Patent Description:
The present invention belongs to the field of biotechnology, and more specifically relates to an mRNA-mannose conjugate as defined in claim <NUM> and use thereof.

Lectin receptors are a class of glycoproteins, glycolipids or glycoconjugates distributed on the surface of the cell membrane, which can specifically recognize and bind to a part of glycosyls. The mannose receptor is the most important and highly efficient endocytic lectin receptor, whose function comprises eliminating endogenous molecules, promoting antigen presentation, and regulating cell activation and trafficking, and is also closely related to the immune escape and metastasis of tumors. It is mainly expressed in macrophages, dendritic cells and tumor cells, and can specifically recognize mannose glycosyl molecules. As the most promising targeted group, mannosyl has many advantages such as non-toxicity, non-immunogenicity, good biocompatibility and biodegradability, and can be widely used in glycosylation modification of nucleic acid drug delivery systems.

Liposome is a bilayer vesicle having a structure similar to a biological membrane, and nanoliposome is a liposome structure with a particle size of less than <NUM> which is the most classic nano-targeted drug delivery carrier. In the prior art, mannose has been coupled to nanoliposomes to improve their targeting.

However, in the process of preparing nanoliposomes, a large amount of organic solvents that are harmful to the human body are used, resulting in residual toxic organic solvents. Moreover, there are problems with these traditional nanoliposome preparation methods, such as low encapsulation rates, easy rupture of liposome membranes, poor stability, insufficient storage stability, difficulty in achieving large-scale production, and high cost. Therefore, if mannose can be directly coupled to nucleic acid drugs, nanoliposomes can be omitted. <NPL>; [<NPL>, report on a mannosylated mRNA nanoparticles, namely mannosylated lipopolyplexes or Man-LPR loaded with mRNA encoding a melanoma antigen.

In view of the above, the purpose of the present invention is to provide an mRNA modification method, by which the modification of mature mRNA molecules with mannose can be realized, so that the mRNAs can be directly and efficiently coupled with mannose, and thus the targeted delivery of the mRNAs can be carried out without the need for a carrier.

To achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:.

The invention provides an mRNA-mannose conjugate as defined in claim <NUM>. Preferred embodiments are characterized in claims <NUM> to <NUM>.

The present invention provides an mRNA-mannose conjugate comprising: an mRNA encoding a polypeptide with at least one <NUM>' cap structure, a <NUM>' UTR with at least one Kozak sequence, a <NUM>' UTR, and a polyA tail, and at least one mannose attached to the mRNA via an amine at a <NUM>' terminus of the mRNA, and wherein the mRNA comprises uridine, cytidine, adenosine, guanosine and/or chemically modified nucleosides thereof, wherein the amine is a secondary amine, and wherein the amine is formed on a last adenine of the polyA tail of the mRNA.

