Chemoprotection of haematopoietic stem cells (HSCs) by gene therapeutic transfer of drug-resistance genes represents the encouraging approach to prevent myelosuppression, which is one of the most severe side effects in tumor therapy. Thus, we cloned and evaluated six different bicistronic lentiviral SIN vectors encoding two transgenes, MGMT(P140K) (an O(6)-benzylguanine-resistant mutant of methylguanine-DNA methyltransferase) and MDR1 (multidrug resistance 1), using various linker sequences (IRESEMCV, IRESFMDV and 2A-element of FMDV (F2A)). Expression of both transgenes in HL-60 and in K562 cells was assayed by quantitative real-time PCR. Combination therapy with ACNU plus paclitaxel in HL-60 cells and with carmustin (BCNU) plus doxorubicin in K562 cells resulted in the most significant survival advantage of cells transduced with the lentiviral vector HR'SIN-MGMT(P140K)-F2A-MDR1 compared with untransduced cells. In human HSCs, overexpression of both transgenes by this vector also caused significantly increased survival and enrichment of transduced cells after treatment with BCNU plus doxorubicin or temozolomide plus paclitaxel. In summary, we could show significant chemoprotection by overexpression of MDR1 and MGMT(P140K) with a lentiviral vector using the F2A linker element in two different haematopoietic cell lines and in human primary HSCs with various combination regimens. Consequently, we are convinced that these in vitro investigations will help to improve combination chemotherapy regimens by reducing myelotoxic side effects and increasing the therapeutic efficiency.

Tumor radiotherapy with large-field irradiation results in an increase of p53-dependent apoptosis of the radiosensitive hematopoietic stem cells. Proapoptotic PUMA is a transcriptional target of p53. Thus suppression of PUMA expression by gene therapy with the transcription repressor SNAI2 as transgene might be a potential approach for normal tissue protection during radiotherapy. SNAI2 cDNA was cloned in a lentiviral SIN vector in a bicistronic expression cassette followed by a foxed IRES-EMCV linker and EGFP as selection gene. Wild-type p53 TK6 cells were used as the cellular model system. We could demonstrate the significant radioprotective effect of SNAI2 overexpression in a cytotoxicity assay after irradiation with 0-5 Gy compared with untransduced or control vector (inverse oriented SNAI2 cDNA)-transduced cells. Additionally, TK6-SNAI2 compared to TK6-SNAI2inv cells showed a survival advantage in a clonogenic assay after irradiation with 0-3 Gy. Determination of the proportion of sub-G(1) cells in TK6-SNAI2 cells revealed an approximately 50% reduction in apoptosis compared with both control entities. In this study using a bicistronic lentiviral vector, we were able to provide proof of principle that lentiviral overexpression of SNAI2 might be used for radioprotective gene therapy to widen the therapeutic range in radiotherapy. (C) 2010 by Radiation Research Society

Unrestricted somatic stem cells support HSC proliferation in vitro and engraftment in vivo and possess no tumorigenic potential K. Jeltsch, T. Radke, S. Laufs, F. Giordano, H. Allgayer, F. Wenz, W.J. Zeller , G. Kögler, P. Maier ________________________________________________________________ DKFZ, G360, Heidelberg, Germany University Medical Center Duesseldorf, Institute fo r Transplantation Diagnostics and Cell Therapeutics , Düsseldorf, Germany Mannheim Medical Center, University of Heidelberg, Department of Experimental Surgery, Mannheim Medica l Center, Mannheim, Germany Mannheim Medical Center, University of Heidelberg, Department of Radiation Oncology, Mannheim, Germany

