CN119661723A

CN119661723A - Growth hormone fusion protein, nucleic acid medicine and application thereof

Info

Publication number: CN119661723A
Application number: CN202311223715.9A
Authority: CN
Inventors: 章雪晴; 邢扬菲
Original assignee: Rongcan Biomedical Technology Shanghai Co ltd
Current assignee: Rongcan Biomedical Technology Shanghai Co ltd
Priority date: 2023-09-20
Filing date: 2023-09-20
Publication date: 2025-03-21
Also published as: WO2025060968A1

Abstract

The invention relates to the field of medicines, in particular to a growth hormone fusion protein, a nucleic acid medicine and application thereof, wherein the growth hormone fusion protein comprises an ABD domain of streptococcal protein G and growth hormone, and the ABD domain and the growth hormone are connected through a connecting peptide (linker). The fusion protein has small molecular weight, avoids the problems that the large molecular weight growth hormone fusion protein is difficult to permeate into target tissues and has low biological activity, and has higher biological activity. Compared with the traditional protein medicine, the mRNA-LNP medicine for encoding the fusion protein can directly utilize the cells of the human body to express the protein medicine, and the protein medicine produced by the human body has high activity, can obviously improve the medicine effect, and can achieve relatively higher biological activity on the premise of lower administration dosage.

Description

Growth hormone fusion protein, nucleic acid medicine and application thereof

Technical Field

The invention relates to the field of medicines, in particular to a growth hormone fusion protein, a nucleic acid medicine and application thereof.

Background

Human growth hormone (Human Growth Hormone, hGH) is a peptide hormone secreted by eosinophils of the anterior pituitary of the human body and consists of 191 amino acids, most of which are present in the form of 22 kDa. hGH promotes protein synthesis and lipolysis of recipient cells, such as expression of insulin-like growth factor-1 (IGF-1), which is involved in cell growth and regeneration, by binding to growth hormone receptors on recipient cells in various tissues, organs and bones of the body, thereby promoting metabolism and growth development in humans. Is mainly applied to the treatment of slow growth and severe burn of children caused by growth hormone deficiency of children, chronic renal failure and Tener's syndrome clinically.

HGH as a small molecule polypeptide is easily denatured or enzymatically digested in the blood, liver or kidney, and thus has a half-life of only a few hours in the blood, and is highly frequently administered (usually daily) during clinical use with corresponding side effects (such as injection site discomfort, transient edema, joint pain, etc.), poor patient compliance, and greatly limiting its clinical use.

To overcome the problem of hGH shortness, methods using fusion proteins have been used for the development of long-acting growth hormone, such as Human Serum Albumin (HSA) fusion growth hormone. HSA, a mature protein consisting of 585 amino acids, participates in FcRn-mediated recycling mechanisms with plasma half-life as long as 14-20 days, thus extending the half-life of hGH after fusion with hGH.

But the structure and size of hGH are strictly conserved in various species, this conservation of size may represent an evolutionary control enabling hGH to pass through less vascular tissues (fat, bone and growth plates) and well-vascular tissues (muscle, heart). Although HSA fusion proteins can prolong half-life and reduce clearance of hGH, fusion proteins, due to a substantial increase in molecular weight, rather prevent absorption of hGH into target tissues and reduce their biological activity. Meanwhile, the half-life of the HSA-hGH fusion protein is 58-80h (published data of CN 109851674B), and there is still room for improvement so as to further reduce the administration frequency.

At present, the traditional fusion protein medicine mainly adopts antibody protein medicines clinically, the molecular structure of the antibody protein medicines is complex, the production cost is high, the medicines generally need to be injected and administrated, and the medicines can only generally act with proteins on the surface of cell membranes or outside the cell, so that the application of the medicines is limited to a certain extent.

Compared with antibody protein medicines or other types of protein medicines, the nucleic acid medicines have incomparable advantages in the aspects of patent medicine target number, medicine design period, targeting specificity, high efficiency, durability and the like, the high-efficiency research and development of mRNA vaccines drives the high-speed development of the whole nucleic acid medicine field, and the nucleic acid medicines have wider application scenes along with the clinical promotion and the maturation of related technologies in the future.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, it is an object of the present invention to provide a growth hormone fusion protein, a nucleic acid drug and uses thereof for solving the problems of the prior art.

To achieve the above and other objects, the present invention provides a fusion protein comprising an ABD domain of streptococcal protein G linked to a growth hormone via a linking peptide (linker), the ABD domain having an amino acid sequence shown in SEQ ID No.11 or having 95% or more similarity to SEQ ID No.11 and having a function of the amino acid sequence defined in SEQ ID No.11, and having an amino acid sequence shown in SEQ ID No.12 or having 95% or more similarity to SEQ ID No.12 and having a function of the amino acid sequence defined in SEQ ID No. 12.

The invention also provides a nucleic acid molecule comprising a nucleotide sequence encoding said fusion protein.

The invention also provides a cell containing or used for preparing any one or more of the fusion protein and the nucleic acid molecule.

The invention also provides a pharmaceutical composition comprising any one or more of the fusion proteins and the nucleic acid molecules, and preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or auxiliary materials.

The invention also provides the use of said fusion protein, said nucleic acid molecule, said pharmaceutical composition or any combination of the above for the manufacture of a medicament for the treatment of a disease, said disease being a disease suitable for treatment with growth hormone.

As described above, the growth hormone fusion protein, the nucleic acid drug and the use thereof of the present invention have the following beneficial effects:

1. The ABD-linker-GH fusion protein of the invention utilizes the interaction of ABD and HSA to improve the recycling mechanism of the fusion protein mediated by FcRn, thereby achieving the purpose of prolonging half-life. Compared with HSA-hGH fusion protein drugs, the binding of ABD and HSA in a natural state can more effectively utilize FcRn-mediated recycling mechanism, further improve half-life of fusion protein and reduce administration frequency.

2. Compared with other fusion protein medicines, the ABD-linker-GH fusion protein has small molecular weight, avoids the problems that high molecular weight hGH is difficult to permeate into target tissues and has low biological activity, and has higher biological activity.

3. Compared with the traditional protein medicine, the ABD-linker-GH fusion protein can directly utilize the cells of the human body to express the protein medicine, and the self-produced protein medicine has high activity, can obviously improve the medicine effect, and can achieve relatively higher biological activity on the premise of lower administration dosage.

Drawings

FIG. 1 shows a schematic structure of a fusion protein of the present invention.

FIG. 2 shows the expression levels of the fusion ABD-linker-GH mRNA of the invention in HEK293T cell supernatants.

FIG. 3 shows the variation of hGH content in rat serum after intravenous injection of different ABD-linker-GH mRNA-LNP.

FIG. 4 shows the change in IGF-1 levels in rat serum after intravenous injection of different ABD-linker-GH mRNA-LNP.

FIG. 5 shows the variation of hGH content in rat serum after intravenous injection of different ABD-linker-GH mRNA-LNP.

FIG. 6 shows the change in IGF-1 levels in rat serum after intravenous injection of different ABD-linker-GH mRNA-LNP.

Detailed Description

The invention provides a fusion protein, which is named as ABD-linker-GH, and comprises an ABD domain of streptococcal protein G and growth hormone, wherein the ABD domain is connected with the growth hormone through a connecting peptide (linker), the amino acid sequence of the ABD domain is shown as SEQ ID NO.11 or has 95% or more similarity with the amino acid sequence of SEQ ID NO.11 and has the function of the amino acid sequence defined by SEQ ID NO.11, and the amino acid sequence of the growth hormone is shown as SEQ ID NO.12 or has 95% or more similarity with the amino acid sequence defined by SEQ ID NO. 12.

LAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALP(SEQ ID NO.11)

MATGSRTSLLLAFGLLCLPWLQEGSAFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF(SEQ ID NO.12)

The ABD domain may be a wild-type domain or a mutant domain derived from streptococcal protein G of a different species, and the present invention is not particularly limited. Specifically, the ABD domain is the GA module of streptococcal protein G (Streptococcal Protein G, SPG for short) (GA module, johansson et al, J Mol Biol 266:859-865,1997; johansson et al, J Bio Chem 277:8114-8120.2002, protein G-related albumin binding module for the triple helix protein domain responsible for albumin binding).

Streptococcal protein G on the surface of streptococcal strains has a structural domain capable of binding with serum albumin, which has been determined to be a triple helix bundle structure, and this motif is designated ABD (albumin binding domain), so that the ABD peptide (46 amino acids in size) of G protein of streptococcal G148 is fused with GH through a connecting peptide (linker) to form an ABD-linker-GH fusion protein, and the interaction of ABD and HSA is utilized to improve the recycling mechanism of the fusion protein through FcRn, so that the half-life is prolonged. Because of the small molecular weight of ABD, the biological activity of hGH is affected very little after fusion.

The motif according to the invention is generally defined as a short sequence or pattern that is repeated in different organisms or molecules, with specific functional or structural characteristics.

