818 Proteasome Inhibitors Enhance Gene Delivery by Adeno Associated Virus Vectors Carrying Large Genomes in Hemophilia Mouse and Dog Models Molecular Therapy Volume 17, Supplement 1, May 2009 Copyrigh[.]
PRECLINICAL AND CLINICAL APPLICATIONS OF AAV containing a CMV-nuclear localized lacZ-PGK-Neo-Ori (CnZPNO) cassette and transduced fibroblasts Again, all three NIFV vectors and the integrase-proficient FV vector expressed the transgene, nuclear localized lacZ, as evidenced by the presence of beta-galactosidasepositive foci In addition, after selecting infected fibroblasts with G418, we found that the NIFV vectors integrated at a frequency several logs lower than integrase-proficient FV vectors NIFV vectorchromosome integration junctions from G418 resistant fibroblast clones will be sequenced to determine the mechanism of integraseindependent vector insertion Since non-integrating viral genomes are known to exist in various forms, including linear, one-LTR or two-LTR circles, we are currently utilizing Southern blot analysis to identify the predominate DNA species Our NIFV vector would be especially advantageous for those applications in which short-term gene expression is required, such as expressing a factor to influence development, inducing a cellular phenotype, or expressing a genomic modifying protein (e.g Cre recombinase) Also, NIFV vectors may be useful for targeted gene repair In summary, our novel NIFV system expresses transgenes, infects different cell types, has applications for gene therapy, and most importantly, does not integrate, greatly reducing the risk of genotoxicity Preclinical and Clinical Applications of AAV 816 AAV Capsid-Specific CD8+ T Cells and Their Responsiveness to Hepatic AAV Gene Transfer Hua Li,1 Katherine High,2 Hildegund C J Ertl.1 Immunology Program, The Wistar Institute, Philadelphia, PA; Children’s Hospital of Philadelphia, Philadelphia, PA Hepatic adeno-associated virus (AAV)-serotype 2-mediated gene transfer results in sustained transgene product expression in experimental animals but not in human subjects We hypothesized that loss of transgene product expression in humans might be caused by immune memory mechanisms that become reactivated upon AAV vector transfer We tested the effect of hepatic AAV2-hF.IX or AAV8-hF.IX gene transfer on AAV capsid-specific CD8+ T cells in mice AAV capsid-specific CD8+ T cells were found to proliferate in response to hepatic AAV gene transfer indicating that degradation of the AAV particles triggered a recall response The kinetics of the recall response analyzed by adoptive transfer of CD8+ T cells into mice that had received AAV-2hF.IX or AAV-8hF.IX vectors previously showed differences in the degradation rates of AAV-2 and AAV-8 vectors Other functional properties of AAV capsid-specific CD8+ T cells were analyzed and the most remarkable finding was that in mice AAV capsid-specific CD8+ T cells require a rather high concentration of their cognate antigen in order to exhibit lysis The implication of these findings on the potential role of AAV capsid-specific CD8+ T cells in limiting hepatic AAV gene transfer in humans will be discussed 817 In Vivo Allele Specific Knockdown of Mutant Alpha One Antitrypsin Using Recombinant AAV Delivered shRNA Christian Mueller,1 Qiushi Tang,1 Brian O’Sullivan Murphy,1 Sofia Braag,1 Terence R Flotte.1 UMass Gene Therapy Center, UMass Medical School, Worcester, MA Alpha-1 antitrypsin (AAT) deficiency is one of the most common genetic diseases in North America, with a carrier frequency of approximately 4% in the US population Homozygosity for the most common mutation caused by a single base pair change (Glu342Lys, PI*Z) leads to the synthesis of a mutant protein, which accumulates and polymerizes within hepatocytes rather than being efficiently secreted This lack of secretion causes severe serum deficiency Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy predisposing patients to chronic lung disease 12 to 15% of patients with PI*ZZ also develop liver disease, which can be severe, even in infancy This is thought