Abstract
Recombinant adeno-associated viral (AAV) vectors have emerged as prominent gene delivery vehicles for gene therapy. In the journey of an AAV vector, AAV vectors can be exposed to different proteolytic environments inside the production cells, during the cell lysis step, within the endosome, and finally inside the cell nucleus. The stability of a modified AAV serotype 2 (AAV2) capsid was evaluated via a proteolytic approach using trypsin and other proteases and both denaturing and non-denaturing analytical methods. Trypsin digestion of the AAV2 capsids resulted in clips of the capsid proteins at the C-terminus as confirmed by denaturing methods including SDS-PAGE, CE-SDS, Western blot, and RPLC-MS. It was found that the AAV2 capsid with clips not only remains structurally intact, as confirmed by non-denaturing methods including SEC, thermostability testing, and cryo-EM, but also remains potent, as confirmed in a cell-based potency assay. This finding reveals that AAV2 capsid with proteolytic cuts remains intact and potent since the icosahedral three-dimensional structural arrangement of AAV capsid proteins can protect the clipped fragment from being released from the capsid, such that the AAV capsid remains intact allowing for the functionality to be maintained to deliver the DNA in the host cell. Evaluation of AAV stability using a proteolytic approach and multiple denaturing and non-denaturing analytical methods can provide valuable information for engineering AAV capsids to develop AAV-based gene therapy.
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Introduction
Recombinant adeno-associated viral (AAV) vectors, known for their nonpathogenic nature and the ability to enable sustained gene expression, have emerged as prominent gene delivery vehicles to treat a variety of human diseases [1, 2]. An AAV is a small nonenveloped virus with a single-stranded DNA genome that is encapsidated in an icosahedral protein capsid shell [3, 4]. The AAV icosahedral capsid is formed by three viral proteins, VP1, VP2, and VP3 at an approximate 1:1:10 ratio [5]. The three capsid proteins are N-terminal splice variants that share a common C-terminus. VP1 is the full-length protein, VP3 is the shortest protein, and VP2 is the protein with size between VP1 and VP3 [5, 6].
There are more than 10 AAV serotypes that share 51–99% capsid amino acid sequence homology [1, 5, 7]. AAV capsid proteins are pivotal in defining tissue specificity and immunogenicity and play significant roles in facilitating receptor binding, aiding the virus in escaping the endosome, and in the transport of viral DNA to the nucleus [8]. As of October 2024, among all serotypes, AAV serotype 2 (AAV2) has been predominantly utilized in gene therapy development according to clinicaltrials.gov. Two AAV2-based gene therapy products, Luxturna and Upstaza, have been commercialized in 2017 and 2022 for the treatment of Retinal dystrophy (RPE65 gene) and Aromatic L-amino acid decarboxylase (AADC) deficiency, respectively.
During production and administration, AAV vectors encounter various proteolytic environments that include intracellular synthesis, cell lysis step, which precedes downstream purification, as well as within the endosome after entering the target cell, then DNA delivery in cell nucleus. Therefore, evaluation of AAV stability using a proteolytic approach and both denaturing and non-denaturing analytical methods can provide valuable insight for engineering AAV vectors for AAV-based gene therapy development.
