In vitro testing of drug combinations employing nilotinib and alkylating agents with regard to pretransplant conditioning treatment of advanced-phase chronic myeloid leukemia.

PURPOSE: The prognosis of patients with advanced-phase chronic myeloid leukemia (CML) remains dismal despite the availability of targeted therapies and allogeneic stem cell transplantation (allo-SCT). Increasing the antileukemic efficacy of the pretransplant conditioning regimen may be a strategy to increase remission rates and duration. We therefore investigated the antiproliferative effects of nilotinib in combination with drugs that are usually used for conditioning: the alkylating agents mafosfamide, treosulfan, and busulfan. METHODS: Drug combinations were tested in vitro in different imatinib-sensitive and imatinib-resistant BCR-ABL-positive cell lines. A tetrazolium-based MTT assay was used for the assessment and quantification of growth inhibition after exposure to alkylating agents alone or to combinations with nilotinib. Drug interaction was analyzed using the median-effect method of Chou and Talalay, and combination index (CI) values were calculated according to the classic isobologram equation. RESULTS: Treatment of imatinib-sensitive, BCR-ABL-positive K562 and LAMA84 cells with nilotinib in combination with mafosfamide, treosulfan, or busulfan resulted in synergistic (CI < 1), additive (CI ~ 1), and predominantly antagonistic (CI > 1) effects, respectively. In imatinib-resistant K562-R and LAMA84-R cells, all applied drug combinations were synergistic (CI < 1) at higher growth inhibition levels. CONCLUSIONS: Our in vitro data warrant further investigation and may provide the basis for nilotinib-supplemented conditioning regimens for allo-SCT in advanced-phase CML.

The disparate twins: a comparative study of CXCR4 and CXCR7 in SDF-1α-induced gene expression, invasion and chemosensitivity of colon cancer.

PURPOSE: In colorectal cancer, increased expression of the CXC chemokine receptor 4 (CXCR4) has been shown to provoke metastatic disease due to the interaction with its ligand stromal cell-derived factor-1 (SDF-1). Recently, a second SDF-1 receptor, CXCR7, was found to enhance tumor growth in solid tumors. Albeit signaling cascades via SDF-1/CXCR4 have been intensively studied, the significance of the SDF-1/CXCR7-induced intracellular communication triggering malignancy is still only marginally understood. EXPERIMENTAL DESIGN: In tumor tissue of 52 patients with colorectal cancer, we observed that expression of CXCR7 and CXCR4 increased with tumor stage and tumor size. Asking whether activation of CXCR4 or CXCR7 might result in a similar expression pattern, we performed microarray expression analyses using lentivirally CXCR4- and/or CXCR7-overexpressing SW480 colon cancer cell lines with and without stimulation by SDF-1α. RESULTS: Gene regulation via SDF-1α/CXCR4 and SDF-1α/CXCR7 was completely different and partly antidromic. Differentially regulated genes were assigned by gene ontology to migration, proliferation, and lipid metabolic processes. Expressions of AKR1C3, AXL, C5, IGFBP7, IL24, RRAS, and TNNC1 were confirmed by quantitative real-time PCR. Using the in silico gene set enrichment analysis, we showed that expressions of miR-217 and miR-218 were increased in CXCR4 and reduced in CXCR7 cells after stimulation with SDF-1α. Functionally, exposure to SDF-1α increased invasiveness of CXCR4 and CXCR7 cells, AXL knockdown hampered invasion. Compared with controls, CXCR4 cells showed increased sensitivity against 5-FU, whereas CXCR7 cells were more chemoresistant. CONCLUSIONS: These opposing results for CXCR4- or CXCR7-overexpressing colon carcinoma cells demand an unexpected attention in the clinical application of chemokine receptor antagonists such as plerixafor.

CXCR4 Expression and Treatment with SDF-1α or Plerixafor Modulate Proliferation and Chemosensitivity of Colon Cancer Cells.

