Therapeutic EphA2 Gene Targeting In vivo Using Neutral Liposomal Small Interfering RNA Delivery

Abstract

Inducing destruction of specific mRNA using small interfering RNA (siRNA) is a powerful tool in analysis of protein function, but its use as a therapeutic modality has been limited by inefficient or impractical delivery systems. We have used siRNA incorporated into the neutral liposome 1,2-dioleoylsn-glycero-3-phosphatidylcholine (DOPC) for efficient in vivo siRNA delivery. In nude mice bearing i.p. ovarian tumors, nonsilencing siRNA tagged with the fluorochrome Alexa 555 was encapsulated into DOPC liposomes and shown to be taken up by the tumor as well as many major organs. Furthermore, DOPC-encapsulated siRNA targeting the oncoprotein EphA2 was highly effective in reducing in vivo EphA2 expression 48 hours after a single dose as measured by both Western blot and immunohistochemistry. Therapy experiments in an orthotopic mouse model of ovarian cancer were initiated 1 week after injection of either HeyA8 or SKOV3ip1 cell lines. Three weeks of treatment with EphA2-targeting siRNA-DOPC (150 Mg/kg twice weekly) reduced tumor growth when compared with a nonsilencing siRNA (SKOV3ip1: 0.35 versus 0.70 g; P = 0.020; HeyA8: 0.98 versus 1.51 g; P = 0.16). When EphA2-targeting siRNA-DOPC was combined with paclitaxel, tumor growth was dramatically reduced compared with treatment with paclitaxel and a nonsilencing siRNA (SKOV3ip1: 0.04 versus 0.22 g; P < 0.001; HeyA8: 0.21 versus 0.84 g; P = 0.0027). These studies show the feasibility of siRNA as a clinically applicable therapeutic modality. (Cancer Res 2005; 65(15): 6910-8)

EphA2 Induces Metastatic Growth Regulating Amoeboid Motility and Clonogenic Potential in Prostate Carcinoma Cells

“…Recently, Nasreen and colleagues (12) showed that silencing EphA2 expression by using short interfering RNA (siRNA) inhibits the proliferation and haptotaxis of malignant mesothelioma cells. In agreement, Landen and colleagues (13) showed that the therapeutic delivery of EphA2 siRNA into an orthotopic mouse model of ovarian cancer reduced tumor growth when compared with a nonsilencing siRNA. Moreover, Brantley-Sieders and colleagues (14) showed that EphA2 has a positive role during mammary tumor onset and growth in the MMTV-Neu transgenic mice model but not in mice overexpressing the polyomavirus middle T antigen, suggesting that, at least in breast carcinoma, EphA2 role in tumor progression depend on oncogene/tumor suppressor context.…”

Effectiveness of siRNA uptake in target tissues by various delivery methods

“…Higher levels of siRNA were noted in ovarian tumors when DOTAP was used compared to naked (unmodified) siRNA. 16 Undoubtedly, the type of vehicle utilized for siRNA delivery will affect tissue uptake and will require further refinement before clinical use.…”

Translation regulatory factor RBM3 is a proto-oncogene that prevents mitotic catastrophe

“…Xenograft tumor model siRNA was administered into the xenografts after incorporation into DOPC (1,2-Dioleoyl-sn-Glycero-3-Phosphocholine) (Avanti Polar Lipids, Alabaster, AL, USA) (Landen et al, 2005) generated by injecting HCT116 cells (6  10 6 cells) subcutaneously into the flanks of female athymic nude mice (NCr-nu) and housed in specific pathogen-free conditions. Tumors were measured with calipers and the volume calculated as (length  width 2 )  0.5.…”