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

MRNA COMPLEXES, THEIR ANUFACTURING, ANDTHEIR USE FOR TREATMENTCROSS-REFERENCE TO RELATED APPLICATIONThis application claims priority to United States Provisional Application Number 63 / 709,855, which was filed on October 21, 2024. The entire content of the application referenced above is hereby incorporated by reference herein.TECHNICAL FIELD

[0001] This disclosure relates to an mRNA complex, its method of manufacturing, and its use for treatment.BACKGROLND

[0002] 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.

[0003] 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.

[0004] 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 short in length (e.g., less than about 100-200 nucleotides in length) due to issues with purity and yield and theseissues 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. Further, means of regulating control (e.g., translational control) of mRNA sequences having 5’ UTRs is needed.SUMMARY OF THE INVENTION

[0005] The engineered oligonucleotides and mRNA complex described in this disclosure can impart desirable properties onto mRNA efficiently, without the need for labor-intensive ligation.

[0006] Accordingly, provided herein is an mRNA complex comprising an mRNA molecule and at least one engineered oligonucleotide hybridized to the 5’ end of the mRNA molecule, wherein said engineered oligonucleotide is hereafter referred to as the Str-O-Nuc.

[0007] It has also been discovered that adding an overhang to a 5'UTR engineered oligonucleotide reduces the translational repression normally observed for 5'UTR oligonucleotides. The exemplary version of this is an oligonucleotide that binds at or near the cap, which normally strongly represses translation but a Str-O-Nuc with a 3 '-overhang (and to a lesser extent 5' overhang) allows translation.

[0008] Opening up just 1 or 2 nucleotides proximal to the cap (e.g., 'AG') allows improved translation.

[0009] The overhang on a 5'UTR oligonucleotide could be used for imparting regulation to mRNA translation (e.g., increasing or decreasing mRNA translation), which provides useful methods for regulating mRNA translation.

[0010] 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.

[0011] 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

[0012] 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. Some examples may be practiced with additional components or steps and / or without all the illustrated components or steps. The drawings are not necessarily to scale.

[0013] 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.

[0014] Figure 1. Figure 1 depicts certain results as described, e.g., in Example 1.

[0015] Figure 2. Figure 2 depicts certain results as described, e.g., in Example 2.

[0016] Figure 3. Figure 3 depicts certain results as described, e.g., in Example 2.

[0017] Figure 4. Figure 4 depicts certain results as described, e.g., in Example 2.

[0018] Figure 5. Figure 5 depicts certain results as described, e.g., in Example 3.

[0019] Figure 6. Figure 6 depicts certain results as described, e.g., in Example 4.

[0020] Figure 7. Figure 7 depicts certain results as described, e.g., in Example 5.

[0021] Figure 8. Figure 8 depicts certain results as described, e.g., in Example 6.DETAILED DESCRIPTION

[0022] Provided herein is an mRNA complex comprising an mRNA molecule and at least one engineered oligonucleotide hybridized to the 5’UTR of the mRNA molecule, wherein said engineered oligonucleotide is hereafter referred to as the Str-O-Nuc.

[0023] Also provided herein is an mRNA complex comprising an mRNA molecule and at least one engineered oligonucleotide hybridized to the 5' region of the mRNA molecule, wherein said engineered oligonucleotide is hereafter referred to as the Str-O-Nuc, wherein the Str-O-Nuc is hybridized downstream of a cap and upstream of an IRES of the mRNA.

[0024] In certain embodiments, the Str-O-Nuc is partially or substantially completely hybridized to the mRNA molecule.

[0025] 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.

[0026] In certain embodiments, the mRNA complex contains a Str-O-Nuc containing an overhang which contains a sequence that is complementary to a sequence on an endogenous mRNA, wherein the overhang would hybridize to the complementary sequence on said endogenous mRNA, and wherein the Str-O-Nuc de-hybridizes from the the exogenous mRNA complex.

[0027] In certain embodiments, the Str-O-Nuc is an oligonucleotide engineered to impart double-strandedness to the mRNA complex.

[0028] In certain embodiments, the mRNA molecule to be hybridized to the Str-O-Nuc has no poly(A) tail.

[0029] In certain embodiments, the mRNA molecule is an mRNA molecule engineered to have no poly (A) tail.

[0030] In certain embodiments, the Str-O-Nuc is hybridized to the mRNA molecule’s 5’UTR.

[0031] In certain embodiments, the Str-O-Nuc contains a poly(A) tail and / or a poly(A) region. In certain embodiments, the Str-O-Nuc contains a poly(A) tail and / or a poly(A) region, wherein the poly(A) tail and / or poly(A) region does not comprise modified adenine nucleotides.

[0032] In certain embodiments, the Str-O-Nuc contains a poly(A) tail and / or a poly(A) region, wherein the poly(A) tail and / or poly(A) region is comprised of at least one adenine nucleotide that has been modified with a 2’-H, 2’-OH, 2’-0Me, 2’-M0E, 2’-F, P-S linkage, 3’-inv-dT, 3’-ddC, LNA or phosphorothioate linkages modification.

[0033] In certain embodiments, the Str-O-Nuc contains a poly(A) tail and / or a poly(A) region, wherein the poly(A) tail and / or poly(A) region is comprised of at least one adenine nucleotide that has been modified with a 2’-0Me.

[0034] In certain embodiments, the Str-O-Nuc contains a poly(A) tail and / or a poly(A) region, wherein the poly(A) tail and / or poly(A) region is comprised of at least one adenine nucleotide that has been modified with a 2’-OH.

[0035] In certain embodiments, a poly(A) tail and / or a poly(A) region is hybridized to the Str-O-Nuc.

[0036] In certain embodiments, a Str-O-Nuc is hybridized to the 5’ 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.

[0037] In certain embodiments, a Str-O-Nuc is hybridized to the 5’ 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.

[0038] 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.

[0039] 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.

[0040] In certain embodiments, the targeting moiety is GalNAc, a C16, a peptide, IgG, Fab, VHH, scFv, an anti-CD5 antibody, a nucleic acid, or a combination thereof.

[0041] 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.

[0042] In certain embodiments, the Str-O-Nuc comprises a 2’ -OH.

[0043] In certain embodiments, the Str-O-Nuc comprises a 2’-H, 2’-OH, 2’-0Me, 2’-M0E, 2’-F, P-S linkage, 3’-inv-dT, 3’-ddC modification, LNA or phosphorothioate linkages.

[0044] 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.

[0045] 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.

[0046] In certain embodiments, the Str-O-Nuc comprises a conjugated protein, a peptide, a nucleic acid, a lipid, a sugar, or a small molecule.

[0047] In certain embodiments, the Str-O-Nuc comprises a modified phosphodiester backbone.

[0048] In certain embodiments, the Str-O-Nuc comprises a modified phosphorothioate linkage.

[0049] In certain embodiments, the Str-O-Nuc comprises a partially or substantially complete 100% uridine modification.

[0050] 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.

[0051] Certain embodiments provide a composition comprising an mRNA complex of described herein.

[0052] 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, a polymer, a polymeric nanoparticle, a protamine, a solid nanoparticle, or a virus-like particle.

[0053] Certain embodiments provide a pharmaceutical composition, comprising an mRNA complex described herein.

[0054] 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 lipidcarrier, a platelet membrane-coated nanoparticle, a protein-based nanoparticle, a polymer, a polymeric nanoparticle, a protamine, a solid nanoparticle, or a virus-like particle.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] Embodiments from claims

[0062] 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.

[0063] 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.

[0064] 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 unengineered mRNA.

[0065] 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 unengineered mRNA, a more durable response compared to an unmodified mRNA, and / or a more durable response compared to an un-engineered mRNA.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] In certain embodiments, the Str-O-Nuc is hybridized to a specific sequence within the mRNA’s poly (A) tail.

[0071] In certain embodiments, the Str-O-Nuc is an oligonucleotide engineered to impart double-strandedness to the mRNA complex.

[0072] In certain embodiments, the mRNA molecule to be hybridized to the Str-O-Nuc has no poly(A) tail.

[0073] In certain embodiments, the mRNA molecule is an mRNA molecule engineered to have no poly (A) tail.

[0074] In certain embodiments, the Str-O-Nuc is hybridized to the mRNA molecule’s 5’UTR.

[0075] In certain embodiments, the Str-O-Nuc contains a poly(A) tail.

[0076] 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.

[0077] In certain embodiments, the Str-O-Nuc contains a poly(A) tail, wherein the poly(A) tail does not comprise modified adenine nucleotides.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] In certain embodiments, a Str-O-Nuc is hybridized to the 5’ 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 un-engineered mRNA.

[0085] 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 anunmodified 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.

[0086] In certain embodiments, a Str-O-Nuc is hybridized to the 5’ 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.

[0087] 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.

[0088] 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.

[0089] In certain embodiments, the targeting moiety is GalNAc, a C16, a peptide, IgG, Fab, VHH, scFv, an anti-CD5 antibody, a nucleic acid, or a combination thereof.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] 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.

[0094] In certain embodiments, the Str-O-Nuc containing a poly(A) tail and / or a poly(A) region 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.

[0095] 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.

[0096] 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 5’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 reverse direction.

[0097] 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 5’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.

[0098] 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 5’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.

[0099] 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 5’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.

[0100] 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 5’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.

[0101] In certain embodiments, the Str-O-Nuc comprises a 2’ -OH.

[0102] In certain embodiments, 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.

[0103] 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.

[0104] 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.

[0105] In certain embodiments, the Str-O-Nuc comprises a conjugated protein, a peptide, a nucleic acid, a lipid, a sugar, or a small molecule.

[0106] In certain embodiments, the Str-O-Nuc comprises a modified phosphodiester backbone.

[0107] In certain embodiments, the Str-O-Nuc comprises a modified phosphorothioate linkage.

[0108] In certain embodiments, the Str-O-Nuc comprises a partially or substantially complete 100% uridine modification.

[0109] 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.

[0110] Certain embodiments provide a composition, comprising an mRNA complex described herein.

[0111] 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, a polymer, a polymeric nanoparticle, a protamine, a solid nanoparticle, or a virus-like particle.

[0112] Certain embodiments provide a pharmaceutical composition, comprising an mRNA complex described herein.

[0113] 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.

[0114] 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.

[0115] 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.

[0116] 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.

[0117] 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.

[0118] Certain embodiments provide 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 intrathecal route.

[0119] Certain embodiments provide 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 intradermal route.

[0120] Certain embodiments provide 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 intravitreal route.

[0121] Certain embodiments provide 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 intracerebroventricular route.

[0122] Certain embodiments provide a method for treating an individual, comprising administering an effective amount of a pharmaceutical composition comprising an mRNAcomplex of any preceding or subsequent claims to an individual, wherein the pharmaceutical composition is administered by a subretinal route.

[0123] 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, a polymer, a polymeric nanoparticle, a protamine, a solid nanoparticle, or a virus-like particle.

[0124] 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.

[0125] Certain embodiments provide the use of the mRNA complexes described herein for the prophylactic or therapeutic treatment of a disease in a subject.

[0126] Certain embodiments provide the mRNA complexes described herein for use in medical therapy.

[0127] Certain embodiments provide the use of the mRNA complexes described herein to prepare a medicament for the treatment of a disease in a subject.

[0128] 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.

[0129] 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 orpreferred; 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 other 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.

[0130] 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.

[0131] 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, i.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.

[0132] 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, i.e., “only A, but not B.”

[0133] 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.

[0134] 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, each range discussed herein can bereadily 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.

[0135] 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.

[0136] 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.

[0137] 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.

[0138] 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.

[0139] 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 the 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.

[0140] 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.

[0141] 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 a 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 phrasepresenting 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 andB.”

[0142] 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.

[0143] 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.

[0144] 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.

[0145] 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.

[0146] 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.

[0147] 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.

[0148] 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.

[0149] The phrase “composed of’ means “including” or “consisting of.” Typically, this phrase denotes that an object is formed from a material.

[0150] 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.”

[0151] 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.

[0152] 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.

[0153] 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.

[0154] 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 be 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.

[0155] In this disclosure, the following acronyms and abbreviations are used.

[0156] 3’ UTR: three prime untranslated region.

[0157] 2’-F: 2' fluoro modification.

[0158] 2’-0Me: 2'-O-methylation modification.

[0159] 2’ -MOE: 2'-O-methoxyethyl modification.

[0160] 3’ -ddC: 3’ Dideoxy cytidine modification.

[0161] 3’ -inv-dT: 3’ inverted deoxythymidine modification.

[0162] P-S: phosphorothioate.

[0163] 5’ UTR: five prime untranslated region.

[0164] A: Adenine

[0165] ApoE: Apolipoprotein E.

[0166] CART: charge-altering releasable transporters.

[0167] C: Cytosine

[0168] CD: Cluster of Differentiation

[0169] DNA: deoxyribonucleic acid

[0170] dsRNA: double-stranded RNA.

[0171] EDV: enveloped delivery vehicle.

[0172] Fab: fragment antigen-binding.

[0173] GalNAc: N-Acetylgalactosamine.

[0174] GNA: glycerol nucleic acids

[0175] gRNA: guide RNA.

[0176] G: Guanine

[0177] HD-RNA: hetero-duplex RNA.

[0178] IgG: Immunoglobulin G.

[0179] LDL receptor: low-density lipoprotein receptor.

[0180] LNA: locked nucleic acid

[0181] LNP: lipid nanoparticles.

[0182] mRNA: messenger RNA.

[0183] nt: nucleotide

[0184] OH: hydroxy modification

[0185] PNA: peptide nucleic acids

[0186] PNP: polymeric nanoparticles.

[0187] RNA: ribonucleic acid

[0188] siRNA: small interfering RNA.

[0189] Str-O-Nuc: an engineered oligonucleotide of this disclosure.

[0190] T: Thymine

[0191] Tm: thermal melting point.

[0192] TNA: threose nucleic acids

[0193] U: Uracil

[0194] VHH: Variable Heavy domain of Heavy chain

[0195] VLP: virus-like particles.

[0196] 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 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 inter-sugar 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.

[0197] 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”.

[0198] 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.

[0199] 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.

[0200] This disclosure’s engineered oligonucleotides are hereafter referred to as Str-O-Nuc, and provide the following advantages.

[0201] 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.

[0202] 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.

[0203] 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 site-specific 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, Nl-methylpseudouridine, in the mRNA manufacturing to limit immunogenicity.

[0204] 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 viruslike particle. mRNA complexes that include a Str-O-Nuc can also be delivered without additional delivery vehicles.

[0205] 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, orRNA 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.

[0206] In some embodiments, a Str-O-Nuc is placed in the coding sequence of an mRNA. In some embodiments, a Str-O-Nuc is placed in the coding sequence of an RNA.

