Your SlideShare is downloading. ×
New advances and future outlook in the management and cure of hemoglobin disorders
Upcoming SlideShare
Loading in...5
×

Thanks for flagging this SlideShare!

Oops! An error has occurred.

×

Introducing the official SlideShare app

Stunning, full-screen experience for iPhone and Android

Text the download link to your phone

Standard text messaging rates apply

New advances and future outlook in the management and cure of hemoglobin disorders

1,019
views

Published on

New advances and future outlook in the management and cure of hemoglobin disorders by Philip Leboulch

New advances and future outlook in the management and cure of hemoglobin disorders by Philip Leboulch

Published in: Health & Medicine, Technology

0 Comments
0 Likes
Statistics
Notes
  • Be the first to comment

  • Be the first to like this

No Downloads
Views
Total Views
1,019
On Slideshare
0
From Embeds
0
Number of Embeds
1
Actions
Shares
0
Downloads
0
Comments
0
Likes
0
Embeds 0
No embeds

Report content
Flagged as inappropriate Flag as inappropriate
Flag as inappropriate

Select your reason for flagging this presentation as inappropriate.

Cancel
No notes for slide

Transcript

  • 1. New advances and future outlook in the management and cure of c re hemoglobin disorders gP. Leboulch – Bangkok TIF 2011
  • 2. Advances in allogeneic hematopoietictransplantationInduction of γ-globin expression γg pInduced pluripotent stem (iPS) cellsProspects for gene therapy
  • 3. Mutations Causing -Thalassemia
  • 4. Advances in allogeneichematopoietic transplantation
  • 5. 2011
  • 6. Overall Survival (OS) Disease Free Survival (DFS)
  • 7. “SCURT” SCURT (Sickle Cell Unrelated Transplant Study)Unrelated Donor Hematopoietic Cell Transplantation for  p p children with severe SCD using a reduced intensity  regimen  (BMT CTN 0601) regimen (BMT‐CTN 0601) A Multicenter Phase II Clinical Trial A Multicenter Phase II Clinical Trial Co‐PIs: Shalini Shenoy, MD y, Naynesh Kamani, MD
  • 8. SCURT Trial: Current status Data provided by DCC (Aug. 2011)• I i i d i A il 2008 Initiated in April 2008• Study now activated at 20 centers • Actively enrolling eligible patients with 8/8 allele matched unrelated Actively enrolling eligible patients with 8/8 allele matched unrelated  bone marrow donors• Target accrual: 45 patients (incl. 8 who had UCBT) Target accrual:  45 patients (incl. 8 who had UCBT)• 21 have undergone BMT – 8 males (median age: 13 1 y) 8 males (median age: 13.1 y) – 13 females (median age: 13.6 y)• Anticipated end date for enrollment: Nov 2011 Anticipated end date for enrollment: Nov 2011 Cord blood arm closed in January 2011 due to a high incidence of graft rejection
  • 9. Induction of γ-globin expression
  • 10. SCIENCE VOL 322 19 DECEMBER 2008SCIENCE VOL 334 18 NOVEMBER 2011
  • 11. BLOOD, 10 MARCH 2011 – VOLUME 117, NUMBER 10
  • 12. γ-globin expression levels obtained in postnatal mouse cells (wild-type, SCD mice) and human thalassemic erythroid cells afterBCL11A KO (floxed) or knocked-down (shRNA) are inferieur orequal to those achieved with γ- or β-globin lentiviral transfer,even after administration of deacetylating agents (i.e., 5-azaD, 5 azaD,SAHA)If therapeutic strategy involves lentiviral transfer of BCL11AshRNA ⇒ same i hRNA issues as with globin l ti i l t ith l bi lentiviral transfer fSmall molecules inhibiting BCL11A (?)
