WO2020262635A1

WO2020262635A1 - Method for introducing polynucleotide into follicular cell, and composition for said method

Info

Publication number: WO2020262635A1
Application number: PCT/JP2020/025302
Authority: WO
Inventors: 隆司篠原; 哲史渡邉; 美都篠原
Original assignee: 国立大学法人京都大学
Priority date: 2019-06-27
Filing date: 2020-06-26
Publication date: 2020-12-30
Also published as: JP2022123154A

Abstract

Provided are: a method for introducing a polynucleotide into an ovarian follicle cell, said method including administering an adeno-associated virus vector including the polynucleotide to a subject; and a composition, etc., including an adeno-associated virus vector including the polynucleotide, for introducing the polynucleotide into an ovarian follicle cell.

Description

Methods for introducing polynucleotides into follicle cells and compositions for them

This application claims priority with respect to Japanese Patent Application No. 2019-12173, which is incorporated herein by reference in its entirety.
The present disclosure relates to methods and compositions for introducing polynucleotides into follicular cells.

The ovary contains follicles composed of an egg and the body cells surrounding it. Follicles are primordial follicles, primary follicles, pre-follicle follicles, follicular follicles, mature follicles due to the action of local factors in the ovary and follicle-stimulating hormone (FSH) and luteinizing hormone (LH), which are gonadotropins secreted from the ovary. It develops in stages with (Graf follicles), causing the release of ova from the ovaries, or ovulation. The somatic cells that make up the follicle include granulosa cells and pod cells, which express FSH and LH receptors, respectively, and produce estrogen and androgen. Folliculogenesis is gonadotropin-independent from primordial follicles to follicular follicles and is regulated by TGF-β family proteins, estrogens, androgens, insulin, IGF-1, and the like. After antral follicles with a diameter of more than 2 mm, follicle development becomes gonadotropin-dependent. In addition, various factors such as activin, activin, follistatin, IGF, BMP, GDF, and IL-6 have been reported to be involved in follicle development and ovulation.

Ovarian dysfunction causes infertility. Drugs such as FSH preparations have been used for many years to treat ovarian dysfunction or infertility, but their effects are currently limited.

It is an object of the present disclosure to provide a method for introducing a polynucleotide into a follicle cell and a composition for that purpose.

In some embodiments, the present disclosure provides a composition comprising an adeno-associated virus vector containing said polynucleotide for introducing a polynucleotide into follicular cells.

In a further aspect, the present disclosure provides a method of introducing a polynucleotide into follicular cells, comprising administering to a subject an adeno-associated virus vector containing the polynucleotide.

In a further aspect, the present disclosure is a method of making a non-human vertebrate in which a polynucleotide has been introduced into a follicular cell, comprising administering to the non-human vertebrate an adeno-associated virus vector containing the polynucleotide. Provide a method.

With this disclosure, it has become possible to easily introduce a polynucleotide into a follicle cell. The adeno-associated virus vector is a safe and easy-to-use vector that has already been used in gene therapy, and is expected to have wide applications. As an example, the introduction of a polynucleotide into a follicular cell can treat a disease or condition, such as ovarian dysfunction or the resulting disease or condition and cancer.

FIG. 1 shows the ovaries of C57BL / 6 (B6) × DBA / 2 F1 (BDF1) mice 7 days after microinjection of mCherry-expressing AAV (AAV1, AAV9, AAVDJ, AAVDJ8, AAV6, AAV6.2). FIG. 2 shows the results of immunostaining of tissue sections of ovaries with anti-mCherry antibody in mCherry-expressing AAV9-administered mice and negative control mice not administered with a vector. Solid arrows indicate granulosa cells, and dashed arrows indicate capsular cells. The scale bar is 50 μm. FIG. 3 shows a tissue section of the ovary of R26R-Eyfp mice 7 days after microinjection of Cre-expressing AAV9. Figure 4 shows a ^{^{Kitl Sl-t / Kitl Sl-}} t mice uterus after microinjection 2 months KITL expression AAV9 (Uterus) and progeny (Offspring). Figure 5 shows the mice born from ^{^{Kitl Sl-t / Kitl Sl-}} t mice after microinjection 62 days KITL expression AAV9.

