Anton P. McCaffrey

RNA interference in adult mice

RNA interference is an evolutionarily conserved surveillance mechanism that responds to double-stranded RNA by sequence-specific silencing of homologous genes. Here we show that transgene expression can be suppressed in adult mice by synthetic small interfering RNAs and by small-hairpin RNAs transcribed in vivo from DNA templates. We also show the therapeutic potential of this technique by demonstrating effective targeting of a sequence from hepatitis C virus by RNA interference in vivo.

Gene expression - RNA interference in adult mice

RNA interference is an evolutionarily conserved surveillance mechanism that responds to double-stranded RNA by sequence-specific silencing of homologous genes. Here we show that transgene expression can be suppressed in adult mice by synthetic small interfering RNAs and by small-hairpin RNAs transcribed in vivo from DNA templates. We also show the therapeutic potential of this technique by demonstrating effective targeting of a sequence from hepatitis C virus by RNA interference in vivo.

Inhibition of hepatitis B virus in mice by RNA interference

Hepatitis B virus (HBV) infection substantially increases the risk of chronic liver disease and hepatocellular carcinoma in humans. RNA interference (RNAi) of virus-specific genes has emerged as a potential antiviral mechanism. Here we show that RNAi can be applied to inhibit production of HBV replicative intermediates in cell culture and in immunocompetent and immunodeficient mice transfected with an HBV plasmid. Cotransfection with plasmids expressing short hairpin RNAs (shRNAs) homologous to HBV mRNAs induced an RNAi response. Northern and Southern analyses of mouse liver RNA and DNA showed substantially reduced levels of HBV RNAs and replicated HBV genomes upon RNAi treatment. Secreted HBV surface antigen (HBsAg) was reduced by 94.2% in cell culture and 84.5% in mouse serum, whereas immunohistochemical detection of HBV core antigen (HBcAg) revealed >99% reduction in stained hepatocytes upon RNAi treatment. Thus, RNAi effectively inhibited replication initiation in cultured cells and mammalian liver, showing that such an approach could be useful in the treatment of viral diseases.

Engineered zinc finger nickases induce homology-directed repair with reduced mutagenic effects

Engineered zinc finger nucleases (ZFNs) induce DNA double-strand breaks at specific recognition sequences and can promote efficient introduction of desired insertions, deletions or substitutions at or near the cut site via homology-directed repair (HDR) with a double- and/or single-stranded donor DNA template. However, mutagenic events caused by error-prone non-homologous end-joining (NHEJ)-mediated repair are introduced with equal or higher frequency at the nuclease cleavage site. Furthermore, unintended mutations can also result from NHEJ-mediated repair of off-target nuclease cleavage sites. Here, we describe a simple and general method for converting engineered ZFNs into zinc finger nickases (ZFNickases) by inactivating the catalytic activity of one monomer in a ZFN dimer. ZFNickases show robust strand-specific nicking activity in vitro. In addition, we demonstrate that ZFNickases can stimulate HDR at their nicking site in human cells, albeit at a lower frequency than by the ZFNs from which they were derived. Finally, we find that ZFNickases appear to induce greatly reduced levels of mutagenic NHEJ at their target nicking site. ZFNickases thus provide a promising means for inducing HDR-mediated gene modifications while reducing unwanted mutagenesis caused by error-prone NHEJ.

Correlation between microRNA expression levels and clinical parameters associated with chronic hepatitis C viral infection in humans.

