RNA interference gene therapy in dominant retinitis pigmentosa and cone-rod dystrophy mouse models caused by GCAP1 mutations

Abstract

RNA interference (RNAi) knockdown is an efficacious therapeutic strategy for silencing genes causative for dominant retinal dystrophies. To test this, we used self-complementary (sc) AAV2/8 vector to develop an RNAi-based therapy in two dominant retinal degeneration mouse models. The allele-specific model expresses transgenic bovine GCAP1(Y99C) establishing a rapid RP-like phenotype, whereas the nonallele-specific model expresses mouse GCAP1(L151F) producing a slowly progressing cone-rod dystrophy (CORD). The late onset GCAP1(L151F)-CORD mimics the dystrophy observed in human GCAP1-CORD patients. Subretinal injection of scAAV2/8 carrying shRNA expression cassettes specific for bovine or mouse guanylate cyclase-activating protein 1 (GCAP1) showed strong expression at 1 week post-injection. In both allele-specific [GCAP1(Y99C)-RP] and nonallele-specific [GCAP1(L151F)-CORD] models of dominant retinal dystrophy, RNAi-mediated gene silencing enhanced photoreceptor survival, delayed onset of degeneration and improved visual function. Such results provide a "proof of concept" toward effective RNAi-based gene therapy mediated by scAAV2/8 for dominant retinal disease based on GCAP1 mutation. Further, nonallele-specific RNAi knockdown of GCAP1 may prove generally applicable toward the rescue of any human GCAP1-based dominant cone-rod dystrophy.

Keywords: RNA interference; cone-rod dystrophy; guanylate cyclase-activating protein 1; photoreceptor guanylate cyclase; retinitis pigmentosa; self-complementary adeno-associated virus; short-hairpin RNA.

FIGURE 1

Phototransduction feedback loop regulates levels of cGMP and Ca²⁺. Dark levels of cGMP and Ca²⁺ are high in rod and cone photoreceptors. Light activation of rhodopsin initiates rod phototransduction, activated PDE6 rapidly hydrolyzes cytoplasmic cGMP, and cGMP-gated cation channels close. Continued extrusion of Ca²⁺ by the light-insensitive Na⁺-K⁺/Ca²⁺ exchanger (NCKX) lowers free Ca²⁺ which activates guanylate cyclase-activating proteins (GCAPs) and guanylate cyclases (GCs). Restoration of cGMP dark levels re-opens cation channels and Ca²⁺ levels equilibrate to dark levels.

FIGURE 2

Structure of myristoylated GCAP1 (PDB 2R2I; Baehr and Palczewski, 2009). N, N-terminal. C, C-terminal. EF hand helix-loop-helix structures are shown: EF1 (inactive and gray), EF2 (red), EF3 (dark blue), and EF4 (turquoise). Ca²⁺-binding loops (green) and approximate positions of relevant missense mutations associated with adCORD (red arrows) are indicated. The N-terminal myristoyl group (orange–brown) is buried and nearly invisible.

FIGURE 3

Schematic representation of GCAP1 transgenes used to generate RP and CORD mouse models. (A), bovine GCAP1(Y99C) transgene. Bovine Guca1a cDNA (blue) is expressed under the control of the Rho promoter (red). Approximate position of the Y99C mutation in EF3 is indicated (Jiang et al., 2011). (B), mouse GCAP1 transgene under the control of its native promoter (red). Exons (blue) are numbered; the L151F mutation is located in exon 4 and EGFP is fused in-frame to the GCAP1 C-terminus [see Figure 2 (Jiang et al., 2013)].

FIGURE 4

In vitro screening strategy to identify a potent shRNA knockdown sequence. (A), a construct expressing the GCAP1-EGFP fusion protein under the control of the CMV promoter. (B), the shRNA expression construct. shRNA (hp yellow box) is under the control of the H1 promoter, and the reporter gene mCherry is under the control of the CMV promoter. Hp consists of a sense-loop-antisense construct (the hairpin). Tts, transcription terminator signal. (C), four candidate shRNAs for suppression of bovine GCAP1, and a 2-nucleotide mismatch control. (D), four candidate shRNAs for suppression of both wild-type and mutant mouse Guca1a mRNA, and a 2-nucleotide mismatch control. See also Figure 1 (Jiang et al., 2011), and Figure 6 (Jiang et al., 2013).

