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WO2025038762A1 - Procédés, dispositifs et systèmes d'amélioration de la qualité du stockage des globules rouges et de l'efficacité des transfusions - Google Patents

Procédés, dispositifs et systèmes d'amélioration de la qualité du stockage des globules rouges et de l'efficacité des transfusions Download PDF

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WO2025038762A1
WO2025038762A1 PCT/US2024/042332 US2024042332W WO2025038762A1 WO 2025038762 A1 WO2025038762 A1 WO 2025038762A1 US 2024042332 W US2024042332 W US 2024042332W WO 2025038762 A1 WO2025038762 A1 WO 2025038762A1
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biomarker
erythrocyte
rsl
hypoxanthine
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Angelo D'alessandro
Travis Nemkov
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University of Colorado System
University of Colorado Colorado Springs
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University of Colorado Colorado Springs
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/80—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood groups or blood types or red blood cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • Red blood cells account for -83% of all the cells in the human body. Ease of collection and shear abundance of RBCs have made them an eligible model for the investigation of metabolism in the early days of biochemistry. Unlike other vertebrates, mammalian RBCs have evolved to maximize oxygen carrying capacity by increasing hemoglobin content per cell, a feature they achieved by losing nuclei, organelles, and the ability to synthesize proteins de novo. Given the absence of de novo protein synthesis capacity, RBC rely on dynamic metabolic reprogramming in response to environmental stimuli, including hypoxia or oxidant stress (e.g. at altitude or during exercise).
  • environmental stimuli including hypoxia or oxidant stress (e.g. at altitude or during exercise).
  • RBCs represent a simplified model of eukaryotic cell metabolism, a cell type that exclusively relies on glycolysis as the main source of energy for the synthesis of ATP. For the reasons above, is not a coincidence that earliest efforts to reconstruct human metabolic networks in silico have been centered on RBC metabolism.
  • erythrocytes or population thereof, wherein the RBCs possess enhanced characteristics related to storage or posttransfusion, as well as other stress-related characteristics such as tolerance to exercise, toxic chemicals such as chemotherapy, irradiation, disease, etc.
  • the RBCs may be stored for longer periods and undergo transfusion with lower rates of hymolysis.
  • the disclosed methods may be used to identify cells with enhanced characteristics, such as stem or progenitor cells for ex -vivo production, such as for manufacture of RBCs with optimal storage and resuscitation performance.
  • the methods comprising steps of assaying the RBC for one or more biomarkers, for example ATP, hypoxanthine, and lactate.
  • the enhanced RBCs may have ATP and/or lactate levels that are higer than about 90% of the comparator RBCs from various donors and/or may have hypoxanthine levels lower than about 90% of comparator RBCs.
  • the at least one genetic marker is a single nucleotide polymorphism selected from rs2388595, rs2388594, rs56882221. rs34538474, rs!0903962, rs872398.
  • the erythrocyte’s characteristics may enhanced if the subject’s genome includes a SNP selected from rs2388595, rs2388594, rs56882221, rs34538474, rs!0903962, rs872398, rs7914782, rsl2249060, rs!
  • rs55845900 rs597808
  • rs2072931 rs9438900
  • rs609320 rs586178
  • a SNP selected from rs9891699, rs2542591, rs2542590. rs2542573.
  • arrays for identifying a subject with superior blood donor properties are also disclosed.
  • the arrays consist of oligonucleotides selected from rs2388595, rs2388594. rs56882221, rs34538474, rs!0903962.
  • FIG. 1A an overview of the study design and donor demographics.
  • FIGs. 1B-E linear discriminant analysis of all the samples as a function of donor age, sex, body mass index (BMI) and ethnicity, respectively.
  • FIG. IF Uniform Manifold Approximation and Projection (uMAP), color-coded as a function of 10 main K mean clusters.
  • FIG. 1G Uniform Manifold Approximation and Projection
  • FIG. 1H network view of metabolite-metabolite correlations (Spearman) as a function of the 10 K means clusters. Each metabolite is a node and a red and blue edge indicates positive or negative correlations higher than 0.5 in module.
