Phage display and experimental brain therapeutics

Doctoral thesis English OPEN
Smith, Mathew Wayne

Phage display, a powerful polypeptide display technology, affords the rapid identification of peptides and proteins that interact with a target of interest The aims of the project were the phage display identification of peptides that interact with a druggable target in a brain disorder (glioblastoma multiforme) and the identification of peptides that serve as targeting vectors for brain delivery. Validation studies were undertaken to qualify the use of a cyclic 7-mer peptide phage library against targets including streptavidin and paracetamol chosen as examples of a large complex and small simple molecule, respectively. With the aim of identifying peptide phages that bind to the luminal surface of brain micro vasculature, a primary in-vitro porcine model of the blood-brain barrier (BBB) comprising primary brain capillary endothelial cells was established and characterised. An in-vivo phage display was undertaken in the rat with the aim of identifying peptide sequences that mediated translocation across the BBB into brain grey matter. A 7-mer cyclic peptide was identified with sequence AC-SYTSSTM-CGGGS that enhanced the uptake of phages into brain grey matter by 4-fold compared to control wild-type phages. This peptide may serve as a novel targeting vector for the delivery of a therapeutic cargo to the brain. Caveolin-1 was identified as a potential new therapeutic target in in-vitro models of grade IV astrocytomas (glioblastoma multiforme), with siRNA knockdown of caveolin-1 associated with reduced glioma cell proliferation and invasiveness. With the caveolin-1 scaffolding domain (aa 81-101 in the caveolin-1 protein) as a target, an in-vitro peptide phage selection was undertaken and identified a series of peptides that bind the scaffolding domain with high affinity. These peptides will serve as a template for the development of low molecular weight peptidomimetics that inhibit caveolin-1 function. In conclusion, the studies in this thesis have demonstrated the utility of phage display in experimental therapeutics of brain disorders.
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    Chapter 1 Phage display and experim ental brain therapeutics ___ 1 1.1 CNSdisorders------------------------------------------------------------------------------ 2 12 Astrocytomas-------------------------------------------------------------------------------3 13 TheBlood-Brain barrier_______________________________________ 6 1.4 Caveolaeand caveolin________________________________________ 7 1.5 Polypeptide display systems___________________________ .________ 9 1.5.1 Phage Display__________________________________________ 10 1.6 Phage Biology---------------------------------------------------------------------------- 11 1.6.1 Phage Structure------------------------------------------------------------------- 11 1.6.2 Phage Genome..................... 14 1.7 Phage Lifetyde--------------------------- 16 1.7.1 Infection.......................................... 16 1.7.3 Assembly______________________________________________19

    1.8 Coatproteins usedfo r display__________________________________ 21 1.8.1 p ill and pVIII display ................................... 22

    1.9 Wild-typephage vectorM13KE -------------- 24

    1.10 Applications o fphage display._________________________________25 1.10.1 Applications of phage display in disease therapeutics----------------- 26 1.10.2 Applications of phage display in drug delivery-------------------------- 28 1.10.3 Phage therapy---------------- 32

    1.11 Scopeo fthesis____________________________________________ 35

    Chapter 2 Phage display method developm ent -------------------------49 2.1 Introduction_________ ._____________________________________ 50 2.1.1 General principles of phage display panning studies-------------------- 50 2.1.2 Affinity selection-------------------- 52 2.1.3 Target presentation______________________________________53 2.13.1 Elution____________________________________________________55 2.1.4 Technology validation------------- 56 2.1.4.1 Streptavidin_______________________________________________ 56 2.1.42 4-AAP____________________________________________________ 57 2.1.5 Objectives..................... 59 22 Materials & Methods - Phage Display------------------- 60 2.2.1 Materials........................ 60 2.2.2 Phage library__________________________________________60 2.2.3 Phage display media and solutions---------------------------------- ------ 61 2.2.4 Maintenance of the bacterial host used for phage propagation..........62 2.2.5 Phage amplification and purification--------------------------------------- 62 2.2.6 Phage plaque formation assay......................... 63 2.2.7 Gene sequencing ........................................................................... 64 2.2.7.1 Phage plaque amplification and DNA extraction ........................... 64 22.72 PCR_______________________________________________ 65 2.2.8 Statistical analysis............................................. 66

    23 Streptavidin Panning________________________________________ 66 2.3.1 Materials & Methods................................. 66

    3.5 Discussion......................... 161

    43 Results:______________________________________ _______ ___203 4.3.1 Analysis of in-vivo phage recovery and peptide sequences_____ 203 4.3.2 Phage amplification errors______________________________ 214 4.3.3 Ex-vivo stability of SYTSSTM-M13 and insertless-M13________ 215 4.3.4 Effects of perfusion on phage numbers____________________ 216 4.3.5 Fifteen minute brain uptake o fselected peptide-phage clones____ 217 4.3.6 In-vivo tissue distribution o fSYTSSTM-M13__________________218 4.3.7 Pharmacokinetic simulations o fbrain uptake________________ 226

    4.4 rilSCUSSMm________________________ ------------------------------------- 233

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