WO2006066247A2

WO2006066247A2 - Methods and compositions for extending telomere length and increasing cell lifespan

Info

Publication number: WO2006066247A2
Application number: PCT/US2005/046134
Authority: WO
Inventors: Gary Alan Suttle
Original assignee: Exvivo Technologies
Priority date: 2004-12-17
Filing date: 2005-12-19
Publication date: 2006-06-22
Also published as: WO2006066247A3

Abstract

Methods of elongating a telomere of a cell, the methods comprising enhancing hTERT expression in the cell and increasing telomerase processivity. Telomerized cells which comprise a cell having a telomere, the telomere being elongated by enhancing hTERT expression in the cell and by increasing the processivity of telomerase in the cell.

Description

METHODS AND COMPOSITIONS FOR EXTENDING TELOMERE LENGTH AND INCREASING CELL LIFESPAN

BACKGROUND

The present invention relates to methods for increasing the length of a cell's telomere and to compositions having cell's with lengthened telomeres.

Cells have a finite lifespan, and thus most cells in the body duplicate and replenish themselves. A progenitor cell, also referred to as a stem cell, is generally considered an undifferentiated cell that can give rise to other types of cells. A progenitor cell has the potential to develop into cells with a number of different phenotypes. Differentiation usually involves the selective expression of a subset of genes, which vary from cell type to cell type, without the loss of chromosomal material. Thus, the lineal descendants of a progenitor cell can differentiate along an appropriate pathway to produce a fully differentiated phenotype. All differentiated cells have, by definition, a progenitor cell type, for example, neuroblasts for neurons and germ cells for gamete cells. Progenitor cells share the three following general characteristics: (1) the ability to differentiate into specialized cells, i.e., not terminally differentiated, (2) the ability to regenerate a finite number of times, and (3) the ability to relocate and differentiate where needed. Progenitor cells may give rise to one or more lineage-committed cells, some of which are also progenitor cells, that in turn give rise to various types of differentiated cells and tissues. Progenitor cells generally constitute a small percentage of the total number of cells present in the body and vary based on their relative level of commitment to a particular lineage. Because progenitor cells have the ability to produce differentiated cell types, they may be useful, among other things, for replacing the function of aging or failing cells in many tissues and organ systems. There are three major classes of progenitor cells, based on what they have the potential to become. The earliest cells, from the fertilized egg through the first few division cycles, are totipotent. A totipotent cell has the gentic potential to create every cell of the body, including the placenta and extra-embryonic tissues.

Next come the pluripotent, or multipotent, cells, which can become more than one kind of cell, but do not have the potential to become all cell types. A pluripotent cell (i.e., an embroyonic progenitor cell) has the potential to create every cell of the body, but not the necessary placenta and extra-embryonic tissues required to form a human being. Pluripotent cells can be isolated from embryos and the germ line cells of fetuses. A multipotent cell, or a multipotent adult progenitor cell ("MAPC"), can give rise to a limited number of other particular types of cells. Multipotent cells are found in both developing fetuses and fully developed human beings and have been observed to develop into a variety of cell types such as cardiomyocytes, hepatocytes, and epithelial cells. For example, hematopoietic cells (blood cells) in the bone marrow are multipotent and give rise to the various types of blood cells, including red blood cells, white blood cells, and platelets. Unlike pluripotent cells, multipotent cells are often present in a fully developed human being. But multipotent cells may only be present in minute quantities and their numbers can decrease with age. Multipotent cells from a specific patient may take time to mature in culture in order to produce adequate amounts for treatment.

And finally there are unipotent cell types, such as the muscle-cell progenitors. These still have the quality of regenerating, but may be more differentiated or committed to a certain cell type. The development of an organism is a strictly regulated program in which controlled gene expression guarantees the establishment of a specific phenotype. The chromosome termini or so-called telomeres preserve the integrity of the genome within developing cells. In the germline, during early development, human telomeres are balanced between shortening processes with each cell division and elongation by telomerase, but once terminally differentiated or mature, the equilibrium is shifted to gradual shortening by repression of the telomerase enzyme. Gradual telomere shortening in normal human somatic cells during consecutive rounds of replication eventually leads to critically short telomeres that may induce replicative senescence.

Thus, somatic progenitor cells have a limited proliferative capacity. On average, it is believed that most progenitor cells have the ability to divide between 7 and 50 times, depending on the type, age, and genotype of donor. And as a mammal ages they have fewer of these progenitor cells. So, a similarly derived 50 mL sample of bone marrow aspirate from a 60 year old, for instance, would be expected to have fewer progenitor cells, and the progenitor cells would have less proliferative potential than those of a 20 year old. Telomeres are the non-encoding regions of DNA capping the ends of chromosomes, in association with various proteins. The DNA that forms the telomere consists of the sequence (TTAGGG)_n, which is referred to as a "telomeric repeat" because it is repeated in tandem over about 5 to about 15 kilobases (kb). K. Collins & J.R. Mitchell, Oncogene 21:564-579 (2002). Telomeres protect the ends of linear eukaryotic chromosomes from degradation, prevent end-to-end fusions, and partake in chromosome localization and segregation. Cooper, Curr. Opin. Genet. Dev. 10: 169-77 (2000); McEachern et al, Annu. Rev. Genet. 34: 331-358 (2000); Price, Cur.r Opin. Gene.t Dev. 9: 218-24, (1999). They allow the complete replication of the 5' ends of chromosomal DNA without the loss of internal sequences and the genes these sequences may encode.

Telomere length is maintained in part by the enzyme telomerase. But in most human somatic cells, telomerase activity is either undetectable or is insufficient, and telomeres shorten with successive cell divisions. In the absence of telomerase activity, about 50-200 bases of DNA, depending on cell type, are lost with each round of cell division. If telomere erosion is not balanced by elongation, telomeres will progressively shorten and may enter a state of arrested growth called replicative senescence. The maintenance of telomere length thus is believed to play a key role in the ability of cells to avoid replicative senescence and to propagate indefinitely. Likewise, aberrant maintenance of telomere length is believed to underlie indefinite cellular proliferation characteristic of cancer cells.

The telomerase enzyme is assembled as a ribonucleoprotein enzyme complex. The human telomerase holoenzyme comprises a reverse transcriptase protein (hTERT) and an RNA template (hTR). Both components are essential for telomerase activity. And hTERT may regulate the transcription of a variety of genes implicated in cell growth, chromatin modification, DNA repair, and chromosome stabilization, without influencing telomere length. L.M. Pirzio, et al., Cytogenet. Genome Res. 104:87-94 (2004).