Preferably, the chemically modified nucleoside is one or more selected from the group consisting of pyridin-<NUM>-one ribonucleoside, <NUM>-aza-uridine, <NUM>-thio-<NUM>-aza-uridine, <NUM>- thiouridine, <NUM>- thiopseudouridine, <NUM>-thio-pseudouridine, <NUM>-hydroxyuridine, <NUM>- methyluridine, <NUM>-carboxymethyl-uridine, <NUM>-carboxymethyl-pseudouridine, <NUM>-propynyl - uridine, <NUM>-propynyl-pseudouridine, <NUM>-taurinomethyl uridine, <NUM>-taurinomethyl- pseudouridine, <NUM>-taurinomethyl-<NUM>-thio-uridine, <NUM>-taurinomethyl-<NUM>-thio-uridine, <NUM>-methyl- uridine, <NUM>-methyl pseudouridine, <NUM>-thio-<NUM>-methyl-pseudouridine, <NUM>-thio-<NUM>-methyl- pseudouridine, <NUM>-methyl-<NUM>-deaza-pseudouridine, <NUM>-thio-<NUM>-methyl-<NUM>-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, <NUM>-thio-dihydrouridine, <NUM>-thio-dihydropseudouridine, <NUM>-methoxyuridine, <NUM>-methoxy-<NUM>-thio-uridine, <NUM>-methoxy- pseudouridine, <NUM>-methoxy-<NUM>-thio-pseudouridine, <NUM>-aza-cytidine, pseudoisocytidine, <NUM>- methyl-cytidine, N4-acetylcytidine, <NUM>-formyl cytidine, N4-methyl cytidine, <NUM>-hydroxymethyl cytidine, <NUM>-methyl pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, <NUM>-thio-cytidine, <NUM>-thio-<NUM>-methyl-cytidine, <NUM>-thio-pseudoisocytidine, <NUM>- thio-<NUM>-methyl-pseudoisocytidine, <NUM>-thio-<NUM>-methyl-<NUM>-deaza-pseudoisocytidine, <NUM>-methyl- <NUM>-deaza-pseudoisocytidine, zebularine, <NUM>-aza-zebularine, <NUM>-methyl-zebularine, <NUM>-aza-<NUM>- thio-zebularine, <NUM>-thio-zebularine, <NUM>-methoxy-cytidine, <NUM>-methoxy-<NUM>-methyl-cytidine, <NUM>- methoxy-pseudoisocytidine, <NUM>-methoxy-<NUM>-methyl-pseudoisocytidine, <NUM>-aminopurine, <NUM>,<NUM>- diaminopurine, <NUM>-deaza-adenine, <NUM>-deaza-<NUM>-aza-adenine, <NUM>-deaza-<NUM>-aminopurine, <NUM>- deaza-<NUM>-aza-<NUM>-aminopurine, <NUM>-deaza-<NUM>,<NUM>-diaminopurine, <NUM>-deaza-<NUM>-aza-<NUM>,<NUM>- diaminopurine-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6- (cis-hydroxyisopentenyl)adenosine, <NUM>-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycylcarbamoyladenosine, N6-threonylcarbamoyladenosine, <NUM>-methylthio-N6- threonylcarbamoyladenosine, N6,N6-dimethyladenosine, <NUM>-methyladenine, <NUM>-methylthio- adenine, <NUM>-methoxy-adenosine, inosine, <NUM>-methyl-inosine, Y nucleoside, wybutosine, <NUM>- deaza-guanosine, <NUM>-deaza-<NUM>-aza-guanosine, <NUM>-thio-guanosine, <NUM>-thio-<NUM>-deaza-guanosine, <NUM>-thio-<NUM>-deaza-<NUM>-aza-guanosine, <NUM>-methyl-guanosine, <NUM>-thio-<NUM>-methyl-guanosine, <NUM>- methylinosine, <NUM>-methoxy-guanosine, <NUM>-methylguanosine, N2-methylguanosine, N2,N2- dimethylguanosine, <NUM>-oxo-guanosine, <NUM>-methyl-<NUM>-oxo-guanosine, <NUM>-methyl-<NUM>-thio- guanosine, N2-methyl-<NUM>-thio-guanosine and N2,N2-dimethyl-<NUM>-thio-guanosine.

Preferably, the at least one <NUM>' cap structure is one or more selected from the group consisting of CapO, Cap1, ARCA, inosine, N1-methyl-guanosine, <NUM>'-fluoro-guanosine, <NUM>- deaza-guanosine, <NUM>-oxo-guanosine, <NUM>-amino-guanosine, LNA-guanosine, <NUM>-azido- guanosine, <NUM>-methyl-guanosine-<NUM>'-triphosphate-<NUM>'-adenosine, guanosine-<NUM>'-triphosphate- <NUM>'-adenosine, <NUM>-methyl-guanosine-<NUM>'-triphosphate-<NUM>'-guanosine, guanosine-<NUM>'- triphosphate-<NUM>'-guanosine, and <NUM>-methyl-guanosine-<NUM>'-triphosphate-<NUM>'-<NUM>-methoxyadenine- guanosine.

The invention also provides a pharmaceutical composition as defined in claim <NUM>.

The invention also provides a method of preparing an mRNA-mannose conjugate as defined in claim <NUM>. Preferred embodiments are characterized in claims <NUM> to <NUM>.

The present invention also provides a kit for preparing an mRNA-mannose conjugate as defined in claim <NUM>.

The present invention also provides a mRNA-mannose conjugate for use as defined in claim <NUM>. Preferred embodiments are characterized in claims <NUM> to <NUM>.

The present invention will be further illustrated below in combination with examples and accompanying drawings.

The present invention presents a method of preparing an mRNA-mannose conjugate as defined in claim <NUM>, specifically, a preparation method of an mRNA composition, see <FIG>, comprising:.