  • FIG. II heat map of the top 50 significant metabolites that discriminate the 10 clusters identifies packed RBCs from donors in cluster 4 as the group with the highest glycolytic intermediates and products, including ATP.
  • Figures 2A-H - Donors in cluster 4 are characterized by elevation in glycolysis.
  • FIG. 2A overview of glycolysis.
  • FIG. 2A overview of glycolysis.
  • FIG. 2A overview of glycolysis.
  • ridge plots for all glycolytic intermediates show elevation of hexoses, hexose phosphate and triose phosphates all the way to pyruvate in donors from cluster 4.
  • uMAP color coded by ATP levels (also used for the z axis).
  • donors in cluster 4 have elevated ATP levels compared to the other 9 clusters independently from their age.
  • Figures 3A-D - Glycolysis is impacted by donor sex, age, ethnicity and BMI.
  • Line plots (median + quartile - Loess smoothing algorithm) as a function of donor age, alone (FIG. 3A) or in combination with sex (FIG. 3B), BMI (FIG. 3C) and ethnicity’ (FIG. 3D).
  • FIG. 4A Genetic underpinnings of glycolytic heterogeneity in end of storage units from 13,091 REDS RBC Omics Index donors. Along with metabolomics analyses, as part of the REDS RBC Omics study donors were tested for -879,000 single nucleotide polymorphisms (SNPs - FIG. 4A).
  • SNPs single nucleotide polymorphisms
  • FIG. 4B A summary Manhattan plot of metabolite Quantitative Trait Loci (mQTL) is shown in FIG. 4B for all glycolytic metabolites merged, or just for ATP (FIG. 4C). For both ATP (FIG. 4D) and lactate (Manhattan plot in FIG. 4E, locus Zoom in FIG. 4F).
  • FIG. 4G Summary 7 table of the most significant SNP hits mapping on PFKP for all glycolytic metabiolites, ATP and hypoxanthine.
  • Figures 5A-H Manhattan plots of glycolytic metabolites upon mQTL analysis of 13,091 end of storage packed RBCs from the REDS RBC Omics Index cohort. Manhattan plots for all glycolytic intermediates and ribose phosphate - end product of the oxidative phase of the pentose phosphate pathway (FIGs. 5A-H).
  • Figures 7A-D Positive and negative association of Glycolysis/ ATP and hypoxanthine to in vitro and in vivo hemolysis in autologous healthy and non-autologous critically ill recipients requiring transfusion.
  • End of storage spontaneous hemolysis one of the two FDA gold standard for the quality of stored blood products - was measured in all 13.091 units. Lactate levels at storage day 42 were the top predictor of spontaneous hemolysis (FIG.
  • FIG. 8 Ridge plots of citrate and mannitol by K means cluster of the 13,091 REDS RBC Omics Index donors. Clusters 3, 6, 7 and 8 showed a subpopulation with higher levels of mannitol, while citrate was the highest in clusters 4-5, suggesting that the former group was stored in mannitol containing additives (AS-1 for this study) and the latter in mannitol-free additives with higher concentrations of citrate (AS-3 in this study).
  • Figure 9 Rainfall plots and uMAP of the 13,091 donors for ATP and hypoxanthine.
  • the z axis is either showing normalized ATP (left) or hypoxanthine (right).
  • the left panel shows a volcano plot of the differences between the two groups with high vs low ATP (ATP is excluded from the plot).
  • heat map of the top 40 metabolites by T-test betw een the two groups.
  • the left panel shows a volcano plot of the differences between the two groups with high vs low hypoxanthine (hypoxanthine is excluded from the plot).
  • the left panel shows a volcano plot of the differences between the two groups with high vs low hypoxanthine and low vs high ATP.
  • Figure 13 Combined Manhattan plots for all the significant SNPs across all glycolytic metabolites as determined by mQTL analysis of 13,091 REDS RBC Omics Index donors
  • FIG. 14 mQTL analysis of ATP. mQTL analyses were performed for ATP and identified HBS1L and PFKP SNPs as significant (QQQ plot on the left panel, bottom row). Locus Zoom for the PFKP coding region in the right panel on the bottom row.
