122. Genetic Modification of Target Cells by Direct Delivery of Active Protein

Abstract

Precise modification of the human genome has been a goal of researchers for over two decades. Currently, genome modification is performed by either transient or stable delivery of nucleic acids that encode genome modifying components to target cells. This nucleic-acid-based approach has a number of drawbacks, including low efficiency, toxicity, prolonged expression, off target effects, and potential delay in modification due to transcription and translation post-delivery. An alternative approach is direct intracellular delivery of genome-modifying proteins to live cells. This method allows both the timing and dose of target protein to be tightly controlled, thereby improving efficiency. Consistent with this, it has been reported that direct Cas9 protein delivery leads to higher levels of on-target editing and fewer off-target effects (eLife 2014;10.7554/eLife.04766). Direct protein delivery, however, is limited by the need to express and purify protein in E. coli, where issues with protein yield, proper folding, lack of post-translational modification may ultimately reduce activity. The use of recombinant protein is further complicated by a lack of efficient and consistent delivery methods into target cells of interest. Improved methods, combining mammalian based protein production and efficient packaging into particles into one step can address these shortcomings. Here we report cellular delivery of DNA modifying proteins using VSV-G induced microvesicles (Gesicles). Gesicles are produced by co-overexpression of the spike glycoprotein of VSV-G with a protein of interest (POI), within a mammalian packaging cell. This leads to production of Gesicles containing active amounts of the POI expressed in mammalian cells. Based on this principle, we have developed a method for actively packaging genome modifying proteins into the Gesicles via ligand dependent dimerization. This approach allowed us package a POI containing a nuclear localization signal (NLS) efficiently into this particle. Analysis of the physical properties of these Gesicles demonstrated that they are highly stable over multiple freeze-thaw cycles, are consistent in size, and demonstrate minimal aggregation. Functionally, these Gesicles could efficiently deliver genome modifying proteins to a variety of cells, ultimately leading to genomic alterations. This effect could be demonstrated in over a dozen different cell lines; in all cases, cells maintained high viability and the results closely mimicked those obtained with viral transduction. Taken together, this work suggests that Gesicles can be considered a novel and universal tool for genome modification, providing a direct, rapid, and transient method for delivering active genome modifying proteins to target cells.