Innovative gene therapies for sickle cell disease successfully tested in vitro

With 350,000 new patients diagnosed each year worldwide, sickle cell disease is the most common genetic disease. It is caused by a mutation in the gene that code for β-globin, one of the proteins that make up haemoglobin, the main component of red blood cells. "In small blood vessels - where oxygen is scarce - this mutated haemoglobin is subject to a polymerisation phenomenon: it leads to the rigidification of the red blood cell, which then takes on the shape of a sickle," explains Sophie Ramadier, post-doctoral researcher in the "Chromatin and gene regulation during development" laboratory, directed by Annarita Miccio. As a result, the red blood cells locally obstruct the blood flow, causing extremely painful attacks called vaso-occlusive crises. Patients also experience severe anaemia, increased susceptibility to infection and progressive loss of organ function.

Today, the only curative treatment for sickle cell disease is a bone marrow transplant from a compatible donor. Most of the time, this is a sibling, a family member or a donor found on a national bank. Unfortunately, the chances of finding the right person remain low. In such cases, a major alternative is blood exchange, which replaces sickle cell red blood cells with healthy ones, thus temporarily replenishing a stock of healthy haemoglobin. Another avenue explored by AP-HP doctors and researchers at Imagine Institute (Inserm, AP-HP, University of Paris) is gene therapy. In 2017, the team of Prof. Marina Cavazzana, a paediatrician, director of the biotherapy department and director of the Clinical Investigation Centre for Biotherapy at Necker-Enfants Malades AP-HP hospital, was able to cure the very first sickle cell patient by gene therapy. Since then, around fifty patients have been treated with this approach with good results for 60% to 70% of them.

Viral vectors based on CrispR-Cas9 molecular scissors

The principle? To limit the deleterious effect of the mutated gene by integrating a new gene into patient stem cells. In practice, the scientists use a viral vector - in this case, a lentivirus - into which a genetic sequence coding for a healthy β-globin has been introduced. In the presence of the patient's stem cells, this vector penetrates the nucleus where it inserts itself and expresses the new healthy sequence. As a result, the cells produce healthy β-globins which, by associating with α-globins, form a functional adult haemoglobin," explains Sophie Ramadier. The defective gene continues to be expressed in parallel, but this is not necessarily a problem. Indeed, α-globin has a better affinity with healthy β-globin than with its pathological counterpart, which increases the probability of forming healthy adult haemoglobin.

In a new study published in the journal Molecular Therapy, Annarita Miccio's team has developed three new viral vectors to increase the efficiency of this process [1]. All three vectors have a genetic sequence encoding a β-globin containing not one but three anti-sickle cell (or AS3) mutations. "In the first vector, we introduced this βAS3 globin gene in combination with CRISPR-Cas9 technology, making it possible to 'cut' very precisely the mutated β globin gene responsible for the disease," explains Sophie Ramadier, the first author of this work. This is a way of reducing or even abolishing the expression of the mutated gene and thus limiting the polymerisation phenomenon, thus favouring the expression of therapeutic AS3 haemoglobin.

Restoring fetal haemoglobin production by reactivating a silent gene

The other two viral vectors also contain the sequence coding for βAS3 globin, coupled with CRISPR-Cas9 technology, but this time with the aim of targeting certain areas of the DNA very finely in order to reactivate a normally silent gene. This is the gene coding for globin ɣ, a protein which, during foetal development, enables the formation of a perfectly functional foetal haemoglobin. The principle of these two vectors is therefore to reactivate the production of this globin which is normally replaced at birth by adult haemoglobin. The use of these two vectors will therefore make it possible to express both therapeutic AS3 haemoglobin and foetal haemoglobin, promoting the production of anti-sickle cell haemoglobin to counter the formation of mutated haemoglobin.

In experiments conducted in vitro on patient stem cells, these new vectors have shown, at the same dose, a better efficacy than the vectors used today in the clinic. Although these basic results are still very preliminary and need to be proven in vivo, they are promising for the future of gene therapy for sickle cell disease.

[1] S. Ramadier et al., Molecular Therapy, https://doi.org/10.1016/j.ymthe.2021.08.019, 2021.