Date of Award

2010

Document type

Thesis

Degree Name

MSc by research (Master of Science by research)

First Supervisor

Dr Marc Devocelle

Keywords

Gene Therapy, RNA, Small Interfacing

Abstract

This work focuses primarily on the synthesis of the cell penetrating peptide poly(L-lysine). This 30mer peptide (K³º) has been shown to be extremely useful in delivery of genetic material to the cell for gene therapy. So far it has shown transgene expression of up to twelve weeks, using DNA, which was delivered to the brain.[l] Lung cells have also been success full transfected by ths method.[2,3]

In this research we aim to use a polymer conjugate of K³º for the delivery of small interfering RNA (siRNA), a new exciting alternative to gene therapy, which operates downstream in the transcription pathway, through modulation of mRNA to produce gene silencing. Once optirnised, it is hoped that PEGylated K³º can be used as a universal delivery agent for any number of siRNA therapeutics in vivo.

K³º can be classified as a 'difficult peptide sequence'. This so-called terminology is used in the literature to describe any peptide which cannot be synthesised by classical means, involving rvutine automated peptide synthesis.[4-7] Generally, automated synthesis is reliable to a length of approxiinately twenty residues[8], so it is not surprising that K³º has been difficult to make by these means.

Various methods were attempted towards the synthesis of K³º, including (i) modification of the standard automated procedure with double coupling from a PEG-polystyrene composite resin, (ii) stepwise synthesis of two unprotected fragments to be ligated orthogonal to amide bond chemistry, and (iii) the synthesis of protected fragments to be coupled in solution to form the amide bond. [8]

In the end, monitoring of the sequence assembly was used to overcome the limitations associated with the solid phase synthesis of this peptide and the polylyshe sequence was successfully elongated by this approach.

After a certain point, about 18 residues, the peptide begins to aggregate on the solid support, preventing the reagents from reaching and reacting with the N-terminal of the peptide. To overcome this aggregation, a resin with a high hydrophilic content and a low substitution was chosen. The hydrophilicity of this resin repels the growing hydrophobic, protected chain. Furthermore the sparsity of linkers reduces the contact between adjacent chains. This NovaPEG Low Loading resin, combined with double coupling chemistry yielded the peptide K³º.

Other work included synthesis of host defence and cell penetrating peptide P17, peptide PEGylation by thiol-maleimide ligation and azo-modified amino acids for use in Click chemistry with alkynyl PEG.

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-Share Alike 3.0 License.

File Size

4,409 KB

Comments

A thesis submitted to the National University of Ireland for the degree of Master of Science through Research, 2010.

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