With development and application of direct gene transfer systems for the potential treatment of neurodegenerative disease, several considerations related to vector safety and gene product expression quickly become apparent. The current proposal will examine whether the innate andl/or cellular arms of the irnmune system are activated following placement of a transgene within the rnouse CNS? Two specific aims will be addressed. The first specific aim seeks to deterrnine the immunopathology within the mouse striatum for 3 HSV-1 based vectors: 1) lHSV-lL plasmid based amplicon generated using a replication deficient helper virus; 2) a plasmid-based HSV-1 amplicon produced in a helper virus-free systen about. In both cases, the vector will express lacZ as a novel reporter gene. 3) A third plasmid based HSV-1 amplicon produced in a helper virus-free system and expressing mouse intestinal alkaline phosphatase (iAP) will be exarnined. The use of iAP as the reporte:r gene will allow for easy histochemical localization of a non CNS protein similar to 1acZ but one that should not be immunogenic. The effects of pre-immunization with HSV will also be examined. Mouse brains will be analyzed using both molecular analysis (quantitative PGR, RT-PCR), immunocytochernistry and enzyme assay to correlate t]he inflammatory :response to the viral o:r transgene proteins to transgene expressior. Analyses will examie pro-inflammatory and inhibitory cytokines about chemokines, adhesion molecules, blood-brain barrier irtegrity and transgene expression and survival. The second specific aim will examine the role of the innate immune response in li aboutting transfection efficiency. The innate immnune response (consisting of non-specific phagocytic neutrophils and monocytes/maGrophages and driven by the alternative complement pathway) will be examined following injection of the HSViAP amplicon. The response will be altered in several ways. First, the selective rernoval of either neutrophils or circulating monocytes/macrophages will be accomplished using specific antibodies to be each cell type injected immediately before during and after infusion of the arnplicon vecto: Secondly, we will attempt to inhibit adhesion and limit diapedesis utrophils/monocytes/macrophages into the transfection area using specific antibodies to the adhesion molecules P-selectin and ICAM- 1. Third, we wil1 attempt to prevent or limit induction of pro-inflammatory cytokines, chemokines and adhesion molecules co-expressing the inhilbitory cytokine IL-10 in our HSVi AP vector. Finally, the role of complement in the innate immune response will be tested by injecting recombinant Crry-Ig ip an attempt to block the role of chemoattractants and activators of irnmune cells. The effectiveness of each manipulation will be assayed with the same methods used in Specific Aim 1.
| Howard, Michael J; Liu, Su; Schottler, Frank et al. (2005) Transplantation of apoptosis-resistant embryonic stem cells into the injured rat spinal cord. Somatosens Mot Res 22:37-44 |
| Hurley, S D; O'Banion, M K; Song, D D et al. (2003) Microglial response is poorly correlated with neurodegeneration following chronic, low-dose MPTP administration in monkeys. Exp Neurol 184:659-68 |
| Olschowka, John A; Bowers, William J; Hurley, Sean D et al. (2003) Helper-free HSV-1 amplicons elicit a markedly less robust innate immune response in the CNS. Mol Ther 7:218-27 |
| Hurley, Sean D; Olschowka, John A; O'Banion, M Kerry (2002) Cyclooxygenase inhibition as a strategy to ameliorate brain injury. J Neurotrauma 19:1-15 |