  • Figure 15 Locus Zoom for all the significant SNPs for PFKP and HK1 across all glycolytic metabolites as determined by mQTL analysis of 13,091 REDS RBC Omics Index donors
  • Figure 16 Metabolic correlates to the degree of vesiculation in 643 units from the REDS RBC Omics Recalled donor population (i.e., a subset of donors with extreme hemolytic propensity - 5th and 95th percentile of the total Index population).
  • Red blood cells account for -83% of all the cells in the human body. Ease of collection and shear abundance of RBCs have made them an eligible model for the investigation of metabolism in the early days of biochemistry. Unlike other vertebrates, mammalian RBCs have evolved to maximize oxygen carrying capacity by increasing hemoglobin content per cell, a feature they achieved by losing nuclei and organelles. Given the absence of de novo protein synthesis capacity, RBC rely on dynamic metabolic reprogramming in response to environmental stimuli, including hypoxia or oxidant stress (e.g., at altitude or during exercise).
  • environmental stimuli including hypoxia or oxidant stress (e.g., at altitude or during exercise).
  • ATP adenosine triphosphate
  • Mitochondria devoid RBCs represent a simplified model of eukaryotic cell metabolism, a cell type that exclusively relies on glycolysis as the main source of energy for the synthesis of ATP. For the reasons above, it is not a coincidence that the earliest efforts to reconstruct human metabolic networks in silico have been centered on RBC metabolism.
  • RBC metabolism may be useful in optimizing storage conditions and predicting post-transfusion performance of blood products.
  • Transfusion of packed RBCs is the most common in hospital medical procedure after vaccination, a life-saving procedure for tens of millions of civilian and military patients whose survival depends solely on chronic or massive transfusion, with no current alternative iatrogenic interventions.
  • Altered energy' metabolism is a hallmark of the so-called storage lesion, a series of biochemical and morphological alterations that ultimately increase the propensity of stored RBCs to hemolyze in the unit or upon transfusion. Rapid depletion of ATP pools, storage-dependent depletion of glucose and accumulation of lactate are biomarkers of the metabolic storage lesion, though the clinical relevance of such lesion is unclear.
  • RBC metabolism under refrigerated storage conditions is impacted by storage temperatures, additive solutions, dietary or other exposures (e.g., smoking, alcohol consumption, drugs that are not grounds for blood donor deferral), but also by factors such as donor age, sex, ethnicity and body mass index (BMI).
  • BMI body mass index
  • blood is a biological product derived from the altruistic gift of millions of volunteers around the world every year and, as such, its quality, storability and post-transfusion performance are tied to donor genetics and non-genetic factors.
  • Applicants show that mQTL studies are useful in the field of transfusion medicine. Specifically, through unsupervised analyses Applicants have surprisingly identified the glycolysis pathway as a pathway that informs clustering of donors as a function of their biology (sex, age. ethnicity, BMI) and genetics. These results are correlated to RBC hemolytic propensity- in-vitro and in-vivo, upon transfusion of stored RBCs in healthy autologous recipients and - through a state-of-the art vein-to-vein database - in thousands of clinical patients receiving transfusion. [0028] In recent years, strategies have been introduced to bioengineer red blood cells and red blood cell precursor ex vivo from hematopoietic stem cells or induced pluripotent stem cells.
  • Generating RBCs with optimal glycolytic capacity of ex vivo generated RBCs can boost both the proliferation/ expansion phase, as well as the efficacy (i.e., ATP synthesis and oxygen offloading) and storability of the end product.
  • Applicants identify genetic polymorphisms that maximize RBC glycolytic potential for these purposes.
  • this invention identifies genetic signature (single nucleotide polymorphisms) associated with elevated glycolysis in human cells, thus making the genetic signatures at the center of the present disclosure suitable markers for the prediction of cancer progression, severity and responses to therapies targeting glycolysis.
