Peggy Marconi

Deletion of multiple immediate-early genes from herpes simplex virus reduces cytotoxicity and permits long-term gene expression in neurons.

Herpes simplex virus type 1 (HSV-1) has many attractive features that suggest its utility for gene transfer to neurons. However, viral cytotoxicity and transient transgene expression limit practical applications even in the absence of viral replication. Mutant viruses deleted for the immediate–early (IE) gene, ICP4, an essential transcriptional transactivator, are toxic to many cell types in culture in which only the remaining IE genes are expressed. In order to test directly the toxicity of other IE gene products in neurons and develop a mutant background capable of long-term transgene expression, we generated mutants deleted for multiple IE genes in various combinations and tested their relative cytotoxicity in 9L rat gliosarcoma cells, Vero monkey kidney cells, and primary rat cortical and dorsal root neurons in culture. Viral mutants deleted simultaneously for the IE genes encoding ICP4, ICP22 and ICP27 showed substantially reduced cytotoxicity compared with viruses deleted for ICP4 alone or ICP4 in combination with either ICP22, ICP27 or ICP47. Infection of neurons in culture with these triple IE deletion mutants substantially enhanced cell survival and permitted transgene expression for over 21 days. Such mutants may prove useful for efficient gene transfer and extended transgene expression in neurons in vitro and in vivo.

Intrathecal Delivery of IFN-γ Protects C57BL/6 Mice from Chronic-Progressive Experimental Autoimmune Encephalomyelitis by Increasing Apoptosis of Central Nervous System-Infiltrating Lymphocytes

The exclusive detrimental role of proinflammatory cytokines in demyelinating diseases of the CNS, such as multiple sclerosis, is controversial. Here we show that the intrathecal delivery of an HSV-1-derived vector engineered with the mouse IFN-γ gene leads to persistent (up to 4 wk) CNS production of IFN-γ and inhibits the course of a chronic-progressive form of experimental autoimmune encephalomyelitis (EAE) induced in C57BL/6 mice by myelin oligodendrocyte glycoprotein (MOG)35–55. Mice treated with the IFN-γ-containing vector before EAE onset showed an earlier onset but a milder course of the disease compared with control mice treated with the empty vector. In addition, 83% of IFN-γ-treated mice completely recovered within 25 days post immunization, whereas control mice did not recover up to 60 days post immunization. Mice treated with the IFN-γ-containing vector within 1 wk after EAE onset partially recovered from the disease within 25 days after vector injection, whereas control mice worsened. Recovery from EAE in mice treated with IFN-γ was associated with a significant increase of CNS-infiltrating lymphocytes undergoing apoptosis. During the recovery phase, the mRNA level of TNFR1 was also significantly increased in CNS-infiltrating cells from IFN-γ-treated mice compared with controls. Our results further challenge the exclusive detrimental role of IFN-γ in the CNS during EAE/multiple sclerosis, and indicate that CNS-confined inflammation may induce protective immunological countermechanisms leading to a faster clearance of encephalitogenic T cells by apoptosis, thus restoring the immune privilege of the CNS.

Development of herpes simplex virus replication-defective multigene vectors for combination gene therapy applications

Some gene therapy applications will require simultaneous expression of multiple gene products to achieve a therapeutic effect. In this study we describe the generation and characterization of replication incompetent herpes simplex virus type 1 (HSV-1) vectors (HX86Z or HX86G) carrying distinct and independently regulated expression cassettes for five transgenes (hIL-2, hGM-CSF, hB7.1, HSV-tk and lacZ or hIFNγ). The transgenes, representing 12 kb of DNA sequence, were recombined into separate loci of a single mutant virus vector deleted for 11.6 kb of vector sequences representing portions of nine viral genes, ICP4, ICP22, ICP27, ICP47, UL24, UL41, UL44, US10 and US11. Deletion of the immediate–early genes ICP4, ICP22 and ICP27 substantially reduced vector cytotoxicity, prevented early and late viral gene expression and left intact MHC class I antigen expression. Simultaneous expression of multiple transgenes was obtained for up to 7 days in primary human melanoma cells with peak expression at 2–3 days after infection. The transgenes were chosen for their potential to function synergistically in tumor destruction and vaccine gene therapy applications, but the method and vector employed could be applied to other multigene therapy strategies. This study demonstrates the potential for engineering large transgene capacity DNA viruses such as HSV-1 for expression of multiple transgenes.

