The assembly-activating protein promotes capsid assembly of different adeno-associated virus serotypes.

Adeno-associated virus type 2 (AAV2) capsid assembly requires the expression of a virally encoded assembly-activating protein (AAP). By providing AAP together with the capsid protein VP3, capsids are formed that are composed of VP3 only. Electron cryomicroscopy analysis of assembled VP3-only capsids revealed all characteristics of the wild-type AAV2 capsids. However, in contrast to capsids assembled from VP1, VP2, and VP3, the pores of VP3-only capsids were more restricted at the inside of the 5-fold symmetry axes, and globules could not be detected below the 2-fold symmetry axes. By comparing the capsid assembly of several AAV serotypes with AAP protein from AAV2 (AAP-2), we show that AAP-2 is able to efficiently stimulate capsid formation of VP3 derived from several serotypes, as demonstrated for AAV1, AAV2, AAV8, and AAV9. Capsid formation, by coexpressing AAV1-, AAV2-, or AAV5-VP3 with AAP-1, AAP-2, or AAP-5 revealed the ability of AAP-1 and AAP-2 to complement each other in AAV1 and AAV2 assembly, whereas for AAV5 assembly more specific conditions are required. Sequence alignment of predicted AAP proteins from the known AAV serotypes indicates a high degree of homology of all serotypes to AAP-2 with some divergence for AAP-4, AAP-5, AAP-11, and AAP-12. Immunolocalization of assembled capsids from different serotypes confirmed the preferred nucleolar localization of capsids, as observed for AAV2; however, AAV8 and AAV9 capsids could also be detected throughout the nucleus. Taken together, the data show that AAV capsid assembly of different AAV serotypes also requires the assistance of AAP proteins.

Recombinant adeno-associated virus for the generation of autologous, gene-modified tumor vaccines: evidence for a high transduction efficiency into primary epithelial cancer cells.

To explore the potential of recombinant vectors based on recombinant adeno-associated virus (rAAV) for cancer vaccination, we investigated the transduction efficiency of rAAV into cancer cells ex vivo. Infection of human epithelial cancer cell lines with rAAV carrying reporter genes encoding beta-galactosidase (rAAV/LacZ) or luciferase (rAAV/Luc) resulted in high levels of reporter gene expression (>90% positive cells). In marked contrast, rAAV poorly transduced all murine tumor cell lines, as well as human hematopoietic cell lines. Either irradiation or adenovirus infection of tumor cells prior to rAAV infection induced a 10- to 100-fold increase of reporter gene expression. To determine the transduction efficiency of rAAV into primary cancer cells, freshly isolated, irradiated tumor cells from malignant melanoma and ovarian carcinoma patients were infected with rAAV/Luc, resulting in up to 6.9-fold higher levels of gene expression than in a HeLa tumor cell line. Time course experiments with freshly isolated tumor cells infected with rAAV/Luc showed maximal levels of luciferase expression between days 3 and 9 posttransduction. Simultaneous infection of primary tumor cells with up to three rAAV vectors containing genes encoding the immunostimulatory proteins B7-2 (CD86), p35 subunit of IL-12, and p40 subunit of IL-12 resulted in high expression of B7-2 in more than 90% of the tumor cells and in the secretion of high levels of IL-12. Taken together, our results demonstrate that rAAV efficiently transduces freshly isolated human, epithelial tumor cells and might therefore be a potent tool to produce improved, gene-modified cancer vaccines.

Recombinant adeno-associated virus vectors.

Recombinant adeno-associated virus (rAAV) is a promising vector for somatic gene therapy due to the ability to transduce terminally-differentiated and non-dividing cells, the lack of any apparent pathogenicity, a low immunogenicity, a relatively high stability of transgene expression, and the potential for targeted integration. Improved methods of rAAV packaging allow the generation of concentrated and highly purified rAAV for clinical trials. Preclinical studies with rAAV are currently in progress for the treatment of a variety of inherited monogenic defects, but also for acquired diseases like HIV infection and cancer. Because of the broad host range of wild type AAV, rAAV is able to transduce a variety of human tissues, preferentially those of epitheloid origin, with high efficiency and therefore may be used for various clinical applications. Whilst several issues including safety, tissue tropism and methods to achieve site-specific integration need further improvement, rAAV certainly has a sufficient number of advantages to be seriously considered as a gene therapy vector.

Mutational analysis of adeno-associated virus Rep protein-mediated inhibition of heterologous and homologous promoters.

The four Rep proteins encoded by adeno-associated virus type 2 (AAV-2) inhibit transcription of their own promoters and of several heterologous promoters. To gain insight into the molecular mechanism of Rep-mediated transcription repression, we studied the effects of the four Rep proteins on the accumulation of mRNA transcribed from the human papillomavirus type 18 upstream regulatory region HPV18 URR, the human immunodeficiency virus long terminal repeat, and the AAV-2 p5 and p19 promoters by transient transfection experiments in HeLa cells. We observed a distinct contribution of the C- and N-terminal sequences in which the four Rep proteins (Rep78, Rep68, Rep52, and Rep40) differ from each other. While Rep78 showed a more than 10-fold inhibition of the four promoters studied, transcriptional repression mediated by Rep68 and Rep52 was reduced and nearly completely abolished for Rep40. The contribution of the C terminus of Rep78 was reduced with respect to the inhibition of the AAV-2 p5 and p19 promoters. Point mutations and deletions showed that a C-terminal zinc binding motif is required for zinc binding in vitro but plays no obvious role in the inhibition of homologous and heterologous promoters. Overall, inhibition of the four different promoters was dependent on the identical Rep protein domains with the exception of the AAV-2 p5 promoter. Expression of the AAV-2 p5 promoter was inhibited by a Rep78 protein with a mutation in the nucleotide binding motif, whereas expression of the AAV-2 p19 promoter, the human immunodeficiency virus long terminal repeat, and the HPV18 URR was not. Mutational analysis of the HPV18 URR showed that several, but not a single, cis regulatory elements are involved in the inhibition process. This finding suggests that transcriptional repression is mediated by protein-protein interactions of the Rep proteins either with multiple transcription factors or with target proteins of sequence-specific transcription factors of the basal transcription machinery.

Cell lines inducibly expressing the adeno-associated virus (AAV) rep gene: requirements for productive replication of rep-negative AAV mutants.

The adeno-associated virus (AAV) rep gene codes for a family of nonstructural proteins which are required for AAV gene regulation and DNA replication. In addition, rep has been implicated in a variety of activities outside the AAV life cycle which have been difficult to study, since attempts to achieve separate and constitutive expression of rep in stable cell lines have failed so far. Here we report the generation of two cell lines which inducibly express Rep78 under the control of the glucocorticoid-responsive mouse mammary tumor virus promoter. In addition, one of the cell lines constitutively expresses relatively high levels of Rep52. Both cell lines showed similar plating efficiencies with and without induction of Rep78 expression, which rules out cytotoxic effects of Rep78. The cell lines efficiently support DNA replication of a rep-negative AAV genome and initiate the formation of AAV particles. However, despite the correct sizes and stoichiometry of the three capsid proteins, the AAV particles were noninfectious. This was found to be due to a defect in the accumulation of single-stranded AAV DNA. Transient transfection of single expression constructs for constitutive, high-level expression of individual Rep proteins (either Rep78, Rep68, Rep52, or Rep40) complemented this defect. Infectious rep-negative AAV progeny was produced at varying efficiencies depending on the rep expression construct used. These data show that functional expression of full-length Rep in recombinant cell lines is possible and that the state of Rep expression is critical for the infectivity of AAV progeny produced.