542 the innate immune response to intravenously injected adenoviral vectors: role of endosomal escape in MAP kinase activation and the cytokinechemokine response

542 The Innate Immune Response to Intravenously Injected Adenoviral Vectors Role of Endosomal Escape in MAP Kinase Activation and the Cytokine/Chemokine Response Molecular Therapy Volume 18, Supplemen[.]

IMMUNOLOGIC & HOST RESPONSES IN GENE & CELL THERAPY suppression of antibody formation upon hepatic gene transfer, while IL-10, although a likely contributor is not absolutely required 541 Abstract Withdrawn 540 Murine Hepatocytes Transduced with Y-F Mutant Capsid Are More Resistant to Killing by Capsid Specic CD8+ T Cells Ashley Martino,1 David M Markusic,1 Arun Srivastava,1 Roland W Herzog.1 Pediatrics, University of Florida, Gainesville, FL In clinical trial, liver gene transfer of an AAV2 vector expressing factor IX resulted in transient correction of hemophilia B due to a CD8+ T cell response against the viral capsid Recently, we found that mutations of surface exposed tyrosine residues to phenylalanine (Y-F) on the AAV2 capsid improved gene transfer efciency and reduced ubiquitination of the capsid protein Poly-ubiquitinated proteins are degraded in the proteasome, and subsequently displayed as peptides on the surface of MHC I molecules We hypothesized that Y-F capsid mutants would be less efciently presented on MHC I molecules because of a reduction in viral dose required for therapeutic expression and in poly-uqibuitination as compared to wtAAV2 To evaluate MHC I presentation of wtAAV2 and AAV2Y444+500+730F (AAV2TRP) capsids, we established an in vitro CTL assay using murine hepatocytes Preliminary experiments showed that an AAV2-Y-F capsid mutant vector induced a similar frequency of IFN-γ + cells as compared to wtAAV2 upon in vitro re-stimulation with VPQYGYLTL peptide, the dominant, Ld-restricted CD8+ T cell epitope for this strain and also a HLA-B*0702 restricted epitope in humans, suggesting comparable T cell activation Subsequently, capsid-specic CD8+ T cells were generated in BALB/c (H-2d) mice using a prime-boost protocol based on IM administration of AAV-2 vector followed by boost with VPQYGYLTL peptide Capsid-specic CD8+ T cells were expanded in vitro and co-cultured at different effector:target (E:T) ratios with BALB/c H2.35 adult hepatocytes, transduced 24 hrs earlier with wtAAV2 or AAV2TRP vectors expressing hF.IX Prior to co-culture, hepatocytes were labeled with a uorescent dye (DiOC18), which labels 100% of cells After 24hrs co-culture, cells were stained with 7-AAD, which specically stains dead cells Flow cytometry was used to determine percent 7- AAD+ hepatocytes of total DiOC18+ hepatocytes Parallel co-cultures were analyzed 48hrs after gene transfer using immunostaining and ow cytometry to determine hF.IX+ DiOC18+ cells In two independent experiments we observed specic killing of AAV transduced hepatocytes, conrming that murine hepatocytes present AAV capsid to CD8+ T cells In order to achieve comparable transduction efciency, an approximately 10fold higher MOI of wtAAV2 was required Although the extent of hepatocyte killing was MOI-dependent, a reduction in the MOI from 105 to 103 vg/cell resulted in only a 23% decrease in killing at the highest E:T ratio tested for wtAAV2 capsid In contrast, a reduction in the MOI from 105 to 103 of AAV2TRP transduced cells resulted in a 60% decrease in killing At equal MOIs, killing of AAV2TRP transduced hepatocytes was reduced 56% and, when adjusted for equal transduction efciency, was reduced by 67% These results strongly suggest that AAV2TRP transduced hepatocytes are more resistant to killing by capsid specic CTL, presumably because of reduced peptide presentation Studies are ongoing to exclude the possibility of shifts in epitope usage Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy 542 The Innate Immune Response to Intravenously Injected Adenoviral Vectors: Role of Endosomal Escape in MAP Kinase Activation and the Cytokine/Chemokine Response Jeffrey S Smith,1 Zhili Xu,1 Jie Tian,1 Donna J Palmer,2 Philip Ng,2 Andrew P Byrnes.1 Division of Cellular and Gene Therapies, Food and Drug Administration, Bethesda, MD; 2Molecular and Human Genetics, Baylor College of Medicine, Houston, TX Innate immune responses, characterized by a cytokine/chemokine burst, are a signicant hindrance to the systemic use of adenoviral vectors for treatment of disease A better understanding of innate signaling pathways may suggest targets