  • 13. Yearly lifelong costs of enzyme replacement for Lysosome Storage Disorders (average 50 kg adult)• Gaucher disease (1991) – $250 000 / year $250,000• Fabry disease (2004) – $290 000 / year $290,000• MPS I (2004) – $520 000 / year $520,000• MPS II (2007) – $850 000 / year $850,000• MPS VI (2007) – $800,000 / year $ , y
  • 14. Cost considerations for widespread application of gene therapy vs. small molecule The developing country of Thailand has decided to reimburse every allogenic CD34+ cell transplant for β th l ll t l t f β-thalassemia because it is so much cheaper than lifelong bl d i b i h h th lif l blood transfusion and chelation.Proposed pricing of one-time gene therapy for β-thalassemia < $200,000 and may dropto < 50,000 with widespread diffusion.Pricing of small molecule $1-$10 per day. When adjusted for inflation, this meansbetween $140,180 and $1,402,800 over an 80 year lifespan.
  • 15. Induced pluripotent stem (iPS) cells
  • 16. SCIENCE VOL 318 21 DECEMBER 2007NATURE BIOTECHNOLOGY VOLUME 29 NUMBER 1 JANUARY 2011
  • 17. Thalassemia patient ‐Induced pluripotent stem cells derivation  (Thal‐iPS) Why iPS cells?1/ An alternative for gene therapy in the future ?- Can grow in culture indefinitely- Can differentiate into any cell types.- Possible correction of any genetic defect by gene transfer or homologous recombination.- Possible selection of safe corrected cells before engraftment to the patient.2/ Our Thalassemia patient-Induced iPS cells is a good model to:- Evaluate the hematopoietic potential of Thal-iPS cells.- Study endogenous globin g y g g gene expression in Thal-iPSC-derived erythroid cells in vitro and in vivo. p y- Study the βA(T87Q)-globin vector efficacy and properties.- Investigate the oncogenic risks of a lentiviral βA(T87Q)-globin vector transferred into βE/β0- thalassaemiapatient.- Compare lentiviral integration sites in corrected THAL-iPS cells with those of the patient. Dr. Leila Maouche‐Chrétien (France) in  . e a aouc e C ét e ( a ce) collaboration with Alisa Tubswan and  Prof. Suthat Fucharoen (Thailand)
  • 18. Study Design ‐thal/HbE patient Measurement of globin expression Pyrosequencing  Lentiviral vector carrying A‐T87Q gene Hematopoietic reconstitution Oct4, Sox2, Klf4,  CFC assay in NOD/SCID mice cMyc retroviruses reprogramming ‐globin transfer differentiation MOI=30 Thal‐iPSTG+CD34‐ cells THAL‐iPS cells Hematopoietic cells iPS cells  S ce s FACs analysis characterization of hematopoietic markers
  • 19. Characterization of Thal‐iPS cells TRA1 60 TRA1-60 SSEA 3 SSEA-3 SSEA 1 SSEA-1 NANOG OCT 4 OCT‐4 SSEA‐4 Intestine DAPI DAPI CNS Bronchea DAPI Epithelium  Cartilage CNS, Muscle M l Retina R ti Adipose Analysis of pluripotent genes Analysis of exogenous genes Analysis of transgene integration by expression by RT-PCR silencing by RT-PCR PCRendo OCT4endo SOX2 pMig OCT4 OCT 4endo KLF4 pMig SOX2 SOX 2endo c-MYC c MYC pMig KLF4 Mi KLF 4NANOG pMig c-MYC c-MYCβ-ACTIN
  • 20. In vitro hematopoietic differentiation of Thal iPS cells before (TG ) and after (TG+)  In vitro hematopoietic differentiation of Thal‐iPS cells before (TG‐) and after (TG+) transduction with  the lentiviral vector A‐T87Q gene.Thal-iPSTG-Thal-iPSThal iPSTG+ Mature BFU-E CFU-MIX CFU-G CFU-M
