664. Identification of TLR2 as a Mediator of the Innate Immune Response to Adenoviral Vectors

662. Study of Adenovirus Vector-Induced Activation of Muscle Dendritic Cells and Analysis of Mouse Dendritic Cell Migration from the Tibialis Anterior Muscle Lei Wang, Paula R. Clemens. 'Neurology. University ofPittsburgh, Pittsburgh, I'll; Weurology Service, Department ofVeterans Affairs Medical Center; Pittsburgh, PA. Dendritic cells (DCs) , known to be increased in skeletal muscle undergoing dystrophic change, are the most potent professional APCs that initiate the adaptive immune response to Ad vectormediated gene delivery. Therefore, these cells may be an excellent target to mediate immune suppression to facilitate successful gene transfer. Toward that end , we have studied the localization and trafficking ofDCs in the setting ofAd vector-mediated gene transfer to skeletal muscle. First, we explored theAd vector-induced activation of muscle DCs. The tibial is anterior muscle (TA) of C57BLllO mice was injected with PBS or a first-generation Ad EOFP vector. Immunohistochemistry revealed that the majority ofactivated DCs (CDI l c" CD86+) were observed in vector-infected muscle at 7 days after gene transfer, when EOFP expression reached its highest level. CDI lc" CD86+ DCs were also found at the margin of white and red pulp in spleen, and in the paracortical area of ILNs on the side of injection. PBS-injected mice did not show activated DCs in TA muscle, LNs or spleen. Although migration of DCs through lymphatics to draining LNs is an important step in the initiation of an adaptive immune response, very little is known about how the muscle DCs migrate from muscle treated with gene transfer. To study DC trafficking we adoptively transferred BM-derived mature DCs labeled with CFSE ex vivo to syngeneic recipient mice, We quantified CFSWDCs in PLNs, ILNs and spleen by flow cytometry alter footpad (FP) orTA injection. On day 2 after adoptive transfer to Fp, most reco vered CFSWDCs were found in PLNs, with fewer found in ILNs. There were very few recovered DCs found in spleen on day I or 2. This study confirmed the results of others showing that CFSE-Iabeled DCs migrated to draining LNs after FP injection, and a vel) ' small fraction ofsuch cells further migrated to spleen through the thoracic duct, In contrast, after adoptive transfer ofCFSWDCs to TA, a few DCs appeared in the spleen within 12 h. Over the next 36 hours, the number decreased and became undetectable. On day I, we recovered the largest number ofCFSE+DCs from PLNs, with fewer from ILNs . The numbers were reduced in PLNs and ILNs after 2 days , and were undetectable thereafter. Interestingly, our study found that the peak number of CFSWDCs recovered in spleen occurred at 12h, not 24h afterTA injection when the largest number of these cells appeared in the PLNs. This finding suggests that a subfraction of the transferred DCs from the muscle injection site could travel through the bloodstream directly to spleen perhaps gaining access through the well-developed capillary network of skeletal muscle. Subsequently, the largest fraction oftransferred DCs trafficked to the PLNs and ILNs, likely through lymphatic vessels. Previous studies have shown that DCs migrating to the spleen and draining LNs is required to induce humoral immunity and primary immune response respectively. Therefore, our study yielded important evidence implicating DCs in the deleterious immunological consequences induced by Ad-mediated gene transfer to skeletal muscle.

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663. Methotrexate Selection of Gene Modified T Cells with an Engineered Human Dihydrofolate Reductase (DHFR) Christine E. Brown,' Wen-Chung Chang,' Christine Wright , 1 Araceli Naranjo,' Jamie Wagner, I Hunsar B. Meechoovet,' Julie R. Ostberg,' Michael C. Jensen .' 'Division a/Cancer lmmunotherapeutics & Tumor Immunology, Beckman Research Institute, City ofHope National Medical Center, Duarte, CA. Endowing T-cells with resistance to Iymphotoxic drugs by their genetic modification affords the opportunity for in vitro and in vivo selection. The clinical usc of bacterial drug resistance genes such as neomycin and hygromycin phophotransferases is problematic due to the immunogcnicity of these transgcnes, as well as, the incompatibility of 0418 and hygromycin tor direct administration to humans for in vivo selection. Here , we report on the utility of a human dihydrofolate reductase double mutant (DHFRdm; Leu22---7 Phe22, Phe31 ---7Ser31 ) transgene for conferring resistance of human 'l-cclls to the drug methotrexate (MTX). Studies using Jurkat T cells , peripheral blood OKT3-stimulated 'l-cells and established human T-celilincs demonstrated their sensitivity to the lympholytic effects of MTX at concentrations of 0.05 JlM or more . The stable expression in Jurkat T cells with DHFR constructs containing either both mutations or each individual mutation alone revealed that the DHFRdm was the most efficient at conferring MTX resistance. Using a OFP-DHFRdm fusion construct introduced into PBMC derived T cells by c1ectrotransfer, we were able to derive OFP' stable integrants in the presence of cyctocidal concentrations of MTX (0.1 JlM). These MTX-resistant T cells retained dependency on antigen receptor/gamma-c cytokines for surviv al and proliferation and displayed T cell phenotypic markers similar to their DHFRdm' counterparts. We next evaluated the utility ofthe selection transgene to enforce the expression ofa second therapeutic transgene, in this case a zetakine chimeric antigen receptor, by fusion using a 2A cleavable linker. Electroporation of a DHFRdm-2A-IL 13zetakine construct into T cells resulted in MTX resistance and coordinated zetakine expression; these CTLs killed glioma target cells (expressing the IL 13zetakine ligand, IL 13R(2), as determined using in vitro chromium release assays. These data demonstrate the utility of this human enzyme transgene selection system for human T cell genetic modification. Our studies are now focusing on the potential to select for transgene expressing MTX-resistant cells in vivo.

