Gene Augmentation Therapy Restores Retinal Function and Visual Behavior in a Sheep Model of CNGA3 Achromatopsia

Abstract

Achromatopsia is a hereditary form of day blindness caused by cone photoreceptor dysfunction. Affected patients suffer from congenital color blindness, photosensitivity, and low visual acuity. Mutations in the CNGA3 gene are a major cause of achromatopsia, and a sheep model of this disease was recently characterized by our group. Here, we report that unilateral subretinal delivery of an adeno-associated virus serotype 5 (AAV5) vector carrying either the mouse or the human intact CNGA3 gene under the control of the red/green opsin promoter results in long-term recovery of visual function in CNGA3-mutant sheep. Treated animals demonstrated shorter maze passage times and a reduced number of collisions with obstacles compared with their pretreatment status, with values close to those of unaffected sheep. This effect was abolished when the treated eye was patched. Electroretinography (ERG) showed marked improvement in cone function. Retinal expression of the transfected human and mouse CNGA3 genes at the mRNA level was shown by polymerase chain reaction (PCR), and cone-specific expression of CNGA3 protein was demonstrated by immunohistochemisrty. The rescue effect has so far been maintained for over 3 years in the first-treated animals, with no obvious ocular or systemic side effects. The results support future application of subretinal AAV5-mediated gene-augmentation therapy in CNGA3 achromatopsia patients.

Figures

Figure 1

Photopic maze passage time before (“day 0”) and after gene-augmentation therapy. (a) Sheep no. 3909 was treated with an AAV5 vector containing mouse (m) CNGA3. (b) Sheep no. 4984 was treated with a vector containing human (h) CNGA3. (c) Average passage time for sheep treated with viral vectors containing either mCNGA3 (blue) or hCNGA3 (red). Each post-treatment data point represents the mean passage time of up to six animals. Gray background represents passage time of normal unaffected sheep (mean ± 2 SD, n = 20). Please note that as treatment with the mouse gene was initiated before using the human gene (see study design), the follow up time for animals treated with the mouse gene was longer. (d,e) Maze navigation time (d) and number of collisions (e) following alternate eye patching in unaffected control sheep (n = 4) and CNGA3-mutant sheep treated in their right eye with an AAV5 vector carrying either the human (n = 8) or the mouse (n = 4) CNGA3 gene.

Figure 2

Photopic electroretinography (ERG) responses. (a) Cone flicker tracings (30–80 Hz, 10 cd•s/m2) of representative CNGA3-mutant sheep eyes treated with mouse (m) (sheep 2998, left panel) or human (h) (sheep 2875, right panel) CNGA3 showing functional efficacy of treatment. Following treatment, marked increase of cone flicker amplitudes was observed in treated eyes. Black tracing: baseline preoperative recording; blue and gray tracings: postoperative recording in treated (blue) and nontreated fellow (gray) eyes. Postoperative recordings were conducted at 4 and 14 months post-op for the mCNGA3- and hCNGA3-treated animals, respectively. Flash stimulus onset is shown by arrows. (b) Critical flicker fusion frequency (CFFF) of CNGA3-mutant sheep treated with mouse (left) and human (right) CNGA3. For both treatments, CFFF values at the three highest stimulus intensities were significantly higher in the treated eyes compared to both the baseline values and the untreated fellow eyes at the first and second follow-up recordings (P ≤ 0.05). Black bars: baseline preoperative CFFF at the four intensities tested; blue bars: first post-op (~2 months) recording (n = 8 in each treatment group); blue hash-marked bars: second post-op recording (4.6 ± 1.2 months for mCNGA3 and 7.6 ± 1.2 months for hCNGA3, n = 8 in each treatment group); dotted blue bars: last recording performed to date (30.4 ± 1.5 months for mCNGA3 and 16.5 ± 2.1 months for hCNGA3, n = 3 in each treatment group); gray bars: CFFF values of the nontreated fellow eye at the first follow-up. Results are presented as mean ± SE, in Hz; last follow-up recording could not be statistically analyzed due to small number of recorded animals. (c) Cone flicker responses of CNGA3-mutant sheep treated with mouse (left) and human (right) CNGA3. Following gene-augmentation therapy, cone flicker responses significantly improved in both mCNGA3- and hCNGA3-treated eyes. Effect of treatment was statistically significant (P ≤ 0.05), and was maintained over the course of follow-up in both groups. Black lines: baseline preoperative flicker amplitudes; blue, dashed blue and dotted blue lines: flicker amplitudes in treated eyes at the first, second and last follow-up, respectively, with recording intervals and number of animals identical to those detailed for panel b. Responses of the nontreated fellow eyes recorded in the same sessions are shown in gray, dashed gray, and dotted gray lines, respectively. Responses to the 10 cd•s/m2 flicker stimulus are plotted as a function of the stimulating frequency (Hz), and results are presented as mean ± SE, in µV.

