RESUMEN
The adeno-associated virus (AAV) vector is one of the most advanced platforms for gene therapy because of its low immunogenicity and non-pathogenicity. The concentrations of both AAV vector empty particles, which do not contain DNA and do not show any efficacy, and AAV vector full particles (FPs), which contain DNA, are important quality attributes. In this study, a dual fluorescence-linked immunosorbent assay (dFLISA), which uses two fluorescent dyes to quantify capsid and genome titers in a single analysis, was established. In dFLISA, capture of AAV particles, detection of capsid proteins, and release and detection of the viral genome are performed in the same well. We demonstrated that the capsid and genomic titers determined by dFLISA were comparable with those of analytical ultracentrifugation. The FP ratios determined by dFLISA were in good agreement with the expected values. In addition, we showed that dFLISA can quantify the genomic and capsid titers of crude samples. dFLISA can be easily modified for measuring other AAV vector serotypes and AAV vectors with different genome lengths. These features make dFLISA a valuable tool for the future development of AAV-based gene therapies.
RESUMEN
Glycosylation of biopharmaceuticals can affect their safety and efficacy. Glycans can occur on recombinant adeno-associated viruses (rAAVs) that are used for gene therapy; however, the types of glycans that attach to rAAVs are controversial. Here, we conducted lectin microarray analyses on six rAAV serotype 6 (rAAV6) preparations that were produced differently. We demonstrate that O-glycans considered to be attached to rAAV6 were recognized by Agaricus bisporus agglutinin (ABA) and that N-glycans were detected in rAAV6 purified without affinity chromatography. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis showed that the N-glycans detected in rAAV6 were derived from host cell proteins. A combination of ABA-based fractionation and LC-MS/MS revealed that rAAV6 was O-glycosylated with the mucin-type glycans, O-GalNAc (Tn antigen), and mono- and di-sialylated Galß1-3GalNAc (T antigen) at S156, T162, T194, and T201 in viral protein (VP) 2 and with O-GlcNAc at T242 in VP3. The mucin-type O-glycosylated rAAV6 particles were 0.1%-1% of total particles. Further physicochemical and biological analyses revealed that mucin-type O-glycosylated rAAV6 had a lower ratio of VP1 to VP2/VP3, resulting in a lower transduction efficiency both in vitro and in vivo compared with rAAV6 without mucin-type O-glycans. This report details conclusive evidence of rAAV glycosylation and its impact on rAAV-based therapeutics.
RESUMEN
Adeno-associated viruses (AAVs) are effective vectors for gene therapy. However, AAV drug products are inevitably contaminated with empty particles (EP), which lack a genome, owing to limitations of the purification steps. EP contamination can reduce the transduction efficiency and induce immunogenicity. Therefore, it is important to remove EPs and to determine the ratio of full genome-containing AAV particles to empty particles (F/E ratio). However, most of the existing methods fail to reliably evaluate F/E ratios that are greater than 90 %. In this study, we developed two approaches based on the image analysis of cryo-electron micrographs to determine the F/E ratios of various AAV products. Using our developed convolutional neural network (CNN) and morphological analysis, we successfully calculated the F/E ratios of various AAV products and determined the slight differences in the F/E ratios of highly purified AAV products (purity > 95 %). In addition, the F/E ratios calculated by analyzing more than 1000 AAV particles had good correlations with theoretical F/E ratios. Furthermore, the CNN reliably determined the F/E ratio with a smaller number of AAV particles than morphological analysis. Therefore, combining 100 keV cryo-EM with the developed image analysis methods enables the assessment of a wide range of AAV products.
