In 1904, Walter Clement Noel was admitted to the Presbyterian Hospital in Chicago with respiratory difficulty and fever (1). When doctors performed a blood smear, they were baffled by what they saw. “The shape of the [red blood cells] was very irregular, but what especially attracted attention was the large number of thin, elongated, sickle-shaped and crescent-shaped forms,” wrote physician James Herrick in 1910 (2). Try as they might, doctors couldn’t figure out what the cause was. “Whether the blood picture represents merely a freakish poikilocytosis or is dependent on some peculiar physical or chemical condition of the blood or is characteristic of some particular disease I cannot at present answer,” Herrick was forced to conclude.

Noel’s condition was eventually named sickle cell disease. The genetic disease disproportionately affects Africans and people with African ancestry, and it can drastically shorten lifespans (3,4). Noel only survived until age 32. In Africa, where the majority of children with sickle cell disease are born, between 50 and 90 percent of patients do not survive past their 18^th birthdays (5). 

People with sickle cell disease produce an altered form of hemoglobin, the red blood cell protein that shuttles oxygen from the lungs to the rest of the body. Instead of floating freely within the red blood cells, the mutant hemoglobin sticks together to form long fibers in a process called hemoglobin polymerization, which transforms the cell from a flexible disc into a hard crescent moon. 

These crescent moon cells only live for a few weeks, compared to the four-month lifespan of a healthy red blood cell, which results in anemia. The hard, sticky cells also get lodged in small blood vessels, blocking blood flow to various tissues. Depending on their location, these blocked blood vessels can result in vision loss, stroke, and damage to the heart, kidneys, and liver. When blood and oxygen don’t reach the lungs, acute chest syndrome, a life-threatening condition involving coughing, fever, and difficulty breathing, can develop. Blockages can also lead to vaso-occlusive crises, episodes of severe pain in oxygen-starved tissues, which can result in hospitalization (6).

Although the cause of the disease has been known since 1949, treatments were slow to emerge (7). John Tisdale, a hematologist at the National Heart, Lung, and Blood Institute, recalls that during his residency at Vanderbilt University in the 1990s, “we had patients coming in with horrible pain from sickle cell disease and we had nothing for them. We had pain medication, but we weren’t very good at administering that because of this incorrect notion that remains today, that patients with sickle cell disease are just drug seeking. But in fact, they’re seeking relief from this horrific pain.”

In 1998, the FDA approved the first drug to treat sickle cell anemia, the most common form of sickle cell disease (5). This drug, called hydroxyurea, is effective in some but not all patients. While hydroxyurea is an effective treatment for pain in sickle cell disease, long-term safety data are still being collected (8). Research shows that the drug is underutilized, especially among those under 18 years of age; prescription rates are relatively low, and patients reported that concerns about side effects were substantial barriers to use (9–11).

Between 2017 and 2019, three more drugs joined the ranks of FDA-approved treatments, including drugs that help prevent hemoglobin polymerization or prevent blood cells from sticking to the blood vessel lining. A major gap in life expectancy remains, however, and better treatments are sorely needed. Researchers, including Tisdale, are working hard to close this gap, developing therapies to ameliorate or even cure this deadly disease.

An incidental cure

In 1984, doctors at St. Jude Children's Research Hospital cured a patient with sickle cell disease almost by accident (12). The patient had been diagnosed with the disease at two years of age and then received an even more serious diagnosis at age eight: acute myeloblastic leukemia. To treat the leukemia, the girl received chemotherapy and a bone marrow transplant from her younger brother. Since hematopoietic stem cells in the bone marrow divide to produce all types of blood cells including red blood cells, receiving healthy bone marrow cells enabled the girl to produce healthy red blood cells. Ultimately, she was cured of both leukemia and sickle cell disease and remains healthy to this day, said Tisdale.

We had patients coming in with horrible pain from sickle cell disease, and we had nothing for them. 
- John Tisdale, the National Heart, Lung, and Blood Institute

Unfortunately, these transplants come with major risks, including graft rejection, graft-versus-host disease, and even death (13). Even if patients were willing to face these risks, it’s estimated that 80 percent or more would not be able to find a compatible donor, rendering this cure impossible for most patients (13).

