Splice Redirection as a Convenient Assay to Monitor CPP–ON Efficiency and Mechanism
Several strategies based on synthetic oligonucleotides (ON) have been proposed to control gene expression. As for most biomolecules, however, delivery has remained a major roadblock for in vivo applications. Conjugation of steric-block neutral DNA mimics, such as peptide nucleic acids (PNA) or phosphorodiamidate morpholino oligonucleotides (PMO), to cell-penetrating peptides (CPP) has recently been proposed as a new delivery strategy. It is particularly suitable for sequence-specific interference with pre-mRNA splicing, thus offering various applications in fundamental research and in therapeutics. The chemical synthesis of these CPP–ON conjugates will be described as well as easy-to-implement assays to monitor cellular uptake, endosome leakage, and efficiency of splicing redirection.
Key wordsCell-penetrating peptides Oligonucleotides Splicing regulation Delivery Liposome leakage
We thank R. Kole (University North Carolina) for providing the HeLa pLuc705 cell line. Amer Saleh was supported by the MRC-Technology Development Gap Fund and also by Action Duchenne. Rachida Abes was supported by Ligue Française contre le Cancer PhD fellowship and Fatouma Said Hassane by Association Française contre les Myopathies.
Studies funded by EC grant QLK3-CT-2002-01989 and CEFIPRA grant 3205.
- 6.Turner, J.J., Ivanova, G.D., Verbeure, B, Williams, D., Arzumanov, A.A., Abes, S., Lebleu, B. and Gait, M.J. (2005) Cell-penetrating peptide conjugates of peptide nucleic acids (PNA) as inhibitors of HIV-1 Tat-dependent trans-activation in cells. Nucleic Acids Res. 33, 6837–6849.CrossRefPubMedGoogle Scholar