Major Advances in the Development of Vectors for Clinical Gene Therapy of Hematopoietic Stem Cells from European Groups over the Last 25 Years

Abstract

The first attempts at hematopoietic stem cell–based gene therapy (HSC-GT) were reported >25 years ago for primary immune deficiencies, marking the beginning of a vibrant field of translational and therapeutic research. Since then, many HSC-GT studies have been conducted in diverse genetic diseases. The approach has been improved over time, showing biological and therapeutic efficacy with an overall excellent safety record. Within a defined regulatory and ethical landscape, the field of HSC-GT has reached industrialization and commercialization stages, with a landmark recent approval by the European Medicines Agency of the first HSC-GT medicine for human use. At such a pivotal stage, it is important to look back at 25 years of European applied research in this field. This review highlights some of the key contributions of European teams to the field of HSC-GT, focusing in particular on the development of safer gene transfer vectors and international cooperation.

Introduction

Hematopoietic stem cell gene therapy (HSC-GT) is a niche specialty that was developed to treat genetic diseases in lieu of allogeneic HSC transplantation. HSC-GT consists in administering to patients their own genetically modified autologous hematopoietic stem and progenitor cells to achieve long-lasting expression of a therapeutic transgene in cells of hematopoietic origin. European researchers have embraced this concept with enthusiasm and for >25 years have translated it to the clinic in several model diseases, starting with severe primary immunodeficiencies,¹ extending it to neurodegenerative storage diseases,² and eventually to globin disorders.^3,4 This was made possible by the availability of performant gene transfer vectors. Over the past 30 years, non-replicative recombinant gene transfer vector systems have been engineered from various members of the Retroviridae family to achieve long-term expression of a transgene in the hematopoietic system.⁵ For clinical applications, essentially two main gene transfer vector systems have been employed: gamma-retroviral vectors (gRV), mostly derived from Moloney murine leukemia virus (MLV), and lentiviral vectors (LVs), derived from human immunodeficiency virus type 1 (HIV-1). While gRV were first used and have permitted remarkable pioneering advances in the field, this vector system has been replaced by LVs in all new trials for HSC-GT because of superior efficacy, safety, and pharmaceutical quality. Many European teams have played a key role in advancing the field of HSC-GT through preclinical studies, technology and analytical development, clinical studies, and industrialization. Many of these activities were enabled by a long-standing support of the European Commission, promoting cooperation and exchange between teams beyond national barriers at all stages throughout the development of gene therapy products.⁶ Currently, the field has reached an industrialization and commercialization stage, with the approval of the first gene and cell therapy drug and many more ongoing clinical trials that provide a rich future perspective.

Advances Made Possible by gRVs

First successes in primary immunodeficiencies

The first attempts at HSC-GT were made with gRV to treat severe combined immunodeficiency diseases (SCID).¹ The first attempt at HSC-GT in man was reported by an Italian team in 1995, aimed at treating adenosine deaminase (ADA)-deficient SCID. A gRV was used to express the ADA gene into the lymphocytes and bone marrow cells of two patients with ADA-SCID with scarce access to enzyme replacement therapy and destined to a fatal evolution.⁷ While encouraging, this study did not obtain very high levels of engraftment of gene-marked cells, prompting changes in the protocol. To increase HSC marking, the small, HSC-containing CD34+ cell fraction was used instead of whole mononuclear cells from the bone marrow. This was made possible by the development and commercialization of a clinically approved CD34 immuno-selection device by the German company Miltenyi. Cytoreductive conditioning of the patients was found to be critical to promote the engraftment of gene-modified cells with multi-lineage differentiation potential. Finally, a selective pressure was applied to gene-corrected cells by the cessation of ADA enzyme replacement therapy. This new protocol successfully restored cellular and humoral immunity and detoxified ADA-SCID patients, improving their clinical condition.⁸ This positive outcome and the key role of marrow conditioning for ADA-SCID were also reported in other centers in London and in the United States using different gRV.^9,10

At about the same time, HSC-GT with a gRV was attempted to treat another form of SCID: the X-linked SCID due to IL2Rg deficiency (SCID-X1). Successful treatment of this disease by gene therapy was first reported in 2000 by a French team, providing the first evidence that gene therapy could restore biological function in humans. Results were confirmed 2 years later, showing sustained marking and restoration of normal lymphocyte counts and T cell immunity in SCID-X1 patients.^11,12 In SCID-X1, contrary to ADA-SCID, prior conditioning was not required to obtain engraftment of marked cells due to the strong selective advantage gained by lymphoid progenitor cells and T cells expressing the IL2Rg transgene. Altogether, these first clinical successes of gene therapy in ADA-SCID and in SCID-X1 patients in Paris, Milan, and later London and the United States¹ not only provided the basic methodology for HSC-GT but were also the first proofs worldwide that genetically corrected cells could treat a human disease.

