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

作者： Amira D. Rghei1, Brenna A. Y. Stevens*1, Sylvia P. Thomas*1, Jacob G. E. Yates*1, Benjamin M. McLeod1, Khalil Karimi1, Leonardo Susta1, Byram W. Bridle1, Sarah K. Wootton1 1Department of Pathobiology,University of Guelph
简介：

Overview

This protocol outlines a cost-effective method for producing high-titer, research-grade AAV viral vectors using adherent HEK293 cells. The approach is designed for efficiency and high purity, making it suitable for preclinical studies in large animal models.

Key Study Components

Area of Science

  • Gene therapy
  • Viral vector production
  • Cell culture techniques

Background

  • AAV vectors are crucial for gene therapy applications.
  • HEK293 cells are commonly used for AAV production.
  • High titer and purity are essential for effective research outcomes.
  • This method minimizes handling disruptions to cells.

Purpose of Study

  • To provide a reliable protocol for AAV vector production.
  • To enhance the efficiency of viral vector generation.
  • To support research in gene therapy and related fields.

Methods Used

  • Utilization of adherent HEK293 cells in cell culture chambers.
  • Affinity chromatography for purification of AAV vectors.
  • Collection of cells at 80% confluency for optimal yield.
  • Time-efficient procedures to reduce cell handling.

Main Results

  • Consistent yield of >1 x 1013 vector genomes/mL.
  • High purity of produced AAV vectors.
  • Protocol suitable for large animal studies.
  • Adaptable for various AAV serotypes.

Conclusions

  • This protocol significantly aids in the production of AAV vectors.
  • It is a valuable tool for advancing gene therapy research.
  • Potential for broader applications in viral vector production.

Frequently Asked Questions

What are AAV vectors used for?
AAV vectors are primarily used in gene therapy to deliver genetic material into cells.
Why use HEK293 cells for AAV production?
HEK293 cells are efficient for producing high-titer AAV vectors due to their robust growth and transfection properties.
How does affinity chromatography purify AAV vectors?
Affinity chromatography separates AAV vectors based on their binding properties, resulting in high purity.
What is the significance of high titer in AAV production?
High titer ensures that sufficient viral vectors are available for effective research and therapeutic applications.
Can this protocol be adapted for other AAV serotypes?
Yes, the protocol can be modified for the production of various AAV serotypes that bind heparan sulfate.
Is this method time-efficient?
Yes, the protocol is designed to minimize handling and disruptions, making it time-efficient.

Here we provide a detailed procedure for large-scale production of research-grade AAV vectors using adherent HEK 293 cells grown in cell stacks and affinity chromatography purification. This protocol consistently yields >1 x 1013 vector genomes/mL, providing vector quantities appropriate for large animal studies.

标签: Adeno-Associated Virus,    AAV Vectors,    Preclinical Studies,    Gene Therapy,    HEK293 Cells,    Cell Culture,    Viral Vector Production,    Cell Density Analysis,    Trypsinization,    Polyethyleneimine-DNA,    Centrifugation,    Cell Confluency,    DMEM,    Incubation Protocols,   
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