Designer gene delivery vectors: molecular engineering and evolution of adeno-associated viral vectors for enhanced gene transfer

Abstract

Gene delivery vectors based on adeno-associated virus (AAV) are highly promising due to several desirable features of this parent virus, including a lack of pathogenicity, efficient infection of dividing and non-dividing cells, and sustained maintenance of the viral genome. However, several problems should be addressed to enhance the utility of AAV vectors, particularly those based on AAV2, the best characterized AAV serotype. First, altering viral tropism would be advantageous for broadening its utility in various tissue or cell types. In response to this need, vector pseudotyping, mosaic capsids, and targeting ligand insertion into the capsid have shown promise for altering AAV specificity. In addition, library selection and directed evolution have recently emerged as promising approaches to modulate AAV tropism despite limited knowledge of viral structure-function relationships. Second, pre-existing immunity to AAV must be addressed for successful clinical application of AAV vectors. "Shielding" polymers, site-directed mutagenesis, and alternative AAV serotypes have shown success in avoiding immune neutralization. Furthermore, directed evolution of the AAV capsid is a high throughput approach that has yielded vectors with substantial resistance to neutralizing antibodies. Molecular engineering and directed evolution of AAV vectors therefore offer promise for generating 'designer' gene delivery vectors with enhanced properties.

Fig. 1

Molecular engineering and evolution approaches have been applied to design AAV viral vectors for tropism change (A–D) and evasion of neutralizing antibodies (NABs) (C–F). A Mosaic capsids, composed of a mixture of capsid subunits from different serotypes, can possess the beneficial features of different serotypes. B Decoration of AAV capsids with either an antibody or a peptide targeting a cellular receptor can yield a targeted vector. C Pseudotyping the AAV2 genome with a capsid from a different serotype can “transfer” the desirable properties of the different serotype to AAV2. D Directed evolution of the AAV capsid, distinct from single round library generation and selection, involves multiple rounds of genetic diversification and selection. AAV libraries containing capsid variants are generated and subjected to selection for novel, diverse properties. Specific variants with desirable properties will be enriched and used for library generation of a next round of selection (denoted by the solid, curved arrows). E Disruption of the epitopes of AAV2 NABs allows evasion of antibody neutralization. F Conjugation of a synthetic polymer to AAV capsid can shield the AAV vector from NABs.