Lipid nanoparticle siRNA systems for silencing the androgen receptor in human prostate cancer in vivo

Abstract

The androgen receptor (AR) plays a critical role in the progression of prostate cancer. Silencing this protein using shorthairpin RNA (shRNA) has been correlated with tumor growth inhibition and decreases in serum prostate specific antigen (PSA). In our study, we have investigated the ability of lipid nanoparticle (LNP) formulations of small-interfering RNA (siRNA) to silence AR in human prostate tumor cell lines in vitro and in LNCaP xenograft tumors following intravenous (i.v.) injection. In vitro screening studies using a panel of cationic lipids showed that LNPs containing the ionizable cationic lipid 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA) exhibited the most potent AR silencing effects in LNCaP cells. This is attributed to an optimized ability of DLin-KC2-DMA-containing LNP to be taken up into cells and to release the siRNA into the cell cytoplasm following endocytotic uptake. DLin-KC2-DMA LNPs were also effective in silencing the AR in a wild-type AR expressing cell line, LAPC-4, and a variant AR expressing cell line, CWR22Rv1. Importantly, it is demonstrated that LNP ARsiRNA systems containing DLin-KC2-DMA can silence AR gene expression in distal LNCaP xenograft tumors and decrease serum PSA levels following i.v. injection. To our knowledge, this is the first report demonstrating the feasibility of LNP delivery of siRNA for silencing AR gene expression in vivo.Prostate cancer is the most frequently diagnosed nonskin cancer and the second-leading cause of cancer-related deaths in males.

siRNA Lipid Nanoparticle Potently Silences Clusterin and Delays Progression When Combined with Androgen Receptor Cotargeting in Enzalutamide-Resistant Prostate Cancer

“…The ionizable cationic lipid, DLin-KC2-DMA, is highly active in the liver (31) and also previously shown to silence AR and reduce PSA levels upon intravenous administration in LNCaP tumor model (16). PC-3M-luc cells showed significant correlation between mean bioluminescence and both the total numbers of these cells in vitro ( Supplementary Fig.…”

“…Although monotargeting of AR or CLU with ASO or siRNA has been reported previously (16,24,35), and combinatorial cotargeting the AR and CLU in ENZ-R CRPC with nucleotide-based therapeutics is biologically rational, combining two antisense drugs in vivo is precluded by toxicity induced by doubling the dose of the oligonucleotide backbone. To circumvent this backbone toxicity limitation, we combined the AR-ASO with a CLU LNP-siRNA formulation.…”

“…To assess the effects of LNP siRNA tumor delivery in vivo, the expression of luciferase in PC-3M-luc stably transfected cells treated with LNP LUC-siRNA containing DLin-KC2-DMA (16,31) was examined by the IVIS imaging system. The ionizable cationic lipid, DLin-KC2-DMA, is highly active in the liver (31) and also previously shown to silence AR and reduce PSA levels upon intravenous administration in LNCaP tumor model (16).…”

“…Propensity of LNP systems to accumulate in the liver has become a major obstacle in the extrahepatic delivery of siRNA-LNP systems. Although preclinical studies in prostate cancer (16) and immune cells (17) are promising, distant tumor delivery has not been tested in the clinic. In order to mitigate this higher Peg-lipid content is used to improve the pharmacodynamic and biodistribution of LNP systems to the tumor site (18)(19)(20).…”

The Niemann-Pick C1 Inhibitor NP3.47 Enhances Gene Silencing Potency of Lipid Nanoparticles Containing siRNA

“…22 The third area of interest concerns whether enhanced gene silencing potency observed in vitro in the presence of NP3.47 can be extended to in vivo situations. As noted elsewhere the gene silencing potency of LNP-siRNA systems for hepatocyte targets is extremely high, requiring doses of as little as 10 μg/kg body weight to achieve 50% gene silencing; 3,4 however, for other tissues dose levels of 1 mg/kg body weight or more are required, [23][24][25] limiting clinical applications. The addition of agents such as NP3.47 to enhance potency would appear an attractive option, however the problems associated with the likely toxicities of In this regard we have recently shown 26 that a hydrophobic prodrug derivative of dexamethasone, when associated with LNP systems is a 20-fold or more potent for suppressing immunostimulatory effects of encapsulated RNA or DNA as compared with free dexamethasone.…”