Multicellular living organisms and unmodified parts thereof and – Method of making a transgenic nonhuman animal – Via microinjection of dna into an embryo – egg cell – or...
Reexamination Certificate
1998-06-12
2004-06-01
Crouch, Deborah (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Method of making a transgenic nonhuman animal
Via microinjection of dna into an embryo, egg cell, or...
C800S021000, C435S325000, C435S455000
Reexamination Certificate
active
06743967
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods for preparing cell lines that contain artificial chromosomes, to methods for isolation of the artificial chromosomes, targeted insertion of heterologous DNA into the chromosomes, isolation of the chromosomes, and delivery of the chromosomes to selected cells and tissues. Also provided are cell lines for use in the methods, and cell lines and chromosomes produced by the methods.
BACKGROUND OF THE INVENTION
Several viral vectors, non-viral, and physical delivery systems for gene therapy have been developed [see, e.g., Mitani et al. (1993)
Trends Biotech.
11:162-166]. The presently available systems, however, have numerous limitations, particularly where persistent, stable, or controlled gene expression is required. These limitations include: (1) size limitations because there is a limit, generally on order of about ten kilobases [kB], at most, to the size of the DNA insert [gene] that can be accepted by viral vectors, whereas a number of mammalian genes of possible therapeutic importance are well above this limit, especially if all control elements are included; (2) the inability to specifically target integration so that random integration is required which carries a risk of disrupting vital genes or cancer suppressor genes; (3) the expression of randomly integrated therapeutic genes may be affected by the functional compartmentalization in the nucleus and are affected by chromatin-based position effects; (4) the copy number and consequently the expression of a given gene to be integrated into the genome cannot be controlled. Thus, improvements in gene delivery and stable expression systems are needed [see, e.g., Mulligan (1993)
Science
260:926-932].
In addition, safe and effective gene therapy methods and vectors should have numerous features that are not assured by the presently available systems. For example, a safe vector should not contain DNA elements that can promote unwanted changes by recombination or mutation in the host genetic material, should not have the potential to initiate deleterious effects in cells, tissues, or organisms carrying the vector, and should not interfere with genomic functions. In addition, it would be advantageous for the vector to be non-integrative, or designed for site-specific integration. Also, the copy number of therapeutic gene(s) carried by the vector should be controlled and stable, the vector should secure the independent and controlled function of the introduced gene(s); and the vector should accept large (up to Mb size) inserts and ensure the functional stability of the insert.
The limitations of existing gene delivery technologies, however, argue for the development of alternative vector systems suitable for transferring large [up to Mb size or larger] genes and gene complexes together with regulatory elements that will provide a safe, controlled, and persistent expression of the therapeutic genetic material.
At the present time, none of the available vectors fulfill these requirements. Some of these characteristics, however, are possessed by chromosomes. Thus, an artificial chromosome would be an ideal vector for gene therapy, as well as for production of gene products that require coordination of expression of numerous genes or that are encoded by large genes, and other uses. Artificial chromosomes for expression of heterologous genes in yeast are available, but construction of a mammalian artificial chromosome has not been achieved. Such construction has been hindered by the lack of an isolated, functional, mammalian centromere and uncertainty regarding the requisites for its production and stable replication. Unlike in yeast, there are no selectable genes in close proximity to a mammalian centromere, and the presence of long runs of highly repetitive pericentric heterochromatic DNA makes the isolation of a mammalian centromere using presently available methods, such as chromosome walking, virtually impossible. Other strategies are required for production of mammalian artificial chromosomes, and some have been developed. For example, U.S. Pat. No. 5,288,625 provides a cell line that contains an artificial chromosome, a minichromosome, that is about 20 to 30 megabases. Methods provided for isolation of these chromosomes, however, provide preparations of only about 10-20% purity. Thus, development of alternative artificial chromosomes and perfection of isolation methods as well as development of more versatile chromosomes and further characterization of the minichromosomes is required to realize the potential of this technology.
Therefore, it is an object herein to provide mammalian artificial chromosomes and methods for introduction of foreign DNA into such chromosomes. It is also an object herein to provide methods for introduction of the artificial mammalian chromosome into selected cells, and to provide the resulting cells, as well as transgenic animals and plants that contain the artificial chromosomes. It is also an object herein to provide methods for gene therapy and expression of gene products using artificial chromosomes. It is a further object herein to provide methods for constructing species-specific artificial chromosomes.
SUMMARY OF THE INVENTION
Mammalian artificial chromosomes [MACs] are provided. Also provided are artificial chromosomes for other higher eukaryotic species, such as insects and fish, produced using the MACS are provided herein. Methods for generating and isolating such chromosomes. Methods using the MACs to construct artificial chromosomes from other species, such as insect and fish species are also provided. The artificial chromosomes are fully functional stable chromosomes. Two types of artificial chromosomes are provided. One type, herein referred to as SATACs [satellite artificial chromosomes] are stable heterochromatic chromosomes, and the another type are minichromosomes based on amplification of euchromatin.
Artificial chromosomes permit targeted integration of megabase pair size DNA fragments that contain single or multiple genes. Thus methods using the MACs to introduce the genes into cells, animals and tissues are also provided. The artificial chromosomes with integrated heterologous DNA are to be used in methods of gene therapy, in methods of production of gene products, particularly products that require expression of multigene biosynthetic pathways, and also are intended for delivery into the nuclei of germane cells, such as embryo-derived stem cells [ES cells] for production of transgenic animals.
Mammalian artificial chromosomes provide extra-genomic specific integration sites for introduction of genes encoding proteins of interest and permit megabase size DNA integration so that, for example, genes encoding an entire metabolic pathway or a very large gene, such as the cystic fibrosis [CF; ~600 kb] gene, several genes, such as a series of antigens for preparation of a multivalent vaccine, can be stably introduced into a cell. Vectors for targeted introduction of such genes, including the tumor suppressor genes, such as p53, the cystic fibrosis transmembrane regulator gene [CFTR], anti-HIV ribozymes, such as an anti-HIV gag ribozyme, into the artificial chromosomes also provided.
The chromosomes provided herein are generated by introducing heterologous DNA that includes DNA encoding a selectable marker into cells, preferably a stable cell line, growing the cells under selective conditions, and identifying from among the resulting clones those that include chromosomes with more than one centromere or that have chromosomes that are fragments of chromosomes that had more than one centromere. The amplification that produces the additional centromere occurs in cells that contain chromosomes in which the heterologous DNA has integrated near the centromere in the pericentric region of the chromosome. The selected clonal cells are then used to generate artificial chromosomes.
In preferred embodiments, the DNA with the selectabl
Hadlaczky Gyula
Szalay Aladar A.
Chromos Molecular Systems Inc.
Crouch Deborah
Seidman Stephanie L.
Ton Thai-an N.
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