3D Artificial Dynamometer Scaffold

Abstract

We provide a 3D artificial kinetic chorodor scaffold. [Solution] A composition is provided comprising a three-dimensional artificial kinetochore scaffold comprising a unit complex containing NDC80 protein and NUF2 protein. The composition may be biodegradable or proteinaceous, and the number of unit complexes contained in the three-dimensional artificial kinetochore scaffold may be 10,000 to 50,000 or 20,000 to 40,000. The three-dimensional artificial kinetochore scaffold of this disclosure has the ability to align at the spindle equator during meiosis of an oocyte, for example. The three-dimensional artificial kinetochore scaffold of this disclosure can preferably be used, for example, to suppress premature chromosome segregation during meiosis of an oocyte.

Description

Technical Field

[0001] This disclosure relates to a three-dimensional artificial kinetochore scaffold. This disclosure also relates to a method of treating oocytes with a three-dimensional artificial kinetochore scaffold.

Background Art

[0002] The chromosomal abnormalities of eggs increase with maternal aging. Chromosomal abnormalities can occur due to chromosomal segregation errors during meiosis and cause infertility, miscarriage, and births with congenital diseases. According to Non-Patent Document 1, the estimated frequency of trisomy in clinically recognized pregnancies increases with maternal aging, being less than 4% in the 10s and exceeding 30% in the 40s. This is considered to be an obstacle in reproductive medicine.

[0003] In Non-Patent Documents 2 and 3, an NDC80 complex fused with GFP was bound to magnetic beads immobilized with an anti-green fluorescent protein antibody. In Non-Patent Document 2, as a result, it was shown that the beads aligned at the spindle equator in metaphase, and in Non-Patent Document 3, it was shown that the rate of early chromosome separation decreased. Non-Patent Documents 2 and 3 are publications by the inventors of the present application.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Summary of the Invention

[0005] The present disclosure provides a three-dimensional artificial kinetochore scaffold. The present disclosure also provides a method of treating an oocyte with a three-dimensional artificial kinetochore scaffold.

[0006] The inventors prepared a three-dimensional artificial kinetochore scaffold having a surface to which microtubules bind. As a result, the three-dimensional artificial kinetochore scaffold was able to align at the spindle equator in metaphase of meiosis of oocytes. It has been found that introduction of the three-dimensional artificial kinetochore scaffold reduces the amount of microtubules binding to chromosomes in metaphase of meiosis and can suppress premature separation of chromosomes. The three-dimensional artificial kinetochore scaffold may be proteinaceous.

[0007] According to the present invention, for example, the following inventions are provided. [1] A composition comprising a three-dimensional artificial kinetochore scaffold comprising a unit complex containing NDC80 protein and NUF2 protein. [2] The composition according to [1] above, wherein the three-dimensional artificial kinetochore scaffold is biodegradable or proteinaceous. [3] The composition according to [1] or [2] above, wherein the number of unit complexes contained in the three-dimensional artificial kinetochore scaffold is 10,000 to 50,000 or 20,000 to 40,000. [4] The composition according to any one of [1] to [3] above, wherein the three-dimensional artificial kinetochore scaffold has the ability to align at the spindle equator at least within an oocyte in metaphase of the first meiotic division. [5] The composition according to any one of [1] to [4] above, wherein the three-dimensional artificial kinetochore scaffold comprises a unit complex and a support, and the unit complex is bound to the support and exposes the unit complex. [6] The composition according to [5] above, wherein the unit complex has a donor tag, the support has an acceptor tag, and the donor tag and the acceptor tag have binding affinity for each other, whereby the donor tag and the acceptor tag are bound. [7] The composition according to [5] above, wherein the donor tag is linked to NDC80. [8] The composition according to [6] or [7], wherein the support comprises a fusion protein including a multimerizing domain and a proteinacceptor tag, the multimerizing domains associate with each other to form a multimer, thereby the support presenting a plurality of proteinacceptor tags, and the unit complex is bound to the proteinacceptor tags via its donor tag. [9] The composition according to any one of [6] to [8] above, wherein the donor tag is an FK506-binding protein (FKBP)-rapamycin-binding domain (FRB) and the acceptor tag is FKBP.

[10] The composition according to any one of [6] to [9] above, wherein the three-dimensional artificial kinetic scaffold includes a linker having two acceptor tags and a spacer connecting the acceptor tags.

[11] A fusion protein of NDC80 or NUF2 and FK506-binding protein (FKBP), or a nucleic acid encoding said fusion protein.

[12] (1)(i) a fusion protein comprising NDC80 and a proteinoid donor tag and NUF2, or (ii) a nucleic acid encoding the fusion protein and a nucleic acid encoding NUF2; (2)(i) a fusion protein comprising NDC80 and a protein-based acceptor tag and NUF2, or (ii) a nucleic acid encoding the fusion protein and a nucleic acid encoding NUF2; (3)(i) a fusion protein containing NUF2 and a proteinoid donor tag and NDC80, or (ii) a nucleic acid encoding the fusion protein and a nucleic acid encoding NDC80; or (4)(i) a fusion protein comprising NUF2 and a protein-based acceptor tag and NDC80, or (ii) a nucleic acid encoding the fusion protein and a nucleic acid encoding NDC80; and The protein-based donor tag is FK506-binding protein (FKBP), and the protein-based acceptor tag is the FKBP-rapamycin-binding domain (FRB). A combination of items.

[13] A combination (first combination) selected from the group consisting of (1)(i), (2)(i), (3)(i), and (4)(i) described in

[12] above, and a combination (second combination) comprising a fusion protein of the proteinial acceptor tag and the polymerization domain.

[14] A combination (first combination) selected from the group consisting of (1)(i), (2)(i), (3)(i), and (4)(i) described in

[12] above, and a combination (third combination) comprising two proteinoid donor tags and a linker protein having a proteinoid spacer between the two proteinoid donor tags.

[15] A combination (fourth combination) comprising the combination described in

[13] or

[14] above and a linker protein having a protein spacer between the two protein donor tags.

[16] A combination (combination 2-1) comprising any one combination selected from the group consisting of (1)(ii), (2)(ii), (3)(ii), and (4)(ii) described in

[12] above, and a nucleic acid encoding a fusion protein of the proteinial acceptor tag and the multimerization domain (combination 2-2).

[17] A combination (combination 2-1) selected from the group consisting of (1)(ii), (2)(ii), (3)(ii), and (4)(ii) described in

[12] above, and a combination (combination 2-3) comprising two proteinoid donor tags and a nucleic acid encoding a linker protein having a proteinoid spacer between the two proteinoid donor tags.

[18] A combination (combination 2-4) comprising the combination described in

[16] or

[17] above and a nucleic acid encoding a linker protein having a protein spacer between the two proteinoid donor tags.

[19] A method for suppressing premature chromosome segregation in oocytes, Introducing a three-dimensional artificial kinetochore scaffold with microtubule-binding ability into oocytes before or during meiosis, and Aligning a 3D artificial kinetochore scaffold with the spindle equator. Methods that include...

[20] The introduction of a 3D artificial kinetic scaffold is This is performed by injecting a three-dimensional artificial kinetochore scaffold constructed extracellularly into the cell, or, The method according to

[19] above, which is carried out by introducing nucleic acids encoding the components of the three-dimensional artificial kinetochore scaffold to cause the three-dimensional artificial kinetochore scaffold to self-assemble in an oocyte.

[21] The method according to

[19] or

[20] , wherein the three-dimensional artificial kinetic scaffold is as defined in any one of claims 1 to 10. [Brief explanation of the drawing]

