Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.566376
Title: Identification of peptides capable of targeting immature dendritic cells using phage display
Author: Ismail, Ida Shazrina Binti
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2012
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Abstract:
The ability of dendritic cells (DC) to be either tolerogenic or immunogenic offers many opportunities to manipulate DC function and genetically modify DC to promote allograft acceptance. However, the greatest challenge in DC gene therapy is the difficulty of achieving successful transfection. At present, DC-targeting gene delivery strategies are hindered by the lack of DC-specific target molecules. Therefore, the essential aim of this study is to isolate and identify peptides capable of targeting immature DC (iDC), in particular those that internalise well as they are likely to be more effective in gene delivery. Phage library of short cyclic peptides expressed on pIII protein of M13 filamentous phage was used first in selection against streptavidin. Phage bearing HPQ and HPM motifs were successfully isolated indicating the feasibility of the library in selecting streptavidin-binding peptides through specific biotin interaction. In order to develop a method for in vitro selection on cell surfaces, the library was used against a cell line expressing αVβ3 integrin. One phage clone with encoded sequence of CLSSPALLC bound to the cell surface and was taken up by the cells expressing the αVβ3 integrin. Using methods established in the previous panning, the library was panned against iDC, after two subtractive pannings against PBMC and CD14+ cells. The phage isolated were characterised by using several methods including flow cytometric analysis and confocal microscopy. Two phage selected, DC4.25 and DC4.44 with encoded peptide sequences of CFPPTFPAC and CTPLLSPFC, respectively, have demonstrated the ability to bind to, and be internalised by, iDC. Soluble biotinylated peptides, which contain the encoded peptide sequences, were synthesised along with sequence-scrambled control peptides. Flow cytometric analysis detected non-specific binding of the monomeric peptides to iDC when used at high concentration. In an attempt to increase the binding avidity of the peptides to iDC, Streptavidin conjugated with Alexa-488® flurophore was used to form complexes with the peptides. However, flow cytometric analysis revealed that the complexes also bound non-specifically to iDC, as binding was also seen in control peptides. Streptavidin conjugated with HRP was then used to form complexes with the peptides, in order to increase the sensitivity of the binding assay. The results corroborated the previous findings, which showed that the peptides bound iDC non-specifically. The failure of the synthetic peptides to bind to iDC might be due to a number of reasons, including loss of cyclised conformation of the peptides during synthesis and insufficient incorporation of sequences that is essential and accountable for the binding, in the synthetic peptides. However, this approach does offer the possibility of identifying cell binding peptides that may allow delivery of genes and other therapeutic or imaging agents.
Supervisor: George, Andrew Sponsor: Kementerian Pengajian Tinggi, Malaysia ; Universiti Sains Malaysia
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.566376  DOI: Not available
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