Raphael Darcy

Therapeutic targeting in the silent era: advances in non-viral siRNA delivery

Gene silencing using RNA-interference, first described in mammalian systems almost a decade ago, is revolutionizing therapeutic target validation efforts both in vitro and in vivo. Moreover, the potential for using short interfering RNA (siRNA) as a therapy in its own right is also progressing at a significant pace. However, the widespread use of such approaches is contingent on having appropriate systems to achieve clinically appropriate, safe, and efficient delivery of siRNA. There are many physicochemical and biological barriers to such delivery, and a growing emphasis on the design and characterisation of non-viral technologies that will overcome these barriers and expedite targeted delivery. This review discusses the considerations and challenges associated with use of siRNA-based therapeutics, including stability and off-target effects. Speculation is made on the properties of an ideal delivery system and the non-viral delivery approaches used to date, both in vitro and in vivo, are classified and discussed. Moreover, the ability of cyclodextrin-based delivery vectors to fulfil many of the criteria of an ideal delivery construct is also elaborated.

Anisamide-targeted cyclodextrin nanoparticles for siRNA delivery to prostate tumours in mice

A hepta-guanidino-β-cyclodextrin (G-CD), its hepta-PEG conjugate (G-CD-PEG), and the corresponding anisamide-terminated PEG conjugate (G-CD-PEG-AA) have been synthesised and compared as delivery vectors for siRNA to prostate cancer cells and tumours in vivo. The G-CD-PEG-AA.siRNA formulations (in which anisamide targets the sigma receptor), but not the non-targeted formulations, induced prostate cell-specific internalisation of siRNA resulting in approximately 80% knockdown in vitro of the reporter gene, luciferase. Following intravenous administration of the anisamide-targeted formulation in a mouse prostate tumour model significant tumour inactivation with corresponding reductions in the level of vascular endothelial growth factor (VEGF) mRNA were achieved, without demonstrating enhanced toxicity. This data imply significant potential for anisamide-conjugated cyclodextrin vectors for targeted delivery of therapeutic siRNAs in the treatment of prostate cancer.

Novel Amphiphilic Cyclodextrins: Graft-Synthesis of Heptakis(6-alkylthio-6-deoxy)-β-cyclodextrin 2-Oligo(ethylene glycol) Conjugates and Their ω-Halo Derivatives

Novel amphiphilic cyclodextrins were synthesised by grafting oligo(ethylene glycol) units onto the secondary side of heptakis(6-alkylthio-6-deoxy)-β-cyclodextrins (alkyl = ethyl, hexyl, dodecyl and hexadecyl). The oligo(ethylene glycol) substituents were introduced by reaction of heptakis(6-alkylthio-6-deoxy)-β-cyclodextrins with ethylene carbonate in the presence of potassium carbonate at elevated temperatures. The resulting oligo(ethylene glycol)-cyclodextrin conjugates were characterised by 1H NMR and 13C NMR, COSY and HSQC spectroscopy, and mass spectrometry. Addition of ethylene carbonate occurs exclusively at OH2 and results in a degree of substitution of 8−22 ethylene glycol units per cyclodextrin. Also, the ω-bromo and ω-iodo derivatives of heptakis[6-deoxy-6-hexylthio-2-oligo(ethylene glycol)]-β-cyclodextrin were prepared as strategic synthetic intermediates. The observation that seven halogen atoms are introduced per cyclodextrin provides strong evidence for the grafting of oligo(ethylene glycol) to each of the seven glucose units in the cyclodextrin ring.

Self-assembling modified β-cyclodextrin nanoparticles as neuronal siRNA delivery vectors: focus on Huntington's disease.

Huntingtons disease (HD) is a rare autosomal dominant neurodegenerative disease caused by the expression of a toxic Huntingtin (HTT) protein. The use of short interfering RNAs (siRNAs) to silence the mutant protein is one of the most promising therapeutic strategies under investigation. The biggest caveat to siRNA-based approaches is the lack of efficient and nontoxic delivery vectors for siRNA delivery to the central nervous system. In this study, we investigated the potential of modified amphiphilic β-cyclodextrins (CDs), oligosaccharide-based molecules, as novel siRNA neuronal carriers. We show that CDs formed nanosize particles which were stable in artificial cerebrospinal fluid. Moreover, these complexes were able to reduce the expression of the HTT gene in rat striatal cells (ST14A-HTT120Q) and in human HD primary fibroblasts. Only limited toxicity was observed with CD·siRNA nanoparticles in any of the in vitro models used. Sustained knockdown effects were observed in the striatum of the R6/2 mouse model of HD after single direct injections of CD·siRNA nanoparticles. Repeated brain injections of CD·siRNA complexes resulted in selective alleviation of motor deficits in this mouse model. Together these data support the utility of modified β-CDs as efficient and safe siRNA delivery vectors for RNAi-based therapies for neuropsychiatric and neurodegenerative disorders.

Cationic β-cyclodextrin bilayer vesicles

Cationic amphiphilic β-cyclodextrins, substituted with hydrophobic n-alkylthio chains at the primary hydroxyl side and hydrophilic ω-amino-oligo(ethylene glycol) units at the secondary side, form bilayer vesicles with a diameter of 30–35 nm (when alkyl = hexadecyl) or nanoparticles with a diameter of ca. 120 nm (when alkyl = hexyl) in water.

