CN110812366B

CN110812366B - mRNA medicine for hormone supplement and preparation method thereof

Info

Publication number: CN110812366B
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: 2023-11-17
Anticipated expiration: 2039-11-18
Also published as: CN110812366A

Abstract

The invention provides an mRNA medicament for hormone supplement and a preparation method thereof, and relates to the technical field of medicaments. Nucleic acid agents include mRNAs encoding a reproductive hormone protein containing at least two subunits. The nucleic acid medicine can prolong half life of genital hormone medicine and prolong medicine action time. The nucleic acid has good pharmaceutical 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 supplement and a preparation method thereof.

Background

The reproductive hormone is hormone related to reproduction, such as estrogen, progestin, follicle stimulating hormone, luteinizing hormone, prolactin, and androgens, etc., wherein the reproductive hormone comprises various proteins. 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 natural reproduction hormone proteins from substances containing the natural reproduction hormone proteins to be used as medicines, and mainly comprises the processes of collecting raw materials and purifying. The defects of the extraction method are mainly as follows: natural material sources are difficult to obtain, the cost is gradually increased, the process is complex, the yield is low, and the ever-increasing market demands cannot be met. The recombinant protein is the target protein obtained by designing recombinant DNA or RNA, expressing the protein in vitro through an expression system and purifying. The recombinant protein technology has the defects that a cell culture mode is needed in production, the technology is complex, and the cost is high; modifications of recombinant proteins such as glycosylation differ from the natural products, and recombinant proteins and natural proteins often have different pharmacokinetic patterns. Meanwhile, protein medicines have the defect of short half-life in vivo, and are required to be repeatedly injected by patients, so that the medicines are inconvenient to use and have poor compliance.

Taking chorionic gonadotrophin (hCG) as an example, chorionic gonadotrophin (hCG) is a glycoprotein hormone secreted by placental syngeneic trophoblastic cells and naturally secreted by the anterior pituitary gland. The main functions of hCG are: maintain corpus luteum and reduce maternal lymphocyte activity, and prevent rejection reaction to fetus. hCG is useful as a medicament for women undergoing superovulation prior to receiving assisted reproductive technologies such as In Vitro Fertilization (IVF): promoting final follicle maturation and luteinisation after stimulating follicle growth; it can also be used for anovulatory or anovulatory women: can promote ovulation and luteinization of anovulatory or anovulatory patients after stimulating follicular growth.

hCG extracted from urine of pregnant women has been used for many years in the treatment of infertility. The extraction of hCG from urine involves the collection and handling of large amounts of urine, pH adjustment to precipitate impurities, and purification by ion exchange column chromatography. Recombinant forms of hCG protein (rhCG) can be expressed using Chinese Hamster Ovary (CHO) cells, followed by purification using ion exchange chromatography, reverse-phase high performance liquid chromatography, and the like. Urine is used as a raw material for purification, and because urine sources are limited, the process is complex, and the yield is low, the mainstream hCG products in the market at present all adopt recombinant protein technology. However, recombinant hCG products have different pharmacokinetic patterns from hCG produced from human urine, and a continuous stimulus is required for the recombinant protein hCG products to function in vivo, and usually hCG is cleared after about 24 hours after a single injection, and even if the administration dose is increased, the action time of protein in vivo cannot be prolonged. Therefore, all recombinant hCG products on the market need continuous and repeated injection to exert the effect, and the compliance of patients is poor. Therefore, improvements in traditional reproductive hormone protein drugs are currently needed in the market.

In view of this, the present invention has been made.

Disclosure of Invention

The first aim of the invention is to provide a nucleic acid medicament which alleviates the problem of poor medicament effect of the genital hormone protein medicaments in the prior art.

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

A third object of the present invention is to provide the use of the above nucleic acid drug, or a method for producing the above nucleic acid drug, for producing a product for treating a reproduction-related disease.

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 an mRNA encoding a reproductive hormone protein comprising at least two subunits.

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

According to another aspect of the invention, the invention also provides the use of a nucleic acid medicament as described above or a method of preparation as described above for the preparation of a product for the treatment of a reproduction-related disorder.

