GMPs for selective delivery of siRNA to phagocytic cells in mice

Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA.
Biochemical Journal (Impact Factor: 4.4). 03/2011; 436(2):351-62. DOI: 10.1042/BJ20110352
Source: PubMed


Phagocytic macrophages and dendritic cells are desirable targets for potential RNAi (RNA interference) therapeutics because they often mediate pathogenic inflammation and autoimmune responses. We recently engineered a complex 5 component glucan-based encapsulation system for siRNA (small interfering RNA) delivery to phagocytes. In experiments designed to simplify this original formulation, we discovered that the amphipathic peptide Endo-Porter forms stable nanocomplexes with siRNA that can mediate potent gene silencing in multiple cell types. In order to restrict such gene silencing to phagocytes, a method was developed to entrap siRNA-Endo-Porter complexes in glucan shells of 2-4 μm diameter in the absence of other components. The resulting glucan particles containing fluorescently labelled siRNA were readily internalized by macrophages, but not other cell types, and released the labelled siRNA into the macrophage cytoplasm. Intraperitoneal administration of such glucan particles containing siRNA-Endo-Porter complexes to mice caused gene silencing specifically in macrophages that internalized the particles. These results from the present study indicate that specific targeting to phagocytes is mediated by the glucan, whereas Endo-Porter peptide serves both to anchor siRNA within glucan particles and to catalyse escape of siRNA from phagosomes. Thus we have developed a simplified siRNA delivery system that effectively and specifically targets phagocytes in culture or in intact mice.

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    • "The hollow and porous material properties of GPs allow for the encapsulation , transport, delivery, and release of electrostatically bound payloads. Previously we have reported the use of GPs for macrophage-targeted delivery of soluble payload macromolecules (i.e., proteins [23], DNA [24], and siRNA [25] [26]), and small drug molecules, such as the antibiotic rifampicin [27]. However, the use of GPs for small drug molecule delivery is limited since the majority of small drug molecules are neutral, monovalent in charge, or insoluble in water, and such payloads are not easily trapped within glucan particles using the polyplex core or LbL encapsulation methods developed for nucleic acids and proteins. "
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    ABSTRACT: Glucan particles (GPs) are hollow, porous 2–4 μ m microspheres derived from the cell walls of Baker's yeast ( Saccharomyces cerevisiae ). The 1,3- β -glucan outer shell provides for receptor-mediated uptake by phagocytic cells expressing β -glucan receptors. GPs have been used for macrophage-targeted delivery of soluble payloads (DNA, siRNA, protein, and small molecules) encapsulated inside the hollow GPs via core polyplex and layer-by-layer (LbL) synthetic strategies. In this communication, we report the incorporation of nanoparticles as cores inside GPs (GP-NP) or electrostatically bound to the surface of chemically derivatized GPs (NP-GP). GP nanoparticle formulations benefit from the drug encapsulation properties of NPs and the macrophage-targeting properties of GPs. GP nanoparticle formulations were synthesized using fluorescent anionic polystyrene nanoparticles allowing visualization and quantitation of NP binding and encapsulation. Mesoporous silica nanoparticles (MSNs) containing the chemotherapeutic doxorubicin (Dox) were bound to cationic GPs. Dox-MSN-GPs efficiently delivered Dox into GP phagocytic cells resulting in enhanced Dox-mediated growth arrest.
    01/2012; 2012(6):143524. DOI:10.1155/2012/143524
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    ABSTRACT: RNA interference (RNAi) is a robust gene silencing mechanism that degrades mRNAs complementary to the antisense strands of double-stranded, short interfering RNAs (siRNAs). As a therapeutic strategy, RNAi has an advantage over small-molecule drugs, as virtually all genes are susceptible to targeting by siRNA molecules. This advantage is, however, counterbalanced by the daunting challenge of achieving safe, effective delivery of oligonucleotides to specific tissues in vivo. Lipid-based carriers of siRNA therapeutics can now target the liver in metabolic diseases and are being assessed in clinical trials for the treatment of hypercholesterolemia. For this indication, a chemically modified oligonucleotide that targets endogenous small RNA modulators of gene expression (microRNAs) is also under investigation in clinical trials. Emerging 'self-delivery' siRNAs that are covalently linked to lipophilic moieties show promise for the future development of therapies. Besides the liver, inflammation of the adipose tissue in patients with obesity and type 2 diabetes mellitus may be an attractive target for siRNA therapeutics. Administration of siRNAs encapsulated within glucan microspheres can silence genes in inflammatory phagocytic cells, as can certain lipid-based carriers of siRNA. New technologies that combine siRNA molecules with antibodies or other targeting molecules also appear encouraging. Although still at an early stage, the emergence of RNAi-based therapeutics has the potential to markedly influence our clinical future.
    Nature Reviews Endocrinology 04/2011; 7(8):473-84. DOI:10.1038/nrendo.2011.57 · 13.28 Impact Factor
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    ABSTRACT: RNA interference (RNAi) has been extensively employed for in vivo research since its use was first demonstrated in mammalian cells 10 years ago. Design rules have improved, and it is now routinely possible to obtain reagents that suppress expression of any gene desired. At the same time, increased understanding of the molecular basis of unwanted side effects has led to the development of chemical modification strategies that mitigate these concerns. Delivery remains the single greatest hurdle to widespread adoption of in vivo RNAi methods. However, exciting advances have been made and new delivery systems under development may help to overcome these barriers. This review discusses advances in RNAi biochemistry and biology that impact in vivo use and provides an overview of select publications that demonstrate interesting applications of these principles. Emphasis is placed on work with synthetic, small interfering RNAs (siRNAs) published since the first installment of this review which appeared in 2006.
    Molecular Therapy 12/2011; 20(3):483-512. DOI:10.1038/mt.2011.263 · 6.23 Impact Factor
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