Mutations on the External Surfaces of Adeno-AssociatedVirus Type 2 Capsids That Affect Transduction andNeutralization

Abstract

Mutations were made at 64 positions on the external surface of the adeno-associated virus type 2 (AAV-2) capsid in regions expected to bind antibodies. The 127 mutations included 57 single alanine substitutions, 41 single nonalanine substitutions, 27 multiple mutations, and 2 insertions. Mutants were assayed for capsid synthesis, heparin binding, in vitro transduction, and binding and neutralization by murine monoclonal and human polyclonal antibodies. All mutants made capsid proteins within a level about 20-fold of that made by the wild type. All but seven mutants bound heparin as well as the wild type. Forty-two mutants transduced human cells at least as well as the wild type, and 10 mutants increased transducing activity up to ninefold more than the wild type. Eighteen adjacent alanine substitutions diminished transduction from 10-to 100,000-fold but had no effect on heparin binding and define an area (dead zone) required for transduction that is distinct from the previously characterized heparin receptor binding site. Mutations that reduced binding and neutralization by a murine monoclonal antibody (A20) were localized, while mutations that reduced neutralization by individual human sera or by pooled human, intravenous immunoglobulin G (IVIG) were dispersed over a larger area. Mutations that reduced binding by A20 also reduced neutralization. However, a mutation that reduced the binding of IVIG by 90% did not reduce neutralization, and mutations that reduced neutralization by IVIG did not reduce its binding. Combinations of mutations did not significantly increase transduction or resistance to neutralization by IVIG. These mutations define areas on the surface of the AAV-2 capsid that are important determinants of transduction and antibody neutralization.Adeno-associated virus type 2 (AAV-2) is a parvovirus belonging to the Dependovirus genus (16). AAV-2 has a linear, single-stranded DNA genome containing 4,679 nucleotides that encodes two genes called rep and cap. The rep gene encodes four proteins that play roles in viral DNA replication, DNA packaging, and the regulation of transcription and splicing. The cap gene encodes three proteins (VP1, VP2, and VP3) that package viral DNA and determine viral tropism. The external surface of the capsid is also the target for neutralizing antibodies. To date, about 180 different AAV capsids have been identified. The three capsid proteins are produced from the same translational reading frame by means of alternative splicing and translation initiation sites.The C termini, each of which is typically about 530 amino acids long, are identical and represent VP3. VP3 is the most abundant capsid protein, representing about 80% of the protein in an assembled capsid. VP2 is typically about 65 amino acids longer than VP3 at the amino terminus and is dispensable for most AAV-2 functions (51). VP1 is typically about 135 amino acids longer than VP2 at the amino terminus. This N-terminal domain is located inside the capsid (26) and encodes the phospholipase A2, which is required f...

Single Amino Acid Modification of Adeno-Associated Virus Capsid Changes Transduction and Humoral Immune Profiles

“…The second approach is to use error-prone PCR to generate a library of AAV variants and select for NAb escape mutants in the presence of NAb in vitro (31,41,56). The third approach is to randomly modify AAV capsids and to escape NAbs (2,36,37) and yield novel capsids with preferred tropism. The fourth approach is to use other types of AAV which have shown no or low NAb cross-reactivity in mice (27,30,38,60,65).…”

“…These findings present a concern to the gene therapy community as to how we can avoid antagonistic NAb activity during systemic delivery of AAV vectors. To overcome this concern, several approaches have been exploited in our lab and by other groups, including (i) the utilization of polymers to cover the AAV capsid and block NAb recognition (11,32,33), (ii) the development of NAb escape mutants by error-prone PCR in vitro (31,41,56), (iii) the application of other types of AAV vectors (27,30,38,60,65), and (iv) the generation of chimeric types via AAV shuffling (2,36,37). In this study, we have systematically explored the possibility of using different types and AAV mutants as alternative vectors for intramuscular gene delivery in mice preimmunized against different AAV types.…”

Immunity to adeno-associated virus vectors in animals and humans: a continued challenge

“…Mutations in the capsid can avoid neutralizing antibody responses. 68 A strategy employed by a number of groups has been to use site-directed mutagenesis to disrupt neutralizing epitopes on AAV capsids. 68,69 The most promising approach was used by Mahesri et al A large (410 6 ) AAV2 library was generated with randomly distributed capsid mutations.…”

“…68 A strategy employed by a number of groups has been to use site-directed mutagenesis to disrupt neutralizing epitopes on AAV capsids. 68,69 The most promising approach was used by Mahesri et al A large (410 6 ) AAV2 library was generated with randomly distributed capsid mutations. A highthroughput approach was then used to select for AAV variants with the ability to evade neutralizing antibodies by delivering genes in the presence of anti-AAV serum.…”

Structural Insights into Adeno-Associated Virus Serotype 5

“…The AAV capsid surface features that are unique to AAV5-shorter 3-fold protrusions, extended VR-VII, and smaller HI loop-contain amino acid residues or are proximate to capsid regions reported to play essential roles in the AAV life cycle. For example, residues within VR-IV, VR-V, and VR-VIII which make up the 3-fold protrusions have been reported to control glycan receptor attachment (VR-V and VR-VIII for AAV2 and VR-V for AAV9) (90)(91)(92)(93)122), transduction (VR-IV, VR-V, and VR-VIII in AAV2, VR-VIII in AAV8, and VR-IV, VR-V, and VR-VIII in AAV9) (94)(95)(96)(97)(98)(99) and antigenic phenotypes (VR-IV, VR-V, and VR-VIII in AAV2 and VR-VIII in AAV8 (97,100,101). These regions have similar roles in AAV5.…”