DESCRIPTION

The present invention is based in the observation that by both enhancing hTERT expression and increasing the processivity of the telomerase holoenzyme, the telomere of a cell can be elongated to a desired length, thereby increasing the proliferative capacity of the cells. The term "proliferative capacity" refers to the number of divisions that a cell can undergo, and to the ability of the cell to continue to divide where the daughter cells of such divisions are not transformed, i.e., they maintain normal response to growth and cell cycle regulation. For example, the telomeres of progenitor cells of mesenchymal lineage may lose between about 50 to about 200 base pairs per cell division, and these cells typically senesce when the telomeres reach between 8-10 kb. Thus, for every 1,000 base pairs the telomere can be elongated, a corresponding 5 to 20 fold increase in the cell's proliferative capability may be expected. The present invention provides methods for extending the proliferative capacity of a cell. Such methods may increase the proliferative capacity of a cell without differentiation and while maintaining genomic stability. The present invention also provides "telomerized cells" and methods of using telomerized cells in applications such as the treatment of arthritis, cartilage and bone injury, tissue engineering, and anti-aging treatments. The term "telomerized," in all its forms, refers to a telomere in which its average length has been increased, and to the increase in the possible number of population doublings remaining for a cell with such a telomere, hi addition, the methods and compositions of the present invention may allow for the generation of autologous and allogenic cells and tissues for therapeutic use. The methods and compositions of the present invention do not require the use of embryonic progenitor cells to obtain a therapeutically effective supply of progenitor cells. The present invention also may include various systems, compositions, and cells or tissues related to these methods and compositions.

In certain embodiments, the present invention provides methods of increasing the proliferative capacity of a cell that comprises enhancing hTERT expression in the cell, and increasing the processivity of telomerase in the cell. hi general, the cells used in the methods of the present invention should be capable of self-renewal and should be able to undergo at least one division in culture. In certain embodiments, any progenitor cell may be used in the methods of the present invention. Suitable progenitor cells include those that are capable of differentiating into any one the different cell types found in a human. In certain embodiments, suitable progenitor cells do not express adequate levels of hTERT to maintain telomere length and therefore may have a limited proliferative capacity.

Progenitor cells suitable for use in the methods of the present invention may be obtained from a number of somatic cell sources. The term "somatic cell" refers to any postnatally derived cell. Such sources include, for example, bone marrow, muscle, lipo aspirates, peripheral blood, and the like. Examples of progenitor cells suitable for use in the present invention include progenitor cells of mesenchyme origin, mesenchymal-like progenitor cells, nonhematopoietic progenitor cells, keratinocyte progenitor cells, neural progenitor cells, and hematopoietic progenitor cells, as well as progenitor cells of epithelial and endothelial origin.

In certain embodiments of the present invention, the cell is a mesenchymal-like progenitor cell. These progenitor cells are somatic cells derived from various sources, including bone marrow stroma, adipose tissue, muscle, and blood. Under appropriate culture conditions, mesenchymal-like progenitor cells can be induced to differentiate into cells of mesenchymal lineage. These include differentiation into cells that become less potent. For example, these cells may differentiate into precursor cells such as osteoblasts, and primary cells such as osteocytes. A similar differentiation pathway exists for the adipocytes, chondrocytes, tenocytes, ligamentogenic cells, myogenic cells, bone marrow stroma cells, and dermogenic cells. Mesenchymal-like progenitor cell induction is described in Pittenger et al., Science. 284:143-7 (1999); Herzog et al., Blood 102:3483-93 (2003); and U.S. Patent No. 5,736,396, the relevant disclosures of which are incorporated herein by reference. In certain embodiments of the present invention, the cell is a keratinocyte progenitor cell. Keratinocyte progenitor cells are derived from epidermis, have the capacity of self- renewal, and give rise to epidermis and hair. Keratinocyte progenitor cells may be isolated from the upper hair follicle, called the bulge, as described in Cotsarelis et al., Exp Dermatol. 8:80-8 (1999) and Morris et al., Nat Biotechnol. 22:411-7 (2004), the relevant disclosure of which is incorporated herein by reference.

In certain embodiments of the present invention, the cell is a neural progenitor cell. Neural progenitor cells are derived from the nervous system, have the capacity for self- renewal, and give rise to neural cell types including neurons, astrocytes, and oligodendrocytes. Neural progenitor cells may be obtained from multiple sources within the mammalian brain, including the hippocampus, subventricular zone, ependymal cells, subgranular zone of the dentate gyrus, and the olfactory bulb, as described in F. H. Gage, Science 287:1433-8 (2000); D.L. Clarke, Bone Marrow Transplant. 32 Suppl. l:S13-7 (2003), the relevant disclosure of which is incorporated herein by reference.

In certain embodiments of the present invention, the cell is a hematopoietic progenitor cell. Hematopoietic progenitor cells are multipotent cells capable of self renewal and differentiation into multiple blood cell types, including erythrocytes, megakaryocytes, monocytes/macrophages, granulocytes, mast cells, B-cells, and T-cells. Hematopoietic progenitor cells may be obtained from adult bone marrow and peripheral blood (with and without mobilization), as described in Kondo, et al., Annu. Rev. Immunol. 21:759-806 (2003) and Zubair, et al, Transfusion 42:1096-101 (2002), the relevant disclosure of which is incorporated herein by reference. Generally, cells used in the methods of the present invention may be isolated using methods known in the art. For example, progenitor cells may be isolated using antigenic determinates, cell adherence, flow cytometry, magnetic beads, and the like. Wognum, et al, Arch Med Res. 34:461-75 (2003), Gimble, et al, Cytotherapy 5:362-9 (2003), Papini, et al, Stem Cells, 21:481-94 (2003), Jones, et al, Arthritis Rheum. 46:3349-60 (2002). Once obtained, the cell may be cultured according to standard cell culture protocols available to those of ordinary skill in the art, for example as described in METHODS IN MOLECULAR MEDICINE: HUMAN CELL CULTURE PROTOCOLS (G. E. Jones, ed.), Humana Press Inc., Totowa, NJ. (1996). For example, in certain embodiments, cells, such as progenitor cells, may be cultured as adherent cells on tissue culture dishes or flasks at about 37°C in an atmosphere having about 5% CO. It is generally accepted in the art that progenitor cells may be cultured many times while maintaining a partially differentiated or undifferentiated state and while retaining the capacity to differentiate into one or more cell and tissue types. Once cultured, the cells may be expanded. Enhancing hTERT Expression Telomerase is a ribonucleoprotein that synthesizes telomeric DNA and is a key component in the regulation of the telomere. Human telomerase is made up of at least two components: (1) an essential structural RNA component (hTR) and (2) a catalytic protein component, (hTERT). Together, hTR and hTERT may be referred to as the telomerase holoenzyme. Telomerase can add multiple telomeric repeats to the 3' end of a telomere by using hTR as a template and the catalytic activity of hTERT. While hTR is relatively ubiquitously expressed in embryonic and somatic tissues, expression of hTERT is tightly regulated and not detectable in most somatic cells. K. A. Kolquist, et al., Nat Genet. 19:182- 86 (1998). And studies by many groups have now demonstrated that hTERT plays a fundamental role in telomere preservation and cell proliferation. In the context of the invention, the term hTERT refers to hTERT or an hTERT-like splice variant such as, for example, an hTERT-like splice variant expressed from an hTERT in which all or a portion of the promoter is deleted. And hTERT refers to at least a portion of a gene encoding an hTERT protein.

