Antisense Reduction of Mutant COMP Reduces Growth Plate Chondrocyte Pathology

Abstract

Mutations in cartilage oligomeric matrix protein cause pseudoachondroplasia, a severe disproportionate short stature disorder. Mutant cartilage oligomeric matrix protein produces massive intracellular retention of cartilage oligomeric matrix protein, stimulating ER and oxidative stresses and inflammation, culminating in post-natal loss of growth plate chondrocytes, which compromises linear bone growth. Treatments for pseudoachondroplasia are limited because cartilage is relatively avascular and considered inaccessible. Here we report successful delivery and treatment using antisense oligonucleotide technology in our transgenic pseudoachondroplasia mouse model. We demonstrate delivery of human cartilage oligomeric matrix protein-specific antisense oligonucleotides to cartilage and reduction of cartilage oligomeric matrix protein expression, which largely alleviates pseudoachondroplasia growth plate chondrocyte pathology. One antisense oligonucleotide reduced steady-state levels of cartilage oligomeric matrix protein mRNA and dampened intracellular retention of mutant cartilage oligomeric matrix protein, leading to a reduction of inflammatory markers and cell death and partial restoration of proliferation. This novel and exciting work demonstrates that antisense-based therapy is a viable approach for treating pseudoachondroplasia and other human cartilage disorders. INTRODUCTIONTreatments of skeletal dysplasia/dwarfing conditions, including pseudoachondroplasia (PSACH), are few and fraught with problems because of the inaccessibility of chondrocytes within the cartilage matrix and the relative avascular nature of the cartilage environment. PSACH is clinically characterized by disproportionate short stature, rhizomelic shortening of the long bones, brachydactyly, and extreme joint laxity. 1,2 Joint pain in childhood and osteoarthritis (OA) in early adulthood are the most debilitating features of PSACH; only symptomatic treatments are available and have limited efficacy. 2,3 The diagnosis is made between 2-3 years of age when linear growth slows and a waddling gait develops. 1,2 Because the diagnosis, in most cases, is made post-natally, treatments aimed at resolving the pathology will have to be started immediately after diagnosis.PSACH is caused by mutations in cartilage oligomeric matrix protein (COMP), a pentameric extracellular matrix (ECM) glycoprotein abundant in musculoskeletal tissues. 4,5 Functionally, COMP facilitates type II collagen fibril assembly, enhances chondrocyte attachment in vitro, and interacts with other ECM proteins, including type II and IX collagens, matrilin 3, and SPARC. [6][7][8][9][10][11][12] In contrast, mutations in COMP cause misfolding of the protein, preventing export from the chondrocyte and resulting in massive retention within the endoplasmic reticulum (ER). 10,[13][14][15] Although this finding has long been appreciated, there was little understanding of the pathologic molecular mechanisms, which are critical to develop mechanism-driven therapeutics. To circumvent this proble...

Genetic Disorders of the Extracellular Matrix

“…Since structural mutations in collagen I in OI (and other ECM proteins in other ECMopathies) can exert such strong dominant gain‐of‐function effects, a goal of gene targeting approaches is complete, or near‐complete, mutant gene expression knock‐down. Antisense oligonucleotides, ribozymes, and small interfering RNAs have shown experimental efficacy for both COL1A1 (Lindahl et al, ; Rousseau et al, ) and COMP (Posey et al, ) mutations in vitro , and in the case of COMP mutations antisense delivery reduces the severity of the growth plate cartilage pathology in a pseudoachondroplasia mouse model (Posey et al, ). However, there are still significant obstacles to clinical delivery, along with uncertainties about possible off‐target effects.…”

Novel therapeutic interventions for pseudoachondroplasia

“…In a second approach, we tested whether systemic administration would deliver COMP targeted antisense oligonucleotides (ASO) to growth plate chondrocytes and decrease MT-COMP growth plate chondrocyte pathology [96]. We found that ASO1 was successfully delivered reducing steady-state levels of cartilage oligomeric matrix protein mRNA dampening intracellular retention of MT-COMP.…”

“…We found that ASO1 was successfully delivered reducing steady-state levels of cartilage oligomeric matrix protein mRNA dampening intracellular retention of MT-COMP. Interestingly, although ASO1 was designed against human COMP, mutant human COMP mRNA was reduced 38% and wild-type mouse COMP mRNA was reduced 60% [96]. Reduction in COMP retention in chondrocytes, led to a reduction of inflammatory markers, interleukin-16 (IL-16) and chitinase-like 3 (Chil3/YM1 a macrophage protein), the number of TUNEL positive chondrocytes, and a partial restoration of DNA proliferation [96].…”

“…Interestingly, although ASO1 was designed against human COMP, mutant human COMP mRNA was reduced 38% and wild-type mouse COMP mRNA was reduced 60% [96]. Reduction in COMP retention in chondrocytes, led to a reduction of inflammatory markers, interleukin-16 (IL-16) and chitinase-like 3 (Chil3/YM1 a macrophage protein), the number of TUNEL positive chondrocytes, and a partial restoration of DNA proliferation [96]. These exciting novel results demonstrate that ASOs can be delivered and knock down mRNAs in growth plate and articular cartilages and open the door for a new antisense treatment approach for PSACH and potentially other human cartilage disorders.…”

Novel mTORC1 Mechanism Suggests Therapeutic Targets for COMPopathies

“…one mutant subunit causing a dominant negative effect that results in the protein being trapped in the ER. 39 Ultimately, the rough ER of affected chondrocytes enlarge, filling the cytoplasmic space and being cytotoxic to growth plate chondrocytes. 2,20,32,34 Previously, a doxycycline-inducible mouse model that expresses MT-COMP (D469del) in growth plate chondrocytes, and that recapitulates the clinical phenotype and chondrocyte pathology of PSACH, was generated, validated, and used to identify the pathologic mechanisms.…”