157. Quantitation of Long-Term Gene Expression from the HSV-1 Latency-Associated Promoter in the Mouse Brain Following Intracranial Injection

Abstract

Top of pageAbstract Introduction: Inability of HSV-1 vectors to sustain long-term expression in the CNS has previously been reported using a variety of different promoters and vector strains (reviewed in Lilley et al, 2001). Vectors using the LAT promoter generally have superior levels of latent-phase gene expression, although even these vectors fail to maintain stable CNS transduction. To date, long-term transduction studies of HSV-1 vectors have suffered from either inadequate quantitation of gene expression (using only histological techniques) or a lack of truly long-term time points. We have performed intracranial injections into the adult mouse striatum with an HSV-1 vector (1716-LAT-hGUSB) that expresses the human beta-glucuronidase (hGUSB) reporter gene under the control of the endogenous viral LAT promoter. We have quantified long-term transduction at both acute and latent time points (up to 52 weeks) at the level of reporter enzyme activity as well as the presence of RNA transcripts. Vector genome maintenance was also measured. Results: Here we show a neuro-attenuated HSV-1 vector (based on 1716, an ICP34.5 mutant) achieves widespread transduction after a single, site-specific intracranial injection. An analysis of long-term expression revealed the following: A histochemical stain reveals a decrease in hGUSB-positive cells over time as measured by digital analysis of the GUSB-positive area. Significant levels of hGUSB are detected in midbrain homogenates by enzyme activity assays at 2 and 13 weeks but not at 26 and 52 weeks. The vector genome is stably maintained in the midbrain throughout latency as measured by quantitative PCR. Further, genome levels are equivalent for both 1716-LAT-hGUSB and 1716 at all time points. In situ hybridization towards LAT-specific transcripts is able to detect transcripts at 2 and 8 weeks post-injection, but not at 26 and 52 weeks. LAT promoter activity during both acute and latent infection was quantified using Q-RT-PCR (primers specific to the LAT intron). We found that LAT transcript levels peaked around 2 weeks post-injection, but were found at significantly lower, albeit stable levels at 13, 26, and 52 weeks post-injection. No significant differences in LAT transcripts were found at 13, 26, and 52 weeks when comparing 1716-LAT-hGUSB to 1716. Conclusions: An HSV-1 ICP34.5 mutant is unable to produce stable long-term reporter enzyme activity in the mouse brain when driven by the LAT promoter. Vector genome is stably maintained throughout latency (2 through 52 weeks). LAT transcript levels in the brain are stable from 13 through 52 weeks by Q-RT-PCR but are at levels undetectable by in situ hybridization. A gene insertion into exon 1 of the LAT gene is not responsible for the lack of long-term LAT promoter activity as the parental virus produces similar LAT transcript levels at latent time points.