Improved poly(a) tails for mRNA and non-coding RNA, and uses thereof

Abstract

The present invention relates to a modified mRNA or non-coding RNA that includes a poly-A region, where (a) greater than 90% of the nucleotides of the poly-A region are modified nucleotides (e.g., at least 91% or 92% of the nucleotides of the poly-A region are modified nucleotides), and (b) 3 or more of the 10 last nucleotides of the poly-A region are modified nucleotides.

Description

IMPROVED POLY(A) TAILS FORMRNA AND NON-CODING RNA, AND USES THEREOFCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Patent Application No. 63 / 736,867, filed December 20, 2024, the contents of which are incorporated by reference herein in their entirety.FIELD OF THE INVENTION

[0002] The present invention relates to a modified mRNA or non-coding RNA that includes a poly-A region, where (a) greater than 90% of the nucleotides of the poly-A region are modified nucleotides (e.g., at least 91% or 92% of the nucleotides of the poly-A region are modified nucleotides), and (b) 3 or more of the 10 last nucleotides of the poly-A region are modified nucleotides.BACKGROUND

[0003] Messenger RNA (mRNA) technology is an emerging alternative to conventional small molecule therapeutics and vaccine approaches because it is potent, programmable, and capable of rapid production of mRNAs with desired sequences. mRNA therapeutics is a rapidly developing field and has been used for the expression of therapeutic proteins, ranging from vascular regeneration factors to vaccines for COVID-19, influenza, and Zika virus. Despite recent clinical successes, mRNA therapeutics still face challenges of instability and short-term efficacy. Increasing the stability of mRNAs to enhance their efficacy in vivo remains an important problem that must be solved to increase the feasibility of mRNA therapeutics for clinical applications.

[0004] International Publication No. WO 2022 / 241045 discloses a modified mRNA comprising an open reading frame encoding a protein, and a poly-A region, where 1% to 90% of the poly-A region are modified nucleotides.SUMMARY OF THE INVENTION

[0005] One aspect of the present invention is a modified mRNA comprising (i) an open reading frame (ORF) encoding a protein, and (ii) a poly-A region which is 3' to the open 1167200.02701 / 151646090v.lreading frame, where (a) greater than 90% of the nucleotides of the poly-A region are modified nucleotides (e.g., at least 91% or 92% of the nucleotides of the poly-A region are modified nucleotides), and (b) 3 or more of the 10 last nucleotides of the poly-A region are modified nucleotides. In one embodiment, the poly-A region comprises 10 or more nucleotides.

[0006] These modified mRNAs have improved stability in cells and thereby enhance protein production. Conventional mRNAs comprise poly-A tails with multiple adenosine nucleotides (also referred to as adenine nucleotides) at the 3' end, which can be degraded by cellular exonucleases, which remove 3' nucleotides. Once exonucleases remove the poly-A tail and begin removing nucleotides of the open reading frame, the mRNA is unable to be translated into an encoded protein. mRNAs that are more resistant to 3' exonuclease activity are degraded more slowly and are thus more stable, having increased half-lives in cells, and more protein can be produced from a given mRNA molecule. Modified nucleotides containing one or more structural modifications to the nucleobase, sugar, or phosphate linkage of the mRNA can interfere with 3' exonuclease activity, rendering the mRNA more stable. However, the same structural modifications that inhibit 3' exonucleases can also hinder the ability of poly adenylating enzymes to incorporate them into a poly-A tail. Surprisingly, the present inventors have been able to synthesize modified mRNA having a poly-A tail in which greater than 90% of the nucleotides in the poly-A region are modified nucleotides, and found them to improve the stability of mRNA. Additionally, modified mRNAs produced by the methods provided herein may be circularized by ligating the terminal ends of a linear mRNA to produce a circular mRNA. The techniques described herein for improving the stability of a mRNA may also be suitable for improving the stability of a non-coding RNA, for the reason that non-coding RNA is also vulnerable to 3' exonuclease activity.

[0007] Accordingly, the present disclosure provides, in one aspect, a modified mRNA comprising:(i) an open reading frame (ORF) encoding a protein; and(ii) a poly-A region, wherein the poly-A region is 3' to the open reading frame and comprises 10 or more nucleotides, wherein (a) greater than 90% of the nucleotides of the poly-A region are modified nucleotides, and (b) 3 or more of the 10 last nucleotides of the poly-A region are modified nucleotides. In one embodiment, the poly-A region comprises 10 or more nucleotides.2167200.02701 / 151646090v.l

[0008] In some embodiments, the poly-A region comprises 25 or more adenosine nucleotides, wherein 23 or more of the nucleotides of the poly-A region are modified nucleotides, and 3 or more of the 25 last nucleotides of the poly-A region are modified nucleotides.

[0009] In some embodiments, 2 or more consecutive nucleotides of the 25 last nucleotides of the poly-A region are linked by a modified internucleotide linkage.

[0010] In some embodiments, 3 or more consecutive nucleotides of the 25 last nucleotides of the poly-A region are modified nucleotides independently selected from a deoxyribonucleotide, a 2'- modified nucleotide, and a phosphorothioate-linked nucleotide.

[0011] In some embodiments, the 3 or more modified nucleotides are consecutive nucleotides located at the 3' terminus of the poly-A region.

[0012] In some embodiments, 6 or more consecutive nucleotides of the 25 last nucleotides of the poly-A region comprise the same type of nucleotide or internucleoside modification.

[0013] In some embodiments, 9 or more of the 10 last nucleotides of the poly-A region are modified nucleotides.

[0014] In some embodiments, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the nucleotides of the poly-A region are modified nucleotides.

[0015] In some embodiments, 20, 21, 22, 23, 24, or 25 of the 25 last nucleotides of the poly-A region are modified nucleotides.

[0016] 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, wherein the 3' UTR is between the ORF and the poly-A region.

[0017] In some embodiments, the modified mRNA is a circular mRNA, wherein the poly-A region is between the 3' UTR and the 5' UTR.

[0018] In some aspects, the present disclosure provides a modified mRNA comprising:(i) an open reading frame (ORF) encoding a protein;(ii) a poly-A region, which is 3' to the open reading frame and comprises 10 or more nucleotides;(iii) one or more copies of a structural sequence comprising at least two nucleotides that are capable of forming a secondary structure, where (a) greater than 90% of the3167200.02701 / 151646090v.lnucleotides of the poly-A region are modified nucleotides, (b) the one or more copies of the structural sequence are 3' to the poly-A region, and (c) the modified mRNA comprises a secondary structure, where the secondary structure comprises one or more copies of the structural sequence.

[0019] In some embodiments, the poly-A region is 3' to the open reading frame and comprises 25 or more nucleotides, wherein the one or more copies of the structural sequence are 3' to the poly-A region, and wherein the modified mRNA comprises a secondary structure, wherein the secondary structure comprises one or more copies of the structural sequence.

[0020] 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, wherein the 3' UTR is between the ORF and the poly-A region.

[0021] In some embodiments, the modified mRNA is a circular mRNA, wherein the one or more copies of the structural sequence are between the poly-A region and the 5' UTR.

[0022] In some embodiments, the structural sequence is a G-quadruplex sequence.

[0023] In some embodiments, the G-quadruplex is an RNA G-quadruplex sequence.

[0024] In some embodiments, the G-quadruplex is a DNA G-quadruplex sequence.

[0025] In some embodiments, the structural sequence is a telomeric repeat sequence.

[0026] In some embodiments, the secondary structure of the mRNA is an aptamer that is capable of binding to a target molecule.

[0027] In some embodiments, the poly-A region of the modified mRNA comprises at least one modified nucleotide.

[0028] In some embodiments, at least one modified nucleotide comprises a modified nucleobase.

[0029] In some embodiments, the modified nucleobase is selected from xanthine, allyaminouracil, allyaminothymidine, hypoxanthine, digoxigeninated adenine, digoxigeninated cytosine, digoxigeninated guanine, digoxigeninated uracil, 6- chloropurineriboside, N6-methyladenine, methylpseudouracil, 2-thiocytosine, 2-thiouracil, 5- methyluracil, 4-thiothymidine, 4-thiouracil, 5,6-dihydro-5-methyluracil, 5,6-dihydrouracil, 5- [(3- Indolyl)propionamide-N-allyl]uracil, 5 -aminoallylcytosine, 5-aminoallyluracil, 5- bromouracil, 5- bromocytosine, 5-carboxycytosine, 5-carboxymethylesteruracil, 5- carboxyuracil, 5-fluorouracil, 5-formylcytosine, 5-formyluracil, 5 -hydroxy cytosine, 5- hydroxymethylcytosine, 5- hydroxymethyluracil, 5-hydroxyuracil, 5-iodocytosine, 5-4167200.02701 / 151646090v.liodouracil, 5 -methoxy cytosine, 5- methoxyuracil, 5 -methyl cytosine, 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, dabcyl-5-3- aminoallyluracil, desthiobiotin- 16-aminoallyl-uracil, desthiobiotin-6-aminoallylcytosine, isoguanine, N1 -ethylpseudouracil, Nl- methoxy methylpseudouracil, N1 -methyladenine, Nl- methylpseudouracil, Nl -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-carboxamide-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-threonyl carbamoyladenine (ms2t6A), N6-methyl-N6-threonylcarbamoyladenine (m6t6A), N6-hydroxynorvalylcarbamoyladenine (hn6A), 2-methylthio-N6-hydroxynorvalyl carbamoyladenine (ms2hn6A), N6,N6- dimethyladenine (m62A), and N6-acetyladenine (ac6A).

[0030] In some embodiments, at least one modified nucleotide comprises a modified sugar.

[0031] In some embodiments, the modified sugar is selected from 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'-0- methylnbose, 5'-aminoribose, 5 '-thioribose, 5-nitro-l-indolyl-2'-deoxyribose, 5'-biotin-ribose, 2'-O,4'-C-methylene-linked, 2'-O,4'-C-amino-linked ribose, and 2'-O,4'-C-thio- linked ribose.

[0032] In some embodiments, at least one modified nucleotide comprises a 2' modification.5167200.02701 / 151646090v.l

[0033] In some embodiments, the 2' modification is selected from a locked-nucleic acid (LNA) modification (i.e., a nucleotide comprising an additional carbon atom bound to the 2' oxygen and 4' carbon of ribose), 2'-fluoro (2'-F) , 2'-O-methoxy-ethyl (2'-M0E), 2'-O- methylation (2'-OMe), and 2'-O-N-methylacetamido (2'-0-NMA). In some embodiments, at least one modified nucleotide comprises a modified phosphate.

[0034] In some embodiments, the modified phosphate is selected from phosphorothioate (PS), phosphorodithioate, thiophosphate, 5'-O-methylphosphonate, 3'-O- methylphosphonate, 5'-hydroxyphosphonate, hydroxyphosphanate, phosphoroselenoate, selenophosphate, phosphoramidate, carbophosphonate, methylphosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidinium ring, triazole ring, boranophosphate (BP), methylphosphonate, and guanidinopropyl phosphoramidate.

[0035] In some embodiments, the poly-A region comprises at least 3, at least 4, at least 5, or at least 6 phosphorothioates.

[0036] In some embodiments, the poly-A region comprises at least 6 phosphorothioates.

[0037] In some embodiments, the poly-A region comprises at least 3 guanine nucleotides and at least 3 phosphorothioates.

[0038] In some embodiments, the poly-A region comprises at least 6 nucleotides comprising a 2' modification.

[0039] In some embodiments, the poly-A region comprises at least 3 deoxyribose sugars.

[0040] In some embodiments, the poly-A region comprises at least 5, at least 10, at least 15, at least 20, or at least 23 deoxyribose sugars.

[0041] In some embodiments, the poly-A region comprises at least 23 deoxyribose sugars.

[0042] In some embodiments, the 3' terminal nucleotide of the mRNA does not comprise hydroxy at the 3' position of the 3' terminal nucleotide.

[0043] In some embodiments, the 3' terminal nucleotide of the mRNA comprises an inverted nucleotide.

[0044] In some embodiments, the 3' terminal nucleotide of the mRNA comprises a dideoxyadenosine, dideoxycytidine, dideoxyguanosine, dideoxythymidine, dideoxyuridine, or inverted-deoxythymidine.6167200.02701 / 151646090v.l

[0045] In some embodiments, the 3' terminal nucleotide of the mRNA comprises a dideoxy cytidine.

[0046] In some embodiments, the mRNA comprises a peptide-binding sequence. In some embodiments, the peptide-binding sequence is a poly-A binding protein (PABP)- binding sequence

[0047] In some embodiments, the modified mRNA comprises a first modified nucleotide and a second modified nucleotide, wherein the first and second modified nucleosides comprise different structures.

[0048] In some embodiments, the poly-A region comprises at least 25-500 nucleotides.

[0049] In some embodiments, the poly-A region comprises at least 50, at least 100, at least 150, or at least 200 nucleotides.

[0050] In some embodiments, 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 95%, at least 96%, at least 97%, at least 98%, or at least 99% of nucleotides of the poly-A region are adenosine nucleotides.

[0051] In some embodiments, the modified mRNA is a linear mRNA, wherein the linear mRNA comprises a 5' cap.

[0052] In some embodiments, the 5' cap comprises a 7-methylguanosine.

[0053] In some embodiments, the 5' cap further comprises one or more phosphates connecting the 7- methylguanosine to an adjacent nucleotide of the modified mRNA.

[0054] In some embodiments, the 5' cap comprises a 3'-O-Me-m7G(5')ppp(5')G.

[0055] In some embodiments, one or more phosphates of the 5' cap is a modified phosphate selected from phosphorothioate, triazole ring, dihalogenmethylenebisphosphonate, imidodiphosphate, and methylenebis(phosphonate).

[0056] In some embodiments, the modified mRNA comprises a 5' UTR comprising 1 or more modified nucleotides. In some embodiments, the modified mRNA comprises an ORF comprising 1 or more modified nucleotides.

[0057] In some aspects, the present disclosure provides a modified non-coding RNA comprising:(i) a non-coding RNA sequence; and(ii) a poly-A region, where (a) the poly-A region is 3' to the non-coding RNA sequence and comprises 10 or more nucleotides, (b) greater than 90% of the nucleotides of7167200.02701 / 151646090v.lthe poly-A region are modified nucleotides, and (c) 3 or more of the 10 last nucleotides of the poly-A region are modified nucleotides.

[0058] In some embodiments, the poly-A region is 3 ' to the open reading frame and comprises 25 or more adenosine nucleotides, greater than 90% of the nucleotides of the poly- A region are modified nucleotides, and 3 or more of the 25 last nucleotides of the poly-A region are modified nucleotides.

[0059] In some embodiments, 4 or more of the 25 last nucleotides of the poly-A region are modified nucleotides.

[0060] In some embodiments, 2 or more consecutive nucleotides of the 25 last nucleotides of the poly-A region are linked by a modified intemucleotide linkage.

[0061] In some embodiments, 3 or more consecutive nucleotides of the 25 last nucleotides of the poly-A region are modified nucleotides independently selected from a deoxyribonucleotide, a 2'- modified nucleotide, and a phosphorothioate-linked nucleotide.

[0062] In some embodiments, the 3 or more modified nucleotides are consecutive nucleotides located at the 3' terminus of the poly-A region.

[0063] In some embodiments, 6 or more consecutive nucleotides of the 25 last nucleotides of the poly-A region comprise the same type of nucleotide or internucleoside modification.

[0064] 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 poly-A region are modified nucleotides.

[0065] In some embodiments, at least 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 of the 25 last nucleotides of the poly-A region are modified nucleotides.

[0066] In some embodiments, the modified non-coding RNA is a circular non-coding RNA, wherein the poly-A region is 5' to the non-coding RNA sequence.

[0067] In some embodiments, the modified non-coding RNA further comprises one or more copies of a structural sequence comprising at least two nucleotides that are capable of forming a secondary structure, wherein the one or more copies of the structural sequence are 3' to the poly-A region, and wherein the modified non-coding RNA comprises a secondary structure, and wherein the secondary structure comprises one or more copies of the structural sequence.8167200.02701 / 151646090v.l

[0068] In some embodiments, the modified non-coding RNA is a circular mRNA, wherein the one or more copies of the structural sequence are between the poly-A region and the non-coding RNA sequence.

[0069] In some embodiments, the structural sequence is a G-quadruplex sequence.

[0070] In some embodiments, the G-quadruplex is an RNA G-quadruplex sequence.

[0071] In some embodiments, the G-quadruplex is a DNA G-quadruplex sequence.

[0072] In some embodiments, the structural sequence is a telomeric repeat sequence.

[0073] In some embodiments, the secondary structure of the non-coding RNA is an aptamer that is capable of binding to a target molecule.

[0074] In some embodiments, at least one modified nucleotide comprises a modified nucleobase.

[0075] In some embodiments, the modified nucleobase is selected from xanthine, allyaminouracil, allyaminothymidine, hypoxanthine, digoxigeninated adenine, digoxigeninated cytosine, digoxigeninated guanine, digoxigeninated uracil, 6- chloropurineriboside, N6-methyladenine, methylpseudouracil, 2-thiocytosine, 2-thiouracil, 5- methyluracil, 4-thiothymidine, 4-thiouracil, 5,6-dihydro-5-methyluracil, 5,6-dihydrouracil, 5- [(3- Indolyl)propionamide-N-allyl]uracil, 5 -aminoallylcytosine, 5-aminoallyluracil, 5- bromouracil, 5- bromocytosine, 5-carboxycytosine, 5-carboxymethylesteruracil, 5- carboxyuracil, 5-fluorouracil, 5-formylcytosine, 5-formyluracil, 5 -hydroxy cytosine, 5- hydroxymethylcytosine, 5- hydroxymethyluracil, 5-hydroxyuracil, 5-iodocytosine, 5- iodouracil, 5 -methoxy cytosine, 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, dabcyl-5-3- aminoallyluracil, desthiobiotin- 16-aminoallyl-uracil, desthiobiotin-6-aminoallylcytosine, isoguanine, N1 -ethylpseudouracil, Nl- methoxymethylpseudouracil, N1 -methyladenine, Nl- m ethylpseudouracil, Nl -propylpseudouracil, N2-methylguanine, N4-biotin-OBEA-cytosine,9167200.02701 / 151646090v.lN4-methylcytosine, N6- methyladenine, 06-methylguanine, pseudoisocytosine, pseudouracil, thienocytosine, thienoguanine, thienouracil, xanthosine, 3 -deazaadenine, 2,6-diaminoadenine, 2,6- daminoguanine, 5-carboxamide-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-threonyl carbamoyladenine (ms2t6A), N6-methyl-N6-threonylcarbamoyladenine (m6t6A), N6-hydroxynorvalylcarbamoyladenine (hn6A), 2-methylthio-N6-hydroxynorvalyl carbamoyladenine (ms2hn6A), N6,N6- dimethyladenine (m62A), and N6-acetyladenine (ac6A).

[0076] In some embodiments, at least one modified nucleotide comprises a modified sugar.

[0077] In some embodiments, the modified sugar is selected from 2'-thioribose, 2', 3 '-di deoxyribose, 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-l-indolyl-2'-deoxyribose, 5'-biotin-ribose, 2'-O,4'-C-methylene-linked, 2'-O,4'-C-amino-linked ribose, and 2'-O,4'-C-thio- linked ribose.

[0078] In some embodiments, at least one modified nucleotide comprises a 2' modification.

[0079] In some embodiments, the 2' modification is selected from a locked- nucleic acid (LNA) modification (i.e., a nucleotide comprising an additional carbon atom bound to the 2' oxygen and 4' carbon of ribose), 2'-fluoro (2'-F) , 2'-O-methoxy-ethyl (2'-M0E), 2'-O- methylation (2'-OMe), and 2'-O-N-methylacetamido (2'-0-NMA).

[0080] In some embodiments, at least one modified nucleotide comprises a modified phosphate.

[0081] In some embodiments, the modified phosphate is selected from phosphorothioate (PS), phosphorodithioate, thiophosphate, 5'-O-methylphosphonate, 3'-O- methylphosphonate, 5'-hydroxyphosphonate, hydroxyphosphanate, phosphoroselenoate, selenophosphate, phosphoramidate, carbophosphonate, methylphosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidinium ring, triazole ring, boranophosphate (BP), methylphosphonate, and guanidinopropyl phosphoramidate.10167200.02701 / 151646090v.l

[0082] In some embodiments, the poly-A region comprises at least 3, at least 4, at least 5, or at least 6 phosphorothioates.

[0083] In some embodiments, the poly-A region comprises at least 6 phosphorothioates.

[0084] In some embodiments, the poly-A region comprises at least 3 guanine nucleotides and least 3 phosphorothioates.

[0085] In some embodiments, the poly-A region comprises at least 6 nucleotides comprising a 2' modification.

[0086] In some embodiments, the poly-A region comprises at least 3 deoxyribose sugars.

[0087] In some embodiments, the poly-A region comprises at least 5, at least 10, at least 15, at least 20, or at least 23 deoxyribose sugars.

[0088] In some embodiments, the poly-A region comprises at least 23 deoxyribose sugars.

[0089] In some embodiments, the 3' terminal nucleotide of the non-coding RNA does not comprise hydroxy at the 3' position of the 3' terminal nucleotide.

[0090] In some embodiments, the 3' terminal nucleotide of the non-coding RNA comprises an inverted nucleotide.

[0091] In some embodiments, the 3' terminal nucleotide of the mRNA comprises a dideoxyadenosine, dideoxycytidine, dideoxyguanosine, dideoxythymidine, dideoxyuridine, or inverted-deoxythymidine.

[0092] In some embodiments, the 3' terminal nucleotide of the mRNA comprises a dideoxy cytidine.

[0093] In some embodiments, the modified non-coding RNA comprises a first modified nucleotide and a second modified nucleotide, wherein the first and second modified nucleosides comprise different structures.

[0094] In some embodiments, the poly-A region comprises at least 25-500 nucleotides.

[0095] In some embodiments, the poly-A region comprises at least 50, at least 100, at least 150, or at least 200 nucleotides.

[0096] In some embodiments, 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 95%, at least 96%, at least 97%, at least 98%, or at least 99% of nucleotides of the poly-A region are adenosine nucleotides.11167200.02701 / 151646090v.l

[0097] Yet another aspect is a modified mRNA comprising:(i) an open reading frame (ORF) encoding a protein; and(ii) a poly region comprising adenosine nucleotides and cytosine nucleotides, wherein (i) the poly region is 3' to the open reading frame, (ii) the poly region has a length of from 30 to 500 nucleotides, (iii) at least 50% of the total number of adenosine and cytosine nucleotides in the poly region are cytosine nucleotides, and (iv) the number of consecutive adenosine nucleotides in the poly region does not exceed 15.

[0098] Yet another aspect is a modified non-coding RNA comprising:(i) a non-coding RNA; and(ii) a poly region, wherein (i) the poly region is 3' to the open reading frame, (ii) the poly region has a length of from 30 to 500 nucleotides, (iii) at least 50% of the total number of adenosine and cytosine nucleotides in the poly region are cytosine nucleotides, and (iv) the number of consecutive adenosine nucleotides in the poly region does not exceed 15.

[0099] The poly-C region may have manufacturing advantages (such as a stable tail on a plasmid and a shorter tail) over a poly-A region. Furthermore, the poly-C tail have improved cell type specific stability and translation kinetics.

[0100] The poly region for the modified mRNA and modified non-coding RNA may have the same modifications as described herein for the poly-A region and have any of the other parameters described herein for the poly-A region.

[0101] In one embodiment of the modified mRNA and modified non-coding RNA, the poly region includes repeating at least 2 or 6 consecutive repeats of an AC monomer, such as from 6 to 20 consecutive repeats (e.g., 8, 9, 10, 11, or 12 consecutive repeats) of an AC monomer. In one embodiment, the poly region includes 10 consecutive repeats of an AC monomer.

[0102] In another embodiment of the modified mRNA and modified non-coding RNA, the poly region includes repeating at least 2 or 6 consecutive repeats (e.g., 6 to 20 consecutive repeats) of a CCA monomer, CCCA monomer, or CCCCA monomer. In one embodiment, the poly region includes repeating at least 2 or 6 consecutive repeats (e.g., 6 to 20 consecutive repeats) of a CCA monomer. In another embodiment, the poly region includes repeating at least 2 or 6 consecutive repeats (e.g., 6 to 20 consecutive repeats) of a CCCA monomer. In yet another embodiment, the poly region includes repeating at least 2 or 6 consecutive repeats (e.g., 6 to 20 consecutive repeats) of a CCCCA monomer.12167200.02701 / 151646090v.l

[0103] In another embodiment of the modified mRNA and modified non-coding RNA, the poly region includes repeating at least 2 consecutive repeats (e.g., 2 to 30 (such as 2, 3, 4, 5, 6 or 7) consecutive repeats) of (C)9A (SEQ ID NO: 78), (C)I9A (SEQ ID NO: 79), A(C)9(SEQ ID NO: 80), or A(C)I9(SEQ ID NO: 81).

[0104] In yet another embodiment of the modified mRNA and modified non-coding RNA, the poly region includes CCC at the 3’ end of the poly region (e.g., the poly region may be (AC)nCCC (SEQ ID NO: 82) where n is 6 to 20).

[0105] In another embodiment of the modified mRNA and modified non-coding RNA, the at least one nucleotide in the poly region comprises a modified sugar.

