Abstract
Previously we reported a new series of highly defective herpes simplex virus type 1 (HSV-1) vectors that were functionally devoid of all viral immediately early (IE) genes, resulting in virtual absence of viral gene expression. Nevertheless, a reporter gene cassette inserted into the vector flanked by boundary elements from the viral latency locus showed high, persistent reporter gene activity in non-neuronal cells while an independent expression cassette inserted into a deleted ICP4 locus remained almost silent. In contrast to non-neuronal cells, we show here that the ICP4 locus cassette permitted robust reporter gene expression in a diversity of neurons following stereotactic injection of different rat brain regions; transgene expression in the hippocampus lasted up to 6 months and was essentially restricted to neurons. No evidence of neuronal cell toxicity or induction of inflammatory cell infiltrates was observed. An independent reporter gene cassette located in an intergenic region remained silent, indicating that the transgene promoter and/or insertion site are critical for sustained expression. These findings suggest the suitability of this vector for therapeutic intervention into diseases of the central nervous system that require the expression of large and/or multiple therapeutic transgenes.
Highlights
Neurological diseases have an enormous medical and social impact and are without effective treatments
The vector contains two expression cassettes for reporter genes: the enhanced green fluorescence protein gene inserted between the UL3 and UL4 genes under control of the CAG promoter, a strong synthetic promoter frequently used to drive high levels of transgene expression in mammalian cells; and the mCherry gene driven by the ubiquitin C (UbC) promoter inserted into the deleted ICP4 locus in the right terminal repeat
Bacterial artificial chromosome (BAC) genes allowing viral genome replication in bacteria, a chloramphenicol-resistance gene, and a β-galactosidase expression cassette are located between loxP sites in the UL37-UL38 intergenic region (Fig. 1)
Summary
Neurological diseases have an enormous medical and social impact and are without effective treatments. Transgene cassette replacement of the essential ICP4 IE gene provided transgene expression for at least 6 months in rat hippocampus and transgene activity was readily documented in a variety of other brain regions that included caudate putamen, substantia nigra, and cortex While this genomic region is normally transcriptionally silent in neurons, changes in vector genome organization and the use of a foreign promoter appeared to contribute to high transcriptional activity at this site. Vector inoculation at high doses in multiple brain regions did not induce neuronal damage or attract an inflammatory infiltrate This is the first HSV vector capable of long-term innocuous transgene expression in the brain and offers a new, highly engineered prototype vector that can be applied to brain gene therapies where large or multiple transgene cassettes are required to achieve a therapeutic outcome
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