Tumor-targeted in vivo gene silencing via systemic delivery of cRGD-conjugated siRNA.

Xiaoxia Liu,Biliang Zhang,Wenqing Zhang,Ping Wu,Zhuomin Wu,Wei Yang,Zhiyong Qiu,Dmitry Samarsky,Wei Wang,Qian Xu,Xiaohui Chen,Zhongyi Zhang,Lingfeng Zhao,Aimin Ji,Guangzu Zhu,Li Liu,Jingjing Zhang,Quan Zeng ,Haiyi Deng ,Xiaoxia Zuo

doi:10.1093/nar/gku831

Abstract

RNAi technology is taking strong position among the key therapeutic modalities, with dozens of siRNA-based programs entering and successfully progressing through clinical stages of drug development. To further explore potentials of RNAi technology as therapeutics, we engineered and tested VEGFR2 siRNA molecules specifically targeted to tumors through covalently conjugated cyclo(Arg-Gly-Asp-d-Phe-Lys[PEG-MAL]) (cRGD) peptide, known to bind αvβ3 integrin receptors. cRGD-siRNAs were demonstrated to specifically enter and silence targeted genes in cultured αvβ3 positive human cells (HUVEC). Microinjection of zebrafish blastocysts with VEGFR2 cRGD-siRNA resulted in specific inhibition of blood vessel growth. In tumor-bearing mice, intravenously injected cRGD-siRNA molecules generated no innate immune response and bio-distributed to tumor tissues. Continuous systemic delivery of two different VEGFR2 cRGD-siRNAs resulted in down-regulation of corresponding mRNA (55 and 45%) and protein (65 and 45%) in tumors, as well as in overall reduction of tumor volume (90 and 70%). These findings demonstrate strong potential of cRGD-siRNA molecules as anti-tumor therapy.

Highlights

RNAi gene silencing technology, with siRNAs as its triggers, is based on natural intracellular mechanisms and has a strong potential as novel therapeutic strategy for a broad range of diseases––from genetic disorders to cancer and viral infection [1,2]
To prepare peptide-siRNA conjugate molecules, peptides were covalently linked to the 3 -end of siRNA passenger strands using thiol-maleimide linker
Sequences for siRNAs against human, mouse and zebrafish VEGFR2 mRNAs, and their precise structures are listed in Supplementary Table S2

Summary

Introduction

RNAi gene silencing technology, with siRNAs as its triggers, is based on natural intracellular mechanisms and has a strong potential as novel therapeutic strategy for a broad range of diseases––from genetic disorders to cancer and viral infection [1,2]. To become widely applied in the clinic this technology has to address several challenges, including stability of siRNA molecules in vivo, delivery and biodistribution to intended tissues/organs, as well as potential non-specific (e.g. off-target and immunostimulatory) effects. While stability and specificity of siRNAs in vivo has been improved using various chemical modifications [2], in vivo delivery remains the biggest difficulty, due to the large size (∼13 kDa) and strong relative negative charge of the molecules.

Methods

Results

Conclusion