Suicide gene therapy

1. Suicide gene therapy Literature discussion – Haematology Biomedical Sciences - Utrecht University 2005 Eric Lammertsma, Tineke Lenstra & Hiljanne van der Meer

2. Contents • Literature • Gene therapy • Suicide gene therapy • Phase 1 study: Suicide gene therapy after allogeneic marrow graft • Discussion

3. Literature • Gene therapy: trials and tribulations; Somia, N. and Verma, I.M.; Nature Reviews; 2000 • Would suicide gene therapy solve the ‘T-cell dilemma’ of allogeneic bone marrow transplantation?; Cohen, J.L., Boyer, O. and Klatzmann, D.; Immunology today; 1999 • Administration of herpes simplex-thymidine kinase-expressing donor T cells with a T-cell-depleted allogeneic marrow graft; Tiberghien, P. et al; Blood; 2001

4. Gene therapy Introduction of a gene into cells to cure or slow down the progression of a disease.

5. Vectors • Non-viral • Naked DNA • Liposomes • large amounts and fewer toxic and immunological problems, • inefficient gene transfer and transient expression • Viral • Retro-virus • Lenti-virus • Adeno-associated virus (AAV) • Adenovirus • integrating and non-integrating

6. Viral vectors • Transfection of packaging cells with DNA • Production of vectors • Transduction of target cells with vectors • Expression of target proteins

7. Retro-virus • 3 genes (RNA): Gag, Pol, Env and packaging sequence

8. Retro-virus • production, storage and distribution on large scale possible • different target cells by changing the env protein • high transduction efficiencies • inability to infect non-dividing cells • on transplantation in the host, transcription often extinguished

9. Lenti-virus • 9 genes (RNA): Gag, Pol, Env, Tat, Rev, Nef, Vif, Vpu, Vpr • recombination and generation of infectious HIV? • lentiviral vector system retains less that 25% of viral genome • Traduction of non-dividing cells • Non-specific integration in the chromosome

10. Adeno-associated virus • Small, non-pathogenic, single-stranded DNA virus • 2 genes: rep, cap and 2 inverted terminal repeats • other genes provided by adenovirus or herpes virus

11. Adeno-associated virus • broad range of target cells • long-term expression • cytostatic and cytotoxic to packaging cells  difficult to scale up production • low coding capacity (4.5 kb)

12. Adenovirus • Pathogenic DNA virus containing a dozen genes • Episomal infection • Transduction of dividing and non-dividing cells • Easy to generate high-titre commercial-grade recombinant vectors • Short time expression, because of immune response • New virus: ‘gutless’  all the viral genes removed and provided in trans

13. Immune response • Cellular: cytotoxic T cells  elimination of transduced cells • Humoral: antibodies  no repeated administration possible • Adenoviral vectors: cytotoxic and humoral response • Retroviral, lentivral and AAV vectors: no cytotoxic T cell response and almost no humoral response

14. Applications • Deficiency of ornithine transcarbamylase (OTC): breakdown of ammonia • X-linked severe combined immunodeficiency (X-SCID): differentiation of T cells and NK cells • Adensine deaminase deficiency (ADA) • Hemophilia

15. Bone Marrow Transplantation • Used following radio-chemotherapy against Hematological malignancies (leukemia) • Reinforcement of hosts weakened/absent immune response • Donor T cells contribute to: • Graft versus Infection • Graft versus Leukemia • Graft versus Host

16. Graft versus Infection (GvI) • Donated mature T cells, including memory T cells, recognize Ag’s presented by HLA molecules shared between the host and the donor • General improvement of immune response

17. Graft versus Leukemia (GvL) • Recognition of mismatched MHC Ag, minor histocompatibility Ag and possibly leukemia-specific Ag • A major component of the efficacy of BMT

18. Graft versus Host Disease (GvHD) • Provides an advantage in hemapoietic stem cell (HSC) engraftment through destruction of competing host cells • T cell recognition of host MHC Ag • Leads to rejection of the host by the donor T cells • Characterized by immunosuppression and multi-organ dysfunction • Full donor T cell depletion increases risk of relapse • Method needed to eliminate only deleterious cells

