HK40007627A - Methods and compositions to increase human somatic cell nuclear transfer (scnt) efficiency by removing histone h3-lysine trimethylation, and derivation of human nt-esc - Google Patents

Description

Methods and compositions for increasing human Somatic Cell Nuclear Transfer (SCNT) efficiency by removing histone H3-lysine trimethylation, and for increasing human NT-ESC derivatives

Cross Reference to Related Applications

Based on 35 u.s.c. § 119(e), the present application claims the benefit of US provisional patent application US62/239,318 filed 2015, 10, 9 and US 62/242,050 filed 2015, 10, 15, the contents of each of which are incorporated herein by reference.

Sequence listing

This application contains a sequence listing that has been submitted in ASCII format through EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy name created 10, 7, 2016 is 701039-.

Technical Field

The present invention relates generally to the field of Somatic Cell Nuclear Transfer (SCNT), and more particularly to the inhibition of methylation of H3K9me3, increasing the efficiency of human SCNT and producing human nuclear transfer ESC (hNT-ESC) by overexpression of the demethylase KDM4 family and/or by inhibition of SUV39H1 and/or SUV39H2 histone methyltransferases.

Background

When the nucleus is exposed to the molecular environment of the oocyte cytoplasm via Somatic Cell Nuclear Transfer (SCNT), the differentiated somatic cell genome can be reprogrammed back into the embryonic state (Gurdon, 1962), enabling the production of pluripotent (pluripotent) Embryonic Stem Cells (ESC) from terminally differentiated somatic cells (Wakayama et al, 2001). Because ESCs derived from SCNT (NT-ESCs) are genetically autologous to the nuclear donor somatic cells, hSCNT has great potential in therapeutic and regenerative medicine, including disease models and cell/tissue replacement therapy (Hochedlinger and Jaenisch, 2003; Yang et al, 2007). Thus, hSCNT can be used to repair mitochondrial gene-related defects that cannot be made by transcription factor-based reprogramming (Ma et al, 2015). Despite the great potential of human NT-ESC, the technical difficulties make it extremely difficult to apply to human therapy (French et al, 2008; Noggle et al, 2011; Simerly et al, 2003).

The first NT-ESCs were generated by the Mitalipov group using differentiated fetal and infant fibroblasts as donors (Tachibana et al, 2013). Using their optimal conditions, the inventors and others successfully obtained NT-ESCs from adult and geriatric patient somatic cells (Chung et al, 2014; Yamada et al, 2014). However, due to the very low probability of SCNT embryos developing to the blastocyst stage, NT-ESC derivatives remain quite difficult. Currently, only oocytes from certain females with the highest quality support SCNT embryonic development up to the blastocyst stage (Chung et al, 2014; Tachibana et al, 2013), limiting the available oocyte donor pools.

Terminally differentiated somatic cells can be reprogrammed to a totipotent state when transferred into an enucleated oocyte by Somatic Cell Nuclear Transfer (SCNT) means (Gurdon, 1962). Because SCNT allows whole animals to be generated from a single nucleus of differentiated somatic cells, it has great potential in agriculture, the biomedical industry, and preservation of endangered species (Y ang et al, 2007). In fact, since the first successful mammalian cloning of sheep in 1997 (Wilmut et al, 1997), more than 20 mammalian species have been cloned by SCNT (Rodriguez-Osorio et al, 2012). Furthermore, because pluripotent embryonic stem cells can be established from blastocysts generated by SCNTs (Wakayama et al, 2001), SCNTs have great promise in human therapy (Hochedlinger and Jaenisch, 2003). This hope is increasingly approaching reality after the first successful recent acquisition of human nuclear transferred embryonic stem cells (hNT-ESCs) (Tachibana et al, 2013) and the generation of human hNT-ESCs from older adult cells or human patient cells (Chung et al, 2014; Yamada et al, 2014). These hNT-ESCs can be used as a valuable cell source in vitro disease models, and also as a cell source for regenerative and cell/tissue replacement therapy.

Despite its great potential, several technical problems have prevented the practical use of SCNT, particularly its extremely low efficiency in producing cloned animals. For example, about half of the murine SCNT embryos showed a cessation of development prior to transfer, with only 1% to 2% of embryos transferred into surrogate mothers developing into a shape (Ogura et al, 2013). The overall reproductive cloning efficiency of all other species, except bovine species with a higher rate of reproductive cloning efficiency (5% to 20%), was very low (1% to 5%) (Rodriguez-Osorio et al, 2012). Moreover, the success rate of establishment of hNT-ESCs is also low due to the very poor development of the embryo prior to implantation (10% to 25% progress to the blastocyst stage; Tachibana et al, 2013; Yamada et al, 2014).

In order to realize the application potential of SCNT, attempts have been made to improve SCNT cloning efficiency. First, it has been reported that transient treatment of 1-cell SCNT embryos with Histone Deacetylase (HDAC) inhibitors, such as trichostatin a (TSA) or scriptaid, improves the reprogramming efficiency of a number of mammalian species, including murine (Kishigami et al, 2006; Van Thuan et al, 2009), porcine (Zhao et al, 2009), bovine (Akagi et al, 2011), and human (Tachibana et al, 2013; Yamada et al, 2014). Second, it has been reported that knock-out (knock-out) or knock-down (knock-down) of Xist improves the development of murine SCNT embryos after implantation into the uterus (Inoue et al, 2010; Matoba et al, 2011). However, none of these methods improves the cloning efficiency of human SCNTs enough to allow human SCNTs to be used to generate human totipotent and pluripotent stem cells (e.g., human NT-ESC) for use in therapeutic cloning or regenerative therapy.

At the time point of activation of the fertilized egg gene (ZGA), developmental defects of the SCNT embryo began to appear at the 2-cell stage for mice and at the 4-8-cell stage for pigs, cattle and humans (Schultz, 2002). SCNT embryos have difficulty at ZGA due to pre-existing undefined epigenetic barriers in the donor cell genome. Although it has been demonstrated that there are a large number of deregulated genes in murine 2-cell SCNT embryos (Inoue et al, 2006; Suzuki et al, 2006; Vassena et al, 2007) and in human SCNT embryos at the later stage of lysis (Noggle et al, 2011), the nature of the "pre-existing epigenetic barrier" in SCNT embryos and its relationship to impaired ZGA is unknown.

Accordingly, there is a need for a method of: human SCNT cloning efficiency is improved by removing these epigenetic barriers in the nuclear genome of the donor cell, so that human SCNT embryos can efficiently progress through Zygote Gene Activation (ZGA) without cessation of development and successfully develop into blastocysts through the 2, 4, and 8 cell stages without developmental defects or loss of viability.

Disclosure of Invention

The present invention is based, at least in part, on the following findings: in human somatic cells, H3K9me3 also serves as a barrier for human SCNT reprogramming. The inventors have demonstrated that KDM4A overexpression (e.g. by injection of exogenous KDM4AmRNA) terminates the developmental arrest at the time point of Zygote Gene Activation (ZGA) and significantly improves human SCNT embryo development, allowing efficient production of patient-specific NT-ESC using human oocytes obtained from donors whose oocytes did not successfully develop into blastocysts in controlled experiments without the help of KDM4A overexpression. Thus, the inventors have discovered a method to expand the availability of human oocyte donors for human SCNTs (hscnts) and to establish histone demethylase-assisted SCNTs, e.g., overexpressing members of the KDM4 family, useful in this method to improve the availability of human SCNTs in therapeutic cloning and human nuclear transfer ESC (NT-ESC), particularly the availability of patient-derived human NT-ESC for therapeutic use and research and disease models. The present invention is not directed to human reproductive cloning.

Mammalian (non-human) oocytes can reprogram somatic cells to a pluripotent state in which animal reproductive cloning by Somatic Cell Nuclear Transfer (SCNT) is permitted, or an ES cell line (NT-ESC) is produced from a blastocyst developing from an SCNT embryo. However, most SCNT embryos fail to develop into blastocysts or developmentally shape due to ambiguous reprogramming defects. The inefficiency of mammalian SCNTs is a key limiting factor in the development of patient-specific hESC lines for regenerative medicine applications.

Although there have been reports of the production of human blastocysts derived from human SCNT using human donor somatic cells, the blastocyst quality and developmental efficiency are insufficient to allow the production of a human embryonic stem cell line (human ntESC, also known as hNT-ESC) (French AJet al, StemCells 26, 485-. Human nuclear transfer embryonic stem cells (hNT-ESCs) have been reported (Tachibana et al, 2013), and the production of human hNT-ESCs from older adult cells or human patient cells (Chunget al, 2014; Yamada et al, 2014). However, the success rate of establishment of human hNT-ESCs is very low due to the very poor development prior to metastasis (only 10 to 25% development to blastocyst stage; Tachibana et al, 2013; Yamada et al, 2014). Therefore, refinement of human SCNT technology is critical to improve the development of the embryo to the blastocyst stage of human SCNT, thereby reducing the number of donor oocytes required for SCNT, and successfully producing human and patient-specific isogenic embryonic stem cell lines for research and cell-based therapies.

The extremely low efficiency of Somatic Cell Nuclear Transfer (SCNT) for obtaining human embryonic stem cells (hNT-ESCs) limits their potential for use. Blastocysts formed from human SCNT embryos occur at low rates and only some oocyte donors can achieve. The very poor developmental potential of SCNT embryos is not limited to humans, but is also common in all mammalian species examined (Rodriguez-Osorio et al, 2012).

Through comparative transcriptomics and epigenetic analysis of murine In Vitro Fertilization (IVF) and SCNT embryos, the inventors have previously demonstrated that the histone H3 lysine 9 trimethylation (H3K9me3) in the somatic genome of donors acts to prevent transcriptional reprogramming of murine cells by SCNT, leading to failure of Zygote Genome Activation (ZGA) and failure of pre-embryo implantation development (Matoba et al, 2014). The inventors have also previously demonstrated that this epigenetic barrier in the somatic cells of murine donors can be removed by ectopic overexpression of murine KDM4d, an H3K9me3 demethylase. Removal of H3K9me3 facilitated ZGA and thus improved development of the murine SCNT embryos to the blastocyst stage, resulting in increased rate and efficiency of murine NT-ESC production (mNT-ESC) (Matoba et al, 2014).

More specifically, the inventors previously demonstrated that in mice, histone H3 lysine 9 trimethylation (H3K9me3) is reduced by ectopic expression of H3K9me3 demethylase KDM4d, greatly improving SCNT murine embryo development, which is disclosed in international patent application WO2016/044271, which is incorporated herein by reference in its entirety.

In contrast to previous studies, herein, the inventors demonstrated that overexpression of H3K9me3 demethylase KDM4A in human cells surprisingly improved human SCNT, while H3K9me3 in the human somatic genome was an SCNT reprogramming barrier that prevented efficient development of human SCNT embryos through Zygote Gene Activation (ZGA). This is unexpected because human ES cells are very different from murine ES cells, and it is impossible to predict that substances that function in murine cells will function in human cells.

More specifically, since Zygote Gene Activation (ZGA) occurs at different time points in murine and human cells, it cannot be expected that reprogramming methods that remove the ZGA barrier in murine cells will also play a role in removing the ZGA barrier within a completely different time frame in human cells. As described herein in fig. 2A and 2E, the process and/or method of increasing the efficiency of SCNT in murine cells (see, fig. 2A) is different from the process and/or method of increasing the efficiency of SCNT in human cells (see, e.g., fig. 2E). Herein, the inventors surprisingly demonstrated that overexpression of KDM4A significantly improves the blastocyst formation rate of human SCNT embryos by promoting transcriptional reprogramming, allowing efficient harvesting of human NT-ESCs from different human patient populations, e.g., the inventors have demonstrated generation of hNT-ESCs from adult age-related macular degeneration (AMD) patient somatic cell nuclear donors. Thus, the discovery herein of methods for increasing the efficiency of human SCNT has potential application in a variety of contexts, including regenerative medicine and therapeutic cloning.

In particular, the inventors have found that histone H3 lysine 9 trimethylation (H3K9me3) in the donor nuclear genome of differentiated human somatic cells is a major pre-existing epigenetic barrier that blocks efficient reprogramming of human cells by SCNTs; it has also been found that reducing H3K9me3 methylation in human donor cell nuclei or in activated SCNT embryos increases the efficiency of human SCNT, particularly the efficiency of human SCNT embryos developing to the 8-cell or blastocyst stage prior to embryo implantation.

More specifically, by comparative analysis, the inventors have discovered genomic regions of human donor nuclei in human SCNT embryos that are resistant to fertilized egg gene activation (ZGA). Unlike other mammals such as ZGA, which occur in the 2-cell stage, ZGA, which occur in the 4-cell stage, pigs and cattle (Schultz, 2002), ZGA in humans occurs in the 8-cell stage (Schultz, 2002). Herein, the inventors have found that Reprogramming Resistant Regions (RRRs) in human donor genetic material are enriched in the inhibitory histone modification H3K9me3, while removing this epigenetic marker in human donor somatic cells can increase the efficiency of human SCNTs. Two approaches to improving the efficiency of human SCNT are contemplated in the methods and compositions disclosed herein, including: (i) increasing expression or activation of an H3K9me 3-specific demethylase in an oocyte or an activated SCNT embryo (e.g., after a hybrid oocyte has been fused or activated), e.g., overexpressing at least one member of the human KDM4 family (e.g., expressing exogenous human KMD4A, KDM2B, KDM4C, KDM4D, or KDM4 EmRNA); and/or (ii) knockdown or inhibition of expression or function of a human H3K9 methyltransferase, such as human SUV39H1 or human SUV39H2 or both (i.e., SUV39H1/2), in a somatic cell nucleus of a human donor. This approach not only attenuates the ZGA defect in the human donor nucleus and reactivates the RRR, but also greatly improves the efficiency of human SCNT, e.g., increases the percentage of SCNT embryos that develop to 2, 4, and 8 cell or blastocyst stages.

Therefore, SUV39H1/2 mediated H3K9me3 is the "epigenetic barrier" of human SCNT, and in the nucleus of a human somatic cell donor cell, recipient human oocyte, hybrid oocyte or human SCNT embryo, inhibition and/or removal of trimethylation of H3K9me3 (via overexpression of any one or more of KDM4A/JHDM3A or any other member of the human KDM4 family, e.g., overexpression of any one or more of the human KDM4A, human KDM4B, human KDM4C, human KDM4D, human KDM4E genes) and/or use of inhibitors of the human SUV39H1/2 protein or gene, may be used in the methods, compositions, and kits disclosed herein, the methods, compositions, and kits are useful for removing the epigenetic barrier that occurs in human cell reprogramming, particularly human somatic reprogramming via human SCNT ZGA, and are encompassed by methods of improving the cloning efficiency of human SCNT.

Accordingly, the present invention is based on the following findings of the inventors: in human cells, H3K9me3 was enriched in RRRs of human somatic cells used to produce SCNT embryos, and the H3K9me3 barrier in human somatic cells could be removed by overexpression of KDM4D family members.

Importantly, the inventors have demonstrated that removal of hSCNT embryos (e.g., between 5 and 10hpa, or between the 2 and 8 cell stages), H3K9me3 in recipient oocytes, by overexpression of at least one member of the human KDM4 family protein, e.g., human KDM4A, human KDM4B, human KDM4C, human KDM4D, human KDM4E (e.g., by introduction of an exogenous mRNA encoding a KDM4 family member, e.g., KDM4A mRNA or cDNA), results in a surprisingly significant increase in the efficiency of human SCNT cloning. In particular, the inventors surprisingly demonstrated that the rate of hSCNT embryos injected with KDM4A developing into blastocysts increased by more than 20% (i.e., from 4.2% to 26.8% by KDM4A injection), and that 14% of hSCNT embryos injected with KDM4A developed to the expanded blastocyst stage (as compared to control hSCNT embryos with no development).

Accordingly, aspects of the present invention are based on the following findings: trimethylation of histone H3 lysine 9 (H3K9me3) in human donor somatic cells prevented efficient human somatic cell nuclear reprogramming (hSCNT). As the inventors previously demonstrated that inhibition of SUV39h1/2 in the somatic cell nucleus of murine donors surprisingly increased mammalian SCNT efficiency (as in international application PCT/US2015/050178 filed 9/2015, 15, which is disclosed as WO2016/044271 and is incorporated herein by reference in its entirety), the methods and compositions disclosed herein encompass two approaches to improve the efficacy of human SCNT, including: (i) promotion of demethylation of H3K9me3 by overexpression (i.e., exogenous expression, or ectopic expression) of a demethylase KDM4 family member, such as KDM4A (also known as JMJD2A or JHDM 3A); and/or (ii) inhibits methylation of H3K9me3 by inhibiting human histone methyltransferases SUV39H1 and/or SUV39H 2. Thus, overexpression of KDM4A/JHDM3a, or other members of the human KDM4 family (e.g., overexpression of one or more of the human KDM4A, human KDM4B, human KDM4C, human KDM4D, human KDM4E genes), and/or suppression of human SUV39h1/2 protein or gene, can be used in the methods, compositions, and kits disclosed herein for removing the epigenetic barrier present in ZGA in human cell reprogramming, particularly human somatic reprogramming via human SCNT.

Accordingly, aspects of the present invention relate to methods, compositions, and kits for increasing human SCNT efficiency by reducing H3K9me3 methylation in human SCNT embryos by any one of: (i) (ii) expression of a histone demethylase capable of demethylating H3K9me3, e.g., a histone demethylase member of the KDM4 family, such as, but not limited to, JMJD2A/KDM4A and/or JMJD2D/KDM4D and/or JMJD2B/KDM4B and/or JMJD2C/KDM4C and/or JMJD2E/KDM 4E; and/or (ii) inhibits human histone methyltransferases involved in methylation of H3K9me3, e.g., inhibits any one of human SUV39H1, human SUV39H2, or human SETDB1, or a combination thereof. In some embodiments, an agent that increases the expression or activity of at least one member of the histone demethylase family of KDM4, e.g., JMJD2A/KDM4A and/or JMJD2D/KDM4D and/or JMJD2B/KDM4B and/or JMJD2C/KDM4C and/or JMJD2E/KDM4E, is injected into or contacted with a human SCNT embryo according to the methods disclosed herein.

Although demethylation of H3K9me3 (by KDM4c/Jmjd2c) has been reported to increase the efficiency of somatic cell reprogramming (e.g., to induce generation of pluripotent stem (iPS) cells (Sridharan et al, 2013)), demethylation of H3K9me3 has not been reported to increase the efficiency of SCNT from terminally differentiated somatic cells. Antony et al reported the use of KDM4B/JMJD2B in SCNTs derived from donor nuclei of pluripotent ES cells (Antony et al, "Transient JMJD 2B-medial Reduction of H3K9me3 Levels Improgramming of embryo Stemnic StemCells in closed embryos," mol.cell biol., 2013, 33(5), 974). Pluripotent ES cells are immature cells that are not identical to terminally differentiated somatic cells. Importantly, there are significant differences in the global epigenetic status of Embryonic Stem (ES) cells or Induced Pluripotent Stem (iPS) cells compared to differentiated somatic cells. Pluripotent ES cells have fewer epigenetic barriers (e.g., less methylation, particularly in the reprogramming confrontation region (RRR)), and thus the efficiency of SCNT embryos produced using ES nuclei as donor nuclei differs greatly from the efficiency of SCNT embryos produced using nuclei from terminally differentiated somatic cells (Rideout et al, 2000, Nature Genetics, 24(2), 109-10).

In contrast to the report by antonym et al, the inventors herein demonstrated that reducing the level of H3K9me3 in a hybrid oocyte, such as an enucleated oocyte containing donor somatic genetic material (e.g., by overexpressing human KDM4AmRNA), either before or after activation, resulted in a surprising increase in development following 8-cell SCNT, e.g., 32% of treated human SCNT embryos developed into morula, 26.8% developed into blastocysts, and 14.3% developed into and beyond the expanded blastocyst stage (as opposed to 0% of untreated human SCNT embryos reaching the expanded blastocyst stage). This is a 14% increase. This is highly unexpected in view of the report by antonym et al that even using ES cells derived from donor nuclei as immature cells in development that do not have the same epigenetic marker as terminally differentiated somatic cells resulted in only about a 9% improvement in development prior to ligand implantation.

Moreover, although there have been a number of reports (e.g., U.S. patent applications US 2011/0136145 and US2012/0034192, which are incorporated herein by reference in their entirety) on the efficiency of H3K9me3 demethylation to increase the efficiency of reprogramming somatic cells to early developmental stages (e.g., inducing the generation of pluripotent stem (iPS) cells), the mechanism by which somatic cells are reprogrammed to generate iPS cells is significantly different from the mechanism by which somatic cells are reprogrammed to generate SCNT embryos (e.g., Pasque et al, 2011, Mechanism of nuclear reprogramming by genes: a specific process?. Rev.mol.cell biol.12, 453-; and Stistoloc, 459E., and Hodlinger, K., 2013, chromosome nucleic acid along with nuclear growth. Thus, what was learned from H3K9me3 demethylation in the generation of iPS cells was not relevant or available and could not be transferred to methods of successfully generating SCNT human embryos or methods of increasing the efficiency of human SCNT embryos before and after embryo implantation.

In particular, there was a significant difference between the barrier present in human SCNT and the barrier present in human iPS reprogramming, and also a significant difference between human SCNT and murine SCNT reprogramming. First, the H3K9me3 barrier in murine iPSC reprogramming was established primarily by SETDB1 (Chen et al, 2013; Sridharan et al, 2013). Second, the downstream gene networks necessary for successful iPSC and SCNT reprogramming are different. For example, in iPSC reprogramming, key core pluripotent network genes such as Nanog and Sox2, which are repressed by the H3K9me3 barrier, are expressed in the relatively late stages of reprogramming (Chen et al, 2013; Sridharan et al, 2013). In contrast, in SCNT reprogramming, the gene repressed by H3K9me3 was enlarged at the 2-cell embryonic stage and had an important function at this stage (discussed below). In each context, this distinction most likely results from differences between the sets of transcription factors required for successful reprogramming. In fact, the core transcription factor Oct4/Pou5fl required for iPSC reprogramming has been shown to be dispensable in SCNT reprogramming (Wu et al, 2013). Thus, although H3K9m3 appears to be a reprogramming barrier common to both iPS cell generation and successful SCNTs, its deposition and how it affects the reprogramming process is vastly different from the methods of reprogramming to generate iPS cells and reprogramming to generate SCNT embryos.

Thus, even though it has been demonstrated that the H3K9me3 barrier in human somatic cell reprogramming to human iPS cells is removed, there is no indication that this method would have an effect on reprogramming human somatic cells in the generation of human SCNT embryos because different reprogramming genes and reprogramming mechanisms are used in iPS cell generation. Indeed, both US 2011/0136145 and US2012/0034192 specifically demonstrate that their methods are only useful for the reprogramming of somatic cells to ipscs, and are not applicable to the generation of totipotent cells or for the production of human SCNT embryos. Thus, the teachings of both US 2011/0136145 and US2012/0034192 are far from the present invention.

Moreover, as summarized in Table 1 below, the mechanism employed in somatic cell reprogramming in the generation of iPSCs is very different from that employed in the generation of SCNT embryos, and stem cells generated from the reprogrammed somatic cells to produce iPSCs are significantly different from those obtained from SCNT embryos (Ma et al, 2014, Abnormal in human pluripotent cell torrerogramming mechanisms, Nature, 511(7508), 177-183).

TABLE 1 summary of key differences between SCNT-and iPS-mediated reprogramming

Accordingly, as described above, since the reprogramming genes and mechanisms for human somatic cells to reprogram into human iPS cells are significantly different from those for human SCNT, and since the resulting cells are also significantly different, there is no sign or reason to believe that the method that functions in reprogramming to produce iPSC will play a role in reprogramming to produce human SCNT. In particular, normal iPSCs retain a residual DNA methylation pattern that is typical of parental somatic cells, whereas DNA methylation and transcriptome profiles of NTES cells are closely related to IVF-derived ES cells (see Ma et al, Nature.2014 Jul 10, 511 (7508): 177-183).

Accordingly, one aspect of the present invention is directed to a method of increasing the efficiency of human somatic cell nuclear transfer (hSCNT), comprising contacting at least one of a donor human somatic cell, a recipient human oocyte, a hybrid oocyte (e.g., an enucleated human oocyte comprising donor genetic material prior to fusion or activation) or a human SCNT embryo (i.e., after fusion of the donor nucleus and the enucleated oocyte) with an agent that reduces methylation of H3K9me3 in the donor human cell, recipient human oocyte or human SCNT embryo, thereby increasing the efficiency of human SCNT, e.g., increasing the efficiency of development of the resulting human SCNT into a blastocyst and beyond, as compared to an untreated human SCNT embryo.

In some embodiments, the present invention provides a method of increasing the efficiency of human somatic cell nuclear transfer (hSCNT), comprising at least one of: (i) contacting a donor human somatic cell or an acceptor human oocyte with at least one agent that reduces methylation of H3K9me3 (e.g., KDM4A mRNA) in the donor human somatic cell or the acceptor human oocyte; wherein the recipient human oocyte is a nucleated or enucleated oocyte; if the recipient human oocyte is nucleated, enucleating the human oocyte; transferring a nucleus from the donor human somatic cell into the enucleated oocyte to form a hybrid oocyte; and, activating the hybrid oocyte to form a human SCNT embryo; or (ii) contacting the oocyte hybrid with at least one agent that reduces methylation of H3K9me3 in the oocyte hybrid, wherein the oocyte hybrid is an enucleated human oocyte comprising human somatic cell genetic material; and, activating the hybrid oocyte to form a human SCNT embryo; or (iii) contacting the activated human SCNT embryo generated from the fusion of an enucleated human oocyte with human somatic genetic material with at least one agent that reduces H3K9me3 methylation in a human SCNT embryo; and, incubating the SCNT embryo for a sufficient time to form a blastocyst. In some embodiments, at least one blastomere is collected from the blastocyst and the blastomere is cultured to form at least one human NT-ESC.

In some embodiments, the agent that reduces methylation of H3K9me3 is at least one of: (i) an agent that increases the expression or activation or function of a histone demethylase member of the KDM4 family, and/or (ii) an H3K9 methyltransferase inhibitor, thereby removing the epigenetic barrier in the RRR and increasing the efficiency of human SCNT.

In some embodiments, increasing the efficiency of human Somatic Cell Nuclear Transfer (SCNT) comprises: contacting an SCNT embryo (e.g., after fusion of an enucleated human oocyte with the human genetic material of the donor cell) at least 5 hours post-activation (5hpa), or between 10 and 12hpa (i.e., at the 1-cell stage), or at about 20hpa (i.e., the early 2-cell stage), or between 20 and 28hpa (i.e., at the 2-cell stage) with at least one of (i) a histone demethylase of the KDM4 family (e.g., KDM4A mRNA) and/or (ii) an inhibitor of H3K9 methyltransferase (e.g., an inhibitor of human SUV39H 1/2).

In some embodiments, the reduction in H3K9me3 methylation occurs by overexpression or exogenous expression of a human KDM4 gene, such as hKDM4A, hKDM4B, hKDM4C, hKDM4D, or hKDM4E, in any one of the human donor oocyte (either pre-enucleated or post-enucleated), or the hybrid oocyte (e.g., an enucleated oocyte comprising donor nuclear genetic material but prior to activation), or in the human SCNT embryo (e.g., at least 5 hours post-activation (5hpa) or at the 1-cell stage, or at the 2-cell stage), or the donor human somatic cell prior to removal of genetic material, or a combination thereof.

In some embodiments, exogenous expression of a human KDM4 gene, such as KDM4A, occurs in a human donor oocyte. In some embodiments, exogenous expression of a human KDM4 gene, such as KDM4A, occurs in an enucleated human donor oocyte, or a hybrid oocyte (e.g., an enucleated oocyte comprising donor nuclear genetic material but prior to activation). In some embodiments, exogenous expression of a KDM4 gene, such as KDM4A, occurs in an SCNT embryo at any of the following stages: between 5hpa, 10hpa and 12hpa (i.e., 1 cell stage), about 20hpa (i.e., early 2 cell stage), or between 20hpa and 28hpa (i.e., 2 cell stage). In some embodiments, if the human SCNT embryo is contacted with an agent that inhibits H3K9me3, such as an agent that increases exogenous expression of a human KDM4 gene, such as KDM4A (e.g., KDM4A mRNA or mod-RNA), the KDM4A activator or over-expression agent is injected into each cell of the SCNT embryo (e.g., 2 cell embryo, or 4 cell embryo) (e.g., KDM4A mRNA is injected into each cell of the SCNT embryo).

In other embodiments, the methods of reducing methylation of H3K9me3 in donor genetic material disclosed herein occur by inhibiting the expression of SUV39H1 and/or SUV39H2, or both (SUV39H1/2) in one or a combination of a human donor oocyte (either pre-enucleation or post-enucleation), or a hybrid oocyte (i.e., an enucleated oocyte comprising donor genetic material prior to activation), or a SCNT embryo (e.g., at least 5 hours (5hpa) after activation or at the 1-cell stage, or at the 2-cell stage, or at the 4-cell stage), or a donor human somatic cell.

In some embodiments, inhibition of SUV39h1 and/or SUV39h2, or both (SUV39h1/2) occurs in a donor human somatic cell, e.g., at a time point of at least about 24 hours, or at least about 48 hours, or at least about 3 days, or at least about 4 days, or more than 4 days, prior to removal of the nucleus or genetic material for transfer to the enucleated human donor oocyte. In some embodiments, the expression of SUV39h1 and/or SUV39h2, or both (SUV39h1/2) is inhibited by siRNA and the inhibition occurs for at least 12 hours, or at least 24 hours or more, within the period of time prior to removal of the nucleus.

Another aspect of the invention relates to a method of increasing the efficiency of human Somatic Cell Nuclear Transfer (SCNT), the method comprising: contacting a human SCNT embryo, human oocyte or hybrid oocyte, or donor human somatic cell with an agent that reduces methylation of H3K9me3 (e.g., KDM4A mRNA), thereby increasing the efficiency of SCNT. In some embodiments, the recipient human oocyte is a human oocyte of very poor quality, the quality of which does not inhibit successful fertilization using IVF procedures. In some embodiments, the human oocyte is contacted prior to injection of the donor human nucleus or genetic material. In some embodiments, the recipient human oocyte is an enucleated human oocyte. In some embodiments, the SCNT embryo is a 1-cell stage, or a 2-cell stage SCNT embryo. In some embodiments, the agent that reduces methylation of H3K9me3 (e.g., KDM4A mRNA) is contacted with an recipient human oocyte or an enucleated human oocyte prior to nuclear transfer using a nucleus or genetic material from a terminally differentiated human somatic cell.

In some embodiments, the agent that contacts a recipient human oocyte, a hybrid oocyte, a somatic cell of a human donor, or a human SCNT embryo increases the expression or activity of at least one member of the histone demethylase enzyme of the KDM4 family, for example, at least one member of the human KDM4(JMJD2) family consisting of human KDM4A (SEQ ID NO: 1), human KDM4B (SEQ ID NO: 2), human KDM4C (SEQ ID NO: 3), or human KDM4D (SEQ ID NO: 4). In some embodiments, the agent that increases expression or activity of a histone demethylase of the KDM4 family increases expression or activity of KDM4D (JMJD2D) or KDM4A (JMJD2A) or KDM4B or KDM 4C. In some embodiments, the agent comprises a nucleic acid sequence of KDM4 from a human, e.g., KDM4A (SEQ ID NO: 1), human KDM4B (SEQ ID NO: 2), human KDM4C (SEQ ID NO: 3) or human KDM4D (SEQ ID NO: 4) or human KDM4E (SEQ ID NO: 45), or a biologically active fragment thereof or homologue thereof having a sequence identity of at least 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98%, or at least about 99%, which biologically active fragment or homologue increases the efficiency of a human SCNT to a level at which the nucleotide sequence of SEQ ID NO: 1 to SEQ ID NO: 4 or SEQ ID NO: 45 (e.g., by at least about 110%, or at least about 120%, or at least about 130%, or at least about 140%, or at least about 150%, or more than 150%).

In some embodiments, the agent that contacts a recipient human oocyte or human SCNT embryo increases the relative abundance of SEQ id no: 9, and/or comprises a sequence corresponding to SEQ ID NO: 1, or a biologically active fragment thereof that increases the efficiency of human SCNT to a level equivalent to SEQ ID NO: 1 (e.g., by at least about 110%, or at least about 120%, or at least about 130%, or at least about 140%, or at least about 150%, or more than 150%).

In some embodiments, the agent that contacts the recipient human oocyte or human SCNT embryo increases the relative abundance of SEQ id no: 12, and/or comprises a sequence corresponding to SEQ ID NO: 4, or a biologically active fragment thereof. In some embodiments, the nucleic acid sequence of SEQ ID NO: 12, a biologically active fragment of KDM4D comprising SEQ ID NO: 12, as disclosed in antonym et al, Nature, 2013. In some embodiments, the nucleic acid sequence of SEQ id no: 12 comprises SEQ ID NO: 12, which also lacks amino acids 1 to 424 of SEQ ID NO: 12, at least 1, or at least 2 to 10, or at least 10 to 20, or at least 20 to 50, or at least 50 to 100 amino acids C-terminal or N-terminal to amino acids 1 to 424 of SEQ ID NO: 12 at least 1, or at least 2 to 10, or at least 10 to 20, or at least 20 to 50, or at least 50 to 100 amino acids from the C-terminus and the N-terminus of amino acids 1 to 424.

In alternative embodiments, the agent that contacts the donor human cell, e.g., the donor nucleus of a terminally differentiated cell, increases the expression or activity of a histone demethylase of the KDM4 family, e.g., but not limited to, the KDM4 family consisting of KDM4A, KDM4B, KDM4C, KDM4D, or KDM4E, as discussed above.

Another aspect of the invention relates to a method of increasing the efficiency of human Somatic Cell Nuclear Transfer (SCNT), the method comprising: contacting the nucleus of a donor human cell, such as a terminally differentiated somatic cell, with an agent that reduces methylation of H3K9me3 in the nucleus of the donor human somatic cell, thereby increasing the efficiency of SCNT.

In some embodiments, in all aspects of the invention, the agent that contacts the donor human somatic cell is an inhibitor of H3K9 methyltransferase, such as, but not limited to, an inhibitor of human SUV39H1, human SUV39H2, or human SETDB1 expression or protein function. In some embodiments, at least one inhibitor of human SUV39h1, human SUV39h2, or human SETDB1, or any combination of such inhibitors, may be used to increase the efficiency of human SCNT in the method. In some embodiments, the inhibitor of H3K9 methyltransferase is not an inhibitor of human SETDB 1.

In some embodiments, the inhibitor of H3K9 methyltransferase is selected from the group consisting of: RNAi agents, siRNA agents, shRNA, oligonucleotides, CRISPR/Cas9, CRISPR/cpf1, neutralizing antibodies or antibody fragments, aptamers, small molecules, proteins, peptides, small molecules, avimidir, functional fragments or derivatives thereof, and the like. In some embodiments, the H3K9 methyltransferase inhibitor is an RNAi agent, e.g., an siRNA or shRNA molecule. In some embodiments, the agent comprises a nucleic acid inhibitor to inhibit the expression of human SUV39H1 protein (SEQ ID NO: 5 or SEQ ID NO: 48). In some embodiments, the agent comprises a nucleic acid inhibitor to inhibit the expression of human SUV39H2 protein (SEQ ID NO: 6). In some embodiments, the siRNA inhibitor of human SUV39h1 comprises at least one of: SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 20. SEQ ID NO: 21. SEQ ID NO: 22 or SEQ ID NO: 23 or a fragment thereof of at least 10 contiguous nucleotides, or a fragment thereof which hybridizes to SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 20. SEQ ID NO: 21. SEQ ID NO: 22 or SEQ ID NO: 23 (or a sequence identity of at least about 85%, or at least about 90%, or at least about 95%, or at least about 98%, or at least about 99%) of the nucleic acid sequence. In some embodiments, the siRNA inhibitor of human SUV39h1 comprises at least one of: SEQ ID NO: 8. SEQ ID NO: 21 or SEQ ID NO: 23 or a fragment thereof of at least 10 contiguous nucleotides, or a fragment of SEQ id no: 8. SEQ ID NO: 21 or SEQ ID NO: 23 (or a sequence identity of at least about 85%, or at least about 90%, or at least about 95%, or at least about 98%, or at least about 99%) of the nucleic acid sequence.

In some embodiments, the siRNA or other nucleic acid inhibitor hybridizes fully or partially to the nucleic acid sequence of SEQ ID NO: 14 or SEQ ID NO: 47 (corresponding to SUV39h1 variant 2 and SUV39h1 variant 1, respectively).

In some embodiments, the siRNA inhibitor of human SUV39h2 comprises at least one of: SEQ ID NO: 18 or SEQ ID NO: 19. or SEQ ID NO: 24. SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO: 27. or a fragment thereof of at least 10 contiguous nucleotides, or a fragment thereof which hybridizes to SEQ ID NO: 18 or SEQ ID NO: 19. or SEQ ID NO: 24. SEQ ID NO: 25. SEQ ID NO: 26. SEQ ID NO: 27 is at least 80% (or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98%, or at least about 99%) of the nucleic acid sequence. In some embodiments, the siRNA inhibitor of human SUV39h2 comprises at least one of: SEQ ID NO: 19. SEQ ID NO: 25. SEQ ID NO: 27. or a fragment thereof of at least 10 contiguous nucleotides, or a fragment thereof which hybridizes to SEQ ID NO: 19. SEQ ID NO: 25. SEQ ID NO: 27 (or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98%, or at least about 99%) less than 80% of the nucleic acid sequence.

In some embodiments, the siRA or other nucleic acid inhibitor hybridizes, in whole or in part, to the nucleic acid sequence of SEQ ID NO: 15. SEQ ID NO: 49. SEQ ID NO: 51. SEQ ID NO: 52 and SEQ ID NO: 53 (hsov 39h2 variants 1 to 5).

In some embodiments, the agent can be contacted with the SCNT embryo prior to activation, or about 5 hours after activation, or when the human SCNT embryo is in the 1-cell stage, the 2-cell stage, or the 4-cell stage. In alternative embodiments, the agent can be contacted with the human SCNT embryo at 5 hours after activation or when the human SCNT embryo is at the 2-cell stage. In some embodiments, the agent is injected into the recipient human oocyte, hybrid oocyte, or human SCNT embryo, e.g., KDM4A mRNA is injected into the nucleus and/or cytoplasm of the recipient human oocyte, hybrid oocyte, or human SCNT embryo. In some embodiments, the agent increases the expression or activity of at least one member of a histone demethylase of the KDM4 family.

In some embodiments, an agent that reduces methylation of H3K9me3 (e.g., KDM4A mRNA) is contacted with or injected into the nucleus or cytoplasm of a donor human cell, such as a terminally differentiated somatic cell, prior to injecting the nucleus of the donor human cell into an enucleated human oocyte. In some embodiments, the agent is contacted with the donor human somatic cell for at least 1 hour, or at least 2 hours or more, wherein the contacting occurs for at least 1 day (24 hours), or at least 2 days, or at least 3 days, or more than 3 days, prior to removing the nucleus from the donor human somatic cell and transferring it into the enucleated human oocyte.

In all aspects of the invention, the human SCNT embryo is prepared by injecting a donor human somatic cell nucleus from a differentiated somatic cell (typically a terminally differentiated cell, but not an ES cell or iPSC), which is not derived from an Embryonic Stem (ES) cell or Induced Pluripotent Stem (iPS) cell, or a fetal cell, into an enucleated human oocyte. In all aspects of the invention, the human SCNT embryo is produced by: donor cell nuclei from terminally differentiated human somatic cells are injected into enucleated human oocytes. In some embodiments, the donor human somatic cell genetic material is injected into a non-human recipient oocyte. In some embodiments, the human SCNT embryo develops following activation (or fusion) of the hybrid oocyte. In some embodiments, the hybrid oocyte comprises an enucleated human oocyte comprising nuclear genetic material from a somatic cell of a human donor, and mitochondrial genetic material (e.g., mitochondrial DNA or mtDNA) from a third human donor (i.e., mtDNA that is non-native to the enucleated oocyte).

In all aspects of the invention, the donor somatic cell, recipient oocyte or SCNT embryo is a human cell, e.g., a human donor cell, recipient human oocyte or human SCNT embryo.

Accordingly, in all aspects of the invention, the method results in an increase in the efficiency of human SCNT of at least about 5%, or at least about 10%, or at least about 13%, or at least about 15%, or at least 30%, or at least 50%, or from 50% to 80%, or more than 80% over human SCNT in the absence of an agent that decreases methylation of H3K9me3 (i.e., in the absence of an agent that increases expression or activation of a KDM4 family member). Stated another way, the methods disclosed herein increase the efficiency of SCNT embryo development prior to transfer, or increase the development of a hSCNT embryo to the blastocyst stage, or increase the development of a hSCNT embryo to the expanded blastocyst stage, whereby at least about 5%, or 7%, or 10%, or 12% or more than 12% develops to the expanded blastocyst stage. In another embodiment, the method increases the efficiency of human SCNT embryo development, e.g., increases the efficiency of successful development to the blastocyst stage by at least 3-fold, or at least 4-fold, or at least 5-fold, or at least about 6-fold, or at least about 7-fold, or at least about 8-fold, or more than 8-fold, compared to hSCNT embryos prepared in the absence of an agent that reduces H3K9me3 methylation. In some embodiments, the methods and compositions provided as disclosed herein increase human SCNT efficiency, meaning increase production or yield of human SCNT embryos derived from embryonic stem cells (human NT-ESC).

Another aspect of the invention relates to a composition comprising at least one of a human SCNT embryo, an acceptor human oocyte, or a hybrid oocyte or a human blastocyst, and at least one of: (i) an agent that increases the expression or activity of a histone demethylase of the KDM4 family (Jmjd2) or (ii) an agent that inhibits H3K9 methyltransferase.

In some embodiments, the composition comprises a recipient human oocyte, which is either an enucleated human oocyte or a human oocyte prior to injection of a donor nucleus obtained from a terminally differentiated somatic cell. In some embodiments, the composition comprises a hybrid oocyte (e.g., an enucleated human oocyte comprising genetic material of a donor nucleus prior to activation). In some embodiments, the human SCNT embryo is a 1-cell stage, or 2-cell, or 4-cell stage human SCNT embryo. In some embodiments, the composition comprises an agent that increases the activity of at least one gene encoding a member of the histone demethylase family of KDM4, or at least one member of the histone demethylase family of KDM4, such as KDM4A, KDM4B, KDM4C, KDM4D, or KDM 4E. In some embodiments, the agent increases the expression or activity of KDM4D (JMJD2D) or KDM4A (JMJD2A), or biologically active fragments or homologs thereof, which increases the efficiency of SCNT to a level similar to or greater than SEQ ID NO: 1 to SEQ ID NO: 4 or SEQ ID NO: 45 to the extent of the corresponding sequence. In some embodiments, the composition comprises a peptide corresponding to SEQ ID NO: 1, or a biologically active fragment thereof, that increases the efficiency of SCNT to a level similar to or greater than SEQ ID NO: 1, or a nucleic acid sequence of the nucleic acid sequence of 1.

In some embodiments, the composition comprises an agent that is an inhibitor of H3K9 methyltransferase, such as, but not limited to, an inhibitor of human SUV39H1, human SUV39H2, or human SETDB 1. In some embodiments, at least one of human SUV39h1, human SUV39h2, or inhibitors of human SETDB1, or any combination thereof, may be used in the method to increase the efficiency of human SCNT.

In some embodiments, the composition comprises an inhibitor of H3K9 methyltransferase selected from the group consisting of siRNA, shRNA, neutralizing antibodies or antibody fragments, aptamers, small molecules, proteins, peptides, small molecules, and the like. In some embodiments, the H3K9 methyltransferase inhibitor is an siRNA or shRNA molecule that inhibits human SUV39H1 or human SUV39H2 or human SETDB 1. In some embodiments, the composition comprises a nucleic acid inhibitor that hybridizes, in whole or in part, to the nucleic acid sequence of SEQ ID NO: 14 or SEQ ID NO: 47 (corresponding to SUV39h1 variant 2 and variant 1, respectively), or human SUV39h2 SEQ ID NOS: 15. SEQ ID NOS: 49. SEQ ID NOS: 51. SEQ ID NOS: 52 and SEQ ID NOS: 53 (hsov 39h2 variants 1 to 5).

In some embodiments, the composition comprises an siRNA inhibitor of human SUV39h1 that binds, in whole or in part, to SEQ ID NO: 7 or a fragment thereof of at least 10 contiguous nucleotides, or a sequence that hybridizes to SEQ ID NO: 7 is at least 80% (or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98%, or at least about 99%) sequence identity. In some embodiments, the composition comprises an siRNA inhibitor of human SUV39h1 comprising the amino acid sequence of SEQ ID NO: 8 or a fragment thereof of at least 10 contiguous nucleotides, or a fragment thereof that hybridizes to SEQ ID NO: 8 is at least 80% (or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98%, or at least about 99%) sequence identity. In some embodiments, the composition comprises an siRNA or other nucleic acid inhibitor that hybridizes, in whole or in part, to the nucleic acid sequence of SEQ ID NO: 14 or SEQ ID NO: 47 (corresponding to SUV39h1 variant 2 and variant 1, respectively).

In some embodiments, the compositions comprise siRNA or other nucleic acid inhibitor that hybridizes, in whole or in part, to the nucleic acid sequence of SEQ ID NOS: 15. SEQ ID NOS: 49. SEQ ID NOS: 51. SEQ ID NOS: 52 and SEQ ID NOS: 53 (hsov 39h2 variants 1 to 5).

In some embodiments, the composition comprises a human SCNT embryo at the 1-cell or 2-cell or 4-cell stage. In some embodiments, the composition comprises an enucleated human oocyte or a hybrid oocyte. In some embodiments, the composition comprises a human SCNT embryo, an acceptor human oocyte, a human hybrid oocyte, or a human blastocyst.

Another embodiment relates to a kit comprising (i) an agent that increases the expression or activity of a histone demethylase of the KDM4 family, e.g., an mRNA comprising a member of the human KDM4 family; and/or (ii) an agent that inhibits H3K9 methyltransferase.

In preferred embodiments, the disclosure herein does not relate to processes for cloning humans, for modifying germline genetic identity of humans, or for using human SCNT embryos for industrial or commercial purposes or for modifying human genetic identity, which may cause the embryo to suffer without any substantial medical benefit to the person receiving the processes.

Drawings

Fig. 1A to 1F show that the human reprogramming confrontation region (RRR) in somatic cells is rich in H3K9me 3. Fig. 1A is a schematic of the experimental procedure. Samples for RNA-seq are marked with a dashed rectangle. FIG. 1B is a graphical representation of a heat map of the transcriptome of IVF human embryos prior to implantation. Each slice represents the average of the peaks within the region obtained by sliding window analysis. 707 regions from 4 to 8 cell stage activation in IVF embryos are shown. The RNA-seq dataset was obtained from an existing publication (Xue et al, 2013). FIG. 1C is a graphical representation of a heat map comparing transcriptomes of donor somatic cells, IVF, and SCNT embryos at the 8-cell stage. The 707 regions identified in fig. 1A are shown. These regions were classified into three groups based on Fold Change (FC) in transcription level between SCNT embryos and IVF 8 cell embryos. FRR, PRR, and RRR indicate a completely reprogrammed region (FC < ═ 2), a partially reprogrammed region (2 < FC < ═ 5), and a reprogramming confrontation region (FC > 5), respectively. FIG. 1D shows the mean ChlP-seq intensities of H3K9me3 and H3K4me3 in human fibroblasts (Nhlf), showing the intensities within FRR, PRR, and RRR compared to the 200kb flanking region. The histone modification ChlP-seq dataset was obtained from The ENCODE Project (Bernstein et al, 2012; The EncodeConsortium Project, 2011). FIGS. 1E and 1F are boxed graphs comparing the mean intensity of H3K9me3-ChIP-seq (FIG. 1E) and DNasel-seq (FIG. 1F) within FRR, PRR and RRR in different somatic cell types. The ChlP-seq dataset and DNasel-seq dataset were obtained from the ENCODE Project (ENCODE Project Consortium, 2011). The median line in the colored region indicates the median value, the edge indicates the 25th/75th percentile (Dercentile), and the whisker line indicates the 2.5th/97.5th percentile. P < 0.001, p < 0.01. See also fig. 5, table 5 and table 6. (abbreviation: RRR ═ reprogramming confrontation region; PRR ═ partial reprogramming region; FRR ═ complete reprogramming region).

Fig. 2A-2H show that injection of human KDM4A mRNA improved development of murine SCNT embryos and human SCNT embryos. Figure 2A is a schematic of the murine SCNT process. Figure 2B shows representative nuclear images of1 cell stage SCNT embryos stained with anti-H3K 9me3 and DAPI 5 hours after mRNA injection. Fig. 2C shows that KDM4A mRNA injection greatly improved the pre-implantation development of murine SCNT embryos. Shown is the percentage of embryos that reached the indicated stage. Error bars indicate standard deviation. FIG. 2D shows representative images of SCNT embryos after 120 hours in vitro culture. Scale bar 100 Jim. Figure 2E is a schematic of the human SCNT process. FIG. 2F is the average developmental efficiency of human SCNT embryos obtained using oocytes from four different donors during 7 days of in vitro culture. The number of embryos reaching the 2-cell stage was used to calculate the efficiency. Blast: blastocyst, ExBlast: the blastocyst is expanded. The development rates were statistically analyzed by the exact probability method (Fisher's exact test). FIG. 2G shows representative images of SCNT embryos after 7 days in vitro culture. FIG. 2H shows a histogram of the rate of development of human SCNT embryos derived from each female donor oocyte. See also tables 3 and 4.

FIGS. 3A to 3J show the establishment and characterization of NTK-ESCs from AMD patients. FIG. 3A is a summary of NT-ESC lines in close proximity to SCNT assisted by KDM4A using AMD patients fibroblasts as nuclear donors. FIG. 3B shows representative phase-contrast and immunohistochemical stain images of NTK-ESCs. Scale bar 100 Jim. FIG. 3C is a bar graph showing the expression levels of pluripotency-specific genes and fibroblast-specific genes based on RNA-seq data. FIG. 3D is a scatter plot comparing gene expression levels between the control ESC line (ESC 15) and a representative NTK-ESC, NTK 6. Differentially expressed genes (FC > 3.0) are shown as black dots. FIG. 3E shows hierarchical clustering of NTK-ESCs, control ESCs and donor skin fibroblasts based on the RNA-seq dataset. FIG. 3F is a representative image of immunohistochemically stained Embryoid Bodies (EBs) spontaneously differentiated for 2 weeks in vitro. Scale bar 100 Jim. Fig. 3G shows representative histological images of teratomas derived from NTK6 at 12 weeks post-metastasis. The scale bar is 100 μm. Figure 3H shows a representative image of cytogenic G-banding analysis of NTK 6. FIG. 3I shows nuclear DNA genotyping using 16STR markers. FIG. 3J shows mitochondrial DNA genotyping of representative Single Nucleotide Polymorphism (SNP) sites. See also fig. 6 and 7. Fig. 3J discloses the sequences as SEQ ID NOs: 58. SEQ ID NO: 58 and SEQ ID NO: rs2853826 (m.10398A > G) sequence of 59, in order of appearance; and as SEQ ID NO: 58. SEQ ID NO: 58 and SEQ ID NO: 59 (m.10400C > T) sequence, in chronological order.

Fig. 4A to 4C show that when KDM4A mRNA was injected into SCNT 8 cell embryos, a portion of the transcription was restored. Fig. 4A shows a heat map comparing the transcript levels of 318 RRRs at the late 8-cell stage. Expression levels of 158 of 318 RRRs were significantly increased (FC > 2) in response to KDM4AmRNA injection. FIG. 4B shows a gene ontology analysis (FC > 2) of the 206 KDM4A response gene. Fig. 4C shows histograms and genome navigation of transcript levels of two representative KDM4A response genes UBTFL1 and THOC5 in IVF or SCNT (with or without KDM4A mRNA injection) 8 cell embryos. See also table 7.

Fig. 5A to 5E are related to fig. 1, and show RRRs (reprogramming confrontation regions) in human somatic cells having heterochromatin characteristics. Fig. 5A shows a boxplot comparing the mean ChlP-seq signals of 6 histone modifications at FRR, PRR and RRR in human fibroblasts (Nhlf). FIGS. 5B and 5C show boxplots comparing the mean intensity of H3K9me3-ChIP-seq (FIG. 5B) and DNasel-seq (FIG. 5C) within FRR, PRR and RRR in different somatic cell types. The ChlP-seq dataset and DNasel-seq dataset were obtained from the ENCODE Project (ENCODE Project Consortium, 2011). Note that H3K9me3 intensity is quite abundant in RRR compared to FRR and PRR; whereas the Dnasel-seq intensity in RRR is considerably poorer compared to FRR and PRR. P < 0.001, p < 0.01, p < 0.05. FIG. 5D shows boxed plots comparing the average percentage of exon sequences representing the density of protein-encoding genes in FRR, PRR and RRR of the human genome. P < 0.001, p < 0.05. FIG. 5E shows a boxplot comparing the average percentage of repeat sequences within FRR, PRR and RRR. P < 0.001, p < 0.05, ns: not significant.

Figures 6A to 6F correlate with figure 1 and show that human NTK-ESC exhibits normal pluripotency. FIG. 6A shows representative immunohistochemical staining images of NTK-ESCs and IVF-derived control ESCs. ESC clones were stained with anti-SOX 2 antibody, anti-SSEA 4 antibody and DAPI. The scale bar is 100 μm. FIG. 6B is a scatter evaluation of RNA-seq reproducibility of different biological replicates of control ESCs and NTK-ESCs. FIG. 6C shows a scatter plot comparing global gene expression patterns between control ESCs and NTK-ESCs. Differentially expressed genes (FC > 3.0) are shown as black dots. Note that the correlation for each pair-wise comparison is extremely high (r ═ 0.95 to 0.99). Fig. 6D shows representative images of immunohistochemically stained Embryoid Bodies (EBs) spontaneously differentiated for 2 weeks in vitro. The EBs were stained with anti-TUJl, anti-BRACHYURY, or anti-AFP antibodies along with DAPI. The scale bar is 100 μm. Figure 6E shows representative histological images of teratomas derived from NTK-ESC #6 at 12 weeks post-metastasis. The scale bar is 100 μm. Fig. 6F shows representative histological images of teratomas derived from the NTK7 and NTK8 cell lines at 12 weeks post-metastasis.

Figures 7A to 7C relate to figure 3 and show that human NTK-ESC contain genome derived from nuclear donor and mitochondria derived from oocyte donor. FIG. 7A shows representative images of cytogenetic G banding analysis showing normal karyotypes with the expected NTK-ESC lines NTK7 and NTK8 sex chromosome composition. FIG. 7B shows nuclear DNA genotyping using 16STR markers. Note that all STR markers for NTK-ESC NTK7 and NTK8 perfectly matched the markers for DFB-6 and DFB-8, respectively, of the original nuclear donor fibroblasts. FIG. 7C shows mitochondrial DNA genotyping of representative Single Nucleotide Polymorphism (SNP) sites. Mitochondria of NTK-ESC are exclusively derived from donor oocytes. Fig. 7C discloses the sequences shown as SEQ ID NOs: 60 to SEQ ID NO: rsl 116907 (m.8468C > T) sequence of 65, in order of occurrence; and as SEQ ID NOs: 66 to SEQ ID NO: 69 and SEQ ID NO: 69 to SEQ ID NO: rsl 116904 (m.8027G > A) sequence of 70, in order of occurrence.

Detailed Description

Despite its great potential in both basic science and therapeutic uses, the efficiency of cloning human somatic cells by Somatic Cell Nuclear Transfer (SCNT) is still extremely low, resulting in poor efficiency of development to blastocysts and a small number of cells in the expanded blastocyst stage. These deficiencies also rarely succeed in establishing human ES cells from cloned human SCNT embryos. The inefficacy of the cloned human embryo is largely due to incomplete nuclear reprogramming and/or epigenetic barriers within the donor human nucleus.

The present invention is based on the following findings: trimethylation of histone H3-lysine 9 (H3K9me3) occurs in the reprogramming confrontation region (RRR) of the nucleus of this human donor cell and is an epigenetic barrier preventing efficient human somatic cell nuclear reprogramming by SCNT. As disclosed herein, the inventors have demonstrated two approaches to improve the performance of human SCNT, first by promoting demethylation of H3K9me3 of the donor nuclear genetic material using exogenous expression or increased expression (e.g., overexpression) of a KDM4 demethylase family member, such as KDM4A or KDM4D, and/or by inhibiting methylation of H3K9me3 by inhibiting histone methyltransferases, such as SUV39H1 and/or SUV39H 2. In some embodiments, a hybrid human oocyte (e.g., an enucleated human oocyte comprising nuclear genetic material from a human donor somatic cell prior to activation) and/or a human SCNT embryo is injected with an agent that increases the expression of KDM4A and/or KDM4D (e.g., mRNA encoding human KDM4A protein or a functional fragment of the KDM4A protein and/or mRNA encoding human KDM4D protein or a functional fragment of the KDM4D protein). In some embodiments, the agent is an mRNA encoding a human KDM4A or KDM4D protein, or a homolog thereof, or another member of the human KDM4 family histone demethylase.

In some embodiments, a donor human somatic cell, a recipient human oocyte, a hybrid oocyte (e.g., an enucleated human oocyte comprising donor genetic material prior to fusion or activation) or a human SCNT embryo (i.e., after fusion of the donor nucleus with the enucleated oocyte) is injected with an mRNA encoding a member of the KDM4 family, or an mRNA or nucleic acid analog, including a modified mRNA (also known as mod-RNA). In some embodiments, a donor human somatic cell, an acceptor human oocyte, a hybrid oocyte, or a human SCNT is injected with mRNA encoding human KDM4A protein or a functional fragment of the KDM4A protein and/or mRNA encoding human KDM4D protein or a functional fragment of the KDM4D protein. In some embodiments, the injection of hSCNT can be performed at any stage after activation, e.g., at 5hpa, or 10 to 12hpa, or 20 to 28hpa, 1 cell stage, 2 cell stage, or 4 cell stage hSCNT embryos.

Accordingly, the present invention relates to methods, compositions and kits comprising H3K9me3 histone demethylase activators such as activators of the human KDM4/JMJD2 family, and/or H3K9me3 methyltransferase inhibitors such as inhibitors of human SUV39H1 or human SUV39H2 or human SETDB1, to remove epigenetic barriers in human nuclear genetic material (e.g., in the human donor genome), thereby increasing the efficiency of successful human SCNTs, including the efficiency of development of a hSCNT embryo to the blastocyst stage and beyond.

Accordingly, aspects of the present invention relate to methods, compositions, and kits directed to increasing the efficiency of human SCNT by reducing H3K9me3 methylation by: (i) (ii) expression of a histone demethylase capable of demethylating H3K9me3, e.g., a member of the KDM4 family of histone demethylases, such as, but not limited to, JMJD2A/KDM4A or JMJD2B/KDM4B, or JMJD2C/KDM4C or JMJD2D/KDM4D or JMJD2E/KDM 4E; and/or (ii) histone methyltransferases involved in methylation of H3K9me3, e.g., inhibition of any one or combination of human SUV39H1, human SUV39H2, or human SETDB 1. In some embodiments, the agent that increases expression or activity of a human KDM4 family histone demethylase increases expression or activity of KDM4E (JMJD2E), KDM4D (JMJD2D), KDM4C (JMJD2C), KDM4B (JMJD2B), or KDM4A (JMJD 2A).

Another aspect relates to the use of human SCNT embryos produced using the methods and compositions disclosed herein to develop into one or more blastomeres that can be removed or biopsied and/or used to generate human ES cells (i.e., human NT-ESC). NT-hescs generated using the methods disclosed herein can be used for a variety of purposes, e.g., for regenerative and/or cell-based therapy, for assays, and in disease models (e.g., if the hNT-ESC is a patient-specific hNT-ESC, the hNT embryo is generated using genomic donor nuclei from a human donor subject having a particular mutation or SNP and/or having a predisposition to develop a particular disease). The hNT-ESCs can also be used in assays, such as drug screening assays, including, but not limited to, personalized drug screening and/or disease-specific drug screening. The hNT-ESC produced using the methods and compositions disclosed herein can be cryopreserved and can be stored in the human NT-ESC bank.

Definition of

For convenience, certain terms employed throughout the application (including the specification, examples, and appended claims) are collected here. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The phrase "somatic cell nuclear transfer" or "SCNT" is also generally referred to as therapeutic or phonomorphic cloning, a process by which somatic cells fuse with enucleated oocytes. The nucleus of a somatic cell provides genetic information, while an oocyte provides nutrients and other energy producing materials necessary for embryonic development. Once fusion has occurred, the cell is pluripotent and eventually develops into a blastocyst, at which time the inner cell mass is isolated.

As used herein, the term "nuclear transfer" refers to a genetic manipulation technique that allows one to obtain the exact same characteristics and qualities by manually combining an enucleated oocyte with the nuclear genetic material or the nucleus of a somatic cell. In some embodiments, the nuclear transfer process is the transfer of nuclear or nuclear genetic material from a donor somatic cell, such as an enucleated egg or oocyte (an egg or oocyte whose nucleus/pronuclei has been removed). The donor nuclei may be derived from autologous cells.

The term "nuclear genetic material" refers to structures and/or molecules found in the nucleus of a cell that comprise a polynucleotide (e.g., DNA) that encodes information about the individual. Nuclear genetic material includes chromosomes and chromatin. The term also refers to nuclear genetic material (e.g., chromosomes) produced by cell division, such as division of a parent cell into daughter cells. Nuclear genetic material does not include mitochondrial DNA.

The term "SCNT embryo" refers to a cell of an enucleated oocyte that has been fused to the nucleus or nuclear genetic material of a somatic cell, or a pluripotent progeny thereof. SCNT embryos can develop into blastocysts and, after implantation, into live offspring. The SCNT embryo can be a1 cell embryo, a 2 cell embryo, a4 cell embryo, or an embryo at any stage prior to becoming a blastocyst.

The term "parent embryo" is used to refer to an SCNT embryo from which a single blastomere has been removed or biopsied. After biopsy, the remaining parent embryo (parent embryo minus the biopsied blastomere) may be cultured with the blastomere to help promote proliferation of the blastomere. Subsequently, the remaining viable parent SCNT embryos can be frozen for long periods of time or stored permanently or for future use. Alternatively, viable parent embryos can be used to create pregnancy.

The term "donor human cell" or "donor human somatic cell" refers to the somatic cell or the nucleus of a human cell that is transferred into an recipient oocyte as a nuclear recipient or recipient.

The term "somatic cell" refers to a plant cell or animal cell that is not a germ cell or germ cell precursor. In some embodiments, the differentiated cell is not an embryonic cell. Somatic cells are not associated with pluripotent or totipotent cells. In some embodiments, a somatic cell is a "non-embryonic somatic cell," which means a somatic cell that is not present in, or cannot be obtained from, an embryo and cannot be caused by the proliferation of such cells in vitro. In some embodiments, a somatic cell is an "adult somatic cell," which means a cell that is present in or obtained from an organism other than an embryo or fetus or that can be caused by in vitro culture of such a cell.

As used herein, the term "differentiated cell" refers to any cell that is in the process of differentiating into a somatic cell lineage or that has been terminally differentiated. For example, embryonic cells can differentiate into epithelial cells that line the intestine. For example, the differentiated cells can be isolated from a fetus or a live newborn animal.

In the context of cellular ontologies, the adjective "differentiated", or "differentiation", is a relative term, meaning that a "differentiated cell" has progressed further downstream in the developmental pathway than the cell it is being compared to. Thus, stem cells can differentiate into lineage-defined precursor cells (e.g., mesodermal stem cells), which in turn can differentiate into other types of precursor cells downstream in the pathway (e.g., cardiomyocyte precursors), followed by differentiation into terminally differentiated cells, which play a characteristic role in certain tissue types and may or may not retain the ability to further proliferate.

Herein, the term "oocyte" refers to a mature oocyte which has reached metaphase II of maturation. Oocytes are also used to reveal female gametes or embryonic cells involved in reproduction and are commonly referred to as eggs. Mature eggs have a set of maternal chromosomes (23, X in human primates) and stop at metaphase II.

"hybrid oocyte" refers to an enucleated oocyte having cytoplasm from a first human oocyte (referred to as an "acceptor") but not having the nuclear genetic material of the acceptor oocyte; it has nuclear genetic material from another human cell called the "donor". In some embodiments, the hybrid oocyte may also include mitochondrial dna (mtdna), which is not from the recipient oocyte, but from a donor cell (which may be the same donor cell as the nuclear genetic material, or from a different donor, such as from a donor oocyte).

Herein, the term "enucleated oocyte" refers to a human oocyte the nucleus of which has been removed.

Herein, the term "enucleation" refers to a process by which nuclear material of a cell is removed, leaving only cytoplasm. When applied to eggs, enucleation refers to removal of maternal chromosomes that are not surrounded by nuclear membranes. The term "enucleated oocyte" refers to an oocyte whose nuclear material or nucleus is removed.

Herein, an "acceptor human oocyte" refers to a human oocyte that, after removal of its original nucleus, receives a nucleus from a human nucleus donor.

As used herein, the term "fusion" refers to the combination of the nuclear donor system with the lipid membrane of the recipient oocyte. For example, the lipid membrane may be the plasma membrane or nuclear membrane of a cell. When the nuclear donor cell and the recipient oocyte are placed adjacent to each other or when the nuclear donor cell is placed in the perivitelline space of the recipient oocyte, fusion may occur if an electrical stimulus is applied between the nuclear donor cell and the recipient oocyte.

As used herein, the term "activation" refers to the stimulation of cell division before, during or after nuclear transfer. Preferably, in the present invention, it means stimulating cell division after nuclear transfer.

Herein, the term "living offspring" means an animal that can survive in utero. It is preferably an animal that can survive for 1 second, 1 minute, 1 day, 1 week, 1 month, 6 months, or more than 1 year. The animal may not require an intrauterine environment for survival.

The term "prenatal" refers to the presence or emergence prior to birth. Likewise, the term "postpartum" refers to the presence or presence after birth.

As used herein, the term "blastocyst" refers to an embryo of about 30 to 150 cells prior to implantation into a placental mammal (about 3 days after fertilization for a mouse; about 5 days after fertilization for a human). The blastocyst stage follows the morula stage and can be distinguished by its unique morphology at the same time. The blastocyst consists of a sphere composed of a cell layer (trophectoderm), a fluid-filled cavity (the blastocyst or blastocyst cavity), and an internal cluster of cells (the inner cell mass, or ICM). If the blastocyst is implanted into the uterus, the ICM, which is composed of undifferentiated cells, results in material that will become fetal. If these same ICM cells are grown in culture, these cells form an embryonic stem cell line. Upon implantation, the murine blastocyst is composed of approximately 70 trophoblast cells and 30 ICM cells.

Herein, the term "blastocyst" refers to an early stage of embryonic development, which consists of a hollow sphere of cells enclosing a fluid-filled cavity, called the blastomere. The term blastocyst is sometimes used interchangeably with blastocyst.

The term "blastomere" is used throughout to refer to at least one blastomere (e.g., 1, 2, 3, 4, etc.) obtained from a pre-implantation embryo. The term "cluster of two or more blastomeres" is used interchangeably with "blastomere-derived product" and refers to cells produced during in vitro culture of blastomeres. For example, after a blastomere is obtained from an SCNT embryo and initially cultured, it typically splits at least once to produce clusters of two or more blastomeres (also referred to as blastomere-derived products). The clusters can be further cultured with embryonic or fetal cells. Eventually, the blastomere-derived product will continue to divide. From these constructs, ES cells, Totipotent Stem (TS) cells, and partially differentiated cell types will develop during the course of the culture process.

Herein, the term "nucleus" refers to the nucleus of a cell, which is obtained from the cell by enucleation, surrounded by the narrow edge of the cytoplasm and the serosa.

As used herein, the term "cell pairing" refers to enucleated oocytes with somatic or fetal nuclei prior to fusion and/or activation.

As used herein, the term "lytic mode" refers to a mode of cell division in a very early embryo; the organisms of each species show a characteristic lysis pattern that can be observed under a microscope. Contrary to this characteristic pattern, embryos are generally indicated to be abnormal, and therefore, the cleavage pattern serves as a pre-implantation screening criterion for embryos.

As used herein, the term "clone" refers to an exact genetic duplication of a DNA molecule, cell, tissue, organ, or whole plant or animal, or an organism having the same nuclear genome as another organism.

As used herein, the term "cloned" refers to a genetic manipulation technique used to prepare a new individual unit having a complete set of genes identical to another individual unit. In the present invention, the term "cloned" herein refers to a cell, embryonic cell, fetal cell, and/or animal cell that has a nuclear DNA sequence that is substantially similar or identical to the nuclear DNA sequence of another cell, embryonic cell, fetal cell, differentiated cell, and/or animal cell. The terms "substantially similar" and "identical" are disclosed herein. The cloned SCNT embryos may be caused by a nuclear transfer, or the cloned SCNT embryos may be caused by a cloning process comprising at least one recloning step.

Herein, the term "transgenic organism" refers to an organism which has been experimentally transferred with genetic material from another organism, and thus, the transferred genetic trait is obtained in the chromosomal composition of the host.

Herein, the term "embryo split" refers to the separation of an early embryo into two or more embryos having identical gene constitution, essentially creating identical twins or multiple embryos (triplets, quadruplets, etc.).

Herein, the term "morula" refers to a preimplantation embryo 3 to 4 days after fertilization, which is a solid mass consisting of 12 to 32 cells (blastomeres). After the 8-cell stage, the cells of the preimplantation embryo begin to adhere more tightly to each other, becoming "compact". The resulting embryos resemble morulae, called morulae (mulberry in latin).