[0008] [Figure 1]Construction of NDC80ΔSPC-GEM. (A) Design of NDC80ΔSPC-GEM. Abbreviations are defined in the text. (BE) NDC80ΔSPC-WT-GEM particles cluster via the loop domain of NDC80. Nocodazole-treated oocytes expressing GEM, NDC80ΔSPC-GEM, and NDC80ΔSPC-DFAA-GEM constructs were imaged. (B) shows the Z projection image of GEM 4 hours after nuclear membrane breakdown (NEBD, a marker of M-phase entry). Scale bar, 10 μm. In (C), the number of NDC80ΔSPC-GEM bright spots in a 40 μm thick cytoplasmic region was counted. Each spot represents an oocyte. Bar graphs show the mean and SEM. P values ​​were calculated using the Kruskal-Wallis test and Dunn's post-hoc test (Bonferroni correction). In D and E), the diameter of individual fluorescent spots was estimated by measuring the full width at half maximum (FWHM) of the signal intensity. Each dot represents the mean value of oocytes. P-values ​​were calculated using an unpaired two-sided t-test. Numbers in parentheses indicate oocyte number. (F) Design of the enlarged NDC80ΔSPC-GEM cluster. (G,H) NDC80ΔSPC-WT-GEM cluster enlarged with a linker. Similar to (B) and (E), z-projection images of nocodazole-treated oocytes and plots of measured FWHM values ​​are shown. Scale bar, 10 μm. P-values ​​were calculated using an unpaired two-sided t-test. Numbers in parentheses indicate oocyte number. [Figure 2]Size-dependent alignment of NDC80ΔSPC-GEM clusters. (AC) NDC80ΔSPC-WT-GEM clusters align with linkers. Live imaging of NDC80ΔSPC-GEM and NDC80ΔSPC-GEM-Linker throughout meiosis I (M phase). Z-projection of chromosome (H2B-mCherry, magenta) and NDC80ΔSPC-GEM (green). Arrows indicate the direction in which NDC80ΔSPC-GEM-Linker moves toward the spindle. Scale bar, 10 μm. In (B), the distance of NDC80ΔSPC-GEM clusters from the metaphase plate (M plate) was measured during metaphase I (6 hours post-NEBD). "Aligned" refers to a position within 5 μm of the M plate. The volume of individual clusters was normalized by the total volume of the oocyte. The number of oocytes is shown in parentheses. In C), each dot represents an oocyte. The bar graph shows the mean and standard deviation. P-values ​​were calculated using Dunn's post-hoc test, which adds Bonferroni correction to the Kruskal-Wallis test. Numbers in parentheses indicate oocyte number. (D) Alignment stability of NDC80ΔSPC-GEM-Linker clusters. NDC80ΔSPC-GEM-Linker clusters and chromosomes were tracked in 3D. The time change in distance from the M plate from 5 to 6 hours after NEBD of the oocyte is shown. (E) Alignment of NDC80ΔSPC-GEM-Linker clusters is not completely stable. The percentage of NDC80ΔSPC-GEM-Linker clusters that maintained a position within 5 μm of the M plate from 5 hours after NEBD until the start of anaphase (average 8.2 hours after NEBD). P-values ​​were calculated using a two-sided t-test without pairing. Numbers of oocytes are shown in parentheses. (F, G) Aligned NDC80ΔSPC-GEM-Linker clusters extended along the spindle axis. The clusters were fitted to an ellipsoid. In (F), the angle between the major axis of the ellipsoid and the spindle axis was measured at metaphase I (6 hours after NEBD). In (G), the aspect ratio was calculated. Each dot represents an aligned NDC80ΔSPC-GEM-Linker cluster. The number in parentheses indicates the number of clusters from 9 oocytes. [Figure 3]Recruitment of SPC24-SPC25 improves the alignment of NDC80-GEM clusters. (A) Aggregates of NDC80ΔSPC-GEM-Linkers and NDC80FL-GEM-Linkers in nocodazole-treated oocytes. Z projection of NDC80-GEMs 4 hours post-NEBD (metaphase). Scale bar, 10 μm. (B) FWHM of NDC80-GEMs in (A). Each dot represents an oocyte. P-values ​​were calculated using an unpaired two-sided t-test. Number of oocytes in parentheses. (C) Live imaging of the dynamics of NDC80ΔSPC-GEM-Linkers and NDC80FL-GEM-Linkers 6 hours post-NEBD. Z projection of chromosomes (H2B-mCherry, magenta) and NDC80-GEMs (green). Scale bar, 10 μm. (D) Relative volumes of the NDC80ΔSPC-GEM-Linker and NDC80FL-GEM-Linker along the spindle axis of (C). Distance from the spindle equator was quantified. Numbers in parentheses indicate oocyte numbers. (E) Alignment score of the NDC80ΔSPC-GEM-Linker, NDC80FL-GEM-Linker, and chromosomes of (C). "Alignment" is defined as being within 5 μm of the spindle equator. P values ​​were calculated using the Kruskal-Wallis test and Dunn's post-hoc test. Numbers in parentheses indicate oocyte numbers. (F) Percentage of NDC80ΔSPC-GEM-Linker, NDC80FL-GEM-Linker, and chromosomes that maintain stable alignment from 5 hours after GVBD until the start of anaphase. P values ​​were calculated using the Kruskal-Wallis test and Dunn's post-hoc test. Numbers in parentheses indicate oocyte numbers. Bipolar microtubule attachment was observed on aligned NDC80-GEM-Linkers 6 hours (metaphase) (G) and 15 hours (extended metaphase) (H) after (GH)NEBD. Single-slice images of NDC80-GEM (magenta) immunostained with HURP (green) from ProTAME-treated oocytes. Scale bar, 10 μm. HURP bundle attachment by NDC80-GEM-Linkers is classified into no attachment (red frame), unipolar attachment (orange frame), and bipolar attachment (green frame), and magnified. Scale bar, 1 μm. The number of oocytes is shown in parentheses. [Figure 4]NDC80-GEM-Linker clusters compete with kinetochores for HURP-decorated microtubule fibers. (A) Top: Diagram showing NDC80FL-GEM-Linker clusters and chromosomes on a metaphase plate (M-plate). Bottom: Live imaging of NDC80FL-GEM-Linker and chromosome dynamics (6 hours post-NEBD). Top view of the M-plate including chromosome (H2B-mCherry, magenta) and NDC80-GEM (green). Scale bar, 10 μm. (B) Localization of NDC80FL-GEM-Linker and chromosomes from (A) on the M-plate. Green dots represent NDC80FL-GEM-Linker clusters, and reddish-purple dots represent chromosomes. Black dots indicate the center of the M-plate. Two dashed circles indicate 5 μm and 10 μm from the center of the M-plate. Numbers in parentheses indicate the number of chromosomes and clusters measured from 24 oocytes. (C) Percentage of NDC80FL-GEM-Linker and chromosome from the spindle axis in (A). Distance from the spindle axis was quantified. The number of chromosomes and clusters measured from 24 oocytes is shown in parentheses. (D) HURP-decorated kinetochore-microtubule fibers in oocytes with and without NDC80-GEM-Linker clusters, 15 hours after NEBD (extended metaphase). Single-slice images immunostained for HURP (green), ACA (magenta), and DNA (Hoechst33342, blue). Representative fibers are shown in red frames and magnified. Microtubule channels of representative fibers are shown in white frames and magnified. Arrows indicate the direction of linear selection in FWHM analysis. Scale bar is 10 μm. (E) Width of HURP-decorated kinetochore-microtubule fibers in oocytes with and without NDC80-GEM-Linker clusters in (D). P-values ​​were calculated using an unpaired two-sided t-test. Numbers in parentheses indicate oocyte count. (F) Percentage of oocytes with shifted chromosomes in (D). Numbers in parentheses indicate oocyte count. (G) Kinetochore-microtubule fibers decorated with HURP in oocytes with NDC80-WT-GEM-Linker and NDC80-9A-GEM-Linker clusters. Single-slice images immunostained for HURP (green), ACA (magenta), and DNA (Hoechst33342, blue). Representative fibers are shown in red frames and magnified. Microtubule channels of representative fibers are shown in white frames and magnified.Arrows indicate the direction of linear selection in FWHM analysis. Scale bar is 10 μm. (H) (G) Width of HURP-decorated kinetochore-microtubule fibers in oocytes with NDC80-WT-GEM-Linker and NDC80-9A-GEM-Linker clusters. P-values ​​were calculated using an unpaired two-sided t-test. Number of oocytes is shown in parentheses. (I) (G) Percentage of oocytes in which sister chromatids separated early. P-values ​​were calculated using Fisher's exact test. Each dot corresponds to data from one experiment. Number of oocytes is shown in parentheses. [Figure 5]NDC80-GEM clusters prevent premature chromosome segregation (premature chromosome segregation) in senescent oocytes. (A) NDC80-GEM clusters prevent premature chromosome segregation 22 hours after NEBD (extension of meiosis I). Z projection image of ProTAME-treated senescent oocytes immunostained with ACA (white) and DNA (Hoechst33342, magenta). Arrows indicate prematurely segregated chromosomes. Scale bar, 10 μm. (B) Percentage of oocytes in (A) where chromosomes segregated prematurely. P-values ​​were calculated using Fisher's exact test. Each dot corresponds to data from one experiment. Number of oocytes in parentheses. (C) Percentage of oocytes in (A) where all aligned chromosomes were intact. P-values ​​were calculated using an unpaired two-sided t-test. Each point represents one oocyte. Number of oocytes in parentheses. (D) Overexpression of HURP increases early chromosome segregation at 22 hours post-NEBD (extension of meiosis I). Z projection image of immunostained ACA (white) and DNA (Hoechst33342, magenta) of ProTAME-treated senescent oocytes. Arrows indicate early segregated chromosomes. Scale bar, 10 μm. (E) Percentage of oocytes with early chromosome segregation in (D). P-values ​​were calculated using Fisher's exact test. Each dot corresponds to data from one experiment. Number of oocytes in parentheses. (F) NDC80-GEM clusters prevent early segregation of sister chromatids at metaphase II in senescent oocytes. Top image: Immunostained images of ACA (white) and DNA (Hoechst33342, magenta) at 20 hours post-NEBD. Arrows indicate early segregated chromosomes. Separated chromatids (red box) and paired chromatids (yellow box). Bottom: Top view of the M plate. Z-projection of 3D reconstructed kinetochores (KT) (separated chromatids, red; paired chromatids, yellow) and DNA (Hoechst33342, white). Scale bar, 10 μm. (G) Early segregation number of sister chromatids in (F). P-values ​​were calculated using an independent two-sided t-test. Each dot corresponds to data from one oocyte. Number of oocytes in parentheses. (H) KT-to-KT distance of paired chromatids in (F). P-values ​​were calculated using an independent two-sided t-test. The number of paired chromatids of 38-oocyte and 32-oocyte cells is shown in parentheses. (I) Percentage of aligned paired chromatids in (F).The p-value was calculated using an independent two-tailed t-test. The number in parentheses represents the oocyte count. [Figure 6] Construction of NDC80ΔSPC-GEM clusters. (A) Nocodazole inhibits microtubule polymerization. Oocytes were fixed 4 hours after NEBD and immunostained for α-tubulin (gray). Scale bar, 10 μm. (B) Co-localized ΔSPC-GEM of NUF2 on NDC80. Oocytes were fixed 4 hours after NEBD and immunostained for HA (magenta) and DNA (Hoechst33342, blue). Z projection image is shown. Scale bar, 10 μm. (C) Maximum value of NDC80-GEM in (Figure 1B). Each dot represents an oocyte. P-values ​​were calculated using an unpaired two-sided t-test. Numbers in parentheses indicate oocyte count. (D) Maximum value of NDC80-GEM in (Figure 1G). Each dot represents an oocyte. P-values ​​were calculated using an unpaired two-sided t-test. Numbers in parentheses indicate oocyte count. [Figure 7] Alignment of NDC80-GEM clusters by size correlation. (A) As the size increases, the alignment of NDC80ΔSPC-GEM-Linker clusters improves. The NDC80-GEM cluster in Figure 2A) was used for distance measurement from the M-plate. [Figure 8]Recruitment of SPC24-SPC25 improved the alignment of the NDC80-GEM cluster. (A) Figure of the NDC80 complex, Mis12 complex, and CENP-T. (B) Co-localization of CENP-T on NDC80 in the FL-GEM-Linker. Oocytes were fixed 4 hours after NEBD and immunostained with mCherry (magenta). Z-projection images are shown. Scale bar is 5 μm. P-values ​​were calculated using an unpaired two-sided t-test. The number of clusters measured from 9 oocytes is shown in parentheses. (C) Localization of DSN1 on the NDC80FL-GEM-Linker. Oocytes were fixed 4 hours after NEBD and immunostained with DSN1 (magenta) and DNA (Hoechst33342, blue). Z-projection images are shown. The kinetochore and NDC80FL-GEM are shown in gray and green frames. The scale bar is 5 μm. P-values ​​were calculated using an unpaired two-sided t-test. The number in parentheses is the number of measurement clusters obtained from 9 oocytes. (D) Maximum value of NDC80-GEM in (Figure 3A). Each dot represents an oocyte. P-values ​​were calculated using an unpaired two-sided t-test. The number in parentheses is the number of oocytes. (E) Recruitment of SPC24-SPC25 improves the alignment of NDC80-GEM clusters. The NDC80-GEM clusters in (Figure 3C) were used to measure the distance from the M plate. (F) Alignment stability of NDC80FL-GEM-Linker clusters. NDC80FL-GEM-Linker clusters and chromosomes were tracked in 3D. The time change of the distance from the M plate from 5 to 6 hours after oocyte NEBD is shown. [Figure 9]NDC80-GEM-Linker clusters do not preferentially reside in the medial region of the metaphase plate. (A) Live imaging of 1 μm and 3 μm NDC80 bead and chromosome dynamics 6 hours post-NEBD. Z projection image of chromosome (H2B-mCherry, magenta) and NDC80 bead (green). Scale bar, 10 μm. (B) Top: Localization of NDC80 beads and chromosomes in (A) on an M-plate. Bottom: Percentage of NDC80 beads and chromosomes from the spindle axis in (A). Numbers in parentheses indicate the number of chromosomes and clusters measured in 11 oocytes and 14 oocytes. (C) Live imaging of 1 μm and 3 μm NDC80 bead and chromosome dynamics 6 hours post-NEBD. 3D reconstructed image of chromosome (H2B-mCherry, magenta) and NDC80 bead (green). Scale bar, 10 μm. (D) Percentage of oocytes with all chromosomes present, as shown in (C). One point represents an oocyte. P-values ​​were calculated using an independent two-sided t-test. Numbers in parentheses indicate the number of oocytes. (E) Both large and small beads have similar NDC80 density. The image in (C) was used to measure the NDC80-GFP fluorescence intensity on the beads. P-values ​​were calculated using an independent two-sided t-test. Numbers in parentheses indicate the number of beads with 11 and 14 oocytes. [Figure 10] NDC80-GEM-Linker clusters compete with kinetochores for HURP-decorated microtubule fibers. (A) Top: NDC80-GEM clusters do not increase the aneuploidy rate of metaphase II oocytes. Immunostaining images of ACA (white) and DNA (Hoechst33342, magenta) 20 hours after NEBD. Bottom: Top view of M plate. Z-projection image using 3D reconstructed kinetochores (KT) (yellow) and DNA (Hoechst33342, white). Scale bar, 10 μm. (B) Percentage of aneuploid metaphase II oocytes in (B). P-values ​​were calculated using Fisher's exact test. Each dot corresponds to data from one experiment. Oocyte count is shown in parentheses. [Figure 11]Rapamycin treatment in the absence of the NDC80FL-GEM-Linker component does not significantly affect the frequency of early chromosome segregation. (A) Rapamycin treatment does not increase the frequency of early chromosome segregation from 22 hours post-NEBD (extended meiosis I). Z projection image of immunostained ACA (white) and DNA (Hoechst33342, magenta) of ProTAME-treated aging oocytes. Arrows indicate early segregated chromosomes. Scale bar, 10 μm. (B) Percentage of oocytes with early chromosome segregation in (A). P-values ​​were calculated using Fisher's exact test. Each dot corresponds to data from one experiment. Number of oocytes in parentheses. [Modes for carrying out the invention]

[0009] In this specification, "oocyte" refers to a type of female germ cell. Oocytes arise from oogonia. During the fetal period, primordial germ cells proliferate in the ovary to become oogonia. Oogonia differentiate into primary oocytes through repeated mitosis. Primary oocytes temporarily arrest in prophase I of meiosis (also called meiotic division I) and remain in the ovary as primordial follicles. After puberty, primary oocytes resume meiosis, releasing their first polar body to become secondary oocytes. Secondary oocytes arrest in metaphase II of meiosis and are ovulated. Upon fertilization, secondary oocytes complete meiosis, releasing their second polar body to become mature oocytes. Errors in chromosome segregation during meiosis can cause miscarriage or congenital disorders (e.g., Down syndrome).