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

the nucleic acid drug provided by the invention comprises mRNA (messenger ribonucleic acid) 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 the mRNA is attenuated, the mRNA can still continuously synthesize new target protein to supplement, so that the protein can be continuously expressed, the half-life period of the encoded protein in the body is prolonged, and the effect of prolonging the drug effect maintaining time is realized. The nucleic acid medicine of the invention can obviously reduce the administration times of patients on the premise of achieving the same effect, improve the medication compliance of the patients and have better treatment effect. After mRNA is delivered into the body, endogenous proteins can be produced by using cells in the human body, and the mRNA has better activity. The preparation method of the nucleic acid medicine 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 that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.

FIG. 1 shows the hCG protein concentration in HEK293 cell supernatant as in example 1 of the invention;

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

FIG. 3 is a graph showing the change of blood hCG concentration with time in mice in example 4 of the present invention;

FIG. 4 is a graph showing the blood concentration of hCG in mice of example 5 of the present invention over time;

FIG. 5 shows the effect of uterine weight gain in mice following administration of hCG mRNA in example 6 of the invention.

Detailed Description

The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

mRNA is a precursor of a protein, and is gradually expressed as a target protein after being introduced into a human body. In the process that the target protein synthesized by the mRNA is attenuated by the natural half-life period, the mRNA can still continuously synthesize new target protein to supplement. The mRNA can be delivered to the body to continuously express the protein within a certain period of time, so that the mRNA is used as an active ingredient of the reproductive hormone medicines to prolong the half-life period of the coded reproductive hormone in the body, thereby realizing long-time drug effect. Therefore, the nucleic acid medicine of the invention can obviously reduce the administration times of patients and improve the medication compliance of the patients and has better treatment effect on the premise of achieving the same effect. mRNA encoding genital hormone protein is used as the active component of the medicine, natural raw materials are not required to be collected, and the production method is simple. After mRNA is delivered into the body, endogenous proteins can be produced by using cells in the human body, and the mRNA has better activity.

In some preferred embodiments, the nucleic acid agent preferably comprises gonadotropin (gonadotrophin) which is a group of heterodimeric glycoprotein hormones that regulate reproductive function in 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 agent may contain only mRNA encoding one of FSH, LH, and CG; in some alternative embodiments, the nucleic acid agent may also contain multiple mrnas encoding different gonadotrophin simultaneously to supplement multiple gonadotrophin simultaneously: for example, but not limited to: simultaneously comprising an mRNA encoding FSH and an mRNA encoding LH; simultaneously comprising an mRNA encoding FSH and an mRNA encoding LH; simultaneously comprising an mRNA encoding LH and an mRNA encoding CG; or, it contains both the mRNA encoding FSH, the mRNA encoding LH and the mRNA of CG.

The invention does not limit the species source of the reproductive hormone, and mRNA encoding the reproductive hormone protein can be the sequence of natural mRNA or the sequence optimized by codons; the coded reproductive hormone protein can be human reproductive hormone protein or other species of reproductive hormone protein; preferably, human reproductive hormone proteins are included for use in supplementing human reproductive hormone.

The reproduction hormone protein of the invention is a protein containing at least two subunits. In some alternative embodiments, a single mRNA contains the coding region for all subunits of the reproductive hormone protein, i.e., one mRNA in a nucleic acid drug is capable of translating into the entire reproductive hormone protein with a multi-subunit structure; in other alternative embodiments, a single mRNA contains only the coding region for one subunit of the reproductive hormone protein; that is, an mRNA is capable of translating only one subunit of the reproductive hormone protein. It will be appreciated that the nucleic acid agent may contain mRNA encoding all of the subunits of the reproductive hormone protein, such that the nucleic acid agent translates the complete reproductive hormone protein with multi-subunit structure in the subject to which it acts; when a subunit of a reproductive hormone protein is also pharmaceutically active, the nucleic acid agent may also contain at least one mRNA encoding a single subunit, such that the nucleic acid agent translates the pharmaceutically active subunit in the subject to which it is acting.

According to the invention, through experiments of nucleic acid medicaments of hCG, the coding region of the alpha subunit and the coding region of the beta subunit of hCG are respectively arranged in different mRNAs, and the therapeutic effect of the nucleic acid medicaments is better than that of arranging the coding regions of two subunits of hCG in the same mRNA, so that in some preferred embodiments, the nucleic acid medicaments are preferably constructed in a mode that a single mRNA only contains the coding region of one subunit in a genital 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 the mRNA from being degraded by exonuclease, and provide a signal for recognition of mRNA by ribosome, preferably m7GpppG is used. (b) a polyadenylation sequence: the polyadenylation sequence is capable of preventing exonuclease degradation of mRNA while terminating transcription. The polyadenylation preferably contains 60 to 120 adenosines, more preferably 80 to 100 adenosines. (c) 5' UTR: the 5 'untranslated region has a regulatory effect on translation of the mRNA, and the 5' UTR is preferably 10 to 200 nucleotides in length, more preferably 15 to 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 shown 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 untranslated region of 3 'preferably comprises 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): IRES allows protein translation initiation independent of the 5' cap structure, directly from the messenger mRNA intermediate initiation of translation. IRES preferably comprises the IRES sequence of Poliovirus type II (Poliovirus type 2), the sequence encoding Poliovirus type II preferably being as shown in SEQ ID NO. 7.

