Gene therapy clinical trials worldwide to 2012 ... - Wiley Online Library

THE JOURNAL OF GENE MEDICINE REVIEW J Gene Med 2013; 15: 65–77. Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/jgm.2698

ARTICLE

Gene therapy clinical trials worldwide to 2012 – an update

Samantha L. Ginn1,2 Ian E. Alexander1,3 Michael L. Edelstein4 Mohammad R. Abedi5 Jo Wixon6* 1

Gene Therapy Research Unit, Children’s Medical Research Institute and The Children’s Hospital at Westmead, NSW, Australia

2

Sydney Medical School, University of Sydney, NSW, Australia

3

Discipline of Paediatrics and Child Health, University of Sydney, NSW, Australia

4

Imperial Healthcare NHS Trust, London, UK

5

Department of Laboratory Medicine, Örebro University Hospital, Örebro, Sweden Health Sciences Journals – Editorial, John Wiley & Sons Ltd, Oxford, UK

6

*Correspondence to: J. Wixon, Health Sciences Journals – Editorial, John Wiley & Sons Ltd, 9600 Garsington Road, Oxford OX4 2DQ, UK. E-mail: [email protected]

Copyright © 2013 John Wiley & Sons, Ltd.

Abstract To date, over 1800 gene therapy clinical trials have been completed, are ongoing or have been approved worldwide. Our database brings together global information on gene therapy clinical trials from official agency sources, published literature, conference presentations and posters kindly provided to us by individual investigators or trial sponsors. This review presents our analysis of clinical trials that, to the best of our knowledge, have been or are being performed worldwide. As of our June 2012 update, we have entries on 1843 trials undertaken in 31 countries. We have analysed the geographical distribution of trials, the disease indications (or other reasons) for trials, the proportions to which different vector types are used, and which genes have been transferred. Details of the analyses presented, and our searchable database are available on The Journal of Gene Medicine Gene Therapy Clinical Trials Worldwide website at: http://www. wiley.co.uk/genmed/clinical. We also provide an overview of the progress being made in clinical trials of gene therapy approaches around the world and discuss the prospects for the future. Copyright © 2013 John Wiley & Sons, Ltd. Keywords database

clinical trials; gene therapy; The Journal of Gene Medicine; worldwide;

Gene therapy trials: meeting the challenges Gene therapy is a relatively new paradigm in medicine with enormous therapeutic potential. However, a number of widely reported adverse events [1–4] have focused attention on associated risks ahead of the exciting therapeutic progress being made. In 2000, the optimism of the gene therapy research community was bolstered by the first report of successful treatment of a genetic disease by gene therapy [5,6]. The condition, X-linked severe combined immunodeficiency (SCID-X1), commonly diagnosed in early infancy, is characterised by recurrent infection as a result of an absence of cell-mediated and humoral immunity. Importantly, the majority of treated infants underwent full immunological reconstitution with eradication of established infections. A total of 20 infants were treated in two initial trials, nine in France: one by a group in Australia in collaboration with the French, and ten by a team in the UK [5–8]. Long-term follow-up of the nine boys treated in the French trial, now aged between 8 and 11 years, has been reported, with eight patients alive after a median period of 9 years [9]. This has established gene therapy as a realistic therapeutic alternative for patients without a suitably matched sibling donor, which is associated with less favourable survival rates. Overshadowing this impressive success, 30 months out from treatment, one of the initial patients in the French trial developed a T cell leukaemia, which was the direct result of the gene transfer vector used [10]. This was shown to be related to retrovirus vector

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integration near the LMO2 proto-oncogene promoter, a phenomenon known as insertional mutagenesis, leading to aberrant transcription and expression of LMO2 [10,11]. The trial was placed on voluntary hold while the cause was investigated and led to a worldwide effort to improve vector safety and reduce the likelihood of similar events in future clinical trials for this and other diseases using integrating vectors. A further three patients in the French trial [3] and one in the British trial for SCID-X1 [4] went on to develop T cell leukaemia, with four of the five patients being successfully treated with chemotherapy and in complete remission. Now, almost a decade later, a multi-national trial utilising a vector with improved safety features is open, and has begun treating patients [12]. Adenosine deaminase-deficiency (ADA-SCID) is another primary immunodeficiency for which gene therapy is showing great promise in the clinic. Initial trials for this disease were unsuccessful for several reasons that included the maintenance of patients on pegylated ADA (PEG-ADA) during gene therapy and the targeting of gene transfer to T lymphocytes [4,13]. Cessation of PEG-ADA treatment during gene therapy facilitated a selective advantage for the gene-corrected cells. In addition, the targeting of haematopoietic stem cells (HSCs) using an improved gene transfer protocol and a myeloablative conditioning regime underpin the more recent success [14,15]. More than 30 patients with ADA-SCID have been treated worldwide with gene therapy since 2000 [16,17], with the majority of those treated at the San Raffaele Telethon Institute for Gene Therapy in Milan [18]. In addition, six patients have been treated at Great Ormond Street Hospital in London [19,20], six at the Children’s Hospital Los Angeles [21,22] and a further three at the University of California Los Angeles [23]. In the majority of cases, reconstitution of immune function has been achieved without the need for supporting enzyme replacement therapy with PEG-ADA. Importantly, adverse events related to the gene transfer technology have not been observed. Another disease involving targeting of the haematopoietic compartment showing promising results in the clinic is X-linked adrenoleukodystrophy (X-ALD). By contrast to SCID-X1 and ADA-SCID, however, no survival advantage is conferred on gene-modified cells by the disease pathophysiology. To overcome this lack of selective advantage, the patients underwent myeloablative conditioning and a lentiviral vector, based on HIV-1, was used to achieve gene transfer [24]. Made famous by the movie Lorenzo’s Oil, X-ALD is a fatal demyelinating disease of the central nervous system, with the only viable treatment option being allogeneic haematopoietic stem cell (HSC) transplantation. In addition, this treatment is only effective if performed at an early stage of disease progression and relies on the replacement of brain microglia by gene-corrected cells of the monocyte lineage present in the bone marrow. At present, the results for only two boys have been reported in the literature, although a further two have been treated [25]. For these two patients, evidence of clinical benefit is clear, with demyelination Copyright © 2013 John Wiley & Sons, Ltd.

