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Control of excess α -chains in β- thalassemia

Control of excess α -chains in β- thalassemia. G. Vassilopoulos MD PhD Associate Professor, Hematology and Internal Medicineci U. of Thessalia Medical School Principal Investigator, BRFAA Division of Genetics & Gene Therapy.

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Control of excess α -chains in β- thalassemia

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  1. Control of excess α-chains in β-thalassemia G. Vassilopoulos MD PhD Associate Professor, Hematology and Internal Medicineci U. of Thessalia Medical School Principal Investigator, BRFAA Division of Genetics & Gene Therapy

  2. Globin Chain Imbalance is the Major Determinat of Disease Severity in β-thalassemia Disease Morbidity β/α = 0.4-0.6 normal β-thalassemia Carrier state β/α = 1 β/α < 0.2 • Excess α chains • Inclusion bodies (can trigger immune destruction) • Membrane damage (Mechanical removal) • Phosphatidyl exposure (Hypercoagulability) • Apoptosis (Ineffective erythropoiesis)

  3. Loss of erythrocyte precursors in the Bone Marrow Chronic Hemolytic Anemia Ineffective Erythropoiesis 60-75% of total erythropoiesis β globin mutations

  4. HYPOTHESIS If alpha - globin excess is ameliorated, a significant portion of ineffective erythropoiesis would be eliminated. Tool : RNAi

  5. Drosha DGCR8 pri-miRNA B. pre-miRNA Exportin-5 DICER cytoplasm 19-23 nt siRNAs 5 3 Ago-2 5’- CAP 5 3 ΑΑΑΑ mRNA Target RISC • Nobel in Medicine 2006: • RNA interference Synthesized Pol II miRNA Pol III shRNA ChemicalsiRNA nucleus

  6. IP FV vector d.pol d.gag d.env R U5 R CMV U5 delU3 Tas Pol Gag Env Tas bel 2&3 SP SU TM U3 U3 R R U5 U5 M - C - NC envelope PR RT IN enzymes • Foamy Virus wt and derived Vectors Deleted Foamy (ΔΦ)del U3-LTR (SIN) Minimal cis-acting sequences No Transactivator

  7. Foamy Virus Vectors can transduce HSC AP-expressing vector GFP-expressing vector GFP+ CFU WBC RBC PLT 2000 39% 51% 59% 1000 Counts 0 104 100 102 100 102 104 100 102 104 GFP

  8. 100 80 75 % 71 68 68 65 60 55 43 40 CFU (preBMT) 39 Engraftment 20 0 BM CD45 BM CD19 BM CD33 BMCD34 PB CD45 SP CD45 GFP+ FV vector mediated transduction of huCD34+ cells Josephson, et al HuGeneTher, 2004

  9. mU6.shRNA CMV R U5 R U5 Mscv H1.shRNA • Design of FV-RNAi vectors FV.MscvGFP/ΔNGFR ΔNGFR or GFP CMV R U5 R U5 Mscv FV.mU6/H1.shRNA.MF/N ΔNGFR or GFP shRNA Promoters mU6 or H1 (5’pol) Reporter genes GFP orΔNGFR

  10. Untx-GFP+ • FV-mU6 or H1 vectors reduce marker gene expression in vitro 100 80 60 40 HT1080 20 HeLa 293T 0 Η1 mU6

  11. Η7 Η5 shNS 22% 23% 120 shNS 100 shbcr.abl 80 2.5% 4.5% 60 % GFP over D3 40 20 shbcr.abl 0 5 7 15 20 30 3 ΗΜΕΡΕΣ FV.H1-RNAi: Targeting bcr.abl induces apoptosis in Κ562 cells Target: GFP+ K562 cells Apoptosis d7 60 40 % Annexin 20 NS bcr.abl