In some specific embodiments, optionally, the mRNA is comprised of coding regions, 5 'utrs, and 3' utrs; alternatively, the mRNA is comprised 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 polyadenylation 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 are modified, for example, by 2' -O-methylation; or other types of nucleotides may be used to replace conventional nucleotides in the mRNA, such as L-nucleotide (L-nucleoside) and pseudouracil (pseudo-UTP). Optionally, the mRNA contains L-nucleosides and pseudo-UTP; optionally, a 2' -O-methylation-modified nucleotide.

In some preferred embodiments, the modification comprises a pseudo-uracil modification, replacing UTP in the mRNA with pseudo-UTP, the pseudo-uracil modification being capable of enhancing stability and emergency response of the mRNA.

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

The location of the modification includes, but is 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; modifying the cap region of the mRNA.

In some alternative embodiments, the nucleic acid drug further comprises a delivery formulation for delivering mRNA, such that mRNA encoding a reproductive hormone protein is better delivered into the body, the delivery formulation including, but not limited to, a cationic liposome, a cationic protein, a cationic polypeptide, or a cationic polymer.

In some preferred embodiments, the nucleic acid drug comprises lipid nanoparticles consisting of mRNA encoding a reproductive hormone-like protein and a lipid component, wherein the mass ratio of lipid component to mRNA is preferably (10-30): 1, such as may be, 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 comprises a protonatable cationic lipid, cholesterol, a co-lipid, and a surfactant. The protonatable cationic lipids preferably include, but are not limited to DODMA, dlin-MC3-DMA, dlin-KC2-DMA or Dlin DMA, preferably including Dlin-MC3-DMA. Preferably, the protonatable cationic lipids comprise 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 an amount of 20-50% by mole 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 helper 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, a PEG modified lipid and/or a water soluble surfactant, which may be used alone or in combination. 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 the water soluble surfactant and the 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 gonadotrophin (hCG) mRNA agent. The main functions of hCG are: maintaining the corpus luteum and reducing maternal lymphocyte activity, preventing rejection to the fetus, for acceptance of assisted reproductive techniques, and for anovulatory or anovulatory women. Preferably, the mRNA encoding hCG encodes human hCG, more preferably expressed in a mammal, particularly a human, and preferably the mRNA encoding hCG is codon optimized.

The hCG protein structure includes two subunits, α and β. The alpha subunit has a large immunological cross-reaction with LH, FSH, TSH, especially LH, and contains 92 amino acids. The beta subunit is unique to hCG and contains 145 amino acids. The mRNA product with the hCG protein expression function is synthesized by mRNA technology, and the mouse experiment shows that the hCG nucleic acid medicine can obviously prolong the action time of hCG in the body and improve the protein expression effect after the medicine is delivered into the body, and the effect which can be achieved by injecting the mRNA product for a single time only can be exceeded by injecting the corresponding protein medicine for a plurality of times in the market; the hCG nucleic acid medicine can produce human chorionic gonadotrophin in mice and has uterine weight increasing effect in mice.

In some alternative embodiments, the nucleic acid agent comprises hCG mRNA comprising a coding region for the α subunit of hCG and a coding region for the β subunit of hCG, capable of encoding both the α subunit and the β subunit. The hCG mRNA preferably contains the coding region for the alpha subunit of hCG, the coding region for the beta subunit of hCG, the 5' -cap structure, the polyadenylation sequence, the 5' UTR, the 3' UTR and the internal ribosome entry sequence, and preferably contains the sequence shown in SEQ ID NO. 1.

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

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

In some preferred embodiments, the hCG beta mRNA comprises the coding region for the beta subunit, a 5' -cap structure, a polyadenylation sequence, 5' UTR and 3' UTR, preferably the hCG beta mRNA comprises the sequence shown as SEQ ID NO. 3.