Nevertheless, the 1984 case provided an important proof-of-concept: replacing mutant bone marrow cells with normal bone marrow cells provided a lasting sickle cell disease cure.

Inspired by the efficacy of these transplants and the then-nascent field of gene therapy, Tisdale and others wondered if they could harvest a patient’s hematopoietic stem cells, tweak the cells’ genetics to enable them to make healthy hemoglobin, and return them to the patient.

The promise of gene therapy

In healthy adults, hemoglobin proteins consist of four subunits: two alpha subunits and two beta subunits. People usually have two normal copies — one from each parent — of the gene that encodes the beta subunit, called HBB. Sickle cell anemia is an autosomal recessive condition; people with the disease have two “misspelled” copies of the HBB gene and are unable to produce any normal beta subunits. This misspelling is very slight — only a single nucleotide is different — but it results in the production of mutant beta subunits that form mutant hemoglobin, called hemoglobin S, which is responsible for red blood cell sickling.

When individuals have one healthy copy and one mutant copy of the HBB gene, they usually experience no ill effects, and in fact, carrying a single copy of the mutant gene appears to protect against severe malaria (14). Thus, the prevalence of malaria in Sub-Saharan Africa likely explains why individuals with origins in this region are at a higher risk for sickle cell disease. Importantly for gene therapy researchers, the good health of these sickle cell carriers indicated that scientists may not need to remove the faulty gene. Adding a working copy might be enough to cure the disease.

“We started working on ways to transfer genes to bone marrow stem cells using viral vectors,” said Tisdale. “Lentivirus is the one that’s most utilized now because those viruses in nature stick themselves into our DNA permanently.”

In the mid-2010s, Tisdale partnered with biotechnology company bluebird bio to test a lentivirus-based gene therapy in patients with sickle cell disease (15). This therapy, lovo-cel (formerly LentiGlobin), involves harvesting patients’ hematopoietic stem and progenitor cells (HSPCs), using a lentiviral vector to insert a modified gene for the beta subunit of hemoglobin, and then transfusing these cells back into the patient. This modified beta subunit results in an antisickling form of the hemoglobin protein called HbA^T87Q.

“In the first cohort of patients, we had only a modest benefit,” said Tisdale. Even though the gene transfer worked — patients produced the antisickling hemoglobin — they didn’t produce the new form of hemoglobin at high enough levels. HbA^T87Q only made up about ten percent of the total hemoglobin in these patients (13).

It’s a real shot in the arm to go to the clinic and see patients who’ve had their lives completely transformed by this. 
- John Tisdale, the National Heart, Lung, and Blood Institute

The team knew they needed higher HbA^T87Q levels than that, so they made some adjustments to the protocol by improving the viral transduction efficiency and increasing the number of cells each patient received.

“This group of patients had a really profound effect on their disease,” said Tisdale. “Their hemoglobin levels increased to normal. We didn't see sickling anymore, and the patients didn't come back to the hospital in severe pain.” Patients reported an increase in health-related quality of life as well (16).

“It’s a real shot in the arm to go to the clinic and see patients who’ve had their lives completely transformed by this,” said Tisdale. This treatment may soon be available to patients outside of clinical trials. In April 2023, bluebird bio applied for FDA approval for lovo-cel as a treatment for sickle cell disease (17).

First blood

Lovo-cel isn’t the only gene therapy for sickle cell disease in the works. Another treatment, called exa-cel (formerly CTX001), uses nonviral CRISPR gene editing instead of lentivirus and targets a different mechanism. The idea to target this mechanism has its origins back in 1948, when Janet Watson, a physician at the Long Island College of Medicine, published her observation that an unexpected group of individuals seemed to be relatively immune to sickle cell disease: babies (18).

Watson correctly hypothesized that infants were protected because they have a different kind of hemoglobin than adults. At birth, infants produce fetal hemoglobin, which is made of alpha and gamma subunits, and since the disease-causing mutation occurs in the beta subunit, fetal hemoglobin does not sickle. Around six months of age, infants start to produce adult hemoglobin, allowing symptoms to appear (19).  

“For decades, researchers have been trying to figure out what the heck causes that switch from fetal to adult hemoglobin,” said Tisdale. Researchers reasoned that flipping the switch back to fetal hemoglobin production held promise for treating sickle cell disease.

