Targeting IL-13Rα2 for effective treatment of malignant peripheral nerve sheath tumors in mouse models.

OBJECTIVEMalignant peripheral nerve sheath tumors (MPNSTs) are aggressive soft tissue sarcomas that harbor a high potential for metastasis and have a devastating prognosis. Combination chemoradiation aids in tumor control and decreases tumor recurrence but causes deleterious side effects and does not extend long-term survival. An effective treatment with limited toxicity and enhanced efficacy is critical for patients suffering from MPNSTs.METHODSThe authors recently identified that interleukin-13 receptor alpha 2 (IL-13Rα2) is overexpressed on MPNSTs and could serve as a precision-based target for delivery of chemotherapeutic agents. In the work reported here, a recombinant fusion molecule consisting of a mutant human IL-13 targeting moiety and a point mutant variant of Pseudomonas exotoxin A (IL-13.E13 K-PE4E) was utilized to treat MPNST in vitro in cell culture and in an in vivo murine model.RESULTSIL-13.E13 K-PE4E had a potent cytotoxic effect on MPNST cells in vitro. Furthermore, intratumoral administration of IL-13.E13 K-PE4E to orthotopically implanted MPNSTs decreased tumor burden 6-fold and 11-fold in late-stage and early-stage MPNST models, respectively. IL-13.E13 K-PE4E treatment also increased survival by 23 days in the early-stage MPNST model.CONCLUSIONSThe current MPNST treatment paradigm consists of 3 prongs: surgery, chemotherapy, and radiation, none of which, either singly or in combination, are curative or extend survival to a clinically meaningful degree. The results presented here provide the possibility of intratumoral therapy with a potent and highly tumor-specific cytotoxin as a fourth treatment prong with the potential to yield improved outcomes in patients with MPNSTs.

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.

Effects of herpes simplex virus vector-mediated enkephalin gene therapy on bladder overactivity and nociception.

We previously reported the effects of herpes simplex virus (HSV) vector-mediated enkephalin on bladder overactivity and pain. In this study, we evaluated the effects of vHPPE (E1G6-ENK), a newly engineered replication-deficient HSV vector encoding human preproenkephalin (hPPE). vHPPE or control vector was injected into the bladder wall of female rats 2 weeks prior to the following studies. A reverse-transcription PCR study showed high hPPE transgene levels in L6 dorsal root ganglia innervating the bladder in the vHPPE group. The number of freezing behaviors, which is a nociceptive reaction associated with bladder pain, was also significantly lower in the vHPPE group compared with the control group. The number of L6 spinal cord c-fos-positive cells and the urinary interleukin (IL)-1ß and IL-6 levels after resiniferatoxin (RTx) administration into the bladder of the vHPPE group were significantly lower compared with those of the control vector-injected group. In continuous cystometry, the vHPPE group showed a smaller reduction in intercontraction interval after RTx administration into the bladder. This antinociceptive effect was antagonized by naloxone hydrochloride. Thus, the HSV vector vHPPE encoding hPPE demonstrated physiological improvement in visceral pain induced by bladder irritation. Gene therapy may represent a potentially useful treatment modality for bladder hypersensitive disorders such as bladder pain syndrome/interstitial cystitis.

Targeted drug delivery to the peripheral nervous system using gene therapy.

Gene transfer to target delivery of neurotrophic factors to the primary sensory afferent for treatment of polyneuropathy, or of inhibitory neurotransmitters for relief of chronic pain, offers the possibility of a highly selective targeted release of bioactive molecules within the nervous system. Preclinical studies with non-replicating herpes simplex virus (HSV)-based vectors injected into the skin to transduce neurons in the dorsal root ganglion have demonstrated efficacy in reducing-pain related behaviors in animal models of inflammatory pain, neuropathic pain, and pain caused by cancer, and in preventing progression of sensory neuropathy caused by toxins, chemotherapeutic drugs or resulting from diabetes. Successful completion of the first phase 1 clinical trial of HSV-mediated gene transfer in patients with intractable pain from cancer has set the stage for further clinical trials of this approach.

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 herpes simplex virus vector system for expression of complex cellular cDNA libraries.