Assessing viral stability for AAV-based gene therapy is a complex task and requires evaluation of whole viral particles, capsid proteins, and viral genomes. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and capillary electrophoresis sodium dodecyl sulfate (CE-SDS) are denaturing methods for the measurement of protein size and impurities. CE-SDS is widely used for evaluation of the ratios of VP1:VP2:VP3 of AAV capsid proteins and purity [9,10,11,12,13,14,15,16]. Western blot is a denaturing method used for the identification of specific protein based on its antibody binding. The denaturation of proteins is done using the combination of heat and SDS for SDS-PAGE, CE-SDS, and Western blot. Reversed-phase liquid chromatography-mass spectrometry (RPLC-MS) is also a denaturing method, it uses a combination of organic solvents in the mobile phases and the heat of the column to denature the proteins, then uses mass spectrometry for identification of proteins and modifications through accurate measurement of protein masses. Several RPLC-MS methods were reported to evaluate AAV capsid proteins at intact protein levels [7, 9, 10, 17,18,19]. Size exclusion chromatography (SEC) is a non-denaturing method for the evaluation of soluble protein purities, monomer, dimmer, oligomers, aggregation, and fragments. A number of papers have addressed using SEC to evaluate AAV capsid purities and capsid concentrations [9, 20,21,22]. The heat-induced degradation of AAV involves two pathways, genome ejection and capsid disruption. An instrument named “Uncle” by Unchained Labs is a multi-modal stability platform that can monitor capsid disruption and genome ejection as a function of temperature [23]. Capsid disruption occurs when capsid proteins denature and capsid structure is lost. In genome ejection, the capsid remains intact, but the viral genome is released. The Uncle instrument can track both degradation pathways of capsid disruption and genome ejection with full-spectrum fluorescence detection [24, 25]. Cryo-electron microscopy (cryo-EM) is also a non-denaturing method for analysis of protein three-dimensional (3D) structures and has been used for evaluation of AAV capsid [26,27,28]. Proteolytic mapping of AAV capsids [29] and comparative analysis of AAV stability and dynamics using limited proteolysis by proteinase K [30] have been previously reported, but whether AAV capsids with clips remain potent has not yet been evaluated.
In this work, we evaluated the stability of AAV2 capsids with a proteolytic approach using trypsin and other protease enzymes and leverage various analytical methods to probe structural and biological impacts. Proteolytic digestion of AAV2 capsids with trypsin results in cuts at the C-terminus of the capsid proteins, which was confirmed by denaturing methods including SDS-PAGE, CE-SDS, Western blot, and RPLC-MS. However, it was found that the AAV2 capsid with proteolytic cuts not only remains intact as confirmed by the non-denaturing methods including SEC, thermostability testing, and cryo-EM, but also remains potent as confirmed in a cell-based potency assay.
This finding reveals that the AAV2 capsid with proteolytic cuts remains intact and potent since the icosahedral 3D arrangement of AAV2 capsid proteins can protect the clipped fragments from being released from the capsid, and the capsid remains its functionality for delivering the DNA in the host cell. This study provides a valuable guidance on how to implement a proteolytic approach and leverage multiple denaturing and non-denaturing analytical methods to evaluate AAV capsids to support engineering AAV vectors for AAV-based gene therapy development.
Materials and methods
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).
Methods
Proteolytic 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).
Results
Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
The SDS-PAGE analysis showed that the AAV2 capsid proteins were cut by trypsin or Asp-N, but not by Cathepsin D (CatD), Cathepsin L (CatL), Cathepsin B (CatB), chymotrypsin, or pepsin under the same conditions, as shown in Fig. 1A. The pH used in this study was a neutral pH around 7.5, which was to mimic the pH used in the harvest step. Since the optimal pH for the proteolytic activities of CatD, CatL, CatB, and pepsin is about 4–5, the neutral pH used for these experiments may have resulted in no apparent cut of the AAV2 capsid by these enzymes. An additional proteolytic experiment was carried out to digest two different lots of the AAV 2 capsids by trypsin for different digestion time of 10, 30, 60 min at room temperature, and it was found that the AAV2 capsids in both lots were clipped by trypsin even in 10 min as shown in Fig. 1B.
