Understanding the biological barriers to Jurkat Transfection using non-viral peptides

  • Yixuan Shen

Student thesis: Masters ThesisMaster of Philosophy

Abstract

The overall objective of this thesis is to examine the feasibility of RALA peptide to manufacture CAR-T cells.

As primary T-cells are hard to transfect, the key barriers to the transfection of primary T-cells should be explored for further rational designs of transfection agents. The low level of heparin sulfate proteoglycans on the cell surface of Jurkat and primary T-cells weakens the cell entry of cationic nanoparticles. Besides, eliminated endosomal acidification in Primary T-cells limits the endosomal escape of transfection agents based on pH sensitiveness.

Next, RALA and RALA-Ks variants were designed and characterised for overcoming identified barriers to T-cell transfection. However, the changes in ellipticities showed RALA-Ks peptides followed an opposite trend in pH-sensitiveness compared to the RALA peptide, which were not suitable for T-cell transfections. Peptide simulations by I-Tasser suggested that salt bridges between glutamic acid and cationic residues may determine the pH sensitiveness of RALA series peptides.

Although RALA peptide has got 25-30% transfection efficiencies in Jurkat cell transfection, such a low rate and an additional pH stability assay suggest it is not rational to extend the RALA system to the transfection of primary T-cells.

Afterwards, the mechanism of the cell entry of RALA/pEGFP-N1 nanoparticles was identified by investigating the main receptors which may be responsible for cellular uptake via bioinformatics. In silico data showed there were correlations between transfection efficiencies (RALA) and two kinds of membrane proteins, syndecan-4 & glypican-1. This hypothesis was verified by transfecting 5 breast cancer cell lines with different levels of syndecan-4 and glypican-1. Results from transfections and RT-PCR illustrated the correlations again.

Finally, an overall summary and the prospects for the future work of investigation of the transduction uptake of arginine rich peptides were proposed, aiming to improve the cell viability and the transcriptional stability of RALA mediated transfections.
Date of AwardJul 2022
Original languageEnglish
Awarding Institution
  • Queen's University Belfast
SupervisorHelen McCarthy (Supervisor) & Niamh Buckley (Supervisor)

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