Prostate cancer (PCa) bone metastases have been reported in ~90% of patients with advanced disease. Bone metastases disrupt tissue homeostasis and weaken the skeleton, resulting in an increased risk of bone fractures and morbidity. Specifically, PCa cells disrupt the crosstalk between critical cells within the tumor/bone microenvironment (osteoblasts, osteoclasts, and immune cells), and utilize this effector-rich environment for cancer survival and growth. Therefore, a key therapeutic objective in malignant skeletal disease management is to eliminate tumors while restoring bone homeostasis. Current treatments include palliative radiotherapy, chemotherapy, or anti-RANK treatments, all of which have considerable side effects such as osteonecrosis of the jaw or enhanced tumor invasion. There remains a critical gap in therapies than can reduce tumor burden and simultaneously restore bone homeostasis. To address this gap, our work explores emerging gene therapy approaches for treating skeletal malignancies by utilizing multifunctional cytokine-based agents that can simultaneously combat tumor growth and promote bone regeneration.<div><br></div><div>We hypothesize that rationally designed cytokine-based gene therapies that can be secreted from skeletal muscle and targeted to the bone/tumor microenvironment, could effectively reduce tumor growth and restore bone cell homeostasis. To test this hypothesis, we adopted two strategies: 1) a second-generation targeted IL-27 cytokine, and 2) a de novodesign of a cytokine-like therapeutic agent (Propeptide) that includes anti-tumorigenic and pro-osteogenic domains. Both strategies share modules with overlapping therapeutic functions, rendering them complementary in their therapeutic application. In this work, we examined the proof of principle for propeptide gene therapy in muscle cells (in vitro models) and assessed the therapeutic efficacy of our cytokine-based biologics in reducing prostate tumor growth and rebalancing bone cell proliferation and differentiation. Our studies resulted in a propeptide construct representative of a cytokine structure comprised of a bundle of helices that we were able to express in cells. Additionally, our work demonstrated the targeting and anti-tumor efficacy of our therapeutic cytokines in cancer and bone cell models. Ultimately, this will provide the framework for innovative peptide and cytokine-based therapeutics that target and treat both the tumor metastases and bone. This approach will facilitate improvement of morbidity and quality of life of prostate cancer patients with bone metastases and could be applicable to other diseases with bone/tumor pathologies. <br><div><br></div></div>