All digestions by proteases were carried out at room temperature. A Proteolytic digestion of AAV2 capsids for 2 h. Lane 1: marker; lane 2: untreated AAV2 capsids as control (C); lane 3: trypsin-treated AAV2 capsids; lane 4: CatD-treated AAV2 capsids; lane 5: CatL-treated AAV2 capsids; lane 6: CatB-treated AAV2 capsids; lane 7: chymotrypsin-treated AAV2 capsids; lane 8: Asp-N-treated AAV2 capsids; lane 9: pepsin-treated AAV2 capsids; lanes 10–16: enzymes only – trypsin, CatD, CatL, CatB, chymotrypsin, Asp-N, or pepsin, respectively. B Proteolytic digestion of AAV2 by trypsin for 10, 30, and 60 min. Lane 1: marker; lanes 2 and 7: untreated AAV2 capsids as control (C); lanes 3 and 8: 10 min trypsin-treated AAV2 capsids; lanes 4 and 9: 30 min trypsin-treated AAV2capsids; lanes 5 and 10: 60 min trypsin-treated AAV2 capsids; lane 6: trypsin only; n = 2, biological replicates.
Western blot
Based on the Western blot analysis, using an antibody against the shared VP C-terminus, VP1, VP2, and VP3 bands were identified in the untreated control AAV2 capsid, but not in the trypsin-treated AAV2 capsid. Instead, a strong positive band around 16 kDa was observed, indicating that the C-termini of VP1, VP2, and VP3 were clipped by trypsin (Fig. 2A). Using the antibody against the VP1-unique region, a VP1 band (82 kDa) was identified in the untreated control AAV2 capsid and a positive band around 65 kDa was identified in the trypsin-treated AAV2 capsid, indicating that the C-terminus of VP1 was clipped by trypsin (Fig. 2B).
The untreated (control, C) or trypsin-treated (T) AAV2 capsids were run in duplicate on the same SDS-PAGE gel and then subjected to immunoblotting with an antibody against the shared VP C-terminus A or an antibody against the VP1-unique region B. Molecular weight standards are given to the left of each blot in kDa (n = 2, technical replicates). A Antibody against the shared VP C-terminus. B Antibody against the VP1-unique region.
Capillary electrophoresis sodium dodecyl sulfate (CE-SDS)
The clips of the AAV2 VP1, VP2, and VP3 by trypsin were also observed in the CE electropherograms as shown in Fig. 3. The peaks of VP1, VP2, and VP3 were observed in the untreated AAV2 capsid, but not in the trypsin-treated AAV2 capsid. Two additional peaks with lower masses were observed in the trypsin-treated AAV2 capsid (Fig. 3). The total peak area of the AAV2 VP1, VP2, VP3, and VP3 variant peaks in the untreated AAV2 capsid was 27% higher than the total peak area of all peaks in the trypsin-treated AAV2 capsid (data not shown) due to the technical limitation of CE-SDS analysis that cannot detect the fragments with masses lower than 10 kDa.
The untreated AAV2 capsids in black trace; trypsin-treated AAV2 capsids in red trace (n = 2, technical replicates).
Reversed-phase liquid chromatography-mass spectrometry (RPLC-MS)
As shown in Fig. 4, AAV2 VP1 (observed mass 82854.77 Da, mass error 2.4 ppm), VP2 (observed mass 67487.45 Da, mass error 7.8 ppm), and VP3 (observed mass 60973.48 Da, mass error 6.1 ppm) were identified in the untreated AAV2 capsids but not in the trypsin-treated AAV2 capsids. Instead, four major peaks (Peak a-d) were identified with the observed masses of 16770.45 Da (Peak a), 16456.08 Da (Peak b), 44218.69 Da (Peak c), and 43205.30 Da (Peak d). These clips of the AAV2 VP1, VP2, and VP3 by trypsin were confirmed by RPLC-MS/MS and are in agreement with the results of SDS-PAGE, Western blot, and CE-SDS analysis.
The untreated AAV2 capsids in black trace; trypsin-treated AAV2 capsids in red trace (n = 2, technical replicates).
In order to identify the clipping sites of the AAV2 capsid proteins, a top-down RPLC-MS/MS experiment was carried out. Figure 5 shows the fragment map and matched spectrum of the two major fragments resulting in Peak a (clip site between R595 and S596) and Peak b (clip site between R598 and Q599) to the C-terminus. These two clipping sites follow the typical cleavage site of trypsin, which cleaves N-terminus of arginine or lysine residues except when arginine or lysine are followed by proline, suggesting that these clipping sites are tryptic clipping sites, not random clips.
