title: Identification and functional validation of PNAs that inhibit murine CD40 expression by redirection of splicing.
abstract: Cognate recognition between the CD40 receptor and its ligand, CD154, is thought to play a central role in the initiation and propagation of immune responses. We describe the specific down regulation of cell surface associated CD40 protein expression by use of a peptide nucleic acid (PNA) antisense inhibitor, ISIS 208529, that is designed to bind to the 3' end of the exon 6 splice junction within the primary CD40 transcript. Binding of ISIS 208529 was found to alter constitutive splicing, leading to the accumulation of a transcript lacking exon 6. The resulting protein product lacks the transmembrane domain. ISIS 208529-mediated CD40 protein depletion was found to be sequence specific and dose dependent, and was dependent on the length of the PNA oligomer. CD40-dependent induction of IL-12 in primary murine macrophages was attenuated in cells treated with ISIS 208529. Oligolysine conjugation to the PNA inhibitor produced an inhibitor, ISIS 278647, which maintained its specificity and displayed efficacy in BCL1 cells and in primary murine macrophages in the absence of delivery agents. These results demonstrate that PNA oligomers can be effective inhibitors of CD40 expression and hence may be useful as novel immuno-modulatory agents.
authors.affiliation: Department of Medicinal Chemistry, ISIS Pharmaceuticals, 2292 Faraday Avenue, Carlsbad, CA 92008, USA.
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Siwkowski
authors.forename: Leila
authors.initials: L
authors.lastname: Malik
authors.forename: Christine C
authors.initials: CC
authors.lastname: Esau
authors.forename: Martin A
authors.initials: MA
authors.lastname: Maier
authors.forename: Edward V
authors.initials: EV
authors.lastname: Wancewicz
authors.forename: Klaus
authors.initials: K
authors.lastname: Albertshofer
authors.forename: Brett P
authors.initials: BP
authors.lastname: Monia
authors.forename: C Frank
authors.initials: CF
authors.lastname: Bennett
authors.forename: Anne B
authors.initials: AB
authors.lastname: Eldrup
mesh_terms: D017398:Alternative Splicing / Q000187:drug effects* / Q000235:genetics; D000818:Animals; D019013:CD40 Antigens / Q000032:analysis / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation / Q000187:drug effects; D005091:Exons / Q000235:genetics; D005260:Female; D005434:Flow Cytometry; D018664:Interleukin-12 / Q000096:biosynthesis; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D020135:Peptide Nucleic Acids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D013997:Time Factors
chemical_list: D019013:CD40 Antigens; C486486:ISIS 208529; D020135:Peptide Nucleic Acids; D012333:RNA, Messenger; D018664:Interleukin-12