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Investigation of a novel cell penetrating protein for the delivery of biomolecules
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Date
2024
Authors
Cantillon, Emer A.
Journal Title
Journal ISSN
Volume Title
Publisher
University College Cork
Published Version
Abstract
This study introduces a newly characterized protein with the ability to cross mammalian cell membranes efficiently and safely, addressing a major challenge in delivering therapeutic macromolecules such as proteins, nucleic acids, and genome‑editing tools. The protein penetrates cells in a time‑dependent manner and remains effective in the presence of serum. Mechanistic experiments show that cellular entry does not rely on endocytosis, a finding supported by successful uptake in red blood cells, which lack endocytic pathways.
Building on this capability, the protein was engineered to deliver biomolecular cargo. Fusion constructs carrying fluorescent protein tags were produced using recombinant DNA methods, enabling rapid purification and visualization of intracellular delivery via confocal microscopy. These experiments confirmed the protein’s capacity to transport protein cargo directly into cells.
To improve therapeutic specificity and reduce off‑target cytotoxicity, the system was further modified to incorporate a peptide targeting the HER2 receptor. This enhanced uptake in HER2‑positive cells compared with HER2‑negative controls, demonstrating potential for cell‑type‑specific delivery.
Finally, the protein was adapted for gene therapy applications by generating variants intended to transport siRNA or CRISPR–Cas9 ribonucleoprotein complexes. While siRNA delivery was unsuccessful, the CRISPR delivery construct showed promising activity, warranting further optimization to assess its viability for therapeutic genome editing.
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Keywords
Cell penetrating protein , Intracellular delivery , Biomolecule delivery
Citation
Cantillon, E. A. 2024. Investigation of a novel cell penetrating protein for the delivery of biomolecules. PhD Thesis, University College Cork.