No abstract

The antitumoral effects that follow the local delivery of the N-terminal fragment of human plasminogen (angiostatin K3) have been studied in two xenograft murine models. Angiostatin delivery was achieved by a defective adenovirus expressing a secretable angiostatin K3 molecule from the cytomegalovirus promoter (AdK3). In in vitro studies, AdK3 selectively inhibited endothelial cell proliferation and disrupted the G 2 ͞M transition induced by M-phasepromoting factors. AdK3-infected endothelial cells showed a marked mitosis arrest that correlated with the downregulation of the M-phase phosphoproteins. A single intratumoral injection of AdK3 into preestablished rat C6 glioma or human MDA-MB-231 breast carcinoma grown in athymic mice was followed by a significant arrest of tumor growth, which was associated with a suppression of neovascularization within and at the vicinity of the tumors. AdK3 therapy also induced a 10-fold increase in apoptotic tumor cells as compared with a control adenovirus. Furthermore, we showed that systemic injection of AdK3 delayed C6 tumor establishment and growth, confirming that angiostatin can function in a paracrin manner. Our data support the concept that targeted antiangiogenesis, using adenovirus-mediated gene transfer, represents a promising alternative strategy for delivering antiangiogenic factors as their bolus injections present unsolved pharmacological problems.

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.

AdmATF is a recombinant adenovirus encoding a secreted within and at the vicinity of the injection site was also supversion of the amino-terminal fragment (ATF) of murine pressed, suggesting that AdmATF inhibited primary tumor urokinase (uPA). This defective adenovirus was used in growth by targeting angiogenesis. AdmATF also interfered three murine models to assess the antitumoral effects with tumor cell establishment at distant sites: (1) lung disassociated with local or systemic delivery of ATF, a broad semination of Lewis lung carcinoma cells was significantly cell invasion inhibitor that antagonizes uPA binding to its reduced following intratumoral injection at the primary site; cell surface receptor (uPAR). A single intratumoral injection and (2) systemic administration of AdmATF inhibited subof AdmATF into pre-established MDA-MB-231 human sequent liver metastasis in a LS174T human colon carcibreast xenografts grown in athymic mice, or into pre-estabnoma xenograft model. These data outline the potential of lished C57/BL6 syngeneic Lewis lung carcinoma resulted using a recombinant adenovirus directing the secretion of an in a specific arrest of tumor growth. Neovascularization antagonist of cell-associated uPA for cancer gene therapy.

We have designed stable pKD1 derivatives for efficient secretion of recombinant human serum albumin (rHSA) by industrial strains of Kluyveromyces yeasts. A comparison of this multi-copy expression system with isogenic cassettes integrated at chromosomal loci demonstrated that high level secretion of rHSA is a function of gene dosage in K. lactis. Various signal sequences could be used, and the secretion levels were independent of the presence of the native pro peptide. The mitotic stability of the pKD1-based expression vectors was found to be species and strain dependent and was influenced by promoter strength and culture conditions. Vector stability was drastically enhanced when the HSA gene was expressed from an inducible promoter: 90% of the transformed cells still harbored the vector after 100 generations of non-selective growth in uninduced culture conditions. Secretion levels in the range of several grams per liter of correctly folded and processed rHSA were obtained at the pilot scale, thus making the industrial production of pharmaceutical-grade, Kluyveromyces-derived rHSA economically feasible.

Ad2 and Ad5 belong to a group of human cytolytic viruses that target the respiratory airways for reproduction, whereas latent infections establish within other tissues. Signals therefore exist that control this dichotomic process in different cell types, perhaps including cis and/or trans elements of viral origin. Since 1993, Ad2- and Ad5-based adenoviruses lacking all or part of the E1 regulatory region have been undergoing evaluation in phase I trials that target cancer and cystic fibrosis. These viruses are extremely attenuated and actually do not reproduce in most human cells. However, they retain most of the virus genetic program and often promote a significant cytotoxicity after infection, emphasizing the need to further cripple the virus biology to extend the duration of transgene expression, if required. We will review the strategies currently followed to engineer a professional lytic virus for epithelial cells into an innocuous gene delivery vehicle. Potential effects on the transducing properties of the vector that may result from the inactivation of viral activities that normally allow/regulate extrachromosomal gene expression during wild-type infection are discussed.

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.

Human adenovirus (HAV) serotypes 2 and 5 are commonly used as vector backbones for adenovirus-mediated gene transfer. However, HAVs were chosen as a backbone for the vectors for historical reasons and have a number of significant disadvantages when used as a shuttle for gene transfer in humans. As an initial trial to circumvent some of the shortcomings of HAV vectors, we have produced an E1-deleted canine adenovirus type 2 (CAV-2) vector for gene transfer. Initially, we demonstrated that CAV-2 undergoes an abortive viral cycle in a wide range of human-derived cell lines. Second, we assayed human sera containing HAV-5 neutralizing antibodies for their ability to inhibit CAV-2-induced plaques on permissive cells. In the cohort tested, our data demonstrate that the humoral response directed against HAV-5 does not inhibit CAV-2 plaque formation in the majority of cases. Canine cell lines expressing the E1 region of CAV-2 were generated and characterized. A recombinant CAV vector (CAVRSVbetagal) deleted in the E1 region and harboring lacZ was constructed. We show that CAVRSVbetagal is able to transduce and direct expression of the transgene in vitro in a variety of mammalian cells, most notably primary human-derived cells. In addition, gene transfer is demonstrated in vivo using chick embryos.