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News, Calendars, and Events » Calendars » Master Calendar » Biochemistry and Molecular & Cellular Biology, Department of
Seminar Series: Dr. Robert Kotin
Schedule information
Event Seminar Series: Dr. Robert Kotin
When Tuesday, February 19, 2013 from 12:00pm to 1:00pm
Where Basic Science Building 341 (Library)
Event details
Details 'A secret to successful gene therapy outcomes is vector production'

Dr. Robert Kotin

Senior Investigator and Head
Laboratory of Molecular Virology and Gene Therapy
Genetics and Developmental Biology Center (GDBC)
National Heart, Lung, and Blood Institute
National Institutes of Health

Abstract: Remediation of genetic and other diseases by gene transfer is conceptually compelling but has lagged far behind (often unrealistic) expectations. However, the fundamentals of gene therapy are sound, supported by numerous successful examples in small animal mod­els of genetic diseases. Recombinant adeno-associated virus (rAAV) has demon­strated utility for delivering genetic information to patients affected either by genetic lesions or acquired diseases. Despite convincing proof-of-principle studies in small ani­mal models of genetic disease, relatively few clinical studies have been initiated due in large part to the economics and feasibility of producing rAAV. All methodologies for produc­ing AAV vectors require co-expression of the AAV non-structural Rep proteins and structural capsid proteins. In addition, a required set of proteins from a helper virus renders the cell permissive for AAV propagation. The AAV vector consists of three capsid proteins VP1, VP2, and VP3 (present in a ratio of approximately 1:1:10) that assemble into a non-enveloped, icosahedral particle. Vector genomes, not exceeding the wild-type virus genome size (4700 nt including 290 nt of terminal repeat sequences necessary for replication), package more efficiently than larger genomes. The conventional method for producing rAAV involves transient co-transfec­tion of mammalian cells with plasmids containing the adenovirus helper genes, rep and cap genes, and the vector genome. This production platform depends on chemi­cal transfection that is most efficient with adherent cells, thereby limiting the potential number of AAV-producer cells. During a wild-type AAV infection, the copy number of the rep and cap genes amplifies geometrically during the early infection phase, whereas the prokaryotic-derived plasmids are not amplified in mammalian cells. In addition, there is no cell-to-cell spread of the plasmids further limiting the process scalability. To over­come this production barrier, our laboratory sought alternative approaches and discovered that in the Spodoptera frugiperda (Sf9) insect cell line, the AAV vector genome was “res­cued” and replicated in a manner reminiscent of a wild-type AAV infection. Recombi­nant baculovirus expression vectors (bevs) efficiently infect the Sf9 cells produc­ing progeny bevs that finally infect all the cells in the suspension culture. The Rep and Cap open reading frames were engineered to improve efficiency and achieve the specific stoichiometry. The Cap open reading frame is transcribed as a single mRNA and translated into VP1, VP2, and VP3 by using non-canonical initiation codons for VP1 and VP2 the desired 1:1:10 stoichiometry of the three capsid proteins was achieved. Using a similar strategy, Rep 78 and Rep 52 were produced from one tran­script. Depending on the serotype and transgene, the yields for several rAAV genotypes and serotypes often approach 1E+14 particles per liter with linear scalability demon­strated in cultures ranging from 0.02 to 500 liters. The vector quantities are now suffi­cient to obtain meaningful outcomes in clinically relevant models of Duchenne muscular dystrophy and for other intractable disease.

Amsterdam Molecular Therapeutics B.V. (re-organized as uniQure B.V.) recently received approval for rAAV1-lipoprotein lipaseS447X (generic name - alipogene tipar­vovec, trade name - Glybera (TM) ) by the European Medicines Agency. The world’s first AAV gene therapy product was produced using the methodologies and materials invented and developed by investigators in our laboratory in the NIH.
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Contact Juanita Chipani Biochemistry and Molecular & Cellular Biology office, x71512
Sponsors Department of Biochemistry and Molecular & Cellular Biology
Calendar Biochemistry and Molecular & Cellular Biology, Department of
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