Abstract Hematotoxicity is a major and frequently dose-limiting side effect of chemotherapy. Retroviral methylguanine-DNA-methyltransferase (MGMT; EC 2.1.1.63) gene transfer to primitive hematopoietic progenitor cells (CD34(+) cells) might allow the application of high-dose alkylator chemotherapy with almost mild to absent myelosuppression. Because gammaretroviral vector integration was found in association with malignant or increased proliferation, novel lentiviral vectors with self-inactivating (SIN) capacity might display a safer option for future gene transfer studies. We assessed the influence of chemoselection on integration patterns in 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)-treated and untreated human CD34(+) cells transduced with an SIN lentiviral vector carrying the MGMT(P140K) transgene, using ligation-mediated PCR (LM-PCR) and next-generation sequencing. In addition, for the first time, the local influence of the lentiviral provirus on the expression of hit and flanking genes in human CD34(+) cells was analyzed at a clonal level. For each colony, the integration site was detected (LM-PCR) and analyzed (QuickMap), and the expression of hit and flanking genes was measured (quantitative RT-PCR). Analyses of both treated and untreated CD34(+) cells revealed preferential integration into genes. Integration patterns in BCNU-treated cells showed mild, but not significant, differences compared with those found in untreated CD34(+) cells. Most importantly, when analyzing the local influence of the provirus, we saw no significant deregulation of the integration-flanking genes. These findings demonstrate that SIN vector-mediated gene transfer might display a feasible and possibly safe option for MGMT(P140K)-mediated chemoprotection of CD34(+) cells.

Myelotoxicity is a dose-limiting effect of many chemotherapeutic regimens. Thus, there is great interest in protecting human hematopoietic stem cells by the transfer of drug resistance genes. The main focus of this study was the simultaneous overexpression of multidrug resistance 1 (MDR1) and the O6-benzylguanine (O6-BG)-resistant mutant MGMTP140K (O6-methylguanine-DNA methyltransferase) with a bicistronic lentiviral vector (HR′SIN–MDR1–IRES–MGMTP140K), with regard to the capability to convey chemoprotection in the leukemia cell line, HL60, and human hematopoietic stem cells (CD34+). Combination therapy with O6-BG/1-(2-chloroethyl)-3-(4-amino-2-methylpyrimidine-5-yl)methyl-1-nitrosourea) (ACNU) plus paclitaxel showed a significant survival advantage of HL60 cells transduced with this combination vector. In CD34+ cells, monotherapy with O6-BG/temozolomide (TMZ) resulted in an increased percentage of MGMT-positive cells (vs untreated cells) after transduction with HR′SIN–MDR1–IRES–MGMTP140K (28.3%). For combination therapy with O6-BG/temozolomide plus paclitaxel the increase was higher with the combination vector (52.8%) than with a vector expressing MGMTP140K solely (29.1%). With regard to MDR1-positive cells the protective effect of the combination vector (88.5%) was comparable to the single vector HR′SIN–MDR1 (90.0%) for monotherapy with paclitaxel and superior for combination therapy with O6-BG/temozolomide plus paclitaxel (84.6 vs 69.7%). In conclusion, the combination vector presents simultaneous protective effects of two drug-resistance genes, offering an opportunity to increase the cancer therapeutic index.

Several events of insertional mutagenesis in pre-clinical and clinical gene therapy studies have created intense interest in assessing the genomic insertion profiles of gene therapy vectors. For the construction of such profiles, vector-flanking sequences detected by inverse PCR, linear amplification-mediated-PCR or ligation-mediated-PCR need to be mapped to the host cell's genome and compared to a reference set. Although remarkable progress has been achieved in mapping gene therapy vector insertion sites, public reference sets are lacking, as are the possibilities to quickly detect non-random patterns in experimental data. We developed a tool termed QuickMap, which uniformly maps and analyzes human and murine vector-flanking sequences within seconds (available at www.gtsg.org ). Besides information about hits in chromosomes and fragile sites, QuickMap automatically determines insertion frequencies in +/− 250 kb adjacency to genes, cancer genes, pseudogenes, transcription factor and (post-transcriptional) miRNA binding sites, CpG islands and repetitive elements (short interspersed nuclear elements (SINE), long interspersed nuclear elements (LINE), Type II elements and LTR elements). Additionally, all experimental frequencies are compared with the data obtained from a reference set, containing 1 000 000 random integrations (‘random set’). Thus, for the first time a tool allowing high-throughput profiling of gene therapy vector insertion sites is available. It provides a basis for large-scale insertion site analyses, which is now urgently needed to discover novel gene therapy vectors with ‘safe’ insertion profiles.

