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System for efficient helper-dependent minimal adenovirus construction and rescue.

Helper-dependent minimal adenoviral vectors deleted for all viral coding sequences are promising vectors for gene therapy. They retain only the adenovirus cis elements for replication and packaging, can accommodate up to 36 kb of foreign DNA, and exhibit prolonged transgene expression and reduced tissue toxicity as compared with first-generation adenoviral vectors. We have developed a system consisting of a set of cosmid cloning vectors (pMV and pMVX) for simple routine construction and efficient rescue of minimal adenoviral vectors. In the cloning vectors the inverted terminal repeats (ITRs) are flanked by recognition sites for the super rare-cutting endonuclease I-SceI. This allows the release of linear minimal adenovirus genomes for rescue of minimal adenovirus regardless of the sequence of the insert DNA. pMV contains a multiple cloning site for the insertion of 26 to 36 kb of therapeutic DNA. pMVX contains a noncoding human X-chromosomal DNA fragment as a vector backbone, which provides endonuclease restriction sites that allow for complete or partial replacement of the vector backbone by 1 to 26 kb of therapeutic DNA sequences, while retaining a packageable final minimal adenovirus genome size between 27 and 37.5 kb. Both vectors exist in two forms, with or without an Escherichia coli lacZ reporter gene cassette. Several minimal adenoviral vectors with insert sizes ranging from 1.5 to 16 kb were constructed with these cloning vectors. Minimal adenoviruses were efficiently rescued and amplified to high titers, using a Cre/lox-based helper system. Vectors containing the X-chromosomal backbone were stable during amplification. This simple and efficient system facilitates the construction of minimal adenoviruses and should be useful for further improvement of these new vectors.