Marina Mata

Deletion of multiple immediate-early genes from herpes simplex virus reduces cytotoxicity and permits long-term gene expression in neurons.

Herpes simplex virus type 1 (HSV-1) has many attractive features that suggest its utility for gene transfer to neurons. However, viral cytotoxicity and transient transgene expression limit practical applications even in the absence of viral replication. Mutant viruses deleted for the immediate–early (IE) gene, ICP4, an essential transcriptional transactivator, are toxic to many cell types in culture in which only the remaining IE genes are expressed. In order to test directly the toxicity of other IE gene products in neurons and develop a mutant background capable of long-term transgene expression, we generated mutants deleted for multiple IE genes in various combinations and tested their relative cytotoxicity in 9L rat gliosarcoma cells, Vero monkey kidney cells, and primary rat cortical and dorsal root neurons in culture. Viral mutants deleted simultaneously for the IE genes encoding ICP4, ICP22 and ICP27 showed substantially reduced cytotoxicity compared with viruses deleted for ICP4 alone or ICP4 in combination with either ICP22, ICP27 or ICP47. Infection of neurons in culture with these triple IE deletion mutants substantially enhanced cell survival and permitted transgene expression for over 21 days. Such mutants may prove useful for efficient gene transfer and extended transgene expression in neurons in vitro and in vivo.

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.

Transgene-mediated enkephalin release enhances the effect of morphine and evades tolerance to produce a sustained antiallodynic effect in neuropathic pain

&NA; We examined the pharmacologic characteristics of herpes simplex virus (HSV) vector‐mediated expression of proenkephalin in the dorsal root ganglion in a rodent model of neuropathic pain. We found that: (i) vector‐mediated enkephalin produced an antiallodynic effect that was reversed by naloxone; (ii) vector‐mediated enkephalin production in animals with spinal nerve ligation prevented the induction of c‐fos expression in second order sensory neurons in the dorsal horn of spinal cord; (iii) the effect of vector‐mediated enkephalin enhanced the effect of morphine, reducing the ED50 of morphine 10‐fold; (iv) animals did not develop tolerance to the continued production of vector‐mediated enkephalin over a period of several weeks; and, (v) vector transduction continued to provide an analgesic effect despite the induction of tolerance to morphine. This is the first demonstration of gene transfer to provide an analgesic effect in neuropathic pain. The pharmacologic analysis demonstrates that transgene‐mediated expression and local release of opioid peptides produce some effects that are distinct from peptide analogues delivered pharmacologically.

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.

HSV-mediated expression of interleukin-4 in dorsal root ganglion neurons reduces neuropathic pain

BackgroundTo examine the role of inflammatory mediators in neuropathic pain, we used a replication-defective genomic herpes simplex virus (HSV)-based vector containing the coding sequence for the anti-inflammatory peptide interleukin (IL)-4 under the transcriptional control of the HSV ICP4 immediate early promoter, vector S4IL4, to express IL-4 in dorsal root ganglion (DRG) neurons in vivo.ResultsSubcutaneous inoculation of S4IL4 in the foot transduced lumbar DRG to produce IL-4. Transgene-mediated expression of IL-4 did not alter thermal latency or tactile threshold in normal animals, but inoculation of S4IL4 1 week after spinal nerve ligation (SNL) reduced mechanical allodynia and reversed thermal hyperalgesia resulting from SNL. Inoculation of S4IL4 1 week before SNL delayed the development of thermal hyperalgesia and tactile allodynia, but did not prevent the ultimate development of these manifestations of neuropathic pain. S4IL4 inoculation suppressed non-noxious-induced expression of c-Fos immunoreactivity in dorsal horn of spinal cord and reversed the upregulation of spinal IL-1β, PGE2, and phosphorylated-p38 MAP kinase, characteristic of neuropathic pain.ConclusionHSV-mediated expression of IL-4 effectively reduces the behavioral manifestations of neuropathic pain, and reverses some of the biochemical and histologic correlates of neuropathic pain at the spinal level.

