Exosome medicine for treating premature ovarian failure as well as preparation method and application of exosome medicine

Abstract

The invention discloses an exosome drug for treating premature ovarian failure as well as a preparation method and application of the exosome drug. The exosome drug is obtained by mixing LNP or TNP encapsulated with an mRNA composition encoding NAMPT, NMNAT3 and COX15 with an exosome of a specific targeting ovarian cell, animal experiments prove that the exosome medicine has a remarkable treatment effect on the premature ovarian failure, a new thought and strategy are provided for the technical field of premature ovarian failure treatment, and the clinical application prospect is wide.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and specifically relates to an exosome drug for treating premature ovarian failure, and a preparation method and application thereof. Background Art

[0002] Premature ovarian failure (POF), also known as ovarian insufficiency (POI) or premature menopause, is characterized by loss of ovarian function, manifested as cessation of ovarian function and premature exhaustion of follicles before the age of 40. The causes of POF include genetic factors, autoimmune diseases, unhealthy lifestyles, etc. Different causes jointly lead to a reduction in the reserve of ovarian follicles and follicular dysfunction. The incidence of familial POF has been reported to be 4%-31% in different populations, and X chromosome abnormalities are recognized as the main cause of POF. Autoimmune diseases are associated with the onset of POF. Surveys and studies have shown that about 9%-40% of POF patients have autoimmune diseases, such as SLE, myasthenia gravis, rheumatoid arthritis, autoimmune thyroiditis, hypoparathyroidism, idiopathic thrombocytopenic purpura, etc. The incidence of POF is about 1‰ in people under 30 years old, and 1-3% in people under 40 years old. Generally, the incidence is higher in economically developed areas, and the incidence is on the rise with the improvement of social development level. POF causes infertility in women of childbearing age, which results in great social and family problems. In addition, the decrease in estrogen levels causes a series of low estrogen symptoms, such as low libido, hot flashes, sweating, osteoporosis and urinary system diseases, which reduces the quality of life of patients.

[0003] Exosomes are a type of extracellular vesicles produced by living cells that can mediate intercellular communication or the excretion of cellular metabolic products. A large number of studies on natural exosomes have shown that exosomes have physiological functions such as immunomodulation, inflammation inhibition, and tissue repair promotion. The functions they play may be related to their contents or to their membrane components. Exosomes from different types of cells have different surface characteristics, which give them tropism for different types of cells. The contents of exosomes mainly include proteins, micro-RNAs, mRNAs, etc. In recent years, through transcriptomics research and other methods, it has been found that the types and contents of micro-RNAs in exosomes produced by cells of different types or physiological states are different, resulting in different regulatory characteristics for target protein expression. Exosomes are lipid bilayer-wrapped vesicles produced by cells. Their surface only expresses a small amount of major histocompatibility complex molecules, and they also carry anti-inflammatory proteins such as CD73. They have good tissue compatibility and low immunogenicity, making them widely studied and developed as small molecule drugs and gene drug delivery carriers. At present, the development directions of exosome drug delivery systems mainly include engineering modification to increase the tropism of exosomes for specific tissues and further reduce immunogenicity, engineering modification to increase the intracellular loading of exosomes for target nucleic acids, and realizing the in vitro loading of exosomes for target small molecules or nucleic acid drugs through process development.

[0004] Lipid nanoparticles (LNPs) are composed of four different components: cationic lipids, phospholipids, polyethylene glycol (PEG)-lipids, and cholesterol. Various drugs, including small molecules, peptides, and nucleic acids, can be loaded into their cavities through high-flow microfluidics. The nucleic acid-containing phase is the aqueous phase, and the lipid mixture is the organic phase. Each lipid component and its ratio play an important role in LNP encapsulation. Negatively charged nucleic acids and positively charged lipids are electrostatically bound and assembled into LNPs through hydrophobic and van der Waals interactions between lipid components. LNPs can protect loaded drugs from destructive proteases, assist in the intracellular delivery of mRNA, and prolong the retention time of drugs in the body. In addition, LNPs can support repeated and instantaneous administration, making up for the shortcomings of current gene delivery drug carriers.

[0005] Ovarian granulosa cells are important cells that protect, nourish, and support the follicles. They play a vital role in follicular development, oocyte maturation, and ovulation through oocyte nutrition, mechanical protection, and the secretion of hormones such as E2 and AMH. POF patients usually experience symptoms such as decreased follicle number, decreased secondary and mature follicles, increased atretic follicles, and inability to ovulate. Histological examinations show that there is a decrease in the number of granulosa cells, increased granulosa cell aging and apoptosis, and weakened secretory capacity in POF patients and animal model tissues. Because restoring the activity and function of granulosa cells is an important way to treat POF.

[0006] At present, there is no good treatment for POF, especially for improving egg quality and restoring fertility. Conventional hormone therapy can only temporarily improve the related symptoms caused by low hormone levels. Estrogen-progesterone replacement therapy (HRT) is mainly for young POF patients. HRT can relieve low estrogen symptoms and urogenital atrophy, and prevent long-term complications (osteoporosis, Alzheimer's disease, etc.). However, long-term HRT may lead to the occurrence of endometrial cancer and breast cancer. Progesterone is divided into natural progesterone and synthetic progesterone. Micronized progesterone can be taken orally, but usually requires a large dose and is expensive. Ovulation induction therapy requires a large dose of hMG and a long duration, and is not effective for patients with long-term and severe POF. Dehydroepiandrosterone (DHEA) is an important substance for the synthesis of testosterone and E2. DHEA for infertile women with normal ovarian reserve but older childbearing age can reduce the miscarriage rate to a certain extent. In addition to the above conventional therapies, immunosuppressive therapy has a certain effect on POF related to autoimmune diseases. There is currently no clear medication standard, and immunosuppression can cause serious side effects, so it is limited in the treatment of autoimmune disease-related POF. In the study of the rodent model of POF treated with oral nicotinamide mononucleotide (NMN), long-term high-dose oral intake is required. Although good results have been achieved in rodent models, the efficacy of NMN as a supplement in the human population is currently widely questioned. In fact, even high-dose oral NMN still has no significant effect on anti-aging. The reason may be that the efficiency of NMN conversion to nicotinamide adenine dinucleotide (NAD) in the human body is too low. Direct administration of NAD has the problem that NAD has a short half-life in the body and is difficult to enrich in the target tissue. Summary of the invention

[0007] In view of this, in order to overcome the technical problems existing in the above-mentioned prior art regarding drugs for treating POF, the object of the present invention is to provide an exosome drug for treating POF and a related preparation method thereof, which improves the stability of NAD or mRNA drugs in the body and their distribution in ovarian tissue through natural or engineered exosomes.