BACKGROUND: Signaling through stromal cell-derived factor-1α (SDF-1α), strongly secreted by bone marrow stromal cells and the CXC chemokine receptor 4 (CXCR4) exposed on tumor cells has pivotal roles in proliferation, metastasis, and tumor cell "dormancy." Dormancy is associated with cytostatic drug resistance and is probably a property of tumor stem cells and minimal residual disease. Thus, hampering the SDF-1α/CXCR4 cross talk by a CXCR4 antagonist like Plerixafor (AMD3100) should overcome tumor cell dormancy bymobilization of tumor cells from "sanctuary" niches. Our aim was to elucidate the direct effects exerted by SDF-1α and Plerixafor on proliferation, chemosensitivity, and apoptosis of CXCR4-expressing tumor cells. METHODS: The ability of SDF-1α and Plerixafor to regulate intracellular signaling, proliferation, and invasion was investigated using two colon cancer cell lines (HT-29 and SW480) with either high endogenous or lentiviral expression of CXCR4 compared to their respective low CXCR4-expressing counterparts as a model system. Efficacy of Plerixafor on sensitivity of these cell lines against 5-fluorouracil, irinotecan, or oxaliplatin was determined in a cell viability assay as well as stroma-dependent cytotoxicity and apoptosis assays. RESULTS: SDF-1α increased proliferation, invasion, and ERK signaling of endogenously and lentivirally CXCR4-expressing cells. Exposure to Plerixafor reduced proliferation, invasion, and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling. Combination of chemotherapy with Plerixafor showed an additive effect on chemosensitivity and apoptosis in CXCR4-overexpressing cells. An SDF-1-secreting feeder layer provideda"protective niche" for CXCR4-overexpressing cells resulting in decreased chemosensitivity. CONCLUSION: CXCR4-antagonistic therapy mobilizes and additionally sensitizes tumor cells toward cytoreductive chemotherapy.

Interferometry with Bose-Einstein condensates in microgravity.

Atom interferometers covering macroscopic domains of space-time are a spectacular manifestation of the wave nature of matter. Because of their unique coherence properties, Bose-Einstein condensates are ideal sources for an atom interferometer in extended free fall. In this Letter we report on the realization of an asymmetric Mach-Zehnder interferometer operated with a Bose-Einstein condensate in microgravity. The resulting interference pattern is similar to the one in the far field of a double slit and shows a linear scaling with the time the wave packets expand. We employ delta-kick cooling in order to enhance the signal and extend our atom interferometer. Our experiments demonstrate the high potential of interferometers operated with quantum gases for probing the fundamental concepts of quantum mechanics and general relativity.

F2A sequence linking MGMT(P140K) and MDR1 in a bicistronic lentiviral vector enables efficient chemoprotection of haematopoietic stem cells.

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.

The impact of a specific aqua-training for adult haemophilic patients--results of the WATERCISE study (WAT-QoL).

Sport is increasingly recommended for haemophilic patients due to physical and psychological benefits. 'WATERCISE' is a specific aqua-training programme for haemophiliacs in which endurance, strength, coordination and mobility are trained. In the WAT-QoL study benefits and risks of regular WATERCISE training sessions were investigated in terms of health-related quality of life (HRQoL), physical functioning (PF), orthopaedic joint status (OJS), bleeding frequency and factor consumption. Patients in the WATERCISE group attended an aqua-training programme once a week for 1 h over 12 months, patients in the control group did not. Patients were matched for clinical and demographic data. Information on clinical data, orthopaedic status, PF (HEP-Test-Q) and HRQoL were collected in both groups at baseline and at follow-up (6 and 12 months). Twenty-eight adult severely affected haemophilic patients (WATERCISE group: 10 haemophilia A (HA), 3 haemophilia B (HB) patients; control group: 12 HA and 3 HB patients) were enrolled (aged 40.68 ± 12.7 years). Baseline data (body mass indices, OJS, sportive activities, HRQoL and PF) were well distributed between groups. After 12 months the WATERCISE group reported a significantly better PF (M(W) = 65.22, SD = 11.3; M(C) = 52.5, SD = 15.0), especially for endurance (P < 0.004). Although always differently reported by the patients within the WATERCISE group, HRQoL did not prove to be significantly different between groups. WATERCISE seems to have a positive effect on the PF of patients suffering from haemophilia. These study findings need to be further investigated in a larger study group.

A Lentiviral CXCR4 overexpression and knockdown model in colorectal cancer cell lines reveals plerixafor-dependent suppression of SDF-1α-induced migration and invasion.

