376 Immunosuppressive Strategies To Protect Transgenic Skin Cells from Immune Rejection Implications for Cutaneous Gene/Cell Therapy Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The[.]
IMMUNOLOGIC & HOST RESPONSES IN GENE & CELL THERAPY neutrophils) in the MDSCsBMP4 group versus the PBS control group At 7d PI, we observed a trend in a decrease of inflammatory cells in the MDSCsBMP4 group compared to the PBS control group At 14d and 21d PI, we found significantly fewer neutrophils and macrophages in the MDSCsBMP4 group versus the PBS control group (4) We found significantly more vascular endothelial cells migrating actively from the dura mater toward the MDSCs transplanted in the MDSCsBMP4 group versus the control group at 3d, 7d, 14d, and 21d PI These endothelial cells were derived from the host (4) We found significantly more vascular endothelial cells migrating actively from the dura mater toward the MDSCs transplanted in the MDSCsBMP4 group but not control group at 3d At all time points PI, we observed significantly more endothelial cells in the MDSCs group versus the PBS group.(5) We observed significantly fewer CD4 and CD8 immune cells at 7d in the MDSCsBMP4 group versus the control group But we found significantly more CD4 and CD8 cells at 14d and 21d PI in MDSCsBMP4 group which indicated the recruitment of bone marrow cells Conclusion: MDSCs expressed SOX-9, BMP receptors and other chemo-attractant genes BMP4expressing MDSCs participated in endochondral bone regeneration during cranial defect healing Chemo-attracted inflammatory cells induced early onset and early resolution of inflammation which facilitated bone regeneration MDSCs also chemo-attracted vascular cells from the host and promoted angiogenesis MDSCs inhibited the immune response at early time point which reduced the immune rejection of transplanted stem cells Immunologic & Host Responses in Gene & Cell Therapy 374 Characterizing AAV1 Capsid Antigenic Regions Yu-Shan Tseng,1 Brittney Gurda,1,2 Wendy Weichert,3 Colin R Parrish,3 John A Chiorini,4 Mavis Agbandje-McKenna.1 Biochemistry and Moleculalr Biology, University of Florida, Gainesville, FL; 2Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA; 3James A Baker Institute for Animal Health, Cornell University, Ithaca, NY; 4MPTB, NIDCR, National Institutes of Health, Bethesda, MD Analyses of human samples and the pre-clinical trial screening of patient serum has detected the prevalence of significant levels of pre-existing capsid-targeted antibodies directed against the Adenoassociated viruses (AAVs) under development as vectors for human gene delivery applications These antibodies are detrimental to the successful outcome of gene delivery procedures However, other than the prototype AAV2 serotype, very little is known about the antigenic regions of the AAV capsids We have used cryo-electron microscopy and image reconstruction obtain data for AAV:FAb complex structures which resulted in the proposal that ocommon antigenic epitopes exist on the capsid surface of a number of AAV serotypes, including AAV1, AAV2, AAV5, and AAV8 These studies point to the protrusions which surround the icosahedral threefold axes as the common target of antibody reactivity for vector neutralization We have extended AAV1 studies using biochemical and molecular approaches to verify the antigenic epitopes for three different neutralizing antibodies mapped onto the capsid These studies confirm pseudo-atomic modeling studies previously used to propose the antigenic epitopes on the AAV1 which overlap with regions of the AAV2 and AAV5 capsids known to interact with receptors, suggesting a possible mechanism of neutralization Significantly, mutant vectors, now able to “escape’ neutralization by parental antibodies, have wild virus titre levels and maintain the ability to transduce cells in vitro Thus these viruses have the potential to be developed as secondary generation vectors with the ability to evade pre-existing host immune responses for improved AAV gene delivery efficacy S146 375 Avoiding Trangene Immune Rejection after AAV Vector Delivery to the Skeletal Muscle: Tolerogenic Dendritic Cell-Based Immunotherapy in a Nonhuman Primate Model Aurélie Moreau,1 Mercedes Segovia,2 Laurence Dubreil,3 Gaëlle Tilly,2 Jack-Yves Deschamps,3 Yan Cherel,3 Ignacio Anegon,2 Philippe Moullier,1,4 Maria Cristina Cuturi,2 Oumeya Adjali.1 INSERM UMR 649, Nantes Hospital, Nantes, France; 2INSERM UMR 643, Nantes Hospital, Nantes, France; 3INRA UMR 703, Nantes National Veterinary School, Nantes, France; 4Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL Recombinant adeno-associated virus (rAAV) provides a clinically relevant platform for efficient and sustained gene therapy However, recent gene transfer studies into animal models, and more recently in humans, indicate that the risk of transgene immune responses is not negligible and depends on multiple factors including the route of vector delivery Even if immunosuppressive regimens have been successfully used, they are associated to adverse effects Immmunomodulation strategies to avoid transgene immune rejection are therefore needed One innovative strategy is based on the delivery of tolerogenic dendritic cells (DC) The successful use of such cells over the ten past years in rodent models of allograft rejection has indeed highlighted the therapeutic potential of this immunotherapy Our aim is to evaluate the tolerogenic potential of DC in the context of a nonhuman primate