RNA interference (RNAi)-based therapeutics have the potential to treat chronic hepatitis B virus (HBV) infection in a fundamentally different manner than current therapies. Using RNAi, it is possible to knock down expression of viral RNAs including the pregenomic RNA from which the replicative intermediates are derived, thus reducing viral load, and the viral proteins that result in disease and impact the immune system's ability to eliminate the virus. We previously described the use of polymer-based Dynamic PolyConjugate (DPC) for the targeted delivery of siRNAs to hepatocytes. Here, we first show in proof-of-concept studies that simple coinjection of a hepatocyte-targeted, N-acetylgalactosamine-conjugated melittin-like peptide (NAG-MLP) with a liver-tropic cholesterol-conjugated siRNA (chol-siRNA) targeting coagulation factor VII (F7) results in efficient F7 knockdown in mice and nonhuman primates without changes in clinical chemistry or induction of cytokines. Using transient and transgenic mouse models of HBV infection, we show that a single coinjection of NAG-MLP with potent chol-siRNAs targeting conserved HBV sequences resulted in multilog repression of viral RNA, proteins, and viral DNA with long duration of effect. These results suggest that coinjection of NAG-MLP and chol-siHBVs holds great promise as a new therapeutic for patients chronically infected with HBV.
The assembly and maturation of box C/D snoRNPs, factors essential for ribosome biogenesis, occur in the nucleoplasm. To investigate this process, we have analyzed non-snoRNP factors associated with the nucleoplasmic human U3 snoRNA. We show that both the precursor and mature length nucleoplasmic U3 snoRNAs are present in larger multiprotein complexes that contain the core box C/D proteins as well as many non-snoRNP factors linked to snoRNP assembly (TIP48, TIP49, Nopp140), RNA processing (TGS1, La, LSm4, hRrp46), and subcellular localization (CRM1, PHAX). Using RNAi, we show that most of these factors are essential for box C/D snoRNA accumulation. Furthermore, we demonstrate that the core proteins undergo a restructuring event that stabilizes their binding to the snoRNA. Importantly, restructuring, which may be mediated by the putative remodeling factor TIP49, appears to be linked to nucleolar localization. We believe that the assembly complex coordinates snoRNA processing, snoRNP assembly, restructuring, and localization.
The Whitehead siRNA (short interfering RNA) Selection Web Server (http://jura.wi.mit.edu/bioc/siRNA) automates the design of short oligonucleotides that can specifically 'knock down' expression of target genes. These short sequences are about 21 nt in length, and when synthesized as double stranded RNA and introduced into cell culture, can reduce or eliminate the function of the target gene. Depending on the length of a gene, there are potentially numerous combinations of possible 21mers. Some experimental evidence has already shown that not all 21mers in a gene have the same effectiveness at silencing gene function. Our tool incorporates published design rules and presents the scientist with information about uniqueness of the 21mers within the genome, thermodynamic stability of the double stranded RNA duplex, GC content, presence of SNPs and other features that may contribute to the effectiveness of a siRNA.
SummaryRetinal photoreceptors entrain the circadian system to the solar day. This photic resetting involves cAMP response element binding protein (CREB)-mediated upregulation of Per genes within individual cells of the suprachiasmatic nuclei (SCN). Our detailed understanding of this pathway is poor, and it remains unclear why entrainment to a new time zone takes several days. By analyzing the light-regulated transcriptome of the SCN, we have identified a key role for salt inducible kinase 1 (SIK1) and CREB-regulated transcription coactivator 1 (CRTC1) in clock re-setting. An entrainment stimulus causes CRTC1 to coactivate CREB, inducing the expression of Per1 and Sik1. SIK1 then inhibits further shifts of the clock by phosphorylation and deactivation of CRTC1. Knockdown of Sik1 within the SCN results in increased behavioral phase shifts and rapid re-entrainment following experimental jet lag. Thus SIK1 provides negative feedback, acting to suppress the effects of light on the clock. This pathway provides a potential target for the regulation of circadian rhythms.
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