[0207] In some embodiments, a Str-O-Nuc is placed in the non-coding sequence of an mRNA. In some embodiments, a Str-O-Nuc is placed in the non-coding sequence of an RNA, e.g., long non-coding RNA, microRNA. Similarly, an RNA complex comprising an RNA molecule and at least one engineered oligonucleotide hybridized to the 3’UTR of the RNA molecule, wherein said engineered oligonucleotide is hereafter referred to as the Str-O-Nuc, can be placed in the non-coding sequence of the RNA.

[0208] 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-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. 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.

[0209] This disclosure’s mRNA complex, comprising Str-O-Nuc and mRNA, schematically shown herein, by way of example, have the following additional advantages over other technologies.

[0210] 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 canrecognize surface receptors on the VLP, which results in antibodies that bind and neutralize the VLP upon the second dose of the VLP.

[0211] 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-pseudouri dine on the mRNA and / or modifications to the ribose 2’ -OH on the oligonucleotide), which allows for better re-dosing and tolerability.

[0212] 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 desired 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.

[0213] 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.

[0214] 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, orRNA 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 5’ UTR of an mRNA may likely not dissociate if engineered to have a high enough melting temperature.

[0215] 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% N1 -Methylpseudouridine to limit the immunogenicity of the unwanted side products, which negatively impacts the function of most naturally occurring functional elements.

[0216] 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 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) 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).

[0217] 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.

[0218] 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.

[0219] In this disclosure, Str-O-Nuc may be engineered to contain accessory elements such as a m7G-cap, RNA aptamers (e.g., eIF4G-binding aptamer), a poly(A) tail, and / or a poly(A) region (successive sequence of adenines occurring anywhere on the Str-O-Nuc other than at the 3’ end). 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 beengineered to have a poly(A) tail in addition to sequences that are complementary to the 5’UTR of the mRNA. A Str-O-Nuc may also be engineered to have a poly(A) region in addition to sequences that are complementary to the 5’UTR of the mRNA. Such Str-O-Nuc may bind to the 5’-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 such a Str-O-Nuc. A Str-O-Nuc having a poly(A) region 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 protein output of the mRNA complex as compared to a mRNA that does not contain such a Str-O-Nuc.

[0220] The Str-O-Nuc’ s poly(A) tail and / or a poly(A) region may also be chemically modified to make them more stable, resulting in a more stable and / or higher 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.

[0221] The mRNA may optionally contain substantially close to 100% uridine modification (e.g., N1 -methylpseudouridine or 5-methyoxyuridine). 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).

[0222] 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’-M0E, 2’-F, 2’-cEt, P-S linkage, 3’-inv-dT, 3’-ddC or LNA modifications.

[0223] 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 cellularbarriers that is necessary for subcutaneous injection. The only approved siRNA drug that utilizes LNPs is administered by IV infusion, while GalN Ac-conjugated siRNAs are administered by subcutaneous administration.

[0224] 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.

[0225] 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 (charge-altering 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.

[0226] In this disclosure, mRNA may encode a therapeutic protein.

[0227] Such mRNAs can be synthesized by in vitro transcription using standard methods and contain an m7G-cap, a 5’UTR, a coding sequence, a 5’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 5’UTR and no poly (A) tail.

[0228] 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 5’UTR of the mRNA. Such Str-O-Nuc may bind to the 5’-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-Nuc already having a poly(A) tail may also be hybridized on the mRNA that does not have a poly (A) tail.

[0229] 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-Nucmay also be engineered to contain a 5’UTR. 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 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.

[0230] 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.

[0231] The mRNA may optionally contain substantially close to 100% uridine modification (e.g., N1 -methylpseudouridine or 5-methyoxyuridine). 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).

[0232] 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’-M0E, 2’-F, 2’-cEt, P-S linkage, 3’-inv-dT, 3’-ddC or LNA modifications.

[0233] 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.

[0234] 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.

[0235] 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.

[0236] A Str-O-Nuc may contain additional structures (i.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. 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.

[0237] 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.

[0238] 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 inproximity. 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 (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.

[0239] The mRNA complexes of this disclosure may form a double-stranded structure.

[0240] 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.

[0241] 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’ -MOE, 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).

[0242] The Str-O-Nucs of this disclosure are 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, 0. ImM 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.

[0243] 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.

[0244] 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.

[0245] A Str-O-Nuc of this disclosure may be synthesized with a region complementary to the mRNA’s 5’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.

[0246] For Str-O-Nucs with a poly(A) tail, these Str-O-Nuc may be oriented in the forward or reverse direction. 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.

[0247] 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.

[0248] 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 5’ UTR of an mRNA with no poly(A) tail.

[0249] 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 5’ 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. 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 5’ 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.

[0250] 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 5’ end of an mRNA with no poly(A) tail. 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 5’ end of an mRNA with no poly(A) tail. 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 5’ end of an mRNA with no poly (A) tail.

[0251] 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 5’ 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. 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 5’ UTR of an mRNA with nopoly(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.

[0252] 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 5’ 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. 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 5’ 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. 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 5’ 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.

[0253] 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. 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. 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. 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 a 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.

[0254] 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.

[0255] 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. 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-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.

[0256] 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. 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’MOEmodifications, wherein the at least two nucleotides are held together with PS linkages, and the second Str-O-Nuc is oriented in the forward direction.

[0257] 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. 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 together with PS linkages, and the second Str-O-Nuc is oriented in the reverse direction.

[0258] 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-methoxyethyl (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-methoxyethyl (MOE) alternate with each other, and that bind to the 3’ terminal nucleotides (nt) in 5’ 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 5’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-m ethoxy ethyl (MOE), that binds to the 18nt of the 3’ terminal end of the 5’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-methoxy ethyl (MOE) backbone modification that binds to the 25 nt of the 3’ terminal end of the 5’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 5’UTR of the mRNA.

[0259] 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 such poly(A) tails or lack of double strandedness. Herein we provide a system to stabilize these templates.

[0260] Double-stranded RNA have intrinsic thermodynamics stability compared to singlestranded 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.].

[0261] 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 codelivered 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 singlestranded 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 the resulting enzymatic protein reverse transcribes the therapeutic mRNA into cDNA, and inserts it in the genome.

[0262] 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 basespecific 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.

[0263] 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.

[0264] 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 DNAsequences 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 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.

[0265] 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 complementary 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.

[0266] 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).

[0267] The Str-O-Nucs may contain am oligonucleotide containing a hairpin loop. One mechanism by which mRNA stability is regulated is by 5’-3’ exonucleases that hydrolyze ribonucleotides of mRNA (e.g. RNA exosome or CCR4-NOT). Introduction of a hairpin loop can result in steric blocking of the mRNA, thus enhancing mRNA half-life and stability. 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.

[0268] 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 hydrolyzeribonucleotides of mRNA (e.g. polynucleotide phosphorylases (PNPases)). Introduction of a modified 3 ’-3’ phosphodiester bond blocks activity of the 3 ’-5’ exonuclease, thus enhancing mRNA half-life and stability. The secondary oligonucleotide that is complementary to the splint oligonucleotide terminates in a 3 ’-3’ inverted dT, thereby inhibiting activity of 3 ’-5’ exonucleases.

[0269] 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.

[0270] 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.

[0271] 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 sequencedependent 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.

[0272] 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 nucleic 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).

[0273] 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.

[0274] 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.

[0275] 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 Kieft 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.

[0276] 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 thatalternative 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.

[0277] 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 (i.e., 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.

[0278] 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.

[0279] 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 alignmentand 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.

[0280] 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.

[0281] 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, 9O 5S'73-5'7S'l (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.

[0282] 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 (i.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 thedisease or disorder is to be prevented. In one embodiment “treat”, “treatment”, or “treating” does not include preventing or prevention.

[0283] 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.

[0284] 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.

[0285] 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 not limited to, carriers, binders, disintegrating agents, lubricants, sweetening agents, flavoring agents, coatings, preservatives, and dyes.

[0286] 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.

[0287] 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 requiredparticle 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.

[0288] 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.

[0289] 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.

[0290] 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.

[0291] 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.

[0292] 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 sub-doses 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.

[0293] An administration route may be made using any therapeutically effective means, including injection, and including intradermal, intrathecal, intraparenchymal,intracerebroventricular, intramuscular, intravenous, intravitreal, intranasal, subretinal and subcutaneous.

[0294] The invention will now be illustrated by the following non-limiting Examples.EXAMPLES

[0295] 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), and 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.

[0296] 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., N1 -methylpseudouridine or 5-methyoxyuridine).

[0297] 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.

[0298] In this disclosure, the mRNA molecule may be partially or substantially (e.g., close to 100%) hybridized to the Str-O-Nuc, leaving minimal to no single stranded region(s).

[0299] 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).

[0300] 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., Nl-methylpseudouridine or 5-methyoxyuridine).

[0301] 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).

[0302] 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, scFv), 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.

[0303] 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 viruslike 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.

[0304] 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 etal., Nature, 621, 396-403 (2023)).

[0305] 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 by nucleases in biofluids, endosomes, and / or the cytoplasm; or a combination of such stabilization methods.

[0306] 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.

[0307] Example 1.

[0308] This study uses mRNA encoding luciferase (SecNanoLuc) with a 120 poly(A) tail. In this study, Str-O-Nuc will be transfected with SecNanoLuc mRNA: Str-O-Nuc _367 - (1) hdRNA167- 5’ untranslated region (UTR) tiling with no overhang on 5'UTR, (2) Str-O-Nuc _368 - hdRNA168 - 5’UTR tiling with cap overhang on 5'UTR, (3) Str-O-Nuc _066 -hdRNA169 - 5’UTR tiling with 5'UTR overhang on 5'UTR, (4) hdRNA170 - coding sequence (CDS) tiling with medium Tm , (5) hdRNA171 - 3’UTR tiling, (6) hdRNA172 - CDS Str-O-Nuc with 3’UTR tiling, (7) Str-O-Nuc _066 - hdRNA173 - 5’UTR with no overhang, CDS engineered oligonucleotides, and 3’UTR tiling, (8) Str-O-Nuc _368 - hdRNA174 - 5’UTR with cap overhang, CDS engineered oligonucleotides, and 3’UTR tiling, and (9) Str-O-Nuc 367 -hdRNA175 - 5’UTR with a 5'UTR overhang, CDS, 3’UTR tiling. All of the aforementioned engineered oligonucleotides have the 2'-O-methoxyethyl (2'-M0E) backbone modification.

[0309] The aforementioned engineered oligonucleotides will be delivered with SecNanoLuc mRNA (hdRNA166). Relevant controls include: (1) untreated condition, (2) Lipofectamine Messenger Max (LMM) vehicle only condition, and (3) hdRNA166- SecNanoLuc mRNA alone.

[0310] Engineered oligonucleotides were annealed to the SecNanoLuc (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.

[0311] 100 nanograms (ng) of the annealed mRNA were transfected into the adherent HEK293T cell line, utilizing the mRNA lipofection reagent LMM. Following the transfection, supernatants containing the secreted luciferase from HEK293T cells were taken 24 hours posttransfection. Luciferin reagent was added to diluted samples (200x) and read on a plate reader for luminescence.

[0312] SecNanoLuc mRNA (hdRNA166) transfected alone had the highest luminescence signal of all conditions tested. Tiling with engineered oligonucleotides of only the CDS (hdRNA170) produced a luminescence signal similar to that of hdRNA166 of approximately 108RLU. The addition of engineered oligonucleotides tiling of the 3’ UTR with the CDS (hdRNA172) results in an approximately 1 log lower luminescence signal compared to CDS tiling alone. Tiling of the 5’ UTR, CDS, and 3’UTR (hdRNAsl73-175) of SecNanoLuc mRNA resulted in the lowest luminescence values of any groups tested, excluding untreated and LMM only controls. Tiling the 5’ UTR of SecNanoLuc mRNA alone gave the luminescence signal relative to transfection of hdRNA166 alone. Within the transfected groups of 5’ UTR tiling, the use of a cap overhang on the 5’ UTR (hdRNA168) gave a 1.5-fold increase in signal over no overhang (hdRNA167). These results are depicted in Figure 1.

[0313] Example 2.

[0314] The engineered oligonucleotides described herein are nucleic acids that contain a region complementary to a particular mRNA (e.g. eGFP) with modifications intended to preserve transfected mRNA from degradation. The purpose of this study is to determine if a Str-O-Nuc and its modifications provide enhanced stability to mRNA containing a poly(A) tail.

[0315] In this study, the following engineered oligonucleotides will be transfected with eGFP mRNA containing a poly(A) tail (120A): (1) Str-O-Nuc _499 - hdRNA148 - a 20 nucleotide (nt) Str-O-Nuc binding at the first nt of the 5’ untranslated region (UTR) of eGFP (120A), (2) Str-O-Nuc _500 - hdRNA149 - a 20 nucleotide (nt) Str-O-Nuc binding at the third nt of the 5’ untranslated region (UTR) of eGFP (120A), (3) Str-O-Nuc 501 - hdRNA150 - a 20 nucleotide (nt) Str-O-Nuc binding at the fifth nt of the 5’ untranslated region (UTR) of eGFP (120A), (4) Str-O-Nuc _502 - hdRNA151 - a 20 nucleotide (nt) Str-O-Nuc binding at the seventh nt of the 5’ untranslated region (UTR) of eGFP (120A), (5) Str-O-Nuc 503 -hdRNA152 - a 20 nucleotide (nt) Str-O-Nuc binding at the ninth nt of the 5’ untranslated region(UTR) of eGFP (120A), (6) Str-O-Nuc _504 - hdRNA153 - a 20 nucleotide (nt) Str-O-Nuc binding at the eleventh nt of the 5’ untranslated region (UTR) of eGFP (120 A), (7) Str-O-Nuc _505 - hdRNA154 - a 20 nucleotide (nt) Str-O-Nuc binding at the thirteenth nt of the 5’ untranslated region (UTR) of eGFP (120A), (8) Str-O-Nuc _506 - hdRNA155 - a 20 nucleotide (nt) Str-O-Nuc binding at the fifteenth nt of the 5’ untranslated region (UTR) of eGFP (120 A), (9) Str-O-Nuc _509 - hdRNA158 - a 19 nt Str-O-Nuc binding at the first nt of the 5’ untranslated region (UTR) of eGFP (120A), (10) Str-O-Nuc _508 - hdRNA157 - a 19 nt Str-O-Nuc binding at the twentieth nt of the 5’ untranslated region (UTR) of eGFP (120A), (11) Str-O-Nuc 507 -hdRNA156 - a 19 nt Str-O-Nuc binding at the thirty-ninth nt of the 5’ untranslated region (UTR) of eGFP (120A), (12) Str-O-Nuc _066 - hdRNA142 - a 1. lx 5’ UTR Str-O-Nuc with a 2'-O-methoxyethyl (2'-M0E) backbone modification, (13) Str-O-Nuc _368 - hdRNA143 - a l.lx 5’ UTR Str-O-Nuc with a 20nt 3’ overhang over the T7 CleanCap with a 2'-M0E backbone modification, (14) Str-O-Nuc _367 - hdRNA144 - a l.lx 5’ UTR Str-O-Nuc with a 20nt 5’ overhang over the 5’ UTR with a 2'-M0E backbone modification, (15) Str-O-Nuc 066 -hdRNA145 - a 2X 5’ UTR Str-O-Nuc with a 2'-M0E backbone modification, (16) Str-O-Nuc _368 - hdRNA146 - a 2X 5’ UTR Str-O-Nuc with a 20 nt 3’ overhang over the T7 CleanCap, with a 2'-M0E backbone modification, (17) Str-O-Nuc _367 - hdRNA147 - a 2X 5’ UTR Str-O-Nuc with a 20 nt 5’ overhang over the 5’ UTR, with a 2'-M0E backbone modification, (18) Str-O-Nuc _400 - hdRNA163 - a 14 nt Str-O-Nuc binding at the first nt of the 5’ UTR with a 5nt 3’ overhang, (19) Str-O-Nuc _399 - hdRNA164 - a 14 nt Str-O-Nuc binding at the first nt of the 5’ UTR with a lOnt 3’ overhang, (20) Str-O-Nuc _398 - hdRNA165 - a 14 nt Str-O-Nuc binding at the first nt of the 5’ UTR with a 15nt 3’ overhang, (21) Str-O-Nuc 513 - hdRNA162 - a 20 nucleotide (nt) Str-O-Nuc binding at the third nucleotide of the 5’ UTR with a 15nt 3’ overhang, (22) Str-O-Nuc 512 - hdRNA159 - a 20 nt Str-O-Nuc binding at the first nt of the 5’ UTR with a 5nt 3’ overhang, (23) Str-O-Nuc _511 - hdRNA160 - a 20 nt Str-O-Nuc binding at the first nt of the 5’ UTR with a lOnt 3’ overhang, and (24) Str-O-Nuc 510 - hdRNA161 - a 20 nt Str-O-Nuc binding at the first nt of the 5’ UTR with a 15nt 3’ overhang.

