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: 6.23). 10/2006; 14(3):336-42. DOI: 10.1016/j.ymthe.2006.04.001
Source: PubMed


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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    • "or pcDNA3.0-IL-10 on TNBS-induced colitis, we intraperitoneally injected these plasmids into mice mixed with liposome 24 h after TNBS injection (Table 1) according to previous studies [23-30]. In brief, 100 μg plasmids were mixed with 30 μl LipofectAMINE 2000 and then incubated for 20 min at the room temperature, and injected into mice. "
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    ABSTRACT: Inflammatory bowel disease (IBD) is characterized by disturbance of pro-inflammatory cytokines and anti-inflammatory cytokines. Previous studies have demonstrated the effect of anti-inflammatory cytokines, such as interleukin-10 (IL-10) or IL-4 on IBD, but their data were controversial. This study further investigated the effect of IL-4 (IL-4), IL-10 and their combination on treatment of trinitrobenzenesulfonic acid (TNBS)-induced murine colitis. pcDNA3.0 carrying murine IL-4 or IL-10 cDNA was encapsulated with LipofectAMINE 2000 and intraperitoneally injected into mice with TNBS-induced colitis. The levels of intestinal IL-4 and IL-10 mRNA were confirmed by quantitative-RT-PCR. Inflamed tissues were assessed by histology and expression of interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha and IL-6. The data confirmed that IL-4 or IL-10 over-expression was successfully induced in murine colon tissues after intraperitoneal injection. Injections of IL-4 or IL-10 significantly inhibited TNBS-induced colon tissue damage, disease activity index (DAI) and body weight loss compared to the control mice. Furthermore, expression of IFN-gamma, TNF-alpha and IL-6 was markedly blocked by injections of IL-4 or IL-10 plasmid. However, there was less therapeutic effect in mice injected with the combination of IL-4 and IL-10. These data suggest that intraperitoneal injection of IL-4 or IL-10 plasmid was a potential strategy in control of TNBS-induced murine colitis, but their combination had less effect.
    BMC Gastroenterology 12/2013; 13(1):165. DOI:10.1186/1471-230X-13-165 · 2.37 Impact Factor
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    • "ΔCt of the gene of interest was compared to the ΔCt of the reference gene to determine relative gene expression. Gene of interest primers were specific for murine Lamin A/C, 5′-GAGAGGCTAAGAAGCAGC-3′ (sense) and 5′-ACGCAGTTCCTCGCTGTAA-3′ (antisense),53 murine TNFα, 5′-CATCTTCTCAAAATTCGAGTGACAA-3′ (sense) and 5′-TGGGAGTAGACAAGGTACAACCC-3′ (antisense), murine IFNβ, 5′-AGCTCCAAGAAAGGACGAACAT-3′ (sense) and 5′-GCCCTGTAGGTGAGGTTGATCT-3′ (antisense), whereas reference gene primers were specific for murine β-actin, 5′-GCTACAGCTTCACCACCACA-3′ (sense) and 5′-TCTCCAGGGAGGAAGAGGAT-3′ (antisense). "
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    ABSTRACT: RNA interference (RNAi) may provide a therapeutic solution to many pulmonary epithelium diseases. However, the main barrier to the clinical use of RNAi remains the lack of efficient delivery vectors. Research has mainly concentrated on the intranasal route of delivery of short interfering RNA (siRNA) effector molecules for the treatment of respiratory diseases. However, this may be complicated in a diseased state due to the increased fluid production and tissue remodeling. Therefore, we investigated our hydration of a freeze-dried matrix (HFDM) formulated liposomes for systemic delivery to the lung epithelium. Here, we show that 45 ± 2% of epithelial murine lung cells receive siRNA delivery upon intravenous (IV) liposomal administration. Furthermore, we demonstrate that liposomal siRNA delivery resulted in targeted gene and protein knockdown throughout the lung, including lung epithelium. Taken together, this is the first description of lung epithelial delivery via cationic liposomes, and provides a proof of concept for the use of IV liposomal RNAi delivery to specifically knockdown targeted genes in the respiratory system. This approach may provide an attractive alternate therapeutic delivery strategy for the treatment of lung epithelium diseases.Molecular Therapy - Nucleic Acids (2013) 2, e96; doi:10.1038/mtna.2013.22; published online 4 June 2013.
    Molecular Therapy - Nucleic Acids 06/2013; 2(6):e96. DOI:10.1038/mtna.2013.22 · 4.51 Impact Factor
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    • "Previous strategies to overcome vaginal delivery barriers have included the simple application of lipoplexes into the vaginal cavity either with [111, 112] or without [113] progesterone treatment, as well as mucus removal in the vaginal cavity prior to siRNA administration [114]. Unsurprisingly, investigations of naked siRNA uptake into vaginal tissues after intravaginal administration reported inefficient delivery [113] under normal physiological conditions. This was likely caused by rapid degradation and inefficient mucosal uptake of naked siRNAs in the vaginal cavity. "
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    ABSTRACT: Human Papillomavirus (HPV)-induced diseases are a significant burden on our healthcare system and current therapies are not curative. Vaccination provides significant prophylactic protection but effective therapeutic treatments will still be required. RNA interference (RNAi) has great promise in providing highly specific therapies for all HPV diseases yet this promise has not been realised. Here we review the research into RNAi therapy for HPV in vitro and in vivo and examine the various targets and outcomes. We discuss the idea of using RNAi with current treatments and address delivery of RNAi, the major issue holding back clinical adoption. Finally, we present our view of a potential path to the clinic.
    The Open Virology Journal 12/2012; 6:204-15. DOI:10.2174/1874357901206010204
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