CONSTRUCTION OF REPLICATION-DEFECTIVE HERPES SIMPLEX VIRAL VECTORS FOR TARGETING THE LHX2 GENE IN THE CENTRAL NERVOUS SYSTEM

Epilepsy is a chronic disorder affecting about 65 million people worldwide and temporal lobe epilepsy (TLE) is among the most frequent types of intractable epilepsy. In most cases the causes of TLE are unknown but it is believed that it may take place after an initial precipitating injury (IPI) such as brain tumor, ictus, head trauma, meningitis, encephalitis, and febrile seizures during childhood. Despite the development of new antiepileptic drugs (AEDs), about 35% of epileptic patients still suffer from pharmacoresistant seizures, with surgical resection of the epileptic locus as possible last option. In addition, AEDs don’t prevent the progression of the disease but they are designed only for treatment of patients with an already established syndrome. Hence, there is an unmet medical need for the prevention of seizures for the patients at high-risk of developing epilepsy. This clarify how urgent is the need to find novel therapeutic concepts to fill this gap. Epilepsy could develop when the intracerebral balance between excitation and inhibitory neurotransmission is impaired. Experimental findings show that after an epileptogenic insult the brain react to the injury with an enhanced hippocampal neurogenesis as a homotypic response to the neuronal loss in an attempt to restore the pre-existing cellular network. However, this plastic remodeling that the brain goes through is usually aberrant, since the cells that undergo the replacement of degenerating neurons upon severe brain injury are mostly high proliferating reactive astrocytes. This leads to important alterations of brain signals and, consequently, high risk of seizure development. Starting from this concept, we hypothesize that controlling the neural stem cells fate after an initial precipitating injury could prevent epileptogenesis or at least improve the clinical picture of the patient. Based on the recent literature, we decided to test the effects of Lhx2 protein overexpression on cells of central nervous system. Lhx2 is a transcription factor that plays a crucial role since early stages in telencephalic patterning but its function is not limited to the early embryonic neuroepithelium: recent evidences have shown a unique role for this protein in the phase of active neurogenesis, when its overexpression may enhances and prolongs the neurogenesis to generate neurons from progenitors that would otherwise give rise to astrocytes. The recent advances of gene therapy promise innovative and revolutionary new treatments for neurological disorders. Various methods have been developed for gene delivery to target cells. However, gene transfer by viral vectors is thus far the widest used approach. In particular, up today the most efficient systems to achieve a long term transgene expression is based upon retroviral and lentiviral vectors. Unfortunately both these viruses cannot be designed for clinical applications since their infections result in insertion of viral DNA into the host chromosomes at an unpredictable position, a dangerous event which can seriously disturbs cellular genes functions potentially leading to cancer transformation of infected cells. It is then important to set up novel tools to safely deliver genes. Herpes simplex virus-1 (HSV-1) offers unique features that support its development as a great candidate viral vector especially for targeting the nervous system: it is a highly infectious, naturally neurotropic virus able to establish life-long latency in neurons, along with the largest capacity for exogenous DNA cloning. Moreover, it doesn’t integrate into the host genome, avoiding any possibility of insertional activation or inactivation of cellular genes. However, some technical problems still need to be overcome, such as the efficient delivery of the vector to target cells, the maintenance and control of foreign gene expression, and the control of unwanted host immune responses. This thesis describes the development of a highly efficient method for in vitro and in vivo targeting of the Lhx2 gene using novel replication-defective herpes simplex viral vectors, named JΔβββ4 and JΔΝΙ, opportunely engineered to reduce the innate toxicity of the virus and to allow a good expression of the transgene. HSV-mediated delivery of Lhx2 resulted in highly effective gene overexpression in several cell types in vitro, including mouse neuronal and non-neuronal cells, along with reduced or null toxicity and a differential transgene expression, depending on the viral backbones. These vectors have been additionally tested in vivo by injection into the hippocampus of naïve rats and of rat models of epilepsy. Ex vivo analyses of injected brains showed good infection pattern from both viruses along with no evident toxicity. Moreover, the hippocampal delivery of Lhx2 by JΔβββ4-based vector was associated with reductions of both astrocyte density and recurring seizures, giving rise to more favorable pathologic features and improved outcomes. Put together, we can finally assess that both the JΔβββ4 and JΔΝΙ-based vectors could represent useful tools for differential purposes: while for in vitro applications the JΔΝΙ vector is the best compromise between transgene expression and low toxicity effects, for in vivo gene transfer it result almost ineffective. On the other hand, the JΔβββ4 vector displayed an opposite behavior, too toxic for in vitro approaches but much more effective for gene delivery in vivo

