Growth hormone fusion protein, nucleic acid medicine and application thereof

Abstract

The invention relates to the field of medicine, in particular to a growth hormone fusion protein, a nucleic acid medicine and application of the growth hormone fusion protein and the nucleic acid medicine, the growth hormone fusion protein comprises an ABD structural domain of streptococcus protein G and a growth hormone, and the ABD structural domain is connected with the growth hormone through a linker. The molecular weight of the fusion protein is small, the problems that a large-molecular-weight growth hormone fusion protein is difficult to permeate into a target tissue and the biological activity is low are solved, and the fusion protein has higher biological activity. Compared with a traditional protein drug, the mRNA-LNP drug for coding the fusion protein can directly utilize human body cells to express the protein drug, and the protein drug generated by the mRNA-LNP drug has high activity, can significantly improve the drug effect, and can achieve relatively high biological activity on the premise of low administration dosage.

Description

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 of the human body. It is composed of 191 amino acids, most of which exist in the form of a molecular weight of 22KDa. hGH binds to growth hormone receptors on receptor cells in various tissues, organs and bones of the body, promoting protein synthesis and fat decomposition in receptor cells, such as the expression of insulin-like growth factor-1 (IGF-1) involved in cell growth and regeneration, thereby promoting human metabolism and growth and development. In clinical practice, it is mainly used to treat slow growth and severe burns in children caused by growth hormone deficiency, chronic renal failure and Turner syndrome.

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

[0004] In order to overcome the problem of short-acting hGH, the method of using fusion protein has been used to develop long-acting growth hormone, such as human serum albumin (HSA) fused growth hormone. Mature HSA is a protein composed of 585 amino acids, which participates in the recycling mechanism mediated by FcRn and has a plasma half-life of up to 14-20 days. Therefore, after fusion with hGH, the half-life of hGH can be extended.

[0005] However, the structure and size of hGH are strictly conserved in various species, and this conservation of size may represent an evolutionary control that allows hGH to pass through tissues with fewer blood vessels (fat, bones and growth plates) and tissues with good blood vessels (muscles, heart). Although HSA fusion protein can prolong the half-life and reduce the clearance of hGH, the fusion protein greatly increases the molecular weight, which hinders the absorption of hGH into target tissues and reduces its biological activity. At the same time, the current half-life of HSA-hGH fusion protein is 58-80h (data published in CN109851674B), and there is still room for improvement to further reduce the frequency of administration.

[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. 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. Summary of the invention

[0008] 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.

[0009] To achieve the above-mentioned object and other related objects, the present invention provides a fusion protein, 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, 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.

[0010] The present invention also provides a nucleic acid molecule, wherein the nucleic acid molecule comprises a nucleotide sequence encoding the fusion protein.

[0011] The present invention also provides a cell, wherein the cell contains or is used for preparing any one or more of the fusion protein and the nucleic acid molecule.

[0012] 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.

[0013] 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.

[0014] As described above, the growth hormone fusion protein, nucleic acid drug and use thereof of the present invention have the following beneficial effects:

[0015] 1. The ABD-linker-GH fusion protein of the present invention utilizes the interaction between ABD and HSA to improve the recycling mechanism mediated by FcRn of the fusion protein, thereby achieving the purpose of extending the half-life. Compared with HSA-hGH fusion protein drugs, the binding of ABD to native HSA can more effectively utilize the recycling mechanism mediated by FcRn, further improve the half-life of the fusion protein, and reduce the frequency of administration.

[0016] 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 is difficult to penetrate into target tissues and has low biological activity, and has higher biological activity.

[0017] 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 use the body's own cells to express protein drugs, and the protein drugs produced by 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

[0018] Figure 1 Shown is a schematic diagram of the structure of the fusion protein of the present invention.

[0019] Figure 2 Shown is the expression level of the fusion ABD-linker-GH mRNA of the present invention in the supernatant of HEK293T cells.

[0020] Figure 3 Shown are the changes in hGH content in rat serum after intravenous injection of different ABD-linker-GH mRNA-LNPs.

[0021] Figure 4 Shown are the changes in IGF-1 levels in rat serum after intravenous injection of different ABD-linker-GH mRNA-LNPs.

