Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Method of regulating cell metabolism or physiology
Reexamination Certificate
2000-07-24
2004-01-27
Lacourciere, Karen A. (Department: 1635)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Method of regulating cell metabolism or physiology
C435S006120, C435S091100, C435S325000, C536S023100, C536S024500, C536S024300, C536S024310, C536S024330
Reexamination Certificate
active
06682930
ABSTRACT:
TECHNICAL SCOPE
This invention involves a new kind of triplex forming oligonucleotide (TFO), particularly a new triplex forming oligonucleotide and their derivatives that form triplex DNA with two similar homopoly purine and pyrimidine fragments. This invention also involves the application of TFO in inhibiting the hepatitis B virus.
TECHNICAL BACKGROUND
In the 1980s, it was found that in vivo double stranded DNA (dsDNA) formed by homopolypurine/homopolypyrimidine sequences can fold over, and one of the strands can then form triplex DNA with the 5′-upstream homopolypurine/homopolypyrimidine sequence of dsDNA. The other strand then becomes single stranded DNA (ssDNA). Furthermore, the triplex DNA can become involved in the regulation of gene expression (Lyamichev V I, Mirkin S M, et al., J Biomol Struct Dyn, 1986, 3: 667-9; Larsen A, Weintraub H et al., Cell, 1982, 29: 609-22; Htun H, Dahlberg J E, Science, 1989, 243: 1571-76). Triplex DNA can be also be formed by the in vitro recognition of oligonucleotides with specific target DNA. A strand of homopolypurine/homopolypyrimidine oligonucleotides can specifically recognise a complementary sequence of homopolypurines/homopolypyrimidines in dsDNA by base pair complementation, and can form a stable triplex DNA structure by linking with purine chains in dsDNA via Hoogsteen bonds or anti-Hoogsteen bonds. The oligonucleotide fragment that forms triplex DNA with dsDNA is named Triplex Forming Oligonucleotide (TFO). The formation of triplex DNA can inhibit the binding of proteins to DNA. TFO carrying a terminal EDTA-Fe
2+
to bind a specific target sequence can form triplex DNA, resulting in the targeted dsDNA being cleaved at that specific position in the triplex (Moser H. E., Dervan P. B., Science, 1987, 238: 645-50). Therefore, it is possible to inhibit gene expression at the DNA level by the formation of triplex DNA, which gives rise to the so-called Antigene Strategy.
This technology is superior to anti-sense and nuclease technologies. TFO uses a specific sequence of dsDNA as a binding site to form triplex DNA or to cleave targeted DNA at a specific location in the triplex DNA. This inhibits transcription or gene replication. Anti-sense nucleic acids and nuclease enzymes both target mRNA. They cause the inhibition of gene expression by binding with mRNA to stop translation, or by promoting the degradation of mRNA. In cells, a single copy of DNA can produce multiple copies of mRNA. Therefore, it may be more efficient at the DNA level to block gene replication or transcription. DNA replication or RNA transcription can be inhibited by two strategies based on the theory of triplex DNA formation and the rules of base pair complementarity. One strategy is to use a TFO fragment based on a promoter of a particular gene, so that it forms triplex DNA by binding to the complementary sequence of the targeted gene, thereby blocking the binding of protein to DNA. In the second strategy, a TFO fragment is designed based on a specific portion of a gene, with which it forms triplex DNA by binding to the complementary sequence of the targeted gene. This inhibits DNA replication or RNA transcription by blocking the movement of the replication-transcription complex. In 1988, Cooney and coworkers proved that the molecular-triplex DNA structure formed at the starting point of c-myc inhibits the transcription of c-myc (Cooney M., Science, 1988, 241:450-9). However, up until now, there have been very few practical applications of TFO in the inhibition of gene expression. In a review of international patents, only one such patent has been found, and this involved the application of TFO to inhibit the expression of androgen receptor gene (U.S Pat. No. 5,556,956, Sep. 17, 1996). The main reason for this lack of applications is that it is very rare to find in promoters or other particular regions of genes homopolypurine/homopolypyrimidine sequences which are long enough. The triplex DNA formed by relatively short TFOs and target dsDNA is not particularly stable or specific, therefore their ability to act as inhibitors is weak, which limits their practical application in the Antigene Strategy. There have only been a few theoretical studies on the range of target dsDNA that can form triplex DNA, but no practical applications have resulted. Horne and Dervan et al designed an alternating triplex DNA containing two fragments of homopolypurine sequences located on the two chains of dsDNA, whereby a part of the TFO sequence matches a purine chain in the double strand, and the other part of the TFO matches the other purine chain in the double strand (Home D A, Dervan P B, J. Am. Chem. Soc., 1990; 112: 2435-37).
