669 involvement of myd88 and TLR9 in the innate immune response elicited by replication incompetent adenovirus vectors

669 Involvement of MyD88 and TLR9 in the Innate Immune Response Elicited by Replication Incompetent Adenovirus Vectors anti adenoviral immunity may not be an obstacle to the application of adenoviral[.]

anti-adenoviral immunity may not be an obstacle to the application of adenoviral gene transfer in islet transplantation A Glucose tol et'~ 25 dsys posl lram plent - ~-"' D B Glucose tolerance mort hS post nepl twt ••· t:l _ _ liI n 00 ,-5) (ro 5) ··00·· - .00 p~ '" ~300 g § 200 :2 i iii O+ o Mi"lul r- ,. - ""T"""- , 30 60 90 120 net g "'CO ~ 1l;c.d1on r;- · 008 ~ « ~ -_.- ~ ~=-~- - - - -) 100 O'+-o ···· · i ·~· · - - ~ -r ,. -, 30 60 Mmutes I fl.« gl.lcOse Il ~ on 90 120 I p&02 15 667 Periocular Triamcinolone Enhances Intraocular Gene Expression Following Delivery by Adenovirus Marisol D Cano, I Choul Y Park.l-' Roy S Chuck, I Margaret Yew,' Vict Nguyen,' Jack Parker,I Kcisuke Mori,' Peter L Gchlbach.' 'Ophthalmology; Johns Hopkins UniversitySchool ofMedicine Baltimore MD; lOphthalmolog)\ Dong-Guk UniversitySchool of Medicine, llsan, Korea; 'Biomedical Engineering Johns Hopkins University Baltimore, MD; "Ophthalmology; Saitama Medical University Iruma, Saitama, Japan Immunogenicity isa recognizeddrawbackofadenovirusmediated gene transfer in vivo While relative immune tolerance is present in the eye, immune responses may damage delicate ocular tissues and alter transgene expression A variety of vector modifications and treatment strategies have been employed to diminish vector induced immune response, ineluding deletion of nonessential viral genes; selective retention of genes for evasion of the host immune response and the use of more efficient vectors in smaller doses Immune modulation is standard component of the treatment of a variety of ocular diseases However, the extent to which local immune modulation affects viral transgene expression is not well understood In this study we monitored expression of the firefly luciferase reporter gene in mouse eyes following intravitreous delivery of adenovirus vcetor Gene expression was assessed by serial measurement of ocular bioluminescence with an IVIS' 200 ImagingSystem(Xenogen).Patternsofluciferase expressionin mice receiving periocular injection of the corticosteroid, Triamcinolone acetonide (TA), were compared to those in mice with no local immune modulation.The resultsindicatethat local immunemodulatory treatmentsmay significantlyprolongthe durationofpeak adenovirus mediated gene expression and the total period of gene expression in previously naive eyes The study also indicates that non-invasive imaging is a useful approach to longitudinal assessment of in vivo ocular gene expression in mice 668 Effect of Pre-Existing Anti-Adenovirus Immunity and Anti-Tumor Immunity on the Efficacy of Adenovirus Vectors in Immunocompetent Syrian Hamster Model Debanjan Dhar,' Jacqueline F Spencer; Karoly Toth,' William S M.Wold ' 'Molecular Microbiology and Immunology, St Louis University, St Louis, MG The field of Oncolytic adenovirus (Ad) vectors needs a proper animal model to study these vectors Due to the species specificity of Ads, such vectors are commonly evaluated in immunodeficient mice bearing human tumor xenografts Because these mice are immunodeficient and nonpermissive for human Ads, this model cannot adequately address the effect of the host immune system on the vector-infected tumor or the toxicity and biodistribution of the S256 vector in normal tissues Wehave recently developed a novel Syrian hamster model for the study of oncolytic Ads (Thomas M.A et al Cancer Res 66:1270-1276) We have been exploring the benefits of direct intratumoral injection of our oncolytic Ad5-based vector VRX-007 intosubcutaneousSyrianhamstertumors.VRX-007 lacks most E3 genes and overexpresses the Adenovirus Death Protein which mediates efficient infected cell lysis and vector spread This vector is very effective in suppressing the growth of tumors A key question regarding the use of Ad5-based oncolytic Ad vectors in humans is whether pre-existing immunity to Ad5 will limit the use of such vectors for cancergene therapy To address this question, we compared the effectiveness ofVRX-007 in naive and Ad5-immune hamsters, We found that, following intratumoral injcction, VRX007 suppressed the growth of tumors equally well in both groups Thus, pre-existing immunity to Ad5 does not affect vector efficacy following intratumoral injection However, pre-existing immunity greatly prevented the dissemination of vector from the tumor to lung and liver Wealso examined the role of pre-existing immunity to Ad in vector replication and toxicity in the liver following i.v, injection of VRX-007 with and without immunosuppression with cyclophosphamide (CP) The CP prevented an immune