A number of key physiological processes such as the relative paucity of dendritic cells (DCs) in the brain , lack of lymphatic drainage, production of anti-inflammatory mediators such as TGF-β and nitric oxide (NO) by cells in the central nervous system (CNS ), as well as low MHCII expression on infiltrating microglia, protect the brain from an immune-mediated attack but at the same time, also contribute to its immune suppressive environment [6–9]. We have succeeded in developing an adenoviral mediated immunotherapy for brain tumors that is dependent on the expression of two genes: Thymidine kinase (TK), that phosphorylates the prodrug Ganciclovir which induces DNA cross-linking followed by cell death and fms-like tyrosine kinase-3 ligand (Flt3L), a potent DC growth factor that recruits DCs within the tumor microenvironment [10–12]. Tumor cell death induced by TK causes the release of intracellular proinflammatory molecules and tumor antigens, which are taken up by surveying DCs that subsequently prime the T cells to elicit an antigen specific cytotoxic antitumor immune response [10–12]. Combination therapy using these two adenoviruses induces tumor regression, long-term survival, and immunological memory in several mouse and rat GBM model s [10, 12, 13].

Gene therapy vectors can also be used for the treatment of neurodegenerative diseases. In order to be useful for gene therapy of chronic neurological disorders, vector systems should allow long-term transduction of brain cells in the absence of undesirable long-term side effects. Recombinant adenovirus vectors are powerful tools for gene delivery to the CNS [14, 15]. Adenoviruses can be easily purified to high titers and efficiently transduce differentiated cells such as neurons and glial cells. Additionally, transgene expression is restricted to the area of vector administration or areas that project to the injection site [16, 17]. The majority of current adenoviral-mediated gene therapy protocols utilize first-generation vectors, which are recombinant vectors that are non-replicative because of the deletion of the E1 region from the viral genome [18, 19]. While in the absence of prior immune priming to adenovirus, first-generation adenoviral vectors injected into the brain parenchyma can sustain prolonged transgene expression for months, activation of antiviral T cells by peripheral immunization leads to loss of vector-mediated transgene expression [17, 20]. To overcome this instability, “gutless,” high-capacity adenoviral (HC-Ad) vectors have been engineered in which all viral encoding genes have been eliminated and replaced with noncoding stuffer DNA sequences. Even in the presence of an anti-adenoviral immune response, expression from HC-Ad vectors remains stable for at least 1 year [21, 22]. Critical parameters that determine the efficiency of transgene expression are (1) dose and volume of adenoviruses administered, (2) vector backbone and the choice of promoter, (3) immune status of the animal, and (4) purity of the adenoviral vector stock [23, 24]. In this unit we provide detailed instructions for the implantation of various syngeneic GBM model s in rodents and adenoviral-mediated gene therapy using Ad-TK and Ad-Flt3L. Adenoviral-mediated transgene expression can be readily detected in treated brain tumors by immunocytochemistry. Figure 3 shows representative images from syngeneic rat CNS-1 model, U251 and U87 xenografts models injected intratumorally with Ad adenoviral vectors encoding TK. TK expression was assessed by immunohistochemistry and immunofluorescence. ELISPOT and T cell proliferation are extremely useful assays for testing the quality and quantity of T cell antitumor responses induced by immunotherapeutic approaches; hence the protocols for setting up these assays are also described [10, 12, 13, 25–29].