[0008] The preparation process of the above-mentioned drug for treating POF provided by the present invention involves the acquisition of exosomes, the preparation of LNP-mRNA, the mixing of NAD or LNP-mRNA with exosomes and electroporation treatment. The present invention also involves using NAD as a target for treating POF. Compared with NMN, NAD has the advantage of directly exerting its effect without relying on the enzymatic reaction conversion in the body. In ovarian tissue, it improves the physiological functions of eggs, corpora lutea, ovarian stromal cells, and ovarian macrophages by promoting sirt2-dependent spindle-kinetochore assembly, PARP-dependent DNA repair, aerobic respiration and other mechanisms, thereby improving ovarian dysfunction such as premature ovarian failure in the long term. In terms of drugability, the drug preparation method mentioned in this method can be used to produce different dosage forms such as intravenous reinfusion preparations, in situ injection preparations, and oral preparations, and has good clinical application prospects.

[0009] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0010] A first aspect of the present invention provides a nucleic acid composition for treating premature ovarian failure.

[0011] Furthermore, the nucleic acid composition includes mRNA, DNA or recombinant protein encoding NAMPT, NMNAT3 and COX15.

[0012] In the present invention, the information of the genes NAMPT, NMNAT3 and COX15 is as follows: the Gene ID of the gene NAMPT (nicotinamide phosphoribosyltransferase [Homo sapiens (human)]) is 10135, the Gene ID of the gene NMNAT3 (nicotinamide nucleotide adenylyltransferase 3 [homo sapien (human)]) is 349565, and the Gene ID of the gene COX15 (cytochrome c oxidase assembly homologCOX15 [Homo sapiens (human)]) is 1355. The detailed information of the genes can be obtained at https: / / www.ncbi.nlm.nih.gov / gene / .

[0013] In a specific embodiment of the present invention, the nucleic acid composition is a nucleic acid composition comprising mRNA encoding NAMPT, NMNAT3 and COX15.

[0014] A second aspect of the present invention provides an ovary targeting peptide.

[0015] Further, the ovary targeting peptide includes LH-Lamp2b fusion protein or FSH-Lamp2b fusion protein;

[0016] The amino acid sequence of the LH-Lamp2b fusion protein is shown in SEQ ID NO: 2;

[0017] The amino acid sequence of the FSH-Lamp2b fusion protein is shown in SEQ ID NO:4.

[0018] Furthermore, the nucleotide sequence of the LH-Lamp2b fusion protein is shown in SEQ ID NO: 1;

[0019] The nucleotide sequence of the FSH-Lamp2b fusion protein is shown in SEQ ID NO:3.

[0020] In the present invention, LH refers to luteinizing hormone, which is a glycoprotein gonadotropin secreted by pituitary cells and can promote the conversion of cholesterol into sex hormones in gonadal cells. For women, it works together with follicle-stimulating hormone (FSH) to promote follicle maturation, secrete estrogen, ovulation, and the formation and maintenance of corpus luteum, secrete progesterone and estrogen. For men, luteinizing hormone promotes the synthesis and release of testosterone by interstitial cells.

[0021] In the present invention, FSH refers to follicle stimulating hormone, which is a hormone secreted by the basophils of the anterior pituitary gland. It is a glycoprotein and its main function is to promote the maturation of follicles. Follicle stimulating hormone can promote the proliferation and differentiation of follicle granulosa cells and promote the growth of the entire ovary. Its action on the seminiferous tubules of the testis can promote sperm formation. Follicle stimulating hormone is secreted in pulses in the human body.

[0022] In the present invention, the Lamp2b is one of the keys of the targeted delivery device of exosomes (exosome membrane protein). Lamp2b is an exosome surface protein commonly used to display targeting motifs. Lamp2b is a member of the lysosome-associated membrane protein (LAMP) family. It is mainly localized in lysosomes and endosomes in cells and is also expressed in large quantities on the surface of exosomes. The target molecule and Lamp2b are usually fused and displayed on the surface of the exosome membrane through Lamp2b.

[0023] The third aspect of the present invention provides exosomes that specifically target ovarian cells.

[0024] Furthermore, the exosomes contain the ovary targeting peptide described in the second aspect of the present invention.

[0025] Further, the exosomes are derived from mesenchymal stem cells;

[0026] Optionally, the exosomes contain the FSH-Lamp2b fusion protein described in the second aspect of the present invention.

[0027] In a specific embodiment of the present invention, the exosomes are exosomes comprising the FSH-Lamp2b fusion protein described in the second aspect of the present invention.

[0028] In addition, the present invention verifies through comparative experiments that it is the first time to find that the FSH ovarian targeting peptide structure is significantly better than the LH ovarian targeting peptide structure. Specifically, the FSH ovarian targeting peptide structure is significantly better than the LH ovarian targeting peptide structure in improving the delivery efficiency of mesenchymal stem cell-derived exosomes to LNP-encapsulated mRNA in ovarian cells and improving the ovarian distribution of mesenchymal stem cell-derived exosomes. This technical effect is a technical effect that is unexpected by those skilled in the art based on the prior art.

[0029] A fourth aspect of the present invention provides any of the following products:

[0030] (1) An exosome drug for treating premature ovarian failure, wherein the exosome drug for treating premature ovarian failure is obtained by mixing LNP or TNP encapsulating the nucleic acid composition of the first aspect of the present invention and the exosomes of the third aspect of the present invention;

[0031] (2) An exosome drug encapsulating NAD, wherein the exosome drug encapsulating NAD is an exosome drug obtained by encapsulating NAD drug in exosomes;

[0032] (3) A pharmaceutical composition comprising the exosome drug for treating premature ovarian failure and / or the exosome drug encapsulating NAD;

[0033] (4) A pharmaceutical preparation, comprising the exosome drug for treating premature ovarian failure and / or the exosome drug encapsulating NAD.

