Bone formation occurs during embryonic development, somatic growth, and during bone remodeling and repair. The ability to form bone throughout an organisms life suggests the continuous presence of a reservoir of cells capable of osteogenesis. Osteoblasts are the differentiated cell type responsible for the biosynthesis, secretion, and orderly deposition of type 1 collagen and other bone matrix elements. A number of studies have demonstrated that the population of adherent marrow stromal cells include stem cells for at least four types of connective tissue: hematopoiesis-supporting stroma, adipose, cartilage, and bone. Osteogenesis imperfecta (01), caused by mutations in type 1 procollagen genes, is one example of a disease that may benefit from somatic gene therapy. Since bone fragility in 01 is caused by structural mutations in the genes that encode type 1 procollagen, candidate cells for 01 gene therapy are osteoblast progenitor cells and/or osteoblasts. Mice with the oim mutation are a valuable tool to investigate this question since these mice provide convenient markers for wild type and oim alleles at the gene, mRNA, and protein levels. The rationale for these studies is that mice heterozygous for the oim mutation have an improved phenotype compared to homozygous mice in terms of increased body weight and bones that are less brittle. Two problems related to somatic gene therapy for 01 are addressed by this proposal: (1) the identification and characterization of the cellular target vehicle using bone marrow transplantation and (2) determining the minimal level of replacement gene expression in transgenic mice that results in phenotypic improvement. The transplantation studies will identify optimal transplantation conditions for engraftment and proliferation of donor osteoprogenitor cells into oim/oim mice. This work will form the basis of future work using genetically modified cells. Our markers for success in this project are (1) allele specific-oligonucleotides that can discriminate wild type from oim gene nucleotide sequence, (2) allele specific oligonucleotides that can discriminate wild type from oim mRNA sequence, and (3) SDS-PAGE analysis for presence of the proalpha2(I) chain in cell culture and tissue samples. Successful engraftment and expression of normal collagen in bone is only one measure of success. A more important test is the demonstration of a functional improvement in bone biomechanical properties. Variable expression of proalpha2(I) cDNA constructs in transgenic mice will establish a relationship between construct expression (assessed at the mRNA and protein levels) and functional improvement determined by ex vivo mechanical testing of femurs.
