Method for introducing nucleic acid into trophectoderm cell

Abstract

Disclosed are: a method for introducing a nucleic acid into a trophectoderm cell, the method comprising a step for introducing the nucleic acid into a blastocyst using an adeno-associated virus vector in vitro; a method for producing a blastocyst in which a nucleic acid has been introduced into a trophectoderm cell, the method comprising a step for introducing the nucleic acid into a blastocyst using an adeno-associated virus vector in vitro; a method for producing a genetically modified non-human animal, the method comprising (a) a step for introducing a nucleic acid into a blastocyst using an adeno-associated virus vector in vitro and (b) a step for transplanting the blastocyst obtained in the step (a) into a recipient; and a nucleic acid-introducing agent used for introducing a nucleic acid into a blastocyst, the nucleic acid-introducing agent comprising an adeno-associated virus vector containing the nucleic acid.

Description

Method for introducing nucleic acids into trophectoderm cells

[0001] The present invention relates to a method for introducing nucleic acid into trophectoderm cells, a method for producing blastocysts in which nucleic acid has been introduced into trophectoderm cells, a method for producing genetically modified non-human animals, and a nucleic acid transfer agent used for introducing nucleic acid into blastocysts.

[0002] The placenta, which serves as the interface between the mother and fetus, is an essential organ for healthy fetal development, performing functions such as nutrient and gas exchange, waste disposal, and hormone synthesis. Mouse studies have demonstrated that placental dysfunction due to genetic abnormalities often leads to fetal growth restriction and miscarriage. Therefore, analyzing placental gene function is important for healthy pregnancy, birth, and postnatal development. However, the causes of placental dysfunction remain unknown, and no clear treatments for these conditions exist. While numerous genes causing placental abnormalities have been identified, much remains unknown about placental formation and function. Therefore, analyzing gene function involved in placental formation is expected to contribute not only to elucidating placental formation and function but also to understanding pregnancy abnormalities and developing treatments for these conditions.

[0003] On the other hand, conventional methods for producing genetically modified animals (transgenic mice, knockout mice, knockin mice, etc.) involve introducing genes into fertilized eggs, which results in genetic modification of both the fetus and placenta, making site-specific analysis difficult.

[0004] Several methods for gene transfer into the placenta have been attempted. For example, one method involves performing an abdominal incision during pregnancy and directly injecting a viral vector into the placenta using a syringe needle, but the surgical procedure is very burdensome and gene transfer throughout the placenta is not expected. Another method involves infecting a blastocyst-stage embryo with a lentiviral vector, which specifically transfers genes to trophoblast cells, resulting in placenta-specific gene transfer (Non-Patent Document 1). Thus, Non-Patent Document 1 reports that by introducing a lentivirus into mouse blastocysts after removal of the zona pellucida, genes are incorporated specifically into trophectoderm cells, and that all placentas analyzed were gene-transfected, but no fetuses were gene-transfected.

[0005] However, the use of lentiviral vectors requires the removal of the zona pellucida surrounding the embryo, which can result in significant damage during embryo manipulation. Thus, lentiviral vectors have the problem of being unable to penetrate the zona pellucida. The zona pellucida is an extracellular matrix that covers the early preimplantation embryo to protect it from infection by viruses and other pathogens. Furthermore, because lentiviral vectors are randomly integrated into the host genome, they pose problems such as mutagenicity due to endogenous gene disruption. Furthermore, lentiviral vectors must be handled at biosafety level 2, which requires specialized laboratory facilities and equipment and places restrictions on the handling of laboratory animals.

[0006] Non-Patent Document 2 reports that adeno-associated virus (AAV) can transfer genes into fertilized eggs by crossing the zona pellucida.

[0007] Nature biotechnology, vol. 25, No. 2, 233-237(2007)Biochemical and Biophysical Research Communication, 526, 85-90(2020)

[0008] The present invention aims to provide a method for introducing nucleic acid into trophectoderm cells, which is simple to operate, causes minimal damage to the embryo, and enables nucleic acid introduction into the entire placenta, a method for producing blastocysts in which nucleic acid has been introduced into trophectoderm cells, a method for producing genetically modified non-human animals, and a nucleic acid introduction agent used to introduce nucleic acid into blastocysts.

[0009] Adeno-associated virus (AAV) vectors are characterized by their small particle size, ability to change serotype depending on infection tropism, and ability to induce transient gene expression without being incorporated into the genome. We investigated whether AAV could be used to induce transient gene expression in early embryos without removing the zona pellucida.

