Genome editing in vivo with Cas9 RNP bound to gold nanoparticles

The synthetic modification of proteins is a powerful method for enhancing their cellular uptake, reducing immunogenicity, and targeting their delivery to diseased tissues. However, covalent modification of proteins, particularly Cas9, frequently leads to protein inactivation and there is great interest in the development of degradable linkages that can be used as a traceless modification to retain protein activity. We report the design and synthesis of a bi-functional self-immolative linker utilizing a disulfide bond, which is chemically inert during synthetic transformations, and undergoes intracellular sulfhydryl-assisted cleavage to release the unmodified protein. The linker was composed of a PEG chain to provide water solubility, a 1,7-eliminating disulfide to prevent protein degradation, and a terminal orthopyridyl discovered to enhance intracellular uptake. This work demonstrates that thiol cleavable self-immolative linkers are a new traceless modification strategy to effectively deliver intact protein, and provides a chemical handle for additional cargo installation. Studies to determine the effects on enhanced editing efficiency and conjugation to endosomal disrupting conjugates are underway. Other Cas9 delivery strategies developed by our group, including the use of nanoparticles for both in vitro and in vivo delivery, will also be discussed.