Experimental gene therapy with special reference to plasma cell tumors

Abstract

Gene therapy may open new perspectives in the treatment of numerous hematopoietic and metabolic disorders. Particularly, hematopoietic cells are very attractive targets for retroviral mediated gene transfer. Both marrow and peripheral blood progenitor cells are readily obtained, manipulated in vitro, returned into a recipient and ultimately repopulate the entire hematopoietic system with progeny of the hematopoietic stem cells. In order to obtain a high transduction rate by retroviral vectors, target cells need to be in active proliferation. Because the majority of hematopoietic stem cells are in the quiescent G0-phase of the cell cycle, these cells may be prestimulated by different growth factors.

The effect of basic fibroblast growth factor (bFGF) in combination with other growth factors has been tested for this purpose. CD34-positive peripheral blood progenitor cells were selected from leukapheresis products, transduced with a retroviral vector containing a marker gene, and their progeny was evaluated for presence of the marker gene. The results indicate that bFGF in combination with IL-3, IL-6, and SCF increases the transduction rate of retroviral-mediated gene transfer into hematopoietic progenitor cells.

The optimal conditions for retroviral-mediated gene transfer of human bone marrow stromal cells without cytokine stimulation have also been investigated. Transduced stromal cells were analysed in vitro, injected into SCID mice and evaluated for homing and surviving time. It was shown that the inserted gene is expressed in vivo for at least two months after transfer of the human cells into SCID mice.

A special field of interest is the transfer of genes into tumor cells with the ultimate goal of a direct therapeutic approach. Another rationale may be to mark tumor cells for detection in vivo after autologous bone marrow transplantation, in an attempt to determine the origin of relapse. It was shown that foreign genes could be inserted into the low-proliferative myeloma cells by retroviral-mediated gene transfer. Myeloma cells from patients with advanced myeloma were isolated by immunomagnetic separation, using the B-B4 monoclonal antibody. Cells were then transduced with a retroviral vector carrying the neoR gene. The results provide evidence that retroviral-mediated gene transfer into human myeloma cells is feasible, and form part of the basis for current clinical studies of gene marking of bone marrow or peripheral blood progenitor cells before autologous stem cell transplantation in multiple myeloma.

Several proposals for the gene therapy of cancer have been made that involve transfer of the therapeutic gene either into tumor cells directly or into the immune cells to enhance their presumed tumoricidal properties. Transfer of the herpes simplex thymidine kinase (HSVtk) gene into the tumor cells using retroviral vectors followed by ganciclovir (GCV) provides a potential strategy for the treatment of some malignancies. GCV is metabolised by the viral thymidine kinase into a monophosphate form and subsequently to GCV-triphosphate by endogenous kinases. GCV-triphosphate competes with normal nucleotides for DNA replication and can cause cell death. During GCV treatment, not only cells that express the HSVtk gene are killed, but frequently also neighbouring tumor cells which are not genetically altered. This phenomenon is called the "bystander effect". We have tried to use this system to treat human plasma cell tumors in SCID mice. Mice were injected subcutaneously (s.c.) with human myeloma cell lines transduced with HSVtk and subsequently treated with GCV. A bystander effect, as well as a "distant" bystander effect, was noticed in mice receiving mixtures of HSVtk-transduced and non-transduced cells.

In recent experiments, the mechanism of the bystander effect has been investigated in SClD-beige mice. A cell line, C6, which exhibits a low level of intercellular communication by gap junctions and connexin43 (Cx43) transfected clones of this cell line forming gap junctions of a moderate to high level were transduced with HSVtk. Transduced and non-transduced cells were mixed in various concentrations and then cultured in vitro or injected s.c. into the mice followed by intraperitoneal injections of GCV. Cx43 transfected clones showed a significant increase of the bystander effect compared with the less coupled C6 parental cell line indicating that gap junctions play an important role in this phenomenon.