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European Biopharmaceutical Review

Gene Therapy

Great Promise

With the help of recombinant DNA technology and a successful partnership between academia and industrial partners, gene therapy is finally demonstrating its potential

Gene therapy can be defined as the introduction of therapeutic genetic material, most commonly a functional copy of a mutated gene, into disease cells to correct their phenotype. With this seemingly straightforward concept, gene therapy holds the promise of a cure for many known monogenic conditions, and has therefore raised high expectations since its beginnings in the 1970s. However, the challenge lies in overcoming a number of critical requirements, including safe administration of the therapeutic gene, correction of the appropriate target tissue and stable expression of the gene at physiological levels in order to produce long-term therapeutic effects. Successful gene therapy has now become possible thanks to the remarkable progress of recombinant DNA technology in the 1980s, the development of viral vectors for efficient gene transfer, and the improvement of cell culture conditions and transduction protocols (1).

These technical developments supported the implementation of gene therapy protocols and led to the opening of the first approved clinical trial in 1990 to treat two patients suffering from Adenosine Deaminase-Severe Combined Immunodeficiency (ADASCID), a rare primary immunodeficiency (PID). PIDs have been the model of choice for gene therapy, as they are generally well-characterised monogenic diseases restricted to the haematopoietic compartment, and there is extensive experience available regarding the harvesting and ex vivo manipulation of haematopoietic stem cells (HSCs). Results of allogeneic haematopoietic stem cell transplantation (HSCT) also demonstrate that the engraftment of functional stem cells into the patient’s bone marrow is sufficient to rescue the disease phenotype and thus provide a rationale for curing the disease through transferring the functional gene into HSCs, especially if the corrected cells have a selective advantage over the deficient patient cells (2).

In the case of ADA-SCID, mutations are found in the gene coding for ADA – an enzyme involved in purine metabolism – resulting in the accumulation of toxic metabolites that affect the development and function of lymphocytes. The first attempts at gene therapy for ADA-SCID using γ-retroviral vectors did not use any pre-conditioning chemotherapy, whether targeting peripheral T cells or HSCs, and were inconclusive – producing only low levels of gene-marked cells in peripheral blood, with no obvious clinical benefit to patients (3). In the following clinical trials, the protocol was modified so that patients received a conditioning regime that was significantly less toxic than that used for an allogeneic HSCT procedure, to facilitate the engraftment of transplanted HSCs and progenitors in the bone marrow. Three Phase 1/2 trials using variations of this protocol, with a total number of 42 patients, were conducted simultaneously in Italy, the UK and the US. Impressively, all patients to date are alive, with a follow-up of greater than 10 years in some patients, and most of them show high levels of gene marking and sustained immunological and metabolic correction of the disease, demonstrating the successful engraftment of corrected multipotent HSCs (4,5). The alternative treatment for these patients would have been a matched or mismatched unrelated transplant, which carries a 30 per cent or greater chance of mortality, and so the safety of using gene-modified autologous cells with this disease is clear (2).

A similar protocol was adapted to treat X-linked SCID patients, another severe combined immunodeficiency resulting from mutations in the IL2RG gene, which is also involved in the development and function of lymphocytes. Two clinical trials were conducted in France and in the UK, including a total number of 20 patients, and in both studies HSCs were transduced with a γ-retroviral vector and reinfused to the patients without preconditioning. Although the engraftment of stem cells was low – as expected from an unconditioned procedure – the high survival advantage of gene-corrected cells resulted in a rapid reconstitution of the T cell lineage, associated with the recovery of T cell function and the ability to clear common viral infections (6,7).

The overall outcome of these trials was at first very encouraging, but regrettably, two to five years after the treatment, five patients who were clinically well developed a T cell acute lymphoblastic leukaemia resulting from mutagenic retroviral insertions (8,9). They were all treated with standard chemotherapy, but one patient died of refractory leukaemia. Similar adverse events were seen using γ-retroviral vectors in clinical trials of gene therapy for Wiskott-Aldrich syndrome (WAS) and X-linked chronic granulomatous disease (X-CGD) (10,11).

Gamma-retroviral vectors are known to integrate preferentially in regions of opened chromatin, often near the transcription start sites of active genes. In the adverse events in the X-SCID, WAS and X-CGD trials, the mapping of the integration sites revealed insertion of the vector in the vicinity of known proto-oncogenes, or genes with leukaemogenic potential.Unfortunately, these unexpected cases of insertional mutagenesis came not long after the death of Jesse Gelsinger, a young volunteer enrolled in an adenoviral gene therapy trial for ornithine transcarbamylase (OT) deficiency, who suffered multiple organ failure due to vector-related toxicity (12). Altogether, these events revived a number of controversies and safety concerns associated with gene therapy, and highlighted the necessity to increase the biosafety of the procedure and provide a better regulatory framework to accompany clinical gene therapy trials.

