Antitumor effects in hepatocarcinoma of isoform-selective inhibition of HDAC2.

Histone deacetylase 2 (HDAC2) is a chromatin modifier involved in epigenetic regulation of cell cycle, apoptosis, and differentiation that is upregulated commonly in human hepatocellular carcinoma (HCC). In this study, we show that specific targeting of this HDAC isoform is sufficient to inhibit HCC progression. siRNA-mediated silencing of HDAC inhibited HCC cell growth by blocking cell-cycle progression and inducing apoptosis. These effects were associated with deregulation of HDAC-regulated genes that control cell cycle, apoptosis, and lipid metabolism, specifically, by upregulation of p27 and acetylated p53 and by downregulation of CDK6 and BCL2. We found that HDAC2 silencing in HCC cells also strongly inhibited PPARγ signaling and other regulators of glycolysis (ChREBPα and GLUT4) and lipogenesis (SREBP1C and FAS), eliciting a marked decrease in fat accumulation. Notably, systemic delivery of HDAC2 siRNA encapsulated in lipid nanoparticles was sufficient to blunt the growth of human HCC in a murine xenograft model. Our findings offer preclinical proof-of-concept for HDAC2 blockade as a systemic therapy for liver cancer.

Definition of ubiquitination modulator COP1 as a novel therapeutic target in human hepatocellular carcinoma.

The development of targeted therapeutics for hepatocellular carcinoma (HCC) remains a major challenge. The ubiquitination modulator COP1 regulates p53 activity by ubiquitination and it is frequently overexpressed in human HCC. In this study, we tested the hypothesis that COP1 blockade by short interfering RNA (siRNA)-mediated inhibition could affect the course of HCC progression. The COP1 isoform COP1-1 was selected as the most effective target for siRNAs in terms of growth inhibition and apoptotic induction in several HCC cell lines. Growth inhibition occurred in HCC cells that retained wild-type p53 or expressed mutant p53 (Y220C or R249S), whereas p53-null Hep3B cells were resistant. Microarray expression analysis revealed that the antiproliferative effects of COP1 blockade were driven by a common subset of molecular alterations including a p53-associated functional network. In an orthotopic mouse xenograft model of HCC, systemic delivery of a modified COP1 siRNA by stable nucleic acid-lipid particles suppressed neoplastic growth in liver without unwanted immune responses. Our findings offer a first proof of principle that COP1 can be a promising target for systemic therapy of HCC.

Confirming the RNAi-mediated mechanism of action of siRNA-based cancer therapeutics in mice.

siRNAs that specifically silence the expression of cancer-related genes offer a therapeutic approach in oncology. However, it remains critical to determine the true mechanism of their therapeutic effects. Here, we describe the preclinical development of chemically modified siRNA targeting the essential cell-cycle proteins polo-like kinase 1 (PLK1) and kinesin spindle protein (KSP) in mice. siRNA formulated in stable nucleic acid lipid particles (SNALP) displayed potent antitumor efficacy in both hepatic and subcutaneous tumor models. This was correlated with target gene silencing following a single intravenous administration that was sufficient to cause extensive mitotic disruption and tumor cell apoptosis. Our siRNA formulations induced no measurable immune response, minimizing the potential for nonspecific effects. Additionally, RNAi-specific mRNA cleavage products were found in tumor cells, and their presence correlated with the duration of target mRNA silencing. Histological biomarkers confirmed that RNAi-mediated gene silencing effectively inhibited the target's biological activity. This report supports an RNAi-mediated mechanism of action for siRNA antitumor effects, suggesting a new methodology for targeting other key genes in cancer development with siRNA-based therapeutics.

RNAi-mediated gene silencing in non-human primates.