The term "embryonic stem cell" (ES cell) refers to a pluripotent cell derived from the inner cell mass of a blastocyst or morula, which has been continuously developed into a cell line. ES cells may result from fertilization of an egg cell using sperm or DNA, nuclear transfer such as SCNT, parthenogenesis, and the like. The term "human embryonic stem cells" (hES cells) refers to human ES cells. The term "ntESC" refers to embryonic stem cells obtained from the inner cell mass of blastocysts or morulae produced from SCNT embryos. "hNT-ESC" refers to embryonic stem cells obtained from the inner cell mass of the blastocyst or morula produced by human SCNT embryos. The generation of ESCs is disclosed in U.S. Pat. Nos. 5,843,780, 6,200,806, and ESCs obtained from the inner cell mass of somatic cell nuclear transfer-derived blastocysts are disclosed in U.S. Pat. Nos. 5,945,577, 5,994,619, 6,235,970, which are incorporated herein by reference in their entirety. The distinguishing characteristics of embryonic stem cells define embryonic stem cell phenotypes. Accordingly, a cell has the phenotype of an embryonic stem cell if it possesses one or more of the unique characteristics of that cell and is therefore distinguishable from other cells. Exemplary differentiating embryonic stem cell characteristics include, without limitation, gene expression profile, proliferative capacity, energy to differentiate, karyotype, ability to respond to specific culture conditions, and the like.

As used herein, the term "pluripotent" refers to a cell that has the ability to differentiate into more than one different differentiated cell type under different conditions, and preferably has the ability to differentiate into the cell type characteristic of all three embryonic cell layers. A primary characteristic of pluripotent cells is the ability to differentiate into more than one cell type, preferably all three germ layers, using, for example, the nude mouse teratoma formation assay. Such cells include hES cells, human embryo-derived cells (hEDC), human SCNT embryo-derived stem cells, and adult-derived stem cells. The pluripotent stem cells may be genetically modified (genetically modified) or not. The genetically modified cells may include a marker, such as a fluorescent protein, to facilitate their identification. Pluripotency is also demonstrated by expression of Embryonic Stem (ES) cell markers, but the preferred pluripotency test is to demonstrate the ability to differentiate into cells in each of the three germ layers. It should be noted that simply culturing such cells does not lend themselves to being pluripotent. The reprogrammed pluripotent cells (e.g., iPS cells, the term of which is defined herein) also have the following characteristics: the reprogrammed pluripotent cells have the ability to expand pathways without loss of growth potential relative to primary cell parents, which typically have only a limited number of divisions in culture.

Herein, the term "pluripotent" with respect to an SCNT embryo refers to an SCNT embryo that can develop into a live-born animal.

Herein, the terms "iPS cell" and "induced pluripotent stem cell" are used interchangeably and refer to a pluripotent stem cell that is artificially derived (e.g., induced or by complete reversal) from a non-pluripotent cell by, for example, inducing forced expression of one or more genes. The non-pluripotent cells are typically adult human cells.

Herein, the term "reprogramming" refers to a process of changing or reversing a differentiated somatic cell, and thus, the developmental clock of the nucleus is reset; for example, adults in a reduced developmental state differentiate nuclei, and thus, may carry genetic programs for early embryonic nuclei, making all the proteins required for embryonic development. In some embodiments, the donor human cells are terminally differentiated prior to reprogramming by SCNT. As disclosed herein, somatic cells in a differentiated state are reprogrammed to a complete reversal of pluripotent or totipotent cells. Reprogramming typically includes alterations such as inversions of at least a portion of nucleic acid modifications (e.g., methylation), chromatin condensation, epigenetic variations, genomic blots, etc., that occur during cellular differentiation as the zygote develops into an adult. In Somatic Cell Nuclear Transfer (SCNT), the grouping of the cytoplasm of the recipient oocyte is thought to play an important role in reprogramming the somatic cell nucleus to achieve the function of the embryonic nucleus.

Herein, the term "culturing" with respect to SCNT embryos refers to a laboratory process involving the placement of embryos in culture medium. SCNT embryos can be placed in culture for a suitable period of time to allow the SCNT embryos to remain static but functional in the culture medium, or to allow the SCNT embryos to grow in culture. Suitable media for culturing embryos are well known to those skilled in the art. See, for example, U.S. Pat. No. 5,25,1993 entitled "In vitro culture of Bovine Embryos" (In vitro culture of Bovine Embryos) US5,213,979 and Rosenkrans, Jr. et al, U.S. Pat. No. 3,17,1992 US5,096,822 entitled "Bovine Embryo culture Medium", which is incorporated herein by reference In its entirety, including all figures, tables, and drawings.

The term "culture medium" is used interchangeably with "appropriate medium" and refers to any medium that allows for the proliferation of cells. The appropriate medium need not promote maximal proliferation, but only measurable cell proliferation. In some embodiments, the medium maintains the cells in a pluripotent or totipotent state.

Herein, the term "implantation" of an SCNT embryo as disclosed herein refers to the placement of an SCNT embryo as disclosed herein into a surrogate female. This technique is well known to those skilled in the art. See, e.g., Seidel and Elsden, 1997, cow Embryo Transfer (Embryo Transfer in Dairy cat), w.d. hoard & Sons, co., Hoards Dairyman. The embryo may be allowed to develop in the uterus, or the fetus may be removed from the uterine environment prior to delivery.

Herein, the term "agent" means any compound or substance, such as, but not limited to, a small molecule, nucleic acid, polynucleotide, peptide, drug, ion, and the like. An "agent" can be any chemical, whole or part, including, without limitation, synthetic and naturally occurring proteinaceous whole and non-proteinaceous whole. In some embodiments, the agent is a nucleic acid, nucleic acid analog, protein, antibody, peptide, aptamer, oligomer of nucleic acid, amino acid, or carbohydrate, including without limitation, proteins, oligonucleotides, enzymatic nucleic acids, dnases, glycoproteins, siRNA, lipoproteins, aptamers, modifications and combinations thereof, and the like. In certain embodiments, the agent is a small molecule having a chemical moiety. For example, unsubstituted or substituted alkyl, aryl, or heterocyclyl moieties including chemical moieties include macrolides, leptin mycins, and related natural products or analogs thereof. The compound may be known to have the desired activity and/or properties, or may be selected from a library of various compounds.

Herein, the term "contacting" (i.e., contacting a human donor cell, a human recipient oocyte, a hybrid oocyte, or a human SCNT embryo with an agent) is intended to include incubating the agent reagent and the human cell, human oocyte, hybrid oocyte, or hSCNT embryo together in vitro (e.g., incubating the agent reagent added to the donor human cell, human oocyte, hybrid oocyte, or hSCNT embryo in a culture medium or in a container). In some embodiments, the term "contacting" is not intended to include exposing a cell to an agent disclosed herein in vitro, which exposure may occur naturally in vivo in a subject (i.e., exposure that may occur as a result of a natural physiological process). The step of contacting the human somatic cell, human oocyte, hybrid oocyte or hSCNT embryo with the agent may be performed in any suitable manner. For example, human somatic cells, human oocytes, hybrid oocytes, or hSCNT embryos may be processed in adherent or suspension culture. It is understood that a human somatic cell, human oocyte, hybrid oocyte or hSCNT embryo may be contacted with an agent as disclosed herein, and may also be contacted simultaneously or subsequently with another agent, such as a growth factor or other differentiation agent or agent, or to otherwise modify, to stabilize the cell or to further differentiate the cell. Likewise, a human somatic cell, a human oocyte, a hybrid oocyte, or a hSCNT embryo may be contacted with an agent disclosed herein (e.g., KDM4 histone demethylase activator or mRNA) followed by a second agent disclosed herein (e.g., an H3K9 methyltransferase inhibitor), or vice versa. In some embodiments, the human somatic cell, human oocyte, hybrid oocyte, or hSCNT embryo is contacted with a contacting agent as disclosed herein or with a second agent as disclosed herein, and the contacting is temporally segregated. In some embodiments, a human donor cell, a human somatic cell, a human oocyte, a hybrid oocyte, or a hSCNT embryo as disclosed herein is contacted with one or more agents substantially simultaneously (e.g., with KDM4 histone demethylase activator (e.g., KDM4D mRNA) and an H3K9 methyltransferase inhibitor substantially simultaneously).

The term "exogenous" refers to a substance that is present in a cell or organism in a manner other than its natural source or level. For example, the term "exogenous nucleic acid" or "exogenous protein" refers to a nucleic acid or protein that has been introduced by involving an artificial entry into a biological system, such as a cell or organism, where the nucleic acid or protein is not normally found in the biological system or where the introduced nucleic acid or protein is normally found in a lower amount. A substance is considered exogenous if it is introduced into a cell or cell progenitor that inherits the substance. In contrast, the term "endogenous" refers to a substance that is native to a biological system or cell at the time. For example, "exogenous KDM 4A" refers to the introduction of KDM4A mRNA or cDNA not normally found or expressed at the level at which it was introduced into the cell or organism.

The term "expression" refers to cellular processes involved in the proper production of RNA and proteins, e.g., transcription, translation, folding, modification, and processing. "expression products" include RNA transcribed from genes and polynucleotides obtained by translation of mRNA transcribed from the gene.

The term "mitochondrial DNA" is used interchangeably with "mtDNA" and refers to DNA of the mitochondria, a structure located within the cytoplasm of a cell and not the nucleus (where all other chromosomes are located). In vivo, all mtDNA inherits from the mother. In each mitochondrion, there are 2 to 10 copies of the mtDNA genome. mtDNA is a double-stranded circular molecule. mtDNA is very small relative to chromosomes within the nucleus and includes only a limited number of genes, such as those encoding a large number of subunits in the mitochondrial respiratory chain complex and genes for certain ribosomal RNAs and transfer RNAs. The cells comprise mitochondrial-based continuously replicating mtDNA derived from the cell texture, which in the case of spindle transfer is based on the acceptor cytoplasm.

The term "mitochondrial disease" refers to diseases and pathologies affecting mitochondrial function and/or caused by mitochondrial DNA. mtDNA is inherited exclusively from the mother line. Typically, these diseases are due to oxidative phosphorylation of the disease. Mitochondrial diseases are generally caused by pathogenic mutations in mitochondrial genes. The mutation is usually heterogeneous, so there is a mixture of normal and mutant DNA, the levels of which are variable in different tissues. However, some mutations are homogeneous, so they are present in 100% of mtDNA. The percent heterogeneity of point mutations in offspring correlates with the percent mutation in the parent. There is a population genetic bottleneck effect that occurs during oocyte development.

A "genetically modified" or "engineered" cell refers to a cell into which exogenous nucleic acid has been introduced by a process involving labor (or the descendent of the word cell having inherited at least a portion of the nucleic acid). For example, the nucleic acid can contain sequences that are exogenous to the cell, can contain native sequences (i.e., sequences naturally found within the cell) but are present in a non-naturally occurring arrangement (e.g., coding regions linked to promoters from different genes), or can be a variant version of a native sequence, etc. The process of transferring the nucleus into the cell may be achieved by any suitable technique. Suitable techniques include calcium phosphate or lipid mediated transfection, electroporation, and transduction or infection with viral vectors. In some embodiments, the polynucleotide or portion thereof is integrated into the genome of the cell. The nucleic acid may then have been removed or excised from the genome, provided that the removal or excision results in a detectable change in the cell relative to an unmodified but otherwise equivalent cell.

The term "identity" refers to the degree to which two or more nucleic acids or polypeptides are identical in sequence. By aligning the sequence of interest with the second sequence; determining the number of residues (nucleotides or amino acids) on opposite sides of the same residue that allow gaps to be introduced to maximize identity over the length of the sequence of interest within the evaluation window; divided by the total number of residues of the sequence of interest or the second sequence (whichever is longer) that fall within the window; and multiplied by 100 to calculate the percent identity of the two sequences. When calculating the number of identical residues required to achieve a particular percent identity,the fraction is rounded to the nearest integer. The percent identity can be calculated using a variety of computer programs known in the art. For example, computer programs such as BLAST2, BLASTN, BLASTP, Gapped BLAST, and the like, generate alignments between sequences of interest and provide percent identities. The reaction mixture was prepared as described by Karlin and Altschul, proc.natl.acad.sci.usa 90: 5873 Alrlin and Altschul's algorithm (Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87: 22264-2268, 1990) modified as described in 5877, 1993, is incorporated into the NBLAST and XBLAST program of Altschul et al (Altschul, et al, J. mol. biol. 215: 403-410, 1990). To obtain Gapped alignments for comparison purposes, Gapped BLAST was used, as disclosed by Altschul et al (Altschul, et al. nucleic Acids Res.25: 3389-. When BLAST and Gapped BLAST programs are used, the default parameters for each program can be used. An APAM250 or BLOSUM62 matrix may be used. Software for the above BLAST analysis is publicly available through the National Center for Biotechnology Information (NCBI). Can be from a website aswww.ncbi.nlm.nih.govThe web site obtains these programs. In a specific embodiment, percent identity is calculated using BLAST2 with default parameters provided by NCBI. In some embodiments, the sequence identity of a nucleic acid or amino acid sequence to another nucleic acid or amino acid sequence is at least 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98%, or at least about 99%.

Herein, the term "isolated" or "partially purified" in the context of a nucleic acid or polypeptide refers to a nucleic acid or polypeptide that is isolated from at least one other component (e.g., a nucleic acid or polypeptide), wherein the at least one other component is found in its natural source to be present with the nucleic acid or polypeptide and/or will be present with the nucleic acid or polypeptide when expressed by a cell or secreted by a cell in the context of secreting a polypeptide. Nucleic acids or polypeptides that are chemically synthesized or synthesized using in vitro transcription/translation are considered "isolated". An "isolated cell" is a cell that has been removed from the organism in which it was originally found or as a descendent of the cell. Optionally, the cell has been cultured in vitro, e.g., in the presence of other cells. Optionally, the cell is subsequently introduced into a second organism or reintroduced into an organism from which the cell was isolated (or as a descendent of the cell).

Herein, the term "isolated population" in reference to an isolated population of cells refers to a population of cells that has been removed and separated from a mixed or heterogeneous population of cells. In some embodiments, the isolated population is a substantially pure population of cells as compared to a heterogeneous population from which the cells were isolated or enriched.

The term "substantially pure" with respect to a particular cell population refers to a cell population that is at least about 75%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% pure with respect to the cells that make up the total cell population. Moreover, with respect to a population of definitive endoderm cells, the term "substantially pure" or "substantially pure" refers to a population of cells that comprise less than about 20%, more preferably less than about 15%, 10%, 8%, 7%, most preferably less than about 5%, 4%, 3%, 2%, 1%, or less than 1% of non-definitive endoderm cells or progeny cells thereof as that term is defined herein. In some embodiments, the invention encompasses methods of expanding a population of definitive endoderm cells, wherein the expanded population of definitive endoderm cells is a substantially pure population of definitive endoderm cells. Likewise, a "substantially pure" or "substantially pure" population of stem cells or pluripotent stem cells derived from SCNT refers to a population of cells having less than about 20%, more preferably less than about 15%, 10%, 8%, 7%, most preferably less than about 5%, 4%, 3%, 2%, 1%, or less than 1% non-stem cells or progeny cells thereof as that term is defined herein.

Herein, the terms "enrichment" or "enriched" are used interchangeably and mean that the yield (fraction) of a type of cell is increased by at least 10% over the fraction of the cell type in the beginning of culture or preparation.

Herein, the terms "renewal" or "self-renewal" or "proliferation" are used interchangeably to refer to the ability of a stem cell to renew itself by dividing into the same non-specialized cell type over an extended period of time and/or over multiple months to years. In some cases, proliferation refers to the expansion of a cell by repeating the process of a single cell dividing into identical daughter cells.

Herein, the term "lineage" discloses cells having a common ancestor or cells having a common developmental fate. In the context of a cell being of endodermal origin or being "endodermal lineage", this term means that the cell is derived from an endodermal cell and can differentiate along an endodermal lineage-restricted pathway, such as one or more developmental lineage pathways leading to definitive endodermal cells, which can differentiate into hepatocytes, thymus, pancreas, lung, and intestine, in turn.

Herein, the term "xenogeneic" refers to cells derived from different species.

Herein, the term "marker" is used to reveal a characteristic and/or phenotype of a cell. The marker may be used for selection of cells comprising the feature of interest. The label will vary with the particular cell. The marker is unique regardless of the morphological, functional or biochemical (enzymatic) characteristics of the cell of a particular cell type or of the molecule expressed by that cell type. Such labels are preferably proteins, more preferably provided with epitopes for antibodies or other binding molecules available in the art. However, the marker may be composed of any molecule found in a cell, including, but not limited to, proteins (peptides and polypeptides), lipids, polysaccharides, nucleic acids, and steroids. Examples of topographical features or characteristics include, but are not limited to, shape, size, and nuclear to cytoplasmic ratio. Examples of functional features or characteristics include, but are not limited to, the ability to adhere to a particular substrate, the ability to incorporate or exclude a particular dye, the ability to migrate under particular conditions, and the ability to differentiate along a particular lineage. The label may be detected by any method available to those skilled in the art. The marker may also be the absence of topographical features or the absence of proteins, lipids, etc. The marker can be a unique set of features and other morphological features that are present and absent from the polypeptide.

The term "modulate" is used consistently with its use in this field, i.e., to mean causing or facilitating qualitative or quantitative changes, variations, or modifications in a process, pathway, or phenomenon of interest. Without limitation, such alteration may be an increase, decrease, or change in different components or branches of a process, pathway, or phenomenon. A "modulator" is an agent that causes or contributes to a qualitative or quantitative change, variation, or modification of a process, pathway, or phenomenon of interest.

The term "RNA interference" or "RNAi" is used consistently herein for its use in the field and refers to the phenomenon by which double-stranded RNA (dsRNA) triggers sequence-specific degradation or translational repression of the corresponding mRNA complementary to the dsRNA strand. It will be appreciated that the complementarity between the dsRNA strand and the mRNA need not be 100%, but need only be sufficient to mediate inhibition of gene expression (also referred to as "silencing" or "knock-out"). For example, the degree of complementarity should be such that the strand can (i) direct the cleavage of mRNA in the RNA-induced silencing complex (RISC); or (ii) causes translational repression of the mRNA. In certain embodiments, the double-stranded portion of the RNA is less than about 30 nucleotides in length, e.g., between 17 and 29 nucleotides in length. In mammalian cells, RNAi can be achieved by introducing an appropriate double-stranded nucleic acid into the cell, or expressing the nucleic acid in the cell, followed by intracellular processing of the cell to obtain dsRNA therein. Nucleic acids capable of mediating RNAi are referred to herein as "RNAi agents". Exemplary nucleic acids capable of mediating RNAi are short hairpin rna (shrna), short interfering rna (sirna), and microRNA precursors. These terms are well known and are consistently used herein in their meaning in the art. siRNA typically comprises two separate nucleic acid strands that hybridize to each other to form a double helix. They can be synthesized in vitro, e.g., using standard nucleic acid synthesis techniques. They may comprise a wide variety of modified nucleosides, nucleoside analogs, and may comprise chemically or biologically modified bases, modified backbones, and the like. Any modification recognized in the art as useful for RNAi may be used. Some modifications result in increased stability, cellular uptake, potency, etc. In certain embodiments, the siRNA comprises a double helix of about 19 nucleotides in length, and one or two 3' overhangs of1 to 5 nucleotides in length, which overhangs may be composed of deoxyribonucleotides. shRNA comprises a single nucleic acid strand containing two complementary portions separated by a region of predominantly non-self-complementarity. The complementary portions hybridize to form a duplex structure, and the non-self-complementary region forms a loop connecting the 3 'end of one strand of the duplex to the 5' end of the other strand. The shRNA is processed intracellularly to generate siRNA.

The term "selectable marker" refers to a gene, RNA, or protein that, when expressed, confers a selectable phenotype on a cell, such as resistance to cytotoxic or cytostatic agents (e.g., antibiotic resistance), prototrophy to nutrients, or expression of a particular protein, which can serve as a basis for distinguishing cells that express a protein from cells that do not express a protein. Proteins whose expression can be easily detected, such as fluorescent or luminescent proteins or enzymes that act on a substrate to produce a colored, fluorescent, or luminescent substance ("detectable markers") constitute a subset of selectable markers. The presence of a selectable marker linked to an expression control element produced from a gene that is normally expressed selectively or exclusively in pluripotent cells makes it possible to identify and select somatic cells that have been reprogrammed to a pluripotent state. Various selectable marker genes can be used, such as neomycin resistance gene (neo), puromycin resistance gene (puro), guanine phosphoribosyltransferase (gpt), dihydrofolate reductase (DHFR), adenosine deaminase (ada), puromycin-N-acetyltransferase (PAC), hygromycin resistance gene (hyg), multidrug resistance gene (mdr), Thymidine Kinase (TK), hypoxanthine-guanine phosphoribosyltransferase (HPRT), and hisD gene. Detectable labels include Green Fluorescent Protein (GFP); blue, cyan, yellow, red, orange and cyan fluorescent proteins; and any variants of these fluorescent proteins. Photoproteins such as luciferase (e.g., firefly luciferase or renilla luciferase) may also be used. It will be apparent to those skilled in the art that, herein, the term "selectable marker" may refer to a gene or to an expression product of a gene, such as an encoded protein.

The term "small molecule" refers to an organic compound having multiple carbon-carbon bonds and a molecular weight of less than 1500 daltons. These compounds typically contain one or more functional groups that mediate structural interactions with the protein, such as hydrogen bonding, and typically include at least an amino, carbonyl, hydroxyl, or carboxyl group, and in some embodiments, at least two chemical functional groups. The small molecule agent may comprise a cyclic carbocyclic or heterocyclic structure and/or an aromatic or polycyclic aromatic structure substituted with one or more chemical functional groups and/or heteroatoms.

As used herein, the term "polypeptide" refers to a polymer of amino acids. The terms "protein" and "polypeptide" are used interchangeably. Peptides are relatively short polypeptides, typically between about 2 and 60 amino acids in length. The polypeptides used herein typically contain amino acids, such as the most common 20L-amino acids in proteins. However, other amino acids and/or amino acid analogs known in the art may be used. One or more amino acids in a polypeptide can be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a phosphate group, a fatty acid group, a linker for conjugation, functionalization, and the like. A polypeptide having a non-polypeptide moiety covalently or non-covalently associated therewith is still considered a "polypeptide". Exemplary modifications include glycosylation and palmitoylation. Polypeptides may be purified from natural sources, produced using recombinant DNA techniques, synthesized by chemical means such as conventional solid phase peptide synthesis methods, and the like. Herein, the term "polypeptide sequence" or "amino acid sequence" may refer to the polypeptide material itself, and/or to the sequence information that biochemically characterizes the polypeptide (i.e., consecutive alphabetic or three-letter codons used as abbreviations for amino acid names). Unless otherwise indicated, polypeptide sequences presented herein are presented N-terminal to C-terminal.

The term "variant" with respect to a polypeptide or nucleic acid sequence can be, e.g., a polypeptide or nucleic acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the full-length polypeptide or nucleic acid sequence. In some embodiments, a variant can be a fragment of a full-length polypeptide or nucleic acid sequence. In some embodiments, the variant can be a naturally occurring splice variant. For example, Suv39h1(Gene ID: 6839) has two splice variants which are not the same, variant 1 produces a Suv39h1 isoform 1 protein (long transcript and encodes a longer isoform) and corresponds to mRNA NM-001282166.1 and protein NP-001269095.1, while variant 2 produces Suv39h1 isoform 2, variant 2 differs in the 5 'UTR, lacks the 5' coding region, and initiates transcription at an alternative start codon as compared to variant 1. The encoded Suv39h1 subtype (2) protein is shorter and has a distinct N-terminus from subtype 1. The mRNA for subtype 2 of Suv39h1 is NM _003173.3, which encodes a subtype 2 protein corresponding to NP _ 003164.1. A variant may be a polypeptide or nucleic acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to a fragment at least 50% of the length of the full-length polypeptide or nucleic acid sequence, wherein the fragment is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% of the length of the full-length wild-type polypeptide or nucleic acid sequence having the activity of interest. For example, a variant of KDM4d has the ability to increase SCNT efficiency to the same or similar extent as KDM4d polypeptide or KDM4d nucleic acid sequences.

Herein, the terms "functional fragment" or "biologically active fragment" are used interchangeably and refer to a polypeptide having an amino acid sequence that is smaller in size than the polypeptide from which it is a fragment, wherein the biological effect of the functional fragment polypeptide is at least 50%, or 60%, or 70%, or 80%, or 90%, or 100% or more than 100%, e.g., 1.5-fold, 2-fold, 3-fold, 4-fold, or more than 4-fold, of the polypeptide from which it is a fragment. Functional fragment polypeptides may have additional functions that may include reduced antigenicity, increased DNA binding (e.g., in transcription factors), or altered RNA binding (e.g., in modulating RNA stability or degradation). In some embodiments, the biologically active fragment is substantially homologous to the polypeptide from which it is a fragment. Without being bound by theory, illustrative examples of functional fragments of KDM4 histone demethylase activator of KDM4A include SEQ ID NO: 9 (e.g., wherein the fragment is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% of SEQ ID NO: 9 in length), the ability of the functional fragment to increase SCNT efficiency using the same method and under the same conditions is a fragment comprising SEQ ID NO: about at least 50%, or 60%, or 70%, or 80%, or 90%, or 100% or more than 100%, e.g., 1.5-fold, 2-fold, 3-fold, 4-fold, or more than 4-fold of the KDM4A polypeptide of amino acid 9. In some embodiments, the nucleic acid sequence of SEQ ID NO: 9 lacks the biologically active fragment of SEQ ID NO: 9, at least 1, or at least 2 to 10, or at least 10 to 20, or at least 20 to 50, or at least 50 to 100 amino acids of the C-or N-terminus. In some embodiments, the nucleic acid sequence of SEQ ID NO: 9 lacks the biologically active fragment of SEQ ID NO: 9 at least 1, or at least 2 to 10, or at least 10 to 20, or at least 20 to 50, or at least 50 to 100 amino acids from the C-and N-terminal ends. In some embodiments, the nucleic acid sequence of SEQ ID NO: 12, a biologically active fragment of KDM4D, e.g., SEQ id no: 12 comprising SEQ ID NO: 12, as disclosed in antonym et al, Nature, 2013. In some embodiments, the nucleic acid sequence of SEQ ID NO: 12 comprises SEQ id no: 12 (e.g., a fragment corresponding to SEQ ID NO: 13). In some embodiments, the nucleic acid sequence of SEQ ID NO: 12 also lacks the biologically active fragment of SEQ ID NO: 12 from amino acids 1 to 424 at least 1, or at least 2 to 10, or at least 10 to 20, or at least 20 to 50, or at least 50 to 100 amino acids from both the C-and N-terminal ends. In some embodiments, the nucleic acid sequence of SEQ ID NO: 12 comprises SEQ ID NO: 12, which also lacks amino acids 1 to 424 of SEQ ID NO: 12 from amino acids 1 to 424 at least 1, or at least 2 to 10, or at least 10 to 20, or at least 20 to 50, or at least 50 to 100 amino acids from both the C-and N-terminal ends.

Herein, the term "functional fragment" or "biologically active fragment" in reference to a nucleic acid sequence refers to a nucleic acid sequence that is smaller in size than the nucleic acid sequence from which it is a fragment, wherein the nucleic acid sequence has a biological effect that is at least 50%, or 60%, or 70%, or 80%, or 90%, or 100% or more than 100%, e.g., 1.5-fold, 2-fold, 3-fold, 4-fold, or more than 4-fold, of the polypeptide from which it is a biologically active fragment. Without being bound by theory, illustrative examples of the nucleic acid sequence of KDM4 histone demethylase activator of KDM4A include SEQ ID NO: 1 (e.g., wherein the fragment is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% of SEQ ID NO: 1 in length), the ability of the nucleic acid sequence to increase SCNT efficiency using the same method and under the same conditions is SEQ ID NO: 1, about at least 50%, or 60%, or 70%, or 80%, or 90%, or 100% or more than 100%, e.g., 1.5-fold, 2-fold, 3-fold, 4-fold, or more than 4-fold of the KDM4A nucleic acid sequence.

The terms "treating", "disposing", and the like, as applied to an isolated cell, include subjecting the cell to various processes or conditions or performing various operations or processes on the cell. When applied to a subject, the term refers to providing medical or surgical attention, care, or management to an individual. The individual is typically ill (suffering from a disease or other medical/surgical concern) or injured, or at increased risk of illness relative to the average members of the population and in need of such attention, care, or management. Herein, "individual" and "subject" are used interchangeably. In any embodiment of the invention, the "individual" may be a human, e.g., a human that suffers from or is at risk of the disease ("indicated") for which the cell therapy is used.

Herein, the term "synchronized" or "simultaneous" with respect to the estrus cycle refers to assisted reproduction techniques well known to those skilled in the art. These techniques are fully disclosed in the references cited in the previous paragraphs. Typically, estrogen cycles of the female are synchronized with the developmental cycles of the embryos using estrogen and progesterone hormones. As used herein, the term "developmental cycle" refers to the embryo of the invention and the period of time that exists between each cell division within the embryo. This period is predictable to the embryo and can be synchronized with the estrus cycle of the recipient animal.

Herein, the phrase "substantially similar" to a nuclear DNA sequence refers to two nearly identical nuclear DNA sequences. The difference between the two sequences is the difference in replication errors that normally occur during nuclear DNA replication. The identity of substantially similar DNA sequences is preferably greater than 97%, more preferably greater than 98%, and most preferably greater than 99%. Consistency was measured by: the number of identical residues in both sequences is divided by the total number of residues and the result is multiplied by 100. Thus, two copies of the identical sequence are 100% identical, while sequences that are less conserved and have deletions, additions, or substitutions have a lower degree of identity. One skilled in the art will recognize that several computer programs can be used to perform sequence alignments and determine sequence identity.

The terms "reduce", "decrease", "alleviate" or "decline" or "inhibit" are all used herein to generally mean a statistically significant amount of decline. However, for the avoidance of doubt, "reduce", "mitigating" or "decline" or "inhibition" means a decrease of at least 10% compared to a reference level, for example, a decrease of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including 100% decrease (i.e., the level of absence compared to a reference sample), or a decrease between 10% and 100% compared to a reference level.

The terms "increased"/"increase" or "boost" or "activation" are all used herein to generally mean an increase in a statistically significant amount; for the avoidance of any doubt, the term "increased"/"increase" or "boost" or "activation" means an increase of at least 10% compared to a reference level, for example, an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including 100% or an increase of between 10% and 100% compared to a reference level, or at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold or at least about 10-fold, or an increase of between 2-fold and 10-fold or more compared to a reference level.

The term "statistically significant" or "significant" refers to statistical significance, and generally means two standard deviations (2SD) or less below the normal concentration of the marker. The term refers to statistical evidence that there is a difference. It is defined as the probability of making a decision to reject a null hypothesis when it is actually true. This decision is typically made using the p-value.

As used herein, the term "comprising" is used in reference to compositions, methods, and individual ingredients thereof, as necessary for the present invention, but the possibility exists of including elements not specifically recited, whether or not such elements are necessary.

The term "consisting essentially of" is used herein to refer to the elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the novel or functional characteristics of this embodiment of the invention.

The term "consisting of" refers to the compositions, methods, and individual components thereof disclosed herein, which term excludes any elements not listed in the description of the embodiments.

As used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless expressly excluded. Thus, for example, reference to "the method" includes one or more methods, and/or types disclosed herein and/or apparent to those skilled in the art upon reading this disclosure and so forth.

It is to be understood that both the foregoing detailed description and the following examples are exemplary and explanatory only and are not restrictive of the scope of the invention, as claimed. Various changes and modifications to the disclosed embodiments, which will be apparent to those skilled in the art, may be made without departing from the spirit and scope of the invention. Further, all patents, patent applications, and publications identified are expressly incorporated herein by reference for the purpose of disclosure and disclosure, e.g., the methods disclosed in these publications can be used in connection with the present invention. Of these publications, only those prior to the filing date of this application are provided for use. In this regard, nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of the forward documents.

KDM4 histone demethylase activating factor

In one aspect, the invention provides a method of increasing the efficiency of human SCNT, the method comprising: contacting the nucleus or cytoplasm of a donor human somatic cell, a recipient human oocyte, a hybrid oocyte (e.g., an enucleated human oocyte comprising donor genetic material prior to fusion or activation) or a human SCNT embryo (i.e., after fusion of the donor nucleus with the enucleated oocyte) with an agent that inhibits histone methylation, particularly inhibition of H3K9 methylation, particularly inhibition of H3H9me3 trimethylation. In some embodiments, the agent is KDM4 histone demethylase activator.

In some embodiments, KDM4 histone demethylase activators useful in the methods, compositions, and kits disclosed herein are agents that increase the expression of a gene encoding a KDM4 family histone demethylase, or increase the activity of a human KDM4 family histone demethylase, e.g., human KDM4A, human KDM4B, human KDM4C, or human KDM 4D. In some embodiments, the agent increases the expression or activity of KDM4D (JMJD2D) or KDM4A (JMJD 2A).

In some embodiments, KDM4 histone demethylase activators useful in the methods, compositions, and kits disclosed herein are nucleic acid agents encoding a KDM4A polypeptide, or a KDM4A polypeptide, or variants or biologically active fragments thereof. Herein, the human KDM4A nucleotide sequence corresponds to gene bank (Genbank) accession No. NM — 014663.2 and refers to seq id NO: 1. KDM4A is also known as lysine (K) -specific demethylase 4A, JMJD2, JMJD2A, "2-containing jumonji domain", or "2A-containing jumonji domain". The human KDM4A protein corresponds to GenBank accession number NP-055478.2 (SEQ ID NO: 9). Accordingly, the protein sequence of KDM4A is as follows:

in some embodiments, the agent comprises a nucleic acid sequence of human KDM4A (SEQ ID NO: 1), or a biologically active fragment or homolog or variant thereof having at least 80% identity (or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98%, or at least about 99% sequence identity) to its sequence, which agent increases the efficiency of human SCNT to a level similar to or greater than the level of sequence identity of SEQ ID NO: 1 degree of the corresponding sequence. In some embodiments, the composition comprises a peptide corresponding to SEQ id no: 1 or a biologically active fragment thereof, which increases the efficiency of human SCNT to a level similar to or greater than SEQ ID NO: 1, or a nucleic acid sequence of the nucleic acid sequence of 1.

In some embodiments, the histone demethylase activator used in the methods disclosed herein is selected from the group consisting of a nucleic acid agent encoding any human KDM4A polypeptide, or a nucleic acid agent encoding any variant or biologically active fragment of a human KDM4A polypeptide. In some embodiments, the histone demethylase activator used in the methods disclosed herein is selected from a human KDM4A polypeptide, or a variant or biologically active fragment of the human KDM4A polypeptide. It is contemplated in the present invention that one of skill in the art can identify suitable human homologs of human KDM4A polypeptide, and nucleic acids encoding the human homologs for use in the methods and compositions disclosed herein.

In some embodiments, KDM4 histone demethylase activator useful in the methods, compositions, and kits disclosed herein are nucleic acid agents encoding the KDM4B polypeptide, or KDM4B polypeptide, or variants or biologically active fragments thereof. Herein, the human KDM4B nucleic acid corresponds to genbank accession No. NM — 015015.2 and refers to seq id NO: 2. KDM4B is also known as lysine (K) -specific demethylase 4B, JMJD2B or "2B-containing jumonji domain", KIAA0876, TDRD 14B, or "14B-containing tudor domain". The human KDM4B protein corresponds to GenBank accession number NP055830.1(SEQ ID NO: 10). Accordingly, the protein sequence of KDM4B is as follows:

in some embodiments, the agent is a nucleic acid sequence of human KDM4B (SEQ ID NO: 2), or a biologically active fragment or homolog or variant thereof having at least 80% sequence identity thereto (or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98%, or at least about 99% sequence identity thereto), that increases the efficiency of human SCNT to a level similar to or greater than the level of sequence identity of SEQ ID NO: 2 degree of corresponding sequence. In some embodiments, the composition comprises a peptide corresponding to SEQ ID NO: 2, or a biologically active fragment thereof, that increases the efficiency of human SCNT to a level similar to or greater than SEQ ID NO: 2, or a nucleic acid sequence of seq id No. 2.

In some embodiments, the histone demethylase activator used in the methods disclosed herein is selected from a nucleic acid agent encoding any human KDM4B polypeptide, or a nucleic acid agent encoding a variant or biologically active fragment of a human KDM4B polypeptide. In some embodiments, the histone demethylase activator used in the methods disclosed herein is selected from any human KDM4B polypeptide, or a variant or biologically active fragment of the human KDM4B polypeptide. It is contemplated in the present invention that one of skill in the art can identify suitable human homologs of human KDM4B polypeptide, and nucleic acids encoding the human homologs for use in the methods and compositions disclosed herein.

In some embodiments, KDM4 histone demethylase activator useful in the methods, compositions, and kits of the invention are nucleic acid agents encoding a KDM4C polypeptide, or a KDM4C polypeptide, or variants or biologically active fragments thereof. Herein, the human KDM4C nucleic acid sequence corresponds to GenBank accession No. NM-015061.3 (SEQ ID NO: 3), as disclosed herein. KDM4C is also known as lysine (K) -specific demethylase C, JMJD2C or "2C-containing jumonji domain", GASC1, KIAA0780, TDRD 14C or "14C-containing tudor domain". The human KDM4C protein corresponds to GenBank accession number NP-055876.2 (SEQ ID NO: 11). Accordingly, the protein sequence of KDM4C is as follows:

in some embodiments, the agent comprises the nucleic acid sequence of human KDM4C (SEQ ID NO: 3), or a biologically active fragment or homolog or variant thereof having at least 80% identity (or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98%, or at least about 99% sequence identity) to its sequence, which agent increases the efficiency of human SCNT to a level similar to or greater than the level of sequence shown in SEQ ID NO: 3 degree of corresponding sequence. In some embodiments, the composition comprises a peptide corresponding to SEQ id no: 3, or a biologically active fragment thereof, that increases the efficiency of human SCNT to a level similar to or greater than SEQ ID NO: 3, or a nucleic acid sequence of seq id no.

In some embodiments, the histone demethylase activator used in the methods disclosed herein is selected from a nucleic acid agent encoding any human KDM4C polypeptide, or a nucleic acid agent encoding a variant or biologically active fragment of a human KDM4C polypeptide. In some embodiments, the histone demethylase activator used in the methods disclosed herein is selected from any human KDM4C polypeptide, or a variant or biologically active fragment of the human KDM4C polypeptide. It is contemplated in the present invention that one of skill in the art can identify suitable human homologs of human KDM4C polypeptide, and nucleic acids encoding the human homologs for use in the methods and compositions disclosed herein.

In some embodiments, KDM4 histone demethylase activator useful in the methods, compositions, and kits of the invention are nucleic acid agents encoding a KDM4D polypeptide, or a KDM4D polypeptide, or variants or biologically active fragments thereof. Herein, the human KDM4D nucleic acid sequence corresponds to genbank accession No. NM — 018039.2 and refers to seq id NO: 4. KDM4D is also known as lysine (K) -specific demethylase 4D, FLJ10251, JMJD2D or "2D-containing jumonji domain". The human KDM4D protein corresponds to GenBank accession number NP-060509.2 (SEQ ID NO: 12). Accordingly, the protein sequence of KDM4D is as follows:

in some embodiments, the agent comprises the nucleic acid sequence of human KDM4D (SEQ ID NO: 4), or a biologically active fragment or homolog or variant thereof having at least 80% identity (or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98%, or at least about 99% sequence identity) to its sequence, which agent increases the efficiency of human SCNT to a level similar to or greater than the level of SEQ ID NO: 4 degree of corresponding sequence. In some embodiments, the composition comprises a peptide corresponding to SEQ id no: 4, or a biologically active fragment thereof, increases the efficiency of human SCNT to a level similar to or greater than SEQ ID NO: 4, or a nucleic acid sequence of seq id no.

In some embodiments, the agent that contacts a donor human somatic cell, an acceptor human oocyte, a hybrid oocyte (e.g., an enucleated human oocyte comprising donor genetic material prior to fusion or activation) or a human SCNT embryo (i.e., after fusion of the donor nucleus with the enucleated oocyte) increases the relative abundance of SEQ ID NO: 9, or the human KDM4A protein of SEQ ID NO: 10, or the human KDM4B protein of SEQ ID NO: 11, or the human KDM2C protein of SEQ ID NO: 12, and/or comprises a sequence corresponding to SEQ ID NO: 1, a human KDM4A nucleic acid sequence corresponding to SEQ ID NO: 2, a human KDM4B nucleic acid sequence corresponding to SEQ ID NO: 3, a nucleic acid sequence of human KDM4C corresponding to SEQ ID NO: 4, a nucleic acid sequence of human KDM4D corresponding to SEQ ID NO: 45, or the human KDM4E nucleic acid sequence of SEQ ID NO: 1 to SEQ ID NO: 4 or SEQ ID NO: 45 that increases the efficiency of human SCNT to a level similar to or exceeding that of SEQ ID NO: 1 to SEQ ID NO: 4 or SEQ ID NO: 45 (e.g., an increase of at least about 110%, or at least about 120%, or at least about 130%, or at least about 140%, or at least about 150%, or more than 150%).

In some embodiments, the nucleic acid sequence of SEQ ID NO: 12 comprises SEQ ID NO: 12, as disclosed in antonym et al, Nature, 2013. In some embodiments, the nucleic acid sequence of SEQ ID NO: 12 comprises SEQ ID NO: 12, which also lacks amino acids 1 to 424 of SEQ ID NO: 12, at least 1, or at least 2 to 10, or at least 10 to 20, or at least 20 to 50, or at least 50 to 100 amino acids of the C-terminus, or the N-terminus of amino acids 1 to 424. In some embodiments, the nucleic acid sequence of SEQ ID NO: 12 comprises SEQ id no: 12, which also lacks amino acids 1 to 424 of SEQ ID NO: 12 from amino acids 1 to 424 at least 1, or at least 2 to 10, or at least 10 to 20, or at least 20 to 50, or at least 50 to 100 amino acids from both the C-and N-terminal ends. In some embodiments, the nucleic acid sequence of SEQ ID NO: 12 comprises SEQ ID NO: 64, wherein, SEQ id no: 13 comprises:

in some embodiments, the nucleic acid sequence of SEQ ID NO: 12 comprises SEQ ID NO: 13, which also lacks the amino acid sequence set forth in SEQ ID NO: 13, or at least 1, or at least 2 to 10, or at least 10 to 20, or at least 20 to 50, or at least 50 to 100 amino acids of the C-terminus. In some embodiments, the nucleic acid sequence of SEQ ID NO: 12 comprises SEQ ID NO: 13, which also lacks the amino acid sequence set forth in SEQ ID NO: 13, or at least 1, or at least 2 to 10, or at least 10 to 20, or at least 20 to 50, or at least 50 to 100 amino acids of the N-terminus.

In some embodiments, the histone demethylase activator used in the methods, compositions, and kits disclosed herein is selected from a nucleic acid agent encoding any human KDM4D polypeptide, or a nucleic acid agent encoding a variant or biologically active fragment of a human KDM4D polypeptide. In some embodiments, the histone demethylase activator used in the methods disclosed herein is selected from any human KDM4D polypeptide, or a variant or biologically active fragment of the human KDM4D polypeptide. It is contemplated in the present invention that one of skill in the art can identify suitable human homologs of human KDM4D polypeptide, and nucleic acids encoding the human homologs for use in the methods and compositions disclosed herein.

In some embodiments, KDM4 histone demethylase activator useful in the methods, compositions, and kits of the invention are nucleic acid agents encoding a KDM4E polypeptide, or a KDM4E polypeptide, or variants or biologically active fragments thereof. Herein, the human KDM4E nucleic acid corresponds to genbank accession No. NM — 001161630.1 and refers to seq id NO: 45. KDM4E is also known as lysine (K) -specific demethylase 4E, JMJD2E or "2E-containing jumonji domain", KDM4DL, or "lysine (K) -specific demethylase 4D-like". The human KDM4E corresponds to GenBank accession number NP001155102.1(SEQ ID NO: 46). Accordingly, the protein sequence of human KDM4E is as follows:

in some embodiments, the agent comprises the nucleic acid sequence of human KDM4E (SEQ ID NO: 45), or a biologically active fragment or homolog or variant thereof having at least 80% identity (or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98%, or at least about 99% sequence identity) to its sequence, which agent increases the efficiency of human SCNT to a level similar to or greater than the level of SEQ ID NO: 45 degree of the corresponding sequence. In some embodiments, the composition comprises a peptide corresponding to seq id NO: 45, or a biologically active fragment thereof, increases the efficiency of human SCNT to a level similar to or greater than SEQ ID NO: 45 to a nucleic acid sequence of seq id no.

In some embodiments, the histone demethylase activator used in the methods disclosed herein is selected from a nucleic acid agent encoding any human KDM4E polypeptide, or a nucleic acid agent encoding a variant or biologically active fragment of a human KDM4E polypeptide. In some embodiments, the histone demethylase activator used in the methods disclosed herein is selected from any human KDM4E polypeptide, or a variant or biologically active fragment of the human KDM4E polypeptide. It is contemplated in the present invention that one of skill in the art can identify suitable human homologs of human KDM4E polypeptide, and nucleic acids encoding the human homologs for use in the methods and compositions disclosed herein.

As used in some embodiments, the histone demethylase activating factor used in the methods disclosed herein is selected from any one of the group consisting of AOF (LSD), FBXL (JHDM 1), 110(JHDM 1), FBXL (JHDM 1), KIAA1718(JHDM 1), PHF (JHDM 1), JMJD 1(JHDM 2), KDM4 (jjd 2; JHDM 3), jmdm 3, KDM4 (jjd 2), RBP (JARID 1), PLU (JARID IB), SMCX (smcy), (jjijd), jiumjimji (jajjd), jrix (jjjd), jjjjjyjjjd (jjd), jjjjjjjjjd (jjd), jjjjjd), jjjjd (jjjd), jjd (jjd), jjjjjjjjd). Such histone demethylase activators are disclosed in U.S. patent application No. US 2011/0139145, which is incorporated by reference herein in its entirety.

In some embodiments, KDM4 histone demethylase activator is a polypeptide variant, or a nucleic acid sequence encoding a polypeptide variant having at least 80%, 85%, 90%, 95%, 98%, or 99% identity to a full-length polypeptide, or SEQ id no: 9 to SEQ ID NO: 12 or SEQ ID NO: 46 or by a sequence corresponding to SEQ ID NO: 1 to SEQ ID NO: 4 or SEQ ID NO: 45 (KDM 4A) to KDM4E) of any of the human KDM4 polypeptides encoded by any of the nucleic acid sequences.

In some embodiments, KDM4 histone demethylase activator is a polypeptide variant; or a variant that encodes a polypeptide variant and has at least 80%, 85%, 90%, 95%, 98%, or 99% identity to the full-length polypeptide; or SEQ ID NO: 9 to SEQ ID NO: 12 or SEQ ID NO: 46 KDM4 polypeptide (human KDM4A to KDM 4E). In some embodiments, KDM4 histone demethylase is SEQ ID NO: 9 to SEQ ID NO: 12 or SEQ ID NO: 46 (human KDM4A to KDM4E) in at least 20 contiguous amino acids; or a fragment of human KDM4A, KDM4B, KDM4C, KDM4D, or KDM4E that is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% of the length of the wild-type polypeptide; or a domain thereof having an activity of interest, such as a polypeptide having a sequence identical to SEQ ID NO: 9 to SEQ ID NO: 12 or SEQ ID NO: 46 (human KDM4A to KDM4E) increases SCNT efficiency by at least 80% or more compared to the efficiency of the protein.

In some embodiments, a biologically active fragment of human KDM4A comprises SEQ ID NO: 9 (e.g., wherein the fragment is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% of SEQ ID NO: 9 in length), the ability of the fragment to increase SCNT efficiency using the same method and under the same conditions is a fragment comprising SEQ ID NO: about at least 50%, or 60%, or 70%, or 80%, or 90%, or 100%, or more than 100%, such as 1.5-fold, 2-fold, 3-fold, 4-fold, or more than 4-fold of the KDM4A polypeptide of amino acid 9.

In some embodiments, a biologically active fragment of human KDM4B comprises SEQ ID NO: 10 (e.g., wherein the fragment is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% of SEQ ID NO: 10 in length), the ability of the fragment to increase SCNT efficiency using the same method and under the same conditions is a fragment comprising SEQ ID NO: 10, about at least 50%, or 60%, or 70%, or 80%, or 90%, or 100%, or more than 100%, such as 1.5-fold, 2-fold, 3-fold, 4-fold, or more than 4-fold of the KDM4B polypeptide.

In some embodiments, a biologically active fragment of human KDM4C comprises SEQ ID NO: 11 (e.g., wherein the fragment is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% of SEQ ID NO: 11 in length), the ability of the fragment to increase SCNT efficiency using the same method and under the same conditions is a fragment comprising SEQ ID NO: 11, about at least 50%, or 60%, or 70%, or 80%, or 90%, or 100%, or more than 100%, such as 1.5-fold, 2-fold, 3-fold, 4-fold, or more than 4-fold of the KDM4C polypeptide.

In some embodiments, a biologically active fragment of human KDM4D comprises SEQ ID NO: 12 (e.g., wherein the fragment is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% of SEQ ID NO: 12 in length), the ability of the fragment to increase SCNT efficiency using the same method and under the same conditions is a fragment comprising SEQ ID NO: 12, about at least 50%, or 60%, or 70%, or 80%, or 90%, or 100%, or more than 100%, such as 1.5-fold, 2-fold, 3-fold, 4-fold, or more than 4-fold of the KDM4D polypeptide. In some embodiments, the nucleic acid sequence of SEQ ID NO: 12 comprises seq id NO: 12, as disclosed in antonym et al, Nature, 2013. In some embodiments, the nucleic acid sequence of SEQ ID NO: 12 comprises SEQ ID NO: 12, which also lacks amino acids 1 to 424 of SEQ id no: 12 from amino acids 1 to 424 at least 1, or at least 2 to 10, or at least 10 to 20, or at least 20 to 50, or at least 50 to 100 amino acids from the C-terminus or the N-terminus. In some embodiments, the nucleic acid sequence of SEQ ID NO: 12 comprises EQ ID NO: 12, which also lacks amino acids 1 to 424 of SEQ ID NO: 12 from amino acids 1 to 424 at least 1, or at least 2 to 10, or at least 10 to 20, or at least 20 to 50, or at least 50 to 100 amino acids from both the C-and N-terminal ends. In some embodiments, the nucleic acid sequence of SEQ ID NO: 12 comprises SEQ ID NO: 13, wherein SEQ ID NO: 13 comprises:

in some embodiments, the nucleic acid sequence of SEQ ID NO: 12 comprises SEQ ID NO: 13, which also lacks the amino acid sequence set forth in SEQ ID NO: 13, or at least 1, or at least 2 to 10, or at least 10 to 20, or at least 20 to 50 amino acids of the C-terminus. In some embodiments, the nucleic acid sequence of SEQ ID NO: 12 comprises SEQ ID NO: 13, which also lacks the amino acid sequence set forth in SEQ ID NO: 13, or at least 1, or at least 2 to 10, or at least 10 to 20, or at least 20 to 50 amino acids from the N-terminus.

In some embodiments, a biologically active fragment of human KDM4E comprises SEQ ID NO: 46 (e.g., wherein the fragment is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% of SEQ ID NO: 46 in length), the ability of the fragment to increase SCNT efficiency using the same method and under the same conditions is a fragment comprising SEQ ID NO: 46, or 60%, or 70%, or 80%, or 90%, or 100%, or more than 100%, such as 1.5-fold, 2-fold, 3-fold, 4-fold, or more than 4-fold of the KDM4E polypeptide.

In some embodiments, a biologically active variant of human KDM4A comprises SEQ ID NO: 9, which is homologous to seq id NO: 9 (e.g., wherein the variant has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID No. 9), and the ability of the fragment to increase the efficiency of SCNT using the same method and under the same conditions is a fragment comprising SEQ ID NO: about at least 50%, or 60%, or 70%, or 80%, or 90%, or 100%, or more than 100%, such as 1.5-fold, 2-fold, 3-fold, 4-fold, or more than 4-fold of the KDM4A polypeptide of amino acid 9.

In some embodiments, a biologically active variant of human KDM4B comprises SEQ ID NO: 10, which is homologous to seq id NO: 10 (e.g., wherein the variant has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID No. 10), and the ability of the fragment to increase the efficiency of SCNT using the same method and under the same conditions is a fragment comprising SEQ ID NO: 10, about at least 50%, or 60%, or 70%, or 80%, or 90%, or 100%, or more than 100%, such as 1.5-fold, 2-fold, 3-fold, 4-fold, or more than 4-fold of the KDM4B polypeptide.

In some embodiments, a biologically active variant of human KDM4C comprises SEQ ID NO: 11, which is homologous to seq id NO: 11 (e.g., wherein the variant has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID No. 11), and the ability of the fragment to increase the efficiency of SCNT using the same method and under the same conditions is a fragment comprising SEQ ID NO: 11, about at least 50%, or 60%, or 70%, or 80%, or 90%, or 100%, or more than 100%, such as 1.5-fold, 2-fold, 3-fold, 4-fold, or more than 4-fold of the KDM4C polypeptide.

In some embodiments, a biologically active variant of human KDM4D comprises SEQ ID NO: 12, which is homologous to seq id NO: 12 (e.g., wherein the variant has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID No. 12), and the ability of the fragment to increase the efficiency of SCNT using the same method and under the same conditions is a fragment comprising SEQ ID NO: 12, about at least 50%, or 60%, or 70%, or 80%, or 90%, or 100%, or more than 100%, such as 1.5-fold, 2-fold, 3-fold, 4-fold, or more than 4-fold of the KDM4D polypeptide.

In some embodiments, a biologically active variant of human KDM4E comprises SEQ ID NO: 46, which is homologous to seq id NO: 46 (e.g., wherein the variant has at least 85%, 90%, 95%, 98%, or 99% identity to SEQ ID No. 46), and the ability of the fragment to increase the efficiency of SCNT using the same method and under the same conditions is a fragment comprising SEQ ID NO: 46, or 60%, or 70%, or 80%, or 90%, or 100%, or more than 100%, such as 1.5-fold, 2-fold, 3-fold, 4-fold, or more than 4-fold of the KDM4E polypeptide.

In some embodiments, KDM4 histone demethylase activator useful in the methods, compositions, and kits disclosed herein is a polypeptide corresponding to SEQ ID NO: 1 to SEQ ID NO: 4 or SEQ ID NO: 45, or a nucleic acid agent encoding a polypeptide corresponding to SEQ ID NO: 9 to SEQ ID NO: 12 or SEQ ID NO: 46 or a functional fragment, or a biologically active variant or fragment thereof, such as an RNA or modified RNA (modrna) as disclosed in US patent application US 2012/03228640. In some embodiments, KDM4 histone demethylase activator comprises a nucleic acid sequence selected from SEQ ID NO: 1 to SEQ ID NO: 4 or SEQ ID NO: 45. or a nucleic acid variant, wherein the nucleic acid variant hybridizes to any one of SEQ ID NOs: 1 to SEQ ID NO: 4 or SEQ ID NO: 45 is at least 80% (or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98%, or at least about 99%) sequence identity. In some embodiments, KDM4 histone demethylase activator comprises a nucleic acid that is SEQ ID NO: 1 to SEQ ID NO: 4 or SEQ ID NO: 45 of any one of SEQ ID NOs: 1 to SEQ ID NO: 4 or SEQ ID NO: 45, or at least 20, or at least 30, or at least 40, or at least 50 nucleic acids. In some embodiments, KDM4 histone demethylase activator, which is a nucleic acid agent useful in the methods, compositions, and kits, is expressed by a vector, such as a viral vector.

In alternative embodiments, the KDM4 histone demethylase activator encompassed for use herein is a polypeptide corresponding to the sequence of SEQ ID NO: 1 to SEQ ID NO: 4 or SEQ ID NO: 45, (modrna) or a nucleic acid sequence encoding a nucleic acid sequence corresponding to EQ ID NO: 9 to SEQ ID NO: 12 or SEQ ID NO: 46 or a functional fragment thereof, (modRNA). Synthetic modified rnas (modrnas) are disclosed in U.S. patent applications US2012/03228640, US2009/0286852 and US2013/0111615 and U.S. patents US 8,278,036, US 8,691,966, US 8,748,089, US 8,835,108, which are incorporated herein by reference in their entirety. In some embodiments, the synthetically modified RNA molecule is not expressed in a vector, and the synthetically modified RNA molecule can be a non-modified synthetically modified RNA molecule. In some embodiments, the composition can comprise at least one synthetic modified RNA molecule present in a lipid complex.

In some embodiments, the synthetic modified RNA molecule comprises at least two modified nucleotides, e.g., at least two modified nucleotides selected from the group consisting of: 5-methylcytidine (5mC), N6-methyladenosine (m6A), 3, 2 '-O-dimethyluridine (m4U), 2-thiouridine (s2U), 2' -fluorouridine, pseudouridine, 2 '-O-methyluridine (Um), 2' -deoxyuridine (2 '-dU), 4-thiouridine (s4U), 5-methyluridine (m5U), 2' -O-methyladenosine (m6A), N6, 2 '-O-dimethyladenosine (m6Am), N6, N6, 2' -O-trimethyladenosine (m62Am), 2 '-O-methylcytidine (Cm), 7-methylguanosine (m7G), 2' -O-methylguanosine (Gm), N2, 7-dimethylguanosine (m2, 7G), N2, N2, 7-trimethylguanosine (m2, 2, 7G), and inosine (I). In some embodiments, the synthetic modified RNA molecule further comprises a 5 ' cap, such as a 5 ' cap analog, such as a 5 ' diguanosine cap. In some embodiments, the synthetic modified RNA molecules used in the methods and compositions disclosed herein do not comprise a 5' triphosphate. In some embodiments, the synthetic modified RNA molecules used in the methods and compositions disclosed herein further comprise a poly (a) tail, a Kozak sequence, a 3 'untranslated region, a 5' untranslated region, or any combination thereof, and, in some embodiments, the synthetic modified RNA molecule can be optionally treated with alkaline phosphatase.

H3K9 methyltransferase inhibitors

In one aspect, the invention provides a method of increasing the efficiency of human SCNT, the method comprising: contacting the nucleus or cytoplasm of the donor human somatic cell, recipient human oocyte, hybrid oocyte (e.g., an enucleated human oocyte comprising the donor genetic material prior to fusion or activation), or human SCNT embryo (i.e., after fusion of the donor nucleus with the enucleated oocyte) with an agent that inhibits histone methylation in human nuclear genetic material, particularly H3K9 methylation, particularly H3H9me3 trimethylation. In certain embodiments of the invention, the agent inhibits histone methyltransferase activity. In certain embodiments of the invention, the agent inhibits the expression of human histone methyltransferase. In certain embodiments of the invention, the inhibitor is an inhibitor of human H3K9 methyltransferase. As discussed herein, the inventors have found that inhibition of the H3K9 methyltransferase protein can be used to increase the efficiency of human SCNT. In some embodiments, the H3K9 methyltransferase inhibitor is a protein inhibitor, and in some embodiments, the inhibitor is any agent that inhibits the function of the H3K9 methyltransferase protein or the expression of the H3K9 methyltransferase from its gene.

In certain embodiments of the invention, the agent inhibits the expression or function of human histone methyltransferase SUV39h1 protein. SUV39h1 has two alternatively spliced variants (variant 1 and variant 2) that produce the SUV39h1 subtype 1 protein and SUV39h1 subtype 2 protein, respectively. In some embodiments, the reagents used in the methods, kits, and compositions disclosed herein inhibit mRNA of variant 1(SEQ ID NO: 47) or variant 2(SEQ ID NO: 14) of SUV39h 1. In some embodiments, the agents used in the methods, kits, and compositions disclosed herein inhibit the function of subtype 1(SEQ ID NO: 48) or subtype 2(SEQ ID NO: 5) of the SUV39h1 protein.

In certain embodiments of the invention, the agent inhibits human histone methyltransferase SUV39h2 protein. In certain embodiments of the invention, the agent is the expression or function of human histone methyltransferase SUV39h2 protein. SUV39h2 has five alternatively spliced variants (variants 1 to 5) that produce four subtypes of SUV39h2 (both variant 2 and variant 3 encode subtype 2). In some embodiments, the agents used in the methods, kits, and compositions disclosed herein inhibit the activity of SEQ ID NO: 15. SEQ ID NO: 49. SEQ ID NO: 51. SEQ ID NO: 52 and SEQ ID NO: translation of any one or more of the mrnas of 53 (hsov 39h2 variants 1 to 5). In some embodiments, the agents used in the methods, kits, and compositions disclosed herein inhibit the activity of a polypeptide corresponding to SEQ ID NO: 6 and SEQ ID NO: 54 to SEQ ID NO: 57, subtypes 1 to 4 of hSuv39h 2.

In certain embodiments of the invention, the agent is an inhibitor of human histone methyltransferase EHMT 1. In certain embodiments of the invention, the agent inhibits human histone methyltransferase SETDB 1. In certain embodiments, at least two H3K9 methyltransferases (e.g., H3K9 methyltransferases 2, 3, 4, etc.) are inhibited. In certain embodiments of the invention, both SUV39h1 and SUV39h2 are inhibited by the same agent (e.g., SUV39h1/2 inhibitor), or by 2 or more separate agents. In certain embodiments of the invention, the agent is an RNAi agent, e.g., an siRNA or shRNA that inhibits expression of any one or more of H3K9 methyltransferase, human SUV39H1, human SUV39H2, or human SETDB 1.

Herein, the term "SUV 39H 1" or "H3K 9-histone methyltransferase SUV39H 1" has its ordinary meaning in the art and refers to histone methyltransferases "suppressor genes of Caulopus 3 to 9 congener 1 (Drosophila)" which methylate Lys-9 of histone H3 "(Aagaard L, Laible G, Selenko P, Schmid M, Dorn R, Schotta G, Kuhfittig S, Wolf A, Lebersorger A, Singh P B, Reuter G, Juwein T (June 1999)," functional mammalian congener Su (Drosophila) PEV modifier 3 to 9 encodes a centromere-related protein complexed with heterochromatin component M31 ", EMBO J18 (7): 1923-38). The histone methyltransferase is also known as MG44, KMT1A, SUV39H, histone lysine N-methyltransferase SUV39H1, H3-K9-HMTase 1, otthumb 00000024298, su (var) cognate 1 3-9, lysine N-methyltransferase 1A, histone H3-K9 methyltransferase 1, position effect variegated 3 to 9 cognates, histone lysine N-methyltransferase, or H3 lysine 9-specific 1. The term encompasses all orthologs of SUV39h1, such as su (var)3 to 9, and includes variants 1 and 2 encoding SUV39h1 subtype 1 and SUV39h1 subtype 2. As summarized in Table 8, and not wishing to be bound by theory, Suv39h1(Gene ID: 6839) has two alternatively spliced variants, variant 1 producing a Suv39h1 subtype 1 protein (long transcript and encoding the longer subtype) and corresponding to mRNANM-001282166.1 (SEQ ID NO: 47), and protein NP-001269095.1 (SEQ ID NO: 48). Variant 2 of Suv39h1 encodes subtype 2, which differs from variant 1 in the 5 'UTR, variant 2 lacks part of the 5' coding region, and translation is initiated at the alternative start codon. Variant 2 encodes a Suv39h1 isoform 2 protein that is shorter and has a distinct N-terminus compared to the isoform 1 protein. The mRNA for subtype 2 Suv39h1 is NM-003173.3 (SEQ ID NO: 14) which encodes a subtype 2 protein corresponding to NP-003164.1 (SEQ ID NO: 5).

Herein, the term "SUV 39H 2" or "H3K 9-histone methyltransferase SUV39H 2" has the usual meaning in the art and refers to histone methyltransferase "suppressor gene of variegated 3 to 9 congener 1 (drosophila)" that methylates Lys-9 of histone H3. The histone methyltransferases are also known as KMT1B, FLJ23414, H3-K9-HMTase 2, histone H3-K9 methyltransferase 2, lysine N-methyltransferase 1B, su (var)3 to 9 congener 2. The term encompasses all orthologues (the Suv39h2 gene is conserved in chimpanzee, macaque, dog, cow, mouse, rat, chicken and frog bodies), as well as alternatively spliced variants of Suv39h2 disclosed in table 8. Without wishing to be bound by theory, Table 8 lists five alternatively spliced human Suv39h2 (Gene ID: 79723) variants, which are: variant 1 encodes the Suv39h2 isoform 1 protein (long transcript and encodes the longer isoform); both variant 2 and variant 3 encoded Suv39h2 subtype 2; variant 4 encodes Suv39h2 subtype 3; whereas variant 5 encodes Sub39h2 subtype 4. Sequence identifiers for the mRNA for the Suv39h2 variant and its corresponding protein are shown in table 8.

TABLE 8 summary of sequences of hSUVh1 and hSUVh2 variants

According to the present invention, the inhibitor of human SUV39H1 is selected from the group consisting of an inhibitor of H3K 9-histone methyltransferase SUV39H1 protein function or an inhibitor of H3K 9-histone methyltransferase SUV39H gene expression.

The term "inhibitor of H3K 9-histone methyltransferase SUV39H 1" refers to any compound (natural or not) having the ability to inhibit methylation of Lys-9 of histone H3 by H3K 9-histone methyltransferase SUV39H 1. The term "inhibitor of H3K 9-histone methyltransferase SUV39H 2" refers to any compound (natural or otherwise) having the ability to inhibit methylation of Lys-9 of histone H3 by H3K 9-histone methyltransferase SUV39H 2.

The inhibitory activity of a compound can be determined using a variety of methods, such as Greiner d.et al.nat chembil.2005 August; 1(3): 143-5 or Eskeland, R.et al biochemistry 43, 3740-.

In some embodiments, inhibition of H3K9 methyltransferase is by an agent. Any agent may be used, such as, but not limited to, nucleic acids, nucleic acid analogs, peptides, bacteriophages, phagemids, polypeptides, peptidomimetics, ribosomes, aptamers, antibodies, small or large organic or inorganic molecules, or any combination thereof.

In some embodiments, the inhibitor of H3K9 methyltransferase is selected from the group consisting of: RNAi agents, siRNA agents, shRNA, oligonucleotides, CRISPR/Cas9, CRISPR/Cpf1 neutralizing antibodies or antibody fragments, aptamers, small molecules, proteins, peptides, small molecules, avimidir, functional fragments or derivatives thereof, and the like. Commercially available sequences for the knockdown of SUV39h1 and/or SUV39h2 via CRISPR/Cas9 or CRISPR/Cpf1 systems are available from Origene (product numbers KN202428 and KN317005) and Santa Cruz Biotechnology (product preference: sc-401717) and can be used in the methods and compositions disclosed herein.

Agents useful in the methods disclosed herein can also inhibit gene expression (i.e., suppress and/or repress expression of the gene). Such agents are referred to in the art as "gene silencing agents" and are well known to those skilled in the art. Examples include, but are not limited to, nucleic acid sequences for RNA, DNA or nucleic acid analogues, which may be single-or double-stranded, and may be selected from the group comprising nucleic acids encoding the protein of interest, oligonucleotides, nucleic acids, nucleic acid analogues, such as, but not limited to, Peptide Nucleic Acids (PNA), pseudo-complementary PNA (pc-PNA), Locked Nucleic Acids (LNA), derivatives thereof, and the like. Nucleic acid agents can also include, for example, but are not limited to, nucleic acid sequences encoding proteins that function as transcription inhibitors, antisense molecules, ribosomes, small inhibitory nucleic acid sequences, such as, but not limited to, RNAi, shRNAi, siRNA, micro RNAi (miRNA), antisense oligonucleotides, and the like.

In some embodiments, in all aspects of the invention, the agent that is contacted with the donor human somatic cell, the recipient human oocyte, the hybridized oocyte (e.g., an enucleated human oocyte comprising the donor genetic material prior to fusion or activation) or the human SCNT embryo (i.e., after fusion of the donor nucleus with the enucleated oocyte) is an inhibitor of H3K9 methyltransferase, such as, but not limited to, an inhibitor of any one of human SUV39H1, human SUV39H2 or human SETDB 1. In some embodiments, at least one or any combination of inhibitors of human SUV39h1, human SUV39h2, or human SETDB1 may be used in the method to increase the efficiency of human SCNT. In some embodiments, an inhibitor of SUV39h1, SUV39h2, or SETDB1 inhibits the activity of human SUV39h1, human SUV39h2, or human SETDB1 nucleic acid sequence (e.g., SEQ ID NO: 14 through SEQ ID NO: 16, or SEQ ID NO: 47 or SEQ ID NO: 49, SEQ ID NO: 51 through SEQ ID NO: 53), or human SUV39h1 protein (SEQ ID NO: 5 or SEQ ID NO: 48), human SUV39h2(SEQ ID NO: 6 or SEQ ID NO: 54 through SEQ ID NO: 57), or human SETDB1 protein (SEQ ID NO: 17).

In the context of the present invention, inhibitors of H3K 9-histone methyltransferase SUV39H1/2 are more selective for H3K 9-histone methyltransferase SUV39H1/2 than other molecules. By "selective" is meant that the inhibitor has an affinity that is at least 10-fold, preferably 25-fold, more preferably 100-fold, and even more preferably 500-fold greater than the selectivity for other histone methyltransferases.

Typically, inhibitors of H3K 9-histone methyltransferases SUV39H1 and/or SUV39H2 are small organic molecules. The term "small organic molecule" refers to a molecule having a size comparable to those organic molecules commonly used in pharmacy. The term does not include biological macromolecules (e.g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 5000Da, more preferably up to 2000Da, and most preferably up to 1000 Da.

