Kevin McClintock

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.

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.

Postexposure Protection of Guinea Pigs against a Lethal Ebola Virus Challenge Is Conferred by RNA Interference

Abstract BackgroundEbola virus (EBOV) infection causes a frequently fatal hemorrhagic fever (HF) that is refractory to treatment with currently available antiviral therapeutics. RNA interference represents a powerful, naturally occurring biological strategy for the inhibition of gene expression and has demonstrated utility in the inhibition of viral replication. Here, we describe the development of a potential therapy for EBOV infection that is based on small interfering RNAs (siRNAs) MethodsFour siRNAs targeting the polymerase (L) gene of the Zaire species of EBOV (ZEBOV) were either complexed with polyethylenimine (PEI) or formulated in stable nucleic acid–lipid particles (SNALPs). Guinea pigs were treated with these siRNAs either before or after lethal ZEBOV challenge ResultsTreatment of guinea pigs with a pool of the L gene–specific siRNAs delivered by PEI polyplexes reduced plasma viremia levels and partially protected the animals from death when administered shortly before the ZEBOV challenge. Evaluation of the same pool of siRNAs delivered using SNALPs proved that this system was more efficacious, as it completely protected guinea pigs against viremia and death when administered shortly after the ZEBOV challenge. Additional experiments showed that 1 of the 4 siRNAs alone could completely protect guinea pigs from a lethal ZEBOV challenge ConclusionsFurther development of this technology has the potential to yield effective treatments for EBOV HF as well as for diseases caused by other agents that are considered to be biological threats

Misinterpreting the Therapeutic Effects of Small Interfering RNA Caused by Immune Stimulation

Activation of innate immunity has direct effects in modulating viral replication, tumor growth, angiogenesis, and inflammatory and other immunological processes. It is now established that unmodified siRNA can activate this innate immune response and therefore there is real potential for siRNA to elicit nonspecific therapeutic effects in a wide range of disease models. Here we demonstrate that in a murine model of influenza infection, the antiviral activity of siRNA is due primarily to immune stimulation elicited by the active siRNA duplexes and is not the result of therapeutic RNA interference (RNAi) as previously reported. We show that the misinterpretation stems from the use of a particular control green fluorescent protein (GFP) siRNA that we identify as having unusually low immunostimulatory activity compared with the active anti-influenza siRNA. Curiously, this GFP siRNA has served as a negative control for a surprising number of groups reporting therapeutic effects of siRNA. The inert immunologic profile of the GFP sequence was unique among a broad panel of published siRNAs, all of which could elicit significant interferon induction from primary immune cells. This panel included eight active siRNAs against viral, angiogenic, and oncologic targets, the reported therapeutic efficacy of which was based on comparison with the nonimmunostimulatory GFP siRNA. These results emphasize the need for researchers to anticipate, monitor, and adequately control for siRNA-mediated immune stimulation and calls into question the interpretation of numerous published reports of therapeutic RNAi in vivo. The use of chemically modified siRNA with minimal immunostimulatory capacity will help to delineate more accurately the mechanism of action underlying such studies.

Abstract 2829: Preclinical characterization of TKM-080301, a lipid nanoparticle formulation of a small interfering RNA directed against polo-like kinase 1

