Poly mRNA

Abstract

Provided is a poly mRNA, comprising a plurality of mRNA structures. Each mRNA structure comprises: a cap region at the 5' end; and an open reading frame (ORF) encoding a polypeptide. The plurality of mRNA structures comprise a first mRNA structure and a second mRNA structure. The 3' terminus of the first mRNA structure is covalently linked to the second mRNA structure.

Description

A multi-mRNA TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to a multi-mRNA. BACKGROUND

[0002] With the rapid development of human gene technology, gene therapy is becoming an emerging revolutionary medical technology, showing a wide application prospect and great potential. mRNA drugs are an important category of gene therapy drugs.

[0003] Because different disease mechanisms involve different action proteins, mRNA technology can achieve symptomatic treatment, making the human body become its own "pharmacy". It is especially suitable for rapid response and situations that require transient protein expression, such as personalized tumor mRNA vaccines that can deliver multiple tumor targets, providing multiple combinations for treatment. The convenience of mRNA vaccine design and the rapidity of production can quickly adapt to different disease needs.

[0004] mRNA drugs only work in the cytoplasm and do not enter the nucleus, so they will not be integrated into the genome, thereby avoiding the risk of genomic instability; however, because the half-life of mRNA in the body is short, the expression time is not persistent enough, so people have been seeking a molecule that can express multiple proteins while significantly increasing its expression and half-life.

[0005] Therefore, there is still a need to improve the half-life of mRNA and improve the efficiency of mRNA expression. SUMMARY

[0006] The present application provides a multi-mRNA, comprising a plurality of mRNA structures with a 5' end cap region and an open reading frame (ORF), and the plurality of mRNA structures are covalently linked through the 3' end.

[0007] In cells, during the initiation stage of translation, the 40S subunit of the small ribosome forms a 43S pre-initiation complex structure with eIF3, eIF1, eIF1A, eIF5, and eIF2 containing GTP and Met-tRNAi or fMet-tRNAf. Once the 43S pre-initiation complex is formed, it binds to the cap structure of mRNA through the eIF4F complex to form a 48S initiation complex, and binds to the PolyA binding protein PABP to form a circular mRNA, which closes the head and tail of the mRNA to maintain stability. The 48S initiation complex moves along the mRNA chain in the 3' direction until it finds the start codon AUG to start the translation expression process.

[0008] Degradation of mRNA usually starts with shortening of the poly-A tail at its 3' end, a process known as deadenylation, deadenylation complex recognizes and binds to the poly-A tail, and through 3'-5' exonuclease CCR4, is able to remove adenosine residues on the poly-A tail step by step, once the poly(A) tail is significantly shortened, the mRNA begins to become unstable, and enters the path of rapid degradation. PolyA shortening, PABP binding to it and polyA tail dissociation, make the polyA-PABP complex separate from the eIF4F complex. This process further induces the removal of the 5' end cap structure of mRNA, becoming the de-capping stage. After the de-capping stage starts, eIF4F associated with the cap structure falls off, and the de-capping enzyme recognizes and cuts the cap structure. De-capping not only starts the degradation of mRNA, but also can prevent further translation of mRNA. Once the tail and cap are removed, the mRNA becomes more unstable. Certain specific RNA binding proteins can recognize the mRNA after de-capping, promoting its further degradation. mRNA is further cleaved by endonuclease and exonuclease.

[0009] The mRNA provided by the present application has its 3' PolyA covalently bound together, which is not exposed outside, effectively reducing the degradation of deadenylase, increasing the stability of the multivalent mRNA, and at the same time, this multimeric mRNA has multiple mRNA with cap structures, effectively improving the binding ability of mRNA to eIF4F complex and 48S translation initiation complex, improving the translation ability, and also reducing the rate of de-capping, making the mRNA more stable.

[0010] In one aspect, the present application provides a multimeric mRNA, comprising a plurality of mRNA structures, the mRNA structure comprising: a cap region at the 5' end; an open reading frame (ORF) encoding a polypeptide; wherein the plurality of mRNA structures comprises a first mRNA structure and a second mRNA structure, the 3' end of the first mRNA structure is covalently linked to the second mRNA structure.

[0011] In some embodiments, the 3' end of the first mRNA structure and the 3' end of the second mRNA structure are covalently linked.

[0012] In some embodiments, the mRNA structure comprises a PolyA region.

[0013] In some embodiments, for the first mRNA structure and the second mRNA structure, the 3' end of the PolyA region of the first mRNA structure is covalently linked to the nucleotide of the second mRNA structure.

[0014] In some embodiments, for the first mRNA structure and the second mRNA structure, the 3' end of the PolyA region of the first mRNA structure and the 3' end of the PolyA region of the second mRNA structure are covalently linked.

[0015] In some embodiments, there is a 5' untranslated region (UTR) between the cap region and the open reading region.

[0016] In some embodiments, there is a 3' untranslated region (UTR) between the PolyA region and the open reading region.

[0017] In some embodiments, the 3' end of the cap region is linked to the 5' end of the ORF or 5' UTR.

[0018] In some embodiments, the 5' end of the PolyA region is linked to the 3' end of the ORF or 3' UTR.

[0019] In some embodiments, the PolyA region of the mRNA structure comprises modified adenosine nucleotides.

[0020] In some embodiments, the covalently linking comprises direct linking or linking through a linking structure.

[0021] In some embodiments, the linking structure is a first linker, which is a group resulting from a coupling reaction.

[0022] In some embodiments, wherein the linking structure comprises a first linker and a dendrimer, wherein for each of the mRNA structures, its 3' end is linked to the dendrimer directly or through its corresponding first linker, forming a branch.

[0023] In some embodiments, the linking structure comprises a first linker, a dendrimer, and a second linker, wherein for each of the dendrimers, the first linker is linked to the dendrimer through the second linker.

[0024] In some embodiments, wherein the second linker is a PolyA sequence, wherein:

[0025] a) the 3' end of the second linker is linked to the dendrimer; or

[0026] b) the 5' end of the second linker is linked to the first linker; or

[0027] c) the 5' end of the second linker is linked to the dendrimer; or,

[0028] d) the 3' end of the second linker is connected to the first linker.

[0029] In some embodiments, there is a PolyA sequence connected to the bifurcation structure only, wherein,

[0030] a) the 3' end of the PolyA sequence connected to the bifurcation structure only is connected to the bifurcation structure; or

[0031] b) the 5' end of the PolyA sequence connected to the bifurcation structure only is connected to the bifurcation structure.

[0032] In some embodiments, the PolyA sequence comprises modified adenosine nucleotides.

[0033] In some embodiments, the poly-mRNA has a structure as described in Formula (1):

[0034] C1— U 11 — O1— U 12 — A1— L— A2— U 22 — O2— U 21 — C2(I), wherein,

[0035] C1 is a cap region of a first mRNA structure, O1 is an ORF of the first mRNA structure, A1 is a PolyA region of the first mRNA structure, U 11 is a bond or is a nucleotide, U 12 is a bond or is a nucleotide;

[0036] L is a linker structure;

[0037] C2 is a cap region of a second mRNA structure, O2 is an ORF of the second mRNA structure, A2 is a PolyA region of the second mRNA structure, U 21 is a bond or is a nucleotide, U 22 is a bond or is a nucleotide.

[0038] In some embodiments, wherein L is a first linker.

[0039] In some embodiments, wherein C1 to A1 are 5'-3' connected, and A2-C2 are 3'-5' connected.

[0040] In some embodiments, wherein the 3' end of C1 is connected to the 5' end of U 11 .

[0041] In some embodiments, wherein the 3' end of U 11 is connected to the 5' end of O1.

[0042] In some embodiments, wherein the 3' end of O1 is connected to the 5' end of U12 The 5' end.

[0043] In some implementations, U 12 The 3' end is connected to the 5' end of A1.

[0044] In some implementations, L is connected to the 3' end of A1.

[0045] In some implementations, L is connected to the 3' end of A2.

[0046] In some implementations, the 5' end of A2 is connected to U. 22 The 3' end.

[0047] In some implementations, U 22 The 5' end is connected to the 3' end of O2.

[0048] In some implementations, the 5' end of O2 is connected to O 21 The 3' end.

[0049] In some implementations, U 21 The 5' end of C2 is connected to the 3' end of C2.

[0050] In some implementations, U 11 This is the 5' untranslated region (UTR) of the first mRNA structure.

[0051] In some implementations, U 12 This is the 3'UTR of the first mRNA structure.

[0052] In some implementations, U 22 This is the 3'UTR of the second mRNA structure.

[0053] In some implementations, U 21 This is the 5'UTR of the second mRNA structure.

[0054] In some embodiments, the structure is as described in formula (II):

[0055] in

[0056] C represents the cap region of the mRNA structure, O represents the ORF of the mRNA structure, A represents the PolyA region of the mRNA structure, U1 represents a bond or a nucleotide, and U2 represents a bond or a nucleotide.

[0057] L represents the connection structure;

[0058] A' is H, or one or more oligonucleotides linked to L;

[0059] n is an integer selected from 2-100.

[0060] In some embodiments, wherein L comprises a bifurcation structure, the first linker and the second linker.

[0061] In some embodiments, the multimeric mRNA according to any one of claims 35-36, wherein the first linker and the second linker are connected to the bifurcation structure.

[0062] a structure according to Formula (III) or (IV):

[0063] wherein D is a bifurcation structure, A' is H, or one or more oligonucleotides attached to D, Li is a first linker, L2 is a second linker.

[0064] In some embodiments, wherein C to A are 5'-3' connected.

[0065] In some embodiments, wherein the 3' end of C is connected to the 5' end of U1.

[0066] In some embodiments, wherein the 3' end of U1 is connected to the 5' end of O.

[0067] In some embodiments, wherein the 3' end of O is connected to the 5' end of U2.

[0068] In some embodiments, wherein the 3' end of U2 is connected to the 5' end of A.

[0069] In some embodiments, wherein Li is connected to the 3' end of A.

[0070] In some embodiments, wherein L2 is a PolyA sequence, wherein:

[0071] a) the 3' end of L2 is connected to the bifurcation structure, or

[0072] b) the 5' end of L2 is connected to the first linker

[0073] c) the 5' end of L2 is connected to the bifurcation structure; or,

[0074] d) the 3' end of L2 is connected to the first linker.

[0075] In some embodiments, wherein the first linker comprises a group generated from a coupling reaction or a click chemistry reaction.

[0076] In some embodiments, wherein the first linker comprises a group: wherein Ra and Rb are optional substituents.

[0077] In some embodiments, wherein Raand Rbform a ring with their adjacent atoms.

[0078] In some embodiments, wherein the first linker is:

[0079] In some embodiments, wherein the branched structure comprises a three-armed star molecule, a four-armed star molecule, a comb molecule, or a dendrimer.

[0080] In some embodiments, wherein the branched structure is C((CH2) n3 O(CH2) n1 CH3) n2 , wherein,

[0081] n1 is an integer selected from 1-10;

[0082] n2 is an integer selected from 2-10;

[0083] n3 is an integer selected from 1-10.

[0084] In some embodiments, wherein the branched structure is:

[0085] In some embodiments, wherein the modified nucleotides comprise one or a combination of base-modified nucleotides, sugar-modified nucleotides, or phosphate-modified nucleotides.

[0086] In some embodiments, the base modification is selected from one or a combination of the following group:

[0087] Xanthine, Allylaminouracil, Allylaminothymidine, Hypoxanthine, Dioxane, Dioxane, Dioxane, Dioxane, Dioxane, 6-Chloropurinyl nucleoside, N6-methyladenine, Methylpseudorazine, 2-Thiocytosine, 2-Thiouracil, 5-Methyluracil, 4-Thiothymidine, 4-Thiouracil, 5,6-Dihydro-5-Methyluracil, 5,6-Dihydrouracil, 5-[(3-indolyl)propionamide-N-allyl]uracil, 5-Aminoallylcytosine, 5-Aminoallyluracil, 5-Bromouracil, 5-Bromocytosine, 5-Carboxycytosine, 5-Carboxycytosine, 5-Carboxyuracil, 5-Fluorouracil Pyridine, 5-formylcytosine, 5-formylureapyrimidine, 5-hydroxycytosine, 5-hydroxymethylcytosine, 5-hydroxymethylureapyrimidine, 5-hydroxyureapyrimidine, 5-iodocytosine, 5-iodouracil, 5-methoxycytosine, 5-methoxyuracil, 5-methylcytosine, 5-methyluracil, 5-propynylaminocytosine, 5-propynylaminouracil, 5-propynylcytosine, 5-propynyluracil, 6-azacytosine, 6-azauracil, 6-chloropurine, 6-thioguanine, 7-deadenine, 7-deadenine, 7-deaden-7-propylaminoadenine, 7-deaden-7-propynylaminoadenine, 8-azaadenine 8-Azideadenine, 8-Chloroadenine, 8-Oxoadenine, 8-Oxoguanine, Aladenine, Oxytetracycline, Alaguanine, Alapyrimidine, Biotin-16-7-deazo-7-propynylaminoguanine, Biotin-16-aminoallylcytosine, Biotin-16-aminoallyluracil, 3-5-propynylaminocytosine, 3-6-propynylaminouracil, Cyano-3-aminoallylcytosine, Cyano-3-aminoallyluracil, Cyano-5-6-propynylaminocytosine, Cyano-5-aminoallylcytosine, Cyano-5-aminoallyluracil, Cyano-5-aminoallyluracil, Cyano-7-aminoalenyluracil Propyluracil, Dabcyl-5-3-aminoallyluracil, dethiobiotin-16-aminoallyluracil, dethiobiotin-6-aminoallylcytosine, isoguanine, N1-ethylpseudouracil, N1-methoxymethylpseudouracil, N1-methyladenine, N1-methylpseudouracil, N1-propylpseudouracil, N2-methylguanine, N4-biotin-OBEA-cytosine, N4-methylcytosine, N6-methyladenine, O6-methylguanine, pseudoisocytosine, pseudouracil, thienocytosine, thienoguanine, thienouracil, xanthine, 3-deazoadenine, 2,6-diaminoadenine, 2,6-diaminoguanine, 5-formamidyluracil, 5-ethynyluracil, N6-iso-pentenyladenine (i6A), 2-methylthio-N6-iso-pentenyladenine (ms2i6A), 2-methylthio-N6-methyladenine (ms2m6A), N6-(cis-hydroxyisopentenyl)adenine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenine (ms2io6A), N6-glycinamidoformyladenine (g6A), N6-threoninamidoformyladenine (t6A), 2-methylthio-N6-threoninamidoformyladenine (ms2t6A), N6-methyl-N6-threoninamidoformyladenine (m6t6A), N6-hydroxynalanylamidoformyladenine (hn6A), 2-methylthio-N6-hydroxynalanylamidoformyladenine (ms2hn6A), N6,N6-dimethyladenine (m62A), and N6-acetyladenine (ac6A).

