The EHA Research Roadmap: Hematopoietic Stem Cell Gene Therapy
Authors: Luigi Naldini, Maria Pia Cicalese, Maria Ester Bernardo, Bernhard Gentner, Michela Gabaldo, Giuliana Ferrari, Alessandro Aiuti
Journal: HemaSphere (2022)

In 2016, the European Hematology Association (EHA) published the EHA Roadmap for European Hematology Research1 aiming to highlight achievements in the diagnostics and treatment of blood disorders, and to better inform European policy makers and other stakeholders about the urgent clinical and scientific needs and priorities in the field of hematology. Each section was coordinated by 1 to 2 section editors who were leading international experts in the field. In the 5 years that have followed, advances in the field of hematology have been plentiful. As such, EHA is pleased to present an updated Research Roadmap, now including 11 sections, each of which will be published separately. The updated EHA Research Roadmap identifies the most urgent priorities in hematology research and clinical science, therefore supporting a more informed, focused, and ideally a more funded future for European hematology research. The 11 EHA Research Roadmap sections include Normal Hematopoiesis; Malignant Lymphoid Diseases; Malignant Myeloid Diseases; Anemias and Related Diseases; Platelet Disorders; Blood Coagulation and Hemostatic Disorders; Transfusion Medicine; Infections in Hematology; Hematopoietic Stem Cell Transplantation; CAR-T and Other Cell-based Immune Therapies; and Gene Therapy. INTRODUCTION Genetic engineering of hematopoietic stem cells (HSCs) has progressed from early stage clinical trials providing evidence for substantial and durable benefits in some genetic deficiencies to the first Advanced Therapy Medicinal Products (ATMP) approved for the EU market (Figure 1).2 As safety and efficacy of HSC gene therapy (HSCGT) is further established by increasingly longer follow-up and a higher number of patients treated for different diseases, it may become a new pillar of treatment for several inherited monogenic affections for which allogeneic HSC transplantation (HSCT) represents a treatment option. Because HSCGT exploits autologous patient-derived cells, it has the following advantages over conventional allogeneic HSCT: (1) is in principle available to every patients; (2) does not entail the risk of graft-versus-host disease; (3) can lower substantially the risk of graft rejection, as the only potentially antigenic element in the administered cells is the therapeutic gene product; (4) can more easily establish partial chimerism with the administered HSC, which, according to disease and protocol, choice may allow lowering the requirement for myeloablative conditioning regimens and still provide substantial therapeutic benefit. Moreover, because of the genetic engineering step, gene correction may be designed to provide an even higher benefit than obtained with allogeneic normal or haploidentical carrier HSC, for instance by increasing therapeutic gene dosage in lysosomal storage diseases. HSCGT may eventually be exploited also for the treatment of some acquired affections, by instructing a new function to HSC or some of its progeny such as in gene-based delivery of a biotherapeutic or by establishing genetic resistance to an infectious agent. On the other hand, HSCGT requires ex vivo culture and manipulation of the cells to enable genetic engineering, which in turn demands for establishing a suitable GMP-compliant multistep process from cells harvest to manufacturing and delivery at the clinical point-of-care. Furthermore, the culture as well as the genetic engineering procedure may affect some fundamental HSC biological properties negatively impacting the safety and efficiency of the procedure. Although this is an active area of investigation, most current protocols for growing HSC ex vivo fail to expand or even maintain for more than a few days the most primitive and long-term engrafting cells and both gene transfer and gene editing procedures have been reported to trigger innate cellular responses that may lead to delayed or halted growth, differentiation or even apoptosis and cell death. Consequently, if these adverse effects individually or cumulatively surpass a threshold during cell manufacturing, they may cause delayed hematopoietic recovery or even failure of engraftment, exposing the patient to high risk of infections, and also compromise the clonal composition of the engineered hematopoietic graft, which may reduce its long-term resilience and safety. Last but not least, every meaningful genetic engineering of HSC requires stable, albeit local, modification of the cellular DNA sequence for ensuring life-long transmission to its cell progeny. This is a mutagenic procedure by definition, which entails a genotoxic risk dependent on the type (gene transfer versus editing), site, extent, and specificity of engineering. Whereas genotoxicity emerged as a significant risk in early HSCGT trials leading to leukemia development in some