The ability of a replication-deficient adenovirus vector to transfer a foreign gene into neural cells of adult rats in vivo has been analysed. A large number of neural cells (including neurons, astrocytes and ependymal cells) expressed an E. coli lacZ transgene for at least 45 days after inoculation of various brain areas. Injecting up to 3 x 10(5) pfu in 10 microliters did not result in any detectable cytopathic effects--these were only observed for very high titres of infection (> 10(7) pfu 10 microliters-1). Adenovirus vectors therefore appear to be a promising means for in vivo transfer of therapeutic genes into the central nervous system.

Duchenne progressive muscular dystrophy is a lethal and common X-linked genetic disease caused by the absence of dystrophin, a 427K protein encoded by a 14 kilobase transcript. Two approaches have been proposed to correct the dystrophin deficiency in muscle. The first, myoblast transfer therapy, uses cells from normal donors, whereas the second involves direct intramuscular injection of recombinant plasmids expressing dystrophin. Adenovirus is an efficient vector for in vivo expression of various foreign genes. It has recently been demonstrated that a recombinant adenovirus expressing the lac-Z reporter gene can infect stably many mouse tissues, particularly muscle and heart. We have tested the ability of a recombinant adenovirus, containing a 6.3 kilobase pair Becker-like dystrophin complementary DNA driven by the Rous sarcoma virus promoter to direct the expression of a 'minidystrophin' in infected 293 cells and C2 myoblasts, and in the mdx mouse, after intramuscular injection. We report here that in vivo, we have obtained a sarcolemmal immunostaining in up to 50% of fibres of the injected muscle.

Motor neuron diseases such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy cause progressive paralysis, often leading to premature death. Neurotrophic factors have been suggested as therapeutic agents for motor neuron diseases, but their clinical use as injected recombinant protein was limited by toxicity and/or poor bioavailability. We demonstrate here that adenovirus-mediated gene transfer of neurotrophin-3 (NT-3) can produce substantial therapeutic effects in the mouse mutant pmn (progressive motor neuronopathy). After intramuscular injection of the NT-3 adenoviral vector, pmn mice showed a 50% increase in life span, reduced loss of motor axons and improved neuromuscular function as assessed by electromyography. These results were further improved by coinjecting an adenoviral vector coding for ciliary neurotrophic factor. Therefore, adenovirus-mediated gene transfer of neurotrophic factors offers new prospects for the treatment of motor neuron diseases.

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.

Liver tropism potentially leading to massive hepatocyte transduction and hepatotoxicity still represents a major drawback to adenovirus (Ad)-based gene therapy. We previously demonstrated that substitution of the hexon hypervariable region 5 (HVR5), the most abundant capsid protein, constituted a valuable platform for efficient Ad retargeting. The use of different mouse strains revealed that HVR5 substitution also led to dramatically less adenovirus liver transduction and associated toxicity, whereas HVR5-modified Ad were still able to transduce different cell lines efficiently, including primary hepatocytes. We showed that HVR5 modification did not significantly change Ad blood clearance or liver uptake at early times. However, we were able to link the lower liver transduction to enhanced HVR5-modified Ad liver clearance and impaired use of blood factors. Most importantly, HVR5-modified vectors continued to transduce tumors in vivo as efficiently as their wild-type counterparts. Taken together, our data provide a rationale for future design of retargeted vectors with a safer profile.

Targeting adenovirus encoding therapeutic genes to specific cell types has become a major goal in gene therapy. Coxsackievirus and adenovirus receptor (CAR) and alpha(V) integrins have been identified as the primary cell surface components that interact with adenovirus type 5 (Ad5)-based vectors during in vitro transduction. Redirecting Ad5-based vectors requires abrogation of the natural interaction between the viral capsid and its cellular receptors and simultaneous introduction of a new binding specificity into the viral capsid. To abrogate native Ad5 tropism, fiber knob mutations Pro409Glu and Lys417Ala were each incorporated into adenoviral vectors, while the RGD motif was deleted from the penton base. In vitro transduction experiments showed that these capsid mutations eliminated Ad5 interactions with CAR and alpha(V) integrins. Moreover, incorporation in the fiber HI loop of a vitronectin-derived ligand (VN4) specific for the uPAR/CD87 receptor provided the Lys417Ala virus with an alternative entry pathway specific for uPAR-expressing cells, indicating a successful in vitro retargeting of the vector. Unexpectedly, however, simultaneous disruption of Ad5 binding to CAR and alpha(V) integrins had no effect on liver gene transfer following systemic administration in mice. This study highlights the need to understand better the molecular determinants involved in adenovirus uptake by the liver to control the fate of adenoviral vectors in vivo.

The nematode-borne Grapevine fanleaf virus, from the genus Nepovirus in the family Comoviridae, causes severe degeneration of grapevines in most vineyards worldwide. We characterized 347 isolates from transgenic and conventional grapevines from two vineyard sites in the Champagne region of France for their molecular variant composition. The population structure and genetic diversity were examined in the coat protein gene by IC-RT-PCR-RFLP analysis with EcoRI and StyI, and nucleotide sequencing, respectively. RFLP data suggested that 55 % (191 of 347) of the isolates had a population structure consisting of one predominant variant. Sequencing data of 51 isolates representing the different restrictotypes confirmed the existence of mixed infection with a frequency of 33 % (17 of 51) and showed two major predominant haplotypes representing 71 % (60 of 85) of the sequence variants. Comparative nucleotide diversity among population subsets implied a lack of genetic differentiation according to host (transgenic vs conventional) or field site for most restrictotypes (17 of 18 and 13 of 18) and for haplotypes in most phylogenetic groups (seven of eight and six of eight), respectively. Interestingly, five of the 85 haplotypes sequenced had an intermediate divergence (0?036-0?066) between the lower (0?005-0?028) and upper range (0?083-0?138) of nucleotide variability, suggesting the occurrence of homologous RNA recombination. Sequence alignments clearly indicated a mosaic structure for four of these five variants, for which recombination sites were identified and parental lineages proposed. This is the first in-depth characterization of the population structure and genetic diversity in a nepovirus.

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