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499 Development of AAV2/8 Mediated Gene Therapy Clinical Trial for Crigler Najjar Syndrome Type I Optimization of Liver Specific Expression Cassette Molecular Therapy Volume 21, Supplement 1, May 2013[.]
GENE & CELL THERAPY OF DIABETES, METABOLIC AND GENETIC DISEASES II 497 Prevention of Neuropathology and Normal Cognitive Development in the Hyperargininemic Mouse with AAV Gene Transfer Ellen K Lee,1 Chuhong Hu,1 Ragini Bhargava,1 Ravi Ponnusamy,2 Hana Park,1 Sarah Novicoff,1 Nora Rozengurt,3 Bart Marescau,4 Peter De Deyn,4 David Stout,5 Lisa Schlichting,6 Wayne W Grody,7 Stephen D Cederbaum,7 Gerald S Lipshutz.1 Surgery, David Geffen School of Medicine at UCLA, Los Angeles, CA; 2Psychology, David Geffen School of Medicine at UCLA, Los Angeles, CA; 3Pathology and Laboratory Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA; 4Laboratory of Neurochemistry and Behavior, University of Antwerp and Institute Born Bunge, Antwerp, Belgium; 5Molecular and Medical Pharmacology, David Geffen School of Medicine at UCLA, Los Angeles, CA; 6Biochemical Genetics Laboratory, University of Colorado, Denver, CO; 7Pediatrics, David Geffen School of Medicine at UCLA, Los Angeles, CA Arginase I deficiency is the least severe urea cycle disorder and is characterized by hyperargininemia and infrequent episodes of hyperammonemia Patients suffer from neurological impairment with cortical and pyramidal tract deterioration, spasticity, loss of ambulation, and seizures, and is associated with intellectual disability In mice, onset is heralded by weight loss beginning around day 15; gait instability follows progressing to inability to stand and development of tail tremor with seizure-like activity and death We developed a serotype rh10 adeno-associated virus expressing arginase under either 1) the CBA promoter or 2) a liver specific promoter (LSP) and followed animals long-term for the development of any evidence consistent with brain dysfunction Survival was greater at months in mice injected with the CBA promoter compared to the LSP promoter With the CBA promoter, arginase is detected in extrahepatic tissues; however it is unclear if functional protein can be produced at these sites An extensive battery of behavioral testing was performed on the AAV-CBA mice including SHIRPA, open field, elevated plus and Morris water maze, hot plate pain assay, and rotarod and demonstrated no differences from controls Furthermore, histopathological evaluation demonstrated that treated mice were indistinguishable from littermates and that putative compounds associated with neurotoxicity (guanidino compounds; GCs) were diminished but not normal later in life In addition, treatment resulted in near complete resolution of amino acid and ammonia levels early in life; however there was development of some metabolic derangement later with decline in transgene expression, more so in the LSP mice Ammonium challenging at months revealed that treated mice were affected by exogenous loading much greater than littermates These results demonstrate that AAV-based therapy for hyperargininemia can be effective and prevent the development of neurological abnormalities and cognitive dysfunction in a mouse model of hyperargininemia As GCs become elevated later in life with no obvious resultant CNS abnormality, these data suggest that the hypothesis that GCs are the cause of the unique neurotoxicity in hyperargininemia may not be correct Finally, nitrogen challenging reveals that these gene therapy treated mice remain impaired in the handling of waste nitrogen and are vulnerable 498 Assessing the Safety of AAV Liver Gene Transfer of Hyperfunctional FIX Padua in InhibitorProne Dogs and Mice Julie M Crudele,1 Jonathan D Finn,1 Joshua I Siner,1 Yifeng Chen,1 Shangzhen Zhou,1 Glenn P Niemeyer,2 Clinton D Lothrop Jr,2 Federico Mingozzi,1 Katherine A High,1 Valder R Arruda.1 Children’s Hospital of Philadelphia, Philadelphia, PA; 2University of Alabama, Birmingham, AL Clinical trials have shown that the safety of adeno-associated viral vector (AAV) gene transfer can be limited by dose-dependent immune responses against the capsid Modifications to the transgene to increase the specific activity of a protein could allow for lower vector doses to be given, provided the neotransgene is not immunogenic Previously we showed that the naturally occurring gain-of-function Factor IX R338L (FIX-Padua) exhibits ˜8-fold increased specific activity in humans (N Engl J Med 2009) In hemophilia B (HB) dogs with a missense mutation in the canine (c) F9 gene, delivery of AAV6-cFIX-Padua to skeletal muscle resulted in similar increased specific activity with no anti-FIX neutralizing antibodies (inhibitors) or FIX-specific T-cell response (Blood 2012) Here we tested the efficacy and immunogenicity of cFIX-Padua in a severe HB dog colony with an early stop codon mutation prone to develop FIX inhibitors A naive dog treated with a liver-specific AAV8-cFIX-Padua showed expression levels of ˜2% antigen and ˜23% activity, with no inhibitor development Whole blood clotting time (WBCT) returned to normal by day post-vector administration and remains stable for >12 months An additional dog had previously being exposed to human (h) FIX protein and developed inhibitors that cross-reacted with cFIX When treated with AAV, anti-cFIX antibodies peaked at day 14 post-AAV, but dropped to undetectable levels by day 70 with a concurrent rise in expression levels to activity levels of ˜30% and normalization of WBCT We also compared the long-term safety and efficacy of hFIX-Padua to hFIX-WT in WT mice Five dose cohorts of AAV8-hFIX-WT (2.5x108–5x1010 vg/kg; n=5/group) led to expression ranging from 600% of normal when measured by antigen or activity, with a linear correlation of activity/antigen In contrast, AAV8-hFIX-Padua doses (1x108–1x1010 vg/kg; n=5/group) led to antigen levels ranging from 200% of normal but activity levels of 1900%, with an elevated specific activity ˜9 