Fragment map and fragment match spectrum of the trypsin-treated AAV2 capsids.
The clipped fragments Peak a and Peak b (Fig. 4) come from the shared C-terminus of any of VP1, VP2, or VP3. In a theoretical AAV capsid, the 60 subunits are composed of 5 VP1 proteins, 5 VP2 proteins, and 50 VP3 proteins. Since the majority of the capsid is VP3, the majority of these clipped fragments were from VP3. The 43–44 kDa clipped fragments Peak c (44218.69 Da) with the sequence from M203 to R598 and Peak d (43205.30 Da) with the sequence from M203 to R595 are unique to the N-terminus of VP3 only (Fig. 4). The masses (kDa) of the clipped fragments that occurred in the N-terminus of VP1 & VP2 are larger than the clipped fragments ~ 43–44 kDa (Peak c and Peak d) of VP3.
In the trypsin-treated AAV2 sample, the peaks with low-intensity in red trace at the retention time of 17–18 min correspond to the N-terminus of VP1 and VP2, similar to Peak c and Peak d around 20–21 min correspond to the N-terminus of VP3. The intensity of the clipped fragments from the N-terminus of VP1 and VP2 (red trace, 17–18 min) are much lower than the intensity of the clipped fragments from the N-terminus of VP3 (Peak c and Peak d; red trace, 20–21 min) because the abundance of VP1 and VP2 is much lower than that of VP3 in an AAV capsid.
Potency assay
In the cell-based potency assay, the percent relative potency was calculated using parallel line analysis of dose response curve generated from serially diluted untreated AAV2 capsids (assay control) and trypsin-treated AAV2 capsids against reference AAV2 capsids. The reference AAV2 capsids was the same AAV2 capsids and kept at −70 oC prior to use for the potency assay. The untreated AAV2 capsids (assay control) and the trypsin-treated AAV2 capsids were prepared by adding same volume of LC-MS grade water or trypsin solution, respectively, then filtered using an Amicon 50 kDa MWCO filter to remove the trypsin (about 24 kDa) after digestion for 10, 30, or 60 min for the trypsin-treated AAV2 capsid and 60 min for the assay control. As shown in Table 1, the potencies of the untreated AAV2 capsids (assay control) and the trypsin-treated AAV2 capsids for 10, 30, and 60 min were all within the acceptance criteria ( ± 30% of the reference potency). The potency results indicate that the AAV2 capsid remains potent even when the capsid proteins were clipped by trypsin with no negative impact on its potency.
Size exclusion chromatography (SEC)
SEC was used to evaluate whether there were any changes of protein purity, monomer, aggregation, and fragments in the AAV2 capsid upon trypsin treatment. The untreated AAV2 capsids showed a major peak (AAV2 capsid monomer peak) at approximately 33 min. The trypsin only injection (in green) showed the trypsin peak at 38 min (Fig. 6). The trypsin-treated AAV2 capsids (in red) showed the trypsin peak at 38 min, and the major peak at around 32.5 min, which is the AAV2 capsid monomer peak. No fragment peaks were observed in the trypsin-treated AAV2 capsid sample (Fig. 6). The monomer peak areas of the untreated and trypsin-treated AAV2 capsids were similar, both greater than 97%. It was noticed that the retention times of the monomer peaks in the untreated and treated-capsids were slightly different, where the trypsin-treated capsid eluted slightly earlier than the untreated capsid. It is possible that the 3D structure of the trypsin-treated capsid may be slightly larger than an untreated capsid. Nonetheless, there are no significant differences of SEC results between untreated and trypsin-treated AAV2 capsids under the native condition, suggesting the trypsin-treated capsid remained intact (Fig. 6).
The untreated AAV2 capsids in black trace; trypsin-treated AAV2 capsids in red trace; trypsin only in formulation buffer in green trace (n = 2, technical replicates).
