Materials

The modified AAV2, wild-type AAV2 and AAV9 vectors were manufactured at Novartis Gene Therapies. AAV5 and AAV8 vectors were purchased from SAB Tech Inc. (Philadelphia, PA). AAV2 capsid used throughout this article means the modified AAV2 capsid.

For RPLC-MS, water and acetonitrile were LC-MS grade and purchased from Honeywell Burdick&Jackson (Muskegon, MI). LC-MS grade difluoroacetic acid (DFA) was purchased from Waters (Milford, MA).

For SDS-PAGE and Western blot, NuPAGE 4–12% Bis-Tris Midi Protein Gels, NuPAGE MOPS SDS Running Buffer (20x), NuPAGE Sample Reducing Agent (10x), NuPAGE LDS Sample buffer (4x), SeeBlue Plus2 Pre-stained protein ladder, iBind Solution Kit (including iBind buffer and 100x additive) were all purchased from Thermo Fisher Scientific (Waltham, MA). Primary antibodies targeting the shared VP C-terminus (Progen 690058) and targeting a VP1-unique region (Progen 61056) were purchased from Progen (Wayne, PA). Secondary antibody goat anti-mouse was purchased from LICOR (Lincoln, NE). Blots were imaged on a LICOR Odyssey DLx imager (Lincoln, NE).

For potency assay, the parental AAV293 cells were purchased from Agilent (Santa Clara, CA). A clonal AAV293 cell line was developed by Novartis Biomedical Research (Cambridge, MA). The primary transgene specific mouse antibody was purchased from Millipore Sigma (Burlington, MA), the secondary antibody, Goat Anti-Mouse IgG H&L (Alexa Fluor® 488 conjugated) was purchased from Abcam (Cambridge, MA), and Hoechst 33342 Nuclear Dye was purchased from Invitrogen (Carlsbad, CA).

MethodsProteolytic digestion of samples

All samples were kept at ≤ −60 oC prior to proteolytic digestion. The proteolytic digestion was carried out by adding the proteolytic enzymes at the ratio of protease:AAV capsid of 1:5 (w/w) in the samples for 10, 30, 60 min, 1 hr, 2 hr, or overnight at room temperature.

Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot

To prepare samples for SDS-PAGE and Western blot, 10x reducing agent and 4xLDS sample buffer were added to untreated control AAV2 and trypsin-treated AAV2 samples to a final concentration of 1x reducing agent and 1xLDS sample buffer. The sample mixture was heated at 70 °C for 10 min, then removed from heat and loaded onto an SDS-PAGE gel. The gel was run in 1x MOPS SDS running buffer, and then stained with Coomassie blue for SDS-PAGE analysis.

For Western blot, upon completion of electrophoresis, the proteins were transferred to PVDF membrane with the iBlot 2 PVDF stack and iBlot 2 Gel Transfer Device. The blot was cut vertically into strips and blocked with iBind buffer for about an hour. Antibodies (2.5 mL of Progen 690058 targeting the shared VP C-terminus, 1:333; 2.5 mL of Progen 61056 targeting a VP1-unique region, 1:500; 5 mL of goat anti-mouse, LICOR IRDye 680 RD; 1:3000) were prepared in blocking buffer. Blotting was performed with an iBind Western Device and iBind Flex card following the manufacturer’s instruction and left overnight. Approximately 18 h later, the blots were imaged with a LICOR Odyssey DLx imager.

Capillary electrophoresis sodium dodecyl sulfate (CE-SDS)

CE-SDS analysis was performed using a PA800 Plus Pharmaceutical Analysis CE system equipped with a photodiode array (PDA) detector and SDS-MW Analysis kit from Sciex, (Framingham, MA) to determine the AAV capsid protein purity by comparing the peak areas of viral proteins and related impurities under denatured and reduced conditions [11]. The reagents used and general procedure were adapted from a Sciex Application Note [12]. Briefly, AAV samples were first buffer exchanged into SDS-MW Sample Buffer from the SDS-MW Analysis kit using Amicon Ultra-0.5 centrifugal filters (EMD-Millipore, Burlington, MA) to reduce salt concentration. The capsids were then denatured at 60 °C for 10 min and reduced with 2-mercaptoethanol (Thermo Fisher Scientific, Waltham, MA). Benzonase (Millipore Sigma, Burlington, MA) was added to remove any residual free DNA and DNA from disrupted AAV capsids by incubating the sample with Benzonase at 37 °C for 30 min prior to CE-SDS analysis. The user guide of the SDS-MW Analysis kit [13] was followed for instrument set up.