Viral vector-based gene expression libraries from normal or diseased tissues offer opportunities to interrogate cellular functions that influence or participate directly in specific biological processes. Here we report the creation and characterization of a herpes simplex virus (HSV)-based expression library consisting of cDNAs derived from PC12 pheochromocytoma cells. A replication-defective HSV vector backbone was engineered to contain both a bacterial artificial chromosome (BAC) and the Invitrogen in vitro Gateway recombination system, creating DBAC-GW. A cDNA library was produced and transferred into the DBAC-GW genome by in vitro recombination and selection in bacteria to produce DBAC-L. DBAC-L contained at least 15,000 unique cDNAs, as shown by DNA array analysis of PCR-amplified cDNA inserts, representing a wide range of cancer- and neuron-related cellular functions. Transfection of the recombinant DBAC-L DNA into complementing animal cells produced more than 1 million DBAC-L virus particles representing the library genes. By microarray analysis of vector-infected cells, we observed that individual members of this vector population expressed unique PC12 cDNA-derived mRNA, demonstrating the power of this system to transfer and express a variety of gene activities. We discuss the potential utility of this and similarly derived expression libraries for genome-wide approaches to identify cellular functions that participate in complex host-pathogen interactions or processes related to disease and to cell growth and development.

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.

Effects of transgene-mediated endomorphin-2 in inflammatory pain.

We examined the analgesic properties of endomorphin-2 expressed in DRG neurons transduced with a non-replicating herpes simplex virus (HSV)-based vector containing a synthetic endomorphin-2 gene construct. HSV-mediated endomorphin-2 expression reduced nocisponsive behaviors in response to mechanical and thermal stimuli after injection of complete Freund's adjuvant (CFA) into the paw, and reduced peripheral inflammation measured by paw swelling after injection of CFA. The analgesic effect of the vector was blocked by either intraperitoneal or intrathecal administration of naloxone methiodide, blocking peripheral and central mu opioid receptors, respectively. Endomorphin-2 vector injection also reduced spontaneous pain-related behaviors in the delayed phase of the formalin test and in both CFA and formalin models suppressed spinal c-fos expression. The magnitude of the vector-mediated analgesic effect on the delayed phase of the formalin test was similar in naïve animals and in animals with opiate tolerance induced by twice daily treatment with morphine, suggesting that there was no cross-tolerance between vector-mediated endomorphin-2 and morphine. These results suggest that transgene-mediated expression of endomorphin-2 in transduced DRG neurons in vivo acts both peripherally and centrally through mu opioid receptors to reduce pain perception.

Protein kinase C epsilon contributes to basal and sensitizing responses of TRPV1 to capsaicin in rat dorsal root ganglion neurons.

Phosphorylation of the vanilloid receptor (TRPV1) by protein kinase C epsilon (PKCepsilon) plays an important role in the development of chronic pain. Here, we employ a highly defective herpes simplex virus vector (vHDNP) that expresses dominant negative PKCepsilon (DNPKCepsilon) as a strategy to demonstrate that PKCepsilon is essential for: (i) maintenance of basal phosphorylation and normal TRPV1 responses to capsaicin (CAPS), a TRPV1 agonist and (ii) enhancement of TRPV1 responses by phorbol esters. Phorbol esters induced translocation of endogenous PKCepsilon to the plasma membrane and thereby enhanced CAPS currents. These results were extended to an in-vivo pain model in which vHDNP delivery to dorsal root ganglion neurons caused analgesia in CAPS-treated, acutely inflamed rat hind paws. These findings support the conclusion that in addition to receptor sensitization, PKCepsilon is essential for normal TRPV1 responses in vitro and in vivo.

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.

An HSV vector system for selection of ligand-gated ion channel modulators.

Pathological alterations of ion channel activity result from changes in modulatory mechanisms governing receptor biology. Here we describe a conditional herpes simplex virus (HSV) replication-based strategy to discover channel modulators whereby inhibition of agonist-induced channel activation by a vector-expressed modulatory gene product prevents ion flux, osmotic shock and cell death. Inhibition of channel activity, in this case, the rat vanilloid (Trpv1 or the glycine receptor (GlyRalpha1), can allow selection of escape vector plaques containing the 'captured' modulatory gene for subsequent identification and functional analysis. We validated this prediction using mixed infections of a wild-type Trpv1 expression vector vTTHR and a nonfunctional 'poreless' Trpv1 subunit-expressing vector, vHP, wherein vHP was highly selected from a large background of vTTHR viruses in the presence of the Trpv1 agonist, capsaicin. The approach should be useful for probing large libraries of vector-expressed cDNAs for the presence of ion channel modulators.