Asunto(s)
Microscopía por Crioelectrón , Dependovirus , Vectores Genéticos , Procesamiento de Imagen Asistido por Computador , Microscopía por Crioelectrón/métodos , Dependovirus/genética , Procesamiento de Imagen Asistido por Computador/métodos , Humanos , Redes Neurales de la Computación , Virión/ultraestructura , Terapia Genética/métodos , Células HEK293RESUMEN
Wnt proteins are thought to be transported in several ways in the extracellular space. For instance, they are known to be carried by exosomes and by Wnt-carrier proteins, such as sFRP proteins. However, little is known about whether and/or how these two transport systems are related. Here, we show that adding sFRP1 or sFRP2, but not sFRP3 or sFRP4, to culture medium containing Wnt3a or Wnt5a increases re-secretion of exosome-loaded Wnt proteins from cells. This effect of sFRP2 is counteracted by heparinase, which removes sugar chains on heparan sulfate proteoglycans (HSPGs), but is independent of LRP5/6, Wnt co-receptors essential for Wnt signaling. Wnt3a and Wnt5a specifically dimerize with sFRP2 in culture supernatant. Furthermore, a Wnt3a mutant defective in heterodimerization with sFRP2 impairs the ability to increase exosome-mediated Wnt3a re-secretion. Based on these results, we propose that Wnt heterodimerization with its carrier protein, sFRP2, enhances Wnt accumulation at sugar chains on HSPGs on the cell surface, leading to increased endocytosis and exosome-mediated Wnt re-secretion. Our results suggest that the range of action of Wnt ligands is controlled by coordination of different transport systems.
Asunto(s)
Exosomas , Proteínas Relacionadas con Frizzled Secretadas , Exosomas/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Vía de Señalización Wnt , Proteínas Portadoras/metabolismo , Azúcares/metabolismoRESUMEN
Environmentally regulated gene expression is critical for bacterial survival under stress conditions, including extremes in temperature, osmolarity and nutrient availability. Here, we dissect the thermo- and osmo-responsory behavior of the transcriptional repressor H-NS, an archetypal nucleoid-condensing sensory protein, ubiquitous in enterobacteria that infect the mammalian gut. Through experiments and thermodynamic modeling, we show that H-NS exhibits osmolarity, temperature and concentration dependent self-association, with a highly polydisperse native ensemble dominated by monomers, dimers, tetramers and octamers. The relative population of these oligomeric states is determined by an interplay between dimerization and higher-order oligomerization, which in turn drives a competition between weak homo- versus hetero-oligomerization of protein-protein and protein-DNA complexes. A phosphomimetic mutation, Y61E, fully eliminates higher-order self-assembly and preserves only dimerization while weakening DNA binding, highlighting that oligomerization is a prerequisite for strong DNA binding. We further demonstrate the presence of long-distance thermodynamic connectivity between dimerization and oligomerization sites on H-NS which influences the binding of the co-repressor Cnu, and switches the DNA binding mode of the hetero-oligomeric H-NS:Cnu complex. Our work thus uncovers important organizational principles in H-NS including a multi-layered thermodynamic control, and provides a molecular framework broadly applicable to other thermo-osmo sensory proteins that employ similar mechanisms to regulate gene expression.
Asunto(s)
Proteínas Bacterianas , Proteínas de Unión al ADN , Enterobacteriaceae , Proteínas Bacterianas/metabolismo , ADN/genética , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Enterobacteriaceae/metabolismo , Temperatura , Factores de Transcripción/metabolismoRESUMEN
Adeno-associated virus (AAV) vectors are produced as a mixture of the desired particle (full particle, FP), which is filled with the designed DNA, product-related impurities such as particle without DNA (empty particle, EP), and aggregates. Cesium chloride or iodixanol equilibrium density gradient ultracentrifugation (DGE-UC) has been used for the purification of AAV vectors. DGE-UC can separate FP from impurities based on the difference in their buoyant densities. Here, we report the applications and limitations of equilibrium density gradient analytical ultracentrifugation (DGE-AUC) using a modern AUC instrument that employs DGE-UC principles for the characterization and quantitation of AAV vectors. We evaluated the quantitative ability of DGE-AUC in comparison with sedimentation velocity AUC (SV-AUC) or band sedimentation AUC (BS-AUC) using AAVs with different DNA lengths and different serotypes. DGE-AUC enabled the accurate quantification of the ratio of FP to EP when the AAV vector primarily contains these particles. Furthermore, we developed a new workflow to identify the components of separated peaks in addition to FP and EP. Ultraviolet absorption spectra obtained by multiwavelength detection can also support peak assignment following component identification. DGE-AUC experiments for AAV vectors have limitations with regard to minor components with low absorption at the detected wavelength or those with a density similar to that of major components of AAV vectors. DGE-AUC is the only analytical method that can evaluate particle density heterogeneity; therefore, SV-AUC or BS-AUC and DGE-AUC are complementary methods for reliable assessment of the purity of AAV vectors.
