1090. Prevention of Type I Diabetes Recurrence Following Islet Transplantation in Diabetic NOD Mice by Gene Therapy

1090. Prevention of Type I Diabetes Recurrence Following Islet Transplantation in Diabetic NOD Mice by Gene Therapy

we assayed response of primary macrophages from C57BLl6 mice to Ad capsid components. We found that I'ID-Ad DNA together with penton proteins had the ...

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we assayed response of primary macrophages from C57BLl6 mice to Ad capsid components. We found that I'ID-Ad DNA together with penton proteins had the highest stimulatory effect. In conclusion, adenoviral vectors activate immunity through TLR-dependent pathways leading to thc production of pro-inflammatory cytokines and chemokines. This is mediated cooperatively by cell surface and endosomal receptors, TLR2 and TLR9 , respectively. Moreove , they are dependent on a common TLR adaptor MyD88-1-. By modulating signaling via these receptors, we may be able to affect the innate and adaptive immune responses to HDY in vivo.

1089. An In Vitro Model of CD8+ T Cell-Mediated Killing of AAV-Transduced Human Hepatocytes

Etiena Basner-Tschakarjan,' Daniel J. Hui; Shangzhen Zhou; Federico Mingozzi,' Katherine A. High.P I Hematology, The Children Hospital ofPhiladelphia, Philadelphia. PA; ]Howard Hughes Medical Institute, Philadelphia. PA.

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Adeno-associated virus (AAY) vectors have been successfully used as gene therapy vectors, mainly to correct monogenic disorders, where an advantage over other delivery systems is the mild immune response evoked in animal models , allowing prolonged expression of the transgene. However, in a recent clinical trial of AAY-mediated hepatic gene transfer for hemophilia B a CD8+ T cell response to the vector capsid was documented. We hypothesize that this CD8+ T cell response eventually led to the destruction of transduced hepatocytes with loss of transgene expression and an accompanying reversible rise in serum transaminases. In order to further elucidate the underlying immune response as well as to try to identify subjects prone to react to AAY capsid before treatment, we established an in vitro system by which AAY capsid epitopeexpanded peripheral blood mononuclear cells (PBMC) can be tested for their cytotoxic capacity towards virus-transduced target cells. We were able to identify several major T cell epitopes from normal donors and from subjects enrolled in AAY gene therapy trials using ELISpot assays and a peptide library derived from theAAY capsid YP I protein sequence. Subsequently we used these peptide epitopes to expand CD8+ T cells in vitro and also used them in a cytotoxicity (CTL) assay with an HLA-matched hepatocyte cell line (an immortalised primary human hepatocyte cell line kindly provided by Arvind H. Patel, University of Oxford, Oxford, UK) as target cells. Target cells were transduced with an AAY2 virus prior to the assay and cytotoxicity could be shown as soon as four hours after treatment (20% specific lysis at an E:T ratio of 10:I) with higher levels after 48 hours (30% specific lysis). Peptide loading of the hepatocytes with the relevant HLA-matched MHC class I epitope led to constant high levels of cytotoxicity (30-40% specific lysis). Our data suggest that human hepatocytes transduced in vitro with AAY2 are able to process capsid antigen and present it in an MHC I context to CD8+ T cells, recapitulating in vitro what we observed in vivo in humans . Additional experiments with alternate serotypes arc currently underway. In conclusion we have established an in vitro system for the expansion of human CD8+ T cells reactive to AAY2 capsid. We have also established an in vitro cytotoxicity assay for AAY2-transduccd human hepatocytes. These results clearly demonstrate that capsid-specific CD8+ T cells can lyse HLA-matched AAY-transduced hepatocytes, providing strong supporting evidence for our hypothesis that loss ofexpression and transaminitis observed after hepatic artery infusion of AAY2-F.lX resulted from CD8+ T cell-mediated lysis oftransdueed hepatoeytes. The same system can be used to test the performance of alternate serotypes. This assay system is particularly valuable since it has so far not been possible to establish an animal model ofCD8+-mediated T cell destruction ofAAY-transduced target cells.

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GENE EXPRESSION IN ANIMAL MODEL SYSTEMS

1090. Prevention of Type I Diabetes Recurrence Following Islet Transplantation in Diabetic NOD Mice by Gene Therapy Chaorui Tian,' Mohammed Javeed Ansari ,' Jesus Paez-Cortez,' Jessamyn Bagley,' Mohamed Sayegh,' John Iacomini.' 'Transplantation Research Center; Brigham and Women s Hospital and Children Hospital Boston, Boston, MA.

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Introduction: We have recently shown that induction of molecular chimerism by reconstitution of young NOD mice with autologous bone marrow cells transduced with retrovirus carrying genes encoding MHC class II ~ chains from protective haplotypes can prevent occurrence of type I diabetes in NOD mice. Here we show that induction of molecular chimerism in mice that have spontaneously developed diabetes can be used to prevent recurrence of type I diabetes following islet transplantation. Methods: Bone marrow cells were harvested from 4-week old NOD mice treated with 5-fluorouracil (150mglkg) 7 days prior and then transduced with retrovirus carrying genes encoding either a protective MHC class II ~ chain fused to GFP (IAW-GFP) or GFP alone as a control. Spontaneously diabetic NOD mice were lethally irradiated and reconstituted the following day with either IA~d-G FP or GFP transduced bone marrow. All mice were maintained normoglycemic with daily subcutaneous insulin injection. Four weeks after reconstitution, all recipient mice were transplanted with 1000 NOD.scm islets under the kidney capsule. Blood glucose levels were measured daily after islet transplantation. Islet grafts were harvested upon hyperglycemia or 90 days after islet transplantation. Results: All of the mice reconstituted with control GFPtransduccd bone marrow developed diabetes by 9 days after islet transplantation (Median onset time (MOT) = 2 days). Immunohistochemical studies showed that there was massive CD3+T cell infiltration and no insulin storage in the islet grafts ofthese mice. In contrast, 5 out of6 mice reconstituted with protective IA~d-GFP transduced'bone remained normoglycemic for 90 days after islet transplantation (MOT= 90 days, n= 6, p
1091. Induction of Molecular Chimerism in Rhesus Macaques Reconstituted with Autologous CD34+ Cells Transduced with the Gene Encoding the a(1-3)galactosyltranferase (aGT) Lorenzo Benatuil , Robert Donahue, Aylin Bonifacino, Cynthia E. Dunbar, John lacomini. 'Transplantation Research Center: Brigham and Women s Hospital and Children s Hospital Boston, Harvard Medical School, Boston. AlA; ]Hemat%gy Branch, NHBLI, NIH. Bethesda, MD. Background: We have previously shown that genetic engineering of autologous bone marrow stem cells to induce molecular chimerism can be used to establish B cell tolerance in mice. In this study, we examined whether a similar approach could be used to induce B cell tolerance in Rhesus macaques, a preclinical animal model. To this end, we investigated whether induction ofmolecular chimerism could be used to affect production of antibodies specific for the carbohydrate epitope Galal-3Gal~I-4GlcNAe-R (aGal). Molecular Therapy Volume 15.Supplement I•.\1,,)" 2007 Copyri ght © ' 111C American Society o f G ene Tllt'r.lP)'