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The transformative potential of HSC gene therapy as a genetic medicine


Hematopoietic stem cells (HSCs) are precursor cells that give rise to blood, immune and tissue-resident progeny in humans. Their position at the starting point of hematopoiesis offers a unique therapeutic opportunity to treat certain hematologic diseases by implementing corrective changes that are subsequently directed through to multiple cell lineages. Attempts to exploit HSCs clinically have evolved over recent decades, from initial approaches that focused on transplantation of healthy donor allogeneic HSCs to treat rare inherited monogenic hematologic disorders, to more contemporary genetic modification of autologous HSCs offering the promise of benefits to a wider range of diseases. We are on the cusp of an exciting new era as the transformative potential of HSC gene therapy to offer durable delivery of gene-corrected cells to a range of tissues and organs, including the central nervous system, is beginning to be realized. This article reviews the rationale for targeting HSCs, the approaches that have been used to date for delivering therapeutic genes to these cells, and the latest technological breakthroughs in manufacturing and vector design. The challenges faced by the biotechnology cell and gene therapy sector in the commercialization of HSC gene therapy are also discussed.


Hematopoietic stem cells (HSCs) are precursor cells with the capacity to generate any of the diverse mature functional hematopoietic cell types that constitute a healthy hematologic system [1]. The vital role played by HSCs in the genesis of hematopoiesis offers an exciting therapeutic opportunity to treat a range of inherited hematologic disorders, including primary immune deficiencies, lysosomal storage, and metabolic disorders, hemoglobinopathies, and congenital cytopenias. Introduction of genetically modified HSCs can provide an ongoing source of self-renewing stem cells and their blood cell progeny of all lineages, providing lifelong therapeutic benefit while circulating in the periphery or in residence within other tissues and organs [2].

HSC gene therapy (HSC GT) is one approach that has been used to deliver stable engraftment of gene-corrected HSCs. It involves transplantation of a patient’s own HSCs following ex vivo genetic modification with viral vectors that integrate the transgene into the target cell’s genomic DNA [2]. More than two decades have now passed since the first clinical trials for severe combined immune deficiency (SCID) illustrated the curative potential of HSC GT [3, 4], and the field is now on the cusp of an exciting new era. HSC GT is already delivering on its promise in several monogenic rare diseases, including licensed medicines for adenosine deaminase deficiency (ADA)-SCID, beta-thalassemia, and metachromatic leukodystrophy (MLD), offering an effective and durable new treatment option for these often-neglected diseases [5,6,7,8]. Moreover, leveraging the durability of HSC GTs and their potential to deliver gene-corrected cells to a range of tissues and organs [2] has led to a realization that this approach may also be used for treatment of multisystem disorders.

This review examines the rationale for targeting HSCs and describes some of the approaches that have been used to date for delivering therapeutic transgene expression to these cells. The potential clinical applications of HSC GT are examined, including an overview of investigational products and their therapeutic target indications under development. Finally, some of the practical challenges faced by the biotechnology sector in delivering commercial cell and gene therapies to wider patient numbers are discussed.

The rationale for hematopoietic stem-cell gene therapy

Hematopoietic stem cells

HSCs are multipotent, which means they have the capacity to self-renew and to develop into the various specialized cellular components of the haemopoietic system [9]. This includes erythrocytes, megakaryocytes, and platelets that interact with blood vessels and coagulation factors, cells of the innate and acquired immune systems that protect against microbial attack, and blood monocyte-derived macrophages that migrate into multiple different tissues to replenish tissue-resident macrophages, which can then fulfill key homeostatic organ-specific functions through immune defense, trophic, regulatory and repair mechanisms. (Fig. 1a, b) [1].

Fig. 1: A combination of hematopoietic stem-cell self-renewal and lentiviral vector-mediated integration of the therapeutic gene into the hematopoietic stem-cell genome offers the potential for permanent correction.

a Lentiviral vector-mediated integration of the therapeutic gene into the hematopoietic stem-cell and progeny genome. b The propensity of blood derived monocytes to migrate and infiltrate most tissues allows the replacement of tissue-resident and embryonically derived macrophages, potentially allowing HSC GT correction of indications affecting multiple different tissues.