Localized delivery of fibroblast growth factor–2 and brain-derived neurotrophic factor reduces spontaneous seizures in an epilepsy model

A loss of neurons is observed in the hippocampus of many patients with epilepsies of temporal lobe origin. It has been hypothesized that damage limitation or repair, for example using neurotrophic factors (NTFs), may prevent the transformation of a normal tissue into epileptic (epileptogenesis). Here, we used viral vectors to locally supplement two NTFs, fibroblast growth factor–2 (FGF-2) and brain-derived neurotrophic factor (BDNF), when epileptogenic damage was already in place. These vectors were first characterized in vitro, where they increased proliferation of neural progenitors and favored their differentiation into neurons, and they were then tested in a model of status epilepticus-induced neurodegeneration and epileptogenesis. When injected in a lesioned hippocampus, FGF-2/BDNF expressing vectors increased neuronogenesis, embanked neuronal damage, and reduced epileptogenesis. It is concluded that reduction of damage reduces epileptogenesis and that supplementing specific NTFs in lesion areas represents a new approach to the therapy of neuronal damage and of its consequences.

Rapid method for construction of recombinant HSV gene transfer vectors

Herpes simplex virus type 1 (HSV-1) is a neurotrophic human pathogen that naturally persists in neurons in a latent state and carries a large number of viral functions which can be replaced by foreign genes to create a vector for gene therapy applications. In this report we describe a two-step method for insertion/deletion mutagenesis of HSV genes and the efficient insertion of transgenes into these locations in the viral genome. The first step is the insertion of a reporter gene (lacZ) cassette flanked by PacI restriction enzyme sites not otherwise found in the viral genome, using standard marker transfer procedures to interrupt a portion of the target HSV gene. The second step is substitution of the reporter gene with other foreign cDNAs by digestion of the vector DNA with PacI to remove the lacZ gene and subsequent repair of the vector genome by homologous recombination with a transgene expression plasmid. Potential recombinants indentified by a ‘clear plaque’ phenotype after X-gal staining arose at high frequency (80–100%). Of these, recombinants containing the transgene in place of the lacZ gene ranged from 19–65%. Insertion of the transgene expression construct into the viral genome eliminates the PacI sites, allowing this method to be used repeatedly for the sequential deletion of multiple HSV genes while inserting multiple transgenes. This procedure was repeated in succession to produce a vector carrying two independent expression cassettes at distinct viral loci.

Fibroblast growth factor-II gene therapy reverts the clinical course and the pathological signs of chronic experimental autoimmune encephalomyelitis in C57BL/6 mice.

The development of therapies aimed to promote remyelination is a major issue in chronic inflammatory demyelinating disorders of the central nervous system (CNS) such as multiple sclerosis (MS), where the permanent neurological impairment is due to the axonal loss resulting from recurrent episodes of immune-mediated demyelination. Here, we show that the intrathecal injection of a herpes simplex virus (HSV) type-1 replication-defective multigene vector, engineered with the human fibroblast growth factor (FGF)-II gene (TH:bFGF vector), was able to significantly revert in C57BL/6 mice the clinicopathological signs of chronic experimental autoimmune encephalomyelitis (EAE), the animal model of MS. The treatment with the TH:bFGF vector was initiated within 1 week after the clinical onset of EAE and was effective throughout the whole follow-up period (ie 60 days). The disease-ameliorating effect in FGF-II-treated mice was associated with: (1) CNS production of FGF-II from vector-infected cells which were exclusively located around the CSF space (ependymal, choroidal and leptomeningeal cells); (2) significant decrease (P < 0.01) of the number of myelinotoxic cells (T cells and macrophages) both in the CNS parenchyma and in the leptomeningeal space; and (3) significant increase (P < 0.01) of the number of oligodendrocyte precursors and of myelin-forming oligodendrocytes in areas of demyelination and axonal loss. Our results indicate that CNS gene therapy using HSV-1-derived vector coding for neurotrophic factors (ie FGF-II) is a safe and non-toxic approach that might represent a potential useful ‘alternative’ tool for the future treatment of immune-mediated demyelinating diseases.

Central nervous system gene therapy with interleukin-4 inhibits progression of ongoing relapsing-remitting autoimmune encephalomyelitis in Biozzi AB/H mice.

