302 Drosha and the Control of Mammalian Hematopoiesis Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy S117 HEMATOLOGIC NOVEL TECHNOLOGIES, LINEAGE[.]
HEMATOLOGIC-NOVEL TECHNOLOGIES, LINEAGE SPECIFIC REGULATION 300 Long-Term Transgene Expression in NewBorn Cynomolgus Macaques Following IntraUterine Gene Transfer of Self-Complementary AAV Citra N Mattar,1 Niraja M Dighe,1 Simon N Waddington,2 Jenny McIntosh,3 Arjit Biswas,1 Nicholas M Fisk,4 Andrew M Davidoff,5 Amit C Nathwani,3 Mahesh A Choolani,1 Jerry Chan.1 Obstetrics and Gynaecology, National University of Singapore, Singapore, Singapore; 2Haematology, Univerisity College London, London, United Kingdom; 3UCL Cancer Institute, University College London, London, United Kingdom; 4University of Queensland’s Centre for Clinical Research, Brisbane, Australia; Surgery, St Jude Children’s Research Hospital, Memphis Introduction: Intra-uterine gene transfer (IUGT) has the potential of averting irreversible organ damage which may lead to severe disability and premature death in a wide range of inherited diseases However, the consequences of IUGT with rAAV, a virus that persists largely in an episomal form, in a context relevant to humans has not been critically evaluated Here we report for the first time the level and stability of gene transfer, as well as the immunological and toxic effects of IUGT of relatively large doses of a self complementary AAV (scAAV) vector encoding human coagulation factor IX, which serves as a useful marker gene Materials and methods: Cynomolgus macaques were time-mated, and at 0.9 gestation, 4x1012 vg of scAAV2/5- or scAAV 2/8-LP1-hFIXco were injected into the fetal intrahepatic vein under ultrasound guidance Maternal blood was sampled serially for viraemia studies Offspring were delivered by caesarean section and maternal tissue biopsies performed for vector biodistribution and transgene expression Maternal and infant blood was sampled serially to quantitate transgene levels Results: Eight fetuses received AAV-hFIX (1.3x 1013 vg/kg) Maternal viraemia (2-32vg/ml) was detected up to 24 hours post-injection, with higher vector copy numbers following AAV5 delivery Widespread maternal tissue transduction was detected at low levels and maternal antiAAV antibody response was transient hFIX was not detected in maternal plasma Post IUGT, plasma hFIX was expressed in offspring between 10-633% of normal, with expression being ten-fold higher after AAV8 administration After an initial decline during the first month of birth, hFIX expression remained stable at µg/ml (100% of normal levels) between 2-11 months after birth (duration of the study) despite a four fold increase in infant weight Although the liver was preferentially transduced with serotype and vectors, the proviral DNA was detectable in all tissues examined, although at substantially lower levels However, the hFIX transcript was only detectable in the liver by a sensitive RT-PCR assay A robust antiAAV antibody response was observed consistent with the relative immunological maturity of the macaque fetus However this did not affect scAAV mediated transgene expression Conclusions: Our data suggests that IUGT of scAAV vectors results in long-term high level stable transgene expression in newborn nonhuman primates without significant toxicity Therefore, this vector holds great promise for the treatment of a variety of congenital disorders, although the mechanisms responsible for stable rAAV mediated expression need to be established 301 Recombinant AAV Vector-Mediated Gene Delivery for the Potential Treatment of Adenosine Deaminase Deficiency Jared Silver, Giridhara R Jayandharan, Pedro Cruz, Li Zhong, Melissa Elder, Arun Srivastava, Terence R Flotte Department of Pediatrics, University of Florida College of Medicine, Gainesville, FL; UMass Gene Therapy Center, University of Massachusetts Medical School, Worcester, MA Adenosine deaminase (ADA) deficiency is a rare (1:500,000) single gene defect resulting in severe combined immunodeficiency Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy (SCID) It is characterized by profound deficiency of T, B, and NK cells, and often leads to death in infancy if left untreated Standard of care for treatment of ADA-SCID is through histocompatible hematopoietic stem cell transplantation or alternately by ADA enzyme replacement therapy if HLA identical donors are not available Although recombinant ADA retroviral vectors have shown efficacy in a recent clinical trial for ADA-deficiency, the safety profile of recombinant adeno-associated virus (rAAV) vectors makes them an attractive alternative for gene therapy of human ADA deficiency Single stranded (ss) and self-complementary (sc) AAV vectors based on serotypes and encoding secretory human ADA (hADA) were designed and tested intravenously for their efficacy in a partial knock out model of ADA-deficient mice All animals were followed up for serum ADA activity and immunologic restoration of T, B, and NK cell populations over time (60-150 days) The efficiency of rAAV transduction in different organs