[0034] Further, the concentration of the nucleic acid composition in the LNP or TNP encapsulating the nucleic acid composition described in the first aspect of the present invention is 10 ng / μL;

[0035] Optionally, the ratio of mRNAs of NAMPT, NMNAT3 and COX15 in the nucleic acid composition in the LNP or TNP encapsulating the nucleic acid composition according to the first aspect of the present invention is 1:1:1;

[0036] Optionally, the final concentration of the exosome drug for treating premature ovarian failure comprising LNP encapsulating the nucleic acid composition described in the first aspect of the present invention is: the exosome NTA described in the third aspect of the present invention is 4×10 10 / mL, the LNP encapsulating the nucleic acid composition according to the first aspect of the present invention is 25 μg / mL;

[0037] Optionally, the final concentration of the exosome drug for treating premature ovarian failure comprising TNP encapsulating the nucleic acid composition described in the first aspect of the present invention is: the exosome NTA described in the third aspect of the present invention is 5×10 10 / mL, the TNP encapsulating the nucleic acid composition according to the first aspect of the present invention is 75 μg / mL;

[0038] Optionally, the exosomes in the NAD-encapsulated exosome drug are 4×10 10 / mL, NAD is 600μg / mL;

[0039] Optionally, the exosomes in the NAD-encapsulated exosome drug are derived from mesenchymal stem cells;

[0040] Optionally, the dosage form of the pharmaceutical preparation includes intravenous reinfusion preparations, in situ injection preparations, oral preparations, tablets, capsule preparations, granule preparations, sustained-release preparations, powders, and pills.

[0041] In the present invention, the NAD (or NAD+) is a biological substance that transfers electrons. Its Chinese name is Nicotinamide Adenine Dinucleotide (NAD), which is an oxidized coenzyme I. This is a coenzyme in many dehydrogenases in the body, such as alcohol dehydrogenase (ADH), which is used to oxidize ethanol. It plays an irreplaceable role in glycolysis, gluconeogenesis, tricarboxylic acid cycle and respiratory chain. Its function is to connect the tricarboxylic acid cycle and the respiratory chain, and transfer the hydrogen removed during the metabolic process to flavoprotein. Its reduced form is NADH and H+.

[0042] In some embodiments, the present invention has no particular limitation on the dosage form of the pharmaceutical preparation, and the dosage form of the pharmaceutical preparation includes, but is not limited to, intravenous reinfusion preparations, in situ injection preparations, oral preparations, tablets, capsule preparations, granule preparations, sustained-release preparations, powders, and pills. The pharmaceutical preparation preparation method known to those skilled in the art can be used to prepare the pharmaceutical preparation.

[0043] In some embodiments, the pharmaceutical composition or pharmaceutical formulation further comprises a pharmaceutically acceptable carrier and / or excipient.

[0044] In some embodiments, suitable pharmaceutically acceptable carriers and / or excipients are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995), and these substances are used to help the stability of the formulation or to help improve the activity or its biological effectiveness or to produce an acceptable taste or smell in the case of oral administration as needed. The pharmaceutical composition or pharmaceutical preparation thus prepared can be administered in any appropriate manner known to those skilled in the art as needed. When using the pharmaceutical composition or pharmaceutical preparation provided by the present invention, a safe and effective amount of the pharmaceutical composition or pharmaceutical preparation of the present invention is applied to a human.

[0045] In some embodiments, the present invention has no particular limitations on the pharmaceutically acceptable carrier and / or excipient, and any pharmaceutically acceptable carrier and / or excipient suitable for combining with the active ingredient provided by the present invention (such as the exosome drug for treating premature ovarian failure, the exosome drug encapsulating NAD as described above) to obtain the pharmaceutical composition or pharmaceutical preparation of the present invention is within the scope of protection of the present invention.

[0046] In some embodiments, specific illustrative examples of the pharmaceutically acceptable carrier and / or excipient include, but are not limited to: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethyl cellulose and methyl cellulose; tragacanth powder; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyols, such as propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifiers, such as wetting agents, such as sodium lauryl sulfate; colorants; flavorings; tableting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solution; and phosphate buffer, etc.

[0047] In some embodiments, the suitable dosage of the pharmaceutical composition or pharmaceutical preparation described in the present invention can be prescribed in a variety of ways according to factors such as formulation method, administration mode, patient's age, weight, sex, morbidity, diet, administration time, administration route, excretion rate and reaction sensitivity. A skilled physician can usually easily determine the prescription and the dosage that is effective for the desired treatment.

[0048] A fifth aspect of the present invention provides any of the following methods:

[0049] (1) A method for preparing the exosome drug for treating premature ovarian failure as described in the fourth aspect of the present invention, the preparation method comprising the following steps: mixing LNP or TNP encapsulating the nucleic acid composition as described in the first aspect of the present invention and the exosomes as described in the third aspect of the present invention to obtain the exosome drug;

[0050] (2) An exosome drug encapsulating NAD as described in the fourth aspect of the present invention, wherein the exosome drug encapsulating NAD is an exosome drug obtained by encapsulating NAD drug in exosomes.

[0051] Furthermore, the LNP or TNP encapsulating the nucleic acid composition of the first aspect of the present invention is prepared by the following method: mixing the mRNAs of NAMPT, NMNAT3 and COX15 to obtain a mixed solution NNC mRNA, loading the NNC mRNA into LNP or TNP to obtain LNP or TNP encapsulating the nucleic acid composition of the first aspect of the present invention;

[0052] Optionally, the final concentration of the mixed solution is 10 ng / μL;

[0053] Optionally, the mRNA ratio of NAMPT, NMNAT3 and COX15 in the mixed solution is 1:1:1;

[0054] Optionally, the NNC mRNA is loaded into LNP or TNP using a high-flow microfluidics method;

[0055] Optionally, in the high-flow rate microfluidic method, the flow rate ratio of the lipid phase to the aqueous phase is 1:3, and the total flow rate is 20 mL / min;

[0056] Optionally, the exosomes described in the third aspect of the present invention are prepared by the following method: constructing an iMSC-FSH / iMSC-LH cell line by lentiviral infection, collecting and purifying the exosomes secreted by the iMSC-FSH / iMSC-LH cells, thereby obtaining the exosomes described in the third aspect of the present invention;

[0057] Optionally, the exosome drug encapsulating NAD is an exosome drug obtained by encapsulating the NAD drug in exosomes using an electroporation method;

[0058] Optionally, the exosomes in the NAD-encapsulated exosome drug are 4×10 10 / mL, NAD is 600μg / mL;

[0059] Optionally, the exosomes in the NAD-encapsulated exosome drug are derived from mesenchymal stem cells.

[0060] A sixth aspect of the present invention provides any of the following applications:

[0061] (1) Use of the nucleic acid composition of the first aspect of the present invention, the ovarian targeting peptide of the second aspect of the present invention, and / or the exosomes of the third aspect of the present invention in the preparation of an exosome drug for treating premature ovarian failure;

[0062] (2) Use of the exosomes described in the third aspect of the present invention in targeted delivery of nucleic acid molecules;

[0063] (3) Use of the exosome drug for treating premature ovarian failure and / or the exosome drug encapsulating NAD described in the fourth aspect of the present invention in the preparation of a pharmaceutical composition or pharmaceutical preparation for treating premature ovarian failure.