Protection of radiosensitive normal tissue (e.g.: peripheral blood progenitor cells or microvascular cells) against radiation-induced damage may potentially increase the therapeutic ratio of radiotherapy. A promising strategy for such an approach is gene therapy-mediated overexpression of radioprotective genes. Novel lentiviral vectors has the advantage of transducing non-proliferating cells and are constructed to be relatively safe. Using a lentiviral-based vector system, the radioprotective potential of superoxide-dismutase (SOD), Catalase (CAT), and the multi-drug resistance 1 (MDR1) genes was determined. Human lymphoblastoid cells (TK6) were stably transduced with novel lentiviral vectors containing the CuZnSOD, MnSOD, an inverse MnSOD ORF or combinations of each gene with catalase. In other experiments, TK6 cells were transduced with MDR1 which codes for P-glycoprotein (P-gp) and confers protection against apoptosis. Transduced cells were irradiated and assayed for gene transfer efficiency, apoptosis, protein expression (western blot) and SOD enzyme activity. Resistance to O2¯ radicals generated by paraquat was determined using a proliferation assay. The surviving fraction (SF) after irradiation was determined using the colony forming assay (CFA). For MDR1, activation of PI3K/Akt and apoptotic signalling pathways was determined by Western blotting. Transduction with the lentiviral vectors resulted in efficient overexpression of the genes of interest, e.g., 6–8-fold for CuZn- and MnSOD. MnSOD provided 1.9–2.5 fold protection against O2¯ radicals produced by 64–128 μM paraquat. Combinations of Catalase and MnSOD tended to be slightly more protective than MnSOD alone. By contrast, CuZnSOD showed no effect. Data from CFA corroborated these findings. Preliminary results with X irradiation (1–4 Gy) indicated a clear protection by MnSOD in the absence as well as in the presence of catalase (2.6–4.0-fold) while protection by CuZnSOD was lower (1.3–2.6-fold). Transfer of a non-coding control gene did not result in radioprotection cells, irrespective of whether catalase was present or not. Overexpression of P-pg was achieved in 10–50% of TK6 cells transduced with MDR1. Fractionated irradiation with 3 × 2 Gy given as daily fractions enhanced the proportion of P-gp-expressing cells to 30–75% and was protective in the CFA, demonstrating that radioprotection may be achieved in a clinically relevant schedule. Mechanistic studies showed radiation-induced activation of the PI3K/Akt pathway. Possible differences in expression patterns between TK6-MDR1 and wild type TK6 are currently being studied. The results show that lentiviral-mediated overexpression of protective genes in p53-wt cells of hematopoietic origin is feasible. MnSOD and MDR1 were shown to protect against radiation, and efficient protection against O2¯ radicals by MnSOD was verified. The results encourage further studies in clinically relevant settings.

Summary 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, IntegrationSeqand 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.

BACKGROUND The objective of multidrug resistance-1 (MDR1) gene therapy is protection of the myeloid cell lineage. It is therefore important to examine the effect of retroviral transduction on myeloid maturation. Transfer of the human MDR1 gene can confer resistance to a variety of cytostatic drugs. For a safe application in humans it is paramount to follow-up the development of transduced cells. METHODS We transduced human mobilized peripheral blood progenitor cells (PBPC) with a viral vector containing the human MDR1 cDNA and transplanted the transduced cells into non-obese diabetic severe combined immunodeficient (NOD/SCID) mice. The progeny of the transduced cells was analyzed in detail by flow cytometry. RESULTS A detailed analysis by four-color flow cytometry showed that MDR1 transgene-expressing CD33+ myeloid cells were preferentially negative for the maturation-associated myeloid markers CD11b and CD10, while the untransduced CD33+ myeloid cells expressed significantly higher proportions of these Ag (P<0.01 each). There was no difference in the expression of B- or T-lymphoid Ag among the MDR1-transduced and untransduced lymphoid cells. DISCUSSION These data indicate that retroviral MDR1 gene transfer results in preferential P-glycoprotein expression in myeloid progenitor cells, which is the target cell population for myelotoxicity of cytostatic drugs.

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.