Targeted chromosomal insertion of large DNA into the human genome by a fiber-modified high-capacity adenovirus-based vector system.

A prominent goal in gene therapy research concerns the development of gene transfer vehicles that can integrate exogenous DNA at specific chromosomal loci to prevent insertional oncogenesis and provide for long-term transgene expression. Adenovirus (Ad) vectors arguably represent the most efficient...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Manuel A F V Gonçalves, Maarten Holkers, Gijsbert P van Nierop, Roeland Wieringa, Maria G Pau, Antoine A F de Vries
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2008
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/7635feeaf02b4cf78c72a82d7fbfa366
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:7635feeaf02b4cf78c72a82d7fbfa366
record_format	dspace
spelling	oai:doaj.org-article:7635feeaf02b4cf78c72a82d7fbfa3662021-11-25T06:18:47ZTargeted chromosomal insertion of large DNA into the human genome by a fiber-modified high-capacity adenovirus-based vector system.1932-620310.1371/journal.pone.0003084https://doaj.org/article/7635feeaf02b4cf78c72a82d7fbfa3662008-08-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/18769728/?tool=EBIhttps://doaj.org/toc/1932-6203A prominent goal in gene therapy research concerns the development of gene transfer vehicles that can integrate exogenous DNA at specific chromosomal loci to prevent insertional oncogenesis and provide for long-term transgene expression. Adenovirus (Ad) vectors arguably represent the most efficient delivery systems of episomal DNA into eukaryotic cell nuclei. The most advanced recombinant Ads lack all adenoviral genes. This renders these so-called high-capacity (hc) Ad vectors less cytotoxic/immunogenic than those only deleted in early regions and creates space for the insertion of large/multiple transgenes. The versatility of hcAd vectors is been increased by capsid modifications to alter their tropism and by the incorporation into their genomes of sequences promoting chromosomal insertion of exogenous DNA. Adeno-associated virus (AAV) can insert its genome into a specific human locus designated AAVS1. Trans- and cis-acting elements needed for this reaction are the AAV Rep78/68 proteins and Rep78/68-binding sequences, respectively. Here, we describe the generation, characterization and testing of fiber-modified dual hcAd/AAV hybrid vectors (dHVs) containing both these elements. Due to the inhibitory effects of Rep78/68 on Ad-dependent DNA replication, we deployed a recombinase-inducible gene switch to repress Rep68 synthesis during vector rescue and propagation. Flow cytometric analyses revealed that rep68-positive dHVs can be produced similarly well as rep68-negative control vectors. Western blot experiments and immunofluorescence microscopy analyses demonstrated transfer of recombinase-dependent rep68 genes into target cells. Studies in HeLa cells and in the dystrophin-deficient myoblasts from a Duchenne muscular dystrophy (DMD) patient showed that induction of Rep68 synthesis in cells transduced with fiber-modified and rep68-positive dHVs leads to increased stable transduction levels and AAVS1-targeted integration of vector DNA. These results warrant further investigation especially considering the paucity of vector systems allowing permanent phenotypic correction of patient-own cell types with large DNA (e.g. recombinant full-length DMD genes).Manuel A F V GonçalvesMaarten HolkersGijsbert P van NieropRoeland WieringaMaria G PauAntoine A F de VriesPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 3, Iss 8, p e3084 (2008)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Manuel A F V Gonçalves Maarten Holkers Gijsbert P van Nierop Roeland Wieringa Maria G Pau Antoine A F de Vries Targeted chromosomal insertion of large DNA into the human genome by a fiber-modified high-capacity adenovirus-based vector system.