to be due to toxic effects of the accumulated mutant Z-AAT within the hepatocyte Thus, an approach to reduce AAT-deficient liver disease will likely require some mechanism to decrease the amount of Z-AAT within hepatocytes Here we describe studies of allele specific small interfering RNAs (siRNAs) designed to down regulate PI*Z specific AAT within hepatocytes Two different siRNA sequences were identified and cloned into a recombinant adeno-associated virus (rAAV) backbone as U6 driven shRNA, one with the single base pair change at position 10 (p10) and one with the change at position 16 (p16) of the targeting siRNA molecule Each construct was able to reduce Z-AAT levels, but experiments on wildtype PiM AAT expressing cell culture models showed the greatest PiZ specific reduction with p10, whereas the p16 construct also significantly down regulated wildtype AAT The rAAV-U6p10 was then packaged into AAV8 capsids and used in vivo to transduce the livers of human Z-AAT over-expressing transgenic mice These studies show a decrease in total human AAT after weeks, and a clearing of Z-AAT accumulation by immunohistochemistry in the livers The rAAV8-U6-p10 vector may hold promise as a potential therapy for patients with AAT liver disease 818 Proteasome Inhibitors Enhance Gene Delivery by Adeno-Associated Virus Vectors Carrying Large Genomes in Hemophilia Mouse and Dog Models Junjiang Sun,1 Paul E Monahan,1 R Jude Samulski,1 Clinton D Lothrop, Jr Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC; 2Biochemistry and Molecular Genetics, School of Medicine, University of Alabama, Birmingham, AL The use of adeno-associated virus (AAV) vectors for correction of many clinically relevant congenital deficiencies has been hampered by the relatively small size of the AAV genome (4680 nt) and inefficient AAV vector delivery of genes that are larger than the wild type (wt) size We previously demonstrated in vitro that proteasome inhibitor (PI) treatment concurrent with AAV delivery improves transduction using larger than wt expression cassettes that exceed the size of the wtAAV genome We have extended these studies using AAV2 or vectors carrying a 5.6 kb factor VIII cassette in hemophilia A mice treated with or without proteasome inhibitor PI enhancement of expression was observed that was increased on average 6-fold (AAV2) and 3-fold (AAV8); enhanced expression persisted throughout the year of observation In addition, a limiting dose of x 1013 vg/kg AAV8.canine FVIII was delivered via portal vein to two hemophilia A dogs after receiving PI I.V.; two hemophilic dogs received vector without PI No toxicity was observed; specifically, there were no abnormalities of liver transaminases, blood platelet, white blood cell or other cell counts, or development of FVIII inhibitory antibodies The Whole Blood Clotting Time (WBCT), which is normally prolonged to greater than 20 minutes in hemophilic dogs, corrected to the normal range (6-10 minutes) by the first timepoint at one week in the dogs receiving PI Over one year after vector delivery dogs receiving AAV8cFVIII alone had a mean WBCT of 13.6 minutes, and experienced bleeding episodes apiece, a bleeding rate not different from untreated hemophilic dogs followed in parallel Dogs receiving vector with PI had a mean WBCT of 9.0 minutes, and correction of the hemophilic phenotype was evidenced by zero bleeding episodes Because male gender may be associated with higher hepatic expression from AAV vectors, a second litter of dogs was subsequently treated with two females receiving AAV + PI and one male receiving vector alone After five months follow-up, the PI-treated females show the same pattern of improved hemostatic correction The first litter has now been followed for 27 months remaining asymptomatic with S313 PRECLINICAL AND CLINICAL APPLICATIONS OF AAV mean WBCT of 12 minutes, whereas the two dogs that received AAV without PI have each suffered fatal hemorrhages In summary, the proteasome inhibitor enhanced persistent AAV-mediated expression of this large therapeutic transgene cassette in two