Stem Cell Editing Repairs Severe Immunodeficiency


The B and T cells of the adaptive immune system recognize unique features on infectious microbes that enter the body. They accomplish this feat using B-cell and T-cell receptors, which take on various shapes to bind to different antigens on foreign invaders. Recombination activating gene 1 (RAG1) is central to this shapeshifting behavior.1 It shuffles the order of DNA sequences in the genes for these receptors, producing multiple versions of the immune receptors that can bind staggering combinations of antigens. However, some people carry mutations in RAG1 that prevent the enzyme from recombining the DNA sequences that code for these receptors. Without properly functioning receptors, B and T cells fail to develop, leading to severe combined immunodeficiency (SCID), a condition in which even the mildest of infections can prove lethal. In a study published in Science Translational Medicine, researchers developed an efficient method to repair RAG1 genes in immune cell progenitors called hematopoietic stem cells (HSC) taken from SCID patients, and revealed that they could restore immune function in mice.2

When you would like to correct the gene, you have to keep in mind that close to the gene, there are a lot of regulatory elements that are relevant for correct gene expression.
-Maria Carmina Castiello, San Raffaele Scientific Institute

Maria Carmina Castiello and Anna Villa, two translational immunologists at the San Raffaele Scientific Institute, set out to overcome some of the challenges with editing the RAG1 gene that researchers previously faced. In the past, scientists have taken healthy, functional HSC and inserted them into SCID-model mice, but they often get destroyed by other types of immune cells that recognize the transplants as foreign.3 Normally, doctors use immunosuppressants like chemotherapy before transplantation to deplete immune cells, but this isn’t an option for SCID patients. “This disease can be associated with severe organ damage, so the critical conditions of the patients do not allow them to receive chemotherapy,” Villa explained. 

Castiello and her colleagues took a different approach, modifying a SCID patient’s own stem cells to express a functional RAG1 gene. While other research groups had successfully added RAG1 to patient HSC, they were unable to properly regulate expression of the gene, and therefore couldn’t ensure that the stem cells were safe or would effectively replenish B and T cells. 

Introducing the gene into the wrong site in the genome may have partly caused this shortcoming. “When you would like to correct the gene, you have to keep in mind that close to the gene, there are a lot of regulatory elements that are relevant for correct gene expression,” Castiello said. 

Rather than adding a functional copy of RAG1, the researchers decided to modify the existing copy, ensuring that the regulatory networks remained intact. In fact, other researchers succeeded when they took a similar approach to edit RAG2.4

Before Castiello and her team could fix the gene, however, they had to choose their editing strategy. Some researchers use base editing, which modifies single letters in the DNA sequence to correct other genetic disorders of these stem cells, like sickle cell disease and β-thalassemia.5

However, RAG-1 mutations can occur at several different sites within the gene, so base editing wouldn’t cover every type of mutation. Instead, the research team used the clustered regularly interspaced short palindromic repeats (CRISPR-Cas9) system to cut out a large section of the mutant gene, and then provided cells with the correct DNA sequence using a lentiviral delivery system. Since the correct sequence was nearly identical to the original gene, the cell could swap the sequences unassisted using homology-directed repair (HDR), a built-in DNA repair pathway that fixes double-strand DNA breaks using complementary DNA as a template. 

Once Castiello and her colleagues swapped the HSC’s old, mutated coding sequence for a fresh one, they had to test whether the gene produced a functional RAG1 protein. They inserted a backwards green fluorescent protein (gfp) gene flanked by sequences that RAG1 recognizes. Promisingly, they found that the edited RAG1 inverted gfp comparably to RAG1 in HSC from healthy donors, thereby switching it to an “on” state, resulting in a functional gfp gene.

They next had to check that these edited cells could restore immune function in the body. They transplanted these edited human cells into SCID-model mice and found that B and T cells spiked to levels similar to those seen in mice that received HSC from healthy donors. 

“What’s intriguing from the study is that we don’t need to correct all the stem cells. If we manage to correct at least 10 percent of the stem cells, this is going to give us a therapeutic benefit,” said Saravanabhavan Thangavel, a geneticist at the Institute of Stem Cell Research and Regenerative Medicine who was not involved with the work. However, he also mentioned, “We need to track the HDR-edited cells long term.” The researchers need to ensure that the modified cells persist in the bodies of people with SCID so that their newly gained immunity doesn’t wane over time. “If, by chance, the HDR-edited cells faded away, they may not have a therapeutic benefit,” Thangavel added.

Down the line, the team aims to refine their protocol. “We are trying to increase the editing efficiency that we achieve,” Castiello said. She also wants to optimize delivery of the gene into the cells by comparing different methods. In this study they used lentiviruses to deliver the DNA template to the stem cells, but they plan to test other strategies like using lipid nanoparticle conduits that conceal the DNA template and fuse with the cell membrane to release the DNA into the cell.

The team will also have to test the safety of this gene editing strategy and find a way to scale up production of the edited stem cells, Castiello added. Then they should be able to test their edited cells in people with the hope of eventually treating the variety of conditions caused by RAG1 defects. “We are really committed to translating our strategy to the clinic,” she said. 

Anna Villa and Maria Carmina Castiello are inventors with two patents involved with editing RAG genes.

References

  1. Lee YN, et al. Characterization of T and B cell repertoire diversity in patients with RAG deficiency. Sci Immunol. 2016;1(6):eaah6109.
  2. Castiello MC, et al. Exonic knockout and knockin gene editing in hematopoietic stem and progenitor cells rescues RAG1 immunodeficiency. Sci Transl Med. 2024;16(733):eadh8162. 
  3. Murphy WJ, et al. Rejection of bone marrow allografts by mice with severe combined immune deficiency (SCID). Evidence that natural killer cells can mediate the specificity of marrow graft rejection. J Exp Med. 1987;165(4):1212-1217. 
  4. Allen D, et al. CRISPR-Cas9 engineering of the RAG2 locus via complete coding sequence replacement for therapeutic applications. Nat Commun. 2023;14(1):6771. 
  5. Antoniou P, et al. Base and prime editing technologies for blood disorders. Front Genome Ed. 2021;3:618406. 

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