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Homologous recombination in multipotent adult progenitor cellsRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Process Of Mutation, Cell Fusion, Or Genetic Modification, Introduction Of A Polynucleotide Molecule Into Or Rearrangement Of Nucleic Acid Within An Animal CellHomologous recombination in multipotent adult progenitor cells description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20060228798, Homologous recombination in multipotent adult progenitor cells. Brief Patent Description - Full Patent Description - Patent Application Claims
RELATED APPLICATIONS [0001] This application claims the benefit of priority to U.S. application Ser. No. 60/429,631, filed on Nov. 27, 2002. This application makes reference to International Application No. PCT/US00/21387, filed Aug. 4, 2000 and to International Application No. PCT/US02/04652, filed Feb. 14, 2002, both of which are hereby incorporated by reference. Each document cited or referenced in each of the foregoing applications, and any manufacturer's instructions or catalogues for any products cited or mentioned in each of the foregoing applications and in any of the cited documents, are hereby incorporated herein by reference. Furthermore, all documents cited in this text, and all documents cited or referenced in documents cited in this text, and any manufacturer's instructions or catalogues for any products cited or mentioned in this text or in any document incorporated into this text, are incorporated herein by reference. Documents incorporated by reference into this text or any teachings therein can be used in the practice of this invention. Documents incorporated by reference into this text are not admitted to be prior art. FIELD OF THE INVENTION [0002] The present invention relates generally to alteration of gene expression in mammalian multipotent adult progenitor cells (MAPCs) [previously referred to as multipotent adult stem cells (MASC)], and more specifically to methods for making homologously recombined MAPCs. The invention further relates to the therapeutic treatment of disease using homologous recombination and gene targeting techniques in MAPCs. BACKGROUND OF THE INVENTION [0003] The alteration of gene expression, by upregulating, downregulating, knocking-in or knocking-out gene products, can be accomplished using gene targeting approaches, such as homologous recombination. Gene targeting approaches are an alternative for both in vitro and in vivo production of proteins. In vitro expression of desired proteins has multiple uses, from production of therapeutic drugs to generating nutrients to providing drug and disease screening and research tools. An aspect of gene targeting involves gene therapy, which has been advanced as a treatment for medical conditions that require alteration of the level of protein production in a cell, whether ex vivo or in vivo, and, if necessary, the delivery of such protein(s) to other cells and tissues. [0004] An aspect of gene targeting ex vivo involves transfection and transplantation of recombinant autologous or allogeneic cells. As one example, stem cells provide promise for improving the results of such cell-based gene targeting. Stem cells can be genetically altered in vitro, then reintroduced in vivo to produce a desired gene product. [0005] The quintessential stem cell is the embryonic stem (ES) cell, as it has unlimited self-renewal and multipotent differentiation potential (Thomson et al., 1995; Thomson et al., 1998; Shamblott et al., 1998; Williams et al., 1988; Orkin, 1998; Reubinoff et al., 2000). These cells are derived from the inner cell mass of the blastocyst (Thomson et al., 1995; Thomson et al., 1998; Martin, 1981), or can be derived from the primordial germ cells from a post-implantation embryo (embryonal germ cells or EG cells). ES and EG cells have been derived from mouse, and more recently also from non-human primates and humans. When introduced into mouse blastocysts, ES cells have been shown to contribute to tissues from all three germ layers of the mouse (Orkin 1998). ES cells are therefore pluripotent. [0006] Stem cells that are not isolated from an embryo are simply referred to as stem cells or sometimes tissue-specific stem cells or adult stem cells. Stem cells have been identified in most organs and tissues. A well-characterized stem cell is the hematopoietic stem cell ("HSC"). This mesoderm-derived cell has been purified based on cell surface markers and functional characteristics. The HSC, isolated from bone marrow ("BM"), blood, cord blood, fetal liver and yolk sac, is the progenitor cell that generates blood cells, or following translation, reinitiates multiple hematopoietic lineages. HSCs can reinitiate hematopoiesis for the life of a recipient. (See Fei et al., U.S. Pat. No. 5,635,387; McGlave et al., U.S. Pat. No. 5,460,964; Simmons et al., U.S. Pat. No. 5,677,136; Tsukamoto et al., U.S. Pat. No.5,750,397; Schwartz et al., U.S. Pat. No. 759,793; DiGuisto et al., U.S. Pat. No.5,681,599; Tsukamoto et al., U.S. Pat. No. 5,716,827; Hill et al., 1996.) Stem cells which differentiate only to form cells of the hematopoietic lineage, however, are unable to provide a source of cells for repair of other damaged tissues, for example, heart or lung tissue damaged by high-dose chemotherapeutic agents. They are also limited in their use