CN110812366A

CN110812366A - mRNA medicine for hormone supplement and preparation method thereof

Info

Publication number: CN110812366A
Application number: CN201911125305.4A
Authority: CN
Inventors: 刘隽; 彭育才; 向晟楠; 苏晓晔; 刘琪; 雷奕欣; 李爽
Original assignee: Zhuhai Lifanda Biotechnology Co Ltd
Current assignee: Zhuhai Lifanda Biotechnology Co Ltd
Priority date: 2019-11-18
Filing date: 2019-11-18
Publication date: 2020-02-21
Anticipated expiration: 2039-11-18
Also published as: CN110812366B

Abstract

The invention provides an mRNA (messenger ribonucleic acid) medicine for hormone supplementation and a preparation method thereof, and relates to the technical field of medicines. The nucleic acid agent includes mRNA encoding a reproductive hormone-like protein having at least two subunits. The nucleic acid medicine can prolong the half-life period of reproductive hormone medicine and prolong the action time of the medicine. The nucleic acid medicine has good activity, can obviously reduce the administration times of patients, improves the medication compliance of the patients, and has better treatment effect. The preparation method of the nucleic acid medicine does not need to collect natural raw materials, and is simple to operate.

Description

mRNA medicine for hormone supplement and preparation method thereof

Technical Field

The invention relates to the technical field of medicines, in particular to an mRNA medicine for hormone supplementation and a preparation method thereof.

Background

The reproductive hormones include hormones related to reproduction, such as estrogen, progestogen, follicle stimulating hormone, luteinizing hormone, prolactin, and androgen, and include various proteinaceous hormones. The traditional preparation method of the reproductive hormone protein medicine comprises two processes of extracting and recombining protein: the extraction process is to extract active substances of the reproductive hormone proteins from substances containing natural reproductive hormone proteins to be used as medicines, and mainly comprises the steps of collecting raw materials and purifying. The extraction method has the following defects: the natural material source is difficult to obtain, the cost is gradually improved, the process is complex, the yield is low, and the ever-increasing market demand cannot be met. The recombinant protein is a target protein obtained by designing recombinant DNA or RNA, expressing the protein in vitro by an expression system and then purifying. The recombinant protein process has the defects that a cell culture mode is required for production, the process is complex, and the cost is high; modifications of recombinant proteins, such as glycosylation, etc., differ from the natural product, and recombinant proteins and natural proteins often have different pharmacokinetic patterns. Meanwhile, protein drugs also have the defect of short half-life in vivo, and need repeated injection for patients, so that the compliance is poor due to inconvenient medication.

In the case of chorionic gonadotropin-based drugs, chorionic gonadotropin (hCG) is a glycoprotein hormone secreted by placental syncytiotrophoblast cells and naturally secreted from the anterior pituitary gland. The main functions of hCG are: maintain corpus luteum and reduce the activity of maternal lymphocytes, prevent rejection of the fetus. hCG as a medicament is useful in women who undergo superovulation before receiving assisted reproductive techniques such as In Vitro Fertilization (IVF): promoting final follicular maturation and luteinization after stimulating follicular growth; it can also be used for anovulatory or anovulatory women: can promote ovulation and luteinization of patients with anovulation or anovulation after stimulating follicle growth.

hCG extracted from urine of pregnant women has been used in infertility treatment for many years. The extraction of hCG from urine includes the collection and treatment of large amount of urine, pH value regulation to separate out impurity, and ion exchange column chromatography for purification. The recombinant form of hCG protein (rhCG) can be expressed using Chinese Hamster Ovary (CHO) cells and subsequently purified using methods such as ion exchange chromatography and reverse phase high performance liquid chromatography. Urine is used as a raw material to carry out a purification mode, and the urine source is limited, the process is complex, and the yield is low, so that the current mainstream hCG products in the market all adopt a recombinant protein technology. However, the recombinant hCG products have a different pharmacokinetic profile from that of hCG produced from human urine, and the recombinant protein hCG products require continuous stimulation to function in vivo, and hCG is usually cleared after about 24 hours after a single injection, and even an increased dose does not prolong the in vivo duration of protein action. Therefore, all recombinant hCG products on the market require repeated injections to be effective and patient compliance is poor. Therefore, improvements to traditional reproductive hormone-like protein drugs are currently needed in the market.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a nucleic acid medicine, which solves the problem of poor drug effect of reproductive hormone protein medicines in the prior art.

The second object of the present invention is to provide a method for producing the above nucleic acid drug.

The third purpose of the invention is to provide the application of the nucleic acid medicament or the preparation method of the nucleic acid medicament in preparing products for treating diseases related to reproduction.

In order to solve the technical problems, the invention adopts the following technical scheme:

according to one aspect of the present invention, there is provided a nucleic acid agent comprising mRNA encoding a reproductive hormone-like protein comprising at least two subunits.

According to another aspect of the invention, the invention also provides a preparation method of the nucleic acid medicament, which comprises the step of mixing mRNA encoding reproductive hormone protein and optional auxiliary materials to obtain the nucleic acid medicament.

According to another aspect of the invention, the invention also provides the application of the nucleic acid medicament or the preparation method in preparing products for treating diseases related to reproduction.

Compared with the prior art, the invention has the following beneficial effects:

the nucleic acid drug provided by the invention comprises mRNA encoding a reproductive hormone protein containing at least two subunits. In the process that the natural half-life period of the target protein synthesized by mRNA is attenuated, mRNA can continuously synthesize new target protein for supplement, so that the protein can be continuously expressed, the half-life period of the encoded protein in vivo is prolonged, and the effect of prolonging the duration of the drug effect is realized. On the premise of achieving the same effect, the nucleic acid medicament can obviously reduce the administration times of patients, improve the medication compliance of the patients and have better treatment effect. After being delivered into the body, mRNA can utilize cells in the human body to produce endogenous proteins, and has better activity. The preparation method of the nucleic acid medicament provided by the invention does not need to collect natural raw materials, and the production method is simple.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 shows the hCG protein concentration in the supernatant of HEK293 cells in example 1 of the present invention;

FIG. 2 shows the effect of different administration modes in example 3 of the present invention;

FIG. 3 is a graph of the plasma concentration of hCG over time in mice according to example 4 of the present invention;

FIG. 4 is a plot of hCG plasma concentration over time in mice according to example 5 of the present invention;

