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

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

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 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