664. Identification of TLR2 as a Mediator of the Innate Immune Response to Adenoviral Vectors Daniel M. Applcdorn, I Jeannine M. Scott ,' Andrea Amalfitano.' 'Microbiology and Molecular Genetics , Mlchlgan State University, East Lansing, MI. The interaction between adenovirus (Ad) and pathogen recognition receptors (PRR) of the toll-like receptor family (TLR) has recently received increased attention. TLR9, found predominantly in endosomes, recognizes viral CpO dsDNA that , in the presence of an agonist, leads to increased NF-KB activation, and increased plasma levels of Type I interferons, IL-6 and IL- I2. Recent reports have identified TLR9 as a PRR activated following high titer Ad injection, and it has been shown that the production of type I IFNs was mediated by TLR9 in a MyD88 dependent manner in plasmacytoid dendritic cells (pDCs), but not conventional DCs (cDCs). However, the induction of numerous additional pro-inflammatory cytokines and chcmokincs, i.e. IL-Ia, RANTES and type II IFN-y among others, were not addressed in those studies. Furthermore, those studies observed a TLR9 independent induction of monocyte chemoattractant protein-I (MCP-I) suggesting the specific anti-Ad innate immune response is multi-faceted. Because of this , we hyMolecular Therapy Yofume 15. Supplement I, .\by 2007 Copyright © '111C American Societyo f Gene TIICr.lpr

pothesized an additional PRR was required to initiate inflammatory signals in conjunction with TLR9 in the context ofAd. Studies utilizing herpes-simplex viruses, Trypanosoma cruzi, and Mycobacterium tuberculosis, have reported cooperation between TLR2 and TLR9 in controlling cytokine and chemokine responses to these pathogens. In conjunction with TLRs I and 6, known agonists ofTLR2 include, but arc not limited to lipoprotein, modulin, porin and peptidoglycan. To determine ifTLR2 was partly responsible for these anti-Ad innate immune responses, we investigated the cytokine/chemokine response , and thrombocytopenia, following high titer adenovirus injection into TLR2 deficient mice. We found that like TLR9, TLR2 is also involved in the production ofIL-12. We also found that Ad induction ofIL-Ia, IFN-y, MCP-I and RANTES were in part, TLR2 dependent. Although we saw reduced levels of IL-6 in Ad treated TLR2 deficient mice, these results were not statistically significant. It is well established that Ad injection induces thrombocytopenia in a complement dependent fashion, and recently we observed that this may be a response that is both mediated by the TLR adaptor TRI F and MyD88 at relatively lower viral particle dosages. However, Ad injection into TLR2-KO mice still elicited thrombocytopenia. Based upon these findings, and those of others , we propose a model where TLR2 and TLR9 synergistically cooperate to detect both viral capsid and viral DNA , and subsequently induce the immune responses noted shortly after intravenous Ad delivery,

665. Host Response to Adenovirus, HelperDependent Adenovirus and Adeno-Associated Virus in Mouse Liver

Paul Fawcett,' Hiroyuki Nakai ,' Anja Ehrhardt,' Mark A. Kay ,~ Anton McCamey.s 'Internal Medicine, Virginia Commonwealth University, Richmond, I~; lMoleclllar Genetics and Biochemistry; University of Pennsylvania, Pittsburgh, PA; JVirology. Ludwig-MaximiliansUniversity Munich, Munich, Bavaria, Germany; "Pediatrics and Genetics, Stanford University, Stanford, CA; Sinternal Medicine, University ofIowa, Iowa City, IA.