Markers of pathological activation of coagulation were assayed 2-8 months post-AAV, with no significant differences seen in thrombinantithrombin complexes or D-dimer as a function of expression of FIX-Padua vs FIX-WT In addition, Kaplan-Meyer survival curves were similar between the groups Finally, mice receiving AAV8hFIX-WT were immunologically challenged 10-15 weeks after gene delivery with recombinant hFIX-Padua protein, and visa versa, alone or with CFA In no instance did mice develop antibodies to FIX Together these data show that mice expressing FIX-Padua exhibit no increased risk of thrombophilia or mortality at the levels studied (up to nearly 2000% activity) compared to FIX-WT FIX-Padua also shows no increase in immunogenicity compared to FIX-WT and is capable of tolerizing HB dogs and mice Thus, FIX-Padua is an attractive transgene that will allow for decreased vector doses in human HB gene therapy 499 Development of AAV2/8-Mediated Gene Therapy Clinical Trial for Crigler-Najjar Syndrome Type I: Optimization of Liver-Specific Expression Cassette Nunzia Pastore,1 Alberto Auricchio,1,2 Nicola Brunetti-Pierri.1,2 Telethon Institute of Genetics and Medicine, Naples, Italy; Department of Pediatrics, Federico II University, Naples, Italy Crigler-Najjar syndrome type I is a severe inborn error of bilirubin metabolism due to mutations of the liver-specific uridine diphosphoS192 Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy CANCER - IMMUNOTHERAPY II glucuronosyl transferase 1A1 (UGT1A1) gene Affected patients have increased serum bilirubin that is treated with daily phototherapy for several hours to prevent irreversible brain damage Despite treatment, patients remain at risk of life-threatening elevations of serum bilirubin and often undergo orthotopic liver transplantation Therefore, alternative therapies are needed to overcome the mortality and morbidity associated with transplantation procedure, and risks of life-long immunosuppression AAV2/8 vectors are very attractive for liver-directed gene therapy and have recently resulted in encouraging results in a human clinical trial for hemophilia B Gene therapy has the potential to provide a definitive cure for patient with Crigler-Najjar syndrome type I and our studies focused on the design and optimization of an AAV2/8 vector for clinical trial Multiple expression cassettes expressing the UGT1A1 gene inserted into the AAV2/8 vectors were generated for in vivo investigation in the Gunn rats, the animal model for Crigler-Najjar syndrome The comparison of the expression cassettes was performed injecting AAV2/8 vectors into the tail vein of 4-6 week-old female Gunn rats We first injected animals with a vector expressing the human UGT1A1 under the control of the liver specific thyroxine-binding globulin (TBG) promoter but only the high dose of 5x1013 genome copies (gc)/kg resulted in a significant 60% reduction of serum bilirubin levels by 12 weeks post-injection Next, we achieved a 50% reduction of baseline serum bilirubin with 5x1012 gc/kg of a vector encoding the human UGT1A1 under the control of the liver-specific LP1 regulatory element that consists of the core domains from the human apolipoprotein hepatic control region and the human alpha-1-antitrypsin gene promoter Interestingly, the injection of the same dose of another vector bearing codon optimized human UGT1A1 cDNA under the control of the same LP1 promoter resulted in complete normalization of serum bilirubin levels by weeks post-injection and the correction was sustained for all the time of the observation In summary, our study shows that AAV2/8 vector with codon optimized UGT1A1 gene under the control of the hepatocyte-specific LP1 promoter results in improved and sustained correction of hyperbilirubinemia in Gunn rats Taken together, these data demonstrate the development of an optimal expression cassette for liver-directed gene therapy of Crigler-Najjar syndrome type I and form the preclinical basis for the development of a gene therapy trial for this severe disorder 500 Stromal Cell-Derived Factor-1 Non-Viral DNA Therapy Accelerates Wound Healing and Decreases Scar Formation in Porcine Surgical Incisions Timothy J Miller,1 Karissa Henson,1 Rahul Aras,1 Evan Facher,1 Marc S Penn.2,3 SironRX Therapeutics, Inc, Cleveland, OH; 2Summa Health System, Akron, OH; 3Northeast Ohio Medical University, Rootstown, OH JVS-100 is a non-viral gene therapy that expresses human Stromal cell-Derived Factor-1, (SDF-1, aka CXCL12) in target tissue SDF-1 is a strong chemoattractant of bone marrow derived stem cells and tissue specific progenitor cells to the site of tissue damage, which promote tissue preservation and blood vessel development SDF-1 triggers a number of protective molecular cascades that are both anti-inflammatory and anti-apoptotic Endogenous SDF-1 expression is increased in damaged tissue and has been demonstrated to be a pro-healing chemokine, but expression lasts for less than a week, and therefore the induced stem cell homing response quickly fades SDF-1 gene transfer to prolong or re-introduce SDF-1 expression at the site of tissue damage has been proposed as a potential therapeutic strategy for treatment of wound healing based on animal studies by several independent laboratories We demonstrate that nonviral gene therapy with JVS-100 to increase the duration of SDF-1 expression in the skin around wounds is a safe and efficacious Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy means to accelerate wound repair and decrease scar formation in clinically relevant porcine wounds Full thickness incisions were created on the backs of juvenile female Yorkshire pigs and JVS-100 was delivered as multiple needle-free subcutaneous injections at the time of wound closure High levels of protein expression were detected at specifically at injections sites days post-injection Wound healing was significantly increased 40-120% in all dose groups, with dose-dependent acceleration of wound closure observed at Day (P