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The use of gene transfer techniques, designed to effect the continuous release of analgesic peptides, offers the possibility to treat what may otherwise be intractable pain. In this chapter, we review the biology underlying this approach, the results of preclinical experiments in animal models, the human trials that have been completed, and prospects for the near-term future.

Neurogenic erectile dysfunction (ED) is one of major causes of ED and is often difficult to treat. In this review, we report significant improvements of ED caused by diabetes mellitus (DM) or cavernous nerve injury by using a gene therapy approach in rat models of ED. Herpes simplex virus (HSV) vectors were used to deliver neurotrophic factors, such as neurotrophin 3 (NT3), glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN), into cavernous nerves. The retrograde transport of HSV vector-mediated neurotrophic factors to pelvic ganglion neurons occurred after the administration of the vector around the cavernous nerves. HSV vector administration around the cavernous nerves can improve DM-induced ED and promote the recovery of erectile function after cavernous nerve injury. The HSV vector-mediated delivery of neurotrophic factors could be applicable for the treatment of neurogenic ED. NT3, GDNF, and NTN would be the possible factors that could be used for this gene therapy application.

Erectile dysfunction (ED) is frequently associated with injury to the cavernous nerve sustained during pelvic surgery. Functional recovery from cavernous nerve injury is generally incomplete and occurs over an extended time frame. We employed a therapeutic gene transfer approach with herpes simplex virus (HSV) vector expressing glial cell line-derived neurotrophic factor (GDNF). Rat cavernous nerve was injured bilaterally using a clamp and dry ice. For HSV-treated groups, 20 microl of purified vector stock was administered directly to and around the damaged nerve. Delivery of an HSV vector expressing both green fluorescent protein (GFP) and lacZ (HSV-LacZ) was used as a control. Intracavernous pressure along with systemic arterial pressure (ICP/AP) was measured 2 and 4 weeks after the nerve injury. Fluorogold (FG) was injected into the penile crus 7 days before killing to assess nerve survival. Approximately 60% of major pelvic ganglion (MPG) cells were GFP positive after viral administration. At 4 weeks after nerve injury, rats treated with HSV-GDNF exhibited significant recovery of ICP/AP compared with control vector or untreated groups. The HSV-GDNF group also yielded more FG-positive MPG cells than the control vector group. HSV vector-mediated delivery of GDNF presents a viable approach for the treatment of ED following cavernous nerve injury.

Our work uses replication-defective genomic herpes simplex virus type-1 (HSV-1)-based vectors to transfer therapeutic genes into cells of the central nervous system and other tissues. Obtaining highly purified high-titer vector stocks is one of the major obstacles remaining in the use of these vectors in gene therapy applications. We have examined the effects of temperature and media conditions on the half-life of HSV-1 vectors. The results reveal that HSV stability is 2.5-fold greater at 33 degrees C than at 37 degrees C and is further stabilized at 4 degrees C. Additionally, a significantly higher half-life was measured for the vector in infection culture conditioned serum medium compared to fresh medium with or without serum. Synchronous infections incubated at 33 degrees C produced 2-fold higher amounts of vector than infected cells incubated at 37 degrees C, but with a lag of 16-24 h. Vector production yielded 3-fold higher titers and remained stable at peak levels for a longer period of time in cultures incubated at 33 degrees C than 37 degrees C. A pronounced negative effect of increased cell passage number on vector yield was observed. Vector production at 33 degrees C yielded similar levels regardless of passage number but was reduced at 37 degrees C as passage number increased. Together, these results contribute to improved methods for high-titer HSV vector production.

