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Gene therapy

(last update: January 2014)

The term gene therapy applies to approaches to treat diseases based on the transfer of genetic material (DNA, or RNA) into the genome of targeted cells or tissues of an individual to either complement or replace the function of a defective gene. Various modulatory and interfering RNA molecules can be used in addition, to obtain a more sophisticated regulation at the transcriptional or post-transcriptional level of gene expression (e.g., exon skipping approach).
The concept of gene therapy was inspired by major discoveries made in basic genetic research since the 1950’s, strategies for gene delivery were matured through the 1980’s and the first clinical trials undertaken at the end of the 1980’s. Today’s gene therapy research may be seen as pursuing intelligent drug design through a logical extension of fundamental biomedical research outcomes on the molecular basis of a disease. Since its beginning, a few thousand of patients participated in clinical trials for gene therapy.
In the early 2000’s one of the most important success was the gene therapy trial for X-linked severe combined immune deficiency (conducted by Drs. M. Cavazzana-Calvo and A. Fischer from the Hôpital Necker Enfants Malades in Paris, France). The trial resulted in effective and life-saving immune reconstitution in 10 out of 11 patients, although it revealed also the potential toxicity of this treatment (see section on adverse events). Other accomplishments in gene therapy include:

  • a successful hematopoietic stem cell gene therapy based on the use of oncoretroviral vectors (i.e., murine leukemia virus, MLV) for the treatment of adenosine deaminase – severe combined immunodeficiency (ADA-SCID), in which 29 out of the 40 treated patients (treated in different centres in Italy, UK and USA) no longer required a replacement therapy with pegademase bovine (PEG-ADA);
  • a therapy for adrenoleukodystrophy (ALD) based on lentiviral vectors, in which 4 patients have been treated up to now, and
  • a therapy for β-thalassemia also based on the use of lentiviral vectors.

Further clinical trials ongoing in the domain of blood disorders using lentiviral vectors, include therapies against the Wiskott-Aldrich syndrome (WAS) and the chronic granulomatous disease (CGD).
Successes have also been achieved using adeno-associated viral vectors (AAV vectors) for the treatment of hemophilia B and for RPE65, a retinal disorder leading to blindness, with encouraging perspectives for the treatment of other metabolic as well as retinal diseases. In particular, the treatment of a very rare metabolic disorder, the familial lipoprotein lipase deficiency (LPLD) using an AAV vector developed by AMT/uniQure (i.e., Glybera®), represents the first gene therapy treatment receiving marketing authorization in 2012 by the European Medicines Agency (EMA).
In the field of non-rare diseases, promising developments have occurred for e.g., the treatment of cancer and cardiovascular diseases. In the field of cancer treatment, the following non-exhaustive advances took place:

  • the first ever authorized gene therapy treatment was approved in China in 2003 (Gendicine, a recombinant Ad-p53 applied via intratumoral injection) for the treatment of head and neck squamous cell cancer (HNSCC);
  • recent studies conducted at different clinical centres in the USA showed that blood cancers such as Acute Lymphoblastic Leukaemia (ALL) can be efficiently treated with gene-modified T-cells rendered specific for antigens expressed on tumour cells, in order to attack and remove malignant B-cells after reinfusion. Of 27 ALL treated patients (young and adults), 22 didn’t have a relapse up to today. Similar treatments are in development for other blood or solid cancers.

In the field of cardiovascular diseases, clinical trials and preclinical studies have been conducted for the treatment of coronary heart disease, heart failure, and arrhythmia. Studies targeting heart failure (HF) have the most advanced findings showing decrease in HF symptoms, increased functional status, and reversal of the negative left ventricular remodelling (i.e., the CUPID trial using AAV1 vector and targeting the SERCA2 sarcoplasmic reticulum calcium ATPase pump, essential for calcium homeostasis, which is decreased in heart failure), and improvement in the 6-minute walk test and quality of life (i.e., a trial using DNA-plasmid vector and targeting the Stromal-Derived Factor-1 (SDF-1) with the proposed mechanism that SDF-1 activates endogenous stem cells via the SDF-1 chemokine receptor type 4 pathway). However, considerable more efforts will be needed before achieving routine gene therapy treatment of heart diseases in the clinic.
Finally, significant advances were also achieved regarding the exon skipping approach using viral vectors (see also paragraph on gene therapy strategies). Using such an approach in an animal model for Duchenne’s muscular dystrophy, showed that defective dystrophine could be repaired by expression of a functional but shorter form of the dystrophine molecule (1) Such an approach can be performed either via a viral vector (AAV) as above-mentioned, which has to be administered loco-regionally or systemically, or via antisense oligonucleotides which are administered systemically at regular intervals. The latter approach, although not being a gene therapy approach, is far advanced in clinical studies using different oligonucleotide chemistries.
Despite these successes, gene therapy is still in its infancy and a long way has still to be undertaken in order to offer gene therapy as a regular treatment for rare and non-rare diseases.

