review

Gene therapy for monogenic disorders of the bone marrow Sujal Ghosh,1,2 Adrian J. Thrasher1 and H. Bobby Gaspar1 1

Infection, Immunity, Inflammation and Physiological Medicine, Molecular and Cellular Immunology Section, University College London – Institute of Child Health, London, UK, and 2Department of Paediatric Oncology, Haematology and Clinical Immunology, Medical Faculty, Centre of Child and Adolescent Health, Heinrich-Heine-University, D€ usseldorf, Germany

Monogenic disorders of the bone marrow consist of a large number of diverse conditions, extending from bone marrow failure syndromes and several anaemic haemoglobinopathies to a broad range of more than 250 primary immunodeficiency syndromes (Al-Herz et al, 2014). Treatment strategies

are different in each disease and include anti-infective prophylaxis and transfusion of blood-derived products, such as immunoglobulins, erythrocytes and platelets (Hoernes et al, 2011; Chandrakasan & Kamat, 2013; Schmid et al, 2014). However, until now haematopoietic stem cell transplantation (HSCT) has been the only way to achieve permanent functional reconstitution. In nearly all conditions, other than certain forms of severe combined immunodeficiency (SCID), conditioning with cytoreductive chemotherapy is necessary and the best results are seen in patients with well-matched stem cell donors. Despite growing numbers of donors in haematopoietic stem cell (HSC) registries, some patients will not be able find a suitable donor and, in these cases, gene therapy using corrected autologous patient cells may offer a potentially safer and equally efficacious strategy. Starting in the early 1990s, a number of initial trials of HSC gene therapy were initiated in primary immunodeficiency conditions (Fig 1). These studies used gammaretroviral vectors to correct autologous HSCs by inserting a functional copy of the mutated gene into the host genome. Despite functional recovery of the patients’ haematological and immunological system, the success of these trials was clouded by the occurrence of leukaemia and myelodysplasia. In a number of patients this was caused by a process termed insertional mutagenesis, which related to the design of the gammaretroviral vectors used. Understanding the mechanism of insertional mutagenesis and also of the biology of retroviruses has led to a new generation of clinical trials using safer vector designs. These trials are showing considerable success, not only in primary immunodeficiencies but also in specific lysosomal storage diseases. The promising early reports emerging from trials of gene therapy for beta thalassaemia also suggest that autologous stem cell gene therapy may have considerable utility for the future treatment of these conditions.

Correspondence: Professor H. Bobby Gaspar, GOSHCC Professor of

Adenosine deaminase deficiency

Summary Ex-vivo gene transfer of autologous haematopoietic stem cells in patients with monogenic diseases of the bone marrow has emerged as a new therapeutic approach, mainly in patients lacking a suitable donor for transplant. The encouraging results of initial clinical trials of gene therapy for primary immunodeficiencies were tempered by the occurrence of genotoxicity in a number of patients. Over the last decade, safer viral vectors have been developed to overcome the risk of insertional mutagenesis and have led to impressive clinical outcomes with considerably improved safety. We review the efforts in specific immunodeficiencies including adenosine deaminase deficiency, X-linked severe combined immunodeficiency, chronic granulomatous disease and Wiskott Aldrich syndrome. Major recent progress has also been made in haemoglobinopathies, such as beta-thalassaemia, sickle cell disease and Fanconi anaemia, and also specific lysosomal storage diseases, which, although not strictly bone marrow specific conditions, have been effectively treated by bone marrow-based treatment. The success of these recent studies and the advent of new technologies, such as gene editing, suggest that gene therapy could become a more generally applied treatment modality for a number of haematopoietic disorders. Keywords: gene therapy, primary immunodeficiency, haemoglobinopathies, metabolic disorders.

Paediatrics and Immunology, Consultant in Paediatric Immunology, Infection, Immunity, Inflammation and Physiological Medicine, Molecular and Cellular Immunology Section, UCL Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK.

Adenosine deaminase (ADA) acts as an essential enzyme in the purine salvage pathway catalysing the deamination of the toxic metabolites into the deoxyinosine and inosine respectively. Mutations in ADA lead to the intra- and extracellular

E-mail: [email protected]

ª 2015 John Wiley & Sons Ltd British Journal of Haematology, 2015, 171, 155–170

First published online 5 June 2015 doi: 10.1111/bjh.13520

Review

Fig 1. Haematopoietic stem cell differentiation and currently investigated clinical and pre-clinical (in brackets) approaches in monogenic diseases. Mutations in genes causing severe and other combined immunodeficiencies affect the lymphoid compartment. Red blood cell disorders comprise b-thalassaemia and sickle cell disease. Although metabolic disorders do not affect haematopoietic differentiation directly, a therapeutic effect is mediated by replacement of microglia derived from the substituted myelomonocyte compartment. In CGD, patients neutrophil function is impaired, while platelets and lymphocytes are affected in WAS patients. HSC, haematopoietic stem cell; CMP, common myeloid progenitor; CLP, common lymphoid progenitor; T, T cell; B, B cell; NK, Natural Killer cell; ADA, Adenosine deaminase; SCID-X1, X-linked severe combined immunodeficiency; CGD, chronic granulomatous disease; WAS, Wiskott Aldrich Syndrome; PRF, perforin deficiency (PRF1); XLP, X-linked lymphoproliferative syndrome (SH2D1A); RAG, RAG deficiency (RAG1/2); Artemis, Artemis (DCLRE1C) protein deficiency (DCLRE1C); Fanconi, Fanconi Anaemia (FANC genes) SCD, sickle cell disease; b-Thal, b-thalassaemia; MLD, metachromatic leucodystrophy; X-ALD, X-linked Adrenoleucodystrophy; MPS-I, Mucopolysaccharidosis type I; MPS-III, Mucopolysaccharidosis type III; LAD, Leucocyte adhesion deficiency.

