Targeted Chromosomal Insertion of Large DNA into the Human Genome by a Fiber-Modified High-Capacity Adenovirus-Based Vector System Manuel A. F. V. Gonc¸ alves1*, Maarten Holkers1, Gijsbert P. van Nierop 1 , Roeland Wieringa1, Maria G. Pau 2 , Antoine A. F. de Vries1 1 Virus and Stem Cell Biology Laboratory, Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands, 2 Crucell Holland BV, Leiden, The Netherlands Abstract 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). Citation: Gonc¸alves MAFV, Holkers M, van Nierop GP, Wieringa R, Pau MG, et al. (2008) Targeted Chromosomal Insertion of Large DNA into the Human Genome by a Fiber-Modified High-Capacity Adenovirus-Based Vector System. PLoS ONE 3(8): e3084. doi:10.1371/journal.pone.0003084 Editor: Sebastian D Fugmann, National Institute on Aging, United States of America Received April 15, 2008; Accepted July 21, 2008; Published August 29, 2008 Copyright: 2008 Gonc¸alves et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by research grants from the Prinses Beatrix Fonds against neuromuscular diseases (PBF; MAR04-0222) and the Association Franc¸aise contre les Myopathies (AFM; 12259-SR-GROUPE E). Competing Interests: The authors have declared that no competing interests exist. * E-mail: m.goncalves@lumc.nl Introduction Experiments in animal models of various human inherited diseases have shown that high-capacity (hc) adenovirus (Ad) vectors are superior to early region-deleted Ad vectors both in terms of safety and persistence of transgene expression [1–3]. However, at present, extensive use of this gene delivery system in in vivo settings is hampered by the difficulty in obtaining the vast amounts of vector particles needed for large animal experiments and the limited understanding of the physiological effects caused by the interaction(s) between vector capsids and, for instance, the innate immune system. In addition, because Ad genomes normally remain episomal, hcAd vectors are of limited use in applications aiming at stable genetic modification of dividing cell populations. Thus, a high demand exists to endow hcAd vectors with mechanisms that allow exogenous DNA persistence. A strategy to achieve this goal consists of incorporating into hcAd vector genomes the DNA integration-promoting elements from naturally occurring integration systems such as (i) DNA transpo- sons/transposases, (ii) retroviral long terminal repeats/integrases, (iii) retrotransposons, (iv) DNA recombinases or (v) the Rep78/68- binding sequences/large Rep proteins of the seemingly non- pathogenic helper-dependent parvovirus adeno-associated virus (AAV) [4,5]. Of these integrating hcAd-based vector systems, those that capitalize on the DNA integration machinery of AAV have the advantage of allowing preferential insertion of foreign DNA into the so-called AAVS1 locus on human chromosome 19 (19q13.3-qter). AAV trans- and cis-acting elements needed for this reaction are the Rep78 and/or Rep68 protein and their cognate binding sequences, respectively [6]. The cis-acting elements are located in the AAV inverted terminal repeats (ITRs), which can fold into a palindromic T-shaped hairpin, and in the AAV p5 promoter, which contains a sequence dubbed the p5 integration efficiency element (p5IEE) [7]. Another shortcoming of hcAd-based gene transfer systems relates to the fact that many clinically important cell types (e.g., human hematopoietic stem and progenitor cells, mesenchymal stem cells PLoS ONE | www.plosone.org 1 August 2008 | Volume 3 | Issue 8 | e3084