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Effective gene editing for a rare immunodeficiency affecting the RAG1 gene

08 February 2024

Researchers from the San Raffaele-Telethon Institute of Milan demonstrate in the laboratory the effectiveness of a CRISPR/Cas9-based system in correcting the responsible genetic defect, borne by the RAG1 gene.

The first results of the gene editing strategy developed by researchers at the San Raffaele-Telethon Institute for Gene Therapy (SR-Tiget) in Milan for primary immunodeficiencies due to defects in the RAG1 gene are positive: they are described in the pages of the journal Science Translational Medicine by the research group led by Anna Villa, who is also a researcher at the Milan unit of the CNR Institute for Genetic and Biomedical Research.

RAG1 deficiency is part of severe combined immunodeficiencies (SCID) and depends on mutations in a gene that is very important for the correct development of the immune system. RAG1 is highly finely regulated, so it must be “turned on” and produce the protein it encodes for only within a short period of time during the lifetime of T and B lymphocytes. Under normal conditions, RAG1 contributes to the production of both types of white blood cells: if, however, it doesn't work, these cells do not form, leaving the body without two fundamental components to defend us from infections.

RAG1 deficiency

Those born with a RAG1 deficiency therefore have a serious immunodeficiency from birth, with recurrent and potentially fatal infections, chronic diarrhea, skin rashes, growth retardation: life expectancy is limited if no action is taken. There are also cases in which the RAG1 protein is not completely absent, but is able to promote the formation of only a few cells: this translates into an unregulated activity of the immune system, characterized by autoimmunity and chronic inflammation (Omenn syndrome and atypical SCID).

The only definitive intervention is a blood stem cell transplant, provided that a compatible donor is available. Unfortunately, time can be a tyrant with respect to the effectiveness of the transplant: therefore it is best to carry it out in the first months of life, in fact in cases of late diagnosis the damage to the various organs can compromise its success. In this sense, newborn screening for SCID can make a difference in avoiding unfortunate outcomes, however it is currently included in the national screening panel of the USA and only some European countries (such as Denmark, Germany, Norway, Iceland, Ireland, Norway, Switzerland). In Italy only some regions or cities have activated pilot projects or dedicated programs (such as in Tuscany, Liguria, Padua, Palermo), but its inclusion in our national panel is still pending.

For this reason, the group led by Anna Villa has been working for many years to develop alternative therapeutic strategies for RAG1 SCID, also thanks to the experience gained by the entire institute in the field of advanced therapies aimed precisely at correcting blood stem cells. As Maria Carmina Castiello, first author of the work and herself a CNR researcher, explains,

“since 2016 we have focused on gene editing, because it allows us to correct the gene defect by leaving RAG1 in its natural location, maintaining its physiological regulation. The correction was carried out in hematopoietic stem cells, capable of generating all lines of the immune system including T and B lymphocytes. The gene editing approach adds to the viral-derived vector-based gene therapy platforms, as has been successfully done in other diseases, such as ADA-SCID or Wiskott-Aldrich syndrome".

Over the years the group has tried various strategies, until they identified the most promising one described in this study. The corrective system exploits the now famous CRISPR/Cas9, the subject of the 2020 Nobel Prize: an enzyme capable of cutting DNA, associated with an RNA sequence that acts as a guide and allows the cut to be directed to the desired point, i.e. where there is 'is the pathological mutation. To introduce the "cut-and-sew" system into the cells, the electroporation method was used, which uses short electrical pulses to open the pores on the cell membrane. Once the cut was made, the researchers provided the cell with the correct sequence with which to repair the DNA, using viral vectors that do not insert themselves into the cellular DNA, to avoid any unwanted modifications. The whole correction strategy is the result of a long collaboration with the group of SR-Tiget director Luigi Naldini, and in particular with Samuele Ferrari and Daniele Canarutto.

On the left, the structure of the Cas9 nuclease (in pink) captured in its active state: in red, the guide RNA necessary for the activity of the enzyme. To such strand, another DNA strand (in green) is paired up, which is unwound and cut, leaving a paired fragment (in light blue) downstream of the cleavage point. In the figure on the right a part of the Cas9 protein (the HNH domain, responsible for part of the Cas9 activity) has been excluded to allow us to appreciate how the target DNA and the guide RNA form a hybrid double helix and favor the opening of the DNA, allowing it to be cut at a specific spot. Courtesy of Prof. Massimo Degano, UniSR Associate of Biochemistry, Group leader of the Biocrystallography Unit of the IRCCS San Raffaele Hospital.

As Anna Villa explains,

“with this strategy we managed to correct between 20 and 30 percent of the target stem cells: a very satisfactory percentage if we consider that, as emerged in our studies conducted in the mouse model, correcting 5 to 10 percent is enough to achieve a therapeutic effect. The next step will be to refine the correction system by conveying the correct sequence through a new nanoparticle-based transport system, similar to that used in anti-COVID vaccines. Our goal is to be able to transfer this therapeutic approach to the clinic: it could potentially prove to be an alternative to transplantation, both to overcome the lack of a donor, but also to limit the risks associated with chemotherapy conditioning".

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