mRNA complexes, their manufacturing, and their use for treatment

Abstract

Provided herein is an mRNA complex comprising an mRNA molecule and at least one engineered oligonucleotide hybridized to the mRNA molecule, wherein said engineered oligonucleotide is hereafter referred to as the Str-O-Nuc.

Description

Ref. ParB 24001; 07800.002W01MRNA COMPLEXES, THEIR MANUFACTURING, AND THEIR USE FOR TREATMENTCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to United States Provisional Application Number 63 / 692,944, which was filed on September 10, 2024, and to United States Provisional Application Number 63 / 638,797, which was filed on April 25, 2024. The entire content of the applications referenced above is hereby incorporated by reference herein.TECHNICAL FIELD

[0002] This disclosure relates to an mRNA complex, its method of manufacturing, and its use for treatment.BACKGROUND

[0003] Current methods of mRNA synthesis are limited in both functionality and control as it is difficult to incorporate certain nucleotide modifications using RNA polymerase and determine with precision where these nucleotide modifications can be specifically incorporated. It is a known problem that incorporation of nucleotides into the mRNA from the nucleotide pool in the reaction can be random and heterogenous. Further, certain modifications may inhibit the in vitro transcription reaction.

[0004] In vitro transcription is the current method of synthesizing mRNA. However, introducing specific sequence modifications at specific locations of an mRNA is not possible through in vitro transcription. For example, 2’-0Me and 2’ -MOE modifications inhibit the in vitro transcription reaction. Currently, the only approach to incorporate specific modifications into an mRNA is through ligation (chemical or enzymatic) of a chemically synthesized RNA molecule. Additionally, in vitro transcription is severely limited in nucleotide chemistries that can be incorporated into mRNA manufacturing. For example, stabilizing modifications to 2’ -OH of the nucleotide are generally not incorporated by the polymerase during mRNA synthesis. Current methods also suffer from a lack of control as to where these nucleotide modifications can be specifically incorporated. That is, the incorporation of nucleotides into the mRNA from the nucleotide pool in the reaction is random and heterogeneous.Ref. ParB 24001; 07800.002W01

[0005] Current methods of chemical synthesis of mRNA known in the art can incorporate nucleotide modifications in a sequence defined way and homogenously. However, these methods of chemical synthesis of mRNA are limited to mRNAs that are less than about 100 nucleotides in length due to issues with purity and yield and these issues are further increased as the mRNAs increase in length. Other known methods in the art include chemical or enzymatic ligation to covalently link an in vitro transcribed mRNA with a chemically synthesized oligonucleotide (or any combination thereof), but these processes can be inefficient and require an extra processing step of ligation.SUMMARY OF THE INVENTION

[0006] The mRNA complex described in this disclosure can impart desirable properties of specific nucleotide modifications onto the mRNA efficiently, without the need for labor- intensive ligation. For example, the mRNA complex in this disclosure can be stabilized with oligonucleotides that are hybridized to specific regions of an mRNA. The stabilized mRNA complex of this disclosure has an increased half-life.

[0007] Accordingly, provided herein is an mRNA complex comprising an mRNA molecule and at least one engineered oligonucleotide hybridized to the mRNA molecule, wherein said engineered oligonucleotide is hereafter referred to as the Str-O-Nuc. Further provided herein is an mRNA complex comprising an mRNA molecule followed by at least one Plus-Nuc at the mRNA’s 3’ end, 3’UTR, or poly(A) tail. Further provided herein is an mRNA complex comprising an mRNA molecule linked to at least one Str-O-Nuc at the mRNA’s 3’ end, 3’UTR, or poly (A) tail.

[0008] The scope of this disclosure includes any combination of inventive features disclosed in the preceding paragraphs of this section or the paragraphs of the following sections.

[0009] These, as well as other components, steps, features, objects, benefits, and advantages, will become apparent after reviewing the detailed description of illustrative embodiments, the accompanying drawings, and the claims.BRIEF DESCRIPTION OF DRAWINGS

[0010] The drawings of this disclosure are illustrative examples. They do not illustrate all examples. Other examples may be used in addition or instead. Details that may be apparent or unnecessary may be omitted to save space or for more effective illustration.Ref. ParB 24001; 07800.002W01Some examples may be practiced with additional components or steps and / or without all the illustrated components or steps. When the same numeral appears in different drawings, it refers to the same or similar components or steps. The drawings are not necessarily to scale.

[0011] For a further understanding of the present disclosure's nature, objects, and advantages, reference should be made to the following detailed description, read in conjunction with this disclosure's drawings.

[0012] Figure 1. Figure 1 (FIG. 1A-1E) illustrates exemplary mRNA complexes of this disclosure.

[0013] Figure 2. Figure 2 (FIG. 2A-2E) illustrates exemplary mRNA complexes of this disclosure.

[0014] Figure 3. Figure 3(FIG. 3A-3E) illustrates exemplary mRNA complexes of this disclosure.

[0015] Figure 4. Figure 4 illustrates an exemplary mRNA complex of this disclosure.

[0016] Figure 5. Figure 5 depicts results as described in Example 1.

[0017] Figure 6. Figure 6 depicts results as described in Example 2.

[0018] Figure 7. Figure 7 depicts aspects of the disclosure as described in Example 3.

[0019] Figure 8. Figure 8 depicts results as described in Example 3.

[0020] Figure 9. Figure 9 depicts aspects of the disclosure as described in Example 4.

[0021] Figure 10. Figure 10 depicts results as described in Example 4.

[0022] Figure 11. Figure 11 depicts results as described in Example 5.

[0023] Figure 12. Figure 12 depicts results as described in Example 6.

[0024] Figure 13. Figure 13 depicts results as described in Example 7.

[0025] Figure 14. Figure 14 depicts results as described in Example 8.

[0026] Figure 15. Figure 15 depicts results as described in Example 9.

[0027] Figure 16. Figure 16 depicts results as described in Example 10.

[0028] Figure 17. Figure 17 depicts results as described in Example 11.

[0029] Figure 18. Figure 18 depicts results as described in Example 11.

[0030] Figure 19. Figure 19 depicts results as described in Example 12.

[0031] Figure 20. Figure 20 depicts results as described in Example 13.Ref. ParB 24001; 07800.002W01

[0032] Figure 21. Figure 21 depicts results as described in Example 14.

[0033] Figure 22. Figure 22 depicts results as described in Example 15.

[0034] Figure 23. Figure 23 depicts results as described in Example 16.

[0035] Figure 24. Figure 24 depicts results as described in Example 17.

[0036] Figure 25. Figure 25 depicts results as described in Example 17.

[0037] Figure 26. Figure 26 depicts results as described in Example 18.

[0038] Figure 27. Figure 27 depicts aspects of the disclosure as described in Example 19.

[0039] Figure 28. Figure 28 depicts results as described in Example 19.

[0040] Figure 29. Figure 29 depicts results as described in Example 19.

[0041] Figure 30. Figure 30 depicts results as described in Example 20.

[0042] Figure 31. Figure 31 depicts results as described in Example 21.

[0043] Figure 32. Figure 32 depicts results as described in Example 22.

[0044] Figure 33. Figure 33 depicts aspects of the disclosure as described in Example23.

[0045] Figure 34. Figure 34 depicts results as described in Example 23.

[0046] Figure 35. Figure 35 depicts aspects of the disclosure as described in Example24.

[0047] Figure 36. Figure 36 depicts results as described in Example 24.DETAILED DESCRIPTION

[0048] Provided herein is an mRNA complex comprising an mRNA molecule and at least one engineered oligonucleotide hybridized to the mRNA molecule, wherein said engineered oligonucleotide is hereafter referred to as the Str-O-Nuc.

[0049] Also provided herein is an mRNA complex comprising an mRNA molecule and at least one engineered oligonucleotide hybridized to the mRNA molecule, wherein the mRNA molecule comprises a coding sequence (CDS) and a 3' untranslated region (UTR), and optionally a poly(A) tail, wherein the engineered oligonucleotide is referred to as the Str-O-Nuc, wherein the Str-O-Nuc is at least partially hybridized to the 3' end of the mRNA molecule.Ref. ParB 24001; 07800.002W01

[0050] Also provided herein is an mRNA complex comprising an mRNA molecule and at least one engineered oligonucleotide hybridized to the mRNA molecule, wherein the mRNA molecule comprises a coding sequence (CDS) and a 3' untranslated region (UTR), and optionally a poly(A) tail, wherein the engineered oligonucleotide is referred to as the Str-O-Nuc, wherein the Str-O-Nuc is at least partially hybridized to the 3' UTR of the mRNA molecule.

[0051] In certain embodiments, the mRNA molecule further comprises at least one additional nucleotide following the end of 3' UTR, which at least one additional nucleotide following the end of the 3'UTR is referred to as the Plus-Nuc,

[0052] In certain embodiments, the mRNA molecule is an mRNA molecule engineered to have no poly(A) tail, wherein a Str-O-Nuc is hybridized to the 3' end of the mRNA molecule, wherein the Str-O-Nuc contains a poly(A) tail, at least one additional nucleotide is linked to the end of the Str-O-Nuc's poly(A) tail, and the Str-O-Nuc is oriented in the forward direction.

[0053] In certain embodiments, the Str-O-Nuc is partially or substantially completely hybridized to the mRNA molecule.

[0054] In certain embodiments, the Str-O-Nuc is hybridized to a specific sequence on the mRNA such that at least 50% of the hybridized section of the Str-O-Nuc sequence is complementary to the said sequence on the mRNA.

[0055] In certain embodiments, the Str-O-Nuc is an oligonucleotide engineered to impart double-strandedness to the mRNA complex.

[0056] In certain embodiments, the mRNA molecule to be hybridized to the Str-O-Nuc has no poly (A) tail.

[0057] In certain embodiments, the mRNA molecule is an mRNA molecule engineered to have no poly (A) tail.

[0058] In certain embodiments, the Str-O-Nuc is hybridized to the mRNA molecule’s 3 ’UTR.

[0059] In certain embodiments, the Str-O-Nuc contains a poly(A) tail.

[0060] Regarding the term “poly(A) tail”, this term should not be interpreted as limiting the location of the poly(A) sequence to the extreme 3 ’-end of the oligonucleotide, e.g., of the Str-O-Nuc. Rather, as described herein, the poly(A) tract may also be located in positions other than the extreme 3 ’-end of the oligonucleotide, e.g., of the Str-O-Nuc.Ref. ParB 24001; 07800.002W01

[0061] In certain embodiments, the Str-O-Nuc contains a poly(A) tail, wherein the poly(A) tail does not comprise modified adenine nucleotides.

[0062] In certain embodiments, the Str-O-Nuc contains a poly(A) tail, wherein the poly(A) tail is comprised of at least one adenine nucleotide that has been modified with a 2’-H, 2’-OH, 2’-OMe, 2’-M0E, 2’-F, P-S linkage, 3’-inv-dT, 3’-ddC, LNA or phosphorothioate linkages modification.

[0063] In certain embodiments, the Str-O-Nuc contains a poly(A) tail, wherein the poly(A) tail is comprised of at least one adenine nucleotide that has been modified with a 2’-0Me.

[0064] In certain embodiments, the Str-O-Nuc contains a poly(A) tail, wherein the poly(A) tail is comprised of at least one adenine nucleotide that has been modified with a 2’ -OH.

[0065] In certain embodiments, a poly(A) tail is hybridized to the Str-O-Nuc.

[0066] In certain embodiments, a Str-O-Nuc is hybridized to the 3’ end of an mRNA and contains a loop-back end blocker, and wherein said mRNA complex has a half-life greater than an unmodified mRNA.

[0067] In certain embodiments, a Str-O-Nuc is hybridized to the 3’ end of an mRNA and contains a splinted end blocker that also hybridizes to another engineered oligonucleotide and wherein said mRNA complex has a half-life greater than an unmodified mRNA.

[0068] In certain embodiments, the mRNA complex further comprises a targeting moiety, and wherein the at least one Str-O-Nuc is conjugated to a targeting moiety.

[0069] In certain embodiments, the targeting moiety is a sugar, a small molecule, a peptide, an antibody, an antibody fragment, a nanobody protein, a mini protein, an anti- CD5 antibody, a nucleic acid, or a combination thereof.

[0070] In certain embodiments, the targeting moiety is GalNAc, a C16, a peptide, IgG, Fab, VHH, svFv, an anti-CD5 antibody, a nucleic acid, cholesterol or a combination thereof.

[0071] In certain embodiments, the targeting moiety is GalNAc, a C16, a peptide, IgG, Fab, VHH, svFv, an anti-CD5 antibody, a nucleic acid, or a combination thereof.

[0072] In certain embodiments, the Str-O-Nuc has a length in a range of 1 nt to 200 nt, or 1 nt to 50 nt, or 2 nt to 25 nt, or 40 nt to 50 nt, or 50 nt to 75 nt, or 75 nt to 100 nt, or 100Ref. ParB 24001; 07800.002W01 nt to 125 nt, or 100 nt to 150 nt, or 125 nt to 150 nt, or 150 nt to 175 nt, or 150 nt to 200 nt, or 175 nt to 200 nt.

[0073] In certain embodiments, the Str-O-Nuc comprises a 2’ -OH.

[0074] In certain embodiments, the Str-O-Nuc comprises a 2’-H, 2’-OH, 2’-0Me, 2’- MOE, 2’-F, P-S linkage, 3’-inv-dT, 3’-ddC modification, LNA or phosphorothioate linkages.

[0075] In certain embodiments, the Str-O-Nuc comprises a sequence complementary to some part of the mRNA, wherein said sequence has a length in a range of 2 nt to 50 nt.

[0076] In certain embodiments, the Str-O-Nuc comprises a sequence complementary to some part of the mRNA, wherein said sequence has a length in a range of 2 nt to 75 nt.

[0077] In certain embodiments, the Str-O-Nuc comprises a conjugated protein, a peptide, a nucleic acid, a lipid, a sugar, or a small molecule.

[0078] In certain embodiments, the Str-O-Nuc comprises a modified phosphodiester backbone.

[0079] In certain embodiments, the Str-O-Nuc comprises a modified phosphorothioate linkage.

[0080] In certain embodiments, the Str-O-Nuc comprises a partially or substantially complete 100% uridine modification.

[0081] In certain embodiments, the Str-O-Nuc comprises a partially or substantially complete 100% thymidine substitution, and wherein the thymidine nucleotides replaced the uridine nucleotides.

[0082] In certain embodiments, the Str-O-Nuc comprises a partially or substantially complete 100% 5 -methyl cytosine substitution, and wherein the 5 -methylcytosine nucleotides replaced the cytosine nucleotides.

[0083] Certain embodiments provide a composition comprising an mRNA complex of described herein.

[0084] Certain embodiments provide a composition comprising an mRNA complex described herein, a dendrimer, an exosome, a lipid nanoparticle, a liposome-like nanoparticle, a lipoprotein particle, a lipid-polymer hybrid nanoparticle, a nanostructured lipid carrier, a platelet membrane-coated nanoparticle, a protein-based nanoparticle, aRef. ParB 24001; 07800.002W01 polymer, a polymeric nanoparticle, a protamine, a solid nanoparticle, or a virus-like particle.

[0085] Certain embodiments provide a pharmaceutical composition, comprising an mRNA complex described herein.

[0086] Certain embodiments provide a pharmaceutical composition comprising an mRNA complex described herein, a dendrimer, an exosome, a lipid nanoparticle, a liposome-like nanoparticle, a lipoprotein particle, a lipid-polymer hybrid nanoparticle, a nanostructured lipid carrier, a platelet membrane -coated nanoparticle, a protein-based nanoparticle, a polymer, a polymeric nanoparticle, a protamine, a solid nanoparticle, or a virus-like particle.

[0087] Certain embodiments provide a method for treating an individual, comprising administering an effective amount of a pharmaceutical composition comprising an mRNA complex described herein to an individual.

[0088] Certain embodiments provide a method for treating an individual, comprising administering an effective amount of a pharmaceutical composition comprising an mRNA complex described herein to an individual, wherein the pharmaceutical composition is administered by an intravenous route.

[0089] Certain embodiments provide a method for treating an individual, comprising administering an effective amount of a pharmaceutical composition comprising an mRNA complex described herein to an individual, wherein the pharmaceutical composition is administered by an intramuscular route.

[0090] Certain embodiments provide a method for treating an individual, comprising administering an effective amount of a pharmaceutical composition comprising an mRNA complex described herein to an individual, wherein the pharmaceutical composition is administered by a subcutaneous route.

[0091] Certain embodiments provide a method for treating an individual, comprising administering an effective amount of a composition comprising an mRNA complex as described herein, a dendrimer, an exosome, a lipid nanoparticle, a liposome-like nanoparticle, a lipoprotein particle, a lipid-polymer hybrid nanoparticle, a nanostructured lipid carrier, a platelet membrane -coated nanoparticle, a protein-based nanoparticle, a polymer, a polymeric nanoparticle, a protamine, a solid nanoparticle, or a virus-like particle.Ref. ParB 24001; 07800.002W01

[0092] Certain embodiments provide any combination of features of the mRNA complexes, the compositions comprising the mRNA complexes, methods of manufacturing of the mRNA complexes and the compositions, and / or methods of using the mRNA complexes for treatment, which are disclosed herein.

[0093] Embodiments from claims

[0094] Certain embodiments provide an mRNA complex comprising an mRNA molecule and at least one engineered oligonucleotide hybridized to the mRNA molecule, wherein said engineered oligonucleotide is hereafter referred to as the Str-O-Nuc.

[0095] Certain embodiments provide an mRNA complex comprising an mRNA molecule containing a poly(A) tail, wherein there is at least one Plus-Nuc following the end of the mRNA’s poly (A) tail.

[0096] Certain embodiments provide an mRNA complex comprising an mRNA molecule and at least one Str-O-Nuc linked to the end of the mRNA’s poly(A) tail.

[0097] In certain embodiments, the at least one Str-O-Nuc is partially or substantially completely hybridized to the mRNA molecule, and wherein said mRNA complex has: a half-life greater than an unmodified mRNA or an un-engineered mRNA, a more durable response compared to an unmodified mRNA, and / or a more durable response compared to an un-engineered mRNA.

[0098] In certain embodiments, the at least one Str-O-Nuc is linked to the mRNA molecule, and wherein said mRNA complex has: a half-life greater than an unmodified mRNA or an un-engineered mRNA, a more durable response compared to an unmodified mRNA, and / or a more durable response compared to an un-engineered mRNA.

[0099] In certain embodiments, the mRNA molecule contains a poly(A) tail, wherein there is at least one Plus-Nuc following the end of the mRNA’s poly(A) tail, and wherein said mRNA complex has: a half-life greater than an unmodified mRNA or an un-engineered mRNA, a more durable response compared to an unmodified mRNA, and / or a more durable response compared to an un-engineered mRNA.

[0100] In certain embodiments, the mRNA does not comprise a poly(A) tail, wherein the at least one Str-O-Nuc is partially or substantially completely hybridized to the mRNA molecule, and wherein said mRNA complex is easier to manufacture than an mRNA complex comprising a poly(A) tail and not comprising a Str-O-Nuc.Ref. ParB 24001; 07800.002W01

[0101] In certain embodiments, the Str-O-Nuc is hybridized to a specific sequence on the mRNA such that at least 50% of the hybridized section of the Str-O-Nuc sequence is complementary to the said sequence on the mRNA.

[0102] In certain embodiments, the Str-O-Nuc is hybridized to a specific sequence on the mRNA such that at least 50% of the hybridized section of the Str-O-Nuc sequence is complementary to the said sequence on the mRNA, and wherein the specific sequence can be located anywhere on the mRNA complex.

[0103] In certain embodiments, the Str-O-Nuc is hybridized to a specific sequence on the mRNA such that at least 50% of the hybridized section of the Str-O-Nuc sequence is complementary to the said sequence on the mRNA, and wherein the specific sequence can be located before the start of the mRNA’s poly(A) tail.

[0104] In certain embodiments, the Str-O-Nuc is hybridized to a specific sequence within the mRNA’s poly(A) tail.

[0105] In certain embodiments, the Str-O-Nuc is an oligonucleotide engineered to impart double-strandedness to the mRNA complex.

[0106] In certain embodiments, the mRNA molecule to be hybridized to the Str-O- Nuc has no poly (A) tail.

[0107] In certain embodiments, the mRNA molecule is an mRNA molecule engineered to have no poly (A) tail.

[0108] In certain embodiments, the Str-O-Nuc is hybridized to the mRNA molecule’s 3’UTR.

[0109] In certain embodiments, the Str-O-Nuc is hybridized to a nucleotide within the region of the 3’UTR following the coding sequence at the 3 ’end.

[0110] In certain embodiments, the Str-O-Nuc contains a poly(A) tail.

[0111] In certain embodiments, the Str-O-Nuc contains at least two poly(A) tails, wherein at least one poly(A) tail is in the forward direction, and wherein at least one poly(A) tail is in the reverse direction.

[0112] In certain embodiments, the Str-O-Nuc contains a poly(A) tail, wherein the poly(A) tail does not comprise modified adenine nucleotides.

[0113] In certain embodiments, the Str-O-Nuc contains a poly(A) tail, wherein the poly(A) tail is comprised of at least one adenine nucleotide that has been modified with aRef. ParB 24001; 07800.002W012’-H, 2’-0H, 2’-0Me, 2’-M0E, 2’-F, P-S linkage, 3’-inv-dT, 3’-ddC, LNA or phosphorothioate linkages modification.

[0114] In certain embodiments, the Str-O-Nuc contains a poly(A) tail, wherein the poly(A) tail is comprised of at least one adenine nucleotide that has been modified with a 2’-0Me.

[0115] In certain embodiments, the Str-O-Nuc contains a poly(A) tail, wherein the poly(A) tail is comprised of at least one adenine nucleotide that has been modified with a 2’ -OH.

[0116] In certain embodiments, the mRNA molecule is an mRNA molecule with a poly(A) tail, wherein a first Str-O-Nuc is linked to the poly(A) tail of the mRNA molecule, wherein a second Str-O-Nuc containing a poly(A) tail is partially or fully hybridized to the first Str-O-Nuc, at least one additional nucleotide is linked to the end of the second Str-O- Nuc’ s poly(A) tail, and the second Str-O-Nuc is oriented in the reverse direction.

[0117] In certain embodiments, the mRNA molecule is an mRNA molecule with a poly(A) tail, wherein a first Str-O-Nuc is linked to the poly(A) tail of the mRNA molecule, wherein a second Str-O-Nuc containing a poly(A) tail is partially or fully hybridized to the first Str-O-Nuc, at least one additional nucleotide is linked to the end of the second Str-O- Nuc’ s poly(A) tail, and the second Str-O-Nuc is oriented in the forward direction.

[0118] In certain embodiments, the mRNA molecule is an mRNA molecule with a poly(A) tail, wherein a first Str-O-Nuc is linked to the poly(A) tail of the mRNA molecule, wherein a second Str-O-Nuc is partially hybridized to the first Str-O-Nuc and contains a loop-back end blocker.

[0119] In certain embodiments, the mRNA molecule is an mRNA molecule with a poly(A) tail that is followed by a Plus-Nuc, wherein a Str-O-Nuc containing a poly(A) tail is partially or fully hybridized to the Plus-Nuc, at least one additional nucleotide is linked to the end of the Str-O-Nuc’ s poly(A) tail, and the Str-O-Nuc is oriented in the reverse direction.

[0120] In certain embodiments, the mRNA molecule is an mRNA molecule with a poly(A) tail that is followed by a Plus-Nuc, wherein a Str-O-Nuc containing a poly(A) tail is partially or fully hybridized to the Plus-Nuc, at least one additional nucleotide is linked to the end of the Str-O-Nuc’ s poly(A) tail, and the Str-O-Nuc is oriented in the forward direction.Ref. ParB 24001; 07800.002W01

[0121] In certain embodiments, the mRNA molecule is an mRNA molecule with a poly(A) tail that is followed by a Plus-Nuc, wherein a Str-O-Nuc is partially hybridized to the Plus-Nuc and contains a loop-back end blocker.

[0122] In certain embodiments, a Str-O-Nuc is hybridized to the 3’ end of an mRNA and contains a loop-back end blocker, and wherein said mRNA complex has a half-life greater than an unmodified mRNA or an un-engineered mRNA, a more durable response compared to an unmodified mRNA, and / or a more durable response compared to an unengineered mRNA.

[0123] In certain embodiments, a Str-O-Nuc is hybridized to a Plus-Nuc and contains a loop-back end blocker, and wherein said mRNA complex has a half-life greater than an unmodified mRNA or an un-engineered mRNA, a more durable response compared to an unmodified mRNA, and / ora more durable response compared to an un-engineered mRNA.

[0124] In certain embodiments, a Str-O-Nuc is hybridized to another Str-O-Nuc and contains a loop-back end blocker, and wherein said mRNA complex has a half-life greater than an unmodified mRNA or an un-engineered mRNA, a more durable response compared to an unmodified mRNA, and / or a more durable response compared to an un- engineered mRNA.

[0125] In certain embodiments, a Str-O-Nuc is hybridized to the 3’ end of an mRNA and contains a splinted end blocker that also hybridizes to another engineered oligonucleotide and wherein said mRNA complex has a half-life greater than an unmodified mRNA or an un-engineered mRNA, a more durable response compared to an unmodified mRNA, and / or a more durable response compared to an un-engineered mRNA.

[0126] In certain embodiments, the mRNA complex further comprises a targeting moiety, and wherein the at least one Str-O-Nuc is conjugated to a targeting moiety.

[0127] In certain embodiments, the targeting moiety is a sugar, a small molecule, a peptide, an antibody, an antibody fragment, a nanobody protein, a mini protein, an anti- CD5 antibody, a nucleic acid, or a combination thereof.

[0128] In certain embodiments, the targeting moiety is GalNAc, a C16, a peptide, IgG, Fab, VHH, svFv, an anti-CD5 antibody, a nucleic acid, cholesterol or a combination thereof.Ref. ParB 24001; 07800.002W01

[0129] In certain embodiments, the Str-O-Nuc has a length in a range of 1 nt to 200 nt, or 1 nt to 50 nt, or 2 nt to 25 nt, or 40 nt to 50 nt, or 50 nt to 75 nt, or 75 nt to 100 nt, or 100 nt to 125 nt, or 100 nt to 150 nt, or 125 nt to 150 nt, or 150 nt to 175 nt, or 150 nt to 200 nt, or 175 nt to 200 nt.

[0130] In certain embodiments, the Str-O-Nuc has a length of 15 nt, at least 16 nt, at least 17 nt, at least 18 nt, at least 20 nt, at least 25 nt, at least 30 nt, at least 35 nt, at least 40 nt, at least 45 nt, at least 50 nt, at least 55 nt, at least 60 nt.

[0131] In certain embodiments, the Plus-Nuc has a length in a range of 1 nt to 200 nt, or 1 nt to 50 nt, or 2 nt to 25 nt, or 40 nt to 50 nt, or 50 nt to 75 nt, or 75 nt to 100 nt, or 100 nt to 125 nt, or 100 nt to 150 nt, or 125 nt to 150 nt, or 150 nt to 175 nt, or 150 nt to 200 nt, or 175 nt to 200 nt.

[0132] Th In certain embodiments, the Str-O-Nuc has a length in a range of 1 nt to 200 nt, or 1 nt to 50 nt, or 2 nt to 25 nt, or 40 nt to 50 nt, or 50 nt to 75 nt, or 75 nt to 100 nt, or 100 nt to 125 nt, or 100 nt to 150 nt, or 125 nt to 150 nt, or 150 nt to 175 nt, or 150 nt to 200 nt, or 175 nt to 200 nt.

[0133] In certain embodiments, the Plus-Nuc has a length of 15 nt, at least 16 nt, at least 17 nt, at least 18 nt, at least 20 nt, at least 25 nt, at least 30 nt, at least 35 nt, at least 40 nt, at least 45 nt, at least 50 nt, at least 55 nt, at least 60 nt.

[0134] In certain embodiments, the Str-O-Nuc has a length of 15 nt, at least 16 nt, at least 17 nt, at least 18 nt, at least 20 nt, at least 25 nt, at least 30 nt, at least 35 nt, at least 40 nt, at least 45 nt, at least 50 nt, at least 55 nt, at least 60 nt.

[0135] In certain embodiments, the Str-O-Nuc containing a poly(A) tail has a length of 15 nt, at least 16 nt, at least 17 nt, at least 18 nt, at least 20 nt, at least 25 nt, at least 30 nt, at least 35 nt, at least 40 nt, at least 45 nt, at least 50 nt, at least 55 nt, at least 60 nt.

[0136] In certain embodiments, the Str-O-Nuc not containing a poly(A) tail has a length of 15 nt, at least 16 nt, at least 17 nt, at least 18 nt, at least 20 nt, at least 25 nt, at least 30 nt, at least 35 nt, at least 40 nt, at least 45 nt, at least 50 nt, at least 55 nt, at least 60 nt.

[0137] In certain embodiments, the mRNA molecule is an mRNA molecule engineered to have no poly(A) tail, wherein a Str-O-Nuc is hybridized to the 3’UTR of the mRNA molecule, wherein the Str-O-Nuc contains a poly(A) tail, at least one additionalRef. ParB 24001; 07800.002W01 nucleotide is linked to the end of the Str-O-Nuc’s poly(A) tail, and the Str-O-Nuc is oriented in the reverse direction.

[0138] In certain embodiments, the mRNA molecule is an mRNA molecule engineered to have no poly(A) tail, wherein a Str-O-Nuc is hybridized to the 3’UTR of the mRNA molecule, wherein the Str-O-Nuc has a length of at least 15 nt, the Str-O-Nuc contains a poly(A) tail, the Str-O-Nuc and the poly(A) tail have a combined length of at least 30 nt, and at least 6 additional nucleotides are hybridized to the end of the Str-O- Nuc’ s poly(A) tail, wherein the at least 6 nt contain phosphorothioate linkages and 2’- MOE modifications, and the Str-O-Nuc is oriented in the forward direction.

