Assessing the Safety, Tolerability, and Efficacy of APR-2020 in Pediatric and Adolescent Subjects With RPS19 Deficient Diamond-Blackfan Anemia

This open-label, single-arm study evaluates the safety and efficacy of APR-2020 in transfusion-dependent, steroid-resistant pediatric and adolescent patients with RPS19-deficient Diamond-Blackfan Anemia (DBA). Disease Background: DBA is a congenital bone marrow (BM) failure syndrome characterized by early-onset hypoplastic anemia secondary to selective erythroid aplasia. The cardinal hematologic manifestation is a severe normochromic, macrocytic anemia in the presence of preserved leukocyte and platelet counts. In approximately 90% of affected individuals, hematologic abnormalities manifest within the first year of life; the median age at clinical presentation is approximately 2 months, with a median age at diagnosis of 3 months (Sieff 2023). Genotype-phenotype data have demonstrated substantial clinical heterogeneity both within and across molecular subtypes. Accordingly, the term DBA syndrome has been adopted to reflect the broader phenotypic spectrum, encompassing classic DBA-estimated to occur at an incidence of 5 to 10 per million live births with no significant sex predilection-as well as non-classical or attenuated presentations. Most patients exhibit a reticulocytopenic (hyporegenerative) anemia, consistent with impaired erythroid progenitor differentiation and maturation, with or without associated congenital anomalies or growth abnormalities (Vlachos et al. 2018). DBA is primarily caused by heterozygous pathogenic variants in genes encoding ribosomal proteins (RPs), resulting in ribosomal haploinsufficiency and defective ribosome biogenesis. The most frequently implicated genes include RPS19 (25-30%), RPL5 (7-12%), RPS26 (6-9%), RPL11 (5-7%), RPS24 (2-3%), and RPS10 (1-3%). Additional RP gene variants have been identified at lower frequencies (Sieff 2023; Wlodarski et al. 2024). RPS19 remains the most commonly mutated gene in DBA (Da Costa et al. 2020; Sieff 2023; Wlodarski et al. 2024). Rare pathogenic variants in non-ribosomal protein genes associated with DBA-like phenotypes-such as GATA1, TSR2, and EPO-have also been described but collectively account for fewer than 1% of cases (Da Costa et al. 2020; Wlodarski et al. 2024). Patients eligible for inclusion in the present study have a confirmed diagnosis of DBA attributable to pathogenic variants in RPS19. Study Population: The protocol will enroll 4 subjects with genetically confirmed RPS19-deficient DBA who demonstrate transfusion-dependent, corticosteroid-refractory disease. Study Drug: The study drug, APR-2020 is a gene therapy that consists of autologous cluster of differentiation (CD)34+ hematopoietic stem and progenitor cells (HSPCs) derived from mobilized peripheral blood (PB) from subjects with RPS19-deficient DBA. These CD34+ cells will be exposed ex-vivo to the Sponsor's highly purified and concentrated third generation self-inactivating (SIN) lentiviral vector (LVV), which provides the functioning RPS19 gene. The efficacy of APR-2020 was demonstrated through a rescue of the DBA phenotype in the RPS19-deficient DBA mouse model and through in vitro and in vivo RPS19 gene transfer to RPS19-deficient mouse hematopoietic stem and progenitor cells using Apriligen's proprietary vector. (Liu et al. 2022). APR-2020 is intended to supplement haploinsufficiency of RPS19 gene expression in patients with DBA. APR-2020 provides gene-corrected multipotent progenitor cells with a fitness advantage which is expected to enable engraftment and clonal expansion of corrected cells without the need for myeloablative conditioning, thereby eliminating conditioning-related toxicity in this vulnerable pediatric population. The aforementioned preclinical studies generated data that support evaluating APR-2020 without any genotoxic conditioning. (Dahl et al. 2021; Garelli et al. 2019; Yoshida et al. 2025) Expression of the RPS19 protein has the potential to restore impaired erythroid differentiation and proliferation of erythroid progenitors potentially eliminating the need for RBC transfusions. After APR-2020 infusion, transduced CD34+ HSPCs are expected to engraft in the BM, which will be measured by presence of RPS19 and vector copy number (VCN). Healthy