Infertility treatment

Abstract

To develop novel therapeutic techniques that do not pose the risk of genotoxicity to help infertility patients. [Solution] A single-stranded negative-strand RNA viral vector containing a nucleic acid sequence encoding a protein associated with the formation or development of germ cells, or a pharmaceutical composition for preventing or treating infertility containing an RNA molecule encoding the protein, is provided.

Description

[Technical Field]

[0001] The present invention relates to a pharmaceutical composition for preventing or treating infertility, which comprises a single-stranded negative-strand RNA viral vector or an RNA molecule. [Background technology]

[0002] Oogenesis is a continuous and complex process regulated by numerous intra- and extra-ovarian factors. Mutations in genes involved in this regulation lead to infertility, affecting one in every five to six couples. Recent next-generation sequencing technologies have identified many candidate genes responsible for infertility. However, to date, there are few infertility treatments based on these causative genes.

[0003] In addition to congenital infertility, the number of cancer survivors who become infertile after birth is also increasing as the number of cancer survivors increases due to the recent development of anticancer drugs in cancer treatment. This is particularly true for prepubertal patients, who remain infertile for approximately half of the time, despite a survival rate of 80-90%.

[0004] Currently, assisted reproductive technologies such as in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) are widely used for infertility treatment, but the application of these technologies is limited to cases where mature gametes are available. Therefore, a significant proportion of cases caused by immature gametes remain untreated. Furthermore, studies in mice have shown the possibility of progeny defects associated with assisted reproductive technologies, raising concerns about human offspring (Non-Patent Document 1).

[0005] Regarding research into gene therapy for infertility, it has been reported that the use of lentivirus, adenovirus, and adeno-associated virus vectors in the treatment of congenitally infertile mice resulted in the birth of normal offspring with appropriate genomic imprinting patterns (e.g., Non-Patent Document 2, Non-Patent Document 3). However, some studies in somatic cells have shown that these gene therapy vectors may integrate into the host genome. For example, detailed analysis of a long-term study of AAV-based therapy for hemophilia A in dogs showed numerous integrations of vectors with partial deletions or rearrangements (Non-Patent Document 4). Therefore, despite the success of infertility treatment experiments in mice, concerns about the integration of gene therapy vectors into the host cell genome have hindered clinical application in humans. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] J. Clin. Invest. 2023;133(22): e170140 [Non-patent document 2] Proc. Natl. Acad. Sci. USA 2002;99(11): 7524-7529 [Non-patent document 3] Cell Rep. Med. 2022;3(5): 100606 [Non-patent document 4] Nat. Biotechnol. 2021;39(1): 47-55 Summary of the Invention [Problem to be solved by the invention]

[0007] In view of the above, there is a need to develop novel therapeutic techniques that do not pose the risk of genotoxicity to help infertility patients. [Means for solving the problem]

[0008] As a result of extensive research, the present inventors have surprisingly found that injecting a single-stranded negative-strand RNA viral vector carrying a nucleic acid encoding a protein associated with germ cell formation or development into the ovaries of congenitally or acquiredly infertile female mice restores the fertility of the infertile mice. Even more surprisingly, they have found that direct injection of an RNA molecule containing a nucleic acid sequence encoding a protein associated with germ cell formation or development into the germ cells of congenitally infertile female and male mice restores the fertility of the infertile mice. Based on these findings, the present invention has been completed.

[0009] Therefore, the present application provides, for example, the following aspects. [1] A pharmaceutical composition for preventing or treating infertility, comprising a single-stranded negative-strand RNA viral vector containing a nucleic acid sequence encoding a protein associated with the formation or development of germ cells. [2] The pharmaceutical composition described in [1], wherein the infertility is acquired infertility. [3] The pharmaceutical composition according to [2], wherein the acquired infertility is infertility caused by administration of an anticancer drug. [4] The pharmaceutical composition described in [1], wherein the infertility is congenital infertility. [5] The pharmaceutical composition according to [4], wherein the protein is a functionally defective protein. [6] The pharmaceutical composition according to any one of [1] to [5], wherein the protein is a protein associated with the formation or development of an ovum. [7] The pharmaceutical composition according to any one of [1] to [5], wherein the protein is a protein associated with sperm formation or development. [8] The pharmaceutical composition according to any one of [1] to [7], wherein the protein is selected from the group consisting of Kitl, Cldn11, FGF family proteins, GDNF family proteins, and AKT family proteins. [9] The pharmaceutical composition according to [8], wherein the protein is Kitl.

[10] The pharmaceutical composition according to any one of [1] to [9], wherein the RNA viral vector is a Sendai viral vector.

[11] A pharmaceutical composition for preventing or treating infertility, comprising an RNA molecule encoding a protein associated with the formation or development of germ cells.

[12] The pharmaceutical composition according to

[11] , wherein the infertility is acquired infertility.

[13] The pharmaceutical composition according to

[12] , wherein the acquired infertility is infertility caused by administration of an anticancer drug.

[14] The pharmaceutical composition according to

[11] , wherein the infertility is congenital infertility.

[15] The pharmaceutical composition according to

[14] , wherein the protein is a defective protein.

[16] The pharmaceutical composition according to any one of

[11] to

[15] , wherein the protein is a protein associated with the formation or development of an oocyte.

[17] The pharmaceutical composition according to any one of

[11] to

[15] , wherein the protein is a protein associated with sperm formation or development.

[18] The pharmaceutical composition according to any one of

[11] to

[17] , wherein the protein is selected from the group consisting of Kitl, Cldn11, FGF family proteins, GDNF family proteins, and AKT family proteins.

[19] The pharmaceutical composition according to

[18] , wherein the protein is Kitl or Cldn11.

[20] The pharmaceutical composition according to any one of

[11] to

[19] , wherein the RNA molecule is mRNA.

[21] A single-stranded negative-strand RNA viral vector containing a nucleic acid sequence encoding a protein associated with the formation or development of germ cells.

[22] The vector according to

[21] , which is a Sendai virus vector.

[23] The vector according to

[21] or

[22] , wherein the protein is a protein associated with the formation or development of an egg.

[24] The vector according to

[21] or

[22] , wherein the protein is a protein associated with sperm formation or development.

[25] The vector according to any one of

[21] to

[24] , wherein the protein is selected from the group consisting of Kitl, Cldn11, an FGF family protein, a GDNF family protein, and an AKT family protein.

[26] The vector according to

[25] , wherein the protein is Kitl.

[27] A method for preventing or treating infertility, comprising administering to an infertile subject a single-stranded negative-strand RNA viral vector comprising a nucleic acid sequence encoding a protein associated with the formation or development of germ cells.

[28] The method according to

[27] , wherein the subject is a subject suffering from acquired infertility.

[29] The method described in

[28] , wherein the acquired infertility is infertility caused by administration of an anticancer drug.

[30] The method according to

[27] , wherein the protein is a protein with a defective function and the subject is a subject with congenital infertility.

[31] The method according to any one of

[27] to

[30] , wherein the protein is a protein associated with the formation or development of an oocyte.

[32] The method according to any one of

[27] to

[30] , wherein the protein is a protein associated with sperm formation or development.

[33] The method according to any one of

[27] to

[32] , wherein the protein is selected from the group consisting of Kitl, Cldn11, FGF family proteins, GDNF family proteins, and AKT family proteins.

[34] The method according to

[33] , wherein the protein is Kitl.

[35] The method according to any one of

[27] to

[34] , wherein the RNA viral vector is a Sendai viral vector.

[36] A method for preventing or treating infertility, comprising administering to an infertile subject an RNA molecule encoding a protein associated with the formation or development of germ cells.

[37] The method according to

[36] , wherein the subject is a subject suffering from acquired infertility.

[38] The method described in

[37] , wherein the acquired infertility is infertility caused by administration of an anticancer drug.

[39] The method according to

[36] , wherein the protein is a protein with a defective function and the subject is a subject with congenital infertility.

[40] The method according to any one of

[36] to

[39] , wherein the protein is a protein associated with the formation or development of an egg.

[41] The method according to any one of

[36] to

[39] , wherein the protein is a protein associated with sperm formation or development.

[42] The method according to any one of

[36] to

[41] , wherein the protein is selected from the group consisting of Kitl, Cldn11, FGF family proteins, GDNF family proteins, and AKT family proteins.

[43] The method according to

[42] , wherein the protein is Kitl or Cldn11.

