LOS ANGELES — That the honor of opening the Presidential Symposium of the American Society of Gene and Cell Therapy (ASGCT) conference should fall to 2020 Nobel laureate Jennifer Doudna, PhD, the CRISPR genome editing pioneer, was no surprise. But the topic of Doudna’s presentation—new research in the arena of T-cell therapy—might have raised a few eyebrows in the packed audience.

Doudna is the Li Ka Shing Chancellor’s Professor of Biomedical Science at the University of California, Berkeley, co-founder of the Innovative Genomics Institute (IGI), and an investigator with the Howard Hughes Medical Institute. She is the co-founder of several genome editing companies, including Caribou Biosciences, Intellia Therapeutics, and Mammoth Biosciences.

More than a decade after the landmark 2012 CRISPR-Cas9 Science paper, co-authored with Emmanuelle Charpentier, PhD, and colleagues, Doudna emphasized the translational aspects of CRISPR-Cas9 gene editing in a talk titled, “CRISPR Chemistry and Applications in the Clinic.”

“One of the reasons I founded the Innovative Genomics Institute [in 2014] was because CRISPR as a technology has incredible potential to impact people’s lives, if we can figure out how to make these tools more broadly available in the clinic,” stated Doudna in a pre-recorded video.

RNA-guided DNA recognition has developed as a powerful toolbox with capabilities, such as gene disruption, gene repair, transcriptional control, diagnostics, and imaging. However, Doudna described two key areas that continue to challenge CRISPR’s clinical potential: effective genome editor delivery and ensuring editing precision.

At a chromosome loss

Doudna focused on the applications of CRISPR editing for adoptive T-cell therapies. She cited a 2020 landmark study from Carl June, MD, and colleagues at the University of Pennsylvania that reported a Phase I clinical trial to assess the safety and feasibility of CRISPR-Cas9 gene editing in three patients with advanced cancer. That study evaluated ex vivo CRISPR gene editing, in which T cells were removed from the patient, edited using CRISPR to disrupt key antitumor immunity genes, and re-introduced into the patient.

Doudna described the challenges of precision editing, as breaks introduced into the genome from CRISPR-Cas9 editing can trigger aberrant effects such as chromosome loss. Key questions include understanding how general this phenomenon is and what factors influence it. In addition, does chromosome loss impact clinical viability and can it be avoided?

In a study posted as a bioRxiv preprint, led by Doudna lab graduate student Connor Tsuchida, the occurrence of chromosome loss was evaluated using a 400 single guide RNA library that included active and inactive genes and targeted every chromosome. The results indicated that more than 3% of targeted primary human T cells had whole or partial chromosome loss as a result of Cas9 cleavage, indicating that chromosome loss occurs regardless of guide RNA or target choice.

But what did these observations mean for clinical viability?

Tsuchida found answers after serendipitously connecting with Howard Chang, MD, PhD, professor of cancer research and genetics at Stanford University, at a conference at Cold Spring Harbor Laboratory last year. Chang had additional data from T-cell infused patients that had not yet been published. And so, a collaboration began.

“What was very interesting was that [analysis of these T cells from clinical trials] indicated very little chromosome loss in these patients. This was a surprising observation!” exclaimed Doudna. “When we took cells that had the same modifications done in the laboratory, we saw that they did result in chromosome loss. So what was going on?”

Doudna presented compelling data that indicated that the difference was explained by the order of operations between the laboratory and clinical trial protocols. In the laboratory, primary T cells are isolated, and activated and stimulated prior to treatment with reagents such as CRISPR-Cas9. Alternatively, the clinical protocol activates and stimulates cells after treatment with CRISPR-Cas9 reagents.

The reason for this protocol difference was entirely practical. In the clinical setting, there was insufficient GMP-produced Cas9 available to use on cells that were first activated and stimulated.

“We found that the [method in which] these cells are handled can minimize chromosome loss while maintaining editing efficiency, which can be a very important change to future [implementation of these therapies],” Doudna remarked.

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