[0064] In addition, the present invention also provides a method for treating premature ovarian failure, the method comprising the following steps: administering a therapeutically effective amount of the exosome drug for treating premature ovarian failure, the exosome drug encapsulating NAD, the pharmaceutical composition and / or the pharmaceutical preparation described in the fourth aspect of the present invention to a subject in need.

[0065] In some embodiments, the subject of the present invention refers to any animal, and also refers to humans and non-human animals. Non-human animals include all vertebrates, for example, mammals, such as non-human primates (particularly higher primates), sheep, dogs, rodents (such as mice or rats), guinea pigs, goats, pigs, cats, rabbits, cattle, and any livestock or pets; and non-mammals, such as chickens, amphibians, reptiles, etc. In a preferred embodiment, the subject is a human.

[0066] In some embodiments, the treatment described herein generally relates to treating humans or animals (e.g., used by veterinarians), where certain desired therapeutic effects can be achieved, such as inhibiting the development of a disease (including reducing the rate of development, stopping the development), improving the disease, and curing the disease. Treatment as a preventive measure (e.g., prevention) is also included. Use in patients who have not yet developed a disease but are at risk of developing the disease is also included in the term treatment as used herein.

[0067] In some embodiments, the effective amount of the present invention refers to an amount having a therapeutic effect or an amount required to produce a therapeutic effect in a subject. For example, a drug therapeutically or pharmaceutically effective amount refers to the amount of the drug required to produce the desired therapeutic effect, and the therapeutic effect can be reflected by the results of clinical trials, model animal studies and / or in vitro studies. The pharmaceutically effective amount depends on several factors, including but not limited to: characteristic factors of the subject (such as height, weight, sex, age and medication history), the severity of the disease, etc.

[0068] In some embodiments, the administration methods of the exosome drug for treating premature ovarian failure, the exosome drug encapsulating NAD, the pharmaceutical composition and / or the pharmaceutical preparation of the present invention include, but are not limited to: intramuscular, subcutaneous, intradermal, oral, topical, intraperitoneal, intravenous, intranasal, intrapulmonary, intrarectal, etc. When administered orally, it can be formulated into a coating that protects the active ingredients in the pharmaceutical composition or pharmaceutical preparation to prevent degradation in the stomach. In addition, the active ingredient can be administered by any device that can be transferred to the target tissue. In a specific embodiment, the pharmaceutical composition or pharmaceutical preparation provided by the present invention can be prepared into various dosage forms according to actual needs, and can be administered by a clinician according to factors such as the type, age, weight and general disease condition of the subject, and the mode of administration to determine the dosage that is beneficial to the patient. The mode of administration can be, for example, injection or any other suitable mode of administration known to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 The HPLC-MS method was used to detect the content of cisplatin in the blood, brain and kidney tissues of mice in each group at 1h and 24h after administration;

[0070] Figure 2 This is the imaging result of MSC-derived exosomes in various organs after tail vein injection of mice;

[0071] Figure 3 This is a statistical result diagram of the weight changes of mice during and after drug administration;

[0072] Figure 4 This is a statistical result diagram of the change in food intake of mice during and after drug administration;

[0073] Figure 5 This is a statistical result diagram of the changes in body temperature of mice during and after drug administration;

[0074] Figure 6 This is the result chart corresponding to the organ coefficient of ovarian test after 21 days of administration;

[0075] Figure 7 The results of the FSH and LH ovarian targeting peptide structures that can improve the ovarian distribution of MSC-derived exosomes and FSH is superior to LH;

[0076] Figure 8 The result graph shows that the FSH and LH ovarian targeting peptide structures can improve the delivery efficiency of MSC-derived exosomes to LNP-encapsulated mRNA in ovarian cells, and the FSH ovarian targeting peptide structure is superior to the LH ovarian targeting peptide structure;

[0077] Fig. 9 This is the result diagram of the survival status of mice in each group after administration of cisplatin;

[0078] Fig.10 The results of the effects of each treatment group on the estrous cycle of mice are shown in the figure;

[0079] Fig.11 The results of each treatment group on the volume of mouse ovarian follicles, the number of granulosa cells, the apoptosis of granulosa cells, and the number of atretic follicles;

[0080] Fig.12 The corresponding result graph shows that exosome-delivered TNP-NNC has a therapeutic effect on mice with premature ovarian failure. DETAILED DESCRIPTION

[0081] The present invention is further described below in conjunction with specific embodiments, which are only used to explain the present invention and cannot be understood as limiting the present invention. It can be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and purpose of the present invention, and the scope of the present invention is defined by the claims and their equivalents.

[0082] The reagents and raw materials used in the present invention are easily available to those of ordinary skill in the art. Unless otherwise specified, they can be obtained from commercial sources. The experimental methods in the present invention that do not specify specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer. In particular, the following examples are only used to illustrate the present invention and should not limit the scope of the present invention in any way. Example 1 Exosomes can increase the half-life of small molecule compounds in the body, thereby increasing the concentration of long-term drugs

[0083] 1. Experimental Materials

[0084] The main experimental materials are shown in Table 1 below.

[0085] Table 1 Main experimental materials

[0086]

[0087]

[0088] 2. Experimental methods

[0089] SPF grade C57BL / 6J male mice, 6-8 weeks old, were used. The supplier is Beijing Weitong Lihua Co., Ltd., and the production license number is SCXK (Su) 20211-0006. The animals were kept in intelligent independently ventilated animal cages (IVCs) with cage specifications of 362mm×154mm×135mm, with no more than 5 animals per box. Artificial lighting, light: dark = 12h:12h. The animals were kept free to eat and move throughout the breeding process. SPF rat growth and breeding feed, produced by Beijing Keao Xieli Feed Co., Ltd. The batch number is 200663313, the production license number is SCXK (Beijing) 2019-0003, and the quality certificate number is 1112622000023997. Full feeding is carried out 1-2 times a week. The drinking water bottle (Watsons) is replaced 1-2 times a week. Use wood shavings litter, sourced from Chenfu Eden Litter Processing Factory in Dachang Hui Autonomous County, batch number 20200622, production license number SCXK (Hebei) 2017-001. Replace once a week, and replace at any time if there is leakage, contamination or other abnormal conditions in the cage box. Mark the animals with ear numbers, and display the ear numbers of the animals in this cage on the cage card. Experimental cage numbering: Before grouping, use temporary cage cards to mark the animal cages before grouping. After grouping, indicate the topic number, cage number, dose, animal group, breed strain, gender, animal number, topic person in charge, start and end dates of the experiment, etc. on cage cards of different colors.