Asunto(s)
Dependovirus , Vectores Genéticos , Dependovirus/genética , Ultracentrifugación/métodos , ADNRESUMEN
Studies of recombinant adeno-associated virus (rAAV) revealed the mixture of full particles with different densities in rAAV. There are no conclusive results because of the lack of quantitative stoichiometric viral proteins, encapsidated DNA, and particle level analyses. We report the first comprehensive characterization of low- and high-density rAAV serotype 2 particles. Capillary gel electrophoresis showed high-density particles possessing a designed DNA encapsidated in the capsid composed of (VP1 + VP2)/VP3 = 0.27, whereas low-density particles have the same DNA but with a different capsid composition of (VP1 + VP2)/VP3 = 0.31, supported by sedimentation velocity-analytical ultracentrifugation and charge detection-mass spectrometry. In vitro analysis demonstrated that the low-density particles had 8.9% higher transduction efficacy than that of the particles before fractionation. Further, based on our recent findings of VP3 clip, we created rAAV2 single amino acid variants of the transcription start methionine of VP3 (M203V) and VP3 clip (M211V). The rAAV2-M203V variant had homogeneous particles with higher (VP1+VP2)/VP3 values (0.35) and demonstrated 24.7% higher transduction efficacy compared with the wild type. This study successfully provided highly functional rAAV by the extensive fractionation from the mixture of rAAV2 full particles or by the single amino acid replacement.
RESUMEN
LI-cadherin is a member of the cadherin superfamily. LI-cadherin mediates Ca2+-dependent cell-cell adhesion through homodimerization. A previous study reported two single nucleotide polymorphisms (SNPs) in the LI-cadherin-coding gene (CDH17). These SNPs correspond to the amino acid changes of Lys115 to Glu and Glu739 to Ala. Patients with colorectal cancer carrying these SNPs are reported to have a higher risk of lymph node metastasis than patients without the SNPs. Although proteins associated with metastasis have been identified, the molecular mechanisms underlying the functions of these proteins remain unclear, making it difficult to develop effective strategies to prevent metastasis. In this study, we employed biochemical assays and molecular dynamics (MD) simulations to elucidate the molecular mechanisms by which the amino acid changes caused by the SNPs in the LI-cadherin-coding gene increase the risk of metastasis. Cell aggregation assays showed that the amino acid changes weakened the LI-cadherin-dependent cell-cell adhesion. In vitro assays demonstrated a decrease in homodimerization tendency and MD simulations suggested an alteration in the intramolecular hydrogen bond network by the mutation of Lys115. Taken together, our results indicate that the increased risk of lymph node metastasis is due to weakened cell-cell adhesion caused by the decrease in homodimerization tendency.
Asunto(s)
Neoplasias Colorrectales , Polimorfismo de Nucleótido Simple , Humanos , Cadherinas/genética , Cadherinas/metabolismo , Adhesión Celular/genética , Neoplasias Colorrectales/patología , Metástasis Linfática/genéticaRESUMEN
Mass photometry (MP) is a label-free, single-molecule technique that can determine molecular mass distribution with very low sample consumption in a short time. Because of the established experimental instrument and analytical software, MP measurements may be readily obtained; thus, the application of MP is expanding, especially in the fields of bioscience and biotechnology. However, because the MP data quality is strongly focus-dependent, optical settings must be intrinsically strict. In this study, we report the importance of the critical calibration of the mass photometer, which is required for the accurate estimation of high-molecular mass samples, such as adeno-associated virus vectors. Additionally, a method for optimizing the instrument settings, including the calibration of the stage, is presented.
