Diamond-Blackfan anemia (DBA) is an intriguing and enigmatic disease that has held the attention of clinicians and scientists alike for close to a century. First described as congenital hypoplastic anemia in 1936 (1, 2), it is characterized by macrocytic anemia, reticulocytopenia, erythroblastopenia, and constitutional anomalies including craniofacial, skeletal, and cardiac abnormalities in about 50% of the patients (3). DBA is an ultra-rare disease, with an incidence of 2-8 per million live births per year (3, 4). Approximately 90% of the patients are diagnosed in the first year of life with 35% presenting in the first 4 months of life with progressive anemia (3, 5). The phenotypic presentation is diverse and can widely vary in between family members with the same mutation (5). Despite profound improvements in our understanding of the molecular mechanisms of this disorder, no newer therapies have met clinical approval since original report of corticosteroids as an effective option in 1951 (6).

DBA is a ribosomopathy caused by loss-of-function mutations or deletions involving ribosomal protein genes (5). Out of the 83 ribosomal protein (RP) genes, 23 RP genes have so far been reported to be associated with DBA, where the clinical features are secondary to impaired ribosomal biogenesis (3). RPS19 mutations account for 25% of cases. Very rarely, mutations in non-RP genes such as EPO and GATA1 have been reported cause a DBA-like disease characterized by erythroblastopenia, without any impairment ribosomal biogenesis (5). Several studies have partially elucidated the mechanisms of DBA-associated anemia. DBA bone marrow shows increased apoptosis and impaired maturation of erythroid progenitors (7–10). Potential explanations for these erythroid defects include reduced translation of the essential erythroid transcription factor GATA1 (11), build-up of cytotoxic free heme due to reduced translation of globin proteins (12), and activation of TP53 via several potential mechanisms (9, 13, 14). In addition to a specific erythroid failure seen in younger patients, bone marrow hypocellularity, progressive multilineage cytopenias and immunodeficiency are noted in older patients with DBA, suggesting impairment of hematopoietic stem and progenitor cells (HSPCs) (15–17). Mechanisms of DBA HSPCs dysfunction are not clear, although TP53 activation appears to play a critical role (18). Additionally, systematic review of published case reports suggested an increased risk of cancer in DBA, which was subsequently confirmed by pioneering studies lead by the DBA Registry of North America (DBAR) (19–21). These prospective studies established DBA as a cancer predisposition syndrome with increased risk of MDS/AML and solid tumors such as colon carcinoma and osteosarcoma (20, 22, 23). While the exact mechanisms underlying this risk of malignancy are unknown, acquired somatic mutations leading to clonal hematopoiesis have been reported in DBA (24). For a more thorough review of the DBA pathophysiology, the readers are referred to recent review articles by Da Costa et al. (3, 5, 25 )

The three major therapeutic options for anemia in DBA are packed red blood cell (pRBC) transfusions, corticosteroids (prednisone), and allogeneic hematopoietic cell transplantation (HCT) ( Figure 1A ) (27). Majority of patients are diagnosed during their infancy and initially supported with pRBC transfusions during the first year of life after which corticosteroid treatment is initiated. Oral corticosteroids have been the mainstay of DBA treatment for well over half a century. Oral prednisone is started at a dose of 2 mg/kg/day (or 80 mg/day in adults) for a 2-week period and then tapered over several weeks to months to a maximum maintenance dose of 0.3 mg/kg/day. Although 60-80% of the patients respond to corticosteroids initially, at least half of them will either lose responsiveness or develop steroid intolerance over time (27). Patients who require a maintenance dose > 0.3 mg/kg/day or those that do not respond to prednisone are supported with chronic pRBC transfusions. A second trial of prednisone may be attempted at least 12 months after the first attempt as anecdotally some children may subsequently become responders (27). Prompt evaluation and treatment for iron overloading are critical in transfusion-dependent patients to avoid secondary organ damage and to improve outcomes for a potential HCT.

