Turner, John J. and Fabani, M.M. and Jones, Simon W. and Moschos, Sterghios A. and Perry, Mark M. and Lindsay, Mark A. and Gait, Michael J. (2006) In vitro and in vivo delivery studies of siRNA-peptide conjugates. Journal of RNAi and Gene Silencing, 2 (2). pp. 171-172. ISSN 1747-0854
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Official URL: http://www.libpubmedia.co.uk/RNAiJ-Issues/Issue-4/...
siRNA is a novel reagent for targeting RNA in cells and reducing gene expression. However, its major limitation is the need for a suitable carrier for specific delivery into cell cultures or animal tissues without associated toxicity. In the work presented here, we use different cell penetrating peptides (CPPs) for in vitro and in vivo delivery of various siRNAs in the absence of other transfection reagents. CPPs were conjugated to the siRNA sense strand (OMe/DNA or RNA) by a disulfide linkage and subsequently hybridized to the complementary antisense RNA strand. Amongst the CPPs studied were Penetratin, Transportan and Tat (residues 48-58). CPP-siRNA conjugates were evaluated initially using either an in vitro model targeting plasmid-encoded firefly luciferase or by targeting a disease-relevant endogenous gene coding for p38 a MAP kinase. The ability to modulate levels of endogenous MAPK14 (p38 a ) expression is important in validating the role of the p38 pathway in disease models and in establishing new drug targets. The results showed that free CPP-5’-siRNA (Luc) conjugates were unable to knockdown firefly luciferase expression at low concentrations (up to 500 nM), when analyzed by luminescence 24 hr post-transfection in HeLa or HepG2 cells. In contrast, it was found that certain CPP-siRNA(p38) conjugates were able to knockdown p38 a MAP kinase expression. HeLa cells were incubated with CPP-siRNA(p38) conjugates in serum-free media for 24 hr and p38 expression normalised to 18S rRNA was determined by quantitative real-time PCR using Taqman probes. Following 24 hr transfection, p38 knockdown was achieved with CPP 5’- and 3’-conjugated siRNA. The highest activity was observed for the Penetratin 3’-conjugate, which gave ~ 50% knockdown (P < 0.01) of p38 expression (n=4) at 10 µM. Certain CPP-siRNA conjugates were evaluated in an in vivo mouse model, by targeting p38 a MAP kinase in lungs following intratracheal instillation. p38 mRNA knockdown efficiency, duration and siRNA recovery from the mouse lung appeared to be dose-dependent over a 24-hour period in the absence of delivery reagents/CPPs. Conjugation of Penetratin or Tat did not appear to improve significantly upon knockdown efficiency. Whilst recovery of siRNA from lung homogenates was impaired due to CPP conjugation, our findings suggest that the knockdown efficiency paradox compared to our in vitro data may relate to sequence-dependent siRNA instability.
|Research Community:||University of Westminster > Life Sciences, School of|
|Deposited On:||08 Mar 2012 12:24|
|Last Modified:||08 Mar 2012 12:24|
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