Paclitaxel-induced peripheral neuropathy (PIPN) is devastating constellation of symptoms that arise as a complication in patients receiving paclitaxel chemotherapy for cancer, most notably breast cancer. It presents as a glove and stocking neuropathy characterized by numbness, tingling, and occasionally burning pain in the feet and hands. Approximately 230,000 women will be diagnosed with breast cancer this year and over half, or about 120,000 women, will develop peripheral neuropathy as a consequence of paclitaxel therapy; this neuropathy will last for 5 years or longer in a quarter of these women. The development of PIPN may result in dose reduction of the paclitaxel, a switch to less efficacious agent, or even cessation of all chemotherapy treatment. Currently there are no approved therapies for the treatment of PIPN.PGN-503 is a new investigation drug designed to prevent and/or treat PIPN. It is a herpes simplex virus type 1 based vector expressing human neurotrophin-3. Following a skin injection, PGN-503 is taken up by sensory nerves, transported back to the cell body, and directs the expression of neurotrophin-3 (NT3). NT3 is a well-studied neurotrophic factor necessary for the growth and maintenance of sensory neurons, and thus represents an ideal candidate for the treatment of primary sensory neuropathies. In a series of preclinical studies, we examined the efficacy of PGN-503 in a mouse model of PIPN.Paclitaxel, dosed at 30 mg/kg/day for 6 days over two weeks, causes a peripheral neuropathy in BALB/c mice characterized by a long-lasting decrease in evoked sensory nerve action potentials (SNAPs) and conduction velocities (SNCVs). Mice, pretreated with a single injection of PGN-503 into the plantar surface of the hind paws 3 days prior to paclitaxel dosing, were completely protected against this decrease in SNAPs and SNCVs for the length of the study (approximately 4 months). This protection was dose-dependent on the total plaque forming units of PGN-503 applied to the paws. In a separate set of animals, we determined that a single application of PGN-503 was completely protective against the development of neuropathy following a second round of paclitaxel dosing beginning at 47 days post vector injection and partially protective against a third round beginning at 99 days post vector injection. Importantly, we have also demonstrated that PGN-503, applied one week after paclitaxel dosing, is capable of reversing the peripheral neuropathy. A Phase I/II trial of PGN-503 in preventing the development of PIPN in adjuvant breast cancer treatment is currently planned to begin next year.

Objective: 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. Methods: We conducted a multicenter, dose-escalation, phase I clinical trial of NP2, a replication-defective HSVbased vector expressing human preproenkephalin (PENK) in subjects with intractable focal pain caused by cancer. NP2 was injected intradermally into the dermatome(s) corresponding to the radicular distribution of pain. The primary outcome was safety. As secondary measures, efficacy of pain relief was assessed using a numeric rating scale (NRS), the Short Form McGill Pain Questionnaire (SF-MPQ), and concurrent opiate usage. Results: Ten subjects with moderate to severe intractable pain despite treatment with >200mg/day of morphine (or equivalent) were enrolled into the study. Treatment was well tolerated with no study agent-related serious adverse events observed at any point in the study. Subjects receiving the low dose of NP2 reported no substantive change in pain. Subjects in the middle- and high-dose cohorts reported pain relief as assessed by NRS and SF-MPQ. Interpretation: Treatment of intractable pain with NP2 was well tolerated. There were no placebo controls in this relatively small study, but the dose-responsive analgesic effects suggest that NP2 may be effective in reducing pain and warrants further clinical investigation. ANN NEUROL 2011;70:207–212

The first human trial of gene therapy for chronic pain, a phase 1 study of a nonreplicating herpes simplex virus (HSV)-based vector engineered to express preproenkephalin in patients with intractable pain from cancer, began enrolling subjects in December 2008. In this article, we describe the rationale underlying this potential approach to treatment of pain, the preclinical animal data in support of this approach, the design of the study, and studies with additional HSV-based vectors that may be used to develop treatment for other types of pain.

Neurturin (NTN), a member of glial cell line-derived neurotrophic factor (GDNF) family, is known as an important neurotrophic factor for penis-projecting neurons. We recently demonstrated significant protection from erectile dysfunction (ED) following a replication-defective herpes simplex virus (HSV) vector-mediated GDNF delivery to the injured cavernous nerve. Herein, we applied HSV vector-mediated delivery of NTN to this ED model. Rat cavernous nerve was injured bilaterally using a clamp and dry ice. For HSV-treated groups, 20 microl of vector stock was administered directly to the damaged nerve. Delivery of an HSV vector expressing both green fluorescent protein and lacZ (HSV-LacZ) was used as a control. Intracavernous pressure along with systemic arterial pressure (ICP/AP) was measured 2 and 4 weeks after the nerve injury. Fluorogold (FG) was injected into the penile crus 7 days before being killed to assess neuronal survival. Four weeks after nerve injury, rats treated with HSV-NTN exhibited significantly higher ICP/AP compared with untreated or control vector-treated groups. The HSV-NTN group had more FG-positive major pelvic ganglion neurons than the control group following injury. HSV vector-mediated delivery of NTN could be a viable approach for the improvement of ED following cavernous nerve injury.