[0010] In this specification, "meiosis I" (or "meiosis I") is a cell division that halves the number of chromosomes during gamete formation, and includes prophase, metaphase, anaphase, and telophase. During prophase, chromosomes condense, and homologous chromosomes pair up to form bivalent chromosomes. During this process, genetic information is exchanged (crossover) between homologous chromosomes, resulting in genetic diversity. In metaphase, bivalent chromosomes align in the center of the cell (equator plane), forming a spindle, which then attaches to the kinetochores of the chromosomes. In anaphase, homologous chromosomes separate and move to opposite poles. In telophase, cytokinesis progresses, forming the oocyte and the first polar body. At this point, the number of chromosomes in both the oocyte and the first polar body is half that of the original cell.

[0011] In this specification, "meiosis II" (or "meiosis II") is a cell division that halves the number of chromosomes and occurs during gamete formation, and includes prophase, metaphase, anaphase, and telophase. During prophase, chromosomes condense and the nuclear membrane disappears. Centrioles move to opposite poles, and the spindle is formed. During metaphase, chromosomes align in the center of the cell (equator plane), and the spindle attaches to the chromosomal kinetochores. During anaphase, homologous chromosomes separate and move to opposite poles. Meiosis II in an egg cell occurs at fertilization, forming the fertilized egg and the second polar body.

[0012] In this specification, a "three-dimensional artificial kinetochore scaffold" is a three-dimensional scaffold that mimics a portion of the kinetochore of a chromosome, has the ability to bind to microtubules extending from both poles of the spindle, and has the ability to align with the spindle equator during metaphase of meiosis. Alignment with the spindle equatorial plane requires the input of equal and a certain level of tension from both poles. The three-dimensional artificial kinetochore scaffold is a scaffold to which microtubules from both poles bind and which allows for the input of tension to the opposing poles by microtubules. The three-dimensional artificial kinetochore scaffold is preferably biodegradable and biocompatible. The three-dimensional artificial kinetochore scaffold is, for example, composed of proteins and is formed within a cell, for example, by supplying its components with mRNA within the cell. The three-dimensional artificial kinetochore scaffold can be formed in the presence of cytoplasmic components.

[0013] In this specification, “kinetochore” refers to a protein complex formed on chromosomes during eukaryotic cell division, playing a crucial role in the precise distribution of chromosomes to daughter cells. The following describes the natural kinetochores. The kinetochore consists of an inner plate, an outer plate, and a coronal region. The inner plate binds tightly to centromere DNA, forming a special chromatin region that persists throughout cell telophase. The outer plate interacts with microtubules and assembles and functions during cell division. The inner plate may contain CENP-A, CENP-C, CENP-H, and CENP-I. CENP-A is a histone H3 variant that binds tightly to centromere DNA to form the kinetochore base. CENP-C, CENP-H, and CENP-I bind to CENP-A to stabilize the inner plate. The outer plate may contain the NDC80 complex, CENP-E, and dynein. The NDC complex is a heterotetramer containing NDC80, NUF2, SPC24, and SPC25. The NDC complex enables the binding of microtubules to kinetochores. CENP-E and dynein are motor proteins involved in chromosome movement. The kinetochore further comprises a corona region, the outermost layer of the kinetochore where spindle checkpoint proteins (Mad1, Mad2, BubR1, etc.) are localized to monitor the binding state between microtubules and kinetochores.

[0014] In this specification, "NDC80" is a protein included in the NDC80 complex that links the kinetochore and spindle microtubules, regulating chromosome movement and segregation. NDC80 has an N-terminal domain, a coiled-coil domain, and a C-terminal domain. The N-terminal domain is responsible for binding to microtubules. The coiled-coil domain promotes heterodimerization with NUF2 and further contributes to ensuring the stability of the heterotetramer complex through interaction with SPC24 and SPC25. The C-terminal domain binds to SPC24 and SPC25, and through this binding, is responsible for linking to the inner region of the kinetochore. NDC80 has a phosphorylation site, particularly in its N-terminal domain. Phosphorylation is mediated by Aurora B kinase. Phosphorylation of NDC80 alters its binding affinity to microtubules. A balance between phosphorylation and dephosphorylation of NDC80 can maintain proper binding between the kinetochore and microtubules and contribute to accurate chromosome segregation. Examples of NDC80 include human NPC80, for example, human NPC80 having the amino acid sequence registered in UniProtKB / Swiss-Prot: O14777.1. In this specification, NDC80 also includes NDC80 having an amino acid sequence that has 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the amino acid sequence registered in UniProtKB / Swiss-Prot: O14777.1, in addition to the full length of NDC80. In this specification, NDC80 also includes NDC80 fragments having an amino acid length of 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more of the full length of NDC80. NDC80 possesses all of the following characteristics: the ability to bind to microtubules, the ability to bind to NUF2, and the ability to bind to SPC24 and SPC25.

[0015] In this specification, "NUF2" is a protein included in the NDC80 complex, which forms a dimer with NDC80 to facilitate microtubule binding and promotes linkage with the inner region of the kinetochore. NUF2 has an N-terminal domain, a coiled-coil domain, and a C-terminal domain. The N-terminal domain, together with NDC80, is responsible for microtubule binding. The coiled-coil domain promotes heterodimer formation with NDC80 and further contributes to ensuring the stability of the heterotetramer complex through interaction with SPC24 and SPC25. The C-terminal domain is responsible for linkage with the inner region of the kinetochore through binding with SPC24 and SPC25. Examples of NUF2 include human NUF2, for example, human NUF2 having the amino acid sequence registered in NCBI Reference Sequence: NP_663735.2. In this specification, NUF2 includes NDC80 having an amino acid sequence having 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the amino acid sequence registered in UniProtKB / Swiss-Prot: O14777.1. In this specification, NUF2 also includes NUF2 fragments having an amino acid length of 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more of the full length of NUF2. NUF2 has all of the following selected from the group consisting of microtubule-binding-promoting ability, NDC80-binding ability, and kinetochore-binding-promoting ability via SPC24 and SPC25.

[0016] In this specification, a “tag” is a peptide that can be attached to a protein. Tags typically have an affinity for a particular molecule. In this specification, any one of two tags that bind to each other is called a donor tag and the other is called an acceptor tag. When two tags bind together, either one may be called the donor tag.

[0017] In this specification, "fusion protein" means a polypeptide formed by linking the N-terminuses and C-terminuses of two proteins. In a fusion protein, a spacer (e.g., a GS linker) may be included between the two proteins. Examples of GS linkers include, but are not limited to, linkers containing flexible G and hydrophilic S, such as ((G) n S) m An example is a repeating array {where n can be a natural number such as 1, 2, or 3, and m can be any natural number such as 3, 4, 5, or 6}.

[0018] In this specification, "multimerizing domain" is also called a multimerizing domain and refers to the entire length of a protein or a portion of it (e.g., a domain) that has the ability to multimerize. Multimerizing domains in proteins can be easily identified by those skilled in the art.

[0019] <3D artificial kinetic scaffold of this disclosure> This disclosure provides a three-dimensional artificial kinetochore scaffold. The three-dimensional artificial kinetochore scaffold is capable of binding microtubules from both poles of a spindle and aligning them to the spindle's equatorial plane due to tension from both poles. Preferably, the three-dimensional artificial kinetochore scaffold has little adverse effect on the fidelity of chromosome segregation.

[0020] The three-dimensional artificial kinetochore scaffold of this disclosure comprises a complex (also referred to as a “unit complex”) containing NDC80 and NUF2. The three-dimensional artificial kinetochore scaffold may comprise multiple unit complexes. NDC80 and NUF2 are preferably human NDC80 and human NUF2. In the three-dimensional artificial kinetochore scaffold, NDC80 and NUF2 form a heterodimer. This is thought to be due to the interaction between the coiled-coil domain of NDC80 and the coiled-coil domain of NUF2. In the three-dimensional artificial kinetochore scaffold of this disclosure, NDC80 and NUF2 are included in a manner that allows them to bind to microtubules. The three-dimensional artificial kinetochore scaffold of this disclosure may further comprise one or both of SPC24 and SPC25.

[0021] In this disclosure, NDC80 has binding affinity to either or both of SPC24 and SPC25 (i.e., binding domains to SPC24 and SPC25, e.g., C-terminal domains).

[0022] The three-dimensional artificial kinetic scaffold of this disclosure is not particularly limited, but may have various shapes such as linear, rod-shaped, spherical, sheet-shaped, or block-shaped.

[0023] The three-dimensional artificial kinetochore scaffolds of this disclosure are preferably biocompatible. The three-dimensional artificial kinetochore scaffolds of this disclosure are preferably biodegradable. The three-dimensional artificial kinetochore scaffolds of this disclosure are preferably biocompatible and biodegradable. Therefore, the three-dimensional artificial kinetochore scaffolds of this disclosure can be removed from egg cells after a certain period of time after introduction. Biocompatibility and / or biodegradability can be provided, for example, by biomolecules (e.g., biopolymers). Biomolecules may include one or more selected from the group consisting of polysaccharides, amino sugars (e.g., chitosan, galactosamine, etc.), peptides, and nucleic acids. A biomolecule is, for example, a peptide.

[0024] The three-dimensional artificial kinetic chamostat scaffold of this disclosure may include solid beads (such as those made of resin, plastic, metal, or magnetic material), but preferably does not include non-biodegradable structures such as solid beads (such as those made of resin, plastic, metal, or magnetic material).

[0025] The three-dimensional artificial kinetochore scaffold of this disclosure may comprise a unit complex containing NDC80 protein and NUF2 protein, and a support. The unit complex is bound to the support and, for example, is exposed on the support. Exposure on the support facilitates microtubule access for the unit complex. While the unit complex is substantially composed of protein, the support does not necessarily have to be protein. However, the support is preferably protein from the viewpoint of biocompatibility and biodegradability. This is because if the main component of the three-dimensional artificial kinetochore scaffold is protein, it can be produced by translation from mRNA. Production by translation from mRNA is advantageous in preparation because the three-dimensional artificial kinetochore scaffold can be prepared intracellularly or extracellularly (e.g., in vitro). The support and the unit complex may be bound by covalent bonds (e.g., peptide bonds), or by non-covalent bonds (e.g., binding between two binding proteins such as a donor tag and an acceptor tag).

[0026] In some embodiments, a three-dimensional artificial kinetochore scaffold comprises, for example, more than 10,000, more than 15,000, more than 20,000, more than 25,000, more than 30,000, more than 40,000, or more than 50,000 unit complexes. In some embodiments, a three-dimensional artificial kinetochore scaffold comprises, for example, less than or equal to 100,000, less than or equal to 90,000, less than or equal to 80,000, less than or equal to 70,000, less than or equal to 60,000, less than or equal to 50,000, or less than or equal to 40,000 unit complexes. In some embodiments, a two-dimensional artificial kinetochore scaffold comprises, for example, more than 10,000 to 100,000, less than or equal to 20,000 to 90,000, less than or equal to 30,000, less than or equal to 80,000, less than or equal to 40,000, less than or equal to 50,000 unit complexes. Each of these unit complexes can bind to microtubules.

[0027] In some embodiments, a three-dimensional artificial kinetochore scaffold comprises a unit complex and a support. The unit complex and the support may be covalently or noncovalently bonded. In the three-dimensional artificial kinetochore scaffold of this disclosure, the unit complex is exposed on the support in a manner accessible to microtubules, so that it binds to microtubules. If both the unit complex and the support are proteins, either a component of the unit complex or a component of the support may be a fusion protein, or the unit complex and the support may be composed of different proteins.

[0028] The support of this disclosure is preferably biocompatible. The support of this disclosure is preferably biodegradable. The support of this disclosure is preferably biocompatible and biodegradable. Therefore, the support of this disclosure can be removed from the oocyte after a certain period of time after introduction. Biocompatibility and / or biodegradability can be provided, for example, by biomolecules (e.g., biopolymers). The biomolecules may include one or more selected from the group consisting of polysaccharides, amino sugars (e.g., chitosan, galactosamine, etc.), peptides, and nucleic acids. The biomolecule is, for example, a peptide.