In some preferred embodiments, the molar ratio of hcgα mRNA to hcgβ mRNA in the nucleic acid drug is (1-2): (1-2); preferably (1 to 1.5): (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 formulation in nucleic acid pharmaceuticals for hCG. And is preferably administered intravenously and subcutaneously, and hCG protein is detected in blood 3 hours after administration in mice and can be expressed continuously for more than 48 hours, with intravenous administration being more effective.

According to one aspect of the invention, the invention further provides a preparation method of the nucleic acid drug, wherein the preparation method comprises the steps of mixing mRNA and optional auxiliary materials to obtain the nucleic acid drug. "optional excipients" refers to excipients that are acceptable in the art, either added optionally or not.

In some preferred embodiments, the mRNA is transcribed from a DNA template. Optionally, the DNA template contains coding regions for all subunits of a reproductive hormone-like protein; alternatively, the DNA template contains only the coding region of one subunit of the reproductive hormone protein. The mRNA is obtained by transcription of the linearized DNA template, preferably by in vitro transcription, and in vitro transcription of the mRNA is produced by in vitro cell-free production, so that the culture of living cells and complex separation engineering are not involved, and the production conditions are convenient to control. Preferably, mRNA is obtained by in vitro transcription under the catalysis of RNA polymerase, and the raw materials for mRNA transcription include ATP, CTP, pseudo-UTP, GTP and ARCA caps. And concentrating the in vitro transcribed product to obtain mRNA encoding the genital hormone protein in the nucleic acid medicine.

In some preferred embodiments, the mRNA is dissolved in the aqueous phase to obtain a solution containing the mRNA, a first mixture is obtained, and the first mixture is mixed with the delivery formulation to obtain the nucleic acid drug; wherein the aqueous phase preferably comprises a buffer, including but not limited to a citrate buffer or an acetate buffer; preferably, a citrate buffer is used, the pH of which is preferably 3.5 to 4.5. The concentration of mRNA in the first mixture is preferably 0.05 to 0.15mg/mL, and may be, for example, but not limited to, 0.05mg/mL, 0.08mg/mL, 0.1mg/mL, 0.12mg/mL, or 0.15mg/mL, preferably 0.1mg/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: the lipid component is dissolved in an organic phase to obtain a second mixture, and the first mixture and the second mixture are then mixed to obtain a third mixture. In some alternative embodiments, the third mixture is diluted and then concentrated to provide the nucleic acid drug, preferably 50-100 fold, 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 agent is obtained after removal of the organic phase in the third mixture. The concentration of lipid nanoparticles in the nucleic acid drug is preferably brought to 50-200. Mu.g/ml by dilution followed by concentration of the third mixture, or by removal of the organic phase in the third mixture, independently, and may be, for example 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.

The organic phase is preferably an anhydrous C1-C4 lower alcohol, more preferably an anhydrous alcohol. The concentration of the lipid in the second mixture is 4 to 8mg/mL, for example, but not limited to, 4mg/mL, 5mg/mL, 6mg/mL, 7mg/mL or 8mg/mL, preferably 6mg/mL. The first mixture and the second mixture are preferably in a volume ratio of 1: (2-4), more preferably in a ratio of 1:3. The first mixture and the second mixture may be mixed manually or by using a microfluidic device, and in order to make the mixing operation more accurate, it is preferable to use a microfluidic device for 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 12mL/min.

In some preferred embodiments, the protein encoded in the nucleic acid agent comprises hCG, the nucleic acid agent comprising hCG a mRNA and hCG β mRNA; and the molar ratio of hCG alpha mRNA to hCG beta mRNA is (1-2): (1-2) mixing; preferably in a molar ratio (1 to 1.5): (1-1.5) mixing; more preferably in a molar ratio of 1:1.

According to another aspect of the invention, the invention also provides the use of the above nucleic acid drug, or a method of preparing the above nucleic acid drug, in the preparation of a product for the treatment of a reproduction-related disease. The nucleic acid medicine can prolong the half life of hormone medicine, prolong the acting 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 products for treating reproduction-related diseases, for example, the products are combined with other medicines or are used in a complete set with other treatment equipment, and the treatment effect of the products is better.

