosine, 5-aminoallyluracil, 5-bromouracil, 5-bromocytosine, 5-carboxycytosine, 5-carboxymethylesteruracil, 5-carboxyuracil, 5-fluorouracil, 5-formylcytosine, 5-formyluracil, 5-hydroxycytosine, 5-hydroxymethylcytosine, 5-hydroxymethyluracil, 5-hydroxyuracil, 5-iodocytosine, 5-iodouracil, 5-methoxycytosine, 5-methoxyuracil, 5-methylcytosine, 5-methyluracil, 5-propargylaminocytosine, 5-propargylaminocytosine, azacytosine, 5-propynyluracil, 6-azacytosine, 6-azauracil, 6-chloropurine, 6-thioguanine, 7-deazaadenine, 7-deazaguanine, 7-deaza-7-propargylaminoadenine, 7-deaza-7-propargylaminoguanine, 8-azaadenine, 8-azidoadenine, 8-chloroadenine, 8-oxoadenine, 8-oxoguanine, araadenine, aracytosine, araguanine, arauracil, biotin-16, 7-deaza-7-propargylaminoguanine, biotin- 16-aminoallylcytosine, biotin-16-aminoallyluracil, cyanine 3-5-propargylaminocytosine, cyanine 3-6-propargylaminouracil, cyanine 3-aminoallylcytosine, cyanine 3-aminoallyluracil, cyanine 5-6-propargylaminocytosine, cyanine 5-6-propargylaminouracil, cyanine 5-aminoallylcytosine, cyanine 5-aminoallyluracil, cyanine 7-aminoallyluracil, dabsyl-5-3-aminoallyluracil, desthiobiotin-16-aminoallyl-uracil,Desthiobiotin-6-aminoallylcytosine, isoguanine, N1-ethylpseudouracil, N1-methoxymethylpseudouracil, N1-methyladenine, N1-methylpseudouracil, N1-propylpseudouracil, N2-methylguanine, N4-biotin-OBEA-cytosine, N4-methylcytosine, N6-methyladenine, O6-methylguanine, pseudo Isocytosine, pseudouracil, thienocytosine, thienoguanine, thienouracil, xanthosine, 3-deazaadenine, 2,6-diaminoadenine, 2,6-daminoguanine, 5-carboxamidouracil, 5-ethynyluracil, N6-isopentenyladenine (i6A), 2-methyl-thio-N6-isopentenyladenine (ms2i6A), 2-methylthio-N6-methyl The modified mRNA of the present invention 1075 is selected from the group consisting of N6-(cis-hydroxyisopentenyl)adenine (ms2m6A), N6-(cis-hydroxyisopentenyl)adenine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenine (ms2io6A), N6-glycinylcarbamoyladenine (g6A), N6-threonylcarbamoyladenine (t6A), 2-methylthio-N6-threonylcarbamoyladenine (ms2t6A), N6-methyl-N6-threonylcarbamoyladenine (m6t6A), N6-hydroxynorvalylcarbamoyladenine (hn6A), 2-methylthio-N6-hydroxynorvalylcarbamoyladenine (ms2hn6A), N6,N6-dimethyladenine (m62A), and N6-acetyladenine (ac6A). , [This invention 1077] The modified mRNA of any of 1074 to 1076, wherein one or more modified nucleotides comprises a modified sugar. [This invention 1078] The modified sugar may be 2'-thioribose, 2',3'-dideoxyribose, 2'-amino-2'-deoxyribose, 2'deoxyribose, 2'-azido-2'-deoxyribose, 2'-fluoro-2'-deoxyribose, 2'-O-methylribose, 2'-O-methyldeoxyribose, 3'-amino-2',3'-dideoxyribose, 3'-azido-2',3'-dideoxyribose, 3'-deoxyribose 2'-O,4'-C-amino-linked ribose, 2'-O,4'-C-amino-linked ribose, and 2'-O,4'-C-thio-linked ribose. [This invention 1079] The modified mRNA of any of 1074 to 1078, wherein one or more modified nucleotides comprises a modified phosphate. [The present invention 1080] 1079. The modified mRNA of the present invention, wherein the modified phosphate is selected from the group consisting of phosphorothioate (PS), thiophosphate, 5'-O-methylphosphonate, 3'-O-methylphosphonate, 5'-hydroxyphosphonate, hydroxyphosphate, phosphoroselenoate, selenophosphate, phosphoramidate, carbophosphonate, methylphosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidinium ring, triazole ring, boranophosphate (BP), methylphosphonate, and guanidinopropylphosphoramidate. [This invention 1081] The modified mRNA of the present invention 1079 or 1080, wherein the 5' cap region contains 1 to 3, 3 to 5, 5 to 10, 10 to 15, 15 to 30, 30 to 50, 50 to 100, or 100 to 200 phosphorothioates. [This invention 1082] 1081. The modified mRNA of the present invention, wherein the 5' cap region comprises 5 to 30 phosphorothioates. [This invention 1083] The modified mRNA of any one of 1074 to 1082 of the present invention, wherein the 5' cap region comprises 3 to 5, 5 to 10, 10 to 15, 15 to 30, 30 to 50, 50 to 100, or 100 to 200 deoxyribose sugars. [This invention 1084] The modified mRNA of the present invention 1083, wherein the 5' cap region comprises 5 to 30 deoxyribose sugars. [This invention 1085] The modified mRNA of any one of 1074 to 1084 of the present invention, wherein the mRNA comprises a 3'-terminal nucleotide, and the 3'-terminal nucleotide is dideoxyadenosine, dideoxycytidine, dideoxyguanosine, dideoxythymidine, dideoxyuridine, or inverted deoxythymidine. [The present invention 1086] The modified mRNA of any one of 1050 to 1085 of the present invention, wherein the 5' cap region comprises 25 to 500 nucleotides. [This invention 1087] 1086. The modified mRNA of the present invention, wherein the 5' cap region comprises 50 to 100, 100 to 150, 150 to 200, 200 to 300, 300 to 400, or 400 to 500 nucleotides. [This invention 1088] The modified mRNA of any one of 1050 to 1087, wherein the 5' cap region comprises 1 to 3, 3 to 5, 5 to 7, or 7 to 10 5' caps. [This invention 1089] The modified mRNA of any of claims 1050 to 1088, wherein the 5' cap region comprises one or more 7-methylguanylic acid caps containing a 5'-5' triphosphate linkage. [The present invention 1090] The modified mRNA of any of claims 1050 to 1089, wherein the 5' cap region comprises one or more 2,2,7-trimethylguanosine caps containing a 5'-5' triphosphate linkage. [This invention 1091] The modified mRNA of any of claims 1050 to 1090, wherein the 5' cap region comprises one or more modified 5' caps. [This invention 1092] 1091. A modified mRNA of the present invention, wherein one or more modified 5' caps are a locked nucleic acid (LNA) modified cap, a 5' cap comprising a 5'-5' triphosphate linkage modified by 5'-phosphorothiolate, or a 5' cap comprising a 5'-5' tetraphosphate linkage. [This invention 1093] The modified mRNA of the present invention 1091 or 1092, wherein the 5' cap region comprises one or more 5' caps comprising one or more modified nucleotides. [This invention 1094] The modified mRNA of the present invention 1093, wherein one or more modified nucleotides are 2'-O-methylated nucleotides. [This invention 1095] Any of the modified mRNAs of 1050 to 1094 of the present invention, wherein the first 5' cap comprises 10 to 50 ribonucleotides, 1 to 10 modified uridine deoxyribonucleotides including a binding point, and a 5' cap, and the second 5' cap comprises 10 to 50 ribonucleotides and a 5' cap. [This invention 1096] the first 5' cap is 5'- / Cap / rGrGrGrArArAdU(c1)rArArGrArGrArArArArGrArArGrArGdU(c1)rArArGrArArGrArArAdU(c1)rA-3' (SEQ ID NO: 4) comprising the nucleotide sequence shown as the second 5' cap is 5'- / Cap / rGrGrGrArGrArCrTrGrCrCrArCrCrA*rA*rA*rA*rA*rA*(d1)-3' (SEQ ID NO: 5) comprising the nucleotide sequence shown as each instance of said second linker independently TIFF2026016516000045.tif14139, "rA" represents adenosine ribonucleotide; "rT" represents thymidine ribonucleotide; "rC" represents cytidine ribonucleotide; "rG" represents guanosine ribonucleotide; "dU(c1)" represents a modified uridine deoxyribonucleotide; "*" represents a phosphorothioate bond, "Cap" represents the 5' cap, c1 in each example of dU(c1) represents the point of attachment of the dU(c1) to the uridine; d1 of rA*(d1) represents the attachment point at * in rA*(d1), Each instance of c1 is connected to one instance of c2, Each instance of d1 is connected to one instance of d2, The modified mRNA of the present invention. [This invention 1097] The modified mRNA of the present invention 1095 or 1096, wherein the modified uridine deoxynucleotide is 5-octadiynyl deoxyuridine. [This invention 1098] Any of the modified mRNAs of 1050 to 1094 of the present invention, wherein the first 5' cap comprises 5 to 10 ribonucleotides and a 5' azide moiety, and the second 5' cap comprises 10 to 50 ribonucleotides, a 3' azide moiety, and a 5' cap. [This invention 1099] the first 5' cap is 5'- / / 5AzideN / rArArA rArA-3' comprising the nucleotide sequence shown as the second 5' cap is 5'- / Cap / rGrGrGrArGrArCrTrGrCrCrArCrCrA*rA*rA*rA*rA*rA* / 3AzideN / -3' (SEQ ID NO: 6) comprising the nucleotide sequence shown as wherein "rA" represents an adenosine ribonucleotide, "rT" represents a thymidine ribonucleotide, "rC" represents a cytidine ribonucleotide, "rG" represents a guanosine ribonucleotide, "5AzideN" represents a 5' azido moiety, "3AzideN" represents a 3' azido moiety, "*" represents a phosphorothioate bond, and "Cap" represents a 5' cap; The modified mRNA of the present invention. [The present invention 1100] A method for producing a modified mRNA of any of claims 1001 to 1099, comprising: Ligating a first RNA comprising an open reading frame (ORF) encoding a protein to a tailing nucleic acid comprising two or more polyA tails and / or a capping nucleic acid comprising two or more 5' caps in the presence of an RNA ligase, whereby the RNA ligase forms a covalent bond between the 3' nucleotide of the first RNA and the 5' nucleotide of the tailing nucleic acid, and / or a covalent bond between the 5' nucleotide of the first RNA and the 3' nucleotide of the capping nucleic acid, to produce the modified mRNA. [The present invention 1101] 1100. The method of claim 1100, wherein the modified mRNA comprises a 5' untranslated region (5'UTR) and a 3' untranslated region (3'UTR), the ORF is between the 5'UTR and the 3'UTR, the 3'UTR is between the ORF and the ligated tailing nucleic acid, and the 5'UTR is between the ORF and the ligated capping nucleic acid. [The present invention 1102] 1102. The method of any one of claims 1100 to 1101, wherein said tailing nucleic acid comprises a first polyA tail and one or more second polyA tails, said first polyA tail being covalently linked to said 3'UTR. [The present invention 1103] A modified mRNA produced by any of the methods of the present inventions 1098 to 1100. [The present invention 1104] A delivery agent comprising the modified mRNA of any one of claims 1001 to 1099 of the present invention, wherein the delivery agent comprises a lipid, a peptide, a protein, an antibody, a carbohydrate, a nanoparticle, or a microparticle. [This invention 1105] The delivery agent of the present invention 1104, wherein the nanoparticles or microparticles are lipid nanoparticles or microparticles, polymer nanoparticles or microparticles, protein nanoparticles or microparticles, or solid nanoparticles or microparticles. [The present invention 1106] A cell comprising any one of the modified mRNAs of the present inventions 1001 to 1099. [This invention 1107] The cell of the present invention 1106, which is a mammalian cell. [This invention 1108] A composition comprising the modified mRNA of any one of the present inventions 1001 to 1099, the delivery agent of the present invention 1104 or 1105, or the cell of the present invention 1106 or 1107. [This invention 1109] The composition of claim 1108, further comprising an additional agent. [The present invention 1110] The composition of claim 1109, wherein the additional agent is an agent that has a therapeutic effect when administered to a subject. [The present invention 1111] The composition of any one of claims 1109 to 1110, wherein the additional agent is a nucleotide, nucleic acid, amino acid, peptide, protein, small molecule, aptamer, lipid, or carbohydrate. [The present invention 1112] The composition of claim 1111, wherein the additional agent is an shRNA, an siRNA, or an ASO. [The present invention 1113] The composition of any one of claims 1109 to 1111, wherein the additional agent is an antigen or an adjuvant. [This invention 1114] The composition of any one of claims 1108 to 1113, wherein the composition is a pharmaceutical composition, and the pharmaceutical composition further comprises a pharmaceutically acceptable excipient. [This invention 1115] A method comprising introducing into a cell the modified mRNA of any one of items 1001 to 1099 of the present invention or the delivery agent of item 1104 or 1105 of the present invention. [The present invention 1116] A method comprising introducing into a subject a modified mRNA of any one of claims 1001 to 1099 of the present invention, a delivery agent of claim 1104 or 1105 of the present invention, a cell of claim 1106 or 1107 of the present invention, or a composition of any one of claims 1108 to 1114 of the present invention. [This invention 1117] A method for preventing a disease in a subject in need thereof, comprising introducing into the subject an effective amount of a modified mRNA of any one of claims 1001 to 1099 of the present invention, a delivery agent of claim 1104 or 1105 of the present invention, a cell of claim 1105 or 1107 of the present invention, or a composition of any one of claims 1108 to 1114 of the present invention, wherein the open reading frame of the modified mRNA encodes a protein. [This invention 1118] A method for treating a disease in a subject in need thereof, comprising introducing into the subject an effective amount of a modified mRNA of any one of claims 1001 to 1099 of the present invention, a delivery agent of claim 1104 or 1105 of the present invention, a cell of claim 1106 or 1107 of the present invention, or a composition of any one of claims 1108 to 1114 of the present invention, wherein the open reading frame of the modified mRNA encodes a protein. [This invention 1119] 1117. The method of claim 1118, wherein said protein is a protein antigen or a fragment thereof derived from a cell or virus that causes said disease. [The present invention 1120] The method of claim 1117 or claim 1118, wherein said protein is a protein that is expressed in said subject at a level lower than a reference level. [This invention 1121] A method for replacing an enzyme in a subject, comprising introducing into the subject a modified mRNA of any one of claims 1001 to 1099 of the present invention, a delivery agent of claim 1104 or 1105 of the present invention, a cell of claim 1106 or 1107 of the present invention, or a composition of any one of claims 1108 to 1114 of the present invention, wherein the open reading frame of the modified mRNA encodes an enzyme. [This invention 1122] The method of any one of claims 1116 to 1117, wherein the subject is a human. [This invention 1123] A modified mRNA of any of claims 1001 to 1099, a delivery agent of claim 1104 or 1105, a cell of claim 1106 or 1107, or a composition of any of claims 1108 to 1114, for use in preventing a disease in a subject in need thereof. [This invention 1124] A modified mRNA of any of claims 1001 to 1099, a delivery agent of claim 1104 or 1105, a cell of claim 1106 or 1107, or a composition of any of claims 1108 to 1114, for use in treating a disease in a subject in need thereof. [Invention 1125] A kit comprising the first RNA of any one of 1100 to 1102 of the present invention and a tailing nucleic acid and / or a capping nucleic acid. [The present invention 1126] The kit of claim 1125, further comprising an RNA ligase. [This invention 1127] A kit comprising the pharmaceutical composition of the present invention 1114, a device for administering said pharmaceutical composition to a subject, and instructions for administering said pharmaceutical composition to a subject. [Brief explanation of the drawings]

[0123] [Figure 1A]Figures 1A-J show schematic diagrams illustrating alternative design pathways and routes for synthesizing poly-tailed and poly-capped mRNA. Figure 1A shows the canonical pathway of eukaryotic translation initiation. Figure 1B shows a pseudo-closed-loop model of actively translating mRNA, in which the poly(A) tail is degraded through exonuclease activity. Figure 1C shows a first orthogonal design pathway for synthesizing poly-tailed mRNA, using crosslinking chemistry (e.g., click chemistry) to generate poly-tailed mRNA. Poly-A branches are attached to the mRNA using site-specific "crosslinking" groups, which are incorporated as modified nucleotides at the 3' end of the mRNA transcript (e.g., by site-specific modification with a polymerase or enzymatic treatment, or via ligation of a molecule / oligo containing the crosslinking group). Figure 1D shows a schematic diagram illustrating a route to synthesize a poly-tailed mRNA construct starting from a poly-A oligo modified with several internal 5-octadiynyl dU sites, to which additional poly-A oligos are attached via click chemistry (triangles) to generate a brush-like poly-A tail. The brush-like poly-A tail is then ligated to the 3' end of an RNA containing a coding sequence (CDS). Figure 1E shows a second orthogonal design route to synthesize a poly-tailed mRNA, in which a poly-A-tailed functionalized dendrimer is attached to the 3' end of a linear mRNA using chemical or enzymatic ligation. Figure 1F shows a schematic diagram illustrating a route to synthesize a poly-tailed mRNA construct starting from a PAMAM dendrimer functionalized with an alkyne group, to which a poly-A oligo is attached via click chemistry (triangles). One of the dendrimer's poly-A oligos is then ligated to the 3' end of an RNA containing a coding sequence (CDS). Figure 1G shows a first orthogonal design pathway for synthesizing poly-capped mRNA, which uses cross-linking chemistry (e.g., click chemistry) to generate poly-capped mRNA. Poly-capped branches are attached to the mRNA using site-specific "cross-linking" groups, which are incorporated as modified nucleotides at the 5' end of the mRNA transcript (e.g., via ligation of a molecule / oligo containing the cross-linking group).Figure 1H shows a schematic diagram illustrating a route to synthesize a polycapped mRNA construct starting from a 5'-capped oligo modified with several internal 5-octadiynyl dU sites, to which additional 5'-capped oligos are attached via click chemistry (triangles) to generate a brush-like 5'-capping sequence. The brush-like 5'-capping sequence is then ligated to the 5'-end of an RNA containing a coding sequence (CDS). Figure 1I shows a second orthogonal design route for synthesizing polycapped mRNA, in which a polycapped functionalized dendrimer is attached to the 5'-end of a linear mRNA using chemical or enzymatic ligation. Figure 1J shows a schematic diagram illustrating a route to synthesize a polycapped mRNA construct starting from a PAMAM dendrimer functionalized with an alkyne group, to which a 5'-capping oligo is attached via click chemistry (triangles). One of the dendrimer's 5'-capping oligos is then ligated to the 5'-end of an RNA containing a coding sequence (CDS). [Figure 1B] See legend to Figure 1A. [Figure 1C] See legend to Figure 1A. [Figure 1D] See legend to Figure 1A. [Figure 1E] See legend to Figure 1A. [Figure 1F] See legend to Figure 1A. [Figure 1G] See legend to Figure 1A. [Figure 1H] See legend to Figure 1A. [Figure 1I] See legend to Figure 1A. [Figure 1J] See legend to Figure 1A. [Figure 1K]Figures 1K and 1L show protein expression from different poly-tailed mRNA constructs. Figure 1K shows GFP production over time from different poly-tailed mRNA constructs. Untreated GFP mRNA: GFP mRNA obtained from in vitro transcription. Mock ligation: Product of the ligation reaction between GFP mRNA and no oligo. Brush oligo-only ligation: Product of the ligation reaction between GFP mRNA and a brush oligo precursor shown in Table 1. Brush 3' azido click and ligation: Product of the ligation reaction between GFP mRNA and a brush oligo synthesized from a click reaction between a brush oligo precursor and a 3' azido poly(A) oligo from Table 1. Brush 5' azido click and ligation: Product of the ligation reaction between GFP mRNA and a brush oligo synthesized from a click reaction between a brush oligo precursor and a 5' azido poly(A) oligo from Table 1. mCherry mRNA was always unligated and therefore used as an internal control, cotransfected with GFP mRNA. The GFP / mCherry ratio was calculated from the intensities of the GFP and mCherry signals, respectively. Figure 1L shows the time course of firefly luciferase production from different poly-tailed mRNA constructs. Untreated luciferase mRNA: firefly luciferase mRNA obtained from in vitro transcription. Mock ligation: product of the ligation reaction between firefly luciferase mRNA and no oligo. Brush oligo-only ligation: product of the ligation reaction between firefly luciferase mRNA and the brush oligo precursor shown in Table 1. Brush 3' azido click & ligation: product of the ligation reaction between firefly luciferase mRNA and the brush oligo synthesized from the click reaction between the brush oligo precursor and the 3' azido poly(A) oligo shown in Table 1. Brush 5' azido click & ligation: Product of the ligation reaction between firefly luciferase mRNA and a brush oligo synthesized from a click reaction between a brush oligo precursor and a 5' azido poly(A) oligo from Table 1.Renilla luciferase mRNA was always unligated and therefore used as an internal control and co-transfected with firefly luciferase mRNA. The firefly / renilla ratio was calculated from the intensity of the bioluminescence signals induced by firefly luciferase and Renilla luciferase. [Figure 1L] See legend to Figure 1K. [Figure 2A] Figures 2A-2D show conceptualizations of chimeric mRNAs with multiple poly(A) tails. Figure 2A shows a pseudo-closed-loop model for actively translating mRNA. Figure 2B shows an overview of the oligonucleotide chemical conjugation method. Screening was performed using a 15-nt dA model substrate at micromolar concentrations. Modification handles were incorporated via solid-phase synthesis, followed by amine-NHS labeling and, if necessary, HPLC purification. Figure 2C shows gel electrophoresis of crude thiol-ene / yne oligonucleotide conjugation of a 15-nt model substrate containing only one conjugation handle. Figure 2D shows gel electrophoresis of crude CuAAC and IEDDA 30-nt oligonucleotides bearing three EU / TCO handles reacting with a 30-nt N3 / Tz-modified oligo. [Figure 2B-1] See legend to Figure 2A. [Figure 2B-2] See legend to Figure 2A. [Figure 2C] See legend to Figure 2A. [Figure 2D] See legend to Figure 2A. [Figure 2E]Preliminary dual-luciferase assays using branched mRNA prepared from unpurified CuAAC mixtures enriched with different equivalent amounts of branched oligos are shown. Branched oligos included natural (5' to 3') or reverse (3' to 5') orientation and were modified in the last six bases. Time course assays were performed as outlined in Figure 1. n = 3 independent transfections in each biological condition. Mean ± sem. P values ​​were calculated by regular two-way ANOVA (alpha = 0.05) (Dunnett's multiple comparison test, comparing means across time points) using multiple comparisons with the linear construct. ****P < 0.0001, nsP > 0.1234. [Figure 3A]Figures 3A-F show the conceptualization of chimeric mRNA molecules with multiple poly(A) tails. Figure 3A shows the synthesis and screening pipeline for branched mRNAs. EU- and azide-containing oligos were chemically conjugated, HPLC-purified, and enzymatically ligated to the 3' end of mRNA to generate translatable branched mRNAs encoding firefly luciferase reporters. Constructs were screened in a dual-luciferase assay using Renilla luciferase as a transfection control over a 72-hour time course. M is a marker. Figures 3B-3D show HPLC purification chromatograms and gel electrophoresis profiles of oligos containing 1, 2, or 3 branched poly(A). Fractions containing the desired product (boxed) were pooled and isolated. Figure 3E shows a representative RNase H characterization of chimeric mRNA branched poly(A) conjugates. The RNase H probe was designed 200 nt upstream from the 3' end of the mRNA, and the conjugation product (boxed) was characterized by a corresponding band shift after RNase H digestion by gel electrophoresis. Figure 3F shows a bar plot of a time-course dual-luciferase assay screening chemical modifications in the multimerized poly(A) tail. Protein expression was measured by Renilla luciferase luminescence and firefly luciferase luminescence normalized to the linear mRNA control at 24, 48, and 72 h posttransfection. Figures are means ± s.e.m. P values ​​were calculated using a two-way ANOVA (alpha = 0.05) with Dunnett's multiple comparison test (comparison of means across time points) using multiple comparisons with the linear construct. *P < 0.0332, **P < 0.0021, ***P < 0.0002, ****P < 0.0001, nsP > 0.1234. [Figure 3B] See legend to Figure 3A. [Figure 3C] See legend to Figure 3A. [Figure 3D] See legend to Figure 3A. [Figure 3E] See legend to Figure 3A. [Figure 3F] See legend to Figure 3A. [Figure 4A]Figures 4A-4K show mechanistic characterization of mRNA stabilization by multimerized poly(A) tails. Figures 4A-4B show kinetic characterization of the firefly-degron reporter mRNA. Firefly RLU for each construct at the corresponding time point was normalized to the Renilla luciferase control by firefly RLU / Renilla RLU*mean(Renilla RLU) and normalized to the normalized 8-hour firefly RLU for each construct. Decay half-lives were calculated by fitting to an exponential curve. P values ​​were calculated by a regular two-way ANOVA (alpha = 0.05) (Dunnett's multiple comparison test, comparing means across time points) with multiple comparisons to the linear construct. ****P<0.0001. Figure 4C shows the decay kinetics of the internal control Renilla luciferase RLU outlined in Figure 2C. P values ​​were calculated by two-way ANOVA (alpha = 0.05) (Dunnett's multiple comparison test, comparing means across time points) using multiple comparisons with linear constructs. nsP > 0.1234. Figure 4D shows a schematic diagram of synergistic in situ sequencing characterization of targeted mRNA transcripts using STARmap + RIBOmap. Six hours after cotransfection of firefly luciferase mRNA and Renilla luciferase mRNA, cells were replated into two wells per condition per time point. RIBOmap and STARmap characterization of engineered firefly luciferase transcripts in each duplicate of ribosome-bound or total mRNA, respectively. Unmodified Renilla luciferase mRNA was sequenced by STARmap in all replicates to account for transfection efficiency. Representative STARmap / RIBOmap images were acquired under the same confocal imaging settings. For each condition, at least three independent transfections were performed, and four FOVs were imaged from each. DAPI (blue), nuclei; firefly luciferase amplicon (magenta); Renilla luciferase amplicon (yellow). Colocalized firefly / renilla amplicons (white dots) in STARmap represent lipid transfection vesicles and were excluded from downstream quantification. Figure 4E shows a representative STARmap / RIBOmap image acquired under the same confocal imaging settings.STARmap / RIBOmap characterization of modified firefly luciferase mRNA and internal control Renilla luciferase mRNA was performed as outlined in Figure 2. DAPI (blue), nuclei; firefly luciferase amplicon (magenta); Renilla luciferase amplicon (yellow). Colocalized firefly / Renilla amplicons (white dots) in STARmap represent lipid transfection vesicles and were excluded from downstream quantification. Figures 4F-4G show violin diagrams of single-cell quantification of firefly luciferase STARmap / RIBOmap amplicons. P values ​​were calculated by ordinary two-way ANOVA (alpha = 0.05). ****P < 0.0001. Figure 4H shows a comparison of translation efficiency (TE) of branched and linear mRNAs across different time points. For each single cell, total firefly mRNA abundance was measured by log10(firefly STARmap / Renilla STARmap), and ribosome-bound firefly mRNA abundance was measured by log10(firefly RIBOmap / Renilla STARmap). TE was calculated by the ratio of ribosome-bound firefly / total firefly, and the median value for each FOV was normalized to linear firefly mRNA. Mean ± sem. P values ​​were calculated by unpaired t-test with Welch's correction. **P<0.004, nsP>0.1234. Figure 4I shows gel shift assays of three branched poly-A oligos with various concentrations of PABPC1-GST. The stem oligos were labeled at the 5' end with Alexa Fluor 546. Both the stem and branched poly-A oligos were 30 nt long and modified with PS-2MOE in the last six bases and ddC at the 3' end (top). Figure 4I also shows a gel shift assay of the same modified poly-A oligo without branching (center). Finally, Figure 4I shows a competitive binding assay of Alexa546-labeled branched poly(A) oligos with unlabeled linear poly(A) oligos for PABCP1-GST (bottom). Figures 4J-4K show the apparent Kd calculations for branched / linear poly(A) oligos.The percent of poly(A) oligos bound to x-mer PABPs was calculated as the total percent of oligos bound to >x-mer PABPs, and the apparent Kd was calculated by plotting the percent of poly(A) oligos bound to PABPs against the PABP concentration using GraphPad Prism (specific binding with Hill slope). [Figure 4B] See legend to Figure 4A. [Figure 4C] See legend to Figure 4A. [Figure 4D] See legend to Figure 4A. [Figure 4E-1] See legend to Figure 4A. [Figure 4E-2] See legend to Figure 4A. [Figure 4F] See legend to Figure 4A. [Figure 4G] See legend to Figure 4A. [Figure 4H] See legend to Figure 4A. [Figure 4I-1] See legend to Figure 4A. [Figure 4I-2] See legend to Figure 4A. [Figure 4J] See legend to Figure 4A. [Figure 4K] See legend to Figure 4A. [Figure 5A]Figures 5A-5C show a comparison of branched mRNAs versus circular mRNAs. Figure 5A shows gel electrophoresis of circRNAs generated by IVT, backsplicing, and enriched by RNase R treatment. Figure 5B shows a comparison of linear / branched / circular mRNAs with optimized UTRs at different doses using secreted nanoluciferase in HeLa cells 24 h after transfection. Both branched and linear mRNAs were cotranscriptionally capped and tailed and contained the human alpha globin UTR. Branched mRNAs were ligated with an optimized branched construct containing a 6*PS-2MOE modification. CircRNAs were designed to encode an HRV IRES (with a proximal loop Apt-eIF4G insertion) and a 3'-PABP binding motif. Figure 5C shows a time course comparison of linear mRNAs, branched mRNAs, and circular mRNAs encoding secreted NanoLuc. Both branched and linear mRNAs were cotranscriptionally capped and tailed and contained the human alpha globin UTR. Branched mRNAs were ligated with an optimized branched construct containing a 6*PS-2MOE modification. CircRNAs were designed to encode an HRV IRES (with a proximal loop Apt-eIF4G insertion) and a 3'-PABP binding motif. Cells were transfected with equimolar amounts (0.2 pmol) of each construct (three independent lipofections for each condition). Cells were cultured in phenol red-free DMEM medium. The medium was completely changed every day, and 50 μL of cultured medium was used for luciferase assays. Mean ± sem. n = 3 independent transfections for each biological condition. [Figure 5B] See the legend to Figure 5A. [Figure 5C] See the legend to Figure 5A. [Figure 6A]Figures 6A-6J show GFP ablation using branched Cas9 mRNA. Figure 6A shows a schematic of the GFP ablation experiment. Branched / linear Cas9 mRNA was co-transfected with sgRNA targeting the GFP CDS. Figure 6B shows representative images acquired under the same confocal imaging settings. Hoechst (blue), nuclei; GFP (green). Figure 6C shows a violin plot of GFP % intensity normalized to the control in HEK293-uGFP cells treated with 100 ng of branched / linear Cas9 mRNA after 72 hours. Figure 6D shows a bar plot of single-cell GFP intensity 72 hours after transfection. The average GFP intensity in individual cells was measured and normalized to the average GFP intensity in untreated cells. Mean ± sem. P values ​​were calculated by ordinary one-way ANOVA (alpha = 0.05) (Tukey's multiple comparison test) with multiple comparisons between groups. ****P < 0.0001. nsP > 0.1234. Figure 6E shows representative images acquired under the same confocal imaging settings 72 hours after transfection. For each condition, the same amount of sgRNA was transfected with varying doses of Cas9 mRNA. Hoechst (blue), nuclei; GFP (green). Figure 6F shows a schematic diagram of the in vivo expression of branched / linear NanoLuc mRNA. mRNA was formulated in LNPs, and equimolar branched / linear mRNA-LNP complexes were administered via retroorbital injection. Mice were imaged at the indicated time points, and serum was collected 15 days after injection. Figure 6G shows in vivo luminescence images of mice treated with polyC / linear / branched mRNA-LNPs at 24, 72, 144, and 240 hours after LNP administration. Figures 6H–6I show the in vivo luminescence signals of linear / branched NanoLuc mRNA. Luminescence was measured by integrating the total flux for each mouse, normalized by subtracting the background (polyC-treated mice). Mean values ​​± s.e.m. n=3 male mice for each condition. Figure 6J shows serum levels of immunogenicity and hepatotoxicity biomarkers. Serum concentrations of TNF-α, AST, and ALT were quantified by ELISA. Mean ± sem. n=3 mice for each biological condition measured in duplicate.P values ​​were calculated by one-way ANOVA (alpha = 0.05) (Tukey's multiple comparison test) with multiple comparisons between groups. **P<0.004, ****P<0.0001, nsP>0.1234. [Figure 6B] See legend to Figure 6A. [Figure 6C] See legend to Figure 6A. [Figure 6D] See legend to Figure 6A. [Figure 6E] See legend to Figure 6A. [Figure 6F] See legend to Figure 6A. [Figure 6G] See legend to Figure 6A. [Figure 6H] See legend to Figure 6A. [Figure 6I] See legend to Figure 6A. [Figure 6J] See legend to Figure 6A. [Figure 7A] Figures 7A-7E show conceptualizations of chimeric mRNAs with multiple 5' caps. Figure 7A shows the chemical structure of the eukaryotic mRNA 5' cap. Figure 7B shows a pseudo-closed-loop model of cap-dependent mRNA translation initiation. The 5' cap is recognized by eIF4E, leading to the formation of a translation initiation complex and ribosome recruitment. Figure 7C shows mocRNAs bearing chemical conjugation handles ligated to capped oligonucleotides via click chemistry. Multiple m7G caps result in increased binding to eIF4E and higher ribosome loading of mRNA transcripts. Figure 7D shows type 1 multi-capped mocRNAs characterized by branching within the 5' UTR via chemical conjugation to capped oligonucleotides. Figure 7E shows type 2 multi-capped mocRNAs characterized by ligation to multivalent small molecule handles conjugated to multiple capped oligonucleotides. [Figure 7B] See the legend to Figure 7A. [Figure 7C]See the legend to Figure 7A. [Figure 7D] See the legend to Figure 7A. [Figure 7E] See the legend to Figure 7A. [Figure 8A] Figures 8A-8D show the general synthesis workflow for type 1 multi-capped mRNA. Figure 8A shows that chemically synthesized mRNA transcripts and oligos bearing click chemistry handles were chemically capped with m7G-imidazolide, HPLC-purified, and finally crosslinked by the corresponding click reaction. Figure 8B shows that chemically synthesized mRNA transcripts and oligos bearing click chemistry handles were crosslinked by the click reaction and HPLC-purified. The conjugation product was then capped using m7G-imidazolide to incorporate multiple caps. Figure 8C shows that chemically synthesized oligos bearing click chemistry handles were crosslinked and HPLC-purified to generate branched oligos, which were then capped with m7G-imidazolide to incorporate multiple caps. The multi-capped oligos were ligated to 5'-monophosphorylated mRNA transcripts generated by IVT and RppH hydrolysis. FIG. 8D shows an alternative workflow to FIG. 8C, where the oligos were first chemically capped and then cross-linked. [Figure 8B] See the legend to Figure 8A. [Figure 8C] See the legend to Figure 8A. [Figure 8D-1] See the legend to Figure 8A. [Figure 8D-2] See the legend to Figure 8A. [Figure 9A]Figures 9A-9D show proof-of-concept experiments using type 1 multi-capped mRNAs encoding HiBit tags. Figure 9A shows synthetic HiBit mRNAs with one EU handle in the 5' UTR were chemically capped and ligated to a 5'-capped, 3'-azide oligonucleotide. The doubly capped HiBit mRNAs were then transfected into HeLa cells and evaluated by luciferase assay. Figure 9B shows the synthesis of the m7G-imidazolide chemical capping reagent. Figure 9C shows representative HPLC traces for purifying uncapped oligos and oligos capped with click chemistry handles. Figure 9D shows agarose gel electrophoresis of single-capped / single-capped and branched / doubly-capped HiBit mRNAs. [Figure 9B] See the legend to Figure 9A. [Figure 9C] See the legend to Figure 9A. [Figure 9D] See the legend to Figure 9A. [Figure 9E] Hibit bioluminescence at 12 hours after mRNA transfection is shown. Mean ± sem. P values ​​were calculated by unpaired t-test with Welch's correction. ****P<0.0001, **P<0.004, nsP>0.1234. DETAILED DESCRIPTION OF THE INVENTION