  • Applicants have identified specific bio and genetic markers the correlate with with various characteristics such as stability in storage, post-perfusion resistance to hemolysis, stress tolerance, oxygen capacity, energy metabolism, etc.
  • the disclosed markers may be used to identity' cells with enhanced characteristics, or identify, and in some cases avoid use of, cells having less desirable characteristics.
  • these genetic signatures can be used to design optimal ex vivo engineered artificial red blood cells from any stem cell source (e.g., hematopoietic stem cells, induced pluripotent stem cells).
  • stem cell source e.g., hematopoietic stem cells, induced pluripotent stem cells.
  • the inclusion of such polymorphisms would make artificial RBC precursors easier to culture (i.e., faster proliferation because of enhanced glycolysis), store better because of improved energy metabolism and decreased susceptibility' to lipid peroxidation and other oxidative stress markers.
  • the subjects may be identified by assessing genetic markers, in one example polymorphic markers, for example small nuclear polymorphisms or SNPs.
  • the genetic markers may be located at, near, or in the locus of a gene.
  • the disclosed polymorphisms may alter coding or non-coding sequences and/or affect regulation, expression, splicing, or post-translational processing.
  • the genes may be associated with glycolysis and/or the glycolytic pathway.
  • the genes may be selected from genes presented at FIGs. 4 and 5.
  • the one or more genetic markers may be associated with one or more of the genes ACYP2, ATXN2, AURKB, BST1, CIS, CPPED1, CTC1, ERLEC1.
  • the genetic marker is located at or near the location of PFKP.
  • the genetic marker may be selected from the markers in Figure 4J. [0032]
  • the disclosed cells and subjects may possess or lack one or more SNPs.
  • the SNP may be one or more of rs2388595, rs2388594, rs56882221, rs34538474, rs!0903962, rs872398, rs7914782, rsl2249060, rsl 1592619, rs55845900, rs597808, rs2072931, rs9438900, rs609320, rs586178, rs9891699, rs2542591, rs2542590. rs2542573, rs2692513, rs2692512. rs4791641. rsl059476.
  • SNPs designated by “rs” numbers, are well known to those of skill in the art, and can be obtained from various sources including the National Library of Medicine's SNP database. dbSNP at ncbi.nlm.nih.gov/snp/.
  • perfusion may include autologous and non-autologous perfusion, reperfusion, and tranfusion.
  • the disclosed cells may be obtained, derived, or genetically engineered.
  • the cells may be obtained or derived from the subjects described above.
  • the cells may be engineered to include one or more polymorphisms as described above.
  • the disclosed cells may possess and may be identified by one or more biomarkers.
  • the biomarker may be a product, intermediate, or metabolite of glycolysis or the glycolytic pathway.
  • the biomarker may be selected from ATP, hypoxanthine, or lactate.
  • cellular compositions and biomarkers useful in identifying cells and subjects having enhanced characteristics related to storage and use of red blood cells are intermediates and products of a metabolic pathway, for example glycolysis.
  • a biomarker may be selected from the intermediates and products shown at Figure 2A, in some embodiments one biomarker may be adenosine triphosphate (ATP), hypoxanthine, or lactate.
  • the enhanced cells possess biomarker levels significantly different than cells with less desirable, average, or poor characteristics.
  • the levels may be elevated.
  • the levels may be lowered.
  • the levels of some biomarkers in the disclosed cells may be elevated while other biomarker levels low ered.
  • the biomarker is hypoxanthine
  • the hypoxanthine levels may be lower than the average for other cells.
  • the biomarker is ATP.
  • the disclosed red blood cells may possess elevated adenosine triphosphate (ATP) levels relative to other cells.
  • the disclosed cells may have high levels of ATP and low' levels of hypoxanthine.
  • the disclosed cells have biomarker levels that are very high and/or very low relative to cells of average characteristics.
  • the biomarker level may' be at or above the 90 th , 91 st , 92 nd , 93 rd , 94 th , 95 th , 96 th , 97 th , 98 th , or 99 th percentile of similar cells, and/or lower than the 10 th . 9 th .
