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. 2018 Oct 3;26(10):2407-2417.
doi: 10.1016/j.ymthe.2018.07.016. Epub 2018 Jul 19.

A Drug-Tunable Gene Therapy for Broad-Spectrum Protection Against Retinal Degeneration

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Free PMC article

A Drug-Tunable Gene Therapy for Broad-Spectrum Protection Against Retinal Degeneration

Clayton P Santiago et al. Mol Ther. .
Free PMC article

Abstract

Retinal degenerations are a large cluster of diseases characterized by the irreversible loss of light-sensitive photoreceptors that impairs the vision of 9.1 million people in the US. An attractive treatment option is to use gene therapy to deliver broad-spectrum neuroprotective factors. However, this approach has had limited clinical translation because of the inability to control transgene expression. To address this problem, we generated an adeno-associated virus vector named RPF2 that was engineered to express domains of leukemia inhibitory factor fused to the destabilization domain of bacterial dihydrofolate reductase. Fusion proteins containing the destabilization domain are degraded in mammalian cells but can be stabilized with the binding of the drug trimethoprim. Our data show that expression levels of RPF2 are tightly regulated by the dose of trimethoprim and can be reversed by trimethoprim withdrawal. We further show that stabilized RPF2 can protect photoreceptors and prevent blindness in treated mice.

Keywords: LIF; destabilization domain; drug regulated gene expression; gene therapy; leukemia inhibitory factor; neuroprotection; retina.

