Long Term Genetic Modification of Neurons, Astrocytes and Ependymocytes In Vivo using a High Capacity Adenovirus Vector

ABSTRACT

For the evaluation of a possible adenovirus-mediated gene transfer into cells of the CNS a replication deficient 3^rd generation gutless adenovirus vector was used.

For cell transduction, vectors with different reporter sequences were applied, resulting in the possibility of measuring the transduction success after infection by the transgene expression. The reporter genes include the green fluorescent protein (GFP), the LacZ (β-galactosidase) as well as the human secreted placenta alkaline phosphatase (SEAP). Applying various infectious particles (10, 50 and 100 MIO) a concentration-dependent transduction rate can be ascertained. The vector was injected into different brain areas including the ventricles. Applying immunohistochemical techniques using antibodies against NeuN (neuronal specific nuclear protein) and the gilal fibrillary acidic protein (GFAP) it can be shown that after vector administration into the striatum a preferential transduction of astrocytes is achieved. Within this brain compartment a diffusion of the vector up to a distance of 1000 ..m from the injection site can be determined. Following injection of vector particles into the corpus callosum, a vector distribution within the entire fibre tract of the white substance including the contralateral hemisphere is observed.

With these results it can be shown that the allocation of the vector is dependent on the structure and features of the receiving tissue. Injecting the adenovirus vector into the cerebrospinal fluid results in transgene expression (GFP, LacZ and SEAP) in the ependymal cells surrounding the entire ventricle.

After installing a permanent catheter in the lateral ventricle cerebrospinal fluid may be removed in a continuous manner in order to analyse/evaluate the marker enzyme SEAP. Using a luminometric screening procedure, the enzyme SEAP can be demonstrated up to a period of 42 days following vector injection into the cerebrospinal fluid. By these data it can be shown that the use of an adenovirus vector at least under experimental circumstances might be a pioneer therapeutical option for diseases of the central nervous system.

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