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Suicide gene therapy Literature discussion – Haematology

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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

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