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Gene Therapy for Primary Immunodeficiency

Gene Therapy for Primary Immunodeficiency. Phase l/ll studies at ICH/GOS X-SCID (9+1 patients) ADA SCID (1 patient) X-CGD (2 patients). X-linked severe combined immunodeficiency (SCIDX1). 1 in 50-100,000 live births Major form of SCID

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Gene Therapy for Primary Immunodeficiency

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  1. Gene Therapy for Primary Immunodeficiency Phase l/ll studies at ICH/GOS X-SCID (9+1 patients) ADA SCID (1 patient) X-CGD (2 patients)

  2. X-linked severe combined immunodeficiency (SCIDX1) 1 in 50-100,000 live births Major form of SCID Severe diarrhoea, pneumonia, septicaemia, fungal infection, failure to thrive, death usually within first year of life.

  3. P P STAT5 STAT5 P P STAT5 STAT5 IL-2 a b g JAK3 JAK1 STAT5 Gene transcription

  4. Blood Bone marrow Stem cells B Thymus T Bone Marrow Transplantation: Use donor bone marrow stem cells to correct the defect NK lymphocytes

  5. Blood Bone marrow Stem cells B Thymus T Gene therapy: Put the new gene into bone marrow stem cells to correct the defect NK lymphocytes

  6. Treatment by bone marrow transplantation • Use of matched donors successful in 90% of cases • for 60% of cases, no matched donors have to use mis-matched donors, reduces success rate to <60% • need to improve success for children with no matched donor

  7. Cumulative probability of survival after related HLA-mismatched HSCT in SCID patients 3 years survival rate 45.2% 53.8%

  8. SCIDX1 morbidity and mortality following HLA-mismatched transplantation… 20% 1 year mortality Long term effects related to chemotherapy, usually with alkylating agents (growth, fertility, secondary malignancy, neuropsychological, hypodontia) Incomplete immunological reconstitution

  9. X-SCID: major selective growth and survival advantage for corrected cells…. Somatic reversion events Animal models

  10. Y+

  11. Gene therapy for X-SCID: phase I study Criteria for entry: No matched sibling donor Molecularly confirmed diagnosis Common gamma chain vector: PG 13 producer cells (GALV envelope) titre approximately 1x10e6 transducing units per ml

  12. Transduction protocol – SCIDX1 Harvest CliniMacs CD34+ bone marrow Pre-activation (40hours) X-Vivo10 (serum free) SCF 300ng/ml, FL 300ng/ml, TPO 100ng/ml, IL-3 20ng/ml Transduction (3 cycles over 72 hours) Nexell gas permeable flexible containers Retronectin coating Virus pre-loading Infusion

  13. D1 D4 D5 gc CD34 P2: Transduction process (days 1-5)

  14. Clinical data Age at therapy (months) Maternal graft Mutation Gamma chain expression Total cells infused (x10e6) Patient details (April 2006) P1 10 ++ R289X ++ 180 P2 10 ++ S238N - 180 P3 4 - Y125C +/- 78 P4 3y - R289X ++ 115 P5 10 - R222C ++ 200 P6 10 - PolyA - 200 P7 6 - M1i - 84 P8 13 - C182Y - 207 P9 7 - S108P - 160

  15. P1 P2 P3 P4 P5 P6 Lymphocyte recovery CD3/4/8 (weeks, April 2006) T cells per l P8 P7

  16. Lymphocyte recovery CD3 (weeks, April 2006) P1 P5 P6 P3 P7 P8 P4 P2 P9

  17. wk4 wk10 wk16 Flow cytometric analysis of CD45RO-CD27+ naïve T cells in P1 wk20 wk24 wk28 CD27 CD45RO Good evidence for recent thymic emigrants

  18. scIg P1 Immunoglobulin levels………

  19. Somatic mutation + CD19 PE CD27 FITC Immune responses to challenge… 10 8 6 4 Ig g/l 2 0 0 20 40 60 80 100 weeks VZV IgG and IgM positive Vaccine antigen positive Development of antigen-experienced B cells (CD19+CD27+)

  20. Lymphocyte proliferation responses

  21. 5’LTR 3’LTR Unknownhost Genomic DNA Proviral gene Unknown host Genomic DNA HIJ Host/Integrant/Junction (HIJ) Integration analysis – efficiency and quality of transduction • RT-PCR, linker adaptor mediated (LAM)-PCR and related techniques • Copy number • Numbers of integration sites • Integration destinations

  22. Efficiency of gene transfer….. CD3 <3 copies CD19 0.01-0.2 copies CD33 0.001-0.1 copies CD34 +ve (P1, P2, P3)

  23. LAM-PCR analysis... #1 #2 100bp 3 19 M G NK 3 G M 19 NK

  24. Summary of results: • Immunological reconstitution  • Immune cell function  • Immunisation  • Children at home, off therapy 

  25. Similar case with another adult patient in Paris

  26. Paris study - update 11 children treated (1 atypical) Good response in 10 patients However: Patients 4, 5 and 10 – Serious adverse event Full immune reconstitution but developed monoclonal T cell lymphoproliferation - CD3+ leukemia All presented at between 30-36 months post-treatment All treated by chemotherapy & BMT Conclusion: P5 AW P10 AW P4 relapsed and died

  27. Mechanism of leukaemogenesis? Insertional mutagenesis P4 & P5 - Single insertions into LMO-2 gene (P4 in intron 1, P5 5’ upstream, promoter region) MFG gc MFG gc 1 2 3 4 5 6 P4 P5 LMO-2 Oncogene, known to be expressed in T cell leukemia, LMO-2 is highly expressed in leukaemic clones Other factors? P10 - 3 different integration sites (Lyl-1, Bmi-1, ?) Synergistic effect of c + LMO-2?

