964. Combinatorial Process for Engineering Specific Meganucleases with Tailored Specificities toward a Natural Target in the XPC Gene

Abstract

Xeroderma pigmentosum (XP) is a rare disease transmitted in an autosomal recessive manner Patients have an extreme sensitivity to sunlight and develop serious sunburns with onset of poikilodermia in the light-exposed skin. Skin cancers already appear in childhood, and the disease can also be associated with neurological defects. XP results from defects in the Nucleotide Excision Repair (NER) pathway, a DNA maintenance system which removes UV induced DNA damage such as cyclobutane pyrimidine dimers. XP Patients were assigned to 7 complementation groups (XP-A to XP-G), each complementation group resulting from mutations in a distinct NER gene. There is no treatment, and the majority of patients die before reaching adulthood because of metastases. However, skin engraftment can be made locally, but with the general limitations of grafts. Thus gene and cell therapy represents a huge hope for this kind of disease. Meganucleases are at the basis of a new kind of gene therapy for inherited monogenic disease, based on gene correction instead of gene complementation. These sequence-specific endonucleases can stimulate homologous gene targeting by several orders of magnitude, thereby enabling the correction of mutated genes with significant efficiency. Recently, meganucleases have been used to induce targeted recombination events in mouse hepatocytes with high efficiency, paving the way for therapeutic applications. One of the major challenges is to tailor artificial meganucleases cleaving the gene of interest, while keeping high levels of specificity. We have used a semi-rational approach to produce meganucleases targeting the XPC gene. These novel meganucleases display high levels of activity and specificity. Such results identify modularity of different functional subdomains in the I-CreI DNA-binding scaffold that can be modified without altering the overall structure, and be combined to achieve novel specificities. Xeroderma pigmentosum (XP) is a rare disease transmitted in an autosomal recessive manner Patients have an extreme sensitivity to sunlight and develop serious sunburns with onset of poikilodermia in the light-exposed skin. Skin cancers already appear in childhood, and the disease can also be associated with neurological defects. XP results from defects in the Nucleotide Excision Repair (NER) pathway, a DNA maintenance system which removes UV induced DNA damage such as cyclobutane pyrimidine dimers. XP Patients were assigned to 7 complementation groups (XP-A to XP-G), each complementation group resulting from mutations in a distinct NER gene. There is no treatment, and the majority of patients die before reaching adulthood because of metastases. However, skin engraftment can be made locally, but with the general limitations of grafts. Thus gene and cell therapy represents a huge hope for this kind of disease. Meganucleases are at the basis of a new kind of gene therapy for inherited monogenic disease, based on gene correction instead of gene complementation. These sequence-specific endonucleases can stimulate homologous gene targeting by several orders of magnitude, thereby enabling the correction of mutated genes with significant efficiency. Recently, meganucleases have been used to induce targeted recombination events in mouse hepatocytes with high efficiency, paving the way for therapeutic applications. One of the major challenges is to tailor artificial meganucleases cleaving the gene of interest, while keeping high levels of specificity. We have used a semi-rational approach to produce meganucleases targeting the XPC gene. These novel meganucleases display high levels of activity and specificity. Such results identify modularity of different functional subdomains in the I-CreI DNA-binding scaffold that can be modified without altering the overall structure, and be combined to achieve novel specificities.