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Targeting the Heart Using in vivo Phage Display

Zahid, Maliha (2010) Targeting the Heart Using in vivo Phage Display. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Abstract

Background: Ischemic heart disease remains the number one killer in the developed world. A protein transduction peptide specific for the heart capable of efficiently delivering agents of therapeutic potential at the time of injury, would be of immense public health significance. This work was undertaken to identify peptide(s) able to transduce heart tissue in vivo in a tissue-specific manner. Biopanning was performed in cell culture followed by in vivo with an M13 phage peptide display library. Using the heart-specific peptide, we delivered nemo-binding domain peptide in a murine infarct model to test if NF-kB inhibition can reduce infarct size.Methods and Results: A cardiomyoblast cell line, H9C2, was incubated with M13 twelve amino acid phage peptide display library. Internalized phage was recovered, amplified and subjected to a total of three rounds of in vivo biopanning where infectious, internalized phage was isolated from cardiac tissue following intravenous injection. After the third round, 60% of sequenced plaques carried the peptide sequence APWHLSSQYSRT, termed cardiac targeting peptide (CTP). This peptide was synthesized either fluorescently labeled, biotinylated, or in combination with a NEMO-binding peptide (NBD), an inhibitor of the inducible NF-kappa B Kinase (IKK). We demonstrate that CTP was able to transduce cardiomyocytes functionally in culture in a concentration and cell-type dependent manner. Mice injected with CTP showed significant transduction of heart tissue with minimal uptake by lung and kidney capillaries, and no uptake in liver, skeletal muscle, spleen or brain. The level of heart transduction by CTP also was not observed with a cationic transduction domain. CTP-NBD was able to inhibit NF-kB activation in cell culture in a dose-dependent fashion. When administered to mice in a murine infarct model, CTP-NBD showed a trend towards smaller infarct size, which did not reach statistical significance. Conclusions: Biopanning using a peptide phage display library identified a peptide, termed CTP, able to transduce cardiomyoblast cell line in vitro, and heart tissue in vivo, efficiently and specifically. Administration of CTP-NBD to post-infarct mice showed a trend towards reduction of infarct size. CTP could be used to deliver therapeutic peptides, proteins and nucleic acid specifically to the heart.


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Details

Item Type: University of Pittsburgh ETD
Status: Unpublished
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Zahid, Malihamaz7@pitt.eduMAZ7
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairRobbins, Paul dprobb@pitt.eduPROBB
Committee MemberMctiernan, Charles Fmctiernanc@upmc.eduMCTIER
Committee MemberFeingold, Eleanorfeingold@pitt.eduFEINGOLD
Committee MemberFerrell, Robertrferrell@pitt.eduRFERRELL
Date: 27 January 2010
Date Type: Completion
Defense Date: 14 October 2009
Approval Date: 27 January 2010
Submission Date: 2 December 2009
Access Restriction: 5 year -- Restrict access to University of Pittsburgh for a period of 5 years.
Institution: University of Pittsburgh
Schools and Programs: Graduate School of Public Health > Human Genetics
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: biopanning; heart; in vivo; Page display; protein transduction domains; transduction
Other ID: http://etd.library.pitt.edu/ETD/available/etd-12022009-155853/, etd-12022009-155853
Date Deposited: 10 Nov 2011 20:07
Last Modified: 19 Dec 2016 14:37
URI: http://d-scholarship.pitt.edu/id/eprint/9964

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