Cross-platform transcriptomic profiling of the response to recombinant human erythropoietin
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ABSTRACT:
SUBMITTER: Wang G
PROVIDER: S-EPMC8568984 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Whole-transcriptome sequencing ('RNA-Seq') has been drastically changing the scale and scope of genomic research. In order to fully understand the power and limitations of this technology, the US Food and Drug Administration (FDA) launched the third phase of the MicroArray Quality Control (MAQC-III) project, also known as the SEquencing Quality Control (SEQC) project. Using two well-established human reference RNA samples from the first phase of the MAQC project, three sequencing platforms were tested across more than ten sites with built-in truths including spike-in of external RNA controls (ERCC), titration data and qPCR verification. The SEQC project generated over 30 billion sequence reads representing the largest RNA-Seq data ever generated by a single project on individual RNA samples. This extraordinarily ultradeep transcriptomic data set and the known truths built into the study design provide many opportunities for further research and development to advance the improvement and application of RNA-Seq.
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Project description:Recombinant human erythropoietin (rHuEPO) is a well-known performance enhancing drug in human athletes, and there is anecdotal evidence of it being used in horse racing for the same purpose. rHuEPO, like endogenous EPO, increases arterial oxygen content and thus aerobic power. Micro-doping, or injecting smaller doses over a longer period of time, has become an important concern in both human and equine athletics since it is more difficult to detect. Horses offer an additional challenge of a contractile spleen, thus large changes in the red blood cell mass occur naturally. To address the challenge of detecting rHuEPO doping in horse racing, we determined the transcriptomic effects of rHuEPO micro-dosing over seven weeks in exercised Thoroughbreds. RNA-sequencing of peripheral blood mononuclear cells isolated at several time points throughout the study identified three transcripts (C13H16orf54, PUM2 and CHTOP) that were significantly (PFDR < 0.05) different between the treatment groups across two or three time point comparisons. PUM2 and CHTOP play a role in erythropoiesis while not much is known about C13H16orf54, but it is primarily expressed in whole blood. However, gene expression differences were not large enough to detect via RT-qPCR, thereby precluding their utility as biomarkers of micro-doping.
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Project description:We present SEEK (search-based exploration of expression compendia; http://seek.princeton.edu/), a query-based search engine for very large transcriptomic data collections, including thousands of human data sets from many different microarray and high-throughput sequencing platforms. SEEK uses a query-level cross-validation-based algorithm to automatically prioritize data sets relevant to the query and a robust search approach to identify genes, pathways and processes co-regulated with the query. SEEK provides multigene query searching with iterative metadata-based search refinement and extensive visualization-based analysis options.
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Project description:Recombinant human erythropoietin (rHuEPO) is a biopharmaceutical drug given to patients who have a low hemoglobin related to chronic kidney disease, cancer or anemia. However, some patients repeatedly receiving rHuEPO develop anti-rHuEPO neutralizing antibodies leading to the development of pure red cell aplasia (PRCA). The immunogenic antibody response activated by rHuEPO is believed to be triggered by T-cells recognizing EPO epitopes bound to MHC molecules displayed on the cell surface of APCs. Previous studies have reported an association between the development of anti-rHuEpo-associated PRCA and the HLA-DRB1*09 gene, which is reported to be entrenched in the Thai population. In this study, we used computational design to screen for immunogenic hotspots recognized by HLA-DRB1*09, and predicted seventeen mutants having anywhere between one through four mutations that reduce affinity for the allele, without disrupting the structural integrity and bioactivity. Five out of seventeen mutants were less immunogenic in vitro while retaining similar or slightly reduced bioactivity than rHuEPO. These engineered proteins could be the potential candidates to treat patients who are rHuEpo-dependent and express the HLA-DRB1*09 allele.