[0124] Detailed Description Provided herein are modified mRNAs containing an additional polyA tail and / or an additional 5' cap to improve the stability of the modified mRNA in cells, thereby enhancing production of the encoded gene product, such as a protein. Also provided herein are methods for producing the modified mRNAs described herein by adding a polyA region containing multiple polyA tails to the 3' end of the mRNA and / or by adding a 5' cap region containing multiple 5' caps to the 5' end of the mRNA. Various methods for producing poly-tailed and poly-capped mRNAs are contemplated herein. In one method, an oligonucleotide containing a polyA tail or an oligonucleotide containing a 5' cap oligonucleotide is ligated to the 3' or 5' end of an mRNA molecule, respectively. The ligated oligonucleotide is then attached to an additional polyA-tailed or 5'-capped oligonucleotide (e.g., via click chemistry with the modified oligonucleotide), depending on whether it is present at the 3' or 5' end of the modified mRNA (Figures 1B and 1D). In an alternative method, dendrimers attached to multiple oligonucleotides containing either a poly-A tail or a 5' cap (e.g., via click chemistry with modified oligonucleotides) are ligated to the 3' or 5' end of an mRNA molecule, respectively (Figures 1C and 1E). Additionally, the present disclosure provides compositions, including pharmaceutical compositions, comprising one or more of the modified mRNAs provided herein. Kits containing reagents for producing modified mRNAs are also described herein. Further provided herein are methods for administering one or more of the described modified mRNAs to a cell or a subject, as well as kits for use in administering any one of the pharmaceutical compositions provided herein to a subject.

[0125] Conventional mRNA contains a poly(A) tail with multiple adenosine nucleotides at the 3' end and a 5' cap at the 5' end. While each of these components serves to protect the mRNA and recruit factors involved in protein translation, the mRNA is subject to degradation by exonucleases. Once exonucleases remove the poly(A) tail and / or 5' cap and begin to remove nucleotides from the open reading frame, the mRNA cannot be translated into the encoded protein. One of the major determinants of mRNA stability within a cell is the time required to degrade the ends of the mRNA. mRNAs that are more resistant to 3' and 5' exonuclease activity are degraded more slowly. The modified mRNAs of the present disclosure typically have a longer half-life in cells and are therefore more stable. Due to their increased stability (i.e., resistance to exonuclease activity), the modified mRNAs of the present disclosure have higher translation efficiency (i.e., the amount of gene product, e.g., protein, translated from the mRNA per second) than their unmodified counterparts. Modified mRNAs may further contain one or more structural changes in the nucleobase, sugar, and / or phosphate linkages of the mRNA that also interfere with exonuclease activity. Furthermore, the additional poly(A) tail and / or 5' cap added to the mRNA described herein may also enhance translation efficiency by introducing more sites into the mRNA for binding by factors involved in eukaryotic protein translation. These effects may be combined by modifying the mRNA molecule to include an additional poly(A) tail and an additional 5' cap. Modified mRNAs with increased stability within a cell, and therefore the ability to produce more of the encoded protein from a given RNA molecule, are useful for a variety of applications, including, for example, use in the treatment and prevention of disease, such as vaccines and other RNA-based therapies, such as the delivery of mRNA encoding essential enzymes, clotting factors, transcription factors, growth factors, cytokines, chemokines, antibodies, protein hormones, signaling proteins, structural proteins, or cell surface receptors to cells of interest.

[0126] definition "Messenger RNA" ("mRNA"), as used herein, refers to a nucleic acid that includes an open reading frame that encodes a gene product, such as a protein, and a polyA region that is 3' to the open reading frame. An mRNA may also include a 5' untranslated region (5'UTR) that is 5' (upstream) to the open reading frame, and a 3' untranslated region that is 3' (downstream) to the open reading frame. An mRNA may also include a 5' cap at the 5' end of the mRNA.

[0127] As used herein, a "protein-encoding open reading frame" refers to a nucleic acid sequence comprising a coding sequence that results in the production of a protein when the open reading frame is translated. The nucleic acid sequence may be an RNA sequence, in which case translation of the RNA sequence produces a polypeptide having the amino acid sequence of the protein. The nucleic acid sequence may be a DNA sequence, in which case a protein is produced when RNA polymerase uses the DNA sequence to transcribe an RNA molecule comprising an RNA sequence that is complementary to the DNA sequence, and translation of the RNA sequence produces a polypeptide having the amino acid sequence of the protein. An open reading frame typically begins with an initiation codon such as AUG in the RNA sequence (ATG in the DNA sequence) and ends with a termination codon such as UAG, UAA, or UGA in the RNA sequence (TAG, TAA, or TGA in the DNA sequence), where the number of bases between the initiation codon G and the termination codon T or U is a multiple of three (e.g., 3, 6, 9).

[0128] RNA molecules that can be translated are called messenger RNAs, or mRNAs. DNA or RNA sequences encode genes through codons. A codon refers to a group of three nucleotides in a nucleic acid sequence, such as DNA or RNA. An anticodon refers to a group of three nucleotides in a nucleic acid, such as a transfer RNA (tRNA), that is complementary to a codon, such that a codon in a first nucleic acid associates with an anticodon in a second nucleic acid through hydrogen bonding between the bases of the codon and the anticodon. For example, a codon 5'-AUG-3' on an mRNA has a corresponding anticodon 3'-UAC-5' on a tRNA. During translation, a tRNA with an anticodon complementary to the codon to be translated generally associates with a codon on an mRNA to deliver the amino acid corresponding to the codon to be translated or to facilitate the termination of translation and release of the translated polypeptide from the ribosome.

[0129] Translation is the process by which an RNA coding sequence is used to direct the production of a polypeptide. The first step in translation is initiation, in which a ribosome associates with an mRNA, and a first transfer RNA (tRNA) carrying a first amino acid associates with the first codon, or initiation codon. The next stage of translation, elongation, involves three steps. First, a second tRNA, carrying an anticodon complementary to the initiation codon or the second codon and carrying a second amino acid, associates with the mRNA. Second, the carbon atom of the non-side chain carboxylic acid moiety at the end of the first amino acid reacts with the nitrogen of the non-side chain amino acid moiety at the end of the second amino acid, forming a peptide bond between the two amino acids. The second amino acid binds to the second tRNA, and the first amino acid binds to the second amino acid, but the first amino acid does not bind to the first tRNA. Third, the first tRNA dissociates from the mRNA, and the ribosome advances along the mRNA, leaving the position of the first tRNA associated with the ribosome occupied by the second tRNA, and the position previously occupied by the second tRNA, free for an additional tRNA carrying an additional amino acid to associate with the mRNA. These three steps—1) association of tRNA carrying an amino acid, 2) peptide bond formation that adds the additional amino acid to the growing polypeptide, and 3) progression of the ribosome along the mRNA—continue until the ribosome reaches a stop codon, resulting in the termination of translation. Generally, the tRNA associated with the stop codon does not carry an amino acid, and therefore, association of a tRNA that does not carry an amino acid during the elongation step results in cleavage of the bond between the polypeptide and the tRNA carrying the final amino acid in the polypeptide, thereby releasing the polypeptide from the ribosome. Alternatively, if a tRNA does not associate with the stop codon, the ribosome may dissociate from the mRNA and release the polypeptide.

[0130] "Nucleic acid," or "polynucleotide," as used herein, refers to an organic molecule comprising two or more covalently linked nucleotides. "Nucleotide," as used herein, refers to an organic molecule comprising: 1) a nucleoside comprising a sugar covalently linked to a nitrogenous base (nucleobase); and 2) a phosphate group covalently linked to the sugar of the nucleoside. The nucleotides in a polynucleotide are typically linked by phosphodiester bonds, with the 3' carbon of the sugar of a first nucleotide linked to the 5' carbon of the sugar of a second nucleic acid by a bridging phosphate group. Typically, the bridging phosphate contains two non-bridging oxygen atoms bonded only to the phosphorus atom of the phosphate and two bridging oxygen atoms, each connecting the phosphorus atom to either the 3' carbon of the first nucleotide or the 5' carbon of the second nucleotide. In a nucleic acid sequence describing the order of nucleotides within a nucleic acid, a first nucleotide is said to be 5' (upstream) relative to a second nucleotide if the 3' carbon of the first nucleotide is connected to the 5' carbon of the second nucleotide. Similarly, if the 5' carbon of a second nucleotide is connected to the 3' carbon of a first nucleotide, the second nucleotide is said to be 3' (downstream) to the first nucleotide. Nucleic acid sequences are typically read in 5'→3' order, beginning with the 5' nucleotide and ending with the 3' nucleotide.

[0131] As used herein, a "modified nucleotide" refers to a nucleotide having a structure that is not the standard structure of an adenosine, cytidine, guanine, or uracil nucleotide. The standard structure of a molecule refers to the structure generally known in the art to be the structure referred to by the name of the molecule. The standard structure of an adenosine nucleotide, containing an adenosine base, a ribose sugar, and one or more phosphate groups, is shown below in the form of adenosine monophosphate. TIFF2026016516000046.tif29128

[0132] The canonical structure of AMP also refers to a structure in which one or more hydroxyl groups of the phosphate and / or one or more hydroxyl groups of the sugar are deprotonated, and in which the oxygen atom of the phosphate and / or the 3' oxygen atom of the sugar are attached to adjacent nucleotides in a nucleic acid sequence.

[0133] The canonical structure of a cytosine nucleotide, containing a cytosine base, a ribose sugar, and one or more phosphate groups, is shown below in the form of cytidine monophosphate. TIFF2026016516000047.tif30128CMP canonical structure also refers to a structure in which one or more hydroxyl groups of the phosphate and / or one or more hydroxyl groups of the sugar are deprotonated, and in which the oxygen atom of the phosphate and / or the 3' oxygen atom of the sugar are bound to adjacent nucleotides in a nucleic acid sequence.

[0134] The canonical structure of a guanine nucleotide, containing a guanine base, a ribose sugar, and one or more phosphate groups, is shown below in the form of guanosine monophosphate. TIFF2026016516000048.tif30128GMP canonical structure also refers to a structure in which one or more hydroxyl groups of the phosphate and / or one or more hydroxyl groups of the sugar are deprotonated, and in which the oxygen atom of the phosphate and / or the 3' oxygen atom of the sugar are bound to adjacent nucleotides in a nucleic acid sequence.

[0135] The standard structure of a uracil nucleotide, containing a uracil base, a ribose sugar, and one or more phosphate groups, is shown below in the form of uridine monophosphate. The canonical structure of TIFF2026016516000049.tif27128UMP also refers to a structure in which one or more hydroxyl groups of the phosphate and / or one or more hydroxyl groups of the sugar are deprotonated, and in which the oxygen atom of the phosphate and / or the 3' oxygen atom of the sugar are bound to adjacent nucleotides in a nucleic acid sequence.

[0136] The structure of a modified nucleotide can differ from the structure of a standard nucleotide by one or more modifications in the sugar, nitrogenous base, or phosphate of the nucleotide. In some embodiments, a modified nucleotide includes a modified nucleoside that is not of the standard structure of an adenine nucleoside, a cytosine nucleoside, a guanine nucleoside, or a uracil nucleoside. As used herein,

[0137] An example of the canonical structure of adenosine, the adenine nucleoside, is reproduced below. TIFF2026016516000050.tif30128 The canonical structure of adenosine also refers to a structure in which one or more hydroxyl groups of the phosphate and / or one or more hydroxyl groups of the sugar are deprotonated, the 5' carbon is attached to the 5' phosphate in a nucleic acid sequence, and the 3' oxygen atom is attached to the 5' phosphate group of an adjacent nucleotide in a nucleic acid sequence.

[0138] An example of the canonical structure of cytidine, the cytosine nucleoside, is reproduced below. TIFF2026016516000051.tif29128 The canonical structure of cytidine also refers to a structure in which one or more hydroxyl groups on the phosphate and / or one or more hydroxyl groups on the sugar are deprotonated, the 5' carbon is attached to the 5' phosphate in a nucleic acid sequence, and the 3' oxygen atom is attached to the 5' phosphate group of an adjacent nucleotide in a nucleic acid sequence.

[0139] An example of the canonical structure of guanosine, guanine nucleoside, is reproduced below. TIFF2026016516000052.tif30128 The canonical structure of guanosine also refers to a structure in which one or more hydroxyl groups of the phosphate and / or one or more hydroxyl groups of the sugar are deprotonated, the 5' carbon is attached to the 5' phosphate in a nucleic acid sequence, and the 3' oxygen atom is attached to the 5' phosphate group of an adjacent nucleotide in a nucleic acid sequence.

[0140] An example of the canonical structure of uridine, uracil nucleoside, is reproduced below. TIFF2026016516000053.tif30128 The canonical structure of uridine also refers to a structure in which one or more hydroxyl groups of the phosphate and / or one or more hydroxyl groups of the sugar are deprotonated, the 5' carbon is attached to the 5' phosphate in a nucleic acid sequence, and the 3' oxygen atom is attached to the 5' phosphate group of an adjacent nucleotide in a nucleic acid sequence.

[0141] "Ligase," as used herein, refers to an enzyme capable of forming a covalent bond between two nucleotides, and the process of "ligation" refers to the formation of a covalent bond between two nucleotides.

[0142] As used herein, a "tailing nucleic acid" refers to a nucleic acid that is ligated to the 3' end of another nucleic acid. The tailing nucleic acid may comprise a polyA tail.

[0143] "Capping nucleic acid," as used herein, refers to a nucleic acid that is ligated to the 5' end of another nucleic acid. The capping nucleic acid may include a 5' cap.

[0144] As used herein, a "poly-A tail" refers to a nucleic acid sequence containing adenosine nucleotides attached to the 3' end of a nucleic acid, such as RNA. A "poly-A region" refers to a nucleic acid containing one or more poly-A tails. A poly-A tail or poly-A region can be composed of nucleotides that are 25-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, 90-100%, 95-100%, 96-100%, 97-100%, 98-100%, or 99-100% adenosine nucleotides. The adenosine nucleotides contained by the poly-A tail or poly-A region can be standard adenosine nucleotides or modified (non-standard) adenosine nucleotides.

[0145] As used herein, a "5' cap" refers to one or more nucleotides covalently attached to the 5' end of a nucleic acid, such as an RNA. A "5' cap region" refers to a nucleic acid comprising one or more 5' caps. A 5' cap can include a 5' capping nucleotide attached to the 5' end of an mRNA 5' to a 5' triphosphate internucleotide linkage. In some embodiments, a nucleotide attached to an mRNA 5' to a 5' triphosphate internucleotide linkage is referred to as a "natural" 5' capping nucleotide. In some embodiments, a natural 5' capping nucleotide is a 7-methylguanosine (m7G) nucleotide. In some embodiments, a 5' cap is a modified 5' cap that includes one or more modified nucleotides, such as a 5' capping nucleotide, or one or more modified internucleotide modifications, such as a modification 5' to a 5' triphosphate internucleotide linkage. In some embodiments, a 5' cap includes one or more nucleotides with a sugar modification, such as a 2'-O-methylation.

[0146] An example of the canonical structure of 7-methylguanosine attached to a ribonucleic acid sequence (eg, mRNA) at a 5' to 5' triphosphate internucleotide linkage is reproduced below. TIFF2026016516000054.tif37128

[0147] "Brush oligonucleotide," as used herein, refers to a nucleic acid sequence that includes an internal covalent linkage to either the 3' or 5' end of one or more second nucleic acid sequences.

[0148] "Dendrimer," as used herein, refers to an organic polymeric compound comprising a core structure to which multiple terminal groups, called "dendrons," are covalently attached. The dendrons of a dendrimer can be modified (e.g., via click chemistry) for conjugation with oligonucleotides.

[0149] modified mRNA In some aspects, the present disclosure provides modified mRNAs comprising i) two or more poly-A tails, and / or ii) two or more 5' caps, wherein each poly-A tail is encompassed by a poly-A region at the 3' end of the mRNA and each 5' cap is encompassed by a 5' cap region at the 5' end of the mRNA. In some embodiments, the modified mRNAs provided herein comprise a poly-A tail and two or more 5' caps. In some embodiments, the modified mRNAs provided herein comprise two or more poly-A tails and a 5' cap. In some embodiments, the modified mRNAs provided herein comprise two or more poly-A tails and two or more 5' caps.

[0150] As defined herein, the "polyA region" of an mRNA, also referred to as the "poly(A) region," refers to the region of an mRNA that is 3' (downstream) of the open reading frame (ORF) and 3' untranslated region (UTR) and contains multiple consecutive adenosine nucleotides (i.e., any adenosine nucleotide covalently linked to at least one other adenosine nucleotide). A polyA region typically contains 50 to 300 consecutive adenosine nucleotides and may include multiple non-adenosine nucleotides upstream, downstream, or interspersed between the consecutive adenosine nucleotides. The "5' cap region" of an mRNA refers to the region of an mRNA that is 5' (upstream) of the ORF and 5' UTR. In cells, after transcription of a DNA sequence to produce precursor messenger RNA (pre-mRNA), a poly(A) tail is added by a polyadenylation enzyme such as poly(A) polymerase (PAP), resulting in a long sequence of multiple consecutive adenosine nucleotides at the 3' end of the RNA, while a 5' cap is added by a 5' capping enzyme such as mRNA guanylyltransferase. The poly(A) tract and the 5' cap each play multiple important roles in the production of the protein encoded by the mRNA. First, the poly(A) tract provides a binding site for poly(A) binding protein (PABP), which associates with the mRNA in the nucleus and promotes its export to the cytoplasm (see, e.g., Tudek et al. Philos Trans R Soc Lond B Biol Sci. 2018.373(1762):20180169). Second, the presence of a polyA tail and 5' cap on an mRNA facilitates translation initiation (see, e.g., Gallie. Genes & Dev. 1991. 5:2108-2116, and Munroe et al. Mol Cell Biol. 1990. 10(7):3441-3455). Finally, the polyA tail and 5' cap stabilize the mRNA by protecting the ORF from the activity of exonucleases such as polynucleotide phosphorylase (PNPase), which can remove 3' and 5' nucleotides from the mRNA.As exonucleases remove nucleotides, mRNAs become progressively shorter. Once all downstream nucleotides of the open reading frame have been removed, the nucleotides removed by exonucleases will be those of the ORF. Removal of nucleotides from the ORF prevents translation of the encoded protein. In addition, association of exonucleases with mRNAs near the ORF can inhibit translation by sterically hindering ribosomes and tRNAs from associating with the mRNA. In particular, removal of the poly(A) tail is often referred to as the rate-limiting step in mRNA degradation, and the lifespan of mRNAs in cells is determined by the time required to remove the poly(A) tail (see, e.g., Dreyfus et al., Cell. 2002, 111(5):611-613). The composition of the poly(A) tail of mRNAs varies, but it contains approximately 75 adenosine nucleotides in yeast cells and 250 adenosine nucleotides in mammalian cells. The composition of the 5' cap in an mRNA is less variable and typically comprises a 5' 7-methylguanosine attached to the mRNA 5' to the 5' triphosphate internucleotide linkage, although certain natural modifications are known to occur naturally at the 5' capped nucleotide, such as 2'-O-methylation at the first nucleotide of an mRNA (Cap 1) and 2'-O-methylation at the first and second nucleotides of an mRNA (Cap 2). A 5' cap without 2'-O-methylation is known as a "Cap 0" cap.

[0151] In some embodiments of the modified mRNAs provided herein, the modified mRNA comprises a polyA region and / or a 5' cap region that does not contain modified nucleotides (i.e., every single nucleotide in the polyA region and / or the 5' cap region is a standard nucleotide). In some embodiments, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, 90% to 95%, 95% to 99%, or 100% of the nucleotides in the polyA region or the 5' cap region are standard nucleotides. In some embodiments, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, 90% to 95%, 95% to 99%, or 100% of the adenosine nucleotides in the polyA region are standard adenosine nucleotides.

[0152] In some embodiments of the modified mRNA provided herein, the modified mRNA comprises one or more modified nucleotides in the polyA region and / or 5' cap region of the mRNA. In some embodiments, the polyA region and / or 5' cap region comprises one or more nucleotides that are not standard adenosine, cytidine, guanosine, or uridine nucleotides. In some embodiments, the polyA region comprises one or more nucleotides that are 3' (downstream) to a nucleic acid sequence that comprises multiple consecutive adenosine nucleotides. In some embodiments, the polyA region comprises 10-25 consecutive adenosine nucleotides, which may be standard or modified adenosine nucleotides. In some embodiments, the polyA region comprises 10-15 consecutive adenosine nucleotides, 15-20 consecutive adenosine nucleotides, or 20-25 consecutive adenosine nucleotides, which may be standard or modified adenosine nucleotides. In some embodiments, the polyA region comprises at least 25 consecutive adenosine nucleotides, which can be standard or modified adenosine nucleotides. In some embodiments, the polyA region comprises 25 to 500 consecutive adenosine nucleotides, which can be standard or modified adenosine nucleotides. In some embodiments, the polyA region comprises 25 to 300 consecutive adenosine nucleotides. In some embodiments, the polyA region comprises at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, or at least 200 consecutive adenosine nucleotides. In some embodiments, the 5' cap region comprises 1 to 3, 3 to 5, 5 to 7, or 7 to 10 5' caps. In some embodiments, the polyA region and / or the 5' cap region comprises between 10 and 500 nucleotides.In some embodiments, the polyA region and / or the 5' cap region comprises 10-15, 15-20, 20-25, 25-50, 50-100, 100-150, 150-200, 200-300, 300-400, or 400-500 nucleotides.

[0153] In some embodiments, every single nucleotide of a modified mRNA contains a standard phosphate group. In some embodiments, a modified mRNA contains one or more nucleotides containing a modified phosphate group. The modified phosphate group connecting two nucleotides of a modified mRNA is referred to as a "modified internucleotide linkage" or "modified internucleoside linkage." A modified phosphate group is a phosphate group that differs from the standard structure of phosphate (phosphodiester). An example of a standard structure of phosphate (phosphodiester) is shown below: TIFF2026016516000055.tif23128In the formula, R5 and R3 are atoms or molecules to which the standard phosphate is attached. For example, for a phosphate in a nucleic acid sequence, R5 can refer to the upstream nucleotide of the nucleic acid, and R3 can refer to the downstream nucleotide of the nucleic acid. The standard structure of the phosphate also refers to a structure in which one or more hydroxyl groups of the phosphate are deprotonated, or a structure in which the oxygen atom of the phosphate is attached to an adjacent nucleotide in the nucleic acid sequence. Non-limiting examples of modified phosphate groups that can replace standard phosphate in nucleic acids include phosphorothioate (PS, R and / or S stereoisomers), thiophosphate, 5'-O-methylphosphonate, 3'-O-methylphosphonate, 5'-hydroxyphosphonate, hydroxyphosphate, phosphoroselenoate, selenophosphate, phosphoramidate, carbophonate, methylphosphonate, phenylphonate, ethylphosphonate, H-phosphonate, guanidinium ring, triazole ring, boranophosphate (BP), methylphosphonate, guanidinopropyl phosphoramidate, peptide bond (e.g., in peptide nucleic acids), thiophosphoamidate.

[0154] In some embodiments of the modified mRNAs comprising modified nucleotides provided herein, at least one modified nucleotide comprises a modified nucleobase. In some embodiments, at least one modified nucleotide comprises a modified sugar. In some embodiments, at least one modified nucleotide comprises a modified phosphate. In some embodiments, at least one modified nucleobase is selected from the group consisting of xanthine, aliaminouracil, aliaminothymidine, hypoxanthine, digoxigenylated adenine, digoxigenylated cytosine, digoxigenylated guanine, digoxigenylated uracil, 6-chloropurine riboside, N6-methyladenine, methylpseudouracil, 2-thiocytosine, 2-thiouracil, 5-methyluracil, 4-thiothymidine, 4-thiouracil, 5,6-dihydro-5-methyl Uracil, 5,6-dihydrouracil, 5-[(3-indolyl)propionamido-N-allyl]uracil, 5-aminoallylcytosine, 5-aminoallyluracil, 5-bromouracil, 5-bromocytosine, 5-carboxycytosine, 5-carboxymethylesteruracil, 5-carboxyuracil, 5-fluorouracil, 5-formylcytosine, 5-formyluracil, 5-hydroxycytosine, 5-hydroxymethylcytosine, 5-hydroxymethyluracil Cytosine, 5-hydroxyuracil, 5-iodocytosine, 5-iodouracil, 5-methoxycytosine, 5-methoxyuracil, 5-methylcytosine, 5-methyluracil, 5-propargylaminocytosine, 5-propargylaminouracil, 5-propynylcytosine, 5-propynyluracil, 6-azacytosine, 6-azauracil, 6-chloropurine, 6-thioguanine, 7-deazaadenine, 7-deazaguanine, 7-deaza-7-propargylaminoadenine, 7-deaza-7-propargylaminoguanine, 8-azaadenine, 8-azidoadenine, 8-chloroadenine, 8-oxoadenine, 8-oxoguanine, araadenine, aracytosine, araguanine, arauracil, biotin-16-7-deaza-7-propargylaminoguanine, biotin-16-aminoallylcytosine, biotin-16-aminoallyluracil, cyanine 3-5-propargylaminocytosine, cyanine 3-6-propargylaminouracil,Cyanine 3-aminoallylcytosine, cyanine 3-aminoallyluracil, cyanine 5-6-propargylaminocytosine, cyanine 5-6-propargylaminouracil, cyanine 5-aminoallylcytosine, cyanine 5-aminoallyluracil, cyanine 7-aminoallyluracil, dabsyl-5-3-aminoallyluracil, desthiobiotin-16-aminoallyl-uracil, desthiobiotin-6-aminoallylcytosine, isoguanine, N1-ethylpseudododecanoate uracil, N1-methoxymethylpseudouracil, N1-methyladenine, N1-methylpseudouracil, N1-propylpseudouracil, N2-methylguanine, N4-biotin-OBEA-cytosine, N4-methylcytosine, N6-methyladenine, O6-methylguanine, pseudoisocytosine, pseudouracil, thienocytosine, thienoguanine, thienouracil, xanthosine, 3-deazaadenine, 2,6-diaminoadenine, 2,6-daminoguanine, 5-Carboxamidouracil, 5-ethynyluracil, N6-isopentenyladenine (i6A), 2-methyl-thio-N6-isopentenyladenine (ms2i6A), 2-methylthio-N6-methyladenine (ms2m6A), N6-(cis-hydroxyisopentenyl)adenine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenine (ms2io6A), N6-glycinylcarbamoyladenine (g6A), N6-threonylcarbamoyl In some embodiments, the at least one modified nucleotide comprises a modified nucleobase selected from the group consisting of 2'-thioribose, 2',3'-dideoxyribose, 2'-amino-2'-deoxyribose, 2'-deoxyribose, 2'-azido-2'-deoxyribose, 2'-fluoro-2'-deoxyribose, 2'-O-methyl ...The modified sugar may be selected from the group consisting of 2'-O-methyldeoxyribose, 3'-amino-2',3'-dideoxyribose, 3'-azido-2',3'-dideoxyribose, 3'-deoxyribose, 3'-O-(2-nitrobenzyl)-2'-deoxyribose, 3'-O-methylribose, 5'-aminoribose, 5'-thioribose, 5-nitro-1-indolyl-2'-deoxyribose, 5'-biotin-ribose, 2'-O,4'-C-methylene-linked, 2'-O,4'-C-amino-linked, and 2'-O,4'-C-thio-linked ribose. In some embodiments, at least one modified nucleotide comprises a 2'-modification, such as a locked nucleic acid (LNA) modification (i.e., a nucleotide containing an additional carbon atom attached to the 2' oxygen and 4' carbon of ribose) or 2'-O-methylation. In some embodiments, at least one modified nucleotide comprises a modified phosphate selected from the group consisting of phosphorothioate (PS), thiophosphate, 5'-O-methylphosphonate, 3'-O-methylphosphonate, 5'-hydroxyphosphonate, hydroxyphosphate, phosphoroselenoate, selenophosphate, phosphoramidate, carbophosphonate, methylphosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidinium ring, triazole ring, boranophosphate (BP), methylphosphonate, and guanidinopropylphosphoramidate.

