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

Background. 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. Methods. 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. Results. 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. Conclusions. C-raf inhibition extends heart allograft survival, and ME-modification potentiates antisense activity. The T cell receptor (TCR) complex is involved in the initiation of signal transduction from the T cell membrane to the nucleus (1). After the engagement of TCR ab chains by alloantigen, the TCR signal is transduced by the CD3 gdeezz chains in association with the Fyn, Lck, and ZAP70 kinase network, leading to the initiation of the Ras/Raf/Mek mitogen-activated protein (MAP) kinase cascade (2‐ 4). In addition to TCR signaling, full activation of T cells requires a second membrane signal delivered through the interaction of CD28 molecules (expressed on T cells) with B7‐1/B7‐2 ligands that are present on antigen presenting cells (APC) (5). The CD28/B7 signal transduction pathway utilizes the MAP kinase pathway to stabilize and increase the production of cytokines via the CD28 response element present in the