If red blood cells are the ferryboats of the bloodstream, hemoglobin is the ferryman. With its two alpha globin and two beta globin subunits working in harmony, hemoglobin helps shuttle oxygen into cells and remove carbon dioxide waste products from them. But even one small defect — such as a single amino acid change — in one of these globin subunits can spell disaster for the body.
Defective hemoglobin results in red blood cells that can’t transport oxygen effectively, which can lead to severe anemia and other complications. Inherited defects in hemoglobin, called hemoglobinopathies, include sickle cell disease, beta thalassemia, and alpha thalassemia, among others (1).
While regular blood transfusions and certain medications can help people with these diseases manage their symptoms, for many years, the only cure has been a bone marrow transplant. This procedure requires a matched donor and chemotherapy to clear out the recipient’s bone marrow. Only about twenty percent of people have a bone marrow match, and even with a good match, there is still a chance for rejection.
Recent advances in gene therapy are now offering patients what may be a one-time cure for these conditions. Just last year, the FDA approved bluebird bio’s gene therapy for beta thalassemia ZYNTEGLO, and the company has another gene therapy for sickle cell disease currently in Phase 3 clinical trials. Closing in on an additional gene therapy for sickle cell disease and beta thalassemia is a partnership between CRISPR Therapeutics and Vertex Pharmaceuticals with the therapy exagamglogene autotemcel (exa-cel). Researchers from both companies just released positive interim data from Phase 3 clinical trials for their gene therapies in both conditions.
These two approaches circumvent the risks of bone marrow transplants by using patients’ own stem cells as the basis of their therapies. Both treatments require isolating patient stem cells and editing the cells to fix the beta globin defect. ZYNTEGLO uses a lentiviral vector to replace the defective beta globin gene with a functional one in patient stem cells, and exa-cel delivers CRISPR/Cas9 components to patient stem cells via electroporation to engineer the cells to produce high levels of fetal hemoglobin, which does not contain beta globin and therefore will not lead to the production of defective hemoglobin in these patients.
While these advances in gene therapies for hemoglobinopathies are very exciting, one disease is conspicuously missing.
“A lot of different gene therapy approaches over the last decades have been tailored for beta thalassemia and sickle cell disease, which is great, but there has been this unmet need for alpha thalassemia,” said Eva Segura, a graduate student researcher in Donald Kohn’s laboratory at the University of California, Los Angeles. “Alpha thalassemia, for gene therapy, has really been historically ignored.”
One of the reasons for this is that alpha thalassemia affects fetuses in utero. Alpha thalassemia arises from the loss of the alpha globin subunit, which is a vital component of fetal hemoglobin. So without in utero blood transfusions, fetuses with alpha thalassemia are unlikely to survive gestation. But with the increased awareness of in utero blood transfusions for alpha thalassemia, more patients are surviving to term and living with regular blood transfusions like their other hemoglobinopathy peers.
“This gene therapy approach really aims to decrease the blood transfusions — if not totally eliminate the requirement for these blood transfusions — as now, the modified or corrected stem cells will naturally be able to produce alpha globin,” said Segura.
Due to the similarities between alpha thalassemia and the other hemoglobinopathies, multiple research groups are looking to extend these gene therapy strategies to alpha thalassemia.
Like beta like alpha
When Kyle Cromer, a stem cell biologist now at the University of California, San Francisco (UCSF), started developing an alpha thalassemia gene therapy, he knew very little about blood disorders.
“I was in it really for the tools,” he said. “CRISPR truly gives us the ability to edit the genome as if it's a Microsoft Word document. I was just enamored by that fact.”
With a strong interest in the gene editing power of CRISPR, Cromer joined Matthew Porteus’ group at Stanford University as a postdoctoral researcher when they were developing a CRISPR-based method to fix beta hemoglobinopathies in patient hematopoietic stem cells (2). Their strategy involved making precise cuts in the DNA of these patient cells and then flooding the cells with a copy of the beta globin gene delivered via an AAV vector. Then, the cells would repair the DNA break by integrating the full-length copy of the beta globin gene.
In Porteus’ group, Cromer and his colleagues further refined this approach by considering that in beta thalassemia, the ratio of alpha globin to beta globin weighs too heavily on the side of alpha globin. To bring the levels of healthy alpha and beta globin in balance with each other, Cromer used this CRISPR method to replace one copy of the alpha globin gene with a healthy copy of the missing beta globin gene in beta thalassemia patient stem cells (3).
Porteus presented this research at a conference, and after his talk, a researcher from UCSF asked him whether this approach might also work for alpha thalassemia.
“They're the number one referral center for this disease, so [there is] incredible expertise resident at UCSF for alpha thal,” said Cromer. “In the same way that beta thal was a leap from sickle cell disease, alpha thal is a leap from beta thal. We have all the assays that we need. We have the genome editing strategy. We just need to come up with a different knock in strategy to cure alpha thal.”
When Cromer started his laboratory at UCSF, he began collaborating with alpha thalassemia expert Tippi Mackenzie at UCSF to develop a CRISPR-based gene therapy approach for alpha thalassemia.
Mirroring his beta thalassemia gene therapy, Cromer and his team developed a strategy to replace one beta globin gene with a healthy copy of an alpha globin gene in alpha thalassemia patient hematopoietic stem cells.
He and his team optimized their approach in stem cells from healthy individuals first and then tested the gene therapy in stem cells from alpha thalassemia patients. When they transplanted the cells into immunodeficient mice, the edited cells functioned as expected and made healthy red blood cells. Cromer and his team are writing up a manuscript describing these results now.
In the same way that beta thal was a leap from sickle cell disease, alpha thal is a leap from beta thal. We have all the assays that we need. We have the genome editing strategy. We just need to come up with a different knock in strategy to cure alpha thal.
