In vivo hematopoietic stem/progenitor cell (HSPC) gene therapy remains limited by low gene-editing efficiency and a lack of clinically applicable selection strategies to enrich therapeutically corrected progeny. We used in vivo base and prime editing to introduce a nonpathogenic EPOR variant into HSPCs, conferring erythropoietin hypersensitivity and promoting preferential expansion of gene-corrected erythroid cells. EPOR editing was combined with three therapeutic approaches for the correction of hemoglobinopathies: γ-globin gene addition, γ-globin reactivation, or correction of the sickle cell disease mutation. Tropism-modified helper-dependent adenoviral vectors (HDAd6/3+) targeting HSPCs were used to simultaneously deliver the EPOR-editing machinery and the corresponding therapeutic components. In vitro studies in an erythroid progenitor cell line and primary CD34+ cells demonstrated that the EPORW439* variant conferred a strong proliferative advantage to therapeutically modified erythroid progenitors. Mice humanized with CD34+ cells from a β[0]/β[0]-thalassemia patient were subjected to EPORW439*-mediated EPO hypersensitivity alongside a therapeutic γ-globin transgene which resulted in >70% HbF-positive erythroid cells and substantial reversion of the disease-associated phenotype, including reduced oxidative stress, near-complete elimination of splenic iron deposition, and reduced splenomegaly. Importantly, these effects were achieved after simple intravenous administration of the vectors following HSPC mobilization and cytokine prophylaxis, without subsequent pharmacologic selection. Together, these findings establish a strategy to amplify the therapeutic benefit of otherwise limited in vivo HSPC gene editing for hemoglobinopathies.

Sickle cell disease (SCD) is a debilitating hereditary hematologic disorder characterized by severe physical complications, including painful vaso-occlusive crises, chronic pain, multi-organ damage, and reduced life expectancy. Diverse medications are prescribed to treat SCD or manage its associated complications. However, no comprehensive resource for concomitant medication and drug-drug interaction (DDI) risk exists for people living with SCD. Here, we utilized real-world claims data from the Merative Commercial MarketScan Research Database to identify the 100 most prescribed drugs for adolescents and adults living with SCD in the United States with commercial health insurance. DDI risks were assessed by leveraging the CERTARA Drug Interaction Database. In total, 43/100 drugs on the list were associated with DDI risks. Inhibitors were more frequently represented than inducers, suggesting that interaction risk in this population may be driven primarily by inhibition-mediated mechanisms. CYP3A4 accounted for the largest proportion of identified interactions. The high prescription rates of the CYP3A4 substrates oxycodone (40%) and fentanyl (18.5%), associated with potentially serious clinical consequences due to DDIs, highlight the importance of considering CYP3A4 interaction risk during development of novel therapies for SCD. Overall, this analysis provides a population-level insight into prescription patterns and subsequent DDI risks for people living with SCD. We expect these findings to (1) create clinical DDI risk awareness for this population and (2) inform strategic planning of DDI assessments during development of novel therapies indicated for SCD.

HbF concentration influences the clinical phenotype of sickle cell disease (SCD) and the prevailing management approach involves pharmacological or genetic stimulation of HbF expression. Understanding how HbF affects the disease's progression, in the context of other potential modifiers, is vital. Kuwaiti patients with SCD predominantly carry the Arab/Indian haplotype, and their average spontaneous HbF concentration varies from ∼15-30%. Our team has conducted a series of clinical studies on these patients between 1994 and 2024 to understand their clinical course; some of these studies are summarized here. Most patients show no symptoms before age 4 or 5, when their HbF level is about 30%. Splenic function remains intact in childhood and in 60-80% of adult patients, which may explain the rarity of severe bacterial infections. Other less common complications include stroke, leg ulcers, and priapism. Most patients do not develop cerebral vasculopathy, as shown by normal transcranial Doppler studies. However, osteonecrosis of the femoral head is relatively common, affecting about 25% of children and over 50% of adults. The factors that predispose to this complication remain unclear. While the overall phenotype in pediatric patients is generally mild, the disease becomes quite severe in adults, indicating that subclinical inflammatory events accumulate over time. The pattern observed in our patients provides insight into what to expect in those treated with HbF inducers or gene therapy. Indeed, increasing HbF alone is not a cure for SCD. The greatest benefit of elevated HbF is seen in younger patients, making early intervention crucial.

Type III CRISPR systems utilize a complex containing Cas10, additional Cas proteins and a crRNA to detect foreign transcripts. Upon detection, Cas10 synthesizes cyclic oligoadenylates (cOA), signaling molecules that coordinate interference by stimulating downstream enzymes with DNase, RNase, protease or other activities. Type III systems possess properties well-suited to their deployment as molecular diagnostics: specific detection and activation of a cascade of multi-turnover enzymatic reactions that can be harnessed for signal generation. We answered key questions concerning the structure and function of Serratia Cas10-Csm to facilitate its use in molecular diagnostics. We determined that Serratia Cas10-Csm (SerrCas10-Csm) synthesizes predominantly cA3 molecules and this synthesis is sensitive to mismatches in the crRNA-target RNA duplex adjacent to Cas10. To understand how Cas10 senses the presence of a bound target RNA, we determined the structure of SerrCas10-Csm unbound and bound to target RNA identifying conformational changes associated with target binding. We programmed SerrCas10-Csm with a crRNA recognizing a single nucleotide polymorphism that occurs in the human HBB transcript and is associated with sickle cell disease. We demonstrate that when the polymorphism occurs in the +1 position of the crRNA-target duplex, SerrCas10-Csm can distinguish it indicating an additional role for type III CRISPR systems in point-of-care diagnostics in low-resource settings.

Nitric oxide (NO)-based therapy represents a promising strategy against multidrug-resistant infections owing to its broad-spectrum antibacterial activity and minimal propensity to induce resistance. Hydroxyurea (HyU), a clinically approved drug for sickle cell disease, has recently emerged as a hydrogen peroxide (H2O2)-responsive NO precursor. However, the hydroxamic acid moiety responsible for NO generation is highly susceptible to hydrolytic degradation in aqueous environments, severely limiting its therapeutic utility. Herein, we report a coordination-driven stabilization strategy that incorporates HyU as a coordinating component within a Cu(II)-based metal-organic framework (CuPH). Coordination with Cu(II) preserves the chemical integrity of HyU for at least 14 days under physiological conditions, enabling its use as a stable NO precursor within a glucose-responsive nanoreactor. Using this stabilized framework, glucose oxidase (GOx) is co-confined within CuPH to construct a nanoreactor (GOx@CuPHcs). Under hyperglycemic wound conditions, GOx catalyzes in situ H2O2 generation from glucose, which subsequently activates Cu-mediated oxidation of coordinated HyU to achieve glucose-gated and on-demand NO release. The generated NO effectively eliminates methicillin-resistant Staphylococcus aureus (MRSA) and disrupts mature biofilms. GOx@CuPHcs was also associated with macrophage phenotype remodeling, enhanced fibroblast migration, and accelerated wound healing in a diabetic murine model with negligible systemic toxicity. This work demonstrates that coordination interactions can improve the stability of hydrolytically labile therapeutic molecules while enabling their integration into stimulus-responsive nanoreactors, highlighting the potential of coordination-based strategies for developing advanced therapeutic systems.

Hereditary hematological disorders, including sickle cell disease, β-thalassemia, and hemophilia, are severe monogenic diseases that impose substantial morbidity and lifelong treatment burdens. Conventional therapies are largely supportive and rarely curative, whereas gene therapy is increasingly transforming the therapeutic landscape by addressing the underlying genetic defects. This review summarizes the major gene therapy strategies currently being developed for hereditary hematological disorders, with a particular focus on gene addition, gene editing, and gene silencing, as well as ex vivo and in vivo delivery platforms. We highlight recent clinical breakthroughs, including approved products for hemoglobinopathies and hemophilia, and discuss emerging approaches such as base editing, prime editing, epigenetic modulation, and lipid nanoparticle-mediated delivery. In addition, we examine the major challenges that continue to limit broader clinical adoption, including immune responses, off-target effects, conditioning-related toxicity, manufacturing complexity, high cost, and limited accessibility. Finally, we outline future directions that may accelerate clinical translation, including improved editing precision, next-generation vector engineering, artificial intelligence-assisted design, scalable manufacturing, and more equitable access to treatment. Together, these advances suggest that gene therapy is steadily moving from experimental innovation toward durable, potentially curative treatment for hereditary hematological disorders.

We developed CRISPR_SCD001, an autologous cellular therapy in which the pathogenic HBB sickle allele is corrected by CRISPR-Cas9-mediated homology-directed repair using a single-stranded oligodeoxyribonucleotide as DNA donor template. The Drug Product (DP) from Plerixafor-mobilized peripheral-blood CD34[+] cells from sickle and healthy donors were used for: 1) pharmacodynamic assessments to interrogate therapeutic potential; 2) evaluation of genotoxicity; and 3) in vivo characterization by a non-GLP toxicology study in NBSGW mice. Large-scale DP lots yielded 89 ± 5 % CD34[+] purity with 84 ± 6 % viability. Mean gene correction at HBB was 22 ± 4 % of alleles, with 51 ± 6 % of allelic disruption. Edited erythroid cultures produced 39 ± 5 % adult hemoglobin and 40 ± 6 % fetal hemoglobin, reducing sickle hemoglobin from 89% to 22 ± 6 %. In NBSGW mice, CRISPR_SCD001 engrafted robustly (59 ± 25%), maintained editing frequencies comparable to injected cells (∼20% gene correction), showed polyclonal distribution of the edited cells, and no noteworthy treatment-related toxicity or pathologic evident were found by a blinded histopathology assessment. Our protocol generates a clinical-grade, cryopreserved CD34[+] cell product that corrects the sickle mutation, restores anti-sickling hemoglobins, and meets pre-clinical safety criteria, establishing a reproducible manufacturing process suitable for clinical translation. These data support initiation of a first-in-human phase I trial of CRISPR_SCD001 for sickle patients presenting with severe pathology.