Asunto(s)
Dependovirus , Fotometría , Dependovirus/genética , Calibración , Exactitud de los Datos , Biotecnología , Vectores GenéticosRESUMEN
Adeno-associated virus (AAV) vector is a promising platform for in vivo gene therapy. The accurate assessment of distribution state of particles contained in AAV vector samples is one of the most important and challenging matters and is necessary because the product-related impurities with the capsid structure (empty particles, intermediate particles, and aggregates) could be a possible cause of reducing the therapeutic efficacy and enhancing the unfavorable immune response. In this study, we report an effective approach for size distribution analysis with component identification. A small amount of AAV vectors were used by the analytical zone centrifugation c(s) analysis of band sedimentation analytical ultracentrifugation (BS-AUC) with multiwavelength detection. Using PBS/H218O, the concentration of each component could be determined in BS-AUC with high resolution. Compared with the sedimentation velocity AUC (SV-AUC), which generally requires 2 × 1012 vg of AAV vectors, BS-AUC could be performed with about 1/25 of the AAV vector amount at 260 nm detection and ideally with about 1/50 of the AAV vector amount at 230 nm detection (4 × 1010 vg), depending on the extinction coefficient of the AAV sample at each wavelength. According to the limit of quantification of this BS-AUC, 6.3 × 1011 cp mL-1 of empty particle (EP) and 4.4 × 1011 vg mL-1 of full particle (FP) could be quantified for 4 × 1010 vg in 15 µL of AAV8-CMV-EGFP. These results demonstrated that proposed BS-AUC approach we established here can compensate for the drawback in terms of the sample amount of SV-AUC.
Asunto(s)
Proteínas de la Cápside , Dependovirus , Dependovirus/genética , Proteínas de la Cápside/genética , Terapia Genética , Ultracentrifugación/métodos , Vectores GenéticosRESUMEN
Colonization of the host intestine is the most important step in Vibrio cholerae infection. The toxin-coregulated pilus (TCP), an operon-encoded type IVb pilus (T4bP), plays a crucial role in this process, which requires an additional secreted protein, TcpF, encoded on the same TCP operon; however, its mechanisms of secretion and function remain elusive. Here, we demonstrated that TcpF interacts with the minor pilin, TcpB, of TCP and elucidated the crystal structures of TcpB alone and in complex with TcpF. The structural analyses reveal how TCP recognizes TcpF and its secretory mechanism via TcpB-dependent pilus elongation and retraction. Upon binding to TCP, TcpF forms a flower-shaped homotrimer with its flexible N terminus hooked onto the trimeric interface of TcpB. Thus, the interaction between the minor pilin and the N terminus of the secreted protein, namely, the T4bP secretion signal, is key for V. cholerae colonization and is a new potential therapeutic target.
Asunto(s)
Cólera , Vibrio cholerae , Proteínas Bacterianas/metabolismo , Cólera/metabolismo , Proteínas Fimbrias/genética , Proteínas Fimbrias/metabolismo , Fimbrias Bacterianas , Humanos , Vibrio cholerae/metabolismoRESUMEN
In this study, we conducted a collaborative study on the classification between silicone oil droplets and protein particles detected using the flow imaging (FI) method toward proposing a standardized classifier/model. We compared four approaches, including a classification filter composed of particle characteristic parameters, principal component analysis, decision tree, and convolutional neural network in the performance of the developed classifier/model. Finally, the points to be considered were summarized for measurement using the FI method, and for establishing the classifier/model using machine learning to differentiate silicone oil droplets and protein particles.
