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Objective: Increasing use of retroviral vector-mediated gene transfer created intense interest to characterize vector integrations on the genomic level. Techniques to determine insertion sites, mainly based on time-consuming manual data processing, are commonly applied. Since a high variability in processing methods hampers further data comparison, there is an urgent need to systematically process the data arising from such analysis., Methods: To allow large-scale and standardized comparison of insertion sites of viral vectors we developed two programs, IntegrationSeq and IntegrationMap. IntegrationSeq can trim sequences, and valid integration sequences get further processed with IntegrationMap for automatic genomic mapping. IntegrationMap retrieves detailed information about whether integrations are located in or close to genes, the name of the gene, the exact localization in the transcriptional units, and further parameters like the distance from the transcription start site to the integration., Results: We validated the method using 259 files originating from integration site analysis (LM-PCR). Sequences processed by IntegrationSeq led to an increased yield of valid integration sequence detection, which were shown to be more sensitive than conventional analysis and 15 times faster, while the specificities are equal. Output files generated by IntegrationMap were found to be 99.8% identical with results retrieved by much slower conventional mapping with the ENSEMBL alignment tool., Conclusion: Using IntegrationSeq and IntegrationMap, a validated, fast and standardized high-throughput analysis of insertion sites can be achieved for the first time.

Graft-versus-host disease (GvHD) is a severe complication in the context of allogeneic stem cell transplantation and adoptive immunotherapy. The transfer of a suicide gene into donor T-lymphocytes (TLCs) allows selective elimination of GvHD-causing cells. As retroviral gene transfer into hematopoietic stem cells can induce leukaemia, there is an urgent need also to analyze retroviral integration sites in TLCs. We examined suicide gene-transduced TLCs in four grafts and from four transplanted patients. One-hundred and fifteen integration sites were detected in vitro. Of these 90 could be mapped to the human genome; 50% (45) were located in genes and 32% (29) were detected 10 kb upstream or downstream of transcription start sites. We found a significant overrepresentation of genes encoding for proteins with receptor activity, signal transducer activity, transcription regulator activity, nucleic acid binding activity and translation regulator activity. Similar data were obtained from patient samples. Our results point to preferred vector integration patterns, which are specific for the target cell population and probably independent of selection processes. Thus, future preclinical analysis of the integration repertoire with abundant amounts of transduced cells could allow a prediction also for the in vivo situation, where target cells are scarce.