ITOP – easy and efficient intracellular delivery of macromolecules

SURE symposium ‘Science Fiction: The impact of scientific research on modern society’

Speaker:     Prof. Dr. Niels Geijsen (Hubrecht Institute Utrecht)
Subject:      ITOP – novel opportunities for safe and efficient gene repair
Location:    Erasmus MC Rotterdam
Date:           Friday, 13.05.2016

Author: Edgar Schönfeld

Introducing a new protein into a cell can be done by transfecting the cells of interest with the appropriate DNA sequence. Direct delivery of protein into cells, however, is more challenging. An example of a currently used protein delivery system are cell-penetrating peptides (CPPs). Currently, it seems that a protein that is fused to a CPP can easily penetrate cell membranes of all types of cells. Yet, such a fusion can negatively influence the protein’s function.
When Prof. Geijsen was thinking about alternative ways of intracellular protein delivery, he found inspiration in the membrane-penetration mechanism of the Anthrax toxin (Fig.1). The Anthrax toxin consists of three proteins: two inducers of apoptosis and one protein (PA83) which binds to the cell membrane and assembles into a simple core complex, allowing the other two proteins to enter the cell.
Fig.1: The 3 Anthrax toxins are Edema Factor (EF), Lethal Factor (LF) and PA83. PA83 binds the Anthrax Toxin Receptor (ATR), reacts with Furin and gives rise to PA63. PA63 forms a heptameric complex that eneables EF and LF to enter the cytoplasm. [1]

Geijsen experimented with the PA protein in an attempt to guide Oct4 into the cytoplasm of cells. After 9 months of ongoing research, however, he was not unamused when he realized that Oct4 also enters the cytoplasm if the PA protein is omitted. Geijsen’s first thought was that he wasted 9 months of research. Then it dawned upon him that the composition of the buffer might have enabled Oct4 to enter the cells. He tested his idea by repeating the experiment several times, each time omitting only one ingredient of the buffer. It became evident that NaCl, as well as a substance named “non-detergent Sulfobetaine-201” (NDSB-201, full name: 3-(1-Pyridino)-1-propane sulfonate), were essential for successful cell membrane penetration. Further investigations revealed that the NaCl-induced hypertonicity triggers macropinocytosis, a process in which cells take up a certain amount of their surrounding fluid, including solute molecules. In this case, NaCl causes the cells to take up Oct4 and NDSB-201, which is encapsulated in intracellular vesicles in the process. NDSB-201 has the ability to dissolve these vesicles, thereby releasing the vesicular contents into the cytoplasm.
Geijsen and colleagues chose the name “iTOP” for their newly developed system, which stands for “induced transduction by osmocytosis and propanebetaine” (Fig.2).
Fig.2: Scheme of the iTOP system. NaCl hypertonicity cause macropinocytosis. Consequently the protein of interest and NDSB-201 are taken up in vesicles. In the cytoplasm, NDSB-201 dissolves the vesicles, which thereby releases the protein of interest.[2]
They went one step further and demonstrated that this system can be employed for super efficient intracellular delivery of the Cas9 protein and its guide RNA. In particular, they targeted the DPH7 gene in a leukemia cell line. If both DPH7 alleles are knocked-out, a cell becomes resistant to diphtheria-toxin-mediated cell death. Geijsen and colleagues found that after 2 rounds of treatment with the diphtheria toxin none of the control cells survived, but 70% of the cells targeted by iTOP-CRISPR/Cas9. Seventy percent are remarkable, given that two alleles have to be knocked out parallelly. Later, it was found that the remaining 30% were also mutated at the DPH7 loci, but the particular mutations did not shield them from DPH7 mediated cell death. For this reason, Geijsen concluded in his talk that he actually achieved a 100% efficiency in targeting the DPH7 gene.
All in all, the iTOP method is simple and efficient and does not seem to disrupt cellular processes. I think it will find wide application in experiments in which abrupt introduction of a protein is more desirable than expressing it via a cloning.



[2] D’Astolfo DS, Pagliero RJ, Pras A, Karthaus WR, Clevers H, Prasad V, Lebbink RJ, Rehmann H, Geijsen N. Efficient intracellular delivery of native proteins. Cell. 2015 Apr 23;161(3):674-90



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