Example 1

And verifying the influence of two different mRNA design ideas on the expression quantity of hCG protein: two different mRNAs were transfected on HEK293 cells using a commercial reagent lipofectamine2000 (hCG mRNA: single-stranded mRNA, two subunits designed on the same strand, having the sequence shown as SEQ ID NO.1, hCG. Alpha. MRNA and hCG. Beta. MRNA, hCG. Alpha. MRNA encoding hCG. Subunit, having the sequence shown as SEQ ID NO.2, hCG. Beta. MRNA encoding hCG. Subunit, having the sequence shown as SEQ ID NO.3, the molar ratio of hCG. Alpha. MRNA to hCG. Beta. MRNA being 1:1 mixed at the time of transfection), and the mRNAs were subjected to cell transfection.

HEK293 cells resuscitated from liquid nitrogen were cultured for one generation and then ready for use. HEK293 cells were seeded into 6-well plates 24 hours prior to the experiment at a cell density of 400000-500000 per well. Culturing in a 37 ℃ incubator for 24 hours to observe the cell state, and starting the experiment when the cell confluence reaches about 80-90%. The nanocomposite of lipofectamine2000 and mRNA encoding HCG gene was prepared according to the method provided in the product description, and the same amount of naked HCG mRNA was used as a negative control group, the volume-mass ratio of lipofectamine2000 to HCG gene encoding mRNA was 2:1, and the two were mixed and allowed to stand at room temperature for 15 minutes, and directly added into a 6-well plate cell culture solution, thereby ensuring that 1 μg of mRNA was added per well. The cells were then incubated in a 37℃incubator for 24 hours, and the cell culture supernatant was collected and assayed for HCG protein expression by ELISA. Experimental results show that when the mRNA respectively codes hCG, hCG protein expression is higher. The results are shown in FIG. 1.

Example 2

The in vivo and in vitro delivery efficiency of hCG-encoding mRNA was evaluated for each type of delivery formulation and for different administrations. In this example, hCG alpha mRNA (sequence shown as SEQ ID NO. 2) and hCG beta mRNA (sequence shown as SEQ ID NO. 3) were used as the hCG-encoding mRNA. The experimental results show that various delivery formulations are capable of efficiently transfecting hCG mRNA and expressing hCG protein on cells in vitro, with the commercial reagent lipofectamine2000 having the highest transfection efficiency. However, lipid nanoparticle formulation LNP (MC 3) prepared using Dlin-MC3-DMA alone in vivo animal experiments can successfully deliver hCG mRNA by intravenous or subcutaneous administration. The experimental result shows that the in vitro cell transfection result has no correspondence with the in vivo animal experimental result, and the in vivo delivery process of the mRNA of hCG is far more complicated than in vitro cell transfection. The experimental results are shown in table 1.

TABLE 1 transfection efficiency of hCG-encoding mRNA and various delivery formulations on HEK293 cells and delivery 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 is detected from cell supernatant or hCG is detected from serum 3h-48h after mouse administration, ++, and: strong positive, -: hCG expression was not detected from cell supernatants or hCG was not detected from serum 3h-48h after mouse dosing, LNP (MC 3): lipid nanoparticles with Dlin-MC3-DMA as core lipid. LNP (DOTAP): lipid nanoparticles with DOTAP as core lipid.

Wherein, the preparation method of the mRNA delivered by each delivery preparation is as follows:

preparation of protamine (protamine) nanoparticles:

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

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

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

d. Mixing for 30 seconds by using a vortex oscillator, and standing for 15 minutes at room temperature.

Preparation of Polyethyleneimine (PEI) nanoparticles:

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

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

Preparation of cationic liposome and cationic lipid nanoparticle:

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

a. lipid chloroform solutions were prepared according to the following formulation: the cationic lipid is selected from DOTAP, and the neutral auxiliary 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 an organic solvent by using a rotary evaporation method, and preparing a dried lipid membrane;

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

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

e. The hydrated liposome is placed in a water bath ultrasonic instrument and is subjected to ultrasonic treatment at 40 ℃ for 40 minutes, and the water suspension product of the cationic liposome is obtained and is named as Tf4.

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

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

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

c. Mixing the two by using a pipetting gun, lightly blowing for 5 times, and standing for 15 minutes to obtain the cationic compound.

d. The particle size analysis analyzes the quality of the product, and the size of the compound is about 180-200nm.

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

a. mRNA was dissolved in OptiMEM (Thermo) to adjust the concentration to 0.1mg/ml.

b. Lipofectamine2000 was dissolved in OptiMEM (Thermo) with 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. Standing for 15 minutes.

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

1. hcgα mRNA and hcgβ mRNA were mixed and dissolved in citrate buffer at ph=4 at a molar ratio of 1:1, and the concentration was adjusted to 0.1mg/ml.

