Growth hormone fusion protein, nucleic acid drug and use thereof

Abstract

The present invention relates to the field of medicine, and in particular to a growth hormone fusion protein, a nucleic acid drug and a use thereof. The growth hormone fusion protein comprises an ABD domain of streptococcal protein G and a growth hormone, the ABD domain and growth hormone being connected by means of a linker. The fusion protein of the present invention has a low molecular weight, which avoids the problems of high molecular weight growth hormone fusion proteins having difficulty penetrating into target tissues and having low biological activity, and has higher biological activity. Compared with a traditional protein drug, an mRNA-LNP drug encoding a fusion protein can directly use human cells to express a protein drug, and the protein drug produced by the body itself has high activity, which can significantly improve drug efficacy and achieve relatively high biological activity at a relatively low dosage.

Description

Growth hormone fusion protein, nucleic acid drug and use thereof Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a growth hormone fusion protein, a nucleic acid drug and uses thereof. Background Art

[0002] Human growth hormone (hGH) is a peptide hormone secreted by eosinophils in the anterior pituitary gland. It consists of 191 amino acids, most of which exist as a 22 kDa molecular weight. By binding to growth hormone receptors on receptor cells in various tissues, organs, and bones, hGH promotes protein synthesis and fat breakdown in these cells, including the expression of insulin-like growth factor-1 (IGF-1), which is involved in cell growth and regeneration. This, in turn, promotes metabolism and growth. Clinically, it is primarily used to treat childhood growth hormone deficiency, chronic renal failure, slow growth caused by Turner syndrome, and severe burns.

[0003] As a small molecule polypeptide, hGH is easily denatured or degraded by enzymes in the blood, liver or kidneys. Therefore, it has a half-life of only a few hours in the blood. In clinical application, the dosage frequency is high (usually daily) and is accompanied by corresponding side effects (such as injection site discomfort, transient edema and joint pain). Patient compliance is poor, which greatly limits its clinical application.

[0004] To overcome the short-lived effects of hGH, fusion proteins have been used to develop long-acting growth hormones, such as growth hormone fused to human serum albumin (HSA). Mature HSA is a 585-amino acid protein that participates in FcRn-mediated recycling and has a plasma half-life of 14-20 days. Therefore, fusion with hGH can extend the half-life of hGH.

[0005] However, the structure and size of hGH are strictly conserved across species. This size conservation may represent an evolutionary control, enabling hGH to pass through both less vascularized tissues (fat, bone, and growth plates) and well-vascularized tissues (muscle and heart). While HSA fusion proteins can extend the half-life and reduce hGH clearance, their significantly increased molecular weight actually hinders hGH absorption into target tissues, reducing its biological activity. Furthermore, the current half-life of HSA-hGH fusion proteins is 58-80 hours (data published in CN109851674B), leaving room for improvement to further reduce dosing frequency.

[0006] At present, traditional fusion protein drugs in clinical practice are mainly antibody protein drugs. Antibody protein drugs have complex molecular structures, high production costs, and generally require injection. In addition, this type of drug can usually only work with proteins on the cell membrane surface or outside the cell, which limits its application to a certain extent.

[0007] Compared with antibody protein drugs or other types of protein drugs, nucleic acid drugs have unparalleled advantages in the number of drug targets, drug design cycle, target specificity, high efficiency and durability. Especially after the outbreak of the new crown epidemic, the efficient research and development of mRNA vaccines has driven the rapid development of the entire nucleic acid drug field. In the future, with the advancement of clinical trials and the maturity of related technologies, nucleic acid drugs will have a wider range of application scenarios.

[0008] Summary of the Invention

[0009] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a growth hormone fusion protein, a nucleic acid drug and uses thereof, so as to solve the problems in the prior art.

[0010] To achieve the above objectives and other related objectives, the present invention provides a fusion protein, comprising the ABD domain of streptococcal protein G and growth hormone, wherein the ABD domain and the growth hormone are connected via a linker peptide, the amino acid sequence of the ABD domain is as shown in SEQ ID NO.11 or has 95% or more similarity to 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 as shown in SEQ ID NO.12 or has 95% or more similarity to SEQ ID NO.12, and has the function of the amino acid sequence defined by SEQ ID NO.12.

[0011] The present invention also provides a nucleic acid molecule, which includes a nucleotide sequence encoding the fusion protein.

[0012] The present invention also provides a cell, wherein the cell contains or is used to prepare any one or more of the fusion protein and the nucleic acid molecule.

[0013] The present invention also provides a pharmaceutical composition, which comprises any one or more of the above-mentioned fusion protein and the above-mentioned nucleic acid molecule; preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0014] The present invention also provides use of the fusion protein, the nucleic acid molecule, the pharmaceutical composition or any combination thereof in preparing a drug for treating a disease, wherein the disease is a disease suitable for treatment with growth hormone.

[0015] As described above, the growth hormone fusion protein, nucleic acid drug and use thereof of the present invention have the following beneficial effects:

[0016] 1. The ABD-linker-GH fusion protein of the present invention utilizes the interaction between ABD and HSA to enhance FcRn-mediated recycling of the fusion protein, thereby extending its half-life. Compared to HSA-hGH fusion proteins, the binding of ABD to native HSA more effectively utilizes FcRn-mediated recycling, further extending the half-life of the fusion protein and reducing dosing frequency.

[0017] 2. Compared with other fusion protein drugs, the ABD-linker-GH fusion protein of the present invention has a small molecular weight, which avoids the problem that large molecular weight hGH has difficulty penetrating into target tissues and has low biological activity, and has higher biological activity.

[0018] 3. The ABD-linker-GH fusion protein of the present invention is developed into an mRNA-LNP drug. Compared with traditional protein drugs, it can directly utilize the human body's own cells to express protein drugs. The protein drugs produced by the body itself have high activity, can significantly improve the efficacy, and can achieve relatively high biological activity at a lower dosage. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG1 is a schematic diagram showing the structure of the fusion protein of the present invention.