Figure 3

Molecular genetic analysis. (a) Transcripts of sheep, mouse, and human CNGA3 orthologs. The causative mutation in CNGA3-mutant sheep is located in exon 8 which is present in the major CNGA3 transcript, but is skipped (together with exons 5–7) in the novel minor transcript reported herein. The agarose gel (upper right panel: lane 1—molecular size marker, lane 2—reverse transcription–polymerase chain reaction (RT-PCR) products from a normal unaffected sheep retina, lane 3—double distilled water negative control) depicts RT-PCR products of a normal sheep retina (lane 2) using primers located in exons 3 and 9. Sanger-sequencing analysis revealed that the upper band represents the major CNGA3 transcript and the lower band represents the novel minor transcript in which exons 5 through 8 are skipped. The same RT-PCR pattern was observed in both normal and affected CNGA3-mutant sheep. The middle panel depicts the mRNA structure in different species. Note that since the full sequence of the sheep CNGA3 gene is currently unknown, exon borders are deduced from those reported in the orthologous genes. Black arrows represent the location of the different primers that were designed to specifically amplify CNGA3 mRNA of each of the three species (our analysis did not include the most 5' exons 1 and 2). The chromatograms demonstrate parts of the mouse and human transcripts amplified from retinas of treated CNGA3-mutant, showing some of the sequence differences that distinguish them from the sheep sequence (highlighted in light blue boxes). (b) Agarose gel electrophoresis of RT-PCR products obtained following use of primers designed to amplify mouse (left panel) or human (right panel) CNGA3 mRNA. Lane 1—molecular size marker, lane 2—retinal cDNA of CNGA3-treated affected sheep, lane 3—retinal cDNA of untreated CNGA3-mutant sheep, lane 4—retinal cDNA of untreated normal sheep, lane 5—double distilled water negative control. A specific band was obtained in lane 2 for both the human (~300 bp) and mouse (~200 bp) CNGA3 cDNA, and verified by sequencing. Lanes 3 and 4 in the left panel contain nonspecific amplification due to the lack of a specific template for PCR.

Figure 4

Gene-augmentation treatment restores CNGA3 protein expression in CNGA3-mutant sheep retinas. (a) Normal sheep retina. Anti-CNGA3 immunohistochemical staining shows strong protein expression in the outer segments of cone photoreceptors, colocalizing with red/green opsin (5-month-old animal). (b) No CNGA3 expression was observed in the retina of CNGA3-mutant day-blind sheep. Anti-red/green opsin staining revealed a large number of red/green cones in the retina, with some mislocalization of opsin to the cone cell bodies and synaptic region (5-month-old animal). (c) Unaffected sheep retina injected with an AAV5 vector carrying the green fluorescent protein (GFP) marker gene under control of the red/green opsin promoter. Double-labeling with anti-GFP and anti-red/green opsin antibodies showed strong GFP expression in the cell bodies and inner segments of red/green cones (age of animal at surgery 7 months, enucleated 6 months post-op). (d,e) CNGA3-mutant sheep retinas following gene-augmentation therapy with either mouse (d) or human (e) CNGA3. In both cases, CNGA3 protein was expressed, mainly in the outer segments of red/green cone photoreceptors (both animals 31 months old at surgery. Mouse CNGA3-treated enucleated 4 months post-op; Human CNGA3-treated enucleated 14 months post-op). INL, inner nuclear layer; IPL, inner plexiform layer; IS+OS, inner and outer segments of photoreceptors; ONL, outer nuclear layer; OPL, outer plexiform layer; RPE, retinal pigment epithelium. Nuclei are counterstained with 4′,6-diamidino-2-phenylindole (blue). Original magnification ×40. Scale bars = 50 µm.