The ability to target abnormal genes by introducing functional copies through gene therapy holds extreme promise for the treatment of human diseases. Since the first human therapeutic gene transfer in 1990, gene therapies have been efficacious in treating diseases like hemophilia and leukemia, and this list is continuously growing. Importantly, gene therapies reflect a shift in modern medicine from the treatment of symptoms to the permanent correction of underlying genetic causes of disease.
As not all diseases are created equal, gene replacement therapies are harder to implement if the genetic mutations resulting in pathologies are unclear. Recent research published by fifth year GTV student Devin McDougald aims to develop a broad-spectrum approach to gene therapy by targeting conserved pathways in disease pathogenesis. As a member of Dr. Jean Bennett’s laboratory in the Department of Ophthalmology, Devin investigates canonical neurodegenerative mechanisms for novel gene therapies in ocular diseases such as optic neuritis.
Visual impairment due to optic neuritis often manifests in patients with Multiple Sclerosis (MS), which can lead to permanent visual dysfunction. In some cases, irreversible loss of vision is caused by the degeneration of retinal ganglion cells, or RGCs- neurons that are important for accurate visual processing. Working with collaborators in Dr. Ken Shindler’s lab at Penn, Devin and colleagues set out to promote neuroprotection in optic neuritis using a powerful mouse model for studying MS pathogenesis. Similar to MS patients, experimental autoimmune (EAE) mice lose RGCs and suffer from visual impairments with age. It is hypothesized that oxidative injury from the accumulation of harmful reactive oxygen species (ROS) are integral to MS pathology and optic neuritis. With this in mind, Devin hypothesized that they could promote neuroprotection by reducing oxidative injury and enhancing redox homeostasis.
Two likely candidate genes to target, NRF2 and SIRT1, both have antioxidant properties and play integral roles in promoting cell survival. Notably, Nrf2-/- mice lose RGCs and are visually impaired, while SIRT1 overexpression promotes the survival of RGCs. To examine their neuroprotective roles in optic neuritis, Devin successfully created AAV2 expression plasmids containing human NRF2 or SIRT1 under a ubiquitous promoter. AAV2-NRF2 or AAV2-SIRT1 were transduced into cell lines to examine transgene expression and cellular localization. After confirming successful RNA and protein upregulation in transduced cell lines, AAV2-NRF2 or AAV2-SIRT1 were transduced into the retinas of 4-week postnatal C57Bl/6J mice via intravitreal delivery, with an approximately 20% RGC transduction rate for both expression vectors. Four weeks after transduction, mice were immunized with MOG to induce EAE, which begins to develop within two weeks of immunization.
To determine if NRF2 or SIRT1 gene transfer can preserve visual function in optic neuritis, transduced EAE mice were evaluated using Optokinetic Response Recordings (OKR) compared to AAV2-eGFP transduced EAE animals. This eye tracking software measures functional response of the eye, with a decline in OKR score denoting a decrease in responsiveness of the eye. Intriguingly, EAE eyes treated with AAV2-SIRT1 but not AAV2-NRF2 showed less decline in visual function, suggesting that SIRT1 can preserve functionality of the eye during EAE-mediated optic neuritis.
Because the decline in vision during optic neuritis is associated with the loss of RGCs, Devin and colleagues evaluated the ability of NRF2 or SIRT1 gene augmentation to improve RGC survival during EAE. Retinal treatment with AAV2-NRF2 showed a statistically significant increase in the number of intact RGCs, suggesting that NRF2 promotes RGC survival. While AAV2-SIRT1 also trended towards an increase in RGC density, it was not statistically significant. Finally, the effects of gene transfer on optic nerve inflammation and demyelination, two hallmarks of autoimmune-mediated lesions in MS contributing to visual decline, were determined in EAE mice transduced with NRF2, SIRT1, or eGFP. In optic nerve sections, no differences in immune cell recruitment or nerve demyelination were observed, suggesting that gene transfer does not preserve visual function through these pathways.
Optic neuritis results in a loss of retinal ganglion cells (RGCs), resulting in irreversible vision loss. Intravitreal injection of AAV2-NRF2 or AAV2-SIRT1 demonstrates neuroprotective effects in experimental autoimmune encephalitis (EAE) mice, where NRF2 promotes RGC survival and proliferation while SIRT2 prevents further loss of vision.
Taken together, Devin and colleagues successfully harnessed the neuroprotective effects of NRF2 and SIRT1 in optic neuritis using their novel gene augmentation strategy. Importantly, this study has set a framework for other experiments in the lab. Devin says, “we are actively pursuing ways to optimize the approaches outlined in the paper as well as applying them to other pre-clinical models of optic neuropathy in collaboration with other Penn investigators. We are particularly interested in the SIRT1 approach because it was able to show some degree of functional preservation in the EAE model.” He also notes that “one of the major limitations in the study surround[s] the very low transduction efficiency of our AAV2 vectors, which likely played a role in observing such small effects on phenotype rescue.” To enhance targeting efficiency, Devin has generated a new panel of AAVs that target RGCs. With these modifications, he hopes to increase the demonstrated neuroprotective effects of targeting oxidative injury and redox homeostasis.
McDougald DS, Dine KE, Zezulin AU, Bennett J, Shindler KS. SIRT1 and NRF2 gene transfer mediate distinct neuroprotective effects upon retinal ganglion cell survival and function in experimental optic neuritis. Invest Opthalmol Vis Sci. 2018, 59: 1212-1220. .