Advances in the understanding of the pathobiology underlying DBA has led to newer medical therapies which are currently being investigated. L-leucine has been shown to improve translational efficiency and activate mTOR pathway resulting in improvement of anemia in animal and cell models of DBA and was recently demonstrated to be safe with modest efficacy in patients with DBA (28–30). Recent studies have suggested that RP deficiency can lead to reduced globin chain production in erythroid cells, resulting in accumulation of free heme that is cytotoxic to erythroid progenitors and precursors (12, 31). Based on this hypothesis, eltrombopag is predicted to improve erythropoiesis in DBA by chelating intracellular iron. Preliminary studies were affected by eltrombopag-induced thrombocytosis and showed low response rate (32). Alternate approaches to inhibit heme accumulation, such as glycine transporter 1 inhibitors (33), are currently being explored (34). Trifluoperazine, which was shown to improve erythroid defect in experimental DBA models by reducing TP53 activity via calmodulin inhibition, is being evaluated for clinical use (14).

Allogeneic HCT remains the only option for hematopoietic cure in patients with DBA. The first HCT for DBA was performed in 1976, proving that hematological cure is possible (35). Numerous case reports since then confirmed the feasibility of HCT in DBA. The current accepted recommendations for HCT in DBA include transfusion dependence, non-responsiveness to steroids, requiring > 0.3 mg/kg/day of steroids, unacceptable adverse effects from steroid therapy, clinically relevant cytopenias such as neutropenia and transfusion-dependent thrombocytopenia, and evolution to hematologic malignancy (36).

The earliest published data from DBAR showed 87% overall survival (OS) after HCT from HLA-matched related donors (MRD) (37). However, in this early report OS after alternative donor HCTs was only 14%. Multiple retrospective studies, across the globe, have since confirmed the good outcomes after MRD HCT ( Table 1 ) (38, 41–44, 46). Outcomes for HLA-matched unrelated donor (MUD) HCTs have shown improvement over the last two decades, mainly due to advances in supportive care, high-resolution HLA typing and better donor-recipient matching. More recent data from the Franco-German DBA registries with 70 DBA patients showed comparable OS between MRD and MUD HCT recipients (91% vs 92%). However, in patients that underwent HCT after year 2000, recipients of MUD HCT had slightly lower chronic graft-versus-host disease (cGVHD)-free survival (GFS) compared to MRD HCT recipients (87% vs. 100%, p=0.06) (47). Similar observations about comparable OS after MRD HCT and MUD HCT were made by the European Society for Blood and Marrow Transplantation (EBMT) and Italian Association of Pediatric Hematology and Oncology Registry ( Table 1 ), although GFS was not reported (41, 46). Overall, these data show that 1) MRD HCT outcomes continue to be excellent and 2) OS after MUD HCT is comparable to those after MRD HCT. In the following sections, we will discuss more specific factors affecting outcomes after HCT in DBA.

Once RPS19 was identified as the first DBA gene (followed by discovery of >20 other DBA genes), gene complementation emerged as a potential therapeutic option (74). Although major advances have been made in the last few decades, several preclinical safety, efficacy and regulatory challenges remain before these transformative therapies are ready for clinical use. Autologous HCT of genetically-manipulated HSPCs could overcome several disadvantages of allogeneic HCT, namely a) lack of suitable donors for many patients b) immune toxicities such GVHD c) risk of graft rejection, and d) risk of donor-related hematopoiesis. In general, autologous gene therapy for hematological diseases is carried out by isolating patient HSPCs, genetically correcting them ex vivo, followed by conditioning chemotherapy and infusion of corrected HSPCs ( Figure 1B ). Genetic correction of HSPCs can be achieved using lentiviral vector to express wild type (healthy) copy of the affected gene or using CRISPR/Cas9 or related nucleases for targeted genetic correction of the mutation in the endogenous locus.

Early preclinical work on DBA gene therapies in laboratory models, primarily used retroviral vectors and subsequently used self-inactivating (SIN) lentiviral vectors (75–78). The third-generation SIN lentiviral vectors incorporate several safety features such as deletion of viral enhancer/promoter sequences within the U3 region of 3’ Long Tandem Repeat (LTR), removal of all viral protein genes from the vector plasmid and deletion of viral Tat sequence (79). These changes improve safety by reducing the risks of insertional gene activation and replication-competent lentiviral vector generation (79). Preclinical DBA gene therapy studies have primarily focused on RPS19, the most common DBA gene (5). One of the major challenges has been limited access to DBA CD34⁺ HSPCs for clinical development, as typically millions of CD34⁺ cells are required for these studies. We recently developed a CRISPR/Cas9-based approach to model RPS19-mutated DBA using CD34⁺ HSPCs from healthy donors (18). Utilizing this model, we demonstrated the efficacy of an RPS19-expressing SIN lentiviral vector (17, 18). Recently, reduced translation of the erythroid transcription factor GATA1 was suggested to cause the characteristic erythroid defect of DBA, regardless of the mutated RP gene (11). This raises the possibility of forced erythroid-specific expression of GATA1 as a DBA gene-agnostic therapeutic strategy. Preliminary data using this approach are promising (80).