[0316] The aforementioned engineered oligonucleotides will be delivered with eGFP mRNA (hdRNA141). Relevant controls include: (1) untreated condition, (2) Lipofectamine Messenger Max (LMM) vehicle only condition, and (3) hdRNA141- eGFP mRNA alone.

[0317] Engineered oligonucleotides were annealed to the eGFP 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. These results are depicted in Figure 2.

[0318] Within the engineered oligonucleotides that have a 20 nt binding sequence, there was no observable pattern of eGFP fluorescence shifting the binding sequence 2 nts along the 5’ UTR. hdRNA155, placed at the fifteenth nt of the 5’ UTR had the highest level of eGFP expression among hdRNAs 148-155, relative to eGFP mRNA alone (hdRNA141). The expression of hdRNA155 was approximately half the expression of hsRNA141. There was also a 13 -fold reduction in expression by placing the engineered oligonucleotide at the third nt of the 5’ UTR versus the first nt. Comparing 20nt versus 19nt binding sequences, hdRNA158 which binds to the first nt of the 5’ UTR had a 100-fold greater expression than hdRNA148 and surpassed expression of eGFP mRNA alone (hdRNA141). hdRNA158 has the same binding site but one addition nt in the binding sequence. These results are depicted in Figure 3.

[0319] When comparing the level of expression of eGFP by using a 3’ overhang that extends over the T7 CleanCap or a 5’ overhang that extends over the 5’ UTR, hdRNA143 and hdRNA146 (which contain 3’ overhangs that extend over the T7 CleanCap) had higher expression than hdRNA144 and hdRNA147 (which contain 5’ overhangs that extend over the 5’ UTR). hdRNA142 and hdRNA145 which lack overhangs had the lowest expression of eGFP amongst the conditions tested. These results are depicted in Figure 4.

[0320] When comparing the effect of overhang length within engineered oligonucleotide constructs, there was a construct-dependent effect on eGFP expression by the number of nts included in the overhang. hdRNA143, hdRNA163, hdRNA164, and hdRNA165 which contain a 14nt binding sequence with a 3’ overhang of varying nt length had no change on the relative level of eGFP expression. hdRNA148, hdRNA159, and hdRNA161 which contain a 20nt binding sequence with a 3’ overhang of varying nt length had little to no detectable expression of eGFP regardless of overhang length. However, hdRNA160 which contains the same 20nt binding sequence with a 3’ overhang length of lOnt had an approximately 14-fold increase in eGFP expression relative to hdRNA161 and hdRNA159.Table 1. Certain sequences of the Examples# hdRNA # Engineered Engineered oligonucleotide sequence oligonucleotide (overhands in lowercase,label hybridization region in UPPERCASE) Str-O- hdRNA142 / 5 ' UTR_4 AGTTTATTTCTCCTNuc _066 hdRNA145 / / hdRNA169 / (SEQ ID hdRNA173NO: 1)# hdRNA # Engineered Engineered oligonucleotide sequence oligonucleotide (overhands in lowercase,label hybridization region in UPPERCASE) Str-O- hdRNA143 / 5 ' UTR_4 with ttcgtctggccgtactttccAGTTTATTTCTCCT Nuc _368 hdRNA146 / 2 Ont 5 'hdRNA168 / overhang(SEQ ID hdRNA174NO: 2)Str-O- hdRNA144 / 5 ' UTR_4 with AGTTTATTTCTCCTttcgtctggccgtactttcc Nuc _367 hdRNA147 / 2 Ont 3 'hdRNA167 / overhang(SEQ ID hdRNA175NO: 3)Str-O- hdRNA148 5 ' UTR_20nt-l AATACTAGTTTATTTCTCCTNuc _499(SEQ IDNO: 4)Str-O- hdRNA149 5 ' UTR_2 Ont-2 AGAATACTAGTTTATTTCTCNuc _500(SEQ IDNO: 5)Str-O- hdRNA150 5 ' UTR_2 Ont-3 GAAGAAT AC TAGTTTATTTCNuc _501(SEQ IDNO: 6)Str-O- hdRNA151 5 ' UTR_2 Ont-4 C AGAAGAAT AC TAGTTTATTNuc _502(SEQ IDNO: 7)Str-O- hdRNA152 5 ' UTR_2 Ont-5 AC C AGAAGAAT AC T AGT T T ANuc _503(SEQ IDNO: 8)Str-O- hdRNA153 5 ' UTR_20nt-6 GGACCAGAAGAATACTAGTTNuc _504(SEQ IDNO: 9)Str-O- hdRNA154 5 ' UTR_2 Ont-7 GGGGACCAGAAGAATACTAGNuc _505(SEQ IDNO: 10)Str-O- hdRNA155 5 ' UTR_20nt-8 GTGGGGACCAGAAGAATACTNuc _506(SEQ IDNO: 11)# hdRNA # Engineered Engineered oligonucleotide sequence oligonucleotide (overhands in lowercase,label hybridization region in UPPERCASE) Str-O- hdRNA156 5 ' GGTGGCGGGTTCTCTCTGANuc _507 UTR 19ntSegments-5(SEQ IDNO: 12)Str-O- hdRNA157 5 ' GTCTGTGGGGACCAGAAGANuc _508 UTR 19ntSegments-6(SEQ IDNO: 13)Str-O- hdRNA158 5 ' ATACTAGTTTATTTCTCCTNuc _509 UTR 19ntSegments-7~(SEQ IDNO: 14)Str-O- hdRNA161 5 ' UTR_20nt-l AATACTAGTTTATTTCTCCTttcgtctggccgtac Nuc _510 with 15nt 3 ' - overhang(SEQ IDNO: 15)Str-O- hdRNA160 5 ' UTR_20nt-l AATACTAGTTTATTTCTCCTttcgtctggc Nuc _511 with lOnt 3 ' - overhang(SEQ IDNO: 16)Str-O- hdRNA159 5 ' UTR_20nt-l AATACTAGTTTATTTCTCCTttcgtNuc _512 with 5nt 3 ' - overhang(SEQ IDNO: 17)Str-O- hdRNA162 5 ' UTR_2 Ont-2 AGAATACTAGTTTATTTCTCttcgtctggccgtac Nuc _513 with 15nt 3 ' - overhang [ free(SEQ ID AG]NO: 18)Str-O- hdRNA163 5 ' UTR_4 with AGTTTATTTCTCCTttcgtNuc _400 5nt 3 ' -overhang(SEQ IDNO: 19)Str-O- hdRNA164 5 ' UTR_4 with AGTTTATTTCTCCTttcgtctggcNuc _399 lOnt 3 ' - overhang(SEQ IDNO: 20)Str-O- hdRNA165 5 ' UTR_4 with AGTTTATTTCTCCTttcgtctggcNuc _398 15nt 3 ' - overhang(SEQ IDNO: 21)(SEQ ID hdRNA406- PO 360 TGCCGCCCACTCAGACTTNO: 22) 410# hdRNA # Engineered Engineered oligonucleotide sequence oligonucleotide (overhands in lowercase,label hybridization region in UPPERCASE) (SEQ ID hdRNA407 / 40 PO066 AGTTTATTTCTCCTNO: 23) 9(SEQ ID hdRNA408 / 41 PO367 AGTTTATTTCTCCTttcgtctggccgtactttcc NO: 24) 0(SEQ ID hdRNA409 / 41 PO064 AGTTTATTTCTCCTttcgtctggccgtactttcc NO: 25) 0(SEQ ID hdRNA409 / 41 POO 65 CCAGAAGAATACTNO: 26) 0(SEQ ID hdRNA409 / 41 PO067 CTGAGTCTGTGGGGANO: 27) 0# hdRNA # Engineered Engineered oligonucleotide sequence oligonucleotide (overhands in lowercase,label hybridization region in UPPERCASE) (SEQ ID hdRNA405- mRNAO 18 AGGAGAAAUAAACUAGUAUU CUUCUGGUCCC CACAG NO: 28) 410 ACU C AGAGAGAAC C C G C C AC C AU G GAAGAU G C C AAA AACAUUAAGAAGGGGCCAGCGCCAUUCUACCCACUC GAAGACGGGACCGCAGGAGAGCAGCUGCACAAAGCC AUGAAGAGAUACGCACUGGUGCCUGGCACUAUCGCC UUUAC C GAC GCACAUAUAGAGGU GGAUAUUAC CUAU GCCGAGUACUUUGAGAUGUCCGUUCGGCUGGCAGAA GCUAUGAAGCGCUAUGGGUUAAAUACAAAUCAUCGG AUU GU C GU GU GUAGC GAAAAUU C CUU GCAGUU CUUU AUGCCCGUCCUUGGGGCCCUCUUCAUCGGUGUGGCU GU G G C C C C AG CU AAC GAC AU CU AC AAC GAG C GU GAG CUGCUGAACUCUAUGGGAAUCAGCCAGCCUACCGUC GUAUUUGUGAGCAAGAAAGGGCUGCAAAAGAUCCUC AAU GUU CAGAAGAAACUAC C GAU CAUACAGAAGAUA AUUAU CAU GGAUU C CAAAAC C GACUAC CAGGGCUU C CAGU CAAU GUAUAC CUU C GU GACUU C C CAUUU GC CA C CU GGCUUUAAU GAGUAC GAUUUU GU GC CU GAAU C G UU C GAU C GGGAUAAAACUAUU GC C CU GAU CAU GAAC AGUUCUGGAAGUACUGGAUUGCCCAAGGGCGUAGCA CUU C C GCAU C GAACU GCUU GU GU C C GAUU CAGU CAU GCUAGAGAU C C CAU CUU C GGCAAC CAGAUUAUU C CU GAU AC C GCUAUACU CU CAGU C GUU C CAUUU CAU CAC GGAUUCGGCAUGUUCACGACGCUGGGCUACUUGAUC UGCGGCUUUAGGGUCGUGCUCAUGUACAGGUUUGAG GAGGAGCUAUUCUUAAGGUCCUUGCAAGACUAUAAG AUUCAAUCUGCCCUGCUGGUGCCCACACUUUUUAGC UUUUUU GCUAAGU CUACU CU CAUU GACAAAUAU GAC CUAUCCAACUUACACGAGAUAGCCAGCGGAGGGGCG CCGCUCUCUAAAGAGGUAGGUGAGGCCGUGGCGAAA AGGUUUCACCUUCCAGGAAUACGCCAGGGGUACGGG CU GACAGAAACAACAU CAGC CAUU CU GAU CAC C C C C GAAGGGGACGACAAACCUGGCGCAGUAGGCAAGGUG GUGCCCUUCUUCGAGGCUAAGGUGGUGGAUUUGGAU ACCGGUAAAACACUGGGUGUGAAUCAGAGAGGGGAA CUGUGCGUCCGUGGACCCAUGAUCAUGUCAGGCUAU GUUAACAAU C C C GAGGCUACAAAU GCU CU CAU C GAC AAGGACGGCUGGCUGCACUCGGGAGACAUUGCAUAU U G G GAC GAAGAU GAGCACUUCUU C AU AGU C GAU C G G CUGAAGAGUUUAAUCAAAUACAAGGGCUACCAGGUA GCAC CAGCU GAACUU GAGAGCAU C CUU CUU CAACAC CCCAACAUUUUCGACGCAGGGGUCGCGGGACUGCCU GACGAUGAUGCCGGCGAACUGCCUGCCGCAGUCGUC GU G CU C GAAC AC G GU AAAACU AU GAC G GAGAAG GAA AUCGUGGACUAUGUGGCCAGCCAGGUUACAACCGCC AAGAAGCUGCGCGGUGGUGUUGUGUUUGUUGAUGAG GUUCCUAAAGGACUGACCGGCAAAUUGGACGCGCGC AAGAUCAGAGAAAUUCUCAUUAAGGCCAAAAAAGGC GGAAAGAUCGCCGUGUGAUGAGCUGGAGCCUCGGUG GCCUAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCC CUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUU UGAAUAAAGUCUGAGUGGGCGGCA# hdRNA # Engineered Engineered oligonucleotide sequence oligonucleotide (overhands in lowercase,label hybridization region in UPPERCASE) (SEQ ID hdRNA404 mRNAO 19 AGGAGAAAUAAACUAGUAUU CUUCUGGUCCC CACAG NO: 29) ACU C AGAGAGAAC C C G C C AC C AU G GAAGAU G C C AAA AACAUUAAGAAGGGGCCAGCGCCAUUCUACCCACUC GAAGACGGGACCGCAGGAGAGCAGCUGCACAAAGCC AUGAAGAGAUACGCACUGGUGCCUGGCACUAUCGCC UUUAC C GAC GCACAUAUAGAGGU GGAUAUUAC CUAU GCCGAGUACUUUGAGAUGUCCGUUCaGGCUGGCAGA AGCUAUGAAGCGCUAUGGGUUAAAUACAAAUCAUCG GAUU GU C GU GU GUAGC GAAAAUU C CUU GCAGUU CUU UAUGCCCGUCCUUGGGGCCCUCUUCAUCGGUGUGGC U GU G G C C C C AG CU AAC GAC AU CU AC AAC GAG C GU GA GCUGCUGAACUCUAUGGGAAUCAGCCAGCCUACCGU CGUAUUUGUGAGCAAGAAAGGGCUGCAAAAGAUCCU CAAU GUU CAGAAGAAACUAC C GAU CAUACAGAAGAU AAUUAU CAU GGAUU C CAAAAC C GACUAC CAGGGCUU C CAGU CAAU GUAUAC CUU C GU GACUU C C CAUUU GC C AC CU GGCUUUAAU GAGUAC GAUUUU GU GC CU GAAU C GUU C GAU C GGGAUAAAACUAUU GC C CU GAU CAU GAA CAGUUCUGGAAGUACUGGAUUGCCCAAGGGCGUAGC ACUUCCGCAUCGAACUGCUUGUGUCCGAUUCAGUCA U GCUAGAGAU C C CAU CUU C GGCAAC CAGAUUAUU C C U GAU AC C GCUAUACU CU CAGU C GUU C CAUUU CAU CA CGGAUUCGGCAUGUUCACGACGCUGGGCUACUUGAU CUGCGGCUUUAGGGUCGUGCUCAUGUACAGGUUUGA GGAGGAGCUAUUCUUAAGGUCCUUGCAAGACUAUAA GAUUCAAUCUGCCCUGCUGGUGCCCACACUUUUUAG CUUUUUU GCUAAGU CUACU CU CAUU GACAAAUAU GA CCUAUCCAACUUACACGAGAUAGCCAGCGGAGGGGC GCCGCUCUCUAAAGAGGUAGGUGAGGCCGUGGCGAA AAGGUUUCACCUUCCAGGAAUACGCCAGGGGUACGG GCU GACAGAAACAACAU CAGC CAUU CU GAU CAC C C C CGAAGGGGACGACAAACCUGGCGCAGUAGGCAAGGU GGUGCCCUUCUUCGAGGCUAAGGUGGUGGAUUUGGA UACCGGUAAAACACUGGGUGUGAAUCAGAGAGGGGA ACUGUGCGUCCGUGGACCCAUGAUCAUGUCAGGCUA U GUUAACAAU C C C GAGGCUACAAAU GCU CU CAU C GA CAAGGACGGCUGGCUGCACUCGGGAGACAUUGCAUA UU GGGAC GAAGAU GAGCACUU CUU CAUAGU C GAU C G GCUGAAGAGUUUAAUCAAAUACAAGGGCUACCAGGU AGCAC CAGCU GAACUU GAGAGCAU C CUU CUU CAACA CCCCAACAUUUUCGACGCAGGGGUCGCGGGACUGCC UGACGAUGAUGCCGGCGAACUGCCUGCCGCAGUCGU C GU G CU C GAAC AC G GU AAAACU AU GAC G GAGAAG GA AAUCGUGGACUAUGUGGCCAGCCAGGUUACAACCGC CAAGAAGCUGCGCGGUGGUGUUGUGUUUGUUGAUGA GGUUCCUAAAGGACUGACCGGCAAAUUGGACGCGCG C AAGAU C AGAGAAAUU CU C AUU AAG G C C AAAAAAG G CGGAAAGAUCGCCGUGUGAUGAGCUGGAGCCUCGGU GGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCC CCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCU UUGAAUAAAGUCUGAGUGGGCGGCAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAExample 3:

