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Sarepta Therapeutics, Inc. Message Board

  • bionerd51 bionerd51 Oct 17, 2013 9:05 AM Flag

    Excitement Over "New" Stearylated {RXR]4 Peptide...

    The inclosed paper summary highlights a "new", low-toxicity translocation peptide that has Sarepta biochemists excited. This peptide is the key to safer PPMO's and PPMO's that are not mortally entrapped in intracellular endosomes. These are stearylated poly-arginine peptides. Delivery of nucleic acids with a stearylated (RxR)4 peptide using a non-covalent co-incubation strategy.
    Lehto T, Abes R, Oskolkov N, Suhorutsenko J, Copolovici DM, Mäger I, Viola JR, Simonson OE, Ezzat K, Guterstam P, Eriste E, Smith CI, Lebleu B, Samir El Andaloussi, Langel U.
    Laboratory of Molecular Biotechnology, Institute of Technology, University of Tartu, Nooruse 1, 50411 Tartu, Estonia.
    In recent years, oligonucleotide-based molecules have been intensely used to modulate gene expression. All these molecules share the common feature of being essentially impermeable over cellular membranes and they therefore require efficient delivery vectors. Cell-penetrating peptides are a group of delivery peptides that has been readily used for nucleic acid delivery. In particular, polyarginine and derivates thereof, i.e. the (RxR)(4) peptide, have been applied with success both in vitro and in vivo. A major problem, however, with these arginine-rich peptides is that they frequently remain trapped in endosomal compartments following internalization. The activity of polyarginine has previously been improved by conjugation to a stearyl moiety. Therefore, we sought to investigate what impact such modification would have on the pre-clinically used (RxR)(4) peptide for non-covalent delivery of plasmids and splice-correcting oligonucleotides (SCOs) and compare it with stearylated Arg9 and Lipofectamine 2000. We show that stearyl-(RxR)(4) mediates efficient plasmid transfections in several cell lines and the expression levels are significantly higher than when using unmodified (RxR)(4) or stearylated Arg9. Although the transfection efficiency is lower than with Lipofectamine 2000, we show that stearyl-(RxR)(4) is substantially less toxic. Furthermore, using a functional splice-correction assay, we show that stearyl-(RxR)(4) complexed with 2'-OMe SCOs promotes significant splice correction whereas stearyl-Arg9 fails to do so. Moreover, stearyl-(RxR)(4) promotes dose-dependent splice correction in parity with (RxR)(4)-PMO covalent conjugates, but at least 10-times lower concentration. These features make this stearic acid modified analog of (RxR)(4) an intriguing vector for future in vivo experiments.

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