Chromosomal context and epigenetic mechanisms control the efficacy of genome editing by rare-cutting designer endonucleases.

The ability to specifically engineer the genome of living cells at precise locations using rare-cutting designer endonucleases has broad implications for biotechnology and medicine, particularly for functional genomics, transgenics and gene therapy. However, the potential impact of chromosomal context and epigenetics on designer endonuclease-mediated genome editing is poorly understood. To address this question, we conducted a comprehensive analysis on the efficacy of 37 endonucleases derived from the quintessential I-CreI meganuclease that were specifically designed to cleave 39 different genomic targets. The analysis revealed that the efficiency of targeted mutagenesis at a given chromosomal locus is predictive of that of homologous gene targeting. Consequently, a strong genome-wide correlation was apparent between the efficiency of targeted mutagenesis (≤ 0.1% to ≈ 6%) with that of homologous gene targeting (≤ 0.1% to ≈ 15%). In contrast, the efficiency of targeted mutagenesis or homologous gene targeting at a given chromosomal locus does not correlate with the activity of individual endonucleases on transiently transfected substrates. Finally, we demonstrate that chromatin accessibility modulates the efficacy of rare-cutting endonucleases, accounting for strong position effects. Thus, chromosomal context and epigenetic mechanisms may play a major role in the efficiency rare-cutting endonuclease-induced genome engineering.

Sequence-specific targeting of IGF-I and IGF-IR genes by camptothecins.

We and others have clearly demonstrated that a topoisomerase I (Top1) inhibitor, such as camptothecin (CPT), coupled to a triplex-forming oligonucleotide (TFO) through a suitable linker can be used to cause site-specific cleavage of the targeted DNA sequence in in vitro models. Here we evaluated whether these molecular tools induce sequence-specific DNA damage in a genome context. We targeted the insulin-like growth factor (IGF)-I axis and in particular promoter 1 of IGF-I and intron 2 of type 1 insulin-like growth factor receptor (IGF-IR) in cancer cells. The IGF axis molecules represent important targets for anticancer strategies, because of their central role in oncogenic maintenance and metastasis processes. We chemically attached 2 CPT derivatives to 2 TFOs. Both conjugates efficiently blocked gene expression in cells, reducing the quantity of mRNA transcribed by 70-80%, as measured by quantitative RT-PCR. We confirmed that the inhibitory mechanism of these TFO conjugates was mediated by Top1-induced cleavage through the use of RNA interference experiments and a camptothecin-resistant cell line. In addition, induction of phospho-H2AX foci supports the DNA-damaging activity of TFO-CPT conjugates at specific sites. The evaluated conjugates induce a specific DNA damage at the target gene mediated by Top1.

The triple helix: 50 years later, the outcome.

Triplex-forming oligonucleotides constitute an interesting DNA sequence-specific tool that can be used to target cleaving or cross-linking agents, transcription factors or nucleases to a chosen site on the DNA. They are not only used as biotechnological tools but also to induce modifications on DNA with the aim to control gene expression, such as by site-directed mutagenesis or DNA recombination. Here, we report the state of art of the triplex-based anti-gene strategy 50 years after the discovery of such a structure, and we show the importance of the actual applications and the main challenges that we still have ahead of us.

Optimized synthesis and enhanced efficacy of novel triplex-forming camptothecin derivatives based on gimatecan.

Sequence-specific camptothecins are useful tools to inhibit specifically gene expression. The camptothecins are attached to the 3' end of triplex-forming oligonucleotides (TFO), sequence-specific DNA ligands that position the camptothecin moiety exclusively in proximity to their binding site. We studied here different gimatecan derivatives or analogues, a potent lipophilic camptothecin compound in clinical trials. We optimized the synthesis procedure in order to increase the yields and the purity and obtain the conjugates on a large scale. The greatly improved synthesis is now based on the conjugation of a bromoalkyl analogue of gimatecan to the 3' phosphorothioate of the TFO. We showed that the most efficient conjugate, both in vitro and in HeLa cells, bears the TFO on position 7 of the gimatecan analogue, and it is more efficient than the previous camptothecin conjugates. In addition, the gimatecan-like moiety at the 3' end of the TFO protects from nuclease degradation.

Exploring the cellular activity of camptothecin-triple-helix-forming oligonucleotide conjugates.