Mechanistic studies on the uptake and intracellular trafficking of novel cyclodextrin transfection complexes by intestinal epithelial cells

Oral delivery of gene therapeutics would facilitate treatment of local intestinal disease, including colon cancer and inflammatory bowel disease, thus avoiding invasive surgery. The aims of this study were to investigate; if the orientation of the lipid tail on the cyclodextrin (CD) influenced the efficacy of a novel poly-6-cationic amphiphilic CD to transfect intestinal enterocytes; the endocytotic uptake pathway(s), and, the intracellular trafficking of the CD·DNA complexes. Inhibitors of clathrin- and caveolae-mediated endocytosis and macropinocytosis were used to determine the mechanism(s) of CD·DNA uptake by both undifferentiated and differentiated Caco-2 cells. Cell surface heparan sulphate proteoglycans were involved in the association of CD·DNA complexes with undifferentiated Caco-2 cells. Complexation of pDNA with CD facilitated significant levels of pDNA uptake and gene expression (comparable to PEI) in both undifferentiated and differentiated Caco-2 cells. Disruption of intracellular vesicular trafficking reduced transfection activity. CD was also capable of transfecting the more physiologically relevant differentiated Caco-2 model. Macropinocytosis was responsible for the uptake of CD·DNA transfection complexes by both undifferentiated and differentiated Caco-2 cells. The ability of this novel CD to transfect differentiated intestinal cells indicates the potential of this vector for oral gene delivery.

Click-modified cyclodextrins as nonviral vectors for neuronal siRNA delivery.

RNA interference (RNAi) holds great promise as a strategy to further our understanding of gene function in the central nervous system (CNS) and as a therapeutic approach for neurological and neurodegenerative diseases. However, the potential for its use is hampered by the lack of siRNA delivery vectors which are both safe and highly efficient. Cyclodextrins have been shown to be efficient and low toxicity gene delivery vectors in various cell types in vitro. However, to date, they have not been exploited for delivery of oligonucleotides to neurons. To this end, a modified β-cyclodextrin (CD) vector was synthesized, which complexed siRNA to form cationic nanoparticles of less than 200 nm in size. Furthermore, it conferred stability in serum to the siRNA cargo. The in vitro performance of the CD in both immortalized hypothalamic neurons and primary hippocampal neurons was evaluated. The CD facilitated high levels of intracellular delivery of labeled siRNA, while maintaining at least 80% cell viability. Significant gene knockdown was achieved, with a reduction in luciferase expression of up to 68% and a reduction in endogenous glyceraldehyde phosphate dehydrogenase (GAPDH) expression of up to 40%. To our knowledge, this is the first time that a modified CD has been used as a safe and efficacious vector for siRNA delivery into neuronal cells.

Cationic cyclodextrin amphiphiles as gene delivery vectors

Amphiphilic polycationic cyclodextrins, heptakis[2-(m-amino-oligoethyleneglycol)-6-deoxy-6-hexylthio]-p-cyclodextrin and heptakis[2-(m- amino-oligoethyleneglycol)-6-deoxy-6-hexadecylthio]-p-cyclodextrin, were assessed for condensation of plasmid DNA and cell transfection. These cyclodextrins can self-assemble into cationic vesicles or nanoparticles and, unlike their amphiphilic non-aminated analogues, form DNA lipoplexes which efficiently transfected COS-7 cells. Levels of luciferase expression with the hexadecylthiopolyamino-CD vector were 2 x 10 4 times greater than for DNA alone. These levels are a significant improvement on non-amphiphilic polyamino CDs as vectors and are of the same order as the widely used transfection agent Lipofectin.

Multivalent binding of galactosylated cyclodextrin vesicles to lectin

Amphiphilic beta-cyclodextrins with alkylthio chains at the primary-hydroxyl side and galactosylthio-oligo-(ethylene glycol) units at the secondary-hydroxyl side, which form nanoparticles and vesicles, show multivalent effects in their binding to lectin.

Gene silencing of TNF-alpha in a murine model of acute colitis using a modified cyclodextrin delivery system

Inflammatory bowel disease (IBD) is a chronic relapsing inflammation of the gastrointestinal tract. The cytokine TNF-alpha (TNF-α) plays a pivotal role in mediating this inflammatory response. RNA interference (RNAi) holds great promise for the specific and selective silencing of aberrantly expressed genes, such as TNF-α in IBD. The aim of this study was to investigate the efficacy of an amphiphilic cationic cyclodextrin (CD) vector for effective TNF-α siRNA delivery to macrophage cells and to mice with induced acute-colitis. The stability of CD.siRNA was examined by gel electrophoresis in biorelevant media reflecting colonic fluids. RAW264.7 cells were transfected with CD.TNF-α siRNA, stimulated with lipopolysaccharide (LPS) and TNF-α and IL-6 responses were measured by PCR and ELISA. Female C57BL/6 mice were exposed to dextran sodium sulphate (DSS) and treated by intrarectal administration with either CD.siRNA TNF-α or a control solution. In vitro, siRNA in CD nanocomplexes remained intact and stable in both fed and fasted simulated colonic fluids. RAW264.7 cells transfected with CD.TNF-α siRNA and stimulated with LPS displayed a significant reduction in both gene and protein levels of TNF-α and IL-6. CD.TNF-α siRNA-treated mice revealed a mild amelioration in clinical signs of colitis, but significant reductions in total colon weight and colonic mRNA expression of TNF-α and IL-6 compared to DSS-control mice were detected. This data indicates the clinical potential of a local CD-based TNF-α siRNA delivery system for the treatment of IBD.