2024 ASGCT Poster Presentation

Efficient Scalable Manufacturing of Virus-like Particles for the Delivery of CRISPR-Cas9 Ribonucleoproteins using a cGMP-Compliant Electroporation Platform

American Society of Gene and Cell Therapy annual meeting
Baltimore, Maryland, USA
May 10, 2024
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Our MaxCyte scientist, Isabel Daher, presents her poster from the 2024 American Society of Gene and Cell Therapy annual meeting

Genome editing tools such as CRISPR-Cas9 nucleases, base editors, and prime editors hold tremendous promise for treating human diseases by being able to specifically modify a targeted region of the DNA genome. However, there are limited in vivo delivery options that are safe, efficient and transient. Moreover, viral vectors such as AAV are hampered by limited cargo size capacity. Virus-like particles (VLPs) offer a solution to these problems. VLPs are derived from retroviral structural proteins, which can be engineered to specifically package a cargo of interest. They are safer than traditional viral vectors because they lack a viral genome but can utilize the traditional virus delivery machinery to target and enter cells. Recently, VLPs have been reported to efficiently package base editors and prime editors for delivery into mice at therapeutically relevant levels. To realize the potential of VLPs for in vivo delivery, alternative methods to scale up their manufacturing will be critical for future clinical applications. Here, we utilized the MaxCyte ExPERT GTx, a cGMP-compliant electroporation instrument, to manufacture VLPs—packaged with CRISPR-Cas9 RNPs for genome editing in target cells—in adherent and suspension HEK293 cells. We found that electroporation consistently produced significantly higher yields of functional VLPs compared to a commercially available transfection reagent, with up to a 20-fold improvement when using an optimized electroporation protocol. Furthermore, the production of VLPs using electroporation exhibited favorable production kinetics compared to other transfection methods, enabling a shorter VLP manufacturing process. Finally, we demonstrated the scalability of VLP production across a 15-fold volume range with minimal re-optimization. In summary, our results show that electroporation is a viable means for consistent, efficient and scalable manufacturing of VLPs for gene-editing applications and has high promise to address needs for future clinical and commercial VLP manufacturing.

Key takeaways

  • Transfect up to 100 billion cells using a cGMP-compliant method that can enable rapid clinical manufacturing
  • Produce functional VLPs in both adherent and suspension HEK293 cells
  • Multiple performance parameters were superior using electroporation over chemical transfection

Watch Isabel's poster presentation

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Presenter

Headshot of Isabel Daher

Isabel Daher

Research Associate at MaxCyte

Isabel Daher graduated from the University of Florida with a bachelor of science in microbiology and cell science. She studied the role of gammaherpesvirus-encoded microRNAs and their role in promoting pathogenesis in primary and persistent infection. She then transitioned to the National Cancer Institute where she completed a two-year post-baccalaureate fellowship focusing on the noncanonical functions of gammaherpesvirus enzymes and their interactions with the host proteome. Now at MaxCyte, Isabel works as a research associate in the Technical Applications Lab where she focuses on developing and improving a virus-like particle production workflow designed to streamline production and maximize yield.