treated patients, it appears to be substantially alleviated by the new generation of lentiviral vectors currently in broader use. In any case, long-term monitoring of HSCGT patients is still required to establish the long-term safety and efficacy of these promising new therapies. In the following sections, we highlight some key features of the genetic engineering platforms under clinical development for HSCGT, the current stage and future outlook of clinical applications of HSCGT in some major diseases families, and the regulatory implications of these novel and revolutionary medicines entering the clinical arena.Figure 1.: Bone marrow-resident hematopoietic stem cells and hematopoietic progenitor cells replenish blood and tissues with new mature cells.2 Both hematopoietic stem cells and hematopoietic progenitor cells express the cell surface marker CD34, which is used to enrich a mixture of hematopoietic stem and progenitor cells for transplantation and gene therapy. Hematopoietic stem cells can be classed as long-term hematopoietic stem cells (LT-HSCs) or short-term hematopoietic stem cells (ST-HSCs). ST-HSCs progressively acquire lineage specifications to differentiate into lineage-committed progenitors and eventually terminally differentiated cells, which are released into the peripheral blood. A simplified scheme of human hematopoiesis is presented here. Alternative models have been postulated on the basis of cell surface marker analyses, in vitro and in vivo functional assays, clonal tracking by insertion analyses in hematopoietic stem and progenitor cell gene therapy studies, and single-cell RNA analyses (reviewed previously22). Mendelian genetic disorders can affect self-renewal, differentiation, and/or the function of different blood and immune cells. Examples of genetic diseases for which gene therapy is under investigation or approved are represented in white boxes below affected cell types. Wiskott–Aldrich syndrome affects platelets and other lineages. CDP = common dendritic progenitor; CID = combined immunodeficiency; CLP = common lymphoid progenitor; CMP = common myeloid progenitor; GMP = granulomonocytic progenitor; LMMP = lymphoid-myeloid primed progenitor; MEP = megakaryocytic–erythroid progenitor; MPP = multipotent progenitor; NK cell = natural killer cell; preB = pre-B cell; preT = pre-T cell; SCID = severe combined immunodeficiency. Reprinted with permission from Nat Rev Genet. 2021;22:216–234.GENE THERAPY PLATFORMS Introduction Most current genetic engineering of HSC exploits 2 major strategies, gene replacement mediated by retroviral gene transfer vectors or targeted gene editing mediated by artificial sequence-specific endonucleases. Gene transfer strategies exploit a couple of well-established platforms that were developed in research laboratories between 15 and 20 years ago. Since then, progressive clinical testing in >300 patients worldwide for the treatment of several diseases have generated 2 ATMPs approved for the EU market with several more expected in the upcoming years (Table 1).2 Gene editing strategies are still in the earliest stage of clinical testing, although they are witnessing a remarkable and constant progress advancing the versatility, precision, and scope of the technological platforms adopted.3,4 European research contributions The EU has represented a privileged theatre for the clinical development of HSC GT based on retroviral gene transfer from the earliest pioneering clinical trials until today's most advanced stages. Retroviral vectors are replication-defective viral particles derived either from γ-retroviruses (γ-retroviral vectors [γRVs]), such as Moloney leukemia virus (MLV), or from the human lentivirus HIV-1 (lentiviral vectors [LVs]). They integrate semi-randomly a reverse transcribed RNA genome into the chromatin of the transduced cells. The vector genome comprises cis-acting signals for packaging, reverse transcription, and integration, lacks full length or open viral genes and comprises the therapeutic gene expression cassette. γRVs were the earliest to be developed, having limited efficiency of gene transfer into the more primitive HSC because productive infection is strictly dependent on concurrent replication of the targeted cell. Moreover, their early and most commonly used design exploited the strong enhancer/promoter sequences embedded in the long terminal repeats (LTRs) of the viral genome to drive transgene expression. This feature, coupled with an integration bias favoring insertion near the promoter of actively expressed genes, result in some likelihood of altering expression of endogenous genes nearby the insertion site through promoter insertion and enhancer-dependent trans-activation. Sporadic insertions near proto-oncogenes may result in activation of their oncogenic potential, thus endowing the transduced cell with a growth advantage leading to progressive clonal expansion in vivo and, in some cases, eventual transformation by further accumulation of mutations. Vector