ABSTRACT Lymphangioleiomyomatosis (LAM) is a Tuberous sclerosis-related disorder. Both occur due to an inherited or sporadic mutation in either the TSC1 or TSC2 gene, which function as negative regulators of the mTOR pathway. Uncontrolled mTORC1 activity leads to the neoplastic proliferation of abnormal smooth muscle cells (LAM cells) in the lungs, progressive shortness of breath, recurrent pneumothoraxes, and loss of pulmonary tissue structure and function primarily in women. The first and only FDA-approved treatment for LAM is the immunosuppressant sirolimus, marketed since 2015 by Pfizer. It is the current standard-of-care and acts by inhibiting mTORC1. Sirolimus has several clinical disadvantages, including a considerable number of non- responders, severe adverse events due to its immunosuppressive properties and pregnancy category C, limiting its use in women of childbearing age. Thus, there is a high unmet medical need to develop alternative and safer treatment options for LAM and TS. We have identified treatment with miRNA302b mimics as a potential novel therapy for LAM/TS. Using a murine lung injury model, our collaborator Hao Shen was able to show that non-targeted treatment with miRNA302b mimics as neutral lipid emulsion improved lung function, host recovery, and alveolar epithelial regeneration mice. We also demonstrated a “stalled” AT2-AT1 transdifferentiation state in human LAM, suggesting that impaired AT2 fitness may contribute to loss of alveolar structure. Our goal for this grant is the investigation of pulmonary-epithelium targeted miRNA302b mimic lipid nanoparticles (LNP) efficacy for the treatment of LAM by enhancing AT2 cell regeneration. Aim 1 is composed of in vitro characterization of the miRNA-302b mimic lipid nanoparticle and investigation of several targeting strategies in vivo. Our PI Dr. Jake Brenner will design and construct the nanoparticles in his bioengineering lab. We will perform 3D organoid experiments with human and mouse AT2 cells in the presence of the LNP[miR302b] followed by an in vivo study comparing different targeting strategies to reach the desired tissue and cell type using a transgenic model of LAM, developed by our other PI Prof. Vera Krymskaya. Using the most effective targeting strategy, Aim 2 will be an in vivo proof-of-concept study using that same transgenic murine model pre and post-pregnancy to answer the question whether our treatment will prevent airspace enlargement and lead to lung recovery. We will observe mice for 4 and 8 weeks after intratracheal LNP[miR302b] administration. Endpoints will include lung function, BALF analysis, qPCR, and histological lung sections stained for the disease related markers and cell types. All collaborators are experts in their respective field and thus well equipped to successfully complete this project, bringing together a number of unique and innovative aspects to treat this devastating rare disease.

Narrative Tuberous sclerosis (TSC) and Lymphangioleiomyomatosis (LAM) are multi-system and often life-threatening rare genetic diseases affecting thousands of people in the United States and approximately 1 million worldwide. We showed that treatment with miRNA302b mimics led to enhanced regeneration of alveolar epithelial cells, tissue repair, and recovery of lung function in a murine lung injury model, which makes it a promising regenerative therapy approach for the treatment of LAM, since the only approved therapy only halts disease progression without curing it. Our proposed in vitro and in vivo research will investigate epithelial-targeted therapy with miRNA302b mimics formulated as a Lipid Nanoparticle in a LAM organoid, cell culture and mouse model to validate our hypothesis and take the first step to fast-track development for clinical use in LAM and TSC.