The technical solution of the present invention allows modifying the <NUM>' end of an mRNA with an amino group, so that the mRNA is linked to a mannose through an isourea bond of the amino group to obtain an mRNA-mannose conjugate. The resulting mRNA- mannose conjugate is able to target specific cells without the need for a carrier, which simplifies the preparation process of mRNA drugs and greatly improves the level of development and utilization of mRNA drugs.

In the practice of the present invention, it will be appreciated that the raw materials obtained from or used by the preparation method comprise a polyadenylic acid of which the <NUM>' end is labeled with an amino group, an mRNA precursor, a mannose and a ligase, and these materials can be present in the form of a kit, so that a kit as defined in claim <NUM> is obtained comprising: an mRNA precursor, the mRNA precursor comprising a coding sequence encoding a specific protein; a polyadenylic acid, the <NUM>' end of the polyadenylic acid being labeled with an amino group; a mannose, the mannose and the amino group being covalently linked through an isourea bond; and a ligase, the ligase being used for linking the mRNA precursor and the polyadenylic acid.

Specifically, the chemical formula of the amino group is -NH<NUM>. Any primary amine with the general formula RNH<NUM> can be used, and secondary amines with the general formula R<NUM>NH can be generated.

Specifically, the length of the polyadenylic acid synthesized can be in a range of <NUM> to <NUM> bp. The polyadenylic acid is usually the poly A tail sequence of an mRNA. When the polyA tail sequence of an mRNA is too long, the polyadenylic acid can also be a part of the polyA tail sequence, and it will be appreciated that the length of the polyadenylic acid can be the length of any part of the polyA tail sequence. The mRNA precursor is a part of an mRNA that does not comprise the polyadenylic acid sequence, and the mRNA precursor and the polyadenylic acid constitute a complete mRNA sequence. The mRNA has at least one <NUM>' cap structure, a <NUM>' non-coding region (UTR) with at least one Kozak sequence, a <NUM>' non-coding region (UTR), and an open reading frame (ORF). in addition to the polyA tail, and the mRNA is composed of uridine, cytidine, adenosine, guanosine and/or chemically modified nucleosides thereof.

More specifically, the chemically modified nucleoside is one or more selected from the group consisting of pyridin-<NUM>-one ribonucleoside, <NUM>-aza-uridine, <NUM>-thio-<NUM>-aza-uridine, <NUM>- thiouridine, <NUM>- thiopseudouridine, <NUM>-thio-pseudouridine, <NUM>-hydroxyuridine, <NUM>- methyluridine, <NUM>-carboxymethyl-uridine, <NUM>-carboxymethyl-pseudouridine, <NUM>- propynyl- uridine, <NUM>-propynyl-pseudouridine, <NUM>-taurinomethyl uridine, <NUM>-taurinomethyl- pseudouridine, <NUM>-taurinomethyl-<NUM>-thio-uridine, <NUM>-taurinomethyl-<NUM>-thio-uridine, <NUM>-methyl- uridine, <NUM>-methyl pseudouridine, <NUM>-thio-<NUM>-methyl-pseudouridine, <NUM>-thio-<NUM>-methyl- pseudouridine, <NUM>-methyl-<NUM>-deaza-pseudouridine, <NUM>-thio-<NUM>-methyl-<NUM>-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, <NUM>-thio-dihydrouridine, <NUM>-thio- dihydropseudouridine, <NUM>-methoxyuridine, <NUM>-methoxy-<NUM>-thio-uridine, <NUM>-methoxy- pseudouridine, <NUM>-methoxy-<NUM>-thio-pseudouridine, <NUM>-aza-cytidine, pseudoisocytidine, <NUM>- methyl-cytidine, N4-acetylcytidine, <NUM>-formyl cytidine, N4-methyl cytidine, <NUM>-hydroxymethyl cytidine, <NUM>-methyl pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, <NUM>-thio-cytidine, <NUM>-thio-<NUM>-methyl-cytidine, <NUM>-thio-pseudoisocytidine, <NUM>- thio-<NUM>-methyl-pseudoisocytidine, <NUM>-thio-<NUM>-methyl-<NUM>-deaza-pseudoisocytidine, <NUM>-methyl- <NUM>-deaza-pseudoisocytidine, zebularine, <NUM>-aza - zebularine, <NUM>-methyl-zebularine, <NUM>-aza-<NUM>- thio-zebularine, <NUM>-thio-zebularine, <NUM>-methoxy-cytidine, <NUM>-methoxy-<NUM>-methyl-cytidine, <NUM>- methoxy-pseudoisocytidine, <NUM>-methoxy-<NUM>-methyl-pseudoisocytidine, <NUM>-aminopurine, <NUM>,<NUM>- diaminopurine, <NUM>-deaza-adenine, <NUM>-deaza-<NUM>-aza-adenine, <NUM>-deaza-<NUM>-aminopurine, <NUM>- deaza-<NUM>-aza-<NUM>-aminopurine, <NUM>-deaza-<NUM>,<NUM>-diaminopurine, <NUM>-deaza-<NUM>-aza-<NUM>,<NUM>- diaminopurine-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6- (cis-hydroxyisopentenyl)adenosine, <NUM>-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycylcarbamoyladenosine, N6-threonylcarbamoyladenosine, <NUM>-methylthio-N6- threonylcarbamoyladenosine, N6,N6-dimethyladenosine, <NUM>-methyladenine, <NUM>-methylthio- adenine, <NUM>-methoxy-adenosine, inosine, <NUM>-methyl-inosine, Y nucleoside, wybutosine, <NUM>- deaza-guanosine, <NUM>-deaza-<NUM>-aza-guanosine, <NUM>-thio-guanosine, <NUM>-thio-<NUM>-deaza-guanosine, <NUM>-thio-<NUM>-deaza-<NUM>-aza-guanosine, <NUM>-methyl-guanosine, <NUM>-thio-<NUM>-methyl-guanosine, <NUM>- methylinosine, <NUM>-methoxy-guanosine, <NUM>-methylguanosine, N2-methylguanosine, N2,N2- dimethylguanosine, <NUM>-oxo-guanosine, <NUM>-methyl-<NUM>-oxo-guanosine, <NUM>-methyl-<NUM>-thio- guanosine, N2-methyl-<NUM>-thio-guanosine and N2,N2-dimethyl-<NUM>-thio-guanosine.