[0029] In some embodiments, the support may be a vesicle, and the unit complex may be exposed on the vesicle. The vesicle is not particularly limited, but may be, for example, a lipid membrane vesicle, liposome, micelle, polyion complex polymerosome, polyion complex micelle, or lipid nanovesicle. When the support is a lipid membrane vesicle, the unit complex has a lipid-soluble portion (such as a fatty acid chain or cholesterol), and can bind to the lipid membrane vesicle via this lipid-soluble portion, allowing the unit complex portion to be presented on the support. When the support is a polyion complex polymerosome or micelle, the unit complex has a polycation or polyanion portion, and can bind to the polyion complex polymerosome or micelle by ionic bonds, allowing the unit complex portion to be presented on the support.

[0030] The unit complex and the support may each have a tag. The unit complex and the support may each have a tag, and they may be bound together via the tags. In this case, the unit complex may have a donor tag, and the support may have an acceptor tag. This allows the unit complex and the support to be bound based on the affinity between the donor tag and the acceptor tag.

[0031] In one embodiment, the unit complex includes NDC80 and NUF2, with NDC80 linked to a donor tag. In another embodiment, the unit complex includes NDC80 and NUF2, with NUF2 linked to a donor tag. In yet another embodiment, the unit complex includes NDC80 and NUF2, with NDC80 and NUF2 each linked to a donor tag.

[0032] In one embodiment, the support exposes an acceptor tag. In another embodiment, the support is proteinaceous and contains a fusion protein comprising a multimerizing domain and a proteinaceous acceptor tag. In this embodiment, the fusion protein can multimerize via the multimerizing domain and expose multiple proteinaceous acceptor tags. With this configuration, a unit complex binds to the exposed acceptor tag via a donor tag, and the unit complex covers the surface of the support.

[0033] In one embodiment, the donor tag is an FK506-binding protein (FKBP)-rapamycin-binding domain (FRB), and the acceptor tag is FKBP. In another embodiment, the donor tag is FKBP and the acceptor tag is FRB. These donor and acceptor tags bind in the presence of an effective amount of rapamycin or its derivatives (e.g., AP21967, AP1903).

[0034] In one embodiment, the donor tag is the DNA gyrase B subunit (GyrB), and the acceptor tag is cumermycin. In another embodiment, the donor tag is cumermycin, and the acceptor tag is GyrB.

[0035] In some embodiments, the donor tag and acceptor tag may be phytochrome B and abyssin, respectively. The binding of these tags can be controlled by light irradiation.

[0036] In one embodiment, the donor tag and acceptor tag may be DmrA and DmrC, respectively. These tags are concatenated in the presence of AP20187.

[0037] In some embodiments, the donor tag and acceptor tag may be FKBP and DmrB, respectively. These tags bind together in the presence of Shield-1.

[0038] In one embodiment, the donor tag and acceptor tag may be avidin (e.g., oocyte avidin, streptavidin, neutravidin, etc.) and biotin, respectively.

[0039] In addition, various combinations of donor tags and acceptor tags can be used as appropriate.

[0040] In one embodiment, the three-dimensional artificial kinetic chorodor scaffold may further include a linker having two acceptor tags and a spacer (particularly a protein spacer) between the two acceptor tags. This linker is advantageous in connecting supports together to form a larger support.

[0041] In some embodiments, a three-dimensional artificial kinetic chorodor scaffold may have an average diameter of approximately 300 nm to 1 μm, 400 nm to 900 nm, 400 nm to 800 nm, or 500 nm to 700 nm. The diameter may be, for example, a hydrodynamic diameter and can be determined by dynamic light scattering (DLS) spectroscopy. The hydrodynamic diameter (Dh) can be calculated, for example, by the following formula.

number

[0042] In one embodiment, the three-dimensional artificial kinetic chorodor scaffold is in sheet form and may have an area of ​​approximately 300 nm to 1 μm squared, 400 nm to 900 nm squared, 400 nm to 800 nm squared, or 500 nm to 700 nm squared. The thickness is not particularly limited but may be, for example, approximately 50 nm to 300 nm.

[0043] In one embodiment, a fusion protein of NDC80 and FKBP, or a nucleic acid encoding the same, is provided. In another embodiment, a fusion protein of NUF2 and FKBP, or a nucleic acid encoding the same, is provided. The nucleic acid may be DNA or RNA (particularly mRNA).

[0044] In this disclosure, a three-dimensional artificial kinetochore scaffold can be produced by introducing nucleic acids encoding a portion of the proteins constituting the kinetochore (particularly including NDC80 and NUF2) into oocytes, thereby expressing the constituent proteins, including NDC80 and NUF2, from the nucleic acids within the oocytes and allowing them to self-assemble. Alternatively, in this disclosure, a three-dimensional artificial kinetochore scaffold may be produced by in vitro translating the constituent proteins from nucleic acids encoding a portion of the proteins constituting the kinetochore (particularly including NDC80 and NUF2), allowing them to self-assemble. The introduction of nucleic acids or proteins into cells is not particularly limited, but can be achieved, for example, by microinjection.

[0045] In some cases, (1)(i) a fusion protein containing NDC80 and a proteinoid donor tag and NUF2, or (ii) a nucleic acid encoding the fusion protein and a nucleic acid encoding NUF2; (2)(i) a fusion protein comprising NDC80 and a protein-based acceptor tag and NUF2, or (ii) a nucleic acid encoding the fusion protein and a nucleic acid encoding NUF2; (3)(i) a fusion protein containing NUF2 and a proteinoid donor tag and NDC80, or (ii) a nucleic acid encoding the fusion protein and a nucleic acid encoding NDC80; or (4)(i) a fusion protein comprising NUF2 and a protein-based acceptor tag and NDC80, or (ii) a nucleic acid encoding the fusion protein and a nucleic acid encoding NDC80; A combination including the above is provided. The combination of acceptor tag and donor tag is as described above. In one embodiment, the proteinoid donor tag is an FK506-binding protein (FKBP), and the proteinoid acceptor tag is an FKBP-rapamycin-binding domain (FRB).

[0046] In one embodiment, a combination (a second combination) is provided which includes one combination (a first combination) selected from the group consisting of (1)(i), (2)(i), (3)(i), and (4)(i), and a fusion protein of the proteinial acceptor tag and the polymerization domain.

[0047] In one embodiment, a combination is provided which includes any one combination selected from the group consisting of (1)(i), (2)(i), (3)(i), and (4)(i) (a first combination), and a combination which includes two proteinoid donor tags and a linker protein having a proteinoid spacer between the two proteinoid donor tags (a third combination).

[0048] In one embodiment, a combination is provided which includes any one combination selected from the group consisting of (1)(i), (2)(i), (3)(i), and (4)(i) (a first combination), a fusion protein of the proteinial acceptor tag and the polymerization domain, and a linker protein having two proteinial donor tags and a proteinial spacer between the two proteinial donor tags (a fourth combination).

[0049] In one embodiment, a combination (combination 2-2) is provided, which includes one combination selected from the group consisting of (1)(ii), (2)(ii), (3)(ii), and (4)(ii) (combination 2-1) and a nucleic acid encoding a fusion protein of the proteinial acceptor tag and the polymerization domain.

[0050] In one embodiment, a combination is provided which includes any one combination selected from the group consisting of (1)(ii), (2)(ii), (3)(ii), and (4)(ii) (combination 2-1), and a combination which includes two proteinoid donor tags and a nucleic acid encoding a linker protein having a proteinoid spacer between the two proteinoid donor tags (combination 2-3).

[0051] In one embodiment, a combination is provided (combination 2-1) selected from the group consisting of (1)(ii), (2)(ii), (3)(ii), and (4)(ii), a nucleic acid encoding a fusion protein of the proteinial acceptor tag and the multimerization domain, and a nucleic acid encoding a linker protein having two proteinial donor tags and a proteinial spacer between the two proteinial donor tags.

[0052] In one embodiment, any of the above combinations may further include either or both of the nucleic acids encoding SPC24 and the nucleic acids encoding SPC25.

[0053] The multimerizing domain is not particularly limited, but for example, it forms a homomultimer. The multimerizing domain is also not particularly limited, but for example, it forms about 50 to 400 units (e.g., about 50 to 300 units, about 60 to 240 units, for example, about 120 units). The multimerizing domain is not particularly limited, but for example, encapsulin can be used. Encapsulin self-assembles to form an icosahedral structure. The structure thus formed may contain 60 to 240 encapsulin units. By linking encapsulin with unit complexes, a three-dimensional artificial kinetochore scaffold can be constructed that expresses a number of unit complexes corresponding to the number of encapsulin units in the structure. Other examples of multimerizing domains include hepatitis B virus core protein (forming about 240 units) and human papillomavirus capsid protein (forming about 360 units).

[0054] The fusion protein of a proteinial acceptor tag and a multimerizing domain may contain a spacer or a fluorescent protein (not particularly limited, but such as green fluorescent protein (GFP) and EGFP). The fluorescent protein may be inserted between the proteinial acceptor tag and the multimerizing domain. The fusion protein of a proteinial acceptor tag and a multimerizing domain may contain the multimerizing domain and the proteinial acceptor tag from the N-terminus to the C-terminus. The N-terminus of the fusion protein may further contain a tag {preferably a tag that does not interact with either the acceptor tag or the donor tag, and not particularly limited, but such as an hAG tag}.

[0055] The linkage between the multimerizing domain (e.g., encapsulin) and the unit complex can be direct. For example, a fusion protein containing a multimerizing domain (e.g., encapsulin) and NDC80 or NUF2 may be provided, and a unit complex containing such a fusion protein may be provided. Preferably, the multimerizing domain (e.g., encapsulin) and the unit complex may be linked via acceptor tags and donor tags attached to each. In this way, the use of linkers can enlarge the size of the three-dimensional artificial kinetochore scaffold, potentially reducing the number of microtubules on the chromosomes by removing more microtubules and thereby increasing the effect of suppressing premature chromosome segregation.

[0056] Encapsulin is widely found in bacteria and archaea. Examples of bacteria include actinomycetes (e.g., Mycobacterium tuberculosis, Streptomyces), Bacillus sativus, Clostridium, Deinococcus teleradiodurans, Rhodococcus, Nitrosomonas, Leptospira, Acidithiobacillus, and Synechococcus. Examples of archaea include methanogenic archaea, haloarchaea, hyperthermophilic archaea, and acidic archaea. Pyrcoccocus furiosus This is another example. Pyrcoccocus furiosus Encapsulin has an amino acid sequence registered, for example, in NCBI Reference Sequence: WP_237718489.1, and forms an icosahedral structure with 120 amino acids.

[0057] When using a multimerizing domain (e.g., encapsulin) as the support portion, in order to display more than 10,000 unit complexes on a three-dimensional artificial kinetochore scaffold, it is possible to form aggregates of numerous linked unit structures formed by the self-assembly of the multimerizing domain (e.g., encapsulin). This aggregate formation can be achieved by linking the unit complexes together. Linking may be done via a linker. For example, aggregate formation can be achieved using a fusion protein of a multimerizing domain (e.g., encapsulin) and an acceptor tag, and a linker protein having two donor tags with a spacer between them. By configuring it in this way, multimerized complexes of multimerizing domains (e.g., encapsulin) expressing many acceptor tags can be obtained, and these multimerized complexes can be further linked by a linker.

[0058] Therefore, the above combination may further include nucleic acids encoding a fusion protein of a multimerization domain (e.g., encapsulin) and an acceptor tag, and a linker protein having two donor tags with a spacer between the donor tags.

[0059] In one preferred embodiment, all nucleic acids are mRNA. mRNA has a structure suitable for translation. For example, mRNA includes a 5' cap structure, a 5' untranslated region (UTR), an open reading frame (ORF), a 3' UTR, and polyadenine (poly-A). In another embodiment, the nucleic acid is expressibly incorporated into the genome of a viral vector. In another embodiment, all nucleic acids are DNA. DNA is operably ligated to a promoter (particularly the promoter of RNA polymerase II). DNA can be circular or linear and can be an expression vector such as a plasmid or a viral genome.

