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CRISPR in Cell Therapy: Will It Be Superseded by Base Editors?

Base editors hold great promise for safer multiplex gene editing

Jonathan Frampton, PhD

Jonathan Frampton is a business development professional who has been working for Horizon Discovery for the past nine years and currently as their corporate development partner. He is always scouting for exciting novel technology that could complement Horizon’s already extensive gene engineering toolkit. In addition to this, he works closely with Horizon’s partners to manage out-licensing opportunities.

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Published:Dec 16, 2020
|2 min read
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Delivering cell therapy to oncology patients with solid tumors has to date been unsuccessful. The main issues include compromised survival of engineered therapeutic primary cells in the toxic tumor microenvironment, as well as poor persistence and a low proliferation rate. An attractive path to overcome these issues involves gene editing the therapeutic primary cell to enable it to survive, proliferate, and successfully target the cancerous cells in the patient. To achieve a modified cell with this phenotype, it is likely that between five and ten simultaneous gene knockouts would be required. 

While CRISPR has been a massively disruptive technology within the gene editing field and has delivered significant scientific breakthroughs since its emergence, it might not be the most suitable gene editing technology for cell therapy.

Off-target CRISPR events

The mode of action of CRISPR is to generate a DNA double-strand break (DSB) to facilitate a gene modification event. In the context of the stability of the whole genome, this is a potentially catastrophic occurrence. The formation of a DSB at the targeted gene editing site is not in itself an issue. However, issues arise when off-target DSBs are generated, potentially resulting in genomic changes that steer an edited cell towards immortality within the transfused patient. 

These off-target CRISPR events are manageable when one or two genes are targeted through downstream quality control processes, such as whole genome sequencing and other screens, to identify cells without off-target activity. But when three or more gene editing events are required, the chances of identifying suitable, safely edited cells for therapy become slim. 

Base editors step forward

Are there other technologies on the horizon that are better suited than CRISPR to deliver the required number of gene edits for cell therapy targeting solid tumors? Base editors hold great promise for safer multiplex gene editing. While there is excitement surrounding the prospect of base editing for second generation cell-based therapeutics, we still need to understand this technology better.

Base editors can be used to introduce a stop codon in a specific gene through a mechanism that uses a deaminase and the generation of a DNA single-strand break (SSB). Introduction of a SSB is substantially more favorable than a DSB because SSBs generate fewer off-target insertions, deletions, and translocations. So, do base editors represent a genuine challenge for CRISPR-mediated gene modification in the clinic? 

Base editors have undergone numerous iterations since their inception in 2016 and early data appear to support the hype. They can be tuned to target a specific sequence and demonstrate high on-target editing efficiency to introduce a single base change. Although the outlook is bright for base editors, data indicate off-target deamination elsewhere in the genome can occur and some deaminase also causes off-target alterations in RNA. This could potentially lead to gene expression changes in the cell, causing unwanted downstream phenotypes. Work is ongoing to understand these changes better and to limit their occurrence prior to mainstream use of this technology in the clinic. 

The next generation of cell-based therapeutics

Although CRISPR has been a disruptive gene editing technology and its application to drive research forward has been revolutionary, its limitations in certain areas, and the need for alternative technologies, should be recognized. One of these areas is cell therapy, where multiplex gene knockout is an attractive option to develop the next generation of cell-based therapeutics. CRISPR is not currently built to provide error-free multiplex editing whereas early data suggest base editing is. Maybe it is time for this new technology to step into the ring.