Collaboration Helps Drive Advancement

September 27, 2023 / by Patrick Paez, Ph.D.

Making use of ultra-low endotoxin Cas9 from Aldevron

The latest research article from the Innovative Genomics Institute (IGI) at the University of California, Berkeley, highlights the collaboration between Aldevron with the laboratories of Nobel Laureate Dr. Jennifer Doudna and Dr. David Savage. Published in Molecular Therapy-Nucleic Acids, “Genome editing in the mouse brain with minimally immunogenic Cas9 RNPs,” shows how Aldevron generated ultra-low endotoxin Cas9 for IGI to enable a novel in vivo CRISPR delivery system, targeted directly into the brain for editing.

The study compared adeno-associated virus 9 (AAV9), the current “gold standard” for central nervous system gene editing, to IGI’s direct injection of the mouse striatum with cell-penetrating Cas9-RNPs termed convection-enhanced delivery (CED). Alternative delivery systems of AAV for CNS disorders remain a theme well entrenched within the field due to:

• Cargo capacity limitations seen with AAV
• Immunogenicity—or the immune system's ability to destroy the virus before it can infect target cells and insert the therapeutic gene intended for the therapy
• High costs in manufacturing the AAV quality level for human treatment using GMP grade materials

Authors of these types of reports often tell stories that required thousands of hours of development and were years in the making. In this publication, readers are guided through the journey using several graphics, summarized as:

Figure 1: Authors show in vivo proof of concept that delivering Cas9 RNPs via direct injection into the brain's striatum can edit neurons like AAV9-mediated delivery. They test two different injection catheters to optimize CED.

• They find that while AAV9 can edit larger brain volumes, Cas9 RNPs delivered via CED significantly edited more neurons in vivo.

Figure 2: Discovered measurable levels of immune responses from both treatment modalities, prompting them to seek a lower endotoxin version of their Cas9.

• Both treatments displayed IgG antibodies against the treatment modalities, suggesting an activated immune response in response to the delivery of Cas9
• Cas9 RNPs displayed an increase in microglia presence—a macrophage resident of the brain, compared to the Cas9 AAV-treated animals
• Cas9 AAV showed increased CD3e expression at 28 days and not in the Cas9 RNP treated, suggesting an increase of adaptive immune response in the Cas9 AAV-treated animals
• The immune responses were notable in both treatment modalities. However, the authors hypothesized that the immune activation might be mitigated by improving the endotoxin levels of the Cas9 RNPs CED method

Figure 3: Cas9 RNPs manufactured using an optimized low endotoxin Cas9 manufactured by Aldevron* reduced local immune response and adaptive immune responses.

• Ultra-low endotoxin Cas9 protein prevented reduced microgial presence after Cas9 RNP injection (figure 3C)
• The authors summarize the benefits of ultra-low endotoxin Cas9 RNPs in Figure S14B, comparing AAV9 Cas9-gRNA, when injected into the brain. The optimized Cas9 RNPs offers GMP-compliant low endotoxin standard levels, and enables high-levels of editing in neurons within a localized region, minimize adaptive immune response, and timely and cost-efficient scale-up

The authors demonstrated a viable alternative in obtaining ultra-low endotoxin nuclease with the delivery of high-grade RNPs to meet the FDA required thresholds and an alternative to viral vector delivery (Supplemental figure 12A). This study shows the work at each step as obstacles were encountered in developing a potentially safe and translational method of delivering Cas9 RNPs via CED. This endeavor to reduce the endotoxin of Cas9 to meet FDA standards for intrathecal injection was a challenge Aldevron was ready to take on.

“Advancing every day means helping amazing researchers. The timeline for developing and manufacturing their Cas9 presented a challenge, but we operate with a sense of urgency. We don’t like to use the word impossible with our client's timelines,” said Tom Foti, VP of the Protein Business Unit, when Aldevron was approached for assistance with the project.

And as Athen Kimberlin, a Group Leader in Aldevron’s Protein Business Unit, points out, the customized protein with a low-endotoxin process was delivered within six weeks of the initial call. “There was definitely a buzz,” he said.

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*Aldevron provides RNPs only to customers who are duly licensed, including to make and have made RNPs, for their intended use.