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Farzin Farzaneh Department of Haematological Medicine King’s College London

Gene Therapy – hype or hope ?. Farzin Farzaneh Department of Haematological Medicine King’s College London. Gene Therapy – Inherited Monogenic disorders. Successful gene therapy of common  Chain cytokine receptor defect (SCID)-X1 Disease: 9 children cured and off treatment!

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Farzin Farzaneh Department of Haematological Medicine King’s College London

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  1. Gene Therapy – hype or hope ? Farzin Farzaneh Department of Haematological Medicine King’s College London

  2. Gene Therapy – Inherited Monogenic disorders • Successful gene therapy of common  Chain cytokine • receptor defect (SCID)-X1 Disease: 9 children cured and off treatment! • Alain Fischer – Institut Pasteur, Paris. • Adrian Thrasher – Institute of Child Health, London. • Science 2000, Vol. 288: 669-672. • Science 2003, Vol. 302: 415-419. • Successful gene therapy of ADA deficiency (SCID) • 2 children cured and off treatment! • Caludio Bordignon – Hospital San Rafael, Milan • Shimuon Slavin – Hadasa University Hospital, Jerusalem • Science 2002, Vol. 296: 2410-2413. • Cure for two fatal genetic disorders!

  3. Clinical trial for X-SCID (Alain Fischer – Paris, Adrian Thrasher - London) • SCID due to deficiency of common • interleukin receptor c chain • lethal at 4 months if untreated • survival prognosis - 10 years under sterile conditions 1998-2000: Successful gene transfer in 10 out of 11 patients Oct 2002:1st report describing development of leukaemic syndrome Jan 2003:2nd report of identical adverse event Jan 2005: 3rd report of leukaemia Mar 2007: 4th report of leukaemia

  4. Viral DNA is integrated into the host cell genome Nucleus Integrated provirus DNA Unintegrated provirus DNA Integrated viral genome is transcribed into genomic RNA and viral mRNA Viral RNA genome is transcribed into provirus DNA by reverse transcriptase ψ Virus receptor Assembly and release of viral particles Infectious virions Virus attachment to receptors on the host cell Protein synthesis, processing and assembly Budding Shed virus Retroviral life cycle

  5. Replication defective (helper dependent) retro-viral vectors cDNA inserts gag pol env LTR LTR   Helper dependent retroviral vector cDNA neo The genome of a typical retrovirus  cDNA neo gagpolenv  Viral RNA Host cell DNA Retrovirus producer Cell gag pol env Retroviral Packaging Cell cDNA neo Helper dependent retrovirus  Infected target cell – no virus production

  6. LTR LTR puro puro LTR LTR Insertional inactivation LTR puro LTR Insertional activation LTR puro LTR Retroviral insertional mutagenesis Provirus DNA Genomic DNA sequence regulatory gene Pseudorandom provirus integration into the host cell genome truncated transdominant products

  7. C antisense LMO2 insertional mutagenesis: 3 of 15 SCID-X1 (C) children developed T-cell leukaemia after the retroviral transfer of C gene to the CD34+ haematopoietic stem cells LMO2 2 kb P4 Integration CHO CHO + MO2 RPMI - 8402 MEL-F4N N. T Cell () P4 N. T Cell () P5 exon 5 exon 1 exon 2 P5 Integration LMO2 antisense C sense LMO2 - From: Hacein-Bey-Abina et al (2003). Science 302: 415-419. Actin -

  8. Possible factors contributing to development of T cell leukaemia in the C clinical trial • Use of retroviral vectors – hence inherent risk of insertional mutagenesis • Selective growth advantage of T cells expressing C • The inherent anti-apoptotic effect of C gene expression • Genetic modification of haematopoieticstem cells • Genetic modification of large numbers of cells – hence increased numbers of cells at risk of mutagenesis • The immune suppressed status of the host • Reduced endogenous numbers of competing T-cells • Potential predisposing cytogenetic abnormalities

  9. Retroviral insertional mutagenesis A problem turned on its head - functional analysis of the genome

  10. Functional analysis of the genome Objective: Identification of phenotypic / physiological function Determination of rate-limiting, regulatory steps Identification of causally associated rather than consequential changes • Strategy: • - Retroviral cDNA library expression cloning • - RNA interference (siRNA) library based inhibition cloning • - Retroviral insertional mutagenesis

  11. Retroviral cDNA library expression cloning  cDNA neo  cDNA neo gag pol env  cDNA neo Infect cells with cDNA library gagpolenv  cDNA neo Select phenotype and expand  cDNA neo gag pol env gag pol env  cDNA neo Introduce viral genes to rescue cDNA vector Confirm cDNA encodes the selected function Identify cDNA Williams & Farzaneh (2004). Cancer Immunol. Immunother. 53: 160-165.

