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Hypervariable region 5 (HVR5) is a hydrophilic, serotypically nonconserved loop of the hexon monomer which extrudes from the adenovirus (Ad) capsid. We have replaced the HVR5 sequence of Ad5 with that of heterologous peptides and studied their effects on virus viability and peptide accessibility. A poliovirus model epitope was first inserted in a series of nine “isogenic” viruses that differed in their flanking spacers. Whereas virus productivity was not profoundly altered by any of these modifications, immunoprecipitation experiments under nondenaturing conditions demonstrated that epitope recognition by its cognate monoclonal antibody (C3 MAb) was strongly linker dependent and correlated perfectly with the ability of C3 MAb to inhibit transgene delivery and expression. An α v -specific ligand (DCRGDCF) was then inserted in a suitable linker context to investigate whether hexon-modified capsids would enhance the transduction of cells displaying limiting amounts of the virus attachment receptors. Interestingly, although hexon has never been implicated in Ad entry, the modified virus significantly increased the transduction of human vascular smooth muscle cells in vitro. Competition experiments with 293 cells saturated with recombinant knob further indicated that the hexon-modified virus could use an additional, knob-independent pathway for entry. We concluded that genetic engineering of the Ad5 hexon monomer constitutes a novel and feasible approach to equip the virus with additional targeting ligands.

The present investigation concerns 80–90 day-old male rats born from morphine-exposed mothers (2×10 mg/kg per day from days 11 to 18 of gestation which showed at birth reduced size and activity of the adrenals). This prenatal treatment did not significantly disturb under resting conditions: (1) the postnatal body growth up to week 10 after birth, (2) the activity of the pituitary gonadal axis (circulating luteinizing hormone (LH) and testosterone (T), weight of the testicles and seminal vesicles), (3) the activity of the hypothalamo-pituitary-adrenal axis (HPA) (hypothalamic corticoliberin (CRF) content, plasma adrenocorticotrophic hormone (ACTH) level, adrenal weight and corticosterone (B) content, plasma B level) as well as B max and K d of mineralocorticoid (type I) and glucocorticoid (type II) receptors to B in both the hippocampus and the hypothalamus. In contrast these rats showed reduced content of adrenals in noradrenaline (NA) and adrenaline (A) but increased circulating levels of A.

Recombinant adenoviruses are widely used for the transfer of foreign genes into various mammalian cells. However, the utilization of these vectors for cancer gene therapy requires the specific and efficient expression of the transferred gene in tumor cells. To obtain targeted expression in hepatoma cells, we constructed recombinant adenoviral vectors containing transcriptional elements from either the rat alpha-fetoprotein (AFP) or the human insulin-like growth factor II (IGFII) genes driving expression of the nuclear beta-galactosidase gene (nls lacZ). In vitro infection revealed that the AFP but not the IGFII transcriptional regulatory sequence controlled nls lacZ expression specifically in hepatoma cells. The same specificity was obtained in vivo in subcutaneous human hepatic tumors generated by engraftment of Huh7 hepatoma cells in nude mice as well as in primary liver tumors developed in rats and mice. No marker gene expression was detectable after AFP-nls lacZ gene transfer to normal rat liver parenchyma despite evidence for the presence of DNA encoding the nls lacZ gene. However, in vivo experiments with primary liver tumors in rats and mice also revealed that primary hepatoma cells were poorly infected by adenoviral vectors. Peritumoral and normal tissues were infected efficiently by adenoviral vectors. We conclude that hepatoma cell-specific expression of a transgene can be achieved with AFP regulatory sequences but that adenoviral vectors may not be the preferable vector for transferring genes in vivo in primary liver tumors.

The Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA-2) has been shown to transactivate both cellular and viral gene promoters including the promoter for the viral terminal protein 1 gene (TP-1). We investigated whether three other EBV nuclear antigens EBNA-3A, -3B, and -3C (which themselves share a degree of primary sequence homology) could also play a role in TP-1 gene regulation. The TP-1 promoter sequence was linked to the chloramphenicol acetyltransferase (CAT) gene and used in cotransfection experiments in an EBV negative cell line with various combinations of vectors expressing individual EBNA-3s. In the absence of other EBV proteins, the EBNA-3s did not stimulate TP-1 promoter activity. In the presence of EBNA-2, the EBNA-3s were shown to be capable of reducing the level of TP-1 promoter-driven CAT activity. The EBNA-3s had no effect on a panel of heterologous promoters, indicating that EBNA-2 and/or transcription elements specific to the TP-1 promoter are essential for the observed activity of the EBNA-3s. The functional antagonism between the EBNA-2 and EBNA-3 proteins may be important in the overall viral strategy.

NV1FGF is an expression plasmid encoding sp.FGF-1(21-154) currently under investigation for therapeutic angiogenesis in clinical trials. NV1FGF plasmid distribution and transgene expression following intramuscular (IM) injection in patients is unknown. The study involved six patients with chronic critical limb ischemia (CLI) planned to undergo amputation. A total dose of 0.5, 2, or 4 mg NV1FGF was administered as eight IM injections (0.006, 0.25, or 0.5 mg per injection) 3-5 days before amputation. Injected sites (30 cm(3)) were divided into equally sized smaller pieces to assess spatial distribution of NV1FGF sequences (PCR), NV1FGF mRNA (reverse transcriptase-PCR), and fibroblast growth factor-1 (FGF-1)-expressing cells (immunohistochemistry). Data indicated gene expression at all doses. The distribution area was within 5-12 cm for NV1FGF sequences containing the expression cassette, up to 5 cm for NV1FGF mRNA, and up to 3 cm for FGF-1-expressing myofibers. All FGF receptors were detected indicating robust potential for bioactivity after NV1FGF gene transfer. Circulating levels of NV1FGF sequences were shown to decrease within days after injection. Data support demonstration of plasmid-mediated gene transfer and expression in muscles from patients with CLI. FGF-1 expression was shown to be limited to injection sites, which supports the concept of multiple-site injection for therapeutic use.

The development of genetically modified adenoviral vectors capable of specifically transducing a given cell population requires the addition and functional presentation of particular tropism determinants within the virus capsid, together with the abrogation of the molecular determinants that dictate their natural tropism in vivo. The human adenovirus serotype 5 (Ad5) first attaches to the cell surface following high-affinity binding of the C-terminal knob of the fiber capsid protein to the coxsackie and adenovirus receptor (CAR). Here we have assessed whether genetic shortening of the fiber shaft (virus BS1), or replacing the Ad5 fiber shaft and knob with their Ad3 counterparts (virus DB6), could cripple this interaction in vitro and in vivo. A 10-fold decrease in the binding of the modified capsids to soluble CAR was evidenced, which correlated with a similar reduction of their ability to transduce CAR-positive cells in vitro. The ability of BS1 to interact with cellular integrins was also impaired, suggesting that the penton base and the short-shafted fiber when embedded in the capsid preclude each other from efficiently interacting with their cognate cell surface receptors (CAR and integrins respectively). BS1 and DB6 intravenous injections in mice further supported a profound impairment of the ability of the capsid-modified viruses to transduce the liver as demonstrated by a 10-fold reduction of intracellular viral DNA and transgene expression. Interestingly enough, the host humoral response was also specifically weakened in BS1- and DB6-inoculated animals. Taken together, these observations indicate that (i) fiber shortening and (ii) pseudo-typing of Ad5-based vectors with the shaft and knob from non-CAR-binding serotypes constitute two promising strategies to successfully attenuate their native tropism in vitro and most importantly in vivo.

