28. Rescue of a Lethal Murine Model of Methylmalonic Acidemia Using rAAV8 Mediated Gene Therapy – One Year Post-Treatment

METABOLIC DISEASES 26. Lentiviral Vector-Mediated Gene Transfer In Vivo in Adenosine Deaminase (ADA)-Deficient Mice and Rhesus Monkeys: Effects of Vector Backbone and Pseudotype

Denise A. Carbonaro, Alice F. Tarental, C. Chang I. Lee, Roger P. Hollis,2 Dianne C. Skelton,1 Cinnamon Hardee,2 Xiangyang Jin,2 Donald B. Kohn.2 1 Keck School of Medicine, Uiversity of Southern California, Los Angeles, CA; 2Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, CA; 3 Center for Fetal Monkey Gene Transfer for Heart, Lung, and Blood, University of California, Davis, CA. 1,2

3

3

Intravenous (IV) delivery of a lentiviral vector expressing human ADA to ADA-deficient murine neonates was sufficient for multisystemic correction. However, correction required 1.0 x 10^8 TU/neonate or 5 x 10^11 TU/kg for survival, presenting a serious challenge for translating this method to the clinic. To determine if vector pseudotype influenced gene transfer efficiency, we compared HIV-1 based vectors pseudotyped with either an amphotropic(n=3) or Vesicular Stomatitis Virus (VSV) envelope(n=4) (5.0x10^10 TU/kg) in ADA-deficient neonates. Marking in lymphoid tissues (thymus, spleen, marrow) and isolated T, B, and myeloid cells (0.05 copies/cell) was similar with both pseudotypes. However, liver(1.82 copies/cell), lung, brain, and gonads showed more marking with VSV. To assess scalability of in vivo gene delivery, studies were performed in neonatal rhesus monkeys (Macaca mulatta). To test if vector backbone species of origin was a contributing factor, HIV-1 based (CCL) and SIV-1 based (CL20c, derived from SIVmac1A11) lentiviral supernatants carrying either of two non-expressed genes (neomycinR or PhiX174) were produced. Neonatal rhesus monkeys (d1) were given an IV injection both vectors (SIV/VSV vs. HIV/VSV; n=4), with alternation of the non-expressed reporter sequences at 2.0 x 10^9 TU/kg. At one month, biodistribution was assessed by qPCR in all tissues including bilateral organs. Gene delivery by the SIV and the HIV vectors was different and the two marker genes were detected with equivalent efficiency. Proviral sequences were highest in liver (0.013 copies/ cell), spleen (0.004 copies/cell), adrenal glands (0.001 copies/cell), lung (0.0001 copies/cell), and myocardium (0.00003 copies/cell) for both the vectors. There was low level sporadic presence of the vector sequences in other tissues. To determine the effect of pseudotype, SIV vectors were packaged with either VSV or Gibbon Ape Leukemia Virus (GALV) envelope. Neonates were administered a mixture of the pseudotypes with alternating marker genes (n=4) at the maximal titer possible for each pseudotype (VSV: 2.0 x 10^9TU/kg, GALV: 2.0 x 10^8 TU/kg). At one month, biodistribution of VSV pseudotyped vector was similar as in the prior study comparing backbones; in contrast, the GALV-pseudotyped vectors were detected in the liver, 100-times lower than with VSV pseudotype, but not in any other tissues. In summary, gene marking in the liver of monkeys was consistent, with 1% of the liver cells transduced; whereas, similar vector dosage/kg in mice yielded 100-fold higher marking. Thus, in vivo gene transfer to the liver was much less effective in monkeys than in the mouse model, suggesting scale-up will require very high vector dosages or high ADA expression in human subjects to achieve comparable levels of ADA protein production in vivo.

Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy

27. Optimizing Gene Therapy for MPSVI: Overcoming Immune Responses and High Vector Doses

Gabriella Cotugno,1,2 Alessandra Tessitore,1 Anita Capalbo,1 Patrizia Annunziata,1 Caterina Strisciuglio,1 Armida Faella,1 Thomas O’Malley,3 Elvira De Leonibus,1 Luigi Aloj,4 Mark Haskins,3 Alberto Auricchio.1,5 1 Telethon Institute of Genetics and Medicine (TIGEM), Naples, Italy; 2S.E.M.M.-European School of Molecular Medicine, Naples, Italy; 3Dept. of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA; 4Dept of Nuclear Medicine, INT G.Pascale, Naples, Italy; 5Medical Genetics, Dept. of Pediatrics, Federico II University, Naples, Italy. Mucopolysaccaridosis VI (MPS VI) or Maroteaux-Lamy syndrome, is a lysosomal storage disease caused by deficient activity of Arylsulfatase B (ARSB) resulting in lysosomal storage of undegraded dermatan sulphate (DS) and characterized by skeletal dysplasia, organomegaly, heart valve thickening, and corneal clouding. Cats and rats affected with MPS VI and bearing missense and null ARSB mutations, respectively, are available for study. We have previously reported that liver-directed AAV-mediated ARSB gene transfer in newborn animals results in secretion of therapeutic ARSB serum levels. The enzyme is taken up by affected tissues, resulting in improvement of the disease phenotype in both models (1). Here we show that in newborn MPS VI cats, normal circulating ARSB levels were achieved only following systemic delivery of high vector doses (6x1013 gc/kg). In contrast, delivery of AAV2/8 doses as low as 6x1011 gc/kg resulted in close to normal serum ARSB levels in juvenile MPS VI cats suggesting that hepatocyte proliferation and vector dilution occurring in newborn animals may limit the therapeutic efficacy at low vector doses. In MPS VI null rats, we have observed a neutralizing humoral immune response to ARSB following gene transfer. To overcome this and to understand whether higher serum ARSB levels could be achieved resulting in higher therapeutic efficacy, we co-administered immunosuppressant drugs (CTLA4-Ig, Rapamycine, Fk506, or Cyclosporine A) with gene transfer both in newborn and in juvenile MPS VI rats. Several, but not all, treated MPS VI rats showed serum ARSB levels up to 75% of normal, resulting in normalization of visceral, skeletal and behavioural defects. Our data suggest that high circulating ARSB levels are required for complete rescue of the MPS VI phenotype in affected rats and that the combination of immune suppression and gene transfer could be required for the treatment of patients showing null but not missense mutations. 1) Tessitore et al., Mol Ther. (2008) 16(1):30-7.

28. Rescue of a Lethal Murine Model of Methylmalonic Acidemia Using rAAV8 Mediated Gene Therapy – One Year Post-Treatment

Randy J. Chandler,1 Charles P. Venditti.1 National Human Genome Research Institute, National Institutes of Health, Bethesda, MD. 1

Methylmalonic acidemia (MMA) is a metabolic disorder caused by deficient activity of the mitochondrial enzyme methylmalonylCoA mutase (MUT). MMA patients exhibit increased methylmalonic acid levels in the plasma, urine and cerebrospinal fluid and display a clinical phenotype of lethal metabolic decompensation, growth retardation, renal failure and metabolic strokes. A MUT null mouse model of MMA, which displays a phenotype of growth retardation and neonatal lethality was used to examine the effectiveness of adenoassociated viral (AAV) gene therapy. A recombinant AAV serotype 8 carrying the murine Mut cDNA (rAAV-mMut) driven by a ubiquitous chicken beta-actin promoter was injected directly into the liver of newborn Mut-/- mice. Greater than 95% of the 27 Mut-/- mice injected S11

METABOLIC DISEASES with 1 or 2x1011GC of rAAV8-mMut have survived for 1 year or longer and are indistinguishable from their control littermates. All the untreated mutants (n=58) perished before day of life 72. Subsequently, a smaller group of Mut-/- mice (n=4) received an intraperitoneal (IP) injection of 3x1011GC of rAAV-mMut, with 75% of the mice rescued for longer than 120 days. rAAV-mMut treated Mut-/- mice achieved normal body weights while untreated mutants reached only 40% of the weight of the wild-type mice before death. Hepatic Mut RNA levels decreased from 37-72% at 90 days post-injection to 10-15% at one year post-treatment. Similarly, the liver and skeletal muscle from a treated Mut-/- mice had significant levels of immunoreactive MUT protein at 90 days post-treatment but MUT protein was not detectable at one year post treatment. Plasma methylmalonic acid levels in the treated mutant mice were significantly reduced compared to uncorrected animals at 24 and 60 days after treatment and remained stable one-year post-treatment, indicating that substantial MUT enzymatic activity was restored and maintained. Whole body MUT enzymatic activity, indirectly measured by in vivo conversion of 1-13C-sodium propionate into 13CO2, was readily detected in mice one-year post-treatment. A single 20 day-old untreated Mut-/- mouse that was lethargic and distressed received an IP injection of 3x1011GC of rAAV-mMut. Following treatment this mouse exhibited increased vigor, weight gain and MUT enzymatic activity that was accompanied by a decreased methylmalonic acid plasma level. These experiments provide the first evidence that gene therapy has clinical utility in the long term and acute treatment for methylmalonic acidemia and provide proof of principle evidence to support the development of gene therapy for other organic acidemias.

29. Combination Therapy Utilizing shRNA and Optimize Alpha-1 Antitrypsin (AAT) Expression Cassette for Treatment and Correction of AAT Liver Deficiency Chengwen Li,1 Richard Jude Samulski.1 1 Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC.