[0090] Exosome (EXO)-cisplatin: Prepare 1 mg / mL cisplatin solution with normal saline, take 0.5 mL cisplatin solution and 0.5 mL NTA to make 9×10 9 / mL exosomes were slowly and thoroughly mixed to prepare a cisplatin concentration of 0.5 mg / mL and an exosome NTA of 4.5×10 9 / mL mixture was transferred to an electroporation cup for electroporation. The electroporator used here was Celtrix, and the electroporation conditions were 1mL, -4 program.

[0091] The grouping and dosing conditions are shown in Table 2. Blood, brain and kidney tissues of mice were collected at 1 h and 24 h after administration, and Zhonglian (Beijing) Pharmaceutical Technology Co., Ltd. was commissioned to detect the content of cisplatin in the above tissues using HPLC-MS.

[0092] Table 2 Grouping and medication

[0093]

[0094] Note: Before sampling, physiological saline needs to be perfused to remove blood from the brain parenchyma and avoid interference of cisplatin in the blood with the test results.

[0095] 3. Experimental results

[0096] The results are as follows Figure 1 As shown, the results showed that in the blood, cisplatin could be detected 1h and 24h after the tail vein injection of cisplatin and exosome-cisplatin, and the cisplatin content in the exosome-cisplatin group was higher than that in the cisplatin group; in the brain tissue, cisplatin could be detected only 1h after the tail vein injection of exosome-cisplatin, and no cisplatin was detected in the cisplatin alone group; in the kidney, cisplatin could be detected 1h and 24h after the tail vein injection of cisplatin and exosome-cisplatin, and the cisplatin content in the cisplatin + EXO group was higher than that in the cisplatin group. The above results show that after exosomes encapsulate cisplatin, cisplatin can pass through the blood-brain barrier, increase the blood concentration of cisplatin in the blood and tissues, and prolong its half-life.

[0097] Example 2 Exosomes from mesenchymal stem cells (MSCs) have ovarian enrichment properties

[0098] 1. Experimental Materials

[0099] The main experimental materials are shown in Table 3 below.

[0100] Table 3 Main experimental materials

[0101]

[0102] 2. Experimental methods

[0103] SPF grade Balb / c Nude mice, female, 6-8 weeks old, were supplied by Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., with a production license number of SCXK (Beijing) 2021-0006. The animals were adapted for 5 days and injected with DIR-stained MSC-derived exosomes (Chengnuo Medical) via the tail vein at a dose of 6×10 10 vg / animal. 48h after caudal vein administration, the animals were dissected and the heart, liver, spleen, lung, kidney and ovary were taken for organ imaging. All data were Lumina IIl ROI analysis was imported into Excel worksheet for organization, and GraphPad Prism 8.0 was used for visualization.

[0104] 3. Experimental results

[0105] The results are as follows Figure 2 As shown, the results showed that after tail vein injection, MSC-derived exosomes were mainly distributed in the liver, lungs, spleen, and ovaries, but not in the heart and kidneys. The above results indicate that MSC-derived exosomes have the characteristics of ovarian enrichment.

[0106] Example 3 Therapeutic effect of NAD-encapsulated exosomes on animal models of premature ovarian failure

[0107] 1. Experimental Materials

[0108] The main experimental materials are shown in Table 4 below.

[0109] Table 4 Main experimental materials

[0110] name factory Part Number EP tube 1.5mL (QSP) Thermo 509-GRD-Q 0.22μm filter pall PALL 4612 SPL10mL pipette SPL SPL91010 Boxed Tips 1250μL (QSP) Thermo T112NXLRS-Q 50mL centrifuge tube (QSP) Thermo 339652 Coenzyme I (NAD) for injection Kaifeng Connor H41024721

[0111] 2. Experimental methods

[0112] The test drug of the present invention is an exosome encapsulated with NAD. The specific preparation method is: the required NAD drug is encapsulated into the exosome by electroporation. The prepared exosome-NAD is used for animal and clinical experiments. The final concentration of the drug used is: exosome NTA (the concentration of exosomes is detected by nanoparticle tracking analysis technology) is 4×10 10 / mL, NAD was 600μg / mL. The exosomes used were derived from mesenchymal stem cells (MSC).

[0113] The specific drug preparation method is as follows: (1) Dissolve the NAD drug in DPBS to a 10 mg / mL stock solution. (2) Take the required NAD stock solution and slowly add it to the exosomes. After fully mixing, incubate the mixture at room temperature for 15 minutes. (3) Electroporate the exosome-NAD mixture using the cellcyte electroporator. The optimal electroporation program for the 1mL system electroporation cup is "-4"; the optimal electroporation program for the 10mL system electroporation cup is "-6". (4) Let the exosome-NAD mixture stand on ice for 30 minutes after electroporation. The preparation is complete.

[0114] POF animal model: 8-week-old C57B / L6 female mice were intraperitoneally injected with cisplatin 2 mg / (kg·d) for 7 consecutive days. The experimental animals were divided into 5 groups, with 5 mice in each group. Group 1 was the healthy control group, and groups 2-5 were cisplatin modeling groups.

[0115] The test drug was administered once every three days, for a total of three times. Group 1, the healthy control group, and Group 2, the vehicle group, were given saline; Group 3 was given exosomes without NAD encapsulation, Group 4 was given NAD alone, and Group 5 was given exosome-NAD. The administration method was tail vein injection, and the main physiological indicators such as body weight, diet, and body temperature were recorded during and after administration to measure safety. The ovaries were taken 21 days after administration to detect the organ coefficient.

[0116] 3. Experimental results

[0117] The results are as follows Figure 3-6 As shown in the results, cisplatin modeling caused the ovarian organ coefficient to decrease by about 50%. The use of exosome-NAD via tail vein injection can restore the organ coefficient, while tail vein injection of NAD alone has no significant effect ( Figure 6). The above results show that exosomes encapsulating NAD (exosome-NAD) have a significant therapeutic effect on the animal model of premature ovarian failure. Example 4 Engineering exosomes to achieve ovarian targeted delivery of drugs

[0118] 1. Experimental Materials

[0119] The main experimental materials are shown in Table 5 below.

