CRISPR Corrects IPEX – a Rare and Life-Limiting Autoimmune Disease of Childhood

CRISPR gene editing technology provides hope for cures for the 10,000 single gene disorders described to date, many of which are incurable. Researchers from Stanford University have now demonstrated that CRISPR is a feasible therapeutic strategy for IPEX Syndrome, a life-threatening paediatric monogenic autoimmune disease with unmet need.

By: Karen O'Hanlon Cohrt - Aug. 6, 2020
Rosa Bacchetta, Associate Professor at the Department for Pediatrics - Stem Cell Transplantation at...
Rosa Bacchetta, Associate Professor at the Department for Pediatrics - Stem Cell Transplantation at Stanford University. Rosa Bacchetta's research team recently used CRISPR-Cas9 to correct the rare and life-limiting paediatric autoimmune disease IPEX.

Paediatrician by training Rosa Bacchetta first became interested in a rare childhood disease called Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome while working at the San Raffaele Scientific Institute in Milan, Italy.

Here, she focused on dissecting the genetic and immunological basis of primary immunodeficiencies with autoimmune aspects that might be treatable by gene therapy.

»I was in Milan and we had gotten the first IPEX patient when a few key papers emerged about FOXP3 and regulatory T cells, and deletion of the FOXP3 gene in mice led to what would become the first animal models for IPEX,« recalls Rosa Bacchetta, who is now Associate Professor at the Department for Pediatrics - Stem Cell Transplantation at Stanford University.

IPEX arises from mutations in the FOXP3 gene, whose product, the FOXP3 transcription factor, controls the regulatory arm of the immune system to prevent autoimmunity and allergy (See Fact Box).

»I already had several years of experience in working with regulatory T cells in the team of Dr. Maria Roncarolo (now at Stanford University), so it was straightforward for us to make connections and start conducting research on IPEX,« says Rosa Bacchetta.

The monogenic nature of IPEX makes it an excellent candidate for gene therapy to correct the FOXP3 sequence, and Rosa Bacchetta’s lab has now shown that CRISPR can correct FOXP3 to restore function in cells from IPEX patients. I interviewed Rosa Bacchetta to hear more about the work that was recently published in Science Advances.

Fact Box: Immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX)

More than 350 monogenic (single-gene) immune diseases have been described to date, and IPEX is in the severe end of the spectrum. IPEX manifests early during childhood and affects only males. IPEX is caused by a lack of functional regulatory T cells (Tregs) due to mutations in the forkhead box protein 3 (FOXP3) protein, which is the master transcription factor that regulates Treg function. Healthy Tregs regulate the activity and proliferation of effector T cells (Teff). Without functional Tregs, Teff cells go unchecked, resulting in an immune system that cannot prevent autoimmunity or allergy. The most common autoimmune manifestations of IPEX include type 1 diabetes, intestinal enteropathy and eczema. Read more about IPEX here.

Gene Therapy for IPEX – From Lentiviral Vectors to CRISPR

Rosa Bacchetta’s research on genetic autoimmunity is multifaceted with the overarching goal of improving the prognosis for IPEX patients. Her work to date has focused on unravelling the pathogenesis of the disease, improving diagnostics, and investigating new therapeutic approaches.

Although in use, drug-based treatments for IPEX are limited, and while allogenic haematopoeitic stem cell transplants may be curative, finding suitable donors is challenging and the transplants themselves are not without complications.

With the surge of gene-transfer and new gene editing technologies, the Bacchetta Lab, located in the Paediatric Division of Stem Cell Transplantation and Regenerative Medicine at Stanford University, began to explore the potential of combining gene therapy with autologous transplantation for IPEX. They approached this using lentiviral vectors to deliver a healthy copy of the FOXP3 gene to patient-derived CD4 T cells (CD4LVFOXP3), therefore generating functional Treg-like cells that are missing in IPEX.

While the lab plans to start clinical studies for lentiviral-based FOXP3 gene therapy in IPEX patients next year, whether this approach might be suitable for long-term IPEX treatment remains an open question. This is partly because the strategy exclusively converts CD4 T cells to Tregs, thus it excludes other cell types that also contribute to IPEX pathology, such as Teff cells.

It is not feasible to solve this issue by combining lentiviral-mediated addition of FOXP3 with haematopoeitic stem cells because consistent FOXP3 overexpression is detrimental to these cells. However, despite the potential shortcomings of lentiviral-based approaches in T cells, Rosa Bacchetta expects that the converted Tregs will likely be extremely useful as adoptive cellular therapy not only for the rare IPEX but also for other more common autoimmune diseases.

Realising that the ideal scenario would be to precisely deliver FOXP3 to its own locus in the genome of blood stem cells to allow endogenous regulation in different immune cells, the Bacchetta lab explored CRISPR-Cas9.

CRISPR Replaces FOXP3 Gene in Native Genomic Context with High Specificity

The group edited FOXP3 at its native locus using single guide RNAs to direct Cas9 to the FOXP3 target site. A donor template carrying the full-length complementary DNA (cDNA) for FOXP3 was used to replace the mutated gene and the presence of a reporter gene on the template allowed the researchers to identify edited cells.

»We used the CRISPR and AAV technology to insert the wild-type FOXP3 gene in its correct physiological location to preserve its endogenous regulatory elements, which are very complex for this gene. Being a transcription factor, FOXP3 is very challenging to modify and engineer. We could only do what we did using CRISPR technology, because we could insert the gene exactly where we wanted.« explains Rosa Bacchetta about the advantages of the CRISPR-based approach.

Using CRISPR, the >70 diverse FOXP3 mutations described so far can be corrected in a single sweep by replacing the entire gene.

