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* Gene Therapy Program, Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104; and
Department of Biochemistry and Molecular Biology, Center for Structural Biology, The McKnight Brain Institute, University of Florida, Gainesville, FL 32610
The immunological sequelae of adeno-associated virus (AAV)-mediated gene transfer in vivo is quite complex. In murine models, most AAV capsids are associated with minimal or dysfunctional T cell responses to antigenic transgene products. In this study we compared T cell activation against AAV2/8 and AAV2/rh32.33 vectors expressing nuclear-targeted LacZ (nLacZ), GFP, or firefly luciferase in murine skeletal muscle. We show that, unlike AAV8, AAVrh32.33 yields qualitatively and quantitatively robust T cell responses to both the capsid and transgene product. AAV2/rh32.33.CB.nLacZ, but not AAV2/8, drives a high degree of cellular infiltration and a loss of detectable transgene expression in C57BL/6 mice. However, cellular immunity to AAVrh32.33 is ablated in the absence of CD4, CD40L, or CD28, permitting stable β-galactosidase expression. Treatment of CD40L–/– mice with the CD40 agonist, FGK45, failed to restore the CD8 response to AAV2/rh32.33.nLacZ, suggesting that additional factors are involved. Our results suggest that specific domains within the AAVrh32.33 capsid augment the adaptive response to both capsid and transgene Ags in a CD4-dependent pathway involving CD40L signaling and CD28 costimulation. Structural comparison of the AAV8 and rh32.33 capsids has identified key differences that may drive differential immunity by affecting tropism, Ag presentation or the activation of innate immunity. This murine model of AAV-mediated cytotoxicity allows us to delineate the mechanism of viral immune activation, which is relevant to the translation of AAV technology in higher order species.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by Grants P01-HL059407, P30-DK47757 (to J.M.W.), T32-AR053461-03 (to L.E.M.), and R01-GM082946-01 (to M.A.M.) from the National Institutes of Health and by GlaxoSmithKline. Part of the information included in this paper was presented in The Eleventh Annual Meeting of the American Society of Gene Therapy held in Boston, MA, May 28 through June 1, 2008, as Oral Abstract no. 425, as well as The Twelfth Annual Parvovirus Workshop held in Cordoba, Spain, June 1 through 5, 2008, as Oral Abstract no. 3.9.
2 Address correspondence and reprint requests to Dr. James M. Wilson, Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104. E-mail address: wilsonjm{at}mail.med.upenn.edu
3 Abbreviations used in this paper: AAV, adeno-associated virus; β-gal, β-galactosidase; GC, genome copy; SFU, spot-forming unit; nLacZ, nuclear-targeted LacZ; VP, viral protein.
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