402. Comparison of Intravesical lnterleukin-12 Gene Therapy and Intravesical BCG Therapy in an Orthotopic Bladder Cancer Model

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402. Comparison of Intravesicallnterleukin-12 Gene Therapy and Intravesical BCG Therapy in an Orthotopic Bladder Cancer Model Minoru Horinaga,' Kelley Harsch ,' Ryuichi Fukuyarna,' Warren Heston,' William Larchian.' 'Cancel' Biology, The Cleveland Clinic Foundation, Cleveland, OH; }Glickman Urological Institute, 111e Cleveland Clinic Foundation. Cleveland, OH. Intravesical immunotherapy with bacillus Calmette-Guerin (BCG) has a high efficacy against superficial bladder cancer. However, 60 to 70 % of superficial bladder tumors recur, and 30 % of these recurrent tumors present with a higher grade with invasive properties. Intravesical BCG treatment was effective in wild type orthotopic bladder cancer model, while BCG was completely ineffective in IFN gamma knock out and interleukin (IL- 12) knock out orthotopic bladder cancer models. This study was designed to evaluate the antitumor effect of intravesical cationic liposome treatment of interleukin-12 (IL-12) in an orthotopic murine bladder cancer model and to compare the protective immunological memory against tumors between intravesical IL-12 gene therapy and intravesical BCG therapy. We used an orthotopic bladder cancer model which established by simple instillation of 5 x 105 MBT-2 eells into the lumen of the bladder offemale C3HIHeJ mouse through a urethral catheter. To assess the antitumor effect of IL-12, intravesical IL-12 therapy was evaluated at various doses: OJlg(control}, 3Jlg, 5Jlg and IOug (n=8). Intravesical treatments were performed at 3day intervals and repeated 6 times starting from day 5 after tumor implantation. To compare the long- term tumor specific immunity between IL12(n=18} and BCG (n=20), intravesical re-challengement ofMBT-2 cells were performed for surviving mice and new control mice (n=IO) at the day 60. These re-challenge survival mice as well as new control did not receive any further treatment. On the dayl20 all surviving mice were sacrificed and necropsied. In our survival analysis of the IL-12 groups at doses of Oug, 3Jlg, 5Jlg and IOJlg, 0,2,3 and 3 mice survived, respectively. Five and 10Jlg of1L-12 group demonstrated significantly longer survival than that ofcontrol group. All mice cured by IL-12 treatment successfully rejected the re-challenged MBT-2 cells, while mice cured by BCG and new control mice succumbed to the re-challenged bladder tumors. Our results suggest that intravesical IL- I2 gene therapy which induced long-lasting tumor-specific immunological memory demonstrated the survival advantage in an orthotopic bladder cancer model. This finding of intravesical IL-12 gene therapy might be potentially advantages compared to intravesical BCG therapy. Molecular Therapy Volume 15 ~ Supplement I. May2007 Copyright © Thc AmericanSociety of Gf,.'Jl1; Therapy

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403. A Novel In-Vivo Approach for AAVMediated Gene Therapy of Hemophilia BUsing Salivary Glands as a Depot Organ Hiroshi Takahashi; Bruce Baum,' Jay Chiorini.' 'Gene Therapy and Therapeutic Branch, National Institute of Dental and Craniofacial Research, National Institutes ofHealth• Department ofHealth and Human Services. Bethesda, MD. Successful gene therapy for hemophilia must combine the right vector and target tissue to allow sustained therapeutic expression, while avoiding an immune response. Both liver and musele have been extensively tested as target sites for coagulation Factor IX (FIX) expression, but have major limitations that may prove difficult to overcome. The salivary gland (SG) in many ways is an ideal organ for gene therapy because of its high secretion activity, well-encapsulated structure, slow cell division rate, and ease of access. To determine if SGs are able to produce biologically active human-FIX (hFIX) at therapeutic levels, we administered 10'" particles ofAAV serotype 2 (AAV2) vectors with different promoters, encoding hFIX, directly to individual Balb/c mouse submandibular SGs. Following vector infusion, biologically active hFIX was easily detected in plasma at levels that would be therapeutic in hemophilia B patients. Recombinant hFIX expression was affected by the dose of vector, choice ofpromoter and codon optimization ofthe hFIX open reading frame. Steady state expression was observed for at least 26 weeks and no significant antibodies against AAV2 and hFIX were detected. Importantly, a repeat SG administration with the same dose of vector was possible and could further increase hFIX expression. These results show that SGs are promising targets for gene therapy of hemophilia B patients.

404. Partial Correction of the FVIII Deficit in Hemophilia A Mice Using Minicircle DNA Vectors Expressing Pre-Trans-Splicing Molecules

S. Gary Mansfield,' Ping DU,I Suja Hiriyanna ,' M. Puttaraju,' Gerard J. McGarrity,3 Mariano A. Garcia-Blanco.' 'Hemophilia RNA Therapy, lntronn Inc. Rockville; }Vector Synthesis, Genl-ec Inc, Gaithersburg; "Sciemific & Regulatory Affairs. VIR--<:SYS Inc. Gaithersburg; "Molecular Genetics & Microbiology, Duke University, Durham.

Previously we demonstrated modest phenotypic correction of Hemophilia A mice using spliceosome mediated RNA trans-splicing (SMaR"fTM ) to repair FVIII RNA. SMaRT uses exogenously supplied constructs called pre-trans-splicing molecules (PTMs) for therapeutic purposes either by repairing or modifying endogenous RNA. In the present study we have developed PTMs that can transsplice to highly abundant albumin pre-mRNA to enhance FVIII production in vivo. A PTM was designed to base-pair with intron I of the mouse albumin pre-mRNA and mediate trans-splicing with exon I of albumin. The PTM binding domain was isolated by high capacity screening and then further optimized to maximize transsplicing efficiency. I-1emophiliaA mice were injected hydrodynamically with the above PTM (in plasmid form) or with saline (negative control) and plasma was analyzed by Coatest 2 d after injection. Whereas all mice that received saline showed no significant activity compared to prebleed levels (p = 0.34), mice that received the PTM showed significant FVIII activity (mean = 14.2 ± 1.6% of normal, p = 0.0004). To rule out the possibility that the PTM could generate activity in the absence of trans-splicing, a mutant PTM was constructed in which the splice acceptor site was mutated from AG to CT. Mice were injected with either the mutant PTM, the active PTM or saline. Miee that received the splice mutant or saline showed very similar Ievcls of FVIII activity compared to prebleed levels SI55