AAV VECTORS: DISEASE APPLICATIONS GALV-αGT was similar to the frequency of CFUs expressing αGT suggesting that expression of αGT is not toxic to primate CD34+ cells Conclusion: These data demonstrate that rhesus macaque CD34+ BM cells can be efficiently transduced with GALV-enveloped viruses carrying the gene encoding αGT and the expression of αGal epitopes in their progeny is not toxic The transduction efficiency was similar to that observed using viruses carrying the marker gene GFP Using the transduction conditions described, the majority of transduced cells expressing αGal or GFP remained CD34+ Thus, it may be possible to use genetic engineering of BM in primates in order to establish tolerance to αGal AAV VECTORS: DISEASE APPLICATIONS 110 Performance of Different AAV Serotype Vectors Following Injection into the Deep Cerebellar Nuclei of ASMKO Mouse Brain James C Dodge,1 Jennifer Clarke,1 Anthony Song,1 Jie Bu,1 Qi Zhao,1 Tatyana V Taksir,1 Denise Griffiths,1 Lamya S Shihabuddin,1 Catherine R O’Riordan,1 Marco A Passini,1 Ed H Schuchman,2 Gregory R Stewart.1 Neuroscience, Genzyme Corporation, Framingham, MA; Department of Human Genetics, Mount Sinai School of Medicine, New York, NY Niemann-Pick A disease (NPA) is a lysosomal storage disorder caused by a deficiency in acid sphingomyelinase (ASM) activity Consequent accumulation of sphingomyelin and other lipids in the CNS results in the development of a rapidly progressive neurodegenerative disease with death occurring by to years of age Previously we have shown that the storage pathology in the ASM knockout (ASMKO) mouse brain is amenable to AAV2/2mediated gene therapy Interestingly, correction of storage pathology occurred not only at the injection site, but also in regions that send and/or receive input from the injection site – suggesting that AAV vector and/or expressed ASM protein underwent transport The present experiment evaluated the relative ability of recombinant AAV2/1, AAV2/2, AAV2/5, AAV2/7 and AAV2/8 serotype vectors encoding human ASM to facilitate gene transduction, express ASM protein, correct cholesterol storage pathology, undergo transport, rescue Purkinje cells, and initiate functional recovery in the ASMKO mouse Male ASMKO mice (∼7 weeks old) were unilaterally injected with the different AAV serotype vectors within the deep cerebellar nuclei of the cerebellum (DCN) The DCN was targeted because it is highly connected with the CNS; and therefore, may provide a means to achieve widespread ASM expression throughout the brain Mice were sacrificed at 14 weeks of age after undergoing rotarod testing Mice injected with AAV2/1 and AAV2/8 demonstrated significant functional improvement on the rotarod, whereas mice injected with AAV2/2, AAV2/5 and AAV2/7 did not Consistent with the behavioral results, cerebellar ASM protein levels (as detected by ELISA) were significantly higher in mice injected with AAV2/1 and AAV2/8, than mice injected with AAV2/2, AAV2/5, AAV2/7 and control mice Preservation of Purkinje cells based on calbindin immunostaining was greatest in mice injected with AAV2/1 and AAV2/8 In all AAVASM treated mice, expression of ASM led to widespread clearance of filipin/cholesterol staining in the cerebellum, brainstem, and midbrain – indicating that AAV vector and/or expressed ASM underwent transport from the DCN Overall, a positive relationship between ASM protein levels, filipin clearance, Purkinje cell survival, and rotarod performance was observed These results support the further evaluation of AAV2/1 and AAV2/8-based vectors for gene therapy of the CNS manifestations in Niemann-Pick A disease Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy 111 AAV7, 8, and Are More Efficient and Less Immunogenic Vectors for Muscle-Directed Gene Therapy for Hemophilia B Lili Wang,1 Jean-Pierre Louboutin,1 Yan Li,1 James M Wilson.1 Gene Therapy Program, University of Pennsylvania, Philadelphia, PA Vectors based on AAV serotype have been tested in a phase I clinical trials for hemophilia B Problems of pre-existing immunity to AAV2 in human population could limit its broad application Another concern is the anti-FIX inhibitor formation which has been observed in the preclinical animal studies using AAV2 and AAV1 vectors Pseudotyped AAV vectors with different tissue tropisms and improved gene transfer efficiencies provide potential alternatives to overcome these problems In this study, we compared the longterm expression of canine factor IX (cFIX) levels and anti-cFIX antibody responses following muscle-directed gene transfer of vectors pseudotyped with AAV1, 2, 5, 7, 8, and in immunocompetent hemophilia B mice We demonstrated that AAV8, 9, and are superior vectors for muscle-directed gene therapy for hemophilia B, compared to AAV2/1, 2/2, and 2/5 vectors Our data indicate that AAV2/8 is about 25-fold more efficient than AAV2/1 vector for muscle-directed gene transfer in immunocompetent hemophilia B mice These novel vectors are not only more efficient in transducing muscle fibers, they are also less immunogenic in eliciting B cell responses to the