In certain embodiments, hTERT expression should be enhanced to a level sufficient to lengthen the telomere of a cell or to maintain the length of the telomere of a cell for a desired period of time. For example, in certain embodiments, in which the cell is a mesenchymal- like progenitor cell, the expression of hTERT may be enhanced to about 20-30% constitutive expression to maintain the length of a telomere.

By enhancing hTERT expression in a cell, not only may the telomere be lengthened or maintained, but also the genomic instability due to short telomeres may be reduced. See L.M. Pirzio, et al, Cytogenet Genome Res. 104:87-94 (2004); V. Gorbunova, et al., J. Biol.

Chem. 277:38540-49 (2002). Expression of hTERT has been shown not to transform cells.

W.C. Hahn, et al., Nature 400:464 (1999). Indeed, highly expressing cell lines showed virtually normal karyotypes, even after extended culture when lower expressing lines exhibit chromosomal abnormalities. W. Cui et al., J. Biol. Chem. 277:38531-39 (2002). And ectopic expression of hTERT may extend the replicative lifespan of human somatic cells (A.G.

Bodnar, et al., Science 279:349-52 (1998); H. Vaziri & S. Benchimol, Curr Biol. 8:279-82

(1998); CM. Counter, et al., Oncogene 16:1217-22 (1998); J. Yang, et al., J Biol Chem.

274:26141-48 (1999)) without altering their karyotype, differentiation characteristics, or activation of known oncogenes (X.R. Jiang, et al., Nat Genet. 21:111-14 (1999); KX. MacKenzie, et al., Exp Cell Res. 259:336-50 (2000); M.A Dickson, et al., MoI Cell Biol.

20:1436-47 (2000)).

Any method of enhancing hTERT expression in a cell may be suitable for use in the methods of the present invention. Suitable methods may transiently enhance hTERT expression, while other methods may enhance hTERT expression for longer periods of time, for example, through integration of hTERT into a cell's genome. In general, expression of hTERT may be enhanced in cells using endogenous methods, exogenous methods, or both.

Examples of suitable endogenous method include, creating hypoxic conditions, such as incubating at 3% O₂ or directly upregulating hypoxia inducible factor (HIF-I α); treatment with histone deacetylator (HDAC) inhibitors, such as TSA; or both. Other examples of suitable endogenous methods include methods that may transiently downregulate or transiently inactivate genes responsible for repressing hTERT expression or any gene that is associated with telomerase processivity or both. Suitable genes are known in the art, or may be identified using methods known in the art, and include menl, madl, britl, sipl, nficl, and rάk. Any method may be used to downregulate or inactivate genes that repress hTERT, suitable for use in the methods of the present invention.

Telomerase processivity may be increased through the modulation of a gene responsible for repressing hTERT expression or a gene that is associated with telomerase processivity or both; ultimately increasing the amount of hTERT produced, hi the context of the present invention, "modulation," means either an increase (stimulation) or a decrease (inhibition) in the expression of a gene, hi certain embodiments, hTERT expression may be increased by modulating genes responsible for repressing hTERT expression using antisense molecules. This is accomplished by providing antisense molecules (e.g., oligonucleotides) that specifically hybridize with one or more nucleic acids encoding an hTERT repressor. As used herein, the terms "target nucleic acid" and "nucleic acid encoding an hTERT repressor" encompass DNA encoding an hTERT repressor, RNA (including pre-niRNA and mRNA) transcribed from such DNA, and also cDNA derived from such RNA. The specific hybridization of an oligomeric compound with its target nucleic acid interferes with the normal function of the nucleic acid. This modulation of function of a target nucleic acid by compounds which specifically hybridize to it is generally referred to as "antisense." The functions of DNA to be interfered with include replication and transcription. The functions of RNA to be interfered with include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in or facilitated by the RNA. The overall effect of such interference with target nucleic acid function is to increase the expression hTERT.

In general, specific nucleic acids are targeted for antisense. "Targeting" an antisense compound to a particular nucleic acid, in the context of this invention, is a multistep process. The process usually begins with the identification of a nucleic acid sequence whose function is to be modulated. This may be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with hTERT expression or repression. The targeting process also includes determination of a site or sites within this gene for the antisense interaction to occur such that the desired effect, e.g., modulation of expression of the protein, will result. The site that may be targeted effectively may be any portion of the nucleic acid sequence whose function is to be modulated, for example, an intragenic site or portion of an open reading frame (ORF), the 5' untranslated region (5'UTR), the 5' cap of an mRNA, which includes the first 50 nucleotides adjacent to the cap.

Once one or more target sites have been identified, oligonucleotides are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect. In the context of this invention, the term "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally-occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target and increased stability in the presence of nucleases.

In the context of this invention, "hybridization" means hydrogen bonding, which may be Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases. "Complementary," as used herein, refers to the capacity for precise pairing between two nucleotides. For example, if a nucleotide at a certain position of an oligonucleotide is capable of hydrogen bonding with a nucleotide at the same position of a DNA or RNA molecule, then the oligonucleotide and the DNA or RNA are considered to be complementary to each other at that position. The oligonucleotide and the DNA or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other. Thus, "specifically hybridizable" and "complementary" are terms which are used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the oligonucleotide and the DNA or RNA target. It is understood in the art that the sequence of an antisense molecule need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. An antisense molecule is specifically hybridizable when binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense molecule to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed.

Antisense molecules which hybridize to the target and inhibit expression of the target are identified through experimentation, and include at least a portion of one or more of the genes associated with hTERT repression. Antisense molecules will generally have a size in the range for from about 7 to about 500 nucleotides in length, depending on efficiency of inhibition, specificity, including absence of cross-reactivity, and the like.