FIG. 5 is a graph showing the effect of uterine weight gain in mice following administration of hCG mRNA in example 6 of the present invention.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

mRNA is a precursor of protein, and is gradually expressed as a target protein after being introduced into a human body. In the process that the target protein white synthesized by mRNA is attenuated by natural half-life, mRNA can still continuously synthesize new target protein for supplement. The mRNA can continuously express protein within a certain period of time after being delivered into the body, so that the mRNA serving as an active ingredient of the reproductive hormone medicine can prolong the half-life period of the encoded reproductive hormone in the body, thereby realizing long-term efficacy. Therefore, the nucleic acid medicament of the invention can obviously reduce the administration times of patients, improve the medication compliance of patients and achieve better treatment effect on the premise of achieving the same effect. mRNA for coding reproductive hormone protein is used as a medicinal active component, natural raw materials do not need to be collected, and the production method is simple. After being delivered into the body, mRNA can utilize cells in the human body to produce endogenous proteins, and has better activity.

In some preferred embodiments, the nucleic acid agent preferably comprises a gonadotropin, a group of heterodimeric glycoprotein hormones that regulate reproductive function in both men and women. The gonadotropins include, but are not limited to, one or more of Follicle Stimulating Hormone (FSH), Luteinizing Hormone (LH), and Chorionic Gonadotropin (CG); in some alternative embodiments, the nucleic acid drug may contain only mRNA encoding one of FSH, LH and CG; in some alternative embodiments, the nucleic acid drug may also simultaneously comprise multiple mrnas encoding different gonadotropins to simultaneously supplement multiple gonadotropins: for example, but not limited to, may be: comprising both mRNA encoding FSH and mRNA encoding LH; comprising both mRNA encoding FSH and mRNA encoding LH; contains both mRNA encoding LH and mRNA encoding CG; or, both the mRNA encoding FSH, the mRNA encoding LH and the mRNA encoding CG.

The species source of the reproductive hormone is not limited, and the mRNA for coding the reproductive hormone protein can be a sequence of natural mRNA and also can be a sequence optimized by a codon; the encoded reproductive hormone protein can be human reproductive hormone protein and also can be reproductive hormone protein of other species; in order to be more suitable for human supplementation with reproductive hormones, human reproductive hormone-like proteins are preferably included.

The reproductive hormone protein provided by the invention is a protein at least comprising two subunits. In some alternative embodiments, a single mRNA contains coding regions for all subunits of the reproductive hormone-like protein, i.e., one mRNA in a nucleic acid drug can translate the entire reproductive hormone-like protein having a multi-subunit structure; in other alternative embodiments, a single mRNA contains coding regions for only one subunit of a reproductive hormone-like protein; that is, only one mRNA can be translated into one subunit of the reproductive hormone-like protein. It is understood that the nucleic acid drug may contain mRNA encoding all subunits of the reproductive hormone-like protein, so that the nucleic acid drug in the subject of its action translates into the complete reproductive hormone protein having a multi-subunit structure; when a subunit of the reproductive hormone-like protein also has pharmaceutical activity, the nucleic acid drug may also contain at least one mRNA encoding a single subunit, so that the nucleic acid drug is translated into the pharmaceutically active subunit in the subject of its action.

According to the invention, through the experiment of the nucleic acid medicament of the hCG, the coding regions of the α subunit and the β subunit of the hCG are respectively arranged on different mRNAs, and the therapeutic effect of the nucleic acid medicament is better than that of the nucleic acid medicament in which the coding regions of the two subunits of the hCG are arranged on the same mRNA, so that in some preferred embodiments, the nucleic acid medicament is preferably constructed in a mode that the single mRNA only contains the coding region of one subunit in the reproductive hormone protein.

In some alternative embodiments, the mRNA further comprises at least one of (a) - (e): (a) 5' -cap structure: the 5' -cap structure can increase the stability of mRNA, prevent mRNA from being degraded by exonuclease and provide a signal for ribosome to recognize mRNA, and m7GpppG is preferably used. (b) Poly (A) sequence: the polyadenylation sequence prevents degradation of the mRNA by exonucleases and terminates transcription. The poly (A) preferably contains 60 to 120 adenosines, more preferably 80 to 100 adenosines. (c) 5' UTR: the 5 'untranslated region has a regulatory effect on the translation of mRNA, and the 5' UTR is preferably 10-200 nucleotides in length, more preferably 15-100 nucleotides in length; the 5 'UTR preferably comprises the 5' UTR of the DNAH2 gene, and the DNA sequence encoding the 5 'UTR comprising the 5' UTR of the DNAH2 gene is preferably as set forth in SEQ ID No. 5; alternatively, the 5' UTR contains a KOZAK sequence, and the DNA sequence encoding the KOZAK sequence is preferably as shown in SEQ ID NO. 4. (d) 3' UTR: the 3 'untranslated region preferably includes the 3' UTR sequence of hemoglobin HBA2, and the DNA sequence encoding the 3 'UTR comprising the 3' UTR sequence of hemoglobin HBA2 is preferably as shown in SEQ ID NO. 6. (e) Internal Ribosome Entry Sequence (IRES): the IRES makes it possible to initiate protein translation independent of the 5' cap structure, directly starting translation from the middle of the messenger mRNA. The IRES preferably comprises the IRES sequence of a Poliovirus type II (Poliovirus type 2), and the sequence encoding the Poliovirus type II is preferably as shown in SEQ ID NO. 7.

In some specific embodiments, optionally, the mRNA consists of a coding region, a 5 'UTR, and a 3' UTR; alternatively, the mRNA consists of a coding region and an IRES sequence; alternatively, the mRNA consists of a coding region, a 5' -cap structure, an IRES sequence, and a poly a sequence.

In some preferred embodiments, the mRNA contains a 5 ' -cap structure, a polyadenylation sequence, a 5 ' UTR, and a 3 ' UTR. In some preferred embodiments, the mRNA contains a 5 ' -cap structure, a polyadenylation sequence, a 5 ' UTR, a 3 ' UTR, and an internal ribosome entry sequence.

In some alternative embodiments, the mRNA contains modified nucleotides, which refers to nucleotides in the mRNA that have been modified, e.g., modified nucleotides by 2' -O-methylation; or replacing conventional nucleotides in the mRNA with other kinds of nucleotides, for example, L-nucleoside and pseudouracil (pseudo-UTP). Alternatively, the mRNA contains L-nucleosides and pseudo-UTP; optionally, a 2' -O-methylated modified nucleotide is contained.