Evaluation of thermostability and DNA leakage of the AAV 2 capsids
Thermal denaturation of AAV involves genome ejection and capsid disruption. The stabilities of the untreated and trypsin-treated AAV2 capsids were compared by using an Uncle instrument (Unchained Labs). Genome ejection from AAV capsids occurs at relatively lower temperatures as DNA starts to escape from intact capsids. Uncle tracks genome ejection with a DNA-binding fluorescent dye, SYBR Gold, to determine a melting temperature (Tm) based on DNA release. The genome ejection melting curve is based on the fluorescence intensity of SYBR Gold bound to DNA. As the capsids are heated along a thermal ramp, SYBR Gold fluorescence increases as more DNA is ejected from the capsid. As shown in Fig. 7A, the untreated and trypsin-treated AAV2 capsids showed similar genome ejection profiles under thermal stress.
The genome ejection melting curves A were based on the fluorescence intensity of SYBR Gold bound to DNA; the capsid unfolding curves B were based on intrinsic fluorescence of capsid proteins. The untreated AAV2 capsids in black trace; trypsin-treated AAV2 capsids in red trace (treated for 10 min), in green trace (treated for 30 min), and in sky blue (treated for 60 min) (n = 2, technical replicates).
AAV capsid disruption occurs when protein unfolding causes the viral capsid to lose structural integrity and break apart. Capsid disruption of AAV can be studied on the Uncle instrument by monitoring the intrinsic protein fluorescence of capsid proteins to determine their unfolding behavior. As capsid proteins are heated and unfold, the local environment of fluorescent amino acids change and as a result, the protein’s total fluorescence behavior changes. As shown in Fig. 7B, the untreated and trypsin-treated AAV2 capsids showed similar protein unfolding curve and similar melting temperatures with only 2 °C difference, indicating that the trypsin-treated AAV2 capsid remains its intact structure well. The lines indicating the Tms of all samples were determined by Uncle Analysis software.
Cryo-electron microscopy (cryo-EM)
Cryo-EM was performed to visualize the shape and structure of the untreated and trypsin-treated capsids. The cryo-EM analysis confirmed that the trypsin-treated AAV2 capsids with clips remain intact as shown in Fig. 8.
A A motion-corrected cryo-EM image showing the trypsin-treated AAV2 capsids; B 2D class averages generated from the cryo-EM images of the trypsin-treated AAV2 particles showing on the left in A providing evidence that the AAV capsids are intact.
Discussion
Since the optimal active temperature for trypsin is 37 °C, the digestion of the modified AAV2 capsids by trypsin at 37 °C for 2 h and 5 h was also carried out as positive controls. The same clipped bands of AAV2 capsids treated with trypsin at 37 °C (data not shown) were observed by SDS-PAGE as the clipped bands of AAV2 capsids treated with trypsin at RT. This result suggests that the incubation temperature at RT or 37 °C did not affect tryptic digestion of intact AAV2 capsids under non-denaturing conditions. SEC results also showed that the AAV2 capsids incubated with trypsin at 37 °C for 2 h and 5 h remain intact (data not shown). A potency assay using AAV2 capsids incubated with trypsin at 37 °C for 2 h and 5 h was not performed because the incubation of AAV capsids at 37 °C increases deamidation levels of asparagine (N) residues, especially N57 or N56 of AAV VPs [18, 34], and the increased deamidation at N57 or N56 decreases transduction efficiency [35] and potency (unpublished data).
