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Suicide gene therapy
Eric Lammertsma, Tineke Lenstra & Hiljanne van der Meer
Literature discussion – Haematology
Biomedical Sciences - Utrecht University 2005
Contents
• Literature
• Gene therapy
• Suicide gene therapy
• Phase 1 study:
Suicide gene therapy after allogeneic marrow graft
• Discussion
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
Gene therapy
Introduction of a gene into cells to cure or slow down the progression of a disease.
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
Viral vectors
• Transfection of packaging cells with DNA
• Production of vectors• Transduction of target
cells with vectors• Expression of target
proteins
Retro-virus
• 3 genes (RNA): Gag, Pol, Env and packaging sequence
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
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
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
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)
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
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
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
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
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
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
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
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’
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
TK/GCV system
• Herpes simplex virus type 1 thymidine kinase (TK)
• Ganciclovir (GCV) monophosphate form triphosphate metabolite inhibition of DNA elongation
• Cell death
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”
Applications
• Hematological Malignancy– Chronic Myeloid Leukemia (CML)
• Other malignancies– Breast Cancer– Prostate Cancer
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
Patients
• 12 patients• Hematological
malignancies• HLA-identical sibling
donor• Female donor - male
recipient mismatch• Risk factors
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
Production Genetic Modified Cells (GMC)
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)
Detection GMCs in vivo
• Competitive PCR assay with the NeoR gene PBMCsPBLSkin biopsy
• Histological examinationSkin bioptSalivary gland (1 patient, suspected GvHD)
Results
• Production GMCs
• Engraftment
• GMC survival and circulation
• GvHD and GCV
• Complications
• Survival patients
Production GMCs
• All quality control criteria were met
• 90.5 T cells: 39.8% CD4+ and 52.5% CD8+
• 13.0 NK cells
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
Engraftment and survival of GMCs• Initial engraftment in all patients
• Two patients with late graft failure
• Circulating GMCs in all patients early after transplantation
GvHD and GCV
• 4 patients with acute GvHD
• 1 patient with chronic GvHD
• 1 patient with CMV infection and acute GvHD
GvHD and GCV
• Variable GMC fractions
• Significant reduction
after GCV treatment:
92.7 % (relative)
85.3 % (absolute)
• GCV susceptibility stable
Complications• 3 patients with EBV-LPD:EBV-lymphoma -> reinfusion GMC -> CR -
> cerebral toxoplasmosisPolyclonal EBV-LDP -> lung aspergillosisLethal EBV-lymphoma
• No vector in tumor cells• No circulating RCR
Survival patients
• After 29-38 months: 4 of 12
• Transplantation in early stage: 4 of 7
• Deaths:3 infections2 relapses1 acute GvHD
Conclusions
• HS-tk-expressing donor T cells produced
• No acute toxicity
• In vivo expansion
• Survival more than 2 years
• Reduction of GMCs with GCV
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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