[0060] Promoters typically bind RNA polymerase II. Promoters include, for example, a TATA box, an initiator (Inr) sequence, and a TFIIB recognition element (BRE). While not particularly limited, examples of promoters include the CMV promoter, SV40 promoter, RSV promoter, HIV promoter, β-actin promoter, EF1α promoter, PGK promoter, GAPDH promoter, CAG promoter, EF1α-HTLV promoter, and SV40-HTLV promoter. Inducible promoters may also be used. Examples of inducible promoters include the tetracycline-inducible promoter (TET promoter) and the GAL promoter.

[0061] The combination may contain each component as a mixture or as separate components. The combination may be a solution or a freeze-dried product.

[0062] Since the composition is introduced into cells, it has a physiologically acceptable composition. In some embodiments, the composition has a medically acceptable composition. The introduction of the composition into cells is not particularly limited, but can be carried out, for example, by microinjection.

[0063] The composition may contain additives in addition to the three-dimensional artificial kinetic chorodor scaffold. The additives are not particularly limited, but examples include solvents (e.g., water), salts, pH buffers, isotonic agents, thickeners, preservatives, stabilizers, and dispersants.

[0064] In some embodiments, the three-dimensional artificial kinetochore scaffold of this disclosure can reduce the amount of microtubule binding to chromosomes during meiosis I and meiosis II of oocytes. In some embodiments, the three-dimensional artificial kinetochore scaffold of this disclosure can prevent premature chromosome segregation during meiosis I and meiosis II of oocytes.

[0065] <Method of using the 3D artificial kinetic chariot scaffold of this disclosure> The three-dimensional artificial kinetochore scaffold of this disclosure can be injected into cells (particularly oocytes). This allows the three-dimensional artificial kinetochore scaffold of this disclosure to be aligned with the spindle equator during cell division (particularly oocytes). This also prevents premature chromosome segregation during meiosis I and II of oocytes.

[0066] Accordingly, the present disclosure provides a method for aligning a three-dimensional artificial kinetochore scaffold to the spindle equator during cell division (particularly in oocytes).

[0067] The three-dimensional artificial kinetochore scaffold possesses at least microtubule-binding ability and sufficient physical strength to withstand tension from both poles. This allows the three-dimensional artificial kinetochore scaffold to align with the spindle equator of a dividing cell. When the three-dimensional artificial kinetochore scaffold aligns with the spindle equator, the amount of microtubules binding to the endogenous chromosome kinetochore is reduced. This reduction has the effect of suppressing premature chromosome segregation.

[0068] Alignment of the spindle to the equator can be determined, for example, by whether or not a three-dimensional artificial kinetic choroid scaffold exists within 5 μm, 4 μm, 3 μm, 2 μm, or 1 μm from the M plate.

[0069] The three-dimensional artificial kinetic scaffold may preferably be one of those described above.

[0070] According to this disclosure, A method for preventing premature chromosome segregation during meiosis I and II of oocytes (e.g., human oocytes); or A method for suppressing chromosomal abnormalities in oocytes (e.g., human oocytes) after meiosis I and meiosis II. These methods are provided. These methods involve introducing an effective amount of a three-dimensional artificial kinetochore scaffold into oocytes. The effective amount is the amount that produces an effect. Early chromosome segregation refers to the phenomenon in which chromosomes segregate during prophase and metaphase of meiosis, which consists of prophase, metaphase, and anaphase.

[0071] The introduction of a three-dimensional artificial kinetochore scaffold into cells can be achieved by introducing a three-dimensional artificial kinetochore scaffold formed extracellularly into the cells, or by introducing nucleic acids containing the elements necessary for the intracellular construction of a three-dimensional artificial kinetochore scaffold into the cells and allowing them to self-assemble within the cells. In this process, the three-dimensional artificial kinetochore scaffold, nucleic acids, or combinations described above can be used.

[0072] The methods described herein may be useful in infertility treatments such as assisted reproductive technology using animal (particularly human) oocytes, especially oocytes from older individuals, for example, women aged 25-30, 30-35, 35-40, or older.

[0073] In some embodiments, the methods described herein may be industrially applicable inventions. In some embodiments, the methods described herein may not involve medical procedures.

[0074] This disclosure provides a method for observing a three-dimensional artificial kinetochore scaffold. This method includes preparing cells (particularly oocytes) into which the three-dimensional artificial kinetochore scaffold has been introduced, and observing the cells (particularly oocytes) during cell division (particularly meiosis). The observation can be performed, for example, under a stereomicroscope. To facilitate observation, the three-dimensional artificial kinetochore scaffold is preferably labeled with a fluorescent dye (particularly a fluorescent protein). For example, this can be advantageously achieved by composing a fusion protein with a fluorescent protein as part of the constituent proteins of the three-dimensional artificial kinetochore scaffold. This is also advantageous because it allows for confirmation of the alignment of the three-dimensional artificial kinetochore scaffold to the spindle equator. If the labeling is proteinaceous, it is further advantageous in terms of high biocompatibility and biodegradability.

[0075] Oocytes that have not yet undergone meiosis I are used in in vitro maturation techniques for immature oocytes. These oocytes can be harvested from the ovary when the follicular diameter reaches 7-10 mm, while monitoring the development of the ovarian follicular cells using ultrasound. Oocyte retrieval can be performed transvaginally by inserting a thin needle into the ovarian follicular cavity using ultrasound guidance and aspirating the follicular fluid.

[0076] Oocytes in metaphase II of meiosis can be collected by administering hormones such as gonadotropins to stimulate the ovaries and develop follicles, monitoring follicle growth and maturity, and then administering human chorionic gonadotropin (hCG) or LH to induce final maturation of the oocytes when they are fully mature. 34 to 48 hours after hCG administration, a thin needle is inserted transvaginally into the follicle in the ovary using ultrasound guidance, and the follicular fluid is aspirated.

[0077] By injecting the three-dimensional artificial kinetochore scaffold of this disclosure into the cytoplasm of these oocytes, the three-dimensional artificial kinetochore scaffold can be aligned with the spindle equator during metaphase. This reduces the amount of microtubules that bind to chromosomes. This reduction in the amount of bound microtubules can prevent premature chromosome segregation.

[0078] As described above, this disclosure provides a method for injecting a three-dimensional artificial kinetochore scaffold into an oocyte. The oocyte may be a cell in the pre-meiotic I stage or a cell in the metaphase of meiotic II. An effective amount of the three-dimensional artificial kinetochore scaffold is injected into the oocyte.

[0079] This can prevent premature chromosome segregation in meiosis I and / or meiosis II. Accordingly, the present disclosure provides a method for treating oocytes, comprising injecting an effective amount of the three-dimensional artificial kinetochore scaffold of the present disclosure into the oocytes and aligning the three-dimensional artificial kinetochore scaffold at the spindle equator in the oocytes. The method of the present disclosure can thereby reduce the amount of microtubule binding to chromosomes compared to the amount of microtubule binding to chromosomes in a control that does not receive the three-dimensional artificial kinetochore scaffold. The reduction may be, for example, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, or 50% or more. In this way, the method of the present disclosure can reduce premature chromosome segregation.

[0080] This disclosure provides a three-dimensional artificial kinetochore scaffold for use in the above method. This disclosure also provides a composition comprising a three-dimensional artificial kinetochore scaffold for use in the above method. This disclosure also provides nucleic acids, or combinations thereof, that encode components of a three-dimensional artificial kinetochore scaffold for use in the above method.

[0081] This disclosure provides the use of the three-dimensional artificial kinetochore scaffold of this disclosure in the manufacture of a pharmaceutical product for use in the above method. It also provides the use of nucleic acids encoding components of the three-dimensional artificial kinetochore scaffold of this disclosure, or combinations thereof, in the manufacture of a pharmaceutical product for use in the above method. [Examples]

[0082] In the embodiment, a three-dimensional artificial kinetochore scaffold is constructed. In particular, a biodegradable three-dimensional artificial kinetochore scaffold is constructed. As an example, by making the three-dimensional artificial kinetochore scaffold composed of protein, all the components of the scaffold can be supplied by nucleic acids, and the three-dimensional artificial kinetochore scaffold can be obtained through protein production and self-assembly within cells that receive nucleic acids.

[0083] Materials and methods mouse All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at the RIKEN Kobe Branch. Female B6D2F1 (C57BL / 6×DBA / 2) mice were used to obtain oocytes. Mice aged 18-20 months were used for aging experiments, and 8-12 weeks were used for other experiments.

[0084] Mouse oocyte culture Mice were intraperitoneally injected with 0.2 ml (10 IU) of pregnant mare serum gonadotropin (PMSG) or 0.1 ml of CARD HyperOva (KYUDO). Fully developed germinal vesicle (GV) oocytes were collected 48 hours after PMSG injection. Oocytes were cultured at 37°C in M2 medium containing 200 μM 3-isobutyl-1-methylxanthine (IBMX, Sigma). Restart of meiosis was induced by washing with IBMX. If instructed, 10 μM nocodazole (Sigma), 2 μM ProTAME (Bio-Techne R&D Systems), or 500 nM rapamycin (Funakoshi) was added to the medium immediately after IBMX washing.

[0085] RNA microinjection mRNA was transcribed and purified using the mMESSAGE mMACHINE T7 kit (Thermo Fisher Scientific). The mRNA was microinjected into fully grown GV oocytes and then incubated at 37°C for 2 hours until the IBMX washout. 0.3 pg of NDC80(ΔSPC-WT / ΔSPC-DFAA / FL-WT / FL-9A)-FKBP, 0.3 pg of NUF2, 0.3 pg of NUF2-HA, 0.45 pg of H2B-mCherry, 3.5 pg of hAG-Pfv(GEM)-Sapphire-GS-FRB, 0.15 pg of FKBP-GS-FKBP (linker), 1 pg of CENP-T-mCherry, and 1 pg of mCherry were injected. In the microbead experiment, 1 pg of NDC80-GFP, 1 pg of NUF2, and 1.5 pg of H2B-mCherry were injected.

[0086] Preparation of anti-GFP beads Rat anti-GFP antibody (D153-3, MBL; 1:30) was immobilized on protein G-conjugated beads (MonoMag Protein G Beads, 1 μm diameter MPG1000 / 3 μm diameter MPG3000, Ocean NanoTech; 1:20) and rotated in PBS at 4°C for 2.5 hours. The beads were washed and stored in PBS (hereinafter referred to as anti-GFP beads).

[0087] Bead microinjection Anti-GFP beads were introduced into mRNA-injected GV-phase oocytes using piezopulse microinjection. Four 3 μm diameter beads or 4 to 11 1 μm diameter beads were microinjected into each oocyte. The microinjected oocytes were cultured at 37°C for 1 hour before meiosis restarted.

[0088] Live video An LSM 900 confocal microscope (Carl Zeiss) equipped with a 40×C-Apochromat 1.2NA water immersion objective lens (Carl Zeiss) was controlled using Zen software and a custom-made multi-position autofocus macro. In the nocodazole treatment experiment, 81 z-confocal sections (at 0.5 μm intervals) consisting of 512×512 pixel xy images covering a total volume of 79.9 μm×79.9 μm×40 μm were recorded at 30-minute intervals. In other experiments, z-confocal sections (at 1 μm or 1.5 μm intervals) of 512×512 pixel xy images were recorded at 10-minute intervals. In the microbead experiment, an LSM780 confocal microscope (Carl Zeiss) equipped with a 40×C-Apochromat 1.2NA water immersion objective lens (Carl Zeiss) was controlled using Zen software with a multi-position autofocus macro PipelineConstructor. For imaging of chromosomes and beads, 25 z-confocal sections (1.25 μm apart) of 512 × 512 pixel xy images were recorded at 5-minute intervals for at least 12 hours after maturation induction, covering a total volume of 38.65 μm × 38.65 μm × 30 μm.