To compare the proteolytic digestion results of the modified AAV2 capsid, the proteolytic digestions of the wild-type AAV2, AAV9, AAV5, and AAV8 capsids were carried out using trypsin and other proteases under the same conditions at room temperature for 2 h. The wild-type AAV2 capsid proteins were cut by trypsin and the two major generated fragments marked with red arrows were same as the fragments generated from the modified AAV2 capsid digested by trypsin (Fig. 9A). However, VP1, VP2, and VP3 bands were slightly visible in the wild-type AAV2 capsid, in contrast, VP1, VP2, and VP3 bands were almost invisible in the modified AAV2 capsid as shown in Fig. 9A. This observation indicates that the modified AAV2 capsid is more susceptible to cleavage by trypsin than the wild-type AAV2 capsid. AAV9 capsid proteins were not susceptible to cleavage by trypsin, CatD, CatL, Asp-N, chymotrypsin, or pepsin, but did show susceptibility to CatB as illustrated by one additional band below VP1 in Fig. 9B. Overnight digestion of AAV9 capsids by trypsin at 37 °C was also carried out, and no fragments were observed (data not shown). AAV5 capsid proteins were not clipped by trypsin, CatD, CatL, or chymotrypsin. AAV8 capsid proteins were cleaved by trypsin or CatL as shown by the additional band below VP2 in the trypsin or CatL treated AAV8 capsid, but not in CatD or chymotrypsin treated AAV8 capsid as shown in Fig. 9C. Based on the above observations, under the same digestion conditions at room temperature for 2 h, the AAV2 capsids showed the least stable profile when exposed to trypsin. Based on SDS-PAGE results, proteolytic digestion of AAV2 by trypsin can distinguish AAV2 from AAV5, AAV8, and AAV9, as seen by unique fragment patterns (Figs. 1 and 9).
A Comparison of the trypsin digestion of the modified and the wild-type AAV2 capsids. Untreated AAV2 capsids as control (C); Trypsin-treated AAV2 capsids (T). The red arrows indicate the major fragments. B Proteolytic digestion of AAV9 capsids. Lane 1: marker; lane 2: untreated AAV9 capsids as control (C); lane 3: trypsin-treated AAV9 capsids; lane 4: CatD-treated AAV9 capsids; lane 5: CatL-treated AAV9 capsids; lane 6: CatB-treated AAV9 capsids; lane 7: chymotrypsin-treated AAV9 capsids; lane 8: Asp-N-treated AAV9 capsids; lane 9: pepsin-treated AAV9 capsids. C Proteolytic digestion of AAV5 and AAV8 capsids. Lane 1: marker; lane 2: untreated AAV5 capsids as control (C); lane 3: trypsin-treated AAV5 capsids; lane 4: CatD-treated AAV5 capsids; lane 5: CatL-treated AAV5 capsids; lane 6: chymotrypsin-treated AAV5 capsids; lane 7: chymotrypsin only; lane 8: untreated AAV8 capsids as control (C); lane 9: trypsin-treated AAV8 capsids; lane 10: CatD-treated AAV8 capsids; lane 11: CatL-treated AAV8 capsids; lane 12: chymotrypsin-treated AAV8 capsids.
In order to understand why AAV2 capsid is more susceptible to cleavage by trypsin than other AAV serotypes, a sequence alignment of amino acids containing R595 and R598 of the modified AAV2 and 13 wild AAV serotypes is shown in Fig. 10. This alignment shows that only AAV2 has a potential trypsin cleavage site in the region containing R588 of the wild-type AAV2 or R598of the modified AAV2. The modified AAV2 capsid used in this study was created by inserting 10 amino acids between N587 and R588 (VP1 numbering) of the wild-type AAV2, resulting in that R588 of the wild-type AAV2 became R598 of the modified AAV2. Among those inserted 10 amino acids in the modified AAV2 capsid, the inserted R595 is an additional potential trypsin cleavage site, which is absent in the wild-type AAV2 capsid. This extra trypsin cleavage site in the modified AAV2 capsid may provide a structural explanation for the observed increase in cleavage in the modified AAV2 capsid than the wildtype AAV2 capsid. In the other serotypes studied here (AAV5, AAV8, AAV9), the structurally homologous R588 is not present, thus explaining why no trypsin cleavage was observed in AAV5, AAV8, and AAV9.
Sequence alignment of amino acids containing R595 and R598 of the modified AAV2 and 13 wild-type AAV serotypes.
