Enabling the immune system to attack tumors
The aim of this project is to develop methodologies for robust, and safe gene editing in human immune cells, particularly T‐cells. The results of the project provide a valuable toolset for researchers around the world to perform gene targeting in a wide variety of cells, enabling the development of personalised gene therapies and specific anti‐tumor immune therapies.
Most primary cell types have developed strong defensive mechanisms that prevent efficient manipulation of their genome. A proprietary technology called induced Transduction by Osmocytosis and Propanebetaine (iTOP) has been developed, a unique method for the intracellular delivery of (large) bioactive molecules that is conceptually very different from other methods. iTOP is vastly superior in its ability to transduce primary cells, its unparalleled high efficiency of cell transduction, the narrow control over dosage and timing of the delivered protein and the nonintegrating nature of protein manipulation, improving safety and minimizing off‐target effects.
iTOP technology was reported in a publication in the prestigious journal Cell last year (D’Astolfo et al., 2015 Cell, 161:674‐690) The advantages of iTOP gene editing were quickly recognized by the field and recently highlighted in an opinion article in the leading journal Nature Methods (Nature Methods 12, 602 (2015) doi:10.1038/nmeth.3464). The iTOP transduction technology developed in the Hubrecht Institute provides an effective backdoor into these cells, and allows efficient intracellular delivery of recombinant proteins into virtually any cell type. However, the iTOP reagent needs to be optimised and validated for the transduction of specific, clinically relevant cell types such as T‐cells.
An iTOP transduction of freshly isolated T‐cells has been performed as well as T‐cells that were cultured for a period of 1‐2 weeks prior to transduction. The goal was to transduce cells and achieve a knockout efficiency of about 20%. It is now possible to achieve 40‐50% knockout, thus exceeding the goals. In addition, new methods have been developed for the transduction of long DNA oligonucleotides into cells and are optimising this technology on primary T‐cells. In addition, shelf life will be tested continously and storage temperature of the reagents, etc. with the ultimate goal to develop an of‐the ‐shelf research tool (‘kit’) for the genetic manipulation of T‐cells.