[0020] FIG2 shows the expression level of the fusion ABD-linker-GH mRNA of the present invention in the supernatant of HEK293T cells.

[0021] FIG3 shows the changes in hGH levels in rat serum after intravenous injection of different ABD-linker-GH mRNA-LNPs.

[0022] FIG4 shows the changes in IGF-1 levels in rat serum after intravenous injection of different ABD-linker-GH mRNA-LNPs.

[0023] FIG5 shows the changes in hGH content in rat serum after intravenous injection of different ABD-linker-GH mRNA-LNPs.

[0024] FIG6 shows the changes in IGF-1 levels in rat serum after intravenous injection of different ABD-linker-GH mRNA-LNPs. DETAILED DESCRIPTION

[0025] The present invention provides a fusion protein named ABD-linker-GH, which comprises the ABD domain of streptococcal protein G and growth hormone, wherein the ABD domain and the growth hormone are connected via a linker peptide. The amino acid sequence of the ABD domain is as shown in SEQ ID NO.11 or has 95% or greater similarity to SEQ ID NO.11, and has the function of the amino acid sequence defined by SEQ ID NO.11. The amino acid sequence of the growth hormone is as shown in SEQ ID NO.12 or has 95% or greater similarity to SEQ ID NO.12, and has the function of the amino acid sequence defined by SEQ ID NO.12.

[0026] The ABD domain can be a wild-type domain or a mutant domain of Streptococcal protein G from different species, and is not specifically limited in the present invention. Specifically, the ABD domain is the GA module of Streptococcal protein G (abbreviated as SPG) (GA module, proposed by Johansson et al., J Mol Biol 266:859-865, 1997; Johansson et al., J Bio Chem 277:8114-8120, 2002, a protein G-related albumin binding module, a triple-helical protein domain responsible for albumin binding).

[0027] Streptococcal protein G on the surface of Streptococcus strains has a domain that binds to serum albumin, a three-helix bundle structure identified as the ABD (albumin binding domain). Therefore, the present invention fuses the 46-amino acid ABD peptide of Streptococcus G148 G protein to GH via a linker to form an ABD-linker-GH fusion protein. By leveraging the interaction between ABD and HSA, the fusion protein enhances FcRn-mediated recycling, thereby extending its half-life. Due to its small molecular weight, the ABD has minimal impact on the biological activity of hGH.

[0028] The motif described in the present invention is generally defined as a short sequence or pattern that appears repeatedly in different organisms or molecules and has specific functional or structural characteristics.

[0029] 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 to the amino acid sequence shown in SEQ ID NO.11, or by adding one or more (specifically 1-50, 1-30, 1-20, 1-10, 1-5 or 1-3) amino acids to the N-terminus and / or C-terminus, and has the function of a polypeptide fragment with an amino acid sequence as shown in SEQ ID NO.11. The amino acid sequence obtained after the substitution, deletion or addition of amino acids may have a sequence similarity of 95%, 96%, 97%, 98% or 99% or more to SEQ ID NO.11.

[0030] In certain embodiments of the present invention, the amino acid sequence of the ABD domain may also be as shown in any of SEQ ID NOs. 3 to 5, or have 95% or greater similarity to any of SEQ ID NOs. 3 to 5. Among them, SEQ ID NO. 3 is the amino acid sequence of protein G from Streptococcus dysenteriae, SEQ ID NO. 4 is the amino acid sequence of G protein GA3 mutant 1 from Streptococcus G148, and SEQ ID NO. 5 is the amino acid sequence of G protein GA3 mutant 2 from Streptococcus G148.

[0031] In certain embodiments of the present invention, the growth hormone is selected from growth hormone of mammals, such as humans, cows, pigs, mice, and monkeys.

[0032] In certain embodiments of the present invention, the growth hormone is human growth hormone (hGH), and the amino acid sequence of the growth hormone can 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 as shown in SEQ ID NO. 12, or by adding one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, or 1-3) amino acids to the N-terminus and / or C-terminus, and has the function of a polypeptide fragment with the amino acid sequence as shown in SEQ ID NO. 12. The amino acid sequence obtained after the substitution, deletion, or addition of amino acids may have a sequence similarity of 95%, 96%, 97%, 98%, or 99% or more to SEQ ID NO. 12.

[0033] In certain embodiments of the present invention, the amino acid sequence of the growth hormone may also be shown as SEQ ID NO.9.

[0034] The connecting peptide (linker) is not specifically limited, as long as it can connect the ABD domain to the growth hormone, and the ABD domain and the growth hormone are not limited to play a role. In certain embodiments of the present invention, the connecting peptide (linker) can be selected from a rigid connecting peptide (linker) or a flexible connecting peptide (linker). The rigid connecting 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.

[0035] In certain embodiments of the present invention, the amino acid sequence of the linker is as shown in any one of SEQ ID NOs. 6 to 8, or has 95% or greater similarity to any one of SEQ ID NOs. 6 to 8, or a combination thereof.

[0036] (GGGGS)1 (SEQ ID NO.6)

[0037] (GGGGS)5 (SEQ ID NO.7)

[0038] (EAAAK)2 (SEQ ID NO.8)

[0039] In certain embodiments of the present invention, the fusion protein comprises, from the N-terminus to the C-terminus, the following sequence: ABD domain, linker peptide, growth hormone. After expression, the fusion protein further comprises a growth hormone signal peptide at the N-terminus before being secreted outside the cell.