Suitable antisense molecules include antisense oligonucleotides and oligonucleotide mimetics, as well as chimeric antisense molecules formed as composite structures of two or more of oligonucleotides, modified oligonucleotides, oligonucleosides and oligonucleotide mimetics. Suitable antisense molecules also include catalytic antisense molecules such as ribozymes, external guide sequence (EGS) oligonucleotides (oligozymes), and other short catalytic RNAs or catalytic oligonucleotides that hybridize to the target nucleic acid and modulate its expression. The antisense molecules used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. Antisense molecules also may be produced by expression of all or a part of the target sequence in an appropriate vector. Antisense molecules, such as ribozymes, may be synthesized in vitro and administered to a cell, or may be encoded on an expression vector, from which the ribozyme is synthesized in the targeted cell.

In certain embodiments, hTERT expression may be enhanced by modulating one or more hTERT repressor genes using RNAi-directed gene silencing. Methods of using RNAi- directed gene silencing are known and routinely practiced in the art, including those described in D.M. Dykxhoorn, et al, Nature Reviews 4:457-67 (2003) and J. Soutschek, et al, Nature 432:173-78 (2004), the relevant disclosures of which are incorporated herein by reference. In certain embodiments, hTERT expression may be increased by transfection of a cell with siRNA or a shRNA vector. Suitable shRNA vectors or siRNA molecules may directed to the rnRNA of proteins that repress hTERT expression, such as the mRNA MENl, MADl₅ BRITl, and RAK. As mentioned above, exogenous methods may be used to enhance hTERT expression in a cell. hTERT expression should be enhanced to a level sufficient to maintain the telomere or elongate the telomere, but overexpression to a level detrimental to the cell should be avoided. In general, exogenous methods involve the integration of hTERT into a chromosome of a cell. Such integrated hTERT should be regulateable, for example, under weak promoter control. Li general, the exogenous methods used in the methods of the present invention should not randomly integrate into the host genome, among other things, to avoid introducing a mutation. Examples of suitable exogenous methods include, site- specifically integrating hTERT into a cell's genome (e.g., thru a viral vector, bacteriophage, or artificial chromosome). See, e.g., Lu, et al., Stem Cells Dev. 13:133-45 (2004), Groth, et al., J MoI Biol. 335:667-78 (2004), Vanderbyl, et al., Stem Cells 22:324-33 (2004).

In certain embodiments, hTERT may be integrated into a cell's genome by methods that use bacteriophage. Such methods are known and routinely practiced in the art and some of these methods are reviewed in Groth, et al., JMo/ Biol. 335:667-78 (2004), the relevant disclosure of which is incorporated herein by reference.

In certain embodiments, hTERT may be integrated into a cell's genome using a serine bacteriophage and a bacteriophage vector of the present invention. In general, the bacteriophage vectors of the present invention comprises an pINT integrase gene, a modified attB site, and a multicloning site. The modified attB site should be designed to increase the specificity of the bacteriophage vector to the attachment site (e.g., to prevent binding to pseudo sites). Any oligonucleotide may be cloned into the bacteriophage vector's multicloning site, for example, hTERT. The bacteriophage vectors of the present invention may be used in conjunction with a serine bacteriophage, such as ρhiC31.

The bacteriophage vectors of the present invention may be used in methods for site- specifically integrating an oligonucleotide (e.g., a gene) into a chromosome. Such methods involve site specifically integrating an oligonucleotide into a modified attP site already existing in a cell's genome using the pFNT integrase. The modified attP site should be designed to increase the specificity of the integrase. The modified attP site may be introduced into the cell using a two-vector AAV system to express the Rep gene in trans under regulatory control, and thereby integrating a modified attP site in the AAVSl site on chromosome 19. Such methods are particularly useful because the AAVSl site is a DNA Hypersensitivity Site (DHS). And the DHS enables easier access for the subsequent integrase activity. This, in conjunction with the modified attP and modified attB, improves efficiency and specificity of subsequent integrations. In certain embodiments, a second modified attP site may be integrated into the genome of a cell with the oligonucleotide. Among other things, this may facilitate the integration of a second oligonucleotide into the cell's genome, and this may be repeated to increase the capability for site-specific integration. In certain embodiments, these methods may be used to site-specifically integrate hTERT into a cell's genome. Accordingly, the oligonucleotide integrated into the cell's genome may be hTERT. In other embodiments, additional oligonucleotides, e.g., a gene, may be site-specifically integrated into the cell's genome after or along with hTERT. In certain embodiments, a viral vector may be used to site-specifically integrate hTERT into the host genome. Suitable viral vectors should be capable of transferring hTERT into a recipient cell and include an attenuated or defective DNA virus, such as herpes simplex virus (HSV), Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), and the like. In certain embodiments, an adeno-AAV hybrid viral vector of the present invention may be used to site-specifically integrate hTERT into the host genome. The adeno-AAV hybrid viral vectors of the present invention comprise specific elements of the AAV virus (i.e. p5 integration efficiency element) and specific elements of the adenovirus (i.e. packaging elements). These vectors should be capable of inserting an oligonucleotide (e.g., a gene) into a cell's genome. Accordingly, in certain embodiments in which the oligonucleotide is hTERT, the adeno-AAV hybrid vector may be used to insert the hTERT in the AAVSl site on chromosome 19.

In other embodiments, an artificial chromosome may be used to enhance hTERT expression. In such embodiments, hTERT is first transfected into a mammalian artificial chromosome (preferably under regulateable control or a weak promoter), and then the artificial chromosome is transfected into the cell. Suitable methods of using mammalian artificial chromosomes are disclosed in Vanderbyl, et al, Stem Cells 22:324-33 (2004), the relevant disclosure of which is incorporated herein by reference. Increasing telomerase processivity Telomerase processivity is regulated at multiple levels, which include transcription, mRNA splicing, post-translational modification, and recruitment and access to the telomeric substrate. See I. Horikawa & J.C. Barrett, Carcinogenesis 24:1167-76 (2003). And the telomerase holoenzyme is not the only activity that affects the length of telomeres. As telomeres become longer, their further extension by telomerase may be progressively inhibited. Studies have shown that telomere length control may involve a feedback loop in which the addition of new telomeric repeats by telomerase creates binding sites for telomere and telomerase regulators. See, e.g., A. Smogorzewska & T. de Lange, Annu. Rev. Biochem. 73:177-208 (2004).

Accordingly, one step of the methods of the present invention is to transiently increase telomerase processivity. By increase telomerase processivity is meant that the telomerase, or telomerase holoenzyme, is not prevented or inhibited from extending the length of a telomere. In certain embodiments, telomerase processivity may be increased through the transient modulation of the function of telomere-associated genes, ultimately modulating the amount of telomere-associated proteins produced. For example, for certain telomere-associated proteins, e.g., POTl, inhibition is the preferred form of modulation of gene expression. For other telomere-associated proteins, e.g., tankyrases, stimulation is the preferred form of modulation of gene expression. By modulating telomere-associated genes, the telomerase should have better access to the telomeres thereby increasing telomerase processivity.