cFIX transgene product The absence of antibody response to transgene by AAV8 is independent of the dose of vector and level of transgene expression Besides, pre-existing immunity to these vectors in human is also less problematic Therefore, these new AAV serotypes provide unique advantages for their future applications in treating hemophilia and other genetics diseases 112 Strong Transgene-Specific Immune Responses Can Be Elicited with Novel AdenoAssociated Virus Vectors in Mice Yan Zhi,1 Julie Johnston,1 Di Wu,1 Joanita Figueredo,1 Martin Lock,1 Peter Bell,1 Guangping Gao,1 James M Wilson.1 Gene Therapy Program, University of Pennsylvania, Philadelphia, PA Vectors based on recombinant adeno-associated virus (rAAV) have been widely appreciated for gene therapy application, in which the sustained expression of self-genes has been successfully achieved in a variety of animal models However, the potential of rAAV vectors as vaccine carriers has only recently been recognized We have isolated several novel AAV serotypes from primates i.e AAVs 7, 8, and Therefore, it is of our interest to develop potential vaccines for human immunodeficiency virus type (HIV-1) based on rAAV vectors of these new serotypes We have constructed a panel of rAAV vectors based on different AAV serotypes (including AAV2/ 1, AAV2, AAV2/5, AAV2/7, AAV2/8, and AAV2/9), which all encoded HIV-1 gag p17 and p24 (designated as gagshort) BALB/c mice were i.m immunized with this panel of AAVHIVgagshort vectors at the dose of E11 genome copies/mouse 2, 3, and weeks after immunization, splenocytes were harvested and stimulated with the H-2d restricted gag-specific CD8 T-cell epitope in intracellular IFNg staining Overall, AAV2/7 and AAV2/8 vaccine vectors elicited the highest levels of gag-specific CD8 T-cell responses; while AAV2/1 and AAV2/9 vaccine vectors were able to induce intermediate levels of gag-specific CD8 T-cell responses Gag-specific CD8 T-cell responses induced with AAV2/5 and AAV2/2 vaccine vectors were barely detectable In addition, serum samples from immunized mice were collected and total IgG responses to gag p24 were measured by ELISA Overall, AAV2/7 and AAV2/9 vaccine vectors yielded the strongest IgG responses against p24; while AAV2/8, AAV2/1, and S45 AAV VECTORS: DISEASE APPLICATIONS AAV2/5 vaccine vectors produced the intermediate IgG responses against p24 Surprisingly, there was no detectable p24-specific IgG response in serum samples from mice immunized with AAV2/ 2HIVgagshort vector, even though this vector yielded very high level of p24 expression in transduced cells in vitro Interestingly, different patterns of p24-specific IgG1 vs IgG2a responses were observed in serum samples from mice immunized with HIVgagshort vaccine vectors based on different AAV serotypes Furthermore, significant inflammation and infiltration in the mouse muscles injected with AAV2/7 and AAV2/8 vaccine vectors were observed at weeks after i.m injection by H/E staining Taken together, these data suggested that strong transgene-specific immune responses can be elicited with novel rAAV vectors in mice The immunogenicity of these vectors based on novel AAV serotypes will be examined in nonhuman primates 113 Manipulating AAV2 Tropism for Enhanced Delivery of AAV Vectors to the Spinal Cord Adam Davis,1 Matthew Stachler,1 Wenfang Shi,1 James Liu,2 Nicholas Boulis,2 Jeffrey Bartlett.1 Gene Therapy Center, Childrens Research Institute, Columbus, OH; 2Pediatrics, The Ohio State University, Columbus, OH; Neuroscience, The Cleveland Clinic Foundation, Cleveland, OH Amyotrophic Lateral Sclerosis (ALS) is a rapidly progressing neurodegenerative disorder Currently, the pathway of motoneuron degeneration, caused by ALS, is poorly characterized and has severely limited the development of viable treatment options Although gene therapy may provide a novel approach for developing effective treatments for ALS the need to deliver therapeutic genes throughout the CNS will require highly efficient and selective gene vector systems On the basis of its extremely efficient uptake and delivery to motor neurons, we have attempted to develop tetanus toxin as a targeting ligand for neurospecific binding of AAV vectors with the goal of increasing the efficiency of retrograde transport and spinal cord delivery of AAV vectors We have previously shown that large peptide ligands can be inserted at the N-terminus of the AAV2 VP2 capsid protein Accordingly, an AAV clone was constructed to encode the C-terminal fragment of the tetanus toxin heavy chain protein (TTHC) as an AAV2 VP2 N-terminal fusion This construct was complemented with an AAV2 VP1, VP3 expression construct to produce infectious AAV2 particles Concurrently a fusion protein was constructed by genetically fusing the coding sequence for E coli streptavidin (SAv) with the TTHC This fusion protein was expressed in bacteria using the pTrcHis-TOPO system and purified by nickel-affinity chromatography AAV vectors displaying TetC peptide and molecular conjugates prepared by binding SAv-TetC to metabolically biotinylated AAV vectors