Asunto(s)
Aceites de Silicona , Siliconas , Tamaño de la Partícula , ProteínasRESUMEN
The interaction between IgG and Fc gamma receptor IIIa (FcγRIIIa) is essential for mediating immune responses. Recent studies have shown that the antigen binding fragment (Fab) and Fc are involved in IgG-FcγRIII interactions. Here, we conducted bio-layer interferometry (BLI) and isothermal titration calorimetry to measure the kinetic and thermodynamic parameters that define the role of Fab in forming the IgG-FcγRIII complex using several marketed therapeutic antibodies. Moreover, hydrogen/deuterium exchange mass spectrometry (HDX-MS) and crosslinking mass spectrometry (XL-MS) were used to clarify the interaction sites and structural changes upon formation of these IgG-FcγRIII complexes. The results showed that Fab in IgG facilitates the interaction via slower dissociation and a larger enthalpy gain. However, a larger entropy loss led to only a marginal change in the equilibrium dissociation constant. Combined HDX-MS and XL-MS analysis revealed that the CL domain of Fab in IgG was in close proximity to FcγRIIIa, indicating that this domain specifically interacts with the extracellular membrane-distal domain (D1) and membrane-proximal domain (D2) of FcγRIIIa. Together with previous studies, these results demonstrate that IgG-FcγRIII interactions are predominantly mediated by the binding of Fc to D2, and the Fab-FcγRIII interaction stabilizes complex formation. These interaction schemes were essentially fucosylation-independent, with Fc-D2 interactions enhanced by afucosylation and the contribution of Fab slightly reduced. Furthermore, the influence of antigen binding on IgG-FcγRIII interactions was also investigated. Combined BLI and HDX-MS results indicate that structural alterations in Fab caused by antigen binding facilitate stabilization of IgG-FcγRIII interactions. This report provides a comprehensive understanding of the interaction between IgG and FcγRIII.
Asunto(s)
Inmunoglobulina G , Receptores de IgG , Glicosilación , Espectrometría de Masas de Intercambio de Hidrógeno-Deuterio , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fc de Inmunoglobulinas/química , Inmunoglobulina G/química , Receptores de IgG/metabolismoRESUMEN
Cancer-specific cell surface antigens are ideal therapeutic targets for monoclonal antibody (mAb)-based therapy. Here, we report that multiple myeloma (MM), an incurable hematological malignancy, can be specifically targeted by an mAb that recognizes a ubiquitously present protein, CD98 heavy chain (hc) (also known as SLC3A2). We screened more than 10,000 mAb clones raised against MM cells and identified R8H283, an mAb that bound MM cells but not normal hematopoietic or nonhematopoietic cells. R8H283 specifically recognized CD98hc. R8H283 did not react with monomers of CD98hc; instead, it bound CD98hc in heterodimers with a CD98 light chain (CD98lc), a complex that functions as an amino acid transporter. CD98 heterodimers were abundant on MM cells and took up amino acids for constitutive production of immunoglobulin. Although CD98 heterodimers were also present on normal leukocytes, R8H283 did not react with them. The glycoforms of CD98hc present on normal leukocytes were distinct from those present on MM cells, which may explain the lack of R8H283 reactivity to normal leukocytes. R8H283 exerted anti-MM effects without damaging normal hematopoietic cells. These findings suggested that R8H283 is a candidate for mAb-based therapies for MM. In addition, our findings showed that a cancer-specific conformational epitope in a ubiquitous protein, which cannot be identified by transcriptome or proteome analyses, can be found by extensive screening of primary human tumor samples.
Asunto(s)
Anticuerpos Monoclonales , Mieloma Múltiple , Anticuerpos Monoclonales/uso terapéutico , HumanosRESUMEN
Recently, an international randomized controlled clinical trial showed that patients with SARS-CoV-2 infection treated orally with the 3-chymotrypsin-like protease (3CLpro) inhibitor PF-07321332 within three days of symptom onset showed an 89% lower risk of COVID-19-related hospital admission/ death from any cause as compared with the patients who received placebo. Lending support to this critically important result of the aforementioned trial, we demonstrated in our study that patients infected with a SARS-Cov-2 sub-lineage (B.1.1.284) carrying the Pro108Ser mutation in 3CLpro tended to have a comparatively milder clinical course (i.e., a smaller proportion of patients required oxygen supplementation during the clinical course) than patients infected with the same sub-lineage of virus not carrying the mutation. Characterization of the mutant 3CLpro revealed that the Kcat/Km of the 3CLpro enzyme containing Ser108 was 58% lower than that of Pro108 3CLpro. Hydrogen/deuterium-exchange mass spectrometry (HDX-MS) revealed that the reduced activity was associated with structural perturbation surrounding the substrate-binding region of the enzyme, which is positioned behind and distant from the 108th amino acid residue. Our findings of the attenuated clinical course of COVID-19 in patients infected with SARS-CoV-2 strains with reduced 3CLpro enzymatic activity greatly endorses the promising result of the aforementioned clinical trial of the 3CLpro inhibitor.
