AAV-Mediated Gene Supplementation Therapy in Achromatopsia Type 2: Preclinical Data on Therapeutic Time Window and Long-Term Effects

Abstract

Achromatopsia type 2 (ACHM2) is a severe, inherited eye disease caused by mutations in the CNGA3 gene encoding the α subunit of the cone photoreceptor cyclic nucleotide-gated (CNG) channel. Patients suffer from strongly impaired daylight vision, photophobia, nystagmus, and lack of color discrimination. We have previously shown in the Cnga3 knockout (KO) mouse model of ACHM2 that gene supplementation therapy is effective in rescuing cone function and morphology and delaying cone degeneration. In our preclinical approach, we use recombinant adeno-associated virus (AAV) vector-mediated gene transfer to express the murine Cnga3 gene under control of the mouse blue opsin promoter. Here, we provide novel data on the efficiency and permanence of such gene supplementation therapy in Cnga3 KO mice. Specifically, we compare the influence of two different AAV vector capsids, AAV2/5 (Y719F) and AAV2/8 (Y733F), on restoration of cone function, and assess the effect of age at time of treatment on the long-term outcome. The evaluation included in vivo analysis of retinal function using electroretinography (ERG) and immunohistochemical analysis of vector-driven Cnga3 transgene expression. We found that both vector capsid serotypes led to a comparable rescue of cone function over the observation period between 4 weeks and 3 months post treatment. In addition, a clear therapeutic effect was present in mice treated at 2 weeks of age as well as in mice treated at 3 months of age at the first assessment at 4 weeks after treatment. Importantly, the effect extended in both cases over the entire observation period of 12 months post treatment. However, the average ERG amplitude levels differed between the two groups, suggesting a role of the absolute age, or possibly, the associated state of the degeneration, on the achievable outcome. In summary, we found that the therapeutic time window of opportunity for AAV-mediated Cnga3 gene supplementation therapy in the Cnga3 KO mouse model extends at least to an age of 3 months, but is presumably limited by the condition, number and topographical distribution of remaining cones at the time of treatment. No impact of the choice of capsid on the therapeutic success was detected.

Keywords: AAV vector; CNGA3; achromatopsia; cone function restoration; gene therapy; non-invasive diagnostic techniques; photoreceptor cells; subretinal delivery.

Figure 1

Monitoring of retinal integrity after subretinal AAV delivery. (A–H) In vivo cSLO and SD-OCT scans of the subretinal bleb 10 min (A–D) and 4 weeks (E–H) after injection at PM1. The dashed line indicates the area of the bleb due to subretinal detachment, arrows mark the injection site. A total volume of 1.0 μl AAV 2/8 (Y733F)-S opsin-Cnga3 was injected into the dorsal-temporal part of the retina. d, dorsal; n, nasal; t, temporal; v, ventral; onh, optic nerve head. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; IS, inner segments; OS, outer segments; RPE, retinal pigment epithelium; CC, choroid complex.

Figure 2

Immunolabeling of flat-mounted treated Cnga3 KO retina. (A–D) Representative montage images of epifluorenscence imaging of a treated Cnga3 KO retina mounted with the photoreceptor side up. The animal was treated at the age of 2 weeks with AAV2/8 (Y733F)-S opsin-Cnga3 and imaged at 4 months after treatment. (A) Cones were stained with the marker peanut agglutinin (PNA, green). In 4-month-old Cnga3 KO mice, the majority of ventrally located cones, specifically of blue opsin expressing (S opsin) cones, are lost in untreated regions. (B) Immunosignal obtained with a Cnga3-specific antibody (red) revealed that about 1/3 of the treated Cnga3 KO retina was transduced with a single injection and expressed the AAV vector-encoded Cnga3 protein. (C) Merged image showing an overlay of Cnga3 (red) and PNA (green) signals. (D) Magnified detail of the treated area as shown in (C). d, dorsal; n, nasal; t, temporal; v, ventral; onh, optic nerve head.

Figure 3

Functional performances of AAV2/5 (Y719F) and AAV2/8 (Y733F) vector serotypes in vivo. (A–C) Cnga3 KO mice were treated at an age of 2 weeks with AAV2/5 (Y719F)- or AAV2/8 (Y733F)-S opsin-Cnga3 vectors and the functional outcome was assessed at 1 and 3 months PI. (A) Representative single flash ERG traces obtained under scotopic (dark-adapted) or photopic (light-adapted) conditions. Top traces: scotopic rod system response, center traces: scotopic mixed rod/cone system response, bottom traces: photopic cone system response. (B) Comparison of photopic 7 Hz flicker ERG responses obtained from representative animals treated with AAV2/5 (Y719F) or AAV2/8 (Y733F) serotypes. Top panel: evaluation at PM1 PI, bottom panel: evaluation at PM3 PI. (C) Box plots summarizing the corresponding group data (n = 10). ERG b-wave amplitudes of TEs are shown in red and those of UEs in black. Boxes indicate the 25–75% quantile range, whiskers the 5 and 95% quantiles and solid lines connect the medians of the data. As no flicker waveform could be detected in any of the UEs, the result are indicated as “n.d.” (non-detectable). SF, single flash.

Figure 4

Long-term outcome of AAV treatment. (A–D) Cnga3 KO mice were treated with AAV2/5 (Y719F)-S opsin-Cnga3 and analyzed according to the scheme depicted in (A). (B) Representative photopic (light-adapted) 7 Hz flicker ERG traces obtained at 1M and 12M PI. (C) Photopic (light-adapted) single flash ERG recordings from a treated (TE, red trace), untreated (UE, black trace), and wild-type (wt) eye (gray trace) obtained at 12M PI. (D) Box plots summarizing the corresponding flicker ERG group data (n = 14). Flicker ERG amplitudes of TEs are shown in red and those of UEs in black. Boxes indicate the 25–75% quantile range, whiskers the 5 and 95% quantiles and solid lines connect the medians of the data. As no flicker waveform could be detected in any of the UEs, the result are indicated as “n.d.” (non-detectable). PM, post natal month; PI, post-injection. SF, single flash.

Figure 5

Immunohistological evaluation of an AAV-treated Cnga3 KO retina at 12M PI. (A–E) Representative confocal scans of vertical cryo-sections obtained from the retina of Cnga3 KO mice at 12 months (12M) after treatment with AAV2/5 (Y719F)-S opsin-Cnga3. Staining was performed using specific antibodies and the cone-specific marker peanut agglutinin (PNA). Cell nuclei were marked with Hoechst 33342. (A–D) Low magnification survey (A,B) and high magnification detail images (C,D) featuring Cnga3 immunosignal (red) alone (A,C) or in combination with the PNA signal (green) (B,D). The region depicted in (A,B) spans from the treated (right) to the untreated (left) part of the retina. (C,D) shows higher magnification details from the treated part. (E) Overview image from a consecutive cryo-section to the section shown in (A,B) stained with an S opsin-specific antibody. The S opsin signal (green) indicates preservation of this subtype of cones at 12M PI.

Figure 6

Dependency of AAV treatment on age at intervention. (A–C) Cnga3 KO mice (n = 4) were treated with AAV2/8 (Y733F)-S opsin-Cnga3 and analyzed according to the scheme depicted in (A). (B) Representative photopic (light-adapted) 7 Hz flicker ERG traces obtained at 1M and 12M PI when treated at PM3. (C) Photopic (light-adapted) single flash ERG recordings from a PM3-treated (TE, red trace), untreated (UE, black trace), and wild-type (wt) eye (gray trace) obtained at 12M PI. PM, postnatal month; PI, post-injection. SF, single flash.