In some preferred embodiments, the modifications include pseudouracil modifications that replace UTP in mRNA with pseudo-UTP, which can enhance mRNA stability and the emergency response.

In some preferred embodiments, the mRNA contains a modified UTP and/or CTP; optionally, the UTP is modified; alternatively, CTP is modified; alternatively, UTP and CTP are modified. UTP modifications include, for example, 5-terminal-substituted UTPs, such as 5-methoxyuridine; 1-terminally substituted pseudouridine, such as 1-methyl-pseudocytidine; CTP modifications include, for example, 5' terminal-substituted CTPs, such as 5-methyl-cytidine.

The modified positions include, but are not limited to, one or more of the coding region, 5 '-UTR region, 3' -UTR region and cap region of the protein or protein subunit, examples include, but are not limited to: modifying the coding region of the mRNA; modifying the coding region and the 5 '-UTR region and the 3' -UTR region of the mRNA; the cap region of the mRNA is modified.

In some alternative embodiments, the nucleic acid drug further comprises a delivery formulation for delivering mRNA, including but not limited to cationic liposomes, cationic proteins, cationic polypeptides or cationic polymers, for better delivery of mRNA encoding reproductive hormone-like proteins into the body.

In some preferred embodiments, the nucleic acid drug comprises a lipid nanoparticle consisting of mRNA encoding a reproductive hormone-like protein and a lipid component, wherein the mass ratio of the lipid component to the mRNA is preferably (10-30):1, such as but not limited to 10:1, 15:1, 20:1, 25:1 or 30:1, preferably 20:1, more preferably 20: 1.

In some preferred embodiments, the lipid component includes a protonatable cationic lipid, cholesterol, a helper lipid, and a surfactant. The protonatable cationic lipid preferably includes, but is not limited to, DODMA, Dlin-MC3-DMA, Dlin-KC2-DMA or DlinDMA, preferably Dlin-MC 3-DMA. Preferably, the protonatable cationic lipid comprises 20-50% of the molar content of the lipid component, such as but not limited to 20%, 30%, 40% or 50%. Preferably, cholesterol or cholesterol derivative is present in a molar amount of 20-50% of the lipid component, such as but not limited to 20%, 30%, 40% or 50%. Helper lipids include, but are not limited to, DSPC, DOPE, DOPC or DOPS, preferably including DSPC. Preferably, the co-lipid comprises 5-20% of the molar content of the lipid component, such as but not limited to 5%, 10%, 15% or 20%. Preferably, the surfactant includes, but is not limited to, PEG-modified lipid and/or water-soluble surfactant, and the surfactant may be PEG-modified lipid alone or water-soluble surfactant alone, or both PEG-modified lipid and water-soluble surfactant. Wherein the PEG-modified lipid includes but is not limited to PEG-DMG or PEG-DSPE, more preferably PEG-DMG. Preferably, the PEG-modified lipid comprises 1-5% of the molar content of the lipid component, such as but not limited to 1%, 2%, 3%, 4% or 5%, more preferably 3%. Water-soluble surfactants include, but are not limited to, Tween 20, Tween 80, P188 or PVA. Preferably, the weight to volume ratio of water-soluble surfactant to lipid component is 0.2-2%, such as but not limited to 0.2%, 0.5%, 1%, 1.5% or 2%, more preferably 1%.

In some preferred embodiments, the nucleic acid agent comprises a human chorionic gonadotropin (hCG) mRNA agent. The main functions of hCG are: maintaining the corpus luteum and reducing the activity of maternal lymphocytes, preventing rejection of the fetus, for use in assisted reproductive technologies, and for anovulatory or anovulatory women. Preferably, the mRNA encoding hCG encodes human hCG, and is more suitable for expression in mammals, particularly humans, and the mRNA encoding hCG is preferably codon optimized.

The hCG protein structure includes α and β subunits, α subunit is similar to LH, FSH and TSH, especially has relatively large immunological cross reaction with LH, contains 92 amino acids, β subunit is unique to hCG and contains 145 amino acids.

In some alternative embodiments, the nucleic acid agent comprises hCG mRNA comprising the coding region for the α subunit of hCG and the coding region for the β subunit of hCG, capable of simultaneously coding for the α subunit and the β subunit, hCG mRNA preferably comprises the coding region for the α subunit of hCG, the coding region for the β subunit of hCG, a 5 ' -cap structure, a polyadenylation sequence, a 5 ' UTR, a 3 ' UTR, and internal ribosome entry sequences, hCG mRNA preferably comprising a sequence as set forth in SEQ ID No. 1.

In some preferred embodiments, the nucleic acid drug comprises hCG α mRNA and hCG β mRNA, hCG α mRNA comprises the coding region for the α subunit of hCG, and hCG β mRNA comprises the coding region for the β subunit of hCG.

In some preferred embodiments, the hCG α mRNA contains the coding region for the α subunit, a 5 ' -cap structure, a polyadenylation sequence, a 5 ' UTR and a 3 ' UTR, and the hCG α mRNA preferably contains the sequence shown in SEQ ID No. 2.

In some preferred embodiments, the hCG β mRNA contains the coding region for the β subunit, a 5 ' -cap structure, a polyadenylation sequence, a 5 ' UTR and a 3 ' UTR, and the hCG β mRNA preferably contains the sequence shown in SEQ ID No. 3.

In some preferred embodiments, the molar ratio of hCG α mRNA to hCG β mRNA in the nucleic acid drug is (1-2): preferably (1-1.5): more preferably 1: 1.

In some preferred embodiments, it has been found experimentally that Dlin-MC3-DMA is preferred as a delivery agent in nucleic acid pharmaceuticals for hCG. And preferably, the administration is carried out by intravenous administration and subcutaneous administration, and the hCG protein can be detected in blood after 3 hours of administration in mice after administration, and can be continuously expressed for more than 48 hours, wherein the intravenous administration has better effect.

According to one aspect of the invention, the invention also provides a preparation method of the nucleic acid medicament, which comprises the step of mixing mRNA and optional auxiliary materials to obtain the nucleic acid medicament. "optional adjuvants" means adjuvants which are acceptable in the art, optionally with or without addition.