[0120] Table 5 Main experimental materials

[0121]

[0122]

[0123] 2. Experimental methods

[0124] The inventors designed FSH and LH ovarian targeting peptide structures, which were connected to the extracellular end of the exosome membrane protein Lamp2b and targeted to the ovarian tissue by ligand-receptor binding. iMSC-FSH / iMSC-LH cell lines were constructed by lentiviral infection, specifically by: (1) constructing a lentiviral vector overexpressing FSH-Lamp2b and LH-Lamp2b; (2) using HEK293T to package the corresponding lentivirus: 3 million cells were inoculated into a 10 cm culture dish, and after 24 hours, the cell confluence was 80%, and the lentiviral packaging plasmid, FSH-Lamp2b or LH-Lamp2b 10 μg, PMD-2G 7 μg, VSV-G 5 μg, REV 4 μg were transfected, and then cultured in a 5% CO2, 37°C incubator. After 48 and 72 hours, the supernatant was collected into a 50 mL centrifuge tube, and centrifuged at 3000 rpm for 5 min to remove cell debris. The supernatant was collected with a 10 mL syringe and filtered using a 0.45 μm filter membrane, and 5X PEG8000+NaCl, 10000g at 4℃ overnight, then centrifuged at 4℃ for 1.5h, the supernatant was removed, and the precipitate was gently washed with 1mL DPBS until the virus precipitate was completely dissolved, and the virus titer was detected by qPCR; (3) Lentivirus-infected cells: The target cells in good condition were inoculated into a 6-well plate to a cell concentration of 10 6cells / well, with a cell density of 30%. Before infection, the original culture medium of the cells was aspirated, 2 mL of new culture medium was added, and the virus suspension was added to the cell culture supernatant at a ratio of 5000 viruses / cell. After that, the culture was continued for 72 hours, and puromycin with a final concentration of 10 ng / mL was added for screening for 72 hours. Total RNA was extracted from the obtained cells, and the expression of the target gene was detected by RT-qPCR. The exosomes secreted by iMSC-FSH / iMSC-LH (i.e., iPSC-derived MSCs stably transfected with FSH-Lamp2b or LH-Lamp2b) cells were collected and purified using the PEG8000 concentration method. The obtained exosomes were stained with DIR dye and injected into the mouse through the tail vein at a dose of 2.68*10 9 Particles / mouse, 48h after tail vein administration, the animals were grossly dissected, and the heart, liver, spleen, lung, kidney, and ovary were removed for organ imaging. SPF grade Balb / c Nude mice, female, 6-8 weeks old, were supplied by Beijing Weitonglihua Experimental Animal Technology Co., Ltd., and the production license number was SCXK (Beijing) 2021-0006.

[0125] The LH-Lamp2b fusion protein DNA sequence, LH-Lamp2b fusion protein amino acid sequence, FSH-Lamp2b fusion protein DNA sequence, and FSH-Lamp2b fusion protein amino acid sequence are shown in Table 6 below.

[0126] Table 6 DNA sequence and amino acid sequence of LH-Lamp2b fusion protein and FSH-Lamp2b fusion protein

[0127]

[0128]

[0129] In addition to the animal level, the present invention also verifies the targeting of FSH and LH to ovarian cells at the cellular level. Rats were intraperitoneally injected with pregnant mare serum gonadotropin at a dose of 40 IU. After 48 hours of administration, rat ovarian granulosa cells GC were isolated and the granulosa cells were cultured at a concentration of 1×10 5 The cells were inoculated at a density of 100 cells / well in a 12-well cell culture plate. After 12 hours, 2 μg of GFP mRNA encapsulated in different ways were added to the cell culture supernatant, namely LNP-GFP, LNP-GFP+iMSC-EXO, LNP-GFP+iMSC-FSH-EXO, and LNP-GFP+iMSC-LH-EXO. Fluorescence imaging of the cells was performed at 24 hours and 48 hours.

[0130] The preparation method of LNP-mRNA is as follows: (1) prepare a GFP mRNA solution with a concentration of 110 ng / μL using a citric acid buffer; (2) load the prepared GFP mRNA into LNP using a high-flow microfluidic method, with a lipid phase to water phase flow rate ratio of 1:3 and a total flow rate of 20 mL / min; (3) centrifuge the encapsulated LNP-GFP using a Milipore 30KD ultrafiltration tube at 3000 g for 10 min, ultrafilter to 1 / 4 of the original volume, then add PBS buffer and ultrafilter the sample, repeat three times to obtain a concentrated sample; (4) mix the obtained LNP-GFP with the above-mentioned different exosomes, let stand at 4°C for 30 min, and the amount of exosomes used is 1×10 9 Particles / 2μg mRNA.

[0131] 3. Experimental results

[0132] The results are as follows Figure 7-8 As shown, the results showed that FSH and LH ovarian targeting peptide structures can improve the ovarian distribution of MSC-derived exosomes, and the effect of FSH is better than that of LH ( Figure 7 ). FSH and LH ovarian targeting peptide structures can improve the delivery efficiency of MSC-derived exosomes to LNP-encapsulated mRNA in ovarian cells, and the FSH ovarian targeting peptide structure is superior to the LH ovarian targeting peptide structure ( Figure 8 ).

[0133] Example 5 Therapeutic effect of FSH ovarian targeting peptide-modified exosomes delivering nucleic acid drugs (LNPs encapsulating NAMPT, NMNAT3, COX15 mRNA) on mice with premature ovarian failure

[0134] 1. Experimental Materials

[0135] The main experimental materials are shown in Table 7 below.

[0136] Table 7 Main experimental materials

[0137] name factory Part Number EP tube 1.5mL (QSP) Thermo 509-GRD-Q 0.22μm filter pall PALL 4612 SPL 10mL Pipette SPL SPL91010 Boxed Tips 1250μL (QSP) Thermo T112NXLRS-Q 50mL Centrifuge Tube QSP Thermo 339652 Cisplatin for injection (dry powder) Qilu Pharmaceutical SM-102 Avituo 2089251-47-6 Distearoylphosphatidylcholine DSPC Avituo S01005 CHO-HP High Purity Cholesterol Avituo O01001 PEG2000-DMG Avituo O02005

[0138] 2. Experimental methods

[0139] The test drug of the present invention is a mixture of LNP (lipid nanoparticles) and exosomes encapsulating NAMPT, NMNAT3, COX15 mRNA (referred to as NNC mRNA).