The opportunity to harness the RNA interference (RNAi) pathway to silence disease-causing genes holds great promise for the development of therapeutics directed against targets that are otherwise not addressable with current medicines. Although there are numerous examples of in vivo silencing of target genes after local delivery of small interfering RNAs (siRNAs), there remain only a few reports of RNAi-mediated silencing in response to systemic delivery of siRNA, and there are no reports of systemic efficacy in non-rodent species. Here we show that siRNAs, when delivered systemically in a liposomal formulation, can silence the disease target apolipoprotein B (ApoB) in non-human primates. APOB-specific siRNAs were encapsulated in stable nucleic acid lipid particles (SNALP) and administered by intravenous injection to cynomolgus monkeys at doses of 1 or 2.5 mg kg(-1). A single siRNA injection resulted in dose-dependent silencing of APOB messenger RNA expression in the liver 48 h after administration, with maximal silencing of >90%. This silencing effect occurred as a result of APOB mRNA cleavage at precisely the site predicted for the RNAi mechanism. Significant reductions in ApoB protein, serum cholesterol and low-density lipoprotein levels were observed as early as 24 h after treatment and lasted for 11 days at the highest siRNA dose, thus demonstrating an immediate, potent and lasting biological effect of siRNA treatment. Our findings show clinically relevant RNAi-mediated gene silencing in non-human primates, supporting RNAi therapeutics as a potential new class of drugs.

Design of noninflammatory synthetic siRNA mediating potent gene silencing in vivo.

Targeted silencing of disease-associated genes by synthetic short interfering RNA (siRNA) holds considerable promise as a novel therapeutic strategy. However, unmodified siRNA can be potent triggers of the innate immune response, particularly when associated with delivery vehicles that facilitate intracellular uptake. This represents a significant barrier to the therapeutic development of siRNA due to toxicity and off-target gene effects associated with this inflammatory response. Here we show that immune stimulation by synthetic siRNA can be completely abrogated by selective incorporation of 2'-O-methyl (2'OMe) uridine or guanosine nucleosides into one strand of the siRNA duplex. These noninflammatory siRNA, containing less than 20% modified nucleotides, can be readily generated without disrupting their gene-silencing activity. We show that, coupled with an effective systemic delivery vehicle, 2'OMe-modified siRNA targeting apolipoprotein B (apoB) can mediate potent silencing of its target mRNA, causing significant decreases in serum apoB and cholesterol. This is achieved at therapeutically viable siRNA doses without cytokine induction, toxicity, or off-target effects associated with the use of unmodified siRNA. This approach to siRNA design and delivery should prove widely applicable and represents an important step in advancing synthetic siRNA into a broad range of therapeutic areas.

Sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA.

Short interfering RNAs (siRNAs) that mediate specific gene silencing through RNA interference (RNAi) are widely used to study gene function and are also being developed for therapeutic applications. Many nucleic acids, including double- (dsRNA) and single-stranded RNA (ssRNA), can stimulate innate cytokine responses in mammals. Despite this, few studies have questioned whether siRNA may have a similar effect on the immune system. This could significantly influence the in vivo application of siRNA owing to off-target effects and toxicities associated with immune stimulation. Here we report that synthetic siRNAs formulated in nonviral delivery vehicles can be potent inducers of interferons and inflammatory cytokines both in vivo in mice and in vitro in human blood. The immunostimulatory activity of formulated siRNAs and the associated toxicities are dependent on the nucleotide sequence. We have identified putative immunostimulatory motifs that have allowed the design of siRNAs that can mediate RNAi but induce minimal immune activation.

Interleukin 15 controls both proliferation and survival of a subset of memory-phenotype CD8(+) T cells.

Previous work has shown that memory-phenotype CD44(hi) CD8(+) cells are controlled by a cytokine, interleukin (IL)-15. However, the dependency of CD44(hi) CD8(+) cells on IL-15 is partial rather than complete. Here, evidence is presented that CD44(hi) CD8(+) cells comprise a mixed population of IL-15-dependent and IL-15-independent cells. The major subset of CD122(hi) CD44(hi) CD8(+) cells is heavily dependent on IL-15 by three different parameters, namely (1) "bystander" proliferation induced via IFN-induced stimulation of the innate immune system, (2) normal "background" proliferation, and (3) T cell survival; IL-15 dependency is most extreme for the Ly49(+) subset of CD122(hi) CD44(hi) CD8(+) cells. In contrast to CD122(hi) cells, the CD122(lo) subset of CD44(hi) CD8(+) cells is IL-15 independent; likewise, being CD122(lo), CD44(hi) CD4(+) cells are IL-15 independent. Thus, subsets of memory-phenotype T cells differ radically in their sensitivity to IL-15.