In some preferred embodiments, the mRNA is transcribed from a DNA template. Optionally, the DNA template contains coding regions for all subunits of reproductive hormone-like proteins; optionally, the DNA template contains coding regions for only one subunit of a reproductive hormone-like protein. The linearized DNA template is transcribed to obtain mRNA, preferably in vitro transcription is adopted to obtain mRNA, in vitro transcription mRNA is produced in vitro without cells, the culture of living cells and complex separation engineering are not involved, and the production condition is convenient to control. Preferably, mRNA is obtained by in vitro transcription catalyzed by RNA polymerase, and the mRNA is obtained from ATP, CTP, pseudo-UTP, GTP and ARCA cap. Then the product of the in vitro transcription is concentrated to obtain mRNA for coding the reproductive hormone protein in the nucleic acid medicament.

In some preferred embodiments, the nucleic acid drug is obtained by dissolving mRNA in an aqueous phase to obtain a solution containing mRNA to obtain a first mixture, and uniformly mixing the first mixture with a delivery formulation; wherein the aqueous phase preferably comprises a buffer, including but not limited to citrate buffer or acetate buffer; preferably, a citrate buffer is used, and the pH of the citrate buffer is preferably 3.5-4.5. The concentration of mRNA in the first mixture is preferably 0.05-0.15 mg/mL, and may be, but is not limited to, 0.05mg/mL, 0.08mg/mL, 0.1mg/mL, 0.12mg/mL, or 0.15mg/mL, preferably 0.1 mg/mL.

In some preferred embodiments, the nucleic acid drug comprises a lipid nanoparticle: the preparation method of the lipid nanoparticle further comprises the following steps: dissolving the lipid component in an organic phase to obtain a second mixture, and then mixing the first mixture and the second mixture to obtain a third mixture. In some alternative embodiments, the third mixture is diluted first and then concentrated to obtain the nucleic acid drug, preferably 50-100 fold diluted, such as but not limited to 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold; in other alternative embodiments, the nucleic acid drug is obtained after removing the organic phase from the third mixture. The concentration of the lipid nanoparticles in the nucleic acid drug is preferably adjusted to 50-200. mu.g/ml, such as but not limited to 50. mu.g/ml, 80. mu.g/ml, 100. mu.g/ml, 120. mu.g/ml, 150. mu.g/ml or 200. mu.g/ml, independently, by using a method of diluting and then concentrating the third mixture, or by removing the organic phase from the third mixture.

The organic phase is preferably anhydrous C1-C4 lower alcohol, and more preferably anhydrous ethanol. The concentration of the lipid in the second mixture is 4-8 mg/mL, and may be, but is not limited to, 4mg/mL, 5mg/mL, 6mg/mL, 7mg/mL, or 8mg/mL, preferably 6 mg/mL. The first mixture and the second mixture are preferably mixed in a volume ratio of 1: (2-4), more preferably 1: 3. In mixing the first mixture and the second mixture, they may be mixed manually or by using a microfluidic device, which is preferred for more precise mixing. When mixing with a microfluidic device, the flow rate control is preferably 3mL/min to 24mL/min, and may be, for example, but not limited to, 3mL/min, 5mL/min, 10mL/min, 12mL/min, 15mL/min, 20mL/min, or 24mL/min, preferably 12 mL/min.

In some preferred embodiments, the protein encoded in the nucleic acid drug comprises hCG, the nucleic acid drug comprises hCG α mRNA and hCG β mRNA, and hCG α mRNA and hCG β mRNA are mixed in a molar ratio of (1-2): (1-2), preferably in a molar ratio of (1-1.5): (1-1.5), and more preferably in a molar ratio of 1: 1.

According to another aspect of the invention, the invention also provides the application of the nucleic acid medicament or the preparation method of the nucleic acid medicament in preparing products for treating diseases related to reproduction. The nucleic acid medicine can prolong the half-life period of hormone medicine, prolong the action time of the medicine, has good medicine activity, can obviously reduce the administration times of patients, improves the medication compliance of the patients and has better treatment effect. The nucleic acid medicine is prepared into a product for treating diseases related to reproduction, for example, the product is used in combination with other medicines or is used in a set with other treatment equipment, and the treatment effect of the product is better.

Example 1

The effect of two different mRNA design ideas on hCG protein expression was verified by transfecting two different mRNAs (hCG mRNA: single-chain mRNA, two subunits designed on the same chain and having the sequence shown in SEQ ID NO.1, hCG α mRNA and hCG β mRNA, hCG α mRNA encoding hCG α subunit, having the sequence shown in SEQ ID NO.2, hCG β mRNA encoding hCG β subunit, having the sequence shown in SEQ ID NO.3, and hCG α mRNA and hCG β mRNA at the time of transfection at a molar ratio of 1:1 mixed) on HEK293 cells with commercial reagent lipofectamine2000, and transfecting the mRNAs.

HEK293 cells recovered from liquid nitrogen were cultured for one generation and then ready for use. HEK293 cells were seeded 24 hours prior to the experiment into 6-well plates at a cell density of 400000 and 500000 per well. The cell state is observed after the culture in the incubator at 37 ℃ for 24 hours, and the experiment can be started when the cell confluence reaches about 80-90%. The nano-composite of lipofectamine2000 and mRNA coding HCG gene is prepared according to the method provided in the product instruction, the same amount of mRNA of naked HCG is used as a negative control group, the volume mass ratio of lipofectamine2000 to mRNA coding HCG gene is 2:1, the lipofectamine2000 and the mRNA coding HCG gene are mixed and then are stood for 15 minutes at room temperature, and the mixture is directly added into 6-hole plate cell culture solution, so that 1 mu g of mRNA is added into each hole. The cells were then incubated at 37 ℃ for 24 hours, and the cell culture supernatant was collected and assayed for HCG protein expression by ELISA. The results of the experiment show that hCG protein expression is higher when the mRNAs respectively code hCG. The results are shown in FIG. 1.

Example 2

The results of experiments using hCG α mRNA (shown as SEQ ID NO. 2) and hCG β mRNA (shown as SEQ ID NO. 3) as hCG-encoding mRNA showed that various delivery agents were able to transfect hCG mRNA efficiently and express hCG protein on cells in vitro, with the commercial agent lipofectamine2000 having the highest transfection efficiency.