[0010] Therefore, the present inventors have conducted extensive research to achieve the above-mentioned object, and as a result have found that gene transfer into the trophectoderm of mouse blastocysts, i.e., gene transfer throughout the placenta, is possible by culturing mouse blastocysts in a medium containing an AAV vector without removing the zona pellucida. Thus, the present inventors have found that gene transfer into blastocysts without removing the zona pellucida is possible using AAV, and further found that gene transfer is placenta-specific using AAV1, and that gene transfer into both the placenta and fetus is possible using AAV6.

[0011] The present invention was completed based on these findings and through further research, and provides a method for introducing nucleic acid into trophectoderm cells, a method for producing blastocysts in which nucleic acid has been introduced into trophectoderm cells, a method for producing genetically modified non-human animals, and a nucleic acid transfer agent used to introduce nucleic acid into blastocysts.

[0012] Item 1. A method for introducing a nucleic acid into a trophectoderm cell ex vivo, comprising the step of introducing the nucleic acid into a blastocyst using an adeno-associated virus vector. Item 2. A method for producing a blastocyst in which the nucleic acid has been introduced into a trophectoderm cell ex vivo, comprising the step of introducing the nucleic acid into a blastocyst using an adeno-associated virus vector ex vivo. Item 3. A method for producing a genetically modified non-human animal, comprising the steps of: (a) introducing a nucleic acid into a blastocyst ex vivo using an adeno-associated virus vector; and (b) implanting the blastocyst obtained in step (a) into a recipient. Item 4. The method according to any one of Items 1 to 3, wherein the blastocyst is one from which the zona pellucida has not been removed. Item 5. The method according to Items 1, 2, or 4, wherein the blastocyst is derived from a mammal selected from the group consisting of mouse, rat, rabbit, dog, cat, horse, cow, pig, goat, sheep, and primate. Item 6. The method of any one of Items 3 and 4, wherein the blastocyst is derived from a mammal selected from mouse, rat, rabbit, dog, cat, horse, cow, pig, goat, sheep, and non-human primates. Item 7. The method of any one of Items 1 to 6, wherein the nucleic acid is introduced specifically into trophectoderm cells. Item 8. The method of any one of Items 1 to 6, wherein the nucleic acid is introduced into both trophectoderm cells and inner cell mass cells. Item 9. The method of any one of Items 1 to 8, wherein the adeno-associated virus vector is AAV1 or AAV6. Item 10. The method of any one of Items 1 to 9, wherein the nucleic acid encodes a protein involved in the formation and / or function of the placenta and / or RNA that controls the expression of the protein. Item 11. A nucleic acid transfer agent used for introducing a nucleic acid into a blastocyst, the agent comprising an adeno-associated virus vector containing the nucleic acid.

[0013] According to the present invention, 1) by using an adeno-associated virus vector, gene transfer to the entire placenta is possible because the burden on the mother and fetus is small as it is not injected directly into the placenta; 2) pre-treatment such as removal of the zona pellucida is not required, making the procedure simple and reducing damage to the embryo; 3) unlike lentivirus vectors, transient gene expression is possible without being incorporated into the genome; 4) gene transfer to the placenta is possible more safely from a biosafety perspective, eliminating the need for specialized laboratory facilities and equipment; and 5) it is possible to transfer genes specific to trophectoderm cells that will form the placenta in the future.