Multiple sclerosis (MS) is an immune-mediated inflammatory disease of the central nervous system (CNS) that might benefit from anti-inflammatory therapies. However, systemic delivery of anti-inflammatory drugs in MS patients has so far been disappointing, mostly due to the limited capacity of these molecules to enter the CNS. We injected into the cisterna magna (i.c.) of Biozzi AB/H mice affected by a relapsing–remitting form of experimental autoimmune encephalomyelitis (EAE), the animal model of MS, a non-replicative herpes simplex virus (HSV) type-1-derived vector containing the interleukin (IL)-4 gene (d120:LacZ:IL-4). CNS delivery of the d120:LacZ:IL-4 vector, after EAE onset, induced the in situ production of IL-4 by CNS-resident cells facing the cerebrospinal fluid (CSF) spaces and reduced by 47% (P < 0.02) the disease-related deaths. compared with mice treated with the control d120:lacz vector, il-4-treated mice also showed a shorter duration of the first eae attack, a longer inter-relapse period, and a reduction in the severity and duration of the first relapse. protection from eae progression in il-4-treated mice was associated with activation of microglia in spinal cord areas where mrna content of the pro-inflammatory chemokines, macrophage chemoattractant protein-1 (mcp-1) and rantes, was reduced and that of the anti-inflammatory cytokine il-4 was increased. finally, cns-infiltrating mononuclear cells from il-4-treated mice produced lower levels of mcp-1 mrna compared with control mice. our results, showing that il-4 gene delivery using hsv-1 vectors induces protection from eae by in situ modulating the cytokine/chemokine-mediated circuits sustaining effector cell functions, indicate that the intrathecal ‘therapeutic’ use of nonreplicative hsv-1-derived vectors containing anti-inflammatory molecules might represent an alternative strategy in inflammatory diseases of the cns.

Hippocampal FGF-2 and BDNF overexpression attenuates epileptogenesis-associated neuroinflammation and reduces spontaneous recurrent seizures.

Under certain experimental conditions, neurotrophic factors may reduce epileptogenesis. We have previously reported that local, intrahippocampal supplementation of fibroblast growth factor-2 (FGF-2) and brain-derived neurotrophic factor (BDNF) increases neurogenesis, reduces neuronal loss, and reduces the occurrence of spontaneous seizures in a model of damage-associated epilepsy. Here, we asked if these possibly anti-epileptogenic effects might involve anti-inflammatory mechanisms. Thus, we used a Herpes-based vector to supplement FGF-2 and BDNF in rat hippocampus after pilocarpine-induced status epilepticus that established an epileptogenic lesion. This model causes intense neuroinflammation, especially in the phase that precedes the occurrence of spontaneous seizures. The supplementation of FGF-2 and BDNF attenuated various parameters of inflammation, including astrocytosis, microcytosis and IL-1β expression. The effect appeared to be most prominent on IL-1β, whose expression was almost completely prevented. Further studies will be needed to elucidate the molecular mechanism(s) for these effects, and for that on IL-1β in particular. Nonetheless, the concept that neurotrophic factors affect neuroinflammation in vivo may be highly relevant for the understanding of the epileptogenic process.

HSV Recombinant Vectors for Gene Therapy

The very deep knowledge acquired on the genetics and molecular biology of herpes simplex virus (HSV), has allowed the development of potential replication-competent and replication-defective vectors for several applications in human healthcare. These include delivery and expression of human genes to cells of the nervous systems, selective destruction of cancer cells, prophylaxis against infection with HSV or other infectious diseases, and targeted infection to specific tissues or organs. Replication-defective recombinant vectors are non-toxic gene transfer tools that preserve most of the neurotropic features of wild type HSV-1, particularly the ability to express genes after having established latent infections, and are thus proficient candidates for therapeutic gene transfer settings in neurons. A replication-defective HSV vector for the treatment of pain has recently entered in phase 1 clinical trial. Replication-competent (oncolytic) vectors are becoming a suitable and powerful tool to eradicate brain tumours due to their ability to replicate and spread only within the tumour mass, and have reached phase II/III clinical trials in some cases. The progress in understanding the host immune response induced by the vector is also improving the use of HSV as a vaccine vector against both HSV infection and other pathogens. This review briefly summarizes the obstacle encountered in the delivery of HSV vectors and examines the various strategies developed or proposed to overcome such challenges.

Development of Replication-Defective Herpes Simplex Virus Vectors

Numerous diseases of the nervous system result from single gene or multifactorial gene defects such as cancer, immune pathological disorders, metabolic diseases, and common neurodegenerative syndromes (Parkinsons and Alzheimers diseases). A greater understanding of the molecular, biochemical, and genetic factors involved in the progression of a specific disease state has led to the development of genetic therapies using direct gene transfer to ameliorate the disease condition or correct a genetic defect in situ. Standard gene therapeutic approaches employing retroviruses have not proven feasible for treating disorders of the central nervous system (CNS) since these vectors require dividing cells for integration and expression of the transgene, whereas CNS neurons are postmitotic, terminally differentiated cells. Thus, methods for delivery and expression of therapeutic gene products to treat CNS disease will require new delivery strategies and vehicles including the development of novel vectors for direct gene transfer. These vectors should: efficiently deliver the therapeutic gene(s) to a sufficient number of nondividing neurons; persist long-term in a nonintegrated state within the nerve cell nucleus without disturbing host cell functions; and be able to regulate therapeutic gene expression for diseases that may either require high-level transient transgene expression or continuous low level synthesis of the therapeutic product.