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Peptide Based Gene Delivery

Rapid progress in molecular biology and proteomics has generated a large number of new biomolecular drugs, such as antisense oligonucleotides and small interfering RNA (siRNA), but none of these molecules can be used as therapeutic agents unless effective delivery systems can be found.  Oligonucleotides tend to degrade in the bloodstream and are not easily taken up into cells.  A potential new delivery system is based on the use of self-assembling peptides. These peptides consist of alternating hydrophobic and hydrophilic (charged) amino acids, and can be designed to act as gene carriers.

Interaction between  peptides and oligonucleotides

Complexation between peptides and oligonucleotides is the first step in developing peptide-mediated gene delivery systems.  Quantitative analysis of UV titration results demonstrated that the peptide interacts with oligonucleotides and forms 1:1 complex with a dissociation constant in the micromole and millimole range for single- and double-stranded oligonucleotides, respectively, at acidic pH, but not neutral or basic pH.  This pH effect shows that the interaction is mainly electrostatic, between negative charges of nucleic acids and positive charges of peptide residues. Further protective quenching studies demonstrated that the formation of complexes could protect oligonucleotides from degradation during transfer.  A wide range of physicochemical methods have been used in this project, including: UV/Vis spectroscopy, dynamic surface tension, steady-state fluorescence, fluorescence anisotropy, Atomic Force Microscopy (AFM), Scanning Electron Microscopy (SEM) and Dynamic Light Scattering (DLS).

Penetration  of oligonucleotide/peptide complexes into cells

Large unilamellar vesicles (LUVs) will be used to mimic artificial cell membranes in the initial study. The translocation of fluorophore-labelled oligonucleotides into LUV with a quencher encapsulated will be studied by fluorescence resonance energy transfer (FRET). Once positive results are obtained, we will move onto cell line and animal model studies. 

Modification of peptide sequence to improve cell penetration and targeting

Sequence of cell targeting or penetrating peptides, such as Penetratin and Tat, can be added to self-assembling peptides to improve the delivery efficacy, safety and targeting.