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New Insight Into Red Blood Cell Machinery Offers Clues to Treating Sickle Cell Disease (Medicine)

Scientists at St. Jude Children’s Research Hospital have uncovered how the body generates protective fetal hemoglobin against sickle cell disease and beta thalassemia

Hematologists at St. Jude Children’s Research Hospital have discovered key molecular details of how genetic variants in blood-forming machinery enable some people to retain the ability to generate red blood cells with a form of hemoglobin that is normally expressed only before birth. Persistence of fetal hemoglobin expression after birth can protect patients from the deleterious effects of mutations that cause beta thalassemia and sickle cell disease. The findings are already aiding development of gene therapies. The work was published online today in Nature Genetics.

Fetuses have a different form of hemoglobin that binds oxygen more strongly, enabling them to thrive in the low-oxygen environment of the womb. Researchers are developing a gene therapy whereby a patient’s own blood-forming cells, called hematopoietic stem cells, or HSCs, are genetically reprogrammed to favor production of protective fetal globin after birth, rather than transitioning to the aberrant adult form.

“More than 50 years ago, researchers had observed that people with beta thalassemia or sickle cell disease mutations who also inherited a genetic variant that gave them persistently high levels of fetal hemoglobin didn’t get as sick,” said corresponding author Mitchell Weiss, M.D., Ph.D., chair of the St. Jude Department of Hematology. “So, it has been a holy grail to figure out how to use drugs or genetic manipulation to enhance fetal hemoglobin therapeutically in these common, serious diseases.

“Our current study builds upon an extraordinary amount of prior research to offer important, therapeutically relevant insights into the mechanisms by which these variants repress the transition from fetal to adult hemoglobin,” Weiss added.

Mapping the genetic machinery of fetal hemoglobin

The researchers mapped the genetic machinery controlling how six fetal hemoglobin-persistent variants inhibit the fetal-to-adult hemoglobin switch. These variants either destroy regulatory DNA that represses fetal hemoglobin or create new regulatory sequences that activate it.

“Genes are controlled by a balance between activation and repression,” said first author Phillip Doerfler, Ph.D., of St. Jude Hematology. “Our work shows how the unique interplay between activators and repressors ultimately controls the expression of fetal hemoglobin.”

The work identified two distinct activator sequences, each paired with a nearby repressor sequence. Fetal hemoglobin expression was turned on by using genome editing to disrupt a repressor sequence and turned off by disruption of an activator.

A gene therapy treatment to turn on fetal hemoglobin

The close proximity of activator and repressor sequences has important therapeutic implications for treating beta thalassemia and sickle cell disease. The researchers are already developing clinical trials of a gene-editing treatment that disrupts the repressor binding, yet still enables the fetal hemoglobin gene to be turned on. This genome editing approach must be precise, so as to not disrupt the adjacent activator sequence.

“Now we understand that because these activator and repressor sites are so close to each other, when we perform gene editing to manipulate them, we have to be careful that by trying to interfere with the repressor we don’t block the activator,” Weiss said.

The researchers’ objective is to take hematopoietic stem cells from sickle cell disease or beta thalassemia patients, edit the cells to disrupt the repressor binding site and return them to the patients. The red blood cells then made by those stem cells will be tipped toward expressing more fetal hemoglobin, which will alleviate the disease.

The same genetic strategy of enhancing fetal hemoglobin can treat both sickle cell disease and beta thalassemia. Although the two diseases are different genetically, both involve mutations of the gene for adult hemoglobin that disrupt its function or expression.

Authors and Funding

Other authors of the paper are Ruopeng Feng, Yichao Li, Lance Palmer, Shaina Porter, Shondra Pruett-Miller and Yong Cheng, all of St. Jude; and Henry Bell and Merlin Crossley of the University of New South Wales.

This work was supported by grants from the National Institutes of Health (P01HL053749, R01HL156647, R35GM133614, F32DK118822), The Assisi Foundation of Memphis, Doris Duke Charitable Foundation, an Australian Government Research Training Program Scholarship, the Australian National Health and Medical Research Council, the St. Jude research collaborative on sickle cell disease, and ALSAC, the fundraising and awareness arm of St. Jude.

Featured image: Corresponding author Mitchell Weiss, M.D., Ph.D., chair of the St. Jude Department of Hematology (left), helped uncover how the body generates protective fetal hemoglobin against sickle cell disease and beta thalassemia © St. Jude Children’s Research Hospital

Read the full text of the article:

Activation of γ-globin gene expression by GATA1 and NF-Y in hereditary persistence of fetal hemoglobin.” Nature Genetics, Published August 2, 2021

Provided by St. Jude Children’s Research Hospital

Genetic Base Editing Treats Sickle Cell Disease in Mice (Biology)

Converting a pathogenic hemoglobin gene to a benign variant enables healthy blood cell production in an animal model of sickle cell disease.

