Engineering and Selection of Shuffled AAV Genomes: A New Strategy for Producing Targeted Biological Nanoparticles

Abstract

We report a DNA shuffling-based approach for developing cell type-specific vectors through directed evolution. Capsid genomes of adeno-associated virus (AAV) serotypes 1-9 were randomly fragmented and reassembled using PCR to generate a chimeric capsid library. A single infectious clone (chimeric-1829) containing genome fragments from AAV1, 2, 8, and 9 was isolated from an integrin minus hamster melanoma cell line previously shown to have low permissiveness to AAV. Molecular modeling studies suggest that AAV2 contributes to surface loops at the icosahedral threefold axis of symmetry, while AAV1 and 9 contribute to two- and fivefold symmetry interactions, respectively. The C-terminal domain (AAV9) was identified as a critical structural determinant of melanoma tropism through rational mutagenesis. Chimeric-1829 utilizes heparan sulfate as a primary receptor and transduces melanoma cells more efficiently than all serotypes. Further, chimeric-1829 demonstrates altered tropism in rodent skeletal muscle, liver, and brain including nonhuman primates. We determined a unique immunological profile based on neutralizing antibody (NAb) titer and crossreactivity studies strongly supporting isolation of a synthetic laboratory-derived capsid variant. Application of this technology to alternative cell/tissue types using AAV or other viral capsid sequences is likely to yield a new class of biological nanoparticles as vectors for human gene transfer.

Immune responses to adeno-associated virus type 2 encoding channelrhodopsin-2 in a genetically blind rat model for gene therapy

“…Intra-vitreous injection caused relatively little antibody production ( Figure 7a) compared with the different route of AAV injection used by Zhang et al 16 Li et al 40 demonstrated that a single intra-vitreous injection of AAV2 causes increased AAV antibody production at 2 months after injection. Our data are consistent with their report in terms of the timing of increase and the amount of antibody production (Figure 7a).…”

Neutralizing antibodies against adeno-associated virus examined prospectively in pediatric patients with hemophilia

“…This understanding may further inform AAV vector technologies such as the use of rational capsid design or DNA shuffling to generate capsid mutants to escape antibody neutralization and enhance transduction. 37,38 One such mutant designed in our lab has been used in patients with Duchenne muscular dystrophy in a Phase I human clinical trial. 39 …”

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

“…[1][2][3][4][5][6] Capsids of adeno-associated virus (AAV)-derived vectors have been modified for receptor targeting basically in three ways: (1) by conjugating receptorbinding ligands to their capsids, (2) by genetic insertion of ligands into the capsid (for reviews see Muzyczka and Warrington 1 and Buning et al 7 ) and (3) by DNA shuffling from different AAV serotypes. [8][9][10] Pseudotyping of AAV vectors with capsids of different serotypes also expanded the range of infectable cells, 11 but has not resulted in cell type-selective transduction after systemic application.…”

“…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.…”

“…A major step forward to improve the target peptide design has been the development of virus display peptide libraries [33][34][35][36][37] or libraries of random capsid chimeras derived from different AAV serotypes. [8][9][10] In such libraries, a multitude of potentialtargeting motifs is presented within the restrictions of capsid assembly and genome packaging. Selection of targeting motifs occurs by amplification of viruses, which have successfully delivered their genomes to the target cell, for example by using not only the right entry receptor but also the appropriate post-entry pathway.…”