response against the vector and allowed us to evaluate thc duration of the original immunization All the non-immunized hamsters that were immunosuppressed and injected with VRX-007 were moribund by day) and had extremely high levels of serum ALT/AST The livers from non-immunized group were infected to a much greater extent than their immunized counterparts These results indicate that preexisting antibody to Ad5 does not affcet the efficacy ofVRX-007 in suppressing tumor growth following intratumoral injection but it does reduce the toxicity ofthe vector Wealso investigated whether an immune response develops against the tumor and if the vector acts as an adjuvant Hamsters were immunizedagainst the tumor by establishingsubcutaneous HaK (hamsterkidneycarcinoma)tumors Tumors were either injected with VRX-007 or vehicle After two weeks,tumorswcre surgicallyremovedfrom both groups HaK cells were injected into the other hind flank of both the groups and also into the hind flank of naive hamsters The growth of tumors in both VRX-007 and vehicle injected groups was suppressed to similar extent whereas in naive animals the tumorgrowth was not restricted Thus, an immune response develops against the tumor.No evidence for an adjuvant effect of the vector was obtained; however further research on this important question is needed, 669 Involvement of MyD88 and TLR9 in the Innate Immune Response Elicited by ReplicationIncompetent Adenovirus Vectors Tomoko Yamaguchi, Kenji Kawabata,' Naoya Koizumi.t-' Fuminori Sakurai, I Kazuko Nakashima,' Tomomi Sasaki, I Naoki Okada,' Hiroyuki Mizuguchi.P JLaboratory ofGene Transferand Regulation, National lnstitute ofBiomedical Innovation, Ibaraki, Japan; "Department of PharmaceuticalSciences, Showa Pharmaceutical University, Machida, Japan; 'Graduate School ofPharmaceuticalSciences, Osaka University Suita, Japan A replication-incompetent adenovirus (Ad) vector is arousing growing interest for both gene therapy and immunotherapy A major limitation of the use of Ad vectors is the innate immune response, whieh causes inflammatory cytokine production and tissue damage; however, the precise mechanism of it remains to be clarified Previous studies demonstrated that antigen-presenting cells express various types of pattern-recognizingreceptors (PRRs), such as toll-like receptors (TLRs), retinoic acid-inducible gene I (RIG-I), and nucleotide binding oligomerization domain (NOD)-I, and that innate immune responses in antigen-presenting cells were augmented by recognition of pathogens by PRRs TLRs recognize Molecular Therapy Volume15 Supplement I, \by 2007 Copyright © '111C American Societyo f Gene TIICr.lpr microbial components and trigger the signal cascade that activates innate immune responses Following the recognition of microbial components by TLRs, they, except for TLR3 , transduce intracellular signaling through the adaptor protein , myeloid differentiating factor 88 (MyD88), which initiates a signaling cascade leading to NF-kB activation Among 13 members of TLRs, TLR3 , TLR4, TLR7, TLR8, and TLR9 have been demonstrated to be involved in the recognition ofviral components Here , we show that Ad vectors elicit innate immune responses through a MyD88 /TLR9-dependent and/or -independent manner according to cell types Following stimulation with Ad vectors, the production of interleukin (IL) -6 was significantly decreased in MyD88- orTLR9-deficient dendritic cells (DCs), compared with wild-type DCs In addition, the surface expression of maturation marker protein, such as CD86 , was lower in Ad-infected MyD88-dcficicnt granulocyte-macrophage colonystimulating factor (GM-CSF) induced DCs than in wild-type DCs On the other hand, MyD88- or TLR9-deficient peritoneal macrophages produced the same level of IL-6 as wild-type macrophages after infection with Ad vectors We did not find any differences in the mRNA expression levels of the molecules involved in innate immunity, such as MyD88, TLR3 , TLR 7, and TLR9, between DCs and macrophages The intravenous injection of luciferase-exprcssingAd vectors into wild-type, MyD88-deficient, orTLR9-deficient mice resulted in almost comparable levels of IL-6 production and luciferase expression These results suggest that Ad vectors can activate the innate immunity via MyD88/TLR9-dependent and -independent mechanisms 670 Administration of a Combination of a Nondepleting CD4 Antibody and Cyclosporine Is Efficient at Attenuating Humoral but Not Cellular Immunity after Adenovirus Serotype Mediated Gene Transfer Jenny H Mclntosh,' Melanie Cochrane,' Andrew M Davidoff.' Amit C Nathwani.P I Department ofHaematology, University College London, London, United Kingdom; 2Haematology, National Blood Service, London United Kingdom; JDepartment ofSurgery, St Jude Children s Research Hospital, Memphis, TN Gene therapy is being explored for chronic disorders such as