Shaohua Huang

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.

Bcl-2 and GDNF Delivered by HSV-Mediated Gene Transfer after Spinal Root Avulsion Provide a Synergistic Effect

Proximal spinal nerve injury results in the death of motor neurons in ventral horn. We have previously demonstrated this cell death can be prevented by HSV-mediated transfer of the gene coding for the antiapoptotic peptide Bcl-2 7 days prior to injury, but that expression of Bcl-2 does not preserve ChAT expression in the lesioned cells. In the current study, we examined two related issues: whether Bcl-2 delivered by HSV-mediated gene transfer 30 min after injury could similarly protect motor neurons from cell death, and whether the additional HSV-mediated expression of the glial cell derived neurotrophic factor (GDNF) could improve the result. At 30 min after avulsion of the L4, L5, and L6 spinal nerves, replication defective genomic HSV-based vectors coding for Bcl-2, GDNF, a reporter transgene (lacZ), or the Bcl-2 and GDNF vectors together were injected into spinal cord. Transduction of motor neurons with either the Bcl-2-expressing vector or the GDNF-expressing vector resulted in a substantial increase in the number of surviving motor neurons, and coinjection of the two vectors together resulted in cell survival that was similar to the result obtained with either vector alone. Neither the Bcl-2-expressing vector nor the GDNF-expressing vector delivered alone protected choline acetyltransferase (ChAT) expression in lesioned neurons. However, simultaneous injection of the Bcl-2- and the GDNF-expressing vectors together resulted in a substantial increase in the number of ChAT in cells in the lesioned ventral horn. Together, these findings suggest an approach to improving cell survival and regeneration following proximal root injury.

Gene transfer of human manganese superoxide dismutase protects small intestinal villi from radiation injury

Small bowel toxicity represents a major dose-limiting side effect of radiation treatment for many malignancies. We examined the effects of overexpressing human manganese superoxide dismutase (MnSOD) in the small intestine in mice to prevent radiation enteritis. Mice were treated with the human MnSOD gene delivered enterally using a nontoxic, replication-defective herpes simplex virus (HSV)-1-based vector. HSV vectors containing the human MnSOD transgene and green fluorescent protein (GFP) transgene, or GFP transgene alone, were constructed and injected intraluminally into a 2cm length of small intestine of C3H/HeNsd mice. Total body irradiation of 15 Gy was delivered to mice inoculated 24 hours earlier with either HSV-MnSOD (103 to 108 plaque-forming units), control HSV-GFP, or no vector. At 24 or 72 hours after irradiation, mice were killed and villi areas were measured from appropriate segments of the small intestine. Control irradiated mice showed a decreased villi area of 82% by day 3 after irradiation, whereas treatment of mice with HSV-MnSOD 108 plaque-forming units led to only a 16% decrease in villi area (P< 0.001) before radiation. Similar findings were seen on day 3 and were associated with a significant (P< 0.001) preservation of enteric protein content in HSV-MnSOD-treated mice. A dose-dependent effect of MnSOD in preventing radiation-induced small bowel injury was evident. These data demonstrate that overexpression of human MnSOD via a replication-defective herpes viral vector is an efficacious method of protecting the small intestine from ionizing radiation damage.

The transcription factor Sox11 promotes nerve regeneration through activation of the regeneration-associated gene Sprr1a

Factors that enhance the intrinsic growth potential of adult neurons are key players in the successful repair and regeneration of neurons following injury. Injury-induced activation of transcription factors has a central role in this process because they regulate expression of regeneration-associated genes. Sox11 is a developmentally expressed transcription factor that is significantly induced in adult neurons in response to injury. Its function in injured neurons is however undefined. Here, we report studies that use herpes simplex virus (HSV)-vector-mediated expression of Sox11 in adult sensory neurons to assess the effect of Sox11 overexpression on neuron regeneration. Cultured mouse dorsal root ganglia (DRG) neurons transfected with HSV-Sox11 exhibited increased neurite elongation and branching relative to naïve and HSV-vector control treated neurons. Neurons from mice injected in foot skin with HSV-Sox11 exhibited accelerated regeneration of crushed saphenous nerves as indicated by faster regrowth of axons and nerve fibers to the skin, increased myelin thickness and faster return of nerve and skin sensitivity. Downstream targets of HSV-Sox11 were examined by analyzing changes in gene expression of known regeneration-associated genes. This analysis in combination with mutational and chromatin immunoprecipitation assays indicates that the ability of Sox11 to accelerate in vivo nerve regeneration is dependent on its transcriptional activation of the regeneration-associated gene, small proline rich protein 1a (Sprr1a). This finding reveals a new functional linkage between Sox11 and Sprr1a in adult peripheral neuron regeneration.

