Building precision disease-responsive gene therapy

Project summary Achieving precise spatiotemporal control of gene expression is critical to maximize the delivery of in vivo gene therapy. Adenoviral-associated viruses (AAVs) drive sustained gene expression, particularly in post- mitotic tissues like the neural retina. However, these systems lack built-in mechanisms to modulate or halt gene expression post-delivery, leading to uncontrolled and long-term expression. To address these limitations, we propose to develop “smart” gene therapies that utilize injury-specific regulatory elements to enable therapeutic gene expression to dynamically adapt to disease conditions in real-time. Central to our approach is harnessing the endoplasmic reticulum (ER) stress response, a pathway that is implicated in various ophthalmic diseases. Furthermore, the application of in vivo gene editing technologies such as CRISPR/Cas systems presents unique challenges. Traditional Cas9 systems are limited in their ability to target multiple genomic sites simultaneously. In contrast, the newer Cas12a system, which processes its own poly-CRISPR RNA (crRNA) arrays, offers significant advantages for multiplexing. We recently developed a hyper-efficient Cas12a variant ("hyperCas12a") that enables multiplexed genome regulation and excels in inducible systems. We propose to use this platform to drive CRISPR activation (CRISPRa) and CRISPR inhibition (CRISPRi) in vivo in RGCs. Our objectives are two-fold. First, we aim to harness ER stress-responsive promoters to achieve injury- inducible gene expression and neuroprotection, perform rational design to optimize these promoters for precise gene regulation, and utilize pharmacologic inhibitors for post-delivery control of expression. Second, we will develop a CRISPR multiplexing platform using a dual-AAV system to deliver hyper-efficient Cas12a for the activation of endogenous neuroprotective pathways, alongside multiplexed CRISPR inhibition to promote optic nerve regeneration. These strategies address critical unmet needs in treatment of ophthalmic disease and hold potential for advancing gene therapy across multiple therapeutic areas.