accumulation of deoxyadenosine and adenosine and through conversion by specific enzymes to the intracellular accumulation of deoxyadenosine triphosphate and adenosine triphosphate. The build up of these metabolites leads to diverse systemic defects, such as skeletal, gastrointestinal, pulmonary and neuronal abnormalities in ADA-deficient patients. However the most profound manifestations are seen in the immune system where the deleterious effects on the lymphocyte development cause a T-B-Natural Killer (NK)-SCID phenotype. Affected infants present early in the first few months of life with persistent severe infections and failure to thrive and will usually succumb without prompt intervention. Weekly or twice-weekly enzyme replacement therapy (ERT) with bovine ADA conjugated to polyethylene glycol (pegadamase bovine/Adagen or PEG-ADA) has been available in high-resource countries and demonstrates efficient detoxification and clinical improvement. However, ERT has limited efficacy in sustained improvement of immune parameters. HSCT had been considered as the only definite treat156

ment for a long time and a human leucocyte antigen (HLA)matched sibling or family donor (MFD) transplant has success rates of c. 90% without the need for cytoreductive conditioning. The transplantation of a matched-unrelateddonor (MUD) or haploidentical graft leads to far inferior results (1-year survival: MFD 90%, MUD 67%, haplo 43%) (Gaspar et al, 2009). Gene correction of autologous HSCs was attempted in the early 1990s with gammaretroviral vectors (Blaese et al, 1995; Bordignon et al, 1995; Onodera et al, 1998; Aiuti et al, 2002). A total of 19 patients received transduced peripheral blood lymphocytes or haematopoietic progenitor cells while continuing ERT. These patients did not show any toxicity and, surprisingly, gene-corrected T cells persisted for several years after infusion. Nevertheless, the lack of substantial immunological improvement and clinical benefit due to the limited engraftment of gene-modified cells led to significant changes in gene transfer protocols. Initial results suggested that concomitant use of PEG-ADA would not lead to a competitive advantage for transduced cells. Hence, subsequent clinical trials in Milan and London ª 2015 John Wiley & Sons Ltd British Journal of Haematology, 2015, 171, 155–170

Review involved the cessation of PEG-ADA prior to gene therapy (Aiuti et al, 2009; Gaspar et al, 2011a) and used a non-myeloablative reduced intensity conditioning regimen consisting of busulfan (4 mg/kg) or melphalan (140 mg/m2); both trials showed long-term correction. In a joint trial between the Children’s Hospital Los Angeles and the National Institutes of Health (NIH), the protocol was amended after treating four patients without conditioning. Implementation of a cytoreductive regimen and cessation of ERT prior to gene therapy improved the clinical and immunological outcome in a further 6 patients (Candotti et al, 2012). Notably, two children with ADA deficiency were treated in Japan in 2003/2004 by gammaretroviral mediated gene transfer to bone marrow CD34+ cells after ERT cessation but without conditioning regimen (Otsu et al, 2006). Although the patients slowly developed gene-corrected T cells, full immune recovery was not achieved during the follow-up period, underlining the need for conditioning as a determining factor for successful gene therapy. Most likely the cytoreduction of autologous ADA-deficient HSC favoured engraftment of the corrected HSC, which was further reflected in recent murine studies (Carbonaro et al, 2012). Interestingly, in contrast to the perception of the early Milan and London trials, continuation of ERT has shown significantly increased levels of gene-modified cells in the thymus, indicating that genemodified cells can develop even when subjects are on ERT. The studies described above in Milan, London and the USA treated over 40 patients with gammaretroviral vectors. All treated patients are alive and c. 75% of patients are off ERT. Transduced cells engrafted permanently and effective metabolic detoxification was observed. T cell reconstitution did not reach normal levels but in most patients cellular and humoral responses improved. The application of a mild nonmyeloablative regimen also led to significantly improved Band NK-lymphocyte and myeloid cell counts. Notably, none of these patients, although treated with a gammaretroviral vector, driving ADA expression from the 50 viral long terminal repeat (LTR), developed insertional mutagenesis, as seen in X-linked severe combined immunodeficiency (SCID-X1), chronic granulomatous disease (CGD) and Wiskott Aldrich Syndrome (WAS) patients (see below). Nevertheless, integration events near proto-oncogenes, including LMO2, have been reported in all trials, which led some groups to develop potentially safer vector designs. To address these safety requirements, two lentiviral (LV) vector designs have been published. A self-inactivating (SIN)LV (deletion of U3 in LTR), driving ADA expression from the phosphoglycerate kinase 1 (PGK1, PGK) promoter, was able to rescue ADA-deficient mice in a pre-clinical study (Mortellaro et al, 2006). The new generation of clinical trials in the USA and UK involves a codon-optimized human cDNA ADA gene under the control of the short form elongation factor-1 alpha (EEF1A1, EF1a) promoter in a SINconfigured lentivirus (LV), which also showed efficacy in the mouse model (Carbonaro et al, 2014). Further modifications ª 2015 John Wiley & Sons Ltd British Journal of Haematology, 2015, 171, 155–170

involve a Woodchuck Hepatitis Virus post-transcriptional regulatory element (WPRE) to enhance the expression in the lentiviral setting. In vitro immortalization assays showed a reduced transformation potential of these new lentiviral compared to gammaretroviral vectors. Thus far, five patients aged between 12 and 45 years have been treated with busulfan (at a single dose of 5 mg/kg) and this vector. The procedure was well tolerated by all patients. At a mean follow-up of 361 d (range: 152–599), there has been significant immunological recovery. All patients have shown a rise in total T cell counts from a mean of 0204 9 109 cells/l pre-gene therapy to a mean of 094 9 109 cells/l at the latest follow-up. Integration site analysis shows some expansions but no persistence of expanded clones, furthermore there were no clones with genes previously associated with insertional mutagenesis (Gaspar et al, unpublished observations).

