title: Delivery of NADPH-cytochrome P450 reductase antisense oligos using avidin-biotin approach.
abstract: Although avidin-mediated intracellular delivery of oligonucleotides or proteins has been shown before, the efficacy studies are lacking. Here, we tested the effectiveness of avidin for delivery of a cytochrome P450 reductase (CPR) antisense oligo in rat liver epithelial cells. A phosphorodiamidate morpholino oligo (PMO) against CPR was biotinylated using four reagents with short, cleavable, or long linkers, followed by conjugation with avidin. The dose-inhibitory response of the unmodified PMO in the presence of a transfection reagent (Endoporter, EP) and the effectiveness of the EP-assisted and avidin-assisted delivery of biotinylated PMOs were tested by Western blot analysis. Additionally, in a preliminary study, the avidin-biotin PMO with a long linker was also tested in vivo in rats. The biotinylated oligos were at least as effective as the unmodified oligo. Whereas the avidin conjugate of biotinylated PMO with the short linker was ineffective, those with the long linkers showed significant reductions in CPR protein expression. Finally, the in vivo study showed modest, but significant, reductions in CPR activity. In conclusion, these studies show for the first time that avidin-mediated intracellular delivery of biotinylated oligos can effectively knock down target genes in vitro, depending on the length of the linker. Additionally, the avidin-biotin approach may be of potential value for in vivo gene knockdown.
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University Health Sciences Center, 1300 Coulter, Amarillo, Texas 79106, USA.
authors.forename: Venkateswaran C
authors.initials: VC
authors.lastname: Pillai
authors.forename: Rekha
authors.initials: R
authors.lastname: Yesudas
authors.forename: Imam H
authors.initials: IH
authors.lastname: Shaik
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Thekkumkara
authors.forename: Ulrich
authors.initials: U
authors.lastname: Bickel
authors.forename: Kalkunte S
authors.initials: KS
authors.lastname: Srivenugopal
authors.forename: Reza
authors.initials: R
authors.lastname: Mehvar
mesh_terms: D000818:Animals; D001360:Avidin / Q000737:chemistry* / Q000378:metabolism; D001710:Biotin / Q000737:chemistry* / Q000378:metabolism; D019921:Biotinylation; D002460:Cell Line; D004337:Drug Carriers / Q000737:chemistry* / Q000378:metabolism*; D008099:Liver / Q000166:cytology / Q000378:metabolism; D008297:Male; D009025:Morpholines / Q000737:chemistry* / Q000378:metabolism*; D060172:Morpholinos; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D004337:Drug Carriers; D009025:Morpholines; D060172:Morpholinos; D012333:RNA, Messenger; D001360:Avidin; D001710:Biotin; D009251:NADPH-Ferrihemoprotein Reductase