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Harnessing the RNA interference pathway offers a new therapeutic modality; however, solutions to overcome biological barriers to small interfering RNA (siRNA) delivery are required for clinical translation. This work demonstrates, by direct northern and quantitative PCR (qPCR) detection, stability, gastrointestinal (GI) deposition, and translocation into peripheral tissue of nonmodified siRNA after oral gavage of chitosan/siRNA nanoparticles in mice. In contrast to naked siRNA, retained structural integrity and deposition in the stomach, proximal and distal small intestine, and colon was observed at 1 and 5 hours for siRNA within nanoparticles. Furthermore, histological detection of fluorescent siRNA at the apical regions of the intestinal epithelium suggests mucoadhesion provided by chitosan. Detection of intact siRNA in the liver, spleen, and kidney was observed 1 hour after oral gavage, with an organ distribution pattern influenced by nanoparticle N:P ratio that could reflect differences in particle stability. This proof-of-concept work presents an oral delivery platform that could have the potential to treat local and systemic disorders by siRNA.

Transcription of retroviruses is initiated at the U3-R region boundary in the integrated provirus and continues unidirectionally to produce genomic and mRNA products of positive polarity. Several studies have recently demonstrated the existence of naturally occurring protein-encoding transcripts of negative polarity in complex retroviruses. We report here on the identification of transcripts of negative polarity in simple murine leukemia virus (MLV). In T-cell and B-cell lymphomas induced by SL3-3 and Akv MLV, antisense transcripts initiated in the U3 region of the proviral 5 long terminal repeat (LTR) and continued into the cellular proto-oncogenes Jdp2 and Bach2 to create chimeric transcripts consisting of viral and host sequence. The phenomenon was validated in vivo using a knock-in mouse model homozygous for a single LTR at a position known to activate Nras in B-cell lymphomas. A 5 rapid amplification of cDNA ends (RACE) analysis indicated a broad spectrum of initiation sites within the U3 region of the 5 LTR. Our data show for the first time transcriptional activity of negative polarity initiating in the U3 region of simple retroviruses and suggest a novel mechanism of insertional activation of host genes. Elucidation of the nature and potential regulatory role of 5 LTR antisense transcription will be relevant to the design of therapeutic vectors and may contribute to the increasing recognition of pervasive eukaryotic transcription.The first base of the R region in the 5Ј long terminal repeat (LTR) defines the main transcription initiation site of retroviruses. In simple retroviruses, two major RNA species are produced by the actions of the cellular RNA polymerase II: an unspliced transcript, which serves as a template for translation of the viral genes gag and pol as well as for genomic RNA in progeny viruses, and a singly spliced transcript, which allows for production of appropriate levels of env mRNA. In addition to these common species, viral mRNA generated from the usage of an alternative splice donor site and the canonical env acceptor site has been described for Friend and Moloney murine leukemia viruses (MLVs) (11). Also, we have recently identified in Akv MLV an alternative exon generated from usage of cryptic splice acceptor and donor sites (45).

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This work demonstrates a versatile procedure for decorating nanoparticle surfaces with hydrophilic "nano-shields". XPS in combination with NMR enabled precise determination of PEG at the outmost surface to predict and optimize the biological performance of nanoparticle-based drug delivery.