Ocular stress enhances contralateral transfer of lenadogene nolparvovec gene therapy through astrocyte networks

Abstract

Lenadogene nolparvovec (GS010) was developed to treat a point mutation in mitochondrial ND4 that causes Leber hereditary optic neuropathy. GS010 delivers human cDNA encoding wild-type ND4 packaged into an rAAV2/2 vector that transduces retinal ganglion cells, to induce allotopic expression of hybrid mitochondrial ND4. GS010 clinical trials improved best-corrected visual acuity (BCVA) up to 5 years after treatment. Interestingly, unilateral treatment improved BCVA bilaterally. Subsequent studies revealed GS010 DNA in visual tissues contralateral to the injected eye, suggesting migration. Here we tested whether unilateral intraocular pressure (IOP) elevation could influence the transfer of viral ND4 RNA in contralateral tissues after GS010 delivery to the IOP-elevated eye and probed a potential mechanism mediating translocation in mice. We found IOP elevation enhanced viral ND4 RNA transcripts in contralateral visual tissues, including retinas. Using conditional transgenic mice, we depleted astrocytic gap junction connexin 43 (Cx43), required for distant redistribution of metabolic resources between astrocytes during stress. After unilateral IOP elevation and GS010 injection, Cx43 knockdown eradicated ND4 RNA transcript detection in contralateral retinal tissues, while transcript was still detectable in optic nerves. Overall, our study indicates long-range migration of GS010 product to contralateral visual tissues is enhanced by Cx43-linked astrocyte networks.

Keywords: Astrocytes; Connexin 43; Intraocular pressure; Leber hereditary optic neuropathy; Lenadogene Nolparvovec; Optic nerve; Retina.

Graphical abstract

Figure 1

RT-qPCR detection of ND4 in GS010 IVT-injected naive mice Retina and optic nerve tissues were analyzed for the gene product ND4 at (A) 3Wk and (B) 5Wk after injection of GS010 in naive WT mice. Retinas from eyes receiving IVT injection of GS010 are denoted by an asterisk. Viral gene product was detected in the ipsilateral retinas, ipsilateral optic nerves, and contralateral optic nerves. Viral products were not detected in contralateral retinas at either 3Wk or 5Wk after injection.

Figure 2

IOP elevation increases ND4 detection in contralateral retinas; after a single injection of microbeads, elevation of IOP was sustained for the duration of the experiment (A) Plot of IOP measurements for saline and microbead injected eyes for the 3Wk cohorts. Arrow indicates time point of GS010 injection. Data plotted as mean ± SEM. (B) For the 3Wk cohort, eyes receiving microbead injection had an IOP increase of 34.7% compared with saline-injected eyes (∗p < 0.01). (C) With IOP elevation, the ND4 viral gene product was detected in 40% of contralateral retinas at 3 weeks after injection. (D) Plot of saline and microbead injected eye IOP measurements for the 5Wk cohort. Arrow indicates injection of GS010. Data plotted as mean ± SEM. (E) For the 5Wk cohort, the IOP increased by 35.9% compared with saline-injected eyes (∗p < 0.01). (F) After IOP elevation, the ND4 viral gene product was detected in 30% of contralateral retinas at 5 weeks post injection. Statistics (B, E) unpaired t-tests.

Figure 3

Cx43 KO decreases transport of ND4 (A) Confirmation of Cx43 KO. Tissues were collected from both cerebellum (CB) and frontal cortex (FC) for genotyping. A positive Ctrl for Cx43 KO (+KO) was included. (B) IOP plot after a single injection of microbeads for both Ctrl and Cx43 KO mice. Data plotted as mean ± SEM. (C) Ctrl and Cx43 mice had IOP increases of 42% and 40%, respectively, compared with saline Ctrls (∗p ≤ 0.001). (D) In Ctrl mice, after IOP elevation, the ND4 viral gene product was detected in the ipsilateral retina, ipsilateral optic nerve, contralateral optic nerve, and the contralateral retina. (E) In IOP elevated Cx43 KO mice, the ND4 viral gene product was detected in the ipsilateral retina, ipsilateral optic nerve, and contralateral optic nerve. However, the ND4 viral gene product was not detected in the contralateral retina. Statistics (C) One-way ANOVA and Tukey’s post hoc test.