Year of publication
- 2010 (4) (remove)
- English (4) (remove)
- Role of SOCS proteins in FLT3-ITD and BCR/ABL mediated leukemogenesis (2010)
- Acute myeloid/lymphoid leukemia is a fatal hematological malignancy characterized by accumulation of nonfunctional, immature blasts, which interferes with the production of normal blood cells. Activating mutations of receptor tyrosine kinases are common genetic lesions in leukemia. FLT3-ITD is a frequent activating mutation found in AML patients, leading to uncontrolled proliferation of leukemic blasts. FLT3-ITD directly activates STAT5, leading to the induction of STAT5 target gene expression like PIM kinases and SOCS genes. STAT5 and PIM kinases have been shown to play a crucial role in the FLT3-ITD mediated transformation. On the other hand, the role of SOCS proteins in FLT3-ITD mediated transformation has not been studied to date. SOCS proteins are part of a negative feedback mechanism that controls Jak kinases downstream of cytokine receptors. One of the SOCS family members, SOCS1 has been reported to suppress oncogenecity of several activating kinases implicated in hematologic malignancies. In this thesis the role of these SOCS proteins in FLT3-ITD mediated transformation (in vitro) and leukemogenesis (in vivo) is systematically explored. Expression of FLT3-ITD in cell lines of myeloid (32D) and lymphoid (Ba/F3) origin, led to CIS, SOCS1 and SOCS2 expression. FLT3-ITD expression in primary murine bone marrow stem/progenitor cells led to a 59 fold induction of SOCS1 expression. Furthermore, FLT3-ITD positive AML cell lines (MV4-11, MOLM-13) show kinase dependent CIS, SOCS1, and SOCS3 expression. Importantly SOCS1 is highly expressed in AML patients with FLT3-ITD compared to healthy individuals. SOCS1 protein was expressed in FLT3-ITD transduced murine bone marrow stem cells and SOCS1 expression was abolished with kinase inhibition in MOLM-13 cell line. In conclusion, SOCS1 was highly regulated by FLT3-ITD in myeloid, lymphoid cell lines, in bone marrow stem/progenitors and in AML patient samples. SOCS1 co-expression did not affect FLT3-ITD mediated signaling and proliferation, but abolished IL-3 mediated proliferation and protected 32D cells from interferon-α and interferon-γ mediated growth inhibition. FLT3-ITD expressing 32D cells showed diminished STAT1 activation in response to interferons (α and γ). Alone, SOCS1 strongly inhibited cytokine induced colony formation of bone marrow stem and progenitors, but not FLT3-ITD induced colony formation. Most importantly, in the presence of growth inhibitory interferon-γ, SOCS1 co-expression with FLT3-ITD led to increased colony formation compared to FLT3-ITD alone. Taken together, FLT3-ITD induced and exogenously expressed SOCS1, shielded cells from external cytokines, signals, while not affecting FLT3-ITD induced proliferation/signaling. In further experiments the in vivo effects of SOCS1 were studied in a bone marrow transplantation model. SOCS1 bone marrow transplants were unable to engraft/proliferate in mice. FLT3-ITD was shown to induce a myeloproliferative disease. Both control (empty vector), SOCS1 transplanted mice were normal and did not show any disease phenotype. FLT3-ITD alone and SOCS1 co-expressing FLT3-ITD developed either myeloproliferative disease or acute lymphoblastic leukemia with equal distribution. SOCS1 co-expression with FLT3-ITD led to a decreased latency. Mice transplanted with FLT3-ITD alone and SOCS1 co-expressing FLT3-ITD displayed enlarged spleens, liver and hypercellular bone marrow indicating infiltration of leukemic cells. Mice were also anemic and showed decreased platelet counts. Importantly SOCS1 co-expression particularly shortened the latency of myeloproliferative disease but not of acute lymphoblastic leukemia. In summary, in the context of FLT3-ITD, SOCS1 acts as a ‘conditional oncogene’ and cooperates with FLT3-ITD in the development of myeloproliferative disease. With these data we propose the following model: FLT3-ITD induces SOCS gene expression, which shields cells against proliferation and differentiation signals from cytokines, while not affecting FLT3-ITD mediated proliferative signals. This leaves cells under the dictate of FLT3-ITD thereby contributing to leukemogenesis. Similar to FLT3-ITD, BCR/ABL (P190) (an oncogenic fusion kinase often found in acute lymphoblastic leukemia) induces SOCS gene expression in K562 and long-term cultured cells from patients with acute lymphoblastic leukemia. SOCS1 co-expression does not affect BCR/ABL mediated proliferation while abrogating IL-3 mediated proliferation. These findings suggest that SOCS proteins may play a general co-operative role in the context of oncogenes which aberrantly activate STAT3/5 independently of JAK kinases. This study reveals a novel molecular mechanism of FLT3-ITD mediated leukemogenesis and suggests SOCS genes as potential therapeutic targets.