[0022] Figure 5 Shown are the changes in hGH content in rat serum after intravenous injection of different ABD-linker-GH mRNA-LNPs.

[0023] Figure 6 Shown are the changes in IGF-1 levels in rat serum after intravenous injection of different ABD-linker-GH mRNA-LNPs. DETAILED DESCRIPTION

[0024] The present invention provides a fusion protein named ABD-linker-GH, which comprises an 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.

[0025] LAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALP(SEQ ID NO.11)

[0026] MATGSRTSLLLAFGLLCLPWLQEGSAFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQS WLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF(SEQID NO.12)

[0027] The ABD domain may be a wild-type domain or a mutant domain of Streptococcal protein G from different species, which is not specifically limited in the present invention. Specifically, the ABD domain is a GA module of Streptococcal protein G (abbreviated as SPG) (GA module, 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).

[0028] Streptococcal protein G on the surface of streptococcal strains has a domain that can bind to serum albumin, which has been determined to be a three-helix bundle structure. This motif is named ABD (albumin binding domain). Therefore, the present invention fuses the ABD peptide (46 amino acids) of the G protein of streptococcus G148 with GH through a linker to form an ABD-linker-GH fusion protein. The interaction between ABD and HSA is used to improve the recycling mechanism of the fusion protein mediated by FcRn, thereby achieving the purpose of extending the half-life. Because the molecular weight of ABD is small, the effect on the biological activity of hGH after fusion is extremely low.

[0029] 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.

[0030] 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 substituting, deleting or adding amino acids may have a sequence similarity of 95%, 96%, 97%, 98% or 99% or more with SEQ ID NO.11.

[0031] 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 a similarity of 95% or more 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 of Streptococcus G148, and SEQ ID NO. 5 is the amino acid sequence of G protein GA3 mutant 2 of Streptococcus G148.

[0032] LAEAKELAIRELKKYGISDYYKNLINKAKTVEGVKALKDEILAALP(SEQ ID NO.3)

[0033] LAEAKVLANRELDKYGVSDFYKRLINKAKTVEGVEALKLHILAALP(SEQ ID NO.4)

[0034] LAEAKEAANAELDSYGVSDFYKRLIDKAKTVEGVEALKDAILAALP(SEQ ID NO.5)

[0035] In certain embodiments of the present invention, the growth hormone is selected from growth hormone of mammals, and the mammals are selected from humans, cattle, pigs, mice, monkeys, etc.

[0036] 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 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 of the amino acid sequence shown in SEQ ID NO.12, and the amino acid sequence obtained after substituting, deleting or adding amino acids can have a sequence similarity of 95%, 96%, 97%, 98% or 99% or more with SEQ ID NO.12.

[0037] 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.

[0038] MATGSRTSLLLAFGLLCLPWLQEGSAFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQS WLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPDPGQIFAQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF(SEQID NO.9)

[0039] The linker is not specifically limited, as long as the ABD domain can be connected 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 linker can be selected from a rigid linker or a flexible linker. The rigid linker is (EAAAK)*n, n=1-5. The flexible linker is selected from (GGGGS)*n, (GSGGG)*n, (GGGSG)*n or (GGSGG)*n, n=1-5.

[0040] 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 more similarity with any one of SEQ ID NOs. 6 to 8 or a combination thereof.

[0041] (GGGGS)1 (SEQ ID NO.6)

[0042] (GGGGS)5 (SEQ ID NO.7)

[0043] (EAAAK)2 (SEQ ID NO.8)

[0044] In certain embodiments of the present invention, the fusion protein includes, from the N-terminus to the C-terminus, the ABD domain, the linker, and the growth hormone. Before the fusion protein is secreted outside the cell after expression, the N-terminus also includes the growth hormone signal peptide.

