title: Targeted integration of functional human ATM cDNA into genome mediated by HSV/AAV hybrid amplicon vector.
abstract: Ataxia-telangiectasia (A-T) is an autosomal recessive disorder characterized by neurodegeneration, immunodeficiency, cancer predisposition, genome instability, and sensitivity to ionizing radiation (IR). We have previously shown that a herpes simplex virus type 1 (HSV-1) amplicon vector carrying the human ataxia-telangiectasia mutated (ATM) complementary DNA (cDNA) is able to correct aspects of the cellular phenotype of human A-T cells in culture, and is also able to transfer the ATM cDNA to the Atm(-/-) mouse cerebellum. In order to achieve stable gene replacement, we have generated an HSV/adeno-associated virus (AAV) hybrid amplicon vector carrying the expression cassettes for the ATM cDNA [(9.2 kilobases (kb)] and enhanced green fluorescent protein (EGFP), flanked by AAV inverted terminal repeats (ITRs). This hybrid vector, in the presence of AAV Rep proteins, mediates site-specific integration into the AAVS1 site on chromosome 19 in human cells and in Atm(-/-) mice carrying that human locus. The functional activity of the vector-derived ATM was confirmed in vitro and in vivo by ATM autophosphorylation at Ser-1981 after IR. This proof-of-principle study establishes the ability of HSV/AAV hybrid amplicon vectors to mediate functional targeted integration of the ATM cDNA into A-T cells in culture and in Atm(-/-) mice in vivo, thus laying a foundation for possible gene therapy approaches in the treatment of A-T patients.
authors.affiliation: Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Maria L
authors.initials: ML
authors.lastname: Cortés
authors.forename: Angelika
authors.initials: A
authors.lastname: Oehmig
authors.forename: Okay
authors.initials: O
authors.lastname: Saydam
authors.forename: Jocelyn D
authors.initials: JD
authors.lastname: Sanford
authors.forename: Katherine F
authors.initials: KF
authors.lastname: Perry
authors.forename: Cornel
authors.initials: C
authors.lastname: Fraefel
authors.forename: Xandra O
authors.initials: XO
authors.lastname: Breakefield
mesh_terms: D000818:Animals; D001260:Ataxia Telangiectasia / Q000235:genetics; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000235:genetics*; D002460:Cell Line; D002461:Cell Line, Transformed; D002522:Chlorocebus aethiops; D018076:DNA, Complementary / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000172:deficiency / Q000235:genetics*; D000229:Dependovirus / Q000235:genetics*; D018390:Gene Targeting; D005822:Genetic Vectors / Q000235:genetics*; D015894:Genome, Human; D006801:Humans; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D017346:Protein Serine-Threonine Kinases / Q000172:deficiency / Q000235:genetics*; D018139:Simplexvirus / Q000235:genetics*; D025521:Tumor Suppressor Proteins / Q000172:deficiency / Q000235:genetics*; D014709:Vero Cells
chemical_list: D018797:Cell Cycle Proteins; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D025521:Tumor Suppressor Proteins; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C576325:Atm protein, mouse; D017346:Protein Serine-Threonine Kinases