Gregory Slobodkin

Treatment of disseminated ovarian cancer using nonviral interleukin‐12 gene therapy delivered intraperitoneally

The poor prognosis associated with ovarian cancer is primarily the result of delayed diagnosis and the lack of an effective treatment for advanced disease. Use of novel immunotherapy strategies are being evaluated that work to enhance local and systemic immune responses against cancer cells and can possibly work together with traditional cytotoxic chemotherapy regimens to produce more effective treatment options.

Versatile cationic lipids for siRNA delivery.

Exploitation of the RNA interference (RNAi) pathway offers the promise of new and effective therapies for a wide variety of diseases. Clinical development of new drugs based on this platform technology is still limited, however, by a lack of safe and efficient delivery systems. Here we report the development of a class of structurally versatile cationic lipopolyamines designed specifically for delivery of siRNA which show high levels of target transcript knockdown in a range of cell types in vitro. A primary benefit of these lipids is the ease with which they may be covalently modified by the addition of functional molecules. For in vivo applications one of the core lipids (Staramine) was modified with methoxypolyethylene glycols (mPEGs) of varying lengths. Upon systemic administration, PEGylated Staramine nanoparticles containing siRNA targeting the caveolin-1 (Cav-1) transcript caused a reduction of the Cav-1 transcript of up to 60%, depending on the mPEG length, specifically in lung tissue after 48h compared to treatment with non-silencing siRNA. In addition, modification with mPEG reduced toxicity associated with intravenous administration. The ability to produce a high level of target gene knockdown in the lung with minimal toxicity demonstrates the potential of these lipopolyamines for use in developing RNAi therapeutics for pulmonary disease.

Abstract 5535: Delivery of siRNA via functionalized lipopolyamine systems

The efficient delivery of siRNA molecules to target cells is critical for the application of RNAi to the treatment of human cancers. To this end, we have developed novel cationic lipopolyamime structures that can be used to complex or encapsulate siRNA into nanoparticles for efficient in vitro or in vivo delivery. Unlike conventional cationic delivery systems, these lipopolyamines are chemically flexible which allow for covalent attachment of functional moieties which can modulate their biological activity. For example, modification of these systems with serum stabilizing agents allows for modulation of physical parameters important for biological properties such as toxicity and biodistribution. Intravenous (iv) injection of these functionalized nanoparticles showed preferential distribution to the lungs and corresponding depletion of targeted mRNA transcripts from lung tissue. We have investigated these systems for use in delivering siRNA targeting VEGF in mice with advanced disseminated ovarian cancer through intraperitoneal (ip) injection of siRNA-lipopolyamine nanoparticles. This model was established by injecting animals (ip) with 2.5×10 6 ID8 cells (malignant transformed ovarian surface epithelial cells that have been further modified to overexpress VEGF protein). Delivery of serum stabilized complexes to the peritoneal cavity of mice with disseminated ovarian cancer resulted in significant target gene knockdown in cells obtained from the malignant ascites fluid of these animals with modest knockdown in the peritoneal tumors themselves. A further modification of the core lipopolyamine structure incorporated attachment of a targeting peptide ligand through a linker molecule. Physico-chemical characterization of this targeted system and in vitro assays with ID8 cancer cells demonstrated specific delivery of siRNAs dependent on the presence of the targeting ligand on the liposome and the targeted receptor on the cell surface. These results have prompted in vivo testing using the targeted delivery systems to assess siRNA distribution as a function of delivered dose. Additional studies are underway that will establish the optimized delivery conditions for use in tumor bearing animals. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5535.

279. A Novel Functionalized Polymeric Formulation for use in Delivering Therapeutic Genes for the Treatment of Solid Tumors

We have described the synthesis, physico-chemical properties and gene therapy application of a novel water soluble gene delivery system PEG-PEI-Chol (PPC). PPC is composed of a low molecular weight branched polyethyleneimine, cholesterol and polyethylene glycol and designed to promote DNA condensation into nanoparticles, enhance membrane uptake and improve biocompatibility and pharmacokinetics, respectively. The chemical composition and molar ratios between the individual components of the polymer were verified by NMR. Formulation of PPC with DNA produced particles of |[sim]|100nm mean diameter and zeta potential of |[sim]|20mV. In vitro optimization studies using a luciferase reporter gene were performed in order to evaluate the effects of varying amounts of PEG and increasing nitrogen to phosphorus ratio (N:P). Use of the PPC delivery system with a plasmid encoding for interleukin-12 (IL-12) for treatment of solid tumors was examined in mice. Significant inhibition in tumor growth rate and/or increased incidence of complete tumor rejection was seen in murine models of breast cancer and head and neck cancer following intratumoral injections. A combination therapy approach was evaluated where the IL-12/PPC treatment was combined with an investigative polymeric formulation of paclitaxel against 4T1 mammary tumors and it was shown that combined therapy led to a synergistic inhibition of tumor growth. This form of paclitaxel had little effect on SCCVII head and neck tumors however, cyclophosphamide strongly inhibited SCCVII tumor growth. Further, when IL-12/PPC intratumoral injections were combined with cyclophosphamide administration, there was a slight additional inhibition in tumor growth over the cyclophosphamide treatment alone and there was a significant increase in the rate of complete tumor rejection. Future studies will be aimed at optimizing dose and dosing regimens of combination therapies for the treatment of solid tumors.