Anionic polymers for decreased toxicity and enhanced in vivo delivery of siRNA complexed with cationic liposomes

Abstract

We recently reported a cationic lipid-based vector of siRNA, termed siRNA lipoplex that was very efficient in specific gene silencing, both in cell culture and in mouse disease models. To be more efficient, this vector included the addition of a plasmid DNA as an anionic “cargo.” Although this plasmid DNA was devoid of any eukaryotic expression cassette, we decided to replace it by an anionic polymer that would be more acceptable for clinical applications. We identified seven anionic polymers, regarded as non-toxic, biodegradable, of various characteristics and nature. The addition of polymers to siRNA lipoplexes led to the formation of particles with similar characteristics to crude siRNA lipoplexes, decreased cellular toxicity and variable in vitro gene silencing efficiency depending on the type of polymer used. Upon i.v. injection in mice, siRNA lipoplexes prepared with the best polymer, polyglutamate, led to significantly increased recovery of siRNA in liver and lung compared with lipoplexes without polymer.

Introduction

Since its discovery by Fire et al. [1], RNA interference (RNAi) has emerged as a promising alternative to the more classical antisense approaches. This interference effect is a sequence-specific gene silencing process that is mediated by 21- to 25-nucleotide RNA cleavage products of longer double-stranded RNAs (dsRNAs). Introduction in mammalian cells of synthetic versions of 21-nucleotide RNA duplexes, termed siRNAs (small interfering RNAs), with perfect homology to their target sequences can induce specific gene silencing after transfection in cells. Currently, hundreds of reports that involve in vivo administration of siRNAs in mammals have been published (reviewed in Ref. [2]). The power of gene silencing with nucleic acid molecules is undisputed as a tool in the laboratory, and RNA-based medicines are now working their way into the clinic [3]. Although the number of technologies showing efficacy in animal models is growing rapidly, some have been associated with either clinically unsustainable dosages [4] or with non specific and adverse side effects due either to the delivery vehicle or immuno-stimulatory effects of the siRNA payload [5], [6], [7]. The principal challenge that remains in achieving the broadest application of siRNA therapeutics is the hurdle of delivery.

It was initially thought that, for in vivo siRNA applications, naked siRNA was biologically stable enough to be effective without requiring a delivery system. However, more recent studies have highlighted both (i) the need for chemical modification to enhance siRNA stability in the biological milieu and decrease immuno-stimulatory effects of siRNA and (ii) the need for specific delivery systems to enhance both biological stability and delivery to target cells and/or organs [8]. Due to its large molecular weight and polyanionic nature, naked siRNA does not freely cross the cell membrane. A wide variety of methods have been used to facilitate the delivery of nucleic acids to cells in culture or to whole organism, from cationic chemical agents (cationic lipids, liposomes, polymers or peptides) to physical methods (electrotransfer). Many vectors used for gene delivery can be adapted for the delivery of siRNA [2], [9], [10]. In addition, unlike DNA that has to reach the nucleus to be transcribed, siRNA only needs to be delivered to cytoplasm.

Cationic lipids have been shown to significantly reduce necessary siRNA doses for efficient gene silencing by enhancing siRNA stability in the serum and improving cellular uptake [11]. They rely on DNA compaction by electrostatic interaction between DNA phosphates and cationic lipids to form lipoplexes. Their efficacy depends on their ability to overcome several extra- and intracellular barriers that the particles encounter between the site of delivery and the intracellular targeted site. We have designed a new vector of cytokine targeted siRNA, based on cationic lipid, which proved to be efficient in restoring immunological balance in a mouse arthritis model following intravenous injection [12], [13], [14]. We showed that weekly injections of siRNAs targeted to IL-1, IL-6 or IL-18, delivered in combination and formulated with cationic liposomes significantly reduced all pathological rheumatoid arthritis features [13]. We determined that the preferential immune cell type targeted by the siRNA complexes was macrophages, the main inflammatory cytokine producer cells in rheumatoid arthritis. This efficient siRNA vector is formed by mixing a lipopolyamine-containing cationic liposome with a premixed solution of siRNA and plasmid DNA (pDNA) acting as an anionic “cargo.” We have recently shown that the addition of pDNA cargo to siRNA prior to form the complex with cationic liposome leads to enhanced gene silencing efficiency with reduced siRNA concentrations [15]. However, we suspected that the addition of a DNA molecule in siRNA lipoplexes will not be acceptable in a clinical application since pDNA is a biomolecule that contains coding sequences.

In the present study, we examined the feasibility to replace pDNA by an anionic polymer that would be clinically approvable. We prepared siRNA lipoplexes with anionic polymers, and assayed their gene silencing efficiency as well as their physico-chemical characteristics. When added to siRNA lipoplexes, these anionic polymers increase their gene silencing efficiency. Among seven candidates, we identified a non toxic and biodegradable polymer, which is already used in biomedical applications. We prepared pegylated and unpegylated siRNA lipoplexes and assayed siRNA recovery after their injection in mice.