[0014] 1 is a diagram showing an outline of the experiment in Test Example 1. It is a photograph showing the results of fluorescence microscopy of blastocyst-stage embryos in Test Example 1 (top: No. Virus, middle: AAV1, bottom: AAV6) (left: tdTomato, center: EGFP, right: merge). It is an enlarged photograph showing the results of fluorescence microscopy of blastocyst-stage embryos in Test Example 1 (top: AAV1, bottom: AAV6) (left: tdTomato, center: EGFP, right: merge) (arrows: inner cell mass). It is a diagram showing an outline of the experiment in Test Example 2. It is a photograph showing the results of fluorescence microscopy of embryos in Test Example 2 (from top: No. Virus, AAV1, AAV6, AAV6) (left: bright field, center: tdTomato, right: EGFP). It is a diagram showing the results of an in vitro experiment in Test Example 4. (A) Schematic diagram of an in vitro rescue experiment using myr-AKT1-AAV1 against celecoxib-induced abnormalities in TE (trophectoderm) cell invasion. AT, acidic Tyrode's solution. (B) Graph showing the rate of embryo attachment to the endometrium after 24 hours. Mean ± SEM. (C) Graph showing the rate of embryo attachment to the endometrium after 48 hours. Mean ± SEM. (D) 3D images of typical implanted embryos in each treatment group. LE: luminal epithelium; Stroma: stroma. Dotted lines indicate the surface of the LE and the border between the LE and Stroma. (E) Quantitative data showing the volume of TE cell invasion. + represents the mean. *: P<0.05; **: P<0.01; ****: P<0.0001; ns: no significant difference. Scale bar: 50 μm. Each plot represents (B, C) the results for each recipient mouse, or (E) the values ​​for each embryo. Figures show the results of an in vivo experiment in Test Example 4. (F) Schematic diagram of an in vivo rescue experiment using myr-AKT1-AAV1 for embryo implantation failure caused by celecoxib. (G) Photograph of the uterus at 5.5 days post coitum (dpc) after intravenous injection of blue dye solution. The implantation site (IS) is indicated by an arrow. (H) Graph showing the number of implantations per mouse when 16 embryos were transferred into each pseudopregnant mouse. Mean ± SEM. (I) Graph showing IS weight. + indicates mean value. (J) Immunofluorescent staining image for CK8 in a tissue section. The arrow indicates an embryo. The dotted line indicates the perimeter of the invasive trophoblast.GE: glandular epithelium; Str: stroma; Tr: trophoblast; *: P<0.05; **: P<0.01; ****: P<0.0001; ns: not significant. Scale bar: 50 μm. Each plot represents the results per recipient mouse (H) or the values ​​per embryo (I).

[0015] Hereinafter, an embodiment of the present invention will be described.

[0016] The method for introducing the nucleic acid of the present invention into trophectoderm cells (hereinafter sometimes referred to as the "introduction method of the present invention") is characterized by including a step of introducing the nucleic acid into a blastocyst ex vivo using an adeno-associated virus vector.

[0017] Furthermore, the method for producing a blastocyst in which the nucleic acid of the present invention has been introduced into a trophectoderm cell (hereinafter sometimes referred to as the "blastocyst production method of the present invention") is characterized by including a step of introducing the nucleic acid into the blastocyst ex vivo using an adeno-associated virus vector.

[0018] Furthermore, the method for producing a genetically modified non-human animal of the present invention (hereinafter, sometimes referred to as the "method for producing a non-human animal of the present invention") is characterized by comprising: (a) a step of introducing nucleic acid into a blastocyst ex vivo using an adeno-associated virus vector; and (b) a step of transplanting the blastocyst obtained in step (a) into a recipient.

[0019] Hereinafter, the above-mentioned introduction method of the present invention, the blastocyst production method of the present invention, and the non-human animal production method of the present invention may be collectively referred to as the "method of the present invention."

[0020] The method of the present invention uses blastocysts. A "blastocyst" refers to an embryo that has completed the cleavage stage in early mammalian development. Mammalian eggs are yolk-free and cleave completely to form a mass of blastomeres, which at the 32-cell stage separate into the trophectoderm that surrounds the outside of the mass and the inner cell mass. The inner cell mass will eventually become the body of the fetus, and the trophectoderm will differentiate into the placenta.

[0021] Fertilized eggs can be produced by collecting eggs and sperm from any animal and fertilizing them using a method known to those skilled in the art. Blastocysts can be produced by culturing the obtained fertilized eggs in KSOM medium for 96 hours using a method known to those skilled in the art. In addition to this, methods commonly used by those skilled in the art (for example, "Manipulating the mouse embryo," a laboratory manual, 3 ｒｄ It is also possible to obtain blastocysts by direct removal from animals according to the method described in "The Methods of Immunoprecipitation" (published in the 1997 American Journal of Immunology, Vol. 1, No. 1, pp. 201-203, Cold Spring Harbor Laboratory Press).

[0022] In the method of the present invention, the use of blastocysts allows the nucleic acid to be introduced specifically into trophectoderm cells. Whether or not the nucleic acid has been introduced specifically into the trophectoderm of the blastocyst by the method of the present invention can be determined by methods known to those skilled in the art, such as PCR to amplify the introduced gene and detecting the expression of a reporter gene such as EGFP or lacZ by fluorescence or color development.

