Advancing the Story of Discovery in Blood Disorders

For St. Jude, the story of discovery in blood disorders — conditions such as leukemia, sickle cell disease, Diamond-Blackfan anemia, β-thalassemia, and GATA2 deficiency — is both the foundation of the institution and an area of continued innovation.

To advance this narrative of exploration, St. Jude researchers have focused their investigations on the underlying mechanisms of disease and the science of gene therapy to advance the story of discovery and treat blood disorders at their source.

Rewriting treatment possibilities for Diamond-Blackfan anemia

Diamond-Blackfan anemia (DBA) is a bone-marrow failure syndrome caused by mutations in genes that encode the cell’s protein factory, the ribosome. These mutations prevent the body from producing enough red blood cells, leading to potentially life-threatening complications. Senthil Bhoopalan, MBBS, PhD, Department of Bone Marrow Transplantation & Cellular Therapy, and Michell J. Weiss, MD, PhD, Department of Hematology chair, have been leading efforts to find a curative treatment for DBA: one that directly alters the ribosome genes that house these mutations.

“Fifty years ago, the standard of care [for DBA] was corticosteroids and blood transfusions across a person’s lifetime, both with significant long-term side effects. The only curative therapy was bone marrow transplantation from a healthy donor, which remains true today, but we are now one step closer to another curative option,” said Bhoopalan.

In results published in Molecular Therapy, Bhoopalan and Weiss focused on the most common dysfunctional gene in DBA, RPS19, which encodes a key component of the ribosome. The researchers developed a gene therapy approach by engineering a lentiviral vector, a genetically modified form of a lentivirus, to integrate functional RPS19 into blood-forming stem cells. The team demonstrated that adding a functional copy of RPS19 to RPS19-mutated human blood–making stem cells rescued ribosome function, increased red blood cell production, and allowed corrected stem cells to survive over noncorrected DBA stem cells.

“The rescued cells had a competitive survival advantage over the uncorrected stem cells,” said first and co-corresponding author Bhoopalan. “In some patients, blood-forming cells that have spontaneously lost the mutated RPS19 gene and duplicated the healthy copy of RPS19 can expand over time. Thus, our data suggest that lentiviral vector–corrected stem cells will expand over time and take over the bone marrow. That means we may be able to use low-intensity conditioning for future gene therapy studies, which will reduce treatment-related toxicity for these patients.”

To test this hypothesis, Bhoopalan is planning a St. Jude–led clinical trial to assess this gene therapy approach in patients with DBA.

Weiss credits the groundbreaking work of the late Brian Sorrentino, PhD, who developed a curative gene therapeutic approach for treating X-linked severe combined immunodeficiency that laid the foundation for this potential DBA therapy.

“As an institution, we have built the expertise and infrastructure to cure devastating blood disorders with lentiviral vector–based gene therapy. We are now positioned to cure DBA using this approach,” said senior and co-corresponding author Weiss.

As an institution, we have built the expertise and infrastructure to cure devastating blood disorders with lentiviral vector–based gene therapy. We are now positioned to cure DBA using this approach.

Michell J. Weiss, MD, PhD

Department of Hematology

Revising gene editing approaches for blood disorders

As technologies have advanced, new treatment solutions for blood disorders have shifted to precision approaches, including the use of CRISPR-Cas9–based gene editing. Sickle cell disease and β-thalassemia, blood disorders caused by mutations in the HBB gene that encodes the β-globin subunit in adult hemoglobin, have a commercially available, Food and Drug Administration (FDA)–approved gene editing approach. However, investigators are working to better understand the mechanisms by which the gene therapy works.

By silencing BCL11A, a gene responsible for switching hemoglobin production from fetal to adult forms, the commercially available CRISPR-Cas9 gene therapy approach allows a patient to maintain a high fetal hemoglobin level. This lessens the severity of both disorders by inhibiting the production of mutant sickle hemoglobin in sickle cell anemia or compensating for deficient β-globin subunit gene production in β-thalassemia.

Leading the way in advancing the understanding of how this CRISPR-Cas9 gene therapy approach works, investigations led by principal investigator Jian Xu, PhD, Department of Pathology, and co-investigator Weiss set out to determine how CRISPR-Cas9 reactivates fetal hemoglobin.

“Our motivation for this study was twofold,” said corresponding author Xu. “First, to find out how CRISPR genome editing effectively inactivates BCL11A for fetal hemoglobin reactivation in this commercially approved gene therapy approach. And second, to identify more accessible therapeutic strategies.”

To examine how CRISPR-Cas9 specifically inactivates BCL11A, the team discovered that the enhancer (a short region of DNA that controls transcription of specific genes) targeted by the gene therapy folds into a three-dimensional structure. “We found that this enhancer forms a chromatin ‘rosette’ structure, making multiple contacts with key regulatory elements of the gene,” said Xu. “This ensures high-level BCL11A expression and prevents its silencing in red blood cell precursors.”