To confirm experimental observations and provide further explanation, 3D structural analysis was performed. Analysis of the modified AAV2 protomer and assembled capsid showed that the experimentally identified trypsin cutting sites (R595 and R598) are located on the surface of the capsid and are thus susceptible to trypsin cleavage (Fig. 11A). The overlaid 3D structures of the modified AAV2, wild-type AAV2, AAV5, AAV8, and AAV9 reveal that the potential trypsin cleavage sites R595 and R598 of AAV2 are likely more susceptible to cleavage by trypsin than other AAV serotypes (Fig. 11B). Figure 11C shows that the different regions of the fragment from the cutting site R595 to the C-terminus are located at different distances from the capsid center, on the inside (650–661, 683–701), midway (607–649, 662–663, 702–712, 732–745) or on the outside (595–606, 664–682, 713–731), respectively. This 3D arrangement of AAV2 capsid proteins potentially protects the clipped fragment from being released from the capsid, such that the AAV capsid remains intact allowing for the functionality to be maintained to deliver the DNA in the host cell.
A Assembled capsid of modified AAV2 with zoom-in of the modified AAV2 protomer. The modified AAV2 protomer is colored gray with insert in red. Structural elements representing five-fold, three-fold, and two-fold axis are colored blue, green, and orange, respectively. R595 and R598 are shown in ball and stick representation. B Overlaid 3D structures of the modified AAV2, wild-type AAV2, AAV5, AAV8, and AAV9. C C1: Assembled capsid of the modified AAV2 with a protomer shown in surface representation. Surface of the protomer is colored according to the distance from capsid center shown as a red sphere. C2: slice halfway through the capsid. C3: Zoom-in of the protomer shown in surface representation. The C-terminal fragment of the modified AAV2 capsid proteins from the clip site R595 to the end of C-terminus is colored black. Regions of the protomer that are on the inside (650–661, 683–701), midway (607–649, 662–663, 702–712, 732–745) or on the outside (595–606, 664–682, 713–731) of the capsid are labeled accordingly. N- and C-terminals of the VP3 are labeled N and C, respectively.
Furthermore, in line with structural analyses, this study suggests that some AAV VP-related impurities, such as VP clips, may be observable using denaturing methods (SDS-PAGE CE-SDS, or RPLC), but cannot be removed from the capsid during downstream purification, as the clipped fragments would not dissociate from the capsids and are buried in the protomer (Fig. 11C).
Moreover, as shown in Fig. 12, two fragment peaks, peak 1 (16456.08 Da) at 5.8 min and peak 2 (43205.30 Da) at 21.7 min were observed in the trypsin-treated wild-type AAV2 capsids (red trace), but not in the untreated wild-type AAV2 (black trace), indicating that there is one potential trypsin cleavage site on the wild-type AAV2. The clip site between R588 and Q589 was identified by the top-down RPLC-MS/MS and a similar fragment map as Peak b and fragment match spectrum as Peak b were observed as shown in Fig. 5. The wild-type AAV2 VP1 (observed mass 81854.65 Da, mass error 3.0 ppm), VP2 (observed mass 66487.72 Da, mass error 2.8 ppm), and VP3 (observed mass 59973.41 Da, mass error 6.2 ppm) were identified in the untreated wild type AAV2 capsids. The observation of slightly visible VP1, VP2, and VP3 peaks in the trypsin-treated wild-type AAV2 capsids is in agreement with the results of SDS-PAGE (Fig. 9A) due to the incomplete cleavage of wild-type AAV2 capsids by trypsin at room temperature for 2 h.
The untreated AAV2 capsids in black trace; trypsin-treated AAV2 capsids in red trace (n = 2, technical replicates). A Overlaid RPLC-MS total ion chromatograms of the untreated and trypsin- treated wild AAV2 capsids. B The fragment map of the identified clip form in the wild-type AAV2.
Based on the potency results, it seems that the clipped C-terminal 147 amino acids (16 kDa) have no impact on the potency of AAV2 based gene therapy and this finding may apply to other serotypes of AAV based gene therapies.