In a particular embodiment, the inhibitor of the H3K 9-histone methyltransferase SUV39H1 is chaetocin (CAS28097-03-2), such as Greiner D, Bonaldi T, Eskeland R, Roemer E, Imhof A. identificatono a specific inhibitor of the histone methyl transferase SU (VAR)3-9.Nat Chembiol.2005 August; 1(3): 143-5.Epub 2005 jul.17; weber, H.P., et al, The molecular structure and absolute configuration of chaetocin. acta Crystal, B28, 2945-; udagawa, s., et al, The production of chaetogenins, stereotaxin, O-methyl stereotaxin, and chaetocin by Chaetomium spp. and related fungi, can. j. microbial, 25, 170-; gardiner, d.m., et al, theopeptyiodoxopiperazine (etp) class of fundamental toxins: distribution, mode of action, functions and biology, Microbiol, 151, 1021-. For example, chaetocin is commercially available from SigmaAldrich.

In another embodiment, an inhibitor of H3K 9-histone methyltransferase SUV39H1 is an aptamer. Aptamers are a class of molecules that can act as a substitute for antibodies in terms of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the ability to recognize almost any class of target molecule with high affinity and high specificity. Such Ligands can be isolated by the Systematic Evolution of Ligands (SELEX) technique of exponential enrichment of random sequence pools, as disclosed in Tuerk c.and Gold l., 1990. The random sequence library was obtained by combinatorial chemical synthesis of DNA. In this library, each member is a final chemically modified linear oligomer of a unique sequence. Possible modifications, uses and advantages of such molecules have been reviewed in Jayasena s.d., 1999. Peptide aptamers consist of conformationally constrained antibody accessible regions displayed by platform proteins such as e.coli thioredoxin a selected from combinatorial libraries by two hybridization methods (Colas et al, 1996).

The expression inhibitors for use in the present invention may be antisense oligonucleotides, including antisense RNA molecules and antisense DNA molecules, which will act by binding thereto to directly block translation of H3K 9-histone methyltransferase SUV39H1 or HP1 α mRNA, thus preventing protein translation or increasing mRNA degradation, thereby reducing the level of H3K 9-histone methyltransferase SUV39H1 or HP1 α, and thus reducing intracellular activity, for example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding H3K 9-histone methyl transferase SUV39H1 may be synthesized by conventional phosphodiester techniques and administered by, e.g., intravenous injection or infusion administration, methods using antisense techniques to specifically inhibit gene expression of genes known to sequences in the art (see, e.g., U.S. patent 6,566,135, US6,566,131, US6,365,354, US 58 6,410,323, US6,107,091, 6,046,321, and 6,046,321, which are disclosed herein by the disclosures of the entire application for human SUV 3539, the disclosure of the subject application for inhibition of SUV 3539, SUV 358, and/3639, which are incorporated herein by the methods disclosed in the entire application for the disclosure of human penicillin inhibitors (see US).

Inhibitors of SUV39h2 and methods of identifying the same are disclosed in U.S. patent application US2014/0094387, which is incorporated by reference herein in its entirety.

RNAi inhibitors of H3K9 methyltransferase

In some embodiments, the H3K9 methyltransferase inhibitor is an RNAi agent, e.g., an siRNA or shRNA molecule. RNAi agents to human SUV39h1, human SUV39h2, human SETDB1, human EHMT1, and human PRDM2 are well known in the art. In some embodiments, the inhibitor of H3K9 methyltransferase is an RNAi agent. In some embodiments, the RNAi agent hybridizes fully or partially to a target sequence located within a nucleotide region of any one of the human SUV39h1 nucleic acid sequence (SEQ ID NO: 14 or SEQ ID NO: 47), human SUV39h2 protein (SEQ ID NO: 15, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53), or human SETDB1 protein (SEQ ID NO: 16), as disclosed herein.

In some embodiments, the RNAi agent inhibits expression of any one of human SUV39h1 protein (SEQ ID NO: 5 or SEQ ID NO: 48), human SUV39h2 protein (SEQ ID NO: 6 or SEQ ID NO: 54 to SEQ ID NO: 57), or human SETDB1 protein (SEQ ID NO: 17), as disclosed herein.

Inhibition of the H3K9 methyltransferase gene can be performed by gene silencing RNAi molecules according to methods well known to those skilled in the art. In some embodiments, the H3K9 methyltransferase inhibitor is an RNAi agent selected from any one or a combination of siRNA agents of table 2.

For example, the gene silencing siRNA oligonucleotide double helix targets a region within human SUV39h1 corresponding to NM-003173.3 (SEQ ID NO: 14) for variant 2, or NM-001282166.1 (SEQ ID NO: 47) for variant 1, and can be readily used to knock down human SUV39h1 expression. Using siRNA, SUV39h1 mRNA was successfully targeted; while other siRNA molecules can be readily prepared by those skilled in the art based on the known sequence of the target mRNA. For the avoidance of doubt, the sequence of human SUV39h1 is provided, for example, under GenBank accession number NM-003173.3 (SEQ ID NO: 14) (variant 2 encoding isoform 1) or NM-001282166.1 (SEQ ID NO: 47) (variant 1, encoding isoform 1). One skilled in the art can select an RNAi agent to be used that inhibits the expression of mRNA encoding human SUV39h1 protein (SEQ ID NO: 5 or SEQ ID NO: 48), or that inhibits the expression of any other mammalian SUV39h1 protein.

For the avoidance of doubt, the sequence of the human SUV39H1 cDNA is provided, for example, under Gene Bank accession number NM-003173.3 (SEQ ID NO: 14) corresponding to variant 2, or under Gene Bank accession number NM-001282166.1 (SEQ ID NO: 47) corresponding to variant 1, and can be used to design a gene silencing RNAi modulator that inhibits the expression of human SUV39H1 mRNA, which can be used as an H3K9 methyltransferase inhibitor in the methods and compositions disclosed herein. In some embodiments, the inhibitor of human SUV39h1 is an siRNA agent, e.g., an siRNA agent comprising at least one or both of GAAACGAGUCCGUAUUGAAtt (SEQ ID NO: 7) or UUCAAUACGGACUCGUUUCtt (SEQ ID NO: 8) and fragments or derivatives thereof having at least 80% sequence identity.

Herein, the term "SUV 39h1 protein" refers to the amino acid sequence of SEQ ID NO: 5 (subtype 2) or SEQ ID NO: 48 (subtype 1) and homologues thereof, and conservative substitutions, additions and deletions which do not negatively affect the functional structure are included therein. In some embodiments, the SUV39h1 protein is encoded by a nucleic acid sequence for variant 2 (encoding a SUV39h1 subtype 2 protein) of the human SUV39h1 transcript (SEQ ID NO: 14) as follows:

in some embodiments, the SUV39h1 protein is encoded by a nucleic acid sequence for variant 1 (encoding a Suv39h1 subtype 1 protein) of the human SUV39h1 transcript (SEQ ID NO: 47) as follows:

in some embodiments, the agent comprises a nucleic acid inhibitor that inhibits or reduces human SUV39h1 mRNA (SEQ ID NO: 14 or SEQ ID NO: 47) by at least 50% (as compared to in the absence of SUV39h1 inhibitor).

In some embodiments, the agent comprises a nucleic acid inhibitor that inhibits or reduces the level or function of human SUV39h1 protein (SEQ ID NO: 5 (isoform 2) or SEQ ID NO: 48 (isoform 1). in some embodiments, the agent comprises a nucleic acid inhibitor that inhibits or reduces the level or function of human SUV39h2 protein (i.e., any one of SEQ ID NO: 6, SEQ ID NO: 54 to SEQ ID NO: 57).

In some embodiments, the siRNA inhibitor of human SUV39h1 is SEQ ID NO: 8 or a fragment thereof of at least 10 contiguous nucleotides, or a fragment thereof that hybridizes to SEQ ID NO: 8 is at least 80% (or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98%, or at least about 99%) sequence identity. In some embodiments, the siRNA or other nucleic acid inhibitor hybridizes fully or partially to SEQ ID NO: 7.

In some embodiments, the siRNA inhibitor of murine SUV39h2 is SEQ ID NO: 19 or a fragment thereof of at least 10 contiguous nucleotides, or a fragment thereof that hybridizes to SEQ ID NO: 19 is at least 80% (or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98%, or at least about 99%) sequence identity. In some embodiments, the siRNA or other nucleic acid inhibitor hybridizes fully or partially to SEQ ID NO: 18.

In some embodiments, the siRNA inhibitor of human SUV39h1 is SEQ ID NO: 21 or a fragment thereof of at least 10 contiguous nucleotides, or a fragment thereof that hybridizes to SEQ ID NO: 21 is at least 80% (or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98%, or at least about 99%) sequence identity. In some embodiments, the siRNA or other nucleic acid inhibitor hybridizes fully or partially to SEQ ID NO: 20.

In some embodiments, the siRNA or other nucleic acid inhibitor hybridizes fully or partially to the nucleic acid sequence of SEQ ID NO: 15. SEQ ID NO: 49. SEQ ID NO: 51. SEQ ID NO: 52 and SEQ ID NO: 53 (hsov 39h2 variants 1 to 5).

Inhibition of the H3K9 methyltransferase gene can be performed by gene silencing RNAi molecules according to methods well known to those skilled in the art. Inhibition of human SUV39h1, human SUV39h2, human SETDB1, human EHMT1, and human PRDM2 are well known in the art. In some embodiments, the H3K9 methyltransferase inhibitor is an RNAi agent selected from any one or a combination of siRNA agents of table 2.

In some embodiments, SUV39H1 can be targeted and inhibited by hsa-miR-98-5p (MIRT027407), hsa-miR-615-3p (MIRT040438), hsa-miR-331-3p (MIRT043442), or a miR that has at least 85% sequence identity thereto. Commercially available siRNA, RNAi and shRNA products that inhibit SUV39h1 and/or SUV39h2 in human cells are available from Origene, Qiagen and Santa Cruz Biotechnology and can be used by those skilled in the art.

For example, gene silencing siRNA oligonucleotides that bind to and partially or fully hybridize to a nucleic acid sequence located in any one of human SUV39H2 variants 1 through 5(SEQ ID NO: 15, SEQ ID NO: 9, SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 53) can be readily used to knock-out SUV39H2 expression. SUV39h2 mRNA could be successfully targeted using siRNA; and other siRNA molecules can be readily prepared by those skilled in the art based on the known sequence of the target mRNA. For the avoidance of doubt, the sequences of the human SUV39h2 variants are shown in table 8. For the avoidance of doubt, the sequence of the human SUV39h2 variant cDNA is provided, for example, in the gene bank deposit: NM-024670.3 (SEQ ID NO: 15), NM 001193425.1(SEQ ID NO: 51), NM 001193426.1(SEQ ID NO: 52), NM OO 1193427.1 (SEQ ID NO: 53), and can be used to design gene silencing RNAi modulators that inhibit human SUV39H2 mRNA expression, useful as H3K9 methyl transfer inhibitors in the methods and compositions disclosed herein. In some embodiments, the inhibitor of SUV39h2 is an siRNA agent, e.g., the siRNA may comprise at least one or both of the following sequences: GCUCACAUGUAAAUCGAUUtt (SEQ ID NO: 18) or AAUCGAUUUACAUGUGAGCtt (SEQ ID NO: 19), and fragments or derivatives thereof having a sequence identity of at least 80%. In some embodiments, the inhibitor of SUV39h2 is an siRNA agent that binds at least to the target sequence of GCUCACAUGUAAAUCGAUUtt (SEQ ID NO: 18). In some embodiments, the inhibitor of SUV39h2 is an siRNA agent comprising at least 5 contiguous nucleotides of a portion of AAUCGAUUUACAUGUGAGCtt (SEQ ID NO: 19) or a fragment or derivative thereof having at least 80% sequence identity.

Herein, the term "SUV 39H2 protein" refers to SEQ ID NO: 54 (subtype 1), SEQ ID NO: 6 or SEQ ID NO: 53 (subtype 2), SEQ ID NO: 56 (subtype 3) or SEQ ID NO: 57 (subtype 4) and homologues thereof, including conservative substitutions, additions, deletions which do not adversely affect functional structure.

The accession numbers for hSUV39h2 variant nucleic acid sequences and their corresponding proteins are shown in table 8. For example, the SUV39H2 subtype 2 protein is encoded by the nucleic acid sequence of the human SUV39H2 variant 3 transcript (SEQ ID NO: 15) as follows:

in some embodiments, the agent inhibits SEQ ID NOS: 15. SEQ ID NOS: 49. SEQ ID NOS: 51. SEQ ID NOS: 52. and SEQ ID NOS: 53 (hsov 39h2 variants 1 to 5). In some embodiments, one of skill in the art can select an RNAi agent to be used that inhibits a polypeptide encoded by SEQ ID NO: 6. SEQ ID NO: 54 to SEQ ID NO: 57 of one or more of human SUV39h2 protein.

Other exemplary siRNA sequences for inhibiting human SUV39H1 and SUV39H2 are disclosed in U.S. patent application US2012/0034192, which is incorporated herein by reference in its entirety.

Table 2 exemplary siRNA sequences that inhibit H3K9 methyltransferase:

for the avoidance of doubt, the sequence of the human SETDB1 cDNA is provided, for example, as deposited under genbank accession no: NM-001145415.1 (SEQ ID NO: 16), and can be used by those skilled in the art to design gene silencing RNAi modulators that inhibit human SETDB1 mRNA expression, useful as H3K9 methyl transfer inhibitors in the methods and compositions disclosed herein.

For the avoidance of doubt, the sequence of the human EHMT1 cDNA is provided, for example, as genbank accession no: NM-024757.4 (SEQ ID NO: 42), and can be used by those skilled in the art to design gene silencing RNAi modulators that inhibit human EHMT1 mRNA expression, useful as H3K9 methyl transfer inhibitors in the methods and compositions disclosed herein.

For the avoidance of doubt, the sequence of the human PRDM2 cDNA is provided, for example, as a genbank deposit number: NM-012231.4 (SEQ ID NO: 43), and can be used by those skilled in the art to design gene silencing RNAi modulators that inhibit human PRDM2 mRNA expression, useful as H3K9 methyl transfer inhibitors in the methods and compositions disclosed herein.

In some embodiments, the inhibitor of H3K9 methyltransferase is selected from the group consisting of: RNAi agents, siRNA agents, shRNA, oligonucleotides, CRISPR/Cas9, CRISPR/Cpf1 neutralizing antibodies or antibody fragments, aptamers, small molecules, proteins, peptides, small molecules, avimidir, functional fragments or derivatives thereof, and the like. In some embodiments, the H3K9 methyltransferase inhibitor is an RNAi agent, e.g., an siRNA or shRNA molecule. In some embodiments, the agent comprises a nucleic acid inhibitor that reduces protein expression of human SUV39H1 protein (SEQ ID NO: 5 or SEQ ID NO: 48) or SUV29H1 mRNA (SEQ ID NO: 14 or SEQ ID NO: 47) or human SUV39H2 protein (SEQ ID NO: 6 or SEQ ID NO: 54 to SEQ ID NO: 57) or SUV39H2 mRNA (SEQ ID NO: 15 or SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53). In some embodiments, the siRNA inhibitor of human SUV39h1 is SEQ ID NO: 8 or a fragment thereof having at least 10 contiguous nucleotides, or a fragment thereof that differs from SEQ ID NO: 8 is at least 80% (or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98%, or at least about 99%) sequence identity. In some embodiments, the siRNA or other nucleic acid inhibitor hybridizes fully or partially to the sequence of SEQ ID NO: 7. In some embodiments, the siRNA inhibitor of human SUV39H2 comprises SEQ ID NO: 19 or a fragment thereof having at least 10 contiguous nucleotides, or a fragment thereof that differs from SEQ ID NO: 19 is at least 80% (or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98%, or at least about 99%) of the nucleic acid sequence. In some embodiments, the siRNA or other nucleic acid inhibitor hybridizes fully or partially to the sequence of SEQ ID NO: 18 or SEQ ID NO: 15, or a targeting sequence of seq id no.

In other embodiments of the above aspects, the H3K9 methyltransferase inhibitor inhibits any one of the following histone methyltransferases selected from the group consisting of: SUV39H1, SUV39H2, G9A (EHMT2), EHMT1, ESET (SETDB 1), SETDB2, MLL2, MLL3, SETD2, NSD1, SMYD2, DOT1L, SETD8, SUV420H1, SUV420H2, EZH2, SETD7, PRDM2, PRMT1, PRMT2, PRMT3, PRMT4, PRMT5, PRMT6, PRMT7, PRMT8, PRMT9, PRMT10, PRMT11, CARM 1.

In some embodiments, the agent that inhibits an H3K9 methyltransferase, such as inhibits human SUV39H1, human SUV39H2, or human SETDB1, is a nucleic acid. Nucleic acid inhibitors of H3K9 methyltransferases such as SUV39H1, SUV39H2, or SETDB1 include, for example, but are not limited to, RNA interference inducing (RNAi) molecules, such as, but not limited to, siRNA, dsRNA, stRNA, shRNA, and modified versions thereof, wherein the RNA interference (RNAi) molecules silence gene expression from any of the human SUV39H1, human SUV39H2, and/or human SETDB1 genes.

Accordingly, in some embodiments, inhibitors of H3K9 methyltransferases, e.g., inhibitors of human SUV39H1, human SUV39H2, or human SETDB1, may be inhibited by any "gene silencing" method known to those skilled in the art. In some embodiments, the nucleic acid inhibitor of H3K9 methyltransferase, e.g., an inhibitor of human SUV39H1, human SUV39H2, or human SETDB1, is an antisense oligonucleotide, or a nucleic acid analog, such as, but not limited to, DNA, RNA, peptide-nucleic acid (PNA), pseudocomplementary PNA (pc-PNA), or Locked Nucleic Acid (LNA), and the like. In alternative embodiments, the nucleic acid is DNA or RNA, and nucleic acid analogs, such as PNA, pcPNA, and LNA. The nucleic acid may be single-stranded or double-stranded, and may be selected from the group consisting of nucleic acids encoding the protein of interest, oligonucleotides, PNAs, and the like. Such nucleic acid sequences include, for example, but are not limited to, nucleic acid sequences encoding proteins that function as transcription repressors, antisense molecules, ribosomes, small inhibitory nucleic acid sequences, such as, but not limited to, RNAi, shRNAi, siRNA, micro RNAi (mrrnai), antisense oligonucleotides, and the like.

In some embodiments, single-stranded RNA (ssRNA), a form of RNA endogenously found in eukaryotic cells, can be used to form RNAi molecules. Cellular ssRNA molecules include messenger RNA (and messenger RNA progenitor), small cell RNA, small nuclear RNA, transfer RNA, and ribosomal RNA. Double-stranded RNA (dsRNA) induces a size-dependent immune response, so dsRNA larger than 30bp activates the interferon response, while shorter dsRNA is injected into the endogenous RNA interference machinery of the cell downstream of the dicer.

RNA interference (RNAi) provides a powerful way to inhibit the expression of a selected target polypeptide. RNAi uses small interfering RNA (sirna) duplexes that target messenger RNA encoding the target polypeptide for selective degradation. siRNA-dependent post-transcriptional silencing of gene expression involves cleavage of the target messenger RNA molecule at a site guided by the siRNA.

RNA interference (RNAi) is an evolutionarily conserved process whereby the expression of or induction of an RNA sequence identical or highly similar to a target gene results in the specific post-transcriptional gene silencing (PTGS) of messenger RNA (mrna) that causes sequence-specific degradation or transcription from the target gene (see Coburn, g.and Cullen, B. (2002) j.of virology 76 (18): 9225), thereby inhibiting the expression of the target gene. In one embodiment, the RNA is double-stranded RNA (dsrna). This process has been revealed in plant cells, invertebrate cells and mammalian cells. In fact, RNAi is initiated by this dsRNA-specific endonuclease, dicer, which promotes progressive cleavage of long dsRNA into double-stranded fragments known as siRNA. The siRNA is incorporated into a protein complex (named "RNA-induced silencing complex" or "RISC") that recognizes and cleaves the target mRNA. RNAi can also be initiated by inducing nucleic acid molecules, such as synthetic sirnas or RNA interference agents, to inhibit or silence the expression of a target gene. Herein, "inhibition of expression of a target gene" includes any reduction in the expression or protein activity or level of the target gene or a protein encoded by the target gene, as compared to a situation in which no RNA interference has been induced. The reduction may be at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% or more as compared to the expression of the target gene or the activity or expression of the protein encoded by the target gene that is not targeted by the RNA interfering agent.

"short interfering RNA (siRNA)," also referred to herein as "small interfering RNA," is defined as an agent that functions to inhibit the expression of a target gene, e.g., by RNAi. The siRNA may be chemically synthesized, may be produced by in vitro transcription, or may be produced in a host cell. In one embodiment, the siRNA is a double stranded rna (dsrna) molecule of about 15 to about 40 nucleotides in length, preferably about 15 to about 28 nucleotides in length, more preferably about 19 to about 25 nucleotides in length, and more preferably 19, 20, 21, 22, or 23 nucleotides in length, and may contain 3 'and/or 5' overhangs of about 0, 1, 2, 3, 4, or 5 nucleotides in length on each strand. The length of the overhang is independent between the two chains, i.e., the length of the overhang on one chain is not linked to the length of the overhang on the second chain. Preferably, the siRNA promotes RNA interference through degradation of target messenger RNA (mrna) or specific post-transcriptional gene silencing (PTGS).

sirnas also include small hairpin (also known as stem-loop) rna (shrna). In one embodiment, the shrnas are composed of a short (e.g., about 19 to about 25 nucleotides) antisense strand followed by a nucleotide loop of about 5 to about 9 nucleotides, and a similar sense strand. Alternatively, the sense strand may precede the nucleotide stem-loop structure, and the antisense strand may follow. These shRNAs can be included in plasmids, retroviruses, and lentiviruses, and can be expressed, for example, from the pol III U6 promoter, or other promoters (see, e.g., Stewart, et al (2003) RNA Apr 9 (4): 493-.

The target gene or sequence of the RNA interfering agent may be a cellular gene or genomic sequence, such as the H3K9 methyltransferase gene sequence of the SUV39H1, SUV39H2, or SETDB1 gene sequences. The siRNA may be substantially homologous to the target gene or genomic sequence, or a fragment thereof. As used in this context, the term "homologous" is defined as substantially identical, sufficiently complementary, or similar to a target mRNA, or fragment thereof, to affect RNA interference with the target. In addition to native RNA molecules, RNAs useful for inhibiting or interfering with expression of a target sequence include RNA derivatives and analogs. Preferably, the siRNA is identical to its target sequence.

The siRNA preferably targets only one sequence. Each RNA interfering agent, such as siRNA, can be screened for potential off-target effects by, for example, expression profiling. Such methods are known to those skilled in the art and are disclosed, for example, in Jackson et al, Nature Biotechnology 6: 635 and 637, 2003. In addition to expression profiling, libraries of sequences can be screened for analogs of potential target sequences to identify potential sequences that may have off-target effects. For example, according to Jackson et al (Id.), a sequence identity of 15 or possibly as few as 11 contiguous nucleotides is sufficient to direct silencing of non-target transcripts. Thus, siRNAs can be initially screened for which sequence identity analysis using any known sequence alignment method, such as BLAST, is identified as avoiding potential off-target silencing.

siRNA molecules need not be limited to those containing only RNA, but may, for example, further encompass chemically modified nucleotides and non-nucleotides, as well as molecules in which a ribose molecule is replaced by another sugar molecule or a molecule that performs a similar function. In addition, non-natural linkers, such as phosphorothioate linkers, between nucleotide residues may be used. For example, siRNAs containing D-arabinofuranosyl structures in place of the naturally occurring D-ribonucleosides in RNA can be used in RNAi molecules according to the invention (U.S. Pat. No. 5,5,177,196). Other examples include RNA molecules containing an O-linker between the sugar of the nucleotide and the heterocyclic substrate, which confers nuclease resistance and tight complementary strand binding to oligonucleotide molecules similar to oligonucleotides containing 2' -O-methyl ribose, arabinose, especially D-arabinose (U.S. Pat. No. 5,177,196).

The RNA strand may be derivatized with a reactive functional group of a reporter group, such as a fluorophore. Particularly useful derivatives are those modified at one or more ends of the RNA, typically at the 3' end of the sense strand. For example, the 2 '-hydroxyl group at the 3' end can be easily and selectively derivatized using a variety of groups.

Other useful RNA derivatives incorporate nucleotides with modified carbohydrate moieties such as2 ' -O-alkylated residues or 2 ' -O-methylribosyl derivatives and 2 ' -O-fluororibosyl derivatives. The RNA bases may also be modified. Any modified base that can be used to inhibit or interfere with expression of a target sequence can be used. For example, halogenated bases such as 5-bromouracil and 5-iodouracil can be combined. The base may also be alkylated, for example, 7-methylguanosine may be incorporated in place of a guanosine residue. Non-natural bases that achieve successful inhibition can also be combined.

Most preferred siRNA modifications include 2 '-deoxy-2' -fluorouracil or Locked Nucleic Acid (LNA) nucleotides and RNA duplexes containing phosphodiester or variable number of phosphorothioate linkages. Such modifications are known to those skilled in the art and are disclosed, for example, in braasco et al, Biochemistry, 42: 7967 and 7975, 2003. Most of the available modifications to siRNA molecules can be introduced using chemical methods constructed for antisense oligonucleotide technology. Preferably, the modification involves minimal 2' -O-methyl modification, preferably no such modification is included. The modification also preferably excludes modification of the free 5' -hydroxyl group of the siRNA.

siRNA and miRNA molecules having various "tails" covalently attached to their 3 '-ends or 5' ends, or both, are also known in the art and can be used to stabilize siRNA and miRNA molecules delivered using the methods of the invention. Generally, insertion groups, various reporter groups, and lipophilic groups attached to the 3 'or 5' end of the RNA molecule are well known in the art and can be used in accordance with the methods of the invention. Synthetic descriptions of 3 '-cholesterol or 3' -acridine modified oligonucleotides suitable for use in preparing modified RNA molecules according to the present invention can be found in articles such as: gamper, H.B., Reed, M.W., Cox, T., Virosco, J.S., Adams, A.D., Gall, A.Scholler, J.K., and Meyer, R.B, (1993) facility preference and Exoneclease Stability of 3' -modified Oligoxygranules, nucleic Acids Res.21145-150; and Reed, M.W., Adams, A.D., Nelson, J.S., and Meyer, R.B., Jr. (1991) Acridine and Cholesterol-Derivatized solid Supports for Improved Synthesis of 3' -modified Oligonuclotides, bioconjugate chem.2217-225 (1993).

Other siRNAs that can be used to target H3K9 methyltransferases such as SUV39H1, SUV39H2 or SETDB1 genes can be readily designed and tested. Accordingly, sirnas useful in the methods disclosed herein include siRNA molecules of about 15 to about 40 or about 15 to about 28 nucleotides in length that are homologous to the specific H3K9 methyltransferase gene, such as the SUV39H1, SUV39H2, or SETDB1 gene. In some embodiments, the H3K9 methyltransferase targeting agent, e.g., SUV39H1, SUV39H2, or SETDB1, targets siRNA molecules from about 25 to about 29 nucleotides in length. In some embodiments, the H3K9 methyltransferase tannar siRNA, e.g., SUV39H1, SUV39H2, or SETDB1 tannar siRNA molecules are about 27, 28, 29, or 30 nucleotides in length. In some embodiments, the H3K9 methyltransferase targeted RNAi, e.g., SUV39H1, SUV39H2, or SETDB1 targeted siRNA molecules may also comprise a 3' hydroxyl group. In some embodiments, the H3K9 methyltransferase targeted siRNA, e.g., SUV39H1, SUV39H2, or SETDB1 targeted siRNA molecules may be single-stranded or double-stranded; such molecules may be blunt-ended or comprise pendent ends (e.g., 5 ', 3'). In particular embodiments, the RNA molecule can be double-stranded, and either blunt-ended or comprise a overhang.

In one embodiment, at least one strand of the RNA molecule targeted with an H3K9 methyltransferase such as SUV39H1, SUV39H2, or SETDB1 has a 3' overhang of about 0 to about 6 nucleotides in length (e.g., pyrimidine nucleotides, purine nucleotides). In other embodiments, the 3' overhang is from about 1 to about 5 nucleotides, from about 1 to about 3 nucleotides, and from about 2 to about 4 nucleotides in length. In one embodiment, the RNA molecule targeted to human SUV39h1/2, SETDB1, EHMT1, or PRDM2 is double stranded: one strand has a 3' overhang and the other strand may be blunt-ended or have an overhang. In embodiments where the H3K9 methyltransferase, such as SUV39H1, SUV39H2, SETDB1, EHMT1 or PRDM2 RNAi agent, is double-stranded and both strands comprise a overhang, the length of the overhang on each strand may be the same or different. In particular embodiments, the RNA of the invention comprises about 19, 20, 21, or 22 paired nucleotides and has a overhang of about 1 to about 3, particularly about 2 nucleotides in length at both 3' ends of the RNA. In one embodiment, the 3' overhang can be stabilized against degradation. In a preferred embodiment, the RNA is stabilized by including purine nucleotides, such as adenosine or guanosine nucleotides. Alternatively, substitution of pyrimidine nucleotides with modified analogs, e.g., substitution of uridine 2 nucleotides with 2 '-deoxythymidine for 3' overhang, is tolerable and does not affect the efficiency of RNAi. The absence of the 2' -hydroxyl group significantly increases the nuclease resistance of the overhang in tissue culture medium.

As disclosed herein, sirnas for the H3K9 methyltransferases SUV39H1, SUV39H2 and SETDB1 have been successfully used to increase the efficiency of murine SCNT. In some embodiments, RNAi agents that inhibit expression of/silence the gene of human SUV39H1, human SUV39H2, human SETDB1, human EHMT1, or human PRDM2, if gene silencing RNAi of H3K9 methyltransferase, are not commercially available, gene silencing RNAi agents targeted to inhibit human SUV39H1, human SUV39H2, human SETDB1, human EHMT1, or human PRDM2 or PRDM2, can be produced by one skilled in the art and according to the methods disclosed herein. In some embodiments, assessment of expression and/or knockdown of human SUV39h1, human SUV39h2, human SETDB1, human EHMT1, or human PRDM2 mRNA and/or protein can be determined using commercially available kits known to those of skill in the art. Others can be readily prepared by those skilled in the art based on the known sequence of the target mRNA.

In some embodiments, the inhibitor of H3K9 methyltransferase is a gene silencing RNAi agent that down-regulates or reduces mRNA levels of any one or more of human SUV39H1, human SUV39H2, human SETDB1, human EHMT1, or human PRDM2, and may be a 25-nt hairpin sequence. In some embodiments, the H3K9 methyltransferase inhibitor is a gene silencing RNAi, e.g., shRNA sequence of any one or more of human SUV39H1, human SUV39H2, human SETDB1, human EHMT1, or human PRDM 2.

In one embodiment, the RNA interfering agents used in the methods disclosed herein are actively taken up by cells in vivo following intravenous injection, such as aqueous hydrodynamic injection, without the use of a carrier, illustratively an RNA interfering agent such as used in the methods of the invention is effective in the in vivo delivery of siRNA.

Other strategies for delivering RNA interfering agents, such as siRNA or shRNA used in the methods of the invention, can also be employed, for example, by delivery via vectors such as therapeutics or viral vectors such as lentiviral vectors. Such vectors may be as for example Xiao-Feng Qinet al proc.natl.acad.sci.u.s.a., 100: 183- & 188. Other delivery methods include the use of a basic peptide to effect delivery of an RNA interfering agent, such as the siRNA or shRNA of the invention, by conjugating or mixing the RNA interfering agent with a fragment of a basic peptide, such as the TAT peptide, and mixing with a cationic liposome or formulating in a particle.

Note that dsRNA such as siRNA or shRNA can be delivered using inducible vectors such as tetracycline inducible vectors. For example, those described in Wang et al proc. natl. acad. sci.100: 5103-5106, using the pTet-On vector (BDbiosciences Clontech, Palo Alto, Calif.). In some embodiments, the vector may be a plasmid vector, a viral vector, or any other suitable vector adapted for insertion of a foreign sequence and introduction thereof into a eukaryotic cell. The vector may be an expression vector capable of directing transcription of the DNA sequence of the agonist or antagonist nucleic acid molecule into RNA. The viral expression vector may be selected from the group comprising: for example, retroviral vectors, lentiviral vectors, E-B viral vectors, bovine papilloma viral vectors, adenovirus and adeno-associated virus based vectors, or hybrid viral vectors of any of the above. In one embodiment, the vector is an episomal vector. The use of a vector of the appropriate episome provides for the addition of chromosomal DNA to the antagonist nucleic acid molecule at high copy numbers in a subject, thereby eliminating potential chromosomal integration effects.

RNA interference molecules and nucleic acid inhibitors useful in the methods disclosed herein can be produced using any known technique, such as direct chemical synthesis, processing of longer double-stranded RNA by exposure to a combination dicer protein or drosophila embryo lysate, delivery from S2 cells by in vitro systems, use of bacteriophage RNA polymerase, RNA-dependent RNA polymerase, and DNA-based vectors. Subsequent isolation of the short siRNA, e.g., from about 21 to 23 nucleotides, from the lysate, etc., can further be involved using cell lysates or in vitro processing. Chemical synthesis is generally carried out by: two single-stranded RNA oligos are prepared, and then the two single-stranded oligos are annealed into double-stranded RNA. Other examples include the methods disclosed in WO 99/32619 and WO 01/68836, which teach chemical and enzymatic synthesis of sirnas. In addition, a number of commercial services are available for designing and manufacturing specific sirnas (see, e.g., QIAGEN inc., Valencia, CA and AMBION inc., Austin, TX).

The terms "antimir", "microRNA inhibitor" or "miR inhibitor" are synonyms and refer to oligonucleotides that interfere with the activity of a particular miRNA. Inhibitors can take a variety of configurations, including single-stranded, double-stranded (RNA/RNA or RNA/DNA double helix), and hairpin designs, typically microRNA inhibitors comprise one or more sequences or portions of sequences that are complementary or partially complementary to the mature strand(s) of the miRNA to be targeted, and in addition, the miRNA inhibitor can also comprise additional sequences located 5 'and 3' to the sequence, which are the inverse complement of the mature miRNA. The additional sequence may be the reverse complement of the sequence adjacent to the mature miRNA in the pri-miRNA from which the mature miRNA is derived, or the additional sequence may be any sequence (having A, G, C, U, or a mixture of dT). In some embodiments, one or both of the additional sequences is any sequence that is capable of forming a hairpin. Thus, in some embodiments, the sequence that is the reverse complement of the miRNA is flanked on the 5 'and 3' sides by hairpin structures. When the MicroRNA inhibitor is double-stranded, it may include mismatches between nucleotides on opposite strands.

In some embodiments, the agent is a protein or polypeptide or an RNAi agent that inhibits human SUV39h1, human SUV39h2, human SETDB1, human EHMT1, or humanExpression of any one or combination of the classes PRDM 2. In such embodiments, the cell can be modified (e.g., by homologous recombination) to provide increased expression of the agent, e.g., by replacing, in whole or in part, the naturally occurring promoter with all or a portion of a heterologous promoter, such that the cell expresses an inhibitor of human SUV39h1, human SUV39h2, human SETDB1, human EHMT1, or human PRDM2, e.g., a protein or an RNAi agent (e.g., a gene silencing-RNAi agent). Typically, the heterologous promoter is inserted in a manner such that it is operably linked to the desired nucleic acid encoding the agent. See, for example, PCT International publication WO 94/12650 to Transkaryotic therapeutics, PCT International publication WO 92/20808 to CellGenesys, Inc., and PCT International publication WO 91/09955 to Applied Research Systems. The cell may also be engineered to express an endogenous gene comprising the inhibitor under the control of an inducible regulatory element, in which case the regulatory sequence of the endogenous gene may be replaced by homologous recombination. Gene activation techniques are disclosed in U.S. Pat. No. 5,272,071 to Chappel, U.S. Pat. No. 5,578,461 to Sherwin et al, International patent PCT/US92/09627(W093/09222) to Selden et al, and PCT/US90/06436(W091/06667) to Skoultchi et al. The agent may be prepared by culturing the transformed host cell under culture conditions suitable for expression of the miRNA. Subsequently, the resulting expressed reagent can be purified from the culture (i.e., from the culture medium or cell extract) using known purification procedures such as gel filtration and ion exchange chromatography. Purification of peptide or nucleic acid agent inhibitors of human SUV39h1, human SUV39h2, human SETDB1, human EHMT1 or human PRDM2, and may further comprise an affinity column containing an agent that will bind to the protein; one or more affinity resins of this type, e.g. concanavalin A-agarose, HEPARIN-TOYOPEARL ^TMOr a column step on a Cibacrom blue 3GA agarose gel; one or more hydrophobic interaction chromatographs involving the use of such resins as phenyl ether resins, butyl ether resins, or propyl ether resins; immunoaffinity chromatography; or complementary cDNA affinity chromatography.

In one embodiment, the nucleic acid inhibitor (e.g., a gene silencing RNAi agent) of human SUV39h1, human SUV39h2, human SETDB1, human EHMT1, or human PRDM2 can be obtained synthetically, e.g., by chemically synthesizing the nucleic acid by any synthetic method known to those of skill in the art. Subsequently, synthetic nucleic acid inhibitors of H3K9 methyltransferases such as human SUV39H1, human SUV39H2, human SETDB1, human EHMT1, or human PRDM2 may be purified by any method known in the art. Chemical synthesis methods for nucleic acids include, but are not limited to, chemical synthesis using phosphotriester, phosphate or phosphoramidite chemistry and solid phase techniques, or in vitro chemical synthesis via deoxynucleotide H-phosphonate intermediates (see, U.S. Pat. No. 5,705,629 to Bhongle).

In some cases, for example, if increased nuclease stability of a nucleic acid inhibitor is desired, nucleic acids having nucleic acid analogs and/or modified internucleotide linkages can be used. Nucleic acids containing modified internucleotide linkages can also be synthesized using reagents and methods well known in the art. For example, the synthesis contains phosphate, phosphorothioate, dithiolin, phosphoramidite, methoxyethyl phosphoramidite, methylal, thiometal, diisopropylsilyl, acetamide, carbamate, dimethylene sulfide (-CH)₂-S-CH₂) Dimethylene sulfoxide (-CH)₂-SO-CH₂) Dimethylene sulfone (-CH)₂-SO₂-CH₂) Methods for nucleic acids, 2 ' -O-alkyl, and 2 ' -deoxy-2 ' -fluorosulphosphate internucleotide linkages are well known in the art (see, Uhlmann et al, 1990, chem. rev. 90: 543-; schneider et al, 1990, tetrahedron lett.31: 335 and references cited herein). Nucleic acid analogs useful for enhancing nuclease stability and cellular uptake are also disclosed in U.S. Pat. Nos. 5,614,617 and 5,223,618 to Cook et al, US5,714,606 to Acevedo et al, US5,378,825 to Cook et al, US5,672,697 and 5,466,786 to Buhr et al, US5,777,092 to Cook et al, US5,602,240 to De Mesmacker et al, US5,610,289 to Cook et al, and US5,858,988 to Wang.

Synthetic siRNA molecules, including shRNA molecules, can also be readily obtained using a number of techniques known to those skilled in the art. For example, the siRNA molecule may be chemically synthesized or recombinantly produced using methods known in the art, such as using suitably protected ribonucleotide phosphoramidites and conventional DNA/RNA synthesizers (see, e.g., Elbashir, S.M.et al (2001) Nature 411: 494-498; Elbashir, S.M., W.Lendeckel and T.Tuschl (2001) Genes & Development 15: 188-200; Harborth, J.et al. (2001) J.cell Science 114: 4557-4565; Masters, J.R.et al (2001) Proc.Natl.Acad.Sci., USA 98: 8012-8017; and Tuschl, T.et al (1999) Genes & Development 13: 3191-3197). Alternatively, several commercial RNA synthesis suppliers are available, including, but not limited to, Prologo (Hamburg, Germany), Dharmacon Research (Lafayette, CO, USA), Pierce Chemical (part of Perbio Science, Rockford, IL, USA), Glen Research (Sterling, VA, USA), Chemgenes (Ashland, MA, USA), and Cruache (Glasgow, UK). As such, siRNA molecules are not very difficult to synthesize and can be readily provided in a quality suitable for RNAi. Furthermore, dsRNA can be expressed as encoded stem-loop structures by plasmid vectors, retroviruses and lentiviruses (Paddison, P.J.et al. (2002) GenesDev.16: 948-. These vectors typically have a polIII promoter located upstream of the dsRNA and can independently express sense and antisense RNA strands, and/or express sense and antisense RNA strands as hairpin structures. In the cell, dicer enzyme processes the short hairpin RNA (shRNA) into effective siRNA.

In some embodiments, the inhibitor of H3K9 methyltransferase is a gene silencing siRNA molecule expressed as human SUV39H1, human SUV39H2, human SETDB1, humanAny one of the genes EHMT1 or human PRDM2 is targeted, and in particular embodiments, the coding RNA sequence of human SUV39h1, human SUV39h2, human SETDB1, human EHMT1, or human PRDM2, starting from about 25 to 50 nucleotides, about 50 to 75 nucleotides, or about 75 to 100 nucleotides downstream of the initiation codon is targeted. One method of designing an siRNA molecule of the invention involves identifying the 29 nucleotide sequence motif AA (N29) TT (where N can be any nucleotide) (SEQ ID NO: 50), and selecting a number of samples having a G/C content of at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75%. The "TT" portion of the sequence is contingent on the situation. Alternatively, if this sequence is not found, the search can be extended using the motif NA (N21), where N can be any nucleotide. In this case, the 3 'end of the sense siRNA can be converted to TT, allowing the generation of a symmetrical double helix with respect to the combination of sense and antisense 3' overhanging sequences. Subsequently, the antisense siRNA molecule can be synthesized as a complement to nucleotide positions 1 to 21 of the 23 nucleotide sequence motif. The use of synthetic 3' TT pendants is advantageous for ensuring the formation of small interfering ribonucleoprotein particles (sirnps) that cleave sirnps with nearly equal sense and antisense target RNAs (ex Elbashir et al, (2001) and ex Elbashir et al, 2001). Analysis of sequence databases, including but not limited to NCBI, BLAST, Derwent, and GenSeq, and conventional oligo-synthesis software such asIt can also be used to select siRNA sequences in EST libraries to ensure that only one gene is targeted.

Sirnas useful in the methods disclosed herein include siRNA molecules of about 15 to about 40 or about 15 to about 28 nucleotides in length that are homologous to any one of the H3K9 methyltransferases, such as human SUV39H1, human SUV39H2, human SETDB1, human EHMT1, or human PRDM 2. The length of the targeted siRNA molecule that is human SUV39h1, human SUV39h2, human SETDB1, human EHMT1, or human PRDM2 is preferably about 19 to about 25 nucleotides. More preferably, the targeted siRNA molecule is about 19, 20, 21, or 22 nucleotides in length. The targeted siRNA molecule can also include a 3' -hydroxyl group. The targeted siRNA molecule can be single-stranded or double-stranded; such molecules may be blunt-ended or comprise pendent ends (e.g., 5 'ends, 3' ends). In particular embodiments, the RNA molecule is double-stranded, and is either blunt-ended or comprises a overhang.

In one embodiment, at least one strand of the H3K9 methyltransferase RNAi-targeting RNA molecule has a 3' overhang of about 0 to about 6 nucleotides in length (e.g., pyrimidine nucleotides, purine nucleotides). In other embodiments, the 3' overhang is from about 1 to about 5 nucleotides, from about 1 to about 3 nucleotides, and from about 2 to about 4 nucleotides in length. In one embodiment, the targeting RNA molecule is double-stranded: one strand has a 3' overhang and the other strand may be blunt-ended or have an overhang. In embodiments where the targeting RNA molecule is double-stranded and both strands comprise a overhang, the length of the overhang on each strand may be the same or different. In particular embodiments, the RNA of the invention comprises about 19, 20, 21, or 22 paired nucleotides and has a overhang of about 1 to about 3, particularly about 2 nucleotides in length at both 3' ends of the RNA. In one embodiment, the 3' overhang can be stabilized against degradation. In preferred embodiments, stabilization is achieved by including purine nucleotides, such as adenosine or guanosine nucleotides. Alternatively, substitution of pyrimidine nucleotides with modified analogs, e.g., substitution of uridine 2 nucleotides with 2 '-deoxythymidine for 3' overhang, is tolerable and does not affect the efficiency of RNAi. The absence of the 2' -hydroxyl group significantly increases the nuclease resistance of the overhang in tissue culture medium.

Oligonucleotide modification

In some applications, unmodified oligonucleotides may be less than ideal, e.g., unmodified oligonucleotides may be demonstrated to be degraded by cellular nucleases. Nucleases can hydrolyze phosphodiester bonds of nucleic acids. However, chemical modification of one or more subunits of an oligonucleotide may impart improved properties thereto and may, for example, render the oligonucleotide more nuclease-stable.

The modified nucleic acids and modified nucleotide substitutes may include one or more of the following: (i) alterations such as substitutions to one or both non-linked phosphate oxygens and/or one or more linked phosphate oxygens in the phosphodiester backbone linkages; (ii) alterations such as substitutions to the ribose backbone, such as the 2' -hydroxyl on the ribose; (iii) the integral replacement of the phosphate moiety with a "dephosphorylated" linker; (iv) modifications or substitutions with bases that occur naturally in non-natural base pairs; (v) replacement or modification of the ribose-phosphate backbone; (vi) modifications such as removal of the 3 'or 5' end of the oligonucleotide, modifications or substitutions to a terminal phosphate group or to the attachment of a moiety, such as a fluorescent label moiety, to the 3 'or 5' end of the oligonucleotide; and (vii) modifications to sugars (e.g., 6-membered rings).

The terms replacement, modification, alteration, and the like as used in this context do not imply any process limitations, e.g., modification does not imply that it is necessary to start with a reference or naturally occurring ribonucleic acid and modify it to produce a modified ribonucleic acid, but rather simply modify it to the extent that it is different from a naturally occurring molecule.

Since oligonucleotides are polymers of subunits or monomers, the modifications disclosed herein can occur mostly at repeated positions within the oligonucleotide, e.g., modifications to the nucleobase, sugar, phosphate moiety, or non-bridging oxygens of the phosphate moiety. It is not necessary that all positions of a given oligonucleotide be uniformly modified, and in fact, more than one of the foregoing modifications may be incorporated into a single oligonucleotide or even into a single nucleotide within an oligonucleotide.

In some cases, the modification will occur at all positions tested on the oligonucleotide, but in most cases, in fact, in most cases, this is not the case. For example, the modification may occur at the 3 'or 5' terminal position, may occur only in the inner region, may occur only in the terminal region, e.g., at the terminal nucleotide position or the last 2, 3, 4,5, or 10 nucleotides of the oligonucleotide. The modification may occur in the double-stranded region, the single-stranded region, or both regions. The modification may occur only in the double-stranded region of the oligonucleotide, or may occur only in the single-stranded region of the oligonucleotide. For example, phosphorothioate modifications at non-bridging oxygen positions may occur only at one or both ends, may occur only in the terminal region, e.g., at the terminal nucleotide position of the strand or the last 2, 3, 4,5, or 10 nucleotides of the strand, or may occur in double-stranded and single-stranded regions, particularly the ends. Either or both of the 5 'and the 5' ends may be phosphorylated.

The modifications disclosed herein may be unique modifications, or a unique type of modification included over multiple nucleotides, or the modifications may be combined with one or more other modifications disclosed herein. The modifications disclosed herein can also be combined on an oligonucleotide, as different nucleotides in an oligonucleotide have different modifications disclosed herein.

In some embodiments, for example, it is particularly preferred to promote stability, include specific nucleic acid bases in the overhang, or include modified nucleotides or nucleotide substitutions in a single-stranded overhang, such as a 5 'or 3' overhang, or both. If desired, it may be desirable to include purine nucleotides in the overhang. In some embodiments, all or some of the bases in the 3 'or 5' overhangs will be modified, e.g., with the modifications disclosed herein. Modifications can include, for example, the use of modifications located on the ribose 2' -OH group; for example, deoxyribonucleotides such as deoxythymidine are used instead of ribonucleotides; and modifications in phosphates, e.g., thiosulfate modifications. The overhang need not be homologous to the target sequence.

Specific modification of oligonucleotides

Phosphoric acid ester group

The phosphate group is a negatively charged group. The charge is equally distributed over two non-bridging oxygen atoms. However, the phosphate group may be modified by replacing one oxygen with a different substituent. One consequence of this modification of the phosphate backbone of RNA is that it is possible to increase the resistance of the oligoribonucleotide to nucleolytic degradation. Thus, while not wishing to be bound by theory, in some embodiments it may be desirable to introduce a change that results in an uncharged linker or a linker with an asymmetric charge distribution.

Examples of modified phosphates include phosphorothioate, phosphoroselenoate, borophosphate, hydrogen phosphate, phosphoramide, alkyl or aryl phosphate, and phosphotriester. In certain embodiments, one non-bridging phosphoester oxygen molecule in the phosphate backbone moiety may be replaced by any of the following: s, Se, BR₃(R is hydrogen, alkyl, aryl), C (i.e., alkyl, aryl, etc.), H, NR₂(R is hydrogen, alkyl, aryl), OR OR (R is alkyl OR aryl). The phosphorus atom in the unmodified phosphate group is achiral. However, replacing one of the non-bridging oxygens with one of the above sources or groups of atoms, makes the phosphorus atom chiral; in other words, the phosphorus atom in the phosphate group modified in this way is a stereocenter. The stereogenic central phosphorus atom may have either the "R" configuration (herein denoted as Rp) or the "S" configuration (herein denoted as Sp).

The phosphorodithioates have two non-bridging oxygen atoms replaced by sulfur. The phosphorus center in the phosphorodithioate is achiral, which prevents the formation of oligoribonucleotide diastereomers. Thus, while not wishing to be bound by theory, modifications to two non-bridging oxygens that eliminate the chiral center, such as the formation of phosphorodithioates, may be desirable in that a mixture of diastereomers cannot be produced. Thus, the non-bridging oxygens may independently be any of S, Se, B, C, H, N, OR OR (R is alkyl OR aryl).

The phosphate linker may also be modified by replacing the bridging oxygen (i.e., the oxygen linking the phosphate to the nucleotide) with nitrogen (bridged phosphoramide), sulfur (bridged phosphorothioate), and carbon (bridged methylene phosphate). The substitution may occur at one or both catenated oxygens. When the bridging oxygen is the 3' -oxygen of the nucleotide, a carbon substitution is preferred. When the bridging oxygen is the 5' -oxygen of the nucleotide, it is preferably replaced by nitrogen.

Replacement of phosphate groups

The phosphate group can be replaced with a non-phosphorus containing linker. While not wishing to be bound by theory, it is believed that since the charged phosphodiester group is the reaction center for nucleolytic degradation, replacing it with a neutral structural mimetic should confer increased nuclease stability. Again, while not wishing to be bound by theory, in some embodiments it may be desirable to introduce a change in which the charged phosphate group is replaced with a neutral moiety.

Examples of moieties that can replace the phosphate group include methyl phosphate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethyleneoxy linkage, sulfonate, sulfonamide, thiometal, methylal, oxime, methyleneimino, methylenemethylimino, methylenehydrazino, methylenedimethylhydrazino, and methyleneoxymethylimino. Preferred substitutions include methylenecarbonylamino and methylenemethyliminomethyl groups.

Modified phosphate linkers in which at least one oxygen linked to the phosphate has been replaced or the phosphate has been replaced with a non-phosphorus group are also referred to as "non-phosphodiester backbone linkers".

Replacement of the ribose phosphate backbone

Oligonucleotide-mimetic scaffolds can also be constructed in which the phosphate linker and ribose are replaced with nuclease-resistant nucleotides or nucleotide substitutes. While not wishing to be bound by theory, it is believed that the absence of a repeatedly charged backbone reduces its binding to proteins that recognize polyanions (e.g., nucleases). Again, while not wishing to be bound by theory, in some embodiments it may be desirable to introduce changes in the bases of which are linked by a neutral surrogate backbone. Examples include N-morpholinyl, cyclobutyl, pyrrolidine, and Peptide Nucleic Acid (PNA) nucleotide substitutes. The preferred substitute is a PNA substitute.

Sugar modification

The oligonucleotide may include modifications to all or part of the sugar groups of the nucleic acid. For example, the 2' -hydroxy (OH) group can be modified or replaced by a number of different "oxy" or "deoxy" substituents. While not wishing to be bound by theory, an increase in stability is expected because the hydroxyl group may no longer be deprotonated to form a 2' -alkoxide. The 2' -alkoxide can be catalytically degraded by intramolecular nucleophilic attack on the phosphorus atom of the linker. Again, while not wishing to be bound by theory, in some embodiments it may be desirable to introduce a modification that does not make alkoxide formation at the 2' position possible.

Examples of "oxy" -2' -hydroxy modifications include alkoxy OR aryloxy (OR, e.g., R ═ H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, OR saccharide); polyethylene glycol (PEG), O (CH)₂CH₂O)_nCH₂CH₂OR; "locked" nucleic acids (LNAs) in which the 2 '-hydroxyl group is linked to the 4' carbon of the same ribose sugar, for example, by a methylene bridge; O-AMINE (AMINE ═ NH)₂Alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, polyamine) and aminoalkoxy, O (CH)₂)_nAMINE (e.g., AMINE ═ NH)₂Alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, polyamine). Notably, containing only Methoxyethyl (MOE) (OCH)₂CH₂OCH₃PEG derivatives) exhibit nuclease stability comparable to those oligonucleotides with robust phosphorothioate modifications.

"deoxy" modifications include hydrogen (i.e., deoxyribose, which is associated with, inter alia, a overhang portion of a partially double-stranded RNA); halogen (e.g., fluorine); amino (e.g., NH)₂An alkylamino group, a dialkylamino group, a heterocyclic group, an arylamine group, a diarylamino group, a heteroarylamino group, a diheteroarylamino group, or an amino acid); NH (CH)₂CH₂NH)_nCH₂CH₂-AMINE(AMINE＝NH₂An alkylamino group, a dialkylamino group, a heterocyclic group, an arylamine group, a diarylamino group, a heteroarylamino group, a diheteroarylamino group, or a diheteroarylamino group); -nhc (o) R (R ═ alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or saccharide); a cyano group; a mercapto group; alkyl-thio-alkyl; a thioalkoxy group; a thioalkyl group; and optionally alkanes substituted, for example, with amino functionalityAlkyl, cycloalkyl, aryl, alkenyl, and alkynyl.

Thus, an oligonucleotide may include nucleotides containing, for example, arabinose as the sugar group, the monomer may have an α linkage at the 1' position on the sugar, e.g., α -nucleotides.

Preferred substituents are 2 '-O-Me (2' -O-methyl), 2 '-O-MOE (2' -O-methoxyethyl), 2 '-F, 2' -O- [2- (methylamino) -2-oxoethyl](2 ' -O-NMA), 2 ' -S-methyl, 2 ' -O-CH₂-(4′-C)(LNA)、2′-O-CH₂CH₂- (4 ' -C) (EN A), 2 ' -O-aminopropyl (2 ' -O-AP), 2 ' -O-dimethylaminoethyl (2 ' -O-DMAOE), 2 ' -O-dimethylaminopropyl (2 ' -O-DMAP) and 2 ' -O-dimethylaminoethoxyethyl (2 ' -O-DMAEOE).

End modification

The 3-terminal (3 ') and 5-terminal (5') ends of the oligonucleotide may be modified. Such modifications may be located at the 3 'end, the 5' end, or both ends of the molecule. They may include modifications or substitutions to the overall terminal phosphate, or to one or more atoms of the phosphate group. For example, the 3 'end and the 5' end of the oligonucleotide can be conjugated to the entirety of other functional molecules, such as a labeling moiety, such as a fluorophore (e.g., pyrene, TAMRA, fluorescein, Cy3 dye, or Cy5 dye) or a protecting group (e.g., sulfur, silicon, boron, or ester based protecting group). The functional molecule as a whole may be attached to the sugar via a phosphate group and/or a linker. The end of the linker is derived from O, N, S or a C group that may be attached to or replace the linking atom of the phosphate group or C-3 'or C-5' of the sugar. Alternatively, the linker may be attached to or replace a terminal atom of a nucleotide substitute (e.g., PNA).

When the linker/phosphate-functional molecule ensemble-linker/phosphate array is placed between the two strands of a dsRNA, this array can replace the hairpin RNA loop in a hairpin RNA agent.

Terminal modifications that may be used to modulate activity include modification of the 5' terminus with a phosphate or phosphate analog. For example, in a preferred embodiment, the antisense strand of the dsRNA is 5 '-phosphorylated, or includes a phosphoryl analog at the 5' terminal end. 5' -phosphate modifications include those that are compatible with RISC-mediated gene silencing. Modifications at the 5' -terminus may also be used to stimulate or suppress the immune system of a subject. Suitable modifications include: 5' -monophosphates ((HO)₂(O) P-O-5'); 5' -bisphosphate ((HO)₂(O) P-O-P (HO) O-5'); 5' -triphosphate ((HO)₂(O) P-O- (HO) (O) P-O-P (HO) (O) -O-5'); 5 ' -guanosine cap (7-methylated or unmethylated) (7m-G-O-5 ' - (HO) (O) P-O- (HO) (O) P-O-P (HO) (O)) O- (5 '); 5 ' -adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N-O-5 ' - (HO) (O) P-O- (HO) (O) P-O-P (HO) (O) O-5 '); 5' -monothiophosphate (thiophosphate; (HO)₂(S) P-O-5'); 5 '-dithiophosphoric acid esters (dithiophosphoric acid esters: (HO) (HS) (S) P-O-5'); 5' -thiophosphate ((HO)₂(O) P-S-5 '), oxygen/sulfur substituted monophosphates, diphosphates and triphosphates (e.g., 5 ' - α -thiophosphate, 5 ' - β -thiophosphate, 5 ' -gamma-thiophosphate, etc.), 5 ' -phosphoramide ((HO)₂(O)P-NH-5′、(HO)(NH₂) (O) P-O-5 '), 5 ' -alkylphosphate (R ═ alkyl ═ methyl, ethyl, isopropyl, propyl, and the like, e.g., rp (OH) (O) -O-5 ' -, (OH)₂(O)P-5′-CH₂-), 5' -alkyl ether phosphate (R ═ alkyl ether ═ methoxymethyl (MeOCH)), and a mixture of these₂-), ethoxymethyl methyl, and the like, e.g., RP (OH) (O) -O-5' -) in any other combination. Other embodiments include the replacement of oxygen/sulfur with BH3, BH 3-and/or Se.

Terminal modifications may also be used to monitor the respective, and in this case, preferred groups to be added include fluorophores such as fluorescein orDyes, e.g. for dyeing488. Terminal modifications may also be used to enhance uptake, and useful modifications for this purpose include cholesterol. End modifications may also be used to crosslink an RNA agent to another moiety; modifications that may be used for this purpose include mitomycin C.

Nucleic acid base

Adenine, guanine, cytosine and uracil are the most common bases found in RNA. These bases may be modified or replaced to provide RNAs with improved properties. For example, nuclease-resistant oligoribonucleotides can be prepared using these bases or synthetic and natural nucleobases (e.g., inosine, thymine, xanthine, hypoxanthine, nucularine, isogenine, or tubercidine) and any of the modifications described above. Alternatively, substituted or modified analogs of any of the bases described above, as well as "universal bases" may be employed. Examples include: 2- (halo) adenine, 2- (alkyl) adenine, 2- (propyl) adenine, 2- (amino) adenine, 2- (aminoalkyl) adenine, 2- (aminopropyl) adenine, 2- (methylthio), N6- (isopentenyl) adenine, 6- (alkyl) adenine, 6- (methyl) adenine, 7- (deaza) adenine, 8- (alkenyl) adenine, 8- (alkyl) adenine, 8- (alkynyl) adenine, 8- (amino) adenine, 8- (halo) adenine, 8- (hydroxy) adenine, 8- (thioalkyl) adenine, 8- (mercapto) adenine, N6- (isopentyl) adenine, N6- (methyl) adenine, N6, N6- (dimethyl) adenine, 2- (alkyl) guanine, 2- (propyl) guanine, 6- (alkyl) guanine, 6- (methyl) guanine, 7- (alkyl) guanine, 7- (methyl) guanine, 7- (deaza) guanine, 8- (alkyl) guanine, 8- (alkenyl) guanine, 8- (alkynyl) guanine, 8- (amino) guanine, 8- (halo) guanine, 8- (hydroxy) guanine, 8- (thioalkyl) guanine, 8- (mercapto) guanine, N- (methyl) guanine, 2- (thio) cytosine, 3- (deaza) -5- (aza) cytosine, 3- (alkyl) cytosine, 3- (methyl) cytosine, 5- (alkyl) cytosine, 5- (alkynyl) cytosine, 5- (halo) cytosine, 5- (methyl) cytosine, 5- (propynyl) cytosine, 5- (trifluoromethyl) cytosine, 6- (azo) cytosine, N4- (acetyl) cytosine, 3- (3-amino-3-carboxypropyl) uracil, 2- (thio) uracil, 5- (methyl) -2- (thio) uracil, 5- (methylaminomethyl) -2- (thio) uracil, 4- (thio) uracil, 5- (methyl) -4- (thio) uracil, 5- (methylaminomethyl) -4- (thio) uracil, 5- (methyl) -2, 4- (dithio) uracil, 5- (methylaminomethyl) -2, 4- (dithio) uracil, 5- (2-aminopropyl) uracil, 5- (alkyl) uracil, 5- (alkynyl) uracil, 5- (allylamino) uracil, 5- (aminoallyl) uracil, 5- (aminoalkyl) uracil, 5- (guanidinoalkyl) uracil, 5- (1, 3-oxadiazol-1-alkyl) uracil, 5- (cyanoalkyl) uracil, 5- (dialkylaminoalkyl) uracil, 5- (dimethylaminoalkyl) uracil, 5- (halo) uracil, 5- (methoxy) uracil, uracil-5-oxyacetic acid, 5- (methylaminomethyl) uracil, 5- (dimethylamino) uracil, 5-amino-2, 4-dimethyl-amino-2, 4- (dithio) uracil, 5- (aminopropyl) uracil, 5- (2-aminopropyl) uracil, 5- (alkyl) uracil, 5- (methoxycarbonylmethyl) -2- (thio) uracil, 5- (methoxycarbonylmethyl) uracil, 5- (propynyl) uracil, 5- (trifluoromethyl) uracil, 6- (azo) uracil, dihydrouracil, N3- (methyl) uracil, 5-uracil (i.e., pseudouracil), 2- (thio) pseudouracil, 4- (thio) pseudouracil, 2, 4- (dithio) pseudouracil, 5- (alkyl) pseudouracil, 5- (methyl) pseudouracil, 5- (alkyl) -2- (thio) pseudouracil, 5- (methyl) -2- (thio) pseudouracil, 5- (alkyl) -4- (thio) pseudouracil, uracil, 5- (methyl) -4- (thio) pseudouracil, 5- (alkyl) -2, 4- (dithio) pseudouracil, 5- (methyl) -2, 4- (dithio) pseudouracil, 1-substituted 2- (thio) -pseudouracil, 1-substituted 4- (thio) pseudouracil, 1-substituted 2, 4- (dithio) pseudouracil, 1- (aminocarbonylvinyl) -2 (thio) pseudouracil, 1- (aminocarbonylvinyl) -4 (thio) pseudouracil, 1 (aminocarbonylvinyl) -2, 4- (dithio) pseudouracil, 1- (aminoalkylaminocarbonylvinyl) -pseudouracil, and pharmaceutically acceptable salts thereof, 1- (aminoalkylamino-carbonylvinyl) -2 (thio) -pseudouracil, 1- (aminoalkylaminocarbonylvinyl) -4 (thio) pseudouracil, 1- (aminoalkylaminocarbonylvinyl) -2, 4- (dithio) pseudouracil, 1, 3- (diaza) -2- (oxo) -phenoxazin-1-yl, 1- (aza) -2- (thio) -3- (aza) -phenoxazin-1-yl, 1, 3- (diaza) -2- (oxo) -phenothiazin-1-yl, 1- (aza) -2- (thio) -3- (aza) -phenothiazin-1-yl, phenoxazin-l, phenoxaz, 7-substituted 1, 3- (diaza) -2- (oxo) -phenoxazin-1-yl, 7-substituted 1- (aza) -2- (thio) -3- (aza) -phenoxazin-1-yl, 7-substituted 1, 3- (diaza) -2- (oxo) -phenothiazin-1-yl, 7-substituted 1- (aza) -2- (thio) -3- (aza) -phenothiazin-1-yl, 7- (aminoalkylhydroxy) -1, 3- (diaza) -2- (oxo) -phenoxazin-1-yl, 7- (aminoalkylhydroxy) -1- (aza) -2- (thio) -3- (aza) -phenoxazin-1-yl, 7- (aminoalkylhydroxy) -1, 3- (diaza) -2- (oxo) -phenothiazin-1-yl, 7- (aminoalkylhydroxy) -1- (aza) -2- (thio) -3- (aza) -phenothiazin-1-yl, 7- (guanidinoalkylhydroxy) -1, 3- (diaza) -2- (oxo) -phenoxazin-1-yl, 7- (guanidinoalkylhydroxy) -1- (aza) -2- (thio) -3- (aza) -phenoxazin-1-yl, 7- (guanidinoalkylhydroxy) -1, 3- (diaza) -2- (oxo) -phenothiazin-1-yl A group, 7- (guanidinoalkylhydroxy) -1- (aza) -2- (thio) -3- (aza) -phenothiazin-1-yl, 1,3, 5- (triaza) -2, 6- (dioxa) -naphthalene, inosine, xanthine, hypoxanthine, nubularine, tubercidin, isogluaniine, inosinyl (inosinyl), 2-aza-inosinyl, 7-deaza-inosinyl, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazolyl, nitroindazolyl, aminoindolyl, pyrrolopyrimidyl, 3- (methyl) isoquinolinyl, 5- (methyl) isoquinolinyl, 3- (methyl) -7- (propynyl) isoquinolinyl, 7- (aza) indolyl, 6- (methyl) -7- (aza) indolyl, 3- (aza) indolyl, Imidazopyridinyl, 9- (methyl) -imidazopyridinyl, pyrrolopiperazinyl, isoquinolinyl, 7- (propynyl) isoquinolinyl, propynyl-7- (aza) indolyl, 2,4, 5- (trimethyl) phenyl, 4- (methyl) indolyl, 4, 6- (dimethyl) indolyl, phenyl, naphthyl, anthryl, phenanthryl, pyrenyl, stilbenyl, tetracenyl, pentacenyl, difluorotolyl, 4- (fluoro) -6- (methyl) benzimidazole, 4- (methyl) benzimidazole, 6- (azo) thymine, 2-pyridone, 5-nitroindole, 3-nitropyrrole, 6- (aza) pyrimidine, 2- (amino) purine, 2, 6- (diamino) purine, 5-substituted pyrimidines, N2-substituted purines, N6-substituted purines, O6-substituted purines, substituted 1, 2, 4-triazoles, or any O-alkylated or N-alkylated derivatives thereof.

Other purines And pyrimidines include those disclosed in U.S. Pat. No. 3,687,808, incorporated herein by reference, those disclosed in convention Encyclopedia Of Polymer Science And Engineering, pages 858- & 859, Kroschwitz, J.I., ed.John Wiley & Sons, 1990, And those disclosed in Englisch et al, Angewandte Chemie, International Edition, 1991, 30, 613.

Cationic group

Modifications to the oligonucleotide may also include one or more cationic groups attached to the sugar, base, and/or phosphate or phosphorus atoms of the modified phosphate backbone moiety. The cationic group may be attached to any atom that can be substituted on the natural non-universal or universal base. Preferred positions are positions that do not interfere with hybridization, i.e., do not interfere with hydrogen bonding interactions required for base pairing. The cationic group may be attached, for example, through the C2' position of the sugar or a similar position in a cyclic or acyclic substitute. The cationic group may include, for example, a deprotonated AMINE group derived from, for example, O-AMINE (AMINE ═ NH)₂Alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, polyamine); aminoalkoxy radicals, e.g. O (CH)₂)_nAMINE (e.g., AMINE ═ NH)₂Alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, polyamine); amino (e.g., NH)₂An alkylamino group, a dialkylamino group, a heterocyclic group, an arylamine group, a diarylamino group, a heteroarylamino group, a diheteroarylamino group, or an amino acid); or NH (CH)₂CH₂NH)_nCH₂CH₂-AMINE(AMINE＝NH₂Alkylamino, dialkylamino, heterocyclic, arylamino, diarylamino, heteroarylaminoOr a diheteroarylamine group).

Arrangement within an oligonucleotide

Some modifications may preferably be included at specific positions of the oligonucleotide, e.g., internal positions of the strand, or at the 5 'or 3' end of the oligonucleotide. The preferred location of the modification on the oligonucleotide may confer preferred properties to the agent. For example, preferred locations for specific modifications may confer optimal gene silencing properties, or increased resistance to endonuclease or exonuclease activity.

One or more nucleotides of the oligonucleotide may have a2 '-5' linkage. One or more nucleotides of the oligonucleotide may have a reverse linkage, such as a3 '-3' linkage, a 5 '-5' linkage, a2 '-2' linkage, or a2 '-3' linkage.

The oligonucleotide may comprise at least one 5 '-pyrimidine-purine-3' (5 '-PyPu-3') dinucleotide, wherein the pyrimidine is modified with modifications independently selected from the group consisting of 2 '-O-Me (2' -O-methyl), 2 '-O-MOE (2' -O-methoxyethyl), 2 '-F, 2' -O- [2- (methylamino) -2-oxoethyl ] (2 '-O-NMA), 2' -S-methyl, 2 '-O-CH 2- (4' -C) (LNA) and 2 '-O-CH 2CH2- (4' -C) (ENA).

