CRISPR/Cas9-based therapeutics, especially those that can correct gene mutations via homology directed repair (HDR), have the potential to revolutionize the treatment of genetic diseases. However, HDR-based therapeutics are challenging to develop because they require simultaneous in vivo delivery of Cas9 protein, guide RNA and donor DNA. Here, we demonstrate that a delivery vehicle composed of gold nanoparticles conjugated to DNA and complexed with cationic endosomal disruptive polymers can deliver Cas9 ribonucleoprotein and donor DNA into a wide variety of cell types, and efficiently correct the DNA mutation that causes Duchenne muscular dystrophy in mice via local injection, with minimal off-target DNA damage.

Protein therapeutics based on transcription factors, gene editing enzymes, signaling proteins and protein antigens, have the potential to provide cures for a wide number of untreatable diseases, but cannot be developed into therapeutics due to challenges in delivering them into the cytoplasm. There is therefore great interest in developing strategies that can enable proteins to enter the cytoplasm of cells. In this review article we will discuss recent progress in intracellular protein therapeutics, which are focused on the following four classes of therapeutics, Firstly, vaccine development, secondly, transcription factor therapies, thirdly, gene editing and finally, cancer therapeutics. These exciting new advances raise the prospect of developing cures for several un-treatable diseases.

Therapeutics based on transcription factors have the potential to revolutionize medicine but have had limited clinical success due to delivery problems1–4. The delivery of transcription factors is challenging because it requires developing a delivery vehicle that can complex transcription factors, target cells, and stimulate endosomal disruption, with minimal toxicity5,6. In this report we present a novel multifunctional oligonucleotide, termed DARTs (DNA Assembled Recombinant Transcription factors), which can deliver transcription factors with high efficiency in vivo. DARTs are composed of an oligonucleotide that contains a transcription factor binding sequence and hydrophobic membrane disruptive chains that are masked by acid cleavable galactose residues. DARTs have a unique molecular architecture, which allows them to bind transcription factors, trigger endocytosis in hepatocytes, and stimulate endosomal disruption. The DARTs target hepatocytes as a result of the galactose residues and can disrupt endosomes efficiently with minimal toxicity, because unmasking of their hydrophobic domains selectively occurs in the acidic environment of the endosome. We show here that DARTs can deliver the transcription factor Nuclear erythroid 2-related factor 2 (Nrf2) to the liver, catalyze the transcription of Nrf2 downstream genes, and rescue mice from acetaminophen induced liver injury.