Gene Therapy for Arterial Restenosis Using AAV Vectors

The work I will describe in this thesis concerns the study of the properties of AAV vectors both as a gene transfer system and as a therapeutic vector in the context of a restenosis model. The gene transfer properties of AAV are evaluated in vitro and in viva, directly exposing recombinant AAV vectors (rAAV) in cells and tissues. Complementary to these studies, the rAAV transfer properties are also evaluated in vitro and in organ culture conditions using intracoronary sents as scaffolds for gene delivery. The main focus of the thesis was to evaluate the effects of two different AAV vectors, AAV-Timpl and AAV-PTX3, in blocking arterial stenosis. AAV-Timpl vector encodes for the human Tissue-inhibitor of metalloproteinases 1, which blocks the metalloproteinases (MMPs) and AAV-PTX3 vector encodes for the human long-pentraxin 3, which inhibits Fibroblast growth factor 2 (FGF-2) induced proliferation. Both MMPs and FGF-2 are involved in the pathogenesis of restenosis and atherosclerosis. Chapter 1 offers an introduction to the characteristics of the normal and atherosclerotic vessels, angioplasty and restenosis. It reviews the available animals models for restenosis and pathogenesis. The functional properties of MMPs, Timps and PTX3 are also reviewed. The history of gene transfer to the vessel wall and other gene therapy approaches for restenosis together with the characteristics of the AAV vectors are described. Chapter 2 covers both the details of experimental techniques and materials. The rat animal model was the main tool for in viva evaluation. In Chapter 3 the experimental results are exposed and Chapter 4 includes their discussion. In Chapter 5 the rationale and the proof of principle of AAV attachment to coated intracoronary stents are described. The novelty of this thesis is three-fold. First, from the point of view of gene therapy, this is the first time that AAV is used as a therapeutic vector in a restenosis model. Second, from the point of view of investigating the role of a new protein, this is the first time that PTX3 is overexpressed in the vascular wall and its effects reported. Third, this is the first time that an AAV gene delivery system using stent-based gene therapy has been reported. AAV gene delivery is a powerful new approach for site-specific expression of genes aiming to block restenosis or in-stent restenosis.