[0088] In some embodiments, the modification of the sugar is selected from one or several of the following group:

[0089] 2'-thioribose, 2',3'-dideoxyribose, 2'-amino-2'-deoxyribose, 2'-deoxyribose, 2'-azido-2'-deoxyribose, 2'-fluoro-2'-deoxyribose, 2'-O-methylribose, 2'-O-methyldideoxyribose, 3'-amino-2',3'-dideoxyribose, 3'-azido-2',3'-dideoxyribose, 3'-deoxyribose, 3'-O-(2-nitrobenzyl)-2'-deoxyribose, 3'-O-methylribose, 5'-aminoribose, 5'-thioribose, 5-nitro-indolyl-2'-deoxyribose, 5'-biotinylribose, 2'-O,4'-C-methylene-bridged ribose, 2'-O,4'-C-amino-bridged ribose, and 2'-O,4'-C-thio-bridged ribose.

[0090] In some embodiments, the modification of the phosphate is selected from one or several of the following group: phosphorothioate (PS), 5'-O-methylphosphonate, 3'-O-methylphosphonate, 5'-hydroxylphosphonate, hydroxylphosphonate, selenophosphonate, phosphonate, carborane phosphonate, methylphosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidine ring, triazole ring, boronophosphate (BP), methylphosphonate, and guanidinopropyl phosphoramidate.

[0091] In some embodiments, the modification of the phosphate is selected from one or several of the following group: phosphorothioate (PS), 5'-O-methylphosphonate, 3'-O-methylphosphonate, 5'-hydroxylphosphonate, hydroxylphosphonate, selenophosphonate, phosphonate, carborane phosphonate, methylphosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidine ring, triazole ring, boronophosphate (BP), methylphosphonate, and guanidinopropyl phosphoramidate.

[0092] In some embodiments, wherein the cap region comprises a cap structure, which has the following structure:

[0093] wherein:

[0094] R3 is selected from guanine, adenine, cytosine, uracil, guanine analog, adenine analog, cytosine analog, uracil analog; np is an integer from 1 to 10; wherein np can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; in some embodiments np is 3;

[0095] R4 is (N1p) x N2, wherein N1 and N2 are ribonucleosides, and N1 and N2 are the same or different;

[0096] p is independently at each position a phospho group, phosphorothioate, phosphorodithioate, alkylphosphonate, arylphosphonate, or N-phosphoramidate linkage, preferably the modification of the phosphate is selected from one or several of the following group: phosphorothioate (PS), 5'-0-methylphosphonate, 3'-0-methylphosphonate, 5'-hydroxylphosphonate, hydroxylphosphonate, seleno-phosphonate, phosphonate, carbon phosphonate, methyl phosphonate, phenyl phosphonate, ethyl phosphonate, H-phosphonate, guanidine ring, triazole ring, boron phosphate (BP), methyl phosphonate, and guanidinopropyl phosphoramidate;

[0097] X is an integer from 0 to 8, wherein if X > 2, the ribonucleosides N1 in (N1p)x are the same or different from each other;

[0098] R1 and R2 groups are independently selected from O-alkyl, halogen, acetylamino (AcNH), hydrogen, or hydroxyl.

[0099] In some embodiments, wherein the sugars in N1 and N2 are independently at each position selected from ribose and deoxyribose, and can contain modifications including 2'-0-alkyl, 2'-0-methoxyethyl, 2'-0-allyl, 2'-0-alkylamine, 2'-fluoro-ribose, and 2'-deoxyribose;

[0100] and / or the bases in N1and N2are independently selected for each position from adenine, uracil, guanine, or cytosine, or an analog of adenine, uracil, guanine, or cytosine, and the nucleotide modified base can be selected from the group consisting of xanthine, allylaminouracil, allylaminothymidine, hypoxanthine, dioxuracil, dioxycytosine, dioxoguanine, dioxouracil, 6-chloropurine nucleoside, N6-methyladenine, methylpseudo-uracil, 2-thiocytosine, 2-thiouracil, 5-methyluracil, 4-thiothymidine, 4-thiouracil, 5,6-dihydro-5-methyluracil, 5,6-dihydrouracil, 5-[(3-indolyl)propionamido-N-allyl]uracil, 5-aminoallylcytosine, 5-aminoallyluracil, 5-bromouracil, 5-bromocytosine, 5-carboxycytosine, 5-carboxycytosine, 5-carboxyuracil, 5-fluorouracil, 5-formylcytosine, 5-formyluracil, 5-hydroxycytosine, 5-hydroxymethylcytosine, 5-hydroxymethyluracil, 5-hydroxyuracil, 5-iodocytosine, 5-iodouracil, 5-methoxycytosine, 5-methoxyuracil, 5-methylcytosine, 5-methyluracil, 5-propynylaminocytosine, 5-propynylaminouracil, 5-propynylcytosine, 5-propynyluracil, 6-azacytosine, 6-azauracil, 6-chloropurine, 6-thioguanine, 7-deazanucleoside, 7-deazaguanine, 7-deaza-7-propylaminoadenine, 7-deaza-7-propynylaminoadenine, 8-azanucleoside, 8-azidoadenine, 8-chloroadenine, 8-oxoadenine, 8-oxoguanine, biotin-16-7-deaza-7-propynylaminoguanine, biotin-16-aminoallylcytosine, biotin-16-aminoallyluracil, 3-5-propynylaminocytosine, 3-6-propynylaminouracil, cyano 3-aminoallylcytosine, cyano 3-aminoallyluracil, cyano 5-6-propynylaminocytosine, cyano 5-6-propynylaminouracil, cyano 5-aminoallylcytosine, cyano 5-aminoallyluracil, cyano 7-aminoallyluracil, Dabcyl-5-3-aminoallyluracil, desulfo-biotin-16-aminoallyluracil, desulfo-biotin-6-aminoallylcytosine, isoguanine, N 1 -methoxy-methyl-pseudo-uracil, N 1 -methyl-adenine, N 1 -methyl-pseudo-uracil, N 1 -propyl-pseudo-uracil, N 4 -biotin-OBEA-cytosine, N 6- methyladenine, 06-methylguanine, pseudoisocytosine, pseudouracil, thienocytosine, thienoguanine, thienouracil, xanthine, 3-deazanole, 2,6-diaminoadenine, 2,6-diaminoguanine, 5- formamidouracil, 5-ethynyluracil, N 6 - isopentenyladenine (i6A), 2-methylthio-N 6 - isopentenyladenine (ms2i6A), 2-methylthio-N 6 - methyladenine (ms2m6A), N 6 - (cis-hydroxyisopentenyl)adenine (io6A), 2-methylthio-N 6 - (cis-hydroxyisopentenyl)adenine (ms2io6A), N 6 - glycylcarbamoylad enine (g6A), N 6 - threonylcarbamoylad enine (t6A), 2-methylthio-N 6 - threonylcarbamoylad enine (ms2t6A), N 6 - methyl-N 6 - threonylcarbamoylad enine (m6t6A), N 6 - hydroxyvalylcarbamoylad enine (hn6A), 2-methylthio-N 6 - hydroxyvalylcarbamoylad enine (ms2hn6A), N 6 ,N 6 - dimethyladenine (m62A) and N 6 - acetyl adenine (ac6A).

[0101] In some embodiments, wherein the cap structure analog is a di-nucleotide cap analog or a tri-nucleotide cap analog.

[0102] In some embodiments, wherein the cap structure analog has the following general formula:

[0103] wherein R5and R6groups are independently selected from O-alkyl (O-methyl), halogen, a tag, hydrogen, or hydroxyl;

[0104] X1and X2are bases, and X1is the same or different from X2.

[0105] In some embodiments, wherein X1and / or X2is cytosine, uracil guanine, or adenine.

[0106] In some embodiments, wherein the cap structure analog is one of the following:

[0107] In another aspect, the present application provides a delivery agent, including a polymeric mRNA, wherein the delivery agent comprises a lipid, a peptide, a protein, an antibody, a carbohydrate, a nanoparticle, or a microparticle.

[0108] In some embodiments, wherein the nanoparticle or microparticle is a lipid nanoparticle or lipid microparticle, a polymer nanoparticle or polymer microparticle, a protein nanoparticle or protein microparticle, or a solid nanoparticle or solid microparticle.

[0109] In another aspect, the present application provides a cell comprising the aforementioned polymeric mRNA.

[0110] In another aspect, the present application provides a pharmaceutical composition comprising the aforementioned polymeric mRNA, or the aforementioned delivery agent or the aforementioned cell, and optionally a pharmaceutically acceptable carrier.

[0111] In another aspect, the present application provides the aforementioned polymeric mRNA, or the aforementioned delivery agent or the aforementioned cell, or the aforementioned pharmaceutical composition, for use in the treatment and / or prevention of a disease.

[0112] In another aspect, the present application provides the aforementioned polymeric mRNA, or the aforementioned delivery agent or the aforementioned cell, or the aforementioned pharmaceutical composition, for use in the preparation of a medicament for the treatment and / or prevention of a disease.

[0113] In another aspect, the present application provides a method comprising introducing the aforementioned polymeric mRNA, or the aforementioned delivery agent into a cell.

[0114] In another aspect, the present application provides a method comprising administering to a subject in need thereof an effective amount of the aforementioned polymeric mRNA, or the aforementioned delivery agent or the aforementioned cell, or the aforementioned pharmaceutical composition.

[0115] In another aspect, the present application provides a method of treating and / or preventing a disease, comprising administering to a subject in need thereof an effective amount of the aforementioned polymeric mRNA, or the aforementioned delivery agent or the aforementioned cell, or the aforementioned pharmaceutical composition.

[0116] Other aspects and advantages of the present application will become apparent to those skilled in the art from the following detailed description in conjunction with the drawings. Only the preferred embodiments of the application and the best mode contemplated by the inventors of carrying out the application are shown and described. The application is capable of modifications in various obvious respects, all of which are intended to be within the scope of the present application. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive. BRIEF DESCRIPTION OF DRAWINGS

[0117] The specific features of the invention involved in the present application are shown in the appended claims. The features and advantages of the invention involved in the present application can be better understood by referring to the detailed description of exemplary embodiments and the attached drawings. The drawings are briefly described as follows:

[0118] Fig. 1A-1C show a poly-mRNA structure described in the present application;

[0119] Fig. 2A-2D show a poly-mRNA structure described in the present application:

[0120] Fig. 3 shows the expression of a poly-mRNA structure described in the present application in HEK293T and A549 cells, specifically, sampling at day 1, 3, 6, respectively, there are 6 groups of data for each day sampling in the figure, and each group of data from left to right is Hibit mRNA (20A), Hibit mRNA PS modification PolyA ddC (20A), Hibit mRNA 3’N3 (20A), Hibit mRNA 3’DBCO (20A), Bi-valence Hibit mRNA (20A), negative control;

[0121] Fig. 4 shows the area calculation (AUC) of the expression amount of a poly-mRNA structure described in the present application, each group of data from left to right is Hibit mRNA (20A), Hibit mRNA PS modification PolyA ddC (20A), Hibit mRNA 3’N3 (20A), Hibit mRNA 3’DBCO (20A), Bi-valence Hibit mRNA (20A), negative control;

[0122] Fig. 5 shows the expression of a poly-mRNA structure described in the present application in HEK293T and A549 cells, specifically, sampling at day 1, 2, 3, respectively, there are 4 groups of data for each day sampling in the figure, and each group of data from left to right is Hibit mRNA (30A), Hibit mRNA PS modification PolyA (30A), Bi-valence Hibit mRNA PS modification PolyA (30A), negative control;

[0123] Figure 6 shows the area under the curve (AUC) of the expression of a multi-mRNA structure described herein. Each group of data from left to right is Hibit mRNA (30A), Hibit mRNA PS modification PolyA (30A), Bi-valence Hibit mRNA PS modification PolyA (30A), negative control.

[0124] Figure 7 shows a bifurcation structure of a multi-mRNA structure described herein.

[0125] Figure 8 shows a multi-mRNA structure described herein.

[0126] Figure 9 shows a multi-mRNA structure described herein.

[0127] Figure 10 shows the expression of a multi-mRNA structure described herein in HEK293T and A549 cells. Specifically, samples were taken at day 1 and day 2. In the figure, each day of sampling has 5 groups of data from left to right, which are Hibit mRNA (30A), Hibit mRNA PS modification PolyA (30A), Tri-valence Hibit mRNA PS modification PolyA (30A) Trembler 5*A, 4-valence Hibit mRNA PS modification PolyA (30A) Trembler 5*A, negative control. DETAILED DESCRIPTION

[0128] The following specific examples illustrate the embodiments of the present application. Those skilled in the art can easily understand other advantages and effects of the present application from the disclosure herein.

[0129] Definitions of Terms

[0130] In the present application, the terms "polynucleotide," "oligonucleotide," "nucleic acid molecule," and "nucleic acid" are used interchangeably and generally include DNA molecules, RNA molecules, analogs of DNA or RNA generated using nucleotide analogs, and hybrids thereof. The nucleic acid molecule can be single-stranded or double-stranded. The following are non-limiting examples of polynucleotides: a gene or gene fragment, e.g., a probe, primer, EST or SAGE tag, exon, intron, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozyme, cDNA, recombinant polynucleotide, branched polynucleotide, plasmid, vector, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probe, siRNA, shRNA, RNAi agent, and primer. A polynucleotide can be modified at one or more bases, sugars and / or phosphates, or substituted with any of a variety of modifications or substitutions known in the art or described herein. A polynucleotide can comprise modified nucleotides, e.g., methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be made before or after assembly of the polymer. The polynucleotide can be modified after polymerization, e.g., by conjugation with a labeling component.