description	A prominent goal in gene therapy research concerns the development of gene transfer vehicles that can integrate exogenous DNA at specific chromosomal loci to prevent insertional oncogenesis and provide for long-term transgene expression. Adenovirus (Ad) vectors arguably represent the most efficient delivery systems of episomal DNA into eukaryotic cell nuclei. The most advanced recombinant Ads lack all adenoviral genes. This renders these so-called high-capacity (hc) Ad vectors less cytotoxic/immunogenic than those only deleted in early regions and creates space for the insertion of large/multiple transgenes. The versatility of hcAd vectors is been increased by capsid modifications to alter their tropism and by the incorporation into their genomes of sequences promoting chromosomal insertion of exogenous DNA. Adeno-associated virus (AAV) can insert its genome into a specific human locus designated AAVS1. Trans- and cis-acting elements needed for this reaction are the AAV Rep78/68 proteins and Rep78/68-binding sequences, respectively. Here, we describe the generation, characterization and testing of fiber-modified dual hcAd/AAV hybrid vectors (dHVs) containing both these elements. Due to the inhibitory effects of Rep78/68 on Ad-dependent DNA replication, we deployed a recombinase-inducible gene switch to repress Rep68 synthesis during vector rescue and propagation. Flow cytometric analyses revealed that rep68-positive dHVs can be produced similarly well as rep68-negative control vectors. Western blot experiments and immunofluorescence microscopy analyses demonstrated transfer of recombinase-dependent rep68 genes into target cells. Studies in HeLa cells and in the dystrophin-deficient myoblasts from a Duchenne muscular dystrophy (DMD) patient showed that induction of Rep68 synthesis in cells transduced with fiber-modified and rep68-positive dHVs leads to increased stable transduction levels and AAVS1-targeted integration of vector DNA. These results warrant further investigation especially considering the paucity of vector systems allowing permanent phenotypic correction of patient-own cell types with large DNA (e.g. recombinant full-length DMD genes).
format	article
author	Manuel A F V Gonçalves Maarten Holkers Gijsbert P van Nierop Roeland Wieringa Maria G Pau Antoine A F de Vries
author_facet	Manuel A F V Gonçalves Maarten Holkers Gijsbert P van Nierop Roeland Wieringa Maria G Pau Antoine A F de Vries
author_sort	Manuel A F V Gonçalves
title	Targeted chromosomal insertion of large DNA into the human genome by a fiber-modified high-capacity adenovirus-based vector system.
title_short	Targeted chromosomal insertion of large DNA into the human genome by a fiber-modified high-capacity adenovirus-based vector system.
title_full	Targeted chromosomal insertion of large DNA into the human genome by a fiber-modified high-capacity adenovirus-based vector system.
title_fullStr	Targeted chromosomal insertion of large DNA into the human genome by a fiber-modified high-capacity adenovirus-based vector system.
title_full_unstemmed	Targeted chromosomal insertion of large DNA into the human genome by a fiber-modified high-capacity adenovirus-based vector system.
title_sort	targeted chromosomal insertion of large dna into the human genome by a fiber-modified high-capacity adenovirus-based vector system.
publisher	Public Library of Science (PLoS)
publishDate	2008
url	https://doaj.org/article/7635feeaf02b4cf78c72a82d7fbfa366
work_keys_str_mv	AT manuelafvgoncalves targetedchromosomalinsertionoflargednaintothehumangenomebyafibermodifiedhighcapacityadenovirusbasedvectorsystem AT maartenholkers targetedchromosomalinsertionoflargednaintothehumangenomebyafibermodifiedhighcapacityadenovirusbasedvectorsystem AT gijsbertpvannierop targetedchromosomalinsertionoflargednaintothehumangenomebyafibermodifiedhighcapacityadenovirusbasedvectorsystem AT roelandwieringa targetedchromosomalinsertionoflargednaintothehumangenomebyafibermodifiedhighcapacityadenovirusbasedvectorsystem AT mariagpau targetedchromosomalinsertionoflargednaintothehumangenomebyafibermodifiedhighcapacityadenovirusbasedvectorsystem AT antoineafdevries targetedchromosomalinsertionoflargednaintothehumangenomebyafibermodifiedhighcapacityadenovirusbasedvectorsystem
_version_	1718413931206148096

Targeted chromosomal insertion of large DNA into the human genome by a fiber-modified high-capacity adenovirus-based vector system.

Ejemplares similares