Development of Patient-specific AAV Vectors After Neutralizing Antibody Selection for Enhanced Muscle Gene Transfer

Abstract

A major hindrance in gene therapy trials with adeno-associated virus (AAV) vectors is the presence of neutralizing antibodies (NAbs) that inhibit AAV transduction. In this study, we used directed evolution techniques in vitro and in mouse muscle to select novel NAb escape AAV chimeric capsid mutants in the presence of individual patient serum. AAV mutants isolated in vitro escaped broad patient-specific NAb activity but had poor transduction ability in vivo. AAV mutants isolated in vivo had enhanced NAb evasion from cognate serum and had high muscle transduction ability. More importantly, structural modeling identified a 100 amino acid motif from AAV6 in variable region (VR) III that confers this enhanced muscle tropism. In addition, a predominantly AAV8 capsid beta barrel template with a specific preference for AAV1/AAV9 in VR VII located at threefold symmetry axis facilitates NAb escape. Our data strongly support that chimeric AAV capsids composed of modular and nonoverlapping domains from various serotypes are capable of evading patient-specific NAbs and have enhanced muscle transduction.

Nab Escaping AAV Mutants Isolated from Mouse Muscles

“…The imaging was carried out by the IVIS Lumina In Vivo imaging system and the photon signal was measured by Living Image software. The results were shown in Figure 2 of the original paper (Li et al , 2016). …”

“…Clone the capsids from DNA shuffling library into wild-type AAV2 plasmid psub201 to generate AAV capsid DNA library (Li et al ., 2008; Li et al ., 2016). …”

High-Density Recombinant Adeno-Associated Viral Particles are Competent Vectors forIn VivoTransduction

“…50 AAV serotype switching and capsid engineering strategies have produced promising results. 51,52 Because the capsid composition is different between rAAV RD and rAAV HD particles, it can be hypothesized that humoral immunity may also have some difference. Immunoelectron microscopy studies indicated that rAAV RD and rAAV…”

Systemic delivery of adeno-associated viral vectors

“…This approach has allowed isolation of neutralizing antibody escaping AAV variants AAV-r2.15 by Maheshri et al and AAV-DJ by Grimm et al [127,128]. More recently, Li et al found that capsid variants isolated following in vitro selection in human serum had poor in vivo transduction strength although they were able to escape neutralization [129]. In vivo selection in the presence of the patient serum may yield escaping-capsids with better in vivo performance [129].…”