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- Title
948. Rapid Production of Specific Artificial Meganucleases for Gene Correction in Patients with Inherited Disease.
- Authors
Duchateau, Philippe; Chames, Patrick; Epinat, Jean-Charles; Sylvain, Arnould; Smith, Julie; Gouble, Agnes; Perez, Christophe; Grizot, Sylvestre; Paques, Frederic
- Abstract
Meganucleases are sequence-specific endonucleases recognizing large (>14 bp) sequences. Because of this unusual specificity, meganuclease have emerged as powerful tools for genome engineering: creation of double-strand breaks by meganucleases at specific loci can be used to enhance homologous gene targeting by several orders of magnitude in mammalian and plant cells. Recently, such proteins could be used to induce gene repair in mouse hepatocytes, paving the way for a novel strategy for inherited diseases therapy based on gene correction instead of complementation. Thus, the generation of meganucleases with tailored specificities is under intense investigation. A major issue is whether it is possible to obtain novel dedicated endonuclease which levels of specificity compatible with the high requirements of therapeutic applications.Homing endonucleases (HEs) are natural meganucleases encoded by mobile genetic elements such as class I introns and inteins. Their exquisite specificity identifies them as ideal scaffolds to derive novel meganucleases with engineered DNA binding domains. HEs from the LAGLIDADG family, the largest and most widespread family of HEs, display two independent domains or units. Moreover, despite the lack of apparent additional modularity at the structural level, we have identified separable functional subdomains within a same LAGLIDADG domain binding distinct parts of the DNA target. Using a semi-rational approach, we have used a two steps strategy to produce meganucleases cleaving natural genes. In a first step, we have derived hundreds of novel meganucleases with novel specificities from I-CreI, a natural homodimeric homing endonuclease. In a second step, a combinatorial approach allows us to assemble these I-CreI variants into novel meganucleases with predictable cleavage site. Importantly, these proteins have retained high levels of specificity. Examples of meganucleases with potential therapeutic applications will be disclosed.Molecular Therapy (2006) 13, S366–S366; doi: 10.1016/j.ymthe.2006.08.1040
- Subjects
NUCLEASES; GENETIC disorders; ENDONUCLEASES; GENETIC engineering; GENE targeting; LIVER cells
- Publication
Molecular Therapy, 2006, Vol 13, pS366
- ISSN
1525-0016
- Publication type
Article
- DOI
10.1016/j.ymthe.2006.08.1040