[0131] The term "messenger ribonucleic acid" (mRNA) refers to a ribonucleic acid (RNA) molecule that is capable of mediating the transfer of genetic information in the cytoplasm to the ribosome, where it serves as a template for protein synthesis. It is synthesized from a DNA template during transcription. In eukaryotes, mRNA is transcribed in vivo by RNA polymerase within the cell in vivo from a gene within a chromosome. A 5' end cap (also known as an RNA cap, an RNA 7-methylguanosine cap, or an RNA m7G cap) is added to the 5' end of the mRNA in vivo during or after transcription in vivo. The 5' end is a terminal 7-methylguanosine residue linked by a 5'-5'-triphosphate linkage to the first transcribed nucleotide. In addition, most eukaryotic mRNA molecules have a polyadenylic acid moiety ("poly(A) tail") at the 3' end of the mRNA molecule. In vivo, the polyadenylic moiety (Poly A) is added post-transcriptionally in eukaryotic cells. Thus, a typical mature eukaryotic mRNA has the following structure: starting with the mRNA cap nucleotide at the 5' end, followed by a 5' untranslated region (5' UTR) of nucleotides, then an open reading frame (ORF, which can be a coding sequence for a protein) starting with a start codon (an AUG triplet of nucleotide bases) and ending at a stop codon (a UAA, UAG, or UGA triplet of nucleotide bases), then a 3' untranslated region (3' UTR) of nucleotides, ending with a polyadenylic moiety. While the features of a typical mature eukaryotic mRNA can be produced naturally in vivo in eukaryotic cells, the same or structurally and functionally equivalent features can also be produced in vitro using molecular biology methods. Thus, any RNA that has a similar structure to a typical mature eukaryotic mRNA can function as an mRNA, and is also included within the scope of the term "messenger ribonucleic acid."

[0132] The term "mRNA structure" is a piece of RNA molecule that has the structure (e.g., with a cap region and an open reading frame ORF) and function (e.g., as a template for protein synthesis in a ribosome) of an mRNA. In some embodiments, the mRNA structure can be an mRNA, including a 5' end cap structure, a 3' end Poly A, and an open reading frame.

[0133] The term "poly-mRNA" is a molecule containing multiple mRNA structures. The multiple mRNA structures can be covalently linked.

[0134] The terms“first mRNA structure” and“second mRNA structure” are merely to distinguish two mRNA structures in a“poly-mRNA”. The first mRNA structure and the second mRNA structure can be the same or different; the cap region (e.g., 5’ cap) of the first mRNA structure and the cap region (e.g., 5’ cap) of the second mRNA structure can be the same or different; the ORF of the first mRNA structure and the ORF of the second mRNA can be the same or different; the PolyA region of the first mRNA structure and the PolyA region of the second mRNA can be the same or different.

[0135] In the present application, the terms“PolyA region” and“PolyA” can be used interchangeably to refer to a nucleic acid sequence consisting of mostly adenosine nucleotides (which can be adenine or deoxy-adenine) attached to the end of a nucleic acid (e.g., the end of a non-5’ cap, and e.g., the 3’ end). The poly-adenosine portion can consist of 10%-100%, 25%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80%-100%, 90%-100%, 95%-100%, 96%-100%, 97%-100%, 98%-100%, or 99%-100% of adenine nucleotides. In some embodiments, the poly-adenosine portion (PolyA region) can consist of all adenosine nucleotides (which can be adenine or deoxy-adenine). And the adenosine nucleotides included in the poly-adenosine portion can be standard adenosine nucleotides or modified (non-standard) adenosine nucleotides. Generally, the PolyA region can have 20-500 consecutive adenine nucleotides, and there can be modified adenine nucleotides upstream, downstream, or between the 20-300 consecutive adenosine nucleotides.

[0136] In the present application, the term“cap structure” can be a 7-methylguanosine (m7G) nucleotide or a stretch of nucleic acid sequence.

[0137] In some embodiments, the cap structure can be any compound of the following general formula:

[0138] wherein:

[0139] R3is selected from guanine, adenine, cytosine, uracil, guanine analog, adenine analog, cytosine analog, uracil analog; npis an integer from 1-10; wherein npmay be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; in some embodiments npis 3;

[0140] R4is (N1p) xN2, wherein N1and N2are ribonucleosides, and N1and N2are the same or different;

[0141] P is independently at each position a phospho group, a phosphorothioate, a phosphorodithioate, an alkylphosphonate, an arylphosphonate, or an N-phosphoramidate linkage, preferably, P can include a modification of the phosphate selected from one or more of the following group: phosphorothioate (PS), phosphorothioate, 5'-O-methylphosphonate, 3'-O-methylphosphonate, 5'-hydroxylphosphonate, hydroxylphosphonate, selenophosphate, phosphonate, carbophosphonate, methylphosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidine ring, triazole ring, boronophosphate (BP), methylphosphonate, and guanylpropylphosphoramidate;

[0142] X is an integer from 0 to 8, wherein if X > 2, the ribonucleosides N1in (N1P)x are the same or different from each other;

[0143] R1and R2groups are independently selected from O-alkyl, halogen, acetylamino (AcNH), hydrogen, or hydroxyl.

[0144] In some embodiments, the cap structure can be any compound of the following general formula:

[0145] The cap structure can include, for example, a dinucleotide cap analog of the formula m7G(5')p3(5')G, wherein the guanine nucleotide (G) is linked via its 5' OH to a triphosphate bridge. In some dinucleotide cap analogs, the 3'-OH group is substituted with hydrogen or OCH3(refer to US 7,074,596; Kore, Nucleotides, Nucleotides, and Nucleic Acids, 2006, 25:307-14; and Kore, Nucleotides, Nucleotides, and Nucleic Acids, 2006, 25:337-40). Dinucleotide cap analogs include m7G(5')p3G, 3'-OMe-m7G(5')p3G (ARCA). The term "cap" also includes tri-nucleotide cap analogs (as defined below) as well as other longer molecules (e.g., cap analogs having four, five, or six or more nucleotides linked to a triphosphate bridge).

[0146] The term "cap region" is a stretch of sequence that contains a cap structure. The 5' end of the cap region can be a 7-methylguanosine (m7G) nucleotide, or a nucleotide linked by a triphosphate bridge.

[0147] The term "covalently linked" refers to the process of forming a covalent bond directly between two moieties to connect them to each other, or to form a covalent bond with the same linking structure to connect them to each other through the linking structure.

[0148] The terms "5'" and "3'" are conventional designations used to describe the features of nucleic acid sequences, and relate to the positions of genetic elements and / or the direction of events such as RNA polymerase transcription or ribosomal translation, which proceed in a 5' to 3' direction. Synonyms are upstream (5') and downstream (3'). Typically, DNA sequences, genetic maps, and RNA sequences can be drawn with the 5' to 3' direction from left to right, or the 5' to 3' direction can be indicated with an arrowhead symbol, where the arrowhead points in the 3' direction. So when following this conventional usage, 5' (upstream) means placing a genetic element to the left, and 3' (downstream) means placing a genetic element to the right.

[0149] The term "terminus" refers to an end nucleotide or a stretch of contiguous nucleotides ending in that nucleotide.

[0150] The term "end nucleotide" refers to the first nucleotide of a polynucleotide in either the 5' to 3' direction or the 3' to 5' direction.

[0151] In some embodiments, "end nucleotide" refers to a nucleotide having a free phosphate group at the 5' position of the nucleotide pentose, or nucleotides linked to one another without dehydration condensation of the hydroxyl groups with adjacent nucleotides, or a nucleotide having a free hydroxyl group at the 3' position of the nucleotide pentose, or nucleotides linked to one another without dehydration condensation of the phosphate groups with adjacent nucleotides.

[0152] In some embodiments, "5' end nucleotide" refers to a nucleotide having a free phosphate group at the 5' position of the nucleotide pentose, or nucleotides linked to one another without dehydration condensation of the hydroxyl groups with adjacent nucleotides. In some embodiments, "3' end nucleotide" refers to a nucleotide having a free hydroxyl group at the 3' position of the nucleotide pentose, or nucleotides linked to one another without dehydration condensation of the phosphate groups with adjacent nucleotides.

[0153] wherein the term "5' end" or "5' terminus" can be the first nucleotide of a polynucleotide in the 5' to 3' direction or a stretch of contiguous nucleotides ending in that nucleotide, and the term "3' end" or "3' terminus" can be the last nucleotide of a polynucleotide in the 5' to 3' direction or a stretch of contiguous nucleotides ending in that nucleotide.

[0154] The term "dendritic structure" or "dendrimer" as used herein refers to an organic polymeric compound having a core structure covalently linked to a plurality of arms or dendrons, the ends of which can be modified to attach to an oligonucleotide (e.g., via a click chemistry reaction).

[0155] DETAILED DESCRIPTION

[0156] Polymeric mRNA

[0157] In an aspect, the present application provides a polymeric mRNA comprising a plurality of mRNA structures.

[0158] In some embodiments, the plurality of mRNA structures comprises a first mRNA structure and a second mRNA structure, and the 3’ end of the first mRNA structure can be covalently linked to the second mRNA structure.

[0159] In some embodiments, the plurality of mRNA structures comprises a first mRNA structure and a second mRNA structure, and the 3’ end of the first mRNA structure can be covalently linked to the second mRNA structure.

[0160] In some embodiments, the 3’ end of the first mRNA structure can be covalently linked to the 3’ end of the second mRNA structure. For example, the 3’ end nucleotide of the first mRNA structure can be covalently linked to the 3’ end nucleotide of the second mRNA structure. The covalent linkage can include direct linkage or linkage through a linking structure.

[0161] In some embodiments, the mRNA structure comprises a PolyA region.

[0162] In certain embodiments, the PolyA region comprises modified adenine nucleotides and unmodified adenine nucleotides. For example, the PolyA region comprises unmodified adenosine nucleotides and modified adenosine nucleotides. The modified adenosine nucleotides can comprise 5-300 nucleotides. For example, the number of modified adenosine nucleotides can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100. For example, the number of modified adenosine nucleotides can range from 5-300, 5-200, 5-100, 5-70, 5-60, 5-50, 5-40, 5-30, 5-20, 5-10, 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 10-20, 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 20-40, 20-30, 30-100, 30-90, 30-80, 30-70, 30-60, 30-50, 30-40, 40-100, 40-90, 40-80, 40-70, 40-60, 40-50, 50-100, 50-90, 50-80, 50-70, 50-60, 60-100, 60-90, 60-80, 60-70, 70-100, 70-90, 70-80, 80-100, 80-90.

[0163] In this case, the 3' end of the PolyA region of the first mRNA structure can be covalently linked to the nucleotide of the second mRNA structure. For example, the 3' end nucleotide of the PolyA region of the first mRNA structure can be covalently linked to the nucleotide of the second mRNA structure. The covalent linkage includes direct linkage or linkage through a linkage structure.

[0164] Further, the 3' end of the Poly A region of the first mRNA structure can be covalently linked to a nucleotide of the Poly A region of the second mRNA structure. For example, the 3' end nucleotide of the Poly A region of the first mRNA structure can be covalently linked to a nucleotide of the Poly A region of the second mRNA structure. The covalently linking includes direct linking or linking through a linking structure.

[0165] Further, the 3' end of the Poly A region of the first mRNA structure can be covalently linked to the 3' end of the Poly A region of the second mRNA structure. For example, the 3' end nucleotide of the Poly A region of the first mRNA structure can be covalently linked to the 3' end nucleotide of the Poly A region of the second mRNA structure. The covalently linking includes direct linking or linking through a linking structure.

[0166] Each mRNA structure can include a terminal end. The terminal end can be a terminal nucleotide or a stretch of contiguous nucleotides ending at a terminal nucleotide.

[0167] The terminal nucleotide of an mRNA structure can be the first nucleotide of the mRNA structure in the 5' to 3' direction or the 3' to 5' direction.

[0168] In some embodiments, the terminal nucleotide of an mRNA structure can be a 5' terminal nucleotide, i.e., the first nucleotide of the mRNA structure in the 5' to 3' direction.

[0169] For example, the 5' terminal nucleotide of an mRNA structure can be the first nucleotide of the mRNA structure in the 5' to 3' direction, which has a free phosphate group at the 5' position of the pentose sugar.

[0170] For another example, the 5' terminal nucleotide of an mRNA structure can be the first nucleotide of the mRNA structure in the 5' to 3' direction, which does not have a 3'-5' phosphodiester bond with the hydroxyl group of the adjacent nucleotide.

[0171] For another example, the 5' terminal nucleotide of an mRNA structure can be the first nucleotide of the mRNA structure in the 5' to 3' direction, which forms a 5'-5' triphosphate bond with the adjacent nucleotide.

[0172] For another example, the 5' terminal nucleotide of an mRNA structure can be a 7- methylguanosine (m7G) nucleotide.

[0173] In some embodiments, the terminal nucleotide of an mRNA structure can be a 3' terminal nucleotide, i.e., the first nucleotide of the mRNA structure in the 3' to 5' direction.

[0174] For example, the 3' terminal nucleotide of the mRNA structure can be the first nucleotide in the 3' to 5' direction of the mRNA structure that has a free hydroxyl group at the 3' position of the pentose sugar.

[0175] For example, the 3' terminal nucleotide of the mRNA structure can be the first nucleotide in the 3' to 5' direction of the mRNA structure that has a free hydroxyl group at the 3' position of the pentose sugar.

[0176] Each mRNA structure can have two ends, and the mRNA structure between the two ends has the structure (e.g., has a cap region and an open reading frame, ORF) and function (e.g., serves as a template for protein synthesis in a ribosome) of an mRNA.

[0177] Each mRNA structure (e.g., the first mRNA structure or the second mRNA structure) can have two ends. In some embodiments, the two ends can be a 5' end and a 3' end, respectively. In some embodiments, the two ends can be an end with a cap region and an end without a cap region, respectively. For example, the end with the cap region can be the 5' end, and the end without the cap region can be the 3' end.