Virus vectors have been employed as gene transfer vehicles for various pre-clinical and clinical gene therapy applications. Replication-competent herpes simplex virus (HSV) vectors that replicate specifically in actively dividing glial tumor cells have been used in Phase I-II human trials in patients with glioblastoma multiforme (GBM), a fatal form of brain cancer. Research during the last decade on the development of HSV vectors has resulted in the engineering of recombinant vectors that are totally replication defective, non-toxic, and capable of long-term transgene expression. 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 studies in animals.

To investigate the neuroprotective effects of erythropoietin (EPO) in a rodent model of Parkinson disease, we inoculated a nonreplicating herpes simplex virus-based vector expressing EPO (vector DHEPO) into the striatum of mice 1 week prior to, or 2 weeks after, the start of continual administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (4 mg/kg intraperitoneally, 5 of 7 days) for 6 weeks. Inoculation with DHEPO prior to MPTP intoxication preserved behavioral function measured by pellet retrieval and the histological markers of tyrosine hydroxylase-immunoreactive (TH-IR) neuronal cell bodies in the substantia nigra (SN) and TH-IR and dopamine transporter-immunoreactive (DAT-IR) terminals in striatum. Inoculation of DHEPO 2 weeks into a 6-week course of MPTP resulted in improvement of behavioral function and restoration of TH-IR cells in SN and TH- and DAT-IR in the striatum. The effects of vector-produced EPO were similar in magnitude to the effects of vector-mediated expression of glial-derived neurotrophic factor in the same model. These results demonstrate that vector-mediated EPO production may be used to reverse dopaminergic neurodegeneration in the face of continued toxic insult.

The vanilloid receptor (TRPV1) is an important pro-nociceptive Ca+2 channel, the inhibition of which attenuates pain. Recent studies have shown that TRPV1 phosphorylation by protein kinase C epsilon (PKCe) causes receptor sensitization and hyperalgesia. Inhibition of PKCe activity in nociceptors can hence be an approach to treat chronic pain. We previously reported replication defective HSV-1 vectors as a means to efficiently deliver analgesic gene products to rat dorsal root ganglion (DRG) neurons in-vivo. In this study, we recombined dominant negative PKCe (DNPKC) deleted for the C2 and C1b domains and the catalytic region into a replication defective HSV-1 vector (called vHDNPKC). DNPKC was fused to a green fluorescent protein (GFP) coding sequence and placed under transcriptional control of the HCMV immediate early promoter. Live cell imaging of vHDNPKC-infected Vero and U2OS cells displayed perinuclear green fluorescence that translocated to the plasma membrane following stimulation with the PKC activator, PMA. Whole-cell electrophysiological recordings of capsaicin-induced TRPV1 currents in vHDNPKC-infected adult rat DRG neurons demonstrated significantly smaller current amplitudes (2.5 fold) that desensitized faster (4 fold) than uninfected or vHG-infected controls. Control capsaicin responses were potentiated by isoform-specific PKCe translocation following stimulation with a specific bradykinin receptor 2 agonist, HYP3-bradykinin. HYP3-bradykinin failed to potentiate capsaicin currents in vHPKCDN-infected neurons, while the non-specific PKC activator PDBu caused a slow drift current that was antagonized by the PKC inhibitor bisindolylmaleimide. This result demonstrates isoform-specific inhibition of PKCe activity. Taken together, the data demonstratean in-vitro inhibition of TRPV1 function in nociceptive neurons by HSV-1 vector-expressed dominant negative PKCe. This vector can therefore be used as a viable approach for the in-vivo induction of analgesia.