[0106] Yet another aspect is a modified non-coding RNA comprising:(i) an open reading frame (ORF) encoding a protein; and(ii) a poly region comprising adenosine nucleotides and cytosine nucleotides, wherein (i) the poly region is 3' to the open reading frame, (ii) the poly region has a length of from 30 to 500 nucleotides, (iii) 5 to 50% of the total number of adenosine and cytosine nucleotides in the poly region are cytosine nucleotides, and (iv) the number of consecutive adenosine nucleotides in the poly region does not exceed 20.

[0107] Yet another aspect is modified non-coding RNA comprising:(i) a non-coding RNA; and(ii) a poly region, wherein (i) the poly region is 3' to the open reading frame, (ii) the poly region has a length of from 30 to 500 nucleotides, (iii) 5 to 50% of the total number of adenosine and cytosine nucleotides in the poly region are cytosine nucleotides, and (iv) the number of consecutive adenosine nucleotides in the poly region does not exceed 20.

[0108] In one embodiment of the modified mRNA and modified non-coding RNA, the poly region includes repeating at least 2 consecutive repeats (e.g., 10 to 30 (such as 12 or 20) repeats) of an AAC, AAAC, or AAAAC monomer. In one embodiment, the poly region includes repeating at least 2 or 6 consecutive repeats (e.g., 6 to 20 consecutive repeats) of an AAC monomer. In another embodiment, the poly region includes repeating at least 2 or 6 consecutive repeats (e.g., 6 to 20 consecutive repeats) of an AAAC monomer. In yet another embodiment, the poly region includes repeating at least 2 or 6 consecutive repeats (e.g., 6 to 20 consecutive repeats) of an AAAAC monomer.

[0109] In another embodiment of the modified mRNA and modified non-coding RNA, the poly region includes repeating at least 2 consecutive repeats (e.g., 2 to 30 (such as13167200.02701 / 151646090v.l2, 3, 4, 5, 6 or 7) consecutive repeats) of (C)9A (SEQ ID NO: 78), (C)I9A (SEQ ID NO: 79), A(C)9(SEQ ID NO: 80), or A(C)I9(SEQ ID NO: 81).

[0110] In another embodiment of the modified mRNA and modified non-coding RNA, the at least one nucleotide in the poly region comprises a modified sugar.

[0111] In some aspects, the present disclosure provides a method of producing a modified mRNA of the present invention, the method comprising ligating a first RNA comprising an open reading frame encoding a protein to a tailing nucleic acid comprising one or more modified nucleotides, in the presence of an RNA ligase, whereby the RNA ligase forms a covalent bond between the 3' nucleotide of the RNA and the 5' nucleotide of the tailing nucleic acid to produce the modified mRNA.

[0112] 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, wherein the 3' UTR is between the ORF and the poly-A region.

[0113] In some embodiments, the method further comprises circularizing the modified mRNA in the presence of a ribozyme, wherein the modified mRNA comprises a 3' intron and a 5' intron, wherein the 3' intron is 5' to the 5' UTR, wherein the 5' intron is 3' to the poly-A region, whereby the ribozyme forms a covalent bond between a nucleotide that is 3' to the 3' intron and a nucleotide that is 5' to the 5' intron to produce a circular mRNA that does not comprise the 5' intron or the 3' intron, wherein the poly-A region is between the 3' UTR and the 5' UTR of the circular mRNA.

[0114] In some embodiments, the method further comprises the steps of:(i) introducing a 5' terminal phosphate group onto the first nucleotide of the modified mRNA;(ii) cleaving one or more 3' terminal nucleotides of the modified mRNA to produce a modified mRNA with a 3' terminal hydroxyl group; and(iii) circularizing the modified mRNA produced in step (ii) in the presence of a circularizing ligase; whereby the circularizing ligase forms a covalent bond between the 3' nucleotide of the modified mRNA and the 5' nucleotide of the modified mRNA to produce a circular modified mRNA, wherein the poly-A region is between the 3' UTR and the 5' UTR.

[0115] In some aspects, the present disclosure provides a method of producing a modified mRNA of the present invention, the method comprising ligating an RNA comprising an open reading frame encoding a protein to a tailing nucleic acid comprising one or more copies of a structural sequence in the presence of an RNA ligase, whereby the ligase14167200.02701 / 151646090v.lforms a covalent bond between the 3' nucleotide of the RNA and the 5' nucleotide of the tailing nucleic acid to produce the modified mRNA.

[0116] 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, wherein the 3' UTR is between the ORF and the poly-A region, wherein the poly- A region is between the 3' UTR and the one or more copies of the structural sequence.

[0117] In some embodiments, the method further comprises circularizing the modified mRNA in the presence of a ribozyme, wherein the modified mRNA comprises a 3' intron and a 5' intron, wherein the 3' intron is 5' to the 5' UTR, wherein the 5' intron is 3' to the one or more copies of the structural sequence, whereby the ribozyme forms a covalent bond between a nucleotide that is 3' to the 3' intron and a nucleotide that is 5' to the 5' intron to produce a circular mRNA that does not comprise the 5' intron or the 3' intron, wherein the one or more copies of the structural sequence are between the poly-A region and the 5' UTR of the circular mRNA.

[0118] In some embodiments, the method further comprises the steps of:(i) introducing a 5' terminal phosphate group onto the first nucleotide of the modified mRNA;(ii) cleaving one or more 3' terminal nucleotides of the modified mRNA to produce a modified mRNA with a 3' terminal hydroxyl group; and(iii) circularizing the modified mRNA produced in step (ii) in the presence of a circularizing ligase; whereby the circularizing ligase forms a covalent bond between the 3' nucleotide of the modified mRNA and the 5' nucleotide of the modified mRNA to produce a circular modified mRNA, wherein the one or more copies of the structural sequence are between the 3' UTR and the 5' UTR.

[0119] In some embodiments, the modified mRNA is circularized in the presence of a scaffold nucleic acid, wherein the scaffold nucleic acid is a nucleic acid that is capable of hybridizing with the modified mRNA, wherein the modified mRNA forms a circular secondary structure when bound to the scaffold nucleic acid,

[0120] In some embodiments, the scaffold nucleic acid comprises:(a) a first hybridization sequence comprising 5 or more nucleotides, wherein the first hybridization sequence is complementary to at least the first five (5) nucleotides of the modified mRNA; and(b) a second hybridization sequence comprising 5 or more nucleotides, wherein the15167200.02701 / 151646090v.lsecond hybridization sequence is complementary to at least the last five (5) nucleotides of the modified mRNA; wherein at least the first five (5) nucleotides of the modified mRNA hybridize with the first hybridization sequence, and at least the last five (5) nucleotides of the modified mRNA hybridize with the second hybridization sequence.

[0121] In some embodiments, a last nucleotide of the first hybridization sequence and a first nucleotide of the second hybridization sequence are adjacent in the scaffold nucleic acid and not separated by any other nucleotides.

[0122] In some embodiments, the modified mRNA comprises:(i) a first self-hybridization sequence that is 5' to the open reading frame;(ii) a second self-hybridization sequence that is 3' to the open reading frame;(iii) a first non-hybridization sequence that is 5' to the first self-hybridization sequence; and(iv) a second non-hybridization sequence that is 3' to the second self-hybridization sequence, where the first and second self-hybridization sequences are capable of hybridizing with each other, and where the first and second self-hybridization sequences are not capable of hybridizing with each other.

[0123] In some embodiments, hybridization of the first and second self-hybridization sequences forms a secondary structure in which the 5' terminal nucleotide and the 3' terminal nucleotide of the modified mRNA are separated by a distance of less than 100 A.

[0124] In some embodiments, the 5' terminal nucleotide and the 3' terminal nucleotide are separated by a distance of less than 90 A, less than 80 A, less than 70 A, less than 60 A, less than 50 A, less than 40 A, less than 30 A, less than 20 A, or less than 10 A.

[0125] In some embodiments, the circularizing ligase is T4 RNA ligase.

[0126] In some embodiments, the structural sequence is a G-quadruplex sequence.

[0127] In some embodiments, the G-quadruplex is an RNA G-quadruplex sequence.

[0128] In some embodiments, the G-quadruplex is a DNA G-quadruplex sequence.

[0129] In some embodiments, the structural sequence is a telomeric repeat sequence.

[0130] In some embodiments, the structural sequence is an aptamer sequence comprising at least two nucleotides that are capable of interacting to form an aptamer, wherein the aptamer is a secondary structure that is capable of binding to a target molecule.

[0131] In some embodiments, the tailing nucleic acid comprises at least one modified nucleotide.16167200.02701 / 151646090v.l

[0132] In some embodiments, the 5' nucleotide of the RNA does not comprise a 5' terminal phosphate group; wherein the 3' nucleotide of the RNA comprises a 3' terminal hydroxyl group; wherein the 5' nucleotide of the tailing nucleic acid comprises a 5' terminal phosphate group; and wherein the 3' nucleotide of the tailing nucleic acid does not comprise a 3' terminal hydroxyl group.

[0133] In some embodiments, the 5' nucleotide of the RNA does not comprise a 5' terminal hydroxyl group; wherein the 3' nucleotide of the RNA comprises a 3' terminal phosphate group; wherein the 5' nucleotide of the tailing nucleic acid comprises a 5' terminal hydroxyl group; wherein the 3' nucleotide of the tailing nucleic acid does not comprise a 3' terminal phosphate group; and wherein the RNA ligase is an RtcB ligase.

[0134] 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.

[0135] In some embodiments, at least 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 of the 25 last nucleotides of the tailing nucleic acid are modified nucleotides.

[0136] In some embodiments, at least one modified nucleotide comprises a modified nucleobase.

[0137] In some embodiments, the modified nucleobase is selected from xanthine, allyaminouracil, allyaminothymidine, hypoxanthine, digoxigeninated adenine, digoxigeninated cytosine, digoxigeninated guanine, digoxigeninated uracil, 6- chloropurineriboside, N6-methyladenine, methylpseudouracil, 2-thiocytosine, 2-thiouracil, 5- methyluracil, 4-thiothymidine, 4-thiouracil, 5,6-dihydro-5-methyluracil, 5,6-dihydrouracil, 5- [(3- Indolyl)propionamide-N-allyl]uracil, 5 -aminoallylcytosine, 5-aminoallyluracil, 5- bromouracil, 5 -bromocytosine, 5-carboxycytosine, 5-carboxymethylesteruracil, 5- carboxyuracil, 5-fluorouracil, 5-formylcytosine, 5-formyluracil, 5 -hydroxy cytosine, 5- hydroxymethylcytosine, 5- hydroxymethyluracil, 5-hydroxyuracil, 5-iodocytosine, 5- iodouracil, 5 -methoxy cytosine, 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- 16-17167200.02701 / 151646090v.laminoallylcytosine, 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, dabcyl-5-3 - aminoallyluracil, desthiobiotin- 16-aminoallyl-uracil, desthiobiotin-6-aminoallylcytosine, isoguanine, N1 -ethylpseudouracil, Nl- methoxymethylpseudouracil, N1 -methyladenine, Nl- methylpseudouracil, Nl- 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-carboxamide-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-threonyl carbamoyladenine (ms2t6A), N6-methyl-N6-threonylcarbamoyladenine (m6t6A), N6-hydroxynorvalylcarbamoyladenine (hn6A), 2-methylthio-N6-hydroxynorvalyl carbamoyladenine (ms2hn6A), N6,N6- dimethyladenine (m62A), and N6-acetyladenine (ac6A).

[0138] In some embodiments, at least one modified nucleotide comprises a modified sugar.

[0139] In some embodiments, the modified sugar is selected from 2'- thioribose, 2', 3 '-di deoxyribose, 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-l-indolyl-2'-deoxyribose, 5'-biotin-ribose, 2'-O,4'-C-methylene-linked, 2'-O,4'-C-amino-linked ribose, and 2'-O,4'-C-thio-linked ribose.

[0140] In some embodiments, at least one modified nucleotide comprises a 2' modification.

[0141] In some embodiments, the 2' modification is selected from a locked-nucleic acid (LNA) modification (i.e., a nucleotide comprising an additional carbon atom bound to the 2' oxygen and 4' carbon of ribose), 2'-fluoro (2'-F) , 2'-O-methoxy-ethyl (2’-M0E), 2'-O- methylation (2’-OMe), and 2'-O-N-methylacetamido (2'-0-NMA).

[0142] In some embodiments, at least one modified nucleotide comprises a modified phosphate.18167200.02701 / 151646090v.l

[0143] In some embodiments, the modified phosphate is selected from phosphorothioate (PS), phosphorodithioate, thiophosphate, 5'-O-methylphosphonate, 3'-O- methylphosphonate, 5'-hydroxyphosphonate, hydroxyphosphanate, phosphoroselenoate, selenophosphate, phosphoramidate, carbophosphonate, methylphosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidinium ring, triazole ring, boranophosphate (BP), methylphosphonate, and guanidinopropyl phosphoramidate.

[0144] In some embodiments, the tailing nucleic acid comprises at least 3, at least 4, at least 5, or at least 6 phosphorothioates.

[0145] In some embodiments, the tailing nucleic acid comprises at least 6 phosphorothioates.

[0146] In some embodiments, the tailing nucleic acid comprises at least 3 guanine nucleotides and least 3 phosphorothioates.

[0147] In some embodiments, the tailing nucleic acid comprises at least 6 nucleotides comprising a 2' modification.

[0148] In some embodiments, the tailing nucleic acid comprises at least 3 deoxyribose sugars.

[0149] In some embodiments, the tailing nucleic acid comprises at least 5, at least 10, at least 15, at least 20, or at least 23 deoxyribose sugars.

[0150] In some embodiments, the tailing nucleic acid comprises at least 23 deoxyribose sugars.

[0151] In some embodiments, the 3' terminal nucleotide of the tailing nucleic acid comprises a dideoxyadenosine, dideoxycytidine, dideoxyguanosine, dideoxythymidine, dideoxyuridine, or inverted-deoxythymidine.

[0152] In some embodiments, the tailing nucleic acid comprises a first modified nucleotide and a second modified nucleotide, wherein the first and second modified nucleotides comprise different structures.

[0153] In some embodiments, 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 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the poly-A region of the modified mRNA are adenosine nucleotides.

[0154] In some embodiments, the poly-A region of the modified mRNA comprises at least 25-500 nucleotides.19167200.02701 / 151646090v.l

[0155] In some embodiments, the poly-A region of the modified mRNA comprises at least 50, at least 100, at least 150, or at least 200 nucleotides.

[0156] In some embodiments, the modified mRNA is a linear mRNA, wherein the linear mRNA comprises a 5' cap.

[0157] In some embodiments, the 5' cap comprises a 7-methylguanosine.

[0158] In some embodiments, the 5' cap further comprises one or more phosphates connecting the 7- methylguanosine to an adjacent nucleotide of the modified mRNA.

[0159] In some embodiments, the 5' cap comprises a 3'-O-Me-m7G(5')ppp(5')G.

[0160] In some embodiments, one or more phosphates of the 5' cap is a modified phosphate selected from phosphorothioate, triazole ring, dihalogenmethylenebisphosphonate, imidodiphosphate, and methylenebis(phosphonate).

[0161] In some embodiments, the RNA ligase is T4 RNA ligase.

[0162] In some aspects, the present disclosure provides a method of producing a modified noncoding RNA of the present invention, the method comprising ligating a first RNA comprising a non-coding RNA sequence to a tailing nucleic acid comprising one or more modified nucleotides, in the presence of an RNA ligase, whereby the RNA ligase forms a covalent bond between the 3' nucleotide of the RNA and the 5' nucleotide of the tailing nucleic acid to produce the modified non-coding RNA.

[0163] In some embodiments, the modified non-coding RNA comprises a poly-A region that is 3' to the non-coding RNA sequence.

[0164] In some embodiments, the method further comprises circularizing the modified non-coding RNA in the presence of a ribozyme, wherein the modified non-coding RNA comprises a 3' intron and a 5' intron, wherein the 3' intron is 5' to the non-coding RNA sequence, wherein the 5' intron is 3' to the poly-A region, whereby the ribozyme forms a covalent bond between a nucleotide that is 3' to the 3' intron and a nucleotide that is 5' to the 5' intron to produce a circular noncoding RNA that does not comprise the 5' intron or the 3' intron, wherein the poly-A region is between the 3' and 5' nucleotides of the non-coding RNA.

[0165] In some embodiments, the method further comprises steps of:(i) introducing a 5' terminal phosphate group onto the first nucleotide of the modified noncoding RNA;(ii) cleaving one or more 3' terminal nucleotides of the modified non-coding RNA to produce a modified non-coding RNA with a 3' terminal hydroxyl group; and20167200.02701 / 151646090v.l(iii) circularizing the modified non-coding RNA produced in step (ii) in the presence of a circularizing ligase; whereby the circularizing ligase forms a covalent bond between the 3' nucleotide of the modified non-coding RNA and the 5' nucleotide of the modified noncoding RNA to produce a circular modified non-coding RNA, wherein the poly-A region is between the 3' and 5' nucleotides of the non-coding RNA.

[0166] In some embodiments, the tailing nucleic acid further comprises one or more copies of a structural sequence.

[0167] In some embodiments, the modified non-coding RNA comprises a poly-A region is between the non-coding RNA sequence and the one or more copies of the structural sequence.

[0168] In some embodiments, the method further comprises circularizing the modified non-coding RNA in the presence of a ribozyme, wherein the modified non-coding RNA comprises a 3' intron and a 5' intron, wherein the 3' intron is 5' to the non-coding RNA sequence, wherein the 5' intron is 3' to the one or more copies of the structural sequence, whereby the ribozyme forms a covalent bond between a nucleotide that is 3' to the 3' intron and a nucleotide that is 5' to the 5' intron to produce a circular non-coding RNA that does not comprise the 5' intron or the 3' intron, wherein the one or more copies of the structural sequence are between the poly-A region and the noncoding RNA sequence of the circular non-coding RNA.

[0169] In some embodiments, the method further comprises the steps of(i) introducing a 5' terminal phosphate group onto the first nucleotide of the modified noncoding RNA;(ii) cleaving one or more 3' terminal nucleotides of the modified non-coding RNA to produce a modified non-coding RNA with a 3' terminal hydroxyl group; and(iii) circularizing the modified non-coding RNA produced in step (ii) in the presence of a circularizing ligase; whereby the circularizing ligase forms a covalent bond between the 3' nucleotide of the modified non-coding RNA and the 5' nucleotide of the modified noncoding RNA to produce a circular modified non-coding RNA, wherein the one or more copies of the structural sequence are between the poly-A region and the non-coding RNA sequence.

[0170] In some embodiments, the modified non-coding RNA is circularized in the presence of a scaffold nucleic acid, wherein the scaffold nucleic acid is a nucleic acid that is21167200.02701 / 151646090v.lcapable of hybridizing with the modified non-coding RNA, wherein the modified non-coding RNA forms a circular secondary structure when bound to the scaffold nucleic acid.

[0171] In some embodiments, the scaffold nucleic acid comprises:(a) a first hybridization sequence comprising 5 or more nucleotides, wherein the first hybridization sequence is complementary to at least the first five (5) nucleotides of the modified non-coding RNA; and(b) a second hybridization sequence comprising 5 or more nucleotides, wherein the second hybridization sequence is complementary to at least the last five (5) nucleotides of the modified non-coding RNA; wherein at least the first five (5) nucleotides of the modified noncoding RNA hybridize with the first hybridization sequence, and at least the last five (5) nucleotides of the modified noncoding RNA hybridize with the second hybridization sequence.

[0172] In some embodiments, a last nucleotide of the first hybridization sequence and a first nucleotide of the second hybridization sequence are adjacent in the scaffold nucleic acid and not separated by any other nucleotides.

[0173] In some embodiments, the modified non-coding RNA comprises:(i) a first self-hybridization sequence that is 5' to the open reading frame;(ii) a second self-hybridization sequence that is 3' to the open reading frame;(iii) a first non-hybridization sequence that is 5' to the first self-hybridization sequence; and(iv) a second non-hybridization sequence that is 3' to the second self-hybridization sequence, wherein the first and second self-hybridization sequences are capable of hybridizing with each other, and wherein the first and second self-hybridization sequences are not capable of hybridizing with each other.

[0174] In some embodiments, hybridization of the first and second self-hybridization sequences forms a secondary structure in which the 5' terminal nucleotide and the 3' terminal nucleotide of the modified non-coding RNA are separated by a distance of less than 100 A.

[0175] In some embodiments, the 5' terminal nucleotide and the 3' terminal nucleotide are separated by a distance of less than 90 A, less than 80 A, less than 70 A, less than 60 A, less than 50 A, less than 40 A, less than 30 A, less than 20 A, or less than 10 A.

[0176] In some embodiments, the circularizing ligase is T4 RNA ligase.

[0177] In some embodiments, the structural sequence is a G-quadruplex sequence.

[0178] In some embodiments, the G-quadruplex is an RNA G-quadruplex sequence.22167200.02701 / 151646090v.l

[0179] In some embodiments, the G-quadruplex is a DNA G-quadruplex sequence.

[0180] In some embodiments, the structural sequence is a telomeric repeat sequence.

[0181] In some embodiments, the structural sequence is an aptamer sequence comprising at least two nucleotides that are capable of interacting to form an aptamer, wherein the aptamer is a secondary structure that is capable of binding to a target molecule.

[0182] In some embodiments, the 5' nucleotide of the RNA does not comprise a 5' terminal phosphate group; wherein the 3' nucleotide of the RNA comprises a 3' terminal hydroxyl group; wherein the 5' nucleotide of the tailing nucleic acid comprises a 5' terminal phosphate group; and wherein the 3' nucleotide of the tailing nucleic acid does not comprise a 3' terminal hydroxyl group.

[0183] In some embodiments, the 5' nucleotide of the RNA does not comprise a 5' terminal hydroxyl group; wherein the 3' nucleotide of the RNA comprises a 3' terminal phosphate group; wherein the 5' nucleotide of the tailing nucleic acid comprises a 5' terminal hydroxyl group; wherein the 3' nucleotide of the tailing nucleic acid does not comprise a 3' terminal phosphate group; and wherein the RNA ligase is an RtcB ligase.

[0184] 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.

[0185] In some embodiments, at least 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 of the 25 last nucleotides of the tailing nucleic acid are modified nucleotides.

[0186] In some embodiments, at least one modified nucleotide comprises a modified nucleobase.

[0187] In some embodiments, the modified nucleobase is selected from xanthine, allyaminouracil, allyaminothymidine, hypoxanthine, digoxigeninated adenine, digoxigeninated cytosine, digoxigeninated guanine, digoxigeninated uracil, 6- chloropurineriboside, N6-methyladenine, methylpseudouracil, 2-thiocytosine, 2-thiouracil, 5- methyluracil, 4-thiothymidine, 4-thiouracil, 5,6-dihydro-5-methyluracil, 5,6-dihydrouracil, 5- [(3- Indolyl)propionamide-N-allyl]uracil, 5 -aminoallylcytosine, 5-aminoallyluracil, 5- bromouracil, 5- bromocytosine, 5-carboxycytosine, 5-carboxymethylesteruracil, 5- carboxyuracil, 5-fluorouracil, 5-formylcytosine, 5-formyluracil, 5 -hydroxy cytosine, 5- hydroxymethylcytosine, 5- hydroxymethyluracil, 5-hydroxyuracil, 5-iodocytosine, 5- iodouracil, 5 -methoxy cytosine, 5- methoxyuracil, 5-methylcytosine, 5-methyluracil, 5-23167200.02701 / 151646090v.lpropargylaminocytosine, 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, dabcyl-5-3- aminoallyluracil, desthiobiotin- 16-aminoallyl-uracil, desthiobiotin-6-aminoallylcytosine, isoguanine, N1 -ethylpseudouracil, Nl- methoxymethylpseudouracil, N1 -methyladenine, Nl- methylpseudouracil, Nl -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-carboxamide-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-threonyl carbamoyladenine (ms2t6A), N6-methyl-N6-threonylcarbamoyladenine (m6t6A), N6-hydroxynorvalylcarbamoyladenine (hn6A), 2-methylthio-N6-hydroxynorvalyl carbamoyladenine (ms2hn6A), N6,N6- dimethyladenine (m62A), and N6-acetyladenine (ac6A).

[0188] In some embodiments, at least one modified nucleotide comprises a modified sugar.

[0189] In some embodiments, the modified sugar is selected from 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-l-indolyl-2'-deoxyribose, 5'-biotin-ribose, 2'-O,4'-C-methylene-linked, 2'-O,4'-C-amino-linked ribose, and 2'-O,4'-C-thio- linked ribose.

[0190] In some embodiments, at least one modified nucleotide comprises a 2' modification.24167200.02701 / 151646090v.l

[0191] In some embodiments, the 2' modification is selected from a locked-nucleic acid (LNA) modification (i.e., a nucleotide comprising an additional carbon atom bound to the 2' oxygen and 4' carbon of ribose), 2'-fluoro (2'-F) , 2'-O-methoxy-ethyl (2’-M0E), 2'- O- methylation (2’-OMe), and 2'-O-N-methylacetamido (2 -0-NMA).

[0192] In some embodiments, at least one modified nucleotide comprises a modified phosphate.

[0193] In some embodiments, the modified phosphate is selected from phosphorothioate (PS), phosphorodithioate, thiophosphate, 5'-O-methylphosphonate, 3'-O- methylphosphonate, 5'-hydroxyphosphonate, hydroxyphosphanate, phosphoroselenoate, selenophosphate, phosphoramidate, carbophosphonate, methylphosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidinium ring, triazole ring, boranophosphate (BP), methylphosphonate, and guanidinopropyl phosphoramidate.