Small interfering RNAs (siRNAs) have tremendous potential for the selective inhibition, or ‘silencing’, of genes involved in cancer cell growth and division. This inhibition occurs through a process known as RNA interference (RNAi). Polo-like kinase 1 (PLK1) is a target that has multiple critical roles in cell cycle regulation and cytokinesis. Here we describe the preclinical characterization of TKM-080301, a lipid nanoparticle (LNP) formulation of an siRNA directed against human PLK1 mRNA. Studies were performed to assess the in vitro pharmacologic activity and inherent immune stimulatory potential of various siRNAs. PLK1 siRNA formulated in LNP resulted in potent anti-proliferative activity and gene-specific silencing in many cancer cell lines; and TKM-080301 exhibited strong anti-tumor activity in several xenograft models of human cancer, including tumors implanted intra-hepatically and subcutaneously. RNAi and the intended pharmacologic effects were confirmed in these models by histopathology, to visualize mitotic disruption, and by molecular methods, to confirm the presence of the RNAi-induced PLK1 mRNA cleavage product, the degree of PLK1 silencing relative to housekeeping genes, and the duration of silencing. In vivo, PLK1 silencing persisted for up to 7-10 days after a single administration and, importantly, occurred in the absence of any measurable stimulation of the innate immune system. Finally, unlike most small molecule PLK1 inhibitors, which are highly myelosuppresive, the toxicity profile of TKM-080301 was governed by the distribution profile of the LNP and toxicity was largely restricted to the liver and spleen. Collectively, these studies support the clinical evaluation of TKM-080301 as a new approach to targeting PLK1 in solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2829. doi:10.1158/1538-7445.AM2011-2829

Abstract B204: Development of ALN‐VSP: An RNAi therapeutic for liver malignancies

Malignancies of the liver, including primary (hepatocellular carcinoma) and secondary (metastatic) tumors, represent a significant unmet medical need. We are developing a therapeutic for solid tumors involving the liver that is comprised of lipid particle‐formulated small interfering RNAs (siRNAs) targeting VEGF and the mitotic kinesin, KSP (Eg5). For each target, potent siRNA duplexes were selected following extensive screening in tissue culture cells. To assess efficacy in vivo , a stable nucleic acid lipid particle (SNALP) formulation was developed based on similar formulations previously shown to silence liver‐expressed genes via systemic administration in multiple species. A SNALP‐formulated combination of the KSP and VEGF siRNAs (referred to as ALN‐VSP) was tested in orthotopic liver tumor models in which human tumor cells are implanted directly into the livers of immunocompromised mice. We show that intravenous administration of ALN‐VSP leads to silencing of both KSP and VEGF expression in established liver tumors. This is accompanied by the formation of numerous aberrant mitotic figures (“monoasters”) in tumor cells indicative of the pharmacologic inhibition of KSP. In addition, we demonstrate that ALN‐VSP treatment provides a clear survival benefit even when treatment is initiated in animals with a significant tumor burden. A Phase 1 clinical trial of ALN‐VSP has recently been initiated. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B204.

798. Anti-Influenza-Viral Efficacy Attributed to siRNA Formulated as Simple Lipoplex or PEI Polyplexes Is a Result of Their Immunostimulatory Properties

A number of groups have published reports of antiviral efficacy associated with the delivery of siRNA directed against the influenza virus using either lipoplex or polyplex mediated siRNA delivery. In order to assess the extent to which the immunostimulatory properties of siRNA may have contributed to these results we developed non- immunostimulatory analogues of the previously published NP1496 and PA2087 siRNA. siRNA were prepared containing minimal chemical modifications previously shown to abrogate immune stimulation while retaining the ability to mediate RNAi. The immunostimulatory properties of all siRNA were confirmed by intravenous injection of liposome encapsulated siRNA followed by determination of interferon alpha in the plasma 6h post-injection in mice. When tested in an in vitro cell-based system the modified, non-stimulatory siRNA potently inhibited influenza virus replication in MDCK cells. However, the modified siRNA lost all previously observed antiviral efficacy when delivered to mice infected with Influenza A/PR/8/34. Mice were treated either intranasally using lipoplex (Oligofectamine TM) at 1 mg/kg, intranasally as ‘naked’ siRNA at 12.5 mg/kg or by intravenous administration as PEI polyplex at 6 mg/kg. This was in contrast to previously reported studies yielding substantial antiviral efficacy when using the immunostimulatory parent molecules. When these studies were repeated using the immunostimulatory parent molecules we observed a significant reduction in viral titre in the mouse lung as measured by HA EIA. The results suggest caution when interpreting results obtained in vivo using immunostimulatory siRNA, in particular within the context of anti-viral applications.