In brief

Pre-existing antibodies and strong T cell responses against RfxCas13d, an RNA-editing enzyme isolated from the bacteria Ruminococcus flavefaciens, highlights safety considerations for using RNA-editing therapies in patients.

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Humans reject helpful RNA-editing enzymes

19 Oct 2023

Gene editing holds promise for treating many conditions, but the bacterial origins of RNA-editing enzymes has been found to activate unwanted immune responses in healthy people.

Much like a Swiss Army knife for genes, CRISPR technology is capable of snipping, removing and inserting the building blocks of genetic material to restore function in faulty cells. However, experimental gene therapies targeting DNA have raised both excitement and alarm—while they can reverse conditions from cancer to inherited diseases, they also carry the risk of making permanent, off-target changes to the genome.

Because RNA molecules have a limited lifespan in cells, targeting them with gene editing technologies would create transient, reversible effects that could be safer for patients, explained Emily Tang, Senior Research Fellow at Institute of Molecular and Cell Biology (IMCB).

Tang and collaborators from the Yong Loo Lin School of Medicine, National University of Singapore were interested in an enzyme that recognises and modifies RNA sequences called RfxCas13d. With its small size and high specificity to RNA, RfxCas13d has a distinct edge over first-generation DNA-editing enzymes such as Cas9.

The problem is that RfxCas13d is extracted from bacteria, and similar Cas enzymes with bacterial origins have triggered unwanted immune reactions in patients when administered. “Testing for pre-existing immunity to RfxCas13d would inform decisions on whether to use this enzyme as a therapeutic, or shed light on what kind of indications may be more appropriate,” said Tang.

To investigate, the team evaluated the presence of antibodies and T cell responses to RfxCas13d in healthy individuals. Using an enzyme-linked immunosorbent assay (ELISA) approach, they found that most donors had RfxCas13d-reactive antibodies in their blood. Moreover, in experiments using T cell culture assays, the researchers detected CD4+ helper T cells and CD8+ cytotoxic T cells that activated a strong immune response when stimulated with recombinant RfxCas13d protein. These immune responses were comparable to those against other Cas family enzymes such as SaCas9 and SpCas9.

These results came as a surprise to the researchers. “We were not expecting this strong response against RfxCas13d, certainly not as strong as SaCas9 or SpCas9,” said Tang, who went on to explain that most people are continually exposed to Cas9 enzymes because they are present in bacteria that colonise the skin, mouth and nose.

This puzzled the team as RfxCas13d originates from bacteria that live in the digestive tracts of livestock, but not in humans. “There may be some cross reactivity with similar bacteria from the same family that are present in the human gut,” suggested Tang.

The team aims to tweak RfxCas13d so that it can be safely deployed as a therapeutic for patients. “A strategy to build immune tolerance could be broadly applicable to any new therapeutic protein discovered or designed and may also help to tame autoimmunity and allergic reactions,” said Tang.

The A*STAR-affiliated researchers contributing to this research are from the Institute of Molecular and Cell Biology (IMCB).

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Tang, X.-Z.E., Tan, S.X., Hoon, S. and Yeo, G.W. Pre-existing adaptive immunity to the RNA-editing enzyme Cas13d in humans. Nature Medicine 28, 1372 - 1376 (2022). | article

About the Researcher

Emily Tang was a Senior Research Fellow in the Molecular Engineering Laboratory at the Institute of Molecular and Cell Biology (IMCB). She was an A*STAR National Science Scholarship recipient who completed her BSc in Immunology at University College London and her PhD at the University of California, San Francisco. While with A*STAR, she worked as an immunologist analysing T cell functions and antibody responses, tissue macrophage characterisation, animal modelling, and developing in vitro assays using human and mouse samples. She has great interest in applying immunological skillsets to achieve translational goals.

This article was made for A*STAR Research by Wildtype Media Group