[0045] In certain embodiments of the present invention, ABD-linker-GH can be formed in any of the following ways:

[0046] 1) The ABD domain shown in SEQ ID NO.11, the linker shown in SEQ ID NO.6 and the growth hormone shown in SEQ ID NO.12;

[0047] 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;

[0048] 3) formed by the ABD domain shown in SEQ ID NO.11, the linker shown in SEQ ID NO.8 and the growth hormone shown in SEQ ID NO.12;

[0049] 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;

[0050] 5) formed by the ABD domain shown in SEQ ID NO.11, the linker shown in SEQ ID NO.7 and the growth hormone shown in SEQ ID NO.9;

[0051] 6) The ABD domain shown in SEQ ID NO.11, the linker shown in SEQ ID NO.8 and the growth hormone shown in SEQ ID NO.9;

[0052] 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;

[0053] 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;

[0054] 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;

[0055] 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;

[0056] 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;

[0057] 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;

[0058] 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;

[0059] 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;

[0060] 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;

[0061] 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;

[0062] 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;

[0063] 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;

[0064] 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;

[0065] 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;

[0066] 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;

[0067] 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;

[0068] 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;

[0069] 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.

[0070] 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.

[0071] LAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALPGGGGSGGGGSFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQ KSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF(SEQ ID NO.14)

[0072] 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.

[0073] LAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALPGGGGSGGGGSGGGGSGGGGSGGGGSFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPS NREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF(SEQ ID NO 20:ABD11-linker7-GH12 amino acid sequence)

[0074] LAEAKELAIRELKKYGISDYYKNLINKAKTVEGVKALKDEILAALPGGGGSGGGGSGGGGSGGGGSGGGGSFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPS NREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPDPGQIFAQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF(SEQ ID NO 21:ABD3-linker7-GH9 amino acid sequence)

[0075] LAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALPGGGGSFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSN LELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPDPGQIFAQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF(SEQ ID NO 22:ABD11-linker6-GH9 amino acid sequence)

[0076] The present invention also provides a nucleic acid molecule, wherein the nucleic acid molecule comprises a nucleotide sequence encoding the fusion protein.

[0077] 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 biological custom oligonucleotide one or more combinations thereof.

[0078] In certain embodiments of the present invention, the nucleic acid molecule is a DNA nucleic acid molecule.

[0079] In certain embodiments of the invention, the nucleic acid molecule is plasmid DNA.

[0080] In certain embodiments of the invention, the nucleic acid molecule is mRNA.

[0081] In certain embodiments of the present invention, the nucleic acid molecule includes nucleotides encoding the signal peptide of growth hormone—the ABD domain—the linker—the growth hormone in sequence from the 5' end to the 3' end.

[0082] In certain embodiments of the present invention, the nucleotide sequence encoding the fusion protein is shown as SEQ ID NO.2.

[0083] In certain embodiments of the present invention, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO.13.

[0084] In certain embodiments of the present invention, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO.10.

[0085] The present invention also provides a cell, wherein the cell contains or is used for preparing any one or more of the fusion protein and the nucleic acid molecule.

[0086] In some embodiments, the cell genome has integrated therein a nucleotide sequence as shown in SEQ ID NO. 2 or 13 or a nucleotide sequence having 95% or more similarity to either of them.

[0087] In some embodiments, the cell is a cell used to prepare the nucleic acid molecule and / or the nucleic acid molecule encodes a fusion protein.

[0088] 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 passage cell or a primary cell, that is, a cell directly isolated from an organism (such as a human). The host cell is an adherent cell or a suspension cell.

[0089] The present invention also provides a method for preparing the fusion protein, which comprises the following steps:

[0090] a) culturing the cell as described above under expression conditions, thereby expressing the fusion protein;

[0091] b) isolating and purifying the fusion protein described in step a).

[0092] The present invention also provides a pharmaceutical composition, which comprises 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.

[0093] In certain embodiments of the present invention, the pharmaceutical composition may include a fusion protein and an adjuvant, or include a nucleic acid molecule and a vector, or include a nucleic acid molecule, a vector and an adjuvant.

[0094] In certain embodiments of the present invention, the pharmaceutical composition is a nucleic acid drug.

[0095] In a more preferred embodiment, the pharmaceutical composition is an mRNA drug.

[0096] In certain embodiments of the present invention, the mRNA drug comprises the nucleic acid molecule and a pharmaceutically acceptable carrier, and the pharmaceutically acceptable carrier is a lipid nanoparticle (LNP). In certain embodiments of the present invention, the mRNA drug further comprises an adjuvant.

[0097] In certain embodiments of the present invention, the lipid nanoparticles comprise at least one ionizable lipid compound.

