# Kinase Multitargeting for Glaucoma Neuroprotection

> **NIH NIH R01** · UNIVERSITY OF CALIFORNIA, SAN DIEGO · 2021 · $383,150

## Abstract

PROJECT SUMMARY – Glaucoma is a neurodegenerative disease in which there is specific loss of retinal ganglion
cells (RGCs). Current therapies center around lowering intraocular pressure (IOP) although this can be
challenging in some patients. In order to advance towards a neuroprotective strategy that could complement
IOP-lowering, we have been identifying potential neuroprotective targets in primary RGCs using high-
throughput functional genomic screening. The first iteration of this work, using RNA interference, identified dual
leucine zipper kinase (DLK) and leucine zipper kinase (LZK) as key mediators of RGC cell death and validated
the biology in rodent models of optic neuropathy, including glaucoma. Since then, we have completed a clustered
regularly-interspaced short palindromic repeat (CRISPR)-based screen in order to identify genes whose
knockout further potentiates the RGC protection conferred by DLK/LZK inhibition. The top new hit in this screen
was glycogen synthase kinase three beta (GSK-3β). Highlighting the utility of our agnostic screening approach,
multiple groups have previously found that while GSK-3β is indeed activated in RGCs after axonal injury, GSK-
3β loss alone does not increase RGC survival. We have shown however, in the setting of DLK/LZK pathway
inhibition, GSK-3β loss does lead to a further increase in RGC survival. Moreover, we found an unexpected
synergy in neurite degeneration with inhibition of DLK/LZK and GSK-3β leading to robust neurite protection.
The central hypothesis of this proposal is that DLK/LZK and GSK-3β cooperate, potentially as a result of their
ability to dually phosphorylate myocyte enhancer factor 2A (MEF2A), to cause somal and axonal degeneration
and that simultaneous inhibition of DLK, LZK and GSK-3β is required for maximal neuroprotection. In order to
test this hypothesis in vivo and to create a generalizable method for gene multitargeting in vivo, we have
developed a novel adeno-associated virus (AAV)/CRISPR vector. This uses a novel insight about the compact H1
promoter which allows both guide RNA (gRNA) and S. pyogenes Cas9 (SpCas9) to be delivered in a single AAV
virus, overcoming a major hurdle in the field of therapeutic gene editing. Specific aim 1 (SA1) will develop
AAV/CRISPR vectors to multitarget DLK/LZK/GSK-3β, validate them in primary RGCs and then use the
resulting cells to explore the role of MEF2A as a key convergence point of GSK-3β and DLK/LZK signaling. SA2
will use AAV/CRISPR vectors in vivo to test whether DLK/LZK/GSK-3β inhibition affects normal retinal
structure/function and whether multitargeting leads to long-term preservation of electrophysiologically-active
RGCs and decreased axon degeneration in the mouse optic nerve crush model. Finally, SA3 will use a more
therapeutically-relevant design, in which the AAV/CRISPR virus delivers all of the CRISPR components, to test
the hypothesis that kinase multitargeting in RGCs improves visual outcomes in a rat glaucoma model. T...

## Key facts

- **NIH application ID:** 10200067
- **Project number:** 5R01EY029342-04
- **Recipient organization:** UNIVERSITY OF CALIFORNIA, SAN DIEGO
- **Principal Investigator:** Derek Stuart Welsbie
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $383,150
- **Award type:** 5
- **Project period:** 2018-09-01 → 2023-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10200067

## Citation

> US National Institutes of Health, RePORTER application 10200067, Kinase Multitargeting for Glaucoma Neuroprotection (5R01EY029342-04). Retrieved via AI Analytics 2026-05-21 from https://api.ai-analytics.org/grant/nih/10200067. Licensed CC0.

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