[0089] Immunofluorescence Oocytes were fixed at room temperature for 30 minutes with fixation buffer (100 mM PIPES (pH 7.0), 1 mM MgCl2, 0.1% Triton-X100, 1.6% formaldehyde). After four washes, oocytes were incubated overnight at 4°C in PBT (PBS + 0.1% Triton-X100). Oocytes were blocked at room temperature for 1 hour with 3% BSA-PBT and incubated overnight at 4°C with the primary antibody in 3% BSA-PBT. Oocytes were washed four times with 3% BSA-PBT and incubated with the secondary antibody and 5 μg / ml Hoechst33342 (Invitrogen) in 3% BSA-PBT at room temperature for 2 hours or overnight at 4°C. The primary antibodies used were mouse anti-HA (BioLegend, MMS-101R; 1:500), rabbit anti-DSN1 (donated by Dr. H. Shibuya; 1:500), rabbit anti-CENP-C (donated by Dr. Y. Watanabe; 1:500), mouse anti-mCherry (Clontech, 632543; 1:500), human anti-centromere antibody (ACA, Antibodies Incorporated, 15-234; 1:500), rabbit anti-HURP (Santa Cruz Biotechnology, sc-98809; 1:500), and rat anti-α-tubulin (YL1 / 2) (Bio-Rad, MCA77G; 1:1500). The secondary antibodies used were Alexa Fluor 555 goat anti-rat IgG(H+L) (A21434), goat anti-mouse IgG(H+L) (A21424), Alexa Fluor 647 goat anti-human IgG(H+L) (A21445), and donkey anti-rabbit IgG(H+L) (A31573) (1:500, Molecular Probes). Oocytes were imaged under a Zeiss LSM880 with Airyscan confocal microscope equipped with a 40×C-Apochromat 1.2NA water immersion objective lens (Carl Zeiss). In the nocodazole treatment experiment, z-confocal sections (at 0.5 μm intervals) were recorded from 512×512 pixel xy images. In the other experiments, z-confocal sections (at 0.2 μm intervals) were recorded from 512×512 pixel xy images.

[0090] 4D analysis Chromosomes and NDC80-GEMs were reconstructed in 3D using Imaris software (Bitplane). The spindle axis was manually determined based on the chromosome distribution at each time point. For 2 hours post-NEBD, before the spindle axis was established, the same spindle axis as at 3 hours post-NEBD was applied. The volume of NDC80-GEMs was calculated using Imaris software, and NDC80-GEMs located outside the spindle were manually excluded.

[0091] Signal strength measurement All fluorescence signal quantification was performed in Fiji. In the FWHM analysis, linear selection was performed through the center of the NDC80-GEM, and the density profile plot of the current selection was displayed. The output data was fitted to a Gaussian distribution, and the full width at half maximum (FWHM) was calculated. Ten NDC80-GEMs were randomly selected from each egg cell. The signal intensity on the NDC80-GEM was measured by randomly selecting 10 rectangular regions (4 pixels each). The signal intensity on the kinetic chorod was also measured by randomly selecting 10 rectangular regions (4 pixels each). For the signal intensity on the NDC80 beads, the average fluorescence intensity on the beads was measured and subtracted by the average intensity of the adjacent cytoplasmic region.

[0092] statistical analysis All graphs and statistical analyses were performed using R. All experiments were conducted independently at least three times.

[0093] result Design of protein-based kinetochore-like particles To design protein-based functional kinetochore-like particles, we referenced the design concept of NDC80 beads. NDC80 beads are spherical beads with a diameter of approximately 2 μm, carrying an NDC80 (SEQ ID NO: 1)-NUF2 (SEQ ID NO: 2) complex on their surface, and can establish a bioreientation-like state independently of SPC24-SPC25 recruitment. As a protein-based scaffold, we used gene-encoded fluorescent multimeric (GEM) nanoparticles with a diameter of ~40 nm. We fused the FKBP (FK506-binding protein)-rapamycin-binding domain (FRB) (SEQ ID NO: 8) to the GEM to obtain a GEM-FRB with the amino acid sequence described in SEQ ID NO: 9, and expressed it on the surface of the GEM particle (Figure 1A). An hAG tag (SEQ ID NO: 9) was added to the N-terminus of the GEM. Furthermore, FKBP (SEQ ID NO: 4) was added to NDC80Δ SPC NDC80Δ, which has the amino acid sequence described in SEQ ID NO: 6, is fused to the C-terminus of (SEQ ID NO: 4). SPC -FKBP was obtained (Figure 1A). NDC80Δ SPC This is a construct lacking the SPC24-SPC25-recruitment domain. mRNA encoding these proteins, along with mRNA encoding untagged NUF2 (SEQ ID NO: 2), were microinjected into the cytoplasm of mouse oocytes arrested in prophase I. Oocytes were synchronously induced into M phase in the presence of rapamycin, which triggers FKBP-FRB binding, thereby inducing GEM to NDC80Δ SPC This bridged the gap (the resulting form is referred to below as NDC80Δ SPC - (referred to as GEM) (Figure 1A). To evaluate microtubule-independent particle self-assembly, oocytes were further treated with nocodazole (Figure 6A), a drug that inhibits microtubule polymerization. Using a confocal microscope, numerous fluorescent spots distributed throughout the cytoplasm were observed (Figure 1B, C; "NDC80Δ SPC -GEM). These bright spots contained NUF2 (Figure 6B). The diameter of each bright spot was approximately 500 nm (Figure 1D, E), which is much larger than the diameter of a single GEM particle (40 nm), suggesting multiple NDC80ΔSPC - It was suggested that it is a cluster of GEM particles. In contrast, NDC80Δ SPC - When GEM was expressed without using FKBP, such fluorescent bright spots were not observed, and a uniform signal diffused throughout the cytoplasm was seen (Figure 1B, C; "GEM"). This is consistent with the formation of freely diffusing 40 nm particles that could not be separated by confocal microscopy. When there is a mutation in the loop domain of NDC80 that promotes the NDC80-NDC80 interaction, NDC80Δ SPC - The formation of GEM fluorescent bright spots was significantly reduced (Figure 1B, C; NDC80Δ SPC - DFAA-GEM). NDC80Δ SPC - The bright spots of DFAA-GEM were significantly smaller in diameter (Figure 1D, E), and NDC80Δ SPC - The bright spots of GEM were less fluorescent than those of NDC80Δ SPC - These results suggest that the NDC80 loop promotes the clustering of NDC80Δ SPC - GEM particles, resulting in the formation of sub-microscale NDC80Δ

[0094] NDC80Δ SPC - Cluster design of GEM particles Based on these observations, it was decided to verify whether larger NDC80-GEM clusters could be obtained by promoting GEM-GEM interactions. For this purpose, a linker protein with FKBP domains at both ends (FKBP-GS-FKBP (SEQ ID NO: 10), hereinafter abbreviated as "linker") was additionally expressed (Figure 1F). When this linker was co-expressed, significantly larger NDC80Δ SPC - GEM clusters were formed with an increase in fluorescence intensity (Figure 1G, H; hereinafter referred to as "NDC80Δ SPC - GEM-Linker") (Figure 6D). These clusters were generally 600 nm to 700 nm in diameter. Thus, the linker serves as a tool to expand the NED80Δ SPC - GEM clusters within the sub-microscale range.

[0095] NDC80Δ SPC - Alignment of GEM cluster size correlations Next, NDC80Δ SPC - We investigated the ability of GEM clusters to align with the spindle equator. Live imaging was performed throughout the M phase of meiosis I in oocytes without nocodazole, followed by 3D analysis. In the absence of a linker, NDC80Δ SPC -It was revealed that the majority of GEM clusters did not align with the spindle equator but were localized at the spindle poles (Figure 2A-C; 7A). This behavior is similar to that of approximately 25 nm particles linked by CENP-T in somatic cells (Sissoko et al., 2024). In contrast, when a linker was used, NDC80Δ SPC The proportion of GEM-linker clusters located within 5 μm of the spindle equator during metaphase significantly increased (Figure 2A-C; 7A), and although the proportion localized at the spindle poles remained considerable, it was shown that alignment ability had been acquired (Figure 2A-C). Aligned NDC80Δ SPC Tracking the GEM-Linker clusters revealed that approximately 80% maintained their position near the spindle equator for more than 60 minutes until the start of anaphase (Figure 2D, E). Aligned NDC80Δ SPC -GEM-Linker clusters often showed extension along the spindle axis by anaphase of metaphase (Figure 2A, F, G), and were shown to be pulled evenly toward the opposite spindle pole by microtubules. Consistent with this idea, these clusters remained in the central region of the spindle even after the chromosomes separated in anaphase (Figure 2A). These results are relevant to submicroscale NDC80Δ SPC -GEM clusters, while having significantly lower alignment capabilities compared to chromosomes, suggest that they can stably align in a size-correlated manner.

[0096] Due to the SPC24-SPC25 interaction, NDC80Δ SPC - GEM cluster alignment has been improved. Next, NDC80Δ SPC-We tested whether the alignment ability of the GEM-Linker cluster could be enhanced by recruiting more kinetochore proteins from the endogenous pool. For this purpose, we used full-length NDC80 (NDC80-NUF2), which includes a C-terminal domain that interacts with the SPC24-SPC25 subcomplex that links the outer kinetochore Mis12 complex to the inner kinetochore protein CENP-T. FL ) was used (Figure 8A). NDC80Δ SPC -Unlike GEM-Linker, NDC80 FL - The GEM-Linker cluster carried CENP-T (Figure 8B), but not DSN1 (Figure 8C), a component of the Mis12 complex. Notably, NDC80 FL -GEM-Linker increased NDC80Δ in nocodazole-treated oocytes. SPC -GEM-Linkers self-assembled into clusters that were slightly but significantly larger than those of GEM-Linkers (Figure 3A, B; 8D). In nocodazole-free oocytes, live imaging throughout the M phase of meiosis I revealed that NDC80 FL -GEM-Linker is NDC80Δ SPC - It aligned more efficiently than the GEM-Linker cluster, but NDC80 FL -A significant portion of the GEM-Linker cluster remained at the spindle pole (Figure 3C-E; 8E). Aligned NDC80 FL - Most GEM-Linker clusters maintained their position near the metaphase plate for 60 minutes leading up to the start of anaphase, but their alignment was not as stable as that of chromosome alignment (Figure 3F; 8F). These results were reported in NDC80. FL -The SPC24-SPC25 binding domain of GEM-Linker suggests that, although its alignment capability is still limited compared to chromosomes, it promotes particle clustering and efficient alignment.

[0097] The NDC80-GEM-Linker cluster establishes a state similar to bioremission. The alignment of NDC80-GEM-Linker clusters suggests the ability to establish a state similar to biorelation due to bipolar microtubule attachment. Immunostaining of the kinetochore microtubule marker HURP (Curr. Biol. 16, 731-742, 2006) revealed the alignment of NDC80 FL -65% of GEM-Linker clusters showed bipolar attachment of HURP-positive microtubule bundles during metaphase (Figure 3G). In contrast, aligned NDC80Δ SPC Only 26% of the -GEM-Linker clusters showed bipolar adhesion (Figure 3G). Thus, the alignment ability of NDC80-GEM-Linker clusters correlates with the efficiency of bipolar attachment of HURP-positive microtubules. Notably, when metaphase was prolonged by treatment with the anaphase inhibitor proTAME, bipolar microtubule attachment was observed in aligned NDC80Δ SPC -GEM-Linker and NDC80 FL -This was observed in the majority of both GEM-linker clusters (Figure 3H). These results are found in NDC80Δ SPC -GEM-Linker and NDC80 FL This suggests that both GEM-Linker clusters can establish a bioreientation-like state, and that the efficiency of this establishment is enhanced by the SPC24-SPC25 binding domain of NDC80.

