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Targeted vectors for cancer gene therapy: receptor and tran-scriptional targeting of retroviral, lentiviral, and adenoviral vectors

Deliverables

A tetracycline conditional expression retroviral vector, which has been validated in vitro, is currently being validated for its efficient and safe transduction of a marker gene in liver tumours in murine and rat models. The vector system is being tested for appropriate propagation and transgene expression within the tumour as well as for any detectable dissemination outside of the tumour.
Tetracycline conditional expression retroviral vectors were validated for their efficient expression of a marker gene in various appropriate cell lines. The feasibility to control retroviral replication with the tetracycline (tet) inducible system was previously described in the context of a lentiviral virus. We choose to adapt this system to our Moloney Murine Leukemia Virus (Mo-MLV) based vectors and designed hybrid MoMLV tet-inducible LTRs by replacing viral enhancer sequences with tet-responsive elements. The tet-inducible transactivator (tTA/tetOFF or rtTA/tetON) was trans-complemented either by the cell line, a replication-defective vector, or the RRV itself. In vitro experiments, in H293 Tet-off or Hela Tet-on human cell lines, were performed to validate these new regulatable replicative vectors. Doxycycline (dox) was given or not at the time of the transfection or 10 days thereafter. Without dox at the time of transfection, the RRV-GFP propagated, but dox treatment prevented its propagation. When dox was added at day 10, the level of GFP expression was reduced in the uninduced state. Also propagation started when dox was withdrawn at day 10. These results demonstrate the functionality of the hybrid LTRs to control RRV-GFP propagation by modulating its level of expression, in a reversible manner. We then aimed to carry the whole tet-inducible system within RRVs: one RRV holding the GFP reporter transgene and another one with the rtTA (tetON) transactivator. NIH3T3 cells were co-transfected with these two RRVs, and, in the dox induced state, propagation was observed as in the previous experiment. The supernatant of these induced cells was then passed onto NIH3T3 cells with or without dox. In the dox- induced state, GFP was clearly propagated with a high level of expression. Without dox, no propagation occurred. These results demonstrate that the whole tet-inducible system could be adapted to a pair of RRVs to achieve their inducible propagation. These regulatable replicative retroviral vectors harbour great potential for gene therapy application and will be further analysed for efficacy and safety in vivo.
A tetracycline conditional expression retroviral vector, which has been validated in vitro, is currently being validated for its efficient and safe transduction of a therapeutic gene in models of murine and rat glioblastoma. The vector system is being tested for appropriate propagation and transgene expression within the tumour as well as for any detectable dissemination outside of the tumour. It is also being tested for its therapeutic efficacy, alone or in combination with retroviral vectors transducing other genes of interest.
A set of adenoviral vectors were designed and engineered such as to provide efficient and specific expression of a marker gene in hepatoma, adenocarcinoma and glioblastoma cell lines, and neuronal and glial primary cell cultures. For marking studies, the GFP expressing vector AdFK7 was constructed. This vector expresses EGFP under the control of the hCMV promoter. To allow the easy switch between liver-specific, glia- or neuron-specific, or constitutively regulatable systems, various shuttle plasmids for HC-Ad vector construction were engineered. In a shuttle plasmid, the specific promoter can be exchanged in a single cloning step, and, in addition, the transgene can be exchanged in a single cloning step. The complete cassette then can be removed from the plasmid backbone as a single fragment and is introduced into an HC-Ad shuttle plasmid in a further cloning step. This plasmid then is ready for rescue by transfection of N52.Cre cells together with loxP helper virus. To allow production of HC-Ad vectors with targeted capsids, different adenoviral helper viruses were engineered. Adenoviral helper viruses with capsids were modified such as to contain targeting ligands in the HI-loop of the knob domain of the adenoviral fiber protein. These targeting ligands include a cyclic RGD peptide, a polylysine peptide and a VCAM1 binding peptide. They allow improved transduction of several primary cell types and of several cancer cell lines. Vectors generated by usage of these helper viruses during production will be used to transduce tumours in vivo or endothelial cells in tumours expressing increased VCAM1.
Semi-explicative retroviral vectors were designed and tested for their efficient expression of marker or therapeutic genes in tumour cell lines and animal cancer models. During the course of this project, it became clear that that the importance of restricting defective retroviral vector expression by means of tissue specific ligands or promoters was somewhat overshadowed by their poor efficiency of gene transfer into cancer cells. Indeed, several cancer gene therapy studies have shown that replication-competent retroviral vectors represent a major improvement over replication-defective ones in terms of transgene propagation efficiency. However, this positive effect is somewhat spoiled by the increased risk of dissemination and oncogenesis that replication-competent retroviral vectors entail. To enhance both their integral safety and their transgene capacity, we developed a semi-replication-competent retroviral vector system. The semi-replication-competent retroviral vector system is based on two trans-complementing replication-defective retroviral vectors termed gag-pol vector (GPv) and env vector (Ev). Vector propagation was monitored in vitro and in solid tumours in vivo, using different reporter transgenes for GPv and Ev. Systemic vector dissemination and leukemogenesis was assessed by direct intravenous vector injection and subsequent bone marrow transplantation, in MLV-sensitive mice. In vitro and in vivo the semi-replication-competent retroviral vectors propagate transgenes almost as efficiently as replication-competent ones. The semi-replication-competent retroviral vector system does not lead to detectable dissemination or leukemogenesis as does the replication-competent vector or the parental virus. Additionally, the vector duo allows co-propagation of different transgenes as well as mobilization of a third replication-defective vector. This study is an initial proof of principle for the use of complementary retroviral vectors to deliver and propagate transgenes in vitro and in solid tumours in vivo, but with reduced pathogenicity compared to its parental virus. In-between replication-defective and replication-competent retroviral vectors, this semi-replicative system offers good grounds for its application in vitro studies and allows envisioning its further development for cancer gene therapy.
A targeted adenoviral vector, HC-AdRS24, was validated for its efficient and safe transduction of a marker gene in murine liver tumours models, i.e. to establish pharmacokinetic data in vivo in mice. The vector expresses hIL12 under RU486 inducible control. Different from the mIL12, which at high doses is toxic in mice, the hIL12 does not have functional activity in mice and thus it can be used to analyse expression kinetics. This vector was used Gene therapy of liver diseases would benefit from systems allowing prolonged, regulatable and tissue-specific transgene expression. We thus attempted to produce a vector fulfilling these requirements and generated gutless adenoviral vectors containing a mifepristone (RU486) inducible system for controlled and liver-specific expression of human interleukin 12 (hIL-12) (GL-Ad/RUhIL-12) and mouse IL-12 (mIL-12) (GL-Ad/RUmIL-12). The properties of these vectors were tested both in vitro and in vivo. We found that infection of cells with GL-Ad/RUhIL-12 resulted in high level of hIL-12 expression in the presence of RU486 only in hepatocytic cells. In animals injected with GL-Ad/RUhIL-12 the administration of RU486 induced a transient rise of serum hIL-12 that peaked at 10h and completely disappeared by 72h. The peak value of hIL-12 was dependent on the doses of the vector and the inducer. High and sustained serum levels of hIL-12 could be attained by continuing administration of RU486 every 12 or 24h. Repetitive induction of hIL-12 could be obtained over, at least, a period of 48 weeks after a single injection of GL-Ad/RUhIL-12. Although the vector was detected in many tissues after systemic injection, transcription of the transgene was only found in the liver. In conclusion, gutless adenoviral vectors allow liver-specific and regulatable transgene expression for prolonged periods of time. Thus, these vectors are promising tools for gene therapy of liver cancer and could also be useful for other forms of hepatic disease. Indeed, treatment of liver metastases with 5x108 iu of GL-Ad/RUmIL-12 plus RU846 resulted in complete tumour regression in all animals.
An adenoviral vector is being evaluated for its efficient and safe therapeutic effect in a model of murine glioblastoma. We generated HC-Ad vectors expressing either the reporter EGFP or the molecule PEDF, which has potent anti-angiogenic activity and which we previously tested in an eye model of neoangiogenesis. In collaboration with the Department of Neurology, University of Tubingen, we have started testing these two vectors in murine models of brain cancer. One approach is based on the intracranial LN229 model in CD1 nude mice. Another approach is based on ex vivo gene transfer into normal carrier cells that will then migrate to the tumour. In proof-of-concept experiments we engrafted F98 rat glioblastoma cells into the right caudatoputamen. Tumours were allowed to develop for 1 week, before 50000 glial restricted precursor cells or ES cell-derived precursor cells were transplanted into the corpus callosum of the left hemisphere. One week later the animals were sacrificed and EGFP expressing cells were counted every 10th serial section of the brains. Interestingly, both glial as well as neural precursor cells migrated to the tumour to contra-lateral hemisphere. After arrival at the bumot, both cell types surrounded and even evaded the tumour. In future experiments we will analyse, whether tumour-inhibitory effects can be observed, if the transplanted cells are loaded with HC-Ad vectors expressing potentially therapeutic molecules including PEDF.
Replicative retroviral vectors (RRVs) were designed and tested for their efficient expression of marker or therapeutic genes in tumour cell lines and animal cancer models. During the course of this project, it became clear that that the importance of restricting defective retroviral vector expression by means of tissue specific ligands or promoters was somewhat overshadowed by another key issue; their poor efficiency of gene transfer into cancer cells. The defective nature of the vectors constituted a major bottleneck of current cancer gene therapy, while we reasoned that because tumours are masses of rapidly dividing cells, they would be most efficiently transduced with vector systems allowing transgene propagation. We thus designed two replicative retrovirus -derived vector systems: one inherently replicative vector, and one defective vector propagated by a helper retrovirus. In vitro, both systems achieved very efficient transgene propagation. In immuno-competent mice, replicative vectors transduced >85% tumour cells, whereas defective vectors transduced <1%under similar conditions. The viral propagation could be efficiently blocked by azido-thymidine, in vitro and in vivo. In a model of established brain tumours treated with suicide genes, RRVs were approximately 1000 times more efficient than defective adenoviral vectors. These results demonstrate the advantage and potential of RRVs and strongly support their development for cancer gene therapy. We are currently evaluating their efficacy using different tumour models (glioblastoma and melanoma) and different suicide genes (HSV-TK and CD).
A tetracycline conditional expression retroviral vector was validated for its efficient expression of a therapeutic gene in various appropriate cell lines. We designed an inducible retroviral vector carrying a therapeutic transgene; the cytocine deaminase (CD). As the room for exogenous DNA sequences is limited in retroviral vectors, we had to choose short sequences and thus used the GTX IRES sequence and the CD suicide gene (shorter than for instance HSV1-TK). Also, the rtTA transactivator was fused to CD, further restricting the size of exogenous DNA to be added to the retroviral vector. In a preliminary experiment, the new GTX-rtTA-CD cassette was assayed in a defective retroviral vector, propagated together with a replicative retroviral vector holding the GFP reporter transgene. Induction of GFP expression was fully reversible upon addition or withdrawal of doxycycline, as measured by mean fluorescence intensity. Furthermore, when 5FC (cytotoxic prodrug of the CD suicide gene) was added in the induced state it was toxic to the cells, but not in the uninduced state. This experiment further demonstrates the functionality of tet-inducible replicative retroviral vectors, and proves that the CD suicide gene fused to the rtTA is functional. These regulatable replicative retroviral vectors harbour great potential for gene therapy application and will be further analysed for efficacy and safety in vivo.
A tetracycline conditional expression retroviral vector, which has been validated in vitro, is currently being validated for its efficient and safe transduction of a marker gene in models of murine and rat glioblastoma. The vector system is being tested for appropriate propagation and transgene expression within the tumour as well as for any detectable dissemination outside of the tumour.
A tetracycline conditional expression retroviral vector, which has been validated in vitro, is currently being validated for its efficient and safe transduction of a therapeutic gene in liver tumours in murine and rat models. The vector system is being tested for appropriate propagation and transgene expression within the tumour as well as for any detectable dissemination outside of the tumour. It is also being tested for its therapeutic efficacy, alone or in combination with retroviral vectors transducing other genes of interest.
