✓ Because accurate regulation of toxin gene expression is critical for safe and effective gene therapy applications, the authors have examined the regulation of diphtheria toxin A (DTA) fragment expression in human glioma cell lines using two transcriptional control systems derived from Escherichia coli: the tetracycline (Tet) system and the lactose (Lac) system. The Tet system includes a tetracycline-controlled transactivator (tTA), a tTA-responsive minimum human cytomegalovirus (hCMV) promoter controlling the expression of the DTA gene, and tetracycline as an allosteric inhibitor. The Lac system includes the lac repressor (lacR), a lacR-regulated Rous sarcoma virus—long terminal repeat (RSV-LTR) promoter controlling the expression of the DTA gene, and isopropyl-thio-β-d-galactoside (IPTG) as an allosteric inducer. Expression plasmids encoding either tTA or lacR were transfected into U-87MG and U-343MG glioma cells along with the responsive DTA plasmid. Cell killing was monitored by the ability of the toxin to abolish protein synthesis and was quantitated using a luciferase reporter gene. In the Tet system, tumor cell killing could be regulated by tetracycline up to 120-fold. In contrast, only a twofold IPTG-dependent regulation was obtained using the Lac system because of an incomplete repression of DTA expression in the uninduced state. Replacement of the RSV-LTR promoter with the heavy metal—inducible mouse metallothionein-1 promoter in the lacR-responsive unit, as well as the generation of a clonal glioma cell line expressing lacR, did not significantly enhance regulation of DTA in the Lac system. In conclusion, this study demonstrates that the Tet system is of potential use in gene therapy applications in which regulated expression of a therapeutic gene is an important issue.
Werner Paulus, Inge Baur, Daniel M. Oberer, Xandra O. Breakefield and Steven A. Reeves
Yoshiaki Takamiya, M. Priscilla Short, Frederick L. Moolten, Christina Fleet, Toshihiro Mineta, Xandra O. Breakefield and Robert L. Martuza
✓ Recent research using rodent models of central nervous system gliomas indicates that a combination of gene transfer and drug treatment may be successful in killing tumor cells. In the present study, a mouse fibroblast-derived packaging cell line, psi 2, which releases a replication-defective retrovirus vector bearing the herpes simplex virus type 1 (HSV)-thymidine kinase (TK) gene, was grown with rat C6 tumor cells in the presence and absence of wild type Moloney murine leukemia virus (MoMLV). Consequently, tumor cells became sensitive to ganciclovir, which is selectively converted to a toxic nucleotide analog by HSV-TK. This killing effect was more effective in the presence than in the absence of wild type retrovirus both in culture and in subcutaneous tumors in nude mice. Tumors regressed in vivo and failed to regrow over a subsequent 10-day observation period after combined treatment with packaging cells, wild type MoMLV, and ganciclovir. This killing effect may be augmented by the ability of the helper retrovirus to package the vector in tumor cells and thus extend delivery of the HSV-TK gene to more tumor cells. This represents significant improvement in tumor therapy in this model system as compared with helper-free systems previously reported by the authors and others. Although additional improvements in the therapy can be envisioned, this approach may prove useful in combination with current modes of therapy for these insidious and lethal tumors.
David M. Frim, M. Priscilla Short, William S. Rosenberg, Joseph Simpson, Xandra O. Breakefield and Ole Isacson
✓ Neurotrophic factors, such as nerve growth factor (NGF), in addition to their role in neuronal development, have protective effects on neuronal survival. Intracerebral implantation of cells genetically altered to secrete high levels of NGF is also found to promote neuronal survival in experimental lesioning models of the brain. The range of activity for such biological delivery systems has not yet been well described either spatially or temporally. Therefore, the authors chose to study the local and distant protective effects of an NGF-secreting rat fibroblast cell line implanted in an excitotoxic lesion model of Huntington's disease. They found that preimplantion of NGF-secreting fibroblasts placed within the corpus callosum reduced the maximum crosssectional area of a subsequent excitotoxic lesion in the ipsilateral striatum by 80% when compared to the effects of a non-NGF-secreting fibroblast graft, and by 83% when compared to excitotoxic lesions in ungrafted animals (p < 0.003). However, NGF-secreting cells placed in the contralateral corpus callosum failed to affect striatal lesion size significantly when compared to contralateral or ipsilateral non-NGF-secreting cell implants. Of note, fibroblasts were clearly visible within the graft site at 7 and 18 days after implantation; however, few cells within the grafts stained positively for NGF peptide or for the messenger ribonucleic acid (mRNA) encoding the transfected NGF gene-construct at either time point. These results show that biological delivery systems for NGF appear to have a profound but local effect on neuronal excitotoxicity, which will necessitate careful neurosurgical placement for maximum effect. Furthermore, the ability of this genetically altered cell line to synthesize NGF mRNA and peptide appears to decrease spontaneously in vivo, a characteristic that will need to be addressed before this method of biological delivery can be utilized as a treatment for chronic degenerative diseases.