  28. Follow up months Patient follow up (April 2006)… P1 AW* 56 P2 AW 52 P3 AW* 47 P4 AW* 39 P5 AW* 27 P6 AW* 24 P7 AW 17 P8 AW 4 P9 AW 0 *off prophylactic therapy

  29. Insertions around Transcription Start Sites 8% TSS 7% 6% 5% 4% Insertions in % 3% 2% 1% 0% 1 to 2 3 to 4 5 to 6 7 to 8 0 to 1 2 to 3 4 to 5 6 to 7 8 to 9 9 to 10 -1 to 0 -9 to -8 -7 to -6 -5 to -4 -8 to -7 -4 to -3 -3 to -2 -2 to -1 -6 to -5 -10 to -9 Distance from Transcription Start Site TSS (kb)

  30. Analysis of Retroviral Integration Sites (IS) Post-Transplantation Absolute Percentage Total number of integration sites (IS) 344 100% Exactly mappable IS 252 IS in RefSeq genes 99 39% IS in RefSeq genes including the region 10 kb upstream and downstream of the gene 148 59% IS more than 10 kb away from RefSeq genes 104 41% IS within +/- 5 kb around transcription start site 58 23%

  31. Lymphocyte recovery CD3 (Paris, March 2005)….. 9000 8000 * P4 7000 6000 P8 5000 T Lymphocytes/µl P10* 4000 3000 P2 P7 * 2000 P5 P6 1000 P1 P9 0 0 10 20 30 40 50 60 70 Months after Gene Therapy

  32. Lymphocyte recovery CD3 (Paris and London, March 2005)….. 9000 8000 * P4 7000 6000 P8 * 5000 * T Lymphocytes/µl * P10 4000 * 3000 P2 P7 * 2000 P5 P6 1000 P1 P9 0 0 10 20 30 40 50 60 70 Months after Gene Therapy

  33. UK SCIDX1 protocol…. (MFG, GALV-pseudotype, no B2 mutation) Harvest CliniMacs CD34+ bone marrow Pre-activation (40hours) X-Vivo10 (serum free) SCF 300ng/ml, FL 300ng/ml, TPO 100ng/ml, IL-3 20ng/ml Transduction (3 cycles over 72 hours) Nexell gas permeable flexible containers Retronectin coating Virus pre-loading No protamine sulphate Infusion

  34. Will it happen to children treated in London? We don’t know, but: • At least 4 of our patients are out of the “time-frame” • We used a slightly different virus • We used a different protocol Development of safer vectors

  35. New Improved Retroviral Vectors • Self-inactivating (SIN) vectors - promoter and enhancer element deletion in 3’LTR - should have no LTR-directed transcription - should reduce the risk of insertional mutagenesis • Tissue specific promoters - viral promoters, SFFV LTR is active in stem and progenitor cells - constitutive eukaryotic promoters, EF1α - haematopoietic cell specific promoters, WASp

  36. MLV LTR IL2RG MLV LTR MLV LTR MLV LTR SFFV EF1α IL2RG IL2RG wpre wpre MLV LTR delU3 MLV LTR delU3 MLV LTR WASP IL2RG wpre MLV LTR delU3 A. B. C. D. • Schematic representation of SIN vectors • Vector used in our current clinical trial • (B-D) SIN vectors in which the IL2RGγc transgene is regulated by internal regulatory sequences: • (B) spleen focus forming virus promoter (SFFV) • (C) elongation factor 1α promoter (EF1α) • (D) Wiskott Aldrich syndrome protein promoter

  37. SIN gammaretroviral reconstitution of murine T and B cell compartments.... Mock wt wt SF EF EF B220 CD8 IgM CD4 5 months after gene transfer

  38. GFP expression from modified lentiviral vectors 100% B cells Myeloid cells 80% Progenitor cells 60% 40% 20% 0% pHR'SIN.CEW pHR'SIN.SEW pHR'SIN.cPPT-SEW pHR'SIN.cPPT-SEW MOI <30 MOI 70

  39. Future gene therapy: • Other primary immunodeficiency disorders • ADA-SCID (1 patient, 3 further planned) • X-CGD (2 patients) • WAS (hope to start 2007?) • Primary haematological disorders, including leukemia • Stem cell protection strategies

  40. The Molecular Immunology Unit at ICH ICH Kate Parsley Kimberly Gilmour Jo Sinclair Steve Howe Doug King Suzy Bailey Fang Zhang Aris Giannakopoulos Meera Ulaganathan Mohammed Osman Mike Blundell Graham Davies Christine Kinnon Bobby Gaspar Adrian Thrasher GOS Nursing Staff Pharmacy Staff Funded by: The Wellcome Trust, MRC, BBSRC, Department of Health, CGD Trust, Primary Immunodeficiency Association, CF Trust, EU, LRF, ARC, SPARKS and others… Frankfurt Manuel Grez Stefan Stein Hannover Christopher Baum EUFETS Klaus Khulke Cincinatti/ Freiberg Manfred Schmidt Christof Von Kalle

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