Gene therapy

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Long-term research now bearing fruit

Gene therapy consists of introducing genetic material into cells in order to treat a disease. Originally, this approach was devised to replace a defective gene in the event of a monogenic disorder (i.e. related to a single deficient gene). However, over the last two decades, rapid developments in knowledge and technology have led to an increase in the number of possible strategies and an extension of their use to incorporate many indications, including some forms of cancer.

Major successes have been achieved in clinical trials and the field is experiencing rapid development. Nowadays, several gene therapy drugs are available, in Europe, the USA and China.

  • Treatment through genetic modification of the patient's cell
  • Clinical success: several medicinal products already on the market
  • 700 clinical trials in progress
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Report produced in collaboration with Anne Galy, from the Integrated genetic approaches and new therapies for rare diseases research unit (Inserm/Université d'Evry Val d'Essonne/Ecole Pratique des Hautes Etudes/Généthon unit 951), Evry, France.

Loupe comprendre Understanding gene therapy

Gene therapy was originally devised as a therapeutic approach in monogenic disorders (i.e. those related to a single defective gene), which involves delivering to the cells a "healthy" gene capable of replacing the "sick" gene. Nowadays, the uses and indications are much broader, with 65% of clinical trials concerning the treatment of cancer. There has been considerable diversification in the techniques, which are based on various corrective strategies, vectors and methods of gene therapy.

Feuilles The various gene therapy strategies

Replace a "sick" gene

This first strategy involves importing a copy of a functional gene into a target cell where it will express itself and lead to the production of the missing protein. The gene is transported using a vector (see below).

This was the first strategy developed in gene therapy, to treat monogenic diseases. The imported therapeutic gene does not modify the sick gene: it is simply added to the genetic heritage of the cells so as to compensate the defective function. Depending on the indications, this procedure can be performed:

  • in vivo, directly in the patient’s body
  • ex vivo, in order to genetically modify the cells in the laboratory before reinjecting them into the patient
Two main strategies of gene therapy
Two main strategies of gene therapy: Gene therapy involves genetically modifying a patient's cells, to treat or prevent disease. The protocols followed vary according to the indications and therapeutic objectives. Cells may be modified in vivo, directly in the patient's body, or ex vivo. In the latter case, stem cells are taken from the patient, modified in the laboratory, then reinjected. © Inserm, F. Koulikoff

Working ex vivo makes it possible to better control the steps, use fewer vectors and avoid dispersal of the treatment in non-target organs. This solution is most often used to treat blood diseases because the cells which need to be corrected can be extracted by means of a simple blood sample. For example, the first ex vivo gene therapy drug (Strimvelis, launched in 2016) involved taking CD34+ hematopoietic cells from patients with severe immune deficiency (ADA-SCID), modifying them in the laboratory so that they express the missing gene, and then readministering them.

For other diseases, such as those affecting the muscle, respiratory, ocular, cardiac or neurological systems, gene transfer is performed in vivo, by injecting the vectorized gene directly into the body or target organ, like a drug. Numerous clinical trials are ongoing with this technique and several products have reached the market launch stage (Glybera, Luxturna).

Delete or repair the impaired gene directly in the cell

With this technique, known as genome editing, genetic mutations can be repaired in a targeted manner. Various tools need to be imported into the cell:

  • specific enzymes which will cleave the genome at the required location (nucleases),
  • a DNA segment to repair the genome and provide a functional gene.

These tools include zinc-finger nucleases, TALEN and CRISPR. Although these approaches are still very much at the experimental stage, the revolution provided by the simplicity of CRISPR is generating great hope. A number of clinical trials are already in progress in the USA and China.

Learn more about Genome editing

Modify RNA to obtain a functional protein

This technique consists of having the cell produce a modified version of the missing protein. This requires the injection of small antisense oligonucleotides which bind to the messenger RNA transcribed from the mutated gene and modify splicing, a major step prior to its translation into a protein.