[44] The method according to any one of

[36] to

[43] , wherein the RNA molecule is mRNA. [Effects of the Invention]

[0010] According to the present invention, an RNA molecule containing a nucleic acid sequence encoding a protein associated with germ cell formation or development, carried by a single-stranded negative-strand RNA viral vector, or without a viral vector, can be administered to a subject suffering from congenital or acquired infertility, thereby restoring the fertility of the subject. Furthermore, according to the present invention, an RNA molecule containing a nucleic acid sequence encoding a protein associated with germ cell formation or development, carried by a single-stranded negative-strand RNA viral vector, or without a viral vector, can be administered to a subject at risk of acquired infertility, thereby preventing infertility in the subject. Because the present invention uses a single-stranded negative-strand RNA viral vector, or does not use a viral vector, there is no risk of genotoxicity, as occurs with conventional vector-based gene delivery. Furthermore, because the single-stranded negative-strand RNA viral vector or RNA molecule does not integrate into the host genome, the risk of germline transmission of the transgene is extremely low. Furthermore, because the in vivo half-life of RNA molecules is short, the method of the present invention minimizes the risk of RNA molecule accumulation in vivo. [Brief explanation of the drawings]

[0011] [Figure 1A] Introduction of SeV into wild-type ovaries. Results of real-time PCR analysis of ovaries (n=3) are shown. [Figure 1B] SeV transduction into wild-type ovaries. The results of histological analysis of ovaries are shown. The bar in the figure indicates 100 μm. The ovaries were stained with hematoxylin and eosin. [Figure 1C] SeV transduction into wild-type ovaries. Immunostaining with a lymphocyte marker is shown. The bar in the figure indicates 50 μm. Staining was performed with Hoechst 33342. [Figure 2A] Immunostaining of wild-type ovaries after microinjection of FITC-dextran. The bar indicates 50 μm. Staining was performed with Hoechst 33342. [Figure 2B]Immunostaining of KitlSl-t / KitlSl-t ovaries after microinjection of FITC-dextran. The bar in the figure indicates 50 μm. Staining was performed with Hoechst 33342. [Figure 3A] Functional analysis of SeV-Kitl injection into wild-type mice. The number of live births per mouse is shown (control n=13, SeV-Kitl n=12). [Figure 3B] Functional analysis of SeV-Kitl injection into wild-type mice. The number of litters per birth is shown (control n=13, SeV-Kitl n=14). [Figure 3C] Functional analysis of SeV-Kitl injection into wild-type mice, showing the rate of offspring birth (control n=9, SeV-Kitl n=11). [Figure 3D] Functional analysis of SeV-Kitl injected wild-type mice. Histological analysis of ovaries. The bar in the figure indicates 100 μm. Stained with hematoxylin and eosin. [Figure 3E] Functional analysis of SeV-Kitl injection into wild-type mice. The number of secondary follicles per ovary is shown (control n=7, SeV-Kitl n=6). [Figure 3F] Functional analysis of SeV-Kitl injected wild-type mice. Immunostaining for MK167 is shown. The bar in the figure indicates 50 μm. Staining was performed with Hoechst33342. [Figure 3G] Functional analysis of SeV-Kitl injection into wild-type mice. The number of MK167+ cells per follicle is shown (n=4). [Figure 3H] Functional analysis of SeV-Kitl injected wild-type mice. TUNEL staining is shown. The bar in the figure indicates 50 μm. Staining was performed with Hoechst 33342. [Figure 3I] Functional analysis of SeV-Kitl injection into wild-type mice. Quantitative results of apoptotic cells are shown (n=8). [Figure 3J] Functional analysis of SeV-Kitl injected wild-type mice. Immunostaining with a lymphocyte marker (arrow) is shown. The bar in the figure indicates 50 μm. Staining was performed with Hoechst 33342. [Figure 4A] Restoration of fertility in KitlSl-t / KitlSl-t mice. RT-PCR analysis of Kitl expression. [Figure 4B] Restoration of fertility in KitlSl-t / KitlSl-t mice. Real-time PCR analysis of Kitl expression is shown (n=3). [Figure 4C] Appearance of the ovary after SeV-Kitl injection. The bar in the figure indicates 1 mm. [Figure 4D] Histological analysis of ovaries. The bar in the figure indicates 100 μm. Stained with hematoxylin and eosin. [Figure 4E] Offspring (arrow) born after mating with KitlSl-t / KitlSl-t mutant male mice. [Figure 4F] Analysis of follicular development 6 months after SeV-Kitl injection (control n = 8, SeV-Kitl n = 17). [Figure 5A] Fertility protection of wild-type mice against busulfan by SeV-Kitl injection. Offspring (arrow) born after mating with wild-type B6 male mice. [Figure 5B] Fertility protection of wild-type mice against busulfan by SeV-Kitl injection. Number of litters per mouse (n=10). [Figure 5C] Fertility protection of wild-type mice against busulfan by SeV-Kitl injection. Number of pups per litter (n=7). [Figure 5D] Fertility protection of wild-type mice against busulfan by SeV-Kitl injection. Offspring production rate (n=7). [Figure 5E] Fertility protection of wild-type mice against busulfan by SeV-Kitl injection. Appearance of ovaries 3 months after SeV-Kitl injection (n=20). The bar in the figure represents 1 mm. [Figure 5F] Fertility protection of wild-type mice against busulfan by SeV-Kitl injection. Ovarian weight 3 months after SeV-Kitl injection (n=20). [Figure 5G]Fertility protection of wild-type mice against busulfan by SeV-Kitl injection. Histological analysis of follicular development. Bars in the figure represent 100 μm. Stained with hematoxylin and eosin. [Figure 5H] Fertility protection of wild-type mice against busulfan by SeV-Kitl injection. Analysis of follicular development (n=8). The number of follicles per ovary is shown. In the figure, "Preantral" indicates preantral follicles, "Antral" indicates antral follicles, "Preovulatory" indicates preovulatory follicles, and "Corpus luteum" indicates luteal phase follicles. [Figure 6A] Genomic imprinting analysis. Results of COBRA are shown. [Figure 6B] Genomic imprinting analysis. Results of COBRA are shown. [Figure 6C] Genomic imprinting analysis. Results of bisulfite sequencing are shown. [Figure 6D] Genomic imprinting analysis. Results of bisulfite sequencing are shown. [Figure 7] Figure 7 shows mRNA delivery into testes. Figure 7A shows luciferase activity 1 day after microinjection into busulfan-treated testes (n=4). Approximately 400 ng of mRNA was injected into each testis. Figures 7B and C show the time course of luciferase activity after naked mRNA microinjection into wild-type (B) or busulfan-treated (C) testes (n=4). Approximately 1 μg of mRNA was injected into each testis. In the figures, WT indicates wild-type. P values ​​are shown from a two-tailed t-test. [Figure 8-1] This figure shows delivery of naked mRNA into Sertoli cells. Quantification of EGFP-positive areas within the total GATA4 (Sertoli cell marker)-positive areas in wild-type testes (n=6) is shown. In the figure, WT indicates wild-type. P values ​​are shown by two-tailed t-test. [Figure 8-2] Figure 1 shows delivery of naked mRNA into Sertoli cells. Immunostaining of busulfan-treated testes with Sertoli cell markers (GATA4 or CLDN11) 2 days after microinjection. Bars represent 50 μm. Staining was performed with Hoechst 33342. [Figure 8-3] This figure shows delivery of naked mRNA into Sertoli cells. This figure shows quantification of EGFP-positive areas within the total GATA4-positive areas in busulfan-treated testes (n=6). In the figure, WT indicates wild-type. P values ​​are shown by two-tailed t-test. [Figure 9A] Figure 1 shows innate immune activation by mRNA delivery. Real-time PCR analysis of wild-type testes 4 hours after microinjection of poly(I:C) is shown (n=3). Approximately 4 μg of mRNA was injected into each testis. In the figure, WT indicates wild-type. P values ​​are shown by two-tailed t-test. [Figure 9B] Figure 1 shows innate immune activation by mRNA delivery. Real-time PCR analysis of busulfan-treated testes 4 hours after microinjection of poly(I:C) (n = 6 for Il6 and Infb1, n = 3 for others). Approximately 4 μg of mRNA was injected into each testis. P values ​​are shown by two-tailed t-test. [Figure 9C] This shows innate immune activation by mRNA delivery. Immunostaining of wild-type testes 2 days after microinjection (n=16) is shown. The bar indicates 50 μm. Staining was performed with Hoechst 33342. In the figure, WT indicates wild-type. P values ​​are shown from a two-tailed t-test. [Figure 9D] This shows innate immune activation by mRNA delivery. Immunostaining of busulfan-treated testes 2 days after microinjection (n=16) is shown. The bar represents 50 μm. Staining was performed with Hoechst 33342. WT indicates wild-type. P values ​​are shown from a two-tailed t-test. [Figure 9E] This shows innate immune activation by mRNA delivery. TUNEL staining of wild-type and busulfan-treated testes 2 days after microinjection is shown (n=8). The bar represents 50 μm. Staining was performed with Hoechst 33342. WT indicates wild-type. P values ​​are shown by two-tailed t-test. [Figure 10-1] Figure 1 shows the rescue of spermatogenesis by Cldn11 mRNA delivery. Figure 2 shows histological analysis of Cldn11 KO mouse testes. The bar indicates 100 μm. Hematoxylin and eosin staining was performed. [Figure 10-2]Figure 1 shows rescue of spermatogenesis by Cldn11 mRNA delivery. Real-time PCR analysis of mRNA expression is shown (n=6). Bars represent 50 μm. In the figure, WT indicates wild-type. P values ​​are shown by two-tailed t-test. [Figure 10-3] Figure 1 shows the rescue of spermatogenesis by Cldn11 mRNA delivery. Immunostaining of CD4- and CD8-positive lymphocytes 1 day after microinjection (n=12). Bars represent 100 μm. Staining was performed with Hoechst 33342. P values ​​are shown by two-tailed t-test. [Figure 10-4] Figure 1 shows the rescue of spermatogenesis by Cldn11 mRNA delivery. Histological analysis of Cldn11 KO mouse testes transfected with Cldn11 mRNA 2 months after microinjection. Approximately 16 μg of mRNA was injected into each testis. Arrows indicate elongated spermatids. Bars indicate 100 μm. Hematoxylin and eosin staining was performed. [Figure 10-5] This shows the rescue of spermatogenesis by Cldn11 mRNA delivery. This image shows the epididymis of a Cldn11 KO mouse testis transfected with Cldn11 mRNA 2 months after microinjection. The bar indicates 100 μm. Hematoxylin and eosin staining was performed. [Figure 11A] This figure shows offspring from Cldn11 KO mice induced by Cldn11 mRNA transfer and ICSI. The figure shows the appearance of offspring born after ICSI. [Figure 11B] The results of PCR of tail DNA from offspring of Cldn11 KO mice induced by Cldn11 mRNA introduction and microinsemination are shown. [Figure 11C] Macroscopic appearance of F2 testes of Cldn11 KO mice born after Cldn11 mRNA transfer and microinsemination. The bar indicates 1 mm. [Figure 11D] Histological analysis of F2 testes from Cldn11 KO mice derived from Cldn11 mRNA transfer and microinsemination. Hematoxylin and eosin staining was performed. The bar indicates 100 μm. [Figure 11E]The results of COBRA in F1 mice are shown. In the figure, "uc" stands for "uncut," "c" stands for "cut," and "GS" stands for germline stem cells. [Figure 11F] The results of bisulfite sequencing of F1 mice are shown. In the figure, WT indicates wild type. DETAILED DESCRIPTION OF THE INVENTION

[0012] As used herein, the term "germ cells" refers to cells that are fertilized to produce the next generation of individuals, and includes, for example, eggs, follicles, and sperm, as well as cells that differentiate into these. Examples of female germ cells include eggs, follicles, and cells that differentiate into these, such as oogonia and oocytes, contained in the ovaries. Examples of male germ cells include sperm, and cells that differentiate into these, such as spermatogonia and spermatids, contained in the testes.

[0013] A protein associated with the formation or development of germ cells may be, for example, a protein associated with infertility or a protein that causes infertility, such as a protein that causes infertility when the protein or function is not expressed or when the expression of the protein or function is reduced (i.e., a protein with a defective function in infertility). As used herein, "proteins associated with the formation or development of germ cells" include, for example, proteins that promote the formation or development of germ cells, and proteins involved in their downstream signals, such as proteins that promote survival and proteins involved in the formation of blood-tissue barriers (e.g., blood-follicle barrier, blood-testis barrier, etc.).

[0014] Specifically, proteins associated with the formation or development of human germ cells include, but are not limited to, Kit ligand (Kitl), Cldn11, FGF family proteins, GDNF family proteins, AKT family proteins, and downstream proteins in these signaling pathways. The present invention can be applied to both female and male infertility. For the treatment or prevention of female infertility, proteins associated with the formation or development of germ cells are preferably proteins associated with the formation or development of oocytes, including, but not limited to, Kitl, FGF family proteins, AKT family proteins, and downstream proteins thereof. Kitl is a cytokine and is a molecule that induces and promotes oocyte formation. For the treatment or prevention of male infertility, proteins associated with the formation or development of germ cells are preferably proteins associated with the formation or development of sperm, including, but not limited to, Cldn11, FGF family proteins, GDNF family proteins, AKT family proteins, and downstream proteins thereof. Cldn11 is a molecule that forms the blood-testis barrier (BTB).

[0015] The nucleic acid sequence encoding the protein associated with germ cell formation or development used in the present invention is an RNA sequence. The amino acid sequence of the protein may be one disclosed in a public database such as GenBank or UniProt. The nucleic acid sequence encoding the protein may be determined based on the amino acid sequence or obtained from a public database such as GenBank or EMBL. For example, a nucleic acid sequence encoding the amino acid sequence of the human Kitl protein registered under UniProt KB accession numbers P21583-1, P21583-2, or P21583-3 (referred to herein as SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, respectively) may be used as a nucleic acid sequence encoding a protein associated with oocyte formation or development. For example, a nucleic acid sequence encoding the amino acid sequence of the human Cldn11 protein registered under UniProt KB accession number 075508 (referred to herein as SEQ ID NO: 4) may be used as a nucleic acid sequence encoding a protein associated with sperm formation or development.

[0016] The above-mentioned germ cell formation or development-related proteins encompass mutants containing amino acid sequences with one or more mutations, as long as they achieve the objectives of the present invention. Such mutants may contain, for example, 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 5, or 4, 3, 2, or 1 amino acid mutations compared to the wild-type amino acid sequence, and preferably conservative mutations. As used herein, "amino acid mutation" refers to the substitution, deletion, addition, or insertion of one or more amino acids. Furthermore, the mutants may contain, for example, an amino acid sequence that has at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to the wild-type amino acid sequence. For example, the protein associated with oocyte formation or development may comprise an amino acid sequence containing 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 5, or 4, 3, 2, or 1 amino acid mutations in the amino acid sequence shown in SEQ ID NO: 1, 2, or 3, or an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 1, 2, or 3. For example, the protein associated with sperm formation or development may comprise an amino acid sequence containing 1 to 50, 1 to 30, 1 to 20, 1 to 10, 1 to 5, or 4, 3, 2, or 1 amino acid mutations in the amino acid sequence shown in SEQ ID NO: 4, or an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 4. Furthermore, the nucleic acid sequence encoding the protein associated with the formation or development of germ cells includes mutant sequences containing one or more base mutations, as long as the object of the present invention is achieved.Such mutant sequences may contain, for example, 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, or 1 to 10 base mutations compared to the wild-type nucleic acid sequence, and preferably may contain silent mutations. As used herein, "base mutations" encompass substitutions, deletions, additions, or insertions of one or more bases. Furthermore, the mutant sequences may be nucleic acid sequences that have, for example, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to the wild-type sequence. For example, the nucleic acid sequence encoding the protein associated with the formation or development of an egg may be a nucleic acid sequence that includes 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, or 1 to 10 base mutations in the nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 1, 2, or 3, or a nucleic acid sequence that has at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO: 1, 2, or 3. For example, the nucleic acid sequence encoding the protein associated with sperm formation or development may be a nucleic acid sequence that contains 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, or 1 to 10 base mutations in the nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO: 4, or a nucleic acid sequence that has at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence encoding the amino acid sequence set forth in SEQ ID NO: 4. Here, sequence identity may be determined by conventional methods.

[0017] Infertility includes congenital infertility and acquired infertility. Congenital infertility refers to infertility symptoms caused by genetic mutations (e.g., gene defects) present in the subject. Acquired infertility refers to infertility that develops later in life due to various reasons. Causes of acquired infertility include, for example, environmental stress, exposure to chemicals, the effects of administering anticancer drugs, and radiation therapy. An example of acquired infertility is iatrogenic infertility caused by anticancer drug therapy, radiation therapy, etc. Examples of anticancer drugs include, but are not limited to, busulfan, cyclophosphamide, doxorubicin, cisplatin, etc.

[0018] The present invention can be applied to both congenital infertility and acquired infertility. Furthermore, acquired infertility can also be prevented by the present invention. For the treatment of congenital infertility, the "protein associated with germ cell formation or development" can be, for example, a protein lacking function in infertility, or a downstream protein in its signal transduction pathway. Even in cases where the causative gene of infertility is unknown or where infertility is not caused by a gene defect, such as acquired infertility, a nucleic acid encoding a protein associated with germ cell formation or development can be introduced into a subject's reproductive tissue (e.g., ovary or testis) to improve germ cell formation or development, thereby preventing or treating infertility.