X-linked severe combined immunodeficiency SCID-X1 is caused by mutations in the IL2RG gene, leading to expression of the common gamma chain, a key subunit of the cytokine receptor complex for interleukin (IL)2, IL4, IL7, IL9, IL15 and IL21 (Noguchi et al, 1993; Kovanen & Leonard, 2004). Mutations lead classically to the absence of T and NK cell development and compromised B cell function. As in most other SCID forms, allogeneic transplantation from an HLA-identical donor is highly successful (Gennery et al, 2010; Pai et al, 2014), although some patients have persistent defects in humoral or cellular functions. The rare observation of patients with spontaneous reversion of the mutation and subsequent correction of immune deficiency (Stephan et al, 1996; Speckmann et al, 2008) supports the concept that even a very few number of ‘naturally’corrected wild type cells have a selective advantage over the mutated lymphocytes and therefore led to SCID-X1 being a candidate disease for gene therapy. After successful preclinical murine in vivo studies, Cavazzana-Calvo et al, initiated the first gene therapy trial for SCID-X1 in 1999 (Hacein-Bey-Abina et al, 2002). A retroviral vector derived from a defective Moloney murine leukaemia virus (Mo-MLV) was applied, in which IL2RG expression was driven by the viral LTR (Hacein-Bey-Abina et al, 2002, 2010). Subsequently, a similar study was initiated at Great Ormond Street Hospital (GOSH), London employing a similar Mo-MLV-based vector (Gaspar et al, 2011b). A total of 20 patients, lacking an HLA-identical donor and suffering from SCID-associated morbidities, were enrolled and treated with ex-vivo transduced CD34+ cells without any conditioning regime. Immune reconstitution was impressive in terms of T cell numbers and function. In most patients, ongoing, long-term thymopoiesis was demonstrated by the detection of T-cell receptor excision circles and a diverse Tcell receptor Vb repertoire. The follow-up evidence demonstrates that the population of transduced progenitors is capable of long lived, probably permament T cell recovery. 157

Review However, as expected from data in HSCT patients, restoration of NK cells was only transient and suggests that the corrected population is not capable of long-term NK cell renewal. Similarly, functional B cell reconstitution was limited with a minority of patients able to stop immunoglobulin replacement therapy. This is most likely explained by the absence of a conditioning regimen and the lack of engraftment of gene-modified B cells. A further five older patients, carrying hypomorphic IL2RG mutations [including three from a 2003 initiated trial at the NIH (Chinen et al, 2007)], were treated with a similar protocol. Despite effective transduction, functional reconstitution was not achieved in these patients, most probably due to loss of thymic function by the time of gene therapy. The promising results in the London and Paris studies were subsequently tempered by the occurrence of genotoxicity (Deichmann et al, 2007; Hacein-Bey-Abina et al, 2008; Howe et al, 2008). Five patients developed acute T cell leukaemia, four of whom entered remission after standard chemotherapy. Another patient died despite an allogeneic HSCT due to refractory leukaemia. Investigation of the leukaemic clones revealed the mechanism of insertional mutagenesis. In all the leukaemias there was viral integration within oncogenes, namely LMO2 (four cases). Enhancer activity of the viral LTR was most likely to cause initial aberrant expression of these oncongenes and dysregulated cell cycling which, with the accumulation of other genetic abnormalities, such as associated rearrangements in BMI1, CCND2 and NOTCH1 led to frank leukaemic transformation. The severe adverse events seen in these trials and also in trials of gene therapy for CGD (see below) led to extensive scientific focus in many different groups to develop newer and safer vectors that would minimize the effects of genotoxicity but still allow effective immunological reconstitution. A new generation of SIN vectors were developed in which the

viral LTRs were deleted and the transgene transcribed by an internal mammalian promoter. SIN-configured gammaretroviral, lentiviral and other vectors have now been studied in a variety of in vitro and in vivo models and it is now well proven that, by deleting the viral LTR, the risk of insertional mutagenesis through enhancer activation of the neighbouring genes is significantly reduced (Fig 2). Thornhill et al (2008) generated a SIN gammaretroviral vector under the control of an internal EEF1A1/EF1a promoter (pSRS11.EFS.IL2RG.pre) (Thornhill et al, 2008), in which the Mo-MLV U3 LTR enhancer was deleted. Following demonstration of efficacy and safety, this vector has been employed in a multi-centre clinical trial involving centres in Boston, Cincinatti, London, Los Angeles and Paris. The outcome of nine boys has been reported recently (Hacein-BeyAbina et al, 2014; Touzot et al, 2015). Conditioning was not given to most patients, but two patients received fludarabine or anti-thymocyte gobulin due to a large number of maternally engrafted T cells or maternal graft-versus-host disease (GvHD). One patient unfortunately died from fatal pre-existing adenoviral infection before gene-corrected T cells could reconstitute. The remaining eight patients were followed up for 121–387 months. One patient did not show any gene marking and was successfully transplanted with a mismatched cord-blood graft. The other 7 patients showed similar T cell reconstitution as in the previous studies using LTR-driven vectors; one patient needed a second infusion due to low absolute T cell numbers. The absence of conditioning in these studies also led to minimal gene marking in B cell and myeloid lineages and all patients remain on immunoglobulin replacement therapy. Patients have seen a significant reduction in infection frequency and are free of isolation and social restriction measures. In terms of genotoxicity, there was a significant decrease in the clustering of viral integrations around previously implicated oncogenes such as LMO2.

Fig 2. Comparison between non-SIN, SIN–gammaretroviral and SIN lentiviral vectors driving expression of the IL2RG gene. (Top) This original gammaretroviral vector design was applied in the first SCID-X1 trials. Viral transcription is driven by the gammaretroviral LTR [promoter of the Moloney Murine Leukemia Virus (MoMLV)]. (Middle) SIN gammaretroviral vectors encoding IL2RG for a recent transatlantic consortium trial use a SRS11 vector backbone. The SIN vectors lack the full-length LTR on both ends of the vector, with deleted trancriptional and enhancer activity. Transgene expression is driven by the short form of the human elongation factor 1-a promoter (EEF1A1/EF1a). A post-transcriptional regulatory element is introduced to enhance titres and gene expression. (Bottom) The CL20i4-EF1a-hccOPT vector is based on a lentiviral vector backbone and encodes a codon-optimized version of the IL2RG cDNA, also driven by a human EEF1A1/EF1a. Further regulatory elements: cPPT, central polypurine tract; RRE, Rev Response Element; LTR, long terminal repeat; WPRE, Woodchuck Hepatitis Virus post-transcriptional regulatory element.

