1075. A Clinically Relevant Gene Therapy Approach for Duchenne Muscular Dystrophy by Vascular Delivery of Micro-Dystrophin

complexes at c/p-ratios between 1/1 and 8/1 had mean diameters ranging from 100-200 nm and zeta-potentials between +20 and +32 mY. Degradation kinetic studies showed that at 37°C (pH 7.4) complete degradationof all ester bonds occurred in approximately 7 days. In vitro transfcction efficiency of complexes with DNA resulted in high reporter gene expression comparable to that of LPEI with lower cytotoxicityat optimizedc/p-ratios. Furthermore, after systemic application of pseudodendrimer/DNA complexes in vivo, gene expression was preferentially observed within the tumor at levels comparableto that of LPEI. LPEI/DNAcomplexes lead in vivo to gene expression which is mainly found in the lung being responsiblefor acute toxic effects. In contrast,after applying pseudodendrimcr/DNA complexes gene expression was dramatically reduced in the lung comparedto LPEI (lOS-fold) and no gene expression was foundinall otherorgansinvestigated. Takentogether these results indicate that pseudodendrimers are well tolerated in vitro and by miceand show an increasedspecificityfortransfection of tumor tissue at levels similar to that of LPEI. GENE AND CELL THERAPIES FOR MUSCULAR DYSTROPHIES

1074. Distinct Transduction Profiles in the Dystrophic Dogs with rAAV Serotype 8

Sachiko Ohshima,' Jin-Hong Shin,' Akiyo Nishiyama,' Katsutoshi Yuasa,' Hiroyuki Nakai.' Takashi Okada,I Shin'ichi Takeda.' I Molecular Therapy. National Institute ofNeuroscience, NOV?, Kodaira, Tokyo, Japan; lMolecular Genetics and Biochemistry, University ofPittsburgh School ofMedicine, Pittsburgh, PA.

Background: Duehenne muscular dystrophy (DMD) is an Xlinked,lethal disorderof the striated musclecaused by mutations in the dystrophin gene, which encodes a large sub-sarcolemmal cytoskeletal protein.The absenceof dystrophinassociatedwith the loss of dystrophin-glycoprotein complex (DGC) from the sarcolemma results in progressivemuscle weakness,cardiomyopathy, and early death. Several treatment modalities have been attempted to correct the dystrophic phenotypes, but effective therapy still awaits to be developed.A recombinantadeno-associated virus (rAAV) serotype 2 (rAAV2) have been utilized in the various preclinicaland clinical studies, but the rAAV serotype 8 (rAAV8) demonstratedthe better gene transferefficiencyin the skeletaland cardiac muscle. Here we investigated the transduction profiles with the rAAV2 and rAAV8 in the muscles of canine Xvlinked muscular dystrophy in Japan (cxmd). Methods: The rAAV2 or rAAV8 encoding the lacZ gene driven by a CMV promoter was injected into the tibialis anterior and extensor carpi ulnaris of the normal beagles at 6-10 weeks old (Ix lO" - 2x1013 vg), We did biopsy of the transduced muscle 2 weeks after the injection and analysed histologically to examine B-galexpression and immuneresponse. Wealso injectedthe rAAV8 encodingthe microdystrophin (M3, c/!"CS2) genedrivenby theCMV promoterintothe tibialisanteriorand extensorcarpi ulnarisof cxmd, at 6-8 weeks old (lx10 12 - 2xl0 13 vg) and examined the efficiency ofrAAV8. The transduced muscle has been sampled 4 weeks after the injection to perform histochemical and immunohistochemical analysis, and Western blotting. Results: We detected the greater number of the Bsgal-positivc fibers in the rAAV8-transduced caninc skeletal muscles than those in the rAAV2-transduced muscles 2 weeks after the injection. Moreover, we can detect much less inflammatory cell infiltration in the rAAV8-transduced muscle than the rAAV2-transduced muscles. Immunohistochemistory of CD4, CD8, CDII b, and MHCclassI, revealed lessCTL-mediated immune responsewith the rAAV8 than those with the rAAV2. However, we can detect a certain degree ofimmune responsewith a high dose of rAAV8. Consequently, extensivemicrodystrophin expressionin the rAAV8-injeeted skeletalmuscleof cxmd, wasachieved. Discussion: S410

