Retinal gene therapy with a large MYO7A cDNA using adeno-associated virus

Abstract

Usher 1 patients are born profoundly deaf and then develop retinal degeneration. Thus they are readily identified before the onset of retinal degeneration, making gene therapy a viable strategy to prevent their blindness. Here, we have investigated the use of adeno-associated viruses (AAVs) for the delivery of the Usher 1B gene, MYO7A, to retinal cells in cell culture and in Myo7a-null mice. MYO7A cDNA, under control of a smCBA promoter, was packaged in single AAV2 and AAV5 vectors and as two overlapping halves in dual AAV2 vectors. The 7.9-kb smCBA-MYO7A exceeds the capacity of an AAV vector; packaging of such oversized constructs into single AAV vectors may involve fragmentation of the gene. Nevertheless, the AAV2 and AAV5 single vector preparations successfully transduced photoreceptor and retinal pigment epithelium cells, resulting in functional, full-length MYO7A protein and correction of mutant phenotypes, suggesting successful homologous recombination of gene fragments. With discrete, conventional-sized dual AAV2 vectors, full-length MYO7A was detected, but the level of protein expression was variable, and only a minority of cells showed phenotype correction. Our results show that MYO7A therapy with AAV2 or AAV5 single vectors is efficacious; however, the dual AAV2 approach proved to be less effective.

Fig. 1

Expression of MYO7A from single AAV2 and AAV5 vectors in cultured cells. (a) Diagram of the viral vector encoding human MYO7A cDNA. (b) Western blot of WT eyecup (lane 1), primary RPE cultures derived from Myo7a-null mice and infected with AAV2-MYO7A (lane 2) or AAV5-MYO7A (lane 3), or not infected (lane 4), and primary RPE cultures derived from Myo7a^+/− mice (lane 5). All lanes were immunolabeled with antibodies against actin (as a loading indicator of relative protein loading) and MYO7A. (c-f) Immunofluorescence images of primary RPE cell cultures. Cells derived from Myo7a-null mice that were not infected (c), from Myo7a^+/− mice (d), or from Myo7a-null mice infected with 1x AAV2-MYO7A (e) or 1x AAV5-MYO7A (f). Scale = 10 μm.

Fig. 2

Expression of MYO7A from single AAV2 and AAV5 vectors in vivo. (a-e) EM images of MYO7A immunogold labelling of the connecting cilium and pericilium from rod photoreceptors in a Myo7a-null retina. (a) Longitudinal section from an untreated Myo7a-null retina (background label only). (b, c) Longitudinal sections from Myo7a-null retinas treated with 1x AAV2-MYO7A (b) or AAV5-MYO7A (c). Scale = 200 nm. (d, e) Transverse sections of connecting cilia from rod photoreceptors in Myo7a-null retinas treated with 1x AAV2-MYO7A (d) or AAV5-MYO7A (e). Scale = 100 nm. (f-g) EM images of RPE cells from Myo7a-null retinas treated with 1x AAV2-MYO7A (f) or AAV5-MYO7A (g). Scale = 2 μm. BM = Bruch’s Membrane, AP = Apical Processes. Areas indicated by rectangles are enlarged in f’ and g’, in order to show MYO7A immunogold labeling (indicated by circles). Scale = 500 nm. (h, i) EM image of a longitudinal section of the connecting cilium and pericilium from a rod (h) and a cone (i) photoreceptor in a Myo7a-null retina, treated with 1x AAV2-MYO7A. The section was double-labeled with MYO7A (12 nm gold) and rod opsin (15 nm gold) antibodies. Rod outer segments were labeled with the opsin antibody, while cones were identified by lack of rod opsin labeling in their outer segments. The sections show just the base of the outer segments. Nearly all the label in the connecting cilium is MYO7A, even in the rod. Scale = 200 nm. (i-l) Bar graphs indicating MYO7A immunogold particle density in the rod photoreceptor cilium and pericilium (j, k) and in the RPE (l, m), following treatment with AAV2-MYO7A (j, l) or AAV5-MYO7A (k, m) of different concentrations. n=3 animals per condition. Bars indicate SEM.