In Duchenne muscular dystrophy, caused by mutations in the dystrophin gene, "exon skipping" techniques involve omitting the sequences of the gene which carry the mutation responsible for the disease. The dystrophin obtained is shorter than the usual protein, but functional. In spinal muscular atrophy, the approach consists of blocking a splicing inhibitor site so as to "reinclude" an exon in the RNA and obtain a normal form of the SMN2 gene.

Exon skipping technique
Flow chart of the exon skipping technique. © Inserm, F. Koulikoff

These splicing modulation approaches have proved themselves in clinical trials and two drugs, Eteplirsen and Nusinersen, have recently obtained marketing authorization in the treatment of Duchenne muscular dystrophy and spinal muscular atrophy, respectively.

Use gene therapy to produce therapeutic cells

For certain complex pathologies, there is no one gene to be repaired or replaced. However, it is possible to devise indirect strategies: by combining cell therapy and gene therapy, cells can be obtained which possess new therapeutic properties.

This is the case, for example, with CAR T cells in the domain of cancer: the T-cells of patients with B-cell leukemia are taken and genetically modified to arm them with a chimeric receptor (CAR). This receptor recognizes the CD19 antigen present on malignant cells, making it possible to eliminate them once the CAR T cells have been reinjected into the patient. Numerous gene and cell therapy clinical trials using redirected anti-tumor lymphocytes are ongoing. The first products of this type (Kymriah and Yescarta) have recently been granted marketing authorization.

Use genetically-modified viruses to kill cancer cells

These are called oncolytic viruses. They are genetically modified to specifically infect the tumor cells, which they destroy. A first oncolytic virus (Imlygic), derived from a herpes strain, was granted marketing authorization in 2015. It is indicated in the treatment of melanoma.

Pictogramme vecteur Vectors: the keys to success in ex vivo and in vivo therapy

One of the difficulties associated with the development of gene therapy is that a therapeutic nucleic acid needs to be delivered to the cells of a patient. Most often used are viral vectors, which work by exploiting the exceptional properties of viruses to deliver their genetic cargo. Viral vectors are involved in over 75% of gene therapy clinical trials.

The beginnings of gene therapy were marked by several accidents related to the use of such vectors. These led to uncontrollable inflammatory reactions or caused cancers related to integration of the therapeutic gene in proximity to oncogenes. While rare, these accidents led scientists to deepen their understanding of the function of these viral vectors, and how they insert their DNA into the host chromosomes. Above all, new generations of safe vectors have been developed. Adeno-associated and lentiviral vectors have to a great extent replaced the original adenoviral and gamma-retroviral vectors.

Viral vectors are:

  • integrative: the DNA of the viral vector is inserted into the host DNA,
  • non-integrative: the therapeutic gene remains in the cell without inserting itself into the host genome.

In all cases, the vectors used undergo extensive engineering to neutralize their toxic potential and replication capacity in order to orient them more specifically 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.

Integrative viral vectors

Integrative viral vectors insert their DNA (which contains the therapeutic gene) into the host’s genome. Consequently, the therapeutic gene is transmitted to the daughter cells when cell division occurs. This is the case of the lentiviral vectors, which are derived from human viruses such as HIV but are rendered harmless.

Non-integrative vectors

When a transgene needs to be inserted into non-dividing cells (post-mitotic cells), non-integrative vectors are preferred as these are considered to be safer in vivo. With these vectors, the therapeutic gene remains in the host cell, without being inserted into its genome. It is expressed throughout the lifespan of the cell and disappears when it dies.

inserm_57756_medium
AAV viral vector (green) at the blood retinal barrier. Inserm/Ophélie Vacca

This is the case, for example, of the vectors derived from adeno-associated virus (or AAV). Over the past fifteen years, their development has strongly increased to become one of the most used vectors for in vivo gene transfer in the treatment of monogenic diseases. These vectors are effective and well-tolerated. However, their use can be limited due to natural exposure to wild-type AAV: against which a large number of individuals possess antibodies. Furthermore, since the vector triggers an immune reaction, its use is often restricted to a single injection. To get around this problem, researchers diversify their capsid so as to increase the variety of serotypes and combine immunomodulatory treatments with the gene therapy.