HSCs are a heterogenous population of cells that differ in their cell surface phenotype, response to extrinsic signals, lineage outputs following transplantation, self-renewal capabilities, and repopulation kinetics [1, 10]. The HSC pool is divided into two broad subpopulations based on reconstituting activity: long-term true HSCs, which can self-renew, and short-term HSCs, comprising multipotent progenitors [11]. HSCs are broadly defined by expression of CD34, a transmembrane glycoprotein of largely unknown function, which is correlated with long-term stem and progenitor cells as its expression progressively decreases with lineage commitment [12]. Several CD34+ subsets have now been identified, based on co/expression of surface markers including CD133+, CD38−/low, CD164+, and CD90+CD45RA, which further define more primitive HSCs [13,14,15,16,17,18].

Therapeutic applications

Initial approaches for exploiting HSCs therapeutically to treat inherited hematologic disorders focused on transplantation of healthy donor allogeneic hematopoietic stem cells (HSCT), with the first successful application of allogeneic HSCT being in severe combined immunodeficiency [19, 20]. Although now widely used for a range of diseases, the overall efficacy and safety of this procedure can be dependent upon challenges such as identifying suitable human leukocyte antigen-matched donors, the risk of graft rejection and graft versus host disease, and the clinical burden of sustained high levels of immune suppression required to reduce immunological risks [21,22,23].

These clinical barriers and risks have driven the development of alternative, potentially safer approaches, such as autologous HSC GT. HSC GT uses the patient’s own cells, thereby avoiding graft versus host disease and other alloreactive complications [24]. As with HSCT, HSC GT offers the opportunity for successful treatment from a single administration. Vectors can be used to integrate therapeutic/ functional copies of genes into the genome of long-term repopulating HSCs, providing a durable lifelong supply of genetically modified cells (Fig. 1a) [2]. Furthermore, the ability of gene-corrected HSCs to differentiate and engraft within tissues as tissue-resident macrophages opens up the possibility of treating multiple organ system disorders, including the brain through engraftment of gene-modified microglia (Fig. 1b).

Targeted gene-editing, defined as site-specific genome modification, is an alternative approach to gene-correction of HSCs. Although a detailed review of this method is outside the scope of this article, promising early clinical data with gene-editing in the treatment of sickle-cell anemia and β-thalassemia have recently been published [25, 26].

Delivering genes into hematopoietic stem cells

Manufacturing ex vivo HSC GT as a genetic medicine requires several key processes as outlined in Fig. 2 and described in detail below.

Fig. 2: Gene therapy manufacturing and administration.

An overview of the treatment timeline is shown. HSC hematopoietic stem cell, LV lentiviral vector.

Genetic modification of HSCs

Genetic modification of HSCs relies on a suitable vector, with efficient tropism and stable integration into the host genome to deliver stable functional gene expression. Viral vector transduction, which exploits the innate ability of viruses to enter cells and express their genes in order to replicate, is the current method of choice [27]. Viral vectors are engineered from wildtype viruses. Due to the removal of most or all of the viral genome, they are rendered replication incompetent, while retaining the ability to perform a single round of transduction to deliver the therapeutic genetic material to the target cell [28]. A range of viral vectors have been evaluated for HSC GT, broadly categorized based on whether they are integrating or non-integrating to the target cell genome. Retroviruses are large, membrane-enclosed (enveloped) viruses with RNA genomes that are reverse transcribed into DNA and can integrate into the target cell genome. They are the optimal approach for HSC GT as the functional gene is permanently integrated and heritable for successive generations of progeny [29]. Commonly investigated retroviral vectors (RVs) include those based on gamma retroviruses (gRV), spumaviruses (foamy), and lentiviruses [29].