[0194] In some embodiments, the tailing nucleic acid comprises at least 3, at least 4, at least 5, or at least 6 phosphorothioates.

[0195] In some embodiments, the tailing nucleic acid comprises at least 6 phosphorothioates.

[0196] In some embodiments, the tailing nucleic acid comprises at least 3 guanine nucleotides and least 3 phosphorothioates.

[0197] In some embodiments, the tailing nucleic acid comprises at least 6 nucleotides comprising a 2' modification.

[0198] In some embodiments, the tailing nucleic acid comprises at least 3 deoxyribose sugars.

[0199] In some embodiments, the tailing nucleic acid comprises at least 5, at least 10, at least 15, at least 20, or at least 23 deoxyribose sugars. In some embodiments, the tailing nucleic acid comprises at least 23 deoxyribose sugars.

[0200] In some embodiments, the 3' terminal nucleotide of the tailing nucleic acid comprises a dideoxyadenosine, dideoxycytidine, dideoxyguanosine, dideoxythymidine, dideoxyuridine, or inverted-deoxythymidine.

[0201] In some embodiments, the tailing nucleic acid comprises a first modified nucleotide and a second modified nucleotide, wherein the first and second modified nucleotides comprise different structures.

[0202] In some embodiments, 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 95%, at least 96%, at least 97%, at25167200.02701 / 151646090v.lleast 98%, or at least 99% of the poly-A region of the modified non-coding RNA are adenosine nucleotides.

[0203] In some embodiments, the poly-A region of the modified non-coding RNA comprises at least 25-500 nucleotides.

[0204] In some embodiments, the poly-A region of the modified non-coding RNA comprises at least 50, at least 100, at least 150, or at least 200 nucleotides.

[0205] In some embodiments, the RNA ligase is T4 RNA ligase.

[0206] In some aspects, the present disclosure provides a modified mRNA produced by any one of the methods provided herein.

[0207] In some embodiments, the mRNA encodes an antigen or a therapeutic protein.

[0208] In some embodiments, the antigen is a viral antigen, bacterial antigen, protozoal antigen, or fungal antigen.

[0209] In some embodiments, the therapeutic protein is an enzyme, transcription factor, cell surface receptor, growth factor, or clotting factor.

[0210] In some embodiments, the open reading frame is codon-optimized for expression in a cell.

[0211] In some embodiments, the modified mRNA is codon-optimized for expression in a mammalian cell.

[0212] In some embodiments, the modified mRNA is codon-optimized for expression in a human cell.

[0213] In some aspects, the present disclosure provides a modified non-coding RNA produced by any one of the methods provided herein.

[0214] In some embodiments, the modified non-coding RNA is a guide RNA (gRNA), a prime editing guide RNA (pegRNA), or a long non-coding RNA (IncRNA).

[0215] In some aspects, the present disclosure provides a lipid nanoparticle comprising any one of the modified mRNAs or modified non-coding RNAs provided herein.

[0216] In some aspects, the present disclosure provides a cell comprising any one of the modified mRNAs or modified non-coding RNAs provided herein.

[0217] In some embodiments, the cell is a mammalian cell.

[0218] In some embodiments, the cell is a human cell.

[0219] In some aspects, the present disclosure provides a composition comprising any of the modified mRNAs, modified non-coding RNAs, lipid nanoparticles, or cells provided herein.26167200.02701 / 151646090v.l

[0220] In some aspects, the present disclosure provides a pharmaceutical composition comprising any of the modified mRNAs, modified non-coding RNAs, lipid nanoparticles, or cells provided herein, and a pharmaceutically acceptable excipient.

[0221] In some aspects, the present disclosure provides a method comprising introducing any of the modified mRNAs, modified non-coding RNAs, or lipid nanoparticles provided herein into a cell.

[0222] In some aspects, the present disclosure provides a method comprising introducing any of the modified mRNAs, modified non-coding RNAs, lipid nanoparticles, cells, or compositions provided herein, into a subject.

[0223] In some aspects, the present disclosure provides a method of vaccinating a subject, the method comprising introducing any of the modified mRNAs, lipid nanoparticles, cells, or compositions provided herein, into a subject, wherein the open reading frame of the mRNA encodes an antigen.

[0224] In some aspects, the present disclosure provides a method of replacing an enzyme in a subject, the method comprising introducing any of the modified mRNAs, lipid nanoparticles, cells, or compositions provided herein, into a subject, wherein the open reading frame of the mRNA encodes an enzyme.

[0225] In some aspects, the present disclosure provides a method of modifying the genome of a subject, the method comprising introducing any of the modified non-coding RNAs or compositions provided herein into a subject

[0226] In some embodiments, the subject is a mammal.

[0227] In some embodiments, the subject is a human.

[0228] In some aspects, the present disclosure provides any of the modified mRNAs, modified noncoding RNAs, lipid nanoparticles, cells, or compositions provided herein, for use as a medicament.

[0229] In some aspects, the present disclosure provides a kit comprising an RNA and a tailing nucleic acid of any of the methods provided herein.

[0230] In some embodiments, the kit further comprises an RNA ligase.

[0231] In some aspects, the present disclosure provides a kit comprising any of the pharmaceutical compositions provided herein and a delivery device.

[0232] In some aspects, the present disclosure provides a method for purifying a modified mRNA or a modified non-coding RNA, comprising contacting a mixture comprising a modified mRNA or a modified non-coding RNA with a purification medium,27167200.02701 / 151646090v.lwherein the modified mRNA or modified non-coding RNA interacts with the purification medium to form a modified RNA- purification medium conjugate, separating the modified RNA-purification medium conjugate from the mixture, and eluting the modified mRNA or modified non-coding RNA from the modified RNA-purification medium conjugate with a solvent.

[0233] In some embodiments, the purification medium comprises a paramagnetic bead.BRIEF DESCRIPTION OF THE DRAWINGS

[0234] Figure 1 is bar graph depicting the nLuc activity in HeLa cells 24 hours posttransfection with modified mRNAs, as described in Example 2.

[0235] Figure 2 is an image of a blot depicting oligo ligation efficiency of purified mRNA samples, as described in Example 2.

[0236] Figure 3 depicts line graphs depicting the nLuc activity in HeLa cells 24 hours (3 A), 48 hours (3B), and 72 hours (3C) post-transfection with the modified mRNA, as described in Example 2.

[0237] Figure 4 is an image of a blot depicting oligo ligation efficiency of purified mRNA samples, as described in Example 3.

[0238] Figures 5 A, 5B, and 5C are bar graphs depicting the Nano-Luc activity in HeLa cells (5 A), U2-OS cells (5B), and HEK293 cells (5C) post-transfection with the modified mRNA, as described in Example 3.

[0239] Figure 6 is an image of a blot depicting oligo ligation efficiency of purified mRNA samples, as described in Example 4.

[0240] Figures 7A and 7B are bar graphs depicting the Nano-Luc activity in HEK293FT cells (7 A) and U2-OS cells (7B), as described in Example 4.

[0241] Figures 8A and 8B are bar graphs depicting the Nano-Luc activity in U2-OS cells (8 A) and HEK293FT cells (8B) as described in Example 5.

[0242] Figures 9A, 9B, and 9C are bar graphs depicting the Nano-Luc activity in HeLa cells (9 A), HEK293FT cells (9B), and U2-OS cells (9C) post-transfection with the modified mRNA, as described in Example 6.

[0243] Figures 10A, 10B, and 10C are line graphs depicting the Nano-Luc activity in HeLa cells 6, 18, and 24 hours post-transfection of HeLa cells with 5 ng (10A), 1 ng (10B), 0.1 ng (10C) of mRNA, as described in Example 7.28167200.02701 / 151646090v.l

[0244] Figures 11 A and 1 IB are line graphs depicting the total flux (p / s) in mice administered with the lipid nanoparticle formulations (0.5 or 0.1 mg / kg) as described in Example 8.DETAILED DESCRIPTION

[0245] Provided herein are modified mRNAs with modified nucleotides and / or structural features in or downstream of the poly-A tail of the mRNA to improve stability in cells and thereby enhance protein production. Also provided are methods of making modified mRNAs by ligating a tailing nucleic acid onto the 3' terminus of an mRNA to introduce a defined number of modified nucleic acids or structural sequences at the 3' of the modified mRNA produced by the ligation. Additionally, the present disclosure provides pharmaceutical compositions comprising one or more of the modified mRNAs provided herein, and kits containing reagents to produce the modified mRNAs described herein.Definitions

[0246] A “messenger RNA” (“mRNA”), as used herein, refers to a nucleic acid comprising an open reading frame encoding a protein, and a poly-A region. An mRNA may also comprise a 5' untranslated region (5' UTR) that is 5' to (upstream of) the open reading frame, and a 3' untranslated region that is 3' to (downstream of) the open reading frame.

[0247] An “open reading frame encoding a protein,” as used herein, refers to a nucleic acid sequence comprising a coding sequence, that leads to the production of the 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 with the amino acid sequence of the protein. The nucleic acid sequence may be a DNA sequence, in which case the protein is produced when an 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 with the amino acid sequence of the protein. An open reading frame typically begins with a START codon, such as AUG in the RNA sequence (ATG in the DNA sequence), and ends with a STOP codon, such as UAG, UAA, or UGA in the RNA sequence (TAG, TAA, or TGA in the DNA sequence), with the number of bases between the G of the START codon and the T or U of the STOP codon being a multiple of 3 (e.g., 3, 6, 9).29167200.02701 / 151646090v.l

[0248] An RNA molecule that can be translated is referred to as a messenger RNA, or mRNA. An DNA or RNA sequence encodes a gene through codons. A codon refers to a group of three nucleotides within a nucleic acid, such as DNA or RNA, sequence. An anticodon refers to a group of three nucleotides within a nucleic acid, such as a transfer RNA (tRNA), that are complementary to a codon, such that the codon of a first nucleic acid associates with the anticodon of a second nucleic acid through hydrogen bonding between the bases of the codon and anticodon. For example, the codon 5'-AUG-3' on an mRNA has the corresponding anticodon 3'-UAC-5' on a tRNA. During translation, a tRNA with an anticodon complementary to the codon to be translated associates with the codon on the mRNA, generally to deliver an amino acid that corresponds to the codon to be translated, or to facilitate termination of translation and release of a translated polypeptide from a ribosome.

[0249] Translation is the process in which the 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 START codon. The next phase of translation, elongation, involves three steps. First, a second tRNA with an anticodon that is complementary to codon following the START codon, or second codon, and carrying a second amino acid, associates with the mRNA. Second, the carbon atom of terminal, non-side chain carboxylic acid moiety of the first amino acid reacts with the nitrogen of the terminal, non-side chain amino moiety of the second amino acid carried, forming a peptide bond between the two amino acids, with the second amino acid being bound to the second tRNA, and the first amino acid bound to the second amino acid, but not the first tRNA. Third, the first tRNA dissociates from the mRNA, and the ribosome advances along the mRNA, such that the position at which the first tRNA associated with the ribosome is now occupied by the second tRNA, and the position previously occupied by the second tRNA is now free for an additional tRNA carrying an additional amino acid to associate with the mRNA. These three steps of 1) association of a tRNA carrying amino acid, 2) formation of a peptide bond, which adds an additional amino acid to a growing polypeptide, and 3) advancement of the ribosome along the mRNA, continue until the ribosome reaches a STOP codon, which results in termination of translation. Generally, tRNAs that associate with STOP codons do not carry an amino acid, so the association of a tRNA that does not carry an amino acid during the elongation step30167200.02701 / 151646090v.lresults in cleavage of the bond between the polypeptide and the tRNA carrying the final amino acid in the polypeptide, such that the polypeptide is released from the ribosome.

[0250] Alternatively, ribosomes may dissociate from the mRNA and release the polypeptide if no tRNA associates with the STOP codon.

[0251] A “nucleic acid,” or “polynucleotide,” as used herein, refers to an organic molecule comprising two or more covalently bonded nucleotides. A “nucleotide,” as used herein, refers to an organic molecule comprising a 1) a nucleoside comprising a sugar covalently bonded to a nitrogenous base (nucleobase); and 2) a phosphate group that is covalently bonded to the sugar of the nucleoside. Nucleotides in a polynucleotide are typically joined by a phosphodiester bond, in which the 3' carbon of the sugar of a first nucleotide is linked to the 5' carbon of the sugar of a second nucleic acid by a bridging phosphate group. Typically, the bridging phosphate comprises two non-bridging oxygen atoms, which are bonded only to a phosphorus atom of the phosphate, and two bridging oxygen atoms, each of which connects 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 in a nucleic acid, a first nucleotide is said to be 5' to (upstream of) a second nucleotide if the 3' carbon of first nucleotide is connected to the 5' carbon of the second nucleotide. Similarly, a second nucleotide is said to be 3' to (downstream of) a first nucleotide if the 5' carbon of the second nucleotide is connected to the 3' carbon of the first nucleotide. Nucleic acid sequences are typically read in 5'->3' order, starting with the 5' nucleotide and ending with the 3' nucleotide.

[0252] A “modified nucleotide,” as used herein, refers to a nucleotide with a structure that is not the canonical structure of an adenosine nucleotide, cytidine nucleotide, guanine nucleotide, or uracil nucleotide. A canonical structure of a molecule refers to a structure that is generally known in the art to be the structure referred to by the name of the molecule. As used herein, a “modified nucleotide” may also refer to a nucleotide which comprises a nucleobase or sugar (ribose or deoxyribose) that is not canonical. A “modified nucleotide” may also refer to a nucleotide that is covalently linked to a second nucleotide through an intemucleoside linkage that is not a canonical intemucleoside linkage (i.e., not a phosphodiester intemucleoside linkage, e.g., a phosphorothioate intemucleoside linkage). A canonical structure of an adenosine ribonucleotide, which comprises an adenine base, ribose31167200.02701 / 151646090v.lsugar, and one or more phosphate groups, is shown below, in the form of adenosine monophosphate:

[0253] The canonical structure of AMP also refers to structures in which one or more hydroxyl groups of the phosphate and / or one or more hydroxyl groups of the sugar aredeprotonated, and structures in which an oxygen atom of the phosphate and / or the 3' oxygen atom of the sugar are bound to an adjacent nucleotide in a nucleic acid sequence.

[0254] The canonical structure of a cytosine nucleotide which comprises a cytosine base, ribose sugar, and one or more phosphate groups, is shown below, in the form of cytidine monophosphate:

[0255] The canonical structure of CMP also refers to structures in which one or more hydroxyl groups of the phosphate and / or one or more hydroxyl groups of the sugar are deprotonated, and structures in which an oxygen atom of the phosphate and / or the 3' oxygen atom of the sugar are bound to an adjacent nucleotide in a nucleic acid sequence.

[0256] The canonical structure of a guanine nucleotide which comprises a guanine base, ribose sugar, and one or more phosphate groups, is shown below, in the form of guanosine monophosphate:

[0257] The canonical structure of GMP also refers to structures in which one or more hydroxyl groups of the phosphate and / or one or more hydroxyl groups of the sugar are167200.02701 / 151646090v.ldeprotonated, and structures in which an oxygen atom of the phosphate and / or the 3' oxygen atom of the sugar are bound to an adjacent nucleotide in a nucleic acid sequence.

[0258] The canonical structure of an uracil nucleotide which comprises an uracil base, ribose sugar, and one or more phosphate groups, is shown below, in the form of uridine monophosphate:

[0259] The canonical structure of UMP also refers to structures in which one or more hydroxyl groups of the phosphate and / or one or more hydroxyl groups of the sugar are deprotonated, and structures in which an oxygen atom of the phosphate and / or the 3' oxygen atom of the sugar are bound to an adjacent nucleotide in a nucleic acid sequence.

[0260] The structure of a modified nucleotide may differ from the structure of a canonical nucleotide due to one or more modifications in the sugar, nitrogenous base, or phosphate of the nucleotide. In some embodiments, the modified nucleotide comprises a modified nucleoside that is not the canonical structure of an adenine nucleoside, cytosine nucleoside, guanine nucleoside, or uracil nucleoside.

[0261] An example of a canonical structure of adenosine, an adenine nucleoside, is reproduced below:

[0262] The canonical structure of adenosine also refers to structures in which one or more hydroxyl groups of the phosphate and / or one or more hydroxyl groups of the sugar are deprotonated, structures in which the 5' carbon is bound to a 5' phosphate in a nucleic acid sequence, and structures in which a 3' oxygen atom is bound to a 5' phosphate group of an adjacent nucleotide in a nucleic acid sequence.167200.02701 / 151646090v.l

[0263] An example of a canonical structure of cytidine, a cytosine nucleoside, is reproduced below:(cytidine).

[0264] The canonical structure of cytidine also refers to structures in which one or more hydroxyl groups of the phosphate and / or one or more hydroxyl groups of the sugar are deprotonated, structures in which the 5' carbon is bound to a 5' phosphate in a nucleic acid sequence, and structures in which a 3' oxygen atom is bound to a 5' phosphate group of an adjacent nucleotide in a nucleic acid sequence.

[0265] An example of a canonical structure of guanosine, a guanine nucleoside, is reproduced below:

[0266] The canonical structure of guanosine also refers to structures in which one or more hydroxyl groups of the phosphate and / or one or more hydroxyl groups of the sugar are deprotonated, structures in which the 5' carbon is bound to a 5' phosphate in a nucleic acid sequence, and structures in which a 3' oxygen atom is bound to a 5' phosphate group of an adjacent nucleotide in a nucleic acid sequence.

[0267] An example of a canonical structure of uridine, an uracil nucleoside, is reproduced below:(uridine), ’

[0268] The canonical structure of uridine also refers to structures in which one or more hydroxyl groups of the phosphate and / or one or more hydroxyl groups of the sugar are deprotonated, structures in which the 5' carbon is bound to a 5' phosphate in a nucleic acid34167200.02701 / 151646090v.lsequence, and structures in which a 3' oxygen atom is bound to a 5' phosphate group of an adjacent nucleotide in a nucleic acid sequence.

[0269] By a “modified sugar” is meant a sugar or moiety other than 2’ -OH ribose sugar. Some exemplary nucleotides comprising a modified sugar are 2’-H ribose, 2’-F ribose, 2’-0Me ribose, 2’-O,4’-C-methylene ribose (locked nucleic acid, LNA), anhydrohexitol (1,5- anhydrohexitol nucleic acid, HNA), cyclohexene (Cyclohexene nucleic acid, CeNA), 2’- methoxyethyl ribose, 2’-O-allyl ribose, 2’-C-allyl ribose, 2'-O-N-methylacetamido (2'-O- NMA) ribose, a 2'-O-dimethylaminoethoxyethyl (2'-0-DMAE0E) ribose, 2'-O-aminopropyl (2'-O-AP) ribose, 2’-F arabinose (2'-ara-F), threose (Threose nucleic acid, TNA), and 2,3- dihydroxylpropyl (glycol nucleic acid, GNA). It is noted that the nucleoside with the modified sugar can be present at any position of the polyA region.

[0270] A “structural sequence,” as used herein, refers to a nucleic acid sequence comprising at least two nucleotides that are capable of interacting with each other to form a secondary structure in a nucleic acid comprising the structural sequence.

[0271] An “aptamer,” as used herein, refers to a nucleic acid comprising a secondary structure that is capable of binding to a target molecule.

[0272] A “ligase,” as used herein, refers to an enzyme that is capable of forming a covalent bond between two nucleotides, and the process of “ligation” refers to the formation of the covalent bond between the two nucleotides.

[0273] A “tailing nucleic acid,” as used herein, refers to a nucleic acid that is ligated onto the 3' end of another nucleic acid.Modified mRNAs

[0274] In some aspects, the present disclosure provides modified mRNAs comprising i) one or more modified nucleotides; and / or ii) one or more copies (repeating units) of a structural sequence, with the modified nucleotides and / or structural sequence being part of or 3' to the poly-A region of the mRNA. The poly-A region, also called the poly(A) region or poly(A) tail, of an mRNA is a region of an mRNA that is 3' to (downstream of) the open reading frame, comprising multiple, consecutive adenosine nucleotides, typically 50-300 consecutive adenosine nucleotides, and may encompass multiple non-adenosine nucleotides downstream of the consecutive adenosine nucleotides. In cells, after transcription of a DNA sequence, which produces a precursor messenger RNA (pre-mRNA), the poly-A tail is added by a polyadenylating enzyme, such as a poly-A polymerase (PAP), resulting in a long sequence of multiple, consecutive adenosine nucleotides, at the 3' end of the RNA. The poly-35167200.02701 / 151646090v.lA region plays multiple roles that are important in the production of proteins encoded by mRNAs. First, the poly-A region provides an attachment site for poly-A binding proteins (PABPs), which associate with the mRNA in the nucleus and promote export into the cytoplasm (see, e.g., Tudek et al. Philos Trans R Soo Loud B Biol Sei. 2018. 373(1762):20180169). Additionally, the presence of a poly-A tail in an mRNA facilitates the initiation of translation (see, e.g., Gallic. Genes & Dev. 1991. 5:2108-2116, and Munroe et al. MolCellBiol. 1990. 10(7):3441-3455). Finally, the poly-A tail stabilizes the mRNA by protecting the open reading frame from the activity of exonucleases, such as polynucleotide phosphorylase (PNPase), which remove 3' nucleotides from an mRNA. As an exonuclease removes nucleotides, the mRNA becomes progressively shorter, and once all of the nucleotides downstream of the open reading frame are removed, the nucleotides removed by the exonuclease will be nucleotides of the open reading frame. Removal of nucleotides from the open reading frame prevents translation of the encoded protein.

[0275] Additionally, the association of an exonuclease with the mRNA near the open reading frame can inhibit translation by sterically hindering ribosomes and tRNAs from associating with the mRNA. Removal of the poly-A tail is often cited as a rate-limiting step in mRNA degradation, with the life span of an mRNA in a cell being determined by the time required to remove its poly-A tail (see, e.g., Dreyfus et al., Cell. 2002. Ul(5):611-613). The composition of a poly-A tail of an mRNA varies, but contains approximately 75 adenosine nucleotides in yeast cells and 250 adenosine nucleotides in mammalian cells.

[0276] In some embodiments of the modified mRNAs provided herein, the modified mRNA comprises one or more modified nucleotides in the poly-A region or 3' to (downstream of) the poly-A region of the mRNA. In some embodiments, the poly-A region includes one or more nucleotides that are not canonical adenosine nucleotides. In some embodiments, the poly-A region includes one or more nucleotides that are not adenosine nucleotides. In some embodiments, the poly-A region comprises one or more nucleotides that are 3' to (downstream of) a nucleic acid sequence comprising multiple, consecutive adenosine nucleotides. In some embodiments, the poly-A region comprises at least 25 consecutive adenosine nucleotides, which may be canonical adenosine nucleotides or modified adenosine nucleotides. In some embodiments, the poly-A region comprises 25-500 consecutive adenosine nucleotides, which may be canonical adenosine nucleotides or modified adenosine nucleotides. In some embodiments, the poly-A region comprises 25-300 consecutive adenosine nucleotides. In some embodiments, the poly-A region comprises at least 30, at36167200.02701 / 151646090v.lleast 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.

[0277] In some embodiments, one or more of the modified nucleotides of the modified mRNA comprise a modified phosphate group. A modified phosphate group is a phosphate group that differs from the canonical structure of phosphate. An example of a canonical structure of a phosphate is shown below:

[0278] where Rs and R3 are atoms or molecules to which the canonical phosphate is bonded. For example, for a phosphate in a nucleic acid sequence, Rs may refer to the upstream nucleotide of the nucleic acid, and R3 may refer to the downstream nucleotide of the nucleic acid. The canonical structure of phosphate also refers to structures in which one or more hydroxyl groups of the phosphate are deprotonated, or in which an oxygen atom of the phosphate is bonded to an adjacent nucleotide in a nucleic acid sequence. Non-limiting examples of modified phosphate groups that can be substituted for a canonical phosphate in a nucleic acid include phosphorothioate (PS), phosphorodithioate, thiophosphate, 5'-O- methylphosphonate, 3'-O- methylphosphonate, 5'-hydroxyphosphonate, hydroxyphosphanate, phosphoroselenoate, selenophosphate, phosphoramidate, carbophosphonate, methylphosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidinium ring, triazole ring, boranophosphate (BP), methylphosphonate, and guanidinopropyl phosphoramidate.