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stabilising between 14 and 16 months post-transplantation. In addition, cells expressing the X-ALD protein were observed in multiple lineages, suggesting that gene-correction of true HSCs had been achieved. Although studies with larger cohorts of patients are needed, this is the first report of a lentiviral vector being successfully used to treat genetic disease in humans, as well as the first time a severe brain disease has been treated with measurable efficiency. Increased follow-up times to establish long-term safety could see this approach become a therapeutic option for patients without a suitably matched bone marrow donor. A similar trial, targeting metachromatic leukodystrophy, is also treating patients in Milan [26]. Much excitement was caused by the report of successful immunotherapy of two patients with metastatic melanoma in September 2006 [27]. The Rosenberg group engineered tumour recognition into autologous lymphocytes from peripheral blood using a retrovirus encoding a T cell receptor. High, sustained levels of circulating engineered cells were retained in two patients up to 1 year after infusion, resulting in regression of metastatic melanoma lesions; a dramatic improvement for patients who had only been expected to live for 3–6 months. Although stable engraftment of the transduced cells was seen for at least 2 months after infusion in 15 other patients, they did not respond to the treatment. It appears that it is critical to obtain an effective tumour infiltrating lymphocyte population for the treatment to be successful, and further work is underway aiming to improve response rates and refine the approach. Recently, in a similar clinical trial, this strategy has been extended to treat patients with metastatic synovial cell carcinoma, which is one of the most common soft tissue tumours in adolescents and young adults. Clinical responses were observed in four of six patients with synovial cell carcinoma and in five of 11 patients with melanoma [28]. Despite achieving similar levels of transduction and administering similar levels of gene-modified T cells to patients, the clinical responses were highly variable and require further investigation. Importantly, two of the 11 patients with melanoma were in complete regression at 1 year post-treatment and a partial response in one patient with synovial cell carcinoma was observed at 18 months. Chimeric antigen receptors (CAR) represent another autologous cell-based therapy targeting tumour-associated cell-surface antigens. This approach is receiving increasing attention and brings together the expansion potential and persistence of cytotoxic T cells with the specificity of monoclonal antibodies. Researchers from the Abramson Cancer Center in Philadelphia have described the treatment of three patients with chronic lymphocytic leukaemia (CLL) with autologous Tcells that were genetically modified to express a CAR with specificity for the B-cell antigen CD19 [29]. This publication builds on an earlier report for one of these patients in which complete remission was achieved following an infusion of 1.42 107 transduced T cells [30]. For these three patients, all carrying a considerable CLL tumour burden, cells were administered following conditioning chemotherapy designed for depletion of J Gene Med 2013; 15: 65–77. DOI: 10.1002/jgm


lymphocytes. Post-infusion complications were limited to a transient and treatable tumour lysis syndrome, occurring between 7 and 21 days post-infusion. At the time of publication, two of the three patients were in complete remission, at 10 and 11 months post-therapy, with the third patient showing a partial response at 7 months post-therapy. The persistence of modified cells was also demonstrated, suggesting that this therapy may provide sustained tumour control in these patients. Until more efficient gene delivery systems and/or in vivo selection strategies are developed, many human diseases, potentially amenable to gene therapy, will remain beyond reach. For diseases such as SCID-X1 and ADA-SCID, genetically modified cells undergo selective expansion as a consequence of in vivo selection conferred by the disease pathophysiology despite the correction of only a modest number of progenitors. For diseases lacking a naturally occurring selection pressure, one promising possibility is to confer an exogenous selection pressure on gene-modified cells using a combination of gene and drug therapy. The potential of this type of strategy has recently been highlighted in a trial seeking to confer chemoprotection on human HSCs during chemotherapy with alkylating agents for glioblastoma [31]. The strategy involves delivery of a mutant form (P140K) of methylguanine methyltransferase (MGMT) to HSC followed by alkylating chemotherapy in concert with a small molecule inhibitor of native MGMT, O6benzylguanine (O6BG). The O6BG suppresses tumour MGMT expression, thereby enhancing sensitivity to alkylating chemotherapy. Gene-modified HSC expressing MGMT P140K are concurrently protected and selectively expanded. In this initial study, an extended survival of patients was reported but, in future, this approach could be used to expand progenitor cells bearing a therapeutic gene and P140K to allow in vivo selection and expansion of gene-modified cell numbers to a clinically useful therapeutic threshold. Another strategy that can be employed is targeting disease states in which gene transfer to a small number of cells at anatomically discrete sites has the potential to confer therapeutic benefit. This has been impressively demonstrated in trials for a form of congenital blindness, Leber congenital amaurosis (LCA), which encompasses a group of incurable autosomal recessive dystrophies affecting the retina. One molecular form, accounting for approximately 10% of affected individuals, is caused by mutations in the gene encoding retinal pigment epithelium-specific 65-kDa protein, RPE65. This condition is characterised by a progressive deterioration of vision, with complete loss of sight by early adulthood. Using sub-retinal administration of recombinant adeno-associated viral vectors (rAAV) expressing RPE65, three independent clinical trials for LCA have been initiated [32–35]. The preliminary results indicate there has been no detectable systemic dissemination of the vector from the treated eye, no evidence of a significant humoral immune response to either the vector or encoded RPE65 and no serious adverse events. Importantly, improvements in both objective and Copyright © 2013 John Wiley & Sons, Ltd.