for improving the safety of adenoviral gene therapy In order to gain insight into which features of the adenovirus virion trigger the innate immune response in vivo, we have investigated the response to a mutant Ad2 virus (ts1) that is defective for endosomal escape The innate response triggered by ts1 was compared to a helper-dependent Ad2 (HDAd2) vector whose genome contains only non-coding mammalian DNA sequences, without a transgene We found that ts1 was unable to activate early kinase pathways in the liver and spleen and that ts1 was defective for induction of some, but not all, cytokines and chemokines Results: We rst investigated the mitogen-activated protein kinase (MAPK) S209 IMMUNOLOGIC & HOST RESPONSES IN GENE & CELL THERAPY response in the liver and spleen at an early timepoint (30 min) MAPKs comprise a group of highly conserved intracellular signaling kinases that are involved in many cellular processes, including cytokine synthesis Of interest, MAPKs are known to be activated by toll-like receptors and other pro-inammatory pathways We injected mice i.v with x 1012 vp/kg of ts1 or HDAd2, and livers and spleens were collected 30 later to examine phosphorylation of MAPKs by Western blot We found that HDAd2 increased phosphorylation of p38 and ERK in mice In contrast, ts1 did not cause any signicant changes in the phosphorylation of these MAPKs as compared to buffer controls We also investigated the serum cytokine response to Ad at h Ts1 failed to induce a number of cytokines and chemokines that were robustly induced by HDAd2, including KC, IL-12p70, IL-6, TNF-alpha, IL-1beta and G-CSF Interestingly, however, ts1 was able to induce IP-10, RANTES and IL-10 to similar levels as did HDAd2 Ts1 was also able to partially induce MCP-1 and IFN-gamma Conclusions: Because we found that ts1-induced kinase and cytokine responses were severely impaired, we conclude that most of the innate immune response to adenovirus in vivo is triggered during or after endosomal escape of the virion However, a subset of cytokines and chemokines appear to be induced even without endosomal escape, suggesting a direct response to the viral capsid 543 AAV8 Vectors Evade CTL Mediated Destruction by Failing To Activate a Potent Inammatory Response Suryanarayan Somanathan, Ekaterina Breous, Peter L Bell, James M Wilson Gene Therapy Program, Department of Pathology & Laboratory Medicine, University of Pennsylvania, Philadelphia, PA We found previously that liver-directed gene transfer in mice using AAV serotype-8 vectors resulted in stable long-term expression of vector encoded transgenes Persistence correlated with absence of cytotoxic T lymphocyte (CTL) response to the transgene Stable expression from AAV8 vectors could not be abolished even when mice were challenged with Ad5; an adenovirus known to induce potent cytolytic T cell responses Most notably, there was an absence of IFN-γ secreting CTLs in mice systemically administered with AAV8 when compared to animals that only received an adenoviral vector Thus, it remains unclear whether AAV8 transduced cells can be cleared upon manifestation of a potent CTL response To study the effect of CTLs on AAV8 transduced hepatocytes, we adoptive transferred Ad5 activated CD8 T cells from donor syngeneic mice (C57B/6) into recipient mice (Rag-/-) stably expressing AAV8 encoded transgene The use of Rag -/- mice allowed us to monitor the effect of donor CTLs without interference from recipient T or B cells Although Ad5 induced CTLs could clear HAdV5 nlacZ expression in Rag-/- mice, they failed to eliminate AAV8 nlacZ expression The absence of CTL killing could be due to insufcient “danger signals” that cause ignorance of AAV8 transduced hepatocytes by circulating CTLs To test our hypothesis we co-administered TLR ligands LPS and CpG at the time of adoptive transfer LPS and CpG activate TLR and respectively, and are potent inducers of a systemic inammatory response Our results show that co-administration of LPS or CpG with the CTLs led to the elimination of AAV8 transduced hepatocytes with a concomitant decrease in AAV8 vector genome in the liver In control experiments we established that CTLs to an irrelevant transgene (eGFP) even when co administered with TLR ligands failed to eliminate transgene expression Livers of C57B/6 mice injected with LPS and CpG showed upregulation of MHC class I and cell adhesion molecules such as ICAM-1 and VCAM-I Similar results were also observed in mice that received adenovirus but not AAV8 Our