[0019] In the present invention, a single-stranded negative-strand RNA virus is used as a vector. RNA viruses have RNA as their genomic nucleic acid, and single-stranded negative-strand RNA viruses express viral proteins by synthesizing complementary RNA in host cells using viral RNA polymerase. Examples of single-stranded negative-strand RNA viruses include viruses belonging to the Paramyxoviridae, Orthomyxoviridae, and Rhabdoviridae families, including, but not limited to, Sendai virus, influenza virus, Newcastle disease virus, vesicular stomatitis virus, and measles virus, with Sendai virus being preferred. In the present invention, the use of an RNA virus vector eliminates the risk of viral vector integration into host genomic DNA, as in conventional technologies using adenovirus or adeno-associated virus. Furthermore, since the vector genome, consisting of single-stranded negative-strand RNA, remains in the cytoplasm of the host cell, it does not affect the host genome and is free of genotoxicity risks.

[0020] Sendai virus (SeV) is a murine parainfluenza type 1 virus belonging to the Paramyxoviridae family. SeV is characterized by a single-stranded RNA genome consisting of a linear, non-segmented, negative-strand RNA of approximately 5.4 kb. In the present invention, SeV has the following advantages: (1) it eliminates the risk of integration into genomic DNA as described above; (2) the infection efficiency of SeV is independent of the cell cycle of target cells; (3) SeV is not pathogenic to humans; (4) SeV infects a wide range of host cells by utilizing sialic acid-binding glycoproteins and glycolipids as binding receptors; and (5) paramyxoviruses, including SeV, exhibit low genetic recombination or instability, and no homologous or heterologous recombination has been reported for SeV to date.

[0021] On the other hand, SeV has several drawbacks. One is that it induces a strong immune response. Innate immunity to SeV is very strong. Previous studies have reported that SeV-infected cultured cells were rapidly rejected not only in wild-type mice but also in nude mice. Another drawback is that although the HN (hemagglutinin-neuraminidase) protein of SeV is essential for cell fusion, it also causes red blood cell agglutination, thereby limiting its intravenous administration. Hemagglutination occurs when the HN protein binds to sialic acid on the surface of red blood cells, while both the F protein and HN protein induce hemolysis. Another drawback is that the cargo size of SeV is similar to that of adeno-associated viruses and much smaller than that of lentiviruses and adenoviruses, limiting the length of nucleic acids it can carry and therefore the range of target diseases. Another drawback is that SeV-mediated gene transfer does not allow transgenes to be expressed in vivo for long periods of time.

[0022] However, some of the above-mentioned disadvantages of SeV have been overcome in the present invention. Specifically, because SeV is injected directly into reproductive tissues, the risk of hemagglutination-induced damage is much lower than with injection into blood vessels. Furthermore, because SeV infection is relatively localized via binding to sialic acid, the problem of unwanted systemic vector delivery to other tissues, which is a common problem with gene therapy vectors, is avoided. Furthermore, although the high immunogenicity of SeV was previously thought to hinder its clinical application, Examples 2 and 4 of this application, which will be described later, demonstrate that despite SeV being immunogenic and exceeding the blood-follicle barrier (BFB), administration of Kitl-expressing SeV restored fertility in congenitally infertile mice. Furthermore, as evidenced by the absence of inflammation due to SeV injection in Example 1 and the rescue and maintenance of long-term oogenesis by SeV-Kitl microinjection in Example 4, the present invention demonstrates that the gene introduced by SeV is expressed in the ovaries for a very long time.

[0023] Generally, stable and persistent gene expression via RNA virus infection in immunocompetent animals is difficult or impossible due to the strong immune response. Most RNA virus vectors are considered unsuitable for stable and persistent gene expression due to the strong cytotoxicity caused by the antiviral response in host cells. However, in the present invention, as demonstrated in the study of gene expression related to innate immunity (Example 1) described below, no obvious inflammatory response was observed against SeV infection despite the upregulation of Rig1.

[0024] The RNA viral vector can be prepared by known methods, or a commercially available RNA viral vector can be used. For example, highly safe Sendai viral vectors, such as F gene-deleted Sendai viral vectors, have been developed and are commercially available, such as non-transmissible and temperature-sensitive Sendai viral vectors. Insertion of the nucleic acid sequence encoding the protein associated with germ cell formation or development into the RNA viral vector can be performed by standard methods. For example, a fragment containing the nucleic acid sequence encoding the protein associated with germ cell formation or development can be chemically synthesized by standard methods and inserted into the desired position of the RNA viral vector. For example, when using a Sendai viral vector, the number of bases in the nucleic acid fragment to be inserted is adjusted to a multiple of six. Preferably, to enhance gene expression, the nucleic acid fragment of interest can be inserted near the 3' end of the minus-strand genome of the Sendai viral vector.

[0025] By administering the thus obtained RNA viral vector carrying a nucleic acid sequence encoding a protein associated with the formation or development of germ cells to a subject, infertility in the subject can be prevented or treated. Therefore, in a first aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating infertility, which comprises a single-stranded negative-strand RNA viral vector containing a nucleic acid sequence encoding a protein associated with the formation or development of germ cells.

[0026] In another embodiment of the present invention, an RNA molecule comprising a nucleic acid sequence encoding a protein associated with the formation or development of germ cells may be administered to a subject directly or without using a viral vector. Accordingly, in a second embodiment of the present invention, there is provided a pharmaceutical composition for preventing or treating infertility, comprising an RNA molecule encoding a protein associated with the formation or development of germ cells. In this specification, the pharmaceutical compositions of the first and second embodiments of the present invention are collectively referred to as the "pharmaceutical composition of the present invention."

[0027] The RNA molecule containing a nucleic acid sequence encoding a protein associated with germ cell formation or development is preferably mRNA. The RNA molecule may be chemically modified, for example, to improve in vivo stability or cellular uptake efficiency. Various RNA modifications are known in the art. For example, the modified RNA may contain a 5' cap structure, one or more modified nucleotides, and / or a 3' polyA tail. The length of the 3' polyA tail may be varied, for example, to improve translation efficiency. Modified nucleotides include modifications of the sugar moiety, the base moiety, and the phosphate moiety (internucleoside linkage), as well as combinations thereof. Modifications of the sugar moiety include, but are not limited to, modifications at the 2-position of the furanose ring, such as 2'-O-methoxyethyl, 2'-O-methyl, and 2'-fluoro modifications; cross-linked modifications, such as 4'-LNA (Locked Nucleic Acid) modifications; and substitution with a morpholino ring. Modifications of the base moiety include, but are not limited to, 5'-methyl modifications and modified nucleosides such as pseudouridine. Modifications of the phosphate moiety include, but are not limited to, phosphorothioate modifications. Such RNA modifications are well known in the art.

[0028] The RNA molecule may be administered as a naked RNA molecule or encapsulated in a non-viral particle. Various non-viral particles are known in the art, including, but not limited to, lipid nanoparticles, polymeric micelles, etc. Circular RNA or self-replicating mRNA may also be used as the RNA molecule.

[0029] The pharmaceutical composition of the present invention is administered to germ cells or germ tissues, or somatic cells associated with germ tissues, of a target animal. Examples of germ tissues include the ovaries and testes. The administration method may be, for example, injection, with microinjection (also known as microinjection) being preferred. The dosage is not particularly limited and can be appropriately determined by those skilled in the art depending on the subject, frequency of administration, route of administration, and other factors. The pharmaceutical composition of the present invention can also be administered in combination (simultaneous administration, sequential administration, etc.) with other therapeutic agents, such as small molecule compounds.

[0030] The subject of the present invention is mammal, preferably human.In this specification, the treatment of infertility includes the improvement, alleviation and reduction of infertility symptoms compared with the subject who is not administered the pharmaceutical composition of the present invention.In addition, in this specification, the prevention of infertility includes the absence of infertility symptoms, and the suppression and reduction of the onset of infertility symptoms compared with the subject who is not administered the pharmaceutical composition of the present invention.

[0031] Most importantly, subjects administered the pharmaceutical composition of the present invention can restore fertility through natural mating. Furthermore, offspring born through natural mating from subjects whose fertility has been restored by administration of the pharmaceutical composition of the present invention exhibit normal genomic imprinting patterns and fertility.

[0032] Currently, IVF and ICSI are widely used techniques for treating human infertility. However, these techniques require mature gametes, limiting the number of patients who can be treated. Furthermore, recent findings with ICSI in mice have shown abnormalities in the subsequent generations (Non-Patent Document 1). Because humans have a long reproductive cycle, it is currently impossible to evaluate the clinical implications of ICSI. In contrast, the successful restoration of infertility through spontaneous reproduction from mice with primordial follicles in this study indicates that the technique of the present invention can also be applied to ovarian defects with immature oocytes.

[0033] The pharmaceutical composition of the present invention can also prevent or treat iatrogenic infertility (acquired infertility) induced by cancer treatments such as anticancer drug administration and radiation exposure. This is a very useful effect for protecting fertility against cancer treatment. The fertility protection provided by the pharmaceutical composition of the present invention offers new possibilities for in vivo protection of oocytes. The damage to oocytes and patient health caused by the method of the present invention is thought to be relatively mild compared to conventional methods such as oocyte cryopreservation and ovarian transplantation.