  • the disclosed cells may possess ATP levels higher than the 90 th percentile when compared with a random sampling of RBCs from various donors or subjects.
  • Biomarker levels of the disclosed cells may be assessed at various times, for one example after 10 or more days of storage, for example more than 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29. 30. 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42 days and less than about 42. 41. 40. 39, 38, 37, 36, 35, 34, 33, 32, 31, 30. 29. 28. 27. 26. 25. 24. 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, or 11 days.
  • the disclosed methods and processes include assessing the cells for various biomarkers and/or genetic markers. In many embodiments if the biomarker level is greater than 90% percent of the levels in other cells, or less than 90% of all other cells, the cell may be identified as a cell with enhanced characteristics.
  • the disclosed methods may be useful in identifying or selecting donors with cells having enhanced characteristics - i.e. in the case of blood donation they may be "super"-donors.
  • RBCs from super-donors are characterized by elevated glycolysis and ATP levels, and low hypoxanthine levels. While ATP-depleted ery throcytes may be rapidly removed from the bloodstream via mechanisms of intra- or extravascular hemolysis, the disclosed cells may be longer lived in the bloodstream and ex-vivo, i.e. when stored.
  • Genetic markers are also disclosed that indicate a subject or pre-cursor cell may possess enhanced characteristics. Applicants show that, in addition to PFKP and HK being possible rate-limiting enzymes of glycolysis, their altered expression or function, resulting from genetic polymorphisms affect RBC metabolism during storage as well as hemolysis in vitro and in vivo.
  • a biological sample is obtained from the subject.
  • the biological sample obtained from the subject may be a cell, for example, an erythrocyte from a unit of previously obtained and processed blood.
  • biological sample 7 refers to a sample derived from a subject, e.g., a patient.
  • Biological samples include, but are not limited to tissue (e.g., skin tissue), cerebrospinal fluid, blood, blood fractions (e.g., serum, plasma), sputum, fine needle biopsy samples, urine, peritoneal fluid, and pleural fluid, or cells therefrom (e.g., blood cells (e.g., white blood cells, red blood cells, erythrocytes)).
  • a biological sample may comprise a tissue, cell or biomolecule (e.g., RNA, protein, metabolite, etc).
  • the biological sample is a sample of peripheral blood, serum, cerebrospinal fluid, urine and tissue.
  • a biological sample may be processed in any appropriate manner to facilitate determining levels of various biomolecules and/or the presence or absence of a genetic marker.
  • biochemical, mechanical and/or thermal processing methods may be appropriately used to isolate a biomolecule of interest, e.g., ATP or nucleic acid, from a biological sample.
  • a biomolecule sample may be isolated from a clinical sample by processing the biological sample using methods well known in the art and levels of the biomolecule may be determined in the biological sample.
  • nucleic acid arrays comprise, or consist essentially of. binding probes for SNPs of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 1, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25. at least 26, at least 'Ll, at least 28, at least 29, at least 30, at least 31, at least 32. at least 33.
  • Such arrays may be obtained or produced from commercial sources. Methods for producing nucleic acid arrays are well known in the art.
  • nucleic acid arrays may be constructed by immobilizing to a solid support large numbers of oligonucleotides, polynucleotides, or cDNAs capable of hybridizing to nucleic acids corresponding to SNP, or portions thereof.
  • the skilled artisan is also referred to Chapter 22 “Nucleic Acid Arrays” of Current Protocols In Molecular Biology (Eds. Ausubel et al. John Wiley and #38; Sons NY, 2000).
  • Kits comprising reagents for detecting the presence or absence of at least one enhanced RBC-characteristic from the biological sample are also provided.
  • the kit may include reagents to detect 2, 3 or more of the biomarkers described herein, for example ATP, hypoxanthine, and lactate.
  • the kit may include reagents to detect 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17. 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33. 34. 35. 36. 37.
  • the kit may include at least 39 nucleic acids to detect all 39 of the SNPs.
