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Electrophoresis-Mediated Characterization of Full and Empty Adeno-Associated Virus Capsids.

ABSTRACT: Adeno-associated virus (AAV) has shown great potential in gene therapy due to its low immunogenicity, lack of pathogenicity to humans, and ability to provide long-term gene expression in vivo. However, there is currently a need for fast, high-throughput characterization systems that require low volumes for the determination of its sample composition in terms of full and empty capsids since empty capsids are a natural byproduct of AAV synthesis. To address this need, the following study proposes a high-throughput electrophoresis-mediated microfluidics approach that is independent of sample input concentration to estimate the composition of a given sample by combining its protein and ssDNA information relative to a standard. Using this novel approach, we were able to estimate the percentage of full capsids of six AAV8 samples with an average deviation from the actual percentage of 4%. The experiments used for these estimations were conducted with samples of varying percentages of full capsids (21-75%) and varying concentrations (5 × 10¹¹-1 × 10¹² VP/mL) with a total volume requirement of 3-10 μL for triplicate analysis of the sample. This method offers a rapid way to evaluate the quality and purity of AAV products. We believe that our method addresses the critical need as recognized by the gene and molecular therapy community.

SUBMITTER: Coll De Pena A

PROVIDER: S-EPMC9280930 | biostudies-literature | 2022 Jul

REPOSITORIES: biostudies-literature

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Electrophoresis-Mediated Characterization of Full and Empty Adeno-Associated Virus Capsids.

Coll De Peña Adriana A Masto Lucy L Atwood James J Tripathi Anubhav A

ACS omega 20220629 27

Adeno-associated virus (AAV) has shown great potential in gene therapy due to its low immunogenicity, lack of pathogenicity to humans, and ability to provide long-term gene expression <i>in vivo</i>. However, there is currently a need for fast, high-throughput characterization systems that require low volumes for the determination of its sample composition in terms of full and empty capsids since empty capsids are a natural byproduct of AAV synthesis. To address this need, the following study pr ...[more]

PMID: 35847322

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Project description:Adeno-associated virus (AAV) vectors have been successfully applied in clinical trials for hemophilic patients. Although promising, the clinical results suggest that the capsid-specific CD8+T cell response has a negative effect on therapeutic success. In an in vitro analysis using an engineered AAV virus carrying immune-dominant SIINFEKL peptide in the capsid backbone, we have previously demonstrated that capsid antigen presentation from full (genome containing) AAV capsids requires endosome escape and is proteasome dependent and that no capsid antigen presentation is induced from empty virions. In the present study, we examined capsid antigen presentation from administration of empty virions in animal models. In wild-type mice, similar to AAV full particles, capsid antigen presentation from AAV empty virion infection was dose dependent, and the kinetics studies showed that antigen presentation was detected from 2 to 40 days after AAV empty virion administration. In the transporter associated with antigen processing 1 deficient (TAP-/-) mice, capsid antigen presentation was inhibited from both AAV full and empty virions, but higher inhibition was achieved from AAV full particle administration than that from empty virions. This indicates that the pathway of capsid antigen presentation from AAV transduction is dependent on proteasome-mediated degradation of AAV capsids (mainly for full particles) and that the endosomal pathway may also play a role in antigen presentation from empty particles but not full virions. The capsid antigen presentation efficiency from AAV preparations was positively correlated with the amount of empty virions contaminated with full particles. Collectively, the results indicate that contamination of AAV empty virions induces efficient antigen presentation in vivo and the mechanism of capsid antigen presentation from empty virions involves both endosomal and proteasomal pathways. The elucidation of capsid antigen presentation from AAV empty virions may allow us to rationally design effective strategies to prevent elimination of AAV transduced target cells by capsid specific CD8+ T cells.

Project description:Adeno-associated virus (AAV) has proven to be a promising candidate for gene therapy due to its nonpathogenic nature, ease of production, and broad tissue tropism. However, its transduction capabilities are not optimal due to the interaction with various host factors within the cell. In a previous study, we identified members of the small ubiquitin-like modifier (SUMO) pathway as significant restriction factors in AAV gene transduction. In the present study, we explored the scope of this restriction by focusing on the AAV capsid and host cell proteins as targets. We show that during vector production, the capsid protein VP2 becomes SUMOylated, as indicated by deletion and point mutations of VP2 or the obstruction of its N terminus via the addition of a tag. We observed that SUMOylated AAV capsids display higher stability than non-SUMOylated capsids. Prevention of capsid SUMOylation by VP2 mutations did not abolish transduction restriction by SUMOylation; however, it reduced activation of gene transduction by shutdown of the cellular SUMOylation pathway. This indicates a link between capsid SUMOylation and SUMOylation of cellular proteins in restricting gene transduction. Infection with AAV triggers general SUMOylation of cellular proteins. In particular, the DAXX protein, a putative host cell restriction factor that can become SUMOylated, is able to restrict AAV gene transduction by reducing the intracellular accumulation of AAV vectors. We also observe that the coexpression of a SUMOylation inhibitor with an AAV2 reporter gene vector increased gene transduction significantly.IMPORTANCE Host factors within the cell are the major mode of restriction of adeno-associated virus (AAV) and keep it from fulfilling its maximum potential as a gene therapy vector. A better understanding of the intricacies of restriction would enable the engineering of better vectors. Via a genome-wide short interfering RNA screen, we identified that proteins of the small ubiquitin-like modifier (SUMO) pathway play an important role in AAV restriction. In this study, we investigate whether this restriction is targeted to the AAV directly or indirectly through host cell factors. The results indicate that both targets act in concert to restrict AAV.

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Electrophoresis-Mediated Characterization of Full and Empty Adeno-Associated Virus Capsids.

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Electrophoresis-Mediated Characterization of Full and Empty Adeno-Associated Virus Capsids.

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