in cell-based therapy to the correction of defects that affect only cells of the hematopoietic lineage. Similarly, their use in in vitro and/or ex vivo protein production is limited to proteins normally expressed in cells of hematopoietic lineage. [0007] A second adult stem cell that has been studied extensively is the neural stem cell ("NSC") (Gage, 2000; Svendsen et al., 1999; Okabe et al., 1996). Several studies in rodents, and non-human primates and humans have shown that stem cells continue to be present in adult brain. These stem cells can proliferate in vivo and continuously regenerate at least some neuronal cells in vivo. When cultured ex vivo, NSCs can be induced to differentiate into different types of neurons and glial cells. Clarke et al. (2000) reported that NSCs from Lac-Z transgenic mice were able to contribute, not only to tissues of the central nervous system, but also to mesodermal derivatives and epithelial cells of the liver and intestine. They Were not found in other tissues, including the hematopoietic system. These studies therefore suggested that adult NSCs may have significantly greater differentiation potential than previously realized, but still do not have the pluripotent capability of ES cells or of the adult derived multipotent adult stem cells (MASCs), also known as MAPCs, described in Furcht et al. (International Application Nos. PCT/US00/21387 and PCT/US02/04652) and herein. [0008] A third tissue specific cell with stem cell properties is the mesenchymal stem cell ("MSC"), initially described by Fridenshtein (1982). MSC, originally derived from the embryonal mesoderm, can be isolated from adult bone marrow ("BM") and can differentiate to form muscle, bone, cartilage, fat, marrow stroma, and tendon. A number of MSCs have been isolated. (See, for example, Caplan et al., U.S. Pat. No. 5,486,359; Young et al., U.S. Pat. No. 5,827,735; Caplan et al., U.S. Pat. No. 5,811,094; Bruder et al., U.S. Pat. No. 5,736,396; Caplan et al., U.S. Pat. No. 5,837,539; Masinovsky, U.S. Pat. No. 5,837,670; Pittenger, U.S. Pat. No. 5,827,740; Jaiswal et al., 1997; Cassiede et al., 1996; Johnstone et al., 1998; Yoo et al., 1998; Gronthos, 1994). [0009] Of the many MSCs that have been described, most have demonstrated limited differentiation potential, only forming cells generally considered to be of mesenchymal origin. One of the most multipotent MSC reported is the cell isolated by Pittenger et al., which is capable of differentiating to form a number of cell types of mesenchymal origin (Pittenger et al., 1999). [0010] Other tissue-specific stem cells have been identified, including gastrointestinal stem cells (Potten 1998), epidermal stem cells (Watt, 1997), and hepatic stem cells, also termed oval cells (Alison et al., 1998). [0011] MAPCs are distinct from these other types of stem cells. They can be culture-isolated from multiple sources, such as bone marrow, blood, muscle, brain, skin, fat, umbilical cord and placenta, and have the same morphology, phenotype, in vitro differentiation ability and a highly similar expressed gene profile as ES cells. (See, for example, Reyes and Verfaillie, 2001; Reyes et al., 2001; Jiang et al., 2002a; Jiang et al., 2002b.) MAPCs constitutively expresses oct4 and high levels of telomerase and are negative for CD44, MHC class I and MHC class II expression. One benefit of MAPCs, in terms of therapeutic applications, is that no teratomas are formed in vivo. Furthermore, MAPCs contribute to multiple organs upon transplantation. [0012] Most presently available approaches of gene delivery make use of infectious vectors, such as retroviral vectors, which include the genetic material to be expressed. These approaches have limitations, such as the potential of generating replication-competent virus during vector production; recombination between the therapeutic virus and endogenous retroviral genomes, potentially generating infectious agents with novel cell specificities, host ranges, or increased virulence and cytotoxicity; limited cloning capacity in the retrovirus (which, inter alia, restricts therapeutic applicability) and short-lived in vivo expression of the product of interest. [0013] Further, in most gene delivery systems, it is not possible to direct or target the donor DNA (i.e., the DNA being delivered to the cell, such as therapeutic DNA) to a preselected site in the genome. In fact, in the widely used retrovirus-mediated gene delivery system, retroviruses integrate randomly into independent chromosomal sites in millions to billions of cells. This mixture of infected cells is problematic in two senses: first, since integration site plays a role in the function of the donor DNA, each cell has a different level of function and, second, since the integration of donor DNA into the genome can trigger undesired events, such as the generation of tumorigenic cells, the likelihood of such events is dramatically increased when millions to billions of independent integrations occur. [0014] The problems of populations consisting of large numbers of independent integrants might be avoided in two ways. First, a single cell with a random integration site can be propagated until sufficient numbers of the cloned cell are available for further use. The cells that make up this clonal population would all function identically. While this is theoretically possible, success rates for