Nucleic Acids Research Advance Access published online on September 16, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn541
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Delivery of steric block morpholino oligomers by (R-X-R)4 peptides: structure–activity studies
1UMR 5235 CNRS, Université Montpellier 2, Place Eugene Bataillon, 34095 Montpellier cedex 5, France, 2AVI BioPharma, 4575 SW Research Way, Corvallis, OR 97333, USA and 3Section on Membrane Biology, Laboratory of Cellular and Molecular Biophysics, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-1855, USA
*To whom correspondence should be addressed. Tel: + 33 467 14 92 03; Fax: + 33 467 14 92 01; Email: bernard.lebleu{at}univ-montp2.fr
Received June 11, 2008. Revised August 4, 2008. Accepted August 7, 2008.
Redirecting the splicing machinery through the hybridization of high affinity, RNase H- incompetent oligonucleotide analogs such as phosphoramidate morpholino oligonucleotides (PMO) might lead to important clinical applications. Chemical conjugation of PMO to arginine-rich cell penetrating peptides (CPP) such as (R-Ahx-R)4 (with Ahx standing for 6-aminohexanoic acid) leads to sequence-specific splicing correction in the absence of endosomolytic agents in cell culture at variance with most conventional CPPs. Importantly, (R-Ahx-R)4–PMO conjugates are effective in mouse models of various viral infections and Duchenne muscular dystrophy. Unfortunately, active doses in some applications might be close to cytotoxic ones thus presenting challenge for systemic administration of the conjugates in those clinical settings. Structure–activity relationship studies have thus been undertaken to unravel CPP structural features important for the efficient nuclear delivery of the conjugated PMO and limiting steps in their internalization pathway. Affinity for heparin (taken as a model heparan sulfate), hydrophobicity, cellular uptake, intracellular distribution and splicing correction have been monitored. Spacing between the charges, hydrophobicity of the linker between the Arg-groups and Arg-stereochemistry influence splicing correction efficiency. A significant correlation between splicing correction efficiency, affinity for heparin and ability to destabilize model synthetic vesicles has been observed but no correlation with cellular uptake has been found. Efforts will have to focus on endosomal escape since it appears to remain the limiting factor for the delivery of these splice-redirecting ON analogs.
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