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subjective measures of vision have been reported and maintained for up to 2 years [36,37], supporting further clinical trials for this and other forms of LCA. Although the vector was initially administered unilaterally, redelivery to the contralateral eye has been safely performed with demonstrated efficacy, even years after the initial treatment [38]. Given the progressive degenerative nature of this disease, the use of gene therapy as a potential treatment option needs to be undertaken before the disease reaches an advanced stage associated with photoreceptor cell death. In April 2006, a report on a Swiss–German phase I/II gene therapy clinical trial aimed at treating chronic granulomatous disease (CGD), an inherited primary immunodeficiency that affects phagocytes, brought new hope of success [39]. In this trial, following non-myeloablative pre-conditioning by busulfan treatment, mobilised CD34+ cells isolated from peripheral blood were retrovirally transduced and infused into the patient. At the time of the report, two of the three patients showed clear benefit from this treatment, as demonstrated by a lack of infections and an improved quality of life. After initial clinical improvement, one of these two patients died from a severe bacterial infection as a result of the return of the CGD symptoms over time [40]. This was later determined to be caused by silencing of the transgene as a result of methylation of the viral promoter [41]. In concert, a three- to five-fold increase in the number of genetically modified cells was observed resulting from insertional activation of the MDS1-EVI1 locus [39]. This led to genome instability, monosomy 7 and the development of myelodysplastic syndrome [41]. A total of 12 patients have been treated worldwide in five independent clinical trials [39,41–45]; however, in the majority of cases, although initial clinical benefit was observed, significant engraftment has not been maintained long-term. Efforts are underway to develop vectors containing alternative promoters not only to prevent silencing of the transgene, but also to achieve an improved safety profile [46]. In a clinical trial performed in Paris, an 18-year-old male with b-thalassaemia was transplanted with autologous CD34+ cells transduced ex vivo with a lentiviral vector expressing a marked b-globin transgene. Consistent with therapeutic benefit, the patient subsequently had a reduced transfusion requirement. Of interest, however, approximately half of the therapeutic effect observed appeared to be mediated by a dominant clone containing an integration site within the HMGA2 gene [47]. Haematopoietic homeostasis is currently being maintained; however, long-term follow up is required to establish whether the presence of this dominant clone might lead to any adverse clinical effects. A final trial targeting the haematopoietic compartment showing promising results is being performed at Hannover Medical School in Germany. In this trial for Wiscott–Aldrich syndrome, the results for ten patients have been reported [48]. Following treatment, the clinical condition of the patients improved and long-term engraftment of gene-corrected cells was observed in the bone marrow; however, the development of an acute T cell J Gene Med 2013; 15: 65–77. DOI: 10.1002/jgm

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leukaemia in one of the ten patients treated has been reported [49]. The preliminary data implicate an insertion upstream of LMO2 and additional chromosomal mutations reminiscent of the genotoxic events occurring in the SCID-X1 trials. Again, gene transfer was achieved using a g-retroviral vector containing transcriptionally active long terminal repeats. Thus, at present, the field is making good progress, particularly in diseases targeting the haematopoietic system. Insertional mutagenesis, however, is now recognised as a clinically established risk associated with the use of integrating vectors. As a result, the field is currently exploring alternative vector systems, lacking strong viral enhancer elements and with safer integration profiles, aiming to reduce the likelihood of such events occurring in the future [50].