Sickle cell disease (SCD) is the most common deadly genetic disorder, affecting more than 300,000 newborns worldwide each year. It leads to chronic pain, organ failure, and early death in patients. A team led by researchers at the Broad Institute of MIT and Harvard and St. Jude Children’s Research Hospital has now demonstrated a base editing approach that efficiently corrects the mutation underlying SCD in patient blood stem cells and in mice. This gene editing treatment rescued the disease symptoms in animal models, enabling the long-lasting production of healthy blood cells.

The root of SCD is two mutated copies of the hemoglobin gene, HBB, which cause red blood cells to transform from a circular disc into a sickle shape — setting off a chain of events leading to organ damage, recurrent pain, and early mortality. In this study, the researchers used a molecular technology called base editing to directly convert a single letter of pathogenic DNA into a harmless genetic variant of HBB in human blood-producing cells and in a mouse model of SCD.

“We were able to correct the disease-causing variant in both cell and animal models using a customized base editor, without requiring double-stranded DNA breaks or inserting new segments of DNA into the genome,” says co-senior author David Liu, Richard Merkin Professor and director of the Merkin Institute of Transformative Technologies in Healthcare at the Broad Institute, professor at Harvard University, and Howard Hughes Medical Institute investigator. “This was a major team effort, and our hope is that base editing will provide a promising basis for a therapeutic strategy down the road for sickle cell disease.”

“Our study illustrates the power and excitement of multidisciplinary collaborations for creating novel mechanism-based cures for genetic diseases,” says co-senior author Mitchell Weiss, chair of the St. Jude Department of Hematology. “In particular, we combined expertise in protein engineering, base editing, and red blood cell biology to create a novel approach for treating and possibly curing sickle cell disease.”

The work appears today in Nature, led by co-first authors Gregory Newby at the Broad Institute and Jonathan Yen, Kaitly Woodard, and Thiyagaraj Mayuranathan at St. Jude Children’s Research Hospital.


Currently, the only established method to cure SCD is a bone marrow transplant — but finding an appropriate bone marrow donor for a patient is difficult, and patients who undergo a transplant can suffer dangerous side effects. While there are a number of gene editing treatments under development that avoid these risks by modifying a patient’s own bone marrow directly, these experimental therapies rely on introducing new DNA or cleaving genomic DNA in cells, which can also cause adverse effects.

For this work, the research team used what’s called an “adenine base editor,” a molecular tool developed in Liu’s lab that can target a specific gene sequence and convert the DNA base pair A•T to G•C, altering a gene at the level of a single pair of nucleotides. The base editor used in this study consists of a laboratory-evolved Cas9 variant — a CRISPR-associated protein that positions the base editor at the mutated HBB site in the genome — and a laboratory-evolved enzyme that converts the target A to a base that pairs like G. The base editor also guides the cell to repair the complementary DNA strand, completing the conversion of the target A•T base pair to G•C.

The single DNA mutation underlying sickle cell disease is an A in the healthy hemoglobin gene that has been altered to a T. While an adenine base editor cannot reverse this change, it can convert that T to a C. This edit transforms the dangerous form of hemoglobin into a naturally occurring, non-pathogenic variant called “hemoglobin Makassar.”


The team first introduced the adenine base editor into isolated blood stem cells from human SCD patients. In these experiments, up to 80 percent of the pathogenic hemoglobin variants were successfully edited into the benign Makassar variant, with minimal instances of the editor causing undesired changes to hemoglobin.

The researchers transferred these edited blood stem cells into a mouse model to observe how they functioned in live animals. After 16 weeks, the edited cells still produced healthy blood cells.

“Sixteen weeks after transplantation, the total frequency of the edit maintained in stem cells — which could contain edits in both copies of their hemoglobin gene, in only one copy, or in neither copy — was 68 percent. And we were particularly excited to see that nearly 90 percent of cells contained at least one edited copy of hemoglobin,” explains Newby. “Even those cells with just one edited copy appeared to be protected from sickling.”

In a separate set of experiments, the researchers took blood stem cells from mice harboring the human sickle cell disease variant, edited them, and transplanted the edited cells into another set of recipient mice. Control mice transplanted with unedited cells showed typical symptoms: sickled red blood cells, consequences of short red blood cell lifetime, and an enlarged spleen. In contrast, mice transplanted with edited cells were improved compared to controls by every tested disease metric, with all measured blood parameters observed at levels nearly indistinguishable from healthy animals.

Finally, to confirm durable editing of the target blood stem cells, the researchers performed a secondary transplant, taking bone marrow from mice that had received edited cells 16 weeks previously and transferring the blood stem cells into a new set of mice. In the new animal cohort, edited cells continued to perform similarly to healthy blood stem cells, confirming that the effects of base editing were long-lasting. The team determined that editing at least 20 percent of pathogenic hemoglobin genes was sufficient to maintain blood metrics in the mice at healthy levels.