BACKGROUND: The development of distant metastasis is associated with poor outcome in patients with colorectal cancer (CRC). The stromal cell-derived factor-1 (SDF-1) and its receptor CXC chemokine receptor 4 (CXCR4) have pivotal roles in the chemotaxis of migrating tumor cells during metastasis. Thus, hampering the SDF-1/CXCR4 cross-talk is a promising strategy to suppress metastasis. METHODS: We investigated the invasive behavior of the lentivirally CXCR4 overexpressing CRC cell lines SW480, SW620 and RKO in chemotaxis and invasion assays toward an SDF-1α gradient. Low endogenous CXCR4 expression levels were determined by quantitative realtime polymerase chain reaction (PCR) and fluorescence-activated cell sorting (FACS) analyses. RESULTS: A lentiviral CXCR4 overexpression and knockdown model was established in these CRC cells. In transwell migration assays, CXCR4 overexpression favored chemotaxis and invasion of cells in all 3 lines depending on an SDF-1α gradient (p < 0.001 vs. untransduced cells). Functional CXCR4 knockdown using lentiviral short hairpin RNA (shRNA) vectors significantly decreased the migration behavior in CRC cell lines (p < 0.001), confirming a CXCR4-specific effect. Pharmacologic inhibition of the SDF-1α/CXCR4 interaction by the bicyclam Plerixafor(TM) at 100 µM significantly abrogated CXCR4-dependent migration and invasion through Matrigel(TM) (SW480, SW620, RKO; p < 0.05). CONCLUSION: Our results indicate that a CXCR4-antagonistic therapy might prevent tumor cell dissemination and metastasis in CRC patients, consequently improving survival.

Overexpression of manganese superoxide dismutase does not increase clonogenic cell survival despite effect on apoptosis in irradiated lymphoblastoid cells.

Gene therapy-mediated overexpression of superoxide dismutases (SOD) appears to be a promising strategy for modulating radiosensitivity based on detoxification of superoxide radicals and suppression of apoptosis. Using recombinant lentiviral-based vectors, the effects of SOD overexpression on both were tested in human lymphoblastoid cells (TK6) that are sensitive to radiation-induced apoptosis. TK6 cells were transduced with vectors containing CuZnSOD, MnSOD or inverted MnSOD (MSODi) cDNA. Gene transfer efficiency, SOD activity, superoxide-radical resistance, apoptosis and clonogenic survival were determined. A six- to eightfold increase in SOD activity was observed after transduction, rendering MnSOD-overexpressing TK6 cells significantly more resistant to paraquat-induced superoxide radical production than controls. Although significant differences in sensitivity to apoptosis were observed for MnSOD, no differences in clonogenic survival after irradiation were detected between any groups. Our data show that efficient cellular SOD overexpression, an increased superoxide radical detoxifying ability and, for MnSOD, decreased apoptosis did not result in increased clonogenic survival after irradiation. This strengthens the hypothesis of differences in the radiation-modulating effects of SOD on normal and malignant cells (protective and nonprotective, respectively), thereby showing its potential to increase the therapeutic index in future clinical SOD-based radioprotection approaches.

Clonal inventory screens uncover monoclonality following serial transplantation of MGMT P140K-transduced stem cells and dose-intense chemotherapy.

Gene transfer of mutant O(6)-methylguanine-DNA-methyltransferase (MGMT(P140K)) into hematopoietic stem cells (HSCs) protects hematopoiesis from alkylating agents and allows efficient in vivo selection of transduced HSCs. However, insertional mutagenesis, high regenerative stress associated with selection, and the genotoxic potential of alkylating drugs represent considerable risk factors for clinical applications of this approach. Therefore, we investigated the long-term effect of MGMT(P140K) gene transfer followed by repetitive, dose-intensive treatment with alkylating agents in a murine serial bone marrow transplant model and assessed clonality of hematopoiesis up to tertiary recipients. The substantial selection pressure resulted in almost completely transduced hematopoiesis in all cohorts. Ligation-mediated PCR and next-generation sequencing identified several repopulating clones carrying vector insertions in distinct genomic regions that were ∼ 9 kb of size (common integration sites). Beside polyclonal reconstitution in the majority of the mice, we also detected monoclonal or oligoclonal repopulation patterns with HSC clones showing vector insertions in the Usp10 or Tubb3 gene. Interestingly, neither Usp10, Tubb3, nor any of the genes located in common integration sites have been linked to clonal expansion in previous preclinical or clinical gene therapy trials. However, a considerable number of these genes are involved in DNA damage response and cell fate decision pathways following cytostatic drug application. Thus, in summary, our study advocates ligation-mediated PCR and next generation sequencing as an effective and reliable method to identify gene products associated with clonal survival in specific experimental settings such as chemoselection using alkylating agents.