Unless otherwise specified, the terms used herein have the meanings commonly understood by those skilled in the art in the fields of organic chemistry, medicine, pharmacy, molecular biology, microbiology and the like. Definitions of some terms used herein are provided below, but these definitions supersede the general understanding herein.

In this disclosure, when a numerical value is accompanied by the term "about", it is intended to include a range of ± 10% of the value. For example, "about 20" is assumed to include "18-22". The range of numbers includes all numbers between the endpoints and the numbers at the endpoints. The "about" for a range applies to both ends of the range. Therefore, for example, "about 20 to 30" includes "18 to 33".

The ovary has the functions of egg development and ovulation and secretion of the reproductive hormones estrogen and progesterone. The ovary consists of the cortex and medulla, in which there are follicles at various stages of development between the connective tissue, the stroma. A follicle is composed of an egg and the body cells that surround it. In the process of developing a follicle from a primordial follicle to a mature follicle, a granulosa cell layer is first formed around the egg cell, then a basement membrane is formed around the follicle, and then a capsule cell layer is formed.

In the present disclosure, adeno-associated virus (AAV) vectors introduce polynucleotides into follicular cells. That is, the AAV vector contains a polynucleotide that is introduced into the cells of the follicle. In certain embodiments, the follicular cells are somatic cells. Examples of follicular somatic cells include follicular cells, granulosa cells, luteal cells, follicular epithelial cells, and vascular endothelial cells. In a preferred embodiment, the follicular somatic cells are capsular cells or granulosa cells.

AAV is a non-enveloped virus that has approximately 4.7 kb of single-stranded DNA in its genome. The wild-type AAV genome has a Cap gene encoding a capsid protein and a Rep gene encoding a protein having helicase activity required for AAV replication between two terminally inverted repeat sequences (ITRs). The genome of the AAV vector is usually replaced with a polynucleotide whose genomic sequence between ITRs should be introduced into the cell.

AAV has a directivity towards a specific tissue or cell depending on the serotype. The directivity of AAV is determined by the capsid protein on the surface of the virus. The AAV vector in the present disclosure may have a natural capsid protein or an artificially modified capsid protein. The AAV vector of the present disclosure may be any AAV vector as long as it has directivity toward the cells of the follicle. The AAV vector may have the ability to cross the basement membrane of the follicle in addition to directing the follicle to the cell. The ability of the follicle to cross the basement membrane is advantageous when introducing polynucleotides into cells located inside the basement membrane. For example, if the AAV vector has such ability, the polynucleotide can be introduced into the cells existing inside the follicle after the formation of the basement membrane, and the polynucleotide can be introduced into the cell regardless of the developmental stage of the follicle. can do. A suitable AAV vector can be selected by examining whether or not it infects the cells of interest when administered to the ovary according to the description of Examples.

In certain embodiments, the AAV vector is AAV1, AAV9, AAVDJ, AAVDJ8, AAV6, or AAV6.2, or a variant thereof having a follicular cell orientation. In a further embodiment, the AAV vector is AAV1, AAV9, AAVDJ, AAVDJ8, AAV6, or AAV6.2, or a variant thereof having a follicle cell orientation and the ability to cross the follicle basement membrane. ..

Methods for obtaining variants of AAV are known in the art. For example, a desired mutant can be obtained by modifying the capsid protein and confirming the properties of AAV thereof according to the description of Examples. A library of AAV in which the capsid protein is mutated may be prepared and screened by DNA shuffling, error prone PCR, or the like.

The polynucleotide contained in the AAV vector is not particularly limited, and examples thereof include polynucleotides encoding proteins or peptides, and polynucleotides encoding nucleic acid molecules such as antisense nucleic acids, siRNA, miRNA, stRNA, ribozyme, and decoy nucleic acids. ..

