Engineering Mucosal RNA Interference in Vivo

Laboratory of Retrovirology, Division of Infectious Diseases, Department of Medicine, Brown Medical School, 55 Claverick Street, Providence, RI 02903, USA.
Molecular Therapy (Impact Factor: 6.43). 10/2006; 14(3):336-42. DOI: 10.1016/j.ymthe.2006.04.001
Source: PubMed

ABSTRACT Mucosal surfaces serve as a gateway to disease. Here, we demonstrate that RNA interference can be used to manipulate mucosal gene expression in vivo. Using a murine model, we show that direct application of liposome-complexed siRNA mediates gene-specific silencing in cervicovaginal and rectal mucosa. A single vaginal or rectal administration of siRNA targeting hematopoietic or somatic cell gene products reduced corresponding mRNA levels by up to 90%. Using a murine model of inflammatory bowel disease, we found that the rectal application of siRNA targeting TNF-alpha led to relative mucosal resistance to experimental colitis. Liposomal siRNA formulations proved nontoxic, did not elicit a nonspecific interferon response, and provide a means for genetic engineering of mucosal surfaces in vivo.

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    • "problem, many strategies have been employed to enhance endosomal escape. Lipid or lipid-like molecules responsive to pH and viral fusogenic proteins and peptides have promoted endosomal escape via membrane destabilization, which can result from pHdependent changes in conformation (Akinc et al., 2008; Hughson, 1995; Morrissey et al., 2005; Pal et al., 2005; Palliser et al., 2005; Ren et al., 1999; Skehel and Wiley, 2000; Sørensen et al., 2003; Zhang et al., 2006; Zimmermann et al., 2006). Ideally, such mediators of cellular entry, and any potential toxicity, would be masked within a complex or via 'shielding' prior to arrival at the target tissue (Sawant et al., 2006). "
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    ABSTRACT: The development of short-interfering RNA (siRNA) offers new strategies for manipulating specific genes responsible for pathological disorders. Myriad cationic polymer and lipid formulations have been explored, but an effective, non-toxic carrier remains a major barrier to clinical translation. Among the emerging candidates for siRNA carriers are cell penetrating peptides (CPPs), which can traverse the plasma membrane and facilitate the intracellular delivery of siRNA. Previously, a highly efficient and non-cytotoxic means of gene delivery was designed by complexing plasmid DNA with CPPs, then condensing with calcium. Here, the CPP TAT and a longer, 'double' TAT (dTAT) were investigated as potential carriers for siRNA. Various N/P ratios and calcium concentrations were used to optimize siRNA complexes in vitro. Upon addition of calcium, 'loose' siRNA/CPP complexes were condensed into small nanoparticles. Knockdown of luciferase expression in the human epithelial lung cell line A549-luc-C8 was high (up to 93%) with no evidence of cytotoxicity. Selected formulations of the dTAT complexes were dosed intravenously up to 1000 mg/kg with minimal toxicity. Biodistribution studies revealed high levels of gene knockdown in the lung and muscle tissue suggesting these simple vectors may offer a translatable approach to siRNA delivery.
    International Journal of Pharmaceutics 08/2011; 427(1):134-42. DOI:10.1016/j.ijpharm.2011.08.012 · 3.65 Impact Factor
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    • "siRNAs were observed in the vaginal epithelial cells and were detectable deep into the lamina propria. Furthermore, targeting an endogenous gene resulted in more durable silencing, with reduced expression of mRNA and protein observed for at least 7 days (Palliser et al., 2006; Zhang et al., 2006). A similar observation had been reported for siRNA-mediated inhibition of HIV-1 replication in macrophages in vitro. "
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    ABSTRACT: RNA interference (RNAi) describes a highly conserved pathway, present in eukaryotic cells, for regulating gene expression. Small stretches of double-stranded RNA, termed small interfering RNAs (siRNAs), utilize this pathway to bind homologous mRNA, resulting in site-specific mRNA cleavage and subsequent protein degradation. The ubiquitous presence of the RNAi machinery, combined with its specificity and efficacy, makes it an attractive mechanism for reducing aberrant gene expression in therapeutic settings. However, a major obstacle to utilizing RNAi in the clinic is siRNA delivery. Administered siRNAs must make contact with the appropriate cell types and, following internalization, gain access to the cytosol where the RNAi machinery resides. This must be achieved so that silencing is maximized, whilst minimizing any undesirable off-target effects. Recently, the utility of siRNAs as a microbicide, usually applied to the genital mucosa for preventing transmission of sexually transmitted diseases including HIV-1 and HSV-2, has been investigated. In this review we will describe these studies and discuss potential strategies for improving gene silencing.
    Discovery medicine 02/2011; 11(57):124-32. · 3.50 Impact Factor
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    • "were effective when administered around the time of viral challenge or within a few hours after exposure. Effective siRNA delivery to the cervicovaginal and anal epithelium using lipoplexed siRNAs was also shown by Ramratnam and colleagues (Zhang et al. 2006). "
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    ABSTRACT: A vaginal microbicide should prevent pathogen transmission without disrupting tissue barriers to infection. Ideally, it would not need to be applied immediately before sexual intercourse, when compliance is a problem. Intravaginal administration of small interfering RNA (siRNA) lipoplexes targeting Herpes Simplex Virus Type 2 (HSV-2) genes protects mice from HSV-2. However, protection is short-lived, and the transfection lipid on its own unacceptably enhances transmission. Here, we show that cholesterol-conjugated (chol)-siRNAs without lipid silence gene expression in the vagina without causing inflammation or inducing interferons. A viral siRNA prevents transmission within a day of challenge, whereas an siRNA targeting the HSV-2 receptor nectin-1 protects for a week, but protection is delayed for a few days until the receptor is downmodulated. Combining siRNAs targeting a viral and host gene protects mice from HSV-2 for a week, irrespective of the time of challenge. Therefore, intravaginal siRNAs could provide sustained protection against viral transmission.
    Cell host & microbe 02/2009; 5(1):84-94. DOI:10.1016/j.chom.2008.12.003 · 12.19 Impact Factor
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