Challenging Safety and Efficacy of Retinal Gene Therapies by Retinogenesis

Abstract

Gene-expression programs modulated by transcription factors (TFs) mediate key developmental events. Here, we show that the synthetic transcriptional repressor (TR; ZF6-DB), designed to treat Rhodopsin-mediated autosomal dominant retinitis pigmentosa (RHO-adRP), does not perturb murine retinal development, while maintaining its ability to block Rho expression transcriptionally. To express ZF6-DB into the developing retina, we pursued two approaches, (i) the retinal delivery (somatic expression) of ZF6-DB by Adeno-associated virus (AAV) vector (AAV-ZF6-DB) gene transfer during retinogenesis and (ii) the generation of a transgenic mouse (germ-line transmission, TR-ZF6-DB). Somatic and transgenic expression of ZF6-DB during retinogenesis does not affect retinal function of wild-type mice. The P347S mouse model of RHO-adRP, subretinally injected with AAV-ZF6-DB, or crossed with TR-ZF6-DB or shows retinal morphological and functional recovery. We propose the use of developmental transitions as an effective mode to challenge the safety of retinal gene therapies operating at genome, transcriptional, and transcript levels.

Keywords: adeno-associated virus (AAV); autosomal dominant; gene therapy; retinal degeneration; retinitis pigmentosa; rhodopsin; transcription; zinc finger.

Figure 1

Early somatic delivery of ZF6-DB transcriptional repressor in the WT and P347S adRP murine model by adeno-associated virus (AAV) vector. (A) AAV8-CMV-ZF6-DB construct representation showing the cytomegaloviruses (CMV) promoter, including the SV40intron, the woodchuck hepatitis posttranscriptional regulatory element (WPRE), and the bovine growth hormone polyA (bGH). (B,C) a- and b-waves amplitudes recorded in scotopic conditions plotted as a function of light intensity (log cd * s/m²) in one-month old WT animals treated at postnatal day 4 (P4, white box; n = 5; controls CTR grey box; n = 4). (D,E) a- and b-waves in one-month old P347S adRP mice treated with AAV8-CMV-ZF6-DB at postnatal day 4 (P4, white box; n = 9) and P14 (grey box; n = 18), compared with sham injected contralateral controls eyes (grey box; n = 9, P4 and n = 18, P14); Statistics: t-test corrected with Holm–Sidak method for multiple comparisons. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. (F) Representative ERG track responses in scotopic and photopic conditions in one-month old P347S adRP mice treated with AAV8-CMV-ZF6-DB at postnatal day 4 and P14 compared with sham injected contralateral controls eyes. (G) Representative cryo-retinal sections co-immunostained with Rhodopsin (green) and ZF6-DB HA-tag (red) antibodies in one month old P347S mice treated at P4 and P14 and contralateral controls, sections were counterstained with Vectashield to visualize nuclei. OS, Outer Segment. IS, Inner Segment. ONL, Outer nuclear Layer. INL, Inner Nuclear Layer.

Figure 2

Transgenic mice expressing the ZF6-DB (TR-ZF6-DB) transcriptional repressor in the murine retina. (A) representation of TR-ZF6-DB transgenic construct showing the human Guanine Nucleotide Binding Protein1 (hGNAT1) promoter fragment including the 5′ UTR cloned upstream the ZF6-DB coding sequence followed by the WPRE and the bovine growth hormone polyA (bGH). (B) qReal Time PCR of mRNA expression levels (2^−∆CT) of the ZF6-DB transgene on postnatal day 10 (P10) retina compared to wild-type match controls. TR-ZF6-DB, n = 4 (2 littermate pups belonging to two independent TR-ZF6-DB F0 litter (4 pulled retina); WT n = 6 (2 littermate pups (4 pulled retina) from the same litter). (C,D) a- and b-waves amplitudes recorded in scotopic conditions plotted as a function of light intensity (log cd * s/m²) in one-month old TR-ZF6-DB (n = 4, white box) and WT controls (n = 4, controls CTR grey box). Statistics: t-test corrected with Holm–Sidak method for multiple comparisons. (E) Rho immunofluorescence (green) histological analysis of one-month old TR-ZF6-DB and WT controls. (F) Glia-derived neurotrophic factor (GFAP) immunofluorescence (green) histological analysis of one-month old TR-ZF6-DB and WT controls. Arrows indicate positive in the ganglion cell layer, GC. OS, Outer Segment. IS, Inner Segment. ONL, Outer nuclear Layer. INL, Inner Nuclear Layer.