In certain embodiments, the polynucleotide encodes a factor involved in follicle development. Factors involved in follicle development include follicle-stimulating hormone (FSH), luteinizing hormone (LH), TGF-β family proteins, estrogen, androgens, insulin, IGF-1, inhibin, activin, follistatin, IGF, BMP, Examples include, but are not limited to, GDF, IL-6 or molecules involved in signal transduction thereof (eg, receptors, transcription factors, etc.). In certain embodiments, the polynucleotide encodes FSH, LH, estrogen, or androgen. In a further embodiment, the polynucleotide encodes FSH.

The polynucleotide may be a polynucleotide encoding a protein or nucleic acid molecule for genome editing by CRISPR / Cas9, TALEN, ZFN or the like. Normally, AAV vectors are not inserted into the cell's genome, but can be combined with genome editing techniques to modify the cell's genome. Protein and nucleic acid molecules for genome editing by CRISPR / Cas9 include Cas9 protein, which is an endonuclease, guide RNA (gRNA) that mobilizes Cas9 protein to the target sequence, and donor vector introduced into the double-stranded cleavage site of the genome. Etc. are exemplified. The donor vector may include, for example, a polynucleotide encoding a factor involved in follicle development as described herein.

The polynucleotide may be a polynucleotide encoding a fluorescent protein such as GFP, eGFP, BFP, YFP, EYFP, CFP, RFP, dsRed, and mCherry.

The polynucleotide may contain regulatory elements such as promoters and enhancers. The promoter is not particularly limited as long as it can regulate the expression of the polynucleotide in the cell. Examples of the promoter include CAG promoter, SRα promoter, EF1α promoter, CMV promoter, PGK promoter, U6 promoter, tRNA promoter and the like, and can be appropriately selected depending on the intended purpose.

The size of the polynucleotide contained in the AAV vector is not particularly limited, but is usually up to about 4.7 kbp. One AAV vector may contain two or more polynucleotides, or a combination of two or more AAV vectors may be administered.

The AAV vector may be prepared by any method known in the art. For example, in packaging cells such as HEK293 cells or AAV-293 cells which are variants thereof, 1) AAV vector plasmid having AVR ITR at both ends and the target polynucleotide inserted between them, 2) replication of AAV. And by transfecting an AAV helper plasmid with Rep and Cap genes required for particle formation, and 3) an adenovirus helper plasmid with an adenovirus helper gene required for AAV proliferation. be able to. The production of such an AAV vector can also be performed using a commercially available kit such as AAV Helper-Free System (Agilent Technologies).

The AAV vector can be administered together with a reagent for increasing its introduction efficiency. Examples of such reagents include proteasome inhibitors such as neurominidase and MG132, Eeyarestatin I, trithymidine, cisplatin, etoposide, calpain inhibitors, ubiquitin ligase inhibitors and the like.

AAV vectors can be used to introduce polynucleotides into follicular cells in vitro or in vivo. In certain embodiments, the AAV vector is administered to the subject for introduction of the polynucleotide into the cells of the follicle in vivo. In another embodiment, the AAV vector is added to cultured cells or tissues for the introduction of polynucleotides into follicular cells in vitro.

The method of administration of the AAV vector may be systemic administration or local administration. The administration method includes, but is not limited to, administration to the ovary and intravenous administration. In certain embodiments, the AAV vector is administered to the ovaries. The AAV vector may be administered to any site of the ovary. For example, AAV vectors can be administered to the ovarian cortex.

In the present disclosure, follicular cells are vertebrate cells, and when the AAV vector is administered to a subject, the subject is a vertebrate. Vertebrates include, for example, mammals, birds, fish, amphibians and reptiles. Mammals include, but are not limited to, for example, mice, rats, hamsters, guinea pigs, rabbits, pigs, cows, goats, horses, sheep, minks, dogs, cats, monkeys, red-tailed monkeys, marmosets, orangutans, chimpanzees, humans, etc. Can be mentioned. Examples of birds include chickens, quails, ducks, geese, turkeys, ostriches, emu, ostriches, guinea fowls, and pigeons. In a preferred embodiment, the vertebrate is a mammal. In a further embodiment, the mammal is a rodent, rabbit, or primate animal (eg, mouse, rat, hamster, guinea pig, rabbit, monkey, red-tailed monkey, marmoset, orangutan, chimpanzee, or human). In some embodiments, the mammal is a human. In another embodiment, the vertebrate is a non-human vertebrate, preferably a non-human mammal.

