Design of AAV Vectors

The ability to generate AAV vectors was facilitated by the observation that molecular cloning of double-strand (ds) AAV DNA into bacterial plasmids followed by transfection into helper virus-infected mammalian cells resulted in rescue and replication of the AAV genome free of any plasmid sequence to yield a burst of infectious AAV particles (5). This rescue may occur by a mechanism analogous to that used in reactivation of a latent provirus after superinfection of cells with ade-novirus. The general principles of AAV vector construction (4,5,12) are based on modifying the molecular clones by substituting the AAV coding sequence with foreign DNA to generate a vector plasmid. In the vector, only the cis-acting ITR sequences must be retained. The vector plasmid is introduced into producer cells that are also rendered permissive by an appropriate helper virus such as adenovirus. To achieve replication and encapsidation of the vector genome into AAV particles, the vector plasmid must be complemented for the transacting AAV rep and cap functions that were deleted in construction of the vector plasmid. AAV vector particles can be purified and concentrated from lysates of such producer cells.

The AAV capsid has 3 important effects for AAV vectors. There is a limit of about 5 kb of DNA that can be packaged in an AAV vector particle. This places constraints on inclusion of very large cDNAs and may also limit the ability to include extensive regulatory control sequences in the vector. The cap-sid also interacts with the AAV receptor and coreceptors on host cells, and thus mediates cell entry. The capsid may also induce humoral immune responses that could limit delivery of AAV vectors for some applications.

Except for the limitation on packaging size and the requirements for ITRs, there are no obvious limitations on the design of gene cassettes in AAV vectors. The ITR can function as a transcription promoter (91) but does not interfere with other promoters. Tissue-specific promoters appear to retain specificity (92,93) and a number of other regulated expression systems have now been used successfully in AAV vectors (94-96). Introns function in AAV vectors and may enhance expression, and more than 1 promoter and gene cassette can be inserted in the same vector. Importantly, transcription from AAV does not seem to be susceptible to in vivo silencing, as shown by expression for over 1 year after intramuscular delivery in rodents (54,55,97).

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