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Definitions

• the present invention relates to methods for rejuvenating normal or modified somatic cells or cellular DNA that is senescent, checkpoint arrested, nearing senescence or has an undesirably short cell life, through nuclear transfer techniques.
• the methods are particularly useful for rejuvenating cells which have reached or are approaching senescence due to clonal expansion following complex genetic manipulations or from tissue chronic tissue injury, and thereby increase the potential of such cells to serve as donors for the generation of cloned transgenic animals or for cell therapy in humans.
• the invention is useful for rejuvenation of cells which are senescent or aged as a result of chronologic aging or because of conditions associated with exacerbated cell senescence such as muscular dystrophy or atherosclerosis, imnumosenescence, BPH, neurodegenerative diseases, Barrett esophagus cirrhosis, AMD osteoarthritis and skin ulcers.
• the patient or animal's cells will be reprogrammed or rejuvenated by nuclear transfer or related technique and regenerated and restored to totipotency.
• These totipotent cells may be used to produce cell types including but limited to pluripotent cells such as mesenchymal or premesenchymal stems cells, hematopoietic cells, vascular cells and so on, which can be transplanted into the patient or animal or suitable donor. These cells will "seed" the patient or animal's tissues with healthy proliferation competent cells of numerous types including bone, blood, muscle, neurons, immune cells, and other types.
• the methods of the invention also include the making of differentiated cells from rejuvenated cells, and teratomas which contain cells from any or all three germ layers and are useful for making primary cells of a different type having the same genotype as a primary cell of interest. Such newly generated primary cells have important significance in the field of tissue engineering and organ replacement therapy. Also encompassed are methods of re- cloning cloned mammals, particularly methods where the offspring of cloned mammals are designed to be genetically altered in comparison to their cloned parent.
• the invention relates to assays for identifying compounds that moderate cell aging and senescence, and genes associated therewith, in particular compounds that affect telomere length, EPC-1 activity, tPA, collagenase activity, gas genes, mitotic index, and other indications of cellular aging and proliferation capacity.
• an embryo and embryonic stem cells may be generated using the nucleus from an adult differentiated cell has significant implications for the fields of organ, cell and tissue transplantation. For instance, embryonic stem cells generated from the nucleus of a cell taken from a patient in need of a transplant could be made, and induced to differentiate into the cell type required in the transplant. By using techniques evolving in the field of tissue engineering, tissues and organs could be designed from the cloned differentiated cells which could be used for transplantation. Because the cells and tissues used for the transplant would have the same nuclear genotype as the patient, the problems of transplant rejection and the dangers inherent in the use of immune-suppressive drugs would be avoided or decreased.
• the engineered cells and tissues could be readily modified with heterologous DNA, or modified such that deleterious genes are inactivated, such that the transplanted cells and tissues are genetically corrected or improved if necessary.
• US Application Serial No. co-owned and filed concurrently with the present invention, discusses methods for genetically modifying both the donor nuclear DNA and the recipient mitochondrial DNA, and is herein incorporated by reference in its entirety.
• telomere loss is linked to the aging process for at least two decades. See, Harley, "Telomere loss: mitotic clock or genetic time bomb?" Mutation Res. (1991) 256: 271-282.
• the hypothesis, originally called the “marginotomy theory,” is that the gradual loss of chromosomal ends, or telomeres, leads to cell cycle exit and as a consequence, cell senescence. See Olovnikov, "A theory of Marginotomy” J. Theor. Biol. (1973) 41: 181-190.
• telomere activity in human cells was first identified in 1989. See Morin, "The human telomere terminal transferase is a ribonucleoprotein that synthesizes TTAGGG repeats" Cell (1989) 59: 521-529. Telomerase acts to build on the ends of chromosomes, restoring telomere length. Other studies have shown that, while telomerase activity is repressed during differentiation of somatic cells, telomerase is active at some stage of germ-line cell replication and thus maintains telomere length in germ cells between generations. In addition, telomerase has also been shown to be active in transformed cells. See Harley (1991) for a review.
• telomere suppression of telomerase in differentiated cells may function to limit the capacity of somatic cells to clonally expand in an uncontrolled manner, as in cancer.
• some tumor cell lines show a telomerase negative immortality that has been designated the 'ALT' pathway.
• the inventors propose that this alternative pathway, like the acquisition of telomerase activity in tomorigenesis, is the reappearance of a germ-line trait.
• the inventors propose that damaged telomeres are repaired in the germ line, not only through the addition of telomeric repeats by telomerase, but also through homologous strand invasion and extension by DNA polymerase.
• somatic cells may be more readily maintained in culture and transfected with transgenes than embryonic stem cells. This property facilitates the production of animals which produce therapeutic proteins, i.e., for instance cows which express transgenes from mammary-specific promoters enabling the production of therapeutic proteins in milk.
• somatic cells may be more readily maintained in culture and transfected with transgenes than embryonic stem cells. This property facilitates the production of animals which produce therapeutic proteins, i.e., for instance cows which express transgenes from mammary-specific promoters enabling the production of therapeutic proteins in milk.
• cells used for nuclear transfer were not able to undergo a series of genetic manipulations because of aging chromosomes, it would be virtually impossible to generate animals, cells and tissues with multiple genetic manipulations.
• telomere regeneration will be dependent on the choice of donor somatic cell types.
• Recent studies have shown that reconstruction of telomerase activity leads to telomere elongation and immortalization of normal human fibroblasts and retinal epithelial cells (Bodnar et al. (1998) Science 279: 349; Vaziri and Benchimol (1998) Curr. Biol.
• telomerase activity may be cell-cycle dependent. For instance, in 1996, Dionne reported the down-regulation of telomerase activity in telomerase-competent cells during quiescent periods (G 0 phases) and hypothesized that telomerase activity may be cell-cycle dependent. See http://telomeres,virtualave.net/regulation.html. Similarly, Kruk et al. reported a higher level of telomerase in the early S phase when compared to other points in the cell cycle (Biochem. Biophys. Res. Commun. (1997) 233: 717-722). However, other researchers have reported conflicting results, and have alternatively suggested that telomerase activity correlates with growth rate, not cell cycle (Holt et al.