[0098] The NDC80-GEM-Linker cluster is not preferentially located in the inner region of the mid-term board. Since the ability of NDC80-GEM-Linker clusters to establish a biolientation-like state is detectable but limited, it was decided to compare them with NDC80 beads, which efficiently and robustly establish a biolientation-like state similar to chromosomes (Asai et al., 2024). The biolientation-like state of NDC80 microbeads preferentially locates in the inner region of the anatomical lamina, displacing chromosomes from the inner region (Asai et al., 2024; Takenouchi et al., 2024). On the other hand, NDC80 FL-GEM-Linker clusters did not preferentially occupy the inner region of the anacleidomastia, nor did they interfere with their positioning within the chromosome (Figure 4A-C). We are NDC80 FL We hypothesized that the microscale size of the NDC80 beads, rather than the submicroscale size of the GEM-Linker cluster, facilitates their preferential internal positioning. Consistent with this hypothesis, NDC80 beads with a diameter of ~1 μm did not preferentially occupy the interior, unlike those with a diameter of ~3 μm (Figure 9A, B), but both showed the ability to align on the metaphase plate (Figure 9C, D). There was no significant difference in average fluorescence intensity between beads of different sizes (Figure 9E), indicating that the density of NDC80 on the bead surface was equivalent. These results suggest that NDC80 FL - The submicroscale size of the GEM-Linker cluster suggests that it enables bioreientation-like states without preferential internal positioning.

[0099] NDC80-GEM-Linker clusters compete with kinetochores for HURP-decorated microtubule fibers. These results allowed us to investigate whether artificial kinetochores affect the state of chromosomal biorelation, separate from their effect on chromosome positioning. Careful evaluation of immunofluorescence signals revealed that NDC80 FL -GEM-Linker clusters significantly reduced the thickness of HURP-decorated microtubule bundles attached to chromosomal centromeres (Figure 4D, E). Nevertheless, NDC80 FL The GEM-linker cluster did not increase chromosomal misalignment (Figure 4F) or aneuploidy rate in metaphase II eggs (Figure 10A, B). Based on these results, NDC80 FL -GEM-Linker clusters reduce the number of microtubule fibers attached to the kinetochore, but this is suggested to have little adverse effect on the fidelity of chromosome segregation.

[0100] Based on these observations, we hypothesized that artificial kinetochores compete with chromosomal kinetochores for a finite number of components of HURP-decorated microtubule fibers. If this hypothesis holds true, then NDC80 FL - Increasing the microtubule binding activity of the GEM-Linker cluster should reduce the number of HURP-decorated microtubules attached to the kinetochore. To test this possibility, we used NDC80-9A (SEQ ID NO: 11), a phosphorylation-deficient mutant with higher microtubule binding affinity. Consistent with the hypothesis, NDC80 FL -9A-GEM-Linker cluster is NDC80 FL -GEM-Linker clusters significantly reduced the thickness of microtubule bundles decorated with HURP attached to chromosomal centromeres (Figure 4G, H). Furthermore, NDC80 FL The -9A-GEM-Linker cluster significantly increased chromosomal shifts (Figure 4I). These results suggest that artificial kinetochores compete with chromosomal kinetochores for a finite pool of HURP-decorated microtubule components in the spindle.

[0101] The NDC80-GEM cluster prevents premature chromosome separation associated with prolonged meiosis I in senescent oocytes. Based on these observations, we investigated whether the NDC80-GEM-Linker cluster can suppress premature chromosome segregation in aged oocytes. We used aged mouse oocytes treated with proTAME and arrested at metaphase I as a model for a highly sensitive assay to detect the effect on premature chromosome segregation (Takenouchi et al., 2024). Notably, rapamycin-induced NDC80 FL -GEM-Linker clusters significantly reduced the frequency of premature chromosome segregation (Figure 5A, B). In contrast, NDC80 FLTreatment with rapamycin in the absence of the GEM-Linker component did not significantly affect the frequency of early chromosome segregation (Figure 11A, B). This suppressive effect is consistent with the idea that it is due to a reduction in microtubule fibers decorated with HURP attached to the chromosomal centromere, and HURP overexpression significantly increased early chromosome segregation in senescent oocytes (Figure 5D, E). These results were presented in NDC80 FL We have shown that the GEM-Linker cluster effectively suppresses premature chromosome segregation during prolonged metaphase I in senescent oocytes by causing competition between chromosomal centromeres and HURP-decorated microtubule fibers without affecting chromosome position or alignment.

[0102] NDC80-GEM clusters prevent premature separation of sister chromatids during metaphase II in senescent oocytes. In aged mouse oocytes, early separation of sister chromatids during metaphase II of meiosis, when the oocyte spontaneously arrests before fertilization, is more frequent than early separation of chromosomes during metaphase I of meiosis. This is utilized in NDC80 FL The effect of GEM-Linker clusters on the early separation of sister chromatids during metaphase II of meiosis could be sensitively evaluated without drug-induced cell cycle arrest. Surprisingly, NDC80 FL -GEM-Linker clusters significantly reduced the premature separation of sister chromatids during metaphase II in senescent oocytes (Figure 5F-H). NDC80 FL Consistent with the idea that the GEM-Linker cluster attenuates the tension mediated by microtubules on chromosomal kinetochores, the distance between paired sister kinetochores was significantly reduced (Figure 5I). Nevertheless, NDC80 FL -GEM-linker clusters did not significantly impair the alignment of paired sister chromatids (Figure 5J). These results were found in NDC80 FL We have shown that the GEM-Linker cluster effectively prevents premature separation of sister chromatids in aging oocytes without affecting chromosomal alignment during metaphase II of meiosis.

[0103] discussion This study demonstrated that genetically engineered, protein-based artificial kinetochore clusters establish a bioreientation-like state in the spindle, effectively preventing premature chromosome segregation in both meiosis I and II. The artificial kinetochore clusters act as decoys, competing with chromosomal kinetochores for a limited number of components of kinetochore microtubules, including HURP. As a result, the artificial kinetochore clusters reduce the tension exerted by microtubules on chromosomes, preventing premature chromosome segregation, a major cause of chromosome segregation errors in aging oocytes.

[0104] The protein-based artificial kinetochore clusters designed in this study appear as fluorescent spots with a diameter of approximately 700 nm, smaller than the previously reported NDC80 beads with a diameter of approximately 2 μm. Individual submicroscale artificial kinetochore clusters are less efficient at establishing something like biorelation compared to larger microscale NDC80 beads. This is consistent with the idea that a larger scaffold promotes NDC80-NUF2-mediated biorelation. Nevertheless, expressing a large number of submicroscale artificial kinetochore clusters can effectively prevent premature chromosome segregation. This is likely due to a protein-based self-assembly design that allows oocytes to produce large quantities of functional clusters. This is in contrast to strategies using NDC80 beads, where the number of beads that can be microinjected is technically limited to about six per oocyte.

[0105] In young oocytes, the strength of the microtubule-induced chromosome pull may have evolved to be optimal for chromosome segregation, but this may not be the case in aged oocytes. As chromosome binding forces weaken with age, the microtubule-induced pull makes chromosomes more prone to premature segregation. In aged oocytes, a weaker pull than that optimal for young oocytes may be better suited to minimizing chromosome segregation errors. This study introduces a strategy to weaken the chromosome-induced pull using artificial kinetochores as decoys. By carefully controlling the amount and strength of the kinetochores without interfering with chromosome alignment or segregation, it was demonstrated that premature chromosome segregation could be prevented in aged oocytes during prolonged meiosis I and metaphase II. This protein-based artificial kinetochore decoy strategy may pave the way for the development of degradable tools to prevent chromosome segregation errors in human oocytes.

