James R. Goss

Antinociceptive effect of a genomic herpes simplex virus-based vector expressing human proenkephalin in rat dorsal root ganglion

Endogenous opiate peptides acting pre- and post-synaptically in the dorsal horn of spinal cord inhibit transmission of nociceptive stimuli. We transfected neurons of the dorsal root ganglion in vivo by footpad inoculation with 30 μl (3 × 107 p.f.u.) of a replication-incompetent (ICP4-deleted) herpes simplex virus (HSV) vector with a cassette containing a portion of the human proenkephalin gene coding for 5 met- and 1 leu-enkephalin molecules under the control of the human cytomegalovirus immediate–early promoter (HCMV IEp) inserted in the HSV thymidine kinase (tk) locus. Vector-directed expression of enkephalin produced a significant antinociceptive effect measured by the formalin footpad test, that was most prominent in the delayed (‘tonic’) phase 20–70 min after the administration of formalin. The magnitude of the antinociceptive effect diminished over 4 weeks after transduction, but reinoculation of the vector reestablished the analgesic effect, without evidence for the development of tolerance. The antinociceptive effect was blocked completely by intrathecal naltrexone. These results suggest that gene therapy with an enkephalin-producing herpes-based vector may prove useful in the treatment of pain.

Astrocytes Are the Major Source of Nerve Growth Factor Upregulation Following Traumatic Brain Injury in the Rat

Previous studies from our group have demonstrated an upregulation in nerve growth factor (NGF) RNA and protein in the cortex 24 h following traumatic brain injury (TBI) in a rat model. This increase in NGF is suppressed if rats are subjected to 4 h of whole-body hypothermia following TBI. In the present study we used in situ hybridization to extend our initial RNA gel-blot (Northern) hybridization findings by demonstrating that NGF RNA is increased in the cortex following TBI and that hypothermia diminishes this response. Further, by combining in situ hybridization with immunocytochemistry for glial fibrillary acidic protein we demonstrate that astrocytes are the major cellular source for the upregulation in NGF and that this upregulation can be observed in the hippocampus as early as 3 h posttrauma. The predominantly astrocytic origin suggests that the NGF upregulation is not related primarily to cholinotrophic activities. We hypothesize that its function is to stimulate upregulation of antioxidant enzymes, as part of an injury-induced cascade, and that supplementation of NGF or antioxidants may be warranted in hypothermic therapies for head injury.

Upregulation of nerve growth factor following cortical trauma

As part of the inflammatory response to brain injury, CSF and tissue levels of interleukin-1 beta (IL-1 beta) are elevated after trauma. This elevation in IL-1 beta initiates a cascade of events among which may be an upregulation in nerve growth factor (NGF) in brain tissue. We infused IL-1 beta into the ventricle of adult rats and found a two- to fourfold increase in NGF in the cerebral cortex, hippocampus, and cerebellum, suggesting that IL-1 beta induced in vivo may also increase NGF in the brain. To test this hypothesis we utilized two models of traumatic brain injury (TBI) in the rat and examined NGF protein and RNA in the cortex over a period of several days. Both weight drop and controlled cortical contusion models of CNS trauma demonstrated large and significant increases in NGF protein in the cortex. NGF RNA was assessed in the controlled cortical contusion model and increased approximately fivefold by 1 day post-trauma. The remarkable elevation of NGF observed following TBI suggests that its role in response to injury may be other than as a target-derived growth substance. We hypothesize that the elevation of NGF in trauma induces upregulation of enzymes which suppress free-radical formation after injury.

Gene therapy for pain: Results of a phase I clinical trial

Preclinical evidence indicates that gene transfer to the dorsal root ganglion using replication‐defective herpes simplex virus (HSV)‐based vectors can reduce pain‐related behavior in animal models of pain. This clinical trial was carried out to assess the safety and explore the potential efficacy of this approach in humans.

Herpes vector-mediated expression of proenkephalin reduces bone cancer pain.

We examined whether a herpes simplex virus vector that expresses human proenkephalin could be used to attenuate nociception in a model of bone cancer pain in mice. Osteolytic sarcoma cells were implanted into the medullary space of the right femur, followed by a subcutaneous inoculation of a replication‐defective herpes simplex virus vector expressing human proenkephalin (vector SHPE) or a lacZ‐expressing control vector (vector SHZ). SHPE‐inoculated mice demonstrated a significant, naltrexone‐reversible decrease in pain‐related behavior assessed during open‐field motor activity. These results suggest that gene transfer with an enkephalin‐expressing vector may be used to treat pain resulting from cancer in bone.

Bcl-2 and GDNF delivered by HSV-mediated gene transfer act additively to protect dopaminergic neurons from 6-OHDA-induced degeneration.

Previous studies have demonstrated that either the neurotrophin glial-derived neurotrophic factor (GDNF) or the antiapoptotic peptide Bcl-2 delivered into striatum by a viral vector protects dopaminergic neurons of the substantia nigra in vivo from degeneration induced by the administration of the neurotoxin 6-hydroxydopamine (6-OHDA). In this study we used recombinant, replication-incompetent, genomic herpes simplex virus-based vectors to deliver the genes coding for Bcl-2 and GDNF into rat substantia nigra (SN) 1 week prior to 6-OHDA injection into the striatum. Vector-mediated expression of either Bcl-2 or GDNF alone each resulted in a doubling in cell survival as measured by retrograde labeling with fluorogold (FG) and a 50% increase in tyrosine hydroxylase-immunoreactive (TH-IR) neurons in the lesioned SN compared to the unlesioned side. Gene transfer of Bcl-2 and GDNF were equivalent in this effect. Coadministration of the Bcl-2-expressing vector with the GDNF-expressing vector improved the survival of lesioned SN neurons as measured by FG labeling by 33% and by the expression of TH-IR by 15%. These results suggest that the two factors delivered together act in an additive fashion to improve DA cell survival in the face of 6-OHDA toxicity.