Sources of data The data reported in this review have been compiled as much as possible from information provided by regulatory agencies. Where this is not possible, data are obtained from the published literature, presentations at meetings and personal contacts with sponsors and investigators. Policy on the public availability of data held by regulatory agencies varies widely from country to country, from total transparency in the USA, to varying degrees of confidentiality in European and Asian countries. In the USA, the National Institutes of Health (NIH) Recombinant DNA Advisory Committee [51] compiles a database of all ongoing or completed gene therapy clinical trials and is our primary source of information for trials performed there. In the UK, the functions of the Gene Therapy Advisory Committee (GTAC) were transferred to the National Research and Ethics Committee in June 2011. A Department of Health page providing a summary table of UK gene therapy research [52] is still available but has not been updated since 2010. No information has been received from GTAC since early 2011. The Belgian Biosafety Server, managed by the Service of Biosafety and Biotechnology, provides very comprehensive and up-to-date information about gene therapy clinical trials in Belgium [53]. In Germany, the Zentrum Klinische Studien at the University Hospital in Freiburg (funded by the German Ministry of Education and Research) set up in 2001 an interactive database of trials being conducted in Germany (DeReG). The database is, however, no longer available and has been incorporated into the German clinical trials database [54]. In Switzerland, the Swiss Expert Committee for Biosafety maintains a Swiss gene therapy clinical trial register, albeit with limited information [55]. The page has not been updated since June 2010. The Dutch Ministry of Housing, Spatial Planning and the Environment’s office for licensing of genetically modified organisms maintain a database in Dutch which includes gene therapy clinical trials [56]. In France, the National Drug Safety agency (AFSSAPS) established a public Copyright © 2013 John Wiley & Sons, Ltd.

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clinical trials database in 2009 [57], including gene therapy clinical trials; however, we have found it difficult to identify gene therapy trials because there is no reliable way to systematically search for them. Trials conducted prior to 2009 can be identified in yearly public reports from the commission responsible for genetically modified organisms. The last published report is from 2007. The European Agency for the Evaluation of Medicinal Products (EMEA) is compiling a database of European Community clinical trials (EudraCT) [58], which includes gene therapy clinical trials. It contains trial sponsor-submitted data on all trials initiated in the European Union (EU) from 1 May 2004 onwards and is now publically accessible. There is no systematic way to identify gene therapy trials in the database, limiting its usability. The European Commission also maintains a register of genetically modified organisms deliberately released within the EU [59], including plant, animal and human trials. So far, the register contains trials from Spain, Sweden, the Netherlands, Romania, Italy, Ireland, Finland, France, Iceland, Estonia, UK, Hungary, Germany and Belgium. It appears to be voluntary rather than compulsory and, although it does not list all trials for each country, it has already increased the numbers of trials that we are able to identify for some of these countries. The trend appears to be that more and more countries include their clinical trials on this database. In Australia, The Gene and Related Therapies Research Advisory Panel is no longer in existence. There is no source containing information specifically on gene therapy trials, however, in 2005 the National Health and Medical Research Council established the Australian New Zealand Clinical Trials Registry [60] that compiles all the clinical trials in these two countries. It is not specific to gene therapy and there is no easy way to rapidly identify all gene therapy trials. In China, a clinical trials registry not specific to gene therapy (ChiCTR), maintained by the West China Medical School at Sichuan University, is now available in English [61]. The translation is limited, and it is difficult to ascertain how comprehensive the searches are. Japan now also maintains an online clinical trials database [62], composed of three separate databases listing different trials depending on where they are conducted. The search options are limited and only partial translated information is available, which makes gene therapy-specific searches challenging. Thus, although gene therapy trials are identifiable; it is difficult to be sure that all trials have been found. Other clinical trials registries can be searched simultaneously via the World Health Organization (WHO) International Clinical Trials Registry Platform [63]. Countries included in the WHO search portal include the USA, the European Union, Australia, New Zealand, Brazil, China, India, South Korea, Cuba, Germany, Iran, Japan, Sri Lanka, the Netherlands and several African countries in a Pan-African registry. None of these registries are gene therapy specific and it is difficult to systematically identify gene therapy trials. Other countries that have regulatory bodies overseeing gene therapy trials do not keep J Gene Med 2013; 15: 65–77. DOI: 10.1002/jgm


publically available registries or databases. This includes Canada and those EU countries not mentioned above. Trials from these countries are, however, sometimes identifiable from other databases such as the US NIH clinical trials database, which also lists foreign trials. Most other countries do not have dedicated bodies for gene therapy, which is usually the responsibility of the Ministry of Health, or National Drug Agencies. These countries probably account for a very small proportion of gene therapy clinical trials, and these trials can generally only be identified after the results are published in the scientific literature. We are seeing a marked trend towards more broad databases, which are less specific for gene therapy (i.e. ge-

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neric clinical trials databases, encompassing all trials). Because there is no way to specifically search these resources for gene therapy trials, this makes systematic identification very challenging and we have to search using a range of keywords, which we are sure will lead to us missing some trials.

Number of trials per year The number of trials initiated each year has tended to drop in those years immediately following reports of adverse reactions, such as in 2003 and 2007; however, 2005, 2006 and 2008 were strong years for gene therapy trials (Figure 1). The most recent years (2011 and 2012 in this case) tend to be underrepresented in the database because it takes time for articles to be published, causing a lag in obtaining information about the most recent trials.

Countries participating in gene therapy trials

Figure 1. Number of gene therapy clinical trials approved worldwide 1989–2012.