[0040] In certain embodiments of the present invention, ABD-linker-GH can be formed in any of the following ways:

[0041] 1) an ABD domain as shown in SEQ ID NO.11, a linker as shown in SEQ ID NO.6, and a growth hormone as shown in SEQ ID NO.12;

[0042] 2) an ABD domain as shown in SEQ ID NO.11, a linker as shown in SEQ ID NO.7, and a growth hormone as shown in SEQ ID NO.12;

[0043] 3) an ABD domain as shown in SEQ ID NO.11, a linker as shown in SEQ ID NO.8, and a growth hormone as shown in SEQ ID NO.12;

[0044] 4) an ABD domain as shown in SEQ ID NO.11, a linker as shown in SEQ ID NO.6, and a growth hormone as shown in SEQ ID NO.9;

[0045] 5) an ABD domain as shown in SEQ ID NO.11, a linker as shown in SEQ ID NO.7, and a growth hormone as shown in SEQ ID NO.9;

[0046] 6) an ABD domain as shown in SEQ ID NO.11, a linker as shown in SEQ ID NO.8, and a growth hormone as shown in SEQ ID NO.9;

[0047] 7) The ABD domain shown in SEQ ID NO. 3, the linker shown in SEQ ID NO. 6, and the growth hormone shown in SEQ ID NO. 12;

[0048] 8) The ABD domain shown in SEQ ID NO. 3, the linker shown in SEQ ID NO. 7, and the growth hormone shown in SEQ ID NO. 12;

[0049] 9) The ABD domain shown in SEQ ID NO. 3, the linker shown in SEQ ID NO. 8, and the growth hormone shown in SEQ ID NO. 12;

[0050] 10) The ABD domain shown in SEQ ID NO. 3, the linker shown in SEQ ID NO. 6, and the growth hormone shown in SEQ ID NO. 9;

[0051] 11) The ABD domain shown in SEQ ID NO. 3, the linker shown in SEQ ID NO. 7, and the growth hormone shown in SEQ ID NO. 9;

[0052] 12) The ABD domain shown in SEQ ID NO. 3, the linker shown in SEQ ID NO. 8, and the growth hormone shown in SEQ ID NO. 9;

[0053] 13) The ABD domain shown in SEQ ID NO. 4, the linker shown in SEQ ID NO. 6, and the growth hormone shown in SEQ ID NO. 12;

[0054] 14) The ABD domain shown in SEQ ID NO. 4, the linker shown in SEQ ID NO. 7, and the growth hormone shown in SEQ ID NO. 12;

[0055] 15) The ABD domain shown in SEQ ID NO. 4, the linker shown in SEQ ID NO. 8, and the growth hormone shown in SEQ ID NO. 12;

[0056] 16) The ABD domain shown in SEQ ID NO. 4, the linker shown in SEQ ID NO. 6, and the growth hormone shown in SEQ ID NO. 9;

[0057] 17) The ABD domain shown in SEQ ID NO. 4, the linker shown in SEQ ID NO. 7, and the growth hormone shown in SEQ ID NO. 9;

[0058] 18) The ABD domain shown in SEQ ID NO. 4, the linker shown in SEQ ID NO. 8, and the growth hormone shown in SEQ ID NO. 9;

[0059] 19) The ABD domain shown in SEQ ID NO. 5, the linker shown in SEQ ID NO. 6, and the growth hormone shown in SEQ ID NO. 12;

[0060] 20) The ABD domain shown in SEQ ID NO. 5, the linker shown in SEQ ID NO. 7, and the growth hormone shown in SEQ ID NO. 12;

[0061] 21) The ABD domain shown in SEQ ID NO. 5, the linker shown in SEQ ID NO. 8, and the growth hormone shown in SEQ ID NO. 12;

[0062] 22) The ABD domain shown in SEQ ID NO. 5, the linker shown in SEQ ID NO. 6, and the growth hormone shown in SEQ ID NO. 9;

[0063] 23) The ABD domain shown in SEQ ID NO. 5, the linker shown in SEQ ID NO. 7, and the growth hormone shown in SEQ ID NO. 9;

[0064] 24) The ABD domain shown in SEQ ID NO. 5, the linker shown in SEQ ID NO. 8, and the growth hormone shown in SEQ ID NO. 9.

[0065] In certain embodiments of the present invention, the amino acid sequence of the fusion protein is shown as SEQ ID NO.1 or SEQ ID NO.14.

[0066] In certain embodiments of the present invention, the amino acid sequence of the fusion protein is shown as SEQ ID NO.20, SEQ ID NO.21 or SEQ ID NO.22.

[0067] The present invention also provides a nucleic acid molecule, which includes a nucleotide sequence encoding the fusion protein.

[0068] The "nucleic acid molecule" of the present invention can 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 RNA (lncRNA), micro non-coding RNA (miRNA and siRNA), telomerase RNA (Telomerase RNA Component), small RNA (snRNA and scRNA), circular RNA (circRNA), synthetic miRNA (miRNA mimics, miRNA agomir, miRNA antagomir), antisense oligonucleotide (ASO), ribozyme, asymmetric interfering RNA (aiRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), guide RNA (gRNA), small guide RNA (sgRNA), locked nucleic acid (LNA), peptide nucleic acid (PNA), morpholino antisense oligonucleotide, morpholino oligonucleotide or a combination of one or more of a custom biological oligonucleotide.

[0069] In certain embodiments of the present invention, the nucleic acid molecule is a DNA nucleic acid molecule.

[0070] In certain embodiments of the invention, the nucleic acid molecule is plasmid DNA.

[0071] In certain embodiments of the invention, the nucleic acid molecule is mRNA.

[0072] In certain embodiments of the present invention, the nucleic acid molecule comprises, from the 5' end to the 3' end, nucleotides encoding the signal peptide of growth hormone - the ABD domain - the linker peptide - the growth hormone.

[0073] In certain embodiments of the present invention, the nucleotide sequence encoding the fusion protein is shown as SEQ ID NO.2.

[0074] In certain embodiments of the present invention, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO.13.

[0075] In certain embodiments of the present invention, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO.10.

[0076] The present invention also provides a cell, wherein the cell contains or is used to prepare any one or more of the fusion protein and the nucleic acid molecule.

[0077] In some embodiments, the cell genome integrates a nucleotide sequence as shown in SEQ ID NO. 2 or 13, or a nucleotide sequence with 95% or greater similarity to either of them.

