Engineering Lipid Nanoparticle-Mediated Delivery of Nucleic Acids to Lymphoid Organs and Immune Cells
Ribonucleic acids (RNA) and deoxyribonucleic acids (DNA) hold promise for continued application in the treatment of genetic diseases. Short interfering RNA (siRNA) and microRNA (miRNAs) have been used to silence disease-causing genes. Longer messenger RNA (mRNA) and plasmid DNA (pDNA) have been used to deliver genes whose absence resulted in disease. RNA and DNA are both large hydrophilic macromolecules that are susceptible to degradation by nucleases and can induce immune responses when injected directly into the bloodstream. Moreover, due to their physicochemical properties, nucleic acids are unable to cross biological membranes to reach the cytosol or the nucleus. Viral and non-viral vectors are needed for the protection and effective delivery of nucleic acids. Lipid nanoparticles (LNPs) are an established essential platform for nucleic acid delivery. Efforts have led to the development of vaccines that protect against SARS-CoV-2 infection using LNPs to deliver mRNA coding for the viral spike protein. Significant challenges with LNP-mediated nucleic acid delivery include avoiding entrapment in the endosome and enabling extrahepatic delivery. This dissertation reports the engineering of LNPs for the delivery of mRNA and DNA to lymphoid organs such as the spleen and immune cells including T cells and neutrophils. Phospholipids containing phosphoethanolamine (PE) head groups likely increase endosomal escape due to their fusogenic properties. Additionally, it was found that negatively charged phospholipids drive spleen tropism. The use of a spleen selective organ targeting (SORT) formulation containing a negatively charged lipid transfected T cells in vivo. This formulation produced in situ Chimeric Antigen Receptor (CAR) T cells by delivery of mRNA encoding an anti-CD19 CAR. An in vivo Design of Experiment (DOE) to optimize LNPs for spleen pDNA delivery indicated ionizable lipids have the highest delivery efficacy of pDNA to the spleen, compared to cationic lipids. Additionally, higher molar ratios of phospholipid in relationship to the ionizable lipid increased pDNA delivery to neutrophils in the spleen. Overall, the results highlight how the chemistry and the composition of LNPs influences endosomal escape, organ tropism and cell targeting to obtain LNPs with unique therapeutic potential.
Liposomes, Nanoparticles, RNA, Messenger, RNA, Small Interfering, Transfection, Lipids
University of Texas Southwestern Medical Center
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