[0321] The purpose of this study is to determine the effect of 3’ end, 5’ end, or tiled 5’ UTR Str-O-Nuc on the expression of firefly luciferase expression, in vitro.

[0322] The Str-O-Nuc used in this study are: (1) hdRNA406 - a 3’ end Str-O-Nuc with a 18 nucleotide binding sequence, (2) hdRNA407 - a 5’ end Str-O-Nuc with a 14 nucleotide binding sequence without an overhang and a 3’ end Str-O-Nuc with a 18 nucleotide binding sequence, (3) hdRNA408- a 5’ end Str-O-Nuc containing a 14 nucleotide binding sequence with an overhang extending over the mRNA cap and a 3’ end Str-O-Nuc with a 18 nucleotide binding sequence, (4) hdRNA409 - a tiled 5’ UTR Str-O-Nuc without an overhang and a 3’ end Str-O-Nuc with a 18 nucleotide binding sequence, (5) hdRNA410 - a tiled 5’ UTR Str-O-Nuc with an overhang extending over the mRNA cap and a 3’ end Str-O-Nuc with a 18 nucleotide binding sequence.

[0323] Str-O-Nucs were hybridized to the hdRNA405_Fluc (0A) 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. Following the hybridization of Str-O-Nuc to the mRNA, the hybridized RNAs were purified by size exclusion chromatography.

[0324] 100 nanograms (ng) of the purified mRNA hybridized to Str-O-Nucs were transfected into the adherent HepG2 cell line, utilizing the mRNA lipofection reagent LMM. hdRNA404_Fluc (120A) mRNA and hdRNA405_Fluc (0A) were transfected without hybridizing to any Str-O-Nucs as positive and negative controls, respectively. Following the transfection, the cells were lysed 24 hours post-transfection, luciferin reagent was added to diluted cell lysates (200x) and read on a plate reader for luminescence.

[0325] Relative to hdRNA404 containing a 120 A poly(A) tail, hdRNA405 that lacks a poly(A) tail has an approximate 100-fold reduction in luminescence. The addition of a 3’ end Str-O-Nuc, as seen in hdRNA406, increases expression relative to hdRNA405 by approximately 1 log. Relative to hdRNA406, the addition of a 5’ end Str-O-Nuc without an overhang (hdRNA407) reduces the expression of firefly luciferase mRNA that was stabilized by the 3’ end Str-O-Nuc. However, a 5 ’end Str-O-Nuc with an overhang extending over the cap of the mRNA (hdRNA408) results in higher expression than an equivalent hdRNA without an overhang on the 5’ most Str-O-Nuc (hdRNA409). Lastly, adding Str-O-Nucs that the 5’ UTR of firefly luciferase lacking a poly(A) tail with a 3’ end Str-O-Nuc (hdRNA409) results in less expression relative to adding just one Str-O-Nuc to extreme 5 ’UTR (hdRNA407). Once again, adding an overhang to the 5 ’-most Str-O-Nuc, in addition to adding Str-O-Nucs that tile the5’UTR, results in higher expression than having no overhang (hdRNA409 vs. hdRNA410). These results are depicted in Figure 5.Example 4: Minimal UTRs with chemically optimized Str-O-Nucs maximize Flue translation of tiled mRNAs

[0326] The purpose of this study was to determine the effect of sequence-optimized 5’ and 3’ UTRs or tiled 5’ and 3’ UTRs with Str-O-Nucs on the expression of FLuc in vitro.

[0327] Str-O-Nucs were hybridized to mRNA by heating the mixture of nucleic acids to 95°C for 3 minutes, followed by a cooling step to 25°C, set at a cooling rate of 0.1°C per second. 100 nanograms (ng) of the mRNA hybridized to Str-O-Nucs were transfected into the adherent HEK293T cell line, utilizing the mRNA lipofection reagent Lipofectamine MessengerMax (LMM). Following transfection, the cells were lysed at 4, 24, 48, and 72 hours, mixed with luciferin reagent, and measured for luminescence on a plate reader. Total area under the curve (AUC) for the resulting time course of expression was determined and plotted (as RLU*hr).

[0328] hdRNAl 104 is a standard comparator mRNA with optimized 5’ and 3’ UTRs and a split (30A-10nt-70A) poly(A) tail. All other hdRNAs contain mRNAs that are bound to one or more Str-O-Nuc. Threse mRNAs do not contain a poly(A) tail, and all are bound to Str517. Str517 is a 3’ end Str-O-Nuc, which binds to the 29nt landing pad immediately after the 3’ UTR and at the extreme 3’ end of the mRNA. Hybridizing Str517 to an mRNA without a poly(A) tail effectively replaces the poly(A) tail of an mRNA with a Str-O-Nuc, resulting in similar expression to the comparator mRNA (hdl 105 vs. hdl 104). It allows for simpler tiling of an mRNA and was thus included in all mRNAs (except the hdl 104 comparator).

[0329] hdl 105, 1106, and 1107 are variations of the same mRNA (mRNAlOl). hdl 105 contains no Str-O-Nuc hybridized to the 5’ UTR, whereas hdl 106 and 1107 both contain 4x Str-O-Nucs bound to the entirety of the 5’ UTR. In hdl 106, Str-O-Nucs (Str064-067) are hybridized to mRNAlOl and are fully 2’-M0E modified. In hdl 107, the Str-O-Nucs (Str964-966) are identical in sequence but are fully 2’-0Me modified. Flue expression is >20x higher in hdl 107 (~1.8xl08) compared to hdl 106 (~6.5xl06), suggesting that 2’-0Me modified Str-O-Nucs allow for more translation compared to 2’ -MOE modified Str-O-Nucs.

[0330] hdl 110-1112 are variations of the same mRNA (mRNA126). mRNA126 has a minimal 5’ UTR sequence (min5’UTR_vl). hdl 111 and 1112 are both bound to a single 5’ Str-O-Nuc (Str968 and 969, respectively) that covers the entirety of the minimal 5’ UTR, whereas hdl 110 contains no 5’ UTR Str-O-Nuc. hdl 111 is bound to a Str-O-Nuc that is fully 2’-M0Emodified, whereas the Str-O-Nuc bound to hdl 112 is identical in sequence but is fully 2’-0Me modified, hdl 112 is translated at a significantly higher level than hdl 111, again suggesting that 2’-0Me modifications of the Str-O-Nuc allow for higher levels of translation compared to 2’-MOE modifications.

[0331] The same conclusions can be drawn from hdl 115-1117 and hdl 120-1122. Both hdRNA sets contain different minimal 5’UTR mRNAs (mRNA127 and mRNA 128, respectively) bound to no Str-O-Nuc, or a single Str-O-Nuc that is either 2’ -MOE or 2’-0Me. In all cases, the 2’-OMe-bound mRNA produces more protein compared to the 2'-MOE-bound version.

[0332] The 3’ UTR sequence, length and Str-O-Nuc chemistry was also optimized (hdl 127-hdl 138). In summary, Str-O-Nucs hybridized to the 3’UTR that were 2’0Me modified generally resulted in higher expression compared to sequence identical Str-O-Nucs that were 2’MOE modified (compare hdl 127 with hdl 128; hdl 131 with hdl 132; hdl 134 with hdl 135; hdl 137 with hdl 138).

[0333] These results indicate that Str-O-Nucs that are hybridized to the 5' UTR result in higher FLuc translation when they are 2’0Me modified compared to 2’MOE modified. These results are depicted in Figure 6.Example 5: Minimal UTRs with chemically optimized Str-O-Nucs maximize HiBiT translation of tiled mRNAs

[0334] The purpose of this study was to determine the effect of sequence optimized 5’ and 3’ UTRs or tiled 5’ and 3’ UTRs with Str-O-Nucs on the expression of HiBiT in vitro.

[0335] Str-O-Nucs were hybridized to mRNA by heating the mixture of nucleic acids to 95°C for 3 minutes, followed by a cooling step to 25°C, set at a cooling rate of 0.1°C per second. 100 nanograms (ng) of the mRNA hybridized to Str-O-Nucs were transfected into the HEK293T cell line, utilizing the mRNA lipofection reagent Lipofectamine MessengerMax (LMM). Following transfection, the cells were lysed at 4, 24, 48, and 72hr, mixed with luciferin reagent and measured for luminescence on a plate reader. Total area under the curve (AUC) for the resulting time course of expression was determined and plotted (as RLU*hr).

[0336] hdRNA1069 is a standard comparator mRNA with optimized 5’ and 3’ UTRs and a split (30A-10nt-70A) poly(A) tail. All other hdRNAs contain mRNAs that are bound to one or more Str-O-Nuc. These mRNAs do not contain a poly(A) tail, and all are bound to Str517. Str517 is a 3’ end Str-O-Nuc, which binds to the 29nt landing pad immediately after the 3’ UTRand at the extreme 3’ end of the mRNA. Hybridizing Str517 to an mRNA without a poly(A) tail effectively replaces the poly(A) tail of an mRNA with a Str-O-Nuc, resulting in similar expression to the comparator mRNA (hdl070 vs. hdl069). It allows for simpler tiling of an mRNA and was thus included in all mRNAs (except the hdl069 comparator).

[0337] hdl070, 1071, and 1072 are variations of the same mRNA (mRNAl 18). hdl070 contains no Str-O-Nuc hybridized to the 5’ UTR, whereas hdl071 and 1072 both contain 4x Str-O-Nucs bound to the entirety of the 5’ UTR. In hd 1071, Str-O-Nucs (Str064-067) are hybridized to mRNAl 18 and are fully 2’-M0E modified. In hdl072, the Str-O-Nucs (Str964-966) are identical in sequence but are fully 2’-0Me modified. HiBiT expression is ~3x higher in hdl072 (4.3xl06) compared to hdl071 (1.3xl06), suggesting that 2’-0Me modified Str-O-Nucs allow for more translation compared to 2’ -MOE modified Str-O-Nucs.

[0338] hdl075-1077 are variations of the same mRNA (mRNAl 19). mRNAl 19 has a minimal 5’ UTR sequence (min5’UTR_vl). hdl076 and 1077 are both bound to a single 5’ Str-O-Nuc (Str968 and 969, respectively) that covers the entirety of the minimal 5’ UTR, whereas hdl075 contains no 5’ UTR Str-O-Nuc. hdl076 is bound to a Str-O-Nuc that is fully 2’-M0E modified, whereas the Str-O-Nuc bound to hdl077 is identical in sequence but is fully 2’-0Me modified. hdl077 is translated at a significantly higher level than hdl076, again suggesting that 2’-0Me modifications of the Str-O-Nuc allow for higher levels of translation compared to 2’-MOE modifications.

[0339] The same conclusions can be drawn from hdl080-1082 and hdl085-1087. Both hdRNA sets contain different minimal 5’ UTR mRNAs (mRNA 120 and mRNA121, respectively) bound to no Str-O-Nuc, or a single Str-O-Nuc that is either 2’ -MOE or 2’-0Me modified. In all cases, the 2’-OMe-bound mRNA produces more protein compared to the 2'-MOE-bound version.