Insertional mutagenesis

Since these historical beginnings, gRV were used to treat at least two other primary immunodeficiencies in the early 2000s—Wiskott–Aldrich syndrome (WAS) and X-linked chronic granulomatous disease (X-CGD)—which have a broader clinical presentation and a more complex pathophysiology compared to SCIDs.¹ Whereas gRV have worked safely in >40 ADA-SCID patients over a long-term follow-up, complications emerged in SCID-X1, X-CGD, and WAS patients. In the case of SCID-X1, >30 children have been treated with gRV in Europe and the United States with therapeutic benefit since the early 2000s. Yet, five of these children have reportedly developed acute T cell leukemia at various time points following gene therapy. The use of the linear amplification mediated polymerase chain reaction technique developed by a German team was instrumental to show that clonal expansions of gene-marked cells had occurred in these patients, driven by the mutagenic insertion of the gRV in the genome and the trans-activation of proto-oncogenes such as LMO2 or CCND2. Secondary mutagenic events eventually resulted in overt leukemic transformation.^13,14 While four SCID-X1 patients remain in remission, one unfortunately died. In the case of X-CGD, many attempts at using a gRV-based HSC-GT remained unsuccessful until patient conditioning was introduced to engraft gene-corrected HSC, as previously shown for ADA-SCID.¹⁵ After myeloablative conditioning, several X-CGD patients experienced detectable gRV gene marking in their neutrophils, but this was not sustained, as three distinct problems were encountered. First, there was a lack of engraftment of gene-marked HSC. Therefore, neutrophil marking did not last. Second, clonal expansion occurred in four X-CGD patients caused by gRV-mediated transactivation of MECOM (MDS1/EVI1 complex locus) and PRDM1 in the myeloid cells. Third, the correction of neutrophil function waned over time because the gRV was silenced through methylation of the LTR promoter. In Frankfurt, two adult X-CGD patients developed myelodysplasia and eventually died from complications. In Zurich, two X-CGD children were rescued by successful allogeneic HSC transplantation.^16,17 In the case of WAS, 10 children were treated by gene therapy in Hannover using a gRV, but almost all of them developed complications: six patients experienced acute T cell leukemia, and three more developed acute myeloid leukemia (one primary and two secondary).^18,19 In the Hannover WAS trial, genomic integrations were found in lymphoid and myeloid proto-oncogenes, including LMO2, CCND2, and MDS/EVI1. So altogether, it became very clear that apart from ADA-SCID (probably for disease-related reasons), the use of gRV in HSC could cause insertional mutagenesis and leukemia, despite providing initial therapeutic benefit.

Such unexpected side effects had not been predicted by preclinical models, and this sparked new interest in defining the mechanisms of genotoxicity and insertional mutagenesis caused by gRV. The transformation potential of gRV was found to be directly related to the vector design, which used the strong viral promoter/enhancer elements in the LTR to express the transgene.⁵ Through these active LTRs, the gRV provirus become capable of transactivating neighboring genes, leading to insertional deregulation of proto-oncogenes, clonal expansion, and eventually secondary transformation of highly proliferative hematopoietic cells. Such oncogenic potential is probably the heritage of a strategy developed by MLV to enhance the spreading of its viral genome through clonal expansion. These oncogenic events can therefore be abrogated in self-inactivated (SIN) vectors in which the enhancer function of the LTR is disabled. Through cooperative efforts between European and U.S. teams within a transatlantic gene therapy consortium, second-phase SCID-X1 trials were launched in Paris, London, and Boston using a SIN-gRV expressing the IL2Rg gene from the short EF1-alpha cellular promoter. So far, these studies have obtained satisfactory results, since most patients have recovered blood T cells and display significantly less clustering of insertion sites within LMO2, MECOM, and other lymphoid proto-oncogenes than in SCID-X1 patients treated with the first-generation gRV vector.²⁰