Integrating retroviral vectors for HSC GT

Gamma retroviruses (gRV) were the first RVs evaluated for GT [30] and are still commonly employed as vectors for T-cell engineering [31] and human stem-cell GT [32]. Derived from murine leukemia virus, gRVs are classified as simple retroviruses and require dissolution of the nuclear envelope during mitosis to enter the nucleus; thus, they can only efficiently transduce dividing cells [33, 34]. In order to promote mitosis, HSC are pre-stimulated with cytokines for 2–3 days prior to transduction to drive quiescent HSC into the cell cycle. While effective at improving transduction of HSCs, extended pre-stimulation may induce differentiation and thus limit HSC self-renewal capacity after engraftment.

Insertional oncogenesis is also a consideration with all RVs, particularly gRVs, as they have a propensity to integrate closer to transcription start sites and have viral promoters with enhancer activity and therein the ability to transactivate adjacent genes [35]. Indeed, the consequences of insertional oncogenesis became apparent in the early 2000s, when several children with X-linked SCID (X-SCID) who were treated with gRV vectors harboring a strong viral enhancer/promoter region developed lymphoproliferative disease as a result of integration in or near oncogenes [36]. Similar complications were seen in clinical trials of gRV vectors for X-linked chronic granulomatous disease (X-CGD) and Wiskott–Aldrich syndrome (WAS) [37, 38]. Moreover, recently, a patient with ADA-SCID treated with an approved gRV transduced cell product was diagnosed with a T-cell leukemia; at time of writing causality is still under investigation [39].

To reduce the potential for insertional oncogenesis, self-inactivating (SIN) lentiviral vectors (LVs) are now the standard approach, as they have been designed to self-inactivate the viral promoter and enhancer activity to prevent transactivation of proto-oncogenes [40]. Lentiviruses are classified as complex retroviruses and those used clinically are most often based on human immunodeficiency virus (HIV) 1 [33]. Wildtype HIV-1 has nine viral genes and upon their removal leaves capacity for a larger (8–9 kb) payload compared with gRV [41, 42]. Furthermore, LVs form a pre-integration complex allowing viral vector DNA entry to the nucleus via nuclear pores which can therefore transduce non-dividing cells, a particularly useful property for gene modification of quiescent HSCs (Fig. 3a) [43]. Thus, LVs permit a reduced time for gene modification of HSC of 2–3 days, compared with extended processing periods of 4–5 days with gRV vectors to allow cell division [44].

Lentiviral vector design

Third-generation LVs have a number of additional design features to improve safety described below, including removal of all HIV-1 accessory genes, a strengthened polyadenylation signal, and self-inactivation of the enhancer/promoter region upon reverse transcription and integration [2]. They also have a deletion in the 3′ long terminal repeat (LTR) U3 region. During reverse transcription and integration, the deleted 3′ U3 region is copied to the 5′ LTR U3, inactivating the 5′ LTR enhancer/promoter region in the proviral DNA and eliminating the enhancer and promoter activities [45]. This alteration reduces the potential for proto-oncogene activation when in proximity to neighboring promoters [46]. The propensity of LVs to integrate further away from the transcription start site is an additional benefit of this approach [47, 48]. Later-generation LVs have demonstrated a >100- to 1000-fold lower risk of transactivation of adjacent genes in preclinical murine models (in vitro) [49,50,51]. They have been used to transfer genes into both stem cells and T cells without significant safety issues and no evidence to date of insertional oncogenesis. The long-term safe use of these vectors is supported by growing evidence from clinical trials across a range of diseases, including MLD, X-CGD, ADA-SCID, X-SCID, artemis-deficient (ART)-SCID, WAS, X-linked adrenoleukodystrophy (X-ALD), leukocyte adhesion deficiency type I (LAD-I), mucopolysaccharidoses I (MPS-I), MPS IIIA, Fabry, cystinosis, pyruvate kinase deficiency, sickle cell disease, and transfusion-dependent beta-thalassemia, with more than 250 patients treated in total, in some cases with up to 8 years’ follow-up (summarized in Table 1).

Table 1 Ex vivo lentiviral vector-based HSC GT applications currently approved and/or under clinical development.