[0155] In some embodiments, the modified mRNA comprises more than one type of modified nucleotide. In some embodiments, the modified mRNA comprises at least a first modified nucleotide and a second modified nucleotide having a structure different from the first modified nucleotide. The nucleotides may differ in structure due to differences in the nucleobase, sugar, and / or phosphate group. In some embodiments, the modified mRNA comprises at least a first modified phosphate and a second modified phosphate having a structure different from the first modified phosphate. In some embodiments, the modified mRNA comprises a first modified nucleoside and a second modified nucleoside.

[0156] Aspects of the present disclosure relate to modified mRNAs comprising a polyA region having 10 or more adenosine nucleotides. An adenosine nucleotide is a nucleotide comprising an adenine nucleoside and a phosphate group. An adenosine nucleoside comprises a sugar and an adenine base. In some embodiments, the polyA region comprises 10 or more standard adenosine nucleotides, e.g., 10-15 standard adenosine nucleotides, 15-20 standard adenosine nucleotides, or 20-25 standard adenosine nucleotides. In some embodiments, the polyA region comprises 25 or more standard adenosine nucleotides, e.g., 25-500 standard adenosine nucleotides. A standard adenosine nucleotide comprises an adenine base, a ribose sugar, and a phosphate group arranged in the structure of adenosine monophosphate (AMP). In some embodiments, one or more of the phosphate hydroxyl group and / or the 3' hydroxyl group of the ribose are deprotonated and contain an oxygen ion in place of the -OH group, resulting in the structure: When present in a nucleic acid sequence of an mRNA, the standard adenosine has the following structure: TIFF2026016516000057.tif28128, and is connected to adjacent nucleotides in the following manner: R5 is the adjacent nucleotide that is 5' (upstream) relative to the adenosine nucleotide in the mRNA, and R3 is the adjacent nucleotide that is 3' (downstream) relative to the adenosine nucleotide in the mRNA. In some embodiments, the standard adenosine nucleotide is the 3'-terminal nucleotide (last nucleotide) of the linear mRNA, R3 is hydrogen, and the 3'-terminal nucleotide contains a 3'-terminal hydroxyl (-OH) group. In some embodiments, the standard adenosine nucleotide is the 3'-terminal nucleotide (last nucleotide) of the linear mRNA, and R3 is electron.

[0157] Aspects of the present disclosure also relate to modified mRNAs comprising a 5' cap region having two or more 5' caps. The 5' cap can comprise a 5' capping nucleotide, which is the 5'-terminal nucleotide (first nucleotide) of a linear mRNA, and is attached to the mRNA via a 5' to 5' triphosphate linkage. In some embodiments, the cap comprises one or more modified nucleotides, such as one or more modified nucleotides comprising a 2' modification, such as a locked nucleic acid (LNA) modification (i.e., a nucleotide comprising an additional carbon atom attached to the 2' oxygen and 4' carbon of ribose) or a 2'-O-methylation. In some embodiments, the 5' cap comprises a 5' capping nucleotide attached to the mRNA via a 5'-5' triphosphate linkage or a non-standard linkage, such as a 5'-5' tetraphosphate, modified by a 5' phosphorothioate. In some embodiments, the 5' capping nucleotide is a standard 5' capping nucleotide, such as the following 7-methylguanosine (m7G) nucleotide: TIFF2026016516000058.tif37128

[0158] In some embodiments of the modified mRNAs provided herein, the mRNA comprises a 5' untranslated region (5'UTR) and a 3' untranslated region (3'UTR). The 5' and 3'UTRs are sequences within the mRNA that do not encode amino acids of the protein encoded by the mRNA and are therefore not part of the open reading frame. The 5'UTR is 5' (upstream) to the open reading frame. The 3'UTR is 3' (downstream) to the open reading frame. In some embodiments, the 3'UTR comprises one or more nucleotides that are 3' to the open reading frame of the mRNA and 5' (upstream) to the polyA region of the mRNA.

[0159] In some embodiments of the mRNA provided herein, the mRNA comprises, in 5' to 3' order, 1) a 5' cap region, 2) a 5' UTR, 3) an open reading frame (ORF), 4) a 3' UTR, and 5) a polyA region. In some embodiments, the first nucleotide of the 5' UTR is 3' (downstream) to the last nucleotide of the 5' cap region, and the last nucleotide of the 5' UTR is 5' (upstream) to the first nucleotide of the open reading frame. In some embodiments, the first nucleotide of the open reading frame is 3' (downstream) to the last nucleotide of the 5' UTR, and the last nucleotide of the open reading frame is 5' (upstream) to the first nucleotide of the 3' UTR. In some embodiments, the open reading frame is between the last nucleotide of the 5' UTR and the first nucleotide of the 3' UTR. In some embodiments, the first nucleotide of the 3' UTR is 3' (downstream) to the last nucleotide of the open reading frame, and the last nucleotide of the 3' UTR is 5' (upstream) to the first nucleotide of the polyA region. In some embodiments, the 5'UTR is between the last nucleotide of the 5' cap region and the first nucleotide of the open reading frame. In some embodiments, the 3'UTR is between the last nucleotide of the open reading frame and the first nucleotide of the polyA region. In some embodiments, the last nucleotide of the 5' cap region is 5' (upstream) to the first nucleotide of the 5'UTR. In some embodiments, the first nucleotide of the polyA region is 3' (downstream) to the last nucleotide of the 3'UTR.

[0160] In some embodiments, the mRNA is a linear mRNA. A linear mRNA is an mRNA having a 5'-terminal nucleotide and a 3'-terminal nucleotide. The 5'-terminal nucleotide of a linear mRNA is covalently linked to only one adjacent nucleotide of the mRNA, where the adjacent nucleotide is located 3' to the 5'-terminal nucleotide in the nucleic acid sequence of the mRNA. The 3'-terminal nucleotide of a linear mRNA is covalently linked to only one adjacent nucleotide of the mRNA, where the adjacent nucleotide is located 5' to the 3'-terminal nucleotide in the nucleic acid sequence of the mRNA. In a nucleic acid sequence that includes all nucleotides of a linear mRNA in 5' to 3' order, the 5'-terminal nucleotide is the first nucleotide of the sequence, and the 3'-terminal nucleotide is the last nucleotide of the sequence.

[0161] In some embodiments of the linear mRNA provided herein, the mRNA comprises a 5' cap. Most mRNAs produced in eukaryotic cells contain a 5' cap that is added during processing of pre-mRNA into mature mRNA. The 5' cap plays multiple roles in the processes of mRNA production, export, and translation. First, assembly of the spliceosome, which mediates the removal of introns from pre-mRNA, requires binding of the nuclear cap-binding complex (CBC) to the 5' cap. Furthermore, interaction between the CBC and nuclear pores mediates mRNA export into the cytoplasm, starting from the 5' end. Finally, the CBC bound to the 5' cap mediates the recruitment of multiple factors required for translation initiation, such as CBP80, CTIF, eIF3g, eIF4III, Met-tRNAi, and ribosomal subunits (see, e.g., Ramanathan et al. Nucleic Acids Res. 2016. 44(16):7511-7526). In some embodiments, the 5' cap comprises a 7-methylguanosine. In some embodiments, the 7-methylguanosine comprises the following structure: TIFF2026016516000059.tif38128

[0162] Cap analogs, also referred to herein as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, differ in their chemical structure from natural (i.e., endogenous, wild-type, or physiological) 5'-caps while retaining cap function. Cap analogs can be synthesized and / or attached to nucleic acid molecules chemically (i.e., non-enzymatically) or enzymatically. For example, the anti-reverse cap analog (ARCA) cap contains two guanines joined by 5'-5'-triphosphate groups, one guanine containing an N7 methyl group as well as a 3'-O-methyl group (i.e., N7,3'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine (m 7 G-3'mppp-G, which may equivalently be termed 3'O-Me-m7G(5')ppp(5')G. The 3'-O atom of the otherwise unmodified guanine is attached to the 5'-terminal nucleotide of the capped nucleic acid molecule (e.g., mRNA or mmRNA). The N7- and 3'-O-methylated guanine provides the terminal portion of the capped nucleic acid molecule (e.g., mRNA or mmRNA).

[0163] Another exemplary cap is mCAP, which is similar to ARCA but has a 2'-O-methyl group on the guanosine (i.e., N7,2'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine, m7Gm-ppp-G). Non-limiting examples of 5' cap structures include 7-benzylguanosine (Bn7G), 7-(4-chlorophenoxyethyl)-guanosine and its analogs (see, e.g., Kore et al. (2013) Bioorganic & Medicinal Chemistry 21(15):4570-4574), 7-ethylguanosine (e7G), 7-propylguanosine (p7G), 7-isopropylguanosine (ip7G), 7-butylguanosine (b7G), 7-isobutylguanosine (ib7G), 7-cyclopentylguanosine (cp7G), 7-(carboxymethyl)guanosine (cm7G), 7-benzylguanosine (bn7G), 7-(2-phenylethyl)guanosine [7-(2-PhEt)G], 7-(1-phenylethyl)guanosine [7-(1-PhEt)G], m 7 Gppp BH3 G (D1 and D2 stereoisomers), m 7 Gpp BH3 G (D1 and D2 stereoisomers), m 7 Gp BH3 G (D1 and D2 stereoisomers), m 7 Gpp BH3 pm 7 G, m2 7,2’-O Gppp BH3 G (D1 and D2 stereoisomers), m27 ,2’-O Gpp BH3 pG (D1 and D2 diastereomers), m2 7,2’-O Gpp SpG (D1 and D2 diastereomers), N-arylmethyl analogs (see, for example, Wojcik et al. (2021) Pharmaceutics 13(11)1941), glyceryl, inverted deoxy abasic residues (moieties), 4',5'-methylene nucleotides, 1-(beta-D-erythrofuranosyl) nucleotides, 4'-thionucleotides, carbocyclic nucleotides, 1,5-anhydrohexitol nucleotides, L-nucleotides, alpha-nucleotides, modified base nucleotides, threo-pentofurosyl nucleotides, acyclic 3',4'-seconucleotides, acyclic 3,4-dihydroxybutyl nucleotides Examples include a nucleotide, an acyclic 3,5-dihydroxypentyl nucleotide, a 3'-3'-inverted nucleotide moiety, a 3'-3'-inverted abasic moiety, a 3'-2'-inverted nucleotide moiety, a 3'-2'-inverted abasic moiety, 1,4-butanediol phosphate, a 3'-phosphoramidate, a hexyl phosphate, an aminohexyl phosphate, a 3'-phosphate, a 3'-phosphorothioate, a phosphorodithioate, or a bridged or non-bridged methylphosphonate moiety. Further modified 5'-cap structures that may be used in the context of the present invention are Cap 1 (additional methylation of the ribose of the nucleotide adjacent to m7GpppN), Cap 2 (additional methylation of the ribose of the second nucleotide downstream of m7GpppN), Cap 3 (additional methylation of the ribose of the third nucleotide downstream of m7GpppN), Cap 4 (additional methylation of the ribose of the fourth nucleotide downstream of m7GpppN), ARCA (anti-reverse cap analog), modified ARCA (e.g., phosphothioate-modified ARCA), inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0164] In some embodiments, the 5' cap comprises one or more phosphates connecting the 7-methylguanosine to adjacent nucleotides of the modified mRNA. In some embodiments, the one or more phosphates of the 5' cap are modified phosphates selected from the group consisting of phosphorothioates, triazole rings, dihalogen methylene bisphosphonates, imidodiphosphates, and methylene bis(phosphonates). In some embodiments, the 7-methylguanosine is connected to adjacent nucleotides of the mRNA by a 5' to 5' triphosphate bridge. In some embodiments, the 5' cap has the structure: TIFF2026016516000060.tif37128, where R is the 5' carbon of the first transcribed nucleotide of the mRNA. In some embodiments, the 5' cap comprises 3'-O-Me-m7G(5')ppp(5')G.

[0165] In some embodiments, the sugar backbone of the mRNA comprises ribose. In some embodiments, the sugar backbone comprises a modification. Non-limiting examples of sugar modifications include 2' modifications on the sugar residue, such as 2'-amino 2'-O-methyl, 2'-O-alkyl, 2'-O-alkyl-O-alkyl, morpholino, or 2'-fluoro modified nucleotides. In some embodiments, the mRNA comprises a locked nucleic acid (LNA). A locked nucleic acid is a nucleotide with a modified ribose moiety that includes an extra bridge connecting the 2' and 4' carbons of the ribose moiety. This structure effectively "locks" the ribose in a 3'-endo structural configuration. In other embodiments, the mRNA backbone comprises deoxyribose. In some embodiments, the mRNA comprises 2'-fluoro-deoxyribose.

[0166] In some embodiments, the mRNA is a circular mRNA. A circular mRNA is an mRNA that does not have a 5'- or 3'-terminal nucleotide. Every nucleotide in a circular mRNA is covalently linked to both 1) the 5'-adjacent nucleotide and 2) the 3'-adjacent nucleotide. In a circular mRNA having a nucleic acid sequence that includes all nucleotides of the circular mRNA in 5'-to-3' order, the last nucleotide of the nucleic acid sequence is covalently linked to the first nucleotide of the nucleic acid sequence. In some embodiments of a circular mRNA having a 5'-cap region, a 5'-UTR, a 3'-UTR, and a polyA region, the polyA region is 3' (downstream) relative to the 3'-UTR and 5' (upstream) relative to the 5'-cap region.

[0167] In some embodiments of the modified mRNAs provided herein, between 1% and 90% of the nucleotides in the polyA region or the 5' cap region are modified nucleotides, hi some embodiments, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 12%, at least 14%, at least 16%, at least 18%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% of the nucleotides in the polyA region or the 5' cap region are modified nucleotides.

[0168] In some embodiments of the modified mRNAs provided herein, three or more of the last 10 nucleotides of the polyA region are modified nucleotides. In some embodiments of the modified mRNAs provided herein, three or more of the last 15 nucleotides of the polyA region are modified nucleotides. In some embodiments of the modified mRNAs provided herein, three or more of the last 20 nucleotides of the polyA region are modified nucleotides. In some embodiments of the modified mRNAs provided herein, three or more of the last 25 nucleotides of the polyA region are modified nucleotides. In some embodiments, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, or 25 of the last 10 to 25 nucleotides of the polyA region are modified nucleotides.

[0169] In some embodiments of the modified mRNAs provided herein, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the nucleotides in the polyA region are adenosine nucleotides. One or more adenosine nucleotides in the polyA region can be a standard adenosine nucleotide or a modified adenosine nucleotide that contains a structure different from a standard adenosine nucleotide. Non-limiting examples of modified adenosine nucleotides include N6-isopentenyladenosine (i6A), 2-methyl-thio-N6-isopentenyladenosine (ms2i6A), 2-methylthio-N6-methyladenosine (ms2m6A), N6-(cis-hydroxyisopentenyl)adenosine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms2io6A), N6-glycinylcarbamoyladenosine (g6A), N6-threonylcarbamoyladenosine (t6A), 2-methylthio-N6-threonylcarbamoyladenosine (ms2t6A), N6-methyl-N6-threonylcarbamoyladenosine (m6t6A), N6-hydroxynorval ... adenosine (hn6A), 2-methylthio-N6-hydroxynorvalylcarbamoyl adenosine (ms2hn6A), 2'-O-ribosyladenosine (phosphate) (Ar(p)), N6,N6-dimethyladenosine (m62A), N6,2'-O-dimethyladenosine (m6Am), N6,N6,O-2'-trimethyladenosine (m62Am), 1,2'-O-dimethyladenosine (m62Am), These include methyladenosine (m1Am), N6-acetyladenosine (ac6A), 2'-thioadenosine (2'SA), 5'-thioadenosine (5'SA), 2'-O-(2-azidoethyl)-adenosine, 2'-azido-adenosine, deoxyadenosine (dA), dideoxyadenosine (ddA), and amino-deoxyadenosine (amino-dA).

[0170] In some embodiments of the modified mRNAs provided herein, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the nucleotides in the polyA region are standard adenosine nucleotides.

[0171] In some embodiments of the modified mRNAs provided herein, the polyA region and / or 5' cap region comprises 10-25 nucleotides. In some embodiments of the modified mRNAs provided herein, the polyA region and / or 5' cap region comprises 10-15 nucleotides, 15-20 nucleotides, or 20-25 nucleotides. In some embodiments of the modified mRNAs provided herein, the polyA region and / or 5' cap region comprises 25-500 nucleotides. In some embodiments, the polyA region and / or 5' cap region comprises at least 25, at least 30, at least 50, at least 100, at least 150, or at least 200 nucleotides. In some embodiments, the polyA region and / or 5' cap region comprises at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290, or at least 300 nucleotides. In some embodiments, the polyA region and / or 5' cap region comprises about 200 to about 300 nucleotides. In some embodiments, the polyA region and / or 5' cap region comprises about 250 nucleotides.

[0172] In some embodiments, the polyA region and / or the 5' cap region comprises at least 3, at least 4, at least 5 phosphorothioates, or at least 6 phosphorothioates. In some embodiments, the polyA region of the mRNA comprises at least 3 phosphorothioates and no 3' terminal hydroxyl. In some embodiments, the polyA region of the mRNA comprises at least 3 guanine nucleotides and at least 3 phosphorothioates and no 3' terminal hydroxyl. In some embodiments, the polyA region of the mRNA comprises at least 3 deoxyribose sugars and no 3' terminal hydroxyl. In some embodiments, the polyA region and / or the 5' cap region comprises at least 20 deoxyribose sugars. In some embodiments, the polyA region of the mRNA comprises at least 20 deoxyribose sugars and no 3' terminal hydroxyl. In some embodiments, the polyA region of the mRNA comprises at least 3 copies of a G-quadruplex sequence and no 3' terminal hydroxyl. In some embodiments, the polyA region of the mRNA comprises at least six phosphorothioates and no 3'-terminal hydroxyl. In some embodiments, the polyA region of the mRNA comprises at least six consecutive phosphorothioates and no 3'-terminal hydroxyl. In some embodiments, the polyA region of the mRNA comprises at least six phosphorothioates and three guanine nucleosides and no 3'-terminal hydroxyl. In some embodiments, the polyA region of the mRNA comprises at least three copies of a G-quadruplex sequence and at least six phosphorothioates and no 3'-terminal hydroxyl. In some embodiments, the polyA region of the mRNA comprises at least three copies of a telomeric repeat sequence and at least six phosphorothioates and no 3'-terminal hydroxyl. In some embodiments, the 3'-terminal nucleotide without a 3'-terminal hydroxyl is dideoxycytidine or inverted deoxythymidine.

[0173] In some embodiments, the polyA region comprises two or more polyA tails, and at least two instances of the polyA tails are covalently attached to a first linker. In some embodiments, the polyA region comprises the following structure: -(first polyA tail)-[(first linker)-(second polyA tail)n1]n2, where each instance of n1 is independently an integer between 1 and 20, inclusive, and n2 is an integer between 2 and 10, inclusive, and the first poly-A tail is covalently attached to the 3'UTR of the mRNA.

[0174] In some embodiments, the polyA region comprises a brush oligonucleotide comprising two or more polyA tails.

[0175] In some embodiments, each instance of n1 is 1. In some embodiments, at least one instance of the first linker is covalently attached to an internal nucleotide of the first poly-A tail. In some embodiments, at least one instance of the first linker is covalently attached to the second poly-A tail at the 3' nucleotide of the second poly-A tail. In some embodiments, at least one instance of the first linker comprises at least one moiety formed by reacting two orthogonal click chemistry handles. Any click chemistry reaction known in the art can be used. Click chemistry is a chemical approach introduced by Sharpless in 2001 and describes chemistry tailored to rapidly and reliably generate substances by linking small units together. See, e.g., Kolb, Finn and Sharpless Angewandte Chemie International Edition (2001) 40:2004-2021; Evans, Australian Journal of Chemistry (2007) 60:384-395). Exemplary click chemistry reactions include the formation of esters, thioesters, amides (e.g., peptide coupling) from activated acids or acyl halides, nucleophilic substitution reactions (e.g., nucleophilic displacement of halides or ring opening of strained ring systems), azide-alkyne Huisgen cycloadditions, thiol-yne additions, imine formation, Michael additions (e.g., maleimide additions), and Diels-Alder reactions (e.g., tetrazine [4+2] cycloadditions).Examples of click chemistry reactions are described in, for example, Kolb, HC; Finn, MG and Sharpless, KB Angew. Chem. Int. Ed. 2001, 40, 2004-2021; Kolb, HC and Shrapless, KB Drug Disc. Today, 2003, 8, 112-1137; Rostovtsev, VV; Green, LG; Fokin, VV and Shrapless, KB Angew. Chem. Int. Ed. 2002, 41, 2596-2599; Tomoe, CW; Christensen, C. and Meldal, MJ Org. Chem. 2002, 67, 3057-3064; Wang, Q. et al. J. Am. Chem. Soc. 2003, 125, 3192-3193; Lee, LV et al. J. Am. Chem. Soc. 2003 125, 9588-9589; Lewis, W. Get al. Angew. Chem. Int. Ed. 2002, 41, 1053-41057; Manetsch, R. et al., J. Am. Chem. Soc. 2004, 126, 12809-12818; Mocharla, V. P. et al. Angew. Chem., Int. Ed. 2005, 44, 116-120, each of which is incorporated by reference in its entirety. In certain embodiments, each instance of a click chemistry handle is a monovalent moiety. In certain embodiments, two orthogonal click chemistry handles (a first click chemistry handle and a second click chemistry handle) react via a click chemistry reaction to form a bivalent moiety. In certain embodiments, at least one instance of a first click chemistry handle comprises C≡C or C═C. In certain embodiments, at least one instance of the first click chemistry handle comprises C≡CH, C═CH, CH═CH, C═CH 2 , or CH═CH 2 .In certain embodiments, at least one instance of the first click chemistry handle is -C≡CH, substituted or unsubstituted cyclooctynyl optionally and independently fused to one or more instances of substituted or unsubstituted phenyl, substituted or unsubstituted cyclopropenyl, substituted or unsubstituted cyclobutenyl, substituted or unsubstituted trans-cyclooctenyl optionally and independently fused to one or more instances of substituted or unsubstituted phenyl, or substituted or unsubstituted. TIFF2026016516000061.tif12128. In certain embodiments, at least one instance of the first click chemistry handle comprises -C≡CH. In certain embodiments, at least one instance of the first click chemistry handle comprises: TIFF2026016516000062.tif20128. In certain embodiments, at least one example of the first click chemistry handle is trans-cyclooctenyl, e.g. TIFF2026016516000063.tif15128. In certain embodiments, at least one instance of the second click chemistry handle comprises -N3. In certain embodiments, at least one instance of the second click chemistry handle comprises: TIFF2026016516000064.tif12128, where R 19 is H, halogen, unsubstituted C 1-6 Alkyl, or -O-(unsubstituted C 1-6 In certain embodiments, R 19 is -CH3. In some embodiments, at least one moiety formed by reacting two orthogonal click chemistry handles has the formula: It is TIFF2026016516000065.tif13128.

[0176] In some embodiments, at least one moiety formed by reacting two orthogonal click chemistry handles has the formula: TIFF2026016516000066.tif30128. In some embodiments, at least one moiety formed by reacting two orthogonal click chemistry handles has the formula: TIFF2026016516000067.tif29128. In some embodiments, each instance of the first linker is independently a substituted or unsubstituted C 1-200 Alkylene, substituted or unsubstituted C 1-200 Alkenylene, substituted or unsubstituted C 1-200 Alkynylene, substituted or unsubstituted C 1-200 Heteroalkylene, substituted or unsubstituted C 1-200 Heteroalkenylene, or substituted or unsubstituted C 1-200 In some embodiments, substituted or unsubstituted C 1-200 Alkylene, substituted or unsubstituted C 1-200 Alkenylene, substituted or unsubstituted C 1-200 Alkynylene, substituted or unsubstituted C 1-200 Heteroalkylene, substituted or unsubstituted C 1-200 Heteroalkenylene, and substituted or unsubstituted C 1-200 One or more carbon atoms in the parent chain of each instance of heteroalkynylene are independently replaced with a substituted or unsubstituted carbocyclylene, a substituted or unsubstituted heterocyclylene, a substituted or unsubstituted arylene, or a substituted or unsubstituted heteroarylene. In some embodiments, a substituted or unsubstituted C 1-200 Alkylene, substituted or unsubstituted C 1-200 Alkenylene, substituted or unsubstituted C 1-200 Alkynylene, substituted or unsubstituted C 1-200 Heteroalkylene, substituted or unsubstituted C 1-200 Heteroalkenylene, and substituted or unsubstituted C 1-200 One or more carbon atoms in the parent chain of each instance of heteroalkynylene independently may be TIFF2026016516000068.tif13128. In some embodiments, substituted or unsubstituted C 1-200 Alkylene, substituted or unsubstituted C1-200 Alkenylene, substituted or unsubstituted C 1-200 Alkynylene, substituted or unsubstituted C 1-200 Heteroalkylene, substituted or unsubstituted C 1-200 Heteroalkenylene, and substituted or unsubstituted C 1-200 One or more carbon atoms in the parent chain of each instance of heteroalkynylene independently may be TIFF2026016516000069.tif30135. In some embodiments, at least one example of the first linker has the formula: TIFF2026016516000070.tif11128, wherein L 1 Each instance of is independently a substituted or unsubstituted C 1-199 Alkylene, or substituted or unsubstituted C 1-199 is heteroalkylene, and L 2 Each instance of is independently a substituted or unsubstituted C 1-199 Alkylene, or substituted or unsubstituted C 1-199 In some embodiments, at least one example of the first linker is a heteroalkylene. It is TIFF2026016516000071.tif16130.

[0177] In some embodiments, the first poly-A tail comprises the nucleotide sequence set forth as 5'-rArArArArArArArArArArArAdU(a1)rArArArArArArArArArArArArArAdU(a1)rArArArArArArArArArArArArAdU(a1)ddC-3' (SEQ ID NO: 1); at least one instance of the second poly-A tail comprises the nucleotide sequence set forth as 5'-rArArArArArArArArArArArArArArArArArArArArArArArArArA*rA*rA*rA*rA*rA*rA*(b1)-3' (SEQ ID NO: 2); and each instance of the first linker is independently TIFF2026016516000072.tif16145, wherein "rA" represents an adenosine ribonucleotide, "dU(a1)" represents a modified uridine deoxyribonucleotide, "ddC" represents a cytosine dideoxyribonucleotide, "*" represents a phosphorothioate bond, a1 in each instance of dU(a1) represents the point of attachment at the uridine of dU(a1), b1 in rA*(b1) represents the point of attachment at the * in rA*(b1), each instance of a1 is bonded to one instance of a2, and each instance of b1 is bonded to one instance of b2. In some embodiments, the modified uridine deoxyribonucleotide is 5-octadiynyldeoxyuridine, which has the structure: Shown as TIFF2026016516000073.tif30128.

[0178] In some embodiments, the polyA region comprises a dendrimer comprising two or more polyA tails.

[0179] In some embodiments, each instance of n1 is independently an integer between 2 and 20, inclusive. In some embodiments, at least one instance of the first linker comprises a dendrimer. In some embodiments, at least one instance of the dendrimer is a polyamidoamine (PAMAM) dendrimer. In some embodiments, at least one instance of the PAMAM dendrimer is TIFF2026016516000074.tif22128, wherein each instance of n3 is independently an integer from 1 to 10 (inclusive); each instance of n4 is independently an integer from 0 to 10 (inclusive); R 2 Each instance of is independently hydrogen or TIFF2026016516000075.tif13128, but R 2 At least three examples of TIFF2026016516000076.tif13128, R 1 Each example is a substituted or unsubstituted C 1-10 Alkylene, substituted or unsubstituted C 1-10Alkenylene, substituted or unsubstituted C 1-10 Alkynylene, substituted or unsubstituted C 1-10 Heteroalkylene, substituted or unsubstituted C 1-10 Heteroalkenylene, or substituted or unsubstituted C 1-10 In some embodiments, substituted or unsubstituted C 1-10 Alkylene, substituted or unsubstituted C 1-10 Alkenylene, substituted or unsubstituted C 1-10 Alkynylene, substituted or unsubstituted C 1-10 Heteroalkylene, substituted or unsubstituted C 1-10 Heteroalkenylene, and substituted or unsubstituted C 1-10 One or more carbon atoms in the parent chain of each instance of heteroalkynylene is independently replaced with a substituted or unsubstituted carbocyclylene, a substituted or unsubstituted heterocyclylene, a substituted or unsubstituted arylene, or a substituted or unsubstituted heteroarylene, and each instance of n5 is independently an integer from 0 to 10, inclusive. In some embodiments, at least one instance of a PAMAM dendrimer has the following formula: It is TIFF2026016516000077.tif55128.

[0180] In some embodiments, substituted or unsubstituted C 1-10 Alkylene, substituted or unsubstituted C 1-10 Alkenylene, substituted or unsubstituted C 1-10 Alkynylene, substituted or unsubstituted C 1-10 Heteroalkylene, substituted or unsubstituted C 1-10 Heteroalkenylene, and substituted or unsubstituted C 1-10 One or more carbon atoms in the parent chain of each instance of heteroalkynylene independently may be TIFF2026016516000078.tif13128. In some embodiments, substituted or unsubstituted C 1-200 Alkylene, substituted or unsubstituted C 1-200 Alkenylene, substituted or unsubstituted C 1-200 Alkynylene, substituted or unsubstituted C1-200 Heteroalkylene, substituted or unsubstituted C 1-200 Heteroalkenylene, and substituted or unsubstituted C 1-200 One or more carbon atoms in the parent chain of each instance of heteroalkynylene independently may be TIFF2026016516000079.tif30137. In some embodiments, at least one example of a first linker has the following formula: It is TIFF2026016516000080.tif62157.

[0181] In some embodiments, the first poly-A tail and the second poly-A tail comprise a nucleotide sequence shown as 5'-rArArArArArArArArArArArArArArArArArArArArArArArArA*rA*rA*rA*rA*rA* / AzideN / -3' (SEQ ID NO: 3), where "AzideN" represents the 3' azide moiety and "*" represents a phosphorothioate linkage.

[0182] In some embodiments, at least one instance of the first linker is covalently attached to the 3' nucleotide of the first poly-A tail. In some embodiments, at least one instance of the first linker is covalently attached to the 3' nucleotide of at least one instance of the second poly-A tail.

[0183] In some embodiments, the 5' cap region comprises two or more 5' caps, and at least two instances of the 5' caps are covalently linked by a second linker. In some embodiments, the 5' cap region comprises the following structure: -(first 5' cap)-[(second linker)-(second 5' cap)m1]m2, where each instance of m1 is independently an integer between 1 and 20, inclusive, and m2 is an integer between 2 and 10, inclusive, and the first 5' cap is covalently linked to the 5' UTR, and the second linker is covalently linked to the first 5' cap and the second 5' cap.

[0184] In some embodiments, the 5' cap region comprises a brush oligonucleotide comprising two or more 5' caps.

