667. Successful Liver Gene Therapy of Hemophilia B Dogs Using Lentiviral Vectors and Hyperfunctional Transgene

HEMATOLOGIC AND IMMUNOLOGIC GENE & CELL THERAPY II SINLV.ET.FIX did not show any evidence of genotoxicity, thus providing a safe vector design for hepatocyte gene transfer.

667. Successful Liver Gene Therapy of Hemophilia B Dogs Using Lentiviral Vectors and Hyperfunctional Transgene

Alessio Cantore,1,2 Patrizia Della Valle,3 Sara Bartolaccini,1 Lucia Sergi Sergi,1 Thierry VandenDriessche,4 Marinee Chuah,4 Dwight Bellinger,5 Armando D’Angelo,3 Timothy Nichols,5 Luigi Naldini.1,2 1 San Raffaele Telethon Institute for Gene Therapy, Milan, Italy; 2 ”Vita Salute San Raffaele” University, Milan, Italy; 3San Raffaele Scientific Institute, Milan, Italy; 4The Free University of Brussels, Brussels, Belgium; 5University of North Carolina, Chapel Hill. Lentiviral vectors (LVs) are attractive tools for liver gene therapy, by virtue of their ability to stably integrate in the genome of target cells and the absence of pre-existing humoral and cellular immunity against vector components in most humans. We previously reported longterm phenotypic correction of hemophilia B and transgene-specific immune tolerance after a single intravenous administration of LVs to mice. This was achieved by stringently targeting vector expression to hepatocytes by a combination of transcriptional and posttranscriptional, microRNA-mediated control. We have then translated this gene therapy strategy in a large animal model of hemophilia B. We produced large-scale purified LV batches using manufacturing processes and quality assessment previously established for ex vivo clinical use of LV. Each batch was administered into the portal vein of one adult hemophilia B dog. Infusion was well tolerated except for mild self-limiting hepatocellular toxicity and acute hypotension, which resolved after a single dose of anti-histamine/corticosteroid. Our results show long-term canine Factor IX (FIX) expression up to 0.5-1% of normal levels and clinical improvement (almost complete prevention of spontaneous bleedings) in the two treated dogs (4 years cumulative follow up), without long-term adverse effects or antitransgene immune responses. We next generated hyper-functional FIX transgenes carrying a R338L amino acid substitution, previously associated with clotting hyperactivity and thrombophilia. The use of codon-optimized and hyper-functional FIX transgenes increased the potency of LVs 15-fold, allowing correction of the disease phenotype at low vector doses in mice, thus improving the therapeutic index of the gene therapy. We have now tested this improvement in a dog. One-day pre treatment with anti-histamine/corticosteroid abrogated any detectable infusion-associated toxicity, including elevation of serum aminotransferases. At the current follow up (2 months) the dog is alive and well, the whole blood clotting time has shortened to the normal range and thromboelastogram is near-normal. Overall, this first-in-dog study of systemic administration of LV provides evidence of safe and effective treatment of hemophilia B and supports further pre-clinical development of this strategy. In the future, LVs may complement other vector platforms to broaden application of liver gene therapy.

668. Engineering of a HIV-Resistant Immune System with Hematopoietic Stem Cells – A Polycistronic Approach

Janet Chung,1 Chy-Anh Tran,2 Angel Gu,2 Lisa Scherer,1 Haitang Li,1 David L. DiGiusto,2,3 John J. Rossi.1,4 1 Department of Molecular and Cell Biology, City of Hope, Duarte, CA; 2Laboratory of Cellular Medicine, City of Hope, Duarte, CA; 3 Department of Virology, City of Hope, Duarte, CA; 4Irell and Manella Graduate School of Biological Sciences, City of Hope, Duarte, CA. Combination antiretroviral therapy (cART) prolongs survival and improves the quality of life of HIV-infected individuals, but is not Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy

curative. Gene therapy using hematopoietic stem and progenitor cells genetically modified with antiviral genes (GM-HSPCs) has been proposed as a way to provide an HIV-resistant immune system and may provide a functional cure. We have previously demonstrated safety and feasibility of transplanting GM-HSPCs in AIDS-related lymphoma patients utilizing a lentiviral vector carrying three anti-HIV small RNAs expressed independently from strong Pol III promoters. We have subsequently created a series (5) of “improved” lentiviral vector constructs using an endogenous polycistronic miRNA cluster (MCM7) to co-express different classes of anti-HIV small RNAs (e.g., shRNA, nucleolar RNA decoys, ribozymes), in a single transcript from the Pol II U1 promoter. We also included a selectable genetic marker (MGMTP140K) to allow prospective chemical enrichment of GM-HSPCs. We demonstrated protection from HIV infection in the progeny of GM-HSPCs created with three of the five “improved” constructs with in vitro HIV challenge assays. Furthermore, we demonstrated enhanced transduction efficiency (3-fold) by including rapamycin during transduction and another 2-3 fold through BCNU drug treatment during in vitro expansion of GM-HPSCs in the presence of an aryl-hydrocarbon receptor antagonist (SR1). Thus, using a combination of technologies, we have demonstrated an approximately 6-fold increase in level of stable genetic modification obtainable in HSPCs. We are currently evaluating antiviral activities of our RNA-based constructs in humanized NSG mice. An optimized combination of improvements in vector construct, genetic modification methods and in vitro drug selection will be used in a proof of principle trial of stem cell based HIV gene therapy.

669. Direct and Indirect Immune Suppressive Effects of High Hepatic Expression Direct Effective Reversal of Factor IX Inhibitors by AAV Gene Transfer David Markusic,1 Roland Herzog.1 Pediatrics, University of Florida, Gainesville, FL.

1

While the incidence of antibody formation against factor IX is comparatively low in treatment of hemophilia B (3-5%), these inhibitors are difficult to eradicate due to hypersensitivity and nephrotic syndrome. Thus, conventional clinical immune tolerance (ITI) protocols often fail in hemophilia B, and patients continue to be at high risk for morbidity and mortality. We have developed the first animal model of anaphylaxis in F.IX replacement therapy, C3H/ HeJ hemophilia B (HB) mice with a F.IX gene deletion. Moreover, hepatic AAV8-F.IX gene transfer successfully and safely reversed inhibitors, eliminated anaphylaxis, and restored hemostasis at a dose of 1x1011 vg/mouse. Therefore, gene transfer is potentially a superior alternative to current ITI. Here, we sought to define the requirements and mechanism of inhibitor reversal. HB mice that had formed inhibitors to F.IX protein were injected with rAAV8-F.IX at 2x109 (low) or 1x1010 (mid) vg. Mice treated at the mid dose showed partial correction of the aPTT and remained free of inhibitors and anaphylaxis even after subsequent repeated IV challenge with F.IX protein. In contrast, low-dose treated mice lacked correction and had fatal anaphylactic reactions to subsequent F.IX infusion, similar to animals that did not receive gene transfer. Using an in vitro assay based on ELISpot, we found that low levels of F.IX protein (0.1 IU/ ml) activated F.IX-specific memory B cells, while higher levels (1 and 10 IU/ml) suppressed. This data mirrors in vivo observations that high vector dose led to a rapid reduction in hF.IX antibodies, while mid dose had a slower reduction, and low dose failed. Adoptive transfer of CD4+CD25+ Treg from vector-treated C3H/HeJ was performed to test for induction of cellular immune suppressive responses. Naive C3H/HeJ mice receiving 1x106 control Treg developed hightiter antibodies after immunization against F.IX, while mice given Treg from mid and high dose transduced animals showed a graded reduction. In contrast, Treg from low-dose treated mice failed to S255