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Show moreField of the invention: The invention relates to the field of gene expression and gene therapy, and to novel vectors for these uses. In particular, the invention relates to the development and use of an artificial or synthetic chromosome as a vector for gene expression and gene therapy, especially in humans. The invention enables the controlled construction of stable synthetic or artificial chromosomes from isolated purified DNA. With this DNA, a functional chromosome is formed in a cell and maintained as an extrachromosomal element. The artificial chromosome performs the essential chromosomal functions of naturally-occurring chromosomes so as to permit the chromosome to function as an effective vector for gene therapy when therapeutic DNA is included in the chromosome. Background of the invention: The genetic manipulation of cells aimed at correcting inherited or acquired disease is referred to as gene therapy. Until now, most clinical studies in this field have focused on the use of viral gene therapy vectors. Based on the results of these studies, it is becoming clear that current viral gene therapy vectors have severe clinical limitations. These include immunogenicity, cytopathicity, inconsistent gene expression, and limitations on the size of the therapeutic gene. For these reasons, much attention has been recently focused on the use of non-viral gene therapy vectors. In particular, synthetic mammalian chromosomes would be useful vectors for facilitating a variety of genetic manipulations to living cells. The advantages of synthetic mammalian chromosomes include high mitotic stability, consistent and regulated gene expression, high cloning capacity, and non-immunogenicity. Artificial chromosomes were first constructed in S. cerevisiae in 1983 (Murray et al., Nature 305:189-193 (1983), and in S. pombe in 1989 (Hahnenberger et al., Proc. Natl. Acad Sci. USA 86:577-581 (1989). For many reasons, it has not been obvious whether similar vectors could be made in mammalian cells.First, multicellular organisms (and thus the progenitors of mammalian cells) diverged from yeast over 1 billion years ago. Although there are similarities among living organisms, in general, the similarities among two organisms are inversely related to the extent of their evolutionary divergence. Clearly, yeast, a unicellular organism, is radically different biologically from a complex multicellular vertebrate. Second, yeast chromosomes are several orders of magnitude smaller than mammalian chromosomes. In S. cerevisiae and S. pombe, the chromosomes are 0.2 to 2 megabases and 3.5-5.5 megabases in length, respectively. In contrast, mammalian chromosomes range in size from approximately 50 megabases to 250 megabases. Since there is a significant difference in size, it is not clear, a priori, whether constructs comparable to yeast artificial chromosomes can be constructed and transfected into mammalian cells.
http://www.google.com/patents?vid=USPAT6348353
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Show moreField of the invention: This invention relates to the field of gene therapy and gene therapy vector technology. It relates to the development and use of synthetic human chromosomes, or synthetic chromosomes in related primates or other mammals. It generally relates to the development and use of large arrays of repetitive DNA. Background of the invention: The ability to clone large, repetitive DNA is an important step toward the development and construction of artificial chromosomes and gene therapy vehicles. In addition, stable cloning of repetitive DNA in microorganisms will be important for generating high resolution physical maps of mammalian chromosomes. A variety of cloning systems have been developed to facilitate the cloning and propagation of foreign DNA in micro-organisms. Plasmids, bacteriophage, and yeast artificial chromosomes (YACs) have been used successfully to clone many mammalian DNA sequences. However, some types of repetitive DNA appear to be unstable in these vectors (Schalkwyk et al., Curr. Opin. Biotechnol. 