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263 Efficient Induction of Immune Tolerance to FIX Following Direct Intramuscular Gene Transfer 260 Tolerance Induction to FIX in Mice with Pre Existing Anti FIX Immunity Following Direct Intramuscula[.]
260 Tolerance Induction to FIX in Mice with Pre-Existing Anti-FIX Immunity Following Direct Intramuscular Injection of AAV Ellen F Cohn; Meagan E Kelly,' Jiacai Zhuo,' Hengjun Chao.' Department ofMedicine Division ofHematology and Oncology Mount Sinai School ofMedicine, New York, NY I A major complication in hemophilia B treatment is the formation of inhibitory anti-FIX antibodies Although continuous infusion of high doses of FIX is capable of inducing FIX tolerance in hemophilia patients, the success rate is not satisfactory High incidence of anaphylactic responses to FIX in patients positive with anti-FIX antibodies further limits application ofthis procedure in FIX tolerance induction A new, more efficient and cost-effective procedure is desirable for treatment of hemophilia patients who are positive with anti-FIX antibodies We recently demonstrated that direct intramuscular injection (1M) ofnai've hemophilic mice with a single dose ofAAVI-FIX led to sustained expression of FIX and specific immune tolerance to FIX in mice with diverse genetic and immune backgrounds In this study, we tested whether 1M of AAVI can overcome pre-existing anti-FIX immunity and induce FIX tolerance We generated anti-hFIX antibodies in cohorts ofhemophilia B mice with different genetic and immunological backgrounds (C57BLl6, Balb/c and C3H) by 1MofAAV2 vectors or intraperitoneal injection of recombinant hFIX protein ELISA and Bethesda inhibitor assay measured the anti-FIX antibodies Wc then treated the mice with 1M of AAVI-hFIX vectors, We observed significant reduction of anti-hFIX antibodies and sustained expression oftherapeutic levels ofhFIX after treatment of1M ofAAVI in all the mice For example, in C57BLl6 mice pre-treated with 1MofAAV2-FIX, the level ofantihFIX antibodies greatly decreased from 3974 nglml before AAVI injection to baseline (109 ng/ml) 16 weeks after 1M ofAAVI·FIX In summary, we demonstrated that 1M ofAAVI-FIX overcame the pre-existing anti-FIX immunity and induced tolerance in mice These results validate the potential application of AAVI-FIX for hemophilia B patients with inhibitors, who are extremely difficult to treat Elucidation of the mechanism responsible for FIX tolerance by direct intramuscular injection ofAAV is ongoing 261 Antigen-Specific Tolerance Induction by Transcriptional Targeting of Dendritic Cells with a Novel Lentiviral Vector Christiane Dresch; Thomas Brocker,' 'Immunology; Ludwig-Maximilltans-University; Munich Germany Dendritic cells (DC) arc the most powerful antigen presenting cells (APC) ofthe immune system Since DC can induce both tolerance and immunity, there is an increased interest in understanding the biology of DC for basic research and clinical applications Different DC subpopulations have been described and several attempts have been made trying to correlate these DC subsets with different functions However, the difficulties to manipulate DC ex vivo or in vitro without changing their original phenotypic and functional characteristics are major obstacles in DC-research For this reason we developed a system in which transgenes can be expressed selectively in DC after hematopoietic stem cell transduction with a selfinactivating lentiviral vector containing a DC-specific promoter We show that this gene-therapy approach yields long-term DC-selective transgene-expression that renders the T cell compartment tolerant to transgenic antigen Ag-specific viral or protein immunizations could not break T cell tolerance induced by DC In addition, our preliminary data in vitro show that this system also targets human DC Delivering transgencs specifically to the DC by using viral vectors might be a promising tool in gene therapy SIOO 262 Suppression of Hyperglycemia in Nod Mice after Inoculation with Recombinant Vaccinia Viruses Bela Denes, lie Yu, Nadja