An important challenge for gene therapy with integrating retroviral vectors has been to develop better assays capable of predicting more precisely insertional adverse events, ideally both in cell culture assays and in small animal models, to allow risk–benefit assessment of gene therapy. Genotoxicity assays were developed in Hannover, Milan, and London that could read out the transforming potential of gRV but not SIN-gRV on murine cells either in vitro or in vivo. ^21
–23 These assays preferentially detect myeloid transformation potential but not lymphoid expansion. Nevertheless, demonstrating that candidate vectors do not read out in such genotoxicity assays contributes to the favorable risk–benefit evaluation of a proposed gene therapy. Technology was also developed to monitor the occurrence of clonal expansion in patients using next-generation sequencing techniques. Proficient genomic laboratories and bioinformatic platforms were developed in Heidelberg, Milan, and Philadelphia to monitor the clonal diversity of gene-marked cells in patients treated by gRV vectors in HSC-GT trials.

While the use of the SIN design is real progress for gRV, it does not change the overall genomic integration pattern of these vectors. It was recently identified that MLV insertion is preferentially directed by BET chromatin-binding proteins according to the epigenetic status of chromatin, landing the provirus preferentially into acetylated, active transcriptional control elements of the host cell genome.²⁴ Thus, to overcome the inherent risk of insertional oncogenesis linked to such insertional preference, active research is ongoing to redirect gRV integrase to regions of chromatin less likely to dysregulate nearby genes.²⁵ As a pragmatic alternative, using vectors with different integration profile such as those derived from alpha-retroviruses or lentiviruses is also a solution. At present, lentiviral vectors derived from the human immunodeficiency lentivirus HIV-1 display an inherently different and safer integration profile than gRV,²⁶ and have replaced worldwide gRV vectors in clinical trials involving genetically modified HSPCs.

LVs for HSC Gene Transfer

Advantages of the LV system

Before the safety problems of gRV became evident in HSC-based clinical trials, other limitations of gRV had already been identified, including their inability to transduce quiescent cells, frequent silencing of their LTRs, and their intolerance to certain intronic sequences, for instance those needed to express large globin genes. This prompted the development of alternative gene transfer vectors for HSCs. HIV-1 had several desirable properties that could be exploited in that regard. HIV-1 can infect non-dividing cells, suggesting that the system could be used to transduce quiescent primitive and pluripotent HSC. The HIV-1 virus has evolved to protect its genome by remaining dormant in the host cell to avoid immune responses, predicting that gene transfer with such vectors could be stable and well tolerated by host cells in the long term. A recent review by the pioneers who have developed this system recapitulates how, by progressively paring down the virulent sequences of the native HIV-1 viral genome, substituting for heterologous viral sequences and using split genome cis-production systems, it was possible to generate non-replicative HIV-1-derived LVs that could be produced at reasonably high titer to enable preclinical and clinical studies.²⁷ At present, the most advanced generation of HIV-1-derived LVs do not code for any HIV-1 or viral gene sequence, have a SIN design with exhaustive deletions in the LTRs that further reduce the likelihood of replication and pathogenicity from the vector. Such LVs are very versatile tools that can be fitted with various internal promoters, including polIII promoters to express short hairpin RNAs, and can incorporate various viral, cellular, or artificial sequences such as microRNAs or chromatin insulator sequences to regulate transgene expression. LVs have a good cargo capacity and tolerance to complex inserts, enabling for instance the design of efficient LV globin vectors that can express reasonably high and potentially therapeutic levels of hemoglobin chains in the erythroid lineage.^28

–32 The cellular tropism of LVs can be modulated by pseudotyping with various viral envelope glycoproteins or artificial molecules targeted with antibody fragments or DARPIN sequences to confer very precise cellular tropism.³³ Yet, the most commonly used envelope glycoprotein for LVs is the VSVg glycoprotein which enables LV entry into a broad cellular population comprising primitive CD34+ HSC. VSVg also permits the production of robust viral particles, capable of withstanding large-scale production and purification schemes that are compatible for clinical-grade manufacture in compliance with good manufacturing practice (GMP) standards.³⁴ Thus, very active “vectorology” research has taken place throughout Europe these past 20 years to design and adapt various features of LVs for diverse applications of HSC-GT.

