title: Hybrid lentivirus-phiC31-int-NLS vector allows site-specific recombination in murine and human cells but induces DNA damage.
abstract: Gene transfer allows transient or permanent genetic modifications of cells for experimental or therapeutic purposes. Gene delivery by HIV-derived lentiviral vector (LV) is highly effective but the risk of insertional mutagenesis is important and the random/uncontrollable integration of the DNA vector can deregulate the cell transcriptional activity. Non Integrative Lentiviral Vectors (NILVs) solve this issue in non-dividing cells, but they do not allow long term expression in dividing cells. In this context, obtaining stable expression while avoiding the problems inherent to unpredictable DNA vector integration requires the ability to control the integration site. One possibility is to use the integrase of phage phiC31 (phiC31-int) which catalyzes efficient site-specific recombination between the attP site in the phage genome and the chromosomal attB site of its Streptomyces host. Previous studies showed that phiC31-int is active in many eukaryotic cells, such as murine or human cells, and directs the integration of a DNA substrate into pseudo attP sites (pattP) which are homologous to the native attP site. In this study, we combined the efficiency of NILV for gene delivery and the specificity of phiC31-int for DNA substrate integration to engineer a hybrid tool for gene transfer with the aim of allowing long term expression in dividing and non-dividing cells preventing genotoxicity. We demonstrated the feasibility to target NILV integration in human and murine pattP sites with a dual NILV vectors system: one which delivers phiC31-int, the other which constitute the substrate containing an attB site in its DNA sequence. These promising results are however alleviated by the occurrence of significant DNA damages. Further improvements are thus required to prevent chromosomal rearrangements for a therapeutic use of the system. However, its use as a tool for experimental applications such as transgenesis is already applicable.
authors: Unit of Biotechnology and Biotherapy, Centre de recherche de l'Institut du Cerveau et de la Moelle Epinière, Pierre-and-Marie-Curie University/Institut National de la Santé et de la Recherche Médicale, Paris, France; NewVectys, Villebon-sur-Yvette, France; Biosource, Paris, France.
authors: Nicolas
authors: N
authors: Grandchamp
authors: Unit of Biotechnology and Biotherapy, Centre de recherche de l'Institut du Cerveau et de la Moelle Epinière, Pierre-and-Marie-Curie University/Institut National de la Santé et de la Recherche Médicale, Paris, France; NewVectys, Villebon-sur-Yvette, France.
authors: Dorothée
authors: D
authors: Altémir
authors: Unit of Biotechnology and Biotherapy, Centre de recherche de l'Institut du Cerveau et de la Moelle Epinière, Pierre-and-Marie-Curie University/Institut National de la Santé et de la Recherche Médicale, Paris, France; NewVectys, Villebon-sur-Yvette, France; Biosource, Paris, France.
authors: Stéphanie
authors: S
authors: Philippe
authors: Unit of Biotechnology and Biotherapy, Centre de recherche de l'Institut du Cerveau et de la Moelle Epinière, Pierre-and-Marie-Curie University/Institut National de la Santé et de la Recherche Médicale, Paris, France; NewVectys, Villebon-sur-Yvette, France; Biosource, Paris, France.
authors: Suzanna
authors: S
authors: Ursulet
authors: Unit of Biotechnology and Biotherapy, Centre de recherche de l'Institut du Cerveau et de la Moelle Epinière, Pierre-and-Marie-Curie University/Institut National de la Santé et de la Recherche Médicale, Paris, France; NewVectys, Villebon-sur-Yvette, France; Biosource, Paris, France.
authors: Héloïse
authors: H
authors: Pilet
authors: Laboratoire de Virologie Moléculaire et Structurale, Gif-sur-Yvette, France.
authors: Marie-Claude
authors: MC
authors: Serre
authors: Commissariat à l'Energie Atomique, Laboratoire de Radiobiologie et Oncologie, Fontenay-aux-Roses, France.
authors: Aude
authors: A
authors: Lenain
authors: Molecular Imaging Research Center - Modélisation des biothérapies, Fontenay-aux-Roses, France.
authors: Che
authors: C
authors: Serguera
authors: Unit of Biotechnology and Biotherapy, Centre de recherche de l'Institut du Cerveau et de la Moelle Epinière, Pierre-and-Marie-Curie University/Institut National de la Santé et de la Recherche Médicale, Paris, France.
authors: Jacques
authors: J
authors: Mallet
authors: Unit of Biotechnology and Biotherapy, Centre de recherche de l'Institut du Cerveau et de la Moelle Epinière, Pierre-and-Marie-Curie University/Institut National de la Santé et de la Recherche Médicale, Paris, France; NewVectys, Villebon-sur-Yvette, France.
authors: Chamsy
authors: C
authors: Sarkis
mesh_terms: D000818:Animals; D001287:Attachment Sites, Microbiological / Q000235:genetics; D001435:Bacteriophages / Q000378:metabolism*; D001483:Base Sequence; D002460:Cell Line; D004249:DNA Damage; D005822:Genetic Vectors / Q000378:metabolism*; D006801:Humans; D006824:Hybridization, Genetic; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D008954:Models, Biological; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000235:genetics; D019913:Nuclear Localization Signals; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic
chemical_list: D019913:Nuclear Localization Signals