| S-EPMC7810742 | biostudies-literature
Project description:BACKGROUND:Human recombinant erythropoietin (rHuEpo) that is used for the treatment of the chemotherapy-induced anaemia in cancer patients was shown to cause detrimental effects on the course of disease due to increased adverse events inflicting patient's survival, potentially related to rHuEpo-induced cancer progression. In this study, we elucidate the effect of rHuEpo administration on breast cancer cell proliferation and gene expression after cisplatin (cDDP) induced cytotoxicity. MATERIALS AND METHODS:Two breast carcinoma models, MCF-7 and MDA-MB-231 cell lines, were used differing in oestrogen (ER) and progesterone (PR) receptors and p53 status. Cells were cultured with or without rHuEpo for 24 h or 9 weeks and their growth characteristics after cDDP treatment were assessed together with expression of genes involved in the p53-signaling pathway. RESULTS:Short-term exposure of breast cancer cells to rHuEpo lowers their proliferation and reduces cDDP cytotoxic potency. In contrast, long-term exposure of MCF-7 cells to rHuEpo increases proliferation and predisposes MCF-7 cells to cDDP cytotoxicity, but has no effect on MDA-MB-231 cells. MDA-MB-231 cells show altered level of ERK phosphorylation, indicating involvement of MAPK signalling pathway. Gene expression analysis of p53-dependent genes and bcl-2 gene family members confirmed differences between long and short-term rHuEpo effects, indicating the most prominent changes in BCL2 and BAD expression. CONCLUSIONS:Proliferation and survival characteristics of MCF-7 cells are reversely modulated by the length of the rHuEpo exposure. On the other hand, MDA-MB-231 cells are almost irresponsive to long-term rHuEpo, supposedly due to the mutated p53 and ER(+)/PR(-) status. The p53 and ER/PR status may predict tumour response on rHuEpo and cDDP treatment.
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Project description:Cisplatin exerts its cytotoxic effect through distinct DNA lesions, leading to peripheral neuropathy. The risk of sensory neuropathy is a common problem during cancer treatment with cisplatin, leading to somatic hyperalgesia. Yet, data focussing on cisplatin-induced impairment of the autonomic nervous system are limited. The present study was aimed to investigate the effect of recombinant human erythropoietin (rhEPO) on cisplatin-induced visceral hyperalgesia.C57BL/6 mice were treated either with cisplatin (2 mg/kg, once per week) or with cisplatin (2 mg/kg, once per week) plus rhEPO (40 microg/kg, 3 times per week) for 8 weeks. Controls were treated with saline. To quantify the visceromotor response (VMR) at week 9, standardized electrodes were implanted into the external oblique musculature for electromyographic recordings. After that, animals were decapitated and dorsal root ganglia (DRG) was removed for transmission electron microscopy studies.Cisplatin-treated mice showed a significant increase of VMR compared to the controls [(7080 +/- 969) vs (2864 +/- 279); P< 0.001], while rhEPO dramatically counteracted this effect [(2962 +/- 336) vs (7080 +/- 969); P< 0.001)]. Transmission electron microscopy revealed cisplatin-induced structural lesions of nuclear membrane in DRG cells, which could be ameliorated by rhEPO.Erythropoietin can significantly ameliorate the cisplatin-induced visceral hyperplasia and DRG nuclear membrane structure damage in mice, indicating a neuroprotective role of erythropoietin.
| S-EPMC5560334 | biostudies-other
Project description:ObjectiveTo evaluate the efficacy and safety of repeated low-dose human recombinant erythropoietin (rhEPO) in the improvement of neurological outcomes in very preterm infants.MethodsA total of 800 infants of ≤32-week gestational age who had been in an intensive care unit within 72 hours after birth were included in the trial between January 2009 and June 2013. Preterm infants were randomly assigned to receive rhEPO (500IU/kg; n = 366) or placebo (n = 377) intravenously within 72 hours after birth and then once every other day for 2 weeks. The primary outcome was death or moderate to severe neurological disability assessed at 18 months of corrected age.ResultsDeath and moderate/severe neurological disability occurred in 91 of 338 very preterm infants (26.9%) in the placebo group and in 43 of 330 very preterm infants (13.0%) in the rhEPO treatment group (relative risk [RR] = 0.40, 95% confidence interval [CI] = 0.27-0.59, p < 0.001) at 18 months of corrected age. The rate of moderate/severe neurological disability in the rhEPO group (22 of 309, 7.1%) was significantly lower compared to the placebo group (57 of 304, 18.8%; RR = 0.32, 95% CI = 0.19-0.55, p < 0.001), and no excess adverse events were observed.InterpretationRepeated low-dose rhEPO treatment reduced the risk of long-term neurological disability in very preterm infants with no obvious adverse effects. Ann Neurol 2016;80:24-34.
