Using Base-Editing to Enhance Genetic Editing Capabilities

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Allogeneic cell immunotherapies have the potential to be useful across a broad range of therapeutic areas; however, these ‘off-the-shelf’ therapies face certain limitations that prevent their wide use. In particular, allogeneic therapies can potentially lead to host immune systems rejecting the donor cells and, in some cases, the donor cells are rapidly eliminated before they can perform their required therapeutic role (1).

To enhance allogeneic cell immunotherapies, genetic engineering approaches are being employed to target gene modifications and knockout genes that may cause elimination of donor cells. Clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (CRISPR-Cas9) has been used to successfully perform targeted editing; however, it does so by creating double-strand breaks (DSBs) in DNA, which may lead to chromosomal loss or structural variation (1).

More recently, a novel technology, base editing, is being used that does not cause DSBs. “Both CRISPR-Cas and base editing systems can be used to target gene modifications with great specificity, but using CRISPR-Cas to do this relies on DSBs, which can be disruptive and deleterious to the genes you're to this all of your editing, especially when you're trying to multiplex targets,” explained Shannon Hinsdale, scientist II—Biology R&D at Revvity, a diagnostics and technology company that supports technology platforms for the advancement of cell therapies into the clinic (2). “However, with the base-editing approach, you can still harness the targeting power of CRISPR-Cas without relying on DSBs. And you can modify genes through a number of ways; two of which we commonly use are through introduction of premature stop codons and alteration of splice sites.”

Focusing on Revvity’s Pin-point base-editing platform for her presentation, Hinsdale revealed that it is “a three-component system that relies on an RNA guided enzyme, an extended RNA guide fused to an RNA aptamer, and then an effector enzyme, such as the cytosine deaminase, that recruits an effector protein.” Hinsdale provided data demonstrating the performance of the base-editing platform in primary T cells as well as in induced pluripotent stem cells (iPSCs), highlighting its viability in multiplexing when compared with CRISPR-Cas9. “In our experience, this [viability] equates to about two times the number of total cells at day three post electroporation,” she said. “Additionally, when we look at fold expansion, whether we're looking at three or four targets, with the Pin-point system, there's no effect versus with the [CRISPR]-Cas9, traditional, wild type Cas9, we see that there is a decrease in fold expansion as the number of targets increases.”

“[With the Pin-point platform,] we can achieve efficient editing in T cells, greater than 75% target based without enrichment, 50% of all four proteins knocked out on the surface. We show a good safety profile for edited cells, and we show that [base-editing] can be used for complex engineering in both T cells and iPSCs,” she concluded.

References

1. Porreca, I.; Blassberg, R.; Harbottle, J.; et al. An Aptamer-Mediated Base Editing Platform for Simultaneous Knockin and Multiple Gene Knockout for Allogeneic CAR-T Cells Generation. Molecular Therapy 2024, 32 (8), 2692–2710.

2. Hinsdale, S. Engineering Allogeneic Cell Immunotherapies in a Single Intervention with the Innovative Pin-point Base Editing Platform. Oral Abstract Presentation at ASGCT’s Advancing Gene + Cell Therapies for Cancer Conference, Philadelphia, PA, Oct. 16, 2024.

Hinsdale’s presentation is available ‘on demand’ through the American Society of Gene + Cell Therapy.