Development of functionalized SARM1 nanobodies for glaucoma

About This Grant

PROJECT SUMMARY Glaucoma is a neurodegenerative disease defined by the injury of retinal ganglion cell (RGC) axons at the optic nerve head, leading to axon degeneration, cell death and irreversible vision loss. There has been a longstanding need to develop neuroprotective therapies that could complement intraocular pressure-lowering therapies as well as biomarkers to assess disease activity and the need for escalated treatment. Sterile alpha and TIR motif containing 1 (SARM1) is a key mediator of the active genetic program underlying axon degeneration in glaucoma and other neurodegenerative disease. This makes SARM1 a potential neuroprotective drug target as well as an indicator of disease activity. While small molecule inhibitors and sensors are being developed for SARM1, use in the eye will require highly optimized formulations and pharmacokinetics and will not have systemic availability. In contrast, genetically-encoded SARM1 inhibitors and biosensors can be robust, readily delivered with adeno- associated virus (AAV) and used across a wide range of animal models. Moreover, they themselves can be the basis for future clinical diagnostics and therapeutics. To develop genetically-encoded SARM1 inhibitors and biosensors, we propose to use nanobodies (Nbs). These small, single chain antibodies from camelids can have high antigen affinity and, because of a protruding complementarity determining region 3 (CDR3), are particularly suited to inhibit the active sites of enzymes like SARM1. Moreover, they are modular, allowing one to add protein domains that can degrade or image target antigens. We have developed a novel bacterial display platform capable of identifying high affinity Nbs and shown that we can find Nbs against purified human SARM1 protein. In Specific Aim 1, we use molecular evolution, including a novel approach based on genetic algorithms, and structural determination to optimize Nb binding. Then, in Specific Aim 2, we use a novel mammalian cell platform in which cell survival is coupled to SARM1 activity in order to evolve Nbs with intrinsic neutralizing ability. As a complementary approach, we will functionalize the Nbs without neutralizing ability using the substrate recognition module of E3 ligases in order to retarget them to degrade SARM1. We will then validate the strategy in human stem cell-derived RGCs as well as a rat model of glaucoma. Finally, in Specific Aim 3, we will take advantage of the in vitro nature of the Nb screen in order to develop a conformational biosensor of SARM1 activity. This will be tested in a rodent model of glaucoma. We expect that the aims proposed here will culminate in the development of robust genetically-encoded inhibitors and biosensors of SARM1 activity. In order to successfully achieve this in a five-year timeframe, we have brought together two PIs with expertise in glaucoma neuroprotection, nanobodies and molecular evolution and included additional expertise in AAV production, retinal organoids and live animal imaging.