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Gene transfer for the cure of ß–thalassemia and sickle cell anemia Stefano Rivella, Ph.D.

Gene transfer for the cure of ß–thalassemia and sickle cell anemia Stefano Rivella, Ph.D. Hematopoietic stem cells. Vector carrying the therapeutic gene. Reinfusion. Transduction. Gene Therapy Schematic Approach. Structure of the TNS9 Lentiviral Vector. ß-globin gene.

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Gene transfer for the cure of ß–thalassemia and sickle cell anemia Stefano Rivella, Ph.D.

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  1. Gene transfer for the cure of ß–thalassemia and sickle cell anemia Stefano Rivella, Ph.D.

  2. Hematopoietic stem cells Vector carrying the therapeutic gene Reinfusion Transduction Gene Therapy Schematic Approach

  3. Structure of the TNS9 Lentiviral Vector ß-globin gene Locus Control Region HS2 HS3 HS4 p e+ LTR LTR RRE SD SA 840 bp 1308 bp 1069 bp 1 Kb May C. et al, Nature 2000

  4. Therapeutic Levels of Hb in Mice Affected by Cooley’s Anemia Hb 13-15g/dL Transfusion independent ß-globin LCR 5’LTR 3´LTR TNS9 Hb 2-4g/dL th3/th3 + TNS9 th3/th3 WT Rivella S. et al, Blood 2003

  5. Correction of ß-thalassemia & Sickle Cell Disease

  6. Potential problems • 1) Phenotypic variability (before and after gene transfer) • The level of endogenous hemoglobin produced by each patient + hemoglobin produced by the vector • 1) Phenotypic variability (before and after gene transfer) • The level of endogenous hemoglobin produced by each patient + hemoglobin produced by the vector • 2) Bone marrow “chimerism” • Number of HSC corrected vs. total number of HSC • 3) Random integration • Potential genome toxicity/leukemia

  7. Beta thalassemia patients: ß0/ß0, ß0/ß+ and ß+/ß+ • Based on the ß-globin protein synthesis, all mutations can be classified as ß0, where no ß-globin protein is produced, or ß+, where some, but not sufficient ß-globin chain is made • Therefore, all patients can be classified as ß0/ß0, ß0/ß+ or • ß+/ß+, according to all possible combinations of these mutations How the genotype of each patient influences the outcome of the gene transfer?

  8. A novel lentiviral vector carrying a genomic element that improves the transcription of the human ß-globin gene ß-globin gene Locus Control Region T9W HS2 HS3 HS4 p e+ LTR sinLTR RRE SD SA 840 bp 1308 bp 1069 bp AnkT9W HS2 HS3 HS4 p e+ LTR Ankyrin-sinLTR RRE SD SA 840 bp 1308 bp 1069 bp 1 Kb Laura Breda

  9. + T9W or AnkT9W Testing the AnkT9W vector in thalassemic mice 12 Gy th3/+ HSCs Bone marrow from th3/+ mice th3/+ mice Laura Breda

  10. AnkT9W is more effective than T9W in rescuing the phenotype of Hbbth3/+ BMT chimeras wt AnkT9W T9W T9W AnkT9W th3/+ wt wt Hbbth3/+ Hbbth3/+ th3/+ & T9W Hbbth3/+ T9W AnkT9W T9W AnkT9W th3/+ & AnkT9W wt wt Hbbth3/+ Hbbth3/+ Laura Breda

  11. Analysis in Human Cells ß0/ß0 and ß+/ß+

  12. Erythroid Differentiation (Phase 2) ExapansionIn vitro (Phase 1) Transduction of human erythroid precursors isolated from peripheral blood ß-globin gene Locus Control Region Ficoll separation HS2 HS3 HS4 p e+ LTR Ankyrin-sinLTR RRE +/- SD SA 840 bp 1308 bp 1069 bp 1 Kb

  13. AnkT9W Increases Significantly the HbA in ß0/0 specimens ß0/ß0 no-vector 2/2 2/2 2/2 ß0/ß0 + T9W Laura Breda

  14. Therapeutic levels of ß-globin in ß+/+ specimens #4A (β0/0) +AnkT9W, VCN=0.9 HbA α-aggregates HbFs ß0/0 HbA2 Carriers Hb levels Hb A= 0% HbF= 62.9% α-agg.= 19.2% Hb A= 63.4% HbF= 22.4% α-agg.= 5.0% before after treatment #2C (β+/+) +AnkT9W, VCN=0.9 HbA HbA ß+/+ HbFs Carriers Hb levels Hb A= 39.8% HbF= 43.2% α-agg.= 1.8% Hb A= 74.6% HbF= 19.4% α-agg.= 0% Laura Breda

  15. AnkT9W Increases Significantly the HbA in SCD specimens SCD HbA HbS Blood HbA1c HbA=0% ctrl CD34+-derived HbA=60% AnkT9W-treated VCN = 1.3 Laura Breda

  16. Conclusions • Gene transfer might offer an alternative approach to bone marrow transplant since utilizing autologous hematopoietic stem cells avoids the limitations of finding a compatible donor and prevents GVHD. However, some issues still need to be addressed. • Use of the Ankyrin insulator element in preclinical studies is extremely promising. • ß+/+ patients are likely to be the best candidates for gene therapy trials. • The same vector also shows promising results in SCD cells. However, analysis of a larger cohort of specimens from SCD patients is required…

  17. Associazione Veneta per la Lotta alla Talassemia Associazione Regionale Sarda per la Lotta alla Talassemia Weill Cornell Medical College-NY Laura Breda Carla Casu Irene Mancini Ella Guy Sara Gardenghi Nermi Parrow Lori Bystrom Robert W. Grady Patricia J. Giardina Collaborators Eitan Fibach University of Hadassa – Israel Deepa Manwani Albert Einstein - Montefiore Narla Mohandas & Karina Yazdanbakhsh NY Blood Center Roberto Gambari Universita’ di Ferrara – Italy Luca Cartegni MSKCC - NY The Carlo and Micòl Schejola Foundation

  18. Phenotypic Variability in ß-Thalassemia: β0 vs β+ mutations IVS1-110 (GA) ß0-39 (118C>T) 1424 bp β-globin gene IVS1 IVS2 1-92 1-130 93-315 131-980 316-444 ATG STOP WT transcript β0 39: a single mutant transcript β+ IVS1-110: two splicing products No Hemoglobin-A produced Some Hemoglobin-A produced

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