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.

Tumor necrosis factor-α contributes to below-level neuropathic pain after spinal cord injury

Our objective was to elucidate the mechanisms responsible for below‐level pain after partial spinal cord injury (SCI).

THE MIXED LINEAGE KINASE DLK UTILIZES MKK7 AND NOT MKK4 AS SUBSTRATE

Mixed lineage kinases DLK (dual leucine zipper-bearing kinase) and MLK3 have been proposed to function as mitogen-activated protein kinase kinase kinases in pathways leading to stress-activated protein kinase/c-Jun NH2-terminal kinase activation. Differences in primary protein structure place these MLK (mixed lineage kinase) enzymes in separate subfamilies and suggest that they perform distinct functional roles. Both DLK and MLK3 associated with, phosphorylated, and activated MKK7 in vitro. Unlike MLK3, however, DLK did not phosphorylate or activate recombinant MKK4 in vitro. In confirmatory experiments performed in vivo, DLK both associated with and activated MKK7. The relative localization of endogenous DLK, MLK3, MKK4, and MKK7 was determined in cells of the nervous system. Distinct from MLK3, which was identified in non-neuronal cells, DLK and MKK7 were detected predominantly in neurons in sections of adult rat cortex by immunocytochemistry. Subcellular fractionation experiments of cerebral cortex identified DLK and MKK7 in similar nuclear and extranuclear subcellular compartments. Concordant with biochemical experiments, however, MKK4 occupied compartments distinct from that of DLK and MKK7. That DLK and MKK7 occupied subcellular compartments distinct from MKK4 was confirmed by immunocytochemistry in primary neuronal culture. The dissimilar cellular specificity of DLK and MLK3 and the specific substrate utilization and subcellular compartmentation of DLK suggest that specific mixed lineage kinases participate in unique signal transduction events.

IL-10 promotes neuronal survival following spinal cord injury

We have previously reported that the anti-inflammatory cytokine IL-10 induces a number of signaling cascades through the IL-10 receptor in spinal cord neurons in vitro to activate NF-kappaB transcription Bcl-2 and Bcl-x(L) and that, after exposure to glutamate IL-10, blocks cytochrome c release and caspase cleavage. In the current study we used a herpes simplex virus (HSV)-based vector to express IL-10 in spinal cord in vivo. Injection of the vector 30 minutes after lateral hemisection injury resulted in increased neuronal survival in the anterior quadrant of the spinal cord and improved motor function up to 6 weeks after injury, that correlated with translocation of p50 and p65 NF-kappaB to the nucleus and increased expression of Bcl-2 and Bcl-x(L) in anterior quadrant neurons. Inhibition of cytochrome c release and caspase 3 cleavage was seen in homogenates of injured spinal cord treated by the IL-10 vector. Taken together with in vitro studies that demonstrate direct neuroprotective effects of IL-10 acting through the neuronal IL-10 receptor, these results suggest that IL-10 may provide direct neuroprotective effects in spinal cord injury separate from and in addition to the known anti-inflammatory effects and point to the possibility that IL-10 delivery by gene transfer may be a useful adjunctive therapy for spinal cord injury.

Gene transfer of glutamic acid decarboxylase reduces neuropathic pain

We tested whether transfer of the gene coding for glutamic acid decarboxylase to dorsal root ganglion using a herpes simplex virus vector to achieve release of GABA in dorsal horn would attenuate nociception in this condition. Subcutaneous inoculation of a replication‐defective herpes simplex virus vector expressing glutamic acid decarboxylase (vector QHGAD67) 7 days after selective L5 spinal nerve ligation reversed mechanical allodynia and thermal hyperalgesia; the antiallodynic effect lasted 6 weeks and was reestablished by reinoculation. QHGAD67 inoculation also suppressed induction of c‐Fos and phosphorylated extracellular signal–regulated kinase 1 and 2 in the spinal cord. Ann Neurol 2005;57:914–918