Silencing of gene expression by small interfering RNAs (siRNAs) is rapidly becoming a powerful tool for genetic analysis and represents a potential strategy for therapeutic product development. However, there are no reports of systemic delivery for siRNAs toward treatment of bone-metastatic cancer. Accordingly, we report here that i.v. injection of GL3 luciferase siRNA complexed with atelocollagen showed effective reduction of luciferase expression from bone-metastatic prostate tumor cells developed in mouse thorax, jaws, and͞or legs. We also show that the siRNA͞ atelocollagen complex can be efficiently delivered to tumors 24 h after injection and can exist intact at least for 3 days. Furthermore, atelocollagen-mediated systemic administration of siRNAs such as enhancer of zeste homolog 2 and phosphoinositide 3-hydroxykinase p110-␣-subunit, which were selected as candidate targets for inhibition of bone metastasis, resulted in an efficient inhibition of metastatic tumor growth in bone tissues. In addition, upregulation of serum IL-12 and IFN-␣ levels was not associated with the in vivo administration of the siRNA͞atelocollagen complex. Thus, for treatment of bone metastasis of prostate cancer, an atelocollagen-mediated systemic delivery method could be a reliable and safe approach to the achievement of maximal function of siRNA.bone metastasis ͉ prostate cancer R NA interference (RNAi) induced by small interfering RNA (siRNA) has recently emerged as a powerful technique that is capable of suppressing expression of individual genes with a high degree of specificity (1). The technique has been used for studies of gene function in vivo, primarily in mice. The first demonstration of RNAi-mediated repression in an adult animal showed effective repression of a luciferase reporter gene after hydrodynamic transfection of siRNA expression plasmids into mouse liver (2, 3). Subsequent studies have delivered siRNA by various methods, including viral vector-mediated delivery (4, 5) and lipid-based delivery (6, 7). A more recent study showed that chemically modified siRNAs can silence an endogenous gene after i.v. injection in mice (8). These findings provide hope for using RNAi technology in disease control.Many studies have used siRNAs as an experimental tool to dissect the cellular pathways that lead to uncontrolled cell proliferation and cancer. To develop siRNAs for cancer therapy, several researchers have investigated them in animal models (9-13). However, reports of RNAi-delivery strategies for bonemetastatic cancer are very limited. For example, in advanced prostate cancer, the sites most frequently affected by metastasis are the bones and regional lymph nodes. Patients with these metastases suffer pain and low limb edema, making it extremely important to explore avenues of treating such bone metastases.We previously demonstrated the efficacy of atelocollagen for delivery of nucleotides, such as plasmid DNA and antisense oligonucleotides, in vitro and in vivo (14)(15)(16)(17)(18)(19). Recently, we also reported that a...
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.
Contact hypersensitivity (CHS) is a common skin disease, presenting clinically as allergic contact dermatitis. At inflammatory sites in a typical CHS model in the mouse ear, elevated expression of monocyte chemoattractant protein-1 (MCP-1) has been reported. MCP-1 is a potent chemotactic factor for many types of leukocytes including monocytes/macrophages and T cells. In this study, we aimed at developing a therapy for CHS involving RNA interference targeting MCP-1. A short interfering RNA (siRNA) to mouse MCP-1 successfully inhibited the secretion of MCP-1 by a fibroblastic cell line, L929, and RAW 264.7 cells derived from macrophages, and strikingly suppressed ear swelling in a CHS model. The siRNA systemically administered inhibited the infiltration of both monocytes/macrophages and T cells in the CHS model. Atelocollagen was used in this therapy as a delivery reagent for siRNA into the animal body. Atelocollagen facilitated the incorporation of the siRNA into macrophages/monocytes and fibroblasts, which vigorously secrete MCP-1 protein at inflammatory sites in CHS. This therapy had no adverse effects such as induction of interferon, or liver or renal damage. Our data indicate that the systemic delivery of siRNA targeting MCP-1 is a potent therapeutic strategy for CHS treatment.
To study the possibility of using atelocollagen as an oligonucleotide (ODN) delivery carrier in vivo, the activity of formulated antisense ODN targeted against the intercellular adhesion molecule-1 (ICAM-1) mRNA was investigated in an allergic dermatitis model in mice. The allergic dermatitis was elicited in one ear of animals sensitized by treatment with 2,4-dinitrofluorobenzene. Antisense ODN was given to the animals as a single intravenous injection of formulation containing atelocollagen. Antisense activity was determined by measurement of ear thickness, histopathology, and immunohistochemistry 24 hr after the initiation of the dermatitis. Antisense activity was found to increase according to the concentration of atelocollagen in the formulation. The effect mediated by the ODN formulated with 0.05% atelocollagen was more than 50 times greater than that provided by ODN infusion, although the levels of ODN formulated with atelocollagen dropped below that of the 24-hr infusion group within 30 min. The formulated ODN could suppress inflammatory progression by treatment at 8 hr after the ear challenge when inflammation had already commenced at the challenged site. Moreover, antisense activity was noted even when the formulated ODN was injected 3 days before the initiation of inflammation. These data demonstrate that atelocollagen can enhance antisense activity remarkably and that the sustainable antisense activity mediated by the formulation of ODN with atelocollagen could completely change the strategy of antisense therapeutics.
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