• telomere activation is mediated by other cellular activation signals, as evidenced by the upregulation of telomerase in B cells in vitro in response to CD4O antibody/antigen receptor binding and exposure to interleukin-4 (Website, id., citing Weng, 1997; see also Hiyama et al. (1995) J. Immunol. 155 (8): 3711-3715,).
• telomemse activity remains poorly understood. See, e.g., Smaglik, "Turning to Telomerase: As Antisense Strategies Emerge, Basic Questions Persist," The Philosoph, January 18, 1999, 13(2): 8).
• telomere activity could have wide-reaching effects in the medical community, and has the potential to profoundly influence many more technologies than the regeneration of telomeres in cloned animals. Having the ability to regulate telomerase will enable the treatment of many age-related and other types of disease processes. For instance, the capability to regulate telomerase could be important for improving the effectiveness of bone marrow transplants in connection with cancer chemotherapy; telomerase therapy may be useful in replacing age-worn cells in the immune system, and in the retina of the eye for example, in treating the lining of blood vessels to help prevent heart attack or stroke, extending the life span of hepatocytes for the treatment of cirrhosis, or myoblasts in muscular dystrophy.
• telomere regulation may permit the control of cancerous cells.
• an in vitro model of telomere and telomerase regulation in particular, a model for the reversal of cellular aging, would enable the design of assays and screens to identify the molecular mechanisms of telomere regulation, aging, and cancer.
• a better understanding of the regulation of telomerase has the potential to lead to a wide range of treatments, in addition to securing the efficacy of cloned tissues for tissue engineering and transplants, and ensuring and even increasing the life span of cloned and non-cloned animals.
• the present invention is based on the surprising discovery, in light of the recent doubts about the genetic age of cloned mammals, that the process of nuclear transfer is capable of rejuvenating senescent or near-senescent cells and repairing tandemly repeating DNA sequence such as that in the telomeres, restoring youthful patterns of gene expression such as increasing EPC-1 activity, and/or increases cell life span or cell proliferation capacity.
• the present invention therefore enables what would not have been deemed possible in light of the recent concerns about nuclear transfer; namely, that cells that are at or near senescence, e.g., those grown in culture until they are near senescence, or obtained from humans or animals having age-related defects or conditions may still be used to generate cloned cells, tissues and animals having telomeres that are at least comparable in length, or longer, than age-matched controls. Also, these cells possess patterns of gene expression of young cells, such as increased EPC-1 activity relation to donor cells. Moreover, the present invention establishes, in contrast to what had been recently suggested, that generating clones of clones, i.e. "re-cloning," is entirely feasible, and may be repeated theoretically indefinitely, thereby resulting in "hyper-young" cells, tissues, organs and animals.
• Telomeres may, however, contain an increasing amount of degenerate or non- telomeric repeat DNA progressing centromeric from the telomere.
• telomeric repeat sequences causes a temporary DNA damage checkpoint. Following repair, such as though exonuclease activity, the cell can re- enter the cell cycle. The growth of a mortal cell to terminal senescence with subsequent nuclear transfer causes the synthesis of an extended array of uniform telomeric repeat sequences that do not always appear in nature.
• Cells and/or animals containing chromosomes with such extended and uniform telomeric repeat sequences will be rejuvenated, and have the unique characteristic of being hyper- young, as a mass population of cells having fewer cells in DNA damage checkpoint at any one period of time.
• the present invention is based on the discovery that nuclear transfer techniques may be used to extend the life span of somatic cells, e.g., senescent or near-senescent or checkpoint arrested cells by activating endogenous (cellular) telomerase activity, and young patterns of gene expression by the repair of tandemly repeated DNA sequence damage.
• somatic cells e.g., senescent or near-senescent or checkpoint arrested cells by activating endogenous (cellular) telomerase activity, and young patterns of gene expression by the repair of tandemly repeated DNA sequence damage.
• telomere shortening in clones researchers at Geron Corporation and the Roslin Institute have recently collaborated to combine Geron's cloned telomerase gene (hTERT) with nuclear transfer in order resolve telomere shortening in clones. See, e.g., Business Wire, May 26, 1999. This announcement preceded the May 27th Nature report by researchers at Roslin Institute that two other sheep (after Dolly) cloned by nuclear transfer also exhibit shorter telomeres than age-matched controls.
• researchers at the University of Massachusetts involved in cloning cattle also believed that transfecting donor cells with an exogenous telomerase gene might be beneficial for the life-span of cloned animals, despite their observation that nuclear transfer seemed to rejuvenate senescent donor cells. See http://abcnews.go.com/sections/science/E> ⁇ z7v New5/clones980522.html (1998).
• the present invention is advantageous over proposed methods to express telomerase from a transfected telomerase gene, in that no genetic manipulations are required to activate telomerase and regenerate telomere length in cloned cells, tissues and animals.
• the up-regulation of telomerase activity achieved with the present invention is transient, and while it is sufficient to extend telomere length, it does not impart constitutive immortality. This advantage is particularly significant given the observation that telomerase is constitutively upregulated in many types of cancer cells and constitutive telomerase expression has been reported to result in the up - regulation of the proto-oncogene c-MYC (D. Bead paper).
• telomere activity may be controlled using the cell's own regulatory mechanisms is therefore preferable to inserting exogenous copies of the telomerase gene.
• the present invention is advantageous over the exogenous expression of telomerase in that the culture of somatic cells leading to telomere shortening with subsequent nuclear transfer to extend telomeres results in a population of rejuvenated cells all of which have more uniform tracts of telomeric repeats.
• individual cells isolated from such a population have a greater probability of being competent for extended proliferation and the population will have the unique property of having fewer checkpoint arrested cells than natural cells, thereby being "hyper young.”
• somatic cells which would benefit from the disclosed methods include any somatic cell, e.g. a cell which is nearing senescence, either by reaching the natural limit on population doublings or as a result of harsh selection conditions for complex genetic alterations or conditions, that have exposed the cell to high oxygen tension or other conditions that have damaged telomeric DNA.
• this includes especially cells from patients or animals with age related deficiencies or conditions such as age related macular degeneration, immune senescence neurodegenerative disorders such as Parkinson's or Alzheimer's diseases, osteoarthritis, muscular dystrophy, skin aging, emphysema, aneunsnis, coronary heart disease, atherosclerosis, hypertension, cataracts, adult onset diabetes.
