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

Synthèse et caractérisation de nouveaux dérivés du closo-decahydro-decaborate par activation de la liaison B-H

In our work, we present a new technique for the activation of the B-H to B-L bond of closodecaborate. Therefore, we followed the pathway of dicages synthesis starting from monocages. New derivatives [B10H9LL'B10H9]4- resulting from the coupling between two monocages [B10H9L]- and [B10H9L']- were successfully synthesized and characterized by IR, 11B NMR, 1H NMR and mass spectrometry.In addition, a functionalization of the cage with bioactive molecules "sulfonamides" was the focus of our research. We were also able to isolate the products resulting from the coupling between (PPh4) [2-B10H9CO] and sulfonamides. The latter were characterized by IR, 11B NMR, 1H NMR, 13C NMR, 31P NMR and mass spectrometry.Finally, a very new method has been exposed in our work. It is based on the functionalization of [B10H10]2- derivatives based on Grignard reactions. Through the coupling between the (PPh4) [2- B10H9CO] derivative and the Grignard RMgX reagents, we synthesized a series of [2-B10H9COR]2- derivatives which were subsequently characterized by IR, 11B NMR, 1H NMR, 13C NMR, 31P NMR and mass spectrometry.Keywords: closo-decahydrodecaborates, BNCT, dicages, bioactive molecule "sulfonamide", Grignard reagents.Dans notre travail, nous exposons une nouvelle technique d’activation de la liaison B-H en B-L du closo-decaborate. De ce fait, nous avons suivi le chemin de synthèse des dicages en partant des monocages. De nouveaux dérivés [B10H9LL’B10H9]4- résultants du couplage entre deux monocages [B10H9L]- et [B10H9L’]- ont été synthétisés avec succès et caractérisés par IR, RMN 11B, RMN 1H et spectrométrie de masse.De plus, une fonctionnalisation de la cage par des molécules bioactives « sulfonamides » a constitué l’objet de notre recherche. Nous avons également pu isoler les produits résultants du couplage entre le (PPh4) [2-B10H9CO] et les sulfonamides. Ces derniers ont été caractérisés par IR, RMN 11B, RMN 1H, RMN 13C, RMN 31P et spectrométrie de masse.Finalement, une méthode toute nouvelle a été exposé dans notre travail. Elle est basée sur la fonctionnalisation des dérivés du [B10H10]2- en se basant sur les réactions de Grignard. Par le biais du couplage entre le dérivé (PPh4) [2-B10H9CO] et les réactifs de Grignard RMgX, nous avons synthétisé une série de dérivés [2-B10H9COR]2- qui ont été par la suite caractérisés par IR, RMN 11B, RMN 1H, RMN 13C, RMN 31P et spectrométrie de masse.Mots-clés : closo-décahydrodécaborates, BNCT, dicages, molécule bioactive « sulfonamide », réactifs de Grignard

Recent Achievements on Functionalization within closo‐Decahydrodecaborate [B 10 H 10 ] 2− Clusters

International audienc

Gastrointestinal cancer cells treatment with bevacizumab activates a VEGF autoregulatory mechanism involving telomerase catalytic subunit hTERT via PI3K-AKT, HIF-1α and VEGF receptors

<div><p>Background</p><p>Targeting angiogenesis has been considered a promising treatment of choice for a large number of malignancies, including gastrointestinal cancers. Bevacizumab is an anti-vascular endothelial growth factor (anti-VEGF) being used for this purpose. However, treatment efficacy is largely questioned. Telomerase activity, responsible for cancer cell immortality, is detected in 85–95% of human cancers and is considered a potential regulator of VEGF. The aim of our study was to investigate the interrelationship between VEGF and hTERT in gastrointestinal cancers and to explore cell response to a combined inhibition of telomerase and VEGF.</p><p>Methods</p><p>AGS (gastric cancer), Caco-2 (colorectal cancer) and HepG2/C3A (hepatocellular carcinoma), were treated with telomerase inhibitors BIBR-1232 (10μM) and costunolide (10μM), with bevacizumab (Avastin® at 5 ng/ml or 100μg/ml) or with a combination of both types of inhibitors. VEGF and hTERT mRNA levels, and telomerase activity were detected by RT-PCR. VEGF levels were quantified by ELISA. Telomerase was knocked down using hTERT siRNA and hTERT was overexpressed in the telomerase negative cell line, Saos-2 (osteosarcoma), using constructs expressing either wild type hTERT (hTERT-WT) or dominant negative hTERT (hTERT-DN). Tube formation by HUVECs was assessed using ECMatrix™ (EMD Millipore).</p><p>Results</p><p>Our results showed that telomerase regulates VEGF expression and secretion through its catalytic subunit hTERT in AGS, Caco2, and HepG2/C3A, independent of its catalytic activity. Interestingly, VEGF inhibition with bevacizumab (100μg/ml) increased hTERT expression 42.3% in AGS, 94.1% in Caco2, and 52.5% in HepG2/C3A, and increased telomerase activity 30-fold in AGS, 10.3-fold in Caco2 and 8-fold in HepG2/C3A. A further investigation showed that VEGF upregulates hTERT expression in a mechanism that implicates the PI3K/AKT/mTOR pathway and HIF-1α. Moreover, bevacizumab treatment increased VEGFR1 and VEGFR2 expression in cancer cells and human umbilical vein endothelial cells (HUVECs) through hTERT. Thus, the combination of bevacizumab with telomerase inhibitors decreased VEGF expression and secretion by cancer cells, inhibited VEGFR1 and VEGFR2 upregulation, and reduced tube formation by HUVECs.</p><p>Conclusions</p><p>Taken together, our results suggest that bevacizumab treatment activates a VEGF autoregulatory mechanism involving hTERT and VEGF receptors and that an inhibition of this pathway could improve tumor cell response to anti-VEGF treatment.</p></div

