Defective herpes simplex virus vectors for gene transfer to cells of the central nervous system: The study of glutamic acid decarboxylase

Defective herpes simplex virus (HSV) vectors have been used to efficiently express foreign gene products in the CNS in vitro and in vivo, but have been limited by insert size, difficulty in generation, and toxicity. A defective HSV vector was generated containing two independent transcription units, termed a double-cassette vector, which was able to express two marker enzymes in neurons in vitro and in vivo. A double-cassette vector encoding E. coli guanine phosphoribosyl transferase (GPT) was packaged with increased efficiency by a neuroattenuated HSV mutant in the presence of mycophenolic acid. Resultant vectors were of reduced toxicity in neuronal cultures, and were useful in tracing neuronal projection pathways in rat brains in vivo.; Double-cassette defective HSV vectors were generated to express increased levels of either isoform of glutamic acid decarboxylase (GAD65 or GAD67) and the marker enzyme {dollar}beta{dollar}-galactosidase in primary cultures of neurons and astrocytes. Novel expression of either GAD isoform resulted in increased levels of intracellular GABA in vector infected cerebellar granule cell (CGC) cultures. Vector derived GAD expression in 6 day old CGCs resulted in only Ca{dollar}sp{lcub}++{rcub}{dollar}-independent GABA release, induced by glutamate or veratridine, while vector infected 10 day old cultures exhibited both Ca{dollar}sp{lcub}++{rcub}{dollar}-dependent, induced by K+ depolarization, and Ca{dollar}sp{lcub}++{rcub}{dollar}-independent GABA release. Similar levels and profiles of GABA release were detected in GAD65 and GAD67 vector infected neurons. In cerebral type 1 astrocytes, GABA levels were two-fold higher in GAD67 than GAD65 vector infected cells. GABA was released constitutively from GAD67 vector infected astrocytes, but no stimulated release was detected. {dollar}beta{dollar}-galactosidase vector infected or uninfected astrocytes expressed no GAD and contained no detectable GABA. These data demonstrate that defective HSV vectors encoding GAD65 or GAD67 are able to increase the synthesis and release of GABA in CNS cultures. Neurologic disorders that result from decreased GABA levels are potential targets for gene therapy using these vectors.