INHIBITION OF C-RAF EXPRESSION BY ANTISENSE OLIGONUCLEOTIDES EXTENDS HEART ALLOGRAFT SURVIVAL IN RATS1

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Abstract
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.