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Selective modification of HK peptides enhances siRNA silencing of tumor targets in vivo

Abstract

Our research has focused on systemic delivery of small interference RNA (siRNA) by branched peptides composed of histidine and lysine. After studying several histidine-lysine (HK) peptides, one four-branched peptide, H3K(+H)4b, with a predominant repeating pattern of -HHHK-, was found to be an effective carrier of siRNA. Although the unmodified H3K(+H)4b carrier of siRNA targeting an oncogene was previously shown to have promise in a tumor-bearing mouse model, we sought to develop a more effective HK carrier of siRNA in this study. Our primary goal was to determine whether different ligand (cyclic RGD)-pegylation patterns on the H3K(+H)4b peptide affect siRNA delivery in vitro and in vivo. We compared the unmodified H3K(+H)4b with two modified H3K(+H)4b peptides for their ability to deliver siRNA in a tumor-bearing mouse model; one modified HK peptide, (RGD-PEG)4-H3K(+H)4b, had four cyclic RGD-polyethylene glycol (cRGD-PEG) conjugates per molecule, whereas the other peptide, (RGD-PEG)-H3K(+H)4b, had one cRGD-PEG per molecule. Although the modified HK peptides by themselves did not form stable nanoplexes with siRNA, combination of a highly charged unmodified HK peptide, H2K4b, with either of the modified HK peptides did form stable siRNA nanoparticles. For in vitro experiments with MDA-MB-435 cells that expressed luciferase (Luc), the H3K(+H)4b siRNA nanoplexes targeting Luc decreased its activity by 90% compared with negligible downregulation by the modified H3K(+H)4b nanoplexes (P<0.01). In contrast, the two modified H3K(+H)4b siRNA nanoplexes administered intravenously were more effective than the H3K(+H)4b nanoplexes in silencing Luc in a tumor xenograft model. The Luc activity in tumor lysates of mice administered H3K(+H)4b, (RGD-PEG)-H3K(+H)4b and (RGD-PEG)4-H3K(+H)4b nanoplexes decreased by 18, 35 and 75%, respectively. Thus, the siRNA nanoplex incorporating the highly modified peptide, (RGD-PEG)4-H3K(+H)4b, was the most effective at silencing its target in vivo (P<0.01). These studies demonstrate that selectively modified HK polymers are promising candidates for targeting oncogenes with siRNA.

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Abbreviations

cRGD:

cyclic RGDfK peptide

cRGD-PEG:

cyclic RGD-polyethylene glycol

H3K(+H)4b:

H2K4b-two unmodified 4-branched HK polymers which differ in their lysine to histidine ratios

HK:

generic term for histidine–lysine peptides

(RGD-PEG)-H3K(+H)4b:

a modified H3K(+H)4b in which one cRGD-PEG is attached to the C-terminal core

(RGD-PEG)4-H3K(+H)4b:

a modified H3K(+H)4b in which cRGD-PEG is attached to each of the four N-terminal branches

(RGD-PEG)-H3K(+H)4b/H2K4b or (RGD-PEG)4-H3K(+H)4b/H2K4b:

combination of modified and unmodified polymers

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Acknowledgements

We thank Dr Pamela Talalay for her helpful suggestions and careful reading of this manuscript. This work was supported by the National Institutes of Health (R01-CA136938).

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Correspondence to A J Mixson.

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A James Mixson has license agreements and/or equity with Aparna Biosciences, Sirnaomics and Silence Therapeutics.

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Chou, ST., Leng, Q., Scaria, P. et al. Selective modification of HK peptides enhances siRNA silencing of tumor targets in vivo. Cancer Gene Ther 18, 707–716 (2011). https://doi.org/10.1038/cgt.2011.40

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