Chemically Synthesized, Self-Assembling Small Interfering RNA-Prohead RNA Molecules Trigger Dicer-Independent Gene Silencing.

RNA interference (RNAi) mediated by small interfering RNA (siRNA) duplexes is a powerful therapeutic modality, but the translation of siRNAs from the bench into clinical application has been hampered by inefficient delivery in vivo. An innovative delivery strategy involves fusing siRNAs to a three-way junction (3WJ) motif derived from the phi29 bacteriophage prohead RNA (pRNA). Chimeric siRNA-3WJ molecules are presumed to enter the RNAi pathway through Dicer cleavage. Here, we fused siRNAs to the phi29 3WJ and two phylogenetically related 3WJs. We confirmed that the siRNA-3WJs are substrates for Dicer in vitro. However, our results reveal that siRNA-3WJs transfected into Dicer-deficient cell lines trigger potent gene silencing. Interestingly, siRNA-3WJs transfected into an Argonaute 2-deficient cell line also retain some gene silencing activity. siRNA-3WJs are most efficient when the antisense strand of the siRNA duplex is positioned 5' of the 3WJ (5'-siRNA-3WJ) relative to 3' of the 3WJ (3'-siRNA-3WJ). This work sheds light on the functional properties of siRNA-3WJs and offers a design rule for maximizing their potency in the human RNAi pathway.

Inosine Substitutions in RNA Activate Latent G-Quadruplexes.

It is well-accepted that gene expression is heavily influenced by RNA structure. For instance, stem-loops and G-quadruplexes (rG4s) are dynamic motifs in mRNAs that influence gene expression. Adenosine-to-inosine (A-to-I) editing is a common chemical modification of RNA which introduces a nucleobase that is iso-structural with guanine, thereby changing RNA base-pairing properties. Here, we provide biophysical, chemical, and biological evidence that A-to-I exchange can activate latent rG4s by filling incomplete G-quartets with inosine. We demonstrate the formation of inosine-containing rG4s (GI-quadruplexes) in vitro and verify their activity in cells. GI-quadruplexes adopt parallel topologies, stabilized by potassium ions. They exhibit moderately reduced thermal stability compared to conventional G-quadruplexes. To study inosine-induced structural changes in a naturally occurring RNA, we use a synthetic approach that enables site-specific inosine incorporation in long RNAs. In summary, RNA GI-quadruplexes are a previously unrecognized structural motif that may contribute to the regulation of gene expression in vivo.

Labeling microRNA precursors for Dicer assays.

Excision of the terminal loop of precursor microRNAs (pre-miRNA) by Dicer generates miRNA duplexes, comprising 5p and 3p strands. Dicer often cleaves the RNA at more than one site, producing mature miRNAs with heterogeneous 3' and 5' ends. As pre-miRNAs are most conveniently labeled at their 5' ends, standard in vitro Dicer assays usually assay only 5p miRNA strands. In this work we present a straightforward protocol using the same [32P] isotope for both strands by placing an internal label into the 3p strand, thereby providing a tool to investigate miRNA processing, strand selection and isomiR formation for any pre-miRNA.

Immunogenic properties of chimeric potato virus X particles displaying the hepatitis C virus hypervariable region I peptide R9.

The immunogenic properties of chimeric potato virus X (PVX) particles engineered to display the synthetic R9 peptide have been evaluated. The R9 peptide is a consensus sequence derived from diverse variants of the hypervariable region 1 from the hepatitis C virus (HCV) envelope protein E2. Two different constructs were designed, with the R9 peptide expressed either as an indirect fusion via the ribosomal skip 2A (PVX(R9-2A)CP) sequence or as a direct PVX coat protein fusion (PVX(R9)CP). Systemic infection of Nicotiana benthamiana plants was only achieved with PVX(R9-2A)CP constructs, and the presence of the R9 peptide was detected in extracts from these plants by ELISA, Western blot and electron microscopy using specific anti-R9 antibodies. The virus particles were recovered at yields of up to 125mg/kg from leaf material. BALB/c mice immunized with purified PVX(R9-2A)CP particles developed specific anti-R9 IgG titers of up to 1:50,000. Monoclonal anti-R9 antibodies were obtained from the spleen of a mouse immunized with PVX(R9-2A)CP particles and characterized by Western blot and electron microscopy. Sera from patients infected chronically with HCV were found to react specifically with PVX(R9-2A)CP particles in 35% of cases.

