We have been working on a CNS delivery system for cytokine genes based on viral vectors. An ideal CNS delivery tool should be able to reach different CNS areas (MS is a multifocal disease with a preferential periventricular distribution), should persist long enough within the CNS, should be completely non-toxic for CNS resident cells and should be unable to induce peripheral side-effects. We developed a novel system to deliver cytokine genes into the CNS based on the use of non-replicative HSV type 1-derived vectors. These vectors are considered an alternative to classical retroviral and adenoviral vectors, mainly because HSV is able to accommodate multiple foreign genes and to infect postmitotic cells, such as neurons.21-24 We used an HSV-1-derived vector named d120,24 25 obtained from the HSV-1 Kos strain by deletion of both copies of the IE ICP4 gene. In this non-replicating mutant, we inserted the murine IL-4 gene, driven by the ICP4 promoter, into the thymi-dine kinase locus. In vivo preliminary experiments indicated that d120 is easily transferred within the CNS, diffuses consistently in all cerebrospinal fluid (CSF) spaces, and is able to efficiently infect the layer of ependymal cells surrounding the ventricles as well as choroidal and leptomeningeal cells. Within the CNS, the HSV-1 vector redirects the infected cell machinery to produce discrete amounts of the cytokine in the CSF of mice up to 28 days post-injec-tion.26 We then used the IL-4-containing vector as a therapeutic tool in EAE mice. IL-4 was administered intracerebrally in Biozzi AB/H mice immunized with myelin oligodendrocyte glycoprotein (MOG)40-55 before27 and after28 the appearance of EAE signs. No toxic reactions were observed. A significant amelioration of the clinical and pathological CNS features of EAE was observed with both therapeutic protocols. The protective effect of this therapy was mostly due to the ability of IL-4 to downregu-late in situ production of pro-inflammatory chemokines (monocyte chemoattractant chemokines, and RANTES) and pro-inflammatory cytokines (i.e. IL-1p, TNF-a). Furthermore, we found that lymph node cells from IL-4-treated versus non-treated mice were able to process and proliferate in response, to the encephalitogenic antigen as well as to drive an appropriate Th1 response, thus indicating a lack of interference of the IL-4 gene therapy approach we used on the proper functioning of the immune system in the periphery.
We also used another non-replicative HSV-1-derived viral vector created in an HSV-1 triple deletion mutant backbone lacking the two copies of the ICP4 gene, the ICP27 gene, and the ICP22 gene and containing the mouse IL-1 receptor antagonist (IL-1ra) gene.23 The deletion of the ICP4, ICP22 and ICP27 genes, which are essential for viral in vitro and in vivo replication and viral cytotoxicity, reduces considerably the intrinsic cytotoxicity of this HSV-1-derived mutant.23 After assessing the proper in vivo distribution of the vector into naive mice, we intracerebrally injected the IL-1ra-containing HSV-1-derived vector into C57/BL6 mice immunized with MOG35-55. The treatment was performed either before or after the onset of EAE. The results (R. Furlan, unpublished) indicate that the IL-1ra vector was able to significantly delay the onset and reduce the severity of EAE when preventively injected, but not when the vector was injected after EAE onset. No interference with the proper functioning of the peripheral immune system has been observed when using the IL-1ra-containing vector.
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