Gene therapy: a "molecular scalpel" corrects the genes of blood stem cells

Based on research carried out at the San Raffaele Telethon Institute for Gene Therapy (TIGET) in Milan, a technique has been developed that allows bad genes to be repaired directly on the DNA strand of human blood stem cells. The study, published in the scientific journal Nature, will make gene therapy more precise. Read more

Naldini''s research group

A group of researchers at the San Raffaele Telethon Institute for Gene Therapy (TIGET) in Milan was able for the first time to rewrite the DNA of human blood stem cells by editing the genome, (a technique first introduced by Nobel laureate Mauro Capecchi), which makes it possible to correct errors directly on the affected gene. More specifically, thanks to a "molecular scalpel," scientists were able to repair the defect responsible for severe hereditary immunodeficiency with absolute precision, thus opening the door for the application of this innovative method on humans.

The study, published in Nature*, was carried out under the supervision of Luigi Naldini, Director of the San Raffaele Telethon Institute for Gene Therapy and professor at the University Vita-Salute San Raffaele in Milan, and Angelo Lombardo, a researcher at the same institutions. Further confirmation of the international value of TIGET's research is the prestigious Outstanding Achievement Award given to Luigi Naldini a few days ago in Washington by the American Society of Gene and Cell Therapy.

After years of studies, gene therapy today is beginning to deliver important results, even in human applications. There are now several clinical trials being carried out throughout the world in which viruses that have been appropriately modified and rendered harmless provide patients with the correct versions of genes that are defective and responsible for certain diseases. Last July, in Science magazine, a research team led by Luigi Naldini showed how this technique gives real hope for the treatment of serious genetic childhood diseases such as metachromatic leukodystrophy and the Wiskott-Aldrich syndrome.

"Until now, gene therapy consisted mainly in adding a functional copy of a gene that was faulty, using a virus that had been suitably manipulated and rendered harmless; a little like using a crutch when you have a broken leg," says Luigi Naldini, director of the San Raffaele Telethon Institute for Gene Therapy. "With the new study published today in Nature we have taken an important step forward. The editing of the genome allows us to correct genetic defects directly in the DNA, a little like repairing a broken bone. It is a tremendous step forward, because it allows us to restore not only the gene's function but also its natural regulation (how, when and where it is expressed), which today we cannot perform faithfully when we introduce a new copy of the gene with a virus from the outside. And we have demonstrated how to do it in hematopoietic stem cells, the mothers of all blood cells."

At the heart of the new molecular technique that has been developed are artificial endonucleases, which are proteins made in the laboratory used to induce the modification of a specific DNA sequence. These proteins are currently the focus of attention of researchers all over the world for their potential applications in research. "The artificial nucleases are constituted by two distinct portions, one capable of binding to a specific sequence of letters (bases) on the DNA, which we choose in the gene to be repaired, and the other able to cut the DNA and thereby trigger the normal repair mechanisms of the cell, which recopy a correct sequence supplied by us to the cutting site in the same cell.This is the explanation of Angelo Lombardo, a researcher at the San Raffaele Telethon Institute for Gene Therapy, who received an award at the end of 2011 as the best young researcher by the European Society of Gene and Cell Therapy, and who already in 2007, together with Naldini, had shown the therapeutic potential of these proteins.

"In recent years," explains further Lombardo, "we have studied how to introduce and make this veritable 'micro-surgical team' work in the stem cells of human blood in order to correct defects responsible for genetic diseases." More specifically, Luigi Naldini and the research group he leads have tried to apply this technology to SCID-X1, a hereditary immunodeficiency. Though "traditional" gene therapy does work, it has given rise to safety issues in the past, notably in the course of an experiment conducted in France. Some of the patients, in fact, developed leukaemia as a result of the uncontrolled expression of the therapeutic gene and the random insertion of the vector that had occurred near an oncogenic gene, activating its tumorigenic potential. SCID-X1 is caused by a defect in a specific gene, IL2RG, which is essential for the development of cells of the immune system: T lymphocytes and Natural Killer (NK) cells. In the absence of the protein IL2RG, the stem cells of the bone marrow are not able to produce these crucial defensive elements of the blood. Patients with SCID-X1 are prone to serious infections in early childhood and their life is constantly at risk. So far, researchers have not been able to apply genome editing technologies to human hematopoietic stem cells, which are rather "reluctant" to accept and use the DNA repair and editing machinery delivered from the outside.

As explained by the first author of the work, Pietro Genovese, a researcher at the San Raffaele Telethon Institute for Gene Therapy, "Normally, these cells are found in the bone marrow in a state of quiescence, and they awaken periodically to replicate and regenerate mature blood cells, to replace those that are consumed and die naturally. We, however, have succeeded in finding the right combination of stimuli to wake them up and use our 'molecular scalpel' to repair the defect in the IL2RG gene. We then demonstrated the safety and efficacy of this therapeutic approach in a mouse model in which we 'recreated' a defective human hematopoietic system: the cells we corrected with genome editing techniques were sufficient to rebuild the immune system, giving rise to fully functional T lymphocytes and NK cells. This shows that it takes only a few corrected stem cells to obtain the therapeutic effect, which is, in other words, the reconstitution of a functioning immune system."

With this strategy, it will be possible in the future not only to overcome some of the greatest obstacles that now stand in the way of the application of gene therapy, but also to engineer stem cells in an increasingly precise and innovative way, creating new strategies for treating diseases. The research study was supported by funding from the Telethon Foundation, the European Union and the Ministry of Health, and also benefited from the collaboration of Sangamo Biosciences, an American biotech company.


STUDY PUBLISHED IN NATURE – May 28, 2014 Targeted Genome Editing in Human Repopulating Hematopoietic Stem Cells

Pietro Genovese1, Giulia Schiroli1,2, Giulia Escobar1,2, Tiziano Di Tomaso1, Claudia Firrito1, Andrea Calabria1, Davide Moi1, Roberta Mazzieri1, Chiara Bonini3, Michael C. Holmes4, Philip D. Gregory4, Mirjam van der Burg5, Bernhard Gentner1,2, Eugenio Montini1, Angelo Lombardo1,2, and Luigi Naldini1,2
1 TIGET, San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milan, Italy
2 Vita Salute San Raffaele University, Milan, Italy
3 Experimental Hematology Unit, San Raffaele Scientific Institute, Milan, Italy
4 Sangamo BioSciences Inc., Richmond, CA, United States.
5Dept. of Immunology Erasmus MC, University Medical Center, Rotterdam, The Netherlands
These Authors share senior authorship.