Understanding host responses to viral gene therapy vectors is necessary for the development of safe and efficacious in vivo gene transfer agents. We describe the usc of high-density spotted cDNA mieroarrays to monitor the in vivo host transcriptional responses in mouse liver upon administration ofeither a "first-generation" adenovirus vector (Ad), a helper-dependent "gutless" adenovirus vector (HD), or an adeno-associated virus vector (AAV) containing identical human factor IX expression cassettes (hFlX). These replicationdefective vectors allowed us to assess the contribution of the viral capsid and leaky viral gene expression to the host response in whole animals , since HD and MV vectors additionally contain no viral genes. While all three vectors induced characteristic temporally-sequenced programs ofgene expression, the gene expression programs induced by the Ad and HD adenovirus vectors were largely similar, including the induction ofa prominent interferon-dependent cluster within 6 hours of administration. Differentially expressed genes were validated by quantitative PCR. Our results show that the viral capsid is the main driver of the host transcriptional response. Upon infection with Ad and HD, we observe modulation ofmembers ofthe AP-I signaling pathway, innate immune pathways, chemokines and cytokines, immune-dampening factors as well as proteins involved in antigen presentation. We observe paraerine signaling events that only occur after cross talk between different cell types. This highlights the importance of virus-host interaction studies in whole mammals. In contrast to infection with Ad and HD, theAAV-based vector caused far fewer alterations ofhost-gene expression, Notably, AAV infection induced significantly fewer genes that arc involved in immunity and defense , signal transduction as well as protein metabolism and modification. AAV infection caused a decrease in a Molecular Therapy Volume15.Supplement I, May 2007

Copyright © T he American Socie ty of Gen e Therap y

number ofAP-I components, again in contrast to Ad and HD. AAV also altered the expression ofa number of'immunornodulatory genes. Taken as a whole, the host response to Ad and HD was rema rkably similar suggesting that the initial innate response is driven largely by the viral capsid. The host response to AAV, in comparison, is blunted. Though they share some common features , the response to AAV differs significantly from that to the Ad and HD. We have identified a number of novel genes involved in the host response to Ad, HD and AAV. These virus-host interaction studies in mice should complement previous studies in cultured cells.

666. Effect of Anti-Adenovirallmmunity on Long-Term Vector and Islet Graft Function

Jun Cheng,' Jianmin Sun,' Randall S. Sung.' 'Transplant Division, General Surgery Section, Department of Surgery. University ofMichigan Health System, Ann Arbon MI. Purpose: Adenoviral gene transfer is a potential ex vivo gene therapy for islet transplantation. However, the inflammatory and immune responses induced by adenovirus may be detrimental to islet survival and transgene expression in transduced islets. We investigated the effect of immune response to adenovirus on longterm vector and islet graft function. Methods: C57BLl6 (n=17 per group) mice were used for syngeneic islet transplantation. Islets were isolated and transduced with AdCMVntp-galaetosidase (Ad, type 5 adenovirus, EI and E3-deleted) at a multiplicity of infection of I x \Os pfu/islet. Islets transduced with Ad or untransduced were implanted under the kidney capsule ofstreptozotocin-induced diabetic syngeneic recipients. Blood glucose and glucose tolerance were utilized to assess to graft function. All grafts were collected at intervals after transplantation. Graft inflammation was estimated by H&E and immunohistochemical staining. Intragraft transgene and cytokine/chemokine expressions were assayed by X-gal staining and quantitative real time PCR, respectively. ELISPOT and ELISA were used to measure systemic cytokines and anti-adenoviral antibodies (IgG I and IgG2a), respectively. Results: Diabetes was reversed in one day in recipients ofa marginal mass ofuntransduced islets, while Ad-transduced islets failed to reverse diabetes until 10 days posttransplant (pS:O.048). A profound and progressive infiltration with CD4+, CD8+lymphocytes and CD II b" maerophages was developed in adenovirus transduced grafts from 10 to 25 days after transplantation and paralleled a decline in p-galactosidase expression. However, transgene expression still persisted in transduced islet grafts over 3 months. Glucose tolerance was impaired in transduced grafts at 25 days (p=O.008) (Fig. A), but restored at 3 months (p'=O.275) (Fig. B) post-transplant. Levels of intragraft intcrfcron-y(IFN-y) and CC chemokine ligand 5 (CCLS) mRNA expression were not different between transduced and untransduced islet grath at 5 and 25 days after transplantation, despite the presence ofincreased expression of CC chemokine receptor 5 (CCR5) at 5 days (pS:O.029). No significant splenocyte IFN-yand interleukin-4 (IL-4) production or serum antiadenoviral antibody was detected in recipients of transduced islet grafts. Conclusions: Although transduction of islets with adenoviral vectors results in overt local inflammation of islet grafts and impairs early graft function, long-term (3 months) graft function is not significantly impaired, and transgene expression persists in syngeneic islet transplant. Systemic anti-adenoviral immunity is minimal. Thus,

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