Look out AAV, Here come the Gutted Ad...Best of Luck, Where'd He Go?
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Program No.: 2002
Reversal of Muscular Dystrophy Using Gutted Adenoviral Vectors
Jeffrey S. Chamberlain, Christiana DelloRusso, Jeannine Scott, Dennis Hartigan-O'Connor, Catherine Barjot, Giovanni Salvatori, Susan Brooks
Department of Human Genetics,
Program in Cellular and Molecular Biology,
Department of Physiology,
Center for Gene Therapy, University of Michigan Medical School, Ann Arbor, MI, USA
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Keywords:
Special Text:
Duchenne muscular dystrophy (DMD) is a progressive, lethal disorder caused by mutations in the dystrophin gene. Correction of this disease might be achieved by dystrophin replacement. However, the dystrophin cDNA is 14 KB, larger than the carrying capacity of many viral vectors. We have been developing "gutted" or helper-dependent Ad vectors for DMD by inserting human and mouse dystrophin expression cassettes into gutted plasmids, and by growing these vectors with modified helper viruses in C7 packaging cells that express the Ad replication genes POL and pTP together with Cre recombinase. C7 cells produce 1x105 to 1x106 transducing units (tu) of gutted virus following transfection of a 60 mm palte, whereas 293 cells yield about 100 tu. With C7 cells, 1010 to 1011 tu can be generated in 5 serial passages, vs. 7 with 293 cells. C7-Cre cells produce evn greater yields of vector. Gutted Ad vectors display increased infection in dystrophic mdx muscle aged 6 weeks to 24 months compared with wt muscle, although infectivity drops 6-fold by 24 months of age. Dystrophin expression persisted in adult, immunocompetent mice for at least 4 months following viral injection. Furthermore, dystrophin-expressing muscle fibers excluded the vital dye Evans blue, demonstrating a correction of the integrity of the muscle sarcolemma. We have also developed an eccentric contraction protocol that reveals a significantly greater susceptibility to injury of dystrophic TA muscles relative to wild-type limbs. Injected of dystrophin-expressing gutted Ad vectors into muscles from young and old mdx mice that are already dystrophic reveals a significant correction of the force deficits resulting from eccentric muscle contractions. Together these results indicate that gutted Ad vectors are able to correct and reverse some of the functional deficits in immunocompetent, adult mdx muscles.
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Program No.: 118
An Alternate System for the Production of Fully Deleted Adenoviral Vectors for Gene Therapy
Helen Romanczuk, Patricia Berthelette, Hillard Rubin, Cathleen Sookdeo, Richard Gregory, Samuel Wadsworth
Genzyme Corporation
Keywords:
Special Text: pseudo-adenovirus, helper, recombinase
Recombinant adenoviruses (Ad) are currently being developed as vectors for gene transfer to a wide variety of cells and tissues in vitro and in vivo. Traditionally, simple Ad vectors are deleted of E1 sequences and carry, instead, a therapeutic gene and corresponding expression cassette. Simple Ad vectors can achieve high levels of gene expression in vivo, but the expression is usually transient. This is most likely due, at least in part, to direct cytotoxicity of virally encoded proteins and a host immune response triggered by residual viral gene expression. The development of vectors whose genomes are deleted of one or more viral gene products has shown some promise in improved vector persistence and encouraged the development of even further deleted vectors. The newest generation of "fully-deleted", "gutless", "helper-dependent" or "pseudo-adenoviral" (PAV) vectors has genomes devoid of all viral coding sequences. In their place, large human genomic fragments encoding therapeutic genes are inserted and subsequently replicated and packaged using trans-acting viral gene products. Several groups have now shown that the use of fully-deleted vectors as therapeutic gene delivery vehicles greatly improves persistence of transgene expression in vivo and also results in a significant reduction of vector toxicity, even at high doses, in comparison to first generation vectors. The improved safety and expression profile of fully-deleted vectors, as well as their increased capacity for DNA insertions, comes at the price of inefficient production. The propagation of the vectors requires the provision of trans-acting factors for efficient replication and packaging of the recombinant genome. Most often and effectively, these gene products are provided by a helper vector whose own packaging ability is severely impaired, as in the case of Cre/loxP-based helper vectors. The packaging sequences within these helper vectors are flanked by loxP sites. Cre-mediated recombination at the loxP sites results in the deletion of the packaging sequences and the generation of a helper vector incapable of packaging. This facilitates the production of the therapeutic vector while reducing the amount of contaminating helper in the final product. We have used a similar recombinase system to propagate a pseudoadenoviral vector (PAV) for gene therapy. PAVgal is fully deleted of adenoviral genes and contains, instead, a large human genomic fragment encoding -galactosidase A, the protein deficient in Fabry diseased patients, and a -galactosidase gene for easily tracking virus amplification. The propagation of PAVgal is achieved using a helper vector whose own packaging is impaired following excision of necessary sequences by the Flpe6 yeast recombinase.
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Program No.: 484
A New Viral Cloning System Used to Capture and Manipulate Large Genomic Regions as Stuffer DNA for Gutless Adenoviral Vectors
M. J. Mendez, C. A. Hokanson, E. Dora
Cell Genesys, Inc., Foster City, CA
Keywords:
Special Text: adenovirus, gutless, genomics
With the age of functional genomics, it is becoming increasingly important to not only clone and deliver the gene but also the promoters, enhancers, and other regulatory elements. This would then offer a far more realistic model of genomic gene expression. There are many vector systems capable of manipulating short sequences, but few which have been developed for the cloning, engineering and delivery of large nucleic acid sequences. We describe a unique system in which larger viral genomes and large regions of genomic DNA can be cloned, manipulated and delivered effectively into cells. This yeast artificial plasmid (YAP) system uses an episomal shuttle vector capable of being maintained in both yeast and bacteria. This hybrid approach combines the high rate of homologous recombination in yeast to capture and modify any sequence and the ease of bacteria for amplification and purification. We have successfully used this system in three settings. In the first this YAP cloning system was used to clone and construct over 250 recombinant adenoviral vectors, in which mutation or transgenes can easily be inserted. We have derived a scheme and vectors that allow us to insert mutations by positive selection (ADE3, HIS2, LEU2) and by using negative selection (5-FOA on the URA3), to remove all markers and non-essential sequences, leaving a clean mutational insert. Secondly, this system was also used to capture a large region of genomic DNA containing the Factor IX gene. With this genomic region cloned into our YAP system, we were then able to gene activate the Factor IX by removing its endogenous promoter and replacing it with either a CMV or PGK promoter. This demonstrated the ease with which large genomic regions can be cloned, manipulated, amplified and purified. Thirdly, with the report of the effect of stuffer DNA on transgene expression in gutless adeno vectors (Parks, et al., 1999), we describe a method to generate random or specific stuffer DNAs. We elected to use the 35kb Factor IX genomic gene as a stuffer DNA to both maintain size for efficient adeno DNA packaging and the convenience of a single open reading frame in which both sequence and function are known. This new viral cloning system will aid in the cloning and modifying of any large viral or non-viral genome and easily facilitate their use in recombinant vectors. |