[0139] In certain embodiments, the mRNA molecule is an mRNA molecule engineered to have no poly(A) tail, wherein a Str-O-Nuc is hybridized to the 3’UTR of the mRNA molecule, wherein the Str-O-Nuc contains a poly(A) tail, at least one additional nucleotide is linked to the end of the Str-O-Nuc’s poly(A) tail, and the Str-O-Nuc is oriented in the forward direction.

[0140] In certain embodiments, the mRNA molecule is an mRNA molecule engineered to have no poly(A) tail, wherein a Str-O-Nuc is hybridized to the 3’UTR of the mRNA molecule, wherein the Str-O-Nuc has a length of at least 18 nt, the Str-O-Nuc contains a poly(A) tail, the Str-O-Nuc and the poly(A) tail have a combined length of at least 30 nt, and at least 6 additional nucleotides are hybridized to the end of the Str-O- Nuc’s poly(A) tail, wherein the at least 6 nt contain phosphorothioate linkages and 2’- MOE modifications, and the Str-O-Nuc is oriented in the forward direction.

[0141] In certain embodiments, the mRNA molecule is an mRNA molecule engineered to have no poly(A) tail, wherein a Str-O-Nuc is hybridized to the 3’UTR of the mRNA molecule, wherein the Str-O-Nuc has a length of at least 18 nt, the Str-O-Nuc contains a poly(A) tail, the Str-O-Nuc and the poly(A) tail have a combined length of at least 33 nt, at least 6 nt is hybridized to the end of the Str-O-Nuc’s poly(A) tail, wherein the at least 6 nt contain phosphorothioate linkages and 2’-M0E modifications, and the Str- O-Nuc is oriented in the reverse direction.

[0142] In certain embodiments, the Str-O-Nuc comprises a 2’ -OH.

[0143] In certain embodiments, the Str-O-Nuc comprises a 2’-H, 2’-OH, 2’-0Me, 2’-MOE, 2’-F, P-S linkage, 3’-inv-dT, 3’-ddC modification, LNA or phosphorothioate linkages.Ref. ParB 24001; 07800.002W01

[0144] In certain embodiments, the Str-O-Nuc comprises a sequence complementary to some part of the mRNA, wherein said sequence has a length in a range of 2 nt to 50 nt.

[0145] In certain embodiments, the Str-O-Nuc comprises a sequence complementary to some part of the mRNA, wherein said sequence has a length in a range of 2 nt to 75 nt.

[0146] In certain embodiments, the Str-O-Nuc comprises a conjugated protein, a peptide, a nucleic acid, a lipid, a sugar, or a small molecule.

[0147] In certain embodiments, the Str-O-Nuc comprises a modified phosphodiester backbone.

[0148] In certain embodiments, the Str-O-Nuc comprises a modified phosphorothioate linkage.

[0149] In certain embodiments, the Str-O-Nuc comprises a partially or substantially complete 100% uridine modification.

[0150] In certain embodiments, the Str-O-Nuc comprises a partially or substantially complete 100% thymidine substitution, and wherein the thymidine nucleotides replaced the uridine nucleotides.

[0151] In certain embodiments, the mRNA further comprises a readenylation element, e.g., a virus-derived 3' readenylation element downstream of the 3' UTR.

[0152] In certain embodiments, the sequence of the Str-O-Nuc that hybridizes to the mRNA comprises an internal poly(A) tract within the sequence that hybridizes to the mRNA, e.g. , a 60-nt poly(A) tract.

[0153] In certain embodiments, the mRNA molecule encodes for an enzyme, a second RNA molecule that serves as a reverse transcription template and which does not have a cap or a poly(A) tail, wherein the second RNA molecule contains a Str-O-Nuc hybridized at the 3’ end of the 3 ’UTR, and wherein the mRNA complex has a half-life greater than an unmodified mRNA or an un-engineered mRNA, a more durable response compared to an unmodified mRNA or an un-engineered mRNA, and / or a more durable response compared to an unmodified mRNA or an un-engineered mRNA.

[0154] Certain embodiments provide a composition, comprising an mRNA complex described herein.

[0155] Certain embodiments provide a composition, comprising an mRNA complex described herein, a dendrimer, an exosome, a lipid nanoparticle, a liposome-like nanoparticle, a lipoprotein particle, a lipid-polymer hybrid nanoparticle, a nanostructuredRef. ParB 24001; 07800.002W01 lipid carrier, a platelet membrane -coated nanoparticle, a protein-based nanoparticle, a polymer, a polymeric nanoparticle, a protamine, a solid nanoparticle, or a virus-like particle.

[0156] Certain embodiments provide a pharmaceutical composition, comprising an mRNA complex described herein.

[0157] Certain embodiments provide a pharmaceutical composition, comprising an mRNA complex described herein, a dendrimer, an exosome, a lipid nanoparticle, a liposome-like nanoparticle, a lipoprotein particle, a lipid-polymer hybrid nanoparticle, a nanostructured lipid carrier, a platelet membrane -coated nanoparticle, a protein-based nanoparticle, a polymer, a polymeric nanoparticle, a protamine, a solid nanoparticle, or a virus-like particle.

[0158] Certain embodiments provide a method for treating an individual, comprising administering an effective amount of a pharmaceutical composition comprising an mRNA complex described herein to an individual.

[0159] Certain embodiments provide a method for treating an individual, comprising administering an effective amount of a pharmaceutical composition comprising an mRNA complex described herein to an individual, wherein the pharmaceutical composition is administered by an intravenous route.

[0160] Certain embodiments provide a method for treating an individual, comprising administering an effective amount of a pharmaceutical composition comprising an mRNA complex described herein to an individual, wherein the pharmaceutical composition is administered by an intramuscular route.

[0161] Certain embodiments provide a method for treating an individual, comprising administering an effective amount of a pharmaceutical composition comprising an mRNA complex described herein to an individual, wherein the pharmaceutical composition is administered by a subcutaneous route.

[0162] Certain embodiments provide a method for treating an individual, comprising administering an effective amount of a composition comprising an mRNA complex described herein, a dendrimer, an exosome, a lipid nanoparticle, a liposome-like nanoparticle, a lipoprotein particle, a lipid-polymer hybrid nanoparticle, a nanostructured lipid carrier, a platelet membrane -coated nanoparticle, a protein-based nanoparticle, aRef. ParB 24001; 07800.002W01 polymer, a polymeric nanoparticle, a protamine, a solid nanoparticle, or a virus-like particle.

[0163] Certain embodiments provide any combination of features of the mRNA complexes, the compositions comprising the mRNA complexes, methods of manufacturing of the mRNA complexes and the compositions, and / or methods of using the mRNA complexes for treatment, which are disclosed herein.

[0164] Certain embodiments provide the use of the mRNA complexes described herein for the prophylactic or therapeutic treatment of a disease in a subject.

[0165] Certain embodiments provide the mRNA complexes described herein for use in medical therapy.

[0166] Certain embodiments provide the use of the mRNA complexes described herein to prepare a medicament for the treatment of a disease in a subject.

[0167] Reference will now be made in detail to certain embodiments of the invention, examples of which are illustrated in the accompanying structures and formulas. While the invention will be described in conjunction with the enumerated embodiments, it will be understood that they are not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the scope of the invention as defined by the claims.

[0168] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. The invention is in no way limited to the methods and materials described. Except in the examples, or where otherwise expressly indicated, all numerical quantities in this description indicating amounts of material or conditions of reaction and / or use are to be understood as modified by the word "about" in describing the broadest scope of the invention. Practice within the numerical limits stated is generally preferred. Also, unless expressly stated to the contrary, percent, "parts of," and ratio values are by weight; the description of a group or class of materials as suitable or preferred for a given purpose in connection with the invention implies that mixtures of any two or more of the members of the group or class are equally suitable or preferred; description of constituents in chemical terms refers to the constituents at the time of addition to any combination specified in the description, and does not necessarily preclude chemical interactions among the constituents of a mixture once mixed; the first definition of an acronym or otherRef. ParB 24001; 07800.002W01 abbreviation applies to all subsequent uses herein of the same abbreviation and applies mutatis mutandis to normal grammatical variations of the initially defined abbreviation; and, unless expressly stated to the contrary, measurement of a property is determined by the same technique as previously or later referenced for the same property.

[0169] It must also be noted that, as used in the specification and the appended claims, the singular form "a," "an," and "the" comprise plural referents unless the context indicates otherwise. That is, reference to a component in the singular is intended to include a plurality of components. That is, in this disclosure, the indefinite article “a” and the phrases “one or more” and “at least one” are synonymous and mean “at least one.” Similarly, an element preceded by an “a” or an “an” does not, without further constraints, preclude the existence of additional elements of the identical type.

[0170] As used herein, the term “about” means that the amount or value in question may be the specific value designated or some other value in its neighborhood. For example, the term “about” denoting a particular value represents a range within ± 5% of the value. For example, the phrase “about 100” indicates a range of 100 ± 5, z.e., the value is in the range of 95 to 105. Generally, when the term “about” is used, it can be expected that similar results or effects according to the invention can be obtained, for example, within a range of ± 5% of the indicated value.

[0171] As used herein, the term “and / or” means that all or only one of the elements of said group may be present. For example, “A and / or B” shall mean “only A, or only B, or both A and B”. In the case of “only A,” the term also covers the possibility that B is absent, z.e., “only A, but not B.”

[0172] Unless otherwise stated, all measurements, values, ratings, positions, magnitudes, sizes, and other specifications outlined in this specification, including in the following claims, are approximate, not exact. They are intended to have a reasonable range consistent with the functions to which they relate and with what is customary in the art to which they pertain.

[0173] As will be understood by one skilled in the art, for any purposes, such as providing a written description, all ranges disclosed herein also encompass any possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range to be broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, eachRef. ParB 24001; 07800.002W01 range discussed herein can be readily broken down into a lower third, middle third, upper third, etc. As will also be understood by one skilled in the art of all languages, such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed herein. Finally, as will be understood by one skilled in the art, a range includes each member. Thus, for example, a group with 1-3 articles refers to groups with 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

[0174] While various aspects and embodiments have been disclosed herein, others will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for illustration purposes and are not intended to be limiting.

[0175] All references cited herein, including but not limited to published and unpublished applications, patents, and literature references, are incorporated herein by reference for the subject matter referenced and, in their entirety, are made a part of this specification. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and take precedence over any such contradictory material.

[0176] Concerning the use of substantially any plural and / or singular terms herein, those with skill in the art can translate from the plural to the singular and / or from the singular to the plural as appropriate to the context and / or application. The various singular / plural permutations may be expressly set forth herein for clarity.

[0177] The phrase “means for” when used in a claim is intended to and should be interpreted to embrace the corresponding structures and materials that have been described and their equivalents. Similarly, when used in a claim, the phrase “step for” is intended to and should be interpreted to embrace the corresponding acts described and their equivalents. The absence of these phrases from a claim means that the claim is not intended to and should not be interpreted as limited to these corresponding structures, materials, or acts, or to their equivalents.

[0178] In at least some of the previously described embodiments, one or more elements used in an embodiment can interchangeably be used in another embodiment unless such a replacement is not technically feasible. It will be appreciated by those skilled in the art that various other omissions, additions, and modifications may be made to theRef. ParB 24001; 07800.002W01 methods and structures described herein without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the disclosed subject matter.

[0179] The scope of protection is limited solely by the following claims. That scope is intended and should be interpreted to be as broad as is consistent with the ordinary meaning of the language used in the claims when interpreted in light of this specification and the prosecution history that follows, except where specific meanings have been set forth, and to encompass all structural and functional equivalents.

[0180] It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). Those within the art will further understand that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and, in the absence of such recitation, no such intent is present. For example, as an aid to understanding, the following appended claims may use the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and / or “an” should be interpreted to mean “at least one” or “one or more”); the same holds for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In those instances where aRef. ParB 24001; 07800.002W01 convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”

[0181] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also described in terms of any individual member or subgroup of members of the Markush group.

[0182] Nothing that has been said or illustrated is intended or should be interpreted to cause a dedication of any component, step, feature, object, benefit, advantage, or equivalent to the public, regardless of whether it is or is not recited in the claims.

[0183] The abstract is provided to help the reader quickly ascertain the nature of the technical disclosure. It is submitted with the understanding that it will not be used to interpret or limit the scope or meaning of the claims. In addition, various features in the detailed description are grouped in various embodiments to streamline the disclosure. This method of disclosure should not be interpreted as requiring claimed embodiments to require more features than are expressly recited in each claim. Instead, as the following claims reflect, inventive subject matter lies in less than all features of a single disclosed embodiment. Thus, the following claims are hereby incorporated into the detailed description, with each claim standing on its own as separately claimed subject matter.

[0184] The scope of this disclosure includes any combination of inventive features disclosed in the preceding paragraphs of this section or the paragraphs of the following sections.

[0185] The term “comprising” is synonymous with “including,” “having,” “containing,” or “characterized by.” These terms are inclusive and open-ended and do not exclude additional, unrecited elements or method steps.Ref. ParB 24001; 07800.002W01

[0186] The phrase “consisting of’ excludes any element, step, or ingredient not specified in the claim. When this phrase appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element outlined in that clause; other elements are not excluded from the claim as a whole.

[0187] The phrase “consisting essentially of’ limits the scope of a claim to the specified materials or steps, plus those that do not materially affect the essential and novel characterise cfs) of the claimed subject matter.

[0188] The phrase “composed of’ means “including” or “consisting of.” Typically, this phrase denotes that an object is formed from a material.

[0189] Concerning the terms “comprising,” “consisting of,” and “consisting essentially of,” where one of these three terms is used herein, the presently disclosed and claimed subject matter can include the use of either of the other two terms. Similarly, the terms “comprises,” “comprising,” and any other variation in connection with a list of elements in the specification or claims indicate that the list is not exclusive and that other elements may be included. For example, “comprising A” includes “consisting of A” or “consisting essentially of A.”

[0190] The term “one or more” means “at least one,” and the term “at least one” means “one or more.” The terms “one or more” and “at least one” include “plurality” as a subset.

[0191] Relational terms such as “first,” “second,” and the like may be used solely to distinguish one entity or action from another without necessarily requiring or implying any actual relationship or order between them.

[0192] The terms “substantially,” “generally,” or “about” may be used herein to describe disclosed or claimed embodiments. The term “substantially” may modify a value or relative characteristic disclosed or claimed in the present disclosure. In such instances, “substantially” may signify that the value or relative characteristic it modifies is within ± 0%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5% or 10% of the value or relative characteristic.

[0193] It should also be appreciated that integer ranges explicitly include all intervening integers. For example, the integer range 1-10 explicitly contains 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Similarly, the range 1 to 100 contains 1, 2, 3, 4 ... 10...20. . . 50 ... 76. . . 83 . . . 97, 98, 99, 100. Similarly, when any range is called for, intervening numbers that are increments of the difference between the upper and lower limits divided by 10 can beRef. ParB 24001; 07800.002W01 taken as alternative upper or lower limits. For example, if the range is 1.1. to 2.1 the following numbers 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, and 2.0 can be selected as lower or upper limits.

[0194] In this disclosure, the following acronyms and abbreviations are used.

[0195] 3’ UTR. three prime untranslated region.

[0196] 2’-F: 2' fluoro modification.

[0197] 2’-OMe: 2'-O-methyl modification.

[0198] 2’ -MOE: 2'-O-methoxyethyl modification.

[0199] 3’ -ddC: 3’ Dideoxy cytidine modification.

[0200] 3’-inv-dT: 3’ inverted deoxythymidine modification.

[0201] P-S: phosphorothioate.

[0202] 5’ UTR: five prime untranslated region.

[0203] A: Adenine

[0204] ApoE: Apolipoprotein E.

[0205] CART : charge-altering releasable transporters.

[0206] C: Cytosine

[0207] CD: Cluster of Differentiation

[0208] DNA: deoxyribonucleic acid

[0209] dsRNA: double-stranded RNA.

[0210] EDV: enveloped delivery vehicle.

[0211] Fab: fragment antigen-binding.

[0212] GalNAc: N-Acetylgalactosamine.

[0213] GNA: glycerol nucleic acids

[0214] gRNA: guide RNA.

[0215] G: Guanine

[0216] HD-RNA: hetero-duplex RNA.

[0217] IgG: Immunoglobulin G.

[0218] LDL receptor: low-density lipoprotein receptor.

[0219] LNA: locked nucleic acidRef. ParB 24001; 07800.002W01

[0220] LNP: lipid nanoparticles.

[0221] mRNA: messenger RNA.

[0222] nt: nucleotide

[0223] OH: hydroxy modification

[0224] PNA: peptide nucleic acids

[0225] PNP: polymeric nanoparticles.

[0226] RNA: ribonucleic acid

[0227] siRNA: small interfering RNA.

[0228] Str-O-Nuc: an engineered oligonucleotide of this disclosure.

[0229] T: Thymine

[0230] Tm: thermal melting point.

[0231] TNA: threose nucleic acids

[0232] U: Uracil

[0233] VHH: Variable Heavy domain of Heavy chain

[0234] VLP: virus-like particles.

[0235] This disclosure relates to engineered oligonucleotides that can be used in the preparation of the mRNA complexes of this disclosure. The term “oligonucleotide” as used herein refers to an oligomer or polymer of either ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), as well as 2’-modified derivatives and non-naturally occurring oligonucleotides. Non-naturally occurring oligonucleotides are oligomers or polymers which contain nucleobase sequences which do not occur in nature, or species which contain functional equivalents of naturally occurring nucleobases, sugars, or intersugar linkages, like aptamers, spiegelmers, peptide nucleic acids (PNA), threose nucleic acids (TNA), locked nucleic acids (LNA), or glycerol nucleic acids (GNA). This term includes oligomers that contain the naturally occurring nucleic acid nucleobases adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U), as well as oligomers that contain base analogs or modified nucleobases. Therefore, the person skilled in the art understands that the term “oligonucleotide” comprises but is not limited to RNA, DNA and mixed oligonucleotides, antisense oligonucleotides, short interfering RNA (siRNA), microRNAs (miRNAs), aptamers and also spiegelmers.Ref. ParB 24001; 07800.002W01

[0236] Oligonucleotides can derive from a variety of natural sources such as viral, bacterial and eukaryotic DNAs and RNAs. Other oligonucleotides can be derived from synthetic sources, and include any of the multiple oligonucleotides that are being manufactured for use as research reagents, diagnostic agents or potential and definite therapeutic agents. The term includes oligomers comprising of a single strand nucleic acid or a double strand nucleic acid. The two strands of a double strand nucleic acid are defined as “sense strand” and “antisense strand”.

[0237] As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. “Complementary” sequences, as used herein, may also include, or be formed entirely from, non-Watson-Crick base pairs and / or base pairs formed from non-natural and modified nucleotides, in as far as the above requirements with respect to their ability to hybridize are fulfilled.

[0238] This includes base-pairing of the oligonucleotide or polynucleotide comprising the first nucleotide sequence to the oligonucleotide or polynucleotide comprising the second nucleotide sequence over the entire length of the first and second nucleotide sequence. Such sequences can be referred to as “fully complementary” with respect to each other herein.

[0239] This disclosure’s engineered oligonucleotides are hereafter referred to as Str-O- Nuc, and provide the following advantages.

[0240] Str-O-Nuc may impart new properties to an mRNA without impacting the sequence or chemistry of the mRNA itself. Hybridizing of a Str-O-Nuc to an mRNA may bring the Str-O-Nuc into proximity of the mRNA, thereby imparting unique features to the mRNA through sequences, structures, and / or modifications that are on the Str-O-Nuc itself.

[0241] Str-O-Nuc may be manufactured by chemical synthesis, which allows for considerable flexibility in nucleotide chemistry and functionalization by means of incorporating chemical reactive groups at well-defined positions into the Str-O-Nuc structure for downstream manipulation.Ref. ParB 24001; 07800.002W01

[0242] In contrast, the current in vitro transcription method of manufacturing mRNA is more constrained due to the requirements of the enzymatic synthesis process. Consequently, limited nucleotide chemistries can be incorporated into an mRNA, and sitespecific modifications may not be incorporated. Contaminations associated with the synthesis process are immunogenic, which has led to widespread use of modified nucleotides in the field, for example, N1 -methylpseudouridine, in the mRNA manufacturing to limit immunogenicity.

[0243] However, Str-O-Nuc may be designed to bind to a specified region of any mRNA and impart properties that are engineered into the Str-O-Nuc onto the mRNA. Such mRNA complexes, which include Str-O-Nuc hybridized to an mRNA, may be delivered using any standard mRNA delivery methods such as a dendrimer, an exosome, a lipid nanoparticle, a liposome-like nanoparticle, a lipoprotein particle, a lipid-polymer hybrid nanoparticle, a nanostructured lipid carrier, a platelet membrane -coated nanoparticle, a protein-based nanoparticle, a polymer, a polymeric nanoparticle, a protamine, a solid nanoparticle, or a virus-like particle. mRNA complexes that include a Str-O-Nuc can also be delivered without additional delivery vehicles.

[0244] The use of Str-O-Nuc may also represent a major advance in engineering mRNA complexes. The Str-O-Nuc is simple to manufacture, easy to hybridize, and readily programable with desired sequences, chemistries, or RNA secondary structures.Additionally, a Str-O-Nuc may be designed in such a way that their binding is easily reversible. For example, a Str-O-Nuc placed in the coding sequence of an mRNA may be dislodged by the ribosome. Str-O-Nuc may also be designed in such a way that their binding is stable. For example, a Str-O-Nuc designed to bind in the 3’ UTR of an mRNA may likely not dissociate if engineered to have a high enough melting temperature.

[0245] Also, since fewer immunogenic side products may be produced as a result of the chemical synthesis of a Str-O-Nuc, this disclosure’s manufacturing technique provides a considerably cleaner technology than that of the in vitro transcription of an mRNA. This may allow the possibility of using unmodified nucleotides in the accessory elements of the Str-O-Nuc, which would enable the use of RNA elements that only retain function when unmodified (e.g., naturally occurring cellular or viral regulatory elements). Examples of accessory elements may include unmodified or chemically modified nucleotides, secondary structures, scaffold for other RNAs, conjugated proteins, peptides, nucleic acids, lipids, sugars, or small molecules that impart desirable properties to the Str-O-NucsRef. ParB 24001; 07800.002W01 or the Str-O-Nuc-mRNA complexes. Further, these accessory elements may include additional structures that can incorporate a linker region. Such desirable properties, for example, may include (a) higher translation output, (b) longer mRNA and / or Str-O-Nuc durability, (c) less immunogenicity, (d) cell-type specific delivery and / or (e) translation regulation. These additional chemical structures of Str-O-Nucs, which may not be included in the mRNA structure, may contain extra nucleotides and / or chemical moieties that function to impart new properties to the mRNA by way of being localized to the mRNA through the Str-O-Nuc hybridization. In contrast, the current technologies of the background art produce mRNA with 100% N1 -Methylpseudouridine to limit the immunogenicity of the unwanted side products, which negatively impacts the function of most naturally occurring functional elements.

[0246] This disclosure’s mRNA complex, comprising Str-O-Nuc and mRNA, schematically shown in FIGs. 1-4 and 9-11, by way of example, have the following additional advantages over the current or prior-art technologies.

[0247] Immunogenicity and Re-dosing: Lipid nanoparticles may themselves be immunogenic, which may limit their tolerability in certain applications (e.g., in re-dose LNP-mRNA therapeutics). Viral-like systems may generally be limited to single-dose applications due to immune recognition of previously exposed antigens. That is, the immune system can recognize surface receptors on the VLP, which results in antibodies that bind and neutralize the VLP upon the second dose of the VLP.

[0248] This disclosure’s mRNA complex may be significantly less immunogenic than the current LNP- or VLP -based complexes. Such mRNA complex, as described herein, may be chemically modified to reduce, or prevent immune detection (e.g., with Nlm- pseudouridine on the mRNA and / or modifications to the ribose 2’ -OH on the oligonucleotide), which allows for better re-dosing and tolerability.

[0249] Manufacturing: Currently mRNA is formulated with LNPs, VLPs, or other delivery vehicles in order to protect the mRNA from degradation and also to allow for the delivery of the mRNA. Therefore, the manufacturing needs of LNP, VLPs or other delivery vehicles are considered when analyzing the manufacturing process required to create a composition containing mRNA and its delivery vehicle. Current LNP-mRNA manufacturing may require additional manufacturing overhead and steps. LNP’s size, composition, and homogeneity may need to be controlled and checked by analytical methods. Viral-like delivery methods are generally achieved by expressing the desiredRef. ParB 24001; 07800.002W01 components in a human cell. This leads to issues with (1) specificity: it must be ensured that only the desired therapeutic cargo is present in the VLP and, (2) scaling: it is difficult and costly to culture large volumes of human cells for VLP production.

[0250] This disclosure’s mRNA complex involves two components that are relatively inexpensive and can be manufactured by simple, established workflows: mRNA that is generated by in vitro transcription, and the engineered oligonucleotides, Str-O-Nucs, described herein that are generated by chemical synthesis. Once generated, the mRNA and the Str-O-Nucs may be hybridized to form the mRNA complexes, optionally purified, and administered. The purification of the mRNA complexes can be any form of purification commonly known in the art and can include HPLC, dialysis, reversed phase, ion exchange (IEX), size exclusion (SEC), hydrophobic interaction (HIC), tangential flow filtration (TFF), and affinity.

[0251] The use of Str-O-Nuc may also represent a major advance in engineering mRNA complexes. The Str-O-Nuc is simple to manufacture, easy to hybridize, and readily programable with desired sequences, chemistries, or RNA secondary structures.Additionally, a Str-O-Nuc may be designed in such a way that their binding is easily reversible. For example, a Str-O-Nuc placed in the coding sequence of an mRNA may be dislodged by the ribosome. Str-O-Nuc may also be designed in such a way that their binding is stable. For example, a Str-O-Nuc designed to bind in the 3’ UTR of an mRNA may likely not dissociate if engineered to have a high enough melting temperature.

[0252] Also, since fewer immunogenic side products may be produced as a result of the chemical synthesis of Str-O-Nuc, this disclosure’s manufacturing technique provides a considerably cleaner technology than that of the in vitro transcription of an mRNA. This may allow the possibility of using unmodified nucleotides in the accessory elements of the Str-O-Nuc, which would enable the use of RNA elements that only retain function when unmodified (e.g., naturally occurring cellular or viral regulatory elements). In contrast, the current technologies of the background art produce mRNA with 100% Nl- Methylpseudouridine to limit the immunogenicity of the unwanted side products, which negatively impacts the function of most naturally occurring functional elements.

[0253] Durability: Even for the liver-targeting applications, the mRNA complex of this disclosure, which can be delivered using the technologies described herein (e.g., using GalNAc modification), may result in more durable responses compared to the responses resulting from the use of an LNP encapsulating mRNA not of this disclosure, due to theRef. ParB 24001; 07800.002W01 stabilization imparted by the mRNA complex of this disclosure (e.g., by providing some stability in endosomes that may allow for slow release into the cytoplasm over time) and / or due to the distinct endocytosis mechanism of GalNAc conjugates (as observed for therapeutic siRNAs). Further, even for the liver-targeting applications, the mRNA complex of this disclosure, which can also be delivered as encapsulated in an LNP, may result in more durable responses compared to an LNP encapsulating mRNA not of this disclosure due to the stabilization imparted by the mRNA complex of this disclosure (e.g., by providing some stability in endosomes that may allow for slow release into the cytoplasm over time).

[0254] Stability: mRNA is known to have inherent stability issues due to its structure, current manufacturing methods, excipients, and its interaction with its delivery vehicle, thereby rendering it increasingly unstable throughout its life cycle. Therefore, new mRNA complexes and methods of making mRNA complexes with increased stability are needed.

[0255] This disclosure’s mRNA complex is advantageous over mRNA constructs of the background art because the addition of at least one Str-O-Nuc confers advantageous characteristics, e.g. stabilizing characteristics, to the mRNA. For example, an mRNA complex of this disclosure may contain one or more Str-O-Nuc that may contain one or more accessory elements. The Str-O-Nuc help confer stability to the mRNA complex resulting in increased half-life, expression, and / or increased durable response compared to the mRNA of the prior art.

[0256] In this disclosure, Str-O-Nuc may be engineered to contain accessory elements such as a m7G-cap, a 5’UTR, a 3’UTR, and / or a poly(A) tail. The mRNA complex of this disclosure may be prepared by hybridizing Str-O-Nuc that has such accessory elements to an mRNA that has no such accessory elements. For example, the mRNA of this disclosure may be engineered with no poly(A) tail. Instead, a Str-O-Nuc may be engineered to have a poly(A) tail in addition to sequences that are complementary to the 3’UTR of the mRNA. Such Str-O-Nuc may bind to the 3’-UTR and impart the properties of the poly(A) tail, which is synthesized as a part of the Str-O-Nuc, to the mRNA. Further, a Str-O-Nuc having a poly(A) tail may also be hybridized to the mRNA already having a poly(A) tail. A Str-O-Nuc having a poly(A) tail hybridized to the mRNA complex, wherein the mRNA has a poly(A) tail or the mRNA does not have a poly(A) tail, may result in at least 1 times, at least 2 times, at least 5 times, and at least 10 times increased stability of the mRNA complex as compared to a mRNA that does not contain a Str-O-Nuc.Ref. ParB 24001; 07800.002W01

[0257] The Str-O-Nuc’s poly(A) tail may also be chemically modified to make them more stable, resulting in a more stable, longer expressing mRNA complex. Additionally, it is difficult to make mRNAs with long poly(A) tails due to inefficiencies associated with template-encoded poly(A) tails as plasmids are known to be unstable when they have long poly(A) stretches. Further, extra steps are required for post-transcriptional polyadenylation, for example, with poly(A) polymerase.

[0258] This disclosure’s mRNA complex is advantageous over mRNA constructs of the background art because the addition of at least one Plus-Nuc or Str-O-Nuc confers advantageous characteristics, e.g. stabilizing characteristics, to the mRNA. For example, an mRNA complex of this disclosure may contain one or more Plus-Nuc or Str-O-Nuc. Further, an mRNA complex of this disclosure may contain one or more Plus-Nuc or Str- O-Nuc comprising a Str-O-Nuc that may contain one or more accessory elements hybridized to the Plus-Nuc or Str-O-Nuc. The Plus-Nuc or Str-O-Nuc help confer stability to the mRNA complex resulting in increased half-life, expression, and / or increased durable response compared to the mRNA of the prior art. A mRNA comprising a Plus- Nuc or Str-O-Nuc may result in at least 1 times, at least 2 times, at least 5 times, and at least 10 times increased stability of the mRNA complex as compared to a mRNA that does not contain a Plus-Nuc or Str-O-Nuc.

[0259] The mRNA may optionally contain substantially close to 100% uridine modification (e.g., N1 -methylpseudouridine or 5-methy oxyuridine). The cap can be added co-transcriptionally (e.g., using CleanCap) or post-transcriptionally (e.g., using Vaccinia Capping Enzyme + Methyltransferase). The poly(A) tail on the mRNA may be encoded into the DNA template and added during transcription, or added post-transcriptionally (e.g., using Poly(A) Polymerase).

[0260] A Str-O-Nuc may hybridize to a region of the mRNA. Generally, Str-O-Nucs may be generated by chemical synthesis and may contain a mix of chemically modified and / or unmodified nucleotides. For example, Str-O-Nucs may contain nucleotides that have one or more of 2’-H, 2 ’-OH, 2’-0Me, 2 ’-MOE, 2’-F, 2’-cEt, P-S linkage, 3’-inv-dT, 3’-ddC or LNA modifications.

[0261] This disclosure further includes mRNA complexes in which the DNA template encodes for an mRNA complex that contains a cap at the 5’ end, 5’UTR, coding sequence, 3’UTR, and a poly(A) tail as well as additional nucleotides after the poly(A) tail. This mRNA complex is generated using an in vitro transcription reaction. The additionalRef. ParB 24001; 07800.002W01 nucleotides at the end of the poly(A) tail serve as the hybridization region to which a Str- O-Nuc may bind.

[0262] Subcutaneous administration: Since mRNA-LNPs currently delivered by subcutaneous administration can be inefficient and have adverse reactions, they are generally administered by intravenous infusions. The reason for this is not entirely clear, but the large size of the LNP, typically about 100 nm in diameter, may limit the LNP’s ability to penetrate cellular barriers that is necessary for subcutaneous injection. The only approved siRNA drug that utilizes LNPs is administered by IV infusion, while GalNAc- conjugated siRNAs are administered by subcutaneous administration.

[0263] mRNA delivery using the technologies described herein through subcutaneous administration represents an advantage over current LNP and VLP delivery systems, for example, in terms of easier re-dosing and less invasiveness.

[0264] Thus, the technologies described herein provide improvements over current therapeutic mRNA delivery methods, for example, those relying on encapsulating mRNA in nanoparticles (e.g., lipid nanoparticles, polymeric nanoparticles, CARTs (chargealtering releasable transporters), dendrimers, hydrophilic nanoparticles, etc.). The current technologies described herein also provide improvements over conjugate-mediated methods (e.g., over GalNAc, Cl 6, antibody fragments) that are chemically conjugated to the small RNA.

[0265] In this disclosure, mRNA may encode a therapeutic protein.

[0266] Such mRNAs can be synthesized by in vitro transcription using standard methods and contain an m7G-cap, a 5’UTR, a coding sequence, a 3’UTR and a poly(A) tail. Such mRNAs can be synthesized by in vitro transcription using standard methods and can also contain an m7G-cap, a 5’UTR, a coding sequence, a 3’UTR and no poly(A) tail.

[0267] In this disclosure, Str-O-Nuc may be engineered to include these components: a m7G-cap, a 5’UTR, a 3’UTR, and / or a poly(A) tail. The mRNA complex of this disclosure may be prepared by hybridizing Str-O-Nuc that has such components to an mRNA that has no such components. For example, the mRNA of this disclosure may be engineered with no poly(A) tail. Instead, a Str-O-Nuc may be engineered to have a poly(A) tail in addition to sequences that are complementary to the 3’UTR of the mRNA. Such Str-O-Nuc may bind to the 3’-UTR and impart the properties of the poly(A) tail, which is synthesized as a part of the Str-O-Nuc, onto the mRNA. Further, a Str-O-NucRef. ParB 24001; 07800.002W01 already having a poly(A) tail may also be hybridized on the mRNA that does not have a poly(A) tail.

[0268] Further, the mRNA complex of this disclosure may be prepared by hybridizing Str-O-Nuc that has components such as a m7G-cap, a 5’UTR, a 3’UTR, and / or a poly(A) tail to an mRNA that also has such components. For example, the mRNA of this disclosure may be engineered with a poly(A) tail and contain a Str-O-Nuc hybridized to it wherein the Str-O-Nuc may also be engineered to contain a poly(A). For example, the mRNA of this disclosure may be engineered with a m7G-cap and contain a Str-O-Nuc hybridized to it wherein the Str-O-Nuc may also be engineered to contain a m7G-cap. For example, the mRNA of this disclosure may be engineered with a 5’UTR and contain a Str- O-Nuc hybridized to it wherein the Str-O-Nuc may also be engineered to contain a 5’UTR. For example, the mRNA of this disclosure may be engineered with a 3’UTR and contain a Str-O-Nuc hybridized to it wherein the Str-O-Nuc may also be engineered to contain a 3’UTR. For example, the mRNA of this disclosure may be engineered with a combination of any of a m7G-cap, a 5’UTR, a 3’UTR, and / or a poly(A) tail, and contain a Str-O-Nuc hybridized to it wherein the Str-O-Nuc may also be engineered to contain any of a m7G- cap, a 5’UTR, a 3’UTR, and / or a poly(A) tail.

[0269] This is advantageous because the Str-O-Nuc’ s poly(A) tails may be chemically modified to make them more stable, resulting in a more stable, longer expressing mRNA complex. Additionally, it is difficult to make mRNAs with long poly(A) tails due to inefficiencies associated with template-encoded poly(A) tails as plasmids are known to be unstable when they have long poly(A) stretches. Further, extra steps are required for post- transcriptional polyadenylation, for example, with poly(A) polymerase.

[0270] The mRNA may optionally contain substantially close to 100% uridine modification (e.g., N1 -methylpseudouridine or 5-methy oxyuridine). The cap can be added co-transcriptionally (e.g., using CleanCap) or post-transcriptionally (e.g., using Vaccinia Capping Enzyme + Methyltransferase). The poly(A) tail on the mRNA may be encoded into the DNA template and added during transcription, or added post-transcriptionally (e.g., using Poly(A) Polymerase).

[0271] A Str-O-Nuc may hybridize to a region of the mRNA. Generally, Str-O-Nucs may be generated by chemical synthesis and may contain a mix of chemically modified and / or unmodified nucleotides. For example, Str-O-Nucs may contain nucleotides thatRef. ParB 24001; 07800.002W01 have one or more of 2’-H, 2 ’-OH, 2’-0Me, 2 ’-MOE, 2’-F, 2’-cEt, P-S linkage, 3’-inv-dT, 3’-ddC or LNA modifications.

[0272] A Str-O-Nuc may have a length in a range of 1 nt to 200 nt, or 1 nt to 50 nt, or 2 nt to 25 nt, or 40 nt to 50 nt, or 50 nt to 75 nt, or 75 nt to 100 nt, or 100 nt to 125 nt, or 100 nt to 150 nt, or 125 nt to 150 nt, or 150 nt to 175 nt, or 150 nt to 200 nt, or 175 nt to 200 nt. There may be at least one Str-O-Nuc per mRNA molecule. Further, a Str-O-Nuc containing a poly(A) tail may have a length in a range of 3 nt to 200 nt, or 1 nt to 50 nt, or 2 nt to 25 nt, or 40 nt to 50 nt, or 50 nt to 75 nt, or 75 nt to 100 nt, or 100 nt to 125 nt, or 100 nt to 150 nt, or 125 nt to 150 nt, or 150 nt to 175 nt, or 150 nt to 200 nt, or 175 nt to 200 nt. There may be at least one Str-O-Nuc containing a poly(A) tail per mRNA molecule.

[0273] Further, in certain embodiments, a Str-O-Nuc containing a poly(A) tail may have a length of 3 nt to 5 nt, or 6 nt to 10 nt, or 11 nt to 15 nt, or 16 nt to 20 nt, or 21 nt to 25 nt, not including the adenosines in the poly(A) tail. Further, in certain additional embodiments, a Str-O-Nuc containing a poly(A) tail may have a length of 3 nt to 5 nt, or 6 nt to 10 nt, or 11 nt to 15 nt, or 16 nt to 20 nt, or 21 nt to 25 nt, including the adenosines in the poly(A) tail. Further, in certain additional embodiments, at least 1 nt, or 6 nt, or 8 nt, or 16 nt of the Str-o-Nuc is hybridized to the mRNA.

[0274] The region of the Str-O-Nuc, which hybridizes to a specific sequence on the mRNA, may be hybridized such that 100% of the hybridized section of the Str-O-Nuc sequence is complementary to the said sequence on the mRNA. The region of the Str-O- Nuc, which hybridizes to a specific sequence on the mRNA, may be hybridized such that at least 50% of the hybridized section of the Str-O-Nuc sequence is complementary to the said sequence on the mRNA. That is, there may be intentional gaps or mismatches to limit immunogenicity and weaken the hybridization strength to promote reversibility.

[0275] A Str-O-Nuc may contain additional structures (z.e., accessory elements) that may be unmodified or chemically modified nucleotides, secondary structures, scaffold for other RNAs, conjugated proteins, peptides, nucleic acids, lipids, sugars, or small molecules that impart desirable properties to the Str-O-Nucs or the Str-O-Nuc-mRNA complexes. Further, these accessory elements may include additional structures that can incorporate a linker region. Such desirable properties, for example, may include (a) higher translation output, (b) longer mRNA and / or Str-O-Nuc durability, (c) less immunogenicity, (d) cell-type specific delivery and / or (e) translation regulation. TheseRef. ParB 24001; 07800.002W01 additional chemical structures of Str-O-Nucs, which may not be included in the mRNA structure, may contain extra nucleotides and / or chemical moieties that function to impart new properties to the mRNA by way of being localized to the mRNA through the Str-O- Nuc hybridization.

[0276] These additional chemical structures may be nucleotides that form desirable secondary structures that impart cellular functions (e.g., TENT elements that lead to readenylation of the poly(A) tail), promote translation initiation (small IRES as unmodified RNA or m7G-capped Str-O-Nuc). These additional elements may recruit protein factors in the cell for desirable outputs. For example, stretches of poly(A) on a Str- O-Nuc may recruit poly(A) binding protein. These additional chemical structures may contain sequence elements that promote some modification of the mRNA itself by cellular machinery.

[0277] Additionally, these additional chemical structures may instead function as scaffolds that may allow for controlled assembly of a specified number of Str-O-Nucs per mRNA molecule. Such functioning may be useful in multi-component RNA systems. For example, we may recruit a specified number of gRNAs to one mRNA molecule to allow for optimal gene editing. Or, we may recruit multiple Str-O-Nucs that only impart their function when they are in proximity. For example, some viral elements that may stabilize an mRNA may be beyond the synthesis limit of a Str-O-Nuc. Therefore, if we recruit two or more Str-O-Nucs to one region, the Str-O-Nucs may then form the desired element to impart new properties to the mRNA. There may be chemical linkers separating the hybridization region of the Str-O-Nuc from the accessory element of the Str-O-Nuc. A linker may be a linear linker or a branched linker. A linker may comprise a hydrocarbon chain. A hydrocarbon chain may comprise from 2 to about 2000 or more carbon atoms. The hydrocarbon chain may comprise an alkylene group, e.g. C2 to about 2000 or more alkylene groups. The hydrocarbon chain may have a general formula of — (CH2)n — wherein n is from 2 to about 2000 or more. The hydrocarbon chain may be optionally interrupted by one or more ester groups (i.e. — C(O) — O — ) or one or more amide groups (z.e. — C(O) — N(H) — ). Any linker may be used selected from the group comprising PEG (e.g. Psoralen-PEG3 -Biotin biotinylation), polyacrylamide, poly(2 -hydroxy ethyl methacrylate), Poly-2-methyl-2-oxazoline (PMOXA), zwitterionic polymers, e.g. poly(carboxybetaine methacrylate) (PCBMA), poly[N-(3-sulfopropyl)-N- methacryloxyethyl-N,N dimethyl ammonium betaine] (PSBMA), glycopolymers, andRef. ParB 24001; 07800.002W01 polypeptides. A linker may comprise a polyethylene glycol (PEG) having a general formula of — (CH2 — CH2 — O)n-, wherein n is from 1 to about 600 or more. A linker may comprise oligoethylene glycol -phosphate units having a general formula of — [(CH2 — CH2 — O)n — PO2 - — O]m — where n is from 1 to about 600 or more and m could be 1- 200 or more. Further, a linker may also be formed using click chemistry, RNA ligase, phosphoimidazolide activation, and 2’-OH acetylation / N-cyanoimidazol, electrophilic phosphorothioester ligation.

[0278] The mRNA complexes of this disclosure may form a double-stranded structure.

[0279] Double-stranded RNA (dsRNA) is recognized by the cellular innate immunity sensors. When dsRNA is detected, the cell mounts an immune response, which can lead to decreased translation and even cell death.

[0280] However, Str-O-Nucs of this disclosure may reduce, substantially reduce, or prevent this immune response because the Str-O-Nucs may have modifications in the region where they hybridize with the mRNA that may lead to immune evasion of the heteroduplex structures formed from such Str-O-Nucs. Further, Str-O-Nucs may have 2’- H, 2’-OH, 2’-0Me, 2’-M0E, 2’-F, 2’-cEt, P-S linkage, 3’-inv-dT, 3’-ddC or LNA modifications in the region where they hybridize with the mRNA that may lead to immune evasion of the heteroduplex structures formed from such Str-O-Nucs. These modifications may prevent the cell from sensing the Str-O-Nuc-mRNA double-stranded structure. The Str-O-Nuc-mRNA double-stranded structure is called HD-RNA (heteroduplex-RNA).

[0281] The Str-O-Nucs of this disclosure can be generated by chemical synthesis and can contain chemically modified nucleotides (e.g., 2’-0Me, 2’-M0E, and / or phosphorothioate linkages). Str-O-Nucs can be about 1-200 nt in length, and there will be at least one Str-O-Nuc in the mixture with the mRNA. After synthesis, the mRNA and the Str-O-Nucs are mixed (generally in 1 : 1 molar ratios, + / - 20% of either), and refolding buffer is added at lx concentration (lx refolding buffer: 5mM Tris pH 8, O.lmM EDTA, 15mM NaCl). The mixture is then rapidly heated to 95 degrees C, held at 95 degrees C for 3 minutes and then slowly cooled to 25 degrees C at a rate of O.lC / second. After hybridizing, the HD-RNA can immediately be used in an experiment or stored at -20 degrees C until use. The refolding buffer and the heating / cooling step may not be required for HD-RNA formation, and simple mixing may be sufficient to hybridize a Str-O-Nucs to an mRNA.Ref. ParB 24001; 07800.002W01

[0282] The technology, disclosed herein, may bridge the flexibility of chemical synthesis with the efficiency of in vitro transcription for longer RNAs, without the need for laborious ligation. Desirable modifications may be introduced into Str-O-Nucs by way of chemical synthesis, and these Str-O-Nucs can impart those properties onto the mRNA by way of hybridizing with specific sequences in the mRNA.

[0283] For example, it may be desirable to introduce chemical modifications into the poly(A) tail of an mRNA. Without such modification, poly(A) tail may be deadenylated in the cell, resulting in reduced translation and ultimately RNA degradation. Such chemical modification of poly(A) tail is not possible with current in vitro transcription, and current efforts to ligate chemically synthesized and modified poly(A) tails has been met with limited success.

[0284] A Str-O-Nuc of this disclosure may be synthesized with a region complementary to the mRNA’s 3’UTR, which may also include a stretch of A’s (e.g., at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, or 150 consecutive A’s) that may contain one or more nucleotides with a desirable chemical modification (e.g., 2’-OH, 2’-0Me, 2’-M0E, 2’-F, 2’-cEt, P-S linkage, 3’-inv-dT, 3’-ddC or LNA) to stabilize the stretch of poly(A) nucleotides that function as a poly(A) tail. Such modification may be in addition to, or instead of, the poly(A) tail on the mRNA.

[0285] Complementary binding or chemical linking to the structures of the 3’ end of the mRNA sequence (e.g. terminal 3’ end, 3’UTR, poly(A) tail) by a Str-O-Nu or a Plus- Nuc each results in greater stability for the mRNA complex than an unmodified mRNA (e.g. mRNA with no chemical modifications) or un-engineered mRNA construct (e.g. mRNA construct with no Str-O-Nuc hybridized to it, or mRNA construct with no Plus- Nuc or Str-O-Nuc linked to it).

[0286] For Str-O-Nucs with a poly(A) tail, these Str-O-Nuc may be oriented in the forward or reverse direction, as shown in Figures 1-3. The orientation refers to the location of the poly(A) tail stretch on the Str-O-Nuc. For example, the forward orientation corresponds to a Str-O-Nuc with a poly(A) on the 3’ end of the hybridization region of the Str-O-Nuc. Further, for example, the reverse orientation corresponds to a Str-O-Nuc with a poly(A) on the 5’ end of the hybridization region of the Str-O-Nuc. Both orientations are advantageous over the background art because they would confer enhanced stability when compared to an unmodified and / or an un-engineered mRNA construct.Ref. ParB 24001; 07800.002W01

[0287] A Str-O-Nuc can comprise more than one poly(A) tail. Where the disclosure references one poly(A), it is understood that more than one poly(A) can also be included. The poly(A) tails may be oriented in the forward or reverse direction. A Str-O-Nuc may be comprised of one poly(A) tail in the forward direction and another poly(A) tail in the reverse direction, as shown in FIG. 4.

[0288] Another embodiment of this invention is a mRNA with a Str-O-Nuc hybridized to it, wherein the Str-O-Nuc is comprised of a region comprising 100% 2’MOE modifications that binds to the complementary region of the 3’ UTR of an mRNA with no poly (A) tail, as shown in FIG. 1 A.

[0289] An embodiment of this invention is a mRNA with a Str-O-Nuc hybridized to it, wherein the Str-O-Nuc is comprised of a region that binds to the complementary region of the 3’ UTR of an mRNA with no poly(A) tail, wherein the Str-O-Nuc is comprised of a poly(A) tail followed by nucleotides containing 2’MOE modifications, wherein the nucleotides are held together with PS linkages, and the Str-O-Nuc is oriented in the reverse direction, as shown in FIG. IB. An embodiment of this invention is a mRNA with a Str-O-Nuc hybridized to it, wherein the Str-O-Nuc is comprised of a 15nt region that binds to the complementary region of the 3’ UTR of an mRNA with no poly(A) tail, wherein the Str-O-Nuc is comprised of a poly(A) tail followed by 6nt comprised of 2’MOE modifications, wherein the 6nt are held together with PS linkages, and the Str-O- Nuc is oriented in the reverse direction, as shown in FIG. IB.

[0290] Another embodiment of this invention is a mRNA with a Str-O-Nuc hybridized to it, wherein the Str-O-Nuc is comprised of a region comprising 100% 2’MOE modifications that binds to the 3’ end of an mRNA with no poly(A) tail, as shown in FIG. 1C. An embodiment of this invention includes a Str-O-Nuc comprised of a 18nt region comprised of 100% 2’MOE modifications that binds to the 3’ end of an mRNA with no poly(A) tail, as shown in FIG. 1C. An embodiment of this invention includes a Str-O-Nuc comprised of a 25nt region comprised of 100% 2’MOE modifications that binds to the 3’ end of an mRNA with no poly (A) tail.

[0291] Another embodiment of this invention is a mRNA with a Str-O-Nuc hybridized to it, wherein the Str-O-Nuc is comprised of a region that binds to the 3’ end of an mRNA with no poly(A) tail, wherein the Str-O-Nuc is comprised of a poly(A) tail followed by at least two nucleotides containing 2’MOE modifications, wherein the nucleotides are held together with PS linkages, and the Str-O-Nuc is oriented in the forward direction, asRef. ParB 24001; 07800.002W01 shown in FIG. ID. An embodiment of this invention is a mRNA with a Str-O-Nuc hybridized to it, wherein the Str-O-Nuc is comprised of a 15nt region that binds to the complementary region of the 3’ UTR of an mRNA with no poly(A) tail, wherein the Str- O-Nuc is comprised of a poly(A) tail followed by 6nt comprised of 2’MOE modifications, wherein the 6nt are held together with PS linkages, and the Str-O-Nuc is oriented in the forward direction.

[0292] An embodiment of this invention is a mRNA with a Str-O-Nuc hybridized to it, wherein the Str-O-Nuc is comprised of a region that binds to the 3’ end of an mRNA with no poly(A) tail, wherein the Str-O-Nuc is comprised of a poly(A) tail followed by nucleotides containing 2’MOE modifications, wherein the nucleotides are held together with PS linkages, and the Str-O-Nuc is oriented in the reverse direction, as shown in FIG. IE. Another embodiment of this invention is a mRNA with a Str-O-Nuc hybridized to it, wherein the Str-O-Nuc is comprised of a 18nt region that binds to the 3’ end of an mRNA with no poly(A) tail, wherein the Str-O-Nuc is comprised of a poly(A) tail followed by 6nt comprised of 2’MOE modifications, wherein the 6nt are held together with PS linkages, and the Str-O-Nuc is oriented in the reverse direction, as shown in FIG. IE. Another embodiment of this invention is a mRNA with a Str-O-Nuc hybridized to it, wherein the Str-O-Nuc is comprised of a 25nt region that binds to the 3’ end of an mRNA with no poly(A) tail, wherein the Str-O-Nuc is comprised of a poly(A) tail followed by 6nt comprised of 2’MOE modifications, wherein the 6nt are held together with PS linkages, and the Str-O-Nuc is oriented in the reverse direction.

[0293] Another embodiment of this invention is a mRNA with a poly(A) tail comprising at least one additional nucleotide following the end of the poly(A) tail that was encoded for in the DNA template used to create the mRNA, hereinafter referred to as the Plus-Nuc, as shown in FIG. 2A-2E. A preferred embodiment of this invention includes a mRNA with a poly(A) tail, comprising 120 adenosines, followed by a Plus-Nuc comprised of at least 16 nt linked to the end of the mRNA’s poly(A) tail, as shown in FIG. 2A. A preferred embodiment of this invention includes a mRNA with a poly(A) tail, comprising 120 adenosines, followed by a Plus-Nuc comprised of at least 18 nt linked to the end of the mRNA’s poly(A) tail. A preferred embodiment of this invention includes a mRNA with a poly(A) tail, comprising 120 adenosines, followed by a Plus-Nuc comprised of at least 25 nt linked to the end of the mRNA’s poly(A) tail. In certain embodiments, theRef. ParB 24001; 07800.002W01 mRNA does not comprise a poly(A) tail. In certain embodiments, when the mRNA does not comprise a poly(A) tail, the mRNA comprises a Plus-Nuc following the 3’ UTR.

[0294] Another embodiment of this invention is a mRNA with a poly(A) tail followed by a Plus-Nuc, and a Str-O-Nuc is hybridized to the Plus-Nuc, wherein the Str-O-Nuc is comprised of a loop-back blocker (e.g. an RNA oligonucleotide comprising a hairpin loop), wherein the nucleotides in the loop-back blocker is comprised of 100% 2’MOE modifications and that folds onto itself and hybridizes to itself to form a terminal hairpin, as shown in FIG. 2B.

[0295] Another embodiment of this invention is a mRNA with a poly(A) tail followed by a Plus-Nuc comprised of at least 1 nucleotide following the end of the mRNA’s poly(A) tail, and a Str-O-Nuc complementary to and bound to the Plus-Nuc, wherein the Str-O-Nuc is comprised of fewer, the same, or more than the number of nucleotides as the Plus-Nuc, as shown in FIG. 2C. An embodiment of this invention includes a mRNA with a poly(A) tail, comprising 120 adenosines, followed by a Plus-Nuc comprised of at least 16 nt following the end of the mRNA’s poly(A) tail, and a Str-O-Nuc complementary to and bound to the Plus-Nuc, wherein the Str-O-Nuc is comprised of fewer, the same, or more than the number of nucleotides as the Plus-Nuc, as shown in FIG. 2C. An embodiment of this invention includes a mRNA with a poly(A) tail, comprising 120 adenosines, followed by a Plus-Nuc comprised of at least 18 nt following the end of the mRNA’s poly(A) tail, and a Str-O-Nuc complementary to and bound to the Plus-Nuc, wherein the Str-O-Nuc is comprised of fewer, the same, or more than the number of nucleotides as the Plus-Nuc. An embodiment of this invention includes a mRNA with a poly(A) tail, comprising 120 adenosines, followed by a Plus-Nuc comprised of at least 25 nt following the end of the mRNA’s poly(A) tail, and a Str-O-Nuc complementary to and bound to the Plus-Nuc, wherein the Str-O-Nuc is comprised of fewer, the same, or more than the number of nucleotides as the Plus-Nuc.

[0296] An embodiment of this invention includes a mRNA with a poly(A) tail, comprising 120 adenosines, followed by a Plus-Nuc comprised of at least 16 nt, 18nt, or 25 nt following the end of the mRNA’s poly(A) tail, and a Str-O-Nuc complementary to and bound to the Plus-Nuc, wherein the Str-O-Nuc is comprised of fewer, the same, or more than the number of nucleotides as the Plus-Nuc, wherein the Str-O-Nuc is comprised of a poly(A) tail followed by at least two nucleotides comprised of 2’MOE modifications, wherein the nucleotides are held together with PS linkages, and the Str-O-Nuc is orientedRef. ParB 24001; 07800.002W01 in the forward direction, as shown in FIG. 2D. An embodiment of this invention includes a mRNA with a poly(A) tail, comprising 120 adenosines, followed by a Plus-Nuc comprised of at least 16 nt, 18nt, or 25 nt following the end of the mRNA’s poly(A) tail, and a Str-O- Nuc complementary to and bound to the Plus-Nuc, wherein the Str-O-Nuc is comprised of fewer, the same, or more than the number of nucleotides as the Plus-Nuc, wherein the Str- O-Nuc is comprised of a poly(A) tail followed by 6nts comprised of 2’MOE modifications, wherein the nucleotides are held together with PS linkages, and the Str-O- Nuc is oriented in the forward direction, as shown in FIG. 2D.

[0297] An embodiment of this invention includes a mRNA with a poly(A) tail, comprising 120 adenosines, followed by a Plus-Nuc comprised of at least 16 nt, 18nt, or 25 nt following the end of the mRNA’s poly(A) tail, and a Str-O-Nuc complementary to and bound to the Plus-Nuc, wherein the Str-O-Nuc is comprised of fewer, the same, or more than the number of nucleotides as the Plus-Nuc, wherein the Str-O-Nuc is comprised of a poly(A) tail followed by at least two nucleotides comprised of 2’MOE modifications, wherein the nucleotides are held together with PS linkages, and the Str-O-Nuc is oriented in the reverse direction, as shown in FIG. 2E. An embodiment of this invention includes a mRNA with a poly(A) tail, comprising 120 adenosines, followed by a Plus-Nuc comprised of at least 16 nt, 18nt, or 25 nt following the end of the mRNA’s poly(A) tail, and a Str-O- Nuc complementary to and bound to the Plus-Nuc, wherein the Str-O-Nuc is comprised of fewer, the same, or more than the number of nucleotides as the Plus-Nuc, wherein the Str- O-Nuc is comprised of a poly(A) tail followed by 6nts comprised of 2’MOE modifications, wherein the nucleotides are held together with PS linkages, and the Str-O- Nuc is oriented in the reverse direction, as shown in FIG. 2E.

[0298] Another embodiment of this invention is a mRNA with a poly(A) tail followed by at least 1 nucleotide linked, e.g. a Str-O-Nuc, to the mRNA after the in vitro transcription reaction or separate from the in vitro transcription (e.g. co-transcriptional linking), and attached to the end of the mRNA’s poly(A) tail, as shown in FIG. 3A-3E. An embodiment of this invention is a mRNA with a poly(A) tail, comprising 120 adenosines, linked to a Str-O-Nuc comprised of at least Int, as shown in FIG. 3A. An embodiment of this invention is a mRNA with a poly(A) tail, comprising 120 adenosines, linked to a Str-O-Nuc comprised of at least 16 nt, as shown in FIG. 3 A. An embodiment of this invention is a mRNA with a poly(A) tail, comprising 120 adenosines, linked to a Str-O-Nuc comprised of at least 18 nt. An embodiment of this invention is a mRNA with aRef. ParB 24001; 07800.002W01 poly(A) tail, comprising 120 adenosines, linked to a Str-O-Nuc comprised of at least 25 nt. A linker that links a Str-O-Nuc to the mRNA may comprise a linear linker or a branched linker. A linker may comprise a hydrocarbon chain. A hydrocarbon chain may comprise from 2 to about 2000 or more carbon atoms. The hydrocarbon chain may comprise an alkylene group, e.g. C2 to about 2000 or more alkylene groups. The hydrocarbon chain may have a general formula of — (CH2)n — wherein n is from 2 to about 2000 or more. The hydrocarbon chain may be optionally interrupted by one or more ester groups (i.e. — C(O) — O — ) or one or more amide groups i.e. — C(O) — N(H) — ). Any linker may be used selected from the group comprising PEG e.g. Psoralen-PEG3 -Biotin biotinylation), polyacrylamide, poly(2-hydroxyethyl methacrylate), Poly-2-methyl-2-oxazoline (PMOXA), zwitterionic polymers, e.g. poly(carboxybetaine methacrylate) (PCBMA), poly[N-(3-sulfopropyl)-N-methacryloxyethyl-N,N dimethyl ammonium betaine] (PSBMA), glycopolymers, and polypeptides. A linker may comprise a polyethylene glycol (PEG) having a general formula of — (CH2 — CH2 — O)n-, wherein n is from 1 to about 600 or more. A linker may comprise oligoethylene glycol-phosphate units having a general formula of — [(CH2 — CH2 — O)n — PO2 - — O]m — where n is from 1 to about 600 or more and m could be 1-200 or more. Further, a linker may also be formed using click chemistry, RNA ligase, phosphoimidazolide activation, and 2’-OH acetylation / N- cyanoimidazol, electrophilic phosphorothioester ligation.

[0299] Another embodiment of this invention is a mRNA with a poly(A) tail linked to a first Str-O-Nuc, and a second Str-O-Nuc is hybridized to the first Str-O-Nuc, wherein the second Str-O-Nuc is comprised of a loop-back blocker e.g. an RNA oligonucleotide comprising a hairpin loop), wherein the nucleotides in the loop-back blocker is comprised of 100% 2’MOE modifications and that folds onto itself and hybridizes to itself to form a terminal hairpin, as shown in FIG. 3B.

[0300] Another embodiment of this invention is a mRNA with a poly(A) tail linked to a first Str-O-Nuc comprised of at least 1 nucleotide, and a second Str-O-Nuc complementary to and bound to the first Str-O-Nuc, wherein the second Str-O-Nuc is comprised of fewer, the same, or more than the number of nucleotides as the first Str-O- Nuc, as shown in FIG. 3B-3E. A preferred embodiment of this invention is a mRNA with a poly(A) tail, comprising 120 adenosines, linked to a first Str-O-Nuc comprised of at least 16 nt, 18nt, or 25 nt and a second Str-O-Nuc complementary to and bound to the first Str-Ref. ParB 24001; 07800.002W01O-Nuc, wherein the second Str-O-Nuc is comprised of fewer, the same, or more than the number of nucleotides as the first Str-O-Nuc, as shown in FIG. 3C.

[0301] Another embodiment of this invention is a mRNA with a poly(A) tail, comprising 120 adenosines, linked to a first Str-O-Nuc comprised of at least Int, and a second Str-O-Nuc complementary to and bound to the first Str-O-Nuc, wherein the second Str-O-Nuc is comprised of fewer, the same, or more than the number of nucleotides as the first Str-O-Nuc, and wherein the second Str-O-Nuc is comprised of a poly(A) tail, followed by at least two nucleotides comprising 2’MOE modifications, wherein the at least two nucleotides are held together with PS linkages, and the second Str-O-Nuc is oriented in the forward direction, as shown in FIG. 3D. Another embodiment of this invention is a mRNA with a poly(A) tail, comprising 120 adenosines, linked to a first Str- O-Nuc comprised of at least 16 nt, 18nt, or 25 nt and a second Str-O-Nuc complementary to and bound to the first Str-O-Nuc, wherein the second Str-O-Nuc is comprised of fewer, the same, or more than the number of nucleotides as the first Str-O-Nuc, and wherein the second Str-O-Nuc is comprised of a poly(A) tail, followed by at least two nucleotides comprising 2’MOE modifications, wherein the at least two nucleotides are held together with PS linkages, and the second Str-O-Nuc is oriented in the forward direction, as shown in FIG. 3D.

[0302] Another embodiment of this invention is a mRNA with a poly(A) tail, comprising 120 adenosines, linked to a first Str-O-Nuc comprised of at least Int, and a second Str-O-Nuc complementary to and bound to the first Str-O-Nuc, wherein the Str-O- Nuc is comprised of fewer, the same, or more than the number of nucleotides as the first Str-O-Nuc, and wherein the second Str-O-Nuc is comprised of a poly(A) tail, followed by at least two nucleotides containing 2’MOE modifications, wherein the at least two nucleotides are held together with PS linkages, and the second Str-O-Nuc is oriented in the reverse direction, as shown in FIG. 3E. Another embodiment of this invention is a mRNA with a poly(A) tail, comprising 120 adenosines, linked to a first Str-O-Nuc comprised of at least 16 nt, 18nt, or 25 nt and a second Str-O-Nuc complementary to and bound to the first Str-O-Nuc, wherein the second Str-O-Nuc is comprised of fewer, the same, or more than the number of nucleotides as the first Str-O-Nuc, and wherein the second Str-O-Nuc is comprised of a poly(A) tail, followed by at least two nucleotides containing 2’MOE modifications, wherein the at least two nucleotides are held togetherRef. ParB 24001; 07800.002W01 with PS linkages, and the second Str-O-Nuc is oriented in the reverse direction, as shown in FIG. 3E.

[0303] An embodiment of this invention is an mRNA with a Str-O-Nuc hybridized to it, wherein the Str-O-Nuc comprises at least one winged LNA. Another embodiment of this invention is an mRNA with a Str-O-Nuc hybridized to it, wherein the Str-O-Nuc is comprised of at least two consecutive winged LNAs on the 5’ and 3’ end of the Str-O- Nuc. Another embodiment of this invention is an mRNA with a Str-O-Nuc hybridized to it, wherein the Str-O-Nuc is comprised of at least one LNA and one 2'-O-methoxyethyl (MOE). Another embodiment of this invention is an mRNA with a Str-O-Nuc hybridized to it, wherein the Str-O-Nuc is comprised of at least one winged LNA and at least one LNA and one 2'-O-methoxyethyl (MOE). Another embodiment of this invention is an mRNA with a Str-O-Nuc hybridized to it, wherein the Str-O-Nuc is comprised of at least two consecutive winged LNA and at least two LNA and two 2'-O-methoxy ethyl (MOE), wherein the at least two LNA and two 2'-O-methoxyethyl (MOE) alternate with each other. Another embodiment of this invention is an mRNA with a Str-O-Nuc hybridized to it, wherein the Str-O-Nuc is comprised of at least two consecutive winged LNA and at least two LNA and two 2 '-O-m ethoxy ethyl (MOE), wherein the at least two LNA and two 2 '-O-m ethoxy ethyl (MOE) alternate with each other, and that bind to the 3’ terminal nucleotides (nt) in 3’ untranslated region (UTR) of an mRNA. A preferred embodiment of this invention is a mRNA with a Str-O-Nuc hybridized to it, wherein the Str-O-Nuc is comprised of a 2'-O-methoxyethyl (MOE) backbone modification with winged LNAs, consisting of 3 consecutive LNAs on the 5’ and 3’ end of the Str-O-Nuc, that binds to the 18nt of the 3’ terminal end of the 3 ’UTR of the mRNA. Another preferred embodiment of this invention is an mRNA with a Str-O-Nuc hybridized to it, wherein the Str-O-Nuc is comprised of winged LNAs, consisting of 3 consecutive LNAs on the 5’ and 3’ end of the Str-O-Nuc, followed by alternating LNAs and 2'-O-methoxyethyl (MOE), that binds to the 18nt of the 3’ terminal end of the 3 ’UTR of the mRNA. Another preferred embodiment of this invention is an mRNA with a Str-O-Nuc hybridized to it, wherein the Str-O-Nuc is comprised of a 2'-O-methoxyethyl (MOE) backbone modification that binds to the 25 nt of the 3’ terminal end of the 3 ’UTR of the mRNA. Another preferred embodiment of this invention is an mRNA with a Str-O-Nuc hybridized to it, wherein the Str-O-Nuc is comprised of a phosphorodiamidate morpholino oligomer (PMO) backbone that binds to the 25nt of the 3’ terminal end of the 3 ’UTR of the mRNA.Ref. ParB 24001; 07800.002W01

[0304] An embodiment of this invention is an mRNA with a Plus-Nuc at the 3’ end, wherein the Plus-Nuc comprises at least one winged LNA. Another embodiment of this invention is an mRNA with a Plus-Nuc at the 3’ end, wherein the Plus-Nuc is comprised of at least two consecutive winged LNAs on the 5’ and 3’ end of the Plus-Nuc. Another embodiment of this invention is an mRNA with a Plus-Nuc at the 3’ end, wherein the Plus-Nuc is comprised of at least one LNA and one 2'-O-methoxyethyl (MOE). Another embodiment of this invention is an mRNA with a Plus-Nuc at the 3’ end, wherein the Plus-Nuc is comprised of at least one winged LNA and at least one LNA and one 2'-O- methoxyethyl (MOE). Another embodiment of this invention is an mRNA with a Plus- Nuc at the 3’ end, wherein the Plus-Nuc is comprised of at least two consecutive winged LNA and at least two LNA and two 2'-O-methoxyethyl (MOE), wherein the at least two LNA and two 2'-O-methoxyethyl (MOE) alternate with each other.

[0305] An embodiment of this invention is an mRNA with a Plus-Nuc at the 3’ end, wherein the Plus-Nuc is bound to a Str-O-Nuc comprising at least one winged LNA. Another embodiment of this invention is an mRNA with a Plus-Nuc at the 3’ end, wherein the Plus-Nuc is bound to a Str-O-Nuc comprising at least two consecutive winged LNAs on the 5’ and 3’ end of the Plus-Nuc. Another embodiment of this invention is an mRNA with a Plus-Nuc at the 3’ end, wherein the Plus-Nuc is bound to a Str-O-Nuc comprising at least one LNA and one 2 '-O-m ethoxy ethyl (MOE). Another embodiment of this invention is an mRNA with a Plus-Nuc at the 3’ end, wherein the Plus-Nuc is bound to a Str-O-Nuc comprising at least one winged LNA and at least one LNA and one 2'-O- methoxyethyl (MOE). Another embodiment of this invention is an mRNA with a Plus- Nuc at the 3’ end, wherein the Plus-Nuc is bound to a Str-O-Nuc comprising at least two consecutive winged LNA and at least two LNA and two 2 '-O-m ethoxy ethyl (MOE), wherein the at least two LNA and two 2'-O-methoxyethyl (MOE) alternate with each other.

[0306] Nucleic acids, and in particular in enzymatic proteins, have been used with different technologies including gene therapy, nuclease editing, base editing, prime editing, and gene writing. In vivo delivery of nucleic acids and enzymatic proteins, in particular single-stranded nucleic acids such as RNA, is challenging due to the instability of the molecules in the host environment (e.g. pH, temperature, charges, nuclease proteins). These types of enzymatic systems frequently use an RNA template and it is known that these templates have stability issues due to their lack of sequences suchRef. ParB 24001; 07800.002W01 poly(A) tails or lack of double strandedness. Herein we provide a system to stabilize these templates.

[0307] Double-stranded RNA have intrinsic thermodynamics stability compared to single-stranded RNA molecules which increases the half-life and stability of the therapeutic RNA molecule. However, extrinsic factors may still be able to degrade or prevent the translation of the therapeutic RNA cargo in the RNA duplex within the host cell environment. RNA-binding proteins (RBPs) and nucleases that target double-stranded RNA molecules are extrinsic host-derived factors that can degrade therapeutic RNA molecules hybridized to stabilizing nucleic acids such as Str-O-Nucs. One method of stabilizing the duplex includes chemical modifications to the backbone of the Str-O-Nuc including but not limited to 2’-F, 2’-0Me, 2’-M0E, 3’-ddC, 3’-inv-dT, P-S linkage, and LNAs. These chemical modifications can prevent recognition of the therapeutic nucleic acids by the nuclease for degradation. Soutschek J., et al. Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs. Nature. 2004;432: 173- 178.].

[0308] Str-O-Nuc of this disclosure can generate additional mechanisms of therapeutic genetic modifications via the delivery single-stranded RNAs, such as mRNA hybridized to a Str-O-Nuc of this disclosure. An example of the type of technology for which a Str-O- Nuc may help stabilize the associated RNA structure is a gene therapy that utilizes the insertion of a protein coding sequence of DNA into the host genome for the purpose of gene replacement therapy. Retrotransposons that convert RNA into DNA that are then inserted in the genome may be co-delivered with a therapeutic mRNA. The therapeutic RNA may be reverse transcribed by the retrotransposon and inserted into the host genome to replace a non-functional protein-coding gene. The formulation of such an RNA molecule would require stabilization of the single-stranded RNAs. Str-O-Nucs of this disclosure may be used to provide stability to these RNA molecules by hybridizing to them, generating a more thermodynamically stable duplex structure for formulations and blocking the unprotected ends of the non-translated RNA from nuclease degradation. The delivery of such an RNA molecule encoding the reverse transcriptase and therapeutic mRNA is possible but challenging as unstable RNA lacking a poly(A) tail or mRNA lacking stabilizing UTRs would be rapidly degraded. Str-O-Nucs of this disclosure may be hybridized to the therapeutic RNA until the retrotransposase mRNA is translated, and theRef. ParB 24001; 07800.002W01 resulting enzymatic protein reverse transcribes the therapeutic mRNA into cDNA, and inserts it in the genome.

[0309] Another example of the type of technology for which a Str-O-Nuc may help stabilize the associated RNA structure is an enzymatically synthesized de novo RNA containing base-specific backbone modifications (e.g. 2’-modifications, 3 ’-modifications). The delivery of such enzymatically synthesized de novo RNA is possible, but may have disadvantages due to stability issues. Str-O-Nucs of this disclosure may hybridize to the synthesized RNA to provide structural stability during the formulation of these RNA molecules. Further, the Str-O-Nuc may hybridize to the RNA template to stabilize it in the absence of sequences such as Poly(A) tails.

[0310] Another example of the type of technology for which a Str-O-Nuc may help stabilize the associated RNA structure is a replicase of alphavirus origin (serving as the enzymatic protein) that may be encoded in an RNA molecule containing an RNA replicon (serving as the template RNA) or on a separate RNA molecule. The delivery of a replicase and corresponding RNA replicon is possible, but may have disadvantages due to stability issues. Str-O-Nuc of this disclosure may be hybridized to either the RNA molecule encoding the alphavirus replicase or the RNA replicon during the formulation of these RNA molecules. Once delivered in vivo, the Str-O-Nuc may help stabilize the RNA replicon molecule while the replicase is translated and subsequently binds that RNA replicon.

[0311] Another example of the type of technology for which a Str-O-Nuc may help stabilize the associated RNA structure is a bispecific RNA bridge that recognizes target and donor DNA sequences for recombinase-mediated bacterial genome insertion (e.g. IS621 recombinase). The delivery of such a bridge RNA molecule is possible, but may have disadvantages due to stability issues. Str-O-Nucs of this disclosure may stabilize the the RNA bridge molecule by hybridization during the formulation of the RNA bridge. Once delivered in vivo, the Str-O-Nuc may help stabilize the RNA bridge molecule while the recombinase is translated and subsequently inserts donor DNA into the target site in the host genome.

[0312] The Str-O-Nucs may contain a sequence (e.g., 2 nt- 1 Ont, 2nt-20nt, 2nt-25nt, 2nt-30nt, 2nt-40nt, 2nt-50nt, 2nt - 75 nt, 2nt-100nt, 2nt-125nt, 2nt-200nt) complementary to some part of the mRNA. This complementary sequence may hybridize to the mRNA and may be chemically modified to evade dsRNA sensors. Further, this complementaryRef. ParB 24001; 07800.002W01 sequence may hybridize to the mRNA and may be chemically modified with 2’-H, 2’ -OH, 2’-0Me, 2’-M0E, 2’-F, 2’-cEt, P-S linkage, 3’-inv-dT, 3’-ddC or LNA modifications to evade dsRNA sensors. The Str-O-Nucs may also contain further nucleotides (unmodified or modified), chemical moieties (small molecules), or conjugated molecules (proteins, peptides, small molecules), that are not complementary to the mRNA. These ‘extra features’ would impart desirable properties to the mRNA itself.

[0313] The Str-O-Nucs may contain no additional sequences other than the complementary sequence. One use case for this may be to stabilize the mRNA through hybridizing of the Str-O-Nucs to the mRNA (duplexed RNA is more stable and more resistant to exonucleases and endonucleases).

[0314] The Str-O-Nucs may contain a loop-back end blocker, which is an oligonucleotide containing a hairpin loop. One mechanism by which mRNA stability is regulated is by 3’-5’ exonucleases that hydrolyze ribonucleotides of mRNA (e.g. RNA exosome or CCR4-NOT). Introduction of a loop-back end blocker oligonucleotide results in steric blocking of the 3 ’OH on the end of an mRNA, thus enhancing mRNA half-life and stability. At the 3’ end of the loop back end blocker oligonucleotide, it can contain nucleotides that hybridize to the 3’ end of the mRNA. Further, at the 3’ end of the loop back end blocker oligonucleotide, it can contain nucleotides that hybridize to the poly(A) tail of the mRNA. Preceding the stretch of nucleotides on the 5’ end of the loop back end blocker, there are additional complementary nucleotides that form a duplex stem with an internal ‘loop’ composed of a series of non-complimentary nucleotides. This internal ‘loop’ on the Str-O-Nuc may also be a chemical spacer instead of, or in addition to, the series of non-complementary nucleotides. Once the oligonucleotide is bound to the mRNA, the hairpin loop is immediately adjacent to the end of the mRNA sequence.

[0315] The Str-O-Nucs may contain a splinted end blocker, which is an RNA ‘splint’ oligonucleotide and a second oligonucleotide. Further, the splinted end blocker’s second oligonucleotide may contain a 3 ’-3’ inverted deoxythymidine (dT), 3 ’-dideoxy cytidine (ddC), or other 3 ’-terminal moiety that is not recognized by exonucleases. The Str-O-Nucs with the splinted end blocker may increase the half-life of the mRNA to which they are hybridized. The 3 ’-3’ inverted deoxythymidine (dT) of a splinted end blocker may block exonuclease activity. One mechanism by which mRNA stability is regulated are by 3’-5’ exonucleases that hydrolyze ribonucleotides of mRNA (e.g. polynucleotide phosphorylases (PNPases)). Introduction of a modified 3 ’-3’ phosphodiester bond blocksRef. ParB 24001; 07800.002W01 activity of the 3 ’-5’ exonuclease, thus enhancing mRNA half-life and stability. The RNA splint oligonucleotide in the 5 ’-3’ direction can contain nucleotides that hybridize to the 3’ end of the mRNA of the mRNA as well as the secondary oligonucleotide containing the inverted 3 ’-3’ dT. Further, the RNA splint oligonucleotide in the 5 ’-3’ direction can contain nucleotides that hybridize to the poly(A) tail of the mRNA as well as the secondary oligonucleotide containing the inverted 3 ’-3’ dT. The secondary oligonucleotide that is complementary to the splint oligonucleotide terminates in a 3 ’-3’ inverted dT, thereby inhibiting activity of 3 ’-5’ exonucleases.

[0316] An “effective amount” or “therapeutically effective amount” of an active agent or therapeutic agent such as the mRNA complex described herein is an amount sufficient to produce the desired effect. For example, such effective amount may cause an increase of expression of a target protein in comparison to the normal expression level detected in the absence relative to the control by about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%. Suitable assays for measuring expression of a target gene or target sequence include, e.g., examination of protein or RNA levels, using techniques known to those of skill in the art such as dot blots, northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, as well as phenotypic assays known to those of skill in the art.

[0317] In this disclosure, “substantial identity” refers to a sequence that hybridizes to a reference sequence under stringent conditions, or to a sequence that has a specified percent identity over a specified region of a reference sequence.

[0318] In this disclosure, the phrase “stringent hybridization conditions” refers to conditions under which a nucleic acid may hybridize to its target sequence, typically in a complex mixture of nucleic acids, but to no other sequences. Stringent hybridization conditions may be sequence-dependent and may be different in different circumstances. Longer sequences may hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, “Techniques in Biochemistry and Molecular Biology — Hybridization with Nucleic Probes, Overview of principles of hybridization and the strategy of nucleic acid assays” (1993). The entire content of this publication is incorporated herein by reference.

[0319] Generally, a stringent hybridization condition may be about 5 °C to 10 °C lower than the thermal melting point (Tm) of a specific sequence at a defined ionic strength, pH. The Tmis the temperature (under defined ionic strength, pH, and nucleicRef. ParB 24001; 07800.002W01 concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium).

[0320] Stringent hybridization conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, preferably 10 times background hybridization.

[0321] Exemplary stringent hybridization conditions may be as follows: about 50% formamide, 5*SSC, and 1% SDS, and incubating at about 42 °C; or, 5*SSC, about 1% SDS, incubating at about 65 °C, with a wash in about 0.2* SSC, and about 0.1% SDS at about 65 °C. For PCR, a temperature of about 36 °C is typical for low stringency amplification, although hybridizing temperatures may vary in a range of about 32 °C to about 48 °C depending on primer length. For high-stringency PCR amplification, a temperature of about 62 °C is typical, although high stringency hybridizing temperatures may be in a range of about 50 °C to about 65° C., depending on the primer length and specificity. Typical cycle conditions for both high and low stringency amplifications include a denaturation phase at about 90 °C - 95 °C for about 30 seconds to about 2 minutes, an hybridizing phase lasting for about 30 seconds to about 2 minutes, and an extension phase at about 72 °C for about 1-2 minutes. Protocols and guidelines for low and high stringency amplification reactions are provided, e.g., in Innis et al., PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. N.Y. (1990). The entire content of this publication is incorporated herein by reference. For denaturing the mRNA and Str-O-Nuc to prepare for hybridization, a temperature of about 90 °C to about 95 °C, preferably about 95 °C, for about 30 seconds to 5 minutes, preferably 3 minutes, is typical. For hybridizing the mRNA and Str-O-Nuc, a temperature of about 25 °C to about 35 °C, preferably 25°C, depending on primer length, at a rate of 1 °C / second, is typical. For denaturing the mRNA and Str-O-Nuc, denaturants may also optionally be used.Denaturants for this purpose can include urea, formamide, formaldehyde, glyoxal, DMSO, mercuric hydroxide, guanidine thiocyanate, guanidinium hydrochloride, and SDS.

[0322] Other methods that do not include denaturing the RNA may also be used in the manufacture of the mRNA complexes of this disclosure. For example, the protocols and guidelines provided, e.g., in Ki eft et al., A General Method for Rapid and Nondenaturing Purification ofRNAs, RNA Society, (2004). The entire content of this publication is incorporated herein by reference.Ref. ParB 24001; 07800.002W01

[0323] Nucleic acids that do not hybridize with each other under stringent conditions may still be substantially identical if the polypeptides that they encode are substantially identical. This may occur, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions. Exemplary “moderately stringent hybridization conditions” include a hybridization in a buffer of about 40% formamide, about 1 M NaCl, about 1% SDS at about 37° C., and a wash in 1 *SSC at about 45° C. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions may be utilized to provide conditions of similar stringency. Additional guidelines for determining hybridization parameters are provided in numerous references, e.g., Ausubel et al., eds, Current Protocols in Molecular Biology. The entire content of this publication is incorporated herein by reference.

[0324] In this disclosure, the terms “substantially identical” or “substantial identity,” in the context of two or more nucleic acids, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides that are the same (ie., at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or or at least about 95% identity over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. This definition, when the context indicates, also refers analogously to the complement of a sequence. Preferably, the substantial identity exists over a region that is at least about 5, or at least about 10, or at least about 15, or at least about 20, or at least about 25, or at least about 30, or at least about 35, or at least about 40, or at least about 45, or at least about 50, or at least about 55, or at least about 60 nucleotides in length.

[0325] For sequence comparison, typically, one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.Ref. ParB 24001; 07800.002W01

[0326] A “comparison window,” as used herein, includes reference to a segment of any one of several contiguous positions from about 5 to about 60, or about 10 to about 45, or about 15 to about 30, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, Adv. AppL Math., 2:482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. Mol. BioL, 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology, Ausubel et al., eds. (1995 supplement)). The entire content of each of these references, including its supplemental content, if available, is incorporated herein by reference.

[0327] A preferred example of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res., 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol., 215:403-410 (1990), respectively. BLAST and BLAST 2.0 are used, with the parameters described herein, to determine the percent sequence identity for the nucleic acids of the invention. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). The entire content of each of these references, including its supplemental content, if available, is incorporated herein by reference.

[0328] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, or less than about 0.01, or less than about 0.001.Ref. ParB 24001; 07800.002W01

[0329] The terms “treat”, “treatment”, or “treating” to the extent it relates to a disease or condition includes inhibiting the disease or condition, eliminating the disease or condition, and / or relieving one or more symptoms of the disease or condition. The terms “treat,” “treatment,” or “treating” also refer to both therapeutic treatment and / or prophylactic treatment or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as, for example, the development or spread of cancer. For example, beneficial or desired clinical results include alleviation of symptoms, diminishment of extent of disease or disorder, stabilized (z.e., not worsening) state of disease or disorder, delay or slowing of disease progression, amelioration or palliation of the disease state or disorder, and remission (whether partial or total), whether detectable or undetectable. “Treat,” “treatment”, or “treating,” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the disease or disorder as well as those prone to have the disease or disorder or those in which the disease or disorder is to be prevented. In one embodiment “treat”, “treatment”, or “treating” does not include preventing or prevention.

[0330] The phrase "therapeutically effective amount" or “effective amount” includes but is not limited to an amount of mRNA complex described herein that (i) treats or prevents the particular disease, condition, or disorder, (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein.

[0331] The term “mammal,” as used herein, refers to humans, higher non-human primates, rodents, domestic animals, cows, horses, pigs, sheep, dogs, and cats. In one embodiment, the mammal is a human. The term “patient,” as used herein, refers to any animal, including mammals. In one embodiment, the patient is a mammalian patient. In one embodiment, the patient is a human patient.

[0332] The pharmaceutical compositions of the invention can comprise one or more excipients. When used in combination with the pharmaceutical compositions of the invention the term “excipients” refers generally to an additional ingredient to provide a corresponding composition. For example, when used in combination with the pharmaceutical compositions of the invention, the term “excipients” includes, but is notRef. ParB 24001; 07800.002W01 limited to, carriers, binders, disintegrating agents, lubricants, sweetening agents, flavoring agents, coatings, preservatives, and dyes.

[0333] The active compound may also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0334] The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient, which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form should be sterile, fluid, and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions, or by using surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it may be preferable to include isotonic agents, for example, sugars, buffers, or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0335] Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.Ref. ParB 24001; 07800.002W01

[0336] Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, alcohols or glycols or water-alcohol / glycol blends, in which the present compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.

[0337] Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the user’s skin.

[0338] Useful dosages of the compounds can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Pat. No. 4,938,949.

[0339] The desired dose may conveniently be presented in a single dose, or as divided doses administered at appropriate intervals, for example, as two, three, four or more subdoses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye.

[0340] An administration route may be made using any therapeutically effective means, including injection, and including intrathecal, intraparenchymal, intracerebroventricular, intramuscular, intravenous, intravitreal, intranasal and subcutaneous.

[0341] The invention will now be illustrated by the following non-limiting Examples.EXAMPLES

[0342] Certain embodiments of the invention provide complexes and methods that may be used to express mRNA in a living cell (e.g., cells within a human body, e.g., cells in specific organs). The mRNA molecules encode one or more polypeptides to be expressed within the living cells. In some embodiments, the polypeptides are to be expressed within a diseased organism (e.g., a mammal, such as a human being), andRef. ParB 24001; 07800.002W01 expression of the polypeptide ameliorates one or more symptoms of a disease. The complexes and methods are useful for treating human diseases caused by the absence, or reduced levels, of a functional polypeptide within the human body.

[0343] Generally, mRNA encoding a therapeutic protein may be synthesized from in vitro transcription using standard methods and can include an m7G-cap, a 5’UTR, a coding sequence, a 3’UTR, and a poly(A) tail. Some of these components (e.g., 5’ UTR or 3’UTR) may be chemically synthesized and ligated onto the rest of the mRNA molecule, e.g., to include stabilizing chemical modifications such as phosphorothioate linkages and / or 2'-OH modifications (e.g., 2’-0Me, 2’-F). The 3’UTR and / or poly(A) tail may not be required. The mRNA may optionally contain 100% uridine modification (e.g, Nl- methylpseudouridine or 5-methyoxyuridine).

[0344] This disclosure’s engineered oligonucleotides, Str-O-Nucs, hybridize to a region of the mRNA. Generally, the Str-O-Nucs may be generated by chemical synthesis and may contain chemically modified nucleotides (e.g, 2’-H, 2’-OH, 2’-0Me, 2’-M0E, 2’-F, P-S linkage, 3’-inv-dT, or 3’-ddC, and / or phosphorothioate linkages). The Str-O- Nucs may be 1-200 nt in length, or 40 nt to 50 nt in length, or 50 nt to 75 nt in length, or 75 nt to 100 nt in length, or 100 nt to 125 nt in length, or 125 nt to 150 nt in length, or 150 nt to 175 nt in length, or 175 nt to 200 nt in length. The Str-O-Nuc may preferably be 15 nt in length. The Str-O-Nuc may preferably be 18 nt in length. The Str-O-Nuc may preferably be 25 nt in length. The Str-O-Nuc may preferably be 30 nt in length, including a poly(A) tail. The Str-O-Nuc may preferably be 60 nt in length, including a poly(A) tail. There may be at least one Str-O-Nuc per mRNA molecule.

