Suicide gene therapy

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

• Literature
• Gene therapy
• Suicide gene therapy
• Phase 1 study
• Suicide gene therapy after allogeneic marrow
  graft
• Discussion

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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

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

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Viral vectors

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

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Retro-virus

• 3 genes (RNA) Gag, Pol, Env and packaging
  sequence

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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

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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

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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

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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)

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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

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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

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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

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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

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Graft versus Infection (GvI)

• Donated mature T cells, including memory T cells,
  recognize Ags presented by HLA molecules shared
  between the host and the donor
• General improvement of immune response

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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

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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

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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

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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

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TK/GCV system

• Herpes simplex virus type 1 thymidine kinase (TK)
• Ganciclovir (GCV) ?
• monophosphate form ?
• triphosphate metabolite ?
• inhibition of DNA elongation ?
• Cell death

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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

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Applications

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

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Suicide gene therapy genetic modified donor T
cells

• Clinical Trial Phase 1 study
• Objectives
• Safety
• Survival and circulation of GMCs
• Effect of GCV on GMC survival

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Patients

• 12 patients
• Hematological malignancies
• HLA-identical sibling donor
• Female donor - male recipient mismatch
• Risk factors

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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

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Production Genetic Modified Cells (GMC)
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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)

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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)

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Results

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

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Production GMCs

• All quality control criteria were met
• 90.5 T cells 39.8 CD4 and 52.5 CD8
• 13.0 NK cells

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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

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Engraftment and survival of GMCs

• Initial engraftment in all patients
• Two patients with late graft failure
• Circulating GMCs in all patients early after
  transplantation

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GvHD and GCV

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

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GvHD and GCV

• Variable GMC fractions
• Significant reduction
• after GCV treatment
• 92.7 (relative)
• 85.3 (absolute)
• GCV susceptibility stable

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Complications

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

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Survival patients

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

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Conclusions

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

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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