Study Of Treating Rheumatoid Arthritis With C-myc Antisense Oligodeoxynucleotides

IntroductionRheumatoid arthritis (RA) is a chronic polyarticular inflammatory disease that is usually characterized by symmetrical synovitis in which hyperplasia of synovial lining cells and pannus formation are presented. The effect of clinical treatment has not been satisfacted. Synovial hyperplasia in rheumatoid arthritis has been characterized as a local tumor - like proliferation. Its fibroblast - like synoviocytes (type B cells) and endothelial cells express c-myc. The expression of c-myc is an important mark of proliferating cells. The c-myc pro to - oncogene encodes a transcription factor that plays critical roles in controlling cell proliferation , differentiation and apoptosis. The c-myc proto - oncogene product acts as a master switch that either activates or represses transcription of several growth - and differentiation - related gene. The deregulated expression of c-myc for proliferation of cancer cells and maintenance of the malignant phenotype has been demonstrated. Therefore, the c-myc has been a potential therapeutic target.Antisense therapeutic strategy is one of rational approach to directly to target. Transcription and translation are the two processes that antisense therapeutic agents interfere with to inhibit the synthesis of protein. Antisense sequences are derivatives of nucleic acids that bind to ( hybridize) cytosolic mRNA sense strands via hydrogen bonds to complementary nucleic acid bases or hybridize double - stranded DNA via Hoogsteen hydrogen bonds in the cell nucleus to form local triple helix. Naturally occurring oligonucleotides are susceptive to degradation by nucleases in blood and cells. Therefore, oligonucleotides as agents must be modified to resistant to the catalytic activity of nucleases and to have adequate concentration in target site, and meantime no toxicity increased. Phospho-rothioate phosphodiester linkages are the most applied. Antisense oligonucleoti-des are big molecules, cell membranes are low permeability to them. Therefore, carrier systems have to be designed to improve the penetration of the agents into cells. Liposome is a well - understood carrier system. Its characteristics favors the use for delivering antisense oligonucleotides.An investigation has presented that c-myc antisense oligodeoxynucleotides (c-myc AS ODN) can induce RA synovial type B cells apoptosis. We have further investigations base on this study.ObjectivesFirst: we investigated the effect of phosphorothioate c-myc AS ODN inhibiting cultured human rheumatoid synoviocytes proliferation, and inducing syno-viocytes apoptosis; Second; we used two motifs of c-myc AS ODN to different sites of c-myc mRNA to investigate if there was difference on inhibiting synoviocytes proliferation for selecting a more effective c-myc AS ODN motif. Third: for understanding the effect of c-myc AS ODN on pannus formation in synovium, weinvestigated the effect of c-myc AS ODN on endothelial cell line__ECV304 cellsproliferation. We tried to prove the prime possibility that using c-myc AS ODN treats RA through above studies.MethodsSynovial cells were isolated from synovial fluid obtained from a knee joint cavity of a RA patient. Through passing several times, synovial type B cells were purified then cultured in DMEM medium. ECV304 cells were cultured in RPMI 1640 medium. Synthesized 2 sequences of c-myc AS OND and their corresponding sense oligodeoxynucleotides (S ODN) as antisense specificity negative control. The first AS ODN sequence which targets the initiation codon (AUG) and the next four codons of the c-myc mRNA is 5'-AAC GTT GAG GGG CAT-3'and the corresponding S ODN sequence is 5'-ATG CCC CTC AAC GTT - 3'; Another AS ODN sequence which targets a binding site called coding region determinant (CRD) is 5' - ATG TAT GOT GTG GCT-3' and its corre-spending S ODN sequence is 5'-AGC CAC AGC ATA CAT-3'. In the investigation to type B cells, all phosphodiester linkages were phosphorothioated, while in the investigation to ECV304 cells, only 3 phosphodiester linkages at the 3'and 5't...