374 Optimization of AAV Vectors for Improved Therapeutic Protein Expression in the Canine Models for Duchenne Muscular Dystrophy Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The Ame[.]
AAV VECTORS II conrm the integrity of Rep activity, excision assays were completed in 293 cells coinfected with either Ad/dRep78 or Ad/sRep78, and an Ad/AAV virus expressing the human Factor VIII gene anked by the AAV terminal repeats As expected, a single ∼8Kb excision product was clearly evident using either virus, conrming the functionality of the dRep78 and sRep78 proteins; further, nearly 3-fold greater excision was seen in the presence of inducer (1µg/ml doxycycline) In summary, these data strongly suggest that Rep78 sequences inhibit Ad replication unrelated to Rep78 protein function, and identify novel strategies for maintaining and propagating Rep78 within single-backbone Ad/AAV viruses for potential site-specic transgene delivery Ongoing structure/function studies using computational methods that build on these initial observations will likely rene the specic sequences and molecular mechanism(s) of this effect 373 Improved and Persistent Expression with Novel AAV Serotypes for Neonatal Gene Therapy Chuhong Hu,1 Fides D Lay,1 Joseph Springer,1 Ronald W Busuttil,1 Gerald S Lipshutz.1 Surgery, UCLA School of Medicine, Los Angeles, CA Neonatal gene therapy is a promising strategy for treating a number of congenital diseases that can be diagnosed either prenatally or shortly after birth Correction of genetic diseases during the neonatal period may be achieved by targeting gene replacement therapies to expanding stem cell populations and to developing organ systems and if succsessful, could limit or abrogate the pathologic consequences of genetic mutations However, stable expression in the face of rapid cell division and growth may lead to failure of long-term therapy The goal of these studies was to examine the distribution of and level of expression into early adulthood of 10 natural serotypes of AAV when administered to the rapidly growing neonate Methods: We developed 10 rAAV serotypes (1-rh10) with the chicken β-actin promoter/CMV enhancer and rey luciferase cDNA 2x10e10 genome copies of rAAV was administered intravenously to 2-day-old mice Expression was followed for the rst 100 days of life both by whole animal bioluminescent imaging (BLI) and by tissue luminometry at selected time points Vector copy number was determined by qPCR Results: Mouse growth was particularly rapid during the rst weeks of life with a 3.5-fold increase in weight from 1.7±0.2 grams at birth to 17.6±2.9 grams at weeks Subsequently, mice grow much more slowly weighing 21.3±2.3 grams at day 50 and 29.0±1.2 at day 100 (males) All ten serotypes demonstrate expression within 72 hours of intravenous administration By whole animal BLI, AAV can be subdivided into high- (8,9 rh10), medium- (1, 5, 6, 7), and lowexpressing (2, 3, 4) serotypes in the neonate The lowest expressing serotype at all time points was AAV3 and the highest was AAVrh10 Liver expression was highest with rh10>>8>9>7>6>1 Cardiac expression was highest with rh10>9>8>7=6=1 Skeletal muscle expression was highest with rh10>9>8>7>6=1 In general, the rh10 serotype provided the highest expression days after injection and remained highest at 100 days of life In most organs, including the liver, there is a rapid decline in level of expression over time, likely in part related to cellular division However in skeletal muscle near terminal differentiation, expression is stable or increases with animal growth Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy Conclusions: Neonatal gene therapy is challenged by the need to transduce rapidly growing and dividing cells as the organism develops Long-term expression in rapidly dividing tissues can be obtained with AAV as stable expression occurs after the rapid period of growth is completed In general this was best obtained in tissues with serotype rh10 However, in tissues where growth is a greater feature than cellular division (i.e skeletal muscle), stable or increasing expression is obtained early after AAV administration 374 Optimization of AAV Vectors for Improved Therapeutic Protein Expression in the Canine Models for Duchenne Muscular Dystrophy Alock Malik,1 Marilyn Mitchell,1 Andrew Mead,1 Mike Petrov,1 Hansell Stedman.1 University of Pennsylvania, Philadelphia, PA A major bottleneck in the development