Fodor, Zsuzsanna Takatsy, Istvan Fodor, William I-I R Langridge Department ofBiochemistry and Microbiology; Loma Linda University Loma Linda CA I In autoimmune (Type I) diabetes, autoreactive lymphocytes destroy pancreatic /3- cells responsible for insulin synthesis To assess the feasibility of gene therapy for Type diabetes, recombinant vaccinia virus vectors werc constructed expressing pancreatic islet autoantigens proinsulin (INS) and a 55 kDa immunogenic peptide from glutamic acid decarboxylase (GAD), and the immunomodulatory cytokine IL-IO To augment the beneficial effects of recombinant virus therapy, the INS and GAD genes were fused to the Cvterminusofthe cholera toxin B subunit (CTB) Five week old non-obese diabetic (NOD) mice were injected once with recombinant vaccinia virus (rVV) and humoral antibody immune responses and hyperglycemia in the infected mice analyzed Only 20% ofthe mice inoculated with rVVexpressing the CTB::INS fusion protein developed hyperglycemia, in comparison to 70% ofthe mice in the un-inoculated animal group Islets from pancreatic tissues isolated from euglycemic mice from this animal group showed no sign of inflammatory lymphocyte invasion Inoculation with rVV producing CTB::GAD or IL-IO was somewhat less effective in reducing diabetes Humoral antibody isotypcs of hyperglycemic and cuglycemic mice from all treated groups possessed similar IgG I11gG2c antibody titer ratios from 19 to 31 weeks after virus inoculation In comparison with uninoculated mice, II week old NOD mice injected with virus expressing CTB::INS were delayed in diabetes onset by more than weeks The experimental results demonstrate the feasibility of using rVV expressing CTB::INS fusion protein to generate significant protection and therapy against Type I diabetes onset and progression 263 Efficient Induction of Immune Tolerance to FIX Following Direct Intramuscular Gene Transfer Ellen F Cohn, I Meagan E Kelly,' Jiacai Zhuo,' Hengjun Chao.' I Department ofMedicine Division ofHematology and Oncology, Mount Sinai School ofMedicine New York, NY Formation of inhibitory anti-FIX antibodies is the major complication of FIX protein replacement-based hemophilia B treatment It is difficult to treat patients with anti-FIX antibodies Although gene therapy is emerging as a potentially effective treatment for hemophilia, development of anti-FIX antibodies may impede its application to patients Direct intramuscular injection (1M) of adeno-associated virus (AAV) is a safe and efficient procedure for hemophilia B gene therapy Our previous studies have shown that 1M ofAAVI, in contrast to AAV2, does not cause antibody formation and leads to expression of therapeutic levels of FIX In this study, we aimed to investigate induction of immune tolerance to human FIX (hFIX) by 1M ofAAVI, further validating 1M ofAAVI for hemophilia B gene therapy Cohorts of hernostatically normal and hemophilia B mice with diverse genetic and MHC backgrounds received 1M of AAV-hFIX Human FIX antigen and anti-hFIX antibodies were examined 1M of IxIO II vector genomes (VG) of AAV2 elicits formation ofanti-hFIX antibodies comparable to those by hFIX protein replacement 1M of Ix IOl , VG ofAAVI results in expression oftherapeutie levels ofhFIX (up to 950 ng/ml, Mean=772 ng/ml, SEM ±35.7) and hFIX-specific immune tolerance in C57BLl6 mice Similar results were obtained for Balblc and C3H mice, with both strains of mice expressing high levels of FIX and low levels of antibody Challenge ofall ofthe mice treated with AAV I-hFIX with rhFIX in CFA, which is highly immunogenic, does not affect the Molecular Therapy Volume 15 Supplement I•.