Non-viral vectors

In parallel, the development of non-viral vectors and non-viral techniques to deliver genes and nucleoprotein complexes for genome editing continues, in order to meet needs of safety and simplicity. These approaches generally remain less effective than those which use viral vectors in the case of systemic in vivo treatment. However, non-viral technologies are continually being perfected.

Currently, nearly 20% of gene therapy trials have been performed by directly injecting DNA, which is modified and protected from nucleases thanks to chemical modifications or incorporated in a plasmid. Another strategy is lipofection: the therapeutic gene is combined with cationic lipids which help it to enter the host cell. In terms of ex vivo approaches, electroporation and nucleofection, in which an electrical field is applied, are widely used, particularly for the delivery of the proteins and oligonucleotides of the CRISPR system.

Vectors used in gene therapy clinical trials (1989-2017)

Pictogramme flacon de médicaments Gene therapy drugs

There are two types of gene therapy drug, each with its own distinct development and pharmaceutical regulation pathway:

  • oligonucleotides, simple molecules which have been in existence for around twenty years,
  • innovative biological products, which are much more recent.

The oligonucleotides used (DNA or RNA) include antisense nucleotides, aptamers, and small interfering RNA (siRNA) presenting diverse chemical modifications. These molecules are administered without a viral vector and their pre-clinical development follows the same path as that of conventional drugs.

The biological products include viruses or recombinant viral vectors, hematopoietic cells or genetically-modified lymphocytes. The development of these advanced therapy products is much more complex. Guided by specific French and European regulations, it is necessary to validate large quantities of biological and pharmaceutical data in order to guarantee the safety and reproducibility of cell sampling, viral-vector handling and targeting, etc.

The first gene therapy oligonucleotides began to arrive on the market in 1998, although only a few of these have proved to be effective in treating genetic diseases. However, Nusinersen (Spinraza) and Eteplirsen – indicated in spinal muscular atrophy and Duchenne muscular dystrophy, respectively – can be cited. Gene therapy biological drugs have recently arrived on the market (see below). Positive and sometimes even spectacular results have been obtained with them in various types of cancer and in rare diseases in immunology, hematology, neurology, myology, infectious diseases and ophthalmology. A number of these drugs are currently available in Europe, the USA and Asia.

The approved innovative gene therapy drugs

  • Gendicine: the first drug of this type to be launched on the market, in China, in 2003. It consists of an adenovirus which expresses gene p53 (coding for a tumor suppressor) and is indicated in the treatment of cancers of the head and neck.
  • Oncorine: launched on the Chinese market in 2005, it consists of an oncolytic adenovirus  used in combination with chemotherapy to treat refractory nasopharyngeal cancers.
  • Glybera: the first drug commercialized in Europe, at the end of 2012, in the treatment of familial lipoprotein lipase deficiency. It consists of an adeno-associated vector  and has recently been withdrawn from the market due to lack of demand.
  • Imlygic: approved in 2015 in the USA and Europe, it consists of an oncolytic virus expressing an immunostimulant protein. It is indicated in adults with unresectable melanoma.
  • Strimvelis: the first ever drug combining gene and cell therapy to have been authorized in the world. It was approved in Europe in 2016. Indicated in ADA-SCID, it consists of autologous CD34+ cells which express the ADA gene.
  • Zalmoxis: indicated against bone marrow graft rejection in Europe since 2016, it is based on the genetic modification of allogeneic T-cells using a retroviral vector.
  • Kymriah and Yescarta: approved in 2017 in the USA, they consist of genetically modified autologous T cells (CAR T cells) to treat resistant forms of acute lymphoma affecting the B cells.
  • Luxturna: this adeno-associated vector is indicated in retinal dystrophy related to the RPE65 mutation and was approved in the USA at the end of 2017.