of effective gene therapy for Duchenne Muscular Dystrophy is the design of vectors that yield the highest possible expression of dystrophin or utrophin in the target tissues The magnitude of this challenge has come into clearest focus during the transition from small to large animal models in the preclinical setting We recently initiated studies of limb-wide, vascular gene transfer in the dystrophic dog using AAV vectors based on internally deleted versions of the wild type canine dystrophin and utrophin cDNA sequences Early in the course of these studies, the need for higher levels of protein expression normalized to the administered vector dose prompted a parallel effort to further optimize the entire vector expression cassette Iterative improvements were made through the analysis and serial modication of plasmids encoding identical proteins using distinct coding sequence and transcriptional cassettes Systematic screening, rst in HEK 293 cells and then in mdx mouse muscle allowed us to determine the single best expression cassette from a pool of plasmids with different promoters (ubiquitous, muscle specific and hybrid-synthetic promoters), S145 AAV VECTORS II introns, poladenylational signals, transcriptional enhancer elements, and peptide coding sequences The best combination of these improvements thus far has provided at least a 10-fold higher level of recombinant protein expression than our rst generation cassette, with further improvements under additional analysis Selected versions of these transcriptional cassettes are being further characterized in the context of myotrophic AAV vectors following administration to mdx mice and GRMD dogs 10-fold or greater improvements due to the optimization process should greatly accelerate the pace of therapeutic trials in the dystrophic dogs 375 rAAV1 Infect Mouse Bone Marrow-Derived Dendritic Cells through Receptors with Į2,3 and Į2,6 Sialic Acids Yuanqing Lu, Sihong Song Pharmaceutics, University of Florida, Gainesville, FL Host immune response to recombinant adeno-associated virus (rAAV) is still a hurdle on the road of gene therapy, although rAAVs may have low immunogenicity compare with other viral vectors Better understanding of the mechanism of immune response against rAAVs is important for the safety and optimal effect of gene therapy We have recently shown transduction of dendritic cells (DC) plays a critical role in host immune response to the transgene product of rAAV vectors Different rAAV serotypes display difference immunogenicity depending on their ability of DC infection rAAV1 efciently infected DC and induced strong immune responses in several stains of mouse However, the receptors on DC that interact with rAAV1 vector remain unknown In this study we investigated the rAAV1 receptor on DC To obtain mouse DCs, mouse bone marrow (BM) was isolated from none-obese diabetes (NOD) mice BM cells were cultured in complete RPMI-1640 medium with 2ng/ml of recombinant murine granulocyte-macrophage colony stimulating factor and 5ng/ml of IL-4 for 3days On day4, cells were infected with rAAV1 with 1x104 of MOI Lectin competition assay showed that α2,3 or α2,6 sialic acids were important for rAAV1 infection of DCs This infection can be inhibited by treatment of DCs with proteinase K indicating glycoprotein may be involved In addition, we showed that LPS pretreated DCs can not be infected by rAAV1 indicating that rAAV1 unable to infect mature DCs Taken together, these results suggest that rAAV1 infect mouse bone marrow derived immature dendritc cells through receptors with α2,3and α2,6 sialic acids 376 Using AAV Vector as Carrier for Biosensor: Detecting microRNA’s Activities by Reverse Infection of AAV Vectors Wenhong Tian,1 Xiaoyan Dong,2 Gang Wang,1 Shuping Tan,1 Xiaobing Wu.1 National Laboratory of Molecular Virology and Genetic Engineering, Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China; 2Beijing Fiveplus Molecular Medicine Institute Ltd., Beijing, China Objective Recombinant adeno-associated virus (rAAV) vectors have been used increasingly for gene therapy However, using rAAV as carrier for a detecting sensor has never been reported Here we describe a novel strategy of using rAAV carried biosensors to detect microRNAs activities in life cells named as ‘A-sensors’ A-sensor is a rAAV containing Gaussia luciferase (Gluc) gene expression cassette with one copy of miRNA-complementary sequence in its 3’UTR Also a rey