[0078] In some embodiments, the cell is a cell used to produce the nucleic acid molecule and / or the nucleic acid molecule-encoded fusion protein.

[0079] In some embodiments, the cells include, but are not limited to, any one of mammalian cells (such as CHO, COS, and N2A), plant cells, human cells (human cervical cancer cells such as HELA and human embryonic kidney cells such as HEK293T), bacterial cells (such as Escherichia coli, Streptomyces, Salmonella typhimurium), fungal cells (such as yeast), and insect cells (such as Sf9). 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 directly isolated from an organism (such as a human). The host cell is an adherent cell or a suspension cell.

[0080] The present invention also provides a method for preparing the fusion protein, which comprises the following steps:

[0081] a) culturing the cells as described above under expression conditions to express the fusion protein;

[0082] b) isolating and purifying the fusion protein described in step a).

[0083] The present invention also provides a pharmaceutical composition comprising any one or more of the above-mentioned fusion protein or the above-mentioned nucleic acid molecule, and a pharmaceutically acceptable carrier and / or excipient.

[0084] In certain embodiments of the present invention, the pharmaceutical composition may include a fusion protein and excipients, or include a nucleic acid molecule and a vector, or include a nucleic acid molecule, a vector, and excipients.

[0085] In certain embodiments of the present invention, the pharmaceutical composition is a nucleic acid drug.

[0086] In a more preferred embodiment, the pharmaceutical composition is an mRNA drug.

[0087] In certain embodiments of the present invention, the mRNA drug comprises the nucleic acid molecule and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier is a lipid nanoparticle (LNP). In certain embodiments of the present invention, the mRNA drug further comprises an adjuvant.

[0088] In certain embodiments of the present invention, the lipid nanoparticles comprise at least one ionizable lipid compound.

[0089] In certain embodiments of the present invention, the lipid nanoparticles comprise at least one ionizable lipid compound and one or more combinations of structural lipids, helper lipids, PEG-lipids, and polymers.

[0090] The ionizable lipid compound is a lipid whose protonation degree, and thus its charge properties, are affected by pH. Preferably, it is a lipid that is generally virtually uncharged at normal neutral physiological pH, but can become positively charged at acidic pH, thereby binding to negatively charged nucleic acids. The ionizable lipid compound described herein also includes its isomers or pharmaceutically acceptable salts thereof.

[0091] The aforementioned “isomers” refer to different compounds with the same molecular formula, including but not limited to enantiomers, diastereomers, cis-trans isomers, etc. known in the art.

[0092] The aforementioned "pharmaceutically acceptable salt" refers to an acid addition salt or a base addition salt. All compounds of the present invention that exist in the form of a free base or free acid can be converted into their pharmaceutically acceptable salts by treating with an appropriate inorganic or organic base or acid according to methods known to those skilled in the art. Salts of the compounds of the present invention can be formed by converting them into their free base or acid form using standard techniques.

[0093] Pharmaceutically acceptable salts of the compounds of the present invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable non-toxic acid addition salts are salts with amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, citrate, dodecylsulfate, ethanesulfonate, formate, fumarate, gluconoheptate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxyethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like. The salt derived from suitable base comprises alkali metal salt, alkaline earth metal salt, ammonium salt.Representational alkali metal salt or alkaline earth metal salt comprises sodium salt, lithium salt, potassium salt, calcium salt, magnesium salt etc.In appropriate cases, other pharmaceutically acceptable salt comprises the non-toxic ammonium, quaternary ammonium and amine cation formed using the counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, sulfonate and aryl sulfonate.Other pharmaceutically acceptable salt comprises the salt formed by the quaternization of amine, and this quaternization is carried out using suitable electrophilic reagent (for example, alkyl halide), to form quaternized alkylated amino salt.

[0094] The type of the ionizable lipid is not limited and can be selected from the ionizable lipids disclosed in the prior art, and exemplarily selected from the ionizable lipids in Chinese patent applications 202210344395.1, 202310086372.X, 202310862227.6, 202211374342.0, and 202310830388.7.

[0095] The structured lipids refer to lipids containing structures that can stabilize the composition, including but not limited to one or more combinations of sterols and their derivatives and non-sterols and their derivatives.

[0096] In some specific embodiments, the structured lipids include, but are not limited to, a combination of one or more of sterols and their derivatives, non-sterols, sitosterol, ergosterol, cholestanone, cholestenone, campesterol, stigmasterol, brassicasterol, tomatine, ursolic acid, coprosterol, α-tocopherol, or corticosteroids. Sterols are preferably cholesterol and its derivatives; non-limiting examples of cholesterol derivatives include: polar analogs such as 5α-cholestanol, 5α-coprosterol, cholesteryl-(2'-hydroxy)-ethyl ether, cholesteryl-(4'-hydroxy)-butyl ether, and 6-ketocholestanol; non-polar analogs such as 5α-cholestane, cholesterenone, 5α-cholestenone, 5α-cholestenone, and cholesterol decanoate; and mixtures thereof. In a preferred embodiment, the cholesterol derivative is a polar analog, such as cholesteryl-(4'-hydroxy)-butyl ether. This is not exhaustive, and the selection of structured lipids is not limited, and any structured lipid can be applied to the present invention.

[0097] In some embodiments, the structured lipid is a combination of one or more of cholesterol, sitosterol, ergosterol, corticosteroids and their derivatives.

[0098] In some embodiments, the structured lipid is cholesterol.

[0099] There is no limitation on the type of the “helper lipid”, and phospholipids are preferred, including but not limited to: a combination of one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, phosphatidylserine, phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, and dimyristoylphosphatidylglycerol.

[0100] In some embodiments, the helper lipid can be selected from: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycerophosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPPC), ), 1,2-heneicosanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), dipalmitoylphosphatidylethanolamine (DPPE), 1 -oleoyl-2-cholesterylhemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-O-hexadecyl-sn-glycero-3-phosphocholine, 1,2-dialinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-docohexanoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-diamino A combination of one or more of oleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dialinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-docohexanoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), diacetyl-phosphatidylethanolamine (DEPE), stearoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, and sphingomyelin.