The stability of triplex DNA has also been studied. It was found that triplex DNA could still be formed even though it contains a single mismatched base, but its stability is markedly decreased. Therefore, problems are still to be resolved in finding new TFO structures that can be used to inhibit the expression of harmful genes. There have also been studies on increasing the stability and inhibitory effect of TFO by chemical modification, including thiophosphate modification (Tu, et al., J. Biol. Chem., 1995, 270: 28402-7), a single amino acid linked to the 3′-end (Orsen F. M., et al. Nucleic Acids Res. 1991, 19: 3435-41; Postel E. H., et al. Proc. Natl. Acad. Sci. USA 1991, 88: 8827-31; McShan W. M., et al. J. Biol. Chem. 1992, 267: 5712-21), cholesterol linked to the 3′-end (Ing N. H. et al., Nucleic Acids Res., 1993, 21: 2789-96) and so on. Although the stability and inhibitory capability of TFO can be increased by chemical modification, the length of TFO is still the key factor in TFO stability.
HBV is a hepatic DNA virus that can cause acute and chronic hepatitis. Eighty percent of patients with Hepatocellular Carcinoma (HCC) have HBV infection. The relative risk of HCC in the population with chronic HBV infection increases at least 100 fold. China is a high epidemic region for hepatitis B. Eight to 10% (100 million) of the population are positive to Hepatitis B (virus) Surface Antigen (HBsAg). Hepatitis B caused by HBV and the associated HCC are one of the major health issues in the world. However, as of the present, there is still no effective therapeutic regimen in the clinic. Hence the development of TFO against HBV as a new therapeutic approach is of contemporary interest. However, as the HBV genes do not contain homopolypurine/homopolypyrimidine sequences which are long enough to be a practicable target for TFO, it is worthwhile to search for a new triplex DNA structure.
PURPOSE OF THE INVENTION
The aim of the invention is to provide a type of TFO which is able to form a triplex DNA structure with two fragments of homologous homopolypurine/homopolypyrimidine. This TFO can inhibit the expression of HBV genes and the replication of the virus. It includes two types of TFO. One can bind to two fragments of homologous homopolypurine/homopolypyrimidine in the DR region of HBV. The other binds to two fragments of homologous homopolypurine/homopolypyrimidine in the promoter region of the pre-S gene of HBV adr subtype. Their stability can also be increased by 3′-monophosphorylation or other chemical modifications. These TFOs can be used as therapeutic agents in the treatment of hepatitis B.
DETAILS
The invention provides a type of TFO which is able to form a triplex DNA structure with two fragments of homologous homopolypurine/homopolypyrimidine. The invention is based on the mechanism of triplex DNA formation and the sequences in HBV genes. It is aimed at two fragments of homologous homopolypurine/homopolypyrimidine sequences in the DR region of HBV and the promoter region of pre-S gene of HBV adr subtype. The invention involves the corresponding TFOs, as well as the synthesis on a DNA synthesiser of these TFOs and 3′-monophosphorylated TFO derivatives (see FIG.
1
and FIG.
2
):
B1 (SEQ ID NO:1)5′
AAG GAG GAG GAT GGA GG 3′ 17nt
Lacourciere Karen A.
Shanghai Institute of Biochemistry, Chinese Academy of Sciences
LandOfFree
Triplex forming oligonucleotides and their use in anti-HBV does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Triplex forming oligonucleotides and their use in anti-HBV, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Triplex forming oligonucleotides and their use in anti-HBV will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3185870