haemophilia B The development of neutralising antibodies to the FIX transgcne would be a potentially serious complication of'hacmophilia B gene therapy We have previously shown that a combination ofcyclosporine and non-depleting CD4 antibody (NDCD4), when co-administered with recombinant adeno-associated virus (rAAV) vectors prevented a humoral response to capsid proteins, enabling successful gene transfer upon re-administration ofvectors based on the same serotype Here we investigate whether the same immunemodulating strategy can be used to prevent the development of a humoral immune response to the FIX transgene using a challenging experimental design that involves intramuscular administration of an early generation (E IA-E3 deleted) adenovirus serotype-5 vector encoding human FIX (hFIX) gene under the control ofthe CMV promoter (Ad5-CMV-hFIX) Adenoviral vectors were chosen in preference to AAV because they are more efficient at mediating humoral and cellular immune responses to the adenoviral and transgenic proteins Balb/c mice were treated on days -1,0 +1, +3, +6, and +8 with NDCD4 (0.5mglmouse) and cyclosporine (CyA) 25mglkg On day 0, Ix I0 1" vector particles ofAd5-CMV-hFIX were administered into the hindlimb muscles Control mice received the same vector dose without immunosuppression hFIX at therapeutic levels was detectable in the plasma of both the control and immunosuppression cohorts immediately after vector administration but rapidly declined to undetectable levels within two weeks of vector administration In the control animals, this decline in expression coincided with Molecular Therapy Volume15 Supplement I ~ br 2007 Copyright © The American Soci ety o r Gene Therapy the development ofncutralizing anti-hFIX antibodies (inhibitors) Inhibitors were, however, not detected in animals treated with immunosuppression Instead these animals had developed a robust T cell response to the transgene, which most likely eliminated Ad5CMV-FIX transduced myocytes, Thus, the combination ofNDCD4 antibody and CyA is cffieient at attenuating humoral immunity to the transgenic hFIX proteins cven after administration ofadcnoviral vectors However, our studies suggest that further optimization of the immunosuppressive regimen is required to moderate cellular responses to the transgene following Ad5-CMV-hFIX mediated gene transfer GENE EXPRESSION IN ANIMAL MODEL SYSTEMS 671 Directing a Transgenic Erythropoietin Fusion Protein into the Regulated Secretory Pathway in Murine and Rat Salivary Gland Yuval Samuni , I Ana P Cotrim , I Changyu Zheng,' Gabor Z Racz, I Niamh X Cawley," Peng Y Loh.? Bruce J Baum.' 'Gene Therapy and Therapeutics Branch, NIDCR, NIH, DHHS, Bethesda, MD; 2LaboratOlY ofDevelopmental Neurobiology, NICHD, NIH, DHHS, Bethesda, MD Salivary glands are considered classical exocrine glands that sort and secrete proteins in both regulated (saliva) and constitutive (bloodstream) directions The mechanisms involved in this differential sorting arc believed to be based on structural determinants composed ofamino acid sequences providing a sorting signal or "zip code " for protein delivery, To direct therapeutic proteins normally secreted via the constitutive secretory pathway into the regulated secretory pathway (RSP) and thus into saliva, we investigated whether we could redirect a constitutively secreted protein into the RSP by fusing it with a RSP protein Human growth hormone (hGH), an endocrine protein normally secreted into the bloodstream via the RSP in somatotrophs of the anterior pituitary, is secreted from salivary glands into saliva by the RSP (Baum et al, Hum Gene Ther, 1999) Conversely, human erythropoietin (hEpo), normally secreted via the constitutive secretory pathway by kidney cells, is secreted constitutively to the bloodstream when expressed in salival)' cells (Voutetakis et al, PNAS, 2004) We therefore constructed a hEpo-hGH fusion protein expression construct and showed that the chimeric product was correctly expressed in HEK 293 cells using ELiSAs and Western blotting specific for both hEpo and hGH Experiments in AtT20 cells , a model endocrine cell line containing both constitutive and regulated secretory pathways, demonstrated significant sorting of the hEpo-hGH fusion protein into the RSP compared to hEpo alone The chimeric eDNA was then packaged in a serotype adenoviral (Ad5CMVEpoGH) vector and delivered to murine and rat submandibular cells in vivo via retroductal cannulation Over multiple experiments with different cohorts of mice (n=48) and rats (n=20) we showed that the hEpo-hGH fusion protein was sorted more frequently into saliva , versus thc bloodstream, than the native hEpo protein , demonstrating a redirection of hEpo from the constitutive to the regulated secretory pathway in salivary cells The serum/saliva ratios of hEpo and hEpo-hGH were shown to be statistically different (p