Gene Therapy for the Treatment of Neurological Disorders: Central Nervous System Neoplasms

Abstract

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults with a median survival of 16.2-21.2 months post diagnosis (Stupp et al., N Engl J Med 352(10): 987-996, 2005). Because of its location, complete surgical resection is impossible; additionally because GBM is also resistant to chemotherapeutic and radiotherapy approaches, development of novel therapies is urgently needed. In this chapter we describe the development of preclinical animal models and a conditionally cytotoxic and immune-stimulatory gene therapy strategy that successfully causes tumor regression in several rodent GBM models.

Keywords: Adenoviral gene therapy; GBM models; Immunotherapy; T cell activation assays.

Figure 1. Time-course analysis of early GL26-Cit glioma growth in the striatum in mice

To visualize perivascular glioma growth GL26-Cit cells were implanted in RA/EGxdelCre mice that express GFP in the brain endothelium. Invasive glioma cells maintain close vascular contact at all time-points analyzed over 120 hrs as they disseminate throughout the brain. GFP⁺ brain microvasculature has been pseudocolored red. Corresponding high-magnification micrographs (insets) detail perivascular invasion at the tumor border. White asterisks (*) relate the image area shown in the high-magnification micrographs with the corresponding area in the low-magnification micrographs. Perivascular tumor invasion begins 24 hrs post-implantation. Inset denoted by the carrot (^) in the 120 hr micrograph is included to demonstrate the trapping of normal brain microvessels within the growing tumor mass as perivascular invasion is followed by tumor cell proliferation with in the perivascular space. Time-points progress from top to bottom and from left to right.

Figure 2. Late-stage GL26-Cit glioma tumor

Scanning fluorescence confocal micrograph of syngeneic GL26-Cit mouse glioma (shown in green) 21-days post-tumor implantation into the striatum. Glioma cells were genetically modified to express mCitrine fluorescent protein prior to tumor implantation to facilitate direct tumor visualization by fluorescence microscopy. Individual and merged channels are shown. Tumor-bearing brain tissue was sectioned 50 μm thick and immunolabeled with anti-myelin basic protein (MBP) antibodies (shown in red) then counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (shown in blue). Note that this late-stage tumor has well-defined borders compared to GL26-Cit tumors at earlier stages, which lack definition in their tumor borders and which exhibit extensive perivascular invasion. Also note that large myelinated axonal bundles that become compressed towards the outside of the growing tumor mass. Scale bar corresponds to 300μm. T denotes tumor area.

Figure 3. Adenoviral-mediated transgene delivery into intracranial GBM in rodent models

Microphotographs show TK expression within intracranial GBMs implanted in the rodent brain. Lewis rats bearing intracranial syngeneic CNS-1 GBMs (A) and nude mice bearing intracranial human U251 or U87 GBM (B) were intratumorally injected with Ad vectors encoding thymidin kinase (TK). TK expression was assessed by immunocytochemistry (A) and immunofluorescence (B). Nuclei were stained with DAPI (B, left panels).

Figure 4. Workflow for T cell proliferation assay

Purify splenocytes, remove RBCs, label with CFSE dye and culture with tumor lysate followed by processing for flow cytometric analysis. Dot plots show gating strategy for CD8 T cells. Histograms show representative images of CFSE stains in unstimulated and stimulated CD8 T cells.