| S-EPMC5084793 | biostudies-literature
Project description:Reliable and reproducible monitoring of the conformational state of therapeutic protein products remains an unmet technological need. This need is amplified by the increasing number of biosimilars entering the drug development pipeline as many branded biologics are reaching the end of their market exclusivity period. Availability of methods to better characterize protein conformation may improve detection of counterfit and unlicensed therapeutic proteins. In this study, we report the use of a set of modified DNA aptamers with enhanced chemical diversity to probe the conformational state of 12 recombinant human erythropoietin (rHuEPO) therapeutic protein products; one FDA-licensed rHuEPO originator biological product, three rHuEPO products that are approved for marketing in the US or EU as biosimilars, and eight rHuEPO products that are not approved for marketing in the US or EU. We show that several of these modified aptamers are able to distinguish rHuEPO reference products or approved biosimilars from non-licensed rHuEPO products on the basis of differences in binding kinetics and equilibrium affinity constants. These reagents exhibit sensitivity to the conformational integrity of various forms of rHuEPO and as such represent powerful, simple-to-use analytical tools to monitor the conformational integrity of therapeutic-proteins during manufacture and to screen for and identify both substandard and counterfeit products.
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Project description:Asialo-erythropoietin, a desialylated form of human erythropoietin (EPO) lacking hematopoietic activity, is receiving increased attention because of its broader protective effects in preclinical models of tissue injury. However, attempts to translate its protective effects into clinical practice is hampered by unavailability of suitable expression system and its costly and limit production from expensive mammalian cell-made EPO (rhuEPO(M)) by enzymatic desialylation. In the current study, we took advantage of a plant-based expression system lacking sialylating capacity but possessing an ability to synthesize complex N-glycans to produce cytoprotective recombinant human asialo-rhuEPO. Transgenic tobacco plants expressing asialo-rhuEPO were generated by stably co-expressing human EPO and ?1,4-galactosyltransferase (GalT) genes under the control of double CaMV 35S and glyceraldehyde-3-phosphate gene (GapC) promoters, respectively. Plant-produced asialo-rhuEPO (asialo-rhuEPO(P)) was purified by immunoaffinity chromatography. Detailed N-glycan analysis using NSI-FTMS and MS/MS revealed that asialo-rhuEPO(P) bears paucimannosidic, high mannose-type and complex N-glycans. In vitro cytoprotection assays showed that the asialo-rhuEPO(P) (20 U/ml) provides 2-fold better cytoprotection (44%) to neuronal-like mouse neuroblastoma cells from staurosporine-induced cell death than rhuEPO(M) (21%). The cytoprotective effect of the asialo-rhuEPO(P) was found to be mediated by receptor-initiated phosphorylation of Janus kinase 2 (JAK2) and suppression of caspase 3 activation. Altogether, these findings demonstrate that plants are a suitable host for producing cytoprotective rhuEPO derivative. In addition, the general advantages of plant-based expression system can be exploited to address the cost and scalability issues related to its production.
| S-EPMC3790672 | biostudies-literature
Project description:Recombinant production of pharmaceutical proteins is crucial, not only for personalized medicine. While most biopharmaceuticals are currently produced in mammalian cell culture, plant-made pharmaceuticals gain momentum. Post-translational modifications in plants are similar to those in humans, however, existing differences may affect quality, safety and efficacy of the products. A frequent modification in higher eukaryotes is prolyl-4-hydroxylase (P4H)-catalysed prolyl-hydroxylation. P4H sequence recognition sites on target proteins differ between humans and plants leading to non-human posttranslational modifications of recombinant human proteins produced in plants. The resulting hydroxyprolines display the anchor for plant-specific O-glycosylation, which bears immunogenic potential for patients. Here we describe the identification of a plant gene responsible for non-human prolyl-hydroxylation of human erythropoietin (hEPO) recombinantly produced in plant (moss) bioreactors. Targeted ablation of this gene abolished undesired prolyl-hydroxylation of hEPO and thus paves the way for plant-made pharmaceuticals humanized via glyco-engineering in moss bioreactors.
| S-EPMC3804855 | biostudies-literature