[0185] In some embodiments, each instance of m1 is 1. In some embodiments, at least one instance of the second linker is covalently attached to an internal nucleotide of the first 5' cap. In some embodiments, at least one instance of the second linker is covalently attached to the second 5' cap at the 3' nucleotide of the second 5' cap. In some embodiments, at least one instance of the second linker comprises at least one moiety formed by reacting two orthogonal click chemistry handles. In certain embodiments, the click chemistry handles are as described herein. In some embodiments, each instance of the second linker is independently a substituted or unsubstituted C 1-200 Alkylene, substituted or unsubstituted C 1-200 Alkenylene, substituted or unsubstituted C 1-200 Alkynylene, substituted or unsubstituted C 1-200 Heteroalkylene, substituted or unsubstituted C 1-200 Heteroalkenylene, or substituted or unsubstituted C 1-200 In some embodiments, substituted or unsubstituted C 1-200 Alkylene, substituted or unsubstituted C 1-200 Alkenylene, substituted or unsubstituted C 1-200 Alkynylene, substituted or unsubstituted C 1-200 Heteroalkylene, substituted or unsubstituted C 1-200 Heteroalkenylene, and substituted or unsubstituted C 1-200 One or more carbon atoms in the parent chain of each instance of heteroalkynylene are independently replaced with a substituted or unsubstituted carbocyclylene, a substituted or unsubstituted heterocyclylene, a substituted or unsubstituted arylene, or a substituted or unsubstituted heteroarylene. In some embodiments, a substituted or unsubstituted C 1-200 Alkylene, substituted or unsubstituted C 1-200 Alkenylene, substituted or unsubstituted C 1-200 Alkynylene, substituted or unsubstituted C1-200 Heteroalkylene, substituted or unsubstituted C 1-200 Heteroalkenylene, and substituted or unsubstituted C 1-200 One or more carbon atoms in the parent chain of each instance of heteroalkynylene independently may be TIFF2026016516000081.tif12128. In some embodiments, substituted or unsubstituted C 1-200 Alkylene, substituted or unsubstituted C 1-200 Alkenylene, substituted or unsubstituted C 1-200 Alkynylene, substituted or unsubstituted C 1-200 Heteroalkylene, substituted or unsubstituted C 1-200 Heteroalkenylene, and substituted or unsubstituted C 1-200 One or more carbon atoms in the parent chain of each instance of heteroalkynylene independently may be TIFF2026016516000082.tif31139. In some embodiments, at least one example of the second linker has the formula: TIFF2026016516000083.tif11128, wherein L 3 Each instance of is independently a substituted or unsubstituted C 1-199 Alkylene, or substituted or unsubstituted C 1-199 is heteroalkylene, and L 4 Each instance of is independently a substituted or unsubstituted C 1-199 Alkylene, or substituted or unsubstituted C 1-199 In some embodiments, at least one example of the second linker is a heteroalkylene. It is TIFF2026016516000084.tif15133.

[0186] In some embodiments, the first 5' cap comprises the nucleotide sequence set forth as 5'- / Cap / rGrGrGrArArAdU(c1)rArArGrArGrArGrArArArGrArArGrArGdU(c1)rArArGrArArGrArArAdU(c1)rA-3' (SEQ ID NO:4), and the second 5' cap comprises the nucleotide sequence set forth as 5'- / Cap / rGrGrGrArGrArCrTrGrCrCrArCrCrA*rA*rA*rA*rA*rA*(d1)-3' (SEQ ID NO:5), wherein each instance of the second linker is independently TIFF2026016516000085.tif16146, where "*" represents a phosphorothioate bond, "Cap" represents a 5' cap, c1 in each instance of dU(c1) represents the point of attachment at U in dU(c1), d1 in rA*(d1) represents the point of attachment at * in rA*(d1), each instance of c1 is bonded to one instance of c2, and each instance of d1 is bonded to one instance of d2.

[0187] In some embodiments, the 5' cap region comprises a dendrimer comprising two or more 5' caps.

[0188] In some embodiments, each instance of m1 is independently an integer from 2 to 20, inclusive. In some embodiments, at least one instance of the second linker comprises a dendrimer. In some embodiments, at least one instance of the dendrimer is a polyamidoamine (PAMAM) dendrimer. In some embodiments, at least one instance of the PAMAM dendrimer has the formula: TIFF2026016516000086.tif22128, wherein each instance of m3 is independently an integer from 1 to 10 (inclusive); each instance of m4 is independently an integer from 0 to 10 (inclusive); R 12 Each instance of is independently hydrogen or TIFF2026016516000087.tif13128, but R 12 At least three examples of TIFF2026016516000088.tif13128, R 11 Each example is a substituted or unsubstituted C 1-10 Alkylene, substituted or unsubstituted C 1-10 Alkenylene, substituted or unsubstituted C 1-10 Alkynylene, substituted or unsubstituted C 1-10 Heteroalkylene, substituted or unsubstituted C 1-10 Heteroalkenylene, or substituted or unsubstituted C 1-10 In some embodiments, substituted or unsubstituted C 1-10 Alkylene, substituted or unsubstituted C 1-10 Alkenylene, substituted or unsubstituted C 1-10 Alkynylene, substituted or unsubstituted C 1-10 Heteroalkylene, substituted or unsubstituted C 1-10 Heteroalkenylene, and substituted or unsubstituted C 1-10 One or more carbon atoms in the parent chain of each instance of heteroalkynylene is independently replaced with a substituted or unsubstituted carbocyclylene, a substituted or unsubstituted heterocyclylene, a substituted or unsubstituted arylene, or a substituted or unsubstituted heteroarylene, and each instance of m5 is independently an integer from 0 to 10, inclusive. In some embodiments, at least one instance of a PAMAM dendrimer has the following formula: Includes TIFF2026016516000089.tif55128.

[0189] In some embodiments, substituted or unsubstituted C 1-10 Alkylene, substituted or unsubstituted C 1-10 Alkenylene, substituted or unsubstituted C 1-10 Alkynylene, substituted or unsubstituted C 1-10 Heteroalkylene, substituted or unsubstituted C 1-10 Heteroalkenylene, and substituted or unsubstituted C 1-10 One or more carbon atoms in the parent chain of each instance of heteroalkynylene independently may be TIFF2026016516000090.tif13128. In some embodiments, substituted or unsubstituted C 1-10 Alkylene, substituted or unsubstituted C 1-10 Alkenylene, substituted or unsubstituted C 1-10 Alkynylene, substituted or unsubstituted C 1-10 Heteroalkylene, substituted or unsubstituted C 1-10 Heteroalkenylene, and substituted or unsubstituted C 1-10 One or more carbon atoms in the parent chain of each instance of heteroalkynylene independently may be TIFF2026016516000091.tif30135. In some embodiments, at least one example of a first linker has the following formula: It is TIFF2026016516000092.tif62157.

[0190] In some embodiments, the first 5' cap is / 5AzideN / rArArArArA-3', and the second 5' cap comprises a nucleotide sequence shown as 5'- / Cap / rGrGrGrArGrArCrTrGrCrCrArCrCrA*rA*rA*rA*rA*rA* / AzideN / -3' (SEQ ID NO: 6), where "rA" represents an adenosine ribonucleotide, "rT" represents a thymidine ribonucleotide, "rC" represents a cytidine ribonucleotide, "rG" represents a guanosine ribonucleotide, "dU(c1)" represents a modified uridine deoxyribonucleotide, "5AzideN" represents a 5' azido moiety, "3AzideN" represents a 3' azido moiety, "*" represents a phosphorothioate linkage, and "Cap" represents a 5' cap.

[0191] In some embodiments, at least one instance of the second linker is covalently attached to the 3' nucleotide of the first 5' cap. In some embodiments, at least one instance of the second linker is covalently attached to the 3' nucleotide of at least one instance of the second 5' cap.

[0192] Methods for producing modified mRNA In some aspects, the disclosure provides a method of producing a modified mRNA, comprising: ligating, in the presence of a ligase, an RNA comprising an open reading frame encoding a protein to a tailing nucleic acid comprising two or more polyA tails and / or a capping nucleic acid comprising two or more 5' caps, whereby the ligase forms a covalent bond between the 3' nucleotide of the RNA and the 5' nucleotide of the tailing nucleic acid, or between the 3' nucleotide of the capping nucleic acid and the 5' nucleotide of the RNA, to produce a modified RNA (e.g., a modified mRNA). In some embodiments, the tailing nucleic acid comprises a polyA region as described herein, and the capping nucleic acid comprises a 5' cap region as described herein. When the ligase forms a covalent bond between two linear nucleic acids, a new nucleic acid is produced, and the produced nucleic acid comprises the nucleic acid sequence of both nucleic acids. Ligation of the 3'-terminal nucleotide of a first nucleic acid to the 5'-terminal nucleotide of a second nucleic acid produces a third nucleic acid, which comprises the sequences of the first and second nucleic acids, with the second nucleic acid sequence being 3' (downstream) relative to the first nucleic acid sequence. Ligation by RNA ligase occurs in several steps. First, the amino (-NH2) group of an amino acid (e.g., lysine) of the ligase binds to the phosphate group of adenosine triphosphate (ATP), resulting in the binding of the adenosine monophosphate (AMP) group to the RNA ligase. Second, the 5'-terminal phosphate of the second nucleic acid replaces the phosphate of the RNA ligase-bound AMP. Finally, the oxygen of the 3'-terminal hydroxyl group of the first nucleic acid binds to the phosphorus atom of the 5'-terminal phosphate of the second nucleic acid. This final step forms a phosphodiester bond between the terminal nucleotides of the nucleic acids, thereby forming a single nucleic acid with a continuous sugar-phosphate backbone. In some embodiments, the ligase is an RNA ligase. In some embodiments, the RNA ligase is T4 RNA ligase.

[0193] In some embodiments of the methods for producing modified mRNA provided herein, the RNA to which the tailing nucleic acid and / or capping nucleic acid is ligated is synthesized by in vitro transcription (IVT). IVT is a process in which RNA, such as precursor mRNA (pre-mRNA) or mRNA, is produced by transcription of a DNA template by RNA polymerase. Generally, the DNA template contains a promoter, such as a bacteriophage promoter, upstream of the DNA sequence to be transcribed. RNA polymerase binds to the promoter and initiates transcription of the DNA sequence, producing an RNA transcript having the nucleic acid sequence present in the template, except that thymidine (T) nucleotides in the DNA sequence are replaced with uracil (U) nucleotides in the RNA sequence. The RNA transcript produced by IVT can be modified before ligation of the tailing nucleic acid, such as by adding a 5' cap, cleaving one or more nucleotides from the RNA, or polyadenylation to extend a polyA region. In some embodiments, the DNA template contains a polyA region, thereby allowing IVT to produce an mRNA with a polyA region. See, e.g., Becker et al. Methods Mol Biol., 2011. 703:29-41.

[0194] In some embodiments of the methods for producing modified mRNA provided herein, the 3' nucleotide of the RNA comprises a 3'-terminal hydroxyl group, and the 5' nucleotide of the tailing nucleic acid comprises a 5'-terminal phosphate group. In some embodiments of the methods for producing modified mRNA provided herein, the 3' nucleotide of the capping nucleic acid comprises a 3'-terminal hydroxyl group, and the 5' nucleotide of the RNA comprises a 5'-terminal phosphate group. The combination of a 3'-terminal hydroxyl group and a 5'-terminal phosphate group enables efficient ligation of two nucleic acids. In some embodiments, the RNA does not comprise a 5'-terminal phosphate group. The RNA may lack a 5'-terminal phosphate group due to the addition of a 5'-cap or another chemical modification. The 5'-terminal phosphate can also be removed from the RNA by a phosphatase enzyme to produce an RNA lacking a 5'-terminal phosphate. The lack of a 5'-terminal phosphate group in the RNA prevents RNA ligase from ligating multiple copies of the mRNA together. In some embodiments, the tailing nucleic acid does not comprise a 3'-terminal hydroxyl group. If the last nucleotide of the tailing nucleic acid contains a modified nucleotide that does not contain a 3' hydroxyl group, such as dideoxyadenosine, dideoxycytidine, dideoxyguanosine, dideoxythymidine, or inverted deoxythymidine, the RNA may lack a 3' hydroxyl group. The lack of a 3' hydroxyl group in the tailing nucleic acid prevents RNA ligase from ligating multiple tailing nucleic acids together. In some embodiments, the 5' nucleotide of the RNA does not contain a 5' phosphate group, the 3' nucleotide of the RNA contains a 3' hydroxyl group, the 5' nucleotide of the tailing nucleic acid contains a 5' phosphate group, and the 3' nucleotide of the tailing nucleic acid does not contain a 3' hydroxyl group. In some embodiments, the tailing nucleic acid and / or capping nucleic acid contains at least 3, at least 4, at least 5, or at least 6 phosphorothioates. In some embodiments, the tailing nucleic acid contains at least 3, at least 4, at least 5, or at least 6 phosphorothioates and does not contain a 3' hydroxyl.In some embodiments, the tailing nucleic acid comprises at least three phosphorothioates and no 3'-terminal hydroxyl. In some embodiments, the tailing nucleic acid comprises at least three guanine nucleotides and at least three phosphorothioates and no 3'-terminal hydroxyl. In some embodiments, the tailing nucleic acid comprises at least three deoxyribose sugars and no 3'-terminal hydroxyl. In some embodiments, the tailing nucleic acid and / or capping nucleic acid comprises at least 20 deoxyribose sugars. In some embodiments, the tailing nucleic acid comprises at least 20 deoxyribose sugars and no 3'-terminal hydroxyl. In some embodiments, the tailing nucleic acid comprises at least three copies of a G-quadruplex sequence and no 3'-terminal hydroxyl. In some embodiments, the tailing nucleic acid comprises at least six phosphorothioates and no 3'-terminal hydroxyl. In some embodiments, the tailing nucleic acid comprises at least six consecutive phosphorothioates and no 3'-terminal hydroxyl. In some embodiments, the tailing nucleic acid comprises at least six phosphorothioates and three guanine nucleosides and does not have a 3'-terminal hydroxyl. In some embodiments, the tailing nucleic acid comprises at least three copies of a G-quadruplex sequence and at least six phosphorothioates and does not have a 3'-terminal hydroxyl. In some embodiments, the tailing nucleic acid comprises at least three copies of a telomeric repeat sequence and at least six phosphorothioates and does not have a 3'-terminal hydroxyl. In some embodiments, the 3'-terminal nucleotide that does not have a 3'-terminal hydroxyl is dideoxycytidine or inverted deoxythymidine. In some embodiments, the ligase used to ligate the tailing nucleic acid to RNA is an RNA ligase. In some embodiments, the RNA ligase is T4 RNA ligase. In some embodiments, the T4 RNA ligase is T4 RNA ligase 1. In some embodiments, the T4 RNA ligase is T4 RNA ligase 2.

[0195] In some embodiments of the methods of producing modified mRNA provided herein, the 5' nucleotide of the RNA does not comprise a 5' terminal hydroxyl group, the 3' nucleotide of the RNA comprises a 3' terminal phosphate group, the 5' nucleotide of the tailing nucleic acid comprises a 5' terminal hydroxyl group, the 3' nucleotide of the tailing nucleic acid does not comprise a 3' terminal phosphate group, the RNA ligase is RtcB ligase, and ligates a first nucleotide comprising a 3' terminal phosphate group to a second nucleotide comprising a 5' terminal hydroxyl group.

[0196] Some embodiments of the methods for making modified mRNA provided herein further include producing a circular mRNA. After a linear modified mRNA is produced by ligating an RNA and a tailing nucleic acid, circularizing the modified mRNA involves several additional steps. First, a 5'-terminal phosphate is introduced to the first nucleotide of the modified mRNA, a process known as phosphorylation. In some embodiments, the 5'-terminal phosphate is introduced by a kinase. "Kinase" refers to an enzyme that introduces a phosphate group to a molecule, forming a covalent bond between the phosphate group and the molecule in a process called "phosphorylation." Second, the modified mRNA is engineered to produce a modified mRNA with a 3'-terminal hydroxyl group. In some embodiments, the modified mRNA is engineered by cleaving one or more of the last nucleotides of the modified mRNA to produce a modified mRNA with a 3'-terminal hydroxyl group. In some embodiments, the modified mRNA is cleaved by a restriction enzyme, ribozyme, or endoribonuclease. In some embodiments, cleavage of one or more of the last nucleotides of the modified mRNA occurs before phosphorylation of the first nucleotide of the modified mRNA. In some embodiments, cleavage occurs after phosphorylation. Modified mRNAs containing a terminal phosphate group at one end and a terminal hydroxyl group at the other end can be circularized by ligation of both terminal nucleotides. An RNA ligase that ligates the terminal nucleotides of a linear nucleic acid to generate a circular nucleic acid can be referred to as a "circularization ligase." In some embodiments, the circularization ligase is an RNA ligase. In some embodiments, the circularization ligase is SplintR ligase. In some embodiments, the circularization ligase is T4 RNA ligase. In some embodiments, the circularization ligase is T4 RNA ligase 1. In some embodiments, the circularization ligase is T4 RNA ligase 2. In some embodiments, the modified mRNA contains a 5'-terminal hydroxyl group and a 3'-terminal phosphate group, and the circularization ligase is RtcB ligase, and can ligate nucleotides at the 3'-terminal phosphate and the 5'-terminal hydroxyl group.For ligation to occur, the 5' and 3' terminal nucleotides of the modified mRNA must be close enough for RNA ligase to form a bond between both nucleotides. Methods for positioning both nucleotides of a linear nucleic acid close enough for ligation to occur and circularizing the RNA are generally known in the art (see, e.g., Petkovic et al., Nucleic Acids Res., 2015. 43(4):2454-2465). In some embodiments, the modified mRNA is incubated with a scaffold nucleic acid that can hybridize (hydrogen bond) with the modified mRNA, thereby forming a circular secondary structure when hybridized (bound) to the scaffold nucleic acid.

[0197] When mRNA forms a circular secondary structure, the 5' and 3' terminal nucleotides are in close physical proximity, which is required for RNA ligase to form a covalent bond between them. In some embodiments of the method of circularizing mRNA, one or more of the last nucleotides of the mRNA are bound to a first hybridization sequence in the scaffold nucleic acid, and one or more of the first nucleotides of the mRNA are bound to a second hybridization sequence in the scaffold nucleic acid that is 3' (downstream) from the first hybridization sequence. In some embodiments, the first hybridization sequence comprises five or more nucleotides, and the first hybridization sequence is complementary to at least the first five nucleotides of the modified mRNA. In some embodiments, the first hybridization sequence comprises 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, or 50 or more nucleotides, and at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to 100% of the nucleotides of the first hybridization sequence are complementary to the last N nucleotides of the modified mRNA, where N is the length of the first hybridization sequence. In some embodiments, the second hybridization sequence comprises 5 or more nucleotides, and the second hybridization sequence is complementary to at least the last 5 nucleotides of the modified mRNA. In some embodiments, the second hybridization sequence comprises 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, or 50 or more nucleotides, and at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or up to 100% of the nucleotides of the second hybridization sequence are complementary to the last N nucleotides of the modified mRNA, where N is the length of the second hybridization sequence. In some embodiments, at least the first 5 nucleotides of the modified mRNA hybridize to the first hybridization sequence.In some embodiments, at least the last five nucleotides of the modified mRNA hybridize with the second hybridization sequence. In some embodiments, at least the first five nucleotides of the modified mRNA hybridize with the first hybridization sequence, and at least the last five nucleotides of the modified mRNA hybridize with the second hybridization sequence. In some embodiments, the last nucleotide of the first hybridization sequence and the first nucleotide of the second hybridization sequence are adjacent within the scaffold nucleic acid and are not separated by any other nucleotides.

[0198] In some embodiments of the methods for producing circular mRNA provided herein, a scaffold nucleic acid is not used to promote the formation of a circular secondary structure by the modified mRNA. Instead, the modified mRNA comprises a first hybridization sequence at the 5' end that is complementary to a second hybridization sequence at the 3' end. In some embodiments, each hybridization sequence comprises at least 5 nucleotides. In certain embodiments, each hybridization sequence comprises at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 nucleotides.

[0199] In some embodiments of the methods for producing circular mRNA provided herein, the modified mRNA is not circularized by the use of a scaffold nucleic acid and a circularization ligase, but rather by a ribozyme, a nucleic acid that catalyzes a reaction such as the formation of a covalent bond between two nucleotides. In some embodiments, prior to circularization, the modified mRNA includes a 3' intron that is 5' (upstream) to the 5' UTR of the mRNA and a 5' intron that is 3' (downstream) to a polyA region and / or one or more structural sequences of the mRNA. Ribozymes and other enzymes that catalyze splicing of pre-mRNA to remove introns can catalyze the formation of a covalent bond between a nucleotide 5' to the 5' intron and a nucleotide 3' to the 3' intron, resulting in the formation of a circular mRNA. See, e.g., Wesselhoeft et al., Nat Commun. 2018.9:2629.

[0200] In some embodiments of the methods for producing circular mRNA provided herein, the modified mRNA is not circularized by the use of a scaffolding core, but rather through the use of complementary sequences that promote the formation of a secondary structure by the mRNA, positioning the 5' and 3' terminal nucleotides of the mRNA in close proximity. In some embodiments, prior to circularization, the modified mRNA comprises (i) a first self-hybridizing sequence 5' to the open reading frame, (ii) a second self-hybridizing sequence 3' to the open reading frame, (iii) a first non-hybridizing sequence 5' to the first self-hybridizing sequence, and (iv) a second non-hybridizing sequence 3' to the second self-hybridizing sequence. The first and second self-hybridizing sequences can hybridize to each other, but the first and second self-hybridizing sequences cannot hybridize to each other. In some embodiments, hybridization of the first and second self-hybridizing sequences forms a secondary structure in which the 5'- and 3'-terminal nucleotides of the modified mRNA are separated by a distance of less than 100 Å. In some embodiments, the 5'- and 3'-terminal nucleotides are separated by a distance of less than 90 Å, 80 Å, 70 Å, 60 Å, 50 Å, 40 Å, 30 Å, 20 Å, or 10 Å. See, e.g., Carmona, Ellese Marie. 2019. Circular RNA: Design Criteria for Optimal Therapeutical Utility. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences; Petkovic et al. Nucleic Acids Res., 2015. 43(4):2454-2465; and WO2020 / 237227.

[0201] In some embodiments of the modified mRNA produced by the methods provided herein, the polyA region of the modified mRNA comprises at least one modified nucleotide. In some embodiments, the tailing nucleic acid comprises at least one modified nucleotide. In some embodiments, at least one modified nucleotide comprises a modified nucleobase. In some embodiments, at least one modified nucleotide comprises a modified sugar. In some embodiments, at least one modified nucleotide comprises a modified phosphate. In some embodiments, the at least one modified nucleobase is xanthine, aliaminouracil, aliaminothymidine, hypoxanthine, digoxigenylated adenine, digoxigenylated cytosine, digoxigenylated guanine, digoxigenylated uracil, 6-chloropurine riboside, N6-methyladenine, methylpseudouracil, 2-thiocytosine, 2-thiouracil, 5-methyluracil, 4-thiothymidine, 4-thiazol-1-yl Auracil, 5,6-dihydro-5-methyluracil, 5,6-dihydrouracil, 5-[(3-indolyl)propionamido-N-allyl]uracil, 5-aminoallylcytosine, 5-aminoallyluracil, 5-bromouracil, 5-bromocytosine, 5-carboxycytosine, 5-carboxymethylesteruracil, 5-carboxyuracil, 5-fluorouracil, 5-formylcytosine, 5-formyluracil cytosine, 5-hydroxymethylcytosine, 5-hydroxymethyluracil, 5-hydroxyuracil, 5-iodocytosine, 5-iodouracil, 5-methoxycytosine, 5-methoxyuracil, 5-methylcytosine, 5-methyluracil, 5-propargylaminocytosine, 5-propargylaminouracil, 5-propynylcytosine, 5-propynyluracil, 6-azacytosine, 6-azacytosine le, 6-chloropurine, 6-thioguanine, 7-deazaadenine, 7-deazaguanine, 7-deaza-7-propargylaminoadenine, 7-deaza-7-propargylaminoguanine, 8-azaadenine, 8-azidoadenine, 8-chloroadenine, 8-oxoadenine, 8-oxoguanine, araadenine, aracytosine, araguanine, arauracil, biotin-16-7-deaza-7-propargylaminoguanine,Biotin-16-aminoallylcytosine, biotin-16-aminoallyluracil, cyanine 3-5-propargylaminocytosine, cyanine 3-6-propargylaminouracil, cyanine 3-aminoallylcytosine, cyanine 3-aminoallyluracil, cyanine 5-6-propargylaminocytosine, cyanine 5-6-propargylaminouracil, cyanine 5-aminoallylcytosine, cyanine 5-aminoallyluracil, cyanine 7-aminoallyluracil, dabsyl-5-3-aminoallyluracil, des Thiobiotin-16-aminoallyl-uracil, desthiobiotin-6-aminoallylcytosine, isoguanine, N1-ethylpseudouracil, N1-methoxymethylpseudouracil, N1-methyladenine, N1-methylpseudouracil, N1-propylpseudouracil, N2-methylguanine, N4-biotin-OBEA-cytosine, N4-methylcytosine, N6-methyladenine, O6-methylguanine, pseudoisocytosine, pseudouracil, thienocytosine, thienoguanine, thienouracil, cytosine Santosine, 3-deazaadenine, 2,6-diaminoadenine, 2,6-daminoguanine, 5-carboxamidouracil, 5-ethynyluracil, N6-isopentenyladenine (i6A), 2-methyl-thio-N6-isopentenyladenine (ms2i6A), 2-methylthio-N6-methyladenine (ms2m6A), N6-(cis-hydroxyisopentenyl)adenine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenine (ms2io6A), N6-glycinylcarbamoyl adenine In some embodiments, the at least one modified nucleotide comprises a modified nucleobase selected from the group consisting of 2'-thioribose, 2',3'-dideoxyribose, 2',3'-dideoxyribose, 2',3'-dihydroxy ...2'-amino-2'-deoxyribose, 2'-deoxyribose, 2'-azido-2'-deoxyribose, 2'-fluoro-2'-deoxyribose, 2'-O-methylribose, 2'-O-methyldeoxyribose, 3'-amino-2',3'-dideoxyribose, 3'-azido-2',3'-dideoxyribose, 3'-deoxyribose, 3'-O-(2-nitro) The modified sugars include those selected from the group consisting of 2'-O,4'-C-methylene-linked, 2'-O,4'-C-amino-linked, and 2'-O,4'-C-thio-linked ribose. In some embodiments, at least one modified nucleotide comprises a modified phosphate selected from the group consisting of phosphorothioate (PS), thiophosphate, 5'-O-methylphosphonate, 3'-O-methylphosphonate, 5'-hydroxyphosphonate, hydroxyphosphate, phosphoroselenoate, selenophosphate, phosphoramidate, carbophosphonate, methylphosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidinium ring, triazole ring, boranophosphate (BP), methylphosphonate, and guanidinopropylphosphoramidate. In some embodiments, the polyA region of the mRNA comprises at least three, at least four, or at least five phosphorothioates and does not have a 3'-terminal hydroxyl. In some embodiments, the polyA region of the mRNA comprises at least three phosphorothioates and does not have a 3'-terminal hydroxyl. In some embodiments, the polyA region of the mRNA comprises at least three guanine nucleotides and at least three phosphorothioates and does not comprise a 3'-terminal hydroxyl. In some embodiments, the polyA region of the mRNA comprises at least three deoxyribose sugars and does not comprise a 3'-terminal hydroxyl. In some embodiments, the polyA region of the mRNA comprises at least 20 deoxyribose sugars and does not comprise a 3'-terminal hydroxyl. In some embodiments, the polyA region of the mRNA comprises at least three copies of a G-quadruplex sequence.In some embodiments, the polyA region of the mRNA comprises at least six phosphorothioates and does not comprise a 3'-terminal hydroxyl. In some embodiments, the polyA region of the mRNA comprises at least six consecutive phosphorothioates and does not comprise a 3'-terminal hydroxyl. In some embodiments, the polyA region of the mRNA comprises at least six phosphorothioates and three guanine nucleosides and does not comprise a 3'-terminal hydroxyl. In some embodiments, the polyA region of the mRNA comprises at least three copies of a G-quadruplex sequence and at least six phosphorothioates and does not comprise a 3'-terminal hydroxyl. In some embodiments, the polyA region of the mRNA comprises at least three copies of a telomeric repeat sequence and at least six phosphorothioates and does not comprise a 3'-terminal hydroxyl. In some embodiments, the 3'-terminal nucleotide that does not comprise a 3'-terminal hydroxyl is dideoxycytidine or inverted deoxythymidine.

[0202] In some embodiments of the modified mRNA produced by the methods provided herein, the modified mRNA comprises two or more types of modified nucleotides. In some embodiments, the modified mRNA comprises at least a first modified nucleoside and a second modified nucleoside having a structure different from the first modified nucleoside. In some embodiments, the modified mRNA comprises at least a first modified phosphate and a second modified phosphate having a structure different from the first modified phosphate. In some embodiments, the modified mRNA comprises a modified nucleoside and a modified nucleoside.

[0203] In some embodiments of the modified mRNA produced by the methods provided herein, 1% to 90% of the nucleotides in the polyA region are modified nucleotides, in some embodiments, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 12%, at least 14%, at least 16%, at least 18%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% of the nucleotides in the polyA region are modified nucleotides.

[0204] In some embodiments of the modified mRNA produced by the methods provided herein, three or more of the last 10-25 nucleotides of the polyA tract are modified nucleotides, hi some embodiments, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, or 25 of the last 25 nucleotides of the polyA tract are modified nucleotides.

[0205] In some embodiments of the modified mRNA produced by the methods provided herein, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the nucleotides in the polyA region are adenosine nucleotides. One or more adenosine nucleotides in the polyA region can be a standard adenosine nucleotide or a modified adenosine nucleotide that includes a structure different from a standard adenosine nucleotide.

[0206] In some embodiments of the modified mRNA produced by the methods provided herein, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the nucleotides in the polyA region are standard adenosine nucleotides.

[0207] In some embodiments of the modified mRNA produced by the methods provided herein, the polyA region comprises 10-25 nucleotides. In some embodiments of the modified mRNA produced by the methods provided herein, the polyA region comprises 10-15 nucleotides, 15-20 nucleotides, or 20-25 nucleotides. In some embodiments of the modified mRNA produced by the methods provided herein, the polyA region comprises 25-500 nucleotides. In some embodiments, the polyA region comprises at least 25, at least 30, at least 50, at least 100, at least 150, or at least 200 nucleotides. In some embodiments, the polyA region comprises at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290, or at least 300 nucleotides. In some embodiments, the polyA region comprises about 200 to about 300 nucleotides. In some embodiments, the polyA region comprises about 250 nucleotides.

[0208] In some embodiments of the methods of producing modified mRNA provided herein, the RNA comprises an open reading frame and a polyA region prior to ligation of the tailing nucleic acid. In some embodiments, the polyA region of the RNA comprises 10-25 nucleotides prior to ligation of the tailing nucleic acid. In some embodiments, the polyA region of the RNA comprises 10-15 nucleotides, 15-20 nucleotides, or 20-25 nucleotides prior to ligation of the tailing nucleic acid. In some embodiments, the polyA region of the RNA comprises 25-500 nucleotides prior to ligation of the tailing nucleic acid. In some embodiments, the polyA region comprises at least 25, at least 30, at least 50, at least 100, at least 150, or at least 200 nucleotides. In some embodiments, the polyA region comprises at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, at least 270, at least 280, at least 290, or at least 300 nucleotides. In some embodiments, the polyA region comprises about 200 to about 300 nucleotides. In some embodiments, the polyA region comprises about 250 nucleotides.

[0209] In some embodiments of the methods of producing modified mRNA provided herein, prior to ligation of the tailing nucleic acid, the RNA comprises, in 5' to 3' order, a 5' UTR, an open reading frame, a 3' UTR, and a polyA region. In some embodiments, the open reading frame is between the 5' UTR and the 3' UTR. In some embodiments, the 3' UTR is between the open reading frame and the polyA region.

[0210] In some embodiments of the methods for producing modified mRNA provided herein, the RNA comprises a 5' cap prior to ligation of the tailing nucleic acid. In some embodiments, the 5' cap comprises 7-methylguanosine. In some embodiments, the 5' cap comprises one or more phosphates connecting the 7-methylguanosine to adjacent nucleotides of the RNA. In some embodiments, the 5' cap is added after ligation of the tailing nucleic acid.