[0101] In some embodiments, the phosphatidylcholine is a combination of one or more of DSPC, DPPC, DMPC, DOPC, and POPC.

[0102] In some embodiments, the helper lipid is phosphatidylcholine, specifically DSPC.

[0103] In some embodiments, the helper lipid is phosphatidylethanolamine, specifically DOPE.

[0104] In some specific embodiments, the auxiliary lipid is selected from one or more combinations of DOTAP ((1,2-dioleyloxypropyl)trimethylammonium chloride), DODAP (1,2-dioleoyl-3-dimethylammonium-propane), 18:1PA (1,2-DI (cis-9-octadecenoyl)-SN-glycerol 3-phosphate sodium salt), HS15 (polyethylene glycol (15)-hydroxystearate), and GL67 (N4-arginine cholesterol carbonylamide).

[0105] The PEG-lipid is formed by chemically linking PEG to lipid molecules, including but not limited to PEG-modified phospholipids and derived lipids, exemplified by one or more combinations of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.

[0106] In some embodiments, the PEG-lipids include but are not limited to PEG-C-DMG, PEG-C-DOMG, PEG-DLPE, PEG-DMPE, PEG-DPPE, PEG-DOPE, PEG-DPPC, Azido-PEG, PEG-distearoylphosphatidylethanolamine (PEG-DSPE), PEG-DAA, Chol (cholesterol)-PEG, 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (PEG-DMG), PEG-S-DMG, PEGylated phosphatidylethanolamine (PEG-PE), PEGylated ceramide , polyethylene glycol-dimethacrylate (PEG-DMA), PEG-distearyl glycerol (PEG-DSG), PEG-dipalmitoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacyl glycamide (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), PEG-1,2-dimyristyloxypropyl-3-amine (PEG-c-DMA), 1,2-distearoyloxypropyl-3-amine-N[methoxy(polyethylene glycol)] (PEG-DSA), methoxypolyethylene glycol ditetradecyl acetamide (ALC0159).

[0107] In some embodiments, the PEG-lipid is PEG-DMG.

[0108] The type 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-β-amino ester (PBAE), poly (α-hydroxy acid), lactide / glycolide copolymer (PLGA or PLG) (including lactide / glycolide copolymer, D-lactide / glycolide copolymer, L-lactide / glycolide copolymer and D,L-lactide / glycolide) copolymer), polyglycolide (PGA), polyorthoester (POE), linear or branched polyethylene glycol (PEG), conjugates of poly (α-hydroxy acid), polyaspirins, polyphosphagenes, D-lactide, D,L-lactide-caprolactone, D,L-lactide

[0014] The present invention also includes but is not limited to the following: lactide-glycolide-caprolactone (DL-G)CL), dextran, vinyl pyrrolidone, polyvinyl alcohol (PVA), methacrylate, poly N-isopropylacrylamide), SAIB (sucrose acetate iso)ate), hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxyethyl methylcellulose, carboxymethyl cellulose or its salts, carboxyvinyl poly, poly (hydroxyethyl methacrylate), poly (methoxyethyl methacrylate), poly (methoxyethoxy-ethyl methacrylate), polymethyl methacrylate (PMMA), methyl methacrylate (MMA), PVA-g-PLGA, PEGT-PBT copolymer, PEO-PPO-PEO (pluronics), PEO-PPO-PAA copolymer, PLGA-PEO-PLGA, PEG-PLG, PLA-PLGA, poloxamer 407, PEG-PLGA-PEG triblock copolymer, or block copolymers thereof with polyethylene glycol (PEG), or a combination of one or more of the above polymers or copolymers.

[0109] In certain embodiments of the present invention, the weight average molecular weight of polyethylene glycol in the polyethylene glycol-modified lipid is 1000-10000, for example, 1000-2000, 2000-4000, 4000-6000, 6000-8000, 8000-10000, preferably 2000.

[0110] In some embodiments, the lipid nanoparticles comprise 20-65% ionizable lipid compounds, 20-60% structural lipids, 3-40% helper lipids, and 0.1-10% PEG-lipids, where % refers to molar percentage.

[0111] In some embodiments, the lipid nanoparticles comprise 35-49% ionizable lipid compounds, 35-50% structural lipids, 5-20% helper lipids, and 1-2% PEG-lipids, where % refers to molar percentage.

[0112] In certain 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, for example, (3-15):1, (3-10):1, (5-10):1, or (5-8):1.

[0113] The particle size of the lipid nanoparticles described in the present invention refers to the hydrated particle size determined by dynamic light scattering (DLS), and the hydrated particle size of the lipid nanoparticles is 50-200 nm, for example, 60-150 nm, 75-140 nm, 85-130 nm or 95-110 nm.

[0114] The nucleic acid drug of the present invention can be prepared using existing technologies.

[0115] In certain embodiments of the present invention, the method for preparing the nucleic acid drug comprises the following steps: 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.

[0116] In certain embodiments of the present invention, the organic solvent is ethanol.

[0117] In certain embodiments of the present invention, the aqueous solvent is a buffer solution, more preferably a sodium acetate buffer solution.

[0118] In certain embodiments of the present 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 0.01-0.05 mg / mL, 0.05-0.1 mg / mL, 0.1-0.5 mg / mL, 0.5-1 mg / mL, 1-1.5 mg / mL, 1.5-2 mg / mL, 2-2.5 mg / mL, 2.5-3 mg / mL, 3-3.5 mg / mL, 3.5-4 mg / mL, 4-4.5 mg / mL, or 4.5-5 mg / mL.

[0119] In certain 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), or 1:(11-15).

[0120] In certain embodiments of the present invention, the method for mixing the lipid molecule organic phase with the drug solution is a microfluidic method.

