Cytosine deaminase/5-fluorocytosine gene therapy and Apo2L/TRAIL cooperate to kill TRAIL-resistant tumor cells.

The death ligand Apo2L/TRAIL (Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand) eradicates many tumor types while sparing most normal tissues. However, some tumors are resistant to TRAIL. We therefore determined if TRAIL cooperates with cytosine deaminase/5-fluorocytosine (CD/5-FC) gene therapy and investigated the mechanisms involved. Transfection of human LAN-5 neuroblastoma cells with CD rendered the cells (LAN-5-CD) sensitive to 5-FC-induced, caspase-dependent apoptosis. Mediated by caspase-3, CD/5-FC and TRAIL cooperated to induce apoptosis in these TRAIL-resistant cells and to cleave X-linked inhibitor of apoptosis protein (XIAP). In established LAN-5-CD tumors growing subcutaneously in mice, intratumorally applied TRAIL did not decrease tumor growth and systemically administered 5-FC only attenuated tumor growth. In contrast, 5-FC together with TRAIL dramatically decreased tumor growth and eradicated a tumor. Assuming sufficient gene transfer of CD in situ, CD/5-FC with TRAIL may be useful for the therapy of tumors resistant to TRAIL.

Human blood late outgrowth endothelial cells for gene therapy of cancer: determinants of efficacy.

Human adult blood late outgrowth endothelial cells (BOECs) are potential yet untested cellular vehicles to target tumor-cytotoxic effectors to tumors. We show that, following intravenous injection into irradiated mice, BOECs home to Lewis lung carcinoma (LLC) lung metastases, but less so to liver or kidney metastases. BOECs targeted most but not all of the lung metastases, to a different degree. While most of the homed BOECs took up an extravascular position, some integrated into tumor vessels. Sequestration into normal tissue was low. Placental growth factor mediated both migration and invasion of BOECs into LLC spheroid masses in vitro, as did VEGF. When armed with a suicide gene, BOECs exerted a bystander effect on LLC cells in vitro and in vivo. Surprisingly, i.v. administration of armed BOECs into mice bearing multi-organ LLC metastases did not prolong survival. In addition to homing efficacy other parameters impacted upon the efficacy of BOECs. These include the ultimate susceptibility of BOECs to suicide gene-induced cell death, their paracrine proliferative effect on LLC cells and their low proliferation rate compared to LLC cells. Addressing these determinants may make BOECs a useful addition to the arsenal of tumor-targeting moieties.

Phenotypic analysis of the sensitivity of HIV-1 to inhibitors of the reverse transcriptase, protease, and integrase using a self-inactivating virus vector system.

Conventional phenotypic analysis of resistance of the human immunodeficiency virus (HIV) to antiviral therapy is time-consuming and requires culture of infectious virus. Although phenotypic analyses may be desirable, rapid generation of test results and decentralized availability of the test system will be important to achieve utility in the clinical practice. This study describes the design of an alternative phenotypic resistance test using replication incompetent viral vectors. Chimeric HIV vectors containing a marker gene were generated. The env and most of the regulatory and accessory genes of HIV were removed. In addition, the 3'U3 region was deleted to obtain a self-inactivating construct. Cotransfection of the plasmid with a plasmid that provided the vesicular stomatitis virus glycoprotein resulted in the production of replication-incompetent virus vectors. Infection of susceptible cells with the vectors led to marker gene expression. Vector production in the presence of protease (PR) inhibitors, or infection in the presence of reverse transcriptase (RT) or integrase (IN) inhibitors reduced marker gene expression in a dose-dependent manner. Marker gene activity was preserved at higher drug levels if vectors contained RT and PR genes from resistant virus isolates. Sensitivity to nucleoside and non-nucleoside RT inhibitors, protease and integrase inhibitors could be determined in 10 working days. The phenotypic drug resistance test using replication-incompetent HIV vectors significantly speeds up drug resistance measurements and allows testing at reduced biosafety levels. This will make clinical use of phenotypic assessment of antiviral resistance more feasible.

Efficient intracellular retrotransposition of an exogenous primate retrovirus genome.

The foamy virus (FV) subgroup of Retroviridae reverse transcribe their RNA (pre-)genome late in the replication cycle before leaving an infected cell. We studied whether a marker gene-transducing FV vector is able to shuttle to the nucleus and integrate into host cell genomic DNA. While a potential intracellular retrotransposition of vectors derived from other retroviruses was below the detection limit of our assay, we found that up to 5% of cells transfected with the FV vector were stably transduced, harboring 1 to approximately 10 vector integrants. Generation of the integrants depended on expression of functional capsid, reverse transcriptase and integrase proteins, and did not involve an extracellular step. PCR analysis of the U3 region of the 5' long terminal repeat and determination of proviral integration sites showed that a reverse transcription step had taken place to generate the integrants. Co-expression of a mutated envelope allowing particle egress and avoiding extracellular infection resulted in a significantly increased rescue of cells harboring integrants, suggesting that accumulation of proviruses via intracellular retrotransposition represents an integral part of the FV replication strategy.