[0178] In some embodiments, the mRNA structure further comprises a PolyA region. The cap region and the PolyA region can be located at the two ends of the mRNA structure, respectively. For example, the mRNA structure comprises, in the 5' to 3' direction, a cap region, an open reading frame, and a PolyA region, respectively. The PolyA region can be located at the 3' end of the mRNA structure, i.e., the PolyA region of the mRNA structure can be a stretch of polynucleotides that ends with the 3' terminal nucleotide of the mRNA structure. The cap region can be located at the 5' end of the mRNA structure, i.e., the cap region of the mRNA structure can be a stretch of polynucleotides that ends with the 5' terminal nucleotide of the mRNA structure.

[0179] In some embodiments, the mRNA structure further comprises an untranslated region (UTR). The cap region and the open reading frame have a 5' UTR therebetween. The PolyA region and the open reading frame have a 3' UTR therebetween. The 3' end of the cap region is connected to the 5' end of the ORF or the 5' UTR. The 5' end of the PolyA region is connected to the 3' end of the ORF or the 3' UTR.

[0180] Specifically, as shown in FIG. 1A. The poly-mRNA 100 can include multiple mRNA structures, such as a first mRNA structure 101 and a second mRNA structure 102. The poly-mRNA 100 can include more mRNA structures (not shown in the figure). The first mRNA structure 101 can include a cap region 1011 and an ORF 1012. In some embodiments, the first mRNA structure 101 can further include a PolyA region 1013. The second mRNA structure 102 can include a cap region 1021 and an ORF 1022. In some embodiments, the first mRNA structure 102 can further include a PolyA region 1023.

[0181] As shown in FIG. 1A, the 3’ end 1014 of the first mRNA structure 101 is covalently connected to the second mRNA 102.

[0182] In some embodiments, the 3’ end 1014 of the first mRNA structure 101 can be covalently connected to the second mRNA 102 directly. For example, the 3’ end 1014 of the first mRNA structure 101 can be connected to the second mRNA 102 by reacting a free hydroxyl group of the 3’ end 1014 of the first mRNA structure 101 with a free hydroxyl group of the second mRNA 102. In some embodiments, the 3’ end 1014 of the first mRNA structure 101 can be covalently connected to the second mRNA 102 through a linker 201.

[0183] In some embodiments, the mRNA structure can include a PolyA region. For example, the first mRNA structure can have a PolyA region 1013, and the second mRNA structure can have a PolyA region 1023.

[0184] As shown in FIG. 1A, the 3’ end 1014 of the PolyA region 1013 of the first mRNA structure 101 is connected to a nucleotide of the second mRNA structure directly or through a linker 201. Further, the 3’ end 1014 of the PolyA region 1013 of the first mRNA structure 101 is connected to a nucleotide of the PolyA region 1023 of the second mRNA structure directly or through a linker 201.

[0185] Further, the 3’ end of the first mRNA structure can be connected to the 3’ end of the second mRNA structure on the same linker.

[0186] As shown in FIG. 1B, in some embodiments, the 3’ end 1014 of the first mRNA structure 101 is connected to the 3’ end nucleotide 1024 of the second mRNA 102 directly or through a linker 201.

[0187] In some embodiments, the mRNA structure can include a PolyA region. For example, the first mRNA structure can have a PolyA region 1013, and the second mRNA structure can have a PolyA region 1023.

[0188] Further, as shown in FIG. IB, the 3’ end 1014 of the PolyA region 1013 of the first mRNA structure 101 is connected to the 3’ end nucleotide 1024 of the second mRNA 102, directly or through a connection structure 201.

[0189] Further, as shown in FIG. IB, the 3’ end 1014 of the PolyA region 1013 of the first mRNA structure 101 is connected to the 3’ end 1024 of the PolyA region 1023 of the second mRNA 102, directly or through a connection structure 201.

[0190] For the end, it can refer to the end nucleotide, or a continuous nucleotide segment with the end nucleotide as the end point. For the latter, a certain nucleotide in the 3’ end 1014 of the first mRNA structure 101 can be connected to a certain nucleotide in the 3’ end 1024 of the second mRNA 102, directly or through a connection structure 201, as shown in FIG. 1C.

[0191] In some embodiments, the connection structure can include a fork structure, and the 3’ end of the mRNA structure (e.g., the first mRNA structure and / or the second mRNA structure) is connected to the fork structure.

[0192] For example, as shown in FIG. 2A, the multi-mRNA can include a connection structure 202, where the connection structure 202 is a fork structure, and the fork structure includes multiple forks, e.g., 202-1, 202-2, 202-3, etc., and multiple mRNA structures (e.g., mRNA structures 101, 102, and 103) can be connected through the 3’ end and the forks of the fork structure, respectively.

[0193] When the connection structure includes a fork structure, there are cases where not all forks are connected by the 3’ end of the mRNA structure. For example, as shown in FIG. 2B, there are cases where the forks are not connected, e.g., the fork 202-3 is not connected to a nucleotide sequence. For another example, as shown in FIG. 2C, there are cases where the forks are connected to any polynucleotide, e.g., the fork 202-3 is connected to any polynucleotide 104. The nucleotides of one or more polynucleotides 104 can be connected to the fork structure 202-3. The 3’ end or 5’ end of one or more polynucleotides 104 can be connected to the fork structure 202-3.

[0194] Connection structure

[0195] Since the 3' end of an mRNA structure itself is not easy to be directly connected to the 3' end of another mRNA structure, the interconnection of different mRNA structures needs the help of a linking structure.

[0196] The linking structure can be a chemical bond.

[0197] The linking structure includes a first linker. The first linker is a group generated from a coupling reaction. Coupling is a process of combining two or more molecules or biological macromolecules through a chemical covalent bond. The first linker can be a functional group generated from a first functional group and a second functional group.

[0198] In some embodiments, the interconnection of different mRNA structures is performed with the help of orthogonal biological reactions. For example, a first functional group modification is performed on one mRNA structure, and a second functional group modification is performed on another mRNA structure, and the multiple mRNA structures are connected with the help of the reaction of the first functional group and the second functional group. In some embodiments, the first linker is generated from the reaction of the first functional group and the second functional group.

[0199] In some embodiments, the first linker can be a chemical group generated from the reaction of an amino group. When the first functional group is -NH2, the second functional group can be N-hydroxysuccinimidyl ester (NHS ester), and the first linker can be

[0200] When the first functional group is -NH2, the second functional group can be imidate, and the first linker can be

[0201] When the first functional group is -COOH, the second functional group can be carbodiimide or dicyclohexyl carbodiimide, and the first linker can be

[0202] When the first functional group is -SH, the second functional group can be maleimide, and the first linker can be

[0203] When the first functional group is -SH, the second functional group can be haloacetyl, and the first linker can be

[0204] When the first functional group is -SH, the second functional group can be pyridyl dimercapto, and the first linker can be -S-S-.

[0205] When the first functional group is -CHO, the second functional group can be hydrazine, and the first linker can be

[0206] When the first functional group is -CHO, the second functional group can be alkoxyamine, and the first linker can be

[0207] When the first functional group is azide, the second functional group can be a group containing alkyne, the first linker is

[0208] wherein Raand Rbare optional substituents. For example, the first functional group is azide, the second functional group is DBCO, the first linker is

[0209] Other reactive groups in relation to orthogonal chemistry can be found in Kondengadan S M, Bansal S, Yang C, et al. Click chemistry and drug delivery: A bird's-eye view [J]. Journal of Pharmaceutical Sciences, 2023, 13(5): 1990-2016, which is incorporated by reference in its entirety.

[0210] The connecting structure can comprise a forked structure. As shown in FIG. 2D, the forked structure can comprise a three-arm star molecule (as shown in 205), a four-arm star molecule (as shown in 206), a comb-like molecule (as shown in 207), a dendrimer (as shown in 208), etc.

[0211] The three-arm star molecule or the four-arm star molecule can comprise a three-arm branched PEG or a four-arm branched PEG.

[0212] The dendrimer can comprise PAMAM, whose general formula is shown as follows:

[0213] wherein n3is any integer from 1 to 10; n4is any integer from 0 to 10; R2is n5is any integer from 0 to 10, and R1is any substituted alkyl or cycloalkyl.

[0214] The end of the forked structure can have the first functional group or the second functional group described above, for forming the first linker with the end of the mRNA structure.

[0215] In some embodiments, the connecting structure comprises a first linker, a forked structure, and a second linker, wherein, for each of the forks, the first linker is connected to the forked structure via the second linker.

[0216] In some embodiments, wherein the second linker is a PolyA sequence, wherein: the 3’ end of the second linker is connected to the forked structure; or the 5’ end of the second linker is connected to the first linker; or the 5’ end of the second linker is connected to the forked structure; or, the 3’ end of the second linker is connected to the first linker.

[0217] In some embodiments, wherein the second linker is a PolyA sequence, the second linker comprises modified nucleotides, for example, the nucleotides of the second linker are linked by phosphorothioate.

[0218] Molecular structure

[0219] In some embodiments, the present application provides a multi-mRNA having a structure as shown in Formula (1):

[0220] C1— U 11 O1— U 12 A1— L— A2— U 22 O2— U 21 C2(I), wherein,

[0221] C1 is a cap region of a first mRNA structure, O1 is an ORF of the first mRNA structure, A1 is a PolyA region of the first mRNA structure, U 11 is a bond or a nucleotide, U 12 is a bond or a nucleotide; L is a linker structure; C2 is a cap region of a second mRNA structure, O2 is an ORF of the second mRNA structure, A2 is a PolyA region of the second mRNA structure, U 21 is a bond or a nucleotide, U 22 is a bond or a nucleotide.

[0222] In some embodiments, L is a first linker.

[0223] In some embodiments, C1 to A1 are 5’-3’ connected, and A2-C2 are 3’-5’ connected.

[0224] In some embodiments, the 3’ end of C1 is connected to the 5’ end of U 11 .

[0225] In some embodiments, the 3’ end of U 11 is connected to the 5’ end of O1.

[0226] In some embodiments, the 3’ end of O1 is connected to the 5’ end of U 12 .

[0227] In some embodiments, wherein the 3’ end of U 12 is connected to the 5’ end of A1.

[0228] In some embodiments, wherein L is connected to the 3’ end of A1.

[0229] In some embodiments, wherein L is connected to the 3’ end of A2.

[0230] In some embodiments, the 5' end of U is connected to the 3' end of A2 22 .

[0231] In some embodiments, the 5' end of U is connected to the 3' end of O2 22 .

[0232] In some embodiments, the 5' end of O2 is connected to the 3' end of O 21 .

[0233] In some embodiments, the 5' end of U is connected to the 3' end of C2 21 .

[0234] In some embodiments, U is a 5' untranslated region (UTR) of the first mRNA structure 11 .

[0235] In some embodiments, U is a 3' UTR of the first mRNA structure 12 .

[0236] In some embodiments, U is a 3' UTR of the second mRNA structure 22 .

[0237] In some embodiments, U is a 5' UTR of the second mRNA structure 21 .

[0238] In some embodiments, the multimeric mRNA provided herein has the following structure

[0239] wherein MRNA represents a mRNA structure, A' is H or one or more oligonucleotides linked to L; and n is an integer selected from 2-100. In some embodiments, n can be an integer selected from 2-50. In some embodiments, n can be an integer selected from 2-10. In some embodiments, n can be 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0240] The MRNA can be C-U1-O-U2-A-. C is a cap region of a mRNA structure, O is an ORF of a mRNA structure, A is a PolyA region of a mRNA structure, U1 is a bond or a nucleotide, U2 is a bond or a nucleotide; L is a linking structure; A' is H or one or more oligonucleotides linked to L; and n is an integer selected from 2-100. In some embodiments, the repeating unit C-U1-O-U2-A- only represents multiple mRNA structures, and does not mean that the n mRNA structures are exactly the same. For example, the O of the n mRNA structures can not be exactly the same, the C of the n mRNA structures can not be exactly the same, the C of the n mRNA structures can not be exactly the same.

[0241] In some embodiments, the application provides a multimeric mRNA having a structure according to Formula (II):

[0242] wherein

[0243] C is a cap region of the mRNA structure, O is an ORF of the mRNA structure, A is a PolyA region of the mRNA structure, U1 is a bond or a nucleotide, U2 is a bond or a nucleotide; L is a linking structure; A’ is H, or one or more oligonucleotides linked to L; and n is an integer selected from 2-100. In some embodiments, n can be an integer from 2-50. In some embodiments, n can be an integer from 2-10. In some embodiments, n can be 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0244] The repeating unit C-U1-O-U2-A merely represents multiple mRNA structures, and does not mean that the n mRNA structures are exactly the same. For example, the O of the n mRNA structures can not be exactly the same, the C of the n mRNA structures can not be exactly the same, and the C of the n mRNA structures can not be exactly the same.

[0245] In some embodiments, wherein L comprises a bifurcated structure, a first linker and a second linker.

[0246] In some embodiments, wherein the structure is according to Formula (III) or (IV):

[0247] wherein D is a bifurcated structure, L1 is a first linker, and L2 is a second linker.

[0248] In some embodiments, wherein C to A is 5’-3’ connected.

[0249] In some embodiments, wherein the 3’ end of C is connected to the 5’ end of U1.

[0250] In some embodiments, wherein the 3’ end of U1 is connected to the 5’ end of O.

[0251] In some embodiments, wherein the 3’ end of O is connected to the 5’ end of U2.

[0252] In some embodiments, wherein the 3’ end of U2 is connected to the 5’ end of A.

[0253] In some embodiments, wherein L1 is connected to the 3’ end of A.

[0254] In some embodiments, wherein L2 is a PolyA sequence, wherein: the 3’ end of L2 is connected to the bifurcation structure; or the 5’ end of L2 is connected to the first linker; or, the 5’ end of L2 is connected to the bifurcation structure; or, the 3’ end of L2 is connected to the first linker. n is an integer selected from 2-100. In some embodiments, n can be an integer from 2-50. In some embodiments, n can be an integer from 2-10. In some embodiments, n can be 2, 3, 4, 5, 6, 7, 8, 9, or 10. The repeat unit C-U1-O-U2-A- only represents multiple mRNA structures, and does not mean that the n mRNA structures are exactly the same. For example, the O of the n mRNA structures can not be exactly the same, the C of the n mRNA structures can not be exactly the same, the C of the n mRNA structures can not be exactly the same.

[0255] In some embodiments, the poly-mRNA comprises modified nucleotides, which comprises one or more of base-modified nucleotides, sugar-modified nucleotides, or phosphate-modified nucleotides.