Fig. 3

Correction of mutant phenotypes, following subretinal injections with AAV2-MYO7A or AAV5-MYO7A. (a-e) Correction of melanosome localization. Light micrographs showing the presence of melanosomes in the apical processes of the RPE in a WT retina (a) and retinas injected with 1x AAV2-MYO7A (b) or AAV5-MYO7A (c). Further away from the injection site (d), melanosomes are present in the apical processes of some RPE cells, but not in others (arrows indicate apical melanosomes; white lines indicate regions where melanosomes are absent from the apical processes). (e) Region distant from injection site, where all RPE cells lack melanosomes in their apical processes. Brackets on left side indicate RPE apical processes. Scale = 8 μm. (f) Diagram of a dorso-ventral section through an eyecup, indicating the relative locations of the images shown in a-e. Arrow indicates the site of injection, ONH indicates the optic nerve head. (g) Correction of abnormal levels of opsin in the connecting cilium and pericilium of rod photoreceptors. Bar graph showing opsin immunogold gold particle density, along the length of the connecting cilium. Ultrathin sections of retinas from Myo7a-null and WT mice were stained with rod opsin antibody. The Myo7a-null retinas had been either not treated or treated with 1x or 1:100 AAV2-MYO7A or AAV5-MYO7A. N = 3 animals per condition. Bars indicate SEM.

Fig. 4

Expression of MYO7A from the overlapping AAV2-MYO7A dual vectors. (a) Diagram of the the overlapping AAV2-MYO7A dual vectors. The overlapping region contains 1365 bases. (b) Western blot of proteins from primary RPE cultures derived from Myo7a-null mice and not infected (lane 1), or infected with AAV2-MYO7A(dual) (lane 2); and primary RPE cultures derived from Myo7a^+/− mice (lane 3). All lanes were immunolabeled with anti-MYO7A and anti-actin. (c) Western blot of primary cultures derived from Myo7a-null mice and not infected (lane 1), or infected with AAV2-MYO7A(dual (lane 2) or with AAV5-MYO7A single vector (1x) (lane 3). Lanes were immunolabeled with anti-MYO7A and anti-actin. Densitometry of the actin labeling showed that lane 2 was loaded with 3-fold more protein than lane 3; the MYO7A to actin ratio is 7-fold greater in lane 3 compared with lane 2 in this blot. (d-g) Immunofluorescence of cultured RPE cells transduced with AAV2-MYO7A(dual). (d-f) Primary RPE cultures derived from Myo7a-null mice and (g) ARPE19 cells. Scale = 10 μm. (h) Bar graph indicating the distribution of MYO7A immunogold particle density among RPE cells from retinas of Myo7a-null mice, injected with AAV2-MYO7A(dual). N = 3 animals.

Fig. 5

Correction of mutant phenotypes, following subretinal injections with AAV2-MYO7A(dual). (a) Light microscopy of a semi-thin section from a treated Myo7a-null mouse retina. Region shown is near the injection site. Arrows indicate melanosomes in the apical processes. White lines indicate cells that still show the Myo7a-null phenotype, with an absence of melanosomes in the apical processes. Scale = 10 μm. (b) Low magnification of an immunoEM image of the RPE from a retina treated with AAV2-MYO7A(dual). As in a, the white line indicates a region that still shows the Myo7a-null phenotype. Rectangle, c, includes melanosomes in the apical region, indicating a corrected RPE cell. Scale = 10 μm. (c-e) Higher magnification of regions outlined by rectangles in b. MYO7A immunogold particles are indicated by circles. Scale = 1 μm. (f) Bar graph illustrating MYO7A immunogold particle density measured in RPE cells from Myo7a-null retinas, WT retinas, or from Myo7a-null retinas treated with AAV2-MYO7A(dual) and determined to be corrected or not corrected by the location of their apical melanosomes. N = 3 animals per condition. Bars indicate SEM. (g) ImmunoEM image of a rod photoreceptor cilium double-labeled with antibodies against MYO7A (small gold particles) and against rod opsin (large gold particles). MYO7A labeling is associated with the connecting cilium and periciliary membrane, indicating expression and correct localization of MYO7A, while this region is devoid of opsin labeling, which is restricted to the disk membranes, consistent with the WT phenotype, thus indicating correction of the mutant phenotype. Scale = 300 nm.