Delivery of siRNA to the mouse lung via a functionalized lipopolyamine.

We have designed a series of versatile lipopolyamines which are amenable to chemical modification for in vivo delivery of small interfering RNA (siRNA). This report focuses on one such lipopolyamine (Staramine), its functionalized derivatives and the lipid nanocomplexes it forms with siRNA. Intravenous (i.v.) administration of Staramine/siRNA nanocomplexes modified with methoxypolyethylene glycol (mPEG) provides safe and effective delivery of siRNA and significant target gene knockdown in the lungs of normal mice, with much lower knockdown in liver, spleen, and kidney. Although siRNA delivered via Staramine is initially distributed across all these organs, the observed clearance rate from the lung tissue is considerably slower than in other tissues resulting in prolonged siRNA accumulation on the timescale of RNA interference (RNAi)-mediated transcript depletion. Complete blood count (CBC) analysis, serum chemistry analysis, and histopathology results are all consistent with minimal toxicity. An in vivo screen of mPEG modified Staramine nanocomplexes-containing siRNAs targeting lung cell-specific marker proteins reveal exclusive transfection of endothelial cells. Safe and effective delivery of siRNA to the lung with chemically versatile lipopolyamine systems provides opportunities for investigation of pulmonary cell function in vivo as well as potential treatments of pulmonary disease with RNAi-based therapeutics.

Synthesis and application of a non-viral gene delivery system for immunogene therapy of cancer.

The synthesis and gene delivery application of a novel lipopolymer, PEG-PEI-CHOL (PPC), is described. PPC is composed of a low molecular weight branched polyethylenimine (PEI) covalently linked with functional groups methoxypolyethyleneglycol (PEG) and cholesterol (CHOL). The potential utility of PPC as a gene delivery polymer was evaluated by showing its ability to form stable nanocomplexes with DNA, protect DNA from degradation by DNase and mediate gene transfer in vitro and in vivo in solid tumors. The ratio of PEG/PEI/CHOL and nitrogen to phosphate (Polymer/DNA) was optimized for physico-chemical properties and gene delivery efficiency of PPC/DNA complexes. The gene therapy application of the polymer was shown following administration of a murine IL-12 plasmid (pmIL-12) formulated with PPC into tumors in mice which resulted in significant inhibition of tumor growth. The inhibitory effects of pmIL-12/PPC were enhanced when combined with specific chemotherapeutic agents, demonstrating the potential usefulness of pIL-12/PPC as an adjuvant therapy for cancer treatment.

Lipid nanoparticle delivery of a microRNA-145 inhibitor improves experimental pulmonary hypertension.

Therapies that exploit RNA interference (RNAi) hold great potential for improving disease outcomes. However, there are several challenges that limit the application of RNAi therapeutics. One of the most important challenges is effective delivery of oligonucleotides to target cells and reduced delivery to non-target cells. We have previously developed a functionalized cationic lipopolyamine (Star:Star-mPEG-550) for in vivo delivery of siRNA to pulmonary vascular cells. This optimized lipid formulation enhances the retention of siRNA in mouse lungs and achieves significant knockdown of target gene expression for at least 10days following a single intravenous injection. Although this suggests great potential for developing lung-directed RNAi-based therapies, the application of Star:Star-mPEG mediated delivery of RNAi based therapies for pulmonary vascular diseases such as pulmonary arterial hypertension (PAH) remains unknown. We identified differential expression of several microRNAs known to regulate cell proliferation, cell survival and cell fate that are associated with development of PAH, including increased expression of microRNA-145 (miR-145). Here we test the hypothesis that Star:Star-mPEG mediated delivery of an antisense oligonucleotide against miR-145 (antimiR-145) will improve established PAH in rats. We performed a series of experiments testing the in vivo distribution, toxicity, and efficacy of Star:Star-mPEG mediated delivery of antimiR-145 in rats with Sugen-5416/hypoxia induced PAH. We showed that after subchronic therapy of three intravenous injections over 5weeks at 2mg/kg, antimiR-145 accumulated in rat lung tissue and reduced expression of endogenous miR-145. Using a novel in situ hybridization approach, we demonstrated substantial distribution of antimiR-145 in the lungs as well as the liver, kidney, and spleen. We assessed toxic effects of Star:Star-mPEG/antimiR-145 with serial complete blood counts of leukocytes and serum metabolic panels, gross pathology, and histopathology and did not detect significant off-target effects. AntimiR-145 reduced the degree of pulmonary arteriopathy, reduced the severity of pulmonary hypertension, and reduced the degree of cardiac dysfunction. The results establish effective and low toxicity of lung delivery of a miRNA-145 inhibitor using functionalized cationic lipopolyamine nanoparticles to repair pulmonary arteriopathy and improve cardiac function in rats with severe PAH.

Synthetic tumor networks for screening drug delivery systems.

Tumor drug delivery is a complex phenomenon affected by several elements in addition to drug or delivery vehicle's physico-chemical properties. A key factor is tumor microvasculature with complex effects including convective transport, high interstitial pressure and enhanced vascular permeability due to the presence of "leaky vessels". Current in vitro models of the tumor microenvironment for evaluating drug delivery are oversimplified and, as a result, show poor correlation with in vivo performance. In this study, we report on the development of a novel microfluidic platform that models the tumor microenvironment more accurately, with physiologically and morphologically realistic microvasculature including endothelial cell lined leaky capillary vessels along with 3D solid tumors. Endothelial cells and 3D spheroids of cervical tumor cells were co-cultured in the networks. Drug vehicle screening was demonstrated using GFP gene delivery by different formulations of nanopolymers. The synthetic tumor network was successful in predicting in vivo delivery efficiencies of the drug vehicles. The developed assay will have critical applications both in basic research, where it can be used to develop next generation delivery vehicles, and in drug discovery where it can be used to study drug transport and delivery efficacy in realistic tumor microenvironment, thereby enabling drug compound and/or delivery vehicle screening.

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.

A safety and efficacy study of local delivery of interleukin-12 transgene by PPC polymer in a model of experimental glioma.

Interleukin-12 (IL-12) triggers an antitumoral immune response and an antiangiogenic effect against cancer. In this study, we tested a novel polymeric vehicle for IL-12 gene therapy along with adjuvant local biodegradable carmustine (BCNU) chemotherapy for the treatment of malignant glioma. Highly concentrated DNA/PPC (polyethylenimine covalently modified with methoxypolyethyleneglycol and cholesterol) complexes were used to deliver a murine plasmid encoding IL-12 (pmIL-12). For toxicity assessment, mice received intracranial injections with different volumes of pmIL-12/PPC. For efficacy, mice with intracranial GL261 glioma were treated with local delivery of pmIL-12/PPC and/or BCNU-containing polymers. Intracranial injections of 5-10 microl of pmIL-12/PPC were well tolerated and led to IL-12 expression in the brains of treated animals. Treatment with pmIL-12/PPC led to a significant increase in survival compared with untreated mice (median survival 57 days; 25% long-term survival >95 vs. 45 days for control; P<0.05). Treatment with BCNU led to a significant increase in survival compared with untreated mice, with 75% of treated mice having a long-term survival >95 days, (P<0.05). Most importantly, the combination of BCNU and pmIL-12/PPC led to a survival of 100% of the mice for 95 days after treatment (P<0.0001). This novel strategy is safe and effective for the treatment of malignant glioma. The synergy resultant from the combination of locally administered pmIL-12/PPC and BCNU suggests a role for this approach in the treatment of malignant brain tumors.