One of the disadvantages of lentiviral vectors is the inability to control gene dosage in transduced HSPCs. While this is not an issue for RPS19 protein, whose levels are tightly regulated (75), RPL5 and RPL11 overexpression can potentially lead to TP53 stabilization by inhibiting MDM2, a negative regulator of TP53 (81). Therefore, overexpression of RPL5 or RPL11 can potentially lead to TP53-dependent cell death. Hence, targeted approaches such as knock-in of wildtype cDNA cassette following CRISPR/Cas9 editing, precise nucleotide base editing and prime editing need to be explored as future therapeutic options. These novel and promising technologies have shown effectiveness in preclinical setting for other inherited hematopoietic disorders (82–84). Gene correction using knock-in approaches have been shown to be effective in preclinical models of diseases such as chronic granulomatous disease and sickle cell anemia (85, 86). Base editing involves Cas9-nuclease-derived proteins that can precisely convert an adenine to guanine or cytosine to thymine (87). Base editing has been shown to be effective in correcting preclinical models of monogenic diseases such as sickle cell disease (83, 88). However, the diversity of pathogenic mutations that cause DBA also means a single base editor-guide RNA combination can only be used for patients with identical mutations, which poses a unique set of challenges from a preclinical, regulatory, ethical and health economics standpoint. An alternative approach could be prime editing to correct a several adjacent mutations within a short segment of DBA using a single prime editing guide RNA (89). This, for instance, could be a viable approach particularly for RPS19 between codons 52 and 62, which is a hotspot for several DBA mutations (90). However, it has to be noted that prime editors are less advanced than base editors in terms of preclinical development. Although there are rapid preclinical advances, several practical challenges remain. These include feasibility of mobilization of adequate HSPCs from patients, optimization of lentiviral transduction or genome editing of DBA HSPCs, thoroughly evaluating safety of gene therapy approaches, and regulatory and financial challenges in developing clinical trials for ultra-rare diseases.

In sum, various approaches are being explored towards development of hematological curative therapy for DBA. These approaches could offer a potentially safe and effective therapeutic option for transfusion-dependent DBA patients, especially for those without an MRD ( Figure 1A ). While MRD HCTs have excellent outcome, there is potential clinical equipoise between MUD HCTs and a future gene therapy approach, given the slightly higher rates of cGVHD following MUD HCTs. Furthermore, gene therapy approaches could ultimately prove to be more cost-effective in the long run compared to MUD HCT due to economies of scale. Therefore, the long-term efficacy and safety will have to be determined through carefully designed clinical trials. In particular, it remains to be seen how the risk of MDS/AML is altered following autologous HCT with gene-corrected HSPCs.

Overall, outcomes after allogeneic HCT have remarkably improved over the last two decades. This has made HCT a safe and viable option for many patients. However, several unanswered questions and unaddressed challenges remain. Outcomes after HCT in adolescent children is improving but remains suboptimal while there is limited data on HCT outcomes in adult patients with DBA. Even less is known about outcomes in DBA patients who develop MDS/AML or aplastic anemia. Other challenging questions include the roles of reduced-intensity conditioning regimen, antibody-based conditioning approaches and haploidentical HCT in DBA. The risk of solid tumors post-HCT is unknown and long-term follow-up studies are needed. Renewed interest in gene therapy has raised hopes for a safer and effective curative option for select DBA patients. Concurrent advances in our understanding of DBA mechanisms have raised interest and hope in other therapeutic agents such as L-leucine (30) and trifluoperazine (14). Progress in medical therapies including iron chelation have the potential to improve life expectancy and quality of life for DBA patients that are unable to undergo HCT. Therefore, outcomes of patients that undergo HCT and gene therapy will have to be continually evaluated and compared with more current outcome data of non-transplanted patients to determine the risk-benefit of these transformative therapies. The critical need for such continual assessment of natural history and outcomes in a rare disease population highlights the important role played by disease registries, patient advocacy groups and global alliances, and the need for synergy and interaction between the various stakeholders.

All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.