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

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

4. The resulting mixture was immediately diluted 100-fold with PBS solution at ph=7.4, the ethanol component of the solution was removed by Tangential Flow Filtration (TFF) and concentrated to 100 μg/ml, and the resulting lipid nanoparticle of hCG-coated mRNA was obtained.

Example 3

The mRNA encoding luciferase is single-stranded mRNA with a length of about 2000nt, and the expressed luciferase can emit fluorescent signals 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. Previous studies have found that mRNA for HCG can be delivered in mice and HCG protein can be produced in mouse serum by subcutaneous and intravenous injection. Therefore, we used subcutaneous and intravenous injection as an alternative administration mode, 5 μg of luciferase mRNA was mixed into 20 μg of HCG (α, β chain split) mRNA, and LNP preparation (MC 3) was prepared by referring to the procedure in example 2, and the delivery and expression of HCG mRNA in vivo was indirectly reflected by injecting LNP preparation into mice by different administration modes and using luciferase expression levels detected by a small animal in vivo fluorescence imaging system.

LNP preparation (MC 3) containing 5. Mu.g of luciferase mRNA and 20. Mu.g of HCG mRNA was introduced by means of tail vein administration and subcutaneous injection. The mice were injected with luciferase substrate after 2 hours, anesthetized after 10 minutes and expression of luciferase in the mice was observed by a fluorescence in vivo imaging system (fig. 2). It was found that luciferase mRNA expression was detected after 2 hours either by intravenous injection or subcutaneous injection, with subcutaneous injection of mouse luciferase expressed only at the injection site and intravenous administration of mouse luciferase expressed at the liver site, with the signal intensity and range of the mouse luciferase being greater than that of subcutaneous administration. However, no fluorescent signal was detected by the liver of mice given intravenously 24 hours after administration, indicating a short hepatic mRNA expression time. Subcutaneously administered mice still have a higher fluorescent signal after 24 hours, and the signal can last more than 48 hours after administration. Analysis mRNA expression is faster but of short duration by intravenous administration but the secreted protein expressed can pass directly through the liver into the peripheral circulation, so it can be speculated that intravenous injection can express HCG protein faster and produce drug effects faster. Subcutaneously administered mRNA is expressed more permanently, however secreted proteins need to be absorbed by local tissues to enter the blood circulation and may have slower onset of action.

Example 4

mRNA drugs can express human chorionic gonadotrophin in mice: LNP preparations (MC 3) encapsulating both sequences of mRNA were delivered intravenously or subcutaneously to mice (each injection containing 20 μg of mRNA) and the hCG protein content in the serum of mice was measured 3 and 48 hours after dosing. The hCG protein in the blood sample is expressed by mouse autologous cells and released into the blood after administration via the hCG single-chain mRNA or hcgα mRNA and hcgβ mRNA system. The hCG protein content in blood was quantified by a protein ELISA method. The experimental results are shown in fig. 3: serum hCG concentrations were compared for different sequence designs and for different routes of administration at 3 hours and 48 hours post-administration. The serum hCG concentration of hCG alpha mRNA and hCG beta mRNA is far higher than that of hCG single-chain mRNA 3 hours after intravenous administration, and the expression amount can be maintained for 48 hours. Serum hCG was not detected 3 hours after subcutaneous administration of hcgα mRNA and hcgβ mRNA, but was detected 48 hours later. The obvious difference of blood entering speed and efficiency after hCG expression in different administration modes is shown. The mRNA drug of hCG is therefore preferably injected intravenously.

Example 5

Comparing the trend of serum hCG concentration change within 24 hours after intravenous administration of hCG mRNA and recombinant protein. The hCG mRNA administration group showed an upward trend after 6 hours of administration with the value of 2 hours after intravenous administration as the standard line, and fallen below the standard line after 12 hours. While the recombinant protein-administered group always showed a decreasing trend, and the decreasing rate was higher than that of the mRNA-administered group. The method shows that the concentration of the recombinant protein is always reduced after the blood is introduced, the protein is continuously expressed after the mRNA is introduced into the blood, and the half-life period is longer, so that the mRNA medicament form can achieve longer-lasting expression effect in human or animal bodies, and has superiority compared with protein medicaments. The experimental results are shown in FIG. 4.