preclinical animal models, directing phenotypic correction of hemophilia with a beneficial safety profile 819 Gastrointestinal Directed AdenoAssociated Virus Serotype and 10 Expressing IL-10 Improves Enterocolitis in a Murine Model of Inflammatory Bowel Disease Steven Polyak,1 Thomas Conlon,2 Stacy Porvasnik,2 Annette Mach,1 Clive Wasserfall,3 Lisa Dixon,3 Cathryn Mah.2 Gastroenterology Medicine, University of Florida, Gainesville, FL; 2Pediatrics, University of Florida, Gainesville, FL; Pathology, Immunology and Laboratory Medicine, University of Florida, Gainesville, FL Inflammatory bowel disease is a chronic relapsing illness that requires lifelong therapy Best treatments to date offer only 40% long term response rates and many of these medications have significant side effects The need for more efficacious long term therapy is clear We have previously identified that AAV pseudotypes and 10 can effectively transduce the small intestine and colon via a selective route of intestinal delivery through superior mesenteric artery (SMA) injection Our aim was to determine if these serotypes and this method of vector administration can clinically improve the Piroxicam-induced IL10 knockout mouse model of enterocolitis IL10 knockout mice were treated with 1x1011 vg of AAV9-IL10, AAV10-IL10 and AAV10GFP via SMA and portal vein (PV) injections (n=5) Enterocolitis was initiated and synchronized with Piroxicam Clinical data (weight, stool consistency and blood in stool) and serum were serially collected Mice were sacrificed 30 days after onset of enterocolitis (60 days post AAV treatment) for histopathologic analysis Serum levels of IL10 were maintained throughout the experiment in SMA injected AAV9-IL10 and AAV10-IL10-treated mice (3704 pg/mL and 5360 pg/mL respectively at day 60) PV injected animals had an early increase in IL10, but then had low levels by day 60 (AAV10-IL10: 15,000 pg/mL at day 15 vs 10 pg/mL at day 60) Clinically, control (AAV10-GFP) and PV delivered AAV9-IL10 animals lost weight throughout while the other groups gained weight This effect was also corroborated in cumulative disease activity index scores Colon weight:length ratios were lower in IL10 treated animals as compared to controls Significant splenomegaly was seen in IL10 treated mice, similar to clinical effects seen with IL10 treatment Overall, SMA delivered AAV10-IL10 improved the clinical course of disease most effectively SMA delivered vector or intestinal transduction may provide better long term gene expression Continued development of intestinal AAV mediated intestinal transduction for the modulation of mucosal inflammation is warranted 820 IGF-I Expression from AAV Vectors Leads to Total Fibrosis Reversion in Cirrhotic Rat Livers Luciano Sobrevals,1 Carlos Rodriguez,1 Astrid Pañeda,1 Gabor Gondi,1 Eric Timmermans,2 Iñaki Monreal,1 José Romero Trevejo,1 Nerea Juanarena,1 Sara Arcelus,1 Nerea Razquin,1 Sander van Deventer,2 Gloria Gonzaléz,1 Harald Petri,2 Jesus Prieto,1 Puri Fortes.1 Hepatology and Gene Therapy, CIMA/UNAV/CUN, Pamplona, Navarra, Spain; 2Amsterdam Molecular Therapeutic´s (AMT), Amsterdam, Netherlands Liver transplantation is the only curative treatment for advanced liver cirrhosis Therapies aimed at halting the progression of the disease are urgently needed Previous studies have shown that the administration of insulin like growth factor-I (IGF-I) from a SV40 S314 vector (SVIGF-I) induces hepatoprotective and antifibrogenic effects in experimental cirrhosis and leads to a decrease of liver fibrosis in rats However, SV40 vectors need further development before translation to the clinic As an alternative therapeutic approach, we have evaluated the effect in liver cirrhosis of AAV vectors delivering IGF-I (AAVIGF-I) First, we have compared infectivity of cirrhotic and healthy livers with AAV vectors expressing luciferase (AAVLuc) The results show that both healthy and cirrhotic livers express luciferase to similar levels after intraportal, intraarterial or intrahepatic administration of AAVLuc As intraarterial administration