Diseases or symptoms can be treated by introducing polynucleotides into follicular cells. Diseases or symptoms include ovarian dysfunction or the resulting diseases or symptoms and cancer. Ovarian dysfunction includes impaired egg development or ovulation and impaired reproductive hormone secretion. Diseases or symptoms resulting from or due to ovarian dysfunction include infertility.

In certain embodiments, the disease or condition is due to a genetic abnormality in the capsule or granulosa cells. In another embodiment, the disease or condition results from a genetic abnormality in a factor involved in follicle development. The genetic abnormality can be, for example, the genetic abnormality described in Persani L et al., J. Mol. Endocrinol. 45, 257-279, 2010. The polynucleotide introduced can be, for example, a polypeptide encoding a protein, peptide, or nucleic acid molecule that supplements or suppresses the function of a protein that has been reduced or deleted by such genetic abnormality.

As used herein, treatment of a disease or symptom includes therapeutic treatment and prophylactic treatment. That is, the subjects to which the AAV vector is administered include those who have a disease or symptom and those who are at risk of developing the disease or symptom but have not yet developed it. For example, subjects to whom the AAV vector is administered are those who have ovarian dysfunction or a disease or symptom caused by it, and those who are at risk of developing ovarian dysfunction or a disease or symptom caused by it (eg, granulosa cells). Also included are subjects who have a genetic abnormality in granulosa cells or a genetic abnormality in a factor involved in follicle development) but have not yet developed ovarian dysfunction or the resulting disease or condition.

The composition containing the AAV vector may contain a pharmaceutically acceptable carrier and / or additive in addition to the active ingredient AAV vector. Pharmaceutically acceptable carriers include saline or other physiologically acceptable buffers. Examples of the additive include a dissolution aid, a pH adjuster, a preservative, a stabilizer and the like. The dosage form is not limited, but is, for example, an injection, and examples thereof include a liquid injection and a solid injection (for example, a lyophilized injection) that is dissolved and used before use. The composition containing the AAV vector may be provided as a kit, and the kit may further include a buffer solution used for dissolution at the time of use, instructions for use, and the like.

The AAV vector can also be used to produce non-human vertebrates in which polynucleotides have been introduced into follicular cells. By introducing a polynucleotide into the cells of a follicle (and optionally genetic modification), for example, in a model animal of a disease or symptom, eg, a model animal of a disease or symptom resulting from ovarian dysfunction or cancer. A model animal can be created.

The dose of AAV vector is appropriately changed depending on the subject. For example, from about ^¹⁰ 9 ^~ ¹⁰ 15 vg per body weight 1 kg (vector genome), preferably may be administered at ^{¹⁰ 10} ^~ ¹⁰ 14 vg or ^{¹⁰ 10} ^~ ¹⁰ 13 vg,.

An exemplary embodiment of the present disclosure is described below.

[1]
A composition comprising an adeno-associated virus vector containing the polynucleotide for introducing a polynucleotide into a follicle cell.
[2]
The composition according to 1 above, wherein the follicle cells are somatic cells.
[3]
2. The composition according to 2 above, wherein the somatic cell is a capsule cell or a granulosa cell.
[4]
The composition according to any one of 1 to 3 above, which is used for treating a disease or a symptom.
[5]
A composition comprising an adeno-associated virus vector containing a polynucleotide to be introduced into follicular cells for treating a disease or condition.
[6]
4. The composition according to 4 or 5, wherein the disease or symptom is ovarian dysfunction or a disease or symptom caused by the ovarian dysfunction.
[7]
The composition according to any one of 4 to 6 above, wherein the disease or symptom is infertility.
[8]
The composition according to any one of 4 to 7, wherein the disease or symptom is caused by a genetic abnormality in a capsule cell or a granulosa cell.
[9]
The composition according to any one of 4 to 8 above, wherein the disease or symptom is caused by a genetic abnormality of a factor involved in follicle development.
[10]
The composition according to any one of 1 to 9, wherein the polynucleotide encodes a factor involved in follicle development.
[11]
The composition according to 10 above, wherein the factor involved in follicle development is FSH.
[12]
The composition according to any one of 1 to 11 above, wherein the adeno-associated virus vector is AAV1, AAV9, AAVDJ, AAVDJ8, AAV6, or AAV6.2.
[13]
The composition according to any one of 1 to 12, which is administered to the ovary.
[14]
The composition according to any one of 1 to 13 above, which is administered to a human.

