Lentiviral gene replacement therapy of retinas in a mouse model for Usher syndrome type 1B - PubMed (original) (raw)

Lentiviral gene replacement therapy of retinas in a mouse model for Usher syndrome type 1B

T Hashimoto et al. Gene Ther. 2007 Apr.

Abstract

One of the most disabling forms of retinal degeneration occurs in Usher syndrome, since it affects patients who already suffer from deafness. Mutations in the myosin VIIa gene (MYO7A) cause a major subtype of Usher syndrome, type 1B. Owing to the loss of function nature of Usher 1B and the relatively large size of MYO7A, we investigated a lentiviral-based gene replacement therapy in the retinas of MYO7A-null mice. Among the different promoters tested, a CMV-MYO7A chimeric promoter produced wild-type levels of MYO7A in cultured RPE cells and retinas in vivo. Efficacy of the lentiviral therapy was tested by using cell-based assays to analyze the correction of previously defined, MYO7A-null phenotypes in the mouse retina. In vitro, defects in phagosome digestion and melanosome motility were rescued in primary cultures of RPE cells. In vivo, the normal apical location of melanosomes in RPE cells was restored, and the abnormal accumulation of opsin in the photoreceptor connecting cilium was corrected. These results demonstrate that a lentiviral vector can accommodate a large cDNA, such as MYO7A, and mediate correction of important cellular functions in the retina, a major site affected in the Usher syndrome. Therefore, a lentiviral-mediated gene replacement strategy for Usher 1B therapy in the retina appears feasible.

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Figures

Figure 1

Schematic drawings of lentiviral vectors encoding the human MYO7A cDNA. The LV-MYO7A(A) vector encodes the CMV promoter upstream of the human MYO7A cDNA. The LV-MYO7A(B) vector contains a chimeric promoter, consisting of a partial CMV promoter (CMVp) and a 160-bp sequence from the human MYO7A (MYO7A_-E). The LV-MYO7A(C) encodes the 160-bp 'MYO7A_-E' fragment only. LTR, long terminal repeat; GAΔ, partial HIV1 GAG gene; RRE, Rev responsive element; PPT, polypurine track; ψ, viral packaging sequence; SD, splice donor; SA, splice acceptor. The arrowhead indicates the deletion within the 3′ LTR that causes self-inactivation of the viral LTR enhancer upon integration.

Figure 2

Lentiviral vector-mediated expression of MYO7A. (a–c) Immunofluorescence shows MYO7A immunolabel in (a) Myo7a+/− RPE, (b) _Myo7a_−/− RPE, 7 days after infection with LV-MYO7A(B), and (c) _Myo7a_−/− RPE, 7 days after infection with LV-MYO7A(A). (d) A phase contrast image of (c). (e) Western blot of MYO7A protein (upper panel) in Myo7a+/− RPE (Ctrl), _Myo7a_−/− RPE (Mut) and _Myo7a_−/− RPE, 5 days after infection with LV-MYO7A(B) (Mut+B). HSP60 labeling (lower panel) was used as a loading control. (f) Alkaline phosphatase (AP) histochemistry of a retinal section from an albino control mice. The retina was injected at P4 with LV-AP(B) and fixed at P14. Arrows and arrowheads indicate AP staining in the RPE and photoreceptor cells, respectively. Area shown is away from the site of injection. (g, h) MYO7A immunostaining (arrows) of the RPE in the central (g) and peripheral (h) regions of the retina from an albino _Myo7a_−/− mouse. The retina was injected centrally at P96 with the LV-MYO7A(B) virus and fixed 6 days later. Faint, non-specific staining is evident in the photoreceptor synaptic layer. RPE, retinal pigment epithelium; ONL, outer nuclear layer. Scale bars = 20 _μ_m (a–d), 50 _μ_m (f–h).

Figure 3

Lentiviral correction of _Myo7a_-mutant phenotypes in RPE primary cultures. (a–c) Immunofluorescence of ROSs remaining in Myo7a+/− RPE (a), _Myo7a_−/− RPE (b) and _Myo7a_−/− RPE infected with LV-MYO7A(B) (c). The ROSs are represented by green dots from opsin labeling (e.g. arrows). Nuclei are stained blue. (d) Bar graph showing the total number of ROSs per cell in Myo7a+/− RPE (Ctrl), _Myo7a_−/− RPE (Mut) and _Myo7a_−/− RPE infected with LV-MYO7A(B) (Mut+B). (e–g) Kymographs (showing distance traveled in relation to time) illustrate the differences in movements of individual melanosomes from Myo7a+/− RPE (e), _Myo7a_−/− RPE (f) and _Myo7a_−/− RPE infected with LV-MYO7A(B) (g). The more constrained movements of melanosomes in control and corrected RPE are evident by less displacement. Each line represents the movement of an individual melanosome. (h) Bar graph showing the average distance per 5 min, traveled by randomly selected individual melanosomes measured from Myo7a+/− RPE (Ctrl), _Myo7a_−/− RPE (Mut) and _Myo7a_−/− RPE infected with LV-MYO7A(B) (Mut+B). Scale bars (a–c) = 20 _μ_m. Error bars in (d, h) represent ±s.e.m.

Figure 4

Correction of melanosome localization in the RPE in vivo. Semithin (a–c) and ultrathin (d–f) LR White sections of (a) a Myo7a+/+ retina, (b) a _Myo7a_−/− retina, and (c–f) a _Myo7a_−/− retina, infected with LV-MYO7A(B) at P1 and analyzed at P16. In a–c, brackets indicate the RPE apical processes. Arrows indicate some of the melanosomes localized in the RPE apical processes. Arrows in (d, e) indicate the RPE cell boundaries. Note that the central RPE cell in the field does not contain any melanosomes in the apical region, whereas the two flanking cells do. The section has been immunogold-labeled for MYO7A. Boxed areas in d and e were enlarged in (e, f) respectively, to show immunogold particles (all have been circled). The cytoskeleton of the zonula adherens is evident in (d) across the entire profile of the central cell (bottom right arrow), indicating that the section is near the periphery of the cell. The lack of melanosomes evident in the apical RPE is not due to the plane of the section. In control RPE, melanosomes are obvious in the apical processes of cells sectioned in this manner. Scale bars: a–c, 10 _μ_m; d–f, 1 _μ_m. Bar graph (g) shows the relationship between the density of MYO7A immunogold particles and the observed correction of melanosome localization (data for each bar were obtained from 15 to 18 cells). Error bars represent ±s.e.m.

Figure 5

Correction of opsin distribution in the connecting cilia (arrows) of photoreceptor cells, following in vivo injection of LV-MYO7A(B). Opsin immunogold labeling of sections of photoreceptors from (a) a Myo7a+/+retina, (b) a _Myo7a_−/− retina, (c, d) a _Myo7a_−/− retina, infected with LV-MYO7A(B) at P1 and analyzed at P16. The photoreceptors in (c) are beneath an RPE cell that had correctly distributed melanosomes. Those in (d) are distant from the injection site, where the RPE melanosomes are all distributed as in MYO7A-null RPE cells. Scale bars: 500 nm. (e) Bar graph showing the concentration of opsin immunogold labeling in the cilia of photoreceptors like those in (a–d) (n = 43, 63, 28, and 17 cells, respectively). Error bars represent ±s.e.m.

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Lentiviral gene replacement therapy of retinas in a mouse model for Usher syndrome type 1B - PubMed (original) (raw)