• the invention has application in conditions associated with accelerated cell turnover such as muscular dystrophy, herpes zoster, AIDS, and cirrhosis.
• the present methods are applicable to any somatic cell of interest, and use of such cells as donors for nuclear transfer.
• the methods of the invention allow one to reprogram the nucleus of a late passage somatic cell to an embryonic state. By allowing the embryonic cell to differentiate and develop into many different cell types, one may re-isolate the primary cell of interest in a rejuvenated or "young" state. Also, since the methods of the invention entail making an embryonic stem cell which differentiates into all different cell types, any type of cell may be generated using any primary cell of interest, so long as the genome of the somatic cell has not been altered as to affect cellular development. Thus, the invention provides an invaluable way to analyze the affect of the same genetic alteration in an isogenic background (i.e., a gene knock-out or expression of a heterologous gene) in different cell types in vitro.
• an isogenic background i.e., a gene knock-out or expression of a heterologous gene
• a patient's somatic cells may be reprogrammed by a NT related technique, regenerated and restored to totipotency.
• pluripotent stem cells can be obtained such as pre-mesenchymal, mesenchymal, enangioblasts, hematopoietic stem cells.
• These pluripotent cells or cells derived therefrom can be transplanted into donors where they will "seed" the patient's tissues with healthy proliferation competent cells, such as immune cells, blood cells, bone, muscle, neural, and other types.
• the methods of the present invention also increase the life-span of a desired cell, preferably a mammalian cell, and more preferably is a human cell, e.g., that is in need of rejuvenation, by using said cell, the nucleus or chromosomes therefrom, as a nuclear transfer donor.
• a desired cell preferably a mammalian cell, and more preferably is a human cell, e.g., that is in need of rejuvenation, by using said cell, the nucleus or chromosomes therefrom, as a nuclear transfer donor.
• the process will be repeated, in that cells, nuclei or chromosomes obtained from the resultant cloned embryo will themselves be used as nuclear transfer donors.
• the donor cells will preferably be transgenic.
• the methods of the present invention further allow one to restore repetitive DNAs in desired cells and to activate or modulate (reduce or increase expression) those genes involved in aging including telomerase in desired cells, e.g., mammalian cells in need of rejuvenation, and checkpoint arrested cells, by using said cell, the nucleus or the chromosomes derived therefrom as a donor during nuclear transfer or exposing the DNA of such cell to an embryonic cell type.
• desired cells e.g., mammalian cells in need of rejuvenation, and checkpoint arrested cells
• the present invention may provide a means for identifying specific molecules that are involved in the aging of cells, and which regulate cell life-span.
• the invention provides assays to identify compounds that restore repetitive DNAs such as telomeres, activate or inhibit genes altered in the course of cell aging such as telomerase, gas, tPA and others.
• nuclear transfer may be used to rejuvenate or increase the life-span of mammalian cells, e.g. cells at or near senescence, it is no longer a concern that cloned mammals, fetuses, teratomas, or embryos, or inner cell masses or blastocysts are of the genetic age of their parents.
• the invention also encompasses methods of re-cloning cloned mammals, fetuses, teratomas, embryos, etc. using nuclear transfer techniques.
• Such re-cloning methods are particularly useful for making transgenic mammals expressing more than one heterologous gene, or having more than one gene knocked out, because such animals can be generated by cloning techniques to generate cloned and re-cloned mammals of the same genetic background. Such methods forego the need for mating or breeding, which often results in other genetic differences and may be impossible for obtaining double knockout or double transgenic mammals having altered genes which are closely linked on the genome such that they are inherited together.
• FIG. 1 Characterization of cell senescence in NT donor cells.
• A Cells were observed by phase contrast microscopy. The donor cells displayed an increased cell size and cytoplasmic granularity (b) as compared to the early passage BFF cells (a).
• B Representative electron micrographs of BEF (a) and donor CL53 (b) cells. Note the convoluted nucleus (n) of CL53 cells. CL53 cells are larger than BFF cells, and their cytoplasm contain abundant lysosomes (arrows) and thick fibrils. Both pictures are at the same magnification. The bar represents 2 microns. Mitochondria (in).
• RNAs isolated from cloned calf dermal fibroblast strains are indicated. RNA was extracted from the cells after they were grown to confluence and growth-arrested in serum free medium for 3 days (P. Chomczynski and N. Sacchi (1987) Anal. Biochem 162: 156). Equal amounts of RNA were treated with glyoxal, separated by electrophoresis on agarose gels, transferred to nitrocellulose filters electrophoretically, and hybridized with the full length EPC-1 cDNA using standard conditions (D.G. Phinney, CL. Keiper, M.K. Francis, K. Ryder (1994) Oncogene 9: 2353).
• Figure 2 Normal cows cloned from senescent somatic cells.
• CLS3-8, CL53-20 9, CL53-10, CL53-11 and CL53-12 (nicknamed Lily, Daffodil, Crocus, Forsythia, and Rose, respectively) at 5 months of age; and
• CL53-1 (Persephone, insert) at 10 months of age.
• Figure 3 Ability of nuclear transfer to restore the proliferative life-span of senescent donor cells.
• A The growth curve of the original BFF cell strain (*) is compared to that of cells derived from fetus (ACT99-002) (o) that was cloned from late passage BFF cells (CL53 cells).
• B The growth curve of the CL53 donor cells demonstrating that the cultures bad approximately 2 population doublings remaining.
• telomere fluorescence of gated mononuclear cells was calculated by subtracting the mean background fluorescence from the mean fluorescence obtained with the FITC-labeled telomere probe. Note that the age-related decline in telomere fluorescence values in normal cows and the relatively long telomeres in the cloned animals.
• Genomic DNA isolated from control cells pre-transfection BFF bovine fibroblasts
• senescent CL53 cells fibroblasts from a 7 week old cloned fetus (ACT99-002) cells obtained by NT with senescent CL53 cells.
• TRF analysis of DNA fragments obtained following digestion with Hinfl/Rsal was performed on a 0.5% agarose gel run for 12 hours as described (Telomere Length Assay Kit, Pharmingen, San Diego, CA).
• Lane 1 controls DNA from CEPH lymphoblastoid human cell line 134105; lane 2: biotinylated markers (Pharmingen); lane 3: TeloLow control DNA (Pharmingen, mean TRF length 3.3 kb); lane 4: senescent CL53 cells; lane 5: BFF fibroblasts pre-transfection; lane 6: ACT99-002 (cloned) cells.