model of rAAV-based gene transfer We first generated immature macaque bone marrow derived-DC with in vitro immunomodulatory effects We then tested their in vivo potential after intramuscular (IM) injection of a rAAV expressing an immunogenic transgene groups of primates were injected with the vector in combination with immature autologuous DC administered either intradermally (ID) or via the intravenous (IV) route When autologuous DCs loaded with the transgene product were not immunogenic per se if injected alone in the absence of gene transfer, we found that their intradermal injection one day before the IM delivery of the vector was not able to prevent anti-transgene immune response Moreover, intradermal DC delivery resulted in an earlier immune trangene rejection as compared to the control group injected with the vector alone In contrast, in primates who received DC via the IV route, we observed a long term expression of the transgene, suggesting a possible immunomodulatory effect of DC immunotherapy When these results are still preliminary, they highlight the potential of tolerogenic DC in vivo in a large animal model of rAAV delivery, and in gene transfer protocols in general 376 Immunosuppressive Strategies To Protect Transgenic Skin Cells from Immune Rejection: Implications for Cutaneous Gene/Cell Therapy Soosan Ghazizadeh,1 Weibing Zhang,1 Li T Huang.1 Oral Biology and Pathology, Stony Brook University, Stony Brook, NY Epidermal keratinocytes and dermal fibroblasts are both potential targets for cutaneous gene/cell therapy for genodermatoses Immune elimination of genetically modified cells, however, presents a major impediment to effective therapy Using ex vivo approaches to gene transfer, we have previously shown that expression of an antigen by either cell type was sufficient to prime T cells and induce immune rejection of transplanted cells The nature of these responses and the kinetics of immune rejection, however, were significantly different for these two cell types suggesting different immunosuppressive strategies to control unwanted immune responses In this study, we explored the potential of local expression of immunosuppressive factors to protect transgenic skin cells from immune rejection Primary cultures of mouse keratinocytes or fibroblasts were transduced with a Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy CELL PROCESSING AND VECTOR PRODUCTION bicistronic vector encoding green fluorescent protein (GFP; as a model antigen) and an immunomodulatory molecule including CTLA4Ig, PDL1-Ig or PDL1, and were transplanted onto the back of a syngeneic mouse to regenerate skin Surface GFP expression in live mice was monitored weekly to assess the fate of transgenic cells Coexpression of transgene with either CTLA4Ig or PDL1 in fibroblasts resulted in long-term survival of transgenic fibroblasts (>20 weeks) despite the presence of systemic transgene-specific immune responses Similar treatment did not, however, protect keratinocytes from immune rejection Long-term protection of transgenic keratinocytes was achieved through transient blockade of CD40/CD154 interactions by administration of anti-CD154 antibody during the first two weeks of cell transplantation Although neither of these strategies induced long-lived antigen-specific tolerance, they were sufficient to prevent loss of genetically modified cells These results thus indicate that different strategies are required to restrict rejection of neoantigenexpressing keratinocytes and fibroblasts and long-term survival of genetically modified cells does not require induction of transgenespecific tolerance 377 Hepatocyte-Targeted Expression by Integrase-Defective Lentiviral Vectors Induces Antigen-Specific Regulatory T Cells and Immune Tolerance to Foreign Antigens Andrea Annoni,1 Alessio Cantore,1,2 Kevin Goudy,1 Lucia Sergi Sergi,1 Luigi Naldini,1,2 Maria Grazia Roncarolo.1,2 San Raffaele Telethon Institute for Gene Therapy, Milan, Italy; ”Vita Salute San Raffaele” University, Milan, Italy Integrase-Defective Lentiviral Vectors (IDLV) are emerging as an attractive gene delivery system These vectors harness the pantropism and proficiency of LV transduction without relying on integration and permanent modification of the cellular genome, thus providing a substantial safety feature It is not known whether IDLV can achieve therapeutically relevant transgene expression in the liver, and induce transgene-specific tolerance exploiting the tolerogenic properties of hepatocyte-targeted expression Here we characterize IDLV performance in primary human hepatocytes and in the mouse liver We show that IDLV efficiently transduce vector genomes, but express the transgene at lower levels on a per copy basis, as compared to their integration-competent (IC) counterparts To investigate whether hepatocyte-targeted IDLV can induce tolerance to a specific antigen, treated mice were re-challenged with the antigen to evaluate the induction of secondary anti-transgene immune response The absence of response to transgene indicated that IDLV treatment generate a state of transgene-specific immunological tolerance in recipient mice, while secondary expansion of transgenespecific CD8+ T cells was detectable in mice injected with control immunogenic IDLV To further confirm tolerance induction by IDLV, Rag2-/-γ-chain-/- mice expressing GFP in hepatocytes were immune reconstituted by adoptive transfer of a pool of splenocytes and liver lymphocytes isolated either from immunized (control IDLV-treated) or tolerized or naïve mice Comparable amounts of GFP-expressing hepatocytes were detected in the mice reconstituted with naïve cells or cells derived from tolerant mice, whereas a complete clearance of GFP+ hepatocytes was observed in recipient mice reconstituted with immune cells derived from control immunogenic IDLV-treated mice, accompanied by loss of vector DNA In addition, monitoring GFP expression in CD4+T CD45.2+ cells we could detect de novo Treg induction in mice that received the tolerogenic IDLV encoding for Ovalbuminin (OVA) in which Treg-depleted OTII-FOXP3GFPCD4+CD45.2+T cells (IAb-OVA323-339 specific tg-TCR and GFP-FOXP3+ knock in) were adoptively transferred Critical factors leading to Treg development in the liver are currently under investigation We previously reported the tolerogenic outcome of ICLV stringently targeted to hepatocytes We did not know, however, Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy whether this outcome depends on sustained high levels of transgene expression within hepatocytes, which requires substantial levels of vector integration, limiting the application of this approach outside of gene replacement strategies for the correction of monogenic diseases Our new findings demonstrate that Tregs induction in vivo can be obtained in a safer way by IDLV, thus opening up new therapeutic opportunities for immune modulation Cell Processing and Vector Production 378 Micro RNA Knockdown Significantly Enhances Adenovirus Replication and Vector Production Christina Rauschhuber,1 Martin Hausl,1 Dirk Nettelbeck,2 Anja Ehrhardt.1 Department of Virology, Max von Pettenkofer-Institute, Munich, Germany; 2Department of Dermatology, German Cancer Research Center, Heidelberg, Germany The use of micro RNAs (miRNAs) fundamentally improved the generation of viral vectors for gene therapy In this study we used our recently established HEK293 cells derived RNAi knockdown cell line B6 based on the RNAi suppressor protein P19 derived from the tomato bushy stunt virus to analyze the influence of the RNAi pathway on adenoviral vectors We investigated replication of wildtype adenovirus (wtAd5) and first generation adenovirus (FgAd) on a genome level An up to 10-fold increase in viral genome copy numbers could demonstrate that there is significantly enhanced viral DNA replication under RNAi knockdown conditions To analyze whether we can also achieve higher viral titers in the B6 cell line, we infected with a common FgAd expressing firefly luciferase (FgAdluc) After re-infection of HEK293 cells we measured up to 5-fold increased luciferase activity indicating a higher titer of FgAdluc derived from B6 cells To reach even stronger effects we hypothesized that the amount of the RNAi suppressor protein P19 may be the limiting factor Thus, we generated a recombinant adenovirus expressing p19 under the major late promoter (Bwtp19∆E3) using an advanced cloning technology based on bacterial artificial chromosomes We detected up to 100-fold increased genome copy numbers compared to wtAd5 and the corresponding control virus Notably, replication of Bwtp19∆E3 was even faster compared to an oncolytic virus that contains a 24 bp deletion in the E1a gene To shed light on the mechanism responsible for up regulation of adenoviral replication, we analyzed expression levels of early and late adenoviral proteins We found that expression levels of the early genes E4Orf6, DBP and E1B55K were significantly enhanced on mRNA and protein levels However, structural proteins like hexon and fiber were nearly unaltered on protein level but increased on mRNA level at late time points of infection Furthermore, we analyzed the fate of the virus-associated RNAs VAI RNA and VAII RNA, which are known to be processed by Dicer into 19-29 nucleotide long small virus-associated RNAs (sva-RNAs) We found that under the presence of P19, sva-RNAs are bound to P19 and are degraded into smaller products Last but no least, we developed a helper-virus expressing p19 (BHVp19) for helperdependent adenovirus (HC-AdV) production HC-AdVs represent the most advanced generation of recombinant adenoviral vectors and hold great promise with respect to safety and efficacy in gene therapeutic applications However, production of HC-AdVs is sophisticated and only a few groups are able to produce these vectors in sufficient amounts Thus, we wanted to further improve the viral titers by taking advantage of our finding that adenovirus replication is enhanced in the presence of P19 We found that in contrast to a conventionally used helper-virus the BHVp19 increased HC-AdV titers up to 12-fold In conclusion, we provide new insights into regulation of adenoviral vectors by RNAi and our RNAi knockdown cell line B6 could be used as a novel producer cell line for adenoviral vectors and in the future also for improvements of oncolytic viruses S147 ... 377 Hepatocyte-Targeted Expression by Integrase-Defective Lentiviral Vectors Induces Antigen-Specific Regulatory T Cells and Immune Tolerance to Foreign Antigens Andrea Annoni,1 Alessio Cantore,1,2... transplanted onto the back of a syngeneic mouse to regenerate skin Surface GFP expression in live mice was monitored weekly to assess the fate of transgenic cells Coexpression of transgene with either... amounts of GFP-expressing hepatocytes were detected in the mice reconstituted with naïve cells or cells derived from tolerant mice, whereas a complete clearance of GFP+ hepatocytes was observed in