A next generation of sclerostin inhibitor with bone anabolic potential and without increasing cardiovascular risk for osteogenesis imperfecta: How does sclerostin loop3-specific aptamer work?

Osteogenesis imperfecta (OI) is a dominantly inherited skeletal fragility disorder, characterized by severe defects in bone mass and architecture, without specifically developed pharmacological therapy. OI affects approximately 1 in 10,000-20,000 people worldwide. An estimated 700-800 people in Hong Kong have OI, while an estimated 5-6 newborns each year in Hong Kong have the condition. Both genetic and pharmacologic evidences indicated that sclerostin inhibition could promote bone formation in OI mice (Col1a2+/G610C). Clinically, sclerostin antibody romosozumab demonstrated bone anabolic potential in postmenopausal osteoporosis, whereas imposed severe cardiac ischemic events.

Cardiac valves abnormalities are associated secondary features of OI patients. Accordingly, there is also a growing cardiovascular safety concerns for OI patients during sclerostin antibody treatment, especially for those with cardiovascular abnormalities or with cardiovascular diseases history. So, it is desirable to develop a next generation of sclerostin inhibitor for OI to promote bone formation without increasing cardiovascular risk. Cardiac ischemic events were contributed by chronic progressive inflammatory diseases such as aortic aneurysm (AA) and atherosclerosis. It was reported that transgenic introduction of human sclerostin could suppress inflammatory cytokines and chemokines to prevent AA and atherosclerosis development in apolipoprotein E-deficient (ApoE-/-) mice with angiotensin II (AngII) infusion. The central residues of sclerostin form three loops (loop1, loop2 and loop3). Notably, our in vivo data showed that sclerostin antibody, which targeted both sclerostin loop2 and loop3, could elevate serum levels of both inflammatory cytokines and chemokines, and aggravate AA and atherosclerosis in Col1a2+/G610C.ApoE-/- mice with AngII infusion. Our in vitro data indicated that either loop2&3 deficiency by genetic truncation or loop2&3 inhibition by sclerostin antibody attenuated the suppression effects of sclerostin on expression of inflammatory cytokines and chemokines in primary macrophages and aortic vascular smooth muscle cells (VSMCs) from Col1a2+/G610C.ApoE-/- mice, whereas loop3 deficiency by genetic truncation maintained the above suppression effects of sclerostin. Consistently, transgenic introduction of loop3 deficient sclerostin and full-length sclerostin, respectively, showed similar suppressing effect on expression of inflammatory cytokines and chemokines, and development of AA and atherosclerosis in Col1a2+/G610C.ApoE-/- mice with AngII infusion. It indicated that the protective effect of sclerostin on cardiovascular system was independent of loop3. Moreover, after normalized by bone formation in Col1a2+/G610C mice, the relative bone formation in Col1a2+/G610C mice with transgenic introduction of loop3 deficient sclerostin was significantly higher than that in Col1a2+/G610C mice with transgenic introduction of full- length sclerostin. It suggested that loop3 in sclerostin played an important role in sclerostin’s antagonistic effect on bone formation. Therefore, specifically targeting sclerostin loop3 should be examined for promoting bone formation without increasing cardiovascular risk in OI mice.

Utilizing tailored selection, we obtained one optimized sclerostin loop3-specific aptamer, aptscl56. The in vitro data indicated that aptscl56 inhibited sclerostin’s antagonistic effect on Wnt signaling and promoted osteogenic potential in osteoblasts from Col1a2+/G610C mice, whereas had no influence on sclerostin’s suppression effect on expression of inflammatory cytokines and chemokines in primary macrophages and aortic VSMCs from Col1a2+/G610C.ApoE-/- mice with AngII treatment in vitro. Sclerostin loop3 mutant loop3m was demonstrated to fail to inhibit Wnt signaling but remain binding to aptscl56. The above effect of aptscl56 in osteoblasts from Col1a2+/G610C mice were attenuated by pretreatment of exogenous loop3m, suggesting that aptscl56 inhibited sclerostin’s antagonistic effect on Wnt signaling via targeting sclerostin loop3 in vitro. After PEG40k conjugation, subcutaneously administrated PEG40k-aptscl56 (Apc001) showed 72 times longer half-life (T1/2=57.798h) than the free aptscl56 (T1/2=0.8h) in Col1a2+/G610C mice. For cardiovascular safety, Apc001 had no influence on AA and atherosclerosis progression in Col1a2+/G610C.ApoE-/- mice with AngII infusion, in both of which the inflammatory cytokines and chemokines expression, macrophages infiltration, VSMCs contractile phenotype loss and apoptosis were not altered by Apc001 administration. For bone anabolic potential, Apc001 demonstrated the efficiency on promoting bone formation, increasing bone mass and improving bone microarchitecture integrity in Col1a2 +/G610C mice. For toxicity evaluation, there were no significant differences in serum levels of liver and kidney function indexes and hematologic parameters between Apc001 groups and vehicle groups, after either a single or multiple administration(s). Excitingly, PEG40k-conjugated aptscl56 for OI in our lab was granted orphan-drug designation by US Food and Drug Administration (FDA, DRU-2019-6966) in 2019. Taken together, an open question is how aptscl56 works in OI.

To address the question, it was hypothesized that Apc001 (PEG40k-aptscl56) could promote bone formation via targeting sclerostin loop3 in a dose-dependent manner, while maintain loop3-independent cardiovascular protective effect of sclerostin for OI. To test the hypothesis above, we have the following three specific aims:
(1) Aim 1: To determine whether Apc001 could promote bone formation in a dose-dependent manner in Col1a2 +/G610C mice by examining the bone anabolic effect at different doses.
(2) Aim 2: To determine whether Apc001 could promote bone formation via targeting sclerostin loop3 in Col1a2+/G610C mice by comparing the differences in bone formation between with and without pretreatment of exogenous loop3m.
(3) Aim 3: To determine whether Apc001 could influence the protective effect of the transgenic human sclerostin on cardiovascular system in hSOSTTg.Col1a2+/G610C.ApoE-/- mice with AngII infusion by both determining serum inflammatory cytokines and chemokines and evaluating cardiac ischemic events.

The proposed work would facilitate understanding how the next generation sclerostin inhibitor (sclerostin loop3- specific aptamer) works with bone anabolic potential and without increasing cardiovascular risk in OI.