The amino acid sequence of the ABD domain can be obtained by substituting, deleting or adding one or more (specifically 1-20, 1-10, 1-5 or 1-3) amino acids on the amino acid sequence shown as SEQ ID NO.11, or adding one or more (specifically 1-50, 1-30, 1-20, 1-10, 1-5 or 1-3) amino acids on the N-terminal and/or C-terminal, and has the function of a polypeptide fragment with the amino acid sequence shown as SEQ ID NO.11, and the amino acid sequence obtained by substituting, deleting or adding the amino acids can have the sequence similarity of 95%, 96%, 97%, 98% or more than 99% with the amino acid sequence shown as SEQ ID NO. 11.

In certain embodiments of the invention, the amino acid sequence of the ABD domain may also be as shown in any of SEQ ID NO. 3-5 or have 95% or more similarity to any of the sequences shown in SEQ ID NO. 3-5. Wherein SEQ ID NO.3 is the amino acid sequence of G protein GA3 mutant 1 derived from Streptococcus dysenteriae protein G, SEQ ID NO.4 is the amino acid sequence of G protein GA3 mutant 2 derived from Streptococcus G148, and SEQ ID NO.5 is the amino acid sequence of G protein GA3 mutant 2 derived from Streptococcus G148.

LAEAKELAIRELKKYGISDYYKNLINKAKTVEGVKALKDEILAALP(SEQ ID NO.3)

LAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVEALKLHILAALP(SEQ ID NO.4)

LAEAKEAANAELDSYGVSDFYKRLIDKAKTVEGVEALKDAILAALP(SEQ ID NO.5)

In certain embodiments of the invention, the growth hormone is selected from mammalian growth hormone. The mammal is selected from human, bovine, porcine, murine, monkey, etc.

In some embodiments of the present invention, the growth hormone is human growth hormone (hGH), the amino acid sequence of the growth hormone may be obtained by substituting, deleting or adding one or more (specifically 1-20, 1-10, 1-5 or 1-3) amino acids to the amino acid sequence shown in SEQ ID NO.12, or adding one or more (specifically 1-50, 1-30, 1-20, 1-10, 1-5 or 1-3) amino acids to the N-terminal and/or C-terminal, and has the function of a polypeptide fragment having the amino acid sequence shown in SEQ ID NO.12, and the amino acid sequence obtained by substituting, deleting or adding the amino acids may have a sequence similarity of 95%, 96%, 97%, 98% or more than 99% to SEQ ID NO. 12.

In certain embodiments of the present invention, the amino acid sequence of the growth hormone may also be as shown in SEQ ID NO. 9.

MATGSRTSLLLAFGLLCLPWLQEGSAFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPDPGQIFAQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF(SEQ ID NO.9)

The linking peptide (linker) is not particularly limited as long as it is capable of linking the ABD domain to the growth hormone, and it is not limited that the ABD domain and the growth hormone function. In certain embodiments of the invention, the connecting peptide (linker) may be selected from a rigid connecting peptide (linker) or a flexible connecting peptide (linker). The rigid linking peptide (linker) is (EAAAK) n, n=1-5. The flexible connecting peptide (linker) is selected from (GGGGS) n, (GSGGG) n, (GGGSG) n or (GGSGG) n, n=1-5.

In certain embodiments of the present invention, the amino acid sequence of the linker peptide (linker) has a similarity of 95% or more as shown in any one of SEQ ID NO.6 to 8 or a combination thereof.

(GGGGS)1(SEQ ID NO.6)

(GGGGS)5(SEQ ID NO.7)

(EAAAK)2(SEQ ID NO.8)

In certain embodiments of the invention, the fusion protein comprises, in order from N-terminus to C-terminus, ABD domain-linker-growth hormone. The fusion protein further comprises a signal peptide of growth hormone at the N-terminus before being secreted outside the cell after expression.

In certain embodiments of the invention, the ABD-linker-GH may be formed in any manner selected from the group consisting of:

1) ABD domain shown in SEQ ID NO.11, linker peptide (linker) shown in SEQ ID NO.6 and growth hormone shown in SEQ ID NO. 12;

2) ABD domain shown in SEQ ID NO.11, linker peptide (linker) shown in SEQ ID NO.7 and growth hormone shown in SEQ ID NO. 12;

3) ABD domain shown in SEQ ID NO.11, linker peptide (linker) shown in SEQ ID NO.8 and growth hormone shown in SEQ ID NO. 12;

4) ABD domain shown in SEQ ID NO.11, linker peptide (linker) shown in SEQ ID NO.6 and growth hormone shown in SEQ ID NO. 9;

5) ABD domain shown in SEQ ID NO.11, linker peptide (linker) shown in SEQ ID NO.7 and growth hormone shown in SEQ ID NO. 9;

6) ABD domain shown in SEQ ID NO.11, linker peptide (linker) shown in SEQ ID NO.8 and growth hormone shown in SEQ ID NO. 9;

7) ABD domain shown in SEQ ID NO.3, linker peptide (linker) shown in SEQ ID NO.6 and growth hormone shown in SEQ ID NO. 12;

8) ABD domain shown in SEQ ID NO.3, linker peptide (linker) shown in SEQ ID NO.7 and growth hormone shown in SEQ ID NO. 12;

9) ABD domain shown in SEQ ID NO.3, linker peptide (linker) shown in SEQ ID NO.8 and growth hormone shown in SEQ ID NO. 12;

10 ABD domain shown in SEQ ID No.3, linker peptide (linker) shown in SEQ ID No.6, and growth hormone shown in SEQ ID No. 9;

11 ABD domain shown in SEQ ID No.3, linker peptide (linker) shown in SEQ ID No.7, and growth hormone shown in SEQ ID No. 9;

12 ABD domain shown in SEQ ID NO.3, linker peptide (linker) shown in SEQ ID NO.8 and growth hormone shown in SEQ ID NO. 9;

13 ABD domain shown in SEQ ID No.4, linker peptide (linker) shown in SEQ ID No.6, and growth hormone shown in SEQ ID No. 12;

14 ABD domain shown in SEQ ID No.4, linker peptide (linker) shown in SEQ ID No.7, and growth hormone shown in SEQ ID No. 12;

15 ABD domain shown in SEQ ID No.4, linker peptide (linker) shown in SEQ ID No.8, and growth hormone shown in SEQ ID No. 12;

16 ABD domain shown in SEQ ID No.4, linker peptide (linker) shown in SEQ ID No.6, and growth hormone shown in SEQ ID No. 9;

17 ABD domain shown in SEQ ID No.4, linker peptide (linker) shown in SEQ ID No.7, and growth hormone shown in SEQ ID No. 9;

18 ABD domain shown in SEQ ID No.4, linker peptide (linker) shown in SEQ ID No.8, and growth hormone shown in SEQ ID No. 9;

19 ABD domain shown in SEQ ID No.5, linker peptide (linker) shown in SEQ ID No.6, and growth hormone shown in SEQ ID No. 12;

20 ABD domain shown in SEQ ID No.5, linker peptide (linker) shown in SEQ ID No.7, and growth hormone shown in SEQ ID No. 12;

21 ABD domain shown in SEQ ID No.5, linker peptide (linker) shown in SEQ ID No.8, and growth hormone shown in SEQ ID No. 12;

22 ABD domain shown in SEQ ID No.5, linker peptide (linker) shown in SEQ ID No.6, and growth hormone shown in SEQ ID No. 9;

23 ABD domain shown in SEQ ID No.5, linker peptide (linker) shown in SEQ ID No.7, and growth hormone shown in SEQ ID No. 9;

24 ABD domain shown in SEQ ID No.5, linker peptide (linker) shown in SEQ ID No.8 and growth hormone shown in SEQ ID No. 9.

In certain embodiments of the invention, the fusion protein has an amino acid sequence as shown in SEQ ID NO.1 or SEQ ID NO. 14.

LAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALPGGGGSGGGGSFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF(SEQ ID NO.14)

In certain embodiments of the invention, the fusion protein has an amino acid sequence as set forth in SEQ ID NO.20, SEQ ID NO.21 or SEQ ID NO. 22.

LAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALPGGGGSGGGGSGGGGSGGGGSGGGGSFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF(SEQ ID NO 20:ABD11-linker7-GH12 Amino acid sequence

LAEAKELAIRELKKYGISDYYKNLINKAKTVEGVKALKDEILAALPGGGGSGGGGSGGGGSGGGGSGGGGSFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPDPGQIFAQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF(SEQ ID NO 21:ABD3-linker7-GH9 Amino acid sequence

LAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALPGGGGSFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPDPGQIFAQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF(SEQ ID NO 22:ABD11-linker6-GH9 Amino acid sequence

The "nucleic acid molecule" according to the present invention may be a nucleotide polymer of any length. Including, but not limited to, single-stranded DNA, double-stranded DNA, plasmid DNA, short isomers, mRNA, tRNA, rRNA, long non-coding RNAs (lncRNA), micronon-coding RNAs (miRNA and siRNA), telomerase RNA (TelomeraseRNA Component), small molecule RNAs (snRNA and scRNA), circular RNAs (circRNA), synthetic mirnas (MIRNA MIMICS, miRNA agomir, miRNA antagomir), antisense oligonucleotides (ASO), ribozymes (ribozyme), asymmetric interfering RNAs (aiRNA), dicer-substrate RNAs (dsRNA), small hairpin RNAs (shRNA), guide RNAs (gRNA), small guide RNAs (sgrnas), locked Nucleic Acids (LNAs), peptide Nucleic Acids (PNAs), morpholino antisense oligonucleotides, morpholino oligonucleotides, or combinations of one or more of the biospecific oligonucleotides.

In certain embodiments of the invention, the nucleic acid molecule refers to a DNA nucleic acid molecule.

In certain embodiments of the invention, the nucleic acid molecule is plasmid DNA.

In certain embodiments of the invention, the nucleic acid molecule is mRNA.

In certain embodiments of the invention, the nucleic acid molecule comprises, in order from the 5 'to the 3' end, nucleotides encoding a signal peptide of growth hormone-an ABD domain-a linker-a growth hormone.

In certain embodiments of the invention, the nucleotide sequence encoding the fusion protein is shown in SEQ ID NO. 2.

In certain embodiments of the invention, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO. 13.

In certain embodiments of the invention, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO. 10.

In some embodiments, the cell genome has incorporated therein a nucleotide sequence having 95% or more similarity as set forth in SEQ ID No.2 or 13 or either.

In some embodiments, the cell is a cell used to make the nucleic acid molecule and/or the nucleic acid molecule encodes a fusion protein.

In some embodiments, the cells include, but are not limited to, any of mammalian cells (e.g., CHO, COS, and N2A), plant cells, human cells (human cervical cancer cells such as HELA and human embryonic kidney cells such as HEK 293T), bacterial cells (e.g., escherichia coli, streptomyces, salmonella typhimurium), fungal cells (e.g., yeast), insect cells (e.g., sf 9). In some preferred embodiments, the host cell is an animal cell, and more preferably a human cell. The host cell is a passaged cell or a primary cell, i.e., a cell isolated directly from an organism (e.g., a human). The host cell is an adherent cell or a suspension cell.

The invention also provides a preparation method of the fusion protein, which comprises the following steps:

a) Culturing the cell as described above under expression conditions, thereby expressing the fusion protein;

b) Isolating and purifying the fusion protein of step a).

The invention also provides a pharmaceutical composition comprising any one or more of the fusion proteins or the nucleic acid molecules, and a pharmaceutically acceptable carrier and/or adjuvant.

In certain embodiments of the invention, the pharmaceutical composition may comprise a fusion protein and an adjuvant, or a nucleic acid molecule and a vector and an adjuvant.

In certain embodiments of the invention, the pharmaceutical composition is a nucleic acid drug.

In a more preferred embodiment, the pharmaceutical composition is an mRNA drug.

In certain embodiments of the invention, the mRNA drug comprises the nucleic acid molecule and a pharmaceutically acceptable carrier, which is a Lipid Nanoparticle (LNP). In certain embodiments of the invention, the mRNA drug further comprises an adjuvant.

In certain embodiments of the invention, the lipid nanoparticle comprises at least one ionizable lipid compound.

In certain embodiments of the invention, the lipid nanoparticle comprises at least one ionizable lipid compound and one or more combinations of structural lipids, helper lipids, PEG-lipids, polymers.

The ionizable lipid compound refers to a lipid of which the pH value influences the protonation degree and further influences the charging property. Preferably, a class of lipids that is generally barely charged at normal neutral physiological pH conditions, but is capable of being positively charged at acidic pH to bind to negatively charged nucleic acids. The ionizable lipid compounds of the present invention also include isomers thereof or pharmaceutically acceptable salts thereof.

The foregoing "isomers" refer to different compounds having the same molecular formula, including but not limited to enantiomers, diastereomers, cis-trans isomers, and the like, as known in the art.

The aforementioned "pharmaceutically acceptable salt" refers to an acid addition salt or a base addition salt. All compounds of the invention in free base or free acid form can be converted into their pharmaceutically acceptable salts by treatment with suitable inorganic or organic bases or acids according to methods known to those skilled in the art. Salts of the compounds of the invention may be formed by conversion to their free base or acid by standard techniques.

Pharmaceutically acceptable salts of the compounds of the invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts with inorganic acids such as hydrochloric, hydrobromic, phosphoric, sulfuric and perchloric acids or with organic acids such as acetic, oxalic, maleic, tartaric, citric, succinic or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipates, alginates, ascorbates, aspartate, benzenesulfonates, benzoates, bisulphates, borates, butyrates, camphorinates, camphorsulphonates, citrates, cyclopentanepropionates, digluconates, citrates, dodecyl sulphates, ethanesulphonates, formates, fumarates, glucoheptanoates, glycerophosphate, gluconate, hemisulphates, heptanoates, caprates, hydroiodinates, 2-hydroxyethanesulphonates, lactates, laurates, lauryl sulphates, malates, maleates, malonates, methanesulfonates, 2-naphthalenesulphonates, nicotinates, nitrates, oleates, oxalates, palmates, pamonates, pectinates, persulphates, 3-phenylpropionates, phosphates, picrates, pivalates, propionates, stearates, succinates, sulphates, tartrates, thiocyanates, p-toluenesulfonates, undecanoates, valerates, and the like. Salts derived from suitable bases include alkali metal salts, alkaline earth metal salts, ammonium salts. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Where appropriate, additional pharmaceutically acceptable salts include nontoxic ammonium, quaternary ammonium and amine cations formed using counter ions such as halides, hydroxides, carboxylates, sulphates, phosphates, nitrates, sulphonates and arylsulphonates. Additional pharmaceutically acceptable salts include salts formed from quaternization of amines with suitable electrophiles (e.g., alkyl halides) to form quaternized alkylated amino salts.

The types of the ionizable lipids are not limited, and can be selected from ionizable lipids disclosed in the prior art, and exemplary ionizable lipids are selected from the group consisting of those in chinese patent applications 202210344395.1,202310086372.X,202310862227.6,202211374342.0, 202310830388.7.

The structural lipid refers to a structure containing a compound that can stabilize the composition, including but not limited to sterols and derivatives thereof and non-sterols and derivatives thereof.

In some embodiments, the structural lipid includes, but is not limited to, sterols and derivatives thereof, non-sterols, sitosterols, ergosterols, cholestanones, cholestenone, campesterols, stigmasterols, brassicasterol, lycorine, ursolic acid, stigmasterols, alpha-tocopherols, or a combination of one or more corticosteroids. Non-limiting examples of cholesterol derivatives include polar analogs such as 5α -cholesterol, 5α -fecal sterols, cholesteryl- (2 '-hydroxy) -ethyl ether, cholesteryl- (4' -hydroxy) -butyl ether and 6-ketocholestanol, non-polar analogs such as 5α -cholestane, cholestenone, 5α -cholestenone and decanoate cholesterol esters, and mixtures thereof. In a preferred embodiment, the cholesterol derivative is a polar analogue, such as cholesteryl- (4' -hydroxy) -butyl ether. It is not intended to be exhaustive and any structural lipid may be used in the present invention.

In some embodiments, the structural lipid is a combination of one or more of cholesterol, sitosterol, ergosterol, corticosteroid, and derivatives thereof.

In some embodiments, the structural lipid is cholesterol.

The "helper lipid" is not limited in kind and preferably comprises a phospholipid lipid including, but not limited to, one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, phosphatidylserine, phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, dimyristoyl phosphatidylglycerol.

In some embodiments, the helper lipid may be selected from the group consisting of 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DPPC), 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DLPC), 1, 2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DPPC), 1, 2-dioleoyl-sn-glycero-3-phosphocholine (DSPC), 1, 2-dioleoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1, 2-dioleoyl-octadecenyl-sn-3-phosphocholine (18:0 diether PC), dimyristoyl phosphoethanolamine (DMPC), distearoyl-3-phosphoethanolamine (DSPE), 1, 2-dioleoyl-sn-3-phosphocholine (DSPC), 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1-dioleoyl-5-glycero-sn-3-phosphoethanolamine (DPPE), 1-O-hexadecyl-sn-glycerol-3-phosphocholine, 1, 2-dioleoyl-sn-glycerol-3-phosphocholine, 1, 2-didodecylhexanoyl-sn-glycerol 3-phosphocholine, 1, 2-dioleoyl-sn-glycerol-3-phosphoethanolamine, 1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine, 1, 2-dioleoyl-sn-glycerol-3-phosphoethanolamine, 1, 2-didodecylhexanoyl-sn-glycerol 3-phosphoethanolamine, 1, 2-dioleoyl-sn-glycerol-3-phospho-rac- (1-glycerol) sodium salt (DOPG), diacetyl-phosphoethanolamine, stearoyl-phosphoethanolamine (pe), or a combination of multiple of phospho-sphingomyelin (pe).