• Lane 1 ACT99-002 cells (mean TRF length 19.3 kb);
• lane 2 BFF056H fibroblasts pre transfection (mean TRF length 17.9 kb);
• lane 3 senescent CL53 cells (mean TRF length 16.2 kb);
• lane 4 TeloHigh control DNA (Pharmingen, mean TRF length 11.3 kb);
• lane 5 control DNA from CEPH lymphoblastoid human cell line 134105;
• lane 6 biotinylated lambda DNA cut with Hind 111 (molecular weight markers).
• TRAP Telomeric Repeat Amplification Protocol
• Normal somatic cells is intended to mean that such cells that are committed to a somatic cell lineage are not tumorgenic or transformed, and are capable of being reprogrammed and of facilitating embryonic development after said cell or a nucleus of such a cell or chromosome from said cell is transferred to an enucleated oocyte or otherwise exposed to factors present in germ line cells.
• Normal somatic cells may or may not be genetically modified.
• the inventors mean at least one of the following: that the possible number of population doublings remaining for said somatic cell is increased, that EPC-1 activity or other markers of cellular aging are reversed to a youthful state; that telomerase is upregulated, and/or that telomeres are increased "Hyper-young” indicates that the population of cells have markers of cellular aging that are younger than normal cells.
• Teotoma refers to a group of differentiated cells containing derivatives of mesoderm, endoderm, or ectoderm resulting from totipotent cells.
• the normal somatic cells to be used for the present invention are senescent cells, checkpoint arrested cells, or cells that are near- senescence.
• the present methods are applicable for any desired normal somatic cell, preferably a human cell.
• Replicative senescence is a physiological state distinguishable from quiescence achieved by either serum starvation or density-dependent inhibition of growth of young cells (West et al. (1989) Exp. Cell Res. 184: 138 ; West et al. (1996) Exp. Gerontol. 31 : 175; and Pignolo et al. (1998) Exp. Gerontol.
• Senescent cells may be identified by a variety of means known in the art. For 15 instance, phase contrast light microscopy, and ultrastructural analysis by electron microscopy may be used to verify features of fibroblast replicative senescence, including prominent and active Golgi apparati, increased invaginated and lobed nuclei, large lysosomal bodies, and an increase in cytoplasmic microfibrils as compared to the young cells (Lipetz and Cristofalo (1972) J. Ultrastruct. Res. 39: 43).
• senescent cells have a reduced capacity to enter S phase as measured by a decrease in the incorporation of H-thymidine and a significant increase in the staining of senescence-associated ⁇ -galactosidase (G.P. Dimri et al (1995) Proc. Natl. Acad. Sci. USA 92: 9363).
• Senescent cells also exhibit a reduction in EPC- 1 (early population doubling level cDNA-1) (Pignolo et al. (1993) J. Biol. Chem. 268: 8949) mRNA levels as compared to early passage cells, and a down-regulation of gasl gene expression as compared to quiescent cells (Cowled et al.
• Senescent cells can be isolated by propagating cells until they reach a state of irreversible growth arrest.
• Near-senescence the present inventors mean that such cells have the capability to divide no more than about three to six times, but are preferably less than two or three population doublings from replicative senescence.
• the preferred means of generating senescent cells for nuclear transfer is to passage normal somatic cells until greater than about 90 to 95% of their life-span is completed
• senescence and senescent- like states can also be induced by exposing cells to various agents, including genotoxic agents and Cdk inhibitors (McConnell et al. (1998) Current Biol. 8: 351-354). Genotoxic agents induce a growth arrest similar to senescence and distinct from quiescence called DNA damage checkpoint arrest.
• near-senescent cells can be obtained from animals or humans, e.g., those with aging associated conditions.
• the methods of the present invention may employ cell rejuvenation to generate cloned animals, or may be used to rejuvenate a normal somatic cell of interest for other purposes.
• Such methods may include: a. transferring said somatic cell, the nucleus from said somatic cell, or chromosomes from said somatic cell to a recipient oocyte or egg or other suitable recipient cell in order to generate an embryo; b. obtaining an embryo having at least one cell, an inner cell mass, embryonic disc and/or stem cell using said embryo; c. allowing said embryo, inner cell mass, embryonic disc and/or stem cell to differentiate into desired cell or tissue types; d. isolating said resulting cells or tissues; e. transplanting said cells or tissues into patient.
• the differentiated cells teratomas, inner cell masses, embryonic disc and embryonic stem cells isolated according to the invention will have telomeres that are at least as long if not longer than those of the donor normal somatic cell, and are also an aspect of the invention. Also, these differentiated cells should possess markers of cellular aging that are young or hyper-young.
• a method whereby the differentiated cells or tissues, teratoma cells, inner mass cells, blastocyst cells or embryonic cells are then used as subsequent nuclear donors is also envisioned. Such a method is particular suitable for isolating normal somatic cells, teratomas, ES cells, etc. having multiple transgenes or genetic alterations, and may be repeated indefinitely until the desired number of genetic changes have been accomplished.
• the normal somatic cell used for the methods of the invention may be any cell 20 type.
• Suitable cells include by way of example immune cells such as B cells, T cells, dendritic cells, skin cells such as keratinocytes, epithelial cells, chondrocytes, cumulus cells, neural cells, cardiac cells, esophageal cells, dermal fibroblasts, cells of various organs including the liver, stomach, intestines, lung, pancrease, cornea, skin, gallbladder, ovary, testes, other reproduction organs, kidneys, etc.
• the most appropriate cells are easily propagatable in tissue culture and can be easily transfected.
• cell types for transfecting heterologous DNA and performing nuclear transfer are fibroblasts. Methods and protocols for effecting nuclear transfer are disclosed in U. S. Patent No.
• the somatic cell may be from any type of animal or mammal, such as pig, goat, cat, dog, rat, mouse, bovine, buffalo, sheep, horse, human, non-human primate, but is preferably an ungulate cell, and most preferably a bovine cell.
• the oocyte or egg used for nuclear transfer will be from similar sources and can be of the same or different species than donor cell or DNA.
• the immune-compromised animal may be any animal capable of supporting teratoma formation, and is immune-compromised to the extent that no rejection of the developing teratoma occurs.
