Efficient conditional gene expression following transplantation of retrovirally transduced bone marrow stem cells.

Retroviral gene therapy combined with bone marrow stem cell transplantation can be used to generate mice with ectopic gene expression in the bone marrow compartment in a quick and cost effective manner when compared to generating and maintaining transgenic mouse lines. However a limitation of this procedure is the lack of cell specificity in gene expression that is associated with the use of endogenous retroviral promoters. Restricting gene expression to specific cell subsets utilising tissue-specific promoter driven retroviral vectors is a challenge. Here we describe the generation of conditional expression of retrovirally encoded genes in specific bone marrow derived cell lineages utilising a Cre-dependent retroviral vector. By utilising Lck and CD19 restricted Cre transgenic bone marrow stem cells, we generate chimeric animals with T or B lymphocyte restricted gene expression respectively. The design of the Cre-dependent retroviral vector enables expression of encoded MOG and GFP genes only in association with Cre mediated DNA inversion. Importantly this strategy does not significantly increase the size of the retroviral vector; as such we are able to generate bone marrow chimeric animals with significantly higher chimerism levels than previous studies utilising Cre-dependent retroviral vectors and Cre transgenic bone marrow stem cells. This demonstrates that the use of Cre-dependent retroviral vectors is able to yield high chimerism levels for experimental use and represent a viable alternative to generating transgenic animals.

Gene therapy delivery of myelin oligodendrocyte glycoprotein (MOG) via hematopoietic stem cell transfer induces MOG-specific B cell deletion.

The various mechanisms that have been described for immune tolerance govern our ability to control self-reactivity and minimize autoimmunity. However, the capacity to genetically manipulate the immune system provides a powerful avenue to supplement this natural tolerance in an Ag-specific manner. We have previously shown in the mouse model of experimental autoimmune encephalomyelitis that transfer of bone marrow (BM) transduced with retrovirus encoding myelin oligodendrocyte glycoprotein (MOG) promotes disease resistance and CD4(+) T cell deletion within the thymus. However, the consequence of this strategy on B cell tolerance is not known. Using BM from IgH(MOG) mice that develop MOG-specific B cell receptors, we generated mixed chimeras together with BM-encoding MOG. In these animals, the development of MOG-specific B cells was abrogated, resulting in a lack of MOG-specific B cells in all B cell compartments examined. This finding adds a further dimension to our understanding of the mechanisms of tolerance that are associated with this gene therapy approach to treating autoimmunity and may have important implications for Ab-mediated autoimmune disorders.

Targeting MOG expression to dendritic cells delays onset of experimental autoimmune disease.

Haematopoietic stem cell (HSC) transfer coupled with gene therapy is a powerful approach to treating fatal diseases such as X-linked severe combined immunodeficiency. This ability to isolate and genetically manipulate HSCs also offers a strategy for inducing immune tolerance through ectopic expression of autoantigens. We have previously shown that retroviral transduction of bone marrow (BM) with vectors encoding the autoantigen, myelin oligodendrocyte glycoprotein (MOG), can prevent the induction of experimental autoimmune encephalomyelitis (EAE). However, ubiquitous cellular expression of autoantigen driven by retroviral promoters may not be the best approach for clinical translation and a targeted expression approach may be more acceptable. As BM-derived dendritic cells (DCs) play a major role in tolerance induction, we asked whether targeted expression of MOG, a target autoantigen in EAE, to DCs can promote tolerance induction and influence the development of EAE. Self-inactivating retroviral vectors incorporating the mouse CD11c promoter were generated and used to transduce mouse BM cells. Transplantation of gene-modified cells into irradiated recipients resulted in the generation of chimeric mice with transgene expression limited to DCs. Notably, chimeric mice transplanted with MOG-expressing BM cells manifest a significant delay in the development of EAE suggesting that targeted antigen expression to tolerogenic cell types may be a feasible approach to inducing antigen-specific tolerance.

A molecular Trojan horse: hijacking the bone marrow to treat autoimmune diseases.

Autoimmune diseases such as multiple sclerosis, type 1 diabetes, systemic sclerosis, and rheumatoid arthritis affect approximately 5% of the population and are characterized by a destructive immune response directed to self-tissues. Treatments are often designed to dampen the immune system and are therefore associated with unwanted side effects. A major challenge is to find a cure that does not compromise normal immune function. From our understanding of how the immune system develops, it is clear that mechanisms designed to eliminate or maintain control over self-reactive clones are critical for normal health. These key concepts form the crux of many experimental strategies currently aimed at abrogating the autoimmune response. In this review, we focus on the strategy of harnessing the bone marrow compartment through genetic manipulation directed at promoting ectopic autoantigen expression. Our experience with this strategy is presented in the context of reports in the literature and we argue for the potential benefit of translating this approach to the treatment of human autoimmune disease.