Detection of vector integration into genomic DNA and establishment of a vector integration database
Final Report Abstract
Our goals were to test if viral integration is non-random and, specifically, to clarify whether host and disease-specific integration patterns do exist. Within the period of funding, we established LM-PCR for lentiviral and oncoretroviral vectors and performed insertion site analysis on these vectors expressing different transgenes in different cell types in vitro and in vivo. In collaborative studies on oncoretroviral and lentiviral vectors, we showed specific vector insertion site preferences, such as predominant insertion into genes, transcription start sites of genes, and SINE elements. We also demonstrated for the first time that integration occurs in genes with specific molecular functions in vitro, which were found to be largely consistent with the patterns in vivo. We also developed two bioinformatical applications, termed IntegrationSeq and IntegrationMap, which allow large scale and standardized analysis of insertion sites of viral vectors. An advanced version of IntegrationMap tool, termed QuickMap, has been made available at the GTSG web site (http://www.gtsg.org). The QuickMap application is currently very frequently used by researchers worldwide. Since insertion site analysis protocols differ from lab to lab, comparative analyses of published and accessible insertion site sequences were lacking. We therefore designed a database, which to date contains more than 14,900 different vector insertion sites derived from previous analysis on different vectors (ASLV, FIV, HIV, FV, MLV, and SIV) and different host cells (293T, CD34, Fibroblasts, HeLa, Macrophages, SUPT1, and T-cells). We set up a web server, storing a sequence database for vector insertion site sequences and a local installation of various ENSEMBL drafts of the human and mouse genome. A random insertion site set consisting of 1,000,000 sequences reliably reflects random distribution and currently serves as our reference for all analyses of experimental data. We subjected all insertion sites stored in the GTSG database to our analysis pipeline and characterized insertions with regard to localisation on a chromosome, in or next to a gene, cancer gene, fragile site, transcription factor binding site, CpG island, or repetitive element (SINE, LINE, LTR). Further information, such as vector type, transduced cell type, transduction cytokines and conditions, transplanted species, or time point of obtainment following transplantation, is planed to be co-annotated and will be linked to data obtained from the insertion site. Using the insertion site profiles provided by QuickMap, quantification protocols to determine individual transplanted HSC clone sizes can be applied as described and established by our group. We analyzed samples from a nonhuman primate model with direct relevance to human biology to investigate the safety of MDR1 gene transfer and quantified for the first time the contribution of MDR1-transduced stem cells. Additionally to our experimental gene therapy studies we applied a novel method termed multiple displacement amplification (MDA). We could demonstrate that MDA is suitable to subsequently quantify stem cell engraftment efficiencies when combined with LM-PCR. Thus, MDA enables large scale sensitive detection and reliable quantification of retrovirally transduced human genomic DNA and therefore facilitates follow up analysis in gene therapy studies even from smallest amounts of sample material.
Publications
- Gene therapeutic overexpression of MDR1 results in upregulation of genes involved in detoxification and delivers radioprotection. Radiat Res. 2006;166:463-473
Maier P, Fleckenstein K, Li L, Laufs S, Zeller WJ, Fruehauf S, Herskind C, Wenz F
- Lentiviral vector integration sites in human NOD/SCID repopulating cells. J Gene Med. 2006;8:1197-1207
Laufs S, Guenechea G, Gonzalez-Murillo A, Nagy KZ, Lozano ML, del Val C, Jonnakuty S, Hotz-Wagenblatt A, Zeller WJ, Bueren JA, Fruehauf S
- Retroviral MDR1 gene transfer into marrow-engrafting human peripheral blood progenitor cells results in preferential transgene expression in the immature myeloid compartment rather than in mature myeloid progeny in vivo. Cytotherapy. 2006;8:562-569
Buss EC, Laufs S, Naundorf S, Kuehlcke K, Nagy KZ, Zeller WJ, Fruehauf S
- Retroviral vector insertions in T-lymphocytes used for suicide gene therapy occur in gene groups with specific molecular functions. Bone Marrow Transplant. 2006;38:229-235
Giordano FA, Fehse B, Jonnakuty S, Del Val C, Hotz-Wagenblatt A, Nagy KZ, Kuehlcke K, Naundorf S, Zander AR, Zeller WJ, Fruehauf S, Laufs S
- Viral determinants of integration site preferences of simian immunodeficiency virus based vectors. J Virol. 2006;80:8145- 8150
Monse H, Laufs S, Kuate S, Zeller WJ, Frühauf S, Überla K
- Myeloprotective retroviral gene transfer to primate long-term bone marrow repopulating cells results in polyclonal hematopoiesis after four year follow-up. Stem cells 2007; Jul 5
Bozorgmehr F, Laufs S, Sellers SE, Zeller WJ, Dunbar CE, Fruehauf S
- New bioinformatic strategies to rapidly characterize retroviral integration sites. Methods Inf Med. 2007;46:542-7
Giordano FA, Hotz-Wagenblatt A, Lauterborn D, Appelt JU, Fellenberg K, Nagy KZ, Zeller JW, Suhai S, Fruehauf S, Laufs S
- MDR1 gene transfer using a lentiviral SIN vector confers radioprotection to human CD34+ hematopoietic progenitor cells. Radiat Res. 2008;169:301-10
Maier P, Herskind C, Fleckenstein K, Spier.I, Laufs S, Zeller WJ, Fruehauf S, Wenz F
- Multiple displacement amplification enables large scale clonal analysis after retroviral gene therapy. Journal of Virology, 2008 Mar;82:2448-55
Bleier S, Maier P, Allgayer H, Wenz F, Zeller WJ, Fruehauf S, Laufs S
- Genes involved in acute leukemia are favored targets of HIV vector integration. Gene Ther. 2009 Jul;16(7):885-93
Appelt JA, Giordano FA, Zimmermann M, Weinhard S, Grund N, Hotz-Wagenblatt A, Zeller WJ, Allgayer H, Fruehauf S, Laufs S