integration studies have allowed ascribing the origin of leukemia emerging in treated patients even several years post HSCGT to such types of genotoxic insertions, and longitudinal monitoring of the clonal composition of reconstituted hematopoiesis have shown the frequent expansion of clones carrying such insertions before any clinical signs of leukemia. Despite these hurdles, one of the earliest application of γRV HSCGT for the treatment of severe combined immunodeficiency caused by deficit of adenosine deaminase (ADA-SCID) has shown efficacy and better safety outcome than observed for other diseases and became the first HSCGT ATMP approved for the EU market in 2016.5 Because of the lower gene transfer efficiency and clinically relevant genotoxicity, the γRV platform has now been almost completely replaced by LV, which have improved both aspects.1,6 The key LV features underlying these improvements are: (1) exploitation of the HIV core capacity to infect nondividing cells to enhance transduction of the more primitive HSC after a short ex vivo stimulation; (2) advanced vector design which completely eliminates transcriptional activity from the vector LTRs. Expression of the therapeutic gene is driven from an internal promoter of choice, either reconstituted from the cellular promoter of the gene to be replaced, thus mimicking its physiological expression pattern, or from a moderately active house-keeping promoter, thus providing for ubiquitous expression. These features, combined with an insertion bias that favors the body rather than the promoter of expressed genes, strongly alleviate the risk of genotoxic effects at the insertion site; (3) exploitation of the HIV mechanism for packaging unspliced viral genome to transfer complex gene expression cassettes, such as used to establish erythroid specific and robust expression of a globin transgene in hemoglobinopathies. An ever-growing number of patients treated by LV HSCGT for several diseases have been showing stable and robust engineering of reconstituted hematopoiesis with highly polyclonal composition and without signs of genotoxicity such as expanding clones or enrichment for insertions at cancer gene. Depending on the disease, conditioning applied and product manufacturing, the engineered cell fraction can reach up to near completion, with evidence for distinct types of progenitors contributing to early recovery or steady-state output, which is mostly driven by engrafted self-renewing multipotent HSC.2 Gene editing strategies allow precise and targeted modification of a DNA sequence of choice, opening up novel and unique opportunities to genetic engineering. In the longest available and clinically more advanced version, it exploits an artificial endonuclease either composed of a sequence-specific DNA binding domain made of Zinc Finger or TALE protein modules coupled to a FokI nuclease half-domain (ZFN or TALEN), or of a target complementary single guide CRISPR RNA assembled with a Cas family nuclease (CRISPR/Cas).4 Either platform can deliver a DNA double-strand break (DSB) specifically in the target sequence, whose repair is exploited for the intended type of edit. Nontemplated DSB repair most commonly occurs by nonhomologous end joining (NHEJ), an error-prone process that often introduces small base insertion or deletion (indels) while sealing the break. If the break is targeted to a coding or an essential regulatory sequence the NHEJ outcome will most often be disruption or inactivation of the sequence.7 This represents the most frequent and, provided that the engineered nuclease is highly specific, better tolerated editing procedure. In most cases, electroporation is exploited to transiently introduce the nuclease mRNA or a preassembled CRISPR/Cas nucleoprotein into the cells to be edited. Transient but robust expression is required for efficient break induction while limiting toxicity. Nuclease specificity is crucial because DNA DSBs trigger a detrimental DNA damage response which, if robust and sustained, can lead to growth arrest, senescence, or apoptosis.8 Moreover, multiple DSBs increase the risk of genotoxicity and genomic rearrangements, such as translocations between different edited sites. Developing nuclease reagents with stringent specificity for the target site and comprehensively assessing off-target activity in preclinical models have been major goals of the field. A second more ambitious editing strategy exploits DNA DSB repair by homologous recombination (HR). This requires codelivery of the nuclease with a DNA template carrying the intended edit framed by homologous sequences to the DNA flanking the nuclease target site. If successful, this strategy will allow correcting mutations in situ, thus restoring both, function and physiological expression of the affected gene, and even inserting longer sequences, such as corrective cDNAs or transgene expression cassettes in safe genomic sites.2 The current challenges in applying HR-mediated editing to HSCGT is the relatively low