Fundamentally, LVs utilize distinct cellular and molecular mechanisms than gRV to enter into the host cell nucleus and to integrate into chromosomes, and this improves the efficiency and safety of the system. Recently, it was found that LV integrations are mostly located in the outer portion of the nucleus in close proximity to the nuclear pore, which is coherent with the notion that different components of the nuclear pore complex play a key role in the HIV-1 life cycle.³⁵ As the HIV-1 pre-integration complex can enter the nucleus in the absence of cell division, LV transduction protocols can be much shorter than those for gRV, which require cell division for nuclear entry and therefore last several days. Typically, to prepare for LV transduction, the target CD34+ cells are pre-activated for a day or less by culturing cells in the presence of cytokines, enabling quiescent cells to enter into the G1 phase of the cell cycle. Such pre-activation period also upregulates the levels of the LDL receptor that is the VSVg receptor, thereby enhancing attachment and transduction by VSVg-pseudotyped LVs.³⁶ Short ex vivo transduction protocols better preserve HSC engraftment and hematopoietic differentiation potency. LEDGF/p75 is the major tethering factor bridging the HIV-1 pre-integration complex to the host cell chromatin, as it directly binds to the body of active genes through histone H3 trimethyl lysine 36 (H3K36me3) chromatin marks. Such a mechanism generates a distinct integration profile for LVs compared to gRV. LV insertions disfavor active promoters and enhancers but are distributed throughout transcription units, as revealed by a high definition mapping following transduction of CD34+ cells.³⁷ As a consequence, LVs appear to be less prone to deregulate genes by enhancer elements, but their propensity to integrate into introns may increase the probability of deregulating post-transcriptional mechanisms such as splicing or polyadenylation. Indeed, the insertional mutagenesis potential of LVs was found to be much reduced compared to LTR-driven gRV in transformation assays.^26,38,39

Current clinical studies using HSC-GT

The efficiency of HSC transduction, the good safety profile of LVs, and the capacity for production of pharmaceutical-grade LVs for clinical use have promoted the use of this vector system in clinical applications. LVs are now used in all active HSC-GT clinical protocols, as shown in Table 1, and have replaced gRV in many indications such as SCID-X1, ADA-SCID, WAS, X-CGD, and also Fanconi anemia A, a HSC disease in which gRV are unable to rescue any function.⁴⁰ LVs enable efficient CD34+ HSC transduction and are particularly useful to achieve sustained myeloid, erythroid, or multi-lineage gene marking in patients, even in the absence of selective advantage. At present, >100 patients have been treated with LVs for HSC-GT without side effects. Successful therapeutic results with sustained gene marking from LVs have been reported in the case of neurodegenerative diseases such as adrenoleukodystrophy⁴¹ or metachromatic leukodystrophy,⁴² in landmark studies in France and Italy. This domain was recently reviewed,² showing that the engraftment of high numbers of highly transduced cells was key to obtain myeloid engraftment and to the success of gene therapy for these pathologies. Similar observations were made in the case of WAS.^43,44 Promising results have already been reported in various scientific meetings for the use of LVs to treat ADA-SCID and X-CGD in ongoing trials. LVs have also enabled the design of effective globin vectors. The first demonstrations of therapeutic efficacy in beta-thalassemia⁴⁵ and sickle cell disease⁴⁶ patients have recently been reported, and more efforts are ongoing to adapt the clinical protocols to these diseases. Because LVs permit the transduction of true long-term repopulating human HSCs, as well as more differentiated hematopoietic progenitor cells, LV trials have generated remarkable observations on the dynamics of human hematopoiesis in vivo through the longitudinal tracking of vector insertion in clonal cell populations and cell subsets.⁴⁷

Table 1.