[0279] 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 nucleotide comprises a modified nucleobase selected from the group consisting of: xanthine, allyaminouracil, allyaminothymidine, hypoxanthine, digoxigeninated adenine, digoxigeninated cytosine, digoxigeninated guanine, digoxigeninated uracil, 6-chloropurineriboside, N6-methyladenine, methylpseudouracil, 2-thiocytosine, 2- thiouracil, 5-methyluracil, 4-thiothymidine, 4-37167200.02701 / 151646090v.lthiouracil, 5,6-dihydro-5-methyluracil, 5,6- dihydrouracil, 5-[(3-Indolyl)propionamide-N- allyl]uracil, 5-aminoallylcytosine, 5- aminoallyluracil, 5 -bromouracil, 5 -bromocytosine, 5- carboxycytosine, 5- carboxymethylesteruracil, 5-carboxyuracil, 5-fluorouracil, 5- formylcytosine, 5-formyluracil, 5 -hydroxy cytosine, 5-hydroxymethylcytosine, 5- hydroxymethyluracil, 5-hydroxyuracil, 5- lodocytosine, 5-iodouracil, 5 -methoxy cytosine, 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-propargyl aminoguanine, 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, dabcyl-5-3-aminoallyluracil, desthiobiotin- 16-aminoallyl-uracil, desthiobiotin-6-aminoallylcytosine, isoguanine, Nl- ethylpseudouracil, N1 -methoxymethylpseudouracil, N1 -methyladenine, Nl- 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-carboxamide-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-threonyl carbamoyladenine (ms2t6A), N6-methyl-N6-threonylcarbamoyladenine (m6t6A), N6-hydroxynorvalylcarbamoyladenine (hn6A), 2-methylthio-N6-hydroxynorvalyl carbamoyladenine (ms2hn6A), N6,N6- dimethyladenine (m62A), and N6-acetyladenine (ac6A). In some embodiments, at least one modified nucleotide comprises a modified sugar selected from 2'-thioribose, 2', 3 '- dideoxyribose, 2'-amino-2'-deoxyribose, 2' deoxyribose, 2'-azido-2'-deoxyribose, 2'-fluoro-2'- deoxyribose, 2'-O-m ethylribose, 2'-O-m ethyldeoxyribose, 3 '-amino-2', 3 '-dideoxyribose, 3 '- azido-2', 3 '-dideoxyribose, 3 '-deoxyribose, 3'-O-(2-nitrobenzyl)-2'-deoxyribose, 3 '-0 - methylribose, 5'-aminoribose, 5'-thioribose, 5-nitro-l- indolyl-2'-deoxyribose, 5'-biotin-ribose,38167200.02701 / 151646090v.l2'-O,4'-C-methylene-linked, 2'-O,4'-C-amino-linked ribose, and 2'-O,4'-C-thio-linked ribose.In certain embodiments, at least one modified nucleobase is a 2'-O-(unsubstituted Cl-6 alkoxy)-(unsubstituted Cl-6 alkyl) nucleobase (e.g., 2'-O-(unsubstituted Cl-6 alkoxy)- (unsubstituted Cl-6 alkyl) RNA nucleobase). In certain embodiments, at least one modified nucleobase is a 2'-O-methoxy-ethyl nucleobase (e.g., 2'-O-methoxy-ethyl RNA nucleobase). In some embodiments, at least one modified nucleotide comprises a 2' modification. In some embodiments, the 2' modification is selected from a locked-nucleic acid (LNA) modification (i.e., a nucleotide comprising an additional carbon atom bound to the 2' oxygen and 4' carbon of ribose), 2'-fluoro (2'-F), 2'-O-methoxy-ethyl (2'- MOE), 2'-O-methylation (2’-0Me), and 2'-0-N-m ethyl acetamido (2'-0-NMA).

[0280] In some embodiments, at least one modified nucleotide comprises a modified phosphate selected from phosphorothioate (PS), phosphorodithioate, thiophosphate, 5'-O- methylphosphonate, 3'-O-methylphosphonate, 5'-hydroxyphosphonate, hydroxyphosphanate, phosphoroselenoate, selenophosphate, phosphoramidate, carbophosphonate, methylphosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidinium ring, triazole ring, boranophosphate (BP), methylphosphonate, and guanidinopropyl phosphoramidate.

[0281] 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 that has a different structure from the first modified nucleotide. 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 that has a different structure from the first modified phosphate. In some embodiments, the modified mRNA comprises a first modified nucleoside and a second modified nucleoside.

[0282] Aspects of the present disclosure relate to modified mRNAs comprising poly- A regions with 25 or more adenine nucleotides. In certain embodiments, the poly-A region is 3' to the open reading frame and comprises 10 or more, 15 or more, 20 or more, 30 or more, 40 or more, or 50 or more adenosine nucleotides. In certain embodiments, the poly-A region is 3' to the open reading frame and comprises between 10 and 15, between 15 and 20, between 20 and 25, between 25 and 35, between 35 and 50, between 50 and 70, or between 70 and 100 adenosine nucleotides, inclusive. An adenine nucleotide is a nucleotide comprising an adenine nucleoside and a phosphate group. An adenine nucleoside comprises a39167200.02701 / 151646090v.lsugar and an adenine base. In some embodiments, the poly-A region comprises 25 or more canonical adenine nucleotides. A canonical adenosine nucleotide comprises an adenine base, ribose sugar, and phosphate group, as arranged in the structure of adenosine monophosphate (AMP) below:

[0283] In some embodiments, the one or more of the hydroxyl groups of the phosphate and / or the 3' hydroxyl group of the ribose are deprotonated, comprising an oxygen ion instead of an -OH group, as shown by the structure:

[0284] When present in a nucleic acid sequence of an mRNA, a canonical adenosine comprises the following structure and is connected to adjacent nucleotides in the following manner:where Rs is an adjacent nucleotide that is 5' to (upstream of) the adenosine nucleotide in the mRNA, and R3 is an adjacent nucleotide that is 3' to (downstream of) the adenosine nucleotide in the mRNA. In some embodiments, the canonical adenosine nucleotide is the 3' terminal nucleotide (last nucleotide) of a linear mRNA, R3 is a hydrogen, and the 3' terminal nucleotide comprises a 3' terminal hydroxyl (-OH) group. In some embodiments, the canonical adenosine nucleotide is the 3' terminal nucleotide (last nucleotide) of a linear mRNA, and R3 is an electron.167200.02701 / 151646090v.l

[0285] 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). 5' and 3' UTRs are sequences within an mRNA that do not encode amino acids of the protein encoded by the mRNA, and are thus not part of the open reading frame. The 5' UTR is 5' to (upstream of) the open reading frame. The 3' UTR is 3' to (downstream of) the open reading frame. In some embodiments, the 3' UTR comprises one or more nucleotides that are 3' to the open reading frame and 5' to (upstream of) the poly-A region of the mRNA.

[0286] In some embodiments of the mRNAs provided herein, the mRNA comprises, in 5'-to- 3' order: 1) a 5' UTR; 2) an open reading frame; 3) a 3' UTR; and 4) a poly-A region (FIG. 2B). In some embodiments, the last nucleotide of the 5' UTR is 5' to (upstream of) the first nucleotide of the open reading frame. In some embodiments, the first nucleotide of the open reading frame is 3' to (downstream of) the last nucleotide of the 5' UTR, and the last nucleotide of the open reading frame is 5' to (upstream of) the first base 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' to (downstream of) the last nucleotide of the open reading frame, and the last nucleotide of the 3' UTR is 5' to (upstream of) the first base of the poly-A region. In some embodiments, the 3' UTR is between the last nucleotide of the open reading frame and the first nucleotide of the poly-A region. In some embodiments, the first nucleotide of the poly-A region is 3' to (downstream of) the last nucleotide of the 3' UTR.

[0287] In some embodiments, the mRNA is a linear mRNA. A linear mRNA is an mRNA with a 5' terminal nucleotide and a 3' terminal nucleotide. The 5' terminal nucleotide of a linear mRNA is covalently bonded to only one adjacent nucleotide of the mRNA, with the adjacent nucleotide occurring 3' to the 5' terminal nucleotide in the nucleic acid sequence of the mRNA. The 3' terminal nucleotide of a linear mRNA is covalently bonded to only one adjacent nucleotide of the mRNA, with the adjacent nucleotide occurring 5' to the 3' terminal nucleotide in the nucleic acid sequence of the mRNA. In a nucleic acid sequence comprising every nucleotide of a linear mRNA in 5'-to-3 ' order, the 5' terminal nucleotide is the first nucleotide in the sequence, and the 3' terminal nucleotide is the last nucleotide in the sequence.

[0288] In some embodiments of the linear mRNAs provided herein, the mRNA comprises a 5' cap. Most mRNAs produced in eukaryotic cells include a 5' cap that is added during processing of the pre-mRNA into a mature mRNA. The 5' cap plays multiple roles in41167200.02701 / 151646090v.lthe process of mRNA production, export, and translation. First, assembly of the spliceosome, which mediates removal of introns from the pre-mRNA requires binding of the nuclear capbinding complex (CBC) to the 5' cap. Furthermore, interactions between the CBC and nuclear pores mediate the export of mRNA from into the cytoplasm, beginning with the 5' end. Finally, CBC bound to the 5' cap mediates the recruitment of multiple factors, such as CBP80, CHF, eIF3g, eIF4in, Met-tRNAi, and ribosomal subunits, which are required for the initiation of translation (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 structure:

[0289] In some embodiments, the 5' cap comprises one or more phosphates connecting the 7- methylguanosine to an adjacent nucleotide of the modified mRNA. In some embodiments, one or more phosphates of the 5' cap is a modified phosphate selected from phosphorothioate, triazole ring, dihalogenmethylenebisphosphonate, imidodiphosphate, and methylenebis(phosphonate). In some embodiments, the 7-methylguanosine is connected to an adjacent nucleotide of the mRNA by a 5'-to-5' triphosphate bridge. In some embodiments, the 5' cap comprises the structure:with R being the 5' carbon of the first transcribed nucleotide of the mRNA. In some embodiments, the 5' cap comprises a 3'-O-Me-m7G(5')ppp(5')G.

[0290] In some embodiments, the mRNA is a circular mRNA. A circular mRNA is an mRNA with no 5' terminal nucleotide or 3' terminal nucleotide. Every nucleotide in a circular mRNA is covalently bonded to both 1) a 5' adjacent nucleotide; and 2) a 3' adjacent nucleotide. In a circular mRNA with a nucleic acid sequence comprising every nucleotide of42167200.02701 / 151646090v.lthe circular mRNA in 5'-to-3' order, the last nucleotide of the nucleic acid sequence is covalently bonded to the first nucleotide of the nucleic acid sequence. In some embodiments of circular mRNAs with a 5' UTR, a 3' UTR, and a poly-A region, the poly-A region is 3' to (downstream from) the 3' UTR and 5' to (upstream of) the 5' UTR.

[0291] In some embodiments of the modified mRNAs provided herein, the modified mRNA comprises one or more copies of a structural sequence that are 3' to the poly-A region of the mRNA. In some embodiments, nucleotides of the secondary structure interact by hydrogen bonding. In some embodiments, the secondary structure is a G-quadruplex. A G- quadruplex, or G-quadruplex, is a secondary structure formed by guanine-rich nucleic acid sequences. A guanine-rich nucleic acid sequence comprises multiple guanine nucleotides. Typically, at least 50% of the nucleotides in a guanine-rich nucleic acid sequence are guanine nucleotides. A G- quadruplex comprises at least one plane containing four guanines (G- tetrad), with each guanine binding to two other guanines by Hoogsteen hydrogen bonding. Hoogsteen hydrogen bonding refers to hydrogen bonding between nitrogenous bases of nucleotides or nucleosides other than canonical base pairing (A:T, A:U, and G:C). The guanines of the G-tetrad surround an empty space, which may comprise a positive cation, such as a potassium ion, to stabilize the G-tetrad. A G-quadruplex comprises at least two G- tetrads arranged in a parallel orientation.

[0292] In some embodiments of modified mRNAs comprising one or more structural sequences, the structural sequence is a G-quadruplex sequence. A nucleic acid comprising a G- quadruplex sequence is capable of forming a G-quadruplex comprising one or more nucleotides of the G-quadruplex sequence. In some embodiments, the G-quadruplex sequence comprises one or more spacer nucleotides that are not guanine nucleotides. In some embodiments, the G- quadruplex sequence is an RNA G-quadruplex sequence. In some embodiments, the RNA G- quadruplex sequence comprises the nucleic acid sequence GGGGCC (SEQ ID NO: 18). In some embodiments, the modified mRNA comprises at least 3 copies of the nucleotide sequence of SEQ ID NO: 18. In some embodiments, the G- quadruplex sequence is a DNA G-quadruplex sequence. In some embodiments, the DNA G- quadruplex sequence comprises the nucleic acid sequence GGGGCC (SEQ ID NO: 19). In some embodiments, the modified mRNA comprises at least 3 copies of the nucleotide sequence of SEQ ID NO: 19. In some embodiments, the structural sequence comprises a telomeric repeat sequence. In some embodiments, the telomeric repeat sequence comprises the nucleic acid sequence set forth as one of SEQ ID NOs: 20 or 21. In some embodiments,43167200.02701 / 151646090v.lthe telomeric repeat sequence comprises the nucleic acid sequence set forth as SEQ ID NO: 20. In some embodiments, the modified mRNA comprises at least 3 copies of the nucleotide sequence of SEQ ID NO: 20.

[0293] In some embodiments, the structural sequence is an aptamer sequence comprising at least two nucleotides that are capable of interacting to form an aptamer. Nonlimiting examples of target molecules that can be bound by aptamers include cytokines, cell surface receptors, and transcription factors. In some embodiments, the secondary structure formed by the one or more copies of the structural sequence is an aptamer that is capable of binding to a target molecule.

[0294] Exemplary aptamers are known in the art and include multiple RNA structures capable of binding cell surface receptors such as CD4, CTLA-4, TGF-0 receptors, and receptor tyrosine kinases. See., e.g., Germer et al. Int J Biochem Mol Biol, 2013. 4(l):27-40.

[0295] In some embodiments, the modified mRNA comprises 1-20 copies of the structural sequence. In some embodiments, the modified mRNA comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9 copies of the structural sequence. In some embodiments, the modified mRNA comprises about 4 copies of the structural sequence. In some embodiments, the modified mRNA comprises multiple different structural sequences. In some embodiments, the modified mRNA comprises at least a first structural sequence, and a second structural sequence comprising a different nucleic acid sequence from the first structural sequence. In some embodiments, the modified mRNA comprises at least one G-quadruplex sequence and at least one telomeric repeat sequence.

[0296] In some embodiments of the modified mRNAs comprising one or more copies of a structural sequence provided herein, the poly-A region of the modified mRNA 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, at least one modified nucleotide comprises a modified nucleobase selected from the group consisting of: xanthine, allyaminouracil, allyaminothymidine, hypoxanthine, digoxygeninated adenine, digoxigeninated cytosine, digoxigeninated guanine, digoxigeninated uracil, 6- chloropurineriboside, N6-methyladenine, methylpseudouracil, 2-thiocytosine, 2-thiouracil, 5- methyluracil, 4-thiothymidine, 4-thiouracil, 5,6-dihydro-5-methyluracil, 5,6-dihydrouracil, 5- [(3-indolyl)propionamide-N-allyl]uracil, 5- aminoallylcytosine, 5-aminoallyluracil, 5-44167200.02701 / 151646090v.lbromouracil, 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 -methoxy cytosine, 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-aminoallyhiracil, cyanine 5-6- propargylaminocytosine, cyanine 5-6-propargylaminouracil, cyanine 5- aminoallylcytosine, cyanine 5-aminoallyluracil, cyanine 7-aminoallyluracil, dabcyl-5-3- aminoallyluracil, desthiobiotin- 16-aminoallyl-uracil, desthiobiotin-6-aminoallylcytosine, isoguanine, Nl- ethylpseudouracil, N1 -methoxymethylpseudouracil, N1 -methyladenine, Nl- methylpseudouracil, Nl -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-carboxamide-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-threonyl carbamoyladenine (ms2t6A), N6-methyl-N6-threonylcarbamoyladenine (m6t6A), N6-hydroxynorvalylcarbamoyladenine (hn6A), 2-methylthio-N6-hydroxynorvalyl carbamoyladenine (ms2hn6A), N6,N6- dimethyladenine (m62A), and N6-acetyladenine (ac6A). In some embodiments, at least one modified nucleotide comprises a modified sugar selected from 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-l- indolyl-2'-deoxyribose, 5'-biotin-ribose, 2'-O,4'-C-methylene-linked, 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 2' modification. In some45167200.02701 / 151646090v.lembodiments, the 2' modification is selected from a locked-nucleic acid (LNA) modification (i.e., a nucleotide comprising an additional carbon atom bound to the 2' oxygen and 4' carbon of ribose), 2'-fluoro (2'-F) , 2'-O- methoxy-ethyl (2’-M0E), 2'-O-methylation (2’-0Me), and 2'-0-N-m ethyl acetamido (2'-0-NMA).

[0297] In some embodiments, at least one modified nucleotide comprises a modified phosphate selected from phosphorothioate (PS), phosphorodithioate, thiophosphate, 5'-O- methylphosphonate, 3'-O-methylphosphonate, 5'-hydroxyphosphonate, hydroxyphosphanate, phosphoroselenoate, selenophosphate, phosphoramidate, carbophosphonate, methylphosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidinium ring, triazole ring, boranophosphate (BP), methylphosphonate, and guanidinopropyl phosphoramidate. In some embodiments, the poly-A region of the mRNA comprises at least 3, at least 4, or at least 5 phosphorothioates, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the mRNA comprises at least 3 phosphorothioates, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the mRNA comprises at least 3 guanine nucleotides and at least 3 phosphorothioates, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the mRNA comprises at least 3 deoxyribose sugars, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the mRNA comprises at least 20 deoxyribose sugars, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the mRNA comprises at least 3 copies of a G-quadruplex sequence, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the mRNA comprises at least 6 phosphorothioates, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the mRNA comprises at least 6 sequential phosphorothioates, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the mRNA comprises at least 6 phosphorothioates and 3 guanine nucleosides, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the mRNA comprises at least 3 copies of a G-quadruplex sequence and at least 6 phosphorothioates, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the mRNA comprises at least 3 copies of a telomeric repeat sequence, and at least 6 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 a dideoxy cytidine or an inverted-deoxythymidine.46167200.02701 / 151646090v.l

[0298] 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 nucleoside, and a second modified nucleoside that has a different structure from the first modified nucleoside. In some embodiments, the modified mRNA comprises at least a first modified phosphate, and a second modified phosphate that has a different structure from the first modified phosphate. In some embodiments, the modified mRNA comprises a modified nucleoside and a modified nucleoside.

[0299] In some embodiments of the modified mRNAs comprising a secondary structure provided herein, the mRNA comprises a 5' UTR and a 3' UTR In some embodiments, the 5' UTR is 5' to (upstream of) the open reading frame. In some embodiments, the mRNA comprises, in 5'-to-3' order, 1) a 5' UTR; 2) an open reading frame; 3) a 3' UTR; 4) a poly-A region; and 5) one or more copies of a structural sequence. In some embodiments, the 3' UTR is 3' to (downstream of) the open reading frame. In some embodiments, the poly-A region is 3' to (downstream of) the 3' UTR. In some embodiments, the one or more copies of the structural sequence, and the secondary structure formed by the structural sequences, are 3' to (downstream of) the poly-A region. In some embodiments, the mRNA is a linear mRNA. In some embodiments, the linear mRNA comprises a 5' cap. In some embodiments, the 5' cap comprises a 7-methylguanosine. In some embodiments, the 5' cap comprises one or more phosphates connecting the 7-methylguanosine to an adjacent nucleotide of the modified mRNA. In some embodiments, the 7-methylguanosine is connected to an adjacent nucleotide of the mRNA by a 5'-to-5' triphosphate bridge. In some embodiments, one or more phosphates of the 5' cap is a modified phosphate selected from phosphorothioate, triazole ring, dihalogenmethylenebisphosphonate, imidodiphosphate, and methylenebis(phosphonate). In some embodiments, the 5' cap comprises a 3'-O-Me- m7G(5')ppp(5')G. In some embodiments, the poly-A region of the mRNA comprises at least 3, at least 4, or at least 5 phosphorothioates, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the mRNA comprises at least 3 phosphorothioates, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the mRNA comprises at least 3 guanine nucleotides and at least 3 phosphorothioates, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the mRNA comprises at least 3 deoxyribose sugars, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the mRNA comprises at least 20 deoxyribose sugars, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A47167200.02701 / 151646090v.lregion of the mRNA comprises at least 3 copies of a G-quadruplex sequence, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the mRNA comprises at least 6 phosphorothioates, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the mRNA comprises at least 6 sequential nucleotides comprising a 2' modification, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the mRNA comprises at least 6 sequential phosphorothioates, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the mRNA comprises at least 6 phosphorothioates and 3 guanine nucleosides, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the mRNA comprises at least 3 copies of a G-quadruplex sequence and at least 6 phosphorothioates, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the mRNA comprises at least 3 copies of a telomeric repeat sequence, and at least 6 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 a dideoxy cytidine or an inverted-deoxythymidine.

[0300] In some embodiments of the modified mRNAs comprising a secondary structure provided herein, the modified mRNA comprises, in 5'-to-3' order, 1) a 5' UTR; 2) an open reading frame; 3) a 3' UTR; 4) a poly-A region; and 5) one or more copies of a structural sequence. In some embodiments, the modified mRNA is a circular mRNA. In some embodiments of the circular mRNA, the one or more copies of the structural sequence are between the poly-A region and the 5' UTR. In some embodiments, the secondary structure is between the poly-A region and the 5' UTR.

[0301] In some embodiments of the modified mRNAs provided herein, at least 91% or 92% of the nucleotides of the poly-A region are modified nucleotides. In some embodiments, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the nucleotides of the poly-A region are modified nucleotides.

[0302] In some embodiments of the modified mRNAs provided herein, 3 or more of the last 25 nucleotides of the poly-A 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 25 nucleotides of the poly- A region are modified nucleotides.48167200.02701 / 151646090v.l

[0303] 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 of the poly-A region are adenosine nucleotides. One or more adenosine nucleotides of the poly-A region may be canonical adenosine nucleotides or modified adenosine nucleotides comprising a different structure from the canonical 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-threonyl carbamoyladenosine (ms2t6A), N6-methyl-N6- threonylcarbamoyladenosine (m6t6A), N6-hydroxynorvalylcarbamoyladenosine (hn6A), 2- methylthio-N6-hydroxynorvalyl carbamoyladenosine (ms2hn6A), 2'-O- ribosyladenosine (phosphate) (Ar(p)), N6,N6-dimethyladenosine (m62A), N6,2'-O- dimethyladenosine (m6Am), N6,N6,O-2'-trimethyladenosine (m62Am), l,2'-O-dimethyladenosine (ml Am), 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).

[0304] 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 of the poly-A region are canonical adenosine nucleotides. In some embodiments, the poly-A region further comprises 1 or more nucleotides that are not adenosine nucleotides (e.g., canonical or non-canonical adenosine 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%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 80%, or at least 90% of the nucleotides of the poly-A region are nucleotides that are not adenosine nucleotides.

[0305] In some embodiments of the modified mRNAs provided herein, the poly-A region comprises at least 25-500 nucleotides. In some embodiments, the poly-A region49167200.02701 / 151646090v.lcomprises at least 25, at least 30, at least 50, at least 100, at least 150, or at least 200 nucleotides. In some embodiments, the poly-A 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 poly-A region comprises about 200 to about 300 nucleotides. In some embodiments, the poly-A region comprises about 250 nucleotides.

[0306] In some embodiments, the poly-A region comprises at least 3, at least 4, or at least 5 phosphorothioates, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the mRNA comprises at least 3 phosphorothioates, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the mRNA comprises at least 3 guanine nucleotides and at least 3 phosphorothioates, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the mRNA comprises at least 3 deoxyribose sugars, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the mRNA comprises at least 20 deoxyribose sugars, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the mRNA comprises at least 3 copies of a G-quadruplex sequence, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the mRNA comprises at least 6 nucleotides comprising a 2' modification, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the mRNA comprises at least 6 phosphorothioates, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the mRNA comprises at least 6 sequential nucleotides comprising a 2' modification, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly- A region of the mRNA comprises at least 6 sequential phosphorothioates, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the mRNA comprises at least 6 phosphorothioates and 3 guanine nucleosides, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the mRNA comprises at least 3 copies of a G-quadruplex sequence and at least 6 phosphorothioates, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the mRNA comprises at least 3 copies of a telomeric repeat sequence, and at least 6 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 a dideoxy cytidine or an inverted-deoxythymidine.50167200.02701 / 151646090v.lModified non-coding RNAs

[0307] The modifications to mRNA described herein are also suitable for improving the stability of an RNA that does not encode protein (a “non-coding” RNA) in a cell. Accordingly, in some aspects, the present disclosure provides modified non-coding RNAs comprising i) one or more modified nucleotides; and / or ii) one or more copies (repeating units) of a structural sequence, with the modified nucleotides and / or structural sequence being part of or 3' to the RNA. A non-coding RNA described herein does not comprise an open reading frame (ORF). A non-coding RNA may or may not comprise a 3' poly-A region. A non-coding RNA that does not comprise a 3' poly-A region may be modified to comprise a 3' poly-A region (e.g., by ligating the non-coding RNA to an oligonucleotide comprising a poly-A region by a method disclosed herein or otherwise known in the art). A non-coding RNA may be an RNA comprising a region of complementarity with part of a mRNA transcript or genomic sequence of a cell. A non-coding RNA may be a non-coding RNA that is suitable for genome editing. Examples of non-coding RNA include, but are not limited to, small interfering RNA (siRNA), short hairpin RNA (shRNA), long non-coding RNA (IncRNA), guide RNA (gRNA) for Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) / Cas9 genome editing, non-CRISPR / Cas9 gRNA (e.g., adenosine deaminases acting on RNA (ADAR)-recruiting gRNA), or prime editing guide RNA (pegRNA). See, e.g., Chen, et al., Acta Pharm Sin B. 2021; ll(2):340-354; Chen, et a\., Adv Drug Deliv Rev. 2021; 168:246-258.; Hendel, et al., Nat Biotechnol. 2015; 33:985-989; Qu, et al., Nat Biotechnol. 2019; 37(9): 1059-1069; Yi, et al., Nat Biotechnol. 2022. Epub ahead of print; and Nelson, et al, Nat Biotechnol. 2022; 40(3):402-410. Any technique described herein for generating a modified mRNA may also be used to generate a modified non-coding RNA, unless specifically noted otherwise.

