Inhibition of C-raf expression by antisense oligonucleotides extends heart allograft survival in rats

• Published in: Transplantation Vol. 70; no. 4; pp. 656 - 661
• Main Authors: STEPKOWSKI, S. M, XEIMEI QU, WANG, M.-E, LING TIAN, WENHAO CHEN, WANCEWICZ, E. V, JOHNSTON, J. F, BENNETT, C. F, MONIA, B. P
• Format: Journal Article
• Language: English
• Published: Hagerstown, MD Lippincott 27-08-2000
• Subjects: Animals; Base Sequence; Biological and medical sciences; C-raf protein; Cell Line; Gene Expression Regulation - drug effects; Graft Survival - drug effects; Graft Survival - genetics; Heart Transplantation - immunology; Heart Transplantation - physiology; Humans; Medical sciences; Mice; Oligodeoxyribonucleotides, Antisense - pharmacology; oligonucleotides; Proto-Oncogene Proteins c-raf - genetics; Proto-Oncogenes; Rats; Rats, Inbred ACI; Rats, Inbred Lew; Surgery (general aspects). Transplantations, organ and tissue grafts. Graft diseases; Surgery of the heart; Thionucleotides; Transplantation, Homologous; Heart; Prognosis; Rat; Enzyme; Transferases; Rodentia; Transplantation; Gene expression; Homotransplantation; Antisense oligonucleotide; In vitro; Survival; In vivo; Vertebrata; Mammalia; Protein kinase; Surgery; Animal; Inhibition
• ISSN: 0041-1337; 1534-6080
• DOI: 10.1097/00007890-200008270-00020

C-raf is a well-characterized serine/ threonine (Ser/Thr) protein kinase that is involved in the transduction of multiple signals of T cells. We demonstrate that the inhibition of C-raf mRNA expression prolongs heart allograft survival.
Three 20-mer C-raf antisense oligonucleotides, each with identical sequences, were synthesized with different chemical modifications: one as a uniform phosphorothioate oligodeoxynucleotide (PS oligo), a second with a PS backbone and 2'-methoxyethyl (ME) substitutions at the 2'-sugar positions in the first and last five nucleotides, and a third with a mixed PS and phosphodiester (PD) backbone and ME modifications on the first and last five nucleotides.
Both ME-modified C-raf antisense oligos were at least 5-fold more effective than the PS C-raf antisense oligo in blocking C-raf mRNA expression in two cell lines. Similarly, each of the ME C-raf antisense oligos produced better heart allograft survival rates than did PS C-raf oligo. Furthermore, although the combination of PS C-raf antisense oligo with sirolimus (SRL) acted synergistically to extend heart allograft survival, the effect was potentiated by either of the ME-modified oligos.
C-raf inhibition extends heart allograft survival, and ME-modification potentiates antisense activity.