The packaging capacity of adeno‐associated virus (AAV) and the potential for wild‐type‐plus AAV gene therapy vectors

Abstract

Because of its ability to integrate chromosomally and its non-pathogenic nature, adeno-associated virus (AAV) has significant potential as a human gene therapy vector. Here we investigate the maximum amount of DNA which can be inserted into the AAV genome and still allow efficient packaging into an infectious virus particle. Altered wild-type AAV genomes were constructed with inserts, which increased in size by 100 bp, ligated at map unit 96. These large wild-type-plus genomes were able to replicate and produce infectious virus, at levels slightly reduced but comparable to normal sized wild type, until the insert size reached 1 kb. These data indicate that the maximum effective packaging capacity of AAV is approximately 900 bp larger than wild type, or 119%. Furthermore, it is demonstrated that these large AAV genomes are able to latently infect cells by chromosomal integration as does wild-type AAV. These data suggest that therapy vectors carrying a foreign gene of 900 bp or less can be generated from AAV, by ligation into non-essential locations, and result in a recombinant AAV virus with a fully wild-type phenotype. Such wild-type-plus AAV vectors will have both advantages and disadvantages over defective recombinant AAV virus -the most important advantages being the ease in which high titers of infectious virus can be generated and the ability to specifically integrate within chromosome 19. Once the concern subsides over the presence of wild-type AAV in clinical applications, wild-type AAV vectors may find specific application niches for use in human gene therapy. © 1997 Federation of European Biochemical Societies.

Rapid induction of cytotoxic T-cell response against cervical cancer cells by human papillomavirus type 16 E6 antigen gene delivery into human dendritic cells by an adeno-associated virus vector

“…High -titer rough ( nonpurified ) rAAV virus stocks were generated in a two -step process, using the complementor plasmid ins96 -0.8, and titered as described previously. 37,49 To generate purified rAAV virus the technique described by Auricchio et al 50 was used. Briefly, the virus solution treated by DNase I ( Promega Co., Madison, WI ) was incubated with 0.5% deoxycholic acid ( Sigma, St. Louis, MO ) for 30 minutes at 378C.…”

Quantitative comparison of expression with adeno-associated virus (AAV-2) brain-specific gene cassettes

“…Previous studies suggested that up to 6.0 kb can be packaged into virus, but these 'over-sized' viruses were not infectious, 32 hence the common assumption is that the packaged genome must be limited to just 110% of wild-type AAV or no greater than ෂ5.1 kb. 33 This limited DNA insert size restricts the range of open reading frames which can be inserted for any given promoter and post-regulatory element and polyadenylation signal. 34 A secondary goal was to test the outer limits of AAV-2 genome size, while maintaining packaging and infectious efficiency.…”

Adeno-associated virus vectors: potential applications for cancer gene therapy

“…112,113 This small packaging size of AAV has always been thought to preclude its use for delivering genes larger than 5 kb, such as dystrophin and factor VIII, or the use of large regulatory elements to enhance or control transgene expression. Recently, a new approach has been developed to overcome this vector size limitation by exploiting the unique heterodimerization ability of AAV DNA.…”