efficiency of the process dependent on the poor permissiveness of primitive HSC to this pathway and the cumulative detrimental impact of inducing DNA DSBs and introducing an exogenous template DNA, which is best achieved by exploiting a viral vector such as adeno-associated vector.9 Proposed research for the roadmap While application of LV gene transfer can be envisaged to be tested in an increasingly larger number of diseases, the following areas of further HSCGT platform development can be proposed. Because autologous HSCGT does not require full ablation and replacement of resident HSC, alleviation of the short-term and long-term morbidity of the procedure can be sought by careful targeting of chemotherapeutic regimens to the minimal required dose and through exploring emerging nongenotoxic conditioning strategies, such as those based on antibodies or immunotoxins targeting HSC.10,11 HSCGT may provide a favorable setting for early testing of these approaches, especially in diseases not requiring high levels of chimerism with functional cells for correction. Further optimization of ex vivo HSC culture conditions, including more faithful reconstitution of signals supporting HSC self-renewal in bone marrow niches and/or their emergence from hemogenic endothelium in the embryo, testing novel combinations of transduction enhancers and uncovering their mechanism of action on HSC, and better standardization and more effective process control in ATMP manufacturing should allow improving the extent, predictability and reproducibility of gene transfer across different patients and treatments. Close monitoring of clonal composition and HSC activity in the engineered hematopoietic graft of HSCGT patients will provide not only novel information on HSC biology in living humans but also help uncover clues to the long-term safety and robustness of therapeutic correction. As we learn more on the clinical relevance of clonal shrinking and skewing in aging hematopoiesis, it will be important to investigate whether ex vivo manipulation and genetic engineering may accelerate or aggravate such events. Delayed emergence of new HSC clones after HSCGT may highlight the capacity of engineered cells to return to long-term latency in vivo, establishing a safe reservoir for sustaining the graft in the case of emergencies or aging. On the other hand, if the growing number of treated patients and increasing time of follow-up were to lead to sporadic emergence of expanding dominant clones, we could learn about the residual extent and mechanism of genotoxicity of current vector design and introduce improved versions which are currently being investigated in stressed experimental models but await strong rational for clinical testing. Gene editing strategies must be carefully monitored upon first clinical testing to uncover any adverse outcome of the procedure in terms of hematopoietic recovery and long-term engraftment, clonal composition of the engineered graft, and preserved long-term maintenance and multipotency of the edited HSC. The occurrence of large-scale genomic alterations in some edited cells and their in vivo fate will be investigated with the goal to decrease their occurrence or purge them from the product, if necessary. Protocols allowing for improved efficiency and tolerability of editing, especially when exploiting HR, will be developed for instance by adopting emerging ex vivo HSC expansion strategies and refined editors, or inhibiting or counteracting the detrimental responses induced by the procedure. New gene editing platforms that bypass the requirement for DNA DSB, such as base editors and prime editors will be tested in preclinical studies for potential application to HSCGT investigating their efficiency, tolerability, and specificity, potentially providing for more precise and uneventful genetic engineering.4 The potential adverse effects of pre-existing immunity to gene transfer and, in particular gene editing reagents, some of which are of bacterial origin, will be investigated. Residual antigenic components might be present in the cell product and trigger cellular responses leading to clearance of the administered cells. The impact of pre-existing effector or regulatory T cells specific for the viral/editor component at different levels in the recipient will be investigated. Anticipated impact of the research We are at the beginning of a new era of medicine exploiting genes and cells as novel therapeutics, leveraging on powerful genetic engineering tools to modify cell function and capture fundamental biological processes, such as HSC-driven reconstitution of intra and extravascular hematopoietic populations for therapeutic purposes. These approaches open unprecedented opportunities and largely unexplored paths for therapeutic interventions. Remarkable and durable benefits in until now orphan and otherwise severe and lethal diseases have been reported and are expected to be further achieved in growing numbers. However, this promise comes also with a