More specifically, the at least one <NUM>' cap structure is one or more selected from the group consisting of CapO, Cap1, ARCA, inosine, N1-methyl-guanosine, <NUM>'-fluoro-guanosine, <NUM>- deaza-guanosine, <NUM>-oxo-guanosine, <NUM>-amino-guanosine, LNA-guanosine, <NUM>-azido- guanosine, <NUM>-methyl-guanosine-<NUM>'-triphosphate-<NUM>'-adenosine, guanosine-<NUM>'-triphosphate- <NUM>'-adenosine, <NUM>-methyl-guanosine-<NUM>'-triphosphate-<NUM>'-guanosine, guanosine-<NUM>'- triphosphate-<NUM>'-guanosine, and <NUM>-methyl-guanosine-<NUM>'-triphosphate-<NUM>'-<NUM>-methoxyadenine- guanosine. The <NUM>' cap structure can improve the stability of mRNAs.

Unlike plasmid DNAs and viral vectors, mRNAs are immediately translated once they enter the cytoplasm from the outside of cells, and do not need to be integrated into the host genome, avoiding the risk of insertion of gene mutations, moreover, exogenous mRNAs entering cells can be completely degraded by physiological metabolism. Furthermore, the production of mRNAs is simple and low-cost, which greatly shortens the cycle of new drug development and reduces its cost, making mRNA drugs have great advantages.

In one aspect, the problem of instability for mRNA drugs needs to be solved, and the activity of exogenous mRNAs can be prolonged by chemically modifying mRNAs and regulating the structural elements related to the translation and metabolism of mRNAs. Another important aspect is how to enable the modified mRNAs to enter specific cells and tissues of the body. The present invention obtains an mRNA drug by linking the <NUM>' end of an mRNA molecule to a mannose, and achieves tissue-specific delivery of the mRNA by interacting with the mannose receptor on macrophages, thereby increasing the potency of the mRNA drug molecule.

Optionally, referring to <FIG> is an approach of the preparation method of the mRNA composition in this example. The synthesized polyadenylic acid of which the <NUM>' end is labeled with an amino group and the mRNA precursor are linked by a T4 RNA ligase to form a mature mRNA, and then the resulting mature mRNA is linked with a mannose to obtain an mRNA-mannose conjugate.