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Review However, as expected from data in HSCT patients, restoration of NK cells was only transient and suggests that the corrected population is not capable of long-term NK cell renewal. Similarly, functional B cell reconstitution was limited with a minority of patients able to stop immunoglobulin replacement therapy. This is most likely explained by the absence of a conditioning regimen and the lack of engraftment of gene-modified B cells. A further five older patients, carrying hypomorphic IL2RG mutations [including three from a 2003 initiated trial at the NIH (Chinen et al, 2007)], were treated with a similar protocol. Despite effective transduction, functional reconstitution was not achieved in these patients, most probably due to loss of thymic function by the time of gene therapy. The promising results in the London and Paris studies were subsequently tempered by the occurrence of genotoxicity (Deichmann et al, 2007; Hacein-Bey-Abina et al, 2008; Howe et al, 2008). Five patients developed acute T cell leukaemia, four of whom entered remission after standard chemotherapy. Another patient died despite an allogeneic HSCT due to refractory leukaemia. Investigation of the leukaemic clones revealed the mechanism of insertional mutagenesis. In all the leukaemias there was viral integration within oncogenes, namely LMO2 (four cases). Enhancer activity of the viral LTR was most likely to cause initial aberrant expression of these oncongenes and dysregulated cell cycling which, with the accumulation of other genetic abnormalities, such as associated rearrangements in BMI1, CCND2 and NOTCH1 led to frank leukaemic transformation. The severe adverse events seen in these trials and also in trials of gene therapy for CGD (see below) led to extensive scientific focus in many different groups to develop newer and safer vectors that would minimize the effects of genotoxicity but still allow effective immunological reconstitution. A new generation of SIN vectors were developed in which the

viral LTRs were deleted and the transgene transcribed by an internal mammalian promoter. SIN-configured gammaretroviral, lentiviral and other vectors have now been studied in a variety of in vitro and in vivo models and it is now well proven that, by deleting the viral LTR, the risk of insertional mutagenesis through enhancer activation of the neighbouring genes is significantly reduced (Fig 2). Thornhill et al (2008) generated a SIN gammaretroviral vector under the control of an internal EEF1A1/EF1a promoter (pSRS11.EFS.IL2RG.pre) (Thornhill et al, 2008), in which the Mo-MLV U3 LTR enhancer was deleted. Following demonstration of efficacy and safety, this vector has been employed in a multi-centre clinical trial involving centres in Boston, Cincinatti, London, Los Angeles and Paris. The outcome of nine boys has been reported recently (Hacein-BeyAbina et al, 2014; Touzot et al, 2015). Conditioning was not given to most patients, but two patients received fludarabine or anti-thymocyte gobulin due to a large number of maternally engrafted T cells or maternal graft-versus-host disease (GvHD). One patient unfortunately died from fatal pre-existing adenoviral infection before gene-corrected T cells could reconstitute. The remaining eight patients were followed up for 121–387 months. One patient did not show any gene marking and was successfully transplanted with a mismatched cord-blood graft. The other 7 patients showed similar T cell reconstitution as in the previous studies using LTR-driven vectors; one patient needed a second infusion due to low absolute T cell numbers. The absence of conditioning in these studies also led to minimal gene marking in B cell and myeloid lineages and all patients remain on immunoglobulin replacement therapy. Patients have seen a significant reduction in infection frequency and are free of isolation and social restriction measures. In terms of genotoxicity, there was a significant decrease in the clustering of viral integrations around previously implicated oncogenes such as LMO2.

Fig 2. Comparison between non-SIN, SIN–gammaretroviral and SIN lentiviral vectors driving expression of the IL2RG gene. (Top) This original gammaretroviral vector design was applied in the first SCID-X1 trials. Viral transcription is driven by the gammaretroviral LTR [promoter of the Moloney Murine Leukemia Virus (MoMLV)]. (Middle) SIN gammaretroviral vectors encoding IL2RG for a recent transatlantic consortium trial use a SRS11 vector backbone. The SIN vectors lack the full-length LTR on both ends of the vector, with deleted trancriptional and enhancer activity. Transgene expression is driven by the short form of the human elongation factor 1-a promoter (EEF1A1/EF1a). A post-transcriptional regulatory element is introduced to enhance titres and gene expression. (Bottom) The CL20i4-EF1a-hccOPT vector is based on a lentiviral vector backbone and encodes a codon-optimized version of the IL2RG cDNA, also driven by a human EEF1A1/EF1a. Further regulatory elements: cPPT, central polypurine tract; RRE, Rev Response Element; LTR, long terminal repeat; WPRE, Woodchuck Hepatitis Virus post-transcriptional regulatory element.

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Review gene marked cells. The loss was caused most probably due to transgene silencing after a series of epigenetic events. Both adult patients died due to complications in the context of MDS (Ott et al, 2006; Stein et al, 2010; Aiuti et al, 2012). Two children were treated in Zurich with the same SFFV vector and also developed clonal expansion, one with further development to MDS. Both patients are still alive after HSCT (Bianchi et al, 2009, 2011). To overcome genotoxicity, further clinical trials are planned using SIN lentiviral vectors and a fully myeloablative conditioning regime prior to gene therapy (12–16 mg/kg busulfan). Notably, the groups in London and Frankfurt have also developed a lentiviral vector, in which gp91phox is driven by a synthetic chimeric promoter, created by the fusion of Cathepsin G and c-Fes minimal 50 flanking regions. In vitro and murine in vivo studies could show that this vector is myeloid-specific, restores NADPH oxidase activity and has a reduced potential regarding insertional mutagenesis (Santilli et al, 2011). This vector is currently to be employed in multicentre trials in Europe and in the USA (NCT01906541, NCT01855685, NCT02234934).

Wiskott Aldrich Syndrome Wiskott Aldrich Syndrome is an X-linked inherited disorder caused by mutations in the WAS gene, classically leading to the triad of microthrombocytopenia, immune dysfunction and eczema. WAS protein constitutes a main actin cytoskeleton regulator protein and mutations affecting WAS protein expression may lead to recurrent infections, immune dysregulation and malignancies (Thrasher, 2009; Blundell et al, 2010; Massaad et al, 2013). Interestingly hypomorphic mutations lead to intermediate forms as X-linked thrombocytopenia (XLT) and gain-of-function mutations have been associated with neutropenia (Thrasher & Burns, 2010). HSCT in a matched-donor setting is the curative option in the majority of patients. Murine in-vivo repopulation experiments, as well as the human subphenotype of XLT with a low-level expression of WAS protein, show a selective survival advantage of wild-type cells indicating that transfer of transduced cells may lead to functional correction of the disease phenotype (Moratto et al, 2011). The first clinical trial was conducted in Hannover in 2006 and included 10 patients with no HLA identical donor available. Conditioning included a reduced intensity regimen of Busulfan and transduction of CD34+ peripheral blood or bone marrow cells was performed by an LTR intact gammaretroviral vector. 9 of 10 showed restored gene expression in the myeloid and lymphoid compartment with increased platelet numbers and normalized T cell count and function and NK and B cell function. Clinical susceptibility to infections, bleeding tendency and autoimmune phenomena, impressively decreased in the first years (Boztug et al, 2010). These results were overshadowed by the development of acute leukaemia in 7 of 9 reconstituted patients and further investigations showed dominant clones with integration in 160

