Gene therapy uses nucleic acids (DNA or RNA) to treat or prevent disease. Depending on the disease, this objective may be achieved by delivering a functional gene into the cells, to replace the defective gene causing the disease (transgene), a gene with a therapeutic action, or RNA capable of regulating or partly blocking the expression of a damaged gene. Nucleic acids are usually transported in the patient's cell, via a viral vector, but may also be injected directly into the cells, in the form of naked DNA.
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Understanding gene therapy
The concept of gene therapy dates back to the 1950s, but truly started to take shape in the 1990s, with the first human trials. In 1995, the first patient to receive stable treatment, via injection of genetically modified lymphocytes and stem cells (by a Milan team) suffered from severe ADA SCID type immunodeficiency. The first step, then transformed in the 2000s by striking therapeutic success, was taken at Hôpital Necker, in patients suffering from another form of immunodeficiency (SCID-X1 type) (see below). At the time, gene therapy was often presented as a means of fighting single-gene disorders (related to dysfunction of a single gene), by delivering a "healthy" gene able to replace the "diseased" gene. In reality, the indications are much more extensive: more than 1,800 clinical trials in gene therapy are currently in progress, including 65% in oncology, 10% in the cardiovascular field, and only 10% in single-gene disorders (notably immunodeficiency and hematological disorders, along with disorders such as cystic fibrosis). Other trials are being conducted on infectious diseases (tetanus, AIDS, etc.), neurological disorders (amyotrophic lateral sclerosis, multiple sclerosis, Alzheimer's disease, and Parkinson's disease), ophthalmological disorders (retinitis pigmentosa, glaucoma, age-related macular degeneration), and in inflammatory diseases such as osteoarthritis and rheumatoid arthritis.
Approximately three-quarters of these trials are phase I or II studies, evaluating the safety and efficacy of the investigational products. Phase III trials (which determine the benefit/risk ratio of a new treatment relative to a reference treatment or placebo) only account for 4.5% of ongoing clinical studies. Nevertheless, this number is constantly growing, with promising prospects, for instance, in the treatment of single-gene disorders, such as Leber's congenital amaurosis, hemophilia B, beta-thalassemia, or in the treatment of cancer, via the transfer of genetically modified T-cells.
Two gene therapy products on the market
Two medicinal products have overcome every hurdle in terms of clinical development and are already on the market. One of these products, Gendicine has been marketed in China since 2004. It is indicated for the treatment of carcinoma of the head and neck. It is a (p53) tumor suppressor gene, transported by an adenovirus. More than 10,000 patients have been treated with this medicinal product to date, without any noteworthy undesirable effects. In Europe, the first gene therapy product was approved at the end of 2012: Glybera, administered intramuscular injection, indicated for familial lipoprotein lipase deficiency.
The arrival of this medicinal product on the European market was a key milestone in this medical field: gene therapy is no longer an experimental strategy studied in a laboratory. It can lead to the development of marketable medications, providing regulatory and industrial obligations can be fulfilled (production of vectors and transgenes under standard, controlled conditions, accurate evaluation of the benefit/risk ratio). This development is not possible without support from medical and industrial experts.
Gene therapy protocols vary according to the indications and therapeutic objectives to be achieved. However, these always involve genetically modifying the patient's cells, ex vivo or in vivo, long term or temporarily.
Hence, in the event of a single-gene disorder which affects the blood cells, hematopoietic stem cells (cells which produce all blood cells) are taken from the patient during a procedure which resembles a simple blood draw. These cells are then modified ex vivo: a vector (see below) is used to deliver a therapeutic transgene, and the cells are then are placed in culture for a few days. When the treated cells start to express the therapeutic gene, they are finally reinjected into the patient via venous infusion. The modified cells will then proliferate in the patient's body and, a priori, help treat the patient. The advantage of this approach is that it modifies a highly specific cell population, without any risk of the vector entering non-target organs.
However, it is not always possible to remove the cells requiring correction: this strategy cannot be used to modify cardiac cells or neurons. Certain protocols thus allow for injection of the vector containing the transgene directly into the target organs, in vivo. For example, as regards Leber's congenital amaurosis, a type of retinal degeneration which causes blindness, the vector containing the transgene is injected directly into the retina. The risk with this strategy lies in the lesser control of transgene dissemination.