  • Kits may include a package housing one or more containers with reagent for measuring the presence of at least one biomarker of genetic marker from the biological sample and instructions for determining the level, presence, or absence thereof. Kits comprising the nucleic acid-based assays, including oligonucleotide arrays described herein are also included.
  • RBC storage is not only a clinically relevant manipulation of the erythrocyte, but also represents a form of stress (especially, oxidative stress) that at least in part recapitulates other biologically relevant stressors, such as exercise, exposure to drugs for example during chemotherapy (e.g., anthracy clines like doxorubicin), irradiation, or disease (e g., hemoglobinopathies like sickle cell disease or chronic kidney disease).
  • chemotherapy e.g., anthracy clines like doxorubicin
  • irradiation e.g., doxorubicin
  • disease e g., hemoglobinopathies like sickle cell disease or chronic kidney disease.
  • Applicants show that the cells with the presently disclosed characteristics perform better after storage.
  • the disclosed methods are useful in identifying cells with decreased hemolytic propensity 7 , and improved reperfusion performance.
  • the disclosed methods and markers are also useful in creating engineered cells with enhanced characteristics. In some cases this may be accomplished by identifying donors of hematopoietic or induced-pluripotent stem cell for manufacture of next-generation ex vivo farmed RBCs with optimal storage and resuscitation performances.
  • the disclosed methods and markers may be useful in the identification of optimal starting genotypes for blood group and rare antigen compatibility.
  • the disclosed methods and markers may also be useful in identifying subjects prior to the subject facing challenges requiring increased oxygen consumption, such as exposure to high altitude hypoxia, strenuous or prolonged physical activities or hemorrhage.
  • ATP fuels important processes in mature RBCs including: hemoglobin allostery to metabolism, from proton pumps to membrane integrity via phosphorylation of structural proteins, from protein stabilization via fueling of transglutaminase 2 to cellular mechanics, from cytoskeletal actin polymerization to vesiculation, from membrane lipid symmetry 7 by fueling phosphatidylserine flippases to proteasomal activity to remove damaged proteins.
  • ATP-depleted erythrocyte is rapidly removed from the bloodstream via mechanisms of intra- or extravascular hemolysis, such as sequestration in the splenic slit and erythrophagocytosis by residential macrophages in the reticuloendothelial system.
  • PFK and HK are rate-limiting enzymes of glycolysis
  • previous work has not shown or suggested the coding regions for these genes are polymorphic in healthy humans, or that these polymorphisms affect stored RBC metabolism and/or influence hemolysis in vitro and in vivo, upon perfusion in autologous healthy volunteers and thousands of non-autologous critically ill patients requiring transfusion.
  • the units tested here were all leukocyte (log4.5) and platelet (log2.5) filtered, making the PFKP hit unlikely to be the result of an enrichment in platelets in a subset of units.
  • RBC storage is not only a clinically relevant manipulation of the erythrocyte, but also a form of stress (especially, oxidative stress) that at least in part recapitulates other biologically relevant stressors, such as exercise, exposure to drugs for example during chemotherapy (e.g., anthracy clines like doxorubicin), irradiation, or disease (e.g., hemoglobinopathies like sickle cell disease or chronic kidney disease).
  • chemotherapy e.g., anthracy clines like doxorubicin
  • irradiation e.g., hematomas like sickle cell disease or chronic kidney disease.
  • Disclosed herein is the identification of advantageous polymorphisms in the determination of "super ‘-donors not just with respect to decreased hemolytic propensity, but also improved reperfusion performances in vivo in patients requiring transfusion. Also disclosed are processes for manufacture of ex vivo farmed RBCs from hematopoietic or induced-pluripotent stem cell Applicant’s results are useful in the identification of optimal starting genotypes not just with respect to blood group and rare antigen compatibility, but also with boosted energy metabolism could inform the engineering of ex vivo farmed blood units with optimal storage and resuscitation performances.
  • RBCs and well-curated cohorts of repeated healthy blood donors represent a perfect model to investigate glycolysis at the population levels.