[0211] In some aspects of the methods of producing a modified mRNA provided herein, which comprise ligating a tailing nucleic acid to an mRNA, the tailing nucleic acid comprises one or more modified nucleotides. In some embodiments, the tailing nucleic acid comprises at least one modified nucleotide comprising a modified nucleoside. In some embodiments, at least one modified nucleotide comprises a modified nucleoside comprising a modified nucleobase and / or a modified sugar. In some embodiments, at least one modified nucleotide comprises a modified nucleoside comprising a modified nucleobase and a modified sugar. In some embodiments, at least one modified nucleotide comprises a modified nucleobase. In some embodiments, at least one modified nucleotide comprises a modified sugar. In some embodiments, at least one modified nucleotide comprises a modified phosphate. In some embodiments, the at least one modified nucleobase is xanthine, allaminouracil, allaminothymidine, hypoxanthine, digoxigenylated adenine, digoxigenylated cytosine, digoxigenylated guanine, digoxigenylated uracil, 6-chloropurine riboside, N6-methyladenine, methylpseudouracil, 2-thiocytosine, 2-thiouracil, 5-methyluracil, 4-thiothymidine, 4-thiouracil, 5,6-dihydro-5-methyluracil, 5,6-dihydrouracil, 5-[(3-indolyl)propionamido-N-allyl]uracil, 5-aminoallylcytosine, 5-aminoallyluracil, 5-bromouracil, 5-bromocytosine , 5-carboxycytosine, 5-carboxymethylesteruracil, 5-carboxyuracil, 5-fluorouracil, 5-formylcytosine, 5-formyluracil, 5-hydroxycytosine, 5-hydroxymethylcytosine, 5-hydroxymethyluracil, 5-hydroxyuracil, 5-iodocytosine, 5-iodouracil, 5-methoxycytosine, 5-methoxyuracil, 5-methylcytosine, 5-methyluracil, 5-propargylaminocytosine, 5-propargylaminouracil, 5-propynylcytosine, 5-propynyluracil, 6-azacytosine, 6-azauracil, 6-chloropurine, 6-thioguanine, 7-deazaadenine, 7-deazaguanine,7-deaza-7-propargylaminoadenine, 7-deaza-7-propargylaminoguanine, 8-azaadenine, 8-azidoadenine, 8-chloroadenine, 8-oxoadenine, 8-oxoguanine, araadenine, aracytosine, araguanine, arauracil, biotin-16-7-deaza-7-propargylaminoguanine, biotin-16-aminoallylcytosine, biotin-16-aminoallyluracil, cyanine 3-5-propargylaminocytosine, cyanine 3-6-propargylaminouracil, cyanine 3-aminoallyl Cytosine, cyanine 3-aminoallyluracil, cyanine 5-6-propargylaminocytosine, cyanine 5-6-propargylaminouracil, cyanine 5-aminoallylcytosine, cyanine 5-aminoallyluracil, cyanine 7-aminoallyluracil, dabsyl-5-3-aminoallyluracil, desthiobiotin-16-aminoallyl-uracil, desthiobiotin-6-aminoallylcytosine, isoguanine, N1-ethylpseudouracil, N1-methoxymethylpseudouracil, N1-methyladenine, N1-methylpseudouracil uracil, N1-propylpseudouracil, N2-methylguanine, N4-biotin-OBEA-cytosine, N4-methylcytosine, N6-methyladenine, O6-methylguanine, pseudoisocytosine, pseudouracil, thienocytosine, thienoguanine, thienouracil, xanthosine, 3-deazaadenine, 2,6-diaminoadenine, 2,6-daminoguanine, 5-carboxamido-uracil, 5-ethynyluracil, N6-isopentenyladenine (i6A), 2-methyl-thio-N6-isopentenyladenine (ms2i6A), 2-methylthio-N6-methyladenine (ms2m6A), N6-(cis-hydroxyisopentenyl)adenine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenine (ms2io6A), N6-glycinylcarbamoyladenine (g6A), N6-threonylcarbamoyladenine (t6A), 2-methylthio-N6-threonylcarbamoyladenine (ms2t6A), N6-methyl-N6-threonylcarbamoyladenine (m6t6A), N6-hydroxynorvalylcarbamoyladenine (hn6A),In some embodiments, the at least one modified nucleotide comprises a modified base selected from the group consisting of 2-methylthio-N6-hydroxynorvalylcarbamoyladenine (ms2hn6A), N6,N6-dimethyladenine (m62A), and N6-acetyladenine (ac6A). In some embodiments, the at least one modified nucleotide is 2'-thioribose, 2',3'-dideoxyribose, 2'-amino-2'-deoxyribose, 2'-deoxyribose, 2'-azido-2'-deoxyribose, 2'-fluoro-2'-deoxyribose, 2'-O-methylribose, 2'-O-methyldeoxyribose, 3'-amino-2',3'-dideoxyribose, 3'-azido-2',3'-dideoxyribose. The modified sugars include those selected from the group consisting of ribose, 3'-deoxyribose, 3'-O-(2-nitrobenzyl)-2'-deoxyribose, 3'-O-methylribose, 5'-aminoribose, 5'-thioribose, 5-nitro-1-indolyl-2'-deoxyribose, 5'-biotin-ribose, 2'-O,4'-C-methylene-linked ribose, 2'-O,4'-C-amino-linked ribose, and 2'-O,4'-C-thio-linked ribose. In some embodiments, at least one modified nucleotide comprises a modified phosphate selected from the group consisting of phosphorothioate (PS), thiophosphate, 5'-O-methylphosphonate, 3'-O-methylphosphonate, 5'-hydroxyphosphonate, hydroxyphosphate, phosphoroselenoate, selenophosphate, phosphoramidate, carbophosphonate, methylphosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidinium ring, triazole ring, boranophosphate (BP), methylphosphonate, and guanidinopropylphosphoramidate.

[0212] In some embodiments of the methods for producing modified mRNA provided herein, the tailing nucleic acid comprises two or more types of modified nucleotides. In some embodiments, the tailing nucleic acid comprises at least a first modified nucleoside and a second modified nucleoside having a structure different from the first modified nucleoside. In some embodiments, the tailing nucleic acid comprises at least a first modified phosphate and a second modified phosphate having a structure different from the first modified phosphate. In some embodiments, the tailing nucleic acid comprises a modified nucleoside and a modified nucleoside.

[0213] In some embodiments, 1% to 90% of the nucleotides of the tailing nucleic acid are modified nucleotides. In some embodiments, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 12%, at least 14%, at least 16%, at least 18%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% of the nucleotides of the tailing nucleic acid are modified nucleotides. In some embodiments, three or more of the last 10 to 25 nucleotides of the tailing nucleic acid are modified nucleotides. In some embodiments, at least 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 of the last 10 to 25 nucleotides of the tailing nucleic acid are modified nucleotides.

[0214] In some embodiments, the tailing nucleic acid comprises a polyA region comprising two or more polyA tails, wherein at least two instances of the polyA tails are covalently linked to a first linker. In some embodiments, the tailing nucleic acid comprises a polyA region comprising the following structure: -(first polyA tail)-[(first linker)-(second polyA tail)n1]n2, where each instance of n1 is independently an integer between 1 and 20, inclusive, and n2 is an integer between 2 and 10, inclusive, and the first poly-A tail is covalently ligated to the 3'UTR of the mRNA.

[0215] In some embodiments, the tailing nucleic acid comprises a polyA region comprising a brush oligonucleotide comprising two or more polyA tails.

[0216] In some embodiments, each instance of n1 is 1. In some embodiments, at least one instance of the first linker is covalently attached to an internal nucleotide of the first poly-A tail. In some embodiments, at least one instance of the first linker is covalently attached to the second poly-A tail at the 3' nucleotide of the second poly-A tail. In some embodiments, at least one instance of the first linker comprises at least one moiety formed by reacting two orthogonal click chemistry handles. In certain embodiments, the click chemistry handles are as described herein. In some embodiments, each instance of the first linker is independently a substituted or unsubstituted C 1-200 Alkylene, substituted or unsubstituted C 1-200 Alkenylene, substituted or unsubstituted C 1-200 Alkynylene, substituted or unsubstituted C 1-200 Heteroalkylene, substituted or unsubstituted C 1-200 Heteroalkenylene, or substituted or unsubstituted C 1-200 In some embodiments, substituted or unsubstituted C 1-200 Alkylene, substituted or unsubstituted C 1-200 Alkenylene, substituted or unsubstituted C 1-200 Alkynylene, substituted or unsubstituted C 1-200 Heteroalkylene, substituted or unsubstituted C 1-200 Heteroalkenylene, and substituted or unsubstituted C 1-200 One or more carbon atoms in the parent chain of each instance of heteroalkynylene are independently replaced with a substituted or unsubstituted carbocyclylene, a substituted or unsubstituted heterocyclylene, a substituted or unsubstituted arylene, or a substituted or unsubstituted heteroarylene. In some embodiments, a substituted or unsubstituted C 1-200 Alkylene, substituted or unsubstituted C 1-200Alkenylene, substituted or unsubstituted C 1-200 Alkynylene, substituted or unsubstituted C 1-200 Heteroalkylene, substituted or unsubstituted C 1-200 Heteroalkenylene, and substituted or unsubstituted C 1-200 One or more carbon atoms in the parent chain of each instance of heteroalkynylene independently may be TIFF2026016516000093.tif12128. In some embodiments, substituted or unsubstituted C 1-200 Alkylene, substituted or unsubstituted C 1-200 Alkenylene, substituted or unsubstituted C 1-200 Alkynylene, substituted or unsubstituted C 1-200 Heteroalkylene, substituted or unsubstituted C 1-200 Heteroalkenylene, and substituted or unsubstituted C 1-200 One or more carbon atoms in the parent chain of each instance of heteroalkynylene independently may be TIFF2026016516000094.tif31140. In some embodiments, at least one example of the first linker has the formula: TIFF2026016516000095.tif11128, wherein L 1 Each instance of is independently a substituted or unsubstituted C 1-199 Alkylene, or substituted or unsubstituted C 1-199 is heteroalkylene, and L 2 Each instance of is independently a substituted or unsubstituted C 1-199 Alkylene, or substituted or unsubstituted C 1-199 In some embodiments, at least one example of the first linker is a heteroalkylene. It is TIFF2026016516000096.tif16131.

[0217] In some embodiments, the first poly-A tail comprises the nucleotide sequence set forth as 5'-rArArArArArArArArArArArAdU(a1)rArArArArArArArArArArArArArAdU(a1)rArArArArArArArArArArArArAdU(a1)ddC-3' (SEQ ID NO: 1); at least one instance of the second poly-A tail comprises the nucleotide sequence set forth as 5'-rArArArArArArArArArArArArArArArArArArArArArArArArArA*rA*rA*rA*rA*rA*rA*(b1)-3' (SEQ ID NO: 2); and each instance of the first linker is independently TIFF2026016516000097.tif16144, wherein "rA" represents an adenosine ribonucleotide, "dU(a1)" represents a modified uridine deoxyribonucleotide, "ddC" represents a cytosine dideoxyribonucleotide, "*" represents a phosphorothioate bond, a1 in each instance of dU(a1) represents the point of attachment at the uridine of dU(a1), b1 in rA*(b1) represents the point of attachment at the * in rA*(b1), each instance of a1 is bonded to one instance of a2, and each instance of b1 is bonded to one instance of b2. In some embodiments, the modified uridine deoxyribonucleotide is 5-octadiynyldeoxyuridine, which has the structure: Shown in TIFF2026016516000098.tif30128.

[0218] In some embodiments, the tailing nucleic acid comprises a polyA region comprising a dendrimer comprising two or more polyA tails.

[0219] In some embodiments, each instance of n1 is independently an integer from 2 to 20, inclusive. In some embodiments, at least one instance of the first linker comprises a dendrimer. In some embodiments, at least one instance of the dendrimer is a polyamidoamine (PAMAM) dendrimer. In some embodiments, at least one instance of the PAMAM dendrimer has the formula: TIFF2026016516000099.tif22128, wherein each instance of n3 is independently an integer from 1 to 10 (inclusive); each instance of n4 is independently an integer from 0 to 10 (inclusive); R 2 Each instance of is independently hydrogen or TIFF2026016516000100.tif13128, but R 2 At least three examples of TIFF2026016516000101.tif13128, R 1 Each example is a substituted or unsubstituted C 1-10 Alkylene, substituted or unsubstituted C 1-10 Alkenylene, substituted or unsubstituted C 1-10 Alkynylene, substituted or unsubstituted C 1-10 Heteroalkylene, substituted or unsubstituted C 1-10 Heteroalkenylene, or substituted or unsubstituted C 1-10 In some embodiments, substituted or unsubstituted C 1-10 Alkylene, substituted or unsubstituted C 1-10 Alkenylene, substituted or unsubstituted C 1-10 Alkynylene, substituted or unsubstituted C 1-10 Heteroalkylene, substituted or unsubstituted C 1-10 Heteroalkenylene, and substituted or unsubstituted C 1-10 One or more carbon atoms in the parent chain of each instance of heteroalkynylene is independently replaced with a substituted or unsubstituted carbocyclylene, a substituted or unsubstituted heterocyclylene, a substituted or unsubstituted arylene, or a substituted or unsubstituted heteroarylene, and each instance of n5 is independently an integer from 0 to 10, inclusive. In some embodiments, at least one instance of a PAMAM dendrimer has the following formula: It is TIFF2026016516000102.tif55128.

[0220] In some embodiments, substituted or unsubstituted C 1-10 Alkylene, substituted or unsubstituted C 1-10Alkenylene, substituted or unsubstituted C 1-10 Alkynylene, substituted or unsubstituted C 1-10 Heteroalkylene, substituted or unsubstituted C 1-10 Heteroalkenylene, and substituted or unsubstituted C 1-10 One or more carbon atoms in the parent chain of each instance of heteroalkynylene independently may be TIFF2026016516000103.tif13128. In some embodiments, substituted or unsubstituted C 1-10 Alkylene, substituted or unsubstituted C 1-10 Alkenylene, substituted or unsubstituted C 1-10 Alkynylene, substituted or unsubstituted C 1-10 Heteroalkylene, substituted or unsubstituted C 1-10 Heteroalkenylene, and substituted or unsubstituted C 1-10 One or more carbon atoms in the parent chain of each instance of heteroalkynylene independently may be TIFF2026016516000104.tif30138. In some embodiments, at least one example of a first linker has the following formula: It is TIFF2026016516000105.tif62157.

[0221] In some embodiments, the first poly-A tail and the second poly-A tail comprise nucleotides shown as 5'-rArArArArArArArArArArArArArArArArArArArArArArArArA*rA*rA*rA*rA*rA* / AzideN / -3' (SEQ ID NO: 3), where "rA" represents an adenosine ribonucleotide, "AzideN" represents a 3' azide moiety, and "*" represents a phosphorothioate linkage.

[0222] In some embodiments, at least one instance of the first linker is covalently attached to the 3' nucleotide of the first poly-A tail. In some embodiments, at least one instance of the first linker is covalently attached to the 3' nucleotide of at least one instance of the second poly-A tail.

[0223] In some embodiments, the capping nucleic acid comprises a 5' cap region comprising two or more 5' caps, at least two instances of the 5' caps being covalently linked by a second linker. In some embodiments, the capping nucleic acid comprises a 5' cap region comprising the following structure: -(first 5' cap)-[(second linker)-(second 5' cap)m1]m2, where each instance of m1 is independently an integer between 1 and 20, inclusive, and m2 is an integer between 2 and 10, inclusive, the first 5' cap is covalently linked to the 5' UTR, and the second linker is covalently linked to the first 5' cap and the second 5' cap.

[0224] In some embodiments, the capping nucleic acid comprises a 5' cap region comprising a brush oligonucleotide comprising two or more 5' caps.

[0225] In some embodiments, each instance of m1 is 1. In some embodiments, at least one instance of the second linker is covalently attached to an internal nucleotide of the first 5' cap. In some embodiments, at least one instance of the second linker is covalently attached to the second 5' cap at the 3' nucleotide of the second 5' cap. In some embodiments, at least one instance of the second linker comprises at least one moiety formed by reacting two orthogonal click chemistry handles. In certain embodiments, the click chemistry handles are described herein. In some embodiments, each instance of the second linker is independently a substituted or unsubstituted C 1-200 Alkylene, substituted or unsubstituted C 1-200 Alkenylene, substituted or unsubstituted C 1-200 Alkynylene, substituted or unsubstituted C 1-200 Heteroalkylene, substituted or unsubstituted C 1-200 Heteroalkenylene, or substituted or unsubstituted C 1-200 In some embodiments, substituted or unsubstituted C 1-200 Alkylene, substituted or unsubstituted C1-200 Alkenylene, substituted or unsubstituted C 1-200 Alkynylene, substituted or unsubstituted C 1-200 Heteroalkylene, substituted or unsubstituted C 1-200 Heteroalkenylene, and substituted or unsubstituted C 1-200 One or more carbon atoms in the parent chain of each instance of heteroalkynylene are independently replaced with a substituted or unsubstituted carbocyclylene, a substituted or unsubstituted heterocyclylene, a substituted or unsubstituted arylene, or a substituted or unsubstituted heteroarylene. In some embodiments, a substituted or unsubstituted C 1-200 Alkylene, substituted or unsubstituted C 1-200 Alkenylene, substituted or unsubstituted C 1-200 Alkynylene, substituted or unsubstituted C 1-200 Heteroalkylene, substituted or unsubstituted C 1-200 Heteroalkenylene, and substituted or unsubstituted C 1-200 One or more carbon atoms in the parent chain of each instance of heteroalkynylene independently may be TIFF2026016516000106.tif12128. In some embodiments, substituted or unsubstituted C 1-200 Alkylene, substituted or unsubstituted C 1-200 Alkenylene, substituted or unsubstituted C 1-200 Alkynylene, substituted or unsubstituted C 1-200 Heteroalkylene, substituted or unsubstituted C 1-200 Heteroalkenylene, and substituted or unsubstituted C 1-200 One or more carbon atoms in the parent chain of each instance of heteroalkynylene independently may be TIFF2026016516000107.tif30141. In some embodiments, at least one example of the second linker has the formula: TIFF2026016516000108.tif11128, wherein L 3 Each instance of is independently a substituted or unsubstituted C 1-199 Alkylene, or substituted or unsubstituted C 1-199 is heteroalkylene, and L 4Each instance of is independently a substituted or unsubstituted C 1-199 Alkylene, or substituted or unsubstituted C 1-199 In some embodiments, at least one example of the second linker is a heteroalkylene. It is TIFF2026016516000109.tif16131.

[0226] In some embodiments, the first 5' cap comprises the nucleotide sequence set forth as 5'- / Cap / rGrGrGrArArAdU(c1)rArArGrArGrArGrArArArGrArArGrArGdU(c1)rArArGrArArGrArArAdU(c1)rA-3' (SEQ ID NO:4), and the second 5' cap comprises the nucleotide sequence set forth as 5'- / Cap / rGrGrGrArGrArCrTrGrCrCrArCrCrA*rA*rA*rA*rA*rA*(d1)-3' (SEQ ID NO:5), wherein: Each instance of the second linker independently can be: TIFF2026016516000110.tif15147, in which "rA" represents an adenosine ribonucleotide, "rT" represents a thymidine ribonucleotide, "rC" represents a cytidine ribonucleotide, "rG" represents a guanosine ribonucleotide, "dU(c1)" represents a modified uridine deoxyribonucleotide, "*" represents a phosphorothioate bond, "Cap" represents a 5' cap, c1 in each instance of dU(c1) represents the point of attachment to U in dU(c1), d1 in rA*(d1) represents the point of attachment to * in rA*(d1), each instance of c1 is attached to an instance of c2, and each instance of d1 is attached to an instance of d2. In some embodiments, the modified uridine deoxynucleotide is 5-octadiynyl deoxyuridine, having the structure: Shown in TIFF2026016516000111.tif29128.

[0227] In some embodiments, the capping nucleic acid comprises a 5' cap region that comprises a dendrimer that includes two or more 5' caps.

[0228] In some embodiments, each instance of m1 is independently an integer from 2 to 20, inclusive. In some embodiments, at least one instance of the second linker comprises a dendrimer. In some embodiments, at least one instance of the dendrimer is a polyamidoamine (PAMAM) dendrimer. In some embodiments, at least one instance of the PAMAM dendrimer has the formula: TIFF2026016516000112.tif22128, wherein each instance of m3 is independently an integer from 1 to 10 (inclusive); each instance of m4 is independently an integer from 0 to 10 (inclusive); R 12 Each instance of is independently hydrogen or TIFF2026016516000113.tif13128, but R 12 At least three examples of TIFF2026016516000114.tif13128, R 11 Each example is a substituted or unsubstituted C 1-10 Alkylene, substituted or unsubstituted C 1-10 Alkenylene, substituted or unsubstituted C 1-10 Alkynylene, substituted or unsubstituted C 1-10 Heteroalkylene, substituted or unsubstituted C 1-10 Heteroalkenylene, or substituted or unsubstituted C 1-10 In some embodiments, substituted or unsubstituted C 1-10 Alkylene, substituted or unsubstituted C 1-10 Alkenylene, substituted or unsubstituted C 1-10 Alkynylene, substituted or unsubstituted C 1-10 Heteroalkylene, substituted or unsubstituted C 1-10 Heteroalkenylene, and substituted or unsubstituted C 1-10One or more carbon atoms in the parent chain of each instance of heteroalkynylene is independently replaced with a substituted or unsubstituted carbocyclylene, a substituted or unsubstituted heterocyclylene, a substituted or unsubstituted arylene, or a substituted or unsubstituted heteroarylene, and each instance of m5 is independently an integer from 0 to 10, inclusive. In some embodiments, at least one instance of a PAMAM dendrimer has the following formula: Includes TIFF2026016516000115.tif55128.

[0229] In some embodiments, substituted or unsubstituted C 1-10 Alkylene, substituted or unsubstituted C 1-10 Alkenylene, substituted or unsubstituted C 1-10 Alkynylene, substituted or unsubstituted C 1-10 Heteroalkylene, substituted or unsubstituted C 1-10 Heteroalkenylene, and substituted or unsubstituted C 1-10 One or more carbon atoms in the parent chain of each instance of heteroalkynylene independently may be TIFF2026016516000116.tif13128. In some embodiments, substituted or unsubstituted C 1-10 Alkylene, substituted or unsubstituted C 1-10 Alkenylene, substituted or unsubstituted C 1-10 Alkynylene, substituted or unsubstituted C 1-10 Heteroalkylene, substituted or unsubstituted C 1-10 Heteroalkenylene, and substituted or unsubstituted C 1-10 One or more carbon atoms in the parent chain of each instance of heteroalkynylene independently may be TIFF2026016516000117.tif31136. In some embodiments, at least one example of a first linker has the following formula: It is TIFF2026016516000118.tif62157.

[0230] In some embodiments, the first 5' cap comprises a nucleotide sequence designated as / 5AzideN / rArArArArA-3' and the second 5' cap comprises a nucleotide sequence designated as 5'- / Cap / rGrGrGrArGrArCrTrGrCrCrArCrCrA*rA*rA*rA*rA*rA* / 3AzideN / -3' (SEQ ID NO: 6), where "rA" represents an adenosine ribonucleotide, "rT" represents a thymidine ribonucleotide, "rC" represents a cytidine ribonucleotide, "rG" represents a guanosine ribonucleotide, "5AzideN" represents a 5' azido moiety, "3AzideN" represents a 3' azido moiety, "*" represents a phosphorothioate linkage, and "Cap" represents the 5' cap.

[0231] In some embodiments, at least one instance of the second linker is covalently attached to the 3' nucleotide of the first 5' cap. In some embodiments, at least one instance of the second linker is covalently attached to the 3' nucleotide of at least one instance of the second 5' cap.

[0232] In some embodiments, the modified mRNA described herein is purified by any method known in the art to remove undesired components from IVT or related reactions (e.g., click chemistry reactions), including unincorporated rNTPs, protein enzymes, salts, metal ions, short RNA oligos, etc. Techniques for isolating mRNA transcripts are well known in the art. Well-known procedures include phenol / chloroform extraction or precipitation with alcohol (ethanol, isopropanol) in the presence of monovalent cations or lithium chloride. Additional non-limiting examples of purification procedures that can be used include size exclusion chromatography (Lukavsky, PJ and Puglisi, JD, 2004, Large-scale preparation and purification of polyacrylamide-free RNA oligonucleotides, RNA v. 10, 889-893), silica-based affinity chromatography, and polyacrylamide gel electrophoresis (Bowman, et al., in RNA in vitro transcription and RNA purification by denaturing PAGE in Recombinant and in vitro RNA synthesis Methods v. 941 Conn GL (ed), New York, NY Humana Press, 2012). Purification can be performed using various commercially available kits, including, but not limited to, the SV Total Isolation System (Promega) and the In Vitro Transcription Cleanup and Concentration Kit (Norgen Biotek). Techniques for removing contaminants such as dsDNA have been developed and are known in the art, including, but not limited to, scalable HPLC purification (see, e.g., Kariko, et al., 2011, Nucl Acid Res, v.39 e142; Weissman, et al., 2012, Synthetic Messenger RNA and Cell Metabolism Modulation v.969 (Rabinovich, PHEd)).In a preferred embodiment, the modified mRNA is purified via HPLC, and HPLC-purified mRNA has been reported to be translated at much higher levels, particularly in primary cells and in vivo, compared to other purification methods.

[0233] Compositions Comprising Modified mRNA and Methods of Use In some aspects, the present disclosure provides a composition comprising any one of the modified mRNAs provided herein. In some embodiments, the modified mRNA is produced by any of the methods provided herein, including ligating a tailing nucleic acid and / or a capping nucleic acid to the RNA. Compositions comprising modified mRNA are useful for delivering the modified mRNA to cells to vaccinate a subject against a foreign antigen or to express a therapeutic protein to treat a condition or disorder. Compositions comprising modified mRNA are also useful for exerting a desired effect on a subject in the absence of disease, such as for agricultural use. For example, mRNAs encoding biological pesticides or growth enhancers can be used to increase plant resistance to pests or regulate growth in a manner that increases crop yield, respectively.

[0234] In some embodiments, the open reading frame (ORF) of an mRNA is codon-optimized for expression in a cell of interest. As used herein, "codon optimization" refers to the preferential use of codons that are more efficiently translated in a cell. Multiple codons can encode the same amino acid, and the translation rate and efficiency of each codon is determined by several factors, such as the intracellular concentration of aminoacyl-tRNAs containing complementary anticodons. Codon optimization of a nucleic acid sequence can involve replacing one or more codons with a codon that encodes the same amino acid as the replaced codon but is translated more efficiently than the replaced codon. For example, the amino acid threonine (Thr) can be encoded by ACA, ACC, ACG, or ACT (ACU in RNA), but in mammalian host cells, ACC is the most commonly used codon, and in other species, a different Thr codon may be preferred for codon optimization. Therefore, an mRNA with a codon-optimized open reading frame is expected to be translated more efficiently and produce more polypeptides in a given amount of time than an mRNA with an open reading frame that is not codon-optimized. In some embodiments, the open reading frame is codon-optimized for expression in human cells.

[0235] In some embodiments of the modified mRNA provided herein, the open reading frame encodes an antigen or therapeutic protein. As used herein, a "therapeutic protein" refers to a protein that, when expressed in a subject, such as a human subject, having or at risk of developing a disease or disorder, prevents, reduces, or alleviates one or more signs or symptoms of the disease. A therapeutic protein can be, for example, an essential enzyme, clotting factor, transcription factor, growth factor, cytokine, chemokine, antibody (or antibody fragment thereof), protein hormone, signaling protein, structural protein, or cell surface receptor encoded by a gene that is mutated in the subject. Mutations in genes encoding such proteins can cause reduced levels of the protein to be expressed in one or more cells of the subject. For example, IPEX syndrome in humans is caused by mutations in the FOXP3 gene, which inhibits the development of FOXP3+ regulatory T cells and leads to increased susceptibility to autoimmune and inflammatory disorders. Thus, expression of an essential enzyme, clotting factor, transcription factor, growth factor, cytokine, chemokine, antibody (or antibody fragment thereof), protein hormone, signaling protein, structural protein, or cell surface receptor from mRNA can compensate for a mutation in the gene encoding such protein in a subject. In some embodiments, a therapeutic protein is a protein that is expressed in one or more cells of a subject at a level that is lower (e.g., significantly lower) than a reference level, such as the level of expression of the protein that is typical in cells of one or more healthy subjects (i.e., subjects who do not have and are not at risk of developing a disease or disorder). As used herein, "antigen" refers to a molecule (e.g., a protein) that, when expressed in a subject, elicits the production of antibodies in the subject that bind to the antigen. In some embodiments, the antigen is a protein derived from a pathogen, such as a pathogenic virus, bacterium, protozoan, or fungus. In some embodiments, the antigen is a protein derived from a virus (viral antigen) or a fragment thereof. In some embodiments, the antigen is a protein derived from a bacterium (bacterial antigen) or a fragment thereof.In some embodiments, the antigen is a protein derived from a protozoan (protozoan antigen) or a fragment thereof. In some embodiments, the antigen is a protein derived from a fungus (fungal antigen) or a fragment thereof. A fragment of a full-length protein refers to a protein that is present in the amino acid sequence of the full-length protein but has an amino acid sequence that is shorter than the amino acid sequence.

[0236] In some embodiments, the compositions (e.g., pharmaceutical compositions) provided herein further comprise one or more additional agents. In some embodiments, the additional agent is a nucleotide, nucleic acid, amino acid, peptide, protein, small molecule, aptamer, lipid, or carbohydrate. In some embodiments, the additional agent is an agent that has a therapeutic effect when administered to a subject. In some embodiments, the additional agent is an agent that can regulate the expression of a gene and / or protein of interest, such as a short hairpin RNA (shRNA), small interfering RNA (siRNA), or antisense oligonucleotide (ASO). In some embodiments, the additional agent is a small molecule inhibitor. In some embodiments, the additional agent is an agent that can induce or enhance an immune response in a subject. In some embodiments, the additional agent is an antigen (e.g., a viral antigen, a bacterial antigen). In some embodiments, the additional agent is an adjuvant, defined as an agent that is sufficient to enhance an immune response in a subject when administered in an effective amount, but does not induce an immune response in a subject when administered alone. In some embodiments, the additional agent is an enzyme, such as an enzyme that can catalyze one or more chemical reactions in a subject or in cells of a subject.

[0237] In some aspects, the present disclosure provides a delivery reagent comprising any of the modified mRNAs provided herein. In some embodiments, any of the modified mRNAs provided herein are conjugated to a delivery agent. Any of the modified mRNAs provided herein can be conjugated to a delivery agent comprising, for example, a lipid, peptide, protein, antibody, or carbohydrate. Lipids used in the conjugation and delivery of modified mRNAs are generally known in the art and include, for example, cholesterol. Peptides, proteins, antibodies, and carbohydrates used in the conjugation and delivery of modified mRNAs are generally known in the art and include, for example, any peptide, protein, antibody, or carbohydrate known to specifically bind to a moiety (e.g., a protein) on the surface of a target cell type. Methods for conjugating lipids, peptides, proteins, antibodies, or carbohydrates to modified mRNAs include, for example, methods of conjugating lipids, peptides, proteins, antibodies, or carbohydrates to modified mRNAs at the 5' or 3' end, and are generally known in the art.