Suitable genes include those that may affect the formation of the telomerase complex or those that may affect the ability of the complex to access the telomere. Suitable genes also include those that encode proteins that may be important for maintaining telomere stability and regulating telomere length. These proteins include TRFl, TRF2, POTl, tankyrase, RAPl, ΗN2, and PTOP.

TRFl is thought to regulate telomere length by preventing the elongation of telomeres once they reach a critical size. B. van Steensel & T. de Lange, Nature 385:740-43 (1997). TRF2 appears to be important for stabilizing the chromosome ends by associating with the 3' overhang and suppressing end-to-end fusions between chromosomes. B. van Steensel, et al, Cell 92:401-13 (1998); J. Karlseder, et al., Science 283:1321-25 (1999). Many additional proteins can bind indirectly to telomeres, often via TRFl and TRF2, and together these proteins may function to regulate telomere homeostasis. K. Collins & J.R. Mitchell, Oncogene 21:564-79 (2002). For example, PINXl may bind to the region of the telomerase complex responsible heterodimerization, thereby inhibiting telomerase processivity. And PTOP may bind to the carboxyl terminus of POTl, heterodimerize with POTl, and regulate POTl telomeric recruitment and telomere length. Liu, et ai, Nat Cell Biol. 6:673-80 (2004).

Any telomere-associated gene or combination of telomere-associated genes may be chosen to transiently increase telomerase processivity. Certain choices or combinations may increase telomerase processivity by increasing the telomere elongation rate faster than other choices or combinations. And other choices or combinations may affect the amount of control over telomerase processivity. So, the telomere-associated gene or telomere- associated protein or both chosen will depend on the desired result. For example, if both PDSfXl and POTl are inhibited the rate of telomere elongation may be rapid, but control of the elongation may be poor. Reducing the level of PINXl, increases the formation of the telomerase holoenzyme, and reducing the level of POTl decreases the potential of POTl binding to the ssDNA of the telomere and therefore the access of the telomerase holoenzyme to the telomere ends. Thus, the choice or combination of the specific telomere-associated genes chosen to modulate may depend on the application (for instance, the need for quick elongation, or the need for tight control). A person of ordinary skill in the art, with the benefit of this disclosure, will recognize the appropriate telomere-associated gene(s) to choose based on the desired application.

Telomere processivity should be increased for a length of time sufficient for the adequate expansion of the cell in culture and corresponding maintenance of the telomere. The length of time telomerase processivity is increased may depend on, e.g., the type of cell and its current telomere length, the telomere-associated gene or telomere-associated protein targeted, and the method(s) used. The length of time telomerase processivity is increased also may depend on the length of telomere desired. For example, when the POTl and PINXl genes are modulated by downregulating expression in mesenchymal-like progenitor cells, approximately 18 days may be a sufficient time to increase telomerase processivity and achieve a desired telomere length.

But increasing telomerase processivity should be limited to a length of time sufficient to prevent or decrease the over-elongation of the telomeres, which can lead to genomic instability, cell death, or both. Thus, the telomere-associated genes and proteins should only be transiently affected. In this way the cell may return to normal homeostasis. By way of explanation and not of limitation, this may be due to telomerase' s playing other roles that support cell growth and survival, apart from its function in maintaining 3' overhangs or overall telomere length. See S.A. Stewart, et al, Proc. Natl. Acad. Sd. USA 99:12606-11 (2002); S.E. Artandi,'et al., Proc. Natl. Acad. Sc.i USA. 99:8191-96 (2002); L.L. Smith, et al., Nat. Cell. Biol. 5:474-79 (2003); P. Zhang, et al., FASEB J. 17:767-69 (2003); Y. Cao, et al., Oncogene 21:3130-38 (2002). In certain embodiments, telomerase processivity may be increased by modulating telomere-associated genes using antisense molecules that specifically hybridize with one or more nucleic acids encoding a telomere-associated protein. Such methods are described above and include the use of RNAi and ribozymes. For example, in certain embodiments, telomerase processivity may be transiently increased by transfection of a cell with siRNA or a shRNA vector. Suitable shRNA vectors or siRNA molecules may directed to the rnRNA of telomere associated proteins, such as the niRNA of TRFl, TTN2, POTl, PINXl, PTOP, or a combination thereof. For example, RNAi mediated inhibition of TRFl may result in loss of TIN2 from telomeres and thereby increase the processivity of telomerase.

In certain embodiments, telomerase processivity may be increased through the use of small molecules. Such small molecules may transiently disrupt binding of a telomere- associated protein to the telomere or to another telomere-associated protein or both. Alternatively, agents that bind to a telomere-associated protein's binding site and inhibit its binding to telomere-associated protein are of interest. Alternatively, agents that disrupt telomere-associated protein-protein interactions with cofactors, e.g., cofactor binding, and thereby inhibit binding of the telomere-associated protein to its binding site are of interest. For example, in certain embodiments, the small molecule may be capable of binding to the OB fold of POTl thereby disrupting the binding of POTl to the telomere. Suitable small molecule agents may be identified using methods known in the art, with the benefit of this disclosure. In certain embodiments, a suitable small molecule may be an oligonucleotide "decoy" for a telomere-associated protein. Such a small molecule may bind to a telomere-associated protein and thereby prevent the telomere-associated protein binding to its target. These duplex oligonucleotide decoys have at least that portion of the sequence of the target site required to bind to the telomere-associated protein and thereby prevent its binding to the target site. Oligonucleotide decoys and methods for their use and administration are further described in general terms in Morishita et al., Circ. Res. 82:1023-28 (1998), the relevant disclosure of which is incorporated herein by reference. Once introduced into a cell, the decoy may bind endogenous transcription factors. Li the context of the present invention, decoys are double-stranded nucleic acid molecules with high binding affinity for a targeted transcription factor. Targeted transcription factors are endogenous, sequence-specific double-stranded DNA binding proteins which modulate (increase or decrease) the rate of transcription of one or more specific telomere-associated genes in the target cell, hi general, a specific decoy should be capable of competitively inhibiting transcription factor binding to the endogenous gene.

The length, structure, and nucleotide sequence of the decoy will vary, among other things, depending on the targeted transcription factor. For example, targeted transcription factors frequently bind with different degrees of affinity to a variety of sequences, normally sharing a high degree of homology. Accordingly, one may choose to use a sequence associated with a particular target gene or use a consensus sequence based on the nucleotide at each site which occurs most frequently in the binding sequences to which the particular transcription factor binds. hi addition to binding affinity, the decoys are also selected for binding specificity, hi some embodiments, the decoys should be highly specific for the target transcription factor(s) such that their effect on nontarget cells and nontargeted metabolic processes of target cells are minimized. Such selection is accomplished in vitro by comparative binding to known transcription factors and nuclear extracts and in culture and in vivo by assaying nontargeted cell function and effects on nontargeted cell types.