Efficient gene transfer with pseudotyped recombinant adeno-associated viral vectors into human chronic myelogenous leukemia cells.

Gene transfer into chronic myelogenous leukemia (CML) cells may become of relevance for overcoming therapy resistance. Single-stranded pseudotyped adeno-associated viruses of serotypes 2/1 to 2/6 (ssAAV2/1-ssAAV2/6) were screened on human CML cell lines and primary cells to determine gene transfer efficiency. Additionally, double-stranded self-complementary vectors (dsAAVs) were used to determine possible second-strand synthesis limitations. On human CML cell lines, ssAAV2/2 and ssAAV2/6 were most efficient. On primary cells, ssAAV2/6 proved significantly more efficient (4.1 ± 2.5% GFP(+) cells, p = 0.011) than the other vectors (<1%). The transduction efficiency could be significantly increased (45.5 ± 13.4%) by using dsAAV2/6 vectors (p < 0.001 vs. ssAAV2/6). In these settings, our data suggest conversion of single- to double-stranded DNA and cell binding/entry as rate-limiting steps. Furthermore, gene transfer was observed in both late and earlier CML (progenitor) populations. For the first time, efficient AAV gene transfer into human CML cells could be shown, with the potential for future clinical application.

SNAI2 as a novel radioprotector of normal tissue by gene transfer using a lentiviral bicistronic SIN vector.

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 floxed 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.

Good manufacturing practice-grade production of unrestricted somatic stem cell from fresh cord blood.

BACKGROUND AIMS: The discovery of unrestricted somatic stem cells (USSC), a non-hematopoietic stem cell population, brought cord blood (CB) to the attention of regenerative medicine for defining more protocols for non-hematopoietic indications. We demonstrate that a reliable and reproducible method for good manufacturing practice (GMP)-conforming generation of USSC is possible that fulfils safety requirements as well as criteria for clinical applications, such as adherence of strict regulations on cell isolation and expansion. METHODS: In order to maintain GMP conformity, the automated cell processing system Sepax (Biosafe) was implemented for mononucleated cell (MNC) separation from fresh CB. After USSC generation, clinical-scale expansion was achieved by multi-layered CellSTACKs (Costar/Corning). Infectious disease markers, pyrogen and endotoxin levels, immunophenotype, potency, genetic stability and sterility of the cell product were evaluated. RESULTS: The MNC isolation and cell cultivation methods used led to safe and reproducible GMP-conforming USSC production while maintaining somatic stem cell character. CONCLUSIONS: Together with implemented in-process controls guaranteeing contamination-free products with adult stem cell character, USSC produced as suggested here may serve as a universal allogeneic stem cell source for future cell treatment and clinical settings.

Analysis of self-inactivating lentiviral vector integration sites and flanking gene expression in human peripheral blood progenitor cells after alkylator chemotherapy.

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.

Overexpression of caveolin-1 in lymphoblastoid TK6 cells enhances proliferation after irradiation with clinically relevant doses.