One of the main factors controlling this switch was discovered by molecular hematologist Swee Lay Thein, now also a researcher at the National Heart, Lung, and Blood Institute, in 2007 (20). Thein started out studying beta thalassemia, another blood disorder caused by any of several mutations in the beta globin gene that reduces the number of beta subunits produced, potentially causing life-threatening anemia.

She noted that some patients seemed to be spared the worst effects of the disease, despite inheriting two mutant copies of the beta subunit gene. These patients captured Thein’s attention. “I like looking at outliers because I think that they provide a lot of insights that can help us find new therapies,” she said.

In studying these patients, Thein discovered that, unlike most adults, they still produced substantial amounts of fetal hemoglobin.  By comparing the genes of patients with milder disease to those with more severe disease, she discovered that the gene BCL11A appeared to be at least partially responsible for this effect (20). Among other functions, this gene represses production of the gamma subunit.

This discovery led research groups all over the world to pursue therapeutics that interfered with BCL11A and subsequently increased fetal hemoglobin as treatments for both beta thalassemia and sickle cell disease.

The CRISPR-based exa-cel is one such therapy. It involves harvesting hematopoietic stem cells from patients, using CRISPR to target BCL11A, and then transfusing the edited cells back into the patient.

The results from the ongoing exa-cel trial, “have also been quite impressive,” said Tisdale, who was not involved in exa-cel’s development. Like lovo-cel, a single exa-cel treatment completely eliminated severe vaso-occlusive events, at least in the short term (21).

Exa-cel makers Vertex Pharmaceuticals and CRISPR Therapeutics submitted an application for FDA approval in April 2023 and were granted priority review in June (22).

According to Tisdale, there are advantages and disadvantages of both gene therapy approaches. Scientists have more experience using lentiviral vectors than CRISPR since the former technique has been used to insert genes since the 1990s (23). The CRISPR-based strategy also requires electroporation in order to enter cells, and Tisdale said that the long-term effects of electroporation on hematopoietic stem cell function are still not fully understood.

On the other hand, said Tisdale, “the disadvantage of lentiviruses is that they land semi-randomly in the genome, so if they land upstream of an oncogene, they could turn it on and cause leukemia.” While one woman in the lovo-cel trial did develop acute myeloid leukemia, prompting a hold on the trial, it was later determined that the therapy was not likely to have caused the leukemia based on its location in the genome.  

While long-term effects for both therapies are still being evaluated, Tisdale said that on the whole, “they both look very promising as potentially curative therapies.” 

Back to basics

Despite their impressive efficacy, gene therapies like lovo-cel and exa-cel have one major limitation: accessibility. “The vast majority of patients with sickle cell disease do not have access to this type of life-altering therapy, even in the United States. It’s going to be very expensive and it’s only going to be available in highly specialized centers,” said Tisdale. “Especially outside the United States, for example in Sub-Saharan Africa where the disease is much more prevalent, the infrastructure currently doesn't exist to do these highly technical kinds of curative approaches.”

Furthermore, said Thein, many adult patients already have too much organ damage to be viable candidates.

“There's a need to continue with drug development,” said Betty Pace, a molecular hematologist at Augusta University. “We need more drugs that are cheap and that you can take by mouth.” Pace is studying drugs and endogenous gene expression regulators that can promote the expression of gamma globin and increase levels of fetal hemoglobin in order to treat sickle cell disease.

Pace’s commitment to helping patients with sickle cell disease began early in her life. Growing up, Pace had a close friend affected by the disease. “She had a stroke and then later passed away,” said Pace. “It really affected me. From that point on, I wanted to research sickle cell disease to find better treatments, and I’ve worked toward that goal pretty much all my life.”

Pace is currently interested in how microRNA influences the production of fetal hemoglobin. Pace and her colleagues discovered a microRNA called miRNA-29b that increases fetal hemoglobin by decreasing the expression of two other genes,  MYB and DNMT3, which themselves repress the expression of gamma globin (24). In support of the importance of this microRNA in sickle cell disease, the research team also found that levels of miRNA-29b were higher in patients with higher levels of fetal hemoglobin (25).