[0345] This disclosure’s engineered mRNAs may comprise Plus-Nucs and Str-O-Nucs which may optionally have Str-O-Nucs hybridized to them. Generally, the Plus-Nucs may be generated by in vivo transcription as part of the DNA template encoding the Plus-Nuc or chemical synthesis and may contain chemically modified nucleotides (e.g., 2’-H, 2’- OH, 2’-0Me, 2’-M0E, 2’-F, P-S linkage, 3’-inv-dT, or 3’-ddC, and / or phosphorothioate linkages). Generally, the Str-O-Nucs are linked (e.g. by chemical linker) to the mRNA after its in vitro transcription has taken place. The Plus-Nucs and Str-O-Nucs may be 1- 200 nt in length, or 40 nt to 50 nt in length, or 50 nt to 75 nt in length, or 75 nt to 100 nt in length, or 100 nt to 125 nt in length, or 125 nt to 150 nt in length, or 150 nt to 175 nt in length, or 175 nt to 200 nt in length. The Plus-Nucs and Str-O-Nucs may preferably be 15 nt in length. The Plus-Nucs and Str-O-Nucs may preferably be 18 nt in length. The Plus-Ref. ParB 24001; 07800.002W01Nucs and Str-O-Nucs may preferably be 25 nt in length. The Plus-Nucs and Str-O-Nucs may preferably be 30 nt in length, including a poly(A) tail. The Plus-Nucs and Str-O-Nucs may preferably be 60 nt in length, including a poly(A) tail. There may be at least one Plus- Nuc and Str-O-Nuc per mRNA molecule.

[0346] In this disclosure, the mRNA molecule may be partially or substantially (e.g., close to 100%, e.g., 95%, 96%, 97%, 98% or 99%) hybridized to the Str-O-Nuc, leaving minimal to no single stranded region(s).

[0347] The Str-O-Nucs that hybridize to a specific sequence on the mRNA may generally have greater than 50% sequence complementary to the given sequence on the mRNA (e.g., there could be intentional gaps or mismatches to limit immunogenicity and weaken the hybridization strength to promote reversibility). The Str-O-Nucs that hybridize to a specific sequence on the mRNA may also generally have more than 70-90% sequence complementary to the given sequence on the mRNA (e.g., there could be intentional gaps or mismatches to limit immunogenicity and weaken the hybridization strength to promote reversibility).

[0348] A Str-O-Nuc may be conjugated to 0 targeting moiety, or targeting moiety 1, or 2 targeting moi eties (e.g., by using the 5’ and 3’ ends for conjugation). Additional targeting moieties on a Str-O-Nucs are possible if internal modifications are utilized. The Str-O-Nuc may optionally contain substantially close to 100% uridine modification (e.g., N1 -methylpseudouridine or 5-methy oxyuridine).

[0349] The Str-O-Nucs may include modifications to the 2’-OH (e.g., 2’-H, 2’-0Me, 2’ -MOE, 2’-F). The Str-O-Nucs may include modifications to the phosphodiester backbone (e.g., phosphorothioate linkages).

[0350] A Plus-Nucs may be conjugated to 0 targeting moiety, or targeting moiety 1, or 2 targeting moieties (e.g., by using the 5’ and 3’ ends for conjugation). Additional targeting moieties on Plus-Nucs are possible if internal modifications are utilized. The Plus-Nucs and may optionally contain substantially close to 100% uridine modification (e.g., N1 -methylpseudouridine or 5-methy oxyuridine).

[0351] The Plus-Nucs may include modifications to the 2’-OH (e.g., 2’-H, 2’-0Me, 2’ -MOE, 2’-F). The Plus-Nucs may include modifications to the phosphodiester backbone (e.g., phosphorothioate linkages).Ref. ParB 24001; 07800.002W01

[0352] Targeting moieties: The targeting moiety may be a sugar (e.g., GalNAc), a small molecule (e.g., Cl 6), a peptide, an antibody (e.g., IgG), an antibody fragment e.g., Fab), a nanobody / miniprotein e.g., VHH, svFv), an anti-CD5 antibody, or a nucleic acid. Generally, the targeting moiety may bind to specific receptors on a given cell type to allow for internalization of the cargo. They may also bind non-specifically to membranes, but still allow for internalization through unknown mechanisms. For example, C16 conjugates allow for internalization of small RNAs in the lung, eye, and the CNS. C16 allows for non-specific uptake of nucleic acid but the specificity is imparted by local administration of the RNA to the location of interest e.g., inhaled into the lung, injected into the eye, or injected into the cerebrospinal fluid). Targeting moieties may be naturally occurring or engineered.

[0353] Delivery methods: The mRNA complexes of this disclosure may be delivered using any standard mRNA delivery methods such as a dendrimer, an exosome, a lipid nanoparticle, a liposome-like nanoparticle, a lipoprotein particle, a lipid-polymer hybrid nanoparticle, a nanostructured lipid carrier, a platelet membrane -coated nanoparticle, a protein-based nanoparticle, a polymer, a polymeric nanoparticle, a protamine, a solid nanoparticle, or a virus-like particle. mRNA complexes that include a Str-O-Nuc can also be delivered without additional delivery vehicles. As stated in the sections related to targeting moieties, these Str-O-Nucs may be conjugated to targeting moieties to allow for specific delivery of the mRNA complex to desired cell types.

[0354] Alternative or additional modes of stabilization: in this disclosure, the entire mRNA may not be covered with the Str-O-Nucs. Regions of the mRNA may be stabilized with the use of the Str-O-Nucs described herein, using (especially the 5’UTR or 3’UTR, for example). Such stabilization methods include (a) engineering structure into the mRNA itself: e.g., by designing hairpins or using LinearDesign-type approaches to maximize overall secondary structure, (see, e.g., Zhang et al., Nature, 621, 396-403 (2023)).

[0355] For example, a 3’UTR that is complementary to the entire coding sequence (with sufficient mismatches to avoid dsRNA sensors) may be designed; or (b) Irreversible chemical modifications: including phosphorothioate linkages and / or 2'-OH modifications (2’-0Me, 2’-F); or (c) reversible chemical modifications, for example, 2’-OH modifications that are chemically reversible, and the use of these are envisioned, which can be reversed in the biofluid, endosome, and / or cytoplasm; or (d) sequence engineering: certain sequence motifs of the mRNA may be more or less prone to degradation byRef. ParB 24001; 07800.002W01 nucleases in biofluids, endosomes, and / or the cytoplasm; or a combination of such stabilization methods.

[0356] Sequences described in the Examples are presented in the Table following Example 24. In the table, each ‘C’ that is described in a sequence with the MOE modification is 5mC.

[0357] The following depicts certain aspects of certain embodiments of the invention wherein the mRNA complex impart stability to a therapeutic mRNA and allows for conjugation of interchangeable targeting moieties on the Str-O-Nucs described herein.

[0358] Example 1: Increased expression of HiBiT-12A mRNA with 3’ UTR Str-O- Nucs

[0359] The purpose of the study in this example was to demonstrate the effect on mRNA translation by hybridizing Str-O-Nucs to HiBiT mRNA containing a 12 nucleotide (nt) poly (A) tail.

[0360] HiBiT mRNA are nucleic acids that contain a 5’ Untranslated Region (UTR), Coding Sequence (CDS), a 33 nucleotide HiBiT tag, 3’ UTR, and a minimal 12 nt poly(A) tail. HiBiT mRNA was synthesized through in vitro transcription of template DNA encoding the HiBiT mRNA using a modified form of uracil, Nl-methyl-pseudouridine (Nlm-y), along with a co-translational 5’ Cap-1 structure (CleanCap). The resulting transcribed mRNA contains 100% Nlm-y modification and a 5’ Cap-1 structure.

[0361] Str-O-Nucs in this example are comprised of nucleic acids that contain 2'-O- methoxy-ethyl (2'-M0E) modifications with bases that are complementary to the HiBiT mRNA sequence at the 3’ UTR. The Str-O-Nucs used in this study are StrO53-O59. Str-O- Nucs were hybridized to the HiBiT mRNA by heating the mixture of nucleic acids to 95 degrees C. for 3 minutes, followed by a cooling step to 25 degrees C., set at a cooling rate of 0.1 degrees C. per second.

[0362] Following the hybridization reaction of Str-O-Nucs to HiBiT mRNA, 100 nanograms (ng) of the hybridized mRNA were transfected into the HepG2 cell line, utilizing the mRNA lipofection reagent Lipofectamine Messenger Max (LMM). As a control, lOOng of HiBit mRNA that was not hybridized to Str-O-Nucs were also transfected into the adherent cell line HepG2. Following the transfection, HepG2 cells were lysed 4, 24, 48, or 72 hours post-transfection utilizing the Nano-Gio HiBiT LyticRef. ParB 24001; 07800.002W01Detection System. Briefly, translated HiBiT protein binds to LgBiT reagent to create a luminescent signal that can be read by a luminescence plate reader. The level of luminescence detected directly correlates with the concentration of HiBiT protein lysed from cells.

[0363] As shown in Figure 5, compared to HiBiT mRNA that was not hybridized to Str-O-Nucs prior to transfection, HiBiT mRNA hybridized to Str-O-Nucs exhibited a higher concentration of HiBiT protein at 24, 48, and 72 hours post-transfection. These results suggest that hybridization of the 3’ UTR Str-O-Nucs increases translational efficiency and durability.

[0364] Example 2: Increased expression of HiBiT- 120 A mRNA with 3’ UTR Str-O- Nucs

[0365] The purpose of the study in this example is to demonstrate the effect on mRNA translation by hybridizing Str-O-Nucs to HiBiT mRNA containing a 120 nucleotide (nt) poly(A) tail.

[0366] HiBiT mRNA are nucleic acids that contain a 5’ Untranslated Region (UTR), Coding Sequence (CDS), a 33 nucleotide HiBiT tag, 3’ UTR, and a 120 nt poly(A) tail. HiBiT mRNA was synthesized through in vitro transcription of template DNA encoding the HiBiT mRNA using a modified form of uracil, Nl-methyl-pseudouridine (Nlm-y), along with a co-translational 5’ Cap-1 structure (CleanCap). The resulting transcribed mRNA contains 100% Nlm-y modification and a 5’ Cap-1 structure.

[0367] Str-O-Nucs in this example are comprised of nucleic acids that contain 2'-O- methoxy-ethyl (2'-M0E) modifications with bases that are complementary to the HiBiT mRNA sequence at the 3’ UTR. The Str-O-Nucs used in this study are StrO53-O58 and Str060. Str-O-Nucs were hybridized to the HiBiT mRNA by heating the mixture of nucleic acids to 95 degrees C. for 3 minutes, followed by a cooling step to 25 degrees C., set at a cooling rate of 0.1 degrees C. per second.

[0368] Following the hybridization reaction of Str-O-Nucs to HiBiT mRNA, 100 nanograms (ng) of the hybridized mRNA were transfected into the HepG2 cell line, utilizing the mRNA lipofection reagent Lipofectamine Messenger Max (LMM). As a control, lOOng of HiBit mRNA that was not hybridized to Str-O-Nucs were also transfected into the adherent cell line HepG2. Following the transfection, HepG2 cellsRef. ParB 24001; 07800.002W01 were lysed 4, 24, 48, or 72 hours post-transfection utilizing the Nano-Gio HiBiT Lytic Detection System. Briefly, translated HiBiT protein binds to LgBiT reagent to create a luminescent signal that can be read by a luminescence plate reader. The level of luminescence detected directly correlates with the concentration of HiBiT protein lysed from cells.

[0369] As shown in Figure 6, compared to HiBiT mRNA that was not hybridized to Str-O-Nucs prior to transfection, HiBiT mRNA hybridized to Str-O-Nucs exhibited a higher concentration of HiBiT-tagged protein at 24, 48, and 72 hours post-transfection. These results suggest that hybridization of the 3’ UTR Str-O-Nucs increases translational efficiency and durability.

[0370] Example 3: Str-O-Nuc length and chemistry optimization increases expression of an mRNA without a poly(A) tail

[0371] The purpose of this study is to determine optimal Str-O-Nuc length and chemistry for an mRNA without a poly (A) tail.

[0372] This study uses mRNA encoding Enhanced Green Fluorescent Protein (eGFP) fused to a degron (eGFP[deg]) with or without a 120 nucleotide (nt) poly(A) tail (denoted as 120 A or 0A, respectively). eGFP [deg] mRNA was synthesized through in vitro transcription of template DNA encoding the eGFP[deg] mRNA using a modified form of uracil, Nl-methyl-pseudouridine (Nlm-y), along with a co-translational 5’ Cap-1 structure (CleanCap). The resulting transcribed mRNA contains 100% Nlm-y modification and a 5’ Cap-1 structure.

[0373] The Str-O-Nuc, as shown in Figure 7, bound to the eGFP[deg] mRNAs without a poly(A) tail in this study are: 1) hdRNA092 - Str061 comprising a 3’ loop-back end blocker with 2'-O-methoxyethyl (2'-M0E) backbone, 2) hdRNA093 - Str355 comprising an unmodified 2-hydroxy (OH) backbone that binds to the 3’ terminal 18 nt in the 3’ untranslated region (3' UTR) of the mRNA, 3) hdRNA094 - Str356 modified with a 2’-O- methyl (OMe) backbone that binds to the 3’ terminal 18 nt in the 3' UTR of the mRNA, 4) hdRNA095 - Str357 modified with a 2'-M0E backbone that binds to the 3’ terminal 18 nt in the 3' UTR of the mRNA, 5) hdRNA096 - Str358 modified with a 2'-M0E phosphorothioate (PS) backbone that binds to the 3’ terminal 18 nt in the 3’ UTR of the mRNA, 6) hdRNA097 - Str359 modified with a 2'-M0E backbone with winged lockedRef. ParB 24001; 07800.002W01 nucleic acids (LNAs), comprised of 3 consecutive LNAs on the 5’ and 3’ end of the Str-O- Nuc, that binds to the 3’ terminal 18 nt in the 3' UTR of the mRNA, 7) hdRNA098 - Str360 modified with winged LNAs, comprised of 3 consecutive LNAs on the 5’ and 3’ end of the Str-O-Nuc, and internal, alternating LNAs and 2'-M0E, that binds to the 3’ terminal 18 nt in the 3 ’-UTR of the mRNA, 8) hdRNA099 - 25 nt Str361 modified with a 2'-M0E backbone that binds to the 3’ terminal 25 nt in 3 ’-UTR of the mRNA, and 9) hdRNAlOO - 25 nt Str362 comprising of a phosphorodiamidate morpholino oligomer (PMO) backbone that binds to the 3’ terminal 25 nt in the 3’ UTR of the mRNA.

[0374] Controls in the experiment included: 1) untreated condition, 2) Lipofectamine Messenger Max (LMM) vehicle only condition, 3) hdRNA102 - a 0 nt poly(A) eGFP mRNA mixed with a random PMO negative control (Str365), 4) hdRNA090 - 120 nt poly(A) eGFP mRNA only (no Str-O-Nuc), and (5) hdRNA091- 0 nt poly(A) eGFP mRNA only (no Str-O-Nuc).

[0375] 100 nanograms (ng) of the hybridized mRNA were transfected into the adherent HEK293T cell line, utilizing the mRNA lipofection reagent LMM. At the indicated time point, cells were lysed using Mammalian Protein Extraction Reagent (MPER) and GFP fluorescence was measured on a plate reader.

[0376] As shown in Figure 8, eGFP mRNA with a 120-nt poly(A) tail had the highest level of expression measured by fluorescence at all four timepoints, while eGFP mRNA without a poly(A) tail showed no expression, as expected. Among all mRNA constructs tested that lacked a poly(A) tail, hdRNA098 enabled the highest expression of eGFP. hdRNA099 had the second highest expression of eGFP over the timecourse, followed by hdRNA097, hdRNA095, and hdRNAlOO.

[0377] Examining Str-O-Nucs modifications that bind to the terminal 18nt of the mRNA 3’ UTR end, mixed LNA / 2’MOE with 3x 5’ and 3’ winged LNA nucleotides (hdRNA098) enabled the highest expression of eGFP. Examining hybridization length of the Str-O-Nuc to the 3’ UTR end of the mRNA, 25nt outperformed 18nt in regard to eGFP fluorescence within mRNAs with the 2’ -MOE modification. Lastly, eGFP expression from 0A mRNA complexed with a PMO-modified Str-O-Nuc was lower than with a 2'-M0E- modified Str-O-Nuc of the same length (25nt) and 3’ UTR-binding site.Ref. ParB 24001; 07800.002W01

[0378] Example 4: Str-O-Nuc binding to 3’ end and adding a poly(A) sequence to the Str-O-Nuc stabilizes an mRNA without a poly(A) tail

[0379] The purpose of this study is to determine how Str-O-Nuc positioning and homopolymer overhangs affect expression from mRNA lacking a poly(A) tail.

[0380] This study uses mRNA encoding Enhanced Green Fluorescent Protein (eGFP) fused to a degron (eGFP[deg]) without a 120 nucleotide (nt) poly(A) tail (denoted as 0A). eGFP[deg] mRNA was synthesized through in vitro transcription of template DNA encoding the eGFP[deg] mRNA using a modified form of uracil, N1 -methylpseudouridine (Nlm-y) along with a co-translational 5’ Cap-1 structure (CleanCap). The resulting transcribed mRNA contains 100% Nlm-y modification and a 5’ Cap-1 structure.

[0381] In this study, the following Str-O-Nucs, as depicted in Figure 9, were hybridized with eGFP[deg] mRNA lacking a poly(A) tail: 1) hdRNA127 - a Str-O-Nuc (Str094) that binds to 15 nucleotide nt within the 3' UTR of eGFP (0A); 2) hdRNA129 - a Str-O-Nuc (Str074) that binds to 15 nt within the 3' UTR of eGFP (0A) and additionally contains 30 unmodified adenosine ribonucleotides (rAs) followed by 6 terminal 2'-O- methoxyethyl (2'-MOE) / phosphorothioate (PS)-modified nucleotides; 3) hdRNA130 - a Str-O-Nuc (Str397) that binds to 15 nt within the 3'-UTR of eGFP (0A) and additionally contains 30 unmodified uridine ribonucleotides (rUs) followed by 6 terminal 2'-MOE / PS- modified nucleotides; 4) hdRNA131 - a Str-O-Nuc (Str096) that binds to 18 nt at the 3’ end of eGFP (0A), 5) hdRNA132 - a Str-O-Nuc (Str392) that binds to 18 nt at the 3' end of eGFP (0A) and additionally contains 30 unmodified rAs followed by 6 terminal 2’- MOE / PS -modi fied nucleotides; the homopolymer is on the 3’ end of the Str-O-Nuc, ‘downstream’ of the hybridization region; 6) hdRNA133 - a Str-O-Nuc (Str393) that binds to 18 nt at the 3' end of eGFP (0A) and additionally contains 30 unmodified rAs followed by 6 terminal 2’-MOE / PS-modified nucleotides; the homopolymer is on the 5’ end of the Str-O-Nuc, 'upstream' of the hybridization region.

[0382] Relevant controls for these modifications are: 1) hdRNA126 - eGFP (0A) mRNA only (no Str-O-Nuc), 2) Lipofectamine Messenger Max (LMM) vehicle only condition, and 3) an untreated condition.

[0383] Str-O-Nuc were hybridized to the eGFP mRNAs by heating the mixture of nucleic acids to 95 degrees C. for 3 minutes, followed by a cooling step to 25 degrees C.,Ref. ParB 24001; 07800.002W01 set at a cooling rate of 0.1 degrees C. per second in refold buffer (50 mM Tris pH 7, 1 mM Ethylenediaminetetraacetic acid (EDTA), 150 mM Sodium Chloride (NaCl)).

[0384] Following the hybridizing of Str-O-Nucs to eGFP mRNA, 100 nanograms (ng) of the hybridized mRNA were transfected into the HEK293T cell line, utilizing the mRNA lipofection reagent LMM. HEK293T cells were lysed 4, 24, 48, or 72 hours posttransfection and GFP fluorescence was measured.

[0385] As shown in Figure 10, the expression of eGFP in cells transfected with only eGFP mRNA lacking a poly(A) tail was not detected, as expected. hdRNA129, containing the 3’UTR poly(A) Str-O-Nuc slightly increased GFP fluorescence over background at 24 hours post-transfection. Notably, hdRNA127 and hdRNA130, which do not contain stretches of 30 rAs on the Str-O-Nuc, did not boost expression of the 0A over background, suggesting the effect in hdRNA129 was due to the presence of the 30 rAs on the Str-O- Nucs. Comparing the Str-O-Nuc with 18nt complementary region to the 3’ end of eGFP with the 30 rAs stretch and a terminal 6nt with PS linkage and 2’-M0E modifications, the reverse orientation (hdRNA133) enabled higher eGFP signal compared to the forward orientation (hdRNA132). hdRNA133 also had the highest overall eGFP fluorescence over all timepoints. The kinetics and magnitude of eGFP fluorescence of hdRNA131, a Str-O- Nuc complementary to the 3’ end of eGFP (0A), was similar to hdRNA132, showing the importance of the orientation of the poly(A) on the Str-O-Nuc.

[0386] Example 5: Further optimization of 3'UTR Str-O-Nucs increases the expression of an mRNA without a poly (A) tail

[0387] The purpose of this study is to further evaluate the effect of Str-O-Nuc length, chemistry, and homopolymer addition on the expression of an mRNA lacking a poly(A) tail.

[0388] This study uses mRNA encoding Firefly luciferase (Fluc[Optl]) with or without a 120 nucleotide (nt) poly (A) tail (denoted as 120 A or 0A, respectively). Fluc[Optl] mRNA was synthesized through in vitro transcription of template DNA encoding the Fluc[Optl] mRNA using a modified form of uracil, N1 -methylpseudouridine (Nlm-y) along with a co-translational 5’ Cap-1 structure (CleanCap). The resulting transcribed mRNA contains 100% Nlm-y modification and a 5’ Cap-1 structure.Ref. ParB 24001; 07800.002W01

[0389] In this study, the following Str-O-Nucs were hybridized with FlucfOptl] mRNA lacking a poly(A) tail: 1) hdRNA423 - a Str-O-Nuc (Str360) with winged locked nucleic acids (LNAs), comprised of 3 consecutive LNAs on the 5’ and 3’ end of the Str-O- Nuc, and internal, alternating LNAs and 2'-O-methoxyethyl (2'-M0E), that binds to the 3’ terminal 18 nt in the 3’ UTR of the Flue mRNA; 2) hdRNA424 - a Str-O-Nuc (Str533) with a 5’ end containing 6 2’-MOE / phosphorothioate (PS)-modified nts, an internal stretch of 30 adenosine ribonucleotides (rA), and a 18nt 3’ end containing 3 consecutive LNAs flanking either end of a 12nt stretch of alternating 2’-M0E and LNA-modified nts that bind the 3’ end of Flue mRNA; 3) hdRNA425 - a Str-O-Nuc (Str551) comprised of 50 2’- MOE-modified nts that bind to the 3’ end of the Flue mRNA; 4) hdRN426 - a Str-O-Nuc (Str552) that is comprised of 6 PS-modified nts at the 5’ end of the Str-O-Nuc, an internal 30 rA stretch, and a 3’ end containing 50 2’-MOE-modified nts that bind the 3’ end of Flue mRNA; 5) hdRNA427 - a Str-O-Nuc (Str549) comprised of 70 2’-MOE-modified nts that bind to the 3 ’ end of the Flue mRNA.

[0390] Relevant controls for these modifications are: 1) hdRNA416 - FlucfOptl] mRNA containing a 120 nt poly(A) tail only (no Str-O-Nuc), 2) hdRNA422 - FlucfOptl] mRNA that does not contain a poly (A) tail only (no Str-O-Nuc) 3) Lipofectamine Messenger Max (LMM) vehicle only condition, and (4) an untreated condition.

[0391] Str-O-Nucs were hybridized to the Flue mRNA lacking a poly(A) tail by heating the mixture of nucleic acids to 95 degrees C. for 3 minutes, followed by a cooling step to 25 degrees C., set at a cooling rate of 0.1 degrees C. per second in refold buffer (50 mM Tris pH 7, 1 mM Ethylenediaminetetraacetic acid (EDTA), 150 mM Sodium Chloride (NaCl)).

[0392] Following the hybridizing reaction of Str-O-Nuc to Flue mRNAs, 100 nanograms (ng) of differentially complexed mRNA was transfected into HEK293T cells with Lipofectamine Messenger Max (LMM). Cells were incubated and lysed 4, 24, 48, and 72 hours (hrs) post-transfection using Promega Gio Lysis Buffer (Cat#E266A). Luciferase signal was quantified using the Promega One-Gio Luciferase Assay System (Cat#E6120).

[0393] As shown in Figure 11, the expression of Flue in cells transfected with only Flue mRNA lacking a poly(A) tail was not detected, as expected. hdRNA425, containing a 50nt Str-O-Nuc composed to 100% 2’-M0E modifications binding to the terminal 50nt of the 3 ’UTR increased Flue expression most, but was still less than an Flue mRNARef. ParB 24001; 07800.002W01 containing a 120-nt poly(A) tail. A similar Str-O-Nuc (Str549) that binds the 3’ terminal 70nt of the 3’UTR did not increase expression as much as Str551, and addition of 30 rA nucleotides to the mixed LNA or the 50nt 2’ -MOE Str-O-Nucs (hdRNA423 compared to hdRNA424, and hdRNA425 compared to hdRNA426) did not noticeably increase expression.

[0394] Example 6: Addition of a ‘Plus Nuc’ to an mRNA without a poly(A) tail for Str-O-Nuc binding increases the expression of an mRNA: Str-O-Nuc complex

[0395] The purpose of this study is to evaluate the effect of attaching a Plus Nuc to a mRNA that does not contain a poly(A) tail and subsequently hybridizing the Plus Nuc- containing mRNA to a Str-O-Nuc.

[0396] This study uses mRNA encoding Firefly luciferase (Fluc[Optl]) with or without a 120 nucleotide (nt) poly (A) tail (denoted as 120 A, 0A, or 0A + 16nt, respectively). Fluc[Optl] mRNA was synthesized through in vitro transcription of template DNA encoding the Fluc[Optl] mRNA with or without an additional sequence (+16nt) added after the 3'-UTR (referred to as a Plus Nuc), using a modified form of uracil, Nl-methyl-pseudouridine (Nlm-y) along with a co-translational 5’ Cap-1 structure (CleanCap). The resulting transcribed mRNA contains 100% Nlm-y modification and a 5’ Cap-1 structure.

[0397] In this study, Str-O-Nucs were hybridized with Fluc[Optl] mRNA lacking a poly(A) tail. The following Str-O-Nucs were hybridized with Fluc[Optl] (0+16nt) mRNA: 1) hdRNA429 - a Str-O-Nuc (Str394) comprised of 16 2'-O-methoxyethyl (2'-M0E)- modified nt sequence complementary to the 3’ end of Fluc[Optl] (0+16nt) mRNA; 2) hdRNA430 - a Str-O-Nuc (Str516) with winged locked nucleic acids (LNAs), comprised of 3 consecutive LNAs on the 5’ and 3’ end of the Str-O-Nuc, and internal, alternating LNAs and 2'-O-methoxy ethyl (2'-M0E) nts complementary to the 3’ end of Fluc[Optl] (0+16nt) mRNA; 3) hdRNA 431 - A Str-O-Nuc (Str395) comprised of 16 2’-M0E- modified nts at the 5’ end of the Str-O-Nuc that are complementary to 3’ end of Fluc[Optl] (0+16nt) mRNA, an internal stretch of 30 adenosine ribonucleotides (rAs), and 6 phosphorothioate (PS)-modified nts on the 3’ end of the Str-O-nuc; 4) hdRNA432 - a Str-O-Nuc (Str396) comprised of 16 2’-MOE-modified nts at the 3’ end of the Str-O-Nuc that are complementary to 3’ end of Flue [Opt 1] (0+16nt) mRNA, an internal stretch of 30Ref. ParB 24001; 07800.002W01 adenosine rAs, and 6 phosphorothioate (PS)-modified nts on the 5’ end of the Str-O-Nuc; 5) hdRNA433 - a Str-O-Nuc (Str538) comprised of an internal 16 2’-MOE-modifed nts complementary to the 3’ end of FlucfOptl] mRNA flanked by stretches of 30 rAs terminating in 6 PS-modified nts; 6) hdRNA434 - a Str-O-Nuc (Str548) comprised of 16 2’-MOE-modified nts at the 3’ end of the Str-O-Nuc that are complementary to 3’ end of FlucfOptl] (0+16nt) mRNA, an internal stretch of 60 rAs, and 6 phosphorothioate (PS)- modified nts on the 5’ end of the Str-O-Nuc. Additionally hdRNA425, FlucfOptl] without a poly(A) tail or Plus Nuc is hybridized to a Str-O-Nuc (Str551) comprised of 50 2’-M0E- modified nts that bind to the 3’ end of the FlucfOptl] (0A) mRNA.

[0398] Relevant controls for these modifications are: 1) hdRNA416 - FlucfOptl] mRNA containing a 120 nt poly(A) tail only (no Str-O-Nuc), 2) hdRNA428 - FlucfOptl] mRNA that does not contain a poly(A) tail but does contain a Plus Nuc only (no Str-O- Nuc), 3) Lipofectamine Messenger Max (LMM) vehicle only condition, and 4) an untreated condition.

[0399] Str-O-Nucs were hybridized to the Flue mRNA lacking a poly(A) tail with or without a 16 nt Plus Nuc by heating the mixture of nucleic acids to 95 degrees C. for 3 minutes, followed by a cooling step to 25 degrees C., set at a cooling rate of 0.1 degrees C. per second in refold buffer (50 mM Tris pH 7, 1 mM Ethylenediaminetetraacetic acid (EDTA), 150 mM Sodium Chloride (NaCl)).

[0400] Following the hybridizing reaction of Str-O-Nuc to Flue mRNAs, 100 nanograms (ng) of differentially complexed mRNA was transfected into HEK293T cells with Lipofectamine Messenger Max (LMM). Cells were incubated and lysed 4, 24, 48, and 72 hours (hrs) post-transfection using Promega Gio Lysis Buffer (Cat#E266A).Luciferase signal was quantified using the Promega One-Gio Luciferase Assay System (Cat#E6120).