[0340] The 3’ UTR sequence, length, and Str-O-Nuc chemistry was also optimized (hdRNAl 092-1103). In summary, Str-O-Nucs hybridized to the 3 ’UTR that were 2’MOE modified generally resulted in at least equal or higher expression compared to sequence identical Str-O-Nucs that were 2’0Me modified (compare hdl092 with hdl093; hdl096 with hdl097; hdl099 with hdl 100; hdl 102 with hdl 103). At times, expression of the mRNAs that were hybridized to Str-O-Nucs exceeded expression of that of the comparator mRNA (hdl 069).Notably, hdl094, which contains no 3’UTR (just the 29nt landing pad for Str517 immediately after the dual stop codons) resulted in the highest expression of all constructs, exceeding expression of the comparator mRNA (hdl 069) by over an order of magnitude.

[0341] These results suggest that 3’ Str-O-Nucs can enhance HiBiT expression beyond that of a standard HiBiT mRNA. These results are depicted in Figure 7.Example 6. Effect of using a Str-O-Nuc to intentionally dampen cap-dependent translation and promote cap-independent translation

[0342] The purpose of this study was to determine the effect of using a Str-O-Nuc to intentionally dampen cap-dependent translation and promote cap-independent translation.

[0343] Str-O-Nucs were hybridized to mRNA by heating the mixture of nucleic acids to 95°C for 3 minutes, followed by a cooling step to 25°C, set at a cooling rate of 0.1°C per second. 100 nanograms (ng) of the mRNA hybridized to Str-O-Nucs were transfected into the HEK293T cell line, utilizing the mRNA lipofection reagent Lipofectamine MessengerMax (LMM). Following transfection, the cells were lysed at 4, 24, 48, and 72hr, mixed with luciferin reagent, and measured for luminescence on a plate reader. Measured luminescence (in RLUs) was plotted.

[0344] hdRNA750 is a standard comparator mRNA (mRNA051) with a split (30A-10nt-70A) poly(A) tail. hdRNA751 is the same mRNA (mRNA051) that was hybridized to Str569. Str569 is a 20-nt Str-O-Nuc that binds to the 5’ most 20nt of the mRNA, directly adjacent to the 7-methylguanosine cap. Flue expression from hdRNA751 is considerably less than hdRNA750 across all timepoints, indicating that Str569 binding to the mRNA reduces cap-dependent translation.

[0345] Based on this observation, it was then tested if this dampening of cap-dependent translation (by Str569 binding to the 5’ end of the mRNA) could promote cap-independent translation. mRNA053 contains a CVB3 -derived Internal Ribosome Entry Site (IRES) that recruits ribosomal machinery to translate protein, independent of a 5’ cap. Expression from hdRNA758 was significantly worse than hdRNA750, suggesting that the CVB3 IRES was interfering with cap-dependent translation and also not efficiently promoting cap-independent translation. When Str569 was added to mRNA053 (hdRNA759), translation was significantly higher than hdRNA758 at all time points. This suggests that blocking cap-dependent translation with Str569 enables more effective IRES-dependent translation.