Gene therapy clinical trials have been performed in 31 countries, with representatives from all five continents (Figure 2). We have located data on trials from three new countries since our last review [64], with these being Ireland (with three trials), as well as Romania and Saudi Arabia (with one trial each). The continental distribution of trials has not changed greatly in the last few years, with 65.1% of trials taking place in the Americas (64.2% in 2007) and 28.3% in Europe (26.6% in 2007), with growth in Asia reaching 3.4% from 2.7% in 2007. The USA has still undertaken the most trials globally (63.7%), with 1174 trials; we have entries on 24 trials from Canada (although we know that this is an underestimate and the actual number may be closer to 60) and, in addition to these, there is one trial from Mexico. Within

Figure 2. Geographical distribution of gene therapy clinical trials (by country). Copyright © 2013 John Wiley & Sons, Ltd.

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Europe, the UK accounts for 11.0% of the world total with 203 trials, Germany 4.4% (81 trials), Switzerland 2.7% (50 trials) and France 2.9% (53 trials). Our entries for trials in the Netherlands have jumped to 31 (from 13 in 2007) representing 1.7% of world trials, with 27 in Belgium (1.5%) and 21 in Italy (1.1%). We have recorded only a single additional trial in Eastern Europe in Russia since 2007, when we had records on six trials in Poland, one in the Czech Republic and one in Russia (we are still unable to obtain good data on Russian trials). From Asia, China has the most entries in our database (26 trials), closely followed by Japan (20 trials) representing 1.4% and 1.1% of world trials, respectively, and South Korea has leaped from four trials in 2007 to 14 to date. Israel comes next with nine trials, followed by Singapore with two and Taiwan with one. There has also been significant growth in trial entries for Australia (rising to 30 from 17 in 2007), whereas there have been no new trials in New Zealand (still at two trials). Other countries where gene therapy trials have been performed are Spain (19 trials), Sweden (ten trials), Finland (six trials), Norway (four trials), Austria and Denmark (two trials each) and Egypt (one trial). Sixteen trials have been reported to us as ‘multi-country’, although we know that a large proportion of the trials initiated in one country do have centres in other countries.

Diseases targeted by gene therapy The vast majority (81.5%) of gene therapy clinical trials to date have addressed cancer, cardiovascular disease and inherited monogenic diseases (Figure 3). Although trials targeting cardiovascular disease outnumbered trials for monogenic disease in our 2007 review of the database, the latter group has returned to being the second most common indication treated by gene therapy. It also

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represents the disease group in which the greatest successes of gene therapy to date have been achieved. The range of indications for which gene therapy trials have been approved has widened since our last review (Table 1).

Cancer Thus far, most of the clinical trials in gene therapy have been aimed at the treatment of cancer (64.4% of all gene therapy trials). Many different cancers have been targeted throughout the years, including lung, gynaecological, skin, urological, neurological and gastrointestinal tumours, as well as haematological malignancies and paediatric tumours. A range of strategies has been applied to treat cancer, from inserting tumour suppressor genes, to immunotherapy, oncolytic virotherapy and gene directed enzyme pro-drug therapy. The p53 gene is by far the most commonly transferred tumour suppressor gene, although others such as BRCA-1, Fus-1 and endostatin have been used in cancer trials. There are also some clinical trials that combine p53 gene transfer with chemotherapy or radiotherapy. Immunotherapy of cancer aims to intensify the normally weak humoral and/ or cellular reactions to tumour antigens in tumour-bearing hosts. A number of different strategies have been employed, including vaccination with tumour cells engineered to express immunostimulatory molecules, vaccination with recombinant viral vectors encoding tumour antigens, vaccination with host cells engineered to express tumour antigens or tumour-derived RNA, naked DNA vaccines, and intra-tumoural injection of vectors encoding cytokines or major histocompatibility molecules. Oncolytic virotherapy utilises viruses capable of specifically targeting and replicating in tumour cells and causing cell lysis, thereby killing the infected tumour cells. The viruses used for this approach can be naturally oncolytic,

Figure 3. Indications addressed by gene therapy clinical trials. Copyright © 2013 John Wiley & Sons, Ltd.

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Table 1. Conditions for which human gene transfer trials have been approved Monogenic disorders Adrenoleukodystrophy a-1 antitrypsin deficiency Becker muscular dystrophy b-thalassaemia Canavan disease Chronic granulomatous disease Cystic fibrosis Duchenne muscular dystrophy Fabry disease Familial adenomatous polyposis Familial hypercholesterolaemia Fanconi anaemia Galactosialidosis Gaucher’s disease Gyrate atrophy Haemophilia A and B Hurler syndrome Hunter syndrome Huntington’s chorea Junctional epidermolysis bullosa Late infantile neuronal ceroid lipofuscinosis Leukocyte adherence deficiency Limb girdle muscular dystrophy Lipoprotein lipase deficiency Mucopolysaccharidosis type VII Ornithine transcarbamylase deficiency Pompe disease Purine nucleoside phosphorylase deficiency Recessive dystrophic epidermolysis bullosa Sickle cell disease Severe combined immunodeficiency Tay Sachs disease Wiskott–Aldrich syndrome Cardiovascular disease Anaemia of end stage renal disease Angina pectoris (stable, unstable, refractory) Coronary artery stenosis Critical limb ischaemia Heart failure Intermittent claudication Myocardial ischaemia Peripheral vascular disease Pulmonary hypertension Venous ulcers Infectious disease Adenovirus infection Cytomegalovirus infection Epstein–Barr virus Hepatitis B and C HIV/AIDS Influenza Japanese encephalitis Malaria Paediatric respiratory disease Respiratory syncytial virus Tetanus Tuberculosis

