2024 ESGCT Poster Presentation

Hello, welcome to MaxCyte! My name is Isabel Daher, and I am a research associate here at the Technical Applications lab. We’re excited to present to you our efficient and scalable manufacturing process for the packaging of gene editor RNPs into virus-like particles.
Our workflow first starts by combining the producer cells, either adherent or suspension HEK293 cells, with a four-plasmid mix encoding for both the structural components of the VLPs as well as the cargo. Here, we package CRISPR RNPs for gene editing.

The cells and plasmid master mix are then loaded into one of our static or flow processing assemblies and then inserted into the ExPERT GTx instrument. A pre-loaded and optimized protocol delivers the appropriate energy to enable plasmid delivery before cells are transferred to their respective culture vessel for a short recovery period. Production media is then added, along with a chemical enhancer to improve yield, and VLPs are harvested from the culture supernatant one or two days post-electroporation.

To assess the VLP yield, VLPs are transduced onto either adherent HEK293T or primary human T cells. After three to five days, cells are harvested for genomic DNA extraction followed by sequencing or analyzed via flow cytometry.

When optimizing electroporation, the main two parameters to consider are cargo concentration and electroporation energy. In adherent HEK293 cells, in a dose-responsive manner, increasing DNA concentration helps to improve the potency of the VLPs, reported here as percent indel formation. Additionally, increasing electroporation energy trends with improved potency. And, to further improve the titer, a chemical enhancer was tested in the production media to better improve the yield.

We can now benchmark against a commercially available chemical transfection reagent. On day two, VLPs produced via electroporation exhibit higher editing activity than those produced with chemical transfection. And this is also reflected by the capsid, or p30, titer measured via ELISA.

When comparing the kinetics of VLP production between methods, it is clear that electroporation can enable rapid one-day harvest of highly functional VLPs compared to chemical transfection, where at best, a day-two harvest is required.
This can be explained by not only higher p30 measurements, but also higher and faster incorporation of the CRISPR RNP cargo into VLPs produced via electroporation.

While we now have a validated process in adherent cells, we wanted to test suspension HEK cells for production to accommodate the scale-up required in clinical and commercial manufacturing. Shown here, the same process validated in adherent cells can be seamlessly applied to suspension culture. And the same enhancer can be used to improve yield.

Electroporation can also enable different gene editor RNPs to be packaged. Shown here is an adenine base editor RNP, compared to the wild-type Cas9. Shifting the producer cell culture helps to improve the titer, and therefore, editing capability.

VLPs packaged with RNPs are amenable for in vivo gene editing, and thus must be capable of safe and efficient editing. Adenine base editor-loaded VLPs do not cause cytotoxicity to activated human T cells but can efficiently edit the B2M locus enough to disrupt B2M surface expression more efficiently in VLPs produced with electroporation compared to chemical transfection.

So, we now have an optimized workflow for both adherent and suspension HEK cells, and we’ve also demonstrated functionality. It’s now time to assess the scalability of MaxCyte’s electroporation platform. Here, one billion cells were electroporated in an R-20K processing assembly, and 120 million cells were cultured per condition. When comparing the same producer cell number, electroporation is seamlessly scalable across a 15-fold volume range, and VLPs from both scales outperform those produced with chemical transfection harvested on day one and day two.

Again, a chemical enhancer was added to the production media to help improve functional titer, or editing, as well as the physical, or capsid, titer.

EC50, or effective concentration 50, is a metric used to identify the concentration or dose of a treatment to induce a 50% response; here, is related to indel formation. On day one harvest, electroporation produces VLPs with an approximate 20-fold improvement in potency compared to chemical transfection and a 4.5-fold improvement on day two.

To summarize, we demonstrate MaxCyte’s GMP-compatible electroporation platform is an efficient and scalable method for VLP production, capable of transfecting one billion cells as shown here. We can enable higher VLP-mediated editing activity on a shortened timeline, and in both adherent and suspension HEK cells as you scale up throughout your drug development journey. MaxCyte’s electroporation is a simple, time-saving alternative to chemical transfection for VLP production.

Thank you for attending and please contact us if you are interested or have questions.