Single amino acid modification of adeno-associated virus capsid changes transduction and humoral immune profiles

Abstract

Adeno-associated virus (AAV) vectors have the potential to promote long-term gene expression. Unfortunately, humoral immunity restricts patient treatment and in addition provides an obstacle to the potential option of vector readministration. In this study, we describe a comprehensive characterization of the neutralizing antibody (NAb) response to AAV type 1 (AAV1) through AAV5 both in vitro and in vivo. These results demonstrated that NAbs generated from one AAV type are unable to neutralize the transduction of other types. We extended this observation by demonstrating that a rationally engineered, muscle-tropic AAV2 mutant containing 5 amino acid substitutions from AAV1 displayed a NAb profile different from those of parental AAV2 and AAV1. Here we found that a single insertion of Thr from AAV1 into AAV2 capsid at residue 265 preserved high muscle transduction, while also changing the immune profile. To better understand the role of Thr insertion at position 265, we replaced all 20 amino acids and evaluated both muscle transduction and the NAb response. Of these variants, 8 mutants induced higher muscle transduction than AAV2. Additionally, three classes of capsid NAb immune profile were defined based on the ability to inhibit transduction from AAV2 or mutants. While no relationship was found between transduction, amino acid properties, and NAb titer or its cross-reactivity, these studies map a critical capsid motif involved in all steps of AAV infectivity. Our results suggest that AAV types can be utilized not only as templates to generate mutants with enhanced transduction efficiency but also as substrates for repeat administration.

Fig 1

In vivo evaluation of NAb cross-reactivity among types 1 to 5. (A) AAV2 NAb against other types. Type AAV1 to -5/FIX vectors were injected into AAV2/GFP-pretreated mice. At 1 week postinjection, the FIX level in the serum was measured. The data represents the average ± standard deviation (SD) from 4 mice. (B) Other AAV types induce NAb against AAV2 vectors. The mice were initially injected with AAV/F9 vectors of types 1 to 5, and 2 months later AAV2/AAT vectors were administered. AAT concentrations were determined at week 6 after injection of AAV2/AAT vectors. The data represent the average from 4 mice. On the x axis, that the first number represents which type of AAV vector was used for primary immunization, while the second number represents the AAV type used for the following injection (e.g., AAV2/1 means that mice were injected with AAV2 vector followed by AAV1). Zero means no injection.

Fig 2

Transgene expression from AAV2 mutants following muscular injection. Mice were injected intramuscularly with1 × 10¹⁰ particles of AAV/luciferase. Four weeks later, images were taken and the total photons (luciferase activity) were calculated as described in Materials and Methods. The data represents the average from 4 mice.

Fig 3

Transgene expression with AAV2 265 insertion mutants following muscular injection. AAV2/luciferase mutants with the indicated single amino acid insertion at residue 265 were injected into mouse muscle at 1 × 10¹⁰ particles. The imaging was taken at 4 weeks postinjection, and luciferase activity was quantitated as described in Materials and Methods.

Fig 4

NAb analysis of immunization of AAV2 265 insertion mutants. Mice were administered 1 × 10¹⁰ particles of AAV2 265 insertion mutants via muscular injection. One month later, the pooled sera were collected from three mice and NAb assay was performed. (A) Sera from AAV2-immunized mice. (B) Sera from mice immunized with the indicated AAV2 265 insertion mutants.