Current, active gene therapy HSC-GT trials (July 2017)

Disease	Autologous CD34+ hematopoietic stem cells transduced with	Centers	ClinicalTrials.gov Identifier
Adenosine deaminase (ADA) severe combined immunodeficiency (SCID)	EF1αS-ADA/VSVg lentiviral vector	London, United Kingdom	NCT01380990
X-linked chronic granulomatous disease	Chim-CYBB/VSVg lentiviral vector transduced patient CD34+ cells	Multicentric, Europe	NCT01855685
Wiskott–Aldrich syndrome (WAS)	WAS promoter WAS/VSVg lentiviral vector	Paris, France London, United Kingdom	NCT01347346 NCT01347242
Fanconi anemia A	PGK-FANCA/VSVg lentiviral vector	Madrid, Spain	NCT03157804
Severe sickle cell disease	LentiGlobin BB305 lentiviral vector encoding the human βA-T87Q-globin gene	Multicentric, United States	NCT02140554
Transfusion-dependent thalassemia	GLOBE (beta-globin/VSVG) lentiviral vector	Milan, Italy	NCT02453477
Transfusion-dependent beta-thalassemia	LentiGlobin BB305 lentiviral vector encoding the human βA-T87Q-globin gene	Multicentric, worldwide	NCT02906202
Cerebral adrenoleukodystrophy	Lenti-D lentiviral vector	Multicentric, Europe and the United States	NCT01896102
Adrenoleukodystrophy or metachromatic leukodystrophy	Lentiviral vector non-specified	Shenzhen, China	NCT02559830

In all the trials, the tolerance to LV treatment has been very good, and no adverse events have been reported linked to a LV. This is particularly striking in the case of WAS gene therapy, since >20 patients have been treated with LVs without side effects with >6 years of follow-up, contrasting sharply with the high percentage of leukemia induced by the gRV in this disease.¹⁹ This does not mean that LVs cannot have genotoxic potential. Clonal dominance has been reported in a patient treated for beta-thalassemia, which was caused by the LV insertion that generated a truncated HMGA2 gene transcript. This over-expressed transcript caused benign erythroid expansion, without loss of hematopoietic homeostasis and contributed to the therapeutic benefit for the patient.^45,48 Thus, careful molecular monitoring remains necessary in patients treated with LVs. Indeed, LVs have the propensity to integrate inside genes and to induce alternative splicing and aberrant transcripts.⁴⁹ However, overall, the efficacy and safety record of LV is excellent, and this vector system has enabled major advancements in HSC-GT.

EU Cooperation in the Field of HSC-GT

For >20 years, the European Union has supported the development of HSC-GT at all stages, ranging from exploratory research, proof of concept and preclinical studies, technological or industrial developments, and clinical trials.⁶ Thanks to the funding of cooperative research programs such as Consert (FP6), Persist (Fp7) CellPID (FP7), and large networks such as Clinigene (FP7), it was possible for European scientists, SMEs, and some experts in regulatory affairs and ethics to explore cooperatively the mechanisms of vector insertional mutagenesis; to develop and evaluate new vectors, tools, and assays; and to develop the biotechnology capacity to support the clinical and industrial demands for HSC-GT. These EU-funded projects supported the initial work, which has led to the registration of the first cell and gene therapy product, Strimvelis, to treat ADA-SCID. They also laid the groundwork that prepared the emergence of LVs by supporting preclinical studies for most of the currently ongoing clinical trials. Many more indications of HSC-GT were initiated in these programs, including Gaucher disease, osteopetrosis, and pyruvate kinase deficiency, which may also materialize into approved clinical studies in the near future. In addition, these programs have supported much innovation, since they have funded for more than a decade the attempts to replace the current gene transfer/gene addition approach by more precise site-directed gene editing technologies. In the last 5 years, the EU has focused on the performance of clinical trials for rare diseases and encouraged partnerships between academia and SMEs. In the field of HSC-GT, this concerns projects such as Net4CGD (FP7), EuroFancolen (FP7), and SCIDNet (H2020), which are dedicated to the development of new orphan drugs through the conduct of LV clinical trials for the gene therapy of X-CGD, Fanconi anemia type A, and various SCID diseases, respectively. The research strategy from the EU aims at satisfying the huge demand for advanced therapy medicinal products for patients suffering from deadly rare diseases and who can now hope to have a gene-based treatment.

Emergence of New Gene Therapy Drugs and the Challenge for Pharmaceutical Industry

The regulatory landscape for HSC-GT is well defined at the European level. The capacity for pharmaceutical production of LVs for clinical use has been developed in several organizations in Europe and in the United States. These pharmaceutical production units generate high-quality and well-defined LVs for use in humans. For ex vivo applications such as for HSC-GT, LVs are considered as a component used to manufacture the cell and gene therapy product, that is, the transduced HSC of the patient.