• the immune-compromised animal may be a SCID or nude mouse.
• cells may be differentiated in vivo or in avian eggs.
• somatic cells having complex or compound manipulations, i.e., more than one transfected heterologous gene and/or gene knockout, where it may be difficult to keep the somatic cell in culture long enough to affect all the desirable genetic alterations.
• somatic cells preferably those made hyper-young by nuclear transfer, are used as a substrate for gene targeting.
• the somatic cell could undergo a first genetic manipulation, could then be rejuvenated according to the methods of the invention, and could then go through a second genetic manipulation once the genetic clock has been "reset.”
• a rejuvenated somatic cell according to the invention may have at least one alteration to the genome depending on the complexity of the genetic manipulation and the number of times it has gone through the rejuvenation process.
• Rejuvenated, genetically altered cells generated by the methods of the invention are also encompassed.
• the invention also includes methods of making somatic cells having the same genotype as a first cell which is of a different cell type. Such a method is made possible by the process of rejuvenation, which is effected by transferring a first somatic cell, the nucleus of a first primary cell, or the chromosomes from a first primary cell into an enucleated recipient oocyte or other suitable recipient cells, or by contacting the somatic cell with proteins in the oocyte to generate a teratoma or other mass of differentiated cells, which contains derivatives of any of the germ layers ectoderm, mesoderm and endoderm.
• An enucleated egg just after fertilization may also be used.
• virtually any type of cell may be isolated from the teratoma or by cells from the teratoma to developmentally differentiate.
• Specific cell markers unique to the particular cell type of interest are known in the art and may be used to identify the cloned primary cell.
• methods of making somatic cells of a different type than the cell used for nuclear transfer comprise: a. transferring a first cell, the nucleus from said first cell, or the chromosomes from a first cell to a recipient oocyte or egg or other suitable recipient cell in order to generate an embryo; b. obtaining an embryo having at least one cell, an inner cell mass, an embryonic disc and/or stem cell using said embryo; c. injecting said inner cell mass, embryonic disc and/or stem cell into an immune compromised animal tissue culture or avian egg to form a teratoma; d. isolating said resulting teratoma; e. separating the different germ layers for the purpose of identifying specific cell types; f. isolating a cell of a different type than the first cell.
• the genome of the primary cell may be modified such that the cell is incapable of producing a viable embryo. This may be affected by inactivating or knocking out one or more genes required for the formation of one of the three germ layers, or by expressing a "suicide" gene from a developmentally regulated promoter specifically expressed in a cell type contained in a germ layer which is not of interest Alternatively, gene knockouts or suicide gene expression could be targeted to genes specifically required for attachment to or development in a mammalian uterus.
• the first (nuclear donor) cell is a fibroblast.
• the method may be formed using any species of cell, and finds particular use in human therapeutic cloning in the generation of cloned organs and tissues for transplantation. Thus, the methods may be performed using human cells, and the primary cells isolated may be used to generate a tissue (for transplantation into a patient in need of a transplant).
• Prefe ⁇ ed types of primary cells to be generated by the disclosed methods are neurons,
• skeletal myoblasts cardiac muscle, skin pancreatic ⁇ cells, endothelial cells, hematopoietic
• the method may further comprise isolating cells from the teratoma and growing said cells in the presence of growth factors to facilitate further differentiation.
• the genome of the first cell is altered prior to nuclear transfer, such that the new primary cells and engineered tissues that are generated express at least one therapeutic protein, or fail to express a native protein that may have been detrimental to the donor patient.
• the cells and tissues generated by the disclosed methods are also encompassed.
• Preferred applications of cells and tissues generated by the methods disclosed herein include the production of neurons, pancreatic islet cells, hepatocytes, cardiomyocytes, hematopoietic cells, and other desired differentiated cell types and tissues containing.
• These cells and tissues may be used for cell, tissue and organ transplantation, e.g., treatment of bums, hair transplantation, cancer, chronic pain, diabetes, dwarfism, epilepsy, heart disease such as myocardial infarction, hemophilic, infertility, kidney disease, liver disease, osteoarthritis, osteoporosis, stroke, affective disorders, Alzheimer's disease, enzymatic defects, Huntington's disease, hypocholesterolemine, hypoparathyroidase, immunodeficiencies, Lou Gehrig's disease, macular degeneration, multiple sclerosis, muscular dystrophy, Parkinson's disease, rheumatoid arthritis, spinal cord injuries and other trauma.
• heart disease such as myocardial infarction, hemophilic, infertility, kidney disease, liver disease, osteoarthritis, osteoporosis, stroke, affective disorders, Alzheimer's disease, enzymatic defects, Huntington's disease, hypocholesterolemine, hypoparathyroidase, immunodefic
• the methods of the present invention are also useful in making cloned mammals having complex or compound genetic alterations.
• the present invention is useful in producing animals that are young or more preferably hyper-young.
• the invention encompasses a method of re-cloning a cloned animal, wherein said re-cloned animal has been genetically altered with respect to the cloned animal. Such a method would not have been attempted without the finding of the present invention, which reveals that nuclear transfer rejuvenates late passage cells and restores telomere length.
• the re-cloned mammal was of the same genetic age as the cloned genetic mammal (which is, in turn, the same genetic age of the first nuclear donor), the feasibility of the method would decline depending on the generation of the clone.
• the results obtained by the present inventors to-date suggest that this is not the case and that in fact re-cloning can be effectuated as many times as desired, and will result in "hyper-young" animals, embryos and cells. Hyper young typically animals have enhanced immune systems useful for generating antibodies, and improved coat pigmentation.
• a preferred method of re-cloning according to the present invention comprises the following steps, and may be used to make a cloned animal having at least two genetic modifications: a. obtaining a primary cell from an animal of interest, b. making a first genetic modification to said primary cell by inserting heterologous DNA and/or deleting native DNA, c. using said first genetically modified primary cell as a nuclear donor for nuclear transfer to an enucleated oocyte or egg or other suitable recipient cell, d. obtaining a cloned embryo, fetus or animal having said first genetic modification, e. obtaining a cloned primary cell from said cloned embryo, fetus or animal, f.
• At least one recloning step utilizes a donor cell that has been propagated to senescence or near- senescence, or checkpoint a ⁇ ested, such that the telomeres of the reclones cell are regenerated or restored upon nuclear transfer.
