By N.J. Clifton

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Additional info for Cardiac Cell and Gene Transfer: Principles, Protocols, and Applications (Methods in Molecular Biology Vol 219)

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2. Role of the Helper Virus The ideal helper virus provides robust adenoviral gene expression yet does not compete with or interfere with packaging of the gutted virus. We use helper viruses that are replication-deficient owing to deletion of viral sequences in the Ad early region 1 (E1A and B genes). These gene products are supplied by the packaging cell line. This strategy ensures that any helper virus that escapes negative selection and is copurified with the gutted virus cannot replicate in a nonpermissive cell.

This small packaging capacity is one of the most challenging barriers in AAV-based gene therapy technologies. Many larger therapeutic genes such as factor VIII in hemophilia A and the mini-dystrophin gene in Duchenne’s muscular dystrophy, cannot be packaged into a single AAV virion without further truncations to the therapeutic genes. Several recent research breakthroughs have made rAAV-mediated gene therapy for larger genes (therapeutic genes larger than 5 kb) a viable reality. Studies on AAV transduction biology demonstrate that AAV genomes can form large circular concatamers through inverted terminal repeat (ITR) mediated intermolecular recombination (20–22).

Begy, C. , and Chamberlain, J. S. (1996) Improved adenovirus packaging cell lines to support the growth of replication-defective gene-delivery vectors. Proc. Natl. Acad. Sci. USA 93, 3352–3356. Amalfitano, A. and Chamberlain, J. S. (1997) Isolation and characterization of packaging cell lines that co-express the adenovirus E1, DNA polymerase, and preterminal proteins: implications for gene therapy. Gene Ther. 4, 258–263. , and Chamberlain, J. S. (1999) Improved production of gutted adenovirus in cells expressing adenovirus preterminal protein and DNA polymerase.

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