James B. Wechuck

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.

Multiple Applications For Replication-Defective Herpes Simplex Virus Vectors

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.

A Clinical Trial of Gene Therapy for Chronic Pain

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.

Herpes simplex virus vector-mediated delivery of glial cell line-derived neurotrophic factor rescues erectile dysfunction following cavernous nerve injury.

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 μl 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.

Immobilized Cobalt Affinity Chromatography Provides a Novel, Efficient Method for Herpes Simplex Virus Type 1 Gene Vector Purification

ABSTRACT Herpes simplex virus type 1 (HSV-1) is a promising vector for gene therapy applications, particularly at 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 that are nearly free of cellular DNA and protein contaminants. HSV-1 is an enveloped virus that requires the development of gentle purification methods. Ideally, such methods should avoid centrifugation and may employ selective purification processes that rely on the recognition of a unique envelope surface chemistry. Here we describe a novel method that fulfills these criteria. An immobilized metal affinity chromatography (IMAC) method was developed for the selective purification of vectors engineered to display a high-affinity binding peptide. Feasibility studies involving various transition metal ions (Cu2+, Zn2+, Ni2+, and Co2+) showed that cobalt had the most desirable features, which include a low level of interaction with either the normal virus envelope or contaminating DNA and proteins. The introduction of a cobalt-specific recognition element into the virus envelope may provide a suitable target for cobalt-dependent purification. To test this possibility, we engineered a peptide with affinity for immobilized cobalt in frame in the heparan sulfate binding domain of HSV-1 glycoprotein B, which is known to be exposed on the surface of the virion particle and recombined into the viral genome. By optimizing the IMAC loading conditions and reducing cobalt ion leakage, we recovered 78% of the tagged HSV-1 recombinant virus, with a >96% reduction in contaminating proteins and DNA.

Herpes simplex virus vector-mediated delivery of neurturin rescues erectile dysfunction of cavernous nerve injury

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 μl 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.

Evaluation of Infection Parameters in the Production of Replication-Defective HSV-1 Viral Vectors

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.

Construction and Production of Recombinant Herpes Simplex Virus Vectors

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.

Herpes Simplex Virus‐Mediated Expression of Pax3 and MyoD in Embryoid Bodies Results in Lineage‐Related Alterations in Gene Expression Profiles

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.

Effect of Protease Inhibitors on Yield of HSV‐1‐Based Viral Vectors

The ability to obtain high titer replication‐defective herpes simplex virus (HSV) recombinant vectors will dramatically affect their use in gene therapy clinical trials. A variety of techniques and reagents have been employed to increase the overall yield of the vector. The effects of protease inhibitors on the yield of an HSV‐1‐based viral vector were examined. Experiments were conducted using a commercial protease inhibitor cocktail typically used in mammalian cell culture for protein production. Contrary to our expectation for enhanced vector yield, the results showed a dramatic reduction in vector yield. Moreover, it was found that AEBSF is the only component in the protease cocktail responsible for the low vector yield. On the basis of our hypothesis regarding the mode of action of AEBSF, we suggest that it should not be included in protease inhibitor cocktails designed for use in cultures aimed at production of viral vectors derived from HSV‐1 or possibly several other vectors.