Herpes simplex virus RNAi and neprilysin gene transfer vectors reduce accumulation of Alzheimer's disease-related amyloid- β peptide in vivo

Accumulation of insoluble aggregates of amyloid-β peptide (Aβ), a cleavage product of amyloid precursor protein (APP), is thought to be central to the pathogenesis of Alzheimers disease (AD). Consequently, downregulation of APP, or enhanced clearance of Aβ, represent possible therapeutic strategies for AD. We generated replication-defective herpes simplex virus (HSV) vectors that inhibit Aβ accumulation, both in vitro and in vivo. In cell culture, HSV vectors expressing either (i) short hairpin RNA directed to the APP transcript (HSV-APP/shRNA), or (ii) neprilysin, an endopeptidase that degrades Aβ (HSV-neprilysin), substantially inhibited accumulation of Aβ. To determine whether these vectors showed similar activity in vivo, we developed a novel mouse model, in which overexpression of a mutant form of APP in the hippocampus, using a lentiviral vector (LV-APPSw), resulted in rapid Aβ accumulation. Co-inoculation of LV-APPSw with each of the HSV vectors showed that either HSV-APP/shRNA or HSV-neprilysin inhibited Aβ accumulation in this model, whereas an HSV control vector did not. These studies demonstrate the utility of HSV vectors for reducing Aβ accumulation in the brain, thus providing useful tools to clarify the role of Aβ in AD that may facilitate the development of novel therapies for this important disease.

The antioxidant enzymes glutathione peroxidase and catalase increase following traumatic brain injury in the rat

The inflammatory response following mechanical brain injury is characterized by an increase in the cytokine interleukin-1 beta (IL-1 beta) followed by a large elevation in the neurotrophin, nerve growth factor (NGF). The substantial upregulation in NGF observed in our previous studies suggests that it may have functions in addition to that of a target-derived neuronal support mechanism. We hypothesize that NGF is a mediator of oxidative homeostasis, by inducing the production of oxygen-free radical scavengers in brain tissue following injury. We tested this hypothesis by measuring the activity of the antioxidant enzymes, glutathione peroxidase (GSH-Px), and catalase in cortical brain tissue following experimentally induced cortical contusion. We observed a twofold increase in GSH-Px and a threefold increase in catalase activities in a time course which reflected the temporal increase in NGF observed following the same cortical contusion model. These findings support the hypothesis and illuminate an important reparative role for NGF following trauma.

In vivo gene therapy for pyridoxine-induced neuropathy by herpes simplex virus-mediated gene transfer of neurotrophin-3

Neurotrophic factors have been demonstrated to prevent the development of peripheral neuropathy in animal models, but the therapeutic use of these factors in human disease has been limited by the short serum half‐life and dose‐limiting side effects of these potent peptides. We used peripheral subcutaneous inoculation with a replication‐incompetent, genomic herpes simplex virus‐based vector containing the coding sequence for neurotrophin‐3 to transduce sensory neurons of the rat dorsal root ganglion in vivo, and found that expression of neurotrophin‐3 from the vector protected peripheral sensory axons from neuropathy induced by intoxication with pyridoxine assessed by electrophysiological (foot sensory response amplitude, and conduction velocity, and H‐wave), histological (nerve morphology and morphometry), and behavioral measures of proprioceptive function. In vivo gene transfer using herpes simplex virus vectors provides a unique option for treatment of diseases of the sensory peripheral nervous system.

Herpes Simplex Virus Type 1 ICP0 Protein Does Not Accumulate in the Nucleus of Primary Neurons in Culture

ABSTRACT Infected-cell protein 0 (ICP0), the product of the herpes simplex virus (HSV) immediate-early (IE) α0 gene, is a promiscuous transactivator of viral early (E) and late (L) gene expression. HSV mutants lacking ICP0 function are severely deficient in viral growth and protein synthesis, particularly at low multiplicities of infection. Early in the infectious process in vitro, ICP0 protein accumulates in distinct domains within the nucleus to form characteristic structures active in the transcription of viral genes. However, following infection of primary trigeminal ganglion cells in vitro with a recombinant HSV mutant that expresses only ICP0, we observed that ICP0 protein accumulated in the characteristic intranuclear distribution only in the nuclei of Schwann cells; neurons in the culture did not accumulate ICP0 despite expression of ICP0 RNA in those cells. The same phenomenon was observed in PC12 cells differentiated to assume a neuronal phenotype. In primary neurons in culture, the amount of ICP0 protein could be increased by pharmacologic inhibition of calcium-activated protease (calpain) activity or by inhibition of protein phosphatase 2B (calcineurin). The failure of ICP0 protein to accumulate in the nucleus of neurons suggests that one mechanism which may impair efficient replication of the virus in neurons, and thus favor the establishment of viral latency in those cells, may be found in the cell-specific processing of that IE gene product.