• the method of the invention further comprises steps where said re-cloned embryo, fetus or animal is again re-cloned, and wherein a third genetic modification is made such that the further re-clone has the first, second and third genetic modifications.
• the method may be used to generate animals having numerous genes knocked out, inserted or substituted, and may be used to generate animals having entire cell systems replaced or modified, i.e., substitution of the human immunological system for that of the bovine, substitution of genes involved in complex enzymatic pathways such as those involving the clotting factors, or the complement cascade, etc.
• the method of re-cloning of the present invention will allow the creation of complex animal models for the study of diseases which involve multiple genes and or cell types, and may not be able to be duplicated by the typical animal model which expresses a single transgene, or has a single gene of interest knocked out. Moreover, such animal models may be used to study the effect of therapeutic genes m a particular complex genetic background. Such animal models may also be used to produce and test products that regulate the expression of different genes, to knock out genes that are involved in eliciting immune responses, to substitute collagen genes or other structural proteins genes with homologous counterparts, etc.
• the present invention involves the su ⁇ rising discovery that senescent cells may be rejuvenated, that EPC-1 activity and other cell markers associated with aging may be increased, that telomorase may be activated and that telomeres are extended, and that tandem repeats are repaired, all by the process of nuclear transfer.
• the present invention involves the discovery of a new way to activate telomerase activity and/or EPC-1 activity, which has applications far beyond that of extending telomeres and replicative life-span. Also, we predict other repairs to tandemly repeated DNA sequences.
• the invention provides a method for isolating the mechanism(s) of telomerase activation, EPC-1 activation, or other aging related genes, as well as a means of regulating telomerase or EPC-1, or other genes using the identified mechanisms.
• the cytoplasm of an oocyte can be fractionated and the fractions placed in association with a mortal cell, or a mortal cell nucleus, or telomeres, to assay for telomerase activation, EPC-1 activation (or other cell markers associated with aging) and telomere extension.
• telomerase activation EPC-1 activation (or other cell markers associated with aging)
• telomere extension EPC-1 activation (or other cell markers associated with aging)
• the active substituent or substituents in oocytes responsible for reactivating telomerase, EPC-1 activity, and/or other age associated cell markers can be identified and isolated.
• RNA or cDNAs can be isolated from the oocyte and transfected into a mortal cell, or expressed in a cell-free system for detecting telomerase activity, and transfected cells or cell-free systems demonstrating telomerase activity may be identified.
• Such methods could be supplemented with subtractive hybridization techniques in order to enrich for RNAs which are expressed during embryogenesis and not during senescence. In this way, genes encoding enzymes potentially involved in telomerase activation may be identified. Oocytes or eggs in the period just following fertilization may contain more than one gene or protein involved in telomerase activation.
• telomeres While not wishing to be held to any specific theory, the present inventors believe that there exists at least one regulatory protein or RNA in oocytes, or in ES cells or germ cells resulting from the development of oocytes, that is involved in the regulation of telomerase activity, and the ALT pathway and responds particularly to some aspect of the senescence cellular environment. It is possible that such protein(s) or RNA(s) activate telomerase or telomerase gene expression directly, but it is also possible that such proteins or RNAs work by inhibiting a suppressor of telomerase or the ALT pathway that exists or is expressed in senescent or near-senescent cells. A possible activator of telomerase is Oct 4 or Rex.
• telomere associated protein 1 Several proteins have also been identified that interact directly with telomerase, such as p23/hsp90 (molecular chaperones) and TEP1 (telomerase associated protein 1). Id.
• telomere binding protein or a telomerase repressor
• telomerase activity by upregulating gene expression or enhancing protein stability.
• the present invention includes methods of identifying at least one gene that either directly or indirectly enhances telomerase activity or the ALT pathway. Such methods could involve screening a cDNA or mRNA library generated from an embryo or embryonic stem cell for members that enhance telomerase or ALT activity in a senescent or near-senescent cell. The methods may also involve identifying at least one gene that either directly or indirectly suppresses telomerase or ALT activity, comprising, screening a cDNA or mRNA library generated from a senescent or near-senescent cell for members that suppress telomerase activity in an embryonic stem cell.
• Telomerase activity may be measured by any one of several methods known in the art, including measurement of reporter gene expression, e.g., a hTRT gene or protein fusion.
• a prefe ⁇ ed reporter molecule is green fluorescent protein (GFP).
• Telomerase activity may also be measured using the TRAPeze assay. Screening methods may be combined with other known methods for the pu ⁇ ose of increasing the effectiveness of the screening procedure, for instance, by subjecting cDNA or mRNA libraries to subtractive hybridization with a cDNA or mRNA library from a senescent cell prior to library screening if the test library is generated from an oocyte or an ES cell, or vice versa.
• the present invention also encompasses methods of identifying a protein that enhances telomerase, youthful patterns of gene expression or ALT activity, comprising (a) collecting fractions from the cytoplasm of an oocyte, embryo, or embryonic stem cell, (b) adding them to a cell-free system designed from a senescent or near-senescent cell, and (c) measuring for changes in telomerase, youthful gene expression or ALT activity that result from exposure to specific oocyte or ES cell cytoplasmic fractions.
• Methods for screening for compounds that inhibit telomerase or youthful gene expression are also included, and would comprise exposing an embryonic stem cell generated by nuclear transfer techniques using a senescent or near-senescent donor cell to a compound to determine whether said compound inhibits telomerase, youthful gene expression or ALT activity.
• the invention involves producing cells that have been transfected with the youthful gene, or regulating sequences, preferably linked to a suitable marker and method of using such cells to identify compounds that upregulate youthful gene expression.
• These screens should identify compounds that will modulate cell proliferation or aging. It is hypothesized that several genes may play a role in regulating cell proliferation and cycling, including EPC-1, the gas genes (Ciccarelli et al, Mci. Cell. Bid. 10(4): 1525-1529 (1990), such as gas-2, -3, -5, -7, PI-3 kinase (Tresini et al, Cancer Res. 58 (i): 1-4 (1998), collagenase, tPA, in regulating cell proliferation and cycling.
• Another screen is the modification of a somatic cell, more preferably an aged somatic cell, with a marker gene, i.e., GFP, where the marker gene is fused to or associated with a gene whose expression is altered with cellular aging, i.e., telomerase.