the LMO2 (six cases of acute T-cell acute lymphoblastic leukaemia), MECOM (also termed MDS1/EVI1) (two cases of acute myeloid leukaemia [AML]) and MN1 (one case of AML) oncogenes (Braun et al, 2014). Several groups have since developed SIN-LV vectors in which different promoters drive WAS protein expression in various model systems (Dupre et al, 2006; Marangoni et al, 2009; Avedillo Diez et al, 2011; Bosticardo et al, 2011). A lentiviral vector consisting of the endogenous 16 kb human WAS promoter has shown promising results in several preclinical studies in terms of efficacy and long-term safety by the Milan group (Bosticardo et al, 2011). This vector is now in clinical trials in Boston, London, Milan and Paris. The short-term results (18 months follow-up) in the Milan study in 4 patients have shown stable and effective engraftment of transduced cells (Aiuti et al, 2013; Castiello et al, 2015) with demonstrable WAS protein expression restored in the myeloid and lymphoid compartment. In contrast, high-throughput analysis of the integration events did not reveal insertions in oncogenic target sites, but a longer observation period will be essential to demonstrate fully the superior safety of these SIN LV. The results of 7 patients treated in Paris and London have been recently published. 6 of 7 show an improved clinical outcome, however one patient unfortunately died seven months after treatment due to preexistent drug-resistant herpes virus infection (Hacein-Bey Abina S et al, 2015). Further primary immunodeficiencies have shown promising results for a gene therapy approach. SAP (signalling lymphocyte activation molecule-associated protein) deficiency, resulting from mutations in SH2D1A, is classically associated with Epstein–Barr virus (EBV)-driven haemophagocytic lymphohistiocytosis (HLH) and EBV-driven lymphoproliferation, while mutation in the PRF1 gene (Perforin deficiency) is usually is restricted to infection-associated HLH. Both diseases reveal reduced cytotoxicity in the NK cell, the latter also in the CD8 compartment. Recent studies have shown the successful transfer of gene-corrected HSCs into Sh2d1a/  and Prf1/ mouse models with subsequent immunological reconstitution (Rivat et al, 2013; Carmo et al, 2015).

Lysosomal storage disorders In recent years, gene therapy of HSCs has also proven its efficacy in metabolic diseases, notably lysosomal storage disorders. Dysfunctional lysosomal transport proteins and degrading enzymes, and subsequent accumulation of various undegraded lipids, glycoproteins and mucopolysaccharides lead to neurological and musculoskeletal degeneration (Parenti et al, 2015). In contrast to primary immunodeficiencies the biggest challenge is the sufficient delivery of the corrected stem cells and protein beyond the blood–brain-barrier, hence other strategies, such as the application of adeno-associated virus and intracerebroventricular administration of corrected cells, have been evaluated. ª 2015 John Wiley & Sons Ltd British Journal of Haematology, 2015, 171, 155–170

Review In recent years, lentiviral gene therapy of HSCs has been clinically investigated in two leucodystrophies. X-linked Adrenoleucodystrophy (X-ALD) is caused by a defective peroxisomal transporter (ABCD) followed by the accumulation of very long chain fatty acids, notably in the adrenal cortex and white matter of central nervous system. Affected boys suffer from progressive cerebelar demyelination leading to a vegetative state and death. A total of 4 boys have been treated with a replication-defective HIV-1-derived lentiviral vector expressing the transgene under the control of an internal MND (myeloproliferative sarcoma virus enhancer) promotor. Transduced CD34+ cells were administered after a fully myeloablative conditioning regimen with cyclophosphamide and busulfan, as a survival advantage of transduced over nontransduced cells was not expected in patients with X-ALD. In the first two treated patients reported (Cartier et al, 2009), an arrest of progressive demyelination was seen between 14 and 16 months, which correlates with the time course in patients after HSCT. The therapeutic effect of HSCs is most probably mediated by the replacement of microglia derived from the substituted myelomonocyte compartment. A longterm engraftment, with only 10% corrected cells, seems to be sufficient to arrest disease progression. High-throughput sequencing integration site analysis did not reveal clonal dominance of any proto-oncogene. A modified version of this vector is further investigated in an international trial under the sponsorship of bluebird bio, Cambridge, MA, USA (‘Lenti D’) (Cartier et al, 2009, 2012) – NCT01896102/EudraCT 2011-001953-10. In patients suffering from metachromatic leucodystrophy (MLD), lack of arylsulfatase A (ARSA) leads to the accumulation of sulfatide in oligodendrocytes, microglia and many other cells in the central and peripheral nervous system and subsequently to early and progressive demyelination and neurodegeneration. HSCT has not been successfully applied in MLD patients, however in addition to the murine model, it seems that overexpression of the ARSA gene is absolutely necessary to arrest widespread degeneration. Nine patients have been treated with lentiviral transduced (PGK1 promotor) HSCs after a myeloablative regimen with busulfan. The outcomes of the first 3 presymptomatic treated patients were recently reported (Biffi et al, 2013). While one patient shows a much milder physiological and cognitive impairment compared to his older siblings, who suffered also from MLD, the two other recruited patients are completely asymptomatic (18 months after treatment, 7–10 months after predicted time of symptom onset). Stable high gene marking levels (VCN 09–19 in patients bone marrow) ensured reconstitution of above-normal ARSA activity in the relevant myeloid compartment (Biffi et al, 2013). Various other lysosomal storage disorders are under investigation. Preclinical studies in Mucopolysaccharidosis type I (MPS-I), MPS-IIIa and Pompe disease (Glycogen storage disease II) have successfully investigated the efficacy of lentiviral gene correction of HSCs in the murine model with promisª 2015 John Wiley & Sons Ltd British Journal of Haematology, 2015, 171, 155–170

ing results that set the scene for future clinical trials (van Til et al, 2010; Visigalli et al, 2010; Langford-Smith et al, 2012).

