Peptide nucleic acid (PNA) cell penetrating peptide (CPP) conjugates as carriers for cellular delivery of antisense oligomers

TY - JOUR

T1 - Peptide nucleic acid (PNA) cell penetrating peptide (CPP) conjugates as carriers for cellular delivery of antisense oligomers

AU - Shiraishi, Takehiko

AU - Nielsen, Peter E

PY - 2011

Y1 - 2011

N2 - We have explored the merits of a novel delivery strategy for antisense oligomers based on cell penetrating peptides (CPPs) conjugated to a carrier PNA with sequence complementary to part of the antisense oligomer. The effect of these carrier CPP-PNAs was evaluated by using antisense PNA targeting splicing correction of the mutated luciferase gene in the HeLa pLuc705 cell line, reporting cellular (nuclear) uptake of the antisense PNA via luciferase activity measurement. Carrier CPP-PNA constructs were studied in terms of construct modification (with octaarginine and/or decanoic acid) and carrier PNA length (to adjust binding affinity). In general, the carrier CPP-PNA constructs including the ones with decanoyl modification provided significant increase of the activity of unmodified antisense PNA as well as of antisense octaarginine-PNA conjugates. Antisense activity, and by inference cellular delivery, of unmodified antisense PNA was enhanced at least 20-fold at 6 μM upon the complexation with an equimolar amount of nonamer carrier decanoyl-CPP-PNA (Deca-cPNA1(9)-(D-Arg) 8). The antisense activity of a CPP-PNA [(D-Arg) 8-asPNA at 2 μM] was improved 6-fold and 8-fold by a heptamer carrier CPP-PNA [cPNA1(7)-(D-Arg) 8] and hexamer carrier decanoyl-CPP-PNA [Deca-cPNA1(6)-(D-Arg) 8], respectively, without showing significant additional cellular toxicity. Most interestingly, the activity reached the same level obtained by enhancement with endosomolytic chloroquine (CQ) treatment, suggesting that the carrier might facilitate endosomal escape. Furthermore, 50% down regulation of luciferase expression at 60 nM siRNA was obtained using this carrier CPP-PNA delivery strategy (with CQ co-treatment) for a single stranded antisense RNA targeting normal luciferase mRNA. These results indicated that CPP-PNA carriers may be used as effective cellular delivery vectors for different types of antisense oligomers and also allows use of combinations of (at least two) different CPP ligands.

AB - We have explored the merits of a novel delivery strategy for antisense oligomers based on cell penetrating peptides (CPPs) conjugated to a carrier PNA with sequence complementary to part of the antisense oligomer. The effect of these carrier CPP-PNAs was evaluated by using antisense PNA targeting splicing correction of the mutated luciferase gene in the HeLa pLuc705 cell line, reporting cellular (nuclear) uptake of the antisense PNA via luciferase activity measurement. Carrier CPP-PNA constructs were studied in terms of construct modification (with octaarginine and/or decanoic acid) and carrier PNA length (to adjust binding affinity). In general, the carrier CPP-PNA constructs including the ones with decanoyl modification provided significant increase of the activity of unmodified antisense PNA as well as of antisense octaarginine-PNA conjugates. Antisense activity, and by inference cellular delivery, of unmodified antisense PNA was enhanced at least 20-fold at 6 μM upon the complexation with an equimolar amount of nonamer carrier decanoyl-CPP-PNA (Deca-cPNA1(9)-(D-Arg) 8). The antisense activity of a CPP-PNA [(D-Arg) 8-asPNA at 2 μM] was improved 6-fold and 8-fold by a heptamer carrier CPP-PNA [cPNA1(7)-(D-Arg) 8] and hexamer carrier decanoyl-CPP-PNA [Deca-cPNA1(6)-(D-Arg) 8], respectively, without showing significant additional cellular toxicity. Most interestingly, the activity reached the same level obtained by enhancement with endosomolytic chloroquine (CQ) treatment, suggesting that the carrier might facilitate endosomal escape. Furthermore, 50% down regulation of luciferase expression at 60 nM siRNA was obtained using this carrier CPP-PNA delivery strategy (with CQ co-treatment) for a single stranded antisense RNA targeting normal luciferase mRNA. These results indicated that CPP-PNA carriers may be used as effective cellular delivery vectors for different types of antisense oligomers and also allows use of combinations of (at least two) different CPP ligands.

M3 - Journal article

C2 - 22567192

SN - 1949-095X

VL - 2

SP - 90

EP - 99

JO - Artificial DNA: PNA and XNA

JF - Artificial DNA: PNA and XNA

IS - 3

ER -