The term gene therapy applies to approaches to disease treatment based upon the transfer of genetic material (DNA, or RNA) into the genome of targeted cells or tissues of an individual to complement or replace the function of a defective gene. In addition, various modulatory and interfering RNA molecules are also used that lead to more sophisticated regulation at the transcriptional or post-transcriptional level of gene expression (e.g. exon skipping approach).
The concept of gene therapy has been inspired by major discoveries made in basic genetic research since the 1950s, and strategies for gene delivery were matured through the 1980s leading to the first clinical trial at the end of the eighties. Today's gene therapy research may be seen as pursuing intelligent drug design through a logical extension of results of fundamental biomedical research on the molecular basis of disease. Since the first clinical trial, a few thousand patients have participated in clinical trials for gene therapy. In the early two-thousands the most important success of this new type of therapies is presented by the gene therapy trial for X-linked severe combined immune deficiency (conducted by Drs. Marina Cavazzana-Calvo and Alain Fischer from the Hôpital Necker Enfants Malades in Paris) resulting in the effective and life-saving immune reconstitution in 10 out of 11 patients, although it revealed also the potential toxicity of this treatment.
Three further real successes concern the successful hematopoietic stem cell gene therapy for the treatment of ADA (Adenosine DeAminase)-SCID (13 patients are off ADA treatment today) by Dr. Alessandro Auiti (HSR-TIGET in Milan/I) using an oncoretroviral vector (MLV — mouse leukemia virus) and of ALD  (adrenoleukodystrophy (3 patients have been treated up to now) by Drs. Patrick Aubourg and Nathalie Cartier (Inserm - Hôpital Saint-Vincent de Paul in Paris) using a lentiviral vector and the treatment of familial lipoprotein lipase deficiency (LPLD) using an adeno-associated virus (AAV) vector. With respect to the treatment of LPL it should be indicated here that the product registration (the first one for a gene therapy treatment based on the use of viral vectors in the Western world) has been initiated by the European Medical Agency (EMA) in January 2010 as a centralized procedure (AMT’s Marketing Authorisation Application for Glybera® Progressing on Schedule - http://www.amtbv.com/news-and-events/news-2010). It should be mentioned here that there is one gene therapy approved in China in 2003. It is Gendicine, a recombinant Ad-p53 gene therapy for head and neck squamous cell carcinoma (HNSCC). It is the only one approved in the world today.
Despite these successes, gene therapy is still in its infancy and a long way has still to be gone in order to offer gene therapy as a regular treatment for any disease.
Another very significant advance is the exon skipping approach using viral vectors (see also: gene therapy strategies). Using such an approach it could be shown in in vivo studies (animal model for Duchenne’s muscular dystrophy) that defective dystrophine could be repaired (= expression of a functional but shorter form of the dystrophine molecule)(Goyenvalle 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 now 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 or AIDS, and inherited diseases, such as cystic fibrosis, muscular dystrophy, hemophilia A and B, severe combined immunodeficiencies, cardiovascular diseases (restenosis, familial hypercholesterolemia, peripheral artery disease), Gaucher disease, lipoprotein lipase deficiency, 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 will increase the availability of molecular targets and is expected to substantially increase our 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 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 gene therapy approach. In the case of an ex vivo gene therapy patient cells to be treated are explanted into culture, eventually amplified, treated with the gene transfer vector, followed by their implantation into the host (locally or systemically). By the in vivo gene transfer principle, genetic material is transferred directly to cells located within the host. With respect to regulatory and biosafety issues, vectors used for in vivo gene therapy approaches require higher quality assessments than in the case of ex vivo gene therapy approaches.  
However, major challenges remain with respect to both types of gene delivery strategies in terms of efficient gene transfer to the desired cell types and proper control of expression of the inserted gene. Moreover, problems of this nature mostly need to be addressed specifically for each individual disease or target cell.
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.
In view of avoiding the problems related to insertional mutagenesis a potential problem known for all classical integrative viral vectors and in addition, of maintaining the natural regulatory networks, intensive work has been put into methods for in situ gene repair, using for instance, zinc finger nucleases in order to directly target the defective gene sequence and repair the specific gene defect.    

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 transfer transiently 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 growth inducer genes, this is not the case for the non-integrative vector systems. However, in this latter case, the new function is only maintained in non-dividing tissues else it will be rapidly lost.
Other gene transfer systems use naked DNA or DNA coupled to chemicals that may facilitate various steps of entry of DNA into cells.