Pace’s team is currently developing an effective delivery system for miRNA-29b therapy since these molecules are rapidly degraded in the body. “We've actually been able to modify our microRNA, adding a short sequence to it, to make it look like cholesterol. And when we do that, we can inject the microRNA intravenously and it lasts longer and induces fetal hemoglobin. So, the next step would be some type of oral formulation.” The team is also investigating off-target effects to determine whether miRNA-29b turns on or off expression of other genes, and if so, what influences that might have.

Pace has also been involved in research that used high throughput screening to identify the drug benserazide, which is currently used in the treatment of Parkinson’s disease, as a potent inducer of fetal hemoglobin and confirmed the efficacy of this drug in mouse and baboon models (26,27). This drug is currently being tested in a Phase 1 clinical trial as a beta thalassemia therapeutic, with plans to expand to sickle cell disease (28).

Unusual patients inspire new therapies

While increasing fetal hemoglobin is a popular strategy for investigational sickle cell disease therapeutics, it’s certainly not the only strategy. Thein is now working on another treatment strategy targeting pyruvate kinase, or PKR.

“The idea occurred to me when I was working at King’s College Hospital. I was referred a patient with sickle cell disease who had all the typical clinical symptoms, but genetically, she didn’t have the disease,” said Thein. The patient only had one copy of the gene with the sickle cell mutation.

“I started wondering about what could be happening. It turned out that she had also inherited a mutation in one of her PKLR genes, the gene that produces pyruvate kinase,” said Thein.

This prompted Thein to undertake a larger study on PKLR mutations in people with sickle cell disease. Her research team found that seven genetic variants in the PKLR gene associated with an increased rate of hospitalizations, further suggesting that this gene plays a role in the severity of the disease (29).

According to Thein, PKR may mediate sickle cell disease severity in two different ways. First, reduced PKR activity increases the concentration of a metabolite called 2,3-diphosphoglycerate (2,3-DPG) in red blood cells.  2,3-DPG is a major factor in sickling because it decreases the oxygen affinity of hemoglobin. Since sickle hemoglobin forms the long fibers that lead to sickling when it is not bound to oxygen, higher levels of 2,3-DPG could promote sickling, leading to pain and tissue damage (29).

Secondly, and perhaps even more importantly, PKR is an essential part of the pathway that allows cells to turn glucose into ATP (29). Low or malfunctioning PKR results in the untimely demise of red blood cells, ultimately leading to chronic anemia.

Fortunately, Thein didn’t have to start from scratch to target this enzyme. Researchers at Agios Pharmaceuticals had already developed a drug called mitapivat (which was FDA approved just last year) to treat individuals with PKR deficiency. Thein wanted to know if activating PKR with mitapivat might help treat people with sickle cell disease too.

Results from a small Phase 1 study, while preliminary, were promising. Researchers found that mitapivat reduced 2,3-DPG and increased ATP, as well as reduced markers of hemolysis (30). At least in the relatively short trial, the drug appeared to be well tolerated.

This is really breaking new ground for treating sickle cell disease. 
- Swee Lay Thein, the National Heart, Lung and Blood Institute

Unpublished work from Thein’s group suggests that mitapivat treatment also helps to strengthen the membranes of red blood cells.

Based on these results, Agios launched a randomized, placebo-controlled Phase 2/3 clinical trial called RISE-UP. Enrollment is complete for the Phase 2 portion of the study. Preliminary results from this first placebo-controlled study showed that patients’ hemoglobin levels increased, and there was a trend towards fewer pain crises (31). Thein said that if all goes well, recruitment for the Phase 3 trial will begin in Fall 2023.

“This is really breaking new ground for treating sickle cell disease,” said Thein. “In the past, everyone was just focusing on how to prevent [hemoglobin] polymerization. But when you improve the red cell membrane and improve the cells’ integrity, that’s more than just antisickling. And it’s also applicable to other hemolytic anemias.”

After decades with so few approved drugs to treat this serious disease, hope is now on the horizon for patients, with dozens of therapies being explored in clinical or preclinical studies. Whether through high-tech gene therapies or accessible, orally bioavailable drugs, researchers hope that their work will alleviate suffering on a global level.  

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