Introduction

In this chapter we summarize the state of development of adeno-associated virus (AAV) vectors and provide an overview of AAV as well as some historical comment on early seminal studies that are generally overlooked in current reviews. We do not attempt to provide an exhaustive collection of references on development of AAV vectors but do discuss the key advances in the last several years, including improvements in vector production and studies on the applications for persistent gene expression. Other references provide general reviews of AAV (1,2) and extensive summaries on earlier development of AAV vectors (3-7).

AAV vectors have a number of advantageous properties as gene delivery vehicles. The parental virus does not cause disease. AAV vectors are the smallest and most chemically defined particulate gene delivery system and potentially could be classified as well-characterized biologics for therapeutic applications. AAV vectors contain no viral genes that could elicit undesirable cellular immune responses and appear not to induce inflammatory responses. The primary host response that might impact use of AAV vectors is a neutralizing antibody response. The vectors readily transduce dividing or non-dividing cells and can persist essentially for the lifetime of the cell. Thus, AAV vectors can mediate impressive long-term gene expression when administered in vivo. Consequently, these vectors may be well suited for applications where the vector is delivered infrequently and where any potential host antibody response to the AAV capsid protein may be less inhibitory. One limitation for AAV vectors is the limited DNA payload capacity of about 4.5 kilobases (kb) per particle.

The lack of good production systems that could generate high titer vectors was an early obstacle to development of AAV vectors, but this has been overcome through significant advances in both upstream production and downstream purification of AAV vectors. Clinical development of AAV vectors has progressed significantly and studies of an AAV vector in cystic fibrosis patients (8-11) have been extended to phase II trials. Other AAV vectors have now entered clinical trials for hemophilia B (12) and limb girdle muscular dystrophy (13).

Since the first edition of this book (14), there have been extensive advances in application of AAV vectors in many animal models and further analysis of AAV vector safety profiles and host cell responses. There have also been remarkable advances in understanding the structure and biology of AAV vectors, including uptake into cells, trafficking to the cell nucleus, and the mechanism of genome persistence. These studies suggest possible ways to modify the biological targeting of AAV vectors, enhance transduction efficiency, and overcome the packaging limitation.

Most of the early studies on AAV used AAV serotype 2, but genomes of several other AAV serotypes have been cloned and sequenced (15). The biological properties of individual serotypes include differences in the interactions with cellular receptors (16). Other studies are now providing information on the structure of the AAV capsid and how its interaction with the cell may be modified. Notably, the crystal structure of AAV2 was recently described (17). Thus, together with additional studies on cellular trafficking pathways, it may be possible to modify the targeting of AAV vectors as well as to enhance their transduction efficiency (18).

Studies on the mechanism of persistence of vector genomes in transduced cells indicate that this involves formation of polymeric DNA structures or concatemers. Concatemers can also be formed between 2 different vector genomes introduced into the same cell. This provides a way to partly circumvent the packaging limit of AAV by dividing a gene expression cassette between 2 AAV vectors (''dual vectors'') and allowing recombination in the cell to generate the intact expression cassette.

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