The major breakthroughs in genetic engineering took place in the 1960s and 1980s, and from these developments emerged the technologies that would allow the isolation, cloning and artificial transfer of genes from one individual to another, even between different species, which is what the term “transgenesis” refers to.

The leap toward its applications for the treatment of human diseases due to gene mutations occurred in 1980, when a corrective gene for severe b-thalassemia was first introduced by a professor of medicine at the University of California, Los Angeles (UCLA) to treat two patients with severe β-thalassemia by transfecting marrow cells with a recombinant human globin gene [1]. However, its developer, Dr. Martin Cline, had not obtained the mandatory US National Institute of Health (NIH) permits, so his treatment was not considered ethically acceptable and its merit was not recognized.

Ten years later, the NIH approved the first clinical trial for the correction of severe combined immunodeficiency (SCID) for so-called “bubble children.” Drs. Michael Blaese and French Anderson tackled this challenge by inserting the ADA gene — responsible for synthesis of the enzyme adenosine deaminase — into cells of the hematopoietic system of newborns with this immune deficiency [2]. After extracting stem cells of the immune system from the bone marrow, they were cultured in the laboratory, genetically modified and returned to the children, who acquired the defenses they previously lacked.

A major turning point in the direction of knowledge of the genetic basis of hereditary diseases came with the completion of the Human Genome Project in 2003. Using knowledge of the details of the nucleotide sequences of our genes, remarkable applications began to be developed in the diagnostic, therapeutic and pharmacological fields of medicine. In the line of therapeutic applications, it opened possibilities for correcting diseases due to monogenic systems in the patients’ somatic cells. It would be a way to resolve about 8,000 human diseases.

Nevertheless, even if the cause is known and the technology is available to do so, the realization of gene therapy is highly complex and the ethical issues that arise are mainly safety-related, due to the unpredictable side effects that any modification of our genes may produce. In addition, the technology needed is extremely costly, which makes it very difficult to apply them in public and even private healthcare systems. It is expected that these problems will be resolved over time.

The first generation of CRISPR-Cas9 gene therapy

After years of trials, many of which failed, gene therapy has had a huge boost following the emergence of “gene editing” technology, after the discovery of the CRISPR-Cas9 system. This system works by exerting a direct action on the DNA in order to “edit”, i.e., modify or correct, the sequence of the DNA nucleotide bases of the altered gene (a detailed description of the discovery and the technique can be found in El mensaje de la vida. Credo de un Genetista [3]).

The medical application of CRISPR-Cas9 technology was posited and developed in 2012 by French researcher Emmanuelle Charpentier and American Jennifer Doudna, who were awarded the Prince of Asturias Award for Science in 2015 and the 2020 Nobel Prize in Chemistry for this important contribution. These researchers described how to apply this technology as a genome editing system in June 2012 in the journal Science [4].

Basically, the technique acts directly on the DNA region of the altered gene by inserting two components into the patient’s cells: a guide RNA, to recognize the target DNA, and an enzyme called Cas9, which acts by making a cut at the recognized site. The CRISPR technique is based on knowledge of the sequences in our genome and the pathological consequences of gene alterations. The operation involves cutting the DNA of the altered gene at the site marked by the guide RNA, and either leaving the gene disabled (knockout) or editing the error by replacing the altered nucleotide bases with the correct ones. It is important to note that, technically, it is easier to knock out a gene than to replace nucleotide bases, but both are possible with greater or lesser ease, depending on the case. Although the technique can be applied in vivo, this operation is easier in the laboratory, acting in vitro on cells previously extracted from the patient, which are subsequently returned by autotransplantation.

In any case, the CRISPR-Cas9 technique is more direct, faster and more user-friendly than other previously tested technologies, such as so-called “Zinc fingers” and “TALEN” (transcription activator-like effector nucleases). Indeed CRISPR has proven to be much more efficient and easier to use, with the result that its application for a number of purposes has spread throughout laboratories worldwide, and has it become the great hope for treating human diseases due to gene alterations.

In 2013, the first clinical application of CRISPR-Cas9 was performed in the Netherlands, at the Hubrecht Institute, to correct a mutation in the CFTR gene that causes cystic fibrosis [5]. Five years later, the first clinical trial in the U.S. was launched by researchers from Stanford, Columbia and other institutions, in order to correct two related diseases: b-thalassemia and sickle cell anemia. By the end of 2023, 89 clinical trials or research using CRISPR for therapeutic purposes had been registered in the large global database clinicaltrials.gov. Many of these trials refer to the CAR-T technique for the treatment of multiple types of cancer, including solid cancers. However, in this article, we shall refer specifically to less complex diseases, such as those due to a single gene.

The most remarkable thing is that, in just over 10 years, CRISPR has become a tool for real therapy potentially applicable to patients suffering from a long list of monogenic diseases, with the first treatments being approved by international drug agencies. This is a dizzying rate compared to what we usually find in the development schedules of new drugs, comparable only to the speed of the development of messenger RNA-based vaccines against SARS-Cov2, responsible for Covid-19.

The best proof of concept for the rapid development of CRISPR-based techniques for therapeutic purposes is provided by the CasgevyTM procedure for the treatment of sickle cell anemia and b-thalassemia developed by CRISPR Therapeutics, a Swiss company co-founded by Dr. Charpentier and the biotechnology company Vertex Pharmaceuticals, based in Boston, Massachusetts (U.S.). This treatment was officially approved on 16 November 2023 by the MHRA (UK Medicines and Healthcare Products Regulatory Authority). Shortly thereafter, on 8 December, it was approved by the EMA (European Medicines Agency), and on 15 December by the FDA (US Medicines Agency).