In one embodiment, the majority of the 5 ' -pyrimidines in the all occurring sequence motif 5 ' -pyrimidine-purine-3 ' (5 ' -PyPu-3 ') dinucleotide of the oligonucleotide are modified by modifications selected from the group consisting of 2 ' -O-Me (2 ' -O-methyl), 2 ' -O-MOE (2 ' -O-methoxyethyl), 2 ' -F, 2 ' -O- [2- (methylamino) -2-oxoethyl ] (2 ' -O-NMA), 2 ' -S-methyl, 2 ' -O-CH2- (4 ' -C) (LNA) and 2 ' -O-CH2CH2- (4 ' -C) (ENA).

The double-stranded oligonucleotide may comprise at least one 5 ' -uridine-adenine-3 ' (5 ' -UA-3 ') dinucleotide, wherein the uridine is a2 ' -modified nucleotide; or a 5 '-uridine-guanine-3' (5 '-UG-3') dinucleotide, wherein the 5 '-uridine is a 2' -modified nucleotide; or a terminal 5 '-cytidine-adenine-3' (5 '-CA-3') dinucleotide, wherein the 5 '-cytidine is a 2' -modified nucleotide; or a terminal 5 '-uridine-3' (5 '-UU-3') dinucleotide, wherein the 5 '-uridine is a 2' -modified nucleotide; or a terminal 5 '-cytidine-3' (5 '-CC-3') dinucleotide, wherein the 5 '-cytidine is a 2' -modified nucleotide; or a terminal 5 '-cytidine-uridine-3' (5 '-CU-3') dinucleotide, wherein the 5 '-cytidine is a 2' -modified nucleotide; or a terminal 5 '-uridine-cytidine-3' (5 '-UC-3') dinucleotide, wherein the 5 '-uridine is a 2' -modified nucleotide. Double-stranded oligonucleotides comprising these modifications are particularly stable against endonuclease activity.

General references

Oligoribonucleotides and oligoribonucleotides used according to the invention can be synthesized using solid phase synthesis methods, see, e.g., Oligonucleotide synthesis pragmatics (Oligonucleotide synthesis, a practical prophach, ed.m.j.gait., IRL Press, 1984); practical methods for Oligonucleotides and analogues (Oligonucleotides and analogs, A Practical Approach, Ed.F. Eckstein, IRL Press, 1991 (in particular Chapter 1, modern machine-assisted methods for oligodeoxyribonucleotide synthesis, Chapter 2 oligoribonucleotide synthesis, Chapter 3,2 '-O-methyl oligoribonucleotides: Synthesis and use, Chapter 4, phosphorothioate Oligonucleotides, Chapter 5, Synthesis of oligonucleotide phosphorodithioates, Chapter 6, Synthesis of oligo-2' -deoxyribonucleoside methylphosphates, and Chapter 7, oligodeoxynucleotides containing modified bases.) other particularly useful synthetic procedures, reagents, blocking groups and reaction conditions are disclosed in Martin, P., Helv.Chim.acta.acta, 1995, 78, 486-504; Beaucage, S.L.and Iyer, R.P., Tetrahedron, 1992, Helv.Chim.22248, and Beaucage, S.3, J. 2223, and S.2313, r.p., Tetrahedron, 1993, 49, 6123-; or references cited therein. Here, modifications disclosed in WO00/44895, WO01/75164, or WO02/44321 may be used. The disclosures of all publications, patents and published patent applications cited herein are hereby incorporated by reference.

Phosphate-based references

The preparation of phosphonate oligoribonucleotides is disclosed in U.S. Pat. No. 5,508,270. The preparation of alkylphosphate oligoribonucleotides is disclosed in U.S. Pat. No. 4,469,863. The preparation of phosphoramide oligoribonucleotides is disclosed in U.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878. The preparation of phosphotriester oligoribonucleotides is disclosed in U.S. Pat. No. 5,023,243. The preparation of borophosphate oligoribonucleotides is disclosed in U.S. Pat. Nos. 5,130,302 and 5,177,198. The preparation of 3 '-deoxy-3' -phosphoramide oligoribonucleotides is disclosed in US patent US5,476,925. The preparation of 3 '-deoxy-3' -methylene phosphate oligoribonucleotides is disclosed in An, H, et al.J.org.chem.2001, 66, 2789-2801. Preparation of the sulfur-bridged Nucleotides is disclosed in Sproat et al, Nucleotides 1988, 7, 651 and Crosstick et al tetrahedron Lett.1989, 30, 4693.

Sugar group references

Modifications to 2' -modifications can be found in Verma, S.et al.Annu.Rev.biochem.1998, 67, 99-134 and all references cited therein. Specific modifications to ribose can be found in the following references: 2' -fluoro (kawasakit.al., j.med.chem., 1993, 36, 831-841); 2' -MOE (Martin, P.Helv.Chim.acta 1996, 79, 1930-; "UNA" (Wengel, J.Acc.chem.Res.1999, 32, 301-310).

References to phosphate group substitutions

Methylenemethyliminonyl-linked oligoribonucleosides are also referred to herein as MMI-linked oligoribonucleosides; methylenedimethylhydrazino-linked oligoribonucleosides are also known as MDH-linked oligoribonucleosides; methylenecarbonylamino-linked oligonucleosides are also known as amide-3-linked oligoribonucleosides; and, methyleneaminocarbonyl linked oligonucleosides, also known as amide-4 linked oligoribonucleosides; and, backbone compounds with, for example, alternating MMI and PO or PS linkages, can be prepared as disclosed in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677 and published PCT applications PCT/US92/04294 and PCT/US92/04305 (published as WO 92/20822 and WO 92/20823, respectively). Methylal and thioacetal linked oligoribonucleosides can be prepared as disclosed in US patents US5,264,562 and US5,264,564. Ethylene oxide linked oligoribonucleosides can be prepared as disclosed in U.S. Pat. No. 5,223,618. Siloxane substitutions are disclosed in Cormier, j.f. et al. nucleic Acids res.1988, 16, 458. Carbonate substitutions are disclosed in Tittensor, j.r.j.chem.soc.c 1971, 1933. Carboxymethyl substitutions are disclosed in Edge, m.d. et al.j.chem.soc.perkin trans.11972, 1991. Carbamate substitutions are disclosed in Stirchak, e.p. nucleic Acids res.1989, 17, 6129.

Reference to replacement of the phosphate-ribose backbone

The cyclobutyl sugar replacement compound can be prepared as disclosed in US patent 5,359,044. The pyrrolidine sugar substitute may be prepared as disclosed in US patent US5,519,134. Morpholino sugar substitutes can be prepared as disclosed in U.S. patents US5,142,047 and US5,235,033 and other related patent publications. Peptide Nucleic Acids (PNAs) are known per se and can be prepared according to the expression "Peptide Nucleic Acids (PNAs): synthesis, Properties and Potential Applications (Peptide Nucleic Acids (PNA): Synthesis, Properties and Potential Applications, Bioorganic & Medicinal Chemistry, 1996, 4, 5-23). They may also be prepared according to US patent US5,539,083, which is incorporated herein by reference in its entirety.

End-modified references

Terminal modifications are disclosed in Manoharan, M.et al.antisense and Nucleic Acid drug development 12, 103-128(2002) and references thereto.

Nucleobase references

N-2 substituted purine nucleoside imides can be prepared as disclosed in U.S. Pat. No. 5,459,255. 3-deazapurine nucleoside imides may be prepared as disclosed in US patent US5,457,191. 5, 6-substituted pyrimidine nucleoside imides may be prepared as disclosed in US patent US5,614,617. 5-propynyl pyrimidine nucleoside imides may be prepared as disclosed in U.S. Pat. No. 5,5,484,908. Other references are disclosed in the paragraphs above for base modification.

Oligonucleotide production

The oligonucleotide compounds of the invention can be prepared using solution phase or solid phase organic synthesis methods. Organic synthesis has the following advantages: oligonucleotide chains comprising non-natural or modified nucleotides can be readily prepared. Any other means known in the art for such synthesis may additionally or alternatively be used. The synthesis of other oligonucleotides such as phosphorothioates, phosphorodithioates, and alkylated derivatives using similar techniques is known. The double-stranded oligonucleotide compounds of the invention can be prepared using a two-step process. First, individual strands of the double-stranded molecule are prepared separately. Subsequently, the chain as a component is annealed.

Regardless of the method of synthesis, the oligonucleotide may be prepared in a solution suitable for formulation (e.g., an aqueous solution and/or an organic solution). For example, the oligonucleotide formulation may be precipitated and dissolved in pure double distilled water and lyophilized. Subsequently, the dried oligonucleotide may be resuspended in a solution suitable for the intended formulation process.

Teachings on the synthesis of specific modified oligonucleotides can be found in U.S. Pat. Nos. 5,138,045 and 5,218,105 on polyamine-conjugated oligonucleotides, U.S. Pat. No. 5,212,295 on monomers for preparing oligonucleotides with chiral phosphorus linkages, U.S. Pat. No. 5,378,825 and U.S. Pat. No. 5,541,307 on oligonucleotides with modified backbones, U.S. Pat. No. 5,386,023 on backbone-modified nucleotides and their preparation by reductive coupling, U.S. Pat. No. 5,457,191 on modified nucleic acid bases based on the 3-deazapurine system and their synthesis methods, U.S. Pat. No. 5,459,255 on modified nucleic acid bases based on N-2-substituted purines, U.S. Pat. No. 5,521,302 on the process of preparing oligonucleotides with chiral phosphorus linkages, U.S. Pat. No. 5,539,082 on peptide nucleic acids, U.S. Pat. No. 5,554,746 on oligonucleotides with β -lactams, U.S. Pat. No. 5,571,902 on synthetic oligonucleotides and materials, U.S. Pat. 36 on nucleotides with alkylthio groups, wherein such groups can be used as linking groups in the position for the preparation of modified oligonucleotides with polyamines, U.S. Pat. 3,893, U.S. 5,223, U.S. Pat. 3-to 3-dean-substituted purine analogues, U.S. 3-2-substituted purine analogues, U.S. 3, U.S. Pat. 3-2-3, U.S. Pat. 3-2 analogues, U.S. 3-2 analogues, U.S. Pat. 3-2-substituted purine analogues, U.S. 3, U.S. Pat. 3-2 analogues, U.S. Pat..

Delivery of RNA interference agents

Methods for delivering RNAi agents, such as sirnas or vectors containing RNAi agents, to target cells (e.g., basal cells, or cells of the lung and/or respiratory system, or other desired target cells) are well known to those skilled in the art. In some embodiments, RNAi agents (e.g., gene silencing-RNAi agents) that are H3K9 methyltransferase inhibitors, such as RNAi agents that inhibit any of human SUV39H1, human SUV39H2, human SETDB1, human EHMT1, and/or human PRDM2, can be administered to a subject via aerosol means, e.g., using a nebulizer or the like. In alternative embodiments, administration of an RNAi agent (e.g., a gene silencing-RNAi agent) that is an inhibitor of H3K9 methyltransferase, such as an RNAi agent that inhibits any one of human SUV39H1, human SUV39H2, human SETDB1, human EHMT1, and/or human PRDM2, can include, for example, (i) injection of a composition comprising an RNA interfering agent, such as an siRNA, or (ii) direct contact of a cell (e.g., a donor human cell, recipient oocyte, or SCNT embryo) with a composition comprising an RNAi agent, such as an siRNA.

In some embodiments, the administration of the cell, oocyte or embryo may be by a single injection or by two or more injections. In some embodiments, the RNAi agent is delivered in a pharmaceutically acceptable carrier. One or more gene silencing RNAi agent inhibitors, which may be used concurrently with one or more RNAi agents, such as H3K9 methyltransferases, e.g., SUV39H1, SUV39H2, SETDB1, EHMT1, and/or PRDM2, may be administered together. The RNA interfering agent, e.g., siRNA that inhibits any of human SUV39h1, human SUV39h2, human SETDB1, human EHMT1, and/or human PRDM2, can be delivered alone or in combination with other RNA interfering agents, e.g., siRNA that is targeted to other cellular genes.

In some embodiments, the specific cells are targets for RNA interference, limiting potential side effects of RNA interference due to non-specific targeting by RNA interference. The methods can use, for example, a complex or fusion molecule comprising a cellular targeting moiety and an RNA interference binding moiety for efficient delivery of RNAi into a cell. For example, when mixed with siRNA, an antibody-protamine fusion protein binds to siRNA and selectively delivers the siRNA into cells expressing the antigen recognized by the antibody, silencing gene expression only in those cells expressing the antigen identified by the antibody.

In some embodiments, the siRNA or RNAi-binding moiety is a protein or nucleic acid-binding domain or fragment of a protein, and the binding moiety is fused to a portion of the targeting moiety. The targeting moiety can be located at the carboxy-terminus or amino-terminus of the construct or in the middle of the fusion protein.

In some embodiments, viral-mediated delivery mechanisms may also be employed to deliver siRNA (e.g., gene silencing RNAi agents) inhibitors of siRNA such as human SUV39h1, human SUV39h2, human SETDB1, human EHMT1, and/or human PRDM2 into cells in vitro, such as Xia, h.et al. (2002) Nat Biotechnol 20 (10): 1006) is disclosed in (1). Plasmid-or virus-mediated delivery mechanisms for shRNAs can also be used to deliver shRNAs into cells in vitro and in vivo, as disclosed in Rubinson, D.A., et al. ((2003) nat. Genet.33: 401-406) and Stewart, S.A., et al. ((2003) RNA 9: 493-501). Alternatively, in other embodiments, gene silencing-RNAi agent inhibitors of RNAi agents, e.g., H3K9 methyltransferases such as SUV39H1, SUV39H2, SETDB1, EHMT1, and/or PRDM2, can also be introduced into cells by culturing the cells, oocytes, or SCNT embryos with RNAi agent inhibitors alone or a viral vector expressing the RNAi agent.

In general, any method of delivering a nucleic acid molecule can be adapted for use with RNAi interfering molecules (see, e.g., Akhtars and Julian RL. (1992) Trends cell. biol.2 (5): 139-144; WO94/02595, both of which are incorporated herein by reference in their entirety).

RNA interfering molecules can be modified by chemical conjugation to lipophilic groups, such as cholesterol, to enhance cellular uptake and prevent degradation. In an alternative embodiment, the RNAi molecule can be delivered using a drug delivery system, such as a nanoparticle, dendrimer, polymer, liposome, or cationic delivery system. The positively charged cation delivery system facilitates the binding of RNA interfering molecules (negatively charged) and also enhances the interaction at the negatively charged cell membrane to allow efficient uptake of siRNA by the cell. Cationic lipids, dendrimers, or polymers can either be conjugated to RNA interfering molecules, or induced to form vesicles or micelles that encapsulate RNAi molecules (see, e.g., Kim SH., et al (2008) Journal of controlled Release129 (2): 107-. When administered systemically, the formation of vesicles or micelles further prevents degradation of the RNAi molecule. Methods for making and administering cation-RNAi complexes are well within the ability of those skilled in the art (see, e.g., Sorensen, DR., et al (2003) J.mol.biol 327: 761-766; Verma, UN., et al (2003) Clin.cancer Res.9: 1291-1300; Arnold, AS et al (2007) J.Hypertens.25: 197-205, which are incorporated herein by reference in their entirety).

The amount of reagent for a particular RNAi agent will be that amount necessary to affect silencing of genes of RNA interference, such as human SUV39h1, human SUV39h2, human SETDB1, human EHMT1, and/or human PRDM2, resulting in a reduction in the gene expression levels of human SUV39h1, human SUV39h2, human SETDB1, human EHMT1, and/or human PRDM2, and a subsequent reduction in the expression levels of the respective proteins.

It is also known that RNAi molecules do not necessarily have to perfectly match their target sequence. However, it is preferable that the 5' and middle portions of the antisense (guide) strand of the siRNA are perfectly complementary to the nucleic acid sequence of any one of the human SUV39h1, human SUV39h2, human SETDB1, human EHMT1, and/or human PRDM2 genes.

Accordingly, as disclosed herein, RNAi molecules that function as inhibitors of gene silencing-RNAi of human SUV39h1, human SUV39h2, human SETDB1, human EHMT1 and/or human PRDM2 are, for example, but not limited to, double-stranded (ds) RNA molecules that are not rare and modified, including short-timing regulatory RNA (stRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), double-stranded RNA (dsRNA) (see, e.g., Baulcombe, Science 297: 2002-2003, 2002). The dsRNA molecule, such as siRNA, can also contain a3 ' overhang, preferably a3 ' UU or 3 ' TT overhang. In one embodiment, the siRNA molecules of the invention do not include RNA molecules comprising ssRNA of greater than about 30 to 40 bases, about 40 to 50 bases, about 50 bases, or more. In one embodiment, the siRNA molecules of the invention are double stranded over more than about 25%, more than about 50%, more than about 60%, more than about 70%, more than about 80%, more than about 90% of their length.

In some embodiments, the gene silencing RNAi nucleic acid inhibitor of human SUV39h1, human SUV39h2, human SETDB1, human EHMT1, and/or human PRDM2 is any agent that binds to and inhibits expression of human SUV39h1, human SUV39h2, human SETDB1, human EHMT1, and/or human PRDM2, wherein expression of each methyltransferase gene is inhibited.

In another embodiment of the invention, the inhibitor of human SUV39h1, human SUV39h2, human SETDB1, human EHMT1 and/or human PRDM2 may be a catalytic nucleic acid construct, such as a ribozyme, that cleaves RNA transcripts and thereby prevents production of wild-type protein. By virtue of the two regions of sequence complementary to the target flanking the ribozyme catalytic site, the ribozyme is targeted by and annealed by a specific sequence. After binding, the ribozyme cleaves the target in a site-specific manner. Designing and testing ribozymes, such as cleaving H3K9 methyltransferases such as human SUV39H1, human SUV39H2, human SETDB1, human EHMT1, and/or human PRDM2, that specifically recognize and cleave the gene product sequences disclosed herein is well known to those of skill in the art (e.g., Lleber and Strauss, (1995) Mol Cell Biol 15: 540.551, the disclosure of which is incorporated herein by reference).

Protein and peptide inhibitors of H3K9 methyltransferase

In some embodiments, the H3K9 methyltransferase inhibitor is any protein and/or peptide inhibitor of an H3K9 methyltransferase such as human SUV39H1, human SUV39H2, human SETDB1, human EHMT1, and/or human PRDM2, such as, but not limited to, muteins, therapeutic proteins, and recombinant proteins of human SUV39H1, human SUV39H2, human SETDB1, human EHMT1, and/or human PRDM2, and dominant negative inhibitors (e.g., a non-functional protein of an H3K9 methyltransferase, or a non-functional ligand of an H3K9 methyltransferase that binds to and competes with an H3K9 methyltransferase). Protein and peptide inhibitors may also include, for example, muteins, genetically modified protein proteins, peptides, synthetic peptides, recombinant proteins, chimeric proteins, antibodies, humanized proteins, humanized antibodies, chimeric antibodies, modified proteins, and fragments thereof.

Herein, an agent that can act as an inhibitor of H3K9 methyltransferase, such as human SUV39H1, human SUV39H2, human SETDB1, human EHMT1 and/or human PRDM2 gene expression and/or human SUV39H1, human SUV39H2, human SETDB1, human EHMT1 and/or human PRDM2 protein function, can be any type of entity, such as, but not limited to, a chemical, a nucleic acid sequence, a nucleic acid analog, a protein, a peptide or a fragment thereof. In some embodiments, the agent is a chemical, whole or part, including, without limitation, synthetic and naturally occurring non-protein whole. In certain embodiments, the agent is a small molecule having a chemical moiety.

In alternative embodiments, the agents useful in the methods disclosed herein are proteins and/or peptides or fragments thereof that inhibit gene expression or function of H3K9 methyltransferases such as human SUV39H1, human SUV39H2, human SETDB1, human EHMT1, and/or human PRDM 2. Such agents include, for example, but are not limited to, protein variants, muteins, therapeutic proteins, truncated proteins, and protein fragments. The proteinaceous agent may also be selected from the group comprising muteins, genetically engineered proteins, peptides, synthetic peptides, recombinant proteins, chimeric proteins, antibodies, intermediate antibodies (midibodies), minibodies (miniboodies), triabodies (triabodies), humanized proteins, humanized antibodies, chimeric antibodies, modified proteins, and fragments thereof.

Alternatively, agents useful in the methods disclosed herein as inhibitors of human SUV39h1, human SUV39h2, human SETDB1, human EHMT1, and/or human PRDM2 may be chemicals, small molecules, macromolecules, or whole or parts, including without limitation, synthetic and naturally occurring non-protein whole. In certain embodiments, the agent is a small molecule having a chemical moiety as disclosed herein.

In some embodiments, the H3K9 methyltransferase inhibitors used in the methods and compositions disclosed herein are dominant negative variants of H3K9 methyltransferases, e.g., truncated non-functional variants of human SUV39H1, human SUV39H2, human SETDB1, human EHMT1, and/or human PRDM2, or a polypeptide comprising SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 48 and SEQ ID NO: 54 to SEQ ID NO: 57, e.g., a dominant negative protein of a contiguous amino acid fragment of any of the amino acids of SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 48 and SEQ ID NO: 54 to SEQ ID NO: 57, or at least about 50, or at least about 60, or at least about 70, or at least about 80, or at least about 90, or a fragment of more than 90 amino acids. In some embodiments, the dominant negative inhibitor of an H3K9 methyltransferase protein, such as human SUV39H1, human SUV39H2, human SETDB1, human EHMT1, and/or human PRDM2 protein, is the soluble extracellular domain of an H3K9 methyltransferase protein.

Protein inhibitors, such as gene products or proteins of the DBC 1(Deleted Breast Cancer 1) gene, bind to the SUV39H1 catalytic domain and inhibit its ability to methylate histone H3 in vitro and in vivo (Luet al, Inhibition of SUV39H1 methylation Activity by DBC 1, JBC, 2009, 284; 10361-10366), and can be used in the methods and compositions disclosed herein.

Antibodies

In some embodiments, H3K9 methyltransferase inhibitors useful in the methods of the invention include, for example, antibodies, including monoclonal antibodies, chimeric humanized antibodies, recombinant antibodies, and antigen-binding fragments thereof. In some embodiments, neutralizing antibodies are useful as H3K9 methyltransferase inhibitors. Antibodies can be readily presented to animals by immunization with antigens. Immunized mice are particularly useful for providing a source of B cells for hybridoma production, which cells are subsequently cultured to produce large quantities of monoclonal antibodies. Commercially available antibody inhibitors of human SUV39h1 and/or SUV39h2 may be used in the present invention, e.g., commercially available from santa cruz biotechnology and the like.

In one embodiment of the invention, the inhibitor of a gene product identified herein can be an antibody molecule or an epitope-binding portion of an antibody molecule, or the like. Antibodies provide high binding activity and unique specificity for a large number of target antigens and haptens. Monoclonal antibodies useful in the practice of the present invention include whole antibodies and fragments thereof, and are generated according to conventional techniques such as hybridoma synthesis, recombinant DNA techniques, and protein synthesis techniques.

Useful monoclonal antibodies and fragments can be derived from any species (including humans), or chimeric proteins can be formed that do not employ sequences from more than one species. Human monoclonal antibodies or "humanized" murine antibodies are also used in accordance with the present invention. For example, a murine monoclonal antibody can be "humanized" by genetic recombination of a nucleotide sequence encoding a murine Fv region (i.e., containing an antigen binding site) or its complementarity determining regions with nucleotide sequences encoding a human constant domain region and an Fc region. The humanised targeting moiety is recognised to reduce the immunoreactivity of the antibody or polypeptide in the host recipient, allowing an increased half-life and possibly a reduced reverse immune response, in a manner similar to that disclosed in european patent EP 0,411,893 a 2. The murine monoclonal antibody should preferably be employed in a humanized form. Antigen binding activity was determined by the amino acid sequence and amino acid configuration of 6 (3 in each chain) Complementarity Determining Regions (CDRs) located on the light and heavy chains of an antibody variable portion (Fv). The 25-kDa single chain fv (scFv) molecule, consisting of the variable region of the light chain (VL) and the variable region of the heavy chain (VH) linked together via a short peptide spacer, is the smallest antibody fragment developed to date. Techniques have been developed to display scFv molecules on the surface of filamentous phage containing genes for scFv. scFv molecules with broad antigen specificity can be present in one large pool of scFv-phage libraries. Some examples of high affinity monoclonal antibodies and chimeric derivatives thereof that can be used in the methods of the invention are disclosed in european patent application EP 186,833, PCT patent application WO 92/16553, and US6,090,923.

A chimeric antibody is an immunoglobulin molecule having two or more segments or portions therein derived from different animal species. Typically, the variable regions of chimeric antibodies are derived from non-human mammalian antibodies, such as murine monoclonal antibodies, while the immunoglobulin constant regions are derived from human immunoglobulin molecules. Preferably both regions and combinations have low immunogenicity as determined by conventional assays.

One limitation of scFv molecules is their monovalent interaction with the target antigen. One of the easiest ways to improve the binding of an scFv to its target antigen is to increase its functional affinity by creating multimers. Identical scFv molecules form a combination of diabodies, triabodies and tetrabodies and may contain a large number of identical Fv modules. These agents are therefore multivalent but monospecific. The association of two scFv molecules that are different and each comprise a VH domain and a VL domain derived from different parent igs will form a fully functional bispecific diabody. A unique application of bispecific scfvs is the simultaneous binding of two sites on the same target molecule via two (adjacent) surface epitopes. These reagents achieve significantly better affinity than single scFv or Fab fragments. Numerous scFv-based multivalent structures have been engineered, including, for example, miniantibodies, chimeric miniantibodies, (scFv)2, diabodies, and triabodies. These molecules span a range of valencies (2 to 4 binding sites), size (50 to 120kDa), flexibility, and ease of production. Single chain Fv antibody fragments (scFv) are predominantly monomeric when the VH and VL domains are linked together by a polypeptide chain linker of at least 12 residues. Monomeric scfvs have a linker length of 12 and 25 amino acids under all conditions and are therefore thermodynamically stable. Non-covalent diabody and triabody molecules are readily engineered and are generated by shortening the peptide linker connecting the variable heavy and variable light chains of a single scFv molecule. The scFv dimers are linked by providing a highly flexible amphipathic helix, and the minibody structure can be modified to create a dimeric bispecific (DiBi) minibody containing two miniantibodies (4 scFv molecules) linked via a double helix. The fused or disulfide-bonded scFv dimers provide a moderate degree of flexibility and are generated by simple cloning techniques with the addition of the C-terminal Gly4Cys (SEQ ID NO: 44) sequence. The scFv-CH3 miniantibody consists of two scFv molecules linked together with an IgG CH3 domain either directly (LD minibody) or via a very flexible hinge region (Flex minibody). These bivalent constructs have a molecular weight of approximately 80kDa, so they bind efficiently to antigen. Flex minibodies exhibited impressive tumor localization in mice. Bispecific and trispecific multimers can be formed by the association of different scFv molecules. Increased functional affinity may be achieved when Fab or single chain Fv antibody fragments (scFv) are complexed as dimers, trimers, or larger aggregates. The most important advantage of multivalent scfvs, compared to monovalent scfvs and Fab fragments, is the gain in functional binding affinity (avidity) to the target antigen. High affinity requires that scFv multimers be able to bind simultaneously to independent target antigens. The gain in functional affinity of scFv diabodies compared to scFv monomers is significant and is first seen in a decrease in dissociation rate, which is the result of multiple binding to two or more target antigens and recombination when one Fv dissociates. When such scFv molecules are combined into multimers, they can be designed to have high affinity for a single target antigen or to have multiple specificities for different target antigens. Multiple binding to antigen depends on the correct alignment and orientation of the Fv modules. For full affinity in multivalent scFv targets, the antigen binding sites must point in the same direction. If multiple binding is not sterically possible, the apparent gain in functional affinity may be due to the effect of increased rebinding, which depends on diffusivity and antigen concentration. Antibodies that bind to portions that improve their shape are contemplated by the present invention. For example, antibodies conjugated to PEG that increase their half-life in vivo may be used in the present invention. An immune profile is prepared by PCR amplification of a gene encoding a variable antibody fragment from B lymphocytes from a protozoan or an immune animal or patient. The Combinations of oligos specific for an immunoglobulin gene or immunoglobulin gene family are used by the Combinations of oligonucleotides. Immunoglobulin germline genes can be used to prepare semi-synthetic antibody profiles in which complementarity determining regions of the variable fragments are amplified by PCR using degenerate primers. These single-pot libraries (single-pot libraries) have the following advantages: antibody fragments against a large number of antigens can be isolated from a single pool. Phage display technology can be used to increase the affinity of antibody fragments, and new libraries can be prepared from already existing antibody fragments by random, codon-based or site-directed mutagenesis, by shuffling the chains of individual domains with those of fragments from protoplasts, or by using bacterial mutator strains.

Alternatively, SCID-hu mice, e.g., a model developed by Genpharm, can be used to generate antibodies or fragments thereof. In one embodiment, a novel affinity binding molecule named a pentachain antibody (peptabody) created using a multivalent interaction effect is envisioned. Short peptide ligands are fused to the coiled-coil assembly domain of the cartilage oligomeric matrix protein via a semi-rigid cross-linking region, resulting in a pentameric multivalent binding molecule. In preferred embodiments of the invention, bispecific antibodies may be used, e.g., those generated by chemical linkage of an anti-ligand antibody (Ab) to an Ab directed to a specific target, the ligand and/or chimeric inhibitor being targeted to a tissue-or tumor-specific target. To avoid this limitation of chemical adapters, molecular adapters of antibodies can be used to generate recombinant bispecific single chain abs directed to ligands and/or chimeric inhibitors located at cell surface molecules. Alternatively, two or more active agents and/or inhibitors attached to a targeting moiety may be administered, wherein each conjugation comprises a targeting moiety, e.g., a different antibody. Each antibody may react with a different target site epitope (associated with the same or different target site antigens). The different antibodies with the agents attached accumulate in superposition at the desired target site. Antibody-based or non-antibody based targeting moieties can be employed to deliver the ligand or inhibitor to the target site. Preferably, natural binders for diseases that are not modulated or associated with antigens are used for this purpose.

Small molecules

All applications set forth in the above paragraphs are incorporated herein by reference. In some embodiments, one skilled in the art can use other agents as H3K9 methyltransferase inhibitors, e.g., antibodies, decoy antibodies, or RNAi, effective in the methods, compounds, and kits disclosed herein for increasing SCNT efficiency.

In some embodiments, H3K9 methyltransferase inhibitors useful in the methods, compositions, and kits disclosed herein are gliotoxin or related cyclic polythiodioxopiprazines (epithiodioxypiperazines), or BIX-01294 (diazepine-quinazolinamine derivatives), as disclosed in Takahashi et al, 2012, J. Antibiotics (Antibiotics)65, 263-265, or Shaabam et al, Chemistry and Biology, Vol.14, No. 3, 3 months 2007, p.242-244 (Chemistry & Biology, Volume 14, Issue 3, March 2007, Pages 242-244), both of which are incorporated herein by reference. BIX-01294 has the following chemical structure:

quinazoline, also known as UNC0638, also inhibits G9a and can be used in the methods and compositions disclosed herein. UNC0638 has the following structure:

small molecule inhibitors of SUV39h1 are disclosed in U.S. patent application US2015/0038496, which is incorporated by reference herein in its entirety. This small molecule, verticillin (verticillin) a, is identified as a selective inhibitor of SUV39h1 and SUV39h2 (i.e., inhibits SUV39h1/2), as disclosed in U.S. patent application US2014/0161785, which is incorporated herein by reference in its entirety, and can be used in the methods, compositions, and kits disclosed herein.

Other small molecule inhibitors of SUV39H1 include chaetocin (chemical name: (3S, 3 ' S, 5aR, 5aR, 10bR, 10 ' bR, 11aS, 11 ' aS) -2, 2 ', 3, 3 ', 5a, 5 ' a, 6, 6 ' -octahydro-3, 3 ' -bis (hydroxymethyl) -2, 2 ' -dimethyl- [10b, 10 ' b (11H, 11 ' H) -bi3, 11 a-cyclodithio-11 aH-pyrazino [1 ', 2 ': 1, 5] pyrrolo [2, 3-6] indol ] -1, 1 ', 4,4 ' -tetraone) (see Bernhard et al, FEBS letters, 2011, 585 (22); 3549-3554) having the following chemical structure that can be used in other methods and compositions disclosed herein.

Compound A-366 (also known as CHEMBL3109630) (PubChem CID: 76285486), has been found to be a potential inhibitor of EHMT2 (euchromatin histone methyltransferase 2), also known as G9a, with IC₅₀Is 3.3nM and is 1000-fold more selective than the 21 other methyltransferases (see, Sweis et al, Discovery and level of stored selective inhibitors of hormone methyl transferase, G9a. ACS mechanical ChemLetts, 2014; 5 (2); 205-209) and may be used in the methods and compositions disclosed herein. Small molecule A-366 has the following structure:

the 3-deazaadenosine analog A (DZNep) (CAS No: 102052-95-9) results in a decrease in SETDB1H3K9me3 HMTase and in a decrease in H3K27me3 levels and H3K9me3 levels (Lee et al, Biochem Biophys ResComm, 2013, 438(4), 647-652) and is useful in the methods and compositions disclosed herein. DZNP has the formula:

HMTase inhibitor IV, UNC0638 (available from Calbiochem) minimally inhibits SUV39h2 (IC)₅₀> 10 μ M) (see Vedadi, M., et al.2011.nat. chem. biol.7, 566; and Liu, f., et al.2011.j.med.chem.54, 6139) and can be used in the methods and compositions disclosed herein. The HMTase inhibitor IV is also known as: 2-cyclohexyl-N- (1-isopropylpiperidin-4-yl) -6-methoxy-7- (3- (pyrrolidin-1-yl) propoxy) quinazolin-4-amine, DNA methyltransferase inhibitor III, DNA MTase inhibitor III, EHMT1/GLP inhibitor II, EHMT2/G9a inhibitor IV, and having the following formula:

SCNT

it is a consistent object of the present invention to provide means to increase the efficiency of human SCNT and to produce human NT-ESCs from human SCNT embryos. The disclosed methods can be used to clone mammals, obtain totipotent or pluripotent cells, or reprogram human cells.

Recipient human oocyte

In certain embodiments, recipient human oocytes used in the methods, kits, and compositions of the invention may be from healthy human donors. In some embodiments, cryopreserved oocytes are used as recipient oocyte cells. In certain embodiments, the recipient oocyte is a human. Cryopreservation and thawing of oocytes is known to those skilled in the art (see, Tucker et al, Curr Opin Obstet Gynecol.1995 June, 7 (3): 188-92). In some embodiments, the human recipient oocyte is obtained from a volunteer human female donor, e.g., an egg cell donor for use in IVF clinics. In some embodiments, the oocyte is obtained from a human female subject who has been ovarian stimulated or ovarian hyperstimulation (i.e., ovulation promotion or superovulation promotion). Methods of superovulation are well known in the art, for example as disclosed in US 8,173,592 and international patent application WO2000/059542, which are incorporated herein by reference in their entirety.

In some embodiments, the recipient human oocyte is an enucleated oocyte. Enucleation of donor oocytes can be performed by known methods, as disclosed in U.S. Pat. No. 4,994,384, which is incorporated herein by reference. For example, metaphase II (MII) oocytes may be enucleated immediately, or placed in HECM containing optionally 7.5mg/ml cytochalasin B, or may be placed in a suitable medium such as CR1aa plus 10% estrus bovine serum and enucleated later. Enucleation may also be performed microscopically using a micropipette to remove the polar body and adjacent cytoplasm. The cells are then screened to identify those of them that are successfully enucleated. This screening can be performed by staining the cells in HECM with 1mg/mL 33342 Hoechst dye, followed by visualization of the cells under uv radiation for less than 10 seconds. Subsequently, the cells that have been successfully enucleated can be placed in an appropriate medium.

In some embodiments, a non-invasive approach to enucleation of oocytes may be used, for example, using ultraviolet radiation as a conventional process (Gurdon Q.J.Microsc. Soc.101299-311 (1960)) similar to the enucleation process of oocytes from amphibians. In some embodiments, oocyte enucleation of human oocytes can be performed using DNA-specific fluorophores, and murine oocytes are exposed to ultraviolet light for more than 30 seconds to reduce the developmental potential of the cells (Tsunoda ei al., j. reprod. fertil.82173 (1988)).

In some embodiments, an enucleated human oocyte has undergone "induced enucleation," which refers to enucleation of an oocyte by disrupting a meiotic spindle through destabilization (e.g., disaggregation) of the microtubules of the meiotic spindle (see, U.S. patent application No. US 2006/0015950, which is incorporated herein by reference in its entirety). The destabilization of the microtubules prevents the chromatids from being isolated (e.g., prevents successful mitosis) and induces uneven segregation (e.g., skewing) of the oocyte genome (e.g., chromatin) during meiotic maturation, whereby nearly all of the endogenous chromatin in the oocyte is concentrated within the second diode.

In some embodiments, the oocyte polyurethane is derived from a healthy female, e.g., a healthy human female oocyte donor. In some embodiments, the human oocytes used in the methods, compositions, and kits disclosed herein are excess oocytes obtained from a reproductive medicine clinic that are no longer needed for an IVF procedure. In some embodiments, the human oocytes used in the methods, compositions, and kits disclosed herein are of poor or sub-optimal quality, and because of their poor quality, they are unlikely to be successfully fertilized by sperm in vitro (e.g., the quality of a human oocyte may be very poor and an IVF procedure may not be successfully performed). In some embodiments, the human oocytes used in the methods, compositions, and kits disclosed herein are selected based on their quality, and in some embodiments, low quality oocytes are selected that are unlikely to be successfully fertilized by sperm in vitro (e.g., during IVF). In some embodiments, oocytes of intermediate quality that may be successfully fertilized in vitro (e.g., during IVF) by sperm are selected. In some embodiments, the human oocyte is donated by a postmenopausal human female, and a person who is not expected to be successful in vitro fertilization is selected and used in the methods, compositions, and kits disclosed herein.

In some embodiments, to circumvent the need for human oocyte donors, cross-species SCNTs have been disclosed in which non-human oocytes have been reported for nuclear reprogramming of human donor somatic Cells (Chung et al, Cloning and stem Cells 11, 1-11 (2009)). Accordingly, in some embodiments, the donor oocyte is from a non-human primate, or bovine oocyte, or any other non-human mammalian species, which may be an acceptor oocyte from the nucleus or nuclear genetic material of a human donor somatic cell.

In some embodiments, when a human is stimulated to produce oocytes (e.g., hormonal stimulated) and these oocytes are harvested, the oocytes collected may be at different stages. Some human oocytes are in Metaphase I (MI) and others are in metaphase II (MII). In these cases, human oocytes in Metaphase I (MI) may be cultured until they reach metaphase II, and then enucleated for use as recipient oocytes. Optionally, human oocytes that have been cultured to metaphase II are pooled with oocytes that have been in metaphase II when harvested for the pool of potential host cells. In other cases, only the harvested human oocytes in metaphase II are used for enucleation. Any of these human oocytes may be frozen for further use. Thus, the donor and/or recipient oocytes may be cryopreserved prior to use.

Accordingly, in some embodiments, the recipient human oocyte is obtained from a different subject or individual from which the donor human somatic cell was obtained. In some embodiments, the recipient human oocyte is obtained from the same subject into which a hNT-ESC derived hNT embryo was implanted. For example, patient-specific hNT-ESC can be obtained from a hSCNT embryo, wherein nuclear genetic material from the patient-donor human somatic cell is injected into a recipient human oocyte.

In some embodiments, the oocyte is obtained from a female subject that has not suffered from a mitochondrial disease. In some embodiments, the oocyte is obtained from a female subject suffering from a mitochondrial disease. Mitochondrial diseases that are secondary to mitochondrial dna (mtdna) defects are well known to those skilled in the art.

In one embodiment, the recipient human oocyte is from a subject that does not have a mitochondrial DNA mutation, such as a cytoplasmic or a heteromytotic mitochondrial disease. This can be determined, for example, by genetic testing, such as by evaluating mitochondrial DNA, or can be determined by clinical evaluation. The nuclear genetic material, such as a chromosome, can be isolated from a donor oocyte of a subject, such as a human subject, suffering from a mitochondrial DNA disease, such as a cytoplasmic or heteromytotic mitochondrial disease.

In some embodiments, the mitochondrial disorder is associated with infertility. Examples of mitochondrial diseases associated with infertility include Leber hereditary optic neuropathy, myoclonic epilepsy, or caenss-seoul syndrome (Kearns-sayresvydrome). Thus, in some examples, the recipient primate oocyte is from a subject not suffering from Leber's hereditary optic neuropathy, myoclonic epilepsy, or cahns-seire syndrome.

In other examples, the nuclear genetic material comprising the chromosome is from a primate donor human oocyte, wherein the primate subject has Leber's hereditary optic neuropathy, myoclonic epilepsy, neuropathy, ataxia and retinitis pigmentosa syndrome, maternal hereditary Leigh syndrome (MILS), myoclonic epilepsy with red tear fibers (MERRF), mitochondrial encephalo-muscular disease syndrome with lactic acidosis and cerebrovascular accident (MELAS), maternal hereditary diabetes with deafness, mitochondrial encephalomyopathy, chronic progressive extraocular paralysis, Pearson's myelopancreatic syndrome, diabetes insipidus, diabetes, optic nerve atrophy and deafness (DIDMOAD), chronic progressive extraocular paralysis, or Carnsyll-Seir syndrome. Thus, the recipient human oocyte was isolated from a subject who did not suffer from the following diseases: mitochondrial diseases such as Leber's hereditary optic neuropathy, myoclonic epilepsy, neuropathy, ataxia and retinitis pigmentosa syndrome, maternal hereditary Leigh syndrome (MILS), myoclonic epilepsy with red tear fibers (MERRF), mitochondrial encephalo-muscular disease with lactic acidosis and cerebrovascular accident (MELAS), maternal hereditary diabetes with deafness, mitochondrial encephalomyopathy, chronic progressive extraocular paralysis, Pearson's myelo-pancreatic syndrome, diabetes, optic nerve atrophy and deafness (DIDMOAD), chronic progressive extraocular paralysis and cahns-seire syndrome.

Leber Hereditary Optic Neuropathy (LHON) or Leber optic atrophy is mitochondrial hereditary (maternal inheritance to all offspring) degeneration of Retinal Ganglion Cells (RGCs) and their axons, which results in acute or subacute loss of central vision; this affects primarily young adult males. However, LHON is only transmitted through the mother, since it is mainly due to mutations in the mitochondrial (rather than nuclear) genome and only the egg cells contribute to the mitochondria of the embryo. LHON is usually due to one of three pathogenic mitochondrial DNA (mtDNA) point mutations. These mutations were located in the ND4, ND1 and ND6 subunit genes of complex I of the oxidative phosphorylation chain in mitochondria, respectively, the G mutation at nucleotide position 11778 to a, the G mutation at nucleotide position 3460 to a, and the T mutation at nucleotide position 14484 to C. Clinically, there is acute onset of vision loss, first of all in one eye, followed by vision loss in the other eye for weeks to months. Onset is generally early in adulthood, but onset in the age range of 8 to 60 is reported. This symptom typically progresses to very severe optic nerve atrophy and permanent decline in visual acuity.

Leigh's disease, also known as Subacute Necrotic Encephalomyelitis (SNEM), is a rare disorder of neurometabolism affecting the central nervous system. It is a genetic lesion that generally affects infants between the ages of three months and two, but in rare cases, also affects adolescents and adults. In the case of this disease, mutations in mitochondrial DNA (mtdna) or nuclear DNA (gene SURF and some COX assembly factors) cause degeneration of motor skills and ultimately death. The most well-known point of the disease is its deterioration in the ability of an individual to control exercise. With its rapid progress, the earliest signs can be extremely poor sucking ability and loss of head control and motor skills. Other symptoms include loss of appetite, vomiting, irritability, successive crying (infants), and cramps. Late signs may also be the onset of lactic acidosis, possibly leading to impaired respiratory and renal function. Some children may have a loss of developmental competence or a decline in development, and there are generally cases where they are unable to thrive. As the disease progresses in adults, it can also cause general weakness, renal failure, and heart problems. The life expectancy after onset of symptoms is often less than one year, but cases that survive only several days after an acute outbreak of disease and cases with survival periods over one year have been reported.

Neuropathy, ataxia, and retinitis pigmentosa (NARP) are conditions that cause a variety of signs and symptoms that primarily affect the nervous system. Starting from children or early adulthood, most people with NARP experience paralysis, tingling, or pain in the arms or legs (sensory neuropathy); muscle weakness; and problems with balance and coordination (ataxia). Many affected individuals also have vision loss due to changes in the light sensitive tissue (retina) located at the back of the eye. In some cases, this vision loss is the result of a condition known as retinitis pigmentosa. This eye disease causes progressive degeneration of the photoreceptor cells of the retina. Neuropathy, ataxia, and retinitis pigmentosa are associated with mutations in mitochondrial DNA, particularly the MT-ATP6 gene.

Muscular neuro-gastrointestinal encephalopathy or MNGIE is another mitochondrial disease that typically occurs between the ages of 10 and 50. MNGIE is a multisystem disease that causes ptosis, progressive external ophthalmoplegia, gastrointestinal dyskinesia (generally pseudo-obstruction), diffuse leukoencephalopathy, lean body mass, peripheral neuropathy, and myopathy.

In some embodiments, mitochondrial transfer can occur if the female subject has a defect in mitochondrial DNA (mtDNA) or a mutation in mtDNA, and thus, the cytoplasm with healthy mitochondrial and wild-type mtDNA can be introduced into an recipient oocyte via cytoplasmic transfer, also known as ovocytoplasmic transfer, to result in a heterocytoplasmic oocyte (see: Sterneckert et al, Nat Reviews Genetics, Genetics 15, 625-. Methods for cytoplasmic transfer are well known, as disclosed in U.S. patent application No. US 2004/0268422, which is incorporated herein by reference in its entirety. Subsequently, this heterogenous cytoplasmic oocyte may be enucleated and used as a recipient oocyte to receive injections of nuclear genetic material from the donor somatic cell. Accordingly, in some embodiments, the resulting SCNT embryos can be derived from 3 independent individuals; i.e., containing nuclear genetic material from the donor somatic cell, cytoplasm from the recipient oocyte, and wild-type or mutant mtDNA from a third individual or donor subject.

Donor human cell

The methods, kits, and compositions disclosed herein comprise a donor human cell from which a nucleus is harvested (harvested) and injected into an enucleated human oocyte to generate a human SCNT embryo. In some embodiments, the donor human cell is a terminally differentiated somatic cell. In some embodiments, the donor human cell is not an embryonic stem cell or an adult stem cell or an iPS cell. In some embodiments, the donor somatic cell is obtained from a male human subject, such as an XY subject. In alternative embodiments, the donor somatic cell is obtained from a female human subject, such as a XX subject. In some embodiments, the donor of human somatic cells is obtained from an XXY human subject.

Human donor somatic cells that may be used in the present invention include, for example, epithelial cells, neural cells, epidermal cells, keratinocytes, hematopoietic cells, melanogenic cells, chondrocytes, lymphocytes (B-lymphocytes and T-lymphocytes), other immune cells, erythrocytes, macrophages, melanogenic cells, monocytes, mononuclear cells, fibroblasts, cardiomyocytes, cumulus cells, and other muscle cells, and the like. In some embodiments, human somatic cells for nuclear transfer can be obtained from various organs such as skin, lung, pancreas, liver, stomach, intestine, heart, reproductive organs, bladder, kidney, urethra, and other urinary organs. These are just some examples of suitable human donor cells. Suitable donor cells, i.e., cells that can be used in a subject of the invention, can be obtained from any cell or organ of the body. This includes all somatic cells, and in some embodiments also includes germ cells such as primordial germ cells, sperm cells. In some embodiments, the human donor cell or a nucleus (i.e., nuclear genetic material) from the human donor cell is actively dividing, i.e., a non-quiescent cell, which has been reported to enhance cloning efficiency. Such donor somatic cells include those at the G1, G2, S or M cell phase. Alternatively, resting stage cells may be used. In some embodiments, such human donor cells will be in the G1 cell cycle. In certain embodiments, the human donor and/or acceptor cells of the present application do not undergo 2-cell blockade.

In some embodiments, the nuclear genetic material (i.e., the nucleus) of the human donor somatic cell is obtained from a cumulus cell, a Sertoli cell, or from an embryonic fibroblast or an adult fibroblast.

In some embodiments, the nuclear genetic material is genetically modified, e.g., to correct a gene mutation or abnormality or to introduce a genetic modification, e.g., to study the effect of the genetic modification in a disease model such as NT-ESC obtained from human SCNT embryos. In such embodiments, the NT-ESCs are patient-specific NT-ESCs that can be used to therapeutically clone and/or study a particular disease, wherein the patient suffers from or has a predisposition to develop the particular disease. In some embodiments, the nuclear genetic material of the human donor cell is genetically modified, for example, to introduce a desired characteristic into the donor somatic cell. Methods for genetically modifying somatic cells are well known to those skilled in the art and may be used in the methods and compositions disclosed herein.

In some embodiments, human donor somatic cells are selected according to the methods disclosed in U.S. patent application No. US 2004/0025193, which is incorporated herein by reference in its entirety, and discloses introducing a desired transgene into a human donor somatic cell and selecting a human somatic cell with the transgene prior to harvesting a nucleus for injection into an recipient oocyte.

In some embodiments, human donor cell nuclei (e.g., nuclear genetic material from the donor somatic cells) can be labeled. Cells can be genetically modified using a transgene encoding an early visualization protein such as green fluorescent protein (Y ang, M., et al, 2000, Proc. Natl. Acad. Sci. USA, 97: 1206-1211), or a derivative thereof, or using a transgene constructed from the firefly (Photinus pyralis) luciferase gene (fluee) (Sweeney, T.J., et al, 1999, Proc. Natl. Acad. Sci. USA, 96: 12044-12049), or using a transgene constructed from the Renilla reniformis luciferase (Rluc) (Bhaunik, S.and, Ghambror, S.S., 2002, Proc. Natl. Acad. Sci. USA, 99: 377-382).

One or more transgenes introduced into the nuclear genetic material of the donor somatic cell may be constitutively expressed using a "housekeeping gene" promoter, so that the transgene is expressed at high levels in many or all cells, or the transgene may be expressed using a tissue-specific and/or developmental stage-specific gene promoter, so that only specific cell lineages or cells that have been at a particular niche and developed to a specific tissue or cell type express and visualize the transgene (if the transgene is a reporter gene). Other agents that include transgenes or labels include, but are not limited to, fluorescently labeled macromolecules, including fluorescent protein analogs and biosensors; fluorescent macromolecular chimeras, including those formed using green fluorescent protein and mutants thereof; a fluorescently labeled primary or secondary antibody that reacts with cellular antigens involved in a physiological response; a fluorescent colorant; a dye; and other small molecules. Labeled cells from mosaic blastocysts can be sorted by, for example, flow cytometry to isolate the cloned population.

In some embodiments, the human donor somatic cells can be from a healthy human donor, such as a healthy human, or a donor with an existing medical condition (e.g., Parkinson's Disease (PD), ALS, alzheimer's disease, huntington's disease, Rheumatoid Arthritis (RA), age-related macular degeneration (AMD), diabetes, obesity, heart disease, cystic fibrosis, an autoimmune disease (e.g., MS, lupus), a neurodegenerative disease, any subject with a hereditary or acquired disease), or any subject in need of regenerative therapy and/or stem cell transfer to treat an existing, or pre-existing, or developing condition or disease. For example, in some embodiments, donor human somatic cells are obtained from a subject that will, in the future, serve as an acceptor for SCNT-derived human ES cells (NT-ESC), thereby allowing autologous transfer of patient-specific hES cells. Accordingly, in some embodiments, the methods and compositions allow for the production of patient-specific isogenic embryonic stem cell lines (i.e., isogenic hNT-ESC lines).

Accordingly, the methods, compositions, and kits disclosed herein enable the obtaining of patient-specific human stem cell lines by functionally enucleating a human oocyte line and fusing it with nuclear genetic material from somatic cells harvested from a human patient donor, thereby generating hscnts that can be used to generate patient-specific NT-ESCs. In some embodiments, methods of treatment by administering the patient-specific hNT-ESC to a patient are contemplated herein, wherein, in some embodiments, the patient is a donor of the human somatic cells whose nuclear genetic material is harvested for the SCNT process.

In some embodiments, human donor somatic cells or cell nuclei (i.e., nuclear genetic material) are treated according to the methods disclosed herein using an H3K9 methyltransferase inhibitor disclosed herein, such as any of human SUV39H1, human SUV39H2, or human SETDB 1. In certain embodiments, the donor human cell or nucleus is not pretreated prior to nuclear transfer, and the hybrid oocyte or hSCNT embryo is treated with an H3K9 methyltransferase inhibitor and/or KDM4 histone demethylase activator according to the methods disclosed herein. In certain embodiments, the donor cell or nucleus is not pretreated with spermine, protamine, or putrescine prior to nuclear transfer or collection of genetic material (or nucleus) for injection into an enucleated recipient oocyte.

Contacting the donor somatic cell, recipient human oocyte, hybrid oocyte or human SCNT with an agent that reduces methylation of H3K9me3

In some embodiments, human donor somatic cells are treated or contacted with an H3K9 methyltransferase inhibitor and/or a KDM4 histone demethylase activator. In some embodiments, the nucleus (or nuclear genetic material) of the donor human cell is treated with or contacted with an H3K9 methyltransferase inhibitor and/or a KDM4 histone demethylase activator. In some embodiments, the donor human cell cytoplasm and/or nucleus is treated with or contacted with an inhibitor of any one or a combination of H3K9 methyltransferase inhibitors disclosed herein, such as human SUV39H1, human SUV39H2, and/or human SETDB 1. In some embodiments, the contacting is performed by microinjecting the H3K9 methyltransferase inhibitor and/or KDM4 histone demethylase activator into the cytoplasm and/or nucleus of the donor human somatic cell.

In some embodiments, the donor somatic cell is contacted with an inhibitor of human SUV39h1 and/or human SUV39h2, or both (SUV39h1/2) for at least about 24 hours, or at least about 48 hours, or at least about 3 days, or at least about 4 days, or more than 4 days, prior to removal of the nucleus for transfer into an enucleated human donor oocyte. In some embodiments, the inhibitor of SUV39h1 and/or SUV39h2, or both (SUV39h1/2) is an siRNA and expression of SUV39h1 and/or SUV39h2, or both (SUV39h1/2) occurs for a period of at least 12 hours, or at least 24 hours or more, prior to removal of the nucleus for transfer into an enucleated human donor oocyte. In some embodiments, inhibition of SUV39h1 and/or SUV39h2, or both (SUV39h1/2) occurs, for example, for at least about 24 hours, or at least about 48 hours, or at least about 3 days, or at least about 4 days, or more than 4 days, in an enucleated human donor oocyte prior to removal of the nucleus for transfer into the donor oocyte. In some embodiments, the expression of SUV39h1 and/or SUV39h2, or both (SUV39h1/2) is inhibited by siRNA for a period of time prior to removal of the nucleus, and the inhibition occurs for at least 12 hours, or at least 24 hours or more.

In some embodiments, human oocytes are treated or contacted with an H3K9 methyltransferase inhibitor and/or a KDM4 histone demethylase activator. In some embodiments, the human oocyte is an enucleated oocyte treated or contacted with an H3K9 methyltransferase inhibitor and/or KDM4 histone demethylase activator, e.g., by direct injection into the cytoplasm of the enucleated oocyte. In some embodiments, the human oocyte or enucleated human oocyte is treated or contacted with KDM4 histone demethylase activating factor, for example, but not limited to, an agent that activates any one or combination of histone demethylase members of the KDM4 family, such as human KDM4A, human KDM4B, human KDM4C, human KDM4D, or human KDM 4E. In some embodiments, the enucleated oocyte is not injected or receives donor nuclear genetic material.

In an alternative embodiment, recipient human oocytes will be treated with H3K9 methyltransferase inhibitors and/or KDM4 histone demethylase activator within a time frame of about 40 hours prior to nuclear transfer (i.e., prior to the start of injection of the donor nuclear genetic material). The contacting may occur about 40 hours prior to nuclear transfer, or more preferably within a time frame of about 12 or 24 hours prior to nuclear transfer, and most preferably within a time frame of about 4 to 9 hours prior to nuclear transfer. In some embodiments, when an recipient human oocyte is crossed (i.e., contains nuclear genetic material from the donor somatic cell, but has not been activated), the recipient oocyte is contacted with an H3K9 methyltransferase inhibitor and/or KDM4 histone demethylase activator. The contacting may occur at about 40 hours after nuclear transfer, or more preferably at any time within a time frame of about 1 to 4 hours, or 4 to 12 hours, or within 24 hours after nuclear transfer, and most preferably within a time frame of about 1 to 4 hours or 4 to 9 hours after nuclear transfer but prior to fusion or activation.

Recipient human oocytes may be treated with H3K9 methyltransferase inhibitors and/or KDM4 histone demethylase activator before, during, or after nuclear transfer of nuclear genetic material obtained from human donor somatic cells. Typically, the recipient human oocyte is treated within 5 hours after nuclear transfer or within 5 hours after activation or fusion (e.g., 5 hpa; 5 hours after activation). In some embodiments, activation (or fusion) occurs within 1 to 2 hours or 2 to 4 hours after injection of genetic material from the donor somatic cell into an enucleated oocyte, and in this case, the SCNT embryo is contacted with an H3K9 methyltransferase inhibitor and/or KDM4 histone demethylase activator.

In some embodiments, human SCNT embryos are treated with H3K9 methyltransferase inhibitors and/or KDM4 histone demethylase activators. Human SCNT embryos were generated by: a nucleus (e.g., nuclear genetic material) from a donor somatic cell is injected into an enucleated recipient oocyte to form a "hybrid oocyte", which is activated (or fused) to produce an SCNT embryo. In some embodiments, the hybrid oocyte (e.g., an enucleated oocyte comprising the donor nuclear genetic material prior to activation) is treated with an H3K9 methyltransferase inhibitor and/or a KDM4 histone demethylase activator, as disclosed herein.

Upon activation (also known as fusion) of the donor nuclear genetic material with the cytoplasm of the recipient oocyte, an SCNT embryo is generated. In some embodiments, as disclosed herein, one or both of the cytoplasm or nucleus from a human donor cell and/or enucleated oocyte has been treated or contacted with an H3K9 methyltransferase inhibitor and/or KDM4 histone demethylase activator. In some embodiments, none of the donor cells and/or enucleated oocytes have been treated with an H3K9 methyltransferase inhibitor and/or KDM4 histone demethylase activator, since the hybrid oocytes have been treated and/or the hSCNT embryo has been treated.

In some embodiments, increasing human somatic cell nuclear transfer (hSCNT) efficiency comprises contacting a human SCNT embryo, such as a human SCNT ligand at least 5hpa, or between 10 and 12hpa (i.e., at the 1-cell stage), or at about 20hpa (i.e., the early 2-cell stage), or between 20 and 28hpa (i.e., the 2-cell stage), with at least one of (i) a histone demethylase of the KDM4 family and/or (ii) an H3K9 methyltransferase inhibitor. In some embodiments, exogenous expression of a KDM4 gene, such as KDM4A, occurs in SCNT embryos at any stage of between 5hpa, 10 and 12hpa (i.e., at the 1-cell stage), about 20hpa (i.e., the early 2-cell stage), or between 20 and 28hpa (i.e., the 2-cell stage). In some embodiments, each cell of the SCNT embryo (e.g., each cell of a 2-cell embryo or a 4-cell embryo) is injected with a KDM4A activator or over-expression agent if the SCNT embryo is contacted with an agent that inhibits H3K9me3, such as an agent that increases exogenous expression of a KDM4 gene, such as KDM4A (e.g., KDM4A mRNA or mod-RNA). In some embodiments, exogenous expression of a KDM4 gene, such as KDM4A, occurs in human SCNT embryos at any stage of 5hpa, 10 to 12hpa (i.e., at the 1-cell stage), about 20hpa (i.e., the early 2-cell stage), or 20 to 28hpa (i.e., the 2-cell stage), or later (e.g., at the 4-cell stage). In some embodiments, each cell of the SCNT embryo (e.g., each cell of a 2-cell embryo or a 4-cell embryo) is injected with a KDM4d activator or over-expression agent if the human SCNT embryo is contacted with an agent that inhibits H3K9me3, such as an agent that increases exogenous expression of a KDM4 gene, such as KDM4A (e.g., KDM4A mRNA or mod-RNA).

Method for transferring cell nucleus

It is an object of the present invention to provide means for cloning human somatic cells more efficiently. The disclosed methods and compositions are useful for therapeutic cloning of humans, e.g., for obtaining human Pluripotent Stem Cells (PSC) and human totipotent cells (TSC), and for reprogramming human somatic cells.

Nuclear transfer techniques or nuclear transfer techniques are known in the literature. See, inter alia, Campbell et al, Theriogenology, 43: 181 (1995); collas et al, mol. report dev., 38: 264-; keefer et al, biol. reprod., 50: 935-939 (1994); sims et al, proc.natl.acad.sci., USA, 90: 6143-6147 (1993); WO 94/26884; WO 94/24274; and WO 90/03432, which is incorporated herein by reference in its entirety. In addition, U.S. Pat. nos. 4,944,384 and 5,057,420 disclose nuclear transfer processes for cattle. See also, Cibeli et al, Science, Vol.280: 1256-1258(1998).

The common nucleus can be transferred into the recipient spermatoblast by microinjection means. In some embodiments, minimal cytoplasm is transferred with the nucleus. When microinjection is used, minimal transfer of cytoplasm can be achieved, as compared to by the cell infusion route. In one embodiment, the microinjection apparatus includes a piezoelectric unit. Typically, the piezoelectric unit is operatively attached to the needle to vibrate the needle. However, any configuration of the piezoelectric unit that can vibrate the needle is included within the scope of the present invention. In some cases, the piezoelectric unit may assist the needle in penetrating the target. In some embodiments, the piezoelectric unit may be used to transfer minimal cytoplasm with the nucleus. Any piezoelectric element suitable for use with this template may be used. In some embodiments, the piezoelectric element is a piezoelectric micromanipulator PMM150(PrimeTech, Japan).

In some embodiments, the method includes the step of fusing the donor nucleus with an enucleated oocyte. Fusion of cytoplasm to nucleus is carried out using a number of techniques known in the art, including Polyethylene Glycol (see Pontervo, Polyethylene Glycol (PEG) in the Production of Mammalian cellular hybrids, cytogene Cell Genet.16 (1-5): 399-400(1976)), direct injection of nuclei, Sendai virus mediated fusion (see U.S. Pat. No. 4,664,097 and Graham Wistar Inst. Symp. monogr.919 (1969)), or other techniques known in the art, such as electrofusion. Electrofusion of cells involves bringing cells into close proximity to each other and exposing them to an alternating electric field. Under appropriate conditions, the cells are pushed together and there is fusion of the cell membranes, followed by the formation of fused or hybrid cells. Electrofusion of cells and apparatus for performing electrofusion of cells are disclosed in, for example, U.S. patents US 4,441,972, US 4,578,168, and US5,283,194; international patent application PCT/AU92/00473[ published as WO 1993/05166 ], Pohl "Dielectrophoresis" (Dielectrophoreses, Cambridge university Press, 1978) and Zimmerman et al, Biochimica et Bioplodica acta 641: 160-165, 1981.

Methods of SCNT, as well as methods of activating (i.e., fusing) donor nuclear genetic material using the cytoplasm of an recipient oocyte, are disclosed in U.S. patent application No. US 2004/0148648, which is incorporated by reference herein in its entirety.

Oocyte collection

Oocyte donors may be synchronized and superovulated as previously disclosed (Gavin w.g., 1996) and mated at 48 hour intervals with vasectomized males. After harvest, oocytes were cultured in M199 equilibrated with 10% FBS supplemented with 2mM L-glutamine and 1% penicillin/streptomycin (10,000 i.u./ml each). Nuclear transfer may also use oocytes that may have been matured in vivo or in vitro. In vivo matured oocytes were obtained as described above, while in vitro matured oocytes were harvested for nuclear transfer after development to a particular cellular stage.

Cytoplasm preparation and enucleation

Typically, the cumulus cell-attached oocytes are discarded. Oocytes without cumulus were divided into two groups: mid-phase-II protocol (one polar body) and end-phase-II protocol (no clearly visible polar body or presence of partially extruded second polar body) of rest. First, oocytes in the resting metaphase-II protocol were enucleated. Oocytes assigned to the activated terminal-II protocol were prepared by culturing in M199/10% FBS for 2 to 4 hours. After this period, all activated oocytes (with partially extruded second polar bodies) were divided into a culture-inducing group, a calcium-activated end-II group of oocytes (end-II-Ca cleavage) and an enucleated group. During the culture process the unactivated oocytes were then incubated in M199/10% FBS containing 7% ethanol for 5 min, followed by another 3h incubation in M199 with 10% FBS to reach end-II (end-II-EtOH protocol). All oocytes were treated with cytochalasin-B for 15 to 30 minutes prior to enucleation. metaphase-II oocytes were enucleated by using a glass pipette to aspirate the first polar body and the adjacent cytoplasm (approximately 30% of cytoplasm) surrounding the polar body to remove the metaphase plate. end-II-Ca and end-II-EtOH oocytes were enucleated by removing the first polar body and surrounding cytoplasm containing a partially extruded second cluster (10 to 30% cytoplasm). Immediately after enucleation, all oocytes were reconstituted.

Nuclear transfer and reconstruction

Injection of the donor cells was performed in the same medium as used for enucleation of oocytes. One donor cell was placed between the zona pellucida and the egg membrane using a glass pipette. The cell-oocyte couplets are incubated in M199 for 30 to 60 minutes, followed by the electrofusion and activation process. The reconstituted oocytes were in fusion buffer (300mM mannitol, 0.05mM CaCl)₂、0.1mM MgSO₄、1mM K₂HPO₄0.1mM glutathione, 0.1mg/ml BSA) for 2 minutes. Electrofusion and activation were performed at room temperature in a chamber with 2 stainless steel electrodes molded into a "fusion slide" (500 μm gap; BTX-Genetronics, San Diego, Calif.) filled with a fusion medium.

Fusion (e.g., activation) is performed using a fusion slide. The fusion slide is placed in a fusion dish, and the fusion dish is flooded with a sufficient amount of fusion buffer to cover the electrodes of the fusion slide. The couplets were removed from the culture incubator and washed by fusion buffer. Using a stereomicroscope, the couplet was placed between and equidistant from the two electrodes, with the nuclear/cytoplasmic junction parallel to the electrodes. It should be noted that the voltage applied to the coupling body to promote activation and fusion may range from 1.0kV/cm to 10.0 kV/cm. But preferably the voltage range of the initial single simultaneous fusion and activation electrical pulse is 2.0 to 3.0kV/cm, most preferably 2.5kV/cm, preferably for a duration of at least 20 microseconds. This voltage was applied to the cell couplet using a BTX ECM 2001 electro-cell manipulator. The duration of the micropulses may vary from 10 microseconds to 80 microseconds. Following this procedure, the treated conjugate is typically transferred to a drop of fresh fusion buffer. Fusion-treated couplets were washed with equilibrated SOF/FBSThen transferred to equilibrated SOF/FBS with or without cytochalasin-B. If cytochalasin-B is used, its concentration may vary from 1 to 15. mu.g/ml, most preferably 5. mu.g/ml. The coupling body contains about 5% CO at 37-39 deg.C₂Hatching in the humid air cavity. It should be noted that mannitol can be used in place of cytochalasin-B (with Ca) in any of the protocols provided in this disclosure⁺²And BSA HEPES-buffered mannitol (0.3mm) line medium). The actual presence of the nuclear/cytoplasmic fusion is determined from 10 to 90 minutes after fusion, most preferably from 30 minutes after fusion, to determine the development of the transgenic embryo for subsequent transfer or for another nuclear transfer cycle.

After treatment with cycloheximide, a thorough wash was performed using an equilibrated SOF medium supplemented with at least 0.1%, preferably at least 0.7%, preferably 0.8% bovine serum albumin plus 100U/ml penicillin and 100. mu.g/ml streptomycin (SOF/BSA). The couplets were transferred to equilibrated SOF/BSA at 37 to 39 ℃ in the presence of about 6% O₂、5％CO₂And the rest is nitrogen gas, and the water is incubated in a moist modular incubation cavity for 24 to 48 hours. Nuclear transfer (1 cell at 24 to 48 hours, up to 8 cells) with appropriate developmental age was transferred into alternative synchronized recipients.

Cell nuclear transfer embryo culture and transfer into an acceptor

Culture of SCNT embryos

It has been suggested that embryos obtained by hSCNT may benefit from or even require in vivo culture conditions (at least in vivo) different from those used to culture embryos in general. In the routine multiplication of bovine embryos, the reconstructed embryo(s) have been cultured in ovine oviducts for 5 to 6 days (as disclosed by Willadsen in Mammalian egg transfer 185 CRC Press, Boca Raton, Fla. (1982)). In some embodiments, SCNT embryos can be embedded in a protective medium, such as agar, prior to transfer, and subsequently removed from the agar after recovery from the temporary recipient. The function of the protective agar or other medium is twofold: first, it acts as a structural aid for the SCNT embryos by containing the zona pellucida together; second, it acts as a barrier to the cells of the recipient immune system. Although this approach increases the proportion of embryos forming blastocysts, it has the disadvantage that a large number of embryos may be lost. In some embodiments, hSCNT embryos can be co-cultured on a monolayer of feeder cells, such as primary goat oviduct epithelial cells in 50 μ l drops. Embryo culture can be maintained in a humidified 39 ℃ incubator with 5% CO2 for 48 hours, after which hSCNT embryos are used to collect blastomeres representative of hNT-ESC production.

