Gene editing for a rare immunodeficiency

Researchers from the San Raffaele-Telethon Institute in Milan demonstrate in the laboratory that a CRISPR/Cas9-based system can correct the genetic defect causing the disease, in 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: the findings are described by the research group led by Anna Villa in the pages of the Science Translational Medicine Journal. Anna Villa is also a researcher at the Milan branch of the Institute of Genetic and Biomedical Research of the CNR.

RAG1 deficiency is part of severe combined immunodeficiencies (SCID) and is due to mutations in a gene that is crucial for the correct development of the immune system. The expression of the RAG1 gene is very finely regulated, as it must be “turned on” and produce the protein it encodes for for only a short time during the life of T and B lymphocytes. Under normal conditions, RAG1 contributes to the production of both types of white blood cells. When the gene is mutated and hence defective, these cells do not form, leaving the body without two fundamental cellular components to defend us from infections.

Those born with a RAG1 deficiency present a serious immunodeficiency from birth, with recurrent and potentially fatal infections, chronic diarrhea, skin rashes, growth retardation: life expectancy is limited without medical intervention. 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 therapeutic 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: it is best to carry out this procedure in the first few months of life, as in cases of late diagnosis the damage already present in the various organs can compromise its success. In this sense, newborn screening for SCID can make a difference in avoiding unfortunate outcomes, however SCID is? currently screened for in 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 the Italian 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 SCID due to RAG1 mutations, also thanks to the experience gained by the entire institute in the field of advanced therapies aimed at correcting blood stem cells. As Maria Carmina Castiello - first author of the work and herself a CNR researcher - explains, "since 2016 we have been focusing on gene editing, because it allows to correct the RAG1 mutation by leaving the gene in its location, maintaining its physiological regulation. The gene correction was carried out in hematopoietic stem cells, cells capable of generating all subpopulations of the immune system including T and B lymphocytes. This gene editing-based approach adds on to gene therapy platforms based on vectors of viral origin, as has been successfully done in other pathologies, such as ADA-SCID or Wiskott-Aldrich syndrome".

Over the years the group has tried various strategies, until the identification of the most promising one described in this study. The corrective system exploits the now famous CRISPR/Cas9 platform, the subject of the 2020 Nobel Prize in Chemistry: 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 target, i.e. where the pathological mutation is located. Electroporation was used in this case to introduce the "cut and sew" system into the cells, a method that 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 undesired modifications. The entire 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.

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As Anna Villa explains, "with this strategy we managed to correct between 20 to 30 percent of the target stem cells: this is a very satisfactory number if we consider that, as emerged in our studies conducted on the mouse model, it is enough to correct the 5- 10 percent of the cell pool to achieve a therapeutic effect. The next step will be to refine the platform by conveying the correct gene 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-based conditioning."

Read the full paper here https://www.science.org/doi/10.1126/scitranslmed.adh8162

This article is a translation of the one originally published in italian by Fondazione Telethon.