[0098] 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, auxiliary lipids, PEG-lipids, and polymers.

[0099] The ionizable lipid compound refers to a lipid whose protonation degree is affected by pH value and thus affects its charge property. Preferably, it is a type of lipid that is usually almost uncharged under normal neutral physiological pH conditions, but can be positively charged at acidic pH to bind to negatively charged nucleic acids. The ionizable lipid compound of the present invention also includes its isomers or pharmaceutically acceptable salts thereof.

[0100] The aforementioned “isomers” refer to different compounds with the same molecular formula, including but not limited to enantiomers, diastereomers, cis-trans isomers, etc., which are well known in the art.

[0101] 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 free base or free acid can be converted into their pharmaceutically acceptable salts by treating with appropriate inorganic or organic bases or acids according to methods known to those skilled in the art. The salts of the compounds of the present invention can be converted into their free bases or acids by standard techniques.

[0102] 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 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 such as ion exchange used in the art. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, citrate, dodecylsulfate, ethanesulfonate, formate, fumarate, gluconate heptanoate, 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. Salts derived from suitable bases include alkali metal salts, alkaline earth metal salts, ammonium salts. Representative alkali metal salts or alkaline earth metal salts include sodium salts, lithium salts, potassium salts, calcium salts, magnesium salts, etc. Where appropriate, other pharmaceutically acceptable salts include non-toxic ammonium, quaternary ammonium and amine cations formed using counterions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, sulfonates and aryl sulfonates. Other pharmaceutically acceptable salts include salts formed by quaternization of amines, which are carried out using suitable electrophilic reagents (e.g., alkyl halides) to form quaternized alkylated amino salts.

[0103] 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.

[0104] The structural 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.

[0105] In some specific embodiments, the structural 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 decanoic acid cholesterol ester; 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, the selection of structural lipids is not limited, and any structural lipid can be applied to the present invention.

[0106] In some embodiments, the structured lipid is a combination of one or more of cholesterol, sitosterol, ergosterol, corticosteroids and their derivatives.

[0107] In some embodiments, the structured lipid is cholesterol.

[0108] The type of the "auxiliary lipid" is not limited, 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.

[0109] 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 (DSPC). ), 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 1-O-hexadecyl-sn-glycero-3-phosphocholine, 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diamidonoyl-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-docosahexanoyl-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.

[0110] In some specific embodiments, the phosphatidylcholine is a combination of one or more of DSPC, DPPC, DMPC, DOPC, and POPC.

[0111] In some specific embodiments, the helper lipid is phosphatidylcholine, specifically DSPC.

[0112] In some specific embodiments, the helper lipid is phosphatidylethanolamine, specifically DOPE.

[0113] In some specific embodiments, the auxiliary lipid is selected from a combination of one or more 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).

[0114] The PEG-lipid is formed by connecting PEG to lipid molecules through chemical bonds, including but not limited to PEG-modified phospholipids and derived lipids, exemplified by a combination of one or more of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.

[0115] In some specific 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) or a combination of one or more thereof.

[0116] In some embodiments, the PEG-lipid is PEG-DMG.

[0117] 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), poly(β-amino ester), poly(β-hydroxy acid ... or PLG) (including lactide / glycolide copolymers, D-lactide / glycolide copolymers, L-lactide / glycolide copolymers and D,L-lactide / glycolide) polymers), polyglycolide (PGA), polyorthoesters (POE), linear or branched polyethylene glycol (PEG), conjugates of poly(α-hydroxy acids), polyaspirins, polyphosphagenes, D-lactide, D,L-lactide-caprolactone, D,L-lactide-glycolide-caprolactone ester (DL-G)CL), dextran, vinyl pyrrolidone, polyvinyl alcohol (PVA), methacrylate, poly N-isopropylacrylamide), SAIB (sucrose acetate iso) acid salt), hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxyethyl methylcellulose, carboxymethyl cellulose or its salt, polycarboxyvinyl, poly (hydroxyethyl methacrylate), poly (methoxyethyl methacrylate), poly (methoxyethoxy-ethyl methacrylate), polymethyl methacrylate (PMMA), methyl methacrylate (MMA), PVA-g-PLGA, PEGT-PBT copolymer (polyact) e), PEO-PPO-PEO (pluronics), PEO-PPO-PAA copolymer, PLGA-PEO-PLGA, PEG-PLG, PLA-PLGA, poloxamer 4O7, 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.