6(1):37-43 (1995); Brutlag, D. et al., Cell 10:509-519 (1977)). This results in gaps in physical genomic maps and precludes the use of these vectors as a means of propagating repetitive DNA, and especially highly repetitive mammalian centromeric DNA. Bacterial Artificial Chromosomes (BACs) have been constructed to allow the cloning of large DNA fragments in E. coli (O'Conner et al., Science 244 (4910):1307-12 (1989); Shizuya et al., Proc. Natl. Acad. Sci. USA 89 (18):8794-7 (1992); Hosoda et al., Nucleic Acids Res. 18(13):3863-9 (1990)). While this system appears to be capable of stably propagating mammalian DNA up to at least 300 kb, relatively few independent mammalian DNA fragments have been analyzed (Shizuya et al., Proc. Natl. Acad. Sci. USA 89 (18):8794-7 (1992)). In addition, the few fragments that have been tested for stability in the BAC vector, have not been extensively characterized with respect to the types of sequences present in each fragment. Thus, it is unknown whether these fragments contain repetitive DNA elements. In particular, it is clear, based on the restriction site and Southern analysis, that these fragments do not contain alpha satellite DNA. Many mammalian DNA sequences appear stable in Yeast Artificial Chromosome (YAC) vectors, and yet certain repetitive elements of similar length are not (Neil et al., Nucleic Acids Res. 18(6):1421-8 (1990)). Knowledge of DNA properties derived from the YAC system thus suggests that large arrays of repeating units are inherently unstable, even under conditions where similar sized DNA composed of non-repeating DNA is stable. Thus, the stability of large (greater than 20-100 kb) arrays of repeating units, such as alpha satellite DNA in a BAC vector, prior to the present invention, was not predictable with any reasonable certainty.
http://www.google.com/patents?vid=USPAT5869294
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Show moreField of the invention: This invention relates to the field of gene therapy and gene therapy vector technology. It also relates to the development and practical use of synthetic human chromosomes, or synthetic chromosomes in related primates or other mammals. Background of the invention: The ability to clone large, highly repetitive DNA is an important step toward the development and construction of a human artificial microchromosome, and gene therapy vehicles. In addition, stable cloning of repetitive DNA in microorganisms will be important for generating high resolution physical maps of mammalian chromosomes. A variety of cloning systems have been developed to facilitate the cloning and propagation of foreign DNA in micro-organisms. Plasmids, bacteriophage, and yeast artificial chromosomes (YACs) have been used successfully to clone many mammalian DNA sequences. However, some types of repetitive DNA appear to be unstable in these vectors (Schalkwyk et al., Curr. Opin. Biotechnol. 6(1): 37-43 (1995); Brutlag, D. et al., Cell 10: 509-519 (1977)). This results in gaps in physical genomic maps and precludes the use of these vectors as a means of propagating highly repetitive mammalian centromeric DNA. Bacterial Artificial Chromosomes (BACs) have been constructed to allow the cloning of large DNA fragments in E. coli (O'Conner et al., Science 244 (4910): 1307-12 (1989); Shizuya et al., Proc. Natl. Acad. Sci. USA 89 (18): 8794-7 (1992); Hosoda et al., Nucleic Acids Res. 18(13): 3863-9 (1990)). While this system appears to be capable of stably propagating mammalian DNA up to at least 300 kb, relatively few independent mammalian DNA fragments have been analyzed (Shizuya et al., Proc. Natl. Acad. Sci. USA 89 (18): 8794-7 (1992)). In addition, the few fragments that have been tested for stability in the BAC vector, have not been extensively characterized with respect to the types of sequences present in each fragment. Thus, it is unknown whether these fragments contain repetitive DNA elements. In particular, it is clear, based on the restriction site and Southern analysis, that these fragments do not contain alpha satellite DNA. Many mammalian DNA sequences appear stable in Yeast Artificial Chromosome (YAC) vectors, and yet certain repetitive elements of similar length are not (Neil et al., Nucleic Acids Res. 18(6): 1421-8 (1990)). Knowledge of DNA properties derived from the YAC system thus suggests that large arrays of repeating units are inherently unstable, even under conditions where similar sized DNA composed of non-repeating DNA is stable. Thus, the stability of large (greater than 100 kb) arrays of repeating units such as is found in alpha satellite DNA in the BAC vector cannot be predictable with any reasonable certainty.
http://www.google.com/patents?vid=USPAT5695967
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