  • RBCs are a cell type that exclusively relies on glycolysis to meet its energy demands.
  • blood storage is a clinically-relevant stress-intervention that exacerbates the reliance on glycolytic fluxes in the absence of compensatory mechanisms from other organs in vivo.
  • We argue that lessons learned from these studies are translationally relevant for many other research endeavors. Indeed, metabolic reprogramming towards glycolysis - often referred to as Warburg effect in the context of cancer - is perhaps one of the hottest areas of research in the last two decades.
  • Glycolytic reprogramming has been linked as one of the etiological contributors to many diseases, from cancer to cardiovascular disease, from immunometabolism to aging and age-related neurodegenerative diseases.
  • Our findings suggest that the severity of glycolytic reprogramming in humans - e.g., upon accumulation of mutations to oncogenes/tumor suppressor genes or in response to other factors (e.g., inflammation) - may be mediated by the same biological and genetic factors that regulate glycolysis in human RBCs as described in the present study, making our findings relevant to other disciplines beyond transfusion medicine.
  • Donor recruitment in the REDS RBC Omics study Index donors A total of 13,758 donors were enrolled in the Recipient Epidemiology and Donor evaluation Study (REDS) RBC Omics at four different blood centers across the United States (biolincc.nhlbi.nih.gov/studies/reds_iii/). Of these, 97% (13,403) provided informed consent and 13,091 were available for metabolomics analyses in this study - henceforth referred to as “index donors”.
  • RDS Recipient Epidemiology and Donor evaluation Study
  • the UHPLC was coupled online to a Q Exactive mass spectrometer (Thermo Fisher).
  • the Q Exactive MS was operated in negative ion mode, scanning in Full MS mode (2 qscans) from 90 to 900 m/z at 70,000 resolution, with 4 kV spray voltage, 45 sheath gas, 15 auxiliary gas.
  • raw files were converted to .mzXML using RawConverter then metabolites assigned and peaks integrated using ElMaven (Elucidata) in conjunction with an inhouse standard library.
  • mQTL analysis The workflow' for the mQTL analysis of ATP, hypoxanthine and glycolytic metabolites is consistent with previously described methods from our pilot mQTL study on 250 recalled donors. Details of the genotyping and imputation of the RBC Omics study participants have been previously described by Page, et al. Briefly, genotyping was performed using a Transfusion Medicine microarray consisting of 879,000 SNPs; the data are available in dbGAP accession number phs001955.vl.pl. Imputation was performed using 811,782 SNPs that passed quality control. After phasing using Shape-IT, imputation was performed using Impute2 with the 1000 Genomes Project phase 3 all-ancestry reference haplotypes.
  • OASIS Omics Analysis, Search & Information a TOPMED funded resources, was used to annotate the top SNPS.
  • OASIS annotation includes information on position, chromosome, alellele frequencies, closest gene, type of variant, position relative to closest gene model, if predicted to functionally consequential, tissues specific gene expression, and other information.
  • HCA hierarchical clustering analysis
  • LDA linear discriminant analysis
  • uMAP uniform Manifold Approximation and Projection
  • correlation analyses and Lasso regression were performed using both MetaboAnalyst 5.0 and inhouse developed code in R (4.2.3 2023-03-15).
  • Leukocyte-filtered RBCs were donated by 13,091 volunteers across four different blood centers in the United States and stored for 42 days (end of the shelf-life) prior to testing via high-throughput metabolomics (Figure 1A). Demographics were available for 13,029 of these donors. The donor population was balanced with respect to sex (6507 males and 6522 females), blood center (from a minimum of 3019 to a maximum of 3564 per center). The age range spanned from 18 to 87 (median age 47). Body mass index (BMI) ranged from 10-78 (median 26.6). Ethnicity was biased heavily towards Caucasians (7037), with good representation of individuals of Asian (1602), African (1542) and Hispanic descent (1153).
