Atelocollagen-mediated synthetic small interfering RNA delivery for effective gene silencing in vitro and in vivo

Abstract

Silencing gene expression by siRNAs is rapidly becoming a powerful tool for the genetic analysis of mammalian cells. However, the rapid degradation of siRNA and the limited duration of its action call for an efficient delivery technology. Accordingly, we describe here that Atelocollagen complexed with siRNA is resistant to nucleases and is efficiently transduced into cells, thereby allowing long-term gene silencing. Site-specific in vivo administration of an anti-luciferase siRNA/Atelocollagen complex reduced luciferase expression in a xenografted tumor. Furthermore, Atelocollagen-mediated transfer of siRNA in vivo showed efficient inhibition of tumor growth in an orthotopic xenograft model of a human non-seminomatous germ cell tumor. Thus, for clinical applications of siRNA, an Atelocollagen-based non-viral delivery method could be a reliable approach to achieve maximal function of siRNA in vivo.

Special delivery: targeted therapy with small RNAs

“…Physical methods, such as microinjection and electroporation, [10][11][12][13] as well as calcium co-precipitation, 14 commercially available cationic polymers and lipids, 1,15-20 and cell-penetrating peptides [21][22][23][24][25] can all be used to knock down genes. Aside from the physical methods (in which siRNAs are directly injected into the cell cytoplasm or enter via membrane pores caused by electrical bursts), all the other methods share a common characteristic complexation with a positive (cationic) charge that enables the siRNAs to interact with the negatively charged plasma membrane.…”

“…Although these particles have not been modified to target tumors, passive targeting due to the enhanced permeability and retention effect, causes the selective accumulation within the cancerous tissues as shown in several studies with tumor xenografts. 23,[37][38][39] Initial studies indicated that atelocollagen particles could be administered safely without induction of cytokines or observed toxicity to the tissues.…”

Atelocollagen-mediated local and systemic applications of myostatin-targeting siRNA increase skeletal muscle mass

“…4 On the other hand, atelocollagen (ATCOL), a pepsin-treated type I collagen lacking in telopeptides in N and C terminals that confer its antigenicity, has been shown to elicit an efficient delivery of chemically unmodified siRNAs to metastatic tumors in vivo. [5][6][7] In this study, we sought to examine the effectiveness of siRNA-ATCOL therapy for a nontumorous systemic disease, targeted against myostatin (growth/differentiation factor 8, GDF8), a negative regulator of skeletal muscle growth. 8 Skeletal muscles are the crucial morphofunctional organs, and their atrophy causes severe conditions for life such as muscular dystrophies.…”

Mutant mouse p53 transgene elevates the chemical induction of tumors that respond to gene silencing with siRNA

“…29,30 When tumors grew to a diameter of 5 mm (63 mm 3 in volume), siRNA no. 220-atelocollagen complex was injected into the site surrounding the subcutaneous solid tumor at 2-3 day intervals for a total of four injections.…”

“…Atelocollagen, an end-cut collagen, was used as a delivery system to protect siRNA from RNase and introduce siRNA efficiently to the target molecules in the tumor tissue. 29,30 We report here the construction of transgenic mice carrying intact wild-type p53 and mutant p53 cDNA, being homozygous for the wild-type p53 allele and hemizygous for mutant p53 (p53 þ / þ ; m ). For the sake of convenience, we refer to the p53 þ / þ ; m mice as mp53 mice.…”