PRODUCTION AND PURIFICATION OF HSV-1 VECTORS AND ITS USE FOR GENE TRANSFER TO HUMAN CD34⁺ CELLS

The delivery of therapeutic DNA to patients with genetic or acquired disorders has evolved into a realistic alternate treatment to surgery, drug delivery, or as an adjuvant to therapies. A specific area of this research is to transduce CD34⁺ cells with therapeutic DNA containing vectors developed from viruses. Explanted CD34⁺ progenitor cells are exposed to these vectors and then are transplanted to a host. Despite the positive advances, major limitations exist in the ability to efficiently transduce cells with current vector systems. In addition, if vectors are proven effective at ameliorating disease, the expense of manufacturing sufficient quantities required to treat large populations of patients may prohibit their widespread applications. Our lab utilizes replication-defective herpes simplex virus type-1 (HSV-1) as a gene delivery vector. HSV-1 possesses the capability to deliver large or multiple genes and infects a wide variety of cell types. In order to optimize transduction of human umbilical cord blood CD34⁺ cells, I examined this population for presence and functionality of the three different HSV receptors required for virus binding and entry. Optimal transduction efficiencies of 75% were obtained by systematically varying vector concentration and adsorption time. Additionally, several vector constructs were developed in order to examine the effects of various promoter systems and additional gene deletions on gene expression and vector-associated toxicity. Throughout these studies we were confronted with the difficulty of obtaining sufficient amounts of high quality vector needed for testing the efficacy of HSV-1 vectors in various applications. To further improve the vector yield, I examined the effect of temperature and media conditions on the stability of an HSV-1 vector. Applying a temperature shift to production conditions resulted in increased vector yields that remained stable at peak levels in cultures incubated at 33°C rather than 37°C. To enhance vector recovery, alternative purification strategies were examined. A two-stage tangential flow filtration system coupled to a chromatography step was developed to isolate vector from large debris such as cells and smaller molecules such as protein and DNA. This new system for vector concentration and purification proved superior to the standard protocols currently in use

Gene therapy for pain: Results of a phase I clinical trial

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 HSV‐based 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 2011Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/86983/1/22446_ftp.pd

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.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/74369/1/j.1526-4637.2009.00720.x.pd

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

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 (Cu(2+), Zn(2+), Ni(2+), and Co(2+)) 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