The decoys should contain sufficient nucleotide sequence to ensure target transcription factor binding specificity and affinity sufficient for therapeutic effectiveness. For the most part, the target transcription factors will require at least six base pairs, usually at least about eight base pairs, for sufficient binding specificity and affinity. Frequently, providing the decoys with flanking sequences (ranging from about 5 to 50 bp) beside the binding site enhances binding affinity or specificity or both. Accordingly, cis element flanking regions may be present and concatemer oligonucleotides may be constructed with serial repetitions of the binding sequences or cis element flanking sequences or both.

The oligonucleotides which are employed may be naturally occurring or other than naturally occurring, where the synthetic nucleotides may be modified in a wide variety of ways as known in the art. Thus, oxygens may be substituted with nitrogen, sulfur, or carbon; phosphorus substituted with carbon; deoxyribose substituted with other sugars, or individual bases substituted with an unnatural base. In each case, any change will be evaluated as to the effect of the modification on the binding of the oligonucleotide to the target transcription factor, as well as any deleterious physiological effects. The strands may be synthesized in accordance with conventional ways using phosphoramidite synthesis, commercially available automatic synthesizes, and the like.

Individual decoys or mixtures of decoys or both may be administered to a cell. The decoys are administered to a cell in a form permitting cellular internalization of the decoys in an amount sufficient to competitively inhibit the binding of the targeted transcription factor to an endogenous gene. The selected method of administration depends principally upon the target cell, the nature of the decoy, the host, the size of the decoy. Examples administration methods include transfection with a retrovirus, viral coat protein-liposome mediated transfection, lipofectin, and the like, and are described in Dzau et al., Trends in Biotech. 11 :205-10 (1993), the relevant disclosure of which is incorporated herein by reference. hi certain embodiments, the expression of a dominant negative allele may be used to inhibit telomere-associated proteins and thereby increase telomerase processivity. For example, when an N-terminal truncation form of POTl (POT1^AOB) is expressed, the endogenous POTl is repressed and telomerase processivity increases, hi other examples, RAPl truncation mutants may be used to increase the processivity of telomerase as described in B. Li & T. de Lange, MoI. Biol. Cell 12:5060-68 (2003), the relevant disclosure of which is incorporated herein by reference. Methods for expressing dominant negative alleles are known in the art, including those described in B. van Steensel & T. de Lange, Nature 385:740-43 (1997); A. Smogorzewska, et al., MoI. Cell Biol. 20:16598-68 (2000); and U.S. Patent No. 5,859,183, which describes a TRFl dominant negative allele, the relevant disclosures of which are incorporated by reference. hi certain embodiments, transient overexpression of a telomere-associated protein may be used to transiently increase the processivity of telomerase. For example, transient overexpression of tankyrase 1 or tankyrase 2 or both may be used to increase the processivity of telomerase. These tankyrases may diminish the ability of TRFl to bind to telomeric DNA, and therefore increase ability of telomerase to access the telomere. S. Smith, et al., Science 282:1484-87 (1998). Accordingly transient overexpression of one or more tankyrases may lead to increased telomerase processivity and telomere elongation. See S. Smith & T. De Lange, Curr. Biol. 10:1299-02 (2000); U.S. Patent No. 6,506,587 In certain embodiments, the methods of the present invention also may comprise determining whether any chromosomal changes have occurred. For example, the cytogenetics of the cell may be examined before and after the methods of the present invention have been used. Methods of detecting chromosomal changes are known in the art and include detecting chromosomal changes by karyotyping and detecting genetic mutations using, for example, PCR amplification, and microarrays. Such methods may be performed on a cell treated with the methods of the present invention before treatment or after treatment or both.

The methods of the present invention may result in cells that when cultured, proliferate significantly better and have an increased average telomere length. Cells treated with the methods of the present invention often have significantly more (in some cases 100's times more) proliferative potential as compared to an untreated cell. Accordingly, the methods of the present invention may be used to rejuvenate a cell with shortened telomeres. And the cells formed using the methods of the present invention may have telomeres that are reset to a more homogenous length. Increasing the length of the telomere, and having fewer cells with short telomeres has been shown to decrease genomic instability. It has been shown that longer telomeres have more proteins involved with double strand break repair mechanisms associated with them (for example Ku proteins). Telomerized cells The present invention also provides telomerized cells, as well as differentiated embodiments of telomerized cells. Telomerized cells include any cell treated in accordance with the methods of the present invention, hi general, telomerized cells are cells in which the length of a telomere has been increased. Such cells usually have an increased number of possible population doublings. The telomerized cells of the present invention can be genetically modified to produce proteins or to correct for genetic defects, hi addition, the telomerized cells of the present invention may be continuously passaged and may survive cryopreservation. The telomerized cells of the present invention also may be used to form engineered tissues for implantation in a human.

The preferred telomere lengths for a given cell will vary depending on, among other things, the specific cell type. In certain embodiments, for example, mesenchymal-like progenitor cells may have telomeres of in the range of between about 15 kb and about 22 kb. Methods of measuring telomere length are known in the art and include, for example, Q- FISH, Flow-FISH, and Southern blots. The G-overhang portion of a telomere can be measured using a telomere-oligonucleotide ligation assay (T-OLA).

One characteristic of senescent cells is genomic instability. And genomic instability is often found when cells are expanded in culture. Cells treated with the methods of the present invention, however, may have less genomic instability because they avoid the senescent pathway. Thus, when telomerized cells of the present invention are cultured, a selection against non-telomerized cells may occur.

The telomerized cells of the present invention may be cultured using methods known in the art, for example, as described in METHODS IN MOLECULAR MEDICINE: HUMAN CELL CULTURE PROTOCOLS (G. E. Jones, ed.), Humana Press Inc., Totowa, NJ. (1996). Culturing telomerized cells may be useful, among other things, increase the number of telomerized cells to a therapeutically sufficient quantity. For example, in the case of certain mesenchymal-like progenitor cells, an average culture population of 300,000 cells will proliferate over a 7-10 day period to, on average, approximately 85 million cells (with ranges of between 25 million and 200 million cells depending on culture conditions and cell donor), with telomeres averaging greater than 15 kb.

In certain embodiments, the telomerized cells of the present invention may be treated to promote differentiation. Such treatments are known in the art and typically involve adding certain differentiation factors to the culture. For example, to promote osteogenic differentiation, a media including DMEM, 10% FBS, dexamethasone, ascorbate-2-phosphate, and δetβ-glycerophosphate may be used for about two to about four weeks.

