Intercellular transfer of proteins and RNA mediatedby cell-cell contact

A challenge in genome editing in vivo is to devise an efficientmeans of delivering editing functions, preferably by a vehiclethat evades immune detection. We sought a means to deliverCas۹ and a gRNA enclosed within an natural extracellular vesicleas a vehicle for efficient and targeted gene editing. Cas۹ wasexpressed in a donor cell tethered noncovalently to an integralmembrane protein, CD۶۳, enriched in exosomes. Exosomeshighly enriched in Cas۹ and a gRNA were isolated by buoyantdensity. Isolated exosomes were incubated with reportercells containing an integrated copy of N-luciferase behind asite which when edited would allow the expression of luciferase.In a control experiment, expression of the Cas۹/gRNAcons truct directly in the reporter cell elicited a ۶۰-۷۰ fold increasein luciferase expression. Exosomes containing a similarlevel of Cas۹ elicited no more than a ۵۰% increase above thebackground of luciferase. The same was true of conditionedmedium containing Cas۹-exosomes and even of donor and acceptorcells incubated together separated by a vesicle-permeablemembrane in a transwell chamber. In contras t, donor andacceptor cells cocultured to near confluence showed a ۶۰-foldincrease in luciferase expression. Transfer of Cas۹ appears tobe mediated by open-end membrane tubular connections, likelydependent on membrane fusion at the point of junction betweena tubule from one cell and the target. A molecular dissection forthe requirements for this transfer may permit the developmentof an efficient means for targeted delivery of Cas۹/gRNA