  12. 120 100 80 60 40 20 0 FV.mU6-RNAi: Stable target downregulationin vivo ♀ ♀ ♀ Hosts: wt females d-4 d-1 d0 BMT Busilvex IP Relative GFP expression (MFI) Donors: GFP+ PB 6w scrambled FV: mU6.shGFP/NS.ΔNGFR PB BM SP 13 w

  13. TSS Mouse cDNA Human cDNA AIM Mild (30-50%) reduction in a-globin expression Β9/C7/D3/E1FV.mU6.shA.MF Η1/2/4/5 FV.H1.shA.MF Assay vector efficiency in eryhtroid lines(MEL, K562) Pick best performers for assaying in primary cells

  14. FV.mU6.shA.MF: reduction in mouse α-globin in ΜEL cells 100 79 53 % aglobin sh AGLO vs CMEL 50 scrambled Control 27 12 B9 C7 D3 E1

  15. α globin 1-4.5 1-4 1-5 1.5-4.5 b-actin SCR H1 H2 H4 H5 FV.Η1.shA: human α-globin reduction in Κ562 120 100 80 mRNAshA/control 60 Control 40 20 0 SCR H1 H4 H5

  16. FV.mU6.shA.MF: α-globin mRNA reduction in murine BFUe Pick GFP+ BFUe Lin- wt cells Grow BFUe Tx o/n with vector 100 80 60 %mRNA shA / control Control 28 15 16 40 21 20 0 D3 B9 C7 E1

  17. 30 25 CD71 20 10 Thal3/+ 10 6 B9 C7 TER119 mU6.shA.MF vectors can improve ineffective erythropoiesis in thal3 mice (in vitro) Nishina et al, BBRC, 2009 Thal3/+FLC Thal3/+ FLC-GFP+ 12.3% 6.8% # BFUe /10E4 thal3 Lin-

  18. 100 80 60 40 20 0 UnTx H1 H2 H4 H5 VCN=10-15 , n=3-5 FV.Η1.shA: human α-globin reduction inCD34+ cells AssayRNARealTimePCRα-globin/GAPDH CD34+ transduction withH1.shA.GFP mRNAshA/control Control

  19. 34.9% 32.8% 9.0% MFI 12.8 MFI 7.44 MFI 13.9 β-globin expression in CD34+ from thal patients transduced with two different therapeutic FV vectors HS2.HS3.β Control HS40.β Human β-globin

  20. Combination FV vector expressing α-globinshRNAand β-globin Anti– αGLO shRNA Η1 β-promoter CMV/LTR 3 2 1 3’UTR β-globin a-HS40 pΔΦ.Η4/HS40.β

  21. Amelioration of β/α ratio in CD34+ cells from a thalassemic patient Comfort zone β/α globin mRNA ratio

  22. Conclusions • FV vectors expressing shRNA can provide sustained gene silencing in vitro and in vivo • Efficient gene silencing of the mouse and human a-globin transcripts • Amelioration of ineffective hematopoiesis • Therapeutic effect with the combination vector • In β-thal, strategies aiming at reducing α-excess are rationale and worth further exploration

  23. There are Howevers….. • RNAi is often unpredicted • There is need for tight regulation of expression • Vector integration is unpredicted and could lead to overactive transgenes with non-desired consequences • Vector-transduced cells are cells and not medicine; once in, tough to take them out

  24. Future Perspectives • RNAi is not water and is here to stay • Since RNAi functions in the cytoplasm, transient effect could be more desirable • Expect smart delivery methods since vectors and genes and stem cells are costly, need sophisticated infrastrucrure and are not accessible to every patient.

  25. ACKNOWLEDGMENTS Magda PAPADAKI – RNAi John MORIANOS - Glo vectors Elena SIAPATI – mom of the lab Collaborators K. STAVROPOULOU - BRFAA A. KATAMIS - Ag.Sofia Hospital DW RUSSELL - U.Washington DW EMERY - U.Washington G. STAMATOYANNOPOULOS D. LOUKOPOULOS Support CONSERT, FP6, EU PENED, GSRT, GR BRFAA, Intramural

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