TABLE 1 transfection efficiency of hCG-encoding mRNA and various delivery formulations on HEK293 cells and delivery efficacy in mice by various routes of administration

	Cell transfection	Intravenous injection	Intramuscular injection	Subcutaneous injection
					lipofectamine2000	++++	-	-	-
DOTAP/DOPE	++	-	-	N/A
					jetPEI	++	-	-	N/A
PEI 25kDa	++	-	-	N/A
					LNP(DOTAP)	-	N/A	-	N/A
protamine	-	N/A	-	N/A
					liposome	-	N/A	-	N/A
LNP(MC3)	+	+++	-	+

+: hCG expression was detected in cell supernatants or hCG was detected in sera 3-48 h after administration to mice +++: strong positive, -: hCG expression was not detected in cell supernatants or hCG, LNP (MC3) was not detected in sera 3h-48h after mouse administration: a lipid nanoparticle with Dlin-MC3-DMA as core lipid. Lnp (dotap): lipid nanoparticles with DOTAP as core lipid.

Wherein, the preparation method of each delivery preparation for delivering mRNA comprises the following steps:

preparation of protamine nanoparticles:

a. the mRNA was dissolved in ultrapure water to adjust the concentration to 0.5 mg/ml.

b. Protamine (protamine) was dissolved in another tube of ultrapure water and the concentration was adjusted to 0.5 mg/ml.

c. Protamine was mixed with the mRNA solution at a volume ratio of 1: 2.

d. Mix for 30 seconds using a vortex shaker and stand at room temperature for 15 minutes.

Preparation of Polyethyleneimine (PEI) nanoparticles:

b. PEI is dissolved in another tube of ultrapure water and the concentration is adjusted so that the ratio of the amount of amino groups on PEI to the amount of nucleic acid phosphate groups is between 2:1 and 20: 1.

c. PEI and mRNA solutions were mixed in equal volumes.

Preparation of cationic liposome and cationic lipid nanoparticles:

the preparation method of the cationic liposome comprises the following steps:

a. lipid chloroform solution was prepared according to the following formulation: the cationic lipid is selected from DOTAP, and the neutral helper lipid is selected from DOPE. The molar ratio of the two is 20: 9.

b. Taking a proper amount of chloroform solution in a round-bottom flask, removing the organic solvent by using a rotary evaporation method, and preparing a dry lipid membrane;

c. the lipid film in the round-bottom flask was dried for 2 hours using a vacuum drying apparatus.

d. Adding the RNase-free ultrapure water into a round-bottom flask to hydrate the liposome membrane, and oscillating and hydrating in a water bath at 37 ℃ for 10 minutes to ensure that the concentration of the hydrated liposome is 3 mg/ml.

e. The hydrated liposome is placed in a water bath ultrasonic instrument for ultrasonic treatment at 40 ℃ for 40 minutes to obtain an aqueous suspension product of the cationic liposome, which is named as Tf4 below.

The preparation method of the cationic liposome and mRNA nano-composite comprises the following steps:

a. an appropriate amount of mRNA solution was diluted in PBS solution at a concentration of 0.1 mg/ml.

b. And taking a proper amount of liposome suspension to dilute the liposome suspension in a PBS solution, and controlling the mass ratio of the liposome suspension to the mRNA to be 1.5:1 to 4: 1.

c. Mixing the two solutions by using a pipette gun, lightly blowing for 5 times, and standing for 15 minutes to obtain the cation complex.

d. The product quality was analyzed by particle size analysis, and the complex size was about 180-200 nm.

The preparation method of the Lipofectamine2000 cation complex comprises the following steps:

a. the mRNA was dissolved in OptiMEM (thermo) and adjusted to a concentration of 0.1 mg/ml.

b. Lipofectamine2000 was dissolved in OptiMEM (thermo) at a ratio (v/w) of volume used to mass of nucleic acid used of between 1:1 and 2:1, preferably 2:1 here.

c. And standing for 15 minutes.

The preparation method of the lipid nanoparticle comprises the following steps:

1. hCG α mRNA and hCG β mRNA were mixed at a molar ratio of 1:1 in a citrate buffer at PH 4, and the concentration was adjusted to 0.1 mg/ml.

2. The delivery formulation ingredients were dissolved separately in absolute ethanol and the total lipid concentration was adjusted to 6 mg/ml. The molar ratio of each lipid component is as follows: 50% Dlin-MC3-DMA/DOTAP, 38.5% cholestrol, 10% DSPC, 1.5% PEG-DMG or 50% Dlin-MC3-DMA/DOTAP, 40% cholestrol, 7% DSPC, 3% PEG-DMG or 40% Dlin-MC3-DMA/DOTAP, 50% cholestrol, 10% DSPC, 1.5% PEG-DMG.

3. The organic phase and the water phase are mixed in a volume ratio of 1:3 by a micro-fluidic device, and the flow rate is controlled to be 12ml/min when the micro-fluidic device is used for mixing.

4. The resulting mixture was immediately diluted 100-fold with PBS solution at PH 7.4, and the ethanol content of the solution was removed by Tangential Flow Filtration (TFF) and concentrated to 100 μ g/ml, to obtain lipid nanoparticles encapsulating mRNA of hCG.

Example 3

The length of the mRNA coding luciferase is about 2000nt, and the expressed luciferase can emit a fluorescent signal when catalyzing corresponding substrates, so that the mRNA can be used as a reporter gene research preparation and an administration mode to influence the in vivo delivery efficiency, distribution and expression of the mRNA.

LNP preparation (MC3) containing 5 μ g luciferase mRNA and 20 μ g HCG mRNA was introduced using tail vein administration and subcutaneous injection. The administration mice were injected with luciferase substrate 2 hours later, and after 10 minutes, the mice were anesthetized and the expression of luciferase in the mice was observed by a fluorescence live imaging system (FIG. 2). It was found that the expression of luciferase mRNA was detected after 2 hours regardless of whether intravenous injection or subcutaneous injection was used, that the mouse luciferase injected subcutaneously was expressed only at the injection site, that the mouse luciferase administered intravenously was expressed at the liver site, and that the signal intensity and range of the mouse luciferase administered intravenously were greater than those of the mouse administered subcutaneously. However, no fluorescent signal was detected in the liver of mice given intravenously 24 hours after dosing, indicating that hepatic mRNA expression was short. Mice given subcutaneously still had a high fluorescence signal after 24 hours, and the signal could persist for more than 48 hours post-dose. Analysis of mRNA expressed more rapidly but for a shorter duration by intravenous administration but the secreted protein expressed could pass directly into the peripheral circulation via the liver, so it was speculated that intravenous injection could express HCG protein faster and produce drug effect faster. Subcutaneous mRNA expression is more durable, whereas secreted proteins need to be absorbed by local tissues to enter the blood circulation, and may be slow to act.

