A novel antisense strategy peptide nucleic acid-based to downregulate CD5 expression in chronic lymphocytic leukemia

Abstract

Chronic Lymphocytic Leukemia (CLL) is characterized by the overexpression of the transmembrane protein CD5 in B cells. To assess the downregulation of the protein and modulate the CLL aggressiveness we have focused on developing an antisense approach Peptide Nucleic Acid (PNA) based. Using bioinformatics tools, we selected a tract of 12 mer of the CD5 transcript and synthesized the corresponding DNA (DNA wild type) tract to be used as a mimetic target. Moreover, we also synthesized the complementary 12 mer PNA strand and the scrambled one, named respectively PNA and PNA scrambled. Both the PNA compounds were functionalized with two residues of Serine Phospate (SerP) at its C-terminus interspaced by two glycine (Gly) spacers to favor the transfection process, lipofectamine mediated, for subsequent in vitro experiments. To evaluate the ability of the PNA to selectively bind its target we performed physical-chemical characterizations of the PNA:DNA and PNA scrambled: DNA complexes. Circular Dichroism (CD), CD melting, TDS, and non-denaturing polyacrylamide gel electrophoresis (PAGE) analyses were performed. Each experiment confirmed that only the PNA and DNA wild type are specifically and selectively able to form a heteroduplex PNA: DNA in vitro, justifying further experiments to accomplish the antisense strategy in cells. To confirm the ability of the PNA to downregulate its complementary mRNA we transfected the Jurkat cell line and peripheral blood mononuclear cells from B-CLL patients with PNAs. Cytofluorimetric assays and real-time PCR analysis demonstrated the downregulation of CD5 expression due to the incubation with the anti-CD5 PNA for both cell lines.