Nr2e3 is a genetic modifier that rescues retinal degeneration and promotes homeostasis in multiple models of retinitis pigmentosa

Sujun Li, Shyamtanu Datta, Emily Brabbit, Zoe Love, Victoria Woytowicz, Kyle Flattery, Jessica Capri, Katie Yao, Siqi Wu, Michael Imboden, Arun Upadhyay, Rasappa Arumugham, Wallace B Thoreson, Margaret M DeAngelis, Neena B Haider, Sujun Li, Shyamtanu Datta, Emily Brabbit, Zoe Love, Victoria Woytowicz, Kyle Flattery, Jessica Capri, Katie Yao, Siqi Wu, Michael Imboden, Arun Upadhyay, Rasappa Arumugham, Wallace B Thoreson, Margaret M DeAngelis, Neena B Haider

Abstract

Recent advances in viral vector engineering, as well as an increased understanding of the cellular and molecular mechanism of retinal diseases, have led to the development of novel gene therapy approaches. Furthermore, ease of accessibility and ocular immune privilege makes the retina an ideal target for gene therapies. In this study, the nuclear hormone receptor gene Nr2e3 was evaluated for efficacy as broad-spectrum therapy to attenuate early to intermediate stages of retinal degeneration in five unique mouse models of retinitis pigmentosa (RP). RP is a group of heterogenic inherited retinal diseases associated with over 150 gene mutations, affecting over 1.5 million individuals worldwide. RP varies in age of onset, severity, and rate of progression. In addition, ~40% of RP patients cannot be genetically diagnosed, confounding the ability to develop personalized RP therapies. Remarkably, Nr2e3 administered therapy resulted in reduced retinal degeneration as observed by increase in photoreceptor cells, improved electroretinogram, and a dramatic molecular reset of key transcription factors and associated gene networks. These therapeutic effects improved retinal homeostasis in diseased tissue. Results of this study provide evidence that Nr2e3 can serve as a broad-spectrum therapy to treat multiple forms of RP.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1. Overexpression of AAV8 -Nr2e3 has…
Fig. 1. Overexpression of AAV8-Nr2e3 has no detrimental effects on the retina.
B6 control AAV8-Nr2e3 treated animals show no abnormalities in a. Fundus, hematoxylin/eosin histology staining, and blue, green, and rhodopsin labeling of photoreceptor cells; and b ERG response of control B6 treated and untreated. Animals injected at P0, tissue collected at P30. c GFP label of AAV8-Nr2e3-GFP injected at P0, GFP expression assessed at P7 and P30. N = 5.
Fig. 2. Expression of AAV8-GFP at P30…
Fig. 2. Expression of AAV8-GFP at P30 in RP models has no effect.
a Immunohistochemistry of AAV8-GFP (rd1, Rho−/−, RhoP23H, rd16, and rd7). All RP models except rd7 have only 0–1 cells in the ONL at P30 and GFP expression is more pronounced in other layers yet has no impact on disease. b Semiquantitative analysis of SV40 (part of AAV8) expression in untreated, AAV8-GFP, and AAV8-Nr2e3 retinas of B6 control and RP models relative to beta-actin. c ERG B-wave amplitudes of uninjected and AAV8-GFP injected RP models and B6 control. Animals injected at P0, tissue evaluated at P30. ERGs recorded at P30. Results are mean ± SEM. N = 7.
Fig. 3. AAV8 -Nr2e3 rescues clinical phenotype…
Fig. 3. AAV8-Nr2e3 rescues clinical phenotype in multiple mouse models of RP.
Fundus of P0 injected AAV8-Nr2e3 treated and untreated animals evaluated at P30 (B6 and rd1) or P90-P120 (Rho−/−, RhoP23H, rd16, and rd7). N = 7.
Fig. 4. AAV8- Nr2e3 treatment preserves retinal…
Fig. 4. AAV8-Nr2e3 treatment preserves retinal morphology and retinal integrity in RP models.
B6, rd1, Rho−/−, RhoP23H, rd16, and rd7 animals injected at P0, evaluated at P30 (B6 and rd1) or P90-P120 (Rho−/−, RhoP23H, rd16, and rd7). a Hematoxylin/eosin staining of AAV8-Nr2e3 treated and untreated retinas with white boxes indicating location of cell count. b Rescued and un-rescued regions in retinas treated with AAV8-Nr2e3. c Cell layer numbers of ONL from AAV8-Nr2e3 treated and untreated animals in different RP models. Results are mean ± SEM. N = 7.