tremendous challenge to the currently established framework for developing, regulating and distributing medicines while maintaining their economic sustainability by public and private healthcare providers and guaranteeing fair access to the patients. To address this challenge and attain the benefits that may come from this research, we must continue to support research establishments that produce innovation, facilitate the creation of start-ups promoting early development of the new technologies, advance education of biomedical trainees to become familiar with the new technologies and treatments, update regulatory requirements to emerging platforms and a rapidly evolving landscape, and foster engagement of pharmaceutical industries in clinical development and commercial deployment of the new ATMPs. GENE THERAPY FOR PRIMARY IMMUNODEFICIENCIES Introduction Primary immune deficiencies (PIDs) are a heterogeneous group of >400 genetic diseases characterized by poor or absent function of one or more components of the immune system, whose main clinical manifestations comprise increased frequency and severity of infection, autoimmunity, and aberrant inflammation and malignancy.12 Early diagnosis and treatment remain a mainstay for all forms of PIDs to prevent organ damage and life-threatening infections and to improve prognosis and quality of life. Allogeneic HSCT is curative for many PIDs, but still carries a risk of mortality and morbidity from rejection, toxicity, and graft-versus-host disease. SCIDs were the first monogenic disorders for which HSCGT has been successfully developed. Clinical trials with promising results are ongoing for at least 6 different PIDs due to genetic defects of adaptive and/or innate immunity while a growing number of diseases are at preclinical stage of development.2,13 European research contributions European academic research played a key role in the development of successful HSCGT approaches for PID. In the past 2 decades, the European Commission funded collaborative research networks such as CONSERT, PERSIST, Clinigene, CELL-PID, NET4CGD, SCIDNET, SUPERSIST, and UPGRADE. Over 100 PID patients have been treated in early trials of HSCGT with γRV, showing reconstitution of immunity in most patients affected by SCID-X1 due to IL2RG deficiency, ADA-deficient SCID, Wiskott–Aldrich Syndrome (WAS), and X-linked Chronic Granulomatous Disease (X-CGD).2 ADA-SCID GT was the first PID for which non myeloablative conditioning was introduced and in 2016 became the first HSCGT approved in the EU (Strimvelis, Orchard Therapeutics Ltd, London, UK), with the indication of standard treatment for ADA-SCID lacking a compatible family donor5 Infusion of ADA gene replaced HSC resulted in persistent (>15 y) engraftment of gene-marked cells ranging from 1% to 10% in the myeloid compartment and reaching up to 100% in the lymphoid compartment, correction of adenosine metabolism and improved T-cell counts, leading to discontinuation of prophylaxis and decreased incidence of severe infections.14 The promising clinical data obtained in γRV trials have been tempered by the later development of T-lymphoblastic leukemia and myelodysplasia as a result of insertional mutagenesis. The incidence varied among disease types, suggesting that disease background, transgene function, and individual genetic predisposition influence tumorigenicity. LV-based platform has been deployed in the past decade enabling more effective and safe insertion of therapeutic genes into HSC, with no evidence of leukemogenesis reported to this date. LV-HSCGT clinical trials for SCID-X1 and ADA-SCID have shown improved lymphocyte counts with clinical benefit.15 HSCGT for WAS resulted in amelioration of immune functions, including autoimmunity, and reduced incidence of severe bleeding events.3,16 Moreover, HSCGT-mediated restoration of oxidase function in X-CGD patients leading to protection from bacterial and fungal infections in most treated patients.17 Proposed research for the roadmap Continuous patients monitoring, also through analyses of vector insertion sites, will be required to confirm long-term safety and efficacy of patients who underwent HSCGT. Clinical trials for artemis deficiency, LAD-1, and osteopetrosis have recently started.2 Preclinical studies are actively pursued for other PIDs in which HSCGT could represent an alternative to allogeneic HSCT, also in the most severe or adult patients presenting with ongoing infections and/or organ damage including JAK3-SCID, PNP deficiency, RAG1/2 deficiency, ZAP70 deficiency, Munc 13-4 deficiency, and DADA2.18 HSCGT could also become available for less severe phenotypes, in which alloHSCT would not be indicated such as BTK deficiency. New gene editing technologies have the potential to circumvent some of the problems associated with viral gene addition and could be suitable for diseases requiring physiological regulation of gene expression or inactivation of dominant alleles. Preclinical proof of concept has been attained in