The telomerized cells of the present invention may be expanded in culture to produce large quantities of cells to be implanted or injected into a subject. In certain embodiments, a telomerized progenitor cell may be used to form a tissue specific autologous progenitor cell. The ability to culture and expand large numbers of telomerized cells may be useful in therapeutic applications because large numbers of cells may be produced for implantation without the risk of immune system mediated rejection. So telomerized cells may be used treat diseases of cellular degeneration or to form ex vivo engineered tissues.

In one embodiment, telomerized progenitor cells that have been differentiated to varying degrees may be injected directly into damaged tissue, such as damaged skin, heart, or joints, hi another embodiment, in telomerized progenitor cells (in some cases differentiated) may be seeded onto bioengineered materials for the purpose of generating organs or tissue replacements. In certain embodiments, the telomerized progenitor cells of the present invention may be differentiated and used for the replacement or repair of damaged cells or tissues for the treatment of, for example, bone fractures, spinal cord injury, liver and heart injury, diabetes, multiple sclerosis, degenerative diseases such as stroke, and Parkinson's disease. Differentiated embodiments of telomerized progenitor cells also may be used as replacement cells to treat injuries which require tissue grafts, such as for, cartilage repair and skin grafts.

The telomerized cells of the present invention can be used in any research or medical application where any human adult progenitor cells, embryonic progenitor cells, or any other multipotent, or lineage precursor cells can be used. hi certain embodiments, the telomerized cells of the present invention may be genetically modified to express one or more specific genes-of-interest or to disrupt the expression of specific genes. The phrase "genetically modified" means any modification or alteration in the sequence of any portion of the entire genomic sequence of a cell, including the mitochondrial and nuclear genome, and further including the addition of ectopic nucleic acids to the cell as in a plasmid or artificial chromosome or portion thereof. Exogenous DNA may be transferred to telomerized cells by electroporation, lipofection, viral or microbial vectors or other means commonly known in the art. Such genetically modified cells may be used in bioreactors to produce pharmaceutical products, or in cell therapy treatments for genetic diseases such as hemophilia, Sickle Cell anemia, at the like.

The present invention also provides pharmaceutical products that comprise a telomerized progenitor cell, or a cell derived from a telomerized progenitor cell, transformed with a gene-of-interest, in which the gene-of-interest encodes a useful gene product. The pharmaceutical products of the present invention also may comprise the transformed cell or a product (e.g., a protein) harvested from such a transformed cell. Treatments

By controlling both telomerase activation and regulation of telomere- associated proteins, the telomere can be elongated to a desired length, thereby 'rejuvenating' progenitor cells. The ability to regulate telomerase 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; telomerized cell therapy may be useful in replacing age-worn cells in the retina of the eye or in treating the lining of blood vessels to help prevent heart attack or stroke. In addition, a gene or combination of genes may be site-specifically integrated into a cell's genome, and then expanded to a number sufficient to be used for therapeutic purposes.

Therefore, the present invention is well adapted to attain the ends and advantages mentioned as well as those that are inherent therein. While numerous changes may be made by those skilled in the art, such changes are encompassed within the spirit of this invention as defined by the appended claims.

EXAMPLES

Example 1

Within mesenchymal progenitor cells, endogenous transient activation of hTERT via shRNA targeting of Menin, followed 24 hours later by transient downregulation via shRNA targeting PINXl and TRFl for about 20 days. Media is treated with several factors (such as Xanthine) to prevent asymmetric cell division, and increase cell division.

Example 2

Within hematopoietic progenitor cells (CD34+), exogenous regulated expression of hTERT by site specific integration into the AAVSl region of chromosome 19 utilizing a Ad-

AAV hybrid vector of the present invention, followed 48 hours later by transient downregulation via siRNA targeting POTl for about 14 days. Media is treated with several factors (such as Xanthine) to prevent asymmetric cell division, and increase cell division.

Example 3 Within osteoblast progenitor cells, exogenous regulated expression of modified hTERT via integration into artificial chromosome (using Chromos ACE system), followed 36 hours later by transient downregulation via shRNA targeting TIN2 and PINXl for about 18 days. Media is treated with several factors (such as Xanthine) to prevent asymmetric cell division, and increase cell division. Example 4

Within dermal fibroblasts, activation of hTERT via treatment of cells with HIF-I under hypoxia conditions (3% oxygen culturing over treatment period), followed 48 hours later by regulated transient downregulation of POTl by small molecule that binds to OB fold of POTl protein for about 14 days. Media is treated with several factors (such as Xanthine) to prevent asymmetric cell division, and increase cell division.

Example 5 Hematopoietic progenitor cells from Example 2 also receive the globin gene into the

AAVSl site on Chromosome 19 prior to expansion to treat patients with sickle cell anemia.

Example 6

Osteoblast progenitor cells from Example 3 are used to seed a femur scaffold specifically designed for a patient. The cells are cultured, treated for strengthening and implanted into patient.

Example 7

Measurement of telomere length in Example 1 utilizing Flow-FISH shows telomere length gradually increasing in length over transient transfection to an optimized telomere length for mesenchymal-like progenitor cells (obtained from Cambrex) increasing capability by on average 24 additional cell divisions. Therefore for each mesenchymal-like progenitor cell harvested from patient, approximately 16.5 million additional mesenchymal progenitor cells could be created. The G-overhang portion of the telomere is measured by T-OLA. This is in contrast to the measurement of the full length of the telomere via Flow-FISH or Southrn blot. Measurement of both is important in this characterization of the telomere. Example 8

Analysis of chromosome karyotype and specific genes of osteoblast progenitor cells from Example 2 using CGH, metaphase spreads and microarray (gene specific) chips both before and after 60 population doublings show no changes, and therefore genomic stability during osteoblast expansion. Example 9

Analysis of mesenchymal-like progenitor cells (described as bone-marrow-derived cells obtained from Tulane Center for Gene Therapy) from Example 1 via flow cytometry showed gene expression profiles both before and after expansion as CD45^low,D7- FIB⁺,LNGFR⁺. Therefore, the present invention is well adapted to attain the ends and advantages mentioned as well as those that are inherent therein. While numerous changes may be made by those skilled in the art, such changes are encompassed within the spirit of this invention as defined by the appended claims.

Claims

What is claimed is:

1. A method of elongating a telomere of a cell, the method comprising: enhancing hTERT expression in the cell; and increasing telomerase processivity.

2. The method of claim 1 , wherein hTERT expression is transient.

3. The method of claim 1, wherein enhancing hTERT expression comprises at least one endogenous method chosen from creating hypoxic conditions, upregulating hypoxia inducible factor, and treatment with histone deacetylator inhibitors.

