975. TLR9 Activation Is Involved in the Immune Response to Helper Dependent Adenoviral Vectors

VECTOR AND TRANSGENE IMMUNOLOGY VECTOR AND TRANSGENE IMMUNOLOGY 974. Constitutive Expression of a Mutated Form of CTLA4Ig after Gene Transfer Is Well Tolerated and Prevents Immune Responses Against an Immunogenic Transgene in Nonhuman Primates Alice Toromanoff,1 Marcelo Hill,2 Yan Cherel,3 Jack-Yves Deschamps,4 Olivier Gauthier,5 Bernard Vanhove,2 Fabienne Rolling,1 Philippe Moullier,1 Ignacio Anegon,2 Caroline Le Guiner.1 1 INSERM, UMR649, Nantes, France; 2INSERM, UMR643-ITERT, Nantes, France; 3INRA UMR703, ENV, Nantes, France; 4Service d’Urgences, ENV, Nantes, France; 5Service de Chirurgie, ENV, Nantes, France. rAAV-mediated gene transfer in skeletal muscle of large animal models can sustain long-term expression of transgene, but when applied to genetic diseases, the newly expressed therapeutic protein often induces a deleterious immune response. We investigated whether such immune response could be prevented by treatment with a mutant form of CTLA4Ig with higher affinity for B7 molecules (LEA29Y), known to induce potent immunosuppression and currently used, in its recombinant form (Belatacept®), in phase II/ III clinical development. Our laboratory has previously demonstrated that after intramuscular (IM) injection of a rAAV vector encoding for the cynomolgus erythropoietin (EPO) gene under the control of the doxycyclin-sensitive rtTA chimeric transactivator, 80% of nonhuman primates developed humoral and destructive cellular immune responses specific to rtTA, resulting in loss of EPO secretion. In this study, eight cynomolgus macaques received LEA29Yencoding rAAV-1 and -8 vectors (3.5x1012 vector genome/kg) by IM or by limb perfusion. These injections were associated with a weak and transient oral immunosupression, in order to prevent antiLEA29Y immune response in the first weeks after injection. LEA29Y reached steady-state levels two months post-rAAV delivery and was stably secreted thereafter. Depending on the rAAV serotypes and the route of administration (IM vs perfusion), LEA29Y levels varied between 10 to around 100 µg/ml. After LEA29Y reached plateau levels, animals were challenged with a rAAV-EPO-rtTA vector intramuscularly and EPO secretion was monitored for several months after repeated Dox inductions. Our preliminary data in three macaques suggested that a minimal level of LEA29Y is required to achieve long-term Dox-induced EPO secretion. We are currently evaluating EPO expression in the five other animals. The immune status of the injected macaques was also further analyzed. After immunisation, production of antibodies against SRBC and viral vector was inhibited in all individuals, but not in mock-injected controls. However, skin allograft rejection was similar to the control group, indicating conserved cellular immune responses. Importantly, no biological and no clinical adverse effect was so far (> one year) observed in the cohort. In conclusion, our study demonstrates that injection of LEA29Yencoding rAAV is a safe and efficient method to prevent immune responses against transgene and viral vector in nonhuman primates. These results finally suggest (i) that preclinical evaluation of therapeutic transgenes in other large animal models would benefit from this simple and straightforward approach to block antitransgene immune responses, providing that LEA29Y is able to block T cell proliferation in these other species, and (ii) that treatment with Belatacept® might overcome, when required, anti-transgene and anti-viral vector responses in human gene therapy trials.

Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright  The American Society of Gene Therapy

975. TLR9 Activation Is Involved in the Immune Response to Helper Dependent Adenoviral Vectors Vincenzo Cerullo,1 Michael P. Seiler,1 Nicola Brunetti-Pierri,1 Viraje Mane,1 Christopher Clarcke,1 Brendan Lee.1,2 1 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX; 2Howard Hughes Medical Institute. Toll-like Receptors (TLRs) are innate receptors that sense microbial products and trigger dendritic cell (DCs) maturation and cytokine production, thus effectively bridging innate and adaptive immunity. Members of the Toll-like receptor 9 (TLR9) subfamily are evolutionarily related and sense viral and bacterial infection at the endosomal subcellular compartment. Whereas TLR9 directly recognizes viral and bacterial CpG-DNA motifs, murine TLR7 and human TLR8 sense viral single stranded RNA motifs. Upon liganddriven activation, members of the TLR9 subfamily bridge innate and adaptive immunity by activating immature antigen-presenting DCs into professional antigen-presenting cells in a T helper cellindependent fashion. In this study we evaluated whether TLR9 contributes to the acute immunopathology typically seen after systemic administration of a Helper-dependent adenoviral vector (HD-Ad) in the p∆28E4 Backbone. To address this question we investigated the degree of activation of the innate immune response following systemic injection of Helper-dependent adenoviral vector (HD-Ad) in mice expressing a structurally aberrant TLR9 receptor (TLR9 CpG1/CpG1). In contrast to wild type mice, we found that TLR9 CpG1/CpG1 mice showed a reduced activation of the innate immune response following i.v. injection of high doses of HD-Ad, as shown by reduced increase in IL-6 and IL-12. Next we tested whether pharmacologically blocking TLR9 signaling in wild type mice could attenuate the innate immune response to HD-Ad. In agreement with the TLR9 CpG1/CpG1 model, we found a similar reduction in innate immune response after TLR9 blockade. In the present study we showed for the first time that TLRs are involved in the innate immune response following systemic administration of adenoviral vectors and we showed that selectively blocking TLRs can be potentially used to reduce the toxicity of Ad-mediated gene therapy.

976. TLR and MyD88 Signaling Mediates Innate Immune Responses to Adenoviral Vectors in Mouse Zhe Zhang,1 Gaung-Ping Gao,1 Lee M. Wetzler,2 Peter L. Bell,1 Di Wu,1 Jianping Lin,1 James M. Wilson.1 1 Gene Therapy Program, Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA; 2Section of Infectious Diseases, Department of Medicine, Evans Biomedical Research Center, Boston University School of Medicine, Boston, MA. Toxicity of adenoviral vectors induced after the acute interaction between the vector and innate immune system is the major hurdle for safe application of the vectors in humans. Toll-like receptors (TLRs) are the sentinels of innate immunity. We investigated the role of TLRs and the major TLR adaptor protein MyD88 in the host acute immune responses to systematic administration of type 5 adenoviral vectors. Following intravenous administration of E1E3deleted Ad5-lacZ (2E11 pt./animal), a brisk accumulation of inflammatory cytokines including IL6, KC and TNF-α was found in the serum of C57B6 mice but remained barely detectable in that of MyD88 KO mice; the pattern of cytokine secretion in the splenic CD11c + dendritic cells purified from the mice after vector administration appeared the same. CD11b immunostaining and neutrophil staining showed a robust infiltration of acute immune cells in liver while few were found in MyD88 KO mouse. Under the same vector treatment, secretion of acute cytokines such as KC and S375