In some embodiments, the phosphatidylcholine is a combination of one or more of DSPC, DPPC, DMPC, DOPC, POPC.

In some embodiments, the helper lipid is phosphatidylcholine, particularly DSPC.

In some embodiments, the helper lipid is phosphatidylethanolamine, particularly DOPE.

In some embodiments, the helper lipid is selected from one or more combinations of DOTAP ((1, 2-dioleoxypropyl) trimethylammonium chloride), DOTAP (1, 2-dioleoyl-3-dimethylammonium-propane), 18:1pa (1, 2-DI (cis-9-octadecenoyl) -SN-glycero 3-phosphate sodium salt), HS15 (polyethylene glycol (15) -hydroxystearate), GL67 (N4-argininocarbonamide).

The PEG-lipids are formed from PEG and lipid molecules joined by chemical bonds, including but not limited to PEG-modified phospholipids and derivatized lipids, such as exemplified by one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol.

In some embodiments, the PEG-lipid includes, but is not limited to, PEG-C-DMG, PEG-C-DOMG, PEG-DLPE, PEG-DMPE, PEG-DPPE, PEG-DOPE, PEG-DPPC, azido-PEG, PEG-distearoyl phosphatidylethanolamine (PEG-DSPE), PEG-DAA, chol (cholesterol) -PEG, 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol (PEG-DMG), PEG-S-DMG, polyethylene glycol phosphatidylethanolamine (PEG-PE), polyethylene glycol ceramide, polyethylene glycol-dimethacrylate (PEG-DMA), PEG-distearyl glycerol (PEG-DSG), PEG-dipentaerythritol, PEG-dioleyl, PEG-distearyl, PEG-diacyl Gan Xianan (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), PEG-1, 2-dimyristoyloxypropyl-3-amine (PEG-C), 1, 2-dimyristoyloxypropyl-3-amine (PEG-DMA), polyethylene glycol [ 1, 2-dioleoyl-propyl-3-methoxypropyl-amine (PEG-01 ] or combinations of polyethylene glycol-01 (DSG).

In some embodiments, the PEG-lipid is PEG-DMG.

The kind of the "polymer" is not limited, and the polymer may include, but is not limited to, amphiphilic block copolymers, which are block copolymers composed of hydrophobic polymers and hydrophilic compounds, including, but not limited to, polylactic acid (PLA), polylactic acid-polyglycolic acid copolymer (PLGA), glycolide-lactide copolymer (PLCG), polycaprolactone (PCL), polyorthoester, polyanhydride (PAH), polyphosphazene, poly beta-amino ester (PBAE), poly (alpha-hydroxy acid), lactide/glycolide copolymer (PLGA or PLG) (which includes lactide/glycolide copolymer, D-lactide/glycolide copolymer, L-lactide/glycolide copolymer and D, L-lactide/glycolide) polymer), polyglycolide (PGA), polyorthoester (POE), linear or branched polyethylene glycol (PEG), conjugates of poly (alpha-hydroxy acids), polyacetirin (polyaspirins), polyphosphagenes, D-lactide, D, L-lactide-caprolactone, D, L-lactide-glycolide-caprolactone (DL-G) CL), dextran, vinylpyrrolidone, polyvinyl alcohol (PVA), methacrylate, poly (N-isopropylenamide), SAIB (sucrose acetate iso) acid salt hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxyethyl methylcellulose, carboxymethyl cellulose or salts thereof, polyethylene, poly (hydroxyethyl methacrylate), poly (methoxyethyl methacrylate), poly (methoxyethoxy-ethyl methacrylate), polymethyl methacrylate (PMMA), methyl Methacrylate (MMA), PVA-g-PLGA, PEGT-PBT copolymer (multi-active (polyact)) e)), PEO-PPO-PEO (pluronics)), PEO-PPO-PAA copolymer, PLGA-PEO-PLGA, PEG-PLG, PLA-PLGA, poloxamer 4O7, PEG-PLGA-PEG triblock copolymers, or block copolymers of these with polyethylene glycol (PEG), or a combination of one or more of the foregoing polymers or copolymers.

In some embodiments of the present invention, the polyethylene glycol in the polyethylene glycol modified lipid has a weight average molecular weight of 1000 to 10000, for example, 1000 to 2000, 2000 to 4000, 4000 to 6000, 6000 to 8000, 8000 to 10000, preferably 2000.

In some embodiments, the lipid nanoparticle comprises 20-65% ionizable lipid compound, 20-60% structural lipid, 3-40% helper lipid, 0.1-10% PEG-lipid,% refers to mole percent.

In some embodiments, the lipid nanoparticle comprises 35-49% ionizable lipid compound, 35-50% structural lipid, 5-20% helper lipid, 1-2% PEG-lipid,% refers to mole percent.

In some embodiments of the present invention, the molar ratio of N atoms in the lipid to P atoms in the mRNA drug is (1-20): 1, and may be (3-15): 1, (3-10): 1, (5-8): 1, for example.

The particle size of the lipid nanoparticle refers to a hydration particle size determined by Dynamic Light Scattering (DLS), and the hydration particle size of the lipid nanoparticle is 50-200 nm, for example, 60-150 nm, 75-140 nm, 85-130 nm or 95-110 nm.

The nucleic acid medicament of the invention can be prepared by the prior art.

In some embodiments of the invention, the nucleic acid drug is prepared by mixing a lipid with an organic solvent to obtain a lipid organic phase, mixing an mRNA drug with an aqueous solvent to obtain a drug solution, and mixing the lipid organic phase with the drug solution to obtain the nucleic acid drug.

In certain embodiments of the invention, the organic solvent is ethanol.

In certain embodiments of the invention, the aqueous solvent is a buffer solution. Further preferred is a sodium acetate buffer solution.

In some embodiments of the invention, the concentration of the drug in the drug solution is 0.01-5 mg/mL. For example, the concentration of the drug in the drug solution is 0.01～0.05mg/mL、0.05～0.1mg/mL、0.1～0.5mg/mL、0.5～1mg/mL、1～1.5mg/mL、1.5～2mg/mL、2～2.5mg/mL、2.5～3mg/mL、3～3.5mg/mL、3.5～4mg/mL、4～4.5mg/mL、4.5～5mg/mL.

In some embodiments of the present invention, the volume ratio of the lipid organic phase to the drug solution is 1 (1-15). For example, the volume ratio of the lipid organic phase to the drug solution is 1 (1-3), 1 (3-6), 1 (6-9), 1 (9-11) and 1 (11-15).

In certain embodiments of the invention, the method of mixing the lipid molecular organic phase with the drug solution is microfluidic.

In the present invention, the term "pharmaceutical composition" comprises any one or more of the fusion proteins, nucleic acid molecules described above. Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or adjuvant. Typically, these materials are formulated in a nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 4 to 8, preferably about 5 to 7, although the pH may vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to, intravenous injection, intravenous drip, subcutaneous injection, topical injection, intramuscular injection, intratumoral injection, intraperitoneal injection (e.g., intraperitoneal), intracranial injection, intracavity injection, inhalation administration, implant administration, and the like.

In the present invention, the preferred "pharmaceutical compositions" are pharmaceutical preparations which ensure the conformational integrity of the amino acid core sequence of the fusion proteins disclosed herein, protect the structure of the proteins from degradation (including, but not limited to, aggregation, deamination or oxidation, etc.), and also protect the biological activity and function of the proteins from destruction, thereby more stably exerting therapeutic effects.

By "pharmaceutically acceptable" is meant that the drug does not produce adverse, allergic or other untoward reactions when properly administered to an animal or human.

The "pharmaceutically acceptable carrier and/or adjuvant" should be compatible with the active ingredient, i.e. it is capable of being blended therewith without substantially reducing the efficacy of the drug in the usual manner. Specific examples of substances which may be pharmaceutically acceptable carriers and/or excipients may be lipids (e.g., lipid nanoparticles), sugars such as glucose, mannitol, sucrose, lactose, trehalose, maltose, and the like, starches such as corn starch and potato starch, and the like, celluloses and derivatives thereof such as sodium methylcellulose, ethylcellulose, and methylcellulose, and the like, tragacanth powder, malt, gelatin, talc, solid lubricants such as stearic acid and magnesium stearate, calcium sulfate, vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa butter, and the like, alcohols such as ethanol, propylene glycol, glycerol, sorbitol, mannitol, and polyethylene glycols, and the like, alginic acid, emulsifying agents such as Tween, and the like, wetting agents such as sodium lauryl sulfate, surfactants, lyoprotectants, colorants, flavoring agents, tableting agents, stabilizers, diluents, excipients, antioxidants, preservatives, pyrogen-free water, isotonic saline solutions, buffers and the like, and combinations thereof. These substances are used as needed to increase the stability of the formulation or to help increase the activity or its bioavailability or to create an acceptable mouthfeel or odor in the case of oral administration.

