C.D. Atreya, Ph.D. Associate Director for Research Office of Blood Research and Review

1. MicroRNA-1246 is a Potential Regulator of Factor 8 Gene* 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 *Sarachana T, Dahiya N, Simhadri VL, Pandey GS, Saini S, Guelcher C, Guerrera MF, Kimchi-Sarfaty C, Sauna ZE, Atreya CD. PLoS One. 2015 Jul 15;10(7):e0132433. >>This presentation reflects the views of Dr. C.D. Atreya and should not be construed to represent FDA’s views or policies<<

2. Introduction • Hemophilia A disease • Symptoms • Severity • microRNAs (mi-RNAs), the gene regulators • Biogenesis • Mechanism of action • New insights into FVIII regulation?

3. Hemophilia A (HA) Disease • An X chromosome-linked bleeding disorder, caused by mutations in the F8 gene that results in a dysfunctional clotting Factor VIII (FVIII) • Symptom Severity (percentage breakdown of overall hemophilia population by severity) • Severe(factor levels < 1% in blood) represent ~ 60% of cases • Moderate(factor levels of 1-5%in blood) represent ~ 15% of cases • Mild(factor levels of 6%-40% in blood) represent ~ 25% of cases • In severe and moderate cases, repeat infusions of FVIII are required, which leads the development of FVIII inhibitors (antibodies) • Inhibitors interfere with the FVIII therapy

4. Frequencies of F8 mutation types and HA disease manifestation *Intron 22 inversion, Frameshift, Nonsense, Splice site, Large deletion, Intron 1 inversion, Small deletion and Duplication **In addition to the 2.6% with no mutations in F8, there are a few HA patients (~1.3%) with synonymous mutations in F8 where codon changes do not change the primary amino acid sequence of the translated FVIII protein (Total ~3.99%) Source: CDC HA Mutation Project, aka CHAMP database

5. Hemophilia A Observation • Over 3.9% of patients had either no mutations in F8 gene or had synonymous mutations, but exhibit the complete range of HA phenotypes -mild to severe, with over 50% of the patients being severe (CDC HA Mutation Project, aka CHAMP) and develop FVIII inhibitors • This suggests that mechanisms other than mutations in the F8 coding region per se may play either a primary or secondary role in the manifestation of HA Possible mechanism • Posttranscriptional regulation of F8 mRNA (i.e. translation control)? Hypothesis • Since microRNAs (miRNAs) are potent post-transcriptional negative regulators of mRNAs, they must be affecting FVIII expression, especially in patients with no mutation in F8 or with synonymous mutations

6. Gene Regulation MicroRNAs

7. MicroRNAs (miRNAs) Short single-stranded noncoding regulatory RNAs (17-22 nts in size) Biogenesis and mechanism of action Drsha Nuclear RNAse III Dicer Cytoplasmic RNAse III DGCR8 protein Digeorge Syndrome Critical Region 8 RISC RNA interfering silence complex http://www.sigmaaldrich.com/

8. Study Methods • 15 confirmed HA patients (9 with and, 6 without inhibitors) • 5 healthy controls • Total RNA was isolated from all 20 samples • Affimetrix GeneChip miRNA 3.0 microarrays were used (19,724 probe set covering over 5,600 human miRNAs, pre-miRNAs, snoRNAs, and scaRNAs • Appropriate bioinformatics and statistical analyses software were used to analyze and validate the data

9. Significantly differentially regulated ncRNAs (6) identified by SAM* analysis (*Significance Analysis of Microarrays) A 3-class SAM analysis of ncRNA microarray data revealed 7 ncRNA probes that were differentially expressed between hemophilia A (HA) patients with inhibitor development (HAI), hemophilia A patients without inhibitor development (HAWI), and controls (C) with the false discovery rate less than 5%. (Sarachana et al, PLOS ONE 2015. 10 (7):e0132433)

10. Fold - Change Fold - Change Fold - Change (all HA vs. C) ( HAI vs. C) (HAWI vs. C) ncRNA qRT - PCR qRT - PCR qRT - PCR Microarray Microarray Microarray (p - value) (p - value) (p - value) - 30.056 3.107 43.534 miR-1246 5.001 1.949 12.834 (0.382) (0.146) (0.297) 2.328 2.173 2.656 miR-4521 2.288 2.885 1.814 (0.014) (0.013) (0.027) 3.357 3.127 3.110 HBII - 13 2.538 3.646 1.766 (0.006) (0.047) (0.066) Validation of microarray results by qRT-PCR

11. miR-1246 and F8 mRNA levels in HA patients compared to normal donors (A) The fold change in miR-1246 was determined by RT-PCR. Total RNA was isolated from the blood of 5 control (CON) donors and 15 HA patients (HA). There is a significant increase in the miR-1246 levels in samples from HA patients compared to controls. (B) The fold change in F8 mRNA was determined by RT-PCR. Total RNA was isolated from the blood of 5 control (CON) donors and 15 HA patients (HA). There is a significant decrease in the F8 mRNA levels in samples from HA patients compared to controls

12. So far…. • 6-fold up-regulation of miR-1246 was observed in HAWI patients relative to HAI patients • miR-1246 target site is present in the F8 mRNA 3’UTR Testable hypothesis: miR-1246 down regulates F8 mRNA expression. If this is true, it could explain why 3.9% of the CHAMP database manifested HA phenotype without a mutation in F8 gene.

13. Suppression of F8 mRNA expression by miR-1246 in a stable lymphoblastoid cell line expressing FVIII P=0.0005 P<0.0001 - + - + miR 1246 Precursor Fold Change in miR 1246 Fold Change in F8 mRNA

14. Conclusions so far…. • miR-1246 has the potential to downregulate FVIII levels in normal F8, which could lead to HA phenotype • miR-1246 is a potential biomarker for • ~3.9% CHAMP subset (no mutations/synonymous mutation in F8) • HAWI subset • Next steps…. • More HA patients will be recruited to validate this study • F8 gene from miR-1246 high expresser (HAWI) patients will be sequenced to confirm their F8 gene status

15. Future work…. Testable hypothesis.. Since high levels of serum FVIII (>150 IU/L) is predictive of deep-vein thrombosis (DVT), miR-1246 might be present at low levels in such patient population and hence, low levels of miR-1246 might serve as a predictive biomarker for DVT!

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