Applications of

Obtaining totipotent cells (TPC)

The SCNT experiment showed that nuclei from adult differentiated somatic cells can be reprogrammed to a totipotent state. Accordingly, hSCNT embryos generated using the methods disclosed herein can be cultured in a suitable in vitro culture medium to generate totipotent or embryonic stem cells, or stem cell-like cells and cell colonies. Suitable media for culturing and mutating embryos are well known in the art. Examples of known media that can be used for bovine embryo culture and maintenance include Ham F-10+ 10% Fetal Calf Serum (FCS), tissue culture medium-199 (TCM-199) + 10% fetal calf serum, Taiwan (Tyrode) albumin-lactate-pyruvate (TALP), Dulbecco's Phosphate Buffered Saline (PBS), Eagle's (Eagle) medium, and Whitten's medium. One medium most commonly used for oocyte collection and mutation is TCM-199, and 1 to 20% serum supplementation including fetal bovine serum, neonatal serum, estrus bovine serum, lamb serum, or bovine serum. Preferred maintenance media include TCM-199 with Earl salt, 10% fetal calf serum, 0.2Ma pyruvate and 50ug/ml gentamicin sulfate. Any of the above media may also be involved in the co-culture of various cell types such as granulosa cells, oviduct cells, BRL cells and uterine cells and STO cells.

In particular, human epithelial cells of the endometrium secrete Leukemia Inhibitory Factor (LIF) before and during embryo implantation. Thus, some embodiments comprise adding LIF to the culture medium to enhance the in vitro response of hSCNT-derived embryos. The use of LIF for the culture of embryonic or stem-like cells has been disclosed in US5,712,156, which is incorporated herein by reference.

Another maintenance medium is disclosed in US patent US5,096,822 to Rosenkrans, jr. This embryo culture medium, known as CR1, contains the nutrients necessary to support the embryo. CR1 contains L-lactic acid hemicalcium salt in an amount of 1.0mM to 10mM, preferably 1.0mM to 5.0 mM. The L-lactic acid hemicalcium salt is an L-lactate salt having a hemicalcium salt incorporated therein. In addition, media suitable for maintaining human embryonic stem cells in culture are described in Thomson et al, family (Science), 282: 1145-: 7844-7848 (1995).

In some embodiments, the feeder cells will comprise murine embryonic fibroblasts. Means for a generally suitable fibroblast feeder cell layer are disclosed below, for example, and within the skill of one of ordinary skill in the art.

Methods for obtaining human ES cells (e.g., human NT-ESCs or hNT-ESCs) from blastocyst-stage human SCNT embryos (or equivalents thereof) are well known in the art. Such techniques can be used to obtain human ES cells (e.g., hNT-ESC) from human SCNT embryos, wherein the levels of H3K9me3 in nuclear genetic material donated from human somatic donor cells are reduced by the hNT embryos used to generate the hNT-ESC compared to hNT that have not been treated with KDM4 demethylase family members and/or inhibitors of histone methyltransferases SUV39H1/SUV39H 2. In addition, or in the alternative, the hNT-ESC may be obtained from a cloned human SCNT embryo at an early developmental stage. In certain embodiments, blastomeres generated from human SCNT embryos generated using the methods, compositions, and kits disclosed herein can be aspirated using a glass pipette to obtain totipotent cells. In some embodiments, the pull-off may occur in the presence of 0.25% trypsin (Collas and Robl, 43 BIOL. REPROD.877-84, 1992; Stice and Robl, 39 BIOL. REPROD.657-664, 1988; Kanka et al, 43MOL. REPROD.DEV.135-44, 1996).

In some embodiments, blastocysts, or blastocyst-like clusters, obtained from hSCNT embryos can be used to obtain embryonic stem cell lines, such as nuclear transfer esc (ntesc) cell lines. Such cell lines can be, for example, in accordance with Thomson et al, family (Science), 282: 1145-: 7544-7848 (1995).

Pluripotent embryonic stem cells can also be generated from a single blastomere taken from an hSCNT embryo, interfering with the normal development of the embryo to birth. See US 60/624,827 filed on about day 11 of 2004, 60/662,489 filed on day 14 of 3 of 2005, 60/687,158 filed on day 3 of 6 of 2005, 60/723,066 filed on day 3 of 10 of 2005, 60/726,775 filed on day 14 of 10 of 2005, 11/267,555 filed on day 4 of 11 of 2005, PCT application PCT/US05/39776 filed on day 4 of 11 of 2005, the disclosures of which are incorporated herein by reference in their entireties; see also Chung et al, Nature, Oct.16, 2005 (electronic Pre-print publishing) and Chung et al, Nature V.439, pp.216-219(2006), the disclosures of each of which are incorporated herein by reference in their entirety. In this case, the hSCNT embryo was not destroyed for the generation of pluripotent stem cells.

Such human Pluripotent Stem Cells (PSCs) or Totipotent Stem Cells (TSCs) can be differentiated into any cell within the body, including, without limitation, skin cells, chondrocytes, skeletal cells, skeletal muscle cells, cardiac muscle cells, kidney cells, liver cells, blood cells and hematoblasts, vascular precursor cells and vascular epithelial cells, pancreatic β cells, neuronal cells, glial cells, retinal cells, inner ear follicular cells, intestinal cells, lung cells.

In another embodiment of the invention, hSCNT embryo or blastocyst, or pluripotent or totipotent cells obtained from hSCNT embryo (e.g., NT-ESC) can be exposed to one or more differentiation-inducing agents to obtain other therapeutically useful cells, such as retinal pigment epithelial cells, hematopoietic precursor cells and hemangioblastoma progenitor cells, and various other cell types of ectodermal, mesodermal and endodermal layers, such as cytokine, such as interleukin- α A, interferon- α A/D, interferon- α, interferon- γ -inducible protein-10, interleukin-1-17, keratinocyte growth factor, leptin, leukemia inhibitory factor, macrophage colony stimulating factor, macrophage immunoprotein-1 α, macrophage immunoprotein-1 α, macrophage immunoprotein-2, macrophage immunoprotein- α, macrophage immunoprotein-3 6865, monocyte protein 1-to 3,6, cytokine-derived from human fibroblast growth factor, macrophage growth factor-2, fibroblast growth factor-2, macrophage immunophilin-2, macrophage immunoprotein-derived from fibroblast growth factor receptor, fibroblast growth factor-2, fibroblast growth factor-receptor, fibroblast growth factor-induced receptor growth factor-induced by binding to human growth factor receptor, hormone receptor growth factor-receptor, such as VEGF-receptor factor-induced by human growth factor-induced growth factor, hormone receptor, hormone-induced cytokine-receptor, hormone-2, hormone-receptor growth factor-receptor, hormone-2, hormone-growth factor-stimulating factor-2, hormone-growth factor, hormone-stimulating factor, hormone-growth factor-2, hormone-stimulating factor, hormone-growth factor, hormone-growth factor-2, hormone-growth factor, hormone-stimulating factor-growth factor-hormone-growth factor-2, hormone-stimulating factor, hormone-growth factor, hormone-2, hormone-stimulating factor, hormone-growth factor, hormone-protein, hormone-2, hormone-hormone, hormone-.

Blastomere culture

In one embodiment, hSCNT embryos can be used to produce blastomeres and, in vitro techniques related to those currently used in pre-embryo transfer gene diagnosis (PGD), to isolate individual blastomeres from hSCNT embryos produced by the methods disclosed herein without destroying the hSCNT embryo or significantly altering its viability. As described herein, pluripotent human embryonic stem (hES) cells and cell lines can be generated from a single blastomere taken from an hSCNT embryo disclosed herein without interfering with the normal development of the embryo to birth.

Therapeutic cloning

The findings of Wilmut et al in cloning sheep "Duoli" (Wilmut, et al, Nature 385, 810(1997), together with the findings of Thomson et al in obtaining hESCs (Thomson et al, Science 282, 1145(1998)), has stimulated great enthusiasm for regenerative Cell transfer based on the construction of patient-specific hESCs derived from hSCNT embryos or hSCNT engineered Cell masses generated from the patient's own nuclei, a strategy to avoid immune rejection by autologous transfer, perhaps the strongest clinical rationale for hSCNT sets forth for the same reason complex disease-specific SCNT-hESC derivatives can accelerate the discovery of disease mechanisms, for Cell transfer, the innovative treatment of murine SCID and PD models with the individual mouse's own derived from SCmNTESC (Riout et al, Cell 109, 17 (2002); Bari, Nat. Biohnol.21, 2003 berec.), creating a pool of SCNT-derived stem cells with extensive histocompatibility would reduce the need for a continuous supply of new oocytes.

In certain embodiments of the invention, to exhibit therapeutic utility, pluripotent or totipotent cells (e.g., hNT-ESC) obtained from hSCNT embryos can optionally be differentiated and introduced into tissues within which the cells normally reside. For example, pluripotent or totipotent cells derived from a hSCNT ligand can be introduced into the tissue. In certain other embodiments, the pluripotent or totipotent cells obtained from the hSCNT ligand may be introduced systemically or at a distance from the site of intended therapeutic use. In some embodiments, the pluripotent or totipotent cells derived from the hSCNT ligand may function at a distance from or may be immediately adjacent to the desired site.

In certain embodiments of the invention, the cloned cells, pluripotent or totipotent cells derived from ligand of hSCNT, can be used to induce differentiation of other pluripotent stem cells, the generation of a population of cells derived from single cells that can be propagated in vitro while maintaining expression of embryonic pattern genes, can be used to induce differentiation of other pluripotent stem cells.

Pluripotent or totipotent cells (e.g., ntESC) derived from hSCNT ligands can be used to obtain any desired differentiated cell type. Therapeutic use of such differentiated human cells is unprecedented. For example, human hematopoietic stem cells may be used in medical treatments requiring bone marrow metastasis. Such procedures are used to treat a variety of diseases such as advanced cancers, e.g., ovarian cancer and leukemia, and diseases that compromise the immune system, e.g., AIDS. Hematopoietic stem cells can be obtained, for example, by: according to the methods disclosed herein, donor adult terminally differentiated somatic cells, such as epithelial lymphocytes, obtained from a human cancer or AIDS patient are fused with recipient enucleated human oocytes to obtain hSCNT embryos, which hSCNT ligands can then be used to obtain patient-specific pluripotent or totipotent or stem cell-like cells as disclosed above, and to culture such cells under conditions conducive to differentiation until hematopoietic stem cells are obtained. The hematopoietic cells are useful in the treatment of diseases including cancer and AIDS. As discussed herein, the human adult donor cell, or recipient human oocyte, hybrid oocyte, or hSCNT embryo may be treated with KDM4 histone demethylase activator and/or H3K9 methyltransferase inhibitor according to the methods disclosed herein.

Alternatively, the donor human cells can be adult somatic cells from a human patient suffering from a neurological disorder, and the generated hSCNT embryos can be used to produce patient-specific or disease-specific pluripotent or totipotent cells that can be cultured under differentiating conditions to produce a neural cell line. Such NT-ESCs can be used in therapeutic clones to treat neurological disorders, or in disease models of neurological disorders and neurodegenerative diseases. Such hNT-ESCs can be directed to differentiate along neuronal lineages by methods generally known to those skilled in the art. Specific diseases that can be treated by cell-based therapy and the transfer of such human nerve cells include, for example, parkinson's disease, alzheimer's disease, ALS, MS, cerebral palsy, and the like. In the specific case of Parkinson's Disease (PD), it has been demonstrated that the transferred fetal brain nerve cells make appropriate connections with peripheral cells and produce dopamine. This can lead to long-term reversal of the symptoms of parkinson's disease. Accordingly, in some embodiments, patient-specific NT-ESCs differentiated along the neural lineage can be used in methods of treating a PD patient, wherein the NT-ESCs are obtained from hSCNT embryos created by fusing nuclear genetic material from somatic cells of a subject afflicted with PD with enucleated human oocytes that have been treated with a KDM4 agonist or mRNA and/or an inhibitor of SUV39h1 and/or SUV39h 2.

In some embodiments, pluripotent or totipotent cells (e.g., NT-ESC) obtained from hSCNT embryos can be differentiated into cells having skin prenatal pattern gene expression that is highly elastin or capable of regeneration without scar formation. Skin fibroblasts of mammalian fetal skin, especially at sites corresponding to where the cortex benefits from a high level of elasticity, such as skin fibroblasts in the area surrounding the joints, are responsible for the complex architecture of the cephalic synthesis of elastic fibrils for many years without inversion. In addition, early embryonic skin can be regenerated without scar formation. Cells from this point in embryonic development of pluripotent or totipotent cells obtained from SCNT embryos can be used to promote scarless regeneration of skin, including the formation of normal elastin architecture. This is particularly useful in treating symptoms caused by normal aging or actinic skin damage in humans, where profound dissociation of the skin's elastic tissue may occur, resulting in an aged appearance, including skin sagging and wrinkles.

To allow for specific selection of differentiated cells after differentiation of the NT-ESC along different lineages, in some embodiments, donor human somatic cells can be transfected with a selectable marker expressed via an inducible promoter, thereby permitting selection or enrichment of a particular cell lineage when differentiation is induced. For example, CD34-neo can be used to select hematopoietic cells, Pw1-neo for muscle cells, Mash-1-neo for sympathetic neurons, Mal-neo for human CNS neurons of the grey matter of the outer cortex of the brain, and the like.

A significant advantage of the present invention is that by increasing the efficiency of hSCNT, it provides an essentially unlimited supply of isogenic or homologous human ES cells, particularly pluripotent ES cells that are not induced pluripotent stem cells (e.g., are not ipscs). Since such NT-ESCs are not partially pluripotent and do not have viral transgenes or forced expression of reprogramming factors that direct their reprogramming, they have advantages over ipscs and are suitable for metastasis.

In some embodiments, the hNT-ESC produced from hSCNT is a patient-specific pluripotent cell obtained from a hSCNT embryo, wherein the donor human cell is obtained from a subject to be treated with the pluripotent stem cell or differentiated progeny thereof. Thus, a significant problem associated with current transfer methods, i.e., rejection of transferred tissue that may occur as a result of host-versus-transfer or transfer-versus-host rejection, would be eliminated. Traditionally, rejection is prevented or reduced by administering anti-rejection drugs such as cyclosporine. However, such drugs have significant side effects such as immunosuppression and carcinogenicity and are very expensive. The present invention should eliminate or at least greatly reduce the need for antiretroviral drugs such as cyclosporine, imulan, FK-506, glucocorticoids, rapamycin, and derivatives thereof.

Other diseases and conditions that may be treated by syngeneic cell therapy include, by way of example, but are not limited to, spinal cord injury, Multiple Sclerosis (MS), muscular dystrophy, diabetes, liver diseases such as hypercholesterolemia, heart disease, cartilage replacement, diabetes, burns, foot ulcers, gastrointestinal diseases, vascular diseases, kidney diseases, urinary tract diseases, and age-related diseases, including age-related macular degeneration (AMD) and the like.

Use of human NT-ESCs such as human Pluripotent Stem Cells (PSCs) and human Totipotent Stem Cells (TSCs)

The methods and compositions disclosed herein for increasing the efficiency of hSCNT have many important uses that will advance the development of the field of stem cell research and developmental biology. For example, hSCNT embryos can be used to produce hES cells, hES cell lines, human Totipotent Stem (TS) cells and cell lines, and cells differentiated therefrom can be used to study fundamental developmental biology as well as specific diseases, and can be used therapeutically to treat a wide variety of diseases and disorders. In addition, these hNT-ESCs can be used in screening assays to identify factors and conditions that can be used to modulate the growth, differentiation, survival, or migration of these cells. The identified agents can be used to modulate cellular behavior in vitro and in vivo, and can form the basis of cell therapy or cell-free therapy.

The isolation of multipotent embryonic stem cells and the breakthrough of SCNTs in mammals have increased potential for implementation of human SCNTs to generate a potentially unlimited source of undifferentiated cells for use in research, and have potential applications in tissue repair and transfer medicine.

This concept, sometimes referred to as "therapeutic cloning", refers to the transfer of somatic cell nuclei into enucleated donor oocytes (Lanza, et al, Nature Med.5, 975 (1999)). Theoretically, by silencing all somatic genes and activating embryonic cells, the cytoplasm of the oocyte will repopulate the transferred nucleus. ES cells (i.e., ntESCs) were isolated from the Inner Cell Mass (ICM) of cloned embryos at the pre-implantation stage. When used in a therapeutic setting, these cells may carry the nuclear genome of the patient; therefore, it is proposed to transfer cells after inducing the differentiation of the cells to treat degenerative diseases such as diabetes, osteoarthritis, and parkinson's disease, etc. without causing immune rejection. Previous reports have revealed the generation of bovine ES-like cells (Cibeli et al, Nature Biotechnol.16, 642(1998)) and murine ES cells from ICMs of cloned blastocysts (Munsie et al, Curro Biol 10, 989 (2000); Kawase, et al, genetics 28, 156 (2000); Wakayama et al, Science 292, 740(2001)), and the development of cloned human embryos into 8-to 10-cell stages and blastocysts (Cibeli et al, Regen. Med.26, 25 (2001); Shu, et al, Fertil. Steril.78, S286 (2002)). Here, the invention can be used to generate patient-specific human ES cells from the SCNT engineered cell mass generated by the methods disclosed herein. Such SCNT-derived ES cells are referred to herein as "ntESCs" and may include patient-specific isogenic embryonic stem cell lines.

The present technology for the production of human hESC lines employs additional IVF clinical embryos and does not produce patient-specific ES cells. Patient-specific, immune-matched hescs are expected to be of great biomedical importance in the study of disease and development, and to facilitate the development of therapeutic stem cell transfer methods. Accordingly, the present invention can be used to construct hESC lines from hscnts produced from human donor skin cells, human donor cumulus cells, or other human donor somatic cells from an informed donor whose nucleus has been inserted into a donated enucleated oocyte. These hESC lines derived from hSCNT will grow on animal protein free media.

The major histocompatibility complex identity of each SCNT-derived hESC (i.e., hNT-ESC) can be compared to the patient himself to show immune compatibility, which is important for the eventual metastasis. By generating these SCNT-derived hescs (i.e., hNT-ESCs), an assessment of genetic and epigenetic stability will be made.

Various human injuries and diseases result in defects in single cell types. If defective cells can be replaced by suitable stem cells, progenitor cells, or cells differentiated in vitro, and if immune rejection of the transferred cells can be avoided, it is clinically possible to treat diseases and injuries at the cellular level (Thomson et al, Science 282, 1145 (1998)). The possibility of immune rejection can be avoided if these cells are used for human therapy by generating hescs from human SCNT embryos or SCNT engineered cell masses, where the nuclei of the somatic cells are from individual patients, i.e. in the case where the nuclear (but not mitochondrial dna (mtdna)) genome is identical to the donor cells (janisch, n.engl.med.351, 2787 (2004); drakker, benveninsty, Trends biotechnol.22, 136 (2004)). In recent years, the murine model of Severe Combined Immunodeficiency (SCID) and Parkinson's Disease (PD) (Barberi et al, nat. biotechnol.21, 1200(2003)), a process also known as "therapeutic cloning", has been successfully treated by transfer of autologous differentiated murine embryonic stem cells (mESCs) derived from NT blastocysts.

Hescs can be generated from human SCNT embryos or SCNT engineered cell masses generated using the methods disclosed herein and their expression of hESC pluripotency markers including Alkaline Phosphatase (AP), stage-specific embryo antigen 4(SSEA-4), SSEA-3, tumor rejection antigen I-81(Tra-I-81), Tra-I-60, and octamer-4 (Oct-4) can be assessed. DNA fingerprinting using human short tandem repeat probes can also be used to show with high certainty that each of the NT-hESC lines originated from a respective donor of human somatic cells and that these lines were not the result of enucleation failure and subsequent parthenogenetic activation. Stem cells self-renew by themselves and from all three embryonic germ layers: the ability of ectoderm, mesoderm and endoderm to differentiate into somatic cells is defined. Differentiation will be analyzed in terms of teratoma formation and Embryoid Body (EB) formation by injecting IM into a suitable animal model.

In summary, the methods of the invention for increasing the efficiency of hSCNT provide an alternative to current methods for obtaining ES cells. However, unlike the currently used approaches, hSCNT can be used to generate ES cell lines that are histocompatible with donor tissue. As such, hSCNT embryos produced by the methods disclosed herein may provide an opportunity to develop cell therapies in the future that are histocompatible with patients in need of such treatment.

In some embodiments, the methods, systems, kits, and devices disclosed herein can be implemented by a service provider, for example, when a researcher may request that the service provider provide hSCNT embryos, or pluripotent stem cells derived from the hSCNT embryos, or totipotent stem cells that have been generated in a laboratory operated by the service provider using the methods disclosed herein. In this embodiment, after obtaining donor human somatic cells, a service provider can perform the methods disclosed herein to generate hSCNT embryos or blastocysts derived from hSCNT embryos, or to generate hNT-ESCs from such hSCNT embryos, and then the service provider can provide the hSCNT embryos or blastocysts derived from the SCNT embryos or hNT-ESCs derived from the hSCNT embryos to researchers. In some embodiments, the researcher may send the donor human somatic cell sample to a service provider via mail, courier, or the like, by any means, or the service provider may provide a service to collect the donor human somatic cell sample from the researcher's hand and transport it to the service provider's laboratory. In some embodiments, the researcher may deposit a sample of donor human somatic cells to be used in the hSCNT method at the service provider laboratory site. In an alternative embodiment, the service provider provides a visit service, at which time the service provider dispatches personnel to the researcher's laboratory, and also provides the researcher laboratory with kits, equipment, and reagents for performing the hSCNT method, as well as the inventive system disclosed herein of the researcher's desired/preferred donor human somatic cells (e.g., patient-specific somatic cells). This service can be used for therapeutic cloning, e.g., for obtaining hNT-ESC and/or pluripotent stem cells from blastocysts from hSCNT embryos, e.g., for obtaining patient-specific pluripotent stem cells for transfer into a subject in need of regenerative cell or tissue treatment.

Also provided herein are therapeutic compositions consisting of transferable cells that have been obtained (produced) from NT-ESC and are configured in a form suitable for administration to a human. In one embodiment, the transferred recipient is a human donor from which the donor somatic cells are derived. In some embodiments, the therapeutic composition comprises pluripotent cells, lineage-specific stem cells, and partially or fully differentiated cells derived from the hNT-ESC provided herein.

The hNT-ESC cell preparation derived from hSCNT enables a method of providing cells to a subject by administering an effective amount of one or more preparations that can transfer cells to the subject in need thereof. The cell will be matched to one or more loci of the Major Histocompatibility Complex (MHC). In one embodiment, there is a perfect match at each MHC locus. In one embodiment, the hNT-ESC cell derived from hSCNT is made by translating a nucleus from a somatic cell of an individual of interest into an enucleated host cell (e.g., oocyte) from a second individual. Subsequently, hNT-ESC cells derived from hSCNT can be cultured as described above to generate pluripotent and multipotent stem cells (MPSC). Subsequently, a therapeutically effective amount of the pluripotent cells may be used in a subject of interest. In one embodiment, cells matched to the subject's MHC genome or genomes are generated using the teachings provided herein, such as by SCNT. In a preferred embodiment, the cells are cultured in a serum-free medium. In another preferred embodiment, the cells are not cultured using allogeneic cells (e.g., non-human fibroblasts, such as murine embryonic fibroblasts).

Methods of treating a disease are provided, the methods comprising transferring hNT-ESC cells derived from hSCNT into a human afflicted with a disease characterized by somatic damage or degeneration. Such cells may be pluripotent cells or any other type of transferable cells.

hNT-ESCs derived from hSCNT disclosed herein can be used to generate cells of a desired cell type. In some embodiments, hNT-ESCs derived from hSCNT are used to obtain mesenchymal cells, neural cells, and/or hematopoietic stem cells. In other embodiments, hNT-ESC derived from hSCNT is used to generate cells, including, but not limited to, pancreatic cells, liver cells, skeletal cells, epithelial cells, endothelial cells, tendon cells, chondrocytes and muscle cells, and progenitors thereof. Thus, transferable hNT-ESC cells derived from hSCNT can be administered to a subject in need of one or more cell types to treat a disease, disorder, or condition. Examples of diseases, disorders, or conditions that can be treated or prevented include neurological, endocrine, structural, skeletal, vascular, urinary, digestive, integumentary, hematologic, immunological, autoimmune, inflammatory, renal, bladder, cardiovascular, cancer, circulatory, hematopoietic, metabolic, reproductive, and muscle diseases, disorders, and conditions. In some embodiments, hematopoietic stem cells derived from hNT-ESC are used to treat cancer, wherein the hNT-ESC is derived from hSCNT. In some embodiments, the cells are used in reconstructive applications, such as for repairing or replacing a tissue or organ.

The hNT-ESC derived from the hSCNT disclosed herein can be used to generate pluripotent stem cells or transferable cells. In one example, the transferable cell is a mesenchymal stem cell. Mesenchymal stem cells result in a very large number of distinct tissues (Caplan, J.Orth. Res 641-650, 1991). Mesenchymal stem cells that differentiate into bone, muscle, tendon, adipose tissue, basal cells and cartilage have also been isolated from bone marrow (Caplan, j. orth. res.641-650, 1991). U.S. Pat. No. 5,226,914 discloses an exemplary method for isolating mesenchymal stem cells from bone marrow. In other examples, epithelial progenitor cells or keratinocytes may be produced for the treatment of skin disorders and disorders of the inner wall of the intestinal tract (Rheinwald, meth. cell Bio.21A: 229, 1980). The cells can also be used to generate liver precursor cells (see PCT publication WO 94/08598) or kidney precursor cells (see Karp et al, Dev. biol.91: 5286-5290, 1994). The cells can also be used to generate inner ear precursor cells (see, Li et al, TRENDS mol. med.10: 309, 2004).

The transferable cell derived from hNT-ESC and from hSCNT can also be a neuronal cell. The volume of cell suspension, e.g., neuronal cell suspension, administered to a subject will vary depending on the site of implantation, the therapeutic target, and the amount of cells in the solution. Typically, the cells administered to the subject will be therapeutically effective amounts of the cells. For example, in the treatment of parkinson's disease, transferring a therapeutically effective amount of cells will typically result in a reduction in the amount and/or severity of symptoms associated with the pathology such as rigidity, loss of mobility and gait pathology. In one example, severe parkinson's disease patients require at least about 100,000 dopamine cells to survive per metastatic site to have substantial beneficial effects from metastasis. Typically, due to the low survival rate of cells transferred from brain tissue (5% to 10%), at least 100 ten thousand cells are administered, e.g., about 100 to about 400 ten thousand dopaminergic neurons are transferred. In one embodiment, the cells are administered to the brain of a subject. The cells may be implanted in the cerebrospinal fluid-containing space within the brain parenchyma, such as the subarachnoid or intracerebroventricular space, or may be implanted in the external nervous system. Thus, in one example, the cells are transferred to a site in the subject that is not within the central or peripheral nervous system, such as the celiac ganglion or sciatic nerve. In another embodiment, the cells are transferred into the central nervous system including all structures within the dura mater. Typically, injection of neuronal cells can be accomplished using a sterile syringe with an 18 to 21 gauge needle. Although the exact dimensions of the needle will depend on the species to be treated, the diameter of the needle should not be greater than 1mm for any species. Those skilled in the art are familiar with techniques for administering cells to the brain of a subject.

Typically, a therapeutically effective amount of hNT-ESC derived from hSCNT is administered to a subject. The cells can be administered in a pharmaceutical carrier. The pharmaceutically acceptable carrier used is a conventional carrier. For example, Remington's pharmaceutical Sciences, by E.W. Martin, Mack Publishing Co., Easton, Pa., 15th edition (1975)) discloses compositions and dosage forms suitable for pharmaceutical delivery of the cells disclosed herein. In general, the nature of the carrier will depend on the particular mode of administration employed. For example, parenteral dosage forms typically contain injectable fluids including pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, and the like as vehicles. For solid compositions (e.g., powders, pills, tablets, or capsules), conventional non-toxic solid carriers can be included, for example, pharmaceutical grades of mannose, lactose, starch, or magnesium stearate. The pharmaceutical compositions to be administered contain, in addition to the biologically neutral carrier, minor amounts of non-toxic adjuvants such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example, sodium acetate or sorbitan monolaurate.

The individual may be any subject of interest. Suitable subjects include those that would benefit from the proliferation of cells derived from stem cells or precursor cells. In one embodiment, the subject is in need of proliferating neuronal precursor cells and/or glial precursor cells. For example, the subject may have a neurodegenerative disorder or have experienced an ischemic event such as a stroke. Specific, non-limiting examples of neurodegenerative disorders are: alzheimer's disease, pantothenate kinase-associated neurodegenerative diseases, Parkinson's disease, Huntington's disease (Dexter et al, Brain 114: 1953-1975, 1991), HIV cerebral stems (Miszkziel et al, Magnetic Res. Imag.15: 1113-1119, 1997), and amyotrophic lateral sclerosis. Suitable individuals also include those aged subjects, such as subjects at least about 65 years of age, at least about 70 years of age, at least about 75 years of age, at least about 80 years of age, or at least about 85 years of age. In other examples, the subject may have spinal cord injury, Batten's disease, or spina bifida. In further examples, the individual may have hearing loss, such as a deaf subject or a subject who may need to proliferate stem cells from the inner ear to prevent hearing loss.

In some embodiments, the hNT-ESC derived from hSCNT produced using the methods disclosed herein can contribute to a germ cell line. Thus, somatic cells from a subject of interest can be used to generate ES cells that can later differentiate into oocytes or sperm. Subsequently, the oocytes or sperm are used for fertilization to allow a subject who is infertile to give birth to a child genetically related to the subject. This method can be used for female subjects with mitochondrial disorders where the female with the disorder is the source of the human donor somatic cells in the method, thereby making it possible to produce NT-ESCs from hscnts, which can differentiate into oocytes that can be used to give birth to children by females that do not have mtDNA defects. In addition, ES cell-derived egg cells can be used in research. For example, these egg cells can then be used to make ES cells derived from human SCNT. The availability of these oocytes may reduce the use of donated human oocytes in research.

hNT-ESCs derived from hSCNT can also be used to generate additional embryonic cells, such as trophectoderm for use in cell culture. In one embodiment, the use of autologous cells (e.g., trophectoderm) as feeder cells may aid in the generation of stem cells that in turn have the ability to differentiate into differentiated organ-specific cells. In other embodiments, autologous feeder cells obtained by culturing totipotent stem cells in a manner that produces this feeder layer component can be used to avoid adventitious contamination and thus make it easier for the FDA to approve differentiated cells cultured thereon for therapeutic purposes.

Cells produced by the methods disclosed herein, such as hNT-ESC derived from hSCNT, are also used to test an agent of interest, such as to determine whether an agent affects differentiation or cell proliferation. For example, hNT-ESC derived from hSCNT is contacted with the agent and the ability of the cell to differentiate or proliferate is assessed in the presence and absence of the agent. Thus, hNT-ESC derived from hSCNT produced by the methods disclosed herein can also be used to screen pharmaceutical agents to select agents that affect specific human cell types, such as agents that affect neuronal cells. hNT-ESCs derived from hSCNT produced by the methods disclosed herein can also be used as screening agents to select for those agents that affect differentiation. The test compound can be any compound of interest, including a chemical compound, a small molecule, a polypeptide, or other biological agent (e.g., an antibody or cytokine). In several examples, a panel of potential agents is screened, such as a panel of cytokines or growth factors.

Methods for preparing combinatorial libraries of molecules that can be tested for a desired activity are known in the art and include, for example, methods for making phage display libraries of peptides, wherein the peptides can be constrained peptides (see, e.g., US5,622,699; US5,206,347; Scott and Smith, Science 249: 386-; a peptide library (US 5,264,563); the peptidomimetic library (Blondelle et al, Trends Anal Chem.14: 83-92, 1995); nucleic acid libraries (O' Connell et al, Proc. Natl Acad. set., USA 93: 5883-; oligosaccharide library (Y ork et al, Carb. Res.285: 99-128, 1996; Liang et al, Science 274: 1520. Biol.1522, 1996; Ding et al, adv. Expt. Med. biol.376: 261. 269, 1995); lipoprotein pool (de Kruif et al, FEBSLett.399: 232-; a glycoprotein or glycolipid library (Karaoglu et al, j.cell biol.130.567-577, 1995); or a chemical library containing, for example, drugs or other pharmaceutical agents (Gordonet et al, J.Med.chem.37.1385-1401, 1994; Ecker and crook, Biotechnology 13: 351-360, 1995). Polynucleotides are particularly useful as agents capable of altering the function of pluripotent or totipotent cells, because nucleic acid molecules with binding specificity for cellular targets, including cellular polypeptides, are naturally occurring, and because synthetic molecules with this specificity can be readily prepared and identified (see, e.g., US5,750,342).

In one embodiment, for high throughput formats, the hNT-ESC or MPSC derived from hSCNT produced by the methods disclosed herein can be introduced into a well of a multi-well plate or into a slide or microchip and contacted with the reagents being tested. Usually, the cells are organized as an array, in particular a readable array, so that robotics can be conveniently used for manipulating the cells and solutions and for monitoring the cells, in particular with respect to the function to be examined. One advantage of using high throughput is that a large number of test agents can be examined in parallel, and if desired, control reactions can also be performed under conditions that are exactly the same as the test conditions. As such, the methods disclosed herein provide a means of screening one, several, or a large number of test agents to identify agents that can alter the function of hNT-ESC derived from hSCNT, e.g., agents that induce the differentiation of hNT-ESC to a desired cell type, or agents that prevent spontaneous differentiation, e.g., by maintaining high levels of expression of regulatory molecules.

Contacting the hNT-ESC with a test compound to a sufficient extent to allow the compound to interact with the cell. When a compound binds to a discrete receptor, the cell is contacted for a time sufficient for the agent to bind to its receptor. In some embodiments, the cells are incubated with the test compound for an amount of time sufficient to affect phosphorylation of the substrate. In some embodiments, the hNT-ESCs were treated in vitro with the test compounds at 37 ℃ in a humidified atmosphere containing 5% CO 2. After treatment with test compounds, with Ca-free²⁺And Mg²⁺The cells were washed with PBS and total protein was extracted as disclosed (Haldar et al, CellDeath Diff 1: 109-115, 1994; Haldar et al, Nature 342: 195-198, 1989; Haldar et al, Cancer Res.54: 2095-2097, 1994). In other embodiments, serial dilutions of the test compounds are used.

Compositions and kits

Another aspect of the invention relates to populations of hNT-ESC obtained from SCNT produced by the methods disclosed herein. In some embodiments, the hNT-ESC is a human ntESC, e.g., a patient-specific hNT-ESC, and/or a patient-specific isogenic hNT-ESC. In some embodiments, the hNT-ESCs are present in a culture medium, such as a medium that maintains the hNT-ESCs in a pluripotent or multipotent state. In some embodiments, the medium is a medium suitable for cryopreservation. In some embodiments, hNT-ESC bodies are cryopreserved. Cryopreservation can be used, for example, to store the hNT-ESC for future use, such as for therapeutic use or other uses, such as research use. The hNT-ESCs can be amplified, and a portion of the amplified hNT-ESCs can be used, while another portion can be cryopreserved. The ability to expand and preserve hNT-ESCs allows considerable flexibility, for example, in the production of a variety of patient-specific human hNT-ESCs and in the selection of donor somatic cells for SCNT procedures. For example, cells from a histocompatibility donor may be expanded and used for more than one recipient. Cryopreservation of hNT-ESCs can be provided by an organization bank. The hNT-ESCs can be cryopreserved along with histocompatibility data. The hNT-ESC produced using the methods disclosed herein can be cryopreserved according to conventional procedures. For example, cryopreservation of about 100 to 1000 ten thousand cells may be performed in a "freezing" medium, which may include appropriate proliferation medium, 10% BSA and 7.5% dimethyl sulfoxide. The hNT-ESC was centrifuged. The growth medium was withdrawn and replaced with frozen medium. The hNT-ESC was resuspended as spheres. Cells are slowly frozen, for example, by being placed in a container at-80 ℃. Frozen hNT-ESC were thawed by vortexing in the 37 ℃ domain, resuspended in fresh stem cell culture medium, and allowed to grow as disclosed above.

In some embodiments, the hNT-ESC is produced from an SCNT embryo produced by injecting nuclear genetic material from a donor somatic cell into the cytoplasm of a recipient oocyte, wherein the recipient oocyte comprises mtDNA from a third donor subject.

The invention also relates to hSCNT embryos produced by the methods disclosed herein. In some embodiments, the hSCNT embryo is a human embryo. In some embodiments, the human SCNT embryo is genetically modified, e.g., at least one transgene in the genetic material of the donor nucleus is modified (e.g., introduced or deleted or altered) prior to the SCNT process (i.e., prior to harvesting the donor nucleus and fusing with the cytoplasm of the recipient oocyte). In some embodiments, the hSCNT embryo comprises nuclear DNA from the human donor somatic cell, cytoplasm from the human recipient oocyte, and mtDNA from a third human donor subject.

Another aspect of the invention relates to a composition comprising: at least one of a human SCNT embryo or blastocyst thereof, or an acceptor human oocyte (nucleated or enucleated); and at least one of (i) an agent that increases the expression or activity of a histone demethylase of the KDM4 family, or (ii) an agent that inhibits H3K9 methyltransferase.

In another embodiment, the invention provides a kit for practicing the methods of the invention. Another aspect of the invention relates to a kit comprising one or more containers comprising (i) an agent that increases the expression or activity of a histone demethylase of the KDM4 family and/or an agent that inhibits H3K9 methyltransferase, and (ii) a human oocyte. The kit may optionally comprise a medium for the recipient oocyte and/or SCNT embryo, and one or more reagents for activating (e.g., fusing) the donor nuclear genetic material using the cytoplasm of the recipient oocyte. In some embodiments, the human oocyte is an enucleated oocyte. In some embodiments, the human oocyte is not enucleated. In some embodiments, the human oocyte is frozen and/or is present in a cryopreservation freezing medium. In some embodiments, the human oocyte is obtained from a donor female subject suffering from a mitochondrial disease or having an mtDNA mutation or abnormality. In some embodiments, the oocyte is obtained from a donor female subject that does not suffer from a mitochondrial disease or does not have an mtDNA mutation. In some embodiments, the oocyte comprises mtDNA from a third subject.

The kit may also optionally include suitable systems (e.g., an opaque container) or stabilizers (e.g., antioxidants) to prevent degradation of the agent that increases the expression or activity of a histone demethylase of the KDM4 family and/or the agent that inhibits H3K9 methyltransferase under light or other adverse conditions.

The kit can optionally include instructional materials comprising directions (i.e., protocols) for performing a hSCNT process (e.g., enucleating an oocyte, and/or injecting nuclear genetic material of the donor somatic cell into a recipient oocyte, and/or fusing/activating, and/or culturing a hSCNT embryo), and instructions for contacting at least one of the donor somatic cell and/or the recipient oocyte, and/or the hSCNT embryo with an agent that increases the expression or activity of a histone demethylase of the KDM4 family and/or an agent that inhibits H3K9 methyltransferase.

The following detailed description is presented for a more complete understanding of the invention disclosed herein.

The invention may be defined as any of the following numbered paragraphs:

1. a method of increasing the efficiency of human somatic cell nuclear transfer (hSCNT), comprising: contacting a hybrid oocyte with an agent that increases the expression of a member of the histone demethylase KDM4 family, wherein the hybrid oocyte is an enucleated human oocyte comprising human somatic genetic material.

2. The method of paragraph 1, wherein the contacting occurs after activation or fusion of the hybrid oocyte, but before activation (ZGA) of the genome of the human fertilized egg begins.

3. A method of increasing the efficiency of human Somatic Cell Nuclear Transfer (SCNT), the method comprising at least one of:

(i) contacting a donor human somatic cell or a recipient human oocyte with at least one agent that reduces methylation of H3K9me3 in the donor human somatic cell or the recipient human oocyte, wherein the recipient human oocyte is a nucleated or enucleated oocyte; if the recipient human oocyte is nucleated, enucleating the human oocyte; transferring a nucleus from the donor human somatic cell to the enucleated oocyte to form a hybrid oocyte; and, activating the hybrid oocyte to form a human SCNT embryo; or

(ii) Contacting a hybrid oocyte with at least one agent that reduces methylation of H3K9me3 in the hybrid oocyte, wherein the hybrid oocyte is an enucleated human oocyte comprising human somatic cell genetic material; and, activating the hybrid oocyte to form a human SCNT embryo; or

(iii) Contacting the activated human SCNT embryo generated from the fusion of an enucleated human oocyte with human somatic genetic material with at least one agent that reduces H3K9me3 methylation in a human SCNT embryo;

wherein decreasing H3K9me3 methylation in any one of the donor human somatic cell, recipient human oocyte, hybrid oocyte or the human SCNT embryo increases the efficiency of the SCNT.

4. A method of producing a human nuclear transferred embryonic stem cell (hNT-ESC), the method comprising:

a. at least one of the following: (i) contacting a donor human somatic cell or a recipient human oocyte with at least one agent that reduces methylation of H3K9me3 in the donor human somatic cell or the recipient human oocyte, wherein the recipient human oocyte is a nucleated or enucleated oocyte; if the recipient human oocyte is nucleated, enucleating the human oocyte; transferring a nucleus from the donor human somatic cell to the enucleated oocyte to form a hybrid oocyte; and, activating the hybrid oocyte to form a human SCNT embryo; or

(ii) Contacting a hybrid oocyte with at least one agent that reduces methylation of H3K9me3 in the hybrid oocyte, wherein the hybrid oocyte is an enucleated human oocyte comprising human somatic cell genetic material; and, activating the hybrid oocyte to form a human SCNT embryo; or

(iii) Contacting the activated human SCNT embryo generated from the fusion of an enucleated human oocyte with human somatic genetic material with at least one agent that reduces H3K9me3 methylation in a human SCNT embryo;

b. incubating the SCNT embryo for a sufficient time to form a blastocyst; collecting at least one blastomere from the blastocyst; and culturing the at least one blastomere to form at least one human NT-ESC.

5. A method of producing a human Somatic Cell Nuclear Transfer (SCNT) embryo, comprising:

contacting at least one of a donor human somatic cell, a recipient human oocyte, or a human Somatic Cell Nuclear Transfer (SCNT) embryo with at least one agent that reduces methylation of H3K9me3 in the donor human somatic cell, the recipient human oocyte, or the human SCNT embryo, wherein the recipient human oocyte is a nucleated or enucleated oocyte; if the recipient human oocyte is nucleated, enucleating the human oocyte;

transferring a nucleus from the donor human somatic cell to the enucleated oocyte to form a hybrid oocyte;

activating the hybridized oocyte; and

incubating the hybrid oocyte for a sufficient time to form a human SCNT embryo.

6. The method of any of paragraphs 2 to 5, wherein the agent that reduces methylation of H3K9me3 is an agent that increases expression of a human histone demethylase member of the KDM4 family.

7. The method of paragraph 6, wherein the agent increases the expression or activity of a human KDM4(JMJD2) family histone demethylase.

8. The method of any of paragraphs 1 to 7, wherein the agent increases the expression or activity of at least one of the following enzymes: KDM4A (JMJD2A), KDM4B (JMJD2B), KDM4C (JMJD2C), KDM4D (JMJD4D) or KDM4E (JMJD 2E).

9. The method of any of paragraphs 1 to 8, wherein the agent increases the expression or activity of KDM4A (JMJD 2A).

10. The method of any of paragraphs 1 to 8, wherein the agent comprises a nucleic acid sequence corresponding to SEQ ID NO: 1 to SEQ ID NO: 4 or SEQ ID NO: 45 or a biologically active fragment thereof, which increases the efficiency of SCNT to a level equivalent to seq id NO: 1 to SEQ ID NO: 4 or SEQ ID NO: 45 to a similar or higher degree compared to the corresponding sequence of 45.

11. The method of paragraph 16, wherein the agent comprises a nucleic acid sequence corresponding to SEQ ID NO: 1 or a biologically active fragment thereof, which increases SCNT efficiency to a level comparable to SEQ ID NO: 1, or a higher degree of similarity or comparison.

12. The method of any of paragraphs 1 to 8, wherein the agent is an inhibitor of H3K9 methyltransferase.

13. The method of paragraph 12 wherein the H3K9 methyltransferase is SUV39H1 or SUV39H 2.

14. The method of paragraph 12, wherein the H3K9 methyltransferase is SETDB 1.

15. The method of paragraph 12 wherein two or more of the enzymes SUV39h1, SUV39h2 and SETDB1 are inhibited.

16. The method of paragraph 12 wherein the agent that inhibits H3K9 methyltransferase is selected from the group consisting of RNAi agents, CRISPR/Cas9, CRISPR/Cpf1 oligonucleotides, neutralizing antibodies or antibody fragments, aptamers, small molecules, peptide inhibitors, protein inhibitors, avimidir, and functional fragments or derivatives thereof.

17. The method of paragraph 16 wherein the RNAi agent is an siRNA or shRNA molecule.

18. The method of any of paragraphs 1 to 17, wherein the agent comprises a nucleic acid inhibitor to inhibit the activity of seq id NO: 14 to SEQ ID NO: 16. SEQ ID NO: 47. SEQ ID NO: 49. SEQ ID NO: 51. SEQ ID NO: 52. or SEQ ID NO: 53 in the presence of a protease.

19. The method of paragraph 17, wherein the RNAi agent hybridizes to SEQ ID NO: 14 to SEQ ID NO: 16. SEQ ID NO: 47. SEQ ID NO: 49. SEQ ID NO: 51. SEQ ID NO: 52. or SEQ ID NO: 53.

20. The method of paragraph 17, wherein the RNAi agent comprises the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8. SEQ ID NO: 18 or SEQ ID NO: 19 or fragments of at least 10 consecutive nucleic acids thereof, or a nucleic acid sequence having a sequence identical to seq id NO: 7 or SEQ ID NO: 8. or SEQ ID NO: 18 or SEQ ID NO: 19 is at least 80% homologous to the sequence.

21. The method of any one of paragraphs 1 to 20, wherein the recipient human oocyte is an enucleated human oocyte.

22. The method of any of paragraphs 1 to 20, wherein the human SCNT embryo is any embryo selected from the group consisting of 1-cell stage SCNT embryos, 5-hour (5hpa) SCNT embryos after activation, between 10-12 hours (10-12hpa) SCNT embryos after activation, between 20-28 hours (20-28hpa) SCNT embryos after activation, and 2-cell stage SCNT embryos.

23. The method of any one of paragraphs 1 to 22, wherein the agent is contacted with the recipient oocyte or enucleated human oocyte prior to nuclear transfer.

24. The method of any of paragraphs 1 to 22, wherein the agent contacts the SCNT embryo before the SCNT embryo is in the 1-cell stage, or about 5 hours after activation, or when the SCNT embryo is in the 1-cell stage.

25. The method of any of paragraphs 1 to 22, wherein the agent contacts the human SCNT embryo at 5 hours post-activation (5hpa), or 12 hours post-activation (12hpa), or 20 hours post-activation (20hpa), or when the SCNT embryo is at the 2-cell stage, or at any time between 5hpa and 28 hpa.

26. The method of any of paragraphs 1 to 22, wherein the step of contacting the recipient human oocyte or hybrid oocyte or human SCNT embryo with the agent comprises injecting the agent into the nucleus or cytoplasm of the recipient human oocyte or hybrid oocyte or human SCNT embryo.

27. The method of any one of paragraphs 1 to 26, wherein the agent increases the expression or activity of a histone demethylase of the KDM4 family.

28. A method as described in any of paragraphs 1 to 22, wherein the agent contacts the cytoplasm or nucleus of the donor human somatic cell prior to removal of the nucleus for injection into the enucleated human oocyte.

29. The method of paragraph 28, wherein the donor human somatic cell is contacted with the agent for at least 24 hours or at least 1 day prior to injecting the nucleus of the donor human somatic cell into the enucleated human oocyte.

30. The method of paragraph 28, wherein the agent is contacted with the donor human somatic cell for at least 24 hours, or at least 48 hours, or at least 3 days prior to injecting the nucleus of the donor human somatic cell into the enucleated human oocyte.

31. The method of any of paragraphs 28 to 30, wherein the agent inhibits H3K9 methyltransferase.

32. The method of any of paragraphs 28 to 30, wherein the H3K9 methyltransferase is SUV39H1 or SUV39H2, or SUV39H1 and SUV39H2(SUV39H 1/2).

33. The method of any of paragraphs 1 to 32, wherein the donor human somatic cell is a terminally differentiated somatic cell.

34. The method of any one of paragraphs 1 to 33, wherein the donor human somatic cell is not an embryonic stem cell, or an Induced Pluripotent Stem (iPS) cell, or a fetal cell, or an embryonic cell.

35. The method of any of paragraphs 1 to 34, wherein the donor human somatic cells are selected from the group consisting of cumulus cells, epithelial cells, fibroblasts, neural cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, erythrocytes, macrophages, monocytes, muscle cells, B lymphocytes, T lymphocytes, embryonic stem cells, embryonic germ cells, fetal cells, placental cells, and adult cells.

36. The method of any of paragraphs 1 to 35, wherein the donor human somatic cells are fibroblasts or cumulus cells.

37. The method of any of paragraphs 1 to 36, wherein the agent contacts the nucleus of the donor human somatic cell to remove the nucleus from the donor human somatic cell and the nucleus is used for injection into an enucleated recipient human oocyte.

38. The method of any of paragraphs 1 to 37, wherein the method results in at least a 10% increase in the efficiency of hSCNT in developing blastocysts as compared to hSCNT performed in the absence of an agent that reduces methylation of H3K9me 3.

39. The method of any of paragraphs 1 to 38, wherein the method results in an increase in the efficiency of hSCNT in developing blastocysts by 10% to 20% over hSCNT performed in the absence of an agent that reduces methylation of H3K9me 3.

40. The method of any of paragraphs 1 to 39, wherein the method results in an increase in the efficiency of hSCNT to develop blastocysts of more than 20% over hSCNT performed in the absence of an agent that reduces methylation of H3K9me 3.

41. The method of any of paragraphs 38 to 40, wherein the increase in SCNT efficiency is an increase in human SCNT embryo development to the blastocyst stage.

42. The method of any of paragraphs 38 to 40, wherein the increase in SCNT efficiency is an increase in embryonic stem cell (hNT-ESC) derivatives derived from human SCNT embryos.

43. The method of any of paragraphs 1 to 42, wherein the donor human somatic cell is a transgenic donor human cell.

44. The method of paragraph 5, further comprising culturing the human SCNT embryo in vitro to form a human blastocyst.

45. The method of paragraph 44, wherein the human SCNT embryo is at least 4 cell human SCNT embryo.

46. The method of paragraph 44, wherein the human SCNT embryo is an SCNT embryo of at least 4 cells.

47. The method of paragraph 44, further comprising isolating cells from the inner cell mass from the human blastocyst; and culturing the cells from the inner cell mass in an undifferentiated state to form human Embryonic Stem (ES) cells.

48. The method of any of paragraphs 1 to 48, wherein any one or more of the donor human somatic cell, recipient human oocyte or human SCNT embryo has been frozen and thawed.

49. A population of embryonic stem cells (hNT-ESC) derived from human SCNT embryos produced using the method of any one of paragraphs 1 to 48.

50. The population of hNT-ESCs of paragraph 49, wherein the hNT-ESCs are genetically modified hNT-ESCs.

51. The population of hNT-ESCs of paragraph 49, wherein the hNT-ESCs are pluripotent or totipotent stem cells.

52. The population of hNT-ESCs of paragraph 49, wherein the hNT-ESCs are present in a culture medium.

53. The population of hNT-ESC of paragraph 52, wherein the culture medium maintains the hNT-ESC in a pluripotent or totipotent state.

54. The population of hNT-ESC of paragraph 52, wherein the culture medium is a suitable medium for cryopreservation and cryopreservation of the hNT-ESC.

55. The population of hNT-ESCs of paragraph 54, wherein the hNT-ESCs are frozen or cryopreserved.

56. A human SCNT embryo produced by the method of any of paragraphs 1 to 55.

57. The human SCNT embryo of paragraph 56, wherein the human SCNT embryo is genetically modified.

58. The human SCNT embryo of paragraph 56, wherein the human SCNT embryo comprises mitochondrial DNA (mtDNA) that is not from the recipient human oocyte.

59. The human SCNT embryo of paragraph 56, wherein the human SCNT embryo is present in culture.

60. The human SCNT embryo of paragraph 59, wherein the culture medium is a medium suitable for cryopreservation and cryopreservation of the human SCNT.

61. The human SCNT embryo of paragraph 60, where the human embryo is frozen or cryopreserved.

62. A composition comprising at least one of a human SCNT embryo, recipient human oocyte, human hybrid oocyte or blastocyst, and at least one of:

a. an agent that increases the expression or activity of a histone demethylase of the KDM4 family; or

b. An agent that inhibits H3K9 methyltransferase.

63. The composition of paragraph 62, wherein the agent that increases expression or activity of a histone demethylase of the KDM4(JMJD2) family increases expression or activity of at least one enzyme of KDM4A (JMJD2A), KDM4B (JMJD2B), KDM4C (JMJD2C), KDM4D (JMJD2D) or KDM4E (JMJD 2E).

64. The composition of paragraph 63, wherein the agent increases the expression or activity of KDM4D (JMJD2D) or KDM4A (JMJD 2A).

65. The composition of paragraph 64, wherein the agent comprises a nucleic acid sequence corresponding to SEQ ID NO: 1 to SEQ ID NO: 4 or SEQ ID NO: 45 or a biologically active fragment thereof, which increases the efficiency of human SCNT to a level equivalent to SEQ id no: 1 to SEQ ID NO: 4 or SEQ ID NO: 45 to a similar or higher degree compared to the corresponding sequence of 45.

66. The composition of paragraph 64, wherein the agent comprises a nucleic acid sequence corresponding to SEQ ID NO: 1 or a biologically active fragment thereof, which increases SCNT efficiency to a level comparable to SEQ ID NO: 1, or a higher degree of similarity or comparison.

67. The composition of paragraph 62, wherein the inhibitor of H3K9 methyltransferase inhibits at least one enzyme of SUV39H1, SUV39H2, or SETDB1, or any combination thereof.

68. The composition of paragraph 62, wherein the human SCNT embryo is a human SCNT embryo at the 1-cell stage, 2-cell stage, or 4-cell stage.

69. The composition of paragraph 62, wherein the recipient human oocyte is an enucleated recipient human oocyte.

70. The composition of paragraph 62, wherein the human SCNT embryo is produced from a nucleus injected with a terminally differentiated human somatic cell, or wherein the blastocyst is developed from a human SCNT embryo produced by injecting a nucleus of a terminally differentiated human somatic cell into an enucleated human oocyte.

71. A kit, comprising: (i) an agent that increases the expression or activity of a histone demethylase of the human KDM4 family, and/or an agent that inhibits H3K9 methyltransferase; and (ii) a human oocyte.

72. The composition of paragraph 92 wherein the human oocyte is an enucleated oocyte.

73. The composition of paragraph 92 wherein the human oocyte is a non-human oocyte.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those disclosed herein can be used in the present invention or in the testing of the present invention, suitable methods and materials are disclosed below. The materials, methods, and examples are illustrative only and not intended to be limiting.

All publications, patents, patent publications and applications, and other documents mentioned herein are incorporated by reference in their entirety.

In summary, the present invention provides methods for obtaining ES cells, ES cell lines, and differentiated cell types from a single blastomere of an early embryo without destroying the embryo. Various features of the method are described in detail below. All combinations of the various aspects and embodiments of the invention detailed above and below are contemplated.

[ examples ]

The embodiments presented herein relate to methods and compositions for reducing or removing H3K9me3 in human SCNT embryos and/or in human donor nuclei of human somatic cells by (i) increasing the expression or activity of a human KDM4 family histone demethylase member, such as KDM4A and/or (ii) inhibiting any one of the human methyltransferases hsov 39H1 or hsov 39H2, thereby increasing human SCNT efficiency. Throughout this application, various publications are referenced. All publications and references cited in these publications are incorporated herein by reference in their entirety for the purpose of more fully disclosing the state of the art to which the invention pertains. The following examples are not intended to limit the scope of the claims but rather to exemplify certain embodiments. Any variations to the exemplary method known to those skilled in the art are intended to fall within the scope of the present invention.

Procedure of experiment

Human SCNT Process and KDM4A mRNA injection

In an inverted microscope equipped with Poloscope: (Cambridge Research&Enucleation and fusion of nuclear donor cells under Instrumentation) all mil stage human oocytes with distinct first polar bodies were enucleated in the presence of caffeine (1.25 mM.) for enucleation, oocytes were preincubated in GlobalHTF medium for 5 minutes using hepes (life Global) containing 0.5 μ g/ml cytochalasin B and caffeine (1.25 mM.) for enucleation, then the spindle complex was removed using a piezoelectric actuator (primeth, japan.) dermal fibroblasts resuspended in a droplet containing HVJ-E extract (Cosmo Bio, USA) were inserted into the perivitelline space of the enucleated oocytes, the reconstituted oocytes were kept in manipulation medium containing caffeine (1.25mM) until fusion was confirmed, then the reconstituted oocytes were transferred to Global medium 10% for 1 to 1.5 hours, after which the reconstituted oocytes were transferred to incubation medium with a transfection medium with a 10% of TSA-12 mM, activated by incubation with a supplemental electrical pulse such as indicated for 10% of activating the incubation medium (TSA) and then the incubation medium was changed by pipetting the addition of TSA 3, 10% of pst 2h, 10% of pst-h, 3, 12 mM, 3.7 h, 3h, 7 h, 3h, and then the incubation of the incubation medium with no additional electrical pulse was applied to no change of the growth medium.

For mRNA injection, activated SCNT embryos were washed and cultured in Global 10% SPS for 1 hour prior to KDM4A mRNA injection. Approximately 10pl of KDM4A mRNA was injected into SCNT embryos in Hepes-HTF 10% SPS medium 5 hours after activation using the previously disclosed piezoelectric actuator (Matoba et al, 2014). More details of donor cell preparation, mRNA preparation, RNA-seq and other processes can be found in supplementary experimental procedures.

Identification of human reprogramming confrontation regions

The whole genome expression levels of 4-cell and 8-cell human fetuses were evaluated using a sliding window (size 20kb, step size 10 kb). For each window, quantification was done using normalized RPM (readings per million of uniquely mapped readings). A strict strategy (FC > 5, RPM > 5 in 8-cell IVF embryos) was used to identify significantly activated regions in 8-cell IVF embryos relative to 4-cell IVF embryos and to merge and overlap the regions. These activation regions were classified into three groups based on their difference in expression in human SCNT and IVF 8 cell embryos.

Mouse

B6D2F1/J (BDF1) mice were produced by crossing C57BL/6J females with DBA/2J males and the resulting mice were used to harvest oocytes and somatic cell nuclear donors used in SCNT. All animal experiments were approved by the Harvard medical college laboratory animal care and use Committee.

In vitro transcription of human KDM4A mRNA

In vitro transcription was performed as previously disclosed (Matoba et al, 2014). Briefly, the full length human KDM4A/JHDM3A cDNA was cloned into pcDNA3.1 plasmid containing poly (A)83 at the 3' end of the cloning site. The primastar mutagenesis kit (TAKARA # R045A) was used to generate KDM4A (H188A) as a catalytic deficient mutant. Synthesized using the mMESSAGE mMACHINE T7 super kit (Life technologies # AM 1345). The synthesized mRNA was dissolved in nuclease-free water. The concentration of mRNA was measured by a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies). An aliquot of mRNA was stored at-80 ℃ until use.

Murine SCNT and KDM4A mRNA injection

Somatic cell nuclear transfer in mice was accomplished as previously disclosed (Matoba et al, 2014). Briefly, both recipient MII oocytes and donor cumulus cells were collected from adult BDF1 female mice and collection was accomplished by superovulation by injection of 7.5IU of pregnant horse serum gonadotropin (PMSG; Millipore #367222) and 7.5IU of human chorionic gonadotropin (hCG; Millipore # 230734). 15 to 17 hours after hCG injection, cumulus-oocyte complexes (COCs) were collected from the oviduct and briefly treated with Hepes buffered elemental potassium optimized medium (KSOM) containing 300U/ml bovine testicular hyaluronidase (Calbiochem #385931) to obtain dissociated MII oocytes and cumulus cells. The isolated MII oocytes were enucleated using a piezo-driven micromanipulator (Primetech # PMM-150FU) in Hepes buffered KSOM medium containing 7.5jig/ml cytochalasin B (Calbiochem # 250233). The nucleus of the common cumulus cell is injected into the enucleated oocyte. After incubation in KSOM for 1 hour, the cells were incubated in a medium containing 2.5mM SrCl₂And 5jig/ml cytochalasin B in calcium-free KSOM to activate the reconstituted SCNT oocytes, followed by another 4-hour incubation in KSOM with cytochalasin B. At the start of SrCl₂After 5 hours of treatment (hours post-activation, hpa), activated SCNT embryos were washed and cultured in KSOM at 37.8 ℃ under a humid atmosphere with 5% CO 2. Approximately 10pl of water (control), 1500 ng/. mu.l of wild type or mutant (H188A) human KDM4A mRNA was injected into the SCNT embryos using a piezo-driven micromanipulator at 5 to 6 hpa. The rate of development of the embryo prior to implantation was analyzed by student t-test.

Preparation of human oocytes

The human oocyte experiment (CHA001) strategy was approved by both the CHA regenerative medicine institute (CHARMI) stem cell research Supervision (SCRO) committee and the ethical Review Board (PIRB). Initial oocyte donor recruitment was performed based on network advertising as previously disclosed (Chung et al, 2014). All donors were voluntary, and were screened according to the guidelines of the American Society for Reproductive Medicine (ASRM) based on their reproductive, medical, and physiological health. According to guidelines set by ASRM, time, effort, lost wages, road fees, inconvenience, and other costs associated with the donation process are economically compensated for oocyte donors.

Ovarian stimulation was accomplished as previously disclosed (Chung et al, 2014). Briefly, ovaries were stimulated for 9 to 11 days with GnRH antagonist (Ganirelix acetate, Merck) inhibition using a combination of human recombinant follicle stimulating hormone (rFSH, 225-300IU, Merck) and human menopausal gonadotropin (Menopur 75IU, Ferring). When 1 or 2 follicles reach 18mm in diameter, 4mg leuprolide (Lupron) was used to simulate LH surges. All drugs were administered by subcutaneous injection. A transvaginal oocyte collection procedure was performed approximately 36 hours after the injection of leuprolide. Collected COCs were ablated with 5080IU/ml hyaluronidase (Sigma-Aldrich) within 1 to 2 hours after egg collection. Subsequently, the treated COCs were kept in Global media (IVFOnline) supplemented with 10% serum protein supplement (SPS; Cooper scientific) for use.

Donated human IVF embryos

IVF embryos for this study were obtained from patients who had received the desired number of children after the standard IVF procedure, and the remaining embryos had been cryopreserved for several years (2 to 6 years). All donors voluntarily donated their embryos (multicellular lysis phase) for study by signing an informed consent. The embryo donation procedure for the study was approved by the medical center IRB of the south of the CHA river.

Somatic cell preparation and characterization of human donors

To prepare human nuclear donor somatic cells, excised small pieces of abdominal skin (0.5cm x0.3cm) were biopsied under local anesthesia and washed 3 times in PBS supplemented with antibiotic/antifungal solution (anti-IX, Invitrogen) to remove any possible contaminants. All somatic donors in this study were AMD patients (AMD subtype: Central Meerweil atrophy). DFB-6 is from a 52 year old female. DFB-7 is from a 42 year old female. DFB-8 is from a 59 year old male.

Somatic cell nuclear donor cell preparation procedures were substantially the same as previously disclosed (Chung et al, 2014). Briefly, skin explants were mechanically minced and treated with collagenase (type I, 200 units/ml, Worthington-biochem) in DMEM supplemented with 10. mu.g/ml penicillin-streptomycin to dissociate the skin tissue. After overnight incubation, dissociated cells were harvested and plated in 60mm dishes containing DMEM (Invitrogen with 10% FBS, 1% non-essential amino acids and 10. mu.g/mL penicillin-streptomycin) solution at 37 ℃ under an atmosphere of 5% CO 2. Once the cells reached 80% confluence, 1/2 from the initial outcome was cryopreserved, the remaining cells were passaged several times, and the cells from each passage were cryopreserved. Next, prior to SCNT, the frozen cells were thawed and cultured in 4-well plates (Nunc) until they reached confluency. Subsequently, the cells were cultured in serum-starved DMEM (0.5% FBS) for 2 to 3 days to allow the cells to be homoleptic prior to use.

Derivation of human NTK-ESCs from KDM 4A-assisted SCNT blastocysts

All expanded blastocysts were treated with acid-bench solution to remove zona pellucida, and then the entire blastocyst (without removal of trophectoderm) was placed on mitotically inactivated mouse fibroblasts (MEFs, Global Stem Inc.) in knockoff DMEM supplemented with knockoff serum replacement (10% SR, Invitrogen), FBS (10% Hyclone), bFGF (30ng/ml), human LIF (2000 units/ml, Sigma-Aldrich), and ROCK inhibitor (1uM, Sigma-Aldrich). The derivative media was not changed for the next 3 days, and then 1/2 media was replaced daily with fresh media without ROCK inhibitor as previously described (Chung et al, 2008). After 3 passages, the amount of FBS was reduced to 2% by which it was replaced with SR. After passage 5, the ES cells were cultured in DMEM/F12 supplemented with FGF (8ng/ml, Invitrogen), SR (18%, Invitrogen), and FBS (2% Hyclone). After 10 passages, the ES cells were maintained in DMEM/F12 supplemented with FGF (8ng/ml) and 20% SR.

Preparation of 8-cell human embryos for ZGA analysis

SCNT embryos for ZGA analysis were generated using an oocyte donated by a healthy female (#64) and dermal fibroblasts from AMD patients (DFB-8). When blastomere comparison was initiated, SCNT and IVF embryos were cultured until the late 8-cell stage, and then these embryos were simply treated with acid-bench solution to remove the zona pellucida. To prepare the 8-cell SCNT embryos, oocytes from an oocyte donor and dermal fibroblasts from a somatic cell nuclear donor are used. All procedures were identical to those disclosed in the section "human SCNT procedure and KMD4A mRNA injection". Embryos that reached the full 8-cell stage completely synchronously only 74 hours after activation were collected and used in this experiment.

For control IVF embryo preparation, several donated early 8 cell stage IVF embryos are thawed, cultured for 5 to 7 hours to bring them to the late 8 cell stage, and processed. After removing the zona pellucida, the denuded embryos were washed 3 times in PBS, placed in PCR tubes without RNAse and DNAse, spin-slowed, and snap-frozen in liquid nitrogen. Subsequently, they were kept at-80 ℃ until use. As a control, skin fibroblasts of somatic cell nuclear donors were also prepared. Those fibroblasts were in the region of 25cm²Flasks were cultured in DMEM 10% FBS, and approximately 10,000 cells/donor were collected, snap frozen, and stored at-80 ℃ until use.

Immunohistochemical staining

Mouse 1-cell SCNT embryos, undifferentiated human ESC colonies or differentiated Embryoid Bodies (EBs) were fixed with 4% Paraformaldehyde (PFA) for 20 minutes at room temperature after 3 washes with PBS (PBS/BSA) containing 10mg/ml BSA, the fixed samples were permeabilized for 15 minutes by incubating with 0.5% Triton-X100 after blocking in PBS/BSA at room temperature for 1 hour, the samples were incubated overnight at 4 ℃ in a mixture of primary antibodies as follows, anti-H3K 9me3(Abeam, ab71604, 1: 500), anti-H3K 9me 2 (Abeam, abl09250, 1: 200), anti-OCT-4 (Santa Cruz, sc-8628, 1: 100), anti-SOX 160(Millipore, MAB4360, 1: 100), anti-SOX 42 (R & D, AF2018, 1: 200), anti-Millia-61705, 1: 32, goat laid-rabbit protein, goat rabbit loopa, goat anti-goat rabbit protein, goat rabbit loopa, goat laid protein, goat laid down, laid down.

In vitro differentiation of ESCs and teratoma assays

For in vitro differentiation assays, ESCs were cultured in ESC media without bFGF in low attachment dishes for 1 week until they formed Embryoid Bodies (EBs). Thereafter, the EBs were transferred to 4-well dishes (Nunc) coated with artificial basement membrane (BD Biosciences) and cultured for an additional 1 week. EBs were washed, blocked and permeabilized in PBS containing 1% BSA and 0.1% Triton-X, and incubated overnight with one antibody. EB was washed 3 times with PBS containing 1% BSA, stained with secondary antibody and DAPI for 1 hour, and observed under a fluorescent microscope. For teratoma testing, about 1x10 will be tested⁵An undifferentiated NTK-ESC was injected into the testis of NOD/SCID mice. For each NTK-ESC line, at least 3 animals were used. After 12 weeks, teratomas were resected and fixed in PFA, embedded in paraffin, sectioned, followed by histological analysis after staining as previously disclosed (Chung et al, 2014).

Chromosome analysis

Chromosomal analysis of two NTK-ESC lines was performed by standard protocol as previously disclosed (Chung et al, 2014). Metaphase extension was stained by GTG (G-banding by trypsin using Giemsa) -banding technique, and 20 metaphases were analyzed by two cytogeneticists and karyotype analysis was performed. The symbols were generated by the Ikaros karyotyping System (Metasystems, Germany).

RNA sequencing analysis

In each group, 5 8-cell embryos were directly lysed and used for cDNA synthesis using the SMART-Seq v4 ultra-low input RNA kit (Clontech). For MEF donors, 10ng of total RNA was used in cDNA synthesis using the SMART-Seq v4 ultra-low input RNA kit. After amplification, the cDNA samples were fragmented to an average size of 150bp (Covaris) using a Covaris sonicator M220. According to the manufacturer's instructions (New England Biolabs), the NEBNext super DNA library from Illumina was used to prepare the kit and the fragmented DNA was used to prepare sequencing libraries with different barcodes. For each RNA-seq analysis of hescs, 1 μ g total RNA was used for mRNA purification. Barcoded RNA-seq pools were generated using the NEBNext super-targeted RNA pool preparation kit from illumina (newengland biolabs). Single-ended 50bp sequencing was performed on a HiSeq2500 sequencer (Illumina). The sequencing reads were mapped to the human genome using Tophat2 (hg 19). All procedures were performed under default settings (unless otherwise noted). For each sequencing library, at least 2200 million uniquely mapped reads were obtained, and then combined into a textual record guided by reference annotation (Refseq gene model) using Cufflinks v2.0.2. The expression level of each gene was quantified using normalized FPKM (number of fragments per kilobase exon per million mapped fragments). Statistical analysis was performed using R (available at www.r-project. Distributions were compared by Wilcoxon test function in R using independent 2 sets of Wilcoxon rank sum test. The Pearson's r coefficients are calculated using the cor function of the default parameters. Hierarchical clustering analysis of global gene expression patterns for different samples was done using the heatmap.2 function in R (gplots software package).

