RNAi reduces expression and intracellular retention of mutant cartilage oligomeric matrix protein.

Mutations in cartilage oligomeric matrix protein (COMP), a large extracellular glycoprotein expressed in musculoskeletal tissues, cause two skeletal dysplasias, pseudoachondroplasia and multiple epiphyseal dysplasia. These mutations lead to massive intracellular retention of COMP, chondrocyte death and loss of growth plate chondrocytes that are necessary for linear growth. In contrast, COMP null mice have only minor growth plate abnormalities, normal growth and longevity. This suggests that reducing mutant and wild-type COMP expression in chondrocytes may prevent the toxic cellular phenotype causing the skeletal dysplasias. We tested this hypothesis using RNA interference to reduce steady state levels of COMP mRNA. A panel of shRNAs directed against COMP was tested. One shRNA (3B) reduced endogenous and recombinant COMP mRNA dramatically, regardless of expression levels. The activity of the shRNA against COMP mRNA was maintained for up to 10 weeks. We also demonstrate that this treatment reduced ER stress. Moreover, we show that reducing steady state levels of COMP mRNA alleviates intracellular retention of other extracellular matrix proteins associated with the pseudoachondroplasia cellular pathology. These findings are a proof of principle and the foundation for the development of a therapeutic intervention based on reduction of COMP expression.

An inducible cartilage oligomeric matrix protein mouse model recapitulates human pseudoachondroplasia phenotype.

Cartilage oligomeric matrix protein (COMP) is a pentameric extracellular protein expressed in cartilage and other musculoskeletal tissues. Mutations in the COMP gene cause pseudoachondroplasia (PSACH), a severe dwarfing condition that has a growth plate chondrocyte pathology. PSACH is characterized by intracellular retention of COMP and other extracellular matrix (ECM) proteins, which form an ordered matrix within large rough endoplasmic reticulum cisternae. This accumulation is cytotoxic and causes premature chondrocyte cell death, thereby depleting chondrocytes needed for normal long bone growth. Research to define the underlying molecular mechanisms of PSACH has been hampered by the lack of a suitable model system. In this study, we achieved robust expression of human mutant (MT) or wild-type (WT) COMP in mice by using a tetracycline-inducible promoter. Normal growth plate distribution of ECM proteins was observed in 1-month-old WT-COMP and C57BL\6 control mice. In contrast, the structure of the MT-COMP growth plate recapitulated the findings of human PSACH growth plate morphology, including (1) retention of ECM proteins, (2) intracellular matrix formation in the rER cisternae, and (3) increased chondrocyte apoptosis. Therefore, we have generated the first mouse model to show extensive intracellular retention of ECM proteins recapitulating the human PSACH disease process at the cellular level.