Research And Progress Of Antisense Technology For Cancer Therapy

The common character of the mutational cells is the appearance of aberrant genes which are related with the cell division and differentiation according the current molecularbiology. In order to replace the genetic defect or to inhibit the expression of aberrant gene ,a natural gene can be transplanted into the target cell and get to prevent and treat the carcinoma. The principle of antisense therapy is transferring the sequence-specific binding of an antisense oligonucleotide to target DNA or mRNA, and selectively silencing of function and expression of disease-relevant genes to resulte in the prevention of the disease. Being with high specificity and low toxicity, antisense therapy has been paid more emphasis in cancerous therapeutics and relevant researches have developed greatly. With the continuous improved and perfected, antisense therapy has come into the clinical trial and shown a promising future. 1 principle Uncontrolled growth , the characteristic of tumor histology consist of excess proliferation and undifferentiation with embryonic characters. Thus cellular cycle can be modulated through inhibiting the function of genes in cellular cycle or enhancing the expression of gene which can induce the cellular differentiation and eventually mutational cells can be stimulated to differentiate toward terminal phase. Antisense oligonucleotides sequence-specifically hybridize to the target mRNA through Watson-Crick base pairing and, therefore, inhibit the expression of the gene and proliferation of the cells and stimulate the apoptosis of the cells to prevent the cancer. The inhibiting mechanism of the antisense oligonucleotides on gene expression is as followed. (1) Inhibition of the gene transcription antisense oligonucleotides can bind to specific sequence of double-strand DNA and form triple-helical DNA to inhibit the transcription.(2) Hindrance of the process on RNA, itis mainly to block the cleavage of pre-mRNA.? Inhibition the translation of mRNA a specific oligonucleotides sequence hybridize with the cytoplasmic target mRNA sequence can resulte in the inhibition of translation by interrupting ribosome activity. ?Induction of ribonuclease H (RNAse H) this mechanism of action, maybe dominating mechanism, is mediated by the induction of ribonuclease H that cleaves the mRNA at the site of ODN binding. RNAse H is a ubiquitous enzyme which catalyses the hydrolysis of the RNA strand in a DNA/RNA heteroduplex. The target mRNA sequence is inactivated as an irreversible event since the antisense DNA may degrade several mRNA molecules, through separate hybridizations, resulting in the dissociation of cleaved products. Therefore expression of protein of relevant gene is restrained.2 category and mechanism of antisense technology Antisense technology has included antisense DNA, antisense RNA and ribozyme. Antisense DNA is a specific-hybridized DNA stretch targeting special sequence of a certain gene, the most common used stretch is antisense oligonucleotides(ODN) are stretches of chemically modified DNA usually no more than 30 nucleotides in length. Antisense oligonucleotides can sequence-specifically hybridize to the target RNA and form DNA-RNA hybrid molechuar. This kind of molecular can further shape an triplex with double-strand DNA which will affect the duplication, transcription and translation. Antisense RNA can interfere with gene function through the hybridization with their specific mRNA, hindrance on its translation and acceleration of its degradation. Ribozyme are a series of mRNAs which has the catalysis of enzyme. They can cleave mRNA in specific point and make the mRNA unfunctional after binding target mRNA. In generally, antisense technology has inhibited expression of target gene through the mechanism as followed: (1)inhibition on the duplication of DNA, (2)inhibition on the transcription of DNA, (3)inhibition on transporation across the nuclear membrance of RNA, (4)inhibition on the cleavage of the pre-mRNA, (5) inhibition on the formation of translating compounds, (6)breakage of mRNA.3 design for antisense sequence From the principle of antisense therapy, it is known that the design of antisense oligonucleotides sequence is vital for the effect ofantisense therapy. So the design should be fit for the conditions as followed: (D antisense oligonucleotides sequence that is designed is merely complementary to target gene or mRNA and is heterosequence refered to other genes. It is commonly believe that any sequences of bases that are more than 13bp in mRNA or more than 17bp in DNA will appear for only once in human genome. Thus the specificity can be ensured if the antisense oligonucleotides sequence is more than 17bp. (D base composition of effective target site should be selected in target gene or mRNA. Then antisense oligonucleotides sequence could be designed according the base sequence on target site. According the theory,the site of initial code AUG of mRNA slide into ribosome during translation initiation, and the mRNA on this site is single-strand which is favourable to the hybridization on antisense oligonucleotides, so this site should be the effective target of antisense oligonucleotides. Several technique are adopted to select the target site. Conventionally, a linear 'shot-gun' approach has been used to select antisense reagents. Evidence that ribonuclease H (RNase H) is involved in antisense-mediated effects has led to the development of several procedures that make use of this enzyme to identify accessible sites. Furthermore secondary structure of target RNA is predicted using RNA folding programs by computer. Despite influence of disturbing on antisense efficiency is not considered, this method can rushly decrease the number of antisense oligonucleotides used in high quantity detection.4 progress of antisense therapy in clinic The genesis of malignant tumor is an accumulation of a series mutants including activation of oncogene, defect of cancer inhibitor and other molecular events. At present the strategies to treat tumor for antisense drugs include blockage of oncogene or inhibition of the signal cycle of antocrine growth factors, inhibiting the metastasis of tumor .activation of immunoresponse and reversing drug-resistance. And Clinical trials with antisense therapeutics include: (D Bcl-2 family, (2)Raf kinase, ?p53, ?Protein kinase C-a, ?Ha-ras, ?Protein kinase A.5 future and prospect More and more antisense oligonucleotides have shown comparative security and specific inhibition in clinical trial. Compared to othermethods, still a distance to usage in clinic, antisense therapy have a better future because clinical trial have proved that combination antisense therapy with traditional drugs is an effective treatment to cancer. Meanwhile antisense technology is proved to be a quite way to overcome the resistance to chemotherapy and radiotherapy. Accompanying the progress of genomics, proteomics and biotechnologic research, antisense technology can get breakthrough and make a big step in application.