[0256] The UTR, ORF, PolyA region, or cap region of the poly-mRNA can all comprise modified nucleotides. The second linker can also comprise modified nucleotides when the second linker is a polyA sequence.

[0257] For example, wherein the base modification is selected from one or more of the following group:

[0258] Xanthine, Allylaminouracil, Allylaminothymidine, Hypoxanthine, Dioxetidine, Dioxycytidine, Dioxoguanine, Dioxouracil, 6-Chloropurine nucleoside, N6-Methyladenine, Methylpseudouracil, 2-Thiocytosine, 2-Thiouracil, 5-Methyluracil, 4-Thiothymidine, 4-Thiouracil, 5,6-Dihydro-5-methyluracil, 5,6-Dihydrouracil, 5-[(3-indolyl)propionamido-N-allyl]uracil, 5-Aminoallylcytosine, 5-Aminoallyluracil, 5-Bromouracil, 5-Bromocytosine, 5-Carboxycytosine, 5-Carboxycytosine, 5-Carboxyuracil, 5-Fluorouracil, 5-Formylcytosine, 5-Formyluracil, 5-Hydroxycytosine, 5-Hydroxymethylcytosine, 5-Hydroxymethyluracil, 5-Hydroxyuracil, 5-Iodocytosine, 5-Iodouracil, 5-Methoxycytosine, 5-Methoxyuracil, 5-Methylcytosine, 5-Methyluracil, 5-Propynylaminocytosine, 5-Propynylaminouracil, 5-Propynylcytosine, 5-Propynyluracil, 6-Azacytosine, 6-Azauracil, 6-Chloropurine, 6-Thioguanine, 7-Deazanucleoside, 7-Deazaguanine, 7-Deaza-7-propylaminoadenine, 7-Deaza-7-propynylaminoadenine, 8-Azadenine, 8-Azidoadenine, 8-Chloroadenine, 8-Oxoadenine, 8-Oxoguanine, 3-5-Propynylaminocytosine, 3-6-Propynylaminouracil, Cyano 3-aminoallylcytosine, Cyano 3-aminoallyluracil, Cyano 5-6-propynylaminocytosine, Cyano 5-6-propynylaminouracil, Cyano 5-aminoallylcytosine, Cyano 5-aminoallyluracil, Cyano 7-aminoallyluracil, Dabcyl-5-3-aminoallyluracil, Desthiobiotin-16-aminoallyluracil, Desthiobiotin-6-aminoallylcytosine, Isoguanine, N1-Ethylpseudouracil, N1-Methoxymethylpseudouracil, N1-Methyladenine, N1-Methylpseudouracil, N1-Propylpseudouracil, N2-Methylguanine, N4-Biotin-OBEA-cytosine, N4-Methylcytosine, N6-Methyladenine, Pseudoisocytosine, Pseudouracil, Thienocytosine, Thienoguanine, Thienouracil, Xanthine, 3-Deazanucleoside, 2,6-Diaminoadenine, 2,6-diaminoguanine, 5-formamidyluracil, 5-ethynyluracil, N6-isopentenyladenine (i6A), 2-methylthio-N6-isopentenyladenine (ms2i6A), 2-methylthio-N6-methyladenine (ms2m6A), N6-(cis-hydroxyisopentenyl)adenine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenine (ms2io6A), N6-glycinamidoformyladenine (g6A), N6-threoninamidoformyladenine (t6A), 2-methylthio-N6-threoninamidoformyladenine (ms2t6A), N6-methyl-N6-threoninamidoformyladenine (m6t6A), N6-hydroxynalanylamidoformyladenine (hn6A), 2-methylthio-N6-hydroxynalanylamidoformyladenine (ms2hn6A), N6,N6-dimethyladenine (m62A), and N6-acetyladenine (ac6A).

[0259] For example, wherein the modification of the sugar is selected from one or several of the following group:

[0260] 2'-thioribose, 2',3'-dideoxyribose, 2'-amino-2'-deoxyribose, 2'-deoxyribose, 2'-azido-2'-deoxyribose, 2'-fluoro-2'-deoxyribose, 2'-O-methylribose, 2'-O-methyldideoxyribose, 3'-amino-2',3'-dideoxyribose, 3'-azido-2',3'-dideoxyribose, 3'-deoxyribose, 3'-O-(2-nitrobenzyl)-2'-deoxyribose, 3'-O-methylribose, 5'-amino-ribose, 5'-thioribose, 5-nitro-indolyl-2'-deoxyribose, 5'-biotinylribose, 2'-O,4'-C-methylene-bridged ribose, 2'-O,4'-C-amino-bridged ribose, and 2'-O,4'-C-thio-bridged ribose.

[0261] For example, wherein the modification of the phosphate is selected from one or several of the following group: phosphorothioate (PS), 5'-O-methylphosphonate, 3'-O-methylphosphonate, 5'-hydroxylphosphonate, hydroxylphosphonate, selenophosphate, phosphonate, carbophosphonate, methylphosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidine ring, triazole ring, boronophosphate (BP), methylphosphonate, and guanidinopropylphosphoramidate.

[0262] In some embodiments, the cap region comprises a cap structure having a structure represented by the following general formula:

[0263] wherein:

[0264] R3 is selected from guanine, adenine, cytosine, uracil, guanine analog, adenine analog, cytosine analog, uracil analog; np is an integer from 1 to 10; wherein np can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; in some embodiments np is 3;

[0265] R4 is (N1p) x N2, wherein N1 and N2 are ribonucleosides, and N1 and N2 are the same or different;

[0266] p is independently at each occurrence a phospho group, phosphorothioate, phosphorodithioate, alkylphosphonate, arylphosphonate, or N-phosphoramidate linkage, preferably the modification of the phospho is selected from one or several of the following group: phosphorothioate (PS), phosphorothioate, 5'-0-methylphosphonate, 3'-0-methylphosphonate, 5'-hydroxyphosphonate, hydroxyphosphonate, seleno-phosphoester, phosphonate, carbonophosphonate, methylphosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidine ring, triazole ring, boronophosphate (BP), methylphosphonate, and guanidinopropyl phosphoramidate; X is an integer from 0 to 8, wherein if X > 2, the ribonucleosides N1 in (N1p)x are the same or different from each other; R1 and R2 groups are independently selected from O-alkyl, halogen, acetylamino (AcNH), hydrogen, or hydroxyl.

[0267] In some embodiments, the cap region comprises a cap structure having a structure shown in the general formula:

[0268] wherein:

[0269] R3 is selected from guanine, adenine, cytosine, uracil, guanine analog, adenine analog, cytosine analog, uracil analog;

[0270] R4 is (N1p) x N2, wherein N1 and N2 are ribonucleosides, and N1 and N2 are the same or different;

[0271] p is independently at each occurrence a phospho group, phosphorothioate, phosphorodithioate, alkylphosphonate, arylphosphonate, or N-phosphoramidate linkage;

[0272] x is an integer from 0 to 8, wherein if x > 2, the ribonucleosides N1 in (N1-p)x are the same or different from each other;

[0273] R1 and R2 groups are independently selected from O-alkyl, halogen, acetylamino (AcNH), hydrogen, or hydroxyl.

[0274] In some embodiments, wherein the sugars in N1and N2are independently selected for each position from ribose and deoxyribose, and can contain modifications including 2'-0-alkyl, 2'-0-methoxyethyl, 2'-0-allyl, 2'-0-alkylamine, 2'-fluororibose, and 2'-deoxyribose;

[0275] and / or the bases in N1and N2are independently selected for each position from adenine, uracil, guanine, or cytosine, or an analog of adenine, uracil, guanine, or cytosine, and the nucleotide modified bases can be selected from xanthine, allylaminouracil, allylaminothymidine, hypoxanthine, dioxuracil, dioxycytosine, dioxoguanine, dioxouracil, 6-chloropurine nucleoside, N6-methyladenine, methylpseudo-uracil, 2-thiocytosine, 2-thiouracil, 5-methyluracil, 4-thiothymidine, 4-thiouracil, 5,6-dihydro-5-methyluracil, 5,6-dihydrouracil, 5-[(3-indolyl)propionamido-N-allyl]uracil, 5-aminoallylcytosine, 5-aminoallyluracil, 5-bromouracil, 5-bromocytosine, 5-carboxycytosine, 5-carboxycytosine, 5-carboxyuracil, 5-fluorouracil, 5-formylcytosine, 5-formyluracil, 5-hydroxycytosine, 5-hydroxymethylcytosine, 5-hydroxymethyluracil, 5-hydroxyuracil, 5-iodocytosine, 5-iodouracil, 5-methoxycytosine, 5-methoxyuracil, 5-methylcytosine, 5-methyluracil, 5-propynylaminocytosine, 5-propynylamino-uracil, 5-propynylcytosine, 5-propynyluracil, 6-azacytosine, 6-azauracil, 6-chloropurine, 6-thioguanine, 7-deazanucleoside, 7-deazaguanine, 7-deaza-7-propylamino nucleoside, 7-deaza-7-propynylamino nucleoside, 8-azanucleoside, 8-azidoadenine, 8-chloroadenine, 8-oxoadenine, 8-oxoguanine, biotin-16-7-deaza-7-propynylaminoguanine, biotin-16-aminoallylcytosine, biotin-16-aminoallyluracil, 3-5-propynylaminocytosine, 3-6-propynylamino-uracil, cyano 3-aminoallylcytosine, cyano 3-aminoallyluracil, cyano 5-6-propynylaminocytosine, cyano 5-6-propynylamino-uracil, cyano 5-aminoallylcytosine, cyano 5-aminoallyluracil, cyano 7-aminoallyluracil, Dabcyl-5-3-aminoallyluracil, desulfo-biotin-16-aminoallyluracil, desulfo-biotin-6-aminoallylcytosine, isoguanine, N 1 -methoxyethylpseudo-uracil, N 1 -methoxymethylpseudo-uracil, N 1 -methyladenine, N1 - propyl pseudouracil, N2-methylguanine, N 4 - biotin-OBEA-cytosine, N4-methylcytosine, N 6 - methyladenine, 06-methylguanine, pseudoisocytosine, pseudouracil, thienocytosine, thienoguanine, thienouracil, xanthine, 3-deazadenine, 2,6-diaminoadenine, 2,6-diaminoguanine, 5- formamidouracil, 5-ethynyluracil, N 6 - isopentenyladenine (i6A), 2-methylthio-N 6 - isopentenyladenine (ms2i6A), 2-methylthio-N 6 - methyladenine (ms2m6A), N 6 - (cis-hydroxyisopentenyl)adenine (io6A), 2-methylthio-N 6 - (cis-hydroxyisopentenyl)adenine (ms2io6A), N 6 - glycylcarbamoylad enine (g6A), N 6 - threonylcarbamoylad enine (t6A), 2-methylthio-N 6 - threonylcarbamoylad enine (ms2t6A), N 6 - methyl-N 6 - threonylcarbamoylad enine (m6t6A), N 6 - hydroxyvalylcarbamoylad enine (hn6A), 2-methylthio-N 6 - hydroxyvalylcarbamoylad enine (ms2hn6A), N 6 ,N 6 - dimethyladenine (m62A) and N 6 - acetyl adenine (ac6A).

[0276] In some embodiments, the cap structure analog is a di-nucleotide cap analog or a tri-nucleotide cap analog.

[0277] In some embodiments, the cap structure analog has the following general formula:

[0278] R2, R5, and R6 groups are independently selected from O-alkyl (O-methyl), halogen, acetyl amino (AcNH), hydrogen, or hydroxyl.

[0279] In some embodiments, R2 is hydroxyl.

[0280] In some embodiments, R5 is hydroxyl or methoxyl.

[0281] In some embodiments, R6 is hydroxyl or methoxyl.

[0282] X1and X2are bases, and X1is the same or different than X2. Wherein X1and / or X2is cytosine, guanine or cytosine, uracil. p is independently at each position a phospho group, phosphorothioate, phosphorodithioate, alkylphosphonate, arylphosphonate or N-phosphoramidate linkage, preferably the modification of the phospho is selected from one or several of the following group: phosphorothioate (PS), 5'-0-methylphosphonate, 3'-0-methylphosphonate, 5'-hydroxylphosphonate, hydroxylphosphonate, seleno-phosphonate, phosphonate, carbophosphonate, methylphosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidine ring, triazole ring, boronophosphate (BP), methylphosphonate and guanylpropylphosphoramidate.

[0283] When P is a phospho group, in some embodiments, the cap structure analog has the following general formula:

[0284] R2, R5and R6groups are independently selected from O-alkyl (O-methyl), halogen, acetylamino (AcNH), hydrogen or hydroxyl.

[0285] In some embodiments, R2is hydroxyl.

[0286] In some embodiments, R5is hydroxyl or methoxyl.

[0287] In some embodiments, R6is hydroxyl or methoxyl.

[0288] X1and X2are bases, and X1is the same or different than X2. Wherein X1and / or X2is cytosine, guanine or cytosine, uracil. p is independently at each position a phospho group, phosphorothioate, phosphorodithioate, alkylphosphonate, arylphosphonate or N-phosphoramidate linkage, preferably the modification of the phospho is selected from one or several of the following group: phosphorothioate (PS), 5'-0-methylphosphonate, 3'-0-methylphosphonate, 5'-hydroxylphosphonate, hydroxylphosphonate, seleno-phosphonate, phosphonate, carbophosphonate, methylphosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidine ring, triazole ring, boronophosphate (BP), methylphosphonate and guanylpropylphosphoramidate.

[0289] wherein the cap structure analog is from one of the following:

[0290] Pharmaceutical compositions and uses

[0291] In another aspect, the present application provides a delivery reagent comprising the aforementioned polymeric mRNA, wherein the delivery reagent comprises a lipid, a peptide, a protein, an antibody, a carbohydrate, a nanoparticle or a microparticle.

[0292] In some embodiments, wherein the nanoparticle or microparticle is a lipid nanoparticle or lipid microparticle, a polymer nanoparticle or polymer microparticle, a protein nanoparticle or protein microparticle, or a solid nanoparticle or solid microparticle.

[0293] In another aspect, the present application provides a cell comprising the aforementioned multi-mRNA.

[0294] In another aspect, the present application provides a pharmaceutical composition comprising the aforementioned multi-mRNA, the aforementioned delivery reagent or the aforementioned cell, and optionally a pharmaceutically acceptable carrier.