Analysis of published ChIP-seq and DNA methylation datasets

To perform the histone modification enrichment analysis in FIGS. 1 and 5, the inventors used the following published ChIP-seq and DNasel-seq datasets: H3K9me3, H3K4me1, H3K4me2, H3K4me3, H3K27me3, H3K36me3, H3K27ac, and H4K20mel ChIP-seq in Nhlf fibroblasts (ENCODE/generalized histone project); H3K9me3ChIP-seq in Hsmm and K562 cells (ENCODE/generalized Histone project); H3K9me3ChIP in Mcf7 cells (ENCODE/Sydh histone project); dnasel-seq in IMR90, Hsmm, K562 and Mcf7 cells (ENCODE/OpenChromDnase project). The inventors also used the whole genome bisulfite sequence dataset for DNA methylation analysis (Roadmap Epigenomics et al 2015) from IMR90 cells of the human epigenomic project (Roadmap Epigenomics project). The processed DNA methylation data in IMR90 was downloaded from the website "egg2. wustl. edu/roadmap/web _ portal/". ChIP-seq intensity was quantified using normalized FPKM. The genomic locations covered by the sequencing reads were determined and visualized as custom tracks in the UCSC genome browser. Test function in R, independent 2 groups Wilcoxon rank sum test was used to compare ChIP-seq distributions between each group.

Example 1

Identification of Reprogramming Resistant Region (RRR) in 8-cell human SCNT embryos

Unlike murine Zygote Genome Activation (ZGA), which occurs at the 2-cell stage, human Zygote Genome Activation (ZGA) occurs during the late 4-to late 8-cell stage (Niakan et al, 2012) (fig. 1A). To identify genomic regions that were activated during ZGA in normal human IVF embryos, the inventors analyzed the public pre-transfer human embryo RNA-sequencing (RNA-seq) dataset (Xue et al, 2013) and identified 707 genomic regions in the size range of 20 to 160kb (table 5) that were at least 5-fold activated at the 8-cell stage as compared to the 4-cell stage (fig. 1B).

To determine if ZGA occurred properly in human SCNT, the inventors collected late 8-cell stage embryos (5/group) derived from SCNT or IVF and performed RNA-seq (fig. 1A). Also, the inventors have conducted RNA-seq for donor skin fibroblasts (DFB-8, see methods). The 707 genomic regions defined above (fig. 1B and table 5) indicate that most of the ZGA regions in the SCNT embryo were activated compared to those in the donor fibroblasts (fig. 1C). However, the level of activation was not comparable to that in IVF embryos (fig. 1C). Of the 707 genomic regions, the 169 regions had activation levels comparable to those in IVF embryos (FC ≦ 2, IVF vs SCNT), and thus were named "complete reprogramming region" (FRR) following our previous definition (Matoba et al, 2014). Similarly, 220 regions of SCNT were partially activated (2 < FC < ═ 5) compared to IVF embryos, and these regions were named "partially reprogrammed regions" (PRR). However, the remaining 318 regions (Table 6) failed to successfully complete activation in SCNT embryos (FC > 5), named "reprogramming confrontation region" (RRR). Thus, comparative transcriptome analysis led us to identify 318 RRRs that were tolerant to transcriptional reprogramming in human 8-cell SCNT embryos.

TABLE 5 expression levels of transcripts from the human ZGA activation region (FIG. 1) are correlated. RNA-seq database, obtained from Xue et al, 2013.

TABLE 5

TABLE 5

TABLE 5

TABLE 5

TABLE 5

TABLE 5

TABLE 5

TABLE 5

TABLE 5

TABLE 5

TABLE 5

TABLE 5

TABLE 5

TABLE 5

TABLE 5

TABLE 5

TABLE 5

TABLE 5

TABLE 5

TABLE 5

^*: the RNA-seq database was obtained from Xue et al, 2013.

TABLE 6 transcript expression levels from human reprogramming confrontation regions (associated with FIG. 1)

The heterochromosomal characteristics of RRR are conserved in human somatic cells

Thereafter, the inventors evaluated whether human RRRs possess heterochromatin characteristics similar to murine RRRs. Analysis of The publicly available 8 major histone modifications of ChIP-seq datasets from human fibroblasts (Bernstein et al, 2012; The Encode Consortium Project, 2011) showed that H3K9me3 was specifically enriched in human RRRs (fig. 1D and fig. 5A). Enrichment for H3K9me3 was unique to RRR, as no similar enrichment was observed in FRR or PRR (fig. 1D and fig. 5A). Similar analysis also showed enrichment of H3K9me3 at RRR in K562 erythroleukemia cells, Hsmm skeletal muscle myoblasts, and Mcf7 breast cancer cells (fig. 1E and 5B), indicating that the enrichment of H3K9me3 in RRR is a common feature of somatic cells.

Thereafter, the inventors analyzed DNaseI hypersensitivity of 4 different somatic cell types using the data set generated by the ENCODE project. This analysis showed that RRR was significantly less sensitive to DNaseI than FRR and PRR in all human somatic cell types analyzed (FIG. 1F and FIG. 5C). Consistent with their heterochromatin characteristics, the inheritance of human RRRs is relatively poor compared to FRRs or PRRs (fig. 5D), and is enriched for specific repeats such as LINEs and LTRs, but not SINEs (fig. 5E). Together, these results indicate that the heterochromatin characteristics of RRRs, enrichment of H3K9me3, and reduced accessibility to dnase i are common to murine and human somatic cells.

Example 2

Human KDM4A mRNA injection for improving mouse SCNT embryo development

The inventors have established the concept of human RRR being H3K9me3 rich, and evaluated whether removing H3K9me3 could help overcome the reprogramming barrier in human SCNT embryos. The inventors previously demonstrated, using the murine SCNT model, that the murine H3K9me3 barrier can be removed by injection of mRNA encoding the H3K9me3 demethylase KDM4d (Matoba et al, 2014). Prior to moving into the human SCNT model, assuming that multiple members of the KDM4 family with H3K9me3 demethylase activity were present in both murine and human (Klose et al, 2006; Krishnan and Trievel, 2013; Whetstine et al, 2006), the inventors did not use KDM4D to facilitate SCNT reprogramming, but instead evaluated whether other members of the KDM4 family, such as KDM4A, could be used. In addition, the inventors also evaluated whether members of the KDM4 family could function across species.

For this purpose, the inventors have implemented the following SCNT: after the same procedure in our previous studies, cumulus cells of adult female mice were used as nuclear donors and injected into human KDM4A mRNA 5 hours (hpa) post-activation (fig. 2A) (Matoba et al, 2014). Immunohistochemical staining showed that human KDM4A mRNA greatly reduced H3K9me3 levels in the nucleus of murine SCNT embryos by injection of wild type rather than catalytic mutant (fig. 1B). Importantly, injection of KDM4A mRNA greatly increased the developmental potential of SCNT embryos, 90.3% of them developed to the blastocyst stage, in sharp contrast to the 26% blastocyst formation rate in the control group (fig. 2C and 2D, table 3). This very high blastocyst formation efficiency was similar to the 88.6% blastocyst formation rate in murine SCNT embryos injected with KDM4d (Matoba et al, 2014). These results surprisingly indicate that the reprogramming disorder, H3K9me3 in the genome of a somatic cell, can be removed by any member of the KDM4 family demethylase, as long as that member has H3K9me3 demethylase activity.

TABLE 3 Pre-implantation development of KDM4A assisted murine SCNT embryos, correlation with FIG. 2

P < 0.01, compared to water injected control.

KDM4A mRNA injection significantly increased human SCNT embryo blastogenesis rate

Thereafter, the inventors evaluated whether injection of KDM4A mRNA could help overcome reprogramming disorders in human SCNT using optimized SCNT conditions, including the use of histone deacetylase inhibitor, trichostatin a (tsa) (Tachibanaet al, 2013). Considering the future clinical use of KDM4A to assist SCNTs, the inventors used skin fibroblasts from patients with age-related macular degeneration (AMD) (Bressler et al, 1988) as nuclear donors.

To confirm again the beneficial effects of KDM4A on human SCNT, the inventors selected oocytes that were not successfully developed into oocytes of donors of expanded blastocysts in previous attempts using conventional IVF procedures (Chung et al, 2014). After enucleation, a total of 114 Mil oocytes harvested from four oocyte donors were fused to donor fibroblasts by FfVJ-E. After activation, 63 reconstituted SCNT oocytes were injected with human KDM4A mRNA, the remainder (51) were used as non-injection controls (fig. 2E, table 4). The inventors monitored the development of these SCNT embryos and found that both groups had similar lysis efficiencies in forming 2-cell embryos (control: 48/51 ═ 94.1%; KDM 4A: 56/63 ═ 88.9%) (table 4). As expected, KDM4A mRNA injection did not show any beneficial effect on SCNT embryo development rate before ZGA was completed at the end of the 8-cell stage (68.8% vs 71.4%) (fig. 2F and table 4). However, at the morula stage, this beneficial effect became apparent (16.7% vs 32.1%) (fig. 2F and table 4). Surprisingly, at day 6, 26.8% (15/56) of embryos injected with KDM4A had successfully reached the blastocyst stage, while only 4.2% (2/48) of the control embryos reached the blastocyst stage. On day 7, 14.3% of the embryos injected with KDM4A developed to the stage of expanding blastula, whereas none of the control embryos developed to this stage (fig. 2F and 2G). Importantly, the beneficial effects of KDM4A were observed in all of the 4 donors examined (fig. 2H). Thus, the inventors clearly demonstrated that KDM4A mRNA injection could improve the developmental potential of human SCNT embryos, particularly beyond ZGA.

Table 4: KDM4A aids in the pre-implantation development of human SCNT embryos, associated with fig. 2.

Concentration of injected human KDM4A mRNA was 1500 ng/. mu.l. Control embryos were not injected, blast: blastocyst, ex-blast: the blastocyst is expanded.

Example 3

Establishment and characterization of human ESCs derived from SCNT blastocysts injected with KDM4A

Thereafter, the inventors obtained nuclear transfer ESCs (NT-ESCs) from SCNT blastocysts injected with KDM 4A. The inventors obtained a total of 8 expanded blastocysts from SCNT embryos injected with KDM4A (fig. 3A and table 4). After removal of the zona pellucida, expanded blastocysts were cultured on irradiated Murine Embryonic Fibroblasts (MEFs) in conventional ESC-derived media. 7 of 8 blastocysts attached to MEF feeder cells and began to develop naturally. After passage 5, the inventors succeeded in obtaining 4 stable NT-ESC lines, designated as NTK (KDM4A assisted NT) -ESC #6 to #9, respectively (FIG. 3A, also named CHA-NT #6 to # 9).

Immunohistochemical staining showed that OCT4, NANOG, SOX2, SSEA-4 and TRA1-60 were all expressed in a pattern similar to control human ESC lines obtained by IVF (fig. 3B, fig. 6A and 6B). RNA-seq (FIG. 6C) showed that NTK-ESCs expressed pluripotency marker genes at a similar level as control ESCs (FIG. 3C). Pairwise comparison of global transcriptomes showed high association between NTK-ESC and control ESC (fig. 3D and fig. 6D). Hierarchical clustering analysis of the transcriptome revealed that NTK-ESCs were clustered together with the control ESCs (FIG. 3E). These results suggest that it is not possible to distinguish NTK-ESCs from control ESCs at the molecular level.

The inventors examined the differentiation ability of NTK-ESC by in vitro differentiation and in vitro teratoma assay. Immunohistochemical staining of Embryoid Bodies (EBs) after 2 weeks in vitro culture showed that NTK-ESCs were effective in causing all three layers of germ cells (FIGS. 3F and 6E). In addition, the NTK-ESC formed teratomas containing all three layers of germ layer cells within 12 weeks of metastasis (fig. 3G and 6F). These results indicate that the NTK-ESC is pluripotent.

Human karyotyping analysis showed that these NTK-ESCs maintained a normal number of chromosomes and had the same expected sex chromosome pairs as the nuclear donor somatic cells (for NTK6/7, 46, XX for; for NTK8, 46, XY; FIGS. 3H and S3A). Short Tandem Repeat (STR) analysis showed that all 16 repeat markers located across the genome showed perfect matches between donor somatic cells and their derived NTK-ESCs (fig. 3I and 7B). Mitochondrial DNA sequence analysis showed that the two SNPs of NTK-ESC exactly match the oocyte SNP donor SNP, but not the nuclear donor SNP (fig. 3J and fig. 7C). These results collectively embody the reliability of our SCNT approach and indicate that injection of KDM4A mRNA improved SCNT-mediated ESC derivation without disrupting the pluripotency or genomic stability of the established NTK-ESC.

KDM4A Propromotion of RRR ZGA in 8-cell SCNT embryos

The fact that hSCNT embryo development after ZGA was significantly improved by KDM4A mRNA injection indicates that H3K9me3 in the donor somatic genome does indeed act as a barrier to ZGA in human SCNT embryos. To confirm to what extent KDM4AmRNA injection can overcome the ZGA deficiency in SCNT embryos, the inventors implemented RNA-seq for 8-cell SCNT embryos with or without KDM4A injection. Comparative transcriptome analysis showed that up to 50% (158) of 318 RRRs were significantly upregulated by KDM4AmRNA injection (fig. 4A, FC > 2), indicating that clearance of H3K9me3 may at least partially promote ZGA in SCNT embryos.

To identify genes that might help explain the developmental improvement of SCNT embryos injected with KDM4A, the inventors focused on the analysis of the identified genes. The expression of 206 genes (Table 7) was significantly up-regulated by injection of KDM4A (FPKM > 5, FC > 2). Gene ontology analysis showed that these genes were enriched for transcriptional regulation, ribosome biosynthesis and RNA processing (fig. 4B), suggesting that these dysregulation in important developmental mechanisms may be the cause of SCNT embryonic development arrest. Although the function of most of the 206 genes in pre-embryo implantation development is currently unknown, two of them, UBTFL1 and THOC5 (fig. 4C), are known to be required for normal development before embryo implantation in mice (Wang et al, 2013; Yamada et al, 2009). Thus, imperfect activation of these genes is at least partially responsible for the poor development of human SCNT embryos.

Table 7: KDM4A response expression level of ZGA Gene (associated with FIG. 4)

Example 5

After decades of attempts, human NT-ESCs have recently been finally obtained (Chung et al, 2014; Tachibana et al, 2013; Yamada et al, 2014). These advances are mainly due to the optimization of SCNT acquisition conditions. However, the intrinsic defect in epigenetic reprogramming that causes the cessation of human SCNT embryonic development has not been identified. Herein, the inventors demonstrated that H3K9me3 in the genome of somatic cells is a barrier for human SCNT reprogramming. By removing this barrier by overexpressing H3K9me3 demethylase KDM4A, transcriptional reprogramming at ZGA was facilitated, allowing human SCNT embryos to develop into normal blastocysts more efficiently, from which the inventors succeeded in establishing multiple AMD patient-specific NT-ESC lines without compromising genomic stability or pluripotency. Therefore, the inventors demonstrated that H3K9me3 is a general reprogramming barrier when human somatic cells are reprogrammed by SCNT, but a practical method for improving cloning efficiency was also established.

It is well known that oocytes differ in their ability to support SCNT embryonic development between different human oocyte donors. In fact, human NT-ESCs are available only when a small group of female donated high quality oocytes are used as recipients (Chung et al, 2014; Tachibana et al, 2013; Yamada et al, 2014), but the reason for the dependency on oocyte quality is still unknown. Consistently, only one (ID #58) of the four donors supported SCNT blastocyst formation without KDM4A mRNA injection, even in the presence of TSA that had been reported to promote blastocyst formation (Tachibana et al, 2013) (fig. 2H and table 4). In contrast, oocytes of 4 donors tested all supported blastocyst formation when KDM4A mRNA was injected, indicating that KDM4A could overcome the donor variation problem. It remains to be determined whether KDM4A improves IVF embryo development.

Although the developmental potential of human SCNT embryos to the blastocyst stage was significantly and consistently improved by injection of KDM4A mRNA, the degree of improvement was less dramatic than in mice (90% vs. 27% of humans). Even between oocytes of the same batch originating from one ovulation, species differences and/or quality of human oocytes may vary greatly, even though only a fraction of them have the ability to support development to the blastocyst stage, with success rates varying between 15% and 60%, by IVF (Shapiro et al, 2002; Stone et al, 2014). This is in sharp contrast to murine IVF, where more than 90% of embryos can develop to the blastocyst stage. Therefore, surprisingly, SCNT efficiency was improved by injection of KDM4A even with human oocytes of lower quality compared to murine oocytes. Some human oocytes used for this experiment may not support blastocyst formation even by IVF.

In addition to demonstrating the efficacy of KDM4A in improving human SCNT efficiency and NT-ESC acquisition, another important finding is that KDM4A can promote human SCNT reprogramming. Given that human KDM4A may function within murine SCNT embryos to achieve similar effects to KDM4d, the inventors have demonstrated that all members of the KDM4 family can be used to promote hscnts as long as they possess H3K9me3 demethylase activity.

In summary, the inventors herein have demonstrated an improved KDM 4-assisted human SCNT method. Using this approach, the inventors have obtained human blastocysts from adult AMD patient cells and subsequently established multiple NT-ESCs (NTK-ESCs) with genomes identical to those of the donor patients. This provides a unique and important source of cells for understanding AMD as well as therapeutic drug screening for the treatment of AMD. Given that the same strategy can be used in the study of other human diseases, the inventors have demonstrated a new approach for generating patient-specific NT-ESCs that would have a general impact on human therapeutics. Furthermore, since hSCNT allows for the replacement of somatic mitochondria with recipient oocyte mitochondria, as demonstrated herein (fig. 3H to 3J and fig. 7), the methods, compositions, and kits disclosed herein provide an opportunity to treat mitochondrial DNA-related diseases. In fact, recent studies have shown that the metabolic syndrome phenotype caused by mtDNA mutations can be corrected by SCNT replacing mtDNA (Ma et al, 2015). Therefore, KDM 4-assisted SCNT methods disclosed herein can also be used for mtDNA replacement therapy.

Reference to the literature

The references disclosed herein are incorporated by reference in their entirety.

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Sequence listing

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<120> methods and compositions for increasing human Somatic Cell Nuclear Transfer (SCNT) efficiency by removing histone H3-lysine trimethylation, and for increasing human NT-ESC derivatives

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tgcttggcac actgaagaca tggacctcta cagcatcaac tacctgcact ttggagaacc 780

aaagtcctgg tactctgttc cacctgagca tggaaagcgg ttggaacgcc tcgccaaagg 840

ctttttccca ggaagtgctc aaagctgtga ggcatttctc cgccacaaga tgaccctgat 900

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agagtttatg atcactttcc cttatggtta ccatgccggc tttaaccatg gttttaactg 1020

tgcggagtct accaattttg ctacccgtcg gtggattgag tacggcaagc aagctgtgct 1080

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aagcgccgct agaagtttca gtgagcggga gctggcagag gttgcagatg aatacatgtt 1920

ttccctagaa gagaataaga agtccaaggg acgccgtcag cctttaagca agctcccccg 1980

ccatcaccca cttgtgctgc aggagtgtgt cagtgatgat gagacatctg aacagctgac 2040

ccctgaggaa gaggctgagg agacagaggc ctgggccaag cctctgagcc aactgtggca 2100

gaaccgacct ccaaactttg aggctgagaa ggaattcaat gagaccatgg cccaacaggc 2160

ccctcactgc gctgtctgta tgatcttcca gacttatcat caggttgaat ttggaggctt 2220

taatcagaac tgtggaaatg cttcagattt agccccccag aagcagagga ccaagccatt 2280

gattccagaa atgtgcttca cttcgactgg ctgcagcacg gacatcaacc tttctactcc 2340

ttatcttgag gaggatggca ccagcatact cgtttcctgc aagaagtgca gcgtccgggt 2400

ccatgccagt tgctatgggg tcccccctgc aaaggcttct gaagactgga tgtgttctcg 2460

gtgttcagccaatgccctag aggaggactg ctgtttatgc tcattacgag gaggggccct 2520

gcagagagca aatgatgaca ggtgggtcca cgtttcatgt gctgtggcaa ttctggaagc 2580

aaggtttgtc aacattgcag aaagaagtcc ggtggatgtg agcaaaatcc ccctgccccg 2640

cttcaaactg aaatgtatct tctgtaagaa gcggaggaaa agaactgctg gctgctgtgt 2700

gcagtgttct cacggccgct gcccaactgc cttccatgtg agctgcgccc aggctgccgg 2760

tgtgatgatg cagcctgacg actggccttt tgtggtcttc attacctgct ttcggcacaa 2820

gattcctaat ttggagcgtg ccaagggggc cttgcaaagc atcactgcag gccagaaagt 2880

cattagcaag cataagaacg ggcgcttcta ccagtgtgaa gtggtcaggc tcaccaccga 2940

gaccttctat gaagtcaact ttgatgatgg ctccttcagc gacaatcttt atcctgagga 3000

catagtgagc caggactgtc tccagtttgg tcctcctgct gaaggggaag tggtccaagt 3060

gagatggaca gacggccaag tctatggagc caagtttgtg gcctcccacc ctatccaaat 3120

gtaccaggtg gagtttgagg atggctcaca acttgtggtt aagagagatg atgtatacac 3180

actggatgaa gagcttccca agagagtcaa atctagactg tcagtagcct cagacatgcg 3240

cttcaatgag attttcacag agaaagaggt taagcaagaa aagaaacggc aacgagttat 3300

caactcaaga taccgggaag attatattga gcctgcacta taccgggcca tcatggagta 3360

ggtgcttcca gggtccaagg gattctcagc catccaggca agagcactct gggttccaca 3420

gcacagcagacatggaacgc tgaagtctct gaaagtgaag ttgtaaaaag aaaaggaatg 3480

aaataaccga cccatcatct tctcacccac cctcattgca ttccgctgta gtgaaaggac 3540

gagccatttc tgggcacgtg gcagcagtcg ctgatctccc agctgagggg ctgagcactg 3600

gaatgctgtg gctgcactgg ccccagtcca tagaggggtc aactatgctg gctggactgg 3660

ctgccttgtt cctggcctag gacttagctt cataactatc acctgcaccg actaggctga 3720

ggtgctggta cttgccccaa cccctacttt tgtatttata tgtgtgtgtg tgtgtgcgtg 3780

cgtgcgtgcg tgcgtgtatg tttggtctgg accagcttct gccagcccct ggcctttact 3840

ttcttccttg cctatgcagg gcaaacaaaa tgtgaaattc tgccctcagc tgagctgagt 3900

aagggctcct gggggttggc tggagatggg tgtggcatct gtccaggcct ggaaccgtct 3960

caagacagtg ctggcaaagc tgcagtattg agatgctaag gagctgatgc cacctctttg 4020

tcttccccta aaggagaaca tggggataac atgggtgtgt gcccacaaca ctctaggtgc 4080

agagcccctg tggcaaagta ttacagggtg tgggtgggga ttaccctgaa tcggggattt 4140

taatgatgga agcaggcaga gcctggtggg tgattctgtc aacagaaaat tgcaatcatg 4200

caggggctgg gagggttagg atgaaaaaac tggggccatt ggaggcccac tgtaggtggg 4260

agggagctga ttttggggtg gggggtggga ctagagggca atactgaagg ggttaaacag 4320

gtttttgctc ctcaagaatt tgtttgcctg ggcccaggat tggagggctt cacaccaata 4380

ccctgtgtatacaagaatca gatttataat acttcccctt ttttgttacg tatgaacact 4440

ataaaccaaa ttattttgaa aactggtgca tcaccttgtc cttagcaata aaatgtgttg 4500

agcagaggaa aaaaaaaaaa aaaaaa 4526

<210>2

<211>5675

<212>DNA

<213> Intelligent people

<400>2

agggctcggt cgccagcaac cgagcggggc ccggcccgag cggggcctgg gggtgcgacg 60

ccgagggcgg gggagagcgc gccgctgctc ccggaccggg ccgcgcacgc cgcctcagga 120

accatcactg ttgctggagg cacctgacaa atcctagcga atttttggag catctccacc 180

caggaacctc gccatccaga agtgtgcttc ccgcacagct gcagccatgg ggtctgagga 240

ccacggcgcc cagaacccca gctgtaaaat catgacgttt cgcccaacca tggaagaatt 300

taaagacttc aacaaatacg tggcctacat agagtcgcag ggagcccacc gggcgggcct 360

ggccaagatc atccccccga aggagtggaa gccgcggcag acgtatgatg acatcgacga 420

cgtggtgatc ccggcgccca tccagcaggt ggtgacgggc cagtcgggcc tcttcacgca 480

gtacaatatc cagaagaagg ccatgacagt gggcgagtac cgccgcctgg ccaacagcga 540

gaagtactgt accccgcggc accaggactt tgatgacctt gaacgcaaat actggaagaa 600

cctcaccttt gtctccccga tctacggggc tgacatcagc ggctctttgt atgatgacga 660

cgtggcccag tggaacatcg ggagcctccg gaccatcctg gacatggtgg agcgcgagtg 720

cggcaccatc atcgagggcg tgaacacgcc ctacctgtac ttcggcatgt ggaagaccac 780

cttcgcctgg cacaccgagg acatggacct gtacagcatc aactacctgc actttgggga 840

gcctaagtcc tggtacgcca tcccaccaga gcacggcaag cgcctggagc ggctggccat 900

cggcttcttc cccgggagct cgcagggctg cgacgccttc ctgcggcata agatgaccct 960

catctcgccc atcatcctga agaagtacgg gatccccttc agccggatca cgcaggaggc 1020

cggggaattc atgatcacat ttccctacgg ctaccacgcc ggcttcaatc acgggttcaa 1080

ctgcgcagaa tctaccaact tcgccaccct gcggtggatt gactacggca aagtggccac 1140

tcagtgcacg tgccggaagg acatggtcaa gatctccatg gacgtgttcg tgcgcatcct 1200

gcagcccgag cgctacgagc tgtggaagca gggcaaggac ctcacggtgc tggaccacac 1260

gcggcccacg gcgctcacca gccccgagct gagctcctgg agtgcatccc gggcctcgct 1320

gaaggccaag ctcctccgca ggtctcaccg gaaacggagc cagcccaaga agccgaagcc 1380

cgaagacccc aagttccctg gggagggtac ggctggggca gcgctcctag aggaggctgg 1440

gggcagcgtg aaggaggagg ctgggccgga ggttgacccc gaggaggagg aggaggagcc 1500

gcagccactg ccacacggcc gggaggccga gggcgcagaa gaggacggga ggggcaagct 1560

gcggccaacc aaggccaaga gcgagcggaa gaagaagagc ttcggcctgc tgcccccaca 1620

gctgccgccc ccgcctgctc acttcccctc agaggaggcg ctgtggctgc catccccact 1680

ggagcccccg gtgctgggcc caggccctgc agccatggag gagagccccc tgccggcacc 1740

ccttaatgtc gtgccccctg aggtgcccag tgaggagcta gaggccaagc ctcggcccat 1800

catccccatg ctgtacgtgg tgccgcggcc gggcaaggca gccttcaacc aggagcacgt 1860

gtcctgccag caggcctttg agcactttgc ccagaagggt ccgacctgga aggaaccagt 1920

ttcccccatg gagctgacgg ggccagagga cggtgcagcc agcagtgggg caggtcgcat 1980

ggagaccaaa gcccgggccg gagaggggca ggcaccgtcc acattttcca aattgaagat 2040

ggagatcaag aagagccggc gccatcccct gggccggccg cccacccggt ccccactgtc 2100

ggtggtgaag caggaggcct caagtgacga ggaggcatcc cctttctccg gggaggaaga 2160

tgtgagtgac ccggacgcct tgaggccgct gctgtctctg cagtggaaga acagggcggc 2220

cagcttccag gccgagagga agttcaacgc agcggctgcg cgcacggagc cctactgcgc 2280

catctgcacg ctcttctacc cctactgcca ggccctacag actgagaagg aggcacccat 2340

agcctccctc ggagagggct gcccggccac attaccctcc aaaagccgtc agaagacccg 2400

accgctcatc cctgagatgt gcttcacctc tggcggtgag aacacggagc cgctgcctgc 2460

caactcctac atcggcgacg acgggaccag ccccctgatc gcctgcggca agtgctgcct 2520

gcaggtccat gccagttgct atggcatccg tcccgagctg gtcaatgaag gctggacgtg 2580

ttcccggtgc gcggcccacg cctggactgc ggagtgctgc ctgtgcaacc tgcgaggagg 2640

tgcgctgcag atgaccaccg ataggaggtg gatccacgtg atctgtgcca tcgcagtccc 2700

cgaggcgcgc ttcctgaacg tgattgagcg ccaccctgtg gacatcagcg ccatccccga 2760

gcagcggtgg aagctgaaat gcgtgtactg ccggaagcgg atgaagaagg tgtcaggtgc 2820

ctgtatccag tgctcctacg agcactgctc cacgtccttc cacgtgacct gcgcccacgc 2880

cgcaggcgtg ctcatggagc cggacgactg gccctatgtg gtctccatca cctgcctcaa 2940

gcacaagtcg gggggtcacg ctgtccaact cctgagggcc gtgtccctag gccaggtggt 3000

catcaccaag aaccgcaacg ggctgtacta ccgctgtcgc gtcatcggtg ccgcctcgca 3060

gacctgctac gaagtgaact tcgacgatgg ctcctacagc gacaacctgt accctgagag 3120

catcacgagt agggactgtg tccagctggg acccccttcc gagggggagc tggtggagct 3180

ccggtggact gacggcaacc tctacaaggc caagttcatc tcctccgtca ccagccacat 3240

ctaccaggtg gagtttgagg acgggtccca gctgacggtg aagcgtgggg acatcttcac 3300

cctggaggag gagctgccca agagggtccg ctctcggctg tcactgagca cgggggcacc 3360

gcaggagccc gccttctcgg gggaggaggc caaggccgcc aagcgcccgc gtgtgggcac 3420

cccgcttgcc acggaggact ccgggcggag ccaggactac gtggccttcg tggagagcct 3480

cctgcaggtg cagggccggc ccggagcccc cttctaggac agctggccgc tcaggcgacc 3540

ctcagcccgg cggggaggcc atggcatgcc ccgggcgttc gcttgctgtg aattcctgtc 3600

ctcgtgtccc cgacccccga gaggccacct ccaagccgcg ggtgccccct agggcgacag 3660

gagccagcgg gacgccgcac gcggccccag actcagggag cagggccagg cgggctcggg 3720

ggccggccag gggagcaccc cactcaacta ctcagaattt taaaccatgt aagctctctt 3780

cttctcgaaa aggtgctact gcaatgccct actgagcaac ctttgagatt gtcacttctg 3840

tacataaacc acctttgtga ggctctttct ataaatacat attgtttaaa aaaaagcaag 3900

aaaaaaagga aaacaaagga aaatatcccc aaagttgttt tctagatttg tggctttaag 3960

aaaaacaaaa caaaacaaac acattgtttt tctcagaacc aggattctct gagaggtcag 4020

agcatctcgc tgtttttttg ttgttgtttt aaaatattat gatttggcta cagaccaggc 4080

agggaaagag acccggtaat tggagggtga gcctcggggg gggggcagga cgccccggtt 4140

tcggcacagc ccggtcactc acggcctcgc tctcgcctca ccccggctcc tgggctttga 4200

tggtctggtg ccagtgcctg tgcccactct gtgcctgctg ggaggaggcc caggctctct 4260

ggtggccgcc cctgtgcacc tggccagggg aagcccgggg gtctggggcc tccctccgtc 4320

tgcgcccacc tttgcagaat aaactctctc ctggggtttg tctatctttg tttctctcac 4380

ctgagagaaa cgcaggtgtt ccagaggctt ccttgcagac aaagcacccc tgcacctcct 4440

atggctcagg atgagggagg cccccaggcc cttctggttg gtagtgagtg tggacagctt 4500

cccagctctt cgggtacaac cctgagcagg tcgggggaca cagggccgag gcaggccttc 4560

ggggcccctt tcgcctgctt ccgggcaggg acgaggcctg gtgtcctcgc tccacccacc 4620

cacgctgctg tcacctgagg ggaatctgct tcttaggagt gggttgagct gatagagaaa 4680

aaacggcctt cagcccaggc tgggaagcgc cttctccagg tgcctctccc tcaccagctc 4740

tgcacccctc tggggagcct tccccacctt agctgtctcc tgccccaggg agggatggag 4800

gagataattt gcttatatta aaaacaaaaa atggctgagg caggagtttg ggaccagcct 4860

gggctatata gcaagacccc atcactacaa attttttaca aattagctag gtgtggtggt 4920

gcgcacctgt ggtcccagct actcgggagg ctgtggtggg aggattgctt gagtccagga 4980

ggttgaggct gcagtcagct cagattgcac cactgcactc cagcctgggc aacagagcga 5040

gaccctgtct ccaaaaaaaa aaaaaagcaa tgtttatatt ataaaagagt gtcctaacag 5100

tccccgggct agagaggact aaggaaaaca gagagagtgt tacgcaggag caagcctttc 5160

atttccttgg tgggggaggg gggcggttgc cctggagagg gccggggtcg gggaggttgg 5220

ggggtgtcag ccaaaacgtg gaggtgtccc tctgcacgca gccctcgccc ggcgtggcgc 5280

tgacactgta ttcttatgtt gtttgaaaat gctatttata ttgtaaagaa gcgggcgggt 5340

gcccctgctg cccttgtccc ttgggggtca cacccatccc ctggtgggct cctgggcggc 5400

ctgcgcagat gggccacaga agggcaggcc ggagctgcac actctcccca cgaaggtatc 5460

tctgtgtctt actctgtgca aagacgcggc aaaacccagt gccctggttt ttccccaccc 5520

gagatgaagg atacgctgta ttttttgcct aatgtccctg cctctaggtt cataatgaat 5580

taaaggttca tgaacgctgc gaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 5640

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaa 5675

<210>3

<211>4687

<212>DNA

<213> Intelligent people

<400>3

gccataggtg cgcgtcggcg cccaggagga cgtgtggcgc gtggactaca tcaggtccag 60

ccctgcggga ccccagccag cgcttccggg caaggttctg tgcacctgtt ttctccttct 120

acgcgagtat ctttcccctc cggaaagaat gggatatgcc tgtgtccaaa ggacaagaag 180

atgcgcgcca gcaagcctaa gttaaccaca gcgcggaagt tgagcccaaa gcaagagcgt 240

gccgggcacc tttaagctgt ttgtaagccc acgtgactca ccaagtgcgg gccccagcgg 300

tcacgtgacg gcgcgcgcgc cctcgcgcag ggagagccgg cggtgcgcgc gccttcgccg 360

ctgcctccca cccaccccct cgacgggagg gtgaggcgcg gcgcagtgat cgggcggccg 420

gggtcctgtg cgcgtgcgca gcgaacagct gtcacctagt gcggaacaag tctcccaaat 480

ttcccaaatc tccctgggcc ggaggccact gtcttctctt cctcctccac cgagtcgtgc 540

tctcgcccca acccgcgcgc cagacactgc cctaaccatc atggaggtgg ccgaggtgga 600

aagtcctctg aaccccagct gtaagataat gaccttcaga ccctccatgg aggagttccg 660

ggagttcaac aaataccttg catacatgga gtctaaagga gcccatcgtg cgggtcttgc 720

aaaggtgatt cctcctaagg agtggaagcc aagacagtgc tatgatgaca ttgataattt 780

gctcattcca gcaccaattc agcagatggt cacagggcag tcaggactgt tcactcagta 840

caacatccag aaaaaagcga tgactgtgaa ggagttcagg cagctggcca acagtggcaa 900

atattgtact ccaagatact tggattacga agatttggag cgcaagtact ggaagaactt 960

aacttttgtg gcacctatct atggtgcaga tattaatggg agcatatatg atgagggtgt 1020

ggatgaatgg aacatagctc gcctcaatac agtcttggat gtggttgaag aagagtgtgg 1080

catttctatt gagggtgtaa ataccccata tctctatttt ggcatgtgga agaccacgtt 1140

tgcatggcac accgaagaca tggacctcta tagcattaat tatctccact ttggagagcc 1200

caagtcttgg tatgctatac ctccggagca tggaaaacga cttgaaagac tagctcaagg 1260

ttttttccca agcagctccc aagggtgtga tgcatttctt cgccacaaga tgacattgat 1320

ttctccatca gtattgaaga aatatggtat tccctttgac aagataaccc aggaggctgg 1380

agaattcatg atcactttcc catatggcta ccatgctggt tttaatcatg gtttcaactg 1440

tgcagaatct acaaattttg ctactgtcag atggattgac tatggaaaag ttgccaaatt 1500

gtgcacttgc aggaaagaca tggtgaagat ttcaatggat atctttgtga ggaaatttca 1560

gccagacaga tatcagcttt ggaaacaagg aaaggatata tacaccattg atcacacgaa 1620

gcctactcca gcatccaccc ctgaagtaaa agcatggctg cagaggagga ggaaagtaag 1680

aaaagcatcc cgaagcttcc agtgtgctag gtctacctct aaaaggccta aggctgatga 1740

ggaagaggaa gtgtcagatg aagtcgatgg ggcagaggtc cctaaccccg actcagtcac 1800

agatgacctc aaggtcagtg aaaagtcaga agcagcagtg aagctgagga acacagaagc 1860

atcttcagaa gaagagtcat ctgctagcag gatgcaggtg gagcagaatt tatcagatca 1920

tatcaaactc tcaggaaaca gctgcttaag tacatctgta acagaagaca taaaaactga 1980

ggatgacaaa gcttatgcat atagaagtgt accttctata tccagtgagg ctgatgattc 2040

cattccattg tctagtggct atgagaagcc cgagaaatca gacccatccg agctttcatg 2100

gccaaagtca cctgagtcat gctcatcagt ggcagagagt aatggtgtgt taacagaggg 2160

agaagagagt gatgtggaga gccatgggaa tggccttgaa cctggggaaa tcccagcggt 2220

ccccagtgga gagagaaata gcttcaaagt ccccagtata gcagagggag agaacaaaac 2280

ctctaagagt tggcgccatc cacttagcag gcctccagca agatctccga tgactcttgt 2340

gaagcagcag gcgccaagtg atgaagaatt gcctgaggtt ctgtccattg aggaggaagt 2400

ggaagaaaca gagtcttggg cgaaacctct catccacctt tggcagacga agtcccctaa 2460

cttcgcagct gagcaagagt ataatgcaac agtggccagg atgaagccac actgtgccat 2520

ctgcactctg ctcatgccgt accacaagcc agatagcagc aatgaagaaa atgatgctag 2580

atgggagaca aaattagatg aagtcgttac atcggaggga aagactaagc ccctcatacc 2640

agagatgtgt tttatttata gtgaagaaaa tatagaatat tctccaccca atgccttcct 2700

tgaagaggat ggaacaagtc tccttatttc ctgtgcaaag tgctgcgtac gggttcatgc 2760

aagttgttat ggtattcctt ctcatgagat ctgtgatgga tggctgtgtg cccggtgcaa 2820

aagaaatgcg tggacagcag aatgctgtct ctgcaatttg agaggaggtg ctcttaagca 2880

aacgaagaac aataagtggg cccatgtcat gtgcgccgtt gcggtcccag aagttcgatt 2940

cactaatgtc ccagaaagga cacaaataga tgtaggcaga atacctttac agaggttaaa 3000

attgaaatgc atcttctgca gacaccgggt taagagggtc tctggagcct gcatccagtg 3060

ttcctacggt cgctgcccgg cctccttcca tgtcacttgt gcccatgctg ctggggtact 3120

gatggagcct gatgactggc cttatgtggt gaacattaca tgctttcgac ataaggtcaa 3180

ccccaacgtg aagtccaagg cttgcgagaa ggtcatttcc gtgggtcaaa cggtcatcac 3240

gaagcatcgg aacacccggt attacagttg cagagtgatg gctgtgacat cgcagacctt 3300

ctatgaggtc atgtttgatg atggctcctt tagcagagac acatttcctg aggatatcgt 3360

gagccgagac tgtctgaagc tgggcccacc tgctgaggga gaagtcgtcc aagtcaagtg 3420

gcccgatggc aaactctatg gagcaaaata ttttggatca aatattgccc acatgtacca 3480

ggttgagttt gaagatggat cccagatagc aatgaagaga gaggacatct acactttaga 3540

tgaagagtta cccaagagag tgaaagctcg attttccaca gcctctgaca tgcgatttga 3600

agacacgttt tatggagcag acattatcca aggggagaga aagagacaaa gagtgctgag 3660

ctccaggttt aagaatgaat atgtggccga ccctgtatac cgcacttttt tgaagagctc 3720

tttccagaag aagtgccaga agagacagta gtctgcatac atcgctgcag gccacagagc 3780

agcttgggtt ggaagagaga agatgaaggg acatccttgg ggctgtgccg tgagttttgc 3840

tggcataggt gacagggtgt gtctctgaca gtggtaaatc gggtttccag agtttggtca 3900

ccaaaaatac aaaatacacc caatgaattg gacgcagcaa tctgaaatca tctctagtct 3960

tgctttcact tgtgagcagt tgtcttctat gatcccaaag aagttttcta agtgaaagga 4020

aatactagtg aatcacccac aaggaaaagc cactgccaca gaggaggcgg gtccccttgt 4080

gcggcttagg gccctgtcag gaaacacacg gggacctctc tctctagctc cagcaggtgg 4140

cacctcggta cccagcgggt agggcgataa tttatatatt ttccacagtc agggaaggac 4200

tctcacttat ttgtttcaaa ttgcagtttt tataaaacat ttttaaaaca caaatggcat 4260

gtatgctaat gagatttacc cgtgtgctat ctgtatttcc cttgtacaga acttttacat 4320

ttttgaatat tcctattact tttgattgtg tctgatggga actgagttgt tggcctttgt 4380

gaaatgaaat ttttggctct tgagaaagaa ttcttatgaa ttgttatgcg aattttatat 4440

atttaaagag ggagatctgg ggctgttatt tttaaacact ttttttcata atacatattc 4500

cgagtagata tttataaaat atatgtttct ttcattatgt gtttgtaaaa ttagagttta 4560

aataaatatg ctttgatgca tagttttgaa ctaatgtaac atgatttttc ttttttaaaa 4620

cagcctgaaa atgtactagt gtttaaaaat aaagatttcc attttctcca aaaaaaaaaa 4680

aaaaaaa 4687

<210>4

<211>1572

<212>DNA

<213> Intelligent people

<400>4

atggaaacta tgaagtctaa ggccaactgt gcccagaatc caaattgtaa cataatgata 60

tttcatccaa ccaaagaaga gtttaatgat tttgataaat atattgctta catggaatcc 120

caaggtgcac acagagctgg cttggctaag ataattccac ccaaagaatg gaaagccaga 180

gagacctatg ataatatcag tgaaatctta atagccactc ccctccagca ggtggcctct 240

gggcgggcag gggtgtttac tcaataccat aaaaaaaaga aagccatgac tgtgggggag 300

tatcgccatt tggcaaacag taaaaaatat cagactccac cacaccagaa tttcgaagat 360

ttggagcgaa aatactggaa gaaccgcatc tataattcac cgatttatgg tgctgacatc 420

agtggctcct tgtttgatga aaacactaaa caatggaatc ttgggcacct gggaacaatt 480

caggacctgc tggaaaagga atgtggggtt gtcatagaag gcgtcaatac accctacttg 540

tactttggca tgtggaaaac cacgtttgct tggcatacag aggacatgga cctttacagc 600

atcaactacc tgcaccttgg ggagcccaaa acttggtatg tggtgccccc agaacatggc 660

cagcgcctgg aacgcctggc cagggagctc ttcccaggca gttcccgggg ttgtggggcc 720

ttcctgcggc acaaggtggc cctcatctcg cctacagttc tcaaggaaaa tgggattccc 780

ttcaatcgca taactcagga ggctggagag ttcatggtga cctttcccta tggctaccat 840

gctggcttca accatggttt caactgcgca gaggccatca attttgccac tccgcgatgg 900

attgattatg gcaaaatggc ctcccagtgt agctgtgggg aggcaagggt gaccttttcc 960

atggatgcct tcgtgcgcat cctgcaacct gaacgctatg acctgtggaa acgtgggcaa 1020

gaccgggcag ttgtggacca catggagccc agggtaccag ccagccaaga gctgagcacc 1080

cagaaggaag tccagttacc caggagagca gcgctgggcc tgagacaact cccttcccac 1140

tgggcccggc attccccttg gcctatggct gcccgcagtg ggacacggtg ccacaccctt 1200

gtgtgctctt cactcccacg ccgatctgca gttagtggca ctgctacgca gccccgggct 1260

gctgctgtcc acagctctaa gaagcccagc tcaactccat catccacccc tggtccatct 1320

gcacagatta tccacccgtc aaatggcaga cgtggtcgtg gtcgccctcc tcagaaactg 1380

agagctcagg agctgaccct ccagactcca gccaagaggc ccctcttggc gggcacaaca 1440

tgcacagctt cgggcccaga acctgagccc ctacctgagg atggggcttt gatggacaag 1500

cctgtaccac tgagcccagg gctccagcat cctgtcaagg cttctgggtg cagctgggcc 1560

cctgtgccct aa 1572

<210>5

<211>412

<212>PRT

<213> Intelligent people

<400>5

Met Ala Glu Asn Leu Lys Gly Cys Ser Val Cys Cys Lys Ser Ser Trp

1 5 10 15

Asn Gln Leu Gln Asp Leu Cys Arg Leu Ala Lys Leu Ser Cys Pro Ala

20 25 30

Leu Gly Ile Ser Lys Arg Asn Leu Tyr Asp Phe Glu Val Glu Tyr Leu

35 40 45

Cys Asp Tyr Lys Lys Ile Arg Glu Gln Glu Tyr Tyr Leu Val Lys Trp

50 55 60

Arg Gly Tyr Pro Asp Ser Glu Ser Thr Trp Glu Pro Arg Gln Asn Leu

65 70 75 80

Lys Cys Val Arg Ile Leu Lys Gln Phe His Lys Asp Leu Glu Arg Glu

8590 95

Leu Leu Arg Arg His His Arg Ser Lys Thr Pro Arg His Leu Asp Pro

100 105 110

Ser Leu Ala Asn Tyr Leu Val Gln Lys Ala Lys Gln Arg Arg Ala Leu

115 120 125

Arg Arg Trp Glu Gln Glu Leu Asn Ala Lys Arg Ser His Leu Gly Arg

130 135 140

Ile Thr Val Glu Asn Glu Val Asp Leu Asp Gly Pro Pro Arg Ala Phe

145 150 155 160

Val Tyr Ile Asn Glu Tyr Arg Val Gly Glu Gly Ile Thr Leu Asn Gln

165 170 175

Val Ala Val Gly Cys Glu Cys Gln Asp Cys Leu Trp Ala Pro Thr Gly

180 185 190

Gly Cys Cys Pro Gly Ala Ser Leu His Lys Phe Ala Tyr Asn Asp Gln

195 200 205

Gly Gln Val Arg Leu Arg Ala Gly Leu Pro Ile Tyr Glu Cys Asn Ser

210 215 220

Arg Cys Arg Cys Gly Tyr Asp Cys Pro Asn Arg Val Val Gln Lys Gly

225 230 235 240

Ile Arg Tyr Asp Leu Cys Ile Phe Arg Thr Asp Asp Gly Arg Gly Trp

245 250 255

Gly Val Arg Thr Leu Glu Lys Ile Arg Lys Asn Ser Phe Val Met Glu

260 265 270

Tyr Val Gly Glu Ile Ile Thr Ser Glu Glu Ala Glu Arg Arg Gly Gln

275 280 285

Ile Tyr Asp Arg Gln Gly Ala Thr Tyr Leu Phe Asp Leu Asp Tyr Val

290 295 300

Glu Asp Val Tyr Thr Val Asp Ala Ala Tyr Tyr Gly Asn Ile Ser His

305 310 315 320

Phe Val Asn His Ser Cys Asp Pro Asn Leu Gln Val Tyr Asn Val Phe

325 330 335

Ile Asp Asn Leu Asp Glu Arg Leu Pro Arg Ile Ala Phe Phe Ala Thr

340 345 350

Arg Thr Ile Arg Ala Gly Glu Glu Leu Thr Phe Asp Tyr Asn Met Gln

355 360 365

Val Asp Pro Val Asp Met Glu Ser Thr Arg Met Asp Ser Asn Phe Gly

370 375 380

Leu Ala Gly Leu Pro Gly Ser Pro Lys Lys Arg Val Arg Ile Glu Cys

385 390 395 400

Lys Cys Gly Thr Glu Ser Cys Arg Lys Tyr Leu Phe

405 410

<210>6

<211>350

<212>PRT

<213> Intelligent people

<400>6

Met Glu Tyr Tyr Leu Val Lys Trp Lys Gly Trp Pro Asp Ser Thr Asn

1 5 10 15

Thr Trp Glu Pro Leu Gln Asn Leu Lys Cys Pro Leu Leu Leu Gln Gln

20 25 30

Phe Ser Asn Asp Lys His Asn Tyr Leu Ser Gln Val Lys Lys Gly Lys

35 40 45

Ala Ile Thr Pro Lys Asp Asn Asn Lys Thr Leu Lys Pro Ala Ile Ala

50 55 60

Glu Tyr Ile Val Lys Lys Ala Lys Gln Arg Ile Ala Leu Gln Arg Trp

6570 75 80

Gln Asp Glu Leu Asn Arg Arg Lys Asn His Lys Gly Met Ile Phe Val

85 90 95

Glu Asn Thr Val Asp Leu Glu Gly Pro Pro Ser Asp Phe Tyr Tyr Ile

100 105 110

Asn Glu Tyr Lys Pro Ala Pro Gly Ile Ser Leu Val Asn Glu Ala Thr

115 120 125

Phe Gly Cys Ser Cys Thr Asp Cys Phe Phe Gln Lys Cys Cys Pro Ala

130 135 140

Glu Ala Gly Val Leu Leu Ala Tyr Asn Lys Asn Gln Gln Ile Lys Ile

145 150 155 160

Pro Pro Gly Thr Pro Ile Tyr Glu Cys Asn Ser Arg Cys Gln Cys Gly

165 170 175

Pro Asp Cys Pro Asn Arg Ile Val Gln Lys Gly Thr Gln Tyr Ser Leu

180 185 190

Cys Ile Phe Arg Thr Ser Asn Gly Arg Gly Trp Gly Val Lys Thr Leu

195 200 205

Val Lys Ile Lys Arg Met Ser Phe Val Met Glu Tyr Val Gly Glu Val

210 215 220

Ile Thr Ser Glu Glu Ala Glu Arg Arg Gly Gln Phe Tyr Asp Asn Lys

225 230 235 240

Gly Ile Thr Tyr Leu Phe Asp Leu Asp Tyr Glu Ser Asp Glu Phe Thr

245 250 255

Val Asp Ala Ala Arg Tyr Gly Asn Val Ser His Phe Val Asn His Ser

260 265 270

Cys Asp Pro Asn Leu Gln Val Phe Asn Val Phe Ile Asp Asn Leu Asp

275 280 285

Thr Arg Leu Pro Arg Ile Ala Leu Phe Ser Thr Arg Thr Ile Asn Ala

290 295 300

Gly Glu Glu Leu Thr Phe Asp Tyr Gln Met Lys Gly Ser Gly Asp Ile

305 310 315 320

Ser Ser Asp Ser Ile Asp His Ser Pro Ala Lys Lys Arg Val Arg Thr

325 330 335

Val Cys Lys Cys Gly Ala Val Thr Cys Arg Gly Tyr Leu Asn

340 345 350

<210>7

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic oligonucleotides

<220>

<223> description of the combination DNA/RNA molecules: synthetic oligonucleotides

<400>7

gaaacgaguc cguauugaat t 21

<210>8

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic oligonucleotides

<220>

<223> description of the combination DNA/RNA molecules: synthetic oligonucleotides

