Cell penetrating peptide conjugates of steric block oligonucleotides

Abstract

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.

Fig. 1

Structures of peptides and PNA and various types of peptide–PNA conjugate linkage.

Fig. 2

Structure of PMO and three types of peptide–PMO conjugate linkages. R = arginine, Ahx = 6-aminohexanoic acid, B = beta-alanine and F = phenylalanine.

Fig. 3

(R-Ahx-R)₄·AhxB–PMO conjugates are internalized by an energy-dependent mechanism. A splicing correction assay was used to assess the energy-dependence by ATP deprivation (lanes 5–8) or by low temperature incubation (lanes 9 to 12). HeLa pLuc 705 cells were pre-incubated for 1 h at 37 °C in OptiMEM, supplemented with 10 mM sodium azide and 6 mM 2-deoxy-d-glucose or at 4 °C in OptiMEM. Cells were then incubated with the PMO conjugates at the indicated concentrations for 4 h in the same conditions. Cells were then washed and incubation was continued at 37 °C for 20 h in DMEM. Total RNA was extracted and amplified by RT-PCR. PCR products from incorrectly (268 bp band) and correctly (142 bp band) spliced luciferase pre-mRNA were separated on a 2% agarose gel. Lanes 1, 5 and 9: controls in the absence of (R-Ahx-R)₄ AhxB–PMO conjugate, 2–4: cells incubated at 37 °C and treated with 0.5, 1 or 2.5 μM (R-Ahx-R)₄ AhxB–PMO, respectively, 6–8: cells incubated at 37 °C in the presence of 10 mM sodium azide and 6 mM 2–deoxy-d-glucose and treated with 0.5, 1 or 2.5 μM (R-Ahx-R)₄ AhxB–PMO, respectively, and 9–12: cells incubated at 4 °C and treated with 0.5, 1 or 2.5 μM (R-Ahx-R)₄ AhxB–PMO, respectively.

Fig. 4

RT-PCR analysis of splice correction by R6Pen–PNA conjugates. (a) HeLa pLuc705 cells were incubated for 4 h in OptiMEM in the absence (control), in the presence of 1 μM PNA705 alone or in the presence of 1 μM CPP–PNA705 conjugates. Total RNA was extracted 20 h later and amplified by RT-PCR. PCR products from incorrectly (268 bp) and correctly (142 bp) spliced luciferase pre-mRNA were analysed on a 2% agarose gel. Lane 1: control, 2: PNA705 alone, 3: Pen-ss-PNA705, 4: R₆Pen-s-s-PNA705, 5: R₆Pen-s-s-scrambled PNA705, 6: R₆Pen(W–L)-s-s-PNA705. (b) Dose dependencies of splice correction using 1 μg of total RNA extracted, amplified by RT-PCR and analyses as in (a). Lane 1: control of untreated cells, 2–6: cells treated with 0.25, 0.5, 1, 2 or 4 μM R6Pen-s-s-PNA705 respectively and 7–11: cells treated with 0.25, 0.5, 1, 2 or 4 μM stably linked R6Pen-PNA705 respectively (This figure is reproduced from Fig. 7 of Abes, S. et al. (2007) Nucleic Acids Res., 35, 4495–4502).