Published in Blood, the results showed that breaking this chromatin rosette structure is necessary for other repressive proteins to enter and silence BCL11A. The researchers also found that the enhancer region’s three-dimensional architecture requires the presence of an enhancer RNA (eRNA), a noncoding regulatory RNA produced from the enhancer region.

To test whether this eRNA could be targeted using a more effective type of therapy that does not modify the genome, the team evaluated antisense oligonucleotides (ASOs). Xu explained that “by delivering ASOs to both normal and sickle red blood cell precursors, we were able to selectively degrade the eRNA, leading to BCL11A silencing and fetal hemoglobin reactivation. We think this could offer a more affordable, accessible, and scalable alternative to current gene therapies.”

By delivering ASOs to both normal and sickle red blood cell precursors, we were able to selectively degrade the eRNA, leading to BCL11A silencing and fetal hemoglobin reactivation.

Jian Xu, PhD

Department of Pathology

Editing the epigenetics of blood

Weiss also led the search for mechanisms vital to regulating fetal hemoglobin, solving a 40-year-old mystery about the connection between methyl groups, small chemical tags that regulate gene expression across the genome, and fetal hemoglobin expression. The scientists sought to understand how the body switches hemoglobin genes on and off through epigenetic regulation, changes to genes or chromosomes that alter gene accessibility to transcription factors and expression. Previous research in this area had only been able to remove methyl groups (a process called demethylation) globally across the genome. These findings showed that demethylation of the fetal γ-globin gene was associated with its expression, but did not prove causality.

Published in Nature Communications, Weiss’s team, in collaboration with the University of Sydney, used advancements in CRISPR-Cas9 technology to precisely modify epigenetics, including the removal or addition of methyl groups at specific genes. By removing methyl groups from the promoter of the γ-globin genes, which encode proteins necessary for fetal hemoglobin production, the team demonstrated that demethylation increases gene expression.

“We found that the association between DNA methylation and expression at the γ-globin promoter is causal,” said co-corresponding author Weiss.

By targeting six specific cytosine bases in the γ-globin promoter of red blood cell precursors, the team showed that demethylation activated fetal hemoglobin gene expression. Removal of the methyl group at these sites marks the first time this type of epigenetic modification has been performed in red blood cells. The scientific feat demonstrated that γ-globin promoter demethylation could increase fetal hemoglobin levels from approximately 10% of all hemoglobin to over 30%, indicating its therapeutic potential.

“This paper provides an example of how we used a new technology, epigenetic editing, to address a long-standing question about the regulation of fetal hemoglobin expression,” Weiss said. “Going forward, we will use epigenetic editors to explore new therapies for sickle cell disease and β-thalassemia, blood disorders that are alleviated by high levels of fetal hemoglobin. These studies are likely to provide general insights into gene regulation that could have a positive impact on treating many diseases.”

Continuing the narrative of scientific discovery for blood disorders

When a new treatment reaches the clinic, a slew of additional research begins. Gene therapies are still relatively new approaches for treating genetic blood disorders, but clinical investigators at St. Jude are leading the way in understanding the secondary impacts and long-term outcomes of these treatments for sickle cell disease and hemophilia B.

Sickle cell disease is caused by an inherited hemoglobin mutation that produces misshapen red blood cells that block blood flow, leading to severe pain and organ damage, including stroke. To compensate for the chronic anemia caused by sickle cell disease and the reduced oxygen-carrying capacity of sickled red blood cells, the blood flow speed increases in the vessels supplying blood to the brain, resulting in decreased time for the oxygen molecules to exit red blood cells and enter brain tissue, potentially leading to ischemia. Increased blood flow to the brain, as measured by Doppler ultrasound, is widely recognized as an indicator of stroke risk in people with sickle cell disease.

To understand whether CRISPR-Cas9 gene editing can help slow blood flow in the brain to normal levels, Akshay Sharma, MBBS, MSc, Department of Bone Marrow Transplantation & Cellular Therapy, led a study, published in the American Journal of Hematology, which imaged the brains of three patients with sickle cell disease using magnetic resonance imaging (MRI). The imaging was captured before gene therapy and at one and two years posttreatment. The results showed that blood flow improved significantly posttreatment, with speed decreasing anywhere from 22% to 43%, and remaining stable over time.

“We saw that after gene therapy, elevated blood flow in the brain came down to normal levels, like we previously observed occurring after a bone marrow transplant,” said first and corresponding author Sharma. “This is the closest physiological evidence we have that gene therapy could be effective for patients with neurovascular disease who are at risk of or have had a stroke.”

The study provides hope for patients with sickle cell disease who are at risk of developing a stroke but are interested in pursuing gene therapy. Historically, this subset of patients has been excluded from gene therapy trials because of the high-risk nature of their condition.