<400>8

uucaauacgg acucguuuct t 21

<210>9

<211>1064

<212>PRT

<213> Intelligent people

<400>9

Met Ala Ser Glu Ser Glu Thr Leu Asn Pro Ser AlaArg Ile Met Thr

1 5 10 15

Phe Tyr Pro Thr Met Glu Glu Phe Arg Asn Phe Ser Arg Tyr Ile Ala

20 25 30

Tyr Ile Glu Ser Gln Gly Ala His Arg Ala Gly Leu Ala Lys Val Val

35 40 45

Pro Pro Lys Glu Trp Lys Pro Arg Ala Ser Tyr Asp Asp Ile Asp Asp

50 55 60

Leu Val Ile Pro Ala Pro Ile Gln Gln Leu Val Thr Gly Gln Ser Gly

65 70 75 80

Leu Phe Thr Gln Tyr Asn Ile Gln Lys Lys Ala Met Thr Val Arg Glu

85 90 95

Phe Arg Lys Ile Ala Asn Ser Asp Lys Tyr Cys Thr Pro Arg Tyr Ser

100 105 110

Glu Phe Glu Glu Leu Glu Arg Lys Tyr Trp Lys Asn Leu Thr Phe Asn

115 120 125

Pro Pro Ile Tyr Gly Ala Asp Val Asn Gly Thr Leu Tyr Glu Lys His

130 135 140

ValAsp Glu Trp Asn Ile Gly Arg Leu Arg Thr Ile Leu Asp Leu Val

145 150 155 160

Glu Lys Glu Ser Gly Ile Thr Ile Glu Gly Val Asn Thr Pro Tyr Leu

165 170 175

Tyr Phe Gly Met Trp Lys Thr Ser Phe Ala Trp His Thr Glu Asp Met

180 185 190

Asp Leu Tyr Ser Ile Asn Tyr Leu His Phe Gly Glu Pro Lys Ser Trp

195 200 205

Tyr Ser Val Pro Pro Glu His Gly Lys Arg Leu Glu Arg Leu Ala Lys

210 215 220

Gly Phe Phe Pro Gly Ser Ala Gln Ser Cys Glu Ala Phe Leu Arg His

225 230 235 240

Lys Met Thr Leu Ile Ser Pro Leu Met Leu Lys Lys Tyr Gly Ile Pro

245 250 255

Phe Asp Lys Val Thr Gln Glu Ala Gly Glu Phe Met Ile Thr Phe Pro

260 265 270

Tyr Gly Tyr His Ala Gly Phe Asn His Gly Phe Asn Cys Ala Glu Ser

275 280285

Thr Asn Phe Ala Thr Arg Arg Trp Ile Glu Tyr Gly Lys Gln Ala Val

290 295 300

Leu Cys Ser Cys Arg Lys Asp Met Val Lys Ile Ser Met Asp Val Phe

305 310 315 320

Val Arg Lys Phe Gln Pro Glu Arg Tyr Lys Leu Trp Lys Ala Gly Lys

325 330 335

Asp Asn Thr Val Ile Asp His Thr Leu Pro Thr Pro Glu Ala Ala Glu

340 345 350

Phe Leu Lys Glu Ser Glu Leu Pro Pro Arg Ala Gly Asn Glu Glu Glu

355 360 365

Cys Pro Glu Glu Asp Met Glu Gly Val Glu Asp Gly Glu Glu Gly Asp

370 375 380

Leu Lys Thr Ser Leu Ala Lys His Arg Ile Gly Thr Lys Arg His Arg

385 390 395 400

Val Cys Leu Glu Ile Pro Gln Glu Val Ser Gln Ser Glu Leu Phe Pro

405 410 415

Lys Glu Asp Leu Ser Ser Glu Gln Tyr Glu Met Thr Glu Cys Pro Ala

420 425 430

Ala Leu Ala Pro Val Arg Pro Thr His Ser Ser Val Arg Gln Val Glu

435 440 445

Asp Gly Leu Thr Phe Pro Asp Tyr Ser Asp Ser Thr Glu Val Lys Phe

450 455 460

Glu Glu Leu Lys Asn Val Lys Leu Glu Glu Glu Asp Glu Glu Glu Glu

465 470 475 480

Gln Ala Ala Ala Ala Leu Asp Leu Ser Val Asn Pro Ala Ser Val Gly

485 490 495

Gly Arg Leu Val Phe Ser Gly Ser Lys Lys Lys Ser Ser Ser Ser Leu

500 505 510

Gly Ser Gly Ser Ser Arg Asp Ser Ile Ser Ser Asp Ser Glu Thr Ser

515 520 525

Glu Pro Leu Ser Cys Arg Ala Gln Gly Gln Thr Gly Val Leu Thr Val

530 535 540

His Ser Tyr Ala Lys Gly Asp Gly Arg Val Thr Val Gly Glu Pro Cys

545 550 555 560

Thr Arg Lys Lys Gly Ser Ala Ala Arg Ser Phe Ser Glu Arg Glu Leu

565 570 575

Ala Glu Val Ala Asp Glu Tyr Met Phe Ser Leu Glu Glu Asn Lys Lys

580 585 590

Ser Lys Gly Arg Arg Gln Pro Leu Ser Lys Leu Pro Arg His His Pro

595 600 605

Leu Val Leu Gln Glu Cys Val Ser Asp Asp Glu Thr Ser Glu Gln Leu

610 615 620

Thr Pro Glu Glu Glu Ala Glu Glu Thr Glu Ala Trp Ala Lys Pro Leu

625 630 635 640

Ser Gln Leu Trp Gln Asn Arg Pro Pro Asn Phe Glu Ala Glu Lys Glu

645 650 655

Phe Asn Glu Thr Met Ala Gln Gln Ala Pro His Cys Ala Val Cys Met

660 665 670

Ile Phe Gln Thr Tyr His Gln Val Glu Phe Gly Gly Phe Asn Gln Asn

675 680 685

Cys Gly Asn Ala Ser Asp Leu Ala Pro Gln Lys Gln Arg Thr Lys Pro

690 695 700

LeuIle Pro Glu Met Cys Phe Thr Ser Thr Gly Cys Ser Thr Asp Ile

705 710 715 720

Asn Leu Ser Thr Pro Tyr Leu Glu Glu Asp Gly Thr Ser Ile Leu Val

725 730 735

Ser Cys Lys Lys Cys Ser Val Arg Val His Ala Ser Cys Tyr Gly Val

740 745 750

Pro Pro Ala Lys Ala Ser Glu Asp Trp Met Cys Ser Arg Cys Ser Ala

755 760 765

Asn Ala Leu Glu Glu Asp Cys Cys Leu Cys Ser Leu Arg Gly Gly Ala

770 775 780

Leu Gln Arg Ala Asn Asp Asp Arg Trp Val His Val Ser Cys Ala Val

785 790 795 800

Ala Ile Leu Glu Ala Arg Phe Val Asn Ile Ala Glu Arg Ser Pro Val

805 810 815

Asp Val Ser Lys Ile Pro Leu Pro Arg Phe Lys Leu Lys Cys Ile Phe

820 825 830

Cys Lys Lys Arg Arg Lys Arg Thr Ala Gly Cys Cys Val Gln Cys Ser

835 840845

His Gly Arg Cys Pro Thr Ala Phe His Val Ser Cys Ala Gln Ala Ala

850 855 860

Gly Val Met Met Gln Pro Asp Asp Trp Pro Phe Val Val Phe Ile Thr

865 870 875 880

Cys Phe Arg His Lys Ile Pro Asn Leu Glu Arg Ala Lys Gly Ala Leu

885 890 895

Gln Ser Ile Thr Ala Gly Gln Lys Val Ile Ser Lys His Lys Asn Gly

900 905 910

Arg Phe Tyr Gln Cys Glu Val Val Arg Leu Thr Thr Glu Thr Phe Tyr

915 920 925

Glu Val Asn Phe Asp Asp Gly Ser Phe Ser Asp Asn Leu Tyr Pro Glu

930 935 940

Asp Ile Val Ser Gln Asp Cys Leu Gln Phe Gly Pro Pro Ala Glu Gly

945 950 955 960

Glu Val Val Gln Val Arg Trp Thr Asp Gly Gln Val Tyr Gly Ala Lys

965 970 975

Phe Val Ala Ser His Pro Ile Gln Met Tyr Gln Val Glu Phe Glu Asp

980 985 990

Gly Ser Gln Leu Val Val Lys Arg Asp Asp Val Tyr Thr Leu Asp Glu

995 1000 1005

Glu Leu Pro Lys Arg Val Lys Ser Arg Leu Ser Val Ala Ser Asp

1010 1015 1020

Met Arg Phe Asn Glu Ile Phe Thr Glu Lys Glu Val Lys Gln Glu

1025 1030 1035

Lys Lys Arg Gln Arg Val Ile Asn Ser Arg Tyr Arg Glu Asp Tyr

1040 1045 1050

Ile Glu Pro Ala Leu Tyr Arg Ala Ile Met Glu

1055 1060

<210>10

<211>1096

<212>PRT

<213> Intelligent people

<400>10

Met Gly Ser Glu Asp His Gly Ala Gln Asn Pro Ser Cys Lys Ile Met

1 5 10 15

Thr Phe Arg Pro Thr Met Glu Glu Phe Lys Asp Phe Asn Lys Tyr Val

20 25 30

Ala Tyr Ile Glu Ser Gln Gly Ala His Arg Ala Gly Leu Ala Lys Ile

35 40 45

Ile Pro Pro Lys Glu Trp Lys Pro Arg Gln Thr Tyr Asp Asp Ile Asp

50 55 60

Asp Val Val Ile Pro Ala Pro Ile Gln Gln Val Val Thr Gly Gln Ser

65 70 75 80

Gly Leu Phe Thr Gln Tyr Asn Ile Gln Lys Lys Ala Met Thr Val Gly

85 90 95

Glu Tyr Arg Arg Leu Ala Asn Ser Glu Lys Tyr Cys Thr Pro Arg His

100 105 110

Gln Asp Phe Asp Asp Leu Glu Arg Lys Tyr Trp Lys Asn Leu Thr Phe

115 120 125

Val Ser Pro Ile Tyr Gly Ala Asp Ile Ser Gly Ser Leu Tyr Asp Asp

130 135 140

Asp Val Ala Gln Trp Asn Ile Gly Ser Leu Arg Thr Ile Leu Asp Met

145 150 155 160

Val Glu Arg Glu Cys Gly Thr Ile Ile Glu Gly Val Asn Thr Pro Tyr

165 170 175

Leu Tyr Phe Gly Met Trp Lys Thr Thr Phe Ala Trp His Thr Glu Asp

180 185 190

Met Asp Leu Tyr Ser Ile Asn Tyr Leu His Phe Gly Glu Pro Lys Ser

195 200 205

Trp Tyr Ala Ile Pro Pro Glu His Gly Lys Arg Leu Glu Arg Leu Ala

210 215 220

Ile Gly Phe Phe Pro Gly Ser Ser Gln Gly Cys Asp Ala Phe Leu Arg

225 230 235 240

His Lys Met Thr Leu Ile Ser Pro Ile Ile Leu Lys Lys Tyr Gly Ile

245 250 255

Pro Phe Ser Arg Ile Thr Gln Glu Ala Gly Glu Phe Met Ile Thr Phe

260 265 270

Pro Tyr Gly Tyr His Ala Gly Phe Asn His Gly Phe Asn Cys Ala Glu

275 280 285

Ser Thr Asn Phe Ala Thr Leu Arg Trp Ile Asp Tyr Gly Lys Val Ala

290 295 300

Thr Gln Cys Thr Cys Arg Lys Asp Met Val Lys Ile Ser Met Asp Val

305310 315 320

Phe Val Arg Ile Leu Gln Pro Glu Arg Tyr Glu Leu Trp Lys Gln Gly

325 330 335

Lys Asp Leu Thr Val Leu Asp His Thr Arg Pro Thr Ala Leu Thr Ser

340 345 350

Pro Glu Leu Ser Ser Trp Ser Ala Ser Arg Ala Ser Leu Lys Ala Lys

355 360 365

Leu Leu Arg Arg Ser His Arg Lys Arg Ser Gln Pro Lys Lys Pro Lys

370 375 380

Pro Glu Asp Pro Lys Phe Pro Gly Glu Gly Thr Ala Gly Ala Ala Leu

385 390 395 400

Leu Glu Glu Ala Gly Gly Ser Val Lys Glu Glu Ala Gly Pro Glu Val

405 410 415

Asp Pro Glu Glu Glu Glu Glu Glu Pro Gln Pro Leu Pro His Gly Arg

420 425 430

Glu Ala Glu Gly Ala Glu Glu Asp Gly Arg Gly Lys Leu Arg Pro Thr

435 440 445

Lys Ala Lys Ser Glu Arg Lys Lys Lys Ser Phe Gly Leu Leu Pro Pro

450 455 460

Gln Leu Pro Pro Pro Pro Ala His Phe Pro Ser Glu Glu Ala Leu Trp

465 470 475 480

Leu Pro Ser Pro Leu Glu Pro Pro Val Leu Gly Pro Gly Pro Ala Ala

485 490 495

Met Glu Glu Ser Pro Leu Pro Ala Pro Leu Asn Val Val Pro Pro Glu

500 505 510

Val Pro Ser Glu Glu Leu Glu Ala Lys Pro Arg Pro Ile Ile Pro Met

515 520 525

Leu Tyr Val Val Pro Arg Pro Gly Lys Ala Ala Phe Asn Gln Glu His

530 535 540

Val Ser Cys Gln Gln Ala Phe Glu His Phe Ala Gln Lys Gly Pro Thr

545 550 555 560

Trp Lys Glu Pro Val Ser Pro Met Glu Leu Thr Gly Pro Glu Asp Gly

565 570 575

Ala Ala Ser Ser Gly Ala Gly Arg Met Glu Thr Lys Ala Arg Ala Gly

580 585 590

GluGly Gln Ala Pro Ser Thr Phe Ser Lys Leu Lys Met Glu Ile Lys

595 600 605

Lys Ser Arg Arg His Pro Leu Gly Arg Pro Pro Thr Arg Ser Pro Leu

610 615 620

Ser Val Val Lys Gln Glu Ala Ser Ser Asp Glu Glu Ala Ser Pro Phe

625 630 635 640

Ser Gly Glu Glu Asp Val Ser Asp Pro Asp Ala Leu Arg Pro Leu Leu

645 650 655

Ser Leu Gln Trp Lys Asn Arg Ala Ala Ser Phe Gln Ala Glu Arg Lys

660 665 670

Phe Asn Ala Ala Ala Ala Arg Thr Glu Pro Tyr Cys Ala Ile Cys Thr

675 680 685

Leu Phe Tyr Pro Tyr Cys Gln Ala Leu Gln Thr Glu Lys Glu Ala Pro

690 695 700

Ile Ala Ser Leu Gly Glu Gly Cys Pro Ala Thr Leu Pro Ser Lys Ser

705 710 715 720

Arg Gln Lys Thr Arg Pro Leu Ile Pro Glu Met Cys Phe Thr Ser Gly

725 730 735

Gly Glu Asn Thr Glu Pro Leu Pro Ala Asn Ser Tyr Ile Gly Asp Asp

740 745 750

Gly Thr Ser Pro Leu Ile Ala Cys Gly Lys Cys Cys Leu Gln Val His

755 760 765

Ala Ser Cys Tyr Gly Ile Arg Pro Glu Leu Val Asn Glu Gly Trp Thr

770 775 780

Cys Ser Arg Cys Ala Ala His Ala Trp Thr Ala Glu Cys Cys Leu Cys

785 790 795 800

Asn Leu Arg Gly Gly Ala Leu Gln Met Thr Thr Asp Arg Arg Trp Ile

805 810 815

His Val Ile Cys Ala Ile Ala Val Pro Glu Ala Arg Phe Leu Asn Val

820 825 830

Ile Glu Arg His Pro Val Asp Ile Ser Ala Ile Pro Glu Gln Arg Trp

835 840 845

Lys Leu Lys Cys Val Tyr Cys Arg Lys Arg Met Lys Lys Val Ser Gly

850 855 860

Ala Cys Ile Gln Cys Ser Tyr Glu His Cys Ser Thr Ser Phe His Val

865870 875 880

Thr Cys Ala His Ala Ala Gly Val Leu Met Glu Pro Asp Asp Trp Pro

885 890 895

Tyr Val Val Ser Ile Thr Cys Leu Lys His Lys Ser Gly Gly His Ala

900 905 910

Val Gln Leu Leu Arg Ala Val Ser Leu Gly Gln Val Val Ile Thr Lys

915 920 925

Asn Arg Asn Gly Leu Tyr Tyr Arg Cys Arg Val Ile Gly Ala Ala Ser

930 935 940

Gln Thr Cys Tyr Glu Val Asn Phe Asp Asp Gly Ser Tyr Ser Asp Asn

945 950 955 960

Leu Tyr Pro Glu Ser Ile Thr Ser Arg Asp Cys Val Gln Leu Gly Pro

965 970 975

Pro Ser Glu Gly Glu Leu Val Glu Leu Arg Trp Thr Asp Gly Asn Leu

980 985 990

Tyr Lys Ala Lys Phe Ile Ser Ser Val Thr Ser His Ile Tyr Gln Val

995 1000 1005

Glu Phe Glu Asp Gly Ser Gln Leu Thr Val Lys Arg Gly Asp Ile

1010 1015 1020

Phe Thr Leu Glu Glu Glu Leu Pro Lys Arg Val Arg Ser Arg Leu

1025 1030 1035

Ser Leu Ser Thr Gly Ala Pro Gln Glu Pro Ala Phe Ser Gly Glu

1040 1045 1050

Glu Ala Lys Ala Ala Lys Arg Pro Arg Val Gly Thr Pro Leu Ala

1055 1060 1065

Thr Glu Asp Ser Gly Arg Ser Gln Asp Tyr Val Ala Phe Val Glu

1070 1075 1080

Ser Leu Leu Gln Val Gln Gly Arg Pro Gly Ala Pro Phe

1085 1090 1095

<210>11

<211>1056

<212>PRT

<213> Intelligent people

<400>11

Met Glu Val Ala Glu Val Glu Ser Pro Leu Asn Pro Ser Cys Lys Ile

1 5 10 15

Met Thr Phe Arg Pro Ser Met Glu Glu Phe Arg Glu Phe Asn Lys Tyr

20 25 30

Leu Ala Tyr Met Glu Ser Lys Gly Ala His Arg Ala Gly Leu Ala Lys

35 40 45

Val Ile Pro Pro Lys Glu Trp Lys Pro Arg Gln Cys Tyr Asp Asp Ile

50 55 60

Asp Asn Leu Leu Ile Pro Ala Pro Ile Gln Gln Met Val Thr Gly Gln

65 70 75 80

Ser Gly Leu Phe Thr Gln Tyr Asn Ile Gln Lys Lys Ala Met Thr Val

85 90 95

Lys Glu Phe Arg Gln Leu Ala Asn Ser Gly Lys Tyr Cys Thr Pro Arg

100 105 110

Tyr Leu Asp Tyr Glu Asp Leu Glu Arg Lys Tyr Trp Lys Asn Leu Thr

115 120 125

Phe Val Ala Pro Ile Tyr Gly Ala Asp Ile Asn Gly Ser Ile Tyr Asp

130 135 140

Glu Gly Val Asp Glu Trp Asn Ile Ala Arg Leu Asn Thr Val Leu Asp

145 150 155 160

Val Val Glu Glu Glu Cys Gly Ile Ser Ile Glu Gly Val Asn Thr Pro

165 170 175

Tyr Leu Tyr Phe Gly Met Trp Lys Thr Thr Phe Ala Trp His Thr Glu

180 185 190

Asp Met Asp Leu Tyr Ser Ile Asn Tyr Leu His Phe Gly Glu Pro Lys

195 200 205

Ser Trp Tyr Ala Ile Pro Pro Glu His Gly Lys Arg Leu Glu Arg Leu

210 215 220

Ala Gln Gly Phe Phe Pro Ser Ser Ser Gln Gly Cys Asp Ala Phe Leu

225 230 235 240

Arg His Lys Met Thr Leu Ile Ser Pro Ser Val Leu Lys Lys Tyr Gly

245 250 255

Ile Pro Phe Asp Lys Ile Thr Gln Glu Ala Gly Glu Phe Met Ile Thr

260 265 270

Phe Pro Tyr Gly Tyr His Ala Gly Phe Asn His Gly Phe Asn Cys Ala

275 280 285

Glu Ser Thr Asn Phe Ala Thr Val Arg Trp Ile Asp Tyr Gly Lys Val

290 295 300

Ala Lys Leu Cys Thr Cys Arg Lys Asp Met Val Lys Ile Ser Met Asp

305 310 315 320

Ile Phe Val Arg Lys Phe Gln Pro Asp Arg Tyr Gln Leu Trp Lys Gln

325 330 335

Gly Lys Asp Ile Tyr Thr Ile Asp His Thr Lys Pro Thr Pro Ala Ser

340 345 350

Thr Pro Glu Val Lys Ala Trp Leu Gln Arg Arg Arg Lys Val Arg Lys

355 360 365

Ala Ser Arg Ser Phe Gln Cys Ala Arg Ser Thr Ser Lys Arg Pro Lys

370 375 380

Ala Asp Glu Glu Glu Glu Val Ser Asp Glu Val Asp Gly Ala Glu Val

385 390 395 400

Pro Asn Pro Asp Ser Val Thr Asp Asp Leu Lys Val Ser Glu Lys Ser

405 410 415

Glu Ala Ala Val Lys Leu Arg Asn Thr Glu Ala Ser Ser Glu Glu Glu

420 425 430

Ser Ser Ala Ser Arg Met Gln Val Glu Gln Asn Leu Ser Asp His Ile

435 440 445

Lys Leu Ser Gly Asn Ser Cys Leu Ser Thr Ser Val Thr Glu Asp Ile

450 455 460

Lys Thr Glu Asp Asp Lys Ala Tyr Ala Tyr Arg Ser Val Pro Ser Ile

465 470 475 480

Ser Ser Glu Ala Asp Asp Ser Ile Pro Leu Ser Ser Gly Tyr Glu Lys

485 490 495

Pro Glu Lys Ser Asp Pro Ser Glu Leu Ser Trp Pro Lys Ser Pro Glu

500 505 510

Ser Cys Ser Ser Val Ala Glu Ser Asn Gly Val Leu Thr Glu Gly Glu

515 520 525

Glu Ser Asp Val Glu Ser His Gly Asn Gly Leu Glu Pro Gly Glu Ile

530 535 540

Pro Ala Val Pro Ser Gly Glu Arg Asn Ser Phe Lys Val Pro Ser Ile

545 550 555 560

Ala Glu Gly Glu Asn Lys Thr Ser Lys Ser Trp Arg His Pro Leu Ser

565 570 575

Arg Pro Pro Ala Arg Ser Pro Met Thr Leu Val Lys Gln Gln Ala Pro

580 585 590

Ser Asp Glu Glu Leu Pro Glu Val Leu Ser Ile Glu Glu Glu Val Glu

595 600 605

Glu Thr Glu Ser Trp Ala Lys Pro Leu Ile His Leu Trp Gln Thr Lys

610 615 620

Ser Pro Asn Phe Ala Ala Glu Gln Glu Tyr Asn Ala Thr Val Ala Arg

625 630 635 640

Met Lys Pro His Cys Ala Ile Cys Thr Leu Leu Met Pro Tyr His Lys

645 650 655

Pro Asp Ser Ser Asn Glu Glu Asn Asp Ala Arg Trp Glu Thr Lys Leu

660 665 670

Asp Glu Val Val Thr Ser Glu Gly Lys Thr Lys Pro Leu Ile Pro Glu

675 680 685

Met Cys Phe Ile Tyr Ser Glu Glu Asn Ile Glu Tyr Ser Pro Pro Asn

690 695 700

Ala Phe Leu Glu Glu Asp Gly Thr Ser Leu Leu Ile Ser Cys Ala Lys

705 710 715 720

Cys Cys Val Arg Val His Ala Ser Cys Tyr Gly Ile Pro Ser His Glu

725 730 735

Ile Cys Asp Gly Trp Leu Cys Ala Arg Cys Lys Arg Asn Ala Trp Thr

740 745 750

Ala Glu Cys Cys Leu Cys Asn Leu Arg Gly Gly Ala Leu Lys Gln Thr

755 760 765

Lys Asn Asn Lys Trp Ala His Val Met Cys Ala Val Ala Val Pro Glu

770 775 780

Val Arg Phe Thr Asn Val Pro Glu Arg Thr Gln Ile Asp Val Gly Arg

785 790 795 800

Ile Pro Leu Gln Arg Leu Lys Leu Lys Cys Ile Phe Cys Arg His Arg

805 810 815

Val Lys Arg Val Ser Gly Ala Cys Ile Gln Cys Ser Tyr Gly Arg Cys

820 825 830

Pro Ala Ser Phe His Val Thr Cys Ala His Ala Ala Gly Val Leu Met

835 840 845

Glu Pro Asp Asp Trp Pro Tyr Val Val Asn Ile Thr Cys Phe Arg His

850 855 860

Lys Val Asn Pro Asn Val Lys Ser Lys Ala Cys Glu Lys Val Ile Ser

865 870 875 880

Val Gly Gln Thr Val Ile Thr Lys His Arg Asn Thr Arg Tyr Tyr Ser

885 890 895

Cys Arg Val Met Ala Val Thr Ser Gln Thr Phe Tyr Glu Val Met Phe

900 905 910

Asp Asp Gly Ser Phe Ser Arg Asp Thr Phe Pro Glu Asp Ile Val Ser

915 920 925

Arg Asp Cys Leu Lys Leu Gly Pro Pro Ala Glu Gly Glu Val Val Gln

930 935 940

Val Lys Trp Pro Asp Gly Lys Leu Tyr Gly Ala Lys Tyr Phe Gly Ser

945 950 955 960

Asn Ile Ala His Met Tyr Gln Val Glu Phe Glu Asp Gly Ser Gln Ile

965 970 975

Ala Met Lys Arg Glu Asp Ile Tyr Thr Leu Asp Glu Glu Leu Pro Lys

980 985 990

Arg Val Lys Ala Arg Phe Ser Thr Ala Ser Asp Met Arg Phe Glu Asp

995 1000 1005

Thr PheTyr Gly Ala Asp Ile Ile Gln Gly Glu Arg Lys Arg Gln

1010 1015 1020

Arg Val Leu Ser Ser Arg Phe Lys Asn Glu Tyr Val Ala Asp Pro

1025 1030 1035

Val Tyr Arg Thr Phe Leu Lys Ser Ser Phe Gln Lys Lys Cys Gln

1040 1045 1050

Lys Arg Gln

1055

<210>12

<211>523

<212>PRT

<213> Intelligent people

<400>12

Met Glu Thr Met Lys Ser Lys Ala Asn Cys Ala Gln Asn Pro Asn Cys

1 5 10 15

Asn Ile Met Ile Phe His Pro Thr Lys Glu Glu Phe Asn Asp Phe Asp

20 25 30

Lys Tyr Ile Ala Tyr Met Glu Ser Gln Gly Ala His Arg Ala Gly Leu

35 40 45

Ala Lys Ile Ile Pro Pro Lys Glu Trp Lys Ala Arg Glu Thr Tyr Asp

50 55 60

Asn Ile Ser Glu Ile Leu Ile Ala Thr Pro Leu Gln Gln Val Ala Ser

65 70 75 80

Gly Arg Ala Gly Val Phe Thr Gln Tyr His Lys Lys Lys Lys Ala Met

85 90 95

Thr Val Gly Glu Tyr Arg His Leu Ala Asn Ser Lys Lys Tyr Gln Thr

100 105 110

Pro Pro His Gln Asn Phe Glu Asp Leu Glu Arg Lys Tyr Trp Lys Asn

115 120 125

Arg Ile Tyr Asn Ser Pro Ile Tyr Gly Ala Asp Ile Ser Gly Ser Leu

130 135 140

Phe Asp Glu Asn Thr Lys Gln Trp Asn Leu Gly His Leu Gly Thr Ile

145 150 155 160

Gln Asp Leu Leu Glu Lys Glu Cys Gly Val Val Ile Glu Gly Val Asn

165 170 175

Thr Pro Tyr Leu Tyr Phe Gly Met Trp Lys Thr Thr Phe Ala Trp His

180 185 190

Thr Glu Asp Met Asp Leu Tyr Ser Ile Asn Tyr Leu His Leu Gly Glu

195 200 205

Pro Lys Thr Trp Tyr Val Val Pro Pro Glu His Gly Gln Arg Leu Glu

210 215 220

Arg Leu Ala Arg Glu Leu Phe Pro Gly Ser Ser Arg Gly Cys Gly Ala

225 230 235 240

Phe Leu Arg His Lys Val Ala Leu Ile Ser Pro Thr Val Leu Lys Glu

245 250 255

Asn Gly Ile Pro Phe Asn Arg Ile Thr Gln Glu Ala Gly Glu Phe Met

260 265 270

Val Thr Phe Pro Tyr Gly Tyr His Ala Gly Phe Asn His Gly Phe Asn

275 280 285

Cys Ala Glu Ala Ile Asn Phe Ala Thr Pro Arg Trp Ile Asp Tyr Gly

290 295 300

Lys Met Ala Ser Gln Cys Ser Cys Gly Glu Ala Arg Val Thr Phe Ser

305 310 315 320

Met Asp Ala Phe Val Arg Ile Leu Gln Pro Glu Arg Tyr Asp Leu Trp

325 330 335

Lys Arg Gly Gln Asp Arg Ala Val Val Asp His Met Glu Pro Arg Val

340 345 350

Pro Ala Ser Gln Glu Leu Ser Thr Gln Lys Glu Val Gln Leu Pro Arg

355 360 365

Arg Ala Ala Leu Gly Leu Arg Gln Leu Pro Ser His Trp Ala Arg His

370 375 380

Ser Pro Trp Pro Met Ala Ala Arg Ser Gly Thr Arg Cys His Thr Leu

385 390 395 400

Val Cys Ser Ser Leu Pro Arg Arg Ser Ala Val Ser Gly Thr Ala Thr

405 410 415

Gln Pro Arg Ala Ala Ala Val His Ser Ser Lys Lys Pro Ser Ser Thr

420 425 430

Pro Ser Ser Thr Pro Gly Pro Ser Ala Gln Ile Ile His Pro Ser Asn

435 440 445

Gly Arg Arg Gly Arg Gly Arg Pro Pro Gln Lys Leu Arg Ala Gln Glu

450 455 460

Leu Thr Leu Gln Thr Pro Ala Lys Arg Pro Leu Leu Ala Gly Thr Thr

465 470 475 480

Cys Thr AlaSer Gly Pro Glu Pro Glu Pro Leu Pro Glu Asp Gly Ala

485 490 495

Leu Met Asp Lys Pro Val Pro Leu Ser Pro Gly Leu Gln His Pro Val

500 505 510

Lys Ala Ser Gly Cys Ser Trp Ala Pro Val Pro

515 520

<210>13

<211>423

<212>PRT

<213> Intelligent people

<400>13

Met Glu Thr Met Lys Ser Lys Ala Asn Cys Ala Gln Asn Pro Asn Cys

1 5 10 15

Asn Ile Met Ile Phe His Pro Thr Lys Glu Glu Phe Asn Asp Phe Asp

20 25 30

Lys Tyr Ile Ala Tyr Met Glu Ser Gln Gly Ala His Arg Ala Gly Leu

35 40 45

Ala Lys Ile Ile Pro Pro Lys Glu Trp Lys Ala Arg Glu Thr Tyr Asp

50 55 60

Asn Ile Ser Glu Ile Leu Ile Ala Thr Pro Leu Gln Gln Val Ala Ser

65 70 75 80

Gly Arg Ala Gly Val Phe Thr Gln Tyr His Lys Lys Lys Lys Ala Met

85 90 95

Thr Val Gly Glu Tyr Arg His Leu Ala Asn Ser Lys Lys Tyr Gln Thr

100 105 110

Pro Pro His Gln Asn Phe Glu Asp Leu Glu Arg Lys Tyr Trp Lys Asn

115 120 125

Arg Ile Tyr Asn Ser Pro Ile Tyr Gly Ala Asp Ile Ser Gly Ser Leu

130 135 140

Phe Asp Glu Asn Thr Lys Gln Trp Asn Leu Gly His Leu Gly Thr Ile

145 150 155 160

Gln Asp Leu Leu Glu Lys Glu Cys Gly Val Val Ile Glu Gly Val Asn

165 170 175

Thr Pro Tyr Leu Tyr Phe Gly Met Trp Lys Thr Thr Phe Ala Trp His

180 185 190

Thr Glu Asp Met Asp Leu Tyr Ser Ile Asn Tyr Leu His Leu Gly Glu

195 200 205

Pro Lys Thr Trp Tyr Val Val Pro Pro Glu HisGly Gln Arg Leu Glu

210 215 220

Arg Leu Ala Arg Glu Leu Phe Pro Gly Ser Ser Arg Gly Cys Gly Ala

225 230 235 240

Phe Leu Arg His Lys Val Ala Leu Ile Ser Pro Thr Val Leu Lys Glu

245 250 255

Asn Gly Ile Pro Phe Asn Arg Ile Thr Gln Glu Ala Gly Glu Phe Met

260 265 270

Val Thr Phe Pro Tyr Gly Tyr His Ala Gly Phe Asn His Gly Phe Asn

275 280 285

Cys Ala Glu Ala Ile Asn Phe Ala Thr Pro Arg Trp Ile Asp Tyr Gly

290 295 300

Lys Met Ala Ser Gln Cys Ser Cys Gly Glu Ala Arg Val Thr Phe Ser

305 310 315 320

Met Asp Ala Phe Val Arg Ile Leu Gln Pro Glu Arg Tyr Asp Leu Trp

325 330 335

Lys Arg Gly Gln Asp Arg Ala Val Val Asp His Met Glu Pro Arg Val

340 345 350

Pro Ala Ser Gln Glu Leu Ser Thr Gln Lys Glu Val Gln Leu Pro Arg

355 360 365

Arg Ala Ala Leu Gly Leu Arg Gln Leu Pro Ser His Trp Ala Arg His

370 375 380

Ser Pro Trp Pro Met Ala Ala Arg Ser Gly Thr Arg Cys His Thr Leu

385 390 395 400

Val Cys Ser Ser Leu Pro Arg Arg Ser Ala Val Ser Gly Thr Ala Thr

405 410 415

Gln Pro Arg Ala Ala Ala Val

420

<210>14

<211>2752

<212>DNA

<213> Intelligent people

<400>14

cgctcttctc gcgaggccgg ctaggcccga atgtcgttag ccgtggggaa agatggcgga 60

aaatttaaaa ggctgcagcg tgtgttgcaa gtcttcttgg aatcagctgc aggacctgtg 120

ccgcctggcc aagctctcct gccctgccct cggtatctct aagaggaacc tctatgactt 180

tgaagtcgag tacctgtgcg attacaagaa gatccgcgaa caggaatatt acctggtgaa 240

atggcgtgga tatccagact cagagagcac ctgggagcca cggcagaatc tcaagtgtgt 300

gcgtatcctc aagcagttcc acaaggactt agaaagggag ctgctccggc ggcaccaccg 360

gtcaaagacc ccccggcacc tggacccaag cttggccaac tacctggtgc agaaggccaa 420

gcagaggcgg gcgctccgtc gctgggagca ggagctcaat gccaagcgca gccatctggg 480

acgcatcact gtagagaatg aggtggacct ggacggccct ccgcgggcct tcgtgtacat 540

caatgagtac cgtgttggtg agggcatcac cctcaaccag gtggctgtgg gctgcgagtg 600

ccaggactgt ctgtgggcac ccactggagg ctgctgcccg ggggcgtcac tgcacaagtt 660

tgcctacaat gaccagggcc aggtgcggct tcgagccggg ctgcccatct acgagtgcaa 720

ctcccgctgc cgctgcggct atgactgccc aaatcgtgtg gtacagaagg gtatccgata 780

tgacctctgc atcttccgca cggatgatgg gcgtggctgg ggcgtccgca ccctggagaa 840

gattcgcaag aacagcttcg tcatggagta cgtgggagag atcattacct cagaggaggc 900

agagcggcgg ggccagatct acgaccgtca gggcgccacc tacctctttg acctggacta 960

cgtggaggac gtgtacaccg tggatgccgc ctactatggc aacatctccc actttgtcaa 1020

ccacagttgt gaccccaacc tgcaggtgta caacgtcttc atagacaacc ttgacgagcg 1080

gctgccccgc atcgctttct ttgccacaag aaccatccgg gcaggcgagg agctcacctt 1140

tgattacaac atgcaagtgg accccgtgga catggagagc acccgcatgg actccaactt 1200

tggcctggct gggctccctg gctcccctaa gaagcgggtc cgtattgaat gcaagtgtgg 1260

gactgagtcc tgccgcaaat acctcttcta gcccttagaa gtctgaggcc agactgactg 1320

agggggcctg aagctacatg cacctccccc actgctgccc tcctgtcgag aatgactgcc 1380

agggcctcgc ctgcctccac ctgcccccac ctgctcctac ctgctctacg ttcagggctg 1440

tggccgtggt gaggaccgac tccaggagtc ccctttccct gtcccagccc catctgtggg 1500

ttgcacttac aaacccccac ccaccttcag aaatagtttt tcaacatcaa gactctctgt 1560

cgttgggatt catggcctat taaggaggtc caaggggtga gtcccaaccc agccccagaa 1620

tatatttgtt tttgcacctg cttctgcctg gagattgagg ggtctgctgc aggcctcctc 1680

cctgctgccc caaaggtatg gggaagcaac cccagagcag gcagacatca gaggccagag 1740

tgcctagccc gacatgaagc tggttcccca accacagaaa ctttgtacta gtgaaagaaa 1800

gggggtccct gggctacggg ctgaggctgg tttctgctcg tgcttacagt gctgggtagt 1860

gttggcccta agagctgtag ggtctcttct tcagggctgc atatctgaga agtggatgcc 1920

cacatgccac tggaagggaa gtgggtgtcc atgggccact gagcagtgag aggaaggcag 1980

tgcagagctg gccagccctg gaggtaggct gggaccaagc tctgccttca cagtgcagtg 2040

aaggtaccta gggctcttgg gagctctgcg gttgctaggg gccctgacct ggggtgtcat 2100

gaccgctgac accactcaga gctggaacca agatctagat agtccgtaga tagcacttag 2160

gacaagaatg tgcattgatg gggtggtgat gaggtgccag gcactgggta gagcacctgg 2220

tccacgtgga ttgtctcagg gaagccttga aaaccacgga ggtggatgcc aggaaagggc 2280

ccatgtggca gaaggcaaag tacaggccaa gaattggggg tgggggagat ggcttcccca 2340

ctatgggatg acgaggcgag agggaagccc ttgctgcctg ccattcccag accccagccc 2400

tttgtgctca ccctggttcc actggtctca aaagtcacct gcctacaaat gtacaaaagg 2460

cgaaggttct gatggctgcc ttgctccttg ctcccccacc ccctgtgagg acttctctag 2520

gaagtccttc ctgactacct gtgcccagag tgcccctaca tgagactgta tgccctgcta 2580

tcagatgcca gatctatgtg tctgtctgtg tgtccatccc gccggccccc cagactaacc 2640

tccaggcatg gactgaatct ggttctcctc ttgtacaccc ctcaacccta tgcagcctgg 2700

agtgggcatc aataaaatga actgtcgact gaacaaaaaa aaaaaaaaaa aa 2752

<210>15

<211>3093

<212>DNA

<213> Intelligent people

<400>15

cggggccgag gcgcgaggag gtgaggctgg agcgcggccc cctcgccttc cctgttccca 60

ggcaagctcc caaggcccgg gcggcggggc cgtcccgcgg gccagccaga tggcgacgtg 120

gcggttcccc gcccgccgcg accccaactc cgggacgcac gctgcggacg cctatcctcc 180

cccaggccgc tgacccgcct ccctgcccgg ccggctcccg ccgcggagga tatggaatat 240

tatcttgtaa aatggaaagg atggccagat tctacaaata cttgggaacc tttgcaaaat 300

ctgaagtgcc cgttactgct tcagcaattc tctaatgaca agcataatta tttatctcag 360

gtaaagaaag gcaaagcaat aactccaaaa gacaataaca aaactttgaa acctgccatt 420

gctgagtaca ttgtgaagaa ggctaaacaa aggatagctc tgcagagatg gcaagatgaa 480

ctcaacagaa gaaagaatca taaaggaatg atatttgttg aaaatactgt tgatttagag 540

ggcccacctt cagacttcta ttacattaac gaatacaaac cagctcctgg aatcagctta 600

gtcaatgaag ctacctttgg ttgttcatgc acagattgct tctttcaaaa atgttgtcct 660

gctgaagctg gagttctttt ggcttataat aaaaaccaac aaattaaaat cccacctggt 720

actcccatct atgaatgcaa ctcaaggtgt cagtgtggtc ctgattgtcc caataggatt 780

gtacaaaaag gcacacagta ttcgctttgc atctttcgaa ctagcaatgg acgtggctgg 840

ggtgtaaaga cccttgtgaa gattaaaaga atgagttttg tcatggaata tgttggagag 900

gtaatcacaa gtgaagaagc tgaaagacga ggacagttct atgacaacaa gggaatcacg 960

tatctctttg atctggacta tgagtctgat gaattcacag tggatgcggc tcgatacggc 1020

aatgtgtctc attttgtgaa tcacagctgt gacccaaatc ttcaggtgtt caatgttttc 1080

attgataacc tcgatactcg tcttccccga atagcattgt tttccacaag aaccataaat 1140

gctggagaag agctgacttt tgattatcaa atgaaaggtt ctggagatat atcttcagat 1200

tctattgacc acagcccagc caaaaagagg gtcagaacag tatgtaaatg tggagctgtg 1260

acttgcagag gttacctcaa ctgaactttt tcaggaaata gagctgatga ttataatatt 1320

tttttcctaa tgttaacatt tttaaaaata catatttggg actcttatta tcaaggttct 1380

acctatgtta atttacaatt catgtttcaa gacatttgcc aaatgtatta ccgatgcctc 1440

tgaaaagggg gtcactgggt ctcatagact gatatgaagt cgacatattt atagtgctta 1500

gagaccaaac taatggaagg cagactattt acagcttagt atatgtgtac ttaagtctat 1560

gtgaacagag aaatgcctcc cgtagtgttt gaaagcgtta agctgataat gtaattaaca 1620

actgctgaga gatcaaagat tcaacttgcc atacacctca aattcggaga aacagttaat 1680

ttgggcaaat ctacagttct gtttttgcta ctctattgtc attcctgttt aatactcact 1740

gtacttgtat ttgagacaaa taggtgatac tgaattttat actgttttct acttttccat 1800

taaaacattg gcacctcaat gataaagaaa tttaaggtat aaaattaaat gtaaaaatta 1860

atttcagctt catttcgtat ttcgaagcaa tctagactgt tgtgatgagt gtatgtctga 1920

acctgtaatt cttaaaagac ttcttaatct tctagaagaa aaatctccga agagctctct 1980

ctagaagtcc aaaatggcta gccattatgc ttctttgaaa ggacatgata atgggaccag 2040

gatggttttt tggagtacca agcaagggga atggagcact ttaagggcgc ctgttagtaa 2100

catgaattgg aaatctgtgt cgagtacctc tgatctaaac ggtaaaacaa gctgcctgga 2160

gagcagctgt acctaacaat actgtaatgt acattaacat tacagcctct caatttcagg 2220

caggtgtaac agttcctttc caccagattt aatattttta tacttcctgc aggttcttct 2280

taaaaagtaa tctatatttt tgaactgata cttgttttat acataaattt tttttagatg 2340

tgataaagct aaacttggcc aaagtgtgtg cctgaattat tagacctttt tattagtcaa 2400

cctacgaaga ctaaaataga atatattagt tttcaaggga gtgggaggct tccaacatag 2460

tattgaatct caggaaaaac tattctttca tgtctgattc tgagatttct aattgtgttg 2520

tgaaaatgat aaatgcagca aatctagctt tcagtattcc taatttttac ctaagctcat 2580

tgctccaggc tttgattacc taaaataagc ttggataaaa ttgaaccaac ttcaagaatg 2640

cagcacttct taatctttag ctctttcttg ggagaagcta gactttattc attatattgc 2700

tatgacaact tcactctttc ataatatata ggataaattg tttacatgat tggaccctca 2760

gattctgtta accaaaattg cagaatgggg ggccaggcct gtgtggtggc tcacacctgt 2820

gatcccagca ctttgggagg ctgaggtagg aggatcacgt gaggtcggga gttcaagacc 2880

agcctggcca tcatggtgaa accctgtctc tactgaaaat acaaaaatta gccgggcgtg 2940

gtggcacacg cctgtagtcc cagctactca ggaggctgag gcaggagaat cacttgaatt 3000

caggaggcgg aggttgcagt gagccaagat cataccactg cactgcagcc tgagtgacac 3060

agtaagactg tctccaaaaa aaaaaaaaaa aaa 3093

<210>16

<211>4449

<212>DNA

<213> Intelligent people

<400>16

ggcactaaag gtttgcttcc gggcgtttct tttgcttccc cttccctctt tcacgcttcc 60

tcccctcccc ctcctccctt atcccttcgc tttcgctctt ttccgtcgag gccgacccct 120

gagttgtgag tctggggtct ggttggtgaa aaagagccct tgaagctgga agacgggaga 180

ggacaaaagc atgtcttccc ttcctgggtg cattggtttg gatgcagcaa cagctacagt 240

ggagtctgaa gagattgcag agctgcaaca ggcagtggtt gaggaactgg gtatctctat 300

ggaggaactt cggcatttca tcgatgagga actggagaag atggattgtg tacagcaacg 360

caagaagcag ctagcagagt tagagacatg ggtaatacag aaagaatctg aggtggctca 420

cgttgaccaa ctctttgatg atgcatccag ggcagtgact aattgtgagt ctttggtgaa 480

ggacttctac tccaagctgg gactacaata ccgggacagt agctctgagg acgaatcttc 540

ccggcctaca gaaataattg agattcctga tgaagatgat gatgtcctca gtattgattc 600

aggtgatgct gggagcagaa ctccaaaaga ccagaagctc cgtgaagcta tggctgcctt 660

aagaaagtca gctcaagatg ttcagaagtt catggatgct gtcaacaaga agagcagttc 720

ccaggatctg cataaaggaa ccttgagtca gatgtctgga gaactaagca aagatggtga 780

cctgatagtc agcatgcgaa ttctgggcaa gaagagaact aagacttggc acaaaggcac 840

ccttattgcc atccagacag ttgggccagg gaagaaatac aaggtgaaat ttgacaacaa 900

aggaaagagt ctactgtcgg ggaaccatat tgcctatgat taccaccctc ctgctgacaa 960

gctgtatgtg ggcagtcggg tggtcgccaa atacaaagat gggaatcagg tctggctcta 1020

tgctggcatt gtagctgaga caccaaacgt caaaaacaag ctcaggtttc tcattttctt 1080

tgatgatggc tatgcttcct atgtcacaca gtcggaactg tatcccattt gccggccact 1140

gaaaaagact tgggaggaca tagaagacat ctcctgccgt gacttcatag aggagtatgt 1200

cactgcctac cccaaccgcc ccatggtact gctcaagagt ggccagctta tcaagactga 1260

gtgggaaggc acgtggtgga agtcccgagt tgaggaggtg gatggcagcc tagtcaggat 1320

cctcttcctg gatgacaaaa gatgtgagtg gatctatcga ggctctacac ggctggagcc 1380

catgttcagc atgaaaacat cctcagcctc tgcactggag aagaagcaag gacagctcag 1440

gacacgtcca aatatgggtg ctgtgaggag caaaggccct gttgtccagt acacacagga 1500

tctgaccggt actggaaccc agttcaagcc agtggaaccc ccacagccta cagctccacc 1560

tgccccacct ttcccacctg ctccacctct atccccccaa gcaggtgaca gtgacttgga 1620

aagccagctt gcccagtcac ggaagcaggt agccaaaaag agcacgtcct ttcgaccagg 1680

atctgtgggc tctggtcatt cctcccctac atctcctgca ctcagtgaaa atgtctctgg 1740

tgggaaacct gggatcaacc agacatatag atcaccttta ggctccacag cctctgcccc 1800

agcaccctca gcactcccgg cccctccagc acccccagtc ttccatggca tgctggagcg 1860

ggccccagca gagccctcct accgtgctcc catggagaag cttttctact tacctcatgt 1920

ctgcagctat acctgtctgt ctcgagtcag acctatgagg aatgagcagt accggggcaa 1980

gaaccctctg ctggtcccgt tactatatga cttccggcgg atgacagccc ggcgtcgagt 2040

taaccgcaag atgggctttc atgttatcta taagacacct tgtggtctct gccttcggac 2100

aatgcaggag atagaacgct accttttcga gactggctgt gacttcctct tcctggagat 2160

gttctgtttg gatccatatg ttcttgtgga ccgaaagttt cagccctata agccttttta 2220

ctatattttg gacatcactt atgggaagga agatgttccc ctatcctgtg tcaatgagat 2280

tgacacaacc cctccacccc aggtggccta cagcaaggaa cgtatcccgg gcaagggtgt 2340

tttcattaac acaggccctg aatttctggt tggctgtgac tgcaaggatg ggtgtcggga 2400

caagtccaag tgtgcctgcc atcaactaac tatccaggct acagcctgta ccccaggagg 2460

ccaaatcaac cctaactctg gctaccagta caagagacta gaagagtgtc tacccacagg 2520

ggtatatgag tgtaacaaac gctgcaaatg tgacccaaac atgtgcacaa accggttggt 2580

gcaacatgga ctacaagttc ggctacagct attcaagaca cagaacaagg gctggggtat 2640

ccgctgcttg gatgacattg ccaaaggctc ttttgtttgt atttatgcag gcaaaatcct 2700

gacagatgac tttgcagaca aggagggtct ggaaatgggt gatgagtact ttgcaaatct 2760

ggaccatatc gagagcgtgg agaacttcaa agaaggatat gagagtgatg ccccctgttc 2820

ctctgacagc agtggtgtag acttgaagga ccaggaagat ggcaacagcg gtacagagga 2880

ccctgaagag tccaatgatg atagctcaga tgataacttc tgtaaggatg aggacttcag 2940

caccagttca gtgtggcgga gctatgctac ccggaggcag acccggggcc agaaagagaa 3000

cggactctct gagacaactt ccaaggactc ccacccccca gatcttggac ccccacatat 3060

tcctgttcct ccctcaatcc ctgtaggtgg ctgcaatcca ccttcctccg aagagacacc 3120

caagaacaag gtggcctcat ggttgagctg caatagtgtc agtgaaggtg gttttgctga 3180

ctctgatagc cattcatcct tcaagactaa tgaaggtggg gagggccggg ctgggggaag 3240

ccgaatggag gctgagaagg cctccacctc aggactaggc atcaaggatg agggagacat 3300

caaacaggcc aagaaagagg acactgacga ccgaaacaag atgtcagtag ttactgaaag 3360

ctctcgaaat tacggttaca atccttctcc tgtgaagcct gaaggacttc gccgcccacc 3420

tagtaagact agtatgcatc aaagccgaag actcatggct tctgctcagt ccaaccctga 3480

tgatgtcctg acactgtcca gcagcacaga aagtgagggg gaaagtggga ccagccgaaa 3540

gcccactgct ggtcagactt cggctacagc ggttgacagt gatgatatcc agaccatatc 3600

ctctggctct gaaggggatg actttgagga caagaagaac atgactggtc caatgaagcg 3660

tcaagtggca gtaaaatcaa cccgaggctt tgctcttaaa tcaacccatg ggattgcaat 3720

taaatcaacc aacatggcct ctgtggacaa gggggagagc gcacctgttc gtaagaacac 3780

acgccaattc tatgatggcg aggagtcttg ctacatcatt gatgccaagc ttgaaggcaa 3840

cctgggccgc tacctcaacc acagttgcag ccccaacctg tttgtccaga atgtcttcgt 3900

ggatacccat gatcttcgct tcccctgggt ggccttcttt gccagcaaaa gaatccgggc 3960

tgggacagaa cttacttggg actacaacta cgaggtgggc agtgtggaag gcaaggagct 4020

actctgttgc tgtggggcca ttgaatgcag aggacgtctt ctttagagga cagccttctt 4080

cccaaccctt cttgaactgt cgtttcctca ggaactgggt cttcctgatt gttgaaccct 4140

gacccgaagt ctctgggcta gctactcccc ccagctccta gttgatagaa atgggggttc 4200

tggaccagat gatcccttcc aatgtggtgc tagcaggcag gatcccttct ccacctccaa 4260

aggccctaaa gggtggggag agatcaccac tctaacctcg gcctgacatc cctcccatcc 4320

catatttgtc caagtgttcc tgcttctaac agactttgtt cttagaatgg agcctgtgta 4380

tctactatct ccagtttgta ttatttcttg aaagtctttt aacaatatga taaaactaag 4440

attgtgaaa 4449

<210>17

<211>1291

<212>PRT

<213> Intelligent people

<400>17

Met Ser Ser Leu Pro Gly Cys Ile Gly Leu Asp Ala Ala Thr Ala Thr

1 5 10 15

Val Glu Ser Glu Glu Ile Ala Glu Leu Gln Gln Ala Val Val Glu Glu

20 25 30

Leu Gly Ile Ser Met Glu Glu Leu Arg His Phe Ile Asp Glu Glu Leu

35 40 45

Glu Lys Met Asp Cys Val Gln Gln Arg Lys Lys Gln Leu Ala Glu Leu

50 55 60

Glu Thr Trp Val Ile Gln Lys Glu Ser Glu Val Ala His Val Asp Gln

65 70 75 80

Leu Phe Asp Asp Ala Ser Arg Ala Val Thr Asn Cys Glu Ser Leu Val

85 90 95

Lys Asp Phe Tyr Ser Lys Leu Gly Leu Gln Tyr Arg Asp Ser Ser Ser

100 105 110

Glu Asp Glu Ser Ser Arg Pro Thr Glu Ile Ile Glu Ile Pro Asp Glu

115 120 125

Asp Asp Asp Val Leu Ser Ile Asp Ser Gly Asp Ala Gly Ser Arg Thr

130 135140

Pro Lys Asp Gln Lys Leu Arg Glu Ala Met Ala Ala Leu Arg Lys Ser

145 150 155 160

Ala Gln Asp Val Gln Lys Phe Met Asp Ala Val Asn Lys Lys Ser Ser

165 170 175

Ser Gln Asp Leu His Lys Gly Thr Leu Ser Gln Met Ser Gly Glu Leu

180 185 190

Ser Lys Asp Gly Asp Leu Ile Val Ser Met Arg Ile Leu Gly Lys Lys

195 200 205

Arg Thr Lys Thr Trp His Lys Gly Thr Leu Ile Ala Ile Gln Thr Val

210 215 220

Gly Pro Gly Lys Lys Tyr Lys Val Lys Phe Asp Asn Lys Gly Lys Ser

225 230 235 240

Leu Leu Ser Gly Asn His Ile Ala Tyr Asp Tyr His Pro Pro Ala Asp

245 250 255

Lys Leu Tyr Val Gly Ser Arg Val Val Ala Lys Tyr Lys Asp Gly Asn

260 265 270

Gln Val Trp Leu Tyr Ala Gly Ile Val Ala Glu Thr Pro Asn Val Lys

275 280 285

Asn Lys Leu Arg Phe Leu Ile Phe Phe Asp Asp Gly Tyr Ala Ser Tyr

290 295 300

Val Thr Gln Ser Glu Leu Tyr Pro Ile Cys Arg Pro Leu Lys Lys Thr

305 310 315 320

Trp Glu Asp Ile Glu Asp Ile Ser Cys Arg Asp Phe Ile Glu Glu Tyr

325 330 335

Val Thr Ala Tyr Pro Asn Arg Pro Met Val Leu Leu Lys Ser Gly Gln

340 345 350

Leu Ile Lys Thr Glu Trp Glu Gly Thr Trp Trp Lys Ser Arg Val Glu

355 360 365

Glu Val Asp Gly Ser Leu Val Arg Ile Leu Phe Leu Asp Asp Lys Arg

370 375 380

Cys Glu Trp Ile Tyr Arg Gly Ser Thr Arg Leu Glu Pro Met Phe Ser

385 390 395 400

Met Lys Thr Ser Ser Ala Ser Ala Leu Glu Lys Lys Gln Gly Gln Leu

405 410 415

Arg Thr Arg Pro Asn Met Gly Ala Val Arg Ser Lys Gly Pro Val Val

420 425 430

Gln Tyr Thr Gln Asp Leu Thr Gly Thr Gly Thr Gln Phe Lys Pro Val

435 440 445

Glu Pro Pro Gln Pro Thr Ala Pro Pro Ala Pro Pro Phe Pro Pro Ala

450 455 460

Pro Pro Leu Ser Pro Gln Ala Gly Asp Ser Asp Leu Glu Ser Gln Leu

465 470 475 480

Ala Gln Ser Arg Lys Gln Val Ala Lys Lys Ser Thr Ser Phe Arg Pro

485 490 495

Gly Ser Val Gly Ser Gly His Ser Ser Pro Thr Ser Pro Ala Leu Ser

500 505 510

Glu Asn Val Ser Gly Gly Lys Pro Gly Ile Asn Gln Thr Tyr Arg Ser

515 520 525

Pro Leu Gly Ser Thr Ala Ser Ala Pro Ala Pro Ser Ala Leu Pro Ala

530 535 540

Pro Pro Ala Pro Pro Val Phe His Gly Met Leu Glu Arg Ala Pro Ala

545 550 555 560

Glu Pro Ser Tyr Arg Ala Pro Met Glu Lys Leu Phe Tyr Leu Pro His

565 570 575

Val Cys Ser Tyr Thr Cys Leu Ser Arg Val Arg Pro Met Arg Asn Glu

580 585 590

Gln Tyr Arg Gly Lys Asn Pro Leu Leu Val Pro Leu Leu Tyr Asp Phe

595 600 605

Arg Arg Met Thr Ala Arg Arg Arg Val Asn Arg Lys Met Gly Phe His

610 615 620

Val Ile Tyr Lys Thr Pro Cys Gly Leu Cys Leu Arg Thr Met Gln Glu

625 630 635 640

Ile Glu Arg Tyr Leu Phe Glu Thr Gly Cys Asp Phe Leu Phe Leu Glu

645 650 655

Met Phe Cys Leu Asp Pro Tyr Val Leu Val Asp Arg Lys Phe Gln Pro

660 665 670

Tyr Lys Pro Phe Tyr Tyr Ile Leu Asp Ile Thr Tyr Gly Lys Glu Asp

675 680 685

Val Pro Leu Ser Cys Val Asn Glu Ile Asp Thr Thr Pro Pro Pro Gln

690 695700

Val Ala Tyr Ser Lys Glu Arg Ile Pro Gly Lys Gly Val Phe Ile Asn

705 710 715 720

Thr Gly Pro Glu Phe Leu Val Gly Cys Asp Cys Lys Asp Gly Cys Arg

725 730 735

Asp Lys Ser Lys Cys Ala Cys His Gln Leu Thr Ile Gln Ala Thr Ala

740 745 750

Cys Thr Pro Gly Gly Gln Ile Asn Pro Asn Ser Gly Tyr Gln Tyr Lys

755 760 765

Arg Leu Glu Glu Cys Leu Pro Thr Gly Val Tyr Glu Cys Asn Lys Arg

770 775 780

Cys Lys Cys Asp Pro Asn Met Cys Thr Asn Arg Leu Val Gln His Gly

785 790 795 800

Leu Gln Val Arg Leu Gln Leu Phe Lys Thr Gln Asn Lys Gly Trp Gly

805 810 815

Ile Arg Cys Leu Asp Asp Ile Ala Lys Gly Ser Phe Val Cys Ile Tyr

820 825 830

Ala Gly Lys Ile Leu Thr Asp Asp Phe Ala Asp Lys Glu Gly Leu Glu

835 840 845

Met Gly Asp Glu Tyr Phe Ala Asn Leu Asp His Ile Glu Ser Val Glu

850 855 860

Asn Phe Lys Glu Gly Tyr Glu Ser Asp Ala Pro Cys Ser Ser Asp Ser

865 870 875 880

Ser Gly Val Asp Leu Lys Asp Gln Glu Asp Gly Asn Ser Gly Thr Glu

885 890 895

Asp Pro Glu Glu Ser Asn Asp Asp Ser Ser Asp Asp Asn Phe Cys Lys

900 905 910

Asp Glu Asp Phe Ser Thr Ser Ser Val Trp Arg Ser Tyr Ala Thr Arg

915 920 925

Arg Gln Thr Arg Gly Gln Lys Glu Asn Gly Leu Ser Glu Thr Thr Ser

930 935 940

Lys Asp Ser His Pro Pro Asp Leu Gly Pro Pro His Ile Pro Val Pro

945 950 955 960

Pro Ser Ile Pro Val Gly Gly Cys Asn Pro Pro Ser Ser Glu Glu Thr

965 970 975

Pro Lys Asn Lys Val Ala Ser Trp Leu Ser Cys Asn Ser Val Ser Glu

980 985 990

Gly Gly Phe Ala Asp Ser Asp Ser His Ser Ser Phe Lys Thr Asn Glu

995 1000 1005

Gly Gly Glu Gly Arg Ala Gly Gly Ser Arg Met Glu Ala Glu Lys

1010 1015 1020

Ala Ser Thr Ser Gly Leu Gly Ile Lys Asp Glu Gly Asp Ile Lys

1025 1030 1035

Gln Ala Lys Lys Glu Asp Thr Asp Asp Arg Asn Lys Met Ser Val

1040 1045 1050

Val Thr Glu Ser Ser Arg Asn Tyr Gly Tyr Asn Pro Ser Pro Val

1055 1060 1065

Lys Pro Glu Gly Leu Arg Arg Pro Pro Ser Lys Thr Ser Met His

1070 1075 1080

Gln Ser Arg Arg Leu Met Ala Ser Ala Gln Ser Asn Pro Asp Asp

1085 1090 1095

Val Leu Thr Leu Ser Ser Ser Thr Glu Ser Glu Gly Glu Ser Gly

1100 1105 1110

Thr Ser Arg Lys Pro Thr Ala Gly Gln Thr Ser Ala Thr Ala Val

1115 1120 1125

Asp Ser Asp Asp Ile Gln Thr Ile Ser Ser Gly Ser Glu Gly Asp

1130 1135 1140

Asp Phe Glu Asp Lys Lys Asn Met Thr Gly Pro Met Lys Arg Gln

1145 1150 1155

Val Ala Val Lys Ser Thr Arg Gly Phe Ala Leu Lys Ser Thr His

1160 1165 1170

Gly Ile Ala Ile Lys Ser Thr Asn Met Ala Ser Val Asp Lys Gly

1175 1180 1185

Glu Ser Ala Pro Val Arg Lys Asn Thr Arg Gln Phe Tyr Asp Gly

1190 1195 1200

Glu Glu Ser Cys Tyr Ile Ile Asp Ala Lys Leu Glu Gly Asn Leu

1205 1210 1215

Gly Arg Tyr Leu Asn His Ser Cys Ser Pro Asn Leu Phe Val Gln

1220 1225 1230

Asn Val Phe Val Asp Thr His Asp Leu Arg Phe Pro Trp Val Ala

1235 1240 1245

Phe Phe Ala Ser Lys Arg Ile Arg Ala Gly Thr Glu Leu Thr Trp

1250 1255 1260

Asp Tyr Asn Tyr Glu Val Gly Ser Val Glu Gly Lys Glu Leu Leu

1265 1270 1275

Cys Cys Cys Gly Ala Ile Glu Cys Arg Gly Arg Leu Leu

1280 1285 1290

<210>18

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic oligonucleotides

<220>

<223> description of the combination DNA/RNA molecules: synthetic oligonucleotides

<400>18

gcucacaugu aaaucgauut t 21

<210>19

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic oligonucleotides

<220>

<223> description of the combination DNA/RNA molecules: synthetic oligonucleotides

<400>19

aaucgauuua caugugagct t 21

<210>20

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic oligonucleotides

<220>

<223> description of the combination DNA/RNA molecules: synthetic oligonucleotides

<400>20

gguguacaac guauucauat t 21

<210>21

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic oligonucleotides

<220>

<223> description of the combination DNA/RNA molecules: synthetic oligonucleotides

<400>21

uaugaauacg uuguacacct g 21

<210>22

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic oligonucleotides

<220>

<223> description of the combination DNA/RNA molecules: synthetic oligonucleotides

<400>22

gguccuuugu cuauaucaat t 21

<210>23

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic oligonucleotides

<220>

<223> description of the combination DNA/RNA molecules: synthetic oligonucleotides

<400>23

uugauauaga caaaggacct t 21

<210>24

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic oligonucleotides

<220>

<223> description of the combination DNA/RNA molecules: synthetic oligonucleotides

<400>24

gcucacaugu aaaucgauut t 21

<210>25

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic oligonucleotides

<220>

<223> description of the combination DNA/RNA molecules: synthetic oligonucleotides

<400>25

aaucgauuua caugugagct t 21

<210>26

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic oligonucleotides

<220>

<223> description of the combination DNA/RNA molecules: synthetic oligonucleotides

<400>26

gugucgaugu ggaccugaat t 21

<210>27

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic oligonucleotides

<220>

<223> description of the combination DNA/RNA molecules: synthetic oligonucleotides

<400>27

uucaggucca caucgacacc t 21

<210>28

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic oligonucleotides

<220>

<223> description of the combination DNA/RNA molecules: synthetic oligonucleotides

<400>28

ggacuacagu aucaugacat t 21

<210>29

<211>21

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic oligonucleotides

<400>29

ugucaugaua cuguaguccc a 21

<210>30

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic oligonucleotides

<220>

<223> description of the combination DNA/RNA molecules: synthetic oligonucleotides

<400>30

ggacgaugca ggagauagat t 21

<210>31

<211>21

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic oligonucleotides

<400>31

ucuaucuccu gcaucguccg a 21

<210>32

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic oligonucleotides

<220>

<223> description of the combination DNA/RNA molecules: synthetic oligonucleotides

<400>32

ggaugggugu cgggauaaat t 21

<210>33

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic oligonucleotides

<220>

<223> description of the combination DNA/RNA molecules: synthetic oligonucleotides

<400>33

uuuaucccga cacccaucct t 21

<210>34

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic oligonucleotides

<220>

<223> description of the combination DNA/RNA molecules: synthetic oligonucleotides

<400>34

gcaccuuugu cugcgaauat t 21

<210>35

<211>21

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic oligonucleotides

<400>35

uauucgcaga caaaggugcc c 21

<210>36

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic oligonucleotides

<220>

<223> description of the combination DNA/RNA molecules: synthetic oligonucleotides

<400>36

gaucaaaccu gcucggaaat t 21

<210>37

<211>21

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic oligonucleotides

<400>37

uuuccgagca gguuugaucc a 21

<210>38

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic oligonucleotides

<220>

<223> description of the combination DNA/RNA molecules: synthetic oligonucleotides

<400>38

gaauuugccu ucuuaugcat t 21

<210>39

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic oligonucleotides

<220>

<223> description of the combination DNA/RNA molecules: synthetic oligonucleotides

<400>39

ugcauaagaa ggcaaauuct t 21

<210>40

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic oligonucleotides

<220>

<223> description of the combination DNA/RNA molecules: synthetic oligonucleotides