gax, a diverged homeobox gene expressed in vascular smooth muscle cells (VSMCs), is down-regulated in vitro by mitogen stimulation and in vivo in response to vascular injury that leads to cellular proliferation. Recombinant Gax protein microinjected into VSMCs and fibroblasts inhibited the mitogen-induced entry into S-phase when introduced either during quiescence or early stages of G1. Overexpression of gax with a replication-defective adenovirus vector resulted in G0/G1 cell cycle arrest of VSMCs and fibroblasts. The gax-induced growth inhibition correlated with a p53-independent up-regulation of the cyclin-dependent kinase inhibitor p21. Gax overexpression also led to an association of p21 with cdk2 complexes and a decrease in cdk2 activity. Fibroblasts deficient in p21 were not susceptible to a reduction in cdk2 activity or growth inhibition by gax overexpression. Localized delivery of the virus to denuded rat carotid arteries significantly reduced neointima formation and luminal narrowing. These data indicate that gax overexpression can inhibit cell proliferation in a p21-dependent manner and can modulate injury-induced changes in vessel wall morphology that result from excessive cellular proliferation.

Transgene expression after the administration of recombinant adenovirus with E1 deleted is constantly transient. It is admitted that E1A-substituting activities of cellular or viral origin allow viral antigen synthesis and trigger cytotoxic lymphocyte-mediated clearance of the recipient cells. Our approach to solving this problem relies on the additional deletion of the E4 region from the vector backbone as this region upregulates viral gene expression at both transcriptional and posttranscriptional levels. As a prerequisite to the construction of E1 E4 doubly defective adenoviruses, we investigated the possibility of transcomplementing both functions within a single cell. In particular, the distal ORF6+ORF7 segment from the E4 locus of adenovirus type 5 was cloned under the control of the dexamethasone-inducible mouse mammary tumor virus long terminal repeat. Following transfection into 293 cells, clone IGRP2 was retained and characterized as it can rescue the growth defect of all E1+ E4- adenoviral deletants tested. DNA and RNA analysis experiments verified that the mouse mammary tumor virus promoter drives the expression of the ORF6+ORF7 unit and permits its bona fide alternative splicing, generating ORF6/7 mRNA in addition to the ORF6-expressing primary transcript. Importantly, IGRP2 cells sustain cell confluence for a period longer than that of 293 parental cells and allow the plaque purification of E1- or E4- defective viruses. The dual expression of E1 and E4 regulatory genes within IGRP2 cells is demonstrated by the construction, plaque purification, and helper-free propagation of recombinant lacZ-encoding doubly defective adenoviruses harboring different E4 deletions. In addition, the emergence, if any, of replicative particles during viral propagation in this novel packaging cell line will be drastically impaired as only a limited segment of E4 has been integrated.

Subgroup C human adenoviruses (Ad2 or Ad5) can transduce a wide array of dividing and quiescent adherent cell types in vitro and in vivo, essentially because their primary receptor (CAR) is widely expressed at the cellular surface. On the other hand, specific target cell types (eg some tumor cells, smooth muscle cells, fibroblasts) display limiting amounts of the CAR receptor so that large viral doses are needed for efficient infection, a constraint which favors vector dissemination in vivo, even after loco-regional injection. Abrogating the native virus-CAR interaction while allowing the virus to use a CAR-independent pathway for entry would thus represent a major milestone in order to better control the vector tropism in vivo. Towards this goal, we first engineered a series of capsid-modified adenoviruses by genetically inserting targeting peptides within protruding loops of the fiber and hexon capsid monomers (Vigne E, et al: J Virol 1999, 73:5156). The most interesting virus – AE43 – displays within the fiber HI loop a vitronectin-derived high affinity peptide for the urokinase-type plasminogen activator (uPAR or CD87), a cell surface receptor upregulated during cellular activation, and which controls cellular migration and invasion. Most interestingly, AE43 could increase transduction of cells normally refractory to adenovirus infection more than 100-fold in vitro. We could also demonstrate that AE43 retained its ability to enter the cell via a CAR-dependent pathway in vitro, and displayed a normal tropism following systemic injection in mice. To cripple the virus-CAR interaction, we then evaluated various strategies, including the introduction of CAR-ablating mutations within the fiber, and the shortening of the fiber shaft. Interestingly, some of these constructs could decrease transduction of CAR-positive cells at least 10-fold in vitro, with a similar reduction in liver transduction following systemic injection in naive mice. We are currently assessing the effect of combining within a single vector the uPAR-binding peptide of AE43 with our best CAR-ablating mutations.