Engineering an endomorphin-2 gene for use in neuropathic pain therapy.

Endomorphin-2 (EM-2) is a carboxy-amidated tetrapeptide that binds the mu-opioid receptor with high affinity and is analgesic in several animal models of pain. Endomorphin peptides have been isolated from bovine and human brain, but no DNA sequences corresponding to a potential preproendomorphin gene have been identified in human genome sequence databases. In this study we designed a tripartite synthetic gene to direct production, cleavage, and amidation of EM-2, and placed the endomorphin gene expression cassette in a replication defective Herpes simplex virus (HSV) vector (vEM2). Biosynthesis of amidated endomorphin-2 peptide was quantified by radioimmunoassay and the identity confirmed by mass spectroscopy following vEM2 transduction of cultured primary dorsal root ganglion neurons. Subcutaneous inoculation of vEM2 resulted in vector delivery to dorsal root ganglion where expression of EM-2 peptide from the engineered gene was confirmed by ELISA. vEM2 delivery provided an analgesic effect in the spinal nerve ligation model of neuropathic pain measured by reduction of mechanical allodynia and thermal hyperalgesia. The analgesic effect of vEM2 was blocked by intrathecal delivery of the mu-receptor antagonist CTOP. The gene construct design described represents a broadly useful platform for biosynthesis and delivery of carboxy-amidated peptides for therapeutic and experimental purposes, and the results demonstrate that HSV-gene transfer to sensory neurons provides an effective means to achieve local biosynthesis of endomorphin peptides for the treatment of chronic pain.

Hyperactive Himar1 transposase mediates transposition in cell culture and enhances gene expression in vivo.

The use of nonviral delivery systems results in transient gene expression, in part because of the low efficiency of DNA integration. Previously, vectors based on transposon systems such as Sleeping Beauty have been shown to be able to increase stable transfection efficiencies in cell culture and in animal models. Himar1, a reconstructed active transposon belonging to the Tc1/mariner superfamily, also has been used as a vector for stable gene delivery, but the rate of transposition after transfection is low. In this paper, we evaluate the potential of the hyperactive Himar1 transposase C9, in combination with the Himar1 inverted repeat transposon, as a gene delivery vector. The C9 transposase is a hyperactive mutant of Himar1 with two amino acid substitutions, Q131R and E137K, that result in an increase in activity relative to wild type. Here we demonstrate that cotransfection of the C9 transposase with a Himar1-based vector increases the frequency of stable gene expression in human cells in a transposase concentration-dependent manner. In addition, we establish that C9 transposase mediates integration of the transgene in mammalian cells at a frequency similar to that of Sleeping Beauty under some of the conditions tested. Last, we show significantly higher levels of reporter gene expression in vivo in mouse liver and in synovium of rabbit knee joints after injection of the transposon plasmid expressing the transgene and the C9 transposase. These data suggest that vectors based on the Himar1 transposable element, in conjunction with the hyperactive mutant transposase C9, may be suitable vectors for gene therapy applications.

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.

Long-term neuroprotection achieved with latency-associated promoter-driven herpes simplex virus gene transfer to the peripheral nervous system.

We examined the ability of the herpes simplex virus (HSV) latency-associated promoter (LAP2) to drive biologically relevant prolonged transgene expression in the peripheral nervous system. Rat dorsal root ganglia were transduced in vivo by subcutaneous inoculation of replication-incompetent HSV-based vectors containing nerve growth factor (NGF) or neurotrophin-3 (NT-3) under the control of LAP2 (vectors SLN and QLNT3, respectively) and vector SHN expressing NGF under the control of the human cytomegalovirus immediate early promoter. Twenty-four weeks later a pure sensory neuropathy was induced by overdose of pyridoxine (PDX), and the animals were assessed 6 months after inoculation. Inoculation of SLN, but not SHN, attenuated the nerve damage caused by PDX and protected foot sensory amplitude, H-wave amplitude, and behavioral measures of proprioceptive function. QLNT3 was more effective than SLN in preserving the largest myelinated fibers from degeneration. These results indicate that expression of NGF or NT-3 driven by LAP2 is sufficient to prevent the development of neuropathy 6 months after vector inoculation in rats.