results demonstrate that the propensity for inammation arising from genetic or environmental factors may negatively inuence AAV gene transfer to the liver S210 544 Robust and Sustained Factor IX Expression by IV Administration of AAV8-Based Vectors in Macaques Hiroaki Mizukami,1 Jun Mimuro,2 Akira Ishiwata,2 Hiroya Yagi,1 Tsukasa Ohmori,2 Seiji Madoiwa,2 Tomonori Tsukahara,1 Masashi Urabe,1 Akihiro Kume,1 Yoichi Sakata,2 Keiya Ozawa.1 Div of Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical University, Shimotsuke, Tochigi, Japan; 2Div of Cell and Molecular Medicine, Center for Molecular Medicine, Jichi Medical University, Shimotsuke, Tochigi, Japan Adeno-associated virus (AAV) vectors are promising for gene therapy approaches especially for hemophilia Among the increasing number of serotypes and strains in AAV, serotype 8-based vectors remain particularly suitable for liver-directed trials In order to evaluate therapeutic efficacy of AAV vectors in hemophilia gene therapy, we have been working on preclinical studies using cynomolgus macaques Earlier results indicate that the AAV8-based vectors are capable of transducing macaque liver efciently by intravenous injection On the other hand, inhibitory actions of preexisting neutralizing antibody (NAb) against vector capsid, even at marginal levels, are also demonstrated, suggesting the necessity of more sensitive assay for NAb detection To solve this issue, we have improved NAb detection system against AAV8 capsid with higher sensitivity Five sero-negative male cynomolgus macaques were selected AAV8-based vector encoding mutant macaque factor IX driven by liver-specic promoter was injected intravenously at × 1012 vg/kg (2 animals) or × 1012 vg/kg (3 animals), which are relevant vector dose in human trials All of the animals exhibited robust and sustained factor IX expression at the therapeutic window (3.0 – 20.2% of normal) No differences were found in factor IX concentration by the vector dose High copy numbers of vector genome (12.9 ± 1.3 vg/dge) were detected in the liver biopsy specimen Our results indicate a prospect of hemophilia gene therapy using AAV8 as well as the sufcient sensitivity of our improved NAb assay, which would be useful in human clinical trials using AAV8-based vectors This study was performed in collaboration with Tsukuba Primate Research Center, National Institute for Biomedical Innovation, and The Corporation for Production and Research of Laboratory Primates, Japan 545 Induction of Transgene Immunological Tolerance by a Single Injection of miR-142Regulated Integrase Defective Lentiviral Vector Andrea Annoni,1 Alessio Cantore,1 Lucia Sergi Sergi,1 Luigi Naldini,1,2 Maria Grazia Roncarolo.1,2 San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Milan, Italy; 2Vita-Salute San Raffaele University, Milan, Italy We have developed a microRNA-regulated lentiviral vector (LV) platform, which can maintain long-term sustained transgene expression in the liver of immune competent mice and induce tolerance to the transgene product In order to reduce the risk of insertional mutagenesis and to broaden its applications, we evaluated the use of integrase-defective lentiviral vectors (IDLV) for liver gene transfer IDLV can drive low-level transgene expression from the non-integrated proviral forms that transiently accumulate in transduced cells Because this episomal DNA is progressively lost in actively dividing cells, transgene expression is only transient in proliferating cells while IDLVs have been reported to drive prolonged transgene expression in non-dividing target cells We administered intravenously to mice matched doses of integrasecompetent lentiviral vectors (LV) or IDLV encoding GFP under the control of the hepato-specic promoters and carrying target sequences for miR-142 (ET.142T) At week post-injection we found detectable Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy ... processes, including cytokine synthesis Of interest, MAPKs are known to be activated by toll-like receptors and other pro -in? ??ammatory pathways We injected mice i.v with x 1012 vp/kg of ts1 or HDAd2, and. .. changes in the phosphorylation of these MAPKs as compared to buffer controls We also investigated the serum cytokine response to Ad at h Ts1 failed to induce a number of cytokines and chemokines... RESPONSES IN GENE & CELL THERAPY response in the liver and spleen at an early timepoint (30 min) MAPKs comprise a group of highly conserved intracellular signaling kinases that are involved in