1. Genes - Where Do We Go From Here? Camp Sunshine Monday, July 13, 2015 Dr. Dave Bodine, Ph.D. Chief, Genetics and Molecular Biology Branch National Institute for Human Genome Research 49 Convent Drive, MSC-4442, Room 4A04 Bethesda, MD 20892 Phone: (301) 402-0902 email: tedyaz@mail.nih.gov

3. MACROPHAGE STEM CELLS ERYTHROCYTES PLATELETS T CELLS B CELLS BASOPHILS EOSINOPHILS NEUTROPHILS COMMITTED PROGENITOR CELL ADA Deficiency: A Combined Immunodeficiency ADA Courtesy of Fabio Candotti ADA

4. MACROPHAGE STEM CELLS ERYTHROCYTES PLATELETS T CELLS B CELLS BASOPHILS EOSINOPHILS NEUTROPHILS COMMITTED PROGENITOR CELL Gene Therapy for ADA Deficiency Virus containing a normal ADA gene Courtesy of Fabio Candotti ADA

5. Collect Marrow Isolate CD34+ Cells SCF MGDF FL Combine Aliquots Backup Marrow MND-ADA GCsapM-ADA Transduce x 3 Days Gene Therapy of ADA deficiency Busulfan 75 mg/m 2 ADA- Discontinue PEG-ADA Reinfuse ADA?

8. PROSPECTS FOR GENE THERAPY FOR DIAMOND BLACKFAN ANEMIA: MIXED RPS19 RPL5 RPL11 RPS26 RPS24 RPL35a RPS10 RPS17 RPS7 Other – inc. GATA1, MCM2 deletions unknown deletions genotype unknown No need to have the gene regulated. Modest expression levels should have positive results. Need to improve gene transfer frequency by 2-3 fold at a minimum. Need to develop multiple gene transfer vectors. +-+-

9. SOME NEW FINDINGS ABOUT DBA

10. COST OF SEQUENCING A GENOME

11. SEQUENCING PIPELINE

13. DECIPHERING THE DATA

14. RPS19 DELETIONS

15. COST OF SEQUENCING A GENOME

16. SEQUENCING PIPELINE New genes that cause DBA Small deletions that cause DBA

17. HUMAN CHROMOSOMES

19. THE DBA DEFECT IS AT THE BFU-E STAGE LT-HSC CLP CMP ST-HSC MEP GMP ERY Plt Neu Eo NK Mono Bas Mac T B MEG MPP LMPP HSC CFU-Mk CFU-E Lipton JM, Kudisch M, Gross R, Nathan DG (1986). “Defective erythroid progenitor differentiation system in congenital hypoplastic (Diamond-Blackfan) anemia”. Blood. 67: 962-8. Iskander D, et al (2015). “Elucidation of the EP defect in Diamond-Blackfan anemia by characterization and prospective isolation of human EPs”. Blood. 125 :2553-7.

20. THE DBA DEFECT IS AT THE BFU-E STAGE Question 1: Where can we get DBA erythroid progenitor cells? Question 2: Can enough DBA erythroid cells be cultured from peripheral blood CD34+ cells for analysis? Lipton JM, Kudisch M, Gross R, Nathan DG (1986). “Defective erythroid progenitor differentiation system in congenital hypoplastic (Diamond-Blackfan) anemia”. Blood. 67: 962-8. Iskander D, et al (2015). “Elucidation of the EP defect in Diamond-Blackfan anemia by characterization and prospective isolation of human EPs”. Blood. 125 :2553-7. LT-HSC CLP CMP ST-HSC MEP GMP ERY Plt Neu Eo NK Mono Bas Mac T B MEG MPP LMPP HSC CFU-Mk CFU-E

21. CULTURE OF CIRCULATING CD34+ CELLS 1. Measure growth and differentiation of patient and control cells in vitro 2. Analyze the transcriptional profiles of phenotypically similar patient and control cells in vitro LT-HSC CLP CMP ST-HSC MEP GMP ERY Plt Neu Eo NK Mono Bas Mac T B MEG MPP LMPP HSC CFU-Mk CFU-E Miyake K et al. (2008). “Ribosomal protein S19 deficiency leads to reduced proliferation and increased apoptosis but does not affect terminal erythroid differentiation in a cell line model of Diamond-Blackfan anemia.” Stem Cells. 26: 323-9. Narla A et al. (2011). “Dexamethasone and lenalidomide have distinct functional effects on erythropoiesis.” Blood. 118: 2296-304. Moniz H, et al. (2012). “Primary hematopoietic cells from DBA patients with mutations in RPL11 and RPS19 genes exhibit distinct erythroid phenotype in vitro.” Cell Death Dis. 3:e356.

22. Kelly O’Brien D REDUCED GENERATION OF ERYTHROID CELLS FROM DBA PATIENTS Control DBA 7x10 6 6x10 6 5x10 6 4x10 6 3x10 6 2x10 6 1x10 6 0 Kelly O’Brien

23. MICROARRAY ANALYSIS OF DBA ERYTHROID CELLS (RP MUTATIONS) Top Upstream Regulator: GATA1 Top Functions: Hematological System Development and Function; Increased Hematocrit Increased RBC DBA – RPS17DBA – RPS24DBA - UnknownDBA – RPL5 DBA - Unknown DBA – RPS17 Control s

24. DBA Patients and Families: YOU ARE NOT ALONE! Each of the investigators in this photo represent teams of dedicated people who are trying to understand, treat and cure DBA. FINAL THOUGHTS

26. THANK YOU Thanks to all of you for being here. You are providing the most valuable contribution of all: Hope, Support and a Sense of Community. No DBA patient or family could feel alone in the presence of people like you. No researcher could not be inspired by people like you.