  • 21. HPLC analysis of hemoglobin in BFU‐Es derived from Thal iPS and Thal iPSTG+ no adult Hb E BFU-E patient Blood BFU-E.1 iPS-THAL TG- 0 5 10 20 25 30 CFU-GM. PS-THAL BFU-E 2 iPS-THAL TG+ HbAT87Q (%) = HbAT87Q /(HbAT87Q + HbF+ embryonic Hb) x 100• BFU-Es from Thal-iPS produce mainly Hb F and embryonic Hb, with clear trend to switching in vivo• Hb A (T87Q): highly expressed in BFU-E containing Lentiviral βA(T87Q)-globin vector (level ranged from 40-85%) - no expression in non-erythroid cells
  • 22. Hematopoietic engraftment of hematopoietic cells derived from Thal‐iPS in immunodeficient‐mice 8 of 26 transplant mice showed engraftment with human cells (low levels) 10 5 10 4 Human CD453 weeks post transplantation 10 3 GpA+ cells sorted (1 mouse) 10 2 Late erythroblast 0 0 10 2 10 3 10 4 10 5 Human glycophorin A 10 5 10 5 20 Human CD19/2 Human CD45 10 4 10 4 3 weeks post transplantation Myeloid cells (1 mouse) 10 3 10 3 10 2 10 2 H 0 0 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 Human glycophorin A Human CD15/33/66b 250K 10 5 Hum CD19/20 200K 10 4 FSC 150K 10 3 8 weeks post transplantation man 100K ( 6 mice) 50K 10 2 B-lineage cells 0 0 0 10 2 10 3 10 4 10 5 0 10 2 10 3 10 4 10 5 Human CD45 Human CD15/33/66b
  • 23. Globin switching in vivo ?/(?+?) 1.000 1.20 1.00 0.100 0.80β/(β+) β/(β+) 0.633 0.60 0.010 n=2 0.96 0.40 n=22 0.011 0.57 0.20 0.42 n=1 0.004 n=2 n=3 0.001 n=2 0.00 Thal‐iPS BFU‐E Thal iPS in vivo GPA+ THAL  THAL patient blood In vivo Thal patient CB CB In vivo GPA+ in vivo GPA+ Adult normal adult normal blood iPS BFU-E GPA+ blood CD 34+ CB 34 blood Thal-iPS • The ε to (γ + β) switch was ≈ complete after in vivo passage of iPS derived cells • The β / (β + γ) ratio was ≈ 3 times higher after in vivo passage of iPS derived cells
  • 24. Screening for clones with ‟safe” integrants in BFU‐Es from iPS THALTG+ g gCriteria for iPS cells clones harboring  safe genomic integration sites.Criteria for iPS cells clones harboring safe genomic integration sites.‐ distance of at least 50 kb from any genes‐ distance of at least 300 kb from cancer‐related gene‐ distance of at least 300 kb from any micro RNA distance of at least 300 kb from any micro RNA ‐ location outside a transcription unit  and ultraconserved regions (UCRs) Total 24 individual BFU-Es 12 BFU-Es containing single copy number of the vector 2 of 12 (16%) BFU-Es containing safe integration site
  • 25. Common Integration Sites (CIS) in ALD, β0/βE‐Thal patient and iPS THAL Total of 4859 IS ( 2146 IS ALD P1, 1282 IS ALD P2, 357 IS Thal patient, and 1074 iPS THAL) • located in CIS of the 3th order or higher 1698 IS • Most of CIS are shared integration in 3 studies Most important CISs 50 IS  • IS found from at least 2 studies • should at least be of 6th order # CIS Cluster # CIS Cluster Shared Data set Shared Data set 30 Shared in all data set 7 ALD & β‐Thal 13 ALD & iPS 0 iPS & β‐Thal * considered 2, 3 or 4 insertions as CIS of 2nd, of 3rd or 4th order if they fell within a 30 kb, 50 kb or 100 kb window of genomic sequence from each other, respectively.
