Nanoformulated CRISPR Ribonucleoproteins for Ultrasound-Facilitated Brain Gene Editing

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Nanoformulated CRISPR Ribonucleoproteins for Ultrasound-facilitated Brain Gene Editing Abstract Emerging CRISPR technologies provide new opportunities to advance gene therapy in treating many intractable genetic diseases, including neuronal degeneration disorders. Given the failures of clinical trials in treating Alzheimer's disease through directly targeting amyloid β and tau, there is an unmet need to develop a different strategy in this space, and gene editing technologies may be of great potential. However, one key barrier in developing CRISPR therapeutics is the brain delivery of CRISPR components. Viral vectors could be effective, but the use of these vectors could potentially raise the concerns in immunogenicity and toxicity, which may lead to severe adverse effects. Conventional nonviral systems, in contrast, could be safer but significantly less effective, possibly due to the suboptimal size, which limits their transport to the target brain region. In light of these challenges, we propose to explore the feasibility of screening more transport-favorable, effective nonviral carriers for brain gene editing to tackle Alzheimer's disease. Different from the conventional nanoparticle designs, we will first create a large nanoformulated CRISPR/Cas9 ribonucleoprotein library through split-and-pool lipid coating and optimize the focused ultrasound (FUS)-mediated blood-brain barrier opening to screen all the possible lipid compositions (Aim 1). Compared with the conventional nanoparticle formulations, direct lipid coating may generate smaller and more transport-favorable “nano editors.” By barcoding each lipid in each split- and-pool round, all the nanoformulated Cas9 ribonucleoproteins can be screened directly in the same animal, which minimizes the variations from animals and operations. Our preliminary studies with a small set of nanoformulations in different models have demonstrated the feasibility and reproducibility of our screening approach. Once having the most potent lipid composition, we will validate its gene editing performance and therapeutic efficacy in both reporter and Alzheimer’s mouse models (Aim 2). Our previous efforts in developing FUS delivery for viral brain gene editing have helped us established the capability and all the pipelines needed for editing performance validations. In this proposed research, we aim to expand the CRISPR delivery toolkits from viral to nonviral systems and to explore the potential of nonviral CRISPR gene editing for treating Alzheimer’s disease. The discoveries and findings will help us gain enough supports for larger, potentially IND- enabling studies.