Improving the efficiency and specificity of gene vectors is critical for the success of gene therapy. In an effort to generate viral mutants with controlled tropism we produced a library of adeno-associated virus (AAV) clones with randomly modified capsids and used it for the selection of receptor-targeting mutants. After several rounds of selection on different cell lines that were resistant to infection by wild-type (wt) AAV, infectious mutants were harvested at high titers. These mutants transduced target cells with an up to 100-fold increased efficiency, in a receptor-specific manner and without interacting with the primary receptor for wt AAV. The results demonstrate for the first time that a combinatorial approach based on a eukaryotic virus library allows one to generate efficient, receptor-specific targeting vectors with desired tropism.

To allow the direct visualization of viral trafficking, we genetically incorporated enhanced green fluorescent protein (GFP) into the adeno-associated virus (AAV) capsid by replacement of wild-type VP2 by GFP-VP2 fusion proteins. High-titer virus progeny was obtained and used to elucidate the process of nuclear entry. In the absence of adenovirus 5 (Ad5), nuclear translocation of AAV capsids was a slow and inefficient process: at 2 h and 4 h postinfection (p.i.), GFP-VP2-AAV particles were found in the perinuclear area and in nuclear invaginations but not within the nucleus. In Ad5-coinfected cells, isolated GFP-VP2-AAV particles were already detectable in the nucleus at 2 h p.i., suggesting that Ad5 enhanced the nuclear translocation of AAV capsids. The number of cells displaying viral capsids within the nucleus increased slightly over time, independently of helper virus levels, but the majority of the AAV capsids remained in the perinuclear area under all conditions analyzed. In contrast, independently of helper virus and with 10 times less virions per cell already observed at 2 h p.i., viral genomes were visible within the nucleus. Under these conditions and even with prolonged incubation times (up to 11 h p.i.), no intact viral capsids were detectable within the nucleus. In summary, the results show that GFP-tagged AAV particles can be used to study the cellular trafficking and nuclear entry of AAV. Moreover, our findings argue against an efficient nuclear entry mechanism of intact AAV capsids and favor the occurrence of viral uncoating before or during nuclear entry.

Adeno-associated virus type 2 (AAV-2) targeting vectors have been generated by insertion of ligand peptides into the viral capsid at amino acid position 587. This procedure ablates binding of heparan sulfate proteoglycan (HSPG), AAV-2's primary receptor, in some but not all mutants. Using an AAV-2 display library, we investigated molecular mechanisms responsible for this phenotype, demonstrating that peptides containing a net negative charge are prone to confer an HSPG nonbinding phenotype. Interestingly, in vivo studies correlated the inability to bind to HSPG with liver and spleen detargeting in mice after systemic application, suggesting several strategies to improve efficiency of AAV-2 retargeting to alternative tissues.Adeno-associated virus type 2 (AAV-2) is gaining increasing attention as a gene therapy vector. However, the wide distribution of its primary receptor, heparan sulfate proteoglycan (HSPG) (11), hampers selective transduction of target tissue. Vectors aiming to redirect AAV-2's tropism have been generated by insertion of ligands at position 587/588 of the capsid (1, 2). This is likely to interfere with the HSPG binding of at least two (R585 and R588) of the five positively charged amino acids of the recently identified HSPG binding motif (5, 7), explaining the ablation of HSPG binding of some targeting vectors (3,4,6,8,10). In some cases, however, binding was only partially affected (12) or was even restored (4,8,14). To investigate molecular mechanisms responsible for these differences, we applied a library of AAV-2 capsids carrying insertions of seven randomized amino acids at position 587 (8) to a heparin affinity column (13) to separate binding from nonbinding mutants. We sequenced and statistically analyzed (Table 1) at least 80 clones from (i) the original DNA-library, (ii) the viral AAV-2 display library, (iii) the flowthrough fraction (nonbinders were designated the NB-AAV-pool), and (iv) the 1 M NaCl eluted fraction (binders were designated the B-AAV-pool).The DNA library showed a higher than expected presence of alanines, which originates from the oligonucleotide synthesis procedure. Occurrence of every other amino acid met statistical expectations for an unselected library.The AAV-2 display library showed an excess of the amino acids P, G, and A and a defect of C, L, F, W, and Y (Table 1B). Since P, G, and A are three of the four smallest amino acids, whereas F, Y, and W are three of the four biggest, this bias suggests that the packaging process selects against bulky inserts which would introduce dramatic structural rearrangements and have a deleterious effect on capsid structure. In addition, prolines could favor spatial accommodation of the peptide by introducing kinks and reducing its bulkiness.The B-AAV-pool showed a significant increase of arginine residues (Table 1B). Strikingly, arginines were particularly frequent at the seventh position of the peptide (30%). In contrast, in the AAV-2 display library and the NB-AAV-pool, the frequency of R at this position (15% and 9%, resp...

Adeno-associated virus (AAV), a single-stranded DNA parvovirus, is emerging as one of the leading gene therapy vectors owing to its nonpathogenicity and low immunogenicity, stability and the potential to integrate site-specifically without known side-effects. A portfolio of recombinant AAV vector types has been developed with the aim of optimizing efficiency, specificity and thereby also the safety of in vitro and in vivo gene transfer. More and more information is now becoming available about the mechanism of AAV/host cell interaction improving the efficacy of recombinant AAV vector (rAAV) mediated gene delivery. This review summarizes the current knowledge of the infectious biology of AAV, provides an overview of the latest developments in the field of AAV vector technology and discusses remaining challenges.