is, probably, the most adequate procedure to be used in patients, we have employed this route to inject saline, AAVLuc, SVIGF-I or AAVIGF-I in healthy or cirrhotic rat livers Healthy animals injected with these vectors were used to induce liver cirrhosis by administration of CCl4 for weeks The results show that treatment with AAVIGF-I before cirrhosis induction leads to a protection against liver damage Even more, treatment of AAVIGF-I in rats with established cirrhosis leads to the activation of a complex repair program that results in the total reversion of liver fibrosis and restoration of liver function We have analyzed the molecular mechanisms involved in this response IGF-I expression is detected in the liver at days post virus administration At this time point expression of some hepatoprotective factors such as HGF and HNF4a is increased and levels of pro-fibrogenic factors such as TGFb are decreased in AAVIGF-I treated animals Surprisingly, at only days post-vector injection, we already detect a decrease in activated HSCs, which are the cells that secrete the collagen deposits that result in liver fibrosis Interestingly, these changes were not observed with SVIGF-I Two weeks after vector administration there is a tremendous activation of matrix metalloproteinases, which could be in charge of removing the collagen deposits Two months after treatment with AAVIGF-I animals showed liver function tests identical to healthy animals and months later liver fibrosis was reverted completely This has never been observed with SVIGF-I treated animals We believe that AAVIGF-I gene therapy should be tested in patients with advanced liver cirrhosis that not have access to timely liver transplantation 821 Neutralizing Antibody Affects LiverMediated Factor IX Expression in Non-Human Primates by AAV Vectors Hiroaki Mizukami,1 Jun Mimuro,2 Akira Ishiwata,2 Fumiko Ono,3 Hiroya Yagi,1 Masashi Urabe,1 Akihiro Kume,1 Keiji Terao,3 Yasuhiro Yasutomi,3 Yoichi Sakata,2 Keiya Ozawa.1 Division of Genetic Therapeutics, Jichi Medical University, Shimotsuke, Tochigi, Japan; 2Division of Cell and Molecular Medicine, Jichi Medical University, Shimotsuke, Tochigi, Japan; 3Tsukuba Primate Research Center, National Institute of Biomedical Innovation, Tsukuba, Ibaraki, Japan Adeno-associated virus (AAV) 1-, 8- and 9-based vectors are promising for muscle and liver-directed gene therapy approaches To estimate the efficacy in humans, we utilized cynomolgus monkey (Macaca fascicularis) for in vivo expression As for the transgene, based on our previous study, macaque factor IX gene with a minimal modification was chosen for detection amongst the native form (J Thromb Haemost 2: 275-280, 2004) For intramuscular (IM) delivery, an AAV1-based vector driven by CMV promoter was used; for portal vein (PV) administration, AAV8 or vector driven by a liver specific promoter was prepared After screening of pre-existing neutralizing antibody (NAb) against corresponding AAV serotype capsid, each vector was injected into young adult male macaques at a dose of × 1012 vg/kg In IM group, all animals exhibited significant and prolonged transgene expression with various plasma levels (3.9 ∼ 45.2 % of normal at peak) The difference in expression levels has not been explained by any of the known factors, including NAb status In PV group, AAV8 was used in macaques, and only Molecular Therapy Volume 17, Supplement 1, May 2009 Copyright © The American Society of Gene Therapy ... this large therapeutic transgene cassette in two preclinical animal models, directing phenotypic correction of hemophilia with a beneficial safety profile 819 Gastrointestinal Directed AdenoAssociated... AAV10-IL10 and AAV10GFP via SMA and portal vein (PV) injections (n=5) Enterocolitis was initiated and synchronized with Piroxicam Clinical data (weight, stool consistency and blood in stool) and serum... Center, National Institute of Biomedical Innovation, Tsukuba, Ibaraki, Japan Adeno- associated virus (AAV) 1-, 8- and 9-based vectors are promising for muscle and liver-directed gene therapy approaches