- TK.007: a novel, optimized HSVtk-variant for suicide gene therapy (2010)
- Suicide genes have been broadly used in gene therapy. They can serve as safety tools for conditional elimination of infused cells or for directed tumor therapy. To date, the Herpes simplex virus thymidine kinase/ ganciclovir (HSVtk/GCV) system is the most prominent and the most widely used suicidegene/prodrug combination. Despite its promising performance, the system displays limitations, which include relatively slow killing kinetics and toxicity of the prodrug GCV. Consequently, several groups have either developed new suicide-gene/prodrug combinations or attempted to improve the established HSVtk/GCV suicide system. The present study also aimed towards optimization of the HSVtk/GCV system. To do so, a novel, codon-optimized point mutant (A168H) of HSVtk was developed. The novel mutant was named TK.007. It was extensively tested for its efficiency in two relevant settings: (1) control of severe graft-versus-host disease (GvHD) after adoptive immunotherapy with Tlymphocytes, and (2) direct elimination of targeted tumor cells. TK.007 was compared to the broadly used wild-type, splice-corrected scHSVtk and to a codon-optimized HSVtk (coHSVtk) not bearing the above point mutation. (1) For experiments related to the adoptive immunotherapy approach, HSVtkvariants were expressed from a γ-retroviral MP71 vector as a fusion construct with the selection and marker gene tCD34. Expression levels for TK.007 in transduced lymphoid and myeloid cell lines were significantly higher at initial transduction and over a 12 week period compared to the commonly used scHSVtk and coHSVtk indicating reduced toxicity of TK.007. Killing kinetics of transduced cell lines (PM1 and K562) and primary human T cells were significantly faster for TK.007 in comparison to scHSVtk and coHSVtk in vitro. In vivo-functionality of TK.007 was assessed in an allogeneic transplantation model. T cells derived from C57BL/6J.Ly5.1 donor mice were transduced with MP71 vectors expressing scHSVtk or TK.007. Transduced cells were selected and transplanted into Balb/c Rag2-/- γ-/- immune-deficient recipient mice. Acute, severe GvHD occurred and was effectively abrogated in all mice transplanted with TK.007- transduced T cells, and in five out of six mice transplanted with scHSVtk-transduced cells. In a slightly modified quantitative allogeneic transplantation mouse model, significantly faster and more efficient in vivo killing was demonstrated for TK.007 as compared to scHSVtk, especially at low doses of GCV. (2) In order to assess TK.007 functionality in cells derived from solid tumors, HSVtk-variants were expressed from lentiviral gene ontology (LeGO) vectors in combination with an eGFP/neo-opt selection cassette. Transduced and selected tumor cell lines that derived from several tissues were eliminated at significantly lower GCV doses and to higher extents when transduced with TK.007 compared to scHSVtk. Moreover, a significantly stronger bystander effect of TK.007 was demonstrated. The superior in vitro efficiency of TK.007 was confirmed in an in vivo subcutaneous xenograft mouse model for glioblastoma in NOD/SCID mice. Mice transplanted with TK.007 transduced cells stayed tumor-free after treatment with different GCV-doses. On the contrary, mice of the scHSVtk group either demonstrated only transiently reduced tumor growth in the low-dose GCV group (10 mg/kg) compared to the control groups or suffered from relatively fast relapses after initial tumor shrinking in the standarddose (50 mg/kg) GCV group. As a result, all mice in the scHSVtk group died from vigorous tumor growth. In summary, in two different applications for suicide gene therapy the present study has demonstrated superior functional performance of the novel suicide gene TK.007 as compared to the broadly used wild-type scHSVtk. Differences became particularly pronounced at low doses of GCV. It can be concluded that the new TK.007-gene represents a promising alternative to the commonly used scHSVtk for gene therapeutic applications.