Figures

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Figure 1
Figure 1
Long-Term, Constitutive Expression of AAV-hLIF Leads Can Have Adverse Effects on Retinal Morphology and Function (A) Schematic of AAV-hLIF construct showing the human LIF (hLIF) transgene under the control of the chicken beta actin (CBA) promoter. The expression construct is flanked by inverted terminal repeats (ITRs) to allow for packaging into AAV vectors. The size of the vector is 4.6 kb. (B) Experimental timeline using the AAV-hLIF vector. At 6 weeks of age, BALB/cJ mice were intravitreally injected with AAV2 (quadY-F+T-V) carrying the hLIF transgene. In vivo imaging and functional analysis was performed 7 weeks and 14 weeks post-injection. (C) Scoring rubric for assessment of retinal morphology. A score of 3 indicated a normal retina that maintains well-defined boundaries of hyper- or hypo-reflectance. A score of 2 indicated retinas with minor ruffling and less-defined retinal layer boundaries. Retinas with a score of 1 had multiple rosettes, retinal thinning, excessive cells in the vitreous, and retinal detachment. (D) Retinal scoring in low titer control and AAV2 (quadY-F+T-V) hLIF-injected animals 7 (top) and 14 (bottom) weeks post-injection. Each filled circle represents one animal (n = 8–9), and the black lines represent the mean for each group. (E) Representative ERG trace. The a-wave, the initial negative deflection, reflects the hyperpolarization of the photoreceptors following light stimulation. The b-wave amplitude was measured from the trough of the a-wave to the adjacent peak, indicative of the depolarization due to voltage changes taking place in the inner retina. (F) Average a-wave amplitudes at 1.0 cd.s/m2 of control injected and AAV2 (quadY-F+T-V) hLIF-injected animals 7 and 14 weeks post-injection. Error bars represent SEM for each group. One-way ANOVA with Sidak post-hoc test was performed; n = 8 animals per group.
Figure 2
Figure 2
TMP-Stabilized RPF2 Is Active In Vitro (A) Schematic of the RPF2 construct showing the synthetic neuroprotective factor linked to the destabilization domain expressed from the CBA promoter. Within the linker region, two FURIN cleavage sites (FCS) separate the domains. (B) Illustration of the RPF2 stabilization strategy. In the absence of TMP, RPF2 is rapidly degraded by the proteasome following translation. TMP stabilizes RPF2, allowing it to be processed and secreted. (C) ELISA measuring cytokine levels in the media of rMC-1 cells transfected with CBA-RPF2 with or without TMP. Dotted line indicates the limit of detection of the ELISA assay at 32 pg/mL. (D) Immunoblot analysis of pSTAT3 Y705 levels in rMC-1 cells following treatment with conditioned media from CBA-RPF2-transfected cells, with and without TMP treatment. Error bars represent SEM (C and D). Two-way ANOVA with Sidak post-hoc test was performed for (C) and (D); n = 4 biological replicates.
Figure 3
Figure 3
RPF2 Is Regulated in a Dose-Dependent Manner and Demonstrates No Toxicity In Vivo (A) ELISA levels of RPF2 in the retinal lysates of animals injected with low-titer AAV2 (quadY-F+T-V) RPF2 at varying concentrations of TMP. Tissue was also collected 7 days after TMP withdrawal (60 > WD). Dotted line indicates the limit of detection of the ELISA assay at 32 pg/mL. One-way ANOVA with Sidak post-hoc test was performed; n = 4 biological replicates. (B) Experimental timeline using the AAV2 (quadY-F+T-V) RPF2 vectors following a similar strategy to that in Figure 1B. From week 13 to week 20, mice were kept on either normal chow or a TMP-supplemented diet. (C) Average outer nuclear layer (ONL) thickness in injected animals before and after TMP diet. (D) Average a-wave amplitudes at 1.0 cd.s/m2 of control-injected and AAV2 (quadY-F+T-V) RPF2-injected animals 7 and 14 weeks post-injection. Two-way ANOVA with Sidak post-hoc test was performed for (C) and (D); n = 8 biological replicates. No statistical significance was found. BSS, balanced saline solution. Error bars represent SEM.
Figure 4
Figure 4
RPF2 Can Reduce Retinal Degeneration in an Acute Light Damage Model (A) Experimental timeline using the AAV2 (quadY-F+T-V) RPF2 vectors with light damage (LD). One week after the start of TMP supplementation, animals were LD treated, followed by a week of recovery to allow of clearance of damaged cells. (B) Average ONL thickness in injected animals after LD treatment. (C) Average a-wave amplitudes at 1.0 cd.s/m2 of control and AAV-RPF2-injected animals after LD treatment. Error bars indicate SEM for (B) and (C). Two-way ANOVA with Sidak post-hoc test was performed for (B) and (C); n = 8–9 biological replicates per group.
Figure 5
Figure 5
RPF2 Can Preserve Cone Morphology in the Inherited rd10 Retinal Degeneration Model (A) Timeline showing the natural history of the rd10 mouse model and therapeutic intervention with AAV2 (quadY-F+T-V) RPF2 vectors. The onset of photoreceptor degeneration begins at week 3. Animals were injected with AAV2 (quadY-F+T-V) RPF2 vectors at 2 weeks of age. At weaning, animals were placed on a TMP diet. Morphological and functional analysis was performed over the subsequent weeks before collecting the eyes for histology. (B) Average ONL thickness in therapy-treated animals over time. Two-way ANOVA with Sidak post-hoc test was performed; n = 6 biological replicates per group. Error bars indicate SEM. (C) Immunofluorescent images of retinal flat mounts showing staining of s and m opsin (green). Images were taken at 40× magnification. White bar indicates 100 μm. (D) Quantification of cones in (C). Error bars indicate SEM. Two-way ANOVA with Sidak post-hoc test was performed; n = 4 biological replicates per group, except C57BL/6 = 3 biological replicates. BSS, balanced saline solution.
Figure 6
Figure 6
RPF2-Treated rd10 Mice Maintain Long-Term, Cone-Dependent Vision (A) Average b-wave amplitudes at photopic 60 cd.s/m2 of control and AAV2 (quadY-F+T-V) RPF2-injected animals over time. (B) Illustration of the OptoMotry system used to measure optokinetic-tracking response in therapy-treated animals. Animals stand on an elevated platform surrounded by computer monitors that display a sine wave grating, creating a virtual cylinder. The animals track the rotation of the cylinder with reflexive head and neck movements, which is detected by an overhead camera. (C) Quantification of optokinetic responses from the OptoMotry analysis described in (B). Error bars indicate SEM for (A) and (C). Two-way ANOVA with Sidak post-hoc test was performed for (A) and (C). For (A), n = 6 biological replicates per group. For (C), n = 4 biological replicates per group, except C57BL/6 = 2 biological replicates. BSS, balanced saline solution.

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