[0023] In the method of the present invention, it is desirable to use blastocysts from which the zona pellucida (chicken membrane) has not been removed. Embryos (early pre-implantation embryos) are surrounded by the zona pellucida, an extracellular matrix, to protect them from infection by viruses and the like. Since the method of the present invention does not require removal of the zona pellucida, the procedure is simplified and it is possible to improve the cell survival rate and development rate.

[0024] The animals from which blastocysts are derived are not particularly limited, and examples thereof include mice, rats, rabbits, dogs, cats, cows, horses, pigs, goats, primates (e.g., humans, monkeys, rhesus monkeys, cynomolgus monkeys, marmosets, orangutans, chimpanzees), etc. In the method for producing a non-human animal of the present invention, blastocysts derived from mammals other than humans are used.

[0025] Examples of nucleic acids to be introduced into blastocysts include DNA, RNA, RNA chimeric nucleic acids, DNA / RNA hybrids, etc. Furthermore, the nucleic acid may be either single-stranded or double-stranded.

[0026] Examples of nucleic acids to be introduced in the methods of the present invention include nucleic acids encoding proteins involved in the formation and / or function of the placenta, and nucleic acids encoding RNAs that regulate the expression of proteins involved in the formation and / or function of the placenta. As used herein, "regulating the expression of proteins involved in the formation and / or function of the placenta" includes enhancing or suppressing the transcription of genes encoding proteins involved in the formation and / or function of the placenta, or enhancing or suppressing the translation of such proteins.

[0027] Examples of "nucleic acids encoding proteins involved in the formation and / or function of the placenta" include Dlx3, Fgfr2, Fra1, Fzd5, Gab1, Gcm1, and Grb2. Examples of such proteins include hypomorph, Gja7, Hgf, Hsp84-1, Itgav, Junb, Lifr, Erk1, Erk2, Erk5, Mek1, Mekk3, p38alpha, p38beta, Met, Pdgfra, Pdgfb, Pparg, Rxra, Rxrb, Sos1, Vhlh, Wnt2, Ets2, Mash2, Egfr, Hsf1, Bmp5, Bmp7, Dnmt1, Itga4, Lhx1, Mrj, Tcf, Lef, Cdx2, Eomes, Fgf4, Esrrb, Hand1, Mdfi, Esx1, Arnt, Tcfeb, and Gjb2.

[0028] Examples of RNA that regulates the expression of proteins involved in the formation and / or function of the placenta include small interfering RNA (siRNA), microRNA (miRNA), double stranded RNA (dsRNA), small hairpin RNA (shRNA), antisense RNA, and RNA with ribozyme activity.

[0029] The nucleic acid introduced in the method of the present invention may be, for example, a nucleic acid encoding a protein and / or RNA for genome editing. Examples of proteins include DNA nucleases, such as Cas (particularly Cas9) and Cpf1. By introducing a complex of DNA nuclease and guide RNA (gRNA) into cells in this way, genome editing using the CRISPR / Cas system can be performed. DNA nuclease is an enzyme that binds to DNA in a gRNA-dependent manner and recognizes and cleaves double-stranded DNA formed by pairing with a portion of the gRNA. The gRNA is an RNA having a base sequence that can pair with a genomic DNA-like target sequence cleaved by the DNA nuclease, and crRNA and tracrRNA can be used as the gRNA. Furthermore, when knock-in is performed by genome editing, a nucleic acid to be knocked in can also be used.

[0030] The nucleic acid to be introduced into the blastocyst may contain a promoter, an enhancer, a terminator, a polyadenylation signal, etc. The promoter is not particularly limited as long as it can regulate the expression of the nucleic acid in the cell.

[0031] The size of the nucleic acid contained in the AAV vector is not particularly limited and is usually up to about 4.7 kbp. One AAV vector may contain two or more types of polynucleotides, or two or more types of AAV vectors may be used in combination.

[0032] The nucleic acid used in the present invention may be purified by a method commonly used by those skilled in the art.

[0033] Nucleic acids can be produced by conventional methods such as PCR, chemical synthesis, biochemical cleavage / reassociation, and the like.

