Project II: """"""""Gene Transfer Vector Safety/Efficacy in Mice"""""""" proposes to optimize gene transfer safety and efficacy using specific murine models. Adenoviral vectors will be emphasized initially because of their imminent use in human trials, but similar protocols will be used to study AAV and conjugate-based vectors.
Six Specific Aims are proposed to accomplish this goal. As part of Specific Aim I, improvements in the CF mouse will be performed to extend its lifespan and increase the severity of lung disease.
Specific Aim 2 will focus on strategies to increase the efficacy of gene transfer in vivo. Issues pertinent to general level of gene transfer, distribution of gene transfer (homogeneous vs non-homogeneous) and airway vs alveolar expression will be explored.
Specific Aims 3 and 4 propose a series of studies on safety issues, combining in vitro (mouse and human) and in vivo (mouse) studies. With respect to adenoviral vectors, studies of vector replication, integration, and ectopic CFTR expression will be pursued (Specific Aim 3). A major effort will focus on the inflammatory/immunologic consequences of a single exposure to adenoviral vectors (Specific Aim 4). To study the early (3-5 d) inflammatory lesion, we shall test the hypothesis that vector-epithelial cell interactions promote cytokine (IL-6, IL-8, GM-CSF) production and consequent chemotaxis of inflammatory cells into the airway wall. To study the later (7-9 d) lymphocytic response to adenovirus, we shall evaluate the relative contributions of the Class I and Class II pathways to the cellular response to vector administration. Both acute depletion approaches (radiotherapy, and CD4, CD8 cells) in C57BL/10 mice and genetic model approaches (MHC I and MHC II gene targeted mice) will be utilized. These studies will be complemented by studies in which the efficacy and safety (emphasizing inflammatory/immunologic consequences) of repetitive vector administration will be tested (Specific Aim 5). Finally, as part of Specific Aim 6, strategies derived from studies performed in Specific Aim 2 will be utilized to test gene transfer approaches with CFTR-containing vectors to correct CF mouse airways, intestines, and hepatic ducts. The overall goal of the project is to develop mouse model systems that accurately predict strategies for safe and effective gene transfer in man. These murine systems will then be used routinely as the interface to speed the transfer of new vectors developed in Project I to the clinical arena.
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