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Sickle Cell Disease Research in the Genome Era

Betty S. Pace, MD Department of Molecular & Cell Biology The University of Texas at Dallas. Sickle Cell Disease Research in the Genome Era. Objectives. Review the accomplishments of the human genome project

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Sickle Cell Disease Research in the Genome Era

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  1. Betty S. Pace, MD Department of Molecular & Cell Biology The University of Texas at Dallas Sickle Cell Disease Research in the Genome Era

  2. Objectives Review the accomplishments of the human genome project Understand the impact of genome research in the development of treatments for sickle cell disease Discuss approaches to develop a universal cure for sickle cell disease

  3. Landmarks in Genetics and Genomics Human Genome Project 1990-2003 Advanced Technology 1972 DNA Structure 1953 Mendel 1865 2003 1990

  4. Dr. Francis Collins – Director Twenty academic research centers in the United States and Europe Major Goals Sequence the entire human genome (3 billion bases) Locate all protein coding genes (30,000 genes) Develop new technology for genomics research The Human Genome Project

  5. The Human Genome • 3 billion bases (A, T, G, C) • 30,000 genes to make proteins • 99.9% of DNA bases are identical for all people • 0.1% difference  ~10 million bases • Single nucleotide polymorphism (SNP) makes up 90% of the differences  cause of human diseases

  6. Vision For Genomic Research • Human Genome Project: Foundation • Three Major Areas of Focus • Genomic to Biology: define genome function • Genomics to Health: genome-based health benefit • Genomics to Society: maximize health benefits, minimize harms Collins et al. Nature, 422:835, 2003

  7. Genomics to Biology • Human Genome Project Lives on… • SNP Consortium • HapMap Project • ENCODE (ENCyclopedia Of DNA Elements) • Public & Private Databases, Resource Sharing • Data Mining Software • Technology • Automated Sequencing • Gene Expression – qPCR • Microarray – RNA, DNA, and protein • Mass Spectrometry • Fluorescent Activated Cell Sorting Data • High Throughput Screens

  8. Genomic to Biology – Sickle Cell Disease • History of Sickle Cell Disease • Research Progress - Four Decades • Effort to Develop a Cure

  9. James Herrick History of Sickle Cell Disease

  10. History of Sickle Cell Disease

  11. Sickle Hemoglobin Protein Discovered

  12. bS-Globin DNA Mutation transcription translation

  13. Sickle Cell Trait & Malaria

  14. AS AS AA AS AS SS Normal Sickle Cell Sickle Cell Sickle Cell Trait Trait Disease Inheritance of Sickle Cell Disease • Autosomal Recessive • Both Parents - sickle cell trait (HbAS) • 25% HbAA or HbSS; 50% HbAS • 8-10% of African Americans have sickle cell trait • 100,000 are affected with sickle cell disease • 2009 – SCDAA Legislation for national surveillance

  15. Sickle Hemoglobin

  16. Why Do Red Blood Cells Sickle? • Hemoglobin S protein polymers • Sickle shaped red blood cells • Vascular (vaso)-occlusion -Hypoxia (low oxygen) & tissue injury

  17. Vaso-occlusion In Sickle Cell Disease • Red blood cell hemolysis • Severe anemia • Recurrent pain • Chronic organ damage and failure

  18. Basic Research Progress Over Four Decades Membrane Transport & Lipids Stem Cell Biology Regenerative Medicine Red Cell Adherence Membrane Proteins Red Cell Rheology Gene Therapy Cure Polymer Formation Bone Marrow Transplant Dense cells Animal Models RESEARCH PROGRESS Fetal Hemoglobin Inducers Kinetics of Delay Time Globin Gene Regulation b-Haplotypes Studies GENOME ERA Globin Genes Sequenced ENLIGHTMENT RENAISSANCE NEW THERAPIES CURE 1970 1980 1990 2000 2010

  19. Cooperative Study of Sickle Cell Disease • Study Period – 1978 to 1998 • Purpose: describe the natural history of sickle cell disease • Subjects – over 4,000 • HbSS • HbSC • HbSb0-thal • HbSb+-thal • Over forty peer-reviewed publications

  20. CSSCD – Impact of Fetal Hemoglobin

  21. LCR e Gg Agdb Chromosome 11 The b-Globin Locus % Globin Chains Age (months)

  22. Fetal Hemoglobin Blocks Hemoglobin S Polymerization

  23. Fetal Hemoglobin Research Fetal Hemoglobin Drug Inducers Mechanisms of Action Signal Transduction Pathways Gene Mapping Efforts Gene Modifiers Genome wide studies

  24. Pace et al. Blood 100:4640, 2002

  25. Cure for Sickle Cell Disease • Hematopoietic Stem Cell Transplantation • Universal Gene Therapy

  26. Somatic Stem Cell Modification somatic cell only

  27. Bone Marrow Transplant Cure • 27 Centers in N. America and Europe • 59 children with sickle cell disease • HLA matched sibling donors • 93% Survival • New Frontiers • Mixed Chimerism • Umbilical Cord Blood • Matched unrelated donors • Oakland Children’s Hospital Sibling Donor Cord Blood Program Walters et al. Blood 95:1918, 2000

  28. d7 d14 d21 d28 • One-Phase Liquid Culture System • Peripheral blood mononuclear cells – 3 donors • Day 0: SCF, IL-3, GM-CSF (100ng/ml) & Epo (4IU/ml) • Illumina 42K microarray gene profiling Functional Genomics Approaches

  29. g/g+b (●) & b/g+b () mRNA Ratios Day 7 10 12 14 16 18 21 25 28 31 34 Hemoglobin Switching in Culture Systems

  30. Microarray Analysis

  31. Universal Cure Gene-Based Approaches • Anti-sense molecules: block sickle hemoglobin production • Viral Vectors: gene delivery systems • Nucleotide Exchange: correction of sickle gene mutation

  32. bGenes bs bs Hemoglobin S a2bs2 Red Blood Cell Flow of Genetic Information DNA Nucleus RNA Cytoplasm Protein Stem Cell

  33. b*-globin Lentivirus Vectors nef GAG env POL LTR 5’ 3’ y LTR vpr vif tat Ins Ins LTR LTR 5’ 3’ STEM CELLS

  34. December 2002 A Universal Gene Therapy Cure • Retrovirus – Lentivirus vector • Leboulch – correction of sickle cell disease in transgenic mouse • French Effort – severe combined immuno-deficiency disease • Leboulch and French collaborators – treat thalassemia & sickle cell disease patients

  35. DNA Nucleotide Exchange SCD normal The DNA Tests show that the Single Base Mutation in Sickle Cell Disease was corrected from T (abnormal) back to A (normal) in bone marrow stem cells Liu H. et al. Gene Therapy 9:118, 2002

  36. Human Gene Therapy Approach STEM CELL MODIFICATION

  37. LentiVirus bS globin bA globin LTR LTR bA*-globin HbA/S TNE Technology Ex Vivo Stem Cell Modification Nucleus RNA Cytoplasm Protein Stem Cells

  38. Human Gene Therapy Approach STEM CELL MODIFICATION

  39. The Bottom Line..… • Sickle cell disease is STILL a major health threat after 50 years of research • We have the technology to correct the mutation • The only thing that stands in the way of a cure for sickle cell disease….is MONEY!

  40. Genomics to Health • High Throughput Drug Screens • Mandatory Genetic Testing • Personalized Medicine • Genome wide genetic profile

  41. Genomics to Society • ELSI (Ethical, Legal, and Social Implications) of Human Genome Project Committee • Genetic & Racial Profiling • Privacy and Confidentiality • Laws to protect society

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