[Show abstract][Hide abstract] ABSTRACT: 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.
The Journal of clinical investigation 03/2009; 119(3):661-73. · 15.39 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Human gene therapy 09/2008; 19(10):991-9. · 4.20 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Stabilized plasmid lipid particles (SPLP) consist of a single copy of DNA surrounded by a lipid bilayer. The particles are small ( approximately 100 nm), stable, monodisperse and have a low surface charge. A diffusible polyethylene glycol (PEG) coating attached to a lipid anchor is critical to the SPLP's functionality. The PEG-lipid exchanges out of the bilayer at a rate determined by the size of the lipid anchor. Here we show that SPLP can be prepared using a series of PEG-diacylglycerol lipids (PEG-S-DAGs). SPLP were prepared incorporating PEG-dimyristoylglycerol (C14), PEG-dipalmitoylglycerol (C16) or PEG-distearoylglycerol (C18) and the rate of PEG-lipid diffusion from the bi-layer determined using a FRET assay. SPLP pharmacokinetics confirm a correlation between the stability of the PEG-lipid component and circulation lifetime. PEG-S-DAGs with longer lipid anchors yield more stable SPLP particles with longer circulation half-lives yielding an increase in tumor delivery and gene expression. PEG-distearoylglycerol (C18) containing SPLP bypass so-called 'first pass' organs, including the lung, and elicit levels of gene expression in distal tumor tissue 100- to 1000-fold greater than that observed in any other tissue. The incorporation of PEG-S-DAG in SPLP confirms that small size, low surface charge and extended circulation lifetimes are prerequisite to the accumulation and tumor selective expression of plasmid DNA following systemic administration.
Biochimica et Biophysica Acta 06/2005; 1669(2):155-63. · 4.66 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A fully scalable and extrusion-free method was developed to prepare rapidly and reproducibly stabilized plasmid lipid particles (SPLP) for nonviral, systemic gene therapy.
Liposomes encapsulating plasmid DNA were formed instantaneously by mixing lipids dissolved in ethanol with an aqueous solution of DNA in a controlled, stepwise manner. Combining DNA-buffer and lipid-ethanol flow streams in a T-shaped mixing chamber resulted in instantaneous dilution of ethanol below the concentration required to support lipid solubility. The resulting DNA-containing liposomes were further stabilized by a second stepwise dilution.
Using this method, monodisperse vesicles were prepared with particle sizes less than 200 nm and DNA encapsulation efficiencies greater than 80%. In mice possessing Neuro 2a tumors, SPLP demonstrated a 13 h circulation half-life in vivo, good tumor accumulation and gene expression profiles similar to SPLP previously prepared by detergent dialysis. Cryo transmission electron microscopy analysis showed that SPLP prepared by stepwise ethanol dilution were a mixed population of unilamellar, bilamellar, and oligolamellar vesicles. Vesicles of similar lipid composition, prepared without DNA, were also <200 nm but were predominantly bilamellar with unusual elongated morphologies, suggesting that the plasmid particle affects the morphology of the encapsulating liposome. A similar approach was used to prepare neutral egg phosphatidylcholine:cholesterol (EPC:Chol) liposomes possessing a pH gradient, which was confirmed by the uptake of the lipophilic cation safranin O.
This new method will enable the scale-up and manufacture of SPLP required for preclinical and clinical studies. Additionally, this method now allows for the acceleration of SPLP formulation development, enabling the rapid development and evaluation of novel carrier systems.
Pharmaceutical Research 03/2005; 22(3):362-72. · 4.74 Impact Factor