Hear from the Marson lab at UCSF

Alexander Marson’s lab at the University of California San Francisco, UCSF, aims to understand gene programs controlling the behavior and properties of immune cells, particularly T cells. His lab leverages genetic engineering strategies to better understand immune cell functions and instruct T cell modification for future cell therapies for a broad range of human disease states.

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We have a requirement for long single-stranded DNA that can be used in a clinical manufacturing process, and this is where we benefited from a wonderful collaboration with GenScript

- Alexander Marson at GCE Summit, GenScript

Project’s Critical Challenge

Marson’s team has been developing CRISPR/Cas9 editing tools for non-viral approaches to gene editing, enabling precise and efficient modification of immune cells. They found that co-electroporating the Cas9 ribonucleoprotein complex with DNA payloads into T cells represented an efficient strategy to knock-in gene sequences of interest. For example, by replacing the TCR alpha and beta sequences to re-write T cell specificity.

To improve non-viral gene knock-in efficiency, Marson’s team engineered modifications to the DNA payloads and Cas9 protein, promoting co-localization and delivery or “shuttling” to the nucleus. One essential modification was the adoption of single-stranded DNA payloads, which allowed them to bypass the toxicity associated with high levels of double-stranded DNA. Therefore, Marson’s group adopted the use of single-stranded DNA donor templates to achieve efficient and safe editing of various T cells for therapies. At this stage, the main challenge confronted by the group was achieving the manufacturing of high-quality and reliable single-stranded DNA at sufficient levels for clinical use while meeting GMP standards.