Position effects and transgene silencing remain major obstacles to the generation of stable high-producing CHO cell lines. Applying genetic regulatory elements, such as a ubiquitous chromatin-opening element (UCOE), can mitigate these limitations by maintaining a transcriptionally permissive chromatin environment and supporting sustained transgene expression. In parallel, EGFP-based fluorescence-activated cell sorting (FACS) enables a rapid and efficient platform for the identification and enrichment of high-producing cell populations. Here, we have used the combined strategy of FACS-based screening and UCOE to accelerate the clonal selection and enhance recombinant Darbepoetin alfa (DPO) productivity in Chinese Hamster Ovary (CHO) cells. Accordingly, two plasmids were designed: pOptiVEC™ (non-UCOE) and CET1019HD (containing a UCOE). Both contained a codon-optimized Darbepoetin alfa-LoxP-IRES-EGFP-LoxP-IRES-DHFR fragment. To achieve a stable cell line, the cassettes were linearized and transfected into CHO DG44 cells. Then, EGFP was used as a selection marker in FACS to enrich cells with the brightest green fluorescence intensity. Subsequently, DPO and EGFP expression were assessed at the transcriptional and protein levels using qRT-PCR, Flow cytometry, western blotting, and ELISA. Expression analysis revealed that all UCOE-containing cell pools exhibited higher DPO yield compared with non-UCOE populations. Indeed, FACS sorting and enrichment of UCOE-containing cells led to a clone with more than an eightfold increase in productivity. Moreover, isolating high-producing cells via FACS with a simple gate yielded a 1.5-fold increase in target protein concentration compared with unsorted cells. This study demonstrated that UCOE-mediated transcriptional stabilization establishes a robust expression framework while EGFP-based FACS screening enables efficient enrichment of high-producing cells. Combining them enhances protein yield with potential for further optimization in larger-scale applications.

The international burden of neonatal brain injury (NBI) remains significant with enormous potential for therapeutic intervention. Recent large trials have demonstrated no neuroprotection with either erythropoietin or darbepoetin for term or pre-term brain injury. Neuroprotective strategies with strong evidence are currently limited to antenatal steroids, antenatal magnesium, and supportive care for pre-term NBI and therapeutic hypothermia for term hypoxic-ischemic encephalopathy. Of the pharmacologic strategies currently being investigated clinically, the agents with the highest likelihood of success and closest to clinical implementation include melatonin, topiramate, and stem cell-based therapies. More therapies to prevent or treat NBI are urgently needed.

Inflammation suppresses erythropoiesis, leading to non-regenerative anemia in dogs. Currently, clinical evidence of this inhibitory effect remains limited. This study provides important clinical insight by demonstrating a negative correlation between C-reactive protein (CRP) concentration and reticulocyte count over time in dogs with inflammatory anemia. This study aimed to leverage inflammatory anemia as a model to examine the longitudinal relationship between CRP levels and reticulocyte counts in dogs. Medical records of 23 dogs diagnosed with various inflammatory conditions and persistently non-regenerative erythropoiesis were retrospectively reviewed. Serial results of hematological and serological parameters were analyzed using linear mixed-effects models to account for intra- and inter-individual variation. Reticulocyte counts in anemic dogs with resolving underlying inflammatory conditions were significantly negatively correlated with CRP levels (standardized β=-0.53, 95% confidence interval (CI) -0.68 to -0.37; P<0.001). Moreover, cross-sectional analysis revealed that higher CRP levels were significantly associated with lower reticulocyte counts, suggesting that the severity of inflammation may be linked to reduced reticulocyte counts (Pearson's r=-0.5; P=0.01). Similarly, erythropoietic responses to darbepoetin alfa appeared to be attenuated in dogs with elevated CRP concentrations. In these dogs, whose mean CRP concentration was 6.15 mg/dL, the onset of reticulocytosis was delayed approximately twofold, and the increase in peak reticulocyte count was approximately 38% lower than that in dogs with normal CRP concentrations. This study provides clinical evidence supporting an association between systemic inflammation and reduced or delayed reticulocytosis in dogs, highlighting the clinical utility of concurrently monitoring CRP and reticulocyte levels to assess disease progression and inflammatory recovery.

Stable and robust production of recombinant therapeutic proteins in mammalian cells remains challenging due to position effects and transcriptional silencing of the transgene following random integration. We investigated whether semi-targeted integration and UCOE-incorporation could enhance Darbepoetin alfa (DPO) transgene expression. A PiggyBac-mediated Darbepoetin alfa-secreting cell pool (PB-DPO) was established by semi-targeted insertion of a DPO-containing expression cassette using pB513B-1, a PiggyBac transposon-derived vector, into CHO DG44 host cells. A UCOE-incorporated Darbepoetin alfa-secreting cell pool (UCOE-DPO) was also used to compare DPO production rates between the two groups and the control (pOptiVEC-DPO) cell pool. DPO expression at both transcript and protein levels was compared across DPO-producing cell pools using real-time quantitative PCR, Western blotting, and ELISA. The PB-DPO showed ~ 1.96- and ~ 1.79-fold increases in mRNA and protein levels, respectively, whereas the UCOE-DPO exhibited even stronger enhancement, with ~ 2.94- and ~ 4.53-fold increases compared with the control. In conclusion, PiggyBac-mediated transgene transposition generated a cell population with increased transgene expression, likely through multiple insertions and preferential integration near transcriptionally active regions. Although both modified pools demonstrated increased DPO transcript and protein yields, UCOE-DPO was more effective at overcoming integration-associated silencing. Compared with the semi-targeted transposition method, UCOE incorporation further increased Darbepoetin alfa production by ~ 2.5-fold.