will be assessed for specific and enhanced binding to differentiated pheochromocytoma (PC12) cells, neuroblastoma cells, and primary motor neurons in vitro We have previously reported a phage display biopanning strategy for isolation of peptides with specific affinity for the trisialoganglioside (GT1b) Clostridial toxin receptor This process identified Tet1, a 12 AA peptide with specific and enhanced binding to differentiated pheochromocytoma (PC12) cells, primary motor neurons, and dorsal root ganglion (DRG) cells in vitro Similarly, we have shown that small peptide insertions following AAV2 VP1 amino acid 588 are well tolerated and can alter AAV2 natural tropism Based upon these findings, an AAV2 construct was made encoding the Tet1 peptide at this position in the viral capsid protein Recombinant viral vectors packaged with this modified AAV2 capsid maintained wild type particle and infectious titers on HeLa cells indicating that endogenous vector tropism and biology had not been affected Tet1AAV2eGFP vector was extremely inefficient at transducing undifferentiated PC12 cells However, PC12 cells that S46 had been differentiated by growth in NGF-supplemented media were readily transduced by this modified virus Unmodifed AAV2based vectors failed to efficiently transduce either undifferentiated or differentiated PC12 cells These findings suggest that modified AAV vectors displaying Tet1 peptide insertions might achieve enhanced AAV spinal cord delivery of packaged transgenes 114 Efficient Arterial Formation at the Sites of Adult Neo-Angiogenesis Requires the Recruitment of Bone Marrow Cells through the Neuropilin-1 (NP-1) Receptor Serena Zacchigna,1 Nikola Arsic,1 Lucia Pattarini,1 Silvia Moimas,1 Alessandro Carrer,1 Lorena Zentilin,1 Alessandro Salvi,2 Gianfranco Sinagra,2 Mauro Giacca.1 Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy; 2Unita’ di Cardiologia, Ospedale di Cattinara, Trieste, Italy The potential use of bone marrow-derived cells (BMCs) to promote new blood vessel formation, recapitulating vasculogenesis in the adult, has recently aroused much excitement, and a rapid transposition to the clinic However, new experimental evidence has challenged this notion, demonstrating that BMCs transplanted into ischemic tissues still adopt mature hematopoietic fates and not transdifferentiate into vascular structures To better define the actual role of bone marrow-derived cells in the formation of new blood vessels, we developed a series of AAV vectors to drive the persistent expression of several factors, differing in their capacity to promote angiogenesis and to recruit BMCs Mice were divided into three groups (n >= 24 per group) and injected, into the tibialis anterior muscle, with AAV-VEGF165, AAVVEGF121 and AAV-Sema3A Injection of AAV-VEGF165 determined the formation of an impressive number of new capillaries and arteriolae with a 20-120 µm diameter, paralleled by a massive muscle infiltration with CD45+, CD11b+ mononuclear cells These cells were found to derive from the bone marrow (as detected by bone marrow transplantation and FISH analysis), but were not incorporated in the newly formed vessels In contrast, VEGF121 induced a potent angiogenic sprouting with the massive formation of new capillaries, but neither cellular infiltrates nor arterial vessels were evident Leading from the consideration that the main difference between the two VEGF isoforms is their ability to bind the co-receptor NP-1, we looked at the effect of Sema3A, another ligand for NP-1 We found that Sema3A, although not angiogenic, is able to recruit mononuclear CD11b+ cells from the bone marrow similar to VEGF, and that this recruitment, in both cases, is mediated by NP-1 Consistent with these findings, we found that mononuclear BMCs were able to migrate in vitro in response to both VEGF and Sema3A, and that migration was impaired after NP-1 knock-down using a specific siRNA Finally, we also showed that CD11b+ cells were able to stimulate the migration of smooth muscle cells in culture and to promote the maturation of VEGF121-induced capillaries to acquire an arterial phenotype in vivo Together, these findings demonstrate that the different isoforms of VEGF are not redundant in their angiogenic properties, and that CD11b+ cells recruited from the bone marrow through NP-1, although not directly incorporated into the newly formed vessels, are required for proper arterial formation Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy ... immune responses can be elicited with novel rAAV vectors in mice The immunogenicity of these vectors based on novel AAV serotypes will be examined in nonhuman primates 113 Manipulating AAV2 Tropism... muscles injected with AAV2/7 and AAV2/8 vaccine vectors were observed at weeks after i.m injection by H/E staining Taken together, these data suggested that strong transgene- specific immune responses. .. develop tetanus toxin as a targeting ligand for neurospecific binding of AAV vectors with the goal of increasing the efficiency of retrograde transport and spinal cord delivery of AAV vectors We have