BACKGROUND AND PURPOSE: The transmembrane protein caveolin-1 (CAV1) is an essential component of caveolae, small membrane invaginations involved in vesicle formation. CAV1 plays a role in signal transduction, tumor suppression and oncogene transformation. Previous studies with CAV1 knockout mice and CAV1 knockdown in pancreatic tumor cells implicated CAV1 in mediating radioresistance. The aim of this work was to test the effect of CAV1 overexpression after irradiation in human cells lacking endogenous CAV1 expression. MATERIAL AND METHODS: Human CAV1 was overexpressed in lymphoblastoid TK6 cells (TK6-wt) using a eukaryotic expression plasmid, pCI-CAV1, or a lentiviral SIN (self-inactivating) vector, HR'SIN-CAV1. CAV1 expression was verified in TK6 cells with Western blot analysis or intracellular FACS (fluorescence-activated cell sorting) staining. The effect of CAV1 on proliferation kinetics after irradiation of TK6 cells was measured with a growth assay. RESULTS: TK6-wt showed no detectable endogenous CAV1 expression. Lentivirally mediated transduction with HR'SIN-CAV1 (TK6-CAV1) resulted in a considerably stronger CAV1 expression in comparison to TK6 cells electroporated with pCI-CAV1. Intracellular FACS analysis showed that 90% of transduced cells expressed CAV1. CAV1 enhanced early proliferation of TK6 cells after irradiation with a dose of 2 Gy, whereas proliferation of unirradiated cells was not affected. CAV1 also protected cells after irradiation with 4 Gy. This radioprotective effect was supported by a reduction of radiation-induced apoptosis. CONCLUSION: A model system for expression of exogenous CAV1 by stable lentiviral transduction of TK6 cells was established. Functional assays demonstrated enhanced early proliferation by CAV1 expression in TK6 cells after irradiation with clinically relevant doses supporting the role of CAV1 as a prosurvival factor.

Synergistic activity of nilotinib and established chemotherapeutic drugs in imatinib-sensitive and -resistant BCR-ABL-positive cells.

We investigated various combination treatment regimens employing nilotinib with established chemotherapeutic agents (daunorubicin, mitoxantrone, etoposide and cytarabine) in imatinib-sensitive and -resistant BCR-ABL-positive cells. Mitoxantrone or cytarabine showed synergism (CI < 1) in combination with nilotinib in imatinib-sensitive LAMA84 cells, whereas in imatinib-resistant LAMA84-R cells synergistic effects could be assessed for daunorubicin, mitoxantrone and etoposide when combined with nilotinib. In both imatinib-sensitive and -resistant K562 cells daunorubicin, mitoxantrone and etoposide demonstrated synergism in combination with nilotinib. Moreover, both daunorubicin and mitoxantrone led to synergistic antiproliferative effects when combined with nilotinib in imatinib-resistant Ba/F3 cells carrying point mutations in the ABL TK domain (E255K, E255V and T315I). Annexin V/propidium iodide staining revealed a significant enhancement of nilotinib-induced apoptosis in imatinib-resistant Ba/F3T315I and LAMA84-R cells upon combination with daunorubicin and mitoxantrone, respectively. Our results demonstrate the efficacy of combination treatment regimens employing nilotinib and established chemotherapeutic agents in improving antileukemic effects in imatinib-sensitive and imatinib-resistant cells. This may be the foundation for further study on the potential of the applied combinations in a clinical setting.

Pseudotyped recombinant adeno-associated viral vectors mediate efficient gene transfer into primary human CD34(+) peripheral blood progenitor cells.

BACKGROUND AND AIMS: Because of their pluripotency, human CD34(+) peripheral blood progenitor cells (PBPC) are targets of interest for the treatment of many acquired and inherited disorders using gene therapeutic approaches. Unfortunately, most current vector systems lack either sufficient transduction efficiency or an appropriate safety profile. Standard single-stranded recombinant adeno-associated virus 2 (AAV2)-based vectors offer an advantageous safety profile, yet lack the required efficiency in human PBPC. METHODS: A panel of pseudotyped AAV vectors (designated AAV2/x, containing the vector genome of serotype 2 and capsid of serotype x, AAV2/1-AAV2/6) was screened on primary human granulocyte-colony-stimulating factor (G-CSF)-mobilized CD34(+) PBPC to determine their gene transfer efficacy. Additionally, double-stranded self-complementary AAV (dsAAV) were used to determine possible second-strand synthesis limitations. RESULTS: AAV2/6 vectors proved to be the most efficient [12.8% (1.8-25.4%) transgene-expressing PBPC after a single transduction], being significantly more efficient (all P<0.005) than the other vectors [AAV2/2, 2.0% (0.2-7.3%); AAV2/1, 1.3% (0.1-2.9%); others, <; 1% transgene-expressing PBPC]. In addition, the relevance of the single-to-double-strand conversion block in transduction of human PBPC could be shown using pseudotyped dsAAV vectors: for dsAAV2/2 [9.3% (8.3-20.3%); P<0.001] and dsAAV2/6 [37.7% (23.6-61.0%); P<0.001) significantly more PBPC expressed the transgene compared with their single-stranded counterparts; for dsAAV2/1, no significant increase could be observed. CONCLUSIONS: We have shown that clinically relevant transduction efficiency levels using AAV-based vectors in human CD34(+) PBPC are feasible, thereby offering an efficient alternative vector system for gene transfer into this important target cell population.