A patient with metastasized prostate adenocarcinoma underwent [18]F-PSMA-11 PET/CT restaging following [177]Lu-PSMA-617 treatment and rising PSA-level. The maximum intensity projection image (right-side of the figure) demonstrated an intense homogenous tracer uptake throughout the bone marrow containing skeleton, whereas CT images showed specific bone metastases but no diffuse involvement, and previously known lymph node and liver metastases exhibited significantly reduced or absent uptake (see also left-side of the image, [18]F-PSMA-11 MIP prior to [177]Lu-PSMA-617 therapy). None of the patients that underwent imaging with the same tracer-batch demonstrated similar findings excluding free [18]F as a potential cause for the imaging findings. Furthermore, the patient did not suffer from any bone marrow disorder previously reported to present with diffuse bone marrow uptake on PSMA-targeting PET/CT imaging [1, 2]. Of interest, the patient had received recombinant human erythropoietin, 100 µg of Aranesp, 24 h prior to imaging to treat his renal failure associated anemia. At this dose, Aranesp reaches a nanomolar concentration in the bone marrow resulting in stimulation of the Tissue Protective Receptor on endothelial cells leading to the creation of a massive systemic reservoir of PSMA-positive neovasculature that outcompetes PSMA-expressing prostate carcinoma lesions for radiotracer availability [3, 4]. This may explain the diffuse marrow uptake and the decreased visibility of known metastases [4]. The effect of erythropoietin on bone marrow mobilization and neovascularization can persist for 7-10 days [5]. This should be taken into consideration in order to avoid erroneous interpretation of [18]F-PMSA-11 PET/CT imaging and unnecessary bone marrow toxicity of [177]Lu-PSMA-617 treatment.

Erythropoietin-hybrid Fc fusion protein (EPO-hyFc), comprising an EPO domain fused to a hybrid IgD-IgG4 Fc, is a next-generation erythropoiesis-stimulating agent with an approximately two-fold longer serum half-life than darbepoetin alfa, a clinically established long-acting EPO formulation. Although sialylation at Asn-38 and Asn-83 is known to modulate serum stability and half-life, the site-specific N-glycosylation of EPO-hyFc has not been characterized. Here, we performed comprehensive N-glycan profiling using LC-MS/MS glycomics combined with nano-LC-MS/MS glycoproteomic analysis of Glu-C-digested peptides. In total, 23 N-glycans (15 sialylated and 8 neutral) were identified. Site-normalized quantification revealed that Asn-24 was mainly occupied by mono- and di-sialylated glycans (64.9%), whereas Asn-38 (76.9%) and Asn-83 (87.7%) were enriched in tri- and tetra-sialylated structures. The Fc site (Asn-261) contained mainly non-sialylated glycans (94.4%). The average number of sialic acids per N-glycan was 2.2, and the sialic acid-capping ratio was 90.9%, indicating extensive terminal sialylation across the EPO sites. These results provide the first site-specific characterization of N-glycosylation in EPO-hyFc and offer structural and quantitative insights for optimizing its stability, pharmacokinetics, and therapeutic efficacy.

Post-marketing surveillance was conducted in Japan (2015-2024, UMIN000056049) to evaluate the long-term safety and effectiveness of darbepoetin alfa for anemia with myelodysplastic syndromes (MDS) for 5 years after treatment initiation. Among 1834 patients (median age: 79.0 years), the distribution of International Prognostic Scoring System (IPSS) risk categories was: Low, 39.3%; Intermediate-1, 45.1%; Intermediate-2, 7.7%; and High, 2.8%. The median (range) administration duration, dosing interval, and weekly dose were 330.0 (1-1863) days, 1.7 (0.2-41.5) weeks, and 240.0 (30.0-250.0) µg, respectively. Treatment discontinuation reasons included inadequate response (36.9%) and adverse events (AEs) (31.2%). AEs were reported in 75.1% and included pneumonia (16.6%) and transformation to acute myeloid leukemia (9.0%). Mean hemoglobin concentration (n = 1821) increased from 7.6 g/dL at baseline to > 9.0 g/dL after 52 weeks. Red blood cell transfusion volume decreased by ≥ 50% compared with baseline in 65.2% of patients. In transfusion-dependent patients (n = 909), the highest transfusion-independence rate was 40.9% at year 4-5. Five-year overall/leukemia-free survival rates (n = 1826/1814) by IPSS risk group were: Low, 49.5%/48.8%, Intermediate-1, 31.1%/30.2%, Intermediate-2, 11.9%/10.4%, and High, 0%/0%. Poor prognostic factors for MDS were IPSS risk category, age, chromosomal abnormalities, and platelet count. No new safety or effectiveness concerns were identified.Trial registration: University Hospital Medical Information Network; study ID: UMIN000056049.