Long-term expression driven by herpes simplex virus type-1 amplicons may fail due to eventual degradation or extrusion of introduced transgenes.

In gene therapy applications employing herpes simplex virus amplicon-based vectors, a prevailing problem is the down-regulation of transgene expression over time. We have applied a combined immunocytochemistry and fluorescent in situ hybridization method to determine whether down-regulation of transgene expression at the single-cell level correlates with loss of vector DNA from the host cell nucleus. Utilizing separate fluorescent labels (i.e., rhodamine, fluorescein, and 4',6'-diamidino-2-phenlindole), we were able to simultaneously detect transgenes, their products, and their locations relative to the nuclear compartment of a single cell. Detection of the reporter gene lacZ and its encoded protein beta-galactosidase (beta-gal) was accomplished in in vivo experiments of the dentate gyrus of rats. A time course study of the expression of the transgene post-stereotactic microinfusion up to 60 days was made. Expression reached maximal levels within 12-24 h after infection, and lacZ presence was reduced to less than 3% of its maximal levels within 36 h after infection. In comparing days 1 and 60 post-stereotactic microinfusion, only one-fifth of the original DNA was observed in the area of a 100-mm radius around the site of microinfusion at day 60. Moreover, by comparing the locations of the reporter gene in cells that expressed the encoded protein versus those that did not, we found that introduced transgenes were preferentially localized in the nuclear periphery of down-regulated host cells, compared to expressing host cells. These results suggest that nuclear compartmentalization may play a role in the down-regulation of our reporter gene by means of peripheralization, extrusion, and/or degradation.

Neuroprotective potential of a viral vector system induced by a neurological insult.

Gene transfer into neurons via viral vectors for protection against acute necrotic insults has generated considerable interest. Most studies have used constitutive vector systems, limiting the ability to control transgene expression in a dose-dependent, time-dependent, or reversible manner. We have constructed defective herpes simplex virus vectors designed to be induced by necrotic neurological insults themselves. Such vectors contain a synthetic glucocorticoid-responsive promoter, taking advantage of the almost uniquely high levels of glucocorticoids-adrenal stress steroids-secreted in response to such insults. We observed dose-responsive and steroid-specific induction by endogenous and synthetic glucocorticoids in hippocampal cultures. Induction was likely to be rapid enough to allow transgenic manipulation of relatively early steps in the cascade of necrotic neuron death. The protective potential of such a vector was tested by inclusion of a neuroprotective transgene (the Glut-1 glucose transporter). Induction of this vector by glucocorticoids decreased glutamatergic excitotoxicity in culture. Finally, both exogenous glucocorticoids and excitotoxic seizures induced reporter gene expression driven from a glucocorticoid-responsive herpes simplex virus vector in the hippocampus in vivo.

Myocardial necrosis following general anesthesia in hemoglobin SC disease.

A 4-year-old girl with hemoglobin SC disease died following general anesthesia. Autopsy showed widespread intravascular sickling; staining with hematoxylin-basic fuchsin-picric acid demonstrated newly developed massive myocardial necrosis, a rarely documented finding. Anesthesia may produce conditions that provoke "crises" in patients with sickle hemoglobinopathies. Preoperative identification of sickle states and careful attention to hydration and oxygenation may minimize anesthetic risks in these patients.