Isolation of Adeno-Associated Viral Vectors Through a Single-Step and Semi-Automated Heparin Affinity Chromatography Protocol

作者： Miguel M. Lopes*1,2,3, Sara M. Lopes*1,2,3, Rafael Baganha1,2,4, Carina Henriques1,2,5, Ana C. Silva1,2,3, Diana D. Lobo1,2,3, Luísa Cortes1,2,3,6, Luís Pereira de Almeida*1,2,4,5, Rui Jorge Nobre*1,2,3,4 1CNC-UC - Center for Neuroscience and Cell Biology,University of Coimbra, 2CIBB - Center for Innovative Biomedicine and Biotechnology,University of Coimbra, 3IIIUC - Institute for Interdisciplinary Research,University of Coimbra, 4ViraVector - Viral Vectors for Gene Transfer Core Facility,University of Coimbra, 5FFUC - Faculty of Pharmacy,University of Coimbra, 6MICC-CNC - Microscopy Imaging Center of Coimbra - CNC,University of Coimbra

简介：

Overview

This manuscript describes a detailed protocol for the generation and purification of adeno-associated viral vectors using an optimized heparin-based affinity chromatography method. The resulting vectors exhibit high purity and biological activity, proving their value in preclinical studies.

Key Study Components

Area of Science

• Gene therapy
• Adeno-associated viruses
• Viral vector production

Background

• Current methods for purifying adeno-associated viruses (AAVs) often involve ultracentrifugation.
• These methods can be time-consuming and have limited scalability.
• Chromatography techniques are emerging as alternatives for AAV purification.
• Heparin-based affinity chromatography is explored for its efficiency and effectiveness.

Purpose of Study

• To develop a scalable and cost-effective protocol for producing high-purity AAVs.
• To eliminate the need for ultracentrifugation in the purification process.
• To enhance the biological activity of the produced viral vectors for preclinical applications.

Methods Used

• Transient transfection of HEK 293T cells for AAV production.
• Use of heparin-based affinity chromatography for purification.
• Step-by-step protocol detailing cell culture, transfection, and purification processes.
• Evaluation of the purity and biological activity of the resulting AAVs.

Main Results

• The heparin-based method yields highly pure and biologically active AAVs.
• The protocol can be completed in six days.
• Scalability and cost-effectiveness are improved compared to traditional methods.
• Potential for further refinement by establishing stable producer cell lines.

Conclusions

• The optimized protocol represents a significant advancement in AAV production.
• It offers a practical solution for researchers in gene therapy and related fields.
• Future work may focus on enhancing scalability and reducing empty particle presence.

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