(1Goyenvalle et al. (2004) Rescue of Dystrophic Muscle Through U7 snRNA-Mediated Exon Skipping. Science 306, 1796-1799

Disease targets

While initially focussing on inherited single-gene disorders, gene therapy research is nowadays directed towards a diverse group of human diseases possibly amenable to therapy by gene transfer. Under current investigation at the preclinical or clinical stage are gene therapy strategies for acquired diseases, such as cancer, AIDS, or cardiovascular diseases (restenosis, familial hypercholesterolemia, peripheral artery disease) and inherited diseases, such as cystic fibrosis, muscular dystrophy, hemophilia A and various severe combined immunodeficiencies, Gaucher disease, alpha1-antitrypsin deficiency, rheumatoid arthritis, high blood pressure, obesity, various ocular diseases, and others.

Gene therapy strategies

Any gene therapy strategy must be based upon the identification or design of a gene that may aid in the management or correction of a disease. The termination of the Human Genome Project as well as new proteomic and metabolomic approaches increase the availability of molecular targets and contribute to the understanding of genetic but also regulatory components of human diseases, thus aiding the identification of candidate therapeutic targets (i.e. genes). However, to go from the identification of a gene to the proposal of a gene therapy strategy requires a detailed knowledge of disease pathophysiology and the biology of relevant target cells.
In principle, gene therapy can be performed as ex vivo and as in vivo approaches. In the case of an ex vivo gene therapy patient cells are explanted into culture, eventually amplified, treated with the gene transfer vector and implanted into the host (locally or systemically). By the in vivo gene transfer principle, genetic material is transferred directly to cells located within the host. Vectors used for in vivo gene therapy approaches have higher quality assessment requirements for regulatory and biosafety purposes than ex vivo gene therapy approaches.
However, major challenges remain for both types of gene delivery strategies in terms of efficient gene transfer to the desired cell types and of proper control of expression of the inserted gene. Such issues mostly require to be addressed specifically for each individual disease or target cell/ target tissue.
The initially developed clinical applications of gene therapy are based on relatively ‘simple’ gene replacement or gene complementation approaches. More advanced approaches like exon skipping and the transfer of siRNA (small interfering RNA) are very promising, because they allow a natural regulation or a more sophisticated regulation of gene expression at the transcriptional and post-transcriptional level.
To avoid issues related to insertional mutagenesis, a known problem for all classical integrative viral vectors, and to maintain the natural regulatory networks, intensive efforts have been invested into methods for in situ gene repair, using for instance, zinc finger nucleases or TALENs, in order to directly target the defective gene sequence and repair the specific gene defect (genome editing approach which is presently one of the most important developments in the field of gene therapy).

Vectors for gene delivery

To allow transfer and proper function in a patient the therapeutic gene must be built into a vector. The most efficient gene delivery systems currently available are based upon the gene transfer machinery used in nature by animal viruses. These viral vector systems either allow the integration of the gene to be transferred into the genome of the target cells (e.g., retroviral and lentiviral vectors), or the transient transfer of the new gene function to the target cells (e.g., adenoviral vectors, AAV vectors) in which the transgene will not be integrated. Whereas integrative vectors are associated with the potential risk of insertional mutagenesis when the expression cassette is integrated close to proliferation inducing genes, this is not the case for the non-integrative vector systems. However, in the latter case, the new function is only maintained in non-dividing tissues else it will be rapidly lost. In addition, in the case of adenoviral vectors, the expression of viral antigens by the transduced cells will lead to the induction of an immune response finally leading to the elimination of these cells. Thus today such vectors are essentially developed for vaccine purposes and cancer therapy (- oncolytic viruses). Other existing gene transfer systems make use of naked DNA or DNA coupled to ligands that may facilitate the various steps of entry of DNA into cells.

Interference with immune system

One of the major issues of current gene therapy research is the reaction of the immune system of the treated individual. While some gene therapy strategies, such as cancer immunotherapy, attempt to stimulate the reactivity of the individual’s immune system towards eliminating the cancer cells, other strategies require that the genetically modified cells be protected from destruction by the immune system, signifying that a functional and well developed gene therapy approach for gene repair or the expression of correct/corrected proteins in patients with inherited disease associated with a missing or a truncated form of a protein will obligatorily be associated with a sophisticated manipulation of the immune system (e.g., induction of immune tolerance). Only in the cases of the different forms of SCID, the activation of the immune response is no problem because the patients are devoid of a functional immune system.
Based on the results of several clinical studies it has been found that the immune-response against vector proteins (e.g. capside proteins of AAV) and antigen (i.e. transgene) presenting cells can jeopardize gene therapy because of the elimination of the target cells. Intensive work is ongoing in order to avoid such problems, for instance, via temporal immune suppression.

Clinical trials in gene therapy

Between 1989 and 2014, 1996 gene therapy clinical trials have been worldwide approved. Figures 1 to 3 show the indications of such trials, the vectors used and the gene types transferred respectively (source: Furthermore their distribution according to the clinical phases addressed were as follows:

  • 59.3% in phase I clinical trial, in which the new drug or treatment is tested in a small group of people (20-80) for the first time to evaluate its safety, determine a safe dosage range, and identify side effects;
  • 19.2% in phase I/II clinical trials;
  • 16.5% in phase II clinical trials, in which the drug or treatment is given to a larger group of people (100-300) to evaluate its efficacy and to further evaluate its safety;
  • 1.0% in phase II/III clinical trials;
  • 3.7% in phase III clinical trial, in which the drug or treatment is given to large groups of people (1,000-3,000) to confirm its efficacy, monitor side effects, compare it to commonly used treatments, and collect information that will allow the drug or treatment to be used safely.

