Tuesday 7 October 1997: Workshops WI2 Gene therapy of atherosclerosis and restenosis 109 GENE THERAPY OF ATHEROSCLEROSIS AND RESTENOSIS w12 pii7 1 Novel approach to somatic gene therapy for dyslipidemia Lawrence Chan. Baylor College of Medicine, Houston, TX, USA in the Czech Republic, with an overall prevalence of 53%. Elevated plasma cholesterol (~5.5 mmol/l) was also common ranging from 36% in Spain to 58% in Italy, with an overall prevalence of 44%. Of the patients receiving blood pressure lowering drugs 50% had a systolic BP > 140 mmHg and 21% > 160 mmHg, and of those receiving lipid lowering drugs 49% had a total cholesterol >5.5 mmol/l and 13% 26.5 mmol/l. So there is considerable vari- ations in the prevalence of modifiable risk factors in patients with established CHD across Europe and therefore a considerable potential for cardiologists and physicians to further reduce CHD morbidity and mortality and improve patients chances of survival. Somatic gene transfer is a potentially valuable option for the treatment of various hyperlipidemias that do not respond adequately to conventional ther- apy. There are two major issues that must be addressed before gene therapy should be tested in patients. The first issue is the choice of the gene transfer vector. Although there are many different types of gene transfer vectors that are being developed, there are only two that are currently used extensively for transferring genes into the liver, the target organ for the treatment of hyperlipi- demia: retroviral vectors and adenoviral vectors. Retroviral vectors integrate into the host cell genome resulting in long-term expression of therapeutic genes. Dr. James Wilson’s group used an LDL receptor retrovirus to transduce liver cells isolated from 5 patients with familial hypercholesterolemia (FH). Reimplantation of the genetically altered liver cells produced relatively poor and inconsistent lipid lowering effects indicating that the approach is not feasible for FH patients in general. To date, adenoviral vectors have been used only in experimental animal models of hyperlipidemia. There are encouraging developments in the design of adenoviral vectors that may allow them to be used clinically in the future. The other issue is the choice of therapeutic genes. Candidate genes for this purpose include the LDL receptor, apoA-I, apoE and cholesterol 7a-hydroxylase genes. For both FH and mixed hyperlipemia, the newly cloned gene for the VLDL receptor offers many potential advantages. Future developments in therapy should enable a physician to select an appro- priate therapeutic gene for the treatment of a specific form of hyperlipidemia in the same way that a physician would select an antibiotic for a particular type of infection. I 2.W12 2 Towards gene therapy for vascular disorders D. Branellec, A. Mahfoudi, A. Le Roux, J. Isner’ , P. De&e, K. Walsh’, RPR Gencell Cardiovascular Dept., Mtry/Seine, France; ‘St. Elizabeth’s Medical Center Boston, USA Local arterial gene transfer has been recently developed as a strategy to target vascular disorders such as excessive smooth muscle cell (SMC) proliferation, one of the key component of post-angioplasty restenosis. Gax (Growth Arrest- specific HomeoboX) gene is specifically expressed in cardiovascular tissues and down-regulated in proliferating SMCs, suggesting a role as a negative regulator of cell growth. To assess the growth arrest potency of Gax, we used a recombinant adenovirus encoding rat Gax under the control of CMV promoter (AV GAX). When primary (rat, rabbit, human) SMCs were transduced with AV GAX, thymidine incorporation was markedly reduced within 24 hours as compared to a control 1acZ adenovirus. Growth arrest in response to AV GAX was also reflected by Gl cell cycle arrest as well as increased expression of the cyclin-dependent kinase inhibitor p2 1. To further extend these data in vivo, we first used the model of rat carotid injury. Local delivery of AV GAX (lo9 pfu) significantly reduced neointima formation (-69%, p < 0.001) as compared to a control virus. When locally delivered in the rabbit iliac artery with a conventional angioplasty balloon, AV GAX reduces neointima formation (-56%, p < 0.02) without affecting reendothelialization at the site of vessel injury, but also significantly increases lumen diameter. Taken together, these data suggest that percutaneous aden- ovirus-mediated transfer of GAX gene may be used as a treatment of vascular pathologies such as restenosis following angioplasty. L 2 W12 3 CKIs and control of vascular smooth muscle cell cycle F. Tanner, J. Tashiro, M. Bohm, E. Duckers, D. Gordon, G.J. Nabel, E.G. Nabel. LJepartments