• the telomerase gene and/or promoter may be fused to the marker gene in a series of truncated forms and the marker constructs may then be transfected into senescent or near - senescent cells. Nuclear transfer may then be used to identify region in the telomerase gene or gene promoter or upstream region involved in activating telomerase expression upon nuclear transfer.
• the invention involves placing the EPC-1 and other youthful genes under the control of a heterologous, e.g., regulatable and preferably strong promoter, and assessing the effect of increases or decreases in expression on telomerase activity and telomeres.
• a heterologous e.g., regulatable and preferably strong promoter
• the present invention also includes the case of genetically modified somatic cells to identify the roles of genes in telomere regulation and the ALT pathway. Genes critical to ALT function can for instance be identified by the loss of ALT function when those genes are knocked out in the somatic cell prior to ALT.
• the present invention also includes the regulatory compounds, proteins and nucleic acids identified by the methods described above and pharmaceutical compositions comprising the same, which may be isolated and employed as exogenous telomerase activating agents according to the methods and pu ⁇ oses described herein, i.e., for the treatment of age-related diseases, the treatment of aged tissues such as retinal cells, the therapy of cancer, and the improving the effectiveness of bone marrow transplants.
• a somatic cell strain was derived from a 45-day-old female bovine fetus (BFF) and transfected with a PGK driven selection cassette. Cells were selected with G418 for 10 days, and five neomycin resistant colonies were isolated and analyzed for stable transfection by Southern blotting using a full length cDNA probe.
• One cell strain (CL53) was identified as 63% [total nuclei] positive for the transgene by FISH analysis, and was chosen for the nuclear transfer studies described in this study.
• the CL53 fibroblast cells which were characterized as negative for cytokeratin and positive for vimentin, were passaged until greater than 95% of their life-span was completed.
• the mo ⁇ hology of the cells was consistent with cells close to the end of their life-span as indicated by the phase contrast pictures of the cells by light microscopy (Fig. 1A).
• Fig. 1A A more detailed ultrastructural analysis by electron microscopy demonstrated that these cells exhibited additional features of replicative senescence, including prominent and active Golgi apparati, increased invaginated and lobed nuclei, large lysosomal bodies, and an increase in cytoplasmic microfibrils as compared to the young cells (Fig. IB) (27).
• these late passage cells exhibited a senescent phenotype in showing a reduced capacity to enter S phase as measured by a decrease in the inco ⁇ oration of H-thymidine (Fig. IC) and a significant
• SA- ⁇ -gal senescence-associated ⁇ -galactosidase
• Dermal fibroblasts were isolated from the cloned calves, and mRNA prepared as described in Figure ID. The cells expressed EPC-1 mRNA levels comparable or higher than the early passage fetal cells. To exclude the possibility that there was a small proportion of nonsenescent cells that gave rise to the cloned animals, CL53 donor cells were seeded at both
• telomere lengths in nucleated blood cells of the cloned animals were compared to age-matched control animals, newborn calves ( ⁇ 2 weeks old) and old cows (10 to 19 years old) using flow cytometric analysis following in situ hybridization with directly FITC-labeled (CCCTAA) peptide nucleic acid probe (flow FISH) (32,33).
• CCCTAA directly FITC-labeled peptide nucleic acid probe
• telomere length (63.4 ⁇ 1.7 vs. 51.0 ⁇ 3.1
• telomere lengths of the old cattle were 47.7 ⁇ 0.7 kMESF
• telomere length dynamics was also studied in the senescent (CL53), control (pre- transfection BFF) and cloned (ACT99-002) cells using Southern analysis of terminal restriction fragments (34).
• the results (Fig. 4B-D) were consistent with the flow FISH analysis of the nucleated blood cells.
• the telomeres were longer in the cells derived from the cloned embryo (19.3 kb) than in the senescent and early-passage donor cells (16.2 and 17.9 kb, respectively) (compare lanes 4, 5 and 6, Figure 4B). These results were confirmed by flow cytometric analysis of telomere length (flow FISH, ref 32) of the same cells (Fig. 4D).
• telomere restoration has not been previously described in cloned animals. Our results differ markedly from the study by Shiels et al. (20), in which telomere erosion did not appear to be repaired after nuclear transfer in sheep.
• the telomere lengths of three cloned animals 6LL3 (Dolly, obtained from an adult donor cell), 6LL6 (derived from an embryonic donor cell) and 6LL7 (derived from a fetal donor cell) were found to be decreased relative to age-matched control animals.
• telomere length did not occur because these animals were generated without germline involvement. They further suggested that the shorter TRF in Dolly was consistent the time the donor cells spent in culture before nuclear transfer.
• the present findings are significant, not only because viable offspring were produced from senescent somatic cells, but because the nuclear transfer procedure appeared to extend the telomeres of the animals beyond that of newborn and age- matched control animals. It is not known whether the longevity of these animals will be reflected by the telomeric measurements, although cells derived from a cloned fetus were observed to have a longer proliferative life-span than those obtained from the original same- age nonmanipulated fetus. Indeed, the mean TRF size observed in the later cells was in agreement with these findings.
• 347/347 Three-hundred and thirty-nine of the 347 cells (98%) underwent less than 3 PDs, whereas 347/347 (100%) underwent 4 or less PDs. Furthermore, the cells were grown in high serum (15%) concentrations, and young cells would have been rapidly proliferating and easily observed in the dish. The probability of a young cell in out sample is therefore ⁇ l/347. Seven animals (6 term animals and 1 fetus) were nevertheless cloned from the population of senescent fetal cells. It is therefore highly improbable that we, by chance, cloned the animals from undetectable young cells (P ⁇ 0.001, Chi-square).
• telomeres Differences between cells in the ability of telomerase to extend telomeres, or in the signaling pathways activated upon adaptation to culture, were proposed to explain the differences (38). Other investigators, however, report that the exogenous expression of hTERT extends telomeres and immortalizes human mammary epithelial cells (J. Shay, personal
• telomere length regeneration and maintenance provide chromosomal stability throughout the events of pre- and post-attachment development.