It is to be noted that for clinical trials of rare diseases, the number of patients per phase is considerably reduced, for instance, for a phase I or I/II clinical trial of a rare disease 5 to 10 patients are enrolled. In addition, due to the very small number of patients phase I/II clinical trials are often sufficient for submitting a dossier for obtaining a marketing authorization.
Based on the number of trials undertaken, a solid database of clinical experience has been built and clinical research has moved away from the proof of concept stage.

Figure 1: Indications of gene therapy clinical trials approved between 1989 and 2014 (n=1996)


Figure 2: Vectors used in gene therapy clinical trials approved between 1989 and 2014 (n=1979 protocols)


Figure 3: Genes transferred in gene therapy clinical trials approved between 1989 and 2014 (n=1996 protocols)


(Severe) adverse events observed in clinical trials for gene therapy

Phase I clinical trials primarily assess safety, determine a safe dosage range, and identify side effects. In the case of experimental protocols such as gene therapy protocols, in which a variety of new biological systems are tested for the first time in humans, (severe) adverse events can occur despite the preclinical tests and toxicology tests conducted in animals to predict the safe dose range and assess potential side effects. However, an animal model is only a model and cannot replace the final test in human beings.
In this context, severe adverse events have been reported in relation to gene therapy trials. In 1999, 18-year-old Jesse Gelsinger died from multiple organ failure 4 days after treatment for ornithine transcarboxylase deficiency. His death was triggered by a fulgurate immune response to the very high dose of adenovirus used as the vehicle, a vehicle that is nowadays replaced by AAVs far less immunogenic. This incident slowed down the field of gene therapy for some years.
The very successful oncoretroviral vector based gene therapy trials for X-linked severe combined immune deficiency (the first one, conducted by Drs. M. Cavazzana-Calvo and A. Fischer from the Hôpital Necker Enfants Malades in Paris, France and the second one conducted by Dr. A. Thrasher from the University College in London, UK) resulted in the effective and life-saving immune reconstitution in 10 out of 11 patients (by end of 2005). These patients have been able to lead a normal life, indicating, that from a clinical point of view they should be considered as cured. However, very sadly, three of the treated patients were found to have a T-cell lymphoproliferative disorder, two of which having the retroviral vector integrated in the proximity of the LMO-2 gene in the proliferating T-cells (known as insertional mutagenesis). Subsequently the patients have been treated by chemotherapy. By 2010, 20 children have been treated, 5 of which have developed leukemia-like adverse effects. To date 18 out of 20 treated SCID-X1 children are alive with full reconstitution of T-cell immune functions, revealing a superior success rate (10%) than conventional allogeneic hematopoietic stem cell transplantation (25% mortality rate). From a clinical point of view, patients, who have been able to lead a normal life for up to 3 years should be considered cured by this pioneering gene therapy treatment. None of these children would be alive today, ten years later, without the gene therapy.
Similar adverse events have been observed for other clinical trials (WAS, CGD) performed using oncoretroviral vectors. These adverse effects led to a considerable rethinking on the safety issues of viral vectors used for gene therapy purposes, and led to the decision to replace oncoretroviral vectors by lentiviral vectors for these indications. However, more pre-clinical investigations are required to assess the risk of gene therapy, including more basic research in the development of safer gene transfer vectors.
A further severe adverse event was reported in the press in 2007 on the death of a woman who received a modified gene in an arthritis trial run using AAV vectors. Of high importance for the field however, investigations showed that a fungal disorder was the cause of the death and not the genetic therapy. Furthermore, apart from this severe adverse event, review of AAV-mediated in vivo gene therapies reveals a remarkable safety (and efficacy) record.
These setbacks demonstrate the difficulties and misconceptions that the field of gene therapy can face. Despite the adverse events, both, the public attitude towards gene therapy and international regulatory requirements have evolved. Furthermore, hurdles to clinical success have been identified, indicating the basic issues to be resolved and how to best have an iterative process between the laboratory and the clinic.

ACTIP and gene therapy

While major problems need to be solved for gene therapy to become a standard way of treating patients, the field is maturing with the first marketing authorization in the EU for a viral vector for the treatment of LPLD (in 2012) and continues to attract strong attention from researchers, clinicians and industry including large pharmaceutical companies, having rediscovered this field probably due to the recent successes. Gene therapy and the production of gene therapy products make use of molecular and cell biological techniques similar to those used for in vitro cultivation of cells in the manufacture of biologicals. It seems therefore logical that ACTIP monitors developments in gene therapy technology and applies its expertise on issues such as regulatory requirements, guidelines and public perception.

Otto-Wilhelm Merten, Crespières, 04.01.14

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