[0346] These results suggest that 5’ Str-O-Nucs can weaken cap-dependent expression, opening up the possibility for alternate mechanisms of translation (e.g., IRES-dependent) on a linear mRNA. These results are depicted in Figure 8.Table 2. Certain sequences of the Examples# Name Sequence 2 ’modification Str064 5 ' UTR_1 GGTGGCGGGTTCTCT MOE( SEQ IDNO : 30 )Str065 5 ' UTR_3 CCAGAAGAATACT MOE( SEQ IDNO : 31 )Str066 5 ' UTR_4 AGTTTATTTCTCCT MOE( SEQ IDNO : 32 )Str067 5 ' UTR_2 CTGAGTCTGTGGGGA MOE( SEQ IDNO : 33 )Str963 5 ' UTR_l_OMe GGUGGCGGGUUCUCU OMe( SEQ IDNO : 34 )Str964 5 ' UTR_2_OMe CUGAGUCUGUGGGGA OMe( SEQ IDNO : 35 )Str965 5 ' UTR_3_OMe CCAGAAGAAUACU OMe( SEQ IDNO : 36 )Str966 5 ' UTR_4_OMe AGUUUAUUUCUCCU OMe( SEQ IDNO : 37 )Str968 min5pUTRvl_MOE ggtGGCAAGtctcct MOE( SEQ IDNO : 38 )Str969 min5pUTRvl_OMe gguGGCAAGucuccu OMe( SEQ IDNO : 39 )Str972 min5pUTRv2_MOE ggtttatttctcct MOE( SEQ IDNO : 40 )Str973 min5pUTRv2_OMe g g u u u a u u u c u c c u OMe( SEQ IDNO : 41 )Str976 min5pUTRv3_MOE ggtAAGCTtctcct MOE( SEQ IDNO : 42 )Str977 min5pUTRv3_OMe gguAAGCUucuccu OMe( SEQ IDNO : 43 )Str092 aGlobin_3 ' UTR_MOE_1 CACCGAGGCTCCAGCTCA MOE( SEQ IDNO : 44 )Str098 aGlobin_3 ' UTR_MOE_2 GGCAAGAAGCTAGGC MOE( SEQ IDNO : 45 )Str093 aGlobin_3 ' UTR_MOE_3 GGGGGAGGCCCAAGG MOE( SEQ IDNO : 46 )Str094 aGlobin_3 ' UTR_MOE_4 GGGGAGGAGGGGCTG MOE( SEQ IDNO : 47 )Str348 aGlobin_3 ' UTR_MOE_5 GTACGGGTGCAGGAA MOE( SEQ IDNO : 48 )# Name Sequence 2 ’modification Str095 aGlobin_3 ' UTR_MOE_6 TATTCAAAGACCACGGGG MOE( SEQ IDNO : 49 )Str096 aGlobin_3 ' UTR_MOE_7 TGCCGCCCACTCAGACTT MOE( SEQ IDNO : 50 )Str075 aGlobin_3 ' UTR_OMe_l CACCGAGGCUCCAGCUCA OMe( SEQ IDNO : 51 )Str076 aGlobin_3 ' UTR_OMe_2 GGCAAGAAGCUAGGC OMe( SEQ IDNO : 52 )Str077 aGlobin_3 ' UTR_OMe_3 GGGGGAGGCCCAAGG OMe( SEQ IDNO : 53 )Str078 aGlobin_3 ' UTR_OMe_4 GGGGAGGAGGGGCUG OMe( SEQ IDNO : 54 )Str079 aGlobin_3 ' UTR_OMe_5 GUACGGGUGCAGGAA OMe( SEQ IDNO : 55 )Str080 aGlobin_3 ' UTR_OMe_6 UAUUCAAAGACCACGGGG OMe( SEQ IDNO : 56 )Str081 aGlobin_3 ' UTR_OMe_7 UGCCGCCCACUCAGACUU OMe( SEQ IDNO : 57 )Str980 min3pUTRvl_MOE cgaggcTccagcTca MOE( SEQ IDNO : 58 )Str981 min3pUTRvl_OMe cgaggcuccagcuca OMe( SEQ IDNO : 59 )Str984 min3pUTRv3_MOE CGTCTAGAGCTCGATTCa MOE( SEQ IDNO : 60 )Str985 min3pUTRv3_OMe C GU CUAGAGCU C GAUU C a OMe( SEQ IDNO : 61 )Str569 5 ' UTR_2 Ont-LP_AG GCACGTACGATATGtctcct MOE( SEQ IDNO : 62 )Table 3. Certain sequences of the Examples# Name SequencemRNA117 HiBiT [ CDSopt ] aggagaaauaaacuaguauucuucugguccccacagacucagagagaa ( 30 / 70A) cccgccaccAUGGUGAGCGGCUGGCGGCUGUUCAAGAAGAUCAGCuaa ( SEQ ID ugagcuggagccucgguggccuagcuucuugccccuugggccuccccc NO : 63 ) cagccccuccuccccuuccugcacccguacccccguggucuuugaaua aagucugagugggcggcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa gcauaugacuaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaamRNAl 18 HiBiT [ CDSopt ] aggagaaauaaacuaguauucuucugguccccacagacucagagagaa ( OA+29nt ) cccgccaccAUGGUGAGCGGCUGGCGGCUGUUCAAGAAGAUCAGCuaa ( SEQ ID ugagcuggagccucgguggccuagcuucuugccccuugggccuccccc NO : 64 ) cagccccuccuccccuuccugcacccguacccccguggucuuugaauaaagucugagugggcggcacgaagagcaucggaucccgggcccgucga# Name SequencemRNAl 19 min5 ' UTR_vl aggagaCUUGCCaccAUGGUGAGCGGCUGGCGGCUGUUCAAGAAGAUC HiBiT [CDSopt] AGCuaaugagcuggagccucgguggccuagcuucuugccccuugggcc ( SEQ ID ( OA+29nt ) uccccccagccccuccuccccuuccugcacccguacccccguggucuu NO : 65 ) ugaauaaagucugagugggcggcacgaagagcaucggaucccgggccc gucgamRNAl 20 min 5 ' UTR_v2 aggagaaauaaaccAUGGUGAGCGGCUGGCGGCUGUUCAAGAAGAUCA HiBiT [CDSopt] GCuaaugagcuggagccucgguggccuagcuucuugccccuugggccu ( SEQ ID ( OA+29nt ) ccccccagccccuccuccccuuccugcacccguacccccguggucuuu NO : 66 ) gaauaaagucugagugggcggcacgaagagcaucggaucccgggcccg ucgamRNAl 21 min 5 ' UTR_v3 aggagaAGCUUaccAUGGUGAGCGGCUGGCGGCUGUUCAAGAAGAUCA HiBiT [CDSopt] GCuaaugagcuggagccucgguggccuagcuucuugccccuugggccu ( SEQ ID ( OA+29nt ) ccccccagccccuccuccccuuccugcacccguacccccguggucuuu NO : 67 ) gaauaaagucugagugggcggcacgaagagcaucggaucccgggcccg ucgamRNAl 22 HiBiT [CDSopt] aggagaaauaaacuaguauucuucugguccccacagacucagagagaa no3 ' UTR cccgccaccAUGGUGAGCGGCUGGCGGCUGUUCAAGAAGAUCAGCuaa ( SEQ ID ( OA+29nt ) ugacgaagagcaucggaucccgggcccgucgaNO : 68 )mRNAl 23 HiBiT [CDSopt] aggagaaauaaacuaguauucuucugguccccacagacucagagagaa min3 ' UTR_vl cccgccaccAUGGUGAGCGGCUGGCGGCUGUUCAAGAAGAUCAGCuaa ( SEQ ID ( OA+29nt ) ugagcuggagccucgcgaagagcaucggaucccgggcccgucga NO : 69 )mRNAl 24 HiBiT [CDSopt] aggagaaauaaacuaguauucuucugguccccacagacucagagagaa min 3 ' UTR_v2 cccgccaccAUGGUGAGCGGCUGGCGGCUGUUCAAGAAGAUCAGCuaa ( SEQ ID ( OA+29nt ) ugagcuggagccucggugcgaagagcaucggaucccgggcccgucga NO : 70 )mRNAl 25 HiBiT [CDSopt] aggagaaauaaacuaguauucuucugguccccacagacucagagagaa min 3 ' UTR_v3 cccgccaccAUGGUGAGCGGCUGGCGGCUGUUCAAGAAGAUCAGCuaa ( SEQ ID ( OA+29nt ) uGAAUCGAGCUCUAGACGcgaagagcaucggaucccgggcccgucgaNO : 71 )# Name SequencemRNAl 00 FLuc [ CDSopt] aggagaaauaaacuaguauucuucugguccccacagacucagagagaa ( 30 / 70A) cccgccaccAUGGAAGAUGCCAAAAACAUUAAGAAGGGGCCAGCGCCA ( SEQ ID UUCUACCCACUCGAAGACGGGACCGCAGGAGAGCAGCUGCACAAAGCC NO : 72 ) AUGAAGAGAUACGCACUGGUGCCUGGCACUAUCGCCUUUACCGACGCA CAUAUAGAGGU GGAUAUUAC CUAU GC C GAGUACUUU GAGAU GU C C GUU CGGCUGGCAGAAGCUAUGAAGCGCUAUGGGUUAAAUACAAAUCAUCGG AUUGUCGUGUGUAGCGAAAAUUCCUUGCAGUUCUUUAUGCCCGUCCUU GGGGCCCUCUUCAUCGGUGUGGCUGUGGCCCCAGCUAACGACAUCUAC AACGAGCGUGAGCUGCUGAACUCUAUGGGAAUCAGCCAGCCUACCGUC GUAUUUGUGAGCAAGAAAGGGCUGCAAAAGAUCCUCAAUGUUCAGAAG AAACUAC C GAU CAUACAGAAGAUAAUUAU GAU GGAUU C CAAAAC C GAG UAC CAGGGCUU C CAGU CAAU GUAUAC GUU C GU GACUU C C CAUUU GC GA CCUGGCUUUAAUGAGUACGAUUUUGUGCCUGAAUCGUUCGAUCGGGAU AAAACUAUU GC C CU GAU CAU GAACAGUU CU GGAAGUACU GGAUU GC C C AAGGGCGUAGCACUUCCGCAUCGAACUGCUUGUGUCCGAUUCAGUCAU GCUAGAGAU C C CAU CUU C GGCAAC CAGAUUAUU C CU GAU AC C GCUAUA CUCUCAGUCGUUCCAUUUCAUCACGGAUUCGGCAUGUUCACGACGCUG GGCUACUUGAUCUGCGGCUUUAGGGUCGUGCUCAUGUACAGGUUUGAG GAGGAGCUAUUCUUAAGGUCCUUGCAAGACUAUAAGAUUCAAUCUGCC CUGCUGGUGCCCACACUUUUUAGCUUUUUUGCUAAGUCUACUCUCAUU GACAAAUAUGACCUAUCCAACUUACACGAGAUAGCCAGCGGAGGGGCG CCGCUCUCUAAAGAGGUAGGUGAGGCCGUGGCGAAAAGGUUUCACCUU CCAGGAAUACGCCAGGGGUACGGGCUGACAGAAACAACAUCAGCCAUU CUGAUCACCCCCGAAGGGGACGACAAACCUGGCGCAGUAGGCAAGGUG GUGCCCUUCUUCGAGGCUAAGGUGGUGGAUUUGGAUACCGGUAAAACA CUGGGUGUGAAUCAGAGAGGGGAACUGUGCGUCCGUGGACCCAUGAUC AUGUCAGGCUAUGUUAACAAUCCCGAGGCUACAAAUGCUCUCAUCGAC AAGGACGGCUGGCUGCACUCGGGAGACAUUGCAUAUUGGGACGAAGAU GAGCACUU CUU CAUAGU C GAU C GGCU GAAGAGUUUAAU CAAAUACAAG GGCUACCAGGUAGCACCAGCUGAACUUGAGAGCAUCCUUCUUCAACAC CCCAACAUUUUCGACGCAGGGGUCGCGGGACUGCCUGACGAUGAUGCC GGCGAACUGCCUGCCGCAGUCGUCGUGCUCGAACACGGUAAAACUAUG ACGGAGAAGGAAAUCGUGGACUAUGUGGCCAGCCAGGUUACAACCGCC AAGAAGCUGCGCGGUGGUGUUGUGUUUGUUGAUGAGGUUCCUAAAGGA CUGACCGGCAAAUUGGACGCGCGCAAGAUCAGAGAAAUUCUCAUUAAG GCCAAAAAAGGCGGAAAGAUCGCCGUGuaaugagcuggagccucggug gccuagcuucuugccccuugggccuccccccagccccuccuccccuuc cugcacccguacccccguggucuuugaauaaagucugagugggcggca aaaaaaaaaaaaaaaaaaaaaaaaaaaaaagcauaugacuaaaaaaaa aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa# Name SequencemRNAl 01 FLuc [ CDSopt] aggagaaauaaacuaguauucuucugguccccacagacucagagagaa ( OA+29nt ) cccgccaccAUGGAAGAUGCCAAAAACAUUAAGAAGGGGCCAGCGCCA ( SEQ ID UUCUACCCACUCGAAGACGGGACCGCAGGAGAGCAGCUGCACAAAGCC NO : 73 ) AUGAAGAGAUACGCACUGGUGCCUGGCACUAUCGCCUUUACCGACGCA CAUAUAGAGGU GGAUAUUAC CUAU GC C GAGUACUUU GAGAU GU C C GUU CGGCUGGCAGAAGCUAUGAAGCGCUAUGGGUUAAAUACAAAUCAUCGG AUUGUCGUGUGUAGCGAAAAUUCCUUGCAGUUCUUUAUGCCCGUCCUU GGGGCCCUCUUCAUCGGUGUGGCUGUGGCCCCAGCUAACGACAUCUAC AACGAGCGUGAGCUGCUGAACUCUAUGGGAAUCAGCCAGCCUACCGUC GUAUUUGUGAGCAAGAAAGGGCUGCAAAAGAUCCUCAAUGUUCAGAAG AAACUAC C GAU CAUACAGAAGAUAAUUAU GAU GGAUU C CAAAAC C GAG UAC CAGGGCUU C CAGU CAAU GUAUAC GUU C GU GACUU C C CAUUU GC GA CCUGGCUUUAAUGAGUACGAUUUUGUGCCUGAAUCGUUCGAUCGGGAU AAAACUAUU GC C CU GAU CAU GAACAGUU CU GGAAGUACU GGAUU GC C C AAGGGCGUAGCACUUCCGCAUCGAACUGCUUGUGUCCGAUUCAGUCAU GCUAGAGAU C C CAU CUU C GGCAAC CAGAUUAUU C CU GAU AC C GCUAUA CUCUCAGUCGUUCCAUUUCAUCACGGAUUCGGCAUGUUCACGACGCUG GGCUACUUGAUCUGCGGCUUUAGGGUCGUGCUCAUGUACAGGUUUGAG GAGGAGCUAUUCUUAAGGUCCUUGCAAGACUAUAAGAUUCAAUCUGCC CUGCUGGUGCCCACACUUUUUAGCUUUUUUGCUAAGUCUACUCUCAUU GACAAAUAUGACCUAUCCAACUUACACGAGAUAGCCAGCGGAGGGGCG CCGCUCUCUAAAGAGGUAGGUGAGGCCGUGGCGAAAAGGUUUCACCUU CCAGGAAUACGCCAGGGGUACGGGCUGACAGAAACAACAUCAGCCAUU CUGAUCACCCCCGAAGGGGACGACAAACCUGGCGCAGUAGGCAAGGUG GUGCCCUUCUUCGAGGCUAAGGUGGUGGAUUUGGAUACCGGUAAAACA CUGGGUGUGAAUCAGAGAGGGGAACUGUGCGUCCGUGGACCCAUGAUC AUGUCAGGCUAUGUUAACAAUCCCGAGGCUACAAAUGCUCUCAUCGAC AAGGACGGCUGGCUGCACUCGGGAGACAUUGCAUAUUGGGACGAAGAU GAGCACUU CUU CAUAGU C GAU C GGCU GAAGAGUUUAAU CAAAUACAAG GGCUACCAGGUAGCACCAGCUGAACUUGAGAGCAUCCUUCUUCAACAC CCCAACAUUUUCGACGCAGGGGUCGCGGGACUGCCUGACGAUGAUGCC GGCGAACUGCCUGCCGCAGUCGUCGUGCUCGAACACGGUAAAACUAUG ACGGAGAAGGAAAUCGUGGACUAUGUGGCCAGCCAGGUUACAACCGCC AAGAAGCUGCGCGGUGGUGUUGUGUUUGUUGAUGAGGUUCCUAAAGGA CUGACCGGCAAAUUGGACGCGCGCAAGAUCAGAGAAAUUCUCAUUAAG GCCAAAAAAGGCGGAAAGAUCGCCGUGuaaugagcuggagccucggug gccuagcuucuugccccuugggccuccccccagccccuccuccccuuc cugcacccguacccccguggucuuugaauaaagucugagugggcggcacgaagagcaucggaucccgggcccgucga# Name SequencemRNA126 min5 ' UTR_vl aggagaCUUGCCaccAUGGAAGAUGCCAAAAACAUUAAGAAGGGGCCA Flue [ CDSopt] GCGCCAUUCUACCCACUCGAAGACGGGACCGCAGGAGAGCAGCUGCAC ( SEQ ID ( OA+29nt ) AAAGCCAUGAAGAGAUACGCACUGGUGCCUGGCACUAUCGCCUUUACC NO : 74 ) GAG GCACAUAUAGAGGU GGAUAUUAC CUAU GC C GAGUACUUU GAGAU G UCCGUUCGGCUGGCAGAAGCUAUGAAGCGCUAUGGGUUAAAUACAAAU GAU C GGAUU GU C GU GU GUAGC GAAAAUU C CUU GCAGUU CUUUAU GC C C GUCCUUGGGGCCCUCUUCAUCGGUGUGGCUGUGGCCCCAGCUAACGAC AUCUACAACGAGCGUGAGCUGCUGAACUCUAUGGGAAUCAGCCAGCCU ACCGUCGUAUUUGUGAGCAAGAAAGGGCUGCAAAAGAUCCUCAAUGUU CAGAAGAAACUAC C GAU CAUACAGAAGAUAAUUAU CAU GGAUU C CAAA AC C GACUAC CAGGGCUU C CAGU CAAU GUAUAC CUU C GU GACUU C C CAU UU GC CAC CU GGCUUUAAU GAGUAC GAUUUU GU GC CU GAAU C GUU C GAU CGGGAUAAAACUAUUGCCCUGAUCAUGAACAGUUCUGGAAGUACUGGA UUGCCCAAGGGCGUAGCACUUCCGCAUCGAACUGCUUGUGUCCGAUUC AGU CAU GCUAGAGAU C C CAU CUU C GGCAAC CAGAUUAUU C CU GAU AC C GCUAUACUCUCAGUCGUUCCAUUUCAUCACGGAUUCGGCAUGUUCACG ACGCUGGGCUACUUGAUCUGCGGCUUUAGGGUCGUGCUCAUGUACAGG UUUGAGGAGGAGCUAUUCUUAAGGUCCUUGCAAGACUAUAAGAUUCAA UCUGCCCUGCUGGUGCCCACACUUUUUAGCUUUUUUGCUAAGUCUACU CU CAUU GACAAAUAU GAC CUAU C CAACUUACAC GAGAUAGC CAGC GGA GGGGCGCCGCUCUCUAAAGAGGUAGGUGAGGCCGUGGCGAAAAGGUUU CACCUUCCAGGAAUACGCCAGGGGUACGGGCUGACAGAAACAACAUCA GCCAUUCUGAUCACCCCCGAAGGGGACGACAAACCUGGCGCAGUAGGC AAGGUGGUGCCCUUCUUCGAGGCUAAGGUGGUGGAUUUGGAUACCGGU AAAACACUGGGUGUGAAUCAGAGAGGGGAACUGUGCGUCCGUGGACCC AUGAUCAUGUCAGGCUAUGUUAACAAUCCCGAGGCUACAAAUGCUCUC AUCGACAAGGACGGCUGGCUGCACUCGGGAGACAUUGCAUAUUGGGAC GAAGAU GAGCACUU CUU CAU AGU C GAU C GGCU GAAGAGUUUAAU CAAA UACAAGGGCUACCAGGUAGCACCAGCUGAACUUGAGAGCAUCCUUCUU CAACACCCCAACAUUUUCGACGCAGGGGUCGCGGGACUGCCUGACGAU GAUGCCGGCGAACUGCCUGCCGCAGUCGUCGUGCUCGAACACGGUAAA ACUAUGACGGAGAAGGAAAUCGUGGACUAUGUGGCCAGCCAGGUUACA ACCGCCAAGAAGCUGCGCGGUGGUGUUGUGUUUGUUGAUGAGGUUCCU AAAGGACUGACCGGCAAAUUGGACGCGCGCAAGAUCAGAGAAAUUCUC AUUAAGGCCAAAAAAGGCGGAAAGAUCGCCGUGuaaugagcuggagcc ucgguggccuagcuucuugccccuugggccuccccccagccccuccuc cccuuccugcacccguacccccguggucuuugaauaaagucugagugggcggcacgaagagcaucggaucccgggcccgucga# Name SequencemRNA127 min 5 ' UTR_v2 aggagaaauaaaccAUGGAAGAUGCCAAAAACAUUAAGAAGGGGCCAG Flue [ CDSopt] CGCCAUUCUACCCACUCGAAGACGGGACCGCAGGAGAGCAGCUGCACA ( SEQ ID ( OA+29nt ) AAGCCAUGAAGAGAUACGCACUGGUGCCUGGCACUAUCGCCUUUACCG NO : 75 ) AC GCACAUAUAGAGGU GGAUAUUAC CUAU GC C GAGUACUUU GAGAU GU CCGUUCGGCUGGCAGAAGCUAUGAAGCGCUAUGGGUUAAAUACAAAUC AU C GGAUU GU C GU GU GUAGC GAAAAUU C CUU GCAGUU CUUUAU GC C C G UCCUUGGGGCCCUCUUCAUCGGUGUGGCUGUGGCCCCAGCUAACGACA UCUACAACGAGCGUGAGCUGCUGAACUCUAUGGGAAUCAGCCAGCCUA CCGUCGUAUUUGUGAGCAAGAAAGGGCUGCAAAAGAUCCUCAAUGUUC AGAAGAAACUAC C GAU CAUACAGAAGAUAAUUAU CAU GGAUU C CAAAA C C GACUAC CAGGGCUU C CAGU CAAU GUAUAC CUU C GU GACUU C C CAUU U GC CAC CU GGCUUUAAU GAGUAC GAUUUU GU GC CU GAAU C GUU C GAU C GGGAUAAAACUAUUGCCCUGAUCAUGAACAGUUCUGGAAGUACUGGAU UGCCCAAGGGCGUAGCACUUCCGCAUCGAACUGCUUGUGUCCGAUUCA GU CAU GCUAGAGAU C C CAU CUU C GGCAAC CAGAUUAUU C CU GAU AC C G CUAUACU CU CAGU C GUU C CAUUU CAU CAC GGAUU C GGCAU GUU CAC GA CGCUGGGCUACUUGAUCUGCGGCUUUAGGGUCGUGCUCAUGUACAGGU UUGAGGAGGAGCUAUUCUUAAGGUCCUUGCAAGACUAUAAGAUUCAAU CUGCCCUGCUGGUGCCCACACUUUUUAGCUUUUUUGCUAAGUCUACUC U CAUU GACAAAUAU GAC CUAU C CAACUUACAC GAGAUAGC CAGC GGAG GGGCGCCGCUCUCUAAAGAGGUAGGUGAGGCCGUGGCGAAAAGGUUUC ACCUUCCAGGAAUACGCCAGGGGUACGGGCUGACAGAAACAACAUCAG CCAUUCUGAUCACCCCCGAAGGGGACGACAAACCUGGCGCAGUAGGCA AGGUGGUGCCCUUCUUCGAGGCUAAGGUGGUGGAUUUGGAUACCGGUA AAACACUGGGUGUGAAUCAGAGAGGGGAACUGUGCGUCCGUGGACCCA UGAUCAUGUCAGGCUAUGUUAACAAUCCCGAGGCUACAAAUGCUCUCA UCGACAAGGACGGCUGGCUGCACUCGGGAGACAUUGCAUAUUGGGACG AAGAU GAGCACUU CUU CAUAGU C GAU C GGCU GAAGAGUUUAAU CAAAU ACAAGGGCUACCAGGUAGCACCAGCUGAACUUGAGAGCAUCCUUCUUC AACACCCCAACAUUUUCGACGCAGGGGUCGCGGGACUGCCUGACGAUG AUGCCGGCGAACUGCCUGCCGCAGUCGUCGUGCUCGAACACGGUAAAA CUAUGACGGAGAAGGAAAUCGUGGACUAUGUGGCCAGCCAGGUUACAA CCGCCAAGAAGCUGCGCGGUGGUGUUGUGUUUGUUGAUGAGGUUCCUA AAGGACUGACCGGCAAAUUGGACGCGCGCAAGAUCAGAGAAAUUCUCA UUAAGGCCAAAAAAGGCGGAAAGAUCGCCGUGuaaugagcuggagccu cgguggccuagcuucuugccccuugggccuccccccagccccuccucc ccuuccugcacccguacccccguggucuuugaauaaagucugagugggcggcacgaagagcaucggaucccgggcccgucga# Name SequencemRNA128 min 5 ' UTR_v3 aggagaAGCUUaccAUGGAAGAUGCCAAAAACAUUAAGAAGGGGCCAG Flue [ CDSopt] CGCCAUUCUACCCACUCGAAGACGGGACCGCAGGAGAGCAGCUGCACA ( SEQ ID ( OA+29nt ) AAGCCAUGAAGAGAUACGCACUGGUGCCUGGCACUAUCGCCUUUACCG NO : 76 ) AC GCACAUAUAGAGGU GGAUAUUAC CUAU GC C GAGUACUUU GAGAU GU CCGUUCGGCUGGCAGAAGCUAUGAAGCGCUAUGGGUUAAAUACAAAUC AU C GGAUU GU C GU GU GUAGC GAAAAUU C CUU GCAGUU CUUUAU GC C C G UCCUUGGGGCCCUCUUCAUCGGUGUGGCUGUGGCCCCAGCUAACGACA UCUACAACGAGCGUGAGCUGCUGAACUCUAUGGGAAUCAGCCAGCCUA CCGUCGUAUUUGUGAGCAAGAAAGGGCUGCAAAAGAUCCUCAAUGUUC AGAAGAAACUAC C GAU CAUACAGAAGAUAAUUAU CAU GGAUU C CAAAA C C GACUAC CAGGGCUU C CAGU CAAU GUAUAC CUU C GU GACUU C C CAUU U GC CAC CU GGCUUUAAU GAGUAC GAUUUU GU GC CU GAAU C GUU C GAU C GGGAUAAAACUAUUGCCCUGAUCAUGAACAGUUCUGGAAGUACUGGAU UGCCCAAGGGCGUAGCACUUCCGCAUCGAACUGCUUGUGUCCGAUUCA