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.