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Wagner DL, Amini L, Wendering DJ, Burkhardt LM, Akyüz L, Reinke P, Volk HD, Schmueck-Henneresse M. High prevalence of Streptococcus pyogenes Cas9-reactive T cells within the adult human population. Nat Med. 2018 Oct 29. doi: 10.1038/s41591-018-0204-6


The discovery of the highly efficient site-specific nuclease system CRISPR-Cas9 from Streptococcus pyogenes has galvanized the field of gene therapy. The immunogenicity of Cas9 nuclease has been demonstrated in mice. Preexisting immunity against therapeutic gene vectors or their cargo can decrease the efficacy of a potentially curative treatment and may pose significant safety issues. S. pyogenes is a common cause for infectious diseases in humans, but it remains unclear whether it induces a T cell memory against the Cas9 nuclease. Here, we show the presence of a preexisting ubiquitous effector T cell response directed toward the most widely used Cas9 homolog from S. pyogenes (SpCas9) within healthy humans. We characterize SpCas9-reactive T cells within the CD4/CD8 compartments for multi-effector potency, cytotoxicity, and lineage determination. In-depth analysis of SpCas9-reactive T cells reveals a high frequency of SpCas9-reactive regulatory T cells that can mitigate SpCas9-reactive effector T cell proliferation and function in vitro. Our results shed light on T cell-mediated immunity toward CRISPR-associated nucleases and offer a possible solution to overcome the problem of preexisting immunity.


In October Dimitrios L. Wagner and his team received the Paper of the Month award. We talked to him about his research and the rewarded publication:

What do you research? What is the main focus of your work?

Our study investigates the potential immune responses to the CRISPR-Cas9 “gene scissors”. I have been working with this targeted gene-editing technology since 2015 as part of my doctoral research. CRISPR-Cas9 enables scientists to make cuts in specific human DNA sequences. During a research stay in the United States I helped establish a very efficient CRISPR-Cas9 protocol, which we are now using here in Berlin. We are employing it in our research into diseases, which involves modeling “sick genes” and examining the effect. We are also developing novel therapeutic approaches by correcting mutations or altering certain genes in cells for specific purposes. The main focus of our research group is to use genetic modifications to make immune cells better at alleviating autoimmune diseases or at fighting cancer and infectious diseases.

What motivates your research?

Genome engineering, or the targeted modification of the genome, is undoubtedly one of the most ingenious methods developed in this century. CRISPR-Cas9 is currently revolutionizing basic biological research around the world. Almost every day, international research groups publish research on new and creative ways to improve the technology itself or to cure diseases. Through our proximity to Charité and patients we are particularly interested in the latter. We would like to help ensure that this powerful technology provides the best benefits for patients. In our view, here it is important to work toward developing practices that are safe and sensible.

That may sound great, but it isn’t always easy. Laboratory research is very time-consuming and often frustrating. If I didn’t have such terrific colleagues, I would have given up many times. One shares the passion and the hardships. The study published in October is a great example of this: The project could not have been realized without the entire team’s commitment. Everyone put a great deal of time and intellectual effort into making it happen.

What is the core message of your publication, and how does your study differ from the work of other scientists in this field?

Our study examines whether the Cas9 gene scissors are recognized by the human immune system. In 95 percent of healthy subjects, we detected cells that could elicit an immune response against the most commonly used gene-editing tool. Therapies that require the gene-editing protein to be in the body for a considerable length of time could possibly cause dangerous immune responses. These inflammatory responses may diminish the efficiency and safety of such techniques. Two other groups in the United States studied the same question, but they focused mainly on antibody responses. We looked in detail at how T cells respond to the Cas9 protein using very sensitive tests. In addition, we identified a certain type of cell – so-called regulatory T cells – that also recognizes Cas9. Such regulatory T cells were able to inhibit immune responses against Cas9 in the petri dish. These findings raise exciting new questions, such as whether such regulatory T cells could be used to make CRISPR-Cas9 therapies safer.

Who did you collaborate with on this publication? Who were the key participants?

The publication was a great team effort of the newly formed junior group led by Dr. Michael Schmueck-Henneresse and members of research groups led by Prof. Hans-Dieter Volk and Prof. Petra Reinke respectively. We also received active support from the Immunology Study Lab at Charité Virchow Klinikum. I would particularly like to mention Leila Amini, Desirée Jacqueline Wendering, and Lisa-Marie Burkhardt. As part of their doctoral research, Leila Amini and Desirée Jacqueline Wendering devised many of the tests that we used in the study. Together with Lisa-Marie Burkhardt, they made it possible to carry out a large portion of the experiments when I couldn’t be in the lab due my commitments at the university. It was only through the dedication of everyone involved that it was possible to complete the study in an excellent and timely manner.

What are the next steps for the project?

The project is continuing in several directions. The key finding we made was that the use of CRISPR-Cas9 in people can lead to immune responses. Such responses are known in the field of gene therapy and must be suppressed in order to ensure treatment success. We are currently trying to find out exactly which parts of the gene scissors trigger the immune responses. This may make it possible to create new enzymes that the immune system cannot recognize.

We are also collaborating with other research groups around the world in order to evaluate other enzymes similar to Cas9 with regard to potential immune responses. In addition, we are developing strategies to suppress dangerous immune responses during gene therapy. Here we are focusing on the use of Cas9-specific regulatory T cells. These may eventually prevent or nip in the bud these dangerous immune responses.

What are the possible implications of your findings for patients?

We have developed tests that can be used clinically to monitor immune responses before and during a CRISPR-Cas9 therapy. We can also examine cell products that were modified by CRISPR-Cas9 for residual Cas9. Both could be relevant for studies. That will help us make our own techniques safer. As mentioned, we are working to improve T cells in order to combat autoimmune responses and viral diseases more effectively. The strategy we use does not leave any residual CRISPR-Cas9 complex in the cells. We hope to soon treat conditions that occur after organ transplantation with a cell product whose genes were modified in such a safe way.