[15]
A method for producing a non-human vertebrate in which a polynucleotide has been introduced into a follicle cell, which comprises administering an adeno-associated virus vector containing the polynucleotide to the non-human vertebrate.
[16]
15. The method of 15. The follicle cell is a somatic cell.
[17]
16. The method of 16 above, wherein the somatic cells are capsular cells or granulosa cells.
[18]
The method according to any of 15 to 17, wherein the adeno-associated virus vector is AAV1, AAV9, AAVDJ, AAVDJ8, AAV6, or AAV6.2.
[19]
The method according to any of 15 to 18, wherein the adeno-associated virus vector is administered to the ovary.
[20]
15. The method of any of 15-19, wherein the non-human vertebrate is a non-human mammal.
[21]
The method of 20 above, wherein the non-human mammal is a rodent, lagomorph, or primate animal.

[22]
A method for introducing a polynucleotide into a follicle cell, which comprises administering to a subject an adeno-associated virus vector containing the polynucleotide.
[23]
22. The method of 22 above, wherein the follicle cells are somatic cells.
[24]
23. The method of 23 above, wherein the somatic cell is a capsule cell or a granulosa cell.
[25]
22-24. The method of any of 22-24, wherein the adeno-associated virus vector is administered to treat a disease or condition.
[26]
A method of treating a disease or condition comprising administering to a subject an adeno-associated virus vector containing a polynucleotide to be introduced into follicular cells.
[27]
25 or 26, wherein the disease or symptom is ovarian dysfunction or a disease or symptom resulting from it.
[28]
25. The method of any of 25-27 above, wherein the disease or symptom is infertility.
[29]
25. The method of any of 25-28, wherein the disease or symptom is due to a genetic abnormality in the capsule or granulosa cells.
[30]
25. The method of any of 25-29, wherein the disease or symptom is due to a genetic abnormality in a factor involved in follicle development.
[31]
22-30. The method of any of 22-30, wherein the polynucleotide encodes a factor involved in follicle development.
[32]
31. The method of 31 above, wherein the factor involved in follicle development is FSH.
[33]
The method according to any of 22 to 32 above, wherein the adeno-associated virus vector is AAV1, AAV9, AAVDJ, AAVDJ8, AAV6, or AAV6.2.
[34]
The method according to any of 22 to 33 above, wherein the adeno-associated virus vector is administered to the ovary.
[35]
The method according to any of 22 to 34 above, wherein the subject is a human.

[36]
An adeno-associated virus vector containing said polynucleotide for use in introducing a polynucleotide into a follicle cell.
[37]
Adeno-associated virus vector containing polynucleotides to be introduced into follicular cells for use in the treatment of diseases or conditions.

[38]
Use of an adeno-associated virus vector containing said polynucleotide to introduce a polynucleotide into follicular cells.
[39]
Use of an adeno-associated virus vector containing a polynucleotide to be introduced into follicular cells to treat a disease or condition.

[40]
Use of an adeno-associated virus vector containing said polynucleotide for the manufacture of a medicament for introducing a polynucleotide into a follicle cell.
[41]
Use of an adeno-associated virus vector containing a polynucleotide to be introduced into follicular cells for the manufacture of a drug for treating a disease or condition.

Example 1
1. 1. METHODS: Virus production AAV was produced as previously reported (Watanabe et al., 2017). To explain, AAV vector plasmids (pAAV-CAG-mCherry, pAAV-CAG-Kitl, or pAAV-CAG-Cre), adenovirus helper plasmids (pHelper; Agilent Technologies, Santa Clara, CA), and AAV helper plasmids [pAAV1, PAAV9, pAAV6, pAAV6.2; donated by Penn Vector Core (University of Pennsylvania, PA); pAAV-DJ, pAAV-DJ8; Cell biolabs, San Diego, CA] were transiently transfected into AAV-293 cells. .. Whistle titers were determined by real-time PCR with FastStart Universal SYBR Green Master Mix (Roche Diagnostic GmbH, Penzberg, Germany) and specific primers. The virus titer was 1.0 × 10 ¹³ vg / mL.