[0118] 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.

[0119] In some specific embodiments, the lipid nanoparticles comprise 20-65% ionizable lipid compounds, 20-60% structural lipids, 3-40% auxiliary lipids, and 0.1-10% PEG-lipids, where % refers to molar percentage.

[0120] In some specific embodiments, the lipid nanoparticles comprise 35-49% ionizable lipid compounds, 35-50% structural lipids, 5-20% auxiliary lipids, and 1-2% PEG-lipids, where % refers to molar percentage.

[0121] 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, (5-8):1.

[0122] 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.

[0123] The nucleic acid drug of the present invention can be prepared by existing techniques.

[0124] 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.

[0125] In certain embodiments of the present invention, the organic solvent is ethanol.

[0126] In certain embodiments of the present invention, the aqueous solvent is a buffer solution, more preferably a sodium acetate buffer solution.

[0127] 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, 4.5-5 mg / mL.

[0128] 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), 1:(11-15).

[0129] In certain embodiments of the present invention, the method for mixing the lipid molecule organic phase with the drug solution is a microfluidic method.

[0130] 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 includes 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.

[0131] In the present invention, the preferred "pharmaceutical composition" is a pharmaceutical preparation, so as to exert the therapeutic effect 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 structure of the protein from degradation (including but not limited to aggregation, deamination or oxidation, etc.), and also protect the biological activity and function of the protein from being destroyed.

[0132] "Pharmaceutically acceptable" means that the drugs do not produce adverse, allergic or other untoward reactions when they are properly administered to animals or humans.

[0133] "Pharmaceutically acceptable carriers and / or excipients" should be compatible with the active ingredient, that is, they can be mixed with it 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, etc.; cellulose and its derivatives, such as sodium methylcellulose, ethyl cellulose and methyl cellulose, etc.; tragacanth powder; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate, etc.; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter, etc.; alcohols, such as ethanol, propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol, etc.; alginic acid; emulsifiers, such as Tween, etc.; wetting agents, such as sodium lauryl sulfate, etc.; surfactants; freeze-drying protectants; colorants; flavorings; tablets; stabilizers; diluents; excipients; antioxidants; preservatives; pyrogen-free water; isotonic saline solution; buffer solution, etc., and combinations thereof. These materials 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.

[0134] 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, and the preferred pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or adjuvant. The pharmaceutical composition of the present invention can be made into an inhalation atomization 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 to prepare 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.

[0135] 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 the improvement of the individual's symptoms.

[0136] 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.

[0137] 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.

[0138] 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.

[0139] 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.

[0140] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways 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 the 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 embodiment gives a numerical range, it should be understood that, unless otherwise specified in the present invention, the two 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. In addition to the specific methods, 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 method, equipment, and material in the embodiments of the present invention can also be used to realize the present invention. The 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 is inserted into the signal peptide of the GH gene, the mature GH gene is connected through a linker to obtain ABD-linker-GH. The whole gene is synthesized ABD-linker-GH, and its amino acid sequence and nucleotide sequence 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] MATGSRTSLLLAFGLLCLPWLQEGSALAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALPGGGGSGGGGSFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSE SIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF(SEQ ID NO.1)

[0147] From the amino acid sequence shown in SEQ ID NO.1, it can be seen that the ABD-linker-GH amino acid sequence has a total 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.

[0148] Nucleotide sequence encoding long-acting recombinant human growth hormone ABD-linker-GH immune fusion protein:

