Development of a long-lasting, bone-targeted gene therapy for osteogenesis imperfecta - PROJECT SUMMARY Osteogenesis imperfecta (OI) is the most common bone fragility disease, with an incidence of approximately 1 in 25,000–50,000 individuals in the US. OI is an inherited genetic disorder caused by mutations in the genes involved in collagen production, processing, and cross-linking. Genetic mutations and phenotypes in OI patients are highly variable, so current treatments mainly focus on symptomatic improvements, such as increase bone mass and strength to reduce bone fragility, fracture, and pain, using the drugs initially developed to treat osteoporosis. One innovative approach to treat OI patients who require life-long treatments is a long-lasting, bone-targeted gene therapy that improves skeletal health using recombinant adeno- associated virus (rAAV). To avoid potential off-target adverse effects of AAV gene therapy in non-skeletal tissues, we improved rAAV9 serotype’s bone-specific tropism and expression by implementing a novel bone- targeted capsid and incorporating miRNA-mediated repression of transgene expression in the liver, muscle, and heart into the vector design. Using this bone-tropic rAAV, we enhanced bone-forming WNT signaling in osteoblasts (OBs) by silencing the expression of two WNT inhibitors, Sclerostin (Sost) and Schnurri3 (Shn3). Here, we propose that with a single treatment, systemic delivery of the bone-tropic rAAV conferring single or dual silencing of Shn3 and/or Sost improves bone mass and strength, and increases grip strength, promotes the healing of fracture, and improves mobility in OI mice. We will determine the molecular mechanisms of how these processes occur using mouse and human OI skeletal organoids. First, we will examine whether bone-tropic AAV:WNT modulators improve skeletal health in OI mice (Aim 1). Given that bone-tropic rAAV-mediated silencing of Shn3 and/or Sost promoted bone formation by augmenting WNT/b-catenin signaling and OB function in osteoporosis, this aim will examine their ability to ameliorate skeletal deformities and increase bone mass and strength throughout the skeleton, promote fracture healing, and enhance grip strength and mobility in mice with three different subtypes of OI forms. OI skeletal phenotypes continuously develop throughout lifetime, so we will examine rAAV’s effectiveness and durability at multiple intervention timepoints. Next, we will determine molecular mechanisms of bone-tropic AAV:WNT modulators using mouse (Aim 2) and human (Aim 3) OI skeletal organoids. The 3D-skeletal organoids seeded with mouse or human OI-OBs will be developed for in vitro culture or implantation into a xenograft mouse model. These organoids will allow us to study the molecular actions of the bone-tropic rAAV reversing abnormalities in WNT/b-catenin signaling, osteoblast and osteocyte development, mineralization activity, and collagen biochemistry. We will perform transcriptome profiling of AAV-transduced OI-OBs to identify the downstream regulators of the WNT pathway contributing to collagen production and processing. Successful completion of these aims will provide proof-of-concept evidence for bone-tropic AAV gene therapy as an alternative long-lasting treatment option for OI patients and improve our understanding of its mechanisms of action in the treatment of OI.
