Gene Therapy Strategies Incorporating Large Transgenes

Abstract

The application of nucleic acid therapeutics sometimes requires the transfer of relatively large transgenes. For example, treatment of diseases such as hemophilia A, sickle cell disease, muscular dystrophy, or cystic fibrosis requires the transfer of coding sequences or complex regulatory elements, which can exceed 4.5 kilobases (kb). In general, transgenes are inserted into expression cassettes that can then be incorporated into either viral or nonviral vectors. The vector chosen depends not only on the therapeutic application but also on the size of the expression cassette. The size of the expression cassette depends on the size of the transgene and the requirement of specific regulatory elements necessary for appropriate transgene expression (for example, internal promoters, enhancers, insulators and other regulatory elements). Larger expression cassettes complicate gene therapy applications using viral vectors by (1) limiting the types of vectors available due to encapsidation limitations, (2) increasing the complexity of the transferred nucleic acid sequence, which, for example, can inadvertently introduce splice donor and acceptor sites, and (3) reducing the titer of viral vector that can be produced. Due to these hurdles, gene therapy strategies utilizing large expression cassettes are limited in vector design. One avenue of decreasing the size of transgenes is to genetically engineer minimal complementary DNA (cDNA) cassettes by eliminating regions that encode for non-functional aspects of the encoded protein. For example, the cDNA of human dystrophin, the missing functional gene in Duchenne’s Muscular Dystrophy, is approximately 11 kb. Due to its large size, a truncated functional version termed, mini-dystrophin, is utilized, thereby reducing the transgene size to 6 kb. A similar strategy has also been evaluated for the cDNA encoding factor VIII (FVIII), mutations of which cause hemophilia A. The transgene size has been reduced by eliminating an entire domain, thus reducing the transgene size by >2.5 kb. A second strategy that has been evaluated involves dividing a larger transgene into smaller sub-transgenes that can then be incorporated into separate vectors to be delivered simultaneously. Nonviral gene-delivery systems, on the other hand, are not as constrained by the size of the expression cassette. Despite this advantage, nonviral vectors are currently not widely used because of inefficient gene transfer. However, rapid progress is being made with transposons circumventing limited transfer by capitalizing on their ability to integrate within the genome. For example, the Sleeping Beauty transposon has been used to insert a number of large expression cassettes into the human genome, including the 6.5 kb ┚-globin expression cassette for the treatment of sickle cell disease. Therefore, these vectors hold much promise for gene therapy applications with large expression cassettes.