[0149] ATGGCTACAGGCTCCCGGACGTCCCTGCTCCTGGCTTTTGGCCTGCTCTGCCTGCCCTGGCTTCAAGAGGGCAGTGCCTTAGCTGAAGCTAAAGTCTTAGCTAACAGAGAACTTGACAAATATGGAGTAAGTGACTATTACAAGAACCTAATCAACAATGCCAAAACTGTTGAAGGTGTAAAAGCACTGATAGATGAAATTTTAGCTGCATTACCTGGCGGAGGCGGTTCCGGCGGCGGGGGCAGCTTCCCAACCATTCCCTTATCCAGGCTTTTTGACAACGCTATGCTCCGCGCCCATCGTCTGCACCAGCTGGCCTTTGACACCTACCAGGAGTTTGAAGAAGCCTATATCCCAAAGGAACAGAAGTATTCATTCCTGCAGAACCCCCAGACCTCCCTCTGTTTCTCAGAGTCTATTCCGACACCCTCCAACAGGGAGGAAACACAACAGAAATCCAACCTAGAGCTGCTCCGCATCTCCCTGCTGCTCATCCAGTCGTGGCTGGAGCCCGTGCAGTTCCTCAGGAGTGTCTTCGCCAACAGCCTGGTGTACGGCGCCTCTGACAGCAACGTCTATGACCTCCTAAAGGACCTAGAGGAAGGCATCCAAACGCTGATGGGGAGGCTGGAAGATGGCAGCCCCCGGACTGGGCAGATCTTCAAGCAGACCTACAGCAAGTTCGACACAAACTCACACAACGATGACGCACTACTCAAGAACTACGGGCTGCTCTACTGCTTCAGGAAGGACATGGACAAGGTCGAGACATTCCTGCGCATCGTGCAGTGCCGCTCTGTGGAGGGCAGCTGTGGCTTCTAGTAA(SEQID NO.2)

[0150] Example 2: Preparation of an mRNA-LNP drug encapsulating mRNA encoding an ABD-linker-GH fusion protein (hereinafter simply referred to as ABD-linker-GH LNP)

[0151] 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 includes a 5' cap structure and a poly-A tail (Poly A) with a length of 80 to 125 adenine nucleotides, and the nucleotide sequence of the mRNA construct is shown in SEQ ID NO.10:

[0152]

[0153]

[0154] hGH mRNA sequence:

[0155]

[0156] HSA-hGH mRNA sequence:

[0157]

[0158] Referring to the mRNA-LNP preparation method disclosed in Patent 202211461172.X, and selecting the ionizable lipids E1-E28 and the corresponding LNP components in Example 1 of the patent, a series of mRNA-LNP drugs encoding ABD-linker-GH fusion proteins were prepared. An exemplary mRNA-LNP drug is to dissolve four lipids (ionizable lipid E1: DOPE: cholesterol: DMG-PEG2000) in an ethanol phase, dissolve ABD-linker-GH mRNA in a sodium acetate buffer solution, and assemble LNPs by a microfluidic process. After HEPES buffer is dialyzed to remove ethanol, 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 LNP were determined by RiboGreen, the mRNA integrity was characterized by capillary gel electrophoresis, and the particle size and dispersion were characterized by dynamic light scattering (Malvern Zetasizer NanoZS), which were 61.54 nm and 0.1788, respectively.

[0159] Example 3: Expression level of ABD-linker-GH mRNA in cells

[0160] HEK293T cells were seeded in 12-well plates one day in advance and when the cells reached 70-90% confluence, the cells were stained with Lipofectamine TM mRNA transfection was performed using the 3000 reagent protocol, 3 μg / well. Supernatants were collected 24h and 48h after transfection.

[0161] Human Growth Hormone (hGH) ELISA Kit was used to measure the growth hormone concentration in the supernatant at each time point. The specific experimental steps refer to the kit instructions. Figure 2 As shown, ABD-linker-GH mRNA can effectively express the fusion protein ABD-linker-GH.

[0162] Example 4: Bioactivity Assay of ABD-linker-GH

[0163] 4-1: Half-life of ABD-linker-GH LNP drug in rats after single intravenous administration

[0164] Male mature Sprague Dawley rats (about 5-6 weeks old, weighing 130-180g) were divided into 4 groups, 3 rats in each group, and injected intravenously with equal volumes of normal saline, 500μg hGH mRNA-LNP (hereinafter referred to as hGH LNP) drug (preparation method is the same as in Example 2, hGH mRNA is prepared by a third party company, sequence number SEQ ID NO.15), 500μg HSA-hGHmRNA-LNP drug (hereinafter referred to as HSA-hGH LNP) (preparation method is the same as in Example 2, HSA-hGH mRNA is prepared by a third party company, sequence number SEQ ID NO.16) and 500μg ABD-linker-GH LNP drug, blood samples were taken 0, 4, 24, 48, 72, 120, 168h after administration, and the hGH content in rat serum was detected by Human Growth Hormone (hGH) ELISA Kit. The specific experimental steps refer to the kit instructions. The test results are shown in the attached Figure 3 As shown, the results showed that the plasma half-life of ABD-linker-GH LNP in rats was longer than that of hGH LNP and HSA-hGH LNP, and the peak blood concentration was increased by 3-4 times.