[0034] In the Examples described below, we have developed a congenitally infertile Kitl mouse that completely lacks Kitl expression in the ovary. Sl-t / Kitl Sl-t Mice were microinjected with Kitl-SeV. KIT ligand (Kitl) is expressed in granulosa cells, whereas its tyrosine kinase receptor KIT is expressed in oocytes and theca cells. The complete absence of Kitl expression in the ovary led to the absence of Kitl. Sl-t / Kitl Sl-tThese mice have only primordial follicles and no secondary follicles in the mature ovaries. Therefore, these mice can be used as a model of human premature ovarian failure, which is estimated to affect 0.3-1.1% of women of reproductive age. The same viral vector also rescued chemotherapy-induced iatrogenic infertility in wild-type mice. Therefore, SeV may be applicable to fertility protection against cancer therapy. Successful offspring production from such an infertile mouse model represents a new possibility in infertility treatment.

[0035] In the Examples described later, congenitally infertile Cldn11 knockout mice and Kitl Sl-t / Kitl Sl-t Mice were microinjected with Cldn11 mRNA and Kitl mRNA, respectively, without the use of a vector. Several issues were anticipated with mRNA delivery. One important issue was the lack of sustained mRNA expression (transient expression). However, as shown in Examples 11 and 12, offspring were successfully produced from mRNA-transfected mice, demonstrating that transient mRNA expression was sufficient to induce haploid cells. Furthermore, surprisingly, Example 10 demonstrated that Cldn11 mRNA delivery induced sustained spermatogenesis. Another issue with mRNA delivery is activation of innate immunity. However, as shown in Example 9, inflammation resulting from mRNA injection was relatively mild in wild-type and busulfan-treated testes, even when chemically unmodified mRNA was used. [Example]

[0036] The present invention will be explained in more detail below using examples, but the present invention is not limited to these examples.

[0037] material and method (1) Animals and injection methods In Examples 1-6 and 12, female mice were used because the disease modeled was associated only with females. Unless otherwise noted, 4- to 5-week-old B6 mice were used. In some experiments, 4- to 8-week-old crossbred B6, B6.Cg-Kitl mice on a DBA / 2 background were used. Sl-t / Rbrc mice were used (RIKEN BRC, Ibaraki, Japan). All animal experiments were approved by the Institutional Animal Care and Use Committee of Kyoto University. Animals were maintained under a constant light-dark cycle with unlimited access to water and food. SeV was obtained from ID Pharma (Egfp-SeV / TSdeltaF and mKitl-SeV / TSdeltaF; Tokyo, Japan).

[0038] Ovarian injections were performed as previously described (Non-Patent Document 3). Briefly, bilateral flank incisions were made approximately 2 mm behind the last rib. A glass needle was inserted into the subalbuminea of ​​the ovary by gently clamping the fat pads around the oviduct and ootheca. Approximately 2 μl of FITC-dextran (50 mg / ml; Sigma, St. Louis, MO) or viral particles were microinjected into the ovarian stroma. For dextran injections, ovaries were collected 30 minutes after injection. For offspring production, males were added at least 2 weeks after microinjection.

[0039] In Examples 7-11, 4- to 8-week-old B6 mice and Cldn11 KO mice on a B6 background were used. Where indicated, animals (wild-type mice) were intraperitoneally injected with busulfan (44 mg / ml; Sigma, St. Louis, MO) to eliminate endogenous germ cells. These animals were used at least 35 days after busulfan injection (when all germ cells had disappeared). Microinjection into the testis was performed via the efferent duct. The fat pad surrounding the epididymis was gently grasped, and a glass needle was inserted into the efferent duct. All animal experiments were approved by the Institutional Animal Care and Use Committee of Kyoto University.

[0040] (2) DNA integration analysis To evaluate SeV DNA integration, tail DNA from the offspring was collected and subjected to PCR analysis using Egfp-specific primers (Table 1).

[0041] (3) Immunostaining Ovarian or testicular samples were fixed in 4% paraformaldehyde for 2 hours at 4°C and embedded in Tissue-Tek OCT compound (Sakura Finetek, Tokyo, Japan) to obtain cryosections. To block nonspecific antibody binding, sections were treated with 3% bovine serum albumin (BSA) and 10% goat serum in phosphate-buffered saline (PBS) supplemented with 0.1% Tween 20 (PBST) for 1 hour at room temperature. The sections were then incubated with the indicated primary antibodies overnight and secondary antibodies for 1 hour with 0.5% BSA in PBST. The sections were washed with PBST. The antibodies used in the analysis are listed in Tables 1 and 2. Hoechst 33342 (Sigma) was used for counterstaining. The total area of ​​EGFP, GATA4, and Hoechst 33342 expression was measured using cellSense v2.3 (Olympus, Tokyo, Japan).

[0042] (4) Real-time PCR analysis Total RNA was isolated using TRIzol (Invitrogen, Carlsbad, CA). First-strand cDNA was generated using the Verso cDNA Synthesis Kit for RT-PCR (Thermo Fisher Scientific, Waltham, MA). For real-time PCR, StepOnePlus cDNA was used. TMA real-time PCR system (Applied Biosystems, Warrington, UK) and Power SYBR Green PCR Master Mix (Applied Biosystems) were used according to the manufacturer's protocol. Transcript levels were normalized to those of Hprt. PCR conditions were as follows: 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. Each PCR was performed at least three times. The primers used are listed in Table 3.

[0043] (5) Western blotting Samples were separated by SDS-PAGE, transferred to Hybond-P membranes (Amersham Biosciences, Buckinghamshire, UK), and incubated with primary antibodies. The antibodies used in these experiments are listed in Table 2.

[0044] (6) COBRA Genomic DNA was treated with sodium bisulfite, which deaminates unmethylated cytosine to convert it to uracil, but does not affect 5-methylated cytosine. The DNA was used as a template to amplify DMRs using specific primers. The PCR products were digested with the indicated restriction enzymes that recognize CpG-containing DNA sequences in the original unconverted DNA. The primers used are listed in Table 3.

[0045] (7) Bisulfite sequencing Bisulfite sequencing was performed as previously described (Non-Patent Document 1). Bisulfite-treated DNA was amplified by PCR using a specific primer set. DNA methylation analysis was performed using the Quantification Tool for Methylation Analysis (QUMA) (http: / / quma.cdb.riken.jp / top / quma_main_j.html). The primers used are listed in Table 3.

[0046] (8)mRNA preparation Luciferase and Egfp-encoding mRNA were purchased from Trilink (San Diego, CA, USA). Cldn11-encoding mRNA was prepared using the mMESSAGE mMACHINE T7 Ultra Kit (Ambion, Carlsbad, CA) and then purified with the RNeasy Mini Kit (Quiagen, Hilden, Germany). A block copolymer consisting of 12 kDa polyethylene glycol (PEG) and poly[N'-[N-(2-aminoethyl)-2-aminoethyl]asparamide] (hereafter referred to as PAsp(DET)) was synthesized by aminolysis of PEG-poly(β-benzyl-L-aspartate). PEG-PLys was prepared by deprotection of PEG-PLys(TFA), which was synthesized by ring-opening polymerization of Lys(TFA)-NCA using PEG-NH2 as the initiator. The degree of polymerization of PEG-PAsp(DET) and PEG-PLys was 1 The nucleotide sequences were determined to be 64 and 61 using H-NMR. Poly(IC) (1 mg / ml) was purchased from InvivoGen (Hong Kong, China). Poly(I:C) was purchased from InvivoGen (Hong Kong, China). The mRNA was suspended in 10 mM HEPES for microinjection.

[0047] (9) Luciferase assay Frozen tissues were lysed in Passive Lysis Buffer (E1941, Promega, Madison, WI) and luciferase substrate (E151A, Luciferase Assay System, Promega) was added for luminescence measurement using a Lumat3 LB9508 luminometer (Berthold technologies, Bad Wildbad, Germany). Luminescence values ​​were measured using a Micro BCA TM The total protein content was normalized using a Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA).

[0048] (10) TUNEL staining Apoptotic cells were detected using the In Situ Cell Death Detection Kit: TMR red (Roche Applied Science, Mannheim, Germany) according to the manufacturer's protocol. Cells were counterstained with Hoechst 33342 (Sigma).