There are still very few approved gene therapy drugs, and prior to last year, only one—Glybera—was registered at the European Medicine Agency (EMA), and none were registered in the United States. The encouraging results in ADA-SCID gene therapy enabled the SR-Telethon Institute of Gene Therapy in Milan to develop this product further toward registration through multiple partnerships. Based on data collected on 18 ADA-SCID children successfully treated by HSC-GT and followed over long periods of time, the pharmaceutical company GSK obtained last year the registration of the product by the EMA under the name of Strimvelis.⁵⁰ This represents the first approval, worldwide, for a gene and cell therapy product. Thus, 25 years have been necessary to develop such a product all the way to registration. The approval process of Strimvelis has delineated a critical path from proof of concept to registered drug. This experience will be useful to advance similar products to registration. Yet, the challenges are not over. The conditions under which Strimvelis will be commercialized remain to be seen, considering the recent announcement that GSK might be re-evaluate its strategy in the rare diseases field. Because HSC-GT generally applies to rare and ultra-rare diseases without much commercial prospect, the field may be facing its biggest challenge today, having developed therapies that are effective but too expensive, too complex, or too risky to be commercialized according to the current regulatory and industrial framework. At present, the majority of the ongoing clinical trials in HSC-GT are still conducted by academic, nonprofit entities. Active partnerships between academics and industry still seems to be the model of choice to develop HSC-GT.⁵¹ Yet, there is no doubt that future efforts will be needed to reduce the complexity and costs of HSC-GT. At the industrial level, efforts are already ongoing to develop scaled-up and automated vector production systems that would increase capacity and reduce GMP manufacturing costs. The processes for pricing and reimbursement of future gene therapy drugs that might be approved is also very novel and may need time to evolve. Policies are in place in some countries to contain the rising costs of new drugs, and this may indirectly affect the perception that gene therapy is costly.⁵² In that respect, it will be important to evaluate correctly the value of gene therapy drugs, which, like HSC-GT, are given in one single administration for a potential lifetime benefit. Real-life full-costs economic studies are now needed to compare approaches of HSC-GT with standard treatment or no treatment.

In conclusion, remarkable progress has been made in the field of HSC-GT over these last 25 years and European groups have significantly contributed to these advances. Consistently driven by the pursuit of scientific quality and through cooperation, the field has reached a rewarding stage where many patients with genetic diseases can hope to have access to a treatment in the not too distant future.

Footnotes

Acknowledgments

The author is very grateful to Fulvio Mavilio for improvements made by critical reading of the manuscript. The author would like to apologize in advance for the inevitable omission of contributors to the field.

Author Disclosure

The author has no conflict of interest.

References

Booth

, Gaspar

, Thrasher

. Treating immunodeficiency through HSC gene therapy. Trends Mol Med, 2016; 22:317–327.

Biffi

. Hematopoietic stem cell gene therapy for storage disease: current and new indications. Mol Ther, 2017; 25:1155–1162.

de Dreuzy

, Bhukhai

, Leboulch

, et al. Current and future alternative therapies for beta-thalassemia major. Biomed J, 2016; 39:24–38.

Drakopoulou

, Papanikolaou

, Georgomanoli

, et al. Towards more successful gene therapy clinical trials for beta-thalassemia. Curr Mol Med, 2013; 13:1314–1330.

Maetzig

, Galla

, Baum

, et al. Gammaretroviral vectors: biology, technology and application. Viruses, 2011; 3:677–713.

Gancberg

. Twenty years of European Union support to gene therapy and gene transfer. Hum Gene Ther, 2017; 28:951–953.

Bordignon

, Notarangelo

, Nobili

, et al. Gene therapy in peripheral blood lymphocytes and bone marrow for ADA-immunodeficient patients. Science, 1995; 270:470–475.

Aiuti

, Slavin

, Aker

, et al. Correction of ADA-SCID by stem cell gene therapy combined with nonmyeloablative conditioning. Science, 2002; 296:2410–2413.

Gaspar

, Bjorkegren

, Parsley

, et al. Successful reconstitution of immunity in ADA-SCID by stem cell gene therapy following cessation of PEG-ADA and use of mild preconditioning. Mol Ther, 2006; 14:505–513.

10.

Candotti

, Shaw

, Muul

, et al. Gene therapy for adenosine deaminase-deficient severe combined immune deficiency: clinical comparison of retroviral vectors and treatment plans. Blood, 2012; 120:3635–3646.