20. Suicide gene therapy • Suicide genes code for enzymes that render cells sensitive to otherwise nontoxic prodrugs. • Adding such genes with the ability to control transcription creates a ‘suicide switch’

21. Affects T-cells • Successful implementation of suicide genes in T-cells has led to an application in allogenic bone marrow transplantation in hematological malignancies (leukemia) • Graft versus Infection • Graft versus Leukemia • Graft versus Host

22. TK/GCV system • Herpes simplex virus type 1 thymidine kinase (TK) • Ganciclovir (GCV)  monophosphate form  triphosphate metabolite  inhibition of DNA elongation  • Cell death

23. TK/GCV system • Administration of GCV affects only dividing TK+ GCV-sensitive cells; does not affect resting TK+ GCV-insensitive cells or TK- cells • Low transfection efficiency • Advantageous “bystander effect”

24. Applications • Hematological Malignancy • Chronic Myeloid Leukemia (CML) • Other malignancies • Breast Cancer • Prostate Cancer

25. Suicide gene therapy: genetic modified donor T cells • Clinical Trial: Phase 1 study • Objectives: • Safety • Survival and circulation of GMC’s • Effect of GCV on GMC survival

26. 12 patients Hematological malignancies HLA-identical sibling donor Female donor - male recipient mismatch Risk factors Patients

27. Vector • Alteration gag start codon • Elimination of viral sequences • Packaging in PA317 cell line • Selected in G418 • G1Tk1SvNa • Retro virus from Moloney murine leukemia virus • G1 backbone

28. Production Genetic Modified Cells (GMC)

29. Quality control GMCs in vitro • GCV sensivity • Il-2 dependence • Phenotype: CD3+, CD4+, CD8+ and CD56+ • Cell viability • Mycoplasma • Sterility and endotoxin • Replication Competent Recombinants (RCR)

30. Detection GMCs in vivo • Competitive PCR assay with the NeoR gene • PBMCs • PBL • Skin biopsy • Histological examination • Skin biopt • Salivary gland (1 patient, suspected GvHD)

31. Results • Production GMCs • Engraftment • GMC survival and circulation • GvHD and GCV • Complications • Survival patients

32. Production GMCs • All quality control criteria were met • 90.5 T cells: 39.8% CD4+ and 52.5% CD8+ • 13.0 NK cells

33. T cell infusion • Patient 1-5: 2 x 105 cells per recipient kg • Patient 6-10: 6 x 105 cells per recipient kg • Patient 11 and 12: 20 x 105 cells per recipient kg • Patient 1 and 5: second GMC infusion to treat EBV-LPD • Patient 7: second GMC infusion for ALL

34. Engraftment and survival of GMCs • Initial engraftment in all patients • Two patients with late graft failure • Circulating GMCs in all patients early after transplantation

35. GvHD and GCV • 4 patients with acute GvHD • 1 patient with chronic GvHD • 1 patient with CMV infection and acute GvHD

36. GvHD and GCV • Variable GMC fractions • Significant reduction after GCV treatment: 92.7 % (relative) 85.3 % (absolute) • GCV susceptibility stable

37. Complications • 3 patients with EBV-LPD: • EBV-lymphoma -> reinfusion GMC -> CR -> cerebral toxoplasmosis • Polyclonal EBV-LDP -> lung aspergillosis • Lethal EBV-lymphoma • No vector in tumor cells • No circulating RCR

38. Survival patients • After 29-38 months: 4 of 12 • Transplantation in early stage: 4 of 7 • Deaths: • 3 infections • 2 relapses • 1 acute GvHD

39. Conclusions • HS-tk-expressing donor T cells produced • No acute toxicity • In vivo expansion • Survival more than 2 years • Reduction of GMCs with GCV

40. Discussion • Phenotype of GMCs unknown • Circulation pattern unknown • Altered lifespan/function possible • Low levels GMC present • HS-tk expression activation dependent • Spliced HS-tk genes can be produced • GCV treatment not enough • Immune dysfunctions despite GMCs