In 2% of gene therapy trials, the technique used is similar to gene surgery: i.e., "exon skipping". This approach involves causing the cell to product a version of the deficient protein in the patient, which is shorter than the normal protein but functional, by "skipping" the part of the gene with the mutation responsible for the disease. Exon skipping was tested in animals (team led by Olivier Danos and Luis Garcia, Généthon, Evry), then humans, to treat Duchenne muscular dystrophy. Several clinical trials are currently in progress, notably at the Institut de Myologie in Paris. This technique is particularly suited to this disease as the gene in question is too large to be transported by a gene transfer vector. Potential applications are envisaged in other genetic disorders.
Another approach, aiming to repair the damaged gene directly at the heart of the cell, is appealing in its precision. This would avoid certain undesirable effects associated with transgene transfer. In practice, this strategy is based on the use of enzymes known as "nucleases", able to identify specific DNA sequences on either side of the mutation to be repaired, and to splice the chromosome at this precise location. The cell machinery is then set in motion to repair its DNA. If a "healthy" copy of the gene to be restored is then delivered into the cell, this will serve as a repair matrix, thus allowing reconstitution of a complete, functional gene. This technique works effectively in vitro, and initial in vivo trials are currently in progress. Cellectis, a French firm, is pioneering this field.
Major challenges facing research
Le choix de l’acide nucléique thérapeutique dépend de l’indication
A priority approach in cancer treatment involves stimulating the patient's immune system against its own tumor, so as to facilitate cancer cell recognition and elimination. With this aim in mind, trials have, for example, involved taking dendritic antigen-presenting cells or T-lymphocytes from patients, with a view to inserting a gene encoding a protein implicated in tumor cell recognition or destruction (tumor antigens, cytokines, tumor suppressor genes or suicide enzymes) and reinjecting the whole modified cell into the patients' body. The results are generally mixed. A number of improvements still need to be made so as to make the vectors more immunogenic or more readily controlled.
In the cardiovascular field, scientists are attempting to use gene therapy to promote vascular tissue regeneration in arterial ischemia. They are thus using genes encoding vascular growth factors. They are also attempting to reduce restenosis (undesirable cell proliferation after insertion of a stent), by injecting substances inhibiting cell growth on the artery walls.
Regarding the management of single-gene disorders, numerous trials are focusing on immune deficiency, such as severe combined immunodeficiency (SCID), adenosine-deaminase deficiency (ADA-SCID), Wiskott-Aldrich syndrome, chronic granulomatous disease, along with hematological disorders, such as hemophilia B or A, Fanconi anemia, and beta-thalassemia. Other research is examining retinal disorders, such as Leber's congenital amaurosis or Leber's hereditary optic neuropathy, lysosomal storage diseases, such as Sanfilippo syndrome or Gaucher's disease, and other neurodegenerative diseases such as adrenoleukodystrophy or metachromatic leukodystrophy. The latest example involves skin disorders, such as epidermolysis bullosa. All of these disorders are related to dysfunction of a single gene.
Regarding hematological disorders, several trials which entail modifying stem cells ex vivo by injecting them with a healthy copy of the gene responsible for the disease, then reinjecting them into the patient's blood, have demonstrated sustainable benefit for patients. In the future, the use of diverse types of stem cells, such as induced pluripotent stem cells, or even new tissue engineering procedures, could be part of the gene therapy armamentarium.
In the field of infectious diseases, curative gene therapy treatment could be envisaged, for example, in the event of HIV infection. Several approaches are being studied. The first involves modifying patients' CD4 T4 lymphocytes (HIV targets) to make them resistant to the virus. To do so, a clinician would take hematopoietic stem cells from the patient's blood, and insert a gene into them, which would make the cells immune to the virus (several genes could be used, as shown in in vitro tests). The modified cells would then be reinjected into the patient's body and would lead to the production of HIV-resistant CD4 T4 lymphocytes, capable of surviving and multiplying. Animal studies and, a recent clinical trial conducted in humans, have been published, with promising results. In the context of a second approach, the scientists are working on the development of vaccines from viral vectors used for gene transfer. Promising results in terms of protection have been obtained in primates and human trials are in preparation.
Vectors, a key to successful therapy
To insert the therapeutic nucleic acid into the patient's cells, a vector is used for transport. Modified viruses (viral vectors) are used in more than two-thirds of the trials. This type of vector is still the reference to date.