322. Benign Herpes Simplex Virus Vector Design for Efficient Delivery of Large or Multiple Transgenes To a Diversity of Cells

Viral vectors derived from herpes simplex virus (HSV) have the potential to revolutionize gene therapy due to their ability to accommodate large and multiple therapeutic transgenes. However, current HSV gene therapy vectors express toxic levels of an immediate-early (IE) protein, ICP0, whose function is required for robust and sustained transgene expression. Here we report the development of a new generation of HSV vectors that are IE-gene independent and non-toxic, yet capable of persistent transgene expression in a variety of human primary non-neuronal cell types. We identified a CTCF motif cluster upstream of the latency promoter and a known long-term regulatory region as key elements for the protection of transgene expression cassettes from global silencing of the viral genome in the absence of all viral IE gene products. Using this new HSV vector system, we have observed vigorous expression of full-length dystrophin cDNA (14 kb) for several weeks in a dystrophin-deficient muscle cell line. We further tested our vectors for transgene expression in rodent brain. While we detected variable persistence of gene expression from the latency locus, we were surprised to observe vigorous long-term reporter gene expression from one other locus despite the absence of gene expression from this locus in non-neuronal cells. These findings demonstrate that transgene expression in neurons is operatively different from that in non-neuronal cells and suggest that multiple loci can be used for expression of foreign genes in the nervous system. In addition, our data raise the prospect that our highly defective HSV vector system will be applicable as a safe delivery tool for large and multiple therapeutic genes to a wide range of non-neuronal tissues

Deletion of the Virion Host Shut-off Gene Enhances Neuronal-Selective Transgene Expression from an HSV Vector Lacking Functional IE Genes

The ability of herpes simplex virus (HSV) to establish lifelong latency in neurons suggests that HSV-derived vectors hold promise for gene delivery to the nervous system. However, vector toxicity and transgene silencing have created significant barriers to vector applications to the brain. Recently, we described a vector defective for all immediate-early gene expression and deleted for the joint region between the two unique genome segments that proved capable of extended transgene expression in non-neuronal cells. Sustained expression required the proximity of boundary elements from the latency locus. As confirmed here, we have also found that a transgene cassette introduced into the ICP4 locus is highly active in neurons but silent in primary fibroblasts. Remarkably, we observed that removal of the virion host shutoff (vhs) gene further improved transgene expression in neurons without inducing expression of viral genes. In rat hippocampus, the vhs-deleted vector showed robust transgene expression exclusively in neurons for at least 1 month without evidence of toxicity or inflammation. This HSV vector design holds promise for gene delivery to the brain, including durable expression of large or complex transgene cassettes

New Tools for Epilepsy Therapy

One third of the epilepsies are refractory to conventional antiepileptic drugs (AEDs) and, therefore, identification of new therapies is highly needed. Here, we briefly describe two approaches, direct cell grafting and gene therapy, that may represent alternatives to conventional drugs for the treatment of focal epilepsies. In addition, we discuss more in detail some new tools, cell based-biodelivery systems (encapsulated cell biodelivery (ECB) devices) and new generation gene therapy vectors, which may help in the progress toward clinical translation. The field is advancing rapidly, and there is optimism that cell and/or gene therapy strategies will soon be ready for testing in drug-resistant epileptic patients

