MicroRNAs in Ex Vivo Stored Blood Cells: What Future Do They Hold in Transfusion Medicine?

1. MicroRNAs in Ex Vivo Stored Blood Cells: What Future Do They Hold in Transfusion Medicine? C.D. Atreya, Ph.D. Associate Director for Research Office of Blood Research and Review Center for Biologics Evaluation and Research US Food and Drug Administration US Department of Health and Human Services 5th World Hematologists Congress, Aug 18-19, 2016 ***This presentation reflects the views of Dr. C.D. Atreya and should not be construed to represent FDA’s views or policies***

2. MicroRNAs • Small Single-stranded noncoding regulatory RNAs • which represent ~4% of the genes in the human genome • Ex Vivo Stored Blood Cells • Platelets and RBCs collected, processed, and stored in bags as PC and pRBC units available for transfusion • Transfusion Medicine • A branch of medicine that is concerned with safe and effective transfusion of blood and blood components (e.g. Platelets and RBC)

3. Topics covered…. • miRNAs –Biogenesis and mechanism of action • Changes associated with ex vivo Storage of blood Cells (PCs and pRBC) • Roll of miRNAs in stored blood cells • Extracellular release of miRNAs in the context of ex vivo stored blood cells • Conclusions

4. miRNAs –Biogenesis and mechanism of action • Changes associated with ex vivo Storage of blood Cells (PCs and pRBC) • Roll of miRNAs in stored blood cells • Extracellular release of miRNAs in the context of ex vivo stored blood cells • Conclusions

5. MicroRNA (miRNA) Biogenesis and mechanism of action http://www.sigmaaldrich.com/ • Single-stranded small noncoding regulatory RNAs • Represent ~4% of the genes in the human genome Drsha Nuclear RNAse III Dicer Cytoplasmic RNAse III DGCR8 protein Digeorge Syndrome Critical Region 8 RISC RNA induced silence complex seed

6. Single miRNA can control multiple mRNAs

7. Multiple miRNAs can effectively control a single mRNA by targeting at different sites

8. miRNAs –Biogenesis and mechanism of action • Changes associated with ex vivo Storage of blood Cells (PCs and pRBC) • Roll of miRNAs in stored blood cells • Extracellular release of miRNAs in the context of ex vivo stored blood cells • Conclusions

9. Blood Cell Lineage = Platelets RBC No nucleus! No nucleus!

10. Model of miRNA pathway in megakaryocyte-erythrocyte progenitor cells (MEPs) and anucleate blood cells Ryan and Atreya 2011 Transfusion Med Reviews , 25:247-251

11. Stored blood cells 1. Packed RBC Ex vivo storage @ 1-6 0C with certain anti-coagulants for 42 days; Can be stored @-65 0C in glycerol by specified methods 2. Platelet Concentrates Ex vivo storage @ Controlled Room temp (20-24 0C)

12. RBC ex vivo storage • Currently licensed RBC additive solutions (AS) have acidic pH (~5.5-5.8) • The acidic intracellular environment rapidly decreases ATP (necessary for RBC survival) and 2,3-DPG (necessary for oxygen delivery) and triggers apoptosis-like processes (eryptosis) • Research on RBC storage has repeatedly demonstrated that fundamental biology of RBC is still not well understood Sparrow, Blood Transfusion 2012

13. RBC Storage Lesion (SL) “Biochemical, morphological and immunologic changes which occur within RBC and in the associated storage media during ex vivo preservation of RBC are collectively known as RBC storage lesion” Biochemical: rapid depletion of 2, 3-diphosphoglycerate (DPG) and Adenosine triphosphate (ATP) levels in RBC Morphological: Change in corpuscle shape affecting microcirculation in the recipients Immunological: Immuno-modulatory effects in the recipients

14. Why Study RBC-SL? Ex Vivo Stored RBC with SL Clinical consequences Acute lung injury Multi-organ failure Mortality Biochemical morphological immunologic changes Modified Fig.1 from Kor et al, BJBMC 2009

15. Human RBC • What we know • Mature RBC is enucleated • During ex vivo storage, RBC undergo changes (SL) • RBC-SL has serious clinical consequences • What we didn’t know • How are the cellular processes regulated in enucleated RBC, especially in RBC-SL? • Are there other ‘command & controls’ to compensate for the loss of nucleus in mature RBC? • Can we manipulate these controls to improve the quality and shelf-life of RBC in storage?