Example 4

The hCG protein content in blood is quantified by a protein ELISA method, experimental results are shown in figure 3, the concentration of serum hCG is not detected 3 hours and 48 hours after different sequence designs and different administration routes are compared 3 hours and 48 hours after administration, the concentration of the serum hCG is not detected 3 hours after 3 hours and 48 hours after the administration, the hCG α mRNA and the hCG β mRNA are administered intravenously, the hCG single-chain mRNA is detected 3 hours after the intravenous administration, and the expression quantity of the hCG is maintained to 48 hours after the hCG α mRNA and the hCG β mRNA is administered subcutaneously.

Example 5

The trend of the serum hCG concentration was compared over 24 hours after intravenous administration of hCG mRNA and recombinant protein. The value 2 hours after intravenous administration was taken as a standard line, and the hCG mRNA administration group showed an upward trend after 6 hours of administration and fell back below the standard line by 12 hours. The recombinant protein administration group always shows a descending trend, and the descending speed is higher than that of the mRNA administration group. The concentration of the recombinant protein is always reduced after the recombinant protein enters the blood, and the mRNA also continuously expresses the protein after the recombinant protein enters the blood, so that the half-life period is longer, therefore, the mRNA medicament form is adopted, a more lasting expression effect can be achieved in the human body or the animal body, and the mRNA medicament has superiority compared with protein medicaments. The results of the experiment are shown in FIG. 4.

Example 6

The increase of the uterine weight of mice caused by hCG is the only index for evaluating the therapeutic effect of hCG, the lipid nanoparticles of hCG α mRNA and hCG β mRNA encapsulated by LNP (MC3) prepared in example 2 are used as nucleic acid drugs, and the uterine weight of the mice is weighed 3 days after intravenous and subcutaneous administration (1 mug/mouse) and compared with the uterine weight of mice in a control group.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

