Morpholino delivery by cell-penetrating peptides
Jon Moulton
Wednesday, 01 August 2007 17:56 UTC
My interest in drug delivery is about the delivery of Morpholino antisense into cells of adult animals. Morpholinos hold great promise for treatment of viral and genetic diseases, but do not enter most cells readily without assistance. Here is the latest paper describing the use of cell-penetrating peptides to help Morpholinos into cells, likely occurring primarily through an endocytotic pathway followed by release from the endosome.
Cell-penetrating peptides as transporters for
morpholino oligomers: effects of amino acid
composition on intracellular delivery and cytotoxicity
Rebecca P. Wu, Derek S. Youngblood, Jed N. Hassinger, Candace E. Lovejoy,Michelle H. Nelson, Patrick L. Iversen and Hong M. Moulton
Nucleic Acids Research, 2007, 1–10
doi:10.1093/nar/gkm478
http://nar.oxfordjournals.org/cgi/reprint/gkm478v1
For information on Morpholino oligos, start with the Wikipedia Morpholino page:
http://en.wikipedia.org/wiki/Morpholino
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Replies
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Another new cell-penetrating-peptide and Morpholino paper:
http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=17673254Arginine-rich cell-penetrating peptides facilitate delivery of antisense oligomers into murine leukocytes and alter pre-mRNA splicing.
Marshall NB, Oda SK, London CA, Moulton HM, Iversen PL, Kerkvliet NI, Mourich DV.
J Immunol Methods. 2007 Jul 26; [Epub ahead of print]
Phosphorodiamidate morpholino oligomers (PMO) are synthetic antisense molecules that interfere with translation, pre-mRNA splicing and RNA synthesis. Like other gene-silencing technologies, PMO are poorly taken up by primary leukocytes without the use of physical or chemical delivery techniques. We sought an alternative delivery mechanism of PMO into immune cells that eliminates the need for such manipulations. Here we demonstrate the first use of arginine-rich cell-penetrating peptides (CPPs) to deliver PMO (P-PMO) directly into primary murine leukocytes for inhibition of gene expression and promotion of altered pre-mRNA splicing. We compared the P-PMO delivery efficacy of four arginine-rich CPPs including HIV Tat and penetratin, and one histidine rich CPP, and found that the (RXR)(4) peptide was the most efficacious for PMO delivery and targeted antisense effect. The delivery and antisense effects of P-PMO are time- and dose-dependent and influenced by the activation and maturation states of T cells and dendritic cells, respectively. Targeted expression of several genes using P-PMO is shown including surface signaling proteins (CD45 and OX-40), a cytokine (interleukin-2), and a nuclear transcription factor (Foxp3). Considering the abundance of naturally occurring alternatively spliced gene products involved in immune regulation, P-PMO offer an effective method for modulating gene activity for immunological research and applications beyond traditional antisense approaches.
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Lebleu B, Moulton HM, Abes R, Ivanova GD, Abes S, Stein DA, Iversen PL, Arzumanov AA, Gait MJ. Cell penetrating peptide conjugates of steric block oligonucleotides. Adv Drug Deliv Rev. 2007 Oct 22; [Epub ahead of print]
Charge neutral steric block oligonucleotide analogues, such as peptide nucleic acids (PNA) or phosphorodiamidate morpholino oligomers (PMO), have promising biological and pharmacological properties for antisense applications, such as for example in mRNA splicing redirection. However, cellular uptake of free oligomers is poor and the utility of conjugates of PNA or PMO to cell penetrating peptides (CPP), such as Tat or Penetratin, is limited by endosomal sequestration. Two new families of arginine-rich CPPs named (R-Ahx-R)(4) AhxB and R(6)Pen allow efficient nuclear delivery of splice correcting PNA and PMO at micromolar concentrations in the absence of endosomolytic agents. The in vivo efficacy of (R-Ahx-R)(4) AhxB PMO conjugates has been demonstrated in mouse models of Duchenne muscular dystrophy and in various viral infections.
http://www.ncbi.nlm.nih.gov/sites/entrez?Db=pubmed&Cmd=ShowDetailView&TermToSearch=18037527
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