- Development of lentiviral vectors for the gene therapy of HIV infection (2010)
- Drug toxicity and viral resistance limit long-term efficacy of antiviral drug treatment for HIV infection. Thus, alternative therapies need to be explored. Previously, group of “Prof. von Laer” tested the infusion of T lymphocytes transduced with a retroviral vector (M87o) that expresses an HIV entry inhibitory peptide (maC46). Gene-modified autologous T cells were infused into 10 HIV-infected patients with advanced disease and multidrug resistant virus during antiretroviral combination therapy. T cell infusions were tolerated well with no severe side effects. A significant increase of CD4 counts was observed post infusion. At the end of the one-year follow-up, the CD4 counts of all patients were still around or above baseline. Gene-modified cells could be detected in peripheral blood, lymph nodes and bone marrow throughout the oneyear follow-up, whereby marking levels correlated with the cell dose. No significant changes of viral load were observed during the first four months. Four of the seven patients that changed their antiviral drug regimen thereafter responded with a significant decline in plasma viral load. In conclusion, the transfer of gene-modified cells was safe, led to sustained levels of gene marking and may improve immune competence in HIV-infected patients with advanced disease and multidrug resistant virus. However, the low level of gene marking and the lack of substantial long-term in vivo accumulation of gene-protected cells observed in this trial clearly demonstrate the requirement for new vectors with new strategy. In this thesis self‐inactivating lentiviral vectors harboring internal promoters and RNA elements were therefore evaluated for their potential use in a clinical gene‐therapy trial. The results from this work provide the basis for the selection of a suitable candidate vector for extensive preclinical testing. Apart from being capable of transducing non‐dividing cells, lentiviral vectors incorporate a number of additional features that are of potential value for gene therapeutic applications. These include a larger packaging capacity, higher titers than γ‐retroviral vectors and, most importantly, a reduced risk of deregulating cellular genes due to its natural integration profile. The use of internal promoters to drive expression of the therapeutic transgene maC46 should further improve the safety profile of these new‐generation vectors, while an additional artificial splice acceptor (SA) into the 5‟UTR of the transgene over all elevate transgene expression. The rationale for this is that hematopoietic stem and progenitor cells will be Summary 98 protected from enhancer‐mediated transactivation effects and also from potential side effects due to the aberrant expression of maC46 while at the same time the full clinical benefit for the patients is maintained. In order to find a suitable candidate for preclinical studies, two candidate therapeutic vectors harboring different regulatory elements were selected based on results from pilot experiments. The internal promoters used to drive expression of codon optimized maC46 were the PGK promoter and MPSV promoter. This work focuses on the transgene expression levels in lymphoid cells and antiviral activity. The issues of long term expression, propensity to methylation mediated silencing of the promoters, and genotoxicity were also touched. In a first step the performance of different vectors was evaluated in the human T cell lines. Based on promising data from ex vivo human peripheral blood mononuclear cells, the vector carrying the MPSV promoter along with intron were selected for in vivo transplantation experiments. In summary, the ex vivo data suggested the long term survival of lentiviral gene modified cells, along with maintained expression of introduced genes. It was observed that the expression of these constructs depends strongly on the activation and differentiation status of the targeted T cells. This regulation was not linked to any specific promotor. In vivo study shows that maC46 can be introduced into murine multiple hematopoietic lineages via lentiviral vector and expressed at high levels in their mulilineage progeny, without altering the hematopoiesis. There was no sign of any kind of hematopoietic or lymphoid malignancies. Although gene-modified lymphocytes persisted in-vivo, the downregulation of transgene expression was consistent with the ex-vivo observation. In contrast to that the T cells transplanted group showed delayed engraftment of donor cells and there was no expression of C46 in blood and lymphatic organs. . In conclusion, when considering HIV gene therapy focusing CD4+ T cells, potential problems of T cell activation status as related to the desired clinical effect must be addressed. These results might open the way for a gene therapy targeting mainly or exclusively activated T cells and could be exploited for immunostimulatory as well as suppressive approaches.
- T cell receptor diversity prevents T cell leukemia, lymphoma development / von Nabil Saleh Ahmed Al-Ghaili (2010)
- Gene therapy is a promising therapeutic strategy that emerged from the attractive idea of targeting therapy at the molecular level. For many patients who suffer from genetic and acquired diseases that cannot be effectively treated by conventional treatment approaches gene therapy remains a huge hope of cure in spite of the hurdles regarding efficacy and safety that need to be overcome. The development of efficient gene transfer vehicles, mainly retroviral vectors, led to the first successful gene therapy trial, to treat patients suffering from X-linked severe combined immunodeficiency syndrome (X-SCID) using gene modified stem cells (Hacein-Bey-Abina, Le Deist et al. 2002). Despite the success of this trial, it revealed the danger of retroviral insertional mutagenesis as a major adverse event of gene therapy using gene-modified stem cells (Hacein-Bey-Abina, von Kalle et al. 2003). In contrast to stem cells, T cells are relatively resistant to insertional mutagenesis and transformation even after transduction with potent oncogenes using retroviral vectors (Newrzela, Cornils et al. 2008). However, mature T cells can self-renew, proliferate and survive for long periods. These criteria are supposed to render T cells prone to transformation. Therefore, the questions of mature T cells transformability and the control mechanism limiting their transformation are still elusive.