[0308] In some embodiments, a modified non-coding RNA provided herein comprises a noncoding RNA that comprises a 3' poly-A region. In some embodiments, a modified non-coding RNA provided herein comprises a non-coding RNA that does not typically comprise a 3' poly-A region (e.g., a gRNA). In some embodiments, a modified noncoding RNA provided herein comprises a non-coding RNA that is ligated at its 3' end to the 5' end of an oligonucleotide comprising a poly-A region, thereby producing a modified noncoding RNA comprising a poly-A region described herein. A non-coding RNA may be ligated to an oligonucleotide comprising a poly-A region by any method disclosed herein or otherwise known in the art.51167200.02701 / 151646090v.l

[0309] In some embodiments of the modified non-coding RNAs provided herein, the modified non-coding RNA comprises one or more modified nucleotides in the poly-A region or 3' to (downstream of) a poly-A region that is present in the non-coding RNA. In some embodiments, the poly-A region includes one or more nucleotides that are not canonical adenosine nucleotides. In some embodiments, the poly-A region includes one or more nucleotides that are not adenosine nucleotides. In some embodiments, the poly-A region comprises one or more nucleotides that are 3' to (downstream of) a nucleic acid sequence comprising multiple, consecutive adenosine nucleotides. In some embodiments, the poly-A region comprises at least 25 consecutive adenosine nucleotides, which may be canonical adenosine nucleotides or modified adenosine nucleotides. In some embodiments, the poly-A region comprises 25-500 consecutive adenosine nucleotides, which may be canonical adenosine nucleotides or modified adenosine nucleotides. In some embodiments, the poly-A region comprises 25-300 consecutive adenosine nucleotides. In some embodiments, the poly- A 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.

[0310] In some embodiments, one or more of the modified nucleotides of the modified noncoding RNA comprise a modified phosphate group. A modified phosphate group is a phosphate group that differs from the canonical structure of phosphate. An example of a canonical structure of a phosphate is shown below:where Rs and R3 are atoms or molecules to which the canonical phosphate is bonded. For example, for a phosphate in a nucleic acid sequence, Rs may refer to the upstream nucleotide of the nucleic acid, and R3 may refer to the downstream nucleotide of the nucleic acid. The canonical structure of phosphate also refers to structures in which one or more hydroxyl groups of the phosphate are deprotonated, or in which an oxygen atom of the phosphate is bonded to an adjacent nucleotide in a nucleic acid sequence. Non-limiting examples of modified phosphate groups that can be substituted for a canonical phosphate in a nucleic acid include phosphorothioate (PS), phosphorodithioate, thiophosphate, 5'-O-methylphosphonate, 3'-O-methylphosphonate, 5'-hydroxyphosphonate, hydroxyphosphanate, phosphoroselenoate,52167200.02701 / 151646090v.lselenophosphate, phosphoramidate, carbophosphonate, methylphosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidinium ring, triazole ring, boranophosphate (BP), methylphosphonate, and guanidinopropyl phosphoramidate.

[0311] In some embodiments, of the modified non-coding RNAs 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 nucleotide comprises a modified nucleobase selected from the group consisting of: xanthine, allyaminouracil, allyaminothymidine, hypoxanthine, digoxigeninated adenine, digoxigeninated cytosine, digoxigeninated guanine, digoxigeninated uracil, 6-chloropurineriboside, N6-methyladenine, methylpseudouracil, 2-thiocytosine, 2- thiouracil, 5-methyluracil, 4-thiothymidine, 4- thiouracil, 5,6-dihydro-5-methyluracil, 5,6- dihydrouracil, 5-[(3-Indolyl)propionamide-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 -methoxy cytosine, 5- methoxyuracil, 5 -methyl cytosine, 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-propargyl aminoguanine, 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, dabcyl-5-3-aminoallyluracil, desthiobiotin- 16-aminoallyl-uracil, desthiobiotin-6-aminoallylcytosine, isoguanine, Nl- ethylpseudouracil, N1 -methoxymethylpseudouracil, N1 -methyladenine, Nl- 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-carboxamide-uracil, 5-ethynyluracil, N6-isopentenyladenine (i6A), 2-53167200.02701 / 151646090v.lmethyl- 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-threonyl carbamoyladenine (ms2t6A), N6-methyl-N6-threonylcarbamoyladenine (m6t6A), N6-hydroxynorvalylcarbamoyladenine (hn6A), 2-methylthio-N6-hydroxynorvalyl carbamoyladenine (ms2hn6A), N6,N6- dimethyladenine (m62A), and N6-acetyladenine (ac6A). In some embodiments, at least one modified nucleotide comprises a modified sugar selected from 2'-thioribose, 2', 3 '- dideoxyribose, 2'-amino-2'-deoxyribose, 2' deoxyribose, 2'-azido-2'-deoxyribose, 2'-fluoro-2'- deoxyribose, 2'-0-methylnbose, 2'-O- methyldeoxyribose, 3 '-amino-2', 3 '-dideoxyribose, 3 '- azido-2', 3 '-dideoxyribose, 3 '-deoxyribose, 3'-O-(2-nitrobenzyl)-2'-deoxyribose, 3 '-0 - methylribose, 5'-aminoribose, 5'-thioribose, 5-nitro-l- indolyl-2'-deoxyribose, 5'-biotin-ribose, 2'-O,4'-C-methylene-linked, 2'-O,4'-C-amino-linked ribose, and 2'-O,4'-C-thio-linked ribose. In certain embodiments, at least one modified nucleobase is a 2'-O-(unsubstituted Cl-6 alkoxy)-(unsubstituted Cl-6 alkyl) nucleobase (e.g., 2'-O- (unsubstituted Cn> alkoxy)- (unsubstituted Cw alkyl) RNA nucleobase). In certain embodiments, at least one modified nucleobase is a 2'-O-methoxy-ethyl nucleobase (e.g., 2'-O-methoxy-ethyl RNA nucleobase). In some embodiments, at least one modified nucleotide comprises a 2' modification. In some embodiments, the 2' modification is selected from a locked-nucleic acid (LNA) modification (i.e., a nucleotide comprising an additional carbon atom bound to the 2' oxygen and 4' carbon of ribose), 2'-fluoro (2'-F) , 2'-O-methoxy-ethyl (2'- MOE), 2'-O-methylation (2’-OMe), and 2'-0-N-m ethyl acetamido (2'-0-NMA).

[0312] In some embodiments, at least one modified nucleotide comprises a modified phosphate selected from phosphorothioate (PS), phosphorodithioate, thiophosphate, 5'-O- methylphosphonate, 3'-O-methylphosphonate, 5'-hydroxyphosphonate, hydroxyphosphanate, phosphoroselenoate, selenophosphate, phosphoramidate, carbophosphonate, methylphosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidinium ring, triazole ring, boranophosphate (BP), methylphosphonate, and guanidinopropyl phosphoramidate.

[0313] In some embodiments, the modified non-coding RNA comprises more than one type of modified nucleotide. In some embodiments, the modified non-coding RNA comprises at least a first modified nucleotide, and a second modified nucleotide that has a different structure from the first modified nucleotide. Nucleotides may differ in structure due54167200.02701 / 151646090v.lto differences in the nucleobase, sugar, and / or phosphate group. In some embodiments, the modified non-coding RNA comprises at least a first modified phosphate, and a second modified phosphate that has a different structure from the first modified phosphate. In some embodiments, the modified noncoding RNA comprises a first modified nucleoside and a second modified nucleoside.

[0314] Aspects of the present disclosure relate to modified non-coding RNAs comprising poly-A regions with 25 or more adenine nucleotides. In certain embodiments, the poly-A region is at the 3' end of the non-coding RNA and comprises 10 or more, 15 or more, 20 or more, 30 or more, 40 or more, or 50 or more adenosine nucleotides. In certain embodiments, the poly-A region is at the 3' end of the non-coding RNA and comprises between 10 and 15, between 15 and 20, between 20 and 25, between 25 and 35, between 35 and 50, between 50 and 70, or between 70 and 100 adenosine nucleotides, inclusive. An adenine nucleotide is a nucleotide comprising an adenine nucleoside and a phosphate group. An adenine nucleoside comprises a sugar and an adenine base. In some embodiments, the poly-A region comprises 25 or more canonical adenine nucleotides. A canonical adenosine nucleotide comprises an adenine base, ribose sugar, and phosphate group, as arranged in the structure of adenosine monophosphate (AMP) below:In some embodiments, the one or more of the hydroxyl groups of the phosphate and / or the 3' hydroxyl group of the ribose are deprotonated, comprising an oxygen ion instead of an -OH group, as shown by the structure:When present in a nucleic acid sequence of a non-coding RNA, a canonical adenosine comprises the following structure and is connected to adjacent nucleotides in the following manner:167200.02701 / 151646090v.lwhere Rs is an adjacent nucleotide that is 5' to (upstream of) the adenosine nucleotide in the non-coding RNA, and R3 is an adjacent nucleotide that is 3' to (downstream of) the adenosine nucleotide in the non-coding RNA. In some embodiments, the canonical adenosine nucleotide is the 3' terminal nucleotide (last nucleotide) of a linear non-coding RNA, R3 is a hydrogen, and the 3' terminal nucleotide comprises a 3' terminal hydroxyl (~OH) group. In some embodiments, the canonical adenosine nucleotide is the 3' terminal nucleotide (last nucleotide) of a linear non-coding RNA, and R3 is an electron.

[0315] In some embodiments of the non-coding RNAs provided herein, the noncoding RNA comprises, in 5'-to-3' order: 1) the non-coding RNA; and 2) a poly-A region present within or ligated to the 3' end of the non-coding RNA 1. In some embodiments, the first nucleotide of the poly-A region that is ligated to the non-coding RNA is 3' to (downstream of) the last nucleotide of the non-coding RNA.

[0316] In some embodiments, the non-coding RNA is a linear non-coding RNA. A linear non-coding RNA is a non-coding RNA with a 5' terminal nucleotide and a 3' terminal nucleotide. The 5' terminal nucleotide of a linear non-coding RNA is covalently bonded to only one adjacent nucleotide of the non-coding RNA, with the adjacent nucleotide occurring 3' to the 5' terminal nucleotide in the nucleic acid sequence of the non-coding RNA. The 3' terminal nucleotide of a linear non-coding RNA is covalently bonded to only one adjacent nucleotide of the non-coding RNA, with the adjacent nucleotide occurring 5' to the 3' terminal nucleotide in the nucleic acid sequence of the non-coding RNA. In a nucleic acid sequence comprising every nucleotide of a linear non-coding RNA in 5'-to-3 ' order, the 5' terminal nucleotide is the first nucleotide in the sequence, and the 3' terminal nucleotide is the last nucleotide in the sequence.

[0317] In some embodiments of the linear non-coding RNA provided herein, the noncoding RNA comprises a 5' cap. In some embodiments, the 5' cap comprises one or more phosphates connecting the 7-methylguanosine to an adjacent nucleotide of the modified noncoding RNA. In some embodiments, one or more phosphates of the 5' cap is a modified phosphate selected from phosphorothioate, triazole ring, dihalogenmethylenebisphosphonate, imidodiphosphate, and methylenebis(phosphonate). In some embodiments, the 7-56167200.02701 / 151646090v.lmethylguanosine is connected to an adjacent nucleotide of the non-coding RNA by a 5 '-to- 5' triphosphate bridge. In some embodiments, the 5' cap comprises the structure:with R being the 5' carbon of the first transcribed nucleotide of the non-coding RNA. In some embodiments, the 5' cap comprises a 3’-O-Me-m7G(5')ppp(5')G.

[0318] In some embodiments, the linear non-coding RNA does not comprise a 5' cap.

[0319] In some embodiments, the non-coding RNA is a circular non-coding RNA. A circular non-coding RNA is an non-coding RNA with no 5' terminal nucleotide or 3' terminal nucleotide. Every nucleotide in a circular non-coding RNA is covalently bonded to both 1) a 5' adjacent nucleotide; and 2) a 3' adjacent nucleotide. In a circular non-coding RNA with a nucleic acid sequence comprising every nucleotide of the circular non-coding RNA in 5'-to-3' order, the last nucleotide of the nucleic acid sequence is covalently bonded to the first nucleotide of the nucleic acid sequence. In some embodiments of circular non-coding RNAs, the last nucleotide of a poly-A region within or ligated to the 3' end of a non-coding RNA is 5' to the first nucleotide of the non-coding RNA.

[0320] In some embodiments of the modified non-coding RNAs provided herein, the modified non-coding RNA comprises one or more copies of a structural sequence that are 3' to a poly-A region within or ligated to the non-coding RNA. In some embodiments, nucleotides of the secondary structure interact by hydrogen bonding. In some embodiments, the secondary structure is a G-quadruplex. A G-quadruplex, or G-quadruplex, is a secondary structure formed by guanine-rich nucleic acid sequences.

[0321] In some embodiments of modified non-coding RNAs comprising one or more structural sequences, the structural sequence is a G-quadruplex sequence. A nucleic acid comprising a G-quadruplex sequence is capable of forming a G-quadruplex comprising one or more nucleotides of the G-quadruplex sequence. In some embodiments, the G-quadruplex sequence comprises one or more spacer nucleotides that are not guanine nucleotides. In some embodiments, the G-quadruplex sequence is an RNA G-quadruplex sequence. In some embodiments, the RNA G-quadruplex sequence comprises the nucleic acid sequence 57167200.02701 / 151646090v.lGGGGCC (SEQ ID NO: 18). In some embodiments, the modified non-coding RNA comprises at least 3 copies of the nucleotide sequence of SEQ ID NO: 18. In some embodiments, the G-quadruplex sequence is a DNA G-quadruplex sequence. In some embodiments, the DNA G-quadruplex sequence comprises the nucleic acid sequence GGGGCC (SEQ ID NO: 19). In some embodiments, the modified non-coding RNA comprises at least 3 copies of the nucleotide sequence of SEQ ID NO: 19. In some embodiments, the structural sequence comprises a telomeric repeat sequence. In some embodiments, the telomeric repeat sequence comprises the nucleic acid sequence set forth as one of SEQ ID NOs: 20 or 21. In some embodiments, the telomeric repeat sequence comprises the nucleic acid sequence set forth as SEQ ID NO: 20. In some embodiments, the modified non-coding RNA comprises at least 3 copies of the nucleotide sequence of SEQ ID NO: 20.

[0322] In some embodiments, the structural sequence is an aptamer sequence comprising at least two nucleotides that are capable of interacting to form an aptamer. Nonlimiting examples of target molecules that can be bound by aptamers include cytokines, cell surface receptors, and transcription factors. In some embodiments, the secondary structure formed by the one or more copies of the structural sequence is an aptamer that is capable of binding to a target molecule. Exemplary aptamers are known in the art and include multiple RNA structures capable of binding cell surface receptors such as CD4, CTLA-4, TGF-P receptors, and receptor tyrosine kinases. See., e.g., Germer et al. Int J Biochem Mol Biol., 2013. 4(l):27-40.

[0323] In some embodiments, the modified non-coding RNA comprises 1-20 copies of the structural sequence. In some embodiments, the modified non-coding RNA comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9 copies of the structural sequence. In some embodiments, the modified non-coding RNA comprises about 4 copies of the structural sequence. In some embodiments, the modified noncoding RNA comprises multiple different structural sequences. In some embodiments, the modified noncoding RNA comprises at least a first structural sequence, and a second structural sequence comprising a different nucleic acid sequence from the first structural sequence. In some embodiments, the modified non-coding RNA comprises at least one G- quadruplex sequence and at least one telomeric repeat sequence.

[0324] In some embodiments of the modified non-coding RNAs comprising one or more copies of a structural sequence provided herein, the poly-A region of the modified non-58167200.02701 / 151646090v.lcoding RNA 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, at least one modified nucleotide comprises a modified nucleobase selected from the group consisting of xanthine, allyaminouracil, allyaminothymidine, hypoxanthine, digoxigeninated adenine, digoxigeninated cytosine, digoxigeninated guanine, digoxigeninated uracil, 6- chloropurineriboside, N6- methyladenine, methylpseudouracil, 2-thiocytosine, 2-thiouracil, 5-methyluracil, 4- thiothymidine, 4-thiouracil, 5,6-dihydro-5-methyluracil, 5,6-dihydrouracil, 5-[(3- Indolyl)propionamide-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 -methoxy cytosine, 5- methoxyuracil, 5 -methyl cytosine, 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, dabcyl-5-3- aminoallyluracil, desthiobiotin- 16-aminoallyl-uracil, desthiobiotin-6-aminoallylcytosine, isoguanine, N1 -ethylpseudouracil, N1 -methoxymethylpseudouracil, N1 -methyladenine, Nl- 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-carboxamide-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-threonyl carbamoyladenine (ms2t6A),59167200.02701 / 151646090v.lN6-methyl-N6-threonylcarbamoyladenine (m6t6A), N6-hydroxynorvalylcarbamoyladenine (hn6A), 2-methylthio-N6-hydroxynorvalyl carbamoyladenine (ms2hn6A), N6,N6- dimethyladenine (m62A), and N6-acetyladenine (ac6A). In some embodiments, at least one modified nucleotide comprises a modified sugar selected from 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-l- indolyl-2'-deoxyribose, 5'-biotin-ribose, 2'-O,4'-C-methylene-linked, 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 2' modification. In some embodiments, the 2' modification is selected from a locked-nucleic acid (LNA) modification (i.e., a nucleotide comprising an additional carbon atom bound to the 2' oxygen and 4' carbon of ribose), 2'-fluoro (2'-F) , 2'-O-methoxy-ethyl (2’-M0E), 2'-O-methylation (2’-0Me), and 2'-O-N-methylacetamido (2'-0-NMA). In some embodiments, at least one modified nucleotide comprises a modified phosphate selected from phosphorothioate (PS), phosphorodithioate, thiophosphate, 5'-O-methylphosphonate, 3'-O- methylphosphonate, 5'- hydroxyphosphonate, hydroxyphosphanate, phosphoroselenoate, selenophosphate, phosphoramidate, carbophosphonate, methylphosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidinium ring, triazole ring, boranophosphate (BP), methylphosphonate, and guanidinopropyl phosphoramidate. In some embodiments, the poly- A region of the non-coding RNA comprises at least 3, at least 4, or at least 5 phosphorothioates, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the non-coding RNA comprises at least 3 phosphorothioates, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the non-coding RNA comprises at least 3 guanine nucleotides and at least 3 phosphorothioates, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the non-coding RNA comprises at least 3 deoxyribose sugars, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the non-coding RNA comprises at least 20 deoxyribose sugars, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the non-coding RNA comprises at least 3 copies of a G-quadruplex sequence, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the non-coding RNA comprises at least 6 nucleotides comprising a 2' modification, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the60167200.02701 / 151646090v.lnon-coding RNA comprises at least 6 phosphorothioates, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the non-coding RNA comprises at least 6 sequential nucleotides comprising a 2' modification, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the non-coding RNA comprises at least 6 sequential phosphorothioates, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the non-coding RNA comprises at least 6 phosphorothioates and 3 guanine nucleosides, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the non-coding RNA comprises at least 3 copies of a G-quadruplex sequence and at least 6 phosphorothioates, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the noncoding RNA comprises at least 3 copies of a telomeric repeat sequence, and at least 6 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 a dideoxy cytidine or an inverted-deoxythymidine.

[0325] In some embodiments, the modified non-coding RNA comprises more than one type of modified nucleotide. In some embodiments, the modified non-coding RNA comprises at least a first modified nucleoside, and a second modified nucleoside that has a different structure from the first modified nucleoside. In some embodiments, the modified non-coding RNA comprises at least a first modified phosphate, and a second modified phosphate that has a different structure from the first modified phosphate. In some embodiments, the modified non-coding RNA comprises a modified nucleoside and a modified nucleoside.

[0326] In some embodiments of the modified non-coding RNAs comprising a secondary structure provided herein, the modified non-coding RNA comprises, in 5'-to-3' order, 1) the 5' non-coding RNA; 2) a poly-A region within or ligated to the 3' end of the non- coding RNA; and 3) one or more copies of a structural sequence. In some embodiments, the one or more copies of the structural sequence, and the secondary structure formed by the structural sequences, are 3' to (downstream of) the poly-A region. In some embodiments, the non-coding RNA is a linear non-coding RNA. In some embodiments, the linear non-coding RNA comprises a 5' cap. In some embodiments, the 5' cap comprises a 7-methylguanosine. In some embodiments, the 5' cap comprises one or more phosphates connecting the 7- m ethylguanosine to an adjacent nucleotide of the modified non-coding RNA. In some embodiments, the 7-methylguanosine is connected to an adjacent nucleotide of the non-61167200.02701 / 151646090v.lcoding RNA by a 5'-to-5' triphosphate bridge. In some embodiments, one or more phosphates of the 5' cap is a modified phosphate selected from phosphorothioate, triazole ring, dihalogenmethylenebisphosphonate, imidodiphosphate, and methylenebis(phosphonate). In some embodiments, the 5' cap comprises a 3'-O-Me-m7G(5')ppp(5')G. In some embodiments, the linear non-coding RNA does not comprise a 5' cap. In some embodiments, the poly-A region of the non-coding RNA comprises at least 3, at least 4, or at least 5 phosphorothioates, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the non-coding RNA comprises at least 3 phosphorothioates, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the non-coding RNA comprises at least 3 guanine nucleotides and at least 3 phosphorothioates, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the non-coding RNA comprises at least 3 deoxyribose sugars, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the non-coding RNA comprises at least 20 deoxyribose sugars, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the non-coding RNA comprises at least 3 copies of a G-quadruplex sequence, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the non-coding RNA comprises at least 6 nucleotides comprising a 2' modification, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the non-coding RNA comprises at least 6 phosphorothioates, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the non-coding RNA comprises at least 6 sequential nucleotides comprising a 2' modification, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the non-coding RNA comprises at least 6 sequential phosphorothioates, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the non-coding RNA comprises at least 6 phosphorothioates and 3 guanine nucleosides, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the non-coding RNA comprises at least 3 copies of a G-quadruplex sequence and at least 6 phosphorothioates, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the non-coding RNA comprises at least 3 copies of a telomeric repeat sequence, and at least 6 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 a dideoxy cytidine or an inverted-deoxythymidine.62167200.02701 / 151646090v.l

[0327] In some embodiments of the modified non-coding RNAs comprising a secondary structure provided herein, the modified non-coding RNA comprises, in 5'-to-3' order, 1) the noncoding RNA; 2) a poly- A region within or ligated to the non-coding RNA; and 3) one or more copies of a structural sequence. In some embodiments, the modified noncoding RNA is a circular non-coding RNA. In some embodiments of the circular non-coding RNA, the one or more copies of the structural sequence are between the poly-A region within or ligated to the non-coding RNA and the 5' nucleotide of the non-coding RNA.

[0328] In some embodiments of the modified non-coding RNAs provided herein, 1% to 90% of the nucleotides of the poly-A 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 of the poly-A region are modified nucleotides.

[0329] In some embodiments of the modified non-coding RNAs provided herein, 3 or more of the last 25 nucleotides of the poly-A 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 25 nucleotides of the poly-A region are modified nucleotides.

[0330] In some embodiments of the modified non-coding RNAs 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 of the poly-A region are adenosine nucleotides. One or more adenosine nucleotides of the poly-A region may be canonical adenosine nucleotides or modified adenosine nucleotides comprising a different structure from the canonical 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-threonyl carbamoyladenosine (ms2t6A), N6-methyl-N6- threonylcarbamoyladenosine (m6t6A), N6- hydroxynorvalylcarbamoyladenosine (hn6A), 2- methylthio-N6-hydroxynorvalyl carbamoyladenosine (ms2hn6A), 2'-O-ribosyladenosine (phosphate) (Ar(p)), N6,N6- dimethyladenosine (m62A), N6,2'-O-dimethyladenosine63167200.02701 / 151646090v.l(m6Am), N6,N6,O-2'- trimethyladenosine (m62Am), l,2'-O-dimethyladenosine (ml Am), N6- acetyladenosine (ac6A), 2'-thioadenosine (2'SA), 5 '-thioadenosine (5'SA), 2'-O-(2- azidoethyl)-adenosine, 2’ -azidoadenosine, deoxyadenosine (dA), dideoxyadenosine (ddA), and amino-deoxyadenosine (amino-dA).

[0331] In some embodiments of the modified non-coding RNAs 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 of the poly-A region are canonical adenosine nucleotides. In some embodiments, the poly-A region further comprises 1 or more nucleotides that are not adenosine nucleotides (e g., canonical or non-canonical adenosine 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%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 80%, or at least 90% of the nucleotides of the poly-A region are nucleotides that are not adenosine nucleotides.

[0332] In some embodiments of the modified non-coding RNAs provided herein, the poly-A region comprises at least 25-500 nucleotides. In some embodiments, the poly-A 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 poly-A 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 poly-A region comprises about 200 to about 300 nucleotides. In some embodiments, the poly-A region comprises about 250 nucleotides.