Cancer Gynaecological – breast, ovary, cervix, vulva Nervous system – glioblastoma, leptomeningeal carcinomatosis, glioma, astrocytoma, neuroblastoma, retinoblastoma Gastrointestinal – colon, colorectal, liver metastases, post-hepatitis liver cancer, pancreas, gall bladder Genitourinary – prostate, renal, bladder, anogenital neoplasia Skin – melanoma (malignant/metastatic) Head and neck – nasopharyngeal carcinoma, squamous cell carcinoma, oesophaegeal cancer Lung – adenocarcinoma, small cell/nonsmall cell, mesothelioma Haematological – leukaemia, lymphoma, multiple myeloma Sarcoma Germ cell Li–Fraumeni syndrome Thyroid Neurological diseases Alzheimer’s disease Amyotrophic lateral sclerosis Carpal tunnel syndrome Cubital tunnel syndrome Diabetic neuropathy Epilepsy Multiple sclerosis Myasthenia gravis Parkinson’s disease Peripheral neuropathy Pain Ocular diseases Age-related macular degeneration Diabetic macular edema Glaucoma Retinitis pigmentosa Superficial corneal opacity Choroideraemia Leber congenital amaurosis Inflammatory diseases Arthritis (rheumatoid, inflammatory, degenerative) Degenerative joint disease Ulcerative colitis Severe inflammatory disease of the rectum Other diseases Chronic renal disease Erectile dysfunction Detrusor overactivity Parotid salivary hypofunction Oral mucositis Fractures Type I diabetes Diabetic ulcer/foot ulcer Graft versus host disease/transplant patients

or engineered from originally non-oncolytic viruses. The actions of oncolytic viruses can also stimulate an immune response to the cancer and, in some cases, these viruses are modified to encode immunostimulatory cytokines to enhance this ability. Gene-directed enzyme prodrug therapy is the targeted expression of genes that encode enzymes (often termed ‘suicide genes’) that convert prodrugs into cytotoxic drugs in situ, only within the tumour and its immediate environment, thereby improving the use of certain chemotherapies. Most commonly, herpes Copyright © 2013 John Wiley & Sons, Ltd.

simplex virus thymidine kinase has been used to convert the nontoxic pro-drug ganciclovir into the cytotoxic triphosphate ganciclovir.

Cardiovascular gene therapy Cardiovascular gene therapy is the third most popular application for gene therapy, at 8.4% of trials (down from 9.1% in 2007). The expectation is that gene therapy will J Gene Med 2013; 15: 65–77. DOI: 10.1002/jgm

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provide a new avenue for therapeutic angiogenesis, myocardial protection, regeneration and repair, prevention of restenosis following angioplasty, prevention of bypass graft failure and risk-factor management. The vast majority of cardiovascular gene therapy trials conducted to date have addressed therapeutic angiogenesis to increase blood flow to ischaemic regions. Two dominant categories of ischaemic diseases have been tested in approximately equal numbers, namely myocardial ischaemia as a result of coronary artery disease and lower limb ischaemia as a result of peripheral artery disease. The fibroblast growth factor and vascular endothelial growth factor families have been widely applied, and a small number of trials have used platelet-derived growth factor to treat foot ulcers resulting from the microvascular disease of diabetes. The induction of hypoxia inducible factor as a trigger to stimulate angiogenesis has been used in 11 trials.

Inherited monogenic diseases The ultimate aim in treating monogenic diseases by gene therapy is the correction of the disorder by the stable transfer of the functioning gene into dividing stem cells to ensure the permanence of the correction, and there has been an array of monogenic disease targeted (Table 1). We have identified 161 trials for inherited monogenic disorders, 22.4% of which targeted cystic fibrosis, the most common inherited genetic disease in Europe and the USA. The average life expectancy of patients with cystic fibrosis is less than 40 years; hence, the interest in this disease as a prime target for gene therapy. The second most common group of inherited diseases targeted are the severe combined immunodeficiency syndromes, representing just over 20% of the trials for monogenic diseases. Another monogenic immunodeficiency, chronic granulomatous disease, has also been the target of several trials. This is a group of diseases in which gene therapy has shown lasting and clinically meaningful therapeutic benefit.

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Other indications There have been 147 trials (8.0% of the total) performed directed against infectious diseases. HIV infection is the major target in this category but trials aimed at tetanus, cytomegalovirus and adenovirus infection have also been conducted. Neurological diseases have also been targeted by gene therapy, with 36 registered phase I, I/II and II trials aimed at a variety of diseases such as amyotrophic lateral sclerosis, multiple sclerosis, myasthenia gravis, neurological complications of diabetes, Alzheimer’s disease and Parkinson’s disease. There have been 28 trials to date aimed at treating ocular pathologies, focussing on conditions including retinitis pigmentosa, glaucoma, age-related macular degeneration and Leber congenital amaurosis. Inflammatory diseases for which gene therapy has been trialled include rheumatoid, inflammatory and degenerative (osteo)arthritis and ulcerative colitis. Finally, there are single trials for a small number of other conditions, including chronic renal disease, peanut allergy and bone fractures.

