ORIGINAL ARTICLE Modulation of TNFa, a determinant of acute toxicity associated with systemic delivery of first-generation and helper-dependent adenoviral vectors VP Mane 1,6 , G Toietta 1,6 , WM McCormack 1 , I Conde 3 , C Clarke 1 , D Palmer 1 , MJ Finegold 2 , L Pastore 5 , P Ng 1 , J Lopez 3 and B Lee 1,4 1 Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX, USA; 2 Department of Pathology, Baylor College of Medicine, One Baylor Plaza, Houston, TX, USA; 3 Department of Medicine-Thrombosis, Baylor College of Medicine, One Baylor Plaza, Houston, TX, USA; 4 Howard Hughes Medical Institute, Baylor College of Medicine, One Baylor Plaza, Houston, TX, USA and 5 Dipartimento di Biochimica e Biotecnologie Mediche and CEINGE-Biotecnologie Avanzate, Universita ` degli Studi di Napoli Federico II, Naples, Italy Understanding the determinants of the host innate immune response to systemic administration of adenoviral (Ad) vectors is critical for clinical gene therapy. Acute toxicity occurs within minutes to hours after vector administration and is characterized by activation of innate immune responses. Our data indicate that in mice, indicators of vector toxicity include elevations of cytokine levels, liver transaminase levels and thrombocytopenia. To discern potential targets for blunting this host response, we evaluated genetic factors in the host response to systemi- cally administered first-generation Ad vectors (FGV) and helper-dependent Ad vectors (HDV) containing b-galactosi- dase expression cassettes. A preliminary screen for modula- tion of vector-induced thrombocytopenia revealed no role for interferon-g, mast cells or perforin. However, vector-induced thrombocytopenia and interleukin 6 (IL-6) expression are less evident in tumor necrosis factor alpha (TNFa)-deficient mice. Moreover, we also demonstrated that TNFa blockade via antibody or huTNFR:Fc pretreatment attenuates both thrombocytopenia (440% increase in platelet count) and IL- 6 expression (480% reduction) without affecting interleukin 12 , liver enzymes, hematological indices or vector transduc- tion in a murine model. Our data indicate that the use of HDV, in combination with clinically approved TNFa immunomodu- lation, may represent an approach for improving the therapeutic index of Ad gene therapy for human clinical trials. Gene Therapy (2006) 13, 1272–1280. doi:10.1038/ sj.gt.3302792; published online 18 May 2006 Keywords: helper-dependent; innate immune response; TNFalpha; Enbrel; thrombocytopenia; toxicity Introduction Adenoviral (Ad) vectors represent an attractive candi- date for gene therapy for a variety of diseases, owing to large transgene carrying capacity and high-level transduction of several cell types. Ad vectors can be distinguished between early generation vectors (most viral genes intact) vs helper-dependent Ad vectors (HDV) (viral genes deleted) depending on whether viral coding sequences remain. 1 Systemic administration of Ad vectors in immunocompetent recipients induces a multiphasic toxicity profile. Immediate-early cytotoxicity can occur within minutes, is vector transcription-inde- pendent and is mediated in part by viral particle interaction with components of the host innate immune response. 2 This phase is characterized by acute produc- tion of proinflammatory cytokines which bears similarity to the early phases of sepsis, a hyperinflammatory response to infection. 3 Intermediate-early toxicity is characterized by an augmentation of this response and includes hepatotoxicity, coagulopathy and further am- plification of inflammatory cytokines and chemokines, and may last for hours to days after vector application. 4 As a proinflammatory cytokine, tumor necrosis factor alpha (TNFa) is heavily implicated in the initiation and progression of autoimmune inflammatory disorders. 5,6 However, its role in Ad-induced toxicity has not been extensively studied. Although some studies found no elevation of TNFa following Ad injection, 7–10 other studies have found that TNFa release is in fact a consequence of higher-dose vector application in mice and nonhuman primates. 4,8 Furthermore, low levels of circulating serum TNFa may not reflect membrane- bound TNFa in the tissues. 10 Indeed, proinflammatory cytokines such as TNFa may demonstrate an immediate peak (corresponding to host/vector interaction) followed by a second peak corresponding to the host response to Ad gene transcription. 11 Received 6 November 2005; revised 27 February 2006; accepted 16 March 2006; published online 18 May 2006 This article is dedicated to the memory of our colleague and friend WM McCormack Jr (12 August 1965 – 29 July 2005). Correspondence: Dr Brendan Lee, Department of Molecular and Human Genetics and Howard Hughes Medical Institute, Baylor College of Medicine, One Baylor Plaza Room 630E, Mail Stop BCM 225, Houston, TX 77030, USA. E-mail: blee@bcm.tmc.edu 6 These two authors contributed equally to this work. Gene Therapy (2006) 13, 1272–1280 & 2006 Nature Publishing Group All rights reserved 0969-7128/06 $30.00 www.nature.com/gt