A set of adenoviral vectors were designed and engineered such as to provide efficient and specific expression of a therapeutic gene in hepatoma, adenocarcinoma and glioblastoma cell lines, and neuronal and glial primary cell cultures. For this various HC-Ad vectors were engineered (described above), and different therapeutic genes were incorporated. These were either expressed from constitutive promoters (human CMV, murine CMV, elongation factor 1 alpha), or regulatable promoters. Generation and characterisation of HC-Ad vector HC-Ad-RS45 - This vector expresses soluble Tie-2 under the control of the MCMV promoter and inhibits angiogenesis. - Inhibition of angiopoietin function by this vector may help to inhibit tumour growth in cancer as an adjuvant treatment. Generation and characterisation of HC-Ad vector HC-Ad-RS46 - This vector expresses the soluble PEX domain of MMP2 under the control of the MCMV promoter and is suitable to inhibit matrix metalloproteinases. - Matrix metalloproteinase are crucially involved in the invasion and metastasis of tumour cells. The expression of the secreted PEX domain in hepatic cancer may inhibit tumour growth by inhibiting the activity of matrix metalloproteinases. - Generation and characterisation of HC-Ad vector HC-Ad-RS24 and RS25 - These two vectors express the murine and the human, respectively, IL12 under the control of the inducible TTRB promoter. - Using these vectors, it is possible to induce expression in hepatocytes by oral administration of low doses of mifepristone. Generation and characterisation of HC-Ad vector HC-Ad-RS23 - This vector expresses the human VEGF Receptor 1 (s-FLT-1) under the control of the inducible TTRB promoter. - VEGF is a critical factor for the generation of new vessels (neoangiogenesis) in tumours. This vector allows the local inhibition of VEGF by genetic modification of normal hepatocytes. Only upon oral administration of mifepristone is VEGF produced in normal hepatocytes, thereby locally preventing function of VEGF expressed from the tumour. Generation of a PEDF expressing HC-Ad vector - The vector was constructed to express the PEDF cDNA from the hCMV promoter. This molecule has anti-angiogenic properties.
A set of targeted adenoviral vectors was evaluated for their efficient and safe therapeutic effects in murine liver tumour models. HC-AdRS25 expresses mIL12 under RU486 inducible control. In an orthotropic liver tumour model, 1 x 10(6) MC-38 mouse colon cancer cells were injected into the left liver lobe of C57BL/6J syngenic mice. Seven days before implantation of tumour cells, mice received different doses of the RU486 inducible mIL12 HC-Ad vector (AdRS25) by intravenous injection. A single tumour nodule with a size of about 8 mm was observed in livers 5 days after inoculation of the tumour cells. At this time induction of IL-12 was initiated by i.p. injection of RU486. The treatment was continued by daily injection for a time of 10 days. Five days after completion of the induction regime, mice were sacrificed and tumour sizes were determined. Control animals that were treated with vector but that received no RU486 showed progressive tumour growth. In contrast, all animals that received 5 x 10(8) infectious units of AdRS25 and were induced with RU486 experienced complete tumour regression. Animals that were injected with a lower vector dose of 1 x 10(8) infectious units followed by RU486 induction showed a significant reduction of the tumour mass, but finally the disease progressed and the animals died as a consequence of massive liver metastases. This inducible vector is currently being adapted for clinical phase I trials. HC-AdRS45 and HC-AdRS46 express sTie2 and sPEX. They were tested in orthotropic and subcutaneous tumour models of liver cancer in mice. Different from our expectations, there were only small tumour inhibitory effects. AdRS44 and HC-AdRS23 expresses sFLT1 either constitutively (AdRS44, PEFBOS promoter) or in a RU486 inducible manner (AdRS23). After injection of the sFLT1 vectors very high levels of the expressed protein were observed in the serum. In the latter case there was no background expression in the absence of RU486 induction. Different from our expectations there were only small tumour inhibitory effects. After injection of the sFLT1 expressing vector significant side effects were observed; significant ascites, hypoalbuminemia and kidney damage. sFLT1 likely led to the kidney damage resulting in renal loss of serum albumin thereby causing hypoalbuminemia and ascites. These studies indicate that long-term inhibition of VEGF function will likely result in unacceptable side effects and may also have implications for studies that aim to inhibit VEGF by other than gene transfer strategies.

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