Non-replicative viral vectors (which cannot multiply), integrative viral vectors (the viral vector DNA inserts itself into the host DNA), non-integrative viral vectors (the transgene remains in the cell without inserting itself into the host's genome) and non-integrative, non viral vectors exist. In all cases, the vectors used undergo extensive engineering to neutralize their toxic potential and, when necessary, to make them as silent as possible with regard to the host's immune system so as to enable long-term therapeutic correction.
The early stages of gene therapy were marred by accidents related to the use of viral vectors which entered non-target organs, or which led to integration of the transgene into "pro-oncogene" sequences of the patient's genome, triggering cancer or even death. These accidents caused scientists to explore the specific function of these viral vectors, and the way in which they insert their DNA into the host chromosomes, etc. These findings largely contributed to the development of gene therapy, owing to the development of safer and more effective vectors. The advent of "high-throughput" techniques for genome sequencing and analysis of the sequences obtained has been an essential milestone in this sector.
Integrative viral vectors insert their DNA (which contains the therapeutic transgene) into the host's genome. Consequently, the therapeutic gene is transmitted to the daughter cells when cell division occurs. These vectors are ideal in the case of cell therapy and gene therapy using stem cells, together with approaches where a permanent effect is sought.
Out of the integrative viral vectors, retroviruses were widely used in the 2000s; however, recourse to this category of viral vectors has gradually declined: these are currently used in less than 20% of ongoing trials. These were, in fact, implicated in the onset of leukemia in trials conducted on children with severe combined immunodeficiency or "bubble boy disease" in the 2000s. These viruses are now better understood and managed, hence there is a lower risk of random insertion into the host's genome. A "self-inactivation" function notably prevents the virus from triggering untimely expression of a gene close to its insertion site.
However, scientists are increasingly using lentiviral vectors to overcome this risk of random insertion. These seem to have a safer genomic integration profile compared to retroviruses. Furthermore, lentiviral vectors are able to enter non-dividing cells, such as neurons or hepatocytes (whereas retroviruses have difficulty inserting themselves into these cells). These viruses are derived from human viruses, such as HIV, but are modified so as to be innocuous. These vectors have allowed trials to be conducted in the treatment of adrenoleukodystrophy (team led by Nathalie Cartier and Patrick Aubourg, Inserm Unit 986, Kremlin-Bicêtre) and in the treatment of hemoglobinopathies (team led by Philippe Leboulch and Yves Beuzard, Paris), in collaboration with the Integrated Biological Therapy Clinical Investigation Center at Hôpital Necker (Paris). Furthermore, given the potential of these vectors, and owing to the work achieved by Anne Galy's team (Inserm Unit 951, Evry), Généthon has set up facilities for industrial production of lentiviral vectors and works in partnership with numerous international teams using them, particularly in the treatment of Wiskott-Aldrich syndrome.
When a transgene needs to be inserted into non-dividing cells, non-integrative vectors are preferred as these are considered to be safer. With these vectors, the transgene remains in the host cell, without being inserted into its genome. It is expressed throughout the life of the cell and disappears when the cell dies. Adenoviruses were widely used in the past, but their use is tending to decline, particularly in the treatment of single-gene disorders. However, they are still preferred vectors in cancer immunotherapy. These are able to transport larger DNA sequences than integrative viruses, even though the maximum size of the transgenes transported can be smaller than for human genes. This type of vector has several advantages: it is able to enter non-dividing cells and is associated with a high level of expression of the vectorized gene.
Vectors derived from adeno-associated virus (or AAV) are able to transfer small genetic sequences (only 4 kilobases versus 13 kilobases with lentiviral vectors). These are of interest, having limited inflammatory properties. These are increasingly used, for instance, in the treatment of Leber's congenital amaurosis. Moreover, the only gene therapy product authorized in Europe (Glybera) uses this type of vector.
At the same time, the development of non-viral vectors is continuing, with the aim of resolving two problems: better vector safety, and transport of larger quantities of DNA. In this respect, nearly 20% of gene therapy trials are based on direct injection of modified naked DNA, protected from cell enzymes (nucleases) due to chemical changes. Another strategy is lipofection: the therapeutic gene is combined with cationic lipids which help it to enter the host cell.
Major success stories of note
France is one of the world leaders in gene therapy, both on an academic and clinical level, particularly through teams associated with Inserm.