• EXAMPLE 3 NUCLEAR TRANSFER USING ADULT DONOR CELLS The above data obtained with fetal fibroblast donors are consistent with experiments performed using senescent cells obtained from adult animals. Dermal fibroblasts were grown from three Holstein steers. Single cell clones were isolated and population doublings counted until senescence. Nuclear transfer was performed using these fibroblast cells that were at or near senescence. Fetuses were removed from the uterus at week 6 of gestation and fibroblasts isolated from them and cultured until senescence. Cells were analyzed by imunohistochemistry and were shown to be fibroblasts. The number of population doublings in the original cells from the adult animals at the time of nuclear transfer (counted as number of PDs before senescence) and from 6-week-old fetuses generated from them are shown in
• Tissue biopsies will be obtained from all three germ layers from an adult cow (obtained at time of slaughter). In particular at least the following cells will be collected: ectoderm - keratinocytes mesoderm - dermal fibroblasts endoderm - gut epithelium
• telomere length A portion of the above three cell types will immediately be evaluated to determine telomere length. This can be affected by various methods. The remaining portion of all three cell types will be cultured until senescence. During culturing, a portion of each population will be retained and frozen. The different frozen cell samples will be labeled based on their particular population doubling.
• bovine clones will be produced using all 3 cell types, and using cells from different population doublings, i.e., from 0.8 population doublings away from senescence.
• the cloned bovine fetuses will be produced substantially according to the methods disclosed in U.S. Patent 5,945,577, inco ⁇ orated by reference herein.
• the cloned fetuses will be removed at forty days and cells of all three types isolated therefrom, e.g., keratinocytes, dermal fibroblasts, and gut epithelial cells.
• two same-age (40 day) wild-type fetuses will also be used to recover the same three types of cells. These cells, as well as those isolated from the cloned fetuses, will be cultured until senescence.
• telomere length of these different types of cultured cells will be determined immediately upon isolation from the animal or from such cells which are frozen upon isolation. Further, cells will again be removed and frozen from different cell populations until senescence. Thereafter, telomere length will be computed for the different cell types obtained at different cell population doublings, for cultured cells derived from cloned and wild-type embryos. The results will be compared to the results of Example 4. These experiments are cu ⁇ ently ongoing.
• EPC-1 EXPRESSION IN YOUNG vs. OLD CELLS EPC-1 expression was compared in human, bovine cells that were young or old, in cloned animals, and in controls. These results are shown below.
• telomere length reflects the shortened telomere of the somatic cell donor nucleus.
• our results suggest the exact reverse. Rather, growing a cell to senesence or near- senescence, or checkpoint a ⁇ ested, allowing the cell to lose T 2 AG 3 , removing minor damage along the way by 3' 5 ' exonuclease may afford an opportunity to then transfer that gene into an enucleated oocyte or other embryonic cell and with a subsequent burst of telomerase activity to rebuild a tract of pure T 2 AG 3 (longer than normally present).
• Gene expression will be compared in these cells by known methods, e.g. northern blots or by labeling using suitable probes.
• telomere extension To investigate the mechanism of telomere extension, the levels of telomerase activity in early embryonic development following nuclear transfer (NT) were examined in the bovine system. Similar to results that were previously published for normal (IVF) bovine embryos (Betts and King, 1999, Dev. Genetics 25:397-403), telomerase was detectable in all of the stages of early development that were analyzed. Levels of telomerase activity decreased at the 8-16 cell sage, and then increased at the morula and blastocyst stages for both the NT and IVF control embryos (data not shown). However, the levels of telomerase in the NT blastocysts were 2-fold higher than co ⁇ esponding IVF blastocysts.
• the significance of differences observed with PHA responses may be determined by testing a larger number of subjects. Differences in response to Con A (a T cell mitogen) were small and not statistically significant. For TSST and PWM, the differences are about 2-fold, with the 72 hr TSST system showing a 2.6x effect. Results are given in the table below. In vivo responses to the mitogens were not tested given the heightened sensitivity of the cloned animals observed following vaccination. However, in vivo tests for skin delayed type hypersensitivity responses to recall antigens would pose a low risk and could be performed to analyze in vivo immune responses.
• telomere extension of telomeres in somatic cells by NT was itself nonobvious in light of Wilmut. But the fact that starting from a senescent cell would lead to even better results (longer telomeres, longer lived cells) is nonobvious even in light of the former result.
• telomeres are nontelomeric sequences where there are more tracts of pure TTAGGG at the very telomere and fewer internally.
• telomeres shorten in somatic cells, the cells increasingly encounter nontelomeric sequences at the telomere that cannot bind TRF2 and eventually this raises the levels of activated p53 and then p21 to cause a slowing and eventual cessation of the cell cycle. The point is that this may not be an all or none phenomenon with a young cell proliferating and suddenly becoming senescent.
• telomere extension may result in the resynthesis of new uniform TTAGGG in the telomeres. While not being bound by their hypothesis, the inventors believe that this may occur via the upregulation of telomerase, EPC-1, alone or in association with other genes such as growth a ⁇ est sequences (gas genes), collagenase, tPA, and others.
• telomeres merely extended telomeres and the life-span of cells.
• all of the published reports showed no evidence that cells could be obtained wherein the overall phenotype of such cells is younger or hyper- young. Indeed, many researchers report that old, but not yet senescent cells that have slowed down, continue to divide slowly, but indefinitely withe telomerase.
• a prefe ⁇ ed application of the invention would be to keep telomeres as short as would allow the desired life-span, but to maximize the uniformity of TTAGGG. This would optimize the delicate balance of longevity vs. cancer risk, that is, the cells would not be constitutively immortal, and they would not have longer telomeres than necessary so as to limit the clonal expansion of abnormal cells.
• Animals cloned from senescent cells using this technology would be predicted to have unique properties. For example, animals raised for their coats, would be predicted to have more uniform coat color, would have an increased immune response would be more disease resistant, and have other advantages.
• age-related disease such as age-related macular degeneration, Parkinson's, Alzheimer's, osteoartbritis, osteoporosis, immune senescence, skin aging, emphysema, aneurisms, coronary heart disease, hypertension, cataracts, adult onset diabetes, and so on.
• diseases associated with an accelerated cell turnover such as muscular dystrophy, he ⁇ es zoster, AIDS, and ci ⁇ hosis could be treated by administering regenerated cells.

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• 2002
  • 2002-02-28 IL IL148457A patent/IL148457A/en active IP Right Grant

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