SEQUENCE LISTING

<110> Zhuhai livan der Biotechnology Ltd

<120> mRNA medicine for hormone supplement and preparation method thereof

<160>7

<170>PatentIn version 3.5

<210>1

<211>1798

<212>RNA

<213> Artificial sequence

<400>1

gggagaccca agcuggcuag cgggagaaag cuuaccaugg agauguucca ggggcugcug 60

cuguugcugc ugcugagcau gggcgggaca ugggcaucca aggagccgcu ucggccacgg 120

ugccgcccca ucaaugccac ccuggcugug gagaaggagg gcugccccgu gugcaucacc 180

gucaacacca ccaucugugc cggcuacugc cccaccauga cccgcgugcu gcaggggguc 240

cugccggccc ugccucaggu ggugugcaac uaccgcgaug ugcgcuucga guccauccgg 300

cucccuggcu gcccgcgcgg cgugaacccc guggucuccu acgccguggc ucucagcugu 360

caaugugcac ucugccgccg cagcaccacu gacugcgggg gucccaagga ccaccccuug 420

accugugaug acccccgcuu ccaggacucc ucuuccucaa aggccccucc ccccagccuu 480

ccaaguccau cccgacuccc ggggcccucg gacaccccga uccucccaca augauuaaaa 540

cagcucuggg guuguuccca ccccagaggc ccacguggcg gccaguacac ugguauugcg 600

guaccuuugu acgccuguuu uauacucccu ucccccguaa cuuagaagca caauguccaa 660

guucaauagg aggggguaca aaccaguacc accacgaaca agcacuucug uucccccggu 720

gaggcuguau aggcuguuuc cacggcuaaa agcggcugau ccguuauccg cucauguacu 780

ucgagaagcc uaguaucacc uuggaaucuu cgaugcguug cgcucaacac ucaaccccag 840

aguguagcuu aggucgauga gucuggacgu uccucaccgg cgacgguggu ccaggcugcg 900

uuggcggccu accuguggcc caaagccaca ggacgcuagu ugugaacaag gugugaagag 960

ccuauugagc uaccugagag uccuccggcc ccugaaugcg gcuaauccua accacggagc 1020

aggcaguggc aauccagcga ccagccuguc guaacgcgca aguucguggc ggaaccgacu 1080

acuuugggug uccguguuuc cuuuuauuuu uacaauggcu gcuuauggug acaaucauug 1140

auuguuauca uaaagcaaau uggauuggcc auccggugag aauuugauua uuaaauuacu 1200

cucuuguugg gauugcuccu uugaaaucuu gugcacucac accuauugga auuaccucau 1260

uguuaagaua ucauggauua cuacagaaaa uaugcagcua ucuuucuggu cacauugucg 1320

guguuucugc auguucucca uuccgcuccu gaugugcagg auugcccaga augcacgcua 1380

caggaaaacc cauucuucuc ccagccgggu gccccaauac uucagugcau gggcugcugc 1440

uucucuagag cauaucccac uccacuaagg uccaagaaga cgauguuggu ccaaaagaac 1500

gucaccucag aguccacuug cuguguagcu aaaucauaua acagggucac aguaaugggg 1560

gguuucaaag uggagaacca cacggcgugc cacugcagua cuuguuauua ucacaaaucu 1620

ugaggacuag uuauaagacu gacuagcccg augggccucc caacgggccc uccuccccuc 1680

cuugcaccga gauuaauaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1740

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaag 1798

<210>2

<211>532

<212>RNA

<213> Artificial sequence

<400>2

ggggcuagcg ccgccaccau ggacuacuac cgcaaauacg cagcuaucuu ucucgucacu 60

cucagugugu uccugcaugu gcugcacagu gcaccagacg uucaagacug cccugaaugc 120

acccuccaag agaacccauu cuucagucag ccuggcgcac ccauacucca gugcauggga 180

uguugcuucu caagggccua cccuacucca cugcggucaa agaagaccau gcuggugcag 240

aagaauguua caagugaauc cacaugcugc guugccaaau ccuauaaccg ggugacugug 300

augggugggu ucaaaguuga gaaucacacu gcaugucacu guucuacuug uuacuaucau 360

aagagcuagu gagcuggagc cucgguagcc guuccuccug cccgcugggc cucccaacgg 420

gcccuccucc ccuccuugca ccggcccuuc cuggucuuug aauaaagucu gagugggcag 480

caaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa ag 532

<210>3

<211>679

<212>RNA

<213> Artificial sequence

<400>3

ggggcuagcg ccgccaccau ggaaauguuu caaggccucc ugcuccugcu ccugcucuca 60

augggaggaa cuugggcuuc uaaggagcca cucagaccaa gguguaggcc uaucaacgca 120

acucucgcug uggagaaaga gggcugcccu gucuguauua cugucaacac aaccaucugu 180

gcagguuauu gcccaaccau gaccagggug cuccagggcg ugcugccagc acugccacag 240

guggugugua acuaccggga ugugcgguuc gaaagcauua gacucccagg gugucccaga 300

ggcgugaacc cagugguuuc auacgcagug gcccugucuu gccagugugc ucugugccgg 360

aggaguacaa ccgauugugg aggcccuaag gaccauccuc ugacuuguga ugauccacgc 420

uuucaagaua gcucuagcuc caaggcuccu ccuccauccc ucccuagucc cagcagacug 480

ccagggccua gugacacucc aauucugcca caguagugag cuggagccuc gguagccguu 540

ccuccugccc gcugggccuc ccaacgggcc cuccuccccu ccuugcaccg gcccuuccug 600

gucuuugaau aaagucugag ugggcagcaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 660

aaaaaaaaaa aaaaaaaag 679

<210>4

<211>16

<212>DNA

<213> Artificial sequence

<400>4

ggctagcgcc gccacc 16

<210>5

<211>34

<212>DNA

<213> Artificial sequence

<400>5

gagacccaag ctggctagcg ggagaaagct tacc 34

<210>6

<211>109

<212>DNA

<213> Artificial sequence

<400>6

gctggagcct cggtagccgt tcctcctgcc cgctgggcctcccaacgggc cctcctcccc 60

tccttgcacc ggcccttcct ggtctttgaa taaagtctga gtgggcagc 109

<210>7

<211>738

<212>DNA

<213> Poliovirus type II virus (Poliovirus type 2 IRES)

<400>7

ttaaaacagc tctggggttg ttcccacccc agaggcccac gtggcggcca gtacactggt 60

attgcggtac ctttgtacgc ctgttttata ctcccttccc ccgtaactta gaagcacaat 120

gtccaagttc aataggaggg ggtacaaacc agtaccacca cgaacaagca cttctgttcc 180

cccggtgagg ctgtataggc tgtttccacg gctaaaagcg gctgatccgt tatccgctca 240

tgtacttcga gaagcctagt atcaccttgg aatcttcgat gcgttgcgct caacactcaa 300

ccccagagtg tagcttaggt cgatgagtct ggacgttcct caccggcgac ggtggtccag 360

gctgcgttgg cggcctacct gtggcccaaa gccacaggac gctagttgtg aacaaggtgt 420

gaagagccta ttgagctacc tgagagtcct ccggcccctg aatgcggcta atcctaacca 480

cggagcaggc agtggcaatc cagcgaccag cctgtcgtaa cgcgcaagtt cgtggcggaa 540

ccgactactt tgggtgtccg tgtttccttt tatttttaca atggctgctt atggtgacaa 600

tcattgattg ttatcataaa gcaaattgga ttggccatcc ggtgagaatt tgattattaa 660

attactctct tgttgggatt gctcctttga aatcttgtgc actcacacct attggaatta 720

cctcattgtt aagatatc 738

Claims

1. A nucleic acid agent comprising mRNA encoding a reproductive hormone-like protein comprising at least two subunits.

2. The nucleic acid drug of claim 1, wherein the reproductive hormone-like protein comprises gonadotropin;

preferably, the gonadotropins comprise one or more of follicle stimulating hormone, luteinizing hormone and chorionic gonadotropin;

preferably, in the nucleic acid drug, a single mRNA contains coding regions of all subunits of the reproductive hormone protein;

preferably, in the nucleic acid drug, a single mRNA contains a coding region of only one subunit of the reproductive hormone-like protein;

preferably, the nucleic acid drug comprises mRNA encoding all subunits of the reproductive hormone protein, and a single mRNA only contains a coding region of one subunit of the reproductive hormone protein.

3. The nucleic acid drug of claim 1, wherein the mRNA further comprises at least one of (a) to (e):

(a) a 5' -cap structure, preferably m7 gppppg;

(b) a polyadenylation sequence; the poly (A) preferably contains 60 to 120 adenosines, more preferably 80 to 100 adenosines;

(c) 5' UTR; the 5' UTR is preferably 10-200 nucleotides in length, preferably 15-100 nucleotides in length; the 5 'UTR preferably comprises a DNAH 25' UTR or KOZAK sequence;

(d) a 3 'UTR, preferably comprising the 3' UTR sequence of hemoglobin HBA 2;

(e) internal ribosome entry sequences.

4. The nucleic acid agent of claim 1, wherein the mRNA contains modified nucleotides;

preferably, the modifications include L-nucleoside modifications and/or 2' -O-methylation modifications;

preferably, the mRNA contains a modified UTP and/or CTP;

preferably, the modified nucleotide comprises a substitution of UTP to pseudo-UTP;

preferably, the modified nucleotide is located in at least one of the coding region, the 5 '-UTR region, the 3' -UTR region and the cap region.