[0121] In the present invention, the term "pharmaceutical composition" includes any one or more of the above-mentioned fusion proteins and nucleic acid molecules. Preferably, the pharmaceutical composition also contains a pharmaceutically acceptable carrier and / or excipient. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 4-8, preferably about 5-7, although the pH value may vary with the properties of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intravenous injection, intravenous drip, subcutaneous injection, local injection, intramuscular injection, intratumor injection, intraperitoneal injection (such as intraperitoneal), intracranial injection, intracavity injection, inhalation administration, implantation administration, etc.

[0122] In the present invention, pharmaceutical preparations are preferred, so as to exert therapeutic effects more stably. These preparations can ensure the conformational integrity of the amino acid core sequence of the fusion protein disclosed in the present invention, protect the protein structure from degradation (including but not limited to aggregation, deamination or oxidation, etc.), and at the same time protect the biological activity and function of the protein from being destroyed.

[0123] "Pharmaceutically acceptable" means that the drugs do not produce adverse, allergic or other untoward reactions when properly administered to animals or humans.

[0124] "Pharmaceutically acceptable carriers and / or excipients" should be compatible with the active ingredient, that is, they can be mixed with the active ingredient without significantly reducing the effect of the drug under normal circumstances. Specific examples of some substances that can be used as pharmaceutically acceptable carriers and / or excipients can be lipids (such as lipid nanoparticles); sugars such as glucose, mannitol, sucrose, lactose, trehalose, maltose, etc.; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methylcellulose, ethyl cellulose and methylcellulose; 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; alcohols such as ethanol, propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; surfactants; lyoprotectants; colorants; flavorings; tableting agents; stabilizers; diluents; excipients; antioxidants; preservatives; pyrogen-free water; isotonic saline solution; buffer solution, etc., and combinations thereof. These substances are used as needed to improve the stability of the formulation or to help increase the activity or its bioavailability or to produce an acceptable taste or flavor in the case of oral administration.

[0125] The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of any one or more of the above-mentioned fusion proteins and nucleic acid molecules of the present invention. The preferred pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient. The pharmaceutical composition of the present invention can be made into an inhalation atomized preparation (such as a dry powder preparation, an aerosol preparation, an inhalation mist droplet preparation, etc.), an implantable gel preparation, a microneedle preparation, and can also be made into an injection form, for example, using physiological saline or an aqueous solution containing glucose and other adjuvants for preparation by conventional methods. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms / kg body weight to about 50 mg / kg body weight per day. In addition, the pharmaceutical composition of the present invention can also be used with other therapeutic agents.

[0126] In the present invention, the term "effective amount" refers to the amount or dosage of the pharmaceutical composition of the present invention that produces the expected effect in the treated individual after being administered to the subject, and the expected effect includes but is not limited to improvement of the individual's symptoms.

[0127] In the present invention, the term "subject" includes but is not limited to mammals, such as humans, non-human primates, dogs, rabbits, rats, mice, etc.

[0128] The present invention also provides use of the fusion protein, the nucleic acid molecule, the pharmaceutical composition or any combination thereof in preparing a drug for treating a disease, wherein the disease is a disease suitable for treatment with growth hormone.

[0129] The present invention also provides a method for treating a disease, comprising administering an effective amount of the fusion protein, the nucleic acid molecule, the pharmaceutical composition, or any combination thereof to a subject in need thereof.

[0130] "Treatment" or "treatment" of a condition includes preventing or alleviating the condition, reducing the rate of occurrence or development of a condition, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or stopping symptoms associated with a condition, causing complete or partial reversal of a condition, curing a condition, or a combination of these.

[0131] The disease can be selected from adult growth hormone deficiency (GHD), childhood dwarfism, idiopathic short stature (ISS), short stature or slow growth due to other factors (such as endogenous growth hormone deficiency, SHOX gene defect, achondroplasia, small for gestational age), Prader-Willi syndrome, chronic renal insufficiency (CRI), renal failure, anti-aging in the elderly, beauty and anti-aging, severe burns, Turner syndrome, intrauterine growth retardation, polycystic ovary syndrome infertility (POCS), infertility, central precocious puberty (ICPP) in children, short bowel syndrome in adults, Prader-Willi syndrome (PWS), chemotherapy treatment and diseases caused by the alienation state during AIDS treatment.

[0132] Specifically, the growth hormone deficiency can be isolated or accompanied by deficiencies of other pituitary hormones. When used to treat growth hormone deficiency in children and adults, it can promote height growth and improve metabolic issues. It can also be used to treat various symptoms caused by growth hormone deficiency in adults, such as decreased physical strength, emotional problems, and decreased bone density.

[0133] Specifically, the childhood dwarfism may be caused by growth hormone deficiency, skeletal abnormalities, malnutrition or other endocrine diseases.

[0134] Specifically, idiopathic short stature (ISS) refers to short stature with a height that is 2 standard deviations below the mean for children of the same age and gender, and there is no obvious cause.

[0135] Specifically, chronic renal insufficiency (CKD) includes G1 (mild) to G5 (renal failure), and when used to treat chronic renal insufficiency, it can improve its nutritional and metabolic status.

[0136] Specifically, renal failure usually refers to a severe decline in renal function, including acute renal failure and chronic renal failure.

[0137] Specifically, when used to treat polycystic ovary syndrome infertility or infertility, it can improve the success rate of fertility treatment.

[0138] Specifically, central precocious puberty in children may be divided into complete precocious puberty and incomplete precocious puberty based on sex hormone levels and physical development.

[0139] Specifically, when used to treat burns, generally for severe burns, it can promote burn healing, reduce recovery time, and improve the patient's overall recovery.

[0140] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

[0141] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms; each possibility described in the present specification and claims represents a separate embodiment of the present invention.

[0142] When the embodiments provide numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. Except for the specific method, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any method, equipment, and material of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention. Equipment, materials, etc. used in the present invention are all commercially available sources unless otherwise specified.

