A novel zinc-finger nuclease platform with a sequence-specific cleavage module.

Zinc-finger nucleases (ZFNs) typically consist of three to four zinc fingers (ZFs) and the non-specific DNA-cleavage domain of the restriction endonuclease FokI. In this configuration, the ZFs constitute the binding module and the FokI domain the cleavage module. Whereas new binding modules, e.g. TALE sequences, have been considered as alternatives to ZFs, no efforts have been undertaken so far to replace the catalytic domain of FokI as the cleavage module in ZFNs. Here, we have fused a three ZF array to the restriction endonuclease PvuII to generate an alternative ZFN. While PvuII adds an extra element of specificity when combined with ZFs, ZF-PvuII constructs must be designed such that only PvuII sites with adjacent ZF-binding sites are cleaved. To achieve this, we introduced amino acid substitutions into PvuII that alter K(m) and k(cat) and increase fidelity. The optimized ZF-PvuII fusion constructs cleave DNA at addressed sites with a >1000-fold preference over unaddressed PvuII sites in vitro as well as in cellula. In contrast to the 'analogous' ZF-FokI nucleases, neither excess of enzyme over substrate nor prolonged incubation times induced unaddressed cleavage in vitro. These results present the ZF-PvuII platform as a valid alternative to conventional ZFNs.

Selection-independent generation of gene knockout mouse embryonic stem cells using zinc-finger nucleases.

Gene knockout in murine embryonic stem cells (ESCs) has been an invaluable tool to study gene function in vitro or to generate animal models with altered phenotypes. Gene targeting using standard techniques, however, is rather inefficient and typically does not exceed frequencies of 10(-6). In consequence, the usage of complex positive/negative selection strategies to isolate targeted clones has been necessary. Here, we present a rapid single-step approach to generate a gene knockout in mouse ESCs using engineered zinc-finger nucleases (ZFNs). Upon transient expression of ZFNs, the target gene is cleaved by the designer nucleases and then repaired by non-homologous end-joining, an error-prone DNA repair process that introduces insertions/deletions at the break site and therefore leads to functional null mutations. To explore and quantify the potential of ZFNs to generate a gene knockout in pluripotent stem cells, we generated a mouse ESC line containing an X-chromosomally integrated EGFP marker gene. Applying optimized conditions, the EGFP locus was disrupted in up to 8% of ESCs after transfection of the ZFN expression vectors, thus obviating the need of selection markers to identify targeted cells, which may impede or complicate downstream applications. Both activity and ZFN-associated cytotoxicity was dependent on vector dose and the architecture of the nuclease domain. Importantly, teratoma formation assays of selected ESC clones confirmed that ZFN-treated ESCs maintained pluripotency. In conclusion, the described ZFN-based approach represents a fast strategy for generating gene knockouts in ESCs in a selection-independent fashion that should be easily transferrable to other pluripotent stem cells.

A novel TALE nuclease scaffold enables high genome editing activity in combination with low toxicity.

Sequence-specific nucleases represent valuable tools for precision genome engineering. Traditionally, zinc-finger nucleases (ZFNs) and meganucleases have been used to specifically edit complex genomes. Recently, the DNA binding domains of transcription activator-like effectors (TALEs) from the bacterial pathogen Xanthomonas have been harnessed to direct nuclease domains to desired genomic loci. In this study, we tested a panel of truncation variants based on the TALE protein AvrBs4 to identify TALE nucleases (TALENs) with high DNA cleavage activity. The most favorable parameters for efficient DNA cleavage were determined in vitro and in cellular reporter assays. TALENs were designed to disrupt an EGFP marker gene and the human loci CCR5 and IL2RG. Gene editing was achieved in up to 45% of transfected cells. A side-by-side comparison with ZFNs showed similar gene disruption activities by TALENs but significantly reduced nuclease-associated cytotoxicities. Moreover, the CCR5-specific TALEN revealed only minimal off-target activity at the CCR2 locus as compared to the corresponding ZFN, suggesting that the TALEN platform enables the design of nucleases with single-nucleotide specificity. The combination of high nuclease activity with reduced cytotoxicity and the simple design process marks TALENs as a key technology platform for targeted modifications of complex genomes.

Matricellular signaling molecule CCN1 attenuates experimental autoimmune myocarditis by acting as a novel immune cell migration modulator.

BACKGROUND: CCN1 is an evolutionary ancient matricellular protein that modulates biological processes associated with tissue repair. Induction at sites of injury was observed in conditions ranging from skin wounds to cardiac diseases, including ischemic and inflammatory cardiomyopathy. Here, we provide evidence of a novel function of CCN1 as a modulator of immune cell migration. METHODS AND RESULTS: to understand the role of CCN1 in cardiomyopathies and to evaluate its therapeutic potential, we overexpressed CCN1 using an adenoviral hepatotropic vector in murine experimental autoimmune myocarditis, a model of human inflammatory cardiomyopathy. CCN1 gene transfer significantly reduced cardiac disease score and immune cell infiltration. In vivo tracking of hemagglutinin epitope-tagged CCN1 revealed binding to spleen macrophages but not to cardiomyocytes. Unexpectedly, CCN1 therapy left cardiac chemokine and cytokine expression unchanged but instead strongly inhibited the migration of spleen macrophages and lymphocytes, as evidenced by ex vivo transwell assays. In accordance with the ex vivo data, in vitro preincubation with CCN1 diminished transwell migration of human monocytes and abrogated their chemotactic response to monocyte chemoattractant protein-1, macrophage inflammatory protein-1α, and stromal cell-derived factor-1α. Further mechanistic studies showed that CCN1-driven modulation of immune cell migration is mimicked in part by cyclic RGD peptides currently in clinical evaluation for cancer therapy. CONCLUSIONS: our proof-of-concept study suggests investigation of CCN1 as a novel, endogenous "parent compound" for chemotaxis modulation and of cyclic RGD peptides as a class of partially CCN1-mimetic drugs with immediate potential for clinical evaluation in cardiac diseases associated with chronic pathogenic inflammation.

Phosphatidylinositol 4,5-bisphosphate-specific AKT1 is oncogenic.

The protein kinase AKT1 (v-akt murine thymoma viral oncogene homolog 1), also referred to as protein kinase B (PKB), is an essential mediator of the phosphatidylinositol 3-kinase signaling pathway. Elevated activity of AKT1 is common in human cancer. Localization at the plasma membrane, leading to enhanced phosphorylation and activation of AKT1, is an important factor determining the oncogenicity of this kinase. Although the phosphatidylinositol 3-kinase signaling pathway is frequently upregulated in cancer, cancer-specific mutations in AKT1 are not common. Recently, such a mutation has been identified in breast, colon and ovarian cancers. The mutation is located in the pleckstrin homology (PH) domain of AKT1 and results in a glutamic acid to lysine substitution at residue 17. The resultant change in the conformation of the PH domain facilitates membrane binding of the mutant protein. Here we show that exchange of the PH domain leading to preferential binding of phosphatidylinositol 4,5-bisphosphate (PIP(2)) over phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) constitutively activates AKT1. AKT1 with this altered PIP affinity induces oncogenic transformation in cultures of chicken embryo fibroblasts and causes neoplastic growth and angiogenesis in the chorioallantoic membrane of the chicken embryo. Gain-of-function mutants of AKT1 may not be affected by PI3K inhibitors that are currently in development. Therefore, AKT1 remains a distinct and important cancer target.