Gene Therapy with a Small CEP290 Piece Slows Eye Cell Loss in a Mouse Model of Leber Congenital Amaurosis

For in vitro experiments, FL or miniCEP290-expressing cDNAs were cloned into a pEGFP-C1 plasmid expressing GFP-tagged proteins under the control of a CMV promoter. For AAV production, the miniCEP290-encoding cDNAs were cloned into a pAAV2 vector plasmid between a CMVenhancer/CBA (chicken β-actin) promoter upstream of the internal ribosome entry site (IRES) GFP and β-globin intron. This expression cassette was flanked with AAV2 inverted terminal repeats (ITRs). The recombinant AAV2 genomes were packaged with AAV8 capsid by HEK293-triple transfection method and purified by CsCl gradient centrifugation method, as previously described.³³

MEFs derived from wild-type (WT) and Cep290^rd16 mice were maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. Transient transfection with GFP-CEP290-FL or GFP-miniCEP290s was performed using Lipofectamine 2000 (Thermo Fisher). The transfected cells were either harvested for immunoblotting or were serum starved to induce cilia growth, as described.³⁴ The ciliated cells were then immunostained and imaged under a Leica microscope (DM5500). The cilia length was measured by using Image J. The pixel values of cilia length were transformed into actual length using the Leica Application Suite. The relationship between pixel length and actual length at 63 × magnification is: 680 pixels = 100 μm.

All animal studies were approved by the Institutional Animal Care and Use Committee of the UMASS Medical School. The WT C57BL6/J mice were obtained from Jackson Laboratories. The Cep290^rd16 mice have been described previously.¹² The Cep290^rd16 mouse pups (P0/P1) were subretinally injected unilaterally with 8 × 10⁹ vg/μL (total volume 1 μL) of the virus.^35,36

Scotopic and photopic electroretinograms (ERGs) were performed, as previously reported.³⁷ For scotopic response, mice were dark adapted overnight, and all procedures were performed under dim red light. The dark-adapted ERG protocol consisted of five steps with increasing stimulus intensities from 0.009 to 100 cd.s/m², with a mixed white light (white 6,500 K; ColorDome; Diagnosys LLC). The flash frequency was 0.07 Hz for bright flashes and up to 0.5 Hz for dim flashes. Averages ranged from 10 trials for dim flashes to five trials for bright flashes. Light-adapted (photopic) ERGs were recorded after light adaptation with a background illumination of 30 cd/m² (white 6,500 K) for 8 min.

Immunofluorescence microscopy was performed essentially as described.³⁷ In brief, retinal cryosections were stained with primary antibodies: rhodopsin (mouse monoclonal; EMD Millipore), M-opsin (rabbit polyclonal; EMD Millipore), and peripherin-retinal degeneration slow (RDS; mouse monoclonal; gift from Dr. Muna Naash), ARL13B (rabbit polyclonal; Abcam), GFP (chicken polyclonal; Abcam), and γ-tubulin (mouse monoclonal; Sigma–Aldrich). After washing with phosphate-buffered saline, Alexa-488 or Alexa-546 or Alexa-633-conjugated secondary antibodies were added, and the sections were further incubated for 1 h. After washing, nuclei were stained with DAPI, and cells were imaged using a Leica microscope (DM5500).

All data are presented as means ± standard error of the mean. Data groups were compared by Student's t-tests using the SigmaPlot software. Differences between groups were considered statistically significant if p < 0.05. The statistical significance is denoted with asterisks (*p < 0.01–0.05; **p < 0.001–0.01; ***p < 0.0001–0.001).

The human CEP290 gene consists of 52 exons, which encode a protein of ∼290 kDa (2,479 amino acids). Based on the previous studies on the involvement of the different CEP290 domains in its interactions and ciliary localization,^{12,14,38–42} plasmids encoding GFP-fused human miniCEP290^580–1695, miniCEP290^1751–2050 and miniCEP290^2037–2479 were constructed (Fig. 1A). To test the functional potential of the miniCEP290s, a surrogate assay system was designed using Cep290^rd16 MEFs. First, the expression and localization of the different miniCEP290s in the Cep290^rd16 MEFs were investigated. The analysis revealed that the FL CEP290 and all CEP290 domains expressed at comparable levels in the MEFs (Fig. 1B). Additional bands observed in the immunoblot most likely represent degradation products of the fusion protein. In addition to GFP-CEP290-FL, miniCEP290^580–1695 showed a predominant localization to the base of cilia (co-localization with γ-tubulin) and proximal cilia (co-localization with ADP-Ribosylation Factor-Like 13B; ARL13B; ciliary marker; Fig. 1C). Other variants showed a relatively diffuse pattern of localization. Next, the ability of the miniCEP290s to modulate cilia length in the Cep290^rd16 MEFs was assessed. Cilia of Cep290^rd16 MEFs are around 1.7 μm in length compared to controls, which have around 2.7 μm-long cilia. Cilia length of the Cep290^rd16 MEFs was significantly increased when either FL GFP-CEP290 or GFP-miniCEP290^580–1695 was expressed. Other variants and the plasmid expressing only GFP did not change the cilia length. No effect on the cilia length of the WT MEFs was observed (Fig. 1D).