Genes transferred into humans There have been a vast number of gene types used in human gene therapy trials (Figure 4), making it impossible to discuss each one in the present review. As would be expected, the gene types transferred most frequently (antigens, cytokines, tumour suppressors and suicide enzymes) are those primarily used to combat cancer (the disease most commonly treated by gene therapy). These categories account for 55.3% of trials, although it should be noted that antigens specific to pathogens are also being used in vaccines. Growth factors were transferred in 7.5% of trials, with almost all of these being aimed at cardiovascular diseases. Deficiency genes were used in 8.0% of trials, and genes for receptors (most commonly used for cancer gene therapy) in 7.2%. Marker

Figure 4. Gene types transferred in gene therapy clinical trials. Copyright © 2013 John Wiley & Sons, Ltd.

J Gene Med 2013; 15: 65–77. DOI: 10.1002/jgm


genes were transferred in 2.9% of trials, whereas 4.3% of trials used replication inhibitors to target HIV infection. In 2.1% of trials, oncolytic viruses were transferred (rather than genes) with the aim of destroying cancer cells, and 1.8% of trials involved the transfer of antisense or short interfering RNA, with the aim of blocking the expression of a target gene.

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alternatives to retroviruses. There have been 39 trials that combined two vectors, 28 used poxvirus and vaccinia virus, three used adenovirus and retrovirus, three used adenovirus and vaccinia virus, three used naked DNA and adenovirus, one used adenovirus and modified vaccinia Ankara virus and one used naked DNA and vaccinia virus.

Nonviral vectors

Vectors used in gene therapy A variety of difference vectors and delivery techniques have been applied in gene therapy trials (Figure 5). Although nonviral approaches are becoming increasingly common, viral vectors remain by far the most popular approach, having been used in approximately two-thirds of the trials performed to date.

Viral vectors Recent years have seen a decline in the use of retroviral vectors (currently, only 19.7% of trials compared to 22.8% in 2007 and 28% in 2004), presumably as a result of the severe adverse events observed in the SCID trials, and research is well underway towards engineering safer retroviral vectors. Adenoviruses are the most commonly used vector (23.3% of all trials). They can carry a larger DNA load than retroviruses, although their capacity is still too small to accommodate certain genes required for clinical applications. The main advantages of adenoviral vectors are their abilities to achieve a high efficiency of transduction, high levels of gene expression (though transient) and to infect nondividing cells. Other viruses have been less widely used and include vaccinia virus (7.9% of trials), poxvirus (5.0%), adeno-associated virus (4.9%) and herpes simplex virus (3.1%). The use of these vectors has increased significantly in recent years as

Safety concerns and the relatively small capacity for therapeutic DNA of viral vectors have prompted the development of synthetic vectors not based on viral systems. The simplest nonviral gene delivery system uses ‘naked’ DNA, which, when injected directly into certain tissues, particularly muscle, produces significant levels of gene expression, although lower than those achieved with viral vectors. The popularity of naked DNA has continued to increase (18.3% of trials compared to 18% in 2007 and 14% in 2004), and it is the most popular nonviral system used in clinical trials, followed by lipofection, which involves cationic lipid/DNA complexes (used in 5.9% of all trials). A small number of trials have used a range of modified bacteria (20 trials) or brewer’s yeast strains (seven trials).

Clinical trial phases As was the case in our last review, more than three quarters of gene therapy clinical trials performed to date are phase I or I/II (Figure 6); the two categories combined represent 78.6% of all gene therapy trials. Phase II trials make up 16.7% of the total, and phase II/III and III represent only 4.5% of all trials. The proportion of trials in phase II, II/III and III continues to grow over time (21.2% compared to 19.1% in 2007 and 15% in 2004), indicating the progress being made with respect to bringing gene therapy closer to clinical application.

Figure 5. Vectors used in gene therapy clinical trials. Copyright © 2013 John Wiley & Sons, Ltd.

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Figure 6. Phases of gene therapy clinical trials.