Example 6

The increase in uterine weight of mice caused by hCG was the only index for evaluating the therapeutic effect of hCG, and the lipid nanoparticles of LNP (MC 3) coated with hCG α mRNA and hCG β mRNA prepared in example 2 were used as nucleic acid drugs, and the uterine weight of mice was weighed 3 days after intravenous and subcutaneous administration (1 μg/mouse) to the mice and compared with the uterine weight of mice of the control group. The experimental results are shown in FIG. 5. An increase in uterine weight of mice over control 3 days after administration indicates that hCG has exerted efficacy. According to the experiment, according to the chorionic gonadotrophin bioassay method recommended by the 2010 edition of Chinese pharmacopoeia, female mice with the age of 3 weeks are taken, the administration is respectively carried out for 1 time in a vein or subcutaneous mode, the uterine weight of the mice is measured after 72 hours, and the drug effect is observed. The study found that the uterus of the mice in the third day of administration was significantly increased compared to the control group. And the intravenous administration and the subcutaneous administration can achieve the effect. Early-stage multiple experiments have determined that uterus weight is about 30-60mg after 3-day continuous three high-dose administration of recombinant hCG protein preparation provided by a middle hospital, thus indicating that one administration of mRNA drug can be equivalent to three high-dose administration effects of protein drug.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

SEQUENCE LISTING

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Claims

1. A nucleic acid agent comprising mRNA encoding chorionic gonadotrophin; the coding region of the alpha subunit of chorionic gonadotrophin and the coding region of the beta subunit of chorionic gonadotrophin are each disposed in a different mRNA;

the nucleic acid drug comprises hCG alpha mRNA and hCG beta mRNA;

the hCG alpha mRNA contains a coding region of chorionic gonadotrophin alpha subunit, a 5' -cap structure, a polyadenylation sequence, a 5' UTR and a 3' UTR, and the nucleotide sequence is shown as SEQ ID NO. 2;

the hCG beta mRNA contains a coding region of chorionic gonadotrophin beta subunit, a 5' -cap structure, a polyadenylation sequence, a 5' UTR and a 3' UTR, and the nucleotide sequence is shown as SEQ ID NO. 3;

the nucleic acid drug comprises lipid nanoparticles consisting of mRNA encoding chorionic gonadotrophin and a lipid component; the molar ratio of the hCG alpha mRNA to the hCG beta mRNA is (1-2): (1-2).

2. The nucleic acid drag of claim 1, wherein the 5' -cap structure is m7GpppG.

3. The nucleic acid agent of claim 1, wherein the poly-a contains 60-120 adenosines.

4. The nucleic acid agent of claim 3, wherein the poly-a contains 80-100 adenosines.

5. The nucleic acid drag of claim 1, further comprising an internal ribosome entry sequence.

6. The nucleic acid drug of claim 1, wherein the mRNA contains modified nucleotides.

7. The nucleic acid agent of claim 6, wherein the modification comprises an L-nucleoside modification and/or a 2' -O-methylation modification.

8. The nucleic acid drag of claim 6, wherein the mRNA contains modified UTP and/or CTP.

9. The nucleic acid agent of claim 6, wherein the modified nucleotide comprises a UTP substitution to pseudo-UTP.

10. The nucleic acid agent of claim 6, wherein 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.

11. The nucleic acid agent according to claim 1, wherein the mass ratio of the lipid component to the mRNA is (10-30): 1.

12. The nucleic acid agent of claim 11, wherein the mass ratio of lipid component to mRNA is 20:1.

13. The nucleic acid drag of claim 1, wherein the lipid component comprises a protonatable cationic lipid, cholesterol, a co-lipid, and a surfactant;

the protonatable cationic lipid is selected from DODMA, dlin-MC3-DMA, dlin-KC2-DMA or Dlin DMA;

the helper lipid is selected from DSPC, DOPE, DOPC or DOPS.

14. The nucleic acid drag of claim 13, wherein the protonatable cationic lipid is selected from Dlin-MC3-DMA.

15. The nucleic acid drag of claim 13, wherein the protonatable cationic lipid comprises 20-50% of the lipid component molar content.

16. The nucleic acid drag of claim 13, wherein said cholesterol comprises 20-50% of the molar content of said lipid component.

17. The nucleic acid drug of claim 13, wherein the helper lipid is selected from DSPC.

18. The nucleic acid drag of claim 13, wherein the helper lipid comprises 5-20% of the molar content of the lipid component.

19. The nucleic acid drug of claim 13, wherein the surfactant comprises a PEG-modified lipid and/or a water-soluble surfactant.

