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The ability of embryonic stem cells to develop into multiple cell lineages provides a powerful resource for tissue repair and regeneration. Gene transfer offers a means to dissect the complex events in lineage determination but is limited by current delivery systems. We designed a high-efficiency replication-defective herpes simplex virus gene transfer vector (JDββ) for robust and transient expression of the transcription factors Pax3 and MyoD, which are known to be involved in skeletal muscle differentiation. JDββ-mediated expression of each gene in day 4 embryoid bodies (early-stage mesoderm) resulted in the induction of unique alterations in gene expression profiles, including the upregulation of known target genes relevant to muscle and neural crest development, whereas a control enhanced green fluorescent protein expression vector was relatively inert. This vector delivery system holds great promise for the use of gene transfer to analyze the impact of specific genes on both regulatory genetic events and commitment of stem cells to particular lineages. Disclosure of potential conflicts of interest is found at the end of this article.

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.

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.

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.

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.

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.

The long-term goal of our laboratory is to create replication defective HSV-1 based viral vectors that are suitable to support the clinical development of therapeutic gene delivery applications. In order to reach this goal three considerations that must be satisfied include; (1) proven efficacy of vector upon delivery to target cells, (2) safety of vector stock preparations, and (3) feasibility of clinical vector stock production. Efficacy is primarily dependent upon effective transgene expression for an appropriate duration without interference with host cell processes that might affect target cell viability. Thus among the most important considerations for vector design are strategies to eliminate vector toxicity and to provide for appropriate transgene expression. Various replication defective HSV-1 viral mutants based on the manipulation of immediate early genes have been described previously. However, the simultaneous evaluation of various combinations of these mutations and the impact on transgene expression has not been reported. We have created an expansive panel of replication defective IE gene mutant HSV-1 viruses and complementing cell lines to produce them. The IE gene ICP0 appears to mediate vector toxicity and also play an important role in the efficient expression of exogenous transgenes from either the ICP0 IE promoter or the widely used HCMV promoter. Large viral genomic deletions have no effect on virus mediated toxicity unless they result in the reduction of ICP0 expression while these large deletions appear to have a deleterious effect on transgene expression in the absence of ICP0 expression. Current productive capabilities of ICP0 mutants are now very similar to the growth characteristics of our previously described IC4/ICP22/ICP27 mutants due to improvements in ICP0/ICP4/ICP27 or ICP0/ICP4/ICP22/ICP27 complementing cells lines and growth conditions. Growth capabilities, vector mediated toxicity, transgene expression, and complementing cell line construction will be described in further detail.

Top of pageAbstract Myogenesis is a complex process involving several distinct pathways in the ordered proliferation, migration, and differentiation of stem cells in the developing embryo. The cascade of gene regulation governing embryonic myogenesis has significant gaps of information which has prompted the development of a model system to fill these gaps using herpes simplex virus vectors. We use murine embryonic stem cells (mES) because their pluripotent characteristics provide an ideal model for differentiation along myogenic lineages. We are particularly interested in the identification of genes active during early stages of myogenesis, prior to the activation of basic helix-loop-helix (bHLH) muscle transcription factors MyoD, Myf5, myogenin, and MRF4. Pax3 is our initial target as it has been shown to activate gene expression of the bHLH muscle markers and induce myogenesis in certain pluripotent stem cells and embryonic tissues. Since activators of Pax3 have not been identified, we have begun exploring the possibility of using a mutant HSV-1 viral vector, JD|[beta]||[beta]|, to study early promoter-activating events of myogenesis. JD|[beta]||[beta]| is deleted for ICP4 and ICP22, ICP0 and ICP27 have been converted into |[beta]| (early) genes, and one full genomic repeat element has been eliminated. Preliminary flow cytometry data show that JD|[beta]||[beta]| drives robust reporter transgene expression in embryonic stem cells and is non-toxic. Characterization of this virus, including toxicity studies, transgene expression levels, and IE gene kinetics, will be reported. Several mES cell lines containing myogenic marker genes have been developed by others and characterized, including a Pax3-lacZ mES cell line. mES cells, differentiated into muscle using ectopic differentiating methods, serve as a control. JD|[beta]||[beta]| will be modified to carry genes potentially involved during early myogenesis including |[beta]|-catenin and Wnt3a. In order to ascertain whether these or other transgenes can functionally activate the Pax3 promoter, we will deliver them to Pax3-lacZ mES cells and assay for reporter gene expression as well as activation of downstream muscle-specific marker genes. This will allow us to validate the use of HSV as a means to introduce transgenes in mES cells and assay for muscle differentiation as a result of transgene expression. Upstream factors with consistent, ordered activity as determined by the proposed model system are of great interest for deciphering stages of myogenesis, and will be potential candidates for committing early progenitor cells into the muscle phenotype for therapeutic purposes.

Embryonic development is a complex process involving integrated pathways directing the ordered proliferation, migration, and differentiation of stem cells into a developing embryo. Murine embryonic stem (mES) lines are immortal and homogeneous, and provide a relevant model of embryogenesis via embryoid body formation. An efficient gene delivery system for mES cells will facilitate investigations into the molecular events of embryonic differentiation. Here we describe the engineering and characterization of a replication-defective Herpes simplex virus-1 (HSV-1) vector, JD|[beta]||[beta]|, as a platform gene delivery vehicle. In this vector, the immediate early (IE, |[alpha]|) genes ICP4 and ICP22, and one full genomic joint element have been deleted. IE genes ICP27 and ICP0, whose low-level expression is necessary for transgene expression, have been converted into early (E, |[beta]|) genes in order to constrain their expression to ICP4-complementing cells, effectively eliminating their expression in normal cells. JD|[beta]||[beta]| does not express toxic viral genes in non-complementing cells including mES cells. JD|[beta]||[beta]| contains a green fluorescent protein (GFP) transgene under the control of the HCMV IE promoter and transduces mES cells with high efficiency. Transduction with JD|[beta]||[beta]|, even at elevated MOI, does not alter subsequent embryoid body formation nor the expression of endogenous germ layer markers during embryoid body formation, as determined by quantitative-PCR analysis. JD|[beta]||[beta]| will be further engineered to express developmentally relevant genes individually or in combination. This vector may be a useful tool for the experimental stem cell and regenerative medicine fields due to the attributes of non-toxicity, large transgene capacity, robust transgene expression, and non-integrating genome.

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.