Topoisomerase I is a ubiquitous DNA-cleaving enzyme and an important therapeutic target in cancer chemotherapy for camptothecins (CPTs). These drugs stimulate DNA cleavage by topoisomerase I but exhibit little sequence preference, inducing toxicity and side effects. A convenient strategy to confer sequence specificity consists of the linkage of topoisomerase poisons to DNA sequence recognition elements. In this context, triple-helix-forming oligonucleotides (TFOs) covalently linked to CPTs were investigated for the capacity to direct topoisomerase I-mediated DNA cleavage in cells. In the first part of our study, we showed that these optimized conjugates were able to regulate gene expression in cells upon the use of a Photinus pyralis luciferase reporter gene system. Furthermore, the formation of covalent topoisomerase I/DNA complexes by the TFO-CPT conjugates was detected in cell nuclei. In the second part, we elucidated the molecular specificity of topoisomerase I cleavage by the conjugates by using modified DNA targets and in vitro cleavage assays. Mutations either in the triplex site or in the DNA duplex receptor are not tolerated; such DNA modifications completely abolished conjugate-induced cleavage all along the DNA. These results indicate that these conjugates may be further developed to improve chemotherapeutic cancer treatments by targeting topoisomerase I-induced DNA cleavage to appropriately chosen genes.

Molecular basis of the targeting of topoisomerase II-mediated DNA cleavage by VP16 derivatives conjugated to triplex-forming oligonucleotides.

Human topoisomerase II (topo II) is the cellular target for a number of widely used antitumor agents, such as etoposide (VP16). These agents 'poison' the enzyme and induce it to generate DNA breaks that are lethal to the cell. Topo II-targeted drugs show a limited sequence preference, triggering double-stranded breaks throughout the genome. Circumstantial evidence strongly suggests that some of these breaks induce chromosomal translocations that lead to specific types of leukaemia (called treatment-related or secondary leukaemia). Therefore, efforts are ongoing to decrease these secondary effects. An interesting option is to increase the sequence-specificity of topo II-targeted drugs by attaching them to triplex-forming oligonucleotides (TFO) that bind to DNA in a highly sequence-specific manner. Here five derivatives of VP16 were attached to TFOs. The active topo II poisons, once linked, induced cleavage 13-14 bp from the triplex end where the drug was attached. The use of triple-helical DNA structures offers an efficient strategy for targeting topo II-mediated cleavage to DNA specific sequences. Finally, drug-TFO conjugates are useful tools to investigate the mechanistic details of topo II poisoning.

[Pyeloplasty: aesthetic benefit of lumboscopy]. / Pyéloplastie: bénéfice pariétal de la lomboscopie.

STUDY OBJECTIVE: To evaluate the aesthetic benefit of lumboscopy versus lumbotomy in a group of patients undergoing pyeloplasty. MATERIAL AND METHOD: A telephone survey was conducted in 103 people operated by pyeloplasty via lumboscopy or lumbotomy. The questions concerned the appearance of the scar (size, relief; visible or invisible), skin complications, satisfaction with the scar, postoperative pain, length of hospital stay and return to physical activity or work. RESULTS: The response rate was 58.2% corresponding to 32 lumboscopies and 29 lumbotomies. Lumboscopy scars were shorter (2.7 vs 15.6 cm for lumbotomies, p<0.001). Lumbotomy scars were palpable in 75% of cases versus 53.1% of lumboscopy scars of (p=0.037). Almost 96.5% of lumbotomy scars were visible versus only 68.7% of lumboscopy scars (p=0.0057). Lumbotomies were complicated by incisional hernia in 14.2% of cases, while no incisional hernias were observed in the lumboscopy group (p=0.02). 85% of patients were satisfied with their scars in the lumboscopy group versus 56% of patients operated by lumbotomy (p=0.0286). CONCLUSION: This study showed a statistically significant difference in terms of a purely aesthetic benefit and scar solidity in favour of lumboscopy, which supports the growing place of this surgical technique in urology.

Triple helix-forming oligonucleotides conjugated to new inhibitors of topoisomerase II: synthesis and binding properties.

Triplex-forming oligonucleotides (TFOs) are among the most specific DNA ligands and represent an important tool for specific regulation of gene expression. TFOs have also been used to target DNA-modifying molecules to obtain irreversible modifications on a specific site of the genome. A number of molecules have been recognized to target topoisomerase II and stabilize double-stranded cleavage mediated by this enzyme thus determining permanent DNA damage. Among these poisons, etoposide (VP16), a 4'-demethylepipodophyllotoxin derivative, is widely used in cancer chemotherapy. In the aim to design DNA site-specific molecules, three analogues of VP16 (1, 2, and 3), recently described (Duca et al. J. Med. Chem. 2005, 48, 596-603), were attached to TFOs, together with a fourth one, of which the synthesis is reported here. Two different oligonucleotides, differing by the length (a 16-mer and a 20-mer), and two different linker arms between the oligonucleotide and the drug were used. The coupling reaction between the drug and the TFO was further improved. For the first time, we also report the synthesis of TFO conjugates bearing two molecules of inhibitor linked to the same oligonucleotide end. In total, 16 new conjugates were synthesized and evaluated for their ability to form triple helices. The loss in triplex stability due to the conjugation of the TFO to compounds that do not interact with DNA is compensated by the presence of the ethylene glycol linker arm. This stabilization effect is more pronounced at the 3' end than at the 5' end. All conjugates form a stable triplex selectively on the DNA target at 37 degrees C and pH 7.2.