GU CAU GCUAGAGAU C C CAU CUU C GGCAAC CAGAUUAUU C CU GAU AC C G CUAUACU CU CAGU C GUU C CAUUU CAU CAC GGAUU C GGCAU GUU CAC GA CGCUGGGCUACUUGAUCUGCGGCUUUAGGGUCGUGCUCAUGUACAGGU UUGAGGAGGAGCUAUUCUUAAGGUCCUUGCAAGACUAUAAGAUUCAAU CUGCCCUGCUGGUGCCCACACUUUUUAGCUUUUUUGCUAAGUCUACUC U CAUU GACAAAUAU GAC CUAU C CAACUUACAC GAGAUAGC CAGC GGAG GGGCGCCGCUCUCUAAAGAGGUAGGUGAGGCCGUGGCGAAAAGGUUUC ACCUUCCAGGAAUACGCCAGGGGUACGGGCUGACAGAAACAACAUCAG CCAUUCUGAUCACCCCCGAAGGGGACGACAAACCUGGCGCAGUAGGCA AGGUGGUGCCCUUCUUCGAGGCUAAGGUGGUGGAUUUGGAUACCGGUA AAACACUGGGUGUGAAUCAGAGAGGGGAACUGUGCGUCCGUGGACCCA UGAUCAUGUCAGGCUAUGUUAACAAUCCCGAGGCUACAAAUGCUCUCA UCGACAAGGACGGCUGGCUGCACUCGGGAGACAUUGCAUAUUGGGACG AAGAU GAGCACUU CUU CAUAGU C GAU C GGCU GAAGAGUUUAAU CAAAU ACAAGGGCUACCAGGUAGCACCAGCUGAACUUGAGAGCAUCCUUCUUC AACACCCCAACAUUUUCGACGCAGGGGUCGCGGGACUGCCUGACGAUG AUGCCGGCGAACUGCCUGCCGCAGUCGUCGUGCUCGAACACGGUAAAA CUAUGACGGAGAAGGAAAUCGUGGACUAUGUGGCCAGCCAGGUUACAA CCGCCAAGAAGCUGCGCGGUGGUGUUGUGUUUGUUGAUGAGGUUCCUA AAGGACUGACCGGCAAAUUGGACGCGCGCAAGAUCAGAGAAAUUCUCA UUAAGGCCAAAAAAGGCGGAAAGAUCGCCGUGuaaugagcuggagccu cgguggccuagcuucuugccccuugggccuccccccagccccuccucc ccuuccugcacccguacccccguggucuuugaauaaagucugagugggcggcacgaagagcaucggaucccgggcccgucga# Name SequencemRNA129 Flue [ CDSopt] aggagaaauaaacuaguauucuucugguccccacagacucagagagaa no3 ' UTR cccgccaccAUGGAAGAUGCCAAAAACAUUAAGAAGGGGCCAGCGCCA ( SEQ ID ( OA+29nt ) UUCUACCCACUCGAAGACGGGACCGCAGGAGAGCAGCUGCACAAAGCC NO : 77 ) AUGAAGAGAUACGCACUGGUGCCUGGCACUAUCGCCUUUACCGACGCA CAUAUAGAGGU GGAUAUUAC CUAU GC C GAGUACUUU GAGAU GU C C GUU CGGCUGGCAGAAGCUAUGAAGCGCUAUGGGUUAAAUACAAAUCAUCGG AUUGUCGUGUGUAGCGAAAAUUCCUUGCAGUUCUUUAUGCCCGUCCUU GGGGCCCUCUUCAUCGGUGUGGCUGUGGCCCCAGCUAACGACAUCUAC AACGAGCGUGAGCUGCUGAACUCUAUGGGAAUCAGCCAGCCUACCGUC GUAUUUGUGAGCAAGAAAGGGCUGCAAAAGAUCCUCAAUGUUCAGAAG AAACUAC C GAU CAUACAGAAGAUAAUUAU GAU GGAUU C CAAAAC C GAG UAC CAGGGCUU C CAGU CAAU GUAUAC GUU C GU GACUU C C CAUUU GC GA CCUGGCUUUAAUGAGUACGAUUUUGUGCCUGAAUCGUUCGAUCGGGAU AAAACUAUU GC C CU GAU CAU GAACAGUU CU GGAAGUACU GGAUU GC C C AAGGGCGUAGCACUUCCGCAUCGAACUGCUUGUGUCCGAUUCAGUCAU GCUAGAGAU C C CAU CUU C GGCAAC CAGAUUAUU C CU GAU AC C GCUAUA CUCUCAGUCGUUCCAUUUCAUCACGGAUUCGGCAUGUUCACGACGCUG GGCUACUUGAUCUGCGGCUUUAGGGUCGUGCUCAUGUACAGGUUUGAG GAGGAGCUAUUCUUAAGGUCCUUGCAAGACUAUAAGAUUCAAUCUGCC CUGCUGGUGCCCACACUUUUUAGCUUUUUUGCUAAGUCUACUCUCAUU GACAAAUAUGACCUAUCCAACUUACACGAGAUAGCCAGCGGAGGGGCG CCGCUCUCUAAAGAGGUAGGUGAGGCCGUGGCGAAAAGGUUUCACCUU CCAGGAAUACGCCAGGGGUACGGGCUGACAGAAACAACAUCAGCCAUU CUGAUCACCCCCGAAGGGGACGACAAACCUGGCGCAGUAGGCAAGGUG GUGCCCUUCUUCGAGGCUAAGGUGGUGGAUUUGGAUACCGGUAAAACA CUGGGUGUGAAUCAGAGAGGGGAACUGUGCGUCCGUGGACCCAUGAUC AUGUCAGGCUAUGUUAACAAUCCCGAGGCUACAAAUGCUCUCAUCGAC AAGGACGGCUGGCUGCACUCGGGAGACAUUGCAUAUUGGGACGAAGAU GAGCACUU CUU CAUAGU C GAU C GGCU GAAGAGUUUAAU CAAAUACAAG GGCUACCAGGUAGCACCAGCUGAACUUGAGAGCAUCCUUCUUCAACAC CCCAACAUUUUCGACGCAGGGGUCGCGGGACUGCCUGACGAUGAUGCC GGCGAACUGCCUGCCGCAGUCGUCGUGCUCGAACACGGUAAAACUAUG ACGGAGAAGGAAAUCGUGGACUAUGUGGCCAGCCAGGUUACAACCGCC AAGAAGCUGCGCGGUGGUGUUGUGUUUGUUGAUGAGGUUCCUAAAGGA CUGACCGGCAAAUUGGACGCGCGCAAGAUCAGAGAAAUUCUCAUUAAG GCCAAAAAAGGCGGAAAGAUCGCCGUGuaaugacgaagagcaucggaucccgggcccgucga# Name SequencemRNA130 Flue [ CDSopt] aggagaaauaaacuaguauucuucugguccccacagacucagagagaa min3 ' UTR_vl cccgccaccAUGGAAGAUGCCAAAAACAUUAAGAAGGGGCCAGCGCCA ( SEQ ID ( OA+29nt ) UUCUACCCACUCGAAGACGGGACCGCAGGAGAGCAGCUGCACAAAGCC NO : 78 ) AUGAAGAGAUACGCACUGGUGCCUGGCACUAUCGCCUUUACCGACGCA CAUAUAGAGGU GGAUAUUAC CUAU GC C GAGUACUUU GAGAU GU C C GUU CGGCUGGCAGAAGCUAUGAAGCGCUAUGGGUUAAAUACAAAUCAUCGG AUUGUCGUGUGUAGCGAAAAUUCCUUGCAGUUCUUUAUGCCCGUCCUU GGGGCCCUCUUCAUCGGUGUGGCUGUGGCCCCAGCUAACGACAUCUAC AACGAGCGUGAGCUGCUGAACUCUAUGGGAAUCAGCCAGCCUACCGUC GUAUUUGUGAGCAAGAAAGGGCUGCAAAAGAUCCUCAAUGUUCAGAAG AAACUAC C GAU CAUACAGAAGAUAAUUAU GAU GGAUU C CAAAAC C GAG UAC CAGGGCUU C CAGU CAAU GUAUAC GUU C GU GACUU C C CAUUU GC GA CCUGGCUUUAAUGAGUACGAUUUUGUGCCUGAAUCGUUCGAUCGGGAU AAAACUAUU GC C CU GAU CAU GAACAGUU CU GGAAGUACU GGAUU GC C C AAGGGCGUAGCACUUCCGCAUCGAACUGCUUGUGUCCGAUUCAGUCAU GCUAGAGAU C C CAU CUU C GGCAAC CAGAUUAUU C CU GAU AC C GCUAUA CUCUCAGUCGUUCCAUUUCAUCACGGAUUCGGCAUGUUCACGACGCUG GGCUACUUGAUCUGCGGCUUUAGGGUCGUGCUCAUGUACAGGUUUGAG GAGGAGCUAUUCUUAAGGUCCUUGCAAGACUAUAAGAUUCAAUCUGCC CUGCUGGUGCCCACACUUUUUAGCUUUUUUGCUAAGUCUACUCUCAUU GACAAAUAUGACCUAUCCAACUUACACGAGAUAGCCAGCGGAGGGGCG CCGCUCUCUAAAGAGGUAGGUGAGGCCGUGGCGAAAAGGUUUCACCUU CCAGGAAUACGCCAGGGGUACGGGCUGACAGAAACAACAUCAGCCAUU CUGAUCACCCCCGAAGGGGACGACAAACCUGGCGCAGUAGGCAAGGUG GUGCCCUUCUUCGAGGCUAAGGUGGUGGAUUUGGAUACCGGUAAAACA CUGGGUGUGAAUCAGAGAGGGGAACUGUGCGUCCGUGGACCCAUGAUC AUGUCAGGCUAUGUUAACAAUCCCGAGGCUACAAAUGCUCUCAUCGAC AAGGACGGCUGGCUGCACUCGGGAGACAUUGCAUAUUGGGACGAAGAU GAGCACUU CUU CAUAGU C GAU C GGCU GAAGAGUUUAAU CAAAUACAAG GGCUACCAGGUAGCACCAGCUGAACUUGAGAGCAUCCUUCUUCAACAC CCCAACAUUUUCGACGCAGGGGUCGCGGGACUGCCUGACGAUGAUGCC GGCGAACUGCCUGCCGCAGUCGUCGUGCUCGAACACGGUAAAACUAUG ACGGAGAAGGAAAUCGUGGACUAUGUGGCCAGCCAGGUUACAACCGCC AAGAAGCUGCGCGGUGGUGUUGUGUUUGUUGAUGAGGUUCCUAAAGGA CUGACCGGCAAAUUGGACGCGCGCAAGAUCAGAGAAAUUCUCAUUAAG GCCAAAAAAGGCGGAAAGAUCGCCGUGuaaugagcuggagccucgcgaagagcaucggaucccgggcccgucga# Name SequencemRNA131 Flue [ CDSopt] aggagaaauaaacuaguauucuucugguccccacagacucagagagaa min 3 ' UTR_v2 cccgccaccAUGGAAGAUGCCAAAAACAUUAAGAAGGGGCCAGCGCCA ( SEQ ID ( OA+29nt ) UUCUACCCACUCGAAGACGGGACCGCAGGAGAGCAGCUGCACAAAGCC NO : 79 ) AUGAAGAGAUACGCACUGGUGCCUGGCACUAUCGCCUUUACCGACGCA CAUAUAGAGGU GGAUAUUAC CUAU GC C GAGUACUUU GAGAU GU C C GUU CGGCUGGCAGAAGCUAUGAAGCGCUAUGGGUUAAAUACAAAUCAUCGG AUUGUCGUGUGUAGCGAAAAUUCCUUGCAGUUCUUUAUGCCCGUCCUU GGGGCCCUCUUCAUCGGUGUGGCUGUGGCCCCAGCUAACGACAUCUAC AACGAGCGUGAGCUGCUGAACUCUAUGGGAAUCAGCCAGCCUACCGUC GUAUUUGUGAGCAAGAAAGGGCUGCAAAAGAUCCUCAAUGUUCAGAAG AAACUAC C GAU CAUACAGAAGAUAAUUAU GAU GGAUU C CAAAAC C GAG UAC CAGGGCUU C CAGU CAAU GUAUAC GUU C GU GACUU C C CAUUU GC GA CCUGGCUUUAAUGAGUACGAUUUUGUGCCUGAAUCGUUCGAUCGGGAU AAAACUAUU GC C CU GAU CAU GAACAGUU CU GGAAGUACU GGAUU GC C C AAGGGCGUAGCACUUCCGCAUCGAACUGCUUGUGUCCGAUUCAGUCAU GCUAGAGAU C C CAU CUU C GGCAAC CAGAUUAUU C CU GAU AC C GCUAUA CUCUCAGUCGUUCCAUUUCAUCACGGAUUCGGCAUGUUCACGACGCUG GGCUACUUGAUCUGCGGCUUUAGGGUCGUGCUCAUGUACAGGUUUGAG GAGGAGCUAUUCUUAAGGUCCUUGCAAGACUAUAAGAUUCAAUCUGCC CUGCUGGUGCCCACACUUUUUAGCUUUUUUGCUAAGUCUACUCUCAUU GACAAAUAUGACCUAUCCAACUUACACGAGAUAGCCAGCGGAGGGGCG CCGCUCUCUAAAGAGGUAGGUGAGGCCGUGGCGAAAAGGUUUCACCUU CCAGGAAUACGCCAGGGGUACGGGCUGACAGAAACAACAUCAGCCAUU CUGAUCACCCCCGAAGGGGACGACAAACCUGGCGCAGUAGGCAAGGUG GUGCCCUUCUUCGAGGCUAAGGUGGUGGAUUUGGAUACCGGUAAAACA CUGGGUGUGAAUCAGAGAGGGGAACUGUGCGUCCGUGGACCCAUGAUC AUGUCAGGCUAUGUUAACAAUCCCGAGGCUACAAAUGCUCUCAUCGAC AAGGACGGCUGGCUGCACUCGGGAGACAUUGCAUAUUGGGACGAAGAU GAGCACUU CUU CAUAGU C GAU C GGCU GAAGAGUUUAAU CAAAUACAAG GGCUACCAGGUAGCACCAGCUGAACUUGAGAGCAUCCUUCUUCAACAC CCCAACAUUUUCGACGCAGGGGUCGCGGGACUGCCUGACGAUGAUGCC GGCGAACUGCCUGCCGCAGUCGUCGUGCUCGAACACGGUAAAACUAUG ACGGAGAAGGAAAUCGUGGACUAUGUGGCCAGCCAGGUUACAACCGCC AAGAAGCUGCGCGGUGGUGUUGUGUUUGUUGAUGAGGUUCCUAAAGGA CUGACCGGCAAAUUGGACGCGCGCAAGAUCAGAGAAAUUCUCAUUAAG GCCAAAAAAGGCGGAAAGAUCGCCGUGuaaugagcuggagccucggugcgaagagcaucggaucccgggcccgucga# Name SequencemRNA132 Flue [ CDSopt] aggagaaauaaacuaguauucuucugguccccacagacucagagagaa min 3 ' UTR_v3 cccgccaccAUGGAAGAUGCCAAAAACAUUAAGAAGGGGCCAGCGCCA ( SEQ ID ( OA+29nt ) UUCUACCCACUCGAAGACGGGACCGCAGGAGAGCAGCUGCACAAAGCC NO : 80 ) AUGAAGAGAUACGCACUGGUGCCUGGCACUAUCGCCUUUACCGACGCA CAUAUAGAGGU GGAUAUUAC CUAU GC C GAGUACUUU GAGAU GU C C GUU CGGCUGGCAGAAGCUAUGAAGCGCUAUGGGUUAAAUACAAAUCAUCGG AUUGUCGUGUGUAGCGAAAAUUCCUUGCAGUUCUUUAUGCCCGUCCUU GGGGCCCUCUUCAUCGGUGUGGCUGUGGCCCCAGCUAACGACAUCUAC AACGAGCGUGAGCUGCUGAACUCUAUGGGAAUCAGCCAGCCUACCGUC GUAUUUGUGAGCAAGAAAGGGCUGCAAAAGAUCCUCAAUGUUCAGAAG AAACUAC C GAU CAUACAGAAGAUAAUUAU GAU GGAUU C CAAAAC C GAG UAC CAGGGCUU C CAGU CAAU GUAUAC GUU C GU GACUU C C CAUUU GC GA CCUGGCUUUAAUGAGUACGAUUUUGUGCCUGAAUCGUUCGAUCGGGAU AAAACUAUU GC C CU GAU CAU GAACAGUU CU GGAAGUACU GGAUU GC C C AAGGGCGUAGCACUUCCGCAUCGAACUGCUUGUGUCCGAUUCAGUCAU GCUAGAGAU C C CAU CUU C GGCAAC CAGAUUAUU C CU GAU AC C GCUAUA CUCUCAGUCGUUCCAUUUCAUCACGGAUUCGGCAUGUUCACGACGCUG GGCUACUUGAUCUGCGGCUUUAGGGUCGUGCUCAUGUACAGGUUUGAG GAGGAGCUAUUCUUAAGGUCCUUGCAAGACUAUAAGAUUCAAUCUGCC CUGCUGGUGCCCACACUUUUUAGCUUUUUUGCUAAGUCUACUCUCAUU GACAAAUAUGACCUAUCCAACUUACACGAGAUAGCCAGCGGAGGGGCG CCGCUCUCUAAAGAGGUAGGUGAGGCCGUGGCGAAAAGGUUUCACCUU CCAGGAAUACGCCAGGGGUACGGGCUGACAGAAACAACAUCAGCCAUU CUGAUCACCCCCGAAGGGGACGACAAACCUGGCGCAGUAGGCAAGGUG GUGCCCUUCUUCGAGGCUAAGGUGGUGGAUUUGGAUACCGGUAAAACA CUGGGUGUGAAUCAGAGAGGGGAACUGUGCGUCCGUGGACCCAUGAUC AUGUCAGGCUAUGUUAACAAUCCCGAGGCUACAAAUGCUCUCAUCGAC AAGGACGGCUGGCUGCACUCGGGAGACAUUGCAUAUUGGGACGAAGAU GAGCACUU CUU CAUAGU C GAU C GGCU GAAGAGUUUAAU CAAAUACAAG GGCUACCAGGUAGCACCAGCUGAACUUGAGAGCAUCCUUCUUCAACAC CCCAACAUUUUCGACGCAGGGGUCGCGGGACUGCCUGACGAUGAUGCC GGCGAACUGCCUGCCGCAGUCGUCGUGCUCGAACACGGUAAAACUAUG ACGGAGAAGGAAAUCGUGGACUAUGUGGCCAGCCAGGUUACAACCGCC AAGAAGCUGCGCGGUGGUGUUGUGUUUGUUGAUGAGGUUCCUAAAGGA CUGACCGGCAAAUUGGACGCGCGCAAGAUCAGAGAAAUUCUCAUUAAG GCCAAAAAAGGCGGAAAGAUCGCCGUGuaauGAAUCGAGCUCUAGACGcgaagagcaucggaucccgggcccgucga# Name SequencemRNA051 FLuc [ optl ] aggagaCAUAUCGUACGUGCaauaaacuaguauucuucugguccccac ( 30 / 70A) agacucagagagaacccgccaccauggaggacgccaagaacaucaaga ( SEQ ID agggccccgcccccuucuacccccuggaggacggcaccgccggcgagc NO : 81 ) agcugcacaaggccaugaagcgguacgcccuggugcccggcaccaucg ccuucaccgacgcccacaucgagguggacaucaccuacgccgaguacu ucgagaugagcgugcggcuggccgaggccaugaagcgguacggccuga acaccaaccaccggaucguggugugcagcgagaacagccugcaguucu ucaugcccgugcugggcgcccuguucaucggcguggccguggcccccg ccaacgacaucuacaacgagcgggagcugcugaacagcaugggcauca gccagcccaccgugguguucgugagcaagaagggccugcagaagaucc ugaacgugcagaagaagcugcccaucauccagaagaucaucaucaugg acagcaagaccgacuaccagggcuuccagagcauguacaccuucguga ccagccaccugccccccggcuucaacgaguacgacuucgugcccgaga gcuucgaccgggacaagaccaucgcccugaucaugaacagcagcggca gcaccggccugcccaagggcguggcccugccccaccggaccgccugcg ugcgguucagccacgcccgggaccccaucuucggcaaccagaucaucc ccgacaccgccauccugagcguggugcccuuccaccacggcuucggca uguucaccacccugggcuaccugaucugcggcuuccggguggugcuga uguaccgguucgaggaggagcuguuccugcggagccugcaggacuaca agauccagagcgcccugcuggugcccacccuguucagcuucuucgcca agagcacccugaucgacaaguacgaccugagcaaccugcacgagaucg ccagcggcggcgccccccugagcaaggaggugggcgaggccguggcca agcgguuccaccugcccggcauccggcagggcuacggccugaccgaga caaccagcgccauccugaucacccccgagggcgacgacaagcccggcg ccgugggcaagguggugcccuucuucgaggccaaggugguggaccugg acaccggcaagacccugggcgugaaccagcggggcgagcugugcgugc ggggccccaugaucaugagcggcuacgugaacaaccccgaggccacca acgcccugaucgacaaggacggcuggcugcacagcggcgacaucgccu acugggacgaggacgagcacuucuucaucguggaccggcugaaguccc ugaucaaguacaagggcuaccagguggcccccgccgagcuggagagca uccugcugcagcaccccaacaucuucgacgccggcguggccggccugc ccgacgacgacgccggcgagcugcccgccgccgugguggugcuggagc acggcaagaccaugaccgagaaggagaucguggacuacguggccagcc aggugaccaccgccaagaagcugcggggcggcgugguguucguggacg aggugcccaagggccugaccggcaagcuggacgcccggaagauccggg agauccugaucaaggccaagaagggcggcaagaucgccguguaaugag cuggagccucgguggccuagcuucuugccccuugggccuccccccagc cccuccuccccuuccugcacccguacccccguggucuuugaauaaagu cugagugggcggcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaagcau augacuaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa# Name SequencemRNA053 CVB3 aggagaCAUAUCGUACGUGCuuaaaacagccuguggguugaucccacc FLuc [ optl ] cacaggcccauugggcgcuagcacucugguaucacgguaccuuugugc ( SEQ ID ( 30 / 70A) gccuguuuuauacccccucccccaacuguaacuuagaaguaacacaca NO : 82 ) ccgaucaacagucagcguggcacaccagccacguuuugaucaagcacu ucuguuaccccggacugaguaucaauagacugcucacgcgguugaagg agaaagcguucguuauccggccaacuacuucgaaaaaccuaguaacac cguggaaguugcagaguguuucgcucagcacuaccccaguguagauca ggucgaugagucaccgcauuccccacgggcgaccguggcgguggcugc guuggcggccugcccauggggaaacccaugggacgcucuaauacagac auggugcgaagagucuauugagcuaguugguaguccuccggccccuga augcggcuaauccuaacugcggagcacacacccucaagccagagggca gugugucguaacgggcaacucugcagcggaaccgacuacuuugggugu ccguguuucauuuuauuccuauacuggcugcuuauggugacaauugag agaucguuaccauauagcuauuggauuggccauccggugacuaauaga gcuauuauauaucccuuuguuggguuuauaccacuuagcuugaaagag guuaaaacauuacaauucauuguuaaguugaauacagcaaaccaugga ggacgccaagaacaucaagaagggccccgcccccuucuacccccugga ggacggcaccgccggcgagcagcugcacaaggccaugaagcgguacgc ccuggugcccggcaccaucgccuucaccgacgcccacaucgaggugga caucaccuacgccgaguacuucgagaugagcgugcggcuggccgaggc caugaagcgguacggccugaacaccaaccaccggaucguggugugcag cgagaacagccugcaguucuucaugcccgugcugggcgcccuguucau cggcguggccguggcccccgccaacgacaucuacaacgagcgggagcu gcugaacagcaugggcaucagccagcccaccgugguguucgugagcaa gaagggccugcagaagauccugaacgugcagaagaagcugcccaucau ccagaagaucaucaucauggacagcaagaccgacuaccagggcuucca gagcauguacaccuucgugaccagccaccugccccccggcuucaacga guacgacuucgugcccgagagcuucgaccgggacaagaccaucgcccu gaucaugaacagcagcggcagcaccggccugcccaagggcguggcccu gccccaccggaccgccugcgugcgguucagccacgcccgggaccccau cuucggcaaccagaucauccccgacaccgccauccugagcguggugcc cuuccaccacggcuucggcauguucaccacccugggcuaccugaucug cggcuuccggguggugcugauguaccgguucgaggaggagcuguuccu gcggagccugcaggacuacaagauccagagcgcccugcuggugcccac ccuguucagcuucuucgccaagagcacccugaucgacaaguacgaccu gagcaaccugcacgagaucgccagcggcggcgccccccugagcaagga ggugggcgaggccguggccaagcgguuccaccugcccggcauccggca gggcuacggccugaccgagacaaccagcgccauccugaucacccccga gggcgacgacaagcccggcgccgugggcaagguggugcccuucuucga ggccaaggugguggaccuggacaccggcaagacccugggcgugaacca gcggggcgagcugugcgugcggggccccaugaucaugagcggcuacgu gaacaaccccgaggccaccaacgcccugaucgacaaggacggcuggcu gcacagcggcgacaucgccuacugggacgaggacgagcacuucuucau cguggaccggcugaagucccugaucaaguacaagggcuaccagguggc ccccgccgagcuggagagcauccugcugcagcaccccaacaucuucga cgccggcguggccggccugcccgacgacgacgccggcgagcugcccgc cgccgugguggugcuggagcacggcaagaccaugaccgagaaggagau cguggacuacguggccagccaggugaccaccgccaagaagcugcgggg cggcgugguguucguggacgaggugcccaagggccugaccggcaagcu ggacgcccggaagauccgggagauccugaucaaggccaagaagggcgg caagaucgccguguaaugagcuggagccucgguggccuagcuucuugc cccuugggccuccccccagccccuccuccccuuccugcacccguaccc ccguggucuuugaauaaagucugagugggcggcaaaaaaaaaaaaaaa aaaaaaaaaaaaaaaagcauaugacuaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa

Claims

CLAIMSWhat is claimed is:

1. An mRNA complex comprising an mRNA molecule and at least one engineered oligonucleotide hybridized to the 5’UTR of the mRNA molecule, wherein said engineered oligonucleotide is hereafter referred to as the Str-O-Nuc.

2. An mRNA complex comprising an mRNA molecule and at least one engineered oligonucleotide hybridized to the 5’ region of the mRNA molecule, wherein said engineered oligonucleotide is hereafter referred to as the Str-O-Nuc, wherein the Str-O-Nuc is hybridized downstream of a cap and upstream of an IRES of the mRNA.

3. 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.

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

5. The mRNA complex of any preceding or subsequent claims, wherein the Str-O-Nuc contains a cap.

6. The mRNA complex of any preceding or subsequent claims, wherein the Str-O-Nuc contains a m7G-cap.

7. The mRNA complex of any preceding or subsequent claims, wherein the Str-O-Nuc contains an RNA aptamer (e.g. an eIF4G-binding aptamer).

8. The mRNA complex of any of the preceding claims, wherein the Str-O-Nuc comprises a 3’ and / or 5’ overhang.

9. The mRNA complex of any preceding or subsequent claims, wherein the Str-O-Nuc contains an overhang that is designed to bind to an endogenous mRNA.

10. The mRNA complex of any preceding or subsequent claims, wherein the Str-O-Nuc contains an overhang which contains a sequence that is complementary to a sequence on an endogenous mRNA, wherein the overhang would hybridize to the complementary sequence on said endogenous mRNA, and wherein the Str-O-Nuc de-hybridizes from the exogenous mRNA complex.

11. 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.

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

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

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

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

16. 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.

17. 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.

18. 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.

19. The mRNA complex of any preceding or subsequent claims, wherein the Str-O-Nuc comprises a 2’ -OH.

20. The mRNA complex of any preceding or subsequent claims, wherein the Str-O-Nuc comprises a 2’-H, 2’-OH, 2’-0Me, 2’-M0E, 2’-F, P-S linkage, 3’-inv-dT, 3’-ddC modification, LNA or phosphorothioate linkages.

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

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

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

24. 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.

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

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

27. 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.

28. 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 intravenous route.

29. 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.

30. 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.

31. 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 intrathecal route.

32. 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 byan intradermal route.

33. 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 byan intravitreal route.

34. 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 byan intracerebroventricular route.

35. 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 subretinal route.

36. 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.

37. The mRNA complex of any preceding 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 to the 5’UTR, and wherein the mRNA complex hasa. a half-life greater than an unmodified mRNA or an un-engineered mRNA, b. a more durable response compared to an unmodified mRNA or an un-engineered mRNA, and / orc. a more durable response compared to an unmodified mRNA or an un-engineered mRNA.

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