Gene editing holds great promise for many applications such as immunomodulation, cancer treatment and therapy of inherited disorders. However, internalisation of gene-editing proteins poses many challenges due to their intrinsic high molecular weight and polarity that prohibit them from crossing the cell membrane.
The iTOP method for gene-editing protein delivery into cells
The EU-funded ENTRANCE project employed a combination of small molecules to efficiently introduce gene-editing proteins into cells. Project partners called this process ‘iTOP’ for induced transduction by osmocytosis and propanebetaine. iTOP uses salt to cause hyperosmolality and, in combination with the transduction compound propanebetaine, triggers macropinocytotic uptake and intracellular release of extracellularly applied macromolecules. “We were intrigued to discover that iTOP Technology is highly efficient in delivery of gene-editing systems in various cell types. The iTOP technology is based on the principle of ‘Fast in, Fast clearance’, which makes gene editing non-toxic, easy to use and highly efficient, and makes it stand out from other approaches,” explains Marco de Boer CEO and co-founder of NTrans Technologies, the project coordinator. The iTOP Technology was originally developed by the research group of Niels Geijsen at the Hubrecht Institute and Utrecht University. Gene editing technologies aim to modify specific parts of the genome for research or therapeutic purposes. At the same time, they can only be effective and safe when delivered to the right cells and tissues. iTOP delivery of CRISPR/Cas9 takes place in the form of a ribonucleoprotein (RNP) complex, which has two important benefits. The CRISPR/Cas9 complex is immediately active and efficiently introduces the desired modification; at the same time it is broken down after a relatively short time, reducing the risk of off-target effects associated with the use of viral vectors. In addition, delivery in the form of an RNP complex avoids immune problems often observed with viral delivery. NTrans researchers have optimised the composition of iTOP and developed protocols for the most effective transfection of various cell types including cell lines, stem cells and T lymphocytes. They have also tested a range of reagents for the delivery of different molecules. “The ENTRANCE funding has been fundamental to the development of the iTOP Technology,” emphasises de Boer. The platform is ready to be marketed and partners are looking for an established CRISPR tool supplier with international sales. Ongoing efforts focus on the development of a proprietary novel CRISPR endonuclease which, in combination with iTOP, will offer a unique therapeutic platform for immune therapy and genomic applications. NTrans is also taking the next steps towards the clinical development for the treatment of Duchenne Muscular Dystrophy. “Our mission is to translate the unique iTOP Technology into new revolutionary therapies for the treatment of genetic diseases and cancer,” concludes de Boer.
ENTRANCE, iTOP, gene delivery, CRISPR/Cas9