[0333] In some embodiments, the poly-A region comprises at least 3, at least 4, or at least 5 phosphorothioates, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the non-coding RNA comprises at least 3 phosphorothioates, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the non-coding RNA comprises at least 3 guanine nucleotides and at least 3 phosphorothioates, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the non-coding RNA comprises at least 3 deoxyribose sugars, and does not64167200.02701 / 151646090v.lcomprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the non-coding RNA comprises at least 20 deoxyribose sugars, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the non-coding RNA comprises at least 3 copies of a G-quadruplex sequence, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the non-coding RNA comprises at least 6 nucleotides comprising a 2' modification, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the non-coding RNA comprises at least 6 phosphorothioates, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the non-coding RNA comprises at least 6 sequential nucleotides comprising a 2' modification, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the non-coding RNA comprises at least 6 sequential phosphorothioates, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the non-coding RNA comprises at least 6 phosphorothioates and 3 guanine nucleosides, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the noncoding RNA comprises at least 3 copies of a G-quadruplex sequence and at least 6 phosphorothioates, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the non-coding RNA comprises at least 3 copies of a telomeric repeat sequence, and at least 6 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 a dideoxy cytidine or an inverted-deoxythymidine.Methods of producing modified mRNAs and modified non-coding RNAs

[0334] In some aspects, the present disclosure provides methods of producing modified mRNAs described herein, comprising ligating an RNA, such as an RNA comprising an open reading frame encoding a protein or a non-coding RNA, to a tailing nucleic acid comprising one or more modified nucleotides in the presence of a ligase, whereby the ligase forms a covalent bond between the 3' nucleotide of the RNA and the 5' nucleotide of the tailing nucleic acid to produce a modified RNA (e.g., a modified mRNA or a modified noncoding RNA). When a ligase forms a covalent bond between two linear nucleic acids, a new nucleic acid is produced, with the produced nucleic acid comprising the nucleic acid sequences 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, with the third nucleic acid comprising the sequence of the first nucleic acid and the second nucleic acid, and the second nucleic acid sequence being 3' to (downstream of) the first nucleic acid 65167200.02701 / 151646090v.lsequence. Ligation by an RNA ligase occurs in several steps. First, an amino (-NH2) group of an amino acid (e.g., a lysine) of the ligase bonds to a phosphate group of adenosine triphosphate (ATP), such that an adenosine monophosphate (AMP) group is bound to the RNA ligase. Second, a 5' terminal phosphate of the second nucleic acid displaces the phosphate of the RNA ligase-bound AMP. Finally, an 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 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 a T4 RNA ligase.

[0335] In some embodiments of the methods of producing modified mRNAs or modified non-coding RNA provided herein, the RNA to which a tailing nucleic acid is ligated is synthesized by in vitro transcription (IVT). IVT is a process in which an RNA, such as a precursor mRNA (pre-mRNA), mRNA, or non-coding RNA, is generated through transcription of a DNA template by an RNA polymerase. Generally, the DNA template comprises a promoter, such as a bacteriophage promoter, that is upstream of the DNA sequence to be transcribed. The RNA polymerase binds to the promoter, and begins transcription of the DNA sequence, producing an RNA transcript with a nucleic acid sequence that is present in the template, with the exception that thymidine (T) nucleotides in the DNA sequence are replaced with uracil (U) nucleotides in the RNA sequence. The RNA transcript produced by IVT may be modified prior to ligation of a tailing nucleic acid, such as by the addition of a 5' cap, cleavage of one or more nucleotides from the RNA, or polyadenylation to extend the poly-A region. In some embodiments, the DNA template comprises a poly-A region, such that IVT produces an mRNA or non-coding RNA with a poly-A region. See, e.g., Becker et al. Methods Mol Biol., 2011. 703:29-41.

[0336] In some embodiments of the methods of producing modified mRNAs or modified non-coding RNAs 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. The combination of a 3' terminal hydroxyl group on the RNA and a 5' terminal phosphate group on the tailing nucleic acid allows for efficient ligation of the two nucleic acids. In some embodiments, the RNA does not comprise a 5' terminal phosphate group. An RNA may lack a 5' terminal phosphate group due to the addition of a 5' cap or another chemical modification. A 5' terminal phosphate may also be removed from an RNA66167200.02701 / 151646090v.lby a phosphatase enzyme to produce an RNA that lacks a 5' terminal phosphate. Lack of a 5' terminal phosphate group on the RNA prevents an RNA ligase from ligating multiple copies of an mRNA or noncoding RNA together. In some embodiments, the tailing nucleic acid does not comprise a 3' terminal hydroxyl group. An RNA may lack a 3' terminal hydroxyl group if the last nucleotide of the tailing nucleic acid comprises a modified nucleotide that does not contain a 3' hydroxyl group, such as a dideoxyadenosine, dideoxy cytidine, dideoxy guanosine, dideoxythymidine, or inverted-deoxythymidine. Lack of a 3' terminal hydroxyl group on the tailing nucleic acid prevents an RNA ligase from ligating multiple tailing nucleic acids together. In some embodiments, the 5' nucleotide of the RNA does not comprise a 5' terminal phosphate group; the 3' nucleotide of the RNA comprises a 3' terminal hydroxyl group; the 5' nucleotide of the tailing nucleic acid comprises a 5' terminal phosphate group; and the 3' nucleotide of the tailing nucleic acid does not comprise a 3' terminal hydroxyl group. In some embodiments, the tailing nucleic acid comprises at least 3, at least 4, or at least 5 phosphorothioates, and does not comprise a 3' terminal hydroxyl. In some embodiments, the tailing nucleic acid comprises at least 3 phosphorothioates, and does not comprise a 3' terminal hydroxyl. In some embodiments, the tailing nucleic acid comprises at least 3 guanine nucleotides and at least 3 phosphorothioates, and does not comprise a 3' terminal hydroxyl. In some embodiments, the tailing nucleic acid comprises at least 3 deoxyribose sugars, and does not comprise a 3' terminal hydroxyl. In some embodiments, the tailing nucleic acid comprises at least 20 deoxyribose sugars, and does not comprise a 3' terminal hydroxyl. In some embodiments, the tailing nucleic acid comprises at least 3 copies of a G- quadruplex sequence, and does not comprise a 3' terminal hydroxyl. In some embodiments, the tailing nucleic acid comprises at least 6 nucleotides comprising a 2' modification, and does not comprise a 3' terminal hydroxyl. In some embodiments, the tailing nucleic acid comprises at least 6 phosphorothioates, and does not comprise a 3' terminal hydroxyl. In some embodiments, the tailing nucleic acid comprises at least 6 sequential nucleotides comprising a 2' modification, and does not comprise a 3' terminal hydroxyl. In some embodiments, the tailing nucleic acid comprises at least 6 sequential phosphorothioates, and does not comprise a 3' terminal hydroxyl. In some embodiments, the tailing nucleic acid comprises at least 6 phosphorothioates and 3 guanine nucleosides, and does not comprise a 3' terminal hydroxyl. In some embodiments, the tailing nucleic acid comprises at least 3 copies of a G- quadruplex sequence and at least 6 phosphorothioates, and does not comprise a 3' terminal hydroxyl. In some embodiments, the tailing nucleic acid comprises at least 3 copies67167200.02701 / 151646090v.lof a telomeric repeat sequence, and at least 6 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 a dideoxy cytidine or an inverted-deoxythymidine. In some embodiments, the ligase used to ligate the tailing nucleic acid to the RNA is an RNA ligase. In some embodiments, the RNA ligase is a T4 RNA ligase. In some embodiments, the T4 RNA ligase is a T4 RNA ligase 1. In some embodiments, the T4 RNA ligase is a T4 RNA ligase 2.

[0337] In some embodiments of the methods of producing modified mRNAs or modified non-coding RNAs 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, and the RNA ligase is an RtcB ligase, which ligates a first nucleotide comprising a 3' terminal phosphate group to a second nucleotide comprising a 5' terminal hydroxyl group.

[0338] Some embodiments of the methods of making modified mRNAs or modified noncoding RNA provided herein further comprise producing a circular mRNA or circular noncoding RNA. After a linear modified mRNA or modified non-coding RNA is produced by ligating an RNA and a tailing nucleic acid, circularization of the modified mRNA or modified non-coding RNA comprises several additional steps. First, a 5' terminal phosphate is introduced onto the first nucleotide of the modified mRNA or modified non-coding RNA, a process known as phosphorylation. In some embodiments, the 5' terminal phosphate is introduced by a kinase. A “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 referred to as “phosphorylation.” Second, the modified mRNA or modified noncoding RNA is manipulated to produce a modified mRNA or modified non-coding RNA with a 3' terminal hydroxyl group. In some embodiments, the modified mRNA or modified noncoding RNA is manipulated by cleaving one or more of the last nucleotides of the modified RNA, to produce a modified mRNA or modified non-coding RNA with a 3' terminal hydroxyl group. In some embodiments, the modified mRNA or modified non-coding RNA is cleaved by a restriction enzyme, ribozyme, or endoribonuclease. In some embodiments, cleavage of one or more last nucleotides of the modified mRNA or modified non-coding RNA occurs before phosphorylation of the first nucleotide of the modified RNA. In some embodiments, cleavage occurs after phosphorylation. A modified mRNA or modified non-68167200.02701 / 151646090v.lcoding RNA comprising 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 terminal nucleotides of a linear nucleic acid to produce a circular nucleic acid may be called a “circularizing ligase.” In some embodiments, the circularizing ligase is an RNA ligase. In some embodiments, the circularizing ligase is a SplintR ligase. In some embodiments, the circularizing ligase is a T4 RNA ligase. In some embodiments, the circularizing ligase is a T4 RNA ligase 1. In some embodiments, the circularizing ligase is a T4 RNA ligase 2. In some embodiments, the modified mRNA or modified non-coding RNA comprises a 5' terminal hydroxyl group and a 3' terminal phosphate group, and the circularizing ligase is RtcB ligase, which is capable of ligating nucleotides with a 3' terminal phosphate and 5' terminal hydroxyl group. For ligation to occur, the 5' and 3' terminal nucleotides of the modified mRNA or modified non-coding RNA must be close enough for the RNA ligase to form a bond between both nucleotides. Methods of placing both nucleotides of a linear nucleic acid close enough for ligation to occur, and of circularizing an RNA, are generally known in the art (see, e.g., Petkovic et at, Nucleic Acids Res., 2015. 43(4):2454~2465). In some embodiments, the modified mRNA or modified non-coding RNA is incubated with a scaffold nucleic acid, which is capable of hybridizing (hydrogen bonding) to the modified RNA so that the modified mRNA or modified non-coding RNA forms a circular secondary structure when hybridized (bound) to the scaffold nucleic acid.

[0339] When an RNA forms a circular secondary structure, the 5' and 3' terminal nucleotides are in close physical proximity, which is required for an RNA ligase to form a covalent bond between them. In some embodiments of methods of circularizing an mRNA or non-coding RNA, one or more of the last nucleotides of the RNA are bound to a first hybridization sequence in the scaffold nucleic acid, and one or more of the first nucleotides of the mRNA or non-coding RNA are bound to a second hybridization sequence in the scaffold nucleic acid that is 3' to (downstream of) the first hybridization sequence. In some embodiments, the first hybridization sequence comprises 5 or more nucleotides, and the first hybridization sequence is complementary to at least the first five (5) nucleotides of the modified mRNA or modified noncoding RNA. 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 are complementary to the last N nucleotides of the69167200.02701 / 151646090v.lmodified mRNA or modified non-coding RNA, 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 five (5) nucleotides of the modified mRNA or modified noncoding RNA. 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 are complementary to the last N nucleotides of the modified mRNA or modified non-coding RNA, where A is the length of the second hybridization sequence. In some embodiments, at least the first five (5) nucleotides of the modified mRNA or modified non-coding RNA hybridize with the first hybridization sequence. In some embodiments, at least the last five (5) nucleotides of the modified mRNA or modified non-coding RNA hybridize with the second hybridization sequence. In some embodiments, at least the first five (5) nucleotides of the modified mRNA or modified non-coding RNA hybridize with the first hybridization sequence, and at least the last five (5) nucleotides of the modified mRNA or modified noncoding RNA 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 in the scaffold nucleic acid, and are not separated by any other nucleotides.

[0340] In some embodiments of the methods of producing circular RNAs provided herein, a scaffold nucleic acid is not used to promote the formation of a circular secondary structure by the modified mRNA or modified non-coding RNA. Instead, the modified mRNA or modified noncoding RNA 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 five (5) nucleotides. In some 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.

[0341] In some embodiments of the methods of producing circular RNAs provided herein, the modified mRNA or modified non-coding RNA is not circularized through the use of a scaffold nucleic acid and circularizing ligase, but rather is circularized 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 or modified70167200.02701 / 151646090v.lnon-coding RNA comprises a 3' intron that is 5' to (upstream of) the 5' UTR of the mRNA or the first nucleotide of the non-coding mRNA, and a 5' intron that is 3' to (downstream of) the poly-A region and / or one or more structural sequences of the mRNA or non-coding RNA. Ribozymes and other enzymes that catalyze splicing of pre-mRNA to remove introns can catalyze the formation of a covalent bond between the nucleotide that is 5' to the 5' intron and the nucleotide that is 3' to 3' intron, resulting in the formation of a circular mRNA or noncoding RNA. See, e.g., Wesselhoeft et al., Nat Commun. 2018. 9:2629.

[0342] In some embodiments of the methods of producing circular RNAs provided herein, the modified mRNA or modified non-coding RNA is not circularized through the use of a scaffold nucleic, but rather is circularized through the use of complementary sequences that promote the formation of a secondary structure by the mRNA of non-coding RNA that places the 5' and 3' terminal nucleotides of the mRNA or non-coding RNA in close proximity. In some embodiments, prior to circularization the modified mRNA comprises (i) a first self-hybridization sequence that is 5' to the open reading frame, or 5' to the non-coding RNA; (ii) a second self-hybridization sequence that is 3' to the open reading frame, or 3' to the non-coding RNA; (iii) a first non-hybridization sequence that is 5' to the first selfhybridization sequence; and (iv) a second non-hybridization sequence that is 3' to the second self-hybridization sequence. The first and second self-hybridization sequences are capable of hybridizing with each other, but the first and second self-hybridization sequences are not capable of hybridizing with each other. In some embodiments, hybridization of the first and second self-hybridization sequences forms a secondary structure in which the 5' terminal nucleotide and the 3' terminal nucleotide of the modified mRNA or modified non-coding RNA are separated by a distance of less than 100 A. In some embodiments, the 5' terminal nucleotide and the 3' terminal nucleotide are separated by a distance of less than 90 A, less than 80 A, less than 70 A, less than 60 A, less than 50 A, less than 40 A, less than 30 A, less than 20 A, or less than 10 A. 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 WO 2020 / 237227.

[0343] In some embodiments of the methods of producing modified mRNAs or modified non-coding RNAs provided herein, the modified mRNA or modified non-coding RNA produced by the method comprises one or more copies of a structural sequence that are 3' to the poly-A region of the mRNA or non-coding RNA. In some embodiments, the tailing71167200.02701 / 151646090v.lnucleic acid comprises the one or more copies of the structural sequence. In some embodiments, nucleotides of the structural sequences interact by hydrogen bonding. In some embodiments, the secondary structure is a G-quadruplex. In some embodiments, the structural sequence is a G-quadruplex sequence. In some embodiments, the G-quadruplex sequence comprises one or more spacer nucleotides that are not guanine nucleotides. In some embodiments, the G-quadruplex sequence is an RNA G-quadruplex sequence. In some embodiments, the RNA G-quadruplex sequence comprises the nucleic acid sequence GGGGCC (SEQ ID NO: 18). In some embodiments, the tailing nucleic acid comprises at least 3 copies of the nucleic acid sequence of SEQ ID NO: 18. In some embodiments, the G- quadruplex sequence is an DNA G-quadruplex sequence. In some embodiments, the DNA G- quadruplex sequence comprises the nucleic acid sequence GGGGCC (SEQ ID NO: 19). In some embodiments, the tailing nucleic acid comprises at least 3 copies of the G-quadruplex sequence of SEQ ID NO: 19. In some embodiments, the structural sequence comprises a telomeric repeat sequence. In some embodiments, the telomeric repeat sequence comprises the nucleic acid sequence set forth as one of SEQ ID NOs: 20 or 21 (TAGGGT or TACCCT, respectively). In some embodiments, the telomeric repeat sequence comprises the nucleic acid sequence set forth as SEQ ID NO: 20. In some embodiments, the tailing nucleic acid comprises at least 3 copies of the nucleic acid sequence of SEQ ID NO: 20. In some embodiments, the structural sequence is an aptamer sequence comprising at least two nucleotides that are capable of interacting to form an aptamer. In some embodiments, the secondary structure formed by the one or more copies of the structural sequence is an aptamer that is capable of binding to a target molecule. Formation of an aptamer by an mRNA or non-coding RNA allows for the mRNA or non-coding RNA to be localized to a given region of a cell containing a target molecule, such as a receptor.

[0344] In some embodiments of the modified mRNAs or modified non-coding RNAs produced by the methods provided herein, the modified mRNA or modified non-coding RNA comprises 1-20 copies of the structural sequence. In some embodiments, the modified mRNA or modified non-coding RNA comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9 copies of the structural sequence. In some embodiments, the modified mRNA or modified non-coding RNA comprises about 4 copies of the structural sequence. In some embodiments, the modified mRNA or modified noncoding RNA comprises multiple different structural sequences. In some embodiments, the modified mRNA or modified non-coding RNA comprises at least a first structural sequence,72167200.02701 / 151646090v.land a second structural sequence comprising a different nucleic acid sequence from the first structural sequence.

[0345] In some embodiments of the modified mRNAs or modified non-coding RNAs produced by the methods provided herein, the poly-A region of the modified mRNA or modified non-coding RNA 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, at least one modified nucleotide comprises a modified nucleobase selected from the group consisting of: xanthine, allyaminouracil, allyaminothymidine, hypoxanthine, digoxigeninated adenine, digoxigeninated cytosine, digoxigeninated guanine, digoxigeninated uracil, 6-chloropurineriboside, N6- methyladenine, methylpseudouracil, 2- thiocytosine, 2-thiouracil, 5-methyluracil, 4- thiothymidine, 4-thiouracil, 5,6-dihydro-5- methyluracil, 5,6-dihydrouracil, 5-[(3 - Indolyl)propionamide-N-allyl]uracil, 5- aminoallylcytosine, 5-aminoallyluracil, 5-bromouracil, 5- bromocytosine, 5 -carboxy cytosine, 5-carboxymethylesteruracil, 5-carboxyuracil, 5-fluorouracil, 5-formylcytosine, 5- formyluracil, 5-hydroxycytosine, 5-hydroxymethylcytosine, 5- hydroxymethyluracil, 5- hydroxyuracil, 5-iodocytosine, 5-iodouracil, 5 -methoxy cytosine, 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, dabcyl-5-3-aminoallyluracil, desthiobiotin- 16-aminoallyl-uracil, desthiobiotin-6-aminoallylcytosine, isoguanine, N1 -ethylpseudouracil, Nl- m ethoxymethylpseudouracil, N1 -methyladenine, N1 -methylpseudouracil, Nl- propylpseudouracil, N2-methylguanine, N4-biotin-OBEA-cytosine, N4-methylcytosine, N6- methyladenine, O6-methylguanine, pseudoisocytosine, pseudouracil, thienocytosine,73167200.02701 / 151646090v.lthienoguanine, thienouracil, xanthosine, 3 -deazaadenine, 2,6-diaminoadenine, 2,6- daminoguanine, 5-carboxamide-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-threonyl carbamoyladenine (ms2t6A), N6-methyl-N6-threonylcarbamoyladenine (m6t6A), N6-hydroxynorvalylcarbamoyladenine (hn6A), 2-methylthio-N6-hydroxynorvalyl carbamoyladenine (ms2hn6A), N6,N6- dimethyladenine (m62A), and N6-acetyladenine (ac6A). In some embodiments, at least one modified nucleotide comprises a modified sugar selected from 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-l- indolyl-2'-deoxyribose, 5'-biotin-ribose, 2'-O,4'-C-methylene-linked, 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 2' modification. In some embodiments, the 2' modification is selected from a locked-nucleic acid (LNA) modification (i.e., a nucleotide comprising an additional carbon atom bound to the 2' oxygen and 4' carbon of ribose), 2'-fluoro (2'-F) , 2'-O-methoxy-ethyl (2’-M0E), 2'-O-methylation (2’-0Me), and 2'-O-N-methylacetamido (2'-0-NMA). In some embodiments, at least one modified nucleotide comprises a modified phosphate selected from phosphorothioate (PS), phosphorodithioate, thiophosphate, 5'-O-methylphosphonate, 3'-O- methylphosphonate, 5'- hydroxyphosphonate, hydroxyphosphanate, phosphoroselenoate, selenophosphate, phosphoramidate, carbophosphonate, methylphosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidinium ring, triazole ring, boranophosphate (BP), methylphosphonate, and guanidinopropyl phosphoramidate. In some embodiments, the poly- A region of the mRNA or non-coding RNA comprises at least 3, at least 4, or at least 5 phosphorothioates, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the mRNA or non-coding RNA comprises at least 3 phosphorothioates, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the mRNA or non-coding RNA comprises at least 3 guanine nucleotides and at least 3 phosphorothioates, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the mRNA or non-coding RNA comprises at least 3 deoxyribose sugars, and74167200.02701 / 151646090v.ldoes not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the mRNA or non-coding RNA comprises at least 20 deoxyribose sugars, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the mRNA or non-coding RNA comprises at least 3 copies of a G-quadruplex sequence, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the mRNA or non-coding RNA comprises at least 6 nucleotides comprising a 2' modification, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the mRNA or non-coding RNA comprises at least 6 phosphorothioates, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the mRNA or non-coding RNA comprises at least 6 sequential nucleotides comprising a 2' modification, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the mRNA or non-coding RNA comprises at least 6 sequential phosphorothioates, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the mRNA or non-coding RNA comprises at least 6 phosphorothioates and 3 guanine nucleosides, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the mRNA or non-coding RNA comprises at least 3 copies of a G-quadruplex sequence and at least 6 phosphorothioates, and does not comprise a 3' terminal hydroxyl. In some embodiments, the poly-A region of the mRNA or non-coding RNA comprises at least 3 copies of a telomeric repeat sequence, and at least 6 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 a dideoxy cytidine or an inverted-deoxythymidine.

[0346] In some embodiments of the modified mRNAs or modified non-coding RNAs produced by the methods provided herein, the modified mRNA or modified non-coding RNA comprises more than one type of modified nucleotide. In some embodiments, the modified mRNA or modified non-coding RNA comprises at least a first modified nucleoside, and a second modified nucleoside that has a different structure from the first modified nucleoside. In some embodiments, the modified mRNA or modified non-coding RNA comprises at least a first modified phosphate, and a second modified phosphate that has a different structure from the first modified phosphate. In some embodiments, the modified mRNA or modified non-coding RNA comprises a modified nucleoside and a modified nucleoside.

[0347] In some embodiments of the modified mRNAs or modified non-coding RNAs produced by the methods provided herein, at least 91% or 92% of the nucleotides of the poly- A region are modified nucleotides. In some embodiments, at least 91%, at least 92%, at least75167200.02701 / 151646090v.l93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the nucleotides of the poly-A region are modified nucleotides.

[0348] In some embodiments of the modified mRNAs or modified non-coding RNAs produced by the methods provided herein, 3 or more of the last 25 nucleotides of the poly-A 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 25 nucleotides of the poly-A region are modified nucleotides.

[0349] In some embodiments of the modified mRNAs or modified non-coding RNAs 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 of the poly-A region are adenosine nucleotides. One or more adenosine nucleotides of the poly-A region may be canonical adenosine nucleotides or modified adenosine nucleotides comprising a different structure from the canonical adenosine nucleotide.

[0350] In some embodiments of the modified mRNAs or modified non-coding RNAs 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 of the poly-A region are canonical adenosine nucleotides.

[0351] In some embodiments of the modified mRNAs or modified non-coding RNAs produced by the methods provided herein, the poly-A region comprises at least 25-500 nucleotides. In some embodiments, the poly-A 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 poly- A 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 poly-A region comprises about 200 to about 300 nucleotides. In some embodiments, the poly-A region comprises about 250 nucleotides.

[0352] In some embodiments of the methods of producing modified mRNAs provided herein, prior to the ligation of a tailing nucleic acid, the RNA comprises an open reading frame and a poly-A region prior to ligation of a tailing nucleic acid. In some embodiments of76167200.02701 / 151646090v.lthe methods of producing modified non-coding RNAs provided herein, prior to the ligation of a tailing nucleic acid, the RNA comprises a non-coding RNA and may or may not comprise a poly-A region prior to ligation of a tailing nucleic acid. In some embodiments, prior to ligation of a tailing nucleic acid, the poly-A region of the RNA comprises at least 25-500 nucleotides. In some embodiments, the poly-A 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 poly- A 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 poly-A region comprises about 200 to about 300 nucleotides. In some embodiments, the poly-A region comprises about 250 nucleotides.

[0353] In some embodiments, prior to ligation of a tailing nucleic acid, the tailing nucleic acid comprises at least 10-500 nucleotides. In some embodiments, the tailing nucleic acid comprises at least 10, at least 15, at least 20, at least 25, at least 30, at least 50, at least 100, at least 150, or at least 200 nucleotides. In some embodiments, the tailing nucleic acid 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 nucleotides. In some embodiments, the poly-A region comprises about 10 to about 50 nucleotides.

[0354] In some embodiments of the methods of producing modified mRNAs provided herein, prior to ligation of a tailing nucleic acid, the RNA comprises, in 5'-to-3’ order, a 5' UTR, an open reading frame, a 3' UTR, and a poly-A 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 poly-A region.