[0106] Explanation of the sequence list Sequence ID 1 (Amino acid sequence of mouse NDC80) MKRSSVSTCGAGRLSMQELRTLDLNKPGLYTPQTKERSTFGKLSTHKPTSERKVSIFGKRTSGHGSRNSQLGIFSSSEKIKDPRPLNDKAFIQQCIRQLYEFLTENGYVYSVSMKSLQAPSTKEFLKIFAFLYGFLCPSYELPGTKCEEEVPRIFKALGY PFTLSKSSMYTVGAPHTWPHIVAALVWLIDCIKIDTAMKESSPLFDDGQLWGEETEDGIKHNKLFLEYTKKCYEKFMTGADSFEEEDAELQAKLKDLYKVDASKLESLEAENKALNEQIARLEEEREREPNRLMSLKKLKASLQADVQNYKAYMSNLESHL AVLKQKSNSLDEEIGRVEQECETVKQENTRLQSIVDNQKYSVADIERINHEKNELQQTINKLTKDLEAEQQQMWNEELKYARGKEAIEAQLAEYHKLARKLKLIPKGAENSKGYDFEIKFNPEAGANCLVKYRTQVYAPLKELLNESEEEINKALNKKRH LEDTLEQLNTMKTESKNTVRMLKEEIQKLDDLHQQAVKEAEEKDKKSASELESLEKHKHLLESGVNDGLSEAMDELDAVQREYQLTVKTTTEERRKVENNLQRLLEMVATHVGSLEKHLEEENAKADREYEEFMSEDLLENIREMAEKYKRNAAQLKAPDK Sequence ID 2 (amino acid sequence of mouse NUF2) METLSFPRYNVAELVVHIRNKLLTGADGKNLSKSDLPNPKSDVLYMIYMKALQLVYGVRLEHFYMMPMNIEVTYPHLMEGLPVRSLFFYMDSFMPICRVNDFEIVDILNPRTN RTSRFLSGIINFIHFRETCLEKCEEFLLQNKSSMVRMQQLSNVHQEALMKLEKLNTVPAEEREEFKQFMDDIQELQHLLNEEFRQKTTLLQEEYAKMKSDISEKTKHLNEQKLSLV SLKEVEDNLKSKIVDSPEKLKNYKDKMKGTVQKLRSAREKVMEQYDIYRDSVDCLPSCQLEVQLYQKKSQDLADNREKLSSLLKESLNLEDQIESDSSELKKLKTEENSLIRMTTV KKEKLATARFKINKKQEDVKHYKQAMIEDCNKVQEKRDAVCEQVTTVNQEIHKIKSAIQQLRDTKKREILKSQEIFVNLKSALEKYHEGIEKVAEERSAKLEEKTAELKKRMVRMV Sequence ID 3 (amino acid sequence of mouse NDC80ΔSPC) MKRSSVSTCGAGRLSMQELRTLDLNKPGLYTPQTKERSTFGKLSTHKPTSERKVSIFGKRTSGHGSRNSQLGIFSSSEKIKDPRPLNDKAFIQQCIRQLYEFLTENGYVYSVSMKSLQAPSTKEFLKIFAFLYGFLCPSYELPGTKCEEEVP RIFKALGYPFTLSKSSMYTVGAPHTWPHIVAALVWLIDCIKIDTAMKESSPLFDDGQLWGEETEDGIKHNKLFLEYTKKCYEKFMTGADSFEEEDAELQAKLKDLYKVDASKLESLEAENKALNEQIARLEEEREREPNRLMSLKKLKASLQA DVQNYKAYMSNLESHLAVLKQKSNSLDEEIGRVEQECETVKQENTRLQSIVDNQKYSVADIERINHEKNELQQTINKLTKDLEAEQQQMWNEELKYARGKEAIEAQLAEYHKLARKLKLIPKGAENSKGYDFEIKFNPEAGANCLVKYRTQVY APLKELLNESEEEINKALNKKRHLEDTLEQLNTMKTESKNTVRMLKEEIQKLDDLHQQAVKEAEEKDKKSASELESLEKHKHLLESGVNDGLSEAMDELDAVQREYQLTVKTTTEERRKVENNLQRLLEMVATHVGSLEKHLEEENAKADREY Sequence ID 4 (amino acid sequence of FKBP) GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE Sequence ID No. 5 (amino acid sequence of NDC80-FKBP fusion protein) MKRSSVSTCGAGRLSMQELRTLDLNKPGLYTPQTKERSTFGKLSTHKPTSERKVSIFGKRTSGHGSRNSQLGIFSSSEKIKDPRPLNDKAFIQQCIRQLYEFLTENGYVYSVSMKSLQAPSTKEFLKIFAFLYGFLCPSYELPGTKCEEEVPRIFKALGYPFTLSKSSMYTVGAPHTWPHIVAALVWLIDCIKIDT AMKESSPLFDDGQLWGEETEDGIKHNKLFLEYTKKCYEKFMTGADSFEEEDAELQAKLKDLYKVDASKLESLEAENKALNEQIARLEEEREREPNRLMSLKKLKASLQADVQNYKAYMSNLESHLAVLKQKSNSLDEEIGRVEQECETVKQENTRLQSIVDNQKYSVADIERINHEKNELQQTINKLTKDLEAEQQ QMWNEELKYARGKEAIEAQLAEYHKLARKLKLIPKGAENSKGYDFEIKFNPEAGANCLVKYRTQVYAPLKELLNESEEEINKALNKKRHLEDTLEQLNTMKTESKNTVRMLKEEIQKLDDLHQQAVKEAEEKDKKSASELESLEKHKHLLESGVNDGLSEAMDELDAVQREYQLTVKTTTEERRKVENNLQRLLEM VATHVGSLEKHLEEENAKADREYEEFMSEDLLENIREMAEKYKRNAAQLKAPDKGPVSGSGSGSGGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLELELKLRILQSTVPRARDPVQVKRPL Sequence ID 6 (amino acid sequence of NDC80ΔSPC-FKBP fusion protein) MKRSSVSTCGAGRLSMQELRTLDLNKPGLYTPQTKERSTFGKLSTHKPTSERKVSIFGKRTSGHGSRNSQLGIFSSSEKIKDPRPLNDKAFIQQCIRQLYEFLTENGYVYSVSMKSLQAPSTKEFLKIFAFLYGFLCPSYELPGTKCEEEVPRIFKALGYPFTLSKSSMYTVGAPHTWPHIVAALVWL IDCIKIDTAMKESSPLFDDGQLWGEETEDGIKHNKLFLEYTKKCYEKFMTGADSFEEEDAELQAKLKDLYKVDASKLESLEAENKALNEQIARLEEEREREPNRLMSLKKLKASLQADVQNYKAYMSNLESHLAVLKQKSNSLDEEIGRVEQECETVKQENTRLQSIVDNQKYSVADIERINHEKNELQ QTINKLTKDLEAEQQQMWNEELKYARGKEAIEAQLAEYHKLARKLKLIPKGAENSKGYDFEIKFNPEAGANCLVKYRTQVYAPLKELLNESEEEINKALNKKRHLEDTLEQLNTMKTESKNTVRMLKEEIQKLDDLHQQAVKEAEEKDKKSASELESLEKHKHLLESGVNDGLSEAMDELDAVQREYQ LTVKTTTEERRKVENNLQRLLEMVATHVGSLEKHLEEENAKADREYGPVSGSGSGSGGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLELELKLRILQSTVPRARDPVQVKRPL Sequence ID 7 (amino acid sequence of GEM) MLSINPTLINRDKPYTKEELMEILRLAIIAELDAINLYEQMARYSEDENVRKILLDVAREEKAHVGEFMALLLNLDPEQVTELKGGFEEVKELTGIEAHINDNKKEESNVEYFEKLRSALLDGVNKGRSLLKHLPVTRIEGQSFRVDIIKF EDGVRVVKQEYKPIPLLKKKFYVGIRELNDGTYDVSIATKAGELLVKDEESLVIREILSTEGIKKMKLSSWDNPEEALNDLMNALQEASNASAGPFGLIINPKRYAKLLKIYEKSGKMLVEVLKEIFRGGIIVTLNIDENKVIIFANTPAV LDVVVGQDVTLQELGPEGDDVAFLVSEAIGIRIKNPEAIVVLEWSGSGSGSGSGSGRRIPGLIKMSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFSYGVQCFARYPDHMKQHDFFKSA MPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSIQSALSKDPNEKRDHMVLLEFVTAAGITHGIDELYK Sequence ID 8 (amino acid sequence of FRB) ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK Sequence ID 9 (amino acid sequence of hAG) MVSVIKPEMKIKLCMRGTVNGHNFVIEGEGKGNPYEGTQILDLNVTEGAPLPFAYDILTTVFQYGNRAFTKYPADIQDYFKQTFPEGYHWERSMTYEDQGICTATSNISMRGD CFFYDIRFDGVNFPPNGPVMQKKTLKWEPSTEKMYVRDGVLKGDVNMALLLEGGGHYRCDFKTTYKAKKDVRLPDYHFVDHRIEILKHDKDYNKVKLYENAVARYSMLPSQAK Sequence ID 9 (amino acid sequence of GEM-FRB fusion protein) MVSVIKPEMKIKLCMRGTVNGHNFVIEGEGKGNPYEGTQILDLNVTEGAPLPFAYDILTTVFQYGNRAFTKYPADIQDYFKQTFPEGYHWERSMTYEDQGICTATSNISMRGDCFFYDI RFDGVNFPPNGPVMQKKTLKWEPSTEKMYVRDGVLKGDVNMALLLEGGGHYRCDFKTTYKAKKDVRLPDYHFVDHRIEILKHDKDYNKVKLYENAVARYSMLPSQAKASATGMLSINPT LINRDKPYTKEELMEILRLAIIAELDAINLYEQMARYSEDENVRKILLDVAREEKAHVGEFMALLLNLDPEQVTELKGGFEEVKELTGIEAHINDNKKEESNVEYFEKLRSALLDGVNK GRSLLKHLPVTRIEGQSFRVDIIKFEDGVRVVKQEYKPIPLLKKKFYVGIRELNDGTYDVSIATKAGELLVKDEESLVIREILSTEGIKKMKLSSWDNPEEALNDLMNALQEASNASAGP FGLIINPKRYAKLLKIYEKSGKMLVEVLKEIFRGGIIVTLNIDENKVIIFANTPAVLDVVVGQDVTLQELGPEGDDVAFLVSEAIGIRIKNPEAIVVLEWSGSGSGSGSGSGRRIPGLI KMSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFSYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTL VNRIELKGIDFKEDGNILGHKLEYNFNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSIQSALSKDPNEKRDHMVLLEFVTAAGITHGIDELYK GGGGSLELKLSGSGSGSGSGSILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKAAALDK Sequence ID No. 10 (linker amino acid sequence) MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLESGSGSGSGLEGVQV ETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEKLRILQSTARDPVQVKRPL Sequence ID 11 (amino acid sequence of NDC80-9A) MKRAAVSACGAGRLAMQELRTLDLNKPGLYTPQTKERSTFGKLATHKPASERKVAIFGKRTAGGHGSRNAQLGIFSSSEKIKDPRPLNDKAFIQQCIRQLYEFLTENGYVYSVSMKSLQAPSTKEFLKIFAFLYGFLCPSYELPGTKCEEEVPRIFKALGY PFTLSKSSMYTVGAPHTWPHIVAALVWLIDCIKIDTAMKESSPLFDDGQLWGEETEDGIKHNKLFLEYTKKCYEKFMTGADSFEEEDAELQAKLKDLYKVDASKLESLEAENKALNEQIARLEEEREREPNRLMSLKKLKASLQADVQNYKAYMSNLESHL AVLKQKSNSLDEEIGRVEQECETVKQENTRLQSIVDNQKYSVADIERINHEKNELQQTINKLTKDLEAEQQQMWNEELKYARGKEAIEAQLAEYHKLARKLKLIPKGAENSKGYDFEIKFNPEAGANCLVKYRTQVYAPLKELLNESEEEINKALNKKRH LEDTLEQLNTMKTESKNTVRMLKEEIQKLDDLHQQAVKEAEEKDKKSASELESLEKHKHLLESGVNDGLSEAMDELDAVQREYQLTVKTTTEERRKVENNLQRLLEMVATHVGSLEKHLEEENAKADREYEEFMSEDLLENIREMAEKYKRNAAQLKAPDK

Claims

1. A composition comprising a three-dimensional artificial kinetochore scaffold containing a unit complex comprising NDC80 protein and NUF2 protein.

2. The composition according to claim 1, wherein the three-dimensional artificial kinetic choroid scaffold is biodegradable or proteinaceous.

3. The composition according to claim 1 or 2, wherein the number of unit complexes contained in the three-dimensional artificial kinetic chorodone scaffold is 10,000 to 50,000 or 20,000 to 40,000.

4. The composition according to any one of claims 1 to 3, wherein the three-dimensional artificial kinetochore scaffold has the ability to align with the spindle equator in an oocyte at least during metaphase I.

5. The composition according to any one of claims 1 to 4, wherein the three-dimensional artificial kinetic chorod scaffold comprises a unit complex and a support, the unit complex being bonded to the support and exposing the unit complex.

6. The composition according to claim 5, wherein the unit complex has a donor tag, the support has an acceptor tag, and the donor tag and the acceptor tag have binding affinity to each other, thereby binding the donor tag and the acceptor tag together.

7. The composition according to claim 5, wherein the donor tag is linked to NDC80.

8. The composition according to claim 6 or 7, wherein the support comprises a fusion protein including a multimerizing domain and a proteinacceptor tag, the multimerizing domains associate with each other to form a multimer, thereby the support presenting a plurality of proteinacceptor tags, and the unit complex is bound to the proteinacceptor tags via its donor tag.

9. The composition according to any one of claims 6 to 8, wherein the donor tag is an FK506-binding protein (FKB)-rapamycin-binding domain (FRB) and the acceptor tag is FKB.

10. The composition according to any one of claims 6 to 9, wherein the three-dimensional artificial kinetic scaffold includes a linker having two acceptor tags and a spacer connecting the acceptor tags.

11. A fusion protein of NDC80 or NUF2 and FK506-binding protein (FKBP), or a nucleic acid encoding said fusion protein.

12. (1) (i) a fusion protein comprising NDC80 and a proteinoid donor tag and NUF2, or (ii) a nucleic acid encoding the fusion protein and a nucleic acid encoding NUF2; (2) (i) a fusion protein comprising NDC80 and a protein-based acceptor tag and NUF2, or (ii) a nucleic acid encoding the fusion protein and a nucleic acid encoding NUF2; (3) (i) a fusion protein comprising NUF2 and a proteinoid donor tag and NDC80, or (ii) a nucleic acid encoding the fusion protein and a nucleic acid encoding NDC80; or (4) (i) a fusion protein comprising NUF2 and a proteinial acceptor tag and NDC80, or (ii) a nucleic acid encoding the fusion protein and a nucleic acid encoding NDC80; and The protein-based donor tag is an FK506-binding protein (FKB), and the protein-based acceptor tag is an FKB-rapamycin-binding domain (FRB). A combination of items.

13. A combination comprising one combination selected from the group consisting of (1)(i), (2)(i), (3)(i), and (4)(i) as described in claim 12 (a first combination), and a fusion protein of the proteinial acceptor tag and the polymerization domain (a second combination).

14. A combination comprising any one combination selected from the group consisting of (1)(i), (2)(i), (3)(i), and (4)(i) described in claim 12 (a first combination), and a combination comprising two proteinoid donor tags and a linker protein having a proteinoid spacer between the two proteinoid donor tags (a third combination).

15. A combination (fourth combination) comprising the combination according to claim 13 or 14 and a linker protein having a protein spacer between the two proteinoid donor tags.

16. A combination comprising any one combination selected from the group consisting of (1)(ii), (2)(ii), (3)(ii), and (4)(ii) as described in claim 12 (combination 2-1), and a nucleic acid encoding a fusion protein of the proteinial acceptor tag and the polymerization domain (combination 2-2).

17. A combination (combination 2-1) comprising any one combination selected from the group consisting of (1)(ii), (2)(ii), (3)(ii), and (4)(ii) as described in claim 12, and a combination (combination 2-3) comprising two proteinoid donor tags and a nucleic acid encoding a linker protein having a proteinoid spacer between the two proteinoid donor tags.

18. A combination (combination 2-4) comprising the combination according to claim 16 or 17 and a nucleic acid encoding a linker protein having a protein spacer between the two proteinaceous donor tags.

19. A method for suppressing premature chromosome segregation in oocytes, Introducing a three-dimensional artificial kinetochore scaffold with microtubule-binding ability into oocytes before or during meiosis, and Aligning a 3D artificial kinetic chorodor scaffold with the spindle equator. Methods that include...

20. The introduction of a 3D artificial kinetic scaffold is This is performed by injecting a three-dimensional artificial kinetochore scaffold constructed extracellularly into the cell, or, The method according to claim 19, which is carried out by introducing nucleic acids encoding the components of the three-dimensional artificial kinetochore scaffold to cause the three-dimensional artificial kinetochore scaffold to self-assemble in an oocyte.

21. The method according to claim 19 or 20, wherein the three-dimensional artificial kinetic chorod scaffold is defined in any one of claims 1 to 10.

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