- Optimisation of retroviral suicide gene vectors and prodrug activation strategies.
- Investigation of novel vectors for somatic SGT.
- Testing of suicide gene/prodrug activation strategies in animal models of graft versus host (GvH) and graft versus leukaemia (GvL).
- Establishment of the requirements to prevent an immune response against the suicide gene vector.
- Molecular evaluation of the efficacy of SGT strategies in animal models.
The objective of this project is to develop somatic gene therapy approaches for treating disseminated malignant diseases such as leukaemia. To achieve this objective, a somatic suicide gene therapy/prodrug activation strategy will be undertaken. Allogeneic stem cell transplantation (SCT) is a highly successful treatment modality for leukaemia, partly due to the anti-tumour activity of donor T cells in the graft. However disease relapse is a significant problem. Allied to the graft versus leukaemia (GvL) effect in SCT, donor T cells also mediate graft versus host disease (GvHD) leading to a reduction in long term survival. In this study somatic suicide gene therapy strategies will be evaluated for their ability to enhance tumour cell killing by allogeneic donor T cells without increasing GvHD.
Retroviral vectors containing the herpes simplex thymidine kinase (HSV-Tk) gene or the cytosine deaminase (CD) gene will be evaluated for their ability to transduce donor T cells with high efficiency. New vectors will be developed using selectable markers that allow immunoaffinity selection. In addition adeno-associated viral vectors containing prodrug gene constructs will be examined for their potential as suicide gene therapy vectors. For all vectors conditions will be established for optimal transduction, selection and expansion of transduced animal and human T cells. Integration of the provirus and expression of prodrug and marker genes will be assessed in all in vitro studies using southern blot and PCR analysis.
Optimised vector systems will be tested in vivo in mouse, rat and dog models. Mouse and dog models will address GvH and HvG effects while in the rat model induction of limited GvHD in an effort to activate the GvL effect will be mediated by gene manipulated T cells. Subsequent treatment with appropriate prodrugs will prevent severe GvHD by in vivo elimination of T cells. PCR based chimerism and minimal residual disease(MRD) studies on bone marrow, peripheral blood and selected cell populations in animal studies will be performed to assess the distribution/efficacy of gene manipulated alloreactive T cells.
Recent studies have indicated that gene therapy approaches may be hindered by the ability of the host immune system to recognise gene transduced cells as foreign. Therefore attempts will be made to reduce the immunogenicity of suicide gene therapy vectors. The immediate early protein ICP 47, which blocks viral antigen presentation will be incorporated into a variety of vector constructs. Vectors developed during the programme, including especially those containing the ICP 47 gene, will be tested for their lack of ability to provoke an immune response. The combined expertise of the partners in (i) gene manipulation of T lymphocytes, (ii) relevant animal models for GvH and GvL (iii) evaluation of engraftment and MRD will be combined in evaluating strategies to prevent disease relapse while controlling GvHD following allogeneic stem cell transplantation for leukaemia.
Funding SchemeCSC - Cost-sharing contracts
3584 CX Utrecht
W12 0NN London