129. Development of Vectors Utilizing Strong Neuronal Cell Type Specific Promoters for High Level Expression of Beta Glucuronidase in the Central Nervous System

Abstract

Recombinant adeno-associated viruses (AAV) are promising vectors for delivery to the central nervous system, e.g. in lysosomal storage disorder such as GUSB-deficient MPS VII. The cellular tropism of AAV gene delivery varies depending on the serotype used. In lysosomal enzyme deficient cells, the transferred normal enzyme is secreted and can be endocytosed by adjacent cells (sphere of correction). Although treatments have been shown to work in mouse brains, enzyme delivery will need to be significantly increased to produce the same effect in larger brains such as humans. Our laboratory has shown that the level of GUSB expression and secretion is proportional to increased transcription from vectors. Various promoters have been known to modulate different levels of transcription in the CNS. If vector-mediated gene delivery could produce higher levels of enzyme secretion from a minimal number of injections, the sphere of correction could be increased for enzyme diffusion, axonal transport, and migrating cross-correcting cells. We have designed a eukaryotic minigene vector driven by the natural promoter. The vector contains the first two introns in addition to all the exon sequences of hGUSB. Mouse brains were stereotactically injected into the left midbrain region with 2.5 |[times]| 1012 viral genome equivalents per each of four injection sites. The major structures injected were the cortex, striatum, hippocampus and thalamus. The pattern of transduction at 2 months post-injection was assessed by in situ hybridization which shows similar patterns of transduction to a control vector without introns in the cortex, striatum, hippocampus and thalamus. Quantitative measurements for transcription are in progress. The enzyme positive cells were detected at the sites of injection and further extended beyond the regions of transduced cells when rostral and caudal sections were examined. GUSB enzyme in the hippocampus was also detected in the contralateral region of the hippocampus. Experiments are also in progress to test the neuron-specific enolase (NSE) and latency associated (LAT) promoters. The NSE promoter has been known to confer high levels of gene expression when used in recombinant AAV2 vectors in transduced neurons, and has been found to express therapeutic levels of enzyme in other MPS models. In herpesviruses, the LAT promoter is the only viral gene sequence expressed in life-long latent infections in post-mitotic neurons. It has also been shown that significantly more GUSB activity is detected when the GUSB cDNA is inserted near the LAT promoter as opposed to other lower transcriptional level configurations. Evaluation of the effects of the NSE promoter and HSV-LAT promoter in the transcriptional high level configuration for GUSB are currently in progress for comparison with the hGUSB minigene and cDNA vector controls.