Buffer Valency Engineering Enables High-concentration and Shelf-stable DNA Transfection Particles for Viral Vector...

Area of Science:

• Biotechnology
• Gene Therapy Manufacturing
• Nanoparticle Engineering

Background:

• Adeno-associated virus (AAV)-mediated gene therapy requires cost-effective and scalable production methods.
• Current transient transfection methods for AAV production face challenges with particle instability and concentration limits, impacting reproducibility and scalability.

Purpose of the Study:

• To develop a streamlined and scalable strategy for producing shelf-stable, highly concentrated plasmid DNA (pDNA)/poly(ethylenimine) (PEI) transfection particles.
• To overcome the limitations of existing methods hindering the clinical translation of AAV gene therapies.

Main Methods:

• Incorporation of trivalent citrate ions into dilution buffers to kinetically modulate electrostatic complexation.
• Achieving uniform nanoparticle assembly and preventing aggregation at high concentrations.
• Utilizing a robust particle assembly process compatible with standard workflows and scalable mixing conditions.

Main Results:

• Generation of shelf-stable, highly concentrated pDNA/PEI transfection particles (200 μg/mL, a tenfold increase).
• Reduction in required dosing volume to 0.5% of cell culture medium.
• Demonstration of equivalent AAV production efficiencies compared to standard methods across various production scales.

Conclusions:

• The citrate-ion-mediated strategy enables robust, scalable, and cost-effective AAV manufacturing.
• This method significantly improves the practicality and efficiency of producing AAV for gene therapy applications.
• The developed process supports the advancement of clinical translation for AAV-based therapies.