11.

Cavazzana-Calvo

, Hacein-Bey

, de Saint Basile

, et al. Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease. Science, 2000; 288:669–672.

12.

Hacein-Bey-Abina

, Le Deist

, Carlier

, et al. Sustained correction of X-linked severe combined immunodeficiency by ex vivo gene therapy. New Engl J Med, 2002; 346:1185–1193.

13.

Hacein-Bey-Abina

, Von Kalle

, Schmidt

, et al. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science, 2003; 302:415–419.

14.

Howe

, Mansour

, Schwarzwaelder

, et al. Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis following gene therapy of SCID-X1 patients. J Clin Invest, 2008; 118:3143–3150.

15.

Grez

, Reichenbach

, Schwable

, et al. Gene therapy of chronic granulomatous disease: the engraftment dilemma. Mol Ther, 2011; 19:28–35.

16.

Ott

, Schmidt

, Schwarzwaelder

, et al. Correction of X-linked chronic granulomatous disease by gene therapy, augmented by insertional activation of MDS1-EVI1, PRDM16 or SETBP1. Nat Med, 2006; 12:401–409.

17.

Stein

, Ott

, Schultze-Strasser

, et al. Genomic instability and myelodysplasia with monosomy 7 consequent to EVI1 activation after gene therapy for chronic granulomatous disease. Nat Med, 2010; 16:198–204.

18.

Boztug

, Schmidt

, Schwarzer

, et al. Stem-cell gene therapy for the Wiskott–Aldrich syndrome. New Engl J Med, 2010; 363:1918–1927.

19.

Braun

, Boztug

, Paruzynski

, et al. Gene therapy for Wiskott–Aldrich syndrome—long-term efficacy and genotoxicity. Sci Transl Med, 2014; 6:227ra233.

20.

Hacein-Bey-Abina

, Pai

, Gaspar

, et al. A modified gamma-retrovirus vector for X-linked severe combined immunodeficiency. New Engl J Med, 2014; 371:1407–1417.

21.

Modlich

, Bohne

, Schmidt

, et al. Cell-culture assays reveal the importance of retroviral vector design for insertional genotoxicity. Blood, 2006; 108:2545–2553.

22.

Montini

, Cesana

, Schmidt

, et al. Hematopoietic stem cell gene transfer in a tumor-prone mouse model uncovers low genotoxicity of lentiviral vector integration. Nat Biotechnol, 2006; 24:687–696.

23.

Knight

, Collins

, Takeuchi

. Insertional mutagenesis by retroviral vectors: current concepts and methods of analysis. Curr Gene Ther, 2013; 13:211–227.

24.

Debyser

, Christ

, De Rijck

, et al. Host factors for retroviral integration site selection. Trends Biochem Sci, 2015; 40:108–116.

25.

El Ashkar

, Van Looveren

, Schenk

, et al. Engineering next-generation BET-independent MLV vectors for safer gene therapy. Mol Ther Nucleic Acids, 2017; 7:231–245.

26.

Cavazza

, Moiani

, Mavilio

. Mechanisms of retroviral integration and mutagenesis. Hum Gene Ther, 2013; 24:119–131.

27.

Naldini

, Trono

, Verma

. Lentiviral vectors, two decades later. Science, 2016; 353:1101–1102.

28.

Papanikolaou

, Georgomanoli

, Stamateris

, et al. The new self-inactivating lentiviral vector for thalassemia gene therapy combining two HPFH activating elements corrects human thalassemic hematopoietic stem cells. Hum Gene Ther, 2012; 23:15–31.

29.

Roselli

, Mezzadra

, Frittoli

, et al. Correction of beta-thalassemia major by gene transfer in haematopoietic progenitors of pediatric patients. EMBO Mol Med, 2010; 2:315–328.

30.

Imren

, Fabry

, Westerman

, et al. High-level beta-globin expression and preferred intragenic integration after lentiviral transduction of human cord blood stem cells. J Clin Invest, 2004; 114:953–962.

31.

Arumugam

, Scholes

, Perelman

, et al. Improved human beta-globin expression from self-inactivating lentiviral vectors carrying the chicken hypersensitive site-4 (cHS4) insulator element. Mol Ther, 2007; 15:1863–1871.

32.

