Deamidation of Amino Acids on the Surface of Adeno-Associated Virus Capsids Leads to Charge Heterogeneity and Altered Vector Function

Abstract

Post-translational modification of the adeno-associated virus capsids is a poorly understood factor in the development of these viral vectors into pharmaceutical products. Here we report the extensive capsid deamidation of adeno-associated virus serotype 8 and seven other diverse adeno-associated virus serotypes, with supporting evidence from structural, biochemical, and mass spectrometry approaches. The extent of deamidation at each site depended on the vector's age and multiple primary-sequence and three-dimensional structural factors. However, the extent of deamidation was largely independent of the vector recovery and purification conditions. We demonstrate the potential for deamidation to impact transduction activity and, moreover, correlate an early time point loss in vector activity to rapidly progressing spontaneous deamidation at several adeno-associated virus 8 asparagines. We explore mutational strategies that stabilize side-chain amides, improving vector transduction and reducing the lot-to-lot molecular variability that presents a key concern in biologics manufacturing. This study illuminates a previously unknown aspect of adeno-associated virus capsid heterogeneity and highlights its importance in the development of these vectors for gene therapy.

Keywords: adeno-associated virus; bioengineering; gene therapy; post-translational modification; structural biology; virus structure.

Figure 1

Electrophoretic Analysis of AAV8 VP Isoforms (A) Diagram illustrating the mechanism by which asparagine residues undergo nucleophilic attack by adjacent nitrogen atoms, forming a succinimidyl intermediate. This intermediate then undergoes hydrolysis, resolving into a mixture of aspartic acid and isoaspartic acid. The beta carbon is labeled as such. The diagram was generated in BIOVIA Draw 2018. (B) 1 μg of AAV8 vector was run on a denaturing one-dimensional SDS-PAGE. (C) Isoelectric points of carbonic anhydrase pI marker spots are shown. (D) 5 μg of AAV8 vector was analyzed by two-dimensional gel electrophoresis and stained with Coomassie blue. Spots 1–20 are carbamylated carbonic anhydrase pI markers. Boxed regions are as follows: a, VP1; b, VP2; c, VP3; and d, internal tropomyosin marker (arrow: tropomyosin spot of molecular weight [MW] = 33kDa, pI = 5.2). Isoelectric focusing was performed with a pI range of 4–8. 1 × 10¹¹ GC of WT AAV8 (E) or mutant (F, N255D; and G, N517D) vector were analyzed by 2D gel electrophoresis and stained with SYPRO Ruby. Protein labeling: A, VP1; B, VP2; C, VP3; D, chicken egg white conalbumin marker; E, turbonuclease marker. Isoelectric focusing was performed with a pI range of 6–10. Primary VP1/2/3 isoform spots are circled, and migration distances of major spots of markers are indicated by vertical lines (turbonuclease, dashed; conalbumin, solid).

Figure 2

Analysis of Asparagine and Glutamine Deamidation in AAV8 Capsid Proteins (A and B) Electrospray ionization (ESI) mass spectrometry and theoretical and observed masses of the 3+ peptide (93–103) containing Asn-94 (A) and Asp-94 (B) are shown. (C and D) ESI mass spectrometry and theoretical and observed masses of the 3+ peptide (247–259) containing Asn-254 (C) and Asp-254 (D) are shown. The observed mass shifts for Asn-94 and Asn-254 were 0.982 and 0.986 Da, respectively, versus a theoretical mass shift of 0.984 Da. (E) Percent deamidation at specific asparagine and glutamine residues of interest are shown for AAV8 tryptic peptides purified by different methods. Bars indicating deamidation at asparagine residues with N+1 glycines are crosshatched. Residues determined to be at least 2% deamidated in at least one prep analyzed were included. Data are represented as mean ± SD.