Generally well-tolerated, gene therapy may replace some reference treatments and be offered on a first-line basis once the comparative studies have been performed. Therefore, what we are seeing today is the establishment of a new therapeutic sector.

Buste souriant et pouce levé A host of clinical trials with some major successes

Since 1989, around 2,000 clinical trials have been conducted or are ongoing, including 65% in cancer, and 11% in monogenic diseases, such as Leber’s congenital amaurosis, hemophilia, beta thalassemia and cystic fibrosis. The other clinical trials concern infectious, cardiovascular, neurological and ocular diseases.

Indications addressed by gene therapy clinical trials (1989-2017)

The immense majority 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, account for less than 4% of clinical studies. Nevertheless, this figure is increasing thanks to the development of new vectors derived from lentivirus and AAV, which are safer and more effective.

In France, around thirty gene therapy trials have been performed or are underway, regardless of pathology or phase.

Hélice ADN Real successes and hopes in a number of monogenic diseases

In neuromuscular diseases

The case of infantile spinal muscular atrophy is a good illustration of what gene therapy can currently achieve in situations of major therapeutic deficiency. This disease is characterized by muscle weakness and atrophy starting from the first months of life. Previously incurable, especially the type 1 form (the most severe), the severity of the disease could be attenuated thanks to several highly encouraging approaches. An already commercialized antisense oligonucleotide (Nusinersen), which is administered by a series of intrathecal injections, increases the quantity of a protein which is lacking in the central nervous system of patients. It reduces symptoms and the probability of mortality in patients with SMN1 gene mutations. A second approach, for which clinical trials are currently underway, uses an AAV9 vector which contains a copy of the healthy SMN1 gene, administered by a single and easy intravenous injection. The results presented at congresses are remarkable and show stable functional recovery in treated children.


Other trials based on similar approaches, with a single injection of the AAV vector, are ongoing in other neuromuscular diseases. Positive results have been obtained in young patients with myotubular myopathy. In this disease, the structure of the muscle fiber is modified. An AAV vector is used to transport the MTM1 gene into the muscles. Other myopathies are also the subject of clinical trials with AAV, notably Duchenne muscular dystrophy.

In ocular diseases

One treatment (Luxturna) is already available for Leber’s amaurosis, a retinal degenerative disease of monogenic origin. It consists of an AAV vector with a functional copy of the altered gene in patients, which is injected directly into the retina and halts the progression of the disease.

Fortified by this success, which was one of the first in gene therapy, this approach is also being tested in a number of other monogenic ocular diseases such as Leber’s optic neuropathy and Stargardt disease. Novel approaches, such as optogenetics, are also being tested in this area in order to render neurons sensitive to light.

In hematologic diseases

The results of two trials against hemophilia A and B published at the end of 2017 are extremely conclusive: after one year, they appear to have cured patients - who no longer present symptoms. Treatment consisted of a single injection of an AAV vector transporting the gene coding for the IX factor (in hemophilia B) or the VIII factor (in hemophilia A). Long-term follow-up should confirm the safety of the treatment and the persistence of the therapeutic effect over time.

Sickle cell disease is a severe form of chronic anemia of genetic origin caused by a mutation in the gene coding for β-globin. French and US teams developed a protocol to correct the disease in a 13-year-old patient suffering from a severe form of it. This phase I/II clinical trial consisted of extracting hematopoietic stem cells from the patient’s bone marrow, genetically modifying them with the aid of a lentiviral vector containing the therapeutic gene and reinjecting them into the patient via the venous route. The outcome was full remission after a follow-up period of 15 months.

A similar approach has also produced highly conclusive results in the treatment of thalassemia. In 22 patients, it reduced or even eliminated the need for transfusion, and without side effects.

