Production and characterization of an AAV1-VP3-only capsid: An analytical benchmark standard

Figures

• 

  Figure 1. Expression of AAV1-VP3-only capsid (A) Schematic depiction of the AAV1 cap ORF showing VP1, VP2, and VP3. The start codons for VP1 and VP2 are knocked out. Two possible start codons corresponding to methionine 203 and 211 express VP3. These two forms are labeled as VP3M (major) and VP3m (minor) and are not distinguishable in a standard SDS-PAGE. Example cryo-electron micrograph of AAV1-VP3-only capsids. The percentage of empty capsids in the sample is given. Scale bar: 50 nm. (B) Depiction as in (A) for AAV1-M211V-VP3-only capsid.

• 

  Figure 2. Capsid structure of AAV1-M211V-VP3-only capsid (A) The capsid surface (left) and cross-sectional (right) density map determined by cryo-EM reconstruction are shown. The reconstructed maps are radially colored (blue to red) according to distance to the particle center, as indicated by the scale bar. The icosahedral 2-, 3-, and 5-fold axes are indicated. (B) Amino acid residues modeled for the βD strand are shown inside their density maps (in black). The amino acid residues are labeled and shown as stick representation and colored according to atom type: C = yellow, O = red, N = blue. (C) Superposition of AAV1-VP1/2/3, PDB: 3NG9 (purple), and AAV1-M211V-VP3-only (orange). The β barrel core motif (βB–βI), βA, and the αA helix are indicated. The VRs (VR-I to VR-IX) and the N and C termini are labeled, and the approximate icosahedral 2-, 3-, and 5-fold axes are represented by an oval, triangle, and pentagon, respectively.

• 

  Figure 3. Characterization of AAV1-VP3-only capsids by strong anion exchange liquid chromatography (A) Fluorescence chromatograms of intact AAV1-VP3 and AAV1-M211V-VP3 using a salt gradient. (B) Fluorescence trace of AAV1-VP3 and AAV1-M211V-VP3 samples from a pH gradient. (C and D) UV chromatograms of intact AAV1-VP3 (E and F) and AAV1-M211V-VP3 from salt and pH gradient respectively. The peak width at half height is given in each panel.

• 

  Figure 4. Mass spectrometry analysis of AAV1 capsids under denaturing conditions (A) RP-LC-MS and (B) deconvoluted spectra of AAV1-VP3 and AAV1-M211V-VP3. Each peak is labeled with the corresponding sequence variant. For example, aVP3-A204 indicates N termini starting at Ala204 with acetylation.

• 

  Figure 5. Native mass spectrometry analysis of AAV1 capsids (A) Ensemble native MS spectra and (B) deconvoluted spectra of intact AAV1-VP3, AAV1-M211V-VP3, and AAV1-WT (containing VP1, VP2, and VP3) particles using 25K @m/z 400 resolution (128 msec transient).

• 

  Figure 6. Calibration for Direct Mass Technology mode analysis Calibration coefficient profiles of (A) AAV1-VP3, (B) AAV1-M211V-VP3, and (C) AAV1-WT. (D–H) Individual ion spectra of calibrants for Direct Mass Technology mode and (I) established linear calibration curve out of the five calibrants. The calibration coefficient is 47,052.

• 

  Figure 7. Utilization of Direct Mass Technology Direct Mass Technology mode analysis of (A) AAV1-M211V-VP3, (B) AAV1-WT (containing VP1, VP2, and VP3), and (C) empty and (D) full AAV8 capsids.