A novel bone-targeting AAV-mediated gene therapy to promote bone formation in osteoporosis

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PROJECT SUMMARY Adult bone mass is determined by the balance between bone formation by osteoblasts (OBs) and bone resorption by osteoclasts (OCs), and disturbances in this equilibrium to favor osteoclast (OC)-mediated resorption leads to osteoporosis. The majority of existing therapeutics for osteoporosis act by inhibiting OCs, but these can not cure osteoporosis and are limited by rare side effects. Current anabolic agents, parathyroid hormone (PTH), parathyroid hormone-related protein (PTHrP), and anti-sclerostin antibody exist for promoting osteoblast (OB) function to treat patients with osteoporosis. However, these agents are also limited by concern for off-target adverse effects and waning efficacy. Previously, we and others demonstrated that inhibition of potent OB suppressors, sclerostin (SOST) and the adaptor protein schnurri-3 (SHN3), promotes bone formation in mouse models of postmenopausal and senile osteoporosis. One innovative approach to treat osteoporosis is RNAi-based bone anabolic gene therapy using recombinant adeno-associated virus (rAAV). Using our engineered rAAV9 vector with bone-specific tropism and transgene expression, we will develop novel gene therapeutics that promote bone formation in osteoporosis with a single systemic administration. Additionally, we will identify potential novel osteogenic and/or angiogenic factors regulated by the SOST/SHN3 pathway that could be useful as therapeutic targets for osteoporosis. Aim 1 will examine whether bone-specific rAAV9-mediated silencing of SHN3 or SOST can reverse bone loss in mouse models of osteoporosis. To avoid potential off-target adverse effects in non-skeletal tissues, rAAV9’s bone- specific tropism and transgene expression were further improved by capsid modification and tissue-specific miRNA-mediated repression of rAAV expression. Using two mouse models of osteoporosis (ovariectomized (OVX) and aged mice), we will determine therapeutic potentials of rAAV9-mediated silencing of SHN3 or SOST in postmenopausal and senile osteoporosis. Aim 2 will identify novel osteogenic and/or angiogenic factors regulated by the SHN3/SOST pathway that promote bone formation in osteoporosis. In vivo silencing accuracy of rAAV9 carrying amiR-shn3 or amiR-sost in bone-residing OB lineage cells will be examined by scatterplot analysis of whole transcriptome data. Once validated, wt, shn3- or sost-deficient OB-lineage cells will be FACS-sorted from AAV-treated, OVX-mice and subjected for transcriptome profiling. As a complimentary approach, proteomics will be performed in AAV-transduced OB-lineage cells isolated from MetRS;Prx1-cre mice with OB-specific labeling of nascent proteins after OVX-surgery. These combinatory approaches will allow us to identify novel osteogenic and/or angiogenic factors commonly or differentially regulated by the pathways of SHN3 and SOST in response to estrogen deficiency-induced osteoporosis. Successful completion of these aims will provide proof-of-concept demonstration and identify potential novel regulators that could be useful as therapeutic targets for osteoporosis.