  12. Phenotypic selection of cellular function (e.g. resistance to differentiation, apoptosis, etc.) Immobilized cells in semi-solid culture (e.g. pluripotent cells in soft agar) Induction of differentiation, apoptosis, or other selectable functions Isolation of clonal population of cells with the selected phenotype - Ligand and antibody mediated selection of cells with specific surface markers Alternative strategies: - Tissue/function specific promoters for drug mediated selection of cells with the appropriate phenotype

  13. Protein Phosphatase 4: an inducer of apoptosis! cDNA library transfer - selection of apoptosis resistant cells

  14. W7.2 + Dex W7.2/4n10 + Dex Protein Phosphatase 4: an inducer of apoptosis! cDNA library transfer - selection of apoptosis resistant cells

  15. W7.2 + Dex W7.2/4n10 + Dex Protein Phosphatase 4: an inducer of apoptosis! cDNA library transfer - selection of apoptosis resistant cells Insert identified: C-terminal catalytic subunit of PP4 – induces apoptosis resistance ( PP4 breakdown)

  16. W7.2 + Dex W7.2/4n10 + Dex 120 100 Vector 80 PP4-Cat. 60 Number of colonies 40 20 0 γ (1000cGy) γ (1000cGy) UV (20J/m2) Dex (60nM) UV (20J/m2) Dex (60nM) W7.2 cells Protein Phosphatase 4: an inducer of apoptosis! cDNA library transfer - selection of apoptosis resistant cells Insert identified: C-terminal catalytic subunit of PP4 – induces apoptosis resistance ( PP4 breakdown)

  17. PP4 – a new apoptosis regulator (member of the superfamily of serine/threonine phosphatases) • Expression of the catalytic subunit of PP4 • (C-terminal fragment*): • - steady-state levels of PP4 RNA and protein • - blocks induction of apoptosis by UV, γ-irradiation or • dexamethasone • - target site: TTCTAATAAAAGAAGAAAAAT- reduces • Over-expression of full-length PP4 induces apoptosis in mouse and human cell lines Mourtada-Maarabouni et al. (2003)Cell Death Differ. 10:1016-24.

  18. Apoptosis control by naturally expressed regulatory RNA species Induction of resistance to UV (254nm, 20J/m2), X-rays (1000cGy), steroids (60nM Dex) and etoposide (1nM) • Growth Arrest Specific transcript 5 (GAS5): • A non-coding regulatory RNA • rFAU: • A non-coding antisense transcript identified both by cDNA expression cloning and expressed by Finkel-Biskis-Reilly sarcoma virus (FBRSV) • PP4: Mourtada-Marabouni et al. (2003)Cell Death & Diff. 10: 1016-1024. • rFau: Mourtada-Marabouni et al. (2004)Oncogene 23: 9419-9426. • RACK1: Mourtada-Marabouni et al. (2005)J Leuk. Biol. 78: 503-514.

  19. Functional studies of the genome(RIM, cDNA, RNAi libraries) Direct identification of controlling genes (i.e.causalrather than consequentialchanges) Rate-limiting regulatory gene products:

  20. Cancer Gene Therapy- some of the main strategies

  21. Expression of tumour-suppressor genes • Expression of p53 induces growth arrest and increased apoptosis in response to chemo/radio-therapy. • p53 expression also blocks angiogenesis by ↓ VEGF and by ↑ expression of thrombospondin and IGF-1 BP.

  22. Anti-sense RNA, ribozyme and RNA interference mediated inhibition of oncogene expression Oncogenes examined: c-erbB2, c-erbB4, K-ras, H-ras, HPV E6/E7, bcl-2, Telomerase, c-met, c-myc.

  23. Suicide gene therapy EnzymeProdrugActive productMechanism HSV-tk GCV/ACV GCV/ACV triphosphate Blocks DNA synthesis Cytosine deaminase 5-Fluorocytosine 5-Fluorouracil (5-FU) Blocks DNA/RNA synth. Nitroreductase Nitrobenzyloxcarbonyl Anthracyclines DNA crosslinking anthracyclines Carboxylesterase CPT-11 SN38 Topoisomerase inhibitor Cytochrome p450 Cyclophosphamide Phosphoramide mustard DNA alkylating agent Purine nucleoside 6-mercaptopurine-DR 6-mercaptopurine Purine antagonist phosphorylase

  24. Conditionally replicating / oncolytic viruses Normal cell: abortive replication Virus kills tumour cell, spreads to neighbours Productive replication, cell lysis Oncolytic virus Tumour cell Replication of a conditionally replicating virus. ONYX-015, in a cancer cell from a patient with head and neck cancer during Phase-II clinical trial. 109 infectious E1B defective Adenovirus particles were injected over a 5 day period. After 8 days biopsy was performed and analysed by electron microscopy. Frank McCormick 2001, Nature Reviews 1: 130-141.