[0143] Example 1: Design and synthesis of ABD-linker-GH fusion protein

[0144] After the ABD gene was inserted into the signal peptide of the GH gene, it was connected to the mature GH gene via a linker to obtain ABD-linker-GH. The amino acid sequence and nucleotide sequence of the fully synthesized ABD-linker-GH are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively.

[0145] Amino acid sequence of long-acting recombinant human growth hormone ABD-linker-GH fusion protein:

[0146] From the amino acid sequence shown in SEQ ID NO.1, it can be seen that the ABD-linker-GH amino acid sequence consists of 273 amino acids, of which the first 26 amino acids are the signal peptide sequence of human growth hormone, the next 46 amino acids are the GA3 sequence (ABD) of protein G of Streptococcus G148, "GGGGSGGGGS" is the linker, and the last 191 amino acids are the mature human growth hormone sequence.

[0147] Nucleotide sequence encoding long-acting recombinant human growth hormone ABD-linker-GH immune fusion protein:

[0148] Example 2: Preparation of mRNA-LNP drug encoding ABD-linker-GH fusion protein (hereinafter referred to as ABD-linker-GH LNP)

[0149] After obtaining a plasmid containing the ABD-linker-GH sequence (nucleotide sequence as 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 a DT chromatography column to remove residual DNA template and double-stranded RNA contamination during the enzymatic reaction of synthesizing mRNA. The mRNA was then concentrated and the buffer was replaced. The mRNA construct also included a 5' cap structure and a poly-A tail (PolyA) with a length of 80 to 125 adenine nucleotides. The nucleotide sequence of the mRNA construct is shown in SEQ ID NO.10:

[0150] hGH mRNA sequence:

[0151] HSA-hGH mRNA sequence:

[0152] With reference to the mRNA-LNP preparation method disclosed in Patent 202211461172.X, and selecting the ionizable lipids E1-E28 and corresponding LNP components in Example 1 of the patent, a series of mRNA-LNP drugs encoding ABD-linker-GH fusion proteins were prepared. The exemplary mRNA-LNP drug is to dissolve four lipids (ionizable lipid E1: DOPE: cholesterol: DMG-PEG2000) in an ethanol phase, and the ABD-linker-GH mRNA is dissolved in a sodium acetate buffer solution. The LNP is assembled by a microfluidic process. After the ethanol is removed by dialysis with HEPES buffer, the mRNA concentration in the preparation is measured by Ribogreen fluorescence assay, and its concentration is adjusted to 0.2 mg / mL, and sterilized by filtration through a 0.22 μm PES filter membrane. The mRNA content and encapsulation efficiency of LNPs were determined by RiboGreen, the mRNA integrity was characterized by capillary gel electrophoresis, and the particle size and dispersity were characterized by dynamic light scattering (Malvern ZetasizerNano ZS), which were 61.54 nm and 0.1788, respectively.

[0153] Example 3: Expression level of ABD-linker-GH mRNA in cells

[0154] HEK293T cells were seeded into 12-well plates one day in advance, and when the cells reached 70-90% confluence, the cells were plated with Lipofectamine TM mRNA transfection was performed using the 3000 reagent protocol at 3 μg / well. Supernatants were collected 24 and 48 hours after transfection.

[0155] The human growth hormone (hGH) ELISA Kit was used to measure the growth hormone concentration in the supernatant at each time point. Refer to the kit instructions for specific experimental steps. As shown in Figure 2, the ABD-linker-GH mRNA can effectively express the fusion protein ABD-linker-GH.

[0156] Example 4: Bioactivity Assay of ABD-linker-GH

[0157] 4-1: Half-life of ABD-linker-GH LNP drug in rats after single intravenous administration

[0158] Male mature Sprague Dawley rats (approximately 5-6 weeks old, weighing 130-180 g) were divided into four groups, each with three rats, and received a single intravenous injection of equal volumes of normal saline, 500 μg of hGH mRNA-LNP (hereinafter referred to as hGH LNP) drug (prepared as in Example 2, hGH mRNA was prepared by a third party company, sequence number SEQ ID NO. 15), 500 μg of HSA-hGH mRNA-LNP drug (hereinafter referred to as HSA-hGH LNP) (prepared as in Example 2, HSA-hGH mRNA was prepared by a third party company, sequence number SEQ ID NO. 16), and 500 μg of ABD-linker-GH LNP drug. Blood samples were collected 0, 4, 24, 48, 72, 120, and 168 hours after administration, and the hGH content in the rat serum was measured using a Human Growth Hormone (hGH) ELISA Kit. For specific experimental procedures, refer to the kit instructions. The test results are shown in Figure 3, which show that the plasma half-life of ABD-linker-GH LNP in rats is longer than that of hGH LNP and HSA-hGH LNP, and the peak blood concentration is increased by 3-4 times.

[0159] 4-2: Testing the effects of a single intravenous dose of ABD-linker-GH LNP in rats

[0160] Mature male Sprague Dawley rats (approximately 5-6 weeks old, weighing 130-180 g) were divided into four groups, with three rats in each group. Each group received a single intravenous injection of equal volumes of normal saline, 500 μg hGH LNP, 500 μg HSA-hGH LNP, and 500 μg ABD-linker-GH LNP. Blood samples were collected 0, 4, 24, 48, 72, 120, and 168 h after administration. The IGF-1 content in rat serum was determined using the Mouse / Rat IGF-1 ELISA Kit. For specific experimental procedures, refer to the kit instructions. The test results are shown in Figure 4 , which indicate that intravenous injection of both ABD-linker-GH LNP and HSA-hGH LNP increased IGF-1 levels in rat serum. The duration of the IGF-1 elevation induced by ABD-linker-GH LNP was longer than that induced by hGH LNP and HSA-hGH LNP, with the peak value being extended by approximately 24 hours. The IGF-1 level was nearly 30-fold higher than that induced by HSA-hGH LNP.