[0034] The blastocysts into which nucleic acids encoding proteins involved in placental formation and / or function and nucleic acids encoding RNAs regulating the expression of proteins involved in placental formation and / or function are introduced are not particularly limited. Examples of blastocysts into which these nucleic acids are introduced include blastocysts of animals having abnormalities in proteins involved in placental formation and / or function or in the regulation of expression of such proteins. "Having an abnormality in a protein involved in placental formation and / or function" refers to a state in which the structure and function of the protein are abnormal. Furthermore, "having an abnormality in the regulation of expression of a protein involved in placental formation and / or function" refers to an abnormality in either or both of the processes of transcription of DNA encoding a protein involved in placental formation and / or function into RNA and translation of the RNA into protein. Furthermore, "having an abnormality in the regulation of protein expression" includes not only cases in which protein production is completely inhibited due to an abnormality in either or both of the processes of transcription and translation, but also cases in which the amount and timing of protein expression are different from normal, and cases in which a protein with an abnormality in part of the structure and function of a normal protein is produced.

[0035] In the method of the present invention, the nucleic acid of interest is introduced into the blastocyst using an adeno-associated virus vector containing the nucleic acid.

[0036] AAV is a non-enveloped virus with a genome of approximately 4.7 kb of single-stranded DNA. The wild-type AAV genome has, between two inverted terminal repeats (ITRs), a Cap gene encoding a capsid protein and a Rep gene encoding a protein with helicase activity necessary for AAV replication. In the genome of an AAV vector, the genomic sequence between the ITRs is usually replaced with a nucleic acid to be introduced into a cell.

[0037] AAV has tropism for specific tissues or cells depending on its serotype (serotype). The tropism of AAV is determined by the capsid protein on the viral surface. The AAV vector used in the present invention may have a natural capsid protein or an artificially modified capsid protein, as long as it has tropism for blastocysts. A suitable AAV vector can be selected by examining whether it infects the target trophectoderm cells and inner cell mass when added to blastocysts.

[0038] Examples of AAV vectors include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVDJ, AAVDJ / 8, and AAV-PHP.eB. Among these, AAV1, AAV6, AAV9, AAVDJ, and AAV-PHP.eB are preferred, and AAV1 and AAV6 are more preferred. Use of AAV1 enables nucleic acid transfer specific to trophectoderm cells that will form the placenta in the future, i.e., placenta-specific nucleic acid transfer. Use of AAV6 enables gene transfer into both trophectoderm cells and inner cell mass, i.e., nucleic acid transfer into both the placenta and fetus.

[0039] AAV mutants can be obtained by known methods. For example, desired mutants can be obtained by modifying the capsid protein and confirming the properties of the resulting AAV by the methods described in the Examples. A library of AAVs with mutated capsid proteins can also be prepared by DNA shuffling, error-prone PCR, or the like, and screened.

[0040] AAV vectors can be prepared using known methods, such as (1) a method using a plasmid, (2) a method using a baculovirus, (3) a method using herpes simplex virus, (4) a method using an adenovirus, and (5) a method using yeast. (1) In the method using a plasmid, packaging cells such as HEK293 cells or their modified AAV-293 cells are transfected with I) an AAV vector plasmid having AAV ITRs at both ends and having a nucleic acid of interest inserted therebetween, II) an AAV helper plasmid having Rep and Cap genes required for AAV replication and particle formation, and III) an adenovirus helper plasmid having adenovirus helper genes required for AAV propagation, thereby producing an AAV vector in the cells. Thereafter, the host cells are disrupted by freezing and thawing or the like, the virus is collected, and the virus fraction is then separated and purified by density gradient ultracentrifugation using cesium chloride, a column method, or the like, to prepare the desired AAV vector.

[0041] The use of adeno-associated virus vectors eliminates the need for pretreatment such as removal of the zona pellucida, simplifying the procedure and minimizing damage to the embryo. Furthermore, unlike lentiviral vectors, adeno-associated virus vectors are not incorporated into the genome and allow transient gene expression. Furthermore, compared to lentiviral vectors, gene transfer into the placenta is safer in terms of biosafety and does not require specialized laboratory facilities or equipment.

[0042] Blastocysts can be infected with an adeno-associated virus vector containing a nucleic acid by, for example, mixing a solution containing the adeno-associated virus vector with the blastocyst and leaving it for 4 to 5 hours. By this method, the nucleic acid of interest contained in the adeno-associated virus vector can be introduced into trophectoderm cells (and the inner cell mass).

[0043] The culture medium used when mixing the adeno-associated virus vector with the blastocyst may be a serum-containing medium or a serum-free medium. The amount of the adeno-associated virus vector added is not particularly limited and can be appropriately determined taking into account the number of cells, etc. The concentration of the adeno-associated virus vector added to the blastocyst is not particularly limited as long as it allows the introduction of the target nucleic acid into the cells.