Specifically, the ligase in the kit corresponding to the materials obtained from or used in the preparation method in the examples of the present invention comprises T4 RNA ligase.

In a preferred example, referring to <FIG> is another approach of the preparation method of the mRNA composition in this example. The synthesized polyadenylic acid of which the <NUM>' end is labeled with an amino group and the mRNA precursor are linked by the T4 DNA ligase to a splint DNA. The splint DNA contains a segment of base sequence at the <NUM>' end of the mRNA precursor and a segment of base sequence at the <NUM>' end of the polyadenylic acid, with the splint DNA acting as a bridge to link the mRNA precursor and the polyadenylic acid, and the link efficiency using the T4 DNA ligase is higher.

It will be appreciated that, in the example of the present invention, the ligase in the kit corresponding to the materials obtained from or used in the preparation method comprises a T4 DNA ligase, and the kit further comprises a splint DNA for linking the mRNA precursor and the polyadenylic acid.

The present invention further presents a method of preparing an mRNA-mannose conjugate as defined in claim <NUM>, specifically, a preparation method of an mRNA composition, see <FIG>, comprising:.

Specifically, the intermediate product of the preparation method is the polyadenylic acid- mannose conjugate, and at this time, a kit as defined in claim <NUM> can be obtained, comprising: an mRNA precursor comprising a coding sequence encoding a specific protein; a conjugate of a polyadenylic acid with an amino group at the <NUM>' end and a mannose; a ligase, the ligase being used for linking the mRNA precursor and the polyadenylic acid. The process of preparing the mRNA-mannose conjugate by the kit is also simpler and convenient to use.

Specifically, referring to <FIG> is an approach of the preparation method of the mRNA composition in this example, showing that the polyadenylic acid-mannose conjugate and the mRNA precursor are linked by a T4 RNA ligase; referring to <FIG> is another approach of the preparation method of the mRNA composition in this example, showing that the polyadenylic acid-mannose conjugate and the mRNA precursor are linked by a T4 DNA ligase to a splint DNA to improve the link efficiency.

It will be appreciated that, in the example of the present invention, the ligase in the kit corresponding to the materials obtained from or used in the preparation method can also be a T4 RNA ligase or a T4 DNA ligase. When the ligase in the kit is a T4 DNA ligase, the kit also comprises the splint DNA for linking the mRNA precursor and the polyadenylic acid-mannose conjugate.

The mRNA-mannose conjugates prepared in all the above-mentioned examples as defined in claim <NUM>, preferred embodiments are characterized in claims <NUM> to <NUM>, comprising: an mRNA encoding a polypeptide with at least one <NUM>' cap structure, a <NUM>' UTR with at least one Kozak sequence, a <NUM>' UTR, and a polyA tail, and at least one mannose attached to the mRNA via an amine at a <NUM>' terminus of the mRNA, and wherein the mRNA comprises uridine, cytidine, adenosine, guanosine and/or chemically modified nucleosides thereof, wherein the amine is a secondary amine, and wherein the amine is formed on a last adenine of the polyA tail of the mRNA.

The mRNA-mannose conjugates obtained in the present invention are endocytosed into cells specifically expressing mannose receptors through the pathway as shown in <FIG> for translation. First, the extracellular mannose ligand binds to the mannose receptor on the membrane surface to form a ligand-receptor complex; the ligand-receptor complex laterally diffuses to form Clathrin-coated pits (coated pits) with the involvement of clathrin; then dynein induces the invagination of the cell membrane to form Clathrin- coated vesicles (coated vesicles); subsequently coated vesicles with clathrin removed fuse with lysosomes, the receptor dissociates from the ligand under the action of the proton sponge effect, and the mRNA-mannose conjugates enter cells and are translated into protein.

The examples of the present invention achieved the coupling between an mRNA and a mannose, so that the mRNA can be targeted to specific cells through the mannose receptor. The method does not rely on physical methods and chemical transfection reagent methods, and is safe and effective.

The embodiments of the present invention will be described in detail below in conjunction with specific examples. Examples in which specific conditions are not specified are carried out according to the conventional conditions or conditions suggested by manufacturers. The reagents or instruments employed are all conventional products commercially available if the manufacturer is not indicated.

The preparation process of the mRNA-mannose conjugate independent of a "splint" DNA, for linking the mannose in the terminal reaction:.