Alpha-1 antitrypsin (AAT) deficiency is unique in that disease caused by the “PiZZ” mutation results in life-threatening lung as well as liver disease in children and adults. The lung disease can be prevented by traditional gene therapy addition approaches, however this strategy has no effect on liver disease caused by the accumulation of mutant PiZZ protein in the endoplasmic reticulum. Gene therapy approaches for the PiZZ-associated liver diseases have been focused on elimination/correction of mutant protein at the DNA and/or RNA levels. Small interference RNA (siRNA) represents an alternative approach to suppress PiZZ mutant protein and liver associated disorders. To this end, we tested the ability of three siRNA candidates to knock down AAT expression in vitro and in vivo. After transfection of these candidates into 293/AAT or 293/PiZZ cells, over 70% knockdown of AAT expression was achieved. Since adeno-associated virus (AAV) vectors have been utilized for the treatment of AAT deficiency in pre- and clinical trials we designed an H1 promoter cassette expressing these shRNA in a double-stranded (ds) type-2 capsid backbone (AAV2/shRNA-AAT). Within three days post-infection two out of three shRNA vectors demonstrated over 90% silencing of PiZZ expression. While successful for PiZZ knockdown, the shRNA cassette cannot distinguish between wt AAT expression and mutant form. To avoid wt AAT mRNA degradation delivered by AAV vector and exploit siRNA/PiZZ knockdown in liver, we designed an optimized AAT gene (AAT-opt) based on the degeneracy of the genetic code. This was done to achieve higher levels of AAT expression and to avoid sequence specific siRNA effect directed towards PiZZ mutation. First, after transduction of AAV2/CBA-AAT-opt in mouse B16 cells, 30-50% higher AAT expression was obtained compared with AAV2/CBA-AAT vector. Second, similar results were obtained S12

in mice with AAV2/CBA-AAT-opt vector administration via portal vein. Finally co-transduction of dsAAV2/shRNA and AAV2/CBAAAT-opt or AAV2/CBA-AAT into 293 cells, showed no decrease of AAT expression with AAV2/CBA-AAT-opt in contrast to significant knockdown of AAV2/CBA-AAT vector. These results indicate that a combination therapy of shRNA for silencing PiZZ transcript while addition of optimized AAT to restore AAT expression in the liver delivered by AAV vector provides a promising approach to treat AAT deficiency associated liver and lung disease.

30. Interruption of Proximal Tyrosine Degradation by shRNA Can Be Used To Select for Integrated Transgenes in Hepatocytes In Vivo

Mitchell B. Sally,1 Markus Grompe.1 1 Oregon Stem Cell Center, Oregon Health and Science University, Portland, OR. Type I tyrosinemia (HT1) is characterized by a deficiency in fumaryl acetoacetate hydrolase (FAH), and deficiency in this enzyme results in build up of fumaryl acetoacetate (FAA), which damages liver cells directly. In the tyrosinemia pathway, there are four upstream enzymes that could potentially be targets for interruption to prevent accumulation of FAA. Using a sleeping beauty (SB) transposon system, we have shown that hepatocytes expressing FAH in an HT1 liver show selective growth advantage. In the FAH-knockout mouse model, we hypothesized that by using shRNA technology to prevent accumulation of toxic compounds through upstream genetic inhibition, we will be able to show selective advantage of genetically modified hepatocytes in vivo. The initial target for shRNA modulation was tyrosine amino transferase (TAT), the rate-limiting step in the HT1 pathway. Unique shRNA sequences were devised using an online shRNA design tool. These constructs were placed into a backbone containing terminal repeats previously used in a SB transposon system in our lab, and placed under the control of the U6 promoter. All constructs contained green fluorescent protein (GFP) as a coupled transgene. These transposon constructs were then hydrodynamically co-injected with a hyperactive SB transposase into FAH-deficient mice. Mice were cycled off NTBC to drive selection. For an initial set of experiments, partial hepatectomy was performed at one month, and mice were sacrificed at two months. Subsequent experiments involved no partial hepatectomy, but sacrifice at 2- and 4-month intervals. Genomic DNA was isolated at both hepatectomy and sacrifice, and microscopy was performed examining for GFP. For the first experiment, two NTBC rescue cycles were needed to maintain normal weights. After the second cycle, these mice maintained weight off NTBC, suggesting that functional repopulation with hepatocytes transduced by an upstream shRNA vector can correct FAH deficiency. Presence of shRNA was confirmed by qPCR for GFP at one month partial hepatectomy, with average integration of shRNA vector constructs at 0.05% compared with controls. At two-month sacrifice, average integration for shRNA-injected animals had risen to 0.22%, suggesting possible selective advantage of shRNA transduced cells. Subsequent experiments had consistent weight data, as mice injected with shRNA constructs maintained weights after two rescue cycles at two months. In addition to cycling off NTBC, mice were also kept on an NTBC diet, eliminating selective pressure. Two-month data from these experiments shows shRNA integration at 1.76% and 1.85% for animals off NTBC, and 1.1% for those maintained on NTBC. This data is consistent with shRNA integration and selective advantage for liver repopulation, as the selective pressure of NTBC withdrawal results in increased expression of GFP. Microscopy for both events suggested periportal clustering of fluorescent cells, consistent with qPCR results. These results suggest that it is possible to interrupt the tyrosine pathway upstream, and these transduced cells are selected for in an FAH-deficient mouse. Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy