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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.
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Publisher
University College Cork
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Abstract
Cellular delivery of therapeutically valuable macromolecules such as proteins and nucleotides, or genome editing systems such as CRISPR-Cas9, are key to advancing the treatment of many diseases. To make this a therapeutic reality however, the safe delivery of cargo across the cellular membrane remains a key obstacle. Furthermore, the development of molecular tools that target intracellular compartments and tissue specific subtypes is necessary in order to improve the therapeutic efficiency of macromolecules and negate off-target effects. In this context, several viral and non-viral systems have been developed to varying degrees, but there remain problems with immunogenicity, carcinogenesis, toxicity and low in-vivo delivery efficiency (1–3).
Here we describe a protein, termed GEENIE, and identify its novel cell penetration capabilities, demonstrating that it can translocate across the plasma membrane of mammalian cells. We show that GEENIE can penetrate cells in a time dependent manner and can proceed in the presence of serum. Additionally, using a combination of biochemical and pharmacological experiments, we demonstrate that the mechanism of GEENIE uptake is not limited by endocytosis and confirm GEENIE uptake in red blood cells that lack endocytic machinery.
Having demonstrated that GEENIE can cross the plasma membrane, we then extended its application to the delivery of biomolecules. As for other non-viral delivery systems, we first assessed the ability of GEENIE to deliver protein cargo such as fluorescent proteins. Using recombinant DNA technology, we expressed GFP tagged GEENIE chimeras. This strategy allowed for the quick and affordable purification of chimeric proteins, whereafter their cellular uptake was visualized using confocal microscopy. We were able to successfully demonstrate the ability of GEENIE to deliver GFP protein cargo intracellularly.
The delivery of biotherapeutics can often be limited by the off-target effects that cause excessive cytotoxic to healthy, non-diseased cells. The cell specific delivery of therapeutics is therefore an important stipulation for delivery vehicles in order to maximize therapeutic output and reduce off target effects. The development of delivery vehicles that contain a moiety to target and deliver to a specific cell surface receptor is a common strategy to improve cell specific targeting. To this end, we investigated a newly identified peptide that has shown specificity to the HER2 receptor. Incorporating the peptide within GEENIE, allowed for increased cell specificity to HER2 expressing cells (SKBR-3) compared to HER2 negative cells (MDA-MB-468).
Having demonstrated that GEENIE can be targeted to cell specific subtypes, can traverse the cellular membrane and deliver protein cargo, we next evaluated a strategy to deliver biomolecules involved in gene therapies. Gene therapies are an important class of therapeutics that have potential in the treatment of a wide variety of diseases, while also providing an opportunity to progress personalized treatment strategies. Two important groups include nucleic acids, such as siRNA, and gene editing toolkits, such as CRISPR. Employing a genetic engineering approach, we produced two separate GEENIE chimeras, R9-GEENIE and Cas9-GEENIE, to achieve delivery of siRNA or Cas9-sgRNA ribonucleotide protein respectively. Despite our optimization attempts we were unable to produce a functional R9-GEENIE to achieve siRNA delivery and protein knockdown. On the other hand, we demonstrated a potential strategy for the delivery of Cas9-RNPs via GEENIE. Future studies to optimize gene knockout may provide extra insight into whether GEENIE can be used for this strategy, and whether it has clinical potential for the delivery of Cas9.
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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.