[0295] In another aspect, the present application provides the use of the aforementioned multi-mRNA, or the aforementioned delivery reagent or the aforementioned cell, or the aforementioned pharmaceutical composition, for treating and / or preventing a disease.

[0296] In another aspect, the present application provides the use of the aforementioned multi-mRNA, or the aforementioned delivery reagent or the aforementioned cell, or the aforementioned pharmaceutical composition, for the preparation of a medicament for treating and / or preventing a disease.

[0297] In another aspect, the present application provides a method comprising introducing the aforementioned multi-mRNA, or the aforementioned delivery reagent into a cell

[0298] In another aspect, the present application provides a method comprising administering to a subject in need thereof an effective amount of the aforementioned multi-mRNA, or the aforementioned delivery reagent or the aforementioned cell, or the aforementioned pharmaceutical composition.

[0299] In another aspect, the present application provides a method for treating and / or preventing a disease, comprising administering to a subject in need thereof an effective amount of the aforementioned multi-mRNA, or the aforementioned delivery reagent or the aforementioned cell, or the aforementioned pharmaceutical composition.

[0300] Embodiments

[0301] Example 1 Preparation of bivalent mRNA (20A) and its expression in cells

[0302] To construct bivalent mRNA, this embodiment designs a method of connecting the PolyA tail of mRNA by click chemistry, and the two ends are complete mRNA with cap structure. The mRNA is spliced by enzymatic ligation, the 3' PolyA end of one mRNA carries N3 group, and the 3' PolyA end of the other mRNA carries DBCO group, and the two mRNAs are coupled by click chemistry. We choose Hibit mRNA to prepare bivalent mRNA, and verify its expression in cells.

[0303] Luciferase NanoBiT can be divided into two fragments, short fragment Hibit and long fragment Lgbit, when the two fragments exist separately, there is no luciferase activity, when the two fragments are mixed, they will automatically assemble into a complete luciferase, which produces fluorescence in the presence of substrate. We use this property to verify the capping and poly A tailing method we developed, synthesize Hibit mRNA, when Hibit mRNA can be normally translated, Hibit fragment can be obtained, in the presence of LgBiT protein and substrate, if chemiluminescence can be detected.

[0304] The Hibit mRNA sequence is: Cap-GGCCACCAUGGUGAGCGGCUGGCGGCUGUUCAAGAAGAUUAGCUGAAAAAAAAAAA AAAAAAAAAA (SEQ ID NO: 1). Split this sequence into two segments, and design the DNA sequences Oligo 6 and Oligo 7 paired with it as shown in Table 1. Chemically synthesize these sequences, and then prepare mRNA by enzymatic ligation.

[0305] Table 1 Oligonucleotide sequence information

[0306] * represents the connection of the phosphorothioate bond.

[0307] First, cap Oligo 1 by T4 RNA ligase 2. The specific experimental steps are as follows:

[0308] The feeding amount of the capping ligation system is 100 nmol, and the capping ligation system is as follows:

[0309] Table 2-1 Capping ligation system

[0310] After preparing the mixed solution according to the above system, it is placed in a constant temperature metal bath, incubated at 1000 r / min and 25°C for 1 h. After the reaction is completed, add 1 / 10 volume of DNase I and 10x buffer, and incubate at 37°C for 15 min. After termination, take 2ul and terminate the reaction with 2x RNA loading buffer, denature at 60°C for 5 min. Desalt the desalted product through a hydroxyl column, and purify it through reverse phase HPLC.

[0311] After reverse phase HPLC purification, desalt the 30 nmol Cap-Oligo 1 raw material through a hydroxyl column, and then use T4 polynucleotide kinase (T4PNK) to treat Cap-Oligo 1 to remove the 3' phosphate group. Then connect Cap-Oligo 1 PNK product and Oligo 2-5 by T4 RNA ligase 2. The experimental steps are as follows:

[0312] T4 polynucleotide kinase system as follows:

[0313] Table 2-2 PNK treatment reaction system

[0314] 0.1 nmol ligation system as follows:

[0315] Table 3 ligation system

[0316] After the mixed solution is prepared according to the above system, it is put into a constant temperature metal bath, and incubated at 25°C for 1 h. After the reaction is completed, 1 / 10 volume of DNase I and 10x buffer are added, and incubated at 37°C for 15 min. After the end, the reaction is terminated with 2x RNA loading buffer, and denatured at 60°C for 5 min. Four kinds of mRNA, Hibit mRNA (20A), Hibit mRNA PS modification PolyA ddC (20A), Hibit mRNA 3'N3 (20A), and Hibit mRNA 3'DBCO (20A) are obtained by purification.

[0317] Finally, Hibit mRNA 3'N3 (20A) and Hibit mRNA 3'DBCO (20A) are coupled by click chemistry in a TEAA system. The steps of the ligation experiment are as follows:

[0318] 0.1 nmol chemical coupling system as follows:

[0319] Table 4 chemical coupling

[0320] After the mixed solution is prepared according to the above system, it is put into a constant temperature metal bath, and incubated at 1400 r / min, 37°C for 20 h. After the end, the reaction is terminated with 2x RNA loading buffer, and denatured at 60°C for 5 min. Bi-valence Hibit mRNA (20A) is recovered by purification.

[0321] Hibit mRNA (20A), Hibit mRNA PS modification PolyA ddC (20A), Hibit mRNA 3'N3 (20A), Hibit mRNA 3'DBCO (20A), Bi-valence Hibit mRNA (20A) 5 kinds of mRNA were transfected into HEK293T and A549 cells, and their expression in cells was verified. The day before transfection, HEK293T and A549 cells were plated in 96-well plates. According to the number of cell plates required for the designed transfection days of the experiment, the corresponding number of cell plates was prepared when the cell density reached 50%-60%, and the transfection operation was performed. 30 ng mRNA was transfected per well of cells, two replicates were set for each group of data, and after transfection, the cells were placed in a constant temperature incubator at 37°C, 5% CO2. After the experimental time, the cell plates were taken out, the culture medium was aspirated, 150 uL of PBS was added for washing, and the operation was repeated twice, then 150 uL of HiBiT Lytic Detection System was added. HiBiT Lytic Detection System, and the fluorescence intensity was detected in a microplate reader. The higher the fluorescence intensity value, the higher the mRNA protein expression.

[0322] In the first detection experiment, Hibit mRNA (20A), Hibit mRNA PS modification PolyA ddC (20A), Hibit mRNA 3'N3 (20A), Hibit mRNA 3'DBCO (20A), Bi-valence Hibit mRNA (20A) 5 kinds of mRNA were transfected, and the fluorescence intensity was detected on days 1, 3 and 6.

[0323] The results are shown in Figure 3. The expression trends of the five different modified Hibit mRNAs in HEK293T and A549 cells are consistent. In HEK293T cells, the expression of Hibit mRNA 3’N3(20A) and Hibit mRNA 3’DBCO(20A) is less different, and the expression of the two is about 1.5 times that of Hibit mRNA(20A). The expression of Hibit mRNA PS modification PolyA ddC(20A) is about 3.5 times that of Hibit mRNA(20A) on the first day of transfection, and about 6 times that of Hibit mRNA(20A) on the third day, the sixth day of transfection. The expression of Bi-valence Hibit mRNA(20A) is about 2 times that of Hibit mRNA(20A) on the first day and the third day of transfection, and about 3.5 times that of Hibit mRNA(20A) on the sixth day of transfection.

[0324] In high immunogenic A459 cells, the expression of Hibit mRNA 3’N3(20A) and Hibit mRNA 3’DBCO(20A) is less different on the first day of transfection, the expression of Hibit mRNA PS modification PolyA ddC(20A) is about 3 times that of Hibit mRNA(20A), the expression of Bi-valence Hibit mRNA(20A) is about 2 times that of Hibit mRNA(20A), the expression of Hibit mRNA 3’DBCO(20A) is about 2 times that of Hibit mRNA(20A) on the third day and the sixth day of transfection, and the expression of Hibit mRNA PS modification PolyA ddC(20A) is about 4.5 times that of Hibit mRNA(20A).

[0325] The expression of Bi-valence Hibit mRNA(20A) is about 8 times that of Hibit mRNA(20A) on the third day of transfection, and the ratio decreases by 25% on the sixth day.

[0326] According to 1, 3, 6 days, 5 Hibit mRNA expression amount calculation area (AUC), the results are shown in Figure 4, in HEK293T cells, Hibit mRNA 3'N3 (20A) is similar to Hibit mRNA (20A), Hibit mRNA 3'DBCO (20A) is about 1.5 times of Hibit mRNA (20A), Bi-valence Hibit mRNA (20A) is about 2.5 times of Hibit mRNA (20A), Hibit mRNA PS modification PolyA ddC (20A) is about 5 times of Hibit mRNA (20A). In high immunogenic A459 cells, Hibit mRNA 3'N3 (20A) is similar to Hibit mRNA (20A), Hibit mRNA 3'DBCO (20A) is about 1.7 times of Hibit mRNA (20A), Bi-valence Hibit mRNA (20A) and Hibit mRNA PS modification PolyA ddC (20A) are about 4 times of Hibit mRNA (20A).

[0327] Example 2 Preparation of Bi-valence mRNA (30A) and its expression in cells

[0328] In order to further verify the advantages of bivalent mRNA, this embodiment increases the PolyA tail of Hibit to 30A, and adds 10 thio-modified at the end to prepare Bi-valence Hibit mRNA (30A).

[0329] The Hibit mRNA sequence is split into two segments, and the DNA sequence Oligo 6 and Oligo 7 paired therewith according to zigzag connection mode is designed as shown in Table 5. These sequences are chemically synthesized, and then mRNA is prepared by enzymatic ligation.

[0330] Table 5 Oligonucleotide sequence information

[0331] * represents the connection of thio-phosphodiester bond.

[0332] First, Oligo 1 is capped by T4 RNA ligase 2. The specific experimental steps are as follows:

[0333] The feeding amount of the capping ligation system is 100 nmol, and the capping ligation system is as follows:

[0334] Table 6 Capping ligation system

[0335] After the above system is prepared into a mixed solution, it is placed into a constant temperature metal bath, incubated at 1000 r / min and 25°C for 1 h. After the reaction is completed, 1 / 10 volume of DNase I and 10x buffer are added, and incubation is carried out at 37°C for 15 min. After termination, 2 ul is taken and terminated with 2x RNA loading buffer, and denaturation is carried out at 60°C for 5 min. Desalting is carried out through a hydroxyl column, and the desalted product is purified through reverse phase HPLC.

[0336] After reverse HPLC purification, 30 nmol of Cap-Oligo 1 raw material is obtained through desalting of a hydroxyl column, and then Cap-Oligo 1 is treated with T4 polynucleotide kinase (T4PNK) to remove the 3' phosphate group. Then, the Cap-Oligo 1 PNK product is respectively connected with Oligo2, Oligo7 and Oligo8 through T4 RNA ligase 2. The experimental steps are as follows:

[0337] The T4 polynucleotide kinase system is as follows:

[0338] Table 7 T4PNK treatment reaction system

[0339] The 0.1 nmol connection system is as follows:

[0340] Table 8 Connection system

[0341] After the above system is prepared into a mixed solution, it is placed into a constant temperature metal bath, incubated at 25°C for 1 h. After the reaction is completed, 1 / 10 volume of DNase I and 10x buffer are added, and incubation is carried out at 37°C for 15 min. After termination, 2 ul is taken and terminated with 2x RNA loading buffer, and denaturation is carried out at 60°C for 5 min. Four kinds of mRNA, Hibit mRNA (30A), Hibit mRNA PS modification PolyA (30A), Hibit mRNA PS modification-N3 (30A) and Hibit mRNA PS modification-DBCO (30A) are obtained through purification.

[0342] Finally, Hibit mRNA PS modification-N3 (30A) and Hibit mRNA PS modification-DBCO (30A) are coupled through click chemistry in a TEAA system. The experimental steps are as follows:

[0343] The 0.1 nmol chemical coupling system is as follows:

[0344] Table 9 Chemical coupling

[0345] After the mixed solution is prepared according to the above system, it is placed in a constant temperature metal bath, incubated at 1400 r / min and 37°C for 20 h. After termination with 2x RNA loading buffer, denaturation is performed at 60°C for 5 min. Bi-valence Hibit mRNA (30A) is recovered and purified.

[0346] Hibit mRNA (30A), Hibit mRNA PS modification PolyA (30A), and Bi-valence Hibit mRNA PS modification PolyA (30A) are transfected into HEK293T and A549 cells to verify their expression in cells. One day before transfection, HEK293T and A549 cells are plated in 96-well plates. According to the number of transfection days designed in the experiment, the corresponding number of cell well plates is prepared. When the cell density reaches 50%-60%, transfection can be performed. 30 ng of mRNA is transfected per well of cells, and two replicates are set for each data group. After transfection, the plates are placed in a constant temperature incubator at 37°C and 5% CO2. After incubation for the experimental time, the cell plates are removed, the culture medium is aspirated, and 150 uL of PBS is added for washing. After repeating the operation twice, 150 uL of HiBiT Lytic Detection System is added, and the plates are placed in a microplate reader to detect the fluorescence intensity. The higher the fluorescence intensity value, the higher the mRNA protein expression.

[0347] In the second detection experiment, Hibit mRNA (30A), Hibit mRNA PS modification PolyA (30A), and Bi-valence Hibit mRNA PS modification PolyA (30A) are transfected, and the fluorescence intensity is detected on days 1, 2, and 3. The results are shown in FIG. 5.

[0348] ​3 different modified Hibit mRNA expression trends in HEK293T and A549 cells are consistent, in HEK293T cells, on the first day of transfection, and on the third day, Hibit mRNA PS modification PolyA(30A) expression is about 2 times that of Hibit mRNA(30A), and Bi-valence Hibit mRNA PS modification PolyA(30A) expression is about 6.5 times that of Hibit mRNA(30A), on the second day, Hibit mRNA PS modification PolyA(30A) expression is about 3 times that of Hibit mRNA(30A), and Bi-valence Hibit mRNA PS modification PolyA(30A) expression is about 8 times that of Hibit mRNA(30A).

