PROJECT SUMMARY / ABSTRACT The experiments in this proposal focus on developing a therapy for Huntington's disease based upon Cas9 RNP formulations that are complexed to amphiphilic delivery peptides (ADPs) and conjugated to PEG. We have chosen Huntington's disease as a disease target because it is a fatal disease with no treatment, caused by a well-defined genetic mutation. CRISPR-based genome editing holds tremendous potential for treating Huntington's disease but has been challenging to develop because of delivery limitations. Two key challenges need to be solved before CRISPR-based genome editing can be used clinically. First, strategies for efficiently and safely delivering Cas9 and gRNA into neurons, after an intracranial injection, need to be developed. Second, strategies that can enable large volumes of brain tissue (>1 cm) to be transfected after an intracranial injection of CRISPR reagents must also be developed. This is particularly important for genome editing in large animals because charged macromolecules, such as Cas9 enzymes or viruses, typically only diffuse 1–2 mm away from the injection site after an intracranial injection. We have recently developed a new strategy for performing genome editing in the brain using a combination of convection-enhanced delivery (CED) combined with the Cas9 RNP complexed to new rationally designed amphiphilic delivery peptides (ADPs). This new strategy for Cas9 RNP delivery has shown remarkable promise, and was able to edit as much as 40% of the neurons in the vicinity of the injection site, after an intracranial injection, using the Ai9 mouse model. In addition, we have also demonstrated that conjugation of PEG to the Cas9 RNP dramatically enhances its distribution through brain tissue. Our Cas9 RNP + ADP formulations have much higher brain transfection ability than other strategies based upon just the Cas9 RNP fused to ADPs or nanoparticle based delivery methods. In addition, our formulation is based upon the Cas9 RNP, PEG and a well- defined peptide, and is anticipated to be significantly easier to manufacture than viral or nanoparticle-based formulations, as all the components – protein, gRNA, PEG, and peptide – can readily be produced under GMP conditions and have a robust clinical track record. In this proposal, we will build upon the momentum of our ongoing studies and will develop a Cas9 RNP formulation that can edit >50% of the mutant Huntington's gene after a single intracranial injection, administered via CED, using an RNP formulation that has the manufacturing and toxicology properties needed for performing IND-enabling studies. We propose the following aims: Specific Aim 1: Develop RNP monoparticles that efficiently transfect large volumes of brain tissue. Specific Aim 2. Rescue mice and rats from HD via editing of HTT using Cas9 RNP monoparticles. Specific Aim 3. Safety and process development of Cas9 RNP monoparticles.