In severe immune deficiencies

Chercheur et bébé
Alain Fischer, Inserm Unit 768, "Normal and pathological development of the immune system", Biological Therapy Department and Pediatric Hematology and Immunology Unit, Hôpital Necker Enfants Malades AP-HP, Université Paris Descartes, Paris © AP-HP, J. Renard

In 1999, preliminary trials on "bubble babies" (suffering from SCID X1) demonstrated that human pathologies could be treated with gene therapy. Thanks to new clinical protocols and improved vectors (to avoid the problems of random insertions responsible for several cases of leukemia), new therapeutic successes have been obtained with these techniques.

One drug (Strimvelis) has been launched for patients with ADA-SCID, a severe immune deficiency characterized by the absence of the ADA protein, essential for lymphocyte production.

Highly encouraging results have also been obtained in the treatment of Wiskott-Aldrich syndrome. This disease, caused by mutation in the WAS gene, is characterized by blood cell dysfunction and a lack of platelets. Trials conducted in Europe and the USA confirm the efficacy of the approach to improve the health of those treated, including one adult.

Approaches targeting other immune deficiencies are currently undergoing clinical trials, notably concerning X-linked chronic granulomatous disease: the initial positive results have recently been presented at congresses.

In neurodegenerative diseases

Thérapie génique de l'adrénoleucodystrophie (ALD)
Gene therapy for adrenoleukodystrophy. CD34+ progenitor cells of treated ALD patients continue to express the therapeutic gene more than 2 years after treatment. In red : expression of the ALD protein ; in blue : cell nucleus © Inserm, P. Aubourg

Major progress has also been made in some neurodegenerative diseases. In adrenoleukodystrophy, a demyelinating disease of the central nervous system, blood stem cells from patients are corrected ex vivo with the aid of a lentivirus, and then reinjected. Some twenty patients have been treated and the results show that their condition stabilized or improved in the majority of cases.

Spectacular results have also been obtained with a similar approach in children with metachromatic leukodystrophy and other research is ongoing in Sanfilippo syndrome. A trial is being conducted in France in four children with this disease. The objective is to obtain the production of the missing enzyme by the brain cells. An AAV gene therapy vector is injected into various regions of the brain. No noteworthy side effects were observed during the 30 months following the treatment and an improvement in intellectual and behavioral development was observed, paving the way for a phase III trial.

Gene therapy approaches also apply to other more common neurological diseases, such as Parkinson’s disease. Promising results have been obtained in preclinical models of Alzheimer’s disease.

In dermatological diseases

A spectacular result has recently been obtained in junctional epidermolysis bullosa. A 7-year-old suffering from this severe rare disease received several autologous genetically-modified skin-cell grafts to correct the mutation responsible for the disease, which affects the LAMB3 gene. With this mutation, the attachment of the epidermis to the dermis is weakened. In the face of a life-threatening emergency, German and Italian doctors successfully performed this procedure on 80% of the body’s surface.

In another form of the disease, a clinical trial is ongoing in the USA to produce genetically-corrected epidermal grafts to treat the skin lesions occurring in dystrophic epidermolysis bullosa.

Pictogramme main cellules Cancer and infectious and cardiovascular diseases

In cancer

A priority approach in oncology involves stimulating the patient's immune system against its own tumor, so as to facilitate cancer cell recognition and elimination. To achieve this, trials involved taking T-cells from patients and arming them with receptors that recognize the tumor antigens to enable them to eliminate the cancerous cells. Two gene therapy drugs have been developed on this basis (Yescarta and Kymriah) targeting B-cell leukemia. They comprise a HIV-1 viral vector which integrates naturally in T-cells. The researchers are now working on new receptors (CAR or T) which will enable this strategy to be applied to a large number of cancers. Other approaches are also being considered, notably to inhibit molecule PD1 which constitutes an obstacle to the activation of antitumor lymphocytes.