Reversed-phase liquid chromatography-mass spectrometry (RPLC-MS)

A Thermo ScientificTM Vanquish UHPLC system (Thermo Fisher Scientific) coupled with Thermo ScientificTM Orbitrap Eclipse™ Tribrid™ Mass Spectrometer (Thermo Fisher Scientific) were used for the reversed-phase liquid chromatography-tandem mass spectrometry (RPLC-MS). AAV2 capsid proteins were separated on an ACQUTY UPLC Protein BEH C4 column, 1.7 µm, 300 Å, 2.1 ×150 mm (Waters) with a column temperature of 80 °C, using mobile phase A of 0.1% difluoroacetic acid in water and mobile phase B of 0.1% difluoroacetic acid in acetonitrile at a flow rate of 0.2 mL/min. The proteins were eluted with a gradient from 20–32% mobile phase B over 2 min, then 32–33.5% mobile phase B over 21 min, 33.5–80% mobile phase B over 6 min, then a hold at 80% mobile phase B for 3 min before changing mobile phase B back to 20%. The total run time was 45 min.

Mass spectrometry data were acquired on an Orbitrap Eclipse™ Tribrid™ Mass Spectrometer fitted with a heated electrospray ionization source (HESI) (Thermo Fisher Scientific). The spray voltage set was static at 3.5 kV. Sheath gas flow rate was set at 35 arb, Auxiliary gas flow rate at 7 arb, and Sweep gas at 0 arb. Ion transfer tube temperature was 300 °C and vaporizer temperature was 275 °C. Orbitrap was used for detection and its resolution was 15,000 at 200 m/z. Scan range was set at 700–3000 m/z and RF Lens at 70%. Normalized AGC target was set at 300% and maximum injection time was set at 200 ms. Source fragmentation energy was 20 V. The pressure mode used was standard pressure.

Mass spectra data analysis

The theoretical average masses of the wild-type AAV2 VP1, VP2, and VP3 are based on its protein sequence from UniProt P03135. Theoretical average masses of the modified AAV2 VP1, VP2, and VP3, for which 10 amino acids were inserted between N587 and R588 (VP1 numbering) in the C-terminal shared region of all VPs in the wild-type AAV2, were based on the values from General Protein Mass Analysis for Windows software (GPMAW, version 12). The loss of the initial methionine residue followed by acetylation of the following alanine residue was reported [31] and was also confirmed in AAV capsid proteins VP1 and VP3 [7, 18]. The loss of the initial amino acid, threonine residue of VP2 is confirmed in the same paper [7, 18]. Therefore, the theoretical masses of AAV2 VP1 and VP3 are calculated with the consideration of methionine loss followed by acetylation of the following alanine residue, and the theoretical mass of AAV2 VP2 is calculated with the consideration of the loss of the initial threonine.

Thermo Scientific BioPharma Finder software v4.0 was used for intact mass spec data analysis to determine observed masses of AAV capsid proteins. Thermo Scientific™ ProSightPD™ software v4.2 was used for top-down proteomics data analysis to identify the cutting sites of AAV capsids by trypsin. The variable modifications set included: oxidation (+ 15.99492 Da) of methionine (M) and tryptophan (W), deamidation (+ 0.98402 Da) of asparagine (N) and glutamine (Q), N-terminal methionine residue loss followed by acetylation of the following alanine residue of AAV2 VP1 and VP3 (−89.02992 Da). The deconvolution mass tolerance was 20 ppm.

