Replacing An Entire Faulty Gene To Reverse The Blood Disease β-Thalassemia

A team of scientists led by Stanford University researchers Daniel Dever and Matthew Porteus developed a possible gene therapy for the blood disorder β-thalassemia — by replacing an entire gene at once.

β-Thalassemia is a complex condition that can stem from a wide array of mutations of various sizes and types. One of the main underlying problems is a dangerous reduction in the production of haemoglobin, the protein in red blood cells that shuttles oxygen throughout the body, due to a loss-of-function mutation in the HBB gene that produces β-globin. To restore those haemoglobin levels, scientists cut out one of the two HBA genes that produces α-globin and replaced it with a full-length HBB transgene within patient-derived cells in mice, according to the study published last month in the journal Nature Medicine.

About one in 100,000 people develop β-thalassemia, making it one of the more common genetic blood diseases in the world, according to the study. Without adequate haemoglobin to carry oxygen throughout their body, patients often experience symptoms including anaemia, muscle weakness and fatigue. They may also develop enlarged organs and undergo delayed physical development. The reduced haemoglobin levels are rough enough, but the disease is further complicated because only one of the two components of haemoglobin is missing. Without enough β-globin to bind to, excess unpaired α-globin comes out of solution and forms red-blood-cell-killing aggregates that can also lead to blood clots.

Even with recent improvements in how the disorder is treated, patients worldwide have a mean life expectancy of just 30 years.

Extraordinary and promising approach

Unlike sickle cell disease, which is caused by a single point mutation in the HBB gene — and which Graphite Bio and the Stanford Porteus lab tackled in a different recent study — it can be hard to determine the best approach for a β-thalassemia gene therapy given the multitude of contributing mutations, explains Dr Alexis Thompson, the Hematology Section Head at the Ann & Robert H. Lurie Children’s Hospital of Chicago and a professor of paediatrics at Northwestern University's Feinberg School of Medicine. Thompson didn't work on the study and isn't affiliated with its authors, but she said she was encouraged by what the team managed to accomplish.

»The notion of the patient being their own [blood] donor and being able to modify stem cells ex vivo and return them to the patient as a process, that part of it I think we understand,« says Thompson.

»I think now it comes down to the details: what aspects of thalassemia do we actually want to approach? We're able to entertain or imagine these modifications because we are grounded in an approach that I believe is extraordinary and quite promising,« she adds.

A CRISPR gene-editing first

Instead of choosing a specific problem area in the gene to target with a CRISPR Cas9 therapy, the Stanford team decided to swap out the entire gene at once through ex-vivo hematopoietic stem cell transplantation (HSCT): taking out stem cells, swapping an HBA for an HBB gene, and transfusing them into the body, essentially turning patients into their own blood donors.

Replacing an entire gene at once is an incredible scientific feat. In fact, it may well be unprecedented* across the entire gene editing field, says study author Daniel Dever, now the co-founder and head of discovery research at the San Francisco-based gene-editing company Graphite Bio.
But aside from bragging rights, the accomplishment allowed the researchers to simultaneously downregulate α-globin and upregulate β-globin so that the two were produced at a healthy balance.

»We had this idea: What if you could overexpress β-globin through the α-globin promoter? It would potentially kill two birds with one stone. It would simultaneously reduce the expression of α-globin and increase expression of b-globin,« says Dever.

»We were able to show that it decreases alpha while it increases beta. That was the goal we were looking for,« he adds.

In theory, the team could have tried to swap out the HBB gene for a functional version, but targeting one of the HBA genes makes more sense for a variety of reasons, Dever explains. Chief among them is the fact that β-thalassemia is a complex disease caused by various mutations, some patients may still have residual, endogenous β-globin production, and it would be counterproductive to knock that out.

Thompson says that β-thalassemia could be considered a manageable chronic disease thanks to improvements in both blood-iron level monitoring techniques and the overall safety of the blood supply. But a curative approach through gene therapy, she suggests, would revolutionize the field — and could be ready in the next few years.

Turning gene-swapping into a cure

The big question, of course, is whether the corrective effects in individual cells and mouse models translate into curative effects for human patients.

»Creating these humanized models in the mouse setting eventually runs up against the realities that there are distinct differences [between mice and people]. At some point, the issue becomes being able to take this proof of concept that's been done so elegantly in these various mouse models and establish that something similar can be achieved when in human cells. And then the next question in the thalassemia patient is 'Can you do this at scale?' Can you do these corrections, avoiding increases in indels, and get them to a level where they're making sufficient haemoglobin in a human to perhaps render them transfusion independent?« says Thompson.

There's reason to suspect it might work — at least more reason to think so than for the other gene therapy approaches that have been floated for β-thalassemia — but it's far too soon to tell.

That's because replacing HBA1 with HBB, using the α-globin promoter to produce β-globin, while leaving HBA2 alone so it can continue to produce α-globin, theoretically stands to rebalance globin levels in a way that other approaches don't. Scientists previously suggested using CRISPR Cas9 to reawaken the fetal precursor to β-globin to make up for low haemoglobin levels. But with both HBA1 and HBA2 in full swing, there could still be dangerous levels of unpaired α-globin that the fetal globin approach couldn't account for.

This is the same decision that Graphite Bio made when developing a possible treatment for Sickle Cell Disease. In both cases, Porteus and his team decided to address the mutation directly rather than rely on upregulated fetal globin to make up the difference. Dever says that the team at Graphite Bio hasn't yet decided whether they want to pursue the β-thalassemia gene therapyfurther. But if they do, the ability to replace an entire gene in a clinical setting would represent a potential paradigm shift in how blood disorders are treated.