INHIBITION OF CD4 EXPRESSION BY ANTISENSE OLIGONUCLEOTIDES IN PMA-TREATED LYMPHOCYTES

M Rabanal 1 , A. Franch1, V. Noé 2 , C. J. Ciudad 2 , M. Castell 1 and C. Castellote 1

Antisense & Nucleic Acid Drug Development 12, 399-410 (2002)

1Department of Physiology &endash; Division IV; Faculty of Pharmacy; University of Barcelona, Spain

2 Department of Biochemistry and Molecular Biology &endash; Division IV; Faculty of Pharmacy;

University of Barcelona, Spain

To decrease CD4 expression in T helper lymphocyte surface, antisense oligonucleotides (AS-ODNs), delivered by the cationic liposome DOTAP, were assayed in vitro on rat spleen lymphocytes. Four 21-mer ODNs (AS-CD4-1, -2, -3 and -4) directed against the translation start region of the cd4 gene were designed. AS-CD4-1 was phosphorothioate (PTO)-modified in each base, and the other three were PTO-modified at both ends and in the internal pyrimidine residues. Four ODN controls (fully PTO-modified ODN-A and partially modified ODN-B, -C and -D) were also assayed. CD4 re-synthesis was stimulated by treatment with phorbol 12-myristate 13-acetate (PMA) at the same time as the incubations with the ODN. After 24 h oftreatment, CD4 expression was measured by immunofluorescence staining and flow cytometry. CD4 reexpression in rat PMA-treated lymphocytes was counteracted by 40% by means of AS-CD4-2 and AS-CD4-4 treatments. On the other hand, AS-CD4-3 produced only 20% inhibition, similar to that produced by ODN-B, and AS-CD4-1 did not have any significant effect compared with control ODNs. Both AS-CD4-2 and AS-CD4-4 decreased CD4 mRNA as determined byRT-PCR, and in addition, they did not affect the expression of other surface lymphocyte molecules. Inhibition of surface CD4 expression remained at least 72 hours. The addition of both AS-ODNs did not further increase the effect obtained separately by each AS-ODN. Treatment of rat PMA-lymphocytes with two concentrations of AS-CD4-2 and AS-CD4-4 added 24 h apart did not further decrease CD4 expression. In summary, AS-CD4-2 and AS-CD4-4 could constitute a good strategy to inhibit CD4 expression on T helper lymphocytes and modulate their function.