Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1996-05-06
2002-02-19
Priebe, Scott D. (Department: 1632)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S320100, C536S023100
Reexamination Certificate
active
06348353
ABSTRACT:
FIELD 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, however, 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.
Third, yeast chromosomes are less condensed than mammalian chromosomes. This implies that mammalian chromosomes rely on more complex chromatin interactions in order to achieve this higher level of structure. The complex structure (both DNA structure and higher order chromatin structure) of mammalian chromosomes calls into question whether artificial chromosomes can be created in mammalian cells.
Fourth, yeast centromeres are far less complex than mammalian centromeres. In
S. cerevisiae
, for example, the centromere is made up of a 125 bp sequence. In
S. pombe
, the centromere consists of approximately 2 to 3 copies of a 14 kb sequence element and an inverted repeat separated by a core region (~7 kb). In contrast, human centromeres are made up of several hundred kilobases to several megabases of highly repetitive alpha satellite DNA. Furthermore, in mammalian centromeres, there is no evidence for a central core region or inverted repeats such as those found in
S. pombe
. Thus, unlike yeast centromeres, mammalian centromeres are extremely large and repetitive.
Fifth, yeast centromeres have far fewer spindle attachments than mammalian centromeres (Bloom,
Cell
73:621-624 (1993)).
S. cerevisiae
, for example, has a single microtubule attached to the centromere. In
S. Pombe
, there are 2-4 microtubules attached per centromere. In humans, on the other hand, there are several dozen microtubules attached to the centromere of each chromosome (Bloom,
Cell
73:621-624 (1993)). This further illustrates the complexity of mammalian centromeres compared to yeast centromeres.
Together, these differences are significant, and do not suggest that a result in yeast can be reasonably expected to be transferable to mammals.
Normal mammalian chromosomes are comprised of a continuous linear strand of DNA ranging in size from approximately 50 to 250 megabases. In order for these genetic units to be faithfully replicated and segregated at each cell division, it is believed that they must contain at least three types of functional elements: telomeres, origins of replication, and centromeres.
Telomeres in mammals are composed of the repeating sequence (TTAGGG)
n
and are thought to be necessary for replication and stabilization of the chromosome ends. Origins of replication are necessary for the efficient and controlled replication of the chromosome DNA during S phase of the cell cycle. Although mammalian origins of replication have not been well-characterized at the sequence level, it is believed that they are relatively abundant in mammalian DNA. Finally, centromeres are necessary for the segregation of individual chromatids to the two daughter cells during mitosis to ensure that each daughter cell receives one, and only one, copy of each chromosome. Like origins of replication, centromeres have not been defined at the sequence level. Alpha satellite DNA may be an important centromeric component (Haaf et al.,
Cell
70:681-696 (1992); Larin et al.,
Hum. Mol. Genet
. 3:689-695 (1994); Willard,
Trends in Genet
. 6:410-415 (1990)). But there are cases of mitotically stable abnormal chromosome derivatives that apparently lack alpha satellite DNA (Callen et al.,
Am. J. Med. Genet
. 43:709-715 (1992); Crolla et al.,
J. Med. Genet
. 29:699-703 (1992); Voullaire et al.,
Am. J. Hum. Genet
. 52:1153-1163 (1993); Blennow et al.,
Am. J. Hum. Genet
. 54:877-853 (1994); Ohashi et al.,
Am. J. Hum. Genet
. 55:1202-1208 (1994)). Thus, at this time, the composition of the mammalian centromere remains poorly understood.
While others have claimed to have produced “artificial” chromosomes in mammalian cells, no one has ever produced an artificial chromosome that contains only exogenous DNA. In each of these previous cases, the investigators either modified an existing chromosome to make it smaller (the “pare-down” approach) or they integrated exogenous DNA into an existing chromosome which then broke to produce a chromosome fragment containing endogenous sequences from the preexisting chromosome (the “fragmentation” approach). In the present invention, exogenous DNA sequences are introduced into human cells and form stable synthetic chromosomes without integration into endogenous chromosomes.
Among the pare-down approaches, three specific strategies have been used: (1) telomere directed truncation via illegitimate recombination (Barnett, M. A. et al.,
Nucleic Acids Res
. 21:27-36 (1993); Farr, C. J. et al.,
EMBO J
. 14:5444-54 (1995)) (2) alpha satellite targeted telomere insertion/truncation via homologous recombination (Brown, K. E. et al.,
Hum Mol. Genet
. 3:1227-37 (1994)) (3) formation/breakage of dicentric chromosomes (Hadlaczlky, G., Mammalian Artificial Chromosomes, U.S. Pat. No. 5,288,625 (1994)).
Barnett et al. (
Nucleic Acids Res
. 21:27-36 (1993)), Farr et al. (
EMBO J
. 14:5444-54 (1995)), and Brown et al. (
Hum Mol. Genet
. 3:1227-37 (1994)) describe methods for fragmenting endogenous chromosomes by transfecting telomeric DNA and a selectable marker into mammalian cells. In each case, a truncated chromosome was created that was smaller than the original chromosome. The resulting truncated chromosom
Harrington John J.
Van Bokkelen Gil B.
Willard Huntington F.
Case Western Reserve University
Priebe Scott D.
Sterne Kessler Goldstein & Fox P.L.L.C.
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