“We now have emerging data to at least evaluate the efficacy of gene therapy in patients with a risk of or history of stroke,” said Shama. “Until now, we have only had one option that has had a long-term impact on blood flow in the brain: bone marrow transplantation. But now we may also have gene therapy as another viable method to protect against neurovascular disease in people with sickle cell disease.”

Until now, we have only had one option that has had a long-term impact on blood flow in the brain: bone marrow transplantation. But now we may also have gene therapy as another viable method to protect against neurovascular disease in people with sickle cell disease.

Akshay Sharma, MBBS, MSc

Department of Bone Marrow Transplantation & Cellular Therapy

Following up on gene therapy for hemophilia B

Patient follow-up can continue for more than a decade after gene therapy, and researchers at St. Jude have conducted the longest follow-up analysis of patients with hemophilia B treated by gene replacement therapy. Hemophilia B is caused by insufficient levels of a circulating protein called factor IX, responsible for blood clotting, leading to spontaneous, sometimes severe hemorrhages. Typically, patients require regular preventative intravenous (IV) infusions of the deficient clotting factor to prevent bleeding. St. Jude investigators recently unveiled data from a 13-year follow-up study of long-term outcomes of gene therapy used to treat hemophilia B.

Published in The New England Journal of Medicine and led by Ulrike Reiss, MD, Department of Hematology, and Andrew Davidoff, MD, Department of Surgery chair, in collaboration with University College London, the study included 10 adults with severe hemophilia B who received gene therapy between March 2010 and November 2012. The patients were followed over 10 years; all maintained a steady level of factor IX.

“For these 10 patients, the factor levels have been stable, and they had significantly fewer bleeding events over these 13 years,” said co-first author Reiss. “Further, we have not seen any side effects or toxic events in the long-term follow-up.”

The curative outcomes and lack of toxic effects of the therapy have profound implications for the quality of life of the patients. Because 90% of the gene therapy delivery vehicle, a modified virus called a vector, ends up in the liver, the team kept a close eye on liver function tests. They limited liver inflammation through a brief course of steroids shortly after they administered the gene therapy.

Overall, how often the patients bled over the course of a year, called their annualized bleeding rate, dropped from 14 to 1.5 episodes per year. Recipients needed fewer factor IX infusions, and seven of the 10 patients discontinued regular factor prophylaxis.

“The key benefit is that gene therapy is a one-time, simple intravenous infusion that’s very straightforward to do and potentially has positive effects for a lifetime,” said co-first author Davidoff.

The resultant improvements in quality of life from gene therapy fuel the ongoing narrative of scientific discovery for blood disorders at St. Jude.

For these 10 patients, the factor levels have been stable, and they had significantly fewer bleeding events over these 13 years. Further, we have not seen any side effects or toxic events in the long-term follow-up.

Ulrike Reiss, MD

Department of Hematology

Drafting disease-specific models for GATA2 deficiency

The narrative of discovery in blood disorders at St. Jude, from clinical and basic research efforts, centers the mechanistic understanding of what makes these diseases form, progress, and even transform.

Some genetic blood disorders, like GATA2 deficiency, hardwire alterations into cells that make them prone to transform into cancer. To better understand the mechanisms that foster this transformation, scientists develop models that allow them to monitor disease development. GATA2 is a blood transcription factor vital for the production and maintenance of blood-forming stem cells. GATA2 deficiency, an inherited blood disorder that presents with symptoms of severe immunodeficiency, has a high risk of blood cancer development.

To understand this malignant transformation, a team of researchers at St. Jude, led by John Crispino, PhD, MBA, Division of Experimental Hematology director and Department of Hematology member, devised a disease-specific preclinical model to incorporate various drivers of the disease process.

The team produced a GATA2 mutant mouse model that had a specific abnormality in R396Q, located in the second zinc finger (a protein domain, or location) of Gata2. This location is most often associated with disease progression toward myelodysplastic syndrome or acute myeloid leukemia (AML). By assessing the model with functional studies and single-cell RNA sequencing, the team showed that the mutation confers the potential to contribute to leukemic development.

Their work, published in Leukemia, demonstrated aberrant creation and reduced function of blood-forming stem cells throughout development, a bias toward myeloid cell production, and signs of accelerated aging. However, the models did not progress all the way to cancer, marking the need for additional studies to discern specific drivers of disease progression.

“This model is a great resource, because now we can get cells from the model that have these known genetic events necessary for disease and begin adding in other mutations to screen for particular genes that lead to cancer,” said corresponding author Crispino. “It is an incredibly useful tool as we seek to identify the mechanisms of malignant transformation in GATA2 deficiency.”

Useful tools that help investigators model and examine the mechanisms of disease progression, and even transformation, showcase the commitment of St. Jude to contribute new avenues of investigation and discovery in blood disorders.

From research investigations to improvements in care that range from the bench to the bedside, the quest to advance the narrative of discovery in blood conditions that were once deemed untreatable offers both investigators and patients the opportunity to continue drafting the next chapter of an unfolding story.