Peptide-mediated delivery of antisense oligonucleotides and chemotherapeutics across biological barriers

Thesis: Ph. D. in Biological Chemistry, Massachusetts Institute of Technology, Department of Chemistry, 2018.Cataloged from PDF version of thesis.Includes bibliographical references.Many nucleic acids, peptides, and small molecules struggle to become clinically viable therapeutics as a result of poor delivery. Biological barriers such as the plasma membrane and the blood-brain barrier (BBB) contribute to this challenge as they can limit the passage of macromolecules. Cell-penetrating peptides (CPPs) that interact with membranes can improve the uptake of macromolecules across biological barriers. Here we explore methods for the peptide-mediated delivery of antisense oligonucleotides (ASOs) and chemotherapeutics. First, we address the issue that the optimal peptide sequence for the delivery of a macromolecular cargo is often context-dependent and specific to that cargo. With one class of ASO, we develop a paradigm that combines systematic screening of known CPPs in a functional assay for ASO delivery with machine learning methods. Using our computational model, we identify five novel sequences that increase ASO activity at least three-fold. Next, we demonstrate that combining CPPs of different classes generates chimeric peptides with synergistic effects on ASO delivery. These chimeras improve ASO activity twenty-fold, which is greater than any literature-reported sequence. Then, we examine peptide cyclization with perfluoroaryl-cysteine SNAr chemistry to improve the stability and delivery of peptide-ASO conjugates. We extend our SNAr chemistry to the synthesis of arginine-rich bicyclic peptides, which are more stable to proteolysis than single cycles. Both perfluoroaryl cyclic and bicyclic arginine-rich peptides improve ASO activity fourteen-fold. Consequently, we demonstrate that peptide cyclization with perfluoroaryl-cysteine SNAr chemistry enhances the ability of peptides to cross the BBB. We prepare macrocyclic analogues of both a CPP and a therapeutic peptide. We show that a subset of the macrocycles cross the BBB in both a cellular spheroid model of the BBB, as well as after intravenous injection in mice. Finally, we conjugate a platinum (IV) prodrug of the chemotherapeutic cisplatin to a brain-penetrating perfluoroaryl macrocycle and show that the amount of platinum in the mouse brain is fifteen-fold greater than cisplatin after five hours. In summary, we explore strategies to improve the peptide-mediated delivery of ASOs and small molecule chemotherapeutics across biological barriers. In the future, we envision extending these approaches to other macromolecular cargos of therapeutic interest.by Colin MacLaine Fadzen.Ph. D. in Biological Chemistr