of Internal Medicine, Pathology, Physiology and Biological Chemistry, Howard Hughes Medical Institute, University of Michigan, Ann Arbor, Michigan, USA Neointima formation is a common response of arteries to injury and results, in part, from vascular smooth muscle cell proliferation, migration and connective tissue formation. The mechanisms by which smooth muscle cells proliferate in response to mitogenic signals are well described, however, the role of cellular gene products which cause vascular smooth muscle cells to shift from a proliferative to a quiescent state during Gl phase of the cell cycle are not well understood. Transit through Gl and entry into S phase of the cell cycle requires the action of cyclin-dependent kinases (CDKs), and CDKs are inactivated by protein phosphorylation in association with regulatory subunits, including the cyclins and the cyclin-dependent kinase inhibitors (CKIs). CKIs directly implicated in mitogen dependent CDK regulation are p21 and ~27. Studies from our laboratory suggest that p21 inhibits vascular smooth muscle cell growth by arrest at the GUS phase of the cell cycle and that p21 and p27 are detected in the neointima of balloon injured pig arteries in vivo in a time pattern that ‘nversely correlates with intimal cell proliferation. Furthermore, the CKIs p21 and p27 are expressed by smooth muscle cells and macrophages in human atherosclerotic coronary arteries and saphenous vein bypass grafts. Additional studies in our laboratory have explored the role of p21 in the pathogenesis of atherosclerosis in ~21: apoE null mice. The mechanisms by which these gene products limit smooth muscle cell proliferation and alter the development of atherosclerosis in this genetically bred murine model will be discussed. An understanding of these mechanisms should lend insight into the pathophysiology of vascular diseases and determine whether enhancement of p21 and p27 expression in arteries may limit excessive vascular smooth muscle cell proliferation in these diseases. IXYI 2 W 12 4 Percutaneous arterial gene therapy for the prevention of restenosis Ph. Gabriel w, Laurent J. Feldman. Faculte’ Bichat, INSERM U-460 Paris, France Cardiovascular gene therapy is becoming a clinical reality due to improved vectors, delivery systems and careful experimental validation studies. Percu- taneous catheter-based delivery is feasible for most applications. The ultimate issues that will decide of the future of gene therapy are safety of the transfer and delivery techniques and cost/effectiveness comparisons with altema- tive therapies, including local delivery of drugs, proteins and/or mechanical devices. Restenosis is an important target for gene therapy since it is frequent (30% of patients), costly, refractory to all pharmacological therapies, and related, at least in part, to smooth muscle cell proliferation (especially after stenting) which is an inviting target for anti-proliferative molecular strategies. Because cell division is ultimately controlled by intranuclear events, the protein prod- uct of genes selected for their antiproliferative effects usually remain inside the cells. Consequently, the transfer of growth inhibitory genes needs to be efficient, i.e. involve a large proportion of smooth muscle cells populating the angioplasty site. To date, gene transfer has been used to demonstrate that therapeutic genes encoding cytolytic (thymidine kinase) or cytostatic (rb, eNOs, gax,...) products successfully inhibit smooth muscle cell proliferation and related intimal hyperplasia. It may be possible to use gene therapy for the prevention of restenosis via other approaches such as prevention of cell migration cr enhanced reendothelialization. Ll 2 W 12 5 Adventitial gene transfer to rabbit carotid arteries S. Yl&Hentuala’5, M. Laitinen’ , I. Zachary’, G. Breieg, T. Pakkanen’ , T. H&kinen’, J. Luoma’, W. Risau3, M. Soma4, M. Laaksos, J.F. Martin2. ‘AI. Krtanen Institute, University of Kuopio, Kuopio; ‘Department of Medicine, University of Kuopio, Finland; 3Max-Planck-Znstitute, Bad Nauheim, Germany; 4 University of Milan, Italy; ‘University College London, England Gene transfer can potentially intervene iu the pathogenesis of intimal hyperpla- sia. Most models of intimal thickening are based on balloon denudation which also affects endothelial (EC) function. In the present study SMC proliferation was induced in rabbit carotid arteries by inserting an innert silicone collar around the arteries. The model does not cause EC denudation and permits studies with factors that act on EC, such as vascular endothelial growth 11th International Symposium on Atherosclerosi?. Paris, October 1997