Designer Gene Delivery Vectors: Molecular Engineering and Evolution of Adeno-Associated Viral Vectors for Enhanced Gene Transfer

Abstract

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, preexisting immunity to AAV must be addressed for successful clinical application of AAV vectors. BShielding^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 Fdesigner_ gene delivery vectors with enhanced properties.

Off-target-free gene delivery by affinity-purified receptor-targeted viral vectors

“…However, libraries consisting of shuffled capsids typically expand, but do not redirect viral tropism and, in addition, do not allow directing the selection towards a particular chosen receptor. Similarly, insertion of peptides into the viral capsid as well as equipping AAV vectors non-genetically with targeting ligands without destruction or shielding of the natural receptor binding sites will result in an expanded tropism 4,24 . With regard to genetic targeting approaches candidate peptides are commonly selected from phage or AAV peptide display libraries, which-due to technical constraints-contain only short peptides [8][9][10]23,25 .…”

Evaluation of the specificity and sensitivity of ferritin as an MRI reporter gene in the mouse brain using lentiviral and adeno-associated viral vectors

“…23,24 As rAAVs can target neurons in the nervous system depending on the (hybrid) serotype, these vectors are valuable tools for CNS disease modeling, for the treatment of neurological disorders or for the study of nervous system biology. 24,25 Gene expression directed by rAAV transgene cassettes occurs mainly without integration into the host genome.…”

Successful target cell transduction of capsid-engineered rAAV vectors requires clathrin-dependent endocytosis

“…Although nongenetic cell surface targeting approaches to alter AAV's tropism have been described, genetic modification strategies are more frequently used. 1,3,4 In these approaches, peptide ligands are either inserted at the N-terminus of the viral capsid proteins (VP1, VP2) or at surfaceexposed positions within the capsid proteins 1,3,4 to mediate target receptor binding (for a review see Büning et al 1 ). To restrict vector tropism to the desired ligand-receptor interaction, natural receptor binding has to be eliminated at the same time.…”