Mutational Analysis of the Adeno-Associated Virus Type 2 (AAV2) Capsid Gene and Construction of AAV2 Vectors with Altered Tropism

Abstract

Adeno-associated virus type 2 (AAV2) has proven to be a valuable vector for gene therapy. Characterization of the functional domains of the AAV capsid proteins can facilitate our understanding of viral tissue tropism, immunoreactivity, viral entry, and DNA packaging, all of which are important issues for generating improved vectors. To obtain a comprehensive genetic map of the AAV capsid gene, we have constructed 93 mutants at 59 different positions in the AAV capsid gene by site-directed mutagenesis. Several types of mutants were studied, including epitope tag or ligand insertion mutants, alanine scanning mutants, and epitope substitution mutants. Analysis of these mutants revealed eight separate phenotypes. Infectious titers of the mutants revealed four classes. Class 1 mutants were viable, class 2 mutants were partially defective, class 3 mutants were temperature sensitive, and class 4 mutants were noninfectious. Further analysis revealed some of the defects in the class 2, 3, and 4 mutants. Among the class 4 mutants, a subset completely abolished capsid formation. These mutants were located predominantly, but not exclusively, in what are likely to be ␤-barrel structures in the capsid protein VP3. Two of these mutants were insertions at the N and C termini of VP3, suggesting that both ends of VP3 play a role that is important for capsid assembly or stability. Several class 2 and 3 mutants produced capsids that were unstable during purification of viral particles. One mutant, R432A, made only empty capsids, presumably due to a defect in packaging viral DNA. Additionally, five mutants were defective in heparan binding, a step that is believed to be essential for viral entry. These were distributed into two amino acid clusters in what is likely to be a cell surface loop in the capsid protein VP3. The first cluster spanned amino acids 509 to 522; the second was between amino acids 561 and 591. In addition to the heparan binding clusters, hemagglutinin epitope tag insertions identified several other regions that were on the surface of the capsid. These included insertions at amino acids 1, 34, 138, 266, 447, 591, and 664. Positions 1 and 138 were the N termini of VP1 and VP2, respectively; position 34 was exclusively in VP1; the remaining surface positions were located in putative loop regions of VP3. The remaining mutants, most of them partially defective, were presumably defective in steps of viral entry that were not tested in the preliminary screening, including intracellular trafficking, viral uncoating, or coreceptor binding. Finally, in vitro experiments showed that insertion of the serpin receptor ligand in the N-terminal regions of VP1 or VP2 can change the tropism of AAV. Our results provide information on AAV capsid functional domains and are useful for future design of AAV vectors for targeting of specific tissues.

Chromatographic purification of recombinant adenoviral and adeno-associated viral vectors: methods and implications

“…Targeting of several cell-specific receptors by inserting corresponding ligands into the virus capsid proteins resulted in the enhancement of cell transduction efficiency. 84,85 Pseudotyping rAAV vector, that is, using an alternative serotype capsid to package the rAAV vector genome, can also substantially increase the vector transduction efficiency and broaden the host range for rAAV-mediated gene transfer (reviewed by Flotte). 9 Utilization of emerging alternative serotypes and receptor targeted capsid mutants demands the developPurification of adenoviral and AAV vectors E Burova and E Ioffe ment of efficient methods for their production and purification.…”

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.…”

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.…”