[0238] In some embodiments, any of the modified mRNAs provided herein is conjugated to or encapsulated by a delivery agent, including, for example, a nanoparticle, a microparticle, or an exosome. Nanoparticles refer to particles having a diameter of approximately 10 nm to 1000 nm. Microparticles are defined as particles having a diameter greater than 1000 nm (1 μm), such as particles having a diameter of approximately 1 μm to 100 μm. In some embodiments, the nanoparticles or microparticles are approximately spherical. In some embodiments, the nanoparticles or microparticles are hollow and comprise an internal core. In some embodiments, the nanoparticles or microparticles are lipid nanoparticles or lipid microparticles, respectively. Lipid nanoparticles or lipid microparticles refer to lipid aggregates or compositions comprising one or more lipids that form an enclosed structure having an interior and exterior surface. In some embodiments, the lipid nanoparticles or lipid microparticles comprise a lipid bilayer surrounding an aqueous core. Lipids used in formulating lipid nanoparticles and lipid microparticles for mRNA delivery are known in the art and include, but are not limited to, ionic amino lipids, non-cationic lipids, sterols, and polyethylene glycol-modified lipids. See, e.g., Buschmann et al. Vaccines. 2021.9(1):65. In some embodiments, the modified mRNA is surrounded by the lipids of the lipid nanoparticles or lipid microparticles and is present inside the lipid nanoparticles or lipid microparticles. In some embodiments, the mRNA is dispersed throughout the lipids of the lipid nanoparticles or lipid microparticles. In some embodiments, the lipid nanoparticles or lipid microparticles comprise ionic amino lipids, non-cationic lipids, sterols, and / or polyethylene glycol (PEG)-modified lipids. Lipid nanoparticles and lipid microparticles containing modified mRNA can be prepared by any means commonly known in the art, such as detergent dialysis, emulsion, centrifugation, evaporation, thin film hydration, or ethanol dilution. See, e.g., Barba et al. Pharmaceuticals. 2019.11(8):360.Exosomes are a type of lipid nanoparticle produced by eukaryotic cells as a result of inward budding of vesicles within multivesicular bodies, generally ranging in diameter from 30 nm to 150 nm. Exosomes contain a heterogeneous mixture of endogenous lipids, such as phospholipids, membrane-anchored proteins, and carbohydrates present in eukaryotic cells, surrounding an aqueous core. Exosomes may possess beneficial characteristics difficult to achieve with synthetically produced lipid nanoparticles, such as the ability to cross the blood-brain barrier and deliver modified mRNA to tissues within the brain. Modified mRNA-containing exosomes can be produced by any means commonly known in the art, such as by sonicating or electroporating isolated exosomes in the presence of modified mRNA, or by mixing exosomes with lipid-conjugated modified mRNA, such as modified mRNA conjugated to cholesterol. See, for example, Roberts et al. Nat Rev Drug Discov. 2020.19(10):673-694.

[0239] In some embodiments, the nanoparticles or microparticles are polymeric nanoparticles or polymeric microparticles, respectively. Polymeric nanoparticles or polymeric microparticles refer to nanoparticle or microparticle compositions, respectively, comprising one or more polymers that form polymer aggregates or enclosed structures having an inner and outer surface. In some embodiments, the polymeric nanoparticles or polymeric microparticles comprise a polymer layer surrounding an aqueous core. Polymers used in formulating polymeric nanoparticles and polymeric microparticles for mRNA delivery are generally known in the art and include cationic polymers such as, but not limited to, polyethyleneimine (PEI), poly-amido-amine (PAA), poly-beta-amino-ester (PBAE), polylysine (PLL), spermine, chitosan, polyurethane, and derivatives thereof (e.g., PEI-stearic acid (PSA) copolymer). See, e.g., Liu et al. Front Bioeng Biotechnol. 2021.9:718753. In some embodiments, the modified mRNA is surrounded by the polymer of the polymeric nanoparticle or polymeric microparticle and is present inside the polymeric nanoparticle or polymeric microparticle. In some embodiments, the mRNA is dispersed throughout the polymer of the polymeric nanoparticles or microparticles.

[0240] In some embodiments, the nanoparticles or microparticles are protein nanoparticles or protein microparticles, respectively. Protein nanoparticles or protein microparticles refer to nanoparticle or microparticle compositions, respectively, that contain one or more proteins forming protein aggregates or enclosed structures with an inner and outer surface. In some embodiments, the protein nanoparticles or protein microparticles contain a protein layer surrounding an aqueous core. Proteins used in the formulation of protein nanoparticles and protein microparticles for mRNA delivery are generally known in the art and include, but are not limited to, viral coat proteins and ferritin. See, for example, Wang et al. Nat Nanotechnol. 2020.15(5):406-416. In some embodiments, the modified mRNA is surrounded by the protein of the protein nanoparticle or protein microparticle and is present inside the protein nanoparticle or protein microparticle. In some embodiments, the modified mRNA is present outside the protein of the protein nanoparticle or protein microparticle and is bound to the outer surface of the protein nanoparticle or protein microparticle. In some embodiments, the modified mRNA is conjugated to the protein of a protein nanoparticle or protein microparticle via a covalent bond, such as formed by a click chemistry reaction, or by fusing the modified mRNA and protein, respectively, to a protein or peptide of a protein / peptide pair (e.g., a SpyCatcher / SpyTag protein / peptide pair) that are known to react to form a covalent bond.

[0241] In some embodiments, the nanoparticles or microparticles are solid nanoparticles or solid microparticles, respectively. Solid nanoparticles or solid microparticles refer to nanoparticle or microparticle compositions, respectively, comprising one or more materials that form a solid structure having an outer surface and which may or may not include an inner surface. Solid nanoparticles or solid microparticles may comprise any suitable material commonly known in the art, such as gold, silver, or silicon dioxide (silica). In some embodiments, the modified mRNA is conjugated to the outer surface of the solid nanoparticle or solid microparticle. Solid nanoparticles and solid microparticles comprising the modified mRNA may be produced by any means commonly known in the art, such as, for example, by attaching the modified mRNA to the surface of the solid nanoparticle or solid microparticle via a thiol bond (e.g., modifying DNA to include a cyclic disulfide anchor group) or by modifying the outer surface of the solid nanoparticle or solid microparticle with one or more cationic materials (e.g., PEI) on which the modified mRNA is present. See, e.g., Roberts et al. Nat Rev Drug Discov. 2020. 19(10):673-694, Lee et al. Nano Lett. 2007, 7(7):2112-2115, and Paris and Vallet-Regi. Pharmaceutics. 2020, 12(6):526.

[0242] In some aspects, the disclosure provides a cell comprising any of the modified mRNAs provided herein. In some embodiments, the cell is a human cell comprising any one of the modified mRNAs provided herein. A "cell" is the basic structural and functional unit of all known independent living organisms. It is the smallest unit of life classified as an organism. Some organisms, such as most bacteria, are unicellular (consisting of a single cell). Other organisms, such as plants, fungi, and animals, including cows, horses, chickens, turkeys, sheep, pigs, dogs, cats, and humans, are multicellular. In some embodiments, the half-life of the modified mRNA in the cell is between 15 and 900 minutes. In some embodiments, the half-life of the modified mRNA in the cell is between 30 and 600 minutes. In some embodiments, the half-life of the modified mRNA in the cell is between 60 and 300 minutes. In some embodiments, the half-life of the modified mRNA is at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, or at least 60 minutes. In some embodiments, the half-life of the modified mRNA in a cell is at least 30, at least 60, at least 90, at least 120, at least 150, at least 180, at least 210, at least 240, at least 270, at least 300, at least 330, at least 360, at least 390, at least 420, at least 450, at least 480, at least 510, at least 540, at least 570, at least 600, at least 630, at least 660, at least 690, at least 720, at least 750, at least 780, at least 810, at least 840, or at least 870 minutes.

[0243] In some aspects, the present disclosure provides a composition comprising any of the modified mRNAs, delivery agents, or cells provided herein. In some embodiments, the composition further comprises one or more additional agents, such as nucleotides, nucleic acids, amino acids, peptides, proteins, small molecules, aptamers, lipids, or carbohydrates. In some embodiments, the additional agent has a therapeutic effect when administered to a subject. In some embodiments, the additional agent is an agent for use in modulating the expression and / or activity of one or more gene products (e.g., proteins) in a subject. In some embodiments, the additional agent is a nucleic acid for use in reducing the expression and / or activity of one or more gene products (e.g., proteins), such as a short hairpin RNA (shRNA), small interfering RNA (siRNA), or antisense oligonucleotide (ASO). In some embodiments, the additional agent is an inhibitor for reducing the activity of one or more gene products (e.g., proteins). In some embodiments, the agent is a small molecule inhibitor. In some embodiments, the additional agent is an agent for enhancing an immune response in a subject. In some embodiments, the additional agent is an antigen, such as a nucleic acid antigen, a protein antigen, or a phospholipid antigen. In some embodiments, the additional agent is an adjuvant, such as, for example, aluminum hydroxide or aluminum potassium sulfate (alum), monophosphoryl lipid A (MPL), an oil-in-water emulsion (e.g., a squalene emulsion), cytosine phosphoguanine (CpG) oligodeoxynucleotide, or another adjuvant known in the art. See, e.g., Di Pasquale, A et al. Vaccines. 2015.3(2):320-343. In some embodiments, the composition is a pharmaceutical composition comprising any one of the modified mRNA, delivery agent, or cells provided herein and a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient, carrier, buffer, stabilizer, tonicity agent, preservative, or antioxidant, or other material known to those skilled in the art, should be non-toxic and should not interfere with the efficacy of the active ingredient.The precise nature of the carrier or other material may depend on the route of administration, for example parenteral, intramuscular, intradermal, sublingual, buccal, ocular, intranasal, subcutaneous, intrathecal, intratumor, oral, vaginal, or rectal.

[0244] In some aspects, the disclosure provides methods of administering any of the modified mRNAs, delivery agents, cells, compositions, or pharmaceutical compositions provided herein to a subject. In some embodiments, the subject is a human. In some embodiments, administration is parenteral, intramuscular, intradermal, sublingual, buccal, ocular, intranasal, subcutaneous, intrathecal, intratumoral, oral, vaginal, or rectal. In some embodiments, the compositions should be stored below 50°C, below 40°C, below 30°C, below 20°C, below 10°C, below 0°C, below -10°C, below -20°C, below -30°C, below -40°C, below -50°C, below -60°C, below -70°C, or below -80°C so that the nucleic acid is relatively stable over time. In some embodiments, the modified mRNA is introduced into cells in the subject by in vivo electroporation. In vivo electroporation is a process of introducing nucleic acids or other molecules into cells of a subject using pulses of electricity to promote passage of the nucleic acid or other molecule through the cell membrane and / or cell wall. See, e.g., Somiari et al. Molecular Therapy., 2000.2(3):178-187. The modified mRNA to be delivered is administered to a subject, such as by injection, and a pulse of electricity is applied to the injection site, whereby the electricity promotes entry of the nucleic acid into cells at the administration site. In some embodiments, the modified mRNA is delivered to and taken up by cells of a subject (e.g., cells local to the administration site or cells throughout the subject) via a delivery agent associated with (e.g., conjugated to) the modified mRNA. In some embodiments, the modified mRNA is administered with other elements, such as buffers and / or excipients, that increase the efficiency of electroporation.

[0245] In some aspects, the present disclosure provides kits comprising any of the RNAs and any of the tailing and / or capping nucleic acids provided herein. The RNA and tailing and / or capping nucleic acid can be combined in the presence of an RNA ligase to produce a modified mRNA, such as one of the modified mRNAs provided herein. In some embodiments, the kit comprises a ligase. In some embodiments, the kit comprises an RNA ligase. In some embodiments, the kit comprises T4 RNA ligase. In some embodiments, the kit comprises T4 RNA ligase 1. In some embodiments, the kit comprises T4 RNA ligase 2. In some embodiments, the kit comprises RtcB RNA ligase. In some embodiments, the kit further comprises a buffer for performing ligation. In some embodiments, the kit further comprises a nucleotide triphosphate, such as ATP, to provide the energy required by the ligase. In some embodiments, the kit should be stored at below 50°C, below 40°C, below 30°C, below 20°C, below 10°C, below 0°C, below -10°C, below -20°C, below -30°C, below -40°C, below -50°C, below -60°C, below -70°C, or below -80°C so that the nucleic acids are relatively stable over time.

[0246] In some aspects, the present disclosure provides kits comprising any of the pharmaceutical compositions provided herein and a delivery device. A delivery device refers to a machine or apparatus suitable for administering a composition to a subject, such as a syringe or needle. In some embodiments, the kit should be stored at temperatures below 50°C, below 40°C, below 30°C, below 20°C, below 10°C, below 0°C, below -10°C, below -20°C, below -30°C, below -40°C, below -50°C, below -60°C, below -70°C, or below -80°C, so that the nucleic acids of the pharmaceutical composition are relatively stable over time. In some embodiments, the kit further comprises instructions for administering any of the pharmaceutical compositions provided herein to a subject. [Example]

[0247] In order that the disclosure may be more fully understood, the following examples are set forth to illustrate the modified mRNAs, pharmaceutical compositions, kits, and methods provided herein, and should not be construed in any way as limiting the scope thereof.

[0248] Materials and Methods Plasmid cloning, characterization, and purification The DNA coding sequence (CDS) for the protein of interest was inserted into an optimized backbone containing (in order): an SP6 or T7 promoter sequence, a 5' human alpha globin UTR, the CDS, a 3' human alpha globin UTR, a 100xA template-encoded poly(A) tail, and an Esp3I linearization site. The CDS-containing plasmid / gene block was PCR amplified, gel-purified, assembled into the optimized backbone using NEBuilder HiFi DNA Assembly Master Mix (NEB, E2621S), transformed into stable cells, and sequence-verified by Sanger sequencing.

[0249] The firefly luciferase construct was obtained from the pmirGLO dual-luciferase miRNA target expression vector (Promega, E1330). The destabilized firefly luciferase construct (i.e., firefly-PEST) was obtained by inserting a GeneBlock (IDT, human codon-optimized) encoding a degron derived from mouse ornithine decarboxylase. The Cas9-encoding construct was obtained from Addgene (plasmid number 52962) registered by the Feng Zhang lab. The nanoluciferase construct was obtained by gene synthesis from Genewiz. The Renilla luciferase construct was obtained from pmirGLO without cloning into an optimized vector.

[0250] Synthesis and characterization of linear mRNA DNA plasmids were linearized with Esp3I (NEB, R0734S). Linearized plasmids were purified with the DNA Clean & Concentrator-25 kit from Zymo Research (D4033) and characterized using agarose gel electrophoresis. mRNA constructs were synthesized by in vitro transcription (IVT) using the mMESSAGE mMACHINE SP6 Transcription Kit [ThermoFisher Scientific, AM1340] (for SP6 promoter constructs) or the HiScribe T7 High-Yield RNA Synthesis Kit [NEB, E2040S] (for T7 promoter constructs) according to the manufacturer's protocol, except for 100% replacement of UTP with N1-methylpseudouridine-5'-triphosphate (Trilink, N-1081-1) and the addition of 1:50 SUPERase-In RNase inhibitor [ThermoFisher Scientific, AM2694]. All linear mRNA constructs were co-transcriptionally capped by replacing GTP with a final concentration of 2 mM GTP and 8 mM 3'-O-Me-m7G(5')ppp(5')G RNA anti-reverse cap analog (ARCA) [NEB, S1411S] for the SP6 promoter construct. T7 constructs were co-transcriptionally capped using CleanCap Reagent AG [Trilink, N-7113]. After the IVT reaction, the DNA template was digested with TURBO DNase and purified using the MEGAclear Transcription Cleanup Kit [ThermoFisher Scientific, AM1908]. mRNA concentration was quantified immediately before use using the Qubit RNA HS Assay [ThermoFisher Scientific, Q32852]. Unless otherwise specified, mRNA products are suspended at 1:50 (v / v) in RNase-free water containing RNase inhibitors (hereafter referred to as RNase-free water) and stored at -80°C.

[0251] Chemical conjugation of modified synthetic poly(A) oligonucleotides Chemically modified poly(A) oligonucleotides were obtained from IDT and suspended in RNase-free water to a final concentration of 100 μM.

[0252] For thiol-ene / ene conjugation, disulfide-protected thiol-oligonucleotides were deprotected with 100 molar amounts of TCEP (tris(2-carboxyethyl)phosphine) and immediately mixed with an equimolar amount of alkene / alkyne-modified oligonucleotides and incubated at 37 °C for 30 min to 1 h. For radical conditions, a substoichiometric amount of 2,2-dimethoxy-2-phenylacetophenone was added and the mixture was incubated at room temperature under 370 nm irradiation [Kessil, KSPR160L370].

[0253] For amine-phosphate conjugation, oligos were mixed with 1-2 equivalents of EDC as an additive in the presence of excess imidazole. The reaction mixture was incubated at 37°C for 30 minutes to 1 hour.

[0254] For IEDDA (inverse electron demand Diels-Alder reaction) conjugation, methyltetrazine (Me-Tz) and trans-cyclooctene (TCO)-labeled oligos were obtained from the corresponding amine-modified oligos by labeling with tetrazine-PEG5-NHS ester (Click Chemistry Tools, 1143) or TCO-PEG4-TFP ester (Click Chemistry Tools, 1198) at a molar ratio of 500:1 (small molecule:oligonucleotide) in 100 mM NaHCO3 at 4°C overnight. The NHS-labeled products were purified using ethanol precipitation. The Me-Tz / TCO-labeled oligos were suspended in RNase-free water and incubated at 55°C for 30 min.

[0255] For CuAAC (copper-catalyzed azide-alkyne conjugation) conjugation, azide / alkyne-containing oligonucleotides were mixed at the indicated molar ratios (1:1 for monoantennary oligos, 2:1 for biantennary oligos, and 3:1 for triantennary oligos). The oligonucleotide mixture was diluted in a modified 1.5x click chemistry buffer (Lumiprobe, 61150, 5% SUPERase inhibitor, 5% DMSO, and 5% 10 mM dNTP mix [ThermoFisher Scientific, 18427089]), which was briefly degassed by argon purging for 20 minutes before reaction. For a typical 100 μL reaction, 33 μL of oligonucleotide solution was mixed with 66 μL of click chemistry buffer, and 2 μL of 100 mM L-ascorbic acid solution [Sigma-Aldrich, A5960] was added immediately before reaction. The mixture was incubated at 37°C for 1 hour and then stopped by adding 1 μL of 500 mM EDTA (pH 8.0). The reaction was first purified using the Monarch RNA Cleanup Kit [NEB, T2040], and the crude product was repurified using RNase-free HPLC on an Agilent 1260 Infinity II HPLC with acetonitrile [Sigma-Aldrich, 34851] and 100 mM hexylamine / acetic acid (pH 7.0, supplemented with 10% urea w / v) as the mobile phase. HPLC fractions were analyzed on Novex TBE Urea gels, stained with 1x SYBR Gold [ThermoFisher Scientific, S11494], and visualized using a BioRad ChemiDoc MP Imaging System [12003154]. The desired fractions were then pooled, desalted, and concentrated using the Monarch RNA Cleanup Kit for small-scale preparations or ethanol precipitation for large-scale preparations.

[0256] Enzymatic ligation of modified oligonucleotides to mRNA Synthetic oligos and mRNA were mixed at a 100:1 molar ratio and diluted with 2.5x 50% PEG-8000, 10x T4 RNA ligase buffer, 8x T4 RNA ligase [Promega, M1051], and RNase-free water. The reaction was incubated at 37°C for 45 minutes and inactivated by adding 50x 500 mM EDTA (pH 8.0). The product was purified using RNA Clean XP [Beckman Coulter] according to the manufacturer's protocol, except that 1 volume of beads was used. For large-scale reactions, the ligation product was purified by HPLC to recover residual modified oligos. The ligation product was characterized by an RNase H assay as previously described. If residual oligonucleotides were detected, a second purification was performed. The eluted mRNA was quantified using Qubit, suspended in RNase-free water, and stored at -80°C. For each batch of mRNA ligation, a "mock ligation" condition was included in which all reaction settings were identical except for the addition of synthetic oligos, and the recovered product was used as a linear mRNA control.

[0257] Screening for mRNA modifications using a time-course dual-luciferase assay HeLa cells (CCL-2, ATCC) were maintained in DMEM medium (ThermoFisher Scientific, 119951) containing 10% FBS and 1% penicillin-streptomycin (ThermoFisher Scientific, 15070063) in a 37°C incubator with 5% CO2 and passaged at a ratio of 1:10 every 3 days. The day before mRNA transfection, HeLa cells were seeded at 75% confluence into individual wells of three white, clear-bottom 96-well plates (Corning, 3610). The next day, 30 ng of Renilla luciferase (internal control) mRNA and 30 ng of modified firefly luciferase mRNA were transfected using Lipofectamine MessengerMAX transfection reagent (ThermoFisher Scientific, LMRNA003) according to the manufacturer's protocol. Additional controls containing Renilla luciferase mRNA alone or lipofection reagent alone were included. Three individual transfections were performed for each condition. 24 / 48 / 72 hours after transfection, the cell culture medium was removed, and the cells were rinsed with 1x DPBS. The cells were lysed, and luciferase activity was measured using the Promega Dual Glo Luciferase Assay System (Promega, E2920). Briefly, 50 μL of PBS and 50 μL of firefly luciferase working solution (prepared according to the manufacturer's protocol) were added to each well using a multichannel pipette and mixed by pipetting. After a 10-minute incubation with gentle shaking and protected from light at room temperature, firefly luciferase luminescence was measured using a microplate reader. 50 μL of freshly prepared Renilla luciferase Stop&Glow working solution (prepared according to the manufacturer's protocol) was added. Renilla luciferase luminescence was similarly measured after 10 minutes of incubation. For both firefly and Renilla luminescence, background was measured and subtracted from cells treated with Lipofectamine reagent alone. The firefly / Renilla luminescence ratio for each well was used as a readout of mRNA activity.

[0258] Firefly luciferase degron assay The day before mRNA transfection, HeLa cells were seeded at 75% confluence into individual wells of a 24-well plate. The following day, 200 ng of unmodified Renilla luciferase (internal control) mRNA and 200 ng of firefly degron mRNA with the designed modifications were transfected into each well using Lipofectamine MessengerMAX according to the manufacturer's protocol. After 6 hours of incubation, the cell culture medium containing the transfection mixture was removed, and the cells were rinsed with 1x DPBS, trypsinized, and replated at an 8:6:4:3 ratio into four white, clear-bottom 96-well plates for luciferase quantification at 8, 24, 48, and 72 hours, respectively. Luciferase was quantified at each time point using the previously described protocol. For each time point, firefly luciferase activity was normalized by cell number (replated ratio) and then by luminescence activity using firefly luminescence / renilla luminescence*average renilla luminescence, and calculated firefly luciferase luminescence was normalized to the 8-hour readout.

[0259] mRNA quantification in transfected cell cultures using STARmap / RIBOmap The day before mRNA transfection, HeLa cells were seeded at 75% confluence into individual wells of a 24-well plate. The following day, 500 ng of unmodified Renilla luciferase (internal control) mRNA and 500 ng of firefly degron mRNA with the designed modifications were transfected into each well using Lipofectamine MessengerMAX according to the manufacturer's protocol. After 6 hours of incubation, the transfection mixture was removed, and the cells were rinsed with 1x DPBS, trypsinized, and replated at a 6:4:3 ratio into six (two for each time point) 96-well glass-bottom plates (MatTek, PBK96G-1.5-5-F, poly-D-lysine [Sigma-Aldrich, A-003-M] coated) for in situ sequencing at 24, 48, and 72 hours posttransfection. Each well of transfected cells was similarly replated into two 96-well plates for each time point, one measured by STARmap(firefly) + STARmap(renilla) and the other measured by RIBOmap(firefly) + STARmap(renilla).

[0260] For quantification of mRNA abundance, we followed the published STARmap / RIBOmap protocol for cell culture (Wang et al., "Three-dimensional intact-tissue sequencing of single-cell transcriptional states" (2018) Science 361(6400), eaat5691; Hu et al., "Spatially Resolved Single-Cell Translatomics at Molecular Resolution" (2022) bioRxiv). Briefly, 24 / 48 / 72 hours after transfection, the culture medium was removed and cells were washed with 150 μL of PBS solution. Cells were then fixed with 1.6% paraformaldehyde (Electron Microscope Sciences, 15710-S) / 1X PBS (Gibco, 10010-023) for 15 minutes at room temperature, followed by further fixation and permeabilization with pre-chilled methanol at -20°C for 1 hour. Subsequently, the methanol was removed, and the cells were rehydrated with PBSTR / glycine / YtRNA (PBS supplemented with 0.1% Tween-20 [TEKNOVA INC, 100216-360], 0.5% SUPERase In [Invitrogen, AM2696], 100 mM glycine, and 1% yeast tRNA) for 5 min at RT, followed by one PBSTR wash. Samples were then hybridized with SNAIL probes targeting firefly and Renilla luciferase mRNA in hybridization buffer (2X SSC [Sigma-Aldrich, S6639], 10% formamide [Calbiochem, 655206], 1% Tween-20, 20 mM RVC [ribonucleoside vanadyl complex, New England Biolabs, S1402S], 0.5% SUPERase In, and 1% yeast tRNA, 100 nM each probe) overnight in a humidified oven at 40 °C with shaking and parafilm wrapping (see Supplementary Table 2 for probe sequences).For RIBOmap quantification, the SNAIL probe targeting firefly luciferase was replaced with a RIBOmap primer / padlock probe and splint probe targeting 18S rRNA, while the remaining protocol remained the same. After hybridization, cells were then washed twice at 37°C with PBSTR (20 min each wash) and once with high-salt wash buffer (PBSTR supplemented with 4X SSC) at 37°C, followed by a single rinse with PBSTR at RT. The ligation reaction was carried out at room temperature for 2 h, followed by circularization of the primer-adjacent padlock probe in ligation buffer (1X T4 DNA ligase buffer, T4 DNA ligase, 0.1 Weiss U / µL [ThermoFisher Scientific, EL0012], 0.5 mg / mL Ultrapure BSA [ThermoFisher Scientific, AM2618], 0.5% SUPERase In). Cells were washed twice with PBSTR, and rolling circle amplification (RCA) was performed in RCA buffer (1X Phi29 buffer, 0.2 Weiss U / μL Phi29 DNA polymerase [ThermoFisher Scientific, EP0094], 250 μM dNTP mix [ThermoFisher Scientific, 18427089], 0.5 mg / mL BSA, 0.5% SUPERase In) at 30°C for 2 h, followed by two washes with PBST. Samples were then stained with fluorescent detection oligos in wash and imaging buffer (2X SSC, 10% formamide, 100 nM per detection probe, 1X DAPI [Molecular Probes, D1306]) for 1 h at room temperature (see Supplementary Table 2 for fluorescent detection probe sequences). Confocal imaging stacks were captured using a Leica Stellaris 8 with a 60X oil objective at a pixel size of 283 × 283 nm. A 14-µm stack was imaged at 7 steps of 2 µm each. Three independent transfections were performed for each condition, with at least four representative FOVs. The same imaging settings were used for all samples to be compared.The excitation / detection wavelengths were as follows: DAPI, diode 405 nm / ~[420~489] nm; Alexa 546, WLL 557 nm / ~[569~612] nm; Alexa 647, WLL 653 nm / ~[668~738] nm.

[0261] MATLAB 2021a and CellProfiler 4.0.7 were used for amplicon count-based fluorescence image analysis. First, the amplicon centroids in each fluorescence channel (firefly, Renilla) were identified by finding the extended maximum on the image. Next, a 3x3x3 voxel volume was defined centered on the centroid of each fluorescent point. Within each voxel volume, the integrated intensity in the firefly and Renilla channels was calculated, and the ratio of firefly intensity to Renilla intensity was used for amplicon classification. For STARmap quantification, all measurements were pooled together, and the distribution of log(firefly / Renilla) values ​​was plotted. The corresponding ratio value at the lowest point (local minimum) on the distribution plot was identified as the cutoff value. The first cutoff value less than 0 was designated as cutoff 1, and the first cutoff value greater than 0 was designated as cutoff 2. For RIBOmap quantification, cutoff 1 was assigned as -0.3, and cutoff 2 was assigned as 0.3. Any amplicon with a log(firefly / renilla) value less than cutoff 1 was identified as a Renilla amplicon. Any amplicon with a log(firefly / renilla) value greater than cutoff 2 was identified as a firefly amplicon. Any amplicon with a log(firefly / renilla) value between cutoff 1 and cutoff 2 was identified as a granule. The amplicon classification information, as well as the location of all amplicons, was saved to a file. In each figure, the ratio between the number of firefly amplicons and the number of Renilla amplicons was calculated and used to reflect the amount of firefly luciferase mRNA. For single-cell STARmap quantification, cell segmentation was performed using the same method as for cell segmentation in single-cell protein quantification (see GFP ablation using Cas9 mRNA), and the segmentation mask was saved as a uint16 image. Amplicons were then assigned to cells according to their location in the mask. The ratio between the number of firefly amplicons and the number of Renilla amplicons in each cell was calculated and used to reflect the amount of firefly luciferase mRNA in a single cell.Cells that did not have the firefly amplicon or did not have the Renilla amplicon were considered not successfully transfected and were therefore excluded from the analysis.

[0262] EMSA (Electrophoretic Mobility Shift Assay / Gel Shift Assay) A codon-optimized DNA sequence (GST-tagged) encoding full-length human PABPC1 (residues 1–636) was cloned into the pBluescript II KS(−) vector using GenScript. Transformed Escherichia coli cells (BL21 competent E. coli, New England Biolabs) were grown in 1 L of Luria-Bertani medium in the presence of 50 mg ampicillin at 37°C to an absorbance at 600 nm of 0.6–0.8. Expression of glutathione S-transferase (GST) fusion proteins was induced by the addition of 0.5 or 1 mM isopropyl β-D-1-thiogalactopyranoside, and cells were grown overnight at 18°C. Cells were harvested by centrifugation and disrupted by sonication in 30 mL of lysis buffer (10 mM NaHPO (pH 7.4), 1.8 mM KHPO, 637 mM NaCl, 2.7 mM KCl, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 0.5 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, 0.15 μM aprotinin, 1 μM E-64, and 1 μM leupeptin) supplemented with 0.15% polyethyleneimine to remove nucleic acids from the lysate. Cell debris was removed by centrifugation. The supernatant was applied to a glutathione Sepharose 4B column (GE Healthcare) equilibrated with PBS at 4°C and washed with PBS at 4°C. The GST fusion protein was eluted with elution buffer (100 mM Tris-HCl (pH 7.8), 300 mM NaCl, and 20 mM glutathione 3 (reduced)). The isolated PABPC1-GST eluate was concentrated using a spin filter, and the protein concentration was quantified using the Qubit Broad Range (BR) Assay Kit [Invitrogen, Q33211]. Branched / linear poly(A) oligonucleotides labeled at the 5' end with AlexaFluor546 were synthesized as described in the preceding section.Poly(A) oligos were incubated in binding buffer (10 mM NaHPO, 1.8 mM KHPO, 137 mM NaCl, 2.7 mM KCl, 5 mM MgSO, 1 mM DTT, Novex high-density TBE sample buffer [Invitrogen, LC6678]) with various PABPC1-GST concentrations prepared by serial dilution from a stock solution. In competitive binding assays, the concentrations of Alexa546-labeled branched oligos and PABPC1-GST were kept constant, and various concentrations of unlabeled linear poly(A) oligos were added. The oligos and PABP were incubated at 4°C for 30 min and characterized by EMSA using Novex TBE gels, 4-20% [Invitrogen, EC6225BOX] at 4°C. Alexa546-labeled oligos were imaged using a BioRad ChemiDoc MP imaging system [12003154], and band intensities were quantified using ImageJ. For K calculations, the percent of poly(A) oligos bound to x-mer PABPs was calculated as the total percent of oligos bound to >x-mer PABPs, and K was calculated by plotting the percent of poly(A) oligos bound to PABPs against PABP concentration (specific binding with a Hill slope) using GraphPad Prism.