LVs contain gene expression cassettes comprising an internal human cellular promoter lacking enhancer activity and the therapeutic transgene, and in some cases, additional regulatory elements (Fig. 3b). Tailored selection of the internal promoter is indication specific and can drive transgene expression in a tissue or cell-specific manner and allow for over-expression depending on the requirement [52,53,54,55]. Generation of supraphysiological levels of protein may be particularly attractive for facilitating cross-correction especially in lysosomal storage diseases (LSDs). Some of the promoters commonly employed in HSC GT are detailed in Table 1. LVs also often contain a woodchuck hepatitis virus post-transcriptional regulatory element, which stabilizes vector mRNA transcripts for increased titers during vector production and transgene mRNA stability during transport to cytoplasm for translation [56].

Fig. 3: Lentiviral vector design and gene transfer.

a Gene transfer by a lentiviral vector. b Self-inactivating lentiviral vector design. LTR long terminal repeat, Prom promoter, SIN self-inactivating vector, WPRE woodchuck hepatitis virus post-transcriptional regulatory element.

Lentiviral vector production

LVs are usually produced by transient transfection of vector packaging cells to assemble the necessary viral components into the lentivirus. The most commonly used vector packaging cells are human embryonic kidney 293T cells due to the ease with which they are transfected [57]. The split packaging LV system is used to separate the viral genes on to packaging plasmids, which have been modified to reduce the risk of recombination and the production of replication-competent virus [58]. The transfer vector plasmid contains the LV backbone with the expression cassette. The viral genes produce the proteins needed to package the viral vectors, remaining in the vector packaging cells, and are not packaged into the vector particle [57]. Typically, there are two packaging plasmids encoding for HIV viral genes (gag/pol and rev) [57]. LVs are typically pseudotyped with a different envelope to increase the ability of the HIV-1 based LV to enter more cell types than the endogenous HIV-1 envelope [57]. The env plasmid encodes for the envelope glycoprotein from the vesicular stomatitis virus (VSV-G) in current clinical applications, although the ability to use alternative envelope proteins is under investigation [59]. The VSV-G protein utilizes the low-density lipoprotein receptor [60], which is ubiquitously expressed on many cells including HSCs. Within the packaging cells, the packaging plasmid and env plasmid DNA are transcribed into mRNA and translated into the viral proteins and viral envelope proteins that will form the particle and package the transfer vector RNA genomes [57]. Transfer vector RNA transcripts are exported from the nucleus as unspliced transcripts and are assembled along the cell membrane with the other components for vector packaging. Two important viral proteins, reverse-transcriptase and integrase, are also packaged into the vector particle for use in the target cell. Viral components are then assembled into viral particles, which bud from the host membrane with the embedded envelope transmembrane glycoproteins [57]. Lentiviral particles are released into the growth media, harvested, purified, and tested for safety and quality, as well as their ability to transduce cells [57].

Transduction of HSC

Two methods are generally employed in the collection of autologous HSCs from the patient, leukapheresis of mobilized blood or extraction from bone marrow harvested from the posterior superior iliac crests under general anesthesia [24]. Although the quality of HSCs collected is comparable between both methods, over recent years, harvesting of stem cells from peripheral blood has largely replaced bone marrow for GT primarily due to the ability to collect larger numbers of cells and ease of collection since anesthesia is not required and there is a lower risk of serious adverse events [61].

One challenge of HSC GT is that CD34+ HSCs only comprise ~1% of adult bone marrow cells [62]. However, efficient HSC mobilization protocols and apheresis processing methods of relatively large numbers of cells have been developed for enrichment of CD34+ HSCs, to ensure levels sufficient for use in a clinical setting. Results from multiple clinical trials in these diseases (discussed in greater detail below) have shown stable long‐term hematopoietic reconstitution in most patients receiving HSC GT. Patients have a substantial proportion of gene‐modified cells throughout all lineages, which are maintained by engrafted transduced short-term and long-term HSC progenitors [63, 64].

Once bone marrow or peripheral blood is collected, HSCs are identified by the CD34+ cell surface marker and enriched by immunomagnetic bead selection [65]. Isolated CD34+ cells are activated with cytokines during a pre-stimulation period in preparation for transduction, which is initiated by the addition of viral vector to the growth media of the cultured HSCs. As described above, LV transduction results in stable integration of the gene expression cassette to enable production of the therapeutic protein.

