Darren Wolfe

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.

Peripherally delivered glutamic acid decarboxylase gene therapy for spinal cord injury pain

Neuropathic pain after spinal cord injury (SCI) represents a difficult problem that is commonly refractory to conventional medical management. To determine if spinal release of gamma-amino butyric acid (GABA) could reduce below-level central neuropathic pain after SCI, we constructed a replication-incompetent herpes simplex virus (HSV)-based vector encoding one isoform of human glutamic acid decarboxylase (GAD67). Dorsal root ganglion (DRG) neurons transduced in vitro or in vivo by subcutaneous inoculation produced GAD and released GABA constitutively. T13 spinal cord hemisection resulted in central neuropathic pain manifested by mechanical allodynia and thermal hyperalgesia. Subcutaneous inoculation of the vector into both feet reduced both manifestations of below-level SCI pain; the vector-mediated effect was partially reversed by intrathecal bicuculline or phaclofen at doses that did not affect thresholds in normal or injured uninoculated animals. Vector-mediated GABA release attenuated the increase in spinal calcitonin gene-related peptide immunoreactivity caused by cord hemisection. These results suggest that HSV-mediated gene transfer to DRG could be used to treat below-level central neuropathic pain after incomplete SCI.

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.

Intra-articular delivery of a herpes simplex virus IL-1Ra gene vector reduces inflammation in a rabbit model of arthritis

To evaluate the use of HSV-based vectors for arthritis gene therapy we have constructed a first-generation, ICP4 deficient, replication defective herpes simplex virus (HSV) vector (S/0−) and a second-generation HSV vector derivative (T/0−) deficient for the immediate–early genes ICP4, 22 and 27, each carrying a soluble TNF receptor or IL-1 receptor antagonist transgene cassette. A rabbit synovial-fibroblast line in culture, infected by either vector enabled high-level expression of the transgene product. However, following a single intra-articular injection of the vectors into rabbit knee joints, only the second-generation, HSV T/0− vector expressed detectable levels of soluble TNFR in synovial fluid. Synovial lavage fluid from inoculated joints con- tained up to 12 ng/ml of soluble receptor that persisted at detectable, but reduced levels for at least 7 days. When tested in an experimental model of arthritis generated by intra-articular overexpression of interleukin-1β using retrovirus transduced synovial cells, the HSV T/0− vector expressing the interleukin-1 receptor antagonist was found to inhibit leukocytosis and synovitis significantly. The improved levels and duration of intra-articular transgene expression achieved via HSV-mediated gene delivery suggest that an HSV vector system could be used for therapeutic applications in patients with rheumatoid arthritis (RA) and other joint-related inflammatory diseases.

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

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.

HSV-mediated gene transfer of the glial cell-derived neurotrophic factor provides an antiallodynic effect on neuropathic pain

Neuropathic pain is a difficult clinical problem that is often refractory to medical management. Glial-derived neurotrophic factor (GDNF) administered intrathecally has been shown to prevent or reduce pain in an animal model of neuropathic pain, but cannot be delivered in the required doses to treat human pain. We have previously demonstrated that peripheral subcutaneous inoculation of a replication-incompetent herpes simplex virus (HSV)-based vector can be used to transduce neurons of the dorsal root ganglion. To examine whether HSV-mediated expression of GDNF could be used to ameliorate neuropathic pain, we constructed a replication-incompetent HSV vector expressing GDNF. Subcutaneous inoculation of the vector 1 week after spinal nerve ligation resulted in a continuous antiallodynic effect that was maintained for 3-4 weeks. Reinoculation of the vector reestablished the antiallodynic effect with a magnitude that was at least equivalent to the initial effect. Vector-mediated GDNF expression blocked the nonnoxious touch-induced increase in c-fos expression in dorsal horn characteristic of the painful state. Gene transfer to produce a trophic factor offers a novel approach to the treatment of neuropathic pain that may be appropriate for human therapy.

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.

HSV-mediated gene transfer of vascular endothelial growth factor to dorsal root ganglia prevents diabetic neuropathy

We examined the utility of herpes simplex virus (HSV) vector-mediated gene transfer of vascular endothelial growth factor (VEGF) in a mouse model of diabetic neuropathy. A replication-incompetent HSV vector with VEGF under the control of the HSV ICP0 promoter (vector T0VEGF) was constructed. T0VEGF expressed and released VEGF from primary dorsal root ganglion (DRG) neurons in vitro, and following subcutaneous inoculation in the foot, expressed VEGF in DRG and nerve in vivo. At 2 weeks after induction of diabetes, subcutaneous inoculation of T0VEGF prevented the reduction in sensory nerve amplitude characteristic of diabetic neuropathy measured 4 weeks later, preserved autonomic function measured by pilocarpine-induced sweating, and prevented the loss of nerve fibers in the skin and reduction of neuropeptide calcitonin gene-related peptide and substance P in DRG neurons of the diabetic mice. HSV-mediated transfer of VEGF to DRG may prove useful in treatment of diabetic neuropathy.