1091 Induction of Molecular Chimerism in Rhesus Macaques Reconstituted with Autologous CD34+ Cells Transduced with the Gene Encoding the α(1 3)galactosyltranferase (αGT) we assayed response ofpr[.]
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 s 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 S416 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 s 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) 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 days after islet transplantation (Median onset time (MOT) = days) Immunohistochemical studies showed that there was massive CD3+T cell infiltration and no insulin storage in the islet grafts ofthese mice In contrast, out of6 mice reconstituted with protective IA~d-GFP transduced'bone remained normoglycemic for 90 days after islet transplantation (MOT= 90 days, n= 6, p70% of undifferentiated neurosphere cells over multiple passages, the isoforms properly localize to heterochromatin, and continue to express after differentiation into beta-tubulin positive neurons Because MeCP2 is not normally expressed in NSC, and MeCP2 over-expression is known to cause neurological disease, we created vectors that employ the neuron-specific mouse MeCP2 promoter (McP) In this case, we did not observe EGFP or the MeCP2 isoforms in undifferentiated neurospheres, but detected EOPP or the isoforms after in vitro differentiation into beta-tubulin positive neurons Overall, our SIN-E1~1 a retrovirus vectors demonstrate long-term expression in NSC without observable silencing The MeCP2-EI-myc and MeCP2-E2-myc vectors will be valuable tools for distinguishing the biological functions of the two MeCP2 isoforms, and the restricted expression pattern by the MeP promoter in neurons is well suited for RTT gene therapy We are currently introducing ex vivo modified fetal NSC into E I0 mouse brains using ultrasound guided intracerebral injection, and are preparing for ex vivo infections of adult NSC derived from MeCP2 null mice Injected mice will be examined for MeCP2-myc isoforms by immunostaining to demonstrate long-term retrovirus expression after in vivo NSC delivery as a first step towards RTT gene therapy 1093 Comparison of dsAAV Serotypes for Therapeutic Gene Transfer to Islets for Abrogation of Autoimmunity in Diabetes Daniel F Gaddy,' Khaja K Rehman,' Xiao Xiao,' Suzanne Bcrtera,' Nick Giannoukakis,' Paul D Robbins.' JDepartment ofMolecular Genetics and Biochemistry University ofPittsburgh School ofMedicine, Pittsburgh, PA; ]Division of Molecular Pharmaceutics UNC School ofPharmacy Chapel Hill, NC ; J Diabetes Institute Children Hospital of Pittsburgh, University ofPittsburgh School ofMedicine Pittsburgh, PA s The potential of self-complementary, double-stranded adenoassociated virus (dsAAV) vectors for efficient and long-term gene delivery into pancreatic cells, ineluding J3 cells within islets, has reeently been demonstrated We have compared the efficiency of dsAAV-mcdiatcd gene delivery to endogenous islets among different dsAAV serotypes (8,9, and rhlO) When delivered via the intra-peritoneal (i.p.) route , all three serotypes efficiently transduce endogenous islets However, when delivered via the pancreatic intraductal (i.d.) route, dsAAV8 and dsAAV9 transduce islets equally well , while dsAAVrhlO is much less efficient The ability to deliver and express therapeutic transgenes in endogenous J3 cells was then evaluated utilizing dsAAV8 via the i.p, and i.d, routes ofadministration , Expression orIL-4 in J3 cells prevented the onset of hyperglycemia in NOD mice, resulting in only mild insulitis as compared to controls Moreover, no disease developed whcn NOD.scid mice received adoptively transferred splcnocytes from 22- or 34-wcckold NOD mice treated with dsAAV-lL-4 Thus, we speculate that J3 cell-specific expression of IL-4 plays an essential role in inhibiting the activation of disease-causing lymphocytes Alternatively, the inhibition may be due to TGF-J3, which is secreted by activated T cells in the presence ofIL-4 These results demonstrate the utility of using the dsAAV8 vector to transfer irnmuno-rcgulatory agents to endogenous J3 cells in NOD mice to examine their role in preventing the onset of type-l diabetes S417 ... T cells in the presence ofIL-4 These results demonstrate the utility of using the dsAAV8 vector to transfer irnmuno-rcgulatory agents to endogenous J3 cells in NOD mice to examine their role in. .. gene therapy ofRTT using retrovirus delivery ofMeCP2 isoforms to transduce cycling Neural Stern Cells (NSC) derived from McCP2 null mice Delivery of ex vivo transduced NSC into the mouse brain... IL-6 and hTPO and then infected with VSVg enveloped simian immunodeficient virus carrying the gene encoding the aGT enzyme (SIV-aGT) After transduction, the cells were infused into lethally irradiated