Procedure through which white blood cells are separated and collected from the blood with an advanced centrifuge while red blood cells and other blood components are returned into the circulation.

No abstract

Meganucleases, or homing endonucleases (HEs) are sequence-specific endonucleases with large (>14 bp) cleavage sites that can be used to induce efficient homologous gene targeting in cultured cells and plants. These findings have opened novel perspectives for genome engineering in a wide range of fields, including gene therapy. However, the number of identified HEs does not match the diversity of genomic sequences, and the probability of finding a homing site in a chosen gene is extremely low. Therefore, the design of artificial endonucleases with chosen specificities is under intense investigation. In this report, we describe the first artificial HEs whose specificity has been entirely redesigned to cleave a naturally occurring sequence. First, hundreds of novel endonucleases with locally altered substrate specificity were derived from I-CreI, a Chlamydomonas reinhardti protein belonging to the LAGLIDADG family of HEs. Second, distinct DNA-binding subdomains were identified within the protein. Third, we used these findings to assemble four sets of mutations into heterodimeric endonucleases cleaving a model target or a sequence from the human RAG1 gene. These results demonstrate that the plasticity of LAGLIDADG endonucleases allows extensive engineering, and provide a general method to create novel endonucleases with tailored specificities.

The importance of safer approaches for gene therapy has been underscored by a series of severe adverse events (SAEs) observed in patients involved in clinical trials for Severe Combined Immune Deficiency Disease (SCID) and Chromic Granulomatous Disease (CGD). While a new generation of viral vectors is in the process of replacing the classical gamma-retrovirus–based approach, a number of strategies have emerged based on non-viral vectorization and/or targeted insertion aimed at achieving safer gene transfer. Currently, these methods display lower efficacies than viral transduction although many of them can yield more than 1% engineered cells in vitro. Nuclease-based approaches, wherein an endonuclease is used to trigger site-specific genome editing, can significantly increase the percentage of targeted cells. These methods therefore provide a real alternative to classical gene transfer as well as gene editing. However, the first endonuclease to be in clinic today is not used for gene transfer, but to inactivate a gene (CCR5) required for HIV infection. Here, we review these alternative approaches, with a special emphasis on meganucleases, a family of naturally occurring rare-cutting endonucleases, and speculate on their current and future potential.

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Meganucleases are sequence-specific endonucleases recognizing large (>12 bp) sequence sites and several laboratories have used these proteins to induce highly efficient gene targeting in mammalian cells. The recent development of artificial endonucleases with tailored specificities has opened the door for a wide range of new applications, including therapeutic ones: redesigned endonucleases cleaving chosen sequences could be used to in gene therapy to correct mutated genes or introduce transgenes in chosen loci. Such "targeted" approaches markedly differ from current gene therapy strategies based on the random insertion of a complementing virus-borne transgene. As a consequence, they should bypass the odds of random insertion. Artificial fusion proteins including Zinc-Finger binding domains have provided important proofs of concept, however the toxicity of these proteins is still an issue. Today custom-designed homing endonucleases, the natural meganucleases, could represent an efficient alternative. After a brief description of the origin of the technology, current systems based on redesigned endonucleases will be presented, with a special emphasis on the recent advances in homing endonuclease engineering. Finally, we will discuss the main issues that will need to be addressed in order to bring this promising technology to the patient.

Sequence-specific endonucleases recognizing long target sequences are emerging as powerful tools for genome engineering. These endonucleases could be used to correct deleterious mutations or to inactivate viruses, in a new approach to molecular medicine. However, such applications are highly demanding in terms of safety. Mutations in the human RAG1 gene cause severe combined immunodeficiency (SCID). Using the I-CreI dimeric LAGLIDADG meganuclease as a scaffold, we describe here the engineering of a series of endonucleases cleaving the human RAG1 gene, including obligate heterodimers and single-chain molecules. We show that a novel single-chain design, in which two different monomers are linked to form a single molecule, can induce high levels of recombination while safeguarding more effectively against potential genotoxicity. We provide here the first demonstration that an engineered meganuclease can induce targeted recombination at an endogenous locus in up to 6% of transfected human cells. These properties rank this new generation of endonucleases among the best molecular scissors available for genome surgery strategies, potentially avoiding the deleterious effects of previous gene therapy approaches.

Background: TALE-based technologies are poised to revolutionize the field of biotechnology; however, their sensitivity to cytosine methylation may drastically restrict their ranges of applications. Results: TALE repeat N* proficiently accommodates 5-methylated cytosine. Conclusion: Sensitivity of TALE to cytosine methylation can be overcome by using TALE repeat N*. Significance: Utilization of TALE repeat N* enables broadening the scope of TALE-based technologies.Within the past 2 years, transcription activator-like effector (TALE) DNA binding domains have emerged as the new generation of engineerable platform for production of custom DNA binding domains. However, their recently described sensitivity to cytosine methylation represents a major bottleneck for genome engineering applications. Using a combination of biochemical, structural, and cellular approaches, we were able to identify the molecular basis of such sensitivity and propose a simple, drug-free, and universal method to overcome it.Transcription activator-like effectors (TALEs), 4 a group of bacterial plant pathogen proteins, have recently emerged as new engineerable scaffolds for production of tailored DNA binding domains with chosen specificities (1). Interest in these systems comes from the apparent simple cipher governing DNA recognition by their DNA binding domain (2, 3). The TALE DNA binding domain is composed of multiple TALE repeats that individually recognize one DNA base pair through specific amino acid di-residues (repeat variable di-residues or RVDs). The remarkably high specificity of TALE repeats and the apparent absence of context-dependent effects among repeats in an array allow modular assembly of TALE DNA binding domains able to recognize almost any DNA sequence of interest. Within the past 2 years, engineered TALE DNA binding domains have been fused to transcription activator (dTALEs) (4), repressor (5), or nuclease domains (TALENs) (6) and used to specifically regulate or modify genes of interest (1). Although successfully used in different cellular contexts, engineered TALE DNA binding domains have recently been reported to be affected by the presence of 5-methylated cytosine (5mC) in their endogenous cognate target (7). Often considered as the fifth base, 5mC is found in about 70% of CpG dinucleotides in mammalian and plant somatic/pluripotent cells (8, 9) and has also been reported in 5-cytosine-phosphoadenine, 5-cytosine-phosphothymine, and 5-cytosine-phosphocytosine dinucleotides (10). Moreover, 5mC has been identified in CpG islands embedded in many promoters (11) and, to a higher extent, in proximal exons of several genes (12). These two critical regulatory regions are generally chosen by investigators to knock out genes of therapeutic and biotechnological interest or to modulate their expression using TALE-based technologies. The ubiquity of 5mC in different cell types and genomic kingdoms, its particular localization, and its negative impact on dTALE activity reported in Ref. 7 make this epigenetic modification a major dra...