[0161] 4-3: Activity data of ABD-linker-GH LNP drug in rats after single subcutaneous administration

[0162] In this example, the administration method was further changed. The in vivo activity of the ABD-linker-GH LNP drug prepared by the present invention was tested in rats by subcutaneous administration. The results showed that compared with intravenous administration of the ABD-linker-GH LNP drug, its plasma half-life was approximately 24 hours longer than that of hGH LNP, and the increased plasma IGF-1 level could be extended by 24 hours. At the same time, compared with subcutaneous administration of hGH LNP and HSA-hGH LNP, it also showed a longer plasma hGH half-life and higher plasma IGF-1 levels.

[0163] Example 5 Preparation and efficacy data of mRNA-LNP drugs containing other ABD-linker-GH fusion proteins

[0164] 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.

[0165] SEQ ID NO 17: ABD11-linker7-GH12 mRNA sequence:

[0166] SEQ ID NO 18:ABD3-linker7-GH9 mRNA sequence

[0167] SEQ ID NO 19: ABD11-linker6-GH9 mRNA sequence

[0168] The experimental results are shown in Figures 5 and 6: Whether administered subcutaneously or intravenously, the time it takes for IGF-1 levels to rise is longer than that of hGH LNP and HSA-hGH LNP, the peak is extended by approximately 24 hours, and the level is nearly 10-30 times higher than that of HSA-hGH LNP.

[0169] In summary, the present invention mainly addresses the problems existing in HSA-hGH fusion protein. The mRNA of the target drug is encapsulated by LNP (lipid nanoparticles) to form a more long-lasting and highly active growth hormone nucleic acid drug, which can achieve relatively high biological activity at a lower dosage.

[0170] The above examples are intended to illustrate the embodiments disclosed herein and are not to be construed as limiting the present invention. In addition, the various modifications listed herein and variations of the methods in the invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications apparent to those skilled in the art as described above to obtain the invention should be included within the scope of the present invention.

Claims

1. A fusion protein, characterized in that The fusion protein includes the ABD domain of streptococcal protein G and growth hormone, wherein the ABD domain is connected to the growth hormone via a connecting peptide, the amino acid sequence of the ABD domain is as shown in SEQ ID NO.11 or has 95% or more similarity to 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 as shown in SEQ ID NO.12 or has 95% or more similarity to SEQ ID NO.12, and has the function of the amino acid sequence defined by SEQ ID NO.

12.

2. The fusion protein according to claim 1, characterized in that The amino acid sequence of the ABD domain is also shown in any one of SEQ ID NOs. 3 to 5 or has a similarity of 95% or more to any sequence shown in SEQ ID NOs. 3 to 5.

3. The fusion protein according to claim 1, characterized in that The amino acid sequence of the growth hormone is also shown as SEQ ID NO.9 or has 95% or more similarity to SEQ ID NO.

9.

4. The fusion protein according to claim 1, characterized in that The connecting peptide is selected from a rigid connecting peptide or a flexible connecting peptide; preferably, the rigid connecting peptide is (EAAAK)*n, n=1-5; preferably, the flexible connecting peptide is selected from (GGGGS)*n, (GSGGG)*n, (GGGSG)*n or (GGSGG)*n, n=1-5.

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

6. The fusion protein according to claim 1, characterized in that The amino acid sequence of the fusion protein is 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, characterized in that The nucleic acid molecule comprises a nucleotide sequence encoding the fusion protein according to any one of claims 1-6.

8. The nucleic acid molecule according to claim 7, characterized in that The 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 a nucleotide sequence as shown in SEQ ID NO.13 or having 95% or more similarity to SEQ ID NO.

13.

9. The nucleic acid molecule according to claim 7, characterized in that The nucleic acid molecule is mRNA.

10. The nucleic acid molecule according to claim 9, characterized in that The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.10 or has 95% or more similarity to SEQ ID NO.

10.

11. A cell, characterized in that The cell contains or is used to prepare any one or more of the fusion protein according to any one of claims 1 to 6 and the nucleic acid molecule according to any one of claims 7 to 10.

12. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises any one or more of the fusion protein described in any one of claims 1 to 6 and the nucleic acid molecule described in any one of claims 7 to 10; preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

13. The pharmaceutical composition according to claim 12, characterized in that 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, the pharmaceutical composition further comprises an excipient.

14. The pharmaceutical composition according to claim 13, characterized in that The lipid nanoparticles contain at least one ionizable lipid compound; preferably, the lipid nanoparticles contain at least one ionizable lipid compound and a combination of any one or more of structural lipids, auxiliary lipids, PEG-lipids, and polymers; preferably, the structural lipids are selected from a combination of one or more of sterols, non-sterols, or their respective derivatives; preferably, the auxiliary lipids are selected from a combination of any one or more of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, ceramide, phosphatidylserine, phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, dimyristoylphosphatidylglycerol, DOTAP, DODAP, 18:1PA, HS15, and GL67; preferably, the PEG-lipids are selected from a combination of one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and methoxypolyethylene glycol ditetradecylacetamide.

15. The pharmaceutical composition according to claim 14, characterized in that The lipid nanoparticles contain 20-65% ionizable lipid compounds, 20-60% structural lipids, 3-40% auxiliary lipids, and 0.1-10% PEG-lipids, wherein % refers to molar percentage.

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

17. A method for treating a disease, characterized in that: The method comprises administering to a subject in need thereof an effective amount of the fusion protein of any one of claims 1-6, the nucleic acid molecule of any one of claims 7-10, the pharmaceutical composition of any one of claims 12-15, or any combination thereof.

18. The use according to claim 16 or the method of treatment according to claim 17, characterized in that The disease is selected from growth hormone deficiency, childhood dwarfism, Prader-Willi syndrome, Turner syndrome, intrauterine growth retardation, idiopathic short stature, chronic renal insufficiency, renal failure, polycystic ovary syndrome infertility, infertility, central precocious puberty in children, short bowel syndrome in adults, Prader-Willi syndrome or burns.

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