No 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.

The adeno-associated virus (AAV) terminal repeats (TR) are cis required, and the AAV encoded Rep78 protein is trans required, for AAV DNA replication. The Rep78 protein recognizes and interacts with at least three regions within the TR DNA. The major binding site, with the highest affinity for Rep78 binding, is within the TR stem (nt 36-16) and includes the 'core' GAGC trimer (GAGC 3, nt 33-22; Fig. 2) sequence. In this study mutations were made within the GAGC trimer and these mutants assayed for their ability to allow for AAV double stranded (ds DNA, prepackaging DNA replication), and single stranded DNA (ss DNA, due to virion packaging) replication. Here, it is shown that when the two inside GAGC motifs are mutated, with only motif no. 1 left intact (see Fig. 2), the resulting AAV (mutA) genome was significantly defective for both ds DNA (17% of wild type) and ss DNA (9%). If the TRs contained only the two outside motifs intact (mutB), motifs no. 1 and 2, the AAV genome had a significant but reduced level of both ds (50%) and ss (34%) DNA replication. Finally, if only the middle motif no. 2 was mutated, with motifs no. 1 and 3 left intact (mutC), the resulting DNA replication for both ds and ss forms was essentially wild type (80% that of wild type). These data suggest that the GAGC trimer plays a role in AAV DNA replication, and that GAGC motif no. 3 is the most important of the three motifs for both ds and ss DNA replication.

IntroductionThe loss of resilient dentine as a result of endodontic therapy often causes some discolouration.This can be of a minor nature or it can be such that the light refraction from the less opalescent dentine warrants labial coverage of an anterior tooth.