of the application) Recombinant adenovirus vectors have many attractive features for gene therapy applications, including high transduction efficiencies in vivo and the ability to transduce non-dividing cells. However, the persistence of these vectors is limited by the response of the host immune system to viral gene products and by the inability of these vectors to replicate in the absence of expression of these viral proteins. We propose that safe, efficient and persistent adenovirus-mediated gene transfer may be achieved through the systemic delivery of self-excising, self-replicating episomes by recombinant adenovirus amplicon vectors in which most or all viral genes have been deleted. The design of the self-excising, self-replicating transgene expression cassette will first be optimized in in vitro experiments. The optimized unit will then be inserted into a recombinant adenovirus vector for in vivo confirmation of the ability of this unit to perform its intended excision and replication functions. As the size of this DNA species exceeds the capacity of E1-deleted adenovirus vectors, in vivo delivery in these initial proof-of-concept studies will be mediated by an E1- and E4-deleted adenovirus vector. To negate any possible interference of the host immune system on the outcome of these studies, they will be performed in SCID) mice. The conversion of the virus to the replicating episome will be monitored by PCR. The structure of the episome will be confirmed by restriction analysis of the plasmid rescued from various organs. Once self-excision and self-replication has been validated in immunodeficient animals, this element will be incorporated into an adenovirus amplicon vector for further testing in immunocompetent animals. The use of an adenovirus amplicon to deliver a replicating vector should minimize any immune response against the adenovirally-transduced cells, allowing the long-term persistence of the replicating vector. High titers of the amplicon vector will be produced using a true adenovirus packaging cell line that does not rely on the use of helper virus. In addition to the tests mentioned above, histopatho-logical and immunohistochemical studies will be performed to determine the efficiency of episome excision and the safety of the gene transfer procedure. If successful, the vector developed in these studies will prove useful in the delivery of potentially therapeutic transgenes to the liver and other target organs for the treatment of a variety of human diseases, genetic and otherwise.
| Krougliak, V A; Krougliak, N; Eisensmith, R C (2001) Stabilization of transgenes delivered by recombinant adenovirus vectors through extrachromosomal replication. J Gene Med 3:51-8 |