[0401] As shown in Figure 12, the expression of Flue in cells transfected with only Flue mRNA lacking a poly(A) tail and containing a 'Plus Nuc' (hdRNA428) was not detected, as expected. hdRNA425 was included for reference for a high expressing hdRNA that does not include a ‘Plus Nuc.’ hdRNA429, containing a 16-nt MOE-modified Str-O-Nuc bound to the Plus Nuc at the mRNA 3' end, had significant expression above background. Addition of LNA modifications (hdRNA430) or 30 'A' residues with MOE / PS-stabilized nucleotides at either or both ends (hdRNA431, 432, and 433), had limited impact on expression. In contrast, addition of 60 'A' residues with 2’-MOE / PS-Ref. ParB 24001; 07800.002W01 stabilized nucleotides at the 5'-end of the Str-O-Nuc (hdRNA434), increased expression to essentially that of an mRNA with a 120-nt poly(A) tail and with slightly improved durability.

[0402] Example 7: Addition of a ‘Plus Nuc’ to an mRNA without a poly(A) tail for Str-O-Nuc binding increases the expression of an mRNA: Str-O-Nuc complex

[0403] The purpose of this study is to evaluate the effect of attaching a Plus Nuc to a mRNA that does not contain a poly(A) tail, and subsequently hybridizing the Plus Nuc- containing mRNA to a Str-O-Nuc.

[0404] This study uses mRNA encoding Firefly luciferase (Fluc[Optl]) with or without a 120 nucleotide (nt) poly (A) tail (denoted as 120 A, 0A + 16nt, or 0A +29nt, respectively). Fluc[Optl] mRNA was synthesized through in vitro transcription of template DNA encoding the Fluc[Optl] mRNA with or without an additional sequence (+16 or +29 nt) added after the 3'-UTR (referred to as a Plus Nuc), using a modified form of uracil, N1 -methyl -pseudouridine (Nlm-y) along with a co-translational 5’ Cap-1 structure (CleanCap). The resulting transcribed mRNA contains 100% Nlm-y modification and a 5’ Cap-1 structure.

[0405] In this study, Str-O-Nucs were hybridized with Fluc[Optl] mRNA lacking a poly(A) tail. The following Str-O-Nuc were hybridized with Fluc[Optl] (0+16nt) mRNA: 1) hdRNA434 - Str-O-Nuc 548 (Str548). The following Str-O-Nucs were hybridized with Fluc[Optl] (0+29nt) mRNA: 1) Str-O-Nuc 517 (Str517), 2) Str-O-Nuc 518 (Str518), 3) Str-O-Nuc 545 (Str545), 4) Str-O-Nuc 546 (Str546), 5) Str-O-Nuc 539 (Str539), 6) Str-O- Nuc 540 (Str540),and 7) Str-O-Nuc 543 (Str543). Additionally hdRNA425, Fluc[Optl] without a poly(A) tail or Plus Nuc is hybridized to a Str-O-Nuc (Str551).

[0406] Relevant controls for these modifications are: 1) hdRNA416 - Fluc[Optl] mRNA containing a 120 nt poly(A) tail only (no Str-O-Nuc), 2) hdRNA422 - Fluc[Optl] mRNA that does not contain a poly(A) tail (no Str-O-Nuc), 3) hdRNA435 - Fluc[Optl] mRNA that does not contain a poly(A) tail but does contain a Plus Nuc (no Str-O-Nuc), 4) Lipofectamine Messenger Max (LMM) vehicle only condition, and 5) an untreated condition.

[0407] Str-O-Nucs were hybridized to the Flue mRNA lacking a poly(A) tail with or without a 16 nt or 29nt Plus Nuc by heating the mixture of nucleic acids to 95 degrees C.Ref. ParB 24001; 07800.002W01 for 3 minutes, followed by a cooling step to 25 degrees C., set at a cooling rate of 0.1 degrees C. per second in refold buffer (50 mM Tris pH 7, 1 mM Ethylenediaminetetraacetic acid (EDTA), 150 mM Sodium Chloride (NaCl)).

[0408] Following the hybridizing reaction of Str-O-Nuc to Flue mRNAs, 100 nanograms (ng) of differentially complexed mRNA was transfected into HEK293T cells with Lipofectamine Messenger Max (LMM). Cells were incubated and lysed 4, 24, 48, and 72 hours (hrs) post-transfection using Promega Gio Lysis Buffer (Cat#E266A). Luciferase signal was quantified using the Promega One-Gio Luciferase Assay System (Cat#E6120).

[0409] As shown in Figure 13, the expression of Fluc[Optl] mRNA in cells transfected with only Fluc[Optl] mRNA lacking a poly(A) tail was not detected, as expected (hdRNA422 and hdRNA435). mRNAs with a +29 Plus Nuc hybridized to an 2’- O-methoxyethyl (MOE)-modified Str-O-Nuc on average had higher expression compared to mRNAs with a 120-nt poly(A) tail (hdRNA436, hdRNA438, hdRNA439, hdRNA440, hdRNA441, hdRNA442). Especially notable was the durability observed for hdRNA441, which contains a “padlock” Str-O-Nuc design in which the hybridization region is split between the 5' and 3' ends of the Str-O-Nuc and flanks an internal 60-nt poly(A) region. Reduced expression was observed when a 30 nt poly(A) region was used instead (hdRNA440) or when the split hybridization regions were oriented outward with exposed termini (hdRNA442). Similarly, addition of a 30 nt poly(A) region to the 29 nt 2'-M0E modified Str-O-Nuc had minimal increase in expression beyond just the 29 nt Str-O-Nuc (comparable expression between hdRNA436, hdRNA438 and hdRNA439).

[0410] Example 8: Addition of a ‘Plus Nuc’ to an mRNA without a poly(A) tail for Str-O-Nuc binding increases the expression of an mRNA: Str-O-Nuc complex

[0411] The purpose of this study is to evaluate the effect of attaching a Plus Nuc to a mRNA that does not contain a poly(A) tail, and subsequently hybridizing the Plus Nuc- containing mRNA to a Str-O-Nuc.

[0412] This study uses mRNA encoding Firefly luciferase (Fluc[Optl]) with or without a 120 nucleotide (nt) poly (A) tail (denoted as 120 A, 0A + 16nt, or 0A + 29nt respectively). Fluc[Optl] mRNA was synthesized through in vitro transcription of template DNA encoding the Fluc[Optl] mRNA with or without an additional sequenceRef. ParB 24001; 07800.002W01(+16 or 29nt) added after the 3'-UTR (referred to as a Plus Nuc), using a modified form of uracil, Nl-methyl-pseudouridine (Nlm-y) along with a co-translational 5’ Cap-1 structure (CleanCap). The resulting transcribed mRNA contains 100% Nlm-y modification and a 5’ Cap-1 structure.

[0413] In this study, Str-O-Nucs were hybridized with FlucfOptl] mRNA lacking a poly(A) tail. The following Str-O-Nucs were hybridized with FlucfOptl] (0+16nt) mRNA: 1) hdRNA452 - a Str-O-Nuc (Str394) comprised of 16 2'-O-methoxyethyl (2'-M0E)- modified nts complementary to the 3’ end of FlucfOptl] (0+16nt) mRNA; 2) hdRNA453 - a Str-O-Nuc (Str396) comprised of 16 2’-MOE-modified nts at the 3’ end of the Str-O- Nuc that are complementary to 3’ end of FlucfOptl] (0+16nt) mRNA, an internal stretch of 30 rAs, and 6 phosphorothioate (PS)-modified nts; 3) hdRNA454 - a Str-O-Nuc (Str548) comprised of 16 2’-MOE-modified nts at the 3’ end of the Str-O-Nuc that are complementary to 3’ end of FlucfOptl] (0+16nt) mRNA, an internal stretch of 60 rAs, and 6 phosphorothioate (PS)-modified nts. The following Str-O-Nucs were hybridized with FlucfOptl] (0+29nt) mRNA: 1) hdRNA456 - a Str-O-Nuc (Str517) comprised of a 29 2’MOE-modified nt sequence complementary to the 29nt Plus Nuc within the FlucfOptl] (0A + 29nt) mRNA; 2) hdRNA457 - a Str-O-Nuc (Str545) comprised of a 29 2’-M0E- modified nt sequence at the 3’ end of the Str-O-Nuc complementary to the 29nt Plus Nuc within the FlucfOptl] (0A + 29nt) mRNA, an internal stretch of 30 rAs, and 6 PS- modified nts; 3) hdRNA458 - a Str-O-Nuc (Str546) comprised of a 29 2’-MOE-modified nt sequence at the 5’ end of the Str-O-Nuc complementary to the 29nt Plus Nuc within the FlucfOptl] (0A + 29nt) mRNA, an internal stretch of 30 rAs, and 6 PS-modified nts; 4) hdRNA459 - a Str-O-Nuc (Str539) comprised of 30 rAs flanked by 16 and 13 2'-O- methoxy ethyl (2'-MOE)-modified nt sequences that bind to the 29nt Plus Nuc at the 3’ end of the FlucfOptl] (0A+29nt) mRNA; 5) hdRNA460 - a Str-O-Nuc (Str540) comprised of 60 rAs flanked by 16 and 13 2'-O-m ethoxy ethyl (2'-MOE)-modified nt sequences that bind to the 29nt Plus Nuc at the 3’ end of the FlucfOptl] (0A+29nt) mRNA.

[0414] Relevant controls for these modifications are: 1) hdRNA446 - FlucfOptl] mRNA containing a 120 nt poly(A) tail only (no Str-O-Nuc), 2) hdRNA452 - FlucfOptl] mRNA that does not contain a poly(A) tail but does contain a +16nt Plus Nuc only (no Str-O-Nuc), 3) hdRNA455 - FlucfOptl] mRNA that does not contain a poly(A) tail but does contain a +29 nt Plus Nuc only (no Str-O-Nuc), and 4) Lipofectamine Messenger Max (LMM) vehicle only condition.Ref. ParB 24001; 07800.002W01

[0415] Str-O-Nucs were hybridized to the Flue mRNA lacking a poly(A) tail with a +16 or 29 nt Plus Nuc by heating the mixture of nucleic acids to 95 degrees C. for 3 minutes, followed by a cooling step to 25 degrees C., set at a cooling rate of 0.1 degrees C. per second in refold buffer (50 mM Tris pH 7, 1 mM Ethylenediaminetetraacetic acid (EDTA), 150 mM Sodium Chloride (NaCl)).

[0416] Following the hybridizing reaction of Str-O-Nuc to Flue mRNAs, 100 nanograms (ng) of differentially complexed mRNA was transfected into HepG2 and HEK293T cells with Lipofectamine Messenger Max (LMM). Cells were incubated and lysed 4, 24, 48, and 72 hours (hrs) post-transfection using Promega Gio Lysis Buffer (Cat#E266A). Luciferase signal was quantified using the Promega One-Gio Luciferase Assay System (Cat#E6120).

[0417] In the context of mRNA with +29 Plus Nuc, the highest expression was consistently observed for hdRNA460 across both cell lines and all timepoints, even exceeding that of an mRNA with a 120-nt poly(A) tail. Shortening the poly(A) region of the Str-O-Nuc from 60 to 30 A's (hdRNA459) led to a slight drop in expression. A Str-O- Nuc that contained only a 29-nt, 2’-MOE-modified hybridization region (hdRNA456) also enabled robust Flue expression exceeding that of an mRNA with a 120-nt poly(A) tail, (see Figure 14).

[0418] Example 9: Plus Nuc: Str-O-Nuc complexes express more protein than a standard poly(A)-containing mRNA in primary human T-cells

[0419] The purpose of this study is to evaluate the effect of the hybridization of a Str- O-Nuc to a Plus Nuc attached to an mRNA lacking a poly(A) tail in primary cells.

[0420] This study uses mRNA encoding Firefly luciferase (Fluc[Optl]) with or without a 120 nucleotide (nt) poly(A) tail (denoted as 120A or 0A + 29nt, respectively). Fluc[Optl] mRNA was synthesized through in vitro transcription of template DNA encoding the Fluc[Optl] mRNA with or without an additional sequence (+29nt) added after the 3'-UTR (referred to as a Plus Nuc), using a modified form of uracil, Nl-methyl- pseudouridine (Nlm-y) along with a co-translational 5’ Cap-1 structure (CleanCap). The resulting transcribed mRNA contains 100% Nlm-y modification and a 5’ Cap-1 structure.

[0421] In this study, Str-O-Nucs were hybridized with Fluc[Optl] mRNA lacking a poly(A) tail. The following Str-O-Nuc were hybridized with Fluc[Optl] (0+29nt) mRNA:Ref. ParB 24001; 07800.002W01 hdRNA525 - a Str-O-Nuc (Str540) comprised of a 60 adenosine ribonucleotides (rAs) flanked by 16 and 13 2' -O-m ethoxy ethyl (2'-MOE)-modified nt sequences that bind to the 29nt Plus Nuc at the 3’ end of the FlucfOptl] (0A+29nt) mRNA.

[0422] Relevant controls for these modifications are: 1) hdRNA523 - FlucfOptl] with a 120 nt poly(A) tail only (no Str-O-Nuc); 2) hdRNA524 - FlucfOptl] lacking a poly(A) tail with a 29nt Plus Nuc only (no Str-O-Nuc).

[0423] Str-O-Nucs were hybridized to the FlucfOptl] mRNA lacking a poly(A) tail by heating the mixture of nucleic acids to 95 degrees C. for 3 minutes, followed by a cooling step to 25 degrees C., set at a cooling rate of 0.1 degrees C. per second in refold buffer (50 mM Tris pH 7, 1 mM Ethylenediaminetetraacetic acid (EDTA), 150 mM Sodium Chloride (NaCl)).

[0424] Following the hybridizing reaction of Str-O-Nuc to Flue mRNAs, 100 nanograms (ng) of complexed mRNA was electroporated into primary human T cells. Cells were incubated and lysed 24, 48, 72, 96 hours (hrs) post-transfection using Promega Gio Lysis Buffer (Cat#E266A). Luciferase signal was quantified using the Promega One- Glo Luciferase Assay System (Cat#E6120).

[0425] While luciferase expression of an mRNA with only a +29 'Plus Nuc' and no poly(A) tail (hdRNA524) was essentially at background, complexing the mRNA with Str540 (hdRNA525) led to expression at or above that of the mRNA with a 120 nt poly(A) tail (hdRNA523). (see Figure 15).

[0426] Example 10: Plus Nuc: Str-O-Nuc complexes express more protein than a standard poly(A) containing mRNA in vivo

[0427] The purpose of this study is to examine the level of translation in vivo of mRNAs containing a standard 120 nt poly(A) trail versus an mRNA containing a Plus Nuc hybridized to a Str-O-Nuc.

[0428] This study uses mRNA encoding Firefly luciferase (FlucfOptl]) with or without a 120 nucleotide (nt) poly(A) tail (denoted as 120 A or 0A + 29nt respectively). FlucfOptl] mRNA was synthesized through in vitro transcription of template DNA encoding the FlucfOptl] mRNA with or without an additional sequence (+ 29nt) added after the 3'-UTR (referred to as a Plus Nuc), using a modified form of uracil, Nl-methyl-Ref. ParB 24001; 07800.002W01 pseudouridine (N 1 m-\p) along with a co-translational 5’ Cap-1 structure (CleanCap). The resulting transcribed mRNA contains 100% Nlm-y modification and a 5’ Cap-1 structure.

[0429] Str-O-Nuc 540 (Str540) was hybridized to the FlucfOptl] mRNA lacking a poly(A) tail but containing a Plus Nuc by heating the mixture of nucleic acids to 95 degrees C. for 3 minutes, followed by a cooling step to 25 degrees C., set at a cooling rate of 0.1 degrees C. per second in refold buffer (50 mM Tris pH 7, 1 mM Ethylenediaminetetraacetic acid (EDTA), 150 mM Sodium Chloride (NaCl)). Following the annealing of FlucfOptl] mRNA to Str-O-Nucs, the complex was purified by size exclusion chromatography on a SRT-SEC 2000 column (7.8 by 300 millimeters). Tris- Ethylenediaminetetraacetic acid (TE) buffer of pH 8 was used as the mobile phase and eluted at a flow rate of 1 milliliter (mL) per minute. The purified hdRNA was then encapsulated into SM-102 lipid nanoparticles using a NanoAssemblr Ignite instrument (Precision NanoSystems) according to the manufacturer’s instructions.

[0430] Female BALB / cAnNCrl (Balb / c) mice between 7 and 9 weeks old from Charles River Laboratories were used in this study. After a minimum of 4 days of acclimation in the vivarium, mice were dosed with 5 micrograms (pg) of the following mRNA lipid nanoparticles (LNPs) via intramuscular (IM) route of administration: 1) FlucfOptl] (120A), 2)Fluc[Opl] (0A+29nt), or 3) FlucfOptl] (0A+29nt) + Str540. A vehicle group (denoted as saline) was included as a negative control. Mice were anesthetized with isofluorane for the procedure.

[0431] Ten minutes prior to In Vivo Imaging System (IVIS) imaging at the indicated time points, mice were intraperitoneally (IP) injected with 200 microliters (pL) of 15 milligrams per milliliter (mg / mL) D-Luciferin (Gold Bio catalog # LUCK- 100; solubilized in sterile normal saline (Quality Biological catalog # 114-055-101), anesthetized with isofluorane, and imaged using a Perkin Elmer IVIS instrument. Mice were imaged in the lateral recumbent orientation. Image analysis was performed using Livingimage software. Animal health checks were conducted daily throughout the study.

[0432] As seen in Figure 16, Flucfoptl] (0A+29nt) + Str540 is the highest expression of translated luciferase protein, exceeding protein levels produced by FlucfOptl] (120A) mRNA across all timepoints following 48 hours (hrs). FlucfOptl] (0A + 29nt) mRNA lacking a poly(A) tail and Str-O-Nuc had the lowest expression of protein as, expected. In this study, a Str-O-Nuc hybridized to an mRNA lacking a poly(A) tail, delivered in a LNPRef. ParB 24001; 07800.002W01 in vivo, enhances protein expression and durability beyond an mRNA with a standard poly(A) tail.

[0433] Example 11: Plus Nuc:Str-O-Nuc complexes limit IP-10 immune response upon IV administration in vivo

[0434] The purpose of this study is to compare the level of circulating IP-10 in vivo when delivering mRNAs, with and without Str-O-Nucs, encapsulated with lipid nanoparticles (LNPs) via different routes of administration.

[0435] This study uses mRNA encoding Firefly luciferase (Fluc[Optl]) with or without a 120 nucleotide (nt) poly(A) tail (denoted as 120 A or 0A + 29nt respectively). Fluc[Optl] mRNA was synthesized through in vitro transcription of template DNA encoding the Fluc[Optl] mRNA with or without an additional sequence (+ 29nt) added after the 3'-UTR (referred to as a Plus Nuc), using a modified form of uracil, Nl-methyl- pseudouridine (Nlm-y) along with a co-translational 5’ Cap-1 structure (CleanCap). The resulting transcribed mRNA contains 100% Nlm-y modification and a 5’ Cap-1 structure.

[0436] Str-O-Nuc 540 (Str540) was hybridized to the Fluc[Optl] mRNA lacking a poly(A) tail but containing a Plus Nuc by heating the mixture of nucleic acids to 95 degrees C. for 3 minutes, followed by a cooling step to 25 degrees C., set at a cooling rate of 0.1 degrees C. per second in refold buffer (50 mM Tris pH 7, 1 mM Ethylenediaminetetraacetic acid (EDTA), 150 mM Sodium Chloride (NaCl)). Following the annealing of Fluc[Optl] mRNA to Str-O-Nucs, the complex was purified by size exclusion chromatography on a SRT-SEC 2000 column (7.8 by 300 millimeters). Tris- Ethylenediaminetetraacetic acid (TE) buffer of pH 8 was used as the mobile phase and eluted at a flow rate of 1 milliliter (mL) per minute. The purified hdRNA was then encapsulated into SM-102 lipid nanoparticles using a NanoAssemblr Ignite instrument (Precision NanoSystems) according to the manufacturer’s instructions.

[0437] Female BALB / cAnNCrl (Balb / c) mice between 7 and 9 weeks old from Charles River Laboratories were used in this study. After a minimum of 4 days of acclimation in the vivarium, mice were dosed with 5 micrograms (pg) of the following mRNA lipid nanoparticles (LNPs) via intramuscular (IM) or intravenous (IV) route of administration: l) Fluc[Optl] (120A), 2)Fluc[Opl] (0A+29nt), or 3)Fluc[Optl] (0A+29nt)Ref. ParB 24001; 07800.002W01+ Str540. A vehicle group (denoted as saline) was included as a negative control. Mice were anesthetized with isofluorane for the procedure.

[0438] At 6 hours (hrs) post-administration, mouse whole blood was collected into serum separator tubes (BD, catalog # 365967), incubated at room temperature for 10 to 20 minutes, then centrifuged at 2,000 x gravity (g) for 5 to 7 minutes at 4 degrees Celsius (C.). Supernatant serum was collected into a new tube and stored at -80 C. until use. Cytokines were quantified with the Milliplex Mouse Cytokine 32-Plex Discovery kit (Millipore catalog # MCYTMAG-70K-PX32) according to the manufacturer's instructions using a Luminex 200 instrument.

[0439] As seen in Figure 17, IV dosing of Fluc[Optl] (120A), an mRNA with a 120 nt poly(A) tail, led to an approximate 125 picogram per milliliter (pg / mL) increase in IP-10 levels relative to the saline control. In contrast, IV dosing of Fluc[Optl] mRNA without a poly(A) tail, with a Plus Nuc, and with or without Str540 led to minimal increases in serum IP- 10 levels above background. Levels of IP- 10 were slightly elevated in all of the IM groups, with no differences observed among the mRNAs tested. The replacement of the native 120 nt poly (A) tail with a 29 nt Plus Nuc, with or without a Str-O-Nuc hybridized to the mRNA, allowed for potential enhanced evasion of innate immune sensors, resulting in lower levels of the Interferon Regulatory Factor 3 (IRF3) / Type I Interferon pathway cytokine IP- 10.

[0440] As seen in Figure 18, levels of IL-6 were dramatically increased in all of the IM groups, with no differences observed among the mRNAs tested. In contrast, levels of IL-6 were unaffected by mRNA-LNP dosing through the IV route of administration (relative to saline control).

[0441] Example 12: 3'-UTR binding of Str-O-Nuc to a poly(A)-containing mRNA decreases expression

[0442] The purpose of this study is to evaluate the effect of hybridizing Str-O-Nucs to an mRNA containing a 120 nt poly (A) tail.

[0443] This study uses mRNA encoding Firefly luciferase (Fluc[Optl]) with a 120 nucleotide (nt) poly(A) tail (denoted as 120A). Fluc[Optl] mRNA was synthesized through in vitro transcription of template DNA encoding the Fluc[Optl] mRNA using a modified form of uracil, Nl-methyl-pseudouridine (Nlm-y) along with a co-translationalRef. ParB 24001; 07800.002W015’ Cap-1 structure (CleanCap). The resulting transcribed mRNA contains 100% Nlm-y modification and a 5’ Cap-1 structure.

[0444] In this study, Str-O-Nucs were hybridized with FlucfOptl] mRNA with a 120 nt poly(A) tail. The following Str-O-Nucs were hybridized with FlucfOptl] (120A) mRNA: 1) hdRNA368 - Str-O-Nuc 360 (Str360); 2) hdRNA369 - Str-O-Nuc 533 (Str533).

[0445] Relevant controls for these modifications are: 1) hdRNA367 - FlucfOptl] mRNA containing a 120 nt poly(A) tail only (no Str-O-Nuc); 2) an untreated condition.

[0446] Str-O-Nucs were hybridized to the FlucfOptl] (120A) mRNA by heating the mixture of nucleic acids to 95 degrees C. for 3 minutes, followed by a cooling step to 25 degrees C., set at a cooling rate of 0.1 degrees C. per second in refold buffer (50 mM Tris pH 7, 1 mM Ethylenediaminetetraacetic acid (EDTA), 150 mM Sodium Chloride (NaCl)).

[0447] Following the hybridizing reaction of Str-O-Nuc to FlucfOptl] (120A) mRNA, 100 nanograms (ng) of differentially complexed mRNA was transfected into HepG2 cells with Lipofectamine Messenger Max (LMM). Cells were incubated and lysed 4, 24, 48, and 72 hours (hrs) post-transfection using Promega Gio Lysis Buffer (Cat#E266A).Luciferase signal was quantified using the Promega One-Gio Luciferase Assay System (Cat#E6120).

[0448] As seen in Figure 19, compared to mRNA without Str-O-Nuc (hdRNA367), binding of Locked Nucleic Acid (LNA)-containing Str-O-Nuc (Str360) to the mRNA immediately upstream of the poly(A) tail in the 3'-UTR leads to a decrease in luciferase expression. This is true for a 2’-O-methoxyethyl (MOE) / LNA-modified Str-O-Nuc (Str533) containing only a hybridization region as well as for a Str-O-Nuc that also contains poly(A) and 6 terminal nucleotides with Phosphorothioate (PS) linkage and 2’- MOE modifications.

[0449] Example 13: Addition of a ‘Plus Nuc’ to a poly(A) containing mRNA for Str- O-Nuc binding increases the expression of an mRNA: Str-O-Nuc complex

[0450] The purpose of this study is to examine the effect of attaching a Plus Nuc to a mRNA with a 120 nt poly(A) tail, and hybridizing the mRNA to a Str-O-Nuc.

[0451] This study uses mRNA encoding Firefly luciferase (FlucfOptl]) with nucleotide (nt) poly(A) tail (denoted as 120A or 120A+16 nt). FlucfOptl] mRNA wasRef. ParB 24001; 07800.002W01 synthesized through in vitro transcription of template DNA encoding the FlucfOptl] mRNA with or without an additional sequence (+ 16nt) added after the 3'-UTR (referred to as a Plus Nuc), using a modified form of uracil, Nl-methyl-pseudouridine (Nlm-y) along with a co-translational 5’ Cap-1 structure (CleanCap). The resulting transcribed mRNA contains 100% Nlm-y modification and a 5’ Cap-1 structure.

[0452] In this study, Str-O-Nucs were hybridized with FlucfOptl] mRNA with a 120 nt poly(A) tail and 16 nt Plus Nuc. The following Str-O-Nucs were hybridized with FlucfOptl] (120A + 16nt) mRNA: 1) hdRNA371 - Str-O-Nuc 395 (Str395); 2) hdRNA372 - Str-O-Nuc 396 (Str396); 3) hdRNA373 - Str-O-Nuc 525 (Str525); 4) hdRNA374- Str-O- Nuc 526 (Str526); 5) hdRNA375 - Str-O-Nuc 531 (Str531); 6) hdRNA376 - Str-O-Nuc 527 (Str527); 7) hdRNA377 - Str-O-Nuc 528 (Str528); 8) hdRNA378 - Str-O-Nuc 529 (Str529); 9) hdRNA379 - Str-O-Nuc 530 (Str530); 10) hdRNA380 - Str-O-Nuc 538 (Str538).

[0453] Relevant controls for these modifications are: 1) hdRNA367 - FlucfOptl] mRNA containing a 120 nt poly (A) tail only (no Str-O-Nuc); 2) hdRNA370 - FlucfOptl] mRNA containing a 120 nt poly(A) tail and 16 nt Plus Nuc only (no Str-O-Nuc); 3) hdRNA375 - FlucfOptl] mRNA containing a 120 nt poly(A) tail and 16 nt Plus Nuc with a Str-O-Nuc that does not bind the mRNA; 4) an untreated condition.

[0454] Str-O-Nucs were hybridized to the FlucfOptl] (120A + 16nt) mRNA by heating the mixture of nucleic acids to 95 degrees C. for 3 minutes, followed by a cooling step to 25 degrees C., set at a cooling rate of 0.1 degrees C. per second in refold buffer (50 mM Tris pH 7, 1 mM Ethylenediaminetetraacetic acid (EDTA), 150 mM Sodium Chloride (NaCl)).

[0455] Following the hybridizing reaction of Str-O-Nuc to FlucfOptl] (120A + 16nt) mRNA, 100 nanograms (ng) of differentially complexed mRNA was transfected into HepG2 cells with Lipofectamine Messenger Max (LMM). Cells were incubated and lysed 4, 24, 48, and 72 hours (hrs) post-transfection using Promega Gio Lysis Buffer (Cat#E266A). Luciferase signal was quantified using the Promega One-Gio Luciferase Assay System (Cat#E6120).

[0456] As seen in Figure 20, compared to hdRNA367, the addition of a 16nt Plus Nuc after the 120 nt poly(A) tail in hdRNA370 leads to an overall decrease in luciferase expression. However, hybridization of a Str-O-Nuc to the Plus Nuc (e.g. hdRNA371)Ref. ParB 24001; 07800.002W01 eliminates the negative impact of the Plus Nuc on expression and exceeds expression compared to hdRNA367 across time points. In contrast, addition of a control Str-O-Nuc that does not bind the mRNA (hdRNA375) has the same expression profile as the absence of Str-O-Nuc (hdRNA370). Compared to Str-O-Nucs with 30 nt poly (A) and 6 terminal PS / MOE nucleotides (hdRNA370), changes to the orientation, poly(A) length, poly(A) sequence, and / or terminal nucleotides have subtle effects on expression. Overall, the addition of a Str-O-Nuc to an mRNA with a 120 nt poly(A) tail and 16 nt Plus Nuc leads to greater protein expression over time compared to an mRNA with only a 120 nt poly(A) tail.