Animals and Transplantation In vivo screening and tracer experiments used 4-5 week old C57BL / 6 (B6) x DBA / 2 F1 (BDF1) mice. In some experiments, 4-8 week old R26R-Eyfp mice [donated by Dr. F. Costantini (Columbia University Medical Center, NY)] (Srinivas et al., 2001) were used. In the fertility recovery experiment, 6 weeks old B6. ^{Cg-Kitl <Sl-t>} / Rbrc (Kitl Sl-t / Kitl Sl-t) mouse (Kuroda et al., 1988) was used (RIKEN BRC, Ibaraki, Japan) a.

Microinjection of virus particles Two ventral incisions 2 mm caudal from the last rib were incised for injection into the ovary. The ovaries were pulled out with fine forceps, being careful not to damage the blood vessels and ovarian capsule. Gently supporting the fat pad around the fallopian tubes and sac, the pipette was advanced by a micromanipulator, and a glass needle was inserted under the ovarian tunica albuginea. Filling was confirmed by adding a small amount of trypan blue. Approximately 2 μl of virus particles were microinjected into the ovarian stroma.

Ovary analysis Mice were killed 7-10 days after microinjection, ovaries were fixed with 4% paraformaldehyde on ice for 30 minutes and embedded in Tissue-Tek OCT Compound for frozen section preparation. In the analysis of R26R-Eyfp mice, the ovaries were analyzed under UV light. Immunostaining of frozen sections was performed as previously reported (Watanabe et al., 2018). An anti-mCherry antibody (Thermo Fisher Scientific, M11217) was used as the primary antibody. Alexa fluor 568 anti-rat IgG (Thermo Fisher Scientific, A11077) was used for the detection of the primary antibody. All sections were stained with Hoechst 33342.

2. 2. Results Screening for AAV serotypes by in vivo microinjection First, mCherry-expressing AAV was microinjected to test for infection of ovarian cells. Six types of AAV (AAV1, AAV9, AAVDJ, AAVDJ8, AAV6, AAV6.2) with different capsids were prepared. All of these express mCherry under the tissue non-specific CAG promoter. Using a glass needle, virus particles were microinjected under the ovarian tunica albuginea. The ovaries were collected 7 days after microinjection and analyzed under UV light. All micro-injected ovaries showed mCherry signals (Fig. 1). Of the AAVs tested, AAV9 showed the strongest signal. Based on this observation, AAV9 was used in the following experiments.

Immunostaining of the ovaries of AAV9-administered mice with an anti-mCherry antibody confirmed that mCherry was expressed in the granulosa cells and capsule cells of follicles (Fig. 2). No clear signal was seen in the oocytes. Microinjection did not appear to impair follicle development, as a similar number of follicles developed. These results indicate that AAV9 infected granulosa cells and capsular cells without affecting normal follicle development.

Next, infection with AAV9 was confirmed using R26R-Eyfp mice. By using R26R-Eyfp mice, more sensitive detection of infected cells becomes possible. In this mouse, the STOP sequence sandwiched between loxP and the subsequent Efyp gene are inserted at the Gt (ROSA) 26Sor locus. Cre expression AAV9 was used to monitor the expression of Efyfp. As expected, EYFP signals were detected in the ovaries recovered 7 days after microinjection. When tissue sections were prepared, EYFP + infected cells were found throughout the ovary, despite being injected into the surface area of the ovarian stroma (Fig. 3).