5. The nucleic acid drug of claim 1, further comprising a delivery agent for delivering mRNA;

preferably, the delivery formulation comprises a cationic liposome, a cationic protein, a cationic polypeptide, a cationic polymer;

preferably, the nucleic acid drug comprises lipid nanoparticles consisting of mRNA encoding a reproductive hormone-like protein and a lipid component;

preferably, the mass ratio of lipid component to mRNA is (10-30) to 1, preferably 20: 1;

preferably, the lipid component comprises a protonatable cationic lipid, cholesterol, a helper lipid, and a surfactant;

preferably, the protonatable cationic lipid comprises DODMA, Dlin-MC3-DMA, Dlin-KC2-DMA or DlinDMA, preferably Dlin-MC 3-DMA;

preferably, the protonatable cationic lipid comprises 20-50% of the molar content of the lipid component;

preferably, said cholesterol or cholesterol derivative represents 20-50% of the molar content of said lipid component;

preferably, the helper lipid comprises DSPC, DOPE, DOPC or DOPS, more preferably DSPC;

preferably, the helper lipid comprises 5-20% of the molar content of the lipid component;

preferably, the surfactant comprises a PEG-modified lipid and/or a water-soluble surfactant;

preferably, the PEG-modified lipid comprises PEG-DMG or PEG-DSPE, more preferably PEG-DMG;

preferably, the PEG-modified lipid represents 1-5%, preferably 3%, of the molar content of the lipid component;

preferably, the water-soluble surfactant comprises Tween 20, Tween 80, P188 or PVA;

preferably, the weight to volume ratio of the water-soluble surfactant to the lipid component is 0.2-2%, preferably 1%.

6. The nucleic acid agent of any one of claims 1-5, wherein the nucleic acid agent comprises mRNA encoding human chorionic gonadotropin;

preferably, the nucleic acid agent comprises hCG mRNA containing the coding region for the α subunit of human chorionic gonadotropin and the coding region for the β subunit of hCG;

preferably, the hCG mRNA further comprises a 5 ' -cap structure, a polyadenylation sequence, a 5 ' UTR, a 3 ' UTR, and an internal ribosome entry sequence;

preferably, the hCG mRNA contains a sequence shown as SEQ ID NO. 1;

preferably, the nucleic acid drug comprises hCG α mRNA and hCG β mRNA, said hCG α mRNA comprising the coding region for the α subunit of human chorionic gonadotropin, said hCG β mRNA comprising the coding region for the β subunit of human chorionic gonadotropin;

preferably, the hCG α mRNA contains the coding region for the α subunit, a 5 ' -cap structure, a polyadenylation sequence, a 5 ' UTR and a 3 ' UTR;

preferably, the hCG α mRNA contains a sequence shown as SEQ ID NO. 2;

preferably, the hCG β mRNA contains the coding region for the β subunit, a 5 ' -cap structure, a polyadenylation sequence, a 5 ' UTR and a 3 ' UTR;

preferably, the hCG β mRNA contains a sequence shown as SEQ ID NO. 3;

preferably, the nucleic acid medicament comprises hCG α mRNA and hCG β mRNA, and the molar ratio of the hCG α mRNA to the hCG β mRNA is (1-2): 1-2, preferably (1-1.5): 1-1.5), and more preferably 1: 1.

7. The nucleic acid drug of claim 6, wherein the nucleic acid drug comprises the hCG α mRNA and the hCG β mRNA, and the delivery formulation for delivery of the hCG α mRNA and the hCG β mRNA comprises Dlin-MC 3-DMA;

preferably, the administration mode of the nucleic acid drug includes intravenous administration and subcutaneous administration, and preferably includes intravenous administration.

8. The method for preparing a nucleic acid drug according to any one of claims 1 to 7, wherein mRNA encoding a reproductive hormone-like protein and optionally an adjuvant are mixed to obtain the nucleic acid drug;

preferably, the mRNA is transcribed from a DNA template;

preferably, the DNA template contains coding regions for all subunits of the reproductive hormone-like protein;

preferably, the DNA template contains a coding region for only one subunit of a reproductive hormone-like protein;

preferably, mRNA is obtained by in vitro transcription;

preferably, the linearized DNA template is catalyzed by RNA polymerase to obtain mRNA by in vitro transcription;

preferably, the starting material for in vitro transcription of mRNA includes ATP, CTP, pseudo-UTP, GTP and the ARCA cap.

9. The method for preparing a nucleic acid drug according to claim 8, comprising mixing the mRNA encoding the reproductive hormone-like protein with the delivery agent to obtain the nucleic acid drug;

preferably, mRNA is dissolved in the water phase to obtain a solution containing mRNA, so as to obtain a first mixture, and then the first mixture is uniformly mixed with the delivery preparation, so as to obtain the nucleic acid drug;

preferably, the aqueous phase comprises a buffer;

preferably, the buffer comprises a citrate buffer or an acetate buffer;

preferably, the buffer preferably comprises a citrate buffer;

preferably, the pH value of the citrate buffer solution is 3.5-4.5;

preferably, the concentration of mRNA in the first mixture is 0.05-0.15 mg/mL, preferably 0.1 mg/mL;

preferably, the nucleic acid drug comprises lipid nanoparticles, and the preparation method of the lipid nanoparticles further comprises the following steps: dissolving the lipid component in an organic phase to obtain a second mixture, mixing the first mixture and the second mixture to obtain a third mixture;

then diluting the third mixture, and then concentrating to obtain the nucleic acid medicament;

or, removing the organic phase from the third mixture to obtain the nucleic acid drug;

preferably, the concentration of the lipid nanoparticles in the nucleic acid drug is 50-200 μ g/ml;

preferably, said diluting said third mixture comprises diluting said third mixture by a factor of 50-100;

preferably, said removing the organic phase in the third mixture comprises removing the organic phase in the third mixture using tangential flow filtration;

preferably, the organic phase comprises anhydrous C1-C4 lower alcohol;

preferably, the anhydrous C1-C4 lower alcohol is anhydrous ethanol;

preferably, the concentration of the lipid component in the second mixture is 4-8 mg/mL, preferably 6 mg/mL;

preferably, the first mixture and the second mixture are mixed in a volume ratio of 1: (2-4), preferably mixing according to a ratio of 1: 3;

preferably, the first mixture and the second mixture are mixed using a microfluidic device;

preferably, the flow rate is controlled to be 3mL/min-24mL/min, preferably 12mL/min when the micro-fluidic device is used for mixing;

preferably, the nucleic acid medicine comprises the hCG α mRNA and the hCG β mRNA, and the hCG α mRNA and the hCG β mRNA are mixed in a molar ratio of (1-2): 1-2, preferably in a molar ratio of (1-1.5): 1-1.5, and more preferably in a molar ratio of 1: 1.

10. Use of a nucleic acid drug as claimed in any one of claims 1 to 7, or a method of manufacture as claimed in claim 8 or 9, in the manufacture of a product for use in the treatment of a reproductive-related disease.