[0457] Example 14: Addition of a ‘Plus Nuc’ to a poly(A) containing mRNA for Str- O-Nuc binding increases the expression of an mRNA: Str-O-Nuc complex

[0458] The purpose of this study is to examine the effect of attaching Plus Nuc to a mRNA with a 120 nt poly(A) tail, and hybridizing the mRNA to one or two Str-O-Nucs.

[0459] This study uses mRNA encoding Firefly luciferase (Fluc[Optl]) with nucleotide (nt) poly(A) tail (denoted as 120A or 120A+29 nt). Fluc[Optl] mRNA was synthesized through in vitro transcription of template DNA encoding the Fluc[Optl] mRNA with or without an additional sequence (+ 29nt) added after the 3'-UTR (referred to as a Plus Nuc), using a modified form of uracil, Nl-methyl-pseudouridine (Nlm-y) along with a co-translational 5’ Cap-1 structure (CleanCap). The resulting transcribed mRNA contains 100% Nlm-y modification and a 5’ Cap-1 structure.

[0460] In this study, Str-O-Nucs were hybridized with Fluc[Optl] mRNA with a 120 nt poly(A) tail and 29 nt Plus Nuc. The following Str-O-Nucs were hybridized with Fluc[Optl] (120A + 29nt) mRNA: 1) hdRNA383 - Str-O-Nuc 395 (Str395); 2) hdRNA384 - Str-O-Nuc 531 (Str531); 3) hdRNA385 - Str-O-Nuc 532 (Str532); 4) hdRNA386 - Str395 and Str531; 5) hdRNA387 - Str395 and Str532.

[0461] Relevant controls for these modifications are: 1) hdRNA367 - Fluc[Optl] mRNA containing a 120 nt poly (A) tail only (no Str-O-Nuc); 2) hdRNA382 - Flue [Opt 1] mRNA containing a 120 nt poly(A) tail and 29 nt Plus Nuc only (no Str-O-Nuc); 3) an untreated condition.

[0462] Str-O-Nucs were hybridized to the Fluc[Optl] (120A + 29t) mRNA by heating the mixture of nucleic acids to 95 degrees C. for 3 minutes, followed by a cooling step toRef. ParB 24001; 07800.002W0125 degrees C., set at a cooling rate of 0.1 degrees C. per second in refold buffer (50 mM Tris pH 7, 1 mM Ethylenediaminetetraacetic acid (EDTA), 150 mM Sodium Chloride (NaCl)).

[0463] Following the hybridizing reaction of Str-O-Nuc to FlucfOptl] (120A + 29nt) mRNA, 100 nanograms (ng) of differentially complexed mRNA was transfected into HepG2 cells with Lipofectamine Messenger Max (LMM). Cells were incubated and lysed 4, 24, 48, and 72 hours (hrs) post-transfection using Promega Gio Lysis Buffer (Cat#E266A). Luciferase signal was quantified using the Promega One-Gio Luciferase Assay System (Cat#E6120).

[0464] As seen in Figure 21, compared to mRNA that ends with a 120 nt poly (A) tail (hdRNA367), addition of 29 nt Plus Nuc after the poly(A) tail (hdRNA382) leads to an overall decrease in luciferase expression. Although binding of a 13 nt Str-O-Nuc to the 3' half of the Plus Nuc (hdRNA384 and 385) partially rescues expression, binding of two Str-O-Nucs that together cover the entire Plus Nuc (hdRNA386 and hdRNA387) is required to completely rescue expression. In contrast, binding of a 16 nt Str-O-Nuc to the 5'-half of the Plus Nuc (hdRNA383) does not rescue expression.

[0465] Example 15: Addition of r(A)60 further enhances expression of an mRNA: Str- O-Nuc complex in HepG2 cells

[0466] The purpose of this study is to examine the effect of attaching a Plus Nuc to a mRNA with a 120 nt poly (A) tail, and hybridizing the mRNA to a Str-O-Nuc containing an internal 30 or 60 nucleotide (nt) poly(A) stretch.

[0467] This study uses mRNA encoding Firefly luciferase (FlucfOptl]) with nucleotide (nt) poly(A) tail (denoted as 120A or 120A+16 nt). FlucfOptl] mRNA was synthesized through in vitro transcription of template DNA encoding the FlucfOptl] mRNA with or without an additional sequence (+ 16nt) added after the 3'-UTR (referred to as a Plus Nuc), using a modified form of uracil, Nl-methyl-pseudouridine (Nlm-y) along with a co-translational 5’ Cap-1 structure (CleanCap). The resulting transcribed mRNA contains 100% Nlm-y modification and a 5’ Cap-1 structure.

[0468] In this study, Str-O-Nucs were hybridized with FlucfOptl] mRNA with a 120 nt poly(A) tail and 16 nt Plus Nuc. The following Str-O-Nucs were hybridized withRef. ParB 24001; 07800.002W01FlucfOptl] (120A + 16nt) mRNA: 1) hdRNA448 - Str-O-Nuc (Str394); 2) hdRNA449 - Str-O-Nuc 396(Str396); 3) hdRNA450 - Str-O-Nuc 548 (Str548).

[0469] Relevant controls for these modifications are: 1) hdRNA446 - FlucfOptl] mRNA containing a 120 nt poly (A) tail only (no Str-O-Nuc); 2) hdRNA447 - FlucfOptl] mRNA containing a 120 nt poly(A) tail and 16 nt Plus Nuc only (no Str-O-Nuc); 3) an untreated condition.

[0470] Str-O-Nucs were hybridized to the FlucfOptl] (120A + 16nt) mRNA by heating the mixture of nucleic acids to 95 degrees C. for 3 minutes, followed by a cooling step to 25 degrees C., set at a cooling rate of 0.1 degrees C. per second in refold buffer (50 mM Tris pH 7, 1 mM Ethylenediaminetetraacetic acid (EDTA), 150 mM Sodium Chloride (NaCl)).

[0471] Following the hybridizing reaction of Str-O-Nuc to FlucfOptl] (120A + 16nt) mRNA, 100 nanograms (ng) of differentially complexed mRNA was transfected into HepG2 cells with Lipofectamine Messenger Max (LMM). Cells were incubated and lysed 4, 24, 48, and 72 hours (hrs) post-transfection using Promega Gio Lysis Buffer (Cat#E266A). Luciferase signal was quantified using the Promega One-Gio Luciferase Assay System (Cat#E6120).

[0472] As seen in Figure 22, compared to hdRNA446, hdRNA447 (a mRNA that contains a 120 nt poly(A) tail followed by a 16 nt Plus Nuc after the poly(A) tail) leads to an overall decrease in luciferase expression. Binding of hybridization-only Str448 to the Plus Nuc eliminates the negative impact of the Plus Nuc on expression. The addition of a 60 nt poly(A) stretch and 6 terminal Phosphorothioate (PS) / 2’-O-methoxy ethyl (MOE) nucleotides to the Str-O-Nuc (hdRNA450) leads to a further increase in expression across timepoints, beyond when the poly(A) stretch is only 30 nt (hdRNA449).

[0473] Example 16: Optimization of Plus Nuc after poly(A) tail for maximal expression in HepG2 Cells

[0474] The purpose of this study is to examine the effect of various Plus Nuc designs following a split poly(A) tail (30A and 70A separated by a 10 nucleotide (nt) linker, denoted as 30 / 70A) on a mRNA.

[0475] This study uses mRNA encoding Firefly luciferase (FlucfOptl]) with 30 / 70 poly(A) tail. FlucfOptl] mRNA was synthesized through in vitro transcription of templateRef. ParB 24001; 07800.002W01DNA encoding the FlucfOptl] mRNA with or without an additional sequence (+ 6 nt, 9 nt, 12 nt, 14 nt, 16 nt, 16 nt v2, or 16 nt v3) added after the 3'-UTR (referred to as a Plus Nuc), using a modified form of uracil, Nl-methyl-pseudouridine (Nlm-y) along with a co- translational 5’ Cap-1 structure (CleanCap). The resulting transcribed mRNA contains 100% Nlm-y modification and a 5’ Cap-1 structure.

[0476] In this study, Str-O-Nucs were hybridized with FlucfOptl] mRNA with a 30 / 70 poly(A) tail and a Plus Nuc. The following Str-O-Nuc were hybridized with FlucfOptl] (30 / 70A + 6nt) mRNA: 1) hdRNA516 - Str-O-Nuc 559 (Str559). The following Str-O- Nucs were hybridized with FlucfOptl] (30 / 70A + 9nt) mRNA: 1) hdRNA515 - Str-O-Nuc 557 (Str557); 2) hdRNA552 - Str-O-Nuc 558 (Str558). The following Str-O-Nucs were hybridized with FlucfOptl] (30 / 70A + 12nt) mRNA: 1) hdRNA514 - Str-O-Nuc 555 (Str555); 2) hdRNA521 - Str-O-Nuc 556 (Str556). The following Str-O-Nucs were hybridized with FlucfOptl] (30 / 70A + 14nt) mRNA: 1) hdRNA513 - Str-O-Nuc 553 (Str553); 2) hdRNA520 - Str-O-Nuc 554 (Str554). The following Str-O-Nucs were hybridized with FlucfOptl] (30 / 70A + 16 nt) mRNA: 1) hdRNA502 - Str-O-Nuc 548 (Str548); 2) hdRNA510 - Str-O-Nuc 394 (Str394); 3) hdRNA517 - Str-O-Nuc 395 (Str395). The following Str-O-Nucs were hybridized with FlucfOptl] (30 / 70A + 16 nt v2) mRNA: 1) hdRNA511 - Str-O-Nuc 560 (Str560); 2) hdRNA518 - Str-O-Nuc 561 (Str561). The following Str-O-Nucs were hybridized with FlucfOptl] (30 / 70A + 16 nt v3) mRNA: 1) hdRNA512 - Str-O-Nuc 562 (Str562); 2) hdRNA519 - Str-O-Nuc 563 (Str563).

[0477] Relevant controls for these modifications are: 1) hdRNA497 - FlucfOptl] mRNA with a 30 / 70 poly(A) tail; 2) hdRNA503 - FlucfOptl] mRNA with a 30 / 70 poly(A) tail with a 16 nt Plus Nuc; 3) hdRNA504 - FlucfOptl] mRNA with a 30 / 70 poly(A) tail with a 16 nt v2 Plus Nuc; 4) hdRNA505 - FlucfOptl] mRNA with a 30 / 70 poly(A) tail with a 16 nt v3 Plus Nuc; 5) hdRNA506 - FlucfOptl] mRNA with a 30 / 70 poly(A) tail with a 14 nt Plus Nuc; 6) hdRNA507 - FlucfOptl] mRNA with a 30 / 70 poly(A) tail with a 12 nt Plus Nuc; 7) hdRNA508 - FlucfOptl] mRNA with a 30 / 70 poly(A) tail with a 9 nt Plus Nuc; 8) hdRNA509 - FlucfOptl] mRNA with a 30 / 70 poly(A) tail with a 6 nt Plus Nuc; 9) Lipofectamine Messenger Max (LMM) only control.

[0478] Str-O-Nucs were hybridized to the FlucfOptl] (30 / 70A + X nt) mRNA by heating the mixture of nucleic acids to 95 degrees C. for 3 minutes, followed by a cooling step to 25 degrees C., set at a cooling rate of 0.1 degrees C. per second in refold buffer (50Ref. ParB 24001; 07800.002W01 mM Tris pH 7, 1 mM Ethylenediaminetetraacetic acid (EDTA), 150 mM Sodium Chloride (NaCl)).

[0479] Following the hybridizing reaction of Str-O-Nuc to FlucfOptl] mRNA, 100 nanograms (ng) of differentially complexed mRNA was transfected into HepG2 cells with Lipofectamine Messenger Max (LMM). Cells were incubated and lysed 4, 24, 48, and 72 hours (hrs) post-transfection using Promega Gio Lysis Buffer (Cat#E266A). Luciferase signal was quantified using the Promega One-Gio Luciferase Assay System (Cat#E6120). Figure 23 represents the total area under the curve of luciferase expression across timepoints.

[0480] As seen in Figure 23, compared to mRNA that ends with a split poly (A) tail (hdRNA497), addition of Plus Nuc after the poly(A) tail (hdRNA503-509) leads to an overall decrease in luciferase expression, with longer Plus Nucs having a greater impact on expression. Binding of a hybridization-only Str-O-Nuc to the Plus Nuc (hdRNA510- 516, in some cases Str-O-Nucs include poly(A) hybridization regions as well) largely rescues expression. The addition of 30 nt poly(A) and 6 terminal phosphorothioate (PS) / 2’-O-methoxy ethyl (MOE) nucleotides to the 3' end of the Str-O-Nuc (hdRNA517- 522) leads to a further increase in expression for the 16 nt Plus Nuc mRNAs. The sequence of the 16 nt Plus Nuc has a modest impact on expression as well (hdRNA503- 505, 510-512, and 517-519).

[0481] Example 17: Incorporation of a Plus Nuc or a Str-O-Nuc in an IVT reaction do not appreciably impact mRNA yield or quality

[0482] The purpose of this study is to determine the impact of the inclusion of Str-O- Nuc in the in vitro transcription (IVT) reaction with mRNA containing a 120 nt poly(A) tail, with or without a 16 nt Plus Nuc sequence.

[0483] This study uses mRNA encoding Firefly luciferase (FlucfOptl]) with 120 nt poly(A) tail. FlucfOptl] mRNA was synthesized through in vitro transcription of template DNA encoding the FlucfOptl] mRNA with or without an additional sequence (+ 16 nt) added after the 3'-UTR (referred to as a Plus Nuc), using a modified form of uracil, Nl- methyl-pseudouridine (Nlm-y) along with a co-translational 5’ Cap-1 structure (CleanCap). The resulting transcribed mRNA contains 100% Nlm-y modification and a 5’ Cap-1 structure.Ref. ParB 24001; 07800.002W01

[0484] mRNAs were produced through IVT from a linearized DNA template, with the Str-O-Nuc included in the transcription reaction mix, as indicated in Figure 24 and 25, at a concentration of 7 micromolar (pM). RNA was purified using a Monarch Spin RNA Cleanup Kit (NEB) and quantified by absorbance at 260 nanometers (nm) using a Nanodrop instrument. RNA integrity was analyzed on an Agilent TapeStation instrument.

[0485] Compared to mRNA with a 120 nt poly(A) tail or a 16 nt Plus Nuc after the tail that was transcribed in the absence of any Str-O-Nuc, inclusion of Str-O-Nuc 395 (Str395) in the IVT reaction had no significant impact on mRNA yield (Figure 24) or mRNA integrity (Figure 25).

[0486] Example 18: Purification of Str-O-Nuc: mRNA complexes by size exclusion chromatography

[0487] The purpose of this study is to compare the purity of mRNA transcribed in an in vitro transcription reaction, in the presence or absence of Str-O-Nuc, following size exclusion chromatography.

[0488] This study uses mRNA encoding Firefly luciferase (Fluc[Optl]) with 120 nt poly(A) tail. Fluc[Optl] mRNA was synthesized through in vitro transcription (IVT) of template DNA encoding the Fluc[Optl] mRNA with or without an additional sequence (+ 16 nt) added after the 3'-UTR (referred to as a Plus Nuc), using a modified form of uracil, Nl-methyl-pseudouridine (Nlm-y) along with a co-translational 5’ Cap-1 structure (CleanCap). The Str-O-Nuc (Str395) was included in the transcription reaction mix, as indicated in Figure 26, at a concentration of 7 micromolar (pM). The resulting transcribed mRNA contains 100% Nlm-y modification and a 5’ Cap-1 structure.

[0489] Following IVT, the hybridized mRNA was purified by size exclusion chromatography on an SRT-SEC 2000 column (7.8x300 millimeters (mm)) using Tris- Ethylenediaminetetraacetic acid (TE) (pH 8) as the mobile phase and flow rate of 1 milliliter per minute (mL / min).

[0490] The chromatogram following size exclusion chromatography is depicted in Figure 26, mRNA_lot041 and 042 (Flucfoptl (120A) and Flucfoptl] (120A+16nt)) run nearly identically on the size exclusion column and have overlapping peaks on the chromatogram. The peak for mRNA_lot043 (Flucfoptl] (120A+16nt) + Str395) is shiftedRef. ParB 24001; 07800.002W01 slightly to the left, reflective of its slightly larger size. No other peaks were observed indicating uniform fractions of RNA in all three conditions.

[0491] Example 19: Quantification of Str-O-Nuc hybridization to Plus Nuc on an mRNA before and after purification shows purification-based enrichment of complexed mRNA

[0492] The purpose of this study is to determine the degree of enrichment for mRNA hybridized to Str-O-Nuc following size exclusion chromatography, as assayed by Rnase T1 digestion. RNase T1 cleaves after guanosine residues in single-stranded RNA. Therefore, mRNAs hybridized to Str-O-Nucs (which are not single stranded) are protected from RNase T1 cleavage, whereas naked mRNA is cleaved by RNase Tl. The mRNA poly(A) tail is naturally resistant to RNase Tl because it lacks guanosine residues, as diagrammed in Figure 27.

[0493] This study uses mRNA encoding Firefly luciferase (Fluc[Optl]) with 120 nt poly(A) tail. Fluc[Optl] mRNA was synthesized through in vitro transcription of template DNA encoding the Fluc[Optl] mRNA with or without an additional sequence (+ 16 nt) added after the 3'-UTR (referred to as a Plus Nuc), using a modified form of uracil, Nl- methyl-pseudouridine (Nlm-y) along with a co-translational 5’ Cap-1 structure (CleanCap). The Str-O-Nuc (Str395) was included in the transcription reaction mix, at a concentration of 7 micromolar (pM). The resulting transcribed mRNA contains 100% Nlm-y modification and a 5’ Cap-1 structure.

[0494] Following IVT, half of the mRNA was purified by size exclusion chromatography on an SRT-SEC 2000 column (7.8x300 millimeters (mm)) using Tris- Ethylenediaminetetraacetic acid) (TE) (pH 8) as the mobile phase and flow rate of 1 milliliter per minute (mL / min).

[0495] The purified and unpurified mRNA was then subject to RNase Tl digestion. 75 nanograms (ng) ofFluc[Optl] (120A), Fluc[Optl] (120A + 16nt), or Fluc[Optl] (120A + 16nt) hybridized to Str395 that was either purified by size exclusion chromatography or unpurified were treated with a solution 20 millimolar (mM) Tris with a pH of 7, 50 mM Ethylenediaminetetraacetic acid (EDTA), and 40 units of RNase Tl or 0 units of RNase Tl for 15 minutes at a temperature of 37 degrees Celsius. To quench the reaction, 1 microliter (uL) of Proteinase K is added to the solution for 15 minutes at a temperature ofRef. ParB 24001; 07800.002W0137 degrees Celsius. Subsequently, urea loading dye is added to a lx concentration and run on a Tris-Borate-EDTA (TBE)-Urea 6 percent polyacrylamide gel. The gel is visualized using a 300 nanometer (nm) ultraviolet gel imager with SYBR Gold reagent.

[0496] As seen in Figure 28 from the gel, in the absence of Str-O-Nuc hybridized to the mRNA, only the poly(A) tail fragment is observed after RNase T1 digestion. In the presence of Str-O-Nuc hybridized to the mRNA, the Plus Nuc sequence is protected from RNase T1 digestion, which leads to the appearance of a larger poly(A) tail fragment. After SEC purification, the excess unbound Str-O-Nuc is largely removed and the fraction of mRNA hybridized to Str-O-Nuc is increased, as quantified in Figure 29. This shows that size exclusion chromatography can increase the fraction of mRNA hybridized to Str-O- Nuc and remove excess Str-O-Nuc.

[0497] Example 20: Str-O-Nucs do not impact encapsulation efficiency into lipid nanoparticles

[0498] The purpose of this study is to determine the impact on encapsulation efficiency between naked mRNA with a 120 nt poly(A) tail, mRNA with a 120 nt poly(A) tail and Plus Nuc, or a Str-O-Nuc hybridized to a mRNA with a 120 nt poly(A) tail and Plus Nuc.

[0499] This study uses mRNA encoding Firefly luciferase (Fluc[Optl]) with 120 nt poly(A) tail. Fluc[Optl] mRNA was synthesized through in vitro transcription of template DNA encoding the Fluc[Optl] mRNA with or without an additional sequence (+ 16 nt) added after the polyA tail (referred to as a Plus Nuc), using a modified form of uracil, Nl- methyl-pseudouridine (Nlm-y) along with a co-translational 5’ Cap-1 structure (CleanCap). The Str-O-Nuc (Str395) was included in the transcription reaction mix, at a concentration of 7 micromolar (pM). The resulting transcribed mRNA contains 100% Nlm-y modification and a 5’ Cap-1 structure.

[0500] The hybridized mRNA was purified by size exclusion chromatography on an SRT-SEC 2000 column (7.8x300 millimeters (mm)) using Tris- Ethylenediaminetetraacetic acid (TE) (pH 8) as the mobile phase and flow rate of 1 milliliter per minute (mL / min).

[0501] The positive control mRNA (denoted as + control' referring to an off-the-shelf mRNA encoding codon-optimized Flue with unmodified uridine [Trilink #L-7602]),Ref. ParB 24001; 07800.002W01 naked FlucfOptl] (120A) mRNA, naked FlucfOptl] (120A + 16nt) mRNA, FlucfOptl] (120 A + 16nt) mRNA hybridized to Str395 were encapsulated into SM-102 lipid nanoparticles using a NanoAssemblr Ignite instrument (Precision NanoSystems) according to the manufacturer’s instructions.

[0502] Encapsulation efficiency was determined using the RiboGreen assay (Invitrogen). Briefly, samples were diluted in Tris-Ethylenediaminetetraacetic acid (TE) buffer to a concentration of 0.6 pg / mL in 90 pL in a microplate. Half of the sample was incubated with lOuL of 1% Triton-X for 10 minutes to break open LNPs. IOOUL of the working solution of Quanti-iT RiboGreen RNA Reagent was added to each sample and incubated for 5 minutes at room temperature, protected from light. Fluorescence of the samples was then measured on a microplate reader at 480 nanometers (nm) excitation wavelength. Encapsulation efficiency was calculated as 100% - (concentration of RNA in the untreated sample) / (concentration of RNA in the Tri ton -treated sample).

[0503] As shown in Figure 30, encapsulation efficiencies across all four samples were comparable, indicating that the Plus Nuc nor the Str-O-Nuc hybridization does not impact encapsulation efficiency.

[0504] Example 21: Str-O-Nucs remain hybridized to an mRNA after formulation into a lipid nanoparticle

[0505] The purpose of this study is to determine the impact of hybridization of the Str- O-Nuc to the mRNA after lipid nanoparticle (LNP) encapsulation.

[0506] This study uses mRNA encoding Firefly luciferase (FlucfOptl]) with 120 nt poly(A) tail. FlucfOptl] mRNA was synthesized through in vitro transcription of template DNA encoding the FlucfOptl] mRNA with or without an additional sequence (+ 16 nt) added after the 3'-UTR (referred to as a Plus Nuc), using a modified form of uracil, Nl- methyl-pseudouridine (Nlm-y) along with a co-translational 5’ Cap-1 structure (CleanCap). The Str-O-Nuc (Str395) was included in the transcription reaction mix at a concentration of 7 micromolar (pM). The resulting transcribed mRNA contains 100% Nlm-y modification and a 5’ Cap-1 structure.

[0507] The hybridized mRNA was purified by size exclusion chromatography on an SRT-SEC 2000 column (7.8x300 millimeters (mm)) using Tris-Ref. ParB 24001; 07800.002W01Ethylenediaminetetraacetic acid (TE) (pH 8) as the mobile phase and flow rate of 1 milliliter per minute (mL / min).

[0508] Subsequently, samples were either treated with RNase T1 before or after LNP encapsulation. 75 nanograms (ng) ofFlucfOptl] (120A), FlucfOptl] (120A + 16nt), or FlucfOptl] (120A + 16nt) hybridized to Str395 that was purified by size exclusion chromatography were treated with a solution 20 millimolar (mM) Tris with a pH of 7, 50 mM Ethylenediaminetetraacetic acid (EDTA), and 40 units of RNase T1 or 0 units of RNase T1 for 15 minutes at a temperature of 37 degrees Celsius. To quench the reaction, 1 microliter (uL) of Proteinase K is added to the solution for 15 minutes at a temperature of 37 degrees Celsius. Subsequently, urea loading dye is added to a lx concentration and run on a Tris-Borate-EDTA (TBE)-Urea 6 percent polyacrylamide gel. The gel is visualized using a 300 nanometer (nm) ultraviolet transillumination gel imager with SYBR Gold reagent.

[0509] As seen from the gel in Figure 31, the Str-O-Nuc Str395 protected mRNA fragment is observed with comparable intensity and pattern in both the RNA only and LNP samples. This indicates that LNP encapsulation does not impact mRNA hybridization to the Str-O-Nuc.

[0510] Example 22: Str-O-Nuc: mRNA complexes do not trigger dsRNA immunogenicity sensors

[0511] The purpose of this study is to determine the level of immunogenicity of mRNA containing a 120 nt poly(A) tail, with a 16 nucleotide (nt) Plus Nuc, hybridized to a Str-O-Nuc.

[0512] This study uses mRNA encoding Firefly luciferase (FlucfOptl]) with 120 nt poly(A) tail. FlucfOptl] mRNA was synthesized through in vitro transcription of template DNA encoding the FlucfOptl] mRNA with or without an additional sequence (+ 16 nt) added after the 3'-UTR (referred to as a Plus Nuc), using a modified form of uracil, Nl- methyl-pseudouridine (Nlm-y), along with a co-translational 5’ Cap-1 structure (CleanCap). The Str-O-Nuc (Str395) was included in the transcription reaction mix at a concentration of 7 micromolar (pM). The resulting transcribed mRNA contains 100% Nlm-y modification and a 5’ Cap-1 structure.Ref. ParB 24001; 07800.002W01

[0513] The hybridized mRNA was purified by size exclusion chromatography on an SRT-SEC 2000 column (7.8x300 millimeters (mm)) using Tris- Ethylenediaminetetraacetic acid (TE) (pH 8) as the mobile phase and flow rate of 1 milliliter per minute (mL / min).

[0514] To evaluate immunogenicity, 30 nanograms (ng) of Fluc[Optl] (120A) mRNA, Fluc[Optl](120A + 16nt) mRNA, or hybridized Fluc[Optl](120A + 16nt) mRNA with Str395 were transfected into the adherent A549-Dual cell line, utilizing the mRNA lipofection reagent Lipofectamine Messenger Max (LMM). Positive (unmodified mRNA) and negative (5-methoxyuridine-modified mRNA) controls were included, along with a vehicle only (LMM) control. A549-Dual cells are a human lung epithelial carcinoma cell line that express a secreted luciferase reporter gene under the control of a fusion promoter of an interferon stimulated gene 54 (ISG54) minimal promoter fused to five interferon- stimulated response elements (ISREs). Briefly, immunogenic nucleic acids sensed by endosomal / intracellular sensors such as TLR3, RIG-I, MDA5, PKR, etc. will signal through a type I interferon (IFN) pathway, leading to a transcriptional complex (e.g. Interferon Stimulated Gene Factor 3 (ISGF3)) binding to the aforementioned fusion promoter stimulating luciferase gene transcription. Following the transfection, A549-Dual cells were lysed 72 hours post-transfection utilizing the Quanti-Luc reagent. The level of luminescence detected directly correlates with the relative level of IFN activation within the cell.

[0515] As shown in Figure 32, naked mRNA or mRNA hybridized to Str-O-Nuc do not produce any signal above the negative control, indicating they are nonimmunogenic.

[0516] Example 23: Design and optimization of multi-Plus Nuc and Str-O-Nuc complexes in HepG2 cells

[0517] The purpose of this study is to optimize the expression of mRNA lacking a poly(A) tail but containing a multi-Plus Nuc site for hybridization with a Str-O-Nuc.

[0518] This study uses mRNA encoding Firefly luciferase (Fluc[Opt2]) with or without a split poly(A) tail (30A and 70A separated by a lOnt linker, denoted as 30 / 70A). Fluc[Opt2] mRNA was synthesized through in vitro transcription of template DNA encoding the Fluc[Opt2] mRNA with or without an additional sequence added after the 3'- UTR (referred to as a multi-Plus Nuc), using a modified form of uracil, N1 -methyl -Ref. ParB 24001; 07800.002W01 pseudouridine (Nlm-y), along with a co-translational 5’ Cap-1 structure (CleanCap). The 4 multi-Plus Nucs used in this study are: 1) hdRNA645, hdRNA641, hdRNA635, and hdRNA629. The resulting transcribed mRNA contains 100% Nlm-y modification and a 5’ Cap-1 structure.

[0519] In this study, Str-O-Nucs were hybridized with Fluc[Opt2] mRNA that are detailed below. Str-O-Nucs were hybridized with Fluc[Opt2] (5A+29nt_x3) mRNA (3 sets of: 5A nucleotides followed by a 29nt Plus Nuc). Each Plus Nuc is a different sequence that serves as a binding site for the indicated Str-O-Nucs. The following Str-O-Nucs were complexed with Fluc[Opt2[ (5A+29nt_x3): 1) hdRNA647 - Str-O-Nucs 540, 590, and 591 (Str540,590,591); 2) hdRNA646 - Str-O-Nucs 517, 588, and 589 (Str517, 588, 598).The following Str-O-Nucs were hybridized with Fluc[Opt2] (30A+29nt_x3) mRNA (3 sets of: 30A nucleotides followed by a 29nt Plus Nuc, where each Plus Nuc is a different sequence that serves as a binding site for the indicated Str-O-Nucs): 1) hdRNA643 - Str-O-Nucs 540, 590, and 591 (Str540,590,591); 2) hdRNA642 - Str-O-Nucs 517, 588, and 589 (Str517, 588, 589). The following Str-O-Nucs were hybridized with Fluc[Opt2] (5A+16nt_x3) mRNA (3 sets of: 5A nucleotides followed by a 16nt Plus Nuc, where each Plus Nuc is a different sequence that serves as a binding site for the indicated Str-O-Nucs): 1) hdRNA638 - Str-O-Nucs 548, 585, and 587 (Str548, 585, 587); 2) hdRNA637 - Str-O- Nucs 583, 584, and 586 (Str583, 584, 586); 3) hdRNA636 - Str-O-Nucs 394, 581, and 582 (Str394, 581, 582). The following Str-O-Nucs were hybridized with Fluc[Opt2] (30A+16nt_x3) mRNA (3 sets of: 30A nucleotides followed by a 16nt Plus Nuc, where each Plus Nuc is a different sequence that serves as a binding site for the indicated Str-O- Nucs):: 1) hdRNA632 - Str-O-Nucs 548, 585, and 587 (Str548, 585, 587); 2) hdRNA631 - Str-O-Nucs 583, 584, and 586 (Str583, 584, 586); 4) hdRNA630 - Str-O-Nucs 394, 581, and 582 (Str394, 581, 582). (Refer to Figure 33).

