Gene therapy with human recombinant osteoprotegerin reverses established osteopenia in ovariectomized mice

Abstract

Osteoporosis is a chronic condition that is typically treated by the long-term repeated administration of antiresorptive agents. Gene therapy has the potential to deliver protein-based antiresorptive agents without the need for repeated administration. Osteoprotegerin (OPG) is a naturally occuring protein that prevents bone resorption by inhibiting osteoclast formation, function and survival. We tested whether adeno-associated virus (AAV) could deliver OPG at levels that are sufficient to reverse established osteopenia in ovariectomized (OVX) mice without causing liver toxicity. Tibial bone mineral density (BMD) was measured by peripheral quantitative computed tomography (pQCT) in 12-week-old CDF1 mice prior to OVX or sham surgery. Six weeks later, BMD was significantly reduced in OVX mice compared to sham controls or pre-surgery values. Sham and OVX mice were then injected once IV with an AAV vector carrying cDNA for recombinant hOPG (AAV-OPG) or β-galactosidase (AAV-βGal). BMD and bone histomorphometry were assessed 10 weeks after treatment. A single injection of AAV-OPG led to the appearance of human OPG (hOPG) in the serum of mice within 7 days, and high serum levels of hOPG were maintained for the duration of the 10-week study. At the end of the study, OVX mice given AAV-OPG had significantly greater tibial BMD compared to age-matched OVX animals given AAV-βGal. In sham-operated mice, AAV-OPG also significantly increased tibial BMD compared to AAV-βGal. The increased BMD in AAV-OPG animals was accompanied by significantly increased bone volume and significantly reduced osteoclast surfaces in the proximal tibial metaphysis. Liver histology was normal, and circulating activities of hepatocyte cytosolic enzymes were unaffected by AAV exposure. In an accompanying experiment, young (3–4 weeks) C57BL/6 mice treated once IV with AAV-OPG maintained pharmacologically active levels of OPG in serum for at least 16 months. In summary, a single AAV-OPG treatment reversed established osteopenia in OVX mice without evidence of liver toxicity. AAV delivery appears to be a safe and effective method for producing sustained systemic exposure to OPG.

Introduction

Osteoprotegerin (OPG) is an endogenous protein of the tumor necrosis factor receptor superfamily that inhibits osteoclast formation, activation, and survival (reviewed in Ref. [1]). OPG acts by binding and neutralizing receptor activator of nuclear factor-κB ligand (RANKL), a cytokine that is critical for both physiological and pathological osteoclast activity [1]. The attributes of endogenous OPG, which are shared by recombinant OPG, are likely to account for the unique ability of recombinant OPG treatment to consistently and dramatically reduce osteoclast numbers in animal studies [2], [3], [4], [5], [6]. A single injection of recombinant human OPG-Fc into intact disease-free rats caused a rapid (12 h) reduction in osteoclast surfaces, which was maintained for 30 days [7]. In postmenopausal women, a single injection of human OPG-Fc caused the rapid (12 h) and sustained (30 days) suppression of bone resorption markers [8]. These results suggest that infrequent subcutaneous dosing may be a therapeutic option for osteoporosis in the future.

Convenience is an important attribute for osteoporosis therapies, due to the chronic nature of the disease. Infrequent dosing of osteoporosis drugs may also promote patient compliance, which for some osteoporosis therapies can drop by 50% within a year of initiation [9]. Patient compliance has been closely related to the prevention of future fractures [10]. The minimum dosing frequency among currently approved osteoporosis therapies is once per week [11], but efforts are underway to test once per year intravenous dosing of a bisphosphonate [12]. Because OPG is a protein, the potential exists for using gene therapy delivery to facilitate even longer treatment intervals. A proof-of-concept study recently demonstrated that OPG could be effectively delivered in mice using gene therapy. A single injection of mice with an adenoviral (Ad) vector containing human OPG-Fc resulted in systemic exposure to therapeutic levels of OPG for at least 18 months [13]. This Ad-OPG treatment was able to prevent the development of osteopenia in acutely ovariectomized (OVX) mice.

Safety is another critical attribute for osteoporosis therapies, because this chronic disease often progresses in an indolent manner before fractures occur. In the aforementioned OVX mouse study, the Ad-OPG vector caused liver toxicity that correlated with the amount of Ad vector administered [13]. It is clear that a nontoxic delivery vector would be required for osteoporosis gene therapy. To establish an adequate preclinical safety margin, a nontoxic vector should also promote expression of OPG at levels that can reverse established osteopenia in an OVX model. Adeno-associated viruses (AAV) produce little to no liver toxicity in primate studies [14]. We therefore engineered AAV-OPG vectors and tested their ability to safely deliver sustained pharmacologic levels of recombinant hOPG in mice. We established that a single injection of AAV-OPG could promote near-lifetime expression of therapeutic levels of hOPG and could also reverse established osteopenia in OVX mice, without evidence of toxicity.