Clinical molecular imaging in intestinal graft-versus-host disease: mapping of disease activity, prediction, and monitoring of treatment efficiency by positron emission tomography.

Gastrointestinal graft-versus-host disease (GVHD) is a common and potentially life-threatening complication after allogeneic hematopoietic stem-cell transplantation (HSCT). Noninvasive tests for assessment of GVHD activity are desirable but lacking. In the present study, we were able to visualize intestinal GVHD-associated inflammation in an allogeneic murine transplantation model by (18)F-fluorodeoxyglucose positron emission tomography (FDG-PET) in vivo. A predominant localization of intestinal GVHD to the colon was verified by histology and fluorescence reflectance imaging of enhanced green fluorescent protein (EGFP)-expressing donor cells. Colonic infiltration by EGFP(+) donor lymphocytes matched increased FDG uptake in PET examinations. These preclinical data were prospectively translated into 30 patients with suspected intestinal GVHD beyond 20 days after transplantation. A total of 14 of 17 patients with a diagnostic histology showed significant FDG uptake of the gut, again predominantly in the colon. No increased FDG uptake was detected in 13 patients without histologic evidence of intestinal GVHD. Our findings indicate that FDG-PET is a sensitive and specific noninvasive imaging technique to assess intestinal GVHD, map its localization, and predict and monitor treatment responsiveness. Novel targeted tracers for PET may provide new insights into the pathophysiology of GVHD and bear the potential to further improve GVHD diagnosis.

Differential requirement for a cellular type-1 immune response in tumor-associated versus alloantigen-targeted GvT effects.

BACKGROUND: The major obstacles that impair successful outcome after allogeneic hematopoietic stem cell transplantation (HSCT) for hematologic malignancies remain graft-versus-host disease (GvHD) and tumor relapse. Improved survival after allogeneic HSCT therefore requires more effective control of GvHD while preserving graft-versus-tumor (GvT) effects. METHODS: Allogeneic parent-into-F1 murine transplant models (BALB/c or C57BL/6 --> F1[BALB/cxC57BL/6]) were used to evaluate the interrelation of GvHD and GvT effects targeting tumor-specific antigens or alloantigens on MethA tumor cells. RESULTS: Compared with syngeneic F1-into-F1 controls (F1[H-2(b/d)] --> F1: MethA[H-2d]), significant T cell-mediated GvT effects occurred in both allogeneic transplant models, even in the absence of histoincompatibilities between donor cells and host tumor (BALB/c[H-2d] --> F1: MethA[H-2d]). Selective inhibition of type-1 (Th-1/Tc1) immune responses with TAK-603 after HSCT nearly abolished GvHD in both allogeneic transplant models. While GvT effects directed against alloantigens (C57BL/6[H-2b] --> F1: MethA[H-2d]) remained unaffected during type-1-immune suppression, GvT effects targeted against tumor-associated antigens (BALB/c[H-2d] --> F1: MethA[H-2d]) were not evident. CONCLUSIONS.: Our data show that GvHD and GvT effects are in principle separable from each other by selective type-1 inhibition in transplantation models with major histocompatibility complex disparities between tumor, host, and donor. In contrast, in situations that only allow for GvT effects that exclusively target tumor-associated antigens (TAAs), type-1 inhibition results in complete abrogation not only of GvHD but also desired GvT reactions. These differences in GvT effects targeting alloantigens or TAAs and their interrelation to GvHD should be considered in future studies aimed at separating GvT reactions from GvHD.