Testing Optimal Gene Editor for an Alzheimer's CRISPR therapeutic.

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Summary/Abstract: The overall goal of this U01 proposal is to advance a unique CRISPR-based therapeutic for Alzheimer’s disease (AD) towards the pre-IND stage, by determining the best genome-editor/gRNA combination that can be ultimately used in clinical trials. Though there is an enormous unmet need for developing therapeutics for AD, we do not have drugs that can unequivocally slow down the relentless course of AD. We have developed a gene-editing based strategy targeting APP – a gene with a central and indisputable role in AD; strongly supported by human genetics. We do not eliminate the APP gene, but edit out a pentapeptide YENPTY endocytic domain at the C- terminus of APP, which blocks the entire pathologic APP β-cleavage pathway (including β-amyloid). The transmembrane domain and the N-terminus remains intact, and the ∆C APP is retained on the cell surface, leading to an increase in APP α-cleavage, which in turn upregulates neuroprotective and neuroregenerative APP fragments. Thus, our strategy shifts the balance of APP cleavage from pathologic to physiologic, without eliminating the gene. Since the targeted events are upstream, our strategy should be applicable to forms of AD – sporadic and familial – and also to early-onset AD in Down syndrome, where APP triplication on Ch.21 invariably causes AD. So far, we have used the classical SaCas9 to demonstrate safety and efficacy of our approach in vivo, but SaCas9 is too big to fit into a single AAV, which will be necessary for ultimate delivery in humans. Moreover, since there is no FDA-approved gene-editing clinical trial in the brain yet, and the best genome-editor is unknown. Here, we propose to first test two promising small genome-editors (and corresponding gRNAs) that can fit into a single AAV – SaCas9 and NmCas9 – using genome-scale on- and off- target assays (Aim 1). The best Lead-Editor/gRNA combinations emerging from these experiments will be tested for efficacy, safety, predictability, and durability in human brain organoids and a novel APP mouse model where the entire mouse APP was replaced with human APP (Aim 2). Collectively, these studies will not only clarify the best genome-editor/RNA combination for editing the APP gene, but also help future trials in the brain gene- editing space by determining the best editor for brain-relevant applications. Towards the end of our projects, we propose to hold an INTERACT meeting with the FDA – in consultation with our Clinical/Translational Advisory Team – to get regulatory input and consensus on pre-IND NHP studies. Upon completion, our studies will not only offer a novel therapeutic in an arena of enormous unmet need and litany of failures, but also provide a clinical path for future gene-editing efforts in this therapeutic space.