High-throughput monitoring of integration site clonality in preclinical and clinical gene therapy studies.

Gene transfer to hematopoietic stem cells with integrating vectors not only allows sustained correction of monogenic diseases but also tracking of individual clones in vivo. Quantitative real-time PCR (qPCR) has been shown to be an accurate method to quantify individual stem cell clones, yet due to frequently limited amounts of target material (especially in clinical studies), it is not useful for large-scale analyses. To explore whether vector integration site (IS) recovery techniques may be suitable to describe clonal contributions if combined with next-generation sequencing techniques, we designed artificial ISs of different sizes which were mixed to simulate defined clonal situations in clinical settings.

Impact of raltegravir on HIV-1 RNA and DNA forms following initiation of antiretroviral therapy in treatment-naive patients.

Objectives: The rapid early-phase decay of plasma HIV-1 RNA during integrase inhibitor-based therapy is not fully understood. The accumulation of biologically active episomal HIV-1 cDNAs, following aborted integration, could contribute to antiviral potency in vivo.

Methods: This prospective, controlled clinical observation study explored raltegravir's impact on the dynamics of HIV-1 RNA in plasma, and concentrations of total HIV-1 cDNA, episomal 2-long terminal repeat (LTR) circles and HIV-1 integrants in peripheral blood mononuclear cells (PBMC).

Long-term episomal transgene expression from mitotically stable integration-deficient lentiviral vectors.

Nonintegrating gene delivery vectors have an improved safety profile compared with integrating vectors, but transgene retention is problematic as nonreplicating episomes are progressively and rapidly diluted out through cell division. We have developed an integration-deficient lentiviral vector (IDLV) system generating mitotically stable episomes capable of long-term transgene expression. We found that a transient cell cycle arrest at the time of transduction with IDLVs resulted in 13-45% of Chinese hamster ovary (CHO) cells expressing the transgene for over 100 cell generations in the absence of selection.

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.

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.

Induced pluripotent mesenchymal stromal cell clones retain donor-derived differences in DNA methylation profiles.

Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) is an epigenetic phenomenon. It has been suggested that iPSC retain some tissue-specific memory whereas little is known about interindividual epigenetic variation. We have reprogrammed mesenchymal stromal cells from human bone marrow (iP-MSC) and compared their DNA methylation profiles with initial MSC and embryonic stem cells (ESCs) using high-density DNA methylation arrays covering more than 450,000 CpG sites.

Look who's talking: deregulated signaling in colorectal cancer.

Background: The phenotypic expression of any given heterogeneous tissue type is the composite of interactions between individual cell types governed principally by inherent, cell-specific molecular mechanisms.

Materials And Methods: Using a combination of laser-capture microdissection, oligonucleotide microarrays, bioinformatic and statistical tools, we analyzed colorectal cancer tissues in order to spot the significant pathways that were differentially deregulated between the epithelial and stromal compartments and compared these alongside the ones of whole tissue dissection.

Results: The stromal pathway profiles were very similar to the ones of whole tissue, in contrast to the epithelial input, with stroma emerging as the major determinant of the cancer phenotype.

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.

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.

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.

Retroviral vectors for gene therapy.

Since their first clinical trial 20 years ago, retroviral (gretroviral and lentiviral) vectors have now been used in more than 350 gene-therapy studies. Retroviral vectors are particularly suited for gene-correction of cells due to long-term and stable expression of the transferred transgene(s), and also because little effort is required for their cloning and production. Several monogenic inherited diseases, mostly immunodeficiencies, can now be successfully treated.

Unrestricted somatic stem cells: interaction with CD34+ cells in vitro and in vivo, expression of homing genes and exclusion of tumorigenic potential.

Background Aims: Transplantation of allogeneic hematopoietic stem cells (HSC) within the framework of hematologic oncology or inherited diseases may be associated with complications such as engraftment failure and long-term pancytopenia. HSC engraftment can be improved, for example by co-transplantation with mesenchymal stem cells (MSC). Recently, a new multipotent MSC line from umbilical cord blood, unrestricted somatic stem cells (USSC), has been described.

A combination of granulocyte-colony-stimulating factor (G-CSF) and plerixafor mobilizes more primitive peripheral blood progenitor cells than G-CSF alone: results of a European phase II study.

Background Aims: Previous studies in xenograft models have shown that human peripheral blood progenitor cells (PBPC) mobilized with the CXCR4 antagonist plerixafor (AMD3100) have a higher bone marrow (BM) reconstitution potential than granulocyte-colony-stimulating factor (G-CSF)-mobilized PBPC.

Methods: PBPC obtained during G-CSF-supported mobilization before and after a supplementary administration of AMD3100 from patients with multiple myeloma and non-Hodgkin's lymphoma (n=15; phase II study) were investigated for co-expression of primitive and lineage-associated markers, their proliferative activity in vitro and repopulation potential after clinical transplantation.

