Gene therapy makes headway in PI treatment

This article was originally published in the Fall 2024 edition of the IDF ADVOCATE newsletter. You can download or request a free print copy of the ADVOCATE.

Gene therapy is a rapidly evolving field that has led to novel treatments for several rare, genetic diseases. Treatments so far have focused on using gene addition. Gene addition involves using a vector, a biological delivery vessel, to introduce a functional version of a gene in two ways: in vivo, where the vector is introduced into the bloodstream or target organ directly, or ex vivo, where cells are taken from the patient, the vector is mixed with these cells, and the modified cells are reintroduced to the patient’s body.

Although gene addition is effective, it does come with drawbacks. Gene addition cannot address certain primary immunodeficiencies (PIs) due to how they present in the body. Researchers are exploring new methods for gene therapy and have made significant advancements in the effectiveness, safety, and accessibility of this technology. Here are some recent breakthroughs and what they mean for the future of medicine and PI.

Prime editing: A technology on the rise

In April, the gene therapy-focused biotech company Prime Medicine announced that the Food and Drug Administration (FDA) granted them permission to begin clinical trials for PM359. PM359 uses a new strategy called prime editing to treat chronic granulomatous disease (CGD), a PI in which white blood cells called phagocytes can’t fight off bacterial and fungal infections. CGD can be caused by variants in any one of six genes that allow phagocytes to produce chemicals that kill bacteria and fungi. PM359 targets a CGD-causing variant in the NCF1 gene. This NCF1 variant has a small deletion and about 25% of people with CGD have the NCF1 variant. 

PM359 is an ex vivo product and modifies a patient's hematopoietic stem cells (the cells in bone marrow that give rise to all types of blood cells; HSCs) by inserting the missing nucleotides instead of adding an entire functional copy of NCF1. Prime editing can be used to make several kinds of edits to DNA, including substitutions, deletions, and big and small insertions. These features make it a more versatile option compared to gene addition. Additionally, prime editing is safer as it makes fewer edits to DNA, whereas gene addition can have unintended consequences like triggering the expression of other genes. 

In preclinical studies, PM359 corrected the variant that causes CGD in over 75% of patient HSCs in the lab. The Phase I/II trial will first focus on adults with stable disease, meaning their condition isn’t increasing or decreasing in extent or severity. Next, Prime Medicine will enroll people with severe inflammation, along with adolescent and pediatric patients with CGD, and expects to report trial data in 2025.

Inactivating genes: A new solution 

Another major advancement in the field comes from the National Institutes of Health (NIH) with the use of gene inactivation to treat warts, hypogammaglobulinemia, immunodeficiency, myelokathexis (WHIM) syndrome in mice. People with WHIM syndrome are susceptible to infections of the respiratory system, ears, and skin. These infections can be life-threatening, as people with WHIM syndrome have a low number of neutrophils (a type of white blood cell that fights infections) in their blood. 

WHIM syndrome is a dominant, “gain of function” disorder, meaning that people with WHIM syndrome have overactive protein receptors that sit on the outside surface of neutrophils. These excess protein receptors stop neutrophils from leaving the bone marrow, resulting in a lack of neutrophils in the bloodstream in those with WHIM syndrome. Gene addition cannot be used to treat this disorder, as the function of the CXCR4 protein needs to be reduced, not restored. However, gene inactivation is a viable strategy.

Researchers first isolated HSCs from mice with WHIM syndrome and inactivated one copy of the CXCR4 gene in each cell using CRISPR/Cas9 editing. This inactivation resulted in three types of cells: cells with the WHIM-causing copy of CXCR4 inactivated, cells with the functional copy of CXCR4 inactivated, and cells with both copies inactivated. All these edited cells were then reintroduced to mice with WHIM syndrome. The data showed that stem cells with the WHIM-causing copy of CXCR4 inactivated (but the other, functioning copy of CXCR4 intact) survive and divide the best in the bone marrow. This led to proper neutrophil function in the WHIM syndrome mice.

Modifying T cells: Revisiting an old strategy

Researchers have opted to focus on using blood stem cells for gene therapy to treat PI disorders. However, gene therapy treatment of PI disorders started with T cells. Researchers successfully modified and reintroduced T cells into the bodies of patients in a landmark gene therapy trial at NIH in 1990 to treat adenosine deaminase deficient severe combined immunodeficiency (ADA-SCID), a condition where the body lacks T cells and B cells. A functional copy of adenosine deaminase (ADA) was added to the T cells and treatment was successful. 

In addition, using genetically modified T cells to treat disease is already employed in medicine; CAR T cell therapy is routinely used to treat certain blood cancers. Recent breakthroughs may bring back gene modification of T cells as a viable option for treating PI disorders. 

Our cells have systems in place that control their growth and function. One protein that regulates T cell activity is forkhead box P3 (FOXP3). FOXP3 controls the production of certain proteins that are crucial in a small subset of T cells called T regulatory cells (Tregs). In turn, Tregs control the immune system by suppressing or encouraging an immune response to germs.

Variants in the FOXP3 gene can lead to immune dysregulation polyendocrinopathy enteropathy (IPEX) syndrome, a disorder characterized by multiple autoimmune diseases. Although gene addition in HSCs successfully repairs Tregs, this method cannot be used long-term to treat IPEX, as having too much FOXP3 in other cells can lead to adverse effects. 

In May 2020, researchers at Stanford University conducted a clinical trial where stem cells were obtained from patients with IPEX and healthy donors. The researchers grew T helper cells from the stem cells and made edits to the FOXP3 gene using CRISPR. This proof-of-concept trial led to further research into using modified T cells as a treatment for IPEX. 

In November 2023, Stanford researchers ran a preclinical safety trial using mice to determine the effects of T helper cell transplantation. First, the mice were transplanted with stem cells in which the FOXP3 gene was completely removed or “knocked out,” using CRISPR. This knockout variant was named FOXP3KO The mice with the FOXP3KO cells had symptoms similar to IPEX syndrome.

Next, the researchers isolated T helper cells from the mice and added in a functional version of the FOXP3 gene via gene addition, creating Tregs. The Tregs were given to the FOXP3KO mice and they regained regulatory functions in their immune system. This experiment leads researchers to believe that this methodology can be employed as a model for other experiments and preclinical trials involving Tregs.

This trial is a significant feat for the field, as it reintroduces the idea of editing T cells directly to treat IPEX syndrome and other primary immunodeficiencies with malfunctioning T cells.

Gene therapy has made great progress over the past four decades. Going to the molecular level and treating a genetic disorder as precisely as possible at its source makes newer strategies more flexible than gene addition to HSCs. Multiple PIs cannot be treated with HSC gene addition due to their complexity and the expression of the disorder.

These new techniques make gene therapy more applicable and accessible to a wider range of patients whose treatment options are limited. Gene therapy has revolutionized healthcare as we know it and these recent breakthroughs show that the future is bright for the treatment of PI disorders.