[0044] After adding an adeno-associated virus vector containing a nucleic acid of interest to the blastocyst, the blastocyst is cultured. ２ The concentration and other factors can be set appropriately. Typically, the temperature is about 37°C, the relative humidity is about 95%, and CO ２ The concentration is approximately 5% by volume. The incubation time can also be set appropriately, but is usually in the range of 0.1 to 76 hours, preferably 0.2 to 24 hours, and more preferably 0.5 to 12 hours. If the incubation time is too short, the nucleic acid may not be sufficiently introduced into the cells, and if the incubation time is too long, the cells may become weak.

[0045] In the method for producing a non-human animal of the present invention, the blastocyst into which the nucleic acid has been introduced is finally transplanted into a recipient. The recipient is preferably the same animal as the animal from which the blastocyst was derived, or an animal of the same species. Transplantation of a blastocyst into a recipient can be routinely performed by those skilled in the art (Manipulating the mouse embryo, a laboratory manual, 3 ｒｄ edition, pp. 263-271, Cold Spring Harbor Laboratory Press.) Non-human animals produced by the method for producing a non-human animal of the present invention include, but are not limited to, mice, rats, rabbits, dogs, cats, horses, cows, pigs, goats, sheep, and non-human primates (e.g., monkeys, rhesus monkeys, cynomolgus monkeys, marmosets, orangutans, and chimpanzees).

[0046] The method for producing a non-human animal of the present invention can be used to rescue embryonic lethality in a non-human animal. In this case, the non-human animal is a non-human animal having an abnormality in a protein involved in placental formation and / or function or in the regulation of expression of the protein. A nucleic acid is then introduced into the blastocyst of a non-human animal having an abnormality in a protein involved in placental formation and / or function or in the regulation of expression of the protein using an adeno-associated virus vector. The nucleic acid to be introduced is, for example, a nucleic acid encoding a protein involved in placental formation and / or function, or a nucleic acid encoding an RNA that normalizes the expression of a protein involved in placental formation and / or function.

[0047] Furthermore, the method for producing a non-human animal of the present invention can also be used to screen for nucleic acids that rescue embryonic lethality. In this case, the blastocyst obtained in step (a) is transplanted into a recipient, and then offspring are born. The nucleic acid that rescues embryonic lethality compared to when no nucleic acid is introduced using an adeno-associated virus vector can be selected as the nucleic acid that rescues embryonic lethality. The nucleic acid selected here can be used to rescue a non-human animal that would otherwise experience embryonic lethality. Furthermore, these nucleic acids can be used as therapeutic or preventive agents for diseases or symptoms caused by placental insufficiency.

[0048] The nucleic acid transfer agent of the present invention (hereinafter sometimes referred to as "the agent of the present invention") is characterized in that it contains an adeno-associated virus vector containing a nucleic acid and is used to transfer the nucleic acid into a blastocyst.

[0049] The agent of the present invention can be used to carry out the method of the present invention, and the adeno-associated virus vector containing nucleic acid and the like are the same as those described above. When the agent of the present invention is provided as a research reagent, it may be a solution or dispersion containing various solvents, or it may be a solvent-free agent. It may also contain known additives as appropriate. When the agent of the present invention is provided as a pharmaceutical, it may contain known pharmaceutically acceptable additives or may not contain additives. It may also be an oral or parenteral agent. When the agent of the present invention is provided as a kit, it may contain, in addition to the adeno-associated virus vector, reagents used during nucleic acid transfer and the like.

[0050] According to the present invention, by using an adeno-associated virus vector, gene transfer to the entire placenta is possible without direct injection into the placenta, with minimal stress on the mother and fetus. Furthermore, because pretreatment such as removal of the zona pellucida is not required, the procedure is simple and causes minimal damage to the embryo. Furthermore, gene transfer specific to trophectoderm cells, which will eventually form the placenta, is possible.

[0051] As a result, the method of the present invention enables specific gene transfer into the placenta, facilitating basic research as a technique for analyzing gene function in implantation and the placenta. It is expected to be applied to gene therapy to prevent implantation failure and placental abnormalities in humans and livestock, as well as to prevent miscarriage and adverse effects on the fetus.

[0052] In this specification, the term "comprising" includes "consisting essentially of" and "consisting of."

[0053] Furthermore, the various characteristics (properties, structures, functions, etc.) described in each embodiment of the present invention may be combined in any way to specify the subject matter encompassed by the present invention, i.e., the present invention encompasses all subject matter consisting of any combination of the combinable characteristics described herein.

