An endogenous retrovirus derived from human melanoma cells.

We show that human melanoma cells produce retrovirus-like particles that exhibit reverse transcriptase activity, package sequences homologous to human endogenous retrovirus K (HERV-K), and contain mature forms of the Gag and Env proteins. We also demonstrate expression of the pol gene and of Gag, Env, and Rec proteins in human melanomas and metastases but not in melanocytes or normal lymph nodes. The data suggest that expression of retroviral genes and production of retroviral particles is activated during development of melanoma.

Retrofitting BACs with G418 resistance, luciferase, and oriP and EBNA-1 - new vectors for in vitro and in vivo delivery.

BACKGROUND: Bacterial artificial chromosomes (BACs) have been used extensively for sequencing the human and mouse genomes and are thus readily available for most genes. The large size of BACs means that they can generally carry intact genes with all the long range controlling elements that drive full levels of tissue-specific expression. For gene expression studies and gene therapy applications it is useful to be able to retrofit the BACs with selectable genes such as G418 resistance, reporter genes such as luciferase, and oriP/EBNA-1 from Epstein Barr virus which allows long term episomal maintenance in mammalian cells. RESULTS: We describe a series of retrofitting plasmids and a protocol for in vivo loxP/Cre recombination. The vector pRetroNeo carries a G418 resistance cassette, pRetroNeoLuc carries G418 resistance and a luciferase expression cassette, pRetroNeoLucOE carries G418 resistance, luciferase and an oriP/EBNA-1 cassette and pRetroNeoOE carries G418 resistance and oriP/EBNA-1. These vectors can be efficiently retrofitted onto BACs without rearrangement of the BAC clone. The luciferase cassette is expressed efficiently from the retrofitting plasmids and from retrofitted BACs after transient transfection of B16F10 cells in tissue culture and after electroporation into muscles of BALB/c mice in vivo. We also show that a BAC carrying GFP, oriP and EBNA-1 can be transfected into B16F10 cells with Lipofectamine 2000 and can be rescued intact after 5 weeks. CONCLUSION: The pRetro vectors allow efficient retrofitting of BACs with G418 resistance, luciferase and/or oriP/EBNA-1 using in vivo expression of Cre. The luciferase reporter gene is expressed after transient transfection of retrofitted BACs into cells in tissue culture and after electroporation into mouse muscle in vivo. OriP/EBNA-1 allows stable maintenance of a 150-kb BAC without rearrangement for at least 5 weeks.

Tumor-targeted gene delivery of tumor necrosis factor-alpha induces tumor necrosis and tumor regression without systemic toxicity.

We have recently developed surface-shielded transferrin-polyethylenimine (Tf-PEI)/DNA delivery systems that target reporter gene expression to distant tumors after systemic application. In the present study, we used surface-shielded Tf-PEI/DNA complexes for delivering the gene for a highly potent cytokine, tumor necrosis factor-alpha (TNFalpha). TNFalpha is known for its ability to induce hemorrhagic tumor necrosis and tumor regression. However, the therapeutic application of TNFalpha is hampered by its high systemic toxicity dictating the need to target TNFalpha activity to the tumor. Systemic application of surface-shielded Tf-PEI complexes with the TNFalpha gene resulted in preferential expression of TNFalpha in the tumor without detectable TNFalpha serum levels, in contrast to the application of nontargeted complexes. Tumor-targeted TNFalpha gene delivery induced pronounced hemorrhagic tumor necrosis and inhibition of tumor growth in three murine tumor models of different tissue origins, Neuro2a neuroblastoma, MethA fibrosarcoma, and M-3 melanoma, with complete tumor regressions observed in the MethA model. No systemic TNF-related toxicity was observed due to the localization of the TNFalpha activity to the tumor. Targeted gene therapy may be an attractive strategy applicable to highly active, yet toxic, molecules such as TNFalpha.