In contrast to traditional drugs that generally act by altering existing gene product function, gene therapy aims to target the root cause of the disease by altering the genetic makeup of the cell to treat the disease. Researchers have adapted several classes of viruses as gene-transfer vectors, taking advantage of natural viral mechanisms designed to efficiently and effectively deliver DNA to the host-cell nucleus. Among these, the human herpesviruses are excellent candidate vectors for a variety of applications. Herpes simplex virus type 1 (HSV-1) is a particularly attractive gene-transfer vehicle because natural infection in humans includes a latent state in which the viral genome persists in a nonintegrated form without causing disease in an immune-competent host. HSV-1 is a large DNA virus with a broad host range that can be engineered to accommodate multiple or large therapeutic transgenes (4). HSV vectors may be generally useful for gene transfer to a variety of tissues in which short-term or extended transgene expression of therapeutic transgenes achieve a therapeutic effect. We have used therapeutic vectors to successfully treat human disease models in animals, including cancer, Parkinson's disease, and nerve damage (5-10).

Herpes simplex virus type-1 (HSV-1) is a neurotrophic human pathogen that establishes life-long latency in the nervous system. Our laboratory has extensively engineered this virus to retain the ability to persist in neurons without expression of lytic genes or disease phenotype. Highly defective, replication-incompetent HSV mutants are thus potentially ideal for transfer of therapeutic transgenes to human nerves where long-term therapy of nervous system disease may be provided. A prerequisite for using recombinant HSV vectors for therapeutic gene delivery to humans is the development of methods for large-scale manufacture of HSV vectors. Here we report studies to identify infection parameters that result in high-yield production of immediate early gene deletion mutant HSV vectors in complementing cells that supply the deleted essential viral functions in trans. Virus yield was correlated with various culture media conditions that included pH, glucose metabolism, and serum levels. The results demonstrated that systematic media exchange to remove lactate derived from high-level glucose consumption, maintenance of tissue culture pH at 6.8, and the use of 5% fetal bovine serum gave the highest yield of infectious virus. The data indicate that these are important parameters to consider for high-yield, large-scale virus production.

Herpes simplex virus type-1 (HSV-1) represents a unique vehicle for the introduction of foreign DNA to cells of a variety of tissues. The nature of the vector, the cell line used for propagation of the vector, and the culture conditions will significantly impact vector yield. An ideal vector should be deficient in genes essential for replication as well as those that contribute to its cytotoxicity. Advances in the engineering of replication-defective HSV-1 vectors to reduce vector-associated cytotoxicity and attain sustained expression of target genes make HSV-1 an attractive gene-delivery vehicle. However, the yield of the less-cytotoxic vectors produced in standard tissue-culture systems is at least three order of magnitudes lower than that achieved with wild-type virus. The low overall yield and the complexity involved in the preparation of HSV vectors at high concentrations represent major obstacles in use of replication-defective HSV-derived vectors in gene therapy applications. In this work, the dependence of the vector yield on the genetic background of the virus is examined. In addition, we investigated the production of the least toxic, lowest-yield vector in a CellCube bioreactor system. After initial optimization of the operational parameters of the cellcube system, we were able to attain virus yields similar to or better than those values attained using the tissue culture flask system for vector production with significant savings with respect to time, labor, and cost.

Chronic pain represents a major medical burden not only in terms of suffering but also in terms of economic costs. Traditional medical approaches have so far proven insufficient in treating chronic pain and new approaches are necessary. Gene therapy with herpes simplex virus (HSV)-based vectors offers the ability to directly target specific regions of the neuraxis involved in pain transmission including the primary afferent nociceptor. This opens up new targets to interact with that are either not available to traditional systemic drugs or cannot be adequately acted upon without substantial adverse off-target effects. Having access to the entire neuron, which HSV-based vector gene therapy enables, expands treatment options beyond merely treating symptoms and allows for altering the basic biology of the nerve. In this paper, we discuss several HSV-based gene therapy vectors that our group and others have used to target specific neuronal functions involved in the processing of nociception in order to develop new therapies for the treatment of chronic pain.