HSC GT administration

Once transduced, the cells are harvested and formulated as the drug product, either for cryopreservation (frozen for future use) or administered as a fresh formulation. For both methods, quality control is vital; the drug product is usually certified for cell potency, viability, and sterility before use for cryopreserved products, while this step usually occurs retrospectively for fresh products. In preparation for administration, the patient is given cytoreductive conditioning, usually with different intensities of non-myeloblative chemotherapy, to clear as many of the genetically defective cells from bone marrow as possible in preparation for receiving the genetically modified cells to engraft; this may also promote immune tolerance to the new therapeutic protein that will be produced [66]. The genetically modified HSCs are then administered to the patient intravenously, and once cells engraft in the bone marrow, they self-renew and produce corrected blood cells of all types.

HSC gene therapy treatments

Since the first clinical trials with LV-mediated HSC GT in the mid-2000s [67, 68], this approach has now been employed safely and effectively across numerous different primary immune deficiencies, hemoglobinopathies, and storage and metabolic diseases, providing proof of its transformative potential (Table 1). HSC GT has consistently achieved stable frequencies of gene-corrected blood cells of all lineages, indicating engraftment, long-term persistence, and ongoing generative capacity of gene-modified HSCs (Table 1). Graft versus host disease is avoided and, in some indications, lower amounts of conditioning chemotherapy are required versus HSCT, resulting in improved safety profiles with autologous HSC GT. Analyses of LV integration sites in particular have shown a remarkably consistent pattern in subjects across different clinical trials, with no evidence of proto-oncogene transactivation and no clinically significant clonal expansions [49, 63]. Furthermore, the ability to drive physiologic or supraphysiological expression of the therapeutic gene through the use of tissue/cell-specific promoters or constitutive cellular promoters has led to the development of indication specific LV design.

SCID is a group of rare and life-threatening genetic disorders characterized by no or very low T-cell counts, combined with the absence or dysfunction of B-lymphocytes and in some forms, natural killer cells [69]. Patients usually present with severe and opportunistic infections, chronic diarrhea, and/or failure to thrive in infancy, and without treatment, most patients die within the first year of life [69]. The seminal work performed on gene therapy for SCID with gRVs and gene-modified HSCs led to the European approval [70] of the first gene therapy for ADA-SCID in 2016, Strimvelis® [71]. Subsequent creation of SIN LVs has seen a number of improved HSC GTs under clinical development for various manifestations of SCID, including ADA-SCID, ART-SCID, and X-SCID (which aim to restore ADA, DCLRE1C, an IL2RG function, respectively) (Table 1), and a number of preclinical developments including subtypes of recombinase-activating gene (RAG)-SCID [72, 73]. Data from clinical trials have shown stable long-term engraftment of HSCs, evidence of cellular and humoral immune reconstitution, and good tolerability, thereby validating this approach (Table 1). Promising clinical data with HSC GTs have also been shown in other primary immunodeficiencies such as X-CGD, WAS, and LAD-I (Table 1).

The two most common erythrocyte development disorders are β-thalassemia, which results from low β-globin expression [74], and sickle cell disease (SCD), which is caused by a point mutation in the HBB gene [75]. Patients with β-thalassemia often have a lifelong dependency on blood transfusions. The red blood cells of patients with SCD display polymerization of hemoglobin tetramers upon deoxygenation, leading to abnormal shape, blockage of blood capillaries, ischemia, multi-organ damage, and severe pain [75]. Since β-thalassemia and SCD are both relatively common disorders, there has been enormous impetus to develop effective GTs. The successful engineering of LV with the globin gene promoter and its locus control region act together to provide globin expression that is restricted to the red cell lineage. Previous attempts to include these elements in gRV resulted in unstable constructs. There are several HSC GTs for hemoglobinopathies currently in clinical development, all aiming to restore functional globin production. To date, these therapies have shown evidence of stable engraftment and strong efficacy in ongoing clinical trials, with the ability to reduce or eliminate transfusion dependency in transfusion-dependent β-thalassemia or reduce vascular crises in SCD (Table 1). Recently, clinical trials with a lentiviral HSC GT for β-thalassemia and SCD were suspended due to adverse events of acute myeloid leukemia and myelodysplastic syndrome in patients with SCD; at the time of writing, causality is still under investigation [76]. However, it is noteworthy that there was no evidence of vector insertion in the initial case of myelodysplastic syndrome, suggesting that it might be related to the SCD background marrow, which is chronically stressed [77]. Furthermore, there is also a known increase in susceptibility to development of leukemias in patients with SCD [78, 79].

