HEK293-based production platform for γ-retroviral (self-inactivating) vectors: application for safe and efficient transfer of COL7A1 cDNA.

The clinical application of self-inactivating (SIN) retroviral vectors requires an efficient vector production technology. To enable production of γ-retroviral SIN vectors from stable producer cells, new targetable HEK293-based producer clones were selected, providing amphotropic, GALV, or RD114 pseudotyping. Viral vector expression constructs can reliably be inserted at a predefined genomic locus via Flp-recombinase-mediated cassette exchange.

TCR-engineered T cells: a model of inducible TCR expression to dissect the interrelationship between two TCRs.

TCR gene modified T cells for adoptive therapy simultaneously express the Tg TCR and the endogenous TCR, which might lead to mispaired TCRs with harmful unknown specificity and to a reduced function of TCR-Tg T cells. We generated dual TCR T cells in two settings in which either TCR was constitutively expressed by a retroviral promoter while the second TCR expression was regulable by a Tet-on system. Constitutively expressed TCR molecules were reduced on the cell surface depending on the induced TCR expression leading to strongly hampered function.

Graded or threshold response of the tet-controlled gene expression: all depends on the concentration of the transactivator.

Background: Currently, the step-wise integration of tet-dependent transactivator and tet-responsive expression unit is considered to be the most promising tool to achieve stable tet-controlled gene expression in cell populations. However, disadvantages of this strategy for integration into primary cells led us to develop an "All-In-One" vector system, enabling simultaneous integration of both components. The effect on tet-controlled gene expression was analyzed for retroviral "All-In-One" vectors expressing the M2-transactivator either under control of a constitutive or a new type of autoregulated promoter.

Improved Tet-responsive promoters with minimized background expression.

Background: The performance of the tetracycline controlled transcriptional activation system (Tet system) depends critically on the choice of minimal promoters. They are indispensable to warrant low expression levels with the system turned "off". On the other hand, they must support high level of gene expression in the "on"-state.

Retroviral and transposon-based tet-regulated all-in-one vectors with reduced background expression and improved dynamic range.

The regulated expression of therapeutic genes may become crucial in gene therapy when their constitutive expression interferes with cell fate in vivo. The efficient regulation of transgene expression requires tightly controlled inducible promoters, as shown for the tetracycline regulatory system (tet-system). However, its application requires the introduction of two components into the target cell genome: the tet-responsive transactivator and the regulated expression cassette.

Tumor rejection by local interferon gamma induction in established tumors is associated with blood vessel destruction and necrosis.

It has been shown that injecting a suspension of IFN-γ-secreting tumor cells results in their rejection. This effect has been attributed to IFN-γ preventing tumor stroma formation but not to a direct effect on the cancer cells. However, it is not known, which influence IFN-γ has on tumors with an established stroma.

Improving transcriptional termination of self-inactivating gamma-retroviral and lentiviral vectors.

Adverse events relating to insertional mutagenesis have reinforced the interest in self-inactivating (SIN) gamma-retroviral and lentiviral vectors without enhancer-promoter sequences in the U3 region of the long terminal repeats. However, SIN vectors suffer from leaky transcriptional termination, increasing the probability of read-through into cellular genes. To improve 3' end processing, we incorporated seven upstream polyadenylation enhancer elements (or upstream sequence elements, USEs) derived from viral or cellular genes into the 3' U3 region of gamma-retroviral and lentiviral SIN vectors.

Retroviral vectors containing Tet-controlled bidirectional transcription units for simultaneous regulation of two gene activities.

In this study retroviral self-inactivating (SIN)-vectors were constructed, that allow simultaneous regulation of two genes by integration of bidirectional Tet controlled transcription units. Marker genes (luciferase and eGFP) were expressed under the control of various bidirectional promoters P(tet)bis, in order to determine (i) the fraction of HtTA-1 cells exhibiting tight doxycycline (Dox) dependent control; (ii) possible effects of the vector backbone on the regulation of gene transcription; (iii) the possibility for crosstalk between different minimal promoters within P(tet)bi. When HtTA-1 cells, constitutively expressing the Tet-Transactivator (tTA), were transduced by S2f-lMCg retroviral vector, a high percentage (40) of the cell population displayed tight regulation (5000 fold) of P(tet)bi activity over a wide range of Dox concentrations.

Simplified generation of high-titer retrovirus producer cells for clinically relevant retroviral vectors by reversible inclusion of a lox-P-flanked marker gene.

Retroviral producer cells are generated by the introduction of a viral genome into "helper" cell lines containing all the necessary components for viral packaging and the release of infectious particles. The selection of high-titer vector producer cells is most efficient if the vector genome encodes a selectable marker, while it is extremely tedious to select high-titer producer clones if the transgene cannot be detected and selected directly. Here we describe the development of a screening system that uses reversible integration of lox-P-flanked eGFP as a qualitative and quantitative marker gene in two different vector systems, greatly facilitating the selection of viral producer cells.

Robust and efficient regulation of transgene expression in vivo by improved tetracycline-dependent lentiviral vectors.

We developed a panel of lentiviral vectors that displayed tetracycline-regulated transgene expression over two orders of magnitude in bulk, non-selected populations of transduced cells in vitro and in vivo. The robust expression and homogeneous response indicated that most transduced vector genomes were transcription competent and responsive to regulation, providing the lentiviral vector with a novel competitive advantage for gene transfer. After ex vivo transduction and transplantation of cord blood CD34+ cells into NOD/SCID mice, reporter gene expression could be switched "on" and "off" in human hematopoietic cells in vivo for prolonged times, proving integration of the regulated expression system into long-term repopulating cells.