Engineering HSV-1 Vectors for Gene Therapy

Virus vectors have been employed as gene transfer vehicles for various preclinical and clinical gene therapy applications, and with the approval of Glybera (alipogene tiparvovec) as the first gene therapy product as a standard medical treatment (Yla-Herttuala, Mol Ther 20: 1831-1832, 2013), gene therapy has reached the status of being a part of standard patient care. Replication-competent herpes simplex virus (HSV) vectors that replicate specifically in actively dividing tumor cells have been used in Phase I-III human trials in patients with glioblastoma multiforme, a fatal form of brain cancer, and in malignant melanoma. In fact, T-VEC (talimogene laherparepvec, formerly known as OncoVex GM-CSF) displayed efficacy in a recent Phase III trial when compared to standard GM-CSF treatment alone (Andtbacka et al. J Clin Oncol 31: sLBA9008, 2013) and may soon become the second FDA-approved gene therapy product used in standard patient care. In addition to the replication-competent oncolytic HSV vectors like T-VEC, replication-defective HSV vectors have been employed in Phase I-II human trials and have been explored as delivery vehicles for disorders such as pain, neuropathy, and other neurodegenerative conditions. Research during the last decade on the development of HSV vectors has resulted in the engineering of recombinant vectors that are totally replication defective, nontoxic, and capable of long-term transgene expression in neurons. This chapter describes methods for the construction of recombinant genomic HSV vectors based on the HSV-1 replication-defective vector backbones, steps in their purification, and their small-scale production for use in cell culture experiments as well as preclinical animal studies.

Enhanced functional recovery after proximal nerve root injury by vector-mediated gene transfer

In order to test the functional implication of herpes simplex virus (HSV) vector-mediated gene transfer after axonal injury, we injected replication-incompetent HSV vectors coding for the anti-apoptotic peptide Bcl-2 and the glial cell-derived neurotrophic factor (GDNF), separately or in combination into ventral spinal cord 30 min after a crush injury to the proximal spinal root that was combined with moderate mechanical traction. HSV-mediated expression of Bcl-2 or GDNF enhanced functional recovery assessed by histologic, electrophysiologic, and behavioral parameters up to 5 months after injury. The most sensitive measure of distal motor function, the sciatic function index, was significantly improved in animals injected with the two vectors together. These results suggest an approach to root trauma that might be used to enhance functional recovery after injury.

HSV Delivery of a Ligand-regulated Endogenous Ion Channel Gene to Sensory Neurons Results in Pain Control Following Channel Activation

Persistent pain remains a tremendous health problem due to both its prevalence and dearth of effective therapeutic interventions. To maximize pain relief while minimizing side effects, current gene therapy-based approaches have mostly exploited the expression of pain inhibitory products or interfered with pronociceptive ion channels. These methods do not enable control over the timing or duration of analgesia, nor titration to analgesic efficacy. Here, we describe a gene therapy strategy that potentially overcomes these limitations by providing exquisite control over therapy with efficacy in clinically relevant models of inflammatory pain. We utilize a herpes simplex viral (HSV) vector (vHGlyRα1) to express a ligand-regulated chloride ion channel, the glycine receptor (GlyR) in targeted sensory afferents; the subsequent exogenous addition of glycine provides the means for temporal and spatial control of afferent activity, and therefore pain. Use of an endogenous inhibitory receptor not normally present on sensory neurons both minimizes immunogenicity and maximizes therapeutic selectivity.

Combination gene therapy for glioblastoma involving herpes simplex virus vector-mediated codelivery of mutant I|[kappa]|B|[alpha]| and HSV thymidine kinase

To improve the effectiveness of herpes simplex virus (HSV) thymidine kinase/ganciclovir (HSV-tk/GCV) suicide gene therapy, the replication-defective HSV vector TOIκB expressing both HSV-TK and a mutant form of the NF-κB inhibitor IκBα (IκBαM) was developed. TOIκB was constructed by recombining the IκBαM gene into the UL41 locus of a replication-defective lacZ expression vector, TOZ.1. Expression of IκBαM was confirmed by Western blotting, and the ability of the mutant protein to inhibit NF-κB nuclear translocation was examined by electrophoretic mobility shift assay. In human glioblastoma U-87MG cells, the p50/p50 dimer of NF-κB was already translocated to the nucleus without receptor-dependent signaling by TNF-α. Following infection with TOIκB, nuclear translocation of NF-κB in U-87MG cells was significantly inhibited and caspase-3 activity increased compared with TOZ.1-infected cells. The cytotoxicity of TOIκB for U-87MG cells was investigated by colorimetric MTT assay. At an MOI of 3, TOIκB infection killed 85% of the cells compared to 20% killed by TOZ.1 infection. In the presence of GCV, these numbers increased to 95–100% for TOIκB and 80–85% for TOZ.1. TOIκB neurotoxicity measured on cultured murine neurons was relatively low and similar to that of TOZ.1. The survival of nude mice implanted into the brain with U-87MG tumor cells was markedly prolonged by intratumoral TOIκB injection and GCV administration. Survival of TOIκB+GCV group was significantly longer (P<.02, Wilcoxon test) than for the control groups (TOZ.1 or TOIκB only, PBS or PBS+GCV). These results suggest that IκBαM expression may be a safe enhancement of replication-defective HSV-based suicide gene therapy in vitro and in vivo.