20. The nucleic acid drag of claim 19, wherein the PEG-modified lipid comprises PEG-DMG or PEG-DSPE.

21. The nucleic acid drag of claim 20, wherein the PEG-modified lipid comprises PEG-DMG.

22. The nucleic acid drag of claim 19, wherein the PEG-modified lipid comprises 1-5% of the lipid component by mole.

23. The nucleic acid drag of claim 22, wherein the PEG-modified lipid comprises 3% of the lipid component molar content.

24. The nucleic acid drag of claim 19, wherein the water soluble surfactant comprises Tween 20, tween 80, P188, or PVA.

25. The nucleic acid drag of claim 19, wherein the weight to volume ratio of the water soluble surfactant and the lipid component is 0.2-2%.

26. The nucleic acid drag of claim 25, wherein the weight to volume ratio of the water soluble surfactant and the lipid component is 1%.

27. The nucleic acid drug of claim 1, wherein the molar ratio of hCG α mRNA to hCG β mRNA is (1-1.5): (1-1.5).

28. A nucleic acid agent according to claim 27, wherein the molar ratio of hCG α mRNA to hCG β mRNA is 1:1.

29. A nucleic acid agent according to claim 1, wherein the nucleic acid agent comprises the hCG a mRNA and the hCG β mRNA and the delivery formulation for delivering the hCG a mRNA and the hCG β mRNA comprises Dlin-MC3-DMA.

30. The nucleic acid agent of claim 1, wherein the mode of administration of the nucleic acid agent comprises intravenous administration and subcutaneous administration.

31. The nucleic acid agent of claim 1, wherein the nucleic acid agent is administered intravenously.

32. A method for preparing a nucleic acid drug according to any one of claims 1 to 31, comprising: dissolving mRNA in an aqueous phase to obtain a solution containing mRNA, and obtaining a first mixture; 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; and then performing (a) or (b):

(a) Diluting the third mixture, and concentrating to obtain the nucleic acid medicine;

(b) Removing the organic phase in the third mixture to obtain the nucleic acid medicine.

33. The method of claim 32, wherein the mRNA is transcribed from a DNA template.

34. The method of claim 33, wherein mRNA is obtained by in vitro transcription.

35. The method of claim 34, wherein the linearized DNA template is transcribed in vitro to obtain mRNA under the catalysis of RNA polymerase.

36. The method of claim 34, wherein the in vitro transcribed mRNA comprises ATP, CTP, pseudo-UTP, GTP and ARCA caps.

37. The method of claim 32, wherein the aqueous phase comprises a buffer.

38. The method of claim 37, wherein the buffer comprises a citrate buffer or an acetate buffer.

39. The method of claim 38, wherein the buffer is a citrate buffer.

40. The method of claim 39, wherein the pH of the citrate buffer is 3.5-4.5.

41. The method of claim 32, wherein the concentration of mRNA in the first mixture is 0.05-0.15 mg/mL.

42. The method of claim 41, wherein the concentration of mRNA in the first mixture is 0.1. 0.1mg/mL.

43. The method of claim 32, wherein the concentration of lipid nanoparticles in the nucleic acid drug is 50-200 μg/ml.

44. The method of claim 32, wherein diluting the third mixture comprises diluting the third mixture 50-100 times.

45. The method of claim 32, wherein removing the organic phase from the third mixture comprises removing the organic phase from the third mixture using tangential flow filtration.

46. The method of claim 32, wherein the organic phase comprises anhydrous C1-C4 lower alcohols.

47. The method of claim 46, wherein the anhydrous C1-C4 lower alcohol is anhydrous ethanol.

48. The method of claim 32, wherein the concentration of the lipid component in the second mixture is 4-8 mg/mL.

49. The method of claim 48, wherein the concentration of the lipid component in the second mixture is 6mg/mL.

50. The method of claim 32, wherein the first mixture and the second mixture are mixed in a volume ratio of 1: (2-4) mixing.

51. The method of claim 50, wherein the first mixture and the second mixture are mixed in a volume ratio of 1:3.

52. The method of claim 32, wherein the first mixture and the second mixture are mixed using a microfluidic device.

53. The method of claim 52, wherein the flow rate is controlled to be 3-24 mL/min when the nucleic acid is mixed by a microfluidic device.

54. The method of claim 53, wherein the flow rate is controlled to 12mL/min when mixing by a microfluidic device.