A Matter of Genes: The Hurdles of Gene Therapy for Epilepsy

Gene therapy has recently advanced to the level of standard of care for several diseases. However, its application to neurological disorders is still in the experimental phase. In this review, we discuss recent advancements in the field that provide optimism on the possibility to have first-in-human studies for gene therapy of some forms of epilepsy in the not so distant future

Gene Therapy Tools for Brain Diseases

Neurological disorders affecting the central nervous system (CNS) are still incompletely understood. Many of these disorders lack a cure and are seeking more specific and effective treatments. In fact, in spite of advancements in knowledge of the CNS function, the treatment of neurological disorders with modern medical and surgical approaches remains difficult for many reasons, such as the complexity of the CNS, the limited regenerative capacity of the tissue, and the difficulty in conveying conventional drugs to the organ due to the blood-brain barrier. Gene therapy, allowing the delivery of genetic materials that encodes potential therapeutic molecules, represents an attractive option. Gene therapy can result in a stable or inducible expression of transgene(s), and can allow a nearly specific expression in target cells. In this review, we will discuss the most commonly used tools for the delivery of genetic material in the CNS, including viral and non-viral vectors; their main applications; their advantages and disadvantages. We will discuss mechanisms of genetic regulation through cell-specific and inducible promoters, which allow to express gene products only in specific cells and to control their transcriptional activation. In addition, we will describe the applications to CNS diseases of post-transcriptional regulation systems (RNA interference); of systems allowing spatial or temporal control of expression [optogenetics and Designer Receptors Exclusively Activated by Designer Drugs (DREADDs)]; and of gene editing technologies (CRISPR/Cas9, Zinc finger proteins). Particular attention will be reserved to viral vectors derived from herpes simplex type 1, a potential tool for the delivery and expression of multiple transgene cassettes simultaneously

NPY and Gene Therapy for Epilepsy: How, When,... and Y

Neuropeptide Y (NPY) is a neuropeptide abundantly expressed in the mammalian central and peripheral nervous system. NPY is a pleiotropic molecule, which influences cell proliferation, cardiovascular and metabolic function, pain and neuronal excitability. In the central nervous system, NPY acts as a neuromodulator, affecting pathways that range from cellular (excitability, neurogenesis) to circuit level (food intake, stress response, pain perception). NPY has a broad repertoire of receptor subtypes, each activating specific signaling pathways in different tissues and cellular sub-regions. In the context of epilepsy, NPY is thought to act as an endogenous anticonvulsant that performs its action through Y2 and Y5 receptors. In fact, its overexpression in the brain with the aid of viral vectors can suppress seizures in animal models of epilepsy. Therefore, NPY-based gene therapy may represent a novel approach for the treatment of epilepsy patients, particularly for pharmaco-resistant and genetic forms of the disease. Nonetheless, considering all the aforementioned aspects of NPY signaling, the study of possible NPY applications as a therapeutic molecule is not devoid of critical aspects. The present review will summarize data related to NPY biology, focusing on its anti-epileptic effects, with a critical appraisal of key elements that could be exploited to improve the already existing NPY-based gene therapy approaches for epilepsy

New tools for epilepsy therapy

One third of the epilepsies are refractory to conventional antiepileptic drugs (AEDs) and, therefore, identification of new therapies is highly needed. Here, we briefly describe two approaches, direct cell grafting and gene therapy, that may represent alternatives to conventional drugs for the treatment of focal epilepsies. In addition, we discuss more in detail some new tools, cell based-biodelivery systems (encapsulated cell biodelivery (ECB) devices) and new generation gene therapy vectors, which may help in the progress toward clinical translation. The field is advancing rapidly, and there is optimism that cell and/or gene therapy strategies will soon be ready for testing in drug-resistant epileptic patients