Asunto(s)
COVID-19 , Proteasas 3C de Coronavirus , Mutación Missense , Gravedad del Paciente , Adulto , Anciano , Sustitución de Aminoácidos , COVID-19/enzimología , COVID-19/genética , Proteasas 3C de Coronavirus/genética , Proteasas 3C de Coronavirus/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Liver intestine (LI)-cadherin is a member of the cadherin superfamily, which encompasses a group of Ca2+-dependent cell-adhesion proteins. The expression of LI-cadherin is observed on various types of cells in the human body, such as normal small intestine and colon cells, and gastric cancer cells. Because its expression is not observed on normal gastric cells, LI-cadherin is a promising target for gastric cancer imaging. However, because the cell adhesion mechanism of LI-cadherin has remained unknown, rational design of therapeutic molecules targeting this cadherin has been hampered. Here, we have studied the homodimerization mechanism of LI-cadherin. We report the crystal structure of the LI-cadherin homodimer containing its first four extracellular cadherin repeats (EC1-4). The EC1-4 homodimer exhibited a unique architecture different from that of other cadherins reported so far, driven by the interactions between EC2 of one protein chain and EC4 of the second protein chain. The crystal structure also revealed that LI-cadherin possesses a noncanonical calcium ion-free linker between the EC2 and EC3 domains. Various biochemical techniques and molecular dynamics simulations were employed to elucidate the mechanism of homodimerization. We also showed that the formation of the homodimer observed in the crystal structure is necessary for LI-cadherin-dependent cell adhesion by performing cell aggregation assays. Taken together, our data provide structural insights necessary to advance the use of LI-cadherin as a target for imaging gastric cancer.
Asunto(s)
Cadherinas/química , Cadherinas/metabolismo , Cadherinas/genética , Adhesión Celular , Agregación Celular , Cristalografía por Rayos X , Dimerización , Humanos , Dominios Proteicos , Estructura Terciaria de ProteínaRESUMEN
Protein aggregate formation in prefilled syringes (PFSs) can be influenced by protein adsorption and desorption at the solid-liquid interface. Although inhibition of protein adsorption on the PFS surface can lead to a decrease in the amount of aggregation, the mechanism underlying protein adsorption-mediated aggregation in PFSs is unclear. This study investigated protein aggregation caused by protein adsorption on silicone oil-free PFS surfaces [borosilicate glass (GLS) and cycloolefin polymer (COP)] and the factors affecting the protein adsorption on the PFS surfaces. The adsorbed proteins formed multilayered structures that consisted of two distinct types of layers: proteins adsorbed on the surface of the material and proteins adsorbed on top of the proteins on the surface. A pH-dependent electrostatic interaction was the dominant force for protein adsorption on the GLS surface, while hydrophobic effects were dominant for protein adsorption on the COP surface. When the repulsion force between proteins was weak, protein adsorption on the adsorbed protein layer was increased for both materials and as a result, protein aggregation increased. Therefore, a formulation with high colloidal stability can minimize protein adsorption on the COP surface, leading to reduced protein aggregation.
Asunto(s)
Proteínas , Jeringas , Adsorción , Interacciones Hidrofóbicas e Hidrofílicas , Aceites de Silicona , Propiedades de SuperficieRESUMEN
Prostaglandin D2 (PGD2), an endogenous somnogen, is a unique PG that is secreted into the cerebrospinal fluid. PGD2 is a relatively fragile molecule and should be transported to receptors localized in the basal forebrain without degradation. However, it remains unclear how PGD2 is stably carried to such remote receptors. Here, we demonstrate that the PGD2-synthesizing enzyme, Lipocalin-type prostaglandin D synthase (L-PGDS), binds not only its substrate PGH2 but also its product PGD2 at two distinct binding sites for both ligands. This behaviour implys its PGD2 carrier function. Nevertheless, since the high affinity (Kd = â¼0.6 µM) of PGD2 in the catalytic binding site is comparable to that of PGH2, it may act as a competitive inhibitor, while our binding assay exhibits only weak inhibition (Ki = 189 µM) of the catalytic reaction. To clarify this enigmatic behavior, we determined the solution structure of L-PGDS bound to one substrate analog by NMR and compared it with the two structures: one in the apo form and the other in substrate analogue complex with 1:2 stoichiometry. The structural comparisons showed clearly that open or closed forms of loops at the entrance of ligand binding cavity are regulated by substrate binding to two sites, and that the binding to a second non-catalytic binding site, which apparently substrate concentration dependent, induces opening of the cavity that releases the product. From these results, we propose that L-PGDS is a unique enzyme having a carrier function and a substrate-induced product-release mechanism.
