Brain-Wide Genome Editing Enabled by Intravenously Administered Non-Viral Nanovectors As a Potential Therapy for Alzheimer’s Disease

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Project Summary Currently, there is no effective way to slow down the progress of Alzheimer’s disease (AD) or prevent it. CRISPR genome editing is a revolutionary and versatile genetic engineering technique, making it possible to treat the root causes of genetic neurodegenerative diseases (NDDs) such as AD. However, the promise of brain gene therapy relies on the efficient delivery of biologics to the brain, which is extremely challenging due to the blood- brain barrier (BBB). To date, in vivo brain gene therapy has mostly been achieved using viral vectors that require laborious customization and have troublesome safety profiles. Non-viral vectors are largely administered via intracranial administration, which is invasive and can only enable gene therapy in a small and localized brain region. Similar to other NDDs, AD affects multiple brain regions. Thus, there is an urgent need to develop efficient non-viral delivery vehicles capable of bypassing the BBB for safe and efficient brain-wide gene therapy. The objectives of this project are (1) to engineer glutathione (GSH)-responsive silica nanocapsules (SNCs) capable of bypassing the BBB and delivering CRISPR genome editors to the whole brain systemically, and (2) to evaluate the therapeutic efficacy and biosafety of brain-wide genome editing enabled by the optimized SNC for the treatment of AD using a novel amyloid precursor protein (APP) knock-in AD mouse model and a unique gene target for APP modulation. The unique SNC possess a long list of desirable properties including versatile payload types, versatile surface chemistry for ligand conjugation, high payload loading content and efficiency, small particle sizes, excellent in vivo stability, good biocompatibility, and scalable production. Our preliminary data has shown that intravenously administered SNCs can efficiently deliver mRNA, DNA, Cas9 mRNA/sgRNA, and Cas9/gRNA ribonucleoprotein (RNP) to the whole brain of healthy mice with intact BBB. We aim to further optimize the amounts of the dual brain-targeting ligands (i.e., glucose and rabies virus glycoprotein (RVG) peptide) and dosages of the SNCs for enhanced brain-wide systemic delivery of two types of CRISPR genome editors (i.e., (1) Cas9 mRNA/sgRNA, and (2) plasmid DNA with a neuron-specific human synapsin 1 (SYN1) promoter and expressing both Cas9 and sgRNA). We will further determine their therapeutic efficacy and biosafety in treating AD using a novel APP knock-in AD mouse model while employing a unique gene target for APP modulation. The gene editing efficiency and biosafety profiles of the SNC, and the pathological and behavior changes of the AD mice will be monitored by various techniques including a novel serial two-photon tomography whole-brain amyloid imaging platform. With promising initial studies, the best- performing SNC will be submitted to the Preclinical Testing Core of the NIA-sponsored STOP-AD program for comprehensive preclinical evaluation. This project will pave the road for a new, safe, non-invasive and effective therapeutic approach for familial AD. Given the modularity and versatility of the SNCs, and ease of targeting different genes by the CRISPR system, we anticipate that our SNCs will be applicable for a wide range of NDDs.