Search research articles

ABOUT JoVE

Overview Leadership Blog JoVE Help Center

AUTHORS

Publishing Process Editorial Board Scope & Policies Peer Review FAQ Submit

LIBRARIANS

Testimonials Subscriptions Access Resources Library Advisory Board FAQ

RESEARCH

JoVE Journal Methods Collections JoVE Encyclopedia of Experiments Archive

EDUCATION

JoVE Core JoVE Business JoVE Science Education JoVE Lab Manual Faculty Resource Center Faculty Site

Terms & Conditions of Use

Related Concept Videos

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by

Same author

Enrichment strategies for mucin-domain O-glycoproteins.

Methods in enzymology·2026

Same author

Characterization of O-glycoproteases for applications in mass spectrometry.

Methods in enzymology·2026

Same author

Sustained clinical remission and relapse of severe eosinophilic asthma following long-term mepolizumab treatment.

Allergology international : official journal of the Japanese Society of Allergology·2026

Same author

The PERK-GADD45A axis is a key driver of hepatic stellate cell activation.

Hepatology communications·2026

Same author

Performance of IBD machine learning classifiers varies across microbiome training data independent of geographic diversity.

bioRxiv : the preprint server for biology·2026

Same author

B3GNT7 regulates mucin glycosylation and protects against colitis and infection.

bioRxiv : the preprint server for biology·2026

Same journal

Characterization and quantitation of baculoviral DNA in rAAV vectors produced in Sf9 cells.

Molecular therapy. Methods & clinical development·2026

Same journal

<i>In vivo</i> delivery of a fluorogenic antisense oligonucleotide into human leukocytes in a humanized mouse model system.

Molecular therapy. Methods & clinical development·2026

Same journal

Development of high-throughput analytical methods for characterization and quantitation of rAAV genome integrity.

Molecular therapy. Methods & clinical development·2026

Same journal

Morphological changes induced by buffer exchange during preparation of mRNA-lipid nanoparticles occur in a stepwise manner as pH is elevated.

Molecular therapy. Methods & clinical development·2026

Same journal

Targeted inhibition of <i>ELANE</i> expression using adenine base editing to treat severe congenital neutropenia.

Molecular therapy. Methods & clinical development·2025

Same journal

AAV-PHP.eB achieves superior neuronal transduction over AAV9 in pigtail macaques following intracerebroventricular administration.

Molecular therapy. Methods & clinical development·2025

See all related articles

Search research articles

Related Experiment Video

Updated: Dec 7, 2025

Production, Purification, and Quality Control for Adeno-associated Virus-based Vectors

Production, Purification, and Quality Control for Adeno-associated Virus-based Vectors

Published on: January 29, 2019

Methods Matter: Standard Production Platforms for Recombinant AAV Produce Chemically and Functionally Distinct

Neil G Rumachik¹, Stacy A Malaker¹, Nicole Poweleit²

¹Department of Chemistry, Stanford University, Stanford, CA 94305, USA.

Molecular Therapy. Methods & Clinical Development

|September 30, 2020

Summary

This summary is machine-generated.

Recombinant adeno-associated virus (rAAV) production methods impact vector performance. Human cell-produced rAAV shows higher potency and distinct post-translational modifications compared to insect cell-produced vectors, with potential clinical implications.

Keywords:

AAV PTM adeno-associated virus baculovirus-Sf9 capsid human mass spectrometry methylation post-translational modification potency

More Related Videos

Production of Adeno-Associated Virus Vectors in Cell Stacks for Preclinical Studies in Large Animal Models

Production of Adeno-Associated Virus Vectors in Cell Stacks for Preclinical Studies in Large Animal Models

Published on: June 30, 2021

Author Spotlight: Advancing Gene Therapy Research with High-Titer Adeno-Associated Virus Vector Production

Author Spotlight: Advancing Gene Therapy Research with High-Titer Adeno-Associated Virus Vector Production

Published on: May 3, 2024

Related Experiment Videos

Last Updated: Dec 7, 2025

Production, Purification, and Quality Control for Adeno-associated Virus-based Vectors

Production, Purification, and Quality Control for Adeno-associated Virus-based Vectors

Published on: January 29, 2019

Production of Adeno-Associated Virus Vectors in Cell Stacks for Preclinical Studies in Large Animal Models

Production of Adeno-Associated Virus Vectors in Cell Stacks for Preclinical Studies in Large Animal Models

Published on: June 30, 2021

Author Spotlight: Advancing Gene Therapy Research with High-Titer Adeno-Associated Virus Vector Production

Author Spotlight: Advancing Gene Therapy Research with High-Titer Adeno-Associated Virus Vector Production

Published on: May 3, 2024

Area of Science:

Biotechnology and Gene Therapy
Molecular Biology
Virology

Background:

Recombinant adeno-associated virus (rAAV) is a key vector for gene therapy.
Existing production methods, primarily human HEK293 cells and Sf9 insect cells, yield rAAV with unexplained performance differences.
Understanding these differences is crucial for optimizing rAAV-based therapeutics.

Purpose of the Study:

To conduct a controlled comparative analysis of rAAV production using human HEK293 cells versus Sf9 insect cells.
To characterize the impact of host cell differences on rAAV capsid modifications, genome methylation, and vector performance.
To elucidate the clinical implications of platform-specific rAAV characteristics.

Main Methods:

Comparative production of rAAV in HEK293 and Sf9 cells, keeping other parameters constant.
Proteomic profiling (mass spectrometry), isoelectric focusing, cryo-EM, and denaturation assays for capsid characterization.
Genomic/epigenomic sequencing, human cytokine profiling, and in vitro/in vivo transduction assays for functional assessment.

Main Results:

Identified distinct post-translational modifications (PTMs) on rAAV capsids, including glycosylation, acetylation, phosphorylation, and methylation, differing between production platforms.
Demonstrated differential methylation of rAAV genomes based on the production host cell.
Showcased higher potency of human-derived rAAV compared to Sf9-derived vectors in vitro and in vivo, including in humanized liver models.

Conclusions:

Host cell type significantly influences rAAV PTMs and genome methylation, affecting vector characteristics.
Differences in host cell protein impurities and their PTMs, such as immunogenic glycans, are platform-dependent.
Human cell-produced rAAV exhibits superior potency, suggesting potential advantages for clinical applications regarding efficacy, immunogenicity, and cost.