The strength and integrity of metabolically active bone depends on the balanced activity of two different cell types, namely osteoclasts, which resorbs the bone and osteoblasts which form new bone. Loss of this homeostasis results in a decrease in bone mass, leading to defects in mineralization of bone, resulting in osteopenic conditions including osteoporosis. Ex vivo gene therapy for osteoporosis is a rational alternative to the existing therapies and the potential of rAAV with long-term expression capability should provide greater benefit in this regard. A potential osteoporosis gene therapy is by using an ex vivo approach with gene modified mesenchymal stem cells (MSC) expressing osteogenic agents, which provide a local supply of precursor cells and a supraphysiological dose of osteoinductive molecules, which may promote bone formation. Towards achieving this goal with rAAV, capable of long term expression, we determined osteoprogenitor-specific expression of rAAV-transduced bone marrow stromal cells in vitro. rAAV vectors containing luciferase gene under the control of CMV, alkaline phosphatase, cbfa-1, collagen type-I, osteopontin and osteocalcin promoters were transduced to mouse MSC in replicates in vitro. Following transduction, cells were either maintained as undifferentiated cultures or induced to undergo early differentiation by the addition of 10 mM β-glycerophosphate, 50 μM ascorbic acid and 100 ng/ml recombinant BMP-2. Luciferase activity was determined at different time points as a measure of promoter activation. Results of these studies indicated that there was no significant difference in CMV activity between undifferentiated and BMP-2 differentiated MSC at different time points. However, luciferase activity from alkaline phosphatase, cbfa-1, collagen type-I, osteopontin and osteocalcin promoters in BMP-2 induced MSC cultures indicated increase in transgene expression over time, whereas no significant increase was observed in undifferentiated cultures. Among the bone-specific promoters tested, expression of collagen type-I and cbfa-1 promoters were more pronounced during early differentiation (48 hours) compared to osteocalcin, alkaline phophatase and osteopontin promoters. Northern blot analysis was done with RNA obtained from BMP-2 differentiated MSC at different time points to confirm BMP-2 induced osteoblastic changes in cultured and differentiated MSC. Results of these studies indicate that by using bone specific promoters that are active during early differentiation of MSC, lineage-restricted expression of therapeutic proteins is possible to enhance osteoblast production for increased bone mass.
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A 2004 study studied this question.