[0165] 4-2: Testing the effect of a single intravenous dose of ABD-linker-GH LNP drug in rats

[0166] Male mature Sprague Dawley rats (about 5-6 weeks old, weighing 130-180g) were divided into 4 groups, 3 rats in each group, and injected with equal volumes of normal saline, 500μg hGH LNP drug, 500μg HSA-hGH LNP drug and 500μg ABD-linker-GH LNP drug once intravenously. Blood samples were taken 0, 4, 24, 48, 72, 120, and 168h after administration. The IGF-1 content in rat serum was detected using Mouse / Rat IGF-1ELISA Kit. For specific experimental steps, please refer to the kit manual. The test results are shown in the attached figure. Figure 4 As shown, the results showed that intravenous injection of ABD-linker-GH LNP and HSA-hGH LNP could cause an increase in IGF-1 levels in rat serum. The IGF-1 level increase caused by ABD-linker-GH LNP lasted longer than that caused by hGH LNP and HSA-hGH LNP, with the peak value extended by about 24h, and the level was nearly 30 times higher than that of HSA-hGH LNP.

[0167] 4-3: Activity data of ABD-linker-GH LNP drug in rats after single subcutaneous administration

[0168] In this example, the administration method was further changed, and the in vivo activity of the ABD-linker-GH LNP drug prepared by the present invention in rats was detected by subcutaneous administration. The results showed that compared with the 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 increase in the plasma IGF-1 level could be extended by 24 hours; at the same time, compared with the subcutaneous administration of hGH LNP and HSA-hGH LNP, it also showed a longer plasma hGH half-life and a higher plasma IGF-1 level.

[0169] Example 5 Preparation and efficacy data of mRNA-LNP drugs containing other ABD-linker-GH fusion proteins

[0170] 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 respectively referred to as SEQ ID NO.17LNP, SEQ ID NO.18LNP and SEQ ID NO.19LNP.

[0171] SEQ ID NO 17: ABD11-linker7-GH12 mRNA sequence:

[0172] AGAAUAAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGGCCACA

[0173] GGCAGCCGGACCUCUCUGCUGCUGGCCUUCGGCCUGCUGUGCCUGCCUUGGCUGCAAGAGGGCUC

[0174] CGCCCUGGCCGAGGCUAAGGUGCUGGCUAAUCGGGAACUGGAUAAGUACGGCGUCUCCGACUAC

[0175] UACAAGAACCUGAUCAACAACGCUAAGACCGUGGAAGGCGUGAAGGCCCUGAUCGACGAGAUCC

[0176] UGGCCGCCCUCCCCGGCGGAGGUGGCUCUGGAGGCGGUGGAUCUGGUGGUGGCGGAUCAGGUGG

[0177] AGGCGGGAGCGGAGGCGGCGGUAGUUUCCCUACAAUCCCACUGAGCAGACUGUUCGAUAACGCC

[0178] AUGCUGCGGGCCCACAGACUGCACCAGCUGGCUUUUGAUACCUACCAGGAGUUCGAGGAAGCCU

[0179] ACAUCCCCAAGGAACAGAAGUACUCUUUUCUGCAGAACCCCCAGACCAGCCUGUGCUUCAGCGAG

[0180] AGCAUCCCUACCCCUAGCAACAGAGAGGAAACCCAGCAGAAAAGCAACCUGGAGCUGCUGCGGAU

[0181] CAGCCUGCUGCUGAUUCAGAGCUGGCUGGAGCCUGUGCAGUUCCUGAGGUCCGUUUUCGCCAAU

[0182] AGCCUGGUGUACGGCGCCAGCGACAGCAAUGUGUACGACCUGCUCAAAGAUCUGGAGGAGGGCA

[0183] UCCAGACACUGAUGGGCAGACUGGAAGAUGGCUCUCCUAGAACCGGCCAGAUCUUCAAGCAGAC

[0184] CUAUAGCAAGUUCGACACCAACAGCCACAACGACGACGCCCUGCUUAAAAACUACGGCCUGCUGU

[0185] ACUGCUUCAGAAAGGACAUGGACAAGGUGGAGACAUUUCUGAGAAUCGUGCAAUGUAGAAGCGU

[0186] GGAAGGCUCUUGCGGCUUCCUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCG

[0187] UCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCAC

[0188] UCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCC

[0189] UAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAG

[0190] CUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUGGAGCUAGCAAAAAAAAAA

[0191] AAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0192] AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0193] SEQ ID NO 18: ABD3-linker7-GH9 mRNA sequence

[0194] AGAAUAAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACCAUGGCCACC

[0195] GGCAGCAGAACAAGCCUGCUGCUUGCCUUCGGCCUGCUGUGCCUGCCUUGGCUGCAGGAGGGCUC

[0196] CGCCCUGGCCGAGGCUAAAGAGCUGGCCAUCCGGGAACUGAAAAAGUACGGCAUCAGCGAUUAC

[0197] UACAAGAACCUGAUUAACAAGGCCAAGACCGUGGAAGGCGUGAAGGCCCUGAAAGAUGAGAUCC

[0198] UGGCCGCUUUGCCUGGCGGAGGUGGCUCUGGAGGCGGUGGAUCUGGUGGUGGCGGAUCAGGUGG

[0199] AGGCGGGAGCGGAGGCGGCGGUAGUUUUCCAACCAUCCCCCUGAGCCCGGCUGUUCGACAACGCCA

[0200] UGCUGCGGGCCCAGACAUGCACCAGCUGGCUUUUGAUACAUACCAGGAGUUCGAGGAAGCCUA

[0201] CAUCCCUAAGGAACAGAAGUAUAGCUUCCUGCAGAACCCCCAGACCAGCCUCCUGUUUUUCUGA

[0202] GCAUCCCUACCCCUUUCUAAUAGAGAGGAGACACAGAAAAUCUAACCUGGAGCUGCUCAGAAU

[0203] CAGCCUGCUGAUCCAGAGCUGGCUGGAACCUGUGCAGUUCCUGAGGUCCGUGUUCGCAAAU

[0204] AGCCUGGUGACGGCGCAGCGACAGCAACGUGUACGACCUGCUGAAGGAUCGUGGAAGAGGGAA

[0205] UCCAAACCCUGAUGGGCAGACUGGAAGAUGGCAGUCCAGACCCGGCCAGAUCUUCGCCCAGACC

[0206] UACAGCAAGUUCGACACCAAACAGCCCAAACGACGACGCCCUGCUGAAGAACUACGGACUGCUGUGA

[0207] CUGCUUCAGAAAGGACAUGGACAAGGUGGAAACAUUCCUGCGGAUCGGUUCAAUGUAGAAGCGUC

[0208] GAGGGCAGCUGCGGCUUCCUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGU

[0209] CCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCCACCUCCACCUGCCCCACU

[0210] CACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCU

[0211] AGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGC

[0212] UAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUGGAGCUAGCAAAAAAAAAAA

[0213] AAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0214] SEQ ID NO 19: ABD11-linker6-GH9 mRNA sequence

[0215]

[0216] The experimental results are as follows Figure 5 and Figure 6 As shown: Whether administered subcutaneously or intravenously, the IGF-1 level increases for a longer time than hGH LNP and HSA-hGH LNP, the peak is extended by about 24 hours, and the level is nearly 10-30 times higher than that of HSA-hGH LNP.

[0217] In summary, the present invention mainly addresses the problems existing in HSA-hGH fusion protein, and encapsulates the mRNA of the target drug through LNP (lipid nanoparticles) to form a more long-acting and highly active growth hormone nucleic acid drug, which can achieve relatively high biological activity at a lower dosage.

[0218] The above examples are for the purpose of illustrating the embodiments disclosed by the present invention and are not to be construed as limiting the present invention. In addition, the various modifications listed herein and the variations of the methods in the invention are obvious 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 as described above that are obvious to those skilled in the art 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 as 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. The use according to claim 16, 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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