[0349] In highly immunogenic A459 cells, on the first day of transfection, and on the third day, Hibit mRNA PS modification PolyA(30A) expression is about 2.5 times that of Hibit mRNA(30A), on the second day, Hibit mRNA PS modification PolyA(30A) expression is about 3 times that of Hibit mRNA(30A), and Bi-valence Hibit mRNA PS modification PolyA(30A) expression is about 9.5 times that of Hibit mRNA(30A).

[0350] According to the expression amount of 1, 2, 3 days, 3 Hibit mRNA area (AUC) is calculated, the results are shown in Figure 6, in HEK293T cells, Hibit mRNA PS modification PolyA(30A) is about 2.5 times that of Hibit mRNA(30A). Bi-valence Hibit mRNA PS modification PolyA(30A) is about 7 times that of Hibit mRNA(30A).

[0351] In highly immunogenic A459 cells, Hibit mRNA PS modification PolyA(30A) is about 2.8 times that of Hibit mRNA(30A). Bi-valence Hibit mRNA PS modification PolyA(30A) is about 6 times that of Hibit mRNA(30A).

[0352] Preparation of trivalent and tetravalent mRNA (30A) and its expression in cells

[0353] To construct trivalent and tetravalent mRNA, we designed a way to conjugate mRNA by click chemistry at the 3’ end of the PolyA tail, and the 5’ end of the mRNA is intact with a cap structure. We spliced the mRNA by enzymatic ligation, the 3’ PolyA end of the mRNA has an N3 group, and the other sequence has three or four DBCO groups, and the two mRNAs are coupled by click chemistry. We chose the mRNA of Hibit to prepare trivalent and tetravalent mRNA, and verified its expression in cells. At the same time, mRNA without modification and mRNA with thio-modified PolyA tail were prepared as controls.

[0354] This sequence is split into two segments, and the DNA sequences Oligo 6 and Oligo 7 paired with it according to the zigzag connection method are designed as shown in Table 10. These sequences are chemically synthesized, and then mRNA is prepared by enzymatic ligation.

[0355] Table 10 Oligonucleotide sequence information

[0356] * represents the connection of a phosphorothioate bond. L is a monomer of a branched structure, the molecular formula of which is shown in Figure 7, which can divide the 5’ end of linear RNA into three branches, and we add DBCO groups (Oligo 9) on the three branches of the 5’ end, conjugate Hibit mRNA by Click it reaction, called trivalent mRNA (3-valence Hibit mRNA PS modification PolyA (30A)), and add DBCO groups (Oligo 10) on the three branches of the 5’ end and the 3’ main stem, conjugate Hibit mRNA by Click it reaction, called tetravalent mRNA (4-valence Hibit mRNA PS modification PolyA (30A))

[0357] The monomer structure of the branched structure is shown in Figure 8, and the structures of 3-valence Hibit mRNA PS modification PolyA (30A) and 4-valence Hibit mRNA PS modification PolyA (30A) are shown in Figure 9.

[0358] First, Oligo 1 is capped by T4 RNA ligase 2. The specific experimental steps are as follows:

[0359] The 100 nmol capping ligation system was prepared as follows:

[0360] Table 11 Capping ligation system

[0361] After the mixed solution was prepared according to the above system, it was placed in a constant temperature metal bath, incubated at 1000 r / min and 25°C for 1 h. After the reaction was completed, 1 / 10 volume of DNase I and 10x buffer were added, and incubated at 37°C for 15 min. After termination, 2ul was taken and terminated with 2x RNA loading buffer, and denatured at 60°C for 5 min. Finally, the desalted product was purified by reverse phase HPLC.

[0362] After reverse phase HPLC purification, 30 nmol of Cap-Oligo 1 raw material was obtained by desalting through a hydroxyl column, and then Cap-Oligo 1 was treated with T4 polynucleotide kinase (T4PNK) to remove the 3' phosphate group. Then, the Cap-Oligo 1 PNK product was connected to Oligo 8 by T4 RNA ligase 2. The experimental steps are as follows:

[0363] The T4 polynucleotide kinase system is as follows:

[0364] Table 12 T4PNK treatment reaction system

[0365] The 0.1 nmol ligation system is as follows:

[0366] Table 13 Ligation system

[0367] After the mixed solution was prepared according to the above system, it was placed in a constant temperature metal bath, incubated at 25°C for 1 h. After the reaction was completed, 1 / 10 volume of DNase I and 10x buffer were added, and incubated at 37°C for 15 min. After termination, 2ul was taken and terminated with 2x RNA loading buffer, and denatured at 60°C for 5 min.

[0368] Finally, the three mRNAs were coupled by click chemistry in a TEAA system. The experimental steps are as follows:

[0369] The 0.1 nmol chemical coupling system is as follows:

[0370] Table 14 Chemical coupling

[0371] After the mixed solution is prepared according to the above system, it is placed in a constant temperature metal bath, incubated at 1400 r / min and 37°C for 20 h. After termination, the reaction is terminated with 2x RNA loading buffer and denatured at 60°C for 5 min. The ligation product is recovered and purified to obtain three kinds of mRNA: Bi-valence Hibit mRNA PS modification PolyA(30A), 3-valence Hibit mRNA PS modification PolyA(30A) and 4-valence Hibit mRNA PS modification PolyA(30A).

[0372] Hibit mRNA(30A), Hibit mRNA PS modification PolyA(30A), 3-valence Hibit mRNA PS modification PolyA(30A) and 4-valence Hibit mRNA PS modification PolyA(30A) are transfected into HEK293T and A549 cells, and their expression in cells is verified. The day before transfection, HEK293T and A549 cells are plated in 96-well plates. According to the number of cell wells prepared according to the experimental design, when the cell density reaches 50%-60%, the transfection operation can be performed. 30 ng of mRNA is transfected per well of cells, and two replicates are set for each group of data. After transfection, the cells are placed in a constant temperature incubator at 37°C and 5% CO2. After the experimental time, the cell well plate is taken out, the culture medium is aspirated, 150 μL of PBS is added for washing, and the operation is repeated twice. Then, 100 μL of HiBiT Lytic Detection System is added, and the fluorescence intensity is detected in a microplate reader. The higher the fluorescence intensity value, the higher the mRNA protein expression. The fluorescence intensity is detected on the 1st and 2nd days.

[0373] ​4 different modified Hibit mRNA expression trends are consistent in HEK293T and A549 cells. In HEK293T cells, on the first day of transfection, the expression of Hibit mRNA PS modification PolyA(30A) is about 1.5 times that of Hibit mRNA(30A), the expression of Tri-valence Hibit mRNA PS modification PolyA(30A) Trembler 5*A is about 3.4 times that of Hibit mRNA(30A) on the first day, and the expression is further increased on the second day, which is 7.9 times that of unmodified mRNA. The expression of 4-valence Hibit mRNA PS modification PolyA(30A) Trembler 5*A is about 2.7 times that of Hibit mRNA(30A), and 3.8 times.

[0374] In highly immunogenic A459 cells, on the first day of transfection, from left to right, the expression of Hibit mRNA PS modification PolyA(30A) is about 1.8 times that of Hibit mRNA(30A). The expression of Tri-valence Hibit mRNA PS modification PolyA(30A) Trembler 5*A is about 1.9 times that of Hibit mRNA(30A). The expression of 4-valence Hibit mRNA PS modification PolyA(30A) Trembler 5*A is about 1.7 times that of Hibit mRNA(30A). On the second day, the expression of Tri-valence Hibit mRNA PS modification PolyA(30A) Trembler 5*A is further increased, which is 13 times that of Hibit mRNA(30A), and the expression of 4-valence Hibit mRNA PS modification PolyA(30A) Trembler 5*A is decreased, which is 8 times that of Hibit mRNA(30A). The expression and stability of tri-valence and quad-valence mRNA are much better than that of unmodified mRNA.

Claims

1. A polymeric mRNA comprising a plurality of mRNA structures, the mRNA structures comprising: a) a cap region at the 5' end; b) an open reading frame (ORF) encoding a polypeptide; wherein, the plurality of mRNA structures comprises a first mRNA structure and a second mRNA structure, the 3' end of the first mRNA structure is covalently linked to the second mRNA structure.

2. The polymeric mRNA of claim 1, wherein the 3' end of the first mRNA structure and the 3' end of the second mRNA structure are covalently linked.

3. The polymeric mRNA of any one of claims 1-2, wherein, the mRNA structure comprises a PolyA region.

4. The polymeric mRNA of claim 2, wherein, for the first mRNA structure and the second mRNA structure, the 3' end of the PolyA region of the first mRNA structure is covalently linked to a nucleotide of the second mRNA structure.

5. The polymeric mRNA of claim 4, wherein, for the first mRNA structure and the second mRNA structure, the 3' end of the PolyA region of the first mRNA structure and the 3' end of the PolyA region of the second mRNA structure are covalently linked.

6. The polymeric mRNA of any one of claims 1-5, wherein there is a 5' untranslated region (UTR) between the cap region and the open reading frame.

7. The polymeric mRNA of any one of claims 3-6, wherein there is a 3' untranslated region (UTR) between the PolyA region and the open reading frame.

8. The polymeric mRNA of any one of claims 3-7, wherein the 3' end of the cap region is linked to the 5' end of the ORF or 5' UTR.

9. The polymeric mRNA of any one of claims 3-8, wherein the 5' end of the PolyA region is linked to the 3' end of the ORF or 3' UTR.

10. The polymeric mRNA of any one of claims 3-9, wherein the PolyA region of the mRNA structure comprises modified adenosine nucleotides.

11. The polymeric mRNA of any one of claims 3-10, wherein the covalent linkage comprises a direct linkage or a linkage through a linking structure.

12. The polymeric mRNA of claim 11, wherein the linking structure is a first linker, the first linker is a group resulting from a coupling reaction.

13. The polymeric mRNA of any one of claims 1-12, wherein the linking structure comprises a first linker and a dendrimer, wherein for each of the mRNA structures, its 3' end is linked to the dendrimer directly or through its corresponding first linker, forming a branch.

14. The polymeric mRNA of claim 13, the linkage structure comprising a first linker, a bifurcation structure, and a second linker, wherein, for each of the dendrimers, the first linker is linked to the dendrimer through a second linker.

15. The polymeric mRNA of claim 14, wherein the second linker is a PolyA sequence, wherein: a) the 3' end of the second linker is linked to the dendrimer; or b) the 5' end of the second linker is linked to the first linker; or c) the 5' end of the second linker is linked to the dendrimer; or d) the 5' end of the second linker is linked to the first linker and the 3' end of the second linker is linked to the dendrimer. ​ d) the 3' end of the second linker is connected to the first linker.

16. The polymeric mRNA of any one of claims 14-15, wherein there is one PolyA sequence connected to the bifurcation structure only wherein, a) the 3' end of the PolyA sequence connected to the bifurcation structure only is connected to the bifurcation structure; or b) the 5' end of the PolyA sequence connected to the bifurcation structure only is connected to the bifurcation structure.

17. The polymeric nucleotide of any one of claims 14-16, wherein the PolyA sequence comprises modified adenosine nucleotides.

18. The polymeric mRNA of any one of claims 1-17, having a structure according to Formula (1): wherein, C1-U 11 -O1-U 12 -A1-L-A2-U 22 -O2-U 21 -C2 (I), wherein, C1 is a cap region of a first mRNA structure, O1 is an ORF of the first mRNA structure, A1 is a Poly A region of the first mRNA structure, U 11 is a bond or is a nucleotide, U 12 is a bond or is a nucleotide; L is a linking structure; C2 is a cap region of a second mRNA structure, 02 is an ORF of the second mRNA structure, A2 is a Poly A region of the second mRNA structure, U 21 is a bond or is a nucleotide, U 22 is a bond or is a nucleotide.

19. The polymeric mRNA of claim 18, wherein L is a first linker.

20. The polymeric mRNA of any one of claims 18-19, wherein C1 to A1 are 5'-3' connected, and A2 to C2 are 3'-5' connected.

21. The polymeric mRNA of any one of claims 18-20, wherein the 3' end of C1 is linked to the 5' end of U 11 .

22. The polymeric mRNA of any one of claims 18-21, wherein the 3' end of U 11 is attached to the 5' end of O1.

23. The polymeric mRNA of any one of claims 18-22, wherein the 3' end of O1 is attached to the 5' end of U 12 .

24. The polymeric mRNA of any one of claims 18-23, wherein the 3' end of U 12 is attached to the 5' end of A1.

25. The polymeric mRNA of any one of claims 18-24, wherein L is connected to the 3' end of A1.

26. The polymeric mRNA of any one of claims 18-25, wherein L is connected to the 3' end of A2.

27. The polymeric mRNA of any one of claims 18-26, wherein the 5' end of A2 is linked to the 3' end of U 22 .

28. The polymeric mRNA of any one of claims 18-27, wherein U 22 is attached to the 3' end of 02 at the 5' end.

29. The polymeric mRNA of any one of claims 18-28, wherein the 5' end of O2 is linked to the 3' end of O 21 .

30. The polymeric mRNA of any one of claims 18-29, wherein U 21 is attached to the 3' end of C2 at the 5' end.

31. The polymeric mRNA of any one of claims 18-30, wherein U 11 is a 5' untranslated region (UTR) of the first mRNA structure.

32. The polymeric mRNA of any one of claims 18-31, wherein U 12 is the 3' UTR of the first mRNA structure.

33. The polymeric mRNA of any one of claims 18-32, wherein U 22 is a 3' UTR of a second mRNA structure.

34. The polymeric mRNA of any one of claims 18-33, wherein U 21 is a 5' UTR of the second mRNA structure.

35. The polymeric mRNA of any one of claims 1-17, wherein the has a structure according to Formula (II): C is a cap region of an mRNA structure, O is an ORF of an mRNA structure, A is a PolyA region of an mRNA structure, U1 is a bond or a nucleotide, U2 is a bond or a nucleotide; L is a linking structure; A' is H, or one or more oligonucleotides linked to L; n is an integer selected from 2-100.

36. The polymeric mRNA of claim 35, wherein L comprises a bifurcation structure, a first linker, and a second linker.

37. The polymeric mRNA of any one of claims 35-36, wherein the has a structure according to Formula (III) or (IV): wherein D is a bifurcation structure, A' is H, or one or more oligonucleotides linked to D, L1 is a first linker, and L2 is a second linker.