Individuals with sickle cell anemia are carriers of a b-globin altered by the substitution of an amino acid in the beta chain of hemoglobin in their red blood cells, so that the cells become distorted and present a crescent- or sickle-shaped appearance, hence the name of the disease. As a result, their ability to transport oxygen decreases significantly and patients experience heart problems, fatigue and pain in different parts of the body, especially the hands, feet, intestines and bones. In general, it manifests with a decrease in normal red blood cells, leading to generalized symptoms of anemia. Sickle cell anemia is the most common serious inherited genetic disease in the U.S., affecting more than 100,000 people. Life expectancy is less than 53 years.

Understanding the gene sequence responsible for this disease and the existence of other alternative genes within the multigene family of human globins led to the application of CRISPR technology to beat the disease.

In the CasgevyTM procedure, blood stem cells are extracted from the patients’ bone marrow and cultured ex vivo. After gene editing, the stem cells are expanded in vitro and returned to the patient by autologous transplantation. It consists of inducing knockout gene editing to inactivate the BCL11A gene, which involves direct knockout to prevent its expression. This gene regulates the synthesis of an alternative globin, fetal globin, which is only expressed during the fetal phase and then stops after birth. Switching off the BCL11A gene induces the synthesis of fetal globin, which when expressed replaces the adult b-globin. The idea that fetal hemoglobin (HbF) can protect against the disease is due to a 1948 discovery, when Dr. Janet Watson of Long Island, New York, observed that newborns never showed symptoms of sickle cell anemia, which is strange in a congenital disease. She therefore concluded that the fetal form of the molecule, which is active during pregnancy, protected babies for a few months after birth, only until it was replaced by the altered version of the adult b-globin.

Bone marrow transplantation involves chemotherapy and making room for the edited stem cells, which means that, at the moment, gene editing using this procedure is only applied to patients with very severe sickle cell disease. The outcome of treatment by this procedure has been satisfactory in the more than thirty patients treated with CasgevyTM, who have shown an increase in HbF and a reduction in disease complications.

So far the good news, but there is a problem. The cost of the CasgevyTM application is about 2 million Euros (about 2.2 million US dollars), making it one of the most expensive medicines in the world. This means that insurers have not yet decided whether they will cover the cost of treatment, although given the expense involved in a chronic and debilitating disease such as sickle cell anemia, it is only a matter of time before it ends up being accepted by public health systems.

The following base-editing therapies include one treatment and a growing list of clinical trials for the treatment of numerous monogenic diseases, such as hypercholesterolemia, cystic fibrosis, retinitis pigmentosa, Duchenne muscular dystrophy, Rett syndrome, etc. We can therefore say that we are in the first generation of therapeutic genome editing. Nevertheless, there are still limitations, especially due to possible off-target DNA breaks or lack of safety due to side effects, since the direct and targeted correction of a therapeutic target does not dispel all doubts about effects elsewhere [5].

Technological innovations and new challenges

The trend to follow seeks to increase the precision of gene editing, to avoid “off-target” editing, so as not to affect other regions in the genome.

From studies conducted so far, it has been concluded that it is easier to delete the activity of a gene (knockout) than to edit it by substituting its bases. It is always easier to destroy than to build. However, in order to improve the situation, innovations have appeared in the technique that have enabled the results to be improved, especially with regard to better precision in the base substitution in the altered genes and avoiding errors due to the natural mechanisms of DNA repair. Thus, the techniques “Base Editing” and “Prime Editing” have emerged [6]. The first is aimed at accurately replacing an adenine (A) with a guanine (G), or a cytosine (C) with a thymine (T). Unlike first-generation CRISPR-Cas9, “Base Editing” cuts both strands at the DNA target. In addition, the Cas9 enzyme guides other enzymes to the selected site, where they can perform the work needed to switch the DNA bases.

Additionally, in 2019, a new CRISPR system called “Prime Editing” was developed, allowing individual DNA bases to be switched and small stretches of DNA to be inserted or removed at specific sites. Unlike first-generation gene editing, this innovation can recognize and correct almost any site in the genome more precisely, despite its greater complexity.

Since the correction of many monogenic diseases involves silencing of the altered gene, another procedure to follow is to switch off gene expression indirectly, through epigenetic modification. Techniques aimed at modifying the epigenome have not advanced as rapidly as genome base editing, probably due to the fact that epigenetic modifications are less consistent and can be erased during cell division. The results of research related to epigenetic modifications are more unpredictable than all the progress in relation to gene sequence editing. It is a new challenge, a field open to new research.

We are living in a time of great expectations. We are on the verge of the second generation of CRISPR technology for therapeutic applications, a new generation seeking to overcome limitations with improved precision and versatility, opening a path of great hope in the treatment of many diseases.

Nicolás Jouve

Professor Emeritus of Genetics

Member of the Bioethics Observatory

Former member of the Spanish Bioethics Committee

 

References

[1] Cline M.J. «Perspectives for gene therapy: inserting new genetic information into mammalian cells by physical techniques and viral vectors». Pharmacol. Ther. 1985; 29: 69-92

[2] Anderson W. F. «Prospects for human gene therapy». Science. 1984, 226 (4673): 401–409.

[3] Jouve, N. El mensaje de la vida. Credo de un genetista. (Ed. Encuentro, Madrid, 2020).

[4] Doudna, J.A., Charpentier, E. «Genome editing. The new frontier of genome engineering with CRISPR-Cas9». Science 2014, 346 (6213), 1258096

[5] Huang, M.E., Qin, Y., Shang, Y. et al. «C-to-G editing generates double-strand breaks causing deletion, transversion and translocation». Nat Cell Biol (2024).

[6] Testa LC, Musunuru K. Base Editing and Prime Editing: Potential Therapeutic Options for Rare and Common Diseases. BioDrugs. 2023 Jul;37(4):453-462.

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