Rational Design and Development of Purely Peptidic Amphiphiles for Gene Delivery

Gene therapy depends on viral and non-viral delivery systems to ferry nucleic acids into target cells 1,2. In recent years, gene insertion and interference therapies have made a ground breaking impact in the treatment of rare inherited diseases, neurological disorders, cardiac diseases, and cancer 3. Several disadvantages associated with viral vectors, such as high toxicity and immunogenicity, limitation in size of transgenic DNA, and high manufacturing cost have triggered the rapid expansion of non-viral delivery systems including peptide-based vectors 4. The advantages of peptides are not only their biocompatibility and biodegradability, but sheer limitless possible combinations and modifications of amino acid residues that are able to promote the assembly of modular, multiplexed delivery systems 5. With the advantages of peptides in mind, we looked into the potential of peptide-based nanoassemblies in developing a non-viral gene delivery system. The thesis is structured to successively address (i) the design and development of purely amphiphilic peptides self-assembling into multicompartment micelles (MCMs), (ii) the efficient DNA cargo entrapment up to 100 nucleotides in length into self-assembled peptide MCMs and the delivery thereof, and (iii) targeting of oligonucleotides to the nucleus via a nuclear localization signal (NLS) integrated in the peptide-based carrier. The challenge was to rationally design the peptides and identify the proper conditions in which the DNA entrapment does not interfere with multicompartment micellar self-assembly. In addition, to fulfil the prerequisites of a successful gene delivery system that overcomes cellular barriers, we incorporated biologically active amino acids in our peptide sequences. A systematic characterization of the physicochemical features of the peptidic nanostructures was carried out to gain insight into the mechanism underlying self-assembly and to shed light on ways to tune these features for prospective biomedical applications. Taking into account our findings on how the size/type of genetic payload together with the peptide amphiphile’s charge and length impact the self-assembly process, we successfully established a non-toxic, purely peptidic delivery system that serves as a cornerstone for developing oligonucleotide therapy platforms.