38. The polymeric mRNA of any one of claims 35-37, wherein C to A are 5'-3' connected.

39. The polymeric mRNA of any one of claims 35-38, wherein the 3' end of C is connected to the 5' end of U1.

40. The polymeric mRNA of any one of claims 35-39, wherein the 3' end of U1 is connected to the 5' end of O.

41. The polymeric mRNA of any one of claims 35-40, wherein the 3' end of O is connected to the 5' end of U2.

42. The polymeric mRNA of any one of claims 35-41, wherein the 3' end of U2 is connected to the 5' end of A.

43. The polymeric mRNA of any one of claims 35-42, wherein L1 is connected to the 3' end of A.

44. The polymeric mRNA of any one of claims 35-43, wherein L2 is a PolyA sequence, wherein: a) the 3' end of L2 is connected to the bifurcation structure, or b) the 5' end of L2 is connected to the first linker c) the 5' end of L2 is connected to the bifurcation structure; or, d) the 3' end of L2 is connected to the first linker.

45. The multimeric mRNA of any one of claims 12-44, wherein the first linker comprises a group generated by a coupling reaction or a click chemistry reaction.

46. The multimeric mRNA of claim 45, wherein the first linker comprises a group: wherein Raand Rbare optional substituents.

47. The multimeric mRNA of claim 46, wherein Raand Rbform a ring with their adjacent atoms.

48. The polymeric mRNA of any one of claims 12-48, wherein the first linker is:

49. The multimeric mRNA of any one of claims 14-48, wherein the forked structure comprises a three-armed star molecule, a four-armed star molecule, a comb molecule, or a dendrimer.

50. The multimeric mRNA of claim 49, wherein the bifurcated structure is C((CH2) n3 O(CH2) n1 CH3) n2 , wherein, n1 is an integer selected from 1-10; n2 is an integer selected from 2-10; n3 is an integer selected from 1-10.

51. The polyadenylated mRNA of claim 50, wherein the bifurcation structure is:

52. The multimeric mRNA of any one of claims 1-51, wherein the modified nucleotides comprise a combination of one or more of base-modified nucleotides, sugar-modified nucleotides, or phosphate-modified nucleotides.

53. The polymeric mRNA of claim 52, wherein, The base modification is selected from one or more of the following group: Xanthine, Allylaminouracil, Allylaminothymidine, Hypoxanthine, Dioxetidine, Dioxycytidine, Dioxoguanine, Dioxouracil, 6-Chloropurine nucleoside, N6-Methyladenine, Methylpseudo-uracil, 2-Thiocytosine, 2-Thiouracil, 5-Methyluracil, 4-Thiothymidine, 4-Thiouracil, 5,6-Dihydro-5-methyluracil, 5,6-Dihydrouracil, 5-[(3-indolyl)propionamido-N-allyl]uracil, 5-Aminoallylcytosine, 5-Aminoallyluracil, 5-Bromouracil, 5-Bromocytosine, 5-Carboxycytosine, 5-Carboxyuracil, 5-Fluorouracil, 5-Formylcytosine, 5-Formyluracil, 5-Hydroxycytosine, 5-Hydroxymethylcytosine, 5-Hydroxymethyluracil, 5-Hydroxyuracil, 5-Iodocytosine, 5-Iodouracil, 5-Methoxycytosine, 5-Methoxyuracil, 5-Methylcytosine, 5-Methyluracil, 5-Propynylaminocytosine, 5-Propynylaminouracil, 5-Propynylcytosine, 5-Propynyluracil, 6-Azacytosine, 6-Azauracil, 6-Chloropurine, 6-Thioguanine, 7-Deazanucleoside, 7-Deazaguanine, 7-Deaza-7-propylaminoadenine, 7-Deaza-7-propynylaminoadenine, 8-Azadenine, 8-Azidoadenine, 8-Chloroadenine, 8-Oxoadenine, 8-Oxoguanine, Biotin-16-7-Deaza-7-propynylaminoguanine, Biotin-16-Aminoallylcytosine, Biotin-16-Aminoallyluracil, Cytosine, Guanine, Uracil,6-diaminoguanine, 5-formamidouracil, 5-ethynyluracil, N6-isopentenyladenine (i6A), 2-methylthio-N6-isopentenyladenine (ms2i6A), 2-methylthio-N6-methyladenine (ms2m6A), N6-(cis-hydroxyisopentenyl)adenine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenine (ms2io6A), N6-glycinamidoformyladenine (g6A), N6-threoninamidoformyladenine (t6A), 2-methylthio-N6-threoninamidoformyladenine (ms2t6A), N6-methyl-N6-threoninamidoformyladenine (m6t6A), N6-hydroxynalanylaminofor- myladenine (hn6A), 2-methylthio-N6-hydroxynalanylaminofor-myladenine (ms2hn6A), N6,N6-dimethyladenine (m62A), and N6-acetyladenine (ac6A).

54. The polymeric mRNA of claim 52, wherein, The sugar modification is selected from one or more of the following group: 2'-thioribose, 2',3'-dideoxyribose, 2'-amino-2'-deoxyribose, 2'-deoxyribose, 2'-azido-2'-deoxyribose, 2'-fluoro-2'-deoxyribose, 2'-O-methylribose, 2'-O-methyldideoxyribose, 3'-amino-2',3'-dideoxyribose, 3'-azido-2',3'-dideoxyribose, 3'-deoxyribose, 3'-O-(2-nitrobenzyl)-2'-deoxyribose, 3'-O-methylribose, 5'-aminoribose, 5'-thioribose, 5-nitro-indolyl-2'-deoxyribose, 5'-biotinylribose, 2'-O,4'-C-methylene-bridged ribose, 2'-O,4'-C-aminobridged ribose, and 2'-O,4'-C-thio bridged ribose.

55. The polymeric mRNA of claim 52, wherein, The phosphate modification is selected from one or more of the following group: phosphorothioate (PS), 5'-O-methylphosphonate, 3'-O-methylphosphonate, 5'-hydroxylphosphonate, hydroxylphosphonate, selenophosphonate, phosphonate, carbophosphonate, methylphosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidinium ring, triazole ring, boronophosphonate (BP), methylphosphonate, and guanidinopropyl phosphoramidate.

56. The polyadenylated mRNA of any one of claims 1-55, wherein the cap region comprises a cap structure having the following structure: wherein: R3 is selected from guanine, adenine, cytosine, uracil, guanine analog, adenine analog, cytosine analog, uracil analog; np is an integer from 1-10; R4 is (N1p) x N2, wherein N1 and N2 are ribonucleosides, and N1 is the same or different from N2; p is independently at each occurrence a phospho, phosphorothioate, phosphorodithioate, alkylphosphonate, arylphosphonate or N-phosphoramidate linkage, preferably the modification of the phosphate is selected from one or more of the group: phosphorothioate (PS), 5'-O-methylphosphonate, 3'-O-methylphosphonate, 5'-hydroxyl phosphonate, hydroxyl phosphonate, phosphoroselenoate, phosphorodithioate, phosphoramidate, boranophosphonate (BP), methylphosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidinium ring, triazole ring, methylphosphonate, and guanidinopropylphosphoramidate; X is an integer from 0-8, wherein if X > 2, the ribonucleosides N1in (N1p)x are the same or different from each other; R1and R2groups are independently selected from O-alkyl, halogen, acetylamino (AcNH), hydrogen, or hydroxyl.

57. The polymeric mRNA of claim 56, wherein the sugars in N1and N2are independently at each occurrence selected from ribose and deoxyribose, and can comprise modifications including 2'-O-alkyl, 2'-O-methoxyethyl, 2'-O-allyl, 2'-O-alkylamine, 2'-fluororibose, and 2'-deoxyribose. and / or the bases in N1and N2are independently selected for each position from adenine, uracil, guanine, or cytosine, or an analog of adenine, uracil, guanine, or cytosine, and the nucleotide modified base can be selected from the group consisting of xanthine, allylaminouracil, allylaminothymidine, hypoxanthine, dioxuracil, dioxycytosine, dioxoguanine, dioxouracil, 6-chloropurine nucleoside, N6-methyladenine, methylpseudo-uracil, 2-thiocytosine, 2-thiouracil, 5-methyluracil, 4-thiothymidine, 4-thiouracil, 5,6-dihydro-5-methyluracil, 5,6-dihydrouracil, 5-[(3-indolyl)propionamido-N-allyl]uracil, 5-aminoallylcytosine, 5-aminoallyluracil, 5-bromouracil, 5-bromocytosine, 5-carboxycytosine, 5-carboxycytosine, 5-carboxyuracil, 5-fluorouracil, 5-formylcytosine, 5-formyluracil, 5-hydroxycytosine, 5-hydroxymethylcytosine, 5-hydroxymethyluracil, 5-hydroxyuracil, 5-iodocytosine, 5-iodouracil, 5-methoxycytosine, 5-methoxyuracil, 5-methylcytosine, 5-methyluracil, 5-propynylaminocytosine, 5-propynylaminouracil, 5-propynylcytosine, 5-propynyluracil, 6-azacytosine, 6-azauracil, 6-chloropurine, 6-thioguanine, 7-deazanucleoside, 7-deazaguanine, 7-deaza-7-propylaminoadenine, 7-deaza-7-propynylaminoadenine, 8-azanucleoside, 8-azidoadenine, 8-chloroadenine, 8-oxoadenine, 8-oxoguanine, biotin-16-7-deaza-7-propynylaminoguanine, biotin-16-aminoallylcytosine, biotin-16-aminoallyluracil, 3-5-propynylaminocytosine, 3-6-propynylaminouracil, cyano 3-aminoallylcytosine, cyano 3-aminoallyluracil, cyano 5-6-propynylaminocytosine, cyano 5-6-propynylaminouracil, cyano 5-aminoallylcytosine, cyano 5-aminoallyluracil, cyano 7-aminoallyluracil, Dabcyl-5-3-aminoallyluracil, desulfo-biotin-16-aminoallyluracil, desulfo-biotin-6-aminoallylcytosine, isoguanine, N 1 -methoxy-methyl-pseudo-uracil, N 1 -methyl-adenine, N 1 -methyl-pseudo-uracil, N 1 -propyl-pseudo-uracil, N 4 -biotin-OBEA-cytosine, N 6 - methyladenine, 06-methylguanine, pseudoisocytosine, pseudouracil, thienocytosine, thienoguanine, thienouracil, xanthine, 3-deazan adenine, 2,6-diaminoadenine, 2,6-diaminoguanine, 5-formamidouracil, 5-ethynyluracil, N 6 - isopentenyladenine (i6A), 2-methylthio-N 6 - isopentenyladenine (ms2i6A), 2-methylthio-N 6 - methyladenine (ms2m6A), N 6 - (cis-hydroxyisopentenyl)adenine (io6A), 2-methylthio-N 6 - (cis-hydroxyisopentenyl)adenine (ms2io6A), N 6 - glycylcarbamoylad enine (g6A), N 6 - threonylcarbamoylad enine (t6A), 2-methylthio-N 6 - threonylcarbamoylad enine (ms2t6A), N 6 - methyl-N 6 - threonylcarbamoylad enine (m6t6A), N 6 - hydroxyvalylcarbamoylad enine (hn6A), 2-methylthio-N 6 - hydroxyvalylcarbamoylad enine (ms2hn6A), N 6 ,N 6 - dimethyladenine (m62A) and N 6 - acetyl adenine (ac6A).

58. The polymeric mRNA of claim 57, wherein the cap structure analog is a dinucleotide cap analog or a trinucleotide cap analog.

59. The polymeric mRNA of any one of claims 56-58, wherein the cap analog has the following general formula: wherein, R5and R6groups are independently selected from O-alkyl (O-methyl), halogen, a tag, hydrogen, or hydroxyl; preferably the modification of the phosphate is selected from one or more of the group: phosphorothioate (PS), 5'-O-methylphosphonate, 3'-O-methylphosphonate, 5'-hydroxyl phosphonate, hydroxyl phosphonate, phosphoroselenoate, phosphorodithioate, phosphoramidate, boranophosphonate (BP), methylphosphonate, phenylphosphonate, ethylphosphonate, H-phosphonate, guanidinium ring, triazole ring, methylphosphonate, and guanidinopropylphosphoramidate; X1and X2are bases, and X1and X2are the same or different.

60. The polymeric mRNA of claim 59, wherein X1and / or X2is cytosine, uracil, guanine, or adenine.

61. The polyadenylated mRNA of any one of claims 56-60, wherein the cap analog is from one of:

62. A delivery agent comprising the polymeric mRNA of claims 1-61, wherein the delivery agent comprises a lipid, a peptide, a protein, an antibody, a carbohydrate, a nanoparticle, or a microparticle.

63. The delivery agent of claim 62, wherein the nanoparticle or microparticle is a lipid nanoparticle or lipid microparticle, a polymeric nanoparticle or polymeric microparticle, a protein nanoparticle or protein microparticle, or a solid nanoparticle or solid microparticle.

64. A cell comprising the polymeric mRNA of claims 1-61.

65. A pharmaceutical composition comprising the polymeric mRNA of any one of claims 1-61, or the delivery agent of any one of claims 62-63 or the cell of claim 64, and optionally a pharmaceutically acceptable carrier.

66. The polymeric mRNA of any one of claims 1-61, or the delivery reagent of any one of claims 62-63 or the cell of claim 64, or the pharmaceutical composition of claim 65, for use in the treatment and / or prevention of a disease.

67. Use of the polymeric mRNA of any one of claims 1-61, or the delivery reagent of any one of claims 62-63 or the cell of claim 64, or the pharmaceutical composition of claim 65, for the manufacture of a medicament for the treatment and / or prevention of a disease.

68. A method comprising introducing into a cell the polymeric mRNA of any one of claims 1-61, or the delivery reagent of any one of claims 62-63.

69. A method comprising administering to a subject in need thereof an effective amount of the polymeric mRNA of any one of claims 1-61, or the delivery reagent of any one of claims 62-63, or the pharmaceutical composition of claim 65.

70. A method of treating and / or preventing a disease comprising administering to a subject in need thereof an effective amount of the polymeric mRNA of any one of claims 1-61, or the delivery reagent of any one of claims 62-63, or the pharmaceutical composition of claim 65.

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