Cell penetrating peptide

Cell penetrating peptides (CPPs) are short polycationic sequences which can facilitate cellular uptake of peptides and proteins which contain them. [Jones SW, Christison R, Bundell K, Voyce CJ, Brockbank SMV, Newham P, Lindsay MA. Characterisation of cell-penetrating peptide-mediated peptide delivery. British Journal of Pharmacology. 2005; 145 :1093-1102] When linked to cargo, CPPs enhance uptake into endosomes of living mammalian cells. [Frankel AD, Pabo CO. Cellular uptake of the tat protein from human immunodeficiency virus. Cell. 1988 Dec 23;55(6):1189-93. PMID: 2849510] In the study done by Franklin, researchers studied the effects of a CPP, chloroquine, on the uptake of the tat protein by the Human Immunodeficiency virus(HIV). In the study, cells of HIV were incubated in mediums containing tat proteins which allowed scientists to measure the level of trans-activation(basically the uptake of material) and found increased levels of trans-activation in the HIV cells with CPPs added to them. They also found that as levels of CPPs increased so did the level of trans-activation. This showed that CPPs did enhance the uptake of genetic material into the cell.

An application of cell penetrating peptides is the delivery of antisense oligos by covalently-linked cell penetrating peptides. In a study done by AVI BioPharma and the University Montpellier [Abes S, Moulton HM, Clair P, Prevot P, Youngblood DS, Wu RP, Iversen PL, Lebleu B. Vectorization of morpholino oligomers by the (R-Ahx-R)(4) peptide allows efficient splicing correction in the absence of endosomolytic agents. J Control Release. 2006 Dec 1;116(3):304-13. Epub 2006 Sep 30.] , researchers compared solutions which contained Morpholino oligos linked to CPPs with other solutions containing Morpholinos without CPPs. To measure the amount of splice-modifying Morpholino oligos being transferred into the cytosol and nucleus, they used cells containing an engineered gene with a mutated splice site from human beta-globin inserted into DNA coding for luciferase protein, a protein which emits light in the presence of the chemical luciferin. This engineered gene would only make functional luciferin if the appropriate splice-modifying Morpholino oligos entered the nucleus and blocked the splicing mutation. By measuring the level of luminescence when luciferase was added to cell lysates, they found that the cells exposed to Morpholinos conjugated to CPPs had much higher levels of functional luciferase protein than cells exposed to solutions without CPPs. Light output increased as concentrations of the CPP conjugates were increased. For antisense to be effective, the oligos must not only be endocytosed but must also reach the cytosol to block protein synthesis or the nucleus to block splicing of pre-mRNA. Research on delivery of peptide nucleic acids [Abes S, Moulton H, Turner J, Clair P, Richard JP, Iversen P, Gait MJ, Lebleu B. Peptide-based delivery of nucleic acids: design, mechanism of uptake and applications to splice-correcting oligonucleotides. Biochem Soc Trans. 2007 Feb;35(Pt 1):53-5. ] and Morpholino oligos [Moulton HM, Nelson MH, Hatlevig SA, Reddy MT, Iversen PL. Cellular uptake of antisense morpholino oligomers conjugated to arginine-rich peptides. Bioconjug Chem. 2004 Mar-Apr;15(2):290-9. ] [Nelson MH, Stein DA, Kroeker AD, Hatlevig SA, Iversen PL, Moulton HM. Arginine-rich peptide conjugation to morpholino oligomers: effects on antisense activity and specificity. Bioconjug Chem. 2005 Jul-Aug;16(4):959-66. ] [Abes S, Moulton HM, Clair P, Prevot P, Youngblood DS, Wu RP, Iversen PL, Lebleu B. Vectorization of morpholino oligomers by the (R-Ahx-R)(4) peptide allows efficient splicing correction in the absence of endosomolytic agents. J Control Release. 2006 Dec 1;116(3):304-13. Epub 2006 Sep 30.] [Youngblood DS, Hatlevig SA, Hassinger JN, Iversen PL, Moulton HM. Stability of cell-penetrating Peptide-morpholino oligomer conjugates in human serum and in cells. Bioconjug Chem. 2007 Jan-Feb;18(1):50-60. ] using cell penetrating peptides has been especially productive, including new work describing delivery in postnatal mice [Burrer R, Neuman BW, Ting JPC, Stein DA, Moulton HM, Iversen PL, Kuhn P, Buchmeier MJ. Antiviral effects of antisense morpholino oligomers in murine coronavirus infection models. J. Virol. 2007 Epub 2007 Mar 5.] [Deas TS, Bennett CJ, Jones SA, Tilgner M, Ren P, Behr MJ, Stein DA, Iversen PL, Kramer LD, Bernard KA, Shi PY. In vitro resistance selection and in vivo efficacy of morpholino oligomers against West Nile virus. Antimicrob Agents Chemother. 2007 May 7; [Epub ahead of print] ] .

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