was assessed by immunohistochemistry and quantitative real-time PCR Injection of 3x10e11 particles of ssAAV1-Igk-hADA provided abundant, persistent gene delivery to murine skeletal muscle, but did not restore lymphocyte numbers in treated ADA-SCID animals However, injection of a high dose (1x10e12 particles) of ssAAV9-Igk-hADA fostered substantial, persistent gene delivery to heart, liver, skeletal muscle, and kidney while also inducing a progressive, prolonged restoration of B and T cell populations in treated mice Injection of scAAV9-IgK-ADA at a log lower dose (3x10e11 particles) provided comparable results as that of ssAAV9 vectors ADA enzyme analysis, while not achieving statistical significance, supported a trend for increased serum hADA activity in treated ADA-SCID mice in comparison to untreated controls Since we have recently shown that mutation of the surface-exposed tyrosine (Y730) residues in AAV2 capsids facilitates viral nuclear transport by limiting proteasomemediated degradation, leading to high-efficiency transduction of murine hepatocytes [Proc Natl Acad Sci., USA, 105: 7827-7832, 2008], we also compared the efficacy of both ssAAV2-Y730F-IgK ADA and scAAV2-Y730F-IgK ADA tyrosine-mutant vectors in the liver Preliminary data from mice injected with scAAV2 Y730F mutant vectors at a dose of 1x10e10 vectors demonstrated increasing trends in lymphocyte populations of treated ADA-SCID knockout mice These studies suggest that systemic delivery of scAAV9, and liver-directed delivery of tyrosine-mutant AAV2 vectors may offer a promising and safe therapeutic tool for the potential gene therapy of ADA deficiency in humans 302 Drosha and the Control of Mammalian Hematopoiesis Krist T Azizian,1 Liang Li,2 Lars Aagaard,3 Ravi Bhatia,2 John J Rossi.1 Department of Molecular Biology, Beckman Research Institute of City of Hope, Duarte, CA; 2Department of Hematology/ HCT, Beckman Research Institute of City of Hope, Duarte, CA; Department of Molecular Biology, University of Aarhus, Aarhus, Denmark We have chosen to employ an inducible system for long-term knock-down of Drosha, the nuclear processor of primary miRNAs, by lentiviral delivery of a short-hairpin into CD34+ hematopoietic stem cells (HSCs) We wish first, to understand how important Drosha is as a central processor of miRNAs during HSC differentiation and, second, to identify new miRNAs that function to control lineage differentiation by studying Drosha-deficient states Knocking-down Drosha in HSCs during differentiation resulted in a suppression of erythropoiesis and a promotion of granulopoiesis Staining against markers for differentiation showed a decreased population of erythroid cells in Drosha-deficient cultures with a lineage shift favoring granulocytes Colony-forming assays showed a reduction of erythroid colonies when knock-down of Drosha was induced with S117 HEMATOLOGIC-NOVEL TECHNOLOGIES, LINEAGE SPECIFIC REGULATION doxycycline treatment All colonies were morphologically similar regardless of vector and treatment, suggesting that the transductions and doxycycline were not harmful and that disturbing the global expression of miRNAs modulated, but did not interfere with normal hematopoietic development These results were unexpected, given that a decrease in the abundance of Drosha-dependent miRNAs should have resulted in the opposite effect, that is, a promotion of erythropoiesis and a suppression of granulopoiesis Examples of Drosha-dependent miRNAs that function in hematopoiesis support this claim Specifically, mir-221 and -222 work by targeting c-kit, a pro-erythroid factor, and thereby suppress erythropoiesis as a switch to granulopoiesis occurs [PNAS, 102 (2005)] Because a promotion of erythropoiesis was tested, a decrease in the levels of mir-221 and -222 should not have resulted in a suppression of erythropoiesis Likewise, mir-223 works by targeting NFI-A, a factor that blocks granulopoiesis [Cell, 123 (2005)] Low mir-223 levels would therefore result in a suppression of granulopoiesis Thus, these observations suggest that a subset of Drosha-independent miRNAs may help to control hematopoiesis It is also possible that a default pathway exists to shift lineage differentiation when the expression of Droshadependent miRNAs decreases This work was supported by NIH grant no RO1HL074704 to JJR Fifty percent inhibition of shHPRT491 transduced Molm13 cells was not achieved, even at 10,000 nM Myeloid immortalization assays, using wild type murine BM, indicate that the combination of 6TG and lentivirus delivered iPAR does not result in more immortalized clones than mock transduction, 6TG treatment alone, or lentiviral transduction alone (Table 1) These data indicate that iPAR and 6TG may be safely and effectively used to select for transduced hematopoietic progenitor cells to enhance gene therapy Mock UT Mock 6TG 0 Day 28 Immortalized wells per 96 well plate 0,2,0 1,1,0,1,0,1 491G UT 19 2,1,0,1,2,1 (3 GFP+) 491G 6TG 49 0,1,0 (0 GFP+) Mll-Af9 >50 96,95 Day 28 GFP+ (%) Frequency of Immortalization in 14,350 in 14,350 in 11,623 (1 in 3638) in 14,662 (