Gene-corrected HSCs have the ability to differentiate into macrophages and microglia, opening up the possibility of targeting organs beyond the hematopoietic system, such as bone, muscle, and brain, via a process of cross-correction (Fig. 1b). Therefore, neurometabolic and neurodegenerative disorders, particularly LSDs, are attractive targets for HSC GT. LSDs are caused by mutations in one of ~50 enzymes that are required to break down molecules such as glycoproteins, lipids, and glycogen leading to severe neurological damage [80].

Distribution into the central nervous system (CNS) has been notoriously challenging with other GT approaches developed to date (reviewed in [81]). Preclinical evidence of cross-correction of neurons by gene-modified microglia overexpressing the therapeutic gene has been shown in mouse models of MLD and lysosomal storage disorders [82,83,84]. Recent data from clinical trials in neurometabolic diseases suggest that there are a population of gene-modified HSCs that can naturally cross the blood–brain barrier, distribute throughout the brain, engraft as macrophages and microglia, and express therapeutic enzymes that can be taken up by local neurons [85, 86]. In a study where 29 patients with pre- or early-symptomatic early-onset MLD were treated with an autologous HSC population transduced ex vivo with a LV encoding the human arylsulfatase A (ARSA) gene (OTL-200), all treated patients achieved hematological recovery and showed stable engraftment of gene-corrected cells [6]. Reconstituted ARSA activity in peripheral blood mononuclear cells within or above normal range was observed by 3 months post treatment, including myeloid cells capable of differentiating into macrophages and microglia (Table 1) [6]. ARSA activity in cerebrospinal fluid, mostly undetectable at baseline, was detected by month 3, reached normal levels by 6–12 months post-treatment, and remained within a normal range through up to 7.5 years’ follow-up [6, 87, 88]. Furthermore, in a GT trial in patients with X-ALD who received autologous CD34+ HSCs genetically corrected ex vivo with a LV encoding wildtype ABCD1 (Lenti-D; elivaldogene autotemcel), 15 of the 17 patients (88%) remained free of major functional disability over a median follow-up of 29.4 months, with minimal clinical symptoms [89]. HSC GTs for a range of other LSDs, including MPS-I, Gaucher disease, Fabry disease, and MPS IIIA, are currently in clinical development (Table 1) with a number of others in preclinical development [90, 91].

Genetically modifying tissue macrophages downstream of HSCs, allowing cross-correction of non-HSCT lineages, also has applications beyond a neurometabolic setting. Indeed, in a recent case report of a patient with MPS-I treated with an autologous HSC population transduced ex vivo with a LV encoding the human α-l-iduronidase (IDUA) gene (OTL-203), supraphysiologic alpha-L-iduronidase levels were observed in osteoclasts after GT, which may allow cross-correction of mesenchymal stromal cells and their progeny in the bone microenvironment [92]. Preliminary results from an ongoing Phase I/II trial with the same treatment showed evidence of extensive metabolic correction of peripheral and central compartments in patients with MPS-I, with no safety concerns to date [85] (Table 1).

