Presentation is loading. Please wait.

Presentation is loading. Please wait.

Control of excess α-chains in β-thalassemia G. Vassilopoulos MD PhD Associate Professor, Hematology and Internal Medicineci U. of Thessalia Medical School.

Similar presentations


Presentation on theme: "Control of excess α-chains in β-thalassemia G. Vassilopoulos MD PhD Associate Professor, Hematology and Internal Medicineci U. of Thessalia Medical School."— Presentation transcript:

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 = 1β/α < 0.2 β/α = Carrier state normal β-thalassemia Excess α chains Inclusion bodies (can trigger immune destruction) Membrane damage (Mechanical removal) Phosphatidyl exposure (Hypercoagulability) Apoptosis (Ineffective erythropoiesis)

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

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

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

6 Tas Pol IP Env RU5U3U3 R U3U3 Gag bel 2&3 Tas PRRTIN enzymes SPSUTM envelope M - C - NC Foamy Virus wt and derived Vectors delU3 CMV R U5 R d.gag d.pol d.env FV vector Deleted Foamy (ΔΦ) del U3-LTR (SIN) Minimal cis-acting sequences No Transactivator

7 Foamy Virus Vectors can transduce HSC AP-expressing vector GFP+ CFU Counts WBCRBCPLT %51%59% GFP GFP-expressing vector

8 Engraftment BM CD45 BM CD19 BM CD33 BM CD34 PB CD45 SP CD45 CFU (preBMT) GFP+ % FV vector mediated transduction of huCD34+ cells Josephson, et al HuGeneTher, 2004

9 FV.MscvGFP/ΔNGFR CMVRU5R Mscv Design of FV-RNAi vectors ΔNGFR or GFP FV.mU6/H1.shRNA.MF/N mU6.shRNA CMVRU5R H1.shRNA Mscv ΔNGFR or GFP shRNA PromotersmU6 or H1 (5’ pol) Reporter genes GFP or ΔNGFR

10 Η1mU6 Untx-GFP+ FV-mU6 or H1 vectors reduce marker gene expression in vitro 293THT1080HeLa

11 Η5Η5 shNS shbcr.abl 22% 4.5% Η7Η7 23% 2.5% % Annexin Apoptosis d7 NSbcr.abl FV.H1-RNAi: Targeting bcr.abl induces apoptosis in Κ562 cells Target: GFP+ K562 cells

12 Busilvex IP d-4 d-1 ♀ ♀♀ Relative GFP expression (MFI) FV.mU6-RNAi: Stable target downregulation in vivo Donors: GFP+ d0 BMT Hosts: wt females FV: mU6.shGFP/NS.ΔNGFR PB 6w PBBMSP scrambled 13 w

13

14 Mouse cDNA Human cDNA TSS AIM Mild (30-50%) reduction in a-globin expression Β9/C7/D3/E1 FV.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

15 % aglobin sh AGLO vs CMEL B9C7D3E1 Control FV.mU6.shA.MF: reduction in mouse α-globin in ΜEL cells scrambled

16 H1SCRH2H4H5 α globin b-actin FV.Η1.shA: human α-globin reduction in Κ SCRH1H4H5 mRNA shA/control Control

17 FV.mU6.shA.MF: α-globin mRNA reduction in murine BFUe % mRNA shA / control B9C7 D3 E1 Control Lin- wt cells Tx o/n with vector Grow BFUe Pick GFP+ BFUe

18 6.8% Thal3/+ FLC 12.3% Thal3/+ FLC-GFP+ mU6.shA.MF vectors can improve ineffective erythropoiesis in thal3 mice (in vitro) TER119 CD71 Nishina et al, BBRC, Thal3/+ B9 C # BFUe / 10E4 thal3 Lin-

19 UnTxH1H2H4H5 VCN=10-15, n= 3-5 Control mRNA shA/control FV.Η1.shA: human α-globin reduction in CD34+ cells CD34+ transduction with H1.shA.GFP Assay RNA RealTimePCR α- globin/GAPD H

20 Human β-globin MFI % MFI % MFI % β-globin expression in CD34 + from thal patients transduced with two different therapeutic FV vectors ControlHS40.β HS2.HS3.β HS40.βHS2.HS3.β VCN

21 Combination FV vector expressing α-globin shRNA and β-globin pΔΦ.Η4/HS40.β  -HS40 β-promoter 123 CMV/LTR 3’UTR Η1 Anti – αGLO shRNA β-globin

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

23 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

24 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

25 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.

26 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

27


Download ppt "Control of excess α-chains in β-thalassemia G. Vassilopoulos MD PhD Associate Professor, Hematology and Internal Medicineci U. of Thessalia Medical School."

Similar presentations


Ads by Google