[0355] In some embodiments of the methods of producing modified non-coding RNAs provided herein, prior to ligation of a tailing nucleic acid, the RNA comprises, in 5'-to- 3' order, a non-coding RNA, and optionally a poly-A region. In some embodiments, the first nucleotide of the poly-A region is 3' to the last nucleotide of the non-coding RNA. In some embodiments, prior to ligation of a tailing nucleic acid, a non-coding RNA does not comprise a poly-A tail. Accordingly, in some embodiments, the tailing nucleic acid comprises a poly-A region described herein that is added to the 3' end of the non-coding RNA by ligating the77167200.02701 / 151646090v.ltailing nucleic acid to the 3' end of the non-coding RNA, thereby producing a modified noncoding RNA comprising a poly-A region.

[0356] In some embodiments of the methods of producing modified mRNAs or modified non-coding RNAs provided herein, prior to ligation of a tailing nucleic acid, the RNA comprises a 5' cap. In some embodiments, the 5' cap comprises a 7-methylguanosine. In some embodiments, the 5' cap comprises one or more phosphates that connect the 7- methylguanosine to an adjacent nucleotide of the RNA. In some embodiments, a 5' cap is added after ligation of the tailing nucleic acid. In some embodiments, prior to ligation of a tailing nucleic acid, the RNA does not comprise a 5' cap (e.g., the RNA is a mRNA or noncoding RNA that does not comprise a 5' cap).

[0357] In some aspects of the methods of producing modified mRNAs or modified noncoding RNAs provided herein comprising ligating a tailing nucleic acid to an mRNA or noncoding RNA, 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, at least one modified nucleotide comprises a modified nucleobase selected from the group consisting of: xanthine, allyaminouracil, allyaminothymidine, hypoxanthine, digoxigeninated adenine, digoxigeninated cytosine, digoxigeninated guanine, digoxigeninated uracil, 6- chloropurineriboside, N6- methyladenine, methylpseudouracil, 2-thiocytosine, 2-thiouracil, 5-methyluracil, 4- thiothymidine, 4-thiouracil, 5,6-dihydro-5-methyluracil, 5,6-dihydrouracil, 5-[(3- Indolyl)propionamide-N-allyl]uracil, 5-aminoallylcytosine, 5-aminoallyluracil, 5- bromouracil, 5- bromocytosine, 5-carboxycytosine, 5-carboxymethylesteruracil, 5- carboxyuracil, 5-fluorouracil, 5-formylcytosine, 5-formyluracil, 5 -hydroxy cytosine, 5- hydroxymethylcytosine, 5- hydroxymethyluracil, 5-hydroxyuracil, 5-iodocytosine, 5- iodouracil, 5 -methoxy cytosine, 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, 7-78167200.02701 / 151646090v.ldeaza-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, dabcyl-5-3- aminoallyluracil, desthiobiotin- 16-aminoallyl-uracil, desthiobiotin-6-aminoallylcytosine, isoguanine, N1 -ethylpseudouracil, N1 -methoxymethylpseudouracil, N1 -methyladenine, Nl- 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-carboxamide-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-threonyl carbamoyladenine (ms2t6A), N6-methyl-N6-threonylcarbamoyladenine (m6t6A), N6-hydroxynorvalylcarbamoyladenine (hn6A), 2-methylthio-N6-hydroxynorvalyl carbamoyladenine (ms2hn6A), N6,N6- dimethyladenine (m62A), and N6-acetyladenine (ac6A). In some embodiments, at least one modified nucleotide comprises a modified sugar selected from 2'-thioribose, 2', 3 '- dideoxyribose, 2'-amino-2'-deoxyribose, 2' deoxyribose, 2'-azido-2'-deoxyribose, 2'-fluoro-2'- deoxyribose, 2'-0-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-l- indolyl-2'-deoxyribose, 5'-biotin-ribose, 2'-O,4'-C-methylene-linked, 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 2' modification. In some embodiments, the 2' modification is selected from a locked-nucleic acid (LNA) modification (i.e., a nucleotide comprising an additional carbon atom bound to the 2' oxygen and 4' carbon of ribose), 2'-fluoro (2'-F) , 2'-O- methoxy-ethyl (2’-M0E), 2'-O-methylation (2’-0Me), and 2'-O-N-methylacetamido (2'-0-NMA). In some embodiments, at least one modified nucleotide comprises a modified phosphate selected from phosphorothioate (PS), phosphorodithioate, thiophosphate, 5'-O-methylphosphonate, 3'-O- methylphosphonate, 5'-79167200.02701 / 151646090v.lhydroxyphosphonate, hydroxyphosphanate, phosphoroselenoate, selenophosphate, phosphoramidate, carbophosphonate, methylphosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidinium ring, triazole ring, boranophosphate (BP), methylphosphonate, and guanidinopropyl phosphoramidate.

[0358] In some embodiments of the methods of producing modified mRNAs or modified non-coding RNAs provided herein, the tailing nucleic acid comprises more than one type of modified nucleotide. In some embodiments, the tailing nucleic acid comprises at least a first modified nucleoside, and a second modified nucleoside that has a different structure from the first modified nucleoside. In some embodiments, the tailing nucleic acid comprises at least a first modified phosphate, and a second modified phosphate that has a different structure from the first modified phosphate. In some embodiments, the tailing nucleic acid comprises a modified nucleoside and a modified nucleoside.

[0359] 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, 3 or more of the 25 last 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 25 last nucleotides of the tailing nucleic acid are modified nucleotides.

[0360] In some embodiments of the methods of producing modified mRNAs or modified non-coding RNAs provided herein, the tailing nucleic acid comprises one or more structural sequences. In some embodiments, the tailing nucleic acid comprises one or more copies of a G- quadruplex sequence. In some embodiments, the G-quadruplex sequence is an RNA G- quadruplex sequence. In some embodiments, the RNA G-quadruplex sequence comprises the nucleic acid sequence GGGGCC (SEQ ID NO: 18). In some embodiments, the G-quadruplex sequence is an DNA G-quadruplex sequence. In some embodiments, the DNA G-quadruplex sequence comprises the nucleic acid sequence GGGGCC (SEQ ID NO: 19). In some embodiments, the tailing nucleic acid comprises one or more copies of a telomeric repeat sequence. In some embodiments, the telomeric repeat sequence comprises the nucleic acid sequence set forth as one of SEQ ID NOs: 20 or 21 (TAGGGT or TACCCT, respectively). In some embodiments, the telomeric repeat sequence comprises the nucleic acid sequence set forth as SEQ ID NO: 20. In some embodiments, the structural sequence is80167200.02701 / 151646090v.lan aptamer sequence comprising at least two nucleotides that are capable of interacting to form an aptamer. In some embodiments, the secondary structure formed by the one or more copies of the structural sequence is an aptamer that is capable of binding to a target molecule.

[0361] In some embodiments of the methods of producing modified mRNAs or modified non-coding RNAs provided herein, the tailing nucleic acid comprises 1-20 copies of a structural sequence. In some embodiments, the tailing nucleic acid comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9 copies of the structural sequence. In some embodiments, the tailing nucleic acid comprises about 4 copies of the structural sequence. In some embodiments, the tailing nucleic acid comprises multiple different structural sequences. In some embodiments, the tailing nucleic acid comprises at least a first structural sequence, and a second structural sequence comprising a different nucleic acid sequence from the first structural sequence. Each of the different first and second structural sequences may be any of the structural sequences provided herein, or different sequences. In further embodiments, the methods of producing modified mRNAs or modified non-coding RNAs also relate to methods for isolating (e.g., purifying, enriching) the modified mRNAs or modified non-coding RNAs provided herein. In some embodiments, a method of isolating (e.g., purifying, enriching) a modified mRNA or modified non-coding RNA comprises contacting a mixture comprising the modified mRNA or modified non-coding RNA (e.g., a ligation mixture) with a purification medium, wherein the modified mRNA or modified non-coding RNA interacts with the purification medium to form a modified RNA- purification medium conjugate. In some embodiments, a purification medium that has formed a modified RNA-purification medium conjugate is separated from the mixture by means of one or more physical or chemical properties, such as, but not limited to, size (mass) or charge. In some embodiments, the modified mRNA or modified non-coding RNA is eluted from the purification medium (i.e., separated from the purification medium) by treating the modified RNA-purification medium conjugate with a solvent. In some embodiments, the solvent is an aqueous solvent (e.g., water). In certain embodiments, the solvent is a mixture of two or more (e.g., three) solvents. In certain embodiments, the solvent is a mixture of water and an organic solvent (e.g., acetonitrile, methanol, ethanol, tetrahydrofuran). In certain embodiments, the solvent further comprises a mobile phase modifying substance. In certain embodiments, the mobile phase modifying substance is an acid (e.g., trifluoroacetic acid, acetic acid, formic acid, phosphoric acid), base (ammonia, ammonium hydroxide, ammonium bicarbonate), or salt (a phosphate, an acetate, a citrate, ammonium formate, or a borate). In81167200.02701 / 151646090v.lsome embodiments, the purification medium is a solid purification medium. In some embodiments, the purification medium comprises a bead. In some embodiments, the purification medium comprises a resin. In some embodiments, the purification medium comprises a paramagnetic bead. Examples of purification media suitable for the purification of RNA are well known to those skilled in the art and include, for example, various commercially available purification media (see, e.g., Beckman Coulter Life Sciences # A63987). In certain embodiments, a step described in this paragraph is performed at a temperature between 0 and 20, between 20 and 25, between 25 and 36, between 36 and 38 °C, inclusive. In certain embodiments, a step described in this paragraph is performed at a pressure between 0.9 and 1.1 atm, inclusive.

[0362] In some embodiments of the present invention, the poly-A region of the modified mRNA or modified non-coding RNA is unbranched. In some instances, the poly-A region of the modified mRNA or modified non-coding RNA is branched. In some instances, the poly-A region includes a poly-A tail having one or more crosslinking groups, which can be linked to a poly-A group resulting in branching. In some instances, the poly-A region comprises modified nucleotides that allow for crosslinking. The crosslinking group are incorporated into the mRNA as modified nucleotides (e.g., added by a polymerase or sitespecific modification by enzymatic treatment or through ligation of molecules / oligos containing crosslinking groups). The branched poly-A region can be prepared by crosslinking chemistry (e.g., click chemistry), such as by the methods described in International Publication No. WO 2023 / 141474, which is hereby incorporated by reference.Compositions comprising modified mRNAs or modified non-coding RNAs and methods of use

[0363] In some aspects, the present disclosure provides compositions comprising any one of the modified mRNAs or modified non-coding RNAs provided herein. In some embodiments, the modified mRNA or modified non-coding RNA is made by any of the methods provided herein comprising ligating a tailing nucleic acid onto an RNA. Compositions comprising a modified mRNA are useful for delivering the modified mRNA to a cell in order to vaccinate the subject against a foreign antigen, or express a therapeutic protein to treat a condition or disorder. Compositions comprising a modified non-coding RNA are useful for modulating the expression of genes in a cell or subject, or for editing the genome of a cell or subject, and may be used to treat a condition or disorder. Compositions comprising modified mRNAs or modified noncoding RNAs are also useful for exerting a82167200.02701 / 151646090v.ldesired effect in a subject in the absence of disease, such as for agricultural uses. For example, an mRNA encoding a biological pesticide or growth augmenting factor or a noncoding RNA for genome editing may be used to increase the tolerance of a plant to pests, or modulate growth in a manner that increases crop yield, respectively. Any of the modified mRNAs or modified non-coding RNA described herein or a composition thereof may be used to enhance the delivery and / or stability of mRNAs or modified non-coding RNA to plants or plant cells, and may be used to augment techniques for plant genome engineering that are well established in the art. See, e.g., Stoddard, et al. PLoS One. 2016, 1 l(5):e0154634.

[0364] In some embodiments, the modified RNA is used to treat a diseased patient. In some instances, the diseased patient is cancer. In some instances, the diseased patient is an auto-immune disease. In some instances, the diseased patient is a neurological disease.

[0365] In some embodiments, the open reading frame of the mRNA is codon- optimized for expression in a cell of a subject. As used herein, “codon-optimized" refers to the preferential use of codons that are more efficiently translated in a cell. Multiple codons can encode the same amino acid, with the translation rate and efficiency of each codon being determined by multiple factors, such as the intracellular concentration of aminoacyl -tRNAs comprising a complementary anticodon. Codon optimization of a nucleic acid sequence may include replacing one or more codons with codons that encode the same amino acid as, but are more efficiently translated than, the replaced codons. For example, the amino acid threonine (Thr) may be encoded by ACA, ACC, ACG, or ACT (ACU in RNA), but in mammalian host cells ACC is the most commonly used codon; in other species, different Thr codons may be preferred for codon-optimized. An mRNA with a codon-optimized open reading frame is thus 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 a human cell.

[0366] In some embodiments of the modified mRNAs provided herein, the open reading frame encodes an antigen or a therapeutic protein. As used herein, a “therapeutic protein” refers to a protein that prevents, reduces, or alleviates one or more signs or symptoms of a disease when expressed in a subject, such as a human subject that has, or is at risk of developing, a disease or disorder. A therapeutic protein may be an essential enzyme or transcription factor encoded by a gene that is mutated in a subject. For example, IPEX syndrome in humans is caused by a mutation in the FOXP3 gene, which hinders development83167200.02701 / 151646090v.lof F0XP3+ regulatory T cells and results in increased susceptibility to autoimmune and inflammatory disorders. Expression of an essential enzyme or transcription factor from an mRNA may therefore compensate for a mutation in the gene encoding the enzyme or transcription factor in a subject As used herein, “antigen” refers to a molecule (e.g., a protein) that, when expressed in a subject, elicits the generation of antibodies in the subject that bind to the antigen. 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 (protozoal 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 with an amino acid sequence that is present in, but shorter than, the amino acid sequence of the frill-length protein.

[0367] In some aspects, the present disclosure provides lipid nanoparticles comprising any of the modified mRNAs or modified non-coding RNAs provided herein. A lipid nanoparticle refers to a composition comprising one or more lipids that form an aggregate of lipids, or an enclosed structure with an interior surface and an exterior surface. Lipids used in the formulation of lipid nanoparticles for delivering mRNA or non-coding RNA are generally known in the art, and include ionizable 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 or modified non-coding RNA is surrounded by the lipids of the lipid nanoparticle and present in the interior of the lipid nanoparticle. In some embodiments, the mRNA or non-coding RNA is dispersed throughout the lipids of the lipid nanoparticle. In some embodiments, the lipid nanoparticle comprises an ionizable amino lipid, a non-cationic lipid, a sterol, and / or a polyethylene glycol (PEG)-modified lipid.

[0368] In some aspects, the present disclosure provides cells comprising any of the modified mRNAs or modified non-coding RNAs provided herein. In some embodiments, the cell is a human cell comprising any one of the modified mRNAs or modified non-coding RNAs provided herein. A “cell” is the basic structural and functional unit of all known independently living organisms. It is the smallest unit of life that is classified as a living thing. Some organisms, such as most bacteria, are unicellular (consist of a single cell). Other organisms, such as plants, fungi, and animals, including cattle, horses, chickens, turkeys, sheep, swine, dogs, cats, and humans, are multicellular. In some embodiments, the half-life of the modified mRNA or modified noncoding RNA in the cell is 15-900 minutes. In some84167200.02701 / 151646090v.lembodiments, the half-life of the modified mRNA or modified non-coding RNA in the cell is 30-600 minutes. In some embodiments, the half-life of the modified mRNA or modified noncoding RNA in the cell is 60-300 minutes. In some embodiments, the half-life of the modified mRNA or modified non-coding RNA 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, at least 60 minutes. In some embodiments, the half-life of the modified mRNA or modified noncoding RNA in the 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.

[0369] In some aspects, the present disclosure provides compositions comprising any of the modified mRNAs, modified non-coding RNAs, lipid nanoparticles, or cells provided herein. In some embodiments, the composition is a pharmaceutical composition comprising any one of the modified mRNAs, modified non-coding RNAs, lipid nanoparticles, or cells provided herein, and a pharmaceutically acceptable excipient. Pharmaceutically acceptable excipients, carriers, buffers, stabilizers, isotonicising agents, preservatives or antioxidants, or other materials well known to those skilled in the art. Such materials 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, e.g., parenteral, intramuscular, intradermal, sublingual, buccal, ocular, intranasal, subcutaneous, intrathecal, intratumoral, oral, vaginal, or rectal.

[0370] In some aspects, the present disclosure provides a method of administering to a subject any of the modified mRNAs, modified non-coding RNAs, lipid nanoparticles, cells, compositions, or pharmaceutical compositions provided herein. In some embodiments, the any of the modified mRNAs or modified non-coding RNAs described herein can be used in conjunction with a variety of reagents or materials (e.g., one or more lipid nanoparticles, cells, compositions, or pharmaceutical compositions) or with certain production, purification, formulation, and delivery processes and techniques known in the art, such as those exemplified in, but not limited to, U.S. Patents Nos. 9950065, 10576146, 11045418, 8754062, 10808242, 9957499, 10155785, 11059841, 10876104, 10975369, 9580711, 9670152, 9850202, 9896413, 10399937, 10052284, 10959953, and 10961184, each of which are incorporated by reference herein.85167200.02701 / 151646090v.l

[0371] In some embodiments, the subject is a human. In some embodiments, the administration is parenteral, intramuscular, intradermal, sublingual, buccal, ocular, intranasal, subcutaneous, intrathecal, intratumoral, oral, vaginal, or rectal.

[0372] In some embodiments, the composition is to 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, such that the nucleic acids are relatively stable over time.

[0373] In some embodiments, the modified mRNA or modified non-coding RNA is introduced into a cell in a subject by in vivo electroporation. In vivo electroporation is the process of introducing nucleic acids or other molecules into a cell of a subject using a pulse of electricity, which promote passage of the nucleic acids or other molecules through the cell membrane and / or cell wall. See, e.g., Somiari et al. Molecular Therapy., 2000. 2(3): 178-187. The nucleic acid or molecule to be delivered is administered to the 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 site of administration. In some embodiments, the nucleic acid is administered with other elements, such as buffers and / or excipients, that increase the efficiency of electroporation.

[0374] In some aspects, the present disclosure provides a kit comprising any of the RNAs and any of the tailing nucleic acids provided herein. The RNA and tailing nucleic acid can be combined in the presence of an RNA ligase to produce a modified mRNA or modified noncoding RNA, such as one of the modified mRNAs or modified non-coding RNAs 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 a T4 RNA ligase. In some embodiments, a kit comprises a T4 RNA ligase 1. In some embodiments, a kit comprises a T4 RNA ligase 2. In some embodiments, the kit comprises an RtcB RNA ligase. In some embodiments, the kit further comprises a buffer for carrying out the ligation. In some embodiments, the kit further comprises a nucleotide triphosphate, such as ATP, to provide energy required by the ligase. In some embodiments, the kit is to 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, such that the nucleic acids are relatively stable over time.

[0375] In some aspects, the present disclosure provides a kit comprising any of the pharmaceutical compositions provided herein and a delivery device. A delivery device refers86167200.02701 / 151646090v.lto machine or apparatus suitable for administering a composition to a subject, such as a syringe or needle. In some embodiments, the kit is to 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, such that the nucleic acids of the pharmaceutical composition are relatively stable over time.EQUIVALENTS AND SCOPE

[0376] In the claims, articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “of’ between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0377] For the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing quantities, percentages or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about.” The use of the term “about” applies to all numeric values, whether or not explicitly indicated. This term generally refers to reasonable amount of deviation to the recited numeric values (i.e., having the equivalent function or result). For example, this term can be construed as including a deviation of ±10 percent, alternatively ±5 percent, alternatively ±1 percent, alternatively ±0.5 percent, and alternatively ±0.1 percent of the given numeric value provided such a deviation does not alter the end function or result of the value. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by the present invention.

[0378] Furthermore, it is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the claims or from relevant portions of the description is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Furthermore, where the claims recite a87167200.02701 / 151646090v.lcomposition, it is to be understood that methods of using the composition for any of the purposes disclosed herein are included, and methods of making the composition according to any of the methods of making disclosed herein or other methods known in the art are included, unless otherwise indicated.

[0379] Where elements are presented as lists, e.g., in Markush group format, it is to be understood that each subgroup of the elements is also disclosed, and any elements) can be removed from the group. It is also noted that the term “comprising” is intended to be open and permits the inclusion of additional elements or steps. It should be understood that, in general, where the invention, or aspects of the invention, is / are referred to as comprising particular elements, features, steps, etc., certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements, features, steps, etc. For purposes of simplicity those embodiments have not been specifically set forth in haec verba herein. Thus for each embodiment of the invention that comprises one or more elements, features, steps, etc., the invention also provides embodiments that consist or consist essentially of those elements, features, steps, etc.

[0380] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated, values that are expressed as ranges can assume any specific value within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also to be understood that unless otherwise indicated, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range.

[0381] In addition, it is to be understood that any particular embodiment of the present invention may be explicitly excluded from any one or more of the claims. Where ranges are given, any value within the range may explicitly be excluded from any one or more of the claims. Any embodiment, element, feature, application, or aspect of the compositions and / or methods of the invention, can be excluded from any one or more claims. For purposes of brevity, all of the embodiments in which one or more elements, features, purposes, or aspects is excluded are not set forth explicitly herein.88167200.02701 / 151646090v.lExamplesExample 1Generation of mRNA

[0382] DNA templates for mRNA production were generated by PCR amplifying a synthetic gBLOCK (nLuc gBLOCK) with the oligos in Table 1. All templates contained a modified T7 promoter to enable co-transcri phonal capping, a 5’ UTR, nLuc ORF and 3’UTR as shown in Table 1. The sequences for nLuc- AO (also referred to as nLuc-noA), nLuc-A30, nLuc-AlOO, nLuc gBLOCK, oFBl, oFB2, oFB3, and oFB83 are provided in Table A. SEQ ID NOs: 15-17 were prepared by this procedure.Table 1

[0383] Purified templates were used in an IVT reaction using a Hi Scribe T7 mRNA Kit with a CleanCap Reagent AG (NEB E2080) and fully Nl-Metyl PseudoUridine substitution. The resulting mRNA was purified over Zymo RNA Clean and Concentrator (Zymo RIO 17) column.167200.02701 / 151646090v.lOligo ligation and purification

[0384] Ligation reactions of in vitro transcription (IVT) generated RNA and oligos were performed as follows: 2 pg of nLuc-AO mRNA; 200 pmol oligo; 2 pL of Superase-In RNase Inhibitor; 20 pL of 50% PEG-8000, 5 pL of 10x T4 RNA ligase buffer; 5 pL of T4 RNA ligase (Promega: M1051); and nuclease-free water to a total reaction volume of 50 pL. Reactions were incubated at 37 °C for 30 minutes, followed by inactivation of the reaction via the addition of 1 pL of 500 mM EDTA at pH 8.0. Ligated mRNA were then purified in two successive rounds using 0.9X volumes RNAClean XP beads (Beckman Coulter: A63987). Bound mRNA were washed twice in 70% ethanol and eluted in 1 / 5 starting volume of nuclease-free water. Samples were resolved on an RNA Flash gel (Lonza 57028) and samples with no visible oligo were advanced to transfection assay. The ligated products are shown in Table 2.Table 2Transfection assay

[0385] Luciferase activity was measured in Hela cells 24 hours post-transfection with 25 ng mRNA by the transfection assay described in Example 2. The results are shown in Figure 1.90167200.02701 / 151646090v.lExample 2Generation of mRNA

[0386] The following procedure was used to prepare nLuc-AO, nLuc-A30, and nLuc- A100 which correspond to SEQ ID NO. 15, 16, and 17.

[0387] DNA templates were prepared as described in Example 1. Purified templates were used in an IVT reaction with CleanCap AG and fully Nl-Metyl Pseudouridine substitution. The resulting mRNA was purified over silica column.Oligo ligation and purification

[0388] Ligation reactions of IVT generated RNA and oligos were performed as follows: 2 pg of nLuc-AO mRNA; 100 pmol oligo; 2 pL of Superase-In RNase Inhibitor; 20 pL of 50% PEG-8000, 5 pL of 10x T4 RNA ligase buffer; 5 pL of T4 RNA ligase (Promega: M1051); and nuclease-free water to a total reaction volume of 50 pL. Reactions were incubated at 37 °C for 30 minutes, followed by inactivation of the reaction via the addition of 1 pL of 500 mM EDTA at pH 8.0. Ligated mRNA were then purified using 1.7X volumes of RNAClean XP beads (Beckman Coulter: A63987) pre-washed twice with 2.5M NaCl, 50mM Tris pH8. Bound mRNA were washed twice in 70% ethanol and eluted in 1 / 5 starting volume of nuclease-free water. Samples were resolved on an RNA Flash gel (Lonza 57028). The ligated products are shown in Table 3.Table 391167200.02701 / 151646090v.lRNaseH assay

[0389] RNaseH digestion assay was used to evaluate oligo ligation efficiency as follows:200 ng of purified mRNA sample, 2 pmol of oFB96, 2 pL of annealing stock solution (50 mM KC1, 2.5 mM EDTA, 1 : 100 Superase-In), and nuclease-free water up to a total volume of 10 pL. Reactions were denatured at 70 °C for 5 min, followed by cooling to RT at a rate of 0.2 °C / s in a benchtop thermalcycler. Following probe annealing, 1 pL of the 10x buffer followed by 1 pL of Thermostable RNase H (NEB: M0523S) were added to each reaction. Reactions were incubated at 50 °C for 30 minutes followed by the addition of 1 pL of Proteinase K (ThermoFisher Scientific: 25530049) and incubated at room temperature (RT) for 5 minutes. Samples were then mixed with one volume of Gel Loading Buffer II (Thermo Fisher Scientific: AM8546G), which had been supplemented with EDTA to a final concentration of 50 mM. Samples in 1 * loading buffer were denatured at 70 °C for 5 minutes prior to loading and resolution on 6% Novex TBE-Urea Gels (ThermoFisher Scientific: EC68655BOX), run in 1 * Tris-borate-EDTA (TBE) buffer using Low Range ssRNA Ladder from NEB (N0364S). The results are shown in Figure 2. Ligation efficiency of the samples was estimated to be around 30%.Transfection assay

[0390] These data were all generated via lipofection of mRNA using the Lipofectamine MessengerMAX reagent from Thermo Fisher Scientific (LMRNA001). HeLa cells were cultured in EMEM supplemented with 10% HI-FBS and 1% Non-Essential Amino Acids. The cells were plated in 96-well plates 16-24 hours prior to transfection. Cells were treated with 3, 10, or 30 ng mRNA.