The target gene sequence was searched in the NCBI library according to the purpose, the corresponding vector sequence was designed, and the mRNA precursor sequence was obtained by in vitro transcription method. In addition, a polyadenylic acid tail sequence was synthesized by commercial sources and - NH<NUM> was linked to the <NUM>' end of the polyadenylic acid, the resulting polyadenylic acid is: <NUM>'- AAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-NH<NUM>-<NUM>'.

The T4 RNA ligase was used to link the above-mentioned <NUM>'-AAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-NH<NUM>-<NUM>' to the <NUM>' end of the mRNA precursor to form an mRNA with an amino group labeled at the <NUM>' end: mRNA-NH<NUM>.

In the sodium bicarbonate solution at pH <NUM>, adding <NUM>µg (<NUM>µg/µl, about <NUM> nmol) of mRNA-NH<NUM> and <NUM>µg (<NUM>µg/µl, about <NUM> nmol) of <NUM>-isothiocyanatophenyl-α-D- mannoside with a purity of ≥<NUM>%, followed by reacting the mixture overnight at <NUM>. After the reaction stopped, the α-D-mannose was covalently bonded to the <NUM>' end of the above-mentioned mRNA-NH<NUM> through an isourea bond to obtain an mRNA-mannose conjugate. After the completion of the reaction, the unreacted mRNA-NH<NUM> and <NUM>-isothiocyanatophenyl-α-D-mannose were removed by purification using a desalting column (MWCO <NUM> kDa).

The preparation process of the mRNA-mannose conjugate dependent on a "splint" DNA, for linking the mannose in the terminal reaction:.

The splint DNA fragment was designed and synthesized by commercial sources, the sequence of which consists of two parts, comprising: a. a sequence part complementary to the <NUM>' end sequence of the target gene sequence; b. a part complementary to the polyadenylic acid tail sequence.

The T4 DNA ligase was used to link the above-mentioned <NUM>'-AAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-NH<NUM>-<NUM>' to the <NUM>' end of the mRNA
precursor under the mediation of a splint DNA fragment to form an mRNA of which the <NUM>' end is labeled with an amino group: mRNA-NH<NUM>.

In the sodium bicarbonate solution at pH <NUM>, adding <NUM>µg (<NUM>µg/µl, about <NUM> nmol) of mRNA-NH<NUM> and <NUM>µg (<NUM>µg/µl, about <NUM> nmol) of <NUM>-isothiocyanatophenyl-α-D- mannoside with a purity of ≥<NUM>%, followed by reacting the mixture overnight at <NUM>. After the reaction stopped, covalently bonding the α-D-mannose to the <NUM>' end of the above-mentioned mRNA-NH<NUM> through an isourea bond to obtain an mRNA-mannose conjugate. After the completion of the reaction, the splint DNA fragment was removed using a DNase, and purifying the products by using a desalting column (MWCO <NUM> kDa).

The preparation process of the mRNA-mannose conjugates independent of a "splint" DNA, for linking the mannose in the initiation reaction:.

In the sodium bicarbonate solution at pH <NUM>, adding <NUM>µg (<NUM>µg/µl, about <NUM> nmol) of polyA-NH<NUM> and <NUM>µg (<NUM>µg/µl, about <NUM> nmol) of <NUM>-isothiocyanatophenyl-α-D- mannoside with a purity of ≥<NUM>%, followed by reacting the mixture overnight at <NUM>. After the reaction stopped, covalently bonding the α-D-mannose to the <NUM>' end of the above-mentioned polyA-NH<NUM> through an isourea bond to obtain an polyA-mannose conjugate. After completion of the reaction, the unreacted polyA-NH<NUM> and <NUM>-isothiocyanatophenyl-α-D-mannose were removed by purification using a desalting column (MWCO <NUM> kDa).

The T4 RNA ligase was used to link the above-mentioned polyA-mannose to the <NUM>' end of the mRNA precursor to obtain an mRNA-mannose conjugate. After the completion of the reaction, purification was performed using a desalting column (MWCO <NUM> kDa).

The preparation process of the mRNA-mannose conjugates dependent on a "splint" DNA, for linking the mannose in the initiation reaction:.

