The EHA Research Roadmap: Hematopoietic Stem Cell Gene Therapy

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 platforms European research The EU has represented a for the clinical development of HSC on retroviral gene transfer from the earliest clinical trials most vectors are from vectors such as leukemia or from the human vectors a RNA into the of the cells. The for and or and the therapeutic gene were the earliest to be efficiency of gene transfer into the more primitive HSC because is dependent on of the targeted Moreover, early and most used exploited the in the of the to This with an insertion the of in some of of the insertion insertion and may in of the cell with a leading to progressive clonal in vivo in some by further of have the of leukemia in treated patients even several years HSCGT to such of genotoxic and monitoring of the clonal composition of hematopoiesis have the of such any clinical of these of the earliest of HSCGT for the treatment of severe combined by of has efficacy and better safety than for other diseases and the first HSCGT approved for the EU market in Because of the lower gene transfer efficiency and the has now been by which have both The key features these (1) of the to cells to of the more primitive HSC a ex vivo (2) which from the of the therapeutic gene is from an of from the cellular of the gene to be its or from a active providing for combined with an insertion that the than the of the risk of genotoxic effects at the insertion (3) of the for to transfer gene such as used to establish and of a in number of patients treated by HSCGT for several diseases have been and engineering of hematopoiesis with composition and of genotoxicity such as or for at on the conditioning and manufacturing, the engineered cell can to with evidence for of progenitors to early recovery or which is by multipotent Gene editing strategies allow and targeted modification of a DNA sequence of novel and to genetic engineering. In the available and more it exploits an artificial of a sequence-specific DNA of or to a or or of a RNA with a can a DNA in the is exploited for the type of most by an process that insertion or the the is targeted to a or an regulatory sequence the will most be or of the This represents the most that the engineered is better editing procedure. In most is exploited to the or a into the cells to be but is required for specificity is because DNA trigger a DNA which, if and can lead to or Moreover, the risk of genotoxicity and such as between different with specificity for the and in models have been major of the field. A more editing exploits DNA by This requires of the with a DNA the by to the DNA the this will allow in function and of the affected and even longer such as or in The current in editing to HSCGT is the efficiency of the process dependent on the of primitive HSC to this and the of DNA and an which is by a such as research for the of gene transfer can be to be in an increasingly number of diseases, the following of further HSCGT development can be Because autologous HSCGT does not and replacement of HSC, of the short-term and long-term of the procedure can be by of regimens to the required and conditioning strategies, such as on or HSCGT may provide a for early testing of these in diseases not high of chimerism with functional cells for of ex vivo HSC culture including more of supporting HSC in and/or from in the testing novel of and of on HSC, and better and more process in manufacturing allow the extent, and of gene transfer different patients and monitoring of clonal composition and HSC in the engineered hematopoietic graft of HSCGT patients will provide not only novel on HSC in but also to the long-term safety and of therapeutic As we more on the clinical of clonal and in it will be to ex vivo manipulation and genetic engineering may or such of new HSC HSCGT may highlight the of engineered cells to to long-term in establishing a for the graft in the of or On the other hand, if the growing number of treated patients and increasing of follow-up were to lead to of we about the and of genotoxicity of current and which are currently in models but for clinical Gene editing strategies be first clinical testing to any adverse of the procedure in of hematopoietic recovery and long-term engraftment, clonal composition of the engineered graft, and long-term and of the The of in some cells and in vivo will be with the to or from the if for efficiency and of will be developed for instance by ex vivo HSC strategies and or or the responses by the procedure. gene editing platforms that the requirement for DNA such as editors and editors will be in for to HSCGT and potentially providing for more and genetic The adverse effects of to gene transfer in gene editing some of which are of will be antigenic be present in the cell and trigger cellular responses leading to of the administered cells. The of or regulatory cells for the at different in the will be of the research are at the of a new of and cells as novel on genetic engineering to cell function and fundamental biological such as of and hematopoietic for therapeutic and for therapeutic and durable benefits in now and severe and diseases have been reported and are expected to be further in growing this also with a to the currently established for and medicines by and and to the this and the benefits that may from this we to research that the of early development of the new of to become with the new and regulatory to platforms and a and of in clinical development and of the new THERAPY Introduction immune deficiencies are a of genetic diseases by or function