Rivella

, May

, Chadburn

, et al. A novel murine model of Cooley anemia and its rescue by lentiviral-mediated human beta-globin gene transfer. Blood, 2003; 101:2932–2939.

33.

Buchholz

, Friedel

, Buning

. Surface-engineered viral vectors for selective and cell type-specific gene delivery. Trends Biotechnol, 2015; 33:777–790.

34.

Merten

, Charrier

, Laroudie

, et al. Large-scale manufacture and characterization of a lentiviral vector produced for clinical ex vivo gene therapy application. Hum Gene Ther, 2011; 22:343–356.

35.

Marini

, Kertesz-Farkas

, Ali

, et al. Nuclear architecture dictates HIV-1 integration site selection. Nature, 2015; 521:227–231.

36.

Amirache

, Levy

, Costa

, et al. Mystery solved: VSV-G-LVs do not allow efficient gene transfer into unstimulated T cells, B cells, and HSCs because they lack the LDL receptor. Blood, 2014; 123:1422–1424.

37.

Cattoglio

, Pellin

, Rizzi

, et al. High-definition mapping of retroviral integration sites identifies active regulatory elements in human multipotent hematopoietic progenitors. Blood, 2010; 116:5507–5517.

38.

Modlich

, Navarro

, Zychlinski

, et al. Insertional transformation of hematopoietic cells by self-inactivating lentiviral and gammaretroviral vectors. Mol Ther, 2009; 17:1919–1928.

39.

Montini

, Cesana

, Schmidt

, et al. The genotoxic potential of retroviral vectors is strongly modulated by vector design and integration site selection in a mouse model of HSC gene therapy. J Clin Invest, 2009; 119:964–975.

40.

Adair

, Sevilla

, de Heredia

, et al. Lessons learned from two decades of clinical trial experience in gene therapy for Fanconi anemia. Curr Gene Ther, 2017 Jan 19 [Epub ahead of print].

41.

Cartier

, Hacein-Bey-Abina

, Bartholomae

, et al. Hematopoietic stem cell gene therapy with a lentiviral vector in X-linked adrenoleukodystrophy. Science, 2009; 326:818–823.

42.

Biffi

, Montini

, Lorioli

, et al. Lentiviral hematopoietic stem cell gene therapy benefits metachromatic leukodystrophy. Science, 2013; 341:1233158.

43.

Hacein-Bey Abina

, Gaspar

, Blondeau

, et al. Outcomes following gene therapy in patients with severe Wiskott–Aldrich syndrome. JAMA, 2015; 313:1550–1563.

44.

Aiuti

, Biasco

, Scaramuzza

, et al. Lentiviral hematopoietic stem cell gene therapy in patients with Wiskott–Aldrich syndrome. Science, 2013; 341:1233151.

45.

Cavazzana-Calvo

, Payen

, Negre

, et al. Transfusion independence and HMGA2 activation after gene therapy of human beta-thalassaemia. Nature, 2010; 467:318–322.

46.

Ribeil

, Hacein-Bey-Abina

, Payen

, et al. Gene therapy in a patient with sickle cell disease. New Engl J Med, 2017; 376:848–855.

47.

Biasco

, Pellin

, Scala

, et al. In vivo tracking of human hematopoiesis reveals patterns of clonal dynamics during early and steady-state reconstitution phases. Cell Stem Cell, 2016; 19:107–119.

48.

Payen

, Leboulch

. Advances in stem cell transplantation and gene therapy in the beta-hemoglobinopathies. Hematology, 2012; 2012:276–283.

49.

Moiani

, Paleari

, Sartori

, et al. Lentiviral vector integration in the human genome induces alternative splicing and generates aberrant transcripts. J Clin Invest, 2012; 122:1653–1666.

50.

Aiuti

, Roncarolo

, Naldini

. Gene therapy for ADA-SCID, the first marketing approval of an ex vivo gene therapy in Europe: paving the road for the next generation of advanced therapy medicinal products. EMBO Mol Med, 2017; 9:737–740.

51.

Mavilio

. Developing gene and cell therapies for rare diseases: an opportunity for synergy between academia and industry. Gene Ther, 2017 May 25 [Epub ahead of print]; DOI: 10.1038/gt.2017.36.

52.

Orkin

, Reilly

. MEDICINE. Paying for future success in gene therapy. Science, 2016; 352:1059–1061.