Haemoglobinopathies In contrast to the primary immunodeficiency diseases, haemoglobin disorders are far more frequent among the worldwide population. More than 300 000 infants are born with beta thalassaemia and sickle cell anaemia (SCD) every year (Modell & Darlison, 2008). In both disorders the beta chain of the haemoglobin (b-globin) is affected. Patients with beta thalassaemia carry various mutations affecting different steps of the expression (initiation of transcription to posttranslational modification) leading to either decreased (b1) or absent beta globin production (b0). Haemolytic microcytic anaemia and subsequent blood transfusions lead to iron overload, ultimately resulting in hepatic, cardiac and endocrine complications, depending on the nature of mutation and disease phenotype. In SCD patients a single point mutation leads to the aberrant HbS form, causing different acute ‘sickling’ and chronic complications, e.g. vaso-occlusion, ischaemia (stroke), spleen sequestration and acute chest syndrome (Chandrakasan & Kamat, 2013; Kanter & Kruse-Jarres, 2013; Porter & Garbowski, 2013; Brousse et al, 2014; Weiss, 2014). HSCT is the only long-term curative option in both disorders, with a favourable outcome of more than 90% in a donor-matched setting (Angelucci et al, 2014). However, 10% are at risk of conditioning toxicity, GvHD and other HSCT-associated complications in a disease setting that does not require immediate correction. Furthermore, more than 50% of patients do not have a matched donor. Considerable effort to develop an effective gene transfer model has been made in the last 30 years. Several challenges have to be considered in haemoglobinopathies. Firstly, the large transgene cargo of the globin gene and its regulatory elements may hinder high-level expression. Additionally, the selective transduction of erythroid lineage cells is a necessity. Interestingly, two approaches are being investigated for gene therapy. It is known that increased levels of fetal haemoglobin (HbF; gamma globin) ameliorates the phenotype of both SCD and beta thalassaemia. Therefore, besides adding a correct version of the dysfunctional beta globin gene (HBB), attempts to increase HbF expression have been investigated (Dong et al, 2013; Chandrakasan & Malik, 2014). Elements upstream of HBB, known as the locus control region (LCR), which confer high level expression of b-globin in the erythroid lineage (Tuan & London, 1984) have been used in the construction of vectors. Vector development has focussed on a LV with HBB (May et al, 2000; Puthenveetil et al, 2004), with a mutation leading to anti-sickling properties (A-T87Q) (Pawliuk et al, 2001), and a LV adding the gamma globin gene (HBG1/2) (Pestina et al, 2009; Wilber et al, 2011). Furthermore, murine data suggest that targeting regulators of HBG1/2 (e.g. BCL11A) to allow increased gamma globin expression can also correct the disease phenotype. 161

Review gene marked cells. The loss was caused most probably due to transgene silencing after a series of epigenetic events. Both adult patients died due to complications in the context of MDS (Ott et al, 2006; Stein et al, 2010; Aiuti et al, 2012). Two children were treated in Zurich with the same SFFV vector and also developed clonal expansion, one with further development to MDS. Both patients are still alive after HSCT (Bianchi et al, 2009, 2011). To overcome genotoxicity, further clinical trials are planned using SIN lentiviral vectors and a fully myeloablative conditioning regime prior to gene therapy (12–16 mg/kg busulfan). Notably, the groups in London and Frankfurt have also developed a lentiviral vector, in which gp91phox is driven by a synthetic chimeric promoter, created by the fusion of Cathepsin G and c-Fes minimal 50 flanking regions. In vitro and murine in vivo studies could show that this vector is myeloid-specific, restores NADPH oxidase activity and has a reduced potential regarding insertional mutagenesis (Santilli et al, 2011). This vector is currently to be employed in multicentre trials in Europe and in the USA (NCT01906541, NCT01855685, NCT02234934).

Wiskott Aldrich Syndrome Wiskott Aldrich Syndrome is an X-linked inherited disorder caused by mutations in the WAS gene, classically leading to the triad of microthrombocytopenia, immune dysfunction and eczema. WAS protein constitutes a main actin cytoskeleton regulator protein and mutations affecting WAS protein expression may lead to recurrent infections, immune dysregulation and malignancies (Thrasher, 2009; Blundell et al, 2010; Massaad et al, 2013). Interestingly hypomorphic mutations lead to intermediate forms as X-linked thrombocytopenia (XLT) and gain-of-function mutations have been associated with neutropenia (Thrasher & Burns, 2010). HSCT in a matched-donor setting is the curative option in the majority of patients. Murine in-vivo repopulation experiments, as well as the human subphenotype of XLT with a low-level expression of WAS protein, show a selective survival advantage of wild-type cells indicating that transfer of transduced cells may lead to functional correction of the disease phenotype (Moratto et al, 2011). The first clinical trial was conducted in Hannover in 2006 and included 10 patients with no HLA identical donor available. Conditioning included a reduced intensity regimen of Busulfan and transduction of CD34+ peripheral blood or bone marrow cells was performed by an LTR intact gammaretroviral vector. 9 of 10 showed restored gene expression in the myeloid and lymphoid compartment with increased platelet numbers and normalized T cell count and function and NK and B cell function. Clinical susceptibility to infections, bleeding tendency and autoimmune phenomena, impressively decreased in the first years (Boztug et al, 2010). These results were overshadowed by the development of acute leukaemia in 7 of 9 reconstituted patients and further investigations showed dominant clones with integration in 160