In the sodium bicarbonate solution at pH <NUM>, adding <NUM>µg (<NUM>µg/µl, about <NUM> nmol) of polyA-NH<NUM> and <NUM>µg (<NUM>µg/µl, about <NUM> nmol) of <NUM>-isothiocyanatophenyl-α-D- mannoside with a purity of ≥<NUM>%, followed by reacting the mixture overnight at <NUM>. After the reaction stopped, covalently bonding the α-D-mannose to the <NUM>' end of the above-mentioned polyA-NH<NUM> through an isourea bond to obtain an polyA-Mannose conjugate. After the completion of the reaction, the unreacted polyA-NH<NUM> and <NUM>-isothiocyanatophenyl-α-D-mannose were removed by purification using a desalting column (MWCO <NUM> kDa).

The T4 DNA ligase was used to link the above-mentioned polyA-mannose to the <NUM>' end of the mRNA precursor under the mediation of a splint DNA fragment to obtain an mRNA-mannose conjugate. After the completion of the reaction, purification was performed using a desalting column (MWCO <NUM> kDa).

The amount of amino groups was determined by the fluorescamine method. Fluorosamine, which itself has no fluorescence properties, reacts with the primary amine to produce fluorescent substances. The amino group contained in mRNA-NH<NUM> is a primary amine.

After mRNA-NH<NUM> was coupled with a mannose, the original primary amine became a secondary amine, so that the amount of -NH<NUM> in the conjugate of mRNA-NH<NUM> and mRNA-Mannose can be determined by the amount of fluorescence.

<NUM>µg of the mRNA-Mannose conjugate synthesized in Example <NUM> was mixed with fluorescamine reagent in a <NUM>/mL acetone solution, and the mixture was incubated for <NUM> minutes, at a volume ratio of mRNA-Mannose conjugate to fluorescamine of <NUM>:<NUM>.

After completion of the incubation, the fluorescence intensity of the solution was measured with a fluorescence spectrophotometer (Gemini EMmicroplate reader, Molecular Device, CA, USA) at excitation wavelength and emission wavelength of <NUM> and <NUM>, respectively, and the amount of -NH<NUM> in the mRNA-mannose conjugate was calculated by using the fluorescence intensity of mRNA-NH<NUM> as the standard. The experimental results are shown in <FIG>. As can be seen from the figure, the mRNA- mannose conjugate obtained in Example <NUM> hardly contains any detectable amino-NH<NUM>, which proves that the linkage effect between mRNA and mannose is very good.

The dried mRNA-Mannose conjugate sample was mixed with <NUM>,<NUM>,<NUM>-trihydroxyacetophenone (THAP) in a <NUM>% acetonitrile (ACN)/<NUM>% trifluoroacetic acid (TFA) solution. The sample was analyzed by using MADI/MS (Ultraflextreme, Bruker, Germany) with smartbeam-IITM laser as ionization source. All spectra needed to be performed in positive mode with an ion source voltage of <NUM> kV, a repetition frequency of <NUM>, and an average emission number of <NUM>. The experimental results are shown in <FIG>, where mRNA-NH<NUM> is used as a control to indicate the position of the mRNA peak when mannose has not been linked. It can be seen from the figure that the preparation method of the present invention is able to efficiently prepare mRNA- Mannose conjugates, and there is no mRNA-NH<NUM> without linking to mannose, unconjugated mRNA, in the mRNA-Mannose conjugate.

Since there exists the mannose receptor CD206 in the phagolysosome, which can bind to the mRNA-mannose conjugate, the delivery of the mRNA-mannose conjugate inside the cell does not require complex intracellular pathways, namely macrophages are treated only with mRNA-mannose conjugates whereas without other carriers.

In order to determine whether the α-D-mannose in the mRNA-mannose conjugate can promote its internalization into macrophages, the fluorescently labeled mRNA and α-D- mannose were coupled together to obtain the fluorescently labeled mRNA-mannose conjugate (its preparation method is referred to Example <NUM>, which is not repeated herein), which was then used to treat RAW264. <NUM> cells (mouse mononuclear macrophage leukemia cells) for <NUM>, and at the same time, as a control, the RAW264. <NUM> cells were treated with fluorescently labeled mRNA-NH<NUM> for <NUM> hours, and after the treatment was completed, the number of fluorescent cells was detected by a flow cytometer.