Next, it was asked whether further shortening of miniCEP290^580–1695 would also result in a cilia length rescue. To this end, plasmids encoding GFP-miniCEP290^580–1180 and miniCEP290^1181–1695 were constructed. Both variants exhibited optimal expression, as determined by immunoblotting using GFP antibody (Fig. 2A). Additional degraded products of the fusion protein were also detected using the GFP antibody. While GFP-miniCEP290^580–1180 localized predominantly to the cilia, miniCEP290^1181–1695 exhibited a relatively diffuse localization in the cytosol with some ciliary localization (Fig. 2B). Cilia rescue assay showed that expression of either variant resulted in a comparable and significant increase in the cilia length of the Cep290^rd16 MEFs (Fig. 2C). Based on these results, miniCEP290^580–1180 and miniCEP290^1181–1695 were selected for further studies.

We cloned miniCEP290^580–1180, miniCEP290^1181–1695, and miniCEP290^2037–2479 (as negative control) into AAV2 vector genome with ubiquitously expressed CMVenhancer/CBA (chicken β-actin) promoter containing the IRES between the gene of interest and GFP. The IRES permits translation of CEP290 and GFP from a single bicistronic mRNA, which assists in identifying the transduced cells using GFP antibody. To assess the effect of GFP expression, we also used AAV2-GFP expressing construct as control. The viruses were packaged in AAV8 capsid, which is shown to transduce PRs efficiently.⁴³ The viruses were injected at 8 × 10⁹ vg/eye into the subretinal space of Cep290^rd16 pups at P0/P1. Similar doses have been successfully used to treat retinal degeneration in mice.⁴⁴ Injections at higher doses resulted in toxicity (data not shown).

Analysis of PR function by ERG at 3 weeks post injection revealed improvement (25–30%) in both scotopic (rod PR-mediated) and photopic (cone PR-mediated) responses of the miniCEP290^580–1180-injected mice (Fig. 3A–C). The miniCEP290^1181–1695-injected mice revealed a relatively mild improvement in the outer nuclear layer preservation (Supplementary Fig. S1A and B; Supplementary Data are available online at www.liebertpub.com/hum↗) and scotopic and photopic ERG amplitudes (Supplementary Fig. S1C and D). No improvement was detected using miniCEP290^2037–2479 or GFP (Fig. 3). Age-matched uninjected mice were also used as controls. Further analysis revealed that the improvement in the ERG was stable up to 4 weeks and exhibited significantly decreased amplitudes at 5 and 8 weeks post injection (Fig. 3D). Although in vitro cilia length rescue assay showed similar rescue effect with both miniCEP290^580–1180 and miniCEP290^1181–1695 minigenes, the miniCEP290^580–1180 is more potent in vivo. Such results show that in vivo experiments are important to validate the in vitro data, and indicate the existence of domain-specific functions of CEP290.

To assess the effect of miniCEP290 injections on retinal morphology, retinal cryosections were used in which the injected regions were identified by staining with GFP antibody. The number of layers of the outer nuclear layer correlates with PR survival. The following were found: around six or seven layers in the Cep290^rd16 retinas injected with miniCEP290^580–1180 and two to three layers in retinas injected with miniCEP290^2037–2472 or GFP (equivalent to uninjected Cep290^rd16 at 3 weeks of age; Fig. 4A). The number of layers in the outer nuclear layer progressively declined with age (Fig. 4B). Morphological analysis using ultrathin sections of the Cep290^rd16 retinas injected with miniCEP290^580–1180 exhibited significant preservation of the outer nuclear layer at 3 weeks of age (Fig. 4C).

Next, the structural preservation of the PR outer segment was examined in the miniCEP290^580–1180-injected mice by staining with peripherin-RDS (PR outer segment marker⁴⁵). RDS is a structural protein that specifically localizes to the outer segment discs and maintains the OS structure. The miniCEP290^580–1180-injected Cep290^rd16 mice exhibited improved RDS localization to the outer segment compared to undetectable RDS expression in the GFP-injected mice (Fig. 4D; red staining). The expression of rhodopsin and cone opsins, two of the key phototransduction proteins, was also examined. As predicted, there was undetectable opsin expression in the miniCEP290^2037–2479-injected retinas. However, the miniCEP290^580–1180-injected retinas revealed detectable expression of rhodopsin and cone opsins in the outer segments (Fig. 5). Some staining of cone opsins in the inner segment and outer nuclear layer was also observed. Overall, the results suggest that the expression of miniCEP290^580–1180 can improve the function, morphology, and opsin trafficking of the Cep290^rd16 retinas.