[0049] (11) Intracytoplasmic sperm injection For cryopreserved samples, Cldn11 KO mouse testes injected with Cldn11 mRNA were frozen at -80°C for one month after microinjection. For fresh samples, Cldn11 KO mouse testes were harvested, refrigerated overnight, and used for ICSI the following day. Seminiferous tubules were dissociated using a fine stainless steel needle, and spermatogonia were released into PBS. Round spermatids were microinjected into BDF1 mouse oocytes using a piezo-driven micropipette (PrimeTech, Ibaraki, Japan). After 24 hours of culture, two-cell stage embryos were transferred into the oviducts of pseudopregnant ICR female mice. Offspring were born after cesarean section.

[0050] (12) Statistical analysis Significant differences between single comparisons were determined by Student's t-test. All data were analyzed using Microsoft Excel. All results are expressed as mean ± SEM. A p<0.05 value was considered statistically significant.

[0051] Table 1: Antibodies used in Examples 1 to 6 [Table 1]

[0052] Table 2: Antibodies used in Examples 7 to 11 [Table 2]

[0053] Table 3: PCR primers used in the examples [Table 3]

[0054] Example 1: Introduction of SeV into wild-type ovaries One problem with the use of SeV for infertility treatment is its high immunogenicity. To evaluate the effects of SeV introduction into the ovaries, SeV expressing Egfp (SeV-Egfp) was microinjected into the ovaries of wild-type C57BL / 6 (B6) mice. One week after microinjection, ovaries were collected and real-time polymerase chain reaction (PCR) was performed to identify changes in gene expression related to innate immunity. Real-time PCR analysis showed that SeV administration did not induce typical inflammation-related molecules, such as Il6, Tnfa, Ifna4, Ifnb1, Ifih1, and Tlr3 (Figure 1A). However, only Rig1, which recognizes 5' triphosphate double-stranded RNA, was significantly upregulated.

[0055] Because Rig1 upregulation suggested potential inflammation, we collected ovaries at various time points (days 2, 7, 14, 21, and 28) to examine the development of an inflammatory response. Histological analysis of the ovaries revealed no obvious inflammation or fibrosis in any of the collected samples (Figure 1B). Oogenesis continued normally in SeV-injected ovaries. To confirm the absence of inflammation, immunostaining was performed to evaluate inflammatory cell infiltration. Consistent with the histological analysis results, immunostaining with anti-CD4 or anti-CD8 antibodies did not reveal any obvious lymphocytic infiltration (Figure 1C). Although CD4 or CD8 lymphocytes were occasionally observed, their numbers remained unchanged throughout the experimental period and were comparable to those in control ovaries. In contrast, immunostaining of the spleen showed a strong positive signal. These findings suggested that the ovaries prevent obvious inflammatory responses despite SeV infection. Therefore, SeV can be used for infertility treatment.

[0056] Example 2: KitlSl-t / Kitl Sl-t Defective blood follicle barrier (BFB) in mice Although SeV induced a limited inflammatory response in the ovary, its size exceeds the molecular sieve of the BFB. The BFB restricts the diffusion of proteins larger than 20 nm. The diameter of SeV is 230 nm, far exceeding the size of the BFB and endocytic vesicles. Therefore, SeV may not be able to penetrate the BFB, which prevents foreign molecules from penetrating into the follicle. Double immunostaining using SeV-EGFP and ovarian cell markers was performed to confirm the infected cell type. EGFP signals were observed in HSD3B-expressing theca cells, but no clear signals were detected in cells within the follicle. Neither AMH-expressing granulosa cells nor MVH-expressing oocytes showed EGFP fluorescence. These results suggested that SeV cannot penetrate the BFB.

[0057] These findings indicated that the application of SeV is limited to intrafollicular cell-cell interactions. However, because several previous studies have suggested a defect in the blood-testis barrier in infertile male mice, we hypothesized that the BFB might be defective in infertile animals. To confirm this hypothesis, we used Kitl expression assays in mice that completely lack Kitl expression in the ovaries. Sl-t / Kitl Sl-t BFB was tested in mice.

[0058] KITL-KIT interaction is essential for oogenesis. KITL, expressed on granulosa cells, binds to the KIT tyrosine kinase receptor on oocytes, triggering a signaling cascade to support oocyte survival and differentiation. As a result, Kitl Sl-t / Kitl Sl-t The mice lack oogenesis but contain significant numbers of primordial follicles with a single layer of granulosa cells. These mice are congenitally infertile and serve as a model of premature menopause.

[0059] The inventors prepared fluorescent isothiocyanate (FITC)-dextran with a molecular size of 10K, which cannot pass through the BFB. FITC-dextran was purchased from Kit 1. Sl-t / Kitl Sl-t The ovaries were microinjected into wild-type and wild-type mice, and their ovaries were harvested 30 minutes after microinjection. Analysis of the ovaries revealed FITC signal only in the interstitial region of wild-type ovaries (Fig. 2A). However, the same solution was used in the Kit. Sl-t / Kitl Sl-t When microinjected into the ovary, FITC signals were found within the follicles (Fig. 2B). Sl-t / Kitl Sl-t We demonstrated that BFB is impaired in the ovary, suggesting that SeV can potentially transduce granulosa cells in patients with congenital infertility.

[0060] Example 3: Introduction of the Kitl gene into wild-type ovaries Although the results of Example 2 demonstrated the possibility that SeV can pass through the BFB, several potential problems may arise when Kitl is overexpressed in the ovary. For example, long-term cytokine overexpression may lead to tumor development. Because Kitl is important for organizing the theca cell layer around developing follicles, SeV expression could potentially interfere with oogenesis. To examine the effect of Kitl overexpression on fertility in wild-type mice, wild-type mice were injected with mouse Kitl-expressing SeV (SeV-Kitl). SeV-Egfp was used as a control. After virus injection, the mice were housed with wild-type males for 2 weeks and maintained for 3 months to evaluate the efficiency of offspring production. The amino acid sequence of mouse Kitl expressed was shown in SEQ ID NO: 5.

[0061] A total of 11 of 12 mice were fertile after SeV-Kitl injection, whereas 9 of 13 control mice produced offspring. Over a 3-month period, 14 births were observed among the 11 SeV-Kitl-transduced females, while a total of 13 births were observed among the 9 SeV-Egfp-transduced control females (Fig. 3A). In the SeV-Kitl-injected mice, the litter size (number of pups born per litter) ranged from 2 to 9 with an average of 6.7 pups, whereas in the SeV-Egfp-injected mice, the litter size ranged from 4 to 9 with an average of 6.5 pups. There was no clear difference in litter size between the two types of mice (Fig. 3B). However, regarding the rate of offspring production, one of the 12 SeV-Kitl-injected females produced offspring as early as 50 days after microinjection. In contrast, the first offspring from 13 SeV-Egfp mice were born 40 days later. The mean time to first offspring production was 67.1 days for SeV-Kitl and 59.4 days for SeV-Egfp (Fig. 3C). There was no statistically significant difference. Because widespread Kitl overexpression can trigger differentiation, Kitl induction could potentially lead to oocyte pool exhaustion and infertility. However, three of the 12 SeV-Kitl-injected mice were able to produce two litters within a 3-month period. Similarly, four of the 13 female mice injected with the control vector produced two litters. These differences were not statistically significant.

[0062] Three months after microinjection, all recipient mice were sacrificed and subjected to ovarian histological analysis. Ovarian size did not change significantly between the two samples. Histological analysis demonstrated normal-appearing oogenesis in all SeV-Kitl-injected mice (Figure 3D). Consistent with the fertility analysis, the number of secondary oocytes showed no obvious difference (Figure 3E). Immunostaining of ovaries showed no obvious change in the number of MKI67+ granulosa cells between SeV-Kitl and SeV-Egfp ovaries (Figure 3F, G). TdT-mediated dUTP nick end labeling (TUNEL) staining was also performed to check whether excessive Kitl expression could reduce apoptosis. However, no obvious difference was found between the two samples (Figure 3H, I). Although CD4 or CD8 lymphocytes were occasionally detected, their numbers did not change significantly (Figure 3J).

[0063] To check for the possibility of SeV infection of oocytes, genomic DNA was collected from the offspring and PCR was performed using SeV-Egfp-specific primers to examine the possibility of integration of the Egfp transgene into the offspring genome. However, no evidence of integration into the offspring genome was observed. These results indicated that SeV-Kitl expression enabled complete oogenesis without the risk of transgene integration into the germline.

[0064] Example 4: Kitl Sl-t / Kitl Sl-t Induction of oogenesis in mice To test whether SeV-Kitl can restore congenital infertility, sexually mature Kitl Sl-t / Kitl Sl-t SeV-Kitl was microinjected into mice. Both ovaries received SeV-Kitl injection. The ovaries were collected the day after injection, and reverse transcription PCR (RT-PCR) was performed to confirm Kitl expression. The results showed a 36.7-fold increase in Kitl expression (Figure 4A, B).