luciferase expression cassette is included as an inner control for calibrating transduction titers between different A-sensors Methods To test if cells could be transduced by rAAV in ‘reverse infection’ way, rAAV2-EGFP in serial dilutions were coated on a cell culture plate and air-dried Different cells were added to the S146 plate and cultured in CO2 incubator at 37°C and investigated EGFP expression in 24 to 48h A-sensor vector was designed as shown in Figure1, with BglII and EcoRI site (downstream of Gluc gene) for inserting miRNA-complementary sequence rAAV was produced and puried by our previous methods with rHSV1-repcap as helper virus A-sensors carrying various miRNA-complementary sequences of equal transduction titer were separately coated on 96-well cell culture plate, and dried A-sensor without any miRNA-complementary sequence was coated as control Different A-sensors were arranged orderly in the culture plate resulting in ‘A-sensors array’ Cells to be detected were added with equal number to wells of the A-sensor plate The Gluc activities in the supernatant were detected after 24∼48h The relative microRNA activities could be presented by comparing Gluc activity of the control A-sensor to that of the specic miRNA A-sensor To verify the method, miR-142 3p was chosen as the rst detecting subject Results Results showed rAAV2-EGFP coated on culture plate in air dried condition transduced various cells effectively and dose dependently Using the A-sensor method, miR-142 3p activity was found high in U937, K562, Sp2/0, and P815 cells, and negative in HEK 293 Conclusion rAAV could transduce cells effectively in a reverse infection way, showing a great advantage for using AAV vector as carrier for biosensor This study provides a novel strategy for detecting miRNA activity proles 377 Clearance of Herpes Simplex Virus by Orthogonal Manufacturing Process of Recombinant Adeno-Associated Virus (rAAV) Vectors Guo-jie Ye,1 David R Knop,1 Darby L Thomas,1 Lijun Wang,2 Jeffrey D Chulay.1 Research & Development, Applied Genetic Technologies Corporation, Alachua, FL; 2Process Development, Florida Biologix, Alachua, FL Purpose: rAAV manufacturing using a recombinant herpes simplex virus (rHSV) complementation system employs two ICP27 mutant rHSV helper viruses that are replication-incompetent in normal cells but can be propagated in complementing V27 cells (Vero cells stably transformed with the HSV-1 UL54 gene encoding ICP27) During rHSV production, a replication-defective ICP27 mutant rHSV can recombine with the UL54 gene in the V27 cell resulting in low levels of replication-competent HSV (rcHSV) revertants which may further propagate during rAAV production and carry through into the unprocessed pre-lysis bulk product We evaluated the ability of the current purication process used for GMP manufacture of a rAAV vector expressing human alpha-1 antitrypsin (rAAV1-CB-hAAT) to effectively inactivate and remove all detectable infectious HSV Methods: A 10 L scale batch of rAAV1-CB-hAAT was initiated for generation of matrix samples to support viral clearance studies, using the same process used for manufacturing clinical grade rAAV1-CBhAAT drug substance Three manufacturing unit operations were evaluated for HSV clearance: detergent lysis, CIM-Q anion exchange chromatography, and AVB affinity chromatography Each unit operation was scaled appropriately to mimic “at scale” production Matrix materials spiked (1/10, v/v) with high-titer wild-type HSV (wtHSV) were subjected to the process unit operations under study and HSV clearance measured using a qualied HSV infectious titer assay Clearance of HSV was assessed at specic steps and cumulatively throughout the manufacturing process Results: The three unit operations (detergent lysis, CIM-Q and AVB column Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy ... Medicine Institute Ltd., Beijing, China Objective Recombinant adeno-associated virus (rAAV) vectors have been used increasingly for gene therapy However, using rAAV as carrier for a detecting... infection of DCs This infection can be inhibited by treatment of DCs with proteinase K indicating glycoprotein may be involved In addition, we showed that LPS pretreated DCs can not be infected by rAAV1... other viral vectors Better understanding of the mechanism of immune response against rAAVs is important for the safety and optimal effect of gene therapy We have recently shown transduction of