Project Summary: CRISPR/Cas9 is a revolutionary and versatile genome editing technique with wide-ranging utility. In vivo genome editing is anticipated to be the next wave of therapeutics for various major health threats, including neurode- generative diseases. However, there is an urgent need to develop efficient, non-viral delivery vehicles for safe and efficient in vivo CRISPR genome editing. Furthermore, delivering CRISPR genome editing machinery to the brain/neuron represents a major hurdle due to its dense structure and the blood–brain barrier (BBB). The objective of this project is to engineer a family of versatile, novel, non-viral Cas9-gRNA ribonucleoprotein (RNP) delivery nanocapsules (NCs) that can robustly and safely generate targeted gene edits in neurons within the brain. We envision that our robust and universal RNP delivery nanoplatforms will enable innovative treatments for devastating neurodegenerative diseases. Towards this goal, we will evaluate the feasibility of our approach, in a demonstration, by targeting the amyloid precursor protein (APP) – relevant to Alzheimer's disease (AD) in healthy mice and monkey models. During our preliminary studies, we developed a PEGylated NC with a high RNP loading content (68 wt.%), versatile surface chemistry, ultrasmall size (dH~13 nm), controllable stoichiometry, excellent biocompatibility, and high genome editing efficiency in vitro and in vivo. In UG3 Aim 1, we will further optimize the design of the NC for brain/neuron-targeted genome editing. In particular, we will investigate the synergistic effects of hybrid targeting ligands, including (1) glucose+RVG peptide for intravenous (i.v.) injection to enhance the crossing of the BBB and neuron-specific editing, and (2) CPP+RVG peptide for intracerebral injection to enhance uptake and neuron-specific genome editing. The effects of different types/amounts of targeting ligands on the cellular uptake, biocompatibility, genome editing efficiency, and functional consequences of the NCs in both Neuro2a and primary neuron cells will be investigated. In UG3 Aim 2, we will evaluate the brain/neuron targeting specificity, genome editing efficiency, and potential immune response and systemic toxicity of the i.v. or intracerebrally administered NCs conjugated with various targeting ligands in healthy mice. In UH3 Aim 1, we will develop the set up and synthesis process to scale up the production of NCs. In UH3 Aim 2, we will further evaluate the genome editing efficiency and biocompatibility of the brain/neuron-targeted NCs in healthy rhesus macaques. Our uniquely designed NCs are expected to achieve high brain accumulation, high penetration depth, and high neuron-specific genome editing efficiency due to their desirable characteristics. Given the modularity and ease of targeting different genes by the CRISPR system, we anticipate that the resulting NCs will be useful for a wide range of human diseases, including debilitating neurodegenerative diseases for which there are no cures.

Project Narrative: In vivo genome editing using CRISPR/Cas9 is anticipated to be the next wave of therapeutics for various major health threats, including neurodegenerative diseases. However, to date, very few Cas9-gRNA ribonucleoprotein in vivo delivery methods have been reported, and delivery to the brain has been particularly challenging. The unique nanocapsules we plan to develop will ultimately enable high efficiency neuron-targeted genome editing in the brain, thereby offering new hope to treat devastating neurodegenerative diseases.