[0065] To evaluate the effects of Kitl overexpression, mice were sacrificed 1, 3, and 12 weeks after SeV injection. The size of the ovaries increased after viral injection (Figure 4C). Histological analysis of the ovaries demonstrated follicular development in SeV-Kitl microinjected mice (Figure 4D). The remaining animals were then mated and allowed to conceive naturally. Sl-t / Kitl Sl-t Males are fertile, so Kitl Sl-t / Kitl Sl-t Kitl mutant females Sl-t / Kitl Sl-t She was housed with a male.

[0066] In three separate experiments, five of 22 mice (22.7%) gave birth to offspring as quickly as 50 days after SeV injection (Fig. 4E). The first birth occurred an average of 68.1 days after virus injection. One female gave birth twice, but no offspring were born 99 days later. A total of 13 offspring were born, consisting of four males and nine females. The average litter size was 2.2 per litter. No obvious morphological abnormalities were observed in these offspring, which grew normally and had a white coat color. This is consistent with Kitl Sl-t / Kitl Sl-t Consistent with this, the mutant mice lack melanocytes, a property that depends on KITL-KIT signaling.

[0067] Next, hormone levels were examined after SeV-Kitl injection. Blood samples were collected at various time points, and enzyme-linked immunosorbent assays (ELISAs) were performed to measure the levels of FSH, LH, and estrogen. However, no significant changes were observed in any of the hormones. In normal females, ovulation of mature follicles is typically induced by an LH secretion surge, so ovulation and pregnancy occurred in the mice in this example despite the apparently abnormal hormone levels (no changes in hormone levels at all).

[0068] Six months after SeV microinjection, all Kitl Sl-t / Kitl Sl-t The mouse was killed. Sl-t / Kitl Sl-tThe size (weight) of the ovaries was smaller than that of wild-type mice, but not of Kitl mice that had not received the virus injection. Sl-t / Kitl Sl-t The ovaries were larger than those of mice injected with SeV-Kitl. Histological sections of the ovaries from SeV-Kitl-injected mice showed a normal appearance with various levels of oogenesis, but no follicles appeared in ovaries not inoculated with virus (Fig. 4D). There was a significant increase in the number of follicles with a diameter of over 100 μm (Fig. 4F). These results indicate that SeV-Kitl microinjection rescues and maintains long-term oogenesis.

[0069] Example 5: Reversal of infertility in busulfan-treated mice Busulfan, a chemotherapy agent widely used in cancer treatment, can impair fertility. In a previous study (J. Funct. Foods 2022; 10.1016 / j.jff.2022.104995), administration of busulfan to wild-type B6 mice resulted in significant loss of ovarian follicles. Because KITL provides survival signals, we hypothesized that overexpression of KITL in busulfan-treated mice might protect fertility by enhancing survival signals.

[0070] Ten mice were microinjected with SeV-Kitl or SeV-Egfp and then subjected to busulfan treatment. The fertility of the injected mice was then assessed by mating the female mice with wild-type males for 3 months. While none of the control mice receiving SeV-Egfp produced offspring, 70% of the mice receiving SeV-Kitl became fertile (Fig. 5A, B) and gave birth once within 3 months. The number of pups ranged from 1 to 4, with an average of 2.6 pups (Fig. 5C). Offspring were born as early as 48 days after busulfan injection (Fig. 5D).

[0071] These mice were sacrificed 3 months after busulfan injection and subjected to histological analysis. The size of the ovaries receiving SeV-Kitl was significantly larger than that receiving SeV-Egfp (Fig. 5E, F). Histological analysis also showed that the ovaries receiving SeV-Kitl contained many developing follicles (Fig. 5G). The number of antral follicles was significantly increased in the ovaries receiving SeV-Kitl (Fig. 5H). Numerous corpora lutea were also observed in these ovaries, reflecting successful ovulation. These results indicate that SeV is useful for protecting fertility against cancer therapy.

[0072] Example 6: Analysis of genomic imprinting patterns in offspring Because oocytes undergo gynogenetic genomic imprinting during oogenesis, it was possible that ectopic expression of SeV-Kitl might induce abnormal genomic imprinting patterns. To investigate this possibility, we collected tail DNA from these offspring and performed bisulfite restriction analysis.

[0073] Analysis of the differentially methylated regions (DMRs) of the H19 and Igf2r imprinted genes demonstrated somatic DNA methylation patterns (Figure 6A, B). Furthermore, normal genomic imprinting patterns were confirmed by additional bisulfite sequencing of the DMRs of the H19 and Igf2r genes (Figure 6C, D). These results demonstrated the maintenance of normal genomic imprinting patterns in the offspring.

[0074] Example 7: Microinjection of mRNA into seminiferous tubules To investigate the utility of mRNA therapy in male infertility treatment, we first explored mRNA delivery methods to achieve efficient protein expression in Sertoli cells. We first used naked mRNA. Naked mRNA is known to efficiently deliver mRNA across tissues with minimal tissue damage in several other tissues. Furthermore, we investigated the effect of a delivery vehicle using polymeric micelles (PMs) as a platform. PMs are prepared by mixing mRNA with poly(ethylene glycol)-polycation block copolymers in an aqueous solution and feature a structure in which concentrated mRNA is surrounded by a polyethylene glycol (PEG) layer. This structure reduces nuclease degradation of mRNA and mitigates the innate immune response to mRNA by inhibiting Toll-like receptor (TLR) recognition of mRNA. In this example, two types of polycations were used in the block copolymer: poly(L-lysine), a standard natural cationic peptide, and poly[N'-N-(2-aminoethyl)-2-aminoethyl[asparamide] (hereinafter abbreviated as [PAsp(DET)]), a degradable polycation with endosomal escape ability.

[0075] Using the three delivery systems described above, luciferase mRNA samples were injected into the testes of busulfan-treated mice. Busulfan is known to specifically kill undifferentiated spermatogonia. Therefore, 35 days after busulfan administration (corresponding to one cycle of spermatogenesis), the mice lost all germ cells and retained only Sertoli cells in the seminiferous tubules. An mRNA-containing solution was introduced into the adluminal compartment of the seminiferous tubules by microinjection. Luciferase expression levels were measured one day after microinjection. Although the luciferase expression level in the PM group tended to be higher than that in the naked mRNA group, there was no significant difference among the three groups (Figure 7A). Therefore, in subsequent experiments, we used naked mRNA, a simpler formulation that does not use cationic materials, for testicular delivery.

[0076] Next, luciferase mRNA was administered by microinjection, and expression levels were examined 4 hours, 1 day, and 2 days later. In this experiment, we investigated the effects of naked mRNA delivery into germ cells using both wild-type and busulfan-treated mice. In wild-type testes, the luciferase assay showed the strongest signal 4 hours after mRNA injection, which significantly decreased 2 days after mRNA injection (Figure 7B). On the other hand, busulfan-treated testes showed a different pattern (Figure 7C). The signal was relatively weak 4 hours after mRNA injection but peaked 1 day later. The signal intensity gradually decreased, but was still detectable 4 days later. More importantly, the signal was significantly stronger than that observed in wild-type testes. These results indicate that busulfan-treated testes, which lack germ cells, are more likely to incorporate mRNA than wild-type testes. This suggests that Sertoli cells are preferred targets for mRNA delivery, rather than germ cells. Since the number of germ cells is low even in actual infertile patients, busulfan-treated mice are thought to better reflect the infertility situation than wild-type mice.

[0077] Example 8: Efficient mRNA delivery to Sertoli cells by naked mRNA microinjection To confirm the kinetics of mRNA delivery and the target cell type, EGFP mRNA was administered by microinjection into the seminiferous tubules of wild-type and busulfan-treated mice. Consistent with the results of the luciferase mRNA injection study (Example 7), EGFP signals were generally weaker in wild-type testes, with no apparent signal visible 4 hours after microinjection. One day after microinjection, a weak but clear signal was observed. A weak signal was also observed 4 days later, but the area of ​​the seminiferous tubule that showed EGFP signal was limited. To identify the target cell type, immunostaining for EGFP was performed. Unexpectedly, the EGFP signal did not colocalize with any germ cell markers, including GFRA1 (a marker for undifferentiated spermatogonia), KIT (a marker for differentiating spermatogonia), SYCP3 (a marker for spermatocytes), and PNA (a marker for haploid cells). The EGFP signal was also localized with GATA4. + Although occasional positive cells were observed in Sertoli cells, peaking 1-2 days after mRNA injection (Figure 8-1), these cells were scattered over the seminiferous tubules, with less than two positive cells in cross sections.