Until now, development of HSC GT has mainly focused on rare monogenic hematologic diseases. However, ex vivo HSC GT also holds great promise in other areas of unmet medical need in less-rare conditions [93]. The ability to modify cells such as macrophages that engraft in multiple tissues raises the possibility of delivering therapeutic or disease modifying genes in multiple different organs systems. The clinical benefit seen from this approach in rare severe forms of neurodegeneration such as MLD and X-ALD [6, 89] raises the question as to whether more common forms of neurodegeneration could also be addressed through HSC GT. Although many forms of adult dementia are not monogenic or clearly genetically defined, some specific genetic susceptibilities are now being identified [94] and could be targeted through this approach. Clearly, there are a number of issues that would need to be addressed, but given the lack of other therapeutic options for these severe and common diseases, the ability to use an approach proven to deliver therapeutic genes and proteins to the CNS is worthy of investigation. Similarly, HSC GT has been shown to ameliorate severe colitis in X-CGD [95], presumably through the migration of gene-modified macrophages to the gastrointestinal tract, which raises the possibility of whether other, more common genetically defined forms of colitis could be addressed through such an approach. Other target organs include the lung or liver through delivery of gene-modified pulmonary alveolar macrophages or Kupffer cells, respectively (Fig. 1b).

Challenges & solutions for the commercial application of ex vivo HSC GT

While the utility of ex vivo HSC GT is built on a solid theoretical foundation, and the benefits observed in a clinical trial setting are significant, transitioning this approach to the commercial arena poses a number of issues. One main challenge is scaling up manufacturing from investigational studies run by academic laboratories to commercial operations to meet the expanding number of clinical indications, with potentially larger global patient cohorts [57, 96]. Improvements within other representative therapeutic sectors (e.g., monoclonal antibodies) demonstrate how technological innovations can transform manufacturing processes, and more importantly, improve the overall costs and scale of medicinal product manufacture [97, 98].

Refining stem-cell subset identification and isolation, generation of commercially scalable stable vector producing cell lines with reduced production costs, and using transduction enhancing compounds to substantially reduce vector requirement are three areas where transformative changes could be implemented.

Cell isolation based on expression of the surface marker CD34 is the predominant method currently used to select and manufacture HSCs for autologous and allogeneic transplantation [65]. However, as described above, CD34+ HSCs are a heterogeneous mixture of progenitors at various stages of differentiation, with only a small fraction of these cells holding true multilineage long-term repopulating potential. Several experimental studies have identified distinct subsets within CD34+ HSC, based on co/expression of markers including CD133+, CD38−/low, CD164+ and CD90+CD45RA among others [13,14,15,16,17,18]. Further research has identified a subpopulation of CD34+ HSCs that can fully support functional hematopoietic reconstitution in an experimental large animal in vivo HSCT setting [15], where engraftment of CD34+CD45RACD90+ HSCs quantitively predicted transplant success. Identifying and selecting more primitive stem-cell populations from within total CD34+ cells would allow reduction of the number of HSCs required for viral transduction and processing, while delivering a more potent cellular GT product (Fig. 4). Development of this approach would likely need selection of both long- and short-term progenitors, which are known to contribute toward stable hematopoiesis following HSCT, as shown by longitudinal clonal tracking of gene-modified HSC clinical transplants [63, 64]. A parallel area for development is through application of supplements or basal media tailored to maintain and further improve HSC progenitor viability, cell expansion, and function, during ex vivo processing [99, 100].

Fig. 4: Innovative approaches for transforming commercial gene therapy.

BAC bacterial artificial chromosome, HSC hematopoietic stem cell, TE transduction enhancing, VSV-G vesicular stomatitis virus glycoprotein.

Conventional manufacturing of LVs is done by transiently transfecting producer cells, via genetic information carried on a plasmid, to produce the lentiviral particles [57]. This approach is labor-intensive, introduces batch-to-batch variation, challenging to scale-up, and requires expensive starting materials through the need to generate Good Manufacturing Practice plasmids on a regular basis. Stable cell producer lines, by contrast, have the potential to revolutionize the generation of LVs and have been the focus of efforts to reduce the vector cost, as they are the most expensive component of HSC GT manufacture (Fig. 4) [101,102,103,104,105]. By introducing all the vector components stably into a clonal cell line, high-titer vector can consistently and efficiently be manufactured without the need for the extra transfection step, delivering a more cost-effective vector manufacturing process. This is further improved by the use of suspension-based vector producing cell systems, which are more conducive to larger scale production platforms and bioreactors [104].