[0263] GFP ablation using Cas9 mRNA Linear / branched Cas9 mRNA was synthesized using the previously described protocol. sgRNAs were designed and synthesized using the IDT sgRNA design tool [IDT, Alt-R CRISPR-Cas9 sgRNA]. GFP-expressing HEK293 cells [GenTarget Inc, SC058] were seeded at 50% confluence in individual wells of a 12-well plate the day before mRNA transfection. The following day, cells were co-transfected with 100 ng of sgRNA and various amounts of linear / branched Cas9 mRNA using Lipofectamine MessengerMAX. After 48 hours of incubation, the cell culture medium was removed, and the cells were rinsed with 1x DPBS, trypsinized, and replated in a glass-bottom 12-well plate [MatTek, P12G-1.5-14-F, poly-D-lysine coated]. Seventy-two hours after transfection, the culture medium was removed, and the cells were washed once with 1x DPBS before being incubated in nuclear staining medium (FluoroBrite DMEM [ThermoFisher, A1896701] supplemented with a 1:2000 dilution of Hoechst 33342 [ThermoFisher, 62249]) at 37°C for 10 minutes. Confocal images of nuclei (Hoechst) and GFP were captured with a Leica Stellaris 8 using a 10x air objective with a pixel size of 1135 × 1135 nm. 14 μm stacks were captured at 2 μm / step × 7 steps. At least six representative FOVs were obtained for each condition tested, and the same imaging settings were used for all samples being compared. The excitation / detection wavelengths were as follows: Hoechst, diode 405nm / ~[430~480]nm, GFP, WLL 489nm / ~[500~576]nm.

[0264] Analysis was performed on maximum projection images of the original image stack. CellProfiler 4.0.7 was used for single-cell protein quantification. For single-cell analysis, Hoechst-stained nuclei were first identified as primary objects. The Hoechst and GFP channels were then merged and converted to a grayscale image. Cells were identified as secondary objects in this grayscale image. After cell segmentation, the GFP intensity in each cell was measured.

[0265] Circular RNA synthesis The sequence of the circular RNA encoding the secreted form of NanoLuc with a translation-enhancing UTR (iHRVB3 + PABPv3) was adopted from a previous study (Chen et al., "Engineering circular RNA for enhanced protein production," Nature biotechnology (2022) 1-11). The DNA template was synthesized from Genewiz, PCR-amplified, gel-purified, and used as the IVT template. The circRNA was synthesized using the HiScribe T7 High-Yield RNA Synthesis Kit [New England Biolabs, E2040S]. After IVT, the DNA template was digested with Turbo DNase [ThermoFisher, AM2238]. The reaction mixture was heated to 70°C for 5 minutes and then immediately placed on ice for 3 minutes. After that, GTP was added to a final concentration of 2 mM, and the reaction mixture was incubated at 55°C for 15 minutes. The circRNA was enriched by treatment with RNase R [Lucigen Corporation, RNR07250] for 1.5 hours, and the product was column-purified. The circRNA products were characterized by gel electrophoresis and, in the case of remaining linear RNA precursors, another round of RNase R digestion. The precursor and product patterns were consistent with previous reports.

[0266] In vitro secreted NanoLuc assay The day before mRNA transfection, HeLa cells were seeded at 75% confluence in individual wells of a 96-well plate. FluoroBrite DMEM supplemented with 10% fetal bovine serum and 4 mM L-glutamine was used for culture to minimize the influence of phenol red on the assay. The following day, equimolar amounts of linear, branched, and circular RNA (corresponding to 50 ng, 59 ng, and 90 ng of mRNA, respectively) were transfected into the cells using Lipofectamine. The medium was replaced with 150 μL of the aforementioned FluoroBrite medium 6 h after transfection. Every day, the entire culture medium was replaced with a fresh aliquot of 150 μL of FluoroBrite medium for each well, and 50 μL of the culture medium was used for bioluminescence measurements using the Nano-Glo Luciferase Assay System (Promega, N1110). At least three independent transfections were performed for each condition tested, and background luminescence was subtracted from each well. The assay was run for a total of 14 days.

[0267] mouse All animal procedures followed animal care guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of the Broad Institute of MIT and Harvard under animal protocol number 0255-08-19. Animal experiments were performed in compliance with IACUC policies and NIH guidelines.

[0268] In vivo delivery of mRNA BALB / c mice (male and female, 6-8 weeks old) used in the in vivo NanoLuc assay in this study were purchased from Jackson Laboratory (JAX). Branched and linear mRNA encoding NanoLuc was formulated into LNPs using the NanoAssemblr Spark Kit-Hepato9 mRNA [Precision Nanosystems, NWS0016] in a NanoAssemblr Spark instrument [Precision Nanosystems] according to the manufacturer's protocol. For formulation, the mRNA-LNPs were diluted with 10 mL of 1x PBS solution, and the buffer was exchanged by concentration with a 30 kDa spin filter [MilliporeSigma, UFC901008] to remove residual ethanol. The mRNA concentration was determined using the Quant-it RiboGreen RNA Assay Kit [ThermoFisher, R11490], and the encapsulation efficiency was greater than 95%. Linear / circular mRNA-LNPs (390 pg per nucleotide, corresponding to 350 ng of linear mRNA) were immediately injected into each mouse via retroorbital injection in an injection volume of 100 μL. Three male and three female mice were used for each condition.

[0269] In vivo imaging of NanoLuc In vivo activity of NanoLuc in mice was measured using a Competent IVIS-Perkin Elmer IVIS Spectrum CT system (Perkin Elmer). At each time point, mice were anesthetized with isoflurane. Fluorofurazine substrate (Promega) was freshly reconstituted with 1.05 ml of PBS per vial and injected into mice at a dose of 44 nmol per gram of body weight. Mice were imaged 5 minutes after injection using default settings. Luminescence activity was quantified using Aura 4.0 imaging software. Background was determined by including mice injected with the polyC-LNP negative control and subtracted from each measurement.

[0270] In vivo toxicity assay To evaluate in vivo toxicity and immune responses, blood was collected from mice on day 15 after mRNA-LNP administration and serum was prepared. AST, ALT, and TNF-alpha were measured using ELISA kits for AST [G-Biosciences, IT5530], ALT [G-Biosciences, IT5508], and TNF-alpha [R&D Systems, MTA00B] according to the manufacturer's protocol.

[0271] Example 1. Production of poly-tailed and poly-capped mRNA. Both a 3' polyA tail and a 5' cap are required for efficient translation of eukaryotic mRNA molecules (Figure 1A). However, both the polyA tail and the 5' cap are vulnerable to degradation by exonucleases, which remove nucleotides from mRNA molecules (Figure 1B). If exonucleases degrade the coding portion of an mRNA (e.g., a protein-coding sequence), the mRNA cannot be translated efficiently. Strategies for improving mRNA stability include modifying mRNA molecules to make them resistant to exonuclease activity. Exonuclease resistance can be conferred to an mRNA molecule by modifying the mRNA to include an additional polyA tail and / or a 5' cap. The additional polyA tail and 5' cap can be introduced into an mRNA molecule via any number of techniques.

[0272] In one technique, for example, an additional polyA tail can be introduced into an mRNA molecule (e.g., mRNA isolated from a cell or organism, or mRNA produced by in vitro transcription (IVT)) by ligating the 3' end of the mRNA to a "brush-like" polyA tail oligonucleotide containing an internal covalent bond to either the 3' or 5' end of one or more second nucleic acid sequences (Figure 1C). Brush-like polyA tail oligonucleotides can be produced by starting with a polyA oligonucleotide modified with several internal nucleotides capable of click chemistry (e.g., 5-octadiynyl dU) and then attaching additional polyA oligonucleotides containing a 3' azide moiety to these nucleotides (Figure 1D). For additional exonuclease resistance, any of the oligonucleotides can be modified with one or more modified nucleotides or internucleotide linkages such as phosphorothioate linkages. For example, a first poly A oligonucleotide may comprise the sequence / 5Phos / rArArArArArA / i5OctdU / rArArArArArArArArArArArArA / i5OctdU / rArArArArArArArArArArArArA / i5OctdU / / 3ddC / (SEQ ID NO: 7), to which is attached an oligonucleotide comprising the sequence / 5Phos / rArArArArArArArArArArArArArArArArArArArArArArArArA*rA*rA*rA*rA*rA*rA* / 3AzideN / (SEQ ID NO: 8), where "*" indicates a phosphorothioate linkage. After assembly via click chemistry (e.g., Cu(I)-catalyzed azide-alkyne cycloaddition), the 5' end of the brush polyA oligonucleotide can then be ligated to the 3' end of an mRNA molecule, which may or may not already contain a polyA tail, using an RNA ligase (e.g., T4 RNA ligase).

[0273] Alternatively, an additional polyA tail can be introduced into an mRNA molecule by ligating the 3' end of the mRNA to a dendrimer modified with a polyA tail (Figure 1E). Dendrimers containing a polyA tail can be generated by first functionalizing a dendrimer (e.g., PAMAM dendrimer, CAS No.: 1174157-65-3) with an alkyne group. This functionalization can be achieved by nucleophilic substitution between propargyl-N-hydroxysuccinimidyl ester (CAS No.: 1174157-65-3) and amino groups on the dendrimer. The functionalized dendrimer can then be conjugated to a polyA-tailed oligonucleotide containing a 3' azide moiety via click chemistry (Figure 1F). For additional exonuclease resistance, either of the oligonucleotides can be modified with one or more modified nucleotides or internucleotide linkages such as phosphorothioate linkages. For example, a poly-A oligonucleotide may comprise the sequence / 5Phos / rArArArArArArArArArArArArArArArArArArArArArArArArArA*rA*rA*rA*rA*rA* / 3AzideN / (SEQ ID NO: 8), where "*" indicates a phosphorothioate linkage. The 5' end of one of the poly-A oligonucleotides attached to a dendrimer via click chemistry (e.g., Cu(I)-catalyzed azide-alkyne cycloaddition) can then be ligated to the 3' end of an mRNA molecule, which may or may not already contain a poly-A tail, using an RNA ligase (e.g., T4 RNA ligase).

[0274] Orthologous pathways can also be used to produce polycapped mRNA. For example, polycapped mRNA can be produced by ligating the 5' end of an mRNA to a "brush-like" 5'-cap oligonucleotide (Figure 1G). Similar to the means by which brush-like polyA tail oligonucleotides are produced, brush-like polycapped oligonucleotides can be produced by starting with a polycapped oligonucleotide modified with several internal nucleotides capable of click chemistry (e.g., 5-octadiynyl dU) and then attaching additional polycapped oligonucleotides containing 3' azide moieties to these nucleotides (Figure 1H). For additional exonuclease resistance, any of the oligonucleotides can be modified with one or more modified nucleotides or internucleotide linkages such as phosphorothioate linkages. The 5' cap can be a 7-methylguansine (m7G) cap or a modified cap, such as a cap containing a modified nucleotide or internucleotide linkage. For example, a first poly-capped oligonucleotide may comprise the sequence 5'- / Cap / rGrGrGrArArA / i5OctdU / rArArGrArGrArGrArArArArGrArArG / i5OctdU / rArArGrArArGrArArA / i5OctdU / rArArGrArArGrArA / i5OctdU / rA (SEQ ID NO: 9), to which is attached an oligonucleotide comprising the sequence 5'- / Cap / rGrGrGrArGrArCrTrGrCrCrArCrCrA*rA*rA*rA*rA*rA* / AzideN / -3' (SEQ ID NO: 6), where "*" indicates a phosphorothioate linkage. After assembly via click chemistry (e.g., Cu(I)-catalyzed azide-alkyne cycloaddition), the 3' end of the brush poly-A oligonucleotide can then be ligated to the uncapped 5' end of an mRNA molecule using an RNA ligase (e.g., T4 RNA ligase).

[0275] Alternatively, an additional 5' cap can be introduced into an mRNA molecule by ligating the 5' end of the mRNA to a dendrimer modified with a 5' cap (Figure 1I). Similar to the procedure for producing dendrimers modified with a poly(A) tail, dendrimers containing a 5' cap can be produced by first functionalizing a dendrimer (e.g., PAMAM dendrimer, CAS No.: 1174157-65-3) with an alkyne group. This functionalization can be achieved by nucleophilic substitution between propargyl-N-hydroxysuccinimidyl ester (CAS No.: 1174157-65-3) and an amino group on the dendrimer. The functionalized dendrimer can then be conjugated to a 5'-capped oligonucleotide containing a 3' azide moiety via click chemistry (Figure 1J). Uncapped oligonucleotides containing a 5' azide can also be conjugated to the functionalized dendrimer. The binding rate between a capped oligonucleotide containing a 3' azide moiety and an uncapped oligonucleotide containing a 5' azide moiety to a dendrimer can be controlled by adjusting the stoichiometry between the oligonucleotides. For additional exonuclease resistance, either of the oligonucleotides can be modified with one or more modified nucleotides or internucleotide linkages, such as phosphorothioate linkages. The 5' cap can be a 7-methylguansine (m7G) cap or a modified cap, such as a cap containing a modified nucleotide or internucleotide linkage. For example, a 5'-capped oligonucleotide can have the sequence 5'- / Cap / rGrGrGrArGrArCrTrGrCrCrArCrCrA*rA*rA*rA*rA*rA* / AzideN / -3' (SEQ ID NO: 6), while an uncapped oligonucleotide can have the sequence / 5AzideN / rArArArArA, where "*" indicates a phosphorothioate linkage. The 3' end of an uncapped oligonucleotide, attached to a dendrimer via click chemistry (e.g., Cu(I)-catalyzed azide-alkyne cycloaddition), can then be ligated to the 5' end of an uncapped mRNA molecule using an RNA ligase (e.g., T4 RNA ligase).

[0276] Modified mRNA molecules containing an additional polyA tail can also be modified to contain an additional 5' cap, and vice versa. In addition to having enhanced resistance to exonucleases, mRNA molecules modified with an additional polyA tail and / or 5' cap can also have enhanced translation efficiency compared to unmodified mRNA molecules. For example, the addition of one or more additional polyA tails or 5' caps to an mRNA molecule can enhance binding between the mRNA molecule and one or more components of the eukaryotic translation machinery, such as polyA-binding protein (PABP) and the eukaryotic initiation factor 4E (eIF4E) cap-binding protein of the eukaryotic initiation factor 4 complex (eIF4F) (Figures 1C, E, G, and I).

[0277] Example 2. Effect of multiple poly(A) tails on mRNA translation in vitro. To preliminarily test the translation efficiency of mRNA molecules modified with an additional poly(A) tail, mRNA encoding a fluorescent or luminescent protein was modified with a brush-like poly(A) tail oligonucleotide as described in Example 1. Briefly, mRNA molecules encoding either a constitutively fluorescent variant of green fluorescent protein (hMGFP) or firefly luciferase were ligated to a brush-like poly(A) oligonucleotide precursor, a brush-like poly(A) oligonucleotide precursor conjugated with a 5' azide-containing poly(A) oligonucleotide, or a brush-like poly(A) oligonucleotide conjugated with a 3' azide-containing poly(A) oligonucleotide (Table 1). Mock ligations performed with mRNA, as well as mRNA without oligo and treatment, were also tested. Each mRNA was transfected into human HeLa cell cultures along with a fixed amount of control mRNA. Transfections with mRNA encoding hMGFP were cotransfected with mRNA encoding mCherry. Transfections with mRNA encoding firefly luciferase were cotransfected with Renilla luciferase. The hMGFP / mCherry fluorescence and firefly luciferase / renilla luciferase ratios were measured 24, 48, and 72 hours after transfection and used to assess the translation efficiency from the transfected mRNA.

[0278] Similar ratios of fluorescence and luminescence were measured in cells transfected with untreated or mock-ligated mRNA, and these remained largely unchanged over the 72-h time course (Figure 1K,L). Cells transfected with mRNA ligated only to brush oligo precursors showed increased fluorescence / luminescence after 48 and 72 h, indicating that ligation to the brush poly(A) tail precursor enhanced mRNA stability and translation efficiency. This effect was further enhanced by the addition of poly(A) oligonucleotides containing a 5' azide moiety to the brush poly(A) tail precursor, indicating that the number of poly(A) tails attached to the mRNA positively correlated with mRNA stability and translation efficiency. However, conjugation of poly(A) oligonucleotides containing a 3' azide moiety to the brush poly(A) tail precursor reduced the observed fluorescence / luminescence. This result was not surprising because conjugation of the poly-A oligonucleotide to the brush-like poly-A tail precursor via the 3' azide results in it having an uncapped 5' phosphate at its end rather than a contiguous set of phosphorothioate-modified adenosine ribonucleotides located at the 3' end. Therefore, orienting the azide-modified poly-A oligonucleotide in this manner would be expected to make the poly-A oligonucleotide vulnerable to exonucleases.

[0279] It should also be noted that the ratio of hMGFP to mCherry fluorescence was observed to be stable 72 hours after transfection, whereas the ratio of firefly to Renilla luciferase fluorescence was still observed to be increasing after 72 hours (Figure 1K, L). These results indicate that translation from mRNA modified with an additional poly(A) tail is long-lasting and therefore may be of use in in vivo applications (e.g., prophylactic and therapeutic uses).

[0280] Table 1. Oligonucleotides for the synthesis of brush-like polyA tail oligonucleotides TIFF2026016516000119.tif45160 where " / 5Phos / " refers to the 5' phosphate group, "rA" refers to an adenosine ribonucleotide, " / i5OctdU / " refers to an internal 5-octadiynyluracil deoxyribonucleotide, "3ddC" refers to a 3' cytosine dideoxyribonucleotide, " / 5AzideN / " refers to a 5' azide moiety, " / 3AzideN / " refers to a 3' azide moiety, and "*" refers to a phosphorothioate internucleotide linkage. Internucleotide linkages not specifically shown are phosphodiester internucleotide linkages.

[0281] Example 3. Development of chemically modified, topologically novel capped, branched mRNA oligoconjugates with multivalent poly(A) tails. In the most widely accepted model of cap-dependent translation, the rate-limiting step is the assembly of a translation initiation complex (TIC) centered around the m7G cap by eukaryotic translation initiation factors (eIFs), including eIF4E and eIF4G (Figure 1A, left). The TIC is further stabilized by the interaction of eIF4G with the cytoplasmic poly(A)-binding protein (PABPC), which recognizes the 3' poly(A) tail, forming a closed loop structure (Figure 1A, right). Thus, PABPC plays a dual role: initiating translation through engagement with eIFs and extending mRNA stability by preventing mRNA deadenylation. Site-specific introduction of exonuclease-resistant modifications at the end of the poly(A) tail has been shown to efficiently increase mRNA stability and protein production in cellulo, likely by stabilizing the nascent poly(A) tail and maintaining the poly(A)-PABPC interaction. Additionally, structural studies reveal that PABPC functions as a multimeric protein complex in which individual PABPC1 proteins bind to poly(A) tails to form stable complexes. This structural insight prompted us to investigate how multimerization of poly(A) tails via branched topologies achieves enhanced multivalent interactions of mRNA transcripts with PABPC1, whereby each individual poly(A) tail bears extensive nuclease-resistant modifications (Figure 2A).

[0282] Branched mocRNAs were developed based on the ligation of chemically synthesized oligonucleotides bearing dense modifications to the 3' end of in vitro transcribed (IVT) mRNA using T4 RNA ligase. Various synthetic strategies for generating branched poly(A) oligonucleotides were tested. Branched RNA topologies can naturally occur in the form of lariat intron RNAs, a by-product of pre-mRNA splicing that possesses a "branched" bond between the 2'-hydroxyl group of an internal nucleotide and the 5' end of the spliced ​​intron, which is rapidly eliminated by debranching enzymes. Therefore, screening focused on unnatural branched linkages that cannot be hydrolyzed intracellularly and have high reaction rates at micromolar concentrations for ease of large-scale synthesis. Several oligonucleotide crosslinking chemistries were tested, including thiol-ene and thiol-yne reactions, phosphate-amine-based reactions, and click chemistry methods such as tetrazine-transcyclooctene (Tz-TCO) and copper-catalyzed azide-alkyne cycloaddition (CuAAC) (Figure 2B). Thiol-ene and thiol-yne reactions had low conversions under nonradical conditions and resulted in oligomerization when radical initiators were used, whereas the phosphate-amine reaction produced trace amounts of product even with EDC / imidazole catalysis. Both the Tz-TCO(IEDDA) and CuAAC reactions performed well with high conversions, indicating that CuAAC is the preferred choice due to its ability to introduce an alkyne handle onto 5-octadiynyldeoxyU(EU) during solid-phase synthesis, whereas Tz-TCO(IEDDA) requires an additional step of functionalization (Figure 2C-D).

[0283] To further enable the generation of poly(A) oligos with multiple branches, we designed a 30-nucleotide (nt) "stem" oligo containing multiple internal EU-modified branch sites, gapped by 12 Å for subsequent enzymatic ligation, and PABPC1 binding. This oligo was paired with a 30-nt "branch" poly(A) oligo containing a 5' azide handle. Both the stem and branch poly(A) oligos contained chain-terminating dideoxycytidine (ddC) at their ends to prevent self-ligation and phosphorothioate (PS) modifications in the last 6 nt to block deadenylation. CuAAC conjugation resulted in a mixture of seven products with one to three branches in different topologies, as shown by gel electrophoresis (Figure 2D). Preliminary tests, performed by ligating the crude branched products with a firefly luciferase reporter, showed a 4.6-5.3-fold increase in bioluminescence in constructs treated with CuAAC compared to unligated controls (p<0.0001, ANOVA). However, reversing the direction of the branched poly(A) completely eliminated the increase in luminescence, indicating that the branched tails still required normal 5' to 3' orientation (Figure 2E).

[0284] Example 4. Establishment of a pipeline for branched mocRNA synthesis and purification. A branched mocRNA synthesis and purification pipeline was established by incorporating HPLC purification for the systematic screening of various chemical modification moieties (Figure 3A-D). In this pipeline, eight synthetic poly(A) oligos (shown in Figure 3F) with different numbers of branches (0-3) and different types of chemical modifications (PS(* / rA / ), DNA(* / dA / ), and 2'-O-methoxyethyl (* / i2MOErA / )) were chemically assembled, HPLC purified, and enzymatically ligated to firefly luciferase reporter mRNA. After quality control of the ligation yield using an RNase H assay (Figure 3E), the ligation products were transfected into HeLa cells with Renilla luciferase mRNA as an internal transfection control. The effect of the modified branched poly(A) tail on protein production was quantified by the ratio of firefly / Renilla bioluminescence. Among all constructs, construct 8 shown in Figure 3F, with three branches and the synergistic incorporation of 2'-O-methoxyethyl (2MOE) and PS in both the stem oligo and the branched poly(A) oligo, had the highest enhancement, resulting in 4-, 11-, and 19-fold higher luminescence signals than linear mRNA at 24, 48, and 72 hours post-transfection, respectively (p<0.0001, ANOVA, Figure 3F).

[0285] Table 2: Branched poly(A) tail variants TIFF2026016516000120.tif162170TIFF2026016516000121.tif205170

[0286] Here, the "backbone" portion of the sequence is the unbranched segment of the polyA tail (see, for example, Figure 2A, horizontally highlighted region polyA tail).

[0287] Example 5. Mechanistic characterization of mRNA stabilization by multimerized poly(A) tails. To gain mechanistic insight into how branched poly(A) tails stabilized mRNA transcripts, we analyzed their overall effect on protein translation capacity at two levels: mRNA stability and translation efficiency. To minimize errors introduced by protein half-life in luciferase assays, we synthesized branched mRNA transcripts encoding degron-tagged firefly luciferase (Firefly-PEST), which have been shown to effectively reduce luciferase half-life in HeLa cells from 20.4 h to approximately 0.92 h. Luminescence decay kinetics revealed that branched mRNA increased the luminescence half-life from an estimated 7.2 h to 17.1 h, confirming the increased mRNA stoichiometry at each time point (Figure 4A-B). Such enhancement was not due to differences in transfection efficiency; decay kinetics of the internal Renilla control appeared similar across all conditions (Figure 4C).

[0288] To analyze mRNA stabilization and translation efficiency at the molecular level, in situ profiling was performed to quantify RNA copy number with subcellular resolution. STARmap detected all transcripts of the target sequence, while RIBOmap detected only the ribosome-bound fraction of mRNA copies (Figure 4D; see Materials and Methods above). Six hours after lipofection, cells treated under the same conditions were replated in two replicates for three time points. One replicate was profiled by STARmap, and the other replicate was profiled by RIBOmap for the modified firefly luciferase construct. Unmodified Renilla luciferase was profiled by STARmap in both replicates to normalize for transfection efficiency. In the STARmap / RIBOmap images, magenta and yell...

Claims

1. (i) an open reading frame (ORF) encoding a protein; and (ii) a polyA region; and (iii) a 5' cap region; and Including, the poly A region is 3' to the ORF and comprises 10 or more nucleotides, and the 5' cap region is 5' to the ORF; the polyA region comprises two or more polyA tails and / or the 5' cap region comprises two or more 5' caps; Modified mRNA.

2. The modified mRNA of claim 1, comprising a polyA region comprising two or more polyA tails and a 5' cap region comprising one 5' cap.

3. The modified mRNA of claim 1, comprising a polyA region comprising one polyA tail and a 5' cap region comprising two or more 5' caps.

4. The modified mRNA of claim 1, comprising a polyA region comprising two or more polyA tails and a 5' cap region comprising two or more 5' caps.

5. The modified mRNA of any one of claims 1 to 4, wherein the modified mRNA comprises a 5' untranslated region (5'UTR) and a 3' untranslated region (3'UTR), the ORF is between the 5'UTR and the 3'UTR, the 3'UTR is between the ORF and the polyA region, and the 5'UTR is between the 5' cap region and the ORF.

6. The modified mRNA of any one of claims 1 to 5, wherein the modified mRNA is a linear mRNA, the 5' cap region is at the 5' end of the modified mRNA, and the polyA region is at the 3' end of the modified mRNA.

7. The modified mRNA of claim 5 or 6, wherein the modified mRNA is a circular mRNA and the polyA region is between the 3'UTR and the 5'cap region.

8. 8. The modified mRNA of any one of claims 1 to 7, wherein the polyA region comprises two or more polyA tails, and at least two instances of the polyA tails are covalently linked by a first linker.

9. The poly A region is -(first poly A tail)-[(first linker)-(second poly A tail) ｎ１ ] ｎ２ Including, each instance of n1 is independently an integer from 1 to 20, inclusive; n2 is an integer between 2 and 10, inclusive; the first poly A tail is covalently linked to the 3'UTR; the first linker is covalently attached to the first polyA tail and the second polyA tail; The modified mRNA of claim 8.

10. 10. The modified mRNA of claim 9, wherein each instance of n1 is 1.

11. 11. The modified mRNA of claim 10, wherein at least one instance of the first linker is covalently attached to an internal nucleotide of the first polyA tail.

12. 12. The modified mRNA of claim 10 or 11, wherein at least one instance of the first linker is covalently attached to the second polyA tail at the 3' nucleotide of the second polyA tail.

13. 13. The modified mRNA of any one of claims 10 to 12, wherein at least one instance of the first linker comprises at least one moiety formed by reacting two orthogonal click chemistry handles.

14. At least one moiety formed by reacting two orthogonal click chemistry handles has the formula: The modified mRNA of claim 13,

15. Each instance of the first linker is independently a substituted or unsubstituted C １－２００ Alkylene, substituted or unsubstituted C １－２００ Alkenylene, substituted or unsubstituted C １－２００ Alkynylene, substituted or unsubstituted C １－２００ Heteroalkylene, substituted or unsubstituted C １－２００ Heteroalkenylene, substituted or unsubstituted C １－２００ heteroalkynylene, substituted or unsubstituted carbocyclylene, substituted or unsubstituted heterocyclylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene; Optionally, the substituted or unsubstituted C １－２００ Alkylene, substituted or unsubstituted C １－２００ Alkenylene, substituted or unsubstituted C １－２００ Alkynylene, substituted or unsubstituted C １－２００ Heteroalkylene, substituted or unsubstituted C １－２００ Heteroalkenylene, and substituted or unsubstituted C １－２００ one or more carbon atoms in the parent chain of each instance of heteroalkynylene are independently replaced with a substituted or unsubstituted carbocyclylene, a substituted or unsubstituted heterocyclylene, a substituted or unsubstituted arylene, or a substituted or unsubstituted heteroarylene; A modified mRNA described in any one of claims 10 to 14.

16. The substituted or unsubstituted C １－２００ Alkylene, substituted or unsubstituted C １－２００ Alkenylene, substituted or unsubstituted C １－２００ Alkynylene, substituted or unsubstituted C １－２００ Heteroalkylene, substituted or unsubstituted C １－２００ Heteroalkenylene, and substituted or unsubstituted C １－２００ One or more carbon atoms in the parent chain of each instance of heteroalkynylene independently The modified mRNA according to any one of claims 10 to 15, wherein the amino acid sequence of the modified mRNA is replaced by the amino acid sequence of the modified mRNA.

17. At least one example of the first linker has the formula: It is of L １ Each instance of is independently substituted or unsubstituted C １－１９９ Alkylene, or substituted or unsubstituted C １－１９９ is heteroalkylene, L ２ Each instance of is independently substituted or unsubstituted C １－１９９ Alkylene, or substituted or unsubstituted C １－１９９ is heteroalkylene, A modified mRNA described in any one of claims 10 to 16.

18. At least one example of the first linker has the formula: The modified mRNA according to any one of claims 10 to 17,

19. 10. The modified mRNA of claim 9, wherein each instance of n1 is independently an integer between 2 and 20, inclusive.

20. 20. The modified mRNA of claim 19, wherein at least one example of the first linker comprises a dendrimer.

21. 21. The modified mRNA of claim 20, wherein at least one example of the dendrimer is a polyamidoamine (PAMAM) dendrimer.

22. At least one example of the PAMAM dendrimer has the formula: wherein: each instance of n3 is independently an integer from 1 to 10, inclusive; each instance of n4 is independently an integer from 0 to 10, inclusive; R ２ each instance of is independently hydrogen or where R ２ At least three examples of and R １ Each instance of １－１０ Alkylene, substituted or unsubstituted C １－１０ Alkenylene, substituted or unsubstituted C １－１０ Alkynylene, substituted or unsubstituted C １－１０ Heteroalkylene, substituted or unsubstituted C １－１０ Heteroalkenylene, or substituted or unsubstituted C １－１０ heteroalkynylene, Optionally, the substituted or unsubstituted C １－１０ Alkylene, substituted or unsubstituted C １－１０ Alkenylene, substituted or unsubstituted C １－１０ Alkynylene, substituted or unsubstituted C １－１０ Heteroalkylene, substituted or unsubstituted C １－１０ Heteroalkenylene, and substituted or unsubstituted C １－１０ one or more carbon atoms in the parent chain of each instance of heteroalkynylene are independently replaced with a substituted or unsubstituted carbocyclylene, a substituted or unsubstituted heterocyclylene, a substituted or unsubstituted arylene, or a substituted or unsubstituted heteroarylene; each instance of n5 is independently an integer from 0 to 10, inclusive; 22. The modified mRNA of claim 21.

23. At least one example of the PAMAM dendrimer is 23. The modified mRNA of claim 22, comprising:

24. Substituted or unsubstituted C １－１０ Alkylene, substituted or unsubstituted C １－１０ Alkenylene, substituted or unsubstituted C １－１０ Alkynylene, substituted or unsubstituted C １－１０ Heteroalkylene, substituted or unsubstituted C １－１０ Heteroalkenylene, and substituted or unsubstituted C １－１０ One or more carbon atoms in the parent chain of each instance of heteroalkynylene independently 22. The modified mRNA of claim 21 , wherein

25. At least one example of the first linker has the formula:

25. The modified mRNA of claim 24,

26. 26. The modified mRNA of any one of claims 8 to 25, wherein at least one instance of the first linker is covalently attached to the 3' nucleotide of the first poly-A tail.

27. 27. The modified mRNA of any one of claims 8 to 26, wherein at least one instance of the first linker is covalently attached to the 3' nucleotide of the second poly-A tail.

28. 28. The modified mRNA of any one of claims 1 to 27, wherein the polyA region of the modified mRNA comprises one or more modified nucleotides.

