This paper is the first report describing the induction of gene targeting in an endogenous gene by a single chain engineered meganuclease. The targeted region is within the human Rag1 gene, in which inactivation results in a Severe Combined Immune Deficiency. Meganuclease-induced gene targeting could be achieved in up to 6% of the treated cells, a level which is compatible with therapeutic applications. Thus, these results constitute an essential milestone in the strategy of Cellectis, to develop meganucleases for therapeutic applications.
In order to achieve highly efficient gene targeting in human cells, with maximal accuracy, Cellectis designed a series of novel meganucleases with improved specificities. Furthermore, specific modifications were used to improve efficacies. Most engineered endonucleases developed for therapeutic purposes today are dimeric proteins, made of two different molecules, which interact to form what is called a heterodimer, but they can also be found as homodimers (i.e., each one of the two molecules can interact with itself, forming additional products). Two different strategies can be used in order to avoid the formation of such homodimers: the making of obligate heterodimers (pairs of molecules that cannot form homodimers anymore) and single-chain meganucleases. The first designs aimed at improving the specificity of meganucleases have been described by in earlier studies, by Cellectis in 2003 for single chain molecules (Epinat et al., 2003, Nucleic Acids Res. 31: 2952-2962) and by the Group of Luis Serrano (EMBL-CRG), in collaboration with Cellectis, in 2008 for obligate heterodimers (Fajardo-Sanchez et al., 2008, Nucleic Acids Res. 36: 2163-2173). In this new report, Cellectis used a series of meganucleases that cleave the same target within the Rag1 gene, and showed that these two strategies can provide ideal tools to surgically edit a human gene. Detailed characterization of the single chain proteins properties demonstrated that they could compare with the I-SceI meganuclease, the most specific natural meganuclease and the gold standard in the field, in terms of specificity and innocuity. ”These results constitute a milestone in our strategy to develop the best molecular tools for genome engineering” declared Frédéric Pâques, the CSO of Cellectis.
Further studies will be conducted with these meganucleases, in order to correct mutations in patient cell lines, and eventually, to cure SCID patients. “The use of these engineered meganucleases in a clinical trial would be a wonderful demonstration of the potential of engineered meganucleases for improving human health”, declared André Choulika, the CEO of Cellectis.
About Cellectis
Cellectis S.A. (www.cellectis.com) is a world leader in genome engineering and genome surgery. For more information on Cellectis, visit our web site: www.cellectis.com
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