NFP37 SOMATIC GENE THERAPY Texts for Laypersons

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Prof. Adriano Fontana;	Innere Medizin; Klinische Immunologie; Uni-spital Zürich; Häldeliweg 4; 8044 Zürich; CH; Tel 01 257 38 13; Fax 01 257 2872; e-mail: immfoa@usz.unizh.ch
Title:	FAS ligand gene transfer in brain tumor therapy.
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ABSTRACT FOR LAYPERSONS 1997:

Gentherapie von bösartigen Hirntumoren

Das Glioblastom multiforme (GBM), einer der häufigsten bösartigen Hirntumore, hat trotz allen chirurgischen, strahlentherapeutischen und medikamentösen Therapien eine schlechte Prognose, indem die meisten Patienten innerhalb der ersten zwei Jahren an ihrem Tumorleiden sterben. Um neue Formen der Therapie zu finden, werden Tumorzellen von GBM im Labor gezüchtet. Im Universitätsspital Zürich werden unter Leitung von Prof. A. Fontana in der Klinischen Immunologie des Departements der Inneren Medizin Versuche durchgeführt, die zum Ziele haben, GBM-Zellen in den sogenannten natürlichen Zelltod, die Apoptose, zu überführen. Im Gegensatz zum normalen Zelltod, der Nekrose, ist die Apoptose eine Eigenleistung der Tumorzelle, an deren Zustandekommen verschiedene Gene aktiv beteiligt sind. Ausgelöst wird die Apoptose durch verschiedene Signale unter anderem auch durch ein immunologisches Hormon, welches Fas Ligand (FasL) genannt wird und normalerweise dazu dient, Blutzellen, die Lymphozyten, bei Nichtbedarf zu eliminieren. Dazu bindet FasL an einen Schalter (Fas) auf der Zelloberfläche, was der Zelle signalisiert, sich zu verabschieden. Es liegt auf der Hand, dass die Einschaltung eines solchen Programmes bei GBM-Tumorzellen therapeutisch genutzt werden sollte. In der Tat tragen GBM-Zellen auf der Zelloberfläche Fas und lassen sich durch FasL eliminieren, sie sterben als Folge der Apoptose. Gegenwärtig wird in Tierversuchen getestet, inwiefern GBM durch FasL in die Apoptose gebracht werden und somit die Tiere vor den Folgen des Tumorwachstums geschützt werden können. Dazu werden Strategien entwickelt, wie mittels gentherapeutischer Methoden das FasL-Gen effizient als Apoptose induzierendes Signal in den GBM exprimiert werden kann. Beispielsweise könnten Zellen, die selbst nicht auf FasL empfindlich sind, aber nach gentechnischer Manipulation FasL bilden, in die GBM-Tumore iniziert werden, um daselbst ihre Waffe, FasL, gegen Fas positive Tumorzellen einzusetzen. Alternativ könnten die Tumore mit Viren, die sich nicht mehr vermehren können, welchen aber das FasL-Gen eingebaut wurden, infiziert werden.

Diese verschiedenen Möglichkeiten werden zur Zeit in der Hoffnung, in den kommenden Jahren, die Therapie bösartiger Hirntumore mittels Gentransfer bereichern zu könnnen, ausgetestet.

We have investigated adenoviruses encoding mouse FasL cDNA for their potential to infect glioma cells and to induce cell death. Initial experiments tested the susceptibility of rodent glioma cell lines which are currently used in experimental therapeutic glioma studies. When adding FasL in membrane form the F98 rat glioma cells were found to be susceptible to FasLmediated killing. The addition of membrane form FasL to the glioma cell lines C6, 9L, D74-RG2, CNS-1, MT539MG, and GL-261 caused a cytotoxicity below 20%. Flow cytometry revealed that F98 glioma cells expressed Fas at a high level (SFI = 5.86). In agreement with the flow cytometry analysis F98 cells but not F98/ZH cells were susceptible to FasL-mediated killing (data not shown). A low level of Fas expression was detected by flow cytometry on the rat glioma cell lines C6 (SFI = 1.47), 9L (SFI = 1.07) and D74-RG2 (SFI = 1.57), as well as on the murine gliomas MT539MG (SFI = 1.7) and GL261 (SFI = 1.34). These expression levels correlate to the low cytotoxicity of membrane form FasL on these cell lines.To assess the potential use of adenovirusderived replication-deficient vectors expressing FasL, we first examined whether F98 cells expressed a reporter transgene when infected with rAd-CM-lacZ. Transduction with rAdCMV-lacZ of F98 cells 24 hours after plating resulted in substantial expression of beta-gal activity in a time- and titre dependent manner. 24 hours after rAd transduction, F98 cells infected with 500 or with 1000 multiplicities of infection (MOI) showed 12 and 20% beta-gal positive cells, respectively. Three days after infection, the corresponding values were 11% and 31%.To explore whether infection of F98 with rAd-CMV-FasL results in FasL-mediated killing, survival of glioma cell cultures was assessed at different time points after infection. No apparent morphological differences among control cells and those infected with adenovirus became evident within the first 24 hours after infection. Thereafter, striking cytotoxicity occured in rAd-CMV-FasL but not in rAd-CMV-lacZ treated F98 cells, the extent depending on the amount of rAd-CMV-FasL virus added. At MOIs of 500 and 1000, cytotoxicity of rAd-CMV-FasL infected F98 cells was 51.9 + 4.1% and 81.2 + 2.6%, respectively. Based on the encouraging in vitro data, we proceeded to evaluate the in vivo sensitivity of F98 glioma cells to rAd-CMV-FasL. F98 glioma cells (5x10exp (+4)) were injected stereotactically into the right frontal lobes of the brains of syngeneic Fischer 344 rats. One day later, 5x10exp(+7) plaque forming units (pfu) of either rAd-CMV-lacZ or rAd CMV-FasL were stereotactically injected at the same coordinates. In the first series of experiments expression of the lacZ gene was analyzed at day 10 and day 20 after injection of rAd-CMV-lacZ. Variable results were obtained at both time ponts, ranging from around 50% beta-gal positive tumor cells to only single tumor cells expressing beta-gal. In the second series of experiments mean survival time of rats inoculated with F98 cells was found to be 21.7 + 2.6 days. Rats inoculated with F98 cells and treated with rAd-CMV-FasL survived much longer: 32.9 + 6.5 days (p= 0.005). The growth of tumors was not influenced by the injection of rAd-CMV-lacZ, the mean survival time of the animals being 21.4 + 2.3 days. In further experiments at day 5 after tumor inoculation, rAd-CMV-FasL treated tumors showed PCNA stained tumor cells and necrotic areas, which were much less pronounced in rAd-CMV-lacZ treated tumors and absent in untreated controls. Additional experiments were designed to investigate whether tumor cells become resistant to FasL either by downregulation of Fas expression or by recruitment of inhibitors of the intracellular pathway leading to apoptosis after ligation of Fas receptors. In two rats inoculated with F98 cells and treated with rAd-CMV-FasL, the tumors were excised at the time the animals became moribund (day 39 and 42). The cells were dissociated and analyzed for Fas expression by flow cytometry analysis and for their sensitivity to FasL. The tumor cells in both animals expressed Fas, at a level comparable to the original F98 cell line culture in vitro (data not shown). Furthermore when adding FasL in membrane form killing of the two ex vivo F98 tumor cells was 90% and 95%, respectively. Taken collectively, tumor growth in rats treated with FasL is not due to the development of resistance to the cytokine. To assess toxic effects by the therapeutic strategy used, histologic examinations were performed on liver, lung, spleen and muscle and found to be normal.

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