correcting gene mutations for IL2RG, WAS, p47-CGD, CD40L deficiency, and IPEX, and clinical testing of these strategies is awaited. One of the key factors in the success of HSCGT for SCID and WAS relies on the selective advantage of functionally corrected lymphoid cells, previously observed in patients with somatic revertants and allogeneic HSCT. Thus, even in patients not receiving conditioning, active thymopoiesis was shown to be maintained for many years after treatment, suggesting durable thymic engraftment of long-lived lymphoid progenitors.2 On the other hand, conditioning is required to achieve polyclonal engraftment of gene-corrected HSC, restore normal B-cell lymphopoiesis and establish corrected myelopoiesis. SCIDs seem ideal candidates to explore the use of conditioning mediated by monoclonal antibodies or immunotoxins for HSCGT while exploiting the selective advantage in the lymphoid lineage. Preliminary results of a clinical trial in the context of allogeneic HSCT for SCID-X1 show sufficient degree of HSC engraftment.11 GT with autologous mature lymphocytes or lymphoid progenitors has also been explored preclinically with the rationale to provide in patients not eligible to allogeneic HSCT or HSCGT, immune responses to infection in diseases such as CD40L or perforin deficiency as well as control of immune dysregulation regulation in FOXP3 deficiency. Anticipated impact of the research Gene therapy for PIDs is moving from being an experimental approach to approved drug products that are routinely beneficial. The expansion of gene addition strategies and implementation of gene editing approaches, together with the standardization of the technology, will be important for increasing the armamentarium of approved therapies as an alternative to allogeneic HSCT. HEMOGLOBINOPATHIES Introduction Hemoglobinopathies are genetic defects of hemoglobin chain production, caused by mutations in the α- or β-globin gene clusters. The most frequent and severe are transfusion-dependent β-thalassemia (TDT) and sickle cell disease (SCD), characterized by a reduced or absent level of adult hemoglobin (HbA) and the production of an abnormal structural variant (HbS), respectively. In TDT, survival of patients depends on chronic blood transfusion associated to iron chelation. In SCD, sickling of deoxygenated red blood cells (RBCs) promotes painful vaso-occlusive crises, acute chest syndrome, stroke, and eventually death. Blood transfusion and fetal hemoglobin (HbF) induction by hydroxyurea represent current treatments. Allogeneic HSCT is curative for both diseases, but with limited availability of suitable donors and variability in clinical outcome depending on patient's age. LV-mediated HSCGT relies on the erythroid specific expression of a normal gene copy and has been recently approved in Europe for patients over 12-years-old affected by less severe β-thalassemia mutations, and it is still in experimental trials for SCD.2 Gene editing strategies in the field of hemoglobinopathies have reached the stage of clinical testing, with ongoing phase 1/2 trials using ZFN or CRISPR/Cas technologies in SCD and in TDT. Reactivation of HbF synthesis by disruption of Bcl11a suppressor led to initial encouraging clinical results19 and longer follow-up will fully disclose the real potentiality as well the caveats of this novel approach.20 European research contributions European researchers have been prime actors in nonclinical and clinical research for the cure of TDT and SCD, contributing to the discovery of the disease molecular cause and the molecular mechanisms of globin genes regulation, introducing allogeneic HSCT and translating basic research to clinical application of gene therapy. Two successful clinical trials in TDT have been conducted in France21 and Italy,22 paving the way for application also in SCD.23 The results showed correction of the disease in most adult patients carrying nonsevere mutations and in young patients with severe mutations. The level of marked engrafted cells positively correlates with the clinical benefit, highlighting a threshold of genetically corrected cells to produce sufficient HbA per cell to rescue anemia and ineffective erythropoiesis in TDT, and to dilute abnormal HbS and preventing sickling in SCD. The functional status of HSC and the BM microenvironment is particularly relevant in TDT, where an impaired HSC-niche cross-talk has a negative impact on HSC functionality.24 Proposed research for the roadmap Gene therapy for hemoglobinopathies poses unique challenges, including high level of transgene expression for therapeutic correction and high global incidence worldwide. A single ATMP on the market (Zynteglo) will not be sufficient for such a prevalent disease but demonstration of dissimilarity of drug products might be challenging, thus discouraging biotech and/or pharma investments in further academic preclinical and clinical research. New rules governing the ATMP market should be introduced to favor the treatment of large p