“In these final experimental phases, we demonstrated an editing threshold of about 20 percent that is necessary to mitigate this disease in mice. This base editing strategy is efficient enough to far exceed that benchmark,” explains Liu. “The approach offers promise as the basis of a potential one-time treatment, or perhaps even a one-time cure, for sickle cell disease.”

The researchers and other partners are working to move this concept safely and effectively into additional preclinical studies, with the eventual goal of reaching patients.

This work was supported in part by the US National Institutes of Health (U01 AI142756, RM1 HG009490, R01 EB022376, R35 GM118062, and P01 HL053749), the Bill and Melinda Gates Foundation, the Howard Hughes Medical Institute, the St. Jude Collaborative Research Consortium, the Doris Duke Foundation, the Assisi Foundation of Memphis, and ALSAC, the fundraising and awareness organization of St. Jude.

Featured image credit : Zayna Sheikh, Broad Communications

Paper(s) cited:

Newby GA, Yen JS, Woodard KJ, Mayuranathan T et al. Base editing of haematopoietic stem cells rescues sickle cell disease in mice. Nature. Online June 2, 2021. DOI: 10.1038/s41586-021-03609-w

Provided by Broad Institute

A Better Treatment For Sickle Cell Disease (Medicine)

An organ-on-a-chip device designed by Texas A&M researchers could provide a more personalized approach to addressing the illness.

Sickle cell disease is the most prevalent inherited blood disorder in the world, affecting 70,000 to 100,000 Americans. However, it is considered an orphan disease, meaning it impacts less than 200,000 people nationally, and is therefore underrepresented in therapeutic research.

A team led by Abhishek Jain from the Department of Biomedical Engineering at Texas A&M University is working to address this disease.

“I’m trying to create these new types of disease models that can impact health care, with the long-term goal of emphasizing on applying these tools and technologies to lower health care costs,” said Jain, assistant professor in the department. “We strategically wanted to pick up those disease systems which fall under the radar in orphan disease category.”

Jain’s research is in organ-on-a-chip, where cells from humans can be grown on USB-sized devices to mimic the way the organ would work inside the body. This sort of system is ideal for testing new drug treatments, as drugs cannot be tested on humans, and animal models have not shown to be a good representation of how a patient and disease would interact with a treatment. For sickle cell disease patients, the organ-on-a-chip would also be beneficial because patients can present with mild to severe cases.

Jain works with Tanmay Mathur, a fourth-year doctoral student who trained as a chemical engineer in his undergraduate years. His research focused on microfabrication techniques and simulations, skills he said merged well into the organ-on-a-chip research he now performs in Jain’s lab. The team collaborates closely with the Texas Medical Center in Houston.

The work was recently published in the journal Bioengineering & Translational Medicine. Their paper builds off a 2019 publication in the journal Lab on Chip, where the team demonstrated that endothelial cells (cells that line the blood vessels) could be used to model the disease physiology of a patient without having to stimulate the model to perform differently than a healthy vessel.

“Traditionally these cells were not used for disease modeling, so in that way our approach is very novel,” Mathur said. “We are one of first to harness these cells and employed them in disease modeling research.”

Mathur and Jain demonstrate that these models can be used to differentiate between patients. The first step: build a blood vessel that mimics a patient’s vessel. For that the team would need two components — patient blood and endothelial cells. Collecting the blood involved a simple blood draw. They faced a challenge with the endothelial cells, however. They would need to take a biopsy of the cells or use stem cells to grow their own, neither of which was ideal.

Then they found the answer was in the blood.

“What we learned is within blood samples are some endothelial cells also circulating,” Jain said. “We call them blood outgrowth endothelial cells that we can harness very easily. That’s what is new about this work. You can get those cells, grow them so that’s there’s enough in number and then you can make blood vessels.”

Now that they could build the vessels, the next step was to see if these models would show how the disease has various biological impacts in different patients. Again, the goal was to be able to test treatments on these models, so the closer they mimiced their human patient, the better.

“We’re able to differentiate a very severe sickle cell patient in terms of their phenotype from very mild patients,” Mathur said. “Moving forward, we can take a larger population of any sickle cell disease patients and assess them using our organ-chip technology and then categorize them into different groups based on symptoms.”

Their findings indicate that these organs-on-a-chip could lead to patient-centric, personalized treatment, improving how clinicians approach this and other cardiovascular diseases.

“When you take it to the field, now it can become a predictive device,” Jain said. “Now you do not have to know whether the patient is mild or severe, you can test for that. You can predict if patient is serious and can dictate their therapeutic needs.”

The next step is to continue to expand the patient cohort to collect more results. A long-term goal would be to use the patient information collected to develop a database to better predict disease progression.

