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Synthesis and Formulation of Acid-responsive Polypeptide Vectors for Gene Therapy Applications


Gene therapy holds great promise for treating diseases that stem from genetic origin. Nevertheless, development of safe and efficient gene delivery vectors is still a major challenge. Multiple hurdles are encountered when nucleic acids are administered into the body; they must avoid nuclease degradation, bypass rapid clearance, limit immune responses, extravasate to desired tissues, get taken up by cells, and traffic to desired intracellular locations. Advances in biotechnology aim to develop nontoxic as well as smart nucleic acid delivery carriers with stimuli-responsive features to overcome these challenges. Peptide-based nanomaterials have become widely used in the field of biotechnology for gene and drug delivery due to their structural versatility and biomimetic properties. Particularly, polypeptide vectors that respond to biological stimuli, such as acidic intracellular environments, have great utility in mediating efficient endosomal escape and drug release. Unfortunately, synthesis strategies for efficient polymerization of acid-labile peptides has been minimal due to conditions that fail to preserve acid-degradable functional groups. In this dissertation, a practical polymerization method was first developed for the synthesis of high molecular weight acid-labile polypeptides. Stable urethane derivatives of acid-labile amino acid, ketalized serine (kSer), were synthesized and polymerized to high molecular weight under permissive conditions independent of elevated temperature, restrictive solvents, or inert atmosphere. The practicality in the synthesis method allows further advancement of acid-labile polypeptide vectors as novel and functional biomaterials. Consequently, a new formulation strategy utilizing solvent-assisted self-assembly of poly(kSer)-derived peptides with small interfering RNA (siRNA) was developed. The nanoparticles were highly monodisperse and precisely spherical in morphology which has significant clinical implications in definitive biodistribution, cellular internalization, and intracellular trafficking patterns. Cross-linked poly(kSer)/siRNA nanoparticles demonstrated efficient nucleic acid encapsulation, internalization, endosomal escape, and acid-triggered cargo release, which tackles multiple hurdles in siRNA delivery. In summary, this dissertation provides a full comprehensive study from synthesis to formulation of acid-responsive poly(kSer)-derived peptides for gene therapy applications.

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