  25. ONYX-015 plus Cisplatin/5-FU Baseline Cycle 1, Day 22 Cycle 3, Day 22 Yoon LTK, Laquerre S, Kasahara N (2001) Curr Cancer Drug Targets 1: 85-106.

  26. ONYX-015 plus Cisplatin/5-FU Baseline Cycle 1, Day 22 Cycle 3, Day 22 Yoon LTK, Laquerre S, Kasahara N (2001) Curr Cancer Drug Targets 1: 85-106.

  27. ONYX-015 plus Cisplatin/5-FU Baseline Cycle 1, Day 22 Cycle 3, Day 22 Yoon LTK, Laquerre S, Kasahara N (2001) Curr Cancer Drug Targets 1: 85-106.

  28. Oncolytic virus therapy – problems: – robust immune response (and other intratumoural barriers): rapid clearance of virus – basis for attenuation / tumor selectivity: not well understood

  29. Retro- and lenti-virus vectors • High-titre vectors for - • functional analysis of the genome • immune gene therapy of poor prognosis acute myeloid leukaemia (AML)

  30. Retro- and lenti-virus vectors • High-titre vectors for - • functional analysis of the genome • immune gene therapy of poor prognosis acute myeloid leukaemia (AML)

  31. Generation of biotinylated retroviral vectors Biotin succinimide ester Hughes et al (2001) Molecular Therapy 3: 623-630.

  32. Paramagnetic labelling and concentration of the vector/s Biotin / avidin mediated attachment of targeting ligands Biotinylated retro- and lenti-virus vectors: Vector concentration Attachment of targeting ligands Casimir et al (2004). J. Gene Medicine 6: 1189-1196. Chan et al (2005) J. Virol. 79: 13190-13194.

  33. Paramagnetic bead concentration of retroviral vectors 125x concentration (i.e. reduction in volume) 1x1010 4200 X 1x109 1x108 Titre (cfu/ml) 1x107 1x106 1x Efficient transduction of primary CD34+ blasts: 71 + 23 % of all cells express transgene after a single round of infection at an MOI of 3 1x105 DMSO Control BiotinSE DMSO conc BiotinSE conc

  34. Paramagnetically targeted retrovirus delivery International Society for Cell & Gene Therapy of Cancer

  35. Biotin BAP Endogenously biotinylated LNGFR LNGFR External domain LNGFR Transmembrane domain BirA Packaging cells producing endogenously biotinylated retrovirus vectors Transmembrane domain Signal peptide Extracellular domain LNGFR BAP SPH-1 SPH-1 …LGGA KEAC GGGLNDIFEAQKIbEWHE ACPTGL… Nesbeth et al. 2006, Mol. Ther. 13: 814-822

  36. Envelope / receptor independent vector concentration & targeting Vector concentration (K562 stable colonies) 1x 1011 1x 1010 1x 109 Titre (cfu/ml) Amphotropic vector 1x 108 1x 107 Amphotropic producer cell 1x 106 Control

  37. Envelope / receptor independent vector concentration & targeting Vector concentration (K562 stable colonies) 1x 1011 1x 1010 1x 109 Titre (cfu/ml) Amphotropic vector 1x 108 1x 107 Amphotropic producer cell Amphotropic vector (surface B7.1) 1x 106 Control -B7.1 CTLA4 B7.1 cDNA transduced packaging cells

  38. Envelope / receptor independent vector concentration & targeting Vector concentration (K562 stable colonies) 1x 1011 1x 1010 1x 109 Titre (cfu/ml) Amphotropic vector 1x 108 1x 107 Amphotropic producer cell Amphotropic vector (surface B7.1) 1x 106 Control -B7.1 CTLA4 -LNGFR B7.1 or LNGFRcDNA transduced packaging cells Amphotropic vector (surface LNGFR)

  39. Targeting to c-kit+/CD34 Bone Marrow Cells 8 6 4 Relative transduction efficiency 2 0 neo ampho SCF-ampho Envelope / receptor independent vector concentration & targeting Vector concentration (K562 stable colonies) 1x 1011 1x 1010 1x 109 Titre (cfu/ml) Amphotropic vector 1x 108 1x 107 Amphotropic producer cell Amphotropic vector (surface B7.1) 1x 106 Control -B7.1 CTLA4 -LNGFR B7.1 or LNGFRcDNA transduced packaging cells Amphotropic vector (surface LNGFR) SCF cDNA transduced packaging cells Producer cell with surface expressed SCF Amphotropic vector (surface SCF) Casimir et al (2004). J. Gene Medicine6: 1189-1196.

