title: Gene silencing nucleic acids designed by scanning arrays: anti-EGFR activity of siRNA, ribozyme and DNA enzymes targeting a single hybridization-accessible region using the same delivery system.
abstract: Gene silencing nucleic acids such as ribozymes, DNA enzymes (DNAzymes), antisense oligonucleotides (ODNs), and small interfering (si)RNA rely on hybridization to accessible sites within target mRNA for activity. However, the accurate prediction of hybridization accessible sites within mRNAs for design of effective gene silencing reagents has been problematic. Here we have evaluated the use of scanning arrays for the effective design of ribozymes, DNAzymes and siRNA sequences targeting the epidermal growth factor receptor (EGFR) mRNA. All three gene silencing nucleic acids designed to be complementary to the same array-defined hybridization accessible-site within EGFR mRNA were effective in inhibiting the growth of EGFR over-expressing A431 cancer cells in a dose dependent manner when delivered using the cationic lipid (Lipofectin) delivery system. Effects on cell growth were correlated in all cases with concomitant dose-dependent reduction in EGFR protein expression. The control sequences did not markedly alter cell growth or EGFR expression. The ribozyme and DNAzyme exhibited similar potency in inhibiting cell growth with IC50 values of around 750 nM. In contrast, siRNA was significantly more potent with an IC50 of about 100 nM when delivered with Lipofectin. The potency of siRNA was further enhanced when Oligofectamine was used to further improve both the cellular uptake and subcellular distribution of fluorescently labelled siRNA. Our studies show that active siRNAs can be designed using hybridization accessibility profiles on scanning arrays and that siRNAs targeting the same array-designed hybridization accessible site in EGFR mRNA and delivered using the same delivery system are more potent than ribozymes and DNAzymes in inhibiting EGFR expression in A431 cells.
authors.affiliation: Centre for Genome-based Therapeutics, The Welsh School of Pharmacy, Cardiff University, Redwood Building, King Edward VII Avenue, Cardiff CF10 3XF, UK.
authors.forename: Gary
authors.initials: G
authors.lastname: Beale
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Hollins
authors.forename: Mustapha
authors.initials: M
authors.lastname: Benboubetra
authors.forename: Muhammad
authors.initials: M
authors.lastname: Sohail
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Fox
authors.forename: Ibrahim
authors.initials: I
authors.lastname: Benter
authors.forename: Saghir
authors.initials: S
authors.lastname: Akhtar
mesh_terms: D015153:Blotting, Western; D045744:Cell Line, Tumor; D004247:DNA / Q000096:biosynthesis*; D021881:DNA, Catalytic / Q000187:drug effects*; D016503:Drug Delivery Systems; D066246:ErbB Receptors / Q000037:antagonists & inhibitors*; D005434:Flow Cytometry; D020868:Gene Silencing / Q000187:drug effects*; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007553:Isotope Labeling; D008081:Liposomes; D009693:Nucleic Acid Hybridization; D009696:Nucleic Acids / Q000138:chemical synthesis / Q000494:pharmacology*; D020411:Oligonucleotide Array Sequence Analysis; D009841:Oligonucleotides / Q000138:chemical synthesis; D016337:RNA, Catalytic / Q000187:drug effects*; D034741:RNA, Small Interfering / Q000378:metabolism*; D014162:Transfection
chemical_list: D021881:DNA, Catalytic; D008081:Liposomes; D009696:Nucleic Acids; D009841:Oligonucleotides; D016337:RNA, Catalytic; D034741:RNA, Small Interfering; D004247:DNA; D066246:ErbB Receptors