Figure 3

Double transgenic animals TR-ZF6-DBxP347S, derived by the cross of TR-ZF6-DB with P347S RHO-AdRP mice. (A) qReal Time PCR of mRNA expression levels (2^−∆CT) of the ZF6-DB in double TR-ZF6-DBxP347S (n = 4 eyes) compared to P347S RHO-AdRP (n = 5 eyes) on P30 retina. (B) qReal Time PCR of mRNA expression levels (2^−∆CT) of murine endogenous alleles (mRho), human P347S RHODOPSIN mutant allele, and mouse cone Arrestin 1 (mArr1) in P30 TR-ZF6-DB-P347S and P347S RHO-AdRP mice (on the same animals of B). (C,D) a- and b-waves amplitudes recorded in scotopic conditions plotted as a function of light intensity (log cd * s/m²) in one-month old TR-ZF6/DB-P347S (n = 5, white box) and P347S RHO-AdRP mice (n = 5, grey box). Statistics: t-test corrected with Holm–Sidak method for multiple comparisons. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. (E) Rho Immunofluorescence (green) histological analysis of one-month old TR-ZF6/DB-P347S and P347S RHO-AdRP mice. OS, Outer Segment. IS, Inner Segment. ONL, Outer nuclear Layer. INL, Inner Nuclear Layer. GC, ganglion cell layer. (F) Retinal “spidergram” showing ONL thickness from the optic nerve head (ONH) to the inferior and superior hemispheres of double TR-ZF6-DBxP347S (n = 3 eyes) and P347S (n = 3 eyes). Statistics: t-test corrected with Holm–Sidak method for multiple comparisons. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Figure 3

Double transgenic animals TR-ZF6-DBxP347S, derived by the cross of TR-ZF6-DB with P347S RHO-AdRP mice. (A) qReal Time PCR of mRNA expression levels (2^−∆CT) of the ZF6-DB in double TR-ZF6-DBxP347S (n = 4 eyes) compared to P347S RHO-AdRP (n = 5 eyes) on P30 retina. (B) qReal Time PCR of mRNA expression levels (2^−∆CT) of murine endogenous alleles (mRho), human P347S RHODOPSIN mutant allele, and mouse cone Arrestin 1 (mArr1) in P30 TR-ZF6-DB-P347S and P347S RHO-AdRP mice (on the same animals of B). (C,D) a- and b-waves amplitudes recorded in scotopic conditions plotted as a function of light intensity (log cd * s/m²) in one-month old TR-ZF6/DB-P347S (n = 5, white box) and P347S RHO-AdRP mice (n = 5, grey box). Statistics: t-test corrected with Holm–Sidak method for multiple comparisons. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. (E) Rho Immunofluorescence (green) histological analysis of one-month old TR-ZF6/DB-P347S and P347S RHO-AdRP mice. OS, Outer Segment. IS, Inner Segment. ONL, Outer nuclear Layer. INL, Inner Nuclear Layer. GC, ganglion cell layer. (F) Retinal “spidergram” showing ONL thickness from the optic nerve head (ONH) to the inferior and superior hemispheres of double TR-ZF6-DBxP347S (n = 3 eyes) and P347S (n = 3 eyes). Statistics: t-test corrected with Holm–Sidak method for multiple comparisons. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.