Antibiotic-mediated chemoprotection enhances adaptation of E. coli PNP for herpes simplex virus-based glioma therapy.

The E. coli PNP suicide gene sensitizes solid tumors to nucleoside prodrugs, such as 6-methylpurine-2'-deoxyriboside (MeP-dR). In this study using lentiviral, MuLv, and HSV-based gene transfer, we quantified thresholds for inhibition of tumor growth and bystander killing by E. coli PNP and tested the role of intestinal flora in this process. Regressions of human glioma tumors following retroviral transduction exhibited dose dependence on both the level of PNP expression and the dose of MeP-dR administered, including strong tumor inhibition when 90-99% bystander cells comprised the tumor mass. A replication competent, non-neurovirulent herpes simplex virus (HSV) deficient in both copies of the gamma-1 34.5 gene was next engineered to express E. coli PNP under the egr-1 promoter (HSV-PNP). HSV-PNP injected intratumorally (17 million pfu/0.05 ml) in nude mice bearing 300 mg human glioma flank tumors produced a delay in tumor growth (approximately 24 days delay to one doubling). MeP-dR treatment after antibiotic therapy (to eliminate enteric flora encoding PNP enzymes) resulted in antitumor enhancement, with arrest of tumor growth (delay to doubling >50 days). Bystander killing of the magnitude described here has been difficult to accomplish with other suicide genes, such as HSV-tk or cytosine deaminase. The results establish a model for applying E. coli PNP to HSV treatment of glioma.

Membrane transplantation to correct integral membrane protein defects.

In this report we show that the tendency of certain viruses to carry host membrane proteins in their envelopes can be harnessed for transplantation of small patches of plasma membrane, including fully functional, polytopic ion channel proteins and their regulatory binding partners. As a stringent model we tested the topologically complex epithelial ion channel CFTR. Initially an attenuated vaccinia virus was found capable of transferring CFTR in a properly folded, functional and regulatable form to CFTR negative cells. Next we generated viruslike particles (VLPs) composed of retroviral structural proteins that assemble and bud at the host cell plasma membrane. These particles were also shown to mediate functional ion channel transfer. By testing the capacity of complex membrane proteins to incorporate into viral envelopes these experiments provide new insight into the permissiveness of viral envelopment, including the ability of incorporated proteins to retain function and repair defects at the cell surface, and serve as a platform for studies of ion channel and membrane protein biochemistry.