FIGURE 5

In vitro knockdown of bGCAP1-EGFP. (A), In vitro transfection of HEK cells expressing bGCAP1-GFP (top) with bG1hp1-4 (bottom). Top panels show that hp1 and hp4 are highly effective. Bottom panels demonstrate fairly uniform transfection of four shRNA expression plasmids. (B), Representative semi-quantitative immunoblot of GCAP1 knockdown. Bar graph (top, n = 3) identifies hp4 as the most potent shRNA. See also Figure 2 (Jiang et al., 2011), and Figure 7 (Jiang et al., 2013).

FIGURE 6

Transduction efficiencies of four gene delivery methods for mouse photoreceptors tested by expression of fluorescent protein reporter. (A), Fluorescence microscopy of retina transverse-sections of mice injected subretinally with pCMV-Zeo-GFP, peptide/polymer nanoparticles. Below, retina section probed with anti-GFP antibody. (B), Fluorescence microscopy of retina transfected with pCAG-GFP using recombinant adenovirus 5, Ad5ΔRGD. Below, section at higher magnification. (C), pCMV-GFP-transfected retina using recombinant AAV vector, AAV2. Underneath, section at higher magnification. (D), pCMV-mCherry-transfected retina using electroporation. Micrographs were recorded at various days post-injection (PI).

FIGURE 7

Long-term expression of shRNA in vivo. (A), Schematic of scAAV2/8 shuttle vector for anti-bGCAP1 shRNA. ITR, inverted terminal repeats; hH1, human H1 promoter; hp, shRNA cassette; pCAG, chicken actin promoter driving mCherry. (B), Upper panel, in vivo fluorescence fundus images of wild-type mice at 1 week and months (indicated) post-subretinal injection of scAAVhp4 viral particles. Lower panel, fluorescence microscopy of corresponding mouse retina transverse cryosections (Jiang et al., 2011). RPE, retinal pigmented epithelium; OS, outer segments; IS, inner segments; ONL, outer nuclear layer.

FIGURE 8

Long-term therapeutic efficiency of allele-specific shRNA vector, scAAV2/8-bG1hp4. (A), Direct fluorescence microscopy of retinal cross-sections examines retinal morphology of the bG1hp4 treated and untreated L52H transgenic mice. Viral vectors were injected subretinally at mouse ages P21–P30. Both treated and untreated eyes were harvested at four representative times from 1–11 months post-injection. Red, mCherry expression demonstrating scAAV2/8 virus transduction. Blue, DAPI staining of nuclei. Note significant preservation of ONL thickness at 11 months post-treatment (~12 months of age) compared to non-treated controls. (B,C). Scotopic and photopic ERG amplitudes recorded from bG1hp4-treated (red) and untreated (blue) L52H transgenic mice (Jiang et al., 2011). Subretinal injection of scAAV2/8-bG1hp4 in the transgenic mouse models delayed progression of both rod and cone dysfunction significantly.

FIGURE 9

Knockdown of mGCAP1 by nonallele-specific shRNA vector, scAAV2/8-mG1hp4. (A), Fluorescence fundus images of mG1(L151F)-GFP transgenic mice with subretinal injection of the virus vectors at P21–P30 (left) and non-injected control (right). GFP signal (top) represents the mG1(L151F)-GFP expression level in photoreceptors, and mCherry (bottom) signal indicates scAAV2/8 virus transduction. (B), Immunoblot of the transgenic mG1(L151F)-GFP and endogenous GCAP1 protein levels in the injected and the non-injected retinas at 30 days PI. Transgenic mG1(L151F)-GFP (~50 kD) and endogenous GCAP1 (~25 kD) proteins were detected by UW101 antibody directed against GCAP1. -actin served as endogenous loading control. See also Figure 9 (Jiang et al., 2013).