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

Fluorinated perhexiline derivative attenuates vascular proliferation in pulmonary arterial hypertension smooth muscle cells

Increased proliferation and reduced apoptosis of pulmonary artery smooth muscle cells (PASMCs) is recognised as a universal hallmark of pulmonary arterial hypertension (PAH), in part related to the association with reduced pyruvate dehydrogenase (PDH) activity, resulting in decreased oxidative phosphorylation of glucose and increased aerobic glycolysis (Warburg effect). Perhexiline is a well-recognised carnitine palmitoyltransferase-1 (CPT1) inhibitor used in cardiac diseases, which reciprocally increases PDH activity, but is associated with variable pharmacokinetics related to polymorphic variation of the cytochrome P450-2D6 (CYP2D6) enzyme, resulting in the risk of neuro and hepatotoxicity in 'slow metabolisers' unless blood levels are monitored and dose adjusted. We have previously reported that a novel perhexiline fluorinated derivative (FPER-1) has the same therapeutic profile as perhexiline but is not metabolised by CYP2D6, resulting in more predictable pharmacokinetics than the parent drug. We sought to investigate the effects of perhexiline and FPER-1 on PDH flux in PASMCs from patients with PAH. We first confirmed that PAH PASMCs exhibited increased cell proliferation, enhanced phosphorylation of AKTSer473, ERK 1/2Thr202/Tyr204 and PDH-E1αSer293, indicating a Warburg effect when compared to healthy PASMCs. Pre-treatment with perhexiline or FPER-1 significantly attenuated PAH PASMC proliferation in a concentration-dependent manner and suppressed the activation of the AKTSer473 but had no effect on the ERK pathway. Perhexiline and FPER-1 markedly activated PDH (seen as dephosphorylation of PDH-E1αSer293), reduced glycolysis, and upregulated mitochondrial respiration in these PAH PASMCs as detected by Seahorse analysis. However, both perhexiline and FPER-1 did not induce apoptosis as measured by caspase 3/7 activity. We show for the first time that both perhexiline and FPER-1 may represent therapeutic agents for reducing cell proliferation in human PAH PASMCs, by reversing Warburg physiology. </p

Expanding LAGLIDADG endonuclease scaffold diversity by rapidly surveying evolutionary sequence space

LAGLIDADG homing endonucleases (LHEs) are a family of highly specific DNA endonucleases capable of recognizing target sequences ∼20 bp in length, thus drawing intense interest for their potential academic, biotechnological and clinical applications. Methods for rational design of LHEs to cleave desired target sites are presently limited by a small number of high-quality native LHEs to serve as scaffolds for protein engineering—many are unsatisfactory for gene targeting applications. One strategy to address such limitations is to identify close homologs of existing LHEs possessing superior biophysical or catalytic properties. To test this concept, we searched public sequence databases to identify putative LHE open reading frames homologous to the LHE I-AniI and used a DNA binding and cleavage assay using yeast surface display to rapidly survey a subset of the predicted proteins. These proteins exhibited a range of capacities for surface expression and also displayed locally altered binding and cleavage specificities with a range of in vivo cleavage activities. Of these enzymes, I-HjeMI demonstrated the greatest activity in vivo and was readily crystallizable, allowing a comparative structural analysis. Taken together, our results suggest that even highly homologous LHEs offer a readily accessible resource of related scaffolds that display diverse biochemical properties for biotechnological applications

Does board independence influence financial performance in IPO firms? The moderating role of the national business system

Prior evidence suggests that board independence may enhance financial performance, but this relationship has been tested almost exclusively for Anglo-American countries. To explore the boundary conditions of this prominent governance mechanism, we examine the impact of the formal and information institutions of 18 national business systems on the board independence-financial performance relationship. Our results show that while the direct effect of independence is weak, national-level institutions significantly moderate the independence-performance relationship. Our findings suggest that the efficacy of board structures is likely to be contingent on the specific national context, but the type of legal system is insignificant

Optimization of in vivo activity of a bifunctional homing endonuclease and maturase reverses evolutionary degradation

The LAGLIDADG homing endonuclease (LHE) I-AniI has adopted an extremely efficient secondary RNA splicing activity that is beneficial to its host, balanced against inefficient DNA cleavage. A selection experiment identified point mutations in the enzyme that act synergistically to improve endonuclease activity. The amino-acid substitutions increase target affinity, alter the thermal cleavage profile and significantly increase targeted recombination in transfected cells. The RNA splicing activity is not affected by these mutations. The improvement in DNA cleavage activity is largely focused on one of the enzyme's two active sites, corresponding to a rearrangement of a lysine residue hypothesized to act as a general base. Most of the constructs isolated in the screen contain one or more mutations that revert an amino-acid identity to a residue found in one or more close homologues of I-AniI. This implies that mutations that have previously reduced the endonuclease activity of I-AniI are identified and reversed, sometimes in combination with additional ‘artificial’ mutations, to optimize its in vivo activity

High-resolution profiling of homing endonuclease binding and catalytic specificity using yeast surface display