[0054] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0055] Production Example 1 An AAV plasmid containing a nucleic acid encoding Cre was introduced into HEK293T cells by the calcium phosphate method. The cells were cultured, and the cells into which the AAV vector was introduced were collected. Using the collected cells, the AAV vector was extracted and purified using a commercially available AAV vector purification kit (e.g., AAVpro Purification Kit, Takara Bio Inc.). The purified AAV vector extract was concentrated using an Amicon Ultra-4 (Merck) or the like. Finally, the titer was measured by real-time PCR or the like.

[0056] Test Example 1: Gene transfer into blastocyst stage embryos using AAV1 or AAV6 Blastocyst stage embryos of mice (mTmG mice) that normally constitutively express tdTomato, but in which the Flox region containing the tdTomato sequence is deleted upon Cre expression and EGFP is expressed were transferred to 1 x 10 blastocyst stage embryos containing a gene encoding Cre. １０ The blastocysts were cultured for 5 hours in KSOM medium containing 1000µg / mL AAV1 or AAV6. After culturing the blastocysts in KSOM medium containing AAV1 or AAV6 for 5 hours, they were washed with KSOM medium without AAV1 or AAV6. Culture was continued for two days, and cells exhibiting red or green fluorescence were observed. An outline of this experimental example is shown in Figure 1, and the results are shown in Figures 2 and 3 and Table 1. Blastocysts are broadly divided into two types: the inner cell mass that differentiates into a future individual, and the trophectoderm that differentiates into a future placenta. As a result of the observation, the trophectoderm was observed to have turned green in the case of AAV1, but all inner cell masses remained red. In contrast, the trophectoderm was observed to have turned green in the case of AAV6, and some inner cell masses also turned green (Figures 2 and 3). These results demonstrate that gene transfer into embryos is possible using AAV without removing the zona pellucida.

[0057]

[0058] Test Example 2: Gene transfer into blastocyst-stage embryos using AAV1 or AAV6 Mouse (mTmG mice) blastocysts were cultured for 5 hours in KSOM medium containing AAV1 or AAV6 containing a gene encoding Cre. After culturing the blastocysts in KSOM medium containing AAV1 or AAV6 for 5 hours, the blastocysts were washed with KSOM medium without AAV1 or AAV6. The blastocysts washed with KSOM medium were then transplanted into the uterus of recipient mice, and embryos at embryonic day 14.5 (E14.5) were collected and observed for red or green fluorescence. An outline of this test example is shown in Figure 4, and the results are shown in Figure 5 and Table 2. The observations showed that AAV1 caused the placenta to turn green, but the fetuses remained red, whereas AAV6 caused the placenta to turn green and some fetuses to turn green as well (Figure 5). These results demonstrate that placenta-specific gene transfer is possible using AAV1, whereas gene transfer to both the placenta and fetus is possible in approximately 20% of cases using AAV6.

[0059]

[0060] Test Example 3: Gene transfer into blastocyst stage embryos using AAV9, AAVDJ, AAVDJ / 8, or AAVPHP.eB. １０ An experiment similar to that in Test Example 1 was performed using AAV9, AAVDJ, AAVDJ / 8, or AAV-PHP.eB at 1000µg / mL. The results of this Test Example are shown in Table 3. As a result of observation, it was confirmed that the trophectoderm turned green even when an AAV serotype different from that in Test Example 1 was used. This result demonstrated that gene transfer into embryos without removing the zona pellucida was possible even when an AAV other than AAV1 and AAV6 was used.

[0061]

[0062] Production Example 2: The original vector construct for AKT1 with a myristoylation signal added (myr-AKT1) was purchased from Addgene (#49186, USA). The target sequence was excised with a restriction enzyme and replaced with the EGFP sequence of the pscAAV-CAG-EGFP vector purchased from Addgene (#83279, USA). The pscAAV-CAG-myr-AKT1 vector was then co-cultured with HEK293T cells using calcium phosphate together with an AAV1 rep / cap vector (gift from the University of Pennsylvania) and a pHelper vector from the AAVpro Helper Free System (Takara Bio Inc.), to generate myr-AKT1-AAV1 with a CAG promoter. Myr-AKT1-AAV1 was purified using the AAVpro Purification Kit (All Serotypes) (Takara Bio Inc.) and concentrated with PBS. The titer was measured by real-time PCR using the AAVpro Titration Kit (for Real Time PCR) Ver. 2 (Takara Bio Inc.). The myr-AKT1-AAV1 stock solution was stored at -80°C, thawed, and then diluted to 1 x 10 in EXiM medium. １０ Cryopreserved in vitro fertilization (IVF)-derived embryos were thawed and cultured in KSOM medium for 4 days, and blastocyst-stage embryos were cultured in KSOM medium containing myr-AKT1-AAV1 on a plastic bottom dish covered with liquid paraffin at 37°C / CO ２ After incubation in an incubator for 6 hours, the embryos were washed three times with KSOM medium and used for in vitro co-culture or blastocyst transfer into pseudopregnant mice.