Figure 3

Structural Modeling of the AAV8 VP3 Monomer and Analysis of Deamidated Sites (A) The AAV8 VP3 monomer (PDB: 3RA8) is shown in a coil representation. The color of the ribbon indicates the relative degree of flexibility (blue = most rigid/normal temperature factor; red = most flexible/high temperature factor). Spheres indicate residues of interest. Expanded diagrams are ball-and-stick representations of residues of interest and their surrounding residues to demonstrate local protein structure (blue, nitrogen; red, oxygen). Underlined residues are those in NG motifs. (B–E) Isoaspartic models of deamidated asparagines with N+1 glycines are shown. The 2FoFc electron density map (1 sigma level) generated from refinement of the AAV8 crystal structure (PDB: 3RA8) with (B) an asparagine model of N410 in comparison with isoaspartic acid models of (C) N263, (D) N514, and (E) N540. Electron density map is shown in the magenta grid. All atoms are colored by atom type: carbon, purple (B)/gree6n (C–E); nitrogen, blue; oxygen, pink. The beta carbon is labeled as such. The arrows indicate electron density corresponding to the R group of the residue of interest.

Figure 4

In Vitro Analysis of the Impact of Genetic Deamidation on Vector Performance (A) Titers of WT AAV8 and genetic deamidation mutant vectors were produced by small-scale triple transfection in 293 cells, as measured by qPCR. Titers are reported relative to the WT AAV8 control. NG sites with high deamidation (patterned bars), sites with low deamidation (white bars), and highly variable sites (black bars) are presented with WT AAV8 and a negative control. (B) The transduction efficiency of mutant AAV8 vectors producing firefly luciferase is reported relative to the WT AAV8 control. Transduction efficiency was measured in luminescence units generated per GC added to HUH7 cells and was determined by performing transductions with crude vector at multiple dilutions. Transduction efficiency data were normalized to the WT reference. All data are represented as mean ± SD.

Figure 5

Vector Activity Loss through Time Is Correlated to Progressive Deamidation (A) Vector production (DNase I-resistant GCs) for a time course of triple-transfected HEK293 cells producing AAV8 vector packaging a luciferase reporter gene. GC levels are normalized to the maximum observed value. (B) Purified time-course vector was used to transduce Huh7 cells. Transduction efficiency (luminescence units per GC added to target cells) was measured as in Figure 4 using multiple dilutions of purified time-course vector samples. Error bars represent the SD of at least 10 technical replicates for each sample time. Deamidation of AAV8 NG sites (C) and non-NG sites (D) for vector collected 1, 2, and 5 days post-transfection.

Figure 6

The Impact of Stabilizing Asparagines on Vector Performance (A) Titers of WT AAV8 and +1 position mutant vectors were produced by small-scale triple transfection in 293 cells, as measured by qPCR. Titers are reported relative to the WT AAV8 control. (B) The transduction efficiency of mutant AAV8 vectors producing firefly luciferase is reported relative to the WT AAV8 control. Transduction efficiency was measured as in Figure 4 using crude vector material. (C) Luciferase expression on day 14 of the study period in the liver region from C57BL/6 mice injected intravenously with WT AAV8 or mutant vectors (n = 3–5) was measured by luciferase imaging and reported in total flux units. (D) The titers and transduction efficiency of multi-site AAV8 mutant vectors producing firefly luciferase are reported relative to the WT AAV8 control. All data are represented as mean ± SD. A two-sample t test (^∗p < 0.005) was run to determine significance between WT AAV8 and mutant transduction efficiency for G264A/G515A and G264A/G541A.

Figure 7

Functional Asparagine Substitutions at Non-NG Sites with High Variability between Lots (A) Titers of WT AAV8 and mutant vectors were produced by small-scale triple transfection in 293 cells, as measured by qPCR. Transduction efficiencies for crude vector material were measured as described in Figure 4. Titers and transduction efficiencies were normalized to the value of the WT AAV8 control. (B) Representative luciferase images at day 14 post-injection are shown for mice receiving WT AAV8.CB7.ffluc and N499Q capsid mutant vector. (C) Luciferase expression on day 14 of the study period from C57BL/6 mice injected intravenously with WT AAV8 or mutant vectors (n = 3 or 4) was measured by luciferase imaging and reported in total flux units. WT control shared with Figure 6C. All data are represented as mean + SD.