[0391] Nano-Luc activity was measured in an enzyme activity assay by Promega (N1110). Data were collected on the ClarioSTAR plate reader from BMG LabTech. The results 24, 48, and 72 hours post-transfection are provided in Figure 3.Example 3Generation of mRNA

[0392] DNA templates for mRNA production were generated by PCR amplifying a synthetic gBLOCK (nLuc gBLOCK) with the oligos in Table 4. All templates contained a modified T7 promoter to enable co-transcriptional capping, a 5’ UTR, nLuc ORF and 3’UTR92167200.02701 / 151646090v.las shown in Table 4. The sequences for nLuc-AO (also referred to as nLuc-noA), nLuc-A30, nLuc-AlOO, nLuc gBLOCK, oFBl, oFB2, oFB83, and oFB158 are provided in Table A. SEQ ID NOs: 16-17, and 23 were prepared by this procedureTable 4

[0393] Purified templates were used in an IVT reaction using a Hi Scribe T7 mRNA Kit with a CleanCap Reagent AG (NEB E2080) and fully Nl-Metyl PseudoUridine substitution. The resulting mRNA was purified over Zymo RNA Clean and Concentrator (Zymo RIO 17) column.Oligo ligation and purification

[0394] Ligation reactions of IVT generated RNA and oligos were performed as follows: 2 pg of nLuc-AO mRNA; 100 pmol oligo; 2 pL of Superase-In RNase Inhibitor; 20 pL of 50% PEG-8000, 5 pL of 10x T4 RNA ligase buffer; 5 pL of T4 RNA ligase (Promega: M1051); and nuclease-free water to a total reaction volume of 50 pL. Reactions were incubated at 37 °C for 30 minutes, followed by inactivation of the reaction via the addition of 1 pL of 500 mM EDTA at pH 8.0. Ligated mRNA were then purified using 1.7X volumes of RNAClean XP beads (Beckman Coulter: A63987) pre-washed twice with 2.5M NaCl, 50mM Tris pH8. Bound mRNA were washed twice in 70% ethanol and eluted in 1 / 5 starting volume of nuclease-free water. Samples were resolved on an RNA Flash gel (Lonza 57028). The ligated products are shown in Table 5.Table 593167200.02701 / 151646090v.lRNaseH assay

[0395] The RNaseH digestion assay of Example 1 was used to evaluate oligo ligation efficiency. The results are shown in Figure 4. Ligation efficiency of the samples was estimated to be around 30%.Transfection assay

[0396] These data were all generated via lipofection of mRNA using the Lipofectamine MessengerMAX reagent from Thermo Fisher Scientific (LMRNA001). HeLa cells were cultured in EMEM supplemented with 10% HI-FBS and 1% Non-Essential Amino Acids. HEK293 and U2-OS cells were cultured in DMEM with 10% HI-FBS. The cells were94167200.02701 / 151646090v.lplated in 96-well plates 16-24 hours prior to transfection. Cells were treated with 10 ng mRNA

[0397] Nano-Luc activity was measured in an enzyme activity assay by Promega (N1110). Data were collected on the ClarioSTAR plate reader from BMG LabTech. The results 24 hours post-transfection are provided in Figures 5A, 5B, and 5C.Example 4

[0398] DNA templates for mRNA production were generated by PCR amplifying a synthetic gBLOCK (nLuc gBLOCK) with the oligos in Table 6. All templates contained a modified T7 promoter to enable co-transcri phonal capping, a 5’ UTR, nLuc ORF and 3’UTR as shown in Table 6. The sequences for nLuc-AO (also referred to as nLuc-noA), nLuc-A30, nLuc-AlOO, nLuc gBLOCK, oFBl, oFB2, oFB3, oFB83, and oFB158 are provided in Table A. SEQ ID NOs: 15-17 and 23 were prepared by this procedure.Table 6

[0399] Purified templates were used in an IVT reaction using a Hi Scribe T7 mRNA Kit with a CleanCap Reagent AG (NEB E2080) and fully Nl-Metyl PseudoUridine substitution. The resulting mRNA was purified over Zymo RNA Clean and Concentrator (Zymo R1017) column.Oligo ligation and purification

[0400] Ligation reactions of IVT generated RNA and oligos were performed as follows: 2 pg of nLuc-AO mRNA; 100 pmol oligo; 2 pL of Superase-In RNase Inhibitor; 20 pL of 50% PEG-8000, 5 pL of 10x T4 RNA ligase buffer; 5 pL of T4 RNA ligase (Promega:95167200.02701 / 151646090v.lM1051); and nuclease-free water to a total reaction volume of 50 pL. Reactions were incubated at 37 °C for 30 minutes, followed by inactivation of the reaction via the addition of 1 pL of 500 mM EDTA at pH 8.0. Ligated mRNA were then purified using 1.7X volumes of RNAClean XP beads (Beckman Coulter: A63987) pre-washed twice with 2.5M NaCl, 50mM Tris pH8. Bound mRNA were washed twice in 70% ethanol and eluted in 1 / 5 starting volume of nuclease-free water. Samples were resolved on an RNA Flash gel (Lonza 57028). The ligated products are shown in Table 7.Table 7RNaseH assay

[0401] The RNaseH digestion assay of Example 1 was used to evaluate oligo ligation efficiency. The results are shown in Figure 6. Ligation efficiency of the samples was estimated to be around 30%.Transfection assay96167200.02701 / 151646090v.l

[0402] These data were all generated via lipofection of mRNA using the Lipofectamine MessengerMAX reagent from Thermo Fisher Scientific (LMRNA001). HEK293 and U2-OS cells were cultured in DMEM with 10% HI-FBS. The cells were plated in 96-well plates 16-24 hours prior to transfection. Cells were treated with 10 ng mRNA.

[0403] Nano-Luc activity was measured in an enzyme activity assay by Promega (N1110). Data were collected on the ClarioSTAR plate reader from BMG LabTech. The results 24 hours post-transfection are provided in Figures 7 A and 7B.Example 5Generation of mRNA

[0404] DNA templates for mRNA production were generated by PCR amplifying a synthetic gBLOCK (nLuc gBLOCK) with the oligos in Table 8. All templates contained a modified T7 promoter to enable co-transcri phonal capping, a 5’ UTR, nLuc ORF and 3’UTR as shown in Table 8. The sequences for final DNA templates and oligos are provided in Table A. SEQ ID NOs: 15-17, 23, and 37-39 were prepared by this procedure.Table 8

[0405] Purified templates were used in an IVT reaction using a HiScribe T7 mRNA Kit with a CleanCap Reagent AG (NEB E2080) and fully Nl-Metyl PseudoUridine substitution. The resulting mRNA was purified over Zymo RNA Clean and Concentrator (Zymo RIO 17) column.97167200.02701 / 151646090v.lTransfection assay

[0406] These data were all generated via lipofection of mRNA using the Lipofectamine MessengerMAX reagent from Thermo Fisher Scientific (LMRNA001). HeLa and U2OS cells were cultured in EMEM supplemented with 10% HI-FBS and 1% Non- Essential Amino Acids. The cells were plated in 96-well plates 16-24 hours prior to transfection. Cells were treated with 10 ng mRNA.

[0407] Nano-Luc activity was measured in an enzyme activity assay by Promega (N1110). Data were collected on the ClarioSTAR plate reader from BMG LabTech. The results 24 hours post-transfection are provided in Figures 8 A and 8B.Example 6Generation of mRNA

[0408] DNA templates for mRNA production were generated by PCR amplifying a synthetic gBLOCK (nLuc gBLOCK) with the oligos in Table 9. All templates contained a modified T7 promoter to enable co-transcri phonal capping, a 5’ UTR, nLuc ORF and 3’UTR as shown in Table 9. The sequences for final DNA templates and oligos are provided in Table A. SEQ ID NOs: 17 and 57-73 were prepared by this procedure.Table 998167200.02701 / 151646090v.l

[0409] Purified templates were used in an IVT reaction using a Hi Scribe T7 mRNA Kit with a CleanCap Reagent AG (NEB E2080) and fully Nl-Metyl PseudoUridine substitution. The resulting mRNA was purified over Zymo RNA Clean and Concentrator (Zymo RIO 17) column.Transfection assay

[0410] These data were all generated via lipofection of mRNA using the Lipofectamine MessengerMAX reagent from Thermo Fisher Scientific (LMRNA001). HeLa cells were cultured in EMEM supplemented with 10% HI-FBS and 1% Non-Essential Amino Acids. The cells were plated in 96-well plates 16-24 hours prior to transfection. Cells were treated with 10 ng mRNA.

[0411] Nano-Luc activity was measured in an enzyme activity assay by Promega (N1110). Data were collected on the ClarioSTAR plate reader from BMG LabTech. The results 24 hours post-transfection are provided in Figures 9A-9C.Example 7Generation of mRNA

[0412] DNA templates for mRNA production were generated by PCR amplifying a synthetic gBLOCK (nLuc gBLOCK) with the oligos in Table 10. All templates contained a99167200.02701 / 151646090v.lmodified T7 promoter to enable co-transcri phonal capping, a 5’ UTR, nLuc ORF and 3’UTR as shown in Table 10. The sequences for final DNA templates and oligos are provided in Table A. SEQ ID NOs: 15-17 and 76-77 were prepared by this procedure.Table 10

[0413] Purified templates were used in an IVT reaction using a Hi Scribe T7 mRNA Kit with a CleanCap Reagent AG (NEB E2080) and fully Nl-Metyl PseudoUridine substitution. The resulting mRNA was purified over Zymo RNA Clean and Concentrator (Zymo R1017) column.Tansfection assay

[0414] These data were all generated via lipofection of mRNA using the Lipofectamine MessengerMAX reagent from Thermo Fisher Scientific (LMRNA001). HeLa cells were cultured in EMEM supplemented with 10% HI-FBS and 1% Non-Essential Amino Acids. The cells were plated in 96-well plates 16-24 hours prior to transfection. Cells were treated with 5, 1 and 0.1 ng mRNA.

[0415] Nano-Luc activity was measured in an enzyme activity assay by Promega (N1110). Data were collected on the ClarioSTAR plate reader from BMG LabTech. The results at 6, 18 and 24 hours post-transfection are provided in Figures 10A, 10B, and 10C.Example 8Generation of mRNA

[0416] DNA templates for mRNA production were generated by PCR amplifying a synthetic gBLOCK (nLuc gBLOCK) with the oligos in Table 10. All templates contained a100167200.02701 / 151646090v.lmodified T7 promoter to enable co-transcriptional capping, a 5’ UTR, nLuc ORF and 3’UTR as shown in Table 11. The sequences for final DNA templates and oligos are provided in Table A. SEQ ID NOs: 16, 17, and 76-77 were prepared by this procedure.Table 11

[0417] Purified templates were used in an IVT reaction using a Hi Scribe T7 mRNA Kit with a CleanCap Reagent AG (NEB E2080) and fully Nl-Metyl PseudoUridine substitution. The resulting mRNA was purified over Zymo RNA Clean and Concentrator (Zymo RIO 17) column.Formulation

[0418] A lipid nanoparticulate (LNP) formulation was prepared from the nanoLuc mRNA with murine CD5 Fab and an ionizable lipid by Ignite dilution cartridge / dialysis / ami con.Mouse study

[0419] C57BL / 6J mice (J AX, 000664) were injected with lipid nanoparticles (LNPs) containing mRNA (SEQ ID NOs: 16, 17, 76, and 77) at two different doses (0.1 mg / kg and 0.5 mg / kg) administered via intravenous (IV) injection. To quantify the nanoLuc signal, 50 pL (0.438 pmoles) of NanoGio Fluorofurimazine Substrate (Promega, N41000) was injected intraperitoneally (IP) 10 minutes prior to imaging. Mice were then imaged using the IVIS imaging system (Revvity, CLS136334). Signal quantification was performed 6 hours postadministration, daily for five days, and then twice weekly until the signal was no longer detectable. The results for each dose (0.5 mg / kg and 0.1 mg / kg) are provided in Figures 11A and 1 IB respectively.101167200.02701 / 151646090v.l

[0420] All references, patent applications, and patents cited herein are hereby incorporated by reference.

[0421] Table A describes the sequences used in the present application. Table B describes the nomenclature used in the sequence listing.Table A102167200.02701 / 151646090v.l103167200.02701 / 151646090v.l104167200.02701 / 151646090v.l105167200.02701 / 151646090v.l106167200.02701 / 151646090v.l107167200.02701 / 151646090v.l108167200.02701 / 151646090v.l109167200.02701 / 151646090v.l110167200.02701 / 151646090v.lIll167200.02701 / 151646090v.l112167200.02701 / 151646090v.l113167200.02701 / 151646090v.l114167200.02701 / 151646090v.l115167200.02701 / 151646090v.l116167200.02701 / 151646090v.l117167200.02701 / 151646090v.l118167200.02701 / 151646090v.lTable B167200.02701 / 151646090v.l

Claims

Claims1. A modified mRNA comprising:(i) an open reading frame (ORF) encoding a protein; and(ii) a poly-A region, wherein the poly-A region is 3' to the open reading frame and comprises 10 or more nucleotides, wherein greater than 90% of the nucleotides of the poly-A region are modified nucleotides, and wherein 3 or more of the 10 last nucleotides of the poly-A region are modified nucleotides.

2. A modified non-coding RNA comprising:(i) a non-coding RNA; and(ii) a poly-A region, wherein the poly-A region is 3' to the open reading frame and comprises 10 or more nucleotides, wherein greater than 90% of the nucleotides of the poly-A region are modified nucleotides, and wherein 3 or more of the 10 last nucleotides of the poly-A region are modified nucleotides.

3. The modified mRNA of claim 1 or the modified non-coding RNA of claim 2, wherein the poly-A region is 3' to the open reading frame and comprises 25 or more adenosine nucleotides, wherein greater than 90% of the nucleotides of the poly-A region are modified nucleotides, and wherein 3 or more of the 25 last nucleotides of the poly-A region are modified nucleotides.

4. The modified mRNA of claim 1 or 3 or the modified non-coding RNA of claim 2 or 3, wherein 4 or more of the 25 last nucleotides of the poly-A region are modified nucleotides.

5. The modified mRNA of any one of claims 1, 3, and 4 or the modified non-coding RNA of any one of claims 2-4, wherein 2 or more consecutive nucleotides of the 25 last nucleotides of the poly-A region are linked by a modified internucleotide linkage.

6. The modified mRNA of any one of claims 1 and 3-5 or the modified non-coding RNA of any one of claims 2-5, wherein the 3 or more modified nucleotides are consecutive nucleotides located at the 3' terminus of the poly-A region.120167200.02701 / 151646090v.l7. The modified mRNA of any one of claims 1 and 3-6 or the modified non-coding RNA of any one of claims 2-6, wherein 6 or more consecutive nucleotides of the 25 last nucleotides of the poly-A region comprise the same type of nucleotide or intemucleoside modification.

8. The modified mRNA of any one of claims 1 and 3-7 or the modified non-coding RNA of any one of claims 2-7, wherein the modified mRNA or the modified non-coding RNA comprises a 5' untranslated region (5' UTR) and a 3' untranslated region (3' UTR), wherein, for the modified mRNA, (i) the ORF is between the 5' UTR and the 3' UTR, and (ii) the 3' UTR is between the ORF and the poly-A region.

9. The modified mRNA of any one of claims 1 and 3-8 or the modified non-coding RNA of any one of claims 2-8, wherein the modified mRNA or modified non-coding RNA is a circular mRNA or circular non-coding RNA, wherein the poly-A region is between the 3' UTR and the 5' UTR10. The modified mRNA of any one of claims 1 and 3-9 or the modified non-coding RNA of any one of claims 2-9, wherein at least one modified nucleotide comprises a modified nucleobase.

11. The modified mRNA or modified non-coding RNA of claim 10, wherein the modified nucleobase is selected from xanthine, allyaminouracil, allyaminothymidine, hypoxanthine, digoxigeninated adenine, digoxigeninated cytosine, digoxigeninated guanine, digoxigeninated uracil, 6-chloropurineriboside, N6-methyladenine, methylpseudouracil, 2- thiocytosine, 2-thiouracil, 5-methyluracil, 4-thiothymidine, 4-thiouracil, 5,6-dihydro-5- methyluracil, 5,6-dihydrouracil, 5-[(3-Indolyl)propionamide-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 -methoxy cytosine, 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- propargylaminoguamne, 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, cyanine121167200.02701 / 151646090v.l3-5-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, dabcyl-5-3-aminoallyluracil, desthiobiotin- 16-aminoallyl-uracil, desthiobiotin-6-aminoallylcytosine, isoguanine, N1 -ethylpseudouracil, Nl- methoxymethylpseudouracil, N1 -methyladenine, N1 -methylpseudouracil, Nl- 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-carboxamide-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-threonyl carbamoyladenine (ms2t6A), N6-methyl-N6-threonylcarbamoyladenine (m6t6A), N6-hydroxynorvalylcarbamoyladenine (hn6A), 2-methylthio-N6-hydroxynorvalyl carbamoyladenine (ms2hn6A), N6,N6- dimethyladenine (m62A), and N6-acetyladenine (ac6A).

12. The modified mRNA or modified non-coding RNA of claim 10 or 11, wherein at least one modified nucleotide comprises a modified sugar.

13. The modified mRNA or modified non-coding RNA of claim 12, wherein the modified sugar is selected from 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-m ethylribose, 5 '-aminoribose, 5'- thioribose, 5-nitro-l-indolyl-2'-deoxyribose, 5'-biotin-ribose, 2'-O,4'-C-methylene-linked, 2'- O,4'-C-amino-linked ribose, and 2'-O,4'-C-thio-linked ribose.

14. The modified mRNA or modified non-coding RNA of claim 12 or 13, wherein at least one modified nucleotide comprises a 2' modification.

15. The modified mRNA or modified non-coding RNA of claim 14, wherein the 2' modification is selected from a locked-nucleic acid (LNA) modification, 2'-fluoro (2'-F), 2'-122167200.02701 / 151646090v.lO-methoxy-ethyl (2'-M0E), 2'-O-methylation (2’-0Me), 2’-deoxy (2’-H), and 2'-O-N- methylacetamido (2'-0-NMA).

16. The modified mRNA of any one of claims 1 and 3-15 or the modified non-coding RNA of any one of claims 2-15, wherein the poly-A region is branched.

17. The modified mRNA of any one of claims 1 and 3-15 or the modified non-coding RNA of any one of claims 2-15, wherein the poly-A region is unbranched.

18. The modified mRNA of any one of claims 1 and 3-17 or the modified non-coding RNA of any one of claims 2-17, wherein the modified mRNA or modified non-coding RNA is used for treatment of a diseased patient.

19. The modified mRNA of any one of claims 1 and 3-17 or the modified non-coding RNA of any one of claims 2-17, wherein the modified mRNA or modified non-coding RNA is used as a growth augmenting factor for growth of plants.

20. A method of producing a modified mRNA of any one of claims 1 and 3-17 or a modified non-coding RNA of any one of claims 2-17, the method comprising ligating a first RNA comprising an open reading frame encoding a protein or a non-coding RNA sequence to a tailing nucleic acid comprising one or more modified nucleotides, in the presence of an RNA ligase, whereby the RNA ligase forms a covalent bond between the 3’ nucleotide of the RNA and the 5’ nucleotide of the tailing nucleic acid to produce the modified mRNA or modified non-coding RNA.

21. A modified mRNA comprising:(i) an open reading frame (ORF) encoding a protein; and(ii) a poly region comprising adenosine nucleotides and cytosine nucleotides, wherein (i) the poly region is 3' to the open reading frame, (ii) the poly region has a length of from 30 to 500 nucleotides, (iii) at least 50% of the total number of adenosine and cytosine nucleotides in the poly region are cytosine nucleotides, and (iv) the number of consecutive adenosine nucleotides in the poly region does not exceed 15.

22. A modified non-coding RNA comprising:(i) a non-coding RNA; and(ii) a poly region,123167200.02701 / 151646090v.lwherein (i) the poly region is 3' to the open reading frame, (ii) the poly region has a length of from 30 to 500 nucleotides, (iii) at least 50% of the total number of adenosine and cytosine nucleotides in the poly region are cytosine nucleotides, and (iv) the number of consecutive adenosine nucleotides in the poly region does not exceed 15.

23. The modified mRNA of claim 21 or the modified non-coding RNA of claim 22, wherein the poly region includes repeating at least 6 consecutive repeats of an AC monomer.

24. The modified mRNA of claim 21 or the modified non-coding RNA of claim 22, wherein the poly region includes from 6 to 20 consecutive repeats (e.g., 10 consecutive repeats) of an AC monomer.

25. The modified mRNA of claim 21 or the modified non-coding RNA of claim 22, wherein the poly region includes repeating at least 6 consecutive repeats (e.g., 6 to 20 consecutive repeats) of a CCA monomer, CCCA monomer, or CCCCA monomer.

26. The modified mRNA of claim 21 or the modified non-coding RNA of claim 22, wherein the poly region includes repeating at least 2 consecutive repeats (e.g., 2 to 30 (such as 2, 3, 4, 5, 6 or 7) consecutive repeats) of (C)9A (SEQ ID NO: 78), (C)I9A (SEQ ID NO: 79), A(C)9(SEQ ID NO: 80), or A(C)I9(SEQ ID NO: 81).

27. The modified mRNA of any one of claims 21 and 23-26 or the modified non-coding RNA of any one of claims 22-26, wherein the poly region includes CCC at the 3’ end of the poly region (e.g., the poly region may be (AC)nCCC (SEQ ID NO: 82) where n is 6 to 20).

28. A modified mRNA comprising:(i) an open reading frame (ORF) encoding a protein; and(ii) a poly region comprising adenosine nucleotides and cytosine nucleotides, wherein (i) the poly region is 3' to the open reading frame, (ii) the poly region has a length of from 30 to 500 nucleotides, (iii) 5 to 50% of the total number of adenosine and cytosine nucleotides in the poly region are cytosine nucleotides, and (iv) the number of consecutive adenosine nucleotides in the poly region does not exceed 20.

29. A modified non-coding RNA comprising:(i) a non-coding RNA; and(ii) a poly region,124167200.02701 / 151646090v.lwherein (i) the poly region is 3' to the open reading frame, (ii) the poly region has a length of from 30 to 500 nucleotides, (iii) 5 to 50% of the total number of adenosine and cytosine nucleotides in the poly region are cytosine nucleotides, and (iv) the number of consecutive adenosine nucleotides in the poly region does not exceed 20.

30. The modified mRNA of claim 28 or the modified non-coding RNA of claim 29, wherein the poly region includes repeating at least 2 consecutive repeats (e.g., 10 to 30 (such as 12 or 20) repeats) of an AAC, AAAC, or AAAAC monomer.

31. The modified mRNA of claim 28 or the modified non-coding RNA of claim 29, wherein the poly region includes repeating at least 2 consecutive repeats (e.g., 2 to 30 (such as 2, 3, 4, 5, 6 or 7) consecutive repeats) of (C)9A (SEQ ID NO: 78), (C)I9A (SEQ ID NO: 79), A(C)9(SEQ ID NO: 80), or A(C)I9(SEQ ID NO: 81).

32. The modified mRNA or modified non-coding RNA of any one of claims 21-31, wherein at least one nucleotide comprises a modified sugar.

33. The modified mRNA or modified non-coding RNA of claim 32, wherein the modified sugar is selected from 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-m ethylribose, 5 '-aminoribose, 5'- thioribose, 5-nitro-l-indolyl-2'-deoxyribose, 5'-biotin-ribose, 2'-O,4'-C-methylene-linked, 2'- O,4'-C-amino-linked ribose, and 2'-O,4'-C-thio-linked ribose.

34. The modified mRNA or modified non-coding RNA of claim 32 or 33, wherein at least one modified nucleotide comprises a 2' modification.

35. The modified mRNA or modified non-coding RNA of claim 34, wherein the 2' modification is selected from a locked-nucleic acid (LNA) modification, 2'-fluoro (2'-F), 2'- O-methoxy-ethyl (2'-M0E), 2'-O-methylation (2’-0Me), 2’-deoxy (2’-H), and 2 -O-N- methylacetamido (2'-0-NMA).

36. The modified mRNA or modified non-coding RNA of any one of claims 21-35, wherein the poly region is branched.125167200.02701 / 151646090v.l37. The modified mRNA or modified non-coding RNA of any one of claims 21-35, wherein the poly region is unbranched.

38. The modified mRNA or modified non-coding RNA of any one of claims 21-37, wherein the modified mRNA or modified non-coding RNA is used for treatment of a diseased patient39. The modified mRNA or modified non-coding RNA of any one of claims 21-37, wherein the modified mRNA or modified non-coding RNA is used as a growth augmenting factor for growth of plants.126167200.02701 / 151646090v.l

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