Inhibition of translation initiation by volatile anesthetics involves nutrient-sensitive GCN-independent and -dependent processes in yeast.

Volatile anesthetics including isoflurane affect all cells examined, but their mechanisms of action remain unknown. To investigate the cellular basis of anesthetic action, we are studying Saccharomyces cerevisiae mutants altered in their response to anesthetics. The zzz3-1 mutation renders yeast isoflurane resistant and is an allele of GCN3. Gcn3p functions in the evolutionarily conserved general amino acid control (GCN) pathway that regulates protein synthesis and gene expression in response to nutrient availability through phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha). Hyperphosphorylation of eIF2alpha inhibits translation initiation during amino acid starvation. Isoflurane rapidly (in <15 min) inhibits yeast cell division and amino acid uptake. Unexpectedly, phosphorylation of eIF2alpha decreased dramatically upon initial exposure although hyperphosphorylation occurred later. Translation initiation was inhibited by isoflurane even when eIF2alpha phosphorylation decreased and this inhibition was GCN-independent. Maintenance of inhibition required GCN-dependent hyperphosphorylation of eIF2alpha. Thus, two nutrient-sensitive stages displaying unique features promote isoflurane-induced inhibition of translation initiation. The rapid phase is GCN-independent and apparently has not been recognized previously. The maintenance phase is GCN-dependent and requires inhibition of general translation imparted by enhanced eIF2alpha phosphorylation. Surprisingly, as shown here, the transcription activator Gcn4p does not affect anesthetic response.

Gene transfer of glutamic acid decarboxylase reduces neuropathic pain.

We tested whether transfer of the gene coding for glutamic acid decarboxylase to dorsal root ganglion using a herpes simplex virus vector to achieve release of GABA in dorsal horn would attenuate nociception in this condition. Subcutaneous inoculation of a replication-defective herpes simplex virus vector expressing glutamic acid decarboxylase (vector QHGAD67) 7 days after selective L5 spinal nerve ligation reversed mechanical allodynia and thermal hyperalgesia; the antiallodynic effect lasted 6 weeks and was reestablished by reinoculation. QHGAD67 inoculation also suppressed induction of c-Fos and phosphorylated extracellular signal-regulated kinase 1 and 2 in the spinal cord.

Improvement in erectile dysfunction after neurotrophic factor gene therapy in diabetic rats.

PURPOSE: Erectile dysfunction (ED) is a common but difficult to treat complication of diabetes mellitus (DM). We have previously reported herpes simplex virus (HSV) vector mediated delivery of nerve growth factor into the bladder to treat diabetic cystopathy and neurotrophin-3 (NT3) gene transfer for pyridoxine treatment. Nerve growth factor and NT3 are neurotrophic factors that may protect nerves from mechanical and metabolic damage. We investigated the effects of HSV mediated delivery of NT3 for the treatment of diabetic ED. MATERIALS AND METHODS: Male Sprague-Dawley rats weighing 300 to 400 gm were injected with 65 mg/kg streptozotocin to induce DM. After 4 weeks 20 microl containing 5 x 10 pfu replication defective HSV vector expressing lacZ (6 rats) or NT3 (6) were injected directly into the cavernous nerve sheath with a 30 gauge needle. Four weeks later the animals underwent measurement of intracavernous pressure under electrical stimulation (20 Hz, 0.5 millisecond and 10 V) of the cavernous nerve. Staining for lacZ and neuronal nitric oxide synthase in the major pelvic ganglia was also performed. RESULTS: beta-Galactosidase staining revealed lacZ positive neurons in the major pelvic ganglia. Maximal intracavernous pressure induced by electrical stimulation showed statistically significant mean values +/- SEM of 15.1 +/- 2.1 and 43.8 +/- 11.1 cm H2O in the lacZ and NT3 vector injected groups, respectively (p = 0.03). The mean number of neuronal nitric oxide synthase positive neurons per section in the NT3 group was significantly higher than that in the lacZ control group at 3.33 +/- 0.23 and 0.64 +/- 0.14 neurons per high power field, respectively (p < 0.001). CONCLUSIONS: We report that gene therapy for the treatment of diabetic ED is feasible with HSV vectors. NT3 gene therapy may be applicable for the treatment of ED induced by DM.