»Instead of correcting single mutations that are very different among patients and often very spread out throughout the gene, we replaced the old coding sequence with a new intact one, in collaboration with Dr Porteus at Stanford University. And, by doing this, we cover the entire spectrum of mutations that lead to IPEX while preserving the endogenous regulation. This way, we can guarantee differential expression of FOXP3 in different cell types.« said Rosa Bacchetta.

Off-target editing is a concern in any CRISPR workflow, and the researchers investigated this possibility through a combination of in silico off-target prediction, an in vitro DNA double-strand break assay, and next-generation sequencing of edited cells. Four off-target edits were detected by all 3 strategies, but comparisons with the targeting rate at the FOXP3 locus revealed the off-target rate to be below 2 %, and none of the off-target edits occurred in areas of the genome expected to impact haematopoeisis or cell cycle regulation.

CRISPR-Edited Cells Display Normal FOXP3 Function in Vitro and in Vivo

Once the team had optimised the conditions for FOXP3 editing in an immortalised cell line, they edited FOXP3 in patient haematopoietic stem/progenitor cells (HSPCs) and in the two main cells types that express FOXP3, namely Tregs, and Teffs. The study included IPEX patient cells that represented scattered and diverse FOXP3 mutations.

The results were very encouraging. CRISPR-edited HSPCs did not express FOXP3, while Teffs transiently expressed FOXP3 upon activation and Tregs expressed FOXP3 consistently, in line with endogenous FOXP3 expression patterns.

Edited HSPCs differentiated normally, while CRISPR-edited Teffs from healthy donors and IPEX patients revealed normal proliferative capacity and cytokine signatures. Furthermore, edited Tregs were able to suppress Teffs and displayed phenotypes comparable to wild-type Tregs.

Although the CRISPR-edited cells behaved comparably to their wild-type counterparts as described above, the researchers noticed that FOXP3 protein expression was less abundant in edited Tregs than in wild-type Tregs. This may be due to the presence of different naturally occurring isoforms (splice variants) in human cells, and is something that the team will investigate further, as it may have implications for FOXP3 and IPEX as well as other situations where CRISPR will be used to edit tightly regulated genes that exist as distinct isoforms.

Humanised Mouse Model

In vivo experiments in humanised mice revealed that the edited human HSPCs engrafted successfully, and then underwent multilineage haematopoietic differentiation to reconstitute the bone marrow and the peripheral blood with immune cells. Studying the fate of these cells in more detail, the researchers found that FOXP3-edited HSPCs retained the capacity to give rise to functional Tregs and Teff cells in vivo. No differences in animal survival were observed following injection of edited and non-edited HSPCs demonstrating the safety of using CRISPR to correct FOXP3 in autologous HSPC transplantation.

Future Perspectives

Commenting on the significance of the findings, Rosa Bacchetta said, »I think our work is extremely useful for IPEX but also more generally, as an example of editing a tightly regulated gene with CRISPR. The humanised mouse model that we describe in the paper is very unique and it will be useful to study gene therapy strategies for other similar genetic immune diseases that are caused by mutations in other genes.«

The researchers envision that the CRISPR-edited HSPCs will be used for autologous transplantation in IPEX patients. In addition they will be important for a number of monogenic diseases that manifest because of dysfunctional Tregs, the so-called Tregopathies, due to mutations in genes other than FOXP3. Experiments performed in isolated T cells indicate that this may be a viable approach and future plans include modifications to the CRISPR constructs to optimise gene expression in selected T cell types.

How Far Away is the Clinic?

The new findings provide hopes that CRISPR-mediated gene editing could cure IPEX. Gene therapies have been approved for a few other immunodeficiencies, such as ADA-SCID (adenosine deaminase severe combined immunodeficiency), as well as metabolic disorders, and the field has learned much during the years about the safety of such approaches. So how far away are we from seeing a gene transfer-based therapy for IPEX?

»The gene therapy field is very advanced. There have also been bad experiences, for example, leukaemic transformation, but we now have a good understanding of why that happened, and additional safety checks have been implemented to prevent this from happening again. There are different safety measurements that we have to take for each disease and gene because every gene has its own characteristics and will provoke different questions on efficacy and safety and so on. So there are many things that have to be taken into consideration, but we will be ready for the IPEX patients next year with the adoptive transfer of converted autologous CD4LVFOXP3 T cells and in maybe 3 to 4 years with an optimal CRISPR-based HSPC product, if all goes well«, said Rosa Bacchetta.

»As soon as we have the T cells ready, we will try to offer this therapeutic approach to patients. It may be that the T cells expressing FOXP3 survive once they are transfused back into a Treg deficient patient, and they may influence other T cells to become regulatory. So, it may turn out to be a curative approach, but we don't know yet. Meanwhile, we will also continue to pursue the CRISPR-based HSPC approach, which is the ultimate goal for these patients.« finishes Rosa Bacchetta.

Link to original article in Science Advances:

CRISPR-based gene editing enables FOXP3 gene repair in IPEX patient cells.

Bacchetta Lab

Rosa Bacchetta PhD is an Associate Professor at the Department for Pediatrics - Stem Cell Transplantation at Stanford University. Here, she leads a group of researchers in the Bacchetta Laboratory working on understanding immune regulation in health and disease. As a paediatrician who specialises in immunology, Rosa Bacchetta’s ultimate goal is to challenge the limits of "inexplicable" and "untreatable" immune diseases, by applying current scientific knowledge and new gene editing tools to understand the mechanisms of impaired cellular immunity that underly the clinical manifestations in order to develop curative treatments.


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