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373. Improved and Persistent Expression with Novel AAV Serotypes for Neonatal Gene Therapy
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 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 5 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 5 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 3 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 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
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 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 5 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 5 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 3 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 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