MicroRNAs (miRNAs) are small RNAs that regulate gene expression pathways. Previous studies have shown interactions between hepatitis C virus (HCV) and host miRNAs. We measured miR-122 and miR-21 levels in HCV-infected human liver biopsies relative to uninfected human livers and correlated these with clinical patient data. miR-122 is required for HCV replication in vitro, and miR-21 is involved in cellular proliferation and tumorigenesis. We found that miR-21 expression correlated with viral load, fibrosis and serum liver transaminase levels. miR-122 expression inversely correlated with fibrosis, liver transaminase levels and patient age. miR-21 was induced ∼twofold, and miR-122 was downregulated on infection of cultured cells with the HCV J6/JFH infectious clone, thus establishing a link to HCV. To further examine the relationship between fibrosis and the levels of miR-21 and miR-122, we measured their expression levels in a mouse carbon tetrachloride fibrosis model. As in the HCV-infected patient samples, fibrotic stage positively correlated with miR-21 and negatively correlated with miR-122 levels. Transforming growth factor β (TGF-β) is a critical mediator of fibrogenesis. We identified SMAD7 as a novel miR-21 target. SMAD7 is a negative regulator of TGF-β signaling, and its expression is induced by TGF-β. To confirm the relationship between miR-21 and the TGF-β signaling pathway, we measured the effect of miR-21 on a TGF-β-responsive reporter. We found that miR-21 enhanced TGF-β signaling, further supporting a relationship between miR-21 and fibrosis. We suggest a model in which miR-21 targeting of SMAD7 could increase TGF-β signaling, leading to increased fibrogenesis.

Zinc-finger Nucleases as a Novel Therapeutic Strategy for Targeting Hepatitis B Virus DNAs

Hepatitis B virus (HBV) chronically infects 350-400 million people worldwide and causes >1 million deaths yearly. Current therapies prevent new viral genome formation, but do not target pre-existing viral genomic DNA, thus curing only approximately 1/2 of patients. We targeted HBV DNA for cleavage using zinc-finger nucleases (ZFNs), which cleave as dimers. Co-transfection of our ZFN pair with a target plasmid containing the HBV genome resulted in specific cleavage. After 3 days in culture, 26% of the target remained linear, whereas approximately10% was cleaved and misjoined tail-to-tail. Notably, ZFN treatment decreased levels of the hepatitis C virus pregenomic RNA by 29%. A portion of cleaved plasmids are repaired in cells, often with deletions and insertions. To track misrepair, we introduced an XbaI restriction site in the spacer between the ZFN sites. Targeted cleavage and misrepair destroys the XbaI site. After 3 days in culture, approximately 6% of plasmids were XbaI-resistant. Thirteen of 16 clones sequenced contained frameshift mutations that would lead to truncations of the viral core protein. These results demonstrate, for the first time, the possibility of targeting episomal viral DNA genomes using ZFNs.

Hepatitis C Virus Infection and Hepatic Stellate Cell Activation Downregulate miR-29: miR-29 Overexpression Reduces Hepatitis C Viral Abundance in Culture

BACKGROUND Chronic hepatitis C virus (HCV)-induced liver fibrosis involves upregulation of transforming growth factor (TGF)-β and subsequent hepatic stellate cell (HSC) activation. MicroRNAs (miRNAs) regulate HCV infection and HSC activation. METHODS TaqMan miRNA profiling identified 12 miRNA families differentially expressed between chronically HCV-infected human livers and uninfected controls. To identify pathways affected by miRNAs, we developed a new algorithm (pathway analysis of conserved targets), based on the probability of conserved targeting. RESULTS This analysis suggested a role for miR-29 during HCV infection. Of interest, miR-29 was downregulated in most HCV-infected patients. miR-29 regulates expression of extracellular matrix proteins. In culture, HCV infection downregulated miR-29, and miR-29 overexpression reduced HCV RNA abundance. miR-29 also appears to play a role in HSCs. Hepatocytes and HSCs contribute similar amounts of miR-29 to whole liver. Both activation of primary HSCs and TGF-β treatment of immortalized HSCs downregulated miR-29. miR-29 overexpression in LX-2 cells decreased collagen expression and modestly decreased proliferation. miR-29 downregulation by HCV may derepress extracellular matrix synthesis during HSC activation. CONCLUSIONS HCV infection downregulates miR-29 in hepatocytes and may potentiate collagen synthesis by reducing miR-29 levels in activated HSCs. Treatment with miR-29 mimics in vivo might inhibit HCV while reducing fibrosis.