erythroid progenitors have a significant capacity to increase output of RBCs: In healthy individuals, the rate of erythropoiesis can be increased \> 10 fold in response to increased demand, involving physiological signals such as tissue hypoxia and increased production of erythropoietin (Flygare et al. 2011, Peslak et al. 2012). With this 10-fold over capacity, it is possible that just 10% of healthy amounts of gene-corrected erythroid progenitors is sufficient to sustain healthy erythropoiesis. Immunologically matched DBA mouse models can be cured by healthy stem cell transplantation without conditioning: Successful disease correction in RPL11 and RPS19 deficient mouse models using non genotoxic antibody-based conditioning prior to HSCT has been demonstrated (Dahl et al. 2021, Swartzrock et al. 2024). These studies showed that even with minimal donor engraftment, unconditioned transplantation effectively normalized hemoglobin levels in DBA mice. Further, the molecular efficacy report by Freiman et al. 2025 demonstrates induction of pro-apoptotic gene expression programs in multipotent progenitors from patients with DBA that is significantly downregulated after gene therapy. This suggests APR 2020 derived multipotent progenitors expressing RPS19 will have a healthy fitness advantage over the uncorrected progenitors, allowing for selective expansion even without myeloablative conditioning. In addition, Venugopal et al. 2017 documented a case of self reverting mutations in a patient with DBA that partially corrected the blood phenotype. The expansion of reversion events arising independently suggests a selective advantage over the defective cell population with likely contribution to correcting the phenotypic defect. This clinical case provides real-world validation that reversion in just a fraction of HSCs can achieve significant clinical improvement, supporting a non-myeloablative approach for APR 2020 gene therapy. Collectively, these findings provide a strong scientific rationale for initially attempting APR 2020 gene therapy without genotoxic conditioning. The converging evidence demonstrates that even limited engraftment of gene-corrected cells can provide clinical benefit in DBA due to the selective advantage of corrected cells, the amplification capacity of erythroid progenitors, and the effectiveness of even low-level RPS19 expression in rescuing cellular function; this approach prioritizes patient safety by avoiding the serious risks associated with myeloablative conditioning, including organ toxicity, infertility, and secondary malignancies, while still offering potential for therapeutic benefit. In RPS19-associated DBA, haploinsufficiency resulting from monoallelic loss-of-function leads to defective ribosome biogenesis and erythroid failure. Therefore, the emergence of circulating reticulocytes following gene-based intervention implies successful in vivo transgene expression and functional rescue of erythroid progenitors. Reticulocyte-based response criteria serve as a direct measure of restored endogenous erythropoiesis and function as an early biological marker that precedes and predicts subsequent increases in hemoglobin (Wlodarski et al 2024). Because DBA is characterized by a primary defect in erythroid progenitor proliferation and differentiation, impaired RBC production occurs upstream of reticulocyte formation. Accordingly, baseline reticulocyte counts in eligible subjects are anticipated to be profoundly reduced or near absent. For these reasons, the Sponsor considers the appearance of reticulocytes to be a critical early indicator of therapeutic activity following treatment with APR-2020, representing mechanistic correction of erythropoiesis and a leading signal of subsequent hemoglobin recovery. Clinical Significance: This study represents a potentially transformative approach for transfusion-dependent, steroid-resistant RPS19-deficient DBA patients who currently face lifelong transfusion dependence with associated iron overload, or high-risk allogeneic HSCT as their only alternatives. The non-genotoxic conditioning strategy, paired with an autologous gene therapy, if successful, could establish a new paradigm for gene therapy in inherited bone marrow failure syndromes where corrected cells possess intrinsic selective advantage.