Future prospects Many gene therapy protocols in clinical or preclinical trials are showing great promise. Two notable examples include the treatment of haemophilia B and lipoprotein lipase deficiency in adults [65,66]. In both of these trials, however, only a transient clinical benefit was observed as a result of the immune responses directed against vector constituents, with resultant cell-mediated destruction of the gene-corrected cells in the liver and muscle, respectively. To prevent these unwanted immune responses, some protocols may require modulation of the immune system or transient immune suppression [67]. The results from a clinical trial for haemophilia B were presented by Amit Nathwani (University College London) at the 2010 American Society of Hematology (ASH) meeting [68] and also discussed at the 2011 American Society of Gene and Cell Therapy meeting held in Seattle [69], have now been published [70]. The study, performed in collaboration with Katherine High (Children’s Hospital of Philadelphia), Mark Kay (Stanford University) and Andrew Davidoff and Art Nienhuis (St Jude Children’s Research Hospital) utilised an improved rAAV transfer vector containing a liver-specific promoter and codon optimised human FIX gene. At present, a total of six patients have been treated at three different vector doses. Vector was delivered in the absence of immunosuppressive therapy and, at the time of publication, patients were monitored for between 6 and 16 months. AAV-mediated expression of FIX resulted in between 2% and 11% of normal levels in all patients. Furthermore, four of the six patients were able to discontinue FIX prophylaxis and remained free of spontaneous haemorrhage. For the other two patients, the time between prophylactic injections was increased. For the two patients receiving the high dose of vector, one had a transient elevation of serum aminotransferase levels with an associated detection of AAV8-specific T cells, and the other had a slight increase in liver-enzyme levels. Both patients were treated with a Copyright © 2013 John Wiley & Sons, Ltd.

short course of glucocorticoid therapy that rapidly returned aminotransferase levels to normal, without the loss of transgene expression. Although long-term follow-up is required in more patients, despite the risk of transient hepatic dysfunction, this approach has demonstrated the potential to convert the severe form of this disease into a milder form or to reverse it completely. Another strategy being considered is the use of regulated expression cassettes containing microRNA (miRNA) sequences. Inclusion of miRNA sequences targeting haematopoietic lineages to eliminate or reduce off-target gene expression in professional antigen presenting cells [71] has allowed the stable correction of a haemophilia B mouse model [72] and also been shown to induce antigen-specific immunologic tolerance [73]. The landmark discovery by Takahashi and Yamanaka [74] that somatic cells can be reprogrammed to a state of pluripotency through the ectopic expression of as little as four transcription factors [75,76] has the potential to be a powerful tool for both gene and cellular therapies and to revolutionise the field of regenerative medicine by developing patient-specific treatments. These cells, termed induced pluripotent stem cells (iPS), closely resemble embryonic stem (ES) cells in their morphology and growth properties, and have also been shown to express ES cell markers. Research in this field is still in its infancy and a number of important issues need to be resolved before these cells appear in the clinical setting. These include improvements in reprogramming efficiency, a more complete understanding of the development potential and quality of the iPS cells produced and the establishment of their safety profile in vivo, particularly with respect to tumour formation. Originally produced by retroviral-mediated delivery, refinements to the system using non-integrating vectors and transient expression systems [77–81] will also address safety concerns by eliminating unwanted long-term expression of the encoded transcription factors and the possibility of insertional mutagenesis. J Gene Med 2013; 15: 65–77. DOI: 10.1002/jgm


Proof-of-principle for combining somatic cell reprogramming with gene therapy for disease treatment already exists. For example, dopaminergic neurones derived from iPS cells have been shown to possess mature neuronal activity and, importantly, to improve behaviour in a rat model of Parkinson’s disease [82]. In another study, utilising a humanised mouse model of sickle cell anaemia, mice were rescued following transplantation with haematopoietic progenitors that were corrected by gene-specific targeting [83]. Gene-corrected iPS cells derived from Fanconi anaemia patients have also been differentiated into haematopoietic progenitors of the myeloid and erythroid lineages [84] and may be useful for overcoming the poor quality of HSCs found in the bone marrow of these patients, which is impeding success in the clinic. A human artificial chromosome, carrying a complete genomic dystrophin gene, has also been used to correct iPS cells derived from a murine model of Duchenne muscular dystrophy and from patient fibroblasts. These cells were able to form all three germ layers and human dystrophin expression could be detected in muscle-like tissues [85]. This approach overcomes one of the main obstacles hampering gene therapy for Duchenne muscular dystrophy, namely the unusually large size of the dystrophin gene that is beyond the packaging capacity of current viral vector systems. Another strategy showing promise for the treatment of Duchenne muscular dystrophy uses synthetic oligonucleotide-induced exon skipping to restore the reading frame of the protein [86]. This approach is currently being trialled [87,88]; however, it requires the use of patient mutation-specific oligonucleotides and repeated administration. Although many issues need to be resolved before we see the therapeutic use of

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iPS cells, they have immediate potential for basic research, disease modelling and drug screening, and hold immense promise for the future.

Concluding comments In summary, the overall trajectory of clinical trial activity in the field of gene therapy is positive, with increasing evidence of clinical benefits across a growing number of disease targets. The field has also reacted positively to the occurrence of vector-related adverse events and other lessons learnt from earlier gene therapy trials, including the challenges posed by the immune system. These lessons are now being accommodated in an iterative manner in ongoing trial activity, which is delivering further exciting progress. Although rigorous preclinical efficacy and safety testing remains a foundation stone of clinical trials, much of the knowledge required to drive further progress can only come from well designed and executed clinical trials.

Acknowledgements The authors wish to thank Margot Latham for her help in the preparation of the manuscript. SLG and IEA declare that they have no conflicts of interest. MLE and MRA receive payment from John Wiley and Sons Ltd for their work on The Journal of Gene Medicine Gene Therapy Clinical Trials Worldwide database, and JW is Managing Editor of The Journal of Gene Medicine and an employee of John Wiley and Sons Ltd.

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