The experimental results are shown in <FIG>, in which the total fluxes of fluorescent cells detected after treatment with mRNA-NH<NUM> and mRNA-mannose conjugates were
<NUM>±<NUM> and <NUM>±<NUM>, respectively. The number of fluorescence detected after the macrophages were treated with the fluorescently labeled mRNA-Mannose was much higher than that detected after the macrophages were treated with the fluorescently labeled mRNA-NH<NUM>, which proves that the uptake amount of mRNA-mannose is much greater than that of mRNA-NH<NUM>, and α-D-mannose can promote the internalization of mRNA-mannose conjugates into macrophages.

In order to verify that the uptake of mRNA-mannose conjugate into RAW264. <NUM> cells is mediated by the mannose receptor CD206 in a mannose-specific manner, D- mannosamine was used to conduct a competitive uptake assay in RAW264. <NUM> cells. After pre-incubating RAW264. <NUM> cells with D-mannosamine, the cells were treated with fluorescently-labeled mRNA-mannose conjugate for <NUM> hour, at the same time, fluorescently-labeled mRNA-NH<NUM> was used as a control, to thereby determine the flux of fluorescent cells. The assay results are shown in <FIG>, in which the amount of mRNA-mannose conjugates in macrophages was gradually reduced by adding D- mannosamine. When the treatment concentration of D-mannosamine was <NUM>, <NUM> and <NUM>, the total flux of fluorescent cells decreased significantly. However, the uptake of mRNA-NH<NUM> into macrophages did not decrease significantly due to the addition of D- mannosamine. This result clearly shows that the mRNA-Mannose conjugate is taken up by RAW264. <NUM> cells through mannose receptor-mediated endocytosis.

In order to determine the targetability of delivery of mRNA-mannose conjugates in mice after being endocytosed into cells, mRNA-mannose encoding luciferase was designed to be injected intravenously into mice, and the mRNA-NH<NUM> encoding luciferase was used as a control. <NUM> hours later, the mice were intraperitoneally injected with luciferin, a luciferase substrate, then fluorescence can be observed at sites with high expression of luciferase. The results indicate that, as shown in <FIG>, the spleen is the main target organ for mRNA-mannose.

In order to compare the anti-tumor effects of mRNA-mannose conjugate (mRNA-Man) and mRNA monomer (mRNA-NH<NUM>) encoding PD-<NUM> antibody, a survival rate experiment of tumor-bearing mice was designed. The PD-<NUM> antibody binds to the PD-<NUM> protein on the surface of immune cells, thereby blocking the binding of PD-<NUM> to the PD-L1 protein on the surface of cancer cells, and then activating the activity of immune cells, therefore preventing tumor cells from immune escape, and finally being killed by immune cells.

The experiment results are shown in <FIG>, where the control is a blank control, besides, injection of PD-<NUM> mRNA-Man and PD-<NUM> mRNA-NH<NUM> significantly improves the survival rate of humanized mice bearing melanoma, and the survival rate of mice injected with PD-<NUM> mRNA-mannose was significantly higher than that of mice injected with PD-<NUM> mRNA-NH<NUM>. This result clearly shows that the mRNA-Man conjugate encoding PD-<NUM> antibody is more easily endocytosed into target cells after injection into mice, to express PD-<NUM> antibody, thereby improving the survival rate of tumor-bearing mice.

To carry out the in vitro natural degradation experiment of mRNA, the PCR tube (Rnase free) containing mRNA (<NUM> ng/µl) and mRNA-mannose (<NUM> ng/µl) was put in a <NUM> incubator, the mRNA was collected and purified on day <NUM>, <NUM>, <NUM>. <NUM>, respectively, and the concentration was measured with NanoDrop ultra-micro spectrophotometer. The experimental results are shown in <FIG>, the results indicate that the half-life of mRNA-Mannose can reach a range of <NUM> to <NUM> days, while the half-life of conventional mRNA is only in a range of <NUM> to <NUM> days, which proves that the mRNA-mannose conjugate is relatively stable.

Claim 1:
An mRNA-mannose conjugate, comprising an mRNA encoding a polypeptide with at least one <NUM>' cap structure, a <NUM>' UTR with at least one Kozak sequence, a <NUM>' UTR, and a polyA tail, and at least one mannose attached to the mRNA via an amine at a <NUM>' terminus of the mRNA, and wherein the mRNA comprises uridine, cytidine, adenosine, guanosine and/or chemically modified nucleosides thereof, wherein the amine is a secondary amine, and wherein the amine is formed on a last adenine of the polyA tail of the mRNA.