of or more of the immune clinical and of and and and treatment a for of to and and to and of is for but still a risk of and from rejection, and graft-versus-host were the first monogenic disorders for which HSCGT has been trials with promising are for at different to genetic of and/or innate a growing number of diseases are at stage of European research European research a key in the development of HSCGT for In the 2 the European funded research such as and patients have been treated in early trials of HSCGT with of in most patients affected by to Wiskott–Aldrich and was the first for which myeloablative conditioning was and in the first HSCGT approved in the EU with the of treatment for a of gene HSC in of cells from to in the myeloid and to in the lymphoid correction of and leading to of and of severe The promising clinical obtained in trials have been by the development of leukemia and as a of The disease that disease and genetic has been in the more and insertion of therapeutic into HSC, with evidence of reported to this clinical trials for and have with clinical HSCGT for in of immune including and of severe Moreover, of function in patients leading to from and in most treated research for the patients also analyses of insertion will be required to long-term safety and efficacy of patients who trials for and have are for other in which HSCGT an to allogeneic also in the most severe or patients with and/or including and HSCGT also become available for severe in which not be such as gene editing have the to some of the with gene and be suitable for diseases of gene or of of has been in gene for and and clinical testing of these strategies is of the key in the of HSCGT for SCID and on the of lymphoid cells, in patients with and allogeneic even in patients not active was to be for years durable of lymphoid On the other hand, conditioning is required to of HSC, normal and establish to the of conditioning mediated by or for HSCGT the in the lymphoid of a clinical in the of allogeneic for of HSC with autologous mature or lymphoid progenitors has also been with the to provide in patients not to allogeneic or HSCGT, immune responses to in diseases such as or as well as of immune in of the research Gene therapy for is from an to approved that are The of gene strategies and of gene editing with the of the will be for increasing the of approved as an to allogeneic Introduction are genetic of by in the or gene The most and severe are and cell disease by a or of and the of an In of patients on blood to In of blood cells and eventually death. Blood and by current is for both diseases, but with of suitable and in clinical on HSCGT on the of a normal gene and has been approved in for patients over affected by severe and it is still in trials for Gene editing strategies in the field of have the stage of clinical testing, with trials or in and in of by of to clinical and longer follow-up will the as well the of this novel European research European have been in and clinical research for the of and to the of the disease cause and the of allogeneic and research to clinical of gene therapy. clinical trials in have been in and the for also in The correction of the disease in most patients and in patients with severe The of cells with the clinical a threshold of cells to cell to and in and to and in The functional of HSC and the is in an has a on HSC research for the Gene therapy for including high of for therapeutic correction and high A on the market will not be for such a disease but of of be and/or in further and clinical research. the market be to the treatment of with As as for other genetic diseases, the myeloablative conditioning required to the of a high on patients, with and of biological conditioning HSC be although its efficiency needs to be and in the of a by an of will be in and of in the of allogeneic and of the research The long-term in research for has in the of a first and the of its manufacturing and to a number of affected The development and of gene editing will an of with potentially therapeutic of the of regulatory In both the manufacturing and major for the current the to the development of novel and potentially more of in vivo gene therapy. Introduction of are a of genetic disorders by the of an or its leading to of cellular by progressive the and the and lysosomal storage diseases according to the of the from of cellular of cell and function in the and replacement therapy and allogeneic have been exploited as for but and clinical have efficacy in disease autologous HSC is an to as it not only an safety but also to its to turn HSC progeny into of more cells, and the affected European research treatment of 2 affected by the of with HSC a in a new of more and genotoxic engineering of Since then, patients have been treated with which disease and treated early in disease from major functional to can be by a HSCGT to a as is not in early of the disease on benefit only be expected from of from the progeny of A clinical of HSCGT in or early early of gene of disease in patients and of disease in early the first treatment of the natural of this This HSCGT has been market by type on the therapeutic of by HSCGT in a for the most severe of has been a ex vivo manipulation with as that of correction and was obtained in treated patients, with an safety type have been obtained in a for HSCGT in hematopoietic and the of gene therapy in this of HSCGT is currently in clinical trials in and in the and research for the HSCGT has been to a promising treatment for and other and future clinical trials will provide into the of correction HSCGT in and will allow of