16. Topics covered…. • miRNAs –Biogenesis and mechanism of action • Changes associated with ex vivo Storage of blood Cells (PCs and pRBC) • Roll of miRNAs in stored blood cells • Extracellular release of miRNAs in the context of ex vivo stored blood cells • Conclusions

17. Recent research advances in RBC and platelets • Enucleated mature RBC and platelets have mRNAs, splicing factors etc. in addition to proteins (Denis et al, 2005) • Seminal observations are, RBC and platelets have abundant and diverse array of microRNAs (Landry et al, 2009; Chen et al, 2009; Kannan et al 2009; Kannan and Atreya, 2010; Sarachana et al 2015; Dahiya et al 2016) • MicroRNAs (miRNAs) are established as the key cellular negative regulators of genes (mRNAs) in eukaryotic cells • In enucleated blood cells by far the miRNA is the only major regulatory nucleic acid identified that can regulate cellular processes! • Therefore, understanding the blood cell SL processes through miRNA regulation in ex vivo storage will provide clues towards enhancing the quality and perhaps shelf-life of these cells during storage

18. Understanding the regulatory mechanisms of RBC storage lesion Some of the measurable parameters during storage • RBC shape change (affects microcirculation) as measured by Mean Corpuscular Volume (MCV) • Cellular ATP loss • Apoptosis-like (eryptosis) symptoms

19. miRNA analysis in stored red blood cells • Study design and methods • Leukocyte-reduced pRBC units collected from 11 donors were stored under conventional blood bank conditions • Samples were collected from each bag at day 0, 7, 14, 28, 42, and 56 and mature RBCs were enriched and isolated • Three known RBC-SL parameters were chosen for the study [Mean Corpuscular Volume (MCV), eryptosis and ATP loss] • Purified RNA from each mature RBC sample was subjected to high-throughput miRNA microarray* differential expression profiling and the data was subjected to various bioinformatics programs • *AffymetrixGeneChip miRNA 3.0 microarrays representing 19,724 probes covering over 5,600 miRNAs, pre-miRNAs, snoRNAs etc.

20. Changes in RBC-SL parameters during storage Sarachana et al, Transfusion 2015 Increased MCV Eryptosis ATP loss

21. Normalized microarray data set was uploaded into the TMeV program* and Pavlidis Template Matching (PTM) analysis was performed using the level of eryptosis, ATP or Mean Corpuscular Volume (MCV) as the template for correlation analysis. 92 common miRNAs Correlates to all 3 RBC-SL parameters miR-196a and miR-1269 were differentially expressed over time Eryptosis ATP MCV * TIGR Multiple Array Viewer software package (TMeV version 3.0) Sarachana et al, Transfusion 2015

22. Top two cellular functions associated with potential targets of miR-196a and miR-1269 were identified These data were obtained using Ingenuity Pathway Analysis (IPA) software. P-values were calculated using Fisher’s exact test, which was performed using the entire set of genes within the Ingenuity Knowledge Base as the reference set. We selected miR-196a and evaluated its effects on cell death and/or survival Sarachana et al, Transfusion 2015

23. Overexpression of hsa-miR-196a reduces early programmed cell death and enhances ATP concentration in a human erythroblast cell line (HEL92.1.7) This suggests that miR-196a has the potential to slow eryptosis in stored RBC Sarachana et al, Transfusion 2015

24. miRNA analysis in stored red blood cells • Summary • Our analysis identified two RNAs (miR-196a and miR-1269) whose changes in the expression levels were correlated with the selected three SL parameters that we evaluated. • Overexpression of one such miRNA, the miR-196a, in a human erythroblast cell line (HEL 97.1.2) confirmed its protective effects against cell death and ATP loss. • In support of our concept that miRNAs can protect RBC from eryptosis, Yu et al, 2010 have demonstrated that miR-451 protects erythrocytes against oxidant stress (Yu et al, Genes and Development, 2010, 24:1620-33)

25. Platelet Storage Lesion (PSL) Shrivastava, 2009. Transfusion and Apheresis Science 41:105-113.

26. Significant mRNAs P<0.01, fdr<0.05 Significant miRNAs P<0.05 Downregulated mRNA targets Upregulated mRNA targets Upregulated miRNAs miRNA target filter analysis using IPA mRNA:miRNA correlation based on opposite expression using IPA using experimental target set Downregulated miRNAs Platelet miRNA and mRNA differential analysis (Day5 vs. Day9 relative to Day0 of storage) 144 differentially expressed miRNAs and 704 differentially expressed mRNA targets forming 5261 miRNA:mRNA pairs mRNA target enrichment analysis for selected mRNAs (704 mRNA targets selected based on opposite correlation with miRNAs) IPA functions, pathways, network Dahiya et al, Platelets 2016 (in press)