29. 29. The modified mRNA of Claim 28, wherein three or more of the last ten nucleotides of at least one of the two or more poly-A tails are modified nucleotides and / or non-adenosine nucleotides.

30. 30. The modified mRNA of claim 28 or 29, wherein one or more modified nucleotides in the polyA region are modified adenosine nucleotides.

31. 31. The modified mRNA of any one of claims 28 to 30, wherein one or more modified nucleotides of the polyA region are modified non-adenosine nucleotides.

32. 32. The modified mRNA of any one of claims 28 to 31, wherein one or more modified nucleotides of the polyA region comprise a modified nucleobase.

33. The modified nucleobase may be xanthine, aliaminouracil, aliaminothymidine, hypoxanthine, digoxigenylated adenine, digoxigenylated cytosine, digoxigenylated guanine, digoxigenylated uracil, 6-chloropurine riboside, N6-methyladenine, methylpseudouracil, 2-thiocytosine, 2-thiouracil, 5-methyluracil, 4-thiothymidine, 4-thiouracil, 5,6-dihydro-5-methyluracil, 5,6-dihydrouracil, 5-[(3-indolyl)propionamido-N-allyl]uracil, 5-aminoaliphatic acid ... cytosine, 5-aminoallyluracil, 5-bromouracil, 5-bromocytosine, 5-carboxycytosine, 5-carboxymethylesteruracil, 5-carboxyuracil, 5-fluorouracil, 5-formylcytosine, 5-formyluracil, 5-hydroxycytosine, 5-hydroxymethylcytosine, 5-hydroxymethyluracil, 5-hydroxyuracil, 5-iodocytosine, 5-iodouracil, 5-methoxycytosine, 5-methoxyuracil, 5-methylcytosine, 5-methyluracil, 5-propargylaminocytosine, 5-propargylaminocytosine, azacytosine, 5-propynyluracil, 6-azacytosine, 6-azauracil, 6-chloropurine, 6-thioguanine, 7-deazaadenine, 7-deazaguanine, 7-deaza-7-propargylaminoadenine, 7-deaza-7-propargylaminoguanine, 8-azaadenine, 8-azidoadenine, 8-chloroadenine, 8-oxoadenine, 8-oxoguanine, araadenine, aracytosine, araguanine, arauracil, biotin-16-7-deaza-7-propargylaminoguanine, biotin- 16-aminoallyl cytosine, biotin-16-aminoallyl uracil, cyanine 3-5-propargyl amino cytosine, cyanine 3-6-propargyl amino uracil, cyanine 3-aminoallyl cytosine, cyanine 3-aminoallyl uracil, cyanine 5-6-propargyl amino cytosine, cyanine 5-6-propargyl amino uracil, cyanine 5-aminoallyl cytosine, cyanine 5-aminoallyl uracil, cyanine 7-aminoallyl uracil, dabsyl-5-3-aminoallyl uracil, desthiobiotin-16-aminoallyl uracil,Desthiobiotin-6-aminoallylcytosine, isoguanine, N1-ethylpseudouracil, N1-methoxymethylpseudouracil, N1-methyladenine, N1-methylpseudouracil, N1-propylpseudouracil, N2-methylguanine, N4-biotin-OBEA-cytosine, N4-methylcytosine, N6-methyladenine, O6-methylguanine, pseudoisocytosine cytosine, pseudouracil, thienocytosine, thienoguanine, thienouracil, xanthosine, 3-deazaadenine, 2,6-diaminoadenine, 2,6-daminoguanine, 5-carboxamidouracil, 5-ethynyluracil, N6-isopentenyladenine (i6A), 2-methyl-thio-N6-isopentenyladenine (ms2i6A), 2-methyl Thio-N6-methyladenine (ms2m6A), N6-(cis-hydroxyisopentenyl)adenine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenine (ms2io6A), N6-glycinylcarbamoyladenine (g6A), N6-threonylcarbamoyladenine (t6A), 2-methylthio-N6-threonylcarbamoyladenine (ms2t6 33. The modified mRNA of claim 32, wherein the modified mRNA is selected from the group consisting of N6,N6-dimethyladenine (m62A), N6-methyl-N6-threonylcarbamoyladenine (m6t6A), N6-hydroxynorvalylcarbamoyladenine (hn6A), 2-methylthio-N6-hydroxynorvalylcarbamoyladenine (ms2hn6A), N6,N6-dimethyladenine (m62A), and N6-acetyladenine (ac6A).

34. 34. The modified mRNA of any one of claims 28 to 33, wherein one or more modified nucleotides comprises a modified sugar.

35. The modified sugar may be 2'-thioribose, 2',3'-dideoxyribose, 2'-amino-2'-deoxyribose, 2'-deoxyribose, 2'-azido-2'-deoxyribose, 2'-fluoro-2'-deoxyribose, 2'-O-methylribose, 2'-O-methyldeoxyribose, 3'-amino-2',3'-dideoxyribose, 3'-azido-2',3'-dideoxyribose, 3'-deoxyribose , 3'-O-(2-nitrobenzyl)-2'-deoxyribose, 3'-O-methylribose, 5'-aminoribose, 5'-thioribose, 5-nitro-1-indolyl-2'-deoxyribose, 5'-biotin-ribose, 2'-O,4'-C-methylene linked, 2'-O,4'-C-amino linked, and 2'-O,4'-C-thio linked ribose.

36. 36. The modified mRNA of any one of claims 28 to 35, wherein one or more modified nucleotides comprises a modified phosphate.

37. 37. The modified mRNA of claim 36, wherein the modified phosphate is selected from the group consisting of phosphorothioate (PS), thiophosphate, 5'-O-methylphosphonate, 3'-O-methylphosphonate, 5'-hydroxyphosphonate, hydroxyphosphate, phosphoroselenoate, selenophosphate, phosphoramidate, carbophosphonate, methylphosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidinium ring, triazole ring, boranophosphate (BP), methylphosphonate, and guanidinopropylphosphoramidate.

38. 38. The modified mRNA of claim 36 or 37, wherein the poly A region comprises 1 to 3, 3 to 5, 5 to 10, 10 to 15, 15 to 30, 30 to 50, 50 to 100, or 100 to 200 phosphorothioates.

39. 39. The modified mRNA of claim 38, wherein the poly A region comprises 5 to 30 phosphorothioates.

40. 40. The modified mRNA of any one of claims 28-39, wherein the poly A region comprises 3-5, 5-10, 10-15, 15-30, 30-50, 50-100, or 100-200 deoxyribose sugars.

41. 41. The modified mRNA of claim 40, wherein the poly A region comprises 5 to 30 deoxyribose sugars.

42. 42. The modified mRNA of any one of claims 28 to 41, wherein the mRNA comprises a 3' terminal nucleotide, and the 3' terminal nucleotide is dideoxyadenosine, dideoxycytidine, dideoxyguanosine, dideoxythymidine, dideoxyuridine, or inverted deoxythymidine.

43. 43. The modified mRNA of any one of claims 1 to 42, wherein the polyA region comprises 25 to 500 nucleotides.

44. 44. The modified mRNA of claim 43, wherein the poly A region comprises 50 to 100, 100 to 150, 150 to 200, 200 to 300, 300 to 400, or 400 to 500 nucleotides.

45. 45. The modified mRNA of any one of claims 1 to 44, wherein the polyA region comprises 10 or more adenosine nucleotides.

46. 46. ​​The modified mRNA of any one of claims 1 to 45, wherein 25 to 100%, 30 to 100%, 40 to 100%, 50 to 100%, 60 to 100%, 70 to 100%, 80 to 100%, 90 to 100%, 95 to 100%, 96 to 100%, 97 to 100%, 98 to 100%, or 99 to 100% of the nucleotides in the polyA region are adenosine nucleotides.

47. 47. The modified mRNA of any one of claims 8 to 46, wherein the first poly-A tail comprises 10 to 50 adenosine ribonucleotides, 1 to 10 modified uridine deoxyribonucleotides that include an attachment point, and a 3'-terminal dideoxyribonucleotide or an inverted deoxyribonucleotide, and the second poly-A tail comprises 10 to 50 adenosine ribonucleotides.

48. the first poly A tail comprises: 5'-rArArArArArArAdU(a1)rArArArArArArArArArArArArArAdU(a1)rArArArArArArArArArArArArArArAdU(a1)ddC-3' (SEQ ID NO: 1) comprising the nucleotide sequence shown as At least one example of the second poly A tail is 5'-rArArArArArArArArArArArArArArArArArArArArArArArArArArArArA*rA*rA*rA*rA*rA*(b1)-3' (SEQ ID NO: 2) comprising the nucleotide sequence shown as each instance of said first linker independently and "rA" represents adenosine ribonucleotide; "dU(a1)" represents a modified uridine deoxyribonucleotide; "ddC" represents cytosine dideoxyribonucleotide; "*" represents a phosphorothioate bond, a1 in each example of dU(a1) represents the point of attachment of said dU(a1) to the uridine; b1 of rA*(b1) represents the attachment point at * in said rA*(b1), each instance of a1 is connected to an instance of a2; each instance of b1 is connected to an instance of b2; 48. The modified mRNA of claim 47.

49. The modified mRNA of claim 47 or 48, wherein the modified uridine deoxynucleotide is 5-octadiynyl deoxyuridine.

50. 47. The modified mRNA of any one of claims 8 to 46, wherein the first poly-A tail and the second poly-A tail comprise 10 to 50 adenosine ribonucleotides and a 3'-terminal azide moiety.

51. the first polyA tail and the second polyA tail are 5'-rArArArArArArArArArArArArArArArArArArArArArArArArArArArArA*rA*rA*rA*rA*rA* / AzideN / -3' (SEQ ID NO: 3) comprising the nucleotide sequence shown as wherein "rA" represents an adenosine ribonucleotide, "AzideN" represents a 3' azide moiety, and "*" represents a phosphorothioate bond.

51. The modified mRNA of claim 50.

52. 47. The modified mRNA of any one of claims 8 to 46, wherein the first poly-A tail and the second poly-A tail comprise 10 to 50 adenosine ribonucleotides and a 5'-terminal azide moiety.

53. the first polyA tail and the second polyA tail are 5'- / AzideN / rArArArArArArArArArArArArArArArArArArArArArArArArArArArArA*rA*rA*rA*rA*rA-3' (SEQ ID NO: 27) comprising the nucleotide sequence shown as wherein "rA" represents an adenosine ribonucleotide, "AzideN" represents a 3' azide moiety, and "*" represents a phosphorothioate bond.

53. The modified mRNA of claim 52.

54. 54. The modified mRNA of any one of claims 1 to 53, wherein the 5' cap region comprises two or more 5' caps, and at least two instances of the 5' caps are covalently linked by a second linker.

55. the 5' cap region is -(first 5' cap)-[(second linker)-(second 5' cap) ｍ１ ] ｍ２ Including, each instance of m1 is independently an integer from 1 to 20, inclusive; m2 is an integer from 2 to 10 (inclusive); the first 5' cap is covalently linked to the 5' UTR; the second linker is covalently attached to the first 5' cap and the second 5' cap; 55. The modified mRNA of claim 54.

56. 56. The modified mRNA of claim 55, wherein each instance of m1 is 1.

57. 57. The modified mRNA of Claim 56, wherein at least one instance of the second linker is covalently attached to an internal nucleotide of the first 5' cap.

58. 58. The modified mRNA of claim 56 or 57, wherein at least one instance of the second linker is covalently linked to the second 5' cap at the 3' nucleotide of the second 5' cap.

59. 59. The modified mRNA of any one of claims 56 to 58, wherein at least one instance of the second linker comprises at least one moiety formed by reacting two orthogonal click chemistry handles.

60. At least one moiety formed by reacting two orthogonal click chemistry handles has the formula:

60. The modified mRNA of claim 59,

61. Each instance of the second linker is independently a substituted or unsubstituted C １－２００ Alkylene, substituted or unsubstituted C １－２００ Alkenylene, substituted or unsubstituted C １－２００ Alkynylene, substituted or unsubstituted C １－２００ Heteroalkylene, substituted or unsubstituted C １－２００ Heteroalkenylene, substituted or unsubstituted C １－２００ heteroalkynylene, substituted or unsubstituted carbocyclylene, substituted or unsubstituted heterocyclylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene; Optionally, the substituted or unsubstituted C １－２００ Alkylene, substituted or unsubstituted C １－２００ Alkenylene, substituted or unsubstituted C １－２００ Alkynylene, substituted or unsubstituted C １－２００ Heteroalkylene, substituted or unsubstituted C １－２００ Heteroalkenylene, and substituted or unsubstituted C １－２００ one or more carbon atoms in the parent chain of each instance of heteroalkynylene are independently replaced with a substituted or unsubstituted carbocyclylene, a substituted or unsubstituted heterocyclylene, a substituted or unsubstituted arylene, or a substituted or unsubstituted heteroarylene; A modified mRNA described in any one of claims 56 to 60.

62. The substituted or unsubstituted C １－２００ Alkylene, substituted or unsubstituted C １－２００ Alkenylene, substituted or unsubstituted C １－２００ Alkynylene, substituted or unsubstituted C １－２００ Heteroalkylene, substituted or unsubstituted C １－２００ Heteroalkenylene, and substituted or unsubstituted C １－２００ One or more carbon atoms in the parent chain of each instance of heteroalkynylene independently 55. The modified mRNA of any one of claims 49 to 54, wherein:

63. At least one example of the second linker has the formula: It is of L ３ Each instance of is independently substituted or unsubstituted C １－１９９ Alkylene, or substituted or unsubstituted C １－１９９ is heteroalkylene, L ４ Each instance of is independently substituted or unsubstituted C １－１９９ Alkylene, or substituted or unsubstituted C １－１９９ is heteroalkylene, A modified mRNA described in any one of claims 56 to 62.

64. At least one example of the second linker has the formula: The modified mRNA of any one of claims 56 to 62,

65. 57. The modified mRNA of claim 56, wherein each instance of m1 is independently an integer from 2 to 20, inclusive.

66. 66. The modified mRNA of Claim 65, wherein at least one example of the second linker comprises a dendrimer.

67. 67. The modified mRNA of claim 66, wherein at least one example of the dendrimer is a polyamidoamine (PAMAM) dendrimer.

68. At least one example of the PAMAM dendrimer has the formula: wherein: each instance of m3 is independently an integer from 1 to 10, inclusive; each instance of m4 is independently an integer from 0 to 10, inclusive; R １２ each instance of is independently hydrogen or where R １２ At least three examples of and R １１ Each instance of １－１０ Alkylene, substituted or unsubstituted C １－１０ Alkenylene, substituted or unsubstituted C １－１０ Alkynylene, substituted or unsubstituted C １－１０ Heteroalkylene, substituted or unsubstituted C １－１０ Heteroalkenylene, or substituted or unsubstituted C １－１０ heteroalkynylene, Optionally, the substituted or unsubstituted C １－１０ Alkylene, substituted or unsubstituted C １－１０ Alkenylene, substituted or unsubstituted C １－１０ Alkynylene, substituted or unsubstituted C １－１０ Heteroalkylene, substituted or unsubstituted C １－１０ Heteroalkenylene, and substituted or unsubstituted C １－１０ one or more carbon atoms in the parent chain of each instance of heteroalkynylene are independently replaced with a substituted or unsubstituted carbocyclylene, a substituted or unsubstituted heterocyclylene, a substituted or unsubstituted arylene, or a substituted or unsubstituted heteroarylene; each instance of m5 is independently an integer from 0 to 10, inclusive; 68. The modified mRNA of claim 67.

69. At least one example of the PAMAM dendrimer is 69. The modified mRNA of claim 67 or 68, comprising:

70. The substituted or unsubstituted C １－１０ Alkylene, substituted or unsubstituted C １－１０ Alkenylene, substituted or unsubstituted C １－１０ Alkynylene, substituted or unsubstituted C １－１０ Heteroalkylene, substituted or unsubstituted C １－１０ Heteroalkenylene, and substituted or unsubstituted C １－１０ One or more carbon atoms in the parent chain of each instance of heteroalkynylene independently 69. The modified mRNA of claim 68, wherein

71. At least one example of the first linker has the formula:

69. The modified mRNA of claim 68,

72. 72. The modified mRNA of any one of claims 50 to 71, wherein at least one instance of the second linker is covalently attached to the 5' or 3' nucleotide of the first 5' cap.

73. 73. The modified mRNA of any one of claims 50 to 72, wherein at least one instance of the second linker is covalently linked to the 5' or 3' nucleotide of at least one instance of the second 5' cap.

74. 74. The modified mRNA of any one of claims 50 to 73, wherein the 5' cap region of the modified mRNA comprises one or more modified nucleotides.

75. 75. The modified mRNA of Claim 74, wherein one or more modified nucleotides comprises a modified nucleobase.

76. The modified nucleobase may be xanthine, aliaminouracil, aliaminothymidine, hypoxanthine, digoxigenylated adenine, digoxigenylated cytosine, digoxigenylated guanine, digoxigenylated uracil, 6-chloropurine riboside, N6-methyladenine, methylpseudouracil, 2-thiocytosine, 2-thiouracil, 5-methyluracil, 4-thiothymidine, 4-thiouracil, 5,6-dihydro-5-methyluracil, 5,6-dihydrouracil, 5-[(3-indolyl)propionamido-N-allyl]uracil, 5-aminoaliphatic acid ... cytosine, 5-aminoallyluracil, 5-bromouracil, 5-bromocytosine, 5-carboxycytosine, 5-carboxymethylesteruracil, 5-carboxyuracil, 5-fluorouracil, 5-formylcytosine, 5-formyluracil, 5-hydroxycytosine, 5-hydroxymethylcytosine, 5-hydroxymethyluracil, 5-hydroxyuracil, 5-iodocytosine, 5-iodouracil, 5-methoxycytosine, 5-methoxyuracil, 5-methylcytosine, 5-methyluracil, 5-propargylaminocytosine, 5-propargylaminocytosine, azacytosine, 5-propynyluracil, 6-azacytosine, 6-azauracil, 6-chloropurine, 6-thioguanine, 7-deazaadenine, 7-deazaguanine, 7-deaza-7-propargylaminoadenine, 7-deaza-7-propargylaminoguanine, 8-azaadenine, 8-azidoadenine, 8-chloroadenine, 8-oxoadenine, 8-oxoguanine, araadenine, aracytosine, araguanine, arauracil, biotin-16-7-deaza-7-propargylaminoguanine, biotin- 16-aminoallyl cytosine, biotin-16-aminoallyl uracil, cyanine 3-5-propargyl amino cytosine, cyanine 3-6-propargyl amino uracil, cyanine 3-aminoallyl cytosine, cyanine 3-aminoallyl uracil, cyanine 5-6-propargyl amino cytosine, cyanine 5-6-propargyl amino uracil, cyanine 5-aminoallyl cytosine, cyanine 5-aminoallyl uracil, cyanine 7-aminoallyl uracil, dabsyl-5-3-aminoallyl uracil, desthiobiotin-16-aminoallyl uracil,Desthiobiotin-6-aminoallylcytosine, isoguanine, N1-ethylpseudouracil, N1-methoxymethylpseudouracil, N1-methyladenine, N1-methylpseudouracil, N1-propylpseudouracil, N2-methylguanine, N4-biotin-OBEA-cytosine, N4-methylcytosine, N6-methyladenine, O6-methylguanine, pseudo Isocytosine, pseudouracil, thienocytosine, thienoguanine, thienouracil, xanthosine, 3-deazaadenine, 2,6-diaminoadenine, 2,6-daminoguanine, 5-carboxamidouracil, 5-ethynyluracil, N6-isopentenyladenine (i6A), 2-methyl-thio-N6-isopentenyladenine (ms2i6A), 2-methylthio-N6-methyl 76. The modified mRNA of claim 75, wherein the modified mRNA is selected from the group consisting of N6-(cis-hydroxyisopentenyl)adenine (ms2m6A), N6-(cis-hydroxyisopentenyl)adenine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenine (ms2io6A), N6-glycinylcarbamoyladenine (g6A), N6-threonylcarbamoyladenine (t6A), 2-methylthio-N6-threonylcarbamoyladenine (ms2t6A), N6-methyl-N6-threonylcarbamoyladenine (m6t6A), N6-hydroxynorvalylcarbamoyladenine (hn6A), 2-methylthio-N6-hydroxynorvalylcarbamoyladenine (ms2hn6A), N6,N6-dimethyladenine (m62A), and N6-acetyladenine (ac6A).

77. 77. The modified mRNA of any one of claims 74 to 76, wherein one or more modified nucleotides comprises a modified sugar.

78. The modified sugar may be 2'-thioribose, 2',3'-dideoxyribose, 2'-amino-2'-deoxyribose, 2'-deoxyribose, 2'-azido-2'-deoxyribose, 2'-fluoro-2'-deoxyribose, 2'-O-methylribose, 2'-O-methyldeoxyribose, 3'-amino-2',3'-dideoxyribose, 3'-azido-2',3'-dideoxyribose, 3'-deoxyribose 78. The modified mRNA of claim 77, wherein the ribose is selected from the group consisting of 3'-O-(2-nitrobenzyl)-2'-deoxyribose, 3'-O-methylribose, 5'-aminoribose, 5'-thioribose, 5-nitro-1-indolyl-2'-deoxyribose, 5'-biotin-ribose, 2'-O,4'-C-methylene linked, 2'-O,4'-C-amino linked, and 2'-O,4'-C-thio linked ribose.

79. 79. The modified mRNA of any one of claims 74 to 78, wherein one or more modified nucleotides comprises a modified phosphate.

80. 80. The modified mRNA of claim 79, wherein the modified phosphate is selected from the group consisting of phosphorothioate (PS), thiophosphate, 5'-O-methylphosphonate, 3'-O-methylphosphonate, 5'-hydroxyphosphonate, hydroxyphosphate, phosphoroselenoate, selenophosphate, phosphoramidate, carbophosphonate, methylphosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidinium ring, triazole ring, boranophosphate (BP), methylphosphonate, and guanidinopropylphosphoramidate.

81. 81. The modified mRNA of claim 79 or 80, wherein the 5' cap region comprises 1 to 3, 3 to 5, 5 to 10, 10 to 15, 15 to 30, 30 to 50, 50 to 100, or 100 to 200 phosphorothioates.

82. 82. The modified mRNA of claim 81, wherein the 5' cap region comprises 5 to 30 phosphorothioates.

83. 83. The modified mRNA of any one of claims 74-82, wherein the 5' cap region comprises 3-5, 5-10, 10-15, 15-30, 30-50, 50-100, or 100-200 deoxyribose sugars.

84. 84. The modified mRNA of claim 83, wherein the 5' cap region comprises 5 to 30 deoxyribose sugars.

85. 85. The modified mRNA of any one of claims 74 to 84, wherein the mRNA comprises a 3' terminal nucleotide, and the 3' terminal nucleotide is dideoxyadenosine, dideoxycytidine, dideoxyguanosine, dideoxythymidine, dideoxyuridine, or inverted deoxythymidine.

86. 86. The modified mRNA of any one of claims 50 to 85, wherein the 5' cap region comprises 25 to 500 nucleotides.

87. 87. The modified mRNA of Claim 86, wherein the 5' cap region comprises 50 to 100, 100 to 150, 150 to 200, 200 to 300, 300 to 400, or 400 to 500 nucleotides.

88. 88. The modified mRNA of any one of claims 50 to 87, wherein the 5' cap region comprises 1 to 3, 3 to 5, 5 to 7, or 7 to 10 5' caps.

89. 89. The modified mRNA of any one of claims 50 to 88, wherein the 5' cap region comprises one or more 7-methylguanylic acid caps containing a 5'-5' triphosphate linkage.

90. 90. The modified mRNA of any one of claims 50 to 89, wherein the 5' cap region comprises one or more 2,2,7-trimethylguanosine caps containing a 5'-5' triphosphate linkage.

91. 91. The modified mRNA of any one of claims 50 to 90, wherein the 5' cap region comprises one or more modified 5' caps.

92. 92. The modified mRNA of claim 91, wherein the one or more modified 5' caps are a locked nucleic acid (LNA) modified cap, a 5' cap comprising a 5'-5' triphosphate linkage modified by a 5'-phosphorothiolate, or a 5' cap comprising a 5'-5' tetraphosphate linkage.

93. 93. The modified mRNA of claim 91 or 92, wherein the 5' cap region comprises one or more 5' caps comprising one or more modified nucleotides.

94. 94. The modified mRNA of claim 93, wherein one or more modified nucleotides is a 2'-O-methylated nucleotide.

95. 95. The modified mRNA of any one of claims 50 to 94, wherein the first 5' cap comprises 10 to 50 ribonucleotides, 1 to 10 modified uridine deoxyribonucleotides comprising an attachment point, and a 5' cap, and the second 5' cap comprises 10 to 50 ribonucleotides and a 5' cap.

96. the first 5' cap is 5'- / Cap / rGrGrGrArArArAdU(c1)rArArGrArGrArArArArArGrArArGdU(c1)rArArGrArArArGrArArAdU(c1)rA-3' (SEQ ID NO: 4) comprising the nucleotide sequence shown as the second 5' cap is 5'- / Cap / rGrGrGrArGrArCrTrGrCrCrArCrCrCrA*rA*rA*rA*rA*rA*(d1)-3' (SEQ ID NO: 5) comprising the nucleotide sequence shown as each instance of said second linker independently and "rA" represents adenosine ribonucleotide; "rT" represents thymidine ribonucleotide; "rC" represents a cytidine ribonucleotide; "rG" represents guanosine ribonucleotide; "dU(c1)" represents a modified uridine deoxyribonucleotide; "*" represents a phosphorothioate bond, "Cap" represents the 5' cap; c1 in each instance of dU(c1) represents the point of attachment of said dU(c1) to the uridine; d1 of rA*(d1) represents the attachment point at * in said rA*(d1), each instance of c1 is bound to an instance of c2; each instance of d1 is bound to one instance of d2; 96. The modified mRNA of claim 95.

97. 97. The modified mRNA of claim 95 or 96, wherein the modified uridine deoxynucleotide is 5-octadiynyl deoxyuridine.

98. 95. The modified mRNA of any one of claims 50-94, wherein the first 5' cap comprises 5 to 10 ribonucleotides and a 5' azide moiety, and the second 5' cap comprises 10 to 50 ribonucleotides, a 3' azide moiety, and a 5' cap.

99. the first 5' cap is 5'- / / 5AzideN / rArArA rArA-3' comprising the nucleotide sequence shown as the second 5' cap is 5'- / Cap / rGrGrGrArGrArCrTrGrCrCrArCrCrCrA*rA*rA*rA*rA*rA* / 3AzideN / -3' (SEQ ID NO: 6) comprising the nucleotide sequence shown as wherein "rA" represents an adenosine ribonucleotide, "rT" represents a thymidine ribonucleotide, "rC" represents a cytidine ribonucleotide, "rG" represents a guanosine ribonucleotide, "5AzideN" represents a 5' azido moiety, "3AzideN" represents a 3' azido moiety, "*" represents a phosphorothioate bond, and "Cap" represents a 5' cap.

99. The modified mRNA of claim 98.

100. 100. A method for producing a modified mRNA according to any one of claims 1 to 99, comprising: Ligating a first RNA comprising an open reading frame (ORF) encoding a protein to a tailing nucleic acid comprising two or more polyA tails and / or a capping nucleic acid comprising two or more 5' caps in the presence of an RNA ligase, whereby the RNA ligase forms a covalent bond between the 3' nucleotide of the first RNA and the 5' nucleotide of the tailing nucleic acid, and / or a covalent bond between the 5' nucleotide of the first RNA and the 3' nucleotide of the capping nucleic acid, to produce the modified mRNA.

101. 101. The method of claim 100, wherein the modified mRNA comprises a 5' untranslated region (5'UTR) and a 3' untranslated region (3'UTR), the ORF is between the 5'UTR and the 3'UTR, the 3'UTR is between the ORF and the ligated tailing nucleic acid, and the 5'UTR is between the ORF and the ligated capping nucleic acid.

102. 102. The method of claim 100 or 101, wherein the tailing nucleic acid comprises a first polyA tail and one or more second polyA tails, the first polyA tail being covalently linked to the 3'UTR.

103. A modified mRNA produced by the method of any one of claims 98 to 100.

104. 100. A delivery agent comprising the modified mRNA of any one of claims 1 to 99, wherein the delivery agent comprises a lipid, peptide, protein, antibody, carbohydrate, nanoparticle, or microparticle.

105. 105. The delivery agent of claim 104, wherein the nanoparticle or microparticle is a lipid nanoparticle or microparticle, a polymer nanoparticle or microparticle, a protein nanoparticle or microparticle, or a solid nanoparticle or microparticle.

106. A cell comprising a modified mRNA according to any one of claims 1 to 99.

107. The cell of claim 106, which is a mammalian cell.

108. A composition comprising a modified mRNA according to any one of claims 1 to 99, a delivery agent according to claim 104 or 105, or a cell according to claim 106 or 107.

109. 109. The composition of claim 108, further comprising an additional agent.

110. 110. The composition of claim 109, wherein the additional agent is an agent that has a therapeutic effect when administered to a subject.

111. 111. The composition of claim 109 or 110, wherein the additional agent is a nucleotide, nucleic acid, amino acid, peptide, protein, small molecule, aptamer, lipid, or carbohydrate.

112. The composition of claim 111, wherein the additional agent is an shRNA, an siRNA, or an ASO.

113. 112. The composition of any one of claims 109 to 111, wherein the additional agent is an antigen or an adjuvant.

114. The composition of any one of claims 108 to 113, wherein the composition is a pharmaceutical composition, and the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

115. A method comprising introducing a modified mRNA according to any one of claims 1 to 99, or a delivery agent according to claim 104 or 105, into a cell.

116. A method comprising introducing into a subject a modified mRNA according to any one of claims 1 to 99, a delivery agent according to claim 104 or 105, a cell according to claim 106 or 107, or a composition according to any one of claims 108 to 114.

117. 114. A method of preventing a disease in a subject in need thereof, comprising introducing into the subject an effective amount of a modified mRNA of any one of claims 1 to 99, a delivery agent of claim 104 or 105, a cell of claim 105 or 107, or a composition of any one of claims 108 to 114, wherein the open reading frame of the modified mRNA encodes a protein.

118. 114. A method of treating a disease in a subject in need thereof, comprising introducing into the subject an effective amount of a modified mRNA of any one of claims 1 to 99, a delivery agent of claim 104 or 105, a cell of claim 106 or 107, or a composition of any one of claims 108 to 114, wherein the open reading frame of the modified mRNA encodes a protein.

119. 119. The method of claim 117 or claim 118, wherein the protein is a protein antigen or fragment thereof derived from a cell or virus that causes the disease.

120. The method of claim 117 or claim 118, wherein the protein is a protein expressed in the subject at a level lower than a reference value level.

121. 114. A method of replacing an enzyme in a subject, comprising introducing into the subject a modified mRNA of any one of claims 1 to 99, a delivery agent of claim 104 or 105, a cell of claim 106 or 107, or a composition of any one of claims 108 to 114, wherein the open reading frame of the modified mRNA encodes an enzyme.

122. The method of any one of claims 116 to 117, wherein the subject is a human.

123. 116. A modified mRNA according to any one of claims 1 to 99, a delivery agent according to claim 104 or 105, a cell according to claim 106 or 107, or a composition according to any one of claims 108 to 114, for use in preventing a disease in a subject in need thereof.

124. 116. The modified mRNA of any one of claims 1 to 99, the delivery agent of claim 104 or 105, the cell of claim 106 or 107, or the composition of any one of claims 108 to 114, for use in treating a disease in a subject in need thereof.

125. A kit comprising the first RNA according to any one of claims 100 to 102 and a tailing nucleic acid and / or a capping nucleic acid.

126. 126. The kit of claim 125, further comprising an RNA ligase.

127. 115. A kit comprising the pharmaceutical composition of claim 114, a device for administering the pharmaceutical composition to a subject, and instructions for administering the pharmaceutical composition to a subject.

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