title: A comparison of synthetic oligodeoxynucleotides, DNA fragments and AAV-1 for targeted episomal and chromosomal gene repair.
abstract: BACKGROUND
  Current strategies for gene therapy of inherited diseases consist in adding functional copies of the gene that is defective. An attractive alternative to these approaches would be to correct the endogenous mutated gene in the affected individual. This study presents a quantitative comparison of the repair efficiency using different forms of donor nucleic acids, including synthetic DNA oligonucleotides, double stranded DNA fragments with sizes ranging from 200 to 2200 bp and sequences carried by a recombinant adeno-associated virus (rAAV-1). Evaluation of each gene repair strategy was carried out using two different reporter systems, a mutated eGFP gene or a dual construct with a functional eGFP and an inactive luciferase gene, in several different cell systems. Gene targeting events were scored either following transient co-transfection of reporter plasmids and donor DNAs, or in a system where a reporter construct was stably integrated into the chromosome.
  
  
  RESULTS
  In both episomal and chromosomal assays, DNA fragments were more efficient at gene repair than oligonucleotides or rAAV-1. Furthermore, the gene targeting frequency could be significantly increased by using DNA repair stimulating drugs such as doxorubicin and phleomycin.
  
  
  CONCLUSIONS
  Our results show that it is possible to obtain repair frequencies of 1% of the transfected cell population under optimized transfection protocols when cells were pretreated with phleomycin using rAAV-1 and dsDNA fragments.
authors.affiliation: Genethon-FRE3087 CNRS-Université d'Evry Val d'Essonne, 1 rue de l'Internationale, Département de Recherche Exploratoire, BP 60, 91002 - Evry Cedex, France. leclerc@genethon.fr
authors.forename: Xavier
authors.initials: X
authors.lastname: Leclerc
authors.forename: Olivier
authors.initials: O
authors.lastname: Danos
authors.forename: Daniel
authors.initials: D
authors.lastname: Scherman
authors.forename: Antoine
authors.initials: A
authors.lastname: Kichler
mesh_terms: D000818:Animals; D016466:CHO Cells; D002875:Chromosomes / Q000235:genetics; D006224:Cricetinae; D003412:Cricetulus; D004249:DNA Damage; D004260:DNA Repair; D004274:DNA, Recombinant / Q000235:genetics*; D000229:Dependovirus / Q000235:genetics*; D004317:Doxorubicin / Q000494:pharmacology; D018390:Gene Targeting; D017930:Genes, Reporter; D015316:Genetic Therapy; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D009838:Oligodeoxyribonucleotides / Q000235:genetics*; D010692:Phleomycins / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics; D014162:Transfection
chemical_list: D004274:DNA, Recombinant; D009838:Oligodeoxyribonucleotides; D010692:Phleomycins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D004317:Doxorubicin