Graduate Thesis Or Dissertation
 

Inhalable Lipid Nanoparticles for Messenger RNA Therapeutics

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  • Messenger RNA (mRNA) therapy has massive potential to treat various genetic disorders by providing effective proteins to the diseased cells and tissues. Its therapeutic applications include protein replacement, genome editing, and immunotherapy. For successful mRNA treatment, mRNA must be delivered to the cytosol of target cells. One of the leading non-viral vectors for the successful delivery of mRNA is lipid nanoparticles (LNPs). LNPs enable safe and efficient gene delivery; however, their innate liver tropism significantly limits access to other organs, such as the lungs. Lungs are the primary organs of the respiratory system and are essential for gas exchange in humans. Therefore, dysfunction in the lungs can be fatal by causing respiratory failure in patients. Despite the need for means to deliver mRNA efficaciously, it remains challenging to deliver therapeutic mRNA to the lungs. This dissertation sought effective LNP formulations capable of delivering mRNA to the lungs, focusing on the inhalation route. We first explored whether naturally occurring membrane lipids can enhance mRNA delivery in vivo. We identified that substitution of DSPC to DGTS in LNP formulations improved hepatic delivery of mRNA; however, it did not help when LNPs were aerosolized or delivered to the murine nasal cavity. Next, we tried to stabilize LNPs during aerosolization by a vibrating mesh nebulizer. We focused on polyethylene glycol (PEG)-anchored lipid (PEG-lipid) and sterol since PEG-lipid is known to improve the colloidal stability of LNPs, and sterol substitution appears to improve endosomal escape of LNPs. We demonstrated that increased PEG density and sterol substitution could enable LNPs to tolerate aerosolization and retain transfection competence. This novel formulation delivered in vivo exhibited robust mRNA transfection in mouse lungs without any off-target transfection. In addition, we did not observe any acute toxicity in animals. In the case of repetitive dosing, mRNA expression was retained in the lungs over three administrations without any statistical compromise. Having confirmed that the inhalation of the optimized LNPs can transfect mouse lungs selectively, we attempted to deliver therapeutic mRNA to treat lung diseases. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a contagious virus, causing lethal pulmonary damage in humans. It contains spike proteins on its envelope that bind human angiotensin-converting enzyme 2 (hACE2) expressed on airway cells, enabling entry of the virus and causing infection. The soluble form of hACE2 binds the spike protein, prevents viral entry into cells, and ameliorates lung injury; however, its short half-life limits therapeutic utilities. We developed LNPs encapsulating the mRNA encoding a soluble form of hACE2 (hsACE2) to prevent viral infection. Upon intravenous injection to mouse, LNP delivered mRNA to the liver, leading to the production of circulatory hsACE2 initiated within two hours and sustained over several days. Inhalation of the LNP led to lung transfection and secretion of mucosal hsACE2 to the lung epithelia, the primary entry site and pathogenesis for the virus. Next, we demonstrated that mRNA-encoded hsACE2 binds to the receptor-binding domain of the spike protein of the virus. Further, hsACE2 strongly inhibited the infection of pseudovirus carrying SARS-CoV-2 wild-type spike protein and its variants, suggesting the therapeutic potential of hsACE2 mRNA against the viral infection of SARS-CoV-2. Taken together, the results in this dissertation show the potential of mRNA as a therapeutic for pulmonary diseases. The modularity of LNP formulations provides expansive design space for fine-tuning the physicochemical properties and transfection efficiency of the nanoparticles for aerosolization. Furthermore, inhalation of LNPs offers selective transfection of the respiratory system, overcoming the innate tropism of the nanoparticles to the liver. This targeted transfection of the lungs can be valuable for treating various lung diseases. Alternatively, engineering mRNA sequences to produce secreted proteins can be effective against respiratory diseases via pulmonary circulation. Finally, we hope that this research will foster translating mRNA therapeutics to treat pulmonary diseases.
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  • Intellectual Property (patent, etc.)
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  • 2021-12-07 to 2024-01-07

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