Genetic capsid modifications allow efficient re-targeting of adeno-associated virus type 2

Abstract

The human parvovirus adeno-associated virus type 2 (AAV2) has many features that make it attractive as a vector for gene therapy. However, the broad host range of AAV2 might represent a limitation for some applications in vivo, because recombinant AAV vector (rAAV)-mediated gene transfer would not be specific for the tissue of interest. This host range is determined by the binding of the AAV2 capsid to specific cellular receptors and/or co-receptors. The tropism of AAV2 might be changed by genetically introducing a ligand peptide into the viral capsid, thereby redirecting the binding of AAV2 to other cellular receptors. We generated six AAV2 capsid mutants by inserting a 14-amino-acid targeting peptide, L14, into six different putative loops of the AAV2 capsid protein identified by comparison with the known three-dimensional structure of canine parvovirus. All mutants were efficiently packaged. Three mutants expressed L14 on the capsid surface, and one efficiently infected wild-type AAV2-resistant cell lines that expressed the integrin receptor recognized by L14. The results demonstrate that the AAV2 capsid tolerates the insertion of a nonviral ligand sequence. This might open new perspectives for the design of targeted AAV2 vectors for human somatic gene therapy.

Heart-targeted adeno-associated viral vectors selected by in vivo biopanning of a random viral display peptide library

“…Genetic modification of the AAV2 capsid by oligonucleotide insertions, random mutagenesis or DNA shuffling is able to circumvent some of these limitations. [8][9][10][17][18][19] Whereas larger peptides up to the size of GFP (238 amino acids) could be accommodated at the VP2 N-terminus 15,[20][21][22][23][24][25] the addition of small peptides up to 34 amino acids is tolerated at several positions of the AAV2 capsid. 1 Among the tolerated insertion sites for small peptides, insertions in the AAV2 heparin binding domain 26,27 adjacent to amino acids 587 or 588 was most successful.…”

“…1 Among the tolerated insertion sites for small peptides, insertions in the AAV2 heparin binding domain 26,27 adjacent to amino acids 587 or 588 was most successful. 17,23,[28][29][30][31][32] An inherent problem of genetic modification of the AAV capsid is the design of the targeting peptide. Our knowledge about cell surface receptors and their ligands is incomplete, and the toleration and performance of ligands identified by phage display are so far not predictable within the constraints of the AAV capsid structure, although it was successful in several instances.…”

Intracellular trafficking of adeno-associated viral vectors

“…15,16 These AAV serotypes share a common genome structure, but have varying abilities to infect different cell types and tissue based on their capsid protein recognition by cell surface receptors. The repertoire of rAAV vectors has also been greatly expanded by the development of technologies to pseudo-package rAAV genomes, [17][18][19][20] package AAV genomes with two different ITR serotypes, 21 generate mosaic rAAV particles with more than one capsid serotype, [22][23][24] retarget AAV by generating rAAV capsid modification [25][26][27][28][29] and generate rAAV with chemically modified capsids. 30 These technologies have greatly expanded the ability to tailor rAAV for specific applications in gene therapy.…”

Gene therapy in the CNS

“…16,[54][55][56][57] Strategies to more specifically target AAV transduction include the incorporation of nonviral ligand sequences into the AAV capsid, and the use of bispecific antibodies to target specific cellular receptors. 58,59 Pharmacological regulation of gene expression with AAV vectors has been obtained when regulatory elements and drug-responsive promoters are included. 60 For example, recent studies utilized a transgene under the control of a recombinant promoter that requires a reconstituted dimeric transcription factor complex to become activated.…”