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. Besides that, using fluorescence resonance energy transfer we detected mispaired TCR dimers and different pairing behaviors of individual TCR chains with a mutual influence on TCR chain expression. The loss of function and mispairing could not be avoided by changing the TCR expression level or by introduction of an additional cysteine bridge. However, in polyclonal T cells, optimized TCR formats (cysteineization, codon optimization) enhanced correct pairing and function. We conclude from our data that (i) the level of mispairing depends on the individual TCRs and is not reduced by increasing the level of one TCR, and (ii) modifications (cysteineization, codon optimization) improve correct pairing but do not completely exclude mispairing (cysteineization).

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. RESULTS: Determination of luciferase activity in transduced cell populations indicated improvement of the dynamic range of gene expression for the autoregulated system. Further differences were observed regarding induction kinetics and dose-response. Most notably, introduction of the autoregulated system resulted in a threshold mode of induction, whereas the constitutive system exhibited pronounced effector-dose dependence. CONCLUSION: Tet-regulated gene expression in the applied autoregulated system resembles a threshold mode, whereby full induction of the tet-unit can be achieved at otherwise limiting doxycycline concentrations.

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. To address this question, the plasmacytoma cell line J558L was transduced with a vector allowing doxycycline-inducible IFN-γ gene expression. After the injection of the tumor cells into mice, IFN-γ was induced at different time points. Tumors did not grow when inducing IFN-γ immediately after tumor cell inoculation, while approximately half of the tumors were rejected when IFN-γ was induced in early established tumors within 2 weeks. Induction of IFN-γ 2-3 weeks after tumor cell inoculation was less efficient (0-17% rejection). IFN-γ induction in established tumors led to a reduction of CD146(+) endothelial cells and massive necrosis. Together, we show that vascularized tumors can be rejected by local IFN-γ expression, but that rejection of established tumors was less efficient over time. This suggests that transplanted tumors became less susceptible to local IFN-γ treatment the better they are established.

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. RESULTS: In this study, we systematically modified the widely used Cytomegalovirus (CMV) minimal promoter to further minimize background expression, resulting in an improved dynamic expression range. Using both plasmid-based and retroviral gene delivery, our analysis revealed that especially background expression levels could be significantly reduced when compared to previously established "standard" promoter designs. Our results also demonstrate the possibility to fine-tune expression levels in non-clonal cell populations. They also imply differences regarding the requirements for tight regulation and high level induction between transient and stable gene transfer systems. CONCLUSIONS: Until now, our understanding of mammalian transcriptional regulation including promoter architecture is limited. Nevertheless, the partly empirical modification of cis-elements as shown in this study can lead to the specific improvement of the performance of minimal promoters. The novel composite Ptet promoters introduced here will further expand the utility of the Tet system.

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. Introduction of a clean-up step, mediated by Cre-recombinase, allows the removal of residual sequences that were required for targeting and selection, but were dispensable for the final producer clones and eliminated homology-driven recombination between the tagging and the therapeutic vector. The system was used to establish GALV and RD114 pseudotyping producer cells (HG- and HR820) for a clinically relevant long terminal repeat-driven therapeutic vector, designed for the transfer of a recombinant TCR that delivered titers in the range of 2×10(7) infectious particles (IP)/ml. Production capacity of the amphotropic producer cell (HA820) was challenged by a therapeutic SIN vector transferring the large COL7A1 cDNA. The final producer clone delivered a titer of 4×10(6) IP/ml and the vector containing supernatant was used directly to functionally restore primary fibroblasts and keratinocytes isolated from recessive dystrophic epidermolysis bullosa patients. Thus, the combinatorial approach (fc-technology) to generate producer cells for therapeutic γ-retroviral (SIN) vectors is feasible, is highly efficient, and allows their safe production and application in clinical trials.

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. In order to facilitate the usage of the tet-system for approaches in gene therapy, both components have to be transferred by a single vector, thus eliminating the preselection of transactivator positive cells. Published "all-in-one" vectors for regulated transgene expression display a relatively low signal-to-noise ratio, resulting in regulatory windows of around 500-fold even in selected clones. In this study, we show that a modified vector architecture combined with the introduction of new tet-responsive promoters, Ptet, improved the dynamic range of such all-in-one vectors to levels up to 14,000-fold for viral and 25,000-fold for nonviral transfer vectors in nonclonal human cell lines, and up to 2,800-fold in murine hematopoietic cell lines. This improved regulation was the result of a strong reduction of background expression in the off-state, even if cells were transduced at high multiplicity of infection, while induction remained at high levels. In addition, the results indicated that successful regulation of gene expression in different target cells depended on vector architecture as well as the choice of the Ptet-promoter.

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. A 100-base-pair sequence representing a recombinant direct repeat of the USE derived from simian virus 40 (2xSV USE) gave the best results, improving both titer and gene expression. In both gamma-retroviral and lentiviral SIN vectors, the 2xSV USE partially substituted for effects provided by the much larger post-transcriptional regulatory element derived from woodchuck hepatitis virus (wPRE). By northern blot and reporter assays, we found that the 2xSV USE greatly improved proper messenger RNA (mRNA) processing at the retroviral termination signal. Importantly, the 2xSV USE was superior to the wPRE in suppressing transcriptional read-through, improving not only vector efficiency but potentially also biosafety.

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. As a result of our comparative study on the activity of virus derived minimal promoters (from MMTV, HIV and CMV), a clear hierarchy of activity as well as a different sensitivity to external influences among the various promoters studied was observed. Furthermore, our results strongly support the idea, that viral elements such as part of the MuLV pol/env region significantly affect the regulation capacity of an integrate. Taking into account our observations as outlined above, we succeeded in generating significantly optimized Tet regulated retroviral vectors. The application of such a one-step transfer system for P(tet) controlled genes would be of particular relevance to applications where cellular systems do not allow prolonged selection procedures as it is the case with primary cells considered for ex vivo gene therapy.

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. By vector injection into established tumor grafts, we achieved efficient delivery and quantitative regulation of transgene expression in vivo. By these approaches, gene function studies can now be performed in in vivo models of human hematopoiesis and cancer. In the future, regulated lentiviral vectors will improve the safety and efficacy of gene therapy.

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. After selection and titration of high-titer viral producer cells based on eGFP expression, the eGFP gene could be removed from the provirus by transient introduction of Cre-recombinase into the producer cells, thus allowing the production of therapeutic relevant vectors expressing solely the gene of interest. However, after removal of the marker gene a slight but consistent increase in viral titers compared to the respective control vectors was found, independent of the transgene or backbone used. The single lox-P site retained in the vector backbone does not affect gene expression level or fidelity of RNA processing.