Chemoprotection of human hematopoietic stem cells by simultaneous lentiviral overexpression of multidrug resistance 1 and O(6)-methylguanine-DNA methyltransferase(P140K).

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 O(6)-benzylguanine (O(6)-BG)-resistant mutant MGMT(P140K) (O(6)-methylguanine-DNA methyltransferase) with a bicistronic lentiviral vector (HR'SIN-MDR1-IRES-MGMT(P140K)), with regard to the capability to convey chemoprotection in the leukemia cell line, HL60, and human hematopoietic stem cells (CD34(+)). Combination therapy with O(6)-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 O(6)-BG/temozolomide (TMZ) resulted in an increased percentage of MGMT-positive cells (vs untreated cells) after transduction with HR'SIN-MDR1-IRES-MGMT(P140K) (28.3%). For combination therapy with O(6)-BG/temozolomide plus paclitaxel the increase was higher with the combination vector (52.8%) than with a vector expressing MGMT(P140K) 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 O(6)-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.

Cold spots in hot spots: transcription start sites of active genes are spared from HIV vector integration.

Recent studies on HIV integration in the human genome have reported on certain preferences for chromosomes, genes, or repetitive elements. We performed a high-resolution meta-analysis of public available HIV vector insertion sites (n = 46 114) and detected that HIV vectors significantly spared a region of 1 kb upstream and downstream to transcription start sites (TSS). Genes with the TSS being located within this 'insertional gap' had significantly lower expression levels than those with the TSS located outside the gap. Our data show an either unfavored and/or sterically inaccessible region located + or - 1 kb around TSS of transcriptionally active genes.

Normal-tissue radioprotection by overexpression of the copper-zinc and manganese superoxide dismutase genes.

BACKGROUND AND PURPOSE: Protection of normal tissue against radiation-induced damage may increase the therapeutic ratio of radiotherapy. A promising strategy for testing this approach is gene therapy-mediated overexpression of the copper-zinc (CuZnSOD) or manganese superoxide dismutase (MnSOD) using recombinant adeno-associated viral (rAAV2) vectors. The purpose of this study was to test the modulating effects of the SOD genes on human primary lung fibroblasts (HPLF) after irradiation. MATERIAL AND METHODS: HPLF were transduced with rAAV2 vectors containing cDNA for the CuZnSOD, MnSOD or a control gene. The cells were irradiated (1-6 Gy), and gene transfer efficiency, apoptosis, protein expression/activity, and radiosensitivity measured by the colony-forming assay determined. RESULTS: After transduction, 90.0% +/- 6.4% of the cells expressed the transgene. A significant fivefold overexpression of both SOD was confirmed by an SOD activity assay (control: 21.1 +/- 12.6, CuZnSOD: 95.1 +/- 17.1, MnSOD: 108.5 +/- 36.0 U SOD/mg protein) and immunohistochemistry. CuZnSOD and MnSOD overexpression resulted in a significant radioprotection of HPLF compared to controls (surviving fraction [SF] ratio SOD/control > 1): CuZnSOD: 1.18-fold (95% confidence interval [CI]: 1.06-1.32; p = 0.005), MnSOD: 1.23-fold (95% CI: 1.07-1.43; p = 0.01). CONCLUSION: Overexpression of CuZnSOD and MnSOD in HPLF mediated an increase in clonogenic survival after irradiation compared to controls. In previous works, a lack of radioprotection in SOD-overexpressing tumor cells was observed. Therefore, the present results suggest that rAAV2 vectors are promising tools for the delivery of radioprotective genes in normal tissue.