27. miR-570 expression in platelets (Plts) ATP5L mRNA levels in miR-570 expressed Plts Two miR-570 target sites in ATP5L mRNA 3’UTR Low levels of miR-570 can be a predictive quality biomarker for stored platelets ATP levels in miR-570 expressed Plts Dahiya et al, Platelets 2016 (in press)

28. Another example of miRNAs in stored platelets Our miRNA analysis (Day 0-Day 9 of storage) identified Ras-related protein1 (Rap1) signaling pathway as one of the potential miRNA targets in stored platelets It is known that during platelet storage Rap1 increases and triggers platelet activation as monitored by Flow Cytometry using the activation marker CD62P (Shubert et al, Transfusion 2009) Testable Hypothesis: RAP1 (mRNA) down-regulation by miRNAs should suppress platelet activation during storage and provide opportunities to improve platelet quality in storage Dahiya et al, Unpublished data

29. RAP 1 expression in megakaryocyte cells is suppressed by miR-181, miR-202 and miR-320 Ground work By using Yoon et al, 2012 method, we have enriched 20 miRNAs that are tightly associated with RAP1 mRNA 3’UTR From this list, 5 randomly selected miRNAs were introduced into a human megakaryocyte cell line and after 48 h, evaluated their effect on RAP1 expression Dahiya et al, Unpublished data

30. Topics covered…. • miRNAs –Biogenesis and mechanism of action • Changes associated with ex vivo Storage of blood Cells (PCs and pRBC) • Roll of miRNAs in stored blood cells • Extracellular release of miRNAs in the context of ex vivo stored blood cells • Conclusions

31. Taken up by other cells/ degraded/excreted Packaged in exosomes RNA pol II/III Export thru’ RNA binding proteins Export thru’ microvesicles During membrane blebbing Extracellular release of miRNAs Etheridge et al, Mutat Res 2011, 717:85-90

32. Mechanism of miRNA release from cells in the peripheral blood (Ago2=Argonaute2; NPM1=nucleophosmin1; HDL=high density lipoproteins) Stefano et al, Vascul Pharmacol 2011 Oct;55(4):111-8. Epub 2011 Aug 6.

33. Platelet MP-derived miR-223 can regulate human umbilical vein endothelial cells (HUVEC) gene expression at the protein level • Regulates FBXW7 (a F-Box protein, subunit of ubiquitin protein) • Regulates EFNA1 protein, a member of ephrin family of proteins • Platelet MPs (containing miRs, e.g. miR-126-3p) reprogram macrophage gene expression and function • Platelet MPs induced up-regulation of 34 miRs and downregulation of 367 mRNAs Laffont et al, Blood 2013, 122:253-261; Thrombosis and Haemostasis 2016, 115:311-323.

34. Topics covered…. • miRNAs –Biogenesis and mechanism of action • Changes associated with ex vivo Storage of blood Cells (PCs and pRBC) • miRNAs’ Roll in stored blood cells • Extracellular release of miRNAs in the context of ex vivo stored blood cells • Conclusions

35. MicroRNAs in Stored Blood Cells: What Future Do They Hold in Transfusion Medicine? • miRNAs regulate physiological events of mature (enucleated) blood cells • reversing this regulation may help enhance their quality and shelf-life during storage • some of the miRNAs could serve as quality biomarkers of storage • Enhancing the quality and shelf-life of stored blood cells will increase inter-donation intervals for repeat donors, especially for women whose iron deficiencies can be mitigated by long inter-donation intervals! • Blood cell-derived MP miRNAs can regulate gene expression of other cells • Can transfusion of ex vivo stored blood cells with miRNA containing MPs develop measurable transient alteration of gene expression in other cells of recipients?

36. Enhancing the quality and shelf-life of stored blood cells will increase inter-donation intervals for repeat donors, especially for women whose iron deficiencies can be mitigated by long inter-donation intervals!

37. Advancing innovation is fundamental to US FDA’s core mission of protecting and promoting the public health • -FDA’s Regulatory Science Priorities (2014-2018) Thank you!