Although proteolytic approaches were used previously in AAV capsid studies [29, 30], however, these studies did not report the effect of enzymatic clipping on potency. One report [29] demonstrated that proteolytic digestion of AAV2 virions with trypsin resulted in cleavage at a specific site on the capsid surface while keeping the capsid intact. This result is in agreement with our results and analysis of the unique AAV2 capsid amino acid sequence. In the report, AAV2 capsids treated with trypsin at 37 °C showed reduced receptor binding and reduced infectivity [29]. This observation differs from our finding that is no impact of the AAV2 capsids with tryptic clips on potency. The likely cause of the different findings is the difference in incubation temperatures that impact on asparagine deamidation, which can be increased by higher temperature, and the increased deamidation of AAV capsids decreases transduction efficiency and potency.
Another report [30] showed a comparative analysis of AAV capsid stability and dynamics. AAV1, AAV2, AAV5, and AAV8 were compared by using differential scanning fluorimetry (DSF), differential scanning calorimetry (DSC), electron microscopy (EM), and proteolysis with proteinase K at 37 °C. No potency assay of the AAV capsids treated with proteinase K was carried out in the report.
The potency assay is critical in the development of AAV-based gene therapy products. An cell-based protein expression potency assay not only inherently assesses vector’s infectivity, but also the ability of the vector to deliver its transgene and produce therapeutic protein that can be expressed at cellular compartment that is reflective of its biological mechanism of action. In contrast, the infectivity assay measures only the entry of vectors into a cell.
The novelty of this study is the discovery that capsids with proteolytic cuts by trypsin at room temperature remain intact and potent. The work presented here shows that clipping of the C-terminus of viral proteins does not significantly perturb the higher order structure of the AAV2 virus and does not result in diminished biological activity via a cell-based potency assay. This study demonstrates utilization of the modern analytical techniques to evaluate the thermostability of AAV2 capsids including genome ejection and capsid unfolding; as well as leveraging modern instrumentation to perform top-down proteomic analysis (RPLC-MS/MS) to streamline identification of clip sites with a high degree of confidence.
Conclusion
Proteolytic digestion of AAV2 capsid with trypsin results in clips of the capsid proteins at the C-terminus. The clips were confirmed by denaturing methods including SDS-PAGE, CE-SDS, Western blot, and RPLC-MS/MS. However, it was found that the AAV2 capsid with clips not only remains intact as confirmed by non-denaturing methods including SEC, capsid protein thermostability, genome ejection testing, and cryo-EM, but also remains potent as confirmed by a cell-based potency assay. This finding reveals that AAV2 capsid with proteolytic cuts remains intact and potent to deliver the DNA in the host cells. This study provides a valuable guidance on how to use proteolytic approach and multiple denaturing and non-denaturing analytical methods to evaluate AAV capsid stability to support engineering AAV vectors for AAV-based gene therapy development.
Data availability
All data used in this paper has been presented in the figures.
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Yu Zhou: designed and conducted the proteolytic study, MS data acquisition and data analysis, original draft preparation, and editing. Tina Sach carried out potency assay, draft review and editing. Joseph Y. Ong carried out Western blot, draft review and editing. Ting-An Lim carried out SEC and thermostability and genome ejection testing, draft review and editing. Zoltan Berecz carried out CE-SDS, draft review and editing. Colin Deniston carried out cryo-EM, draft review and editing. Goran Milicic carried out 3D structural analysis, draft review and editing. Connie Y. Tsai, Taryn Kandepalli, Derek Langeslay, and Qiang Qin for supervision, draft review and editing. All authors have made contributions to the final approval of the version to be submitted.
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Zhou, Y., Sach, T., Ong, J.Y. et al. Adeno-associated virus serotype 2 capsids with proteolytic cuts by trypsin remain intact and potent. Gene Ther 32, 121–131 (2025). https://doi.org/10.1038/s41434-024-00507-4
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DOI: https://doi.org/10.1038/s41434-024-00507-4
