Dystrophin expression in the mdx mouse restored by stem cell transplantation.

The development of cell or gene therapies for diseases involving cells that are widely distributed throughout the body has been severely hampered by the inability to achieve the disseminated delivery of cells or genes to the affected tissues or organ. Here we report the results of bone marrow transplantation studies in the mdx mouse, an animal model of Duchenne's muscular dystrophy, which indicate that the intravenous injection of either normal haematopoietic stem cells or a novel population of muscle-derived stem cells into irradiated animals results in the reconstitution of the haematopoietic compartment of the transplanted recipients, the incorporation of donor-derived nuclei into muscle, and the partial restoration of dystrophin expression in the affected muscle. These results suggest that the transplantation of different stem cell populations, using the procedures of bone marrow transplantation, might provide an unanticipated avenue for treating muscular dystrophy as well as other diseases where the systemic delivery of therapeutic cells to sites throughout the body is critical. Our studies also suggest that the inherent developmental potential of stem cells isolated from diverse tissues or organs may be more similar than previously anticipated.

Mutagenesis analysis of the murine leukemia virus matrix protein: identification of regions important for membrane localization and intracellular transport.

We have created two sets of substitution mutations in the Moloney murine leukemia virus (Mo-MuLV) matrix protein in order to identify domains involved in association with the plasma membrane and in incorporation of the viral envelope glycoproteins into virus particles. The first set of mutations was targeted at putative membrane-associating regions similar to those of the human immunodeficiency virus type 1 matrix protein, which include a polybasic region at the N terminus of the Mo-MuLV matrix protein and two regions predicted to form beta strands. The second set of mutations was created within hydrophobic residues to test for the production of virus particles lacking envelope proteins, with the speculation of an involvement of the membrane-spanning region of the envelope protein in incorporation into virus particles. We have found that mutation of the N-terminal polybasic region redirected virus assembly to the cytoplasm, and we show that tryptophan residues may also play a significant role in the intracellular transport of the matrix protein. In total, 21 mutants of the Mo-MuLV matrix protein were produced, but we did not observe any mutant virus particles lacking the envelope glycoproteins, suggesting that a direct interaction between the Mo-MuLV matrix protein and envelope proteins either may not exist or may occur through multiple redundant interactions.

A transient three-plasmid expression system for the production of high titer retroviral vectors.

We have constructed a series of MLV-based retroviral vectors and packaging components expressed from the CMV promoter and carried on plasmids containing SV40 origins of replication. These two features greatly enhanced retroviral gene expression when introduced into cell lines carrying the SV40 large T antigen. The two packaging components, gag-pol and env, were placed on separate plasmids to reduce helper virus formation. Using a highly transfectable human cell line and sodium butyrate to further increase expression of each component, we achieved helper-free viral stocks of approximately 10(7) infectious units/ml by 48 h after transient co-transfection with the three plasmid components. This system can be used both for the generation of high titer retroviral stocks for transduction and for the rapid screening of a large number of MLV gag-pol or env mutants.