A two-step gene replacement procedure was developed that generates infectious adenoviral genomes through homologous recombination in Escherichia coli. As a prerequisite, a human adenovirus serotype 5 (Ad5)-derived genome was first introduced as a PacI restriction fragment into an incP-derived replicon which, in contrast to ColE1-derivatives (e.g., pBR322 or pUC plasmids), is functional in a polA mutant of E. coli. Any modification can be introduced at will following two consecutive homologous recombinations between the incP͞Ad5 replicon and the ColE1 plasmid. The overall procedure requires only the in vitro engineering of the ColE1-derivative by f lanking the desired modification with small stretches of identical sequences. In the first step, a cointegrate between the tetracycline-resistant incP͞Ad5 replicon and the kanamycin-resistant ColE1-derivative is selected by growing the polA host in the presence of both antibiotics. Resolution of this cointegrate is further selected in sucrose growth conditions due to the loss of a conditional suicide marker (the sacB gene of Bacillus subtilis) present in the ColE1 plasmid, leading to unmodified and modified incP͞Ad5 replicons that can be differentiated upon restriction analysis. Consecutive rounds of this two-step cloning procedure allowed the introduction of multiple independent modifications within the virus genome, with no requirement for an intermediate virus. The potential of this procedure is demonstrated by the recovery of several E1E3E4-deleted adenoviruses following transfection of the corresponding E. coli-derived genomes in IGRP2 cells.

We have developed an anion-exchange high-performance liquid chromatography (HPLC) method using Q Sepharose XL (Amersham Pharmacia Biotech) as adsorbent to analyze samples containing adenovirus. This method has several major advantages over the HPLC method previously described for quantitating particles, namely (1) a Ͼ10-fold improvement in the detection limit of adenovirus in crude preparations; (2) absence of interferences originating from nucleic acids and proteins which usually contaminate crude samples; (3) unprecedented sharpness and symmetry of adenovirus peak, rendering the identification of the viral peak unambiguous, even in extremely crude and dilute prep-

A new adenoviral vector (Ad-GFAP-GDNF) (Ad-¼ adenovirus, GFAP ¼ glial fibrillary acidic protein, GDNF ¼ glial cell line-derived neurotrophic factor) was constructed in which (i) the E1,E3/E4 regions of Ad5 were deleted and (ii) the GDNF transgene is driven by the GFAP promoter. We verified, in vitro, that the recombinant GDNF was expressed in primary cultures of astrocytes. In vivo, the Ad-GFAP-GDNF was injected into the striatum of rats 1 week before provoking striatal 6-OHDA lesion. After 1 month, the striatal GDNF levels were 37 pg/mg total protein. This quantity was at least 120-fold higher than in nontransduced striatum or after injection of the empty adenoviral vector. At 3 months after viral injection, GDNF expression decreased, whereas the viral DNA remained unchanged. Furthermore, around 70% of the dopaminergic (DA) neurons were protected from degeneration up to 3 months as compared to about 45% in the control groups. In addition, the amphetamine-induced rotational behavior was decreased. The results obtained in this study on DA neuron protection and rotational behavior are similar to those previously reported using vectors with viral promoters. In addition to these results, we established that a high level of GDNF was present in the striatum and that the period of GDNF expression was prolonged after injection of our adenoviral vector.

The transfer of the Glial cell line-derived neurotrophic factor (GDNF) gene to the central nervous system by a recombinant adenoviral vector (Ad) was studied. We constructed the adenovirus vector Ad-NSE-GDNF from which the E1, E3/E4 regions of Ad5 have been deleted and in which the GDNF gene was under the control of a neuron-specific enolase (NSE) promoter. The vector was injected into the striatum of a rat model of Parkinson's disease. We found that (i) the NSE promoter can restrict transgene expression in neurons; (ii) Ad-NSE-GDNF significantly protected dopaminergic (DA) neurons in the substantia nigra (SN) but did not reverse the impairments of amphetamine-induced rotational behavior in lesioned rats.

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