The pharmaceutical composition of the invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80 wt%) of any one or more of the fusion proteins, nucleic acid molecules of the invention described above, and the preferred pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or adjuvant. The pharmaceutical composition of the present invention can be formulated into inhalable atomized formulations (e.g., dry powder formulations, aerosol formulations, inhalable aerosol droplet formulations, etc.), implantable gel formulations, microneedle formulations, and also can be formulated into injectable forms, for example, using physiological saline or aqueous solutions containing glucose and other adjuvants by conventional methods. The pharmaceutical compositions, such as injections, solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example, from about 10 micrograms per kilogram of body weight to about 50 milligrams per kilogram of body weight per day. In addition, the pharmaceutical compositions of the present invention may also be used with other therapeutic agents.

In the present invention, the term "effective amount" refers to an amount or dose that produces a desired effect in a treated individual, including but not limited to an improvement in a disorder in the individual, following administration of the pharmaceutical composition of the present invention to the subject.

In the present invention, the term "subject" includes, but is not limited to, mammals, such as humans, non-human primates, dogs, rabbits, rats, mice, and the like.

The disease may be selected from the group consisting of adult Growth Hormone Deficiency (GHD), childhood dwarfism, idiopathic Short Stature (ISS), short stature or slow growth due to other factors (e.g. endogenous growth hormone deficiency, SHOX gene deficiency, achondroplasia, less than gestational age), prader-willi syndrome, chronic Renal Insufficiency (CRI), renal failure, aging retardation in elderly, beauty and anti-aging, severe burns, tenna syndrome, intrauterine growth retardation, polycystic ovary syndrome infertility (POCS), infertility, childhood central precocity (ICPP), adult short bowel syndrome, prader-willi syndrome (PWS), chemotherapy treatment and diseases caused by the catabolic state during AIDS treatment.

The invention also provides a method of treating a disease comprising administering to a subject in need thereof an effective amount of the fusion protein, the nucleic acid molecule, the pharmaceutical composition, or any combination thereof.

Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.

Before further describing embodiments of the invention, it is to be understood that the scope of the invention is not limited to the specific embodiments described below, and that the terminology used in the examples of the invention is for the purpose of describing particular embodiments and is not intended to be limiting of the invention, as in the description and claims of the invention, the singular forms "a", "an" and "the" include plural forms, unless the context clearly dictates otherwise, and each possibility described in the description and claims of the invention represents a separate embodiment of the invention.

Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention may be used to practice the present invention according to the knowledge of one skilled in the art and the description of the present invention. The equipment, materials, etc. used in the present invention are commercially available sources unless otherwise specified.

Example 1 design and Synthesis of ABD-linker-GH fusion protein

After the ABD gene is inserted into the signal peptide of the GH gene, the mature GH gene is connected through a linker, so that the ABD-linker-GH is obtained. The amino acid sequence and the nucleotide sequence of the total gene synthesis ABD-linker-GH are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2.

Amino acid sequence of long-acting recombinant human growth hormone ABD-linker-GH fusion protein:

MATGSRTSLLLAFGLLCLPWLQEGSALAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALPGGGGSGGGGSFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF(SEQ ID NO.1)

As is clear from the amino acid sequence shown in SEQ ID NO.1, the ABD-linker-GH amino acid sequence is 273 amino acids in total, wherein the first 26 amino acids are the signal peptide sequence of human growth hormone, the 46 amino acids are the GA3 sequence (ABD) of protein G of Streptococcus G148, "GGGGSGGGGS" is the connecting peptide (linker), and the last 191 amino acids are the mature human growth hormone sequence.

Nucleotide sequence encoded by long-acting recombinant human growth hormone ABD-linker-GH immune fusion protein:

ATGGCTACAGGCTCCCGGACGTCCCTGCTCCTGGCTTTTGGCCTGCTCTGCCTGCCCTGGCTTCAAGAGGGCAGTGCCTTAGCTGAAGCTAAAGTCTTAGCTAACAGAGAACTTGACAAATATGGAGTAAGTGACTATTACAAGAACCTAATCAACAATGCCAAAACTGTTGAAGGTGTAAAAGCACTGATAGATGAAATTTTAGCTGCATTACCTGGCGGAGGCGGTTCCGGCGGCGGGGGCAGCTTCCCAACCATTCCCTTATCCAGGCTTTTTGACAACGCTATGCTCCGCGCCCATCGTCTGCACCAGCTGGCCTTTGACACCTACCAGGAGTTTGAAGAAGCCTATATCCCAAAGGAACAGAAGTATTCATTCCTGCAGAACCCCCAGACCTCCCTCTGTTTCTCAGAGTCTATTCCGACACCCTCCAACAGGGAGGAAACACAACAGAAATCCAACCTAGAGCTGCTCCGCATCTCCCTGCTGCTCATCCAGTCGTGGCTGGAGCCCGTGCAGTTCCTCAGGAGTGTCTTCGCCAACAGCCTGGTGTACGGCGCCTCTGACAGCAACGTCTATGACCTCCTAAAGGACCTAGAGGAAGGCATCCAAACGCTGATGGGGAGGCTGGAAGATGGCAGCCCCCGGACTGGGCAGATCTTCAAGCAGACCTACAGCAAGTTCGACACAAACTCACACAACGATGACGCACTACTCAAGAACTACGGGCTGCTCTACTGCTTCAGGAAGGACATGGACAAGGTCGAGACATTCCTGCGCATCGTGCAGTGCCGCTCTGTGGAGGGCAGCTGTGGCTTCTAGTAA(SEQ ID NO.2)

Example 2 preparation of mRNA-LNP drug (hereinafter abbreviated as ABD-linker-GH LNP) comprising an encoded ABD-linker-GH fusion protein

After obtaining a plasmid comprising the ABD-linker-GH sequence (nucleotide sequence shown in SEQ ID No. 13), the ABD-linker-GH mRNA construct was synthesized in vitro using T7 RNA polymerase-mediated DNA-dependent RNA transcription. In the transcription reaction, modified Uridine Triphosphate (UTP) was used. The mRNA was purified using DT chromatography columns to remove residual DNA template and double stranded RNA contamination during the enzymatic reaction of the synthetic mRNA. The mRNA was then concentrated and the buffer replaced. The mRNA construct also included a 5' cap structure and a Poly-a tail (Poly a) of 80 to 125 adenine nucleotides in length, the mRNA construct having a nucleotide sequence as shown in SEQ ID No. 10:

AGAAUAAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGGCUACAGGCUCCCGGACGUCCCUGCUCCUGGCUUUUGGCCUGCUCUGCCUGCCCUGGCUUCAAGAGGGCAGUGCCUUAGCUGAAGCUAAAGUCUUAGCUAACAGAGAACUUGACAAAUAUGGAGUAAGUGACUAUUACAAGAACCUAAUCAACAAUGCCAAAACUGUUGAAGGUGUAAAAGCACUGAUAGAUGAAAUUUUAGCUGCAUUACCUGGCGGAGGCGGUUCCGGCGGCGGGGGCAGCUUCCCAACCAUUCCCUUAUCCAGGCUUUUUGACAACGCUAUGCUCCGCGCCCAUCGUCUGCACCAGCUGGCCUUUGACACCUACCAGGAGUUUGAAGAAGCCUAUAUCCCAAAGGAACAGAAGUAUUCAUUCCUGCAGAACCCCCAGACCUCCCUCUGUUUCUCAGAGUCUAUUCCGACACCCUCCAACAGGGAGGAAACACAACAGAAAUCCAACCUAGAGCUGCUCCGCAUCUCCCUGCUGCUCAUCCAGUCGUGGCUGGAGCCCGUGCAGUUCCUCAGGAGUGUCUUCGCCAACAGCCUGGUGUACGGCGCCUCUGACAGCAACGUCUAUGACCUCCUAAAGGACCUAGAGGAAGGCAUCCAAACGCUGAUGGGGAGGCUGGAAGAUGGCAGCCCCCGGACUGGGCAGAUCUUCAAGCAGACCUACAGCAAGUUCGACACAAACUCACACAACGAUGACGCACUACUCAAGAACUACGGGCUGCUCUACUGCUUCAGGAAGGACAUGGACAAGGUCGAGACAUUCCUGCGCAUCGUGCAGUGCCGCUCUGUGGAGGGCAGCUGUGGCUUCUAGUAACUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUGGAGCUAGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA(SEQ ID NO.10).

GACTCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATAGACGAATTTAATACGACTCACTATAAGAATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACCATGGCTACAGGCTCCCGGACGTCCCTGCTCCTGGCTTTTGGCCTGCTCTGCCTGCCCTGGCTTCAAGAGGGCAGTGCCTTAGCTGAAGCTAAAGTCTTAGCTAACAGAGAACTTGACAAATATGGAGTAAGTGACTATTACAAGAACCTAATCAACAATGCCAAAACTGTTGAAGGTGTAAAAGCACTGATAGATGAAATTTTAGCTGCATTACCTGGCGGAGGCGGTTCCGGCGGCGGGGGCAGCTTCCCAACCATTCCCTTATCCAGGCTTTTTGACAACGCTATGCTCCGCGCCCATCGTCTGCACCAGCTGGCCTTTGACACCTACCAGGAGTTTGAAGAAGCCTATATCCCAAAGGAACAGAAGTATTCATTCCTGCAGAACCCCCAGACCTCCCTCTGTTTCTCAGAGTCTATTCCGACACCCTCCAACAGGGAGGAAACACAACAGAAATCCAACCTAGAGCTGCTCCGCATCTCCCTGCTGCTCATCCAGTCGTGGCTGGAGCCCGTGCAGTTCCTCAGGAGTGTCTTCGCCAACAGCCTGGTGTACGGCGCCTCTGACAGCAACGTCTATGACCTCCTAAAGGACCTAGAGGAAGGCATCCAAACGCTGATGGGGAGGCTGGAAGATGGCAGCCCCCGGACTGGGCAGATCTTCAAGCAGACCTACAGCAAGTTCGACACAAACTCACACAACGATGACGCACTACTCAAGAACTACGGGCTGCTCTACTGCTTCAGGAAGGACATGGACAAGGTCGAGACATTCCTGCGCATCGTGCAGTGCCGCTCTGTGGAGGGCAGCTGTGGCTTCTAGTAACTCGAGCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCCTGGAGCTAGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACGAGACCTAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTACTGGGCGGTTTTATGGACAGCAAGCGAACCGGAATTGCCAGCTGGGGCGCCCTCTGGTAAGGTTGGGAAGCCCTGCAAAGTAAACTGGATGGCTTTCTCGCCGCCAAGGATCTGATGGCGCAGGGGATCAAGCTCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAAGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGAGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAATTATTAACGCTTACAATTTCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATACAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATAGCACGTGCTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGGCTTTTGCTGGCCTTTTGCTCACATGTTCTT(SEQ ID NO.13).

hGH mRNA sequence:

GAGAAUAAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGUUCCCCACCAUCCCCCUGAGCAGACUGUUCGACAACGCCAUGCUGAGAGCCCACAGACUGCAUCAGCUGGCCUUCGACACCUACCAAGAGUUCGAGGAGGCCUACAUCCCCAAGGAGCAGAAGUACAGCUUCCUGCAGAACCCUCAGACAAGCCUGUGCUUCAGCGAGAGCAUCCCCACCCCUAGCAACAGAGAGGAGACACAGCAGAAGAGCAACCUGGAGCUGCUGAGAAUCAGCCUGCUCCUGAUUCAGAGCUGGCUGGAGCCCGUGCAGUUCCUGAGAAGCGUGUUCGCCAACAGCCUGGUGUACGGCGCUAGCGACAGCAACGUGUACGACCUGCUGAAGGACCUGGAGGAGGGCAUUCAGACCCUGAUGGGCAGACUGGAGGACGGCAGCCCUAGAACCGGGCAGAUCUUCAAGCAGACCUACAGCAAGUUCGACACCAACAGCCACAACGACGACGCCCUGCUGAAGAACUACGGCCUGCUGUACUGCUUCAGAAAGGACAUGGACAAGGUGGAAACCUUCCUGAGAAUCGUGCAGUGCAGAAGCGUGGAGGGCAGCUGCGGCUUCUGAUGACUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUGGAGCUAGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA(SEQ ID NO.15)

HSA-hGH mRNA sequence:

GAGAAUAAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGAAGUGGGUGACCUUCAUCAGCCUGCUGUUCCUGUUCAGCAGCGCCUACAGCAGAGGCGUGUUCAGAAGAGACGCCCACAAGAGCGAGGUGGCCCACAGAUUCAAGGACCUGGGCGAGGAGAACUUCAAGGCCCUGGUGCUGAUCGCCUUCGCUCAGUACCUGCAGCAGUGCCCCUUCGAGGACCACGUGAAGCUGGUGAACGAGGUGACCGAGUUCGCCAAGACCUGCGUGGCCGACGAGAGCGCCGAGAACUGCGACAAGAGCCUGCACACCCUGUUCGGCGACAAGCUGUGCACCGUGGCCACCCUGAGAGAAACCUACGGCGAGAUGGCCGACUGCUGCGCCAAGCAAGAGCCCGAGAGAAACGAGUGCUUCCUGCAGCACAAGGACGACAACCCCAACCUGCCUAGACUGGUCAGACCCGAGGUGGACGUGAUGUGCACCGCCUUCCACGACAACGAGGAAACCUUCCUGAAGAAGUACCUGUACGAGAUCGCUCGGAGACACCCCUACUUUUACGCCCCCGAGCUGCUGUUCUUCGCCAAGAGAUAUAAAGCCGCUUUUACAGAAUGCUGCCAAGCCGCCGACAAGGCCGCCUGCCUGCUGCCCAAGCUGGACGAGCUGAGAGACGAGGGCAAGGCUAGCAGCGCCAAGCAGAGACUGAAGUGCGCUAGCCUGCAGAAGUUCGGCGAGAGAGCCUUCAAGGCCUGGGCCGUGGCUAGACUGAGCCAAAGAUUCCCCAAGGCCGAGUUCGCCGAGGUGAGCAAGCUGGUGACCGACCUGACCAAGGUGCACACCGAGUGCUGCCACGGCGACCUGCUGGAGUGCGCCGACGACAGAGCCGACCUGGCCAAGUACAUCUGCGAGAACCAAGACAGCAUCAGCAGCAAGCUGAAGGAGUGCUGCGAGAAGCCCCUGCUGGAGAAGAGCCACUGCAUCGCCGAGGUGGAGAACGACGAGAUGCCCGCCGACCUGCCUAGCCUGGCCGCCGACUUCGUGGAGAGCAAGGACGUGUGCAAGAACUACGCCGAGGCCAAGGACGUGUUCCUGGGCAUGUUCCUGUACGAGUACGCUAGAAGACACCCCGACUACAGCGUCGUGCUGCUCCUGAGACUGGCCAAGACCUACGAAACCACCCUGGAGAAGUGCUGUGCCGCUGCCGACCCCCAUGAAUGUUACGCCAAGGUGUUCGACGAGUUCAAGCCUCUGGUGGAGGAGCCCCAAAACCUGAUCAAGCAGAACUGCGAGCUGUUCGAGCAGCUGGGCGAGUACAAGUUUCAGAACGCCCUGCUGGUGAGAUACACCAAGAAGGUGCCCCAAGUGAGCACACCCACCCUCGUGGAGGUGAGCAGAAACCUGGGCAAGGUGGGCAGCAAGUGUUGUAAGCACCCUGAGGCCAAGAGAAUGCCCUGCGCCGAGGACUACCUGAGCGUGGUGCUGAAUCAGCUGUGCGUGCUGCACGAGAAGACCCCCGUGAGCGACAGAGUGACCAAGUGCUGCACCGAGAGCCUGGUGAACAGAAGACCCUGCUUCAGCGCCCUGGAGGUGGACGAAACCUACGUGCCCAAGGAGUUCAACGCCGAAACCUUCACCUUCCACGCCGACAUCUGCACCCUGAGCGAGAAGGAGAGACAGAUCAAGAAGCAGACCGCCCUGGUGGAGCUGGUGAAGCACAAGCCCAAGGCCACCAAGGAGCAGCUGAAGGCCGUGAUGGACGACUUCGCCGCCUUCGUGGAGAAGUGUUGUAAGGCCGACGACAAGGAAACCUGCUUCGCCGAGGAAGGCAAAAAGCUCGUGGCCGCUAGCCAAGCCGCUCUGGGCCUCGGCGGGGGCGGCUCCGGGGGCGGGGGCAGCGGGGGCGGGGGGAGCUUCCCCACCAUCCCCCUGAGCAGACUGUUCGACAACGCCAUGCUGAGAGCCCACAGACUGCAUCAGCUGGCCUUCGACACCUACCAAGAGUUCGAGGAGGCCUACAUCCCCAAGGAGCAGAAGUACAGCUUCCUGCAGAACCCUCAGACAAGCCUGUGCUUCAGCGAGAGCAUCCCCACCCCUAGCAACAGAGAGGAGACACAGCAGAAGAGCAACCUGGAGCUGCUGAGAAUCUCCCUGCUCCUGAUUCAGAGCUGGCUGGAGCCCGUGCAGUUCCUGAGAAGCGUGUUCGCCAACAGCCUGGUGUACGGCGCUAGCGACAGCAACGUGUACGACCUGCUGAAGGACCUGGAGGAGGGCAUUCAGACCCUGAUGGGCAGACUGGAGGACGGCAGCCCUAGAACCGGGCAGAUCUUCAAGCAGACCUACAGCAAGUUCGACACCAACAGCCACAACGACGACGCCCUGCUGAAGAACUACGGCCUGCUGUACUGCUUCAGAAAGGACAUGGACAAGGUGGAAACCUUCCUGAGAAUCGUGCAGUGCAGAAGCGUGGAGGGCAGCUGCGGCUUCUGAUGACUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUGGAGCUAGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA(SEQ ID NO.16)