  40. Paramagnetically labelled / concentrated lentivirus 1 m particles with attached vector Nesbeth et al. 2006, Mol. Ther. 13: 814-822

  41. Immune gene therapy of cancer A genetically modified autologous cell vaccine for Acute Myeloid Leukaemia (AML)

  42. Human cancer antigens recognized by T lymphocytes Cancer-testis antigens: MAGE-3, BAGE, GAGE, NY-ESO-1 Melanocyte differentiation antigens: Melan-A/MART-1, Tyrosinase, gp100 Point mutations: β-catenin, MUM-1, CDK-4, p53, ras Overexpressed ‘self’ antigens: Her-2/neu. P53, MUC-1 Viral antigens: HPV, HBV, HCV, EBV

  43. Tumour cells can be immunogenic • There are tumour associated and tumour specific antigens • Cancer is not the product of immune incompetence • - ELISPOT and MHC/antigen tetramers show increased • presence of tumour targeted T cells • Tumour editing of the immune system AND immune editing of the tumour • - a clinical tumour has already escaped immune surveillance

  44. Professional antigen presenting cells: Schwartz 1992

  45. Professional antigen presenting cells: Schwartz 1992 Acute Myeloid Leukaemia (AML): • AML blasts express both HLA class-I, and class-II • Express AML associated antigens (WT1, PRAME, GP250, etc) • Common lineage with APCs – efficient antigen presentation • Express many surface markers present on DC – but not B7.1 (CD80) !

  46. ■ Leukaemogenecity of 32DP210bcr/abl cells modified to express B7.1, IL-2 or both ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ 32D/B7.1/IL-2 ● ● ● ● ● ● 100 ■ ■ 32D/B7.1 ● ■ ■ ● ● ● 80 32D/IL-2 ● ● ● ■ 32D/M3P (vector) 60 % Survival 40 ■ ■ 20 ■ ■ 0 ■ 80 40 60 0 20 Days post-challenge 2x107 leukaemic cells iv

  47. ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ● ● ● ● ● ● ■ Rejection of established myeloid leukemia (32Dp210) in mice, by genetically modified leukemia cells expressing B7.1 and IL-2 100 Cell vaccine ■ ■ ■ ■ ● ● ● 80 32D/B7.1/IL-2 32D/B7.1 ● ● ● ● ● 60 % Survival 32D/IL-2 40 ■ ■ ■ 32D/M3P (Vector) 20 ■ ■ ■ 0 ■ 0 20 40 60 80 100 Leukemia initiation (105 32Dp210 cells iv) Time (days) Vaccination (106 irradiated cells)

  48. Important questions for the clinical application of immune gene therapy: Can B7.1/IL-2 expressing AML cells induce T cell proliferation? If so, are the stimulated T cells functionally competent (Cytokine release, cytolytic activity)? Are AML cells susceptible to T cell mediated lysis? Can post-chemotherapy ,“remission” T cells, stimulate cytolytic activity? Is there any specificity in the cytolytic activity of the stimulated T cells against the leukaemic cells?

  49. IL-2 IL-2/B7 B7 GFP uninfected 0 50 100 150 200 250 300 In vitro stimulation of T cells with autologous primary AML blasts (MLR) AJ – remission, post BMT PW – at presentation CY – remission (no BMT) IL-2 IL-2 IL2 B7/IL-2 IL-2/B7 B7/IL-2 B7 B7 B7 uninfected Uninfected uninfected 0 200 400 600 800 1000 1200 0 2 4 6 8 10 12 14 0 10 20 30 40 50 Stimulation Index Stimulation Index Stimulation Index HM –remission, post BMT MB – at presentation MB – remission (no BMT) IL-2 IL-2 L-2/B7 B7/IL-2 B7 B7 GFP Uninfected uninfected 0 100 200 300 400 500 0 5 10 15 20 25 30 Stimulation Index Stimulation Index Stimulation Index

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