Herpes simplex virus (HSV) is a neurotropic DNA virus. The viral genome is large (152 kb), and many genes are dispensable for viral function, allowing insertion of multiple or large transgene expression cassettes. The virus life cycle includes a latent phase, during which the viral genome remains as a stable episomal element within neuronal nuclei for the lifetime of the host, without disturbing normal function. We have exploited these features of HSV to construct a series of nonpathogenic gene therapy vectors that efficiently deliver therapeutic and experimental transgenes to neural and non-neural tissue. Importantly, transgene expression may be sustained long term; reporter gene expression has been demonstrated for over a year in the nervous system. This article discusses the generation of replication-defective HSV vectors and reviews recent studies investigating their use in several animal models of human disease. We have demonstrated correction or prevention of a number of important neurological phenotypes, including neurodegeneration, chronic pain, peripheral neuropathy, and malignancy. In addition, HSV-mediated transduction of non-neurological tissues allows their use as depot sites for synthesis of circulating and locally acting secreted proteins. New applications for this vector system include the genetic modification of stem cell populations; this may become an important means to direct cellular differentiation or deliver therapeutic genes systemically. Replication-defective HSV vectors are an effective and flexible vehicle for the delivery of transgenes to numerous tissues, with multiple applications.

INTRODUCTIONEngineering effective vectors has been crucial to the efficient delivery and expression of therapeutic gene products in vivo. Among these, HSV-1 represents an excellent candidate vector for delivery to the peripheral and central nervous systems. The natural biology of HSV-1 includes the establishment of a lifelong latent state in neurons in which the viral genome persists as an episomal molecule. Genomic HSV vectors can be produced that are completely replication-defective, nontoxic, and capable of long-term transgene expression. Herpes simplex virus (HSV) vectors are constructed by using a replication-deficient vector backbone (TOZ.1) for homologous recombination with a shuttle plasmid containing a cassette expressing the gene of interest inserted into the UL41 gene sequence. The TOZ.1 vector expresses a reporter gene (lacZ) in the UL41 locus, such that recombination of the transgenic cassette into the UL41 locus results in the loss of the reporter gene activity. The TOZ.1 vector also contains a unique PacI endonuclease site for digestion of parental viral DNA that substantially reduces the nonrecombinant background. Following homologous recombination of the shuttle plasmid into the PacI-digested TOZ.1 genome, the recombinants are identified as clear plaques. After three rounds of limiting dilution analysis, the structure of the recombinants can be confirmed by Southern blot or by polymerase chain reaction (PCR) analysis.

Heart transplantation is the preferred therapy for patients with a variety of end-stage heart diseases. Developments in the use of potent immunosuppressive drugs, technical innovations in transplant surgery, and improvements in postoperative care have significantly improved outcomes of heart transplantation. However, donor organs suffer from perturbations in endothelial cell function during required cold storage and subsequent warm reperfusion that can lead to organ injury (ischemia-reperfusion [I-R]). Thus development of methods for preserving endothelial cell function would contribute to transplant success. Considerable evidence supports an important role for heme oxygenase-1 (HO-1) in protection against I-R injury. HO-1 catabolizes heme into biliverdin, free iron and CO, the latter acting as an anti-inflammatory and anti-apoptotic agent. These actions presumably help suppress deleterious effects associated with transplant rejection, endotoxemia and hyperoxia.

Herpes simplex virus type 1 (HSV-1) is a promising vector for gene therapy applications particularly to peripheral nerves, the natural site of virus latency. Many gene vectors require large particle numbers for even early phase clinical trials emphasizing the need for high yield, scalable manufacturing processes that result in virus preparations nearly free of cellular DNA and protein contaminants. HSV is an enveloped virus that requires the development of gentle purification methods. Ideally, such methods should avoid centrifugation and employ selective purification processes that rely on the recognition of unique envelope surface chemistry. Here we describe a novel method that fulfills these criteria. An immobilized metal affinity chromatography (IMAC) method is described for the selective purification of vectors engineered to display a high affinity binding peptide. Feasibility studies involving various transitional metal ions (Cu2+, Zn2+, Ni2+, and Co2+) showed that cobalt had the best features in terms of its low interaction with virus particles or contaminating DNA and proteins, suggesting that the introduction of a cobalt specific recognition element into the virus envelope would provide a suitable target for cobalt dependent purification. A peptide with affinity for immobilized cobalt was engineered in-frame in the heparan sulfate (HS) binding domain of HSV-1 glycoprotein B (gB) known to be surface exposed on the virion particle and recombined into the viral genome. By optimizing cobalt IMAC loading conditions and reducing cobalt ion leakage, we recovered 78% of the tagged HSV-1 recombinant virus with greater than 96% reduction in contaminating cellular products. This methodology may have broad application to the purification of this and other gene vectors currently in use for gene transfer applications.