Transduction enhancers are chemical agents that boost uptake of LV into HSCs, which have historically been difficult to transduce, necessitating the application of large quantities of viral vectors [106]. Ongoing research aims to identify transduction enhancers that can facilitate the same output of gene-transduced cells using a much smaller amount of viral vector, which can translate to a dramatic reduction in cost. Indeed, various enhancers are already applied in routine commercial gene and cell therapy manufacture (Fig. 4). Transduction enhancer activity can be mediated at several cellular levels, such as viral cellular attachment, entry or integration [107,108,109,110] and can deliver significant fold improvements in HSC transduction efficiency and number of lentiviral integrations [100]. As compounds with enhancer activity may act through several pathways, it is imperative to ensure that HSC engraftment and multilineage commitment are not compromised by their application.

The current method of cell handling is manual, requiring multiple steps of cell manipulation in sterile environments. Recent technological advances in closed system manufacturing and automated cell handling could offer significant efficiencies in the manufacturing process, while ensuring adherence to exacting manufacturing standards [111].

It is also important to understand what regulatory pathways are available for the GT being developed. If the GT fills an unmet need or addresses a serious, life-threatening condition, several expedited pathways may be available [112, 113].

Ensuring rigorous clinical trial designs that capture the necessary data required to convince payers and providers that these potentially higher priced therapies are better options for patients compared with traditional therapies, and possibly less expensive over the lifetime of the patient as they have the potential to require only a single treatment, are vital. Carefully planned preclinical studies can also provide essential information on a range of variables such as gene expression, phenotype correction, and toxicity in murine models, while optimal transduction conditions for engraftment, biodistribution, and genotoxicity can be assessed using xenotransplantation of patient CD34+ cells into immune-deficient mice.

Keeping the cost of gene therapies as reasonable as possible is a key consideration. Payers consider not only the efficacy and safety of a GT but also its cost-effectiveness relative to the current standards of care available, their level of efficacy, and overall impact on patient clinical burden and wellbeing. In this respect, treating patients with one-time, potentially curative treatments represents a radically different approach to the chronic care model that has traditionally been adopted.

Finally, improvements in diagnosis and more comprehensive newborn screening to ensure rapid and accurate diagnoses and better disease education are especially important in rare diseases. This is particularly pertinent for genetic diseases, which progress rapidly and need to be treated before they cause irreparable damage. Identifying patients early also maximizes the number of patients eligible to receive this potentially curative treatment.


Great strides have been made in the development of HSC GT over the last few decades. A wealth of clinical evidence now suggests that it offers a stable, durable, efficacious, and safe treatment for a number of rare diseases. Through the genetic modification of multipotent autologous HSCs that retain the capacity to engraft, self-renew, and produce multilineage blood cell progeny that express the therapeutic gene, HSC GT has the potential to transform the lives of patients with genetic diseases that affect multiple organs and without the complications of allogeneic HSCT. The future of HSC GT could also potentially include therapeutic targets beyond rare disease, such as more common forms of neurodegeneration or disorders of other organ systems. Moving forwards, innovation, investment, collaboration, and rigorous clinical development will be required to realize the transformative potential of HSC GT.


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We would like to thank Robin LeWinter, PhD, Leslie Meltzer, PhD and Denise Sarracino, PhD from Orchard Therapeutics (Europe) Ltd for their help coordinating the writing of the paper. Editorial support, based on authors’ direction, was provided by Ben Drever PhD from Comradis, UK, and was paid for by Orchard Therapeutics Ltd.


This research was supported by Orchard Therapeutics Ltd and medical writing assistance was provided by Ben Drever, PhD, of Comradis, UK and funded by Orchard Therapeutics Ltd.

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Correspondence to H. Bobby Gaspar.

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PS and HBG are employees of Orchard Therapeutics Ltd.

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Sagoo, P., Gaspar, H.B. The transformative potential of HSC gene therapy as a genetic medicine. Gene Ther (2021).

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