Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2000-03-13
2004-04-06
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C424S093510, C530S387300, C530S388210, C536S023100
Reexamination Certificate
active
06716608
ABSTRACT:
TECHNICAL FIELD
The present invention relates to the field of genetic engineering, more specifically, it relates to an artificial mammalian chromosome prepared by genetic engineering techniques.
BACKGROUND ART
Various methods of treating diseases attributed to an inherited or an acquired genetic defect, namely a genetic disease, have been developed. Gene therapy is one such method for treating a genetic disorder fundamentally by replacing a defective gene with a normal gene or complementing a normal gene.
At present, the most clinically and fundamentally developed method of introducing a gene comprises linking a short DNA fragment like cDNA to a downstream site of an ectopic enhancer or promoter that originally does not exist upstream of the gene, introducing the resulting DNA fragment into a cell using a virus or liposome, and allowing the cell to express the gene. This method is easy to manipulate because a short DNA fragment is used. Furthermore, it has a relatively high success rate of introducing a gene into a cell. However, there are some disadvantages. First, it is difficult to control the expression of an introduced gene. In this method, the expression control of a desired gene in a human in vivo is difficult because the promoter and enhancer used are derived from viruses. Second, the existing pattern of an introduced gene in a cell is not stable. Untargeted genes may be destroyed or an excessive number of introduced genes may exist. This is because the introduced gene in a cell may be randomly integrated into a chromosomal DNA. Alternatively, the introduced gene may independently exist extrachromosomally and be maintained without being controlled by DNA synthesis during the S phase or chromosome separation during the M phase of a cell. This fact makes it difficult to control expression of a therapeutic gene in gene therapy and to exhibit therapeutic effects continuously.
For example, sickle cell anemia and thalassemia drew the most attention in the early 1980s as targets for gene therapy. In spite of numerous patients with these diseases, it is not being studied much at present because it is difficult to strictly control the expression of the therapeutic gene (a globin gene) to be introduced. Furthermore, since processing of a huge DNA molecule like a globin gene using restriction enzymes is limited, homologous recombination using yeast is more effective than recombination using an
E. coli
plasmid. A stable chromosome can thus be prepared in a yeast cell. If such a chromosome is capable of replicating in a human cell, it can be used for the most ideal gene therapy. In the field of gene therapy, it is essential to develop a vector system in which a human gene with an expression control region can function and be stably maintained in a human cell.
An “artificial chromosome” that is a yeast artificial chromosome (YAC) vector has been developed. A long-chain DNA molecule such as a gene ligated to a promoter region and/or an enhancer region can be introduced into this vector and stably maintained following the mechanisms of DNA replication and separation in yeast. A DNA fragment requires three functional structures, a centromere, a DNA replication origin, and a telomere, to function as a chromosome. Based on this fact, the YAC vector has been constructed to contain genes for these three functional structures.
However, the above yeast functional structures do not function in mammals, including humans. Therefore, the functional structures from a mammal or those modified to a mammalian type structure must be used to constitute an artificial chromosome that functions in a mammalian cell.
A centromere, a DNA replication origin, and a telomere of yeast each consist of a several kb DNA sequence whose functions have been well analyzed. In contrast, a centromere of a mammal, especially of a human, is a huge DNA molecule in which a repetitive sequence called the alphoid sequence repeats over several hundred kb or more. In addition, even the primary structure of a mammalian DNA replication origin has not been clarified. Thus, the analysis of functional structures in a mammal is far behind that in yeast, and an artificial mammalian chromosome has yet to be constructed.
Mammals, including humans, commonly have a 5′-TTAGGG-3′ sequence as a repetitive unit of a telomere sequence.
DISCLOSURE OF THE INVENTION
An objective of the invention is to provide an artificial mammalian type chromosome, more specifically, an artificial chromosome having a mammalian type telomere sequence added to both its ends. The current invention provides a yeast strain capable of having a mammalian type telomere sequence connected to both ends of its chromosome.
A chromosome requires minimum functional structures of three elements to function. The first is a DNA replication origin region that ensures a single DNA replication during the S phase following mitosis. The second is a centromere that ensures the correct separation of each set of replicated DNA into daughter cells. The third is a telomere that caps the ends of linear chromosomes to ensure their stable existence in a nucleus.
It is evident that a centromere sequence of a mammal, especially of a human, is a huge DNA molecule containing a specific DNA sequence called the repetitive alphoid sequence that repeats over a few hundred kilo base pairs in tandem (M. Ikeno, H. Masumoto & T. Okazaki, Hum Mol Genet 3: 1245-1257 (1994)). Such a huge DNA molecule cannot be manipulated by means of-conventional recombinant DNA techniques. Therefore, the present inventors employed a CEPH artificial yeast chromosome library containing a human genome inserted into a yeast YAC vector into which a long DNA molecule can be inserted. Clones containing the repetitive human alphoid sequence were identified from this library to serve as the basis of an artificial mammalian chromosome. The primary structure of a DNA replication origin in a human genome has not been identified, however, it is estimated that one DNA replication origin exists on average in each 100 kb of a human genome DNA (Molecular biology of cell, third edition, Kyoikusha). Therefore, a YAC clone containing the above-described repetitive alphoid sequence is thought to contain a DNA replication origin.
However, the YAC clone itself does not function as an artificial mammalian chromosome due to the lack of a telomere sequence. Thus, the present inventors engaged in searching for a method of adding a mammalian telomere sequence to the ends of DNA of the thus-obtained YAC clone.
Specifically, the present inventors engaged in investigating a method for adding a telomere sequence not in vitro but in vivo (in yeast), in other words, a method for preparing a yeast strain in which a mammalian telomere sequence can be added to a yeast genome, considering that a desired artificial chromosome is a long-chain DNA molecule and is easily damaged and physically decomposed by nonspecific enzymes.
A telomerase consists of a protein component and an RNA component as a template for extending the telomere sequence. The telomere sequence is added to a chromosome as a complementary sequence to the template RNA. The present inventors modified a template RNA encoding a yeast telomere sequence (TG
1-3
)
n
so as to encode a mammalian telomere sequence (TTAGGG)
n
by in vitro mutagenesis, cloned the modified template RNA in an expression plasmid, then introduced the plasmid into yeast. A hybrid telomerase (composed of a mammalian template RNA derived from the plasmid and protein derived from host yeast) was constructed in yeast. Thus, a yeast strain that replaces the yeast chromosomal telomere sequence with a mammalian telomere sequence was prepared. The mammalian telomere sequence was confirmed to be added to the yeast chromosome in the strain.
In summary, the present invention relates to:
(1) An artificial chromosome having a mammalian telomere sequence added to its ends.
(2) The artificial chromosome of (1), wherein said chromosome comprises an alphoid sequence.
(3) The artificial chromosome of (1) or (2),wherein said chrom
Hasegawa Mamoru
Ishikawa Fuyuki
Clark & Elbing LLP
DNAVEC Research Inc.
Prouty Rebecca E.
Walicka Malgorzata A
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