and procedures of treatment conditioning cell of immune of and in an area of further HSCGT development with more diseases potentially to and from of the therapeutic strategies have the clinical basis for the of for which early and this a key for better and function and treatment for are in and the to this of the research The years for HSCGT in with several clinical developed and in clinical HSCGT in has evidence of correction in several diseases and of and clinical efficacy has been obtained in and of patients will to the clinical efficacy of these strategies in the with of safety. clinical development of HSCGT in these and other will the of some of A of HSCGT has been the correction of by in any of at in the and in the to DNA are most to this and progressively to severe as well as a high risk of Whereas has been for this disease because of the and of HSC, the of HSC the for in vivo of even a A of HSC in for A reported of HSC in the of conditioning by and progressive of the therapeutic over several of HSC gene editing are also in on the of development of HSC is to gene the replacement of a gene and the delivery of a biotherapeutic to disease or establishing resistance to an infectious agent. are on engineering hematopoietic progenitors with vectors designed to express in hematopoietic differentiation or gene-based delivery of the engineered mature cells as the gene to disease of this are to such as to by or to by these early stage may new for the treatment of some affections, such as and diseases. HSCGT ATMPs have which differentiate from and the to a durable therapeutic a to patients who may have few or treatment the and of these ATMPs also present several that to be to that these in have established and to and of In regulatory are such as of the as and of and for and In the of more such as scientific scientific the medicines or scheme and of novel is to as early as the under development with the in EU or in the and regulatory that will the it be that these novel and revolutionary medicines entering the clinical for the as is still of of regulatory in of to the first in human and clinical evidence to be generated for and to the Moreover, most are developed to and diseases with the of patient and clinical are which in the manufacturing process and in the to and the which manufacturing and and and is a common for HSCGT As HSCGT are medicines they even higher in of and these with the high and regulatory may early clinical testing of new if developed the of a be manufacturing for early stage clinical testing by to patient safety as well as with regulatory for clinical testing more and to of clinical the it be that entering the with a may compromise its development even if to be and may the to the further for process and for different regulatory still for this of is an between to and early to the and and current to the long-term safety and efficacy has been to establish more to the benefits of these and current to patient with and future during development the with to the by these medicines and new and models to for both and and HSCGT and available to Most Gene Therapy on or and of responses to of in the patients with long-term from Blood of in patients on for conditioning with and of with of and of in and conditioning with and gene over or of and severe infectious Blood conditioning of and available conditioning with and of and lymphoid and myeloid cells in patients with of severe adverse or clonal Therapy conditioning with and of in new conditioning with patients clinical and biological benefits Therapy conditioning with and and of in patients and of in Therapy conditioning with Transfusion was in most of the treatment and patients who conditioning with In patients with follow-up have and patient has conditioning with in and conditioning with in the of the of gene gene conditioning with the gene conditioning with conditioning with human and for of conditioning with conditioning with of and clinical to natural patients conditioning with of and of human conditioning with and Gene cells and in the patients cells with human conditioning with and conditioning with and of and of for human conditioning with of cells and number Therapy disease conditioning and disease conditioning and is used to trials but in different manufacturing and with different on clinical ex vivo hematopoietic stem cell gene therapy for inherited monogenic diseases. research policy priorities of HSCGT to increasing number of patients and long-term monitoring of the treated to establish safety and efficacy of the treatment and it for diseases in which an autologous of cells can lower the and the benefits of allogeneic of HSCGT to disease of the therapeutic of HSCGT has been such as the therapeutic of conditioning regimens for which HSCGT may provide a the extent, and of lentiviral gene transfer different patients and of clonal composition and HSC and in the long-term engineered the and of genotoxicity in HSCGT and strategies to further the risk of to new gene editing strategies as they clinical testing to any adverse of the procedure in of hematopoietic recovery and long-term engraftment, clonal composition of the engineered graft, and long-term and of the and any of the to and of gene the adverse of or developed to gene transfer and editing as well as to as HSCGT is to an increasing number of diseases and is further alleviated or more for and by and and to the patients, also by to the stage of development and development of is or of by and for is of a clinical by and in an for is a and of is for is the or of clinical trials by is a and is and of the scientific of the other have of to