Accordingly, a considerable amount of work was undertaken to increase the efficiency and reduce the toxicity of therapeutic gene transfer (13). The potent retroviral enhancers were removed from the vector in a new self-inactivating (SIN) γ-retroviral vector design that has recently entered clinical testing, and is showing early promise in patients with SCID-X1 (14). Interest has also has shifted to HIV1-derived lentiviral vectors, which have been designed with deleted viral enhancer sequences and the use of internal mammalian promoters. Such vectors may have a number of advantages, including their capacity to integrate in a more random manner into the genome, and a decreased ability to activate neighbouring genes.

Additional elements, such as DNA insulators or de-targeting microRNA sequences, can also be added to the viral backbone to customise the vectors and further improve their specificity and safety. Once again, early reports in a number of monogenic conditions, including primary immunodeficiency conditions and metabolic diseases, are showing encouraging efficacy with low toxicity, although the follow-up time is so far relatively short (15,16). The long term success and safety of these trials may pave the way for the increased use of gene therapy in a wider range of conditions.

Another approach made possible by the latest advances in genetic engineering is to use the capacity of the cell machinery 

to repair double-stranded breaks in the DNA by homologous recombination. This will replace the disease-causing gene with a functional copy at its own locus, with the hope that the in situ correction of the genetic defect will allow a physiological regulation of the newly inserted gene. This gene editing strategy has been used successfully in vitro, using artificial site-specific zinc-finger nucleases to excise the mutated gene, but the clinical translation of the technique is made difficult by the requirement for multiple components to be transferred and the added risk of off-target cleavage (17).

A vast effort has been made to minimise the risks of severe side effects associated with gene transfer technologies, but it is also crucial to be able to anticipate the emergence of dominant clones in patients posttreatment. Various strategies have been developed, with a particular emphasis on the mapping of vector insertion sites in the genome of gene-corrected cells, given that vector-related leukemias have so far been associated with integration near genes with oncogenic properties. The distribution of these insertion sites defines the clonal repertoire of the cells, and longitudinal monitoring may help to predict the outgrowth of potential malignant clones (18).

More generally, the systematic monitoring of the gene therapy patients is one of the many requirements put in place by the regulatory authorities to ensure patients’ safety. Gene-modified cells are unconventional medicinal products, and therefore careful review of preclinical data – including testing in relevant animal models, control of the production processes and the assessment of the risk-benefit balance of the treatment – are also required to obtain regulatory approval for new trials (19).

Despite many regulatory setbacks, the field of gene therapy has expanded rapidly, with over 160 trials for inherited monogenic conditions registered, using variations of the technology to adapt to the variety of diseases. Several more conditions involving the targeting of HSCs have now been treated successfully, including beta-thalassemia – a betaglobin deficiency resulting in severe anaemia that has recently been treated by lentiviral gene therapy – and X-linked adrenoleukodystrophy (X-ALD) – a demyelinating disease of the nervous system, which can be addressed at early stages using gene-corrected monocyte precursors from the bone marrow to restore the defective microglia (16,20). As a result of these successful studies, and in recognition of the limited therapeutic options available for many monogenic rare diseases, gene therapy has started to attract many investors from the industry and now benefi ts from the fi nancial, logistical and technical support of a number of specialist pharmaceutical companies.

In July 2012, Glybera, in which an adenoviral vector carries a copy of the lipoprotein lipase gene, was the fi rst gene therapy treatment approved for commercialisation in Europe for lipoprotein lipase deficiency, demonstrating that after many highs and lows, gene therapy has now evolved from experimental to established medicine (21). It is certainly the combination of the huge research effort undertaken, the early pioneering proof-of-principle studies, successful collaboration between academia and its industrial partners, and the completion of numerous clinical trials within an appropriate regulatory setting, that has allowed gene therapy to finally live up to its promise.

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Bobby Gaspar is a Physician/Scientist working in paediatric immunology at the Institute of Child Health (ICH) and Great Ormond Street Hospital, London. He initially trained in paediatrics and then became interested in primary immunodeficiencies at an early stage in his career, undertaking a PhD at the Molecular Immunology Unit at ICH. Over the last decade, his team has conducted numerous clinical trials to show that gene therapy can successfully correct the immune defect in specifi c immunodeficiency conditions.


Christine Rivat is a Senior Healthcare Scientist at Great Ormond Street Hospital. She obtained a PhD from Pierre and Marie Curie University, France and then completed a postdoctoral fellowship in the Gustave Roussy Institute studying haematopoietic stem cell biology. This was followed by a research fellowship in Bobby Gaspar’s laboratory at the Institute of Child Health undertaking preclinical studies of XLP gene therapy. She is now responsible for monitoring patients post gene-therapy at Great Ormond Street Hospital, London.

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