Le principal défi des techniques de transfert de gènes est de garantir l’expression du gène d’intérêt tout en assurant l’innocuité des cellules génétiquement modifiées. La plupart des systèmes d’intégration, dérivés des transposons, assurent une intégration aléatoire au sein du génome de la cellule. L’enjeu de ce travail est de développer des outils permettant de cibler l’intégration du transgène au niveau d’un locus choisi dans le but d’améliorer la biosécurité. L’étude s’est portée sur l’utilisation des protéines à ZFD (Zinc Finger Domain) pour leur aptitude à être conçues à façon, in silico, en utilisant les nombreuses ressources disponibles sur Internet. Pour comparer, les propriétés de deux domaines de liaison, NterR2P, provenant de deux rétrotransposons R2 de type non-LTR, ont été étudiées pour leur capacité naturelle à reconnaître spécifiquement une région de 100 pb située dans l’ADN ribosomique 28S. Les résultats obtenus ont montré que les domaines NterR2P reconnaissent spécifiquement leur ADN cible avec une forte affinité de liaison. Deux protéines de fusion, utilisant le domaine NterR2P, ont ensuite été synthétisées dans le but d’intégrer le transgène au niveau de l’ADNr en utilisant le transposon Sleeping Beauty. L’idée originale de ce travail est de réaliser l’intégration du transgène via le ciblage indirect du transposon, ou de la transposase sans que celle-ci ne soit modifiée. L’impact de ces systèmes de ciblage sur les cellules nécessite de réexaminer une telle stratégie. / The main challenge of gene transfer technologies is to maintain and to sustain transgene expression and to confer innocuity on the genetically-modified cells. Most integration systems, derived from transposons, integrate randomly within the genome of cells. The issue of this work is to develop tools to target transgene integrations in a selected locus in order to improve biosecurity. This study consists in using ZFD (Zinc Finger Domain) proteins for their capability to be in silico synthesize, in using many bioinformatic sites. For compare, the properties of two DNA binding domains (DBD), NterR2P, originating from the endonucleases encoded by R2 non-LTR retrotransposons, are able to bind specifically within a 100-bp region of the 28S rRNA genes. The results show that NterR2P DBDs specifically recognize their DNA target with high affinity. Two fusion proteins, using NterR2P DBD, are synthesized in order to integrate the transgene within rDNA, using Sleeping Beauty transposon. The original idea of this work is to realize transgene integration via indirect targeting of transposon, or transposase without its modification. The use of such a targeting system will have to be extensively studied to determine its impacts on cells before it can be considered as safe for use.

GWAS have identified several chromosomal loci associated with ovarian cancer risk. However, the mechanism underlying these associations remains elusive. We identify candidate functional Single Nucleotide Polymorphisms (SNPs) at the 9p22.2 ovarian cancer susceptibility locus, several of which map to transcriptional regulatory elements active in ovarian cells identified by FAIRE-seq (Formaldehyde assisted isolation of regulatory elements followed by sequencing) and ChIP-seq (Chromatin Immunoprecipitation followed by sequencing) in relevant cell types. Reporter and electrophoretic mobility shift assays (EMSA) determined the extent to which candidate SNPs had allele specific effects. Chromosome conformation capture (3C) reveals a physical association between Basonuclin 2 (BNC2) and SNPs with functional properties. This establishes BNC2 as a major target of four candidate functional SNPs in at least two distinct elements. BNC2 codes for a putative transcription regulator containing three pairs of zinc finger (ZF) domains. Furthermore, bnc2 mutation in zebrafish leads to developmental defects including dysmorphic ovaries and sterility, clearly implicating this protein in cellular processes associated with ovarian development. We show that BNC2 is a transcriptional regulator with a specific DNA recognition sequence of targets enriched in genes involved in cell communication through DNA binding assays, ChIP-seq, and expression analysis. This study reveals a comprehensive regulatory landscape at the 9p22.2 locus and indicates that a likely mechanism of susceptibility to ovarian cancer may include multiple allele-specific changes in DNA regulatory elements some of which alter BNC2 expression. This study begins to identify the underlying mechanisms of the 9p22.2 locus association with ovarian cancer and aims to provide data to support advances in care based on one’s genetic composition.