Restoration of fertility in a congenital infertility mouse model by AAV transduction To confirm the infection of AAV into granulosa cells, Kitl ^Sl-t / Kitl ^Sl-t mutant mice lacking the expression of functional KITL were used. Male ^{^{Kitl Sl-t / Kitl Sl-}} t mutant mice have fertility, female mutant mice, the lack of signaling between the granulosa cells expressing oocytes and KITL expressing KIT , Congenitally lacks fertility. The ovaries of Kitl ^Sl-t / Kitl ^Sl-t mutant mice contain only primordial follicles. Kitl-expressing AAV9 was microinjected into the ovaries of two 6-week ^- old Kitl ^Sl-t / Kitl ^Sl-t mutant female mice.

After microinjection of Kitl-expressing AAV9, these female mice were placed in the same cage as heterozygous male mice. When two female mice were killed and analyzed two months after the microinjection, a total of four foets were observed in the womb (Fig. 4). These foets were alive and in normal morphology, with no apparent abnormalities in placental size and morphology. This data indicates that Kitl-expressing AAV9 infected granulosa cells and the ovaries of mutant mice were able to produce fertile oocytes that developed into viable fetuses.

Example 2
In the same manner as in Example 1, produces KITL expression AAV9, was microinjected into ovarian stroma of five 6-week-old ^{^{Kitl Sl-t / Kitl Sl-}} t mutant female mice. After microinjection of Kitl-expressing AAV9, these female mice were placed in the same cage as heterozygous male mice. A total of 7 mice were born 62 days after microinjection (Fig. 5). This data indicates that Kitl-expressing AAV9 restored fertility in mutant mice and allowed normal fetal development.

From the above results, it was shown that the AAV vector can infect follicular cells and treat ovarian dysfunction or diseases or symptoms caused by it.

References
Kuroda, H., Terada, N., Nakayama, H., Matsumoto, K., and Kitamura, Y. (1988). Infertility due to growth arrest of ovarian follicles in Sl / Slt mice. Dev. Biol. 126, 71 -79.
Srinivas, S., Watanabe, T., Lin, CS, William, CM, Tanabe, Y., Jessell, TM, and Costantini, F. (2001). Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus. BMC. Dev. Biol. 1, 4.
Watanabe, S., Kanatsu-Shinohara, M., Ogonuki, N., Matoba, S., Ogura, A., and Shinohara, T. (2017). Adeno-associated virus-mediated delivery of genes to mouse spermatogonial stem cells . Biol. Reprod. 96, 221-231.
Watanabe, S., Kanatsu-Shinohara, M., Ogonuki, N., Matoba, S., Ogura, A., and Shinohara, T. (2018). In vivo genetic manipulate on of spermatogonial stem cells and their microenvironment by adeno- assciated viruses. Stem Cell Reports 10, 1551-1564.

Claims

A composition containing an adeno-associated virus vector containing the polynucleotide for introducing a polynucleotide into a follicle cell.
The composition according to claim 1, wherein the follicle cells are somatic cells.
The composition according to claim 2, wherein the somatic cell is a capsule cell or a granulosa cell.
The composition according to any one of claims 1 to 3, which is used for treating a disease or a symptom.
A composition comprising an adeno-associated virus vector containing a polynucleotide to be introduced into follicular cells for treating a disease or condition.
The composition according to claim 4 or 5, wherein the disease or symptom is ovarian dysfunction or a disease or symptom caused by the ovarian dysfunction.
The composition according to any one of claims 4 to 6, wherein the disease or symptom is infertility.
The composition according to any one of claims 4 to 7, wherein the disease or symptom is caused by a genetic abnormality in a capsule cell or a granulosa cell.
The composition according to any one of claims 4 to 8, wherein the disease or symptom is caused by a genetic abnormality of a factor involved in follicle development.
The composition according to any one of claims 1 to 9, wherein the polynucleotide encodes a factor involved in follicle development.
The composition according to claim 10, wherein the factor involved in follicle development is FSH.
The composition according to any one of claims 1 to 11, wherein the adeno-associated virus vector is AAV1, AAV9, AAVDJ, AAVDJ8, AAV6, or AAV6.2.
The composition according to any one of claims 1 to 12, which is administered to the ovary.
The composition according to any one of claims 1 to 13, which is administered to humans.
A method for producing a non-human vertebrate in which a polynucleotide has been introduced into a follicle cell, which comprises administering an adeno-associated virus vector containing the polynucleotide to the non-human vertebrate.