Engineered HSV vector achieves safe long-term transgene expression in the central nervous system

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 nonneuronal 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.Neurological diseases have an enormous medical and social impact and are without effective treatments. Gene therapies provide a potential means of genetic intervention but, in most cases, treatment requires the availability of high capacity non-integrating gene transfer tools that provide durable, regulated expression of large or multi-gene cassettes in specific neuronal cell subpopulations 1 . In addition, many diseases are autosomal dominant requiring either knock out or knock down of the mutant gene product and either repair or complementing gene addition. We suggest that HSV vectors provide the best opportunity to meet these complex demands since HSV vectors can accommodate large inserts and are well adapted for life-long persistence in neurons as non-integrating, circular episomes.We recently created a safe, high capacity recombinant HSV vector that is highly stable, unable to replicate, but capable of persisting in human primary cells without evidence of cytotoxicity at very high multiplicities of infection 2 . This vector expresses extremely low levels of viral lytic genes that can be detected only by RT-PCR, a result of functional deletion of all immediate early (IE) genes. The reiterated joint region separating the unique long (U L ) and unique short (U S ) genome components is also removed, providing a total of ~25 kb of space for transgene insertion. The challenge has been to further engineer this vector to provide durable transgene expression in both neuronal and non-neuronal cell types since transgenes are rapidly silenced in the absence of expression of one of the IE genes, ICP0, due to heterochromatin formation 3, 4 . We have recently overcome genome silencing through the application of moveable geneti...

Cellular Antisilencing Elements Support Transgene Expression from Herpes Simplex Virus Vectors in the Absence of Immediate Early Gene Expression

“…The suggestion that rDRGs in culture are relatively supportive of gene expression from highly defective HSV backbones agrees with our previous observation that an IE gene-inactive vector containing CAG-EGFP in the UL3-UL4 intergenic region produced substantially more EGFP-positive cells per input gc in rDRGs at 3 dpi than in nonneuronal cells (7). In contrast, we did not detect EGFP expression in rat hippocampus from the UL3-UL4 locus in previous work (31). Likewise, while CAG-EGFP in the LAT locus was active in cultured DRG neurons (reference 7 and this study), hippocampal expression was not observed (but see below).…”

“…In contrast, very little, if any, EGFP signal was detected at any time point. These results indicated that A2UCOE can enhance transient, independent expression of a second transgene in the central nervous system to complement the previously described durable expression of a first transgene positioned in the deleted ICP4 locus (31,32). It remains to be determined what mechanisms can account for the rapid or immediate silencing of the LAT-based EGFP cassette of these vectors in mouse hippocampus.…”

“…We hypothesize that the unexpected long-term reporter gene expression from the terminal ICP4 locus of our defective vectors in central nervous system neurons in vivo (Fig. 9C) (31,32) and the simultaneous lack of reporter expression from the LAT locus reported here are consequences of the disruption of CTCF interactions and the loss of LAT and ICP0 functions. However, it is uncertain whether A2UCOE activity is influenced by the same mechanisms.…”

New Tools for Epilepsy Therapy

“…In addition, at variance with the previous results mentioned above, the deletion of ICP0 does not prevent transgene expression from a specific locus, ICP4. When the expression cassette was inserted in ICP4, a robust, sustained and neuron-specific transgene expression was observed in several rat brain regions, and this was clearly detectable for at least 6 months after vector injection with no detectable sign of neuronal toxicity or immune response (Verlengia et al, 2017 ). A further refinement of this vector was obtained through the additional removal of the virion host shutoff (vhs) gene (Figure 2A ), a strategy that further increased both the levels of transgene expression in neurons and the safety profile of the vector (Miyagawa et al, 2017 ).…”

Herpes Simplex Virus Vectors for Gene Transfer to the Central Nervous System

“…Our studies demonstrated prolonged, non-toxic transgene expression from a foreign promoter inserted directly downstream from the LAT promoter/enhancer region in non-neuronal cells. Interestingly, although expression from the LAT region was transient in rDRG cultures and largely undetectable in mouse hippocampus [ 104 , 105 ], reporter expression from the deleted ICP4 locus without adjacent LAT regulatory elements persisted for months in mouse hippocampus [ 106 ]. Furthermore, we observed that this expression could be further enhanced by deletion of the virion host shutoff gene (vhs or UL41) [ 104 ].…”