\b)' 20m Copyright © The American Society o t Gene Therapy level of FIX and docs not increase the level of antibodies, indicating tolerance induction In conclusion, a single 1M ofAAVI can result in efficient expression of therapeutic levels ofhFIX and induction ofhFIX tolerance in hemostatically normal and hemophilia B mice Our results substantiate the prospect ofiM of AAVI for hemophilia B gene therapy and FIX tolerance induction 264 Induction of FIX Tolerance by Direct Intramuscular Injection of AAV Is AAV-Dose/FIXLevel Dependent Ellen F Cohn; Meagan E Kelly,' Jiaeai Zhuo,' Hengjun Chao.' Department ofMedicine, Division ojHematology and Oncology, Mount Sinai School ofMedicine, New York, NY I We recently reported successful induction ofhFIX-specific immune tolerance by direct intramuscularinjectionofAAVl-hFIX vector in mice, The mechanism accounting for FIX tolerance following intramusculardelivery ofM V is unclear WehypothesizethatAAVI dose and the resulting expression of high levels ofhFIX is critical to induction of FIX tolerance In this study, we investigated the correlation between AAV-hFIX doses and hFIX expression as well as induction of FIX tolerance Cohorts ofC57BLl6 mice received direct intramuscular injection ofAAVl-hFIX vector with escalating doses (Ix l O'", 2xlO lO, Sx l O!" and Ix 10" vector genomes of MVI per mouse, n=5 per cohort) Human FIX antigen and anti-hFIX IgG antibodies in the plasma oftheAAV-injected mice were examined We observed that the levels ofhFIX antigen in the mice after AAV injection is proportional to the amount of AAVI-hFIX injected Direct injection of as little as 2x I 10 vector genomes ofAAVI to 8-week old C57BLl6 mice resulted in expression of therapeutic levels ofhFlX (mean=333 ng/ml, SEM +1-62.6,5.4-7.9% ofnormal hFIX Icvcl) without detection of anti-hFIX antibody High levels of antibodies against hFIX rather than hFIX antigen were detected in plasma of mice that received IxlO 10 vector genomes of AAVIhFIX vector, which was similar to mice that received intramuscular injection of AAV2 Extension ofthese investigations in hemophilia B mice with null FIX mutation reproduced and confirmed our findings in the hemostatically normal mice These results support our hypothesis that MV dose and FIX level is a determining factor for FIX tolerance induction in FIX gene transfer by intramuscular injection of M V In summary, we reported that direct intramuscular injection of a modest dose (2x I01U) ofAAVI expressed therapeutic levels of FIX and induced FIX tolerance in mice We defined the minimum dose of AAVI and the lowest level ofhFIX essential for FIX tolerance induction.Our resultswill contributeto the elucidation of mechanisms underlying induction of FIX tolerance Our results will also provide a new, alternative procedure for FIX tolerance induction, as well as potential adjustments in FIX doses used in current protocols for FIX tolerance pCBLacZ, and the last five mice received same volume of phosphate buffer The animals were immunized four times Seven days after the last immunization, they received intramuscular injection of 50 ug ofpCBLacZ at the left anterior tibialis Animals were sacrificed at 6th days after intramuscular injection to check the expression of beta-galactosidase in the left anterior tibialis Result: Mean numbcrs of muscle cells expressing bcta-galacosidase were much less in the animals immunized with protein or DNA as compared with the littermates Those were 0.833, 2.0 and 68.4 (figure I) ELISA for anti-galactosidase IgGI and IgG2a showed that the animals treated with protein developed a Th2 predominant immunity and the mice receiving DNA had a preferential Th I response (figures 2) Histochemical study shows there are prominent inflammatory changes in muscle of the mice preimmunized with either DNA or protein Discussion: Gene delivery to muscle is a potential therapy for protein deficiencies such as hemophilia or cnzyme defects such as Pornpe's disease Patients may have ever received protein therapy years prior to the application of gene therapy, or they may need repeated gene therapy to keep long-term expression of the transgene, In this study, we demonstrate that pre-existing immunity with either Th I or Th2 predominance inhibits transgene expression in muscle cells 120 ~ 100 OJ c 80 l;::: OJ ::J :0 60 - c E 40 - Z 20 - L OJ ::J - - , :;: Con trol IgG2a 09 OS 07 06 g 0.6 An-Bang Liu.' I Department ofNeurology Buddhist Tzu Chi General Hospital Hualien, Taiwan 1l 0.2 q: 0' /' 0.8 beta -gal IgGl E ~ 1.8