The role of microRNAs miR-200b and miR-200c in TLR4 signaling and NF-κB activation.

Recognition of microbial products by members of the Toll-like receptor (TLR) family initiates intracellular signaling cascades that result in NF-κB activation and subsequent production of inflammatory cytokines. We explored the potential roles of microRNAs (miRNAs) in regulating TLR pathways. A target analysis approach to the TLR4 pathway adaptor molecules identified several putative targets of miR-200a, miR-200b and miR-200c. miRNA mimics were co-transfected with a NF-κB activity reporter plasmid into HEK293 cells stably expressing TLR4 (HEK293-TLR4). Mimics of both miR-200b and miR-200c, but not miR-200a, decreased NF-κB reporter activity in either untreated cells or in cells treated with endotoxin:MD2 as a TLR4 agonist. Transfection of HEK293-TLR4 cells with miR-200b or miR-200c significantly decreased expression of MyD88, whereas TLR4, IRAK-1 and TRAF-6 mRNAs were unaffected. When miR-200b or miR-200c mimics were transfected into the differentiated monocytic THP-1 cell line, the abundance of MyD88 transcripts, as well as LPS-induced expression of the pro-inflammatory molecules IL-6, CXCL9 and TNF-α were diminished. These data define miRNAs miR-200b and miR-200c as factors that modify the efficiency of TLR4 signaling through the MyD88-dependent pathway and can thus affect host innate defenses against microbial pathogens.

Advancing Molecular Therapies through In Vivo Bioluminescent Imaging

Effective development of therapeutics that target the molecular basis of disease is dependent on testing new therapeutic moieties and delivery strategies in animal models of human disease. Accelerating the analyses of these models and improving their predictive value through whole animal imaging methods, which provide data in real time and are sensitive to the subtle changes, are crucial for rapid advancement of these approaches. Modalities based on optics are rapid, sensitive, and accessible methods for in vivo analyses with relatively low instrumentation costs. In vivo bioluminescent imaging (BLI) is one of these optically based imaging methods that enable rapid in vivo analyses of a variety of cellular and molecular events with extreme sensitivity. BLI is based on the use of light-emitting enzymes as internal biological light sources that can be detected externally as biological indicators. BLI has been used to test spatio-temporal expression patterns of both target and therapeutic genes in living laboratory animals where the contextual influences of whole biological systems are preserved. BLI has also been used to analyze gene delivery, immune cell therapies, and the in vivo efficacy of inhibitory RNAs. New tools for BLI are being developed that will offer greater flexibility in detection and analyses. BLI can be used to accelerate the evaluation of experimental therapeutic strategies and whole body imaging offers the opportunity of revealing the effects of novel approaches on key steps in disease processes.

ZFN-site searches genomes for zinc finger nuclease target sites and off-target sites.

BackgroundZinc Finger Nucleases (ZFNs) are man-made restriction enzymes useful for manipulating genomes by cleaving target DNA sequences. ZFNs allow therapeutic gene correction or creation of genetically modified model organisms. ZFN specificity is not absolute; therefore, it is essential to select ZFN target sites without similar genomic off-target sites. It is important to assay for off-target cleavage events at sites similar to the target sequence.ResultsZFN-Site is a web interface that searches multiple genomes for ZFN off-target sites. Queries can be based on the target sequence or can be expanded using degenerate specificity to account for known ZFN binding preferences. ZFN off-target sites are outputted with links to genome browsers, facilitating off-target cleavage site screening. We verified ZFN-Site using previously published ZFN half-sites and located their target sites and their previously described off-target sites. While we have tailored this tool to ZFNs, ZFN-Site can also be used to find potential off-target sites for other nucleases, such as TALE nucleases.ConclusionsZFN-Site facilitates genome searches for possible ZFN cleavage sites based on user-defined stringency limits. ZFN-Site is an improvement over other methods because the FetchGWI search engine uses an indexed search of genome sequences for all ZFN target sites and possible off-target sites matching the half-sites and stringency limits. Therefore, ZFN-Site does not miss potential off-target sites.