Fig. 5. AAV8- Nr2e3 preserves cone and…
Fig. 5. AAV8-Nr2e3 preserves cone and rod opsin expression in multiple mouse models of RP.
Immunohistochemistry of P0 injected AAV8-Nr2e3 treated and untreated retinas labeled with green opsin, blue opsin and rhodopsin evaluated at P30 (rd1) or P90-P120 (Rho−/−, RhoP23H, rd16, and rd7) and B6 control. N = 7.
Fig. 6. Cell counts of blue and…
Fig. 6. Cell counts of blue and green opsin confirm preservation following AAV8-Nr2e3.
Semiquantitative analysis of cell counts of blue and green opsin-positive photoreceptor cells per 100 μm. Results are mean ± SEM. N = 7.
Fig. 7. Cone Opsin topography improved in…
Fig. 7. Cone Opsin topography improved in AAV8-Nr2e3 RP retinas.
Whole mounts of green opsin and blue opsin were evaluated at 1-month old C57Bl6/J control, as well as 1-month rd1, Rho−/−, RhoP23H, and rd16 animals treated with AAV8-Nr2e3 at P0 and untreated animals. N = 7.
Fig. 8. Improved ERG responses in AAV8-…
Fig. 8. Improved ERG responses in AAV8-Nr2e3 treated RP retinas.
a Scotopic and photopic ERG B-wave amplitudes were evaluated at P30 (rd1) or P90-P120 (Rho−/−, RhoP23H, and rd16) AAV8-Nr2e3 treated and untreated animals; B6 control ERGs shown. b Percent increase in ERG B-wave responses in the treated RP models. Results are mean ± SEM. N = 7.
Fig. 9. AAV8- Nr2e3 treated retinas exhibit…
Fig. 9. AAV8-Nr2e3 treated retinas exhibit differential expression in multiple key homeostasis gene networks.
a–c Differentially expressed genes from Nr2e3-directed networks with ≥1.5-fold variance in Nr2e3 treated vs untreated retinas in rd7, rd1, and Rho−/−, respectively. Networks P phototransduction, S survival, A apoptosis, I immunity, N neuroprotection, O oxidative stress, E ER stress, M metabolic. Potential target genes of Nr2e3 are highlighted. d Potential Nr2e3 targets by Chromatin IP—real time PCR. P0 injected, samples collected at 3 months. Results are mean ± SEM. N = 7.
Fig. 10. AAV8- Nr2e3 rescues RP degeneration…
Fig. 10. AAV8-Nr2e3 rescues RP degeneration by recruiting key transcription factors.
Relative expression levels of Nr2e3, Nrl, Rora, Thrb, Nr1d1, and Crx at P30 Nr2e3 treated mutant strains (rd7, Rho−/−, RhoP23H, and rd16) and rd1 at P7 compared with the corresponding untreated controls and normalized to beta-actin. Results are mean ± SEM. N = 7.
Fig. 11. AAV8 -Nr2e3 rescues RP degeneration…
Fig. 11. AAV8-Nr2e3 rescues RP degeneration after disease onset.
Animals injected with AAV8-Nr2e3 at P21 and evaluated at 2–3 months post injection. a Fundus of Rho−/−, RhoP23H, rd16, and rd7. b Hematoxylin/eosin staining shows partial preservation of photoreceptor cells in treated mutant animals. c Cell layer numbers of outer nuclear layer were compared between AAV8-Nr2e3 treated and untreated animals in the four RP models and B6 control. Results are mean ± SEM. N = 7.
Fig. 12. AAV8- Nr2e3 rescues rod and…
Fig. 12. AAV8-Nr2e3 rescues rod and cone opsin expression after disease onset.
Animals were injected with AAV8-Nr2e3 at P21 and evaluated at 2–3 months after injection. Immunohistochemistry of green opsin, blue opsin and rhodopsin of treated and untreated animals in Rho−/−, RhoP23H, rd16, and rd7.
Fig. 13. Cell counts of blue and…
Fig. 13. Cell counts of blue and green opsin confirm rescue following AAV8-Nr2e3.
Semiquantitative analysis of cell counts of blue and green opsin-positive photoreceptor cells per 50 μm of the retina. Results are mean ± SEM. N = 7.

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Source: PubMed

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