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

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