Using a tropism-modified adenoviral vector to circumvent inhibitory factors in ascites fluid

Peritoneal compartmentalization of advanced stage ovarian cancer provides a rational scenario for gene therapy strategies. Several groups are exploring intraperitoneal administration of adenoviral (Ad) vectors for this purpose. We examined in vitro gene transfer in the presence of ascites fluid from ovarian cancer patients and observed significant inhibition of Ad-mediated gene transfer. The inhibitory activity was not identified as either complement or cellular factors, but depletion of IgG from ascites removed the inhibitory activity, implicating neutralizing anti-Ad antibodies. A wide range of preexisting anti-Ad antibody titers in patient ascites fluid was measured by ELISA. Western blot analysis demonstrated that the antibodies were directed primarily against the Ad fiber protein. To circumvent inhibition by neutralizing antibodies, a genetically modified adenoviral vector was tested. The Ad5Luc.RGD vector has an Arg-Gly-Asp (RGD) peptide sequence inserted into the fiber knob domain and enters cells through a nonnative pathway. Compared with the conventional Ad5 vector, Ad5Luc.RGD directed efficient gene transfer to cell lines and primary ovarian cancer cells in the presence of ascites fluid containing high-titer neutralizing anti-Ad antibodies. These results suggest that such modified Ad vectors will be needed to achieve efficient gene transfer in the clinical setting.     

Flex-Hets differentially induce apoptosis in cancer over normal cells by directly targeting mitochondria

Flex-Het drugs induce apoptosis in multiple types of cancer cells, with little effect on normal cells. This apoptosis occurs through the intrinsic mitochondrial pathway accompanied by generation of reactive oxygen species (ROS). The objective of this study was to determine if direct or indirect targeting of mitochondria is responsible for the differential sensitivities of cancer and normal cells to Flex-Hets. Mitochondrial effects and apoptosis were measured using JC-1 and Annexin V-FITC dyes with flow cytometry. Bcl-2, Bcl-x(L), and Bax were measured by Western blot. Flex-Hets induced mitochondrial swelling and apoptosis in ovarian cancer cell lines but had minimal to no effects in a variety of normal cell cultures, including human ovarian surface epithelium. Effects on inner mitochondrial membrane (IMM) potential were variable and did not occur in normal cells. Two different antioxidants, administered at concentrations shown to quench intracellular and mitochondrial ROS, did not alter Flex-Het-induced mitochondrial swelling, loss of IMM potential, or apoptosis. Inhibition of protein synthesis with cycloheximide also did not prevent Flex-Het mitochondrial or apoptosis effects. Bcl-2 and Bcl-x(L) levels were decreased in an ovarian cancer cell line but increased in a normal culture, whereas Bax expression was unaffected by Flex-Hets treatment. In conclusion, ROS seems to be a consequence rather than a cause of mitochondrial swelling. The differential induction of apoptosis in cancer versus normal cells by Flex-Hets involves direct targeting of mitochondria associated with alterations in the balance of Bcl-2 proteins. This mechanism does not require IMM potential, ROS generation, or protein synthesis.     

Targeted adenovirus vectors

Recombinant adenovirus (Ad) vectors continue to be the preferred vectors for gene therapy and the study of gene function because they are relatively easy to construct, can be produced at high titer, and have high transduction efficiency. However, in some applications gene transfer with Ad vectors is less efficient because the target cells lack expression of the primary receptor, coxsackievirus and adenovirus receptor (CAR). Another problem is the wide biodistribution of vector in tissue following in vivo gene transfer because of the relatively broad tissue expression of CAR. To overcome these limitations, various approaches have been developed to modify Ad tropism. In one approach, the capsid proteins of Ad are modified, such as with the addition of foreign ligands or the substitution of the fiber with other types of Ad fiber, in combination with the ablation of native tropism. In other approaches, Ad vectors are conjugated with adaptor molecules, such as antibody and fusion protein containing an anti-Ad single-chain antibody (scFv) or the extracellular domain of CAR with the targeting ligands, or chemically modified with polymers containing the targeting ligands. In this paper, we review advances in the development of targeted Ad vectors.     

Lentivirus-mediated gene transfer to the respiratory epithelium: a promising approach to gene therapy of cystic fibrosis

Gene therapy of cystic fibrosis (CF) lung disease needs highly efficient delivery and long-lasting complementation of the CFTR (cystic fibrosis transmembrane conductance regulator) gene into the respiratory epithelium. The development of lentiviral vectors has been a recent advance in the field of gene transfer and therapy. These integrating vectors appear to be promising vehicles for gene delivery into respiratory epithelial cells by virtue of their ability to infect nondividing cells and mediate long-term persistence of transgene expression. Studies in human airway tissues and animal models have highlighted the possibility of achieving gene expression by lentiviral vectors, which outlasted the normal lifespan of the respiratory epithelium, indicating targeting of a 'stem cell' compartment. Modification of the paracellular permeability and pseudotyping with heterologous envelopes are the strategies currently used to overcome the paucity of specific viral receptors on the apical surface of airway epithelial cells and to reach the basolateral surface receptors. Preclinical studies on CF mice, demonstrating complementation of the CF defect, offer hope that lentivirus gene therapy can be translated into an effective treatment of CF lung disease. Besides a direct targeting of the stem/progenitor niche(s) in the CF airways, an alternative approach may envision homing of hematopoietic stem cells engineered to express the CFTR gene by lentiviral vectors. In the context of lentivirus-mediated CFTR gene transfer to the CF airways, biosafety aspects should be of primary concern.     

Preclinical evaluation of a class of infectivity-enhanced adenoviral vectors in ovarian cancer gene therapy

Ovarian carcinoma cells are often infected inefficiently by adenoviruses (Ad) due to low expression of coxsackie-adenovirus receptors (CAR), hindering the application of adenovirus-mediated gene therapy in ovarian cancer. In this study, we explored a class of infectivity-enhanced Ad vectors, which contain CAR-independent targeting motifs RGD (Ad5.RGD), polylysine (Ad5.pK7), or both (Ad5.RGD.pK7), for their utility in ovarian cancer gene therapy using in vitro and in vivo model systems. We found that these vectors infected established ovarian carcinoma cell lines and primary ovarian cancer cells with significantly enhanced infectivity. Among them, Ad5.RGD.pK7 appeared to be most efficient. Further, we evaluated their gene delivery efficiency using two different ovarian cancer mouse models--subcutaneous and intraperitoneal human ovarian cancer xenografts. All of the modified vectors appeared to be more efficient than the unmodified Ad5 vector in both models, although some of the differences are not statistically significant. Of these, Ad5.RGD.pK7 exhibited the highest efficacy in the subcutaneous tumor model, while Ad5.pK7 worked most efficiently in the intraperitoneal tumor model. These preclinical results suggest that Ad5.RGD.pK7 and Ad5.pK7 may be very useful in ovarian cancer gene therapy.