Research is also being performed into the use of oncolytic viruses genetically modified to selectively infect and eliminate tumor cells. One drug (Imlygic) is already indicated against melanoma. Other oncolytic viruses are currently undergoing clinical trials, including an attenuated measles virus or a mixture of poliovirus and rhinovirus currently being developed for advanced gliomas. In addition to the direct efficacy of the virus, the release of tumor cell debris produced by the destruction of the cancer cells stimulates the patient's immune system against his or her own tumor by producing new anticancer T-cells.

In cardiovascular diseases

In the cardiovascular domain, researchers are developing gene therapy to promote regeneration of the vascular tissues in arterial ischemia or to fight restenosis (narrowing of an artery following the insertion of a stent) aided by genes coding for proteins which impede or, on the contrary, stimulate these processes. Positive results were reported in the treatment of chronic heart failure with an adeno-associated vector coding for Serca2, but these have not been confirmed in subsequent studies. Nevertheless, efforts are ongoing to improve the cardiac tropism of the vectors.

In infectious diseases

Curative gene therapy treatment could be envisaged in the event of HIV infection. Several approaches are being studied. One approach consists of rendering the patient’s CD4 T-cells (cells targeted by HIV) resistant to the virus: hematopoietic stem cells are taken from the patient and genetically modified with the aid of zinc-finger nucleases (genome editing) in order to alter the gene coding for a CCR5 surface receptor. The absence of this receptor prevents the virus from entering the cells. When reinjected into the patient, these modified cells multiply and differentiate into immune cells which are resistant to the virus, making it possible to restore the subject’s immune system. A phase I/II trial is ongoing in the USA.

Progress made possible thanks to the contributions of various stakeholders

Major progress is possible thanks to the efforts and partnerships between academic and clinical research, patient associations, biotechs and pharmaceutical companies.

Various patient associations have for many years been committed to the development of gene therapy through their financial support of research. These include the French Muscular Dystrophy Association (AFM-Téléthon), which pursues a particularly active policy in the domain of rare diseases.

In parallel, industrial stakeholders are developing in the field of gene therapy, particularly in the establishment of an advanced drugs production sector. The possibilities for applying gene therapy beyond rare diseases and in common diseases such as cancer or neurodegenerative, infectious and cardiovascular diseases have attracted strong interest from industry players, thereby boosting the sector. Many encouraging preclinical results are currently being observed in animal models, for example in type 2 diabetes. These could lead to clinical trials which would concern large numbers of patients. Furthermore, the progress made to register the first gene therapy drugs provides firm examples for future products to follow.

Rouages Some obstacles remain

Despite the successes already achieved, researchers remain prudent as to the use of gene therapy and the potential onset of side effects over time. Monitoring the patients treated, over a number of years, will enable more to be known about the safety and efficacy of these drugs. The development of new vectors must also be pursued in order to circumnavigate the problem of immune response which can develop in patients, particularly with the AAV vectors, and the impossibility of reinjecting the treatment a second time. The increasing number of clinical trials in a variety of indications should make it possible to learn a lot more in the years to come in order to improve procedures yet further.

The bioproduction of living products (viruses, vectors, autologous cells) on an industrial scale remains a major obstacle for the development of innovative gene therapy drugs. The procedures are derived from academic research and are not always suitable for large-scale deployment in accordance with the Good Manufacturing Practices applied in pharmaceutical production. Technological and industrial innovations remain necessary if production yields are to be improved. This is all the more relevant given that the doses needed to treat a patient sometimes only enable clinical trials to be conducted on a small number of people.

The cost of these drugs is also a new subject for consideration in the domain of public health. Glybera costs around one million euros, Strimvelis over 600,000 euros per treatment, and Nusinersen has been announced as costing several hundreds of thousands of euros per year - for life. The price of a drug can be justified by its therapeutic value and the resulting reduction in the cost of ongoing treatment administered to people with rare genetic diseases. Nevertheless, full-cost economic studies need to be performed. How to provide access to gene therapy drugs to underprivileged populations is also one of the questions that we need to start thinking about.