Cell penetrating peptides: overview and applications to the delivery of oligonucleotides

Abstract

Crossing biological barriers represents a major limitation for clinical applications of biomolecules such as nucleic acids, peptides or proteins. Cell penetrating peptides (CPP), also named protein transduction domains, comprise short and usually basic amino acids-rich peptides originating from proteins able to cross biological barriers, such as the viral Tat protein, or are rationally designed. They have emerged as a new class of non-viral vectors allowing the delivery of various biomolecules across biological barriers from low molecular weight drugs to nanosized particles. Encouraging data with CPP-conjugated oligonucleotides have been obtained both in vitro and in vivo in animal models of diseases such as Duchenne muscular dystrophy. Whether CPP-cargo conjugates enter cells by direct translocation across the plasma membrane or by endocytosis remains controversial. In many instances, however, endosomal escape appears as a major limitation of this new delivery strategy.

Fig. 1

Splicing redirection assay. a A mutated β-globin gene intron carrying a cryptic splice site has been inserted in the coding sequence of a luciferase reporter gene and the construct has been stably transfected in HeLa pLuc 705 cells. Abnormal splicing does not allow the complete removal of the intron and no functional luciferase is produced. The nuclear delivery of a steric block ON analogue (ON ₇₀₅) and its binding to the mutated site redirects the cellular splicing machinery towards complete elimination of this intron, leading to the production of the correctly spliced luciferase mRNA and to the production of active luciferase protein. b Luciferase up-regulation by PNA conjugates at low and high temperatures. HeLa pLuc 705 cells were preincubated for 30 min at 4 or 37°C before treatment. Cells were then incubated with 1 μM R6Pen-ss-PNA or R6Pen-sc-PNA in OptiMEM for 1 h at low or high temperatures and washed twice with PBS buffer. Luciferase quantification was processed after 23 h supplementary incubation in DMEM at 37°C. These two PNA conjugates differ in the nature of the linker between the peptidic and the oligonucleotidic parts of the conjugates (reducible disulfide—ss—or stable thioester—sc—covalent bond). c RT–PCR analysis of splicing redirection. Total RNA was purified from the same cellular lysates and amplified by RT–PCR. The correctly spliced mRNA can be distinguished from the aberrant one for each conjugate at the two temperatures

Fig. 2

Restoration of muscle and cardiac dystrophin expression in adult mdx mice following single 25 mg/kg intravenous injection of (R-Ahx-R)₄-Ahx-βAla-PMO (reproduced from Fig. 1 from Ref. [54], with permission). a Immunostaining of muscle tissue cross sections to detect dystrophin protein expression and localization in normal (C57BL6) mice (top) untreated mdx mice (middle) and peptide-PMO treated mice (lower) showing near normal levels of dystrophin in the treated mice. TA Tibialis anterior muscle. b RT–PCR to detect exon skipping efficiency at the RNA level demonstrating almost complete exon 23 skipping in the peripheral skeletal muscles and ca. 50% in heart in treated mdx mice. This is shown by the shorter exon-skipped bands (indicated by the box numbered 22–24 for exon 23 skipping). c Western blotting for dystrophin expression in peripheral skeletal muscles showed ca. 100% dystrophin restoration in all skeletal muscles except the diaphragm