rAAV8-mediated gene transfer showed effective transgeneexpression with less immuneresponsesthan those in the rAAV2-mediated transduction. Even though rAAV8-mediated gene transfer is less immunogenic, it is indispensableto clarify the appropriate condition of virus titer and dose accordingto the encoding gene. Weare currently attempting either the limb perfusion or systemic delivery through saphenous vein in cxmd, to examine the rAAV8-mediated transduction efficiencyof microdystrophin. Conclusion: The rAAV8 is the efficienttool for the therapeuticgene delivery into the dystrophic canine skeletal muscle. The systemic microdystrophin transduction with the rAAV8 would be promising for the future DMD gene therapy.

1075. A Clinically Relevant Gene Therapy Approach for Duchenne Muscular Dystrophy by Vascular Delivery of Micro-Dystrophin

Louise R. Rodino-Klapac,' Paul M. Janssen.' Chyrstal L. Montgomcry,' Ryan Jensen; Louis G. Chicoine, I K. Rced Clark,' Jerry R. Mendell.' 'Centerfor Gene Therapy, Columbus Childrens Research Institute , Columbus, OH; lphysiology and Cell Biology, The Ohio State University. Columbus, OH. Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder with monogenicmutationssetting the stage for successful gene therapy treatment. Long-term therapeutic goals include systemic delivery of a small dystrophin transgene, micro-dystrophin, delivered by adeno-associated virus (AAV) via the vasculature. Current and past clinical trials have limited gene delivery to direct intramuscular injection. As we proceed toward our goal of widespread muscle transduction, we anticipate progress in a stepwise fashion, where the transgene is delivered by selective catheterization to branches of the femoral artery. This approach of isolated lower limb perfusion (ILP) permits a clinical trial for muscular dystrophy assessing both safety and efficacy as for the following reasons: I) selective delivery of vector to lower limb muscles can produce clinically meaningful results; 2) the lower limb can be compartmentalized to preventspreadof virusto otherorgansystems providingan important measure of safety; 3) delivery of virus in a compartmentalized system providessale passage lor the virus since pre-existing immunity to AAV may preclude muscle transduction. We first tested ILPof micro-dystrophin in the mdx mouse with the goal of comparing the efficiency of AAV serotypes [AAV I, 6, or AAV8] in crossing the vascular barrier leadingto widespreadgene expression in muscleina mannerdifferent frompreviousstudies,not relying on excessive pressure, volume, or pharmacologic agents to combat vascular resistance. A micro-dystrophin construct was used with featurespreviouslydescribed by Harper et al (2002), deleting the untranslated regionsand C-terminus, retainingspectrin repeats 1-3 and 24, and hinges 1,2, and 4 of full-length dystrophin, under control of a musclespecific promoter, and the addition of an intron to enhance gene expression. Comparative studies conclude that AAV6 and AAV8 deliver and transduce micro-dystrophin by ILP moreefficiently thanAAV I, with micro-dystrophin levels> 85% for both scrotypcs,Functionalsignificancewas established in extensor digitorum longus(EDL)by demonstrating increased maximum force generation and protectionagainst eccentric contractions (P< 0.05). Extending thesestudiesto non-human primate; we havesuccessfully translated ILPvasculardeliveryusingAAV8 to crossthe endothelial barrierof musclevasculature by specifically targetingthe lowerlimb muscles with a fluoroscopy guided catheter to deliver AAV8.GFP (green fluorescent protein).These findings and ongoing studies set the stage lor a future clinical trial in DMD patients with vascular delivery of the micro-dystrophin transgene.

Molecular Therapy Yofume 15. Supplement I, .\by 2007 Copyright © '111C AmericanSocietyo f Gene TIICr.lpr