Asunto(s)
Dominio Catalítico , Oxidorreductasas Intramoleculares/metabolismo , Lipocalinas/metabolismo , Prostaglandina D2/metabolismo , Prostaglandina H2/metabolismo , Animales , Sitios de Unión , Biocatálisis , Oxidorreductasas Intramoleculares/química , Oxidorreductasas Intramoleculares/genética , Cinética , Lipocalinas/química , Lipocalinas/genética , Espectroscopía de Resonancia Magnética , Ratones , Estructura Molecular , Mutación , Prostaglandina D2/química , Prostaglandina H2/química , Unión Proteica , Conformación Proteica , Especificidad por SustratoRESUMEN
During the manufacturing of recombinant adeno-associated virus vectors, it is generally difficult to purify out vectors that lack nucleic acids (empty particles, EPs), contain incomplete nucleic acids (intermediate particles, IPs) or aggregates. These impurities may cause side effects and therefore it is essential to both quantify and reduce them; however, comprehensive identification of the size distribution and components of virus vectors have been lagging. We developed multiwavelength sedimentation velocity analytical ultracentrifugation to characterize EPs, full particles, IPs, and aggregates in adeno-associated virus vector samples. The wavelength-dependent ultraviolet (UV) absorption of capsid protein and encapsulated single-stranded DNA could be deduced from the multiwavelength detection followed by size distribution analysis and peak area integration. Subsequently, a spectral deconvolution analysis using the wavelength-dependent UV absorption data enabled the identification of the protein-nucleic acid ratio of all species. A comprehensive approach for quantifying the viral vector particles and related impurities was established.
Asunto(s)
Dependovirus , Vectores Genéticos , Proteínas de la Cápside , Dependovirus/genética , Ultracentrifugación , ViriónRESUMEN
Recombinant adeno-associated virus is a leading platform in human gene therapy. The adeno-associated virus (AAV) capsid is composed of three viral proteins (VPs): VP1, VP2, and VP3. To ensure the safety of AAV-based gene therapy products, the stoichiometry of VPs of AAV vector should be carefully monitored. In this study, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, capillary gel electrophoresis (CGE), and liquid chromatography-UV-mass spectrometry (LC-UV-MS) were performed to evaluate the VP components of AAV1, AAV2, and AAV6. Two types of VP3-related components, VP3 variant and VP3 fragment, were identified. The VP3 variant was the N-terminal shorter VP3, of which the translation started at M211, not at the conventional initiation codon, M203. The VP3 variant could be generated by leaky scanning of the first initiation codon of VP3. We also showed that the VP3 variant was identified in a minor peak before VP3 in CGE measurement. Meanwhile, the VP3 fragment was the C-terminal cleaved VP3, of which the sequence of VP3 ended at D590 or D626, indicating that cleavage occurred between D590 and P591, or D626 and G627. The cause of the cleavage of the DP or DG sequence was hydrolysis due to low pH of the mobile phase and high temperature of the column oven in the LC system, which was necessary to clearly separate the peak of VPs. VP3 fragments, detected only in LC-UV-MS in small amount account with less than 3% of total peak area, should be included in the quantification of VP3. Finally, the relationship of VP stoichiometry determined by the above three methods was discussed. From this study, we proposed that the VP components of AAV should be complementarily evaluated by CGE and LC-UV-MS.