Shiraishi Takehiko, Nielsen Peter E
Department of Cellular and Molecular Medicine, Faculty of Health Sciences, The Panum Institute, University of Copenhagen, Copenhagen N, Denmark.
Artif DNA PNA XNA. 2011 Jul-Dec;2(3):90-9. doi: 10.4161/adna.18739.
We have explored the merits of a novel delivery strategy for the antisense oligomers based on cell penetrating peptide (CPP) 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% downregulation 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.
我们已经探索了一种基于细胞穿透肽（CPP）与载体肽核酸（PNA）缀合的新型反义寡核苷酸递送策略的优点，该载体PNA的序列与部分反义寡核苷酸互补。通过使用针对HeLa pLuc705细胞系中突变荧光素酶基因的剪接校正的反义PNA，通过荧光素酶活性测量报告反义PNA的细胞（核）摄取，来评估这些载体CPP-PNA的效果。从构建体修饰（用八聚精氨酸和/或癸酸）和载体PNA长度（以调节结合亲和力）方面研究了载体CPP-PNA构建体。一般来说，包括具有癸酰基修饰的载体CPP-PNA构建体，能显著提高未修饰反义PNA以及反义八聚精氨酸-PNA缀合物的活性。未修饰反义PNA与等摩尔量的九聚体载体癸酰基-CPP-PNA（Deca-cPNA1(9)-(D-Arg)8）复合后，在6 μM时反义活性以及由此推断的细胞递送能力至少提高了20倍。一种CPP-PNA（(D-Arg)8-asPNA）（2 μM）的反义活性，分别被七聚体载体CPP-PNA（cPNA1(7)-(D-Arg)8）和六聚体载体癸酰基-CPP-PNA（Deca-cPNA1(6)-(D-Arg)8）提高了6倍和8倍，且未显示出明显的额外细胞毒性。最有趣的是，该活性达到了通过溶酶体溶解剂氯喹（CQ）处理增强所获得的相同水平，这表明该载体可能促进内体逃逸。此外，对于靶向正常荧光素酶mRNA的单链反义RNA，使用这种载体CPP-PNA递送策略（与CQ共同处理）在60 nM siRNA时可实现荧光素酶表达下调50%。这些结果表明，CPP-PNA载体可用作不同类型反义寡核苷酸的有效细胞递送载体，并且还允许使用（至少两种）不同CPP配体的组合。