“You take a history of a lot of these patients and their cardiovascular health with this device, and you can predict which patient might have better chance of having a stroke and you start treating them early on,” Jain said.

Mathur said even with future challenges, he looks forward to continuing their research.

“I think even though it may take 10, 15 years, we will at least push forward some of the research that we’re doing and get it out in the clinical field,” he said. “We are one of the only groups in the world that have started this field of personalized treatment. I feel that our impact is pretty high, and I’m sure we will be able to expand the same treatment to other cardiovascular diseases and attract more attention and deeper insights into the biology that we are looking at.”

This work is funded by a Trailblazer Award Jain received from the National Institute of Biomedical Imaging and Bioengineering.

Featured image: Doctoral student Tanmay Mathur (left) and Abhishek Jain review photos of blood cells formed on the organ-on-a-chip in their lab. © Texas A&M Engineering

Reference: Mathur, T, Flanagan, JM, Jain, A. Tripartite collaboration of blood‐derived endothelial cells, next generation RNA sequencing and bioengineered vessel‐chip may distinguish vasculopathy and thrombosis among sickle cell disease patients. Bioeng Transl Med. 2021;e10211. https://doi.org/10.1002/btm2.10211

Provided by Texas A&M University

New Technology Diagnoses Sickle Cell Disease In Record Time (Medicine)

Researchers at the University of Colorado Boulder and the University of Colorado Anschutz Medical Campus have developed a new way to diagnose diseases of the blood like sickle cell disease with sensitivity and precision and in only one minute. Their technology is smaller than a quarter and requires only a small droplet of blood to assess protein interactions, dysfunction or mutations.

An Acousto Thermal Shift Assay “lab-on-a-chip” device shown next to a quarter for size comparison. ©CU Boulder College of Engineering and Applied Science.

The team published its results Oct. 15 in the journal Small.

“In Africa, sickle cell disease is the cause of death in 5% of children under 5-years-old for lack of early diagnosis,” said Angelo D’Alessandro, a coauthor of the study and associate professor in the departments of Biochemistry and Molecular Genetics and Medicine, Division of Hematology at CU Anschutz. “This common, life-threatening genetic disorder is most prevalent in poor regions of the world where newborn screening and diagnosis are rare.”

Sickle cell disease affects hemoglobin, the molecule in red blood cells that delivers oxygen to cells throughout the body. In some areas of the world where malaria is endemic, variants of hemoglobin have evolved that can cause red blood cells to assume a crescent, or sickle, shape.

“Almost all life activities involve proteins,” said study coauthor Xiaoyun Ding, an assistant professor in the Paul M. Rady Department of Mechanical Engineering at CU Boulder. “We thought if we could measure the protein thermal stability change, we could detect these diseases that affect protein stability.”

Proteins have a specific solubility at a specific temperature. When one bonds to another, or when the protein is mutated, the solubility changes. By measuring solubility at different temperatures, researchers can tell whether the protein has been mutating.

Before recent developments, study coauthor Michael Stowell, an associate professor in the Department of Molecular, Cellular and Developmental Biology (MCDB) at CU Boulder, and his colleagues used Thermal Shift Assays (TSAs) to assess protein stability under varying conditions. Such tests took about a day to run. Now, with the new technology, an Acousto Thermal Shift Assay (ATSA), they can do the same but faster and with greater sensitivity.

The ATSA utilizes high-amplitude sound waves, or ultrasound, to heat a protein sample. The tool then measures data continuously, recording how much of the protein has dissolved at every fraction of change in degrees Celsius.

“Our method is seven to 34 times more sensitive,” said Ding. “The ATSA can distinguish the sickle cell protein from normal protein, while the traditional TSA method cannot.”

Another benefit of the ATSA is cost reduction in terms of human labor and equipment.

“The traditional methods for thermal profiling require specialized equipment such as calorimeters, polymerase chain reaction machines and plate readers that require at least some technical expertise to operate,” said Kerri Ball, a coauthor of the new study and a researcher who works with Stowell at CU Boulder. “These instruments are also not very portable, requiring samples to be transported to the instruments for analysis.”

Ball said the ATSA requires only a power source, a microscope and a camera as simple as the one on your smart phone. Because the protein is concentrated, there is also no need to apply a florescent dye as is sometimes required to highlight protein changes in a traditional TSA.

References: Ding, Y., Ball, K.A., Webb, K.J., Gao, Y., D’Alessandro, A., Old, W.M., Stowell, M.H.B. and Ding, X. (2020), Protein Thermodynamic Stability: On‐Chip Acousto Thermal Shift Assay for Rapid and Sensitive Assessment of Protein Thermodynamic Stability (Small 41/2020). Small, 16: 2070224. doi:10.1002/smll.202070224

Provided by University Of Colorado at Boulder