– Kyle Cromer, University of California, San Francisco
“We showed for the first time that we were able to correct the defect and restore hemoglobin production to patient-derived stem cells that had been differentiated into red blood cells, so really mimicking how they behave in the patient. These cells look functional again,” said Cromer. “We were really over the moon when we saw that.”
Cromer is eager to move this approach to the clinic, but he is proceeding with caution based on the gene editing company Graphite Bio’s experience using a similar genome editing platform. The company recently paused their Phase 1/2 clinical trial when the first patient they dosed had serious and prolonged low blood counts.
“The consensus seems to be that when you expose your hematopoietic stem cells to a virus like this, they kind of freeze. They think that something's wrong, and they undergo this cell cycle arrest and then don't function the way that they need to,” said Cromer.
Because of this, he is considering pivoting to a next generation CRISPR tool: base editors. “Those have given every indication they're going to perform very well in clinic. They also don't create double stranded DNA breaks, which are also bad news for the most part,” said Cromer.
Cromer is excited about the prospect of using base editors in the context of in vivo genome editing. Rather than isolating patient stem cells, treating them with the gene therapy ex vivo, and delivering the edited cells back into the patient, in in vivo genome editing, all of the gene therapy components would be injected directly into a patient’s bloodstream to make the appropriate genome edits independently.
In vivo genome editing would be particularly useful for alpha thalassemia because the disease begins before birth. Researchers could deliver the in vivo genome editing therapy to fetuses with alpha thalassemia, potentially curing them before they’re born.
“Pivoting to these base editor technologies, which are more easily packaged into in vivo genome editing delivery vehicles… that's exactly the path forward that we want,” said Cromer. “We also have collaborations starting up to test some of the best-in-class lipid nanoparticles, LNPs, for in utero delivery, and we're going to be testing those out really soon.”
While Cromer and his team focus on integrating CRISPR tools into their alpha thalassemia gene therapies, researchers like Segura are building off of the success of lentiviral vector-based gene therapies to develop theirs.
A lentiviral strategy
Segura has always been interested in translational research, and she knew that for her doctoral research, she wanted to work on a project that could really change people’s lives. About six months after Segura joined Kohn’s laboratory, the perfect project came across her desk.
“This project came from Dr. Tippi Mackenzie — a longtime friend of Dr. Kohn — who is a researcher but also a fetal surgeon who works on alpha thalassemia. She asked us to join her on her alpha thalassemia project… as we're very big in gene therapy for blood disorders such as sickle cell disease,” said Segura.
Rather than replacing the beta globin gene with alpha globin like Cromer, Segura uses a lentiviral vector to deliver a healthy copy of the alpha globin gene into the genome of alpha thalassemia patients’ stem cells before reinfusing the “fixed” cells back into the patient.
“Instead of integrating the viral genome, we are integrating the gene that is missing along with the regulatory elements that allow this gene to be expressed correctly in the correct cells. So, for alpha thalassemia, in red blood cells,” said Segura. But, she added, “one of the biggest challenges of gene therapy using lentiviral vectors is their size.”
It would be ideal to put the entire alpha globin gene along with its regulatory sequences onto the lentiviral vector to ensure that the edited stem cells produce high levels of the alpha globin gene. But, when lentiviral vectors get too long, the concentration of functional viruses carrying this piece of DNA is low, and therefore the transfer of the therapy into patient stem cells is very low.
Because researchers can’t reduce the size of the alpha globin gene, the regulatory sequences, which enhance the expression of the alpha globin gene, are the ones on the chopping block.
To develop something from my own hands, that's what I love the most. I am developing a therapy that could potentially help so many people who are suffering.
- Eva Segura, University of California, Los Angeles
“It's kind of a tug of war,” Segura said. Though as luck would have it, a former graduate student in the Kohn lab, Richard Morgan, who had been working on a gene therapy for beta-thalassemia, spent years determining the core regulatory elements that needed to be present in a lentiviral vector to ensure proper beta globin expression.
“The vectors that I'm creating, they're based on the beta globin regulatory elements and on beta globin vectors,” said Segura. “Because it's the same protein expressed in the same red blood cells, the alpha globin gene is really well regulated using the beta globin regulatory elements.”
Helpfully, the alpha globin gene is also smaller than the beta globin one, allowing for even more lentivirus production and high gene transfer efficiency into stem cells. Segura has been working on this project for two years so far, and using these beta globin vectors as a starting point, she has now designed multiple promising gene therapy vectors for alpha thalassemia.
Working with alpha thalassemia patient cells provided by Mackenzie’s team, Segura found that she could deliver her alpha globin lentiviral vectors to the patient stem cells and differentiate them into red blood cells.
“These red blood cells had not only alpha globin expression, but they had proper hemoglobin protein complex, so [the fixed alpha globin chain was] assembling well with the endogenous beta globin chain,” she said. “So far, very, very encouraging and promising data.”
She has recently begun in vivo studies to see if her lentiviral vectors can be used to treat a mouse model of alpha thalassemia.
“To develop something from my own hands, that's what I love the most,” said Segura. “I am developing a therapy that could potentially help so many people who are suffering.”
- Kohne, E. Hemoglobinopathies: Clinical Manifestations, Diagnosis, and Treatment. Dtsch Arztebl Int 108, 532–540 (2011).
- Dever, D. et al. CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells. Nature 539, 384–389 (2016).
- Cromer, M.K. et al. Gene replacement of α-globin with β-globin restores hemoglobin balance in β-thalassemia-derived hematopoietic stem and progenitor cells. Nat Med 27, 677–687 (2021).