[0140] The specific preparation method of LNP-NNC is as follows: (1) the three mRNAs mentioned above are mixed with citric acid buffer to prepare a solution (NNC) with a final concentration of 10 ng / μL, with a ratio of 1:1:1; (2) the prepared NNC is placed in a high-flow microfluidic system. mRNA was loaded into LNP (SM-102, Avituo, 2089251-47-6; distearoylphosphatidylcholine DSPC, Avituo, S01005; CHO-HP high purity cholesterol, Avituo, O01001; PEG2000-DMG, Avituo, O02005), the lipid phase and water phase flow rate ratio was 1:3, and the total flow rate was 20 mL / min; (3) The encapsulated LNP-NNC was centrifuged at 3000 g for 10 min using Milipore30KD ultrafiltration tube, ultrafiltered to 1 / 4 of the original volume, and then PBS buffer was added to ultrafilter the sample, and the concentrated sample was obtained after three times; (4) The obtained LNP-NNC was mixed with FSH ovarian targeting peptide modified exosomes (FSH-EXO) at a volume ratio of 1:1, and the concentration of the FSH-EXO was 8×10 10 / mL, and FSH-EXO-LNP-NNC was obtained. The final concentration of the drug FSH-EXO-LNP-NNC used was: exosome (FSH-EXO) NTA was 4×10 10 / mL, LNP-NNC was 25μg / mL.

[0141] POF animal model: 8-week-old C57B / L6 female mice were intraperitoneally injected with cisplatin 2 mg / (kg·d) for 7 consecutive days. The experimental animals were divided into 4 groups, group 1 was a healthy control group with 12 mice, groups 2-4 were cisplatin modeling groups, 12 mice in the vehicle group, 4 mice in the estradiol E2 group, and 15 mice in the NNC group.

[0142] Grouping and administration: Group 1 is the healthy control group; Group 2 is the solvent group, which is injected with normal saline through the tail vein; Group 3 is the positive drug estradiol E2 group, which is subcutaneously injected with E2, 8μg / kg / time, for 3 consecutive days, and stopped for one day until the end of the experiment; Group 4 is FSH-EXO-LNP-NNC, with a dose of exosome NTA of 8×10 9 / mouse, NNC mRNA was 5μg / mouse / time, and the mice were given three times on DAY1, DAY3, and DAY5, each time by combined tail vein and intraperitoneal injection. During and after the administration, the main physiological indicators such as body weight, diet, and body temperature were recorded to measure safety. Vaginal smears were performed every day, fixed with anhydrous ethanol and stained with methylene blue solution to analyze the estrous cycle of mice. After 15 days of administration, the mice were dissected, the organ coefficients were detected, and the ovarian tissue was stained with HE.

[0143] 3. Experimental results

[0144] The results showed that after cisplatin administration, one animal died in the MC group on DAY8 and DAY9, one animal died in the E2 group on DAY9, and the HC and NNC groups had a good survival status ( Fig. 9 ).

[0145] Observation of mouse vaginal cell sections showed that the estrous cycle of the HC group was at a normal level. After POF modeling, the estrous cycles of all mice in the NC and E2 groups began to become disordered to varying degrees on DAY5. Three-quarters of the mice in the NNC group were at a normal level ( Fig.10 ), the above results indicate that FSH-modified exosomes delivered LNP-NNC have a therapeutic effect on the premature ovarian failure model caused by cisplatin.

[0146] The results of observation of mouse ovarian sections showed that compared with the HC group, the volume of ovarian follicles in animals after POF modeling was significantly reduced, the number of granulosa cells decreased, the apoptosis of granulosa cells increased, and the number of atretic follicles increased. FSH-modified exosomes delivered LNP-NNCs could significantly improve the above phenotypes ( Fig.11 ). The above results once again prove that FSH-modified exosomes delivered LNP-NNC have a significant effect in treating premature ovarian failure.

[0147] Example 6 Therapeutic effect of exosome-delivered nucleic acid (TNP encapsulating NAMPT, NMNAT3, COX15 mRNA) on mice with premature ovarian failure

[0148] 1. Experimental Materials

[0149] The main experimental materials are shown in Table 8 below.

[0150] Table 8 Main experimental materials

[0151]

[0152]

[0153] 2. Experimental methods

[0154] The test drug of the present invention is a mixture of TNP (new mRNA delivery system) encapsulating NAMPT, NMNAT3, COX15 mRNA (referred to as NNC mRNA) and exosomes.

[0155] Preparation method of TNP-NNC:

[0156] (1) The three mRNAs were mixed with RNase-free ultrapure water to prepare a solution (NNC) with a final concentration of 1 mg / mL, with a ratio of 1:1:1; (2) The prepared mRNA was loaded into TNP (Deng Hongzhang Laboratory, Xidian University) using a high-flow microfluidic method, with the lipid phase and water phase flow rates of 0.1 mL / min and 0.3 mL / min, respectively; (3) The encapsulated TNP-NNC was ultrafiltered to 1 / 4 of the original volume using a Milipore 30KD ultrafiltration tube and centrifuged at 3000 g for 10 min, and then PBS buffer was added to ultrafilter the sample. The concentrated sample was obtained after three times; (4) The obtained TNP-NNC was mixed with exosomes (the exosomes used were from wild-type mesenchymal stem cells (MSC)) at a volume ratio of 1:1, and the concentration of the exosomes was 10 11 / mL, and let stand at 4℃ for 30min to obtain EXO-TNP-NNC. The final concentration of the drug EXO-TNP-NNC used was: exosome (EXO) NTA was 5×10 10 / mL, TNP-NNC was 75μg / mL.

[0157] POF animal model: 8-week-old C57B / L6 female mice were intraperitoneally injected with 4.5 mg / (kg·d) of cisplatin for 4 consecutive days. The experimental animals were divided into 4 groups, group 1 was the healthy control group, and groups 2-4 were cisplatin modeling groups.

[0158] The test drugs were administered as follows: Group 1 was the healthy control group; Group 2 was the vehicle group, which was injected with normal saline via the tail vein; Group 3 was the positive drug estradiol E2 group, which was injected subcutaneously at 8 μg / kg / time; Group 4 was EXO-TNP-NNC, which was injected with 15 μg / mouse via the tail vein, and the exosome NTA was 10 10 / mouse, and the drugs were administered on DAY2, 3, 4, 5, and 7. During and after the administration, the main physiological indicators, such as body weight, diet, and body temperature, were recorded to measure safety. Vaginal smears were performed every day, fixed with anhydrous ethanol, and stained with methylene blue solution to analyze the estrous cycle of the mice. The mice were dissected 10 days after the administration, and the organ coefficients were detected and the ovarian tissue was stained with HE.