[0063] Statistical analysis was performed using GraphPad Prism 9.2.0 (MDF Co., Ltd.) by one-way ANOVA followed by Tukey's post-hoc test. Statistical significance was determined when P was less than 0.05.

[0064] Experimental Example 4: Enhancement of AKT1 in Embryos We investigated whether celecoxib-induced implantation failure could be reversed by embryo treatment with adeno-associated virus type 1 (AAV1) carrying AKT1 with an N-terminal myristoylation signal (myr-AKT1). Myr-AKT1 is a constitutively active form of mutant AKT1. Embryos obtained by in vitro fertilization (IVF) were pretreated at the blastocyst stage with AAV1 containing myr-AKT1 (myr-AKT1-AAV1) for 6 hours (Figure 6A). pAKT immunoreactivity, indicating successful AKT1 introduction and activation, was higher in myr-AKT1-AAV1-treated, monocultured embryos than in untreated embryos. This activation began 24 hours later and remained positive for 48 hours. The pretreated embryos were then evaluated for implantation potential in vitro. The results showed that the number of embryos remaining on the endometrium after 48 hours was significantly higher with myr-AKT1-AAV1 treatment than with the control EGFP-AAV1 treatment (Fig. 6B and C).Furthermore, myr-AKT1-AAV1 treatment significantly improved the degree of invasion (Fig. 6D and E).

[0065] Finally, we tested whether these findings also apply to in vivo situations. Using a previously established celecoxib treatment protocol to induce trophoblast invasion failure and decidualization, we attempted to ameliorate celecoxib-induced implantation failure by treating embryos with myr-AKT1-AAV1 before blastocyst transfer (Fig. 6F). Myr-AKT1-AAV1 significantly improved implantation site weight loss and trophoblast invasion (Fig. 6G-J).

[0066] The method of the present invention can be applied to gene therapy to prevent miscarriage and adverse effects on fetuses due to implantation failure and placental abnormalities in humans and livestock. Furthermore, since the method of the present invention enables nucleic acid transfer into both the placenta and fetus and the nucleic acid is transferred into part of the fetus, it can also be applied to the production of mosaic mice, chimeric mice, etc.

Claims

1. A method for introducing a nucleic acid into trophectoderm cells, comprising the step of introducing the nucleic acid into a blastocyst ex vivo using an adeno-associated virus vector.

2. A method for producing blastocysts in which nucleic acid has been introduced into trophectoderm cells, comprising the step of introducing nucleic acid into blastocysts ex vivo using an adeno-associated virus vector.

3. A method for producing a genetically modified non-human animal, comprising: (a) introducing nucleic acid into a blastocyst ex vivo using an adeno-associated virus vector; and (b) implanting the blastocyst obtained in step (a) into a recipient.

4. The method according to any one of claims 1 to 3, wherein the blastocyst has not had the zona pellucida removed.

5. The method of claim 1 or 2, wherein the blastocyst is derived from a mammal selected from mouse, rat, rabbit, dog, cat, horse, cow, pig, goat, sheep and primate.

6. The method of claim 3, wherein the blastocyst is derived from a mammal selected from mouse, rat, rabbit, dog, cat, horse, cow, pig, goat, sheep and non-human primate.

7. The method according to any one of claims 1 to 3, wherein the nucleic acid is introduced specifically into trophectoderm cells.

8. The method of any one of claims 1 to 3, wherein the nucleic acid is introduced into both the trophectoderm cells and the inner cell mass.

9. The method of any one of claims 1 to 3, wherein the adeno-associated viral vector is AAV1 or AAV6.

10. The method of any one of claims 1 to 3, wherein the nucleic acid encodes a protein involved in the formation and / or function of the placenta and / or an RNA that controls the expression of said protein.

11. A nucleic acid transfer agent used to transfer a nucleic acid into a blastocyst, the agent comprising an adeno-associated virus vector containing the nucleic acid.

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