Clinical trial links oncolytic immunoactivation to survival in glioblastoma.

Immunotherapy failures can result from the highly suppressive tumour microenvironment that characterizes aggressive forms of cancer such as recurrent glioblastoma (rGBM)1,2. Here we report the results of a first-in-human phase I trial in 41 patients with rGBM who were injected with CAN-3110-an oncolytic herpes virus (oHSV)3. In contrast to other clinical oHSVs, CAN-3110 retains the viral neurovirulence ICP34.5 gene transcribed by a nestin promoter; nestin is overexpressed in GBM and other invasive tumours, but not in the adult brain or healthy differentiated tissue4. These modifications confer CAN-3110 with preferential tumour replication. No dose-limiting toxicities were encountered. Positive HSV1 serology was significantly associated with both improved survival and clearance of CAN-3110 from injected tumours. Survival after treatment, particularly in individuals seropositive for HSV1, was significantly associated with (1) changes in tumour/PBMC T cell counts and clonal diversity, (2) peripheral expansion/contraction of specific T cell clonotypes; and (3) tumour transcriptomic signatures of immune activation. These results provide human validation that intralesional oHSV treatment enhances anticancer immune responses even in immunosuppressive tumour microenvironments, particularly in individuals with cognate serology to the injected virus. This provides a biological rationale for use of this oncolytic modality in cancers that are otherwise unresponsive to immunotherapy (ClinicalTrials.gov: NCT03152318 ).

Herpes simplex virus-based nerve targeting gene therapy in pain management.

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.

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.

Generation of replication-competent and -defective HSV vectors.

INTRODUCTION: Engineering 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.

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-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 (JDbetabeta) for robust and transient expression of the transcription factors Pax3 and MyoD, which are known to be involved in skeletal muscle differentiation. JDbetabeta-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.

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.

HSV-mediated delivery of erythropoietin restores dopaminergic function in MPTP-treated mice.

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.

Inactivation of herpes simplex type 1 gene vector on immobilized metal affinity chromatography: oxidative damage by hydroxyl free radicals and its prevention.

Metal catalyzed oxidation (MCO), which typically involves oxygen free radical generation, is an important pathway that leads to the deterioration of many biological molecules in solution. The occurrence of MCO in immobilized metal affinity chromatography (IMAC) systems and its potential for inactivating biological products has not been well recognized. In this study, we report the inactivation of herpes simplex virus type 1 (HSV-1) gene therapy vector on immobilized cobalt affinity chromatography. We observed that purification of KgBHAT, an HSV-1 mutant bearing cobalt affinity tags (HAT) on the surface, on an IDA-Co2+ column using crude supernatant as starting material resulted in signification loss in virus infectivity (<5% recovery). Electron spin resonance (ESR) revealed that the virus inactivation was caused by hydroxyl free radicals generated from the interactions between cellular impurities and the metal ions on the column. Inclusion of 20 mM ascorbate, a free radical scavenger, in the chromatography mobile phase effectively scavenged the hydroxyl radicals and dramatically augmented the infectivity recovery to 70%. This finding is the first demonstration of oxygen free radical-mediated biological inactivation in an actual IMAC purification and the way on how to effectively prevent it.

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 (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.

Delivery of herpes simplex virus-based vectors to stem cells.

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).

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.

Effect of genetic background and culture conditions on the production of herpesvirus-based gene therapy vectors.

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.

Effect of temperature, medium composition, and cell passage on production of herpes-based viral vectors.

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.