In 1999, French teams (Salima Hacein-Bey Abina, Marina Cavazzana and Alain Fischer, Inserm Unit 768, Hôpital Necker, Paris), in collaboration with English teams, pioneered the gene therapy treatment of children with "bubble boy disease" (suffering from SCID X1). Despite the development of several cases of leukemia in the 19 patients included, the therapeutic effects of treatment continue to persist. Out of the nine children treated in France ten years ago, eight are still alive, living at home, and having a normal education. Without this treatment, their life expectancy would have been very limited.
Leber's congenital amaurosis has also featured in trials with outstanding results. This disease involves retinal pigment degeneration which can cause blindness. It is caused by a mutation affecting the RPE65 gene. Injection of an AAV-type vector containing a functional copy of this gene, directly into the retina, has made it possible to halt disease progression and preserve patients' remaining eyesight. The first successful trials took place in England and in the United States in 2007. A trial is currently in progress in Nantes (team led by Fabienne Rolling and Philippe Moullier, Inserm Unit 1089, Nantes). An American company has recently been created to develop this strategy (Spark Therapeutics) and another exists in France, GenSight, founded by José-Alain Sahel, Director of the Institute of Vision (Inserm Unit 968), Paris.
Adrenoleukodystrophy, a neurodegenerative genetic disorder related to demyelination of the central nervous system, has also been the focus of promising research. A trial was conducted in four children in 2009, by French teams (Nathalie Cartier and Patrick Aubourg, Inserm Unit 986, Kremlin-Bicêtre), in collaboration with an American biotech firm and Hôpital Necker (Paris). The strategy used entailed removing stem cells from bone marrow (mesenchymal stem cells), genetically correcting them ex vivo by means of a lentiviral vector, then reinjecting them into the blood circulation. This treatment was able to halt disease progression in these children, who are now living practically normal lives. This trial paved the way for the development of this strategy for numerous other neurodegenerative diseases. Absolutely spectacular results have recently been achieved by an Italian team (Alessandra Biffi and Luigi Naldini, Milan) in children suffering from metachromatic leukodystrophy, another neurodegenerative genetic disorder. Other approaches are also in development for the treatment of lysosomal storage diseases, such as Sanfilippo syndrome, with research, for instance, conducted by Jean-Michel Heard at Institut Pasteur (Institut Pasteur/Inserm Unit 1115), Marc Tardieu at Hôpital Bicêtre and Michel Zerah at Hôpital Necker, Paris.
A trial, initiated in 2010, moreover demonstrated the efficacy of gene therapy in the treatment of hemophilia B. This involved an Anglo-American protocol (team led by Amit Nathwani, London). The scientists used an AAV vector containing a FIX gene, able to restore blood coagulation. Six patients were included. The gene was expressed in all participants and enabled four of them (those having received the highest doses of vector) to discontinue their prophylaxis for spontaneous bleeding. Long-term follow-up should confirm the safety of treatment and the persistence of the therapeutic effect over time.
Patients suffering from beta-thalassemia, a major form of anemia, could also benefit from gene therapy in the future, based on the results of a French trial conducted in 2010 (team led by Philippe Leboulch and Marina Cavazzana in Paris). This is a pioneering trial which allowed an 18-year-old patient to be treated. A first worldwide. The patient was transplanted with his own CD34 hematopoietic cells corrected ex vivo by means of a lentiviral vector, allowing them to express a beta-globin transgene. The young man is now able to lead a normal life, without needing monthly blood transfusions.
In the field of cancer, the results are more uncertain; however, some research is promising. In 2010, an American team, for instance, demonstrated the efficacy of modified T-cells in the treatment of leukemia (team led by Carl June and Bruce Levine, in Philadelphia). The scientists used an HIV-1 type lentiviral vector as it is naturally inserted into T-lymphocytes. This vector enabled the transfer of genes encoding proteins which facilitate the recognition of tumor cells to eliminate them. Novartis has invested in this sector, and several start-ups have been created, such as Juno Therapeutics in Seattle.
The industrial sector, surrounding gene therapy and the associated service industries, is growing throughout the world and in France. Several key figures in French research associated with Inserm have pioneered these approaches: for instance, David Klatzmann with the creation of Genopoietic in 1993, and also Pierre Charneau with Theravectys, David Sourdive with Cellectis, Philippe Leboulch with Bluebird bio, and, more recently, José-Alain Sahel with the GenSight Biologics Foundation in 2012. AFM Téléthon has, moreover, invested in gene therapy for many years. Also, Généthon BioProd, the first not-for-profit pharmaceutical company which manufactures gene and cell therapy medications, recently opened in Evry.