4. The method of claim 1, wherein enhancing hTERT expression comprises one or more transiently downregulating and transiently inactivating at least one a gene chosen from a responsible for repressing hTERT expression and a gene that is associated with telomerase processivity.

5. The method of claim 1 , wherein enhancing hTERT expression comprises one or more transiently downregulating and transiently inactivating at least one a gene chosen from menl, madl, britλ, sipl, nfxl, and rak.

6. The method of claim 1, wherein enhancing hTERT expression comprises modulating a gene responsible for repressing hTERT expression or a gene that is associated with telomerase processivity, or both.

7. The method of claim 1 , wherein enhancing hTERT expression comprises at least one exogenous method chosen from integration of at least a portion of hTERT into a chromosome of the cell.

8. The method of claim 1, wherein the cell is autologous or allogenic.

9. The method of claim 1 , wherein the cell is a progenitor cell.

10. The method of claim 1, wherein the cell is at least one progenitor cell chosen from a cell of mesenchyme origin, a mesenchymal-like cell, a nonhematopoietic cell, a keratinocyte, a neural cell, a hematopoietic cell, a cell of epithelial origin, and a cell of endothelial origin.

11. The method of claim 1 , wherein hTERT expression is enhanced to a level sufficient to lengthen the telomere of the cell or to maintain the length of the telomere of the cell.

12. The method of claim 1, wherein increasing telomerase processivity comprises transiently modulating the function of a telomere-associated gene.

13. The method of claim 1 , wherein increasing telomerase processivity comprises transiently modulating the function of a telomere-associated gene, wherein the telomere- associated gene affects the formation of a telomerase complex or affects the ability of a telomerase complex to access the telomere.

14. The method of claim 1, wherein increasing telomerase processivity comprises transiently modulating the function of at least one telomere-associated gene that encodes a protein chosen from a gene TRFl, TRF2, POTl, tankyrase, RAPl, TIN2, and PTOP.

15. A method of treating a disease, the method comprising: obtaining a cell from a donor enhancing TERT expression in the cell; increasing telomerase processivity; and administering the cell to a recipient.

16. The method of claim 15, further comprising introducing a gene into the cell.

17. The method of claim 15, wherein the donor and the recipient are the same.

18. The method of claim 15, wherein the disease is a disease of cellular degeneration.

19. The method of claim 15, wherein the disease is chosen from macular degeneration, osteoporosis, diabetes, multiple sclerosis, myocardial infarct, a burn, a bone break, a neurodegenerative disease, a stroke, Alzheimer's disease Parkinson's disease, liver injury, and cancer.

20. The method of claim 15, wherein hTERT expression is transient.

21. The method of claim 15, wherein enhancing hTERT expression comprises at least one endogenous method chosen from creating hypoxic conditions, upregulating hypoxia inducible factor, and treatment with histone deacetylator inhibitors.

22. The method of claim 15, wherein enhancing hTERT expression comprises one or more transiently downregulating and transiently inactivating at least one a gene chosen from a responsible for repressing hTERT expression and a gene that is associated with telomerase processivity.

23. The method of claim 15, wherein enhancing hTERT expression comprises modulating a gene responsible for repressing hTERT expression or a gene that is associated with telomerase processivity, or both.

24. The method of claim 15, wherein enhancing hTERT expression comprises at least one exogenous method chosen from integration of at least a portion of hTERT into a chromosome of the cell.

25. The method of claim 15, wherein the cell is autologous or allogenic.

26. The method of claim 15, wherein the cell is a progenitor cell.

27. The method of claim 15, wherein hTERT expression is enhanced to a level sufficient to lengthen the telomere of the cell or to maintain the length of the telomere of the cell.

28. The method of claim 15, wherein increasing telomerase processivity comprises transiently modulating the function of a telomere-associated gene.

29. A method of site-specifically integrating an oligonucleotide into the genome of a cell, the method comprising: introducing the oligonucleotide into a bacteriophage vector, the bacteriophage vector comprising: a pINT integrase gene; and a modified attB site, wherein the modified attB site increases the specificity of . the bacteriophage vector to the attachment site; introducing the bacteriophage vector into a serine bacteriophage to form a serine bacteriophage with the bacteriophage vector; infecting the cell with the serine bacteriophage with the bacteriophage vector; and allowing the oligonucleotide to integrate into the cell's genome.

30. A method of site-specifically integrating an oligonucleotide into the genome of a cell, the method comprising: introducing the oligonucleotide into a hybrid adeno-AAV viral vector, the hybrid adeno-AAV viral vector comprising: a p5 integration efficiency element from an adeno virus; and a packaging element from an AAV virus; infecting the cell with the hybrid adeno-AAV viral vector; and allowing the oligonucleotide to integrate into the cell's genome.

31. A telomerized cell which comprises a cell having a telomere, the telomere being elongated by enhancing hTERT expression in the cell and by increasing the processivity of telomerase in the cell.

32. The telomerized cell of claim 31, wherein the cell has an increased proliferative capacity.

33. The telomerized cell of claim 31, wherein the average length of the telomere is increased.

34. The telomerized cell of claim 31 , wherein the cell is a progenitor cell.

35. The telomerized cell of claim 31, wherein the cell is autologous or allogenic.

36. The telomerized cell of claim 31, wherein the cell is at least one progenitor cell chosen from a cell of mesenchyme origin, a mesenchymal-like cell, a nonhematopoietic cell, a keratinocyte, a neural cell, a hematopoietic cell, a cell of epithelial origin, and a cell of endothelial origin.

37. The telomerized cell of claim 31 , wherein hTERT expression is enhanced to a level sufficient to lengthen the telomere of the cell or to maintain the length of the telomere of the cell.

38. The telomerized cell of claim 31 , wherein hTERT expression is transient.

39. The method of claim 31 , wherein enhancing hTERT expression comprises at least one endogenous method and one exogenous method.

40. The telomerized cell of claim 31 , wherein increasing telomerase processivity comprises transiently modulating the function of a telomere-associated gene.

41. The telomerized cell of claim 31 , wherein increasing telomerase processivity comprises transiently modulating the function of at least one telomere-associated gene that encodes a protein chosen from a gene TRFl, TRF2, POTl, tankyrase, RAPl, TIN2, and PTOP.

42. A bacteriophage vector comprising: a pINT integrase gene; and a modified attB site, wherein the modified attB site increases the specificity of the bacteriophage vector to the attachment site.

43. A hybrid adeno-AAV virus vector comprising: a p5 integration efficiency element from an adeno virus; and a packaging element from an AAV virus.