  • Additive solution 1 and 3 were the two only additives used in this study. Consistent with prior publications on two-three log smaller cohorts. Linear discriminant analysis of all the samples as a function of donor age, sex, body mass index (BMI) and ethnicity confirmed a strong impact of all these factors on the metabolic heterogeneity of end of storage units (Figure 1B-E). K mean clustering was then used to identity' latent factors influencing clustering of end of storage units upon unsupervised uniform Manifold Approximation and Projection (uMAP - Figure IF). Network view analysis of metabolites across the whole population revealed a central core of metabolites involved in glycolysis, the only energy generating pathway in mature RBCs (Figure 1G).
  • Example 3 - Donors in cluster 4 are characterized by elevation in glycolysis
  • Example 4 - Glycolysis is impacted by donor sex, age, ethnicity and BMI
  • RBCs from donors with the highest BMIs were characterized by significantly higher levels of lactate compared to all the other BMI groups within the age window of 30-60 (Figure 3C).
  • Ethnicity showed a measurable impact on glycolysis, with end of storage RBCs from donors with Hispanic and other ethnicity being characterized by higher levels ATP and 2,3-DPG, and of all glycolytic intermediates through pyruvate, but not lactate - which instead was highest in donors of Caucasian or East Asian descent (Figure 3D).
  • This analysis also identified multiple common hot spot areas in the regions coding for other ratelimiting enzymes of glycolysis, hexokinase 1 (HKL chromosome 10), phosphoglucomutase 2 like 1 (PGM2L1, chromosome 11), pyruvate kinase (PKLR, chromosome 1 - Figure 5). Similarly, end of storage levels of pyruvate showed a strong association with the rate-limiting enzyme of the pentose phosphate pathway, glucose 6-phosphate dehydrogenase (G6PD - Figure 5).
  • HSB1L-MYB chromosome 6
  • CPPED1 chromosome 16
  • Example 6 - RBCs from donors with high end of storage ATP and low hypoxanthine are characterized by lower hemolysis in vitro and in vivo in autologous healthy and non-autologous critically ill recipients requiring transfusion

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Abstract

La présente invention concerne des dispositifs, des compositions, des procédés et des systèmes pour identifier des cellules de mammifères, en particulier les érythrocytes, avec des caractéristiques améliorées, telles que l'hémolyse, la tolérance au stress, la capacité d'oxygène et le métabolisme énergétique. Dans certains modes de réalisation, les dispositifs, les compositions, les procédés et les systèmes peuvent être utiles pour identifier un sujet pour donner des érythrocytes, avec une résistance au stress améliorée, avec une performance physique améliorée telle que la consommation d'oxygène, et des activités physiques intenses ou prolongées, une hémorragie ou une résistance à l'hypoxie de haute altitude. Des sujets peuvent également être identifiés pour donner des cellules destinées à être utilisées dans la fabrication de la prochaine génération de globules rouges cultivés ex vivo avec des performances de stockage et de réanimation optimales. Les dispositifs, compositions, procédés et systèmes de l'invention sont également utiles dans la bio-ingénierie des globules rouges et des précurseurs de globules rouges ex vivo à partir de cellules souches hématopoïétiques ou de cellules souches pluripotentes induites, ainsi que dans la prédiction de la progression du cancer, de sa gravité et des réponses à des thérapies ciblant la glycolyse.
PCT/US2024/042332 2023-08-14 2024-08-14 Procédés, dispositifs et systèmes d'amélioration de la qualité du stockage des globules rouges et de l'efficacité des transfusions Pending WO2025038762A1 (fr)

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ANGELO D'ALESSANDRO, XIAOYUN FU, TAMIR KANIAS, JULIE A. REISZ, RACHEL CULP-HILL, YUELONG GUO, MARK T. GLADWIN, GRIER PAGE, STEVEN : "Donor sex, age and ethnicity impact stored red blood cell antioxidant metabolism through mechanisms in part explained by glucose 6-phosphate dehydrogenase levels and activity", HAEMATOLOGICA, FERRATA STORTI FOUNDATION, vol. 106, no. 5, pages 1290 - 1302, XP093283179, ISSN: 0390-6078, DOI: 10.3324/haematol.2020.246603 *

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