the LMO2 (six cases of acute T-cell acute lymphoblastic leukaemia), MECOM (also termed MDS1/EVI1) (two cases of acute myeloid leukaemia [AML]) and MN1 (one case of AML) oncogenes (Braun et al, 2014). Several groups have since developed SIN-LV vectors in which different promoters drive WAS protein expression in various model systems (Dupre et al, 2006; Marangoni et al, 2009; Avedillo Diez et al, 2011; Bosticardo et al, 2011). A lentiviral vector consisting of the endogenous 16 kb human WAS promoter has shown promising results in several preclinical studies in terms of efficacy and long-term safety by the Milan group (Bosticardo et al, 2011). This vector is now in clinical trials in Boston, London, Milan and Paris. The short-term results (18 months follow-up) in the Milan study in 4 patients have shown stable and effective engraftment of transduced cells (Aiuti et al, 2013; Castiello et al, 2015) with demonstrable WAS protein expression restored in the myeloid and lymphoid compartment. In contrast, high-throughput analysis of the integration events did not reveal insertions in oncogenic target sites, but a longer observation period will be essential to demonstrate fully the superior safety of these SIN LV. The results of 7 patients treated in Paris and London have been recently published. 6 of 7 show an improved clinical outcome, however one patient unfortunately died seven months after treatment due to preexistent drug-resistant herpes virus infection (Hacein-Bey Abina S et al, 2015). Further primary immunodeficiencies have shown promising results for a gene therapy approach. SAP (signalling lymphocyte activation molecule-associated protein) deficiency, resulting from mutations in SH2D1A, is classically associated with Epstein–Barr virus (EBV)-driven haemophagocytic lymphohistiocytosis (HLH) and EBV-driven lymphoproliferation, while mutation in the PRF1 gene (Perforin deficiency) is usually is restricted to infection-associated HLH. Both diseases reveal reduced cytotoxicity in the NK cell, the latter also in the CD8 compartment. Recent studies have shown the successful transfer of gene-corrected HSCs into Sh2d1a/  and Prf1/ mouse models with subsequent immunological reconstitution (Rivat et al, 2013; Carmo et al, 2015).

Lysosomal storage disorders In recent years, gene therapy of HSCs has also proven its efficacy in metabolic diseases, notably lysosomal storage disorders. Dysfunctional lysosomal transport proteins and degrading enzymes, and subsequent accumulation of various undegraded lipids, glycoproteins and mucopolysaccharides lead to neurological and musculoskeletal degeneration (Parenti et al, 2015). In contrast to primary immunodeficiencies the biggest challenge is the sufficient delivery of the corrected stem cells and protein beyond the blood–brain-barrier, hence other strategies, such as the application of adeno-associated virus and intracerebroventricular administration of corrected cells, have been evaluated. ª 2015 John Wiley & Sons Ltd British Journal of Haematology, 2015, 171, 155–170

Los Angeles Cincinatti Charleston Paris

SCD

ª 2015 John Wiley & Sons Ltd British Journal of Haematology, 2015, 171, 155–170 9

4

NCT01560182

NCT01896102

NCT01331018 EudraCT 2011-001953-10

2010

2013

2012 2013

2012

2013

2014 2014 2014 2013

Trial start

LV

LV

LV

LV LV

LV

LV

LV LV LV LV

Viral vector (8 mg/kg) (8 mg/kg) (8 mg/kg) (RIC/MAC)

None Busulfan, Cyclophosphamide (MAC) Busulfan, Cyclophosphamide (MAC) Busulfan, Cyclophosphamide (MAC) Busulfan (MAC)

Busulfan (8 mg/kg) Busulfan (128 mg/kg)

Busulfan (RIC/MAC)

Busulfan Busulfan Busulfan Busulfan

Conditioning

Biffi et al (2013)

Cartier et al (2009)

Cavazzana-Calvo et al (2010, unpublished observations)

Cavazzana-Calvo et al (unpublished observations)

Published results

Numbers of current studies include unpublished observations. R, recruiting or not yet recruiting; ADA, Adenosine deaminase; SCID-X1, X-linked severe combined immunodeficiency; CGD, chronic granulomatous disease; WAS, Wiskott Aldrich Syndrome; SCD, sickle cell disease; B-Thal, b-thalassaemia; FA, Fanconi Anaemia; X-ALD, X-linked Adrenoleucodystrophy; MLD, metachromatic leucodystrophy; LV, lentivirus; RV, retroviral; SIN, self-inactivating; ERT, enzyme replacement therapy; C, completed/terminated/not recruiting any longer; RIC, reduced intensity conditioning (busulfan 8 mg/kg); MAC, myeloablative conditioning with targeted area under curve (AUC) dosing – 14–16 mg/kg busulfan, 120–200 mg/kg cyclosphosphamide.

R

C

Paris

Milan

R

Boston, Los Angeles, Minneapolis

MLD

R R

Seattle Paris, London

FA X-ALD

NCT01639690 4

R C

NCT02247843 NCT02186418 NCT02140554 NCT02151526

Trial number

NCT01745120

2

Patients (n)

R

Chicago, Oakland, Philadelphia, Sydney New York Paris

R R R R

Status

B-Thal

B-Thal/SCD

Centre

Disease

Table I. (Continued)

Review

163

Review The successful clinical correction of beta thalassaemia was first reported in 2010 (Cavazzana-Calvo et al, 2010). A SIN lentiviral vector with two copies of the cHS4 chromatin insulator in the U3 region and encoding the mutated adult beta globin with anti-sickling properties (A-T87Q) achieved transfusion independence in one adult patient with severe bE/b0thalassemia that has been maintained for more than 7 years now. Myeloablative conditioning (busulfan) was given prior to gene transfer. Only one-third of the total haemoglobin levels were therapeutically corrected, indicating that the endogenous production has been a necessity for success in this patient. The level of nucleated blood cells containing the integrated vector stabilized at 11% (187% in the myeloid compartment). Furthermore, about 10% of bone marrow erythroblasts were marked. In terms of safety, 24 chromosomal integration sites were found; one of the sites caused transcriptional activation of HMGA2 conferring clonal dominance, however the clone stabilized 5 years post-gene therapy. This vector was further refined and is now under the sponsorship of bluebird bio as LentiGlobin BB305, utilized in different trials in SCD and beta thalassemia in centres worldwide (see Table I). Cavazzana-Calvo presented preliminary data of the first two patients at the 19th Annual Congress of the European Haematology Association; both patients with beta-thalassemia major and the bE/b0 genotype, treated with the new vector, remained blood transfusion independent after 45 and 2 months, respectively, post-gene therapy. Integration site analysis did not show any dominant clone in Patient 1; data for Patient 2 was not available (unpublished observations). The Memorial Sloan-Kettering Cancer Center group published their experience with an alternative lentiviral vector encoding HBB, which was shown to be effective in different preclinical models after non-myeloablative conditioning (Sadelain et al, 2010; Boulad et al, 2014). They have shown that, after efficient transduction of adult CD34+ HSCs, these were able to engraft in NSG mice. A clinical trial for patients with beta thalassaemia over 18 years is currently open (Sadelain et al, 2010; Boulad et al, 2014). The group at the University of California, Los Angeles (UCLA) has shown effective transduction of human CD34+ cells with a LV carrying the recombinant ‘anti-sickling’ beta globin transgene HBBAS3 (Romero et al, 2013). This vector will be applied in the current UCLA study for adult patients with SCD. The Cincinatti group will focus more on correcting the disease phenotype by applying a LV targeting the gamma globin in patients older than 5 years (Perumbeti et al, 2009).

