Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1997-09-29
1998-11-24
Elliott, George C.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435375, 435325, 514 44, 536 231, 536 241, 536 245, 536 243, C12Q 168, C07H 2100, C12N 510
Patent
active
058404970
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a method for the silencing of specific genes by DNA methylation. The method involves the introduction into cells of oligonucleotides containing 5-methyl cytosine residues, the oligonucleotide having a sequence complementary to a portion of the DNA of the gene to be silenced.
It is known that the methylation of cytosine in DNA by the enzyme DNA methyl transferase results in the inactivation of gene expression in many biological contexts. Furthermore, once a cytosine residue is methylated in a CpG doublet, the enzyme recognises this substrate after DNA replication and methylates the new strand, whereas non-methylated CpG doublets remain unmethylated. Thus a given pattern of DNA methylation is heritable, and the methylated genes remain silent. Non-specific gene silencing in CHO cells has been achieved by uptake of 5-methyl deoxycytosine triphosphate (Holliday & Ho, 1991, Somatic Cell and Molecular Genetics, 17:537-542; Nyce, 1991, Somatic Cell and Molecular Genetics, 17:543-550). The new method developed by the present inventors can silence specific genes, provided the sequence of bases in the promoter region is known. The method developed by the present inventors involves the use of single stranded oligonucleotides containing 5-methyl deoxycytosine in appropriate CpG doublets.
Accordingly, in a first aspect the present invention consists in a method of silencing a specific gene in a cell comprising introducing into the cell a single stranded oligonucleotide containing 5-methyl deoxycytosine, the single stranded oligonucleotide having a sequence complementary to a portion of the DNA sequence of the gene to be silenced.
In a preferred embodiment of the present invention the single stranded oligonucleotide has a sequence complementary to a sequence within the promoter region of the gene to be silenced or within CpG islands of the gene to be silenced.
As will be readily appreciated provided some or all of the cells are actively synthesising DNA (S phase), the methylated oligonucleotide can anneal or hybridize to single stranded DNA at the replication fork. Accordingly, it is presently preferred that the cells are synchronized and treated during DNA synthesis. Synchrony can be achieved by serum starvation, followed by addition of serum which stimulates DNA synthesis, or other methods of synchronization.
As is clear from the above discussion the method of the present invention depends on the synthesis of single stranded oligonucleotides containing 5-methyl deoxycytosine in appropriate CpG doublets or at other sites. The synthesis is a standard procedure substituting 5-methyl deoxycytosine phosphoroamidate for cytosine phosphoroamidate (ABI systems oligonucleotide synthesiser). The target gene is unmethylated in the promoter region and the gene is, or can be, expressed in the cells to be treated. The oligonucleotide can be introduced into the cells by electroporation, Transfectam or by any other means of permealizing cells or by other methods of introducing DNA into cells. Provided some or all of the cells are actively synthesising DNA (S phase), the methylated oligonucleotide can anneal or hybridize to single stranded DNA at the replication fork. This creates a hemimethylated substrate for the DNA methyltranferase. The enzyme methylates the cellular DNA in the region of hybridization. This methylation imprint in a short region of the promoter silences the specific gene, and the effect is permanent because the methylation is subsequently inherited. In order that the target gene is specifically recognize the oligonucleotide is of sufficient length (20-40 bases) to hybridize only with this gene and no other in the genome. Alternatively, the oligonucleotide could hybridise to the gene during active transcription, because there is some unwinding of the DNA during this process. The end result is the same.
The cells can be primary human cells, or permanent lines of human or animal origin. The cells should be synchronized and treated during DNA synthesis. Synchrony can be achieved by serum
REFERENCES:
Rojanasakul, Antisense Oligonucleotide Therapeutics: Drug Delivery and Targeting. Advanced Drug Delivery Reviews, vol. 18 1996 pp. 115-131.
Robin Holliday et al, Gene Silencing in Mammalian Cells by Uptake of 5-Methyl Deoxycytidine-5'-triphosphate, Somatic Cell Molecular Genetics, vol. 17, No. 6, 1991, pp. 537-542.
Jonathan Nyce, Gene Silencing in Mammalian Cells by Direct Incorporation of Electroporated 5-Methyl-2'-deoxycytidine 5'-triphosphate, Somatic Cell Molecular Genetics, vol. 17, No. 6, 1991, pp. 543-550.
W. French Anderson, Human Gene Therapy, Science, vol. 256, pp. 808-813.
Fritz Eckstein et al, Phosphorothioates in Molecular Biology, TIBS 14, Mar. 1989, pp. 97-100.
Elliott George C.
Garry Sean M.
The Commonwealth of Australia Commonwealth Scientific and Indust
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