Potency assay

The clonal AAV293 cells were plated in a 96-well plate for 24 h prior to transduction with the AAV2 capsid. Five concentrations of the trypsin-treated and untreated AAV2 samples were prepared in triplicate. The cells in 96-well plates were transduced with trypsin-treated, untreated AAV2, and untransduced cells-only control (no AAV2 vector), then incubated for 72 h. At 72 h, cells were fixed, permeabilized, and incubated with the primary transgene specific mouse antibody for 2 h and followed by incubating with the secondary anti-mouse Alexa Fluor® 488 antibody and Hoechst 33342 Nuclear Dye for 1 h. The stained cells were then imaged using a high content imaging system to measure dose-dependent protein expression. Relative potency was calculated using parallel line analysis.

Size exclusion chromatography (SEC)

A Waters ACQUITY H-Class UPLC system with a fluorescence detector and an SRT-5 SEC-1000 HPLC column (4.6 × 300 mm, 1000 Å pore size, 5 µm) from Sepax Technologies (Newark, DE) were used for SEC analysis. The untreated control AAV2 sample and the trypsin-treated AAV2 sample at 1E13 vg/mL (20 µL) were loaded on the SEC HPLC column at 25 °C with the flow rate of 0.2 min/mL. The sample was eluted isocratically with mobile phase composition of 0.1 M sodium phosphate, 0.5 M sodium chloride, 0.002 M magnesium chloride, pH 7.0. AAV2 capsid was detected with fluorescence at the wavelength of λex = 280 nm and λem = 330 nm. The total run time was 60 min.

Thermostability evaluation

An Uncle instrument from Unchained Labs (Pleasanton, CA) was used for thermostability evaluation. The 10,000x SYBR™ Gold Nucleic Acid Gel Stain in DMSO from Thermo Fisher Scientific (Waltham, Massachusetts) was used to detect the DNA leakage of AAV2 capsids. An equal volume (8.8 µL) of the untreated control AAV2 capsids and the trypsin-treated AAV2 capsids at 1E13 vg/mL were loaded per well. For both capsid protein thermostability and genome ejection experiments, the temperature gradient started with 180 s initial incubation at 25 °C before temperature increasing at 0.6 °C/min ramp rate from 25 °C to 95 °C. For the capsid protein thermostability experiment, the detector settings were UV-266 Filter 1 (0.50, blocking 50% light source), Blue Laser Filter 3 (0.25, blocking 25% light source), and exposure time 1500 ms. For the genome ejection experiment using SYBR gold, detector settings were UV-266 Opaque (0.00), Blue Laser Filter 3 (0.25), and exposure time 1500 ms; 10,000x SYBR Gold in DMSO was diluted to 400x in formulation buffer and 2.67 µL of 400x SYBR Gold was added into 24.03 µL of AAV2 sample.

Cryo-electron microscopy (cryo-EM)

For cryo-EM, sample was applied to glow-discharged GOQ400R1213 grids (EMS), which were plunge frozen using a Thermo Fisher Vitrobot (8 s blot, 20 force). Data were collected using EPU software on a Glacios (Thermo Fisher Scientific). Movies were recorded using a Falcon 3 in counting mode with a physical pixel size of 0.86 Å and a dose rate of 1.21 e-/pixel/frame. Each movie was a stack of 29 subframes making the total dose ~35 e-/Å2. Defocus values ranged between −0.8 and −2.0 µm.

Over 2530 movies were collected on the trypsin-treated AAV2 capsids. Movies were aligned and summed using Relion 4 motion correction, then imported into CryoSPARC where subsequent processing steps were carried out. The final 2D classification consisted of 50,621 individual capsid particles.

AAV capsid protein three-dimensional structural analysis

For the three-dimensional (3D) structural analysis, the modified AAV2 protomer was generated with AlphaFold 2 [32]. The structural analysis was conducted in Chimera 1.16 [33]. The modified AAV2 capsid was assembled by applying symmetry operators in Chimera. To compare the experimentally identified trypsin cleavage sites, the structure of the modified AAV2 protomer was overlaid with the following AAV protomer structures; AAV2 (PDB:1LP3), AAV5 (PDB: 7KP3), AAV8 (PBD: 2QA0), AAV9 (PBD: 3UX1).