108 escaping immune activation through the use of cpg depleted AAV vectors

108 Escaping Immune Activation through the Use of CpG Depleted AAV Vectors Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy S44 IMMUNOLOGIC &[.]

IMMUNOLOGIC & HOST RESPONSES IN GENE & CELL THERAPY serotype rh.10 adeno-associated vector expressing the human 1AT cDNA, mediates persistent therapeutic levels of human 1AT to the lung Based on these preclinical studies and in support of a clinical trial, we evaluated AAVrh.10h1AT for toxicity and efficacy in C57Bl/6 mice and African green nonhuman primates (NHP) after a one-time intrapleural administration Three groups of 40 mice were intrapleurally administered phosphate-buffered saline (PBS control) or AAVrh.10h1AT at 1010 and 1011 genomic copies (gc)/mouse Two additional groups received PBS or 1011 gc AAVrh.10h1AT vector directly into lung parenchyma, as worst case scenario for misplaced vector administration The mice were evaluated for months, and serum chemistry, neutralizing AAVrh.10 antibodies, gross pathology and microscopic histopathology on major organs assayed at 4, 29, 92 and 183 days post-dosing AAVrh.10h1AT-intrapleurally treated mice showed no vector related adverse effects nor did the group receiving vector to the lung parenchyma Neutralizing antibody titers to AAVrh.10 increased in a dose-responsive manner Titers remained high after day 29, except for 1011 gc dose group females, for which the average titers decreased by 50% at day 92 and remained at that level through day 183 For the NHP study, a total of 36 monkeys prescreened for 98% purity of murine Treg, splenic GFP+ cells were flow sorted after extraction from BALB/c mice containing a GFP reporter linked to FoxP3 message Sorted cells were stimulated in culture using anti-CD28/-CD3 beads and high-level IL-2 (1000 U/ml), which was replenished every days Stimulation was repeated at week After weeks, 20- to 50-fold expansion was repeatedly accomplished For our dose of 1x10^6 Treg/mouse, expansion was sufficient to treat 10 recipient mice per donor The frequency of peripheral Treg approximately doubled at day post-transplant but returned to the normal range wihin month Treg isolated from BALB/c F9 -/- x FoxP3-GFP mice and expanded in vitro were transferred to (BALB/c F9 -/-) hemophilia B mice Two days later, these mice (n=4) were treated by IM injection of AAV1-human FIX vector The resulting lack of inhibitor formation was maintained in of mice for >5 months despite challenge with hF.IX in adjuvant at week 16 Control animals (gene transfer but no Treg) formed high-titer anti-hF.IX (20 g IgG1/ml,  10 BU at week 6), and maintained inhibitor titers of >5 BU While total anti-hFIX IgG declined over time and circulating hF.IX antigen eventually emerged in the controls, IgG titers increased again after challenge Coagulation times were consistently prolonged compared to Tregtreated mice In other experiments, ex vivo expanded Treg were transferred to hemophilia A mice (BALB/c F8e16 -/-), which were then treated IV with human F.VIII (1 IU, once per week) for months Treg transplant again effectively suppressed inhibitor formation Inhibitor titers in control mice were 15-20 BU at month and 30-40 BU at months Treg treated mice (n=5) formed at most low-titer inhibitors (0-3 BU) Secondary Treg transfer confirmed induction of a suppressive response against F.VIII Treg therapy was also able to control inhibitor titers in hemophilia A mice with pre-existing inhibitors (n=6, 25 BU due to F.VIII replacement therapy) Half of these (n=3) received Treg, and all mice received more weeks of F.VIII treatment While inhibitor titers in control mice increased to 100 BU, Treg therapy effectively controlled the response (to 15-20 BU, P90% of an AAV8 vector expressing coagulation factor IX (AAV8-FIX) injected intravenously at a dose of 5x109 vg/mouse The same vector dose was then formulated in increasing amounts of AAV8 empty capsids, showing complete rescue of liver transduction at pre-treatment titers up to 1:100 Rescue of transgene expression correlated with the detection of antibody:capsid immune complexes in plasma of animals 24 hours after vector delivery, at levels up to 3.8X108 complexes/ml, which suggests that the empty capsids act as decoys for anti-AAV NAb Safety and efficacy of the approach was further validated in non-human primate studies, showing that formulation of AAV-FIX vectors in a 9-fold excess of empty capsids results in enhanced FIX levels (371±43 vs 129±82, p