Results: A significant increase in primitive CD34+ CD38(-) cells was observed in intraindividual comparisons of all patients after administration of G-CSF+AMD3100 (peripheral blood: median 8-fold, range 2,4-fold - 39-fold) compared with G-CSF alone.

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.

Plerixafor inhibits chemotaxis toward SDF-1 and CXCR4-mediated stroma contact in a dose-dependent manner resulting in increased susceptibility of BCR-ABL+ cell to Imatinib and Nilotinib.

Despite Imatinib's remarkable success in chronic myelogenous leukemia treatment, monotherapy frequently causes resistance, underlining the rationale for combination chemotherapy. A potential approach would be interrupting the SDF-1/CXCR4 axis using the selective CXCR4 antagonist Plerixafor (previously AMD3100), as this axis has been reported to provide survival-enhancing effects to myeloid progenitor cells. By efficient CXCR4 blocking in the CXCR4(+)/BCR-ABL(+) cell line BV-173, plerixafor (1-100 muM) significantly inhibits SDF-1alpha-mediated chemotaxis and cell migration toward the murine stroma cell line FBMD-1.

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.

Application of a haematopoetic progenitor cell-targeted adeno-associated viral (AAV) vector established by selection of an AAV random peptide library on a leukaemia cell line.

Background: For many promising target cells (e.g.: haematopoeitic progenitors), the susceptibility to standard adeno-associated viral (AAV) vectors is low.

Generation of efficient human blood progenitor-targeted recombinant adeno-associated viral vectors (AAV) by applying an AAV random peptide library on primary human hematopoietic progenitor cells.

Objective: Currently standard recombinant adeno-associated virus serotype 2(rAAV2)-based vectors lack the efficiency for gene transfer into primary human CD34(+) peripheral blood progenitor cells (PBPC).

Materials And Methods: An advancement in vector development now allows the generation of rAAV capsid mutants that offer higher target cell efficiency and specificity. To increase the gene transfer into hematopoietic progenitor cells, we applied this method for the first time on primary human CD34(+) PBPC cells.

MDR1 gene transfer using a lentiviral SIN vector confers radioprotection to human CD34+ hematopoietic progenitor cells.

Tumor radiotherapy with large-field irradiation results in an increase in apoptosis of the radiosensitive hematopoietic stem cells (CD34(+)). The aim of this study was to demonstrate the radioprotective potential of MDR1 overexpression in human CD34(+) cells using a lentiviral self-inactivating vector. Transduced human undifferentiated CD34(+) cells were irradiated with 0-8 Gy and held in liquid culture under myeloid-specific maturation conditions.

Differences in functional activity and antigen expression of granulocytes primed in vivo with filgrastim, lenograstim, or pegfilgrastim.

Background: Granulocyte-colony-stimulating factor (G-CSF) is known to affect functional activity and antigen expression of neutrophil granulocytes. Beside nonglycosylated filgrastim and glycosylated lenograstim, pegylated filgrastim (pegfilgrastim) has recently been introduced for single administration into clinical use.

Study Design And Methods: Here, granulocytes from 27 patients with nonmyeloid malignancies were compared functionally (migration, reactive oxygen species production, and G-CSF serum levels) and phenotypically (cell surface antigen expression) before and after G-CSF administration.

Lentiviral vector integration sites in human NOD/SCID repopulating cells.

Background: Recent observations of insertional mutagenesis in preclinical and clinical settings emphasize the relevance of investigating comprehensively the spectrum of integration sites targeted by specific vectors.

Methods: We followed the engraftment of lentivirally transduced human cord blood (CB) progenitor cells after transplantation into NOD/SCID mice using a self-inactivating HIV-1-derived vector expressing the enhanced green fluorescent protein (EGFP).

Results: The mean of transduction of CD34(+) CB cells was 41%, as deduced from the percentage of EGFP(+) cells before transplantation.

Overexpression of MDR1 using a retroviral vector differentially regulates genes involved in detoxification and apoptosis and confers radioprotection.

Overexpression of P-glycoprotein (P-gp), the product of the MDR1 (multidrug resistance 1) gene, might complement chemotherapy and radiotherapy in the treatment of tumors. However, for safety and mechanistic reasons, it is important to know whether MDR1 overexpression influences the expression of other genes. Therefore, we analyzed differential gene expression in cells of the human lymphoblast cell line TK6 retrovirally transduced with MDR1 using the GeneChip Human Genome U133 Plus2.

Urokinase-receptor (u-PAR): an essential player in multiple games of cancer: a review on its role in tumor progression, invasion, metastasis, proliferation/dormancy, clinical outcome and minimal residual disease.

The relevance of the u-PA system in mediating tumor-associated proteolysis, invasion and metastasis, amongst other phenomena associated with tumor progression, has been clearly demonstrated in diverse cancer entities. This review will update on the biological and clinical relevance of the urokinase-receptor (u-PAR). Specifically, the article focuses on the potential importance of u-PAR for the development of minimal residual disease in solid cancer, and in this context reviews the biological relevance of the u-PAR for tumor cell dormancy.