Telomerase regulates VEGF secretion and expression independently of telomerase activity.

<p>(A, B) VEGF secretion was assessed by ELISA following a 48-h treatment with telomerase inhibitors BIBR-1532 (10 <i>μ</i>M) and costunolide (10 <i>μ</i>M), and after 72 h of hTERT knockdown with siRNA. (C, D) Total RNAs were isolated from the cells treated in (A), and VEGF expression was analyzed by real-time PCR with GAPDH as the internal control. (E, F, G) Saos-2 cells were transiently transfected with an empty vector, hTERT-WT, and hTERT-DN. hTERT (E) and VEGF expression (F) were quantified by real-time PCR. Secreted VEGF was quantified by ELISA (G). Results were expressed as the mean ±SD from a minimum of three experiments.</p

VEGF induces hTERT expression via PI3K/AKT/mTOR and HIF-1α.

<p>AGS (A), Caco2 (B), and HepG2/C3A (C) were treated with PI 828 (10<i>μ</i>M), GSK 690693 (100nM), Rapamycin (200nM), and KC7F2 (40<i>μ</i>M) with or without bevacizumab for 48 h. The amount of hTERT transcripts was quantified by real-time PCR. (D) EMSA was performed on nuclear cell extracts incubated with oligonucleotides of two HIF-1α binding sites, HRE-1 and HRE-2. (E) VEGF secretion was quantified with ELISA. (F) Total RNAs from the cells treated in (E) were collected, and the amount of hTERT transcripts was quantified by real-time PCR. Results were expressed as the mean ± SD from three experiments.</p

Telomerase regulates VEGF, VEGFR1, and VEGFR2 expression.

<p>AGS (A), Caco2 (B), and HepG2/C3A (C) cells were treated with control siRNA or hTERT siRNA with or without bevacizumab, and VEGF secretion was evaluated by ELISA. (D, E) Cells were treated with BIBR-1532, costunolide, or transiently transfected with hTERT siRNA for 72h. VEGFR1 (D) and VEGFR2 (E) transcript amounts were quantified with real-time PCR. (F, G) Saos-2 cells were transiently transfected with an empty vector, hTERT-WT or hTERT-DN. VEGFR1 (F) and VEGFR2 (G) were then quantified with real-time PCR. Results were expressed as the mean ± SD from three experiments.</p

The effect of bevacizumab treatment on PI3K, AKT, and mTOR expression.

<p>The effect of bevacizumab treatment on PI3K, AKT, and mTOR expression.</p

Bevacizumab increases VEGFR1 and VEGFR2 expression.

<p>(A, B) Telomerase inhibitors were combined to bevacizumab (100 <i>μ</i>g/ml) and then VEGFR1 (A) and VEGFR2 (B) were quantified by real-time PCR. (C, D) Cells were transiently transfected with either control siRNA or hTERT siRNA and treated with bevacizumab (100 <i>μ</i>g/ml). VEGFR1 (C) and VEGFR2 (D) were quantified by real-time qPCR. (E, F) HUVECs were cultured in the presence of telomerase inhibitors for 48 h and hTERT siRNA for 72 h. The expression of VEGFR1 (E) and VEGFR2 (F) were quantified by real-time PCR. Results were expressed as the mean ± SD from three experiments.</p