[0520] Relevant controls for these modifications are: 1) hdRNA618 - Fluc[Opt2] mRNA with a split 30 / 70 poly(A) tail; 2) hdRNA645; 3) hdRNA641; 4) hdRNA635; 5) hdRNA629; 6) Lipofectamine Messenger MAX (LMM).

[0521] Str-O-Nucs were hybridized to the Fluc[Opt2] mRNAs by heating the mixture of nucleic acids to 95 degrees C. for 3 minutes, followed by a cooling step to 25 degrees C., set at a cooling rate of 0.1 degrees C. per second in refold buffer (50 mM Tris pH 7, 1 mM Ethylenediaminetetraacetic acid (EDTA), 150 mM Sodium Chloride (NaCl)).Ref. ParB 24001; 07800.002W01

[0522] Following the hybridizing reaction of Str-O-Nuc to Fluc[Opt2] mRNAs, 100 nanograms (ng) of differentially complexed mRNA was transfected into HepG2 cells with Lipofectamine Messenger Max (LMM). Cells were incubated and lysed 4, 24, 48, and 72 hours (hrs) post-transfection using Promega Gio Lysis Buffer (Cat#E266A). Luciferase signal was quantified using the Promega One-Gio Luciferase Assay System (Cat#E6120).

[0523] As seen in Figure 34, for all multi-Plus Nuc designs in the absence of any Str- O-Nuc (hdRNA645, 641, 635, and 629), expression was lower than the control mRNA containing a split poly(A) tail (hdRNA618). Addition of 3 distinct Str-O-Nucs to the multi-Plus Nuc mRNAs led to an increase in expression, in many cases exceeding that of the control mRNA by more than two-fold. These data indicate that multiple Str-O-Nuc hybridization sequences following a 5A or 30A linker on an mRNA, hybridized to three Str-O-Nucs, increases protein expression.

[0524] Example 24: A virus-derived 3 '-readenylation element can synergize with Str- O-Nucs on an mRNA lacking a poly(A) tail

[0525] The purpose of this study is to determine if a virus-derived readenylation element can improve protein expression when utilized in an mRNA lacking a poly(A) tail but containing a 16 nucleotide (nt) Plus Nuc hybridized to a Str-O-Nuc.

[0526] This study uses mRNA encoding Firefly luciferase (Fluc[Opt2]) without a poly(A) tail. Fluc[Opt2] mRNA was synthesized through in vitro transcription of template DNA encoding the Fluc[Opt2] mRNA with an additional sequence (+ 16nt) added at the 3’ end of the mRNA (referred to as a Plus Nuc). An additional sequence in some constructs is added immediately downstream of the standard 3’ UTR that is either a virus- derived 3’ readenylation element as described in Seo et al., Cell (2023) (denoted as K5) or a scrambled sequence of the K5 element (denoted as Scr). All constructs use unmodified uracil and a co-translational 5’ Cap-1 structure (CleanCap).

[0527] Str-O-Nuc 526 (Str526) was hybridized to the Fluc[Opt2] mRNAs (hdRNA738, 740, and 742) (as seen in Figure 35) by heating the mixture of nucleic acids to 95 degrees C. for 3 minutes, followed by a cooling step to 25 degrees C., set at a cooling rate of 0.1 degrees C. per second in refold buffer (50 mM Tris pH 7, 1 mM Ethylenediaminetetraacetic acid (EDTA), 150 mM Sodium Chloride (NaCl)).Ref. ParB 24001; 07800.002W01

[0528] Following the hybridizing reaction of Str-O-Nuc to Fluc[Opt2] mRNAs, 100 nanograms (ng) of differentially complexed mRNA was transfected into HepG2 cells with Lipofectamine Messenger Max (LMM). Cells were incubated and lysed 4, 24, 48, and 72 hours (hrs) post-transfection using Promega Gio Lysis Buffer (Cat#E266A). Luciferase signal was quantified using the Promega One-Gio Luciferase Assay System (Cat#E6120). As seen in Figure 36, in the absence of any Str-O-Nuc, inclusion of the K5 element in the mRNA (hdRNA739) led to an approximate 2-fold increase in expression (compared to the absence of K5 element (hdRNA737) or the scrambled version (hdRNA741)), though overall expression was relatively low. Binding of mRNA to a Str-O-Nuc led to an increase in expression of all tested constructs, but the mRNA containing the Str-O-Nuc and the K5 element (hdRNA740) had the highest expression level across all time points. These data indicate that inclusion of the K5 element in a mRNA lacking a poly(A) tail, containing a Plus Nuc, and hybridized to a Str-O-Nuc increases protein expression.Ref. ParB 24001; 07800.002W01Ref. ParB 24001; 07800.002W01Ref. ParB 24001; 07800.002W01Ref. ParB 24001; 07800.002W01Ref. ParB 24001; 07800.002W01Ref. ParB 24001; 07800.002W01Ref. ParB 24001; 07800.002W01Ref. ParB 24001; 07800.002W01Ref. ParB 24001; 07800.002W01Ref. ParB 24001; 07800.002W01Ref. ParB 24001; 07800.002W01Ref. ParB 24001; 07800.002W01Ref. ParB 24001; 07800.002W01Ref. ParB 24001; 07800.002W01Ref. ParB 24001; 07800.002W01Ref. ParB 24001; 07800.002W01Ref. ParB 24001; 07800.002W01Ref. ParB 24001; 07800.002W01Ref. ParB 24001; 07800.002W01Ref. ParB 24001; 07800.002W01Ref. ParB 24001; 07800.002W01- Ill -Ref. ParB 24001; 07800.002W01Ref. ParB 24001; 07800.002W01Ref. ParB 24001; 07800.002W01Ref. ParB 24001; 07800.002W01Ref. ParB 24001; 07800.002W01Ref. ParB 24001; 07800.002W01Ref. ParB 24001; 07800.002W01Ref. ParB 24001; 07800.002W01Ref. ParB 24001; 07800.002W01Ref. ParB 24001; 07800.002W01Ref. ParB 24001; 07800.002W01Ref. ParB 24001; 07800.002W01Ref. ParB 24001; 07800.002W01Ref. ParB 24001; 07800.002W01Ref. ParB 24001; 07800.002W01Ref. ParB 24001; 07800.002W01Ref. ParB 24001; 07800.002W01Ref. ParB 24001; 07800.002W01

Claims

Ref. ParB 24001; 07800.002W01CLAIMSWhat is claimed is:

1. An mRNA complex comprising an mRNA molecule and at least one engineered oligonucleotide hybridized to the mRNA molecule, wherein the mRNA molecule comprises a coding sequence (CDS) and a 3’ untranslated region (UTR), and optionally a poly (A) tail, wherein the engineered oligonucleotide is referred to as the Str-O-Nuc, wherein the Str-O-Nuc is at least partially hybridized to the 3’ UTR of the mRNA molecule.

2. The mRNA complex of claim 1, wherein the mRNA molecule further comprises at least one additional nucleotide following the end of 3’ UTR, which at least one additional nucleotide following the end of the 3 ’UTR is referred to as the Plus-Nuc.

3. The mRNA complex of claim 1 or 2, wherein the mRNA molecule comprises at least one Str-O-Nuc linked to the end of the mRNA’s poly(A) tail.

4. The mRNA complex of any preceding or subsequent claims, wherein the at least one Str-O-Nuc is partially or substantially completely hybridized to the mRNA molecule, and wherein said mRNA complex has: a. a half-life greater than an unmodified mRNA or an un-engineered mRNA, b. a more durable response compared to an unmodified mRNA, and / or c. a more durable response compared to an un-engineered mRNA.

5. The mRNA complex of any preceding or subsequent claims, wherein the at least one Str-O-Nuc is linked to the mRNA molecule, and wherein said mRNA complex has: a. a half-life greater than an unmodified mRNA or an un-engineered mRNA, b. a more durable response compared to an unmodified mRNA, and / or c. a more durable response compared to an un-engineered mRNA.

6. The mRNA complex of any preceding or subsequent claims, wherein the mRNA molecule contains a poly(A) tail, wherein there is at least one Plus-Nuc following the end of the mRNA’s poly(A) tail, and wherein said mRNA complex has: a. a half-life greater than an unmodified mRNA or an un-engineered mRNA, b. a more durable response compared to an unmodified mRNA, and / orRef. ParB 24001; 07800.002W01 c. a more durable response compared to an un-engineered mRNA.

7. The mRNA complex of any preceding or subsequent claims, wherein the mRNA does not comprise a poly(A) tail, wherein the at least one Str-O-Nuc is partially or substantially completely hybridized to the mRNA molecule, and wherein said mRNA complex is easier to manufacture than an mRNA complex comprising a poly(A) tail and not comprising a Str-O-Nuc.

8. The mRNA complex of any preceding or subsequent claims, wherein the Str-O- Nuc is hybridized to a specific sequence on the mRNA such that at least 50% of the hybridized section of the Str-O-Nuc sequence is complementary to the said sequence on the mRNA.

9. The mRNA complex of any preceding or subsequent claims, wherein the Str-O- Nuc is hybridized to a specific sequence on the mRNA such that at least 50% of the hybridized section of the Str-O-Nuc sequence is complementary to the said sequence on the mRNA, and wherein the specific sequence can be located anywhere on the mRNA complex.

10. The mRNA complex of any preceding or subsequent claims, wherein the Str-O- Nuc is hybridized to a specific sequence on the mRNA such that at least 50% of the hybridized section of the Str-O-Nuc sequence is complementary to the said sequence on the mRNA, and wherein the specific sequence can be located before the start of the mRNA’s poly (A) tail.

11. The mRNA complex of any preceding or subsequent claims, wherein the Str-O- Nuc is hybridized to a specific sequence within the mRNA’s poly(A) tail.

12. The mRNA complex of any preceding or subsequent claims, wherein the Str-O- Nuc is an oligonucleotide engineered to impart double-strandedness to the mRNA complex.

13. The mRNA complex of any preceding or subsequent claims, wherein the mRNA molecule to be hybridized to the Str-O-Nuc has no poly(A) tail.

14. The mRNA complex of any preceding or subsequent claims, wherein the mRNA molecule is an mRNA molecule engineered to have no poly(A) tail.

15. The mRNA complex of any preceding or subsequent claims, wherein the Str-O- Nuc is substantially hybridized to the mRNA molecule’s 3’UTR.Ref. ParB 24001; 07800.002W0116. The mRNA complex of any preceding or subsequent claims, wherein the Str-O- Nuc is hybridized to a nucleotide within the region of the 3’UTR following the coding sequence at the 3 ’end.

17. The mRNA complex of any preceding or subsequent claims, wherein the Str-O- Nuc contains a poly (A) tail.

18. The mRNA complex of any preceding or subsequent claims, wherein the Str-O- Nuc contains at least two poly(A) tails, wherein at least one poly(A) tail is in the forward direction, and wherein at least one poly(A) tail is in the reverse direction.

19. The mRNA complex of any preceding or subsequent claims, wherein the Str-O- Nuc contains a poly(A) tail, wherein the poly(A) tail does not comprise modified adenine nucleotides.

20. The mRNA complex of any preceding or subsequent claims, wherein the Str-O- Nuc contains a poly(A) tail, wherein the poly(A) tail is comprised of at least one adenine nucleotide that has been modified with a 2’-H, 2’-OH, 2’-OMe, 2’-M0E, 2’-F, P-S linkage, 3’-inv-dT, 3’-ddC, LNA or phosphorothioate linkages modification.

21. The mRNA complex of any preceding or subsequent claims, wherein the Str-O- Nuc contains a poly(A) tail, wherein the poly(A) tail is comprised of at least one adenine nucleotide that has been modified with a 2’-0Me.

22. The mRNA complex of any preceding or subsequent claims, wherein the Str-O- Nuc contains a poly(A) tail, wherein the poly(A) tail is comprised of at least one adenine nucleotide that has been modified with a 2’-OH.

23. The mRNA complex of any preceding or subsequent claims, wherein the mRNA molecule is an mRNA molecule with a poly(A) tail, wherein a first Str-O-Nuc is linked to the poly(A) tail of the mRNA molecule, wherein a second Str-O-Nuc containing a poly(A) tail is partially or fully hybridized to the first Str-O-Nuc, at least one additional nucleotide is linked to the end of the second Str-O-Nuc’ s poly(A) tail, and the second Str-O-Nuc is oriented in the reverse direction.

24. The mRNA complex of any preceding or subsequent claims, wherein the mRNA molecule is an mRNA molecule with a poly(A) tail, wherein a first Str-O-Nuc is linked to the poly(A) tail of the mRNA molecule, wherein a second Str-O-Nuc containing a poly(A) tail is partially or fully hybridized to the first Str-O-Nuc, at least one additional nucleotideRef. ParB 24001; 07800.002W01 is linked to the end of the second Str-O-Nuc’s poly(A) tail, and the second Str-O-Nuc is oriented in the forward direction.

25. The mRNA complex of any preceding or subsequent claims, wherein the mRNA molecule is an mRNA molecule with a poly(A) tail, wherein a first Str-O-Nuc is linked to the poly(A) tail of the mRNA molecule, wherein a second Str-O-Nuc is partially hybridized to the first Str-O-Nuc and contains a loop-back end blocker.

26. The mRNA complex of any preceding or subsequent claims, wherein the mRNA molecule is an mRNA molecule with a poly(A) tail that is followed by a Plus-Nuc, wherein a Str-O-Nuc containing a poly(A) tail is partially or fully hybridized to the Plus- Nuc, at least one additional nucleotide is linked to the end of the Str-O-Nuc’s poly(A) tail, and the Str-O-Nuc is oriented in the reverse direction.

27. The mRNA complex of any preceding or subsequent claims, wherein the mRNA molecule is an mRNA molecule with a poly(A) tail that is followed by a Plus-Nuc, wherein a Str-O-Nuc containing a poly(A) tail is partially or fully hybridized to the Plus- Nuc, at least one additional nucleotide is linked to the end of the Str-O-Nuc’s poly(A) tail, and the Str-O-Nuc is oriented in the forward direction.

28. The mRNA complex of any preceding or subsequent claims, wherein the mRNA molecule is an mRNA molecule with a poly(A) tail that is followed by a Plus-Nuc, wherein a Str-O-Nuc is partially hybridized to the Plus-Nuc and contains a loop-back end blocker.

29. The mRNA complex of any preceding or subsequent claims, wherein a Str-O-Nuc is hybridized to the 3’ end of an mRNA and contains a loop-back end blocker, and wherein said mRNA complex has a. a half-life greater than an unmodified mRNA or an un-engineered mRNA, b. a more durable response compared to an unmodified mRNA, and / or c. a more durable response compared to an un-engineered mRNA.

30. The mRNA complex of any preceding or subsequent claims, wherein a Str-O-Nuc is hybridized to a Plus-Nuc and contains a loop-back end blocker, and wherein said mRNA complex has a. a half-life greater than an unmodified mRNA or an un-engineered mRNA,Ref. ParB 24001; 07800.002W01 b. a more durable response compared to an unmodified mRNA, and / or c. a more durable response compared to an un-engineered mRNA.

31. The mRNA complex of any preceding or subsequent claims, wherein a Str-O-Nuc is hybridized to another Str-O-Nuc and contains a loop-back end blocker, and wherein said mRNA complex has a. a half-life greater than an unmodified mRNA or an un-engineered mRNA, b. a more durable response compared to an unmodified mRNA, and / or c. a more durable response compared to an un-engineered mRNA.

32. The mRNA complex of any preceding or subsequent claims, wherein a Str-O-Nuc is hybridized to the 3’ end of an mRNA and contains a splinted end blocker that also hybridizes to another engineered oligonucleotide and wherein said mRNA complex has a. a half-life greater than an unmodified mRNA or an un-engineered mRNA, b. a more durable response compared to an unmodified mRNA, and / or c. a more durable response compared to an un-engineered mRNA.

33. The mRNA complex of any preceding or subsequent claims, wherein the mRNA complex further comprises a targeting moiety, and wherein the at least one Str-O-Nuc is conjugated to a targeting moiety.

34. The mRNA complex of any preceding or subsequent claims, wherein the targeting moiety is a sugar, a small molecule, a peptide, an antibody, an antibody fragment, a nanobody protein, a mini protein, an anti-CD5 antibody, a nucleic acid, or a combination thereof.

35. The mRNA complex of any preceding or subsequent claims, wherein the targeting moiety is GalNAc, a C16, a peptide, IgG, Fab, VHH, svFv, an anti-CD5 antibody, a nucleic acid, cholesterol or a combination thereof.

36. The mRNA complex of any preceding or subsequent claims, wherein the Str-O- Nuc has a length in a range of 1 nt to 200 nt, or 1 nt to 50 nt, or 2 nt to 25 nt, or 40 nt to 50 nt, or 50 nt to 75 nt, or 75 nt to 100 nt, or 100 nt to 125 nt, or 100 nt to 150 nt, or 125 nt to 150 nt, or 150 nt to 175 nt, or 150 nt to 200 nt, or 175 nt to 200 nt.Ref. ParB 24001; 07800.002W0137. The mRNA complex of any preceding or subsequent claims, wherein the Str-O- Nuc has a length of 15 nt, at least 16 nt, at least 17 nt, at least 18 nt, at least 20 nt, at least 25 nt, at least 30 nt, at least 35 nt, at least 40 nt, at least 45 nt, at least 50 nt, at least 55 nt, at least 60 nt.

38. The mRNA complex of any preceding or subsequent claims, wherein the Plus-Nuc has a length in a range of 1 nt to 200 nt, or 1 nt to 50 nt, or 2 nt to 25 nt, or 40 nt to 50 nt, or 50 nt to 75 nt, or 75 nt to 100 nt, or 100 nt to 125 nt, or 100 nt to 150 nt, or 125 nt to 150 nt, or 150 nt to 175 nt, or 150 nt to 200 nt, or 175 nt to 200 nt.

39. The mRNA complex of any preceding or subsequent claims, wherein the Str-O- Nuc has a length in a range of 1 nt to 200 nt, or 1 nt to 50 nt, or 2 nt to 25 nt, or 40 nt to 50 nt, or 50 nt to 75 nt, or 75 nt to 100 nt, or 100 nt to 125 nt, or 100 nt to 150 nt, or 125 nt to 150 nt, or 150 nt to 175 nt, or 150 nt to 200 nt, or 175 nt to 200 nt.

40. The mRNA complex of any preceding or subsequent claims, wherein the Plus-Nuc has a length of 15 nt, at least 16 nt, at least 17 nt, at least 18 nt, at least 20 nt, at least 25 nt, at least 30 nt, at least 35 nt, at least 40 nt, at least 45 nt, at least 50 nt, at least 55 nt, at least 60 nt.

41. The mRNA complex of any preceding or subsequent claims, wherein the Str-O- Nuc has a length of 15 nt, at least 16 nt, at least 17 nt, at least 18 nt, at least 20 nt, at least 25 nt, at least 30 nt, at least 35 nt, at least 40 nt, at least 45 nt, at least 50 nt, at least 55 nt, at least 60 nt.

42. The mRNA complex of any preceding or subsequent claims, wherein the Str-O- Nuc containing a poly(A) tail has a length of 15 nt, at least 16 nt, at least 17 nt, at least 18 nt, at least 20 nt, at least 25 nt, at least 30 nt, at least 35 nt, at least 40 nt, at least 45 nt, at least 50 nt, at least 55 nt, at least 60 nt.

43. The mRNA complex of any preceding or subsequent claims, wherein the Str-O- Nuc not containing a poly(A) tail has a length of 15 nt, at least 16 nt, at least 17 nt, at least 18 nt, at least 20 nt, at least 25 nt, at least 30 nt, at least 35 nt, at least 40 nt, at least 45 nt, at least 50 nt, at least 55 nt, at least 60 nt.

44. The mRNA complex of any preceding or subsequent claims, wherein the mRNA molecule is an mRNA molecule engineered to have no poly(A) tail, wherein a Str-O-Nuc is hybridized to the 3’UTR of the mRNA molecule, wherein the Str-O-Nuc contains aRef. ParB 24001; 07800.002W01 poly(A) tail, at least one additional nucleotide is linked to the end of the Str-O-Nuc’s poly(A) tail, and the Str-O-Nuc is oriented in the reverse direction.

45. The mRNA complex of any preceding or subsequent claims, wherein the mRNA molecule is an mRNA molecule engineered to have no poly(A) tail, wherein a Str-O-Nuc is hybridized to the 3’UTR of the mRNA molecule, wherein the Str-O-Nuc has a length of at least 15 nt, the Str-O-Nuc contains a poly(A) tail, the Str-O-Nuc and the poly(A) tail have a combined length of at least 30 nt, and at least 6 additional nucleotides are hybridized to the end of the Str-O-Nuc’s poly(A) tail, wherein the at least 6 nt contain phosphorothioate linkages and 2’-M0E modifications, and the Str-O-Nuc is oriented in the forward direction.

46. The mRNA complex of any preceding or subsequent claims, wherein the mRNA molecule is an mRNA molecule engineered to have no poly(A) tail, wherein a Str-O-Nuc is hybridized to the 3’UTR of the mRNA molecule, wherein the Str-O-Nuc contains a poly(A) tail, at least one additional nucleotide is linked to the end of the Str-O-Nuc’s poly(A) tail, and the Str-O-Nuc is oriented in the forward direction.

47. The mRNA complex of any preceding or subsequent claims, wherein the mRNA molecule is an mRNA molecule engineered to have no poly(A) tail, wherein a Str-O-Nuc is hybridized to the 3’UTR of the mRNA molecule, wherein the Str-O-Nuc has a length of at least 18 nt, the Str-O-Nuc contains a poly(A) tail, the Str-O-Nuc and the poly(A) tail have a combined length of at least 30 nt, and at least 6 additional nucleotides are linked to the end of the Str-O-Nuc’s poly(A) tail, wherein the at least 6 nt contain phosphorothioate linkages and 2’-M0E modifications, and the Str-O-Nuc is oriented in the forward direction.

48. The mRNA complex of any preceding or subsequent claims, wherein the mRNA molecule is an mRNA molecule engineered to have no poly(A) tail, wherein a Str-O-Nuc is hybridized to the 3’UTR of the mRNA molecule, wherein the Str-O-Nuc has a length of at least 18 nt, the Str-O-Nuc contains a poly(A) tail, the Str-O-Nuc and the poly(A) tail have a combined length of at least 33 nt, at least 6 nt is hybridized to the end of the Str-O- Nuc’s poly(A) tail, wherein the at least 6 nt contain phosphorothioate linkages and 2’- MOE modifications, and the Str-O-Nuc is oriented in the reverse direction.

49. The mRNA complex of any preceding or subsequent claims, wherein the Str-O- Nuc comprises a 2’-OH.Ref. ParB 24001; 07800.002W0150. The mRNA complex of any preceding or subsequent claims, wherein the Str-O- Nuc comprises a 2’-H, 2’-OH, 2’-OMe, 2’-M0E, 2’-F, P-S linkage, 3’-inv-dT, 3’-ddC modification, LNA or phosphorothioate linkages.

51. The mRNA complex of any preceding or subsequent claims, wherein the Str-O- Nuc comprises a sequence complementary to some part of the mRNA, wherein said sequence has a length in a range of 2 nt to 50 nt.

52. The mRNA complex of any preceding or subsequent claims, wherein the Str-O- Nuc comprises a sequence complementary to some part of the mRNA, wherein said sequence has a length in a range of 2 nt to 75 nt.

53. The mRNA complex of any preceding or subsequent claims, wherein the Str-O- Nuc comprises a conjugated protein, a peptide, a nucleic acid, a lipid, a sugar, or a small molecule.

54. The mRNA complex of any preceding or subsequent claims, wherein the Str-O- Nuc comprises a modified phosphodiester backbone.

55. The mRNA complex of any preceding or subsequent claims, wherein the Str-O- Nuc comprises a modified phosphorothioate linkage.

56. The mRNA complex of any preceding or subsequent claims, wherein the Str-O- Nuc comprises a partially or substantially complete 100% uridine modification.

57. The mRNA complex of any preceding or subsequent claims, wherein the Str-O- Nuc comprises a partially or substantially complete 100% thymidine substitution, and wherein the thymidine nucleotides replaced the uridine nucleotides, and / or the Str-O-Nuc comprises a partially or substantially complete 100% 5-methylcytosine substitution, and wherein the 5-methylcytosine nucleotides replaced the cytosine nucleotides.

58. The mRNA complex of any preceding or subsequent claims, wherein the mRNA molecule encodes for an enzyme, a second RNA molecule that serves as a reverse transcription template and which does not have a cap or a poly(A) tail, wherein the second RNA molecule contains a Str-O-Nuc hybridized at the 3’ end of the 3’UTR, and wherein the mRNA complex has a. a half-life greater than an unmodified mRNA or an un-engineered mRNA,Ref. ParB 24001; 07800.002W01 b. a more durable response compared to an unmodified mRNA or an unengineered mRNA, and / or c. a more durable response compared to an unmodified mRNA or an unengineered mRNA.

59. The mRNA complex of any preceding or subsequent claims, wherein the mRNA further comprises a readenylation element, e.g., a virus-derived 3’ readenylation element downstream of the 3 ’ UTR.

60. The mRNA complex of any preceding or subsequent claims, wherein the sequence of the Str-O-Nuc that hybridizes to the mRNA comprises an internal poly(A) tract within the sequence that hybridizes to the mRNA, e.g., a 60-nt poly(A) tract.

61. A composition, comprising an mRNA complex of any preceding or subsequent claims.

62. A composition, comprising an mRNA complex of any preceding or subsequent claims, a dendrimer, an exosome, a lipid nanoparticle, a liposome-like nanoparticle, a lipoprotein particle, a lipid-polymer hybrid nanoparticle, a nanostructured lipid carrier, a platelet membrane -coated nanoparticle, a protein-based nanoparticle, a polymer, a polymeric nanoparticle, a protamine, a solid nanoparticle, or a virus-like particle.

63. A pharmaceutical composition, comprising an mRNA complex of any preceding or subsequent claims.

64. A pharmaceutical composition, comprising an mRNA complex of any preceding or subsequent claims, a dendrimer, an exosome, a lipid nanoparticle, a liposome-like nanoparticle, a lipoprotein particle, a lipid-polymer hybrid nanoparticle, a nanostructured lipid carrier, a platelet membrane -coated nanoparticle, a protein-based nanoparticle, a polymer, a polymeric nanoparticle, a protamine, a solid nanoparticle, or a virus-like particle.

65. A method for treating an individual, comprising administering an effective amount of a pharmaceutical composition comprising an mRNA complex of any preceding or subsequent claims to an individual.

66. A method for treating an individual, comprising administering an effective amount of a pharmaceutical composition comprising an mRNA complex of any preceding orRef. ParB 24001; 07800.002W01 subsequent claims to an individual, wherein the pharmaceutical composition is administered by an intravenous route.

67. A method for treating an individual, comprising administering an effective amount of a pharmaceutical composition comprising an mRNA complex of any preceding or subsequent claims to an individual, wherein the pharmaceutical composition is administered by an intramuscular route.

68. A method for treating an individual, comprising administering an effective amount of a pharmaceutical composition comprising an mRNA complex of any preceding or subsequent claims to an individual, wherein the pharmaceutical composition is administered by a subcutaneous route.

69. A method for treating an individual, comprising administering an effective amount of a composition comprising an mRNA complex of any preceding or subsequent claims, a dendrimer, an exosome, a lipid nanoparticle, a liposome-like nanoparticle, a lipoprotein particle, a lipid-polymer hybrid nanoparticle, a nanostructured lipid carrier, a platelet membrane -coated nanoparticle, a protein-based nanoparticle, a polymer, a polymeric nanoparticle, a protamine, a solid nanoparticle, or a virus-like particle.

70. Any combination of features of the mRNA complexes, the compositions comprising the mRNA complexes, methods of manufacturing of the mRNA complexes and the compositions, and / or methods of using the mRNA complexes for treatment, which are disclosed in any preceding claims.

71. An mRNA complex comprising an mRNA molecule and at least one engineered oligonucleotide hybridized to the mRNA molecule, wherein the mRNA molecule comprises a coding sequence (CDS) and a 3’ untranslated region (UTR), and optionally a poly (A) tail, wherein the engineered oligonucleotide is referred to as the Str-O-Nuc, wherein the Str-O-Nuc is at least partially hybridized to the 3’ end of the mRNA molecule.

72. The mRNA complex of any preceding or subsequent claims, wherein the mRNA molecule is an mRNA molecule engineered to have no poly(A) tail, wherein a Str-O-Nuc is hybridized to the 3’ end of the mRNA molecule, wherein the Str-O-Nuc contains a poly(A) tail, at least one additional nucleotide is linked to the end of the Str-O-Nuc’ s poly(A) tail, and the Str-O-Nuc is oriented in the forward direction.

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