Abstract
Gene therapies for progressive degenerative disorders of the nervous system will require the long-term stable expression of therapeutic transgenes. As a step towards this goal, we have re-engineered a self-inactivating FIV-based vector to include features that are designed to optimize transgene expression in neurons, and to potentially increase vector safety. These features include elimination of the major splice donor site and mutation of the gag start site of translation. An internal promoter drives neural-specific transgene expression, and comprises an optimized TATA box and the initiator regulator from the Dntt gene coupled to enhancer sequences from the Syn1 gene. A shortened DNA central flap and the woodchuck hepatitis virus post-transcriptional regulatory element increase virus production and transgene expression. To reduce position effects the avian beta-globin locus HS4 insulator was inserted into the downstream LTR in place of the U3 region, and a CpG island element from the Aprt gene was added to the internal promoter. Using enhanced yellow fluorescent protein (EYFP) targeted to mitochondria as a marker gene, we have obtained robust EYFP expression in 84% of post-mitotic neurons from embryonic rat cortex in vitro. We are in the process of assessing the long-term stability of transgene expression in these neurons.
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