Interference with immune system

Other major issues of current gene therapy research concern 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.
In the recent years, as results of several clinical studies it was established that the immune-response against vector proteins (e.g. capside proteins of AAV) and antigen (i.e. capside protein) 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 2010, 1644 gene therapy clinical trials have been worldwide approved.
At the end of August 2010, there have been (worldwide) approximately (source: http://www.wiley.uk.co/genmed/clinical/):
* More than 3,500 patients that received gene therapy treatment;
* 1644 gene therapy protocols, of which

1060 addressed cancer,
143 for vascular diseases,
134 for monogenic diseases,
131 for infectious diseases (predominantly AIDS),
50 for gene marking studies,
30 for neurological diseases,
18 for ocular diseases, and
40 for other disease.
38 protocols involved healthy volunteers.

The vectors used were:

adenoviral vectors (in 392 protocols),
retroviral vectors (in 341 protocols),
naked/plasmid dna (301 protocols),
lipofection (in 109 protocols),
vaccinia virus vectors (102 protocols),
AAV (adeno-associated virus) (75 protocols),
pox virus vectors (66 protocols),
herpes simplex virus (56 protocols), and
a variety of other vectors or vector combinations.

The gene types transferred were the following:

Antigen (325 protocols),
Cytokine (302 protocols),
Tumor suppressor (173 protocols),
Growth factor (127 protocols),
Suicide gene (118 protocols),
Deficiency (118 protocols),
Receptor (95 protocols),
Replication inhibitor (68 protocols), and
others (318 protocols).

The clinical phases addressed were (protocols/phase):
phase I (In Phase I clinical trials, researchers test a new drug or treatment 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): 60.5%
phase I/II: 18.7%
phase II, (In Phase II clinical trials, the study drug or treatment is given to a larger group of people (100-300) to see if it is effective and to further evaluate its safety): 16.2%
phase II/III: 0.8%
phase III (In Phase III studies, the study drug or treatment is given to large groups of people (1,000-3,000) to confirm its effectiveness, monitor side effects, compare it to commonly used treatments, and collect information that will allow the drug or treatment to be used safely): 3.5 %
Remark: For clinical trials of rare diseases, the number of patients per phase is considerably reduced, for instance, for a phase I clinical trial of a rare disease 5 to 10 patients are enrolled.
The result of all these protocols has been that a solid database of clinical experience has been built and that clinical research has moved away from the proof of concept stage.

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

As mentioned, phase I trials primarily assess its safety, but determine also a safe dosage range, and identify side effects; and in such experimental protocols as gene therapy protocols, in which a lot of new biological systems are tested for the first time in humans, (severe) adverse events can occur although, the preclinical tests and toxicology tests in animals have for objective to establish the safe dose range and assess potential side effects. However, an animal model is only a model and can never replace the final test in human beings.  
In this context, several severe adverse events have been reported in relation to gene therapy trials, of which the more discussed are shortly presented in the following:
In 1999, 18-year-old Jesse Gelsinger died from multiple organ failure 4 days after treatment for ornithine transcarboxylase deficiency. His dead was triggered by his heavy immune response to the very high dose of adenovirus used as the vehicle; a vehicle that is nowadays replaced by the AAVs, that are far less immunogenic. This incident slowed down the field for years.  
The very successful oncoretroviral vector based gene therapy trials for X-linked severe combined immune deficiency (the first one, conducted by Drs. Marina Cavazzana-Calvo and Alain Fischer from the Hôpital Necker Enfants Malades in Paris, and the second one conducted by Dr. A. Trasher from the University College in London/U.K.) should be mentioned. It 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 came down with a T-cell lymphoproliferative disorder and it could be established for two of these cases that the retroviral vector was found 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. As of today (2010), 20 children have been treated, 4 of them have developed leukemia-like adverse effects and one patient has unfortunately died from leukemia. From a clinical point of view, patients, who have been able to lead a normal life for periods up to 3 years, should be considered cured by this pioneering gene therapy treatment. Otherwise, ten year later, none of these 20 children would be alive today without gene therapy.
This adverse effect led to a considerable rethinking on the safety issues of viral vectors used for gene therapy purposes. However, it is also evident that more pre-clinical investigations in assessing the risk of gene therapy, including more basic research in the development of safer gene transfer vectors will be needed.
A further severe adverse event was reported in the press in 2007 of the death of a woman who received a modified gene in an arthritis trial run using AAV vectors. Of high importance for the field, the investigation showed that a fungal disorder was the cause and not the genetic therapy.
These setbacks demonstrate the difficulties and misconceptions that the field of gene therapy was/is facing.
Despite this adverse event, both, the public attitude towards gene therapy and international regulatory requirements have evolved. In addition, hurdles to clinical success have been identified, indicating which basic issues still need to be resolved and how to start 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 people, the field is maturing with the first demand for marketing authorization in the EU for a viral vector for the treatment of LPL and continues to attract strong attention from researchers, clinicians and industry, large pharmaceutical industry included, having rediscovered this field probably due to recent successes. Gene therapy and the production of gene therapy products use molecular and cell biological methods similar to those used for in vitro cultivation of cells in the manufacture of biologicals. It therefore seems 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, 24.08.10