Fanconi anaemia Fanconi anaemia (FA) is an inherited bone marrow failure syndrome with an incidence of 1 per 350 000 births. A defect in 1 of at least 16 DNA repair genes leads to aplasia and enhanced risk for malignancies, especially AML and MDS. Additionally, the risk for adenoma, adenocarcinomas and 164

squamous cell carcinomas is increased. Most patients also have a short stature, various morphological abnormalities and developmental disorders. Supportive treatment includes regular transfusions of blood products and growth hormone substitution due to concomitant endocrinopathies in FA patients (Smith & Wagner, 2012). HSCT in the donor-matched setting has been the only curative option. Despite the heterogeneity in genes affected, more than 60% of the patients have mutations in the FANCA gene. An international working group has been established to assemble the worldwide experience with gene therapy in FA patients (Tolar et al, 2011). FA patients differ from patients with most primary immunodeficiency diseases and haemoglobinopathies by having a low number of HSCs as a result of bone marrow hypoplasia and chemosensitivity to myelosuppressive regimens because of the underlying abnormalities in DNA repair. Several protocols have been investigated to harvest HSCs (Kelly et al, 2007; Tolar et al, 2011, 2012). However, the first study targeting the FANCA gene was initiated in Seattle to employ a LV in which the FANCA gene is driven by an internal human PGK1 promoter and stabilized by an optimized WPRE. Given the chemosensitivity, the initial phase will not use any conditioning until it is evident that LV corrected stem cells are safe to administer. Besides numerous studies investigating different viral vectors to transduce haematopoietic progenitors, given the low number of haematopoietic precursors in the bone marrow of FA patients, strategies also focus on generating induced pluripotent stem cells (iPSC). The successful application of artificial zinc finger nucleases in fibroblasts from FA patients has been reported recently (Rio et al, 2014). The generation of disease-free iPSCs and subsequent disease-free haematopoietic progenitors might be a new strategy for further clinical trials (Rio et al, 2014).

Gene editing Current and past clinical trials of gene therapy rely on viral delivery and addition of a functional copy of the defective gene. Over the past decades target-specific nuclease enzymes have been investigated to follow a strategy of targeted insertion of a functional copy and correction of the dysfunctional gene. Engineered nuclease enzymes comprise Zinc-finger nuleases (ZFN), transcriptor activator-like effector nucleases (TALENs) and clustered regulatory interspaced short palindromic repeats CRISPR-associated (CRISPR/Cas) based RNA-guided DNA endonucleases. These nucleases catalyse a site-specific double-strand break and the addition of a target gene/sequence by means of homologous recombination, which allows the integration of the correct gene as its endogenous locus or genomic safe harbour site (Gaj et al, 2013) (Fig 3). Usual adverse effects of lenti- or gammaretrovirusmediated gene addition as random insertion and potential insertional mutagenesis are therefore avoided. Furthermore, addition of the corrected gene transcript adjacent to its endogenous promotor should lead to physiological gene ª 2015 John Wiley & Sons Ltd British Journal of Haematology, 2015, 171, 155–170

Review

ZFN

TALEN

CRISPR/Cas

Fig 3. Gene editing tools. Zing-finger nucleases (ZFN), transcriptor activator-like effector nucleases (TALEN) and clustered regulatory interspaced short palindromic repeats CRISPR-associated (CRISPR/Cas) generate specific double-strand breaks and initiate the cell’s own repair mechanism. The addition of a donor template faciliates precise homology directed repair to integrate or modify the desired sequence, while error-prone nonhomologous end joining might lead to small deletions or insertions and mutate or truncate the encoded protein.

expression and regulation. The group of Naldini in San Raffaele, Milan was able to correct the IL2RG gene with zinc finger nucleases (Genovese et al, 2014). They inserted a cDNA comprising exons 5–8 of IL2RG together with a GFP cassette into the IL2RG gene of CD34+ cells from healthy male donors. Transplantation into NSG mice proved the long-term multilineage repopulation capacity of the targeted cells. Finally, based on these results, this strategy was applied on bone marrow CD34+ cells from a symptomatic 4-monthold SCID-X1 patient bearing a missense mutation in the IL2RG gene (exon 7) showing efficient gene correction. However the therapeutic impact of this promising approach still needs to be shown. Gene-targeting approaches have been recently published, applying ZFN, TALEN and CRISPR/Cas9 technology to iPSCs from beta thalassaemic, sickle cell and CGD patients (Sun & Zhao, 2014; Xie et al, 2014).

Conclusion The latest wave of gene therapy trials now employs safer vectors and favours engraftment by the addition of myeloablaª 2015 John Wiley & Sons Ltd British Journal of Haematology, 2015, 171, 155–170

tive and non-myeloablative conditioning regimes. Mortality in the application of the gammaretroviral vectors was mainly associated with the occurrence of insertional mutagenesis and subsequent malignancies. Current vectors are designed in a SIN configuration, which lack the LTR in U3, thereby avoiding enhancer-mediated activation and can employ different internal promoters to drive transgene expression. Incorporation of further elements, as the ubiquitous chromatin opening element or chromatin insulators elements lead to enhanced vector safety. Although the short-term results are encouraging in terms of both safety and efficacy, only longterm clinical and molecular monitoring will show whether lentiviral-mediated gene transfer keeps its promises. Given numerous further genetic defects in primary immunodeficiencies, preclinical studies are focussing on DCLRE1C (Artemis), RAG1/2, SHD2D1A (SAP), Perforin, UNC13D (Munc13-4), CD40LG, FOXP3, BTK, BLNK and Leucocyte adhesion deficiency. Furthermore, gene editing, in contrast to the current applied gene-adding approach, will certainly lead to new perspectives in the treatment of these severely affected patients. 165

Review

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