Reference is made to the method for preparing mRNA-LNP disclosed in patent 202211461172.X, and a series of mRNA-LNP drugs encoding ABD-linker-GH fusion proteins are prepared by selecting the ionizable lipids E1-E28 and the corresponding LNP components in example 1 of this patent. An exemplary mRNA-LNP drug was prepared by dissolving four lipids (ionizable lipid E1: DOPE: cholesterol: DMG-PEG 2000) in ethanol phase, dissolving ABD-linker-GH mRNA in sodium acetate buffer, assembling LNP by microfluidic technology, measuring mRNA concentration in the formulation by Ribogreen fluorometry after removal of ethanol by HEPES buffer dialysis, adjusting its concentration to 0.2mg/mL, and filter-sterilizing by 0.22 μm PES filter. The mRNA content and encapsulation efficiency of LNP were determined by RiboGreen, mRNA integrity was characterized by capillary gel electrophoresis, particle size and dispersity by dynamic light scattering (Malvern Zetasizer Nano ZS) were 61.54nm and 0.1788, respectively.

Example 3 expression level of ABD-linker-GH mRNA in cells

HEK293T cells are inoculated into a 12-well plate one day in advance, and when the cells reach 70-90% confluence, mRNA transfection is carried out according to a Lipofectamine ^TM reagent experimental scheme, and the volume of the mRNA transfection is 3 mug/well. Supernatants were collected 24h,48h post transfection.

Growth hormone concentrations in supernatants at each time point were measured using Human Growth Hormone (hGH) ELISA Kit, specific experimental steps are referred to the Kit instructions. As a result, the ABD-linker-GH mRNA expressed the fusion protein ABD-linker-GH efficiently, as shown in FIG. 2.

EXAMPLE 4 biological Activity assay of ABD-linker-GH

Half-life in rats after single intravenous administration of ABD-linker-GH LNP drug

Male mature Sprague Dawley rats (about 5-6 weeks old, weight 130-180 g) were divided into 4 groups of 3, each of which were respectively administered by intravenous injection with equal volumes of physiological saline, 500. Mu.g of hGH mRNA-LNP (hereinafter referred to as hGH LNP) drug (preparation method was prepared as in example 2, hGH mRNA commission third party company, SEQ ID NO. 15), 500. Mu. gHSA-hGH mRNA-LNP drug (hereinafter referred to as HSA-hGH LNP) (preparation method was prepared as in example 2, HSA-hGH mRNA commission third party company, SEQ ID NO. 16) and 500. Mu.g of ABD-linker-GH LNP drug, and blood was sampled after administration of 0, 4, 24, 48, 72, 120, 168h, and H content in serum of rats was detected by means of Human Growth Hormone (hGH) ELISA Kit for specific experimental procedures. The detection result is shown in figure 3, and the result shows that the plasma half-life of the ABD-linker-GH LNP in the rat body is longer than that of hGH LNP and HSA-hGH LNP, and the peak blood concentration is improved by 3-4 times.

4-2 Test of Effect of ABD-linker-GH LNP drug after Single dose intravenous administration Using rats

Male mature Sprague Dawley rats (about 5-6 weeks old, weight 130-180 g) were divided into 4 groups of 3 animals each, each of which was individually and intravenously injected with an equal volume of physiological saline, 500 μg hGH LNP drug, 500 μg HSA-hGH LNP drug and 500 μg gABD-linker-GH LNP drug, and blood was sampled after administration for 0, 4, 24, 48, 72, 120, 168 hours, and IGF-1 content in Rat serum was detected using a Mouse/Rat IGF-1ELISA Kit, for specific experimental procedures, reference Kit instructions. As shown in FIG. 4, the results show that the intravenous injection of both ABD-linker-GH LNP and HSA-hGH LNP can cause the rise of IGF-1 level in rat serum, wherein the rise of IGF-1 level caused by ABD-linker-GH LNP is longer than that caused by hGH LNP and HSA-hGH LNP, the peak value is prolonged by about 24 hours, and the level is increased by nearly 30 times than that of HSA-hGH LNP.

4-3 Activity data in rats after single subcutaneous administration of ABD-linker-GH LNP drug

The administration mode is further replaced in the example, and the in vivo activity of the ABD-linker-GH LNP medicine prepared by the invention in rats is detected by subcutaneous administration mode, and the result shows that compared with intravenous administration of the ABD-linker-GH LNP medicine, the plasma half-life period of the medicine is prolonged by about 24 hours, the IGF-1 level in the plasma is improved by 24 hours, and simultaneously, the half-life period of the plasma hGH and the IGF-1 level in the plasma are longer compared with subcutaneous administration of the hGH LNP and the HSA-hGH LNP.

Example 5 preparation of drug and efficacy data containing mRNA-LNP encoding other ABD-linker-GH fusion proteins

Referring to the preparation method and activity test experiment in example 4, the mRNA sequences of other ABD-linker-GH fusion proteins were replaced with SEQ ID NO.17, SEQ ID NO.18 and SEQ ID NO.19, and the prepared mRNA-LNP drugs were abbreviated as SEQ ID NO.17LNP, SEQ ID NO.18LNP and SEQ ID NO.19LNP, respectively.

SEQ ID NO 17 ABD11-linker7-GH12 mRNA sequence:

AGAAUAAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGGCCACA

GGCAGCCGGACCUCUCUGCUGCUGGCCUUCGGCCUGCUGUGCCUGCCUUGGCUGCAAGAGGGCUC

CGCCCUGGCCGAGGCUAAGGUGCUGGCUAAUCGGGAACUGGAUAAGUACGGCGUCUCCGACUAC

UACAAGAACCUGAUCAACAACGCUAAGACCGUGGAAGGCGUGAAGGCCCUGAUCGACGAGAUCC

UGGCCGCCCUCCCCGGCGGAGGUGGCUCUGGAGGCGGUGGAUCUGGUGGUGGCGGAUCAGGUGG

AGGCGGGAGCGGAGGCGGCGGUAGUUUCCCUACAAUCCCACUGAGCAGACUGUUCGAUAACGCC

AUGCUGCGGGCCCACAGACUGCACCAGCUGGCUUUUGAUACCUACCAGGAGUUCGAGGAAGCCU

ACAUCCCCAAGGAACAGAAGUACUCUUUUCUGCAGAACCCCCAGACCAGCCUGUGCUUCAGCGAG

AGCAUCCCUACCCCUAGCAACAGAGAGGAAACCCAGCAGAAAAGCAACCUGGAGCUGCUGCGGAU

CAGCCUGCUGCUGAUUCAGAGCUGGCUGGAGCCUGUGCAGUUCCUGAGGUCCGUUUUCGCCAAU

AGCCUGGUGUACGGCGCCAGCGACAGCAAUGUGUACGACCUGCUCAAAGAUCUGGAGGAGGGCA

UCCAGACACUGAUGGGCAGACUGGAAGAUGGCUCUCCUAGAACCGGCCAGAUCUUCAAGCAGAC

CUAUAGCAAGUUCGACACCAACAGCCACAACGACGACGCCCUGCUUAAAAACUACGGCCUGCUGU

ACUGCUUCAGAAAGGACAUGGACAAGGUGGAGACAUUUCUGAGAAUCGUGCAAUGUAGAAGCGU

GGAAGGCUCUUGCGGCUUCCUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCG

UCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCAC

UCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCC

UAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAG

CUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUGGAGCUAGCAAAAAAAAAA

AAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

SEQ ID NO 18 ABD3-linker7-GH9 mRNA sequence

AGAAUAAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGGCCACC

GGCAGCAGAACAAGCCUGCUGCUUGCCUUCGGCCUGCUGUGCCUGCCUUGGCUGCAGGAGGGCUC

CGCCCUGGCCGAGGCUAAAGAGCUGGCCAUCCGGGAACUGAAAAAGUACGGCAUCAGCGAUUAC

UACAAGAACCUGAUUAACAAGGCCAAGACCGUGGAAGGCGUGAAGGCCCUGAAAGAUGAGAUCC

UGGCCGCUUUGCCUGGCGGAGGUGGCUCUGGAGGCGGUGGAUCUGGUGGUGGCGGAUCAGGUGG

AGGCGGGAGCGGAGGCGGCGGUAGUUUUCCAACCAUCCCCCUGAGCCGGCUGUUCGACAACGCCA

UGCUGCGGGCCCACAGACUGCACCAGCUGGCUUUUGAUACAUACCAGGAGUUCGAGGAAGCCUA

CAUCCCUAAGGAACAGAAGUAUAGCUUCCUGCAGAACCCCCAGACCAGCCUCUGUUUUUCUGAGA

GCAUCCCUACCCCUUCUAAUAGAGAGGAGACACAGCAGAAAUCUAACCUGGAGCUGCUCAGAAU

CAGCCUGCUGCUGAUCCAGAGCUGGCUGGAACCUGUGCAGUUCCUGAGGUCCGUGUUCGCAAAU

AGCCUGGUGUACGGCGCCAGCGACAGCAACGUGUACGACCUGCUGAAGGAUCUGGAAGAGGGAA

UCCAAACCCUGAUGGGCAGACUGGAAGAUGGCAGUCCAGACCCCGGCCAGAUCUUCGCCCAGACC

UACAGCAAGUUCGACACCAACAGCCACAACGACGACGCCCUGCUGAAGAACUACGGACUGCUGUA

CUGCUUCAGAAAGGACAUGGACAAGGUGGAAACAUUCCUGCGGAUCGUUCAAUGUAGAAGCGUC

GAGGGCAGCUGCGGCUUCCUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGU

CCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACU

CACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCU

AGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGC

UAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUGGAGCUAGCAAAAAAAAAAA

AAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

SEQ ID NO 19 ABD11-linker6-GH9 mRNA sequence

AGAAUAAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGGCCACAGGCUCUCGGACCUCCCUCCUGCUGGCCUUCGGACUGCUGUGCCUGCCUUGGCUGCAGGAGGGCAGCGCCCUGGCUGAAGCCAAAGUGCUGGCUAAUAGGGAACUGGACAAGUACGGCGUGAGCGAUUACUACAAGAACCUGAUCAACAACGCCAAGACAGUGGAAGGCGUGAAGGCCCUGAUCGACGAGAUCCUGGCCGCCCUGCCCGGCGGAGGAGGCAGCUUUCCAACCAUCCCUCUGUCUAGACUGUUCGACAACGCCAUGCUGCGGGCCCACCGGCUGCACCAGCUGGCCUUUGAUACAUACCAGGAGUUCGAGGAAGCCUACAUCCCCAAGGAACAGAAGUACAGCUUCCUGCAAAAUCCUCAGACCUCUCUGUGUUUUAGCGAGUCCAUCCCCACCCCUAGCAACAGAGAGGAGACACAGCAGAAAUCUAAUCUGGAGCUGCUGAGAAUCAGCCUGCUCCUGAUUCAGAGCUGGCUGGAGCCAGUUCAGUUCCUGCGGAGCGUGUUCGCUAACUCUCUGGUGUACGGCGCCAGCGACAGCAACGUGUACGACCUGCUGAAGGACCUGGAGGAAGGCAUCCAGACCCUGAUGGGCAGACUGGAGGAUGGCUCCCCUGAUCCUGGACAGAUCUUCGCUCAGACCUAUAGCAAGUUCGAUACCAACAGCCACAACGACGACGCCCUUCUGAAAAACUACGGCCUGCUGUACUGCUUCAGAAAGGACAUGGACAAGGUGGAAACCUUCCUGAGAAUCGUGCAAUGCAGAAGCGUCGAGGGCAGCUGCGGCUUCCUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUGGAGCUAGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

The results of the experiment are shown in FIGS. 5 and 6, in which IGF-1 levels were elevated for longer than hGH LNP and HSA-hGH LNP, with peak values of approximately 24h prolonged and levels approximately 10-30 fold higher than HSA-hGH LNP, both subcutaneously and intravenously.

In summary, the invention mainly aims at the problems of HSA-hGH fusion protein, and the mRNA of the target drug is encapsulated by LNP (lipid nanoparticle) to form a growth hormone nucleic acid drug with longer acting and high activity, so that the relatively high biological activity can be achieved under the premise of lower administration dosage.

The above examples are provided to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. Further, various modifications of the methods set forth herein, as well as variations of the methods of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the present invention.

Claims

1. A fusion protein comprising an ABD domain of streptococcal protein G linked to a growth hormone via a linker peptide, said ABD domain having an amino acid sequence as shown in SEQ ID No.11 or having 95% or more similarity to SEQ ID No.11 and having the function of the amino acid sequence defined by SEQ ID No.11, and having an amino acid sequence as shown in SEQ ID No.12 or having 95% or more similarity to SEQ ID No.12 and having the function of the amino acid sequence defined by SEQ ID No. 12.

2. The fusion protein of claim 1, wherein the amino acid sequence of the ABD domain is further shown as any one of SEQ ID nos. 3 to 5 or has 95% or more similarity to any one of SEQ ID nos. 3 to 5.

3. The fusion protein of claim 1, wherein the amino acid sequence of the growth hormone is further as shown in SEQ ID No.9 or has 95% or more similarity to SEQ ID No. 9.

4. The fusion protein of claim 1, wherein the linker peptide is selected from the group consisting of a rigid linker peptide and a flexible linker peptide, preferably wherein the rigid linker peptide is (EAAAK) x n, n=1-5, and wherein the flexible linker peptide is selected from the group consisting of (GGGGS) x n, (GSGGG) x n, (GGGSG) x n, and (GGSGG) x n, n=1-5.

5. The fusion protein of claim 1, wherein the amino acid sequence of the linker peptide is as shown in any one of SEQ ID nos. 6 to 8 or a combination thereof or has a similarity of 95% or more with any one of SEQ ID nos. 6 to 8 or a combination thereof.

6. The fusion protein of claim 1, wherein the fusion protein has an amino acid sequence as shown in SEQ ID No.1 or has 95% or more similarity to SEQ ID No.1 or as shown in SEQ ID No.14 or has 95% or more similarity to SEQ ID No. 14.

7. A nucleic acid molecule comprising a nucleotide sequence encoding the fusion protein of any one of claims 1-6.

8. The nucleic acid molecule of claim 7, wherein said nucleic acid molecule comprises a nucleotide sequence as shown in SEQ ID No.2 or having 95% or more similarity to SEQ ID No.2 or as shown in SEQ ID No.13 or having 95% or more similarity to SEQ ID No. 13.

9. The nucleic acid molecule of claim 7, wherein the nucleic acid molecule is mRNA.

10. The nucleic acid molecule of claim 9, wherein the nucleotide sequence of said nucleic acid molecule is as set forth in SEQ ID No.10 or has 95% or more similarity to SEQ ID No. 10.

11. A cell comprising or used in the preparation of the fusion protein of any one of claims 1-6, the nucleic acid molecule of any one or more of claims 7-10.

12. A pharmaceutical composition comprising any one or more of the fusion protein of any one of claims 1-6, the nucleic acid molecule of any one of claims 7-10, preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and/or adjuvant.

13. The pharmaceutical composition according to claim 12, wherein the pharmaceutical composition comprises the nucleic acid molecule according to any one of claims 9-10 and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier is a lipid nanoparticle, preferably wherein the pharmaceutical composition further comprises an adjuvant.

14. The pharmaceutical composition of claim 13, wherein the lipid nanoparticle comprises at least one ionizable lipid compound, preferably the lipid nanoparticle comprises at least one ionizable lipid compound in combination with any one or more of a structural lipid, a helper lipid, a PEG-lipid, a polymer, preferably the structural lipid is selected from one or more combinations of sterols, non-sterols or respective derivatives, preferably the helper lipid is selected from one or more combinations of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, phosphatidylserine, phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, dimyristoyl phosphatidylglycerol, DOTAP, dolap, 18:1pa, HS15, GL67, preferably the PEG-lipid is selected from one or more combinations of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-diacylglycerol, PEG-modified dialkylglycerol, methoxypolyethylene glycol diacetyl acetamide.

15. The pharmaceutical composition of claim 14, wherein the lipid nanoparticle comprises 20-65% ionizable lipid compound, 20-60% structural lipid, 3-40% helper lipid, 0.1-10% peg-lipid, wherein% refers to mole percent.

16. Use of the fusion protein according to any one of claims 1-6, the nucleic acid molecule according to any one of claims 7-10, the pharmaceutical composition according to any one of claims 12-15 or any combination thereof for the manufacture of a medicament for the treatment of a disease which is suitable for treatment with growth hormone.

17. The use according to claim 16, wherein the disease is selected from growth hormone deficiency, dwarfism in children, prairie-wegener's syndrome, tenna's syndrome, intrauterine growth retardation, idiopathic stump, chronic renal insufficiency, renal failure, polycystic ovary syndrome infertility, central precocity in children, short bowel syndrome in adults, prader-willi syndrome or burns.