Experimental analysis and manipulation of protein–DNA interactions pose unique biophysical challenges arising from the structural and chemical homogeneity of DNA polymers. We report the use of yeast surface display for analytical and selection-based applications for the interaction between a LAGLIDADG homing endonuclease and its DNA target. Quantitative flow cytometry using oligonucleotide substrates facilitated a complete profiling of specificity, both for DNA-binding and catalysis, with single base pair resolution. These analyses revealed a comprehensive segregation of binding specificity and affinity to one half of the pseudo-dimeric interaction, while the entire interface contributed specificity at the level of catalysis. A single round of targeted mutagenesis with tandem affinity and catalytic selection steps provided mechanistic insights to the origins of binding and catalytic specificity. These methods represent a dynamic new approach for interrogating specificity in protein–DNA interactions

DNA Nicks Promote Efficient and Safe Targeted Gene Correction

Targeted gene correction employs a site-specific DNA lesion to promote homologous recombination that eliminates mutation in a disease gene of interest. The double-strand break typically used to initiate correction can also result in genomic instability if deleterious repair occurs rather than gene correction, possibly compromising the safety of targeted gene correction. Here we show that single-strand breaks (nicks) and double-strand breaks both promote efficient gene correction. However, breaks promote high levels of inadvertent but heritable genomic alterations both locally and elsewhere in the genome, while nicks are accompanied by essentially no collateral local mutagenesis, and thus provide a safer approach to gene correction. Defining efficacy as the ratio of gene correction to local deletion, nicks initiate gene correction with 70-fold greater efficacy than do double-strand breaks (29.0±6.0% and 0.42±0.03%, respectively). Thus nicks initiate efficient gene correction, with limited local mutagenesis. These results have clear therapeutic implications, and should inform future design of meganucleases for targeted gene correction

Systematic characterization of deubiquitylating enzymes for roles in maintaining genome integrity.

DNA double-strand breaks (DSBs) are perhaps the most toxic of all DNA lesions, with defects in the DNA-damage response to DSBs being associated with various human diseases. Although it is known that DSB repair pathways are tightly regulated by ubiquitylation, we do not yet have a comprehensive understanding of how deubiquitylating enzymes (DUBs) function in DSB responses. Here, by carrying out a multidimensional screening strategy for human DUBs, we identify several with hitherto unknown links to DSB repair, the G2/M DNA-damage checkpoint and genome-integrity maintenance. Phylogenetic analyses reveal functional clustering within certain DUB subgroups, suggesting evolutionally conserved functions and/or related modes of action. Furthermore, we establish that the DUB UCHL5 regulates DSB resection and repair by homologous recombination through protecting its interactor, NFRKB, from degradation. Collectively, our findings extend the list of DUBs promoting the maintenance of genome integrity, and highlight their potential as therapeutic targets for cancer.This is the author's accepted manuscript. The final version is available from Nature Publishing Group via http://dx.doi.org/10.1038/ncb302

The Social Case as a Business Case: Making Sense of Social Entrepreneurship from an Ordonomic Perspective

This article discusses how the theoretical perspective of ordonomics provides a framework for better understanding and advancing the practice of social entrepreneurship. From an ordonomic perspective, the concept of social entrepreneurship offers a semantic innovation (at the ideas level) whose potential for social innovation can be fully reaped only if it is used as a heuristics for social structural change (on the institutions level). Social entrepreneurs recognize relevant social problems, interpret them as an entrepreneurial challenge, and succeed in turning what was a social case into a business case in a broader sense. Using the real-life example of a successful eco-social entrepreneur, the article demonstrates that such win-win solutions can be reconstructed as the sophisticated management of social dilemmas. It sketches a strategy matrix for the practice of social entrepreneurship and distinguishes four paradigmatic strategies social entrepreneurs can employ to create win-win scenarios by changing the rules of the game to overcome undesirable social dilemmas. The article concludes by discussing social entrepreneurship in the context of new governance processes and highlights key similarities and differences to the concept of corporate citizenship.Dieser Artikel nutzt die theoretische Perspektive der Ordonomik, um einen konzeptionellen Beitrag zum Verständnis von Social Entrepreneurship zu entwickeln. Aus ordonomischer Sicht verbindet sich mit dem Konzept von Social Entrepreneurship eine semantische Innovation (auf der Ebene der Ideen), deren Potential für gesellschaftlichen Wandel jedoch daran geknüpft ist, sozialstrukturellen Wandel (also Reformen auf der Ebene der Institutionen) heuristisch anzuleiten. Social Entrepreneurs identifizieren relevante gesellschaftliche Probleme, interpretieren sie als unternehmerische Herausforderungen und vermögen es, einen „social case“ in einen „business case“ im weiteren Sinne zu transformieren. Am Beispiel eines realen Öko-Sozialunternehmers wird gezeigt, dass derartige Win-Win-Lösungen als das differenzierte Management sozialer Dilemmata rekonstruiert werden können. Der Beitrag entwirft eine Strategiematrix für Social Entrepreneurship und unterscheidet vier paradigmatische Strategien, wie Social Entrepreneurs Win-Win-Potentiale erschließen können, indem sie durch Bindungen unerwünschte soziale Dilemmata überwinden. Abschließend verortet der Artikel das Phänomen Social Entrepreneurship im Kontext von New Governance und diskutiert Unterschiede und Gemeinsamkeiten zum Konzept von Corporate Citizenship