[0159] 3. Experimental results

[0160] Observation of mouse vaginal cells and statistics of estrus on DAY15 showed that the mice in the HC group were in different cycles, and the estrus cycle was in line with normal rules; after POF modeling, most of the mice in the NaCl group were in proestrus and could not enter the estrus; all mice in the E2 group were in estrus; the estrus cycle of the mice in the NNC group was similar to that of the HC and was in line with normal rules ( Fig.12 ), indicating that exosome-delivered TNP-NNC has a therapeutic effect on mice with premature ovarian failure.

Claims

1. A nucleic acid composition for treating premature ovarian failure, characterized in that: The nucleic acid composition includes mRNA, DNA or recombinant protein encoding NAMPT, NMNAT3 and COX15.

2. An ovary targeting peptide, characterized in that: The ovarian targeting peptide includes LH-Lamp2b fusion protein or FSH-Lamp2b fusion protein; The amino acid sequence of the LH-Lamp2b fusion protein is shown in SEQ ID NO: 2; The amino acid sequence of the FSH-Lamp2b fusion protein is shown in SEQ ID NO:

4.

3. The ovary targeting peptide according to claim 2, characterized in that The nucleotide sequence of the LH-Lamp2b fusion protein is shown in SEQ ID NO: 1; The nucleotide sequence of the FSH-Lamp2b fusion protein is shown in SEQ ID NO:

3.

4. An exosome specifically targeting ovarian cells, characterized in that: The exosomes contain the ovary targeting peptide according to claim 2 or 3.

5. The exosome according to claim 4, characterized in that The exosomes are derived from mesenchymal stem cells; Optionally, the exosomes contain the FSH-Lamp2b fusion protein described in claim 2 or 3.

6. Any of the following products: (1) An exosome drug for treating premature ovarian failure, characterized in that: The exosome drug for treating premature ovarian failure is an exosome drug obtained by mixing LNP or TNP encapsulating the nucleic acid composition of claim 1 and the exosomes of claim 4 or 5; (2) An exosome drug encapsulating NAD, characterized in that the exosome drug encapsulating NAD is an exosome drug obtained by encapsulating NAD drug in exosomes; (3) A pharmaceutical composition, characterized in that the pharmaceutical composition comprises the exosome drug for treating premature ovarian failure and / or the exosome drug encapsulating NAD; (4) A pharmaceutical preparation, characterized in that the pharmaceutical preparation comprises the exosome drug for treating premature ovarian failure and / or the exosome drug encapsulating NAD.

7. The product according to claim 6, characterized in that The concentration of the nucleic acid composition in the LNP or TNP encapsulating the nucleic acid composition of claim 1 is 10 ng / μL; Optionally, the ratio of mRNAs of NAMPT, NMNAT3 and COX15 in the nucleic acid composition in the LNP or TNP encapsulating the nucleic acid composition of claim 1 is 1:1:1; Optionally, the final concentration of the exosome drug for treating premature ovarian failure comprising LNP encapsulating the nucleic acid composition of claim 1 is: the exosomes of claim 4 or 5 are 4×10 10 / mL, the LNP encapsulating the nucleic acid composition of claim 1 is 25 μg / mL; Optionally, the final concentration of the exosome drug for treating premature ovarian failure comprising TNP encapsulating the nucleic acid composition of claim 1 is: the exosomes of claim 4 or 5 are 5×10 10 / mL, the TNP encapsulated with the nucleic acid composition of claim 1 is 75 μg / mL; Optionally, the exosomes in the NAD-encapsulated exosome drug are 4×10 10 / mL, NAD is 600μg / mL; Optionally, the exosomes in the NAD-encapsulated exosome drug are derived from mesenchymal stem cells; Optionally, the dosage form of the pharmaceutical preparation includes intravenous reinfusion preparations, in situ injection preparations, oral preparations, tablets, capsule preparations, granule preparations, sustained-release preparations, powders, and pills.

8. Any of the following methods: (1) A method for preparing an exosome drug for treating premature ovarian failure as claimed in claim 6 or 7, characterized in that: The preparation method comprises the following steps: mixing LNP or TNP encapsulating the nucleic acid composition of claim 1 and the exosomes of claim 4 or 5 to obtain an exosome drug; (2) An exosome drug encapsulating NAD as described in claim 6 or 7, characterized in that the exosome drug encapsulating NAD is an exosome drug obtained by encapsulating NAD drug in exosomes.

9. The method according to claim 8, characterized in that The LNP or TNP encapsulating the nucleic acid composition of claim 1 is prepared by the following method: mixing the mRNAs of NAMPT, NMNAT3 and COX15 to obtain a mixed solution NNCmRNA, and loading the NNC mRNA into LNP or TNP to obtain LNP or TNP encapsulating the nucleic acid composition of claim 1; Optionally, the final concentration of the mixed solution is 10 ng / μL; Optionally, the mRNA ratio of NAMPT, NMNAT3 and COX15 in the mixed solution is 1:1:1; Optionally, the NNC mRNA is loaded into LNP or TNP using a high-flow microfluidics method; Optionally, in the high-flow rate microfluidic method, the flow rate ratio of the lipid phase to the aqueous phase is 1:3, and the total flow rate is 20 mL / min; Optionally, the exosomes described in claim 4 or 5 are prepared by the following method: constructing an iMSC-FSH / iMSC-LH cell line by lentiviral infection, collecting and purifying the exosomes secreted by the iMSC-FSH / iMSC-LH cells, thereby obtaining the exosomes described in claim 4 or 5; Optionally, the exosome drug encapsulating NAD is an exosome drug obtained by encapsulating the NAD drug in exosomes using an electroporation method; Optionally, the exosomes in the NAD-encapsulated exosome drug are 4×10 10 / mL, NAD is 600μg / mL; Optionally, the exosomes in the NAD-encapsulated exosome drug are derived from mesenchymal stem cells.

10. Any of the following applications: (1) Use of the nucleic acid composition of claim 1, the ovarian targeting peptide of claim 2 or 3, and / or the exosomes of claim 4 or 5 in the preparation of an exosome drug for treating premature ovarian failure; (2) Use of the exosomes according to claim 4 or 5 in targeted delivery of nucleic acid molecules; (3) Use of the exosome drug for treating premature ovarian failure and / or the exosome drug encapsulating NAD as claimed in claim 6 or 7 in the preparation of a pharmaceutical composition or pharmaceutical preparation for treating premature ovarian failure.

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