<400>40

gaggaauucu agucccguat t 21

<210>41

<211>21

<212>RNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic oligonucleotides

<400>41

uacgggacua gaauuccuca a 21

<210>42

<211>5123

<212>DNA

<213> Intelligent people

<400>42

gcgcgggagg ggcggggcca cgctgcgggc ccgggccatg gccgccgccg atgccgaggc 60

agttccggcg aggggggagc ctcagcagga ttgctgtgtg aaaaccgagc tgctgggaga 120

agagacacct atggctgccg atgaaggctc agcagagaaa caggcaggag aggcccacat 180

ggctgcggac ggtgagacca atgggtcttg tgaaaacagc gatgccagca gtcatgcaaa 240

tgctgcaaag cacactcagg acagcgcaag ggtcaacccc caggatggca ccaacacact 300

aactcggata gcggaaaatg gggtttcaga aagagactca gaagcggcga agcaaaacca 360

cgtcactgcc gacgactttg tgcagacttc tgtcatcggc agcaacggat acatcttaaa 420

taagccggcc ctacaggcac agcccttgag gactaccagc actctggcct cttcgctgcc 480

tggccatgct gcaaaaaccc ttcctggagg ggctggcaaa ggcaggactc caagcgcttt 540

tccccagacg ccagccgccc caccagccac ccttggggag gggagtgctg acacagagga 600

caggaagctc ccggcccctg gcgccgacgt caaggtccac agggcacgca agaccatgcc 660

gaagtccgtc gtgggcctgc atgcagccag taaagatccc agagaagttc gagaagctag 720

agatcataag gaaccaaaag aggagatcaa caaaaacatt tctgactttg gacgacagca 780

gcttttaccc cccttcccat cccttcatca gtcgctacct cagaaccagt gctacatggc 840

caccacaaaa tcacagacag cttgcttgcc ttttgtttta gcagctgcag tatctcggaa 900

gaaaaaacga agaatgggaa cctatagcct ggttcctaag aaaaagacca aagtattaaa 960

acagaggacg gtgattgaga tgtttaagag cataactcat tccactgtgg gttccaaggg 1020

ggagaaggac ctgggcgcca gcagcctgca cgtgaatggg gagagcctgg agatggactc 1080

ggatgaggac gactcagagg agctcgagga ggacgacggc catggtgcag agcaggcggc 1140

cgcgttcccc acagaggaca gcaggacttc caaggagagc atgtcggagg ctgatcgcgc 1200

ccagaagatg gacggggagt ccgaggagga gcaggagtcc gtggacaccg gggaggagga 1260

ggaaggcggt gacgagtctg acctgagttc ggaatccagc attaagaaga aatttctcaa 1320

gaggaaagga aagaccgaca gtccctggat caagccagcc aggaaaagga ggcggagaag 1380

tagaaagaag cccagcggtg ccctcggttc tgagtcgtat aagtcatctg caggaagcgc 1440

tgagcagacg gcaccaggag acagcacagg gtacatggaa gtttctctgg actccctgga 1500

tctccgagtc aaaggaattc tgtcttcaca agcagaaggg ttggccaacg gtccagatgt 1560

gctggagaca gacggcctcc aggaagtgcc tctctgcagc tgccggatgg aaacaccgaa 1620

gagtcgagag atcaccacac tggccaacaa ccagtgcatg gctacagaga gcgtggacca 1680

tgaattgggc cggtgcacaa acagcgtggt caagtatgag ctgatgcgcc cctccaacaa 1740

ggccccgctc ctcgtgctgt gtgaagacca ccggggccgc atggtgaagc accagtgctg 1800

tcctggctgt ggctacttct gcacagcggg taattttatg gagtgtcagc ccgagagcag 1860

catctctcac cgtttccaca aagactgtgc ctctcgagtc aataacgcca gctattgtcc 1920

ccactgtggg gaggagagct ccaaggccaa agaggtgacg atagctaaag cagacaccac 1980

ctcgaccgtg acaccagtcc ccgggcagga gaagggctcg gccctggagg gcagggccga 2040

caccacaacg ggcagtgctg ccgggccacc actctcggag gacgacaagc tgcagggtgc 2100

agcctcccac gtgcccgagg gctttgatcc aacgggacct gctgggcttg ggaggccaac 2160

tcccggcctt tcccagggac cagggaagga aaccttggag agcgctctca tcgccctcga 2220

ctcggaaaaa cccaagaagc ttcgcttcca cccaaagcag ctgtacttct ccgccaggca 2280

aggggagctt cagaaggtgc tcctcatgct ggtggacgga attgacccca acttcaaaat 2340

ggagcaccag aataagcgct ctccactgca cgccgcggca gaggctggac acgtggacat 2400

ctgccacatg ctggttcagg cgggcgctaa tattgacacc tgctcagaag accagaggac 2460

cccgttgatg gaagcagccg aaaacaacca tctggaagca gtgaagtacc tcatcaaggc 2520

tggggccctg gtggatccca aggacgcaga gggctctacg tgtttgcacc tggctgccaa 2580

gaaaggccac tacgaagtgg tccagtacct gctttcaaat ggacagatgg acgtcaactg 2640

tcaggatgac ggaggctgga cacccatgat ctgggccaca gagtacaagc acgtggacct 2700

cgtgaagctg ctgctgtcca agggctctga catcaacatc cgagacaacg aggagaacat 2760

ttgcctgcac tgggcggcgt tctccggctg cgtggacata gccgagatcc tgctggctgc 2820

caagtgcgac ctccacgccg tgaacatcca cggagactcg ccactgcaca ttgccgcccg 2880

ggagaaccgc tacgactgtg tcgtcctctt tctttctcgg gattcagatg tcaccttaaa 2940

gaacaaggaa ggagagacgc ccctgcagtg tgcgagcctc aactctcagg tgtggagcgc 3000

tctgcagatg agcaaggctc tgcaggactc ggcccccgac aggcccagcc ccgtggagag 3060

gatagtgagc agggacatcg ctcgaggcta cgagcgcatc cccatcccct gtgtcaacgc 3120

cgtggacagc gagccatgcc ccagcaacta caagtacgtc tctcagaact gcgtgacgtc 3180

ccccatgaac atcgacagaa atatcactca tctgcagtac tgcgtgtgca tcgacgactg 3240

ctcctccagc aactgcatgt gcggccagct cagcatgcgc tgctggtacg acaaggatgg 3300

ccggctcctg ccagagttca acatggcgga gcctcccttg atcttcgaat gcaaccacgc 3360

gtgctcctgc tggaggaact gccgaaatcg cgtcgtacag aatggtctca gggcaaggct 3420

gcagctctac cggacgcggg acatgggctg gggcgtgcgg tccctgcagg acatcccacc 3480

aggcaccttt gtctgcgagt atgttgggga gctgatttca gactcagaag ccgacgttcg 3540

agaggaagat tcttacctct ttgatctcga caataaggac ggggaggttt actgcatcga 3600

cgcgcggttc tacgggaacg tcagccggtt catcaaccac cactgcgagc ccaacctggt 3660

gcccgtgcgc gtgttcatgg cccaccagga cctgcggttc ccccggatcg ccttcttcag 3720

cacccgcctg atcgaggccg gcgagcagct cgggtttgac tatggagagc gcttctggga 3780

catcaaaggc aagctcttca gctgccgctg cggctccccc aagtgccggc actcgagcgc 3840

ggccctggcc cagcgtcagg ccagcgcggc ccaggaggcc caggaggacg gcttgcccga 3900

caccagctcc gcggctgccg ccgaccccct atgagacgcc gccggccagc ggggcgctcg 3960

ggagccaggg accgccgcgt cgccgattag aggacgagga ggagagattc cgcacgcaac 4020

cgaaagggtc cttcggggct gcgccgccgg cttcctggag gggtcggagg tgaggctgca 4080

gcccctgcgg gcgggtgtgg atgcctccca gccaccttcc cagacctgcg gcctcaccgc 4140

gggcccagtg cccaggctgg agcgcacact ttggtccgcg cgccagagac gctgggagtc 4200

cgcactggca tcaccttctg agtttctgat gctgatttgt cgttgcgaag tttctcgttt 4260

cttcctctga cctccgaggt ccccgctgca ccacggggtt gctctgttct cctgtccggc 4320

ccagactctt ctgtgtggcg ccgccgaagc caccgttagc gcgagctgct ccgttcgccc 4380

tgcccacggc ctgcgtggct ggggccgagt cccaggggcc gcacggaggg cacagtctcc 4440

tgtcaggctc ggagaggtca ggagaccgac cccaccacta actttggaga aaatgtgggt 4500

ttgcttttta aaggaatcct atatctagtc ctatatatca aacctctaac tgacgtttct 4560

tttcgaggaa gtggcttggt gggtgcagcc cccgccggtt ccgttgacgc tggcaccttc 4620

tgttgatttt ttaagccaca tgctatgatg aataaactga tttattttct accattactg 4680

aacattagga caaacacaaa ataaaaaaca aaacacagac aacggtgctg attctggtgt 4740

ggtttctact caccacgtga aataaactat caactgtata aagagaacaa agtgatttta 4800

gaataaaatg caggaaaaac ttttttaaag atgttagtct tgtagcgtga ataaatttgc 4860

catcaccttt tgtgtggtgg cctggcaggt catatacttt tttttggcat ataccttttt 4920

aaagactgta attagtgcag taacagtggg gttttttttg tgcaactctt ctaaaaacat 4980

tcataatgca gtcatgttta tttttttctg ttaaaatgtt tttgacagtt ttaagagcag 5040

tcttttggct ctgaccattt cttgttctgt ttccaatgaa atcaataaaa aaaaagaagt 5100

actttaaaaa aaaaaaaaaa aaa 5123

<210>43

<211>7970

<212>DNA

<213> Intelligent people

<400>43

gtaaaagtga cattctaaat gttcctcact ctgcgaggct tattttttag ggactttgct 60

ataattctga aagacttagt tttacagtac atctgaaagt aggagttttc agaagtatgg 120

ctcttgggat aaatttagat tcttaattgt gaagctctgt taccacttgt tagaaggcag 180

gtcagctcac ctgcttgggg aggtaaatat atgaatgcac tctcgagtaa tttaatggag 240

ccctacctca atgtacagaa tgacagtatc acagatcaag aatggagtac gagtgatttt 300

cggctatggt gggggtaggt aggtcacttg tcccctgttg tctcttacta tttgtaaagt 360

gaagactatg attagtcttt ttgatcggga tggtttgaga tgaataaaga ataggcaggc 420

aatttggata ctttaggctt ttcaagaaca ttagtaacat tttttcttag atatttctcc 480

taatacaatg agtgttgtga aataacatgg cagttattgt tgagagaaaa gccttcccag 540

ttatgtattg agtccttagg cgttttgacc ttccctccac tcttacagaa cttggtggaa 600

ggggccacta tgttttctac ctccttccgt gcctttcaca aagccacatc ctgcaccgtc 660

tacccttctc tgtggatatt tttccgcttg gcaatttcct ttcctgaggc acccacttgg 720

gacatctgaa tctccatctc catgttgatg gcccgtttgt gcttggacgt gttcttccac 780

ttgagactga gggttcatgt aatcaaagaa gtttctttgt tgtgtgtatc tttacagaac 840

acaacaggaa ttgaaaatga atcagaacac tactgagcct gtggcggcca ccgagaccct 900

ggctgaggta cccgaacatg tgctgcgagg acttccggag gaagtgaggc ttttcccttc 960

tgctgttgac aagacccgga ttggtgtctg ggccactaaa ccaattttaa aaggcaaaaa 1020

atttgggcca tttgttggtg ataagaaaaa aagatctcag gttaagaata atgtatacat 1080

gtgggaggtg tattacccaa atttgggatg gatgtgcatt gatgccactg atccagagaa 1140

gggaaactgg ctgcgatatg tgaattgggc ttgctcagga gaagagcaaa atttattccc 1200

actggaaatc aacagagcca tttactataa aactttaaag ccaatcgcgc cgggcgagga 1260

gctcctggtc tggtacaatg gggaagacaa ccctgagata gcagctgcga ttgaggaaga 1320

gcgagccagc gcccggagca agcggagctc ccccaagagc cggaaaggga agaaaaaatc 1380

ccaggaaaat aaaaacaaag gaaacaaaat ccaagacata caactgaaga caagtgagcc 1440

agatttcacc tctgcaaata tgagagattc tgcagaaggt cctaaagaag acgaagagaa 1500

gccttcagcc tcagcacttg agcagccggc caccctccag gaggtggcca gtcaggaggt 1560

gcctccagaa ctagcaaccc ctgcccctgc ctgggagcca cagccagaac cagacgagcg 1620

attagaagcg gcagcttgtg aggtgaatga tttgggggaa gaggaggagg aggaagagga 1680

ggaggatgaa gaagaagaag aagatgatga tgatgatgag ttggaagacg agggggaaga 1740

agaagccagc atgccaaatg aaaattctgt gaaagagcca gaaatacggt gtgatgagaa 1800

gccagaagat ttattagagg aaccaaaaac aacttcagaa gaaactcttg aagactgctc 1860

agaggtaaca cctgccatgc aaatccccag aactaaagaa gaggccaatg gtgatgtatt 1920

tgaaacgttt atgtttccgt gtcaacattg tgaaaggaag tttacaacca aacaggggct 1980

tgagcgtcac atgcatatcc atatatccac cgtcaatcat gctttcaaat gcaagtactg 2040

tgggaaagcc tttggcacac agattaaccg gcggcgacat gagcggcgcc atgaagcagg 2100

gttaaagcgg aaacccagcc aaacactaca gccgtcagag gatctggctg atggcaaagc 2160

atctggagaa aacgttgctt caaaagatga ttcgagtcct cccagtcttg ggccagactg 2220

tctgatcatg aattcagaga aggcttccca agacacaata aattcttctg tcgtagaaga 2280

gaatggggaa gttaaagaac ttcatccgtg caaatattgt aaaaaggttt ttggaactca 2340

tactaatatg agacggcatc agcgtagagt tcacgaacgt catctgattc ccaaaggtgt 2400

acggcgaaaa ggaggccttg aagagcccca gcctccagca gaacaggccc aggccaccca 2460

gaacgtgtat gtaccaagca cagagccgga ggaggaaggg gaagcagatg atgtgtacat 2520

catggacatt tctagcaata tctctgaaaa cttaaattac tatattgatg gtaaaattca 2580

aactaataac aacactagta actgtgatgt gattgagatg gagtctgctt cggcagattt 2640

gtatggtata aattgtctgc tcactccagt tacagtggaa attactcaaa atataaagac 2700

cacacaggtc cctgtaacag aagatcttcc taaagagcct ttgggcagca caaatagtga 2760

ggccaagaag cggagaactg cgagcccacc tgcactgccc aaaattaagg ccgaaacaga 2820

ctctgacccc atggtcccct cttgctcttt aagtcttcct cttagcatat caacaacaga 2880

ggcagtgtct ttccacaaag agaaaagtgt ttatttgtca tcaaagctca aacaacttct 2940

tcaaacccaa gataaactaa ctcctgcagg gatttcagca actgaaatag ctaaattagg 3000

tcctgtttgt gtgtctgctc ctgcatcaat gttgcctgtg acctcaagta ggtttaagag 3060

gcggaccagc tctcctccca gttctccaca gcacagtcct gcccttcgag actttggaaa 3120

gccaagtgat gggaaagcag catggaccga tgccgggctg acttccaaaa aatccaaatt 3180

agaaagtcac agcgactcac cagcatggag tttgtctggg agagatgaga gagaaactgt 3240

gagccctcca tgctttgatg aatataaaat gtctaaagag tggacagcta gttctgcttt 3300

tagcagtgtg tgcaaccagc agccactgga tttatccagc ggtgtcaaac agaaggctga 3360

gggtacaggc aagactccag tccagtggga atctgtctta gatctcagtg tgcataaaaa 3420

gcattgtagt gactctgaag gcaaggaatt caaagaaagt cattcagtgc agcctacgtg 3480

tagtgctgta aagaaaagga aaccaaccac ctgcatgctg cagaaggttc ttctcaatga 3540

atataatggc atcgatttac ctgtagaaaa ccctgcagat gggaccagga gcccaagtcc 3600

ttgtaaatcc ctagaagctc agccagatcc tgacctcggt ccgggctctg gtttccctgc 3660

ccctactgtt gagtccacac ctgatgtttg tccttcatca cctgccctgc agacaccctc 3720

cctttcatcc ggtcagctgc ctcctctctt gatccccaca gatccctctt cccctccacc 3780

ctgtcccccg gtattaactg ttgccactcc gccccctccc ctccttccta ccgtacctct 3840

tccagccccc tcttccagtg catctccaca cccatgcccc tctccactct caaatgccac 3900

cgcacagtcc ccacttccaa ttctgtcccc aacagtgtcc ccctctccct ctcccattcc 3960

tcccgtggag cccctgatgt ctgccgcctc acccgggcct ccaacacttt cttcttcctc 4020

ctcttcatct tcctcctcct cttcgttttc ttcttcatct tcctcctctt ctccttctcc 4080

acctcctctc tccgcaatat catctgttgt ttcctctggt gataatctgg aggcttctct 4140

ccccatgata tctttcaaac aggaggaatt agagaatgaa ggtctgaaac ccagggaaga 4200

gccccagtct gctgctgaac aggatgttgt tgttcaggaa acattcaaca aaaactttgt 4260

ttgcaacgtc tgtgaatcac cttttctttc cattaaagat ctaaccaaac atttatctat 4320

tcatgctgaa gaatggccct tcaaatgtga attttgtgtg cagcttttta aggataaaac 4380

ggacttgtca gaacatcgct ttttgcttca tggagttggg aatatctttg tgtgttctgt 4440

ttgtaaaaaa gaatttgctt ttttgtgcaa tttgcagcag caccagcgag atctccaccc 4500

agataaggtg tgcacacatc acgagtttga aagcgggact ctgaggcccc agaactttac 4560

agatcccagc aaggcccatg tagagcatat gcagagcttg ccagaagatc ctttagaaac 4620

ttctaaagaa gaagaggagt taaatgattc ctctgaagag ctttacacga ctataaaaat 4680

aatggcttct ggaataaaga caaaagatcc agatgttcga ttgggcctca atcagcatta 4740

cccaagcttt aaaccacctc catttcagta ccatcaccgt aaccccatgg ggattggtgt 4800

gacagccaca aatttcacta cacacaatat tccacagact ttcactaccg ccattcgctg 4860

cacaaagtgt ggaaaaggtg tcgacaatat gccggagttg cacaaacata tcctggcttg 4920

tgcttctgca agtgacaaga agaggtacac gcctaagaaa aacccagtac cattaaaaca 4980

aactgtgcaa cccaaaaatg gcgtggtggt tttagataac tctgggaaaa atgccttccg 5040

acgaatggga cagcccaaaa ggcttaactt tagtgttgag ctcagcaaaa tgtcgtcgaa 5100

taagctcaaa ttaaatgcat tgaagaaaaa aaatcagcta gtacagaaag caattcttca 5160

gaaaaacaaa tctgcaaagc agaaggccga cttgaaaaat gcttgtgagt catcctctca 5220

catctgccct tactgtaatc gagagttcac ttacattgga agcctgaata aacacgccgc 5280

cttcagctgt cccaaaaaac ccctttctcc tcccaaaaaa aaagtttctc attcatctaa 5340

gaaaggtgga cactcatcac ctgcaagtag tgacaaaaac agtaacagca accaccgcag 5400

acggacagcg gatgcggaga ttaaaatgca aagcatgcag actccgttgg gcaagaccag 5460

agcccgcagc tcaggcccca cccaagtccc acttccctcc tcatccttca ggtccaagca 5520

gaacgtcaag tttgcagctt cggtgaaatc caaaaaacca agctcctcct ctttaaggaa 5580

ctccagcccg ataagaatgg ccaaaataac tcatgttgag gggaaaaaac ctaaagctgt 5640

ggccaagaat cattctgctc agctttccag caaaacatca cggagcctgc acgtgagggt 5700

acagaaaagc aaagctgttt tacaaagcaa atccaccttg gcgagtaaga aaagaacaga 5760

ccggttcaat ataaaatcta gagagcggag tggggggcca gtcacccgga gccttcagct 5820

ggcagctgct gctgacttga gtgagaacaa gagagaggac ggcagcgcca agcaggagct 5880

gaaggacttc agctacagcc tccgcttggc gtcccgatgc tctccaccag cggccccgta 5940

catcaccagg cagtatagga aggtcaaagc tccagctgca gcccagttcc agggaccatt 6000

cttcaaagag tagacactct ggctgctccc tgacagcacc tgaagtgacc tggaatcagt 6060

gaagccaaag ggactggcag tctgccctgc agggagtacc gacctatccc agttgtgtga 6120

ggctgcgaga gaaagggagt gcatgtgcgc gcgtgcatgt gtgcgtgcgt gtgtgttcac 6180

gtgttctcgt gcgggcgcgt gagtggtctt caaacgaggg tcccgatccc cggggcggca 6240

ggaagggggc cgactccacg ctgtcctttg ggatgatact tggatgcagc tcttgggacc 6300

gtgttctgca gcccagcctt cctgttgggg tggggcctct cctactatgc aatttttcaa 6360

gagctccttg accctgcttt ttgcttcttg agttgtcttt tgccattatg gggactttgg 6420

tttgacccag gggtcagcct taggaaggcc ttcaggagga ggccgagttc cccttcagta 6480

ccacccctct ctccccacct tccctctccc ggcaacatct ctgggaatca acagcatatt 6540

gacacgttgg agccgagcct gaacatgccc ctcggcccca gcacatggaa aacccccttc 6600

cttgcctaag gtgtctgagt ttctggctct tgaggcattt ccagacttga aattctcatc 6660

agtccattgc tcttgagtct ttgcagagaa cctcagatca ggtgcacctg ggagaaagac 6720

tttgtcccca cttacagatc tatctcctcc cttgggaagg gcagggaatg gggacggtgt 6780

atggagggga gggatctcct gcgcccttca ttgccacact tggtgggacc atgaacatct 6840

ttagtgtctg agcttctcaa attagctgca ataggaaaaa aacaaattgg gaaatgaaaa 6900

aaaaatggga agattaaaaa gcacaggggg aagaagaaga gatttcggag gccatcctgc 6960

caggggcgga cggggctgac tcctgctctc tggaggacgg tcagtccatg tctcggagaa 7020

acgggtgagc tgagcttggc gtttggaccc agttcagtga ggttcttggg ttttgtgcct 7080

ttggggcaga ccccaggcaa ggatgtctga gaccacttgg gcgctgtttt ctcagctcca 7140

atttcaagag tgagctatca aacccagagc ggaaggaggg agctctgatg agcacggttt 7200

gtcacacgat aaagggattt tttttttcag ggctactacg gttgatcttg caactctgta 7260

aatatgtatg tagacacttt taaaagcacg tatttatgtc cctgactgta aatgctccat 7320

ttttaaagtt ttataacttg tgttatttaa tgagtcagtc aatcggctgc agtatgggat 7380

ctgataagga tctaggagaa gggtctcatg cggaccctca catgggcaga aaaatggtgg 7440

tcattggccg acatcacagt tttcctgttt cccacccagc taaaaaccgt tgtttgcttt 7500

aaattttcat aaactggaat cctttcaccc gctcctacag ctaaccctca caagcatgaa 7560

gtgctgtggc tgttccttat cctaatgatg cgcttttgtc ccgtaaatgt taacactcat 7620

gaagcatacc ccggcctctc agttcttgag ggcctcccca ccgcagcagc aaggaaagct 7680

cacgaacccc aaacctggca agtcacctgc agcccatggt gagctctggg aagtgtggtt 7740

gaggccttgg ggtcactcct tttttgcatg tgcaaatgtg ctggtcaccc ttcaacgctc 7800

ccagacggtc aggaaaactg ttccaatcat gaaaaggggg gatgattttg taaaagtggc 7860

atttcctggt cagtggtggt cttcaagacg acagctctgt atctgccatg tgaagagaat 7920

taacaataaa agtgtgaaga gcgattgtga ggaacaaaaa aaaaaaaaaa 7970

<210>44

<211>5

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

<400>44

Gly Gly Gly Gly Cys

1 5

<210>45

<211>2339

<212>DNA

<213> Intelligent people

<400>45

ccaagcctga gaatcaggag aagcctcaca gtgacacccc caactgagga aactcacaga 60

gctgggacat actctacttc ttcagaaaaa agtatactga ctagagtgga gtccccctgg 120

ggagtcagaa agcctgtgaa agatctcact tgttcaaaag tccaagtgtg aattactgtc 180

tcacagataa accaaagtat tttgaaaaac aaaggggaga aaagaaatta ctccccagaa 240

ctctcaggca tctagaggac acccaagaac gtgggagtca gctgcttctt gtgtgcagcc 300

atgaagtctg tgcactccag tccccagaac acgagtcata ccatcatgac gttttaccca 360

accatggaag aatttgcaga tttcaacaca tatgttgctt acatggagtc ccaaggcgca 420

catcaagctg gccttgccaa ggtaattcca cccaaggaat ggaaagccag acagatgtat 480

gatgatatcg aagacatctt aatagccact cccctccagc aggtgacctc tgggcaggga 540

ggtgtgttta ctcaatacca taaaaagaag aaagccatga gggtggggca gtatcgccgc 600

ttggcaaaca gtaaaaaata tcagactccg ccacaccaga attttgcaga tttggagcaa 660

cgatactgga agagccaccc cggtaatcca ccaatttatg gtgctgatat cagcggctcc 720

ttatttgaag aaagcactaa acaatggaac ctaggacacc tgggaacaat tctggacctg 780

ttggagcagg aatgtggggt tgtcatcgag ggtgtcaaca caccctacct gtactttggc 840

atgtggaaga ccacgtttgc ctggcacaca gaggacatgg acctttacag catcaactac 900

ctgcactttg gggagcccaa aacttggtac gtggtgcccc cagaacatgg tcagcacctg 960

gaacgcctgg ccagggagct cttcccagac atttctcggg gctgtgaggc cttcctgcgg 1020

cacaaagtgg ccctcatctc gcctacagtt ctcaaggaaa atgggattcc cttcaattgc 1080

atgactcagg aggctgggga gttcatggtg acctttccct atggctacca tgctggcttc 1140

aatcacggct tcaactgcgc agaagccatt aattttgcca ctccacgatg gattgattat 1200

ggcaaaatgg cctctcagtg tagctgtggg gagtcgacag tgaccttttc catggacccc 1260

tttgtgcgca ttgtgcaacc cgagagttat gagctctgga aacacaggca agacttggcc 1320

attgtggaac acacagagcc cagggttgca gaaagccaag agctgagcaa ctggagagat 1380

gatatagtac ttagaagagc tgctctgggc ctgaggcttc tcccaaacct cacagcccag 1440

tgtcccacac agcctgtgtc ctcagggcac tgttacaacc caaaaggctg tggcactgat 1500

gctgtgcctg gatccgcatt ccaaagctct gcatatcata cccagaccca gtcacttacc 1560

ctggggatgt cagccagggt tcttctccct tccactggaa gctggggttc tggtcgtggt 1620

cgtggtcgtg gtcaaggtca aggtcgaggt tgcagtcgtg gtcgtggtca tggttgttgt 1680

actcgagaac tggggactga ggagccaact gttcagcctg catccaagag gcgcctttta 1740

atgggtacaa ggagtagagc tcaaggccac aggcctcagc tcccgcttgc caatgatttg 1800

atgacaaatc tgtccctttg agtggtggcc ttcagcatct tgccaaggct tctggctgct 1860

gctgtgtccc tgatcttcaa ctcctggggc ccccactgga tcgtgatgaa accatgcacc 1920

ctggcctgtg cctgctatcc ctcaacagca ctactagtaa tctccctgat gttgtctgca 1980

tgactcctcc caatgtcatt gtgcctttga ttaagttttc cagggacact ggtggggact 2040

ggaactgatt aagttcacca gggacacttg cctggtgaac atgggcaagg ctgtagcaat 2100

ggaccacttt tacggctcta gggttctgac tccaactaag ttttccagaa tctcctgggc 2160

tcctgactca tctgctgggt ctaaagacac tgagtttagg gatattttcc tccaatacat 2220

gatcaatcct ctggatccac ggctatggaa tatggtgaca aatgtcagtg tctctcttat 2280

tccaacccca ggatcagaga agattcttta cctgcagtaa ctgacacatt tccaaggcc 2339

<210>46

<211>506

<212>PRT

<213> Intelligent people

<400>46

Met Lys Ser Val His Ser Ser Pro Gln Asn Thr Ser His Thr Ile Met

1 5 10 15

Thr Phe Tyr Pro Thr Met Glu Glu Phe Ala Asp Phe Asn Thr Tyr Val

20 25 30

Ala Tyr Met Glu Ser Gln Gly Ala His Gln Ala Gly Leu Ala Lys Val

35 40 45

Ile Pro Pro Lys Glu Trp Lys Ala Arg Gln Met Tyr Asp Asp Ile Glu

50 55 60

Asp Ile Leu Ile Ala Thr Pro Leu Gln Gln Val Thr Ser Gly Gln Gly

65 70 75 80

Gly Val Phe Thr Gln Tyr His Lys Lys Lys Lys Ala Met Arg Val Gly

85 90 95

Gln Tyr Arg Arg Leu Ala Asn Ser Lys Lys Tyr Gln Thr Pro Pro His

100 105 110

Gln Asn Phe Ala Asp Leu Glu Gln Arg Tyr Trp Lys Ser His Pro Gly

115 120 125

Asn Pro Pro Ile Tyr Gly Ala Asp Ile Ser Gly Ser Leu Phe Glu Glu

130 135 140

Ser Thr Lys Gln Trp Asn Leu Gly His Leu Gly Thr Ile Leu Asp Leu

145 150 155 160

Leu Glu Gln Glu Cys Gly Val Val Ile Glu Gly Val Asn Thr Pro Tyr

165 170 175

Leu Tyr Phe Gly Met Trp Lys Thr Thr Phe Ala Trp His Thr Glu Asp

180 185 190

Met Asp Leu Tyr Ser Ile AsnTyr Leu His Phe Gly Glu Pro Lys Thr

195 200 205

Trp Tyr Val Val Pro Pro Glu His Gly Gln His Leu Glu Arg Leu Ala

210 215 220

Arg Glu Leu Phe Pro Asp Ile Ser Arg Gly Cys Glu Ala Phe Leu Arg

225 230 235 240

His Lys Val Ala Leu Ile Ser Pro Thr Val Leu Lys Glu Asn Gly Ile

245 250 255

Pro Phe Asn Cys Met Thr Gln Glu Ala Gly Glu Phe Met Val Thr Phe

260 265 270

Pro Tyr Gly Tyr His Ala Gly Phe Asn His Gly Phe Asn Cys Ala Glu

275 280 285

Ala Ile Asn Phe Ala Thr Pro Arg Trp Ile Asp Tyr Gly Lys Met Ala

290 295 300

Ser Gln Cys Ser Cys Gly Glu Ser Thr Val Thr Phe Ser Met Asp Pro

305 310 315 320

Phe Val Arg Ile Val Gln Pro Glu Ser Tyr Glu Leu Trp Lys His Arg

325 330 335

Gln Asp Leu Ala Ile Val Glu His Thr Glu Pro Arg Val Ala Glu Ser

340 345 350

Gln Glu Leu Ser Asn Trp Arg Asp Asp Ile Val Leu Arg Arg Ala Ala

355 360 365

Leu Gly Leu Arg Leu Leu Pro Asn Leu Thr Ala Gln Cys Pro Thr Gln

370 375 380

Pro Val Ser Ser Gly His Cys Tyr Asn Pro Lys Gly Cys Gly Thr Asp

385 390 395 400

Ala Val Pro Gly Ser Ala Phe Gln Ser Ser Ala Tyr His Thr Gln Thr

405 410 415

Gln Ser Leu Thr Leu Gly Met Ser Ala Arg Val Leu Leu Pro Ser Thr

420 425 430

Gly Ser Trp Gly Ser Gly Arg Gly Arg Gly Arg Gly Gln Gly Gln Gly

435 440 445

Arg Gly Cys Ser Arg Gly Arg Gly His Gly Cys Cys Thr Arg Glu Leu

450 455 460

Gly Thr Glu Glu Pro Thr Val Gln Pro Ala Ser Lys Arg Arg Leu Leu

465 470475 480

Met Gly Thr Arg Ser Arg Ala Gln Gly His Arg Pro Gln Leu Pro Leu

485 490 495

Ala Asn Asp Leu Met Thr Asn Leu Ser Leu

500 505

<210>47

<211>2999

<212>DNA

<213> Intelligent people

<400>47

gatcaactat ccacgctgct cgaatcacag catgctggag ggcctggctg ggtgctctga 60

ctgactgatc acctgacaga cggtgcggtc agtcggatgc tgagaatgac tgacgatgtg 120

atgaggggcg gattgaacga gtcacaggcc agctggccag gagcaaaatc ggcatagctg 180

tctgactcga tggctgtacg tggttacgga ctgtctgccc tgatagaatc tcagcttcaa 240

cgcatcagag gagactgact tgaccaatgg tggggatgag tcgcctgaga aatgacagac 300

tggctgaccc actgacaggc tgcagcgtgt gttgcaagtc ttcttggaat cagctgcagg 360

acctgtgccg cctggccaag ctctcctgcc ctgccctcgg tatctctaag aggaacctct 420

atgactttga agtcgagtac ctgtgcgatt acaagaagat ccgcgaacag gaatattacc 480

tggtgaaatg gcgtggatat ccagactcag agagcacctg ggagccacgg cagaatctca 540

agtgtgtgcg tatcctcaagcagttccaca aggacttaga aagggagctg ctccggcggc 600

accaccggtc aaagaccccc cggcacctgg acccaagctt ggccaactac ctggtgcaga 660

aggccaagca gaggcgggcg ctccgtcgct gggagcagga gctcaatgcc aagcgcagcc 720

atctgggacg catcactgta gagaatgagg tggacctgga cggccctccg cgggccttcg 780

tgtacatcaa tgagtaccgt gttggtgagg gcatcaccct caaccaggtg gctgtgggct 840

gcgagtgcca ggactgtctg tgggcaccca ctggaggctg ctgcccgggg gcgtcactgc 900

acaagtttgc ctacaatgac cagggccagg tgcggcttcg agccgggctg cccatctacg 960

agtgcaactc ccgctgccgc tgcggctatg actgcccaaa tcgtgtggta cagaagggta 1020

tccgatatga cctctgcatc ttccgcacgg atgatgggcg tggctggggc gtccgcaccc 1080

tggagaagat tcgcaagaac agcttcgtca tggagtacgt gggagagatc attacctcag 1140

aggaggcaga gcggcggggc cagatctacg accgtcaggg cgccacctac ctctttgacc 1200

tggactacgt ggaggacgtg tacaccgtgg atgccgccta ctatggcaac atctcccact 1260

ttgtcaacca cagttgtgac cccaacctgc aggtgtacaa cgtcttcata gacaaccttg 1320

acgagcggct gccccgcatc gctttctttg ccacaagaac catccgggca ggcgaggagc 1380

tcacctttga ttacaacatg caagtggacc ccgtggacat ggagagcacc cgcatggact 1440

ccaactttgg cctggctggg ctccctggct cccctaagaa gcgggtccgt attgaatgca 1500

agtgtgggac tgagtcctgc cgcaaatacc tcttctagcc cttagaagtc tgaggccaga 1560

ctgactgagg gggcctgaag ctacatgcac ctcccccact gctgccctcc tgtcgagaat 1620

gactgccagg gcctcgcctg cctccacctg cccccacctg ctcctacctg ctctacgttc 1680

agggctgtgg ccgtggtgag gaccgactcc aggagtcccc tttccctgtc ccagccccat 1740

ctgtgggttg cacttacaaa cccccaccca ccttcagaaa tagtttttca acatcaagac 1800

tctctgtcgt tgggattcat ggcctattaa ggaggtccaa ggggtgagtc ccaacccagc 1860

cccagaatat atttgttttt gcacctgctt ctgcctggag attgaggggt ctgctgcagg 1920

cctcctccct gctgccccaa aggtatgggg aagcaacccc agagcaggca gacatcagag 1980

gccagagtgc ctagcccgac atgaagctgg ttccccaacc acagaaactt tgtactagtg 2040

aaagaaaggg ggtccctggg ctacgggctg aggctggttt ctgctcgtgc ttacagtgct 2100

gggtagtgtt ggccctaaga gctgtagggt ctcttcttca gggctgcata tctgagaagt 2160

ggatgcccac atgccactgg aagggaagtg ggtgtccatg ggccactgag cagtgagagg 2220

aaggcagtgc agagctggcc agccctggag gtaggctggg accaagctct gccttcacag 2280

tgcagtgaag gtacctaggg ctcttgggag ctctgcggtt gctaggggcc ctgacctggg 2340

gtgtcatgac cgctgacacc actcagagct ggaaccaaga tctagatagt ccgtagatag 2400

cacttaggac aagaatgtgc attgatgggg tggtgatgag gtgccaggca ctgggtagag 2460

cacctggtcc acgtggattg tctcagggaa gccttgaaaa ccacggaggt ggatgccagg 2520

aaagggccca tgtggcagaa ggcaaagtac aggccaagaa ttgggggtgg gggagatggc 2580

ttccccacta tgggatgacg aggcgagagg gaagcccttg ctgcctgcca ttcccagacc 2640

ccagcccttt gtgctcaccc tggttccact ggtctcaaaa gtcacctgcc tacaaatgta 2700

caaaaggcga aggttctgat ggctgccttg ctccttgctc ccccaccccc tgtgaggact 2760

tctctaggaa gtccttcctg actacctgtg cccagagtgc ccctacatga gactgtatgc 2820

cctgctatca gatgccagat ctatgtgtct gtctgtgtgt ccatcccgcc ggccccccag 2880

actaacctcc aggcatggac tgaatctggt tctcctcttg tacacccctc aaccctatgc 2940

agcctggagt gggcatcaat aaaatgaact gtcgactgaa caaaaaaaaa aaaaaaaaa 2999

<210>48

<211>423

<212>PRT

<213> Intelligent people

<400>48

Met Val Gly Met Ser Arg Leu Arg Asn Asp Arg Leu Ala Asp Pro Leu

1 5 10 15

Thr Gly Cys Ser Val Cys Cys Lys Ser Ser Trp Asn Gln Leu Gln Asp

20 25 30

Leu Cys Arg Leu Ala Lys Leu Ser Cys Pro Ala Leu Gly Ile Ser Lys

35 40 45

Arg Asn Leu Tyr Asp Phe Glu Val Glu Tyr Leu Cys Asp Tyr Lys Lys

50 55 60

Ile Arg Glu Gln Glu Tyr Tyr Leu Val Lys Trp Arg Gly Tyr Pro Asp

65 70 75 80

Ser Glu Ser Thr Trp Glu Pro Arg Gln Asn Leu Lys Cys Val Arg Ile

85 90 95

Leu Lys Gln Phe His Lys Asp Leu Glu Arg Glu Leu Leu Arg Arg His

100 105 110

His Arg Ser Lys Thr Pro Arg His Leu Asp Pro Ser Leu Ala Asn Tyr

115 120 125

Leu Val Gln Lys Ala Lys Gln Arg Arg Ala Leu Arg Arg Trp Glu Gln

130 135 140

Glu Leu Asn Ala Lys Arg Ser His Leu Gly Arg Ile Thr Val Glu Asn

145 150 155 160

Glu Val Asp Leu Asp Gly Pro Pro Arg Ala Phe Val Tyr Ile Asn Glu

165 170 175

Tyr Arg Val Gly Glu Gly Ile ThrLeu Asn Gln Val Ala Val Gly Cys

180 185 190

Glu Cys Gln Asp Cys Leu Trp Ala Pro Thr Gly Gly Cys Cys Pro Gly

195 200 205

Ala Ser Leu His Lys Phe Ala Tyr Asn Asp Gln Gly Gln Val Arg Leu

210 215 220

Arg Ala Gly Leu Pro Ile Tyr Glu Cys Asn Ser Arg Cys Arg Cys Gly

225 230 235 240

Tyr Asp Cys Pro Asn Arg Val Val Gln Lys Gly Ile Arg Tyr Asp Leu

245 250 255

Cys Ile Phe Arg Thr Asp Asp Gly Arg Gly Trp Gly Val Arg Thr Leu

260 265 270

Glu Lys Ile Arg Lys Asn Ser Phe Val Met Glu Tyr Val Gly Glu Ile

275 280 285

Ile Thr Ser Glu Glu Ala Glu Arg Arg Gly Gln Ile Tyr Asp Arg Gln

290 295 300

Gly Ala Thr Tyr Leu Phe Asp Leu Asp Tyr Val Glu Asp Val Tyr Thr

305 310 315 320

Val Asp Ala Ala Tyr Tyr Gly Asn Ile Ser His Phe Val Asn His Ser

325 330 335

Cys Asp Pro Asn Leu Gln Val Tyr Asn Val Phe Ile Asp Asn Leu Asp

340 345 350

Glu Arg Leu Pro Arg Ile Ala Phe Phe Ala Thr Arg Thr Ile Arg Ala

355 360 365

Gly Glu Glu Leu Thr Phe Asp Tyr Asn Met Gln Val Asp Pro Val Asp

370 375 380

Met Glu Ser Thr Arg Met Asp Ser Asn Phe Gly Leu Ala Gly Leu Pro

385 390 395 400

Gly Ser Pro Lys Lys Arg Val Arg Ile Glu Cys Lys Cys Gly Thr Glu

405 410 415

Ser Cys Arg Lys Tyr Leu Phe

420

<210>49

<211>3148

<212>DNA

<213> Intelligent people

<400>49

aacaagcccc ggcccccaag tcccgcgcgg gccggccagg ggcggggcgt cgggccagct 60

gagctatccc gtcagaccgc gccagtttga atgaaagctc tacaagatgg cggcggtcgg 120

ggccgaggcg cgaggagctt ggtgtgtgcc ttgcctagtt tcacttgata ctcttcagga 180

attatgtaga aaagaaaagc tcacatgtaa atcgattgga atcaccaaaa ggaatctaaa 240

caattatgag gtggaatact tgtgtgacta caaggtagta aaggatatgg aatattatct 300

tgtaaaatgg aaaggatggc cagattctac aaatacttgg gaacctttgc aaaatctgaa 360

gtgcccgtta ctgcttcagc aattctctaa tgacaagcat aattatttat ctcaggtaaa 420

gaaaggcaaa gcaataactc caaaagacaa taacaaaact ttgaaacctg ccattgctga 480

gtacattgtg aagaaggcta aacaaaggat agctctgcag agatggcaag atgaactcaa 540

cagaagaaag aatcataaag gaatgatatt tgttgaaaat actgttgatt tagagggccc 600

accttcagac ttctattaca ttaacgaata caaaccagct cctggaatca gcttagtcaa 660

tgaagctacc tttggttgtt catgcacaga ttgcttcttt caaaaatgtt gtcctgctga 720

agctggagtt cttttggctt ataataaaaa ccaacaaatt aaaatcccac ctggtactcc 780

catctatgaa tgcaactcaa ggtgtcagtg tggtcctgat tgtcccaata ggattgtaca 840

aaaaggcaca cagtattcgc tttgcatctt tcgaactagc aatggacgtg gctggggtgt 900

aaagaccctt gtgaagatta aaagaatgag ttttgtcatg gaatatgttg gagaggtaat 960

cacaagtgaa gaagctgaaa gacgaggaca gttctatgac aacaagggaa tcacgtatct 1020

ctttgatctg gactatgagt ctgatgaatt cacagtggat gcggctcgat acggcaatgt 1080

gtctcatttt gtgaatcaca gctgtgaccc aaatcttcag gtgttcaatg ttttcattga 1140

taacctcgat actcgtcttc cccgaatagc attgttttcc acaagaacca taaatgctgg 1200

agaagagctg acttttgatt atcaaatgaa aggttctgga gatatatctt cagattctat 1260

tgaccacagc ccagccaaaa agagggtcag aacagtatgt aaatgtggag ctgtgacttg 1320

cagaggttac ctcaactgaa ctttttcagg aaatagagct gatgattata atattttttt 1380

cctaatgtta acatttttaa aaatacatat ttgggactct tattatcaag gttctaccta 1440

tgttaattta caattcatgt ttcaagacat ttgccaaatg tattaccgat gcctctgaaa 1500

agggggtcac tgggtctcat agactgatat gaagtcgaca tatttatagt gcttagagac 1560

caaactaatg gaaggcagac tatttacagc ttagtatatg tgtacttaag tctatgtgaa 1620

cagagaaatg cctcccgtag tgtttgaaag cgttaagctg ataatgtaat taacaactgc 1680

tgagagatca aagattcaac ttgccataca cctcaaattc ggagaaacag ttaatttggg 1740

caaatctaca gttctgtttt tgctactcta ttgtcattcc tgtttaatac tcactgtact 1800

tgtatttgag acaaataggt gatactgaat tttatactgt tttctacttt tccattaaaa 1860

cattggcacc tcaatgataa agaaatttaa ggtataaaat taaatgtaaa aattaatttc 1920

agcttcattt cgtatttcga agcaatctag actgttgtga tgagtgtatg tctgaacctg 1980

taattcttaa aagacttctt aatcttctag aagaaaaatc tccgaagagc tctctctaga 2040

agtccaaaat ggctagccat tatgcttctt tgaaaggaca tgataatggg accaggatgg 2100

ttttttggag taccaagcaa ggggaatgga gcactttaag ggcgcctgtt agtaacatga 2160

attggaaatc tgtgtcgagt acctctgatc taaacggtaa aacaagctgc ctggagagca 2220

gctgtaccta acaatactgt aatgtacatt aacattacag cctctcaatt tcaggcaggt 2280

gtaacagttc ctttccacca gatttaatat ttttatactt cctgcaggtt cttcttaaaa 2340

agtaatctat atttttgaac tgatacttgt tttatacata aatttttttt agatgtgata 2400

aagctaaact tggccaaagt gtgtgcctga attattagac ctttttatta gtcaacctac 2460

gaagactaaa atagaatata ttagttttca agggagtggg aggcttccaa catagtattg 2520

aatctcagga aaaactattc tttcatgtct gattctgaga tttctaattg tgttgtgaaa 2580

atgataaatg cagcaaatct agctttcagt attcctaatt tttacctaag ctcattgctc 2640

caggctttga ttacctaaaa taagcttgga taaaattgaa ccaacttcaa gaatgcagca 2700

cttcttaatc tttagctctt tcttgggaga agctagactt tattcattat attgctatga 2760

caacttcact ctttcataat atataggata aattgtttac atgattggac cctcagattc 2820

tgttaaccaa aattgcagaa tggggggcca ggcctgtgtg gtggctcaca cctgtgatcc 2880

cagcactttg ggaggctgag gtaggaggat cacgtgaggt cgggagttca agaccagcct 2940

ggccatcatg gtgaaaccct gtctctactg aaaatacaaa aattagccgg gcgtggtggc 3000

acacgcctgt agtcccagct actcaggagg ctgaggcagg agaatcactt gaattcagga 3060

ggcggaggtt gcagtgagcc aagatcatac cactgcactg cagcctgagt gacacagtaa 3120

gactgtctcc aaaaaaaaaa aaaaaaaa 3148

<210>50

<211>33

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic oligonucleotides

<220>

<221> modified base

<222>(3)..(31)

<223> a, c, t, g, unknown or others

<400>50

aannnnnnnn nnnnnnnnnn nnnnnnnnnn ntt 33

<210>51

<211>3106

<212>DNA

<213> Intelligent people

<400>51

agtttgaatg aaagctctac aagatggcgg cggtcggggc cgaggcgcga ggaggtgagg 60

ctggagcgcg gccccctcgc cttccctgtt cccagcttgg tgtgtgcctt gcctagtttc 120

acttgatact cttcaggaat tatgtagaaa agaaaagctc acatgtaaat cgattggaat 180

caccaaaagg aatctaaaca attatgaggt ggaatacttg tgtgactaca aggtagtaaa 240

ggatatggaa tattatcttg taaaatggaa aggatggcca gattctacaa atacttggga 300

acctttgcaa aatctgaagt gcccgttact gcttcagcaa ttctctaatg acaagcataa 360

ttatttatct caggtaaaga aaggcaaagc aataactcca aaagacaata acaaaacttt 420

gaaacctgcc attgctgagt acattgtgaa gaaggctaaa caaaggatag ctctgcagag 480

atggcaagat gaactcaaca gaagaaagaa tcataaagga atgatatttg ttgaaaatac 540

tgttgattta gagggcccac cttcagactt ctattacatt aacgaataca aaccagctcc 600

tggaatcagc ttagtcaatg aagctacctt tggttgttca tgcacagatt gcttctttca 660

aaaatgttgt cctgctgaag ctggagttct tttggcttat aataaaaacc aacaaattaa 720

aatcccacct ggtactccca tctatgaatg caactcaagg tgtcagtgtg gtcctgattg 780

tcccaatagg attgtacaaa aaggcacaca gtattcgctt tgcatctttc gaactagcaa 840

tggacgtggc tggggtgtaa agacccttgt gaagattaaa agaatgagtt ttgtcatgga 900

atatgttgga gaggtaatca caagtgaaga agctgaaaga cgaggacagt tctatgacaa 960

caagggaatc acgtatctct ttgatctgga ctatgagtct gatgaattca cagtggatgc 1020

ggctcgatac ggcaatgtgt ctcattttgt gaatcacagc tgtgacccaa atcttcaggt 1080

gttcaatgtt ttcattgata acctcgatac tcgtcttccc cgaatagcat tgttttccac 1140

aagaaccata aatgctggag aagagctgac ttttgattat caaatgaaag gttctggaga 1200

tatatcttca gattctattg accacagccc agccaaaaag agggtcagaa cagtatgtaa 1260

atgtggagct gtgacttgca gaggttacct caactgaact ttttcaggaa atagagctga 1320

tgattataat atttttttcc taatgttaac atttttaaaa atacatattt gggactctta 1380

ttatcaaggt tctacctatg ttaatttaca attcatgttt caagacattt gccaaatgta 1440

ttaccgatgc ctctgaaaag ggggtcactg ggtctcatag actgatatga agtcgacata 1500

tttatagtgc ttagagacca aactaatgga aggcagacta tttacagctt agtatatgtg 1560

tacttaagtc tatgtgaaca gagaaatgcc tcccgtagtg tttgaaagcg ttaagctgat 1620

aatgtaatta acaactgctg agagatcaaa gattcaactt gccatacacc tcaaattcgg 1680

agaaacagtt aatttgggca aatctacagt tctgtttttg ctactctatt gtcattcctg 1740

tttaatactc actgtacttg tatttgagac aaataggtga tactgaattt tatactgttt 1800

tctacttttc cattaaaaca ttggcacctc aatgataaag aaatttaagg tataaaatta 1860

aatgtaaaaa ttaatttcag cttcatttcg tatttcgaag caatctagac tgttgtgatg 1920

agtgtatgtc tgaacctgta attcttaaaa gacttcttaa tcttctagaa gaaaaatctc 1980

cgaagagctc tctctagaag tccaaaatgg ctagccatta tgcttctttg aaaggacatg 2040

ataatgggac caggatggtt ttttggagta ccaagcaagg ggaatggagc actttaaggg 2100

cgcctgttag taacatgaat tggaaatctg tgtcgagtac ctctgatcta aacggtaaaa 2160

caagctgcct ggagagcagc tgtacctaac aatactgtaa tgtacattaa cattacagcc 2220

tctcaatttc aggcaggtgt aacagttcct ttccaccaga tttaatattt ttatacttcc 2280

tgcaggttct tcttaaaaag taatctatat ttttgaactg atacttgttt tatacataaa 2340

ttttttttag atgtgataaa gctaaacttg gccaaagtgt gtgcctgaat tattagacct 2400

ttttattagt caacctacga agactaaaat agaatatatt agttttcaag ggagtgggag 2460

gcttccaaca tagtattgaa tctcaggaaa aactattctt tcatgtctga ttctgagatt 2520

tctaattgtg ttgtgaaaat gataaatgca gcaaatctag ctttcagtat tcctaatttt 2580

tacctaagct cattgctcca ggctttgatt acctaaaata agcttggata aaattgaacc 2640

aacttcaaga atgcagcact tcttaatctt tagctctttc ttgggagaag ctagacttta 2700

ttcattatat tgctatgaca acttcactct ttcataatat ataggataaa ttgtttacat 2760

gattggaccc tcagattctg ttaaccaaaa ttgcagaatg gggggccagg cctgtgtggt 2820

ggctcacacc tgtgatccca gcactttggg aggctgaggt aggaggatca cgtgaggtcg 2880

ggagttcaag accagcctgg ccatcatggt gaaaccctgt ctctactgaa aatacaaaaa 2940

ttagccgggc gtggtggcac acgcctgtag tcccagctac tcaggaggct gaggcaggag 3000

aatcacttga attcaggagg cggaggttgc agtgagccaa gatcatacca ctgcactgca 3060

gcctgagtga cacagtaaga ctgtctccaa aaaaaaaaaa aaaaaa 3106

<210>52

<211>2608

<212>DNA

<213> Intelligent people

<400>52

aacaagcccc ggcccccaag tcccgcgcgg gccggccagg ggcggggcgt cgggccagct 60

gagctatccc gtcagaccgc gccagtttga atgaaagctc tacaagatgg cggcggtcgg 120

ggccgaggcg cgaggagctt ggtgtgtgcc ttgcctagtt tcacttgata ctcttcagga 180

attatgtaga aaagaaaagc tcacatgtaa atcgattgga atcaccaaaa ggaatctaaa 240

caattatgag gtggaatact tgtgtgacta caaggtagta aaggatatgg aatattatct 300

tgtaaaatgg aaaggatggc cagattctac aaatacttgg gaacctttgc aaaatctgaa 360

gtgcccgtta ctgcttcagc aattctctaa tgacaagcat aattatttat ctcaggtaat 420

cacaagtgaa gaagctgaaa gacgaggaca gttctatgac aacaagggaa tcacgtatct 480

ctttgatctg gactatgagt ctgatgaatt cacagtggat gcggctcgat acggcaatgt 540

gtctcatttt gtgaatcaca gctgtgaccc aaatcttcag gtgttcaatg ttttcattga 600

taacctcgat actcgtcttc cccgaatagc attgttttcc acaagaacca taaatgctgg 660

agaagagctg acttttgatt atcaaatgaa aggttctgga gatatatctt cagattctat 720

tgaccacagc ccagccaaaa agagggtcag aacagtatgt aaatgtggag ctgtgacttg 780

cagaggttac ctcaactgaa ctttttcagg aaatagagct gatgattata atattttttt 840

cctaatgtta acatttttaa aaatacatat ttgggactct tattatcaag gttctaccta 900

tgttaattta caattcatgt ttcaagacat ttgccaaatg tattaccgat gcctctgaaa 960

agggggtcac tgggtctcat agactgatat gaagtcgaca tatttatagt gcttagagac 1020

caaactaatg gaaggcagac tatttacagc ttagtatatg tgtacttaag tctatgtgaa 1080

cagagaaatg cctcccgtag tgtttgaaag cgttaagctg ataatgtaat taacaactgc 1140

tgagagatca aagattcaac ttgccataca cctcaaattc ggagaaacag ttaatttggg 1200

caaatctaca gttctgtttt tgctactcta ttgtcattcc tgtttaatac tcactgtact 1260

tgtatttgag acaaataggt gatactgaat tttatactgt tttctacttt tccattaaaa 1320

cattggcacc tcaatgataa agaaatttaa ggtataaaat taaatgtaaa aattaatttc 1380

agcttcattt cgtatttcga agcaatctag actgttgtga tgagtgtatg tctgaacctg 1440

taattcttaa aagacttctt aatcttctag aagaaaaatc tccgaagagc tctctctaga 1500

agtccaaaat ggctagccat tatgcttctt tgaaaggaca tgataatggg accaggatgg 1560

ttttttggag taccaagcaa ggggaatgga gcactttaag ggcgcctgtt agtaacatga 1620

attggaaatc tgtgtcgagt acctctgatc taaacggtaa aacaagctgc ctggagagca 1680

gctgtaccta acaatactgt aatgtacatt aacattacag cctctcaatt tcaggcaggt 1740

gtaacagttc ctttccacca gatttaatat ttttatactt cctgcaggtt cttcttaaaa 1800

agtaatctat atttttgaac tgatacttgt tttatacata aatttttttt agatgtgata 1860

aagctaaact tggccaaagt gtgtgcctga attattagac ctttttatta gtcaacctac 1920

gaagactaaa atagaatata ttagttttca agggagtggg aggcttccaa catagtattg 1980

aatctcagga aaaactattc tttcatgtct gattctgaga tttctaattg tgttgtgaaa 2040

atgataaatg cagcaaatct agctttcagt attcctaatt tttacctaag ctcattgctc 2100

caggctttga ttacctaaaa taagcttgga taaaattgaa ccaacttcaa gaatgcagca 2160

cttcttaatc tttagctctt tcttgggaga agctagactt tattcattat attgctatga 2220

caacttcact ctttcataat atataggata aattgtttac atgattggac cctcagattc 2280

tgttaaccaa aattgcagaa tggggggcca ggcctgtgtg gtggctcaca cctgtgatcc 2340

cagcactttg ggaggctgag gtaggaggat cacgtgaggt cgggagttca agaccagcct 2400

ggccatcatg gtgaaaccct gtctctactg aaaatacaaa aattagccgg gcgtggtggc 2460

acacgcctgt agtcccagct actcaggagg ctgaggcagg agaatcactt gaattcagga 2520

ggcggaggtt gcagtgagcc aagatcatac cactgcactg cagcctgagt gacacagtaa 2580

gactgtctcc aaaaaaaaaa aaaaaaaa 2608

<210>53

<211>2566

<212>DNA

<213> Intelligent people

<400>53

agtttgaatg aaagctctac aagatggcgg cggtcggggc cgaggcgcga ggaggtgagg 60

ctggagcgcg gccccctcgc cttccctgtt cccagcttgg tgtgtgcctt gcctagtttc 120

acttgatact cttcaggaat tatgtagaaa agaaaagctc acatgtaaat cgattggaat 180

caccaaaagg aatctaaaca attatgaggt ggaatacttg tgtgactaca aggtagtaaa 240

ggatatggaa tattatcttg taaaatggaa aggatggcca gattctacaa atacttggga 300

acctttgcaa aatctgaagt gcccgttact gcttcagcaa ttctctaatg acaagcataa 360

ttatttatct caggtaatca caagtgaaga agctgaaaga cgaggacagt tctatgacaa 420

caagggaatc acgtatctct ttgatctgga ctatgagtct gatgaattca cagtggatgc 480

ggctcgatac ggcaatgtgt ctcattttgt gaatcacagc tgtgacccaa atcttcaggt 540

gttcaatgtt ttcattgata acctcgatac tcgtcttccc cgaatagcat tgttttccac 600

aagaaccata aatgctggag aagagctgac ttttgattat caaatgaaag gttctggaga 660

tatatcttca gattctattg accacagccc agccaaaaag agggtcagaa cagtatgtaa 720

atgtggagct gtgacttgca gaggttacct caactgaact ttttcaggaa atagagctga 780

tgattataat atttttttcc taatgttaac atttttaaaa atacatattt gggactctta 840

ttatcaaggt tctacctatg ttaatttaca attcatgttt caagacattt gccaaatgta 900

ttaccgatgc ctctgaaaag ggggtcactg ggtctcatag actgatatga agtcgacata 960

tttatagtgc ttagagacca aactaatgga aggcagacta tttacagctt agtatatgtg 1020

tacttaagtc tatgtgaaca gagaaatgcc tcccgtagtg tttgaaagcg ttaagctgat 1080

aatgtaatta acaactgctg agagatcaaa gattcaactt gccatacacc tcaaattcgg 1140

agaaacagtt aatttgggca aatctacagt tctgtttttg ctactctatt gtcattcctg 1200

tttaatactc actgtacttg tatttgagac aaataggtga tactgaattt tatactgttt 1260

tctacttttc cattaaaaca ttggcacctc aatgataaag aaatttaagg tataaaatta 1320

aatgtaaaaa ttaatttcag cttcatttcg tatttcgaag caatctagac tgttgtgatg 1380

agtgtatgtc tgaacctgta attcttaaaa gacttcttaa tcttctagaa gaaaaatctc 1440

cgaagagctc tctctagaag tccaaaatgg ctagccatta tgcttctttg aaaggacatg 1500

ataatgggac caggatggtt ttttggagta ccaagcaagg ggaatggagc actttaaggg 1560

cgcctgttag taacatgaat tggaaatctg tgtcgagtac ctctgatcta aacggtaaaa 1620

caagctgcct ggagagcagc tgtacctaac aatactgtaa tgtacattaa cattacagcc 1680

tctcaatttc aggcaggtgt aacagttcct ttccaccaga tttaatattt ttatacttcc 1740

tgcaggttct tcttaaaaag taatctatat ttttgaactg atacttgttt tatacataaa 1800

ttttttttag atgtgataaa gctaaacttg gccaaagtgt gtgcctgaat tattagacct 1860

ttttattagt caacctacga agactaaaat agaatatatt agttttcaag ggagtgggag 1920

gcttccaaca tagtattgaa tctcaggaaa aactattctt tcatgtctga ttctgagatt 1980

tctaattgtg ttgtgaaaat gataaatgca gcaaatctag ctttcagtat tcctaatttt 2040

tacctaagct cattgctcca ggctttgatt acctaaaata agcttggata aaattgaacc 2100

aacttcaaga atgcagcact tcttaatctt tagctctttc ttgggagaag ctagacttta 2160

ttcattatat tgctatgaca acttcactct ttcataatat ataggataaa ttgtttacat 2220

gattggaccc tcagattctg ttaaccaaaa ttgcagaatg gggggccagg cctgtgtggt 2280

ggctcacacc tgtgatccca gcactttggg aggctgaggt aggaggatca cgtgaggtcg 2340

ggagttcaag accagcctgg ccatcatggt gaaaccctgt ctctactgaa aatacaaaaa 2400

ttagccgggc gtggtggcac acgcctgtag tcccagctac tcaggaggct gaggcaggag 2460

aatcacttga attcaggagg cggaggttgc agtgagccaa gatcatacca ctgcactgca 2520

gcctgagtga cacagtaaga ctgtctccaa aaaaaaaaaa aaaaaa 2566

<210>54

<211>410

<212>PRT

<213> Intelligent people

<400>54

Met Ala Ala Val Gly Ala Glu Ala Arg Gly Ala Trp Cys Val Pro Cys

1 5 10 15

Leu Val Ser Leu Asp Thr Leu Gln Glu Leu Cys Arg Lys Glu Lys Leu

20 25 30

Thr Cys Lys Ser Ile Gly Ile Thr Lys Arg Asn Leu Asn Asn Tyr Glu

35 40 45

Val Glu Tyr Leu Cys Asp Tyr Lys Val Val Lys Asp Met Glu Tyr Tyr

50 55 60

Leu Val Lys Trp Lys Gly Trp Pro Asp Ser Thr Asn Thr Trp Glu Pro

65 70 75 80

Leu Gln Asn Leu Lys Cys Pro Leu Leu Leu Gln Gln Phe Ser Asn Asp

85 90 95

Lys His Asn Tyr Leu Ser Gln Val Lys Lys Gly Lys Ala Ile Thr Pro

100 105 110

Lys Asp Asn Asn Lys Thr Leu Lys Pro Ala Ile Ala Glu Tyr Ile Val

115 120 125

Lys Lys Ala Lys Gln Arg Ile AlaLeu Gln Arg Trp Gln Asp Glu Leu

130 135 140

Asn Arg Arg Lys Asn His Lys Gly Met Ile Phe Val Glu Asn Thr Val

145 150 155 160

Asp Leu Glu Gly Pro Pro Ser Asp Phe Tyr Tyr Ile Asn Glu Tyr Lys

165 170 175

Pro Ala Pro Gly Ile Ser Leu Val Asn Glu Ala Thr Phe Gly Cys Ser

180 185 190

Cys Thr Asp Cys Phe Phe Gln Lys Cys Cys Pro Ala Glu Ala Gly Val

195 200 205

Leu Leu Ala Tyr Asn Lys Asn Gln Gln Ile Lys Ile Pro Pro Gly Thr

210 215 220

Pro Ile Tyr Glu Cys Asn Ser Arg Cys Gln Cys Gly Pro Asp Cys Pro

225 230 235 240

Asn Arg Ile Val Gln Lys Gly Thr Gln Tyr Ser Leu Cys Ile Phe Arg

245 250 255

Thr Ser Asn Gly Arg Gly Trp Gly Val Lys Thr Leu Val Lys Ile Lys

260 265 270

Arg Met Ser Phe Val Met Glu Tyr Val Gly Glu Val Ile Thr Ser Glu

275 280 285

Glu Ala Glu Arg Arg Gly Gln Phe Tyr Asp Asn Lys Gly Ile Thr Tyr

290 295 300

Leu Phe Asp Leu Asp Tyr Glu Ser Asp Glu Phe Thr Val Asp Ala Ala

305 310 315 320

Arg Tyr Gly Asn Val Ser His Phe Val Asn His Ser Cys Asp Pro Asn

325 330 335

Leu Gln Val Phe Asn Val Phe Ile Asp Asn Leu Asp Thr Arg Leu Pro

340 345 350

Arg Ile Ala Leu Phe Ser Thr Arg Thr Ile Asn Ala Gly Glu Glu Leu

355 360 365

Thr Phe Asp Tyr Gln Met Lys Gly Ser Gly Asp Ile Ser Ser Asp Ser

370 375 380

Ile Asp His Ser Pro Ala Lys Lys Arg Val Arg Thr Val Cys Lys Cys

385 390 395 400

Gly Ala Val Thr Cys Arg Gly Tyr Leu Asn

405 410

<210>55

<211>350

<212>PRT

<213> Intelligent people

<400>55

Met Glu Tyr Tyr Leu Val Lys Trp Lys Gly Trp Pro Asp Ser Thr Asn

1 5 10 15

Thr Trp Glu Pro Leu Gln Asn Leu Lys Cys Pro Leu Leu Leu Gln Gln

20 25 30

Phe Ser Asn Asp Lys His Asn Tyr Leu Ser Gln Val Lys Lys Gly Lys

35 40 45

Ala Ile Thr Pro Lys Asp Asn Asn Lys Thr Leu Lys Pro Ala Ile Ala

50 55 60

Glu Tyr Ile Val Lys Lys Ala Lys Gln Arg Ile Ala Leu Gln Arg Trp

65 70 75 80

Gln Asp Glu Leu Asn Arg Arg Lys Asn His Lys Gly Met Ile Phe Val

85 90 95

Glu Asn Thr Val Asp Leu Glu Gly Pro Pro Ser Asp Phe Tyr Tyr Ile

100 105 110

Asn Glu Tyr Lys Pro Ala Pro Gly Ile Ser Leu Val Asn Glu Ala Thr

115 120 125

Phe Gly Cys Ser Cys Thr Asp Cys Phe Phe Gln Lys Cys Cys Pro Ala

130 135 140

Glu Ala Gly Val Leu Leu Ala Tyr Asn Lys Asn Gln Gln Ile Lys Ile

145 150 155 160

Pro Pro Gly Thr Pro Ile Tyr Glu Cys Asn Ser Arg Cys Gln Cys Gly

165 170 175

Pro Asp Cys Pro Asn Arg Ile Val Gln Lys Gly Thr Gln Tyr Ser Leu

180 185 190

Cys Ile Phe Arg Thr Ser Asn Gly Arg Gly Trp Gly Val Lys Thr Leu

195 200 205

Val Lys Ile Lys Arg Met Ser Phe Val Met Glu Tyr Val Gly Glu Val

210 215 220

Ile Thr Ser Glu Glu Ala Glu Arg Arg Gly Gln Phe Tyr Asp Asn Lys

225 230 235 240

Gly Ile Thr Tyr Leu Phe Asp Leu Asp Tyr Glu Ser Asp Glu Phe Thr

245 250255

Val Asp Ala Ala Arg Tyr Gly Asn Val Ser His Phe Val Asn His Ser

260 265 270

Cys Asp Pro Asn Leu Gln Val Phe Asn Val Phe Ile Asp Asn Leu Asp

275 280 285

Thr Arg Leu Pro Arg Ile Ala Leu Phe Ser Thr Arg Thr Ile Asn Ala

290 295 300

Gly Glu Glu Leu Thr Phe Asp Tyr Gln Met Lys Gly Ser Gly Asp Ile

305 310 315 320

Ser Ser Asp Ser Ile Asp His Ser Pro Ala Lys Lys Arg Val Arg Thr

325 330 335

Val Cys Lys Cys Gly Ala Val Thr Cys Arg Gly Tyr Leu Asn

340 345 350

<210>56

<211>230

<212>PRT

<213> Intelligent people

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Met Ala Ala Val Gly Ala Glu Ala Arg Gly Ala Trp Cys Val Pro Cys

1 5 10 15

Leu Val Ser Leu Asp Thr Leu Gln Glu Leu Cys Arg Lys Glu Lys Leu

20 25 30

Thr Cys Lys Ser Ile Gly Ile Thr Lys Arg Asn Leu Asn Asn Tyr Glu

35 40 45

Val Glu Tyr Leu Cys Asp Tyr Lys Val Val Lys Asp Met Glu Tyr Tyr

50 55 60

Leu Val Lys Trp Lys Gly Trp Pro Asp Ser Thr Asn Thr Trp Glu Pro

65 70 75 80

Leu Gln Asn Leu Lys Cys Pro Leu Leu Leu Gln Gln Phe Ser Asn Asp

85 90 95

Lys His Asn Tyr Leu Ser Gln Val Ile Thr Ser Glu Glu Ala Glu Arg

100 105 110

Arg Gly Gln Phe Tyr Asp Asn Lys Gly Ile Thr Tyr Leu Phe Asp Leu

115 120 125

Asp Tyr Glu Ser Asp Glu Phe Thr Val Asp Ala Ala Arg Tyr Gly Asn

130 135 140

Val Ser His Phe Val Asn His Ser Cys Asp Pro Asn Leu Gln Val Phe

145 150 155160

Asn Val Phe Ile Asp Asn Leu Asp Thr Arg Leu Pro Arg Ile Ala Leu

165 170 175

Phe Ser Thr Arg Thr Ile Asn Ala Gly Glu Glu Leu Thr Phe Asp Tyr

180 185 190

Gln Met Lys Gly Ser Gly Asp Ile Ser Ser Asp Ser Ile Asp His Ser

195 200 205

Pro Ala Lys Lys Arg Val Arg Thr Val Cys Lys Cys Gly Ala Val Thr

210 215 220

Cys Arg Gly Tyr Leu Asn

225 230

<210>57

<211>170

<212>PRT

<213> Intelligent people

<400>57

Met Glu Tyr Tyr Leu Val Lys Trp Lys Gly Trp Pro Asp Ser Thr Asn

1 5 10 15

Thr Trp Glu Pro Leu Gln Asn Leu Lys Cys Pro Leu Leu Leu Gln Gln

20 25 30

Phe Ser Asn Asp Lys His Asn Tyr Leu Ser Gln ValIle Thr Ser Glu

35 40 45

Glu Ala Glu Arg Arg Gly Gln Phe Tyr Asp Asn Lys Gly Ile Thr Tyr

50 55 60

Leu Phe Asp Leu Asp Tyr Glu Ser Asp Glu Phe Thr Val Asp Ala Ala

65 70 75 80

Arg Tyr Gly Asn Val Ser His Phe Val Asn His Ser Cys Asp Pro Asn

85 90 95

Leu Gln Val Phe Asn Val Phe Ile Asp Asn Leu Asp Thr Arg Leu Pro

100 105 110

Arg Ile Ala Leu Phe Ser Thr Arg Thr Ile Asn Ala Gly Glu Glu Leu

115 120 125

Thr Phe Asp Tyr Gln Met Lys Gly Ser Gly Asp Ile Ser Ser Asp Ser

130 135 140

Ile Asp His Ser Pro Ala Lys Lys Arg Val Arg Thr Val Cys Lys Cys

145 150 155 160

Gly Ala Val Thr Cys Arg Gly Tyr Leu Asn

165 170

<210>58

<211>33

<212>DNA

<213> Intelligent people

<400>58

aaggattaga ctgaaccgaa ttggtatata gtt 33

<210>59

<211>32

<212>DNA

<213> Intelligent people

<400>59

aaggattaga ctgagctgaa ttggtatata gt 32

<210>60

<211>33

<212>DNA

<213> Intelligent people

<400>60

caaactacca cttacctccc tcaccaaagc cca 33

<210>61

<211>34

<212>DNA

<213> Intelligent people

<400>61

caaactacca cttacctccc tcaccaaagc ccat 34

<210>62

<211>33

<212>DNA

<213> Intelligent people

<400>62

caaactacca cctacctccc tcaccaaagc cca 33

<210>63

<211>32

<212>DNA

<213> Intelligent people

<400>63

attaatgcaa acaataccta acagacccac ag 32

<210>64

<211>31

<212>DNA

<213> Intelligent people

<400>64

attaatgcaa acaataccta acagacccac a 31

<210>65

<211>32

<212>DNA

<213> Intelligent people

<400>65

attaatgcaa acagtaccta acaaacctac ag 32

<210>66

<211>33

<212>DNA

<213> Intelligent people

<400>66

gtactcccga ttgaaacccc cattcgtata ata 33

<210>67

<211>34

<212>DNA

<213> Intelligent people

<400>67

gtactcccga ttgaaacccc cattcgtata ataa 34

<210>68

<211>33

<212>DNA

<213> Intelligent people

<400>68

gtactcccga ttgaagcccc cattcgtata ata 33

<210>69

<211>33

<212>DNA

<213> Intelligent people

<400>69

ctccctagga ggcctgcccc cgctaaccgg ctt 33

<210>70

<211>33

<212>DNA

<213> Intelligent people

<400>70

ctccctagga ggcctacccc cgctaaccgg ctt 33

Claims (73)

1. A method of increasing the efficiency of human somatic cell nuclear transfer (hSCNT), the method comprising:

contacting a hybrid oocyte with an agent that increases the expression of a member of the histone demethylase KDM4 family, wherein the hybrid oocyte is an enucleated human oocyte comprising human somatic genetic material.

2. The method of claim 1, wherein the contacting occurs after activation or fusion of the hybrid oocyte, but before activation (ZGA) of the human fertilized egg genome begins.

3. A method of increasing the efficiency of human Somatic Cell Nuclear Transfer (SCNT), the method comprising at least one of:

(iv) contacting a donor human somatic cell or a recipient human oocyte with at least one agent that reduces methylation of H3K9me3 in the donor human somatic cell or the recipient human oocyte, wherein the recipient human oocyte is a nucleated or enucleated oocyte; if the recipient human oocyte is nucleated, enucleating the human oocyte; transferring a nucleus from the donor human somatic cell to the enucleated oocyte to form a hybrid oocyte; and, activating the hybrid oocyte to form a human SCNT embryo; or

(v) Contacting a hybrid oocyte with at least one agent that reduces methylation of H3K9me3 in the hybrid oocyte, wherein the hybrid oocyte is an enucleated human oocyte comprising human somatic cell genetic material; and, activating the hybrid oocyte to form a human SCNT embryo; or

(vi) Contacting the activated human SCNT embryo generated from the fusion of an enucleated human oocyte with human somatic genetic material with at least one agent that reduces H3K9me3 methylation in a human SCNT embryo;

wherein decreasing H3K9me3 methylation in any one of the donor human somatic cell, recipient human oocyte, hybrid oocyte or the human SCNT embryo increases the efficiency of the SCNT.

4. A method of producing a human nuclear transferred embryonic stem cell (hNT-ESC), the method comprising:

a. at least one of the following: (i) contacting a donor human somatic cell or a recipient human oocyte with at least one agent that reduces methylation of H3K9me3 in the donor human somatic cell or the recipient human oocyte, wherein the recipient human oocyte is a nucleated or enucleated oocyte; if the recipient human oocyte is nucleated, enucleating the human oocyte; transferring a nucleus from the donor human somatic cell to the enucleated oocyte to form a hybrid oocyte; and, activating the hybrid oocyte to form a human SCNT embryo; or

(ii) Contacting a hybrid oocyte with at least one agent that reduces methylation of H3K9me3 in the hybrid oocyte, wherein the hybrid oocyte is an enucleated human oocyte comprising human somatic cell genetic material; and, activating the hybrid oocyte to form a human SCNT embryo; or

(iii) Contacting the activated human SCNT embryo generated from the fusion of an enucleated human oocyte with human somatic genetic material with at least one agent that reduces H3K9me3 methylation in a human SCNT embryo;

b. incubating the SCNT embryo for a sufficient time to form a blastocyst; collecting at least one blastomere from the blastocyst; and culturing the at least one blastomere to form at least one human NT-ESC.

5. A method of producing a human Somatic Cell Nuclear Transfer (SCNT) embryo, comprising:

contacting at least one of a donor human somatic cell, a recipient human oocyte, or a human Somatic Cell Nuclear Transfer (SCNT) embryo with at least one agent that reduces methylation of H3K9me3 in the donor human somatic cell, the recipient human oocyte, or the human SCNT embryo, wherein the recipient human oocyte is a nucleated or enucleated oocyte; if the recipient human oocyte is nucleated, enucleating the human oocyte;

transferring a nucleus from the donor human somatic cell to the enucleated oocyte to form a hybrid oocyte;

activating the hybridized oocyte; and

incubating the hybrid oocyte for a sufficient time to form a human SCNT embryo.

6. The method of any one of claims 2-5, wherein the agent that reduces H3K9me3 methylation is an agent that increases the expression of a human histone demethylase member of the KDM4 family.

7. The method of claim 6, wherein the agent increases human KDM4

(JMJD2) family of histone demethylases expression or activity.

8. The method of any one of claims 1 to 7, wherein the agent increases the expression or activity of at least one of the following enzymes: KDM4A (JMJD2A), KDM4B (JMJD2B), KDM4C (JMJD2C), KDM4D (JMJD4D) or KDM4E (JMJD 2E).

9. The method of any one of claims 1 to 8, wherein the agent increases the expression or activity of KDM4A (JMJD 2A).

10. The method of any one of claims 1 to 9, wherein the agent comprises a nucleic acid sequence corresponding to SEQ ID NO: 1 to SEQ ID NO: 4 or SEQ ID NO: 45 or a biologically active fragment thereof, which increases the efficiency of SCNT to a level equivalent to seq id NO: 1 to SEQ ID NO: 4 or SEQ ID NO: 45 to a similar or higher degree compared to the corresponding sequence of 45.

11. The method of claim 6, wherein the agent comprises a nucleic acid sequence corresponding to SEQ ID NO: 1 or a biologically active fragment thereof, which increases SCNT efficiency to a level comparable to SEQ ID NO: 1, or a higher degree of similarity or comparison.

12. The method of any one of claims 1 to 11, wherein the agent is an inhibitor of H3K9 methyltransferase.

13. The method of claim 12, wherein the H3K9 methyltransferase is SUV39H1 or SUV39H 2.

14. The method of claim 12, wherein the H3K9 methyltransferase is SETDB 1.

15. The method of claim 12, wherein two or more of SUV39h1, SUV39h2, and SETDB1 are inhibited.

16. The method of claim 12, wherein the agent that inhibits H3K9 methyltransferase is selected from the group consisting of RNAi agents, CRISPR/Cas9, CRISPR/Cpfl oligonucleotides, neutralizing antibodies or antibody fragments, aptamers, small molecules, peptide inhibitors, protein inhibitors, avimidir, and functional fragments or derivatives thereof.

17. The method of claim 16, wherein the RNAi agent is an siRNA or shRNA molecule.

18. The method of any one of claims 1 to 17, wherein the agent comprises a nucleic acid inhibitor to inhibit the activity of seq id NO: 14 to SEQ ID NO: 16. SEQ ID NO: 47. SEQ ID NO: 49. SEQ ID NO: 51. SEQ ID NO: 52. or SEQ ID NO: 53 in the presence of a protease.

19. The method of claim 17, wherein the RNAi agent hybridizes to SEQ ID NO: 14 to SEQ ID NO: 16. SEQ ID NO: 47. SEQ ID NO: 49. SEQ ID NO: 51. SEQ ID NO: 52. or SEQ ID NO: 53.

20. The method of claim 17, wherein the RNAi agent comprises the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8. SEQ ID NO: 18 or SEQ ID NO: 19 or fragments of at least 10 consecutive nucleic acids thereof, or a nucleic acid sequence having a sequence identical to seq id NO: 7 or SEQ ID NO: 8. or SEQ ID NO: 18 or SEQ ID NO: 19 is at least 80% homologous to the sequence.

21. The method of any one of claims 1 to 20, wherein the recipient human oocyte is an enucleated human oocyte.

22. The method of any one of claims 1-20, wherein the human SCNT embryo is any embryo selected from the group consisting of 1-cell stage SCNT embryos, 5-hour (5hpa) SCNT embryos post-activation, between 10-12 hours (10-12hpa) SCNT embryos post-activation, between 20-28 hours (20-28hpa) SCNT embryos post-activation, 2-cell stage SCNT embryos.

23. The method of any one of claims 1 to 22, wherein the agent is contacted with the recipient oocyte or enucleated human oocyte prior to nuclear transfer.

24. The method of any one of claims 1-22, wherein the agent contacts the SCNT embryo before the SCNT embryo is at the 1-cell stage, or about 5 hours after activation, or when the SCNT embryo is at the 1-cell stage.

25. The method of any one of claims 1-22, wherein the agent contacts the human SCNT embryo at 5 hours post-activation (5hpa), or 12 hours post-activation (12hpa), or 20 hours post-activation (20hpa), or when the SCNT embryo is at the 2-cell stage, or at any time between 5hpa and 28 hpa.

26. The method of any one of claims 1 to 22, wherein the step of contacting the recipient human oocyte or hybrid oocyte or human SCNT embryo with the agent comprises injecting the agent into the nucleus or cytoplasm of the recipient human oocyte or hybrid oocyte or human SCNT embryo.

27. The method of any one of claims 1-26, wherein the agent increases the expression or activity of a histone demethylase of the KDM4 family.

28. The method of any one of claims 1 to 22, wherein the agent contacts the cytoplasm or nucleus of the donor human somatic cell prior to removing the nucleus for injection into the enucleated human oocyte.

29. The method of claim 28, wherein the donor human somatic cell is contacted with the agent for at least 24 hours or at least 1 day prior to injecting the nucleus of the donor human somatic cell into the enucleated human oocyte.

30. The method of claim 28, wherein the agent is contacted with the donor human somatic cell for at least 24 hours, or at least 48 hours, or at least 3 days prior to injecting the nucleus of the donor human somatic cell into the enucleated human oocyte.

31. The method of any one of claims 28 to 30, wherein the agent inhibits H3K9 methyltransferase.

32. The method of any one of claims 28-30, wherein the H3K9 methyltransferase is SUV39H1 or SUV39H2, or SUV39H1 and SUV39H2(SUV39H 1/2).

33. The method of any one of claims 1 to 32, wherein the donor human somatic cell is a terminally differentiated somatic cell.

34. The method of any one of claims 1 to 33, wherein the donor human somatic cell is not an embryonic stem cell, or an Induced Pluripotent Stem (iPS) cell, or a fetal cell, or an embryonic cell.

35. The method of any one of claims 1-34, wherein the donor human somatic cell is selected from the group consisting of cumulus cells, epithelial cells, fibroblasts, neural cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, erythrocytes, macrophages, monocytes, muscle cells, B lymphocytes, T lymphocytes, embryonic stem cells, embryonic germ cells, fetal cells, placental cells, and adult cells.

36. The method of any one of claims 1 to 35, wherein the donor human somatic cell is a fibroblast or a cumulus cell.

37. The method of any one of claims 1 to 36, wherein the agent contacts the nucleus of the donor human somatic cell to remove the nucleus from the donor human somatic cell and use the nucleus for injection into an enucleated recipient human oocyte.

38. The method of any one of claims 1 to 37, wherein the method results in at least a 10% increase in the efficiency of hSCNT to develop blastocysts as compared to hSCNT performed in the absence of an agent that reduces methylation of H3K9me 3.

39. The method of any one of claims 1-38, wherein the method results in an increase in the efficiency of hSCNT developing blastocysts by 10% to 20% over hSCNT performed in the absence of an agent that reduces H3K9me3 methylation.

40. The method of any one of claims 1 to 39, wherein the method results in an increase in the efficiency of development of blastocysts of hSCNT by more than 20% over that effected in the absence of an agent that reduces methylation of H3K9me 3.

41. The method of any one of claims 38-40, wherein the increase in SCNT efficiency is an increase in development of a human SCNT embryo to the blastocyst stage.

42. The method of any one of claims 38-40, wherein the increase in SCNT efficiency is an increase in embryonic stem cell (hNT-ESC) derivatives derived from human SCNT embryos.

43. The method of any one of claims 1 to 42, wherein the donor human somatic cell is a genetically modified donor human cell.

44. The method of claim 5, further comprising culturing the human SCNT embryo in vitro to form a human blastocyst.

45. The method of claim 44, wherein the human SCNT embryo is at least 4 cell human SCNT embryo.

46. The method of claim 44, wherein the human SCNT embryo is at least 4-cell SCNT embryo.

47. The method of claim 44, further comprising isolating cells from the inner cell mass from the human blastocyst; and culturing the cells from the inner cell mass in an undifferentiated state to form human Embryonic Stem (ES) cells.

48. The method of any one of claims 1 to 48, wherein any one or more of the donor human somatic cell, recipient human oocyte or human SCNT embryo has been frozen and thawed.

49. A population of embryonic stem cells (hNT-ESCs) derived from human SCNT embryos produced using the method of any one of claims 1 to 48.

50. The population of hNT-ESCs of claim 49, wherein the hNT-ESCs are genetically modified hNT-ESCs.

51. The population of hNT-ESCs of claim 49, wherein the hNT-ESCs are pluripotent or totipotent stem cells.

52. The population of hNT-ESCs of claim 49, wherein the hNT-ESCs are present in a culture medium.

53. The population of hNT-ESC of claim 52, wherein the culture medium maintains the hNT-ESC in a pluripotent or totipotent state.

54. The population of hNT-ESC of claim 52, wherein the culture medium is a medium suitable for cryopreservation and cryopreservation of the hNT-ESC.

55. The population of hNT-ESCs of claim 54, wherein the hNT-ESCs are frozen or cryopreserved.

56. A human SCNT embryo produced by the method of any one of claims 1-55.

57. The human SCNT embryo of claim 56, wherein the human SCNT embryo is genetically modified.

58. The human SCNT embryo of claim 56, wherein the human SCNT embryo includes mitochondrial DNA (mtDNA) that is not from the recipient human oocyte.

59. The human SCNT embryo of claim 56, wherein the human SCNT embryo is present in culture.

60. The human SCNT embryo of claim 59, wherein the culture medium is a medium suitable for cryopreservation and cryopreservation of the human SCNT.

61. The human SCNT embryo of claim 60, where the human embryo is frozen or cryopreserved.

62. A composition comprising at least one of a human SCNT embryo, recipient human oocyte, human hybrid oocyte or blastocyst, and at least one of:

a. an agent that increases the expression or activity of a histone demethylase of the KDM4 family; or

b. An agent that inhibits H3K9 methyltransferase.

63. The composition of claim 62, wherein the agent that increases expression or activity of a histone demethylase of the KDM4(JMJD2) family increases expression or activity of at least one enzyme of KDM4A (JMJD2A), KDM4B (JMJD2B), KDM4C (JMJD2C), KDM4D (JMJD2D) or KDM4E (JMJD 2E).

64. The composition of claim 63, wherein the agent increases the expression or activity of KDM4D (JMJD2D) or KDM4A (JMJD 2A).

65. The composition of claim 64, wherein the agent comprises a nucleic acid sequence corresponding to SEQ ID NO: 1 to SEQ ID NO: 4 or SEQ ID NO: 45 or a biologically active fragment thereof, which increases the efficiency of human SCNT to a level equivalent to SEQ id no: 1 to SEQ ID NO: 4 or SEQ ID NO: 45 to a similar or higher degree compared to the corresponding sequence of 45.

66. The composition of claim 64, wherein the agent comprises a nucleic acid sequence corresponding to SEQ ID NO: 1 or a biologically active fragment thereof, which increases SCNT efficiency to a level comparable to SEQ ID NO: 1, or a higher degree of similarity or comparison.

67. The composition of claim 62, wherein the inhibitor of H3K9 methyltransferase inhibits at least one enzyme of SUV39H1, SUV39H2, or SETDB1, or any combination thereof.

68. The composition of claim 62, wherein the human SCNT embryo is a human SCNT embryo at the 1-cell stage, 2-cell stage, or 4-cell stage.

69. The composition of claim 62, wherein the recipient human oocyte is an enucleated recipient human oocyte.

70. The composition of claim 62, wherein the human SCNT embryo is produced from a nucleus injected with a terminally differentiated human somatic cell, or wherein the blastocyst is developed from a human SCNT embryo produced by injecting a nucleus of a terminally differentiated human somatic cell into an enucleated human oocyte.

71. A kit, comprising: (i) an agent that increases the expression or activity of a histone demethylase of the human KDM4 family, and/or an agent that inhibits H3K9 methyltransferase; and (ii) a human oocyte.

72. The kit of claim 92, wherein the human oocyte is an enucleated oocyte.

73. The kit of claim 92, wherein the human oocyte is a non-human oocyte.