  • 26. Conclusions from our Thal‐iPS study from β0/βE‐thalassemia gene therapy patientThal-iPS cells are able to differentiate into multiple blood cell types both in vitro and invivo in immunodeficient-mice but level of multilineage engraftment very low in NSG mice g g y16% of transduced Thal-iPS cells with βA(T87Q)-globin lentiviral vector satisfied thestringent criteria for “safe” areas of vector integration g gBFU-Es derived from genetically corrected Thal-iPS cells show high levels of Lentivector-derived βA(T87Q)-globin expression - Clear trend to globin class switching in vivo g p g gComparative analysis combining IS data show shared CIS between Thal-iPS, Thalgene therapy patient and 2 ALD gene therapy patients with no bearing of thetherapeutic DNA insert (lentivector-backbone only)CIS of 3rd order or higher occur at least 100 times more often than expected under therandom lentiviral distribution (evidence of non-random integration rather than in vivoselection?)Multiple and formidable hurdles remain for the use of iPS cells (both safety and efficacy)
  • 27. Prospects for gene therapy
  • 28. Conversion to transfusion independence of the first βE/β0-thalassemia thalassemia(major) patient for > 3.5 years at ≈ 9 g/dL Hb, > 4.5 years post-gene therapy
  • 29. Intrinsic integration bias independent from DNA insertsBLOOD, 3 JUNE 2010 – VOLUME 115, NUMBER 22 October 2011 / Volume 6 / Issue 10 / e24247October 2010 / Volume 6 / Issue 11 / e1001008
  • 30. Second E/0-thalassemia (major) gene therapy patient transplanted on November 24 2011 24, Patient 1 = PLB Patient 2 = MHV Globin chains in PLB reticulocytes Globin chains in MHV reticulocytes 26 days 32 days post‐transplantation y p p p post‐transplantation p87Q)/(+) 4.4% 8.9%87Q)/() 3.6% 8.2%(87Q)/(E) 9.5% 9 5% 20.7% 20 7%(87Q)/() 9.0% 18.5%(E)/() 46.4% 43.0%()/() 49.2% 48.1%
  • 31. PROSPECTSOptimized βA(T87Q)-globin lentiviral vector validated (higher transduction globinpotency) – New or amended trial to be filed in France and US (early 2012)Continuation Phase I/II for 0-thalassemia (major Cooley) and sickle cell (major,diseasePilot Phase IIb/III for E/0-thalassemia (major) thalassemiaEx vivo or In vivo selection for transduced HSCsConditional suicide for enhanced safetyHSC and progenitor expansionDecreased α-globin (β-thalassemia) by shRNAs(coll.(coll with Dr. Jim VADOLAS, Australia) Dr VADOLAS
  • 32. SCIENCE VOL 329 10 SEPTEMBER 2010
  • 33. BLOOD, 19 MAY 2011 – VOLUME 117, NUMBER 20
  • 34. Naturally occuring truncated (t)Epo-R variants in humansTruncation of the C-terminal moiety of Epo-R is responsible for « dominant benign hereditaryerythrocytosis » (de la Chapelle et al. PNAS 1993) « … cross-country skier having won three Olympic gold medals and two world championships »Hypersensitivity of erythroid progenitors to Epo (Juvonen et al. Blood 1991; Prchal et al. Eur JHaematol 1996) Epo Epo Epo Jak2(P) Jak2(P) Jak2(P) Y343 STAT5(P) STAT5(P) - Y401 Y1 Y429 SHP-1(P) Y12 Y18
  • 35. Co-expression of truncated Epo-R results in cure of thalassemia mice at very low vector copy levelsA B 100 40 80 30 human Hb (%) huRBC (%) 60 20 40 LG P= LG/HA-Y1 LG P= LG/HA-Y1 0,006 r2 = P = 0,079 r2 0,005 r2 = P = 0,144 r2 0,690 = 0,336 10 20 0,696 = 0,247 0 0 0 0,2 0,4 0,6 0,8 0 0,2 0,4 0,6 0,8 Copy per WBC Copy per WBC C 1,9 1,7 - Phenotypic correction with 1,5 < 0.01vector copy in WBCs log Epo 1,3 1,1 - No long-term leukemogenesis 0,9 P = 0,0000007 r2 = 0,857 and organ pathology (>10 months 0,7 and secondary transplants) 0,5 0 20 40 60 80 100 huRBC (%)
  • 36. Thank you for your attention 37