creating a clonal population from a single cell can be low and the number of passages required to amass a usable number of transfected cells can be deleterious. Alternatively, gene targeting can be used, wherein the donor DNA is introduced into a population of cells such that the DNA sequence integrates into a preselected site in the genome. In this case, all the cells function identically and the risk of a deleterious integration event is eliminated. [0015] A number of approaches to gene targeting have been described including chimeroplasty (Bandyopadhyay et al., 1999), triple helix formation (Casey et al., 2001) and short-fragment homologous recombination (Goncz, et al., 2001), all of which may increase the rate of gene targeting. A preferred method of gene targeting by homologous recombination is that of Treco et al., described, for example, in the U.S. Pat. Nos. 6,270,989 and 5,641,670 or in Selden et al., U.S. Pat. No. 6,303,379. [0016] Another alternative approach, based on AAV-mediated gene transfer and targeting, has been described (Inoue et al., 1999; Hirata et al., 2000; Hirata et al., 2002). AAV is a dependent parvovirus with a single-stranded linear DNA genome, from which vectors can be made by replacing the viral genes with foreign DNA between the cis-acting inverted terminal repeats. AAV vectors genetically alter cells by chromosomal integration of the vector genome at the site-specific integration locus of wild-type AAV located on human chromosome 19 (Carter et al., 2000; Inoue et al., 1999; Hirata et al., 2000; Hirata et al., 2002). The gene targeting rates produced by AAV vectors approach 1% at the single-copy HPRT locus in normal human cells, 3 to 4 logs higher than can typically be achieved in human cells with conventional gene targeting methods. [0017] The use of gene targeting has been proposed in ES cell, germ cell and somatic cell systems. Germ cell gene targeting refers to the modification of sperm cells, egg cells, zygotes, or early stage embryos. From a practical standpoint, and due to ethical concerns, germ cell gene targeting is inappropriate for human use. ES cell gene targeting is also controversial, and the availability of ES cells for these purposes is severely limited. In somatic cell gene therapy, targeting somatic cells (e.g., fibroblasts, hepatocytes, or endothelial cells) are removed from a donor organism, cultured in vitro, transfected with the gene(s) of interest, characterized, and used for a desired purpose. However, the practical use of somatic cells is limited to conditions that affect only one cell type. Therefore, for example, an altered somatic cell cannot be induced to differentiate into cells of various tissue types. In addition, somatic cells are generally limited in their potential to propagate in vitro. Hence, an approach that overcomes the drawbacks and limitations of the currently available methods and provides safe, efficacious, long-term protein production and delivery would be valuable. [0018] Historically, transfer (or introduction) of exogenous DNA into stem cells has been challenging, with most of the known transfection methods giving sub-optimal transfer rates. To achieve efficient gene targeting, a good transfer rate is necessary. Achieving high rates of transfer in stem cells has been hindered by the fact that optimal transfection occurs when cells are cultured at high density, while lower cell densities are required to maintain stem cells in an undifferentiated state. In order to effectively use genetically altered stem cells in protein expression, including therapeutic applications, a method is needed that will result in optimal transfer efficiency, for example, under conditions that support undifferentiated stem cells. Moreover, such methods would be ideally suited for use with adult stem cells having pluripotent capacity, such as MAPCs. SUMMARY OF THE INVENTION [0019] The ability to repair, alter, replace, delete or express desired nucleotide sequences in the genome of MAPCs would expand the potential usefulness of MAPCs in the in vitro, ex vivo, or in vivo expression of a gene of interest in order to arrive at a multitude of desired outcomes, such as nutritional and/or therapeutic protein production, non-protein gene expression (e.g. generation, up/down regulation, or knock-out/-in of ribozymes), as well as treatment of disease. Use of gene targeting in MAPCs can provide methods for altering gene expression (i.e., by increasing or decreasing the production of a gene product) not only to produce a novel or enhanced gene product, but also for investigation of gene expression patterns and/or gene function. [0020] The invention provides a method of altering a first gene expression pattern in an isolated multipotent adult progenitor cell (MAPC), the method comprising: [0021] a) introducing into a MAPC an exogenous polynucleotide molecule, wherein the polynucleotide molecule comprises i) a targeting nucleotide sequence and ii) a donor nucleotide sequence; and [0022] b) culturing the MAPC under conditions sufficient to homologously recombine the exogenous polynucleotide molecule, such that a resultant MAPC has a second expression pattern; wherein the introducing and culturing are done simultaneously or consecutively. Continue reading about Homologous recombination in multipotent adult progenitor cells... 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