The main objective of this experimental project is to induce and maintain specific and indefinite tolerance to alloantigens and/or primarily vascularized allografts by DNA mediated gene transfer. In order to induce allotolerance, genes encoding cytokines or second-signal blockers and/or MHC molecules will be transfected either in the organ itself prior to grafting or in recipient bone marrow and stem cells by using and comparing the efficiency of three gene vectors i.e. adenovirus, retrovirus and liposomes. The ultimate goal of the project is to develop innovative therapies for inducing tolerance in human allograft transplantation. The importance of these intercellular mediators (cytokines such as TGF-Beta, IL-4 and IL-10), co-stimulatory (CD28/B7-1.2) apoptotic (Fas/Fas-L) or MHC molecules in the T cell response suggests that the expression of these proteins which regulate the immune response, at a high level locally in the allograft itself or in recipient bone marrow and stem cells prior to grafting might prolong the graft survival and/or induce specific tolerance to allograft.
The following complementary experiments based on the latest developments in Transplantation Immunology are proposed:
I) Transfection of the graft itself by viral or non viral vectors expressing genes encoding
(1) down-regulating cytokines such as IL-4, IL-10, TGF-beta; (2) blockers of co-stimulatory molecules such as CTLA-4Ig or (3) Apoptotic molecules such as FAS/FAS-L.
A) viral vector
Transfection of genes encoding (1) cytokines and/or (2) down-regulating factors such as CTLA_4Ig by adenoviral vector in rats (M.Dallman London)
Transfection of genes encoding cytokines such as TGF-beta, IL-4 and IL-10 in miniatures swine by LIPOSOMAL VECTOR (M.Baru and P.Gianello Brussels)
II)production of transgenic mice expressing TH2 cytokines (IL-10 and IL-4) under the control of inducible promoter (P.Vieira Oieras)
III) transfection of recipient bone marrow and stem cells by retroviral vectors expressing genes encoding mhc molecules.
A) Induction of tolerance to vascularized allografts by inoculation of transfected recipient bone marrow or stem cells expressing various MHC molecules. (K.J. Wood, Oxford)
B) Tolerance to human CD34+ cells in SCID mice after gene transfection by retroviral vectors (JL. Touraine, Lyon)
IV) tolerance induction to alloantigens and/or primarily vascularized allografts by using gene delivery in both the graft itself and recipient bone marrow or stem cells (M. Baru, M. Dallman, P. Gianello, JL Touraine, P. Vieira, K. Wood).
The most efficient vectors for gene transfection into the graft itself (transgene, Lip, adenovirus) and most appropriate retroviral vectors for expressing genes into the recipient bone marrow or stem cells will be selected. Protocols combining both approaches will be investigated in vivo by using the most relevant models of cells or primarily vascularized allograft.
Funding SchemeCSC - Cost-sharing contracts
SW7 2AZ London
OX3 9DU Oxford