Inhibition of CD40-mediated endothelial cell activation with antisense oligonucleotides.
BACKGROUND
CD40-CD154 interactions play a pivotal role in the amplification of immune responses and, as such, represent an attractive target for immune intervention in a number of disease indications. We have previously shown that binding of human CD154 expressed on the Jurkat D1.1 cell line to porcine CD40 on pig aortic endothelial cells (PAECs) can lead to up-regulation of vascular cell adhesion molecule (VCAM)-1 and MHC class II. This activation can be completely inhibited by the addition of a monoclonal antibody (mAb) to human CD154. In this study, we explore an alternative approach to blocking this pathway with antisense oligonucleotides (ASOs).


METHODS
Ten ASOs were generated on the basis of the porcine CD40 cDNA sequence. The ASOs that were found to reduce CD40 expression on PAECs were analyzed for their ability to reduce CD40-mediated PAEC activation.


RESULTS
Four ASOs were found to significantly lower surface expression of porcine CD40 on PAECs 48 hr after transfection. Eight of the ASOs were seen to lead to mRNA cleavage products by ribonuclease protection assay. Of the four ASOs tested in the PAEC activation assay, one (ASO-9) showed a dramatic inhibition of PAEC activation (IC50 approximately 1 nM) results comparable to the use of a blocking mAb. Furthermore, we compared the effect of CD40 ASO on tumor necrosis factor alpha receptor signaling, in which we observed no effect, which confirmed ASO specificity.


CONCLUSIONS
These results indicate that a CD40-dependent activation pathway can be inhibited with an ASO with high potency and specificity. ASO could be an attractive alternative therapy to the use of mAbs.
Rushworth
S A
SA
Imutran Ltd, Cambridge, England.
Bravery
C A
CA
Hall
J
J
Natt
F
F
Parsons
N J
NJ
Weiler
J
J
Häner
R
R
Thompson
S
S
D000818:Animals; D001483:Base Sequence; D019013:CD40 Antigens / Q000235:genetics* / Q000378:metabolism; D023201:CD40 Ligand / Q000502:physiology; D018076:DNA, Complementary / Q000235:genetics; D004730:Endothelium, Vascular / Q000187:drug effects / Q000502:physiology*; D005786:Gene Expression Regulation; D006801:Humans; D019169:Jurkat Cells; D008969:Molecular Sequence Data; D020319:Oligodeoxyribonucleotides, Antisense / Q000138:chemical synthesis / Q000494:pharmacology*; D013552:Swine; D014162:Transfection; D019010:Vascular Cell Adhesion Molecule-1 / Q000235:genetics
D019013:CD40 Antigens; D018076:DNA, Complementary; D020319:Oligodeoxyribonucleotides, Antisense; D019010:Vascular Cell Adhesion Molecule-1; D023201:CD40 Ligand