[0078] On the other hand, macroscopic analysis of busulfan-treated testes clearly demonstrated more efficient transduction of EGFP. + The signal was not clear 4 hours after microinjection, but strong signals were observed 1 and 2 days later. However, the signal was weaker 4 days later. Direct comparison with wild-type testes revealed that the EGFP signal was more intense in the Sertoli cells of busulfan-treated mice (Figure 8-2). Transduced GATA4 +Quantification of Sertoli cells showed a significant increase in the number of transduced Sertoli cells 2 days after injection (Figure 8-3). EGFP signals were observed primarily in the cytoplasm of Sertoli cells, but occasionally in the nuclei. They also overlapped with CLDN11 signals, providing additional evidence of Sertoli cell transduction. Collectively, these results raise the possibility that mRNA delivery to Sertoli cells is more efficient in infertile mice with reduced spermatogonia numbers.

[0079] Example 9: Limited immunological response after mRNA delivery In general, the introduction of mRNA into cells induces a strong immune response by activating innate immunity. However, because the testis is considered an immune-privileged organ, mRNA delivery may not cause severe side effects. To examine the extent of the immune response, real-time polymerase chain reaction (PCR) was performed to monitor gene changes related to innate immunity. In this example, we used a synthetic double-stranded RNA, polyinosinic-polycytidylic acid [poly(I:C)], which activates Toll-like response 3 (TLR3). Wild-type testes and busulfan-treated testes were compared. Previous studies have suggested that TLR3 activation induces innate immunity in germ cells, but none of the four genes (Il6, Tnfa, Infa4, and Infb1) showed significant changes in wild-type testes (Figure 9A). In contrast, busulfan-treated testes showed significant increases in gene expression levels of Il6, Tnfa, and Infb1 (Figure 9B). These results suggested activation of the innate immune response in Sertoli cells.

[0080] Next, we examined lymphocyte infiltration after microinjection of poly(I:C). Immunostaining of wild-type testes revealed no significant changes in the number of CD4- or CD8-positive lymphocytes (Figures 9C and 9D). Similarly, immunostaining of busulfan-treated testes also revealed no significant increases in the number of CD4- or CD8-positive lymphocytes, despite increased expression of several innate immune genes (Figures 9C and 9D). In contrast, many CD4- and CD8-positive lymphocytes were observed in control staining of the spleen.

[0081] Although inflammatory cell infiltration did not occur in either testis, a significant increase in apoptotic Sertoli cells was observed in wild-type testes, as indicated by an increased number of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive Sertoli cells (Figure 9E). Because such an increase was not observed in busulfan-treated testes, the increased Sertoli cell apoptosis in wild-type testes was likely indirectly induced by the presence of germ cells. However, given that the extent of apoptosis was limited (~1%) in both testes, it appears that Sertoli cells are relatively resistant to potential damage caused by naked mRNA delivery.

[0082] Example 10: Rescue of spermatogenesis in Cldn11 knockout (KO) mice Since the results of Examples 7-9 suggested successful protein expression by naked mRNA delivery, we next attempted to restore fertility in congenitally infertile Cldn11 KO mice. CLDN11 is one of the claudin proteins that form the BTB and is essential for male fertility in mice. CLDN11 is also involved in human infertility and various human testicular disorders, although it is unknown whether impaired spermatogenesis is the cause or consequence of BTB structural changes. In mice, the BTB is thought to create an optimal microenvironment for meiosis and also protect haploid germ cells from systemic immunity. Consequently, Cldn11 KO mice lacking the BTB were congenitally infertile and completely lacked secondary spermatocytes and spermatids (Figure 10-1).

[0083] To investigate the possibility that mRNA delivery might provide a solution to spermatogenesis arrest in Cldn11 KO mice, we microinjected naked Cldn11 mRNA into the seminiferous tubules. Mouse Cldn11 mRNA was synthesized by chemical synthesis based on the sequence information (NCBI Reference Sequence: NM_008770.3) (SEQ ID NO: 6). Real-time PCR analysis of testes showed that Cldn11 mRNA levels were significantly increased 2 days after microinjection (Figure 10-2). However, compared with wild-type testes, the expression level was only ∼0.4%. This result indicated that CLDN11 expression was limited, although CD4-positive, but not CD8-positive, lymphocyte infiltration was observed after Cldn11 mRNA injection (Figure 10-3). In contrast, such an increase was not observed in control testes administered with Egfp mRNA. Because EGFP expression was restricted to the cytoplasm, the above immune response generated after Cldn11 mRNA expression indicated that Cldn11 mRNA was translated into protein and exposed on the cell membrane.

[0084] These results suggested that CLDN11 expression was successful. However, because Egfp mRNA expression only lasted for the first four days, it was expected that spermatogenesis might decline or disappear after a few days. Surprisingly, however, mouse studies one month after microinjection showed signs of spermatogenesis restoration. Although elongated spermatids were observed in only one of three testes analyzed, all samples showed progression of spermatogenesis with multiple layers of germ cells (Figure 10-4). Control mice injected with Egfp mRNA showed no obvious changes. Despite signs of haploid cell appearance, immunostaining of injected mice with lymphocyte markers revealed clear inflammation at this time point. Therefore, it was suggested that the BTB is not important for immune privilege. As predicted by the limited recovery of spermatogenesis in the testes, no sperm were observed in the epididymis of Cldn11 KO mice, despite increased spermatogenesis (Figure 10-5).

[0085] Example 11: Generation of offspring from Cldn11 KO mice Although the results of Example 10 indicated that naked Cldn11 mRNA injection initiated spermatogenesis, it was unclear whether the Cldn11 KO mice completed spermatogenesis and produced mature sperm. Nevertheless, due to the presence of elongated spermatids, we attempted to produce offspring by ICSI in this example. One testis was collected one month after injection and freezing, while three testis were collected two months after microinjection, refrigerated overnight, and used for ICSI. Of the four testes, a cell suspension from one refrigerated testis contained round spermatids. This suspension was then microinjected into oocytes from C57BL / 6 × DBA / 2F1 (BDF1) mice using a micromanipulator. A total of 18 embryos were produced. The following day, these embryos were transferred into the uterus of pseudopregnant mother mice. Three offspring, one male and two females, were born by cesarean section (Figure 11A). All of these animals developed into adults, and PCR analysis of tail DNA confirmed that they were heterozygous for the Cldn11 KO allele (Fig. 11B).

[0086] Male and female F1 mice were mated to confirm their fertility through natural mating. As expected, the testes of homozygous Cldn11 KO F2 offspring were significantly smaller (Figure 11C), and histological analysis confirmed reduced spermatogenesis (Figure 11D). Combined bisulfite analysis (COBRA) of tail DNA from F1 mice revealed a normal somatic genomic imprinting pattern. In contrast, germline stem cells (GS) (cultured spermatogonial stem cells) showed a typical androgenic genomic imprinting pattern, encompassing differentially methylated regions (DMRs) with hypermethylation of H19 and hypomethylation of Igf2r (Figure 11E). The normal DNA methylation pattern was further confirmed by bisulfite sequencing (Figure 11F). These results demonstrate that mRNA delivery can rescue congenital male infertility in Cldn11 KO mice.

[0087] Example 12: Kitl Sl-t / Kitl Sl-t Producing offspring from female mice Kitl is expressed in the granulosa cells that surround the oocyte. Kitl-deficient mice are infertile because they only have primordial follicles in the ovaries. Sl-t / Kitl Sl-t Kitl mRNA was administered to the ovaries of female mice three times every other day. Mouse Kitl mRNA was synthesized based on the sequence information (NCBI Reference Sequence: NM_013598.2) (SEQ ID NO: 7). After mating with wild-type male mice, two offspring were born.

Claims

1. A pharmaceutical composition for preventing or treating infertility, comprising a single-stranded negative-strand RNA viral vector containing a nucleic acid sequence encoding a protein associated with the formation or development of germ cells, or an RNA molecule encoding said protein.

2. The pharmaceutical composition according to claim 1, wherein the infertility is acquired infertility.

3. The pharmaceutical composition according to claim 2, wherein the acquired infertility is infertility caused by administration of an anticancer drug.

4. The pharmaceutical composition according to claim 1, wherein the infertility is congenital infertility.

5. The pharmaceutical composition of claim 2 , wherein the protein is a defective protein.

6. The pharmaceutical composition of claim 1 , wherein the protein is a protein associated with the formation or development of eggs.

7. The pharmaceutical composition of claim 6, wherein the protein is Kitl.

8. The pharmaceutical composition of claim 1 , wherein the protein is a protein associated with sperm formation or development.

9. The pharmaceutical composition of claim 8, wherein the protein is Cldn11.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the RNA viral vector is a Sendai virus vector.

11. The pharmaceutical composition according to any one of claims 1 to 9, wherein the RNA molecule is mRNA.

12. A single-stranded negative-strand RNA viral vector comprising a nucleic acid sequence encoding a protein associated with the formation or development of germ cells.

13. The vector of claim 12, which is a Sendai virus vector.

14. The vector of claim 12 or 13, wherein the protein is a protein associated with the formation or development of an egg.

15. The vector of claim 14 , wherein the protein is Kitl.

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